Characterizing the role of Extended Synaptotagmin-2 at contact sites between the ER and plasma membrane

 描述(申请人提供)：神经系统由巨大的神经元回路组成，这些回路受到高度调控，以确保适当的突触传递。突触上蛋白质功能的破坏会损害神经元之间的交流，在某些疾病状态下会导致神经元细胞死亡。在许多神经退行性疾病中，轴突退行性变发生在神经元死亡之前，在几种内质网(ER)驻留蛋白中发现了与神经退行性疾病相关的突变。特别是，网状蛋白、atlastin和受体辅助蛋白1(REEP1)与遗传性痉挛性截瘫(HSP)有关，囊泡相关膜蛋白-B(VAP-B)与肌萎缩侧索硬化症(ALS)有关。这些蛋白定位于内质网和质膜之间的接触部位。内质膜接触部位是被认为在钙信号和脂质运输中具有重要作用的特殊区域。然而，关于接触蛋白如何影响轴突质膜完整性、突触功能和轴突变性的研究还不多见。扩展突触素-2(ESYT-2)是一种位于内质网-质膜接触部位的内质网驻留蛋白。哺乳动物有三个esyt基因，而蠕虫只有一个esyt基因，这使得蠕虫成为表征这种蛋白质作用的一个很好的模型系统。使用esyt-2(Tm5783)功能缺失等位基因的初步数据表明，esyt-2突变体存在神经系统和寿命缺陷。对esyt-2突变体中单个轴突的成像显示轴突形态的加速变化，这让人想起已知的神经变性突变体。以前在酵母中的研究已经证明，酵母esyt-2同系物(三碳联素)在建立和维持ER-质膜接触部位方面起着重要作用。有趣的是，在ER-质膜接触位点缺失的酵母中表达ESYT-2可以挽救接触部位，表明其功能是保守的。我们将使用酿酒酵母和秀丽线虫作为模型系统来研究ESYT-2是否调节轴突膜磷脂和二酰甘油的水平，以及这些脂类的失调如何扰乱轴突的完整性，缩短寿命，最终导致神经变性。

 DESCRIPTION (provided by applicant): The nervous system is composed of vast neuronal circuits that are highly regulated to ensure proper synaptic transmission. Disruption of protein function at the synapse can impair communication between neurons and in some disease states cause neuronal cell death. In many of these neurodegenerative diseases, axon degeneration occurs prior to neuronal death, and neurodegenerative disease-associated mutations have been found in several endoplasmic reticulum (ER) resident proteins. In particular reticulon, atlastin and receptor accessory protein 1 (REEP1) are implicated in hereditary spastic paraplegia (HSP) and vesicle-associated membrane protein-associated protein-B (VAP-B) is implicated in amyotrophic lateral sclerosis (ALS). These proteins localize to contact sites between the ER and plasma membrane. ER-plasma membrane contact sites are specialized regions thought to be important in calcium signaling and lipid transport. However, not much is known about how contact proteins affect axonal plasma membrane integrity, synaptic function and axon degeneration. Extended Synaptotagmin-2 (ESYT-2) is an ER resident protein found at ER-plasma membrane contact sites. Mammals have three esyt genes whereas worms have a single esyt gene, making worms an excellent model system to characterize the role of this protein. Preliminary data using the loss of function allele of esyt-2 (tm5783) suggest that esyt-2 mutants have nervous system and lifespan defects. Imaging of single axons in esyt-2 mutants reveal accelerated changes in axon morphology that is reminiscent of known mutants of neuro- degeneration. Prior studies in yeast have demonstrated a major role for the yeast esyt-2 homolog (tricalbins) in establishing and maintaining ER-plasma membrane contact sites. Interestingly, expression of worm ESYT-2 in an ER-plasma membrane contact site deficient yeast can rescue contact sites suggesting its function is conserved. We will use Saccharomyces cerevisiae and Caenorhabditis elegans as model systems to examine if ESYT-2 regulates axonal membrane phospholipid and diacylglycerol levels and how dysregulation of these lipids perturbs axon integrity, shortens lifespan and ultimately causes neuro-degeneration.