Neuroendocrinology, Volume 182: Pathological Situations and Diseases

PROGRESS IN BRAIN RESEARCH

VOLUME 182

NEUROENDOCRINOLOGY: PATHOLOGICAL

SITUATIONS AND DISEASES

EDITED BY

LUCIANO MARTINI Department of Endocrinology,

University of Milano, Milano, Italy

GEORGE P. CHROUSOS First Department of Pediatrics

Athens University Medical School, Athens, Greece

FERNAND LABRIE Molecular Endocrinology

Laval University, Quebec City, Canada

KAREL PACAK Section on Medical Neuroendocrinology

NICHD-NIH, Bethesda, MD, USA

DONALD W. PFAFF Laboratory of Neurobiology and Behavior,

Rockefeller University, New York, NY, USA

AMSTERDAM – BOSTON – HEIDELBERG – LONDON – NEW YORK – OXFORD

PARIS – SAN DIEGO – SAN FRANCISCO – SINGAPORE – SYDNEY – TOKYO

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands Linacre House, Jordan Hill, Oxford OX2 8DP, UK 360 Park Avenue South, New York, NY 10010-1710 First edition 2010 Copyright � 2010 Elsevier B.V. All rights reserved No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: [email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at http://elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-444-53616-7 ISSN: 0079-6123 For information on all Elsevier publications visit our website at elsevierdirect.com

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List of Contributors

K. Ashida, Department of Endocrinology and Diabetes Mellitus, School of Medicine, Fukuoka University, Fukuoka, Japan H.A. Bimonte-Nelson, Department of Psychology, Arizona State University, Tempe, AZ, USA R. Diaz Brinton, Departments of Pharmacology & Pharmaceutical Sciences, Biomedical Engineering and Neurology, University of Southern California, Los Angeles, CA, USA H.S. Chahal, Centre for Endocrinology, William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK G.P. Chrousos, Division of Endocrinology, Diabetes and Metabolism, First Department of Pediatrics, University of Athens, “Agia Sophia” Children’s Hospital, Goudi, Athens, Greece J.A. Cidlowski, Molecular Endocrinology Group, Laboratory of Signal Transduction, NIEHS, NIH, DHHS, Research Triangle Park, NC, USA A. Colao, Department of Molecular & Clinical Endocrinology and Oncology, “Federico II” University of Naples, Naples, Italy C. Conrad, Department of Psychology, Arizona State University, Tempe, AZ, USA M.C. De Martino, Department of Internal Medicine, Division of Endocrinology, Erasmus Medical Center, Rotterdam, The Netherlands A. Faggiano, Department of Molecular & Clinical Endocrinology and Oncology, “Federico II” University of Naples, Naples, Italy W. Fan, Division of Endocrinology and Metabolism, School of Medicine, University of California, San Diego, La Jolla, CA, USA A.B. Grossman, Centre for Endocrinology, William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK V.W. Henderson, Departments of Health Research & Policy (Epidemiology) and of Neurology & Neurological Sciences, Stanford University, Stanford, CA, USA L.J. Hofland, Department of Internal Medicine, Division of Endocrinology, Erasmus Medical Center, Rotterdam, The Netherlands C. Kanaka-Gantenbein, Division of Endocrinology, Diabetes and Metabolism, First Department of Pediatrics, University of Athens, “Agia Sophia” Children’s Hospital, Goudi, Athens, Greece V. Kantorovich, Division of Endocrinology and Metabolism, Department of Internal Medicine, College of Medicine, University of Arkansas for Medical Sciences, Arkansas Cancer Research Center, AR, USA B. Kola, Centre for Endocrinology, William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK M. Korbonits, Centre for Endocrinology, William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK F. Labrie, Research Center in Molecular Endocrinology, Oncology and Human Genomics, Laval University and Laval University Hospital Research Center (CRCHUL), Québec, Canada S.W.J. Lamberts, Department of Internal Medicine, Division of Endocrinology, Erasmus Medical Center, Rotterdam, The Netherlands C.T. Lim, Centre for Endocrinology, William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK v

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S. Melmed, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA L. Min, Department of Medical Biochemistry, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan S. Molatore, Institute of Pathology, Helmholtz Zentrum München-German Research Center for Environmental Health, Neuherberg, Germany H. Nawata, Graduate School of Medical Science, Kyushu University; Fukuoka Prefectural University, Tagawa City, Fukuoka, Japan M. Nomura, Department of Medicine and Bioregulatory Science, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan K. Pacak, Section on Medical Neuroendocrinology, Reproductive and Adult Endocrinology Program, NICHD, NIH, Bethesda, MD, USA N.S. Pellegata, Institute of Pathology, Helmholtz Zentrum München-German Research Center for Environmental Health, Neuherberg, Germany P. Pervanidou, Developmental and Behavioral Pediatrics Unit, First Department of Pediatrics, Athens University Medical School, “Agia Sophia” Children’s Hospital, Goudi, Athens, Greece R. Pivonello, Department of Molecular & Clinical Endocrinology and Oncology, “Federico II” University of Naples, Naples, Italy S. Sakka, Division of Endocrinology, Diabetes and Metabolism, First Department of Pediatrics, University of Athens, “Agia Sophia” Children’s Hospital, Goudi, Athens, Greece L.K. Smith, Molecular Endocrinology Group, Laboratory of Signal Transduction, NIEHS, NIH, DHHS, Research Triangle Park, NC, USA A. Tahir, Centre for Endocrinology, William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK T. Watanabe, Department of Internal Medicine, Nakatsu Municipal Hospital, Nakatsu, Oita, Japan T. Yanase, Department of Endocrinology and Diabetes Mellitus, School of Medicine, Fukuoka University, Fukuoka, Japan R. Yu, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA

“en guise d’introduction …’’

As presented to me by Luciano Martini, Neuroendocrinology: volume 181 the normal neuroendocrine system and volume 182 pathological situations and diseases will be two virtual volumes of over 500 virtual pages written by a group of distinguished colleagues all well qualified, many of these friends of old. It is not my intention to present or discuss in any shape or form the enormous sum that these few lines will precede and accompany. I would need proficiency in several foreign languages, foreign to me certainly. I am referring to the languages of molecular biology and the immensely intellectual country they come from and serve. Reading the list of titles of the many chapters, these two volumes will be made of, is in itself the description of what happened to that field of knowledge since the word neuro-endocrinology (with the hyphen) was coined and used for the first time in 1946 as the title of the also (already) enormous book (1106 pages) Traité de Neuro-endocrinologie by Gustave Roussy and Michel Mosinger. With the well-established knowledge of neurotransmitters as small molecules (acetylcholine, catechola­ mines, etc.) between nerve endings (synapses) (Sherrington, von Euler, etc.) and that of hormones (coining of the word by Starling in 1904 for secretin out of extracts of duodenal tissue), the concept of – and the word – neurosecretion appeared in the 1940s with the stunning images by the Scharrers (Ernst and Berta) of protein granules (Gomori stain) in neuronal cell bodies and moving along with axoplasmic flow. In vertebrates, that was essentially and originally dealing with observations in the hypothalamus and the posterior pituitary. But similar images were also found in invertebrates, leading to a major extension of the concept. And when the interest started about the mechanisms involved in the control of regulation of the anterior pituitary functions, involving both hypothalamic centres and unusual capillary connections, the very concept of specific molecules of neuronal origin travelling to the pituitary became the question of the day (see Geoffrey W. Harris’s Neural Control of the Pituitary Gland, 1955), which was eventually answered after over 17 years of research with the characterization of all the suspected releasing factors plus an unexpected inhibitory factor, somatostatin, all first characterized in extracts of hypothalamic tissues. So far, a rather linear way of thinking, so to speak. But all along and more and more intriguing were the observations generated by the molecular biology approach I mentioned above, that followed: syntheses of analogues of the original sequences with agonist or antagonist activities, recognition of multiple receptors for each and all of these peptides, cDNA cloning of all, etc. So much so that the conclusion was reached that each and all of these ligands and their receptors were actually quite ubiquitous and functional throughout the organ­ ism and not only located in the hypothalamus and other classical structures of the nervous system.

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One remarkable example, out of a large library of the same: presence of the full CRH system – peptides, mRNAs, receptors, binding proteins … in adipose tissues (e.g. Seres, J., Bornstein, S. R., Seres, P., Willenberg, H. S., Schulte, K. M., Scherbaum, W.A., et al. (2004). Corticotropin-releasing hormone system in human adipose tissue. Journal of Clinical Endocrinology and Metabolism, 89, 965– 970). Also, that several of these peptides and their chemistry are involved in psychological events normal and abnormal is another of these now unquestionable conclusions as discussed in several chapters here; David DeWied (1926–2004) was a precursor. Let me close here these simple opening lines for what will be major reference volumes – if the word may apply to a virtual entity. We do indeed live in a world where even the virtual is real … Roger Guillemin, MD Nobel Laureate The Salk Institute for Biological Studies, La Jolla, California, United States of America

L. Martini (Eds.)

Progress in Brain Research, Vol. 182

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 1

Glucocorticoid-induced apoptosis of healthy and malignant lymphocytes Lindsay K. Smith and John A. Cidlowski
Molecular Endocrinology Group, Laboratory of Signal Transduction, NIEHS,

NIH, DHHS, Research Triangle Park, NC, USA

Abstract: Glucocorticoids exert a wide range of physiological effects, including the induction of apoptosis in lymphocytes. The progression of glucocorticoid-induced apoptosis is a multi-component process requiring contributions from both genomic and cytoplasmic signaling events. There is significant evidence indicating that the transactivation activity of the glucocorticoid receptor is required for the initiation of glucocorticoid-induced apoptosis. However, the rapid cytoplasmic effects of glucocorticoids may also contribute to the glucocorticoid-induced apoptosis-signaling pathway. Endogenous glucocorticoids shape the T-cell repertoire through both the induction of apoptosis by neglect during thymocyte maturation and the antagonism of T-cell receptor (TCR)-induced apoptosis during positive selection. Owing to their ability to induce apoptosis in lymphocytes, synthetic glucocorticoids are widely used in the treatment of haematological malignancies. Glucocorticoid chemotherapy is limited, however, by the emergence of glucocorticoid resistance. The development of novel therapies designed to overcome glucocorticoid resistance will dramatically improve the efficacy of glucocorticoid therapy in the treatment of haematological malignancies. Keywords: glucocorticoid; apoptosis; lymphocyte; haematological malignancy; glucocorticoid resistance

environmental stress, nociception and emotion. Stimulation of hypothalamic corticotropin-releasing hormone (CRH) secretion prompts the release of adreno-corticototropic hormone (ACTH) from the pituitary, which induces glucocorticoid synth­ esis within the zona fasiculata of the adrenal cortex. Glucocorticoids auto-regulate their secretion though negative feedback inhibition of CRH and ACTH synthesis and release. In humans, cortisol is the predominant circulating glucocorticoid. Once in circulation, natural glucocorticoids are predominately bound to corticosteroid-binding globulin (CBG). Due to their lipophilic nature,

Introduction Glucocorticoids are a class of essential stressinduced steroid hormones regulating a variety of cardiovascular, metabolic, homeostatic and immunological functions. Endogenous glucocorticoids are synthesized and secreted under the control of the hypothalamic–pituitary–adrenal axis in response to stressors including


Corresponding author. Tel.: (919)541-1564; Fax: (919)541-1367; E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)82001-1

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of physiological actions. For example, in the liver and adipose tissue, glucocorticoids positively regulate metabolism through the stimulation of gluconogenesis and lipolysis, respectively. Con­ versely, in the immune compartment, glucocorti­ coids are largely inhibitory, causing immune suppression through the induction of apoptosis and cell cycle arrest and the inhibition of inflam­ mation via the repression of pro-inflammatory cytokines (Fig. 1) (Hermoso and Cidlowski, 2003; Rhen and Cidlowski, 2005). Given their broad bioavailability and diverse physiological effects, synthetic glucocorticoids are among the most commonly prescribed drugs for

endogenous glucocorticoids are widely bioavail­ able and easily cross the cell membrane via pas­ sive diffusion (Hermoso and Cidlowski, 2003; Rhen and Cidlowski, 2005). Glucocorticoids exert their physiological effects through the ubiquitously expressed glucocorticoid receptor (GR), a member of the nuclear hormone receptor super family of ligand-activated tran­ scription factors. Upon ligand binding, the GR translocates to the nucleus where it activates or represses the transcription of glucocorticoidresponsive genes. Due to the broad distribution of both glucocorticoids and their cognate recep­ tors, glucocorticoid signaling exerts a wide range

Cerebrum Emotion via limbic system

Cytokines

Thalamus Nociceptive pathways

Hypothalamus CRH

Cerebellum

Anterior pituitary Dorsal root ganglion

Pain receptors

Adrenal gland

Ventral root

ACTH Spleen

Cortisol

Lymph nodes

Thymus

Sympathetic

ganglion

Leukocytes Immune system

Adipose tissue

Cardiovascular system Liver Musculoskeletal system

Fig. 1. Pleitrophic effects of glucocorticoids in responsive tissues. Endogenous glucocorticoids are generated in response to various stressors, including emotion and nociception. The subsequent physiolgical actions of glucocorticoids in responsive tissues are denoted as stimulatory (dashed line) or inhibitory (dotted line); (adapted from Rhen and Cidlowski, 2005).

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the treatment of inflammatory disorders, autoim­ mune diseases and sepsis. They are also a main­ stay in the treatment of haematological malignancies. Numerous high-affinity synthetic glucocorticoids are clinically available, including prednisone and dexamethasone. However, pro­ longed use of these compounds is complicated by numerous deleterious side effects such as osteo­ porosis, hypertension, psychosis, Cushing’s syndrome and leucopenia (Rhen and Cidlowski, 2005). Glucocorticoid use in chemotherapy is lim­ ited by the development of glucocorticoid resis­ tance. Glucocorticoid resistance in leukemia and lymphoma is correlated with a poor prognosis (Dordelmann et al., 1999; Irving et al., 2005; Riml et al., 2004; Schmidt et al., 2004). The mechanisms governing glucocorticoid resistance in these malignancies are an area of considerable interest to both the scientific and medical commu­ nities. This chapter will address the role of gluco­ corticoids in the induction of apoptosis of healthy and malignant lymphocytes as well as the molecu­ lar determinants of glucocorticoid resistance in haematological malignancies.

Glucocorticoid-induced apoptosis of lymphoid cells

region houses the activating function (AF-1) transactivation domain (amino acids 77–262), which interacts with the basal transcription machinery in order to induce transcription (Wright et al., 1993). The central DBD is encoded by exons 3–4 and mediates receptor binding to glucocorticoid response elements (GREs) within the promoters of responsive genes. The DBD also consists of two conserved zinc fingers, which facil­ itate interaction with nuclear factor-kB (NF-kB) and AP-1 transcription factor (first zinc finger) as well as receptor dimerization (second zinc finger) (Heck et al., 1994; Liden et al., 1997; Tao et al., 2001). The region between the two zinc fingers houses a nuclear export signal (NES) (Black et al., 2001; Miesfeld et al., 1987; Tao et al., 2001). A hinge region adjacent to the DBD contains a nuclear localization signal at amino acids 491–498 (Freedman and Yamamoto, 2004). Finally, exons 5–9 encode the C-terminal LBD. This region is responsible for ligand binding and cofactor binding, and also contains a weak AF-2 transactivation domain (Bledsoe et al., 2002; Dahlman-Wright et al., 1992; Hollenberg et al., 1987; Schaaf and Cidlowski, 2002b) (Fig. 2). Following translation, the mature GR resides in the cytosol complexed with an hsp90 dimer, a p23 stabilizing protein and a variety of co-chaperones (Cheung and Smith, 2000; Pratt and Toft, 1997).

Glucocorticoid receptor Structure

Expression

The GR is a member of the type I nuclear hor­ mone receptor super family. Members of this super family are characterized by the formation of homodimers and the presence of three distinct functional domains: the C-terminal ligand-binding domain (LBD), the internal zinc-finger DNAbinding domain (DBD) and the N-terminal transactivation domain (NTD) (Escriva et al., 2004; Escriva et al., 1997; Giguere et al., 1986; Laudet et al., 1992; Weinberger et al., 1985). The GR gene (NR3C1) is located on chromosome 5q31.3 and encodes nine exons (Theriault et al., 1989). Exon 1 represents the 50 -untranslated region while exons 2–9 are protein coding (Duma et al., 2006). Exon 2 encodes the majority of N-terminal domain. This

The predominant GR expressed in human tissues is the full-length GRa isoform (Pujols et al., 2002). However, there are numerous additional GR pro­ tein isoforms generated from the single GR gene via alternative splicing and the use of alternative translation initiation sites (Fig. 3). Alternative splicing of GR pre-mRNA generates five distinct GR protein isoforms, namely GRa, GRb, GRg, GR-A and GR-P. Of these, GRa and GRb are the most widely expressed. These two receptor iso­ forms differ in their carboxyl termini due to the use of alternative splicing sites within exon 9 (Hollenberg et al., 1985). GRa is produced from the splicing of exon 8 to the proximal end of exon 9, thus generating a 777-amino acid protein. Exon

4 Domain structure of GRα protein DNA binding Hinge domain

N-Terminal domain (NTD) AF-1 1

77

DBD 282

421

488 527

Ligand-binding domain (LBD) NLS

AF-2 777

Fig. 2. Domain structure of human glucocorticoid receptor (GR) protein. The GR contains three major functional regions: the N-terminal transactivation domain, the central DNA-binding domain and the C-terminal ligand-binding domain.

9 contributes an additional 50 residues to the LBD of the GRa receptor. GRb is created from the splicing of exon 8 to the distal end of exon 9, generating a shorter protein of 742 residues, including 15 unique C-terminal residues contribu­ ted by exon 9. This splice site is predominantly found in humans and has not been identified in the mouse, explaining the lack of GRb expression in mice (Otto et al., 1997). The 15 C-terminal residues contributed by exon 9 render GRb unable to bind glucocorticoids or transactivate glucocorticoid-responsive promoters (Oakley et al., 1999; Yudt et al., 2003). Initially, GRb was described solely as a dominant negative inhibitor of GRa transactivation (Bamberger et al., 1995; Oakley et al., 1999; 1996). This inhibition occurs through both direct interaction with GRa and competitive recruitment of transcriptional coacti­ vators (de Castro et al., 1996). Deletion of the 15 unique C-terminal amino acids rendered GRb unable to repress GRa transactivation (Oakley et al., 1996). In contrast to earlier findings, recent studies have described a direct transcriptional role for GRb. For example, human glucocorticoid receptor b (hGRb), expressed in the absence of hGRa, possesses intrinsic transcriptional activity. Furthermore, GRb can selectively bind the GR antagonist RU-486 and this binding diminishes its intrinsic transcriptional activity (Lewis-Tuffin et al., 2007). More recently, Kelly et al. found that GRb is capable of repressing transcription from the cytokine interleukin (IL)-5 and (IL)-13 promoters via recruitment of histone deactylase I (Kelly et al., 2008). The GRg splice variant harbours an additional three base insertion in the DBD, resulting in the

insertion of an arginine residue between the two zinc fingers, thus reducing its transcriptional capa­ city. The GRg isoform is largely expressed in lym­ phocytes (3.8–8.7% of total GR mRNA) (Rivers et al., 1999). However, the evaluation of GRg protein expression in lymphocytes is hampered by the absence of a specific GRg antibody. The GR-A splice variant lacks exons 5–7, resulting in a truncated LBD and impaired transactivation activity. Finally, the GR-P splice variant lacks exons 8 and 9, resulting in a truncated LBD lacking the ability to bind glucocorticoids (Moalli et al., 1993). Additional GR isoforms are generated through the use of alternative translation initiation sites. These isoforms were first identified by immuno­ blotting as a GRa doublet migrating at 94 and 91 kDa (Lu and Cidlowski, 2005; Yudt and Cidlowski, 2001). The existence of additional GRa isoforms was confirmed by the identification of an in-frame internal translation initiation site at methionine 27. Use of this site is responsible for the generation of the 91-kDa species of GRa, now termed GRa-B (Yudt and Cidlowski, 2001). Further scanning for internal translational start sites revealed the presence of six additional GRa translational isoforms: GRa-C1, C2, C3, D1, D2 and D3 (Lu and Cidlowski, 2005). These iso­ forms differ only in the length of their N-termini. All bind glucocorticoids with similar affinity, but possess unique gene expression profiles and transcriptional activities. For example, GRa-C3 is the most transcriptionally active, even enhan­ cing the basal transcriptional activity of GRa-A (Lu and Cidlowski, 2005). Conversely, the GRaD isoform is the least transcriptionally active and

5 The glucocorticoid receptor: one gene yields many proteins Chromosome 5

GR gene 1

2

3 4

5

6 7

8

9α

9β

(a) GR precursor mRNA

(b) GR splice variants

1 1

777

GRα

742

GRβ

778

GRγ

R453 1 Δ Exons 5–7 1

592 Δ Exons 8–9

GR-A

1

676

GR-P

(c) GRα translational isoforms

777 A

1 27

B

86

C1

90

C2

98

C3

316

D1

331

D2

336

D3

Fig. 3. The glucocorticoid receptor (GR): one gene yields many proteins. (a and b) The hGR pre-mRNA undergoes alternative splicing, generating the dominant GRa isoform and the lower abundance GRb, GRg, GR-A and GR-P isoforms. (c) The GRa isoform is also subject to alternative translation initiation, giving rise to translational isoforms differing in the length of the N-terminal domain.

unable to induce apoptosis in a stably overexpressing osteosarcoma cell line (Lu et al., 2007). Given the distinct actions of these diverse GR

protein isoforms, alterations in their intracellular ratios may influence the lymphoid response to glucocorticoids.

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GR expression is under the control of three distinct promoters: 1A, 1B and 1C. Alternative promoter usage results in transcripts bearing exons 1A, 1B and 1C. Alternative splicing of these exons leads to the generation of nine alter­ native exon 1 splice variants (1A, 1B, 1C, 1D, 1E, 1F, 1H, 1I and 1J) (Breslin et al., 2001; Presul et al., 2007; Turner and Muller, 2005). Further splicing of exons 1A and 1C yields exons 1A1, 1A2 and 1A3 and exons 1C1, 1C2 and 1C3, respectively (Breslin et al., 2001; Turner and Muller, 2005). These assorted transcripts all encode the same protein; however, selective promoter usage may influence downstream splicing and translation initiation events as well as GR protein levels and, ultimately, responsiveness to glucocorticoid treatment (Breslin et al., 2001; Pedersen et al., 2004; Presul et al., 2007; Russcher et al., 2007).

Glucocorticoid receptor signaling Genomic effects In the cytoplasm, unliganded GR exists as a multiprotein heterocomplex associated with an Hsp90 dimer, a p23 stabilizing protein and various immu­ nophilin chaperone proteins (Cheung and Smith, 2000; Pratt et al., 1996). Upon ligand binding, the GR heterocomplex undergoes a conformational change, releasing the GR from cytoplasmic sequestration, promoting receptor homodimeriza­ tion and nuclear translocation of the GR homodimer (Davies et al., 2002; Elbi et al., 2004; Freedman and Yamamoto, 2004; Hager et al., 2004; Nagaich et al., 2004). In the nucleus, the activated GR homodimer binds specific DNA ele­ ments or GREs within the promoter regions of glucocorticoid-responsive genes. The consensus GRE is composed of two hexamer half-sites sepa­ rated by three random nucleotides. A majority of GREs contain the hexamer half-site sequence TGTTCT. The number and location of these GREs influences the intensity of the transcrip­ tional response (Freedman and Luisi, 1993). Once bound to the GRE, the GR homodimer recruits transcriptional coactivators as well as the basal transcriptional machinery to the

transcription start site. These co-activators include cAMP response element-binding (CREB)-bind­ ing protein (CBP), steroid receptor co-activator-1 (SRC-1), GR-interacting protein-1 (GRIP-1), p300 and SWI/SNF (Adcock, 2001; Rogatsky et al., 2002; Wallberg et al., 2000). These co-activators induce histone acetylation, thus allowing for transactivation of glucocorticoidresponsive genes. The activated GR homodimer is also capable of gene repression. GR can directly interact with DNA via negative GREs within the promoter regions of target genes (Dostert and Heinzel, 2004; Sakai et al., 1988). This interaction inhibits the transcription of genes associated with these promoters. Promoters with described nGREs include the corticotropin-releasing factor (CRF), osteocalcin and prolactin promoters (Drouin et al., 1993; Malkoski and Dorin, 1999; Meyer et al., 1997). Alternatively, GR can regulate tran­ scription independent of GR binding through direct interaction with other nuclear transcription factors such as NF-�B, AP-1, STAT5 and STAT3, thus modulating the transcription of genes under the control of these transcription factors (Fig. 4) (McKay and Cidlowski, 2000; Ray and Prefontaine, 1994; Scheinman et al., 1995; Stocklin et al., 1996; Yang-Yen et al., 1990; Zhang et al., 1997).

Cytoplasmic effects GR signaling has been reported to induce rapid effects in the cytoplasm within minutes of ligand binding. For example, upon ligand binding, Src kinase is released from the cytosolic GR hetero­ complex, resulting in lipocortin-1 activation and inhibition of arachidonic acid release. The activa­ tion of Src required ligand-bound GR, but was independent of transactivation (Croxtall et al., 2000). Furthermore, glucocorticoids have long been known to alter cytoplasmic ion content, causing rapid alterations in calcium, sodium and potassium concentrations (Bortner et al., 1997; McConkey et al., 1989a, 1989b). Glucocorticoids also cause rapid increases in the mitochondrial production of reactive oxygen species, ceramide

7 Glucocorticoid signaling

Glucocorticoids

Extracellular Space

Cytoplasm

p23 HSP90

p23

GR

0

P9

HS

HSP9

0

GR

Gene regulation Nucleus Induction

Repression

GILZ

GR

Osteocalcin

GR

GRE

nGRE

P

GR

Stat5 IGF-1

GR p65 p50

IL-1β

P

STAT

NF-κB

Fig. 4. Mechanisms of glucocorticoid-regulated gene expression. Ligand binding liberates the glucocorticoid receptor (GR) from cytosolic sequestration, leading to rapid nuclear translocation and homodimerization. In the nucleus, GR can activate gene transcription through direct binding to GREs in the DNA or through stimulatory interactions with transcription factors such as STAT5. GR can also repress gene expression by directly interacting with nGREs in the DNA, or through inhibitory protein–protein interactions with transcription factors including NF-kB.

and hydrogen peroxide, and the lysosomal release of cathepsin B (Cifone et al., 1999; Tonomura et al., 2003; Wang et al., 2006a; Zamzami et al., 1995). Interestingly, glucocorticoids have been reported

to induce the translocation of GR to the mitochon­ dria in both thymocytes and lymphoma cells (Sio­ nov et al., 2006). This mitochondrial GR may mediate mitochondrial production of reactive

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oxygen species and ceramide as well as the rapid calcium mobilization following glucocorticoid treat­ ment (Gavrilova-Jordan and Price, 2007). Recently, membrane-bound GR (mGR) has been suggested in T lymphocytes. Glucocorticoid treatment inhibits TCR signaling via the mGR. This inhibition occurs through the disruption of the lymphocyte-specific protein tyrosine kinases Lck and Fyn. These kinases, anchored to Hsp90, are components of the TCR-linked mGR–multi­ protein complex. Glucocorticoid treatment results in the rapid dissociation of Lck and Fyn from the multi-protein complex, leading to reduced phos­ phorylation of Lck/Fyn substrates and impaired initiation of TCR signaling. This diminished TCR signaling suppresses downstream cytokine synthesis, cellular migration and proliferation of T-lymphocytes (Lowenberg et al., 2005, 2006).

Glucocorticoid-induced apoptosis Genomic signaling The progression of glucocorticoid-induced apop­ tosis is a multi-faceted process requiring contribu­ tions from both genomic and cytoplasmic signaling events. Genomic events alter the protein content of the cell, creating an environment favorable to the execution of the apoptotic pathway. There is significant evidence indicating that the transacti­ vation activity of the GR is required for the initia­ tion of glucocorticoid-induced apoptosis. For example, glucocorticoid-induced apoptosis of lym­ phocytes does not progress in the presence of actinomycin D or cycloheximide, indicating a requirement for de novo transcription and transla­ tion in the execution of the apoptotic cascade (Cifone et al., 1999; Mann and Cidlowski, 2001; Mann et al., 2000; McConkey et al., 1989b; Wang et al., 2006b). The finding that an activation-defi­ cient GR mutant possessing unaltered transre­ pression capability fails to initiate glucocorticoidinduced apoptosis further supports this observa­ tion (Ramdas and Harmon, 1998). Finally, thymo­ cytes isolated from a knock-in mouse harbouring a point mutation presumably preventing receptor dimerization, and thus GR transactivation, also

failed to undergo glucocorticoid-induced apopto­ sis (Reichardt et al., 1998). Numerous laboratories have performed gen­ ome-wide microarray analysis in order to identify genes differentially regulated during glucocorti­ coid-induced apoptosis (Medh et al., 2003; Schmidt et al., 2006; Thompson and Johnson, 2003; Thompson et al., 2004; Tissing et al., 2007; Wang et al., 2003a; Yoshida et al., 2002). How­ ever, to date, only a few genes have been assigned a functional role in the regulation of glucocorti­ coid-induced apoptosis. Most notably, the expres­ sion of the pro-apoptotic BH3-only Bcl-2 family member Bim is induced by glucocorticoid treat­ ment in murine lymphoma cell lines, human leu­ kemic cell lines, mouse primary thymocytes and human primary chronic lymphoblastic leukemia (CLL) and acute lymphoblastic leukemia (ALL) samples (Distelhorst, 2002; Iglesias-Serret et al., 2007; Schmidt et al., 2004, 2006; Wang et al., 2003a). The mechanism of induction is likely indir­ ect, as there is no GRE in the promoter region of the Bim gene (Bouillet et al., 2001; Wang et al., 2003a). One potential mechanism of Bim induc­ tion is via the induction of the Fox03A/FKHRL1 transcription factor, which is up-regulated by glu­ cocorticoids (Dijkers et al., 2000; Planey et al., 2003). A more recent study found that the activity of the serine/threonine kinase GSK3 is a key med­ iator of glucocorticoid-induced Bim up-regulation (Nuutinen et al., 2009). The induction and activa­ tion of Bim leads to downstream activation of the apoptotic mediators Bax and Bak (Kim et al., 2009). Once activated, these mediators mediate the destabilization of the mitochondrial mem­ brane potential, a hallmark of the intrinsic mito­ chondrial apoptosis pathway (Kim et al., 2009). The up-regulation of Bim is likely an important mediator of glucocorticoid-induced apoptosis. For example, thymocytes from homozygous Bim knock-out mice exhibit decreased sensitivity to glucocorticoid-induced apoptosis (Bouillet et al., 1999). Furthermore, in vitro studies utilising shRNA or siRNA targeting the various Bim tran­ scripts confirm a substantial role for Bim induction in the progression of glucocorticoid-induced apop­ tosis (Abrams et al., 2004; Lu et al., 2006; Ploner et al., 2008).

9

The role of glucocorticoid-induced leucine zip­ per (GILZ) is an area of expanding interest in the study of glucocorticoid-induced apoptosis. GILZ was first identified as a glucocorticoid-responsive gene by a systemic screen for genes responsive to glucocorticoids in the thymus (D’Adamio et al., 1997). Due to the presence of three GREs in the GILZ promoter, the glucocorticoid induction of GILZ expression is direct and robust (Wang et al., 2004). The strongest evidence that GILZ may mediate glucocorticoid-induced apoptosis is provided by studies of GILZ transgenic mice. In these mice, the GILZ transgene is specifically targeted to the T-cell compartment. Primary thymocytes from these mice were resistant to TCR-induced apoptosis. However, they exhibited augmented glucocorticoid-induced apoptosis due to reduced expression of the Bcl-2 family member, Bcl-XL, as well as increased activation of caspases 8 and 3 (Delfino et al., 2004). GILZ also mediates glucocorticoid-induced cell cycle arrest through direct interaction with and inhibition of the prolif­ erative Ras and Raf oncogenes (Ayroldi et al., 2007). In addition to Bim and GILZ, glucocorticoids rapidly transactivate the stress gene dexametha­ sone-induced gene 2 (Dig2) in murine lymphoma cell lines. Interestingly, Dig2 overexpression reduced the sensitivity of these cells to glucocorti­ coid-induced apoptosis, suggesting a pro-survival function for this gene (Wang et al., 2003b). Gran­ zyme A is up-regulated following glucocorticoid treatment in B-ALL cells. Pharmacological inhibi­ tion of granzyme A blunted the apoptotic response, indicating that this enzyme is an effector of glucocorticoid-induced apoptosis (Yamada et al., 2003). T-cell death-associated gene (TDAG8) is rapidly induced by glucocorti­ coids in thymocytes. Thymocytes from TDAG8 transgenic mice exhibited increased activation of caspases 3, 8 and 9 following glucocorticoid expo­ sure (Tosa et al., 2003). However, thymocytes from TDAG8-deficient mice remained sensitive to glucocorticoid-induced apoptosis, suggesting a minor role for TDAG8 in glucocorticoid-induced apoptosis (Radu et al., 2006). Glucocorticoid exposure represses the pro-survival oncogene c­ myc in human leukemic CEM cells (Wang et al.,

2003a). Furthermore, glucocorticoids repress the glycolytic hexokinase II enzyme (Tonko et al., 2001). Hexokinase II acts as a stabilizer of the mitochondrial voltage-dependent anion channel (VDAC). Interestingly, hexokinase II overexpres­ sion abrogated glucocorticoid-induced apoptosis via inhibition of mitochondrial destabilization (Sade et al., 2004). In summary, glucocorticoids regulate the transcription of several genes, the expression of which influences cellular progression through the glucocorticoid-induced apoptotic pathway.

