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

描述(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(SNARES)是进化上保守的蛋白质，对细胞内囊泡在分泌和内吞途径中的运输和融合是必要的。SNARs广泛存在于突触小泡中，在那里它们是突触小泡融合和神经递质释放所必需的。两种质膜蛋白SNAP-25和Synaxin-1以及突触小泡蛋白Synaptobrevin-2(Syb2/VAMP2)介导了这一过程(Jahn和Scheller，2006)。此外，Syb2在突触小泡外吞作用中起着特殊的作用，而SNAP-25没有。Syb2和SNAP-25都是正常的自发和诱发神经传递所必需的，但它们对刺激诱发的传递相对更重要(Schoch等人，2001；Bronk等人，2007)。因此，生理传递必须部分依赖于其他未知的圈套蛋白，因为一些传递仍然存在于缺乏Syb2或SNAP-25的动物的突触中，而这些替代的圈套可能优先支持自发传递。Syb2是结构上相关的SNARE蛋白家族的成员，其中许多也存在于突触小泡中，尽管水平降低(Takamori等人，2006年)。该家族的一个亚群，包括Vti1a和VAMP7，具有独特的N末端延伸，可能调节这些蛋白质支持神经传递的能力(Wang和Tang，2006；Rossi等人，2004)。根据它们与典型胞外陷阱的结构域同源性以及它们在突触前小泡中的定位，VAMP7和Vti1a很可能在缺乏Syb2或SNAP-25的情况下支持信号传递。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，使用增益法和功能损失法进行研究。VAMP7和Vti1a与pH敏感的绿色荧光蛋白偶联的融合蛋白将被用来测量这些蛋白在突触小泡中的刺激依赖或独立运输以及支持外吞饮耦合的能力(目标1)。全细胞膜片钳记录将直接显示这些蛋白质在突触传递中的功能(目标2)。关键的一系列实验将评估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，Vti1a的特定运输，推测这种蛋白质是一种独特的陷阱，能够支持特定形式的神经传递。重要的是，突触小泡运输的改变可能通过改变突触前或突触后的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。 公共卫生相关性：突触传递是神经元的基本属性，这一过程中的变化与许多病理性神经退行性和精神障碍有关，包括但不限于溶酶体储存障碍、帕金森病、双相情感障碍和精神分裂症。这些实验将进一步阐明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疾病的新治疗靶点。

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.