- Email: [email protected]
The 1998 Neuroendocrine Workshop on Stress: a Report on the American Neuroendocrine Society Annual Workshop
J. Pasqualini (Paris, France) discussed the remarkable development in this synthesis of new progestins over recent years. With regard to breast cancer, information on the action of progestins is limited to the positive response observed with the progestins, medroxyprogesterone acetate and megestrol acetate, in postmenopausal patients with advanced breast cancer. In hormone-dependent breast cancer cells, various progestins (nomegestrol acetate, tibolone and promegestone) are potent sulfatase inhibitory agents. It was also demonstrated that these progestins are active in inhibiting the conversion of estrone to estradiol by 17β-hydroxysteroid dehydrogenase. Consequently, blockade of the formation of estradiol via sulfatase by progestins might provide interesting, new possibilities for clinical applications in breast cancer. J. Cidlowski (Research Triangle Park, NC, USA) emphasized the
importance of apoptosis in the development and homeostasis of total organism cell number, as well as in the editing of damaged or mutant cells. Current studies are aimed at identifying the molecular entities that carry out apoptotic processes, and the understanding of their activation by various apoptotic signals. T lymphocytes serve as an excellent model for such studies because they undergo apoptosis in response to glucocorticoids and other compounds. In T lymphocytes, DNA fragmentation and caspase activation occur only in cells that have undergone a reduction in cell volume. In addition, recent evidence has indicated that early ion fluxes are critical for the activation of apoptotic pathways. L.B. Hendry (Augusta, GA, USA) reported that a key step in gene regulation by steroids is the binding of hormone to receptors that interact with DNA. Steroids insert stereospecifically between base pairs, and this fit
correlates with activity. It was proposed that receptors mediate insertion of hormone into DNA, and that this step is rate limiting. A.M. Soto (Boston, MA, USA) described the manner in which the endocrine and reproductive effects of environmental agents such as pesticides and industrial chemicals are believed to affect wildlife and humans. Such compounds have the ability to: (1) mimic the effects of endogenous hormones; (2) antagonize the effects of endogenous hormones: (3) disrupt the synthesis and metabolism of endogenous hormones; and (4) disrupt the synthesis and metabolism of hormone receptors. A single chemical can produce neurotoxic, estrogenic and antiandrogenic effects. The systematic identification of these compounds before they are introduced into widespread use will require the collaboration of the scientific community, government agencies, the industrial sector and the public at large.
•
The 1998 Neuroendocrine Workshop on Stress: a Report on the American Neuroendocrine Society Annual Workshop James I. Koenig
Since Hans Selye first defined the stress response1, investigators have pursued the identification of the major biochemical mediators and the loci of their effects within the brain tirelessly. In recent years, these efforts have culminated in the identification of corticotropin-releasing hormone (CRH) and vasopressin (AVP) as the main mediators of the response to stress. A newly added mediator whose physiological function in the stress response
J.I. Koenig is at the Maryland Psychiatric Research Center, University of Maryland School of Medicine, PO Box 21247, Baltimore, MD 21228, USA.
TEM Vol. 10, No. 1, 1999
is yet to be established is urocortin2. CRH and urocortin exert their effects by binding to four different CRH receptors in brain and pituitary, as well as binding to CRH-binding protein, which modifies the actions of CRH. The 1998 Neuroendocrine Workshop on Stress was held in New Orleans, LA, USA, from 21–23 June 1998, and was attended by over 125 investigators. The workshop focused on the latest developments in the neuroendocrinology of stress and on the identification, function and clinical utility of these moieties for central nervous system (CNS) and peripheral disorders.
