SNARE-dependence of the exocytosis at the inner hair cell ribbon synapse

感官系统为生物体提供有关外部世界的信息。例如，声音信息由听觉系统处理，涉及耳蜗毛细胞传递机械刺激。最后，声学信息在内毛细胞(LHC)的带状突触处编码。尽管分子生物学和生物物理学的研究工作在带状突触的研究方面取得了重大进展，但对其分子生理学仍知之甚少。这个由DFG资助的项目的工作提出了一个挑衅性的问题，即这种突触是否使用了典型的神经元陷阱复合体。在大多数突触的胞吐过程中，三种SNARE蛋白Synaxin 1、Synaptobrevin 2和SNAP-25之间形成了一个四螺旋卷曲的蛋白质复合体。它在囊泡和质膜之间架起桥梁，在胞吐作用的对接到融合过程中起重要作用。在第一个资助期，雷吉斯·努维安做出了惊人的观察，发现IHC的胞吐作用对梭状芽胞杆菌神经毒素E和D具有抵抗力，这两种毒素在许多其他神经分泌制剂中分别通过裂解SNAP-25和Synaptobrevin来干扰胞吐作用。此外，突触素1基因功能突变缺失的小鼠突变体表现出正常的IHC胞吐功能，这是意想不到的，因为突触素1已经在豚鼠IHC中被明确识别。在探索SNARs在小鼠肝细胞中的表达时，我们没有发现一致的证据来证明神经元SNARs的表达。这次拨款续期的目的是通过对SNARS表达的进一步实验(单细胞聚合酶链式反应、免疫组织化学和对敲入小鼠的SNARS荧光标记变体的分析)以及通过进一步测试梭状芽胞杆菌神经毒素的作用，来确定参与IHC胞吐作用的SNARE。这项工作将在哥廷根与雷吉斯·努维安合作进行，诺维安发起了这项研究，但将于2008年底在蒙彼利埃担任国家研究中心的职位，然后才能完成这一项目。

The sensory systems provide the organism with Information about the extemal world. Acoustic information, for example, is processed by the auditory system involving transduction of mechanical stimuli by hair cells of the cochlea. Finally acoustic information is encoded at the ribbon synapses of the inner hair cells (lHCs). Although molecular biological and biophysical works have initiated major progress in investigating ribbon synapse, its molecular physiology is still poorly understood. Work of this DFG-funded project has raised the provocative question whether this synapse employs the archetypical neuronal SNARE complex. During exocytosis in most synapses, a four-helical coiled-coil protein complex is formed between the three SNARE proteins syntaxin 1, synaptobrevin 2 and SNAP-25. It bridges vesicle and plasma membrane and has been implied from docking to fusion step of exocytosis. During the first funding period Regis Nouvian has made the striking observation that exocytosis in IHCs is resistant to clostridial neurotoxins E and D, which in many other neurosecretory preparations interfere with exocytosis by cleaving SNAP-25 and synaptobrevin, respectively. Moreover, a mouse mutant with a loss of function mutafion in the synaptobrevin 1 gene showed normal IHC exocytosis, which was unexpected, given that synaptobrevin 1 had been formely identified in guinea pig IHCs. When exploring the expression of SNAREs in mouse IHCs we did not find consistent evidence for the expression of neuronal SNAREs. The objective of this grant renewal is to identify the SNAREs involved in IHC exocytosis by further experiments on the expression of SNAREs (single-cell PCR, immunohistochemistry and analysis of knock-in mice for fluorescently tagged variants of SNAREs) and by further testing the effects of clostridial neurotoxins. This work will be performed in Göttingen in collaboration with Regis Nouvian, who initiated this study but will move on to take a CNRS position in Montpellier at the end of 2008 before this project can be completed.