Shusaku Uchida

Epigenetic Status of Gdnf in the Ventral Striatum Determines Susceptibility and Adaptation to Daily Stressful Events

Stressful events during adulthood are potent adverse environmental factors that can predispose individuals to psychiatric disorders, including depression; however, many individuals exposed to stressful events can adapt and function normally. While stress vulnerability may influence depression, the molecular mechanisms underlying the susceptibility and adaptation to chronic stress within the brain are poorly understood. In this study, two genetically distinct mouse strains that exhibit different behavioral responses to chronic stress were used to demonstrate how the differential epigenetic status of the glial cell-derived neurotrophic factor (Gdnf) gene in the ventral striatum modulates susceptibility and adaptation to chronic stress. Our results suggest that the histone modifications and DNA methylation of the Gdnf promoter have crucial roles in the control of behavioral responses to chronic stress. Our data provide insights into these mechanisms, suggesting that epigenetic modifications of Gdnf, along with genetic and environmental factors, contribute to behavioral responses to stress.

Early Life Stress Enhances Behavioral Vulnerability to Stress through the Activation of REST4-Mediated Gene Transcription in the Medial Prefrontal Cortex of Rodents

There is growing evidence suggesting that early life events have long-term effects on the neuroendocrine and behavioral developments of rodents. However, little is known about the involvement of early life events in the susceptibility to subsequent stress exposure during adulthood. The present study characterized the effect of maternal separation, an animal model of early life adversity, on the behavioral response to repeated restraint stress in adult rats and investigated the molecular mechanism underlying behavioral vulnerability to chronic stress induced by the maternal separation. Rat pups were separated from the dams for 180 min per day from postnatal day 2 through 14 (HMS180 rats). We found that, as young adults, HMS180 rats showed a greater hypothalamic-pituitary-adrenal axis response to acute restraint stress than nonseparated control rats. In addition, repeatedly restrained HMS180 rats showed increased depression-like behavior and an anhedonic response compared with nonrestrained HMS180 rats. Furthermore, HMS180 rats showed increased expression of REST4, a neuron-specific splicing variant of the transcriptional repressor REST (repressor element-1 silencing transcription factor), and a variety of REST target gene mRNAs and microRNAs in the medial prefrontal cortex (mPFC). Finally, REST4 overexpression in the mPFC of neonatal mice via polyethyleneimine-mediated gene transfer enhanced the expression of its target genes as well as behavioral vulnerability to repeated restraint stress. In contrast, REST4 overexpression in the mPFC of adult mice did not affect depression-like behaviors after repeated stress exposure. These results suggest that the activation of REST4-mediated gene regulation in the mPFC during postnatal development is involved in stress vulnerability.

Characterization of the vulnerability to repeated stress in Fischer 344 rats: possible involvement of microRNA-mediated down-regulation of the glucocorticoid receptor.

In the present study, we established and characterized an animal model of vulnerability to repeated stress. We found that control Sprague–Dawley (SD) rats showed a gradual decrease in the HPA axis response following 14 days of repeated restraint stress, whereas Fischer 344 (F344) rats did not show such HPA axis habituation. Similar habituation was observed in the expression of c‐fos mRNA, corticotropin‐releasing hormone hnRNA, and phospho‐CREB and phospho‐ERK proteins in the hypothalamic paraventricular nucleus (PVN) of SD rats, but not in the F344 rats. In addition, repeatedly restrained F344 rats exhibited decreased cell proliferation in the dentate gyrus of the hippocampus and increased anxiety‐related behaviours, while repeatedly restrained SD rats exhibited a selective enhancement of hippocampal cell proliferation in the ventral area. Moreover, we found a lower expression of glucocorticoid receptor (GR) protein, but not mRNA, in the PVN of F344 rats compared to SD rats. We also identified that microRNA (miR)‐18a inhibited translation of GR mRNA in cultured neuronal cells and that increased expression of miR‐18a in the PVN was observed in F344 rats compared with SD rats. These strain differences in GR protein levels were not found in the hippocampus and prefrontal cortex, and the expression of miR‐18a was much lower in these brain regions than in the PVN. Our results suggest that F344 rats could be a useful animal model for studying vulnerability to repeated stress, and that miR‐18a‐mediated down‐regulation of GR translation may be an important factor to be considered in susceptibility to stress‐related disorders.

Altered expression of neurotrophic factors in patients with major depression.

There is an abundance of evidence suggesting the involvement of altered levels of expression of neurotrophic factors in the pathophysiology of neuropsychiatric disorders. Although postmortem brain studies have indicated the alterations in the expression levels of neurotrophic factors in mood disorder patients, it is unclear whether these changes are state- or trait-dependent. In this study, we examined the expression levels of the members of the glial cell line-derived neurotrophic factor (GDNF) family (GDNF, artemin (ARTN), neurturin, and persephin), brain-derived neurotrophic factor, nerve growth factor, neurotrophin-3 (NT-3), and neurotrophin-4 mRNAs by using quantitative real-time PCR method in peripheral blood cells of patients with major depressive and bipolar disorders in both a current depressive and a remissive states. Reduced expression levels of GDNF, ARTN, and NT-3 mRNAs were found in patients with major depressive disorder in a current depressive state, but not in a remissive state. Altered expressions of these mRNAs were not found in patients with bipolar disorder. Our results suggest that the changes in the expression levels of GDNF, ARTN, and NT-3 mRNAs might be state-dependent and associated with the pathophysiology of major depression.