Molecular Mechanisms of Central Stress Integration
The 41-amino acid peptide CRH, isolated and characterized originally by Vale et al.3, is the most prominent stimulatory factor controlling pituitary adrenocorticotropin (ACTH) secretion. Previous studies have documented clearly that expression of the CRH gene and the subsequent secretion of CRH by hypothalamic neurons are both under the strong negative influence of the adrenal glucocorticoid hormones, cortisol and corticosterone. However, in non-hypothalamic regions of the brain, such as the amygdala, glucocorticoids exert a stimulatory effect on CRH gene expression. This tissue-specific regulation could be a direct effect of the activated glucocorticoid receptor on the CRH gene, or as discussed by Dr A. Seasholtz (Ann Arbor, MI, USA), both the positive and negative effects of glucocorticoids might arise as a result of novel interactions between the glucocorticoid receptor and cAMP-dependent mechanisms, most likely involving novel interactions with cAMP response
1043-2760/99/$ – see front matter © 1999 Elsevier Science. All rights reserved. PII: S1043-2760(98)00114-3
5
element-binding protein (CREB). This finding is very important for investigators exploring the molecular basis of fear and anxiety behaviors. However, CREB has many roles in CRH neurons and during the stress response. For example, Dr P. Sawchenko (San Diego, CA, USA) showed that in response to ether inhalation stress the phosphorylated form of CREB (pCREB) rises very quickly (within 5 min) in CRH neurons. This rapid rise in pCREB is followed closely by the appearance of CRH heteronuclear RNA (hnRNA). On the other hand, in AVP neurons AVP hnRNA does not increase until 2 h after the ether exposure and the increase is preceded selectively by the appearance of c-Fos. Interruption of protein synthesis by cycloheximide blocks the stress-induced elevation in c-Fos and AVP hnRNA but not the induction of pCREB and CRH hnRNA. Interestingly, however, adrenalectomy to remove the glucocorticoid signal had little effect on the ether-induced appearance of CRH hnRNA but delayed the induction of AVP hnRNA. These studies suggest that stress activates CNS neural pathways that impinge upon CRH cells in the paraventricular nucleus (PVN) and that these cells are able to respond rapidly to stressful events without needing to produce new protein moieties. Other stress-responsive cells, like AVP cells, are not necessary for the initial phases of the response and therefore use slower, protein synthesis-dependent mechanisms to activate factors responsible for more sustained responses to a challenge. The neural mechanism(s) involved in responding to challenges are beginning to be elucidated. Dr J. Herman (Lexington, KY, USA) described studies demonstrating that the disinhibition of γ-aminobutyric acid (GABA)-containing neurons around the PVN might play a critical role in the response to stress. GABAcontaining neurons in the bed nucleus of the stria terminalis (BNST), the preoptic area (POA) and the dorsomedial nucleus project to the PVN and modulate hypothalamic–pituitary–adrenal (HPA) function. Additionally, chronic stress exposure induces GAD65 mRNA 6
production in these cells and decreases GABAB receptor mRNA in the PVN, confirming a role for GABA. Pathological alterations in HPA axis function occur in some psychiatric disorders, but dysfunction of the HPA axis also occurs during normal aging. The decline of cognitive functions seen during aging is related to increased glucocorticoid levels4 and changes in the hippocampal glucocorticoid receptor to mineralocorticoid receptor ratio. However, treating elderly animals with antidepressant agents augments serotoninergic activity and induces the production of nerve growth factor 1A (NGF1A) mRNA. Increased levels of NGF1A upregulate glucocorticoid receptor expression and, thereby, reverse aging-induced cognitive decline (J. Yao, Edinburgh, UK). •
Developmental Stress and the HPA Axis
The interplay of genetic and environmental factors shapes the neurocircuitry of the developing CNS. Stressful events and the concomitant increase in glucocorticoids might alter the connectivity of CNS cells or may disrupt cellular genesis in the developing brain. For example, granule cells of the hippocampus develop during the stress hyporesponsive period (SHRP). During this time, circulating glucocorticoids are maintained at very low levels, so as not to disrupt the hippocampal development. Experiments performed by Dr E. Gould (Princeton, NJ, USA) demonstrated that administering glucocorticoids to animals during this critical period results in a reduction in the number of new cells added to the hippocampus. Moreover, exposure to extremely stressful situations, which raise glucocorticoid levels, also yields the same result. However, the SHRP that was proposed originally to span from postnatal Days 5–14 is a misnomer. Dr S. Levine (Wilmington, DE, USA), performing studies in neonatal rats, demonstrated that the ACTH responses to stress, even mild stress, during the SHRP were very robust, while glucocorticoid responses were minimal. Stronger stresses, like ether exposure or
