Advances in Cell Aging and Gerontology, Volume 5: Programmed Cell Death, Volume I

VOLUME I PROGRAMMED C E L L DEATH

CELLULAR

AND MOLECULAR

MECHANISMS

PREFACE

This first installment of the two volume book on Programmed Cell Death provides a series of concise review articles presenting our current understanding of the cascades of events involved in apoptosis or programmed cell death and signaling pathways that can be engaged to prevent apoptosis. The past five years have seen a virtual explosion in the number of laboratories studying, at the molecular and cellular levels, the mechanisms that lead to cell death of the form called apoptosis which manifests specific morphological, biochemical, and molecular features that distinguish this form of cell death from necrosis. The morphological features of cells undergoing apoptosis include cell shrinkage, surface membrane blebbing, cytoskeletal perturbation, and nuclear chromatin condensation and fragmentation. In addition, organellar structure, (i.e. structure of mitochondria, endoplasmic reticulum, and golgi) are largely preserved in cells undergoing apoptosis. Biochemical features of apoptosis include activation of a class of cysteine proteases called caspases, mitochondrial membrane depolarization, and release of factors, such as cytochrome c, that seem to be critical for nuclear manifestations of apoptosis. In addition, a relatively stereotyped sequence of gene and protein expression occurs involving upregulation of proteins involved in inducing mitochondrial dysfunction and caspase activation, on the one hand, and anti-apoptotic genes that may be involved in suppressing the apoptotic cascade, on the other hand. The first chapter of this book by Jason Mills describes some of the hallmark characteristics of the execution-phase of apoptosis, that is the phase subsequent to initiation of the cell death process but prior to final nuclear changes. The second chapter by Anna Bruce-Keller describes a well-characterized death signaling cascade involving receptors, such as Fas and TNF receptors, that are linked through death domain motifs to the activation of cascades involving caspase activation and certain kinases that ultimately lead to cell death. Carol Troy covers, in some detail, the multiple caspases involved in various paradigms of apoptosis and how caspase cascades may effect the cell death process under various conditions. The next chapter focuses on events occurring in mitochondria during the cell death process that appear to be critical mediators of the final execution-phases of the cell death process. This chapter by Bernard Mignotte and Jean-Luc Vayssiere describes critical roles of membrane permeability transition and release of apoptotic factors. Manicini and colleagues then discuss a quite intriguing concept concerning the role of mitochondria in apoptosis. Namely, the evidence suggesting that mitochondrial proliferation induced by apoptoic signals contributes to mitochondrial dysfunction and cell death. The Bcl-2 family of proteins plays a powerful vii Cellular and Molecular Mechanisms Ed. by M.P. Mattson, S. Estus and V.M. Rangnekar. v i i - - v i i i © 2 0 0 1 Elsevier Science. Printed in the Netherlands.

viii

and key role in either promoting or preventing cell death. Qing Guo and colleagues describe the members of this emerging family of death-related proteins and describe what is known about their sites of action within the cell and their roles in various cell death paradigms in non-neuronal cells and neurons. The next chapter by Steve Tammariello, Gary Landreth, and Steven Estus focuses on the involvement of Jun kinases in programmed cell death in neurons. Work done in models of trophic factor withdrawal suggests a critical active role for these kinases in the cell death process. Vivek Rangnekar describes a novel and important death-promoting protein called Par-4, which appears to act very early in the cell death process prior to mitochondrial dysfunction and caspase activation. Par-4 contains both a leucine zipper domain and a death domain and can be induced at the translational level very early stages in the cell death process. It has been known for some time that cytoskeletal alterations occur in cells undergoing apoptosis. Rakesh Srivastava and Dan Longo describe the possible role of cytoskeleton alterations in the cell death process. The next two chapters consider two prominent anti-apoptotic signaling pathways. Mark Mattson describes the evidence supporting an anti-apoptoic role for the transcription factor NF-•B in a variety of cell death paradigms in different mitotic and post-mitotic cell types. NF-~B appears to be linked to genes that suppress oxidative stress and preserve cellular ion homeostasis. Quinn Deveraux and John Reed then cover an intriguing and emerging family of inhibitor of apoptosis proteins, IAP. The IAPs can suppress cell death in a remarkable array of programmed cell death paradigms. In the final chapter, Marcel Leist and Pierluigi Nicotera describes the roles of exicitatory amino acids and nitric oxide in programmed cell death in neurons. Emerging data suggest the overriding importance of activation of glutamate receptors in neuronal apoptosis in many different in vivo and cell culture settings. The links of calcium influx and nitric oxide production to the apoptotic machinery are discussed. The findings presented in this first volume lay the framework for the second volume of Programmed Cell Death which focuses on the involvement of apoptosis or anti-apoptosis in a variety of disease conditions ranging from neurodegenerative disorders to cancer to cardiovascular disease.

M E C H A N I S M S U N D E R L Y I N G THE H A L L M A R K F E A T U R E S OF THE E X E C U T I O N - P H A S E OF APOPTOSIS

JASON C. MILLS

Table of Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evolution o f the Execution-Phase Concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A Brief History o f the Study o f the Apoptotic Time Course . . . . . . . . . . . . . . . . . . . . . . . Asynchrony . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Circumventing A s y n c h r o n y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Defining the T e r m s of the Apoptotic Time-Course . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Importance o f the Individual Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stricter Definitions o f Time Course Phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A Brief Discussion of the C o m m i t m e n t Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Execution-phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nuclear Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Extranuclear Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary ................................................................

1 2 2 4 8 9 9 9 11 12 12 17 28

Introduction

Natural selection has favored the evolution of a process that allows organisms to dispose efficiently of defective, infected, or surplus cells. This process is apoptosis, the individual cell's consummate act of altruism. It is cell death characterized by: (1) lack of subsequent inflammation (which would damage neighboring, healthy cells); (2) inhibition of replication and spread of potentially dangerous genetic information from exogenous or endogenous sources; and (3) prevention of leakage of potentially injurious, normally sequestered, intracellular substances. To date, much more is understood about how, when, and where, apoptosis is initiated than about what happens once a cell has started the evolutionarily conserved apoptotic process itself. The reasons for this bias toward the study of upstream events in apoptosis are manifold, including simple technical limitations of assays for downstream events. Recently, however, with some technical advances and a general increase in interest in the process of apoptosis, large strides have been made toward understanding downstream events. The downstream, post-initiation phases of apoptosis begin with the commitment phase, when a cell uses highly evolutionarily conserved machinery to commit irreversibly to death and subsequently invoke the multiple pathways that characterize the final stage of apoptosis: the execution-phase. The execution-phase represents the culmination of apoptosis when the committed cell fulfills the evolutionarily driven purpose of apoptosis (namely, altruistic self-elimination). The telltale morphologic patterns that have defined 1 Cellular and Molecular Mechanisms Ed. by M.P. Mattson, S. Estus and V.M. Rangnekar. 1 -- 38 © 2001 Elsevier Science. Printed in the Netherlands.

2

J.C. Mills

apoptosis (e.g. dynamic membrane blebbing and chromatin condensation) since its initial recognition (Kerr et al., 1972) occur during the execution-phase and show remarkable evolutionary conservation, with the final stages of apoptosis looking about the same no matter the organism, tissue, or apoptotic inducing agent. It has been presumed that the conserved morphologic patterns reflect similar conservation of the underlying execution-phase intracellular pathways. As these execution-phase pathways drive the characteristic, altruistic elimination process that defines apoptosis, they are of considerable interest. The aim of this chapter is to review recent significant advances in understanding the intracellular pathways mediating the execution-phase. As some of the progress (namely much of that having to do with nuclear execution-phase events) has been reviewed elsewhere, this chapter will highlight extranuclear events, which have drawn, up until very recently, less focused attention.

Evolution of the Execution-Phase Concept A Brief History of the Study of the Apoptotic Time Course To understand the execution-phase, it helps to understand the concept of the stages of apoptosis as they have developed so far. To this end, this section represents a brief, broad, by no means comprehensive, history of the evolution of the time course of apoptosis. Initially, apoptosis was defined by its morphologic features. It was distinguished from necrosis as noninflammatory death affecting individual cells showing: chromatin condensation, nuclear envelope convolution, cytoplasmic blebbing or process formation, and cell shrinkage with ultrastructural preservation of organelles (see e.g. Wyllie et al., 1980). The bulk of early studies, for the most part, were concerned with characterizing hallmark features (e.g. Chu-Wang and Oppenheim, 1978; or for reviews: Clarke, 1990; Uchiyama, 1995) and identifying the conditions that lead to apoptosis in various cell types. These early studies established apoptosis as a common, if not fundamental, process, and increased interest in identifying the biochemical mechanisms involved. To investigate such mechanisms, cellular model systems were developed and characterized (see e.g. Wyllie et al., 1984; Martin et al., 1988; Pittman et al., 1993; Mills et al., 1995a). The bulk of apoptotic research switched to the factors responsible for initiating apoptosis within a cell population. If the execution-phase is the goal or end-stage of apoptosis, these studies were of what can be called the "initiation phase" of apoptosis: the first stage of the apoptotic time course, characterized by the cell's recognition and response to an apoptotic signal. General categories of inducers of apoptosis that were studied included growth factor withdrawal (e.g. interleukins or nerve growth factor; NGF), treatment with radiation (UV or X-ray), and pharmacological agents (e.g. chemotherapeutic drugs) (see Figure 1). The initiation phase lends itself easily to study because, for one, induction is a binary process. Either apoptosis ensues, or it is inhibited. Experiments can be designed so that inducing agents or their inhibitors can be applied and, 24 h later, cell death can be assessed. Another advantage of studying this phase is that most of the biochemical pathways involved are shared with already defined cellular processes and, thus, had previously been characterized. The overlap of pathways occurs because

Mechanisms in the Execution-phase

3

the initiation phase is essentially the study of upstream, signal transduction processes that a cell uses to respond to any of a variety of inputs. However, the pathway promiscuity that facilitates study of the initiation phase is no aid in understanding the apoptotic process per se, as the characteristic features of apoptosis occur downstream of initiation. Furthermore, apoptotic initiation phase processes tend to be cell- and usually inducer- specific with limited cross-talk; thus, results are of little general importance outside the specific model system being used. Clearly, to understand the specific process of apoptosis, investigations had to move further downstream from the initiation events. Two relatively early discoveries helped start investigation of downstream events, events that seemed specific to apoptosis and were beyond the initiating signal transduction machinery in the time course. One of these was the watershed discovery and characterization of the bcl-2 family of genes whose products seemed directly involved with the control of apoptosis in perhaps every cell in every species (Vaux et al., 1988; Hockenberry et al., 1990; Korsmeyer, 1992; Vaux et al., 1992). The second was the ongoing characterization of genes involved in C. elegans programmed death, among these the bcl-2 homologue, ced-9, along with ced-3 and ced-4, genes whose importance is in the execution-phase (Ellis et al., 1991; Hengartner and Horvitz, 1994). The finding that Bcl-2 and its related proteins (which, depending on the gene, are either pro- or anti-apoptotic: Adams and Cory, 1998) can initiate or inhibit death in almost every cell suggests that they are downstream of the multiple, promiscuous-initiating pathways. But the fact that, in most cases, initiation or inhibition of apoptosis by the bcl-2 family is still binary suggests that this family is still upstream of or right around the time of commitment of an individual cell to death. In other words, the bcl-2 family is involved with a stage between initiation and execution that has been called the "commitment phase". Study of this phase has been very active during the last several years, with considerable advances made in our understanding of the Bcl-2 family and the role of the mitochondrion (see later this chapter for discussion). Soon after an apoptotic cell commits to death, it undergoes a series of evolutionarily conserved, morphological, and biochemical changes. As mentioned, the stage when these occur has come to be called the execution-phase. The execution-phase is post commitment and, thus, downstream of Bcl-2 and its kin. Investigation into the execution-phase has stemmed primarily from efforts to characterize the mammalian family of ced-3 homologues: the caspases. A simple schematic of the relationship of the three active phases of apoptosis in a given apoptotic cell is identified in Figure 1. Note the initiation phase has many inputs; there are many pathways that will induce apoptosis. The commitment phase is the neck of the apoptotic funnel ', the gateway into the process. It is here modeled as only one common pathway that all cellular apoptotic inducers must invoke if the ultimate goal of apoptosis, the execution-phase, is going to be initiated. Once the execution-phase begins, the processes fan out again, as several relatively independent nuclear and nonnuclear pathways occur. Since the execution-phase is the primary concern of this chapter, these pathways will be discussed at length later. Up until very recently, research into the execution-phase has been limited relative to the other phases of apoptosis. One of the principal reasons for this delay is a technical problem that has long plagued study of execution-phase-specific pathways. This problem is discussed in the next section.

4

J.C. Mills

INITIATION

COMMITMENT

EXECUTION ~hromatin ondensation I

*,.

Nuclear

-''''"

isassembly .,Y . IDNAFrag- I ~,,,~mentation

I /

~ytoskeletal hanges I

,'

Extranuclear

Figure 1. Apoptosis can be divided into three distinct phases. The initiation phase is defined as the stage wherein the signal from any of a variety of inducers of apoptosis is reversibly transduced to the commitment phase. The commitment phase, which seems to involve a single evolutionarily conserved pathway, involves the cell's irreversible commitment to death, ending in the execution phase. The execution phase is irreversible and characterized by the numerous pathways (nuclear and extranuclear) that result in the apoptotic morphology of cell death. Abbreviations used: GF - growth factor; PM - plasma membrane.

Asynchrony To understand why studying the execution-phase is inherently problematic, one must understand that apoptosis is a remarkably asynchronous process. Following a given inducer, onset of the characteristic morphologic features within each cell occurs with marked temporal variability across a population (Oppenheim, 1991; Barres et al., 1992; Raff, 1992; Earnshaw, 1995; Collins et al., 1997). This asynchrony was one of the early defining features of the process in vivo. Wyllie and coworkers (1980), for example, stressed that apoptosis, unlike necrosis, affects individual cells and not populations. Indeed, this feature of apoptosis is one of the key reasons why the role of apoptosis in many human disease processes may have been '. and may continue to be '. underestimated by pathologists (see e.g. Camp and Martin, 1996, and Staunton and Gaffney, 1998, for recent discussions). Despite a presumably clonal population of cells under controlled conditions, apoptosis is asynchronous in the vast majority of in vitro model systems as well (Evan et al., 1992; Earnshaw, 1995; Mills et al., 1997; Messam and Pittman, 1998), although it should be noted that Fas-receptor-mediated apoptosis shows somewhat more synchrony. Asynchrony dictates that cells showing the morphologic features of apoptosis (i.e. those in the execution-phase) represent only a small percentage of the population at any given time. Thus, because biochemical assays generally require large numbers of viable cells, asynchrony hinders biochemical study of the execution-phase (Pittman et al., 1998). To illustrate the point, suppose one wanted to assess the role of specific, key aspects of energetics and metabolism in apoptosis. A typical time course of a population following an apoptotic stimulus is shown in Figure 2A. The curve crudely approximates first-order kinetics (i.e. a consistent proportion of the remaining

Mechanisms in the Execution-phase

5

A. 100

~

5o

25

I I Hrs Following Induction of Apoptosis

B. 100

75

g. 25 ~ATPtADP

I

I

I

Hrs Following Induction of Apoptosis

Figure 2. Apoptosis is asynchronous, and decrease in ATP levels (as well as many other biochemical parameters) parallels general loss of viability in the population. A) Idealized curve of viability loss following induction of apoptosis. The approximate first-order kinetics of the curve is typical of apoptosis in most model systems. Depending on the system, the x-axis would range from a maximum of 4 h to 4 days. B) Solid line --- plot of ATP levels (idealized from multiple experiments) in a population of apoptotic cells. Note that the curve follows similar kinetics to that in (A). Dashed line -- plot of ATP/ADP ratio in the same cell population. Note there is little change in this parameter (an estimate of energetic viability) across the population, indicating that loss of ATP merely parallels loss of viability across the culture. Thus, intracellular de-energizadon is likely a late-stage event in individual cells involving only a small portion of the viable population at any one time.

population is lost in each block of time). D e p e n d i n g on the cell system, the x-axis can range f r o m a p p r o x i m a t e l y 4 hours to several days, but the shape of the c u r v e is always essentially the same. In the current illustrative e x a m p l e , let us a s s u m e the c u r v e represents N G F - d e p r i v e d neuronal P C 1 2 cells and, thus, the abscissa scale ranges up to 36 h (Pittman et al., 1993; Mills et al., 1995a). A f e w years ago we (the author, with Mr. D a v i d Nelson, Drs. Maria Erecinska and R a n d y Pittman) p e r f o r m e d studies of various energetic parameters on populations of cells, with the hope o f delineating h o w cellular energetics c h a n g e d during early (i.e., a p p r o x i m a t e l y the first 6 h), middle

6

J.C. Mills

(approximately 6 to 14 h), and late (14 to 24 h) apoptosis. However, the resulting plot of ATP (and other triphosphate nucleotide) levels in the population almost directly paralleled the viability plot (Figure 2B, and see Mills et al., 1995b). The naive interpretation would be that ATP and energy decrease as cells progress through apoptosis, but this is true only for the population of cells. If the ATP data are replotted as a function of viable cells, a drop to about 80% of control levels occurs around when the first cells in the population begin to die. This level is then maintained more or less until quite late in the time course of death (not shown here; see Mills et al., 1995b). To reinforce the conclusions suggested by these results, we took the ATP-ADP ratio at each timepoint from the entire population of dying cells. This ratio remains essentially constant at around 85 to 90% of control nonapoptotic cultures, so that at 24 h when around half the cells have died, and the cultures are strewn with debris, the remaining cells still have energy levels similar to cells in untreated, healthy control cultures (not shown). The point of this example is that the asynchrony of apoptosis means that only a small percentage of cells will be undergoing characteristic execution-phase events at any time. These execution-phase cells will (no matter when the assay is done in the time course) thus be statistically overrun by unaffected cells, which always constitute the vast majority of the population. Therefore, our experiments demonstrate that changes in cellular energetics occur only late (i.e. possibly during execution-phase) within the time course of apoptosis of individual cells. There will be additional discussion about execution-phase energetics later in the chapter. Our early energetic studies also highlight the importance of specifically isolating execution-phase cells to study apoptosis-specific processes. To characterize the asynchrony observed by other investigators and highlighted by our early energetics studies, the same model system (withdrawal of NGF from neuronal differentiated PC12 cells: Greene and Tischler, 1976) was used to generate long-term time-lapse videotape of fields of apoptotic cells. An example of the type of results obtained using these methods is depicted in Figure 3A. Onset of the morphologic (execution) phase of death, as assessed by membrane blebbing, is highly variable across a population of cells. However, as can be seen in Figure 3B, once the execution-phase begins, it is highly temporally invariant, lasting approximately 45 min in this system (regardless of time following NGF withdrawal or differentiation state of the population at the time of withdrawal; Mills et al., 1997). Similar results were seen with nondifferentiated PCI2 cells (Messam and Pittman, 1998) and have been reported by others in a wide variety of systems (Evan et al., 1992; Earnshaw, 1995; Vidair et al., 1996; Simm et al., 1997). Thus, in most systems, the execution-phase lasts about an hour. One could see how such a short duration relative to the comparably long duration time course of death of the population would tend to skew any biochemical analyses. Graphically speaking, if one were to draw a vertical line through the time course in Figure 3A at any point after about 4 h, one would intersect approximately 85% of the morphologically normal cells. Thus, a harvest at that time to assess energetics or other biochemical parameters (e.g. protein phosphorylation) would be skewed to represent largely the stages of apoptosis upstream of the execution-phase. Only relatively recently have methods to study events specific to the execution-phase been developed and widely applied (and, similarly, only relatively recently have

Mechanisms in the Execution-phase

7

A. Cell# 1 2 3 4 5 6 7 8 9 10 11 12 13 14

==

-4

I

I~ I 0 4 Hrs After Apoptosis

I 8 Induction

12

I

16

I

I

20

24

I

B.

2

I

1.

I •

1

•

•

O.

Ip •

I

• B

•

•

•

•

• m

8

•

i •

•

•

w 0 D

D

m

I -4

• • •

•

I 0

Hrs After

4

Apoptosis

I 8

I 12

I 16

I 20

I 24

Induction

Figure 3. Time-lapse video confirms t h a t o n s e t o f t h e e x e c u t i o n p h a s e is a s y n c h r o n o u s b u t s h o w s t h a t t h e e x e c u t i o n p h a s e , o n c e i n i t i a t e d is of h i g h l y i n v a r i a n t d u r a t i o n . A) Hypothetical data characteristic of time-lapse video microscopy studies of apoptotic cultures. 14 individual ceils in a microscopic field are depicted. Shaded horizontal bars indicate when cells show execution phase morphology (as identified by dynamic membrane blebbing); thin straight lines indicate time spent showing normal morphology. Dashed vertical line indicates time of apoptosis induction. Note that cells can undergo apoptosis prior to induction (indicative of normal background cell loss). Note also that, at any given time, only a small percentage of cells ever show morphologic abnormalities. B) Hypothetical data characteristic of multiple videomicroscopic studies, representing duration of execution phase (as identified by blebbing) relative to time before or after induction of apoptosis in the population (time of induction represented by dashed vertical line). Each point represents a single cell. Note the execution phase lasts approximately 45 rains to an hour in the vast majority of cells regardless of time of onset.

e n o u g h d a t a e m e r g e d to w a r r a n t a r e v i e w o f w h a t is k n o w n specifically a b o u t the e x e c u t i o n - p h a s e ) . S u c h m o d e l s will b e d i s c u s s e d in the n e x t section.

8

J.C. Mills

Circumventing Asynchrony The technical problem asynchrony presents to the study of the execution-phase cannot be ignored. Several methods of directing studies specifically to the execution-phase have been developed recently. Some investigators have used differential centrifugation (e.g. Casciola-Rosen et al., 1994) or flow cytometry (e.g. MacFarlane et al., 1997) to isolate actively apoptotic (i.e. execution-phase) cells and study biochemical patterns. These techniques, similar to mechanical methods used to synchronize cells in mitosis, were successfully used in these studies to identify caspase substrates and activation patterns. The limitation of such mechanical isolation methods is that the execution-phase is of such short duration that most cells that have begun the execution-phase will be well into it (or dead) by the time cell sorting is completed. Also, it isn't clear what effect subjecting the (by definition) morphologically altered execution-phase cells to strong mechanical forces like centrifugation would have on many execution-phase processes. Lazebnik et al. (1993, 1994), Solary et al. (1993), and Newmeyer et al. (1994) developed cell-free systems to look at the effect of certain cytoplasmic factors on nuclear features of apoptosis. These systems manipulate cytoplasmic constituents to study the effects on isolated nuclei and have been very helpful in delineating nuclear execution-phase events. From these studies, the mitochondrion has emerged as the crux of the commitment phase. Cell-free systems have also been successfully employed to characterize the nuclear execution-phase. Most of the results detailing such nuclear events to be discussed later in this section were achieved in cell-free systems. Of course, the limitation of cell-free studies is that the nucleus is the endpoint. These systems don't lend themselves to study of cytoplasmic execution-phase events, and plasma membrane-associated events are impossible to study in cell-free systems (at least as they have been used to date). A few other techniques to circumvent asynchrony have been for the biochemical study of nonnuclear execution-phase events. One method is to scan cultures for execution-phase ceils (using nuclear executionphase morphology as a marker) and collect data from this small subset (see e.g. Oberhammer et al., 1994). We used this technique in NGF-deprived neuronal PC12 cells to study phosphorylation of the microtubule-associated protein tau and then used tau phosphorylation state to assess protein phosphatase 2A activity (PP2A; Mills et al., 1998a; see also later this chapter). This technique is limited because only a relatively small subset of biochemical experiments can be performed on individual cells. Another technique for studying nonnuclear execution-phase events was introduced by McCarthy et al. (1997) who found that cultures of fibroblasts overexpressing c-myc could be halted in early execution-phase by inhibitors of caspases (such as z-Val-Ala-Asp-fluoromethylketone, z-VAD-fmk). This technique was further developed to study mediators of apoptotic blebbing (Mills et al., 1998b). Its limitations are the expense of the inhibitors and the fact that, in some cells, z-VAD inhibits blebbing (and presumably other upstream execution-phase events) as well as the nuclear executionphase. Before discussing specific advances in our understanding of nuclear and nonnuclear execution-phase events, it is worth refocusing this discussion on definitions of the phases of death. With the recent mushrooming of research, there has been a concomitant Babelesque mushrooming of terms, and the execution-phase has come to

Mechanisms in the Execution-phase

9

mean different things to different investigators. In the next section, the phases will be as narrowly defined as possible. The definitions should be generally useful, but barring that, will at least be employed consistently to categorize the studies discussed in the remainder of this chapter.

Defining the Terms of the Apoptotic Time-Course The Importance of the Individual Cell To understand the time course of apoptosis, the three phases of initiation, commitment, and execution, one has to remember that these are stages of apoptosis within an individual cell, not within the population. For example, the "commitment point" in apoptosis was initially defined several years ago as the point in a population of dying cells, when 50% of the original population had irreversibly committed to die. That definition is useful for characterizing death kinetics in apoptotic cultures. But to understand, biochemically, what constitutes the initiation, commitment, and executionphases of apoptosis, one must restrict definitions to individual cells. Thus, in Figure 3A, if one follows the timeline of each individual cell, defining the execution-phase becomes relatively straightforward: The commitment and initiation phases are upstream and not represented by outward, morphologic changes; the execution-phase is downstream and characterized by morphologic changes like membrane blebbing. "Commitment point", for the purposes of the time course of apoptosis in an individual cell is the time when that cell commits irreversibly to death. Similarly, the terms "upstream" and "downstream" (or "early" and "late") should refer to progression through the phases of apoptosis in an individual cell. To illustrate a cell that begins blebbing (i.e. enters execution-phase) 6 h following NGF removal (Figures 2A, 3A) is still in a downstream portion of the apoptotic time course, even though 95% of the original population of cells would be alive at that timepoint. On the other hand, a cell that eventually dies is still in the initiation or commitment phase if it has not begun to bleb, even at 48 h, when 75% of the original population has already died. Stricter Definitions of Time-Course Phases So far, the initiation, commitment, and execution-phases have been introduced. In this section, they will be specifically defined (Table 1). The initiation phase comprises multiple pathways that are cell-type and inducing agent specific. NGF withdrawal, for example, induces apoptosis via jun kinase activation (Xia et al., 1995). Ceramidemediated (Cuvillier et al., 1996), PI3-kinase-mediated (Dudek et al., 1997; KauffmanZeh et al., 1997), and stress-activated protein kinase-mediated (Johnson et al., 1996; Verheij et al., 1996; Ichijo et al., 1997) pathways also have all been described. These are initiation events, because the initiation phase is what takes a given cell from recognition of an apoptotic stimulus to the evolutionarily conserved, bottleneck pathway (or gatekeeper) pathway of the commitment phase. So little is known about the onset of the commitment phase that it is difficult to define the transition from initiation, but it can

10

J.C. Mills

be assumed that the commitment phase begins once conserved, predominately deathspecific cellular machinery is engaged. Because the commitment phase is evolutionarily conserved, cytoplasm from any cell that has begun the commitment phase should be able to initiate apoptosis in a cell-free system or in another cell. As certain Bcl-2 family members can induce apoptosis-like death even in yeast and other unicellular eukaryotes, there is evidence for such conservation (see e.g. Ink et al., 1997; Madeo et al., 1997). Note: as we have defined it in this chapter, transition into the commitment phase is not equivalent to commitment to death. Rather, the commitment phase is when the various initiation pathways funnel into evolutionarily conserved machinery that, at some point during this phase, commit a cell to death. The commitment phase, as it is currently understood, hinges on mitochondrial mechanisms, and Bcl-2 family members play the principal role in commitment-phase regulation. Table 1.

Cellular Pathways Location in Cell Duration Cell-type Specificity Reversibility

Initiation P h a s e

Commitment Phase

Numerous(convergent) Plasma membrane, cytoplasm Highly variable (usually long) Cell-typedependent Reversible

One or a small number Numerous(divergent) Mitochondrion Whole cell

Evolution Conservation Variable

??? (probablyshort)

Execution-phase

Invariant,short (~1 hour)

Cell-type independent Cell-typeindependent Switch from reversible Irreversible to irreversibleoccurs during this phase Evolutionarily conserved Evolutionarilyconserved

The execution-phase begins with the pathways leading directly to the hallmark morphologic changes. In many, if not most, cells, it seems to begin with the pathway leading to dynamic membrane blebbing and is well past the actual cellular commitment point (McCarthy et al., 1997; Mills et al., 1997, Brunet et al., 1998; Messam and Pittman, 1998). Note that this is a simple but relatively more restrictive definition than many have adopted, but it serves the purpose of delineating execution from commitment. The execution-phase involves a branching out of several more or less parallel pathways from the bottleneck of commitment. These pathways, which lead to the various hallmark apoptotic changes, are likely much more conserved than those in the initiation phase, because apoptosis (by definition) looks more or less the same regardless of inducer or celltype. However, like the initiation phase, multiple concomitant parallel pathways can be ongoing. It is not binary with regard to death (unlike the commitment phase and the initiation phase). In other words, one can inhibit the morphologic changes individually or as a group and cell death still results. Staurosporine, for example, or inhibitors of myosin light-chain kinase can inhibit blebbing but not death (Mills et al., 1998b). Cytoplasmic and membrane execution

Mechanisms in the Execution-phase

I1

events can occur in cells without a nucleus (Jacobson et al., 1994). Indeed biochemical apoptotic cascades can be activated locally in synaptic compartments. Thus, Mattson et al. (1998a,b) showed that exposure of synaptosomes to staurosporine, or oxidative insults that induce apoptosis in intact neurons, can induce caspase activation, loss of plasma membrane phospholipid asymmetry, mitochondrial membrane depolarization, and release of factors into the cytosol capable of inducing nuclear chromatin condensation and fragmentation. Moreover, Par-4 (prostate apoptosis response-4), a novel apoptosis related protein, can be induced locally in_synaptic compartments. Par-4 is a critical mediator of mitochondrial dysfunction and caspase activation (Guo et al., 1998; Duan et al., 1999). Further, the various apoptotic nuclear events such as DNA fragmentation, chromatin condensation, and lamin disassembly are interrelated but distinct from one another; inhibition of any one pathway does not necessarily affect death (Lazebnik et al., 1993; Oberhammer et al., 1993; Tomei et al., 1993; D.Y. Sun et al., 1994; Hara et al., 1996; Mpoke and Wolfe, 1996; Allera et al., 1997; Hirata et al., 1998). With these definitions, the remainder of the chapter will discuss what constitutes the execution-phase but, first, a brief discussion of recent advances in the study of the commitment phase to aid in further delineating it from execution. A Brief Discussion of the Commitment Phase Commitment-phase research has been prolific and exciting in recent years. To study the commitment phase is to study the mitochondrion (Hirsch et al., 1997; Green and Reed, 1998; Mignotte and Vayssiere, 1998). Mitochondria are the source for two factors known to activate caspases in non-Fas-mediated apoptosis. These two factors are apoptosis-inducing factor (AIF) and cytochrome c. AIF is a protein that has been shown to activate procaspase-3 in vitro and leads to z-VAD-inhibitable initiation of executionphase events (Susin et al., 1996, 1997). Cytochrome c is an inner mitochondrial membrane protein involved in electron transport during oxidative phosphorylation. Disruption of mitochondrial membrane potential leads to release of cytochrome c and/or AIF (Marzo et al., 1998). Cytochrorne c is also released following a variety of apoptotic stimuli I with or without loss of membrane potential (Kluck et al., 1997; Yang et al., 1997) I and microinjected cytochrome c can induce apoptosis in many cell types (Li, F. et al., 1997; Brustugun et al., 1998). Large-scale cytochrome c release is thought by many to mark the commitment point of an individual cell to apoptosis (Green and Reed, 1998). Many Bcl-2 family members have long been known to localize to the mitochondrial outer membrane (Krajewski et al., 1993; Riparbelli et al., 1995) or to reside there under certain conditions (Zha et al., 1996; Wolter, 1997). Anti-apoptotic Bcl-2 family members can inhibit permeability transition, which leads to mitochondrial membrane potential loss (Zamzami et al., 1996; Hirsch et al., 1997). They can also inhibit cytochrome c release (Kluck et al., 1997; Yang et al., 1997). Bax (an apoptosisinducing Bcl 2 bomologue) can directly activate the permeability transition pore (a complex of several trans-mitochondrial membrane proteins that, when activated, forms pores, leading to permeability transition and eventual membrane potential collapse) by binding a key pore protein, the adenine nucleotide translocator (Marzo et al., 1998).

12

J.C. Mills

These various commitment-phase interactions have been linked to the subsequent execution-phase events via AIF and cytochrome c. As discussed above, AIF leads to caspase-3 activation and nuclear execution-phase events. Cytochrome c binds APAF1, the mammalian homologue of the ced-4 C. elegans death gene (Ellis and Horvitz, 1986), in a complex with pro-caspase-9 (P. Li et al., 1997; Zou et al., 1997). The binding of cytochrome c and APAF1 is thought to activate pro-caspase-9, which, in turn, activates caspase-3 and leads to the hallmark events of the execution-phase (P. Li et al., 1997; Wilson, 1998). Bcl-2 family members seem only to be involved with the commitment phase, and have little effect once the execution-phase is in progress (see e.g. Ellerby et al., 1997). Multiple studies of a variety of systems, however, suggest that caspase activation is a postcommitment event (excluding Fas-mediated death, where caspases play a role in the initiation phase). Inhibition of caspases in multiple-cell types delays or alters the morphology of death but does not prevent death from occurring (Xiang et al., 1996; McCarthy et al., 1997; Brunet et al., 1998; Mills et al., 1998b). On the other hand, some non-Fas-dependent apoptotic systems show a slightly more upstream role for a caspase or caspases (Deshmukh et al., 1996; Levkau et al., 1998). Clearly, this issue is not fully resolved (and caspase activation may in fact be different in different cell types), but for the purposes of this chapter, caspase induction will be considered an execution-phase (i.e. postcommitment phase) event.

The Execution-phase Nuclear Events The execution-phase, as discussed, seems to involve multiple nonintersecting pathways that lead from the relative bottleneck of commitment to the many distinctive, presumably critical features that distinguish apoptosis from nonphysiologic death. The data to date suggest that the mitochondrion mediates the commitment phase, but execution-phase transduction of the apoptotic signal must eventually involve pathways to the cytoplasm, cell membrane, and nucleus (Figure 4). So far, the majority of what is known about the execution-phase concerns nuclear events largely because cell-free systems have proved the most valuable for execution-phase studies so far, and these use nuclei as the target of study. Nuclear execution-phase events can be classified readily as: (1) upstream, involving transduction of the signal from the mitochondrion into the nucleus which is mediated predominately by caspases; and (2) downstream, the events or endpoints themselves (DNA laddering, lamin disassembly, chromatin condensation).

Upstream Events: The Caspases Caspases have been discussed and reviewed in numerous recent articles as well as elsewhere in this volume, so the discussion here will be brief. There are at least 13 different caspases (Thornberry and Lazebnik, 1998), and some of these have been shown to have several forms (Faleiro et al., 1997; Martins et al., 1997). Caspases are cysteine

Mechanisms in the Execution-phase

13

o

6 0~

oE ~

":"

~

0

~

~.,

o ~ o ~ ~.~,~ . ~ = ~ ~ ~ 0~,~ ~ _ ~ ~- ~

:.~

~

o

"~.o ~'=

~'~,

¢~.E = ' - ~

~

o

~

~.

~

o

.o

o

o o

" ~

o

~

~

o

~

~

I

~

on

Z 0i

~ c ~ c~ ," = ~-=~ ~ ~~ ~ . ~

~ ,.0 " ~ ,,~ c~ ~

~ ~

"~

n~n

.~:

~

N

. . . .

