New Research on Alzheimer's Disease

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NEW RESEARCH ON ALZHEIMER’S DISEASE

No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

NEW RESEARCH ON ALZHEIMER’S DISEASE

EILEEN M. WELSH EDITOR

Nova Science Publishers, Inc. New York

Copyright © 2006 by Nova Science Publishers, Inc.

All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Library of Congress Cataloging-in-Publication Data New research on Alzheimer's disease / Eileen M. Welsh (editor). p. ; cm. Includes index. ISBN 978-1-60876-520-1 (E-Book) 1. Alzheimer's disease--Research. I. Welsh, Eileen M. [DNLM: 1. Alzheimer Disease. WT 155 N5315 2006] RC523.R472 616.8'310072--dc22

Published by Nova Science Publishers, Inc. New York

2006 2005035992

Contents Preface Chapter I

vii Insights on Alzheimer’s Disease from the Intracerebral Injection and Infusion of Amyloid-β Glenda M. Bishop and Stephen R. Robinson

Chapter II

The Role of Phospholipase A2 in Alzheimer's Disease Wagner F. Gattaz, Evelin L. Schaeffer and Orestes V. Forlenza

Chapter III

Low Molecular Weight Glycosaminoglycans and Apoptosis: Potential Treatment of Neurodegenerative Disorders Including Alzheimer’s Disease Bertalan Dudas

Chapter IV

Chapter V

Chapter VI

Chapter VII

Low Density Lipoprotein Receptor-Related Protein 1 (LRP1): One of the Hallmarks ofAlzheimer’s Disease and Other Complex Degenerative Processes Christiane Gläser, Gerd Birkenmeier, Susanne Schulz and Klaus Huse

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The Role of Peroxisome Proliferator-Activated Receptors in Alzheimer’s Disease Dokmeci Dikmen

125

Role of Sphingomyelin Cycle Signaling System in Alzheimer’s Disease A. V. Alessenko

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Ischemia-Reperfusion Factors in Sporadic Alzheimer’s Disease Ryszard Pluta

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Chapter VIII

Index

Eileen M. Welsh

Amyloid Beta (Aβ) Peptides in Plasma as Biochemical Markers of Alzheimer's Disease and Mild Cognitive Impairment Tomasz Sobow, Marcin Flirski, Pawel P. Liberski and Iwona Kloszewska

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Preface Dementia is a brain disorder that seriously affects a person's ability to carry out daily activities. The most common form of dementia among older people is Alzheimer's Disease (AD), which involves the parts of the brain that contol memory, thought and language. Age is the most important known risk factor for AD. The number of people with the disease doubles every 5 years beyond age 65. AD is a slow disease, starting with mild memory loss and ending with severe brain damage. The course the disease takes and how fast changes occur vary from person to person. On average, AD patients live from 8 to 10 years after they are diagnosed, though the disease can last for as many as 20 years. Current research is aimed at understanding why AD occurs and who is at greatest risk for developing it, improving the accuracy of diagnosis and ability to identify who is at risk, developing, discovering and testing new treatments for behavioral problems in patients with AD. This new book gathers state-of-the-art research from leading scientists throughout the world which offers important information on understanding the underlying causes and discovering the most effective treatments for Alzheimer's Disease. The notion that the amyloid-beta (Aβ) peptide plays a role in the pathogenesis of Alzheimer’s disease (AD) has received strong support from in vitro demonstrations that Aβ is toxic to cultured neurones, but its support from in vivo models has been equivocal. In order to understand the reasons for this difference, the authors of chapter I have critically appraised the numerous in vivo studies that have investigated the toxicity of Aβ by injecting or infusing Aβ into the brains of rodents or primates. Taken together, these studies indicate that neuronal loss is not a consistent response to Aβ, yet it does induce subtle changes in neuronal function by altering several neurotransmitter systems, particularly the cholinergic system. Behavioural impairments are evident on spatial and discriminative learning tasks and are paralleled by a reduced capacity to induce LTP. In addition, Aβ induces astrocytic and microglial reactivity which may be associated with increased indices of oxidative stress. The Aβ injection/infusion models have also shown some brain regions to be more susceptible to the neuroactive properties of Aβ than others. The superior spatial and temporal resolution of injection/infusion models allows comparisons of the effects of individual isoforms or fragments of Aβ, and of agents that enhance or reduce the bioactivity of this peptide. They conclude that the Aβ injection/infusion models are a valuable adjunct to transgenic mouse

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models, and they may help to elucidate the physiological actions of Aβ and further our understanding of the pathogenesis of AD. Phospholipase A2 (PLA2) is a key enzyme in phospholipid metabolism. In the brain, PLA2 is involved in the liberation of membrane choline for acetylcholine synthesis, as well as in the processing of the amyloid precursor protein to form the beta-amyloid, the major component of senile plaques in Alzheimer's disease (AD) as described in chapter II. In previous post-mortem studies the authors reported on decreased PLA2 activity in the frontal and in the parietal cortex of patients with AD, suggesting reduced phospholipid metabolism in the disease. Interestingly, they found also a reduction of PLA2 activity in platelet membranes of AD patients, suggesting that reduced phospholipid turnover may also be observed in peripheral cells of AD patients. In an in vivo study they investigated the intracerebral phospholipid metabolism in 16 patients with probable AD and 18 age-matched controls through 31P-Magnetic Resonance Spectroscopy of the pre-frontal cortex. Cognitive assessment was performed by the CAMDEX schedule. PME resonance was significantly higher and PDE was lower among AD than controls. PME correlated negatively with cognitive performance. Higher PME indicates reduced breakdown of membrane phospholipids, which is in line with reduced PLA2 activity. The findings in the pre-frontal cortex suggest that reduced membrane phospholipid turnover may be a widespread cerebral phenomenon in AD brains, since the pre-frontal cortex is a region not as severely affected by the neurodegenerative process as temporal and parietal areas, specially in early stages of dementia. To investigate further the implications of reduced PLA2 in the neurobiology of AD, they carried out a series of in vitro and animal experiments, to respectively test the effects of the inhibition of PLA2 on neuronal homeostasis in cultured neurons, and in memory formation in rats. In primary cultures of cortical and hippocampal neurons, the inhibition of PLA2 precluded neurite outgrowth, and the sustained inhibition of the enzyme in mature cultures lead to loss of viability. These findings reinforce the involvement of PLA2 enzymes in neurodevelopment and neurodegeneration. In early stages of AD there is an impairment of episodic short-term (STM) and long-term memory (LTM). In rat hippocampal slices, PLA2 has been implicated in mechanisms of synaptic plasticity involved in memory formation. Using adult Wistar rats, we investigated the effects of injections of PLA2 inhibitors into rat hippocampus on the formation of STM and LTM of a one-trial step-down inhibitory avoidance task. Infusion of the PLA2 inhibitors impaired both STM and LTM in a dosedependent manner. Additionally, because memory function is largely dependent on the fluidity of brain membranes, they investigated the effects of in vivo PLA2 inhibition on the fluidity of hippocampal membranes from rats subjected to inhibitory avoidance training. Hippocampal tissue from rats injected with PLA2 inhibitor showed reduced membrane fluidity, and this reduction was highly correlated with PLA2 inhibition. Taken together, the findings suggest that reduced PLA2 activity, probably reducing membrane phospholipids breakdown, may contribute to the memory impairment in AD. Previous studies have shown that apoptosis (programmed cell death) plays a crucial role in the pathomechanism of neurodegenerative disorders. It is a general consensus that apoptosis can be triggered by dysregulation of nerve growth factor physiology and/or oxidative stress processes. As reported in chapter III, since both of these pathological changes are characteristic to AD, it is conceivable that influencing apoptosis can be a valuable tool in

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the treatment of neurodegenerative disorders. Glucosaminoglycans (GAGs) exhibit neuroprotective properties in several animal models of AD. Low molecular weight GAG, C3, attenuates Aβ(25-35)-induced tau-2 immunoreactivity, and AF64A-induced cholinergic lesion in rat brain. Recent studies have also revealed that C3 influences p75 nerve growth factor receptor expression. However, the exact mechanism of the neuroprotective attributes of C3 is not elucidated yet. Since apoptosis is believed to play a pivotal role in numerous neurodegenerative disorders, C3 may induce neuroprotection/neurorepair via affecting apoptotic processes. The neuroprotective effect of glucosaminoglycans including C3 thus may involve multiple mechanisms, including an influence on processes that lead to programmed cell death. Since apoptosis is a crucial event not only in neurodegenerative diseases but also in the general process of aging, C3 may be a valuable therapeutic agent in Alzheimer’s disease-related senile dementia and in other age-related malfunctions. Susceptibility to Alzheimer´s disease (AD) is governed by multiple factors. Remarkably, LDL receptor-related proteins (LRPs) and several of their numerous ligands were associated to AD. Alpha2-macroglobulin (A2M), apolipoprotein E (Apo E), but also amyloid precursor protein (APP) belong to the functionally and genetically interesting molecules involved in the complex degenerative processes resulting in AD as suggested by the findings in chapter IV. LRP1 (CD91) (MIM 107770) is a 4,544-amino acid protein containing a single transmembrane segment and is located on chromosome 12q13.1-12q13.3 within a region shown to be linked to AD. It consists of 84 exons and belongs to the largest human proteins and is highly evolutionary conserved. It could be demonstrated that several genomic variants of LRP1 were associated to AD but also to other degenerative diseases like atherosclerosis and cancer suggesting a complex role in fundamental cellular processes. LRP1 functions as a clearance receptor for several ligands, including amyloid ß (Aß), and mediates signaling pathways affecting the processes of neurite outgrowth and calcium influx. While clearance of Aß diminishes the extracellular Aß-load the interaction with APP may enhance formation of Aß. LRP1 is expressed in brain, blood vessels as well as in endothelial cells of the brainblood-barrier mediating the transcytosis of Aß between blood circulation and brain. Internalization via LRP1 concerns also extracellular proteases and protease-inhibitor complexes involved in Aß degradation. In AD as well as in other diseases genetic variations and structure-caused failure of LRP1 leading to dysfunctions as well as alteration in the expression of LRP1 ligands may determine common degenerative processes. Age-related changes in the level of LRP1 and its ligands may increase the risk for degenerative diseases. The modulation of LRP1 and its ligands by cytokines and growth factors was detected in and outside the brain. There are indications that also inflammatory processes play an important role in the development of degenerative diseases. Alzheimer’s disease (AD) is characterized by a progressive loss of cognitive function with cerebral deposits of amyloid-β (Aβ) senile plaques and neurofibrillary tangles (NFTs) surrounded by inflammatory cells. Neuroinflammation, oxidative stress and impaired glucose and lipid metabolism are postulated to be the mechanisms playing role in the pathophysiology of AD. Recent evidence suggests that inflammatory events are associated with plaque formation in the brains of patients with AD and non-steroid anti-inflammatory drugs (NSAIDs) might influence central nervous system (CNS) inflammation and AD pathology. Reactive oxygen and nitrogen species production may also participate in part to

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neurodegeneration in AD brains; antioxidant and/or free radical scavengers reduce inflammation in AD via free radical quenching and possess neuroprotective activity. In addition, AD patients show hyperinsulinemia and reduced insulin sensitivity during fasting and glucose tolerance tests; and insulin gene polymorphism might act as a modifier of AD progression. An increased number of insulin receptors are also observed in post-mortem brain tissues of AD patients. Furthermore, augmented plasma insulin levels and reduced cerebrospinal fluid (CSF) levels and CSF/plasma insulin ratio are observed in AD patients. Chapter V discusses treatment of insulin resistance may reduce the risk or retard the progression of AD. Peroxisome proliferator-activated receptors (PPARs), members of the nuclear receptor family, are ligand-activated transcription factors. To date, three isoforms, encoded by separated genes, have been identified: PPARα [NR1C1], PPARβ (NUC-1 or PPARδ) [NR1C2] and PPARγ [NR1C3]. All PPAR isotypes are detected and found to exhibit specific patterns of localization in the different areas of the brain. PPARs may have specific functions in regulating the expression of genes involved in neurotransmission, and therefore play roles in complex processes, such as neurodegeneration, learning and memory. It is likely that at least some effects of PPAR agonists and NSAIDs on AD pathology are mediated through PPARs, since both PPARs and cyclooxygenase (COX) expression are increased in AD brains. Recently, NSAIDs, activating PPARα and γ, have been identified. In addition, NSAIDs and PPARα and γ agonists have anti-inflammatory, antioxidant and neuroprotective effects. This neuroprotective effect correlates with the modulation of β-catenin levels, inhibition of glycogen synthase kinase-3β (GSK-3β) activity and increased mRNA levels of the Wnt-target genes engrailed-1, cyclin D1 and PPARδ. Thiazolidinediones (TZDs) such as pioglitazone and rosiglitazone are insulin sensitizers, used to control glucose concentrations in patients with type 2 diabetes. These are potent synthetic PPARγ agonists, which exert both anti-inflammatory and antioxidant effects. PPARα and γ ligands, NSAIDs and TZDs inhibit activation of nuclear factor (NF)-κB, signal transducers and activators of transcription-1 (STAT-1), the nuclear factor of activated T-cells (NFAT) and activating protein-1 (AP-1). Moreover, they prevent the expression of COX-2 and inducible nitric oxide synthase (iNOS) and production of inflammatory cytokines such as interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α in human monocytes and arrest the differentiation of monocytes into activated macrophages. In conclusion, the development of new drugs targeting PPARs would be a great interest for future treatments of AD. Alzheimer’s disease (AD) is characterized by a progressive decline in cognition, memory and intellect. Possible mechanisms for AD neurotoxicity include: alteration in Ca2+ homeostasis, activation of specific receptors affecting cellular homeostasis, activation of oxidative processes, direct disruption of membrane integrity and disorders in lipid metabolism, or a combination of two or more of the above mechanisms. It was established that conformational changes in amyloid-beta peptide (Aβ) may occur in lipid rafts under the control of specific sphingolipids. Chapter VI demonstrates a novel mechanism for development of AD. It has been hypothesized that Aβ and TNF-α may have a prominent role in neurodegeneration. It is well-known that neuronal death is developed according to apoptotic program. Most signaling pathways that trigger apoptosis remain unknown, but the sphingomyelin pathway has been recognized as an ubiquitous signaling system that links specific cell-surface receptors and environmental stresses to the nucleus. This pathway is

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initiated by the hydrolysis of sphingomyelin via the action of sphingomyelinases to generate ceramide. Ceramide then serves as a second messenger in this system, leading to apoptosis. Oxidative mechanisms have been implicated in this pathway. TNF-α activates receptors linked to multiple effector systems, including a sphingomyelin pathway and peroxide oxidation. Recently, it was shown that superoxide radicals are used as signaling molecules within the sphingomyelin pathway. It means that cross-talk between the oxidation system and sphingomyelin cycle exists in cells and could have important implications for the induction phase and evolution of AD. The role of TNF-α in the pathogenesis of AD is unclear, because it has been shown to be involved in both neuroprotection and neurodegeneration, depending on doses and the age of animals. Small doses of this cytokine induced a protective effect against Aβ neurotoxicity. It was suggested that using low doses of TNF-α in the clinic for the prevention of AD development might be a perspective. Identification of the N-SMaseceramide pathway may lead to the development of more effective therapeutic strategies, aimed at preventing Aβ-induced cell death. The study of neurobiology of Alzheimer’s disease, now more than ever, needs an infusion of a new concept. Despite ongoing interest in Alzheimer’s disease, the basis of this entity is not yet clear. For now, the best-established and accepted “culprit” in Alzheimer’s disease pathology by most scientists is the amyloid, as the main molecular factor of neurodegeneration in Alzheimer’s disease. Abnormal upregulation of amyloid production or a disturbed clearance mechanism may lead to pathological accumulation of amyloid in the brain. Chapter VII will critically review these observations and highlight inconsistencies between the predictions of the “amyloid hypothesis” and the published data. There is still controversy over the role of amyloid in the pathological process – is it responsible for the neurodegeneration or does it accumulate because of the neurodegeneration? Recent evidence suggests that the neuropathology of Alzheimer’s disease comprises more than amyloid accumulation, tau protein pathology and finally brain atrophy. At least one third of Alzheimer types of dementia cases exhibit different cerebrovascular diseases. In addition, micro- and macroinfarctions and ischemic white matter changes are also evident in brains of Alzheimer’s disease patients. The presence of vascular abnormalities seems usually ignored and regarded by researchers as insignificant or considered incidental in Alzheimer’s disease etiology. Interestingly, that Alzheimer, in his own report presenting changes in the brain of the first patient had described, that besides “storage of peculiar material in the cortex, one sees endothelial proliferation and also occasionally neovascularisation”. Endothelial proliferation and angiogenesis and moderate arteriosclerosis in the brain arteries of the first case, provide evidence that cerebrovascular diseases were also evident in Alzheimer’s original case, which now defines Alzheimer’s disease. These raise the question of what was the first cerebrovascular disease as trigger of Alzheimer’s disease or neurodegeneration of Alzheimer’s type itself? New findings propose an early and significant role for ischemiareperfusion factors contributing to the neurodegenerative processes in Alzheimer’s disease. The ischemia hypothesis was primarily aimed at stimulating research and redirecting the focus of studies towards ischemic cellular mechanisms of Alzheimer’s disease. In this review, the authors will show that experimental brain ischemia-reperfusion episode produces neurochemical and neuropathological changes that simulate the early stage of Alzheimer’s disease process. Presented data suggest that ischemic mechanisms of neuronal death with β-

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amyloid peptide from circulatory network modulate neuropathology of cerebral ischemiareperfusion injury via molecular events in common with Alzheimer-type neuropathology. These results indicate that ischemic brain processes might be a key factor in the development of the picture of Alzheimer-type dementia over years. The collective data summarized in this review strongly support the idea that sporadic from of Alzheimer’s disease is a neurovascular disorder. Considerable progress has been made in recent years by a handful of researchers in understanding the role of ischemia in the aging process generally and in contributing to the development of Alzheimer’s disease. To accommodate the recent progress of study in Alzheimer’s disease there is a need to synthesize all the divergent pieces of data into a coherent story. This review provides a synopsis of current information about ischemic cellular and molecular mediators involved in Alzheimer’s neuropathology as well as interactions between these mediators that influence pathology. In this review, current knowledge on the relation between ischemia-reperfusion factors and Alzheimer’s-type dementia will be reviewed. They will summarize the results with a special focus on Alzheimer lesions in experimental brain ischemia. Taken all together, evidence presented in this review suggests a scheme for Alzheimer’s pathogenesis with ischemia-reperfusion playing a crucial role in influencing and linking β-amyloid deposition to neuronal damage and clinical disease. Plasma levels of amyloid β peptides (Aβ) represent a potentially attractive biomarker of Alzheimer’s disease (AD). Although plasma Aβ levels are increased in patients with familial AD mutations, results of the studies encompassing sporadic AD cases are equivocal. In several studies elevated plasma Aβ42 levels could be detected long before the onset of symptoms, though the value of that effect in predicting progression to dementia in mildly cognitively impaired (MCI) subjects is not known. It has recently been proposed that plasma Aβ levels increase merely with age and are neither sensitive nor specific for AD or MCI. Additionally, an increase of Aβ42 plasma levels in women with MCI has been reported and thus suggested to represent a biologic explanation for the increased prevalence of females in the population of late-onset AD patients identified by epidemiologic studies. In chapter VIII the authors have assessed the levels of Aβ peptides in plasma of carefully selected AD patients and MCI subjects compared to healthy controls matched for age, gender and education. The selection procedure employed in the study has been aimed at excluding patients with mixed AD as well as patients fulfilling current diagnostic criteria for both AD and other specific forms of dementia (e.g. dementia with Lewy bodies). No difference in any of the evaluated parameters (Aβ40, Aβ42, Aβ40/Aβ42 ratio) was observed between the AD and control groups. In subjects with MCI plasma Aβ42 levels were significantly elevated versus both AD (p 0.1). After training, in animals injected with vehicle, latencies for STM and LTM significantly increased by 9 and 11-fold, respectively (p = 0.001). Injections of 10 µM MAFP (but not of 1 µM; p > 0.5) significantly prevented the increments of latencies for STM and LTM (p < 0.02, as compared to vehicle) (Figure 6A). (b) Training session latencies were similar in animals injected with vehicle or with 10 µM or 100 µM PACOCF3 (p > 0.1). After training, in animals injected with vehicle, latencies for STM and LTM significantly increased by 7 and 8-fold, respectively (p = 0.001). Injections of 100 µM PACOCF3 (but not of 10 µM; p > 0.5) significantly prevented the increments of latencies for STM and LTM (p < 0.02, as compared to vehicle) (Figure 6B). (c) Training session latencies were similar in animals injected with vehicle or with 1 µM or 10 µM BEL (p > 0.2). After training, in animals injected with vehicle, latencies for STM and LTM significantly increased by 11-fold (p = 0.001). Injections of 10 µM BEL (but not of 1 µM; p > 0.5) significantly prevented the increments of latencies for STM and LTM (p < 0.001 and p < 0.01, respectively, as compared to vehicle) (Figure 6C) (Schaeffer and Gattaz, 2005). As pre-training injections of MAFP, PACOCF3, and BEL impaired memory acquisition, we investigated whether the amnesic effects of the PLA2 inhibitors in inhibitory avoidance task were due to their action on general activity (locomotor and exploratory activities) during training performance. At 30 min after injections into the CA1 region of (a) vehicle or 10 µM MAFP, of (b) vehicle or 100 µM PACOCF3, or of (c) vehicle or 10 µM BEL (n = 10 per group), the rats were submitted to 3 min of free exploration of inhibitory avoidance apparatus. Crossings of three imaginary lines on the floor of inhibitory avoidance apparatus were counted. We found that the number of crossings was similar in animals injected with vehicle or with 10 µM MAFP, 100 µM PACOCF3, or 10 µM BEL (p > 0.1) (data not shown), indicating that the impairing effects of the pre-training injections of the PLA2 inhibitors were not caused by gross behavioral alterations on training performance (Schaeffer and Gattaz, 2005). After behavioral procedures, histological analyses were carried out to verify the injection placements and to investigate whether the observed effects of PLA2 inhibition on test session latencies in inhibitory avoidance task were a consequence of neuronal death. Rats injected with vehicle (n = 8) or with 10 µM MAFP (n = 6), 100 µM PACOCF3 (n = 6), or 10 µM BEL (n = 6) were deeply anaesthetized, transcardially perfused with saline and fixed with a 10% formaldehyde solution. The brains were withdrawn and fixed in the same fixative, embedded in paraffin, cut into consecutive 5 µm sections with a microtome, and placed on glass slides. The sections were stained with haematoxylin-eosin for histological localization of the injection sites as well as for histological analysis of neuronal death in the injection sites. Using a light microscope at 400X magnification, neuronal death was determined as the decrease in the percentage of purple-stained cell nuclei by haematoxylin-eosin in a delimited

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area of 2.0 mm2 corresponding to the injection sites in the CA1 region. In the remaining rats submitted to behavioural procedures a 10% trypan blue solution was injected into the brain sites implanted with cannulae. After 30 min the animals were killed by decapitation and the brains were withdrawn, fixed in a 10% formaldehyde solution, and cut manually for histological localization of the injection sites. Injections spread with a radius of 1.0-1.5 mm3 in each cannula-implanted site. On the whole, injections were found to be correct in 94% of the animals. We found that injections of the PLA2 inhibitors did not cause neuronal death as compared to vehicle (10 µM MAFP, p = 0.1; 100 µM PACOCF3 and 10 µM BEL, p > 0.5) (data not shown) (Schaeffer and Gattaz, 2005). Additionally, we investigated the degree of inhibition of PLA2 activity in the CA1 region of another group of rats injected with PLA2 inhibitors. Hence, at 30 min after injections into the CA1 region of (a) vehicle (n = 8) or 1 µM MAFP (n = 6) or 10 µM MAFP (n = 9), of (b) vehicle (n = 9) or 10 µM PACOCF3 (n = 9) or 100 µM PACOCF3 (n = 8), or of (c) vehicle or 1 µM or 10 µM BEL (n = 8 per group), the rats were submitted to inhibitory avoidance task as described above, but they were killed by decapitation immediately after receiving the footshock in order to dissect the CA1 region for PLA2 activity determination around the time of training. The brains were rapidly withdrawn and about 2.0 mm3 of the CA1 region corresponding to the injection sites were bilaterally dissected, homogenized in 5 mM TrisHCl (pH 7.4, 4°C), and stored at –70°C until use. PLA2 activity was determined by a radioenzymatic assay. Briefly, as enzyme substrate we used L-α-1-palmitoyl-2-arachidonylphosphatidylcholine labelled with [1-14C] in the arachidonyl tail at the sn-2 position (arachidonyl-1-14C-PC). We determined optimal assay conditions ([Ca2+] and pH value) for measuring cPLA2 plus iPLA2 activities in CA1 tissue homogenates of rats injected with MAFP or PACOCF3, and for measuring only iPLA2 activity in CA1 tissue homogenates of rats injected with BEL (data not shown). Hence, the assay samples (500 µl) contained 100 mM Tris-HCl (pH 8.5 for cPLA2 plus iPLA2; pH 7.5 for iPLA2), 1 µM CaCl2 (for cPLA2 plus iPLA2) or 100 µM EDTA (for iPLA2), 300 µg of protein from homogenates, and 0.06 µCi arachidonyl-1-14C-PC. After an incubation time of 30 min at 37°C the reaction was stopped and the liberated [1-14C]AA was extracted. The radioactivity of [1-14C]AA was measured in a liquid scintillation counter and used for calculating the PLA2 activity, which is expressed in pmol⋅mg protein⋅min– 1. The activity of cPLA2 plus iPLA2 in post-mortem CA1 region was significantly reduced by 28% by injections of 10 µM MAFP (p < 0.01) and by 26% by injections of 100 µM PACOCF3 (p = 0.002), but was not significantly reduced by injections of 1 µM MAFP (p > 0.2) or 10 µM PACOCF3 (p > 0.05), as compared to vehicle. The activity of iPLA2 in post-mortem CA1 region was significantly reduced by 17% by injections of 10 µM BEL (p = 0.002), but was not significantly reduced by injections of 1 µM BEL (p > 0.2), as compared to vehicle (Figure 7) (Schaeffer and Gattaz, 2005). As inhibition of hippocampal PLA2 activity impaired memory acquisition of inhibitory avoidance task, we carried out biochemical assays to evaluate directly whether the inhibitory avoidance is accompanied by changes in hippocampal PLA2 activity around the time of training. Nonimplanted male Wistar rats (2.5-3 months old) were divided into three groups: (a) naïve controls (n = 8), killed by decapitation immediately after withdrawal from their home cages; (b) shocked animals (n = 8), placed directly over the electrified grid, given a 0.4

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Figure 6. Acute PLA2 inhibition in rat CA1 region impairs acquisition of short- and long-term memory. Step-down latencies (seconds) are given as median (interquartile interval). Injections of 1 µM MAFP (A) (n = 14), 10 µM PACOCF3 (B) (n = 11), or 1 µM BEL (C) (n = 11) did not significantly prevent the increments of latencies for STM and LTM as compared to vehicle (n = 15, 15, 16, respectively). Injections of 10 µM MAFP (A) (n = 17), 100 µM PACOCF3 (B) (n = 13), or 10 µM BEL (C) (n = 12) significantly prevented the increments of latencies for STM and LTM as compared to vehicle (*p < 0.05 and **p < 0.01).


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mA, 4.0 s scrambled foot-shock, and immediately killed by decapitation; and (c) animals trained in the step-down inhibitory avoidance task (n = 10) as described above, and killed by decapitation immediately after receiving the foot-shock. Brain tissue preparation and PLA2 assay were carried out as described above. In this experiment, PLA2 assay was prepared using Tris-HCl pH 8.5 and 1 µM CaCl2. We found that inhibitory avoidance training increased significantly PLA2 activity during acquisition (14%; p = 0.002, as compared to naïve values). Additionally, inhibitory avoidance training increased significantly PLA2 activity as compared to shocked values (13%; p = 0.003), indicating that increments in PLA2 activity were specifically caused by the inhibitory avoidance training. Shocked animals had similar values of PLA2 activity as naïve controls (p > 0.5) (Figure 8) (Schaeffer and Gattaz, 2005). 8

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Figure 7. Dose-dependent inhibition of PLA2 activity in rat CA1 region. PLA2 activity (pmol⋅mg protein⋅min– 1) is given as mean (± standard deviation). Injections of 1 µM MAFP (n = 6), 10 µM PACOCF3 (n = 9), or 1 µM BEL (n = 8) did not significantly reduce PLA2 activity as compared to vehicle (n = 8, 9, 8, respectively). Injections of 10 µM MAFP (n = 9), 100 µM PACOCF3 (n = 8), or 10 µM BEL (n = 8) significantly reduced PLA2 activity as compared to vehicle (**p < 0.01).

Because memory function is largely dependent on the fluidity of brain membranes (Hong, 1995; Müller et al., 1997; Clarke et al., 1999; Scheuer et al., 1999), we investigated the effects of in vivo PLA2 inhibition on the fluidity of hippocampal membranes from rats subjected to inhibitory avoidance training. At 30 min after injections into the CA1 region of vehicle or 100 µM PACOCF3 (n = 24 per group), the rats were placed on the platform in the inhibitory avoidance box; when stepping-down the animals received a 0.4 mA, 4.0 s scrambled foot-shock. Rats were killed after 2.5 min, which was the time found to produce significant AA release mediated by PLA2 activity after in vivo LTP induction (Clements et al., 1991). The CA1 region at the injection sites was bilaterally dissected, homogenized (pools of 3 rats) in 5 mM Tris-HCl (pH 7.4, 4°C), and aliquots for PLA2 activity determination were separated and frozen at –70°C. The rest of homogenates was additionally diluted in 5 mM Tris-HCl (pH 7.4, 4°C) and centrifuged at 48,000 g for 20 min at 4°C (2x). The final membrane pellet was resuspended in 5 mM Tris-HCl (pH 7.4, 4°C) and aliquots for membrane fluidity determination were frozen at –20°C. Membrane fluidity was determined

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Wagner F. Gattaz, Evelin F. Schaeffer and Orestes V. Forlenza


by anisotropy measurements. Briefly, we used the non-polar fluorescence probe 1,6diphenyl-1,3,5-hexatriene (DPH), which provides a measurement of anisotropy from the hydrophobic core of membranes. DPH anisotropy is inversely correlated with the fluidity of this membrane region. The assay samples (2 ml) containing 5 mM Tris-HCl (pH 7.4), 30 µg of protein from membrane suspensions, and 33 µM DPH were incubated for 45 min at 37°C. The steady-state anisotropy was measured in a Photon Technology International – PTI spectrofluorometer, using excitation and emission wavelengths of 360 nm and 450 nm, respectively. As expected, PACOCF3 significantly inhibited PLA2 activity in CA1 homogenates as compared to vehicle (p < 0.01; Figure 9A). Moreover, CA1 membranes from rats injected with PACOCF3 showed significantly reduced fluidity at the hydrophobic core, as indicated by increased DPH anisotropy, as compared to vehicle (p < 0.01; Figure 9B). Membrane fluidity was highly correlated with PLA2 activity in animals injected with PACOCF3 (r = 0.76, p < 0.03; Figure 10A) or with vehicle (r = 0.84, p < 0.01; Figure 10B) (Schaeffer et al., 2005). 6.0 5.5

**

5.0 4.5 4.0 3.5 naive

shocked

trained

Figure 8. Effect of inhibitory avoidance training on PLA2 activity in rat CA1 region. PLA2 activity (pmol⋅mg protein⋅min– 1) is given as mean (± standard deviation). Animals trained in inhibitory avoidance task (n = 10) showed significant increase in PLA2 activity around the time of training as compared to naïve and shocked animals (n = 8 per group). Shocked animals had similar values of PLA2 activity as naïve controls (**p < 0.01).

The findings described above show that the inhibition of PLA2 activity in the CA1 region of the dorsal hippocampus of rats impaired the acquisition of STM and LTM of step-down inhibitory avoidance task. It should be noticed that impairment of memory was only observed when the concentrations of the inhibitors were enough to cause a significant inhibition of PLA2 activity. Memory impairment was not due to effects of the inhibition of PLA2 activity on locomotion and exploratory behaviour during training performance. Moreover, memory impairment did not result from neuronal death in the CA1 region after inhibition of PLA2 activity, suggesting thus a functional effect of PLA2 on memory neurochemistry. Our data confirm the previous findings showing that inhibitors of PLA2 impaired learning in chicks, rats, and mice (Hölscher and Rose, 1994; Hölscher et al., 1995; Fujita et al., 2000), and extend them by demonstrating that injections of PLA2 inhibitors directly into the hippocampus impaired memory acquisition of rats. The diversity of PLA2 family and its


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varied regulatory and functional properties emphasize the importance of considering different groups of PLA2 when investigating their physiological and pathological roles. The present findings show that the selective inhibition of iPLA2 activity by BEL in hippocampal CA1 region of rats impaired the acquisition of STM and LTM of inhibitory avoidance task. This result confirms and extends the previous finding showing that intraperitoneal injections of BEL in mice impaired spatial learning tested in the Morris water maze (Fujita et al., 2000). In our study we optimized some assay conditions for PLA2 activity determination. During these assays we found a dominant activity of iPLA2 in the dorsal hippocampus of rats towards 1palmitoyl-2-[1-14C]arachidonyl-phosphatidylcholine. The remaining PLA2 activity, likely of cPLA2 because of the assay conditions used (micromolar Ca2+ concentration), was about 11fold lower than the iPLA2 activity (data not shown). This finding is in line with previous data showing low levels of mRNA of cPLA2 (Molloy et al., 1998; Kishimoto et al., 1999) and a dominant iPLA2 activity over cPLA2 activity in rat hippocampus (Yang et al., 1999). Despite the low levels of mRNA and low activity of cPLA2, there are some evidences for an involvement of cPLA2 in LTP induction in rat hippocampus (Bernard et al., 1994; Weichel et al., 1999). The development of selective cPLA2 inhibitors is required for a better understanding of the involvement of cPLA2 in memory processes.

5

A

4

**

3 2 1 0 vehicle

PACOCF3

0,220

B

**

Anisotropy (DPH)

0,215 0,210 0,205 0,200 0,195 0,190 ve hicle

PACOCF3

Figure 9. PLA2 activity and anisotropy measurements in post-mortem CA1 region of rats. PLA2 activity (pmol⋅mg protein⋅min–1) and DPH anisotropy are given as mean (± standard deviation). All determinations were done in triplicate. Injections of 100 µM PACOCF3 (n = 8) significantly decreased PLA2 activity (A) and reduced membrane fluidity at the hydrophobic core, as indicated by increased DPH anisotropy (B), as compared to vehicle (n = 8) (**p < 0.01).

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5

A

4

r = -.76

3 2 1 0 0,200

0,205

0,210

0,215

0,220

0,225

Anisotropy (DPH)

B


5,0

r = -.84 4,5

4,0

3,5

3,0 0,194

0,196

0,198

0,200

0,202

0,204

0,206

Anisotropy (DPH)

Figure 10. Correlation between PLA2 activity and anisotropy in post-mortem CA1 region of rats. PLA2 activity (pmol⋅mg protein⋅min–1) and DPH anisotropy are given as the mean value from triplicate determinations. PLA2 activity correlated negatively with anisotropy (= positively with fluidity) at the hydrophobic core in rats injected with PACOCF3 (A; r = -.76, p < 0.03) or with vehicle (B; r = -.84, p < 0.01).

With the current experimental design (i.e. pre-training injections) our findings suggest that PLA2 inhibition blocked memory acquisition. Because STM and LTM are acquired at the same moment, one could argue that blocking acquisition there is no point to analyze the effects of drugs on both STM and LTM. However, it has been clearly shown that STM and LTM for the inhibitory avoidance task are established through partially independent molecular pathways in the hippocampus and related brain areas (Izquierdo et al., 1998). A recent study evaluating possible differential effects of drug injections before acquisition and during the early consolidation period of STM and LTM of inhibitory avoidance supports the view that consolidations of STM and LTM for inhibitory avoidance are parallel, independent processes (Quevedo et al., 2004). Further studies are required to clarify whether acquisition of STM and LTM would involve different mechanisms.


