Subroto Ghose

Molecular adaptations underlying susceptibility and resistance to social defeat in brain reward regions.

While stressful life events are an important cause of psychopathology, most individuals exposed to adversity maintain normal psychological functioning. The molecular mechanisms underlying such resilience are poorly understood. Here, we demonstrate that an inbred population of mice subjected to social defeat can be separated into susceptible and unsusceptible subpopulations that differ along several behavioral and physiological domains. By a combination of molecular and electrophysiological techniques, we identify signature adaptations within the mesolimbic dopamine circuit that are uniquely associated with vulnerability or insusceptibility. We show that molecular recapitulations of three prototypical adaptations associated with the unsusceptible phenotype are each sufficient to promote resistant behavior. Our results validate a multidisciplinary approach to examine the neurobiological mechanisms of variations in stress resistance, and illustrate the importance of plasticity within the brains reward circuits in actively maintaining an emotional homeostasis.

Antidepressant Actions of Histone Deacetylase Inhibitors

Persistent symptoms of depression suggest the involvement of stable molecular adaptations in brain, which may be reflected at the level of chromatin remodeling. We find that chronic social defeat stress in mice causes a transient decrease, followed by a persistent increase, in levels of acetylated histone H3 in the nucleus accumbens, an important limbic brain region. This persistent increase in H3 acetylation is associated with decreased levels of histone deacetylase 2 (HDAC2) in the nucleus accumbens. Similar effects were observed in the nucleus accumbens of depressed humans studied postmortem. These changes in H3 acetylation and HDAC2 expression mediate long-lasting positive neuronal adaptations, since infusion of HDAC inhibitors into the nucleus accumbens, which increases histone acetylation, exerts robust antidepressant-like effects in the social defeat paradigm and other behavioral assays. HDAC inhibitor [N-(2-aminophenyl)-4-[N-(pyridine-3-ylmethoxy-carbonyl)aminomethyl]benzamide (MS-275)] infusion also reverses the effects of chronic defeat stress on global patterns of gene expression in the nucleus accumbens, as determined by microarray analysis, with striking similarities to the effects of the standard antidepressant fluoxetine. Stress-regulated genes whose expression is normalized selectively by MS-275 may provide promising targets for the future development of novel antidepressant treatments. Together, these findings provide new insight into the underlying molecular mechanisms of depression and antidepressant action, and support the antidepressant potential of HDAC inhibitors and perhaps other agents that act at the level of chromatin structure.

[Delta]FosB in brain reward circuits mediates resilience to stress and antidepressant responses

In contrast with the many studies of stress effects on the brain, relatively little is known about the molecular mechanisms of resilience, the ability of some individuals to escape the deleterious effects of stress. We found that the transcription factor ΔFosB mediates an essential mechanism of resilience in mice. Induction of ΔFosB in the nucleus accumbens, an important brain reward-associated region, in response to chronic social defeat stress was both necessary and sufficient for resilience. ΔFosB induction was also required for the standard antidepressant fluoxetine to reverse behavioral pathology induced by social defeat. ΔFosB produced these effects through induction of the GluR2 AMPA glutamate receptor subunit, which decreased the responsiveness of nucleus accumbens neurons to glutamate, and through other synaptic proteins. Together, these findings establish a previously unknown molecular pathway underlying both resilience and antidepressant action.

Human postmortem tissue: what quality markers matter?

Postmortem human brain tissue is used for the study of many different brain diseases. A key factor in conducting postmortem research is the quality of the tissue. Unlike animal tissue, whose condition at death can be controlled and influenced, human tissue can only be collected naturalistically. This introduces potential confounds, based both on pre- and postmortem conditions, that may influence the quality of tissue and its ability to yield accurate results. The traditionally recognized confounds that reduce tissue quality are agonal factors (e.g., coma, hypoxia, hyperpyrexia at the time of death), and long postmortem interval (PMI). We measured tissue quality parameters in over 100 postmortem cases collected from different sources and correlated them with RNA quality (as indicated by the RNA Integrity Number (RIN)) and with protein quality (as measured by the level of representative proteins). Our results show that the most sensible indicator of tissue quality is RIN and that there is a good correlation between RIN and the pH. No correlation developed between protein levels and the aforementioned factors. Moreover, even when RNA was degraded, the protein levels remained stable. However, these correlations did not prove true under all circumstances (e.g., thawed tissue, surgical tissue), that yielded unexpected quality indicators. These data also suggest that cases whose source was a Medical Examiners office represent high tissue quality.