Cytoplasmic signaling Glucocorticoids cause rapid and sustained increases in cytosolic calcium concentrations in thymocytes, lymphoma cells and B lymphoblasts (Bian et al., 1997; Distelhorst and Dubyak, 1998; Hughes et al., 1997; Lam et al., 1993; McConkey et al., 1989b; Orrenius et al., 1991). Buffering of cytosolic calcium or culture in calcium-free media prevented glucocorticoidinduced apoptosis of primary thymocytes (McConkey et al., 1989b). Interestingly, phar­ macological inhibition of the calcium-binding protein calmodulin inhibited DNA fragmentation without interfering with cytosolic calcium increase, indicating that calmodulin mediates the downstream apoptotic effect of glucocorticoid-induced calcium mobilization (Dowd et al., 1991). However, the precise role of calcium mobilization in glucocorticoid-induced apoptosis remains controversial. A subsequent study reported that chelation of intracellular cal­ cium inhibits DNA fragmentation, but not glucocor­ ticoid-induced apoptosis in primary thymocytes (Iseki et al., 1993). Thus, calcium mobilization is likely required for endonuclease activation, but not other aspects of glucocorticoid-induced apoptosis. Glucocorticoids also cause a net potassium efflux in thymocytes and CEM T-ALL cells (Benson et al., 1996; Bortner and Cidlowski, 2000). This potassium loss enhances apoptosis in thymocytes and normal intracellular potassium levels inhibit DNA fragmentation and caspase-3 activation in lymphocytes (Bortner et al., 1997;

10

Hughes et al., 1997). Furthermore, pharmacologi­ cal inhibition of plasma membrane potassium channels in primary thymocytes effectively inhib­ ited glucocorticoid-induced apoptosis through the prevention of cytosolic potassium loss and inhibi­ tion of mitochondrial membrane destabilization (Dallaporta et al., 1999). Glucocorticoids (ranging from 107 to 1012 M) induce a rapid increase in intracellular ceramide concentrations in primary thymocytes within 15 min of treatment (Cifone et al., 1999). This increase is receptor dependent, as determined through co-treatment with the GR antagonist RU-486 (Cifone et al., 1999). Furthermore, phar­ macological inhibition of ceramide biosynthesis prevented apoptosis in glucocorticoid-treated thymocytes (Cifone et al., 1999). The generation of reactive oxygen species is also dramatically increased in response to glucocorticoids (Zamzami et al., 1995). This increase accompanies a decrease in the mRNA levels of anti-oxidant enzymes and contributes to glucocorticoid-induced cell death (Baker et al., 1996). Furthermore, co-treatment with an exogenous thiol anti-oxidant inhibits gluco­ corticoid-induced apoptosis (Tonomura et al., 2003). Moreover, in the absence of oxygen, cell death is prevented, indicating that the generation of reactive oxygen species is an important step in the progression of glucocorticoid-induced apopto­ sis (Torres-Roca et al., 2000). Finally, glucocorti­ coids induce a rapid translocation of GR to the mitochondria in glucocorticoid-sensitive cells (Sio­ nov et al., 2006). Overexpression of a GR species specifically targeted to the mitochondria is suffi­ cient to induce apoptosis, perhaps through the mediation of mitochondrial reactive oxygen species and ceramide production as well as the rapid cal­ cium mobilization following glucocorticoid expo­ sure (Sionov et al., 2006). These rapid cytoplasmic effects are likely participants in the complex, multi­ component glucocorticoid-induced apoptosis-sig­ naling pathway.

Execution of glucocorticoid-induced apoptosis The glucocorticoid-induced apoptosis pathway culminates in the activation of the caspase

cascade. Caspases are a family of proteases that cleave substrates at aspartate residues, mediating the dramatic morphological and biochemical changes occurring during apoptosis (Thornberry and Lazebnik, 1998). Studies utilising broad cas­ pase inhibitors have found that glucocorticoidinduced apoptosis requires caspase activation (Bellosillo et al., 1997; Hughes and Cidlowski, 1998; Sarin et al., 1996; Weimann et al., 1999). Caspase activation occurs through two broadly conserved pathways: the extrinsic and intrinsic apoptosis pathways. In the extrinsic pathway, ligands such as Fas or tumor necrosis factor (TNF) bind cognate receptors on the cell surface, initiating the caspase cascade via activation of caspase 8 (Ashkenazi and Dixit, 1998). The sec­ ond pathway, the intrinsic or mitochondrialmediated pathway, involves disruption of the mitochondrial membrane by the pro-apoptotic Bcl-2 family members Bim, Bax and Bak, leading to the release of cytochrome c and apoptotic peptidase-activating factor 1 (Apaf-1) and subse­ quent activation of caspase 9 (Saleh et al., 1999). Both pathways culminate in the activation of downstream of effector caspases (caspases 3, 7 and 6) (Green, 2000). There is evidence suggesting that glucocorti­ coid-induced apoptosis proceeds via the intrinsic mitochondrial pathway. For example, thymocytes from Apaf-1 and caspase 9 deficient mice exhibit reduced sensitivity to glucocorticoid-induced apoptosis (Hakem et al., 1998; Kuida et al., 1998; Yoshida et al., 1998). Therefore, in a simplified model of glucocorticoid-induced apoptosis, gluco­ corticoid exposure leads to the regulation of genes involved in the initiation of apoptosis, namely the pro-apoptotic Bcl-2 family member, Bim. Bim transactivation leads to the activation of down­ stream apoptotic mediators, Bax and Bak. Upon activation, Bax and Bak mediate the disruption of the mitochondrial membrane potential and the subsequent release of Apaf-1 and cytochrome c into the cytosol. The mitochondria is also respon­ sible for some of the rapid, glucocorticoidmediated cytoplasmic effects including calcium mobilization and the production of reactive oxygen species and ceramide, all of which may contribute to the progression of glucocorticoid­

11

induced apoptosis. Apaf-1 and cytochrome c release leads to the activation of caspase 9, the subsequent activation of downstream effector cas­ pases and the execution of glucocorticoid-induced apoptosis (Fig. 5).

Glucocorticoid-induced apoptosis of healthy lymphocytes In the highly coordinated process of T-lymphocyte development, bone marrow progenitors migrate to the thymus where they undergo a transition to double negative (CD48) immature thymocytes (Radtke et al., 2004). Following random rearran­ gement of the TCR a and b genes, cells become double positive (CD4þ8þ) and undergo selection based on the specificity of the TCR for self-pep­ tides bound to major histocompatibility complex (MHC)-encoded molecules (Kisielow and von Boehmer, 1995). Double positive thymocytes with TCRs bearing low avidity for self-antigen/ MHC undergo ‘death by neglect’. Thymocytes with TCRs bearing high avidity for self-antigen/ MHC undergo TCR activation-induced apoptosis (negative selection). Only cells bearing TCRs with intermediate avidity for self-antigen/MHC are res­ cued from the neglect or negative selection cell death programmes. These cells undergo positive selection, becoming either CD4þ or CD8þ cells. Upon maturation, single positive T cells enter the periphery (Huesmann et al., 1991). How the TCR induces cell death during neglect and negative selection while simulta­ neously rescuing cells from cell death during posi­ tive selection is an area of considerable research. A role for systemic glucocorticoids in the regula­ tion of thymocyte development was first suggested in 1924, when it was demonstrated that bilateral adrenalectomy leads to thymic hypertrophy (Jaffe, 1924). It has been proposed that glucocorticoids interact with TCR signaling in a relationship termed ‘mutual antagonism’. The first evidence in support of the mutual antagonism model reported that stimulation of the TCR protected T cells from glucocorticoid-induced apoptosis. Conversely, glucocorticoids prevented TCR activation-induced cell death in the same model

system, thus coining the phrase ‘mutual antagon­ ism’ (Iwata et al., 1991; Zacharchuk et al., 1990). In this model, glucocorticoids are key modulators of the ‘death by neglect’ of low avidity TCRbearing thymocytes (Zilberman et al., 1996, 2004). In addition, glucocorticoids interfere with the TCR-induced death signaling in cells with intermediate avidity TCRs, allowing these cells to escape TCR-induced apoptosis and undergo positive selection (Iwata et al., 1991; Zacharchuk et al., 1990). However, the TCR activationinduced apoptotic signaling in double positive thy­ mocytes bearing high avidity TCRs overwhelms glucocorticoid antagonism, allowing for the dele­ tion of these cells during negative selection (Ashwell et al., 2000). TCR signaling activates extracellular signalrelated kinase (ERK) signaling (Tsitoura and Rothman, 2004). ERK is responsible for rapid phosphorylation and inactivation of Bim, a key modulator of the glucocorticoid-induced apoptotic pathway (Ley et al., 2003). Therefore, TCR may antagonize glucocorticoid signaling via ERK acti­ vation and Bim inactivation. Alternatively, gluco­ corticoid signaling results in the rapid dissociation of the lymphocyte-specific kinases LCK and FYN from the TCR complex, thus inactivating TCR signaling (Lowenberg et al., 2006). There­ fore, one potential mechanism of glucocorticoid antagonism of TCR is through disruption of the TCR complex. In summary, in this model of mutual antagonism, glucocorticoids act as a rheostat, modulating the threshold for TCR acti­ vation-induced apoptosis during thymocyte selection. In vitro evidence for the mutual antagonism model was generated by experiments in fetal thy­ mic organ culture (FTOC). Pharmacological inhibition of glucocorticoid production or gluco­ corticoid signaling sensitized cells to TCR-induced apoptosis, ultimately altering the T-cell repertoire (Vacchio et al., 1998, 1994). Furthermore, in vivo knock-down of GR in the T-cell compartment dramatically reduced thymus size due to a decrease in the number of double positive and single positive thymocytes. Thus, the absence of glucocorticoid signaling in vivo sensitized double positive thymocytes to negative selection (King

12 Glucocorticoid-induced apoptosis Plasma membrane Glucocorticoid Cytoplasm

Apoptosis Ceramide

p23

ROS

90

p23

P HS

HSP90

Ca2+

HSP90

Effector caspases (3, 6, 7)

GR Destablization Apaf1 GR

Active caspase 9

VDAC Nucleus

GR

Cyto c Activation Bax/Bak

Bim

Fig. 5. Glucocorticoid-induced apoptosis signaling cascade. In this abbreviated model, glucocorticoids regulate the expression of apoptosis-effector genes, namely the pro-apoptotic Bcl-2 family member, Bim. Bim transactivation leads to the activation of downstream apoptotic mediators, Bax and Bak. Upon activation, Bax and Bak mediate disruption of the mitochondrial membrane potential and the subsequent release of cytochrome c and Apaf-1 into the cytosol. Apaf-1 and cytochrome c release leads to the activation of caspase 9, the subsequent activation of downstream effector caspases and the execution of glucocorticoid-induced apoptosis. The mitochondria is also responsible for some of the rapid, glucocorticoid-mediated cytoplasmic effects including calcium mobilization and the production of reactive oxygen species and ceramide, all of which may contribute to the progression of glucocorticoid-induced apoptosis.

et al., 1995). However, more recent studies of T-cell specific GR knock-out mice detected no abnormality in thymocyte development (Baumann et al., 2005; Brewer et al., 2003). Conversely, additional studies modulating GR expression in the thymus report dramatic alterations in thymic cellularity and thymocyte maturation (Jondal et al., 2004; King et al., 1995; Pazirandeh et al., 2002). Therefore, the precise role of glucocorticoids in thymocyte development remains controversial.

Glucocorticoid-induced apoptosis of haematological malignancies The therapeutic potential of synthetic glucocorti­ coids for the treatment of haematological malig­ nancies was first suggested in 1943, when the administration of hydroxycorticosterone resulted in apoptosis of malignant lymphocytes in the mouse (Dougherty and White, 1943; Heilman FR, 1944). Subsequently, synthetic glucocorti­ coids were implemented in the clinical treatment

13

of leukemia and lymphoblastomas (Pearson, 1949; Stickney, 1950). Today, glucocorticoids are central in the treatment of haematological malig­ nancies and are utilized as adjuvants in a majority of chemotherapeutic regimens. Chemotherapy of haematological malignancies consists of three phases: induction of remission, intensification (or consolidation) and maintenance of remission. The induction phase involves the rapid destruc­ tion of malignant cells in order to achieve remis­ sion. Remission is defined as the presence of fewer than 5% malignant cells in the bone marrow and periphery. The intensification phase utilizes chemotherapy to further reduce tumor burden and may consist of bone marrow stem cell transplantation. Finally, maintenance of remission utilizes combination therapies to main­ tain remission and prevent relapse (Hoffbrand et al., 2001). This section will review the efficacy and limitations of synthetic glucocorticoids in the treatment of individual haematomalignancies.

Glucocorticoid therapy of acute lymphoblastic leukemia ALL is characterized as an accumulation of bone marrow-derived malignant, immature lympho­ cytes. ALL is predominantly a disease of children and adolescents, with this age group comprising two-thirds of the 4000 cases diagnosed annually in the United States (Pui and Evans, 1998). Histori­ cally, the use of prednisone monotherapy in the induction phase was efficacious, resulting in remission of 45–65% of the primary childhood ALL cases reported (Hyman et al., 1959). Today, prednisone is widely used in conjunction with the alkylating agent vincristin, the topoi­ somerase II inhibitor anthracycline and catalytic asparaginase in the induction phase of ALL che­ motherapy. This combination regimen induces complete remission in 98% of children and 85% of adults (Pui and Evans, 2006). Following the induction of remission, prednisone is commonly utilized in the maintenance therapy of ALL (Gokbuget and Hoelzer, 2006). The response to prednisone therapy is a strong predictor of prog­ nosis in infant ALL. Infants with a poor response

following a 7-day course of prednisone are less responsive to conventional chemotherapy and require more intensive induction therapies (Dordelmann et al., 1999). Furthermore, early response to combined chemotherapy is also a predictor of outcome in childhood ALL (Schrappe et al., 1996). Therefore, the prednisone response in childhood and infant ALL is currently a prognostic factor utilized in the adaptation of chemotherapy treatment protocols.

Glucocorticoid therapy of chronic lymphoblastic leukemia CLL is characterized as a malignant neoplastic proliferation of the B-cell (common) or T-cell (rare) compartment. CLL is the most commonly diagnosed lymphoproliferative disorder of the Western world (30% of cases) and is primarily an adult disease, with a majority of cases occurring in individuals over 50 (Rozman and Montserrat, 1995). Historically, synthetic glucocorticoids were not added to the conventional chemotherapy regi­ men of CLL. This exclusion was based on the observations of clinical studies performed in the 1970s and 1980s, which found that the addition of prednisone to the conventional CLL regimen con­ ferred no additional benefit (Pettitt, 2008). How­ ever, in the late 1990s an in vitro study of cultured CLL cells from patients with relapsed or resistant disease found that these cells were sensitive to apoptosis induced by high doses of the synthetic glucocorticoid methylprednisolone (Bosanquet et al., 1995). Subsequent clinical trials of highdose methylprednisolone (HDMP) in CLL patients achieved a response rate of 43% (Bosan­ quet et al., 1995). Further trials have been per­ formed in CLL patients with primary or relapsed/ resistant disease utilizing combination chemother­ apy consisting of HDMP and the chimeric mono­ clonal CD20 antibody, rituximab. This study observed a response rate of 93% for primary CLL and 83% for relapsed/refractory CLL (Cas­ tro et al., 2009). The success of HDMP therapy may be due to the fact that glucocorticoid-induced apoptosis is p53 independent. Accordingly, in a separate trial, CLL patients with mutations in the

14

p53 tumor suppressor responded well to HDMP treatment (Thornton et al., 2003). Currently, the use of HDMP in conjunction with conventional chemotherapy or rituximab is gaining popularity in the treatment of CLL.

Glucocorticoid therapy of multiple myeloma Multiple myeloma (MM) is characterized by an increase of monoclonal plasma cells in the bone marrow, resulting in anaemia, hypercalcaemia and renal failure. MM accounts for 10% of all haematomalignancies (Kyle and Rajkumar, 2008). Use of alkylating agent melphalan in con­ junction with the synthetic glucocorticoid predni­ sone has been the foundation of first-line MM chemotherapy for decades. The overall response rate for this regimen was 50% (Rajkumar et al., 2002). Recently, the FDA approved the use of a more aggressive combination chemotherapy con­ sisting of the anti-angiogenic drug thalidomide and high doses of the synthetic glucocorticoid dexamethasone (Thal/Dex). Randomized trial of this induction regimen resulted in a response rate of 63% (Rajkumar et al., 2006). The Thal/ Dex or Thal/Dex/melphalan regimen is currently the most commonly prescribed remission induc­ tion regimen for the treatment of MM (Kyle and Rajkumar, 2008). Therefore, synthetic glucocor­ ticoids remain a cornerstone in the chemother­ apy of MM.

Glucocorticoid therapy of Hodgkin's and non-Hodgkin's lymphoma Hodgkin’s lymphoma, or Hodgkin’s disease, is characterized by the orderly spread of malignant lymphocytes through the lymphatic system and the presence of multi-nucleated lymphocytes known as Reed–Sternberg cells (Kuppers et al., 2002). Historically, Hodgkin’s disease has been treated with a combination chemotherapy consist­ ing of mechlorethamine, vincristine, procarbazine and prednisone (MOPP), resulting in remission induction of 80% of patients (DeVita et al.,

1980). However, the MOPP programme has recently been replaced in favour of more tailoured chemotherapy regimens. These regimens include the Stanford V (doxorubicin, bleomycin, vinblastine, vincristine, mechlorethamine, etoposide and prednisone) and the BEACOPP (doxorubicin, bleomycin, vincristine, cyclophosphamide, procar­ bazine, etoposide and prednisone), both of which incorporate the synthetic glucocorticoid predni­ sone (Evens et al., 2008). However, the impor­ tance of synthetic glucocorticoids in the remission induction of Hodgkin’s disease is unclear as the gold standard chemotherapy regi­ men, ABVD (doxorubicin, bleomycin, vinblastine and dacarbazine), does not include a synthetic glucocorticoid (Evens et al., 2008). Nevertheless, the Stanford V and BEACOPP regimens repre­ sent important glucocorticoid-inclusive treatment alternatives in the chemotherapy of Hodgkin’s disease. Non-Hodgkin’s lymphoma is comprised of a diverse group of haematomalignancies character­ ized by the absence of Reed–Sternberg cells. Induction chemotherapies vary depending on the type of lymphoma; however, the first-line che­ motherapy regimen in a majority of lymphomas consists of cyclophosphamide, vincristine, doxor­ ubicin and prednisone (CHOP). This combined chemotherapy results in remission induction of 50–60% of primary disease (Fisher et al., 1993). The remaining patients either fail to respond or have relapsed. Recent studies find that these patients may be rescued by a salvage therapy con­ sisting of dexamethasone, etoposide, ifosfamide and cisplatin (DVIP), especially when this therapy is combined with stem cell transplantation (Lazar et al., 2009).

Limitations of glucocorticoid chemotherapy Given their pleiotrophic physiological effects, the prolonged use of glucocorticoids in chemotherapy is complicated by numerous injurious side effects including muscle wasting, osteoporosis in adults, inhibition of longitudinal bone growth in children and increased susceptibility to opportunistic infec­ tions (Rhen and Cidlowski, 2005). These side

15

effects are dose and duration dependent. For example, cancer patients receiving high dose glu­ cocorticoid therapy (more than 400 mg dexa­ methasone) and prolonged therapy (more than 3 weeks) reported a 76 and 75% incidence of toxi­ city, respectively (Weissman et al., 1987). Glucocorticoids promote the degradation of muscle protein to free amino acids, resulting in muscle wasting (Mitch, 2000). In children, gluco­ corticoids induce apoptosis and inhibit prolifera­ tion of chondrocytes, the cells responsible for longitudinal bone growth (Chrysis et al., 2005). In adults, glucocorticoids induce apoptosis of mature osteoblasts and increase the bone resorp­ tion activity of osteoclasts, leading to the onset of osteoporosis (Chrysis et al., 2005; Rhen and Cidlowski, 2005). Glucocorticoids are immuno­ suppressive due to their interference with the NF-�B and AP-1 signaling pathways. These tran­ scription factors are key modulators of the inflammatory response and their repression results in decreased expression of inflammatory mediators including TNFa, IL-1b and numerous inflammatory cytokines (Rhen and Cidlowski, 2005). The glucocorticoid-induced immunosup­ pression renders patients vulnerable to opportu­ nistic infections such as oral candidiasis, an infection common in patients undergoing longterm glucocorticoid therapy (Walsh and Avashia, 1992). Another limitation of glucocorticoid che­ motherapy is the emergence of glucocorticoidresistant clonal populations during prolonged glucocorticoid therapy, glucocorticoid resistance upon relapse and the existence of inherently resis­ tant haematomalignancies. Leukemias of the mye­ logenous lineage are often innately resistant to glucocorticoid therapy (Zwaan et al., 2000). Furthermore, patients with relapsed ALL exhibit increased resistance to glucocorticoid therapy (Schrappe et al., 2000). Glucocorticoid resistance in these cancers is associated with a poor prog­ nosis (Dordelmann et al., 1999; Hongo et al., 1997; Kaspers et al., 1997). Therefore, a more compre­ hensive understanding of the factors governing glucocorticoid resistance in haematomalignancies may improve the efficacy of glucocorticoid therapy.

Mechanisms of glucocorticoid resistance in haematomalignancies Altered expression of glucocorticoid receptor isoforms There is compelling clinical and empirical evi­ dence suggesting that altered expression of GR isoforms contributes to the varied responses of haematomalignancies to glucocorticoid therapy. For example, increased expression of the ‘domi­ nant-negative’ GRb isoform has been reported in several haematomalignancies. Under normal con­ ditions, GRb is expressed at extremely low levels (0.16% in healthy lymphocytes) (Honda et al., 2000). However, the levels of GRb expression are increased in T-ALL CEM cells (0.22%), primary ALL patient samples (0.5–1.2%) and glucocorticoid-resistant CLL (Haarman et al., 2004; Shahidi et al., 1999). Interestingly, the ability of prednisone to induce apoptosis of pediatric ALL cells is inversely correlated with GRb levels (Koga et al., 2005). Furthermore, human neutro­ phils exhibit increased GRb levels and decreased glucocorticoid responsiveness, and the in vitro transfection of mouse neutrophils with GRb conferred partial glucocorticoid resistance (Strickland et al., 2001). However, the functional significance of increased GRb expression in haematomalignancies remains controversial, as other studies of GRb in primary cells from ALL patients have not reported a correlation between GRb expression and glucocorticoid resistance (Haarman et al., 2004; Tissing et al., 2005a). In the absence of conclusive evidence, the gen­ eration of a GRb transgenic mouse model would be of great value in determining the precise role of GRb in the development of glucocorticoid resistance. Altered expression of the additional GR iso­ forms may also contribute to glucocorticoid resis­ tance. For example, elevated levels of GRg mRNA are associated with glucocorticoid resistance in a study of primary versus relapsed ALL samples (Haarman et al., 2004). Several studies have iden­ tified increased expression of GR-P or GR-A mRNA in glucocorticoid-resistant malignancies (de Lange et al., 2001; Krett et al., 1995; Moalli

16

et al., 1993). However, these findings are contra­ dicted by more recent studies of patient-derived primary ALL cells (Tissing et al., 2005a). Further­ more, altered ratios of GR translational isoform expression may influence glucocorticoid respon­ siveness. For example, overexpression of the GRa-D isoform, the least transcriptionally active sub-type, confers resistance to glucocorticoidinduced apoptosis in osteosarcoma cells (Lu et al., 2007). Future studies investigating the complement of various GR translational isoforms in glucocorti­ coid-resistant patient samples would clarify the role of these variants in the development of glucocorti­ coid resistance. Finally, alternative GR promoter usage has been associated with glucocorticoid resistance. For example, exon 1A contains a GRE, which promotes the auto-induction of GR transcription upon glucocorticoid treatment (Geng and Vedeckis, 2004). This auto-induction has been associated with increased responsiveness to gluco­ corticoid treatment (Purton et al., 2004). Further­ more, cell-type-specific expression of GR promoter transcripts has been reported in a vari­ ety of haematomalignant cell lines (Breslin et al., 2001; Geng and Vedeckis, 2004; Nunez and Vedeckis, 2002; Pedersen and Vedeckis, 2003). This variable expression of GR promoter tran­ scripts has been correlated with the diverse responsiveness of haematologic malignancies to glucocorticoid therapy (Breslin et al., 2001; Purton et al., 2004). However, other reports contradict these findings and cite no differences in GR pro­ moter usage or differential induction of various GR promoter transcripts in glucocorticoid-resis­ tant primary ALL cells (Tissing et al., 2006).

Glucocorticoid-induced alterations in GR expression In lymphoid cells sensitive to glucocorticoids, GR auto-induction in response to steroid treatment is a common observation. This auto-regulatory action can be attributed to the presence of a GRE in the GR promoter region. GR auto-induc­ tion has been observed in the glucocorticoid-sen­ sitive CEM T-ALL cell lines, primary ALL cells

and immature thymocytes (Barrett et al., 1996; Tissing et al., 2006). Accordingly, the extent of GR auto-induction following glucocorticoid treat­ ment has been directly correlated with the degree of cell death in glucocorticoid-sensitive cells (Geley et al., 1996). However, the importance of GR auto-induction in glucocorticoid-induced apoptosis is challenged by studies of primary ALL xenografts in immune-deficient mice and primary thymocytes. Glucocorticoid-sensitive xenografts contained high levels of basal GR and failed to auto-induce GR upon hormone treat­ ment. Furthermore, glucocorticoid-sensitive pri­ mary thymocytes expressing high-basal GR levels also undergo apoptosis in the absence of auto-induction (Oldenburg et al., 1997). These results suggest that in sensitive cells with high basal GR expression, GR auto-induction is not required to induce apoptosis (Bachmann et al., 2007). However, GR auto-induction is essential for apoptosis in cells harbouring low basal levels of GR (Miller et al., 2007; Ramdas et al., 1999; Riml et al., 2004). Conversely, in cells resistant to glucocorticoids, homologous down-regulation of GR following glucocorticoid treatment is a common observa­ tion. For example, in leukemias of myelogenous origin, GR is down-regulated by glucocorticoids, perhaps accounting for their inherent resistance to glucocorticoid therapy (Kfir et al., 2007). Further­ more, homologous GR down-regulation is an indi­ cator of poor prognosis in ALL (Bloomfield et al., 1981; Pui and Costlow, 1986). Therefore, pro­ longed glucocorticoid therapy, in certain cellular contexts, may result in the emergence of glucocor­ ticoid-resistant cells harbouring low levels of basal GR expression. Homologous down-regulation of GR may employ multiple mechanisms including diminished promoter activity or destabilization of mRNA or protein expression. Homologous downregulation of GR may be due to decreased activity of the GR promoter. However, use of a hetero­ logous promoter to drive GR expression found that the repressive action of glucocorticoids on GR expression remained intact. Further in vitro studies determined that the region of GR encoded by amino acids 550–697 is essential to the homo­ logous down-regulation of GR (Alksnis et al.,

17

1991; Burnstein et al., 1990, 1994). Additionally, decreased GR mRNA stability may contribute to homologous down-regulation. For example, the presence of AUUUA motifs in the 30 -UTR of GR might be involved in the destabilization of GR mRNA. These motifs are common RNAbinding protein motifs and the action of these RNA binding proteins may contribute to the destabilization of GR mRNA (Chen and Shyu, 1995; Schaaf and Cidlowski, 2002a; Shaw and Kamen, 1986). Finally, homologous down-regula­ tion could be due to decreased GR protein stabi­ lity. Studies have indicated that the proteosome complex degrades the GR. Pre-treatment of GR overexpressing cells with the proteosomal inhibi­ tor MG-132 impedes homologous down-regula­ tion of GR protein (Wallace and Cidlowski, 2001). Furthermore, GR contains a conserved proline, glutamic acid, serine and threonine (PEST) degradation motif, also contributing to the destabilization of GR protein (Wallace and Cidlowski, 2001). Additionally, non-coding microRNAs have recently been identified as negative regulators of gene expression through transla­ tional repression of target mRNAs. Hormonal induction of specific microRNAs targeting GR is an attractive model of homologous GR down-reg­ ulation. However, microRNAs targeting GR have only been identified in neurons and their function in the regulation of GR mRNA translation in the lymphoid compartment remains unaddressed (Vreugdenhil et al., 2009). The precise role of these proposed mechanisms in the homologous down-regulation of GR observed in glucocorti­ coid-resistant haematomalignancies remains unclear and additional experiments are war­ ranted. Furthermore, studies of primary ALL xenografts in immune-deficient mice found that the glucocorticoid-resistant tumors possessed suf­ ficient basal levels of GR and did not undergo homologous GR down-regulation upon steroid treatment (Bachmann et al., 2007). These obser­ vations suggest that in these neoplasms, glucocor­ ticoid resistance occurs downstream of GR. Clearly, the precise mechanisms governing gluco­ corticoid resistance in haematomalignancies are divergent and cell-type specific. However, for many haematomalignant cell types, the

directionality of GR expression in response to glucocorticoid treatment modulates the efficacy of glucocorticoid therapy.

Mutations in the glucocorticoid receptor Numerous mutations affecting GRa signaling have been identified in laboratory-derived gluco­ corticoid-resistant leukemic cell lines. For exam­ ple, the CEM T-ALL cell line harbours a mutation in the GR LBD (L753F) that hinders GR ligand binding and subsequent transactivation (Hillmann et al., 2000). Accordingly, the T-ALL 6TG1.1 cell line bearing this mutation is resistant to glucocorticoid-induced cell death (Liu et al., 1995; Powers et al., 1993). Additional mutations in the GR LBD, including the G679S, F737L, I559N, V571A, D641V, V729I and I747M also impair the transcriptional activity of GR (Charmandari et al., 2004). Furthermore, a R477H mutation in one allele of the GR gene inhibits DNA binding in glucocorticoid-resistant Jurkat T-ALL cells. This mutation renders the GR unable to transactivate, thus eliminating GR auto-induction and contribut­ ing to glucocorticoid resistance in these cells (Riml et al., 2004). However, the majority of studies in patient-derived samples have failed to associate glucocorticoid resistance with mutations in the GR. Mutations in the GR were absent in both glucocorticoid-resistant primary cells derived from ALL patients or glucocorticoid-resistant pri­ mary ALL xenografts (Bachmann et al., 2007; Beesley et al., 2009; Tissing et al., 2005b). There­ fore, there are key differences in the mechanisms of glucocorticoid resistance in long-term cultured cells and patient-derived samples and the lesions responsible for impaired GR signaling and gluco­ corticoid resistance in vitro do not contribute to the development of glucocorticoid resistance in vivo.

Aberrant expression of Bcl-2 Aberrant expression of the anti-apoptotic Bcl-2 protein is frequently reported in B-cell follicular lymphomas. In these lymphomas, translocation of

18

Bcl-2 places this oncongene adjacent to the immu­ noglobulin heavy chain locus, resulting in rampant expression of the Bcl-2 fusion gene. This fusion gene is identified in over 80% of B-cell lympho­ mas and Bcl-2 transgenic mice develop diffuse large-cell lymphomas at old age (Cleary et al., 1986; McDonnell et al., 1989; McDonnell and Korsmeyer, 1991; Tsujimoto and Croce, 1986). Bcl-2 functions as a stabilizer of the mitochondrial membrane, thereby preventing the loss of mito­ chondrial membrane potential and the release of cytochrome c and Apaf-1 in response to glucocor­ ticoids (Susin et al., 1999). In addition, the phos­ phorylation of Bcl-2 at specific residues is required for the establishment of glucocorticoid resistance in cultured B lymphocytes (Huang and Cidlowski, 2002). A number of in vitro studies have found that Bcl-2 overexpression confers varying degrees of resistance to glucocorticoid-induced apoptosis, while in vivo knock-down of Bcl-2 expression sen­ sitizes haematopoietic cells to glucocorticoidinduced apoptosis, supporting its role as an impor­ tant mediator of glucocorticoid resistance in haematomalignancy (Kamada et al., 1995; Kfir et al., 2007; Memon et al., 1995; Nakayama et al., 1993; Veis et al., 1993a, 1993b). In addition, the in vitro and in vivo manipulation of the Bcl-2 family members Bcl-XL and Mcl-1 also alters the efficacy of glucocorticoid-induced apoptosis (Opferman et al., 2003; Wei et al., 2006). How­ ever, defining the role of these two family mem­ bers in the development of haematomalignancy and their contribution to glucocorticoid resistance requires further study.