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It is noteworthy that in our study PLA2 activity increased in the CA1 region of rat hippocampus immediately after inhibitory avoidance training. Such increase may be caused by NMDA-stimulated Ca2+ influx (Yoshihara and Watanabe, 1990; Bernard et al., 1994; Underwood et al., 1998) and by phosphorylation mediated by mitogen-activated protein kinase and protein kinase C (Lin et al., 1993; Nemenoff et al., 1993) if considering an involvement of cPLA2. In the case of iPLA2, increased activity may also be mediated by protein kinase C-directed phosphorylation (Underwood et al., 1998; Akiba et al., 1999). Activation of PLA2 may result in increased glutamate binding to AMPA (Bernard et al., 1994) and metabotropic receptors (Collins et al., 1995). All these biochemical mechanisms (NMDA, AMPA, and metabotropic receptors, MAPK, PKC) in the hippocampus participate in the early stages of memory formation of inhibitory avoidance task in rats (Bianchin et al., 1994; Bernabeu et al., 1995; Cammarota et al., 1995, 2000; Alonso et al., 2002). The findings from anisotropy measurements suggest that apart from PLA2 cleaving membrane phospholipids generating arachidonic acid (Farooqui et al., 1997) – an important mediator in synaptic plasticity underlying memory processing (Nishizaki et al., 1999; Fujita et al., 2001) – it also influences membrane fluidity at the hydrophobic core. The increment of hydrophobic core fluidity of rat hippocampal membranes was found to improve memory function (Müller et al., 1997; Scheuer et al., 1999). In our experiments we used crude preparations of hippocampal membranes that probably contains plasma membranes as well as intracellular membranes. The inhibition of cPLA2 and iPLA2 by PACOCF3 (Ackermann et al., 1995; Balsinde and Dennis, 1997) in our study may reduce fluidity of both plasma and intracellular membranes, since cPLA2 translocates to nuclear and endoplasmic reticulum membranes where it releases AA (Schievella et al., 1995; Gijón et al., 1999; Hirabayashi et al., 1999; Perisic et al., 1999), and iPLA2 was found to release AA from the plasma membrane (Mizuno-Kamiya et al., 2001). At the plasma membrane, the effect of increased membrane fluidity on memory improvement (Müller et al., 1997; Scheuer et al., 1999) may be mediated by increased densities of NMDA and muscarinic receptors (Muccioli et al., 1996; Scheuer et al., 1999), which are both involved in memory formation (Izquierdo et al., 1997). Additionally, an increment in nuclear membrane fluidity was found to increase mRNA nucleocytoplasmic transport, whereas a reduction in nuclear membrane fluidity was found to decrease the mRNA efflux (Tomassoni et al., 1999). The mRNA-mediated gene expression is crucially involved in memory formation (Igaz et al., 2002, 2004). Taken together with these data, the present results suggest that the mechanism by which reduced PLA2 activity impairs memory formation involves also changes in membrane fluidity.

Conclusion The results of our study showing that PLA2 inhibition impaired memory acquisition and reduced membrane fluidity are of interest in face of previous reports from our and other laboratories showing decreased PLA2 activity in post-mortem brain, including the hippocampus, of patients with AD (Gattaz et al., 1995; Ross et al., 1998; Talbot et al., 2000), as well as reduced membrane fluidity in post-mortem hippocampus of AD patients (Eckert et al., 2000). In face of these data, we speculate that reduced membrane fluidity mediated by

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decreased PLA2 activity may contribute to memory impairment in AD. Further studies should clarify whether the stimulation of PLA2 does improve memory performance in animals, which could open new avenues for the development of treatment strategies in AD.

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In: New Research on Alzheimer’s Disease Editor: Eileen M. Welsh, pp. 87-98

ISBN 1-59454-939-7 © 2006 Nova Science Publishers, Inc.

Chapter III

Low Molecular Weight Glycosaminoglycans and Apoptosis: Potential Treatment of Neurodegenerative Disorders Including Alzheimer’s Disease Bertalan Dudas Neuroendocrine Organization Laboratory (NEO), Lake Erie College of Osteopathic Medicine, Erie, PA 16509, USA.

Abstract Previous studies have shown that apoptosis (programmed cell death) plays a crucial role in the pathomechanism of neurodegenerative disorders. It is a general consensus that apoptosis can be triggered by dysregulation of nerve growth factor physiology and/or oxidative stress processes. Since both of these pathological changes are characteristic to AD, it is conceivable that influencing apoptosis can be a valuable tool in the treatment of neurodegenerative disorders. Glucosaminoglycans (GAGs) exhibit neuroprotective properties in several animal models of AD. Low molecular weight GAG, C3, attenuates Aβ(25-35)-induced tau-2 immunoreactivity, and AF64A-induced cholinergic lesion in rat brain. Recent studies have also revealed that C3 influences p75 nerve growth factor receptor expression. However, the exact mechanism of the neuroprotective attributes of C3 is not elucidated yet. Since apoptosis is believed to play a pivotal role in numerous neurodegenerative disorders, C3 may induce neuroprotection/neurorepair via affecting apoptotic processes. The neuroprotective effect of glucosaminoglycans including C3 thus may involve multiple mechanisms, including an influence on processes that lead to programmed cell death. Since apoptosis is a crucial event not only in neurodegenerative diseases but also

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Pathology of Alzheimer’s Disease: Introductory Comments Alzheimer’s disease (AD) is a progressive and irreversible brain disorder that is manifested in dementia, motor lesions and behavioral deficits. Since as many as 10% of the population above 65 years of age is affected by AD in the United States, AD is becoming a growing concern in health care. The unequivocal diagnosis of AD is based on histopathological evidence at brain autopsy or biopsy. The major pathological hallmarks of AD include abnormal biochemical alteration of regular proteins that leads to accumulation of the modified molecules in the brain. Intracellular deposition of hyperphosphorylated, polymerized tau protein, forming flame-like tangles disrupts axonal and dendritic transport by destroying the normal microtubular structure. Pathological cleavage of the amyloid precursor protein (APP) normally present in the brain results in beta amyloid fragments that are aggregated to form extracellular build-up of amyloid plaques. These histological hallmarks are associated with extensive loss of cholinergic neurons, which is believed to be the major cause of symptoms in AD. However, the exact patho-mechanism leading to neuronal death is not yet elucidated.

Apoptosis in Neurodegenerative Disorders Apoptosis (programmed cell death) is a natural process that ensures that damaged cells are packaged and removed by the surrounding cells cleanly, in order to prevent inflammation typical in necrotic processes. Apoptosis (Figure 1) is typically triggered by external stimuli (extrinsic pathway) and internal events (intrinsic pathway). The extrinsic pathway is initiated outside of the cell, when conditions in the extracellular environment determine that a cell must die. Extrinsic pathway is triggered by binding ligands to specific "death receptors" on the surface of the cells, leading to activation of cysteinyl proteases that are called initiator caspases. The intrinsic apoptosis pathway begins when an injury occurs within the cell. Mitochondrial damage, for example, can initiate the intrinsic pathway, when cytochrome-c, released from damaged mitochondria, activates an initiator caspase. The extrinsic and intrinsic pathways merge at caspase-3 that is activated by initiator caspases. Subsequent processes result in multiple events, including degradation of DNA, and packaging of the cell into small units that are easily taken up by neighboring cells. Several studies indicated that apoptosis is one of the crucial factors that may be responsible for neuronal loss, characteristic for neurodegeneratory disorders including AD. Caspases are believed to be involved in generation of toxic fragments of protein substrates, that can trigger the earlier stages of neuronal dysfunction including protein aggregation in Huntington's and Alzheimer's disease [1]. Thus, caspase inhibition may be a valuable adjunct

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in the treatment of neurodegenerative disorders. Previous findings also demonstrated dysregulation of apoptotic proteins in AD brain, that may play a crucial role in the neuropathology of AD [2,3]. Since the level of cytochrome c has been reported to be unchanged in the frontal cortex of patients suffering from AD [3], apoptosis may occur via the extrinsic death receptor pathway independent of cytochrome c. However, it is generally believed that multiple factors are responsible for the neuronal cell death in AD. For example, neurofibrillary tangles and amyloid plaques are not associated with caspase-3 induction, in contrast to hippocampal neurons, that exhibit vacuolar degeneration and caspase-3 activation [4]. Thus, caspase-3 may not have a significant role in the widespread neuronal apoptosis that occurs in AD, but may contribute to the specific loss of hippocampal neurons involved in learning and memory.

Figure 1. Major processes of the apoptotic pathway. Apoptosis is a natural process that ensures that damaged cells are packaged and removed by the surrounding cells thoroughly, in order to prevent inflammation typical in necrotic processes. Apoptosis is triggered by external stimuli (extrinsic pathway) and internal events (intrinsic pathway). The extrinsic pathway is activated by binding ligands (DL) to specific "death receptors" (DR) on the surface of the cells, leading to activation of initiator caspases of the external pathway (EIC). The intrinsic apoptosis pathway begins with an intracellular lesion that is typically mitochondrial damage, when cytochrome-c (Cc), released from damaged mitochondria, activates initiator caspases of the internal pathway (IIC). The extrinsic and intrinsic pathways merge at caspase-3 (CA3) that is activated by initiator caspases (EIC and IIC). Subsequent processes result in multiple events, including degradation of DNA in the nucleus, and packaging of the cell into small units that are easily taken up by neighboring cells.

It is a common consensus that there are two major pathways playing roles in triggering apoptosis in neurodegenerative disorders: oxidative stress and trophic factor dysregulation.

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Oxidative Stress in Alzheimer’s Disease Neuronal cell loss is believed to be responsible for the majority of the clinical symptoms of AD including dementia, motor lesions and behavioral deficits. One of the most important pathways known to trigger apoptosis in the brain is oxidative stress. Oxidative stress refers to the situation of a serious imbalance between production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), and antioxidant defense. ROS/RNS cause cell damage and neuronal death through lipid peroxidation (LPO), oxidation of nucleic acids, and oxidation and nitrosylation of proteins and aminoacids. It is generally thought that the amyloid deposit (i.e. the senile plaque), which accumulates in the brain of Alzheimer’s disease (AD) patients, is the cause of the neurodegenerative changes characteristic of AD, and that oxidative stress induced by amyloid beta peptide (Aβ) is a prominent feature of amyloid-induced neuronal cell death in the AD brain. Indeed, post-mortem studies have shown that 4-hydroxy-nonenal (4-HNE), a lipid peroxidation (LPO) end–product accumulates in the AD brain [5]. Moreover, a recent study has shown that iPF(2α)–IV, another marker of LPO (i.e. oxidative stress) was increased in the urine of patients with mild to moderate dementia and probable AD [6]. Studies have also demonstrated that Aβ can directly cause increases in reactive oxygen species (ROS) and produce 4-HNE [7]. These findings indicate that Aβ-induced oxidative stress is a key feature in AD. The contention that AD pathology is associated with increased levels of oxidative stress has also been confirmed in an animal model of Alzheimer amyloidosis [8]. Levels of urinary and plasma iPF(2α)–IV were higher in APP-transgenic mice than in littermate wild type animals as early as eight months of age, and remained high for the rest of the study. Furthermore, homogenates from the cerebral cortex and the hippocampus of APP-mice had higher levels of iPF(2α)–IV, and this increase in LPO preceded amyloid plaque formation. That increased oxidative damage may, indeed, be the earliest event in AD is suggested by findings showing that increases in 8-hydroxyguanosine, an oxidized nucleoside derived from DNA, and nitrotyrosine, an oxidized amino acid, were quantitatively greatest early in the disease [9]. Data confirming that oxidative stress-induced damage precedes the deposition of amyloid in the brain also come from a study of Down’s syndrome brains [10]. Oxidized nucleic acid, 8-hydroxy guanosine (8-OHG), oxidized protein and nitrotyrosine were found to be increased significantly in the teens and in the twenties, while Aβ deposits only occurred after age 30. These findings suggest that brain oxidative damage must contribute to AD pathogenesis before Aβ (i.e. senile plaques) accumulates in the AD brain. Several risk factors may facilitate oxidative stress in the AD brain. One study has showed reduced antioxidant enzyme activity in AD brain [11], and brains of transgenic mice expressing different Alzheimer-linked presenilin-1 gene (PS1) mutations have been shown to have decreased antioxidant enzyme activity [12]. Another risk factor that may facilitate Aβinduced oxidative brain damage is the epsilon4 allele of apolipoprotein E (APOE), an established risk factor for AD. Levels of LPO were found to be significantly increased in tissues from Alzheimer’s cases which were homozygous for the epsilon4 allele of APOE, compared to AD epsilon3/epsilon3 cases and controls [13], suggesting that oxidative stressinduced injury in the frontal cortex of AD cases is related to the APOE genotype.


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Activated astrocytes (and microglia) are observed in a wide range of CNS pathologies, including brain inflammation, trauma, ischemia, and stroke; neurodegenerative diseases including AD, and normal aging [14,15]. In AD, activated microglia and astrocytes expressing the inducible form of nitric oxide synthase (iNOS) are found in the amyloid plaques surrounded by dead and dystrophic neurites [16,17], and a growing body of evidence suggests that these activated glia contribute to neurotoxicity through the production of reactive nitrogen species (RNS) derived from increased production of iNOS. Moreover, aggregated Aβ peptide can also stimulate iNOS production in rat glial cell cultures [18]. The concentration of 3-nitrotyrosine, a marker of oxidative damage mediated by peroxynitrite, and the 3-nitrotyrosine/tyrosine ratio was also increased six-fold in AD patients when compared with cerebrospinal fluid samples from age matched controls, and increased significantly with decreasing cognitive function [19]. Strong nitrotyrosine (NT) immunoreactivity was also observed in the absence of tangle formation in most terminal deoxynucleotidyl transferase (TdT)-positive neurones in the visual cortex of AD patients, suggesting that neuronal DNA damage may precede tangle formation and is associated with up-regulation of nitrotyrosine in AD brain [20]. These studies indicate that increased oxidative damage must contribute to the pathogenesis of AD before amyloid deposits occur in the brain. Thus, oxidative stress and increased production of iNOS are early events that appear to be central in determining cell death in AD.

Neurotrophic Factors in Alzheimer’s Disease Since nerve growth factor (NGF) induces neuronal growth and regeneration [21-25], it is generally accepted that NGF may play a pivotal role in the repair processes of neurodegenerative disorders [26] and may be a crucial element of the neuronal loss in AD[27-30]. Several studies revealed that deficiency of NGF may directly trigger apoptosis. Neuronal PC12 cells underwent apoptosis within two days following withdrawal of nerve growth factor (NGF) from the culture medium. [31]. Anti-NGF transgenic mice develop neurodegeneratory lesions characteristic for AD including amyloid plaques and neurofibrillary tangles in cortical and hippocampal neurons associated with extensive neuronal loss [32]. Although NGF is commonly accepted as a major factor promoting neuronal regeneration and survival, recent findings indicated that NGF may also play a role in triggering apoptosis. The ambivalent nature of NGF may be explained by the physiology of the NGF receptors, the tropomyosin receptor kinase A (TrkA) and the 75 kDa NGF receptor (p75NTR). TrkA induces survival and regeneration, whereas p75NTR appears to be a death receptor mediating the apoptotic effect of NGF. Indeed, NGF induced apoptosis in p75NTR-expressing human neuroblastoma SK-N-MC cells, but neurotrophin-3 (NT-3) or brain-derived neurotrophic factor (BDNF) lacked apoptotic effect [33]. Moreover, betaamyloid peptide, commonly present in amyloid plaques in AD, specifically binds the p75NTR in rat cortical neurons, NIH-3T3 cell line and neuronal hybrid cells, and triggers apoptosis [34-36] indicating that neuronal death in Alzheimer's disease may be mediated by the interaction of beta-amyloid with p75NTR. Since p75NTR is up-regulated in

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neurodegeneratory disorders, p75NTR might be involved in neuronal cell death related to AD [33].

Alzheimer’s Disease and Glycosaminoglycans There is a growing body of evidence that glycosaminoglycans (GAGs) play a pivotal role in the pathogenesis of Alzheimer’s disease. Glycosaminoglycans are highly sulfated polysaccharides that are common constituents of the extracellular matrix. GAGs bind to Aβ, and attenuate the proteoglycan-mediated protection of Aβ against proteolysis. It has been also shown that GAGs inhibit the aggregation and toxicity of Aβ itself [37]. AteroidR, a mixture of low molecular weight GAGs recently used in Europe to reverse the symptoms of AD, improves age-related behavioral deficits in rats. Moreover, human studies revealed that AteroidR significantly improves dementia [38,39]. C3 is a novel low molecular weight GAG exerting neuroprotective property. C3 is synthesized from unfractionated heparin using a standardized process of γ-irradiation. The chemical and biological properties of C3 have been well characterized including its ability to protect against various lesions in rat animal models. Stereotaxic injection of Aβ fragments (Aβ[25-35]) into the amygdala increases tau-2 immunoreactivity in the rat hippocampus [40]. This phenomenon was blocked by administration of C3 [41]. Moreover, C3 attenuated the cholinergic lesions in the septum and cingulum bundle induced by intraventricular administration of a specific cholinotoxin, ethylcholine aziridinium (AF64A; [42,43]. It has been also shown that C3 increase axonal growth and arborization in the rat hippocampus [44]. Recent studies suggested that GAGs may act via modulating the release of nerve growth factor (NGF) by stimulating the effect of growth factors in cell culture [45,46] and/or reducing the expression of cholinotoxin-stimulated growth factor receptor expression in the rat septum [47]. Glial cell line-derived neurotrophic factor (GDNF), that is vital to the development and maintenance of neural tissues, requires heparan sulphate glycosaminoglycan to promote neuronal survival [48]. However, the exact mechanism of the neuroprotective/neurorepair effect of GAGs is not known. The bioavailability of various GAGs depends on their potential to traverse the blood bran barrier (BBB). Since C3 has been demonstrated to cross the BBB, it may be a valuable adjunct in the treatment of neurodegenerative disorders including AD. Indeed, C3 is in phase I clinical trials at the time of this writing.

Glycosaminoglycans and Apoptosis: Potential Treatment of Alzheimer’s Disease Previous studies revealed that GAGs express neuroprotective properties against various lesions in cell culture [49] and in vivo [41-43,50]. Since neural damage is often associated with apoptotic processes, it is possible that GAGs may protect against neural lesions via


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influencing stressor-induced apoptosis. Moreover, C3, the low molecular GAG derived from heparin has been shown to penetrate the blood-brain-barrier [51], thus, it is conceivable that C3 and similar molecules can be used as therapeutic agents to protect against neuronal apoptotic processes, that are major hallmarks of neurodegenerative disorders. It is a common premise that GAGs play a pivotal role in the molecular processes of programmed cell death in several cell types. However, the data are ambiguous. Numerous studies indicate that GAGs may participate in the inhibition of apoptotic pathways. Hyaluronan, a major glycosaminoglycan component of the extracellular matrix of the mammalian bone marrow, antagonizes dexamethasone-induced apoptosis of malignant multiplex myeloma (MM) cells [52]. Since GAGs are major constituents of the bone marrow, these findings support the idea that GAGs could play a major role in the survival of MM cells in vivo, and could explain why MM cells accumulate in the bone marrow of patients with MM and escape conventional chemotherapy. Apoptosis in fibroblasts was also attenuated by administration of chondroitin sulfate, and heparan sulfate. Platelet-derived growth factor (PDGF) and beta-D-xyloside treatment protected fetal lung fibroblasts against serum starvation-induced apoptosis probably by stimulating the GAG synthesis/secretion of the cells. Indeed, application of sulfated GAGs, chondroitin sulfate, and heparan sulfate, also resulted in diminished apoptosis in fetal lung fibroblasts, possibly via increasing Bclassociated death promoter phosphorylation and diminished caspase-3 and caspase-7 cleavage [53] Heparin inhibits mesangial cell apoptosis induced by staurosporine, pyrrolidine dithiocarbamate, and ultraviolet light, and also suppresses oxidant-induced apoptosis of NRK49F fibroblasts and Madin-Darby canine kidney epithelial cells. Furthermore, heparin attenuated spontaneous apoptosis of podocytes in explanted glomeruli. These results indicate the novel potential of heparin as an inhibitor of apoptosis in several cell types, including glomerular cells [54]. Moreover, heparin and aspirin has been shown to inhibit trophoblast apoptosis providing an additional mechanism to explain the clinical benefits of heparin and aspirin on recurrent pregnancy loss [55]. In contrast to these findings, induction of apoptosis by GAGs has been reported by several studies, suggesting that GAGs may be used for eliminating tumor cells. Heparin exerts a significant anti-proliferative and an apoptotic effect on human hepatoma cells in vitro [56], and induces apoptosis of human nasopharyngeal carcinoma CNE2 cells [57]. Internalized heparin induces apoptotic cell death probably by interfering with transcription factor function [58]. Derivatives of heparin and chondroitin sulphate substantially reduce cell viability by induction of apoptosis of myeloma and breast cancer cells in vitro [59]. Heparin also induces apoptosis in peripheral blood neutrophils. This phenomenon may help explain the anti-inflammatory effects resulting from the interaction between vessel wall heparan sulphate and chemoattracted peripheral blood neutrophils [60]. Moreover, heparin is not the only GAG exhibiting apoptotic features. A low molecular weight proteoglycan, lumican induced and/or increased the apoptosis of B16F1 cells, probably via the sulphated GAG component of the molecule [61]. Chondroitin sulphate also induces apoptosis of articular chondrocytes [62].

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Discussion and Summary Apoptosis is believed to play a pivotal role in the pathogenesis of neurodegenerative disorders including AD. Although the exact function of apoptotic processes in brain pathology is not known, it is a common consensus that two major pathways are responsible in triggering apoptosis in neurodegenerative disorders: (1) oxidative stress and (2) trophic factor dysregulation. Oxidative stress appears to be the major pathological hallmark of AD. Moreover, dysregulation in the release of nerve growth factors, and expression of their receptors are characteristic pathological changes attributed to AD. GAGs are known to exhibit neuroprotective properties in numerous animal models of AD. One possible explanation of the GAG-induced neuroprotection is that GAGs may affect apoptotic processes characteristic in neurodegenerative lesions. The pathomechanism of this phenomenon is not entirely understood; GAGs may affect apoptosis either directly, or via modulating growth factor pathology and/or oxidative stress, that are major hallmarks of AD. Thus, GAGs can be extremely veluable adjunct in the treatment of neurodegenerative disorders. Since a low molecular GAG, C3, is able to cross the blood-brain-barrier, C3 treatment may represent an entirely novel avenue in the therapy of AD. Indeed, C3 has successfully passed the toxicology tests as a low-risk compound, and it is currently in phase I clinical trials.

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[37] Kisilevsky R, Lemieux LJ, Fraser PE, Kong X, Hultin PG, Szarek WA. Arresting Amyloidosis In Vivo Using Small-Molecule Anionic Sulphonates Or Sulphates: Implications For Alzheimer's Disease Nat Med 1995; 1: 143-148. [38] Conti L, Re F, Lazzerini F, Morey LC, Ban TA, Santini V, Modafferi A, Postiglione A. Glycosaminoglycan Polysulfate (Ateroid) In Old-Age Dementias: Effects Upon Depressive Symptomatology In Geriatric Patients Prog Neuropsychopharmacol Biol Psychiatry 1989; 13: 977-981. [39] Conti L, Placidi GF, Cassano GB. Ateroid In The Treatment Of Dementia: Results Of A Clinical Trial Mod Probl Pharmacopsychiatry 1989; 23: 76-84. [40] Sigurdsson EM, Lorens SA, Hejna MJ, Dong XW, Lee JM. Local And Distant Histopathological Effects Of Unilateral Amyloid-Beta 25-35 Injections Into The Amygdala Of Young F344 Rats Neurobiol Aging 1996; 17: 893-901. [41] Dudas B, Cornelli U, Lee JM, Hejna MJ, Walzer M, Lorens SA, Mervis RF, Fareed J, Hanin I. Oral And Subcutaneous Administration Of The Glycosaminoglycan C3 Attenuates Abeta(25-35)-Induced Abnormal Tau Protein Immunoreactivity In Rat Brain Neurobiol Aging 2002; 23: 97-104. [42] Rose M, Dudas B, Cornelli U, Hanin I. Protective Effect Of The Heparin-Derived Oligosaccharide C3, On AF64A-Induced Cholinergic Lesion In Rats Neurobiol Aging 2003; 24: 481-490. [43] Rose M, Dudas B, Cornelli U, Hanin I. Glycosaminoglycan C3 Protects Against AF64A-Induced Cholinotoxicity In A Dose-Dependent And Time-Dependent Manner Brain Res 2004; 1015: 96-102. [44] Mervis RF, Mckean J, Zats S, Gum A, Reinhard R, Dudas B, Cornelli U, Lee JM, Lorens SA, Fareed J, Hanin I. Neurotrophic Effects Of The Glycosaminoglycan C3 On Dendritic Arborization And Spines In The Adult Rat Hippocampus: A Quantitative Golgi Study CNS Drug Reviews 2000; 44-46. [45] Damon DH, D'Amore PA, Wagner JA. Sulfated Glycosaminoglycans Modify Growth Factor-Induced Neurite Outgrowth In PC12 Cells J Cell Physiol 1988; 135: 293-300. [46] Lesma E, Di Giulio AM, Ferro L, Prino G, Gorio A. Glycosaminoglycans In Nerve Injury: 1. Low Doses Of Glycosaminoglycans Promote Neurite Formation J Neurosci Res 1996; 46: 565-571. [47] Dudas B, Rose M, Cornelli U, Hanin I. Low Molecular Weight Glycosaminoglycan C3 Attenuates AF64A-Stimulated, Low-Affinity Nerve Growth Factor ReceptorImmunoreactive Axonal Varicosities In The Rat Septum Brain Res 2005; 1033: 34-40. [48] Barnett MW, Fisher CE, Perona-Wright G, Davies JA. Signalling By Glial Cell LineDerived Neurotrophic Factor (GDNF) Requires Heparan Sulphate Glycosaminoglycan J Cell Sci 2002; 115: 4495-4503. [49] Kisilevsky R, Lemieux LJ, Fraser PE, Kong X, Hultin PG, Szarek WA. Arresting Amyloidosis In Vivo Using Small-Molecule Anionic Sulphonates Or Sulphates: Implications For Alzheimer's Disease Nat Med 1995; 1: 143-148. [50] Sigurdsson EM, Lorens SA, Hejna MJ, Dong XW, Lee JM. Local And Distant Histopathological Effects Of Unilateral Amyloid-Beta 25-35 Injections Into The Amygdala Of Young F344 Rats Neurobiol Aging 1996; 17: 893-901.

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[51] Ma Q, Dudas B, Hejna M, Cornelli U, Lee JM, Lorens S, Mervis R, Hanin I, Fareed J. The Blood-Brain Barrier Accessibility Of A Heparin-Derived Oligosaccharides C3 Thromb Res 2002; 105: 447-453. [52] Vincent T, Molina L, Espert L, Mechti N. Hyaluronan, A Major Non-Protein Glycosaminoglycan Component Of The Extracellular Matrix In Human Bone Marrow, Mediates Dexamethasone Resistance In Multiple Myeloma Br J Haematol 2003; 121: 259-269. [53] Cartel NJ, Post M. Abrogation Of Apoptosis Through PDGF-BB-Induced Sulfated Glycosaminoglycan Synthesis And Secretion Am J Physiol Lung Cell Mol Physiol 2005; 288: L285-L293. [54] Ishikawa Y, Kitamura M. Inhibition Of Glomerular Cell Apoptosis By Heparin Kidney Int 1999; 56: 954-963. [55] Bose P, Black S, Kadyrov M, Weissenborn U, Neulen J, Regan L, Huppertz B. Heparin And Aspirin Attenuate Placental Apoptosis In Vitro: Implications For Early Pregnancy Failure Am J Obstet Gynecol 2005; 192: 23-30. [56] Karti SS, Ovali E, Ozgur O, Yilmaz M, Sonmez M, Ratip S, Ozdemir F. Induction Of Apoptosis And Inhibition Of Growth Of Human Hepatoma Hepg2 Cells By Heparin Hepatogastroenterology 2003; 50: 1864-1866. [57] Li HL, Ye KH, Zhang HW, Luo YR, Ren XD, Xiong AH, Situ R. Effect Of Heparin On Apoptosis In Human Nasopharyngeal Carcinoma CNE2 Cells Cell Res 2001; 11: 311-315. [58] Berry D, Lynn DM, Sasisekharan R, Langer R. Poly(Beta-Amino Ester)S Promote Cellular Uptake Of Heparin And Cancer Cell Death Chem Biol 2004; 11: 487-498. [59] Pumphrey CY, Theus AM, Li S, Parrish RS, Sanderson RD. Neoglycans, Carbodiimide-Modified Glycosaminoglycans: A New Class Of Anticancer Agents That Inhibit Cancer Cell Proliferation And Induce Apoptosis Cancer Res 2002; 62: 37223728. [60] Manaster J, Chezar J, Shurtz-Swirski R, Shapiro G, Tendler Y, Kristal B, Shasha SM, Sela S. Heparin Induces Apoptosis In Human Peripheral Blood Neutrophils Br J Haematol 1996; 94: 48-52. [61] Vuillermoz B, Khoruzhenko A, D'Onofrio MF, Ramont L, Venteo L, Perreau C, Antonicelli F, Maquart FX, Wegrowski Y. The Small Leucine-Rich Proteoglycan Lumican Inhibits Melanoma Progression Exp Cell Res 2004; 296: 294-306. [62] Bali JP, Cousse H, Neuzil E. Biochemical Basis Of The Pharmacologic Action Of Chondroitin Sulfates On The Osteoarticular System Semin Arthritis Rheum 2001; 31: 58-68.

In: New Research on Alzheimer’s Disease Editor: Eileen M. Welsh, pp. 99-123


Chapter IV

Low Density Lipoprotein ReceptorRelated Protein 1 (LRP1): One of the Hallmarks ofAlzheimer’s Disease and Other Complex Degenerative Processes Christiane Gläser(1,*), Gerd Birkenmeier(2), Susanne Schulz(1,3) and Klaus Huse(4) (1)

Institute of Human Genetics and Medical Biology, University of Halle, Halle, Germany (2) Institute of Biochemistry, University of Leipzig, Leipzig, Germany (3) Department of Medicine III, University of Halle, Halle, Germany (4) Leibniz Institute for Age Research - Fritz Lipmann Institute, Jena, Germany

Abstract Susceptibility to Alzheimer´s disease (AD) is governed by multiple factors. Remarkably, LDL receptor-related proteins (LRPs) and several of their numerous ligands were associated to AD. Alpha2-macroglobulin (A2M), apolipoprotein E (Apo E), but also amyloid precursor protein (APP) belong to the functionally and genetically interesting molecules involved in the complex degenerative processes resulting in AD. LRP1 (CD91) (MIM 107770) is a 4,544-amino acid protein containing a single transmembrane segment and is located on chromosome 12q13.1-12q13.3 within a region shown to be linked to AD. It consists of 84 exons and belongs to the largest human proteins and is highly evolutionary conserved. It could be demonstrated that several genomic variants of LRP1 were associated to AD but also to other degenerative diseases like atherosclerosis and cancer suggesting a complex role in fundamental cellular processes.

*

Correspondence: Dr. Christiane Gläser, Institute of Human Genetics and Medical Biology, Magdeburger Str. 2, D-06097 Halle, Phone +49-345-557 4296, Fax: +49-345-557 4293 e-mail: [email protected]

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Christiane Gläser, Gerd Birkenmeier, Susanne Schulz and Klaus Huse LRP1 functions as a clearance receptor for several ligands, including amyloid ß (Aß), and mediates signaling pathways affecting the processes of neurite outgrowth and calcium influx. While clearance of Aß diminishes the extracellular Aß-load the interaction with APP may enhance formation of Aß. LRP1 is expressed in brain, blood vessels as well as in endothelial cells of the brain-blood-barrier mediating the transcytosis of Aß between blood circulation and brain. Internalization via LRP1 concerns also extracellular proteases and protease-inhibitor complexes involved in Aß degradation. In AD as well as in other diseases genetic variations and structure-caused failure of LRP1 leading to dysfunctions as well as alteration in the expression of LRP1 ligands may determine common degenerative processes. Age-related changes in the level of LRP1 and its ligands may increase the risk for degenerative diseases. The modulation of LRP1 and its ligands by cytokines and growth factors was detected in and outside the brain. There are indications that also inflammatory processes play an important role in the development of degenerative diseases.

Abbreviations AD Aß α1-PI BACE1 CAD C. elegans CNS D. melanogaster EGF IDE IL LPS MMP NCBI NEP NPxY PAI-1 PDGF RAP u-PA t-PA TFPI HLA-DR

Alzheimer`s Disease amyloid ß protease inhibitor-1 (PI) ß site amyloid beta A4 precursor protein-cleavage enzyme coronary artery disease Caenorhabditis elegans central nervous system Drosophila melanogaster epidermal growth factor insulin-degrading enzyme interleukin lipopolysaccharide matrix metalloprotease National Center for Biotechnology Information neprilysin, membrane metalloendopeptidase (MME) amino acid motif asparagine-proline-x-tyrosine plasminogen activator-inhibitor-1 (PAI1) platelet-derived growth factor low density lipoprotein receptor-related protein-associated protein 1 (LRPAP1) plasminogen activator urinary (PLAU) plasminogen activator tissue (PLAT) tissue factor pathway inhibitor human leucocyte antigens-DR locus

Low Density Lipoprotein Receptor-Related Protein 1 (LRP1)

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Common Features of Alzheimer´s Disease To date, the following four main risk markers have been characterized and related to Alzheimer´s disease (AD): amyloid precursor protein (APP, chromosome 21), apolipoprotein E (Apo E, chromosome 19), presenilin-1 (PSEN1, chromosome 14) and presenilin-2 (PSEN2, chromosome 1). It has been suggested that other genetic loci mapped to chromosome 10, 12, and 20, - including the polymorphic genetic variants of alpha-2 macroglobulin (A2M, chromosome 12) and its receptor the low density lipoprotein receptor-related protein (LRP1, chromosome 12), as well as to the mitochondrial genome, contribute to neurodegenerative processes (for review see Tanzi and Bertram, 2005).

Amyloid Precursor Protein The widely expressed cell surface protein, amyloid precursor protein (APP), in particular its proteolytic fragment amyloid ß (Aß) produced by γ-secretase cleavage, has been identified as playing a role in the formation of the plaque core in AD patients and also in older patients with Down syndrome (Masters et al., 1985). Investigations have described that different APP isoforms (Sandbrink et al., 1994) are created by alternative splicing (Yoshikai et al., 1990). Moreover, several mutations within the APP gene were found in familial AD, which denotes APP as being a significant candidate gene. Under physiological conditions clearance and degradation of soluble APP and Aß is mediated by an efficient “cargo” system, including LRP1 and its ligand A2M (Kounnas et al., 1995, Narita et al., 1997). And von Arnim et al. (2005) could demonstrate that LRP1 functions as a novel ß-secretase (BACE1) substrate.

Apolipoprotein E Apo E is an integral part of several lipoproteins involved in the lipid metabolism (Mahley 1988) and it is inherited in three common alleles, ε2, ε3, ε4 (Strittmatter et al., 1993). The Apo E ε4 allele is strongly associated with an increased amyloid burden in AD while Apo E ε2 considerably decreases the risk for AD (Hyman, 1996). The Apo E ε4 isoform was more frequently found in AD patients and was strongly related to a higher risk for late-onset familial AD and sporadic AD (Lendon et al., 1997; Pericak-Vance et al., 1997; Craig et al., 2004).

Presenilin 1 and 2 PSEN1 and PSEN2 are different in gene structure but display similar expression patterns in tissues and intracellular localization (Kovacs et al. 1996). PSEN1 and PSEN2 represent the fundamental features of a γ-secretase (Li et al., 2000) which is responsible for the APP cleavage producing Aß. Mutations found in PSEN1 and PSEN2 that result in functional

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Christiane Gläser, Gerd Birkenmeier, Susanne Schulz and Klaus Huse

aberration are considered to be the most common cause of familial AD (Berezowska et al., 2005).

