RESUMO
Background: Post-traumatic stress disorder (PTSD) is a highly prevalent psychiatric disorder that can become chronic and debilitating when left untreated. Available pharmacotherapies are limited, take weeks to show modest benefit and remain ineffective for up to 40% of patients. Methylone is currently in clinical development for the treatment of PTSD. Preclinical studies show rapid, robust and long-lasting antidepressant-like and anxiolytic effects. The mechanism of action underlying these effects is not yet fully understood. This study investigated the downstream gene expression changes and signaling pathways affected by methylone in key brain areas linked to PTSD and MDD. It also sought to determine whether neuroplasticity-related genes were involved. We compared effects of methylone with MDMA to explore similarities and differences in their brain effects because MDMA-assisted psychotherapy has recently shown benefit in clinical trials for PTSD and methylone is a structural analog of MDMA. Methods: Monoamine binding, uptake and release studies were performed and a high-throughput-screen evaluated agonist/antagonist activities at 168 GPCRs in vitro. We used RNA sequencing (RNA-seq) to probe drug-induced gene expression changes in the amygdala and frontal cortex, two brain areas responsible for emotional learning that are affected by PTSD and MDD. Rats were treated with methylone or MDMA (both 10 mg/kg, IP), and their responses were compared with controls. We performed functional enrichment analysis to identify which pathways were regulated by methylone and/or MDMA. We confirmed changes in gene expression using immunohistochemistry. Results: Methylone, a monoamine uptake inhibitor and releaser, demonstrated no off-target effects at 168 GPCRs, unlike MDMA, which showed activity at 5HT2A and 5HT2C receptors. RNA-seq results revealed significant regulation of myelin-related genes in the amygdala, confirmed by immunohistochemistry. In the frontal cortex, methylone significantly upregulated genes implicated in neuroplasticity. Conclusion: Results suggest that (1) methylone is a rapid-acting neuroplastogen that affects key brain substrates for PTSD and MDD and that (2) methylone appears to exhibit higher specificity and fewer off-target effects than MDMA. Together, these results are consistent with the reported clinical experiences of methylone and MDMA and bolster the potential use of methylone in the treatment of PTSD and, potentially, other neuropsychiatric disorders.
RESUMO
The signal transduction protein, regulator of G protein signaling 4 (RGS4), plays a prominent role in physiologic and pharmacological responses by controlling multiple intracellular pathways. Our earlier work identified the dynamic but distinct roles of RGS4 in the efficacy of monoamine-targeting versus fast-acting antidepressants. Using a modified chronic variable stress (CVS) paradigm in mice, we demonstrate that stress-induced behavioral abnormalities are associated with the downregulation of RGS4 in the medial prefrontal cortex (mPFC). Knockout of RGS4 (RGS4KO) increases susceptibility to CVS, as mutant mice develop behavioral abnormalities as early as 2 weeks after CVS resting-state functional magnetic resonance imaging I (rs-fMRI) experiments indicate that stress susceptibility in RGS4KO mice is associated with changes in connectivity between the mediodorsal thalamus (MD-THL) and the mPFC. Notably, RGS4KO also paradoxically enhances the antidepressant efficacy of ketamine in the CVS paradigm. RNA-sequencing analysis of naive and CVS samples obtained from mPFC reveals that RGS4KO triggers unique gene expression signatures and affects several intracellular pathways associated with human major depressive disorder. Our analysis suggests that ketamine treatment in the RGS4KO group triggers changes in pathways implicated in synaptic activity and responses to stress, including pathways associated with axonal guidance and myelination. Overall, we show that reducing RGS4 activity triggers unique gene expression adaptations that contribute to chronic stress disorders and that RGS4 is a negative modulator of ketamine actions. SIGNIFICANCE STATEMENT: Chronic stress promotes robust maladaptation in the brain, but the exact intracellular pathways contributing to stress vulnerability and mood disorders have not been thoroughly investigated. In this study, the authors used murine models of chronic stress and multiple methodologies to demonstrate the critical role of the signal transduction modulator regulator of G protein signaling 4 in the medial prefrontal cortex in vulnerability to chronic stress and the efficacy of the fast-acting antidepressant ketamine.