Failure to induce Bim expression It is well established that the induction of Bim expression is important for glucocorticoid-induced apoptosis. Lymphocytes derived from Bim transgenic mice exhibit reduced sensitivity to glucocorticoid-induced apoptosis (Bouillet et al., 1999). Furthermore, inhibition of Bim expression via siRNA and shRNA inhibits caspase-3 activa­ tion and glucocorticoid-induced apoptosis in ALL cell lines (Abrams et al., 2004). A recent study of ALL xenografts in immune-deficient mice

revealed that the resistant xenografts all failed to induce Bim expression upon glucocorticoid treat­ ment (Bachmann et al., 2007). This was not due to lack of transactivation activity, as these xenografts were able to induce the expression of GILZ, sug­ gesting that the failure of glucocorticoid-resistant xenografts to induce Bim is downstream of transactivation. Several mechanisms of glucocorticoidinduced Bim expression have been proposed including (1) transactivation of Bim by the gluco­ corticoid-responsive transcription factors Foxo3A/ FKHRL1, RUNX3 and E2F1 (Dijkers et al., 2000; Yamamura et al., 2006; Zhao et al., 2005), (2) release of Bim from cytoskeletal sequestration (Puthalakath et al., 1999) and (3) repression of Src-mediated inhibitory phosphorylation of Bim (Ley et al., 2003). Therefore, targeting one or more of the pathways governing glucocorticoidinduced Bim expression may prove a beneficial in the development of therapies to overcome glu­ cocorticoid resistance in haematomalignancies.

Interactions with the kinome Glucocorticoid signaling involves complex com­ munication between multiple signaling pathways. For example, the mitogen-activated protein (MAP) kinases ERK and c-Jun N-terminal kinase (JNK) inhibit glucocorticoid-induced apoptosis, while p38 stimulates glucocorticoid-induced apop­ tosis in the CEM leukemic cell line (Miller et al., 2007; Wada and Penninger, 2004). Glucocorticoidresistant CEM clones possess high constitutive JNK activity and glucocorticoid stimulation results in increased ERK activity accompanied by the weak induction of p38 (Miller et al., 2007). The cAMP-driven protein kinase A (PKA) pathway also contributes to glucocorticoid-induced apopto­ sis. Stimulation of PKA with forskolin inhibits JNK signaling and sensitizes resistant CEM cells to glucocorticoid-induced apoptosis by increasing the transcriptional activity of GR (Medh et al., 1998). These results suggest that the GR and PKA pathways exert cooperative effects on GRmediated gene transcription. Additionally, inhibi­ tion of the mammalian target of rapamycin (mTOR)-signaling pathway with rapamycin

19

inhibits JNK signaling, sensitizing CEM and MM cell lines as well as primary MM cells to glucocor­ ticoid-induced apoptosis. This increased sensitiv­ ity is associated with decreased expression of cyclin D2 and the anti-apoptotic protein survivin. The PI3K–AKT pathway has also been shown to prevent glucocorticoid-induced cell death through the inhibitory phophorylation of key apoptotic mediators including Bcl-2-associated death pro­ moter (BAD), caspase 9, Fox03A/FKHRL and CREB (Maddika et al., 2007). Furthermore, the AKT-mediated phospho-inhibition of Fox03A/ FKHRL inhibits the transcription of Bim and con­ tributes to glucocorticoid resistance (Maddika et al., 2007). Finally, Src kinase is released from Hsp90 sequestration upon glucocorticoid treat­ ment and pharmacological inhibition of Src activa­ tion ameliorates glucocorticoid resistance in MM cells (Ishikawa et al., 2003). Given the intricate cross-talk between various kinase pathways and GR signaling, the modulation of the kinome is a compelling avenue in the development of novel therapies to ameliorate glucocorticoid resistance in haematomalignancies.

Novel therapies targeting glucocorticoid resistance Targeting the kinome Previous studies have shown that MAP kinase signaling modulates the response of various leu­ kemic cell lines to glucocorticoids (Miller et al., 2007, 2005). Direct pharmacological inhibition of JNK and ERK signaling sensitizes resistant cells to glucocorticoid-induced apoptosis (Miller et al., 2007). A more recent study of five diverse gluco­ corticoid-resistant haematomalignant cell lines confirmed that inhibition of ERK and JNK signal­ ing results in glucocorticoid sensitivity (Garza et al., 2009). This increased susceptibility to gluco­ corticoid-induced cell death was directly corre­ lated with increases in total and phospho-GR as well as Bim. Conversely, pharmacological inhibi­ tion of p38, one of the kinases responsible for GR phosphorylation at serine 211, protects leukemic cells from glucocorticoid-induced apoptosis

(Miller et al., 2005). Mutation of this residue abro­ gates glucocorticoid-induced apoptosis, suggesting that p38-mediated GR phosphorylation at site 211 is a key component in the glucocorticoid-induced apoptosis signaling cascade (Miller et al., 2005). Indirect inactivation of JNK signaling via the mTOR inhibitor rapamycin also sensitizes resis­ tant cells to glucocorticoid-induced apoptosis through the relief of JNK-mediated phosphoinhibition of Bim (Miller et al., 2007; Stromberg et al., 2004). Additional studies have found that mTOR inhibition leads to decreases in anti-apop­ totic Mcl-1 protein expression, further contribut­ ing to the sensitization potential of mTOR inhibitors (Wei et al., 2006). Encouragingly, mTOR inhibition sensitizes inherently resistant MM cells and primary MM xenografts to gluco­ corticoid-induced apoptosis and glucocorticoid sensitivity persists despite the addition of exogen­ ous survival signals, suggesting that rapamycin inhibition of mTOR signaling may be a suitable option for the circumvention of glucocorticoid resistance in vivo (Miller et al., 2007; Stromberg et al., 2004). Stimulation of the cAMP PKA signaling path­ way with forskolin sensitizes glucocorticoidresistant ALL cells to apoptosis (Miller et al., 2007). Furthermore, recent studies have found that inhibiting the activity of three cAMP phosphodiesterases (PDEs) overexpressed in glucocorticoid-resistant CEM cells (PDE3, PDE4 and PDE7) ameliorates glucocorticoid resistance in these cells (Dong et al., 2009). Activation PI3K– AKT signaling is correlated with the expansion of glucocorticoid-resistant MM cells in the bone marrow (Hideshima et al., 2007). Accordingly, inhibition of AKT signaling with perifosine sensitizes resistant MM cells to glucocorticoidinduced apoptosis through the reduced expression survival factors including IL-6 and survivin (Hideshima et al., 2007). Currently, a phase III clinical trial is evaluating the efficacy of perifosine and dexamethasone combined chemotherapy in the treatment of drug-resistant MM (Richardson, 2009). In addition, the efficacy of glucocorticoid treatment combined with JNK/ERK inhibitors or mTOR inhibitors is being evaluated clinically in order to overcome the dilemma of glucocorticoid

20

resistance in haematological malignancies (Davies et al., 2007; Mita et al., 2008).

future therapies interfering with glucocorticoidinduced Noxa down-regulation may improve the efficacy of glucocorticoid therapy in childhood ALL.

Targeting Bcl-2 family members Recently, small-molecule BH3 mimics have been developed to impede the anti-apoptotic effects of Bcl-2 family members. These molecules directly interact with anti-apoptotic Bcl-2 proteins at their BH3-binding groove, eliminating their ability to sequester pro-apoptotic BH3 domain Bcl-2 family members (Kang and Reynolds, 2009). For instance, the BH3 mimic gossypol (AT-101) binds directly to Bcl-2 and inhibits its function. Co-treat­ ment with gossypol potentiates dexamethasoneinduced cell death in glucocorticoid-resistant MM cell lines and patient-derived primary cells (Kline et al., 2008). The Bcl-2-specific BH3 mimic ABT­ 737 enhanced dexamethasone-induced cell death via the mitochondrial pathway in five of seven ALL cell lines evaluated (Kang and Reynolds, 2009). Furthermore, addition of the Bcl-2-specific BH3 mimic TW-37 to the CHOP regimen enhanced apoptosis in diffuse large-cell lymphoma xenografts (Mohammad et al., 2007). In addition to Bcl-2-targeted BH3 mimics, the Mcl-1-specific inhibitor obatoclax also enhanced glucocorticoidinduced apoptosis (Trudel et al., 2007). Co-admin­ istration of obatoclax and dexamethasone over­ came glucocorticoid resistance in 15 of 16 patientderived MM primary cell lines. In these cells, Mcl-1 inhibition was associated with an increase in Bim expression. Finally, anti-sense antagonism of Bcl-2 with the G3139 oligonucleotide resulted in decreased Bcl-2 protein expression and an enhanced response to dexamethasone/thalidomide chemotherapy in phase II clinical trials (Badros et al., 2005). The efficacy of G3139/dexamethasone combined therapy in the treatment of MM is cur­ rently being evaluated in phase III clinical trials (Kang and Reynolds, 2009). Interestingly, glucocorticoid monotherapy results in the decreased expression of the pro­ apoptotic Bcl-2 family member Noxa in pediatric ALL samples. Conditional overexpression of Noxa in CEM ALL cells accelerated glucocorticoidinduced apoptosis (Ploner et al., 2009). Therefore,

Concluding remarks Glucocorticoids exert pleiotrophic physiological effects, including the induction of apoptosis in the lymphocyte compartment. Glucocorticoid effects are mediated through the GR, a ligand-activated transcription factor. GR transactivation is required for the induction of apoptosis, primarily, the induction of Bim expression is important for the apoptotic activity of glucocorticoids. However, glucocorticoid-induced apoptosis is a highly coordi­ nated, multi-component process. The rapid cytoplasmic effects of glucocorticoids may also con­ tribute to the progression of apoptosis. However, the precise role of these cytoplasmic effects in the advancement of glucocorticoid-induced cell death in lymphocytes requires further investigative scru­ tiny. Endogenous glucocorticoids shape the T-cell repertoire through the induction of apoptosis by neglect as well as the antagonism of TCR-induced apoptosis during positive selection. Owing to their ability to induce apoptosis in lymphocytes, synthetic glucocorticoids are widely used in the treatment of haematological malignancies. Gluco­ corticoid chemotherapy is limited by the emergence of glucocorticoid-resistant clonal populations follow­ ing prolonged glucocorticoid therapy, glucocorticoid resistance upon relapse and the existence of inher­ ently resistant haematomalignancies. The mechan­ isms involved in the development of glucocorticoid resistance are complex and cell-type specific. Altered expression ratios of GR isoforms, homologous down-regulation of GR, the inability to autoinduce GR, mutations in the GR, dysregulation of Bcl2 family members, the failure to induce Bim and interactions with the kinome may all contri­ bute to the formation of glucocorticoid resistance in haematomalignancies. The development of novel therapies to overcome glucocorticoid resistance in haematomaligancy, including specific targeting of the kinome and Bcl-2 family members, will dramatically improve the efficacy of

21

glucocorticoid therapy in the treatment of haema­ tological malignancies.

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L. Martini (Eds.)

Progress in Brain Research, Vol. 182

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 2

Impact of the hypothalamic–pituitary–adrenal/ gonadal axes on trajectory of age-related cognitive decline Cheryl D. Conrad
and Heather A. Bimonte-Nelson Department of Psychology, Arizona State University, Tempe, AZ, USA

Abstract: Life expectancies have increased substantially in the last century, dramatically amplifying the proportion of individuals who will reach old age. As individuals age, cognitive ability declines, although the rate of decline differs amongst the forms of memory domains and for different individuals. Memory domains especially impacted by aging are declarative and spatial memories. The hippocampus facilitates the formation of declarative and spatial memories. Notably, the hippocampus is particularly vulnerable to aging. Genetic predisposition and lifetime experiences and exposures contribute to the aging process, brain changes and subsequent cognitive outcomes. In this review, two factors to which an individual is exposed, the hypothalamic–pituitary–adrenal (HPA) axis and the hypothalamic–pituitary–gonadal (HPG) axis, will be considered regarding the impact of age on hippocampal-dependent function. Spatial memory can be affected by cumulative exposure to chronic stress via glucocorticoids, released from the HPA axis, and from gonadal steroids (estrogens, progesterone and androgens) and gonadotrophins, released from the HPG axis. Additionally, this review will discuss how these hormones impact age-related hippocampal function. We hypothesize that lifetime experiences and exposure to these hormones contribute to the cognitive makeup of the aged individual, and contribute to the heterogeneous aged population that includes individuals with cognitive abilities as astute as their younger counterparts, as well as individuals with severe cognitive decline or neurodegenerative disease. Keywords: spatial memory; declarative memory; hippocampus; stress; glucocorticoid; estrogen; proges­ terone; testosterone; menopause; ovariectomy percentage is expected to substantially increase to 20% by the year 2020 due to the aging of the ‘baby boomer’ generation (U.S. Census Bureau, 2007). As individuals age, it is well documented that some memory loss is observed (Erickson and Barnes, 2003; Tulving and Craik, 2000). However, age influences some forms of learning and mem­ ory differently than others, with those mediated by the hippocampus and frontal cortex particu­ larly susceptible to age-related disruption.

Introduction: rationale for studying aging and hippocampal function In the United States, the proportion of the population that is over 65 is increasing. Today, about 12% of the population is over 65 and this


Corresponding author.

Tel.: þ480-965-7761; Fax: 480-965-8544; E-mail: [email protected] and [email protected]

DOI: 10.1016/S0079-6123(10)82002-3

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Learning and novel memory formation allows adaptability in an organism. It allows acquisition and updating of knowledge and skills within dif­ ferent and overlapping neurobiological domains (Kausler, 1994): strangers become familiar friends, new facts are learned and skill-sets for playing sports, musical instruments, cooking or computer programs are acquired. Within the specific domain of spatial navigation, an individual learns to navi­ gate through a novel environment so that a route to the target eventually becomes familiar, and cues in the environment form associations to help with overall navigation. For a human, this allows one to learn to find their way to a new coffee shop, and, after learning occurs, to navigate to this new location whether one starts the journey from home or the grocery store. For an animal, this allows it to learn the route to a new food source or reward, regardless of its starting loca­ tion. While progression into old age is sometimes associated with a decreased proficiency in learning or plasticity, learning still occurs within many types of memory functions and cognitive domains. Indeed, decades of research have resulted in the general consensus that not all domains of learning and memory are equally affected by aging. Nor­ mally aged humans usually retain the knowledge of how to drive an automobile and brush their teeth (non-declarative memories) and typically remember the name of the President (semantic memories, Balota et al., 2000; Erickson and Barnes, 2003), but demonstrate an age-associated decline in the recall of personal experiences and events, a type of memory referred to as episodic declarative memory, which shows greater ageassociated decline than semantic memory (Balota et al., 2000; Erickson and Barnes, 2003). It is gen­ erally thought that by the fifth decade of life in humans, learning occurs more slowly for difficult tasks such as the declarative memory delayed recall task (Albert, 2002; Albert et al., 1987). Per­ haps most considerably altered with age is spatial learning and memory, which depends upon medial temporal lobe structures such as the hippocampus, and involves the ability to navigate effectively through an environment, acquiring, integrating and retaining environmental features such as land­ marks and other prominent cues (Barnes, 1998).

Since the historic work of Tolman, learning to navigate through a new environment has been referred to as the formation of a cognitive map (Tolman, 1948). Additionally, working memory, requiring manipulation of information kept ‘on­ line’, is intimately related to neocortex, frontal cortex more specifically, and is also affected by normal aging revealing a memory decline that becomes more severe as task difficulty increases (Balota et al., 2000). The hippocampus facilitates the formation of a cognitive map and declarative memories, as well as the knowledge of facts and their relationships (Eichenbaum, 2000; O’Keefe and Nadel, 1978), and the neocortex is the loca­ tion of long-term memory storage (Eichenbaum, 2000). Notably, both the hippocampus and the frontal cortex appear to be particularly vulnerable to age-related changes (Burke and Barnes, 2006). Spatial learning and memory requires an animal to navigate through space to locate a goal and can be readily tested in rodent models. Many experi­ mental tests of rodent spatial memory aim to assess spatial working memory, a form of shortterm memory which requires a subject to retain spatial information which must be updated and is useful for only a short period of time (trial-specific information, Baddeley and Hitch, 1974). In gen­ eral, working memory is distinguished from refer­ ence memory, which is necessary to remember information that remains constant over time (task-specific information, for discussion, see Olton et al., 1979). Rodent studies evaluating aging agree with human studies that spatial navi­ gation decreases in normally aged individuals, for both spatial working and reference memory, and that both elderly humans and aged rodents show individual variability in age-related cognitive change (Kausler, 1994). Many researchers study­ ing aging effects on learning and memory divide aged rodent subjects into impaired and nonimpaired groups to optimize differences in various physiological measurements (Armstrong et al., 1993; Croll et al., 1998; Diana et al., 1995; Fischer et al., 1991; Hasenohrl et al., 1997; Lindner et al., 1996; Nilsson and Gage, 1993; Quirion et al., 1995; Rapp and Gallagher, 1996; Sugaya et al., 1998). While the operational definitions for these classi­ fications are not homogeneous amongst studies, as

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some classify age impairment as those scores that are beyond the mean of the young group by one standard deviation (Croll et al., 1999, 1998), two standard deviations (Armstrong et al., 1993; Fischer et al., 1991; Nilsson and Gage, 1993; Quirion et al., 1995) or outside the range of the young (Rapp and Gallagher, 1996; Sugaya et al., 1998), they all concur that individual expression of age-related memory change exhibits a large range of variability. How that age-related variability in cognition comes about is a topic of intense inquiry. The progression of age-related decline in hip­ pocampal-dependent spatial function is embedded within the context of an individual’s life experi­ ences and exposures. These experiences and expo­ sures span a range of an infinite number of individual and interactive factors, including, but not limited to, genetic predisposition, early orga­ nizational effects of stress and gonadal hormones, enriching experiences, dietary factors (e.g. choline exposure), stress history and activational actions of stress and gonadal hormones. For the purpose of this review, we will focus on the life experiences of the cumulative exposure to stress and gonadal hormones in the context of activational effects in the adult. The underlying theme and main tenet driving discussion are the links amongst aging, gonadal hormones and cognition, with the hypoth­ esis that cumulative stress impacts the trajectory of age-related cognitive changes, with a poten­ tially interactive effect with gonadal hormones.

Aging influences on hippocampal plasticity Age increases risk for Alzheimer's disease and depression A misconception regarding the natural progres­ sion of cognitive function across the lifespan is that cognitive decline is synonymous with neuro­ nal loss. This perception is partially driven by epidemiological data showing that neuron loss occurs in many neurodegenerative disorders, especially those with dementia, with age increas­ ing risk. Alzheimer’s disease (AD) is the cause of about 70% of dementia cases, and studies report an alarming 5–10% prevalence in persons over 65

and 47–50% prevalence for those over 85 (Drachman, 2006). AD is a devastating disease character­ ized by cognitive decline, with initial deficits most pronounced in working memory and visuospatial functioning (Tulving and Craik, 2000). Function­ ing progressively worsens so that all effective cog­ nitive functioning is lost by the latter stage. Unfortunately, AD has no cure and there are scarce drug treatment options. However, aging and AD are not synonymous. While most studies concur that brains unaffected by neurodegenera­ tive disease will show some age-related memory loss, defining the boundaries between normal agerelated changes and neuropathologies has proven to be difficult. To date, age is recognized as the strongest risk factor for developing sporadic AD (Drachman, 2006; Yankner et al., 2008), but AD is not simply ‘accelerated’ aging (Drachman, 2006; Terry, 2006; Yankner et al., 2008). One recent hypothesis regarding the transition to sporadic AD suggests an accumulation of multiple and varied agerelated changes that burden the brain in a summative as well as interactive fashion (Drachman, 2006). Therefore, understanding and deciphering factors affecting the trajectory of age-related changes seen in normal aging may eventually lead to further understanding of neurodegenerative processes such as those seen in AD. Older adults are also at risk for cognitive decline associated with major affective disorder, or commonly discussed as depression. According to the American Psychiatric Association’s Diag­ nostic and Statistical Manual of Mental Disorders, 4th Edition Text Revision (DSM-IV-TR), depres­ sion is characterized by changes in mood, appetite, weight, and increased anhedonia, as well as impaired cognitive function and disrupted sleep, motivation and hypothalamic–pituitary–adrenal (HPA) axis activity. Following teenagers, adults over the age of 65 have the highest rate of suicide, identifying this as a significant public health issue and a major cause of death worldwide (Carballo et al., 2008; WHO, 2008). Moreover, the risk for depression substantially increases in women and those with certain genetic predispositions (i.e. polymorphisms for the gene that transports sero­ tonin) (Altemus, 2006; Caspi et al., 2003; Kessler et al., 1994), suggesting important gonadal/sex

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hormone influences for depression. Finally, potent environmental risk factors include lifetime expo­ sure to stress (Caspi et al., 2003; Levinson, 2006; Nestler et al., 2002), especially when initiated dur­ ing childhood (Andersen and Teicher, 2008). Therefore, genetics factors, including sex, confer a risk to developing depression, but environmen­ tal factors, such as stress, also play an important role. The HPA axis may contribute to AD onset, pro­ gression and accelerated aging (Phillips et al., 2006). Studies investigating AD-related disorders (i.e. dementia of the Alzheimer type) or depression show evidence for dysregulated HPA axis activity or elevated glucocorticoids, steroids released by the adrenal glands in response to stressors (Csernansky et al., 2006; Davis et al., 1986; Gomez et al., 2006; Magri et al., 2006; Rasmuson et al., 2002; Rubinow et al., 1984; Swaab et al., 2005; Umegaki et al., 2000; Weiner et al., 1997). In some cases, greater symp­ tom severity correlates with more HPA axis dysre­ gulation (Dong and Csernansky, 2009; Miller et al., 1998) and decreased hippocampal volume (de Leon et al., 1988; Keller et al., 2006; Magri et al., 2006; O’Brien et al., 1996). Nonetheless, there are some longitudinal data that fail to support the interpreta­ tion that HPA axis dysregulation corresponds with severity of AD symptoms (Swanwick et al., 1998) or depression (Adler and Jajcevic, 2001; O’Brien et al., 2004). It is intriguing that one report indicated that higher HPA activity predicts rapid disease progres­ sion as opposed to the disease state per se (Cser­ nansky et al., 2006). Moreover, evidence that disease etiology differs in young and aged subjects is revealed by findings that older subjects are inclined to show hyper- or hypo-HPA axis activity with depressive symptoms (Bremmer et al., 2007; Chida and Hamer, 2008). These seemingly mixed outcomes may be a consequence of older indivi­ duals having increased risk(s) for other conditions (i.e. cardiovascular disease, cancer, to name a few, Evans et al., 2005; Everson-Rose et al., 2004; Gump et al., 2005; Penninx et al., 1998; Wassertheil-Smol­ ler et al., 2004), which could further moderate HPA axis activity with AD or depressive symptoms. As previously noted, genetic and environmental factors increase the risk for developing AD or depression (Caspi et al., 2003; de Geus et al., 2007), with a

history of chronic stress being particularly impor­ tant (Hammen et al., 2009; Paykel, 2003; Weber et al., 2008). Therefore, young and aged individuals may both carry genetic predispositions, but the aged population has lifetime experiences that can further moderate risk for developing AD and depression, with a history of chronic stress and HPA axis activity playing a prominent role.

Why investigate hippocampal structural changes beyond neuronal loss? The mammalian brain shows considerable plasti­ city, especially in regions related to learning and memory such as the hippocampus and neocortex. Indeed, the hippocampus contains one of the rare sites for neurogenesis that persists long after early development (Eriksson et al., 1998; Kaplan and Hinds, 1977; Kornack and Rakic, 1999). However, the hippocampus is also susceptible to neurode­ generation. The pathological hallmarks of AD include presence of neurofibrillary tangles and amyloid plaques, which occur to a lesser degree in the normal aging brain and are particularly pervasive within the hippocampus and prefrontal cortex (PFC) (Roth et al., 1966; Yankner et al., 2008). Importantly, the hippocampus and neocor­ tex of the AD brain show extensive neuronal loss (Gomez-Isla et al., 1996; Price et al., 2001; West et al., 1994), whereas accounts of neuronal loss with normal aging are mixed. Early research reported age-related loss of neurons in the hippo­ campus (Landfield et al., 1981; West et al., 1994). A recently published study quantifying magnetic resonance imaging (MRI) scans from over 2200 healthy persons showed the largest age-related changes in brain volume after age 50, with the frontal and temporal lobes showing the largest decreases (~12% and ~9%, respectively), with modest age-related alterations elsewhere (DeCarli et al., 2005; Raz and Rodrigue, 2006), which corroborates other reports (Miyahira et al., 2004); however, brain volume changes do not necessarily indicate neuronal loss. Studies report­ ing neuronal counts suggest decreases in neuronal number with normal aging in the hippocampus and the subiculum (Simic et al., 1997; West,

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1993), and that PFC neuron death is related to working memory decline in aged monkeys (Smith et al., 2004). However, other reports sug­ gest that the majority of neuron loss during nor­ mal aging occurs in the neocortex, and not the hippocampus (Drachman, 2006), or that neither cortex nor hippocampus express neuronal loss with normal aging (Peters et al., 1998; Terry, 2006), even when age-related memory deficits are found (Gallagher et al., 2003). Most studies do agree, however, that while brains of normally aging humans may not show neuron loss, AD brains consistently do (Hof and Morrison, 2004). To summarize, the literature consistently shows age-related changes in function, such as spatial memory, but the effects on brain structure and micro-structure during aging are equivocal. Age-related cognitive decline may involve neo­ plastic changes within the brain, such as altera­ tions in dendritic structure, spine number and spine shape. Such changes would be consistent with studies showing that aging is not necessarily synonymous with neuronal loss. Stereological counting methods that allow for unbiased quanti­ fication of cell numbers show that hippocampal neuron numbers can remain relatively constant with aging, even when aged subjects are further categorized into cognitively impaired and cogni­ tively unimpaired based on performance assess­ ments from a spatial navigation task (Rapp and Gallagher, 1996). Indeed, the majority of studies evaluating synaptic alterations (e.g. synaptic den­ sity as revealed via synaptophysin evaluations) in healthy individuals show an age-related decline, resulting in a potentially interesting dissociation between synapse loss and neuron loss, if the latter in fact does not occur (Hof and Morrison, 2004; Terry, 2006). Evidence from updated stereology techniques show that hippocampal synapse num­ ber decreases with the progression from young adulthood to normal aging, and that synapse num­ ber in AD hippocampi is fewer than in those of the normally aged (Bertoni-Freddari et al., 2003). Moreover, factors influencing synaptic transmis­ sion, including the reductions mentioned above in synaptic density, as well as in dendritic spines and number and length of dendrites, have been noted (Esiri, 2001). These findings indicate that

many age-related alterations may involve neoplas­ tic changes that are flexible and dynamic. Studies investigating changes in brain structure of patients suffering from depression suggest that dynamic and reversible alterations may be impor­ tant. Individuals with depression show decreased volumes in the hippocampus (Bremner et al., 2000; Feldmann et al., 2007; Janssen et al., 2004; MacMaster et al., 2008; Neumeister et al., 2005; Saylam et al., 2006; Sheline et al., 2003; 1999; Sheline et al., 1996) and PFC (Bremner, 2002; Coryell et al., 2005; Rajkowska et al., 1999). While neuron death received early attention (Sapolsky, 2000), permanent neuronal loss was inconsistent with the observation that post-mor­ tem hippocampal tissue from patients diagnosed with depression showed reductions in neuropil without neuron loss (Stockmeier et al., 2004). Although cell loss was found in some tissue of patients with depression (Rajkowska, 2000), a likely interpretation is that cell loss represents a small proportion of the etiology underling depres­ sion. Moreover, signs that volumetric changes can be dynamic are revealed from studies showing that antidepressant treatment increases volumes of the hippocampus and PFC (Neumeister et al., 2005; Sheline et al., 2003; Vermetten et al., 2003), and hippocampal volumes increase in depressed patients who are in remission (Frodl et al., 2008). Indeed, patients on long-term antidepressant treatment may be protected against hippocampal volume decreases from cumulative depressive epi­ sodes (Sheline et al., 2003). Consequently, some structural changes occurring in depression most likely include reversible and/or dynamic processes without necessarily involving neuronal loss.

Stress via the HPA axis and gonadal steroid influences on the hippocampus Dendritic retraction and changes in synapse num­ ber/shape may provide a putative mechanism through which hippocampal-dependent cognitive decline occurs with normal aging. Dendritic retrac­ tion involves the pruning of dendrites within the hippocampus, a phenomenon that essentially ren­ ders the hippocampus less sensitive to converging

36

afferents, which in turn could modulate cognition. Importantly, hippocampal dendritic retraction does not represent neuronal loss per se, but a reduction in the neuropil that contains primarily dendrites and a small proportion of glial processes (Tata and Anderson, 2009), perhaps via decreases in cytoskeletal proteins (Cereseto et al., 2006). Chronic stress acting through elevated glucocorti­ coids reliably produces hippocampal synapse loss (Tata et al., 2006) and dendritic retraction (Conrad et al., 1999b, 2007; Fuchs et al., 2001; Kole et al., 2004; Magariños and McEwen, 1995a, 1995b; Magariños et al., 1996; McKittrick et al., 2000; Watanabe et al., 1992b), which can be detected by functional magnetic resonance imaging (fMRI) without necessarily producing noticeable changes in forebrain volume and body weight (Lee et al., 2009). Dendritic retraction is most pronounced within the CA3 region of the hippocampus (Con­ rad, 2006), perhaps reflecting this region’s high sensitivity to stress or glucocorticoids. It is noted that other hippocampal regions can express dendri­ tic retraction when chronic stress or glucocorticoid elevations are severe or persistent (Lambert et al., 1998; Sousa et al., 2000). Importantly, stress-pro­ duced hippocampal dendritic retraction is highly dynamic, demonstrating complete reversal to nonstress control levels following the termination of the stressor and with sufficient time for recovery (Conrad et al., 1999b; Sousa et al., 2000; Vyas et al., 2004). Moreover, antidepressant treatment can prevent stress-induced hippocampal dendritic retraction (Luo and Tan, 2001; Magariños et al., 1999; Watanabe et al., 1992a) and volumetric reductions (Czéh et al., 2001), even when neuro­ genesis is blocked (Bessa et al., 2009). Therefore, stress- or glucocorticoid-induced hippocampal den­ dritic retraction provides a model to study neoplas­ tic changes that are consistent with the types of structural brain dynamics observed in normal aging and depression. Gonadal hormones, and especially estrogens, have been demonstrated to have significant effects on hippocampal morphology. Estrogens were first shown to influence brain regions historically known for roles in reproduction, such as the hypothala­ mus, in rodents (McEwen, 1981; Parsons et al., 1982; Pfaff and McEwen, 1983) and then also

found to alter regions involved with cognition (Foy, 2001; Terasawa and Timiras, 1968). Subse­ quent studies extended these findings to show that estrogens altered neuron morphology by increasing hippocampal CA1 spine density in rats (Gould et al., 1990; Silva et al., 2000; Woolley and McEwen, 1992, 1993). Dendritic spines have long been theorized to be a structural component of memory (Geinisman, 2000; Kasai et al., 2003; Moser, 1999; Nimchinsky et al., 2002; Sorra and Harris, 2000), leading to wide-reaching implications regarding estrogen’s influence on cognitive func­ tion. This tenet is supported by recent work show­ ing that spatial working and reference memory performance is enhanced after 17b-estradiol injec­ tions (McLaughlin et al., 2008; Sandstrom and Williams, 2001, 2004) within the timeframes corre­ sponding to 17b-estradiol-induced increases in hip­ pocampal dendritic spines (Gould et al., 1990; Silva et al., 2000; Woolley and McEwen, 1992, 1993) and other markers of synaptic plasticity (Foy, 2001). Stress and glucocorticoids can also modify spine density and shape within the hippocampus (Diamond et al., 2006; Fuchs et al., 2006; Komatsu­ zaki et al., 2005; McLaughlin et al., 2009; 2005; Shors et al., 2001; Sunanda et al., 1995), with 17b-estradiol, and even cholesterol, the precursor to estrogens, protecting against stress-induced CA3 dendritic retraction in females (McLaughlin et al., 2010). Consequently, morphological alterations in neuronal dendritic structure and synapses may con­ tribute to and/or underlie functional outcomes on hippocampal-dependent cognition.

Influence of the HPA axis on hippocampaldependent learning and memory: inverted U-shaped function Spatial learning and memory How glucocorticoids are thought to influence spa­ tial learning is consistent with the original report by Yerkes and Dodson (1908), stating that perfor­ mance declines in challenging tasks when arousal falls along the extremes of being too low or high (Fig. 1A). However, the manner by which gluco­ corticoids alter spatial memory are complex, and

37 A.

Yerkes–Dodson Law (Hebbian version) Optimal arousal Optimal performance

Strong

Impaired performance because of strong anxiety

Performance

Increasing attention and interest

Weak Low

B.