Putative Role of LRP1 in Alzheimer´s Disease The Lipoprotein Receptor Family The low-density lipoprotein receptors are comprised of a family of cell-surface receptors which recognize multiple, structurally diverse extracellular ligands and internalize them for degradation by lysosomes (Hussain, 2001; Howell and Herz, 2001, Gliemann, 1998). They are also considered to be recruiting receptors and are able to interact with other proteins for signalling, realized by their cytoplasmic domains. The low-density lipoprotein receptor LDLR is the prototype of this family, whose paralogous members in the human genome are listed in Table 1. Most of these genes also have their orthologs in other vertebrates. Members of the family are even found in D. melanogaster and C. elegans. As the results of several duplications, deletions and subsequent alterations the human proteins are evolutionary distant (Figure 1) and their genes are scattered throughout the human genome. All proteins contain four major structural modules: variable numbers (between two and eleven) of cysteine-rich complement-type repeats, EGF-like repeats, a small cytoplasmic domain, and a membranespanning domain. They serve very different functions in distinct physiological systems among them are lipoprotein metabolism, vitamin transport, virus infection, fibrinolysis, angiogenesis, protease clearance, and intercellular communication. Mutations in these different genes, therefore, cause very different phenotypes. Mutations in the LDLR, for instance, which is mainly responsible for the control of cholesterol metabolism, cause familial hypercholesterolemia and premature coronary artery disease. Several of these types of mutations have been described including missense, some that affect the splice site and expression-relevant ones in the LDLR promoter region. With respect to AD it is noteworthy to mention that alterations in lipoprotein and cholesterol metabolism have been linked to brain Aß levels. Kuo et al. (1998) described that there is a correlation between LDL cholesterol and total serum cholesterol as regards to the amounts of Aß 1-42 in the brains of patients with AD and also reported that receptors involved in cholesterol homeostasis and lipoprotein metabolism are putatively and pathogenetically relevant. LRP2 (megalin) is a major receptor in the proximal tubules absorptive epithelial cells and also functions as an antigenic determinant for Heymann nephritis in rats. It also binds cubilin, which is the receptor for the intrinsic factor complex in vitamin B12, thus providing an uptake mechanism for dietary vitamin B12. Furthermore, homozygous knockout mice manifested abnormalities in epithelial tissues e.g. lung and kidney and died perinatally from respiratory insufficiency (Willnow et al., 1996). This has been suspected to be caused by an insufficient cholesterol supply to the brain; these animals also showed impaired brain development. It has also been suggested that megalin mediates cellular uptake and transport of apolipoprotein J, which is able to bind Aß and mediates transport of this protein across the blood-brain barrier and the blood-cerebrospinal fluid barrier (Zlokovic et al., 1996). The functional importance of


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individual receptors may lie in their differential tissue expression. Most of the LRP family members are tissue-specific expressed and some of them are highly expressed in the brain. This is the case concerning LRP8 which is predominantly expressed in brain and is also an apoliprotein E receptor. Although this has not yet been confirmed by independent studies, one synonymous polymorphism in LRP8 has been identified showing a difference in its allele frequencies between AD patients and controls (Ma et al., 2002). The expression regulation of these receptors occurs at the transcriptional level in different ways, for instance, expression of LDLR is regulated by intracellular sterol concentration. However, other members of the family are not regulated by sterols. Equally notable is that all the receptors are regulated by many hormones and growth factors, but the mechanisms of regulation involving hormones are not fully understood. In this context the most frequent studied member of the LRP family is LRP1.

LRP3 LRP12 LRP10 LRP11 LDLR VLDLR LRP8 LRP1 LRP1B LRP2 LRP5 LRP6 SORL1

Figure 1. Evolutionary relationship of the human paralogous members of the LRP protein family. The phylogenetic tree was constructed using ClustalW (Thompson et al., 1994)

LRP1: A Multi-ligand Clearance Receptor The binding of more than 30 different ligands to LRP1 suggests that this receptor affects complex degenerative cellular processes which proceed the onset of AD, Parkinson disease, coronary artery disease or cancer (Hussain 2001; Myllykangas et al., 2000; Boucher at al., 2002; May et al., 2003; Strickland et al., 2003). All these degenerative diseases could be described as cellular dysregulations resulting in the pathogenic accumulation of diverse risk factors.

Table 1: Database summary of the members of the LRP family

Gene symbol

alternative symbols

LDLR

low density lipoprotein receptor precursor

VLDLR LRP1

LRP, A2MR, APR, APOER, CD91

LRP1B

LRP-DIT

LRP2

gp330, megalin

LRP3 LRP5

LR3, LRP7

LRP6 LRP8

APOER2

OMIM

Chromosomal location

mRNA RefSeq

protein RefSeq

606945

19p13.2

NM_000527

NP_000518

very low density lipoprotein receptor

192977

9p24

NM_003383

NP_003374

low density lipoprotein receptor-related protein 1

107770

12q13.1-q13.3

NM_002332

NP_002323

low density lipoprotein receptor-related protein 1B

608766

2q22.1-2q22.2

NM_018557

NP_061027


600073

2q31.1

NM_004525

NP_004516


603159

19q13.11

NM_002333

NP_002324


603506

11q13.2

NM_002335

NP_002326


603507

12p13.2

NM_002336*

NP_002327


602600

1p32.3

NM_004631

NP_004622 NP_054764

LRP10


14q11.2

NM_014045

LRP11


6q25.1

NM_032832

NP_116221

LRP12


8q22.3

NM_013437

NP_038465

11q23.3

NM_003105

NP_003096

SORL1 *

gene product

SORLA1, SORLA, LR11

the major mRNA isoforms only

sortilin-related receptor containing LDLR class

602005


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The domain structure of LRP1 has been described in detail (Herz et al., 1988, Brown et al., 1991; Gaeta et al., 1994). Obviously, its huge size (600 kD) enables the binding of diverse ligands such as proteins, peptides and drugs to take place independently and simultaneously. To carry out functional studies the first attempts were made to create LRP1 knock-out mice. (Herz et al., 1992). However, these experiments failed due to impaired embryonic implantations. Now, new approaches involving short interfering RNA (siRNA) techniques were used and hence a selective decline in LRP1 expression was achieved (Laatsch et al., 2004). It has been shown that a decrease in LRP1 levels significantly reduced LRP1-mediated ligand uptake and degradation (Li and Bu, 2003). As a result of the proposed "cargo" function, it is not surprising that LRP1 is expressed in almost all cells with regards to regulating ligand homeostasis and most importantly, on cells of the vasculature, which enables the transcytosis of ligands. LRP1 ligands fall into several functional categories: (i) Pericellular proteolysis (e.g. A2M; α1-PI, MMPs, u-PA, t-PA, PAI-1); (ii) Lipid metabolism (Apo E, Lipoprotein lipase); (iii) Blood coagulation (Factor IXa, Factor VIIIa, TFPI, Antithrombin III; Heparin cofactor); (iv) Inflammation/immune response (complement factors, chaperons, beta2-integrins) (v) Drugs (suramin, saponin) and (vi) Membrane or intracellular scaffold proteins (Fe65, Disabled, PDGF-receptor, G-proteins, APP). It is well known that the deposition of Aß is a critical pathological feature in AD. A recent study has suggested that improper clearance of Aß is as important as Aß production in amyloid deposition (Glabe, 2001). Moreover, there are several interfaces where LRP1 may determine the balance between Aß synthesis and clearance mechanism:

LRP1 and Pericellular Proteolysis Lowering Aß levels may be achieved by inhibiting its generation from APP or by promoting its clearance by transport or degradation and to date three major proteases have been identified in playing a role in degrading Aß. They are as follows: the neutral endopeptidase, neprilysin (NEP), is located on the cell surface which allows hydrolysis of peptides on the extracellular side including Aß 1-40 (Howell et al., 1995) and Aß 1-42 (Iwata et al., 2000). In vivo inhibition of neprilysin by specific inhibitors results in an accumulation of endogenous Aß (Iwata et al., 2000). It has been suggested that age-related decreases of the neprilysin could lead to increased brain concentrations of Aß, plaque formation, and AD (Newell et al., 2003). A second important protease is the insulin-degrading enzyme (IDE); a zinc-containing metalloprotease found in the cytosol, on the cell surface and in the extracellular space (Qiu et al., 1996; Qiu et al., 1998). It cleaves small proteins many of them share the property to form amyloid fibrils, e.g. Aß, insulin, glucagon and amylin (Farris et al., 2003). Both NEP and IDE mediate degradation of soluble Aß but have less ability to degrade Aß once it becomes insoluble or oligomeric (Selkoe, 2001). The only protease that is capable of degrading soluble and fibrilar Aß seems to be plasmin (Tucker et al., 2002). In brain, tPA, uPA and plasminogen are present in low concentrations. Upregulation of plasminogen activators by aggregated Aß indicates that the

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plasmin system is activated in response to Aß deposition. Thus, plasmin confers neuroprotection which may continue until upregulation of the plasmin system is exhausted during the course of the disease (Ledesma et al., 2000). These findings are supported by a recent report showing an association of a single nucleotide polymorphism in the uPA gene with elevated Aß 1-42/40 (Ertekin-Taner at al., 2005). There are indications of a general proteases deficiency in the brain and blood of AD patients (Aoyagi et al., 1992), which has been found to be similar in healthy but elderly patients (Aoyagi et al., 1994). Although, so far a direct link of LRP1 to regulation of NEP and IDE metabolism has not yet been established, it can be anticipated that the activities of both enzymes are regulated by protease inhibitors as it is the case with the pan-protease inhibitor A2M and plasmin (Tucker et al., 2000). The inhibitory action of A2M is mediated by a unique trapping mechanism (SottrupJensen, 1989). Proteases are sterically trapped but retain their activity toward small proteins and peptides. Accompanying conformation changes make A2M ready to accommodate such peptides and to bind to LRP1 for endocytosis (Ashcom et al., 1990). A2M-protease complexes have been shown to degrade bound Aß initiating extracellular amyloid depletion and to continue intracellular lysosomal Aß degradation following receptor-mediated uptake of A2M-protease-Aß complexes (Qiu et al., 1996). This process is very unique and seems to be a common mechanism to down-modulate pathologically elevated levels of bioactive peptides such as Aß or other growth factors (Birkenmeier, 2001). It has recently been shown that the administration of small proteases such as trypsin or chymotrypsin to human blood leads to degradation of radiolabeled Aß mediated by formation of A2M-protease-Aß complexes (Lauer et al., 2001). Up-regulation of Aß catabolism via proteases delivery could probably reduce the risk of developing AD by preventing Aß accumulation in the brain and in the vasculature. Thus, clearance of Aß strongly depends on the presence of extracellular proteases as well as the level of its vehicle, A2M. The plasma concentration of A2M showed significant individual variations ranging from approximately 50mg/100 ml to 500mg/100ml (Birkenmeier et al., 2003). The reason for this diversity is not yet known, but is assumed to be related to different promoter activity. Most interestingly, a steep decline of the A2M level was manifested at an age when late-onset AD starts to develop (Figure 2). Since AD develops slowly over years, a progressive exhaustion of proteases and A2M in combination with an altered modulation of LRP1 would have a tremendous impact on the clearance system of AD-related ligands. Thus, one can speculate that a decreased A2M level in blood is a risk factor for AD. Various attempts to associate the level of A2M to different polymorphisms in the A2M gene have failed and genetic linkage and association studies of polymorphisms with AD have all provided conflicting results (Saunders et al., 2003). However, it cannot be excluded that known variants in A2M might be linked to pathogenic mutations/polymorphism in the A2M gene or in its neighbourhood that have not yet been identified (Saunders and Tanzi, 2003).


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Total A2M in plasma (mg/100ml)

600 males females

500

400

300

200

100

0 0

20

40

60

80

100

Age (years)

Figure 2. Distribution of A2M level dependent on age and gender in a population of 400 healthy individuals. The concentration of total A2M was determined by ELISA using the test kits MacroNat (BioMac, Leipzig). Females (open circles), males (black circles) (with permission from Exp Neurol. 2003,184:153-61).

LRP1 and APP LRP1 interacts with APP and its expression facilitates APP processing via the amyloidogenic pathway (Kounnas et al., 1995). This is because receptor-mediated endocytosis brings APP into close proximity to the APP-cleaving enzyme, ß-secretase, which results in elevated generation and secretion of Aß (Ehehalt et al., 2003). Thus, dynamic interactions of APP with its clearance receptors determine the amyloid burden in the extracellular space. This is corroborated by recent results showing that expression of LRP1B, a homologue of LRP1, which also binds APP, produces less Aß probably due to the lower rate of endocytosis when compared to LRP1 (Cam et al., 2004). Similar effects were seen after treating cells with RAP, which blocks the binding of ligands to LRP1 (Ulery et al., 2000). Thus, the contribution of LRP1 to AD is complex because it influences both the production and the clearance of Aß. In addition to recognizing numerous extracellular ligands with the aid of the LRP1 ectodomain, the intracellular domain of LRP1 contains binding sites for many adaptor and scaffold proteins (Gotthardt et al., 2000). Phosphorylated NPxY motifs act as anchors for Fe65, Dab, Jip and Shc and are found in both the cytoplasmic tail of LRP1 as well as in APP. Tyrosine phosphorylation of LRP1 was found to result from interaction with the plateletderived growth factor BB which creates docking sites for Shc. By subsequent phosphorylation, different signalling pathways involving Ras and c-Myc are initiated (Loukinova et al., 2002).

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Similarities between LRP1 and APP also concern the proteolysis of the extra- and intracellular domain. Soluble APP ectodomains are obtained by the action of α- or ßsecretase, whereas the shedding of LRP1 is caused by an unknown metalloprotease (Quinn et al., 1999). Analogously to APP processing, the LRP1 cytoplasmic domain is cleaved by a γsecretase-like activity (May et al., 2002). The released proteolytic products may modulate signalling or may travel to other compartments of the cell.

LRP and ApoE It has been shown that ApoE binds to Aß and the isoform-specific effects of ApoE are related to this action with regards to fibrillogenesis and sequestration (Strittmatter et al., 1993; Sanan et al. 1994; Castano et al., 1995; Carter 2005). Cellular uptake of ApoE is mediated by LRP1, which also mediates the effect of ApoE on neurite outgrowth (Beisiegel et al., 1989; Nathan et al., 2002; Gylys et al. 2003).

LRP1 and Ligand Transcytosis at Blood-Brain-Barrier The expression of LRP1 in brain cells, vessel walls and in brain capillary endothelium has been well documented (Shibata et al., 2000). Late-onset AD patients, who do not have increased Aß production or APP overexpression, are likely to exhibit (i) a failure in Aß clearance from the brain to plasma, (ii) defects in Aß degradation in the CNS or (iii) an increased influx of circulating Aß into the CNS. Although the level of free Aß in extracellular brain fluid is higher than that found in plasma, the absolute amounts of Aß in body fluid exceeds the Aß level in brain fluid by factor 10. Thus, body fluids provide a large reservoir for accommodation and degradation of excessive Aß. Two main receptors are involved in shuttling Aß through the blood-brain barrier between both compartments: (i) AGER (advanced glycosylation end product-specific receptor) mediates influx of Aß into the brain (Mackic et al., 1998) and (ii) LRP1 mediates efflux of Aß into circulation (Shibata et al., 2000). In contrast to the brain, uptake and degradation of Aß in blood seems to be chaperoned by A2M or ApoE, two main ligands of LRP1.

LRP1 and Inflammation In AD and APP transgenic mouse models of AD LRP1 is down-regulated (Shibata et al., 2000). However, little information is available on factors that affect LRP1 expression in the brain. Considerable evidence which has been gained over the past decade has led scientists come to the conclusion that neuroinflammation is associated with AD. The main cellular players in inflammatory processes are microglia and the astrocytes (Akiyama et al., 2000). LRP1 is expressed in both cell types, but expression varies with regards to the state of the cellular activation (Hussaini et al., 1999).


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Microglial cells e.g. can be activated by Aß, LPS or other cytokines and they respond to the following: reactive oxidant species, expression of NFkappaB, inflammatory cytokines and HLA-DR (Streit, 2004). Expression of IL-1, α1-antichymotrypsin and complement factors in the AD brain is another indication of ongoing inflammatory processes which take place during the development of AD (Shuguang and Janciauskiene, 2002; Mrak and Griffin, 2001). LPS decreases expression of LRP1 in monocyte lineage cells (LaMarre et al., 1993) and interferon-gamma was found to up-regulate LRP1 and A2M in astrocytoma cell lines (Businaro et al., 1997). Furthermore, LRP1 is a receptor for chaperoned peptides trafficking immunogenic peptides into antigen presenting cells for presentation (Binder et al., 2000). Thus, inflammatory processes will most likely affect the function of LRP1 in the brain and in the blood-brain-barrier, as well. Recent data have suggested that reduced LRP1 expression is a contributing risk factor for AD, possibly because it impedes the clearance of Aß by ApoE and A2M (Kang et al., 2000). Thus, conditions to increase LRP1 expression in the brain could systemically exert beneficial effects on the prevention of AD.

Expression of LRP1 in Alzheimer´s Disease and in Related Degenerative Diseases At present, only limited experimental data have been published describing the in vivo expression of LRP1 with regards to degenerative diseases. Most expression studies have been done on transgenic animals or on post mortem material. Handschug et al. (1998) first described results on in vivo expression of LRP1 in humans by measuring the LRP1 transcripts obtained from patients’ blood monocytes. A mild age-dependent increase in LRP1 expression within a group of healthy controls was recognized. LRP1 transcripts increased from 117.7 ag/cell to 150.2 ag/cell (attogram/cell) in healthy controls ranging in ages from 20-60r, respectively. Surprisingly, Schulz et al. (2003) could show that a high transcription rate was paralleled by a decrease in LRP1 protein concentration and vice versa. Furthermore, LRP1 gene and protein expression were shown to follow a circadian- and gender-specific rhythm, which have to be considered in studies investigating the LRP1 in vivo function. A similar expression profile was also corroborated for disease-related expression studies in patients with degenerative coronary atherosclerosis. A disease-associated significant increase in the LRP1 gene-expression in patients (223 ag/cell) versus controls (122,3 ag/cell) was associated with low LRP1 protein levels in the patient group (1.6 pg/cell) versus controls (6 pg/cell). These data suggest that low LRP1 protein levels are tried to be functionally compensated by a dramatically increased LRP1 transcription rate, acting as an incomplete in vivo feed back mechanism. Motivated by these results, we have done a comparative study of in vivo LRP1 gene expression in patients with degenerative diseases, AD and coronary artery disease (CAD), and compared our results to healthy controls. The impact of age, gender and circadian rhythm on LRP1 expression was taken into account in the statistical analysis (detailed description of materials and methods can be requested from the authors).

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In the whole group of 97 individuals a mean LRP1-mRNA expression of 187.20 ag/cell was detected (Table 2). AD patients showed the highest LRP1 gene expression levels (290.96 ag/cell) followed by patients with coronary degenerative disease (174.49 ag/cell). The healthy controls revealed the lowest levels of LRP1-mRNA expression among all of the three subgroups (158.79 ag/cell). The differences among the three patient groups were statistically significant (Kruskal-Wallis test, p=0.015). A correlation between age and LRP1 gene expression was not detected in any of the three groups (controls: p=0.453, CAD: p=0.650, AD: p=0.871) nor was a correlation found between gender and expression (controls: p=0. 348, CAD: p=0.123, AD: p=0.581); this suggests that the subgroup specific difference in the LRP1-mRNA level is really disease-related. The 83.2 % higher expression of LRP1 found in AD patients indicates that the disordered metabolism of patients suffering from this degenerative disease probably activates mechanisms to help compensate for the loss of function at the protein level. The molecular mechanism causing this inverse relation between the levels of LRP1 transcript and protein is not fully understood. It has been suggested that feed-back control mechanisms do exist (up-regulation of mRNA) but the translation into a protein may be blocked. Unravelling the underlying mechanism will be a challenge concerning therapeutic intervention. It is noteworthy that this is the first report demonstrating a relationship between a cellular blood marker and AD. Further studies are needed to test the feasibility of blood LRP1 as a marker for the onset, the severity or the susceptibility to develop AD. Table 2: Results of a study on in vivo gene expression of LRP1 in AD patients compared with patient suffering from (degenerative) coronary artery disease (CAD) versus healthy controls. There was neither a correlation between LRP1 gene expression and gender nor age.

Number Male Age SD Age min.-max. LRPgene expression (ag/cell) SD Correlation* age/LRP1 expression P Correlation* gender/LRP1 expression P *

All individuals

controls

CAD patients

AD patients

97 71 59.7 7.55 54-86 187.20 80.43

20 18 58.9 3.39 54-65 158.79† 52.12 0.178

68 49 58.4 4.09 54-68 174.49† 66.17 -0.056

9 4 71 8.62 54-86 290.96† 126.83 0.064

0.453 0.222

0.650 0.190

0.871 -0.214

0.348

0.123

0.581

Pearson. † The patient groups differ significantly in their expression levels: p=0.015 (Kruskall-Wallis).


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Genetic Predisposition to Alzheimer’s Disease While investigating the possible genetic causes of AD, a variety of different genes was found to be involved in the pathogenesis of this degenerative disease (Bertram and Tanzi, 2004; Williamson and LaRusse, 2004). The autosomal dominant early onset AD was shown to be associated with genes coding for presenilin-1 and -2 as well as amyloid precursor protein. Mutations in these genes lead to the main clinical feature of AD, which is characterized by an increased generation of Aβ and amyloid deposition. However, genomic variations in these genes have been proven to be fully penetrant, but they are very rare events accounting for only 1% to 2% of all AD cases (Gaskell and Vance, 2004). On the other hand, genetic variations of the Apo E lead to an increased risk of late-onset sporadic and familial AD. The ε4 allele of this gene has been established worldwide as a genomic variant predisposing for AD and this allele has been demonstrated to increase for the risk of developing AD in a dose-dependent fashion (Corder et al., 1993). Since the gene encoding ApoE is highly polymorphic the influence of other single nucleotide polymorphisms on the risk of developing AD was studied. However, only a weak effect concerning other SNPs independent of ε4 allele could be demonstrated (Nicodemus et al., 2004). Since Apo E is reported to account for less than 50% of the genetic effects in AD it implies that the onset as well as the progression of the complex disorder AD is triggered by additional genetic and environmental factors which have already been suggested by Sjogren et al. (1952) and St George-Hyslop et al. (1990). A number of genome wide screens have been published investigating the genetic causes of AD. These studies used linkage- or association-based evaluation procedures and reported on genetic locations of interest on different chromosomes (for review see Bertram and Tanzi, 2004). The LRP1 locus on chromosome 12 has also been shown in different studies to be associated with a late-onset form of familial AD (OMIM 602096). Interestingly, a full genome scan for late onset AD which included 292 affected siblings revealed linkage of the locus on chromosome 12 with similar lod scores as for APP (Kehoe et al., 1999). As of June 2005 128 genomic variants of the LRP1 gene have been reported on in dbSNP database of the NCBI Entrez system. Numerous clinical studies were conducted in order to evaluate possible associations of these genomic variants of LRP1 with AD, however, results are conflicting and are controversially discussed (Table 3) (for review see Gläser et al., 2004). Nevertheless, ethnicity and population-specific differences in genotype distributions were shown for a variety of these genomic variants (Gläser et al., 2004) and may account at least in part for the reported conflicting results. To date, only a few studies have been conducted investigating the possible disease related functional consequences of genomic variants of LRP1 (Schulz et al., 2002; Kang et al., 2000). A polymorphism in the promoter region of the gene (c.1-25C>G) has been proven to increase the LRP1 expression on a transcriptional level due to the creation of a CG-box, which is recognized by the SP1 transcription factor (Schulz et al., 2002). For another polymorphism in exon 3 (c.766C>T) a significant increase in LRP1 level could be described for T-allele carriers suffering from AD (Kang et al., 2000).

Table 3: Published important clinical studies evaluating AD related to genomic variants of LRP1. Genomic Variant 5’ Tetranucleotidpolymorphism

Individuals investigated French: 144 sporadic late onset AD patients ↔ 153 controls Caucasians: 182 AD patients ↔ 118 controls French Caucasians: 600 AD patients ↔ 646 controls Americans: 168 AD patients ↔ 157 controls American Caucasians: 62 AD patients ↔ 282 controls American Whites: 216 sporadic late onset AD patients ↔ 106 controls 179 Familial multiplex late onset AD patients, 436 sporadic AD patients ↔ 240 controls Spaniards: 188 sporadic late onset AD patients ↔ 226 controls Japanese: 100 AD patients ↔ 246 controls North Irish: 219 AD patients ↔ 237 controls French Caucasians: 274 AD patients ↔ 290 controls American Whites: 505 late onset AD patients ↔ 522 controls

c.766C>T US Euorpean Whites: 157 late-onset AD patients ↔ 102 polymorphism in exon 3 controls Chinese: 143 AD patients ↔ 129 controls

Whites: 234 AD patients ↔ 103 controls American Whites: 432 AD patients ↔ 106 controls Whites: 558 AD patients ↔ 596 controls

Clinical association to AD Citations Significant decrease of 87bp-allele in AD patients Wavrant –DeVrieze et al., 1997 Significant increase of 87bp-allele in AD patients Lendon et al., 1997 Significant increase of 91bp-allele in AD patients Lambert et al., 1998 None Clathworthy et al., 1997 None Fallin et al., 1997 None Kamboh et al., 1998 None

Scott et al., 1998

None

Bullido et al., 2000

None None None None

Hatanaka et al., 2000 Mc Ilroy et al., 2001 Verpillat et al., 2001 Luedecking-Zimmer et al., 2003 Kang et al., 1997

Significant increase of CC-genotype in AD patients (more pronounced among AD patients with positive familial history of senile dementia Significantly decreased T-allele in AD patients diagnosed pathologically; no association in AD patients diagnosed clinically Significant increase of CC-genotype in AD patients, no correlation to age of onset Significant increase of TT-genotype in controls Increase of CC-genotype and C-allele in AD patients

Baum et al., 1998

Hollenbach et al., 1998 Kamboh et al., 1998 Lambert et al., 1998

Table 3: Published important clinical studies evaluating AD related to genomic variants of LRP1. (Continued) Genomic Variant Individuals investigated Clinical association to AD c.766C>T Caucasians: 225 neuropathologically confirmed AD patients Weak increase of CC-genotype in AD patients polymorphism in exon 3 ↔ 187 controls Japanese : 100 AD patients ↔ 246 controls Association with age of onset Spaniards: 305 sporadic AD patients ↔ 304 controls Weak increase of CC-genotype in AD patients in meta-analysis Caucasians : 212 AD patients ↔ 337 controls Increase of C-allele in controls Higher age of onset in C-allele carriers 133 late-onset AD patients ↔ 70 controls None Spaniards: 199 sporadic late-onset AD patients ↔ 243 None controls Taiwan Chinese: 82 AD patients ↔ 110 controls None North Irish: 219 AD patients ↔ 237 controls None French Caucasians: 274 AD patients ↔ 290 controls None A217V Northern French Caucasians: 648 AD patients ↔ 670 Weak increase of T-allele in controls polymorphism in exon 6 controls US Whites: 536 late-onset AD patients ↔ 511 controls None A775P mutation in exon 14 D2080N polymorphism in exon 39 D2632E mutation in exon 48 G4379S Mutation in exon 85

Citations Beffert et al., 1999 Hatanaka et al., 2000 Sanchez-Guerra et al., 2001 Kölsch et al., 2003 Woodward et al., 1998 Bullido et al., 2000

29 AD patients ↔ 22 controls

No carrier identified

Hu et al., 2000 Mc Ilroy et al., 2001 Verpillat et al., 2001 Wavrant-DeVrieze et al., 1999 Luedecking-Zimmer et al., 2003 Van Leuven et al., 2001



Van Leuven et al., 2001


Only 1 carrier in control group






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Brain (intracellular)

Brain (interstitial)

Blood

7 Aß

1 6

APP

4 8

10

5

11

A2M

11 LRP1

9

2

3

Figure 3. Schematic diagram of possible interventions by modulation of the LRP1-A2M axis of amyloid ß metabolism. (1)Aß processed via the amyloidogenic pathway (secretases) is released into the interstitial fluid for degradation by IDE, NEP or plasmin. (2) Soluble Aß is bound to proteolytically modified A2M by the action of proteases (e.g. plasmin) that mediates binding to LRP1 for endocytosis and lysosomal degradation (3). Soluble Aß (4) or A2M-Aß complexes (5) are transported via LRP1mediated efflux from the brain into the circulatory system. In blood, soluble Aß is subsequently degraded by proteases (6) or eliminated by LRP1-mediated endocytosis in conjunction with proteaseactivated A2M (9). Possible check points of intervention are the modulation of cytoplasmic binding of scaffold protein to LRP1/APP (10); increasing the expression of LRP1 (11) increasing the expression of A2M in brain and blood (7) and increasing the proteolytic potential in brain (1), and in blood (8) and (9) to support A2M-mediated elimination of Aß.

Clinical and Therapeutic Significance It seems indisputable that LRP1 is one of the most interesting candidate genes, not only for AD and other neurodegenerative diseases such as Parkinson disease but also for other degenerative processes affecting other organs, tissues and vessel-walls causing cellular dysfunctions as in CAD or cell degeneration as in cancer (Gläser et al., 2004). In current consolidated findings significant increased levels of LRP1-mRNA which correspond to a decrease in LRP1-protein expression could be a diagnostic marker in recognizing improper supply and/or disposal processes which are dangerous for cells, tissues and organs. Since an alteration in LRP1 expression is directly transmitted to the level of LRP1-ligands to be cleared in the blood, investigation should be done to determine whether or not a more timesaving analysis of LRP1 plasma ligands is to be preferred. Such a candidate ligand could be


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plasma A2M. It exists in two different forms, native A2M and transformed A2M (Birkenmeier, 2001). The latter is the receptor-recognizable species which can easily be determined by immunological assays (Birkenmeier and Stigbrand, 1993; Sinnreich et al., 2004). With respect to LRP1 and its Aß-clearing ligands possible therapeutic interventions should focus on (i) increasing the clearance capacity of LRP1 by elevating its peripheral expression, (ii) increasing the proteolytic activity in the brain and the blood circulation , (iii) increasing the level of A2M in blood and (iv) promoting the LRP1-mediated transfer of Aß from the brain to circulatory system. In addition to current trials and activities to tame AD progression with secretase inhibitors, inhibition of Aß-fibril formation or with vaccinations, the modulation of the LRP1-ligand system seems to be a promising approach (Figure 3).

Data Bases GeneCardsR, Weizman Institute of Science http://bioinfo.weizmann.ac.il/cardsbin/cardsearch.pl OMIMTM - Online Mendelian Inheritance in ManTM: http://www.ncbi.nlm.nih.gov/entrez/ query.fcgi?db=OMIM

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In: New Research on Alzheimer’s Disease Editor: Eileen M. Welsh, pp.


Chapter V

The Role of Peroxisome Proliferator-Activated Receptors in Alzheimer’s Disease Dokmeci Dikmen∗ Department of Pharmacology, Faculty of Medicine, Trakya University, Edirne, Turkey

Abstract Alzheimer’s disease (AD) is characterized by a progressive loss of cognitive function with cerebral deposits of amyloid-β (Aβ) senile plaques and neurofibrillary tangles (NFTs) surrounded by inflammatory cells. Neuroinflammation, oxidative stress and impaired glucose and lipid metabolism are postulated to be the mechanisms playing role in the pathophysiology of AD. Recent evidence suggests that inflammatory events are associated with plaque formation in the brains of patients with AD and non-steroid antiinflammatory drugs (NSAIDs) might influence central nervous system (CNS) inflammation and AD pathology. Reactive oxygen and nitrogen species production may also participate in part to neurodegeneration in AD brains; antioxidant and/or free radical scavengers reduce inflammation in AD via free radical quenching and possess neuroprotective activity. In addition, AD patients show hyperinsulinemia and reduced insulin sensitivity during fasting and glucose tolerance tests; and insulin gene polymorphism might act as a modifier of AD progression. An increased number of insulin receptors are also observed in post-mortem brain tissues of AD patients. Furthermore, augmented plasma insulin levels and reduced cerebrospinal fluid (CSF) levels and CSF/plasma insulin ratio are observed in AD patients. Accordingly, treatment of insulin resistance may reduce the risk or retard the progression of AD.

∗

Correspondence: Assoc. Prof. Dikmen Dokmeci, M.D. Department of Pharmacology, Faculty of Medicine, Trakya University, 22030, Edirne, Turkey. Tel. no.: 90-284-2359742; Fax no.: 90-284-2352476; e-mail: [email protected], [email protected]

Dokmeci Dikmen

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Peroxisome proliferator-activated receptors (PPARs), members of the nuclear receptor family, are ligand-activated transcription factors. To date, three isoforms, encoded by separated genes, have been identified: PPARα [NR1C1], PPARβ (NUC-1 or PPARδ) [NR1C2] and PPARγ [NR1C3]. All PPAR isotypes are detected and found to exhibit specific patterns of localization in the different areas of the brain. PPARs may have specific functions in regulating the expression of genes involved in neurotransmission, and therefore play roles in complex processes, such as neurodegeneration, learning and memory. It is likely that at least some effects of PPAR agonists and NSAIDs on AD pathology are mediated through PPARs, since both PPARs and cyclooxygenase (COX) expression are increased in AD brains. Recently, NSAIDs, activating PPARα and γ, have been identified. In addition, NSAIDs and PPARα and γ agonists have anti-inflammatory, antioxidant and neuroprotective effects. This neuroprotective effect correlates with the modulation of β-catenin levels, inhibition of glycogen synthase kinase-3β (GSK-3β) activity and increased mRNA levels of the Wnt-target genes engrailed-1, cyclin D1 and PPARδ. Thiazolidinediones (TZDs) such as pioglitazone and rosiglitazone are insulin sensitizers, used to control glucose concentrations in patients with type 2 diabetes. These are potent synthetic PPARγ agonists, which exert both anti-inflammatory and antioxidant effects. PPARα and γ ligands, NSAIDs and TZDs inhibit activation of nuclear factor (NF)-κB, signal transducers and activators of transcription-1 (STAT-1), the nuclear factor of activated T-cells (NFAT) and activating protein-1 (AP-1). Moreover, they prevent the expression of COX-2 and inducible nitric oxide synthase (iNOS) and production of inflammatory cytokines such as interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α in human monocytes and arrest the differentiation of monocytes into activated macrophages. In conclusion, the development of new drugs targeting PPARs would be a great interest for future treatments of AD.

Keywords: Alzheimer’s disease, PPARs, neuroprotective effect, neuroinflammation, antioxidant effect, fibrates, thiazolidinediones, NSAIDs, Wnt signaling

Abbreviations AP-1 AGEs AD Aβ APP ApoE JNK CAT CNS CSF CoA COX DAT

activating protein-1 advanced glycation end-products Alzheimer’s disease amyloid-β amyloid precursor protein apolipoprotein E c-jun-NH2-terminal kinase catalase central nervous system cerebrospinal fluid coenzyme A cyclooxygenase dementia of Alzheimer type

The Role of Peroxisome Proliferator-Activated Receptors… DHA FDA GSH-Px GSSGR GSK HDL iNOS IRS IL LPS LDL LMWAs MMP MCI MAPK NFTs NADPH NO NSAIDs (NF)-κB NFAT PPARs PI3K PUFAs PS PG RXR RNS ROS STAT-1 SOD TZDs TNF VLDLs

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docosahexaenoic acid Food and Drug Administration glutathione peroxidase glutathione reductase glycogen synthase kinase high-density lipoprotein inducible nitric oxide synthase insulin receptor substrate interleukin lipopolysaccaride low-density lipoprotein low-molecular-weight antioxidants matrix metalloproteinase mild cognitive impairment mitogen-activated protein kinase neurofibrillary tangles nicotinamide adenine dinucleotide phosphate nitric oxide non-steroid anti-inflammatory drugs nuclear factor nuclear factor of activated T-cells peroxisome proliferator-activated receptors phosphatidylinositol 3-kinase polyunsaturated fatty acids presenilin prostaglandin retinoid X receptor reactive nitrogen species reactive oxygen species signal transducers and activators of transcription-1 superoxide dismutase thiazolidinediones tumor necrosis factor very-low-density lipoproteins

Introduction Alzheimer’s disease (AD), the most common form of dementia among the elderly population in the world, is a progressive, degenerative disorder of the central nervous system (CNS). Clinically, AD is characterized by impairment in memory, visuospatial skills, complex cognition, language, emotion and personality. Currently, AD affects ~5 million people in the United States and >30 million people worldwide. The prevalence of AD is expected to quadruple by the year 2047. Economically, AD is extremely costly to patients,

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their families, and society. In fact, the cost of caring for patients with AD was over $110 billion per year in the early 1990s in the United States, and the average annual cost per patient is about $45,000 [1]. There are no definitive diagnostic tests or biological markers of AD. Furthermore, there are no known cures or preventions for AD. Therefore, scientists from different disciplines have been trying to find new drugs for the prevention and treatment of AD for a long time. On the other hand, drug discovery for AD is complicated by the fact that the exact cause(s) of the disease is still unknown and early diagnosis of the disease is not currently available [2]. The early-onset, familial form of AD accounts for nearly 5-10% of all cases of AD and is characterized by early manifestations of dementia, in some cases in patients ~40 years of age. It is caused by multiple mutations in the major genes, such as amyloid precursor protein (APP) on chromosome 21, presenilin (PS)-1 on chromosome 14, and PS-2 on chromosome 1 [2, 3]. In contrast, although the initiating events are still unknown, late onset AD, which is sporadic and accounts for ~90-95% of all patients, results from the combination of genetic risk factors such as aging, apolipoprotein E (ApoE), and α2 macroglobulins, with different epigenetic events [4]. Diabetes, cardiovascular complications, arteriosclerosis, and hypertension are considered to be the risk factors in the development of AD [5, 6]. Neuroinflammation, oxidative stress and impaired glucose and lipid metabolism are also postulated to be the mechanisms playing role in the pathophysiology of AD. Furthermore, peroxisome proliferators-activated receptor (PPAR) expression is increased in AD brains and PPARs seem to play an important role in AD etiopathogenesis [7]. PPARs, a member of the nuclear hormone receptor superfamily, are involved in several physiological processes, such as glucose homeostasis, cellular differentiation, regulation of lipid and lipoprotein metabolism, neurodegeneration, learning and memory, as well as in pathological states including atherosclerosis, inflammation, cancer, diabetes, obesity, infertility, and demyelinization [8, 9]. Hipolipidemic fibrates, antidiabetic thiazolidinediones (TZDs) and non-steroid anti-inflammatory drugs (NSAIDs) are PPAR agonists. In addition, to date, several studies have confirmed the insulin sensitizer, hipolipidemic, antiinflammatory and antioxidant properties of various PPAR agonists in vivo and in vitro. Hence, it is of importance to determine if the efficacy of fibrates, TZDs and NSAIDs in reducing AD risk is a result of the action of these drugs on PPARs.