lipopolysaccharide injection, did elicit a corticosterone response, albeit very meager. Thus, this SHRP is more probably a glucocorticoid hyporesponsive period. However, the presence of the dam during this time also diminishes the reactivity to stress5. Taking advantage of this facet of the mother–offspring relationship, Dr P. Plotsky et al. (Atlanta, GA, USA) developed the maternal separation stress, where pups are separated from their mothers for 180 min on Days 2–10 of life. At adulthood, these separated pups show enhanced anxiety, moderate anhedonia, alcohol preferences and dysregulation of the HPA axis, with increased expression of CRH in the PVN, amygdala and BNST. These animals also have increased acoustic startle behavior, defensive withdrawal and decreased water maze performance. Clearly, environmental factors and maternal factors strongly influence the activity of the HPA axis, causing alterations in the connectivity of the brain, and potentially changing adult behavioral patterns. •
Steroid Hormone Interactions and Stress
Gonadal steroid hormones are known to have both activational and organizational effects within the CNS. Sexually dimorphic brain structures are well documented in the literature. The HPA axis is also sexually dimorphic as a consequence of the gonadal steroid milieu. During the neonatal period, gonadectomy enhances corticosterone responsiveness to stress in later life, while either estrogen or testosterone treatment eliminates the enhanced responsiveness (C. McCormick, Lewiston, ME, USA). In adult animals, estrogens cause enhancement of the HPA axis responsiveness and testosterone diminishes the response of the axis to stress. The effect of estrogen might arise as a result of the presence of the beta form of the estrogen receptor in PVN neurons (R. Handa, Maywood, IL, USA). On the other hand, testosterone’s inhibitory effects might be mediated by androgen receptors in cells of the BNST and POA, which are known to contain GABA and to project TEM Vol. 10, No. 1, 1999
to the PVN. Estrogen might also modulate HPA axis activity via actions within the hippocampus (N. Weiland, New York, NY, USA) and its ability to increase synaptic densities of pyramidal cells. At the molecular level, interplay between estrogen receptors and glucocorticoid receptors is known to take place. In general, estrogen stimulates transcriptional events involving AP-1 sites, while glucocorticoids are inhibitory. Although the molecular interplay involves formation of a complex of activated receptors with c-Jun, alternative pathways involving inactivators have also been reported. Estrogen appears to coactivate the CREB-binding protein and prevents the inhibitory effects of the glucocorticoid receptor interacting protein (GRIP) (R. Uht, San Francisco, CA, USA). A careful evaluation of the physiological relevance of these novel molecular interactions remains to be undertaken. •
Molecular Biology of CRH Receptors and the Biology of CRH Antagonists
It was thought originally that CRH was the sole ligand of the CRH receptor. Recent work from Vale et al. demonstrated that a second ligand for the CRH receptors is present in brain, called urocortin2. These peptides bind to a cohort of four receptor subtypes, notably the CRHR1 receptor present in the pituitary corticotrophic cells, the neocortex, BNST and amygdala. The R1 receptor appears to play a major role in the endocrine effects of CRH as well as the anxiogenic and cognitive effects of the peptide. The CRHR2 receptor has three isoforms known as α, β and γ. CRHR2α receptors are present in the lateral septum, dorsal raphe and ventromedial nucleus. This anatomical distribution suggests that the CRHR2α receptor may mediate CRH effects on feeding, body weight and depression. The CRH2β receptor is expressed in the vasculature of the brain and in the choroid plexus. CRH binds preferentially to the CRHR1 receptor while urocortin binds to both R1 and R2 subtypes (E. DeSouza, San Diego, CA, TEM Vol. 10, No. 1, 1999
USA). There are marked species differences in the distribution of receptors. In the human cortex, both CRHR1 and CRHR2 receptors are expressed, whereas CRH binding in the rat cortex is exclusively to the R1 subtype. Therefore, caution must be exercised when using brain tissue to test agents for potential therapeutic efficacy. Transgenic knockout mice lacking the CRHR1 receptor exhibit reduced anxiety, disrupted stress responses and alterations in the adrenal zona fasciculata. CRH mRNA expression in the PVN is greatly augmented, without a change in vasopressin expression or basal ACTH secretion, but upon administering anti-AVP serum to the knockout animals, ACTH levels fall, suggesting that AVP compensates for the lack of CRH. Given the potential clinical usefulness of CRH antagonists in anxiety and depressive disorders, considerable energy has been expended on developing antagonists for CRH. While a number of effective peptide CRH antagonists have been created, their usefulness is limited by their dependence on a vascular