Mechanism of Stress-induced Translocation of the Serotonin Transporter

描述（由申请方提供）：反复应激暴露在小鼠中产生了类似于焦虑的反应，其在行为上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激活了脑中的一系列神经化学反应，包括CRF激活强啡肽释放，强啡肽激活κ阿片受体，κ刺激p38 α MAPK，以及随后的5-羟色胺转运体（SERT）从内体区室转移到质膜。条件性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别需要强啡肽/Kappa/p38 α MAPK/SERT在中缝背核神经元能神经末梢支配腹侧纹状体;中断这些组件中的任何一个基因或神经元阻断应激诱导的烦躁反应，并赋予这些动物模型的压力恢复。因为压力脆弱性是临床上已知的风险因素， 在人类抑郁症中，所提出的研究旨在进一步表征负责多巴胺能神经末梢中SERT易位的机制。具体来说，我们建议表达人SERT基因的慢病毒构建体含有正常的hSERT编码序列的SERT（-/-）小鼠中缝背核神经元的局部注射。我们之前表明，这恢复了小鼠的SERT功能和压力脆弱性。目的1：通过系统的定点突变方法改变lenti-hSERT中的hSERT编码区，以确定胁迫诱导易位所必需的SERT结构特征。已通过基因组测序在人群中鉴定出hSERT的天然变体，并且这些变体中的一些已被描述为向受影响的个体赋予疾病风险。在目标2中，我们将设计lenti-hSERT来表达这些天然变体，然后确定由此产生的结构变化对小鼠应激反应的影响。表达不同形式的lenti-hSERT的SERT（-/-）小鼠将被评估1）行为上的应激诱导的厌恶反应，2）神经化学上的应激诱导的SERT蛋白向分离自腹侧纹状体的突触体的质膜的易位，和3）生物药理学上使用旋转盘伏安法来确定应激对5-羟色胺转运动力学的影响。拟议的研究将更好地定义腹侧纹状体中5-羟色胺转运的压力诱导变化如何控制小鼠抑郁样行为的风险，并且对SERT的天然人类变体的拟议分析可能有助于描述压力弹性个体差异的可能遗传基础。

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.