Neuropathology of AD AD is characterized by a progressive loss of cognitive function with senile plaques, vascular amyloid-β (Aβ) deposits and neurofibrillary tangles (NFTs) as the pathologic hallmarks of the disease. AD brains exhibit a number of other abnormalities: synaptic reduction, neuronal loss, volume loss (atrophy), gliosis, microglial activation, impaired energy metabolism, mitochondrial function, signs of inflammation, and damage secondary to oxygen radicals [10, 11]. NFTs are cytoskeletal lesions largely composed of paired helical and straight filaments, which are intraneuronal aggregates of hyperphosphorylated, microtubule-associated tau protein. After the neurofibrillary tangle-laden neurons die,

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extracellular "tombstone" tangles remain visible [12]. Aβ are deposited as extracellular senile plaques composed of the 39-42 amino-acid-long peptide, derived from the β-APP, caused by cleavage with β-secretase and γ-secretase. The α-secretase cleaves the middle of the Aβ region, releasing a secreted ectodomain that contains the first 16 amino acids of the Aβ (sAPPα). In contrast, β-secretase, the novel transmembrane asparatic protease, BACE, cleaves between Met-671 and Asp-672, producing the amino terminal end of the Aβ and ending with Met-671 (sAPPβ). Further processing of the carboxyl-terminal end of sAPPβ, by the activity of γ-secretase, leads to the release of Aβ [12, 13]. Brain levels of both 42-aminoacid form (Aβ42) and 40-amino-acid form (Aβ40) are increased in AD. The Aβ40 is the most abundantly produced Aβ peptide, whereas a slightly longer and less abundant Aβ42 has been implicated as the more pathogenic species. Despite being a minor Aβ species, Aβ42 is deposited earlier and more consistently than Aβ40 in the AD brain. Collectively, these observations provide a strong rationale for selective targeting of Aβ42 and indicate that reducing Aβ42 levels by as little as 20-30% might retard the development of AD [14]. Interestingly, Kitamura and collegues reported that PPARγ expression could be detected by Western analysis in the temporal cortex of the human brain. Significantly, they found approximately a 50% increase in the amount of immunoreactive PPARγ protein in the brains of AD patients [7]. In conclusion, PPARs play an important role in AD etiopathology and may be a promising new therapeutic approach for the treatment of AD.

PPARs PPARs are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors, which includes retinoid, steroid, vitamin D and thyroid hormone receptors that function as heterodimers with the retinoid X receptor (RXR) and contain a highly conserved DNA-binding domain and a carboxy-terminal ligand-binding domain. These receptors regulate the expression of specific target genes by binding as heterodimers with RXR to specific DNA sequences, which are referred to as PPAR response elements [8]. Intensive study of PPARs during recent years has suggested an important role for these nuclear receptors both in normal physiology and the pathology of various tissues [15]. There are three PPAR isotypes, namely PPARα [NR1C1], PPARβ (PPARδ, NUC-1 or FAAR) [NR1C2] and PPARγ [NR1C3], localized on chromosomes 22, 6 and 3, respectively, with distinct tissue distribution and biological activities [8, 9, 15, 16]. As a result of alternative splicing and alternative promoter usage generates three transcripts from the human PPARγ gene; PPARγ1, PPARγ2 and PPARγ3. Both PPARγ1 and PPARγ3 mRNA encode the same protein product expressed in most tissues, whereas the PPARγ2 mRNA encodes the PPARγ2 protein that contains an additional 28 amino acids and is specific to adipocytes [16]. PPARs have been found and identified so far in amphibians, cyclostoma, teleosts, rodents and humans. PPARs are implicated in many cellular processes, from cell cycle to cell proliferation, from inflammation to apoptosis, and crucial for energy homeostasis. In addition, PPARs act as transcription factors, and regulate the expression of numerous genes and affect glycaemic control, lipid metabolism, inflammation and oxidant status [17-19]. Meanwhile, all PPAR isotypes are detected and found to exhibit specific patterns of

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localization in the different areas of the brain. PPARs have specific functions in regulating the expression of genes involved in neurotransmission, and therefore play roles in complex processes, such as aging, neuroinflammation, neurodegeneration, learning and memory [20]. PPARs may act for a range of compounds including hypolipidemic, antihyperglycaemic drugs, insulin sensitizers, and NSAIDs.

PPARα The first discovered PPAR was PPARα (Issemann and Green, 1990) [21], followed by PPARβ and PPARγ. PPARα is expressed primarily in liver, kidney, heart, adipose tissue, intestine, skeletal muscle and vascular cells [18, 22, 23]. PPARα can be activated by a wide variety of saturated and unsaturated fatty acids including arachidonic acid, oleic acid, linoleic acid, palmitic acid, docosahexaenoic acid (DHA), and eicosapentaenoic acid. Recently, PPARα has also been shown to be activated by very-low-density lipoproteins (VLDLs) in the presence of enzymatically active lipoprotein lipase [24, 25]. PPARα has been shown to regulate lipid metabolism through the control of apolipoprotein expression. Ligand activation of PPARα in vivo lowers triglyceride levels, presumably through its effects on fatty acid and lipoprotein metabolism. PPARα activation is now recognized to be the mechanism of action of the fibrate class of hypolipidemic drugs. The fibrates, amphipathic carboxylic acids that have been proven useful in the treatment of hypertriglyceridemia, are PPARα ligands. Clofibrate is a prototype for this class. Clofibrate and fenofibrate have been shown to activate PPARα with a 10-fold selectivity over PPARγ. Bezafibrate is a pan-agonist that exerts similar potency on all three PPAR isoforms [8]. PPARα transcriptionally regulates fatty acid catabolism and the production of enzymes such as acyl-coenzyme A (CoA) oxidase, the key enzyme in peroxisomal β-oxidation pathway, carnitine palmitoyl transferase I, implicated in the translocation of fatty acids across the inner mitochondrial membrane, as well as CYP4A6, which is an important microsomal ωhydroxylase. Interestingly fatty acids directly affect transcription by activating PPARα [26]. In addition, PPARα is involved in inflammation and in the cell response to reactive oxygen and nitrogen species. PPARα activators also reduce the risk of atherosclerosis, decrease progression of atherosclerotic plaques, and reduce the incidence of mortality from cardiovascular disease [20, 22, 25]. Moreover, PPARα ligands inhibit the inflammatory response in aortic smooth muscle cells, an effect that is lost in the PPARα null mice. This occurs through the inhibition of interleukin (IL)-1-mediated expression of IL-6 and cyclooxygenase (COX)-2. The transcriptional inhibition of this key enzyme of the inflammation process occurs as a result of a PPARα-dependent repression of the nuclear factor (NF)-κB pathway [27].

PPAR β/δ PPARδ is a less well known PPAR isoform, widely distributed in the organism; it is abundant especially in skeletal muscle, skin, placenta and brain. PPARδ interacts with


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saturated and unsaturated fatty acids; its ligand selectivity is intermediate between PPARγ and PPARα. Arachidonic acid, palmitic acid, prostaglandin (PG) A1 and PGD2, eicosapentaenoic acid, dihomo-γ-linolenic acid and carbaprostacyclin have been shown to activate PPARδ [8, 25]. Recently, the antiepileptic drug valproic acid has also been shown to activate PPARδ [28-30]. Its physiological role is not well understood, but it has been suggested to play a role in lipid metabolism, myelination and neuronal signaling, particularly in CNS, carcinogenesis, wound repair, epithelial inflammatory response, obesity, reproductive function, fertility, embryo implantation, cell proliferation, apoptosis, keratinocyte, adipocyte, and oligodendrocyte differentiation [8, 20, 22, 25].

PPARγ PPARγ, the best characterized of the PPARs, is expressed in white adipose tissue, pancreas, intestine, spleen, lymphoid tissue, bone marrow, kidney, heart, ovary, testis, liver, bladder, skeletal muscle and vasculature, epithelial keratinocytes and brain [19]. Fatty acids and eicosanoid derivatives bind and activate PPARγ at micromolar concentrations. PPARγ clearly prefers polyunsaturated fatty acids (PUFAs) including the essential fatty acids linoleic acid, linolenic acid, arachidonic acid and eicosapentaenoic acid. LTD4 antagonists, such as pranlukast, are selective agonists for PPARγ [8, 16]. PPARγ is a key regulator of glucose homeostasis and adipogenic differentiation [31]. PPARγ also promotes lipogenesis and exerts anti-inflammatory and antiproliferative actions [20, 25]. PPARγ is also involved in atherosclerosis [9]. It was reported that PPARγ is markedly upregulated in activated macrophages and that natural and synthetic PPARγ ligands inhibit the induction of indicuble nitric oxide syntase (iNOS), matrix metalloproteinase (MMP, called also gelatinase B) and scavenger receptor A gene transcription [32]. Promoter studies have revealed that PPARγ inhibits the transcriptional activity of these genes by interfering with the activating protein-1 (AP-1), NF-κB, the nuclear factor of activated Tcells (NFAT) and signal transducers and activators of transcription-1 (STAT-1) transcription factors. Furthermore, Jiang et al. [33] have shown that incubation of human monocytes with the natural PPAR γ ligand PGJ2 or with synthetic agonists inhibits the production of proinflammatory cytokines, including tumor necrosis factor (TNF)-α, IL-1β, and IL-6. The pharmacological ligands for PPARγ are TZDs and a variety of NSAIDs. TZDs were primarily developed in an effort to improve the antidiabetic actions of the fibrate hypolipidemic agents [8]. These compounds are used as therapeutic agents for treatment of type 2 diabetes. There are currently two members of the TZD class (pioglitazone and rosiglitazone) that have been approved by Food and Drug Administration (FDA) for treatment of Type 2 diabetes [31]. TZDs decrease plasma triglyceride and free fatty acids levels by enhancing their catabolism via the induction of lipoprotein lipase gene expression in adipose tissue. TZDs such as troglitazone, pioglitazone, rosiglitazone, englitazone and ciglitazone also have been postulated to be promising drugs for the treatment of cancer, hypertension, atherosclerosis, multiple sclerosis, psoriasis, ulcerative colitis, asthma, atopic dermatitis, and

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chronic inflammatory processes. They are also potent neuroprotector compounds, which that inhibit inflammatory activation of cultured brain astrocytes and microglia by diminishing lipopolysaccharide (LPS)-induced IL-6, TNF-α, iNOS, and inducible COX-2 expression. In addition, glycogen synthase kinase (GSK)-3α/β has been recognized as a therapeutic target for the treatment of neuropathological disorders, such as AD. TZDs were first described as GSK-3β inhibitors and were postulated that could be of potential therapeutic use for the treatment of AD [9, 15, 18, 19, 25]. Nitric oxide (NO) is overproduced in adipose tissue and muscle by iNOS in conditions such as chronic inflammation and insulin resistance. Recently, it was shown that PPARγ results in a decrease in iNOS expression in mesangial cells and macrophages via its effects on the NFκB, AP-1 and STAT-1 pathways [31]. NSAIDs are well known COX inhibitors, and some have been indicated recently to activate the PPARs. Hence, NSAIDs were classified into the following theree groups: The first group including aspirin, acetaminophen, tiaramide, and piroxicam shows no agonistic activity for any subtype of PPAR. The second group including indomethacin and diclofenac shows high selectivity for the PPARγ. The last group, e.g. ibuprofen, exerts agonistic activity for all type PPARs [34-36]. NSAIDs exerting PPAR agonistic activity, inhibit activation of NF-κB, STAT-1 and AP-1, and prevent expression of iNOS and production of inflammatory cytokines such as TNF-α and IL-1β. PPAR activators inhibiting COX expression and inflammatory events may have beneficial effects in the treatment of AD patients [7].

The Roles of Glucose Metabolism and PPARs in Alzheimer’s Disease Diabetes mellitus is a metabolic disorder divided into two main types: insulin-dependent and non-insulin–dependent. The various subtypes of diabetes mellitus differ with respect to etiology, pathogenesis, and insulin availability, but share the same consequences of chronic hyperglycemia and impaired insulin actions. Insulin dependent diabetes mellitus (called also Type 1 diabetes) is caused by insulin deficiency as a result of autoimmune destruction of pancreatic islet beta cells. Non-insulin-dependent diabetes mellitus (called also Type 2 diabetes) is the most common form of diabetes that is characterized by hyperinsulinemia and insulin resistance in peripheral tissues. Individuals with Type 2 diabetes have hyperglycemia and hyperinsulinemia. The pathogenesis of insulin resistance in Type 2 diabetes is not completely understood. However, evidence suggests that the insulin resistance is partly mediated by reduced levels of insulin receptor expression (down regulation), insulin receptor tyrosine kinase activity, insulin receptor substrate (IRS)-1 expression, and/or phosphatidylinositol 3-kinase (PI3K) activation in skeletal muscle and adipocytes [37, 38]. Type 2 diabetes is also associated with hippocampal and amygdalar athropy and increasing insulin resistance is associated with more amygdalar atrophy on magnetic resonance imaging [39]. Insulin synthesis and insulin gene expression have been demonstrated in mammalian neuronal cells. Insulin mRNA is distributed in a highly specific pattern with the highest density in pyramidal cells of the hippocampus and a high density in the medial prefrontal


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cortex, the enthorinal cortex, perirhinal cortex, thalamus and the granule cell layer of the olfactory bulb. Neither insulin mRNA nor insulin synthesis is observed in glia cells [40]. In 1978, Havrankova et al. [41] localized the insulin receptor in the CNS for the first time, and later insulin receptors are expressed in neurons throughout the CNS, particularly in the cerebral cortex, cerebellar cortex, hippocampus, olfactory bulbs, thalamus, hypothalamus, brainstem nuclei, spinal cord, and retina. In neurons, the binding of insulin to its receptor initiates tyrosine kinase mediated intracellular signaling and this leads to autophosphorylation of the receptor, followed by tyrosine phosphorylation of IRS proteins, which induce the activation of downstream pathways such as the PI3K and the mitogen-activated protein kinase (MAPK) cascades [42-44]. Binding of tyrosine phosphorylated-IRS to p85 stimulates glucose transport and inhibits apoptosis by activating Akt/Protein kinase B and inhibiting GSK-3β. Akt kinase inhibits apoptosis by phosphorylating GSK-3β [45]. Low levels of Akt kinase and high levels of GSK-3β activity are associated with increased neuronal death [46, 47]. Furthermore, insulin inhibits neuronal apoptosis via activation of protein kinase B in vitro, and regulates phosphorylation of tau, metabolism of the APP and clearance of Aβ from the brain in vivo [44]. Glucose, transported from peripheral blood, is the primary nutrient for neurons and the major fuel for oxidative metabolism, and function in the CNS. However, insulin enters the cerebrospinal fluid (CSF) through the blood-brain barrier by insulin receptor-mediated transport process, and exerts neuromodulator, neuroendocrine and neurotropic effects [5, 47]. Margolis et al. [48] showed that peripheral infusion of insulin leads to an increase in insulin levels in CSF. However, the augmentation of the peripheral insulin concentration acutely increases the concentration in the brain and CSF, whereas prolonged peripheral hyperinsulinaemia downregulates blood-brain barrier insulin receptors and reduces insulin transport into the brain [49]. The transport of insulin across the blood-brain barrier shows a saturable character; however, the molecular identity of this transport mechanism remains obscure [50]. Insulin regulates growth, food intake, sympathetic activity, reproductive endocrinology, glucose homeostasis and peripheral insulin action through the inhibition of hepatic gluconeogenesis [44, 51, 52]. In addition, insulin interferes neuronal growth, survival, differentiation and signal transduction; and promotes neurite outgrowth, migration, protein synthesis, neuronal cytoskeletal protein expression, and nascent synapse formation [53]. The brain may utilize insulin from both locally produced and peripheral (pancreatic) sources for different functional requirements including learning and memory. Furthermore, intranasal administration of insulin rapidly augments the insulin concentration in CSF without affecting blood glucose levels [54], and facilitates brain cognitive functions and working memory [55, 56], suggesting that it influences brain function directly and independently of changes in peripheral glucose. Epidemiologic and clinical studies suggest an important interaction between diabetes mellitus, hyperinsulinemia, insulin resistance and the development of AD, and diabetes mellitus significantly increases the risk for the development of AD [5, 57-64]. Furthermore, AD patiens are also more vulnerable to Type 2 diabetes [65]. There is compelling clinical and pre-clinical evidence that abnormalities of glucose metabolism are related to cognitive impairment and specifically to the development of AD. It is likely that insulin and insulin

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resistance play important roles in this process [5]. Moreover, insulin dysregulation may contribute to AD pathology through several mechanisms including decreased cortical glucose utilization particularly in the hippocampus and entorhinal cortex; increased oxidative stress through the formation of advanced glycation endproducts; increased tau phosphorylation and NFT formation; and increased Aβ aggregation through inhibition of IDE (called also unsulysin) [5]. In addition, pathological filaments with histochemical properties of amyloid are found in human AD pancreas [66], and Aβ-immunoreactive intracellular deposits have been reported in the pancreases of mice overexpressing the 99-amino acid C-terminal fragment of APP [67]. Thus, accumulation of Aβ in islet cells may be an underlying cause of, or at least potentiate the development of, insulin resistance and impaired glucose tolerance in AD [68]. Brain glucose metabolism reduces in AD patients [47]. Individuals suffering from AD have higher plasma insulin and lower CSF insulin concentrations as well as the insulin insensitivity or resistance, indicating impairment in insulin metabolism in the brain [69-71]. Moreover, administration of insulin to Alzheimer’s patients has been demonstrated to result in an improvement on memory and performance [72]. Consistent with the insulin resistant pattern, insulin tyrosine kinase, an insulin transducer, was reduced in the AD brains. Frolich et al. [73] found that brain insulin concentration and insulin receptor densities both decrease with age. Moreover, insulin receptor density is up-regulated in AD disease, and the authors interpreted the increased receptor density as a compensatory upregulation for defective insulin signal transduction [73, 74]. Schubert et al. [75] showed that disruptions of the insulin receptor tyrosine kinase signaling leads to an accumulation of phosphorylated tau in the mice hippocampus. In fact, the reduced CNS expression of genes encoding insulin and its receptor, as well as related signaling molecules, in the AD brain prompted the researchers to consider sporadic AD as a neuroendocrine disease that resembles Type 2 diabetes which was termed ‘’brain diabetes or Type 3 diabetes’’ [47, 63, 76-78]. Aβ40 and Aβ42, the main physiological C-terminal cleavage products of APP, reduce insulin binding and insulin receptor auto-phosphorylation due to reduced affinity of insulin binding to its own receptor, thereby impairing insulin signaling [79]. Aβ accumulations can promote tau hyperphosphorylation and the formation of the AD dementia associated paired helical filament-containing neuronal cytoskeletal lesions (NFTs, neuritic plaques, and neuropil threads) through functional impairment of the insulin signaling cascade, leading to increased levels of GSK-3β activity. It has been shown that Aβ40 and Aβ42 reduce insulin binding to and autophosphorylation of its receptor [80]. Furthermore, insulin protects against Aβ-peptide toxicity in brain mitochondria of diabetic rats [81]. Insulin seems to be involved in clearing Aβ from the brain [82]; on the other hand, inhibits the breakdown of Aβ by competitively blocking IDE. IDE activity is influenced by insulin concentration. IDE is a protease that can cleave a variety of small proteins, such as insulin and glucagons, and regulates the levels of insulin, Aβ -protein, and the Aβ precursor protein [83, 84]. IDE can degrade both intra- and extracellular Aβ as well as APP intracellular domain fragment produced by γ-secretase cleavage of APP [85]. IDE is decreased in Tg2576 mice which will also enhance the accumulation of Aβ [86]. Since both insulin and Aβ are substrates of IDE, insulin may compete with Aβ for IDE and therefore prevent Aβ degradation [61]. IDE deficiency or hypofunction may play a role in the disease


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processes of both AD and diabetes mellitus [87], and the use of IDE inhibitors block the degradation of Aβ and may be beneficial in diabetes mellitus and AD therapy. Advanced glycation end-products (AGEs) are sugar-derived protein modifications formed through a series of non-enzymatic glycosylation reactions [5]. Both diabetes mellitus and AD are characterized by accumulation of extra- and intracellular AGEs with the glucose hypometabolism and the metabolic consequences of oxidative stress [88]. There is CNS neuronal insulin resistance in AD brains which is associated with reduced levels of IRS mRNA, IRS-associated PI3K, reduced activation of Akt, and increased activation of GSK-3β activity and APP mRNA expression [89]. Insulin modulates other enzymes involved in tau phosphorylation, such as GSK-3. GSK3 is a serine/threonine kinase originally identified as a protein destined to phosphorylate and inactivate glycogen synthase. There are two GSK-3 isoforms, termed GSK-3α and GSK-3β. The latter is specifically expressed in CNS [90]. Oxidative stress can result in the phosphorylation of GSK-3β and neuronal injury. Hence, Aβ exposure in cultured hippocampal neurons can activate GSK-3β [91]. In addition to the relationship of GSK-3β with Aβ toxicity, GSK-3β can also alter the processing of APP. Involvement of GSK-3β in AD has also been linked to the microtubule-associated protein tau. The hyperphosphorylated tau is the major component of NFT that are composed of paired helical filaments. On the other hand, GSK-3β and PS1 may have an additional relationship through β-catenin. PS-1 can regulate the stability of β-catenin, a substrate of GSK-3β. GSK-3β negatively regulates βcatenin through phosphorylation and degradation. The loss of β-catenin results in an increase in the susceptibility of cells to apoptosis [90]. TZDs were first described as GSK-3β inhibitors and were postulated that could be of potential therapeutic use for the treatment of AD [92]. In conclusion, the precise role of insulin in the pathophysiology of AD is as yet undetermined. Impaired insulin signal transduction pathways, insulin dysregulation and insulin resistance in AD brain play important role in development and progression of AD. Antidiabetic agents including TZDs that reduce hyperinsulinemia might have the effect of diminishing circulating insulin in the CNS and freeing IDE to metabolize Aβ. TZD class of agents is FDA-approved for diabetes mellitus [31]. They bind to the PPARγ, which regulate fat cell differentiation and glucose uptake in adipose tissue. TZDs improve insulin sensitivity, reduce lipid content in skeletal muscle and other non-adipose tissues, and reduce peripheral glucose levels in patients with Type 2 diabetes mellitus. TZDs may also reduce insulin resistance. The use of insulin sensitizers (preferably ones are CNSspecific) would probably provide the best form of therapeutic rescue in the early and intermediate stages of AD [8, 22]. It is possible that the reported salutary effects of PPARγ might arise from improved brain glucose utilization in AD. Interestingly, some studies suggest that treatment for insulin resistance may reduce the risk or retard the progression of AD. Since insulin resistance increases amyloidosis in AD brain, the amyloid-lowering effect of TZDs may also be due to enhanced insulin sensitivity [47, 61]. Activation of this receptor also results in inhibition of a broad range of proinflammatory genes [93]. Treatment of microglial cultures with PPARγ agonists reduces the neurotoxic Aβ-induced inflammatory response [47]. These agents might prove to be disease-modifying treatments in AD [18, 25].

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Adiponectin is a fat-derived hormone and reduced circulating adiponectin concentrations were found to be strongly associated with the progression or insulin resistance in humans. PPARγ activation enhances adiponectin expression and production in humans and insulin resistant mice, which could contribute to the mechanisms by which PPARγ ligands improve the sensitivity of glucose metabolism to insulin’s action [31]. Uryu et al. [94] suggested that troglitazone, TZD drug and PPARγ agonist, inhibits both post-glutamate neurotoxicity and low-potassium-induced apoptosis in cerebellar granule neurons. Gemma et al. [95] demonstrated that administration of a PPARγ agonist, rosiglitazone, reversed an age-related deficit in contextual fear conditioning. Furthermore, PPARγ activation might modulate the beneficial effects of NSAIDs on cognition. The authors attribute the improvement in verbal memory recovery to the insulin-sensitizing effects of rosiglitazone. The Wnt signaling pathway is one of the best-studied signaling cascades implicated in neural development and maintenance of the CNS [96]. Defects of Wnt signaling appears to play role in neurodegenerative disorders, such as AD, and some of these events may be related to the modulation of β-catenin and GSK-3. Moreover, a loss of function of the Wnt signaling pathway has also been found to play role during Aβ neurotoxicity, and the key components of such pathway are affected in AD, that is, β-catenin is reduced and GSK-3β is activated in preneurofibrillary lesions. Engrailed-1 is a Wnt target gene related with the activation of Wnt signaling pathway in hippocampal neurons. It was demonstrated that PPARγ agonists modulate Wnt signaling pathway components; hence, activation of PPARγ receptors increases the expression of the Wnt-target genes engrailed-1, cyclin D1 and PPARδ, and inhibits the GSK-3β activity [97, 98]. Inestrosa et al. [98] showed that PPARγ agonist troglitazone treatment of rat hippocampal neurons results in the modulation of Wnt signaling pathway components and prevents the neurodegenerative changes induced by Aβ. Furthermore, GSK-3β negatively regulates β-catenin through phosphorylation and degradation. The loss of β-catenin results in an increase in the susceptibility of cells to apoptosis. Farias et al. [99] showed that ibuprofen (NSAID and PPAR pan-agonist) and octyl-pyridostigmine (cholinesterase inhibitor) protects rat hippocampal neurons, inhibits GSK-3β and stabilized β-catenin in rat and mouse brain neurons exposed to Aβ as well as cortical neurons derived from transgenic mice that overexpressed GSK-3β. In addition, these drug combinations enhanced the non-amyloidogenic APP cleavage by increasing secreted APP and decreasing endogenous Aβ40 in rat hippocampal neurons. It has been recently shown that all three PPAR subtypes are activated by valproic acid and some of its analogues in a structure-dependent manner. Kim et al. [100] demonstrated that valproate protects cells from ER stress-induced lipid accumulation and apoptosis by inhibiting GSK-3α/β, a kinase involved in a diverse number of signaling pathways including those controling neuronal gene expression and survival. In addition, valproat has been shown to modulate gene expression by activating the AP-1 family of transcription factors. Loy et al. [101] suggested that pan-agonist valproat has potential benefit for AD and tauopathies by neuroprotective properties. In conclusion, PPAR agonists have potent neuroprotective effect and directly promote neuroprotection in primary neurons exposed to different pro-apoptotic stimuli in vitro. This neuroprotective effect correlates with the modulation of β-catenin levels, inhibition of GSK-


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3β activity and increased mRNA levels of the Wnt-target genes engrailed-1, cyclin D1 and PPARδ [98].

The Roles of Lipid Metabolism and PPARs in Alzheimer’s Disease Brain membranes have a very high content of essential PUFAs and cholesterol. Both PUFA and cholesterol metabolisms play important roles in brain development, maturation, differentiation and functioning. Almost three decades ago, it was realized that cholesterol metabolism is different in the brain than in the other tissues, limiting penetration of lipids into the brain through the blood-brain barrier. It is well known that PUFAs alter the blood–brain barrier, therefore PUFAs or cholesterol, which are present in the diet, modulate brain functions. It was shown that dietary cholesterol induces the transcription of genes coding for acute inflammatory proteins, adhesion molecules, cytokines and stress proteins [102, 103]. Recent studies suggest a link between lipid metabolism and the development of neurodegenerative disorders such as AD, but the mechanisms for these associations are unknown. AD patients show elevated serum total cholesterol levels in their midlives [104, 105], and hypercholesterolaemic rabbits have increased neuronal Aβ accumulation. Since high cholesterol was implicated in the pathogenesis of amyloid plaques in AD, high intake of dietary cholesterol and saturated fat increases the risk of AD and the deposition of Aβ in brains [106]. In addition, dietary factors and the use of cholesterol-reducing drugs may be associated with a low risk of AD and can be protective against the disease [107]. PPARs play a role in lipoprotein metabolism and regulate target genes involved in uptake, transport, catabolism and storage of fatty acids [108]. Synthetic agonists of PPARα, fibrates such as clofibrate, fenofibrate, bezafibrate, ciprofibrate are used in the treatment of hyperlipidemia. The use of fibrates results in a substantial decrease in plasma triglycerides and is usually associated with a moderate decrease in low-density lipoprotein (LDL) cholesterol and an increase in high-density lipoprotein (HDL) cholesterol concentrations. Recent investigations indicate that the effects of fibrates are mediated, at least in part, through alterations in transcription of genes encoding for proteins that control lipoprotein metabolism. Fibrates activate specific transcription factors belonging to the nuclear hormone receptor superfamily, termed PPARs. The PPARα form mediates fibrate action on HDL cholesterol levels via transcriptional induction of synthesis of the major HDL apolipoproteins, apoA-I and apoA-II. Fibrates lower hepatic apoC-III production and increase lipoprotein lipase-mediated lipolysis via PPARα. Fibrates also stimulate cellular fatty acid uptake, conversion to acyl-CoA derivatives, and catabolism by the β-oxidation pathways, which, combined with a reduction in fatty acid and triglyceride synthesis, results in a decrease in VLDL production [109-111]. The PPARα agonist fenofibrate reduces atherosclerosis in animal models of atherosclerosis [112], and also protects against cerebral injury by neuroprotective, antioxidant and anti-inflammatory mechanisms [113]. Brune at al. [114] showed that there is an association between the PPARα L162V V-allele and AD, however the mechanism is unclear.

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PPARα L162V polymorphism, especially in carriers of the insulin gene-1 allele, influences the risk for AD, possibly by altering Aβ42 CSF levels. ApoE is a lipid and cholesterol transport protein that is synthesized within the CNS. Furthermore, functions of ApoE include neuronal repair, dendritic growth, maintenance of synaptic plasticity, and anti-inflammatory activities. Of three principal alleles that exist for this protein, people with the ApoE ε4 allele, who are at a high risk of AD, have high cholesterol concentrations, whereas those with the ε2 allele, who are at a low risk of AD, have low cholesterol concentrations [115]. ApoE has also been shown to colocalize with neuropathological lesions of AD such as Aβ and NFTs. In addition, it has also been demonstrated that ApoE is essential for amyloid deposition in the APP(V717F) transgenic model of AD [2]. The risk for AD has been reported to correlate with transcriptional activity of the ApoE gene. Binding sites for putative transcriptional factors, such as AP-1, AP-2 and NF-κB, are present in the ApoE promoter. The promoter also contains sites for the inflammatory response transcription factors IL-6, STAT-1 and STAT-2. A functional PPARγ has been detected in the ApoE/ApoCI intergenic region. ApoE mRNA levels were shown to be regulated by ciglitazone, a PPARγ agonist [2]. ApoE is expressed in macrophages, where its expression increases when macrophages develop into foam cells [2]. The PPARγ is involved in this conversion. Galetto et al. [116] showed that ciglitazone doubled the levels of ApoEmRNA. In addition, the identification of a PPARδ-selective agonist allowed to link the action of PPARδ with reverse cholesterol transport. GW501516 is a potent and subtype-selective PPARδ agonist. In macrophages, fibroblasts, and intestinal cells, GW501516 increases expression of the reverse cholesterol transporter ATP-binding cassette A1 and induces apolipoprotein A1-specific cholesterol efflux [117]. 24S-hydroxycholesterol is the major elimination product of brain cholesterol and is released into the blood circulation. Most of plasma 24S-hydroxycholesterol originates from the brain and thus plasma 24S-hydroxycholesterol levels may reflect cholesterol turnover in the CNS. Altered 24S-hydroxycholesterol levels in the periphery might point to a neurodegenerative process in the CNS, as detected in AD. The levels of plasma 24Shydroxycholesterol are significantly lower in AD patients compared to healthy controls [118]. Sauder et al. [119] found that PPARγ Pro12Ala polymorphism influenced plasma 24Shydroxycholesterol/cholesterol ratios in AD patients in that carrier of the Ala allele presented with higher ratios than homozygote carriers of the Pro-allele. Furthermore the Pro allele of the PPAR γ Pro12Ala polymorphism is associated with an increased risk of Type 2 diabetes [120]. The unsaturated fatty acids bind to all three PPARs with PPARα exhibiting the highest affinity, while saturated fatty acids are poor PPAR ligands in general. On the other hand, there is a recent increase in the level of interest in the possible role of dietary fatty acids in age-related cognitive decline, and cognitive impairment of both degenerative (AD) or vascular origin. Intake of non-hydrogenated unsaturated fats, low intake of hydrogenated and saturated fats and high intake of PUFAs from fish or vegetable sources can lower the risk of AD and could act synergistically in improving cognitive functioning [106, 107]. In addition, epidemiological studies on the association between diet and cognitive decline also suggested


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a possible role of fatty acids intake in maintaining adequate cognitive functioning and possibly in preventing or delaying the onset of dementia, both of degenerative or vascular origin [121]. Dietary fats may also influence AD through other mechanisms. Higher intakes of hydrogenated and saturated fats are related to insulin resistance, and high insulin concentrations may be related to a high risk of AD. High intake of fats may also cause oxidation, which can result in AD [107]. Appropriate dietary measures or supplementation with specific micro- and macronutrients might open new ways for the prevention and management of cognitive decline and dementia [121]. Hashimoto et al. [122] investigated whether administration of DHA, PPAR agonist and major fatty acid of the brain, ameliorates the impairment of learning ability in an animal model of AD, in rats infused with Aβ40 into the cerebral ventricle. DHA administered for 12 weeks significantly reduced the increase in the number of reference and working memory errors in the Aβ-infused rats, and increased both the cortico-hippocampal level of DHA and the molar ratio of DHA/arachidonic acid, suggesting an amelioration of the impaired spatial cognition learning ability. Furthermore, DHA suppressed the increases in the levels of lipid peroxide and reactive oxygen species (ROS) in the cerebral cortex and the hippocampus of Aβ-infused rats, suggesting that DHA increases antioxidative defenses. DHA is thus a possible therapeutic agent for ameliorating learning deficiencies due to AD [122].

