Hiromasa Funato

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.

Enhanced Orexin Receptor-2 Signaling Prevents Diet-Induced Obesity and Improves Leptin Sensitivity

The hypothalamic orexin neuropeptide acutely promotes appetite, yet orexin deficiency in humans and mice is associated with obesity. Prolonged effects of increased orexin signaling upon energy homeostasis have not been fully characterized. Here, we examine the metabolic effects of orexin gain of function utilizing genetic and pharmacologic techniques in mice. Transgenic orexin overexpression confers resistance to high-fat diet-induced obesity and insulin insensitivity by promoting energy expenditure and reducing consumption. Genetic studies indicate that orexin receptor-2 (OX2R), rather than OX1R signaling, predominantly mediates this phenotype. Likewise, prolonged central administration of an OX2R-selective peptide agonist inhibits diet-induced obesity. While orexin overexpression enhances the anorectic-catabolic effects of central leptin administration, obese leptin-deficient mice are completely resistant to the metabolic effects of orexin overexpression or OX2R agonist infusion. We conclude that enhanced orexin-OX2R signaling confers resistance to diet-induced features of the metabolic syndrome through negative energy homeostasis and improved leptin sensitivity.

An essential role for orexins in emergence from general anesthesia

The neural mechanisms through which the state of anesthesia arises and dissipates remain unknown. One common belief is that emergence from anesthesia is the inverse process of induction, brought about by elimination of anesthetic drugs from their CNS site(s) of action. Anesthetic-induced unconsciousness may result from specific interactions of anesthetics with the neural circuits regulating sleep and wakefulness. Orexinergic agonists and antagonists have the potential to alter the stability of the anesthetized state. In this report, we refine the role of the endogenous orexin system in impacting emergence from, but not entry into the anesthetized state, and in doing so, we distinguish mechanisms of induction from those of emergence. We demonstrate that isoflurane and sevoflurane, two commonly used general anesthetics, inhibit c-Fos expression in orexinergic but not adjacent melanin-concentrating hormone (MCH) neurons; suggesting that wake-active orexinergic neurons are inhibited by these anesthetics. Genetic ablation of orexinergic neurons, which causes acquired murine narcolepsy, delays emergence from anesthesia, without changing anesthetic induction. Pharmacologic studies with a selective orexin-1 receptor antagonist confirm a specific orexin effect on anesthetic emergence without an associated change in induction. We conclude that there are important differences in the neural substrates mediating induction and emergence. These findings support the concept that emergence depends, in part, on recruitment and stabilization of wake-active regions of brain.

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.

Reduced Glucocorticoid Receptor α Expression in Mood Disorder Patients and First-Degree Relatives

BACKGROUND Individuals with mood disorders exhibit altered function of the hypothalamic-pituitary-adrenal (HPA) axis in response to stress. The glucocorticoid receptor (GR) plays an important role in the negative feedback regulation of the HPA axis. There are two protein isoforms of GR, GRalpha and GRbeta, which have distinct biological activity. It has not been examined whether GRalpha messenger RNA (mRNA) and GRbeta mRNA expressions are altered in peripheral blood cells of mood disorder patients. METHODS Using quantitative reverse transcription polymerase chain reaction (RT-PCR), GRalpha mRNA and GRbeta mRNA were measured in peripheral blood cells of major depressive disorder patients (depressive n = 18; remissive n = 38), bipolar disorder patients (depressive n = 13; remissive n = 35), normal control subjects (n = 31), and first-degree relatives of major depressive (n = 17) and bipolar (n = 15) disorder patients. RESULTS Reduced expression of GRalpha mRNA was shown in both bipolar and major depressive disorder patients in a current depressive state as well as in remission. First-degree relatives of bipolar disorder patients also showed GRalpha mRNA reduction. Altered GRbeta mRNA expression was not found in mood disorder patients. CONCLUSIONS Our results suggest that reduced GRalpha mRNA expression might be trait-dependent and associated with the pathophysiology of mood disorders.

Fasting and high-fat diet alter histone deacetylase expression in the medial hypothalamus.

Increasing attention is now being given to the epigenetic regulation of animal and human behaviors including the stress response and drug addiction. Epigenetic factors also influence feeding behavior and metabolic phenotypes, such as obesity and insulin sensitivity. In response to fasting and high-fat diets, the medial hypothalamus changes the expression of neuropeptides regulating feeding, metabolism, and reproductive behaviors. Histone deacetylases (HDACs) are involved in the epigenetic control of gene expression and alter behavior in response to a variety of environmental factors. Here, we examined the expression of HDAC family members in the medial hypothalamus of mice in response to either fasting or a high-fat diet. In response to fasting, HDAC3 and −4 expression levels increased while HDAC10 and −11 levels decreased. Four weeks on a high-fat diet resulted in the increased expression of HDAC5 and −8. Moreover, fasting decreased the number of acetylated histone H3- and acetylated histone H4-positive cells in the ventrolateral subdivision of the ventromedial hypothalamus. Therefore, HDACs may be implicated in altered gene expression profiles in the medial hypothalamus under different metabolic states.