route of administration. However, three highly useful small nonpeptide CRH antagonists have been produced. NBI 27914 (D. Grigoridias, San Diego, CA, USA) has high affinity for the CRHR1 receptor. In pituitary cultures, this molecule decreases CRHinduced cAMP accumulation and ACTH release. This compound reverses swim stress-induced anxiety, restores mounting behavior in CRHtransgenic female mice, and prevents middle cerebral artery occlusioninduced neurodegeneration and infarct, suggesting a novel role for CRH in strokes. This molecule does not block CRH-induced vasodilation and therefore is inactive at the CRH2 receptor. Similar to NBI 27914, CP-154,526 (P. Iredale, Groton, CT, USA) is another small molecule antagonist that inhibits stress-induced ACTH. This compound blocks CRH-induced locus coeruleus neuron firing, inhibits the fear-potentiated startle response, and alleviates learned helplessness. CP-154,526 also blocks naltrexone-induced withdrawal in rats treated chronically with
morphine and prevents footshockinduced reinitiation of cocaine-seeking behavior. A number of studies point to a role for the HPA axis in drug abuse and using a CRH antagonist might be a highly effective and novel preventative agent against relapse. A final molecule of interest is DMP696 (R. Zacacek, Wilmington, DE, USA). This molecule, like NBI 27914, has no CRHR2 affinity and exhibits anxiolytic effects. Interestingly, each of these small molecule antagonists intercalates into the membrane near the active binding site of the receptor. While they successfully disrupt CRH receptor activation, mutations of the receptor do not interfere with antagonist binding, suggesting a novel, and yet to be described, molecular mechanism for CRH receptor antagonism. •
Clinical Aspects of Stress and Psychiatric Disorders
The existence of non-peptide CRH antagonists causes one to focus on the clinical usefulness of these molecules in melancholia, obesity or cancer. However, the diverse array of potential functions of CRH in humans first requires closer scrutiny. For example, knocking out CRH gene expression in experimental animals causes a preservation of basal ACTH secretion and a compromised but functional adrenal cortex. The presence of a functional cortex is the result of chromaffin cells residing in the cortex (S. Bornstein, Bethesda, MD, USA). These cells secrete catecholamines that act in a paracrine manner to elicit cortisol synthesis and release. The presence of chromaffin cells in the cortex might account for adrenal tumor release of cortisol in situations where ACTH action is blocked. Further studies are necessary to understand the pathophysiological significance of cortical chromaffin cells. Obesity is characterized by insulin resistance, overactivity of the HPA axis and inhibition of the hypothalamic–pituitary–gonadal axis. The newly described adipose tissue hormone, leptin, is a central player in obesity. Many obese individuals are resistant to the actions of leptin or lack this hormone completely. Leptin also 7
inhibits the HPA axis by acting on hypothalamic targets. The interplay between leptin and the HPA axis is important in understanding the metabolic regulation of the HPA axis (J. Flier, Boston, MA, USA). Finally, a large literature supports the notion that depressed patients have dysfunctional HPA axis activity. However, closer scrutiny of depressed patients over a longer period reveals that the hypersecretion of cortisol in these patients occurs for only several days out of each nine days. Nevertheless, the ACTH increment may be markedly increased. Sampling blood in depressed individuals does not indicate adrenal insensitivity or a pituitary ACTH defect. Therefore, the primary defect in
depressed patients might reside at CRH producing CNS loci other than the hypothalamus (J. Licinio, Bethesda, MD, USA). While our understanding of the regulation of the stress response has advanced considerably since the discovery of CRH in 1981, a number of questions about the interplay between the HPA axis and the function of the brain remain to be addressed. For example, what is the molecular basis for aging-induced cognitive decline and how do CRH mechanisms in the amygdala participate in anxiety behaviors? Hopefully, by the time the next Neuroendocrine Workshop on Stress takes place the answers to these questions will be available.
References 1 Selye, H. (1936) Syndrome produced by diverse nocuous agents. Nature 138, 32–35 2 Vaughan, J. et al. (1995) Urocortin, a mammalian neuropeptide related to fish urotensin I and to corticotropinreleasing factor. Nature 378, 287–292 3 Vale, W., Speiss, J., Rivier, C. and Rivier, J. (1981) Characterization of a 41residue ovine hypothalamic peptide that stimulates secretion of corticotropin and beta-endorphin. Science 213, 1394–1397 4 Lupien, S.J. et al. (1998) Cortisol levels during human aging predict hippocampal atrophy and memory deficits. Nat. Neurosci. 1, 69–73 5 Liu, D. et al. (1997) Maternal care, hippocampal glucocorticoid receptors and hypothalamic–pituitary–adrenal responses to stress. Science 277, 1659–1662
