Alexandre V. Patchev

Dynamic DNA methylation programs persistent adverse effects of early-life stress

Adverse early life events can induce long-lasting changes in physiology and behavior. We found that early-life stress (ELS) in mice caused enduring hypersecretion of corticosterone and alterations in passive stress coping and memory. This phenotype was accompanied by a persistent increase in arginine vasopressin (AVP) expression in neurons of the hypothalamic paraventricular nucleus and was reversed by an AVP receptor antagonist. Altered Avp expression was associated with sustained DNA hypomethylation of an important regulatory region that resisted age-related drifts in methylation and centered on those CpG residues that serve as DNA-binding sites for the methyl CpG–binding protein 2 (MeCP2). We found that neuronal activity controlled the ability of MeCP2 to regulate activity-dependent transcription of the Avp gene and induced epigenetic marking. Thus, ELS can dynamically control DNA methylation in postmitotic neurons to generate stable changes in Avp expression that trigger neuroendocrine and behavioral alterations that are frequent features in depression.

Depletion of the Neural Precursor Cell Pool by Glucocorticoids

Glucocorticoids (GCs) are indicated for a number of conditions in obstetrics and perinatal medicine; however, the neurodevelopmental and long‐term neurological consequences of early‐life GC exposure are still largely unknown. Preclinical studies have demonstrated that GCs have a major influence on hippocampal cell turnover by inhibiting neurogenesis and stimulating apoptosis of mature neurons. Here we examined the fate of the limited pool of neural progenitor cells (NPCs) after GC administration during neonatal development; the impact of this treatment on hippocampal structure was also studied.

Methylation at the CpG island shore region upregulates Nr3c1 promoter activity after early-life stress

Early-life stress (ELS) induces long-lasting changes in gene expression conferring an increased risk for the development of stress-related mental disorders. Glucocorticoid receptors (GR) mediate the negative feedback actions of glucocorticoids (GC) in the paraventricular nucleus (PVN) of the hypothalamus and anterior pituitary and therefore play a key role in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis and the endocrine response to stress. We here show that ELS programs the expression of the GR gene (Nr3c1) by site-specific hypermethylation at the CpG island (CGI) shore in hypothalamic neurons that produce corticotropin-releasing hormone (Crh), thus preventing Crh upregulation under conditions of chronic stress. CpGs mapping to the Nr3c1 CGI shore region are dynamically regulated by ELS and underpin methylation-sensitive control of this regions insulation-like function via Ying Yang 1 (YY1) binding. Our results provide new insight into how a genomic element integrates experience-dependent epigenetic programming of the composite proximal Nr3c1 promoter, and assigns an insulating role to the CGI shore.

Insidious adrenocortical insufficiency underlies neuroendocrine dysregulation in TIF-2 deficient mice.

The transcription‐intermediary‐factor‐2 (TIF‐2) is a coactivator of the glucocorticoid receptor (GR), and its disruption would be expected to influence glucocorticoid‐mediated control of the hypothalamopituitary‐adrenal (HPA) axis. Here, we show that its targeted deletion in mice is associated with altered expression of several glucocorticoid‐dependent components of HPA regulation (e.g., corticotropin‐releasing hormone, vasopressin, ACTH, glucocorticoid receptors), suggestive of hyperactivity under basal conditions. At the same time, TIF‐2‐/‐mice display significantly lower basal corticosterone levels and a sluggish and blunted initial secretory response to brief emotional and prolonged physical stress. Subsequent analysis revealed this discrepancy to result from pronounced aberrations in the structure and function of the adrenal gland, including the cytoarchitectural organization of the zona fasciculata and basal and stress‐induced expression of key elements of steroid hormone synthesis, such as the steroidogenic acute regulatory (StAR) protein and 3β‐hydroxysteroid dehydrogenase (3(‐HSD). In addition, altered expression levels of two nuclear receptors, DAX‐1 and steroidogenic factor 1 (SF‐1), in the adrenal cortex strengthen the view that TIF‐2 deletion disrupts adrenocortical development and steroid biosynthesis. Thus, hyperactivity of the hypothalamo‐pituitary unit is ascribed to insidious adrenal insufficiency and impaired glucocorticoid feedback. Patchev, A. V., Fischer, D., Wolf, S. S., Götz, F., Gehin, M., Chambon, P., Patchev, V. K., and Almeida O. F. X. Insidious adrenocortical insufficiency underlies neuroendocrine dysregulation in TIF‐2 deficient mice. FASEB J. 21, 231–238 (2007)