~-~ ~ < ~

o

~

""

0

"~

~=

o'~

~

'

0

.1=

~'

0

~'~

~ ~ o ~ ~.~

.° 6

14

J.C. Mills

proteases that exist in the cytoplasm in proenzyme form and have specific (relative to other protease families) substrate requirements: they cleave only after aspartate residues in certain configurations (Porter et al., 1997). Pro-caspases are excellent substrates for activated caspases; thus, it is generally thought that apoptosis may involve a cascade of caspase activation with potential positive feedback amplification. Like all proteases, caspase substrate specificity is easiest studied in vitro, but in vitro systems cannot account for compartmentalization, concentration, natural inhibitors, and competitors that occur in cells. Thus, there is still controversy about the in vivo "significance" of many proteins that have been identified as caspase substrates. Another complication of caspase study is that, although good inhibitors exist to differentiate intracellular caspases from other proteases, there are no well-characterized inhibitors that are specific to individual caspases (Villa et al., 1997). Despite the difficulty in studying normal intracellular caspase activity, the most important upstream execution-phase pathways have been recently established. As discussed in the previous section, cells leave the commitment phase with the release of cytochrome c, which binds APAF-1 and then apparently binds and activates caspase-9 (Figure 4). Activated caspase-9, in turn, activates caspase-3 (P. Li et al., 1997; Zou et al., 1997; Wilson, 1998). In Fas-mediated apoptosis, caspase-8, which is directly linked to the cell surface signaling transduction machinery, seems to play a role like that of caspase-9 (Srinivasula et al., 1996; Medema et al., 1997). What role the mitochondrion and commitment-phase machinery have in the Fas system is unclear, although the downstream, execution-phase events are similar to those in every other system. Inhibition of caspase-8 appears to inhibit Fas apoptosis completely, so caspase-8 is an initiation- or commitment-phase protease. Caspase-3, on the other hand, is downstream of commitment and seems to have the same execution-phase role it does in non-Fas-mediated apoptosis (Hirata et al., 1998). Caspase-3 is a key transducer and effector of execution-phase nuclear events in almost every system. Inhi~bition of caspase-3 in dozens of studies abrogates nuclear apoptosis, and caspase-3 has a broad range of biologically relevant substrates (some of which will be discussed). The other caspase with a well-characterized role in nuclear apoptosis is caspase-6. Caspase-6 seems to be the principal lamin caspase in intact cells (see below and Takahashi et al., 1996). Though in some systems caspase-6 was thought possibly to be upstream of caspase-3, recent studies in intact monocytes (MacFarlane et al., 1997), a cell-free Xenopus system (Farschon et al., 1997), and Fas-stimulated Jurkat cells (Hirata et al., 1998), place caspase-3 as an upstream activator of caspase-6. Corroborating a slightly downstream role for caspase-6, the nuclear lamin breakdown mediated by caspase-6 has generally been shown to be a later step that precedes chromatin condensation but follows initial DNA fragmentation (Oberhammer et al., 1994; Ghibelli et al., 1995; Lazebnik et al., 1995). Accordingly, inhibition of lamin breakdown either by caspase-resistant mutant lamins (Rao et al., 1996), or by specific caspase-6 (but not caspase-3) inhibition (Hirata et al., 1998), leads to DNA fragmentation and death, but not lamin breakdown and chromatin condensation. Summarizing, the upstream regulation of nuclear apoptosis appears to depend on activation of caspase-3, which cleaves a wide variety of substrates itself and also results in activation of caspase-6, which cleaves a few of its own, mainly downstream substrates. The next section

Mechanisms in the Execution-phase

15

discusses what is known about how the cleavage of some of these substrates may result in the nuclear features that have come to define apoptosis. Downstream Nuclear Apoptotic Events DNA fragmentation DNA fragmentation is one of the hallmarks of the execution-phase. The characteristic cleavage of DNA into fragments that are the size of multiples of the intemucleosomal length occurs in the vast majority of apoptotic cells and is very specific for apoptosis. This fragmentation appears to be both an early and late execution-phase event, as many cells show a sequence of fragmentation stages, and fragmentation can be inhibited at various intermediate steps by protease inhibitors or chelators (Weaver et al., 1993; Sun and Cohen, 1994; Lagarkova et al., 1995; Pandey et al., 1997). Generally, it is convenient to distinguish two phases. The first involves the formation of high molecular weight DNA fragments, (2 Mb down to 50 kb), and is thought to occur early in the execution-phase, upstream or concomitant to chromatin condensation (Ghibelli et al., 1995; Hara et al., 1996). The second and final phase involves cleavage of DNA into the characteristic, intemucleosomal-sized fragments (DNA "laddering"); this phase appears to be downstream of chromatin condensation and may, in fact, be among the last things a cell does before the end of the execution-phase (Mpoke and Wolfe, 1996). Numerous proteases and endonucleases have been implicated in DNA fragmentation in both upstream and downstream roles. The topic has been one of intense debate (see Montague and Cidlowski, 1996 for review and see also Pandey et al., 1997). However, there have been recent large strides forward in characterizing the mechanisms underlying this hallmark nuclear execution-phase event. For example, the recent identification of DNA fragmentation factor (DFF), a protein heterodimer of 40 and 45 kDa subunits that can be cleaved by caspase-3 to induce DNA fragmentation (Liu et al., 1997), represents the first direct link between the upstream event of caspase activation and one of the hallmark, downstream nuclear features of apoptosis. Two recent articles substantially strengthen the links between the caspases and internucleosomal fragmentation (Enari et al., 1998; Sakahira et al., 1998). These authors describe a caspase-activated deoxyribonuclease (CAD) and its inhibitor (ICAD). ICAD, which is a murine homologue of the human DFF 45 protein, appears to act as a chaperone necessary for CAD cytoplasmic stability until caspase-3 activation mediates ICAD cleavage. ICAD cleavage presumably frees CAD to enter the nucleus in the execution-phase, where the authors show specifically that internucleosomal cleavage is dependent on CAD. It isn't clear what role, if any, CAD has in the upstream, large molecular weight fragmentation. Thus, critical caspase-linked, upstream endonucleases may still be undiscovered. Other indirect links between DNA fragmentation and the caspases also exist. DNA-dependent protein kinase (DNA-PK), for example, is cleaved by caspase-3 and thereby inactivated (Song et al., 1996; McConnell et al., 1997). DNA-PK is a DNA repair enzyme whose inactivation would be expected to promote fragmentation by ceasing normal restorative efforts. Poly(ADP-ribose) polymerase (PARP) is one of the earliest and most well-characterized substrates of caspases. PARP is cleaved by caspase-3

16

J.C. Mills

(Takahasi et al., 1996) and/or caspase-7 (Lippke et al., 1996; Hirata et al., 1998). PARP is also involved in DNA repair and is deactivated by caspase cleavage, which, as for DNA-PK, may promote DNA fragmentation by hindering normal attempts to stop DNA damage (for review, see Porter et al., 1997). Chromatin Condensation

Chromatin condensation is the process characterized by the formation of globular and marginated, hemispheric aggregates of chromatin that are easily recognizable by light and electron microscopy. This phenomenon is perhaps the most universal, specific feature of apoptosis. Yet, very little is known about its underlying mechanisms. In addition to the technical hindrance of asynchrony (see above discussion), a factor that might have impeded study is that condensation was long thought to be a byproduct of DNA fragmentation. However, as discussed above, the two processes, if not wholly independent can be largely separated from each other and almost certainly involve many independent mechanisms. Two studies have addressed the issue of condensation itself and shown it to be associated with histone alterations. One group describes a specific deubiquination of histone H2A (Marushige and Marushige, 1995). Condensation of chromatin in metaphase of mitosis also involves a similar (albeit reversible) deubiquination of H2A. The authors state that ubiquination is thought to inhibit higher ordering of chromatin. It remains to be seen how H2A deubiquination might be mediated or how the process might be tied to already characterized executionphase pathways. The other histone study shows that histone H4 becomes deacetylated as condensing chromatin undergoes fundamental changes in nucleosome arrangement (Allera et al., 1997). They report progressive "clumping" of chromatin into dense central bodies with tortuous 11 nM fibers rosetting around each core. Nucleosomes were seen to lose their normal, 30 nM solenoid-ordered radial configuration and adopt tight face-to-face packaging. Again, it is unclear how these changes relate to known execution pathways. The only other significant headway into the problem of chromatin condensation involves a link to lamin disassembly. Lamins, which provide the structural support for the nuclear envelope, are disassembled in apoptosis by a process that requires caspases, most likely caspase-6 (Takahashi et al., 1996; Martelli et al., 1997; Hirata et al., 1998). As lamin disassembly and chromatin condensation are late execution-phase events and chromatin condensation involves substantial margination to the nuclear membrane, it was proposed several years ago that dismantling of the lamin network may be critical for condensation (Lazebnik et al., 1993). Although no direct biochemical evidence links the two processes, several other studies support the theory that lamin disassembly precedes and is required for normal condensation (Rao et al., 1996; Hirata et al., 1998). As caspase-mediated cleavage of lamins is well established, the dependence of chromatin condensation on lamin disassembly represents the closest link from chromatin condensation to the upstream execution-phase pathways.

Mechanisms in the Execution-phase

17

Extranuclear Events

Although there have been quite a few studies in recent years, the investigation of extranuclear execution-phase events has lagged that of nuclear events. Extranuclear events cannot be effectively studied in cell-free systems, so investigators have used a number of model systems for circumventing asynchrony. This variation in cell systems and the current lack of focused approach of the field toward any specific extranuclear apoptotic event complicate somewhat the review of this aspect of the execution-phase. Also, to date, there are no direct links between the commitment phase and actual downstream morphologic processes, as can be shown for caspase-mediated activation of CAD and DNA fragmentation. However, a number of potentially important caspase substrates have been identified that seem to play key roles in many cell types in the execution-phase. There has been some success in the study of membrane blebbing and apoptotic body formation. Namely, a few of the important cytoskeleton-associated proteins have been identified and a putative mechanism identified for cellular blebbing. Certain caspase-dependent pathways have been identified as critical for apoptotic body formation. Study of these cell shrinkage-related processes seems on the verge of important advances, and there are also some promising recent discoveries in other aspects of extranuclear execution-phase. The remainder of this chapter will consist of a relatively comprehensive review of what is known about extranuclear execution-phase events. Upstream Extranuclear Events

The upstream and downstream distinction that was made for nuclear execution-phase events is less useful in the extranuclear setting, largely because the events upstream of extranuclear execution-phase events are much less well-characterized. As will be discussed, there is a role for caspase-3 in several downstream extranuclear events; thus, presumably, activation of caspase-3 (the mechanism of which has already been discussed) is a key event. Interestingly, however, dynamic plasma membrane blebbing, a key feature of extranuclear apoptosis, one of the hallmarks of apoptosis, and one of the earliest signs of the execution-phase, does not require caspases in many model systems (Xiang et al., 1996; McCarthy et al., 1997; Mills et al., 1998b). One study using a Fas-dependent model death implicated caspase-7 in membrane blebbing, because specific inhibition of caspases 3 and 6 had no effect on the process (Hirata et al., 1998). However, because plasma membrane changes were only abrogated by inhibition of all caspases, and Fas-mediated death requires caspase-8 as an initiation or commitment phase event, caspase-7 could not be specifically implicated. Another group reports that caspase-3 is required for blebbing in TNF-mediated (i.e., Fas-dependent) death, as tumor cells homozygous for loss of caspase-3 function only bleb when caspase-3 is reintroduced (Janicke et al., 1998). There have been no studies to date of how cytochrome c might affect membrane blebbing or other extranuclear events independent of caspases, and this avenue of exploration might be promising. Until upstream events are better characterized, the best way to review and analyze the extranuclear executionphase is to examine the downstream processes.

18

J.C. Mills

Downstream Extranuclear Events Cell shrinkage, membrane blebbing, apoptotic body formation One of the hallmark, apoptosis-specific, execution events is plasma membrane blebbing (also termed "zeiosis" or "cytoplasmic boiling"). Necrotic cells also extrude blebs, but these are unidirectional (i.e., they are extruded but not retracted), large, and represent an end-stage change (see e.g. Coakley, 1987; Phelps et al., 1989; Herman et al., 1990). Membrane blebbing also sometimes occurs as a byproduct of mitosis, though, unlike apoptotic blebbing, mitotic blebbing is preceded by microvilli formation, is characterized by smaller blebs that are more varied in size, and shows a different pattern of underlying F-actin organization (Laster and Mackenzie, 1996). Apoptotic blebbing is one of the first signs of the execution-phase and is characterized by dynamic extrusion and retraction of surface protrusions. Apoptotic blebbing can be specifically inhibited without inhibition of nuclear changes or death (Cotter et al., 1992; Endresen et al., 1995; Levee et al., 1996; McCarthy et al., 1997; Mills et al., 1998b). This is to be expected, because, as previously discussed, execution-phase events are post-commitment to death, so inhibition of any execution-phase event does not stop a cell from dying. In addition, because postcommitment events seem to depend on multiple, non-intersecting pathways, inhibition of one pathway does not necessarily inhibit another. For example, as has been mentioned, DNA fragmentation and chromatin condensation, two nuclear phase execution-phase hallmarks, depend on parallel, mostly independent pathways, and neither is critical for a cell to die (Lazebnik et al., 1993; Sun et al., 1994; Rao et al., 1996; Brunet et al., 1998). Before discussing some of the mechanics underlying blebbing, it will help to organize and redefine some of the associated terminology. Blebbing is part of a broader category of extranuclear apoptotic change: reduction in cell volume. Almost all apoptotic cells shrink, the vast majority bleb, and many (though possibly not the majority) form apoptotic bodies. The onset of the execution-phase is marked by centripetal contraction and retraction from the substrate; the cell "rounds up". Next, an execution-phase cell blebs for a while, eventually stops, and then undergoes a final condensation or forms apoptotic bodies. These bodies are the result of the pinching of the endexecution-phase cell into multiple small, membrane-enclosed fragments (Kerr and Harmon, 1991). Blebbing and apoptotic body formation may be related, but they are not identical processes. For example, agents (such as the cytochalasins, inhibitors of actin polymerization, and staurosporine, a general kinase inhibitor) that inhibit blebbing (Tanaka et al., 1994; Endresen et al., 1995; Mills et al., 1998b) also inhibit apoptotic body formation (Cotter et al., 1992; Tanaka et al., 1994; Levee et al., 1996). However, early plasma membrane/cytoplasm changes, such as initial cell body shrinkage and blebbing, seem to be upstream of apoptotic body formation in most cells. One recent study showed that specific caspase-3 and 6 inhibition prevents apoptotic body formation but does not affect the blebbing that occurs earlier in the executionphase (Hirata et al., 1998). These data correlate well with the finding that apoptotic body formation depends on DEVD-inhibitable (i.e. caspase-3-1ike) caspases via a mechanism that involves caspase-mediated cleavage of p21-activated kinase 2 (PAK2) (Rudel and Bokoch, 1997;

Mechanisms in the Execution-phase

19

Lee et al., 1997; reviewed in Bokoch, 1998). Hirata et al. (1998) further define PAK2 cleavage (and apoptotic body formation) as being caspase-3 (and not 6 or 7) dependent. The PAKs are a family of kinases whose activity is modulated by the binding of the small G-proteins Rac and Cdc42 (for review: Sells and Chernoff, 1997). Caspasemediated cleavage results in activation of PAK2, which can then mediate apoptotic morphological changes, the best characterized of which seems to be apoptotic body formation. It is also of interest that PAKs have been found to phosphorylate myosin light chain in vitro (see Bokoch, 1998 for review and references), as conventional non-muscle myosin (myosin II) is regulated by specific phosphorylation of its light chain. As will be discussed later in this section, actin/myosin II interactions have been shown to be important in execution-phase cytoplasmic morphologic changes. The dramatic, dynamic shape changes that characterize apoptotic blebbing suggest that the cytoskeleton plays a prominent role in the process. And, indeed, it has been shown that blebbing, like apoptotic body formation depends on polymerized actin, as actin disassembly promoters, namely, the cytochalasins, prevent it (Endresen et al., 1995; Mills et al., 1998b). Furthermore, alterations in actin binding proteins have been associated with a blebbing morphology. For example, tumor cells lacking actin binding protein (also known as filamin) bleb continuously (Cunningham et al., 1992), and peroxideinduced blebbing correlates with alpha-actinin and talin cleavage (Miyoshi et al., 1996). Similarly, one of the best-characterized substrates of caspase-3-1ike proteases is alphafodrin, which is an actin-binding protein that helps stabilize actin-membrane interactions (Martin et al., 1995; Cryns et al., 1996; Nath et al., 1996; Vanags et al., 1996). And actin itself has been purported to be cleaved by caspases in apoptosis (Mashima et al., 1995; Kayalar et al., 1996; McCarthy et al., 1997), although many other groups report seeing no caspase-mediated actin proteolysis in multiple systems (Levee et al., 1996; Song et al., 1997; Brancolini et al., 1997). Still another group sees actin cleavage mediated by calpains but not caspases (Brown et al., 1997). In any case, neither actin nor fodrin cleavage has been strictly correlated with the onset of execution-phase (when blebbing occurs), and both may be late-stage execution-phase events. Furthermore, polymerized actin seems indisputably necessary for blebbing to occur, and proteolysis would counteract the process. And, finally, blebbing occurs in many systems even in the presence of broad-spectrum caspase inhibition (McCarthy et al., 1997; Mills et al., 1998b). Perhaps actin cleavage is an end-stage means of shutting offblebbing. Another cytoskeletal-associated protein, Gas2, is also caspase-cleaved in apoptosis and has been reported to play a role in apoptosis-associated extranuclear morphologic changes (Brancolini et al., 1995). Gas2, when hyperphosphorylated during GO to G1 transition, correlates with membrane ruffle formation. Overexpression of a caspasetruncated Gas2 leads to cell-shrinkage and process formation. Both the cell body and the processes in the shrinking cell become filled with prominent filamentous (i.e. phalloidin-stainable) actin, suggesting a role for caspase-cleaved Gas2 in apoptotic morphology changes. The authors note, however, that not all cells contain appreciable Gas2, and those that don't still undergo normal apoptosis. Furthermore, the authors do not report that the cytoskeletal reorganization seen during Gas2 overexpression involves blebbing, and other studies of actin in normal apoptosis do not correlate with the actin changes seen in the Gas2 cells (see below and Laster and MacKenzie, 1996;

20

J.C. Mills

Pitzer et al., 1996). Also, as mentioned, blebbing occurs in many cells in the absence of, or upstream of, caspase-3 activation. Thus, the role of Gas2 is likely to be primarily downstream of blebbing, perhaps to further promote shrinkage or to assist in apoptotic body formation. Recently, gelsolin, another protein with actin-interacting properties was identified as a caspase substrate (Kothakota et al., 1997). Gelsolin is a calcium-activated, polyphosphoinositide-inhibited actin regulatory protein that can sever and cap the fastgrowing end of filamentous actin (promoting disassembly of long filaments) but also can serve as a nucleator to promote reassembly (see e.g. Kwiatkowski et al, 1989; Weeds et al., 1991). Thus, gelsolin is often thought of as an actin reorganizer, breaking down long, older filaments in favor of new polymerization sites. Kothakota et al. (1997) have recently shown that gelsolin is specifically cleaved by caspase-3 in vitro and in vivo. The caspase-truncated gelsolin shows greatly increased actin severing in vitro, so that severing predominates over monomer binding (i.e. nucleating). Over-expression of cDNA designed to encode a protein mimicking post-caspase-cleaved gelsolin leads to rapid actin depolymerization and unspecified cytoplasmic morphologic changes. Neutrophils from gelsolin knockout animals (i.e. gelsolin -/- mice) take twice as long to initiate blebbing and DNA fragmentation as controls. HeLa cells, which are normally devoid of gelsolin, initiate apoptosis much more rapidly when they are transfected with full-length gelsolin. Interestingly, Kothakota et al. found no effect on caspase-3 activity by expression of gelsolin. This is in contrast to another recent study (Ohtsu et al., 1997), whose authors report inhibition of apoptosis by gelsolin overexpression in a mechanism that seems to be upstream of caspase-3 activation. In other words, gelsolin overexpression leads to blocking of both apoptosis and caspase-3 activation. The differing results suggest the need for additional, clarifying experiments. Both studies use predominately Fas-mediated apoptosis as a model system; thus, the implications for morphologic changes in non-Fas systems have not yet been explored. The implications of the studies with regard to the mechanisms underlying cell blebbing also await further study, as, although Kothakota et al. report "morphological changes" following overexpression of the activated, truncated gelsolin, they do not specify whether this means normal blebbing (e.g. with the same kinetics and duration). Also, they report that cells devoid of gelsolin (either HeLa cells or neutrophils from knockout animals) still bleb, even though onset is delayed, so gelsolin must not be absolutely necessary for blebbing. Finally, blebbing also occurs in caspase-3-inhibited cells, which further suggests that gelsolin truncation is not critical for the process of blebbing. It would be interesting to determine whether gelsolin expression has any effect on duration of blebbing. If not, one would expect that gelsolin may be involved either directly or indirectly in initiation, rather than maintenance of, blebbing. A direct role in initiation is a tempting hypothesis, because gelsolin is known to mediate switching of cytoplasm from a firmer ("gel") state to a more soluble ("sol") state; thus, in apoptosis, it might reorganize the cytoskeleton so that it is in a "bleb-ready" sol state (Yin and Stossel, 1979; see also: Janson and Taylor, 1993, for discussion of gel-sol switching). Presumably, as most cells continue to bleb for about an hour, some sort of steady state is reached, and gelsolin might not be expected to have any role in the maintenance of this state. The fact that homozygous

Mechanisms in the Execution-phase

21

gelsolin null cells still bleb could mean that another reason why blebbing might occur even in gelsolin's absence is that other actin severing proteins can compensate. Recent studies of neurons from gelsolin knock-out mice suggest an interesting role for this actin-severing protein in modulating neuronal cell death. Previous studies have shown that treatment of cultured hippocampal neurons with cytochalasin D, an actin-disrupting agent, can protect those neurons against excitotoxic and oxidative injury (Furukawa et al., 1995; Furukawa and Mattson, 1995). Data in the latter study suggested that the protective mechanism of actin depolymerization involved stabilization of intracellular calcium levels. Studies of hippocampal neurons cultured from gelsolin knock-out mice have shown that neurons lacking gelsolin exhibit increased vulnerability to excitotoxic cell death (Furukawa et al., 1997). Whole cell patch-clamp analyses of ion currents in neurons containing or lacking gelsolin showed that currents through voltagedependent calcium channels and NMDA type glutamate receptor channels are enhanced in neurons lacking gelsolin. Specifically, the rundown of currents seems to be reduced in neurons lacking gelsolin, which is correlated with enhanced calcium influx through voltage-dependent calcium channels and NMDA receptors (Furukawa et al., 1997). Administration of the seizure inducing excitotoxin, kainic acid, to wild-type and gelsolin knock-out mice demonstrated increased vulnerability of hippocampal neurons to seizureinduced injury in the gelsolin knock-out mice. Collectively, these studies have led to the proposal that gelsolin plays an important role in a feedback pathway in which increased intracellular calcium levels lead to active depolymerization, which in turn suppresses calcium influx through voltage-dependent channels and NMDA receptors. Recently, we also undertook experiments that helped elucidate some of the mechanisms underlying blebbing (Mills et al., 1998b). Z-VAD-fmk, a broad-spectrum caspase inhibitor, was used to arrest apoptosis of serum-deprived PC12 cells. Z-VAD delays death and nuclear apoptosis for several days in this system (similar to those reported in other models of apoptosis by McCarthy et al., 1997) without preventing normal progression into the blebbing phase. Thus, because z-VAD-arrested cells seem to stall in an early, pre-nuclear, execution-phase, much of the asynchrony inherent to apoptosis is avoided, and biochemical determinations of mediators can be performed. Using several kinase inhibitors, a critical role for myosin light chain kinase (MLCK) activation was demonstrated, and, in other experiments, myosin light chain phosphorylation was shown to correlate with a blebbing morphology. As mentioned earlier, conventional, smooth-muscle or non-muscle myosin (myosin II) is activated to contract against actin by phosphorylation of its myosin light chain, as mediated, in part, by myosin light chain kinase (Kohama et al., 1996; Gallagher et al., 1997). Myosin light chain (MLC) phosphorylation can also be stimulated by the small G protein, Rho, which activates Rho-associated kinase (ROK), which, in turn, both phosphorylates MLC directly (Amano et al., 1996) and inactivates the MLC phosphatase (Noda et al., 1995; Kimura et al., 1996). C3 transferase, an enzyme which inactivates Rho (Sekine et al., 1989; Aktories et al., 1990; Paterson et al., 1990) also inhibits blebbing. Cytochalasin D, similar to earlier reports (Endresen et al., 1995) also stops blebbing, and a general inhibitor of motor function of both conventional and non-conventional myosins (2,3-butanedione monoxime, BDM) greatly slows blebbing kinetics (i.e. both extrusion and retraction of blebs). A report from another group demonstrates blebbing in response

22

J.C. Mills

to microinjection of catalytically active MLC (Fishkind et al., 1991). Taken together, the data suggest that blebbing depends on myosin II-actin-mediated contraction. Myosin II in non-muscle cells has been implicated in multiple, critical forcegeneration-dependent functions (for reviews: Grebecki, 1994; Maciver, 1996; Mitchison and Cramer, 1996), but it is a large, filamentous protein, which is not thought to be membrane-associated. It is, on the other hand, thought to interact with the cortical actin ring, which is linked to the plasma membrane, regulating membrane structural stability and motility. Myosin contraction of the cortical actin is thought to produce a centripetal force that compresses the cytoplasm (see e.g. Grebecki, 1994). Although there are some decreases in the levels of detergent-soluble and insoluble, F-actin in the execution-phase (Levee et al., 1996), filamentous, polymerized (and polymerizable) actin is necessary for blebbing (Tanaka et al., 1994; Endresen et al., 1995; Levee et al., 1996; Mills et al., 1997). In addition, execution-phase F-actin has been shown to re-organize, concentrating specifically at the base (but not inside) of blebs (Laster and MacKenzie, 1996; Levee et al., 1996; Pitzer et al., 1996; Brancolini et al., 1997). A recent report indicates that cholecystokinin-mediated blebbing (which resembles apoptotic blebbing) in pancreatic acinar cells also appears to depend on actin reorganization along with myosin II relocation and activation (Torgerson and McNiven, 1998). To explain how a ring of myosin-II-generated actin contraction can cause extrusion of a bleb without being directly associated with the bleb itself, a model of blebbing was proposed. The model postulates that an as yet unidentified upstream execution-phase signal induces MLCK and/or Rho activation, which causes myosin II-mediated cell contraction and shrinkage (Figure 5). In non-apoptotic cells, just such a Rho-mediated contraction has been shown to occur without cell blebbing (Jalink et al., 1994; Tigyi et al., 1996; Gebbink et al., 1997). In apoptotic cells, however, membrane-actin interactions might not be as sturdy (due to weaknesses in membrane-cytoskeletal links) so that when myosin contracts the actin ring centripetally, the plasma membrane may not contract uniformly. Blebs may form in these regions of weakness as cytoplasm is extruded through a focal opening in the cortical cytoskeleton (Figure 5). At least two good candidates for proteins critical for maintaining actin-membranecytoskeletal links that are broken down in the execution-phase have been described. Fodrin, for example, is cleaved by caspases in apoptosis (as was noted above), but it also can be cleaved by calpains (Miyoshi et al., 1996; Nath et al., 1996). Interestingly, preliminary experiments in the PC12 system show that calpain inhibition prevents blebbing in apoptosis in the absence of caspases (Mills et al., 1998b). Another group has recently shown that ezrin/moesin/radixin (a family of closely related proteins that, like fodrin, normally help link the membrane to the actin cytoskeleton) translocate from the plasma membrane to the cytoplasm in an early stage of the execution-phase (Kondo et al., 1997). The translocation in these experiments was prevented by caspase inhibition, but apoptosis was induced in this system by the Fas receptor, and, as discussed above, caspase activation is an initiation (i.e. upstream) event in Fas-mediated apoptosis. Thus, ERM translocation may not directly involve caspases. One question not addressed by the model in Figure 5 is how apoptotic blebs retract. Given that the general inhibitor of myosin motor function, BDM, seems to inhibit both extrusion and retraction of blebs (whereas cytochalasins and MLCK inhibitors

23

Mechanisms in the Execution-phase

>

-~

..

m

~.~ ~

~

~°

, E . ~ ~ ~. N&

~ O=

_~0

/'/

./

/°

./

./

I

='=

~: ~

~

..~ ~

/

/

~-~

/

•- ~

~ ~~

~i. ~ ~~

~

~ ~ °°~ ~~ ~ a~ ~ ~

./ /"

/"

/"

./"

~'~

~.~

/./" /" /./

I

=~ ~ ._~

24

J.C. Mills

seem to inhibit extrusion only), there may be a role for non-conventional myosins in bleb retraction (Mills et al., 1998b). It is also possible that blebs simply recoil from some inherent elastic properties of the membrane. Blebbing is a dynamic process and, therefore, requires energy, but it may only need energy in one direction (in this case, i.e. for extrusion). A final note on blebbing: Although caspase inhibition does not stop blebbing in several non-Fas-mediated systems, it apparently does stop blebbing in normal endothelial cells induced to die by growth factor withdrawal (Levkau et al., 1998), in COS7 cells induced to die by tamoxifen (Nicole Stone and Randy Pittman, personal communication), and in HL-60 human leukemia cells treated with camptothecin (Shimizu and Pommier, 1997). Also, primary cultures of sympathetic neurons shrink over a long time course but don't bleb when treated with z-VAD under an apoptotic stimulus and apparently recover completely when the apoptotic stress is removed (Deshmukh et al., 1996). And dorsal root ganglion neurons in culture shrink but don't die for up to 11 days following an apoptotic stimulus when micro-injected with cDNA encoding crmA (Gagliardine et al., 1994), a viral gene whose product blocks caspases 1 and 8 and also possibly 3 and 6 (Villa et al., 1997). Thus, caspase activation may be upstream of blebbing in many cell types, just as it seems to be upstream of nuclear execution-phase events in almost all cell types. Perhaps in those cell types not dependent on caspases for blebbing and initial non-nuclear execution-phase changes, other proteases (such as calpains) may play the role caspases typically play in other cells (or even in the same cells under conditions when caspases are not being actively inhibited). Or put another way, maybe many cells have no means of compensating for caspase blockade in the initiation of extranuclear execution-phase events. Cytoskeletal events not necessarily related to cell shrinkage and miscellaneous cytoplasmic events

Given the dramatic shape changes in the execution-phase, most investigators have approached the cytoskeleton in apoptosis with the intent to understand blebbing and shrinkage. And, as discussed, actin and myosin seem to play an important role in cellular blebbing. However, other structural and cytoskeletal changes have been described that don't necessarily have a direct role in hallmark execution-phase morphologic features. For example, intermediate filaments, such as cytokeratin and vimentin, have been shown to aggregate and eventually be degraded in the executionphase (van Engeland et al., 1997). And it has long been known that the activation of transglutaminase (an enzyme that catalyzes protein-protein crosslinking) in apoptosis can lead to profound structural changes within cytoplasmic proteins (Fesus, 1993). Of the non-actin cytoskeletal proteins, most is known about microtubules. The microtubular system has been shown to undergo execution-phase reorganization by some groups (Pittman et al., 1994; Ireland and Pittman, 1995; van Engeland et al., 1997) and specific disassembly by others (Bonfoco et al., 1996; Canu et al., 1998; Mills et al., 1998a). Further temporal analysis of microtubules in apoptosis indicate that microtubule disassembly is one of the first execution-phase events in neuronally differentiated PC12 ceils (Mills et al., 1998a). Microtubule assembly in this system appears to be upstream of chromatin condensation. Two other early extranuclear

Mechanisms in the Execution-phase

25

execution-phase events were also noted: protein phosphatase 2A activation (PP2A) and resulting dephosphorylation of the microtubule-stabilizing protein tau. Microtubule disassembly also occurs in apoptotic Chinese hamster ovary (CHO) cells, along with PP2A-mediated activation of exogenously expressed tau (Mills et al., 1998a). Inhibition of microtubule disassembly (with the microtubule stabilizing agent taxol) inhibits PP2A activation and tau dephosphorylation, suggesting that microtubule disassembly occurs first, leading to release of microtubule-bound PP2A, which, in turn, mediates tau dephosphorylation. In cerebellar granule neurons, tau is not only dephosphorylated in the execution-phase but also cleaved in a caspase-dependent fashion (Canu et al., 1998). Tau dephosphorylation may lead to easier degradation, as hyperphosphorylated tau is not as easily proteolyzed and can accumulate in the characteristic paired helical filaments of Alzheimer's disease (Litersky and Johnson, 1992). Experiments from another group indicate that PP2A, in Fas-mediated death in Jurkat cells, can be a substrate for Caspase-3 (Santoro et al., 1998). The Caspase-3 cleavage site is on the regulatory Act subunit and results in apparent dissociation of the regulatory subunit from the PP2A trimer. Furthermore, the authors show a general increase in PP2A activity late in the time course of death of the population of cells. Although the increase was not correlated specifically to the execution-phase, the results suggest a model whereby Caspase-3 activates PP2A during the execution-phase. As execution-phase PP2A activation appears to occur in three vastly different cell types (PC12 neuronal, CHO stromal, and Jurkat lymphoid), it seems to be conserved and likely leads to other important execution-phase changes. It is known, for example, that the ezrin/moesin/radixin family of plasma membrane-actin linking proteins becomes enzymatically dephosphorylated early in apoptosis, which leads to their translocation away from the plasma membrane to the cytoplasm (see earlier section and Kondo et al., 1997). Although inhibition of microtubule disassembly with taxol does not prevent eventual apoptotic body formation (Levee et al., 1996), its effect on blebbing has not been reported. However, it has been shown that taxol can protect cultured hippocampal neurons against excitotoxic and apoptotic cell death (Furukawa and Mattson, 1995). An association is suggested because microtubule assembly by itself can initiate or augment blebbing even in non-apoptotic cells (Keller et al., 1985; Keller and Zimmerman, 1986; Mills et al., 1998a). Thus, microtubule disassembly could be one of the first stages in extranuclear apoptosis and might be a fruitful avenue of study to link commitment phase mitochondrial events to the extranuclear execution-phase. Changes in cell-cell and cell-matrix interaction in execution-phase In an organism, apoptosis is an act of cellular altruism that does not occur in a vacuum. The apoptotic cell communicates its status to neighboring cells. The vast majority of studies of apoptosis (and almost all those reviewed so far in this chapter), on the other hand, have only dealt with what the cell does to itself. One way a cell presumably aids in its own clearance is by shrinking and, in many cases, forming apoptotic bodies. But there is also evidence that execution-phase cells help induce their own phagocytosis by altering cell surface components. For example, one well-characterized change is the expression of phosphatidyl serine (PS) on the surface of dying cells. PS is a

26

J.C. Mills

phospholipid that is normally maintained on the internal plasma membrane leaflet. Apoptotic cells show altered activity of a flippase enzyme, whose activity is needed to maintain asymmetric PS localization, and extracellular exposure of PS has been shown to mediate recognition of apoptotic ceils by phagocytes (Fadok et al., 1992). In at least one system, PS exposure is downstream of blebbing and caspase activation (McCarthy et al., 1997). Several types of apoptotic ceils have also been shown to express cell-surface molecules which bind the macrophage vitronectin receptor (Savill et al., 1990) and/or thrombospondin (CD36) receptor (Ren and Savill, 1995; Ren et al., 1995). The nature of the execution-phase event that underlies these changes in cell surface expression remains to be characterized. Apoptotic cells also show execution-phase changes in cell-cell interactions with neighbors that are not necessarily associated with phagocytosis. As already discussed, one theory of blebbing is that it is simply a marker of increased centripetal contraction in cells that have lost much of their cell-cell and cell-matrix anchors (i.e. show the characteristic apoptotic "rounding up") and are on their way to shrinking and/or forming apoptotic bodies. It has been proposed that this process of shrinking and rounding up could play an important role at least in epithelial cells, which must preserve their sheetlike barrier function. Thus, it is likely epithelia would have mechanisms for preserving the integrity of the monolayer barrier when single cells die, rather than simply having large holes form that are gradually repaired by neighboring cells. It is possible, then, that the shrinkage of apoptosis, as mediated by actinomyosin contraction, actually pulls neighboring cells in to cover the space that would have been formed if the cell had simply died. Thus, the hallmark shrinkage (with its associated blebbing) seen in the execution-phase might represent a dying apoptotic cell exhausting its energy to pull on neighboring cells in an altruistic attempt to preserve the epithelial barrier without forcing neighboring cells to expend their own energy (Peralta-Soler et al., 1996; Mills et al., 1998b). In support of this hypothesis, in tissue culture monolayers of renal epithelial cells, apoptotic cell shrinkage in the execution-phase coincides with stretching of neighboring cells toward the center of the apoptotic cell. This process ensures that only very small (if any) holes form in the monolayer as the result of apoptosis (Peralta-Soler et al., 1996). As expected given the model for execution-phase shrinkage, cytochalasin-mediated filamentous actin disruption prevents this "rosetting" of neighboring cells around the apoptotic cell and leads to large gaps in the monolayer. The apoptotic cells show focal concentration of actin and the cadherin-catenin complex of epithelial cell adhesion proteins at the plasma membrane sites where the apoptotic and neighboring cells interact. Another group of investigators show similar shrinkage and rosette formation in apoptosis in retinal pigment epithelium (Nagai and Kalnins, 1996). These results are interesting in light of data from another group in a non-epithelial system showing caspase-mediated cleavage of 13-catenin in fibroblastic (non-epithelial) cells (Brancolini et al., 1997). 13-catenin (among other functions) links the transmembrane E-cadherin, which interacts on its extracellular surface with E-cadherin from neighboring cells, to the cytoplasmic ct-catenin, which interacts with the actin cytoskeleton. Presumably, cleavage of 13-catenin would disrupt cadherin/cytoskeletal interaction and lead to loss of cell-cell interaction in the execution-phase, a finding that would seem

Mechanisms in the Execution-phase

27

contrary to that seen in epithelial cells. The two systems might, in fact, be different, as fibroblasts do not form barrier functions and don't need to maintain monolayers. However, it should be noted that the authors in the fibroblast study were not specifically examining whether apoptotic shrinkage correlated with drawing in of neighboring cells. Thus, it is possible that, despite cleavage of cadherins, enough cell-cell contact is maintained for the shrinkage occurring in the apoptotic cell to mediate pulling on neighbors. A final example of execution-phase cell communication-related changes is the finding that caspases mediate cleavage of focal adhesion kinase (FAK) in endothelial cells (Levkau et al., 1998). FAK is a tyrosine kinase that plays an important role in integrating signaling from integrin receptors (which link the extracellular matrix with the actin cytoskeleton). Dismantling of FAK could be an important step in the very early rounding up of apoptosis. Cells centripetally contracting their actinomyosin cytoskeleton at the same time they lose matrix attachments would tend to round. Indeed, if epithelial cells cleaved FAK as they began to contract (while maintaining cell-cell interactions), the force of contraction would be applied all the more directly toward neighboring cells (rather than the matrix). Cellular energetics Another group of potentially interesting non-nuclear execution-phase events involves the changes in energy metabolism seen in apoptosis. ATP and cellular energy levels are maintained in cultures of asynchronous apoptotic cells (Mills et al., 1995b; for review, see also Nicotera and Leist, 1997), suggesting any substantial loss of cellular energetics occur in the execution-phase or perhaps the commitment phase. Cell-free models for execution-phase events require ATP (see e.g. Lazebnik et al., 1993; Ellerby et al., 1997), and the dramatic cytoskeletal blebbing and cell shrinkage involves myosin-mediated force generation and, therefore, must be energy-dependent. Furthermore, some groups report a direct requirement of ATP for the execution-phase (Nicotera and Leist, 1997; Tsujimoto, 1997). There may be substantial impairment of oxidative-phosphorylationmediated ATP generation in the execution-phase, though this is still an issue of active debate (Zhivotovsky et al., 1998 for review). Thus, it is possible that a larger portion of the ATP required for execution-phase events might be generated by glycolysis. Studies with apoptotic neuronal cells showed consistent, relatively pronounced increases in rate of lactate production in apoptotic cultures (Mills et al., 1995b), suggesting increased glycolytic flux possibly secondary to oxidative phosphorylation impairment (i.e. the "Pasteur effect"). An inhibitor of glycolysis, 2-deoxyglucose, has been shown to inhibit apoptosis (Thakkar and Potten, 1993). Some studies have shown that nonapoptotic cells might preferentially use glycolytic pathways for possibly focal generation of ATP in processes involving the actin cytoskeleton and the plasma membrane (see e.g. Pagliaro and Taylor, 1992). Thus, one intriguing, though entirely speculative hypothesis, for the data suggesting increased glycolysis in the execution-phase is that blebbing and cell shrinkage rely heavily on glycolysis to generate ATP for the actin-myosin force generation.

28

J.C. Mills

Summary As can easily be assessed from the other chapters in this volume (or by any literature search with "apoptosis" as a key word), the study of apoptotic death has mushroomed in the last few years, and many processes and mechanisms specific to apoptosis have been discovered and characterized. The explosion of interest has also naturally led to an impressive proliferation of terms. As it can be helpful to organize all the new findings within a few set categories, the first aim of this chapter was to define more specifically the terms relating to the progression of an individual cell through the time course of death. The time course can be summarized as follows: the initiation phase - (during which the cell responds to numerous types of signals, with cell-type-specific signal transduction pathways, to transduce the signal for apoptosis to the commitment phase); the commitment phase (during which the cell uses a limited number of evolutionarily conserved pathways to integrate the various initiation phase signals into a decision to refrain from apoptosis or commit irreversibly to it, thereby triggering the execution-phase), and the execution-phase l~during which the cell invokes multiple parallel pathways that lead to the hallmark features of apoptosis). The second aim of this chapter was to review recent advances in study of specifically the execution-phase, as this is the stage when all the defining (evolutionarily important) features of apoptosis occur, and there is great potential for fruitful research into identifying the mechanisms responsible for these features. Thus, in this chapter the execution-phase was grouped into nuclear and extranuclear events, and those were further subdivided into upstream and downstream processes. It was noted that the caspase family of proteases comprises the best-characterized upstream regulators in extranuclear and especially nuclear events. Downstream nuclear events were also discussed. These included: DNA fragmentation, which has been well studied and has seen recent great advances with the identification of caspase-inducible endonucleases, and chromatin condensation, which is a process that is still largely uncharacterized. Downstream extranuclear events were discussed, with special attention paid to cell blebbing and apoptotic body formation as part of general cell shrinkage. These morphologic changes have seen recent advances with the identification of caspase-activated kinases necessary for apoptotic body formation and characterization of the possible cellular mechanism underlying apoptotic contraction and blebbing. Other downstream extranuclear events were discussed and included: changes in the cell's interaction with its environment, a field that has seen some progress with the identification of several caspase-mediated changes in membrane-associated proteins; changes in cytoskeleton and cytoskeleton-associated proteins, which have been recently shown to occur in the form of microtubule disassembly and protein phosphatase 2A activation; changes in cellular energetics, which have not been as well studied in the execution-phase itself but the importance of which is hinted at by the dependence of the execution-phase on ATP (as possibly generated preferentially by glycolysis). All the recent advances in the study of the execution-phase discussed in this chapter serve only to highlight how complicated and multifaceted this stage of apoptosis is. Many of the mechanisms underlying the telltale execution-phase morphologic events, especially those mediating extranuclear events, have undoubtedly not even been identified, let alone characterized. Thus, the future seems bright for those interested in discovering how a cell brings about this fascinating, final act of altruism.

Mechanisms in the Execution-phase

29

Acknowledgments I am deeply indebted to Dr. Randy Pittman. The studies cited on which I am an author were performed in his laboratory under his supervision. I also would like to thank Jesse Mills, who helped to edit this chapter and provided useful and insightful comments.