The Roles of Inflammation and PPARs in Alzheimer’s Disease AD is a progressive age-related neurodegenerative disorder that is the most common form of dementia affecting people 65 years and older. The exact etiologic factor of AD is unknown; however, evidence has been growing that supports the hypothesis that neuroinflammation may play a central role in the development of AD. It is not known whether neuroinflammation is primary in the disease process or a result of the disease, but it does appear that it is a part of the pathophysiologic process. Also, the CNS differs from other organs in that it contains a blood-brain barrier, a system of tight junctions at the capillaries within the CNS that obstructs the entry of inflammatory cells, pathogens, and some macromolecules into the subarachnoid space. Although not complete, this barrier acts to protect the sensitive, fragile, and post-mitotic neurons from the damages typically associated with inflammation [87, 92, 123]. Inflammatory activation of neuronal, as well as glial cells is believed to contribute to cell death and damage during this neurological disease. Inflammation develops as a consequence of activation of astrocytes and microglial cells. Over the last two decades, much evidence has accumulated which has demonstrated numerous markers for inflammation associated with plaques in the AD brain. Inflammatory components related to AD neuroinflammation include brain cells such as activated astrocytes, reactive microglia; cytokines; chemokines; interleukins; Aβ; pentraxins such as C-reactive protein; acute-phase proteins; reactive oxygen and nitrogen species; proteases; arachidonic acid derivatives; classical pathway complement proteins from C1q right through to membrane attack complex C5b-9; neuronal-type nicotinic acetylcholine receptors as well as PPARs which could be mediators of the so-called

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secondary damage [12, 124]. Both the microglia and astrocytes have been shown to generate Aβ, one of the main pathologic features of AD. Aβ itself has been shown to act as a proinflammatory agent causing the activation of many of the inflammatory components. Both in vitro and in vivo studies suggest that Aβ deposition induces abnormal τ immunoreactivity, and precedes NFT formation, and plays an important physiological function in the etiology of AD. Aβ aggregates appear to be capable of inciting an inflammatory response, and there is evidence that inflammation can promote increased Aβ production and also enhance Aβ deposition. Thus, an Aβ-induced inflammatory response could promote further Aβ accumulation and increased inflammation. On the basis of the notion that the inflammatory response to Aβ is harmful, anti-inflammatory drugs have been proposed as beneficial agents in AD therapy [125]. Accumulating evidence suggests that PPARs may play a role in inflammation. However, the molecular mechanisms mediating the anti-inflammatory action of PPARs, at present, are not fully understood. Several studies have shown that activation of these receptors may interfere with several signaling pathways regulating the expression of pro-inflammatory genes. More recently, PPAR activators were shown to inhibit the activation of inflammatory response genes (such as IL-1β, IL-2, IL-6, IL-8, iNOS, ICAM-1, COX-2, TNF-α and MMP) by negatively interfering with the NF-κB, C/EBPβ, STAT-1 and NFAT and AP-1 signalling pathways. Furthermore, PPARs interact with p65. PPARs, also induce a decrease in expression of pro-inflammatory cytokines by antagonizing the activities of c-jun-NH2terminal kinase (JNK) and p38 MAPK [9, 126-129]. PPAR activators exert these anti-inflammatory activities in different immunological and vascular wall cell types such as monocyte/macrophages, endothelial, epithelial and smooth muscle cells in which PPARs are expressed. These recent findings indicate a modulatory role for PPARs in the control of the inflammatory response with potential therapeutic applications in inflammation-related diseases, such as atherosclerosis [130], psoriasis [131], rheumatoid arthritis [132, 133], asthma [134], acute myocardial infarction [135] and inflammatory bowel disease [136]. PPAR agonists may also be effective in the treatment of chronic inflammatory disorders of the CNS including AD [137, 138], autoimmune encephalomyelitis [139], multiple sclerosis [140] and Parkinson’s disease [141]. A great amount of experimental evidence indicates that PPARs are related with the pathophysiology of AD [87, 137, 142]. On the contrary, the role of PPARs in the AD-related neuroinflammation process is still not well known. It has been recently shown that PPAR ligands, including fibrates, TZDs and NSAIDs have potent anti-inflammatory effects [143]. Of relevance to AD is that PPAR agonists have been demonstrated to have similar antiinflammatory effects on neurons, microglial cells and astrocytes. All PPAR isotypes are detected and found to exhibit specific patterns of localization in different areas of the brain. PPARs may have specific functions in regulating the expression of genes involved in neurotransmission and neuroinflammation. Kitamura et al. [7] suggested that PPARγ expression could be detected by Western analysis in the temporal cortex of the human brain. Significantly, they found an approximately 50% increase in the amount of immunoreactive PPARγ protein in brains of AD patients. Since PPARγ expression are increased in Alzheimer brains, it is likely that at least some effects of PPAR activators on AD pathology are mediated through changes in the activities of PPARs.


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It has been demonstrated that inflammatory processes play a critical role in AD risk and progression [144]. Furthermore, a large number of experimental, clinical and retrospective and prospective epidemiological studies indicate that chronically NSAIDs administered groups of individuals have a reduced risk for AD, delayed disease onset, ameliorated symptomatic severity and slowed cognitive decline. NSAIDs can also lower amyloidogenic Aβ42 [125, 145-147]. In addition, NSAID treatment has positively correlated with a reduction of plaque-associated, activated microglia in humans and transgenic mice [148-150]. The PPARs have been shown to play a role in the regulation of inflammatory processes in the body [151], and some NSAIDs directly bind to PPARs and activate its transcriptional regulatory activities. In addition, PPARs has been shown to inhibit the expression of wide range of proinflammatory genes, these receptors appears to be a good candidate to mediate the observed anti-inflammatory effects [34, 35, 126, 152]. It is very important that, Lehmann et al. [34] have identified a novel target of NSAID actions, the ligand-activated nuclear receptor PPARs. These findings suggest that the anti-inflammatory effects of NSAIDs may not occur exclusively through their inhibition of COXs, but also as a consequence of the ability of these drugs to directly activate PPARs and inhibit proinflammatory gene expression [126, 153-156]. Combs et al. [126] reported that a variety of PPARγ agonists, including the NSAIDs indomethacin and ibuprofen, the TZDs troglitazone and ciglitazone, and 15d-PGJ2, inhibited the Aβ-induced stimulation of IL-6 and TNF-α expression by monocytes/microglia. Heneka et al. [154] demonstrated that, in cerebellar granule cell cultures, the NSAID PPARγ agonists indomethacin and ibuprofen, as well as the natural PPARγ ligand 15d-PGJ2, inhibited LPSstimulated neuron death. Kim et al. [157] showed that troglitazone, ciglitazone and 15d-PGJ2 inhibited LPS-induced death of neurons in cortical neuron-glia cocultures. Furthermore, troglitazone inhibited glutamate toxicity of cerebellar granule neurons [94]. Specifically, indomethacin, fenoprofen, flufenamic acid and ibuprofen act as PPARγ agonists. Recent studies in AD mouse models have shown that chronic treatment with a subset of NSAIDs (e.g. ibuprofen, flurbiprofen, indomethacin) reduced brain inflammation and Aβ levels in addition to the deposition of Aβ in brain [158-161]. NSAID ibuprofen, a COX-1 and COX-2 inhibitor as well as a PPAR pan-agonist, decreases the production of NO, protects neurons against glutamate toxicity and decreases the production of proinflammatory cytokines. Ibuprofen crosses the blood-brain barrier and suppresses neuritic plaque pathology and inflammation in AD brain. Furthermore, it is a potent free radical scavenger, and could reduce lipid peroxidation and free radical generation [162]. Hence, ibuprofen treatment reduced the amount of Aβ deposited in the brains of an animal model of AD and acts to reduce microglial activation and cytokine production in transgenic mice overexpressing APP [158, 164, 165]. Sastre et al. [165] showed in vitro that NSAIDs, such as indomethacin and ibuprofen, as well as several structurally different PPARγ agonists, reduce immunostimulated Aβ production in a PPARγ-dependent manner. Similarly, Yao et al. [166] demonstrated that the combination of antiinflammatory (indomethacin) and antioxidant (vitamin E) drugs significantly decreased soluble and insoluble Aβ40 and Aβ41 in neocortex and hippocampus and suppressed brain inflammatory and oxidative stress responses in transgenic mice model of AD. Morihara et al. [167] demonstrated that PPAR pan-agonist and NSAID ibuprofen

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suppresses IL-1β induction of pro-amyloidogenic α1-antichymotrypsin to ameliorate Aβ pathology in APPsw (Tg2575) mice of Alzheimer's models. TZDs are potent neuroprotective and anti-inflammatory compounds in neuronal cells through, at least in part, activation of the nuclear receptor PPARγ. TZDs inhibit inflammatory activation of cultured brain astrocytes and microglia by diminishing LPS-induced IL-6, TNFα, iNOS, and inducible COX2 expression. In addition, TZDs inhibits TNF-α and NO production and, concomitantly, protects cortical neurons from cell death induced by cell-free supernatant from activated microglia. These effects are completely inhibited by GW9662, a PPARγ antagonist [92]. Heneka et al. [168] reported that 7 day oral treatment of the specific PPARγ agonist pioglitazone or PPAR pan-agonist and NSAID ibuprofen resulted in a reduction in the number of activated microglia and reactive astrocytes in the hippocampus and cortex in APPV7171 mice. Drug treatment also reduced the expression of the proinflammatory enzymes COX-2 and iNOS. In parallel to the suppression of inflammatory markers, pioglitazone and ibuprofen treatment decreased β-secretase-1 mRNA and protein levels. Importantly, the researchers observed a significant reduction of the total area and staining intensity of Aβ42-positive amyloid deposits in the hippocampus and cortex. Additionally, animals treated with pioglitazone revealed a 27% reduction in the levels of soluble Aβ42 peptide. These findings demonstrate that antiinflammatory drugs can act rapidly to inhibit inflammatory responses in the brain and negatively modulate amyloidogenesis. Bernardo et al. [169] demonstrated that 15d-PGJ2 attenuated TNF-α and NO production in primary cultures of microglia stimulated with LPS, as well as reducing INFγ-induced MHC class II expression. The effects of 15d-PGJ2 on LPS-induced NO and TNF-α production were found to be mediated by PPARγ, in that ciglitazone, a specific synthetic PPARγ agonist, induced similar responses in microglia. These researchers showed that PPARγ is activated in rat microglial cells by the anti-inflammatory drug HCT1026, a derivative of flurbiprofen [161].

The Roles of Oxidative Stress and PPARs in Alzheimer’s Disease Free radicals are atoms or molecules that have a single unpaired electron in their outer orbit. They are chemically unstable and highly reactive compounds, which are formed during normal cellular metabolism. The most important free radicals produced are ROS and reactive nitrogen species (RNS) [90, 170]. The average cell utilizes 1013 O2 per day. It is estimated that 1% of respired molecular oxygen will form O2−, thus approximately 1011 free radical species are produced by each cell in a day [171]. Free radicals may also be produced by endogenous and environmental sources. Due to their reactivity, they can be responsible for cellular and tissue damage anytime their generation exceeds the endogenous ability to destroy them. This condition is also known as oxidant or oxidative stress. ROS can attack PUFAs that lead to lipid peroxidation [87].


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Oxidative stress is very important in the CNS. Compared with other organs or tissues, the brain is more sensitive to ROS- and RNS-induced damage due to its high rate of oxygen consumption, high polyunsaturated lipid content, and relatively low antioxidant defense enzymes and antioxidants [172-175]. In addition, it contains high levels of transition metals, especially iron, which can catalyze the formation of oxygen radicals [173, 174]. The brain uses glucose for energy production and needs about 4 × 1021 molecules every minute. As the mitochondria in aerobes, are the fount of ATP synthesis, this provides an explanation as to why deep hypoglycemia and inhibitors of ATP synthesis can cause neuronal cell death [176, 177]. The production of radicals is thought to be higher in neuronal cells due to heightened oxygen metabolism. Although the brain only constitutes 2–3% of total body mass, it utilizes 20% of basal oxygen supplied to the body. While most of the radicals are sequestered in the mitochondria, oxidative damage is exacerbated by age, metabolic demand, and disease conditions such as AD [171]. The adult brain contains about 1011-1012 neurons, which are supported and protected by at least twice as many neuroglial cells. There are several types of glial cells, oligodendrocytes, microglia and astrocytes. Microglia are the equivalent in the nervous system as monocytes-macrophages [175]. ROS and RNS are the cause of oxidative stress in nervous system. Classically oxidative stress is described as an imbalance between generation and elimination of ROS and RNS. This imbalance between cellular production of ROS and the inability of cells to defend against them is called oxidative stress [178]. Oxidative stress can cause cellular damage and subsequent cell death because ROS oxidizes cardinal cellular components, such as lipids, proteins, and DNA [4, 170, 171]. When oxidative stress occurs, cells function to counteract the resulting oxidant effect and to restore the redox balance. Furthermore, detoxifying enzymes, e.g., superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx) and glutathione reductase (GSSGR) may be altered in their activity or protein synthesis [179, 180]. All organisms have adaptive responses to oxidative stress, such as activation of genes encoding defensive enzymes, transcription factors, and structural proteins. Antioxidants are exogenous (natural or synthetic) or endogenous compounds acting in several ways, including removal of O2, scavenging ROS or their precursors, inhibiting ROS formation, and binding metal ions needed for the catalysis of ROS generation. In summary, the natural antioxidant system can be classified into two major groups: enzymatic (e.g., SOD, CAT), and nonenzymatic or low-molecular-weight antioxidants (LMWAs). The LMWA group of molecules can be further classified into directly acting antioxidants (e.g., scavengers and chain-breaking antioxidants) and indirectly acting antioxidants (e.g., chelating agents) [4]. Oxidative stress and free radical damage have been suggested to play role in several pathological processes including ischemia-reperfusion, inflammatory response and AD. In addition, oxidative stress increases with aging [181]. Lipids, proteins and nucleic acids that have been damaged by free radicals accumulate in the brain and other organs with age, and are implicated in many age-related diseases, including neurodegenerative diseases. Free radical theory of aging is postulated by Harman (1956) [182]. Many antioxidants such as vitamins have shown beneficial effects in different biological systems in which they were

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able to prevent age-associated damage, to lower the incidence of age-associated diseases and to increase lifespan [183]. AD is the most common, complex and challenging form of neurodegenerative disease with dementia in the elderly. Although oxidative damage is well documented in AD [185189], the sources of reactive oxygen and nitrogen species triggering such damage to various macromolecules is yet to be identified. It is not known whether oxidative damage is primary in the disease process or a result of the disease, but it is apparent that it is a part of the pathophysiologic process. Disturbance in the prooxidant/antioxidant balance increases during aging; moreover, the production of free radicals appear to play a significant role in this neurodegenerative process. These processes suggest that oxidative damage is one of the factors in neuronal death underlying the loss of cognition [171]. This is based on the premise that if oxidative stress occurs when there is an imbalance between free radical generation and antioxidant availability, then antioxidants may be used to scavenge excess free radicals and modulate the development and the progression of the AD [190]. Indeed, individuals affected by AD show significantly increased oxidative damage to every class of biological macromolecules including sugar (i.e., glycosylation and glycation), lipid (i.e., lipid peroxidation), protein (i.e., protein oxidation/ nitration) and nucleic acid (i.e., DNA/RNA oxidation) in the brain [171, 191]. There is considerable evidence indicating that oxidative stress is an early event in AD, occurring prior to cytopathology [192, 193]. Indeed, these oxidative mechanisms are involved in the cell loss and other neuropathology associated with AD is evidenced by the large number of metabolic signs of oxidative stress as well as by markers of oxidative damage [171, 192, 194, 195]. Moreover, both NFTs and Aβ-containing plaques seem to be closely associated with markers of oxidative stress, but the exact interrelations between oxidative stress and its pathology have not been determined. The Aβ peptide is the major component of the senile plaques that are hallmark findings in the AD-affected brain. Aβ is the proteolytic product of APP. Large soluble fragments of APPs that are the result of APP cleavage within its Aβ domain are secreted into the extracellular medium. Overexpression of APP can accelerate Aβ secretion, which can form insoluble amyloid aggregates in the presence of amyloidotrophic factors, contributing to the development of AD [189]. Free radicals are produced by the Aβ peptide once it is formed outside the neurons, and were found to be neurotoxic to hippocampal cells and the synaptosomal membranes. As a result, oxidative damage in AD pathogenesis may involve two known pathways. In vitro, direct Aβ application to neuronal cells increase hydrogen peroxide production through metal ion reduction [196], leading to Aβ neurotoxicity. Indirect Aβ neurotoxicity from microglia stimulates superoxide and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, iNOS and NO [197]. In summary, on one hand, Aβ was shown to be sensitive to the action of free radicals, contributing to aggregation. On the other hand, the effect of Aβ accumulation, including that arising from an Aβ-initiated inflammatory response, may include excessive generation of free radicals [197, 198]. Evidence for oxidative stress derives from both human (post-mortem and living patients) studies, and transgenic mouse models of the disease. There is a long list of surrogate markers of ROS-mediated injury that have been found to be increased in the brain, CSF and urine of


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AD patients. It includes malondialdehyde, 4-hydroxynonenal, F2-isoprostanes (lipid peroxidation); protein carbonyls, nitrotyrosine (protein oxidation); 8-hydroxy-2'deoxyguanosine (DNA oxidation) [170, 193, 199]. Transgenic animals show the same type of oxidative damage that is found in AD, and it directly correlates with the presence of Aβ deposits. Many studies also demonstrated that the prevalence of hippocampal atrophy increased with age and was very common in AD [200]. Moreover, several investigators detected changes in the antioxidant enzyme activity of SOD, CAT, GSH-Px, GSSGR and heme oxygenase-1 in several areas of the brain [201]. The direct measurement of a product of lipid peroxidation in the brain tissue and ventricular fluid of patients with AD provides important evidence of the occurrence of oxidative stress in the brain of AD patients. Assessment of free radical-induced oxidative stress in the brains of patients with AD has revealed an increase in thiobarbituric acidreactive substances in the hippocampus [202] and the temporal cortex [203, 204], indicative of significant lipid peroxidation. The aldehyde product of lipid peroxidation, 4hydroxynonenal, is significantly increased in the amygdala, the hippocampus, and the parahippocampal gyrus of the AD brain and coincides in these areas with histopathology characteristic of AD [205]. Elevated levels of 4-hydroxynonenal are also found in the cerebrospinal fluid of patients with probable AD [206]. Isoprostanes are members of a complex family of lipids, isomers of conventional enzymatically derived PGs, which are produced by oxygen radical-catalyzed peroxidation of PUFAs. Most of the work has been focused on a group of isomers of the PGF2α, called iPF2α, and an abundant literature has established that their measurement provides a reliable marker of in vivo lipid peroxidation and oxidative stress [188, 207]. Pratico et al. [209] found that, compared with controls, patients with a clinical diagnosis of AD had increased CSF, plasma, and urinary levels of a major isoprostane, 8,12-iso-iPF2α-VI. In addition, CSF tau protein levels increase and the percentage ratio of Aβ42 decreases with the progression of AD. These researchers showed a direct correlation between CSF 8,12-iso-iPF2α-VI levels and CSF tau and an inverse correlation with CSF Aβ42 in AD patients [208]. These findings suggest that elevation of isoprostane not only reflects an increase in CNS oxidative damage but also correlates with the progression of the disease. Levels of antioxidant enzymes were also measured in the AD-affected brain. Enzymes such as CAT, GSH-Px and GSSGR are important in preventing excessive build-up of free radicals. It was shown that levels of GSH-Px were elevated in the hippocampus; levels of GSSGR were elevated in the hippocampus and amygdala; and levels of CAT were elevated in the hippocampus and temporal neocortex of AD-affected brains as compared with controls [201]. These elevations correlated with increased lipid peroxidation in the same regions. This could indicate that the brain of a patient with AD is attempting to respond to the challenge of increased oxidative stress by increasing the availability of antioxidant enzymes. Upregulation of antioxidant enzymes found in vulnerable neurons in AD indicates that ROS not only cause damage to cellular structures but also provoke cellular response. Gsell et al. [210] measured activities of the enzymes, SOD and CAT, which detoxicate ROS. Enzyme activities were measured postmortem in basal ganglia, cortical and limbic brain regions of patients with dementia of Alzheimer type (DAT) and age-matched controls. SOD activity increased with age in basal nucleus of Meynert. However, there was no

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significant difference in SOD activity between DAT and controls. CAT activity was independent of age and postmortem time. There were significant reductions in CAT activity in parietotemporal cortex, basal ganglia, and amygdala in DAT compared to controls. Their findings are in line with the assumption that ROS could contribute to the pathogenesis of DAT. Over the past 15 years, numerous studies have identified hippocampal atrophy as a predictor of the decline from mild cognitive impairment (MCI) to AD [10, 200]. AD-related volume losses are most readily detected in the hippocampus in MCI, and indicate that in predicting the transition to dementia, it is important to consider both hippocampal and lateral temporal lobe volume reductions. Studies by Hampel et al. [211] suggest that elevated Ptau231 measurements may be specific for AD. The p-tau proteins in CSF come closest to fulfilling the criteria of a biological marker of AD. Antioxidants of widely varying chemical structures have been investigated as potential therapeutic agents for neurodegenerative diseases such as AD. In vitro and animal studies suggest that various compounds with antioxidant ability can attenuate the oxidative stress induced by Aβ [212-214]. Recently, clinical trials have demonstrated potential benefits for treatments with the antioxidants, vitamin E, ascorbic acid, carotenes, selegiline, curcumin, Gingko biloba, L-carnitine and idebenone [107, 215, 216]. Vitamin E, a fat-soluble vitamin, has been the most widely studied antioxidant [217]. Recent prospective studies have indicated that dietary intake of several exogenous antioxidants is associated with a lower risk for AD, suggesting that intervention with antioxidants might be beneficial in the early phases of AD or in people at risk for developing AD [218, 219]. Generally, to achieve efficacy, a candidate antioxidant must penetrate the blood-brain barrier to attain a critical therapeutic level within the CNS and must be given as early as possible, before the irreversible neuronal loss. It also should fit the precise oxidative stress physiology, for example, the type of ROS involved, the place of generation, and the severity of the damage. Thus, antioxidant cocktails or antioxidants combined with other drugs may have more successful synergistic effects [4]. Several studies demonstrated that PPARγ agonists ameliorated the lesions associated with ischemia-reperfusion of gastric tissue, heart, brain, lung, kidney and intestine possibly due to the antiinflammatory and antioxidant properties. Meanwhile, PPARs agonists have potent antioxidant effect [220-223]. Yao and colleagues examined whether the simultaneous administration of an antioxidant, vitamin E, with an anti-inflammatory drug, indomethacin, would exert an additive anti-amyloidogenic effect in the Tg2576 mouse model of AD-like Aβ brain amyloidosis, one of the most extensively studied mouse models of AD [166]. Villegas et al. [222] investigated the effects of the rosiglitazone on gastric injury caused by ischemia following reperfusion in rats. Rosiglitazone significantly reduced the myeloperoxidase, as a marker of neutrophils infiltration, and the levels of TNF-α. Rosiglitazone did not revert the reduced GSH-Px activity but enhanced significantly the decreased xanthine oxidase and SOD activities in gastric mucosa of ischemic rats. In conclusion, rosiglitazone reduces the damage in ischemia-reperfusion gastric injury and alleviates the inflammatory response and the oxidative events. Liu et al. [224] demonstrated that phenylbutyric acid activates PPARs in astrocytes, and binds to PPARα and γ. Phenylbutyric acid maintaines CAT protein levels in brain of ts1-infected mice, and delays the hindlimb paralysis caused by ts1 infection. The


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authors suggest that ts1-induced oxidative stress in infected astrocytes that is alleviated by phenylbutyric acid is mediated via PPARα and/or PPARγ. Uryu et al. [94] suggested that troglitazone inhibits both post-glutamate neurotoxicity and low-potassium-induced apoptosis in cerebellar granule neurons, and has neuroprotective and antioxidant effect; it acts as a dual-inhibitor against both exicotoxicity and apoptosis. Furthermore, in other studies rosiglitazone, PPARγ agonist, improved endothelium-dependent vasodilatation, preserved PVASP, suppressed gp91phox and iNOS expression, reduced superoxide and total NOx production, and inhibited nitrotyrosine formation most likely by attenuation of oxidative and nitrative stresses [108]. Tao et al. [108] showed that rosiglitazone enhanced PPAR expression, improved endothelium-dependent vasodilatation, preserved the phosphorylation of vasodilator-stimulated phosphoprotein, suppressed gp91phox and iNOS expression, reduced superoxide and total NOx production, and inhibited nitrotyrosine formation in hypercholesterolemic rabbits. Moreover, Klucis et al. [225] reported that administration of PPARα activators results in a drastic increase of the activity of CAT, an antioxidant enzyme. In conclusion, since oxidative damage is a key phenomenon in AD, treatment with antioxidants such as PPAR agonists seems to be a promising approach for slowing disease progression to the extend that oxidative damage may be responsible for the cognitive and functional decline observed in AD [226].

Conclusion AD is the most common, complex and challenging form of neurodegenerative disease with dementia in the elderly. Because of their anti-inflammatory and antioxidant activity, and modulating effects of glucose and lipid metabolism PPAR agonists are currently being developed for clinical use, and they may be a promising new therapeutic approach for the treatment of AD.

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Chapter VI

Role of Sphingomyelin Cycle Signaling System in Alzheimer’s Disease A. V. Alessenko∗ Institute of Biochemical Physics of the Russian Academy of Sciences, Moscow, Russia

Abstract Alzheimer’s disease (AD) is characterized by a progressive decline in cognition, memory and intellect. Possible mechanisms for AD neurotoxicity include: alteration in Ca2+ homeostasis, activation of specific receptors affecting cellular homeostasis, activation of oxidative processes, direct disruption of membrane integrity and disorders in lipid metabolism, or a combination of two or more of the above mechanisms. It was established that conformational changes in amyloid-beta peptide (Aβ) may occur in lipid rafts under the control of specific sphingolipids. This review demonstrates a novel mechanism for development of AD. It has been hypothesized that Aβ and TNF-α may have a prominent role in neurodegeneration. It is well-known that neuronal death is developed according to apoptotic program. Most signaling pathways that trigger apoptosis remain unknown, but the sphingomyelin pathway has been recognized as an ubiquitous signaling system that links specific cell-surface receptors and environmental stresses to the nucleus. This pathway is initiated by the hydrolysis of sphingomyelin via the action of sphingomyelinases to generate ceramide. Ceramide then serves as a second messenger in this system, leading to apoptosis. Oxidative mechanisms have been implicated in this pathway. TNF-α activates receptors linked to multiple effector systems, including a sphingomyelin pathway and peroxide oxidation. Recently, it was shown that superoxide radicals are used as signaling ∗

Telephone: 7(495) 939 71 59; email: [email protected]

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A. V. Alessenko molecules within the sphingomyelin pathway. It means that cross-talk between the oxidation system and sphingomyelin cycle exists in cells and could have important implications for the induction phase and evolution of AD. The role of TNF-α in the pathogenesis of AD is unclear, because it has been shown to be involved in both neuroprotection and neurodegeneration, depending on doses and the age of animals. Small doses of this cytokine induced a protective effect against Aβ neurotoxicity. It was suggested that using low doses of TNF-α in the clinic for the prevention of AD development might be a perspective. Identification of the N-SMase-ceramide pathway may lead to the development of more effective therapeutic strategies, aimed at preventing Aβ-induced cell death.

Introduction Alzheimer’s disease (AD) affects about 15 million individuals worldwide. The prevalence of this disease doubles every 5 years after age 65 and approaches 50% by age 85. Because of the ongoing increase in life expectancy, the number of people affected by this disease is rapidly increasing. The major risk factor for late-onset AD is aging [1]. The molecular events that mediate the effect of aging on AD are the subjects of intensive study. AD is characterized neuropathologically by amyloid deposits, neurofibrillary tangles, and selective neuronal loss [2]. The major component of the amyloid deposits is β-amyloid peptide (Αβ - a family of 39-43-residue peptides proteolytically derived from the amyloid precursor protein (APP). APP is first cleaved by β-site APP –cleaving enzyme 1 (BACE1) at the N terminus of Aβ (β-cleavege), producing a C-terminal fragment (β-APP-CTF) of12 kDa, and subsequently in the transmembrane domain (γ-cleavage) by a presenilin-harboring protease complex. The two major sites of γ-cleavage are located at position 40 and 42 of Αβ, generating Aβ40 and Aβ42, respectively. Aβ in AD brains exist either as amorphous deposits called diffuse amyloid or as dense cores of deposits called senile plaques [3]. While diffuse amyloid is present in both normal-aged and AD brains, senile plaques are found only in AD brains, where they are associated with dystrophic neurons [4]. Increasing evidence from genetic [5], pathological [6] and cell culture [7] studies have implicated Aβ as a toxic agent in AD. The membrane lipid ceramide is the backbone of all complex sphingolipids and acts as a second messenger in many biological events. Ceramide is involved in stress-activated apoptosis. The intracellular levels of ceramide increase progressively during aging in both cultered cells and the whole organ [8-11]. In addition, brains from AD patients contain approximately three times more ceramide when compared with a normal brain [12]. The AD brain is subjected to increased oxidative stress resulting from free radical damage and the resulting cellular dysfunctions are widely believed to be responsible for neuronal degeneration in this disorder [13, 14]. Aβ-induced oxidative stress alters membrane lipid metabolism, resulting in increased amounts of ceramides [15]. Possible mechanisms for Aβ neurotoxicity include: alteration in Ca2+ homeostasis, activation of specific receptors affecting cellular homeostasis, activation of oxidative processes, direct disruption of membrane integrity and disorders in lipid metabolism, or a combination of two or more of the above mechanisms.

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Role of Membrain Sphingolipids and Cholesterol in Conformational Transition of the β −amyloid Peptide To understand the neurotoxic action of Aβ, it is essential to identify specific cellular components that interact with the peptide and mediate a biological response of the affected cells. A likely primary target of Aβ is the neuronal plasma membrane. Indeed, a rapidly growing number of observations indicate that the peptide may alter important physical and biological properties of the membranes [16, 17]. The mechanisms of Aβ-membrane interaction remain, however, elusive. It was demonstrated [18] that Aβ40 forms cationselective channels in membranes and have been speculated that these channels disrupt ion homeostasis and thus cause toxicity. Aβ underwent a conformational transition from an alpha-helix-rich structure to a betasheet-rich structure with the increase in protein density on the membrane. The experimental studies provide evidence suggesting that Aβ neurotoxicity stems from interactions of Aβ with membrane proteins or lipids. It was shown by biophysical techniques that phosphatidylcholine (PC) / phosphatidylglycerol (PG) vesicles interact with Aβ-peptide [19]. Several results indicate that Aβ neurotoxicity may be mediated, at least in part, by direct interaction between Aβ and membrane phospholipids and gangliosides. The major conformational transition of the Alzheimer beta-amyloid peptide has been observed upon interaction with sphingolipid-containing membranes. Alzheimer Aβ contains a mutation domain that was found to interact with monomolecular films of galacosylceramide and sphingomylin at the air-water interface. [20] It has been found that Aβ undergoes a conformational transition from random coil to ordered structure rich in β-sheet after the addition of lipid vesicles containing negatively charge lipids and when it binds to the membrane containing the ganglioside GM1 [21]. Aβ binds with high affinity and selectivity to gangliosides, which belong to the sphingomyelin family. Furthermore, in the presence of ganglioside-containing membrane vesicle, there is a dramatic increase in the rate of fibril formation by the peptide [22, 23]. It was postulated that the membrane-bound Aβ may act as a template that catalyzes the fibrogenesis reaction in vivo. Ganglioside-bound Aβ species were found in brains exhibiting early pathological changes of Alzheimer’s disease [24]. This form was identified as Aβ42 and speculated to be localized on the cell surface where it can act as a seed for amyloid fibril formation [22, 24]. Identification of factors that initiate formation of GM1/Aβ may be crucial for determination of the pathogenesis of AD and for development of preventive and curative treatment strategies. It was established that the gandlioside species-specificity is responsible for a conformational changes of Aβ by which ganglioside-bound Aβ acts as a seed for Aβ fibrillogenesis. Aβ recognized ganglioside clusters composed of major gangliosides occurring in brains (GM1, GD1a, GD1b and GT1b), the density of which increased with the number of sialic acid residue. Interestingly, however, mixing of gangliosides inhibited cluster formation. GM1- Aβ exhibited the strongest seeding potential, especially under beta-sheet-forming

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conditions [25]. This study suggested that lipid composition of rafts including gangliosides, sphingomyelin, cholesterol and ceramide strictly controls amyloid formation. So, it appears that the peptides β-sheet structure may facilitate the association with lipid membranes. Ganglioside forms clusters that Aβ can recognize. [21-25]. An important role in Aβ aggregation and neurotoxicity may play caveolae membrane domaines or rafts, specialized transmembrane exchange zones implicated in cell signaling. They are enriched in glycosphingolipids, cholesterol, sphingomyelin (SPM) and lipidenchored membrane proteins. [26]. It was established that conformational changes in Aβ may occur in lipid rafts under the control of specific sphingolipids [20]. Membrane rafts composed of sphingomyelin have van der Waals interactions and hydrogen bonds between the sugar head groups and the sphingosine backbones leading to stronger lateral cohesion resulting in a higher gel-to-liquid phase transition. The amidebounded fatty acid in ceramide ranges from 20 to 26 carbon atoms in length, compared with 16 to 22 for the fatty acids of glycerolipids. In addition, SPH fatty acids are more saturated compared with PtdCho fatty acids, resulting in less lateral packing of the PtdCho molecules. A putative galactosylceramide (GalCer)-binding motif was indentified in Alzheimer’s βamyloid peptide. Interaction of Aβ with GalCer and sphingomyelin was studied by monomolecular film binding assay [20]. Aβ recognizes sphingomyelin as well as GalCer. The ability of Aβ-peptide to recognize both sphingolipids is likely due to the presence of both sugar-binding residues (Tyr10, His13, His14, Phe20, Phe21) and acid residues (Asp7, Glu11) within peptide [20]. It is known that cell surface SM in rafts is mostly found in cholesterol-rich domains [27, 28]. The planar steroid ring of cholesterol can interact more closely with saturated fatty acids of SPH than with the fatty acids of PtdCho, which contains the kink at C9-C10. Hydrolysis of SM has been reported to reduce the clustering of cholesterol into cholesterol-rich domains and to induce retrotransport of cholesterol to the endoplasmic reticulum [27]. Cholesterol level has been reported to be reduced in the brains of AD patients [29]. The 30% reduction in cholesterol level was associated with 3-4 A reduction in the bilayer width of membrane prepared from AD brain [29]. However, it should be noted that post-mortern of an AD brain reflects only the last stage of a progressive neurodegenerative process that started decades before. But recently it was shown that treatment with cholesterol-lowering statins drastically lowered the risk of AD developing [30]. Changes in membrane cholesterol disrupts the formation and function of caveolae, suggesting that these membrane microdomains containing cholesterol are involved in a range of biological processes including the trafficking and proteolytic processing of embedded proteins. Biophysical properties of SPH/cholesterol enriched rafts probably influence the lipidlipid and lipid-protein interactions in the rafts [31, 32]. It is not known if amyloid precursor protein (APP) is transported and proteolytically processed by secretases (αβ or γ while inserted into SPH/cholesterol rafts, but if so, an alteration in rafts lipid composition could conceivably alter both the transport and processing of APP.


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To elucidate the molecular mechanisms underlying GM1/ Aβ formation, the effects of cholesterol and ethers lipid on the binding of Aβ to GM1-containing lipid bilayers were examined in detail using fluorescent dye-labeled human Aβ [33]. Increases in not only GM1 but also cholesterol contents in the lipid bilayers facilitated the binding of Aβ to the membrane by altering the binding capacity, but not binding affinity. An increase in membrane-bound Aβ concentration triggered its conformational transition from helix-rich to β-sheet rich structures [33]. It was suggested that increases in intramembrane cholesterol content, which are likely to occur during aging, appear to be a risk factor for amyloid fibril formation. It was reported [15] that alterations in sphingolipid and cholesterol metabolism during normal brain aging and in the brain of AD patients results in the accumulation of long-chain ceramides and cholesterol. Analysis of 10 different brain regions from patients affected by Alzheimer’s disease /senile demetia of Alzheimer’s type have shown that sphingomyelin content decreased in regions rich in myelin. Cholesterol amounts were highly variable, whereas ubiquinone concentrations increased by 30-100% in most regions of the brain was affected by AD. [34]. Treatment of neurons with alpha-tocopherol or an inhibitor synthesis prevents accumulation of ceramides and cholesterol and protects them against death induced by Aβ. In the endoplasmic reticulum excess cholesterol activates the enzyme acyl-coenzyme A:cholesterol acyltransferase which was implicated in Aβ generation [35]. At the same time it was shown that intracellular levels of ceramide- product of SM digestion regulate Aβ generation by modulation β-secretase cleavage of APP. N-SMase, fumonisin1 and NB-DGJ, general inhibitor of the SM/glycosphingolipid metabolic pathway, caused changes in the ceramide levels, which were consistently paralleled in Aβ generation. It was found that ceramide controls the processing of APP by affecting the molecular stability of the βsecretase, BACE1 [36]. Presented studies suggest that lipid composition of rafts including gangliosides, sphingomyelin, cholesterol and ceramide strictly controls amyloid processing and aggregation.