Epigenetic regulation of RAC1 induces synaptic remodeling in stress disorders and depression

Depression induces structural and functional synaptic plasticity in brain reward circuits, although the mechanisms promoting these changes and their relevance to behavioral outcomes are unknown. Transcriptional profiling of the nucleus accumbens (NAc) for Rho GTPase–related genes, which are known regulators of synaptic structure, revealed a sustained reduction in RAS-related C3 botulinum toxin substrate 1 (Rac1) expression after chronic social defeat stress. This was associated with a repressive chromatin state surrounding the proximal promoter of Rac1. Inhibition of class 1 histone deacetylases (HDACs) with MS-275 rescued both the decrease in Rac1 transcription after social defeat stress and depression-related behavior, such as social avoidance. We found a similar repressive chromatin state surrounding the RAC1 promoter in the NAc of subjects with depression, which corresponded with reduced RAC1 transcription. Viral-mediated reduction of Rac1 expression or inhibition of Rac1 activity in the NAc increases social defeat–induced social avoidance and anhedonia in mice. Chronic social defeat stress induces the formation of stubby excitatory spines through a Rac1-dependent mechanism involving the redistribution of synaptic cofilin, an actin-severing protein downstream of Rac1. Overexpression of constitutively active Rac1 in the NAc of mice after chronic social defeat stress reverses depression-related behaviors and prunes stubby spines. Taken together, our data identify epigenetic regulation of RAC1 in the NAc as a disease mechanism in depression and reveal a functional role for Rac1 in rodents in regulating stress-related behaviors.Depression involves plasticity of brain reward neurons, although the mechanisms and behavioral relevance are unknown. Transcriptional profiling of nucleus accumbens (NAc) for RhoGTPase related genes, known regulators of synaptic structure, following chronic social defeat stress, revealed a long-term reduction in Rac1 transcription. This was marked by a repressive chromatin state surrounding its proximal promoter. Inhibition of class 1 HDACs with MS-275 rescued both decreased Rac1 transcription and social avoidance behavior. A similar repressive chromatin state was found surrounding the Rac1 promoter in human postmortem NAc from depressed subjects, which corresponded with reduced Rac1 transcription. We show Rac1 is necessary and sufficient for social avoidance and anhedonia, and the formation of stubby excitatory spines by redistributing synaptic cofilin, an actin severing protein downstream of Rac1. Our data identifies epigenetic regulation of Rac1 in NAc as a bona fide disease mechanism in depression and reveals a functional role in regulating stress-related behaviors.

AKT Signaling within the Ventral Tegmental Area Regulates Cellular and Behavioral Responses to Stressful Stimuli

BACKGROUND The neurobiological mechanisms by which only a minority of stress-exposed individuals develop psychiatric diseases remain largely unknown. Recent evidence suggests that dopaminergic neurons of the ventral tegmental area (VTA) play a key role in the manifestation of stress vulnerability. METHODS Using a social defeat paradigm, we segregated susceptible mice (socially avoidant) from unsusceptible mice (socially interactive) and examined VTA punches for changes in neurotrophic signaling. Employing a series of viral vectors, we sought to causally implicate these neurotrophic changes in the development of avoidance behavior. RESULTS Susceptibility to social defeat was associated with a significant reduction in levels of active/phosphorylated AKT (thymoma viral proto-oncogene) within the VTA, whereas chronic antidepressant treatment (in mice and humans) increased active AKT levels. This defeat-induced reduction in AKT activation in susceptible mice was both necessary and sufficient to recapitulate depressive behaviors associated with susceptibility. Pharmacologic reductions in AKT activity also significantly raised the firing frequency of VTA dopamine neurons, an important electrophysiologic hallmark of the susceptible phenotype. CONCLUSIONS These studies highlight a crucial role for decreases in VTA AKT signaling as a key mediator of the maladaptive cellular and behavioral response to chronic stress.

