Mood disorders such as bipolar disorder and major depressive disorder have remarkably high prevalence rates in the United States and worldwide. For a large percentage of patients, outcome measures are quite poor, with high rates of suicide, relapse, and residual symptoms (cognitive and functional impairment, psychosocial disability, and diminished well being). Available treatments for mood disorders are far from ideal -- there is a clear need to develop novel treatments for these devastating disorders. This project is focused on discovering genes, gene expression levels, regulators of gene expression, proteins, and molecular cascades thought to influence the activity of neuronal networks associated with mood regulation. The analyses are done using integrated genomic, proteomic, neurochemical, and behavioral approaches. 1) MicroRNA studies MicroRNAs (miRNAs) are a class of small, non-peptide-coding RNAs, which bind to target messenger RNAs (mRNAs) in a sequence-selective manner and cause protein translation suppression and mRNA degradation. MiRNAs are known to play critical roles in cellular stress and early development. Accumulating data indicate that miRNA function influence the molecular regulation underlying neuronal development and dendritic spine morphogenesis, synaptic and circuitry plasticity, and circadian rhythms. This raises the intriguing possibility that they may play a role in the biological mechanisms of stress coping, or conversely, maladaptation. We discovered that chronic treatment with mood stabilizer, lithium or valproate altered hippocampal levels of eight miRNAs and that chronic restraint stress regulated hippocampal levels of sixteen miRNAs. Currently, we are investigating whether altered expression of some of these miRNAs can influence animal behaviors related to stress responses and mood disorders. The targets (mRNA) of miRNAs can be predicted. Based on the predictions, we have discovered that mood stabilizers modulate protein levels of Grm7, ChrM1, STEP and a calcineurin subunit in some brain regions. The roles of these targets in behaviors related to mood disorders are currently being investigated. 2) Antidepressive mechanism of ketamine Recent clinical studies demonstrate that the ketamine, a NMDAR blocker, produces antidepressive effects with three unique features: rapid onset, sustained acting (for days), and effective treatment for resistant depressed patients. The precise molecular mechanisms underlying ketamines unique antidepressive profile are still largely unknown. Previously, we demonstrated that ketamine reduces immobility in the forced swim test administered immediately and a few days after the treatment;antidepressants are only effective immediately following the treatment. Ketamine alleviates helplessness after a single injection, while repeated, but not single, antidepressant administrations produce similar effects. These two results capture in part the sustained acting and rapid onset features of ketamines behavioral effects related to depression. Serotonin depletion causes transient depression episode in subjects with personal or family history of depression. PCPA has been used to deplete serotonin in rodent. A previous study found that PCPA treatment alone does not cause immobility changes in the forced swim test;however, it blocks immobility reduction by SSRIs, suggesting that serotonin depletion can cause resistance to some behavioral effects of antidepressants. We refine the serotonin depletion paradigm and incorporated two hedonic activity tests as the outcome measures. The PCPA treatment reduced serotonin contents in the brain and resulted in the reductions of sucrose preference and female urine sniffing in male rats or mice. A single ketamine treatment improves hedonic activity in depleted males, suggesting that ketamine works through mechanism beyond serotonin transmission. Neurochemical analyses revealed unique AMPAR and NMDAR changes by the behavioral induction and ketamine treatment. The roles of these changes in behavioral induction and ketamine treatment are currently under investigation. 3) Behavioral and neurochemical evaluation of potential therapeutic targets In last fiscal year, we established and validated the female urine sniffing test for monitoring sexually-based hedonic activity in rodents for mood disorder study. We also refined the learned helplessness paradigm for C57 mice. In collaboration with other investigators, we examined whether a Bid inhibitor, GSK-3 inhibitor, CREB potentiator, PDE4 inhibitor, antagonist of voltage-dependent L-type calcium channel, and GRM7 agonist produce antidepressant-like or mood stabilizer-like behavioral actions in our behavioral paradigms and neurochemical assays. 4) Hippocampal PSD proteomic profile of chronic mood stabilizer treatment Increasing evidence supports the hypothesis that symptoms of Bipolar disorder arise from abnormalities in cellular plasticity cascades, leading to aberrant information processing in synapses and circuits. The postsynaptic density (PSD) is an elaborate cytoskeletal and signaling complex that provides anchors for synaptic proteins close to the region of presynaptic neurotransmitter release, and therefore mediates signaling in a host of divergent signal transduction pathways. We investigated modifications of the PSD following chronic treatment with mood stabilizers. Hippocampal PSD complexes were isolated from rats chronically treated with either lithium or valproate by differential centrifugation. The proteomic part of study was carried out by staff in Dr. Sanford Markeys lab.The results showed nine commen targets of chronic lithium and valproate treatments, including the protein Ankyrin G (encoded by the ANK3 gene). The findings are now being further validated with different methods. These findings have provided an important profile of how mood stabilizers alter the expression of target proteins in the hippocampal PSD. This information will be informative for those attempting to design future studies that examine the roles of these PSD proteins in behavioral regulation related to Bipolar disorder. 5) Bcl-2 family and ER stress proteins in mood regulation B cell lymphoma 2 (Bcl-2) family proteins includes anti-apoptosis and pro-apoptosis members. These proteins primarily present in mitochondrial and endoplasmic reticulum (ER). Our previous studies show that treatments with mood stabilizers up-regulate brain regional Bcl-2 levels. Clinical findings, although need to be replicated, suggest the dysfunctional ER stress response and mitochondrial disturbances in mood disorders. Bax inhibitor 1 (BI-1) primarily resides in ER and associated with Bcl-2 and Bcl-XL. This protein blocks apoptosis induced by ER stress, but not by mitochondrial disturbance. BI-1 modulates ER stress pathways including the unfolded protein response (UPR) leading to phosphorylation-inactivation of Bcl-2 and suppressing IRE1alpha, a serine/threonine kinase involved in transmitting the UPR. We found that BI-1 overexpressing mice were protected against both serotonin and catecholamine depletion-induced behavioral displays related to anhedonia. Ten days after learned helplessness induction, BI-1 transgenic mice exhibited enhanced spontaneous recovery rates, as assessed by decreased escape latencies in the learned helplessness paradigm.These behavioral results suggest that targeting BI-1 may provide another mechanism for modulating cell survival, calcium signaling, and ER stress mechanisms, and ultimately enhance affective resilience. We are currently investigating the molecular mechanisms whereby BI-1 may achieve these effects, for instance by modulating factors involved in cell survival or by modulating ER calcium.
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