Role of the Neutral Sphingomyelinase-Ceramide Pathway in Neurodegenerative Damages Numerous molecules involed in cell signaling have been identified in caveolae, suggesting that these structures may serve to compartimentalize, modulate and integrate signaling events at the cell surface. Alteration in the expression of caveolin genes of signaling enzymes has been implicated in Alzheimer’s disease. One of such kind of enzyme is sphingomyelinase (SMase) that controls metabolism of sphingomyelin- the main component of membrane sphingolipids in the brain. Sphingolipids (SLs) are ubiquitous components of eukariotic cell membranes. Relatively high levels of them are located in the membranes of neuronal tissues [37]. Sialylated glycosphingolipids (GSLs), the gangliosides are essential membrane components. Except

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gangliosides, a large number of individual SL species exist, resulting from differences in both the hydrophobic ceramide moiety and in the polar head group. Ceramide consists of a long chain base (sphingosine, sphinganine or phytosphingosine) to which a variety of fatty acids are attached via N-acylation. The polar head group consists of phosphorylcholine, sugar or sulfatide residues. Equilibrium of balance between specific SLs is essential for normal neuronal function [37]. This becomes apparent when considering the SL storage diseases, such as Gaucher, Tay-Sachs and Nieman-Pick A and B diseases in which GleCer, ganglioside GM2 and sphingomyelin accumulate respectively due to a decreased activity of the lysosomal enzymes responsible for their degradation [38, 39]. Although SLs were generally assumed to be little more than inert components of cell membranes, it is now clear that they also act as important signaling molecules, and moreover, individual SL and GSL species are known to function in regulating specific biochemical events in signaling pathways. Over the past decade, relatively simple SLs, such as ceramide, sphingosine, sphingosine-1-phosphate and glucosylceramide play important roles in neuronal functions by regulating rates of neuronal growth, differentiation and death [37]. Ceramides can be generated either from de novo synthesis or by the action of sphingomyelinase (ceramide-phosphocholine phosphodiesterase, EC 3.1.4.12), which cleaves phosphorylcholine from SPH resulting in the formation of ceramide. Five major isoforms of SMases are known, classified primarily according to their pH optima, localization in cell structures (plasma membrane, lysosomas, inner membranes) and cation-dependency [40]. The best characterized Smase is the lysosomal acid SMase (A-SMase), whose deficiency is responsible for Nieman-Pick disease [39]. A-SMase is highly expressed in brain cells and its expression level does not change significantly during development. Neutral membrane-bound SMase (N-SMase) is expressed at the highest level in the brain, and its expression increases during neuronal development [41]. Various factors such as cytokines, growth factors, hormones, oxidative stress and radiation are known to activate SMases in non-neuronal tissues. Less is known about the mechanisms by which SMases are activated in neurons and glial cells. In our experiments we have shown that intracerebral and interperitoneal injection of TNF-α induced activity of NSMase in hippocampus [42, 43]. The mechanisms involved in the activation of N-SMase in response to TNF-α are becoming clear. TNF-α initiates the pathway through TNFR1 (55-kda receptor) leading to phospholipase A2 activation, generation of arachidonic acid, and subsequent activation of N-SMase [44]. In addition, proteases have also been implicated in the pathway leading from TNF-α to the activation of N-SMase. Nerve growth factor (NGF) caused an increase in ceramide levels in T9 glioma cells and hippocampal neurons [45]. Furthermore, in hippocampal neurons, it was shown that the response to NGF after the first 24 h in culture undergoes a switch from acceleration of axonal outgrowth [46] to apoptotic cell death [47], due to an increased level of ceramide generation. In hippocampal neurons, ceramide is generated via N-SMase in respose to NGF. Thus, in these primary neuronal cultures, N-SMase appears to be mainly responsible for ceramide generation in response to trophic factor [47]. In contrast, an excitotoxic stress-activated pathway of ceramide generation does involve A-SMase in hippocampal neurons [48]. It means that different SMases in the same neuron are activated for the generation of different ceramide pools in response to different stimuli.


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Ceramide is a lipid second messenger involved in many biological events that regulate terminal differentiation of neurons, cellular senescence, proliferation and death [37, 49, 50]. Chronic increase in intracellular ceramide can inhibit axonal elongation and receptormediated internalization of nerve growth factor [51]. Ceramide has been implicated in several apoptotic paradigms including trophic factor withdrawal and exposure to proinflammatory molecules [47]. Ceramide can stimulate both protein kinase and phosphatase pathways in neuronal cells. [37]. Ceramidase cleaves the acyl-amide bond of ceramide resulting in sphingosine and free fatty acid. Sphingosine can be phosphorylated by ATP and ceramide kinase to form sphingosine 1-phosphate. Sphingosine and sphingosine1-phosphate also are cell signaling molecules [52]. It is presently not known if sphingosine and sphingosine 1-phosphate levels are altered in the AD brain. Aβ and ceramide share cell death signaling characteristics. Aβ-induced apoptosis involves TNF-α p75 neurotrophin receptor, and FAS ligand [37, 53], which are cell surface receptors that transmit death signals through the sphingomyelin-ceramide pathway. [53, 54, 55, 56, 57]. Moreover, both Aβ [58] and ceramide [59] cause mitochondrial disfunction and induce oxidative stress. Cellular ceramide synthesis increases in response to stress or death signals [37, 55]. It was indicated that membrane-associated oxidative stress occurs in association with lipid alterations, and exposure of hippocampal neurons to Aβ induces membrane oxidative stress and the accumulation of ceramide species and cholesterol [15]. These findings suggest a sequence of events in the pathogenesis of AD in which Aβ induces membrane-associated oxidative stress, resulting in perturbed ceramide and cholesterol metabolism which, in turn, triggers neurodegenerative cascade that leads to clinical disease. [15]. Lower sphingomyelin level [34, 60] and higher ceramide levels [12, 34] in the AD brain have been reported. Examination of ceramide levels in the cerebrospinal fluid (CSF) or brain tissues from neurodegenerative disorders have been shown that ceramide was significantly increased in patients with Alzheimer’s disease than in patients with age-matched amyotrophic lateral sclerosis (ALS) and other neurological controls [6, 61]. In AD brains, ceramide was aberrantly expressed in astroglia in the frontal cortices, but not detected in ALS and control brains [62]. Maximal changes in ceramide contents after single intracerebral administration of TNF-α and Aβ were found in the hippocampus, and were less expressed in the cerebral cortex and cerebellum [42]. Remarkable increases in sphingomyelinase activity and content of ceramide were found after 7 days of injection of Aβ. In this study a link was found between activation of sphingomyelinase and accumulation of ceramide after 7 days injection of fibrillar A25−35 [ 42 ]. Aβ1-42 peptides, but not reverse (Aβ42-1) induce activation of sphingomyelinases and the production of ceramide in neurons [63]. Within 15 min of treatment, fibrillar Aβ1−42 peptides were able to induce the level of ceramide by more than 2-fold. After 10 h of treatment of Aβa 12-fold increase in ceramide production was observed. In contrast to fibrillar form, soluble Aβ1-42 peptides were weakly efficient in inducing the level of ceramide [63]. Several lines of evidence support the suggestion that ceramide mediates at least in part, Aβ-induced oligodenrocytes (OLG) death [64]. Both Aβ and C2-ceramide caused OLG death

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in time dependent manner. Aβ treatment increased ceramide formation in OLGs. In addition, increasing cellular ceramide release from sphingomyelin by exogenous bacterial SMase, which mimics cellular N-SMase action, also induced OLG death. Furthermore, the N-SMase inhibitors, 3-0-methyl-sphingomyelin was effective in protecting OLGs against Aβ cytotoxicity. Aβ 25-35 and Aβ 1-40 activated N-SMase, but not A-SMase, in OLGs. Antisense oligonucleotides specific for N-SMase also attenuated Aβ-induced OLG cell death, further implicating N-SMase as a mediator. Glutathione (GSH) depletion has been shown to activate N-SMase activity [66]. Buthonine sulfoximine (BSO) and diethyl maleate (DEM) have been shown to deplete GSH in various cell types [65]. Both BSO and DEM selectively increased N-SMase activity, increased ceramide level, decreased cellular GSH levels and were cytotoxic to OLGs [64]. Inhibition of ceramide degradation by ceramidase inhibitors, enhanced Aβ cytotoxicity in OLGs [65]. Although the local concentration of fibrillar Aβ peptides present in the brain of AD patients may differ from the concentration of Aβ in primary neurons used in the experiments of this study, nevertheless, these results cleary point out N-SMase as a possible therapeutic target for the prevention of neuronal damage in AD and other neurodegenerative disorders. The physiological response of neurons and other neuronal cells to ceramide generation very little is known. However, research in this area will be stimulated by recent observation that ceramides, together with cholesterol esters appear to mediate oxidative stress-induced death of neurons and apoptosis of oligodendrocytes.

Involvement of Oxidative Stress-induced Abnormalities in Sphingomyelinase Activity and Ceramide Level in Alzheimer’s Disease Oxidative stress plays a prominent role in Aβ-mediated neuronal [66] and OLG death [67]. The involvement of free radicals in the pathogenesis of AD is accepted for the following reasons: 1. Brain metabolism requires substantial quantities of oxygen. 2. Neurons are particularly sensitive to free radicals attack, because their glutathione content is very low, but their membranes contain a high proportion of polyunsaturated fatty acids. 3. β-Amyloid is sensitive to the action of free radicals, contributing to its aggregation. 4. Traces of metals (iron, copper, zinc and aluminium) capable of catalyzing reactions that produce free radicals have been found in the brain of AD patients. 5. AD is related to mitochondrial anomalies, particularly for cytochrome-c-oxidase, and these anomalies may explain the abnormal production of free radicals. 6. Aging is the principal AD risk factor and is itself related to accumulated free radical attacks. 7. Free radical scavengers reduce the toxicity of β-amyloid.


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8. The clinical use of many free scavengers (vitamin E, Ginkgo biloba extract drugs, estrogens, and the iron-chelating agents is quite successful. This evidence clearly supports the involvement of free radicals and ROS in AD. However, it is unknown whether free radicals are really one of the basic causes of the pathogenesis and neuronal damage in AD. Brain tissue is especially sensitive to oxidative injury because of its higher metabolic rate driven by glucose, lower concentration of protective antioxidants, and higher level of polyunsaturated fatty acids that are susceptible to lipid peroxidation. The Aβ cascade hypothesis postulates that Aβ generates free radicals and, thereby, oxidative disorders in cellular constituents [13]. The lipoperoxidation phenomena could have a major, or even causal, influence on the pathogenesis of the AD [68, 69]. Using postmortem material, it was reported that oxidative cellular damage (lipid peroxidation, oxidative damage of protein and DNA) is increased in certain AD brain areas [68]. Free-radical mediated long-lasting oxidative stress might play a primary role in the aggregation of Aβ and formation of extracellular amyloid plaques [68]. The effect of ROS on membrane phospholipids aggregation of Aβ could prove to be important because some alterations to membrane phospholipids may be specific to the pathogenesis of AD [14, 70]. It was shown [71] that lipid peroxidation is a major cause of the depletion of membrane phospholipids in AD. One of the products of lipid peroxidation, 4hydroxynonenal, which is found in high concentration in AD patients, proved to be toxic to hippocampal cells in culture [72]. This highly reactive product, β-aldehyde is thought to cause neuronal death by altering the ATPases involved in ionic transfers and calcium homeostasis [71]. The involvement of reactive oxygen species in the pathogenesis of AD is indicated by activation of catalase, superoxide dismutase and glutathione reductase in the hippocampus and amygdala (regions of the brain that are most affected in AD pathogenesis) [73, 74]. The increased calcium concentration could itself cause of intracellular events, resulting in increased ROS and cellular death. Recently, it was shown [75] that cerebrospinal fluid F2-isoprostane concentrations are elevated in AD patients. These compounds are produced by free radical-catalyzed peroxidation of arachidonic acid, independent of cyclooxygenase enzyme. This discovery is important because it confirms that lipid peroxidation is elevated in AD, but also because it suggests the possible use of the quantification of cerebrospinal fluid F2-isoprostane concentrations as a biomarker of this disease. Although Aβ-mediated oxidative stress induces DNA damage and activates selected transcription factors, including NF-κB and AP-1 the mechanism, by which Aβ induces oxidative stress in the AD brain remains unknown. Nitric acid and peroxynitrite appear to play a crucial role in pathogenesis of AD [76]. The observed increased expression of inducible nitric oxide synthase (iNOS) in the microglia and astrocytes surrounding the Aβ plagues and the significant increase in peroxynitrite in neurons suggest a role for peroxynitrite (ONOO-; a reaction product of NO. and O2 -.) mediated pathology in the AD brain [77]. Peroxynitrite is known to be cytotoxic because it inactivates various cellular processes by nitrosylating proteins, lipids and nucleic acids.

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Aβ(1-42 fibrous form) was reported to upregulate the induction of iNOS in astrocytes [77]. Cytokines, as well as Aβ peptides are known to induce iNOS. Cytokine-induced iNOS were reported to activate the sphingomyelin-ceramide, signal transduction pathway, generating ceramide, which, in turn, was reported to mediate the induction of manganese superoxide dismutase (MnSOD) [78]. It is by direct indications that ceramide induces expression of iNOS [79]. Ceramide-mediated upregulation of iNOS by NFκB activation and its inhibition by antioxidants as N-acetylcysteine and vitamin E suggest a role for cellular redox in the SM-ceramide cascade and induction of MnSOD and iNOS [61, 77, 78]. Presented reports demonstrating an increase in intracellular levels of ceramide in Alzheimer’s disease and the other, showing that ceramide can promote β-peptide biogenesis, substantiate the possible role of ceramide in the pathobiology of AD. Several mechanisms for induction of apoptosis by ROS have been proposed, but an integral model is not established. A growing body of evidence is slowly emerging, indicating that oxidative stress and activation of sphingomyelin pathway are intimately connected in cell cycle and apoptotic signaling [15, 61, 77, 78]. The sphingomyelin pathway has been recognized as an ubiquitous signaling system that links specific cell-surface receptors and environmental stresses to the nucleus [80]. This pathway is initiated by the hydrolysis of sphingomyelin via the action of sphingomyelinases to generate ceramide. Ceramide then serves as a second messenger in this system, leading to apoptosis. Biochemical analysis of recombinant and partially purified mouse N-SMase showed that the enzyme has reactive cysteine residues and that its enzymatic activity is redox-regulated. Indeed, N-SMase is reversibly activated by H2O2 and reversibly inhibited by GSH [81]. It means that cross-talk between the oxidation system and sphingomyelin cycle exists in living cells and could have important implications for developing apoptosis which plays an important role in many kinds of diseases including Alzheimer’s disease. It was shown that fibrillar Aβ induces the activation of N-SMase but not A-SMase in human primary neurons through NADPH-oxidase-sensitive superoxide production [63]. It was shown that, antioxidants inhibited Aβ-induced neuronal activation of N-SMase, but not A-SMase. Aβ1-42, but not Aβ42-1 peptides induced the production of superoxide in neurons. Then DPI, a specific inhibitor of NADPH oxidase, inhibited the Aβ1-42-induced production of superoxide. Primary neurons expressed p22phox, a subunit of NADPH oxidase, and antisense knockdown of p22phox strongly inhibited Aβ induced production of superoxide, suggesting that Aβ induces the production of superoxide via NADPH oxidase. Aβ1-42 peptides also induced the production of H2O2 in neurons, and antisense knockdown of p22phox reduced Aβ-induced production of H2O2, suggesting that superoxide generated from NADPH oxidase was actually converted to H2O2 [63]. Because H2O2 is responsible for the activation of N-SMase, but not A-SMase [81], antisense knockdown of p22phox inhibited Aβ1-42-induced activation of N-Smase, but not A-SMase. The generation of superoxide radicals by hypoxanthine and xanthine oxidase was sufficient to induce the activation of N-SMase in neurons via H2O2. Furthermore, fibrillar Aβ peptides were unable to induce apoptosis and cell death inp22phox knockout human primary neurons. Taken together, these studies [63] support the model in which Aβ peptides induce neuronal apoptosis via the NADPH oxidase-superoxide-H2O2-N-SMase pathway.


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The exact mechanism underlying Aβ-mediated N-SMase activation remains to be elucidated but may involve changes in the cellular redox state and/or GSH metabolism [81]. N-SMase enzymatic activity is directly regulated by cellular GSH content [81]. Aβ has been shown to deplete GSH in cultured cortical neurons [82] and the depletion of cellular GSH stores by oxidative stress has been proposed as a prime mechanism underlying the Aβ cytotoxicity [82]. Although the above sets of observation link sphingomyelin pathway and oxidative stress in cell death, relatively little is known regarding specific mechanisms through which oxidative stress acts on sphingolipid signaling. One could speculate that ROS have direct protein targets. Whether this protein is SMase or a protein with phospholipase, protein kinase, or protease activity, which, in turn regulate SMase, still remains unknown. Many of the relationships between oxidative stress and SM-signaling still remain obscure. It will be of special interest to determine the exact molecular mechanism and the subcellular localization of these interactions. This will allow a better understanding of the roles of ROS and ceramide production as mediators of cell death and of their functions in the pathophysiology of AD and various disease implicating apoptosis. In summary, this review demonstrates a novel mechanism for the development of AD. Presented results of different groups of scientists reveal a causal relationship between Aβ toxicity to various types of brain cells and activation of the N-SMase-ceramide pathway, which is likely to involve oxidative stress. Identification of this pathway may lead to the development of more effective therapeutic strategies, aimed at preventing Aβ-induced cell death. Pharmacological modulation of N-SMase activity can block the Aβ-activated death signaling process and prevent development neurodegenerative damage of the brain during AD disease.

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Chapter VII

Ischemia-Reperfusion Factors in Sporadic Alzheimer’s Disease Ryszard Pluta∗ Department of Neurodegenerative Disorders, Medical Research Centre, Polish Academy of Sciences, 02-106 Warsaw, Pawinskiego 5 Str., Poland

Abstract The study of neurobiology of Alzheimer’s disease, now more than ever, needs an infusion of a new concept. Despite ongoing interest in Alzheimer’s disease, the basis of this entity is not yet clear. For now, the best-established and accepted “culprit” in Alzheimer’s disease pathology by most scientists is the amyloid, as the main molecular factor of neurodegeneration in Alzheimer’s disease. Abnormal upregulation of amyloid production or a disturbed clearance mechanism may lead to pathological accumulation of amyloid in the brain. We will critically review these observations and highlight inconsistencies between the predictions of the “amyloid hypothesis” and the published data. There is still controversy over the role of amyloid in the pathological process – is it responsible for the neurodegeneration or does it accumulate because of the neurodegeneration? Recent evidence suggests that the neuropathology of Alzheimer’s disease comprises more than amyloid accumulation, tau protein pathology and finally brain atrophy. At least one third of Alzheimer types of dementia cases exhibit different cerebrovascular diseases. In addition, micro- and macroinfarctions and ischemic white matter changes are also evident in brains of Alzheimer’s disease patients. The presence of vascular abnormalities seems usually ignored and regarded by researchers as insignificant or considered incidental in Alzheimer’s disease etiology. Interestingly, that Alzheimer, in his own report presenting changes in the brain of the first patient had

∗

Address correspondence: Prof. Ryszard Pluta, MD, PhD, Department of Neurodegenerative Disorders, Medical Research Centre, Polish Academy of Sciences, 02-106 Warsaw, Pawinskiego 5 Str., Poland; Tel: 48 (22) 6086-540; Fax: 48 (22) 668-55-32; E-mail: [email protected]

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Ryszard Pluta described, that besides “storage of peculiar material in the cortex, one sees endothelial proliferation and also occasionally neovascularisation”. Endothelial proliferation and angiogenesis and moderate arteriosclerosis in the brain arteries of the first case, provide evidence that cerebrovascular diseases were also evident in Alzheimer’s original case, which now defines Alzheimer’s disease. These raise the question of what was the first cerebrovascular disease as trigger of Alzheimer’s disease or neurodegeneration of Alzheimer’s type itself? New findings propose an early and significant role for ischemiareperfusion factors contributing to the neurodegenerative processes in Alzheimer’s disease. The ischemia hypothesis was primarily aimed at stimulating research and redirecting the focus of studies towards ischemic cellular mechanisms of Alzheimer’s disease. In this review, we will show that experimental brain ischemia-reperfusion episode produces neurochemical and neuropathological changes that simulate the early stage of Alzheimer’s disease process. Presented data suggest that ischemic mechanisms of neuronal death with β-amyloid peptide from circulatory network modulate neuropathology of cerebral ischemia-reperfusion injury via molecular events in common with Alzheimer-type neuropathology. These results indicate that ischemic brain processes might be a key factor in the development of the picture of Alzheimer-type dementia over years. The collective data summarized in this review strongly support the idea that sporadic from of Alzheimer’s disease is a neurovascular disorder. Considerable progress has been made in recent years by a handful of researchers in understanding the role of ischemia in the aging process generally and in contributing to the development of Alzheimer’s disease. To accommodate the recent progress of study in Alzheimer’s disease there is a need to synthesize all the divergent pieces of data into a coherent story. This review provides a synopsis of current information about ischemic cellular and molecular mediators involved in Alzheimer’s neuropathology as well as interactions between these mediators that influence pathology. In this review, current knowledge on the relation between ischemia-reperfusion factors and Alzheimer’s-type dementia will be reviewed. We will summarize the results with a special focus on Alzheimer lesions in experimental brain ischemia. Taken all together, evidence presented in this review suggests a scheme for Alzheimer’s pathogenesis with ischemia-reperfusion playing a crucial role in influencing and linking β-amyloid deposition to neuronal damage and clinical disease.

Qui vivra verra

Introduction It is estimated that almost 400 million individuals suffer from neurological diseases in the world. These disorders consist of both acute and chronic neurodegenerative diseases that e.g. include brain ischemia, brain trauma, presenile and senile dementia and Alzheimer’s disease. In addition, some endogenous and exogenous toxin exposure has also become increasingly prominent as a precipitant of degenerative disorders in the central nervous system with dementia (Table 1).

Ischemia-Reperfusion Factors in Sporadic Alzheimer’s Disease Table 1. Some causes of dementia in human clinic. Degenerative − Alzheimer’s disease − Parkinson’s disease − Down’s syndrome − Huntington’s disease − Wilson’s disease − Pick’s disease − Lewy body disease − Steel-Richardson-Olszewski syndrome − Frontotemporal dementia − Batten disease Vascular − Cardiac arrest − Multi-infarct dementia − Binswanger’s disease − Lacunar state Neurological lesions − Cerebral hypoxia/anoxia − Brain trauma − Dementia pugilistica Space increasing lesions − Intracranial tumors − Metastatic intracranial tumors − Chronic subdural hematoma Toxic − Alcoholism Metabolic − Diabetes mellitus − Hypothyroidism − Hyperthyroidism − Addison’s disease − Deficit of vitamin B12 − Deficit of vitamin B1 − Deficit of folic acid Infective − HIV infection − Neurosyphilis − Creutzfeldt-Jakob disease − Gerstmann-Sträussler-Scheinker syndrome Other − Normal pressure hydrocephalus

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Dementia is a general term for symptoms exhibited by people with various kinds of cognitive impairment [Burns et al., 1990a; Carlyle et al., 1993; Clark et at., 1998; Lyketsos et al., 2000]. These symptoms may include impaired mental functioning in areas such as memory, learning, judgment, attention, concentration, language and thinking. They are often accompanied by personality and behavioral changes [Burns et al., 1990b; Carlyle et al., 1993; Stewart, 1995; Lyketsos et al., 2000]. There seem to be peaks in the incidence of dementia; one, in patients in their early 40s and another in patients in their 70s and 80s. Dementia may be caused by more than 60 different diseases (Table 1), and affects a significant proportion of the elderly population. Alzheimer’s disease is the most common, accounting for over 60 percent of all cases of dementia over the age of 65. The second most common form is vascular dementia, usually resulting from different neurovascular disorders. Although dementia is not caused by aging, nor is it an inevitable part of the aging process, it is age-related. Between the ages of 65 and 95, the prevalence of all dementias rises with age and nearly doubles every five years. Population projections indicate that the number of aging persons will increase significantly over the next 50 years. The number of European Union elderly with dementia is expected to double by the middle of the 21st century. Fifty years ago, people with dementia were considered an annoyance to be locked away in institutions or hidden at home. Attitude has changed considerably in the past half-century due in no small part to the changing demographics. Now we recognize the serious problems posed by Alzheimer’s-type dementia and vascular dementia to the planet’s population [Barclay et al, 1985; Villardita, 1993; Schumock, 1998; Groves et al., 2000; Vuorinen et al., 2000; Erkinjuntti, 2001]. This review is intended to provide a profile of these dementias and possible etiology. Dementia is already responsible for significant social and economic costs [Schumock 1998], projected to rise exponentially in the coming decades as the elderly part of the society continues to increase. Knowledge of neurological disorder processes, especially among the aging part of the population is increasing rapidly. Dementia is a rising tide and a neglected problem at the same time. Dementia is a ticking bomb in the 21st century. In most countries, dementia has not been a policy priority. There is still a great deal of ignorance about Alzheimer’s disease and other dementias. Primary care physicians may know little about diseases of aging [Barrett et al., 1997]. Diagnosis may be late. Although most primary care physicians are able to diagnose the disease in the moderate stage, it is still very difficult to detect early dementia with the result that treatment and support may be delayed. The substantial increase in the absolute and relative numbers of older people, both in developed and developing countries, can be considered as one of the main features characterizing the world society in the 21st century. By 2025, the population of world aged, 65 years and older will exceed one billion with more than 700 million living in developing countries. It is estimated that the total number of people actually affected by the different forms of dementia stands at 29 million. Yet, by 2025 this number may well surpass 55 million. In some of the oldest age groups, dementia can reach the astonishing prevalence of 25 percent. Society aging has become an important development issue requiring urgent action. As the population ages and people live longer, these findings indicate that the developed countries can expect to have nearly half a million-dementia patients every year. Taken together, dementia affects more than one-quarter of the 85 and over population and a one-third to a half of those aged 90 and over.

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Alzheimer-type Dementia Named for Alois Alzheimer who first described the disease in 1907 [Alzheimer, 1907] Alzheimer’s disease is a chronic and silent neurodegenerative disorder characterized by widespread neuronal loss in specific areas of the brain [Hyman et al., 1984] and synaptic pathology, deposition of the β-amyloid peptide into plaques in parenchyma and blood vessel walls, tau protein pathology and finally brain atrophy. Patients with Alzheimer’s disease show a number of abnormalities on brain imaging. These include cortical atrophy, ventricular dilation and leukoaraiosis. Leukoaraiosis is frequently reported as evidence of cerebrovascular disease. Alzheimer’s disease, the most common of the dementias [Kasckow, 2002], is estimated to account for approximately 60 percent of all dementia [Small et al., 1997]. Increasing age is the greatest risk factor for Alzheimer’s disease and 10 percent of society over 65, and nearly half of those over 85 years old are affected by dementia [Bird et al., 1989; Small et al., 1997; Brookmeyer et al,. 1998; Hebert et al., 2003b]. Although there are rare cases with familial forms of Alzheimer’s disease, the majority of patients have the sporadic (> 90%) form of the disease [Smith, Perry, 1998; Blennow and Skoog, 1999]. However, rare forms of Alzheimer’s disease can strike patients as early as their 30s and 40s [Bird et al., 1989]. Now there are an estimated 4.5 million cases of Alzheimer’s disease in the United States [Fratiglioni et al, 1999; Hebert et al., 2003b]. Due to the increase in longevity, the prevalence of Alzheimer’s disease will rise dramatically within the next few decades so that an estimated 14 to 30 million people in the USA will be living with Alzheimer’s disease by the year 2030-2050 [Fratiglioni et al., 1999; Hebert et al., 2003b]. The degenerative process probably starts 20 to 30 years before the clinical onset of Alzheimer’s disease [Davies et al., 1988]. After the preclinical phase of the disease, the first symptoms generally affect episodic memory. The clinical course of Alzheimer’s disease is characterized by gradual onset and slow progression. Disease progression may be far more rapid in patients with early onset illness. Conversely, the duration of illness of 15-20 years has been reported in alter onset cases [Spisacka and Pluta, 2003]. Not surprisingly, survival is generally the shortest in patients with early onset forms of Alzheimer’s disease and tends to be longer in patients with late onset forms. Alzheimer’s disease is accompanied by the impairment of short- and long-term memory and other intellectual capabilities [Cummings et al., 1987; Deutsch et al., 1991; Paulsen et al., 2000; Rizzo et al., 2000; Vuorinen et al., 2000]. As the neuronal cells die slowly, the individual’s ability to function worsens progressively leading eventually to a loss of most functional skills, including the ability to read, reason, communicate and understand spatial relationships. Alzheimer’s disease is the most common cause of dementia and the fourth most common cause of death [Kuhlman et al., 1991]. Development of Alzheimer’s disease now is looking like the silent epidemic. Despite considerable resources now targeted on research of Alzheimer’s disease little is known about causes [Doraiswamy, 2003] and treatment [Hebert et al., 2003b] for this disorder. After nearly a century of inquiry the cause of Alzheimer’s disease remains to be found [Pluta et al., 1996b; Glabe, 2000; Selkoe, 2001b; Ghiso and Frangione, 2002; Hardy and Selkoe, 2002; Mudher and Lovestone, 2002; Rosenblum, 2002; Silverberg et al., 2003; Zlokovic, 2004]. Even diagnosis is difficult. With this diverse background and in the absence of sensitive and reliable biological markers it is perhaps not surprising that the clinical

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diagnosis of Alzheimer’s disease presents many problems for the practitioner. At this time there are no tests that predict whether a person will develop Alzheimer’s disease. The disorder is diagnosed by a process of elimination and by examination of a patient’s physical and mental state. Although early diagnosis is difficult, in many cases it can allow some patients to make necessary arrangements for care. Some can also benefit from new treatments that may provide a measure of symptomatic relief. Unfortunately, many general practitioners are not trained to recognize the disease [Barrett et al., 1997] and do not diagnose it until the condition has become severe. In addition, the availability and quality of specialist care varies greatly from country to country as well as within countries. A physical examination can rule out other dementias (Table 1). Physicians look for signs of deterioration in memory, language [Vuorinen et al., 2000] and thought. For example, people suffering from Alzheimer’s disease tend to lose short-term memory, forget what words mean, become unable to recognize familiar people and objects, and are unable to orient them spatially. As deterioration continues, their ability to carry out such simple activities of daily living as dressing, bathing, and toileting or even chewing and swallowing is increasingly impaired. Changes in mood [Burns et al., 1990a], behavior [Burns et al., 1990b; Lyketsos et al., 2000] and personality are all too common. People with Alzheimer’s disease are prone to wander at any time of the day or night. They may be incontinent, disoriented in time and space and/or demonstrate uncharacteristically aggressive activity [Carlyle et al., 1993; Clark et al., 1998]. These patients tend to lose weight and muscle tone. Vision is also often affected [Rizzo et al., 2000]. The role of the physician will be fundamentally changed by the introduction of effective reasonable tests and treatment for Alzheimer’s disease. A definitive diagnosis depends on presence neurofibrillary tangles in neurons and senile plaques in brain parenchyma after the death of patients. A careful clinical examination, however, can result in a possible or probable diagnosis based on deficits in cognitive abilities and the presence or absence of a second illness that can cause dementia. There is no known cure for Alzheimer’s disease, but symptomatic treatments are being developed [Sandman, 1990]. Drugs designed to interfere with progression of the disease and to alleviate some symptoms are available, but they have been of limited benefit and only for short periods of time. It has been projected that finding a treatment that could delay the onset of Alzheimer’s disease by two years could reduce the number of patients with Alzheimer’s disease by nearly 50 percent after 50 years [Brookmeyer et al., 1998]. Alzheimer’s disease now is the single most important and probably in the future, most successful area of research in human clinic and neurobiology.

Neurovascular Changes in the First Case of Alzheimer’s Disease Alzheimer in his own report presenting changes in the brain of Augusta D., the first patient, had described that, besides “numerous minute miliary foci which are caused by the deposition of a special substance in the cortex”, “a growth appears on the endothelia, in some places also a proliferation of vessels” and “the larger vascular tissues show arteriosclerotic changes” [Alzheimer 1907]. It seems that the endothelial proliferation and neovascularisation


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were a consequence of the brain ischemia. Endothelial proliferation and angiogenesis and moderate arteriosclerosis in the brain arteries of the first case provide evidence that cerebrovascular diseases were also evident in the original case of Alzheimer [Alzheimer 1907], which now defines Alzheimer’s disease.

Vascular Dementia Cardiovascular diseases still represent the leading cause of death worldwide. Here the morbidity and mortality due to different acute and chronic cerebrovascular diseases play a major role. A further contributing factor is the increasing proportion of aged population with geriatric diseases. Maintenance of cerebral blood flow on physiological range is essential to the preservation of normal mental activity, consciousness and life. There are age-related decreases for cerebral blood flow, measured in gray matter and to a less marked degree for white matter. These declines are accentuated by the presence of risk factors, long before symptoms of brain ischemia become evident. Sudden changes in blood pressure due to impairment of heart activity may provoke complete brain ischemia. However, the major factor in the pathophysiology of acute and/or chronic neurovascular disease is not the total blood flow, but rather a regional circulatory impairment, i.e., the regional and/or focal brain ischemia with a tendency to recur. Another frequent cause is hemodynamic crises due to hypertension and diabetes mellitus and arteriosclerosis (Table 2) that are the major risk factors contributing to the incidence of complete and focal ischemia-reperfusion injury. Lacunar lesions in the brain are small, well-defined neurovascular lesions, which may lead to vascular dementia and/or multiinfarct dementia. Ischemic diffuse white matter disease in subcortical and periventricular area is also associated with low density of the white matter (leukoaraiosis) and dementia. Stroke is the third leading cause of mortality in the United States and other Western countries and is the leading cause of chronic disability [Vukovic et al., 2003] and secondary cause of dementia [Gorelick, 1997]. One study has shown that cerebrovascular disease may be an important determinant in the manifestation of dementia in the presence of Alzheimerlike pathology [Snowdon et al., 1997]. Accumulating evidences from clinical studies suggest that brain stroke is a major risk factor of dementia, ranking only second to age [Gorelick, 1997]. The incidence of dementia in the first year following a cerebral infarct is nine times greater than expected and after the first year, a 50 percent increase is observed in Alzheimer’s disease [Kokmen et al., 1996]. Yearly, approximately 700 thousand patients in the United States are afflicted with a new stroke. Most victims survive the initial stroke, but are left with some degree of significant sensory, motor and cognitive disability. Women account for approximately 60 percent of stroke deaths in the United States due to the fact that women live longer than men. A majority of strokes (approx. 80 percent) are ischemic in nature and the aging population in Western countries suggests that a decline in stroke-related deaths and disabilities will not be seen in the near future. As effect of this, the socioeconomic burden of stroke will increase every year.