β-catenin mediates stress resilience through Dicer1/microRNA regulation

β-catenin is a multi-functional protein that has an important role in the mature central nervous system; its dysfunction has been implicated in several neuropsychiatric disorders, including depression. Here we show that in mice β-catenin mediates pro-resilient and anxiolytic effects in the nucleus accumbens, a key brain reward region, an effect mediated by D2-type medium spiny neurons. Using genome-wide β-catenin enrichment mapping, we identify Dicer1—important in small RNA (for example, microRNA) biogenesis—as a β-catenin target gene that mediates resilience. Small RNA profiling after excising β-catenin from nucleus accumbens in the context of chronic stress reveals β-catenin-dependent microRNA regulation associated with resilience. Together, these findings establish β-catenin as a critical regulator in the development of behavioural resilience, activating a network that includes Dicer1 and downstream microRNAs. We thus present a foundation for the development of novel therapeutic targets to promote stress resilience.

A Novel Role of the WNT-Dishevelled-GSK3β Signaling Cascade in the Mouse Nucleus Accumbens in a Social Defeat Model of Depression

Based on earlier gene expression and chromatin array data, we identified the protein, dishevelled (DVL)-2, as being regulated in the nucleus accumbens (NAc), a key brain reward region, in the mouse social defeat model of depression. Here, we validate these findings by showing that DVL2 mRNA and protein levels are downregulated in NAc of mice susceptible to social defeat stress, effects not seen in resilient mice. Other DVL isoforms, DVL1 and DVL3, show similar patterns of regulation. Downregulation of DVL was also demonstrated in the NAc of depressed humans examined postmortem. Interestingly, several members of the WNT (Wingless)-DVL signaling cascade, including phospho-GSK3β (glycogen synthase kinase-3β), also show significant downregulation in the NAc of susceptible, but not resilient, mice, demonstrating concerted regulation of this pathway in the NAc due to social defeat stress. By using viral-mediated gene transfer to overexpress a dominant-negative mutant of DVL in NAc, or by using a pharmacological inhibitor of DVL administered into this brain region, we show that blockade of DVL function renders mice more susceptible to social defeat stress and promotes depression-like behavior in other assays. Similar prodepression-like effects were induced upon overexpressing GSK3β in the NAc, while overexpressing a dominant-negative mutant of GSK3β promoted resilience to social defeat stress. These findings are consistent with the knowledge that downregulation of DVL and phospho-GSK3β reflects an increase in GSK3β activity. These studies reveal a novel role for the DVL-GSK3β signaling pathway, acting within the brains reward circuitry, in regulating susceptibility to chronic stress.

Differential Expression of Metabotropic Glutamate Receptors 2 and 3 in Schizophrenia: A Mechanism for Antipsychotic Drug Action?

OBJECTIVE Preclinical and clinical data implicate the group II metabotropic glutamate receptors mGluR2 and mGluR3 in the pathophysiology of schizophrenia. Moreover, a recent phase II clinical trial demonstrated the antipsychotic efficacy of a mGluR2/mGluR3 agonist. The purpose of the present study was to distinguish the expression of mGluR2 and mGluR3 receptor proteins in schizophrenia and to quantify glutamate carboxypeptidase II (GCP II) in order to explore a role for the metabotropic receptors in schizophrenia therapeutics. GCP II is an enzyme that metabolizes N-acetyl-aspartyl-glutamate (NAAG), which is the only known specific endogenous agonist of mGluR3 in the mammalian brain. METHOD The normal expression levels of mGluR2, mGluR3, and GCP II were determined for 10 regions of the postmortem human brain using specific antibodies. Differences in expression levels of each protein were examined in the dorsolateral prefrontal cortex, temporal cortex, and motor cortex in 15 postmortem schizophrenia subjects and 15 postmortem matched normal comparison subjects. Chronic antipsychotic treatment in rodents was conducted to examine the potential effect of antipsychotic drugs on expression of the three proteins. RESULTS Findings revealed a significant increase in GCP II protein and a reduction in mGluR3 protein in the dorsolateral prefrontal cortex in schizophrenia subjects, with mGluR2 protein levels unchanged. Chronic antipsychotic treatment in rodents did not influence GCP II or mGluR3 levels. CONCLUSIONS Increased GCP II expression and low mGluR3 expression in the dorsolateral prefrontal cortex suggest that NAAG-mediated signaling is impaired in this brain region in schizophrenia. Further, these data implicate the mGluR3 receptor in the antipsychotic action of mGluR2/mGluR3 agonists.