High

Arousal Yerkes–Dodson Law (Original version)

Strong

Simple task Focused attention, flashbulb memory fear conditioning Difficult task Impairment of: Divided attention, working memory, Decision-making and multi-tasking

Performance

Weak Low

Arousal

High

Fig. 1. Representation of cognitive performance based on: (A) Hebbian version of the Yerkes–Dodson law and (B) the actual findings from Yerkes and Dodson (1908). (A) Cognitive performance is curvilinear with arousal with optimal performance occurring when arousal is not too low or high. (B) Under very simple learning conditions, performance is linear with arousal until a plateau is reached, while complex learning conditions show curvilinear influences with arousal as shown in (A). (From Diamond et al. (2007) with permission.)

involve ascertaining when glucocorticoids are ele­ vated (or absent) relative to the learning processes of acquisition, consolidation and retrieval, and whether the learning event is intrinsically aversive (for review, see Conrad, 2005; Diamond et al., 2007; Sandi and Pinelo-Nava, 2007). For the pur­ pose of this review, the focus will be on whether glucocorticoids are elevated upon the start of the acquisition phase in spatial tasks because gluco­ corticoid elevations often precede learning events in many of the clinical populations discussed in aging, as well as AD and depression. With regard to the type of task, spatial tasks are intrinsically more difficult to perform than simple passive

avoidance or fear conditioning. Spatial tasks require the integration of spatial relationships and demonstrate a non-linear learning function with arousal. In contrast, passive avoidance and fear conditioning motivate subjects by incorporat­ ing aversive stimuli, such as footshock, and exhibit a linear learning function with arousal (Fig. 1B). This review will focus on spatial learning in water or land-navigation tasks that require the integra­ tion of multiple cues to navigate using allocentric strategies for optimal performance (Paul et al., 2009). In healthy young adults, many studies show that the HPA axis has a non-linear relationship with

38

hippocampal-dependent spatial learning, with very low or high corticosterone levels impairing spatial learning and moderate corticosterone levels being optimal. In rodents, the removal of glucocorticoids via adrenalectomy (ADX) impairs spatial learning on the water maze (Oitzl and de Kloet, 1992; Roozendaal et al., 1996) and spatial recognition on the Y-maze (Conrad et al., 1999a; 1997). At the other end of the spectrum, subjects administered with stress levels of glucocorticoids or glucocorticoid receptor agonists exhibit impaired spatial learning on the Y-maze (Conrad et al., 1999a, 1997). In young rats, basal levels of glucocorticoids during the morning positively cor­ relate with spatial learning (Yau et al., 1995). Basal glucocorticoid levels averaged about 2 mg/dl, which represent non-stress levels, reveal­ ing that when glucocorticoids increase within this range, spatial memory improves. Exogenously manipulating glucocorticoid levels in juvenile ground squirrels also show that very high or low glucocorticoids impairs spatial memory, while moderate glucocorticoid levels provide the best spatial performance (Mateo, 2008). The context by which stress or glucocorticoids influence spatial learning and memory is also critical (Yang et al., 2003), as elegantly demonstrated in a study that compared the exposure of rats to a context with a predator and comparing the outcome to context containing a sexually receptive conspecific female (Woodson et al., 2003). In both situations, gluco­ corticoid levels were similarly elevated; however, only the predator exposure effectively and signifi­ cantly impaired spatial memory and showed a sig­ nificant positive correlation between corticosterone levels with spatial memory deficit (Woodson et al., 2003). Moderate corticosterone replacement in animals with their adrenals removed to eliminate the source of glucocorticoids improves spatial memory deficits caused by ADX (Conrad and Roy, 1993, 1995). Work investigating long-term potentiation (LTP) or primed burst potentiation (PBP), processes that are believed to underlie forms of learning and memory (Bliss and Collingridge, 1993; Heynen et al., 1996), cor­ roborate the curvilinear effects of stress and glu­ cocorticoids on hippocampal plasticity (Avital et al., 2006; Diamond et al., 1992; 2005; Joéls,

2006; Kerr et al., 1994). Taken together, these studies demonstrate that elevated glucocorticoids are necessary, but not sufficient to modulate spa­ tial learning and memory.

Influence of chronic stress and glucocorticoids on spatial learning and memory in the young adult The majority of studies show that chronic stress impairs spatial learning and memory in young adults under a variety of manipulations, proce­ dures, durations and assessment. Spatial learning and/or memory using the land radial-arm maze, the land Y-maze or water mazes is worsened fol­ lowing systematic, chronic exposure to the same (homotypic) immobilization stressor over many days to months (Conrad et al., 1996; Kitraki et al., 2004; Luine et al., 1994; Moosavi et al., 2007; Radecki et al., 2005; Srikumar et al., 2006; Sunanda et al., 2000; Venero et al., 2002; Wright and Conrad, 2005), social disruption (Aleisa et al., 2006; Alzoubi et al., 2009; Gerges et al., 2004; Krugers et al., 1997, Bodnoff et al., 1995; Srivar­ eerat et al., 2009), intruder paradigms (Ohl and Fuchs, 1999; Ohl et al., 2000; Touyarot et al., 2004), loud noise (Manikandan et al., 2006) or cold water immersion (Nishimura et al., 1999). Chronic unpredictable stressors, or other forms of heterotypic stressors, also reveal detri­ mental outcomes on spatial learning and memory when using the land radial-arm maze (Park et al., 2001; Zoladz et al., 2008), the land Y-maze (Orsetti et al., 2007) and the Morris water maze (Cerqueira et al., 2007; Song et al., 2006; Sousa et al., 2000) The aforementioned studies suggest that a vari­ ety of chronic stress durations can lead to similar detrimental outcomes on spatial ability in young adults, but there are some important considera­ tions. Both the elevation of glucocorticoids and a compromised hippocampus are hypothesized to underlie the chronic stress-induced spatial learn­ ing and memory deficits. Nearly all of the chronic stress paradigms have demonstrated that they can successfully elevate glucocorticoids. However, hippocampal dendritic retraction requires time to develop with chronic stress. Although the CA3

39

region of the hippocampus is most prone to exhi­ biting atrophied dendrites, it has a relatively slow time course of development: In response to wire mesh restraint, CA3 dendritic retraction occurs following 6 h/day of restraint for 21 days, but not with shorter durations of 2 h/day for 10 or 21 days, nor with wire mesh restraint for 6 h/day for 10 days (Luine et al., 1996; McLaughlin et al., 2007). Importantly, chronic stress conditions that impair spatial memory also produce CA3 dendritic retraction within the same subjects (Fig. 2). In the other chronic stress paradigms, hippocampal dendritic retraction may show a different develop­ mental time course, depending upon the severity of the stressor. For example, CA3 dendritic retrac­ tion can be found in as little as 6 days following activity stress combined with food restriction (Lambert et al., 1998), 10 days after complete immobilization for 2 h/day (Vyas et al., 2002) and 14 days following continual competition for resources (Vyas et al., 2002). Therefore, chronic stress alters hippocampal dendritic structure slowly and different types of stressors can have their own time course of development for impos­ ing structural alterations on hippocampal dendrites.

Role of CA3 dendritic retraction on the hippocampus As found with glucocorticoid elevations, the pre­ sence of CA3 dendritic retraction reveals suscept­ ibility to impaired spatial memory, but is insufficient alone. When early work reported that chronic stress or glucocorticoids potentially harmed the hippocampus (Sapolsky, 1992; Sapolsky and Pulsinelli, 1985), subsequent research followed to investigate the functional out­ comes on spatial ability, but with mixed results. Some studies found 3 weeks to nearly 3 months of chronic glucocorticoid treatment impaired spa­ tial ability in young adults (Bardgett et al., 1994; Dachir et al., 1993; Endo et al., 1996; Luine et al., 1993; McLay et al., 1998), whereas others found no effect (Bodnoff et al., 1995; Clark et al., 1995). The duration of glucocorticoid exposure may be impor­ tant because 3–4 weeks of glucocorticoid

treatment did not alter spatial ability unless gluco­ corticoid treatment continued for another month (Coburn-Litvak et al., 2003). One interpretation is that corticosterone-produced structural alterations within the hippocampus were not evident in the shorter (3–4 weeks) durations of glucocorticoid exposure. However, a study found that chronic glucocorticoid treatment failed to impair spatial memory despite confirmation from the same rats that CA3 dendritic retraction occurred (Conrad et al., 2007). Therefore, the presence of CA3 den­ dritic retraction does not necessarily indicate impaired hippocampal function as measured by spatial navigation. Given that CA3 dendritic retraction can exist without necessarily showing negative conse­ quences on spatial ability, then what role, if any, does CA3 dendritic retraction have in hippocam­ pal function? We hypothesize that the presence of CA3 dendritic retraction indicates susceptibility to compromised function, as well as to metabolic and neurotoxic challenges. Evidence that CA3 dendri­ tic retraction compromises hippocampal function comes from work investigating strategy use and from studies manipulating glucocorticoid levels during behavioral assessment. Several reports show that chronic stress shifts the strategy used during learning. In one study, chronically stressed rats exhibited impaired spatial recognition memory, a hippocampal-dependent task, but the same rats navigated quite well under conditions that were independent of the hippocampus (Wright and Conrad, 2005). In a direct test of which strategy was favoured, chroni­ cally stressed mice implemented a hippocampalindependent stimulus–response strategy significantly more than did the non-stressed controls, which favoured a hippocampal-dependent spatial strategy 100% of the time (Schwabe et al., 2008). Moreover, humans who self-reported a history of chronic stress also favoured stimulus–response strategies more than did subjects reporting a low chronic stress history (Schwabe et al., 2008). These findings were corroborated by another study showing that chronic stress has a tendency to impair learning on a spatial T-maze, but not on a response version (Sadowski et al., 2009). Current thinking is that chronic stress biases behavioral

40 Control

2 h–10 days

2 h–21 days

Basal

Apical

A.

6 h–10 days

6 h–21 days

B. Novel Other

Arm entries in first minute

2.0

1.5

1.0

0.5

0.0

Control

2 h–10 days

2 h–21 days

6 h–10 days

6 h–21 days

Fig. 2. Effects of different durations of chronic restraint stress on hippocampal CA3 dendritic retraction and hippocampaldependent spatial recognition memory on the Y-maze. (A) Chronic stress produced dendritic retraction in the apical CA3 region following 6 h/day for 21 days of restraint, but failed to produced CA3 apical dendritic retraction at a shorter restraint duration (2 h/day for 10 or 21 days) or for the same duration each day, but fewer days (6 h/day/10 days). (B) The status of the CA3 apical dendritic arbors corresponded to the performance on the hippocampal-dependent Y-maze task: chronic restraint for 6 h/day for 21 days impaired spatial recognition of the novel arm compared to the other arm (6 h–21 days), but spatial recognition memory was intact in the other conditions as rats entered the novel arm significantly more than the other arm.
p < 0.05 for novel vs. other for control, 2 h–10 days, 2 h–21 days and 6 h–10 days. (Reprinted from McLaughlin et al. (2007), with permission from Elsevier.)

strategies towards habit (Dias-Ferreira et al., 2009), or processes that rely heavily upon the striatum rather than the hippocampus.

Therefore, chronic stress compromises spatial ability through influences that may include hippocampal CA3 dendritic retraction, but

41

facilitates neural networks that underlie response/habit that likely involve striatal net­ works (for review, see Conrad, 2010). Manipulations of glucocorticoid levels during spatial navigation has demonstrated that chroni­ cally stressed subjects with a compromised hip­ pocampus are capable of spatial learning and memory. Rats that were chronically stressed by restraint for 6 h/day for 21 days, a procedure and timeframe that reliably produces hippocam­ pal CA3 dendritic retraction (Conrad, 2006), showed spatial memory deficits as expected (Wright et al., 2006). However, chronically stressed rats that were injected once on the day of spatial assessment with metyrapone, to attenuate stress levels of glucocorticoids, showed functional spatial abilities despite condi­ tions that produced hippocampal CA3 dendritic retraction (Fig. 3, Wright et al., 2006). Another report found that rats with chemical lesions tar­ geting the CA3 region also showed spatial def­ icits that were prevented with a single injection of metyrapone on the training day and deficits, and then were reinstated with a single corticos­ terone injection (Roozendaal et al., 2001). These studies reveal that manipulations that compromise the CA3 region of the hippocam­ pus can negatively impact spatial ability, but a compromised CA3 region alone is not sufficient because spatial ability can be modified further by the presence or absence of glucocorticoids during spatial learning and/or recall. In addition to compromising spatial ability, hip­ pocampal CA3 dendritic retraction is thought to enhance susceptibility to metabolic challenges. Nearly 25 years ago, glucocorticoid hypersecre­ tion was hypothesized to endanger the hippocam­ pus to contribute to age-related cognitive decline (Sapolsky, 1992; Sapolsky et al., 1986). Key con­ cepts were that glucocorticoid elevations exacer­ bated damage to the hippocampus caused by neurotoxicity or ischemic challenges as compared to the effects with glucocorticoid elevations or the challenges when presented alone (Sapolsky, 1985b; Sapolsky and Pulsinelli, 1985). Repeated exposure to glucocorticoids was proposed to down-regulate hippocampal glucocorticoid recep­ tors, which would hinder HPA axis regulation and

potentiate glucocorticoid elevations with age, and hence, the potential for hippocampal damage (Sapolsky et al., 1983, 1984a, 1984b). Enthusiasm for this hypothesis began to wane when prolonged glucocorticoid elevations failed to consistently produce hippocampal cell loss in adults (Bodnoff et al., 1995; Coburn-Litvak et al., 2004; Leverenz et al., 1999; Müller et al., 2001; Sousa et al., 1998) and evidence suggested that non-human primates expressed insufficient numbers of glucocorticoid receptors to effectively mediate ‘glucocorticoid toxicity’ (Sánchez et al., 2000). Recent findings, however, suggest that hippocampal CA3 neurons can be harmed without concurrent glucocorticoid elevations during the neurotoxic challenge, pro­ vided that stress- or glucocorticoid-induced CA3 dendritic retraction was present during the chal­ lenge (Conrad et al., 2004, 2007). Consequently, hippocampal neurons expressing dendritic retrac­ tion are susceptible to damage beyond the window of when glucocorticoids are elevated. Since stress­ or glucocorticoid-induced CA3 dendritic retrac­ tion is reversible (Conrad et al., 1999b; Sousa et al., 2000; Vyas et al., 2004), then CA3 neurons can recover to their pre-stress/glucocorticoid con­ dition when a metabolic or neurotoxic incident fails to occur. Therefore, CA3 dendritic retrac­ tion conveys susceptibility to neuronal loss, but is not damage per se, which contributed to a revised hypothesis, called the ‘Glucocorticoid Vulnerability’ hypothesis (Conrad, 2008), and provides insight into differences in susceptibility to age-related cognitive decline.

Model for chronic stress and CA3 dendritic retraction in spatial ability Under chronic stress conditions that produce CA3 dendritic retraction, we hypothesize that the hip­ pocampus will be susceptible to spatial memory deficits following chronic glucocorticoid eleva­ tions at levels normally not disruptive to spatial memory in non-stressed controls. Chronically stressed subjects respond to a novel spatial mem­ ory testing paradigm with slightly potentiated glucocorticoid levels. Under these conditions that are combined with a compromised hippocampus

42 A. Percentage of arm entries in first minute

80 Novel Other 60

40

20

0

Total serum corticoterone (µg/100 ml)

B.

CV

CM35

CM75

SV

SM35

35

SM75 Train Test

30 25 20 15 10 5 0

CV

CM35

CM75

SV

SM35

SM75

Fig. 3. Effects of a single metyrapone injection on the day of spatial recognition memory assessment in chronically stressed rats. (A) Rats that were chronically stressed by restraint (6 h/day/21 days) and then tested on the Y-maze showed impaired spatial recognition memory (SV), but those chronically stressed rats given a single injection of metyrapone (75 mg/kg) to attenuate stress-induced release of glucocorticoids prior to the Y-maze showed functional spatial recognition memory (SM75). Spatial recognition memory of controls (CV, CM35, CM75) was unaltered by metyrapone at either dose (
p < 0.05 for novel arm entries vs. other arm entries). (B) Total serum corticosterone levels were measured after the first training trial (Train) and the second testing trial (Test) in a separate cohort of rats. While the corticosterone levels of the chronically stressed rats were higher than those of controls (‡ = significant main effect of stress), metyrapone injections dose-dependently lowered corticosterone in both controls and chronically stressed rats (
p < 0.05 for CM75 and SM75 compared to the remaining groups given vehicle or 35 mg/kg). Moreover, the corticosterone levels of the chronically stressed rats injected with vehicle or 35 mg/kg of metyrapone (SV, SM35) were statistically similar to the controls injected with vehicle or 35 mg/kg of metyrapone (CV, CM35). Therefore, these data support the interpretation that both corticosterone levels and brain sensitivity to corticosterone contribute to outcomes on spatial recognition memory. (From Wright et al. (2006), with permission from Wiley-Blackwell Publishing).

expressing CA3 dendritic retraction, chronically subjects will express spatial learning and memory impairments under conditions that would

normally facilitate optimal spatial abilities in non-stressed controls (Fig. 4). This hypothesis assumes that the spatial navigation paradigm is

43 A. Effect of acute stress on spatial ability in normal controls Acute stress

Hippocampus

Spatial ability (solid line in graph ‘C’)

HPA axis B. Effect of acute stress on spatial ability after chronic stress Hippocampus w/ CA3 dendritic retraction

Acute stress

Spatial ability (dotted line in graph ‘C’)

HPA axis C. Non-linear function for glucocorticoids and spatial ability

Spatial ability

Optimal

Poor ADX Basal Moderate

High

Glucocorticoid levels Fig. 4. Model for chronic stress actions via elevated glucocorticoids influencing spatial ability. (A) Without a chronic stress history, an acute stressor triggers the HPA axis and elevates glucocorticoids, which could feedback to influence the hippocampus and its functions, such as spatial ability. The hippocampus also provides negative feedback on the HPA axis. The up and down arrows illustrate the reciprocal relationship between the hippocampus and the HPA axis. Under these conditions, glucocorticoids influence spatial ability by the inverted U-shaped function, and illustrated by the solid curve in graph C. (B) Following chronic stress, the hippocampus is compromised and exhibits CA dendritic retraction, along with other neurochemical and neuromorphological changes. Consequently, the hippocampus has poor regulation of the HPA axis, indicated by the dashed arrow, whereas the HPA axis and adrenals are over-active, indicated by the two large upward pointing arrows, creating an imbalance. Under these chronic stress conditions, a change in the sensitivity of the hippocampus to novel stress and glucocorticoids is hypothesized to narrow the inverted U-shaped function, as illustrated by the dotted curve in graph C. (C) Consequently, glucocorticoid elevations that optimize spatial ability in non-stressed animals will impair spatial ability in those following chronic stress: compare the curves under the line thin downward arrow. The thick arrowhead illustrates that at low glucocorticoid levels, functional spatial ability is possible following chronic stress.

not overtly arousing or aversive, as would be found in fear conditioning or water maze paradigms, conditions that activate the amygdalar regions and can mask hippocampal function (Conrad, 2006; McLaughlin et al., 2009), perhaps by shifting to well-learned behaviors (Dias-Ferreira et al., 2009). However, when subjects are navigating in a spatial environment under non-threatening conditions, then spatial abilities are hypothesized to differ between controls and

chronically stressed subjects based upon HPA activity and a compromised hippocampus. There are several lines of evidence supporting the interpretation that both the HPA axis and hippocampus contribute to impaired spatial navi­ gation in chronically stressed subjects. Even when rats are tested on a relatively benign spatial navi­ gation task that taps into rats’ innate tendency to explore novelty, chronically stressed rats release higher levels of glucocorticoids than the controls

44

(Fig. 3B). Prior work showing that chronically stressed rats release more glucocorticoids in response to a novel stressor compared to a famil­ iar stressor supports these findings (Bhatnagar et al., 2002; Dallman, 2007; Dallman et al., 2000; Herman et al., 2005). For spatial assessment on the Y-maze, the repeated, same stressor (homo­ typic) is the daily restraint and the novel stressor (heterotypic) is the Y-maze. Consequently, chronically stressed rats release higher levels of glucocorticoids in the Y-maze compared to con­ trols, even though the Y-maze is a relatively benign task that does not require water or food deprivation, or water escape. However, glucocor­ ticoid levels alone do not directly correspond with spatial performance, as tested on the Ymaze. Indeed, we found that circulating glucocor­ ticoid levels released during Y-maze exploration in the chronically stressed rats injected with the moderate dose of metyrapone (35 mg/kg), were statistically similar to the glucocorticoid levels in the controls injected with either vehicle or 35 mg/ kg of metyrapone. In spite of comparable circu­ lating glucocorticoid levels, the controls showed optimal spatial ability whereas the chronically stressed rats injected with the moderate metyra­ pone dose did not (Wright et al., 2006). More­ over, biologically active levels of circulating glucocorticoids were similar among controls and chronically stressed rats (Wright et al., 2006). That circulating glucocorticoid levels did not dif­ fer among groups, while spatial maze perfor­ mance did, supports the hypothesis that changes in brain sensitivity to glucocorticoids contribute to the spatial ability outcomes in chronically stressed rats. How the hippocampus and the HPA axis contri­ bute to spatial ability following chronic stress is illustrated in Fig. 4B. The compromised hippocam­ pus expressing CA3 dendritic retraction is unable to regulate the HPA axis effectively, as illustrated by the downward dotted arrow, and the hippocam­ pus is susceptible to glucocorticoid elevations (indicated by two solid upward arrows targeting the hippocampus). Consequently, the influence between the hippocampus and the HPA axis is no longer balanced, which we hypothesize is revealed by a change in sensitivity of the hippocampus to

novel stress and glucocorticoid elevations. The consequence of the enhanced hippocampal sensi­ tivity to glucocorticoids and glucocorticoid hyper­ secretion is that levels of glucocorticoids that optimize spatial ability in controls lead to impaired spatial ability in chronically stressed rats. Specifi­ cally, chronic stress is hypothesized to narrow the inverted U-shaped function for glucocorticoid effects on spatial ability, by shifting the descending right limb of the inverted U-shaped function to the left to represent an enhanced sensitivity to gluco­ corticoid levels (Fig. 4C). Consequently, moderate levels of glucocorticoids that do not impair spatial ability in controls are predicted to do so following chronic stress conditions that produce CA3 dendritic retraction.

The influence of stress and the HPA axis on age-related cognitive decline The aging population adds another dimension underlying cognitive function as cognitive deficits progressively worsen, with hippocampal-dependent functions being particularly vulnerable (Barnes’, 1979; Fischer et al., 1992; Gallagher and Pelleymounter, 1988b; Geinisman et al., 1995; Ingram, 1988; Luine et al., 1990; Moss et al., 1988; Winocur and Gagnon, 1998). Despite the increased risk for cognitive decline with age, many older indi­ viduals show cognitive performance similar to younger counterparts (Gage et al., 1984; Gallagher and Pelleymounter, 1988a; Issa et al., 1990; Mar­ kowska et al., 1989; Matzel et al., 2008). Indeed, cognitive decline represents the increased variability of the aged population’s performance (Matzel et al., 2008; Rapp and Amaral, 1992), and findings such as these contributed to the characterization that aging may represent a continuum of cognitive decline (Brayne and Calloway, 1988). Understanding why some individuals are able to successfully retain their cognitive abilities with age, whereas others do not, will be essential for facilitating healthy aging. Many factors can contribute to non-optimal aging, and HPA axis dysregulation may be an important component underlying unsuccessful aging and related cognitive declines. As described earlier, a critical risk factor for poor cognitive

45

function associated with aging is life-long expo­ sure to stressful events (McEwen, 1999; Pardon and Rattray, 2008). An individual exposed to a chronically stressful situation will likely suffer poor cognitive outcomes, but can potentially recover quickly when the stressful situation ends. Indeed, some chronic stress situations can opti­ mize cognitive ability later in life, as illustrated by mild stress during the postnatal period in rat (Meaney et al., 1988), while other postnatal manipulations accelerate cognitive decline (Brunson et al., 2005). Importantly, these stressful episodes do not occur in isolation, as they are overlaid upon the individual’s genetic predisposi­ tion and other mitigating factors. As examples, an individual’s cardiovascular status (Raz et al., 2005), caloric intake and/or nutrition (Baran et al., 2005; Joseph et al., 2009), immune function (Segerstrom and Miller, 2004) and aerobic activ­ ity/environmental enrichment (Cotman et al., 2007; Winocur, 1998; Wright and Conrad, 2008) can all impact how chronic stress could influence brain health (Conrad et al., 2009; Raz and Rodrigue, 2006). Moreover, aged individuals have an accumulation of a lifetime of experiences that could further alter the progression of brain aging. This concept has been described as ‘allostatic load’, and refers to the price the body pays for adapting to adverse situations (McEwen, 2000a, 2000b, 2000c; Stewart, 2006). Essentially, the body alters itself to compensate for the immediate threat, but with potentially debilitating long-term ramifications. To illustrate how chronic stress could influence brain health when additional risk factors are involved, the example of hypertension will be dis­ cussed. Undiagnosed hypertension increases the risk of structural alterations in the brain (den Hei­ jer et al., 2005; Raz et al., 2003a, 2003b; Raz et al., 2003c). If this hypertensive person is concurrently experiencing chronic stress, then the stressinduced morphological alterations within the hip­ pocampus could become exacerbated. A severe outcome may even involve cell loss as we recently found that chronic stress increases susceptibility to hippocampal damage (Conrad et al., 2004, 2007). This susceptibility corresponds to brain regions that show stress-induced dendritic retraction and

is not likely attributed to acute elevations of glu­ cocorticoids (Conrad et al., 2004, 2007). While acute stress or glucocorticoid elevations can exacerbate hippocampal damage caused by neu­ rotoxic or metabolic insults (Sapolsky, 1985a, 1985b; Sapolsky and Pulsinelli, 1985), as proposed in the ‘Glucocorticoid Cascade Hypothesis’ (Sapolsky et al., 1986), the current findings suggest that glucocorticoid elevations are not required to confer susceptibility to damage. Instead, suscept­ ibility is revealed by conditions surrounding dendritic retraction, which persists for many days or longer than the hours that glucocorticoids are elevated from a stress episode. Consequently, individuals with a history of stressful life events have broader window by which opportunistic challenges could shift the reversible morpho­ logical changes into permanent hippocampal damage and continued HPA dysfunction. The ‘Glucocorticoid Vulnerability Hypothesis’, recognizes the distinction between glucocorticoids facilitating dendritic retraction without needing to be elevated to create a susceptible brain to poten­ tial damage (Conrad, 2008). Aged individuals are particularly susceptible to alterations in brain health following chronic stress, based upon neuromorphological and cognitive evidence. As eloquently stated by Burke and Barnes (2006), neuronal loss does not have a sig­ nificant role in age-related cognitive decline, but rather, small region-specific changes in dendritic branching and spine density may be critical. The normal aging hippocampus appears to maintain or even increase dendritic complexity in CA1 (Hanks and Flood, 1991; Turner and Deupree, 1991), CA3 (Flood et al., 1987) and dentate gyrus (Flood, 1993), while dendritic complexity decreases within the PFC (de Brabander et al., 1998; Grill and Riddle, 2002; Markham and Juraska, 2002). More­ over, hippocampal spine density does not appear to change with age in the CA1 (Markham et al., 2005) or dentate gyrus (Curcio and Hinds, 1983). However, the aged hippocampus shows regionspecific decreases in synapse number at the mole­ cular layer of the dentate gyrus (Bondareff and Geinisman, 1976; Geinisman et al., 1977), decreases in the number of perforated synapses (Geinisman et al., 1986) and increases in calcium

46

conductance (Foster and Norris, 1997; Landfield, 1988). Indeed, the hippocampus of aged rats with cognitive deficits is unable to update encoding information when environmental cues are manipulated, demonstrating reduced plasticity (Tanila et al., 1997) and that chronic stress can accelerate this process (Kerr et al., 1991). These findings show that brain plasticity and function is already reduced in the aged brain, which can be hindered further by an additional mitigating factor, such as chronic stress. Studies investigating the effects of chronic stress or glucocorticoids on spatial learning show that the aged hippocampus is at a disadvantage. When young and middle-aged rats were directly compared, exposure to glucocorticoids for 3 months impaired spatial learning in middle-aged rats without affecting the performance of the younger cohorts (Bodnoff et al., 1995). Chronic stress accelerated spatial cognitive decline in aged rats (18 months), when 4 weeks of unpredict­ able stress was initiated at 12 months of age and then administered sporadically thereafter (Borcel et al., 2008). Spatial memory positively correlated with neurogenesis in the dentate gyrus (Borcel et al., 2008) and glucocorticoid exposure deter­ mined extent of age-related cognitive decline (Heffelfinger and Newcomer, 2001; Hibberd et al., 2000; Montaron et al., 2006). Moreover, chronic stress or glucocorticoids predicted hippo­ campal atrophy and hippocampal-dependent memory deficits in aged humans (Lupien et al., 1998), and facilitated cognitive decline in middleaged rats (Arbel et al., 1994), including those that were predisposed for high reactivity to novelty (Sandi and Touyarot, 2006). A distinction between how chronic stress influences the learn­ ing of new information and retrieving previously learned information is illustrated by a study that trained rats on a water maze, then administered corticosterone for several weeks, followed by assessment of retention (Hebda-Bauer et al., 1999). Under these conditions, corticosterone facilitated retention in the very old rats (31 months), which is consistent with the interpreta­ tion that chronic stress via the HPA axis shifts behavior towards habits: aged rats may be predis­ posed to shift towards well-learned behaviors

given their reduced hippocampal plasticity. These studies demonstrate a relationship among gluco­ corticoid exposure, hippocampal plasticity and cognitive ability with aging. The synopsis of these findings is that while the aged population consists of a heterogeneous popu­ lation, the aged brain and especially the aged hip­ pocampus will have difficulty rebounding from chronic stress and elevated glucocorticoids. The aged hippocampus is capable of plasticity, but its baseline differs compared to the young hippocam­ pus. Consequently, the inverted U-shaped relation­ ship between glucocorticoids and cognition described for the young may already be narrowed for the aged, perhaps revealing a function that is closer to the dashed curve than the solid one (Fig. 4C). The aged population may have more difficulty recovering from chronic stress than the young, which would provide an even larger window of opportunity for vulnerability. One’s life experi­ ences and exposures to stress, and the resulting effects on the HPA axis, impact the trajectory of aging. This results in an aged population composed of a diverse group of individuals with varied cogni­ tive and brain aging outcomes.

HPG axis and aging: influences on hippocampaldependent functions The HPG axis, age and spatial memory: overarching relationships Several decades of research have converged to indicate that gonadal hormones are potent mod­ ulators of brain structure and function, including in brain regions known to be intimately linked to learning and memory. Many of these cognitive brain regions are sensitive to changes as aging ensues. As represented in Fig. 5, there is a tertiary model representing interactions between aging, spatial learning and memory, and gonadal hor­ mones wherein research within each domain has identified strongly supported tenets: (1) gonadal hormones change with age, (2) age influences spa­ tial learning and memory and (3) gonadal hor­ mones influence spatial learning and memory. While it is clear these individual tenets are strongly

47

ac Im p

cts

Gonadal hormones

Stress and Resulting HPA Change

pa Im

ts

Age

Memory Impact

Fig. 5. Tertiary model representing interactions among aging, spatial learning and memory, and gonadal hormones. Research within each point has been strongly supported by empirical data: (1) aging impacts gonadal hormone milieu, (2) aging impacts spatial learning and memory and (3) gonadal hormones impact spatial learning and memory. Stress can impact trajectory of change across time for each of these three points. How these three tenets interact with each other, and with glucocorticoid release due to stress, is a key area of research that will yield insight into life experiences and exposures that influence age-related brain and cognitive change to ultimately impact phenotype of the aged individual.

supported by empirical data, a fundamental ques­ tion in aging research regarding interactions has yet to be clearly answered. How do gonadal hormone changes that occur with age relate to memory changes that occur with age? There is also evidence that stress can impact trajectory of change across time for each of these three points, as discussed and exemplified above. How these three tenets interact with each other, and with glucocorticoid release and HPA axis alterations due to stress, is an impor­ tant area of research that will yield insight into life experiences and exposures that influence agerelated brain and cognitive change to ultimately impact phenotype of the aged individual. Below we discuss each of these three tenets in turn, with examples of interactions presented when available from the literature.

Reproductive senescence Menopause, occurring typically in the fifth decade of life, is characterized by loss of ovarian-derived

circulating hormones, including estrogen and pro­ gesterone (Timaras et al., 1995). The majority of women undergo menopause not from oophorect­ omy (i.e. surgical removal of the ovary), but as a transitional hormone loss following age-related alterations of the hypothalamus, pituitary and ovary, ultimately resulting in follicular depletion (Timaras et al., 1995). Early in the aging process, neuronal changes in the hypothalamus are hypothesized to initiate transition into reproduc­ tive decline, leading to reproductive senescence (Downs and Wise, 2009). The ultimate hormone profile of the older reproductively senescent female rat and woman differ, limiting the use of ovary-intact female rat as an optimal model of human menopause when testing cognition. None­ theless, the rodent model provides exciting, illu­ minating insights to understand mechanisms of menopause itself since there are some commonal­ ities regarding reproductive physiology (Downs and Wise, 2009). As aging ensues in women, estro­ gen and progesterone decline due to decreased ovarian follicular reserves (Timaras et al., 1995). Thus, ovarian follicle depletion ultimately causes hormone loss during menopause. In contrast, the aging rat undergoes estropause, a persistent estrus state due to chronic anovulation rendering intermediate estrogen levels, or a pseudopreg­ nant/persistent diestrus state characterized by high progesterone levels due to increased ovula­ tion and corpora lutea (Meites and Lu, 1994). These changes in ovarian-derived hormone release in the rat are primarily due to hypothala­ mic/pituitary axis alterations (Meites and Lu, 1994). Thus, the primary mechanism that ulti­ mately results in reproductive senescence and cir­ culating hormone alterations in the woman is ovarian follicle depletion, while in the rat it is the hypothalamic–pituitary axis.