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Table 2. The same proven risk factors in vascular dementia and Alzheimer’s disease Vascular dementia − − − − − − − − − − − − − − − − − − − − − − − −

Aging Silent brain infarcts Arteriosclerosis Ischemic stroke Head injury Transient ischemic attacks Microvessel pathology Cardiac disease Atrial fibrillation Hypertension Hypotension Migraine Diabetes mellitus High serum cholesterol levels High serum viscosity Thrombotic episodes High fibrinogen concentration High fat intake Menopause Smoking Alcoholism Hemorheologic abnormalities Gender Low level of education

Alzheimer’s disease − − − − − − − − − − − − − − − − − − − − − − − −

Aging Silent brain infarcts Arteriosclerosis Ischemic stroke Head injury Transient ischemic attacks Microvessel pathology Cardiac disease Atrial fibrillation Hypertension Hypotension Migraine Diabetes mellitus High serum cholesterol levels High serum viscosity Thrombotic episodes High fibrinogen concentration High fat intake Menopause Smoking Alcoholism Hemorheologic abnormalities Gender Low level of education

Chronic and silent forms of cerebrovascular diseases include neuromicrovascular dementia. Etiology and neuropathogenesis show that vascular dementia does not occur with brain arteriosclerosis alone, but in association with organic brain parenchyma lesions [Kalaria, 2000; Kalaria, 2002]. Neurovascular dementia is related to a single, large infarction, to multiple middle-sized and/or small infarcts and/or to diffuse white matter lacunes [Kalaria, 2000; Kalaria, 2002]. Advancing age with different vascular brain injuries [Kalaria, 2000; Kalaria, 2002] may often be associated with impairment of higher brain function [Cummings et al., 1987; Vuorinen et al., 2000]. Numerous studies have indicated that age related decrease in cerebral blood flow becomes slowly accentuated to a significant degree by the presence of risk factors (Table 2) long before symptoms of brain ischemia become evident. Maintenance of physiological cerebral blood flow is essential for the maintenance of normal mental function and good life. One of the major problems in an aged society is that of memory [Cummings et al., 1987] and behavior deficits [Vuorinen et al., 2000; Erkinjuntti, 2001]. The severe forms are described as dementia and actually present a considerable social and medical problem. Chronic cerebrovascular diseases afflict more frequently the elderly population of the globe. Assessment of the intellectual and psychological stage of aged patients, however, is


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difficult due to the great number of variables involved. The symptoms of chronic cerebrovascular disease can be subdivided as follows: 1) Disorders of brain activity: impairment of memory (immediate and deferred) [Cummings et al., 1987], concentration difficulty and orientation (space and time). 2) Behavioral problems: low drive, resignation, emotional instability, aggressivity, anxiety, depression, poor cooperation and poor sociability [Vuorinen et al., 2000]. 3) Somatic symptoms: sleep disorders, headache, tinnitus, fatigue and vertigo [Erkinjuntti, 2001]. The clinical condition in vascular dementia shows unexpected and sudden onset with step-wise deterioration. The sudden occurrence of an episode of fleeting and recurring amnesia is mostly the first symptoms that are not forcing the patient to visit the physician. Impairment of memory is an early characteristic feature [Cummings et al., 1987]. The shortterm memory is particularly affected. Impairment of long-term memory is involved only at a later phase. The most alarming symptom is the loss of orientation (space and time) that forces the patient to go to the physician. Under neuropathological conditions the patients are unable to recall recent events, past experience and even important data: the amnesic syndrome can be characterized by loss of recent memory, involving retrograde and anterograde memory recall which leads to difficulties in concentration and orientation [Cummings et al., 1987]. Forgetfulness of the physiologically aging brain can be described as the transient inability to recall relatively unimportant data and/or parts of an experience, these missing mosaics may, however, be recalled at another later time. The deterioration of memory retrieval involves the acquisition and retention of new information. The behavioral problems in vascular dementia, which also arise at an early stage, imply emotional disorders and personality changes [Erkinjuntti, 2001]. Emotional disorders are characterized by low drive and emotional liability, including the increased predisposition to tears and to impatience. Personality changes are usually due to an impairment of higher control. Thus, a moody person becomes depressed and an impatient one aggressive [Erkinjuntti, 2001]. Somatic problems very frequently occur in vascular dementia as well as in chronic cerebrovascular diseases and in chronic and silent brain ischemia. The most common complaints are headache and vertigo. The neuropathological findings in patients with vascular dementia have been associated with the characteristic changes in the brain in Alzheimer’s disease patients with the Alzheimer-type dementia [Jendroska et al., 1995; Wisniewski and Maslinska, 1996; Jendroska et al., 1997; Snowdon et al., 1997; Erkinjuntti, 2001]. More recent evidence suggests that the neuropathology of post-mortem brain of Alzheimer’s disease comprises more than amyloid accumulation, tau protein pathology and finally brain atrophy. At least one-third of Alzheimer type of dementia cases exhibit different neurovascular diseases [Kalaria, 2000; Kalaria, 2002]. In addition, micro- and macroinfarctions and ischemic diffuse white matter changes are also evident in brains of Alzheimer’s disease patients [Kalaria 2000, Kalaria 2002]. Epidemiological data revealed that the prevalence of severe dementia in people aged 65 years and older was estimated at 4 to 10 percent, increasing to more than 20 percent above the age of 80 years. About 10 percent of people aged 65 years and over were estimated to suffer from mild to moderate forms of memory loss. No figures are known about the prevalence of presenile dementia, either in severe and/or in mild to moderate forms. Memory loss and dementia may be caused either by neurodegenerative processes of brain parenchyma (Alzheimer’s disease) and/or by changes in neurovasculature including acute and

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chronic transient and complete/global brain ischemia as well as cerebrovascular disease (multiple infarcts) (Table 1). Alzheimer’s disease and vascular dementia, which may both arise from chronic post ischemia-reperfusion injury, are the most common causes of cognitive decline and dementia in the elderly part of the population. Current thinking dictates that cerebromicrovascular factors are associated directly with vascular dementia, several next studies solely suggest that there may be a statistically increased risk of developing Alzheimer’s disease following ischemia-reperfusion injury and/or a previous history of neurovascular risk factor normally associated with brain ischemia, including arteriosclerosis and different forms of cardiovascular diseases [Kokmen et al., 1996; Gorelick, 1997; Snowdon et al., 1997]. It has been presented that about 80 percent of patients with Alzheimer’s disease have cardiovascular problems defined by the presence of arteriosclerosis of the different arterial vessels [Honig et al., 2005]. These effects may be compounded by profound injuries involving pathological changes in cellular components of the cerebral blood vessel wall. Conversely, at least one third of patients with vascular dementia will bear Alzheimer’s neuropathology in brains after autopsy [Wisniewski and Maslinska, 1996; Jendroska et al., 1997]. It is looking that now is the beginning of a new era for vascular dementia in solving the mystery of Alzheimer’s disease.

Vascular Risk Factors in Alzheimer’s Disease and Vascular Dementia Some data present that Alzheimer’s disease and vascular dementia share similar risk factors (Table 2) [De la Torre 2002]. Other studies show of overlapping clinical symptoms in Alzheimer’s disease and vascular dementia [Kalaria and Ballard; 1999] as effect of this is mixed dementia [Korczyn, 2002]. Epidemiological investigation linking vascular factors to cerebromicrovascular pathology that can set in motion neurodegenerative pathology and cognitive changes in patient’s brains with Alzheimer’s disease. In effect, Alzheimer’s disease and vascular dementia shows parallel neuromicrovascular and neurodegenerative etiology that is developing over years (Table 2). In the end, some studies suggest that brain hypoperfusion can trigger cognitive and degenerative changes [De la Torre, 2000]. Based on the epidemiological data it can be concluded that vascular risk factors and indicators of neurovascular disease have an established association with vascular and Alzheimer’s-type dementia (Table 2). The risk factors for Alzheimer’s disease and vascular dementia include silent brain infarcts [Vermeer et al., 2003], diabetes mellitus [Ott et al., 1996; Ott et al., 1999], thrombotic episodes [Bots et al., 1998], smoking [Graves et al., 1991; Van Duijn et al., 1995; Ott et al., 1998], alcoholism [Graves et al., 1991], atrial fibrillation [Ott et al., 1997], low level of education [Ott et al., 1995; Letenneur et al., 2000], high serum cholesterol levels [Breteler et al., 1998; Breteler, 2000], arteriosclerosis [Hofman et al., 1997], high fat intake [De la Torre, 2002], migraine [Tyas et al., 2001], transient ischemic attacks [Meyer et al., 2000], menopause [Ohkura et al., 1994, Waring et al., 1999], high fibrinogen concentration [Ajmani et al., 2000], hemorheologic abnormalities [Fioravanti et al., 1998; Solerte et al., 2000], hypertension [Breteler 2000; Launer et al., 2000] and


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hypotension [Morrison et al., 1996; Guo et al., 1997; Passant et al., 1997; Fioravanti et al., 1998], ischemic stroke [Wisniewski and Maslinska, 1996; Jendroska et al., 1997; Snowdon et al., 1997], head injury [Guo et al., 2000; Lye and Shores, 2000; Plassman et al., 2000], cardiac disease including arrhythmias [Deklunder et al., 1998; Kilander et al., 1998], gender [Fratiglioni et al., 1997] and aging [Kawas et al., 2000] (Table 2). Most of presented risk factors are present not only in the early phases of reported dementias, but often decades before any cognitive abnormalities develop. All presented risk factors are vascular related and all are known to impair and/or reduce cerebral blood flow [De la Torre, 2000]. The presence of silent brain infarcts at base line more than doubled the risk of dementia [Vermeer et al., 2003]. Elderly people with silent brain infarcts have an increased risk of dementia and a steeper decline in cognitive function than those without such lesions [Vermeer et al., 2003]. The relationship of risk factors in vascular and Alzheimer’s dementia (Table 2) suggests that these two dementias share a common etiology/origin. It should be noted that about 30 percent of all Alzheimer’s disease brains show neurovascular pathology [Kalaria, 2000; Kalaria, 2002] and practically all Alzheimer’s disease brains reveal periventricualar diffuse white matter changes, microcirculation degeneration, cerebral amyloid angiopathy or combinations of these pathologies in this situation the connection between Alzheimer’s- and vascular-type dementia appears more than coincidental [Kalaria and Ballard, 1999]. Interestingly, about 40 percent of brains with clinical vascular dementia diagnosis have concurrent Alzheimer’s-type dementia neuropathology hallmarks involving diffuse and senile plaques and tau protein pathology [Jendroska et al., 1995; Wisniewski and Maslinska, 1996; Jendroska et al., 1997; Snowdon et al., 1997; Skoog et al., 1999; Erkinjuntti, 2001]. Upon final reflection of interest to us in these correlations is the fusing of these two dementias as mixed dementia [Korczyn, 2002]. However, this argument today does not explain directly and completely why pure Alzheimer’s dementia still retains a powerful vascular basis. Reported by us, risk factors appear to convert just as easily to vascular dementia as they do to Alzheimer’s dementia (Table 2) [Villardita, 1993; Hofman et al., 1997; Launer et al., 2000; Kivipelto et al., 2001].

Hypothesis It is clear that dementia of Alzheimer’s- and vascular-type continue to burden the planet’s population with not only increasing morbidity and mortality, but also with a significant financial drain [Schumock, 1998] through increasing medical care costs coupled with a progressive loss in economic productivity. A considerable and growing body of evidence suggests that hallmarks of ischemic mechanism(s) are present in Alzheimer’s disease [Pluta et al., 1995b; Pluta et al., 1996c; Kalaria, 2000]. Introducing a human wild type or mutant amyloid precursor protein gene to rodent models of Alzheimer’s disease does not result in clear and complete neurodegeneration, suggesting that contributory factors lowering the threshold of neuronal death may be present in Alzheimer’s disease. Recently, brain ischemia-reperfusion has been recognized as a factor in lowering the threshold of neuronal death [Koistinaho et al., 2002]. Other studies from transgenic animals are surprising too since they suggest neuronal death may be a common feature of Alzheimer’s disease that is not dependent on β-amyloid peptide [Games et al., 1995]. The next study suggests that the

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secreted amyloid precursor protein fragments has acted as a growth factor that protects neurons in the CA1 sector against ischemic injury [Smith-Swintosky et al., 1994]. Transgenic mice expressing different fragments of human amyloid precursor protein exhibited decreased changes to the hippocampus after excitotoxic injuries [Masliah et al., 1997]. Not surprisingly, β-amyloid peptide is produced in response to neuronal injury [Ishimaru et al., 1996a] and probably produces its effects by interacting with cell surface molecules commonly encountered in the ischemic nervous system. The neuropathogenesis of Alzheimer’s-type dementia is not fully understood although two main theories have been proposed: traditional “amyloid hypothesis” and new “ischemia-reperfusion hypothesis”. More recent work has begun to focus on elucidating the complexities of Alzheimer’s disease that involve ischemic mechanisms in killing neurons and developing dementia. I would like to propose a theoretical scheme that fits very well with the ischemia basis of Alzheimer’s disease. In my proposal, Alzheimer’s disease would start to develop when at least two neuropathological events converge: brain ischemia and ischemic chronic opening of blood-brain barrier for serum βamyloid peptide. These two events create two main pathologies that brain ischemia is responsible for; neuronal death in hippocampus and ischemic chronic blood-brain barrier insufficiency which creates amyloid pathology in brain parenchyma. Transgenic mice that accumulate amyloid without neuronal loss in hippocampus support this idea. Additionally, we found overlapping features in both disorders of vascular dementia and Alzheimer’s disease. For example, Alzheimer’s disease and vascular dementia share hallmarks like brain hypoperfusion, diffuse white matter changes, neuropathological markers and overlapping symptoms. Constantly growing evidence indicating that the neuropathogenesis of Alzheimer’s disease is rooted in vascular pathology [Pluta et al., 1995b; Pluta 1997b; Kalaria, 2000; Pluta, 2004a; Pluta, 2004b]. The “amyloid toxicity hypothesis” of Alzheimer’s disease and the “ischemic-reperfusion theory” of Alzheimer’s disease may together explain Alzheimer-type neurodegeneration in brain. Therefore, the ischemic overexpression of amyloid precursor protein and ischemia alone may constitute a vicious cycle that leads to neurodegeneration. Progressing death of neurons following ischemia-reperfusion may be caused not only by a degeneration processes of neurons destroyed during ischemia insult but also by ischemic opening of blood-brain barrier vessels with deposition and influence of cytotoxic fragments of amyloid precursor protein on increase vulnerability of neurons to ischemic excitotoxicity. In this article, we discuss the role of novel cellular pathways that are invoked during ischemia-reperfusion process and may potentially mediate/develop injury in Alzheimer’s disease brain. The fundamental thesis of my proposal is that the neuropathology seen in Alzheimer’s disease is a continuous process from initial ischemic neuronal changes [Pluta, 1997b; Pluta, 2000a] to the well-established extravasations of β-amyloid protein across ischemic blood-brain barrier culminating in the formation of amyloid plaques and dystrophic neurites.


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Mosaic of Alzheimer’s Proteins in Ischemia-reperfusion Injury DNA, an extraordinary molecule present in the nucleus of every body and brain cell, controls the growth and development of the human and animal body and brain as genes. DNA carries all the genetic information necessary for developing not just a brain but also a complete human and animal being. DNA controls the processes of learning and maturation that turn the young brain into an adult brain. Once maturity is reached, DNA controls the metabolic processes that maintain the brain in its homoeostatic balance with some physiological correction during aging. It is also possible that DNA may hold a darker secret. It may hold the codes for the neurodegeneration of the brain with age for loss of memory and intelligence and for the clinical syndrome termed dementia. The neuropathological changes of some proteins seen in brain parenchyma from demented patients (e.g. widespread βamyloid peptide accumulation as diffuse and senile plaques and tau protein pathology as neurofibrillary tangles) may be a reflection of this dark and secret information in specific brain genes.

Amyloid Precursor Protein β-amyloid peptide is derived from the larger parent transmembrane molecule amyloid precursor protein. The gene coding for amyloid precursor protein has been identified on chromosome 21. Amyloid precursor protein mRNA increased over 200 percent and 150 percent in the penumbra and core ischemic regions, respectively on the fourth day after focal brain ischemia and remained high through the seventh day of ischemic insult. This study suggests focal brain ischemic insult enhances amyloid precursor protein mRNA expression and may contribute to the progression of cognitive impairment after ischemic injury [Shi et al., 2000]. After focal ischemia, the Kunitz protease inhibitor-bearing isoforms were increased whereas amyloid precursor protein 695 that lacks Kunitz protease inhibitor domain was decreased. These results show that focal ischemia alters Kunitz protease inhibitor-amyloid precursor protein/amyloid precursor protein 695 ratio in cerebral cortex and this shift in amyloid precursor protein isoforms could be related to neurodegeneration and/or activation of astrocytes during the ischemic process [Kim et al., 1998]. In persistent focal ischemia amyloid precursor protein mRNA species that contain a Kunitz-type protease inhibitor domain were induced in the rat cortex from 1 to 21 days after the injury with maximum at 4 days, while total amounts of amyloid precursor protein mRNA did not change. These results suggest a selective role of amyloid precursor protein species that contain the Kunitz protease inhibitor domain in focal brain ischemia [Abe et al., 1991]. At 7 days postischemia amyloid precursor protein 770 and amyloid precursor protein 751 mRNAs were induced in the infarct core and in a thin perifocal zone [Koistinaho et al., 1996]. Brain samples from ischemic core and penumbra of cortex at one hour and 24 hours following focal ischemia with ovariectomia were investigated for amyloid precursor protein

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mRNA. At one hour after ischemia rats had significant increase in amyloid precursor protein mRNA in the penumbra and core. Estrogen treatment reduced the amyloid precursor protein mRNA over-expression in these two areas [Shi et al., 1998]. This data demonstrates that estrogen may have an important role in reducing the over-expression of amyloid precursor protein mRNA following transient focal ischemia. Thus, these studies suggest a profound effect of estrogen on the brain that may be able to interrupt a vicious cycle of ischemia and neurodegeneration [Shi et al., 1998]. Animals with global and/or focal brain ischemia and followed by reperfusion showed strong, abnormal brain staining to the N-terminal of amyloid precursor protein and to the βamyloid peptide and as well as to the C-terminal of amyloid precursor protein. The staining was noted not only intracellularly (Figure 1B-D) [Pluta et al., 1994b; Hall et al., 1995; Horsburgh and Nicoll, 1996a; Tomimoto et al., 1995; Yokota et al., 1996; Pluta et al., 1997a; Pluta et al., 1998a; Lin et al., 1999; Pluta, 2000a; Lin et al., 2001; Sinigaglia-Coimbra et al., 2002], but also extracellularly (Figure 1A, C, D) [Pluta et al., 1994b; Ishimaru et al., 1996a; Pluta et al., 1997a; Pluta et al., 1998a; Pluta 2000a; Fujioka et al., 2003].

Figure 1. Extracellular β-amyloid peptide plaque-like deposits in enthorhinal cortex (A). Strong immunoreactivity to β-amyloid peptide in astrocytes in hippocampus. Other astrocytes exhibited immunostained processes in contact with capillary (B). Perivascular β-amyloid peptide- (C) and Cterminal of amyloid precursor protein-positive (D) material in hippocampus and frontal cortex respectively. 10-min brain ischemia – 6 months survival. Magnifications; A, B, C, D. x400. Reprinted from Pluta (2004) Curr Neurovasc Res, 1, 441-453 with permission from Bentham Science Publishers Ltd.


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Various fragments of amyloid precursor protein were observed within neuronal cells. Immunoreactivity was seen mainly in undamaged neurons. Animals after ischemia with short-term survival up to 7 days manifested the strongest labeling to the β-amyloid peptide and C-terminal of amyloid precursor protein and weaker staining to the N-terminal of amyloid precursor protein [Pluta et al., 1994b]. Rats with very long survival after ischemia 6 and 12 months presented abnormal brain staining only to the C-terminal of amyloid precursor protein as well as to the β-amyloid peptide domain (Figure 1A-D) [Pluta et al., 1997a; Pluta et al., 1998a; Pluta, 2000a]. Amyloid precursor protein may be expressed abnormally by microglia, astrocytes and oligodendrocytes (Figure 1B, C) [Banati et al., 1995; Palacios et al., 1995; Pluta et al., 1997c; Nihashi et al., 2001; Pluta, 2002a; Pluta, 2002b; Badan et al,. 2003; Badan et al., 2004]. Postischemic amyloid precursor protein overexpression may occur in activated microglial cells [Banati et al., 1995] and reactive astrocytes [Palacios et al., 1995; Pluta et al., 1997c; Nihashi et al., 2001; Pluta, 2002a; Pluta, 2002b]. Following focal brain ischemia and reperfusion amyloid precursor, protein increased expression has been noted mainly in reactive astrocytes [Nihashi et al., 2001]. After complete brain ischemia amyloid precursor protein staining occurs predominantly in reactive astrocytes in the hippocampus (Figure 1B) [Pluta et al., 1997c; Pluta, 2002a; Pluta, 2002b]. Following global brain ischemia with longterm survival, animals showed increased staining in the reactive astrocytes only to the cytotoxic C-terminal of amyloid precursor protein as well as to the β-amyloid peptide region (Figure 1B) [Pluta, 2000a; Pluta, 2002a; Pluta, 2002b]. Especially perivascular astrocytes showed very intense labeling of numerous very long, delicate, thin and widespread processes, which embraced or adjoined mainly the capillaries (Figure 1B) [Pluta, 2002b; Pluta, 2004b]. The reactive astrocytes with different fragments of amyloid precursor protein might be involved in the development of glial scars [Nihashi et al., 2001; Pluta, 2002b; Badan et al., 2003; Badan et al., 2004]. Reactive astrocytes with abnormal level of β-amyloid peptide (Figure 1B) might play an important role in pathological repairing of the host tissue after ischemia including amyloid accumulation and astrocytic death [Pluta et al., 1994b; Pluta, 2002b; Wyss-Coray et al., 2003; Takuma et al., 2004]. Increased amyloid precursor protein staining has been observed within white matter following transient focal brain ischemia. Amyloid precursor protein immunoreactivity was frequently noted in swollen axons between core and penumbra [Yam et al., 1997]. Abnormal amyloid precursor protein expression has been found in the white matter after embolic stroke in animals [Dietrich et al., 1998]. After global brain ischemia in subcortical and periventricular areas was frequently localized labeling for C-terminal of amyloid precursor protein and β-amyloid peptide in rats with very long survival [Pluta et al., 1997a; Pluta, 2003a]. Moreover, the larger and more intense ischemic insult, the more extensive staining of amyloid precursor protein in white matter [Yam et al., 1997]. Amyloid precursor protein staining has been used to detect axonal injury at the side of ischemic injury. The presence of amyloid precursor protein within axons after ischemic brain damage is thought to be due to its accumulation following inhibition of axoplasmic flow. The amyloid precursor protein associated swelling reflect a disturbance of flow in intact axons, but the development of amyloid precursor protein positive granule-like aggregation presents that axotomy and

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irreversible injury has occurred. These processes may be secondary to neuronal degeneration of axons. Extracellular amyloid precursor protein deposits ranged from numerous widespread small dots to irregular diffuse plaques (Figure 1A) [Pluta et al., 1994b; Pluta et al., 1997a; Pluta et al., 1998a; Pluta, 2000a; Pluta, 2002b; Pluta, 2003a]. Widespread and multifocal diffuse plaques predominated in the hippocampus, brain and entorhinal cortex and corpus callosum and around the lateral ventricles. Inside diffuse plaques have been found frequently glial and neuronal cells.

Brain ischemia

Blood-brain barrier Macrophages Microglial cells

Cytokines

Cytokines

Amyloid precursor protein Soluble β-amyloid peptide

Neurotoxicity

Neuronal death

Fibrillar β-amyloid peptide

Figure 2. The potential role of brain ischemia on activated microglial cells and microglia-derived proinflammatory factors in the ischemic pathogenesis of amyloid precursor protein and neuronal death.

The localization of the C-terminal of amyloid precursor protein within ischemic neurons and especially in astrocytes underscores the likely importance of the C-terminal of amyloid precursor protein in the neuropathogenesis of brain ischemia as in Alzheimer’s disease [Pluta et al., 1994b; Yokota et al., 1996; Pluta et al., 1997a; Pluta, 2002b; Badan et al., 2003; Badan et al., 2004]. This supports the possibility that the C-terminal of amyloid precursor protein is activated because the C-terminal of amyloid precursor protein is critical for the seeding and development of amyloid plaques in the brain [Estus et al., 1992]. Additionally, this fragment accumulation indicates that this part of amyloid precursor protein may promote synaptic degeneration and neuronal death [Oster-Granite et al., 1999]. These observations indicate that late C-terminal of amyloid precursor protein and β-amyloid peptide accumulation/ aggregation postischemia may represent a secondary injuring factor that could exacerbate insult outcome [Pluta et al., 1997e; Pluta et al., 1998a]. Not surprisingly, β-amyloid peptide is


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produced in response to neuronal damage [Ishimaru et al., 1996a] and probably produces its effects by interacting with ischemic nerve (Figure 2) and glial cells (Figure 3) surface molecules in the brain adding additional pathology as well as dementia. It is generally believed that β-amyloid peptide participates in the neuronal killing (Figures 2, 3) [Cotter et al., 1999]. β-amyloid peptide is a strong and direct neurotoxic protein and it develops ischemic cascade of intracellular processes including activation of these processes in microglial cells and astrocytes that leads to nerve and glial cells damage (Figures 2, 3) [Giulian et al., 1995]. This supports the additional role of the “amyloid toxicity hypothesis” in Alzheimer’s disease. Extracellular accumulation of β-amyloid peptide is detected only upon neuronal cell death [Ishimaru et al., 1996a] initially as halos of β-amyloid peptide staining around individual dying neurons and subsequently as β-amyloid peptide plaques containing numerous neuronal cell ghosts. Based on presented data we conclude that neuronal cell death likely occurs before the extracellular accumulation of β-amyloid peptide in Alzheimer’s disease brains [LaFerla et al., 1997].

Brain ischemia

Blood-brain barrier β-amyloid peptide

Astrocytes β-amyloid peptide

Astrocytic death

Protection

β-amyloid peptide

No protection

Ischemic neurons

Neuronal death Figure 3. Role of astrocytes in influence of β-amyloid peptide on neurons following ischemiareperfusion brain injury.

Apolipoproteins The apolipoprotein E gene is on chromosome 19. Since β-amyloid peptide binds with high affinity to apolipoprotein E, it has been suggested that apolipoprotein E may promote

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formation of β-amyloid peptide fibrils by acting as a “pathological chaperone” [Wisniewski and Frangione, 1992]. In an experimental study on gerbils, there was an increase in astrocytic apolipoprotein E mRNA with the levels being the highest on 7 days postischemia. This suggests that neuronal injury or insult results in the induction of certain genes in the surrounding reactive astrocytes and these proteins may contribute to post injury amyloidogenesis [Ali et al., 1996]. Apolipoprotein E mRNA expression in glial cells, but not in neurons was seen in ischemic penumbra with a peak at 21 days. In core marked apolipoprotein E mRNA expression was observed in macrophages [Kamada et al., 2003]. Some data present increased expression in apolipoprotein J, particularly by astrocytes that provide neuroprotection against brain ischemia as an anti-stress protein chaperone [Wiggins et al., 2003]. Increased expression of clusterin mRNA was noted in permanent focal cerebral ischemia in the mouse. Clusterin mRNA was located in the perifocal area. Reactive astrocytes within the cortex were found to be strongly immunoreactive for clusterin. It was concluded that local expression of apolipoprotein J mRNA might contribute to the inflammation representing an important process in secondary injury mechanisms after focal cerebral ischemia [Van Beek et al., 2000]. The severe ischemic insult leads primarily to necrotic neuronal death and showed very little if any clusterin mRNA. However, following the moderate insult there was a dramatic time-dependent accumulation of clusterin protein in neurons of the CA1 and CA2 sector in the hippocampus and cortical layers 3-5 regions undergoing delayed neuronal death. Clusterin mRNA expression in contrast to neuronal protein accumulation appeared to be glial in origin with increases in mRNA in and around the hippocampal fissure and only a weak signal over the CA1 and CA2 pyramidal cell layer. These results support the theory that the clusterin protein is synthesized in the astrocytes, secreted and then taken up by dying neurons [Nishio et al., 2003]. Clusterin was accumulating in neurons destined to die by programmed cell death. However, clusterin expression suggests that clusterin production was a result of the selective delayed neuronal death rather than being involved in the neurochemical cascade of events that cause it [Walton et al., 1996]. Apolipoprotein E immunoreactivity was recognized in astrocytes and neurons 3-14 days after transient focal cerebral ischemia. Apolipoprotein E was also detected in macrophages in the ischemic core. In contrast, apolipoprotein E mRNA was expressed in astrocytes and macrophages, but not in neurons. These results suggest that neuronal apolipoprotein E was not synthesized in neurons, but derived from astrocytes [Nishio et al., 2003]. Recent findings support this observation that astroglial cells regulate apolipoprotein E expression in neurons [Harris et al., 2004]. The abnormal staining for apolipoproteins E, A1 and J was noted intracellularly [Kida et al., 1995; Hall et al., 1995; Pluta et al., 1995a; Horsburgh and Nicoll, 1996a; Horsburgh and Nicoll 1996b; Ishimaru et al., 1996b; Pluta, 2000a; Kamada et al., 2003] and extracellularly [Kida et al., 1995; Pluta, 2000a]. Intracellular staining was observed in both slightly as well as markedly damaged neurons, exhibiting signs of ischemic changes [Pluta, 2000a]. Less often immunoreactivity for apolipoproteins was found in glial cells [Kamada et al., 2003]. Apolipoprotein extracellular deposits were well delineated and irregular. Diffuse, broad but faint areas were also seen. Strong staining was found also in irregular, spider-like,


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acellular, necrotic foci [Kida et al., 1995; Pluta et al., 1995a; Ishimaru et al., 1996a]. Extracellular apolipoprotein E and apolipoprotein J deposits were heavily labeled by antibody to apolipoproteinn A1, stronger than by apolipoprotein E antibody [Kida et al., 1995]. They were stained stronger by antibody to apolipoprotein E than apolipoprotein J [Kida et al., 1995]. It is of interest to notice that deposits of apolipoproteins colocalize with deposits of different fragments of amyloid precursor protein [Kida et al., 1995; Pluta et al., 1995c]. Apolipoprotein E can modulate and/or promote the aggregation of soluble β-amyloid peptide into the β-pleated sheet conformation. It is proposed that apolipoprotein E can function as a “pathological chaperone” in formation amyloid deposits [Wisniewski and Frangione, 1992]. Apolipoprotein J is implicated in the transport of β-amyloid peptide across the blood-brain barrier. On other hand, clusterin production was a result of the selective delayed neuronal death [Walton et al., 1996]. The main role of apolipoproteins E, A1 and J in controlling the levels of soluble β-amyloid peptide in the extracellular and intracellular compartments of brain as well as their influence on fibrillar β-amyloid peptide formation is suggested. It is also possible that stable apolipoprotein E and β-amyloid peptide complexes or two proteins separately adhere to the neuronal remnant after neuronal death and develop amyloid plaques. Interestingly we found in experimental brain ischemia faint staining for apolipoprotein E remaining in the pyramidal cell layer of hippocampus even long after their disappearance [Ishimaru et al., 1996a]. On the other hand, apolipoprotein E fragments present in Alzheimer’s disease brains induce neurofibrillary tangle-like intracellular inclusions in neurons [Huang et al., 2001]. Apolipoprotein E potentates β-amyloid peptide-induced lysosomal leakage and apoptosis in neuronal cells [Ji et al., 2002]. Apolipoprotein E-derived peptide produces neuronal toxicity [Moulder et al., 1999]. Neuronal cell death in Alzheimer’s disease correlates with apolipoprotein E uptake and intracellular β-amyloid peptide stabilization [LaFerla et al., 1997]. Apolipoprotein E and J can influence structure, toxicity and accumulation of the βamyloid peptide in the ischemic brain. Both proteins may also be involved in β-amyloid peptide metabolism prior to its deposition. These data demonstrate additive effects of both apolipoproteins on influencing β-amyloid peptide deposition and that they play an important role in regulating extracellular brain β-amyloid peptide metabolism independent of β-amyloid peptide synthesis. Another function, especially for apolipoprotein E in the brain is suggested clearance of ischemic tissue that would be an important process for tissue repair [Kitagawa et al., 2001]. Delayed clearance may hamper healing and reconstruction of the blood-brain barrier and glial boundary formation. The recent study supports current thinking that apolipoprotein E is a key protein for brain parenchyma remodeling after injury. Clearance of irreversible damaged tissue may be one of the important activities of apolipoprotein E in the central nervous system [Kitagawa et al., 2001]. These observations indicate that apolipoproteins E and J accumulation postischemia may represent a secondary injuring factor that could exacerbate healing of ischemic neurons. Extracellular apolipoproteins E and J deposition is noted mainly following neuronal death [Ishimaru et al., 1996a; Walton et al., 1996].

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Presenilins The protein products of the genes on chromosomes 14 and 1 were subsequently termed presenilin 1 and presenilin 2, respectively. Information regarding the genetic factors and environmental conditions that influence presenilins gene expression is essential for the elucidation of its pathophysiological role in Alzheimer’s disease. In one paper, the changes of presenilin 1 mRNA expression in the gerbil hippocampus following complete brain ischemia were studied [Tanimukai et al., 1998]. After ischemia, the selective induction of presenilin 1 gene in neurons of CA3 and dentate gyrus were observed which might be related to the resistant areas after ischemia. In this investigation, presenilin 1 mRNA was induced 1 day postischemia and reached the greatest levels at day 3 in the presented regions. These observations suggest that the expression of presenilin 1 mRNA may be associated with some response of nerve cells injured by ischemia insult. In another study, the induction of presenilins mRNA was examined in the rat hippocampus, cortex, striatum and cerebellum in experimental model of transient focal brain ischemia [Pennypacker et al., 1999]. The levels of presenilins mRNA exhibited the maximal expression in the hippocampus and cortex regions of plaque development in Alzheimer’s disease while the presenilin 1 and 2 genes content in cerebellum and striatum, regions unaffected by Alzheimer’s disease, presented generally no significant increase. The greatest expression for presenilins mRNA was observed in the cortex. Presenilins genes in the hippocampus and striatum show a pattern of expression similar to that seen in the cortex but with smaller intensities. Generally, the expressions were larger on the contralateral side to the focal ischemic injury. This difference may reflect a loss of brain cells expressing presenilins genes on the ipsilateral side. Staining of presenilin was more marked in glial than in neuronal cells and in a trace of the pyramidal cells of hippocampus after global brain ischemia-reperfusion injury [Pluta, 2001]. Presenilin 1 mutation increases neuronal cells’ vulnerability to focal ischemia by elevation of intracellular calcium [Mattson et al., 2000]. A recent study showed that presenilin 1 and intracellular calcium stores regulate neuronal glutamate uptake [Yang et al., 2004]. Taken together, these findings indicate that presenilins and intracellular calcium stores may play a significant role in regulating glutamate uptake and therefore, may be important in limiting glutamate toxicity in the brain. The finding of presenilins in ischemic brain suggest that presenilins may be regulated by conditions of neurodegeneration, a condition which both the ischemic brain and the brain in Alzheimer’s disease may experience.

Tau Protein Neurons expressing tau protein were noted in the hippocampus and brain cortex after cerebral ischemia with hyperthermia during reperfusion [Sinigaglia-Coimbra et al., 2002]. It was reported that tau immunoreactivity within glial cells is increased and that tau protein is degraded in axons in insult region after focal brain ischemia [Dewar and Dawson, 1995; Irving et al., 1997]. There was a noted increase of full-length tau protein staining in oligodendrocytes following focal ischemia-reperfusion injury of the rat brain [Irving et al., 1997]. Another study presented pathological modification of tau protein by microglia around


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ischemic foci [Uchihara et al., 2004]. The somal accumulation of tau protein in neuronal cell bodies throughout the ischemic hippocampal formation has been shown [Geddes et al., 1994]. Other data showed changes in tau protein and ubiquitin in some ischemic neurons [Dewar et al., 1993; Dewar et al., 1994]. Both proteins are components of neurofibrillary tangles in Alzheimer disease brains. These results indicate that only in some neurons are changes in tau protein shortly following brain ischemia [Dewar and Dawson, 1995] that may reflect an early phase of the ischemic processes in these cells [Irving et al., 1997]. Other investigations presented a complete dephosphorylation of microtubule-associated tau proteins after brain ischemia with incomplete rephosphorylation during recirculation period [Mailliot et al., 2000]. The dephosphorylation/phosphorylation of tau protein may alter its distribution between axon and cell body and affect its susceptibility to proteolysis [Shackelford and Yeh, 1998]. The next study presented that tau protein itself blocking transport of amyloid precursor protein from body of neurons into their axons and dendrites causing amyloid precursor protein deposition in the neuronal cell body [Stamer et al., 2002]. The recent study presented that after a brain ischemia, hyperphosphorylated tau accumulates in cortical neurons and colocalizes largely with sings of apoptosis. This process may be involved in the pathogenesis of neurodegenerative disorders. Further, these data indicate that ischemic neuronal damage and apoptosis associates with tau hyperphosphorylation and potentially neurofibrillary tangles formation [Wen et al., 2004b]. Wen et al. [2004c] reported for the first time that reversible brain ischemia induces aberrant mitotic proteins and hyperphosphorylation of tau protein with neurofibrillary tangle-like conformational epitopes in the adult female rat cortex. These data provide a pathological ground for development of dementia in patients with ischemic brain and support the theory that apoptosis and aberrant mitosis are integrated neuropathological events in neuronal cells that may play a important role in the formation of Alzheimer’s disease and other taupathy-related neuropathological changes [Wen et al., 2004c].

β- and γ-secretases The pathological processing of the amyloid precursor protein by β- and γ-secretases is key to β-amyloid peptide plaque development in Alzheimer’s disease. Recent data present that both experimental cerebral ischemia and experimental traumatic brain injury stimulate the expression, production and activity of Alzheimer’s disease β-secretase in animal brain [Blasko et al., 2004; Chen et al., 2004; Wen et al., 2004a]. Additionally, presenilin is involved in the processing of amyloid precursor protein to produce β-amyloid peptide through the γ-secretase complex [Wolfe et al., 1999]. The idea that presenilin the elusive γsecretase was proposed [Wolfe et at., 1999]. Another data has shown that full-length presenilin interacts with immature β-secretase. This observation suggests that presenilin regulates β-secretase activity via direct interaction and facilitated trafficking of β-secretase to different compartments of cells [Hebert et al., 2003a].