References Adams, J.M. & Cory, S. (1998). The Bcl-2 protein family: arbiters of cell survival. Science 281, 1322-1326. Aktories, K., Mohr, C. & Koch, G. (1992). CIostridium botulinum C3 ADP-ribosyltransferase. Curt. Top. Micro. Immunol. 175, 115-131. Allera, C., Lazzarini, G., Patrone, E., Alberti, I., Barboro, P., Sanna, P., Melchiori, A., Parodi, S. & Balbi, C. (1997). The condensation of chromatin in apoptotic thymocytes shows a specific structural change. J. Biol. Chem. 272, 10817-10822. Amano, M., Ito, M., Kimura, K., Fukata, Y., Chihara, K., Nakano, T., Matsuura, Y. & Kaibuchi, K. (1996). Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase). J. Biol. Chem. 27 l, 20246-20249. Barres, B.A., Hart, I.K., Coles, H.S., Burne, J.F., Voyvodic, J.T., Richardson, W.D. & Raft, M.C. (1992). Cell death and control of cell survival in the oligodendrocyte lineage. Cell 70, 31-46. Bokoch, G.M. (1998). Caspase-mediated activation of PAK2 during apoptosis: proteolytic kinase activation as a general mechanism of apoptotic signal transduction? Cell Death Diff. 5,637-645. Bonfoco, E., Leist, M., Zhivotovsky, B., Orrenius, S., Lipton, S.A. & Nicotera, P. (1996). Cytoskeletal breakdown and apoptosis elicited by NO donors in cerebellar granule cells require NMDA receptor activation. J. Neurochem. 67, 2484-2493. Brancolini, C., Benedetti, M. & Schneider, C. (1995). Microfilament reorganization during apoptosis: the role of Gas2, a possible substrate for ICE-like proteases. EMBO J. 14, 5179-5190. Brancolini, C., Lazarevic, D., Rodrigquez, J. & Schneider, C. (1997). Dismantling cell-cell contacts during apoptosis is coupled to a caspase-dependent proteolytic cleavage of B-catenin. J. Cell Biol. 139, 759-771. Brown, S.B., Bailey, K. & Savill, J. (1997). Actin is cleaved during constitutive apoptosis. Biochem. J. 323,233-237. Brunet, C.L., Gunby, R.H., Benson, R.S.P., Hickman, J.A., Watson, A.J.M. & Brady, G. (1998). Commitment to cell death measured by loss of clonogenicity is separable from the appearance of apoptotic markers. Cell Death Diff. 5, 107-115. Brustugun, O.T., Fladmark, K.E., Doskeland, S.O., Orrenius, S. & Zhivotovsky B. (1998). Apoptosis induced by microinjection of cytochrome e is caspase-dependent and is inhibited by Bcl-2. Cell Death Diff. 5,660-668 Camp, V. & Martin, P. (1996). The role of macrophages in clearing programmed cell death in the developing kidney. Anat. Embryol. 194, 341-348. Canu, N., Dus, L., Barbato, C., Ciotti, M.T., Brancolini, C., Rinaldi, A.M., Novak, M., Cattaneo, A., Bradbury, A. & Calissano, P. (1998). Tau cleavage and dephosphorylation in cerebellar granule neurons undergoing apoptosis. J. Neurosci. 18, 7061-7074. Casciola-Rosen, L.A., Miller, D.K., Anhalt, G.J. & Rosen, A. (1994). Specific cleavage of the 70-kDa protein component of the U1 small nuclear ribonucleoprotein is a characteristic biochemical feature of apoptotic

30

J.C. Mills

cell death. J. Biol. Chem. 269, 30757-30760. Chu-Wang, I.-W. & Oppenheim, R.W. (1978). Cell death of motoneurons in the chick embryo spinal cord: I. A light and electron microscopic study of naturally occurring and induced cell loss during development. J. Comp. Neurol. 177, 59-86. Clarke, P.G.H. (1990). Developmental cell death: morphological diversity and multiple mechanisms. Anat. Embryol. 181,195-213. Coakley, W.T. (1987). Hyperthermia effects on the cytoskeleton and on cell morphology. Symp. Soc. Exp. Biol. 41,187-211. Collins, J.A., Schandl, C.A., Young, K.K., Vesely, J. & Willingham, M.C. (1997). Major DNA fragmentation is a late event in apoptosis. J. Histochem Cytochem. 45,923-934. Cotter, T.G., Lennon, S.V., Glynn, J.M. & Green, D.R. (1992). Microfilament disrupting agents prevent the formation of apoptotic bodies in tumor cells undergoing apoptosis. Cancer Res. 52, 997-1005. Cryns, V.L., Bergeron, L., Zhu, H., Li, H. & Yuan J. (1996). Specific cleavage of alpha-fodrin during Fas- and tumor necrosis factor-induced apoptosis is mediated by an interleukin-lbeta-converting enzyme/Ced-3 protease distinct from the poly(ADP-ribose) polymerase protease. J. Biol. Chem. 271, 31277-31282. Cunningham, C.C, Gorlin, J.B., Kwiatkowski, D.J., Hartwig, J.H., Janmey, P.A., Byers, H.R. & Stossel, T.P. (1992). Actin-binding protein requirement for cortical stability and efficient locomotion. Science 255, 325-327. Cuvillier, O., Pirianov, G., Kleuser, B,.Vanek, P.G., Coso, O.A., J.,Gutkind, S. & Spiegel, S. (1996). Suppression of ceramide-mediated programmed cell death by sphingosine-l-phosphate. Nature 381, 800-803. Deshmukh, D., Vasilakos, J., Dechwerth, T.L., Lampe, P.A., Shivers, B.D. & Johnson, E.M., Jr. (1996). Genetic and metabolic status of NGF-deprived sympathetic neurons saved by an inhibitor of ICE family proteases. J. Cell Biol. 135, 1341-1354. Duan, W., Rangnekar, V. & Mattson, M.P. (1999). Par-4 production in synaptic compartments folllowing apoptotic and excitotoxic insults: evidence for a pivotal role in mitochondrial dysfunction and neuronal degeneration. J. Neurochem. In press. Dudek, H., Datta, S.R., Franke, T.F., Birnbaum, M.J., Yao, R., Cooper, G.M., Segal, R.A., Kaplan, D.R. & Greenberg, M.E. (1997). Regulation of neuronal survival by the serine-threonine protein kinase Akt. Science 275,661-665. Earnshaw, W.C. (1995). Nuclear changes in apoptosis. Curr. Opin. Cell Biol. 7, 337-343. Ellerby, H.M., Martin, S., Ellerby, L.M., Nalem, S.S., Rabizadeh, S., Salvesen, G.S., Casiano, C.A., Cashman, N.R., Green, D.R. & Bredesen, D.E. (1997). Establishment of a cell-free system of neuronal apoptosis: comparison of premitochondrial, mitochondrial, and postmitochondrial phases. J. Neurosci. 17, 6165-6178. Ellis, H.M. & Horvitz, H.R. (1986). Genetic control of programmed cell death in the nematode C. elegans. Cell 44, 817-829. Ellis, R.E., Yuan, J. & Horvitz, H.R. (1991). Mechanisms and functions of cell death. Annu. Rev. Cell Biol. 7,663-698. Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A. & Nagata, S. (1998). A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391, 43-50. Endresen, P.C., Fandrem, J., Eide, T.J. and Aarbakke, J. (1995). Morphological modification of apoptosis in HL-60 cells: effects of homocystein and cytochalasins on apoptosis initiated by 3-deazaadenosine. Virch. Arch. 426, 257-266. Evan, G.I., Wyllie, A.H., Gilbert, C.S., Littlewood, T.D., Land, H., Brooks, M., Waters, C.M., Penn, L.Z. &

Mechanisms in the Execution-phase

31

Hancock, D.C. (1992). Induction of apoptosis in fibroblasts by c-myc protein. Cell 69, 119-128. Fadok, V.A. Voelker, D.R., Campbell, P.A., Cohen, J.J., Bratton, D.L., & Henson, P.M. (1992). Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J. lmmunol. 48, 2207-2216. Faleiro, L., Kobayashi, R., Fearnhead H. & Lazebnik Y. (1997). Multiple species of CPP32 and Mch2 are the major active caspases present in apoptotic cells. EMBO J. 16, 2271-2281. Farschon, D.M., Couture, C., Mustelin, T. & Newmeyer, D.D. (1997). Temporal phases in apoptosis defined by the actions of Src homology 2 domains, ceramide, Bcl-2, interleukin-lB converting enzyme family proteases, and a dense membrane fraction. J. Cell Biol. 137, 1117-1125. Fesus, L. (1993). Biochemical events in naturally occurring forms of cell death. FEBS Letters 328, 1-5. Fishkind, D.J., Cao, L. & Wang, Y. (1991). Microinjection of the catalytic fragment of myosin light chain kinase into dividing cells: effects on mitosis and cytokinesis. J. Cell Biol. 114,967-975. Furukawa, K., Smith-Swintosky, V.L. & Mattson, M.P. (1995). Evidence that actin depolymerization protects hippocampal neurons against excitotoxicity by stabilizing [Ca2+]i. Exp. Neurol. 133, 153-163. Furukawa, K. & Mattson, M.P. (1995). Cytochalasins protect hippocampal neurons against amyloid b-peptide toxicity: evidence that actin depolymerization suppresses Ca 2+ influx. J. Neurochem. 65,1061-1068. Furukawa, K. & Mattson, M.P. (1995b. Taxol stabilizes [Ca2+] and protects hippocampal neurons against excitotoxicity. Brain Res. 689, 141-146. Furukawa, K. Fux, W. Witke, W., Kwiatkowski, D.J. & Mattson, M.P. (1997). The actin-severing protein gelsolin modulates calcium channel and NMDA receptor activities and vulnerability to excitotoxicity in hippocampal neurons. J. Neurosci. 17, 8178-8186. Gagliardini, V., Fernandez, P.-A., Lee, R.K.K., Drexler, H.C., Rotello, R.J., Fishman, M.C. & Yuan, J. (1994). Prevention of vertebrate neuronal death by the crmA gene. Science 263,826-828. Gallagher, P.J., Herring, B.P. & Stull, J.T. (1997). Myosin light chain kinases. J. Muscle Res. Cell Motil. 18, 1-16. Gebbink, M.F.B.G., Kranenburg, O., Poland, M., van Horck, F.P.G., Houssa, B. & Moolenaar, W.H. (1997). Identification of a novel, putative Rho-specific GDP/GTP exchange factor and a RhoA-binding protein: control of neuronal morphology. J. Cell Biol. 137, 1603-1613. Ghibelli, L., Maresca, V., Coppola, S. & Gualandi, G. (1995). Protease inhibitors block apoptosis at intermediate stages: a compared analysis of DNA fragmentation and apoptotic nuclear morphology. FEBS Letters 377, 9-14. Grebecki, A. (1994). Membrane and cytoskeleton flow in motile cells with emphasis on the contribution of free-living amoebae. Int. Rev. Cytol. 148, 37-80. Green, D.R. & Reed, J.C. (1998). Mitochondria and apoptosis. Science 281, 1309-1312. Greene, L.A. & Tischler, A. (1976). Establishment ofa noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc. Natl. Acad. Sci. USA 73, 2424-2428. Guo, Q., Fu, W., Xie, J., Luo, H., Sells, S.F., Geddes, J.W., Bondada, V., Rangnekar, V. & Mattson, M.P. (1998). Par-4 is a novel mediator of neuronal degeneration associated with the pathogenesis of Alzheimer's disease. Nature Med. 4, 957-962. Hara, S., Halicka, D., Bruno, S., Gong, J., Traganos, F. & Darzynkiewicz, Z. (1996). Effect of protease inhibitors on early events of apoptosis. Exp. Cell Res. 223, 372-384. Hengartner, M.O. & Horvitz, H.R. (1994). Programmed cell death in Caenorhabditis elegans. Curr. Opin. Genetics Development. 4,581-6. Herman, B., Gores, G.J., Nieminen, A.L., Kawanishi, T., Harman, A. & Lemasters, J.J. (1990). Calcium and pH in anoxic and toxic injury. Crit. Rev. Toxicol. 21,127-148.

J.C. Mills

32

Himta, H., Takahashi, A., Kobayashi, S., Yonehara, S., Sawai, H., Okazaki, T., Yamamoto, K. & Sasada, M. (1998). Caspases are activated in a branched protease cascade and control distinct downstream processes in Fas-induced apoptosis. J. Exp. Med. 187,587-600. Hirsch, T., Matzo, I. & Kroemer, G. (1997). Role of the mitochondrial permeability transition pore in apoptosis. Biosci. Reports 17, 67-75. Hockenberry, D., Nunez, G., Milliman, C., Schreiber, R.D. 8,: Korsmeyer, S.J. (1990). Bcl-2 is an inner mitochondrial protein that blocks programmed cell death. Nature 348,334-336. Ichijo, H., Nishida, E., Irie, K., ten Dijke, P., Saitoh, M., Mofiguchi, T., Takagi, M., Matsumoto, K., Miyazono, K. & Gotoh, Y. (1997). Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science 275, 90-94. Ink, B., Zornig, M., Baum, B., Hajibagheri, N., James, C., Chittenden, T. & Evan, G. (1997). Human Bak induces cell death in Schizosaccharomyces pombe with morphological changes similar to those with apoptosis in mammalian cells. Mol. Cell. Biol. 17, 2468-2472. Ireland, C.M. & Pittman, S.M. (1995). Tubulin alterations in taxol-induced apoptosis parallel those observed with other drugs. Biochem. Pharmacol. 49, 1491-499. Jacobson, M.D., Burne, F.F. & Raft, M.C. (1994). Programmed cell death and Bcl-2 protection in the absence of a nucleus. EMBO J. 13, 1899-1910. Jalink, K., van Corven, E.J., Hengeveld, T., Morii, N., Narumiya, S. & Moolinaar, W.H. (1994). Inhibition of lysophosphatide- and thrombin- induced neurite retraction and neuronal cell rounding by ADP ribosylation of the small GTP-binding protein Rho. J. Cell Biol. 126, 801-810. Janicke, R.U., Sprengart, M.L., Wati, M.R. & Porter, A.G. (1998). Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J. Biol. Chem. 273, 9357-9360. Janson, L.W. & Taylor, D.L. (1993). In vitro models of tail contraction and cytoplasmic streaming in amoeboid cells. J. Cell Biol. 123, 345-356. Johnson, N.L., Gardner, A.M., Diener, K.M., Lange-Carter, C.A., Gleavy, J., Jarpe, M.B., Minden, A., Kafin, M., Zon, L.I. & Johnson, G.L.. (1996). Signal transduction pathways regulated by mitogenactivated/extracellular response kinase kinase kinase induce cell death. J Biol. Chem. 271, 3229-3237. Kauffmann-Zeh, A., Rodriguez-Viciana, P., Ulrich, E., Gilbert, C., Coffer, P., Downward, J. & Evan, G. (1997). Suppression of c-Myc-induced apoptosis by Ras signaling through PI(3)K and PKB. Nature 385, 544-548. Kayalar, C., Ord, T., Testa, M.P., Zhong, L.-T. & Bredesen, D.E. (1996). Cleavage of actin by interleukin lfS-converting enzyme to reverse DNaseI inhibition. Proc. Natl. Acad. Sci. USA 93, 2234-2238. Keller, H.U. & Zimmermann, A. (1986). Shape changes and chemotaxis of Walker 256 carcinosarcoma cells in response to colchicine, vinblastine, nocodazole and taxol. Invasion Metastasis 6, 33-43. Keller, H.U., Zimmermann, A., & Cottier, H. (1985). Phorbol myristate acetate (PMA) suppresses polarization and locomotion and alters F-actin content of Walker carcinosarcoma cells. Int. J. Cancer 36, 495-501. Kerr, J. F.R., Wyllie, A.H. & Curie, A.R. (1972). Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239. Kerr, J.F.R. & Harmon, B.V. (1991) Definition and incidence of apoptosis: an historical perspective. In: Apoptosis: the Molecular Basis of Cell Death, Current Communications in Cell & Molecular Biology, vol. 3. Cold Spring Harbor Laboratory Press, New York, pp. 5-29. Kimura, K., Iho, M., Amano, M., Chihara, K., Fukata, Y., Nakafuku, M., Yamamori, B., Feng, J., Nakano, T., Okawa, K., Iwamatsu, A. & Kaibuchi, K. (1996). Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science 273,245-248. Kluck, R.M., Bossy-Wetzel, E., Green, D.R. & Newmeyer, D.D. (1997). The release of cytochrome c from

Mechanisms in the Execution-phase

33

mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275, 1132-1136. Kohama, K., Ye, L.H., Hayakawa, K. & Okagaki T. (1996). Myosin light chain kinase: an actin-binding protein that regulates an ATP-dependent interaction with myosin. Trends Pharmacol. Sci. 17,284-287. Kondo, T., Takeuchi, K., Doi, Y., Yonemura, S., Nagata, S., Tsukita, S. & Tsukita, S. (1997). ERM (Ezrin/Radixin/Moesin)-based molecular mechanism of microvillar breakdown at an early stage of apoptosis. J. Cell Biol. 149, 749-758. Korsmeyer, S.J. (1992). Bcl-2 initiates a new category of oncogenes: regulators of cell death. Blood 80, 879-86. Kothakota, S., Azuma, T., Reinhard, C., Klippel, A., Tang, J., Chu, K., McGarry, T.J., Kirschner, M.W., Koths, K., Kwiatkowski, D.J. & Williams, L.T. (1997). Caspase-3-generated fragment of gelsolin: effector of morphological change in apoptosis. Science 278,294-298. Krajewski, S., Tanaka, S., Takayama, S., Shibler, M.J., Fenton, W. & Reed, J.D. (1993). Investigation of the subcellular distribution of the Bcl-2 oncoprotein: residence in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membranes. Cancer Res. 53, 4701-4714. Kwiatkowski, D.J. Janmey, P.A. & Yin, H.L. (1989). Identification of critical functional and regulatory domains in gelsolin. J. Cell Biol. 108, 1717-1726. Lagarkova, M.A., Iarovaia, O.V. & Razin, SV. (1995). Large-scale fragmentation of mammalian DNA in the course of apoptosis proceeds via excision of chromosomal DNA loops and their oligomers. J. Biol. Chem. 270, 20239-20241. Laster, S.M. & Mackenzie, J.M. (1996). Bleb formation and F-actin distribution during mitosis and tumor necrosis factor-induced apoptosis. Micro. Res. Tech. 34, 272-280. Lazebnik, Y.A., Cole, S., Cooke, C.A., Nelson, W.G. & Earnshaw, W.C. (1993). Nuclear events of apoptosis

in vitro in cell-free mitotic extracts: a model system for analysis of the active phase of apoptosis. J. Cell Biol. 123, 7-22. Lazebnik, Y.A., Kaufmann, S.H., Desnoyers, S., Poirier, G.G. & Earnshaw, W.C. (1994). Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE. Nature 371,346-347. Lazebnik, Y.A., Takahashi, A., Moir, R.D., Goldman, R.D., Piurier, G.G., Kaufman, S.H. & Earnshaw, W.C. (1995). Studies of the lamin proteinase reveal multiple parallel biochemical pathways during apoptotic execution. Proc. Natl. Acad. Sci. USA 92, 9042-9046. Lee, N., MacDonald, H., Reinhard, C., Halenbeck, R., Roulston, A., Shi, T. & Williams, L.T. (1997). Activation of hPAK65 by caspase cleavage induces some of the morphological and biochemical changes of apoptosis. Proc. Natl. Acad. Sci. USA 94, 13642-13647. Levee, M.G., Dabrowska, M.I., Lelli, J.L. & Hinshaw, D.B. (1996). Actin polymerization and depolymerization during apoptosis in HL-60 cells. Am. J. Physiol. 271 (Cell Physiol. 40) C1981-C1992. Levkau, B., Herren, B., Koyama, H., Ross, R. & Raines, E.W. (1998). Caspase-mediated cleavage of focal adhesion kinase pp125FAK and disassembly of focal adhesions in human endothelial cell apoptosis. J. Exp. Med. 187,579-586. Li, F., Srinivasan, A., Wang, Y., Armstorng, R.C., Tomaselli, K.J. & Fritz, L.C. (1997). Cell-specific induction of apoptosis by microinjection of cytochrome c. Bcl-xl has activity independent of cytochrome c release. J. Biol. Chem. 272, 30299-30305. Li P., Nijhawan, D., Budihardjo, I., Srinivasula, S.M., Ahmad, M., Alnemri, E.S. & Wang, X. (1997). Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91,479-489. Lippke, J.A., Gu, Y., Sarnecki, C., Caron, P.R. & Su, M.S. (1996). Identification and characterization of CPP32/Mch2 homolog 1, a novel cysteine protease similar to CPP32. J. Biol. Chem. 271, 1825-1828. Litersky, J.M. & Johnson, G.V. (1995). Phosphorylation of tau in situ: inhibition of calcium-dependent

34

J.C. Mills

proteolysis. J. Neurochem. 65,903-911. Liu, X., Zou, H., Slaughter, C. & Wang, X. (1997). DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell 89, 175-184. MacFarlane, M., Cain, K., Sun, X.-M., Alnemri, E.S. & Cohen, G. M. (1997). Processing/activation of at least four intedeukin-lB converting enzyme-like proteases occurs during the execution-phase of apoptosis in human monocyte tumor cells. J. Cell Biol. 137,469-479. Maciver, S.K. (1996). Myosin II function in non-muscle cells. BioEssays 18, 179-182. Madeo, F., Frohlich, E. & Frohlich, K.-L. (1997). A yeast mutant showing diagnostic markers of early and late apoptosis. J. Cell Biol. 139, 729-734. Martelli, A.M., Bareggi, R., Bortul, R., Grill, V., Narducci, P. & Zweyer, M. (1997). The nuclear matrix and apoptosis. Histochem. Cell Biol. 108, 1-10. Martin, D.P., Schmidt, R.E., DiStefano, P.S., Lowry, O.H., Carter, J.G. & Johnson, E.M. (1988). Inhibitors of protein synthesis and RNA synthesis prevent neuronal death caused by nerve growth factor deprivation. J. Cell Biol. 106, 829-844. Martin, S.J., O'Brien, G.A., Nishioka, W.K., McGahon, A.J., Mahboubi, A., Saido, T.C. & Green, D.R. (1995). Proteolysis of fodrin (non-erythroid spectrin) during apoptosis. J. Biol. Chem. 270, 6425-6428. Martins, L.M., Kottke, T., Mesner, P.W., Basi, G.S., Sinha, S., Frigon, N. Jr., Tatar, E., Tung, J.S., Bryant, K., Takahashi, A., Svingen, P.A., Madden, B.J., McCormick, D.J., Earnshaw, W.C. & Kaufmann, S.H. (1997). Activation of multiple interleukin-lbeta converting enzyme homologues in cytosol and nuclei of HL-60 cells during etoposide-induced apoptosis. J. Biol. Chem. 272, 7421-7430. Marushige, Y. & Marushige, K. (1995). Disappearance of ubiquinated histone H2A during chromatin condensation in TGFBl-induced apoptosis. Anticancer Res. 15,267-272. Marzo, I., Brenner, C., Zamzami, N., Jurgensmeier, J.M., Susin, S.A., Vieira, H.L.A., Prevost, M.-C., Xie, Z., Matsuyama, S., Reed, J.C. & Kroemer, G. (1998). Bax and adenine nucleotide translocator cooperate on the mitochondrial control of apoptosis. Science 281, 2027-2031. Mashima, T., Naito, M. Fujita, N., Noguci, K. & Tsuruo, T. (1995). Identification of actin as a substrate of ICE and an ICE-like protease and involvement of an ICE-like protease but not ICE in VP-16-induced U937 apoptosis. Biochem. Biophys. Res. Comm. 217, 1185-1192. Mattson, M.P., Keller, J.N. & Begley, J.G. (1998). Evidence for synaptic apoptosis. Exp. Neurol. 153, 35-48. Mattson, M.P., Partin, J. & Begley, J.G. (1998). Amyloid B-peptide induces apoptosis-related events in synapses and dendrites. Brain Res. 807, 167-176. McCarthy, N.J., Whyte, M.K.B., Gilbert, C.S. & Evan, G.I. (1997). Inhibition of Ced-3/ICE-related proteases does not prevent cell death induced by oncogenes, DNA damage, or the Bcl-2 homologue Bak. J. Cell Biol. 136, 215-227. McConnell, K.R., Dynan, W.S. & Hardin, J.A. (1997). The DNA-dependent protein kinase catalytic subunit (p460) is cleaved during Fas-mediated apoptosis in Jurkat cells. J. lmmunol. 158, 2083-2089. Medema, J.P., Scaffidi, C., Kischkel, F.C., Shevchenko, A., Mann, M., Krammer, P.H. & Peter, M.E. (1997). FLICE is activated by association with the CD95 death-inducing signaling complex (DISC). EMBO J. 16, 2794-2804. Messam, C.A. & Pittman, R.N. (1998). Asynchrony and commitment to die during apoptosis. Exp. Cell Res. 238, 389-398. Mignotte, B. & Vayssiere, J.-L. (1998). Mitochondria and apoptosis. Eur. J. Biochem. 252, 1-15. Mills, J.C., Wang, S.L., Erecinska, M. & Pittman, R.N. (1995a). Use of cultured neurons and neuronal cell lines to study morphological, biochemical, and molecular changes occurring in cell death. Methods Cell Biol. 46, 217-242.

Mechanisms in the Execution-phase

35

Mills, J.C., Nelson, D., Erecinska, M. & Pittman, R.N. (1995b). Metabolic and energetic changes during apoptosis in neural cells. J. Neurochem. 65, 1721-1730. Mills, J.C., Kim, L.H, & Pittman, R.N. (1997). Differentiation to an NGF-dependent state and apoptosis following NGF removal both occur asynchronously in cultures of PC12 cells. Exp. Cell Res. 231, 337-345. Mills, J.C., Lee, V. M.-Y. & Pittman, R.N. (1998a). Activation ofa PP2A-like phosphatase and dephosphorylation of tau protein characterize onset of the execution-phase of apoptosis. J. Cell Sci. 111,625-626. Mills, J.C., Stone, N.L., Erhardt, J. & Pittman, R.N. (1998b). Apoptotic membrane blebbing is regulated by myosin light chain phosphorylation. J. Cell Biol. 140, 627-636. Mitchison, T.J. & Cramer, L.P. (1996). Actin-based cell motility and cell locomotion. Cell 84, 371-379. Miyoshi, H., Umeshita, K., Sakon, M., lmajoh-Ohmi, S., Fujitani, K., Gotoh, M., Oiki, E., Kambayashi, J. & Monden, M. (1996). Calpain activation in plasma membrane bleb formation during tert-butyl hydroperoxide-induced rat hepatocyte injury. Gastroenterol. 110, 1897-1904. Montague, J.W. & Cidlowski, J.A. (1996). Cellular catabolism in apoptotic DNA degradation and endonuclease activation. Experentia 52,957-962. Mpoke, S. & Wolfe, J. (1996). DNA digestion and chromatin condensation during nuclear death in tetrahymena. Exp. Cell Res. 225,357-365. Nagai, H. & Kalnins, V. (1996). Normally occurring loss of single cells and repair of resulting defects in retinal pigment epithelium in situ. Exp. Eye Res. 62, 55-61. Nath, R., Raser, K.J., Stafford, D., Hajimohammadreza, I., Posner, A., Allen, H., Talanian, R.V., Yuen, P., Gilbertsen, R.B. & Wang, K.K. (1996). Non-erythroid alpha-spectrin breakdown by calpain and interleukin 1 beta-converting-enzyme-like protease(s) in apoptotic cells: contributory roles of both protease families in neuronal apoptosis. Biochemical J. 319, 683-690. Newmeyer, D.D., Farschon, D.M. & Reed, J.C. (1994). Cell-free apoptosis in Xenopus egg extracts: inhibition by Bcl-2 and requirement for an organelle fraction enriched in mitochondria. Cell 79, 353-364. Nicotera, P. & Leist, M. (1997). Energy supply and the shape of death in neurons and lymphoid cells. Cell Death Diff. 4, 435-442. Noda, M., Yasuda-Fukazawa, C., Moriishi, K., Kato, T., Okuda, T., Kurokawa, K.& Takuwa, Y. (1995). Involvement of Rho in GTP'/S-induced enhancement of phosphorylation of 20 kDa myosin light chain in vascular smooth muscle cells: inhibition of phosphatase activity. FEBS Letters. 367,246-250. Oberhammer, F., Wilson, J.W., Dive, C., Morris, I.D., Hickman, J.A., Wakeling, A.E., Walker, P.R. & Sikorska, M. (1993). Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO J. 12, 3679-3684. Oberhammer, F.A., Hochegger, K., Froschl, G., Tiefenbacher, R. & Pavelka, M. (1994). Chromatin condensation during apoptosis is accompanied by degradation of lamin A+B, without enhanced activation of cdc2 kinase. J. Cell Biol. 126, 827-837. Ohtsu, M., Sakai, N., Fujita, H., Kashiwag, M., Gasa, S., Shimizu, S., Eguchi, Y., Tsujimoto, Y., Sakiyama, Y., Kobayashi, K. & Kuzumaki, N. (1997). Inhibition of apoptosis by the actin-regulatory protein gelsolin. EMBO J. 16, 4650-4656. Oppenheim, R.W (1991). Cell death during development of the nervous system. Ann. Rev. Neurosci. 14, 453-501. Pagliaro, L. & Taylor, D.L. (1992). 2-Deoxyglucose and cytochalasin D modulate aldolase mobility in living 3T3 cells. J. Cell Biol.118, 859-863. Pandey, S., Walker, R. & Sikorska, M. (1997). Identification of a novel 97 kDa endonuclease capable of internuclesosmal DNA cleavage. Biochem. 36, 711-720.

36

J.C. Mills

Paterson, H.F., Self, A., Garrett, M.D., Just, I., Aktories, K. & Hall, A. (1990). Microinjection of recombinant p21 Rho induces rapid changes in cell morphology. J. Cell. Biol. 111, 1001-1007. Peralto Soler, A., Mullin, J.M., Knudsen, K.A. & Marano, C.W. (1996). Tissue remodeling during tunor necrosis factor-induced apoptosis in LLC-PK1 renal epithelial cells. Am. J. Physiol. 270, F869-F879. Phelps, P.C., Smith, M.W. & Trump, B.F. (1989). Cytosolic ionized calcium and bleb formation after acute cell injury of cultured rabbit renal tubule cells. Lab Investigation 60, 630-642. Pittman, R.N., Wang, S.L., DiBenedetto, A.J. & Mills, J.C. (1993). A system for characterizing cellular and molecular events in programmed neuronal cell death. J. Neurosci. 13, 3669-3680. Pittman, R.N., Messam, C.A. & Mills, J.C. (1998). Asynchronous death as a characteristic feature of apoptosis. In: Cell Death in Diseases of the Nervous System (Koliatsos, V.E and Rataned., R., eds.) Humana Press, Totowa, N J, 29-43. Pittman, S.M., Strickland, D. & Ireland, C.M. (1994). Polymerization of tubulin in apoptotic ceils is not cell cycle dependent. Exp. Cell Res. 215,263-272. Pitzer, F., Dantes, A., Fuchs, T. Baumeister, W. & Amsterdam, A. (1996). Removal of proteasomes from the nucleaus and their accumulation in apoptotic blebs during programmed cell death. FEBS Letters 394, 47-50. Porter, A.G., Ng, P. & Janicke, R.U. (1997). Death substrates come alive. Bioessays 19, 501-507. Raft, M.C. (1992). Social controls on cell survival and cell death. Nature 356, 397-400. Rao, L., Perez, D. & White, E. (1996). Lamin proteolysis facilitates nuclear events during apoptosis. J. Cell Biol. 135, 1441-1455. Ren, Y. & Savill, J. (1995). Proinflammatory cytokines potentiate thrombospondin-mediated phagocytosis of neutrophils undergoing apoptosis. J. Immunol. 154, 2366-2374. Ren, Y., Silverstein, R.L., Alien, J. & Savill, J. (1995). CD36 gene transfer confers capacity for phagocytosis of cells undergoing apoptosis. J. Exp. Med. 181, 1857-1862. Riparbelli, M.G., Callaini, G., Tripodi, S.A., Cintorino, M., Tosi, P. & Dallai, R. (1995). Localization of the Bcl-2 oncoprotein to the outer mitochondrial membrane by electron microscopy. Exp. Cell Res. 221,363-369. Rudel, T. & Bokoch, G.M. (1997). Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2. Science 276, 1571-1574. Sakahira, H., Enari, M. & Nagata, S. (1998). Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature 391,96-99. Santoro, M.F., Annand, R.R., Robertson, M.M., Peng, Y.W., Brady, M.J., Mankovich, J.A., Hacket, M.C., Ghayur, T., Walter, G., Wong, W.W. & Giegel D.A. (1998). Regulation of protein phosphatase 2A activity by caspase-3 during apoptosis. J. Biol. Chem. 273, 13119-13128. Savill, J., Dransfield, I., Hogg, N. & Haslett C. (1990). Vitronectin receptor-mediated phagocytosis of cells undergoing apoptosis. Nature 343, 170-173. Sekine, A., Fujiwara, M. & Narumiya, S. (1989). Asparagine residue in the Rho gene product is the modification site for botulinum ADP-ribosyltransferase. J. Biol. Chem. 264, 8602-8605. Sells, M.A. & Chernoff, J. (1997). Emerging from the PAK - the p21-acitvated protein kinase family. Trends Cell Biol. 7, 162-167. Shimizu, T. & Pommier, Y. (1997). Camptothecin-induced apoptosis in p53-null human leukemia HL60 cells and their isolated nuclei: effects of the protease inhibitors z-VAD-fmk and dichloroisocoumarin suggest an involvement of both caspases and serine proteases. Leukemia 11, 1238-1244. Simm, A., Bertsch, G., Frank, H., Zimmermann, U. & Hoppe, J. (1997). Cell death of AKR-2B fibroblasts after serum removal: a process between apoptosis and necrosis. J. Cell Sci. 110, 819-828.

Mechanisms in the Execution-phase

37

Solary, E., Bertrand, R., Kohn, K.W. & Pommier, Y. (1993). Differential induction of apoptosis in undifferentiated and differentiated HL-60 cells by DNA topoisomerase 1 and II inhibitors. Blood 81, 1359-1368. Song, Q., Lees-Miller, S.P., Kumar, S., Zhang, Z., Chan, D.W., Smith, G.C., Jackson, S.P., Alnemri, E.S., Litwack, G., Khanna, K.K. & Lavin, M.F. (1996). DNA-dependent protein kinase catalytic subunit: a target for an ICE-like protease in apoptosis. EMBO J. 15, 3238-3246. Song, Q., Wei, T., Lees-Miller, S., Alnemri,E., Watters, D. & Lavin, M.F. (1997). Resistance of actin to cleavage during apoptosis. Proc. Natl. Acad. Sci. USA 94, 157-162. Srinivasula, S.M., Ahmad; M., Fernandes-Alnemri, T., Litwack, G. & Alnemri, E.S. (1996). Molecular ordering of the Fas-apoptotic pathway: the Fas/APO-1 protease Mch5 is a CrmA-inhibitable protease that activates multiple Ced-3/ICE-like cysteine proteases. Proc. Natl. Acad. Sci. USA 93, 14486-14491. Staunton, M.J. & Gaffney, E.F. (1998). Apoptosis: basic concepts and potential significance in human cancer. Arch. Pathol. Lab. Med. 122, 310-319. Sun, D.Y., Jiang, S., Zheng, L.M., Ojcius, D.M. & Young, J.D. (1994). Separate metabolic pathways leading to DNA fragmentation and apoptotic chromatin condensation. J. Exp. Med. 179, 559-568. Sun, X.M. & Cohen, G.M. (1994). Mg(2+)-dependent cleavage of DNA into kilobase pair fragments is responsible for the initial degradation of DNA in apoptosis. J. Biol. Chem. 269, 14857-14860. Susin, S.A., Zamzami, M., Castedo, T., Hirsch, P., Marchetti, A., Macho, E., Daugas, M., Geuskens, M. & Kroemer, G. (1996). Bcl-2 inhibits the mitochondrial release of an apoptogenic protease. J. Exp. Med. 184, 1331-1342. Susin, S.A., Zamzami, N., Castedo, M, Daugas, E., Wang, H.-G., Geley, S., Fassy, F., Reed, J.C. & Kroemer, G. (1997). The central executioner of apoptosis: multiple connections between protease activation and mitochondria in Fas/APO-1/CD95- and ceramide-induced apoptosis. J. Exp. Med. 86, 25-37. Takahashi, A., Alnemri, E.S., Lazebnik, Y.A., Fernandes-Alnemri, T., Litwack, G., Moir, R.D., Goldman, R.D., Poirier, G.G., Kaufmann, S.H. & Earnshaw, W.C. (1996). Cleavage of lamin A by Mch2 alpha but not CPP32: multiple interleukin 1 beta-converting enzyme-related proteases with distinct substrate recognition properties are active in apoptosis. Proc. Natl. Acad. Sci. USA. 93, 8395-8400. Tanaka, Y., Yoshihara, K., Tsuyuki, M. & Kamiya, T. (1994). Apoptosis induced by adenosine in human leukemia HL-60 ceils. Exp. Cell Res. 213,242-252. Thakkar, N.S. & Potten, C.S. (1993). Inhibition of doxorubicin-induced apoptosis in vivo by 2-deoxy-Dglucose. Cancer Res. 53, 2057-2060. Thornberry, N.A. & Lazebnik, Y. (1998). Caspases: enemies within. Science 281, 1312-1316. Tigyi, G., Fischer, D.J., Sebok, A., Yang, C., Dyer, D.L. & Miledi, R. (1996). Lysophosphatidic acid-induced neurite retraction in PC12 cells: control by phosphoinositide-fa2÷ signaling and Rho. J. Neurochem. 66, 537-548. Tomei, L.D., Shapiro, J.P. & Cope, F.O. (1993). Apoptosis in C3H/10T1/2 mouse embryonic cells: evidence for internucleosomal DNA modification in the absence of double-strand cleavage. Proc. Nat. Acad. Sci. USA 90, 853-857. Torgerson, R.R. & McNiven, M.A. (1998). The actin-myosin cytoskeleton mediates reversible agonist-induced membrane blebbing. J. Cell Sci. 111,2911-2922. Tsujimoto,Y. (1997). Apoptosis and necrosis - intracellular ATP levels as a determinant for cell death modes. Cell Death Diff. 4, 429-434. Uchiyama, Y. (1995). Apoptosis: the history and trends of its studies. Arch. Histol. Cytol. 58, 127-137. Van Engeland, M., Kujipers, H.J.H., Ramaekers, F.C.S., Reutelingsperger, C.P.M. & Schutte, B. (1997). Plasma membrane alterations and cytoskeletal changes in apoptosis. Exp. Cell Res. 236, 421-430.

38

J.C. Mills

Vanags, D.M., Porn-Ares, M.I., Coppola, S., Burgess, D.H. & Orrenius, S. (1996). Protease involvement in fodrin cleavage and phosphatidylserine exposure in apoptosis. J. Biol. Chem. 271, 31075-31085. Vaux, D.L., Cory, S. & Adams, J.M. (1988). Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature 335,440-~42. Vaux, D.L., Weissman, I.L. & Kim, S.K. (1992). Prevention of programmed cell death in Caenorhabditis elegans by homan bcl-2. Science 258, 1955-1956. Verheij, M., Bose, R., Lin, X.H., Yao, B., Jarvis, W.D., Grant, S., Birrer, M.J., Szabo, E., Zon, L.I., Kyriakis, J.M., Haimovitz-Friedman, A., Fuks, Z. & Kolesnick, R.N. (1996). Requirement for ceramide-initiated SAPK/JNK signaling in stress-induced apoptosis. Nature 380, 75-79. Vidair, C.A., Chen, C.H., Ling, C.C. & Dewey, W.C. (1996). Apoptosis induced by X-irradiation of rec-myc ceils is postmitotic and not predicted by the time after irradiation or behavior of sister cells. Cancer Res. 56, 4116-4118. Villa, P., Kaufmann, S.H. & Earnshaw, W.C. (1997). Caspases and caspase inhibitors. Trends Biochem. Sci. 22, 388-393. Weaver, V.M., Lach, B., Walker, P.R. & Sikorska, M. (1993). Role of proteolysis in apoptosis: involvement of serine proteases in internucleosomal DNA fragmentation in immature thymocytes. Biochem. Cell Biol. 71,488-500. Weeds, A.G., Gooch, J., Hawkins, M., Pope, B. & Way M. (1991). Role of actin-binding proteins in cytoskeletal dynamics. Biochem. Soc. Trans. 19, 1016-1020. Wilson, M.R. (1998). Apoptosis: unmasking the executioner. Cell Death Diff. 5,646-652. Wolter, K.G., Hsu, Y.T., Smith, C.L., Nechushtan, A., Xi, X.G. & Youle, R.J. (1997). Movement of Bax from the cytosol to mitochondria during apoptosis. J. Cell Biol. 139, 1281-1292. Wyllie, A.H., Kerr, J.F.R. & Currie, A.R. (1980). Cell death: the significance of apoptosis. Int. Rev. Cytol. 68, 251-306. Wyllie, A.H., Morris, R.G., Smith, A.L. & Dunlop, D. (1984). Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis. J Pathol. 142, 67-77. Xia, Z., Dickens, M., Raingeaud, J., Davis, R.J. & Greenberg, M.E. (1995). Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270, 1326-1331. Xiang, J., Chao, D.T. & Korsmeyer, S.J. (1996). Bax-induced cell death may not require interleukin lf3-converting enzyme-like proteases. Proc. Natl. Acad. Sci. USA 93, 14559-14563. Yang, J., Liu, X., Bhalla, K., Kim, C.N., lbrado, A.M., Cai, J., Peng, T.I., Jones, D.P. & Wang, X. (1997). Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275, 1129-1132. Yin, H.L. & Stossel, T.P (1979). Control of cytoplasmic actin gel-sol transformation by gelsolin, a calciumdependent regulatory protein. Nature 281,583-586. Zamzami, N., Susin, S.A., Marchetti, P., Hirsch, T., Gomez-Monterrey, I., Castedo, M. & Kroemer, G. (1996). Mitochondrial control of nuclear apoptosis. J. Exp. Med. 183, 1533-1544. Zha, J., Harada, H., Yang, E., Jockel, J. & Korsmeyer, S.J. (1996). Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not Bcl-xL. Cell 87,619-628. Zhivotovsky, B., Wade, D., Gahm, A., Orrenius, S. & Nicotera P. (1994). Formation of 50 kbp chromatin fragments in isolated liver nuclei is mediated by protease and endonuclease activation. FEBS Letters 351,150-154. Zou, H., Henzel, W.J., Liu, X., Lutschg, A. & Wang, X. (1997). Apaf-1, a human protein homologous to C. elegans Ced-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90, 405-413.