Aging effects on spatial learning and memory One of the most consistent findings in the animal cognition literature is that aged rodents exhibit poor scores on working and/or reference memory tasks that require spatial navigation compared to young counterparts. The majority of the studies

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testing age-related changes in spatial cognition used male rodents, with studies reporting agerelated memory decline on a multitude of tasks using various protocols and procedures (Arendash et al., 1995; Barnes et al., 1980; Beatty et al., 1985; Bond et al., 1989; Chrobak et al., 1995; Frick et al., 1995; Kadar et al., 1994; Lebrun et al., 1990; Lindner et al., 1992; Noda et al., 1997; Pitsikas and Algeri, 1992; Rapp et al., 1987; Shukitt-Hale et al., 2004; Stewart et al., 1989; Wallace et al., 1980; Wellman and Pelleymounter, 1999; Wyss et al., 2000). In the last decade, there have been increased efforts to study the effects of age-related spatial cognitive decline in female rodents, with the driving force of many studies being interest in the relationships with concurrent gonadal hor­ mone change. Aged females show spatial working memory deficiency compared to young females (Bimonte et al., 2003; Bimonte-Nelson et al., 2003b, 2004; Kobayashi et al., 1988; Luine et al., 1990), with age-related changes in spatial memory likely related to circulating ovarian hormone levels. For example, spatial reference memory decline on the Morris maze emerged by 12–16 months of age, when ovarian hormone levels start to change and estropause ensues (Markowska, 1999; Talboom et al., 2008). When evaluating the vast cognitive aging litera­ ture in the rodent, age-related changes in spatial learning and memory are multi-dimensional and complex, with, for example, age-related changes being dependent upon which phase of learning is being tested and the demand level of the task. Age-related cognitive deficits seem to be greatest during task acquisition, as young and aged rodents eventually reach comparable asymptotic levels on spatial working and reference memory tasks when given extended training (Ikegami, 1994; Rapp et al., 1987). This effect may interact with degree of cognitive impairment in the aged animal at the onset of testing sessions (Ikegami, 1994). It is noted that age-related deficits have been observed after spatial task acquisition as well, with the greatest age-associated cognitive decrements seen when spatial memory demand is high. In humans, age-related deficits are exacerbated with greater working memory complexity, an effect shown on multiple tasks and within non-spatial

(e.g. verbal) and spatial domains (Salthouse et al., 1989). In animals, age-related spatial deficits become more pronounced as memory demand increases. This has been shown for age-associated interference-related deficits (Lebrun et al., 1990) and for memory capacity deficits (Aggleton et al., 1989; Bimonte et al., 2003; Bimonte-Nelson et al., 2003b; 2004). For example, in the spatial water radial-arm maze task, as trials progress within any given session, animals need to hold a greater number of items of information in spatial working memory. Young and aged female rats did not differ in the ability to handle an increasing spatial working memory load during the initial portion of testing (Fig. 6A, Bimonte et al., 2003). However, as testing progressed across days young animals learned to handle more spatial working memory information. This resulted in a significant learning curve for young animals on all trials, whether spatial working memory load was low, moderate or high. In contrast, aged animals exhibited a sig­ nificant learning curve only on the earliest trials, when spatial working memory load was low. As spatial working memory load increased aged rats had difficulty remembering which arms they had visited within a session, and they could not learn to handle this increasing amount of information across testing sessions. Aged rats also made dis­ proportionately more errors on the latter trials, when spatial working memory load was highest. Thus, aged female rats exhibited progressive per­ formance deterioration as the number of items to be remembered, or working memory load, increased (Fig. 6B). There is other evidence that aged animals have difficulty sustaining successful performance as other types of memory demands increase, such as when a delay is imposed between trials. Such findings that aged rats exhibit delay-dependent deficiencies in performance is typically interpreted as a faster rate of forgetting (Beatty et al., 1985; Dunnett et al., 1988; Winocur, 1986; Zornetzer et al., 1982), although not all studies find age differences in rates of forgetting, which may be related to overtraining or animals reaching criter­ ion performance (Beatty et al., 1985; Wallace et al., 1980; Willig et al., 1987). This latter point is underscored by research in humans touching on

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the boundaries between what is, and what is not, normal cognitive aging. For example, research indicates that if younger and older people learn information to the same level, even though the older individuals may take longer to get to this point, the older people will not forget the informa­ tion more rapidly (Albert, 2002; Albert et al., 1987). Thus, while there are clear age-related changes in learning and memory in humans and animal models, there is great variability across individuals. Moreover, decrements associated with aging are typically seen regarding only certain types of information, with spatial memory one of the most robust deficits noted, as well as an age-related selective vulnerability to acquisition and demand task components.

Gonadal hormones and spatial learning and memory: general context and clinical implications By the beginning of the 20th century, specific ‘internal secretions’, now referred to as steroid hormones, were known to be chemical media­ tors of the phenotype (Adler, 1981). Steroid hormones released from the gonads have since been shown to be important for not just classical reproductive actions (Beach, 1947), but also for providing neural plasticity and influencing brain functions such as learning and memory. In humans and animal models, estrogens, proges­ terone and testosterone have each been shown to impact spatial cognition. To evaluate steroid hormone levels and cognitive effects in humans, researchers have been creative in their assess­ ments and have reported effects: across meno­ pause transition stages (Luetters et al., 2007), with sex-change operations and concomitant sex hormone treatment (Gomez-Gil et al., 2009), and before versus after hormone therapy treatment in surgically menopausal women (Sherwin, 2006). To test the cognitive effects of steroid hormones in rodent or monkey models, the traditional procedure is to remove the source of major endogenous synthesis and release, the testes in the male (gonadectomy, or GDX) or the ovaries in the female (ovariect­ omy, or OVX), and give the exogenous steroid

of question as a treatment regimen. Over the last decade, both the human and animal litera­ ture evaluating the potential influence of gona­ dal hormones on brain health and function during aging have increased in breadth and depth. Much of this is because of the recent intense discussion and debate about whether hormone therapies impact normal aging or AD, emanating in part from reports including a meta­ analysis that estrogen-containing hormone therapies decrease the risk of AD by 29% (Yaffe et al., 1998), that placebo-controlled stu­ dies showed that estrogens improved memory or dementia scores in female AD patients (Asthana et al., 2001, 1999; Ohkura et al., 1994, 1995), that menopause might exacerbate age-related cogni­ tive changes in several domains, including visuospatial abilities (Halbreich et al., 1995), and then more recently, the outcome of the Women’s Health Initiative (WHI) studies show­ ing null or detrimental effects on cognition or dementia from the most commonly used hor­ mone therapies (for discussion, see Sherwin and Henry, 2008). Further, the clinical implica­ tions and health-related importance of under­ standing the effects of ovarian hormone loss and replacement has been underscored by reali­ zation that life expectancy of women has increased from an average of 54 years in 1900, to recent values estimating expectancy to about 80 years (Singh et al., 1996). Since age of spon­ taneous menopause has remained stable, women are now living approximately one-third of their lives in a hypo-estrogenic menopausal state (Amundsen and Diers, 1970, 1973; Sherwin, 2003). The emerging findings in the literature have been illuminating, diverse and exciting, showing that multiple parameters impact the extent, and even the direction, of cognitive effects of gonadal hormones, underscoring the impact of gonadal hormone effects on brain function and its rich plasticity. The discussion here will not exhaustively cover this immense and growing literature, and will be limited to points necessary to provide context for the dis­ cussion of gonadal hormone effects on cognitive aging, with special emphasis on the spatial domain.

50 A. Aged animals cannot learn to handle an increasing spatial working memory load, while young animals can. 5 Aged Young

Working memory errors

4

3 High memory Load block 2

1

Moderate memory Load block

Low memory Load block 12

11

10

9

8

7

6

5

4

3

2

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Day of maze testing

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Aged rats

3 Young rats

2 1

gh Hi

M

od er a

te

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0 Lo

Working memory errors

B. Aged animals exhibit an impaired ability to handle an increasing spatial working memory load.

Working memory load Fig. 6. (legend continued)

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HPG axis and spatial learning and memory: estrogens and progesterone Accumulating evidence supports the interpreta­ tion that ovarian hormone loss contributes to cog­ nitive decline in women. Clinical findings show women exhibit cognitive decline after surgical menopause (Farrag et al., 2002; Phillips and Sherwin, 1992; Sherwin, 1988), including on global cognitive function as tested 3 or 6 months postsurgery (Farrag et al., 2002). Further, surgically menopausal women exhibit lower memory scores relative to naturally menopausal women, and age of oopherectomy (surgical ovary removal in the woman) and greater years since surgery correlated with poorer performance (Nappi et al., 1999). Pre­ clinical rodent model evaluations also show that ovarian hormone loss can induce cognitive changes, an effect depending on many factors, including age (Bimonte-Nelson et al., 2006; Markowska and Savonenko, 2002; Savonenko and Markowska, 2003; Talboom et al., 2008). Sur­ gical ovarian hormone loss (e.g. OVX) produces cognitive decrements in young adult female rats (Bimonte and Denenberg, 1999; Daniel et al., 1999; El-Bakri et al., 2004; Feng et al., 2004; Gibbs and Johnson, 2008; Talboom et al., 2008), while enhancing cognition in old age (discussed in more detail below; Bimonte-Nelson et al., 2003b, 2004; Braden et al., 2010). To date, only four studies evaluated OVX effects on maze learning and memory in middle-aged rats (Bimonte-Nelson et al., 2006; Markowska and Savonenko, 2002; Savonenko and Markowska, 2003; Talboom

et al., 2008). In middle-aged females 12–16 months-old, OVX did not impact spatial reference memory (Bimonte-Nelson et al., 2006; Markowska and Savonenko, 2002; Talboom et al., 2008) or spatial working memory (Markowska and Savonenko, 2002; Savonenko and Markowska, 2003). However, spatial working memory deficits were detected in 17-month-old OVX rats following high-demand time-delayed memory retention tests (Markowska and Savonenko, 2002). These data suggest that OVXrelated memory changes in middle-age may become evident when working memory demands are more challenging. In this regard, we previously demonstrated that OVX alters memory in both young and old rats (Bimonte and Denenberg, 1999; Bimonte-Nelson et al., 2003b, 2004; Braden et al., 2010), effects which were more pronounced as working load increased by escalating the num­ ber of items to remember. Thus, elevating work­ ing memory demand either by extending time delays to challenge retention, or by increasing the number of items to remember, allows a broader scope of evaluations to realize OVXinduced memory changes across the ages. It is noted that exacerbated deficits in aged animals are also seen with a higher memory demand, as described above, further indicating that incremen­ tal alterations in task demand could yield insights into changes that may not be seen otherwise. In the rodent, transitional hormone loss can be induced via the industrial chemical 4-vinylcyclo­ hexene diepoxide (VCD), which produces follicu­ lar depletion by selectively destroying primordial

Fig. 6. (A) Mean number of spatial working memory errors committed by Aged and Young rats averaged into the Low (Trials 2 and 3), Moderate (Trials 4 and 5) and High (Trials 6–8) working memory load blocks. Also depicted is the linear trend for each of the working memory load blocks, for each group. Both Young and Aged groups exhibited significant linear trends on the Low working memory load block (regression equations: Aged: y = 0.042x þ 0.600, Young: y = 0.058x þ 0.639), while only the Young group showed a significant linear trend on Moderate and High working memory load blocks (ys = 0.058x þ 1.12 and 0.176x þ 3.76, respectively). These data suggest that while both Young and Aged animals could learn to handle a low spatial working memory load, the young animals could also learn to handle moderate and high spatial working memory loads, while the aged animals could not. (B) Number of spatial working memory errors + SE on Low (Trials 2 and 3), Moderate (Trials 4 and 5) and High (Trials 6–8) load trial blocks, averaged across the latter days of testing, for Aged and Young rats. As trials increased, the number of elements of information to be remembered increased. The significant Age × Trial block interaction reflects that Aged animals committed disproportionately more errors on the latter trials, when the spatial working memory load was highest.

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and primary follicles via acceleration of the nat­ ural atresia process, resulting in hormone profiles more similar to naturally menopausal women ver­ sus OVX (Mayer et al., 2004; Springer et al., 1996; Timaras et al., 1995). Data from work using this novel menopause model, like data from work using the OVX model, have indicated that ovarian hormone loss impacts cognition. VCD-induced transitional menopause impaired learning of a spatial recent memory task, and transitional menopause before OVX was better for spatial memory than an abrupt loss of hormones via OVX only (Acosta et al., 2009a). These results correspond with findings from Rocca et al. (2007) showing women that had undergone oopherectomy prior to menopause onset had elevated cognitive impairment risk compared to age-matched women without oopherectomy. Col­ lectively, the findings suggest that initiation of transitional menopause before surgical ovary removal might benefit mnemonic function and could obviate some negative cognitive conse­ quences of surgical menopause alone, and that surgical menopause may be worse for cognition than transitional menopause. Future studies addressing this important question of cognitive effects of type of ovarian hormone loss, combined with evaluation of potential interactions between type of ovarian loss and subsequent hormone therapy, should yield valuable insight and new interpretations of variables impacting cognitive outcome due to ovarian hormone loss and therapy in menopausal women. Estrogens are a class of hormones including 17b-estradiol, estrone and estriol; 17b-estradiol is the most potent naturally circulating estrogen, followed by estrone and estriol, in order of receptor affinity (Kuhl, 2005; Sitruk-Ware, 2002). To date, 17b-estradiol has been the pri­ mary estrogen used to test cognitive effects of hormone therapy in the animal model. The majority of studies evaluating activational effects of estrogens on a zero-level circulating hormone background (i.e. OVX animals) for spatial learn­ ing and memory have been performed in young rodents, with many showing enhancements due to treatment (Bimonte and Denenberg, 1999; Daniel et al., 1997, 1999; Dohanich et al., 1994;

Galea et al., 2001; Holmes et al., 2002; Luine et al., 2003, 1998; Marriott and Korol, 2003; McLaughlin et al., 2008; Packard and Teather, 1997; Sandstrom and Williams, 2001; Singh et al., 1996). Studies in young OVX monkeys have shown no benefits for specific aspects of learning and memory (Lacreuse and Herndon, 2003; Voytko, 2000), but benefits due to 17b­ estradiol treatment for certain measures such as visuospatial attention (Voytko, 2002). The context of studies experimentally evaluat­ ing effects of estrogens and progestins in aging animal models is usually to understand whether estrogen-containing hormone therapies impact cognition within this older cohort, so that findings might be eventually translated to understand and optimize hormone therapies given to women (e.g. see Daniel, 2006; Frick, 2009). Since the first controlled clinical evaluation showing that 17b-estradiol injections given to a 75-year-old women enhanced memory (Caldwell and Watson, 1952), there have been numerous stu­ dies showing cognitive decline after ovarian hor­ mone loss, and enhancement after treatment with various types of preparations containing estro­ gens, in menopausal women (for review, see Sherwin, 2006). Premarin, the most commonly prescribed hormone therapy given to women (Hersh et al., 2004), is conjugated equine estro­ gens (CEEs), which contains the sulphates of at least 10 estrogens, is over 50% estrone sulphate, 20–25% equilin sulphate and has only trace amounts of 17b-estradiol; after metabolism, the resulting biologically active circulating hormones are primarily estrone and, after estrone’s conversion, 17b-estradiol, as well as equilin (Bhavnani, 2003; Sitruk-Ware, 2002). It is hypothesized that these three metabolites are pri­ marily responsible for the estrogenic effects of CEE (Sitruk-Ware, 2002), although there are other estrogens and related metabolites present that could initiate effects on their own; these hor­ mones include, but are not limited to D8,9 Dehydro-estrone, dihydroequilin-17b and equile­ nin (Bhavnani, 2003; Kuhl, 2005). CEE-contain­ ing therapy improved memory via self-report (Campbell and Whitehead, 1977), case studies (Ohkura et al., 1995) and randomized

53

psychometric evaluations (Kantor et al., 1973). Yet, findings evaluating global cognitive function in the large placebo-controlled WHI Memory Study (WHIMS), conducted by the National Institutes of Health, showed an increase in prob­ able dementia risk and no effect on mild cognitive impairment in women 65 years or older taking the combination therapy CEE plus the synthetic pro­ gestin, medroxyprogesterone acetate (MPA, Shu­ maker et al., 2003). CEE alone showed a non­ significant increase in incidence of probable dementia and mild cognitive impairment (Espeland et al., 2004; Shumaker et al., 2004). An ancillary study to the WHI testing more specific cognitive functions, the WHI Study of Cognitive Aging (WHISCA), reported that CEE plus MPA therapy had a negative effect on verbal memory and a trend for positive effects on figural memory in women 65 and over free of probable dementia (Resnick et al., 2006). Most recently, the WHIMS-MRI study found that CEE use with or without MPA was associated with small but measurable atrophy in the frontal cortex and hippocampus (Resnick et al., 2009). At the pre­ sent time, the studies in women have little con­ sensus, although new studies will be crucial to the understanding and clarification of the complex effects of ovarian hormone loss and hormone therapies. Identifying the effects of the various components of hormone therapies, including detailed evaluations using basic science and sys­ tem approaches, is the optimal approach to con­ verge the many findings that appear contradictory. In fact, as new data emerge it may become clear that the cognitive effects of hor­ mone therapy are not contradictory at all. Rather, they may be dependent on numerous variables not yet taken into account in many studies. One way to further our understanding of gonadal hor­ mone effects on spatial cognition is by using ani­ mal models. For example, female animal models can provide insight into the cognitive effects of ovarian hormones, allowing evaluative changes in cognition due to ovarian hormone withdrawal and treatment, while enabling experimental con­ trol not possible in clinical research. With this in mind, we now ask, do aged animals show cognitive enhancements after treatment with

estrogens? The answer to this question is vague, ‘Yes, but …’ Indeed, effects depend on a multi­ tude of factors. It has become clear that the cog­ nitive effects of estrogens are rich with complexity, and that they have a multi-dimensional nature that we are just starting to understand. In addition to age, estrogenic effects appear to be influenced by innumerable factors including, but not limited to, timing of hormone administration relative to hor­ mone loss, dose, mode of treatment and whether progestins are given concurrently. Two of the most interesting questions driving much of the newer animal research in the field of estrogenic actions on brain health during aging have been spawned from the WHIMS findings. Women who participated in the WHIMS were between 65- and 79-years-old, and many had experienced ovarian hormone deprivation for a substantial amount of time before receiving CEE-containing treatment (Shumaker et al., 1998). This presents the intriguing question of whether the older age of the women, and/or whether the extended window of time from menopause (which occurs in the 50s, on average), impacted outcome. Data collected within the last few years indicate that whether cognitive benefits of estrogen ther­ apy are realized is influenced by the delay between OVX and hormone treatment, with limited benefits seen when there is an extended window between ovarian hormone loss and 17b­ estradiol treatment. This has been seen in the rat on tests of spatial memory, whereby 17b-estradiol replacement initiated immediately after OVX enhanced spatial memory performance in mid­ dle-aged rats, but imparted no benefit when given 5 months after OVX (Daniel et al., 2006). These behavioral findings correspond with neuro­ chemical data from both young and middle-aged rats showing that 17b-estradiol treatment given immediately after OVX increased choline acetyltransferase (ChAT) levels in the hippocampus, while this increase was not seen when initiated 5 months after OVX; it is noteworthy that this pattern was opposite of that seen in PFC (Bohacek et al., 2008). There may also be a critical window for the well-established findings that 17b-estradiol regulates dendritic spines in the hippocampus (Woolley, 2000), as a 10-week

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delay after OVX decreased the effectiveness of 17b-estradiol to increase CA1 apical spine density as compared to treatment given immediately, in young rats (McLaughlin et al., 2008). In the mon­ key, the important question of a critical window for cognitive efficacy of estrogens has not yet been directly addressed, but there is evidence that the surgically menopausal monkey is still sensitive to estrogenic effects in old age even if there is a substantial window after ovarian hormone loss. Indeed, benefits due to treatment with estrogens were seen when treatment was initiated immedi­ ately (Voytko et al., 2008; 2009) within 30 weeks (Rapp et al., 2003), or even when given 10–16 years (Lacreuse et al., 2002) after OVX, although it is noted this latter study used a within-subjects repeated measures design of placebo or estrogenic treatments, and effects were transiently expressed, somewhat limiting interpretation of this specific critical window question since there were windows of absence and exposure across the timeframes of the study. A number of studies evaluated estrogenic effects on spatial ability in middle-aged or older female rodents, most using 17b-estradiol (Bimonte-Nelson et al., 2006; Foster et al., 2003; Frick et al., 2002; Gibbs, 2000; Luine and Rodri­ guez, 1994; Markham et al., 2002; Markowska and Savonenko, 2002; Savonenko and Markowska, 2003; Talboom et al., 2008; Ziegler and Gallagher, 2005), or the synthetic or semi-synthetic estradiol preparations ethinyl estradiol or estradiol cypio­ nate, in monkeys (Lacreuse et al., 2002; Rapp et al., 2003). In general, studies indicate that 17b-estradiol treatment can enhance performance on cognitive tests when treatment is initiated dur­ ing middle-age or in old age, as seen in rodents (Aenlle et al., 2009; Foster et al., 2003; Frick et al., 2002; Gibbs, 2000; Markham et al., 2002; Markowska and Savonenko, 2002; Talboom et al., 2008) and non-human primates (Lacreuse et al., 2002; Rapp et al., 2003; Voytko et al., 2008), although the effect may be transient in monkeys for visual recognition, with benefits seen 12 weeks following treatment initiation, but not another 12 weeks later when tested 24 weeks after treatment initiation (Voytko et al., 2008). A similar transient effect of ethinyl estradiol

treatment was seen in surgically menopausal aged monkeys for spatial delayed recognition, a test that shows hippocampal lesion (Beason-Held et al., 1999) and age-related (Moss et al., 1997) impairments, with benefits seen for months 2–4, but not 6–8, of the study (Lacreuse et al., 2002). There is also new evidence from our laboratory that both tonic and cyclic CEE, at doses relevant to what women take as hormone therapy, can enhance spatial memory and retention, and pro­ tect against cholinergic challenge on spatial tasks in middle-aged OVX rats (Acosta et al., 2009b; Engler-Chiurazzi et al., in press). Age-related changes in responsiveness to treat­ ment with estrogens have been shown for spatial cognition in studies directly testing multiple ages after 17b-estradiol as compared to vehicle control treatment. Aged OVX rats were not responsive to the 17b-estradiol treatment regimen that effec­ tively enhanced spatial reference memory in young and middle-aged OVX rats (Talboom et al., 2008), concurring with age-related interac­ tions with 17b-estradiol replacement for spatial memory shown by others, described below (Foster et al., 2003). Why, then, have some studies shown that aged female rodents can exhibit cognitive enhancements in response to 17b-estradiol treat­ ment? For example, 17b-estradiol injections enhanced spatial reference memory in 27–28­ month-old ovary-intact mice (Frick et al., 2002). The difference in results may relate to type of 17b-estradiol administration as cyclic versus tonic, as priming with cyclic 17b-estradiol­ enhanced responsiveness to tonic 17b-estradiol in older OVX rats (Markowska and Savonenko, 2002). Whether 17b-estradiol replacement improves performance of aged animals may also relate to memory type and demand. Working memory enhancements due to tonic 17b-estradiol treatment have been reported in aged rats, although this effect is more pronounced when memory demand is high (Gibbs, 2000; Luine and Rodriguez, 1994). We and others have shown 17b­ estradiol-induced spatial working memory improvements in young OVX rats as well, an effect most pronounced when spatial working memory demand is high (Bimonte and Denenberg, 1999), which depends on

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administered 17b-estradiol dose (Bimonte and Denenberg, 1999; Daniel et al., 1997; Holmes et al., 2002; Sandstrom and Williams, 2001). Further, a higher supraphysiological 17b-estradiol dose may be necessary to enhance spatial refer­ ence memory retention in rats approaching old age (Foster et al., 2003). These findings corre­ spond with data showing that higher serum levels of exogenous 17b-estradiol treatment correlates with better spatial reference memory performance in young and middle-aged OVX rats (Talboom et al., 2008). It also appears that sensitivity and responsiveness to ovarian hormone loss does not predict sensitivity and responsiveness to 17b-estra­ diol treatment. Indeed, Talboom et al. (2008) found that young animals were responsive to both ovarian hormone removal and replacement, middle-aged animals were not responsive to ovar­ ian hormone removal but were responsive to estrogen replacement and aged animals were not responsive to ovarian hormone removal or repla­ cement for the test trials for spatial reference memory. Estrogens’ effects on cognition may be impacted by the presence of progesterone, sug­ gesting, again, that gonadal hormone effects on cognition are multi-dimensional and complex. Dis­ cussion regarding, and research testing, the com­ bined regimen of estrogens plus progestins has increased as of late, mostly due to the findings that menopausal women taking CEE alone did not differ significantly from those taking placebo for dementia diagnoses (Shumaker et al., 2004), while, in contrast, twice as many women receiving CEE plus the synthetic progestin MPA were diag­ nosed with dementia as compared to the placebo group, a significant effect (Shumaker et al., 2003). Of note, women with a uterus that are taking estrogens must include progestin in their regimen because of increased risk of endometrial hyper­ plasia associated with unopposed estrogen treat­ ment (Smith et al., 1975). We have shown that progesterone abolished the 17b-estradiol-induced benefits on the spatial reference memory Morris maze in middle-aged rats (Bimonte-Nelson et al., 2006). Accordingly, progesterone plus 17b-estra­ diol injections impaired performance on the spa­ tial reference memory Morris water maze, while

17b-estradiol or progesterone treatment alone did not influence performance, and this pattern of effects was not seen on the non-spatial Morris maze suggesting this combination treatment has specific effects within the spatial domain (Chesler and Juraska, 2000). These effects may not trans­ late to working memory tasks, however, as pro­ gesterone treatment enhanced 17b-estradiol’s effects on a delayed-match-to-position spatial T-maze (Gibbs, 2000). Progesterone alone has been associated with detrimental cognitive effects, in both clinical and preclinical studies. The ‘maternal amnesia’ phe­ nomenon in pregnant women is hypothesized to result from high circulating progesterone levels during late pregnancy (Brett and Baxendale, 2001). In healthy women, a large oral progester­ one dose is detrimental to memory (Freeman et al., 1992). High circulating progesterone levels are also observed in most rats following estro­ pause (Lu et al., 1979), and many endocrine stu­ dies have shown that aged female rats enter a pseudopregnant estropause state, whereby pro­ gesterone values become significantly elevated but estradiol levels remain relatively unchanged (Huang et al., 1978; Wise and Ratner, 1980). This pseudopregnant state has in fact been associated with poorer spatial cognition (Warren and Juraska, 2000). Correspondingly, one study has shown that in young, cycling rats spatial maze performance was worse during the proestrus phase, when estrogen and progesterone levels are at their highest, and best during the estrus phase, when estrogen and progesterone are at their lowest (Warren and Juraska, 1997) (but see Berry et al., 1997; Stackman et al., 1997). OVX in aged rats improves cognition (Bimonte-Nelson et al., 2003b), which is likely related to progester­ one removal, since progesterone administration has large detriments on spatial working and refer­ ence memory, reversing the beneficial effects of OVX (Bimonte-Nelson et al., 2004). This effect was recently found in our laboratory using the synthetic progestin MPA, contained in the com­ monly used hormone therapy Prempro, as well. MPA impaired spatial memory retention and exa­ cerbated overnight forgetting on a spatial task (Braden et al., 2010). Noting the same pattern

56

seen with detrimental effects of aging, and bene­ ficial effects of 17b-estradiol treatment, progester­ one and MPA supplementation had the most marked performance effects on the water radialarm maze at the highest working memory load, with progestin-treated aged OVX rats showing disproportional impairments as working memory load reached its highest demand (Bimonte-Nelson et al., 2004; Braden et al., 2010).

HPG axis and spatial learning and memory: testosterone Similar to the postulated relationship between estrogen loss and memory decline in aging women (Sherwin, 1988), recent studies suggest that a decline in testosterone levels is related to age-asso­ ciated memory changes in men (Tan, 2001). In men, testosterone levels decline slowly with age and to only about 40% lower than levels seen in younger men, as compared to the more drastic loss of estro­ gens and progesterone seen in women after meno­ pause (Davidson et al., 1983; Hijazi and Cunningham, 2005). One of the most striking rela­ tionships regarding this research area was discov­ ered in the last decade, with reports that lower testosterone levels are linked with a higher risk of AD (Hogervorst et al., 2001; Moffat et al., 2002, 2004; Rosario et al., 2004), and that, for example, lower serum testosterone levels are seen in male AD patients versus controls (Hogervorst et al., 2001). This work spawned many hypothesis-driven stu­ dies targeting the question of whether testoster­ one enhances cognition in AD patients or in individuals with normal age-associated memory impairment. Relationships between endogenous testoster­ one levels and cognition have been observed in younger and older individuals, with, in general, the strongest relationships seen in the older popu­ lation in retrospective and randomized treatment studies. Testosterone was related to cognitive per­ formance in young men and women, and spatial ability was related to the change in seasons in accordance with seasonal alterations in testoster­ one levels (Kimura and Hampson, 1994; Neave et al., 1999; Silverman et al., 1999). Retrospective

studies have suggested there is a relationship between a greater age-related cognitive decline and lower bioavailable testosterone levels (Barrett-Connor et al., 1999; Moffat et al., 2002; Yaffe et al., 2002). In general, in older men, higher endogenous testosterone levels have been linked to better cognitive function, while an inverted Ushaped dose–response relationship between circu­ lating testosterone and cognition has also been noted, with most beneficial effects with moderate circulating testosterone levels (Barrett-Connor et al., 1999; Gouchie and Kimura, 1991; Yaffe et al., 2002). One of the most convincing pieces of evidence that testosterone effects on cognition hold to an inverted U-shaped function for older men was recently reported. This randomized, pla­ cebo-controlled study evaluating healthy older men found that weekly testosterone injections resulting in moderate increases in serum testoster­ one or its metabolites yielded enhanced spatial and verbal memory, while the testosterone injec­ tions resulting in relatively smaller, or relatively larger, testosterone increases yielded no signifi­ cant change (Cherrier et al., 2007). Of note, the moderate increases due to the testosterone treat­ ment that enhanced memory resulted in circulat­ ing levels that were normal to high normal levels seen in young men, and the large increases due to the testosterone treatment that did not enhance memory were pushed into the supraphysiological range. Work evaluating endogenous levels also suggest a non-linear U-shaped function for testos­ terone levels and spatial ability with moderate levels optimal, as lower relative levels of salivary testosterone in men, and higher relative levels of salivary testosterone in women, were related to the highest performance scores for spatial ability (Gouchie and Kimura, 1991). It is possible that accumulating data will continue to support this inverted U-shaped quadratic relationship, and will help to synthesize the range of findings includ­ ing, for example, newer work showing that agerelated declines in endogenous testosterone levels did not directly correlate with age-related cogni­ tive declines in spatial abilities, such as that seen on the mental rotation test (Martin et al., 2008). As well, the first study testing the effects of testosterone supplementation in monkeys was

57

published this year, and found that supraphysiolo­ gical testosterone levels did not yield benefits on spatial cognition in young male monkeys; whether lower physiological levels within the postulated optimal range of the inverted U-function would have yielded benefits is yet to determined (Lacreuse et al., 2009). Placebo-controlled testosterone treatment stu­ dies have reported enhanced spatial cognition in healthy older men (Cherrier et al., 2001, 2007; Gray et al., 2005; Janowsky et al., 1994), an effect not seen in younger men (Bhasin et al., 2001). Men with AD or mild cognitive impairment who received testosterone supplementation showed improved spatial cognition (Cherrier et al., 2005; Tan and Pu, 2003), although benefits of testoster­ one replacement were not seen in a pilot study evaluating old men with early- to mild-cognitive impairment and pre-treatment low levels of bioa­ vailable testosterone (Kenny et al., 2004). Since testosterone can be converted to either dihydrotestosterone (DHT), which binds to androgen receptors, or to estrogen via the aroma­ tase enzyme, testosterone’s mnemonic effects could be due to either DHT or estrogen (Becker, 1995). In fact, 80% of circulating estradiol is not of testicular origin in men; it is from aromatization of testosterone occurring in the periphery or brain areas, including the hippocampus (Becker, 1995; Naftolin, 1994). However, recently, an elegant study showed that the spatial memory benefits of testosterone were seen in men even when aroma­ tization to 17b-estradiol was pharmacologically blocked, and blood levels confirmed that the tes­ tosterone plus aromatase inhibitor reduced 17b­ estradiol levels by 50%, thereby indicating the testosterone-induced spatial memory improve­ ments occurred in the absence of concomitant 17b-estradiol increases (Cherrier et al., 2005). However, this does not preclude interactions between testosterone and estrogens for spatial cognitive performance. We have shown that tes­ tosterone supplementation given to gonad-intact aged male rats enhanced learning on a spatial working and reference memory task, and improved the ability to handle an increasing spa­ tial working memory load (Bimonte-Nelson et al., 2003a). These effects may be due to an interaction

between estrogens and testosterone; DHT, which is not converted to estrogens, had no effect on spatial maze scores in aged male rats (BimonteNelson et al., 2003a). Accordingly, others have shown enhanced spatial memory retention after testosterone, but not DHT, treatment in aged mice (Benice and Raber, 2009). Of note, in the Bimonte-Nelson et al. (2003a) study, the vehicletreated gonad-intact aged sham rats exhibited elevated serum 17b-estradiol levels and showed compromised maze performance. Conversely, testosterone treatment decreased serum 17b­ estradiol levels and improved maze performance. These combined findings suggest that testosterone did not initiate its effects by increasing serum 17b-estradiol levels, and that the group of aged rats that exhibited the best performance, the tes­ tosterone-treated group, showed relatively higher testosterone and lower 17b-estradiol circulating levels. Similarly, in older men, better cognitive performance was related to higher testosterone and lower 17b-estradiol levels (Barrett-Connor et al., 1999) and testosterone treatment improved spatial cognition in older men, while at the same time it decreased 17b-estradiol levels (Janowsky et al., 1994). It is clear that testosterone can impact spatial cognition, and that these effects may involve estrogenic interactions.