Non-receptor tyrosine kinases integrate fast glucocorticoid signaling in hippocampal neurons

Background: The intracellular signaling cascades through which corticosterone rapidly alters neuronal activity are poorly defined. Results: Corticosterone alters glutamatergic transmission by activating diverse GPCR-dependent signaling pathways. Conclusion: Corticosterone-induced changes in neuronal excitability are initiated at the plasma membrane. Significance: The sequential recruitment and integration of diverse signaling cascades by corticosterone adds to the understanding of how steroids rapidly alter neuronal function. Despite numerous descriptions of rapid effects of corticosterone on neuronal function, the intracellular mechanisms responsible for these changes remain elusive. The present comprehensive analysis reveals that signaling from a membrane-located G protein-coupled receptor activates PKC, Akt/PKB, and PKA, which subsequently trigger the phosphorylation of the tyrosine kinases Pyk2, Src, and Abl. These changes induce rapid cytoskeletal rearrangements (increased PSD-95 co-clustering) within the post-synaptic density; these events are accompanied by increased surface NMDA receptor expression, reflecting corticosterone-induced inhibition of NMDA receptor endocytosis. Notably, none of these signaling mechanisms require de novo protein synthesis. The observed up-regulation of ERK1/2 (downstream of NMDA receptor signaling) together with the fact that c-Abl integrates cytoplasmic and nuclear functions introduces a potential mechanism through which rapid signaling initiated at the plasma membrane may eventually determine the long term integrated response to corticosterone by impacting on the transcriptional machinery that is regulated by classical, nuclear mineralocorticoid, and glucocorticoid receptors.

Androgen receptor-mediated regulation of adrenocortical activity in the sand rat, Psammomys obesus

The wild sand rat, Psammomys obesus, displays seasonal variations in adrenocortical activity that parallel those of testicular activity, indicating functional cross-talk between the hypothalamo-pituitary-adrenal and hypothalamo-pituitary–gonadal axes. In the present study, we examined androgen receptor (AR)-mediated actions of testicular steroids in the regulation of adrenocortical function in the sand rat. Specifically, we examined the expression of AR in the adrenal cortex, as well as adrenal apoptosis in male sand rats that had been surgically castrated or castrated and supplemented with testosterone; biochemical indices of adrenocortical function and hormone profiles were also measured. Orchiectomy was followed by an increase in adrenocorticotropic hormone secretion from the anterior pituitary and subsequently, increased adrenocortical activity; the latter was evidenced by orchiectomy-induced increases in the adrenal content of cholesterol and lipids as well as adrenal hypertrophy (seen as an elevation of the RNA/DNA ratio). Further, androgen deprivation respectively up- and downregulated the incidence of apoptosis within the glucocorticoid-producing zona fasciculata and sex steroid-producing zona reticularis. Interestingly, orchiectomy resulted in increased expression of AR in the zona fasciculata. All of the orchiectomy-induced cellular and biochemical responses were reversible after testosterone substitution therapy. Together, these data suggest that adrenocortical activity in the sand rat is seasonally modulated by testicular androgens that act through AR located in the adrenal cortex itself.

Restoring serotonergic homeostasis in the lateral hypothalamus rescues sleep disturbances induced by early-life obesity