TEM changes in style of citations to go into effect as of 1/1/99 (in order to match the style of other Trends journals published in Cambridge) • References are cited in the text by consecutive numbers in superscript. When two are cited in order the format is11,12; where more than two11–14. The only exception to this rule is where the reference immediately follows an abbreviation, acronym or chemical name, at which point the references should be cited as (Refs 11, 12). References in reviews are quoted as (reviewed in Ref. 13). • If unpublished material is cited, it is referred to as (M.K. Smith and B. Jones, unpublished) or (R. Brines, pers. commun.). Keep such citations to a minimum, and avoid them for data important to the gist of the review. Numbered references must be at least in press. • A reference list of not more than 40 references should be provided. • Not alphabetical, but in numerical order (same order as in text). Introduce each with the appropriate number (1 to 54 etc.) in bold Roman typeface. • One author: Blair, T.W. (1994). Title (bold Roman). Endocrinology 125, 334–337 • Two authors: Blair, T.W. and Thatcher, M. (1996). Title. J. Endocrinol. 39, 45–51 • Up to six authors: Blair, T.W., Thatcher, M., Carroll, H. and Woo, P.T.K. (1993). Title. J. Biol. Chem. 239, 23356–23382 • More than six authors: Blair, T.W. et al. (1998). Title. Proc. Natl. Acad. Sci. U. S. A. 101, 1111–1121 • Chapter in book: Woo, P.T.K. and Wehnert, S.D. (1993). Title. in Parasitic Protozoa (Vol. 2) (2nd edn) (Kreier, J.F. and Baker, J.R., eds) pp 157–256, Academic Press • Abstract: instead of last page, write (abstr.). • Common abbreviations: Eur. J. Immunol. Proc. Natl. Acad. Sci. U. S. A. J. Exp. Med. • One word titles are spelt out in full, e.g. Nature, Cell, Science etc. • Note: several issues ago, TEM switched from mg/mL to mg ml21 (in all similar expressions). • Cite figures as (Fig. 1 or Fig. 2a).
8
TEM Vol. 10, No. 1, 1999
importance of apoptosis in the development and homeostasis of total organism cell number, as well as in the editing of damaged or mutant cells. Current studies are aimed at identifying the molecular entities that carry out apoptotic processes, and the understanding of their activation by various apoptotic signals. T lymphocytes serve as an excellent model for such studies because they undergo apoptosis in response to glucocorticoids and other compounds. In T lymphocytes, DNA fragmentation and caspase activation occur only in cells that have undergone a reduction in cell volume. In addition, recent evidence has indicated that early ion fluxes are critical for the activation of apoptotic pathways. L.B. Hendry (Augusta, GA, USA) reported that a key step in gene regulation by steroids is the binding of hormone to receptors that interact with DNA. Steroids insert stereospecifically between base pairs, and this fit
correlates with activity. It was proposed that receptors mediate insertion of hormone into DNA, and that this step is rate limiting. A.M. Soto (Boston, MA, USA) described the manner in which the endocrine and reproductive effects of environmental agents such as pesticides and industrial chemicals are believed to affect wildlife and humans. Such compounds have the ability to: (1) mimic the effects of endogenous hormones; (2) antagonize the effects of endogenous hormones: (3) disrupt the synthesis and metabolism of endogenous hormones; and (4) disrupt the synthesis and metabolism of hormone receptors. A single chemical can produce neurotoxic, estrogenic and antiandrogenic effects. The systematic identification of these compounds before they are introduced into widespread use will require the collaboration of the scientific community, government agencies, the industrial sector and the public at large.
•
The 1998 Neuroendocrine Workshop on Stress: a Report on the American Neuroendocrine Society Annual Workshop James I. Koenig
Since Hans Selye first defined the stress response1, investigators have pursued the identification of the major biochemical mediators and the loci of their effects within the brain tirelessly. In recent years, these efforts have culminated in the identification of corticotropin-releasing hormone (CRH) and vasopressin (AVP) as the main mediators of the response to stress. A newly added mediator whose physiological function in the stress response
J.I. Koenig is at the Maryland Psychiatric Research Center, University of Maryland School of Medicine, PO Box 21247, Baltimore, MD 21228, USA.
TEM Vol. 10, No. 1, 1999
is yet to be established is urocortin2. CRH and urocortin exert their effects by binding to four different CRH receptors in brain and pituitary, as well as binding to CRH-binding protein, which modifies the actions of CRH. The 1998 Neuroendocrine Workshop on Stress was held in New Orleans, LA, USA, from 21–23 June 1998, and was attended by over 125 investigators. The workshop focused on the latest developments in the neuroendocrinology of stress and on the identification, function and clinical utility of these moieties for central nervous system (CNS) and peripheral disorders.