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.

Forward-genetics analysis of sleep in randomly mutagenized mice

Sleep is conserved from invertebrates to vertebrates, and is tightly regulated in a homeostatic manner. The molecular and cellular mechanisms that determine the amount of rapid eye movement sleep (REMS) and non-REMS (NREMS) remain unknown. Here we identify two dominant mutations that affect sleep and wakefulness by using an electroencephalogram/electromyogram-based screen of randomly mutagenized mice. A splicing mutation in the Sik3 protein kinase gene causes a profound decrease in total wake time, owing to an increase in inherent sleep need. Sleep deprivation affects phosphorylation of regulatory sites on the kinase, suggesting a role for SIK3 in the homeostatic regulation of sleep amount. Sik3 orthologues also regulate sleep in fruitflies and roundworms. A missense, gain-of-function mutation in the sodium leak channel NALCN reduces the total amount and episode duration of REMS, apparently by increasing the excitability of REMS-inhibiting neurons. Our results substantiate the use of a forward-genetics approach for studying sleep behaviours in mice, and demonstrate the role of SIK3 and NALCN in regulating the amount of NREMS and REMS, respectively.

Hand2 controls osteoblast differentiation in the branchial arch by inhibiting DNA binding of Runx2

Members of the basic helix-loop-helix (bHLH) family of transcription factors regulate the specification and differentiation of numerous cell types during embryonic development. Hand1 and Hand2 are expressed by a subset of neural crest cells in the anterior branchial arches and are involved in craniofacial development. However, the precise mechanisms by which Hand proteins mediate biological actions and regulate downstream target genes in branchial arches is largely unknown. Here, we report that Hand2 negatively regulates intramembranous ossification of the mandible by directly inhibiting the transcription factor Runx2, a master regulator of osteoblast differentiation. Hand proteins physically interact with Runx2, suppressing its DNA binding and transcriptional activity. This interaction is mediated by the N-terminal domain of the Hand protein and requires neither dimerization with other bHLH proteins nor DNA binding. We observed partial colocalization of Hand2 and Runx2 in the mandibular primordium of the branchial arch, and downregulation of Hand2 precedes Runx2-driven osteoblast differentiation. Hand2 hypomorphic mutant mice display insufficient mineralization and ectopic bone formation in the mandible due to accelerated osteoblast differentiation, which is associated with the upregulation and ectopic expression of Runx2 in the mandibular arch. Here, we show that Hand2 acts as a novel inhibitor of the Runx2-DNA interaction and thereby regulates osteoblast differentiation in branchial arch development.

Monoaminergic and Neuropeptidergic Neurons Have Distinct Expression Profiles of Histone Deacetylases

Monoaminergic and neuropeptidergic neurons regulate a wide variety of behaviors, such as feeding, sleep/wakefulness behavior, stress response, addiction, and social behavior. These neurons form neural circuits to integrate different modalities of behavioral and environmental factors, such as stress, maternal care, and feeding conditions. One possible mechanism for integrating environmental factors through the monoaminergic and neuropeptidergic neurons is through the epigenetic regulation of gene expression via altered acetylation of histones. Histone deacetylases (HDACs) play an important role in altering behavior in response to environmental factors. Despite increasing attention and the versatile roles of HDACs in a variety of brain functions and disorders, no reports have detailed the localization of the HDACs in the monoaminergic and neuropeptidergic neurons. Here, we examined the expression profile of the HDAC protein family from HDAC1 to HDAC11 in corticotropin-releasing hormone, oxytocin, vasopressin, agouti-related peptide (AgRP), pro-opiomelanocortin (POMC), orexin, histamine, dopamine, serotonin, and noradrenaline neurons. Immunoreactivities for HDAC1,-2,-3,-5,-6,-7,-9, and -11 were very similar among the monoaminergic and neuropeptidergic neurons, while the HDAC4, -8, and -10 immunoreactivities were clearly different among neuronal groups. HDAC10 expression was found in AgRP neurons, POMC neurons, dopamine neurons and noradrenaline neurons but not in other neuronal groups. HDAC8 immunoreactivity was detected in the cytoplasm of almost all histamine neurons with a pericellular pattern but not in other neuropeptidergic and monoaminergic neurons. Thus, the differential expression of HDACs in monoaminergic and neuropeptidergic neurons may be crucial for the maintenance of biological characteristics and may be altered in response to environmental factors.