Reversal of Depressed Behaviors in Mice by p11 Gene Therapy in the Nucleus Accumbens

Reduction of the 5-HT1B receptor–binding protein p11 in the mouse nucleus accumbens induces depression-like behaviors, and gene therapy to restore p11 expression in this region reverses depression-like behaviors. Dialing Down Depression Despite much progress in the study and treatment of depression, the mechanisms underlying this debilitating disease are still unclear. Altered activity of several major neurotransmitters in the brain including serotonin is involved, but pinpointing the parts of the brain affected in depression has proved challenging. Alexander and colleagues now implicate a brain region called the nucleus accumbens and a protein called p11 expressed in this region as important mediators of depression in humans and mice. The authors were alerted to the potential importance of p11 in depression because mice that lack this protein show depressive-like behavior. This protein is involved in the activation of two receptors for serotonin, 5-HT1B and 5-HT4. Alexander et al. decided to down-regulate expression of p11 specifically in the nucleus accumbens by injecting a viral vector containing a small interfering RNA against p11 directly into this brain region in healthy mice. They then tested the treated mice to see if they exhibited depressive-like behaviors in response to two stress tests (suspension by the tail and swimming in a water tank). In both tests, treated mice showed greater immobility compared with control animals, a sign of depressive-like behavior. To show that these depressive symptoms were indeed caused by loss of p11 in the nucleus accumbens, the investigators overexpressed p11 in the nucleus accumbens of mice that completely lacked this protein. They demonstrated restoration of normal immobility times on the two stress tests and an increased desire to sip sucrose solution (a treat that rodents normally enjoy but depressed animals do not). They also showed increased activity of 5-HT1B serotonin receptors expressed by striatal neurons in the nucleus accumbens of mice overexpressing p11. But do these results have any relevance to depression in humans? Alexander and colleagues tackled this question by comparing postmortem nucleus accumbens brain tissue from individuals with and without depression at the time of death. They discovered that expression of p11 was much lower in the nucleus accumbens of depressed individuals compared with healthy persons. These new findings pinpoint the nucleus accumbens and the p11 protein as important mediators of depression and provide new therapeutic targets for drug development. The etiology of major depression remains unknown, but dysfunction of serotonergic signaling has long been implicated in the pathophysiology of this disorder. p11 is an S100 family member recently identified as a serotonin 1B [5-hydroxytryptamine 1B (5-HT1B)] and serotonin 4 (5-HT4) receptor–binding protein. Mutant mice in which p11 is deleted show depression-like behaviors, suggesting that p11 may be a mediator of affective disorder pathophysiology. Using somatic gene transfer, we have now identified the nucleus accumbens as a key site of p11 action. Reduction of p11 with adeno-associated virus (AAV)–mediated RNA interference in the nucleus accumbens, but not in the anterior cingulate, of normal adult mice resulted in depression-like behaviors nearly identical to those seen in p11 knockout mice. Restoration of p11 expression specifically in the nucleus accumbens of p11 knockout mice normalized depression-like behaviors. Human nucleus accumbens tissue shows a significant reduction of p11 protein in depressed patients when compared to matched healthy controls. These results suggest that p11 loss in rodent and human nucleus accumbens may contribute to the pathophysiology of depression. Normalization of p11 expression within this brain region with AAV-mediated gene therapy may be of therapeutic value.