Molecular Mechanisms of Central Stress Integration
The 41-amino acid peptide CRH, isolated and characterized originally by Vale et al.3, is the most prominent stimulatory factor controlling pituitary adrenocorticotropin (ACTH) secretion. Previous studies have documented clearly that expression of the CRH gene and the subsequent secretion of CRH by hypothalamic neurons are both under the strong negative influence of the adrenal glucocorticoid hormones, cortisol and corticosterone. However, in non-hypothalamic regions of the brain, such as the amygdala, glucocorticoids exert a stimulatory effect on CRH gene expression. This tissue-specific regulation could be a direct effect of the activated glucocorticoid receptor on the CRH gene, or as discussed by Dr A. Seasholtz (Ann Arbor, MI, USA), both the positive and negative effects of glucocorticoids might arise as a result of novel interactions between the glucocorticoid receptor and cAMP-dependent mechanisms, most likely involving novel interactions with cAMP response
1043-2760/99/$ – see front matter © 1999 Elsevier Science. All rights reserved. PII: S1043-2760(98)00114-3
5
element-binding protein (CREB). This finding is very important for investigators exploring the molecular basis of fear and anxiety behaviors. However, CREB has many roles in CRH neurons and during the stress response. For example, Dr P. Sawchenko (San Diego, CA, USA) showed that in response to ether inhalation stress the phosphorylated form of CREB (pCREB) rises very quickly (within 5 min) in CRH neurons. This rapid rise in pCREB is followed closely by the appearance of CRH heteronuclear RNA (hnRNA). On the other hand, in AVP neurons AVP hnRNA does not increase until 2 h after the ether exposure and the increase is preceded selectively by the appearance of c-Fos. Interruption of protein synthesis by cycloheximide blocks the stress-induced elevation in c-Fos and AVP hnRNA but not the induction of pCREB and CRH hnRNA. Interestingly, however, adrenalectomy to remove the glucocorticoid signal had little effect on the ether-induced appearance of CRH hnRNA but delayed the induction of AVP hnRNA. These studies suggest that stress activates CNS neural pathways that impinge upon CRH cells in the paraventricular nucleus (PVN) and that these cells are able to respond rapidly to stressful events without needing to produce new protein moieties. Other stress-responsive cells, like AVP cells, are not necessary for the initial phases of the response and therefore use slower, protein synthesis-dependent mechanisms to activate factors responsible for more sustained responses to a challenge. The neural mechanism(s) involved in responding to challenges are beginning to be elucidated. Dr J. Herman (Lexington, KY, USA) described studies demonstrating that the disinhibition of γ-aminobutyric acid (GABA)-containing neurons around the PVN might play a critical role in the response to stress. GABAcontaining neurons in the bed nucleus of the stria terminalis (BNST), the preoptic area (POA) and the dorsomedial nucleus project to the PVN and modulate hypothalamic–pituitary–adrenal (HPA) function. Additionally, chronic stress exposure induces GAD65 mRNA 6
production in these cells and decreases GABAB receptor mRNA in the PVN, confirming a role for GABA. Pathological alterations in HPA axis function occur in some psychiatric disorders, but dysfunction of the HPA axis also occurs during normal aging. The decline of cognitive functions seen during aging is related to increased glucocorticoid levels4 and changes in the hippocampal glucocorticoid receptor to mineralocorticoid receptor ratio. However, treating elderly animals with antidepressant agents augments serotoninergic activity and induces the production of nerve growth factor 1A (NGF1A) mRNA. Increased levels of NGF1A upregulate glucocorticoid receptor expression and, thereby, reverse aging-induced cognitive decline (J. Yao, Edinburgh, UK). •
Developmental Stress and the HPA Axis
The interplay of genetic and environmental factors shapes the neurocircuitry of the developing CNS. Stressful events and the concomitant increase in glucocorticoids might alter the connectivity of CNS cells or may disrupt cellular genesis in the developing brain. For example, granule cells of the hippocampus develop during the stress hyporesponsive period (SHRP). During this time, circulating glucocorticoids are maintained at very low levels, so as not to disrupt the hippocampal development. Experiments performed by Dr E. Gould (Princeton, NJ, USA) demonstrated that administering glucocorticoids to animals during this critical period results in a reduction in the number of new cells added to the hippocampus. Moreover, exposure to extremely stressful situations, which raise glucocorticoid levels, also yields the same result. However, the SHRP that was proposed originally to span from postnatal Days 5–14 is a misnomer. Dr S. Levine (Wilmington, DE, USA), performing studies in neonatal rats, demonstrated that the ACTH responses to stress, even mild stress, during the SHRP were very robust, while glucocorticoid responses were minimal. Stronger stresses, like ether exposure or