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Cytokines Brain ischemia resulted in a concomitant overexpression of cytokines in astroglial cells especially in the CA1 sector of hippocampus [Orzyłowska et al., 1999]. This study suggests that regional increase in cytokines may be involved in the pathology in selective regions of brain [Yamasaki et al., 1995; Orzyłowska et al., 1999; Touzani et al., 1999; Boutin et al., 2001; Touzani et al., 2002]. The main sources of interleukin-1β are astrocytes, oligodendrocytes, microglia and scattered perivascular macrophages (Figure 2) and monocytes. Instead, neurons and some microvessels endothelial cells showed interleukin-1R staining [Saito et al., 1996; Sairanen et al., 1997; Davies et al., 1999; Orzyłowska et al., 1999; Touzani et al., 2002]. These data indicate that neuronal cells in vulnerable areas (Figure 2) and probably the endothelial cells are target cells for ischemia-induced glial interleukin-1β production. It is clear that interleukin-1β may play an important function in ischemic cell damage and development of brain edema [Yamasaki et al., 1995]. Brain ischemia itself produces the glial cytokines that are important in repairing and healing and/or remodeling of ischemically injured parenchyma [Orzyłowska et al., 1999]. However, chronic stimulation of these processes may produce a self-sustaining cycle that gives rise to the characteristic brain pathological changes of Alzheimer’s disease (Figure 2). In this process interleukin-1 is a key agent that promotes neuronal abnormal processing of the amyloid precursor protein [Griffin et al., 1998] thus favoring constant deposition of β-amyloid peptide and release of a number of inflammatory and neurotoxic molecules (Figure 2). As effect of these is noted neuronal dysfunction and death (Figure 2). Neuronal damage arising from these cytokine-induced nerve cell injuries activate microglia with further amplification and self-propagation of this cytokine cycle (Figure 2). Taken together, these results suggest that interleukin-1 is an important factor in ischemia-reperfusion brain injury [Yamasaki et al., 1995; Touzani et al., 1999; Boutin et al., 2001; Touzani et al., 2002]. Several lines of evidence have been found that β-amyloid peptide stimulate microglial cells to release neurotoxins (Figure 2) [Giulian et al., 1995; Giulian et al., 1996]. Microglial cells produce multiple proinflammatory and neurotoxic factors including cytokines. Hypothetically, these factors exert direct neurotoxic effects and may trigger a positive feedback loop that ultimately results in the propagation of a local brain inflammatory response and a further dysregulation of amyloid precursor protein metabolism the conversion of soluble β-amyloid peptide into fibrillar β-amyloid peptide and hence to an aggregation of the disease process (Figure 2).

α-synuclein Transient brain ischemia developed changes in a presynaptic protein α-synuclein in the hippocampus [Ishimaru et al., 1998; Kitamura et al., 2001]. Strong α-synuclein immunoreactivity was observed in perivascular area in the CA1 sector of hippocampus in animals with long survival after injury [Kitamura et al., 2001]. In neurodegenerative areas after ischemia glial cells presented strong staining for α-synuclein [Ishimaru et al., 1998]. These observations support the idea that α-synuclein may be an important protein in the


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molecular ischemic processes [Goedert, 2001]. Abnormal α-synuclein deposition might stop synaptic activity, resulting in memory problems [Hashimoto and Masliah, 1999]. Studies have showed that disruption in the synaptic system is associated with cognitive disturbance and is an early phase in Alzheimer’s disease. Abnormal metabolism of α-synuclein impairs synaptic function, which additionally promote/support neuronal death in ischemic brain and Alzheimer’s disease [Goedert, 2001]. Neurodegeneration may be caused by the loss of αsynuclein function [Han et al., 1995]. In the Alzheimer’s disease, neocortex α-synuclein was colocalized with the dystrophic neuritic component of the plaques [Masliah et al., 1996]. This study suggests that there is a connection between metabolism of presynaptic proteins and amyloid formation and that fragments of α-synuclein might follow diffuse β-amyloid peptide accumulation resulting in the formation of compact amyloid and mature plaques [Masliah et al., 1996]. Thus, the induction of amyloid precursor protein, apolipoproteins and presenilin 1 and 2 and probably other Alzheimer’s disease-related genes during brain ischemia may be the molecular link between Alzheimer’s disease and ischemia-reperfusion brain injury [Pluta, 2001]. As an effect of induction of these genes it was noted long-term abnormal coaccumulation of amyloid precursor protein with β-site amyloid precursor protein-cleaving enzyme and presenilin-1 in damaged axons. The pathological concentration of these proteins may lead to amyloid precursor protein proteolysis and β-amyloid peptide formation within the axonal membrane compartment. It is rather clear that amyloid abnormal metabolism and tau protein pathology can both be triggered by vascular factors, such as ischemia-reperfusion brain injury. It is also interesting to note that microischemic brain insults and small, silent strokes in Alzheimer’s disease patients develop because of chronic brain hypoperfusion due to vascular risk factors. These observations raise a possibility that a neurovascular diseases can participate and trigger/amplify the β-amyloid peptide and tau protein changes and could be an important additional neuropathogenic mechanism contributing to neuropathology and cognitive decline in Alzheimer’s disease. In sum experimental data on ischemic brains show that Alzheimer’s disease-related changes render the brain more susceptible to ischemic damage and in consequence developing neurodegenerative changes.

Alzheimer’s Neuropathological Picture in Ischemia-reperfusion Injury Blood-brain Barrier In short, survival after cerebral ischemia-reperfusion injury blood-brain barrier microvessels showed plenty of changes: vasospastic events, increased numbers of endothelial microvillars, cellular invaginations, microthrombe formation and focal permeability for cellular and uncellular blood elements (Table 3) [Pluta et al., 1991b; Pluta et al., 1994a; Pluta et al., 1994b; Pluta et al., 1994c; Wisniewski et al., 1995; Shinnou et al., 1998; Ueno et al., 2002]. Instead, animals with long-term survival after ischemic brain insults demonstrated chronic insufficiency of blood-brain barrier for cellular and acellular blood components

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(Table 3). Permeability changes were spotty and dispersedly and involved arterioles, microcirculation, venules and veins [Pluta, 2003a; Pluta et al., 2003b]. Blood-brain barrier alterations predominate in hippocampus, brain cortex and white matter.

Table 3. Immunoreactivity for horseradish peroxidase (HRP), rat’s N- and C-terminal of amyloid precursor protein (NAPP, CAPP) and β-amyloid peptide (βA) and human βamyloid peptide 1-42 (βA1-42) in perivascular space following experimental 10-min brain ischemia. Groups Control Short-term survival Long-term survival Brain ischemia Short-term survival Long-term survival

HRP

NAPP

βA

CAPP

βA1-42

-

-

-

-

-

+ +

++ -

++ ++/+++

++ ++/+++

+++ +++

The staining intensity was categorized into four grades as follows: - no staining; + a single and diffuse areas; ++ a few and diffuse areas; +++ many strong and diffuse areas.

Other studies revealed numerous platelet aggregates of varying sizes within both venous and arterial brain tissue vessels following ischemia-reperfusion injury [Pluta et al., 1994c; Pluta 2003a]. Some platelets were observed outside cerebral vessels in the perivascular space [Pluta et al., 1994c; Pluta, 2003a]. Abnormally aggregating platelets like blood-brain barrier alterations were focal, widespread, random and dispersedly. Changes of blood-brain barrier and platelets predominated in vessel branches and bifurcations. Recent evidence suggests that platelets play a major role in ischemia-reperfusion injury not only through thrombus formation but also through participation in inflammatory reactions. Platelets may contribute to the recruitment of inflammatory cells to tissue after ischemia as effect of short life and source of β-amyloid peptide. Platelets pathology and microvascular deficiency [Grammas, 2000] was observed in brains early during the course of Alzheimer’s disease thus supporting a further link between related processes and Alzheimer’s disease [Borroni et al., 2002]. In short-term survival, animals after brain ischemia in the perivascular space were observed widespread and multifocal diffuse N-terminal of amyloid precursor protein, βamyloid peptide and C-terminal of amyloid precursor protein deposits (Table 3) predominantly in the hippocampus, brain cortex and white matter [Pluta et al., 1994b]. Multiple, abundant, extracellular deposits embraced or adjoined the blood-brain barrier vessels. Perivascular deposits formed irregular, often asymmetric, well-delineated areas that frequently encircled microvessels, forming round, perivascular cuffs or hallo. Diffuse, broad, but faintly positive perivascular zones were also found. Endothelial and pericyte cells were stained, too. In long-term survival after brain ischemia perivascular deposits ranged from numerous small-diffused areas to irregular diffuse plaques [Pluta et al., 1997a; Pluta et al., 1997c; Pluta et al., 1997e; Pluta, 2003a]. Deposits in brain vessels and around them stained only for C-terminal of amyloid precursor protein and β-amyloid peptide (Table 3) [Pluta et al., 1997a Pluta et al., 1997c; Pluta et al., 1997e; Pluta, 2003a]. Immunoreactivity in the


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perivascular space suggested diffusion of different fragments of amyloid precursor protein out of the vascular system. Especially staining for C-terminal of amyloid precursor protein in long-lived animals underscores the likely importance of the C-terminal of amyloid precursor protein in the neuropathogenesis of ischemia as in Alzheimer’s disease [Estus et al., 1992]. In other experiments it was tested permeability for human β-amyloid peptide 1-42 in ischemically open blood-brain barrier after single or repeated episodes of global brain ischemia (Table 3). In this study, it was seen multifocal and widespread accumulation of amyloid around and in contact with blood-brain barrier vessels (Table 3) especially in hippocampus and brain cortex and occasionally in white matter [Pluta et al., 1996a; Pluta et al., 1997d; Pluta et al., 1999a; Pluta et al., 2000c]. Amyloid peptide penetration involved arterioles, veins and venules. Endothelial, pericyte, glial and neuronal cells were observed filled with human β-amyloid peptide [Pluta et al., 1996a; Pluta et al., 1997d; Pluta et al., 1999a; Pluta et al., 2000c]. Direct evidence that soluble β-amyloid peptide crosses the ischemic blood-brain barrier and enters the brain parenchyma from the circulation is thus provided.

Cell Death In ischemic brain pathological focus centers on the hippocampus because it is part of the brain selectively vulnerable to ischemia-reperfusion injury. In Alzheimer’s disease, brain structural abnormalities centers on hippocampus, too. Many small areas of pyramidal neuronal cell death were observed in the CA1 region of hippocampus two days after ischemic episode in rats [Pluta, 2000a]. In these cases complete disappearance of vulnerable neurons in the CA1 sector was noted from 7 to 14 days later [Butler et al., 2002]. About one-third of brains after ischemia-reperfusion injury did not present complete disappearance of CA1 sector in short-term survival time. These animals developed death of all nerve cells of CA1 area in late stages (Figure 4) [Pluta, 2000a; Pluta, 2002a; Pluta, 2002b]. Additionally, some data presented distinct pathology in brain regions considered to be resistant to ischemia, such as: CA2, CA3 and CA4 sectors of hippocampus and dentate gyrus. These regions showed early postischemic changes in nerve cells at 1, 6, 9 and 12 months after ischemic insult [Pluta, 2000a]. Pyramidal neurons are more frequently targeted in hippocampus [Pluta, 2000a; Butler et al., 2002; Pluta, 2002a; Pluta, 2002b]. These nerve cells have very long axons that connect different structures of the brain together through many synaptic connections. Pathology in pyramidal neurons may be of significant relevance to Alzheimer’s disease development. Actually, it has become recognized that pathological processes in ischemic nerve cells continue well beyond the acute stages [Pluta, 2000a; Pluta, 2002a]. Data indicate that brain ischemia regardless of survival time is followed by acute neuronal changes in brain parts belonging or not, to selectively vulnerable areas. In ischemic brains neurodegenerative alterations in neurons took the form of “burn faintly phenomenon” [Pluta, 2000a; Pluta, 2002a].

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Figure 4. Dilatation of subarachnoid space around and between brain hemispheres. Enlargement of the ventricular system with atrophy of hippocampus and in atrophic hippocampus complete disappearance of the CA1 sector. 10-min brain ischemia – 1 year survival. H&E. x20.

Recent studies show that astrocytes apoptosis may contribute to neuropathogenesis of many acute and chronic neurodegenerative diseases such as brain ischemia and Alzheimer’s disease [Takuma et al., 2004]. Astrocytes have many functions, such as formation of bloodbrain barrier, angiogenesis, immune response and finally formation of scar tissue after neuronal death (Figure 5). Common astrocytic reactions that occur in the brain ischemia and Alzheimer’s disease are cellular swelling, hypertrophy-hyperplasia (astrogliosis) and proliferation (astrocytosis) [Bernaudin et al., 1998; Stoltzner et al,. 2000]. It is widely believed that reactive astrocytes at the early stage of brain injury have a beneficial effect on neurons by participating in several biological processes, such as the repair of the extracellular matrix, control of the blood-brain interface, and trophic support of neurons (Figure 3). However, whether prolonged reactive astrocytic response is beneficial in neuronal recovery is still controversial. Some experiments showed after brain ischemia the early import from brain parenchyma and circulatory network to the astrocytes different fragments of amyloid precursor protein and in the late stage the export from astrocytes to the brain tissue of the


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toxic C-terminal of amyloid precursor protein and β-amyloid peptide [Pluta, 2002b; Pluta, 2004a; Pluta, 2004b]. We considered that in early stages astrocytes were removed from neuronal cells and their neighborhood through phagocytosis toxic fragments of amyloid precursor protein. In their cytoplasm is going degradation of various fragments of amyloid precursor protein. In the late stages when ischemic abnormal metabolism of amyloid precursor protein took on a chronic form and astrocytic metabolism began to be inefficient, we can observe first disruption of astrocyte processes [Pluta et al., 1994b; Pluta, 2002b]. On the other hand, disruption of astrocyte processes indicated degradation of different toxic fragments of amyloid precursor protein in the astroglial cytoplasm. The late observations of astroglial behavior support the idea that astrocytes produce amyloid precursor protein, a source of β-amyloid peptide. When this process developed, atrophy of the brains began at the same time. In an early stage, noted was vigorous incorporation of N-terminal of amyloid precursor protein into the developing astroglial scar around the infarct core [Badan et al., 2004]. Functional recovery after ischemia may reflect the balance between growth phenomena and degenerative processes, including an inflammatory reaction leading to glial scar formation (Figure 5) as well as the release of neurotoxic factors such as the C-terminal of amyloid precursor protein. At the early stages of infarct development the N-terminal of amyloid precursor protein may be synthesized by cells derived from the vascular endothelium that became fragmented after injury [Badan et al., 2004]. Over time, these cells change shape and size to become incorporated into the glial scar in a close spatial relationship with astrocytes and surprisingly formed new brain vessels that penetrated the scar. Concurrently, with the expression of scar-forming N-terminal of amyloid precursor protein there is expression of potentially neurotoxic factors such as C-terminal of amyloid precursor protein [Hsiao et al., 1996; Uetsuki et al., 1999]. Evidence derived from mice overexpressing the Cterminal of amyloid precursor protein indicates that this fragment may promote synaptic degeneration and neuronal death [Oster-Granite et al., 1996]. Some experiments clearly show that C-terminal of amyloid precursor protein steadily accumulates in neurons in the infarcted region as the infarct progresses the C-terminal of amyloid precursor protein immunoreactive elements become increasingly larger in the core even though the neurons are dying and the core becomes largely acellular [Badan et al., 2004]. The same study suggests that C-terminal of amyloid precursor protein identified in microglial cells could be due to the phagocytosis of dead, C-terminal of amyloid precursor protein containing neurons by microglia [Badan et al., 2004; D’Andrea et al., 2004]. Most interestingly, there are recent reports showing that astrocytes, not microglial cells can take up β-amyloid peptide [Matsunaga et al., 2003; WyssCoray et al., 2003]. Additionally, more recent work shows that C-terminal of amyloid precursor protein induces the death of astrocytes whereas the loss of neurons is a secondary consequence of the neuronal dependence on astrocytes for antioxidant protection [Abramov et al., 2003]. The localization of some fragments of amyloid precursor protein to astrocytes may be of significant relevance to Alzheimer’s disease in which chronic astrocytosis is thought to play a key role in the evolution of amyloid plaques and in repairing host tissue through development of glial scars (Figure 5). The study of astrocytes is particularly important considering the co-existence of the apoptotic death of neurons and astrocytes in damaged brains suffering from ischemia [Pluta, 1991a] and neurodegenerative disorders.

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Furthermore, significant astrocyte death occurs after reactive astrocytosis [Eddleston and Muckel, 1993] and dying astrocytes kill neighboring cells in brain injury [Lin et al., 1998]. Brain ischemia β-secretase Tau Cytokines

Apolipoproteins Presenilins

βA

BBB

Gliosis Ischemic neurons Platelets βA

Neuronal death

Neuronal remnants

Scar

CAMA

Physiological scar

Pathological scar

Diffuse plaques

Senile plaques

VD

MD

AD

Figure 5. Propose role of brain ischemia in the development of sporadic Alzheimer’s disease. βA - βamyloid peptide; BBB – blood-brain barrier; CAMA – cerebral amyloid microangiopathy; VD – vascular dementia; MD – mixed dementia; AD – Alzheimer’s disease.

Brain Atrophy Gross examination of animal brains performed about 1 year after ischemia-reperfusion injury revealed hydrocephalic features of brain ventricles, dilatation of the subarachnoid space around and between the hemispheres (Figure 4) [Pluta et al., 1997a; Pluta, 2003a;


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Pluta, 2004a; Pluta, 2004b]. Atrophy of the dorsal hippocampus with complete disappearance of the CA1 area was found after ischemia (Figure 4) [Hossmann et al., 1987; Pluta, 2000a; Pluta, 2002b; Pluta, 2004b]. The presence of new infarcts in experimental brain ischemiareperfusion injury with very long survival has been shown [Pluta, 2003a]. The cortex of the brain was narrow, expressing increased neuronal density. White matter was narrow and in some places with advanced spongiosis [Pluta, 2000a]. Generally, the atrophic brain is indicative of an active progressing neurodegenerative processes.

Treatment of Brain Amyloidosis The primary treatment is not pharmacological and care related. There is as yet no known cure for Alzheimer’s-type amyloidosis but symptomatic treatments are now available. These symptomatic therapies only provide symptomatic relief. These treatments are of modest benefit and do not prevent progression of the disease. These drugs that work by inhibiting the production of acetylcholinesterase an enzyme implicated in Alzheimer’s disease have been of benefit to some patients for short periods of time but side effects often prove difficult. Longterm outcomes of drug therapy are not yet known. A further difficulty is the importance of introducing drug therapy early in the disease. Most general practitioners, however, do not diagnose Alzheimer’s disease early enough for any treatment to be effective. In addition, too many doctors do not treat their older patients aggressively attributing diseases amenable to cure to old age. Although there is as yet no definitive evidence of its effectiveness against Alzheimer’s disease, some investigators are also researching the preventative value of vigorous mental and physical activity. Keeping the brain and the body active particularly in old age may help the brain become more resistant to damage and can reduce the negative effects of stress. Extensive research on new treatments is ongoing. Investigators are evaluating other potentially promising approaches including looking at drugs now used to treat other diseases studying the female hormone estrogen, NMDA receptor antagonist and trying combinations of treatments and studying relationships between Alzheimer’s and vascular dementia

Symptomatic Therapy Four approved drugs in the USA and European Union such as Aricept, Cognex, Exelon and Reminyl are therapies for Alzheimer’s disease. These drugs have a similar mechanism of action the inhibition of acetylcholinesterase activity [De la Torre, 2004]. They may help slow the progression of symptoms for a limited period. A recently approved drug memantine (Namenda) is an NMDA antagonist [Reisberg et al., 2003; Tariot et al., 2004]. The main effect of Namenda is to delay progression of the symptoms of moderate to severe Alzheimer’s disease.

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β-amyloid Peptide Therapy β-amyloid peptide clearance treatments had remarkable results in ischemic model of Alzheimer’s disease [Pluta et al., 1998b; Pluta et al., 1998c; Pluta et al., 1999a; Pluta et al., 1999b] and fewer effects in transgenic mice with overexpression of amyloid precursor protein in experimental immunization trials [Schenk et al., 1999]. In clinical trials, vaccination effects were less conclusive [Nicoll et al., 2003]. Recently, it has been shown that antibodies against β-amyloid peptide are present in human immunoglobulin, which specifically recognize and inhibit the neurotoxic effects of β-amyloid peptide [Dodel et al., 2004]. The paper by Dodel and coworkers highlights a novel and interesting therapeutic option for Alzheimer’s disease. In the past decade, strategies of treatment have been focused on inhibitors of β-secretase and γ-secretase responsible for proteolytic process of amyloid precursor protein and formation of β-amyloid peptide [Dovey et al., 2001; Selkoe, 2001a; Roberts, 2002]. Other investigations have used small particle libraries to screen for molecules that either interfere with assembly of β-amyloid peptide particles into fibrils [Lashuel et al., 2002; De Felice et al., 2004] or disaggregate existing fibrils [Soto, 2001; Gong et al., 2003; Blanchard et al., 2004].

Tau Protein Therapy Experimental therapies have been directed against hyperphosphorylation of tau protein either by inhibiting various protein kinases or promoting phosphatase activities [Lau et al., 2002; Iqbal and Grudke-Iqbal, 2004]. A recent in vitro study presented a small molecule that inhibited tau protein filament nucleation and fibrillization making this molecule a promising candidate to test in experimental animal models [Chirita et al., 2004]. A new interesting fact about β-amyloid peptide vaccination has been noted lastly in experimental condition, in which were passively immunized triple transgenic mice with antibodies to β-amyloid peptide [Oddo et al., 2004]. β-amyloid peptide immunotherapy leads to clearance of early but not late hyperphosphorylated tau protein aggregates via the proteasome [Oddo et al., 2004].

Other Therapies Drugs that protect neurovascular network could be important in the prevention and treatment of Alzheimer’s disease [Pluta et al., 1997c; Pluta, 2000b]. Recently, statins [Deane et al., 2004a] present some influence on blood-brain barrier vessels. Other drugs used in Alzheimer’s disease treatment may help to control some behavioral symptoms [Ohkura et al., 1994; Waring et al., 1999; Vukovic et al., 2003], which have an influence on daily activity. Using this treatment makes patients’ care easier. However, all these presented drugs today do not stop the progression of the Alzheimer’s disease.


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Table 4. Comparison of pathological cascade of events leading to development of dementia in experimental brain ischemia and Alzheimer’s disease. Brain ischemia Alzheimer’s disease Neuropathophysiological abnormalities N-terminal of amyloid precursor protein N-terminal of amyloid precursor protein C-terminal of amyloid precursor protein C-terminal of amyloid precursor protein β-amyloid peptide β-amyloid peptide β-secretase β-secretase γ-secretase γ-secretase Apolipoprotein E Apolipoprotein E Apolipoprotein J Apolipoprotein J Apolipoprotein A1 Apolipoprotein A1 Presenilins Presenilins Tau protein Tau protein Cytokines Cytokines α-synuclein α-synuclein Blood-brain barrier Blood-brain barrier Neuropathological abnormalities Pathology of platelets Pathology of platelets Diffuse amyloid plaques Diffuse amyloid plaques Gliosis Gliosis Microglial activity Microglial activity Necrosis Necrosis Apoptosis Apoptosis Neuronal death Neuronal death Astrocytic death Astrocytic death Leukoaraiosis Leukoaraiosis Final consequences Atrophy of hippocampus Atrophy of hippocampus Brain atrophy Brain atrophy Dementia Dementia

Conclusion Though Alzheimer’s disease is primarily a human disorder, other animal models need to be developed in order to study the mechanism of Alzheimer’s disease etiology. Over the years, attempts have been made to develop several experimental models that at the best display some but not all neuropathological hallmarks of Alzheimer’s disease. Nevertheless, these models are quite useful to study the cascade of neurochemical processes leading to neurodegeneration and to discover potential therapeutic agents. Among them is ischemic model of Alzheimer’s disease that has shed new light on neurovascular dysfunction as a likely contributor to dementia and chronic neurodegenerative situation in Alzheimer’s disease

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(Figure 5, Table 4). In Alzheimer’s disease, we should explain two main problems: first is neuronal death in a specific area, such as hippocampus and the next is amyloid accumulation in the brain. Neuronal death can be explained by direct influence of brain ischemia on selective vulnerable areas such as hippocampus and cortex and it is rather clear. In this condition, Alzheimer’s disease may be primarily centered on neuronal cells and originate from a primary neuronal disease (Figure 5). The integrity of the microvessels and the supply of the nutritious substances to the ischemic brain, as well as impede catabolic outflow of waste products are crucial for the future pathology and for the recovery and survival after that. Brain ischemia induces vascular changes and a sequence of processes that probably promote in early stage healing of bloodbrain barrier vessels [Atwood et al., 2003]. These alterations include β-amyloid peptide accumulation and its clearance during the healing processes. Mechanisms of sealing the vessels that rely on the platelets aggregation and sticking and disaggregation in the site of a leak would lead to temporal integrity of some vessels with the blood-brain barrier function [Pluta et al., 1994c]. Some data suggest that such process also involves the amyloid precursor protein from disaggregated platelets and its cleavage product β-amyloid peptide. Both proteins are known to accumulate in the cerebrovasculature wall following ischemia and Alzheimer’s disease. β-amyloid peptide aggregates and deposits form vessel scab for structural integrity of the blood-brain barrier and brain tissue (Figure 5) [Nihashi et al., 2001; Pluta 2002b]. Such a mechanism would allow delivery of blood to the ischemic brain by sealing cerebrovascular injuries and preventing cellular and acellular pathological blood components entering the brain parenchyma. It can be physiologically purposive deposits of βamyloid peptide [Pluta et al., 1996b], when this situation is going to be chronic we probably can observe pathological deposits in vessels as effects of inefficient process in cells especially vulnerable to ischemic injury in this situation endothelial [Thomas et al., 1996], pericyte [Lupo et al., 2001; Anfuso et al., 2004] and astoglial cells (Figure 5) [Takuma et al., 2004]. We suggest that chronic sealing of β-amyloid peptide may lead especially to the degeneration and disappearance of the blood-brain barrier vessels. It is also possible that decreased number of blood-brain barrier vessels can diminish the brain capillary network and surface area available for β-amyloid peptide influx and efflux and may further amplify the insufficiency of the neurovascular system to filter β-amyloid peptide from the brain parenchyma in to systemic circulation. Thus, β-amyloid peptide in senile plaques can originate primarily from the neurovascular tissue and either escape into the parenchyma across damaged blood-brain barrier or the β-amyloid peptide-laden small vessels as an effect of the blood-brain barrier sealing finally remains there following degeneration of the brain vessels (Figure 5). Given the extensive network of vessels in the brain, it seems improbable that neuritic plaques are not associated with such vessels. β-amyloid peptide that remain there after degeneration of the vessel become core of neuritic plaques. Massive vascular deposition of β-amyloid peptide results in recurrent ischemia-reperfusion injury. Another factor that supports this idea is the pattern of accumulation of β-amyloid peptide perpendicularly to the axis of the microvessels. Another source from bloodstream of β-amyloid peptide [Mehta and Pirttila, 2002] may be released directly from platelets that carry more than 90 percent of the


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plasma amyloid [Davies et al., 1997]. During degradation of platelet, β-amyloid peptide may be delivered and deposited in the brain vessel wall and parenchyma (Figure 5). On the other hand, the above processes can partly explain chronic blood-brain barrier opening in ischemia. Since leaking blood-brain barrier vessels were found inside the diffuse plaques, it is likely that they are the cause of senile plaques. This observation supported the possibility that the plasma β-amyloid peptide [Mehta and Pirttila, 2002] can be the source of amyloid fibers in Alzheimer’s disease. Other elements of the plaques that showed strong positive reaction for β-amyloid peptide were the astrocytes and their processes inside the area of the diffuse plaques and around blood-brain barrier vessels (Figure 1B) [Nihashi et al., 2001 Pluta 2002b; Pluta, 2004b]. The functional consequences of the chronic disturb leakage of the blood-brain barrier vessels following ischemia are not known. However, it can be expected that the chronic flooding of ischemic neurons with plasma β-amyloid peptide would affect their performance. Deposits in ischemic neurons of β-amyloid peptide for a long time following transient brain injury would be an important additional factor that probably limits the survival of terminally injured neurons. Accumulation of two overlapping pathogenic processes/mechanisms may produce selective delayed vulnerability of hippocampal neurons after ischemia. The increased accumulation of β-amyloid peptide in Alzheimer’s disease suggests excessive and pathological remodeling [Nihashi et al., 2001; Pluta, 2002b] within the sick brain triggered by e.g. repetitive ischemic episodes (Figure 5). Therefore, it is likely that fragments of amyloid precursor protein are involved in the initial healing and subsequent rebuilding of brain tissue [Nihashi et al., 2001; Pluta, 2002b] especially in blood-brain barrier vessels after ischemic injury. Finally, if β-amyloid peptide mediated remodeling is going to be abnormal in susceptible individuals very likely results in neurite and synapse withdrawal and hence the loss of neuronal networks activity (Figure 5). In this review, we presented several lines of evidence that relate ischemia-reperfusion injury of the brain to the development of sporadic Alzheimer’s disease (Table 4). The importance of microischemic episode in the etiology of Alzheimer’s disease has been supported by many recent observations. These observations presented abnormal amyloid precursor protein metabolism and expression as effect of transient brain ischemia [Kim et al., 1998; Lin et al., 1999; Bennett et al., 2000; Pluta, 2000a; Shi et al., 2000; Nihashi et al., 2001; Pluta, 2002b]. This affect was influenced by various risk factors such as age [BaidenAmissah et al., 1998; Popa-Wagner et al., 1998; Kawarabayashi et al., 2001, Badan et al. 2003, Badan et al. 2004] female gander [Pluta 2000a, Pluta 2002b, Pluta 2003b; Wen et al., 2004a; Wen et al., 2004c] hyperthermia [Sinigaglia-Coimbra et al., 2002] hyperglycemia [Lin et al., 2001] and menopause lack of estrogens [Shi et al., 1998]. As for the mechanism that ischemic episode influence Alzheimer’s disease it is suggested that β-amyloid peptide deposition in brain parenchyma may be caused mainly by permeability of ischemic blood-brain barrier for blood β-amyloid peptide (Figure 1C, D, Figure 5). New evidence supports this mechanism that transport β-amyloid peptide between the blood and brain and across the blood-brain barrier regulates brain tissue β-amyloid peptide deposition [Pluta, 2003a; Zlokovic, 2004]. Ischemic insult destabilizes neurons and synapses and makes them more sensitive for toxic parts of amyloid precursor protein. Repeated brain circulatory insufficiency probably explains the heterogeneic profile of Alzheimer’s disease [Etiene et al., 1998]. This data supports the idea of late impairment of

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brain parenchyma and function even though the first acute transient ischemia is followed by temporal neurological recovery. The general knowledge suggests that ischemia plays a principal role in the neuronal death in Alzheimer’s disease brains (Figure 5). On the other hand, associations between the ischemic neurons and aberrant ischemic β-amyloid peptide homeostasis have been shown in animal ischemic model of Alzheimer’s disease (Figure 5). We present that ischemic neuronal injury results at first in the induction of brain ischemia and next in the induction by ischemia certain genes and therefore protein synthesis and these proteins may contribute to postischemic amyloidogenesis as additional repairing hallmark of Alzheimer’s disease brain (Figure 5). These Alzheimer’s disease-related genes are induced in ischemic-reperfusion brain injury suggesting that these Alzheimer’s disease-related genes may be complex components of post-ischemic response. Tge relationship between ischemic neuronal and glial death and synaptic alteration and amyloidogenesis is rather clear. Recent studies have strongly suggested the possible pathogenic interactions between ischemic neurons and Alzheimer’s proteins such as βamyloid peptide, presenilin and α-synuclein. For example, data suggest that presenilin may promote neuronal degeneration in brain ischemia and Alzheimer’s disease by increasing the sensitivity of neurons to ischemia. As effects, we have molecular cascades that result in synaptic dysfunction and neuronal degeneration. An increased level of soluble β-amyloid peptide renders ischemic neurons more vulnerable to apoptosis and excitotoxicity. Presenilin additionally disturbs calcium regulation in cells in a way that sensitizes ischemic nerve cells to apoptosis and excitotoxicity. Links between aberrant calcium homeostasis and altered amyloid precursor protein metabolism are emerging in brain ischemia and Alzheimer’s disease pathology. In summary, the acute generation of β-amyloid peptide has evolved as a probable sealant molecule required during early times after ischemic injury for physiological repairing e.g. leaking blood-brain barrier vessels. The chronic generation of β-amyloid peptide has evolved probably in the pathological healing process in ischemic brain with subsequent damage of blood-brain barrier vessels and deposition/maturation of β-amyloid peptide as senile plaques (Figure 5). In late onset so-called sporadic no genetic Alzheimer’s disease the amassment of βamyloid peptide is not related to amyloid precursor protein neuronal overexpression and overproduction [Selkoe 2001a, Tanzi et al. 2004, Zlokovic 2004]. In addition, a relatively small number of Alzheimer’s disease brains have increased β-amyloid peptide production in the brain that is limited to familial cases of the disease [Zlokovic et al., 2005]. Studies presented that neurovascular dysregulation may result in increased β-amyloid peptide accumulation strongly support the view that neuronal alterations are primarily and observed β-amyloid peptide deposition may be rather secondary to neurovascular problems associated with Alzheimer’s disease. β-amyloid peptide is destroying the blood-brain barrier vessels leaving the brain without its important nutrition and clearance mechanisms. Impaired vascular clearance of β-amyloid peptide across the blood-brain barrier leads to an increased β-amyloid peptide capillary deposition and formation of amyloid lesions [Davis et al., 2004; Deane et al., 2004b]. As an effect of neurovascular β-amyloidosis noted was a decrease of LRP1 receptor levels in brain capillaries [Deane et al., 2004b]. What is creating an increase in the levels of soluble β-amyloid peptide in the brain by regulating its bi-directional


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trafficking across the blood-brain barrier [Pluta et al., 1996a; Pluta et al., 1997d; Pluta et al., 1999a; Pluta et al., 2000c; Deane et al., 2004b; Tanzi et al., 2004]. On the other hand, neuroinflammatory response increased cytokine expression and neuronal co-localization of circulating β-amyloid peptide mediates transcytosis of β-amyloid peptide through RAGE receptor across the blood-brain barrier under pathological conditions such as brain ischemia. At later stages in the Alzheimer’s disease development these two processes may work synergistically to form an interrelated and irreversible pathogenic circle that completely devastates the brain. In summary, it is possible that microneurovascular β-amyloidosis in Alzheimer’s disease develops slowly as a result of a progressive β-amyloid peptide clearance disorder rather than due to its uncontrolled production from amyloid precursor protein. Recent knowledge regarding the neuropathophysiology and neuropathology of brain ischemia and Alzheimer’s disease indicates that similar processes contribute to neuronal death and brain parenchyma disintegration (Table 2, 4, Figure 5).

Acknowledgements This review is being dedicated to three prominent internationally recognized neuropathologists who have departed from our world of neuroscience and with whom I had the great pleasure of working and knowing personally. Professors Henry M. Wisniewski MD, PhD, Mirosław J. Mossakowski MD, PhD, and Józef Kałuża MD, PhD, met untimely deaths in September 1999, December 2001 and July 2003, respectively. These neuroscientists were internationally acclaimed neuropathologists and served as mentors and professional advisors to me and who played prominent roles in influencing my career in the field of neurodegenerative diseases, such as brain ischemia and Alzheimer’s disease. This study was supported in part by funds from the European Community, Polish Comity of Scientific Research and Medical Research Centre. I would like to express my deep gratitude to Mr. Sławomir Januszewski, a collaborator in most experiments described in this review article.

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Chapter VIII

Amyloid Beta (Aβ) Peptides in Plasma as Biochemical Markers of Alzheimer's Disease and Mild Cognitive Impairment Tomasz Sobow1∗, Marcin Flirski1, Pawel P. Liberski2 and Iwona Kloszewska1 1 2

Department of Old Age Psychiatry & Psychotic Disorders; Department of Molecular Pathology and Neuropathology, Medical University of Lodz, Poland

Abstract Plasma levels of amyloid β peptides (Aβ) represent a potentially attractive biomarker of Alzheimer’s disease (AD). Although plasma Aβ levels are increased in patients with familial AD mutations, results of the studies encompassing sporadic AD cases are equivocal. In several studies elevated plasma Aβ42 levels could be detected long before the onset of symptoms, though the value of that effect in predicting progression to dementia in mildly cognitively impaired (MCI) subjects is not known. It has recently been proposed that plasma Aβ levels increase merely with age and are neither sensitive nor specific for AD or MCI. Additionally, an increase of Aβ42 plasma levels in women with MCI has been reported and thus suggested to represent a biologic explanation for the increased prevalence of females in the population of late-onset AD patients identified by epidemiologic studies. In our study we have assessed the levels of Aβ peptides in plasma of carefully selected AD patients and MCI subjects compared to healthy controls matched for age, gender and education. The selection procedure employed in the study has been aimed at excluding patients with mixed AD as well as patients fulfilling current diagnostic criteria for both ∗

Corresponding author, Czechoslowacka 8/10, 92-216 Lodz, Poland. [email protected]

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Tomasz Sobow, Marcin Flirski, Pawel P. Liberski and Iwona Kloszewska AD and other specific forms of dementia (e.g. dementia with Lewy bodies). No difference in any of the evaluated parameters (Aβ40, Aβ42, Aβ40/Aβ42 ratio) was observed between the AD and control groups. In subjects with MCI plasma Aβ42 levels were significantly elevated versus both AD (p

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