HPG axis and spatial learning and memory: gonadotrophins While it is well established that the gonadotropins follicle stimulating hormone (FSH) and lutenizing hormone (LH) are involved in regulating repro­ ductive functions via negative and positive feed­ back loops, it is becoming increasingly apparent that gonadotropins might, directly or indirectly, impact cognitive function as well, including within the spatial domain. Although there have been few links between FSH and cognition (e.g. Acosta et al., 2009a; Luetters et al., 2007), there is strong evidence that LH is related to cognition, with perhaps the strongest evidence from the neurode­ generative disease literature (Webber et al., 2007). Supporting plausibility of LH effects on the brain and spatial cognition, the highest density of LH

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receptors in the brain are found in the hippocam­ pus (Lei et al., 1993; Zhang et al., 1999), a region intimately involved in spatial learning and mem­ ory and affected by aging and AD. Further, LH can cross the blood–brain barrier (Lukacs et al., 1995). In a recent study by our laboratory evaluating VCD-induced follicular depletion and OVX effects on cognition in the middle-aged rat model, there was a very clear inverted U-shaped function for serum LH and number of spatial memory errors, with highest and lowest levels associated with the best performance, an effect not seen with FSH (Acosta et al., 2009a). This relationship with LH became apparent in scatterplots including all treatment groups so that the range of values across groups could be noted. Indeed, this range was broad, because, as expected, OVX increased LH levels due to a lack of ovarian hormone negative feedback after ovarian hormone loss, while Sham Control ani­ mals showed LH values in the relatively lower range. When LH levels ranged from ~0 to 2 ng/ ml, higher LH levels correlated with worse maze performance to reveal a positive relationship with errors. However, when LH levels ranged from ~2 to 10 ng/ml, higher LH levels correlated with bet­ ter maze performance to reveal a negative rela­ tionship with errors. When the groups were put in the same scatterplot the effect was a striking inverted U-shaped function (Fig. 7). This pattern was seen for multiple measures, including spatial working and reference memory. While limitations exist in interpreting this relationship between LH and memory scores in this study because LH levels were confounded by group membership, this quadratic relationship is nonetheless striking, especially given the increasing evidence that LH levels are linked to cognition and pathologies associated with neurodegenerative disorders (Webber et al., 2007). Other studies report higher LH levels are related to better cognitive perfor­ mance, similar to the effects seen in our OVX animals. Tonic treatment with LH-releasing hor­ mone, elevating LH concentrations to OVX levels, enhanced performance on visual discrimi­ nation in young rats (Nauton et al., 1992) and enhanced non-spatial working memory in aged

rats (Alliot et al., 1993). That higher LH levels were associated with better memory in these stu­ dies is likely related to LH levels being increased to that of OVX animals. On the other hand, cor­ responding with the Acosta et al. (2009a) findings in ovary-intact animals that higher LH levels correlated with worse cognitive performance, in ovary-intact aged female mice, experimentally induced LH reductions decreased amyloid-b concentrations and enhanced cognition, while LH increases promoted biochemical brain changes consistent with AD, although none of these studies correlated circulated LH levels with memory scores in individual animals (Bowen et al., 2004; Casadesus et al., 2007; 2006). Also, men and women with AD had higher circulating LH levels than controls (Bowen et al., 2004; Short et al., 2001). In sum, over the last decade, there is increasing evidence that LH levels are related to cognition and possibly pathologies associated with neurodegenerative diseases such s AD. The grow­ ing literature evaluating the relationship between LH and cognition suggests that it may subserve an inverted U-shaped function, with an intermediate level resulting in optimal brain function. This is an important area that will require further study, with results possibly revealing important media­ tors of cognitive function.

Summary and conclusion Cognitive function is multi-dimensional and com­ plex, with hippocampal-dependent spatial mem­ ory being particularly susceptible to age-related decrements, especially when task demand or load is elevated. The presence of age-related memory changes are some of the most consistent seen in the human and animal literature, although there is variability in age-related decrements across indi­ viduals, with the boundaries between normal and pathological age-associated impairments just beginning to be defined. The neurobiology under­ lying age-related spatial memory deficits are not likely caused by neuronal loss, although age increases the risk for brain damage attributed to AD and other disorders. Age-related spatial mem­ ory decrements most likely engage potentially

Distance swum on the spatial reference memory morris maze (higher distance scores reflects poorer performance)

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Fig. 7. Findings from a study assessing the cognitive effects of two experimentally induced forms of ovarian hormone loss, VCD-induced follicular depletion and surgical menopause (OVX), in the middle-aged rat model (Acosta et al., 2009a). There was an inverted U-shaped function for serum LH and spatial memory, with the highest and lowest LH levels associated with the best performance. Regression analysis indicated that in rats without ovaries (OVX and VCD followed by OVX), higher LH was associated with better spatial reference memory as tested on the Morris maze (A, with less distance swum associated with better performance), and the water radial-arm maze (B, with fewer errors associated with better performance). In rats with ovaries (SHAM and VCD), higher LH was associated with worse spatial working memory as tested on the water radial-arm maze (C). Graphically, the significant regressions for these analyses are shown as solid lines; the dashed lines of the other groups are shown for comparison purposes to aid interpretation. The inverted U-shaped, quadratic relationship with LH became apparent in scatterplots including all treatment groups so that the range of values across groups could be noted. When LH levels ranged from ~0 to 2, the relationship was positive with higher LH levels associated with worse maze performance. When LH levels ranged from ~2 to 10, the relationship was negative with higher LH levels associated with better maze performance. It is noteworthy that this pattern was seen for multiple domains of spatial memory, including both spatial working and spatial reference memory.

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reversible neoplastic changes. Morphological changes could include dendritic reorganization and alterations in spine density and/or even spine shape, which could have far-reaching effects on neuronal function without obvious cell loss. Hormones from both the HPA and the HPG axes can substantially alter spatial ability. Chronic stress, via elevated glucocorticoids, attenuates hippocam­ pal plasticity by causing substantial hippocampal CA3 dendritic retraction, which is proposed to impair spatial memory and increase hippocampal vulnerability to metabolic challenges in the young individual. In the aged individual, chronic stressinduced CA3 dendritic retraction is hypothesized to become more severe or slower to recover than in the young adult. Consequently, the inverted U-shaped relationship between glucocorticoids and spatial ability described for the young may be narrowed following chronic stress, which may also represent the process that occurs during aging. Moreover, the window by which opportunistic chal­ lenges can potentially trigger hippocampal neuronal death may be broadened in the aged because of the relative slow rate at which the neoplastic changes may recover from chronic stress. Gonadal hormones are potent modulators of brain structure and function, including in brain regions well documented to modulate spatial learn­ ing and memory. Many of these cognitive brain regions are affected by aging. The gonadal hor­ mones estrogens, progesterone and testosterone each impact spatial learning and memory, and there are some indications that aging alters respon­ sivity or sensitivity to these hormones for spatial cognition and postulated underlying neurobiological mechanisms. We propose a tertiary model repre­ senting interactions between aging, spatial learning and memory and gonadal hormones given that: (1) gonadal hormones change with age, (2) aging impacts spatial learning and memory and (3) gona­ dal hormones impact spatial learning and memory. While these individual tenets are driven and sup­ ported by empirical data, a fundamental question in aging research regarding interactions of these three points has yet to be clearly answered. It is, however, clear that the lifetime experiences, exposures and differences in hormonal milieu from the HPA and HPG axes across individuals create an aged

population that is heterogeneous with diverse cog­ nitive and brain aging outcomes. This tremendous brain plasticity can be interpreted within a frame­ work of allowing one the ability to ‘optimize their aging’. By aligning scientific discoveries with clinical interpretations, we can maximize opportunities for interventions so that individuals can optimize their potential for brain health as aging ensues, even in the context of lifetime experiences and stressor exposures.

Acknowledgements The authors gratefully acknowledge the following individuals for their constructive feedback: Jazmin Acosta, Blair Braden, Elizabeth Engler-Chiurazzi and Joshua Talboom.

Abbreviations AD ADX CEE ChAT DHT fMRI FSH GDX HPA HPG LH LTP MPA MRI OVX PBP PFC VCD WHI WHIMS WHISCA

Alzheimer’s disease adrenalectomy conjugated equine estrogen choline acetyltransferase dihydrotestosterone functional magnetic resonance imaging follicle stimulating hormone gonadectomy hypothalamic–pituitary– adrenal hypothalamic–pituitary– gonadal lutenizing hormone long-term potentiation medroxyprogesterone acetate magnetic resonance imaging ovariectomy primed burst potentiation prefrontal cortex 4-vinylcyclohexene diepoxide Women’s Health Initiative Women’s Health Initiative Memory Study Women’s Health Initiative Study of Cognitive Aging

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L. Martini (Eds.)

Progress in Brain Research, Vol. 182

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 3

Menopause and mitochondria: Windows into Estrogen effects on Alzheimer’s disease risk and therapy Victor W. Henderson1,
and Roberta Diaz Brinton2 1

Departments of Health Research & Policy (Epidemiology) and of Neurology & Neurological Sciences, Stanford

University, Stanford, CA, USA

2 Departments of Pharmacology & Pharmaceutical Sciences, Biomedical Engineering and Neurology, University of

Southern California, Los Angeles, CA, USA

Abstract: Metabolic derangements and oxidative stress are early events in Alzheimer’s disease pathogenesis. Multi-faceted effects of estrogens include improved cerebral metabolic profile and reduced oxidative stress through actions on mitochondria, suggesting that a woman’s endogenous and exogenous estrogen exposures during midlife and in the late post-menopause might favourably influence Alzheimer risk and symptoms. This prediction finds partial support in the clinical literature. As expected, early menopause induced by oophorectomy may increase cognitive vulnerability; however, there is no clear link between age at menopause and Alzheimer risk in other settings, or between natural menopause and memory loss. Further, among older post-menopausal women, initiating estrogen-containing hormone therapy increases dementia risk and probably does not improve Alzheimer’s disease symptoms. As suggested by the ‘critical window’ or ‘healthy cell’ hypothesis, better outcomes might be expected from earlier estrogen exposures. Some observational results imply that effects of hormone therapy on Alzheimer risk are indeed modified by age at initiation, temporal proximity to menopause, or a woman’s health. However, potential methodological biases warrant caution in interpreting observational findings. Anticipated results from large, ongoing clinical trials [Early Versus Late Intervention Trial with Estradiol (ELITE), Kronos Early Estrogen Prevention Study (KEEPS)] will help settle whether midlife estrogen therapy improves midlife cognitive skills but not whether midlife estrogen exposures modify latelife Alzheimer risk. Estrogen effects on mitochondria adumbrate the potential relevance of estrogens to Alzheimer’s disease. However, laboratory models are inexact embodiments of Alzheimer pathogenesis and progression, making it difficult to surmise net effects of estrogen exposures. Research needs include better predictors of adverse cognitive outcomes, biomarkers for risks associated with hormone therapy, and tools for monitoring brain function and disease progression. Keywords: Alzheimer’s disease; estrogen; hormone therapy; memory; menopause; mitochondria


Corresponding author. Tel.: 1-605-723-5456; Fax: 1-605-725-6951; E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)82003-5

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Introduction Gonadal steroid hormones – which include estro­ gens (e.g. 17b-estradiol and estrone), androgens (e.g. testosterone and dihydrotestosterone) and progestagens (e.g. progesterone) – modulate cognitive function and non-reproductive beha­ viors in a variety of mammalian species, including humans. With respect to cognitive aging and dementia, estrogens are of greatest interest to scientists and clinical investigators, because of the striking change in estrogenic hormonal milieu associated with the menopause (Burger et al., 1999) and because estrogen-containing hormone therapy remains among the most commonly prescribed medications. Steroid hormone receptors function as intracel­ lular transcription factors. After ligand activation, they translocate to the cell nucleus, where they bind response elements on the genome to modu­ late expression of target genes. Estrogen, andro­ gen and progesterone receptors are found in human brain, where they are expressed on sub-sets of neurons and glia in topographic distri­ butions unique to each receptor and receptor sub-type. The two types of classic intranuclear recep­ tors for estrogen are estrogen receptor alpha (ERa) and estrogen receptor beta (ERb). They are encoded by different genes on sepa­ rate chromosomes and are expressed on glia and neurons in brain areas involved with cogni­ tive function. A number of ERa and ERb splice variants have been identified in human brain, which are area specific (Taylor et al., 2009) and whose expression may be modified by Alzheimer’s disease (Ishunina and Swaab, 2009). Forebrain cholinergic neurons of the nucleus basalis, believed to play important roles in memory and attention, express ERa (Shughrue et al., 2000). ERb is the predominant estrogen receptor in the neocortex, and both receptor types are expressed on pyramidal neu­ rons and dentate granule cells in the hippocam­ pus (González et al., 2007), an archicortical structure critical to memory encoding. In the mitochondria, estrogen receptors play a pivotal role in regulating energy expenditures and

protecting against oxidative stress (Brinton, 2008; Simpkins et al., 2009). As discussed below, mitochondrial actions provide a model for gauging relations among menopause, estro­ gen exposures and Alzheimer’s disease. Estro­ gen receptors are also associated with the plasma membrane, where G-protein-coupled receptors may serve to regulate intracellular signaling cascades and to mediate rapid effects that do not require genomic activation (Pross­ nitz and Maggiolini, 2009; Raz et al., 2008).

Alzheimer’s disease Dementia can be defined as a decline in cogni­ tive skills that substantially interferes with occupational activities, social activities or inter­ personal relationships. Decline affects more than a single cognitive domain, usually memory plus at least one other area of mental function­ ing. An estimated 24 million people have dementia, and this number is expected to double over the next 20 years (Ferri et al., 2005). Alzheimer’s disease, by far the most common cause of dementia occurring later in life, affects an estimated 2.5–4.5 million older Americans (Hebert et al., 2003; Plassman et al., 2007). More women than men have Alzheimer’s disease, in part because there are more women than men in the oldest segment of the popula­ tion. Studies from Europe (Launer et al., 1999) but not from the United States (Edland et al., 2002) suggest that Alzheimer incidence is increased among women compared to men. Cognitive decline in Alzheimer’s disease begins insidiously and worsens gradually over a period of a decade or more. The earliest manifestation is usually impairment in episodic memory (Small et al., 2000). This form of memory is reflected in one’s ability to learn information and then to recall this information after some interval of time, be it minutes, hours or days. Recollection is explicit (conscious) rather than implicit (without conscious awareness). A decline in episodic memory occurs in dementing disorders other than Alzheimer’s disease, but deficits are less often an early

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or prominent feature. Over time, many Alzheimer patients show behavioral symptoms such as apathy or depression, and they inevitably evince deficits in other cognitive domains. Pathological features of Alzheimer’s disease include neurofibrillary tangles and neuritic pla­ ques. Tangles are intraneuronal inclusions formed of paired helical filaments. These in turn are com­ posed largely of a hyperphosphorylated form of tau, a microtubule-associated protein. Plaques, which are extracellular structures, typically consist of a core of b-amyloid protein, dystrophic nerve processes and activated microglia. Astrocytes are distributed circumferentially around this core. Plaques are associated with a robust chronic inflammatory response (Schwab and McGeer, 2008). Soluble b-amyloid oligomers are neurotoxic (De Felicea et al., 2008), but amyloid in plaque cores is sequestered in an inert b-pleated sheet configuration. Gross cerebral atrophy becomes apparent dur­ ing the course of Alzheimer’s disease. Atrophy is preceded by regional metabolic decline, as demonstrated by positron emission tomography (PET) imaging of the resting brain using a radio­ labeled glucose analogue, 18F-fluorodeoxyglucose (FDG). As revealed by FDG–PET, metabolism in Alzheimer’s disease is typically reduced in neocor­ tical association areas of the parietal and temporal lobes, hippocampus and cortex of the posterior cingulate gyrus (Mosconi et al., 2008).

Estrogens and the brain During a woman’s reproductive years, estrogens (predominately b-estradiol, but also estrone) and progesterone are produced cyclically by develop­ ing ovarian follicles. Menstrual irregularity and fluctuating hormone levels begin on an average about 2 years before the final menstrual period, which is the defining event of the natural meno­ pause. Mean levels of estradiol and estrone fall during this transitional stage, reaching a nadir about 2 years after the final menstrual period (Burger et al., 1999). A distinction is sometimes made between neu­ rosteroids (steroid hormones synthesized within

the central nervous system) and neuroactive steroids (steroid hormones that affect neuronal function independently of origin) (Baulieu, 1997). Estradiol, although clearly neuroactive, was originally not classified as a neurosteroid. It was believed that the central nervous system estrogens were derived solely from steroids pro­ duced in peripheral tissues and circulated to the brain. More recently, however, it has been appre­ ciated that some neurons synthesize estradiol directly from cholesterol, and there may be local effects on synaptic plasticity and other neuronal functions (Hojo et al., 2008). Progesterone is also a neurosteroid (Baulieu, 1997). Thus, effects of menopause on brain activities modulated by ovar­ ian steroids may be less precipitous or calamitous than sometimes envisioned.

Effects on metabolism Metabolic derangements in Alzheimer’s disease brain are evident on FDG-PET images long before the onset of cognitive impairment (Mosconi et al., 2009). Estrogen effects on mitochondrial function seem particularly germane to this reduc­ tion. Energy demands of the healthy brain are extraordinarily high. The brain represents only about 2% of body weight yet consumes 20% of body energy. Energy production is a key function of mitochondria. An outer membrane encloses an intermembrane space, and a folded inner membrane surrounds the mitochondrial matrix (Kroemer and Reed, 2000). These organelles are found in the neuron soma, dendrites, axons and nerve terminals. The density of these orga­ nelles varies within compartments of a single neuron and among different neuronal types (Dubinsky, 2009). In addition to their role in energy generation, mitochondria are also involved in free radical formation, protection against oxidative stress and the determination of cell death through apoptosis. Under normal circumstances, glucose is the near-obligatory substrate for energy required to maintain ionic gradients across cell membranes, to synthesize neurotransmitters and to fuel other metabolic activities in the brain. For each mole

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of glucose oxidized to carbon dioxide and water, about 30 moles of adenosine triphosphate (ATP) are ultimately generated during a set of sequential reactions in the cell cytoplasm and mitochondria (Rich, 2003). As the initial step, blood-borne glucose crosses the blood–brain endothelial bar­ rier to enter astrocytes and neurons. This process of facilitated transport, which is mediated by specific glucose transporters and associated with an increase in insulin growth factor-1 expression, is modulated by estradiol (Cheng et al., 2001; Shi and Simpkins, 1997). Within the cytosol, glucose is converted to pyruvate in the familiar biochemical sequence known as glycolysis. Pyruvate is then decarboxylated, yielding acetyl-coenzyme A, which enters the Krebs tricarboxylic acid cycle. Tricarboxylic acid cycle reactions take place in the matrix of the mitochondrion. Most ATP is generated through the associated process of oxi­ dative phosphorylation coupled to an electron transport chain, whose enzyme complexes are embedded within the inner membrane of the mitochondrion. ERb is found within mitochondrial matrix (Yang et al., 2004), and many of the genes regu­ lated by this receptor sub-type, in contradistinc­ tion to those regulated by ERa, are involved with mitochondrial electron transport and remediation of oxidative stress (O’Lone et al., 2007). Key gly­ colytic enzymes – hexokinase, phosphofructokinase and pyruvate kinase – are up-regulated by estra­ diol during the generation of pyruvate from glucose (Kostanyan and Nazaryan, 1992). Sub­ units of the pyruvate dehydrogenase complex, which regulate the generation of acetyl-coenzyme A from pyruvate, are similarly up-regulated (Nilsen et al., 2007). In addition, estradiol increases activity of complex IV sub-units in the electron transport chain and of ATP synthetase (Nilsen et al., 2007), the final steps in the oxidative phosphorylation of adenosine diphosphate to make ATP. These estrogen effects involve proteins encoded by the nuclear genome (for oxi­ dative phosphorylation) and by the mitochondrial genome (for the electron transport chain). Lasercapture micro-dissection of neurons from autopsy brains confirms under-expression of key sub-units of the electron transport chain in Alzheimer’s

disease patients compared to healthy controls (Liang et al., 2008). Reductions are particularly evident in brain regions where glucose metabo­ lism is known to be diminished in this disorder (posterior cingulum, middle temporal gyrus and the CA1 region of the hippocampus). Functional magnetic resonance imaging shows that short-term use of an estrogen can enhance regional blood flow as cognitive tasks are per­ formed (Joffe et al., 2006; Shaywitz et al., 1999). In this setting, blood flow change is thought to parallel metabolic change. In a 2-year longitudinal study of healthy post-menopausal women, region­ al blood flow was assessed with 15O-PET during performance of memory tasks (Maki and Resnick, 2000). In comparisons between long-term hor­ mone therapy users and women not currently using hormone therapy, the pattern of change in brain activation differed; differences generally reflected increased activation among hormone users in hippocampus and other temporal lobe regions involved with memory. Functional neuroimaging studies also confirm estrogen effects on glucose utilization. In one study of post-menopausal women, acute estra­ diol infusion increased FDG-PET activity in portions of the right frontal cortex and right hippocampus. Path analysis suggested that estradiol had enhanced connectivity within a pre-frontal-hippocampal circuit (Ottowitz et al., 2008). Another small study compared FDG­ PET metabolism between healthy hormone users and non-users (mean age 65 years) (Ras­ gon et al., 2005). Although there were no base­ line metabolic differences between the two groups, 2 years later women not using hor­ mones showed significant declines in FDG­ PET glucose utilization in one of two defined regions of interest (the posterior cingulate but not the lateral temporal cortex). Declines in the same regions were not significant for the hor­ mone users. Another investigative group com­ pared regional metabolism in healthy older women (including both hormone users and non-users) and women with Alzheimer’s disease (mean age about 74 years) (Eberling et al., 2000). Regional metabolic rates were lowest in the Alzheimer group, highest in the hormone user

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group and intermediate for healthy women not using hormone therapy. A follow-up report found a greater metabolic rate in the inferior frontal cortex and temporal cortex among hormone users compared to non-users (mean age 67 years) (Eber­ ling et al., 2004). Among older women with Alzheimer’s disease, spinal fluid concentrations of estradiol are reported to correlate with cerebral glucose metabolism in the left hippocampus but not other brain areas (Scho¨ nknecht et al., 2003). These data, although primarily observational and based on small sample sizes, imply that estrogen-containing hormone therapy improves brain bioenergetics among older post-menopausal women who in most instances have presumably used hormone therapy over long periods of time. Randomized clinical trial data coupled with cogni­ tive outcomes would provide more direct support for this inference, but longer term consequences of hormone therapy are more feasibly studied within an observational framework than with an experimental design.

Other effects A variety of other estrogen effects are potentially relevant to Alzheimer pathogenesis. Mitochon­ drial enzymes within the tricarboxylic acid cycle and the electron transport chain are capable of transferring electrons to oxygen, generating super­ oxide anions. During the vital process of oxidative phosphorylation, reactive oxygen species are thus generated as a by-product of ATP formation. They arise from enzymatic reactions in the mitochondrial outer membrane, inner membrane and matrix. These compounds damage cellular proteins, lipids and nucleic acids. Metabolically active tissues, such as neural tissues, are more vulnerable to oxidative stress. Indeed, oxidative stress is an early event in Alzheimer pathogenesis (Nunomura et al., 2001). Mitochondria are increasingly recognized as key determinants of cell survival or death. A num­ ber of mitochondrial systems are involved in detoxifying reactive oxygen species (Andreyev et al., 2005), and estradiol can reduce free radical formation (Nilsen et al., 2007). Anti-oxidant

effects of estrogens are well documented in sev­ eral model systems (Dykens et al., 2003; Moos­ mann and Behl, 1999). Programmed cell death, or apoptosis, is activated by a wide range of signals and can be initiated through an intrinsic pathway involving release of cytochrome c and other pro­ teins from the mitochondrial membrane space (Kroemer and Reed, 2000). Estradiol increases expression of B-cell lymphoma (Bcl) anti-apopto­ tic proteins Bcl-2 and Bcl-XL (Garcia-Segura et al., 1998; Nilsen and Diaz Brinton, 2003; Pike, 1999), located mainly in the outer membrane, rendering neurons less vulnerable to apoptosis. Calcium sequestration within mitochondria in response to estradiol reduces neuronal vulnerabil­ ity to glutamate excitotoxicity (Brewer et al., 2006; Nilsen and Diaz Brinton, 2003), another potential trigger for apoptosis. Anti-inflammatory actions of estrogens (Pozzi et al., 2006) would be expected to reduce oxidative stress, although estrogen actions are pro-inflammatory as well as anti-inflammatory (Zegura et al., 2003; Hu et al., 2006). Mitochondrial dysfunction is associated with accelerated formation of b-amyloid (Busciglio et al., 2002), although the mechanism is not known. b-Amyloid also contributes to mitochon­ drial toxicity (Lustbader et al., 2004). It is recog­ nized that estradiol speeds up trafficking of the amyloid precursor protein within the Golgi apparatus, reducing the generation of b-amyloid from its precursor (Greenfield et al., 2002). Ovariectomy leads to accumulation of b-amyloid in brains of wild-type laboratory animals (Beach, 2008; Petanceska et al., 2000) and trans­ genic mice that express features of Alzheimer’s disease (Carroll et al., 2007; Zheng et al., 2002). Concentrations of b-amyloid are lowered by treatment with estradiol. Estrogens modulate several neurotransmitter systems. Effects on cholinergic neurons are par­ ticularly relevant to memory and dementia. Magnocellular cholinergic neurons of the basal forebrain nuclei project widely to the hippocam­ pus and neocortex. These neurons express estrogen receptors (Shughrue et al., 1998). They are also selectively vulnerable to neurofi­ brillary tangle formation during the course of Alzheimer’s disease (Rasool et al., 1986) in a

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manner correlated with tangle density in other brain areas (Samuel et al., 1991). In the labora­ tory, estradiol administration after ovariectomy elevates choline acetyltransferase activity, a mar­ ker of acetylcholine synaptic activity, in the basal forebrain and in cortical projection areas (Luine, 1985; Ping et al., 2008; Yamamoto et al., 2007). Estrogen actions on basal forebrain cholinergic neurons mediate physiological effects of estro­ gen on the hippocampus (Rudick et al., 2003) and on performance enhancement on certain kinds of learning tasks (Gibbs, 2002; Gibbs et al., 2009; Markowska and Savonenko, 2002).

Implications for Alzheimer’s disease Given multi-faceted effects of estrogen – includ­ ing improved metabolic profile, lower oxidative stress, reduced b-amyloid formation and enhanced cholinergic transmission – endogenous estrogen and exogenous exposures in the form of estrogen-containing hormone therapy would be expected to influence Alzheimer pathogenesis and symptoms. Predictions include the following: Early menopause should be attended by greater risk of Alzheimer’s disease later in life; estrogen therapy should improve symptoms of Alzheimer’s disease; and hormone therapy should reduce Alzheimer risk. Because episodic memory impairment is a recognized risk factor for Alzheimer’s disease (Elias et al., 2000), it might also be predicted that natural menopause would be attended by memory decline and that hormone therapy would benefit memory perfor­ mance in women without dementia. As consid­ ered below, some of these predictions find support in the clinical data, but others are not supported at all.

Early menopause and cognitive risk In the following discussion, early menopause is used in a general way to describe menopause occurring before the mean age of natural menopause, about 51 years. Little research regarding cognitive out­ comes has included women with premature

menopause, usually defined as menopause occur­ ring before 40 years of age. Statements on early menopause or statements regarding younger age of hormone therapy initiation should, therefore, not be generalized to women with premature menopause. If exposures to endogenous estrogens reduce the risk of developing Alzheimer’s disease, then early menopause should be associated with elevated risk. There is partial support for this prediction. The largest group of women undergoing early meno­ pause are those whose menopause is induced by oophorectomy. By definition, surgical menopause occurs prior to when natural menopause would have otherwise occurred. In a large case–control study from Olmstead County, Minnesota, oophor­ ectomy was associated with elevated risk later in life of cognitive impairment or dementia (relative risk 1.5, 95% confidence interval 1.1–1.9) (Rocca et al., 2007). Risk in this study increased with younger age at the time of surgery. When compared to risks of women not undergoing surgical menopause, bilat­ eral oophorectomy before age 43 years was asso­ ciated with a relative risk of 1.7, between the ages of 43 and 48 with the same risk (namely 1.7), and after age 48 years with a relative risk (1.1) similar to that of the reference group. Early menopause is also associated with increased Alzheimer’s disease risk in women with Down’s syndrome, a chromosomal disorder where pathological features of Alzheimer’s disease appear at an unusually early age. In a community-based sample of women aged 40–60 years, the risk of Alzheimer’s disease for women undergoing menopause before the age of 46 years was 2.7 (95% confidence interval 1.2–5.9) times that of women experiencing later menopause (Schupf et al., 2003). Similar findings are reported in Down’s syndrome cohorts from Ireland and the Nether­ lands, where early age at menopause was signifi­ cantly associated with younger age at diagnosis of dementia (Coppus et al., 2009; Cosgrave et al., 1999). The predicted association between menopause age and Alzheimer’s risk, however, is challenged by findings from several cohorts of older women, where no significant relations between age at menopause and Alzheimer risk were observed (Baldereschi et al., 1998; Paganini-Hill and

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Henderson, 1996; Roberts et al., 2006; Tang et al., 1996). In the Leisure World retirement commu­ nity, for example, when compared to women reporting menopause before age 45 years, the relative risk of Alzheimer’s disease for women undergoing menopause between ages 45 and 54 years was 1.0, and for women older than 54 years, risk was 1.2 (test for trend p = 0.6) (Paganini-Hill and Henderson, 1996).

Hormone therapy and symptoms of Alzheimer’s disease Early, very small open-labelled studies of hormone use among women with Alzheimer’s disease raised the hope that estrogen treatment might improve dementia symptoms (e.g. Fillit et al., 1986; Honjo et al., 1989; Ohkura et al., 1994). This expectation has since been assessed in randomized placebocontrolled, double-blind trials (Asthana et al., 1999, 2001; Henderson et al., 2000; Honjo et al., 1993; Mulnard et al., 2000; Rigaud et al., 2003; Wang et al., 2000; Zhang et al., 2006) (Table 1). Most blinded trials have been relatively small and of relatively short duration. Most, but not all, suggest no cognitive, functional or global benefit. In particular, the largest, longest trial reported no benefit of hormone therapy in this setting (Mulnard et al., 2000), and a recent systematic review concluded that hormone therapy is not indicated for cognitive improvement or main­ tenance in women with Alzheimer’s disease (Hogervorst et al., 2009).