lipopolysaccharide injection, did elicit a corticosterone response, albeit very meager. Thus, this SHRP is more probably a glucocorticoid hyporesponsive period. However, the presence of the dam during this time also diminishes the reactivity to stress5. Taking advantage of this facet of the mother–offspring relationship, Dr P. Plotsky et al. (Atlanta, GA, USA) developed the maternal separation stress, where pups are separated from their mothers for 180 min on Days 2–10 of life. At adulthood, these separated pups show enhanced anxiety, moderate anhedonia, alcohol preferences and dysregulation of the HPA axis, with increased expression of CRH in the PVN, amygdala and BNST. These animals also have increased acoustic startle behavior, defensive withdrawal and decreased water maze performance. Clearly, environmental factors and maternal factors strongly influence the activity of the HPA axis, causing alterations in the connectivity of the brain, and potentially changing adult behavioral patterns. •
Steroid Hormone Interactions and Stress
Gonadal steroid hormones are known to have both activational and organizational effects within the CNS. Sexually dimorphic brain structures are well documented in the literature. The HPA axis is also sexually dimorphic as a consequence of the gonadal steroid milieu. During the neonatal period, gonadectomy enhances corticosterone responsiveness to stress in later life, while either estrogen or testosterone treatment eliminates the enhanced responsiveness (C. McCormick, Lewiston, ME, USA). In adult animals, estrogens cause enhancement of the HPA axis responsiveness and testosterone diminishes the response of the axis to stress. The effect of estrogen might arise as a result of the presence of the beta form of the estrogen receptor in PVN neurons (R. Handa, Maywood, IL, USA). On the other hand, testosterone’s inhibitory effects might be mediated by androgen receptors in cells of the BNST and POA, which are known to contain GABA and to project TEM Vol. 10, No. 1, 1999
to the PVN. Estrogen might also modulate HPA axis activity via actions within the hippocampus (N. Weiland, New York, NY, USA) and its ability to increase synaptic densities of pyramidal cells. At the molecular level, interplay between estrogen receptors and glucocorticoid receptors is known to take place. In general, estrogen stimulates transcriptional events involving AP-1 sites, while glucocorticoids are inhibitory. Although the molecular interplay involves formation of a complex of activated receptors with c-Jun, alternative pathways involving inactivators have also been reported. Estrogen appears to coactivate the CREB-binding protein and prevents the inhibitory effects of the glucocorticoid receptor interacting protein (GRIP) (R. Uht, San Francisco, CA, USA). A careful evaluation of the physiological relevance of these novel molecular interactions remains to be undertaken. •
Molecular Biology of CRH Receptors and the Biology of CRH Antagonists
It was thought originally that CRH was the sole ligand of the CRH receptor. Recent work from Vale et al. demonstrated that a second ligand for the CRH receptors is present in brain, called urocortin2. These peptides bind to a cohort of four receptor subtypes, notably the CRHR1 receptor present in the pituitary corticotrophic cells, the neocortex, BNST and amygdala. The R1 receptor appears to play a major role in the endocrine effects of CRH as well as the anxiogenic and cognitive effects of the peptide. The CRHR2 receptor has three isoforms known as α, β and γ. CRHR2α receptors are present in the lateral septum, dorsal raphe and ventromedial nucleus. This anatomical distribution suggests that the CRHR2α receptor may mediate CRH effects on feeding, body weight and depression. The CRH2β receptor is expressed in the vasculature of the brain and in the choroid plexus. CRH binds preferentially to the CRHR1 receptor while urocortin binds to both R1 and R2 subtypes (E. DeSouza, San Diego, CA, TEM Vol. 10, No. 1, 1999
USA). There are marked species differences in the distribution of receptors. In the human cortex, both CRHR1 and CRHR2 receptors are expressed, whereas CRH binding in the rat cortex is exclusively to the R1 subtype. Therefore, caution must be exercised when using brain tissue to test agents for potential therapeutic efficacy. Transgenic knockout mice lacking the CRHR1 receptor exhibit reduced anxiety, disrupted stress responses and alterations in the adrenal zona fasciculata. CRH mRNA expression in the PVN is greatly augmented, without a change in vasopressin expression or basal ACTH secretion, but upon administering anti-AVP serum to the knockout animals, ACTH levels fall, suggesting that AVP compensates for the lack of CRH. Given the potential clinical usefulness of CRH antagonists in anxiety and depressive disorders, considerable energy has been expended on developing antagonists for CRH. While a number of effective peptide CRH antagonists have been created, their usefulness is limited by their dependence on a vascular route of administration. However, three highly useful