Regulation effect of koumine on T-helper cell polarization in rheumatoid arthritis.

Koumine, an alkaloid, exerts therapeutic effects against rheumatoid arthritis (RA), and thus may have a potential application in novel treatment strategies against this disease. Herein, we investigated the regulatory effect of koumine on Th cell polarization using a "pyramid" structure model to elucidate the mechanism underlying its therapeutic effect on RA. The third layer of the model comprises the cytokine secretion layer, in which the effects of koumine on the balance of Th-related cytokines were investigated in mice with collagen-induced arthritis (CIA). Koumine showed significant therapeutic effects and reversed the imbalance of Th1/Th2 and Th17/Treg cytokines. In the Th cell polarization layer, the effects of koumine on the relative numbers of Th cell subsets in splenocytes of rats with CIA were examined. Koumine attenuated both of the increased Th1/Th2 and Th17/Treg subset ratios accompanied with its therapeutic effects. Finally, the primary cultured splenocytes from BALB/c mice were used to further investigate the effect of koumine on Th cell activation by evaluating cell proliferation induced by concanavalin A (Con A), lipopolysaccharides (LPS) and phytohemagglutinin (PHA). Koumine inhibited the cell proliferation responses and its effects on proliferation induced by Con A and PHA were greater than those by LPS, showing the relatively selective inhibition on the proliferation of Th cells. Our results suggest that koumine might restore the homeostasis of the network system with Th subsets and cytokines by inhibiting the activation of T cells, subsequently regulating the polarization of Th subsets and the downstream imbalance of pro/anti-inflammatory cytokines in RA.

APT1-Mediated Depalmitoylation Regulates Hippocampal Synaptic Plasticity.

Palmitoylation may be relevant to the processes of learning and memory, and even disorders, such as post-traumatic stress disorder and aging-related cognitive decline. However, underlying mechanisms of palmitoylation in these processes remain unclear. Herein, we used acyl-biotin exchange, coimmunoprecipitation and biotinylation assays, and behavioral and electrophysiological methods, to explore whether palmitoylation is required for hippocampal synaptic transmission and fear memory formation, and involved in functional modification of synaptic proteins, such as postsynapse density-95 (PSD-95) and glutamate receptors, and detected if depalmitoylation by specific enzymes has influence on glutamatergic synaptic plasticity. Our results showed that global palmitoylation level, palmitoylation of PSD-95 and glutamate receptors, postsynapse density localization of PSD-95, surface expression of AMPARs, and synaptic strength of cultured hippocampal neurons were all enhanced by TTX pretreatment, and these can be reversed by inhibition of palmitoylation with palmitoyl acyl transferases inhibitors, 2-bromopalmitate and N-(tert-butyl) hydroxylamine hydrochloride. Importantly, we also found that acyl-protein thioesterase 1 (APT1)-mediated depalmitoylation is involved in palmitoylation of PSD-95 and glutamatergic synaptic transmission. Knockdown of APT1, not protein palmitoyl thioesterase 1, with shRNA, or selective inhibition, significantly increased AMPAR-mediated synaptic strength, palmitoylation levels, and synaptic or surface expression of PSD-95 and AMPARs. Results from hippocampal tissues and fear-conditioned rats showed that palmitoylation is required for synaptic strengthening and fear memory formation. These results suggest that palmitoylation and APT1-mediated depalmitoylation have critical effects on the regulation of glutamatergic synaptic plasticity, and it may serve as a potential target for learning and memory-associated disorders.SIGNIFICANCE STATEMENT Fear-related anxiety disorders, including post-traumatic stress disorder, are prevalent psychiatric conditions, and fear memory is associated with hyperexcitability in the hippocampal CA1 region. Palmitoylation is involved in learning and memory, but mechanisms coupling palmitoylation with fear memory acquisition remain poorly understood. This study demonstrated that palmitoylation is essential for postsynapse density-95 clustering and hippocampal glutamatergic synaptic transmission, and APT1-mediated depalmitoylation plays critical roles in the regulation of synaptic plasticity. Our study revealed that molecular mechanism about downregulation of APT1 leads to enhancement of AMPAR-mediated synaptic transmission, and that palmitoylation cycling is implicated in fear conditioning-induced synaptic strengthening and fear memory formation.

mTORC1 Signaling Pathway Mediates Chronic Stress-Induced Synapse Loss in the Hippocampus.

Background: The mechanistic target of rapamycin complex 1 (mTORC1) signaling has served as a promising target for therapeutic intervention of major depressive disorder (MDD), but the mTORC1 signaling underlying MDD has not been well elucidated. In the present study, we investigated whether mTORC1 signaling pathway mediates synapse loss induced by chronic stress in the hippocampus. Methods: Chronic restraint stress-induced depression-like behaviors were tested by behavior tests (sucrose preference test, forced swim test and tail suspension test). Synaptic proteins and alternations of phosphorylation levels of mTORC1 signaling-associated molecules were measured using Western blotting. In addition, mRNA changes of immediate early genes (IEGs) and glutamate receptors were measured by RT-PCR. Rapamycin was used to explore the role of mTORC1 signaling in the antidepressant effects of fluoxetine. Results: After successfully establishing the chronic restraint stress paradigm, we observed that the mRNA levels of some IEGs were significantly changed, indicating the activation of neurons and protein synthesis alterations. Then, there was a significant downregulation of glutamate receptors and postsynaptic density protein 95 at protein and mRNA levels. Additionally, synaptic fractionation assay revealed that chronic stress induced synapse loss in the dorsal and ventral hippocampus. Furthermore, these effects were associated with the mTORC1 signaling pathway-mediated protein synthesis, and subsequently the phosphorylation of associated downstream signaling targets was reduced after chronic stress. Finally, we found that intracerebroventricular infusion of rapamycin simulated depression-like behavior and also blocked the antidepressant effects of fluoxetine. Conclusion: Overall, our study suggests that mTORC1 signaling pathway plays a critical role in mediating synapse loss induced by chronic stress, and has part in the behavioral effects of antidepressant treatment.