Role of the SNARE proteins Vti1a and VAMP7 in central synaptic transmission

描述（由申请人提供）：可溶性n -乙基丙烯酰亚胺敏感因子附着蛋白受体（SNAREs）是在分泌和内吞途径中细胞内囊泡运输和融合所必需的进化保守蛋白。SNAREs在突触囊泡中广泛存在，它们是突触囊泡融合和神经递质释放所必需的。两个质膜蛋白SNAP-25和syntaxin- 1以及突触囊泡蛋白synaptobrevin-2 （Syb2/VAMP2）介导这一过程（Jahn and Scheller, 2006）。此外，Syb2在突触囊泡外胞吞耦合中发挥了特定的作用，而SNAP-25则没有。Syb2和SNAP-25都是正常自发神经传递和诱发神经传递所必需的，但它们在刺激诱发神经传递中相对更重要（Schoch et al., 2001; Bronk et al., 2007）。因此，生理传播必须部分依赖于其他未知的SNARE蛋白，因为一些传递仍然存在于缺乏Syb2或SNAP-25的动物突触中，这些替代的SNARE可能优先支持自发传播。Syb2是结构相关SNARE蛋白家族的成员，其中许多也存在于突触囊泡中，尽管水平较低（Takamori等人，2006）。该家族的一个亚群，包括Vti1a和VAMP7，具有独特的N端延伸，可以调节这些蛋白质支持神经传递的能力（Wang和Tang, 2006； Rossi等人，2004）。基于VAMP7和Vti1a与典型胞外SNAREs的结构域同源性以及它们在突触前囊泡中的定位，VAMP7和Vti1a可能是在Syb2或SNAP-25缺失的情况下支持传播的候选基因。Vti1a和VAMP7在中枢突触传递中的作用将结合最先进的活细胞成像技术和电生理学，使用功能增益和功能损失方法来研究。将使用VAMP7和Vti1a的融合蛋白与ph敏感的GFP偶联来测量这些蛋白的刺激依赖性或非依赖性运输以及支持突触囊泡外胞吞偶联的能力（目的1）。全细胞膜片钳记录将直接证明这些蛋白在突触传递中的功能（目的2）。关键系列实验将评估VAMP7或Vti1a在功能上替代Syb2或SNAP-25的能力，以及它们的n端在这一过程中的调节作用。初步的成像数据显示Vti1a在自发活动期间的特异性运输，推测该蛋白是一种独特的SNARE，能够支持特定形式的神经传递。重要的是，突触囊泡运输的改变可能通过改变突触前或突触后可塑性来促进某些神经退行性或精神疾病的病理影响。对这一基本过程的深入了解将补充这些领域正在进行的研究，并可能阐明新的治疗靶点。

DESCRIPTION (provided by applicant): Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are evolutionarily conserved proteins necessary for intracellular vesicular trafficking and fusion throughout the secretory and endocytic pathways. SNAREs have been extensively characterized in synaptic vesicles, where they are required for synaptic vesicle fusion and neurotransmitter release. Two plasma membrane proteins, SNAP-25 and syntaxin- 1, and the synaptic vesicle protein synaptobrevin-2 (Syb2/VAMP2) mediate this process (Jahn and Scheller, 2006). Furthermore, a specific role in synaptic vesicle exo-endocytosis coupling has been ascribed to Syb2, but not SNAP-25. Both Syb2 and SNAP-25 are required for normal spontaneous and evoked neurotransmission, but each are relatively more important for stimulus-evoked transmission (Schoch et al., 2001; Bronk et al., 2007). Therefore, physiological transmission must partially depend on additional unknown SNARE proteins since some transmission remains in synapses from animals lacking Syb2 or SNAP-25, and these alternative SNAREs may preferentially support spontaneous transmission. Syb2 is a member of a family of structurally related SNARE proteins, many of which are also present in synaptic vesicles, albeit at reduced levels (Takamori et al., 2006). A subgroup of this family, including Vti1a and VAMP7, possesses unique N- terminal extensions that may regulate these proteins' ability to support neurotransmission (Wang and Tang, 2006; Rossi et al., 2004). Based on their domain homologies to the canonical exocytic SNAREs and their localization in presynaptic vesicles, VAMP7 and Vti1a are likely candidates to support transmission in the absence of Syb2 or SNAP-25. The roles of Vti1a and VAMP7 in central synaptic transmission will be studied using both gain- and loss-of-function approaches in combination with state of the art live-cell imaging technologies and electrophysiology. Fusion proteins of VAMP7 and Vti1a coupled to pH-sensitive GFP will be used to measure these proteins' stimulation-dependent or -independent trafficking and ability to support exo- endocytosis coupling in synaptic vesicles (Aim 1). Whole-cell patch-clamp recordings will directly demonstrate functions of these proteins in synaptic transmission (Aim 2). Key series of experiments will assess the ability of VAMP7 or Vti1a to functionally substitute for Syb2 or SNAP-25 as well as the regulatory roles of their N-termini in this process. Preliminary imaging data show specific trafficking of Vti1a during spontaneous activity, putatively identifying this protein as a unique SNARE capable of supporting a specific form of neurotransmission. Importantly, alterations in synaptic vesicle trafficking may contribute to the pathological effects of some neurodegenerative or psychiatric diseases by modifying presynaptic or postsynaptic plasticity. A thorough understanding of this basic process will complement ongoing research in these areas and may elucidate novel therapeutic targets. PUBLIC HEALTH RELEVANCE: Synaptic transmission is a fundamental property of neurons, and alterations in this process are associated with a number of pathological neurodegenerative and psychiatric disorders, including but not limited to lysosomal storage disorders, Parkinson's disease, bipolar disorder, and schizophrenia. The proposed experiments will further elucidate molecular mechanisms underlying presynaptic vesicle trafficking and fusion under normal conditions and promote the discovery of novel therapeutic targets for a variety of human neurological diseases.