Altered sirtuin deacetylase gene expression in patients with a mood disorder.

Sirtuins are a family of NAD+-dependent enzymes that regulate cellular functions through deacetylation of various proteins. Although recent reports have suggested an important role of deacetylases (i.e., histone deacetylases) in mood disorders and antidepressant action, the involvement of sirtuins in the pathophysiology of mood disorders is largely unknown. In this study, we aimed to determine whether there are alterations in sirtuin mRNA expression in peripheral white blood cells of patients with a mood disorder. Also, to examine whether the altered sirtuin mRNA expression is state- or trait-dependent, mood disorder patients who were in a remissive state were assessed. We used quantitative real-time polymerase chain reaction to measure the mRNA levels of seven sirtuin isoforms (SIRT1-7) in peripheral white blood cells of patients with major depressive disorder (MDD) or bipolar disorder (BPD) during depressive and remissive states and in normal healthy subjects. The SIRT1, 2 and 6 mRNA levels in MDD and BPD patients decreased significantly in those who were in a depressive state compared to healthy controls, whereas the expression of those mRNAs in both MDD and BPD of patients in a remissive state were comparable to those in healthy controls. Thus, our data suggest that altered SIRT1, 2 and 6 expression is state-dependent and might be associated with the pathogenesis and/or pathophysiology of mood disorders.

Reduced expression of glyoxalase-1 mRNA in mood disorder patients.

Glyoxalase-1 (Glo1) is an antioxidant enzyme which detoxifies alpha-ketoaldehydes to prevent the accumulation of pro-oxidant compounds, such as methylglyoxal, in all cell types. Glo1 has been suggested to be involved in anxiety disorders, autism, and Alzheimers disease. Mood disorders have a high rate of comorbidity with anxiety disorders although, to date, little is known of the involvement of Glo1 in the pathophysiology of these conditions. In the present study, we examined the expression levels of Glo1 mRNA in peripheral white blood cells of mood disorder patients to understand the role of Glo1 in mood disorders. Quantitative real-time polymerase chain reaction experiments revealed that reduced expression of Glo1 mRNA was observed in major depressive and bipolar disorder patients in a current depressive state, as compared with healthy control subjects. In contrast, the expression of Glo1 mRNA in major depressive and bipolar patients, in a remissive state, showed no significant alteration when compared with healthy control subjects. These results suggest that the aberrant expression of Glo1 might be involved in the pathophysiology of mood disorders.

State-dependent changes in the expression of DNA methyltransferases in mood disorder patients.

Aberrant transcriptional regulation may be one of the key components of the pathophysiology of mood disorders. DNA methylation generally acts as an epigenetic gene silencing mechanism and is catalyzed by a group of enzymes known as DNA methyltransferases (DNMTs). Several lines of evidence have suggested aberrant DNA methylation in patients with neuropsychiatric disorders and in animal models for psychiatric disorders. However, the involvement of DNMTs in the pathophysiology of mood disorders is not completely understood. In this study, we aimed to determine whether there are alterations in the expression of DNMTs mRNA in mood disorder patients. We used quantitative real-time PCR to measure the mRNA expression of four DNMT isoforms in the peripheral white blood cells of major depressive disorder (MDD) and bipolar disorder (BPD) patients during a depressive and a remissive episode. We found that the levels of DNMT1 mRNA were significantly decreased in a depressive but not in a remissive state of MDD and BPD. In addition, the levels of DNMT3B mRNA in MDD were significantly increased in a depressive but not in a remissive state. Thus, our data suggest that the altered expression of DNMTs is state dependent and that the aberrant epigenetic gene regulations caused by the altered expression of DNMT1 and DNMT3B may be associated with the pathophysiology of mood disorders.

Impaired hippocampal spinogenesis and neurogenesis and altered affective behavior in mice lacking heat shock factor 1