DEATH DOMAIN SIGNALING AND ITS ROLE IN THE CENTRAL NERVOUS SYSTEM A N N A D O R A J. B R U C E - K E L L E R

Table of Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Death Domain Signaling Components -- Pathways to Cell Death . . . . . . . . . . . . . . . . . . . Initiation of the Death Signal -- Death Receptor Associated Proteins . . . . . . . . . . . . . . . Execution of the Death Signal -- Caspase Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . Alternative Death Receptor Signaling -- Pathways to Cell Life . . . . . . . . . . . . . . . . . . . . . Inhibitors of Apoptosis -- FLIPs and lAPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . NFKB Activation -- Survival Gene Induction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Death Receptors in the Central Nervous System -- Physiology and Pathophysiology . . . . Fas and the CNS: Tumorigenesis and Chronic Neurodegenerative Disease . . . . . . . . . . . TNF and Neuronal Resistance to Injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

39 40 41 44 46 46 48 50 51 52 56

Introduction

Apoptosis or programmed cell death is a controlled death process that eliminates excess cells for the greater good of multicellular organisms. Apoptosis occurs during both development and adulthood, and in addition to physiological roles in development and tissue remodeling, alterations in apoptosis contribute to such pathophysiological disease states such as cancer, Alzheimer's disease and stroke. Higher organisms have developed the means to rapidly and effectively eliminate unwanted cells by apoptosis through the activation of death-inducing receptors. While these receptors can transmit cytotoxic signals from the extracellular space and rapidly induce apoptosis in cells, in many cases these receptors are also involved in unrelated processes such as cell activation or differentiation. Whether death receptor activation ultimately results in life or death for a cell is both tightly regulated and cell-type specific, but is still a poorly understood process. The elucidation, therefore, of the receptor mechanisms underlying these divergent effects could augment scientific understanding o f cell death and survival in general, and also highlight potential clinical therapeutic strategies for human disease states. An important family of receptors with roles in cell death, differentiation, and survival in mammalian cells is the tumor necrosis factor (TNF) receptor superfamily. These death-inducing receptors are structurally similar in that each possesses two to six extracellular domains of imperfect repeats of 40 amino acids containing approximately six Cys residues (Figure 1). Fas (Apol/CD95), was the first family member to 39 Cellular and Molecular Mechanisms Ed. by M.P. Mattson, S. Estus and V.M. Rangnekar. © 2 0 0 1 Elsevier Science. Printed in the Netherlands.

39 -- 65

40

A.J. Bruce-Keller

be described exclusively in term of its death-inducing function (Itoh et al., 1991; Oehm et al., 1992). Comparisons of the intracellular domains of Fas with tumor necrosis factor receptor-1 (TNF-R1) revealed that both receptors contain a conserved cytoplasmic sequence of 80 amino acids. These conserved, C-terminal residues have since termed "death domains" and the ability of these receptors to transduce a death signal is strictly dependent on an intact death domain (DD) (Tartaglia et al., 1993a; Itoh and Nagata, 1993). Members of the DD receptor family that have been identified on the basis of sequence, structural, and functional similarity include to date (Figure 1): TNF-R1, Fas, TRAMP (death receptor 3/DR3/APO-3/LARD/wsl-1), TRAIL-R1 (death receptor 4/DR4/APO-2), TRAIL-R2 (death receptor 5/DR5), and DR6 (death receptor 6) (Bodmer et al., 1997; Chinnaiyan et al., 1996; Pan et al., 1997a,b, 1998; Sheridan et al., 1997; Walczak et al., 1997; Chaudhary et al., 1997; Schneider et al., 1997). The TRAIL subfamily also contains the truncated decoy receptors DcR1 (TRAIL-R3/TRID/LIT) and DcR2 (TRAIL-R4), which contain the external TRAIL-binding motif and the membrane anchor domain, but lack the full intracellular DD-containing region (Figure 1) (Pan et al., 1997a; Sheridan et al., 1997; Degli-Esposti et al., 1997; Marsters et al., 1997). The decoy receptors bind TRAIL, and thus limit the biological actions of the full length death receptors, an observation that confirms the importance of the intracellular DD motif in the transduction of apoptotic signals. Cognate ligands for many of the DD-containing receptors have been identified, and include Fas ligand (CD95L), TNFct, lymphotoxin-B (TNFB), and TRAIL (TNF-related apoptosis inducing ligand). Interestingly, the ligands themselves display some structural similarity, including a receptor-recognition motif composed of anti-parallel b-sheets (Schulze-Osthoff et al., 1998). It is generally assumed that biologically active ligands are made up of a trimer of identical proteins that activate their receptors by oligomerization (Banner et al., 1993; Dhein et al., 1992).

Death Domain Signaling Components -- Pathways to Cell Death

In general, apoptosis caused by DD-containing receptors is initiated by oligomerization of ligands and receptors, followed by intracellular protein-protein interactions that elicit the recruitment and physical association of caspases, followed finally by caspase activation and resultant cell death. Subsequent to receptor occupation, DD motifs can both self-associate and bind the DD of other proteins through electrostatic interactions. While DD sequences mediate receptor/adaptor interactions, truncated adapter proteins that lack DD's can still transduce a death when overexpressed to high enough levels. Hence, its seems that the DD acts to stabilize protein-protein interaction and facilitate the necessary local increase in death signaling molecules, a situation that is bypassed by overexpression. Since no post-translation modifications have been shown to be required for apoptotic signaling through DD-containing receptors, the oligomerization of receptors and recruitment of their associated "adapter" proteins seem to be the determining factors in executing the death cascade.

41

Death Domains and the Brian

TRAMP

Figure l.

TNF-R1 (~

Fas (CD95/APO-1) TRAIL-R1 (DR4) TRAIL-R2 DcR1 (DR5) Dr6 ~-~ (TRAIL-R3) DcR2 . (TRAIL-R4)

Schematic illustration of death domain-containing receptors. Members of this receptor family

are characterized by the presence of a conserved, 80 amino acid intracellular sequence known as the death domain, here depicted as a striped, upright rectangle. Additionally, these receptors contain similarities in their extracellular domain, consisting of two to four cystiene-rich repeats, depicted here as open ovals. The decoy receptors DcR1 and DcR2 either lack an intracellular domain, or contain a truncated, inactive death domain, and serve to limit activtion of functional TRAIL receptors.

Initiation of the Death Signal -- Death Receptor Associated Proteins The best characterized DD-containing signal transduction pathway is the Fas system (Figure 2), activated by Fas ligand or specific Fas antibodies. FADD(MORT-1), originally cloned by the two-hybrid method using Fas as a bait (Boldin et al., 1995; Chinnaiyan et al., 1995), interacts with Fas through its DD, and can induce death by itself when overexpressed in cell lines (Chinnaiyan et al., 1995). Together with the death receptor (Fas), these proteins form the death-inducing signaling complex (DISC). FADD can be dissociated into an N-terminal "MORT" domain and the C-terminal DD. The MORT domain contains a motif called the death effector domain (DED), as it can transduce the death signal even in the absence of the C-terminal DD (Schulze-Osthoff et al., 1998). On the other hand, a C-terminal truncated protein with only the DD acts as a dominant negative inhibitor of Fas-induced death, suggesting that the DD is required for proper association of FADD with the intracellular region of Fas, while the DED is coupled to intracellular death machinery. FADD was found to associate with a second adapter molecule called FLICE(MACH/caspase 8), again using the yeast two-hybrid system (Boldin et al., 1996; Muzio et al., 1996). FLICE (for FADD-like ICE) contains two N-terminal DEDs that associate with the DED of FADD, and a C-terminal domain that has the typical structure of a cystein protease like interleukin b-converting enzyme (ICE). FLICE belongs to the cystein proteases of the caspase family, and is generally referred to as caspase 8 (Almenri et al., 1996). Following Fas activation, FADD and caspase 8 are requited to the DISC within seconds, and binding of caspase 8 to FADD causes structural modification that result in autoproteolytic activation of the caspase.

42

A.J. Bruce-Keller

The active subunits pl0 and p18 are released into the cytoplasm, while portions of the procaspase domain remain bound to the DISC. Presently, it is assumed that active caspase 8 subunits cleave subsequent caspases and additional death substrates leading to execution of apoptosis (Schulze-Osthoff et al., 1998). Thus, caspase 8 provides a direct link from the membrane signal to the proteolytic death execution stage (see following section -- Execution of the Death Signal -- Caspase Activation). Again, the DED domains of caspase 8 are responsible for the association with FADD, and can act as dominant-negative inhibitors of Fas-induced death when overexpressed in the absence of the C-terminal end; and several natural isoforms which act as dominant-negative inhibitors have been described (Boldin et al., 1996). Death signaling through TNF-R1 shares many similarities with the Fas pathway (Figure 2). Upon cross-linking, TNF-R1 rapidly recruits a molecule called TRADD (TNF receptor associated death domain-containing protein), which in turn interacts with FADD. (Schulze-Osthoff et al., 1998). Death signaling through TRADD requires an ICE-family protease activity to induce cell death (Hsu et al., 1995; Miura et al., 1995), likely reflecting the association with caspase 8 through FADD. Similar to FADD, the C-terminus of TRADD contains a DD which enables self-association and interaction with the DD's of FADD and other signaling proteins. However, TRADD does not contain the typical N-terminal DED, and hence death signals are propagated through interactions with other DD adapter proteins. In addition to FADD, TRADD also associates with the DD containing protein RIP (receptor interacting protein) as well as the RING domain adapter protein TRAF2 (TNF receptor associated protein 2) which does not have a DD (Hsu et al., 1995, 1996a,b). Both TRADD and RIP can induce apoptosis, but also cause the activation of NFKB, which is a common component of TNF signaling (Hsu et al., 1995; Park and Baichwal, 1996; Ting et al., 1996). The TRAF2-associated kinase NIK (NFKB-inducing kinase) has recently been shown to be important in TNF-Rl-mediated NFKB activation, in that it facilitates the activation of IKB kinase (Verma and Stevenson, 1997) and subsequent I~B phosphorylation and degradation (see NFKB Activation -- Survival Gene Induction). RIP contains an N-terminal kinase domain and a C-terminal DD, and was originally thought to play a role in Fas signaling (Stanger et al., 1995). However, later studies demonstrated that RIP does not bind directly to Fas, and is recruited to TNF-R1 through TRADD and TRAF2 (Hsu et al., 1996a). TRAF2 leads to the activation of the NFr~B, which elicits anti-apoptotic signaling in cells (see NF~B Activation -- Survival Gene Induction) and hence may protect cells from apoptosis. TNF-R1 activation has also been reported to induce NFKB activity through ceramide generation (Kolesnick and Golde, 1994), by activation of both neutral and acidic sphingomyelineases (SMase's) (Figure 2). Acidic SMase (aSMase) has a pH optimum of 5.5, is activated by diacylglyceral, is located in lysosomes, and is generally thought to transduce death signals. The DD-containing region of TNF-R1 activates aSMase by prior activation of phosphotidylcholine-specific phospholipase C. Increases in lysosomal ceramide can trigger apoptosis, but it has also been shown that TNF-Rl-mediated apoptosis can occur when this pathway is blocked, indicating that aSMase activation is not necessary for death-signaling by TNF (Santana et al., 1996). Neutral SMase (nSMase) has a pH optimum of 7.4, is Mg+ dependent, and is localized intracellularly

43

Death Domains and the Brian

Activated TNF-R1 C~ ~

Activated Fas

\

,

Sequentional ~ " caspase

V/f, O

TRAMP (DR3)

Activationof NF~cB

//

l¥ Cell Survival

/

~ '"/

/

///

/ /

???

/

Programmed Cell Figure 2. Apoptotic signal transduction pathways of death domain-containing receptors. The initial step in Fas-induced cell death is trimerization of the Fas receptor, followed by recruitment of the adapter protein FADD, which interacts with the death domain of Fas through its own death domain (depicted as a striped, upright rectangle). The death effector domain (DED -- depicted as a shaded rectangle) of FADD in turn interacts with the DED of procaspase 8, which together make up the death-inducing signaling complex (DISC). Cleavage and activation of caspase 8 takes place at the DISC, after which caspase 8 presumably initiates a caspase cascade that results in death of the cell. An alternate pathway of Fas-induced death may involve interactions of FADD with Apaf-1 and caspase 9, which bind through their respective caspase recruitment domains (CARD), depicted here as striped rectangles. Caspase activation through CARD interactions is facilitated by mitochondrial damage. Death signaling through the TNF receptor may proceed through interactions of the adapter protein TRADD (TNF receptor associated death domain) with FADD, and subsequent activation of the FADD/caspase 8 pathway. Alternatively, TNF-RI also activates neutral and acidic sphingomyelinases (aSM and nSM), leading to the production of ceramide. Specifically, i n c r e a s e s in ceramide levels in lysosomes following acidic sphingomyelinase activation has been shown to induce apoptosis. TNF-RI can also cause activation of the transcription factor NFKB, either through ceramide production, or activation of NFKB-inducing kinase (N1K), which associates with TRAF2 (TNF receptor associated factor 2). The role of NFKB in TNF-mediated apoptosis is not clear, but evidence indicates that NFKB may be anti-apoptotic. The signal transduction pathways of the TRAMP, TRAIL, and DR6 receptors have not been characterized, but may involve interactions with FADD and NFKB.

n e a r the p l a s m a m e m b r a n e . A m e m b r a n e p r o x i m a l r e g i o n o f the intracellular r e g i o n o f T N F - R 1 ( w h i c h is u p s t r e a m o f the DD, and is h o m o l o g o u s to a s e q u e n c e in the c o r r e s p o n d i n g r e g i o n o f Fas) b i n d s a m o l e c u l e called F A N ( f a c t o r - a s s o c i a t e d neutral S M a s e ) w h i c h in turn i n c r e a s e s n S M a s e activity. T h e s u b s e q u e n t p r o d u c t i o n o f c e r a m i d e

44

A.J. Bruce-KeHer

can trigger multiple transduction pathways, including activation of the translocatable transcription factor NFKB. Neutral SMase signaling is independent of death signaling by TNF-R1 (Adam-Klages et al., 1996), but the role of ceramide production in death receptor signaling (apoptosis and otherwise) is not yet clear. It is known, however, that once activated NFKB translocates to the nucleus, where it can activate target cytoprotective genes (see NFKB aActivation -- Survival Gene Induction). TRAIL (TNF-related apoptosis inducing ligand) binds the two apoptosis signaling receptors TRAIL-R1 and TRAIL-R2, as well as the two decoy receptors DcR1 and DcR2 (Bodmer et al., 1997; Pan et al., 1997a; Sheridan et al., 1997; Marsters et al., 1997; Walczak et al., 1997; Chaudhary et al., 1997; Schneider et al., 1997) (Figure 2). As is the case for Fas and TNF-R 1, TRAIL-induced death signaling is contingent upon caspase activation, as caspase inhibitors can block apoptosis mediated by TRAIL receptors. Caspase 8 has been reported to be activated by TRAIL receptor activation, but a structurally similar caspase, caspase 10 (FLICE-2) seems to be the preferential caspase activated by TRAIL (Pan et al., 1997b). The signaling aspects of the TRAIL receptors are not wello characterized, but initial reports indicated that TRAIL receptors can signal death through a FADD-independent pathway (Marsters et al., 1996; Pan et al., 1997b). However, it has since been shown that overexpression of dominant negative FADD (DN-FADD) can block TRAIL-induced apoptosis (Walczak et al., 1997; Chaudhary et al., 1997; Schneider et al., 1997). This discrepancy amongst reports may be a reflection of the distinct cell lines used in experiments, and the differences in relative concentrations of overexpressed and endogenous genes. For instance, the concentration of endogenous cellular FADD relative to the expression of the transfected DN-FADD construct will have profound quantitative effects on the ability of dominant negative FADD to block apoptosis. Hence, further investigation is required to determine whether FADD is directly or indirectly involved in TRAIL-induced apoptosis. Likewise, the signal transduction and biological function of TRAMP are also largely unknown. TRAMP is structurally related to TNF-R1, and overexpression of TRAMP leads to both NFKB activation and apoptosis (Bodmer et al., 1997; Chinnaiyan et al., 1996). TRAMP is abundantly expressed in thymocytes and lymphocytes, and may play a role in their development (Bodmer et al., 1997; Chinnaiyan et al., 1996), but the putative ligand for TRAMP has not yet been identified. Execution of the Death Signal -- Caspase Activation Activation of members of the caspase family may be the critical final common pathway mediating most (if not all) apoptotic signals (for review see Cohen, 1997; Nicholson and Thornberry, 1997). Caspases are synthesized as dormant precursor proteins that are activated by proteolytic cleavage, and once activated, cleave substrates after a P1 Asp residue. Based on phylogenetic analyses, the more than 10 mammalian caspase family members can be divided in three main groups. The ICE-like protease family includes caspase 1 (ICE), caspase 4 (ICH-2/ICEreIII), caspase 5 (TY/ICE jlII), and caspase 11 (ICH-3). The CED-3 family includes caspase 3 (CPP32/YAMA/apopain), caspase 6 (Mch2), and caspase 7 (Mch3/ICE-LAP3), caspase 8 (FLICE/MACHI/Mch5), caspase 9 (Mch6/ICE-LAP6), and caspase 10 (Mch4/FLICE2). The third family contains only caspase 2 (Nedd2/ICH-1).

Death Domains and the Brian

45

The importance of caspases in DD receptor signaling was first demonstrated in experiments in which TNF-R1- and Fas-mediated cell killing was inhibited by the caspase inhibitors YVAD and DEVD (Enari et al., 1995; Los et al., 1995; Tewari and Dixit, 1995). Measurement of caspase enzymatic activity also confirmed increased caspase activity within minutes following Fas activation (Los et al., 1995). Caspases can be divided into initiators and executors, based on their structure and relative order of induction in the death pathway. Caspase 8 was cloned as part of the DISC (Figure 2), and hence is the initial upstream caspase in DD receptor signaling. Caspase 8 is known to directly cleave (and hence activate) caspase 3, 4, 7, 9, and 10 in vitro (Muzio et al., 1977). Some reports have since demonstrated caspase 6 to be upstream of caspase 3 and 7 (Orth et al., 1996) in mammalian apoptosis, but it has also been shown that caspase 3 can activate caspase 6, 7, and 9 (Srinivascula et al., 1996; Fernandes-Alnemri et al., 1995, 1996). Hence, while it is known that caspase activation following death receptor occupation is sequential, the exact order of participating caspases is still obscure. An increasing number of proteins have been found to be cleaved by caspases in the apoptotic cascade (for review see Cohen, 1997; Nicholson and Thornberry, 1997). Specific substrates include proteins involved in genomic function, including the DNA repair enzyme poly (ADP-ribose) polymerase (PARP), DNA-polymerase kinase, heteronuclear ribonucleoproteins, and the 140 kDa component of the DNA replication complex. Regulators of cell cycle progression are also cleaved, including the p53 regulator MDM-2, retinoblastoma protein, the nuclear mitotic-associated protein NuMA, and the kinases PKC-~5 and MEKK1. Reorganization of cellular morphology during apoptosis is likely mediated by cytoskeletal alterations, and cytoskeletal proteins that are cleaved by caspases include b-catenin, keratin-18, and spectrin. Additionally, the caspase 6-dependent cleavage of laminins may be important in disassembling the nuclear cytoarchitecture during apoptosis. A direct link between caspase activation and DNA fragmentation has also been demonstrated by the cloning of the murine endonuclease CAD (caspase activated DNAse (Enari et al., 1998). Inactive CAD is sequestered in the cytosol by association with the inhibitory subunit ICAD, which is a specific substrate of caspase 3. Upon induction of apoptosis, ICAD is cleaved, and CAD translocated into the nucleus where it can enzymatically degrade DNA. Interestingly, overexpression of ICAD can block the chromatin alterations characteristic of apoptosis, but does not effect other manifestations (Enari et al., 1998). Besides the FADD/caspase 8 pathway, a novel apoptotic pathway initiated by DD-containing receptors has been identified which is controlled by Apaf-1 (apoptotic protease-activating factor-l). Apaf-1 is the mammalian homolog of the C. Elegans death regulator ced-4. It is not clear whether the Apaf-1 pathway is independent of the caspase 8 pathway, as direct binding of Apaf-1 to mammalian caspase 1 and caspase 8 has been observed. The N-terminal domain of Apaf-1 shares some similarity with other caspases, and serves as the so-called caspase recruitment domain (CARD). Through CARD interactions, Apaf-1 can bind capase 9 (Figure 2)(Hoffman et al., 1997; Pan et al., 1998). The CARD mediates the associations (i.e. binding) of caspases that have similar CARD motifs at their N-terminus (Hoffman et al., 1997). The CARD domain also links this pathway to mitochondrail alterations in apoptosis, as binding of ATP and cytochrome c, both released from damaged mitochondria (Liu et al., 1996a),

46

A.J. Bruce-Keller

induce a conformational change in Apaf-1 to unmask the CARD domain. This event can culminate in the recruitment and activation of caspase 9 (Li et al., 1997), and potentially also caspase 1 and caspase 2, which contain CARD domains. Thus it is possible that this pathway may act independently of FADD and other adaptor proteins, but the link between death receptors, mitochondrial alterations, and Apaf-1 remains to be firmly established.

Alternative Death Receptor Signaling -- Pathways to Cell Life

Although the signaling through death domains is a rapid way to induce cell death, there are alternative signaling pathways of DD containing receptors, and regulatory mechanisms that can modulate or inhibit death signaling. Modulation can affect DD-induced apoptosis at various steps in the pathway, from initiation to execution of the death signal. While the expression of downstream death-domain adaptor protein is generally thought to be constitutive, the presence of "cell life" pathways helps to explain the reported variations in sensitivity to death receptor activation. For example, the presence of Fas on a cell surface is not sufficient to cause Fas-ligand sensitivity. In vitro induction of Fas expression in T cells can precedes Fas-based killing by several days (Klas et al., 1993), and while Fas is expressed on most thymocyte subsets, only double positive cells are sensitive to anti-Fas antibodies (Ogasawara et al., 1995). Hence, apoptotic signaling through DD receptors is subject to multiple intracellular resistance mechanisms that can counteract the apoptotic signal. In addition to blocking apoptosis, these alternate pathways likely mediate such functions as differentiation, activation or proliferation following DD-receptor activation. Inhibitors of Apoptosis -- FLIPs and IAPs Following the discovery of caspase 8 which contains two N-terminal domains related to DED of FADD, homologous viral proteins were discovered that lacked caspase activity. Called v-FLIPs (viral FLICE inhibitory proteins), these proteins bind to the DISC, but prevent caspase 8 recruitment and activation (Bertin et al., 1997; Hu et al., 1997a; Thome et al., 1997). Shortly following v-FLIP identification, a cellular homolog (c-FLIP) was cloned and shown to have important regulatory functions for death signaling in mammalian cells (Irmler et al., 1997). c-FLIP was initially identified by several groups independently, and hence is called c-FLIP, CASPER, I-FLICE, FLAME-l, CASH, CLARP, and MRIT (Shu et al., 1997; Hu et al., 1997b; Srinivasula et al., 1997; Goltsev et al., 1997; Inohara et al., 1997; Han et al., 1997). c-FLIP protein is expressed as two splice variants, including a short form that contains two N-terminal DED motifs through which it associates with other DED proteins (Figure 3). The long form of c-FLIP contains an additional C-terminal region that resembles the proteolytic domain of caspase 8 and caspase 10, but has an amino acid substitution in the active site (Schulze-Osthoff et al., 1998). Hence, c-FLIP is thought to be proteolytically inactive but has been shown to interact with DD adaptor protein including FADD, TRAF2, caspase 8 and caspase 3 (Shu et al., 1997). It has been demonstrated that following transfection into cells, c-FLIP can antagonize

Death Domains and the Brian

47

apoptotic signaling induced by either TNF-R1, Fas, TRAIL, or TRAMP (h'mler et al., 1997; Shu et al., 1997; Srinivasula et al., 1997).

Activated Fas ~ ~ ¢&¢~ ~

Decoy Receptors Activated ~ RTR~AIpLor A~¢'¢ ~

;°

Activated TNF-R1 ('~ kr~r) (~ O

7////

~

/

-"

\IKiBase CA-Ki.ase

r~Caspase8 / I I _1.~/

gWT'~ ~1 ~ "

~.....~ Activated ~ , ' ~ NFKB

t...a ~f~

m

Cell Death

/

/

~

~

9 foteins

Cell Life ~ Figure 3.

Anti-apoptotic signal transduction pathways of death domain-containing receptors.

The initial step of ligand binding to death domain-containing receptors can be blocked by truncated receptors that lack an intracellular death domain (Decoy receptors). The adapter protein c-FLIP (FLICE inhibitory protein) can block interaction of FADD with procaspase 8, and thereby prevent activation of the caspase cascade following Fas or TNF-R1 occupation. Likewise, interactions of TRAF2 (TNF receptor associated protein 2) with IAP (inhibitor of apoptosis protein) family members can block downstream sites caspase activation. Additionally, IAP has been shown to increase NFKB activity. Activation of the transcription factor NF~:B can prevent apoptosis in many cell types, and is induced following TNF-R1 occupation. Several mechanisms exist that cause dissociation of NFKB from the inhibitory subunit I~:B. Increases in neutral sphingomyelinase activity cause increases in ceramide concentrations, which activate NFKB through the action of the ceramide activated kinase (CA-Kinase). Likewise, activation of NFKB-inducing kinase (NIK), following association with TRAF2 (TNF receptor associated factor 2) also activates NFKB. Once dissociated from IKB, NFKB translocates to the nucleus where it can increase expression of protective proteins, thereby preventing apoptotic cell death.

T h e first m e m b e r o f the h u m a n i n h i b i t o r o f a p o p t o s i s p r o t e i n ( l A P ) f a m i l y w a s o r i g i n a l l y i s o l a t e d b a s e d o n its c o n t r i b u t i o n to n e u r o n a l d e g e n e r a t i o n in spinal m u s c u l a r a t r o p h y ( R o y et al., 1995). T h i s n e u r o n a l i n h i b i t o r o f a p o p t o s i s p r o t e i n ( N A I P ) was

48

A.J. Bruce-Keller

shown to be part of a conserved family of proteins originally discovered in baculovirus. In addition to NAIP, four additional human family members have been identified, c-IAP1, c-IAP2, X-lAP, and survivin, and all members have the ability to counteract apoptotic cell death (Rothe et al., 1995; Duckett et al., 1996; Ambrosini et al., 1997). Additionally, c-IAP1 and c-IAP2 have been shown to interact with TNF-R1 through TRAF2 (Rothe et al., 1995; Shu et al., 1996), but the role of c-IAP1/2 recruitment to TNF-R1 is unclear. A common feature of the lAP family is the Cys/His rich domain, repeated in the protein from one to three times, called the baculovirus lAP repeat (BIR). The fact that all family members have this domain suggests that it may be important in inhibiting apoptosis. Additionally, c-IAP1 and c-IAP2 also contain a zinc-finger RING domain at their C-terminus. In vitro studies of the caspase inhibitory properties of these proteins demonstrate that the BIR domains are sufficient for caspase inactivation, but that the proteins that also contain the RING structures are more effective (Deveraux et al., 1997; Cheng et al., 1996). Lastly, it has been demonstrated that c-IAP2 may be associated with activation of NF~B, an effect requiring an intact RING domain of c-IAP2 (Chu et al., 1997). Hence, IAPs could modulate DD-receptor induced apoptosis directly by inhibiting caspase activation, or indirectly, through increases in NFKB-mediated gene activation (see below). NFKB Activation -- Survival Gene Induction NFnB can be rapidly activated by DD-containing receptors by a variety of pathways, both direct and indirect. TNF-R1 causes activation of NFKB directly through TRAF2 and RIP, and indirectly through ceramide generation (Figure 3) (see above section Death domain signaling components -- pathways to cell death). Activation of nSMase causes the release of ceramide and sphingosine from sphingomyelin (Kolesnick and Golde, 1994; Cuvillier et al., 1996), and once formed, ceramide and sphingosine have diverse biological activities that can promote cell survival. Sphingosine and sphingosine-l-phosphate have been recently shown to promote cell survival in Jurkat T cells (Cuvillier et al., 1996), through a mechanism that involves activation of MAP kinase and protein kinase C (Cuvillier et al., 1996). Ceramide release, on the other hand, can also induce translocation of NFKB, via activation of a serine/threionine specific ceramide-activated kinase (CA-kinase), which induces phosphorylation and proteolysis of the NFKB inhibitory factor, IxB (Kolesnick and Golde, 1994). NFKB has also been reported to be activated by Fas (Ravi et al., 1998), and TRAMP (Bodmer et al., 1997; Chinnaiyan et al., 1996), indicating that NFnB may represent a broad-spectrum cellular mechanism to counteract DD-induced apoptotic signals. Additionally, TNF receptors mediate multiple pro-inflammatory pathways in immunocompetent cells (for review see Vassalli, 1992; Bazzoni and Beutler, 1996), and NFnB is an important mediator of inflammatory reactions. NFrB was originally identified in B lymphocytes where it stimulates transcription of the immunoglobulin K light chain (Sen and Baltimore, 1986). NFKB exists in the cytosol as an inducible 3 subunit complex consisting of two active KB subunits and an inhibitory subunit called IrB, and activation occurs when IKB is induced to dissociate from the complex. The active dimer then translocates to the nucleus and binds to 5' regulatory elements of genes responsive to NFKB; consisting of

Death Domains and the Brian

49

a decameric sequence (5'-GGGACTTTCC-3'). As NF~B is a preformed transcription factor with regulated activity, it differs from those in the AP-1 family (for example, whose members are regulated by gene induction). Similar to nuclear hormone receptors, NFKB activation directly transduces an extracellular signal to the nucleus, ensuring a rapid transcriptional response. Until recently, the prevailing view was that NF~zB may be a pro-apoptotic transcription factor, based its association with DD containing receptors. However, while the exact role(s) of NFKB in programmed cell death is not firmly established, recent evidence clearly demonstrates that NFKB activation can prevent apoptosis, both in neurons (Barger et al., 1995; Goodman and Mattson, 1996; Mattson et al., 1997), and in non-neuronal cells (Wu et al., 1996; Beg and Baltimore, 1996; Wang et al., 1996). Furthermore, in experiments in which downstream effectors of TNF-R 1 (FADD, TRAF2 and RIP) were manipulated, data obtained indicated that NFKB activation counteracts FADD-induced death (Liu et al., 1996b). Collectively, these data suggest that TNF-R 1 occupation simultaneously activates a cell death pathway and a cytoprotective pathway involving NFKB. Studies in different laboratories have identified several genes induced by NFKB that likely play important roles in increasing cellular resistance to injury. The antioxidant enzyme MnSOD is induced in several different cell types by TNF receptor stimulation in an NF~B-dependent manner (Eddy et al., 1992; Mattson et al., 1997; Bruce-Keller et al., 1999). In tumor cells resistant to TNF-induced apoptosis, MnSOD activity is increased following TNF treatment, whereas cells vulnerable to TNF-induced apoptosis did not increase their levels of MnSOD (Wong and Goeddel, 1988). MnSOD is a mitochondrially localized antioxidant enzyme that specifically detoxifies superoxide radicals. Superoxide production is increasingly recognized as a critical event in the cell death cascade, as it interacts with nitric oxide resulting in formation of peroxynitrite, a damaging free radical that induces membrane lipid peroxidation (Keller et al., 1998) and causes apoptosis (Beckman and Crow, 1993; Estevez et al., 1995). Peroxynitrite formation in neurons following amyloid B-peptide or iron treatment can be decreased by TNF-R1 stimulation (Mattson et al., 1997), presumably through NFKB-mediated MnSOD activation (Mattson et al., 1997; Furukawa and Mattson, 1998; Bruce-Keller et al., 1999). The existence of calcium dependent, mitochondrially localized nitric oxide synthase isotypes has been reported (Nichol et al., 1995; Buchwalow et al., 1997; Holmqvist and Ekstrom, 1997), supporting a role of peroxynitrite formation in mitochondrial dysfunction. The role of mitochondrial alterations in DD signaling is still disputed, but the role of mitochondrial secretions (ATP and cytochrome c) in Apaf-1 activation (see Execution of the Death Signal -- Caspase Activation) compel further investigation into the role of mitochondrial alterations in the FADD/caspase 8 pathway. For instance, it may be possible that caspase 8 has substrates in the outer mitochondrial membrane, the cleavage of which contributes to mitochondrial swelling and dysfunction, and may be modulated by MnSOD activity. A second gene target of NFI(B that may play a role in its anti-apoptotic action is the calcium-binding protein calbindin D28k. Activation of TNF-R1 can increase expression of calbindin D28k in neurons (Cheng and Mattson, 1994) and in astrocytes (Mattson et al., 1994), and the induction of calbindin can be associated with increased cellular resistance to death induced by agents that elevate intracellular calcium levels

50

A.J. Bruce-Keller

(i.e. excitatory amino acids in neurons and a calcium ionophore in astrocytes). Overexpression of calbindin D28k in pheochromocytoma cells protects those cells against apoptosis induced by calcium ionophore, trophic factor withdrawal, and an apoptosis-enhancing mutation in the presenilin-1 gene linked to Alzheimer's disease (Guo et al., 1998), but the role of calbindin in FADD-induced apoptosis has not been explored. Additional KB responsive genes linked to regulation of cell survival are emerging. In the nervous system, overactivation of glutamate receptors can induce neuronal apoptosis by a mechanism involving calcium overload and free radical production (Ankarcrona et al., 1995; Bonfoco et al., 1995). It was recently reported that NF~:B can modulate neuronal excitability by altering whole-cell currents through voltagedependent calcium channels and ionotropic glutamate receptor channels (Furukawa and Mattson, 1998). Whole-cell perforated patch camp recordings in cultured rat hippocampal neurons showed that long-term treatment (24-48 h), but not acute exposure to TNF results in an increase in Ca 2+ current density and a decrease in currents induced by glutamate. These effects were mimicked by pharmacological agents that specifically activate NFKB, and were prevented by agents that specifically block NFKB, demonstrating the important role of NF~:B activation in these TNF receptor pathways. Calcium imaging studies showed that neurons pretreated with TNF exhibit increased [Ca2+]i following membrane depolarization, but reduced [Ca2+]i responses to glutamate, compared to neurons in untreated control cultures. These findings demonstrate transcription-dependent modulation of voltage-dependent Ca 2÷ channels and glutamate receptors by TNF (Furukawa and Mattson, 1998), and suggest mechanisms whereby NF~:B may regulate neuronal survival, particularly in injury settings where both calcium and NFKB activation are known to be increased.

Death Receptors in the Central Nervous System -- Physiology and Pathophysiology The role of DD-containing receptors in mammalian physiology and pathophysiology is coming to light. It is quite clear, for example, that DD-containing proteins, in particular Fas, is necessary for the down-regulation of the immune system (Depraetere and Golstein, 1997). The elimination of T cells through apoptosis is very important for regulation of the immune response and maintenance of self-tolerance. Mutations of Fas or FasL in mice leads to severe autoimmune pathologies (Watanabe-Fukunaga et al., 1994; Takahashi et al., 1994). Both lpr (for lymphoproliferative) and gld mice (for generalized lymphoproliferative disease) which lack functional Fas and FasL, respectively, show severe autoimmune pathologies that highly resemble systemic lupus erythematosus in humans. Additionally, there is considerable evidence that inappropriate expression of TNF and TNF receptors can play a crucial role in both acute and chronic inflammatory disorders (for review see Vassalli, 1992; Bazzoni and Beutler, 1996). There is, however, considerably less clarity concerning the role of DD receptors in the central nervous system (CNS). TNF receptors are abundantly expressed in the mammalian brain, and as described below, TNF may be an important aspect of neuronal resistance to injury under pathophysiological conditions. In the case of Fas, however, there are conflicting reports concerning even the presence of

Death Domains and the Brian

51

Fas in normal mammalian brain, with some reports describing a low but detectable constitutive level of Fas and FasL expression in the CNS (French et al., 1996; Saas et al., 1997), while others describe the brain as completely devoid of Fas (Nishimura et al., 1995; Leithhauser et al., 1993). However, Fas expression known to be increased in post-ischemic brain, and in brain tissue from multiple sclerosis, Parkinson's disease, and Alzheimer's disease patients (see below), suggesting a role for Fas in causing or exacerbating neuronal injury. Additionally, since the brain is an immune-privileged organ, it is possible, as is the case for the eye and the testis, Fas and FasL are involved in maintaining immune privilege of the CNS. Fas and the CNS: Tumorigenesis and Chronic Neurodegenerative Disease While excessive inflammatory reactions in the brain are generally considered harmful (Giulian et al., 1990, 1993, 1995) and isolation from peripheral immune cells may be beneficial to neurons, the same situation could paradoxically facilitate tumor growth in the CNS. Recent studies have shown that many CNS tumor cells express Fas (Leithhauser et al., 1993; Saas et al., 1997; Gratas et al., 1997), through which the cells may counteract the immune system, and evade immune-mediated detection and elimination (Hahne et al., 1996). In support of this scenario, it has been shown that in contrast to normal brain, astrocytoma and gliobastomas are infiltrated to various degrees by peripheral inflammatory cells (Burger and Scheithauer, 1994), raising the possibility that FasL expression on these cells may prevent cytotoxic T cells or NK cells from removing tumorigenic cells. Moreover, monocytes, neutrophils, and activated T cells all express Fas (Lynch et al., 1995; Iwai et al., 1994), and are susceptible to death signals delivered by active FasL. Hence the same Fas-based mechanisms that protect cells from autoimmune destruction may be used by tumors, especially in immune-privileged organs such as the brain, to eliminate activated immune cells that may attempt to attack and remove the tumor cells. On the other hand, tumor cells in some cases express Fas and, hence, could be susceptible to FasL-induced death (in contrast to the low levels of Fas in normal brain tissue). A modified Fas-based anti-tumor therapy is under study, in many clinical settings, for the treatment of malignant brain tumors (Friesen et al., 1997; Fulda et al., 1997). Based on the roles of Fas and FasL in self-tolerance and immune system regulation, it was hypothesized that this pathway may have a role in CNS autoimmune disorders. Both Fas and FasL have been shown to be expressed in glial cells in chronic inflammatory lesions in multiple sclerosis (MS) (Dowling et al., 1996). Microglial cells at sites of CNS lesions express FasL, and these cells have been proposed to be mediators of apoptotic cell death in MS (D'Souza et al., 1996). Further in vitro studies demonstrate that oligodendrocytes, which are the cellular target in MS, are the main cells in the adult CNS that express Fas, while human neurons, in particular, do not have detectable Fas expression (Becher et al., 1998). These data suggest a mechanism whereby inflammatory cells could selective induce cell-type specific (i.e. oligodendrocyte) injury in the CNS. In further support of this scenario, a significant percentage of immunocompetent cells in MS lesions do indeed express FasL (Dowling et al., 1996). The colocalization of TUNEL-positive dying cells with FasL, and the presence of numerous

52

A.J. Bruce-KeUer

Fas-positive corpses within activated macrophages support the theory that Fas expressing oligodendrocytes are indeed a target of FasL attack in MS lesions (Dowling et al., 1996). Based on the role of caspase activation in Fas-mediated cell death and in vitro evidence for caspase mediated oligodendrocyte death (Enari et al., 1996), inhibition of caspases may have some therapeutic value in MS. The role of neuroimmune interactions in age-related neurodegeneration in the CNS is not well understood, but is has been suggested that chronic neurodegenerative disorders of aging such as Alzheimer's (AD) and Parkinson's disease (PD) may be, in part, mediated by altered immune responses (Fiszer et al., 1991; McGreer and McGreer, 1995, 1996). Based on observations that neuronal loss in AD and PD may be apoptotic, and the reported beneficial effects of anti-inflammatory regimens in especially AD, one could hypothesize a role for Fas in neuronal death in these diseases. Indeed, Fas-positive astrocytes have been documented in Alzheimer's brain samples, and data indicate that Fas expression is associated with astrocyte activation (Nishimura et al., 1995). The presence of Fas in astrocytes surrounding senile plaques is also consistent with this view, but it is not clear at this point what signals induce Fas expression on these cells, and there is as of yet no indication that Fas positive astrocytes are subject to apoptosis in AD brain. In PD brain samples, on the other hand, studies have demonstrated a significant increase in soluble Fas in the dopaminergic nigro-striatal pathway of affected individuals (Mogi et al., 1996). Soluble Fas (sFas) was first identified by molecular cloning and nucleotide sequence analysis that detected a Fas mRNA variant capable of encoding a soluble molecule that lacked the transmembrane anchor due to an exon deletion (Cheng et al., 1994). While the physiological function of sFas are unknown, many features of Fas are shared by the soluble molecule, especially in relation to autoimmune abnormalities (Knipping et al., 1995), indicating that an idiopathic alteration in sFas production may somehow be involved in the specific pattern of dopaminergic neurodegeneration in PD. Collectively, these observations suggest that while the majority of CNS neurons and glial cells have low levels of Fas expression, Fas and FasL are increased in specific, discrete regions under injury conditions, and may contribute to local tissue destruction. In further support of this, detectable increases in FasL have been found in cerebrospinal fluid following severe head injury, and in microglial cells following hypoxia/reoxygenation (Ertel et al., 1997; Vogt et al., 1998). However, only a correlative association can be made between neuronal apoptosis and increases in Fas and FasL expression in brain cells under injury conditions, and delineation of the exact consequences of Fas expression in the brain await further studies. TNF and Neuronal Resistance to Injury TNF levels are known to be increased in human brain in cases of AD, PD, or head trauma, but the consequences are still unclear. Unlike Fas, the role of TNF-R1 in neuronal physiology may be unrelated to its death-inducing functions, as accumulating experimental evidence indicates that TNF acts as a neuroprotective agent in the brain. Studies have demonstrated that TNF-R1 receptor signaling affords significant protection against subsequent glutamate toxicity, oxidative insults, and amyloid f5 peptide toxicity in hippocampal neurons in vitro (Cheng and Mattson, 1994; Barger et al., 1995).