Hormone therapy and risk of Alzheimer’s disease Many, but not all, case–control and cohort stu­ dies have associated hormone therapy use with lower risks of Alzheimer’s disease. Modest but significant protective associations are reported from Leisure World (Paganini-Hill and Hender­ son, 1996), northern Manhattan (Tang et al., 1996), the Baltimore Longitudinal Study of Aging (Kawas et al., 1997),and Cache County, Utah (Zandi et al., 2002). Meta-analyses suggest overall risk reductions of about a third

(Hogervorst et al., 2000). This estimate, if valid, has obvious public health implications. Disappointing clinical trial results reported from the Women’s Health Initiative Memory Study (WHIMS) (Shumaker et al., 2004) chal­ lenge a sanguine interpretation of the observa­ tional findings on hormone therapy and Alzheimer risk. WHIMS was designed as an ancil­ lary study embedded within the Women’s Health Initiative clinical trials. WHIMS eligibility was restricted to participants who were at least age 65 years. The primary outcome was incident dementia, and a total of 108 women developed dementia during the parallel WHIMS trials (Table 2). Half of the dementia cases were attrib­ uted to Alzheimer’s disease, but separate out­ comes were not reported for this diagnosis. In the estrogen–progestin trial of women with a uterus, the relative risk of dementia for women who were assigned to hormone treatment was double that of women assigned to placebo (Shumaker et al., 2003). In the estrogen-alone trial of women with prior hysterectomy, the rela­ tive risk for women assigned to estrogen was also increased, but not significantly so (Shumaker et al., 2004). Why do WHIMS findings not support observa­ tional inferences of estrogen protection against Alzheimer’s disease? A common explanation is that observational findings are methodologically flawed by difficulty to resolve biases (Henderson, 2006). Prior to hormone therapy initiation, hor­ mone users are often healthier than non-users and are more likely to engage in health-promoting behaviors (Matthews et al., 1996). These differ­ ences might reduce Alzheimer risk independently of hormone use (healthy user bias). Recall bias in some reports might also contribute to apparent benefit (Petitti et al., 2002). A second candidate for the discrepancy con­ cerns generalization of WHIMS outcomes to the broader population of women likely to consider hormone therapy (Henderson, 2006). This consid­ eration is relevant if key characteristics of WHIMS participants differed from those of women in observational studies and, equally important, if effects of hormone therapy on dementia risk are modified by these

Table 1. Randomized, double-blind, placebo-controlled trials of hormone therapy in women with Alzheimer’s disease

Reference

Age mean (years)

Menopause typea

Number

Duration

Primary cognitive outcome

Functional outcomeb

Global outcomeb

Asthana et al. (1999) Henderson et al. (2000) Mulnard et al. (2000) Wang et al. (2000) Asthana et al. (2001) Rigaud et al. (2003)e Zhang et al. (2006)f

79 78 75 72 80 76 55

Natural Both Surgical Natural Both Both Not stated

12 42 120 50 20 117 41

8 weeks 16 weeks 12 months 12 weeks 8 weeks 28 weeks 16 weeks

þEstrogenc NS NS NS þEstrogenc NS þEstrogen

– NS NS – NS NS þEstrogen

– NS NSd NS NS NS NSf

Trials with a duration of at least 1 month and an objective measure of cognitive outcome. Active treatment was with oral conjugated equine estrogens (Henderson et al., 2000; Honjo et al., 1993; Mulnard et al., 2000; Wang et al., 2000; Zhang et al., 2006), oral estradiol (Rigaud et al., 2003) or transdermal estradiol (Asthana et al., 1999; 2001). Women in Rigaud et al. (2003) randomized to estradiol also received oral progesterone. þEstrogen = significant difference in favour of active treatment with an estrogen. NS = non-significant probability p < 0.05. a Natural menopause is inferred from statements that all participants underwent Papanicolaou examinations; surgical menopause is based on hysterectomy status. b Functional outcomes assess activities of daily living; global outcomes reflect overall change. c No cognitive outcome was defined as primary; results favoured women in the estrogen group on a sub-set of cognitive tasks. d No difference on the primary global outcome (Clinical Global Impression of Change); significant difference favoured placebo on the Clinical Dementia Rating scale. e Women in both groups received a cholinesterase inhibitor. f Participants were younger than 65 years of age; dosing schedules differed for hormone (once daily) and vitamin B1 placebo (3 times daily), implying the possibility of unblinding. Betweengroup comparisons were not provided; presented data imply significant differences favouring conjugated equine estrogens for cognition (revised Hasegawa Dementia Scale) and activities of daily living but probably not for global performance (Functional Activities Questionnaire).

85 Table 2. Randomized, double-blind, placebo-controlled trials of hormone therapy in older women without dementia: dementia outcomes in the Women’s Health Initiative Memory Study

Reference Shumaker et al. (2003)

Shumaker et al. (2004)

Age range (years)

Menopause typea

Number

Duration (years)

Number of events

Hazard ratio (95%

confidence interval)

Probability

65–79

Surgical

4532

4.1

61

2.1 (1.2–3.5)

0.01

65–79

Natural

2947

5.2

47

1.5 (0.8–2.7)

0.18

Active treatment was with conjugated equine estrogens (0.625 mg/d) plus medroxyprogesterone acetate (2.5 mg/d) in a continuous combined oral formulation (Shumaker et al., 2003) or conjugated equine estrogens alone (Shumaker et al., 2004). a Surgical menopause based on hysterectomy status; in the parent Women’s Health Initiative, 41% of women with hysterectomy reported bilateral oophorectomy (Stefanick et al., 2003).

characteristics. One especially salient difference is the age when hormone therapy was initiated and used. Vasomotor symptoms are most preva­ lent near the time of menopause, and most hor­ mone therapy is prescribed to reduce these bothersome symptoms. Hormone use in observa­ tional studies thus tended to be at a younger age in closer proximity to the menopause. Hormone initiation (or re-initiation among prior hormone users) in WHIMS occurred at age 65 years or older.

Atherosclerosis as a model for the critical window hypothesis Is it reasonable to consider that effects of estrogen therapy might be modified by age, proximity to menopause (i.e. duration of ovarian hormone deprivation) or some health-related factor asso­ ciated with age? One potential mechanism for a so-called ‘critical window’ (Resnick and Hender­ son, 2002) or ‘timing’ (Clarkson and Mehaffey, 2009) effect is the down-regulation of estrogen receptors after a prolonged period of ligand depri­ vation (Toran-Allerand, 2000). Prolonged hypo­ estrogenemia might also alter the number or type of estrogen receptor, or its splice variants, leading to a different effect when estrogen expo­ sures recur. A somewhat different model for the critical window hypothesis – one tied more closely to health factors – relates to atherosclerosis, an

age-associated pathological alteration chiefly affecting walls of large elastic and muscular arteries. Atherosclerosis is an inflammatory, pro­ liferative lesion, which, when advanced, includes endothelial disruption, extracellular lipid deposits, lipid-laden macrophages, an acute-phase inflam­ matory response, smooth muscle proliferation, disruption of the intercellular matrix and collagen deposition (Stary et al., 1992). Experimental and clinical data indicate that an exogenous estrogen could help prevent atherosclerosis when adminis­ tered during one temporal window (younger age with less atherosclerosis, close to menopause) but have no effect or be deleterious when adminis­ tered during another window of time (older age with more atherosclerosis, remote from meno­ pause). In vitro, a similar phenomenon has been proposed as the ‘healthy cell bias’ hypothesis of estrogen action, suggesting that healthy neurons respond differently to an estrogen than stressed neurons (Chen et al., 2006). Genomic and non-genomic effects of estradiol on vascular endothelial and smooth muscle cells promote endothelial restoration after vascular injury and enhance vasodilation (Mendelsohn and Karas, 1999). These salubrious effects are antagonized by 27-hydroxycholesterol, a choles­ terol metabolite found in atherosclerotic lesions (Umetani et al., 2007). In animal models, estradiol can inhibit new fatty deposits without inhibiting progression of established vascular lesions (Rosenfeld et al., 2002); this protective effect may depend on an intact vascular endothelium

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(Hanke et al., 1999). In a primate model, large artery atherosclerosis is reduced when estrogens are given immediately after ovariectomy but not when treatment is delayed (Clarkson and Mehaf­ fey, 2009). Indeed, rupture of an established atherosclerotic plaque is more likely, rather than less likely, in the presence of estrogens. In humans, oral estradiol reduces progression of sub-clinical atherosclerosis in healthy post-meno­ pausal women (mean age 62 years) (Hodis et al., 2001) but has no effect on atherosclerosis progres­ sion in women of about the same age with estab­ lished coronary artery disease (Hodis et al., 2003). In the Women’s Health Initiative clinical trials, women who initiated hormone therapy closer to menopause tended to have less coronary heart dis­ ease risk compared with increased risk among women more distant from menopause (Rossouw et al., 2007). Among surgically menopausal partici­ pants in the Women’s Health Initiative, prior use of hormone therapy close to the time of bilateral oophorectomy was associated with a lower preva­ lence of coronary artery calcification, a risk factor for coronary heart disease (Allison et al., 2008).

Critical window and cognitive outcomes The atherosclerosis model might be directly rele­ vant to Alzheimer’s disease pathogenesis. Risk factors for Alzheimer’s disease and vascular disease overlap substantially (Stampfer, 2006), and cerebrovascular disease potentiates clinical manifestations of Alzheimer neuropathology (Schneider et al., 2007). However, here is no direct evidence that adverse vascular consequences of hormone therapy led to dementia in the WHIMS trials (Coker et al., 2009). Clinical evidence supporting the critical window hypothesis for cognitive outcomes remains limited. In randomly selected households of women over age 60 years, self-reported early initiation of hor­ mone therapy was associated with better perfor­ mance on some cognitive tasks, whereas initiation in late post-menopause was associated with worse performance (MacLennan et al., 2006). Follow-up

analysis of participants in studies of hormone ther­ apy for osteoporosis found that middle-age women randomly assigned to hormone treatment for 2 or 3 years were less likely to be cognitively impaired after a mean interval of 11 years than women in placebo groups (Bagger et al., 2005). Use of hor­ mone therapy at a younger age, i.e. past use but not current use, was associated with reduced Alzheimer risk in the Cache County cohort (Zandi et al., 2002). In the Multi-Institutional Research in Alzheimer Genetic Epidemiology (MIRAGE) study, hormone therapy was associated with reduced Alzheimer risk among younger, but not older, post-menopausal women (Henderson et al., 2005); hormone use necessarily occurred at a younger age among younger women. In animal models of learning, specific effects of estrogens depend on the behavioral paradigm, animal age, interval between ovariectomy and estrogen replacement and mode administration (e.g. Markowska and Savonenko, 2002; Savonenko and Markowska, 2003; Zurkovsky et al., 2007). Given these experimental complex­ ities, it is interesting that behavioral enhancement in some paradigms is observed only if an estrogen is initiated soon after ovariectomy (Daniel et al., 2006; Gibbs, 2000), as predicted by the critical window hypothesis. Menopause and episodic memory Women cannot be randomly ‘assigned’ to undergo menopause, and thus it is obvious that cognitive consequences of the natural menopause in humans cannot be studied experimentally. That is not to say that important questions cannot be rigorously addressed and reasonably answered. Further, cognitive consequences of hormone ther­ apy can be addressed experimentally in rando­ mized clinical trials, although such trials are more feasible for short-term than long-term outcomes. One emerging conclusion from cohorts of midlife women seems to be that cognitive function is not substantially impacted by the natural meno­ pause, despite pronounced changes in hormonal milieu during this midlife transition. At least, no

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important short-term decline is readily discernible. It is more difficult to know whether potential cognitive consequences might become evident only some decades later. Many women complain of forgetfulness around the time of the menopausal transition (Mitchell and Woods, 2001). Because of the association between episodic memory loss and Alzheimer’s disease, this symptom is of course worrisome. However, forgetfulness is a common symptom at other ages as well, and self-reported poor memory is often more strongly linked to low mood than to objective loss of memory per­ formance (Weber and Mapstone, 2009). Crosssectional and longitudinal findings from midlife cohorts in Australia, the United Kingdom, Taiwan, Sweden and the United States are con­ sistent in suggesting that the natural menopausal transition probably has no important effect on episodic memory or on other cognitive skills (Fuh et al., 2006; Henderson et al., 2003; Herlitz et al., 2007; Kok et al., 2006; Luetters et al., 2007). Analyses from the multi-ethnic Study of Women’s Health Across the Nation (SWAN) sample suggests a mild learning deficit during the menopausal transition compared to pre­ menopause, inferred from annual trends in prac­ tice effects (Greendale et al., 2009). However, this small reduction in practice effect was not statistically significant, and there was no reduc­ tion in practice effect when midlife women prior to entering the menopausal transition were com­ pared to women in the early post-menopause. Clinical relevance may confined to the transition per se, a time when estrogen levels are charac­ terized by large variability (Burger et al., 1999).

Hormone therapy and episodic memory Because cognitive outcomes of estrogen-contain­ ing hormone therapy could vary depending on age of initiation or use, the following discussion, which emphasizes findings from randomized clinical trials, separates hormone use by midlife women and by older post-menopausal women. Age 65 years is often taken as a convenient dividing line for this purpose.

Midlife women without dementia Two small, short-term randomized clinical trials in women with surgical menopause suggest that estradiol improves verbal episodic memory when initiated in this setting (Sherwin, 1988; Phillips and Sherwin, 1992). After natural menopause, however, randomized clinical trials in midlife women have generally not reported significant effects of hormonal treatment (reviewed by Henderson and Sherwin, 2007). Treatment durations have been relatively short and sample sizes relatively small. The largest trial involved 180 post-menopausal women aged 45–55 years randomized to conjugated equine estrogens combined with medroxypro­ gesterone acetate, or placebo. Four months of treatment provided no benefit for memory or other cognitive skills (Maki et al., 2007). Two much larger clinical trials currently in progress, the KEEPS (ClinicalTrials.gov identifier NCT00154180) and the ELITE (NCT00114517) will provide clearer evidence regarding cogni­ tive outcomes in this age group after treatment with oral estradiol (ELITE), oral conjugated equine estrogens (KEEPS) and transdermal estradiol (KEEPS). There are several possibilities for differences in study outcomes when trials after surgical menopause are compared to trials after natural menopause (Henderson and Sherwin, 2007). One is that differences are due to chance. Another possibility is reporting bias; results of a small trial with a significant outcome are more likely to be submitted and accepted for publica­ tion than findings from a small trial where between-group comparisons are not significant. An interesting possibility concerns the younger age of women in the two studies of surgical menopause (mean ages of 45 and 48 years) (Phillips and Sherwin, 1992; Sherwin, 1988), compared to women studied after natural meno­ pausal. Other considerations are the prompt initiation of treatment at the time of bilateral oophorectomy, the particular hormone formula­ tion used in these surgical menopause trials and unique physiological changes associated with surgical menopause.

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The hormone formulation is probably not key, even though biological effects of estradiol differ from those of other estrogens and biological effects of progesterone differ from those of syn­ thetic progestins (Brinton et al., 1997; Nilsen and Brinton, 2003). Although surgically menopausal women in these two trials were treated with high doses of parenteral estradiol, similar findings regarding verbal memory in younger women are reported with the addition of a standard dose of oral conjugated equine estrogens after pharmaco­ logical suppression of ovarian function (Sherwin and Tulandi, 1996). With respect to the hormone milieu, natural menopause is attended, of course, by loss of ovarian estrogens and progesterone. In addition to loss of these gonadal steroids, surgical menopause is also accompanied by reduced levels of testosterone (Davison et al., 2005). After nat­ ural menopause, testosterone is derived in part from androgen precursors produced by residual ovarian stromal cells, and hormonal losses are

thus exacerbated among surgically menopausal women. A potential modulating role for testoster­ one is plausible but remains to be explored fully.

Older women without dementia Although clinical trial findings on hormone ther­ apy are limited for middle-age women, more sub­ stantial data exist for older women (Henderson and Sherwin, 2007). Findings from larger rando­ mized trials of late post-menopausal women are summarized in Table 3. The table separates hor­ mone effects on episodic memory tasks from hor­ mone effects on other types of cognitive tasks, because of the important relation between mem­ ory decline and Alzheimer’s disease. As shown in this table, randomized assignment to hormone therapy in these trials did not notably improve episodic memory and did not show consistent effects in other areas.

Table 3. Large randomized, double-blind, placebo-controlled trials of hormone therapy in older women without dementia: cognitive outcomes

Reference

Age mean or range (years)

Menopause typea

Number

Duration

Episodic memory

Other cognitive outcomes

Grady et al. (2002)b Rapp et al. (2003)d Espeland et al. (2004)d Viscoli et al. (2005)b Almeida et al. (2006) Resnick et al. (2006)d Yaffe et al. (2006) Resnick et al. (2009)d

67 65–79 65–79 70 74 71 67 74

Both Natural Surgical Both Surgical Natural Natural Surgical

1063 4381 2808 461 115 1416 417 886

4 years 4 years 5 years 3 years 5 months 4 years 2 years 6 years

NS – – NS NS Variablee NS NS

Most NSc NS NS NS NS NS NS Most NSf

Trials with sample size of at least 100, mean age of at least 60 years, trial duration of at least 1 month and an objective measure of cognitive outcome.

Active treatment was with conjugated equine estrogens (Espeland et al., 2004; Grady et al., 2002; Rapp et al., 2003; Resnick et al., 2006, 2009), oral

estradiol (Almeida et al., 2006; Viscoli et al., 2005), or very low-dose transdermal estradiol (Yaffe et al., 2006). Some women randomized to an estrogen

also received a progestagen (medroxyprogesterone acetate) (Rapp et al., 2003; Resnick et al., 2006).

NS = non-significant probability p > 0.05.

a Surgical menopause based on hysterectomy status.

b Participants had coronary heart disease (Grady et al., 2002) or cerebrovascular disease (Viscoli et al., 2005).

c Significant difference in verbal fluency favoured the placebo group; other cognitive outcomes did not differ.

d Women’s Health Initiative Memory Study of women with (Rapp et al., 2003; Resnick et al., 2006) or without (Espeland et al., 2004; Resnick et al.,

2009) a uterus. Rapp et al. (2003) and Espeland et al. (2004) report global cognition on the Modified Mini-Mental State examination. In the Women’s Health Initiative Study of Cognitive Aging, Resnick et al. (2006, 2009) report more detailed cognitive analyses on sub-sets of women included in reports of Rapp et al. (2003) and Espeland et al. (2004). e Based on annual rates of change, significant differences on verbal memory favoured the placebo group, and significant differences on non-verbal memory favoured the hormone group. f Significant differences on a mental rotation task 3 years after treatment randomization favoured the placebo group, but thereafter the estrogen group showed greater improvement over time.

89

Inferences and conclusions It is difficult to disentangle the relation between menopause – a normal, universal midlife event for women – and Alzheimer’s disease, a common late-life disorder affecting both women and men. We begin with a physiological process character­ ized by loss of ovarian hormone production and end with a series of clinical events that are in some instances difficult to reconcile with each other and with experimental findings from the basic laboratory. This focussed review has emphasized laboratory effects of estrogens acting on and through mitochondria, recognizing that a number of relevant actions involve other biolo­ gical targets. The clinical tableau seems to be the following: Early menopause induced by oophorectomy (sur­ gical menopause) may increase cognitive vulner­ ability (Phillips and Sherwin, 1992; Rocca et al., 2007; Sherwin, 1988), but in other settings there is no clear link between menopause age and Alzhei­ mer risk (Baldereschi et al., 1998; Paganini-Hill and Henderson, 1996; Roberts et al., 2006; Tang et al., 1996). Estrogen therapy initiation probably does not improve Alzheimer’s disease symptoms (Table 1); one of the small trials reporting cogni­ tive benefit to women with Alzheimer’s disease involved only participants younger than 65 years (Zhang et al., 2006). Importantly, hormone ther­ apy initiated at an older age is linked to increased – not decreased – dementia risk (Table 2). Whether effects of hormone therapy on Alzhei­ mer risk are modified by age at initiation, or by prolonged use after early initiation, is implied by some observational results but is not answered with certainty by current evidence. WHIMS find­ ings of increased dementia risk with late life initia­ tion counsel caution in interpreting the observational data. Further, contrary to prediction, natural meno­ pause is not attended by persistent memory decline across the menopause transition, and hor­ mone therapy does not appear to boost memory performance, certainly not when initiated during the late post-menopause (Table 3). Results from the ELITE and KEEPS trials will inform us whether similar results are expected from

hormone use initiated at younger ages, and estro­ gen effects on memory after early menopause merit further study. Despite disappointing outcomes in short- and medium-term clinical trials of estrogens (Tables 1–3), other clinical data raise the possibi­ lity that long-term outcomes might differ. With respect to dementia, increasing duration of hor­ mone use by healthy women is associated with decreasing risk of Alzheimer’s disease (PaganiniHill and Henderson, 1996), although prolonged use is not associated with better cognition when initiated at older ages (Kang et al., 2004). Some observational studies imply that hormone users who develop Alzheimer’s disease experience milder symptoms than women who develop Alzheimer’s disease but are not taking hormones (Doraiswamy et al., 1997; Henderson et al., 1994). Hormone use by these patients likely began years prior to the onset of overt dementia, a situation different from that of initiating ther­ apy after the onset of cognitive impairment. Long-term hormone users, however, are often relatively healthier than never users and former users (reflecting healthy user bias and compli­ ance bias), and any inference concerning longterm estrogen use, Alzheimer symptom ameliora­ tion or other cognitive benefit is at best speculative. The important role of estrogens on brain metabolism and the relation between metabolic decline and Alzheimer’s disease risk suggest a role for FDG-PET as a surrogate marker, for example, after the randomized assignment of an estrogen to recently menopausal women. The predicted response would be an increase in resting brain metabolism, in comparison to pla­ cebo. According to the critical window or healthy cell hypothesis, no discernible response or even a reduced metabolic response would be expected in older post-menopausal women remote from the menopause. This prediction for older women will be more difficult to assess experimentally, given accumulating evidence that hormone initiation in this age group is attended by competing health risks (Anderson et al., 2004; Rossouw et al., 2002; Shumaker et al., 2004).

90

An important consideration, but still not yet well investigated, is that neuronal synthesis of estradiol and other neurosteroids might moder­ ate central nervous system consequences of menopausal loss. In a small post-mortem series, significant concentrations of estradiol were identified in brains of post-menopausal women, suggesting a role for local neuronal production (Bixo et al., 1995). In the hippocampus, neuronal metabolism after menopause may be augmented by locally synthesized estrogens (Ishunina et al., 2007). Nevertheless, regional brain estradiol levels appeared to reflect peripheral concentrations, sug­ gesting that brain levels also depend in part on ovarian production (Bixo et al., 1995). In the pre-clinical laboratory, healthy cellular and animal models acutely exposed to toxic insults provide an inexact model of human neurodegen­ eration, where the process is one of incremental damage over a period of years (Brinton, 2008). A related difficulty in translating laboratory findings to a clinical arena is that sex steroid effects are complex, affect brain functions directly as well as indirectly through actions on non-neural tissues and are almost certainly modified by a variety of health-related and age-related factors (Table 4). Net effects on Alzheimer’s disease risk or on memory performance are therefore difficult to predict. Estrogen effects on mitochondrial function are likely to decrease risk for Alzheimer’s disease. Other actions – including increased levels of

inflammatory proteins and proteins that play roles in coagulation (Katayama et al., 2009) – may affect cognition adversely, leading to out­ comes different from those anticipated on the basis of simpler in vitro experiments and in vivo models. These actions may account, for example, for increased incidence of ischaemic stroke reported in some trials of hormone therapy (Rossouw et al., 2007). Outcomes could also vary according to estrogen type (Brinton et al., 1997), dose (Chen et al., 2006), dosing schedule (Mar­ kowska and Savonenko, 2002) and route of administration (Zegura et al., 2003). Finally, in the laboratory (e.g. Rosario et al., 2006; Tanapat et al., 2005) and perhaps in the clinic (Kang et al., 2004; Rice et al., 2000), nervous system effects of an estrogen differ from those of an estrogen com­ bined with a progestagen. The challenges are daunting but not unsolvable. As suggested by participants in a National Insti­ tute on Aging workshop on estrogen and the aging female brain (Asthana et al., 2009), there remains pressing need for pre-clinical and clinical research on the relation between the menopausal transition and midlife exposures to estrogens, progestagens and related compounds, and on risks for age-asso­ ciated cognitive disorders such as Alzheimer’s dis­ ease. Research needs include better predictors of adverse cognitive outcomes, biomarkers for risks associated with hormone therapy and tools for monitoring brain function and disease progression.

Table 4. Potential factors contributing to net estrogen effects on Alzheimer’s disease Specific effects Different estrogens have different neural and non-neural effects Different progestagens have different neural and non-neural effects Class effects Wide-ranging effects on neural tissues Wide-ranging effects on non-neural tissues that affect brain function Effects modified by dose, dosing schedules (e.g. continuous or sequential) and route (e.g. oral or transdermal) Effects modified by progestagens and androgens Effects modified by age at time of hormone exposure or by timing with respect to menopause (critical window hypothesis) Effects modified by other physiological parameters (e.g. presence of atherosclerosis) (healthy cell bias hypothesis, critical window hypothesis) Menopausal hormonal losses mitigated by local effects of neurosteroids

91

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L. Martini (Eds.)

Progress in Brain Research, Vol. 182

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 4

DHEA, important source of sex steroids in men and even more in women Fernand Labrie
Research Center in Molecular Endocrinology, Oncology and Human Genomics, Laval University and

Laval University Hospital Research Center (CRCHUL), Qu�ebec, Canada

Abstract: A major achievement from 500 million years of evolution is the establishment of a high secretion rate of dehydroepiandrosterone (DHEA) by the human adrenal glands coupled with the indroduction of menopause which stops secretion of estrogens by the ovary. Cessation of estrogen secretion at menopause eliminates the risks of endometrial hyperplasia and cancer which would result from non-opposed estrogen stimulation during the post-menopausal years. In fact, from the time of menopause, DHEA becomes the exclusive and tissue-specific source of sex steroids for all tissues except the uterus. Intracrinology, a term coined in 1988, describes the local formation, action and inactivation of sex steroids from the inactive sex steroid precursor DHEA. Over the past 25 years most, if not all, the genes encoding the human steroidogenic and steroid-inactivating enzymes have been cloned and sequenced and their enzymatic activity characterized. The problem with DHEA, however, is that its secretion decreases from the age of 30 years and is already decreased, on average, by 60% at time of menopause. In addition, there is a large variability in the circulating levels of DHEA with some post-menopausal women having barely detectable serum concentrations of the steroid while others have normal values. Since there is no feedback mechanism controlling DHEA secretion within ‘normal’ values, women with low DHEA will remain with such a deficit of sex steroids for their remaining lifetime. Since there is no other significant source of sex steroids after menopause, one can reasonably believe that low DHEA is involved, in association with the aging process, in a series of medical problems classically associated with post-menopause, namely osteoporosis, muscle loss, vaginal atrophy, fat accumulation, hot flashes, skin atrophy, type 2 diabetes, memory loss, cognition loss and possibly Alzheimer’s disease. A recent randomized, placebo-controlled study has shown that all the signs and symptoms of vaginal atrophy, a classical problem recognized to be due to the hormone deficiency of menopause, can be rapidly improved or corrected by local administration of DHEA without systemic exposure to estrogens. In addition, the four domains of sexual dysfucntion are improved. For the other problems of menopause, although similar large scale, randomized and placebo-controlled studies usually remain to be performed, the available evidence already strongly suggests that they could be improved, corrected or even prevented by exogenous DHEA.
Corresponding author. Tel.: 418-652-0197; Fax: 418-651-1856; E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)82004-7

97

98

In men, the contribution of adrenal DHEA to the total androgen pool has been measured at 40% in 65–75-year-old men. Such data stress the necessity of blocking both the testicular and adrenal sources of androgens in order to achieve optimal benefits in prostate cancer therapy. On the other hand, the comparable decrease in serum DHEA levels observed in both sexes has less consequence in men who continue to receive a practically constant supply of testicular sex steroids during their whole life. In fact, in men, the appearance of hormone-deficiency symptoms common to women is observed at a later age and with a lower degree of severity. Consequently, DHEA replacement has shown much more easily measurable beneficial effects in women. Most importantly, despite the non-scientific and unfortunate availability of DHEA as a food supplement in the United States, a situation that discourages rigorous clinical trials on the crucial physiological and therapeutic role of DHEA, no serious adverse event related to DHEA has ever been reported in the world literature (thousands of subjects exposed) or in the monitoring of adverse events by the FDA (millions of subjects exposed), thus indicating, as expected from its known physiology, the excellent safety profile of DHEA. With today’s knowledge, one can reasonably suggest that DHEA offers the promise of a safe and efficient replacement therapy for the multiple problems related to hormone deficiency after menopause without the risks associated with estrogen-based or any other treatments. Keywords: DHEA; intracrinology; menopause; osteoporosis; vaginal atrophy; type 2 diabetes; Alzheimer’s disease

Introduction The unique physiological importance of DHEA in men was first recognized in the early 1980s when it was discovered that after complete medical castration achieved with gonadotropin-releasing hormone (GnRH) agonists in men suffering from prostate cancer (Labrie et al., 1980), 40–50% of active androgens were left in the prostate, thus indicating an important extratesticular source of androgens (Labrie et al., 1985). When pooling data obtained by various laboratories around the world, values of residual intraprostatic dihydrotestosterone (DHT) range from 25 to 50% for an average of 40% of DHT left in the prostate after castration (Labrie et al., 2009b). That originally surprising observation is now well explained by the transformation of DHEA into androgens and/or estrogens by specific steroidogenic enzymes in each cell type in each peripheral target tissue according to the process of intracrinology, an expression coined in 1988 (Labrie, 1991; Labrie et al., 1988; Labrie et al., 2005; Luu-The and Labrie, 2010). While the supply of sex steroids from DHEA decreases in both men and women in a comparable fashion from the age of 30 years, men receive a

practically continuous supply of testosterone, estrone (E1) and estradiol (E2) from the testicles during their whole life (Fig. 1) while, in women, E2 secretion by the ovaries stops at menopause (Fig. 2). Consequently, after menopause, all estrogens and androgens are derived from DHEA which has already decreased by an average of 60% at time of menopause (Labrie et al., 2006) and continues to decrease thereafter (Fig. 3), with some women hav­ ing barely detectable serum levels (Labrie, 2010). Since DHEA is the only source of sex steroids after menopause, it is reasonable to believe that such a decrease in DHEA-derived sex steroid availability, coupled with aging, is at least partially responsible for the numerous symptoms of hormone deficiency observed after menopause. These pertain to vaginal atrophy, bone loss, fat accumulation, type 2 diabetes, skin atrophy, cognition problems, memory loss and possibly Alzheimer’s disease (Labrie, 2007). A major problem with DHEA, the only source of sex steroids after menopause, is that there is no feedback control of its secretion. In other words, there is no endogenous mechanism in either women or men to increase DHEA secretion when serum DHEA concentrations become low. Consequently, the only possibility to correct a

99 A.

Women

B.

GnRH

GnRH CRH

Anterior pituitary

E2

Men

LH

ACTH

Cortisol

Ovary

Adrenal

Estradiol (E2)

CRH

Anterior pituitary

DHEA

Testosterone

LH

ACTH

Adrenal

Testis

Testosterone

DHEA

Intracrinology

E2 Testo DHT Peripheral target tissues

Cortisol

Intracrinology

E2

Testo

DHT

Peripheral target tissues

Fig. 1. (A) Schematic representation of the role of the ovarian and adrenal sources of sex steroids in pre-menopausal women. (B) Schematic representation of the role of testicular and adrenal sources of sex steroids in men. ACTH, adrenocorticotropin; CRH, corticotropin-releasing hormone; DHEA, dehydroepiandrosterone; DHT, dihydrotestosterone; E2, 17b-estradiol; LH, luteinizing hormone; GnRH, gonadotropin-releasing hormone.

clinically significant lack of DHEA availability in post-menopausal women is to administer exogen­ ous DHEA in order to replace the amount of DHEA missing in these women. Such a replace­ ment with DHEA can thus improve or even make the symptoms of hormone deficiency disappear as observed recently in women treated for vaginal atrophy, a classical consequence of hormone defi­ ciency during post-menopause (Buster, 2009; Labrie et al., 2009a, 2009b, 2009c). In fact, the progressive and variable fall in serum DHEA starting in the thirties has been associated with a series of medical problems including cardiovascu­ lar mortality (Barrett-Connor et al., 1986), malig­ nancy (Ebeling and Koivisto, 1994), osteoporosis

(Sambrook et al., 1992) and a series of other med­ ical problems to be discussed later. Due to the marked decrease in the serum levels of DHEA starting in the thirties, a relatively long series of clinical studies have administered DHEA to women and men in order to correct various symptoms of hormone deficiency (Tables 1 and 2). One should be careful, however, about going as far as considering DHEA as the response to all problems of aging or a ‘fountain of youth’. Unfortunately, for more than 15 years, DHEA has been available over the counter and/or on the Internet, especially in the United States. This uncontrolled availability of DHEA raises serious concerns about self-administration of a series of

100 Postmenopause

New findings intracrinology GnRH CRH –

ACTH Intracrinology

DHEA E2

Adrenal

Cortisol Aldosterone

DHT

Peripheral tissue

Fig. 2. Schematic representation of the unique source of sex steroids in post-menopausal women, namely adrenal DHEA. At menopause, the secretion of E2 by the ovaries ceases. Consequently, after menopause, all estrogens and practically all androgens are made locally from DHEA in peripheral target intracrine tissues. The amount of sex steroids made in peripheral target tissues depends upon the level of the steroid-forming enzymes specifically expressed in each tissue.

