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

描述（由申请人提供）：可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）是在整个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。SNARE在突触囊泡中具有广泛的特征，在那里它们是突触囊泡融合和神经递质释放所必需的。两种质膜蛋白SNAP-25和突触融合蛋白-1以及突触囊泡蛋白小突触蛋白-2（Syb 2/VAMP 2）介导该过程（Jahn和舍勒，2006）。此外，突触囊泡外吞-内吞偶联中的特定作用已归因于Syb 2，而不是SNAP-25。Syb 2和SNAP-25都是正常的自发和诱发的神经传递所需要的，但每一个对于刺激诱发的传递都相对更重要（Schoch et al.，2001; Bronk等人，2007年）。因此，生理传递必须部分依赖于额外的未知SNARE蛋白，因为一些传递仍然存在于缺乏Syb 2或SNAP-25的动物的突触中，并且这些替代SNARE可能优先支持自发传递。Syb 2是结构相关的SNARE蛋白家族的成员，其中许多也存在于突触囊泡中，尽管水平降低（Takamori et al.，2006年）。该家族的一个亚组，包括Vtila和VAMP 7，具有独特的N-末端延伸，其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年）。基于它们与典型的胞吐SNARE的结构域同源性及其在突触前囊泡中的定位，VAMP 7和Vti 1a可能是在缺乏Syb 2或SNAP-25的情况下支持传输的候选者。Vti 1a和VAMP 7在中枢突触传递中的作用将使用功能获得和功能丧失方法结合最先进的活细胞成像技术和电生理学进行研究。将使用与pH敏感性GFP偶联的VAMP 7和Vtila的融合蛋白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中的外吞-内吞偶联的能力（Aim 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VAMP 7或Vti 1a在功能上替代Syb 2或SNAP-25的能力，以及它们的N-末端在这一过程中的调节作用。初步的成像数据显示，在自发活动期间Vti 1a的特定运输，pupelium将这种蛋白质鉴定为能够支持特定形式的神经传递的独特SNARE。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而导致一些神经退行性疾病或精神疾病的病理效应。对这一基本过程的透彻理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。 公共卫生相关性：突触传递是神经元的基本性质，并且该过程中的改变与许多病理性神经变性和精神障碍相关，包括但不限于溶酶体贮积症、帕金森病、双相情感障碍和精神分裂症。拟议的实验将进一步阐明在正常条件下突触前囊泡运输和融合的分子机制，并促进发现各种人类神经系统疾病的新的治疗靶点。

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.