Death Domains and the Brian

53

While two membrane associated TNF receptors (TNF-R1 and TNF-R2) have been characterized (Kinouchi et al., 1991; Wolvers et al., 1993; Lewis et.al., 1991), in vitro studies indicate that TNF-R1 is both necessary and sufficient to for TNF-mediated neuroprotection (Figure 4). Furthermore, most in vitro studies employ a combination of human recombinant TNF applied to rodent cells, and as human TNF will activate only TNF-RI in rodent cells, it is clear that TNF-R1 activation in neurons is neuroprotective. The role of TNF-R2 in protective paradigms is unclear, but may be limited to facilitating TNF-R1 occupancy (Tartaglia et al., 1993b). The protective role of endogenous TNF has been demonstrated through the use of transgenic mice deficient in TNF receptors. Transgenic mice lacking both TNF receptors show significantly increased damage excitotoxic and ischemic insults when compared to wild-type controls (Bruce et al., 1996), indicating that the reported increases in TNF following brain injury may stimulate neuroprotective signaling pathways. Subsequent studies using mice lacking either TNF-R 1 or TNF-R2 demonstrated again that TNF-R 1 is responsible for the neuroprotective effects of TNF in the brain (Gary et al., 1998), and data further indicate that the signal transduction pathway elicited by TNF-R1 in neuroprotection likely involves NF~cB and increases in MnSOD activity (Mattson et al., 1997; Bruce et al., 1996). As mentioned previously, the divergent effects of DD-containing receptor activation is a tightly regulated, cell type specific process. Therefore, the neuroprotective effects of TNF in the brain must also be considered from the standpoint of cell-cell (i.e. neuron-glial) interactions. While TNF-R1 signaling is trophic to neurons, it can be toxic to oligodendrocytes, induce astrocyte and microglial proliferation, and also stimulate trophic factor release from these cells (Goossens et al., 1995; Jaattela, 1991; Gadient et al., 1990; Le and Vilcek, 1987). Experiments aimed at determining the signals elicited by TNF-RI in different cell types in the brain again demonstrate that induction of MnSOD is critical for TNF-R1 mediated cytoprotection (Figure 5). Data indicate that while TNF application is not directly toxic to neurons, astrocytes, or oligodendrocytes, TNF-R1 signaling is beneficial only in cell types in which it can increase MnSOD activity (Figure 5) (Bruce-Keller et al., 1999). It is not clear, at this point, what factors are responsible for the divergent TNF signaling pathways in different cell types in the brain, and further experimentation is necessary to determine if these pathways are hard-wired or can be altered. Expression of TNF in the brain is increased following neuronal injury (Minami et al., 1991; Taupin et al., 1993; Tchelingerian et al., 1993; Liu et al., 1994; Kim, 1996; Buttini et al., 1994; Szaflarski et al., 1995), and the profile of TNF expression following injury is biphasic, with both an early (minutes to hours) peak and a sustained (days) plateau in TNF levels (Liu et al., 1994; Kim, 1996). The immediate and delayed pattern of TNF expression in these studies likely reflects different sources of TNF production, as TNF mRNA is rapidly (1-4 h) increased in affected neurons following injury (Minami et al., 1991; Taupin et al., 1993; Tchelingerian et al., 1993; Bruce et al., 1996), while the protracted expression (2-5 days) is detected in microglial and macrophage cells surrounding the damaged tissue (Liu et al., 1994; Kim, 1996). These observations support the following scenario: immediate neuronal alterations in response to injury conditions leads to early production of TNF in neurons, which when secreted aids in the recruitment and proliferating of resident microglia, which also secrete TNF. The resulting cascade of TNF autocrine and paracrine

54

A.J. Bruce-Keller

"Z

~0

.o .~ ;-

o ~ Z

tm

~

o

~o~

o

t~

@aqmau lel~.~. %) I ~ ! ~ n ~ pmon'~N

,-~

~.~

._~

..~ ~

E

g

if+

°~

~

|

o

! o

°"~ m

(~qumu i~.~m! %) l~!,~m~ pmem~N

omlm~u I~.q.~. %) [~^!~-~ p m o a ~ N

-~ ~. ~ . ~

m

E N.~ ~ ~ .~ "~-~= ~.~.=

Death Domains and the Brian

55

~.~

~ Z

Ca

~

e'~

._v~og -=-'-= ~o 2, "= O0

Ca

' o"==Eg

.+

ee8 ~,.~ = ' -

o

o.a

g.

~.-

m~'= ,4

¢J

o

,4

m

(,,:I gus/~l) ,£1!,%'~e QOSUl, q

"

U.., ©

(lOXlUo:' %) +SeelaX Ll~l"l

,~

-:+

" a{a ~.-~

o

g a .~

(u!~xd

g

=

g

t~m/r'l) ~.u,.qa~ C I O S U ~

I~

~:*u~:~s~onlJ ~4DG

-

= Z

~.-~"

•~

~

~a ~ . . . -

a=

~=.~- ~

.~

~,.~_

~

=.o~===g. ~

=

o

~,-~

~= u

56

A.J. Bruce-Keller

signaling can provide trophic support to neurons while propagating a brain-resident inflammatory cascade that promotes resolution of injury and functional recovery. This hypothesis is supported by experimental observations, in mice lacking TNF receptors, which show significantly increased neuronal loss but remarkably decreased reactive inflammatory gliosis following excitotoxicity or ischemia (Bruce et al., 1996), Furthermore, additional studies have also confirmed that microglial activation can attenuate excitotoxic or ischemic injury in rodents (Berezovskaya et al., 1995), prevent apoptosis in vitro (Toku et al., 1998), and increase neurite outgrowth and functional recovery following brain injury (Lazarov-Spiegler et al., 1996; Prewitt et al., 1997; Rabchevsky and Streit, 1997).

Concluding Remarks This review summarizes the different death receptors and the intracellular pathways they activate to induce cell life or death. Unfortunately, the cloning and characterization of death receptors and their myriad of control mechanisms proceeds well ahead of the understanding of their roles in human physiology or pathophysiology. Hence, a major goal of death receptor research in the future will be to determine the biological function of the death-receptor signaling, and to delineate what additional intracellular signals dictate signaling for life or death for a cell. Such an understanding is crucial if therapeutic interventions are to be based on death receptor signaling. Pharmacological targeting of death-inducing pathways could be very important for cancer therapy, in which resistance to apoptosis is often associated with chemotherapeutic drug resistance. Additionally, a death-domain based therapy would have promising clinical applications for autoimmune disorders such as multiple sclerosis and systemic lupus erythematosus. On the other hand, the potent neuroprotective properties of TNF suggests that mimicry of these pathways could be of significant therapeutic importance in neurodegenerative disorders, including AD, PD, epilepsy and stroke. The neuroprotective signal transduction pathway utilized by TNF-R1 highlights several sites for potential pharmacological intervention. One mode would be to activate TNF-R1 receptor directly, either through application of TNF itself, or through design of a TNF-Rl-specific agonist. However, potentially harmful side effects of this paradigm could occur upon TNF receptor stimulation of other cell types, including microglia, oligodendrocytes, and endothelial cells. The successful harnessing of events downstream of TNF-R 1 receptor activation would preclude such effects, and would include ceramide application, NFKB activation, or the up-regulation of neuroprotective genes such as MnSOD. The observation that Fas and TNF-R1 receptors, which have such extensive overlap in intracellular mediators, could have such divergent functional roles in the CNS further illustrates the highly pleiotrophic nature of these remarkable receptors, and should drive meaningful research for years to come.

Death Domains and the Brian

57

References Adam-Klages, S., Adam, D., Wiegmann, K., Struve, S., Kolanus, W., Schneider-Mergener, J. & Kroenke, M. (1996). Fan, a novel WD repeat protein, couples the p55 TNF-receptor to neutral sphingomyelinase. Cell 86, 937-947. Alnemri, E.S., Livingston, D.J., Nicholson, D.W., Salvesen, G., Thornberyy, N.A., Wong, W.W. & Yuan, J. (1996). Human IVE/CED-3 protease nomenclature. Cell 87, 171. Ambrosini, G., Adida, C. & Altieri, D. (1997). A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nature Med. 3,917-921. Ankarcrona, M., Dypbukt, J.M., Bonfoco, E., Zhivotovsky, B., Orrenius, S., Lipton, S.A. & Nicotera, P. (1995). Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function. Neuron 15,961-973. Banner, D.W., D'Arcy, A., Janes, W., Gentz, R., Schoenfeld, H.J., Broger, C., Loetscher, H. & Lesslauer, W. (1993). Crystal structure of the soluble human p55 TNF receptor-human TNFbeta complex: implications for TNF receptor activation. Cell 73,431-445. Barger, S.W., Horster, D., Furukawa, K., Goodman, Y., Krieglstein, J. & Mattson, M.P. (1995). Tumor necrosis factors alpha and beta protect neurons against amyloid B-peptide toxicity: Evidence for involvement of a kappa B-binding factor and attenuation of peroxide and Ca2+ accumulation. Proc. Nat. Acad. Sci. U.S.A. 92, 9328-9332. Bazzoni, F. & Beutler, B. (1996). The tumor necrosis factor ligand and receptor families. New Engl. J. Med. 334, 1717-1725. Becher, B., D'Souza, S.D., Trout, A.B. & Antel, J.P. (1998). Fas expression on human fetal astrocytes without susceptibility to fas-mediated cytotoxicity. Neuroscience 84, 627-634. Beckman, J.S. & Crow, J.P. (1993). Pathological implications of nitric oxide, superoxide and peroxynitrite formation. Biochem. Soc. Trans. 21,330-334. Beg, A.A. & Baltimore, D. (1996). An essential role for NF-KB in preventing TNF-a-induced cell death. Science 274, 782-784. Berezovskaya, O., Maysinger, D. & Fedoroff, S. (1995). The hemapoietic cytokine, colony stimulating factor 1, is also a growth factor in the CNS: congenetal absence of CSF-1 in mice results in abnormal microglial response and increased neuron vulnerability to injury. Int. J. Dev. Neurosci. 13,285-299. Bertin, J., Armstrong, R.C., Ottilie, S., Martin, D.A., Wany, Y., Banks, S., Wang, G.H., Senkevich, T.G., Alnemri, E.S., Moss, B., Lenardo, M.J., Tomaselli, K.J. & Cohen, J.I. (1997). Death effector domaincontaining herpesvirus and poxvirus proteins inhibit both Fas- and TNFRl-induced apoptosis. Proc. Nat. Acad. Sci. U. S. A. 94, 1172-1176. Bodmer, J.L., Burns, K., Schneider, P., Hofman, K., Steiner, V., Thome, M., Bornand, T., Hahne, M., Schroter, M., Becker, K., Wilson, A., Frence, L.E., Browning, J.L., MacDnoald, H.R. & Tschopp, J. (1997). TRAMP, a novel apoptosis mediating receptor with sequence homology toTNF-R1 and Fas. Immunity 6, 79-88. Boldin, M.P., Goncharov, T.M., Goltsev, Y.V. & Wallach, D. (1996). Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1 and TNF receptor-induced cell death. Cell 85, 803-815. Boldin, M.P., Varfolomeev, E.E., Pancer, Z., Mett, I.L., Camonis, J.H. & Wallach, D. (1995). A novel protein that interacts with the death domains of Fas/APO1 contains a sequence motif related to the death domain. J. Biol. Chem. 270, 7795-7798. Bonfoco, E., Krainc, D., Ankarcrona, M., Nicotera, P. & Lipton, S.A. (1995). Apoptosis and necrosis: two

58

A.J. Bruce-Keller

distinct events induced, respectively, by mild and intense insults with N-methyl-D- aspartate or nitric oxide/superoxide in cortical cell cultures. Proc. Natl. Acad. Sci. U.S.A. 92, 7162-7166. Bruce, A.J., Boling, W.W., Kindy, M.S., Peschon, J., Kraemer, P.J., Carpenter, M.K., Holtzberg, F.W. & Mattson, M.P. (1996). Altered neuronal and microglial responses to excitotoxic and ischemic brain injury in mice lacking TNF receptors. Nat. Med. 2, 788-794. Bruce-Keller, A.J., Geddes, J.W., Knapp, P.E., McFall, R.W., Keller, J.N., Holtsberg, F.W., Steiner, S.M., Partharasy, S. & Mattson, M.P. (1999). Anti-death properties of TNF against metabolic poisoning: mitochondrial stabilization by MnSOD. J. Neuroimmunol. 93, 53-71. Buchwalow, I.B., Schulze, W., Kostic, M.M., Wallukat, G. & Morwinski, R. (1997). Intracellular localization of inducible nitric oxide synthase in neonatal rat cardiomyocytes in culture. Acta. Histochem. 99, 231-40. Burger, P.C. & Scheithauer, B.W. (1994). Tumors of the central nervous system. (Washington, DC: Armed Forces Institute of Pathology.). Buttini, M., Sauter, A. & Boddeke, H.W. (1994). Induction of interleukin-1 beta mRNA after focal cerebral ischaemia in the rat. Mol. Brain Res. 25, 126-134. Chaudhary, P.M., Eby, M., Jasmin, A., Bookwater, A., Murray, J. & Hood, L. (1997). Death receptor 5, a new member of the TNFR family & DR4 induce FADD-dependent apoptosis and activate the NF-kappa B pathway. Immunity 7, 821-830. Cheng, B. & Mattson, M. (1994). Tumor necrosis factors protect neurons against excitotoxic/metabolic insults and promote maintenance of calcium homeostasis. Neuron 12, 139-153. Cheng, E.H., Levine, B., Boise, L.H., Thompson, C.B. & Hardwick, J.M. (1996). Bax-independent inhibition of apotosis by BcI-XL. Nature 379, 554-556. Cheng, J., Zhou, T., Liu, C., Shapiro, J.P., Brauer, M.J., Keifer, M.C., Barr, P.J. & Mountz, J.D. (1994). Protection from Fas-mediated apoposis by a soluble form of the Fas molecule. Science 263, 1759-1762. Chinnaiyan, A.M., O'Rourke, K., Tewari, M. & Dixit, V.M. (1995). FADD, a novel death domain-contianing protein, interacts with the death domain of Fas and initiates apoptosis. Cell 81,505-512. Chinnaiyan, A.M., O'Rourke, K., Yu, G., Lyons, R.H., Gear, M., Duan, D.R., Xing, L., Gebtz, R., Ni, J. & Dixit, V.M. (1996). Signal transduction by DR3, a death domain containing receptor related to TNF-R1 and CD95. Science 274, 990-992. Chu, Z.L., McKinsey, T.A., Liu, L., Gentry, J.J., Malim, M. & Ballard, D.W. (1997). Suppression of tumor necrosis fcator-induced cell death by inhibitor of apoptosis c-lAP is under NF-KB control. Proc. Nat. Acad. Sci. U.S.A. 94, 10657-10667. Cohen, G.M. (1997). Caspases: the executioners of apoptosis. Biochem. J. 326, 1-16. Cuvillier, O., Pirianiv, G., Kleuser, B., Vanek, P.G., Coso, O.A., Gutkind, J.S. & Spiegel, S. (1996). Supression of ceramide-mediated programmed cell death by sphingosine-1-phosphate. Nature 381,800-803. D'Souza, S.D., Bonetti, B., Balasingam, V., Cashman, N.R., Barker, P.A., Troutt, A.B., Raine, C.S. & Antel, J.P. (1996). Multiple sclerosis: Fas signaling in oligodendrocyte cell death. J. Exp. Med. 184, 2361-70. Degli-Esposti, M.A., Smolak, P.J., Walczak, H., Waugh, J., Huang, C.P., DuBose, R.F., Goodwin, R.G., & Smith, C.A. (1997). Cloning and characterization of TRAIL-R3, a novel member of the emerging TRAIL receptor family. J. Exp. Med. 186, 1165-1170. Depraetere, V. & Golstein, P. (1997). Fas and other cell death signaling pathways. Sere. Immunol. 9, 93-107. Deveraux, Q., Takahashi, R., Salvesen, G.S. & Reed, J.C. (1997). X-linked lAP is a direct inhibitor of cell death processes. Nature 388, 300-303. Dhein, J., Daniel, P.T., Trauth, B.C., Oehm, A., Moeller, P. & Krammer, P.H. (1992). Induction of apoptosis by monoclonal antibody anti-APO-1 class switch variants is dependent on cross-linking of APO-1 cell surface receptors. J. Immunol. 149, 3166-3173.

Death Domains and the Brian

59

Dowling, P., Shang, G., Raval, S., Menonna, J., Cook, S. & Husar, W. (1996). Involvement of the CD95 (APO-I/Fas) receptor/ligand system in multiple sclerosis brain. J. Exp. Med. 184, 1513-1518. Duckett, C.S., Nava, V.E., Gedrich, R.W., Clem, R.J., Van Dongen, J.L., Gilfillan, M.C., Shiels, H., Hardwick, J.M. & Thompson, C.B. (1996). A conserved family of cellular genes related to the baculovirus iap gene and encoding apoptosis inhibitors. EMBO J. 15, 2685-2694. Eddy, L., Goeddel, D. & Wong, G. (1992). Tumor necrosis factor-alpha pretreatment is protective in a rat model of myocardial ischemia-reperfusion injury. Biochem. Biophys. Res. Commun. 184, 1056-1059. Enari, M., Hug, H. & Nagata, S. (1995). Involvement of an ICE-like protease in Fas-mediated apoptosis. Nature 375, 78-81. Enari, M., Sakahiera, H., Yokoyama, H., Okawa, K., Iwamatsu, A. & Magata, S. (1998). A caspase activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391, 43-50. Enari, M., Talanian, R., Wong, W. & Nagata, S. (1996). Sequential activation of ICE-like and CPP32-1ike proteases during Fas-mediated apoptosis. Nature 380, 723-726. Ertel, W., Keel, M., Stocker, R., Imhof, H. G., Leist, M., Steckholzer, U., Tanaka, M., Trentz, O. & Nagata, S. (1997). Detectable concentrations of Fas ligand in cerebrospinal fluid after severe head injury. J. Neuroimmunol. 80, 93-96. Estevez, A.G., Radi, R., Barbeito, L., Shin, J.T., Thompson, J.A. & Beckman, J.S. (1995). Peroxynitriteinduced cytotoxicity in PC12 cells: evidence for an apoptotic mechanism differentially modulated by neurotrophic factors. J. Neurochem. 65, 1543-1550. Fernandes-Alnemri, T., Armstrong, R.C., Krebs, J., Srinivasula, S.M., Wang, L., Bullrich, F., Fritz, L.C., Trapani, J.A., Tomaselli, K.J., Litwack, G. & Alnemri, E.S. (1996). In vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-Iike domains. Proc. Natl Acad. Sci. U. S. A. 93, 7464-7469. Fernandes-Alnemri, T., Takahashi, A., Armstrong, R.C., Krebs, J., Tomaselli, K.J., Wang, L., Yu, Z., Croce, C.M., Salveson, G., Earnshaw, W.C., Litwack, G. & Alnemri, E.S. (1995). Mch3, a novel human apoptotic cysteine protease related to CPP32. Cancer Res. 55, 6045-6052. Fiszer, U., Piotrowska, K., Kodak, J. & Czlonkowska, A. (1991). The immunological status in Parkinson's disease. Med. Lab. Sci. 48, 196-200. French, L.E., Hahne, M., Viard, I., Radlgruber, G., Zanone, R., Becker, K., Muller, C. & Tschopp, J. (1996). Fas and Fas ligand in embryos and adult mice: ligand expressin in several immune-privileged tissues and coexpression in adult tissues characterized by apoptotic cell turnover. J. Cell. Biol. 133,335-343. Friesen, C., Herr, I., Krammer, P.H. & Debatin, K.M. (1997). Involvement of the CD95 (APO-1/Fas) receptor ligand system in drug-induced apoptosis in leukemia ceils. Nat. Med. 2, 574-577. Fulda, S., Seiverts, H., Friesen, C., Herr, 1. & Debatin, K.M. (1997). The CD95 (APO-I/Fas) system mediates drug-induced apoptosis in neuroblastoma cells. Cancer Res. 57, 3823-3829. Furukawa, K. & Mattson, M.P. (1998). The transcription factor NF-KB mediates increases in calcium currents and decreases in NMDA and AMPA/kainate-induced currents in response to TNFa in hippocampal neurons. J. Neurochem. 70, 1876-1886. Gadient, R.A., Cron, K.C. & Otten, U. (1990). Interleukin-1 13and tumor necrosis factor-alpha synergistically stimulate nerve growth factor (NGF) release from cultured rat astrcytes. Neurosci. Lett. 117, 335-340. Gary, D.S., Bruce-Keller, A.J., Kindy, M.S. & Mattson, M.P. (1998). Ischemic and excitotoxic brain injury is enhanced in mice lacking the p55 tumor necrosis factor receptor. J. Cereb. Blood Flow Metab. 12, 1283-1287. Giulian, D., Haverkamp, L.J., Li, J., Karshin, W.L., Yu, J., Tom, D., Li, X. & Kirkpatrick, J.B. (1995). Senile plaques stimulate microglia to release a neurotoxin found in Alzheimer brain. Neurochem. Int. 27, 119-137.

60

A.J. Bruce-Keller

Giulian, D., Vaca, K. & Corpuz, M. (1993). Brain glia release factors with opposing actions upon neuronal survival. J. Neurosci. 13, 29-37. Giulian, D., Vaca, K. & Noonan, C. (1990). Secretion of neurotoxins by mononuclear phagocytes infected with HIV-1. Science 250, 1593-1596. Goltsev, Y.K., Kovalenko, A.V., Arnold, E., Varfolomeev, E.E., Brodianskii, V.M. & Wallach, D. (1997). CASH, a novel caspase homolog with death effector domains. J. Biol. Chem. 272, 19641-19644. Goodman, Y. & Mattson, M.P. (1996). Ceramide protects hippocampal neurons against excitotoxic and oxidative insults, and amyloid B-peptide toxicity. J. Neurochem. 66, 869-872. Goossens, V., Grooten, J., De Vos, K. & Fiers, W. (1995). Direct evidence for tumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity. Proc. Natl. Acad. Sci. U.S.A. 92, 8115-8119. Grams, C., Tohma, Y., Van Mier, E.g. Klein, M., Tenan, M., Ishii, N., Tachibana, O., Kleihues, P. & Ohgaki, H. (1997). Fas ligand expression in glioblastoma cell lines and in primary astrocytic brain tumors. Brain Pathol. 7, 863-869. Guo, Q., Robinson, N. & Mattson, M.P. (1998). Secreted APPa counteracts the pro-apoptotic action of mutant presenilin-1 by activation of NF-KB and stabilization of calcium homeostasis. J. Biol. Chem. 273, 12341-12351. Hahne, M., Rimoldi, D., Schroter, M., Romero, P., Schreier, M., French, L.E., Schneider, P., Borland, T., Fontana, A., Lienard, D., Cerottini, J. & Tschopp, J. (1996). Melanoma cell expression of Fas ligand: immplications for tumor immune escape. Science 274, 1363-1366. Han, D.K.M., Chaudry, P.M., Wright, M.E., Friedman, C., Trask, B.J., Reidel, R.T., Baskin, D.G., Schwartz, S.M. & Hood, L. (1997). MRIT, a novel death-effector domain-containing protein interacts with caspases and BclXL and initiates cell death. Proc. Nat. Acad. Sci. U. S. A. 94, 11333-11338. Hoffman, K., Bucher, P. & Tschopp, J. (1997). The CARD domian, a new apoptotic signaling motif. Trends Biochem. Sci. 22, 155-156. Holmqvist, B. & Ekstrom, P. (1997). Subcellular localization of neuronal nitric oxide synthase in the brain of a teleost; an immunoelectron and confocal microscopical study. Brain Res. 745, 67-82. Hsu, H., Huang, J., Shu, H.B., Baichwal, V. & Goeddel, D.V. (1996a). TNF-dependent recruitment of the protein kinase RIP to the TNF receptor-1 signaling complex. Inmmunity 4, 387-396. Hsu, H., Shu, H.B., Pan, M.G. & Goeddel, D.V. (1996b). TRADD-TRAF2 and TRADD-FADD interactions define two distinct TNF receptor 1 signal tmnsduction pathways. Cell 84, 299-308. Hsu, H., Xiong, J. & Goeddel, D.V. (1995). The TNF receptor 1-associated protein TRADD signals cell death and NF-~:B activation. Cell 81,495-504. Hu, S., Vincenz, C., Buller, M. & Dixit, V.M. (1997a). A novel family of viral death effector domiancontaining molecules that inhibit both CD95- and TNF-Rl-induced apoptosis. J. Biol. Chem. 272, 9621-9624. Hu, S., Vincenz, C., Ni, J., Gentz, R. & Dixit, V.M. (1997b). I-FLICE, a novel inhibitor of tumor necrosis factor receptor-l- and CD95-induced apoptosis. J. Biol. Chem. 275, 17255-17257. Inohara, N., Koseki, T., Hu, Y., Chen, S. & Nunez, G. (1997). CLARP, a death effector domain-containing protein interacts with caspase 8 and regulates apoptosis. Proc. Nat. Acad. Sci. U. S. A. 94, 1017-1022. Irmler, M., Thome, M., Hahne, M., Schneider, P., Steiner, V., Bodme, J.L., Schroeter, M., Burns, K., Mattmann, C., Hofmann, C., Rimoldi, D., French, L.E. & Tschopp, J. (1997). Inhibition of death receptor signals by cellular FLIP. Nature 388, 190-195. Itoh, N. & Nagata, S. (1993). A novel protein domain required for apoptosis. Mutational analysis of human Fas antigen. J. Biol. Chem. 268, 10932- 10937.

Death Domains and the Brian

61

Itoh, N., Yonehara, S.I.A., Yonehara, M., Mizushima, S., Sameshima, M., Hase, A., Seto, Y. & Nagata, S. (1991). The polypeptide encoded for the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 66, 233-243. Iwai, K., Miaywaki, T., Takizawa, T., Konno, A., Ohta, K., Yachie, A., Seki, H. & Taniguchi, N. (1994). Differential expression of bcl-2 and susceptability to anti-Fas-mediated cell death in peripheral blood lymphocytes, monocytes & neutrophils. Blood 84, 1201-1208. Jaattela, M. (1991). Biological activities and mechanisms of action of tumor necrosis factor alpha / cachectin. Lab. Invest. 64, 724-742. Keller, J.N., Kindy, M.S., Holtsberg, F.W., St Clair, D.K., Yen, H.C., Germeyer, A., Steriner, S.M., Bruce-Keller, A.J., Hutchins, J.B. & Mattson, M.P. (1998). Mitochondrial MnSOD prevents neuroal apoptosis and reduces ischemic brian injury: supression of peroxynitrite production, lipid peroxidation & mitochondrial dysfunction. J. Neurosci. 18, 687-697. Kim, J.S. (1996). Cytokines and adhesion molecules in stroke and related diseases. J. Neurol. Sci. 137, 69-78. Kinouchi, K., Brown, G., Pasternak, G. & Donner, D.B. (1991). Identification and characterization of receptors for tumor necrosis factor-alpha in the brain. Biochem. Biophys. Res. Commun. 181, 1532-1538. Klas, C., Debatin, K.M., Jonker, R.R. & Krammer, P.H. (1993). Activation interferes with the APO-I pathway in mature human T cells. Immnuol. 5,625-630. Knipping, E., Debatin, K.M., Strucker, K., Heilig, B., Eder, A. & Krammer, P.H. (1995). Identification of soluble APO-1 in supernatents of human B- and T-cell leukemias. Blood. 85, 1562-1569. Kolesnick, R. & Golde, D.W. (1994). The sphingomyelin pathway in tumor necrosis factor and interleukin-I signaling. Cell 77,325-328. Lazarov-Spiegler, O., Solomon, A.S., Zeev-Brann, A.B., Hirschberg, D., Lavie, V. & Schwartz, M. (1996). Transplantation of activated macrophages overcomes central nervous system regrowth failure. FASEB J. 10, 1296-302. Le, J. & Vilcek, J. (1987). Tumor necrosis factor and interleukin 1: cytokines with multiple overlapping biological activities. Lab. Invest. 56, 234-248. Leithhauser, F., Dhein, J., Mechtersheimer, G., Koretz, K., Bruderlein, S., Henne, C., Schmidt, A., Debatin, K.M., Krammer, P.H. & Moiler, P. (1993). Constituative and induced expression of APO-1, a new member of the NGF/TNF receptor family superfamily, in normal and neoplastc cells. Lab. Invest. 69, 415-429. Lewis, M., Tartaglia, L.A., Lee, A., Bennett, G.L., Rice, G.C., Wong, G.H., Chen, E.Y. & Goeddel, D.V. (1991). Cloning and expression of cDNAs for two distinct murine tumor necrosis factor receptors demonstrate one receptor is species specific. Proc. Nat. Acad. Sci. U. S. A. 88, 2830-2834. Li, P., Hijhawan, D., Budihardjo, I., Srinivasula, S.M., Ahmad, M., Alnemri, E.S. & Wang, X. (1997). Cytochrome c and dATP dependent formation of Apaf-l/Caspase-9 complex initiates an apoptotic protease cascade. Cell 91,155-156. Liu, T., Clark, R.K., McConnell, P.C., Young, P.R., White, R.F., Barone, F.C. & Feuerstein, G.Z. (1994). Tumor necrosis factor in ischemic neurons. Stroke 25, 1481-1488. Liu, X., Kim, C.N., Yang, J., Jemmerson, R. & Wang, X. (1996a). Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86, 147-157. Liu, Z.-G., Hsu, H., Goeddel, D.V. & Karin, M. (1996b). Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-•B activation prevents cell death. Cell 87,565-576. Los, M., Van de Craen, M., Penning, L.C., Schenk, H., Westendorp, M., Baeuerle, P.A., Drodge, W., Krammer, P.H., Fiers, W. & Shultz-Osthoff, K. (1995). Requirement of an ICE/CED-3 protease for Fas/APO-l-mediated apoptosis. Nature 375, 81-83.

62

A.J. Bruce-Keller

Lynch, D.H., Ramsdell, F. & Alderson, M.R. (1995). Fas and FasL in the homeostatic regulation of the immune system. Immunol. Today 16, 569-574. Marsters, S.A., Pitti, R.M., Donaheu, C.J., Ruppert, S., Bauer, K.D. & Ashkenazi, A. (1996). Activation of apoptosis by Apo-2 ligand is independent of FADD but blocked by CrmA. Curr. Biol. 6, 750-752. Marsters, S.A., Sheridan, J.P., Pitti, R.M., Skubatch, M., Baldwin, D., Yuan, J., Gurney, A., Goddard, A.D., Godowski, P. & Ashkenazi, A. (1997). A novel receptor for Apo21/TRAIL contains a truncated death domain. Curr. Biol. 7, 1003-1006. Mattson, M., Cheng, B., Baldwin, S., Smith-Swintosky, V., Keller, J., Geddes, J., Scheff, S. & Christakos, S. (1994). Brain injury and tumor necrosis factors induce expression of calbidin D-28 in astrocytes: evidence for a cytoprotective response. J. Neurosci. Res. 42, 357-370. Mattson, M.P., Goodman, Y., Luo, H., Fu, W. & Furukawa, K. (1997). Activation of NFKB protects hippocampal neurons against oxidative stress-induced apoptosis: evidence for induction of MnSOD and supression of peroxynitrie production and protein tyrosine nit~:ation. J. Neurosci. Res. 49, 681-697. McGeer, P.L. & McGeer, E.G. (1996). Immune mechanisms in neurodegenerative disorders. Drugs Today 32, 149-158. McGreer, P.L. & McGreer, E.G. (1995). The inflammatory response system of brain: impliications for therapy of Alzheimer and other neurodegenerative diseases. Brain Res. Rev. 21, 195-218. Minami, M., Kuraishi, Y. & Satoh, M. (1991). Effects of Kainic acid on messenger rna levels of IL-1B, IL-6, TNFct and LIF in the rat brain. Biochem. Biophys. Res. Commun. 176, 593-598. Miura, M., Friedlander, R.M. & Yuan, J. (1995). Tumor necrosis factor-induced apoptosis is mediated by a CrmA-sensitive cell death pathway. Proc. Natl. Acad. Sci. U. S. A. 92, 8318-8322. Mogi, M., Harada, M., Kondo, T., Mizuno, Y., Narabayashi, H., Riederer, P. & T.N. (1996). The soluble form of Fas molecule is elevated in Parkinsonian brain tissues. Neurosci Lett. 220, 195-198. Muzio, M., Chinnaiyan, A.M., Kischkel, F.C., O'Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J.D., Zhang, M., Gentz, R., Mann, M., Krammer, P.H., Peter, M.E. & Dixit, V. M. (1996). FLICE, a novel FADD-homologous ICE/CED-3 like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell 85, 817-827. Muzio, M., Salvesen, G.S. & Dixit, V.M. (1977). FLICE induced apoptosis in a cell-free system. Cleavage of caspase zymogens. J. Biol. Chem. 272, 2952-2956. Nichol, K.A., Chan, N., Davey, D.F. & Bennett, M.R. (1995). Location of nitric oxide synthase in the developing avian ciliary ganglion. J. Auton. Nerv. Syst. 51, 91-102. Nicholson, D.N. & Thornberry, N.A. (1997). Caspases: killer proteases. Trends Biochem. Sci. 22, 299-306. Nishimura, T., Akiyama, H., Yonehara, S., Kondo, H., lkeda, K., Kato, M., Iseki, E. & Kosaka, K. (1995). Fas antigen expression in brains of patients with Alzheimer-type dementia. Brain Res. 695, 137-145. Oehm, A., Behrmann, I., Falk, W., Pawlita, M., Maier, G., Klas, C., Li Weber, M., Richards, S., Dhein, J., Trauth, B.C., Pansting, I.K. & Krammer, P.H. (1992). Purification and molecular cloning of the APO-1 cell surface antigen, a member of the tumor necrosis factor/nerve growth factor superfamily. Sequence identity with the Fas antigen. J. Biol.Chem. 267, 10709- 10715. Ogasawara, J., Suda, T. & Nagata, S. (1995). Selective apoptosis in CD8+ thymocytes by the anti-Fas antibody. J. Exp. Med. 181,485-491. Orth, K., O'Rourke, K., Salvesen, G.S. & Dixit, V.M. (1996). Molecular ordering of apoptotic mammalian CED-3/ICE-Iike proteases. J. Biol. Chem. 271, 20977-20980. Pan, G., Bauer, J.H., Haridas, V., Wang, S., Lui, D., Yu, G., Vincenz, C., Aggarwal, B.B., Ni, J. & Dixit, V.M. (1998). Identification and functional characterization of DR6, a novel death domain-containing TNF receptor. FEBS Lett. 431,351-356.