Serum DHEA

30

Serum DHEA-S

15

25

12

20

(μmol/l)

(nmol/l)

9 15

6 10

3

5

0

0

20

40

60

80

20

40

60

80

Age (years) Fig. 3. Effect of age on DHEA and DHEA-S levels in women. The graph shows the serum concentrations of (A) DHEA and (B) DHEA-S in women aged 20–80 years. Data are expressed as means + SEM. DHEA, dehydroepiandrosterone; DHEA-S, DHEA sulphate (Labrie, 2007).

DHEA formulations not submitted to the quality control process of pharmaceuticals required by regulatory agencies, with the result that the

quantity and quality of the DHEA available in these formulations is very uncertain (Parasram­ puria et al., 1998).

101 Table 1. Summary of the different studies performed with DHEA in women

Authors

Daily dose

Findings

Negative effects

Abrahamsson and Hackl (1981)

200 mg 3 months n = 17 50 mg 4 months placebo/cross-over n = 24 Adrenal insufficiency 50 mg, placebo 3 months n = 30 Perimenopausal 50 mg ! 600 mg 6 months n = 23 400 mg, n = 14 600 mg, n = 3 Systemic lupus erythematosus 50 mg 4 weeks n = 22 Pilot study 50 mg, placebo 12 months n = 70

Decreased serum LH

No

Improved mood, well-being and increased frequency of sexual thoughts and interest

No

No significant effect on mood, perimenopausal symptoms, cognition, memory or well-being

No

Improvement Acne

No

Hot flash score decreased by 50%

No

Increase in BMD at femoral neck and Ward’s triangle in 60–69-year-old women and increased radius BMD in 70–79-year­ old women Improved libido and sexual function, skin hydration, epidermal thickness, pigmentation and increased sebum secretion Trend for decreased body fat. Improved life satisfaction

No

Improved quality of life, social functioning and general health perception

No

Leptin decreased Osteocalcin slightly increased No change in glucose, insulin, body mass index or exercise capacity Insulin sensitivity increased

No

No significant effect on BMD. Insulin sensitivity improved. Triglycerides decreased.

No

Arlt et al. (1999)

Barnhart et al. (1999)

Barry et al. (1998)

Barton et al. (2006)

Baulieu et al. (2000)

Bilger et al. (2005)

Brooke et al. (2006a, 2006b)

Callies et al. (2001)

Casson et al. (1995)

Casson et al. (1998)

50 mg, placebo 12 months n=5 Hypopituitarism 50 mg, placebo 6 months followed by 6 months open n = 26 Hypopituitarism 50 mg 4 months n = 24 Adrenal insufficiency 50 mg, placebo 3 weeks n = 11 25 mg, placebo 6 months n=7

No

No

(Continued)

102 Table 1. (Continued ) Authors

Daily dose

Findings

Chang et al. (2002); Genelabs Briefing Document, 2001 – Taiwan Same study

200 mg 6 months n = 61 ! 58 completed Systemic lupus erythematosus

Diamond et al. (1996); Labrie et al. (1997) Same study

4–6 g of 10% DHEA cream, placebo/cross-over 12 months n = 15

Dhatariya et al. (2005)

50 mg, placebo/cross-over 3 months n = 28 Adrenal insufficiency 175 mg 8 days n = 5 women þ 3 men Hypercholestorelemia 50 mg 6 months n = 31 25 mg 12 months n = 20

Triglycerides decreased Mild acne Improved flare Acne and seborrhoea No hirsutism Infection and headache decreased Sub-cutaneous fat decreased Increase in femoral muscle area Decreased serum insulin and glucose Increased BMD of total hip Vaginal maturation stimulated Sebum production increased Well-being and energy improved Fasting insulin and glucagon lowered Increased insulin sensitivity Decreased total, HDL and LDL cholesterol Total cholesterol decreased

Felt & Starka (1966)

Genazzani et al. (2001)

Genazzani et al. (2003); Genazzani et al. (2006) Same study

Gebre-Medhin et al. (2000)

Gordon et al. (2002)

50 –200 mg 3 months n = 5(50 mg), n = 4(200 mg) Addison’s disease 50 mg 12 months n = 61 Anorexia nervosa

Gurnell et al. (2008)

50 mg, parallel group 12 months n = 30 Addison’s disease

Hackbert and Heiman (2002)

300 mg Acute study n = 16 50 mg 3 months n = 24 Addison’s disease

Hunt et al. (2000)

Negative effects No

No

No

No

Osteocalcin increased Levels of GH and IGF-1 increased

No

Improved vasomotor symptoms No change in endometrial thickness The response to ACTH was increased, thus neutralizing the effect of age on adrenal enzymatic activities HDL and LDL cholesterol decreased Lean mass increased

No

No

Hip BMD increased Bone markers improved Improvement of psychological parameters Increased muscle mass and strength Improved health-related quality of life No change in fat mass lipids Increased BMD at femoral neck Increased lean body mass No change in cognitive function Mental and physical arousal to erotic video improved

No

Mood and fatigue improved No significant effect on cognitive or sexual function Significantly improved self-esteem Mild acne and facial hair growth

No

No

No

(Continued )

103 Table 1. (Continued ) Negative effects

Authors

Daily dose

Findings

Igwebuike et al. (2008)

50 mg, placebo 3 months n = 17

No

Jankowski et al. (2006)

50 mg 12 months n = 34

Johannsson et al. (2002)

20 or 30 mg, placebo 6 months, followed by 6 months DHEA in placebo group n = 38 Pituitary deficiency 25 or 50 mg every 2 or 3 days 3–29 months n=4 Hereditary angioedema 6g of 0.1 to 2.0% DHEA cream, placebo 13 weeks n = 60 4g of 10% DHEA gel or cream or 100 mg oral 14 days n = 36 6.5 ! 23.4 mg intravaginal, placebo, 7 days n = 30 Vaginal atrophy 3g of 0.3% DHEA cream, placebo, 12 months n = 73

Body fat decreased Skeletal muscle weight significantly increased when DHEA was added to exercise Increased BMD at total hip, trochanter, femoral shaft and lumbar spine No significant effect on fat or fat-free mass and handgrip strength Sexual function, mood and behavior improved Appearance of normal skin and/or pubic and/or axillary hair Increased muscle strength Dramatic improvement in clinical state

DHEA metabolism following percutaneous administration Modulation of several genes involved in collagen biosynthesis Bioavailability and metabolism of DHEA after oral and percutaneous administration

No

Metabolism of DHEA after intravaginal administration Beneficial effects against vaginal atrophy

No

Metabolism of DHEA following longterm percutaneous administration Prevention of the appearance of new skin wrinkles Stimulation of sebaceous gland activity Metabolism of DHEA following 12-week intravaginal administration Reversal of all the symptoms and signs of vaginal atrophy Improvement of all four domains of sexual dysfunction and libido Insulin sensitivity improved HDL cholesterol increased LDL cholesterol and triglycerides decreased Glucose tolerance unchanged Fat mass decreased Total cholesterol decreased No change in glucose Fat mass decreased

No

Koo et al. (1983)

Labrie et al. (2007a) Calvo et al. 2008 Same study Labrie et al. (2007b)

Labrie et al. (2008a, 2008b)

Labrie et al. (2008c) Labrie et al. unpublished

Labrie et al. (2009a, 2009b, 2009c)

3.25 ! 13 mg intravaginal, placebo, 3 months n = 163 Vaginal atrophy

Lasco et al. (2001)

25 mg, placebo 12 months n = 20

Libe et al. (2004)

50 mg 4 months n=7 Addison’s disease

No

No

No

No

No

No

No

(Continued)

104 Table 1. (Continued ) Authors

Daily dose

Findings

Negative effects

Lovas et al. (2003)

25 mg, parallel group 9 months n = 19 Adrenal failure 50 mg 3 months n = 17

No statistically significant effect.

No

Insulin sensitivity and percentage of body fat unchanged Improved physical and psychological well-being No significant change in libido Muscle strength unchanged Lean body mass increased No significant effect on fat mass Insulin resistance decreased Total and HDL cholesterol decreased

No

No change in ‘insulin action’ Increased lean body mass’.

No

Improved mental health-related quality of life and McCoy’s sex scale No significant effect on bone density

No

No effect on hand grip and knee muscle strength. Muscle area at thigh unchanged. Acne in 41% at both doses versus 19% in controls HDL cholesterol decreased with 200 mg Hirsutism in 11 and 7.8% at 100 and 200 mg doses and 4.1% in placebo

No

Mild acne in 33% and hirsutism in 16% versus 14 and 2% in controls No therapy withdrawal required HDL cholesterol, triglycerides and C3 complement decreased Myalgia and stomatitis decreased Increased lumbar and spine BMD Some subjective improvement but not in tests of neurological dysfunction

No

Improved Kupperman score – quality of life

No

Improved Kupperman score – quality of life Vasomotor and psychological symptoms

No

Morales et al. (1994)

Morales et al. (1998)

Mortola and Yen (1990)

Nair et al. (2006) Basu et al. (2007) Same study Nordmark et al. (2005)

Percheron et al. (2003)

Petri et al. (2002)

Petri et al. (2004)

Roberts and Fauble (1990)

Stomati et al. (1999)

Stomati et al. (2000)

100 mg 6 months n = 10 1600 mg, placebo 28 days n=6 50 mg, placebo 23 months n = 27 20–30 mg, placebo 6 months followed by 6 months open n = 91 Systemic lupus erythematosus 50 mg, placebo 12 months n = 70 ! 51 (end) 100 mg n = 63 ! 6 (end) 200 mg n = 64 ! 47 (end) 7–9 months Systemic lupus erythematosus 200 mg 12 months n = 189 ! 124 (end) Systemic lupus erythematosus

90 ! 180 mg 6 months n=9 Multiple sclerosis 50 mg (DHEA-S) 3 months n=8 50 mg 6 months n = 31

No

No

No

No

(Continued )

105 Table 1. (Continued ) Negative effects

Authors

Daily dose

Findings

Suh-Burgmann et al. (2003)

150 mg, intravaginal n = 12 for 3 months n = 7 for 6 months 50 mg, placebo/cross-over 4 months n = 16 Hypopituitarism 200 mg 3–6 months n = 10 Systemic lupus erythematosus 100 mg, placebo 3 months, n = 14 Followed by 50–200 mg 3 months, open, n = 21 Systemic lupus erythematosus 200 mg, placebo 6 months (n = 50 ! 34) 12 months, n = 21, 29 months, n = 21 200 mg, placebo 6 months n=9 Systemic lupus erythematosus 50 mg 6 months n = 18 50 mg 6 months n = 28 50 mg, placebo 12 months n = 58 50 mg 6 months n = 17 100 mg 12 months n=8 100 mg 3 months n = 36

DHEA promotes regression of low-grade cervical lesions

No

Depression score and health perception slightly improved

No

Possible improvement

No

Improved flare Modest benefits

No

Improvement of lupus Mild acne and hirsutism

No

Improvement of lupus

No

Improved muscle mass and strength and lumbar BMD

No

Decreased visceral and subcutaneous fat Sensitivity to insulin increased

No

Lumbar spine BMD increased

No

Serum IGF-1 increased

No

No significant effect on knee muscle strength

No

Total cholesterol decreased Flow-mediated dilatation and lower Doppler velocimetry in brachial artery increased Endothelium-dependent, cutaneous blood flow increased Tendency to increased well-being

No

Suggestion of anti-depressant effect

No

Van Thiel et al. (2005)

van Vollenhoven et al. (1994)

van Vollenhoven et al. (1995)

van Vollenhoven et al. (1998)

van Vollenhoven et al. (1999)

Villareal et al. (2000)

Villareal and Holloszy (2004)

Von Muhlen et al. (2008)

Yen et al. (1995)

Yen et al. (1995)

Williams et al. (2004)

Wolf et al. (1997)

Wolkowitz et al. (1997)

50 mg 2 weeks n = 15 30–90 mg 4 weeks n=3

No

106 Table 2. Summary of the different studies performed with DHEA in men Authors

Dose

Findings

Serious side effects

Arlt et al. (2001)

50 mg 4 months n = 22 50 mg placebo n = 70 n = 70 50 mg placebo 6 months followed by 6 months open n = 18 Hypopituitarism 175 mg 8 days n = 5 women þ 3 men Hypercholestorelemia 100 mg, placebo 3 months n = 39 25 mg 12 months n = 10 50 mg, placebo 12 months n = 24 Addison’s disease 50 mg 3 months n = 15 Addison’s disease 50 mg 12 months n = 35 25 mg 3 months 24 men with hypercholesterolemia 50 mg 5 months n=9 25 or 50 mg every 2 or 3 days 3–29 months n=4 Hereditary angioedema 50 mg 4 months n = 13 Addison’s disease

No significant effect

No

No significant change of BMD Skin hydration and pigmentation improved

No

No statistically significant effect

No

Total cholesterol decreased

No

No significant change in body composition Total and HDL cholesterol decreased

No

Improvement in mood, fatigue and joint pain

No

BMD of femoral neck improved Total body and truncal lean mass improved No significant change in fat mass or lipids Well-being partially improved Mood and fatigue improved No significant effect on cognitive or sexual function

No

BMD increased at total hip, trochanter and shaft No significant effect on fat or fat-free mass

No

Glucose decreased but not insulin Insulin sensitivity improved and plasminogen activator inhibitor type 1 decreased

No

Stimulation of immune function

No

Dramatic improvement in clinical state

No

Total cholesterol decreased No significant change in glucose or insulin Fat mass decreased

No

Baulieu et al. (2000)

Brooke et al. (2006a), JCEM

Felt and Starka (1966)

Flynn et al. (1999)

Genazzani et al. (2004)

Gurnell et al. (2008)

Hunt et al. (2000)

Jankowski et al. (2006)

Kawano et al. (2003)

Khorram et al. (1997)

Koo et al. (1983)

Libe et al. (2004)

No

(Continued )

107 Table 2. (Continued ) Authors

Dose

Findings

Serious side effects

Morales et al. (1994)

50 mg 3 months n = 13 100 mg 6 months n=9 75 mg, placebo 23 months n = 29 1600 mg 28 days n=5 50 mg, placebo 12 months n = 70 ! 60 (end) 90 ! 180 mg 6 months n=9 Multiple sclerosis 100 mg 6 months n = 86 Osteoporosis 50 mg, placebo 4 months n = 15 Hypopituitarism 50 mg, placebo 6 months n = 28 50 mg 12 months n = 55 1600 mg 4 weeks n=8 50 mg 2 weeks n = 25 30–90 mg 4 weeks n=3 50 mg 6 months n = 13 100 mg 12 months n=8

Improved physical and psychological well-being Insulin sensitivity and percentage of body fat not significantly changed Muscle strength increased Fat mass decreased No significant effect on BMD Increase in femoral neck BMD No significant change in insulin action

No

Decreased total and LDL cholesterol Decreased body fat No significant change in insulin sensitivity No effect on handgrip and knee muscle strength Muscle area at thigh not significantly changed

No

Some subjective improvement but not in tests of neurological dysfunction

No

BMD increased at lumbar spine and femoral neck

No

Depression score and health perception slightly improved

No

Decrease in visceral and sub-cutaneous fat Sensitivity to insulin increased

No

No significant effect on BMD or bone marker CTX

No

No significant effect

No

No significant effect on cognitive functions

No

Suggestion of antidepressant effect

No

Serum IGF-1 increased

No

Increased knee muscle strength

No

Morales et al. (1998)

Nair et al. (2006) Basu

et al. (2007)

Same study

Nestler et al. (1988)

Percheron et al. (2003)

Roberts and Fauble

(1990)

Sun et al. (2002)

Van Thiel et al. (2005)

Villareal and Holloszy

(2004)

Von Muhlen et al.

(2008)

Welle et al. (1990)

Wolf et al. (1997)

Wolkowitz et al.

(1997)

Yen et al. (1995)

Yen et al. (1995)

No

No

No

108

On the other hand, it should be mentioned that many studies reported as negative do not have the statistical power to reach any conclu­ sion. One has always to consider that data which do not reach the statistical level of significance should not be interpreted as demonstration of an absence of effect. Critical evaluation must always be made of the design of the study, the precision of the parameters used to assess the effect(s) of treatment, the true placebo component, the population studied and, most importantly, the duration of treatment and the number of subjects investigated. As can be seen in Tables 1 and 2, the majority of studies examined the effect of DHEA in a too small number of subjects and/ or the duration of treatment was too short to truly assess the potential changes induced by DHEA. This chapter is an opportunity to briefly mention the beneficial effects of DHEA already observed in the literature and, as expected from the physiological mechanisms involved, to realize that no significant negative effect has ever been observed in any study reported, despite the high doses sometimes used. This lack of significant side effects could, up to an unknown extent, be related to the fact that a natural saturation mechanism limits the transformation of DHEA into andro­ gens and/or estrogens in post-menopausal women at a serum DHEA concentration of about 7 ng/ml, a value well within the physiologi­ cal range (Labrie et al., 2006).

Intracrinology Changes in DHEA secretion with age The foetal adrenal gland secretes high levels of DHEA which is transformed in the placenta into the E2 required for maintenance of pregnancy (Chakravorty et al., 1999). Secretion of DHEA then declines to very low levels immediately after parturition (Parker, 1991). Strong DHEA secretion resumes during the pre-pubertal years at about 8–10 years of age, thus permitting the growth of pubic and axillary hair, a physiological phenomenon called adrenarche (Parker et al.,

1978). After a peak in the early twenties, serum DHEA [and DHEA sulphate (DHEA-S)] levels then decline at the rate of about 5% per year starting in the early thirties (Orentreich et al., 1984) (Fig. 3). The decline in serum DHEA levels in both women and men is highly variable and is driven by unknown factors.

Intracrine system It is remarkable that man, in addition to posses­ sing very sophisticated endocrine and paracrine systems, has largely invested in sex steroid forma­ tion in peripheral tissues (Labrie, 1991; Labrie et al., 1997; Labrie et al., 1985, 1988) (Figs. 1, 2 and 4). The ovaries and testicles have traditionally been considered the exclusive sources of estro­ gens and androgens in women and men, respec­ tively. For example, the fall in serum E2 to extremely low levels at menopause coupled with the beneficial effects of exogenous estrogens on menopausal symptoms (Archer et al., 1999) has focussed the efforts of hormone replacement ther­ apy almost exclusively on various forms of estro­ gens to compensate for the cessation of estrogen secretion by the ovaries. In men, on the other hand, the 95% (or more) fall in serum testoster­ one induced by castration and the clinical benefits of this partial elimination of androgens in men with advanced prostate cancer (Huggins and Hodges, 1941) have led the urological community to erroneously believe that castration eliminates 95% (or more) of androgens and that castration alone is an acceptable treatment for prostate cancer. The scientific findings recently provided by intra­ crinology are quite different (Labrie, 1991; Labrie et al., 1985; Bélanger et al., 1989). For example, since ovarian estrogen secretion ceases at meno­ pause, the exclusive role of peripheral estrogen formation in post-menopausal women is clearly demonstrated, as mentioned above, by the obser­ vation of the major benefits of aromatase inhibitors in advanced breast cancer in post-menopausal women (Goss et al., 2003; Howell et al., 2005; Mouridsen et al., 2003; Nabholtz et al., 2000) as

109

well as by the findings of a 76% decrease in breast cancer incidence in post-menopausal osteoporotic women who received the selective estrogen recep­ tor modulator (SERM) raloxifene for 3 years (Cummings et al., 1999). These effects of estro­ gen blockade are the consequence of the rela­ tively high levels of estrogens which are made locally by intracrine mechanisms in breast can­ cer tissue after menopause (Poortman et al., 1983). In men, on the other hand, the finding that 25–50% of androgens are left in the prostate after castration (Bélanger et al., 1989; Labrie et al., 1985; Mostaghel et al., 2007; Nishiyama et al., 2004) explains why the addition of a pure (non-steroidal) anti-androgen to castration achieves a more complete blockade of androgens and has been the first treatment shown to prolong life in prostate cancer (Caubet et al., 1997; Labrie et al., 1982; Labrie et al., 1985; Labrie et al., 2005; Prostate Cancer Triallists’ Collaborative Group, 2000). The androgens remaining at relatively high levels after castration also explain why com­ bined androgen blockades or the blockade of the androgens of both testicular and adrenal origins at start of treatment can provide cure for most patients when the treatment is started at the loca­ lized stage of the cancer (Akaza, 2006; Labrie et al., 2002; Ueno et al., 2006), thus clearly demon­ strating the major role of extratesticular andro­ gens or intracrinology in men. Transformation of the adrenal precursor steroid DHEA into androgens and/or estrogens in periph­ eral target tissues depends upon the levels of expression of the various steroidogenic and meta­ bolizing enzymes in each cell of these tissues. This situation of a high secretion rate of adrenal pre­ cursor sex steroids in men and women is thus completely different from all animal models used in the laboratory (namely rats, mice, guinea pigs and all others except monkeys), where the secre­ tion of sex steroids takes place exclusively in the gonads (Bélanger et al., 1989; Labrie et al., 1985, 1988, 1997). The androgens testosterone and DHT as well as E2 made in peripheral tissues from DHEA of adrenal origin exert their action locally in the same cells where their synthesis takes place

(Fig. 4). This sophisticated mechanism permits to maintain biologically active levels of intracellular estrogens and/or androgens in specific tissues in need of these sex steroids while the same steroids leak in the blood at very low levels, thus sparing the other tissues from a potentially negative influ­ ence. Following their local formation and immedi­ ate availability for local intracellular action, testosterone and DHT (the most active natural androgen) and E2 are inactivated and transformed in the same cells into water-soluble glucuronide or sulphate derivatives which can then diffuse quan­ titatively into the general circulation where they can be measured by mass spectrometry (Labrie et al., 2006) before their elimination by the kidneys. In women, as mentioned earlier, the role of the adrenal precursor steroid DHEA in the periph­ eral formation of sex steroids is even more impor­ tant than in men. In fact, in men, sex steroid secretion by the testicles continues at an almost constant and high level through life while, in women, estrogen secretion by the ovaries comple­ tely ceases at menopause, thus leaving the adre­ nals as the exclusive source of sex steroids (Fig. 2). Accordingly, the best estimate is that the intracrine formation of estrogens in peripheral tissues in women accounts for 50% of all estro­ gens before menopause and 100% after meno­ pause (Labrie et al., 2003). It should also be noted that the importance of the intracrine formation of androgens and estro­ gens extends to non-malignant diseases such as acne, seborrhoea, hirsutism and androgenic alo­ pecia as well as to osteoporosis and vaginal atrophy (Cusan et al., 1994; Labrie et al., 1997; Labrie et al., 2009a, 2009b, 2009c). Most tissues possess, at various levels, a battery of steroido­ genic enzymes that can transform DHEA.

In men, 40% of total androgens are made from DHEA While the serum levels of testosterone are reduced by 97.4% following castration in 69–80­ year-old men (Labrie et al., 2009b), the sum of the metabolites of androgens, the only accurate and

110

Steroidogenic enzymes adrenal – intracrine tissues Exogenous DHEA

Adrenal

Cholesterol

DHEA-S

P450scc

Sult

P450c17

PREG 3β-HSD

DHEA

20OH-PROG

P450c11β

17-OH-PROG

4-DIONE 17β-HSD 2, 10 5α-red 1, 2, 3

ALDOSTERONE

TESTO 5α-red 1, 2, 3

17β-HSD 5, 15

A-DIONE

P450c11β

DHT 17β-HSD 2, 9, 10 3α-HSD

3α-HSD Aromatase

CORTICO STERONE

3β-HSD

17β-HSD 5

11-DEOXY CORTISOL

11-deoxycorTicosterone

5-DIOL 17β-2, 4

3β-HSD

P450c21

P450c21

P450c11α

17β-HSD 1

P450c17

3β-HSD P450c17

PROG 20α-HSD

17-OH-PREG

17β-HSD 5, 15

ADT

CORTISOL

E1

3α-DIOL

Aromatase

17β-HSD 2, 9, 10, 14 17β-HSD 1, 7, 12

E2

17β-HSD 2, 4, 8, 14

Adrenal

Intracrine tissues

Fig. 4. Schematic representation of the adrenal and intracrine steroidogenic pathways. DHEA, dehydroepiandrosterone; DHEA-S, DHEA sulphate; DHT, dihydrotestosterone; HSD, hydroxysteroid dehydrogenase.

valid parameter of total androgenic activity mea­ surable in the circulation (Labrie et al., 2006), is only reduced by 58.9% (Labrie et al., 2009b), thus indicating that a very important proportion (41.1%) of androgens remains in men after com­ plete elimination of testicular androgens. Such data are in close agreement with the concentration of intraprostatic DHT which shows that, on aver­ age, 39% of DHT is left in the prostate after castration in various studies, namely 45% (Labrie et al., 1985), 51% (Bélanger et al., 1989), 25% (Nishiyama et al., 2004) and 35% (Mostaghel et al., 2007) (see Fig. 4 in Labrie, this volume, Chapter 14).

Comparable amounts of sex steroids of adrenal origin are made in men and women With the knowledge of the major importance of androgens of adrenal origin in men, it is of interest to compare the data mentioned above for men

with the serum levels of the same steroids mea­ sured in intact post-menopausal women. As can be seen in Fig. 5A and B, the serum levels of testosterone and of the total androgen metabolites are almost superimposable in castrated men and post-menopausal women of comparable age. Most interestingly, it can be observed that the serum levels of estrone sulphate (E1S) are also compar­ able (Fig. 5C). It could also be seen that the serum levels of E1 and E2 are also comparable, thus indicating that similar amounts of estrogens of adrenal origin are found in both men and women (Labrie et al., 2009b). The above-summarized data show that ~40% of androgens are made in peripheral tissues in the absence of testicles in 69–80-year-old men. Since serum DHEA decreases markedly with age start­ ing in the thirties (Labrie et al., 2005), and testi­ cular androgen secretion decreases only slightly, it is most likely that androgens of adrenal origin have an even greater relative and absolute impor­ tance at younger ages. The same conclusion

111

applies to women with respect to the androgens synthesized from DHEA.

Women synthesize 40–50% as much androgens as men The data summarized above show that post-meno­ pausal women synthesize androgens and estrogens in quantities similar to castrated men of compar­ able age (Fig. 5). The 50–60% higher androgen formation in men is essentially attributable to the androgens of testicular origin.

High circulating levels of DHEA and menopause are unique to women As mentioned above, protection of the endome­ trium is the most obvious reason why evolution over 500 million years has succeeded in building an intracrine system unique to the human species and able to protect women from systemic expo­ sure to estrogens after menopause. It is remark­ able that while the steroidogenic enzymes appeared ~500 million years ago with the verte­ brates, it is only about 50 million years ago that

3 A.

the adrenals of primates gained the property to secrete large amounts of DHEA (Baker, 2004). DHEA becomes the exclusive source of sex steroids at menopause Recently, it has been more evident that DHEA of adrenal origin became practically the only source of sex steroids in women with the appearance of meno­ pause which corresponds to the arrest of significant sex steroid secretion by the ovaries. It took more than 500 million years of evolution to separate the role of gonadal and DHEA-derived sex steroids, thus per­ mitting women to be free during all their post-meno­ pausal years from the negative systemic effects of estrogens and benefit from a strictly local formation and action of sex steroids made according to the specific age-related needs of each cell type in each tissue by the process of intracrinology (Labrie, 1991). Systemic estrogens are not physiological after menopause Since all women are no more exposed to systemic estrogens after menopause, it is reasonable to believe that the non-physiological situation created

40 B.

250

C.

200

30 2 pg/ml

ng/ml

ng/ml

150 20

100

1 10

0

Castrated men

Post-meno­ pausal women

Testosterone

0

50

Castrated men

Post-meno­ pausal women

ADT – G + 3α – diol – 3G + 17G

0

Castrated men

Post-meno­ pausal women

Estrone sulphate

Fig. 5. Comparison of the serum concentrations of testosterone (A), total androgenic pool (sum of ADT-G, 3a-diol-3G and 17G) (B) and E1S (C) in castrated 69–80-year-old men (n = 34) and intact 55–65-year-old post-menopausal women (n = 377) (Labrie et al., 2006; Labrie et al., 2009b).

112

by the administration of estrogens could be respon­ sible, up to an unknown extent, for the side effects reported in women receiving traditional estrogen replacement therapy (ERT) and HRT (estrogen þ progestin replacement therapy) (Beral, 2003; Beral et al., 2005; Grodstein et al., 2006; Hsia et al., 2006; Pines et al., 2007; Riman et al., 2002; Rossouw et al., 2002; Ruttimann, 2008). The recent observations indicating the risks associated with exogeneous estrogens as replacement therapy should normally help to focus our attention on DHEA, the only physiological source of sex steroids after menopause (Labrie, 2010), and better understand the mechan­ ism of action of DHEA and its preventive and therapeutic roles.

suffering from vaginal atrophy symptoms (~75% of women) and those without symptoms (~25%) is not related to estrogens. In fact, the only remaining hormonal difference between these two groups of women is the difference in the availability of DHEA, the exclusive source of sex steroids (Fig. 2). In addition to markedly decreasing with age (Fig. 3), the serum levels of DHEA are highly variable with some women having barely detectable levels while others have values up to 9–10 ng/ml (Labrie et al., 2006, 2010) (Fig. 5).

High variability of serum DHEA

It should be mentioned that saturation of the enzymatic systems which transform DHEA into active androgens and/or estrogens is observed at serum DHEA levels of about 7 ng/ml, thus pro­ tecting women against potential excess levels of sex steroids (Labrie et al., 2007a) (Fig. 6). We believe that the presence of this natural saturation

An important observation is that despite the arrest of estrogen secretion in all women at time of meno­ pause, not all post-menopausal women suffer from menopausal symptoms. Consequently, the hormo­ nal difference between post-menopausal women

Rapid saturation of the enzymatic mechanisms transforming DHEA into sex steroids: Prevention of overexposure and potential side effects

Highly variable serum levels of DHEA Premenopausal Women (n = 47)

10

Postmenopausal Women (n = 442)

DHEA (ng/mL)

8

6

4

2

0 30

40

50

60

70

Age (Years) Fig. 6. Illustration of the wide variability of serum DHEA levels in normal women aged 30–40 years and 50–75 years. Data are presented individually as well as means and 5–95% centiles (Labrie, 2010).

113

of the enzymatic mechanism makes practically impossible to administer a dose of DHEA which could lead to excess tissue exposure to estrogens and/or androgens. The fact that no serious adverse event has ever been reported with exo­ genous DHEA (Tables 1 and 2) is likely to be explained, up to an unknown extent, by this selfprotecting mechanism. In addition, when replace­ ment with DHEA is limited to physiological amounts of this tissue-specific sex steroid prehor­ mone free of intrinsic sex steroid activity, no adverse effect can be logically expected. In fact, all our pharmacokinetic studies per­ formed in post-menopausal women indicate that doses of prasterone much larger than those used in our efficacy studies could only have a limited effect on global sex steroid formation (Labrie et al., 2007a; Labrie et al., 2007b; Labrie et al., 2008c). As illustrated in Fig. 7, it would be extremely difficult or most likely impossible to administer a dose of DHEA intravaginally (or otherwise) able to increase the serum levels of

the androgen metabolites above the normal post­ menopausal range.

Changes in serum DHEA following treatment with DHEA are overestimates of the true changes in sex steroid formation Before looking at efficacy data, it is important to mention that recent information has shown that following administration of DHEA, the observed changes in serum DHEA are at least twice as high as the ‘true’ changes in sex steroid forma­ tion (Labrie et al., 2008c). This effect is even more pronounced when comparing serum levels of DHEA with ‘true’ levels of estrogens. This observation is of major importance in analyzing the data from DHEA studies where, almost always, changes in serum DHEA are used as the parameter of reference to assess the choice of dose and the efficacy of replacement therapy. Consequently, the dose of DHEA used is

123.6 1.0% DHEA

ADT−G + 3α−DIOL−3G + 3α−DIOL−17G (ng/ml)

50

2.0% DHEA

95th Centile 40

30

0.3 % DHEA

20 0.1 % DHEA

(Basal

post-menopausal)

10

5th Centile 0 0

5

10 DHEA (ng/ml)

Pre-meno­ pausal women

Fig. 7. Effect of increasing serum concentrations of DHEA induced by twice daily percutaneous administration of 3 g of 0% (placebo), 0.1, 0.3, 1.0 or 2.0% DHEA cream on the sum of the serum levels of the androgen metabolites androsterone glucuronide (ADT-G), androstane-3a, 17b-diol-3 glucuronide (3a-diol-3G) and androstane-3a, 17b-diol-17 glucuronide (3a-diol-17G) expressed in ng/ml (Labrie et al., 2007a).

114

frequently too low compared with the additional intra-tissular sex steroid levels needed to show efficacy.

Tissue-specific effects of DHEA reported in the literature Limitations of the available clinical data As mentioned above and indicated by Arlt and Allolio (2003), a problem with the available clin­ ical DHEA studies (Tables 1 and 2) is that a large proportion of these studies are underpowered, thus not contributing to increasing our knowledge in the field. On the contrary, many of these studies add confusion by stating that DHEA has no effect on such and such parameter(s) while, in fact, the study performed did not have the statistical power needed to reach any valid conclusion: A common problem is that a lack of statistical power leads to the erroneous conclusion that there is no effect of DHEA because the p value does not reach