Early-life obesity predisposes to obesity in adulthood, a condition with broad medical implications including sleep disorders, which can exacerbate metabolic disturbances and disrupt cognitive and affective behaviors. In this study, we examined the long-term impact of transient peripubertal diet-induced obesity (ppDIO, induced between 4 and 10 weeks of age) on sleep–wake behavior in male mice. EEG and EMG recordings revealed that ppDIO increases sleep during the active phase but reduces resting-phase sleep quality. This impaired sleep phenotype persisted for up to 1 year, although animals were returned to a non-obesiogenic diet from postnatal week 11 onwards. To better understand the mechanisms responsible for the ppDIO-induced alterations in sleep, we focused on the lateral hypothalamus (LH). Mice exposed to ppDIO did not show altered mRNA expression levels of orexin and melanin-concentrating hormone, two peptides that are important for sleep–wake behavior and food intake. Conversely, the LH of ppDIO-exposed mice had reduced contents of serotonin (5-hydroxytryptamine, 5-HT), a neurotransmitter involved in both sleep–wake and satiety regulation. Interestingly, an acute peripheral injection of the satiety-signaling peptide YY 3–36 increased 5-HT turnover in the LH and ameliorated the ppDIO-induced sleep disturbances, suggesting the therapeutic potential of this peptide. These findings provide new insights into how sleep–wake behavior is programmed during early life and how peripheral and central signals are integrated to coordinate sleep. SIGNIFICANCE STATEMENT Adult physiology and behavior are strongly influenced by dynamic reorganization of the brain during puberty. The present work shows that obesity during puberty leads to persistently dysregulated patterns of sleep and wakefulness by blunting serotonergic signaling in the lateral hypothalamus. It also shows that pharmacological mimicry of satiety with peptide YY3–36 can reverse this neurochemical imbalance and acutely restore sleep composition. These findings add insight into how innate behaviors such as feeding and sleep are integrated and suggest a novel mechanism through which diet-induced obesity during puberty imposes its long-lasting effects on sleep–wake behavior.

Antidepressant responsiveness in adulthood is permanently impaired after neonatal destruction of the neurogenic pool

The dynamic turnover of hippocampal neurons is implicated in the regulation of cognitive and affective behavior. Extending our previous demonstration that administration of dexamethasone (ND) to neonatal rats depletes the resident population of neural precursor cells (NPC) and restrains the size of the neurogenic regions, we now show that the adverse effects of ND persist into adulthood. Specifically, ND impairs repletion of the neurogenic pool and neurogenesis; ND also compromises cognitive performance, the ability to actively adapt to an acute stressor and, the efficacy of glucocorticoid (GC) negative feedback. Interestingly, although ND depletes the neurogenic pool, it does not permanently abolish the proliferative machinery of the residual NPC population; however, ND increases the susceptibility of hippocampal granule neurons to apoptosis. Although the antidepressant fluoxetine (FLX) reverses the latter phenomenon, it does not replenish the NPC pool. Treatment of ND-treated adult rats with FLX also improves GC negative feedback, albeit without rescuing the deleterious effects of ND on behavior. In summary, ND leads to protracted disruption of mental functions, some of which are resistant to antidepressant interventions. We conclude that manipulation of the NPC pool during early life may jeopardize the therapeutic potential of antidepressants in adulthood.

S29-01 Sex-specific long-term behavioral and neuroendocrine consequences oftransient peripubertal metabolic challenges

Interaction between neuroendocrine stress and metabolic signals has become increasingly linked to the pathogenesis of affective and cognitive disorders. Independent evidence shows that nuclear receptors represent an essential interface of communication between these signals and the central nervous system. On the premise that lifelong brain functions are subject to programming by stressful and metabolic signals during early life, we induced voluntary hyperphagia in prepubertal male and female rats by providing them with a cafeteria-like choice between normal chow and foods with high motivational value (palatable, high fat/high energy). The cafeteria diet was withdrawn from one subset of animals during early adulthood (postnatal day, PND 80), and several endpoints of interest were monitored until PND 200. While both sexes displayed similar metabolic outcomes during exposure to the cafeteria diet, there were substantial sex differences in the metabolic, behavioral and endocrine consequences of withdrawal of palatable food. Consistently increased food consumption and body mass gain were recorded in males, whereas females that were pre-exposed to the cafeteria diet showed clear signs of adrenocortical hyperactivity. Thus, besides verification of sex-specific mal-programming of appetitive behavior and metabolic set-points by early life events, we also demonstrate evidence for differential translation of metabolic challenges during peripubertal development into aberrations suggestive of altered neuroendocrine responsiveness to stress.