Death Domains and the Brian

63

Pan, G., Ni, J., Wei, Y.F., Yu, G., Gentz, R. & Dixit, V.M. (1997a). An antagonist decoy receptor and a death domian-containing receptor for TRAIL. Science 277, 815-818. Pan, G., O'Rourke, K., Chinnaiyan, A.M., Gentz, R., Ebner, R., Ni, J. & Dixit, V.M. (1997b). The receptor for the cytotoxic ligand TRAIL. Science 276, 815-818. Pan, G., O'Rourke, K. & Dixit, V.M. (1998). Caspase 9, BcI-XL, and Apaf-1 form a tertiary complex. J. Biol. Chem. 273, 5841-5845. Park, A. & Baichwal, V.R. (1996). Systematic mutational analysis of the death domain of the tumor necrosis factor receptor 1-associated protein TRADD. J. Biol.Chem. 271, 9858-9862. Prewitt, C.M., Niesmann, I.R., Kane, C.J.M. & Houle, J.D. (1997). Activated macrophage/microglial cells can promote the regeneration of sensory axons into the injured spnial cord. Exp. Neurol. 148,433-443. Rabchevsky, A.G. & Streit, W.J. (1997). Grafting of cultured microglial cells into the lesioned spinal cord of adult rats enhances neurite outgrowth. J. Neurosci. Res. 47, 34-48. Ravi, R., Bedi, A., Fuchs, E.J. & Bedi, A. (1998). CD95 (Fas)-induced caspase mediated proteolysis of NF-•B. Cancer Res. 58,882-886. Rothe, M., Pan, M.-G., Henzel, W.J., Ayres, T.M. & Goeddel, D.V. (1995). The TNFR2-TRAF signaling complex contains two novel proteins related to baculoviral inhibitor of apoptosis proteins. Cell 83, 1243 - 1252. Roy, N., Mahadevan, M.S., McLean, M., Shutler, G., Yaraghi, Z., Farahani, R., Baird, S., Besner-Johnston, A., Lefebvre, C., Kang, X., Salih, M., Aubry, H., Tamai, K., Guan, X., Ioannou, P., Cmwford, T.O., de Jong, P.J., Surh, L., Ikeda, J.-E., Korneluk, R.G. & MacKenzie, A. (1995). The gene for neuronal apoptosis inhibitory protein is partially deleted in individuals with spinal muscular atrophy. Cell 80, 167-178. Saas, P., Walker, P.R., Hahne, M., Quiquerez, A., Schnuriger, V., Perrin, G., French, L., Van Meir, E.G., de Tribolet, N., Tschopp, J. & Dietrich, P. (1997). Fas expression by astrocytoma in vivo: maintaining immune privilege in the brain? J. Clin. Invest. 99, 1173-1178. Santana, P., Pena, L.A., Haimovitz Freidman, A., Martin, S., Green, D., McLoughlin, M., Cordon Cardo, C., Schuchman, E.H., Fuks, Z. & Kolesnick, R. (1996). Acid sphingomyelinase-deficient human lymphoblasts and mice are defective in radiation-induced apoptosis. Cell 86, 189-199. Schneider, P., Thome, M., Burs, K., Bodmer, J.L., Hofman, K., Kataoka, T., Holler, N. & Tschopp, J.(1997). TRAIL receptors 1 (DR4) and 2 (DR5) signal FADD-dependent apoptosis and activate NF- Kappa B. Immunity 7, 831-836. Schulze-Osthoff, K., Ferrari, D., Los, M., Wesselborg, S. & Peter, M.E. (1998). Apoptosis signaling by death receptors. FEBS Lett. 254, 439-459. Sen, R. & Baltimore, D. (1986). Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell 46, 705 - 16. Sheridan, J.P., Marsters, S., Pitti, R.M., Gurney, A., Skubatch, M., Badwin, D., Ramakrishnan, L., Gray, C.L., Baker, K., Wood, W.I., Goddard, A.D., Godowski, P. & Ashkenazi, A. (1997). Control of TRIAL-induced apoptosis by a family of signaling of decoy receptors. Science 277, 818-821. Shu, H.B., Halpin, D.R. & Goeddel, D.V. (1997). Casper is a FADD- and caspase-related inducer of apoptosis. Immunity 6, 751-763. Shu, H.B., Takauchi, M. & Goeddel, D.V. (1996). The tumor necrosis factor receptor 2 signal transducers TRAF2 and c-IAPI are componets of tumor necrosis factor receptor 1 signaling complex. Proc. Nat. Acad, Sci. U. S. A. 93, 13973-13978. Srinivascula, S.M., Ahmad, M., Fernandes Alnemri, T., Litwack, G. & Alnemri, E.S. (1996). Molecular ordering of the Fas-apoptotic pathway: the Fas/APO-1 protease Mch5 is a CrmA-inhibitable protease that activaters multiple Ced-3/ICE-like cysteine proteases. Proc. Natl. Acad. Sci. U. S. A. 93, 14486-14491.

64

A.J. Bruce-Keller

Srinivasula, S.M., Ahmad, M., Ottilit, S., Bullrich, F,, Banks, S., Wang, Y., Femandes-Alnemri, T., Croce, C.M., Litwack, G., Tomaselli, K.J., Armstrong, R.C. & Alnemri, E.S. (1997). FLAME-l, a novel FADD-Iike apoptotic molecule that regulates Fas/TNFRl-induced apoptosis. J. Biol. Chem. 272, 18542-18545. Stanger, B.Z., Leder, P., Lee, T.H., Kim, E. & Seed, B. (1995). RIP: a novel protein containing a death domain that interacts with Fas/APO-1 (CD95) in yeast and causes cell death. Cell 81,513-523. Szaflarski, J., Burtrum, D. & Silverstein, F.S. (1995). Cerebral hypoxia stimulates cytokine gene expression in perinatal rats. Stroke 26, 1093-1100. Takahashi, T., Tanaka, M., Brannan, C.I., Jenkins, N.A., Copeland, N.G., Suda, T. & Nagata, S. (1994). Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand. Cell 76, 969-976. Tartaglia, L.A., Ayes, T.M., Wong, G.H. & Goeddel, D.V. (1993a). A novel domain within the 55kD TNF receptor signals cell death. Cell 74, 845-853. Tartaglia, L.A., Pennica, D. & Goeddel, D.V. (1993b). Ligand passing: the 75-kDa tumor necrosis factor (TNF) receptor recruits TNF for signaling by the 55-kDa TNF receptor. J. Biol. Chem. 268, 18542-18548. Taupin, V., Toulmond, S., Serrano, A., Beuavides, J. & Zavala, F. (1993), Increases in IL-6, IL-1 and TNF levels in rat brain following traumatic lesion. J. Neuroimmunol. 42, 177-186. Tchelingerian, J., Quinonero, J., Booss, J. & Jacque, C. (1993). Localization of TNFct and IL-lct immunoreactivities in striatal neurons after surgical injury to the hippocampus. Neuron 10, 213-224. Tewari, M. & Dixit, V.M. (1995). Fas- and tumor necrosis factor induced apoptosis in inhibited by the poxvirus crmA gene product. J. Biol. Chem. 270, 3255-3260. Thome, M., Schneider, P., Hofmann, K., Fickenscher, H., Meinl, E., Neipel, F., Mattmann, C., Bums, K., Bodmer, J.-L., Schroter, M., Scaffidi, C., Krammer, P.H., Peter, M.E. & Tschopp, J. (1997). Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors. Nature 386, 517-521. Ting, A.T., Pimentel Muinos, F.X. & Seed, B. (1996). RIP mediates tumor necrosis factor receptor 1 activation of NF-KB but not Fas/APO-l-initiated apoptosis. EMBO J. 15, 6189-6196. Toku, K., Tanaka, J., Yano, H., Desaki, J., Zhang, B., Yang, L., lshihara, K., Sakanaka, M. & Maeda, N. (1998). Microglial cells prevent nitric oxide-induced neuronal apoptosis in vitro. J. Neurosci. Res. 53, Vassalli, P. (1992). The pathophysiology of tumor necrosis factors. Ann. Rev. Immunol. 10, 411-452. Verma, I.M. & Stevenson, J. (1997). IKB kinase: beginning, not the end. Proc, Nat. Acad. Sci. U.S.A. 94, 11758-11760. Vogt, M., Bauer, M.K., Ferrari, D. & Schulze-Osthoff, K. (1998). Oxidative stress and hypoxiaJreoxygenation trigger CD95 (APO-1/Fas) ligand expression in microglial cells. FEBS. Lett. 429, 67-72. Walczak, H., Delgi Esposti, M.A., Johnson, R.S., Smolak, P.J., Waugh, J.Y., Boiani, N., Timour, M.S., Gerhart, M.J., Schooley, K.A., Smith, C.A., Goodwin, R.G. & Rauch, C.T. (1997). TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL. EMBO J. 16, 5386-5397. Wang, C.-Y., Mayo, M.W. & Baldwin Jr., A.S. (1996). TNF- and cancer therapy-induced apoptosis: Potentiation by inhibition of NF-KB. Science 274, 784-787. Watanabe-Fukunaga, R., Brannan, C.I., Copeland, N.G., Jenkins, N.A. & Nagata, S. (1994). Lymphoproliferation disorder in mice explained by deficits in Fas antigen that mediates apoptosis. Nature 356, 314-317. Wolvers, D.A., Marquette, C., Berkenbosch, F. & Haour, F. (1993). Tumor necrosis factor alpha: specific binding sites in the rodent brain and pituitary gland. Eur. Cytokine Netw. 4, 377-381. Wong, G.H. & Goeddel, D.V. (1988). Induction of MnSOD by TNF: possible protective mechanism. Science 242, 941-944.

Death Domains and the Brian

65

Wu, M., Lee, H., Bellas, R.E., Schauer, S.L., Arsura, M., Katz, D., Fitzgerald, M.F., Rothstein, T.L., Sherr, D.H. & Sonenshein, G. E. (1996). EMBO J. 15, 46824690.

Inhibition of NF-kB/Rel induces apoptosis of murine B cells.

DIVERSITY OF CASPASE I N V O L V E M E N T IN N E U R O N A L CELL D E A T H C A R O L M. TROY

Table of Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Caspase Family of Proteases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Classification of Caspases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Caspase Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Caspase Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Assessment of Caspase Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Measurement of Caspase Activation in Cells and Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . Models of Neuronal Cell Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Paradigms of Programmed Cell Death in Cultured Neuronal Cells . . . . . . . . . . . . . . . . . . . . Caspase Specificity in Neuronal Cell Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Caspases Essential for Normal Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Caspases Necessary for Other Types of Neuronal Cell Death . . . . . . . . . . . . . . . . . . . . . . . . Death Receptors and Neuronal Cell Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . All Caspase Activity Does Not Produce Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

67 68 69 70 71 72 73 73 74 76 78 78 83 84 85

Introduction Programmed cell death (or apoptosis) in neurons is an active, genetically regulated suicide mechanism which is essential for normal neuronal development and is a commonly observed feature in neurodegenerative diseases (Bains and Shaw, 1997). The responses of cells to apoptotic stimuli are morphologically and biochemically similar across cell-types and species. Classically, morphologic hallmarks of apoptosis include cell shrinkage, nuclear condensation, clumping of chromatin and formation of apoptotic bodies (Wyllie, 1980; Wyllie et al., 1984). The dying cells disappear without any accompanying inflammation (Kerr et al., 1972). Associated DNA fragmentation can be visualized as a ladder by gel electrophoresis (Wyllie et al., 1984; Batistatou and Greene, 1991). The machinery employed in cell death is evolutionarily conserved. Genetic studies on development in C. Elegans identified three genes which regulated cell death and survival, demonstrating that specific molecular pathways governed programmed cell death (Hengartner et al., 1992; Yuan and Horvitz, 1992; Yuan et al., 1993). Both proapoptotic (ced-3 and ced-4) and antiapoptotic (ced-9) regulators of cell death were identified in the nematode; all have mammalian homologs. Ced-9 is a homolog of the bcl-2 family of proteins (Vaux et al., 1988, 1992), ced-4 is a homolog of Apaf-1 (Zou et al., 1997). The positive regulator ced-3 was found to be a homolog of the 67 Cellular and Molecular Mechanisms Ed. by M.P. Mattson, S. Estus and V.M. Rangnekar. 67 © 2001 Elsevier Publishers. Printed in the Netherlands.

-

-

92

68

C.M. Troy

previously identified mammalian enzyme, interleukin-1 converting enzyme (Cerretti et al., 1992; Thornberry et al., 1992; Yuan et al., 1993), which is now known as caspase-1. Both ced-3 and caspase-1 induce apoptosis after transient expression in mammalian cells (Miura et al., 1993), providing evidence that a conserved family of proteases is a critical factor in cell death. Soon after the discovery of ced-3 other mammalian caspases were identified and also shown to induce death. At the time of preparation of this chapter 14 members of the caspase family have been identified (Ahmad et al., 1998; Hu et al., 1998; Humke et al., 1998). For recent reviews see: Cohen, 1997; Nicholson and Thornberry, 1997; Bergeron and Yuan, 1998; Cryns and Yuan, 1998; Green, 1998; Thornberry and Lazebnik, 1998).

Caspase Family of Proteases Caspases are cysteine aspartases, each contains a conserved QACXG pentapeptide containing the active site cysteine residue (for a review of structural and enzymatic features see Nicholson and Thornberry, 1997). They are constitutively present in most cells and exist in the cytosol as catalytically inactive proenzymes (zymogens) containing a variable length amino terminal prodomain, a large subunit and a small subunit. They can be activated by autocleavage, cleavage by another caspase or cleavage by granzyme B. Cleavage occurs in two steps, shown in Figure 1. First, the chain is cleaved between the large and small subunits and then a second cleavage removes the prodomain from the large subunit. Crystallographic studies show that the subunits assemble into a heterodimer, and two heterodimers assemble to form a tetramer with two active sites (Walker et al., 1994; Wilson et al., 1994; Rotonda et al., 1996). Once activated there can be additional caspase-caspase interactions and cascades of activation. However, the initial zymogen must be activated to begin these events. An oligomerization model has been proposed to explain the onset of activation of caspases with long prodomains. Procaspase-8 has been shown to possess 1-2% of the activity of mature caspase 8; aggregation of procaspase-8 initiates autoactivation (Muzio et al., 1998; Yang et al., 1998). The aggregation of procaspases is mediated by the prodomain which interacts with the activating apparatus. Two different types of interaction domains have been identified: caspase recruitment domains (CARDs), found in caspases -1, -2, -4, -5, and -9 (Ahmad et al., 1997; Duan and Dixit, 1997; Hofmann et al., 1997); and death effector domains (DEDs), found in caspases -8 and -10 (Boldin et al., 1995; Chinnaiyan et al., 1995; Boldin et al., 1996; Muzio et al., 1996; Vincenz and Dixit, 1997). The procaspases also bind to adaptor molecules containing similar domains. For examples, caspase -8 associates with FADD (Fas-associated protein with death domain) through the DED while caspase -9 forms a complex with APAF- 1 through the CARD. Recruitment of the caspases via the adaptor molecules results in formation of an apoptosome that mediates caspase activation. Details of this model are discussed in other chapters in this book as well as in the review by Green (1998).

Diversity of Caspase Involvement

69

SCHEMATIC DIAGRAM OF CASPASE ACTIVATION

Asp Asp ¢ Largesubunit ~ Smallsubunit

Prodomain

I

!!iiiiiii iiiiii iiiiiii l lst CLEAVAGE

I

iiiiiiiiiiiiiiiiiiiiiiiiiiiii l

2nd CLEAVAGE

i...======........m.l===l. iumnnnmn •mmnnguiH| u u u n n m i n N u l m m n H l | H u n n u a n n n . i u n H m u

lnlllmWlllnHm!=~=llllla

+

l ActiveCaspaseh e t e r o d i m e r ~ ,,,,,,,,=,,, ...... ,, ......... ......... i[, H .... ~, ..... Figure 1.

Schematic diagram of caspase activation. A representative pro-caspase is illustrated.

Cleavage occurs in two steps: The first cleavage releases the small subunit and the second cleavage removes the prodomain from the large subunit. The subunits form a heterodimer, and two heterodimers combine to form the active tetramer. The large and small subunits are extensively conserved throughout the family. The prodomain is not conserved among family m e m b e r s .

Classification of Caspases Several classifications of caspases have been used, including subsets based on structure as well as those based on presumed function. The latter divides the caspases into initiators (caspases with long prodomains) and effectors (caspases with short prodomains) with the initiators acting upstream of the effectors (Green, 1998; Thornberry and Lazebnik, 1998). However, in neuronal systems it is not clear whether this classification strictly applies. Structural considerations, which encompass cleavage specificity as well as sequence homologies, allow the separation of caspases into 3 families (Figure 2).

70

C.M. Troy

The Caspase Family

t

Caspase-l," (.ICEI

Caspase- 12 Caspass-11 (Ich-3) - - -

_• m

Caspase-13 (ERICE) Caspase-4 (ICE rel II, TX, ICH2) Caspass-5 (ICE rel III, TY) Caspase-1 (ICE) Caspasa-3* (Yama, CPP32, Apopain) Caspase-7* (ICE-LAP3, Mch3, CMH-1) Caspass-6* (Mch-2) Caspase-8 (FLICE, MACH, Mch5) Caspase-10 (FLICE2, Mch4) Caspass-9 (ICE-LAP6, Mch6)

Caspase-2 (Ich-1, Nedd2)

Caspase-1 subfamily

Caspase-3 subfamily

Caspase-2 subfamily

*short prodomain Figure 2. The caspase family. The three caspase subfamilies, arranged by structural homologies. * indicates caspases with small prodomains, the rest have large prodomains.

The caspase l-like family, which includes caspases -1, -4, -5, -11, -12 -13; the caspase-3-1ike family, composed of caspases -3, -6, -7, -8, -9, -10 and caspase-2, which is the only member of its family. The most recently identified, caspase 14, is unique in that it is not processed in response to several different death signals but does interact physically with several long prodomain caspases (1, 2, 4, 8, and 10) and does induce death (Hu et al., 1998). It is also the only short prodomain member of the caspase-1 subfamily. The other caspases with short prodomains are members of the caspase-3 family, caspases-3, -6 and -7. Caspase Substrates Active caspases cleave their substrates on the carboxy-terminal side of an aspartate residue, the P1 site, illustrated in Figure 3. However, individual caspases differ in their substrate specificities. These differences are determined by the residues immediately amino-terminal to the P1 site, especially the P4 site. The substrate cleavage specificities have been defined using a positional scanning substrate combinatorial library (Thornberry et al., 1997) or synthetic peptides (Talanian et al., 1997). Three subgroups of caspases were defined based on the substrate preference using positional scanning. The first subgroup (caspases-1, -4 and -5) prefers bulky hydrophobic residues in P4 and has the optimal substrate cleavage site WEXD. The second subgroup (caspases-2, -3, -7 and ced-3) prefers an aspartate at P4 and cleaves optimally carboxy terminal to DEXD, with X=V for caspases-3 and -7 and X=H

Diversity of Caspase Involvement

71

Asp Prodomain

Large subunit

Asp ¢ Small subunit

iiiii!iillii !iill iiiii;///YY/',l Q

A

C

X

~

P4 P3 P2 P l Figure 3.

Schematic of pro-caspase. Conserved QACXG active site region and P1-P4 amino acids are

illustrated.

for caspase-2. The third group (caspases-6, -8 and -9) is less specific in P4 preference with an optimal cleavage sequence of (L/V)EXD. These predicted specificities agree well with the known substrates of the caspases. Caspase-1 cleaves pro-IL-lf5 at two sites, FEAD and YVHD, caspase-3 cleaves the DNA repair enzyme PARP at a DEVD site and caspase-6 cleaves lamin A at a VEID site. Many caspase substrates have been identified but it is still unclear how many of these substrates are directly involved in death. Other known substrates include inhibitors of apoptosis (IcAD (Enari et al., 1998), Bcl-2 (Cheng et al., 1997; Xue and Horvitz, 1997; Adams and Cory, 1998)) and cytoskeletal components (nuclear lamina (Lazebnik et al., 1995; Orth et al., 1996; Takahashi et al., 1996), actin (Mashima et al., 1995)) but the relation of cleavage of these substrates to cell death is not yet understood. Caspase Inhibitors Caspases have differential responses to both naturally occurring and synthetic inhibitors, further supporting uniqueness in the cleavage specificity of the individual members of this family. Three naturally occurring inhibitors have been described. Cytokine responsive modifier A (crmA) is a serpin from cowpox virus which is an excellent inhibitor of caspase-l-like proteases, but not particularly effective against caspase-3-1ike family members (Nicholson et al., 1995). p35, from baculovirus, is another viral gene product which prevents apoptosis and is an irreversible inhibitor of caspases-1 through -4 and ced-3 (Bump et al., 1995; Xue and Horvitz, 1995). The IAP (inhibitor of apoptosis) family of gene products are a mammalian group of polypeptides which inhibit caspases (Deveraux et al., 1997, 1998) which are discussed in detail in another chapter in this book. Synthetic pseudosubstrate inhibitors were designed as broad range or specific inhibitors and most are available as reversible (aldehyde) or irreversible (ketone) forms. The broad range inhibitors zVAD and BAF were developed to inhibit as many caspases as possible, although affinities of the different caspases for these inhibitors do vary (Margolin et al., 1997). YVAD and DEVD were designed as specific inhibitors of caspases-1 and -3 respectively, based on knowledge of the substrates of caspase-1 (pro-IL-1B) and caspase-3 (PARP). As the family members identified increased, kinetic studies of substrate and inhibitor specificities showed that DEVD had a broad

72

C.M. Troy

spectrum of action and inhibited most caspases except for caspase-2, at the concentration commonly used (50 /~M). The broad spectrum of DEVD action requires that caution be used in interpreting earlier work attributing apoptosis to caspase-3 on the basis of DEVD inhibition. YVAD was shown to inhibit caspases-1 and -4 but not caspases-3 or -7, so it appears to be more specific for caspase-l-like family members (Margolin et al., 1997). Since the different caspases have different affinities for the inhibitors, dose-response curves with the reversible versions of the inhibitors can provide indications about caspases involved in a particular death paradigm, as will be shown below. A novel strategy for ICE family inhibition is based on the conservation of the active site (QACXG) in all ICE family proteases, from ced-3 to the human homologs (Wang et al., 1994). In a variation on the pseudosubstrate approach to enzyme inhibition, we reasoned that a peptide, IQACRG, comprised of this active site might compete with the enzyme for binding to substrate, and thus inhibit all caspases present in a cell by acting as a pseudoenzyme, regardless of substrate specificity. In vitro, the IQACRG peptide inhibits the cleavage of recombinant pro-IL-1B by recombinant caspase-1 (Troy et al., 1996b), confirming that the peptide blocks caspase-I activity. The peptide is linked by a disulfide bond to a delivery peptide, Penetratinl, which allows rapid translocation through cell membranes (Prochiantz and Theodore, 1995; Theodore et al., 1995; Troy et al., 1996a). Once in the cytosol the inhibitory peptide is released from Penetratin I and can then interfere with the binding of any of the caspases to their substrates. Other broad spectrum inhibitors include zVAD and BAF. All of these are good for general detection of caspase activity, but do not yield specific information about individual caspases. Individual caspases can also be manipulated directly by overexpression, antisense transfection, antisense oligonucleotides and in transgenic and knockout animals. In a redundant family, such as the caspases, it is useful to manipulate the expression of caspases in several different ways to understand their function. The use of these approaches will be discussed below. Assessment of Caspase Activity Several artificial substrates exist which can measure caspase activity. These include the most commonly used DEVD and YVAD, linked either to AMC or pNA, for fluorometric or colorimetric determinations respectively. Fluorometric measurements are more sensitive than colorimetric. Kinetic studies of substrate cleavage by caspases-1, -2, -3, -4, -6 and -7 show that there is considerable overlap in the cleavage specificity of the different caspases (Talanian et al., 1997). The most commonly used substrate, DEVD, is cleaved by all of the caspases tested except caspase-2. Thus DEVD-cleaving activity can be taken as a general measure of caspase activity, excepting caspase-2 activity, but certainly not as a specific measure of caspase-3 activity or even of activity of caspase-3-1ike family members. A 5 amino acid substrate, VDVAD, is cleaved by caspase-2 but is also cleaved by caspases-3 and -7 (Talanian et al., 1997). Differential use of these two substrates could provide data about caspase-2 activity. Another commonly used caspase substrate is YVAD which is cleaved well by caspases-1 and -4, poorly by caspase-3 and not cleaved by caspases-2, -6 and -7 (Talanian et al., 1997;

Diversity of Caspase Involvement

73

Thornberry et al., 1997). Thus, the use of multiple substrates can provide more specific data about individual caspases than the use of only a single substrate. Another measure of caspase-1 activity is the release of interleukin-16. Caspase-1 is the only family member which cleaves pro-IL-113 in cells, although caspase-4 can cleave pro-IL-1B in vitro with 2 orders of magnitude less activity than caspase-1 (Margolin et al., 1997). Cleavage of cellular substrates are also measures of caspase activity. Cleavage of tamin is indicative of caspase-6 activity (Lazebnik et al., 1995; Orth et al., 1996; Takahashi et al., 1996) and PARP, originally proposed as a specific substrate of caspase-3, can also be cleaved by caspases-1, -4 and -7 though less efficiently by caspases-1 and -4 than by caspases-3 and 7 (Margolin et al., 1997). Measurement of Caspase Activation in Cells and Tissues Since caspases exist as inactive zymogens, activation of a particular caspase can be detected by Western blotting when appropriate antibodies are available to detect the active subunits, which range from 17-20 kDa for the large subunit and 10-12 kDa for the small subunit. Usually there is a large amount of the zymogen present relative to the active subunits, even at timepoints where there is detectable caspase activity. It is likely that once there is caspase activation in a cell that it does not survive for long. Therefore, in a population of cells, at any moment only a small proportion will contain activated caspase. Antibodies have been developed which detect only the active p20 fragment of caspase-3, not the proform (Armstrong et al., 1997). These are very useful for studies of cell death in tissues where detection of the active caspase-3 can be combined with TUNEL labeling to detect dying cells (Namura et al., 1998).

Models of Neuronal Cell Death

Much still remains to be determined about the death pathways. A question of great interest is why there are so many caspases. Multiple family members can be present in one cell. Does each have a particular function or is there biologic redundancy? Data from targeted deletions of individual caspases support different functions for specific caspases. Knockouts of caspase-1 (Kuida et al., 1995; Li et al., 1997), -2 (Bergeron et al., 1998) and -11 (Wang et al., 1998) do not have an obvious neuronal phenotype, indicating that these are not critical for early development and/or there is functional redundancy for these caspases. Caspase-3 (Kuida et al., 1996), -8 (Varfolomeev et al., 1998) and -9 (Hakem et al., 1998; Kuida et al., 1998) knockouts have devastating phenotypes, supporting a critical role for these caspases in developmental cell death. The investigation of other individual caspase knockouts are underway and this approach has yielded much data about the roles of specific caspases in developmental neuronal death. However, the effect of knockouts is often influenced by the genetic background of the animals, and it is possible that the redundancy of caspases allows functional substitution at early developmental stages. The availability of "conditional knockouts" will allow a more complete assessment of the question of which caspases are responsible for the aberrant cell death which occurs in models of

74

C.M. Troy

adult neurodegenerative disorders. The ensuing section will present data from model systems of cell death, as well as the specific phenotypes of the null mice and other in vivo data, which address the specific functions of some of the caspases. Caspase activity has been shown to occur during experimental ischemia (Friedlander et al., 1996, 1997b; Namura et al., 1998) and caspases are likely involved in apoptotic neuronal cell death that has been reported in amyotrophic lateral sclerosis (ALS), Alzheimer's and other neurodegenerative diseases. While it is an open question whether apoptosis represents a key step in the disease process, or simply an agonal event eliminating cells which have long before been rendered useless, there is a possibility that caspase inhibition, or blockade of other steps in the apoptotic cascade, might halt the progress of one or more of these diseases. However, a broad spectrum blockade would also pose the risk of perpetuating excessive growth of mutated ceils, which might go on to form tumors. The data suggesting that parallel paths to cell death are taken by neurons, depending on the type of injury they sustain, raises the possibility that therapies could be crafted that are quite specific for a given disease process. Hence, an important question, which remains to be answered, is which caspases are necessary for progression of different neurodegenerative diseases? Among the most promising results is the delay in progression of symptoms in a mouse model of ALS by expression of the dominant negative caspase-1 (Friedlander et al., 1997a), discussed in more detail below. We, and others, have begun to address the question of caspase specificity using models of neuronal cell death. By studying models of apoptotic neuronal death, the progression to death in response to different stimuli can be defined to determine where these pathways converge and diverge. We have employed multiple models for the induction of death in PC12 cells and primary cultures of sympathetic neurons. These models test key theories about cell death in human neurodegeneration. The stimuli we use include oxidative stress, trophic factor deprivation, and DNA damage. Initiating death in the same cell-type with different stimuli allows us to address whether the molecular mechanisms utilized when the cell is exposed to each stimulus are the same or different. Utilizing the same cell-type ensures that the same repertoire of molecules is available. Paradigms of Programmed Cell Death in Cultured Neuronal Cells The three models of cell death which we have studied include: oxidative stress induced by the downregulation of SOD1 (Troy and Shelanski, 1994; Troy et al., 1996a, 1996b); trophic factor withdrawal, in which NGF is withdrawn from cell-types which are dependent on it for survival (Greene, 1978; Rukenstein et al., 1991; Troy et al., 1996b; 1997b; Stefanis et al., 1998); and DNA damage induced by radiation or drugs (Park et al., 1998b). Details of these models follow. Free radicals represent a class of biologically generated species that have been suggested to play a role in many neuronal disorders (Coyle and Puttfarcken, 1993; Brown, 1995; Schapira, 1996). However, the actual mechanism by which they cause neuronal degeneration has not yet been defined. Cu/Zn superoxide dismutase (SOD1) is among the key cellular enzymes by which neurons and other cells detoxify free radicals and protect themselves from damage (McCord and Fridovich, 1969; Fridovich, 1995).

Diversity of Caspase Involvement

75

The observation that a subset of cases of familial amyotrophic lateral sclerosis (FALS) are associated with missense mutations of SOD1 provided the first molecular evidence for involvement of this enzyme in neuronal degenerative disorders (Rosen et al., 1993). While the initial report associated FALS mutations with a loss of SOD activity, other disease causing mutations in the enzyme have normal levels of activities. Therefore, the mechanism by which these missense mutations lead to cell death is not clear, though there is evidence to support a role for free radical mediated death (Srinivasan et al., 1996). There is also the possibility that, by analogy with SODI null yeast, copper buffering is disrupted (Culotta et al., 1995). As a model for oxidative stress-mediated cell death, we have employed antisense oligonucleotides to specifically downregulate SOD1. Delivery of the antisense is facilitated by linking the oligonucleotide (ASOD1) to Penetratinl (V-), the delivery peptide discussed above, which allows rapid uptake of the oligonucleotide in serumcontaining medium and increases efficacy by 100 fold over non-vector linked oligonucleotides (Troy et al., 1996a). Within 24 h of downregulation, there is 50-60% apoptotic cell death in PCI2 cells (Troy and Shelanski, 1994; Troy et al., 1996a) which can be prevented by vitamin E and overexpression of bcl-2, but not by insulin (Troy and Shelanski, 1994). In sympathetic neurons downregulation of SOD1 is not sufficient to induce death. However, addition of exogenous nitric oxide concurrent with SOD1 decrease induces death. Nitric oxide by itself is not toxic to the sympathetic neurons or to PCI2 cells (Farinelli et al., 1996; Troy et al., 1996a) and, in fact, protects against trophic factor deprivation (Farinelli et al., 1996). Moreover, the SODl-depleted PC12 cells can be rescued by nitric oxide synthase inhibitors (Troy et al., 1996a). These results, among others, point to the production of peroxynitrite -- which is formed from nitric oxide and superoxide -- as the damaging agent. Manganese superoxide (SOD2) has also been shown to protect from NO-mediated toxicity (Gonzalez-Zulueta et al., 1998; Keller et al., 1998), supporting a role for formation of peroxynitrite in the mechanism of the cell death mediated by an increase in superoxide. Trophic factors represent another influence on neuronal survival. While trophic factors are known to be essential in the developing nervous system (Silos-Santiago et al., 1995), mature neurons also require these substances for their maintenance. There has been much speculation that neurodegenerative disorders may involve disturbances in trophic factor supply or in the ability of cells to respond to them (Williams, 1995). Moreover, even if variations in neurotrophic factors, or the responses to them, are not causally involved in neurodegenerative disorders, there is evidence that these agents can ameliorate or protect neurons from degeneration (Coyle and Puttfarcken, 1993). As a model for trophic-deprivation mediated cell death, removal of serum from naive PC12 cells, or of serum and NGF from NGF-primed PC12 ceils, or NGF from sympathetic neurons, results in 50-85% apoptotic cell death after one day (Greene, 1978). This death can be prevented by multiple agents including NGF (Greene, 1978), insulin (Rukenstein et al., 1991), overexpression of the proto-oncogene bcl-2 (Batistatou et al., 1993) and NO (Farinelli et al., 1996). DNA damage can also lead to apopotosis. Clinically, DNA damage is seen after radiation therapy for tumors as well as in diseases with excessive sensitivity to irradiation,

C.M. Troy

76

such as ataxia-telangiectasia (Enns et al., 1998; Lavin, 1998; Park et al., 1998a). In modeling D N A damage several different agents have been used. These include irradiation, arabinoside C, and camptothecin (Park et al., 1998a, 1998b). The most straightforward is irradiation, either ultraviolet or gamma, leading to direct damage o f the DNA. W e have used neuronal (NGF-primed) PC12 cells and sympathetic neurons to study the path to death after D N A damage.

Caspase Specificity in Neuronal Cell Death Knowledge o f the potential players is important in determining the individual caspase(s) responsible for death. Recent work has begun to identify the caspases present in PC12 cells (J. Angelastro, personal communication). Caspases 1-3, 6-9, I 1 are all present (Angelastro et al., 1998). Caspases- 4 and -5 have been isolated only from humans and m a y not have rodent homologs. Table 1.

Caspase Detection in PC12 cells

CASPASE

mRNA

Cas ~ase-1

Yes Yes Yes No No Yes Yes Yes Yes n.d. Yes

Cas ~ase-2 Cas )ase-3 Cas 9ase-4 Cas ~ase-5 Cas ~ase-6 Cas )ase-7 Cas )ase-8 Cas ~ase-9 Cas )ase-10 Cas ~ase-11

PROTEIN Yes Yes Yes

Not determined Yes Yes Not determined Not determined Not determined

Caspase mRNAs were detected by RT-PCR using probes prepared from rat caspase sequence data, when available, or from mouse or human sequences. Protein was detected by Western blotting of whole cell lysates using available antibodies. Adapted from (Angelastro et al., 1998).

Thus, it appears that most, if not all, the caspases are present in one cell-type. This again raises the question o f whether there is a specific function for each caspase. If not, why is there so much redundancy within a given cell-type? Granted, PC12 cells are a cultured tumor cell line and thus may have more caspases expressed than a normal cell. Now that rodent sequences are known for the different family members normal neurons will be evaluated for presence o f the different caspases.

Diversity of Caspase Involvement

77

As an initial approach to determining the caspase requirements in our three death paradigms, several different types of caspase inhibitors were used to generate protection profiles in each of the cell death models using PC12 cells and sympathethic neurons (Stefanis et al., 1996, 1997, 1998; Troy et al., 1996b, 1997a; b; Park et al., 1998b). Y V A D is the most selective inhibitor, V-ICEinh is the least selective. The data support the thesis that different caspases are operative in the different death paradigms and that the death stimulus, not the cell-type, determines the caspase utilized. This is summarized in Table 2. Table 2.

Differentialprotection by caspase inhibitors in three paradigms of cell death YVAD-FMK

ZVAD-FMK DEVD-FMK

downregulation

100% survival

100% survival

100% survival

100% survival

Trophic factor deprivation

No

100%

100%

survival

No protection

100%

protection

survival

survival

No protection

No protection

No protection

100% survival

100% survival

SOD1

DNA damage

BAF

V-ICEinh 100% survival

Cultures of PC12 cells and sympathetic neurons were treated with the various death producing stimuli in the presence of the indicated caspase inhibitors. Survival was quantified as previouslydescribed (adapted from Troy et al., 1996b; Park et al., 1998b; Stefanis et al., 1998).

Despite the effectiveness of the caspase inhibitors in preventing death, there were marked differences in the potency of the inhibitors in the different paradigms. Cells undergoing trophic deprivation-mediated death required higher doses of both the V-ICEInh and zVAD-FMK inhibitors than cells exposed to V-ASOD1. In addition, the inhibitory peptide AcYVAD-CMK protected against SOD1 downregulation but had little effect against trophic factor deprivation or DNA damage in cultures of PC12 cells or sympathetic neurons (Troy et al., 1996b; Park et al., 1998b). DNA damage induced death was prevented only by the broadest spectrum inhibitors, BAF and V-ICEinh. This differential effect of the inhibitors raised the possibility of differences in the substrate(s) utilized in the different paradigms of death or of differences in the caspase(s) involved, or in both. As stated above, the knockout data point to two groups of caspases, those which are essential for normal development and those whose absence has little or no effect on normal development. Data from other systems point to a role for this second group of caspases in neuronal degeneration, which may have pathways that differ from those required for developmental cell death. We will first briefly describe the phenotypes of the mice lacking caspases necessary for normal development and then we will present data, which support roles for caspases-1 and -2 in death paradigms with relevance to neurodegenerative diseases.

78

C.M. Troy

Caspases Essential for Normal Development Mice null for caspases-3, -8 or-9 all have abnormal development. Null deletions of caspases-8 and-9 are both embryonic lethal (Varfolomeev et al., 1998; Hakem et al., 1998; Kuida et al., 1998), while the caspase-3 knockout mice die at 1-3 weeks of age (Kuida et al., 1996). In both caspase-3 and -9 knockouts brain, development is profoundly abnormal with a variety of hyperplasias, disorganized cell distribution, and absence of the pyknotic clusters of cells normally seen in CNS, pointing to a failure of morphogenetic death. These caspases are not necessary for all types of cell death. This is illustrated by the sensitivity of thymocytes from animals lacking caspase-3 to induction of apoptosis by anti-Fas antibody, dexamethasone, C2-ceramide, staurosporine and gamma-irradiation, and thymocytes from animals lacking caspase-9 are sensitive to apoptosis induced by UV irradiation or anti-CD95. Caspase processing, particularly of caspase-3, is inhibited in the caspase-9 null ES cells but not in thymocytes or splenocytes, suggesting different apoptotic pathways in mammalian cells. Although caspase-9 has been found to be activated via a complex with Apaf-1, the caspase-9 knockouts do not accurately mimic the Apaf-1 knockouts, indicating that Apaf-1 can act via another caspase than caspase-9, although none of the other known caspases appear to be activated by Apaf-1 (Cecconi et al., 1998; Yoshida et al., 1998). Caspases Necessary for Other Types of Neuronal Cell Death In the caspase- 1, -2, and- 11 null mice, brain development is grossly normal, suggesting that these caspases do not play a critical role in neuronal development (Kuida et al., 1995; Li et al., 1997) Caspase-1 and -11 null mice are deficient in production of interleukin113, as expected, and intedeukin-la, which has yet not been explained. These mice are resistant to lipopolysaccharide-induced endotoxic shock. Caspase-ll physically interacts with caspase-1, suggesting that caspase-11 is a component of the caspase-1 complex and required for the activation of caspase-1. In vitro thymocytes from these caspase-I null mice are resistant to Fas-mediated apoptosis, in contract to caspase-3 mice, and cultured dorsal root gangion cells are resistant to trophic factor deprivation. A dominant-negative mutant caspase-1 (C285G) has also been expressed in mice (Friedlander et al., 1997b). As in the null mouse, neural development is normal, and the mice are deficient in interleukin-ll3 production. Trophic factor deprivation mediated death is inhibited in cultures of dorsal root ganglion cells from these animals. The mice also have a decrease in injury post-ischemia via middle cerebral artery occlusion. It is possible that expression of this construct might inhibit caspases other than caspase-1. However, the phenotype is in remarkable accord with that of the caspase-1 null mouse. Cells from caspase-2 null mice also have differential responses to death stimuli. Their ovaries have an increase in the number of germ ceils and the oocytes were found to be resistant to cell death following exposure to chemotherapeutic drugs. Apoptosis mediated by granzyme B and perforin was defective in the B lymphoblasts. In contrast, cell death of motor neurons during development was accelerated in the mice. In addition, caspase-2-deficient sympathetic neurons underwent apoptosis more quickly

Diversity of Caspase Involvement

79

than wild-type neurons when deprived of NGF. Thus, caspase-2 acts both as a positive and negative cell death effector, depending upon cell lineage and stage of development.

The Case for Caspase-I Although caspase-1 and its relatives caspases-4, -5 and -11 have been proposed to have roles only in inflammation and not in apopotosis (Thornberry and Lazebnik, 1998), there is considerable evidence pointing to a selective role for caspase-1 in mediating neuronal cell death. The caspase-1 knockout and caspase-1 dominant negative mice are each resistant to apoptosis induced by middle cerebral artery occlusion, and cultured DRGs from both mutant animals are resistant to trophic factor deprivation. The lack of a developmental phenotype can be attributed to either lack of a role for caspase-I in development or to compensation by other caspases during development. Action of interleukin-lB (IL-113) at its cell surface receptor is crucial for caspase-1 mediated death. IL-113 itself does not induce death, it must be acting on a cell which has a redox milieu which allows IL-lf3 to be apoptotic. All of the caspase-1 mediated deaths appear to involve excessive free radical production which leads to alterations of the redox setting of the cells. In our death paradigm, the downregulation of SOD1 leads to production of superoxide. Death can be blocked by YVAD treatment or by blocking the action of IL-1B, either with specific blocking antibodies or treatment with the IL-1 receptor antagonist (IL-1Ra), a naturally occurring peptide inhibitor of the IL-1 receptor (Troy et al., 1996b). Similar treatment protects DRG cultures from trophic factor mediated death and reduces the size of ischemic infarcts after MCA occlusion (Friedlander et al., 1996). Hippocampal neurons treated with staurosporine undergo apoptosis which can be blocked by YVAD (Krohn et al., 1998). In this case, there is an increased production of superoxide which is blocked by inhibition of caspase-l-like proteases. The time course of YVAD-cleaving activity parallels the production of superoxide supporting a pivotal role for a caspase-l-like protease. There is also later induction of DEVD-cleaving activity which does not appear necessary for death, as will be discussed below. The requirement for caspase-1 activation in cell death involving the generation of superoxide agrees with our data from PC12 cells and sympathetic neurons (Troy et al., 1996b). The protective effects of blocking IL-1B action led us to investigate the potential role of IL-1B in these systems in trophic factor deprivation. As shown in Figure 4, a blocking antibody to IL-1B protected cells from V-ASOD1 mediated cell death but not from death induced by trophic factor withdrawal (Troy et al., 1996b). Moreover, IL-1RA completely protected against SOD1 downregulation and had only a very modest effect against trophic factor deprivation, and then only at a much higher dose (Troy et al., 1996b). These differential responses to blockade by antibody and by a receptor antagonist raised the question of what changes occurred in IL-1B processing in response to the two stimuli. Measurement of IL-1B secretion in the medium of cells, in the two death paradigms, show that V-ASOD1 elicits an almost three-fold increase in IL-1B secretion which is blocked by V-ICEinh, while trophic factor withdrawal has no effect on IL- 1B secretion.