small nonpeptide CRH antagonists have been produced. NBI 27914 (D. Grigoridias, San Diego, CA, USA) has high affinity for the CRHR1 receptor. In pituitary cultures, this molecule decreases CRHinduced cAMP accumulation and ACTH release. This compound reverses swim stress-induced anxiety, restores mounting behavior in CRHtransgenic female mice, and prevents middle cerebral artery occlusioninduced neurodegeneration and infarct, suggesting a novel role for CRH in strokes. This molecule does not block CRH-induced vasodilation and therefore is inactive at the CRH2 receptor. Similar to NBI 27914, CP-154,526 (P. Iredale, Groton, CT, USA) is another small molecule antagonist that inhibits stress-induced ACTH. This compound blocks CRH-induced locus coeruleus neuron firing, inhibits the fear-potentiated startle response, and alleviates learned helplessness. CP-154,526 also blocks naltrexone-induced withdrawal in rats treated chronically with
morphine and prevents footshockinduced reinitiation of cocaine-seeking behavior. A number of studies point to a role for the HPA axis in drug abuse and using a CRH antagonist might be a highly effective and novel preventative agent against relapse. A final molecule of interest is DMP696 (R. Zacacek, Wilmington, DE, USA). This molecule, like NBI 27914, has no CRHR2 affinity and exhibits anxiolytic effects. Interestingly, each of these small molecule antagonists intercalates into the membrane near the active binding site of the receptor. While they successfully disrupt CRH receptor activation, mutations of the receptor do not interfere with antagonist binding, suggesting a novel, and yet to be described, molecular mechanism for CRH receptor antagonism. •
Clinical Aspects of Stress and Psychiatric Disorders
The existence of non-peptide CRH antagonists causes one to focus on the clinical usefulness of these molecules in melancholia, obesity or cancer. However, the diverse array of potential functions of CRH in humans first requires closer scrutiny. For example, knocking out CRH gene expression in experimental animals causes a preservation of basal ACTH secretion and a compromised but functional adrenal cortex. The presence of a functional cortex is the result of chromaffin cells residing in the cortex (S. Bornstein, Bethesda, MD, USA). These cells secrete catecholamines that act in a paracrine manner to elicit cortisol synthesis and release. The presence of chromaffin cells in the cortex might account for adrenal tumor release of cortisol in situations where ACTH action is blocked. Further studies are necessary to understand the pathophysiological significance of cortical chromaffin cells. Obesity is characterized by insulin resistance, overactivity of the HPA axis and inhibition of the hypothalamic–pituitary–gonadal axis. The newly described adipose tissue hormone, leptin, is a central player in obesity. Many obese individuals are resistant to the actions of leptin or lack this hormone completely. Leptin also 7
inhibits the HPA axis by acting on hypothalamic targets. The interplay between leptin and the HPA axis is important in understanding the metabolic regulation of the HPA axis (J. Flier, Boston, MA, USA). Finally, a large literature supports the notion that depressed patients have dysfunctional HPA axis activity. However, closer scrutiny of depressed patients over a longer period reveals that the hypersecretion of cortisol in these patients occurs for only several days out of each nine days. Nevertheless, the ACTH increment may be markedly increased. Sampling blood in depressed individuals does not indicate adrenal insensitivity or a pituitary ACTH defect. Therefore, the primary defect in
depressed patients might reside at CRH producing CNS loci other than the hypothalamus (J. Licinio, Bethesda, MD, USA). While our understanding of the regulation of the stress response has advanced considerably since the discovery of CRH in 1981, a number of questions about the interplay between the HPA axis and the function of the brain remain to be addressed. For example, what is the molecular basis for aging-induced cognitive decline and how do CRH mechanisms in the amygdala participate in anxiety behaviors? Hopefully, by the time the next Neuroendocrine Workshop on Stress takes place the answers to these questions will be available.
References 1 Selye, H. (1936) Syndrome produced by diverse nocuous agents. Nature 138, 32–35 2 Vaughan, J. et al. (1995) Urocortin, a mammalian neuropeptide related to fish urotensin I and to corticotropinreleasing factor. Nature 378, 287–292 3 Vale, W., Speiss, J., Rivier, C. and Rivier, J. (1981) Characterization of a 41residue ovine hypothalamic peptide that stimulates secretion of corticotropin and beta-endorphin. Science 213, 1394–1397 4 Lupien, S.J. et al. (1998) Cortisol levels during human aging predict hippocampal atrophy and memory deficits. Nat. Neurosci. 1, 69–73 5 Liu, D. et al. (1997) Maternal care, hippocampal glucocorticoid receptors and hypothalamic–pituitary–adrenal responses to stress. Science 277, 1659–1662
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TEM Vol. 10, No. 1, 1999