Mechanism of Stress-induced Translocation of the Serotonin Transporter

描述（由申请人提供）：重复的应激暴露会在小鼠中产生类似烦躁不安的反应，其行为表现为应激诱导的不动，厌恶和社交回避。在最近的一系列研究中，我们发现暴露于不同形式的持续应激会激活脑内一系列神经化学反应，包括CRF激活促啡肽释放，促啡肽激活kappa阿片受体，kappa刺激p38alpha MAPK，以及随后的5 -羟色胺转运体（SERT）从内体室转移到质膜。条件基因缺失方法和神经化学研究表明，应激诱导的厌恶反应特异性地需要dynorphin/Kappa/p38alpha MAPK/SERT在支配腹侧纹状体的背中叶5 -羟色胺能神经末梢；在这些动物模型中，任何一种成分的遗传或药理学破坏都可以阻断应激诱导的烦躁反应，并赋予应激恢复能力。因为压力易感性是已知的临床风险因素

DESCRIPTION (provided by applicant): Repeated stress exposure produces a dysphoria-like response in mice that manifests behaviorally as stress-induced immobility, aversion and social avoidance. In a series of recent studies, we found that exposure to different forms of sustained stress activates a cascade of neurochemical responses in brain involving CRF activation of dynorphin release, dynorphin activation of kappa opioid receptors, kappa stimulation of p38alpha MAPK, and subsequent translocation of the serotonin transporter (SERT) from an endosomal compartment to the plasma membrane. Conditional gene deletion approaches and neurochemical studies revealed that the stress-induced aversion response specifically required the dynorphin/Kappa/p38alpha MAPK/SERT in the dorsal raphe serotonergic nerve terminals innervating the ventral striatum; disruption of any one of these components genetically or pharmacologically blocked the stress-induced dysphoric response and conferred stress-resilience in these animal models. Because stress-vulnerability is a known risk factor for clinical depression in humans, the proposed studies are designed to further characterize the mechanisms responsible for SERT translocation in the serotonergic nerve terminals. Specifically, we propose to express the human-SERT gene by local injection of a lentiviral construct containing the normal hSERT coding sequence into dorsal raphe neurons of SERT(-/-) mice. We previously showed that this restores SERT functionality and stress-vulnerability in the mice. In aim 1, we would alter the hSERT coding region in the lenti-hSERT by a systematic site-directed mutagenesis approach to define the structural features of SERT necessary for stress-induced translocation. Natural variants of hSERT have been identified in the human population by genome sequencing, and some of these variants have been described as conferring disease risk to the affected individuals. In aim 2, we would engineer lenti-hSERT to express these natural variants, then determine the effects of the resulting structural changes on the stress-response in mice. SERT(-/-) mice expressing different forms of the lenti-hSERT would be assessed 1) behaviorally for stress-induced aversion responses, 2) neurochemically for stress-induced translocation of SERT protein to plasma membrane of synaptosomes isolated from ventral striata, and 3) biophysically using rotating disk electrovoltammetry to define the effects f stress on serotonin transport kinetics. The proposed studies would better define how stress-induced changes in serotonin transport in the ventral striatum might control the risk of depression-like behaviors in mice, and the proposed analysis of the natural human variants of SERT might help describe a possible genetic basis for individual differences in stress-resilience.