C.M. Troy

80

Trophic deprivation

V-ASOD1

treatment

T

100-

i

"o

E

7S-

Q =3

Q

SO-

Q ¢ 0

25-

9

8. T NGF

none

T

r

antML.1B

none

antl-lL-18

~r

Figure 4.

Interleukin-lB is differentially involved in death mediated by oxidative stress and by

trophic deprivation. PC12 cells were either treated with V-ASOD1 (50 nM) or trophic factor deprived in the presence or absence of the indicated concentrations of anti-IL-1B. Cell survival was determined at one day. Adapted from (Troy et al., 1996b).

_o G) o

g

15-

o

O

im

10-

e.,

5-

O. -NGF

-NGF

V-ASOD1 V-ASOD1

+V-ICEInh

+V-ICEIn h

w,-

Figure 5. Interleukin-lg levels are increased by by SOD1 downregulation, not by trophic factor withdrawal. PC12 cells were either treated with V-ASOD1 (50nM) or trophic factor deprived in the presence or absence of V-ICEinh. After 20 h treatment, medium was removed and IL-1B measured by ELISA. Adapted from (Troy et al., 1996b).

Diversity of Caspase Involvement

81

Therefore, the lack of response to blocking antibody and receptor block in the trophic deprivation model is not due to an overwhelming release of IL-1B upon trophic factor deprivation. These observations indicated the necessary involvement of IL-1B and its release in apoptosis triggered by SOD1 downregulation. Our findings further indicated that death caused by trophic factor deprivation might involve a different substrate(s) and possibly a different caspase. A second observation of our IL-1B secretion measurements was that NGF treatment provokes the largest effect on IL-IB secretion, a four-fold increase (Troy et al., 1996b). This indicated that IL-1B alone is not toxic to PC12 cells. This was confirmed by the observation that addition of recombinant IL-1B to PC12 cell cultures had no effect on cell survival in the presence of NGF, insulin or serum. Thus, cell death upon SOD 1 downregulation is not due to an increase in IL-1B alone, but rather to an enhanced vulnerability to this cytokine. Consistent with this, addition of IL-1B to cell cultures treated with V-ASOD1 potentiated cell death (Troy et al., 1996b). Several lines of evidence now point to a role for caspase-1 in the pathology associated with the missense mutations of SOD1. As discussed above, missense mutations of SOD1 are found in a subset of FALS cases. There are now more than 50 mutations described. The mechanism by which mutant SOD1 causes motor neuron degeneration is not yet understood. Expression of the human SOD1 mutation G93A in mice causes the development of fatal motor neuron disease (Gurney et al., 1994). Friedlander and colleagues bred the SODG93A mouse with a mouse they had engineered to overexpress a dominant negative mutation of caspase-1, and found that mice expressing both the SODG93A and the dominant negative caspase-1 had a delay in progression of symptoms, although onset of the disease was unchanged (Friedlander et al., 1997a). This indicates a role for caspases in the death induced by the SODG93A mutation. However, the dominant negative may not be completely selective for caspase-1. This possibility is argued against by the phenotypic concordance of the caspase knockout and the caspase-1 dominant negative mice. Caspase-1 has been shown to be activated in neurons expressing mutant SOD1 (Pasinelli et al., 1998). The processing is seen in spinal cords from mice expressing the G37R and G85R mutations, as well as in N2A cells expressing several missense mutations (G37R, G85R, or G41D). The activation of caspase-1 was enhanced by treatment with xanthine/xanthine oxidase, which increases superoxide and hydrogen peroxide production, and triggers cleavage and secretion of IL-1B and apoptosis. Apoptosis was blocked completely by zVADFMK and partially by Ac-YVAD-CHO, suggesting the involvement of caspase-1 and others caspases. It appears that both superoxide and hydrogen peroxide play roles in this death, and block of caspase-1 only affects the superoxide portion of the paradigm. This is consistent with the above data.

Proposed mechanism of caspase-1 mediated neuronal cell death The death mediated by caspase-1 is somewhat different from those induced by the other caspases.

82

C.M. Troy

PROPOSED PATHWAY OF FREE RADICAL MEDIATED DEATH

Increase superoxide

.~._-----~ 0 Activate caspase-1

pro-lL-113

~ tL-113

/ IL-1 receptor

/

activate JNK

activate other caspases

/

Apoptotic cell death

Figure 6. Proposed pathwayof free radical mediatedneuronal cell death.

The data would support an autocrine component of this death, namely the action of IL-1B at the IL-1 receptor. Activation of the receptor may then lead to induction of nitric oxide synthase and activation of c-jun kinase (JNK). Induction of NOS would increase NO which would then be available to combine with superoxide leading to peroxynitrite, which would lead to an increase in IL-1B production, as well as cause damage to DNA and other cell components. JNK activation could lead to activation of other caspases and thus to apoptotic death. Blocking the action of IL-1B at the IL-1 receptor would block all of these effects and thus confer complete protection. Caspase-2 and neuronal cell death Lack of protection against trophic factor deprivation by manipulations of IL-1B effects, as well as differences in the dose-response curves of the various caspase inhibitors in protectiong from trophic factor deprivation and SOD1 downregulation induced deaths, led us to examine which caspase functions in trophic factor deprivation mediated death. There are high levels of caspase-2 (Nedd2) mRNA and protein in PC12 cells (Kumar et al., 1994; Troy et al., 1997b). Caspase-2 exists as a long and short form, with the long form having pro-apoptotic activity and the short form anti-apoptotic activity. We chose to downregulate caspase 2-long in our death paradigms. To block the expression of caspase-2-1ong in neuronal cells, we designed an antisense oligonucleotide (ANedd)

Diversity of Caspase Involvement

83

which corresponds to the last 9 bases in the 5' UTR and the first 12 in the coding region of the caspase-2-1ong transcript (Kumar et al., 1994; Troy et al., 1997b). This sequence is not homologous to caspase-2-short or to any other mRNAs, including those of the other known caspases. For a control, a scrambled oligonucleotide (same base composition, scrambled) was used. The oligonucleotide was linked to the vector peptide Penetratinl (V-) (Troy et al., 1996a) to enhance its cellular uptake. To show that V-ANedd does decrease caspase-2 protein levels we generated an antibody to an N-terminal peptide of caspase-2 and used both immunohistochemistry and Western blotting to show that there is about 70% downregulation of caspase-2 within 24 h of V-ANedd treatment (Troy et al., 1997b). This downregulation was confirmed using a commercial antibody generated to a C-terminal peptide of caspase-2. The scrambled oligonucleotide (V-SNedd) did not downregulate caspase-2. V-ANedd did not affect the protein levels of caspase-3, nor did it alter the DEVD-AFC cleavage seen after trophic factor withdrawal (Stefanis et al., 1997). Treatment with V-ANedd protected PC12 cells from trophic factor deprivation but not from SOD1 downregulation. Protection from trophic deprivation has been confirmed by other investigators using stably expressed antisense constructs for caspase-2 in PC12 cells (Haviv et al., 1998). Western blots of PC12 cells after trophic deprivation show that there is a decrease in pro-caspase-2 and appearance of processed fragments (Stefanis et al., 1997, 1998). Parallel results were obtained with sympathetic neurons. However, as discussed above, sympathetic neurons from a caspase-2 knockout mouse were as susceptible to NGF withdrawal as wild-type neurons (Bergeron et al., 1998), raising the issue of the differences between our work using an antisense oligonucleotide to caspase-2 and the knockout mouse. In the knockout mouse both forms of caspase-2 (long (pro-apoptotic) and short (anti-apoptotic)) are deleted; in our study it is likely that only the long form is downregulated. These apparently contradictory results allow us the opportunity to compare the effects of deleting a gene, as in a knockout animal, with acute downregulation of protein expression in a mature neuron, as with antisense oligonucleotides. Death Receptors and Neuronal Cell Death Most of the data concerning death receptors and cell death is from non-neuronal systems. Very recent data support a role for Fas ligand induction in trophic factor deprivation death (Le-Niculescu et al., 1999). In this study, JNK activation followed by induction of Fas ligand expression and apoptosis were observed after withdrawal of trophic factors from PC12 cells and after KC1 removal from cerebellar granule neurons (CGNs). Apoptosis was blocked by treatment with a Fas-Fc decoy which sequesters Fas ligand. CGNs from gld mice, whose FasL gene encodes a nonfunctional protein, are resistant to KC1 withdrawal. The pathway proposed is stress signals leading to JNK to cJun to FasL to apoptosis. It is unlikely that this path is important during neuronal development as both the gld mice and jnk3 knockout mice have normal neuronal development and behavior. This pathway may be important in various neurodegenerative disorders. The role of caspases and IL-1 in this pathway have not yet been determined. In the primary cultures of CGNs the glial cells, although a minor contaminant, expressed FasL mRNA first after incubation in 5mM KCI. The glial cells are a major source of

84

C.M. Troy

IL-1 and IL-1, while not known to bind to death receptors, is a potent activator of JNK and NFkB and can induce TNF family members (Gupta et al., 1995; DiDonato et al., 1996). This further points to a role for IL-1 in vivo in certain types of neuronal cell injury and death. All Caspase Activity Does Not Produce Death In naive and neuronally differentiated PC12 cells deprived of trophic factors, there is a rapid induction of DEVD-AFC-cleaving, but not YVAD-AFC cleaving, activity. Cleavage of tPARP is also seen in naive and neuronally-differentiated PC12 cells prior to their death following trophic factor deprivation. However, the concentrations of caspase inhibitors which block the DEVD-AFC cleaving activity, as well as tPARP cleavage, do not protect from cell death. 100 -

[]

relative DEVD-AFC cleavage

•

relative survival

75-

50-

25-

0

1

5

10

DEVD-FMK Figure 7.

25

100

,uM

Inhibition of DEVD-cleaving activity does not protect against trophic factor withdrawal. PC12

cells were trophic factor deprived in the presence or absence of the indicated concentrations of DEVD-FMK. After 6 h cells were harvested and DEVD-AFC cleavage was measured. Sister cultures were used for assessment of survival at 20 h (Stefanis et al., 1998).

Protection from cell death required concentrations of the inhibitors (zVAD-FMK or DEVD-FMK) approximately 10-fold higher than the concentrations that inhibited the DEVD-AFC cleaving activity. Our data would, therefore, support the idea that although regulation of DEVD-AFC/tPARP cleaving activity correlates with cell death, it is not the major determinant of apoptosis in trophic factor deprivation. This agrees with the studies of the substrate and inhibitor profiles for caspase-2.

Diversity of Caspase Involvement

85

Similar results have been obtained in another death paradigm, staurosporine induced apoptosis of cultured hippocampal neurons. As discussed above, activation of caspase- 1 and subsequent superoxide production are necessary for cell death in this paradigm. However, there is induction of DEVD-AMC cleaving activity which parallels the increase in superoxide production. But block of the activity by DEVD-CHO prevented the nuclear fragmentation, but not the rise in superoxide production and subsequent death of the cells. These data reinforce the necessity of correlating all parameters of cell death and caspase activation before claiming causalty of a particular caspase.

Summary Much still remains to be determined about the roles of the individual caspases in cell death or in other, as yet, undetermined functions. It is clear, however, that different stimuli do employ different caspases to cause death. It is also clear that caspases may be activated and not lead to cell death. While it is not yet clear what these activated, but non-essential caspases are doing, it is possible that they are involved in cytoplasmic remodeling via cleavage of the cytoskeletal components. CASPASE SPECIFIC[TIES FOR DIFFERENT PARADIGMS OF CELL DEATH

Free radical

1

Caspase I

Trophic factor deprivation

l

Caspese 2

l

Caspase 3

DNA damage

BAF-sensitive caepese

APOPTOTIC CELL DEATH Figure 8. Caspase specificities in three paradigms of neuronal cell death.

In Figure 8 we present a simplistic schematic diagram of our current knowledge about the caspase requirements in several death paradigms. The data we have presented is compatible with either single caspases acting to produce death or with a cascade of caspases, since blocking one element of the cascade will block the entire cascade. Further elucidation of the caspases required for individual death pathways would require careful attention to the answers our tools produce. At present, a combination of knockouts, expression of antisense constructs and antisense oligonucleotides offer the best option of determining caspase specificity. Since caspases are the executors of cell death, this data, together with our ongoing work on the mechanisms of neuronal death, will enable the rational design of drugs for specific neurodegenerative diseases.

C.M. Troy

86

Acknowledgements I thank Michael L. Shelanski and Wilma J. Friedman for invaluable critical commentary and editorial assistance. CMT is supported by grants from NINDS and the Muscular Dystrophy Association.

References Adams, J.M. & Cory S. (1998). The Bcl-2 protein family: arbiters of cell survival. Science 281, 1322-1326. Ahmad, M., Srinivasula S.M., Hegde R., Mukattash R., Fernandes-Alnemri T. & Alnemri E.S. (1998). Identification and characterization of murine caspase-14, a new member of the caspase family. Cancer Res. 58, 5201-5205. Ahmad, M., Srinivasula, S.M., Wang L., Talanian R.V., Litwack G., Fernandes-Alnemri T. & Alnemri E.S. (1997). CRADD, a novel human apoptotic adaptor molecule for caspase-2, and FasL/tumor necrosis factor receptor-interacting protein RIP. Cancer Res. 57, 615-619. Angelastro, J.M., Qi H., Stefanis, L., Park, D.S. & Troy, C.M. (1998). Identification and determining the functional roles of caspases in apoptosis and PC12 cells. In ISDN 12tb Biennial Meeting, Vancouver, B.C. Armstrong, R.C., Aja, T.J., Hoang K.D., Gaur S., Bai X., Alnemri E.S., Litwack G., Karanewsky D.S., Fritz L.C. & Tomaselli K.J. (1997). Activation of the CED3/ICE-related protease CPP32 in cerebellar granule neurons undergoing apoptosis but not necrosis. J. Neurosci. 17, 553-562. Bains, J.S. & Shaw C.A. (1997). Neurodegenerative disorders in humans: the role of glutathione in oxidative stress-mediated neuronal death. Brain. Res. Brain Res Rev. 25,335-358. Batistatou, A. & Greene L.A. (1991). Aurintricarboxylic acid rescues PC12 cells and sympathetic neurons from cell death caused by nerve growth factor deprivation: correlation with suppression of endonuclease activity. J. Cell Biol. 115,461-471. Batistatou, A., Merry D.E., Korsmeyer S.J. & Greene L.A. (1993). Bcl-2 affects survival but not neuronal differentiation of PC 12 cells. J. Neurosci. 13, 4422-4428. Bergeron, L., Perez G.I., Macdonald, G., Shi, L., Sun, Y., Jurisicova, A., Varmuza S., Latham K.E., Flaws, J.A., Salter, J.C., Hara, H., Moskowitz, M.A., Li E., Greenberg, A., Tilly, J.L. & Yuan, J. (1998). Defects in regulation of apoptosis in caspase-2-deficient mice. Genes Dev. 12, 1304-1314. Bergeron, L. & Yuan, J. (1998). Sealing one's fate: control of cell death in neurons. Curr. Opin. Neurobiol. 8, 55-63. Boldin, M.P., Goncharov,T.M., Goltsev,Y.V. & Wallach,D. (1996). Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell 85, 803-815. Boldin, M.P., Varfolomeev,E.E., Pancer, Z., Mett, I.L., Camonis, J.H. & Wallach, D. (1995). A novel protein that interacts with the death domain of Fas/APO1 contains a sequence motif related to the death domain. J. Biol. Chem. 270, 7795-7798. Brown, R.H., Jr. (1995). Amyotrophic lateral sclerosis: recent insights from genetics and transgenic mice. Cell 80, 687-692. Bump, N.J., Hackett, M., Hugunin, M., Seshagiri, S., Brady, K., Chen, P., Ferenz, C., Franklin, S., Ghayur, T., Li, P. & et al. (1995). Inhibition of ICE family proteases by baculovirus antiapoptotic protein p35. Science 269, 1885-1888.

Diversity of Caspase Involvement

87

Cecconi, F., Alvarez-Bolado, G., Meyer, B.I., Roth, K.A. & Gruss, P. (1998). Apafl (CED-4 homolog) regulates programmed cell death in mammalian development. Cell 94, 727-737. Cerretti, D.P., Kozlosky, C.J., Mosley, B., Nelson, N., Van Ness, K., Greenstreet, T.A., March, C.J., Kronheim, S.R., Druck, T., Cannizzaro, L.A. & et al. (1992). Molecular cloning of the interleukin-1 beta converting enzyme. Science 256, 97-100. Cheng, E.H., Kirsch, D.G., Clem, R.J., Ravi, R., Kastan, M.B., Bedi, A., Ueno, K. & Hardwick, J.M. (1997). Conversion of Bcl-2 to a Bax-like death effector by caspases. Science 278, 1966-1968. Chinnaiyan, A.M., O'Rourke, K., Tewari, M. & Dixit, V.M. (1995). FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell 81,505-512. Cohen, G. M. (1997). Caspases: the executioners of apoptosis. Biochem. J. 326, 1-16. Coyle, J.T. & Puttfarcken, P. (1993). Oxidative stress, glutamate, and neurodegenerative disorders. Science 262, 689-695. Cryns, V. & Yuan, J. (1998). Proteases to die for. Genes Dev. 12, 1551-1570. Culotta, V.C., Joh, H.D., Lin, S.J. Slekar, K.H. & Strain, J. (1995). A physiological role for Saccharomyces cerevisiae copper/zinc superoxide dismutase in copper buffering. J. Biol. Chem. 270, 29991-29997. Deveraux, Q.L., Roy, N., Stennicke, H.R., Van Arsdale, T., Zhou, Q., Srinivasula, S.M., Alnemri, E.S., Salvesen, G.S. & Reed, J.C. (1998). lAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases. Embo J. 17, 2215-2223. Deveraux, Q.L., Takahashi, R., Salvesen, G.S. & Reed, J.C. (1997). X-linked lAP is a direct inhibitor of cell-death proteases. Nature 388, 300-304. DiDonato, J., Mercurio, F., Rosette, C., Wu-Li, J., Suyang, H., Ghosh, S.& Karin, M. (1996). Mapping of the inducible IkappaB phosphorylation sites that signal its ubiquitination and degradation. Mol. Cell. Biol. 16, 1295-1304. Duan, H. & Dixit, V.M. (1997). RAIDD is a new 'death' adaptor molecule. Nature 385, 86-89. Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A. & Nagata, S. (1998). A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD [see comments] [published erratum appears in Nature 1998 May 28;393(6683):396]. Nature 391,43-50. Enns, L., Barley, R.D., Paterson, M.C. & Mirzayans, R. (1998). Radiosensitivity in ataxia telangiectasia fibroblasts is not associated with deregulated apoptosis. Radiat Res. 150, 11-16. Farinelli, S.E., Park, D.S. & Greene, L.A. (1996). Nitric oxide delays the death of trophic factor-deprived PC 12 cells and sympathetic neurons by a cGMP-mediated mechanism. J. Neurosci. 16, 2325-2334. Fridovich, 1. (1995). Superoxide radical and superoxide dismutases. Annu. Rev. Biochem. 64, 97-112. Friedlander, R.M., Brown, R.H., Gagliardini, V., Wang, J. & Yuan, J. (1997a). Inhibition of ICE slows ALS in mice [letter] [published erratum appears in Nature 1998 Apr 9;392(6676):560]. Nature 388, 31. Friedlander, R.M., Gagliardini, V., Hara, H., Fink, K.B., Li, W., MacDonald, G., Fishman, M.C., Greenberg, A.H., Moskowitz, M.A. & Yuan, J. (1997b). Expression of a dominant negative mutant of interleukin-1 beta converting enzyme in transgenic mice prevents neuronal cell death induced by trophic factor withdrawal and ischemic brain injury. J. Exp. Med. 185,933-940. Friedlander, R.M., Gagliardini, V., Rotello, R.J. & Yuan, J. (1996). Functional role of interleukin 1 beta (IL-1 beta) in IL-1 beta- converting enzyme-mediated apoptosis. J. Exp. Med. 184, 717-724. Gonzalez-Zulueta,, M., Ensz, L.M., Mukhina, G., Lebovitz, R.M., Zwacka, R.M., Engelhardt, J.F., Oberley, L.W., Dawson, V.L. & Dawson, T.M. (1998). Manganese superoxide dismutase protects nNOS neurons from NMDA and nitric oxide-mediated neurotoxicity. J. Neurosci. 18, 2040-2055. Green, D.R. (1998). Apoptotic pathways: the road to ruin. Cell 94, 695-698. Greene, L.A. (1978). Nerve growth factor prevents the death and stimulates the neuronal differentiation of

88

C.M. Troy

clonal PC12 pheochromocytoma cells in serum-free medium. J. Cell Biol. 78, 747-755. Gupta, S., Campbell, D., Derijard, B. & Davis, R.J. (1995). Transcription factor ATF2 regulation by the JNK signal transduction pathway. Science 267, 389-393. Gurney, M.E., Pu, H., Chiu, A.Y., Dal Canto, M.C., Polchow, C.Y., Alexander, D.D., Caliendo, J., Hentati, A., Kwon, Y.W., Deng, H. X. & et al. (1994). Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation [see comments] [published erratum appears in Science 1995 Jul 14;269(5221): 149]. Science 264, 1772-1775. Hakem, R., Hakem, A., Duncan, G.S., Henderson, J.T., Woo, M., Soengas, M.S., Elia, A., de la Pompa, J.L., Kagi, D., Khoo, W., Potter, J., Yoshida, R., Kaufman, S.A., Lowe, S.W., Penninger, J.M. & Mak, T. W. (1998). Differential requirement for easpase 9 in apoptotic pathways in vivo.Cell 94, 339-352. Haviv, R., Lindenboim, L., Yuan, J. & Stein, R. (1998). Need for caspase-2 in apoptosis of growth-factordeprived PC12 cells [In Process Citation]. J Neurosci Res. 52, 491-497. Hengartner, M.O., Ellis, R.E. & Horvitz, H.R. (1992). Caenorhabditis elegans gene ced-9 protects cells from programmed cell death. Nature 356, 494-499. Hofmann, K., Bucher, P. & Tschopp, J. (1997). The CARD domain: a new apoptotic signalling motif. Trends Biochem Sci. 22, 155-156. Hu, S., Snipas, S.J., Vincenz, C., Salvesen, G. & Dixit, V.M. (1998). Caspase-14 is a novel developmentally regulated protease. J. Biol. Chem. 273, 29648-29653. Humke, E.W., Ni, J. & Dixit, V.M. (1998). ERICE, a novel FLICE-activatable caspase. J. Biol. Chem. 273, 15702-15707. Keller, J.N., Kindy, M.S., Holtsberg, F.W., St. Clair, D.K., Yen, H.-C., Germeyer, A., Steiner, S.M., BruceKeller, A.J., Hutchins, J.B. & Mattson, M.P. (1998). Mitochondrial MnSOD prevents neural apoptosis and reduces ischemic brain injury: Suppression of peroxynitrite production, lipid peroxidation and mitochondrial dysfunction. J. Neurosci. 18:687-697. Kerr, J.F., Wyllie, A.H. & Currie, A.R. (1972). Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239-257. Krohn, A.J., Preis, E. & Prehn, J.H. (1998). Staurosporine-induced apoptosis of cultured rat hippocampal neurons involves caspase-l-like proteases as upstream initiators and increased production of superoxide as a main downstream effector. J. Neurosci. 18, 8186-8197. Kuida, K., Haydar, T.F., Kuan, C.Y., Gu, Y., Taya, C., Karasuyama, H., Su, M. S., Rakic, P. & Flavell, R.A. (1998). Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9. Cell 94, 325-337. Kuida, K., Lippke, J.A., Ku, G., Harding, M.W., Livingston, D.J., Su, M.S. & Flavell, R.A. (1995). Altered cytokine export and apoptosis in mice deficient in interleukin-1 beta converting enzyme. Science 267, 2000-2003. Kuida, K., Zheng, T.S., Na, S., Kuan, C., Yang, D., Karasuyama, H., Rakic, P. & Flavell, R.A. (1996). Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice. Nature 384, 368-372. Kumar, S., Kinoshita, M., Noda, M., Copeland, N.G. & Jenkins, N.A. (1994). Induction of apoptosis by the mouse Nedd2 gene, which encodes a protein similar to the product of the Caenorhabditis elegans cell death gene ced-3 and the mammalian IL-1 beta-converting enzyme. Genes Dev. 8, 1613-1626. Lavin, M.F. (1998). Radiosensitivity and oxidative signalling in ataxia telangiectasia: an update [In Process Citation]. Radiother. Oncol. 47, 113-123. I..azebnik, Y.A., Takahashi, A., Moir, R.D., Goldman, R.D., Poirier, G.G., Kaufmann, S.H. & Earnsbaw W.C. (1995). Studies of the lamin proteinase reveal multiple parallel biochemical pathways during apoptotic execution. Proc Natl Acad Sci U S A. 92, 9042-9046.

Diversity of Caspase Involvement

89

Le-Niculescu, H., Bonfoco, E., Kasuya, Y., Claret, F.X., Green, D.R. & Karin, M. (1999). Withdrawal of survival factors results in activation of the JNK pathway in neuronal cells leading to fas ligand induction and cell death [In Process Citation]. Mol. Cell Biol. 19, 751-763. Li, P., Allen H., Banerjee, S. & Seshadri, T. (1997). Characterization of mice deficient in interleukin-1 beta converting enzyme. J. Cell Biochem. 64, 27-32. Margolin, N. & Raybuck S.A., Wilson K.P., Chen W., Fox T., Gu Y. & Livingston D.J. (1997). Substrate and inhibitor specificity of interleukin-1 beta-converting enzyme and related caspases. J. Biol. Chem. 272, 7223-7228. Mashima, T., Naito, M., Fujita, N., Noguchi, K. & Tsuruo, T. (1995). Identification of actin as a substrate of ICE and an ICE-like protease and involvement of an ICE-like protease but not ICE in VP-16-induced U937 apoptosis. Biochem. Biophys. Res. Commun. 217, 1185-1192. McCord, J.M. & Fridovich, I. (1969). Superoxide dismutase: an enzymic function for erythrocuprein (hemocuprein). J. Biol. Chem. 244, 6049-6055. Miura, M., Zhu, H., Rotello, R., Hartwieg, E.A. & Yuan, J. (1993). Induction of apoptosis in fibroblasts by IL-1 beta-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3. Cell 75,653-660. Muzio, M., Chinnaiyan, A.M., Kischkel, F.C., O'Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J.D., Zhang, M., Gentz, R., Mann, M., Krammer, P.H., Peter, M.E. & Dixit V.M. (1996). FL1CE, a novel FADD-homologous ICE/CED-3-1ike protease, is recruited to the CD95 (Fas/APO-1) death--inducing signaling complex. Cell 85, 817-827. Muzio, M., Stockwell, B.R., Stennicke, H.R., Salvesen, G.S. & Dixit V.M. (1998). An induced proximity model for caspase-8 activation. J. Biol. Chem. 273, 2926-2930. Namura, S., Zhu, J., Fink, K., Endres, M., Srinivasan, A., Tomaselli, K.J., Yuan, J. & Moskowitz, M.A. (1998). Activation and cleavage of caspase-3 in apoptosis induced by experimental cerebral ischemia. J. Neurosci. 18, 3659-3668. Nicholson, D.W., Ali, A., Thornberry, N.A., Vaillancourt, J.P., Ding, C.K., Gallant, M., Gareau, Y., Griffin, P.R., Labelle, M., Lazebnik, Y.A. & et al. (1995). Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis [see comments]. Nature 376, 37-43. Nicholson, D.W. & Thornberry, N.A. (1997). Caspases: killer proteases. Trends Biochem Sci. 22, 299-306. Orth, K., Chinnaiyan, A.M., Garg, M., Froelich, C.J. & Dixit, V.M. (1996). The CED-3/ICE-Iike protease Mch2 is activated during apoptosis and cleaves the death substrate lamin A. J. Biol. Chem. 271, 16443-16446. Park, D.S., Morris, E.J., Padmanabhan, J., Shelanski, M.L., Geller, H.M. & Greene, L.A. (1998a). Cyclindependent kinases participate in death of neurons evoked by DNA- damaging agents. J. Cell Biol. 143,457-467. Park, D.S., Morris, E.J., Stefanis, L., Troy, C.M., Shelanski, M.L., Geller, H.M. & Greene, L.A. (1998b). Multiple pathways of neuronal death induced by DNA-damaging agents, NGF deprivation, and oxidative stress. J. Neurosci. 18,830-840. Pasinelli, P., Borchelt, D.R., Houseweart, M.K., Cleveland, D.W. & Brown, R.H., Jr. (1998). Caspase-1 is activated in neural cells and tissue with amyotrophic lateral sclerosis-associated mutations in copper-zinc superoxide dismutase. Proc. Natl. Acad. Sci. U S A. 95, 15763-15768. Prochiantz, A. & Theodore, L. (1995). Nuclear/growth factors. Bioessays. 17, 39-44. Rosen, D.R., Siddique, T., Patterson, D., Figlewicz, D.A., Sapp, P., Hentati, A., Donaldson, D., Goto, J., O'Regan, J.P., Deng, H.X. & et al. (1993). Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis [published erratum appears in Nature 1993 Jul

90

C.M.

Troy

22;364(6435):362] [see comments]. Nature 362, 59-62. Rotonda, J., Nicholson, D.W., Fazil, K.M., Gallant, M., Gareau, Y., Labelle, M., Peterson, E.P., Rasper, D.M., Ruel, R., Vaillancourt, J.P., Thornberry, N.A. & Becker, J.W. (1996). The three-dimensional structure of apopain/CPP32, a key mediator of apoptosis. Nat. Struct. Biol. 3,619-625. Rukenstein, A., Rydel, R.E. & Greene, L.A. (1991). Multiple agents rescue PC12 cells from serum-free cell death by translation- and transcription-independent mechanisms. J. Neurosci. 11, 2552-2563. Schapira, A.H. (1996). Oxidative stress and mitochondrial dysfunction in neurodegeneration. Curr. Opin. Neurol. 9, 260-264. Silos-Santiago, I., Greenlund, L.J., Johnson, E.M., Jr. & Snider, W.D. (1995). Molecular genetics of neuronal survival. Curr. Opin. Neurobiol. 5, 42-49. Srinivasan, A., Foster, L.M., Testa, M.P., Ord, T., Keane, R.W., Bredesen, D.E. & Kayalar, C. (1996). Bcl-2 expression in neural cells blocks activation of ICE/CED-3 family proteases during apoptosis. J. Neurosci. 16, 5654-5660. Stefanis, L., Park, D.S., Yan, C.Y., Farinelli, S.E., Troy, C.M., Shelanski, M.L. & Greene, L.A. (1996). Induction of CPP32-1ike activity in PC12 cells by withdrawal of trophic support. Dissociation from apoptosis. J. Biol. Chem. 271, 30663-30671. Stefanis, L., Troy, C.M., Qi, H. & Greene, L.A. (1997). Inhibitors of trypsin-like serine proteases inhibit processing of the caspase Nedd-2 and protect PC12 cells and sympathetic neurons from death evoked by withdrawal of trophic support. J. Neurochem. 69, 1425-1437. Stefanis, L., Troy, C.M., Qi, H., Shelanski, M.L. & Greene, L.A. (1998). Caspase-2 (Nedd-2) processing and death of trophic factor-deprived PC12 cells and sympathetic neurons occur independently of caspase-3 (CPP32)- like activity [In Process Citation]. J. Neurosci. 18, 9204-9215. Takahashi, A., Alnemri, E.S., Lazebnik, Y.A., Fernandes-Alnemri, T., Litwack, G., Moir, R.D., Goldman, R.D., Poirier, G.G., Kaufmann, S.H. & Eamshaw, W.C. (1996). Cleavage of lamin A by Mch2 alpha but not CPP32: multiple interleukin 1 beta-converting enzyme-related proteases with distinct substrate recognition properties are active in apoptosis. Proc. Natl. Acad. Sci. U S A. 93, 8395-8400. Talanian, R.V., Quinlan, C., Trautz, S., Hackett, M.C., Mankovich, J.A., Banach, D., Ghayur, T., Brady, K.D. & Wong, W.W. (1997). Substrate specificities of caspase family proteases. J. Biol. Chem. 272, 9677-9682. Theodore, L., Derossi, D., Chassaing, G., Llirbat, B., Kubes, M., Jordan, P., Chneiweiss, H., Godement, P. & Prochiantz, A. (1995). Intraneuronal delivery of protein kinase C pseudosubstrate leads to growth cone collapse. J. Neurosci. 15, 7158-7167. Thornberry, N.A., Bull, H.G., Calaycay, J.R., Chapman, K.T., Howard, A.D., Kostura, M.J., Miller, D.K., Molineaux, S.M., Weidner, J.R., Aunins, J. & et al. (1992). A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes. Nature 356, 768-774. Thomberry, N.A. & Lazebnik, Y. (1998). Caspases: enemies within. Science 281, 1312-1316. Thornberry, N.A., Rano, T.A., Peterson, E.P., Rasper, D.M., Timkey, T., Garcia-Calvo, M., Houtzager, V.M., Nordstrom, P.A., Roy, S., Vaillancourt, J.P., Chapman, K.T. & Nicholson, D.W. (1997). A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J. Biol. Chem. 272, 17907-17911. Troy, C.M., Derossi, D., Prochiantz, A., Greene, L.A. & Shelanski, M.L. (1996a). Downregulation of Cu/Zn superoxide dismutase leads to cell death via the nitric oxide-peroxynitrite pathway. J. Neurosci. 16, 253-261. Troy, C.M. & Shelanski, M.L. (1994). Downregulation of copper/zinc superoxide dismutase causes apoptotic death in PC12 neuronal cells. Proc. Natl. Acad. Sci. U S A. 91, 6384-6387.

Diversity of Caspase Involvement

91

Troy, C.M., Stefanis, L., Greene, L.A. & Shelanski, M.L. (1997a). Mechanisms of neuronal degeneration: a final common pathway? Adv. Neurol. 72, 103-111. Troy, C.M., Stefanis, L., Greene, L.A. & Shelanski, M.L. (1997b). Nedd2 is required for apoptosis after trophic factor withdrawal, but not superoxide dismutase (SOD1) downregulation, in sympathetic neurons and PC12 cells. J. Neurosci. 17,1911-1918. Troy, C.M., Stefanis, L., Prochiantz, A., Greene, L.A. & Shelanski, M.L. (1996b). The contrasting roles of ICE family proteases and interleukin-lbeta in apoptosis induced by trophic factor withdrawal and by copper/zinc superoxide dismutase downregulation. Proc. Natl. Acad. Sci. U S A. 93, 5635-5640. Varfolomeev, E.E., Schuchmann, M., Luria, V., Chiannilkulchai, N., Beckmann, J.S., Mett, I.L., Rebrikov, D., Brodianski, V.M., Kemper, O.C., Kollet, O., Lapidot, T., Softer, D., Sobe, T., Avraham, K.B., Goncharov, T., Holtmann, H., Lonai, P. & Wallach, D. (1998). Targeted disruption of the mouse Caspase 8 gene ablates cell death induction by the TNF receptors, Fas/Apol, and DR3 and is lethal prenatally. Immunity. 9, 267-276. Vaux, D.L., Cory, S. & Adams, J.M. (1988). Bcl-2 gene promotes haemopoietic cell survival and cooperates with c- myc to immortalize pre-B cells. Nature 335,440-442. Vaux, D.L., Weissman, I.L. & Kim, S.K. (1992). Prevention of programmed cell death in Caenorhabditis elegans by human bcl-2. Science 258, 1955-1957. Vincenz, C. & Dixit V.M. (1997). Fas-associated death domain protein interleukin-lbeta-converting enzyme 2 (FLICE2), an ICE/Ced-3 homologue, is proximally involved in CD95- and p55-mediated death signaling. J. Biol. Chem. 272, 6578-6583. Walker, N.P., Talanian, R.V., Brady, K.D., Dang, L.C., Bump, N.J., Ferenz, C.R., Franklin, S., Ghayur, T., Hackett, M.C., Hammill, L.D. & et al. (1994). Crystal structure of the cysteine protease interleukin-1 beta- converting enzyme: a (p20/p10)2 homodimer. Cell 78, 343-352. Wang, L., Miura, M., Bergeron, L., Zhu, H. &, Yuan J. (1994). Ich-1, an Ice/ced-3-related gene, encodes both positive and negative regulators of programmed cell death. Cell 78,739-750. Wang, S., Miura, M., Jung, Y.K., Zhu, H., Li, E. & Yuan, J. (1998). Murine caspase-11, an ICE-interacting protease, is essential for the activation of ICE. Cell 92, 501-509. Williams, L.R. (1995). Oxidative stress, age-related neurodegeneration, and the potential for neurotrophic treatment. Cerebrovasc. Brain Metab. Rev. 7, 55-73. Wilson, K.P., Black, J.A., Thomson, J.A., Kim, E.E., Griffith, J.P., Navia, M.A., Murcko, M.A., Chambers, S.P., Aldape, R.A., Raybuck, S.A. & et al. (1994). Structure and mechanism of interleukin-1 beta converting enzyme [see comments]. Nature 370, 270-275. Wyllie, A.H. (1980). Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284, 555-556. Wyllie, A.H., Morris, R.G., Smith, A.L. & Dunlop D. (1984). Chromafin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis. J. Pathol. 142, 67-77. Xue, D. & Horvitz, H.R. (1995). Inhibition of the Caenorhabditis elegans cell-death protease CED-3 by a CED-3 cleavage site in baculovirus p35 protein. Nature 377,248-251. Xue, D. & Horvitz, H.R. (1997). Caenorhabditis elegans CED-9 protein is a bifunctional cell-death inhibitor. Nature 390, 305-308. Yang, X., Chang, H.Y. & Baltimore, D. (1998). Autoproteolytic activation of pro-caspases by oligomerization. Mol. Cell 1,319-325. Yoshida, H., Kong, Y.Y., Yoshida, R., Elia, A.J., Hakem, A., Hakem, R., Penninger, J.M. & Mak, T.W. (1998). Apafl is required for mitochondrial pathways of apoptosis and brain development. Cell 94, 739-750.

92

C.M. Troy

Yuan, J. & Horvitz, H.R. (1992). The Caenorhabditis elegans cell death gene ced-4 encodes a novel protein and is expressed during the period of extensive programmed cell death. Development. 116, 309-320. Yuan, J., Shaham, S., Ledoux, S., Ellis, H. M. & Horvitz, H.R. (1993). The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1 beta-converting enzyme. Cell 75, 641-652. Zou, H., Henzel, W.J., Liu, X., Lutschg, A. & Wang, X. (1997). Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3 [see comments]. Cell 90, 405-413.