Aberrant transcriptional regulation in the brain is thought to be one of the key components of the pathogenesis and pathophysiology of neuropsychiatric disorders. Heat shock factors (HSFs) modulate cellular homeostasis through the control of gene expression. However, the roles of HSFs in brain function have yet to be elucidated fully. In the present study, we attempted to clarify the role of HSF1-mediated gene regulation in neuronal and behavioral development using HSF1-deficient (HSF1−/−) mice. We found granule neurons of aberrant morphology and impaired neurogenesis in the dentate gyrus of HSF1−/− mice. In addition, HSF1−/− mice showed aberrant affective behavior, including reduced anxiety and sociability but increased depression-like behavior and aggression. Furthermore, HSF1 deficiency enhanced behavioral vulnerability to repeated exposure to restraint stress. Importantly, rescuing the HSF1 deficiency in the neonatal but not the adult hippocampus reversed the aberrant anxiety and depression-like behaviors. These results indicate a crucial role for hippocampal HSF1 in neuronal and behavioral development. Analysis of the molecular mechanisms revealed that HSF1 directly modulates the expression of polysialyltransferase genes, which then modulate polysialic acid–neural cell adhesion molecule (PSA-NCAM) levels in the hippocampus. Enzymatic removal of PSA from the neonatal hippocampus resulted in aberrant behavior during adulthood, similar to that observed in HSF1−/− mice. Thus, these results suggest that one role of HSF1 is to control hippocampal PSA-NCAM levels through the transcriptional regulation of polysialyltransferases, a process that might be involved in neuronal and behavioral development in mice.

Hippocampal Sirtuin 1 Signaling Mediates Depression-like Behavior

BACKGROUND Although depression is the leading cause of disability worldwide, its pathophysiology is poorly understood. Recent evidence has suggested that sirtuins (SIRTs) play a key role in cognition and synaptic plasticity, yet their role in mood regulation remains controversial. Here, we aimed to investigate whether SIRT function is associated with chronic stress-elicited depression-like behaviors and neuronal atrophy. METHODS We measured SIRT expression and activity in a mouse model of depression. We injected mice with a SIRT1 activator or inhibitor and measured their depression-like behaviors and dendritic spine morphology. To assess the role of SIRT1 directly, we used a viral-mediated gene transfer to overexpress the wild-type SIRT1 or dominant negative SIRT1 and evaluated their depression-like behaviors. Finally, we examined the role of extracellular signal-regulated protein kinases 1 and 2, a potential downstream target of SIRT1, in depression-like behavior. RESULTS We found that chronic stress reduced SIRT1 activity in the dentate gyrus of the hippocampus. Pharmacologic and genetic inhibition of hippocampal SIRT1 function led to an increase in depression-like behaviors. Conversely, SIRT1 activation blocked both the development of depression-related phenotypes and aberrant dendritic structures elicited by chronic stress exposure. Furthermore, hippocampal SIRT1 activation increased the phosphorylation level of extracellular signal-regulated protein kinases 1 and 2 in the stressed condition, and viral-mediated activation and inhibition of hippocampal extracellular signal-regulated protein kinase 2 led to antidepressive and prodepressive behaviors, respectively. CONCLUSIONS Our results suggest that the hippocampal SIRT1 pathway contributes to the chronic stress-elicited depression-related phenotype and aberrant dendritic atrophy.

Hippocampal MicroRNA-124 Enhances Chronic Stress Resilience in Mice.

Chronic stress-induced aberrant gene expression in the brain and subsequent dysfunctional neuronal plasticity have been implicated in the etiology and pathophysiology of mood disorders. In this study, we examined whether altered expression of small, regulatory, noncoding microRNAs (miRNAs) contributes to the depression-like behaviors and aberrant neuronal plasticity associated with chronic stress. Mice exposed to chronic ultra-mild stress (CUMS) exhibited increased depression-like behaviors and reduced hippocampal expression of the brain-enriched miRNA-124 (miR-124). Aberrant behaviors and dysregulated miR-124 expression were blocked by chronic treatment with an antidepressant drug. The depression-like behaviors are likely not conferred directly by miR-124 downregulation because neither viral-mediated hippocampal overexpression nor intrahippocampal infusion of an miR-124 inhibitor affected depression-like behaviors in nonstressed mice. However, viral-mediated miR-124 overexpression in hippocampal neurons conferred behavioral resilience to CUMS, whereas inhibition of miR-124 led to greater behavioral susceptibility to a milder stress paradigm. Moreover, we identified histone deacetylase 4 (HDAC4), HDAC5, and glycogen synthase kinase 3β (GSK3β) as targets for miR-124 and found that intrahippocampal infusion of a selective HDAC4/5 inhibitor or GSK3 inhibitor had antidepressant-like actions on behavior. We propose that miR-124-mediated posttranscriptional controls of HDAC4/5 and GSK3β expressions in the hippocampus have pivotal roles in susceptibility/resilience to chronic stress. SIGNIFICANCE STATEMENT Depressive disorders are a major public health concern worldwide. Although a clear understanding of the etiology of depression is still lacking, chronic stress-elicited aberrant neuronal plasticity has been implicated in the pathophysiology of depression. We show that the hippocampal expression of microRNA-124 (miR-124), an endogenous small, noncoding RNA that represses gene expression posttranscriptionally, controls resilience/susceptibility to chronic stress-induced depression-like behaviors. These effects on depression-like behaviors may be mediated through regulation of the mRNA or protein expression levels of histone deacetylases HDAC4/5 and glycogen synthase kinase 3β, all highly conserved miR-124 targets. Moreover, miR-124 contributes to stress-induced dendritic hypotrophy and reduced spine density of dentate gyrus granule neurons. Modulation of hippocampal miR-124 pathways may have potential antidepressant effects.