MITOCHONDRIAL CONTROL OF APOPTOSIS BERNARD M I G N O T T E and JEAN-LUC VAYSSIERE

Table of Contents Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 The Actors of the Execution Stage of the Apoptotic Cascade . . . . . . . . . . . . . . . . . . . . . . . . 93 Bcl-2 Family Proteins Act at Least in Part at the Mitochondrial Level . . . . . . . . . . . . . . . . . 95 Permeability Transition Marks A Point of No-Return of Cells Condemned to Die . . . . . . . . . 97 A Decrease in Mitochondrial Membrane Potential Precedes DNA Fragmentation During Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Mitochondrial Permeability Transition Provokes a AWmDecrease . . . . . . . . . . . . . . . . . . . . 98 Direct Interventions on the Mitochondrial Permeability Transition Modulate Apoptosis... 98 Mitochondria of Cells Undergoing Apoptosis Release Pro-Apoptotic Factors . . . . . . . . . . . . . 99 Mitochondria Undergoing Permeability Transition Liberate AIF Factor . . . . . . . . . . . . . . . . 99 Cytochrome C is Released by Mitochondria During Apoptosis . . . . . . . . . . . . . . . . . . . . . . 101 The Cytochrome C Pathway is Distinct from that of AIF . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 Bcl-2 Family Proteins Dock Various Proteins to the Mitochondria . . . . . . . . . . . . . . . . . . . 103 What is the Mechanism Involved in Cytoplasmic Release of Mitochondrial Apoptogenic Factors? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Mitochondrial ROS and Apoptosis Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 ROS as Mediators of PCD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 PCD-Mediating ROS are Produced by Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 Mechanisms Of ROS Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113

Introduction The Actors of the Execution Stage of the Apoptotic Cascade The programmed cell death cascade can be conveniently divided into several phases. During the activation phase, multiple signaling pathways lead from the various deathtriggering signals to the central control of the cell death machinery and activate it. This is followed by the execution stage, in which the activated machinery acts on multiple cellular targets, and, finally, the destruction phase in which the dead or dying cell is broken down. A most important clue to the molecular nature of the death program came initially from genetic studies in C. elegans that led to the identification of a dozen cell death genes (ced) that are responsible for one aspect or another of cell death processes (Ellis et al., 1991). Three of these genes stand out. Two, ced-3 and ced-4 are essential for cell death. The third, ced-9, antagonizes the death activities of ced-3 and ced-4. 93 Cellular and Molecular Mechanisms Ed. by M.P. Mattson, S. Estus and V.M. Rangnekar. 93 -- 122 © 2001 Elsevier Science. Printed in the Netherlands.

94

B. Mignotte and J.-L. Vayssiere

Mutational analyses of these genes placed ced-4 between ced-9 and ced-3 in the pathway leading to cell death, suggesting that CED-4 might act as an adaptator, linking the upstream regulator CED-9 to the downstream death effector CED-3 (Shaham and Horvitz, 1996). Biochemical approaches have provided insight into the mechanism by which programmed cell death is regulated in the nematode. CED-4 interacts directly and simultaneously with both CED-3 and CED-9 forming a multimeric protein complex (Chinnaiyan et al., 1997b; Wu et al., 1997a). Furthermore, oligomerized CED-4, acting as a context dependent ATPase, promotes CED-3 activation and its ability to induce apoptosis (Chinnaiyan et al., 1997a). CED-9 binding to CED-4, mutually exclusive with CED-4 oligomerization, prevents it from activating CED-3, thereby blocking cell death (Yang et al., 1998). Remarkably, all three of these C. elegans cell death genes have mammalian counterparts that are likely to play similar, albeit more complex, roles in mammalian cell death (Table 1). CED-3 protein turned out to be a member of a family of cystein proteases, known as caspases (cystein as__laartases). The mammalian caspase family now comprises at least ten known members, most of which have been definitively implicated in PCD (for review see Cryns and Yuan, 1998)). All cleave their substrates after specific aspartic acids and are themselves activated by cleavage at specific aspartic acids (Nicholson and Thornberry, 1997). Caspases mediate PCD by cleaving selected intracellular proteins, including proteins of the nucleus, nuclear lamina, cytoskeleton, endoplasmic reticulum, and cytosol. However, which of these targets is, if any, responsible for the cell blebbing, condensation and fragmentation that characterize PCD remains uncertain. As specific protein or peptide caspase inhibitors can block PCD in all animal and invertebrate cells and in most cell death inducing conditions, it seems likely that caspases form the core of the death program. Table 1.

Programmed cell death in nematodes and mammals is controlled by homologous proteins. Mammalian caspases act either during the activation or the execution-phase of PCD (Nicholson and Thornberry, 1997). Recently, a CED-4 homologue has been identified in human cells (Hofmann et al., 1997; Zou et al., 1997). In mammals some members of the Bcl-2 related proteins are death antagonists while other are death agonists. For a recent review on the structure-function relations of Bcl-2 related proteins see (Kroemer, 1997).

C. elegans

Mammals

CED-3

Activation caspases: caspase-1 (ICE), -4 (ICH-2), -6 (Mch2), -8 (MACH/FLICE)... Execution caspases: caspase-2 (ICH-I), -3 (CPP32), -4 (ICH-2), -7 (ICE-LAP3)...

CED-4 CED-9

Apaf-1 Anti-apoptotic: Bcl-2, Bcl-xe, Bcl-w, Bfl-1, Brag-l, Mcl-1, A1, NR13... Pro-apoptotic: Bax, Bak, Bcl-xs, Bad, Bik, Hrk...

Recent studies have identified and partially characterized Apaf-1 (Apoptotic 12roteaseactivating _factor-I), a mammalian homologue of C. elegans CED-4 (Zou et al., 1997). The N-terminal region of Apaf-1 shares amino acid homology with CED-4 and several caspases with long prodomains, including, caspase-4, -8, -9, and CED-3. This conserved

Mitochondrial Control of Apoptosis

95

region, that has been termed caspase recruitment domain (CARD), mediates the physical association of CARD-containing proteins (Hofmann et al., 1997). Thus, Ced-4 binding to Ced-3 involves their respective amino-terminal protein interaction modules. In such way, Apaf-1 was shown to interact with caspase-9, a mammalian caspase containing a N-terminal long prodomain, and to trigger caspase-9 proteolytic self-activation (Li et al., 1997b). Caspase-9 subsequently proteolyses and activates caspase-3, one of the execution caspases which kill the cell by cleaving key intracellular death targets. Bcl-2 Family Proteins Act at Least in Part at the Mitochondrial Level CED-9 protein is homologous to a family of many members termed the Bcl-2 family in reference to the first discovered mammalian cell death regulator (for review see Adams and Cory, 1998)). Some members, such as Bcl-2 or Bcl-x L, are negative regulators of cell death, able to prevent cells from undergoing apoptosis induced by various stimuli in a wide variety of cell-types (Korsmeyer, 1992; Zhong et al., 1993), whereas others, such as Bax, Bid and Egl-1 promote or accelerate cell death. All members possess at least one of four conserved motifs known as Bcl-2 homology domains (BH1 to BH4). Most pro-survival members contain at least BH1 and BH2, and those most similar to Bcl-2 have all four BH domains. Two pro-apoptotic subfamilies can be define according to their relatedness to Bcl-2. Bax, Bak and Bok, which contain BH1, BH2 and BH3, resemble Bcl-2 fairly closely. In contrast, the other known mammalian and nematode EGL-1 "killers" possess only the central BH3 domain. These "BH3 domain" proteins may represent the physiological antagonists of the pro-survival proteins, because programmed cell death in C. elegans requires Egl-1, which binds to and acts via CED-9. BH3 is essential for the function of the "killers", including Egl-1. The various family members (referred to here as Bct-2s) can heterodimerize and seemingly titrate one another's function, suggesting that their relative concentration may act as a rheostat for the suicide program. Heterodimerization is not required for pro-survival function. For pro-apoptotic activity, heterodimerization is essential in the BH3 domain group, but less so for those of the Bax group, which may have an independent cytotoxic impact. Indeed whether Bax binds to Bcl-2 inside cells has become controversial. Some death agonists may preferentially target subsets of the death repressors. Bok, for example, interacts with Mcl-1 and the Epstein-barr viral protein BHFR1 but not with Bcl-2 or Bcl-x L. Within the BH3 group, Bid is promiscuous, binding to Bax and Bak as well as to the anti-apoptotic proteins, but the others bind only to certain of the death inhibitors. However, the mechanism(s) by which proteins of the Bcl-2 family modulate apoptosis remains elusive and several conflicting theories have been proposed. A widely accepted model postulates that homodimers of Bax promote apoptosis, and that the functional effect of Bcl-2 related proteins is to form competing heterodimers with Bax that cannot promote apoptosis (Oltvai et al., 1993; Sedlak et al., 1995). However, in some systems, Bax binding by Bcl-2 was not sufficient to prevent apoptosis and the overexpression of Bcl-2 or Bcl-x L can repress apoptosis in the absence of Bax (Cheng et al., 1996; Knudson and Korsmeyer, 1997). Thus, while an in vivo competition exists between Bax and Bcl-2, each is able to regulate apoptosis independently.

96

B. Mignotte and J.-L. Vayssiere

CED-9, Bcl-2 (Akao et al., 1994; Chen et al., 1989; de Jong et al., 1994; Hockenbery et al., 1990; Janiak et al., 1994; Krajewski et al., 1993; Nakai et al., 1993; Nguyen et al., 1993), Bcl-x L (Gonzalez-Garcia et al., 1994), Mcl-1 (Wang and Studzinski, 1997; Yang et al., 1995), the BHRF1 Epstein-Barr virus protein (Hickish et al., 1994) and probably other members of the Bcl-2 family are localized to the cytoplasmic surfaces of the nuclear envelope, the endoplasmic reticulum and the outer mitochondrial membrane. However, it must be underlined that only a fraction of Bcl-x L resides on membranes, and that Bax seems to be cytosolic before an apoptotic stimulus, even though both, like most other family members, bear a C-terminal membrane anchor. The membrane association of Bcl-2 is of functional significance as mutant Bcl-2 molecules lacking this membrane anchorage capacity are less effective at preventing apoptosis in some systems (Borner et al., 1994; Nguyen et al., 1994; Zhu et al., 1996). Indeed, it has been reported that, in inhibiting apoptosis of MDCK cells, a mutant Bcl-2 molecule whose anchorage is targeted specifically to the mitochondria is as effective as the wild type protein, whereas mutant Bcl-2 targeted to the ER loses this capacity (Zhu et al., 1996). In contrast, Bcl-2 targeted to the ER in the Rat-1/myc fibroblasts proved to be more active than when targeted to mitochondria. Thus, Bcl-2 mutants with restricted subcellular location reveal distinct pathways for apoptosis depending on celltype. When associated to the endoplasmic reticulum membrane, Bcl-2 could be involved in maintenance of the calcium homeostasis (Distelhorst et al., 1996; He et al., 1997; Lam et al., 1994), while it could modulate protein subcellular trafficking through nuclear pores (Ryan et al., 1994). Recent reports provide spectacular advancements in the understanding of the mechanism of action of antiapoptotic Bcl-2s. It was shown that, in both worm and mammalian cells, the antiapoptotic Bcl-2s act upstream of the "execution caspases" somehow preventing their proteolytic processing into active killers (Golstein, 1997; Shaham and Horvitz, 1996). How these proteins perform this feat remains unknown, although two main mechanisms of action have been proposed to connect Bcl-2s to caspases. In the first one, pro-survival proteins would act by regulating the release of some caspases activators usually sequestered in intracellular compartments. Apoptosis (in vivo and in vitro) involved the preliminary shift to the cytosol of regulatory components, namely cytochrome c or AIF (Apoptosis-Inducing-Factor), previously sequestered in mitochondrial intermembrane space (Kluck et al., 1997a; Susin et al., 1996; Yang et al., 1997). Cytosolic cytochrome c forms an essential part of the vertebrate "apoptosome" which is composed of cytochrome c, Apaf-1 and procaspase-9 (Li et al., 1997b). The result is the activation of caspase-9, which then activates other caspases to orchestrate execution of cell death. AIF is another caspase-activating protein which promotes nuclear apoptosis in vitro probably via the activation of procaspase-3, a major executive caspase. Overexpression of Bcl-2/Bcl-xL in cells or addition of recombinant Bcl-2 to cell-free systems containing mitochondria prevented cytochrome c/AIF exodus from the mitochondria that was triggered normally by a wide variety of apoptogenic stimuli. In contrast, the pro-apoptotic member Bax stimulates mitochondrial cytochrome c release. The ability of Bax, Bcl-2/Bcl-x L to form membranes pores with distinct ionconducting properties may provide a clue to how Bcl-2s can regulate the permeability of the intracellular membranes (Reed, 1997).

Mitochondrial Control of Apoptosis

97

An alternative model proposes that the pro-survival proteins may function downstream of the release of apoptogenic factors by directly inhibiting the ability of CED-4 like proteins to activate caspases. This model arose from the elucidation of the role of the somewhat mysterious CED-4 protein (Hengartner, 1997). Indeed, it was first shown that CED-9 interacts with CED-3 via the bridging protein CED-4 that binds simultaneously to CED-9 and CED-3. CED-9 prevents CED-4 from inducing proteolytic processing and activation of CED-3 (Chinnaiyan et al., 1997b; Wu et al., 1997b). These observations, concerning developmental cell death in C. elegans, were extended to mammalian PCD (Chinnaiyan et al., 1997b). An equivalent ternary complex was found to be present in mammalian cells involving Apaf-1, caspase-9 and Bcl-x L, in which Bcl-x L inhibits Apaf-1-mediated maturation of caspase-9. In this paradigm, pro-apoptotic relatives like Bik may free CED-4/Apaf-I from the death inhibitor. Furthermore, there is some evidence that binding of pro-survival proteins to the apoptosome complex alter its location in cells, pulling it from the cytosol to the intracellular membranes where Bcl-2s often reside (Chinnaiyan et al., 1997b; Wu et al., 1997b). Thus, the mitochondrion seems to constitute a pivotal component of all the mechanisms of activation either as docking sites, via Bcl-2 family proteins, of caspases or as sequestering organelles of caspase activators. This crucial position of mitochondria in PCD control may be reinforced by the results obtained from distinct approaches establishing that mitochondria can contribute to PCD via the production of cell death signaling ROS. This chapter examines the data concerning the mitochondrial features of PCD and the way by which Bcl-2 family proteins participate in the modulation of these mitochondrial events (recent reviews on similar subjects:Mignotte and Vayssi~re, 1998; Zamzami et al., 1998).

Permeability Transition Marks A Point of No-Return of Cells Condemned to Die A Decrease in Mitochondrial Membrane Potential Precedes DNA Fragmentation During Apoptosis Several changes in mitochondrial biogenesis and function are associated with the commitment to apoptosis. A fall of the membrane potential (AqJm) occurs before the fragmentation of the DNA in oligonucleosomal fragments (Kroemer et al., 1995; Petit et al., 1995; Vayssi~re et al., 1994; Zamzami et al., 1995a; Zamzami et al., 1995b). This drop of Attt m is responsible for a defect of maturation of mitochondrial proteins synthesized in the cytoplasm (Mignotte et al., 1990), cessation of mitochondrial translation and an uncoupling of oxidative phosphorylation (Vayssi~re et al., 1994). The drop of AqJm is detectable whatever the apoptosis induction signal, physiological (absence of growth factor, glucocorticoids, TNF) or non-physiological (irradiation, chemotherapy). These data showed, on the one hand, that the nuclear fragmentation is a late event as compared to the drop of the Aq/m and, on the other hand, that this drop marks the point of no-return of a cell condemned to die.

98

B. Mignotte and J.-L. Vayssiere

Mitochondrial Permeability Transition Provokes a AY/m Decrease What is the mechanism involved in the Au.-/mdisruption? The permeability transition (trF) is a phenomenon that is characterized by the opening of large conductance channels known as the mitochondrial PT pores and by its sensitivity to very low concentration of cyclosporine A. These pores, permeable to compounds of molecular mass up to 1500 Da are formed under specific conditions. The opening of these PT pores in the inner membrane allows for an equilibration of ions within the matrix and intermembrane space of mitochondria, thus dissipating the H+ gradient across the inner membrane and uncoupling the respiratory chain. These events lead to the decrease of the AI¥m and the arrest of ATP synthesis (Bernardi et al., 1992; Petronilli et al., 1994). Perhaps more importantly, PT pore opening results in a volume dysregulation of mitochondria due to the hyperosmolarity of the matrix, which cause the matrix space to expand. Because the inner membrane is a larger surface area than the outer membrane, this matrix volume expansion can eventually provoke outer membrane rupture, releasing intermembrane space molecules into the cytosol. Furthermore, permeability transition has properties of self-amplification: the drop of the m ~ m that is linked to depletion of non-oxidized glutathione (Macho et al., 1997), and that result from the opening of the PT pores, would increase the permeability transition in a retrograde manner. We have, therefore, proposed that the opening of the PT pore may constitute an irreversible state of apoptosis and could account for the apparent synchronization in the drop of ALI/m that takes place simultaneously in all the mitochondria of a same cell (Kroemer et al., 1995). The molecular composition of these PT pores remains elusive. The peripheral benzodiazepin receptor, that has recently been implicated in the protection against ROS (Carayon et al., 1996), and the translocase of adenine nucleotides (ANT) are probable components of the PT pore. Indeed, protoporphyrine IX and PK11195, that are ligands of the benzodiazepin receptor, induce a drop of Aq/mand consequently apoptosis (Hirsch et al., 1998; Zamzami et al., 1996a). On the contrary, N-methyl Val-4-cyclosporin A (a derivative of cyclosporine that is not immunosuppressor) and bongkrekic acid that bind to the matrix side of the ANT, prevent the drop of the mitochondrial potential. Altogether, these results sustain the hypothesis that the opening of PT pores is involved in the disruption of AI'I/m observed during apoptosis. Direct Interventions on the Mitochondrial Permeability Transition Modulate Apoptosis Direct alterations of mitochondria can induce apoptosis (Hartley et al., 1994; Wolvetang et al., 1994). The links between mitochondrial perturbations and nuclear alterations have been studied by means of acellular systems where purified nuclei and purified mitochondria are confronted (Newmeyer et al., 1994). Such a system allows study of reciprocal and direct effects of one organelle on another and to characterize at the biochemical level the factors involved. These experiments have shown that when mitochondria are treated with substances capable to induce PT pores opening, they provoke nuclear apoptosis (condensation of the chromatin and fragmentation of the DNA) (Zamzami et al., 1996b). A correlation between induction of the trF and nuclear apoptosis has been observed by using a

Mitochondrial Control of Apoptosis

99

variety of known inductors of the PT such as atractyloside, pro-oxidants, calcium, protonophores and substances that provoke linkage of thiol groups such as diamide. These substances, that have no direct effect on nuclei in absence of mitochondria, confer pro-apoptotic properties upon mitochondria. The pro-apoptotic character (induction of nuclear apoptosis) of the mitochondria treated with atractyloside is altered by inhibitors of the PT such as bongkrekic acid, cyclosporine A and substances like monochlorobimane that block the cross-linking of the thiols. Cyclosporine A can be replaced by its non-immunosuppressor analogue, N-methyl Val-4-cyclosporine A, which shows that its inhibitory effect on PT and nuclear apoptosis is independent from its calcineurine activity. These results suggest the implication of the PT pores opening in the regulation of apoptosis induced via the mitochondria. Nuclei and mitochondria have been purified from hybridomas of T cells transfected by bcl-2 to study how Bcl-2 suppresses apoptosis in in vitro experiments (Zamzami et al., 1996b). Upon treatment with atractyloside, in contrast to mitochondria purified from control cells, mitochondria purified from cells transfected by bcl-2 do not provoke nuclear apoptosis. On the contrary, nuclei purified from cells transfected by bcl-2 show a condensation of the chromatin and a fragmentation of the DNA when they are confronted to control mitochondria treated with atractyloside. Furthermore, bcl-2 inhibits the induction of permeability transition by agents such as atractyloside, oxidants and protonophores. These results show that, even if Bcl-2 intervenes also during latter apoptotic events (Gurnal et al., 1997; Marton et al., 1997) and o n A q J m loss induced by other mechanisms (Shimizu et al., 1998), at least a part of its activity is exerted by acting on the mitochondrial permeability transition (Decaudin et al., 1997). Moreover, the structure of a protein of the Bcl-2 family (Bcl-XL) has been established (Muchmore et al., 1996). It recalls that of bacterial toxins, especially the diphtheria toxin, that form a pH-sensitive transmembrane channel. Furthermore, the pro-apoptotic Bax protein can form channels (Antonsson et al., 1997), as reported also for the anti-apoptotic proteins Bcl-x e (Minn et al., 1997) and Bcl-2 (Schendel et al., 1997). However, the intrinsic properties of Bax and those of Bcl-x e and Bcl-2 reveal differences. The channel forming activity of Bcl-x L and Bcl-2 is observed at highly acidic pH while Bax forms channels at a wide range of pH including at pH=7, those found in cells. Furthermore, Bcl-2 can block the pore-forming activity of Bax. Indeed, recent results suggest that Bax might produce cell death by inducing PT (Pastorino et al., 1998). Bcl-2 might counteract this effect of Bax on mitochondrial membranes (Gross et al., 1998).

Mitochondria of Cells Undergoing Apoptosis Release Pro-Apoptotic Factors Mitochondria Undergoing Permeability Transition Liberate AIF Factor The experiments described above showed that mitochondria from apoptotic cells can effectively control nuclear apoptosis and suggest the involvement of mitochondria derived products in the apoptotic cascade. Acellular experiments have shown that mitochondria contain a pre-formed approximately 50-kD protein, which is released upon PT pores opening and causes isolated nuclei to undergo apoptotic changes,

100

B. Mignotte and J.-L. Vayssiere

such as chromatin condensation and intemucleosomal DNA fragmentation (Figure 1). This apoptosis-inducing factor (AIF) derived from the mitochondria is a protease (or a protease-activating protein), which is blocked by the general caspase inhibitor Z - V A D and is capable of activating purified procaspase-3 (Susin et al., 1996). As expected, when preventing mitochondrial permeability transition, Bcl-2 overexpressed in the outer mitochondrial membrane also impedes the release of A I F from isolated mitochondria. In contrast, Bcl-2 does not affect the formation of AIF, which is contained in comparable quantities in control mitochondria and in mitochondria from Bcl-2-hyperexpressing cells. Furthermore, the presence of Bcl-2 in the nuclear membrane does not interfere with the action of AIF on the nucleus, nor does Bcl-2 overexpression protect cells against AIF. It thus appears that Bcl-2 prevents apoptosis by favoring the retention of an apoptogenic protein in mitochondria (Susin et al., 1996).

.... i

J

Execution caspases activation

Apoptosis

Simplified model of events occurring during apoptosis. Numerous signals can lead to apoptosis. The induction pathways seem to converge to events involving mitochondria. Proteins of the Bcl-2 family act on mitochondrial membrane permeability and regulate the release of pro-apoptotic factors from the intermembrane space to the cytosol.. These factors, directly or indirectly activate execution caspases responsible for the apoptotic phenotype.

Figure 1.

Mitochondrial Control of Apoptosis

101

Cytochrome C is Released by Mitochondria During Apoptosis Another mitochondrial killer component has been demonstrated through the role of cytochrome c in PCD. Indeed, translocation of cytochrome c from mitochondria to cytosol has been shown to be a crucial step in the activation of the PCD machinery in various death models, including Fas-, UV-, staurosporine or etoposide-treated mammalian cells, in a cell-free system using Xenopus egg extracts or dATP-primed cytosols of growing cells (Kluck et al., 1997a; Krippner et al., 1996; Liu et al., 1996; Yang et al., 1997). Furthermore, direct microinjection of cytochrome c into the cytosol can induce apoptosis in various cell-types (Li et al., 1997a; Zhivotovsky et al., 1998). Cytochrome c is an essential component of the mitochondrial respiratory chain: it accepts an electron from cytochrome c reductase and passes it on to cytochrome c oxidase. It is a soluble protein that is located in the intermembrane space and is loosely attached to the surface of the inner mitochondrial membrane. Cytochrome c is translated on cytoplasmic ribosomes as apocytochrome c and follows a unique pathway into mitochondria that does not require the signal sequence, electrochemical potential, and general protein translocation machinery (Mayer et al., 1995). The apoprotein, on entry into the intermembrane space, gains an heine group, to become the fully folded holocytochrome c. This globular, positively charged protein can no longer pass through the outer mitochondrial membrane and is thought to become electrostatically attached to the inner membrane. Upon its release into the cytoplasm during the initiation of apoptosis, cytochrome c promotes the assembly of a multiprotein complex, the apoptosome, that induces proteolytic processing and activation of executive caspases (Li et al., 1997b). Biochemical studies have shown that, besides cytochrome c, caspase-9, Apaf-1 and (d)ATP constituted the activating components of this complex. Apaf-1 and caspase-9 interact via their CARD domains, an association that requires dATP and cytochrome c. Binding of dATP and cytochrome c to Apaf-1 likely alters it's conformation and renders its CARD domain more available to caspase-9. Once bound to caspase-9, Apaf-1 triggers caspase-9 proteolytic self-activation; caspase-9 subsequently proteolyses and activates caspase-3. Many data suggested that the pro-survival Bcl-2 proteins prevent cell death acting upstream of the release of cytochrome c. Overexpresssion of Bcl-2 or Bcl-x L in cells blocks the release of cytochrome c and aborts the apoptotic response triggered by a wide variety of killing signals (Bossy-Wetzel et al., 1998; Kharbanda et al., 1997; Van der Heiden et al., 1997; Yang et al., 1997). On the contrary, caspases inhibitors have no effect on this process. Similarly, addition of recombinant Bcl-2 to Xenopus egg extracts containing mitochondria slows down the release of cytochrome c from these mitochondria and inhibits nuclear apoptosis observed in this cell-free system (Kluck et al., 1997a). Moreover, co-immunoprecipitation studies have shown that Bcl-x L can bind cytochrome c and may thereby act to sequester it in the mitochondria (Kharbanda et al., 1997). Bcl-x s, a pro-apoptotic derivative of Bcl-x L, was found to prevent the formation of the Bcl-XL-Cytochrome c complex. In contrast, overexpression of the pro-apoptotic Bcl-2 family member Bax in cells stimulates both the release of cytochrome c and apoptosis. By using isolated mitochondria and recombinant Bax,

102

B. Mignotte and J.-L. Vayssiere

it was shown that Bax can directly induce cytochrome c release from mitochondria. Similarly, recent reports show that Bid, a BH3 domain-containing pro-apoptotic Bcl-2 family member, induces cytochrome c release and apoptosis after its caspase8-dependent cleavage and subsequent translocation onto mitochondria (Li et al., 1998; Luo et al., 1998). Even though, altogether, these results provide evidence that Bcl-2 family members can modulate the cell death process by directly controlling the availability of cytochrome c in the cytosol (Figure 1), it must underlined that pro-survival actors, as Bcl-2/ Bcl-x L, may also act downstream of cytochrome c to prevent caspase activation under certain circumstances (see below) (Rosse et al., 1998; Zhivotovsky et al., 1998). Although efflux of cytochrome c from mitochondria appears to be a crucial step in the killing cascade initiated by a wide range of apoptogenic stimuli, it must be underlined that this event is not an universal requirement for death signal transduction. Indeed, on the one hand, apoptosis can occur in the absence of detectable cytochrome c release (Chauhan et al., 1997; Tang et al., 1998) and, on the other hand, under certain circumstances, cytochrome c release is not sufficient to promote cell death (Li et al., 1997a). Moreover, it was reported that efflux of cytochrome c could constitute only a later event of the cell death, in other words, a side effect of the terminal dismantelment of the cell (Adachi et al., 1998; Krippner et al., 1996). However, in this Fas-mediated apoptosis model, an inactivation of cytochrome c, correlated to the inhibition of mitochondrial respiration at the cytochrome c level, constitutes an very early causal event in the apoptotic program. It was suggested that inactivation of cytochrome c is associated to its release from the outer surface of the inner mitochondrial membrane where normally it functions as a shuttle connecting respiratory chain energy transducers (Skulachev, 1998). More intriguing, the authors suggest that efflux of cytochrome c which had been associated with early events of apoptosis could be, in some instances, the result of a methodological artifact (Adachi et al., 1998). Indeed, most studies are based on subcellular fractionations, i.e., after cell disruption, centrifugation allows to separate soluble cytosolic elements in the supernatant from heavy membranes, including mitochondria, included in the pellet. In this approach, the in vivo cytochrome c redistribution is inferred from its appearance into the cytosolic fraction during the in vitro procedure. In fact, this method could be more properly considered as a measure of cytochrome c extractability from mitochondria rather than as a exact view of its in situ localization. For instance, some minor alterations of the outer mitochondrial membrane could be compatible with a mitochondrial localization of cytochrome c in the cell and its outflow from mitochondria as fractionation provoked breakdown of the fragilized outer membrane. However, these considerations refute neither the early occurrence of cytochrome c release in various apoptotic models as observed by in situ immunolocalization nor its crucial role in the apoptotic program, ascertained by direct microinjection of cytochrome c in the cytosol. They must be correlated to the evidenced existence of alternative pathways, i.e. cytochrome c independent, in the transduction of apoptogenic stimuli (Chauhan et al., 1997; Li et al., 1997a; Tang et al., 1998).

Mitochondrial Control of Apoptosis

103

The Cytochrome C Pathway is Distinct from that of AIF The above described AIF could be a key regulator of such a cytochrome c independent pathway inasmuch as these two mitochondrial components seem involved in distinct signaling route. Indeed, while AIF release occurs after the PT-associated mitochondrial depolarization, several reports indicates that cytochrome c release is dissociable from PT (Bossy-Wetzel et al., 1998; JiJrgensmeier et al., 1998; Kluck et al., 1997a; Li et al., 1998; Van der Heiden et al., 1997; Yang et al., 1997). Furthermore, cytosolic cytochrome c requires additional cytosolic factors to promote apoptotic changes, via the activation of caspase-3, whereas AIF, once released from mitochondria, directly induces nuclear apoptosis without cytosol. Beyond their differences, these two "execution caspase" activating pathways illustrate the sophistication and the apparent molecular redundancy which characterize the mammalian cell death. They may correspond to alternative and independent links between death-triggering stimuli and the execution machinery or, in contrast, they may work together to induce complete PCD (Golstein, 1997). Bcl-2 Family Proteins Dock Various Proteins to the Mitochondria Even though, increasing reports provide evidence that Bcl-2 family members can modulate the cell death process by directly controlling the availability of cytochrome c in the cytosol, it must be underlined that pro-survival actors, such as Bcl-2/Bcl-xL, may also act downstream of cytochrome c to prevent caspase activation under certain circumstances (Rosse et al., 1998; Zhivotovsky et al., 1998). Moreover, effects of Bcl-2s cannot be reduced merely to models that envision Bcl-2s as regulators of killing caspase activation, inasmuch as Bcl-2 can also block caspase-independent cell deaths such as oxidant and hypoxia-induced necrosis, or Bax-induced yeast killing (Jtirgensmeier et al., 1997; Manon et al., 1997). The ability of Bcl-2 to bind (at least in two hybrid or co-immunoprecipitation experiments) and to dock several cellular proteins that do not belong to the Bcl-2 related proteins family, can provide a clue to understand how the Bcl-2 family governs PCD. First, Bcl-2 has been found to interact with Nip l, Nip2, and Nip3, the function of which is unknown (Boyd et al., 1994), the GTPase R-ras p23 (Fernandez and Bischoff, 1993), Raf-1 (Ali et al., 1997; Wang et al., 1994), BAG-1 (Takayama et al., 1995), the cellular prion protein (PrP) (Kurschner and Morgan, 1995), the p53 binding-protein p53-BP2 (Naumovski and Cleary, 1996), the protein phosphatase calcineurin (Shibasaki et al., 1997) and the mitochondrial membrane protein carnitine palmitoyltransferase I (Paumen et al., 1997). At least some of these interactions could reflect the ability of Bcl-2 to relocalize cellular proteins to mitochondrial membranes. Bcl-2 binds to BAG-1 (Takayama et al., 1995) that can also interact with Raf-1 (Wang et al., 1996b). Active Raf-1 fused with targeting sequences from an outer mitochondrial membrane protein protect cells from apoptosis and phosphorylate BAD, a proapoptotic Bcl-2 homologue (Wang et al., 1996a). Furthermore, plasma membranetargeted Raf-1 did not protect from apoptosis and resulted in phosphorylation of ERK-I and ERK-2 while Raf-1 improved Bcl-2-mediated resistance to apoptosis.

104

B. Mignotte and J.-L. Vayssiere

Bcl-2 can therefore target Raf-1 to mitochondrial membranes, allowing this kinase to phosphorylate BAD. However, the link between Raf-1 and the mitochondrial changes occurring during apoptosis are not yet known and bcl-2 does not always require c-raf-1 kinase activity and an associated mitogen-activated protein kinase signaling pathway for its survival function (Olivier et al., 1997). Second, in accord with C. elegans genetics, biochemical evidence shows that the pro-survival Bcl-2 family proteins may function by directly inhibiting the activation of killing caspases. It was initially shown that binding of CED-9 to CED-4 prevents CED-4 from inducing proteolytic processing and activation of CED-3 (Chinnaiyan et al., 1997b; Wu et al., 1997b). The CED-9 protein, like the mammalian Bcl-2 related proteins, is localized to intracellular membranes and the perinuclear region, whereas CED-4 was distributed in the cytosol. Expression of CED-9, but not a mutant lacking the carboxy-terminal hydrophobic domain, targeted CED-4 from the cytosol to intracellular membranes in mammalian cells (Wu et al., 1997b). A similar mechanism exists in mammalian cells as first suggested by the ability of Bcl-x L to interact with and inhibit the function of CED-4 (Chinnaiyan et al., 1997b). A ternary complex involving Apaf-1, caspase-9 and Bcl-x L, in which Bcl-x L inhibits Apaf-l-mediated maturation of caspase-9, was found to be present in mammalian cells (Hu et al., 1998; Pan et al., 1998). Figure 2 illustrates that pro-apoptotic relatives like Bik, Bak and Bax can promote cell death by disrupting interaction between Bcl-x L and Apaf-1 (Chinnaiyan et al., 1997b; Pan et al., 1998). Such data explain how pro-survival actors, as Bcl-2/Bcl-x L, may also act downstream of cytochrome c to prevent caspase activation under certain circumstances. For instance, overexpression of Bcl-2 and Bcl-x L inhibits apoptosis induced by direct injection of cytochrome c in cytosol (Li et al., 1997a; Zhivotovsky et al., 1998). Similarly, Bcl-2 delays Bax-induced caspase activation and cell death even when cytochrome c is already in the cytosol (Rosse et al., 1998). However, it must underlined that today there is no evidence concerning a physical interaction of Bcl-2 with apoptosome complex. Lastly, there is some evidence that binding of pro-survival proteins to the Apaf-1/caspase-9 complex alters its location in ceils, dragging it from the cytosol to the intracellular membranes where Bcl-2s often reside (Chinnaiyan et al., 1997b; Wu et al., 1997b). In murine thymocytes, Bcl-2 is exclusively membrane-bound, whereas Bcl-x L is present in both soluble and membranebound forms and Bax is present predominantly in the cytosol. Induction of apoptosis by dexamethasone or gamma-irradiation shifts the subcellular locations of Bax and Bcl-x L from soluble to membrane-bound forms. Inhibition of apoptosis with cycloheximide inhibits the movement of Bax and Bcl-x L from the cytosol into intracellular membranes (Hsu et al., 1997). Since Bax and Bik can disrupt the association between CED-9 (or Bcl-xL) and CED-4 (Chinnaiyan et al., 1997b), it is tempting to speculate that Bcl-x L and possibly other members of the Bcl-2 protein family inhibit apoptosis by maintaining the procaspases/Apaf-1 complexes associated to mitochondrial membranes, and that Bax and Bik by dissociating the complexes permit the activation of procaspases.

Mitochondrial Control of Apoptosis

105

"

~caspase-9J1

Survival

Caspase-3 activation

Cell death

Figure 2.

Bcl-2 family proteins regulate the intracellular localization and the activity of caspases

containing complexes. In C. elegans, CED-9 might maintain CED-4/CED-3 complexes at the level of intracellular membranes by its simultaneous binding to mitochondrial membrane and CED-4. Similarly, in mammals, Bcl-xL might anchor Apaf-1/caspase complexes and keep them in an inactive state. The dissociation of the Bcl-XL/Apaf-1interaction by Bax or Bak (or other proapoptotic Bcl-2 family protein) leads to the activation of caspases.

What is the Mechanism Involved in Cytoplasmic Release of Mitochondrial Apoptogenic Factors? The mechanism by which AIF and cytochrome c are released from mitochondria is largely unknown. Although apoptosis can occur in the absence of detectable cytochrome c release (Chauhan et al., 1997; Tang et al., 1998), efflux of cytochrome c from mitochondria appears to be a critical coordinating step in the killing program towards which converge the multiple signaling pathways and beyond which are initiated the entire panel of apoptotic features, pro-caspase 3 expressing cells being then irreversibly committed to die (Li et al., 1997a). With this perspective, the possible mechanisms involved in activation of this central control may be envisaged from data concerning mediators of the death signal transduction cascades. For instance, the release of cytochrome c might result from oxidative imbalance, an upstream event in the apoptotic transduction cascade (see below). This phenomena might lead to the alteration of some redox sensitive crucial regulatory elements of the outer mitochondrial membrane permeability, e.g. by a shift of the redox state of some sulfhydryl groups to a more inactivating oxidized state.

106

B. Mignotte and J.-L. Vayssiere

Another possibility comes from the ability of caspases to promote the release of intermembrane proteins, including cytochrome c and AIF, through the outer mitochondrial membrane. These observations suggested that mitochondria carry a caspase substrate that, when cleaved, promotes cytochrome c release (Mignotte and Vayssi~re, 1998). Recent reports concerning Fas and TNF-ct receptor signaling could provide insight into how caspase induces cytochrome c efflux. Previously, it had been established that activation of caspases, predominantly caspase-8, constitutes an early step in these apoptotic pathways (Nagata, 1997), and that inhibition of these proteases prevents both the release of cytochrome c from mitochondria and the execution of the cell death program (Schulze-Osthoff et al., 1998; Van der Heiden et al., 1997). Recently, it has been shown that caspase-8, activated by cell surface death receptors such as TNF and Fas, cleaves Bid, a death agonist of the Bcl-2 family, which in turn transduces the apoptotic signal from the cell surface to mitochondria (Li et al., 1998; Luo et al., 1998). Thus, while full-length Bid is localized in cytosol, the C-terminal part caspase-8-truncated BID translocates to mitochondria and then induces cytochrome c release and the downstream caspase-dependent apoptotic program. These results point to a model where mitochondria might act as apoptotic amplifiers, fostering a positive feedback loop between cytochrome c-efflux and caspase activation. Any event that primes the loop will initiate the vicious "circle of death", leading to large-scale caspase activation and apoptotic cell death. However, this model does not prevail in all systems as, in numerous PCD, caspase inhibitors have no effect on the loss of cytochrome c (Bossy-Wetzel et al., 1998; Kluck et al., 1997a,b). Alternatively, opening of PT pores, which is a common event of apoptosis, might be involved in AIF and cytochrome c outflow (Skulachev, 1998). However, PT pores, which are thought to connect the mitochondrial matrix to the cytosol, within the contact sites between inner and outer mitochondrial membranes (Beutner et al., 1996) are only permeable to small compounds (molecular mass -

[]

JC-1 red

[]

JC-1 green

[]

clonogenic survival

1

n-

ooo o

S

"S

.
"

>


o

14'

1o

I~

II C°ntr°l

I | .

BII

a~

7~

6

.k

0 Hippocampus

Figure 5.

Cerebellum

Inferior parietal cortex

Par-4 expression is significantly increased in Alzheimer's disease patient brain. (A).

Double label immunohistochemical analysis of hippocampal sections from an Alzheimer's disease (AD) patient and a neurologically normal control patient stained with antibodies against Par-4 and PHF-1 (an antibody that recognizes hyperphosphorylated tau in neurofibrillary tangles). Note Par-4 levels are significantly increased in AD brain and that approximately 40% of PHF-1 immunoreactive neurons (brown, white arrowhead) are also immunoreactive with the Par-4 antibody (black, white arrow). Dystrophic neurites associated with neuritic plaques (black arrow) showed PHF-1 immunoreactivity but little or no Par-4 immunoreactivity. (B). Densometric analysis of Par-4 amounts in three brain regions from control and AD patients. Values are the mean and SD of determinations made in samples from six control and six AD patients. ***P~

_o

.o o D.