Investigating the role of WDR81 in progressive neurological dysfunction

全面了解神经功能障碍进行性形式的生理学是社会面临的一个关键挑战。本博士项目旨在了解含有神经元蛋白WDR81的WD40-BEACH结构域在这些条件下的作用。我们进行了正向基因抑制筛选，以确定绕过神经递质释放中必需蛋白质的新基因突变。从这个筛选中，我们发现WDR81的新突变可以绕过这种必需蛋白，恢复野生型的胞吐水平。尽管WDR81的功能在很大程度上尚未被研究，但该蛋白的特异性突变与人类小脑共济失调、脑小脑发育不全、智力迟钝和四足运动等严重的病理生理缺陷有关(1)。与这些症状一致，WDR81突变小鼠表现出震颤、步态异常以及进行性神经变性。尽管越来越多的证据指向WDR81在细胞内运输和自噬中的作用，但WDR81在这些情况下的功能机制目前尚不清楚(2)。这项研究将使我们能够确定这种新蛋白在膜融合中的确切功能，并从机制上确定WDR81突变如何成为人类复杂神经缺陷的基础。参与该项目的学生将接受多学科培训，包括遗传、细胞和分子方法以及体外生化技术和电生理记录的广泛结合。利物浦大学巴克莱实验室将使用Cas9-CRISPR基因编辑技术，开展一项靶向遗传方法。随着动物年龄的增长，基因编辑的菌株将使用化学感觉和运动神经元输出的功能测定来分析神经功能的进行性恶化。结合这些实验，将采用生物化学方法使用邻近依赖生物素标记来确定WDR81的结合伙伴。最后，该学生将在谢菲尔德大学的苏沃德实验室继续工作，利用膜片钳电生理学、电化学和TIRF（全内反射荧光）技术确定WDR81对胞吐功能的分子机制，以精确量化囊泡对接和融合。通过这个项目，学生将熟悉现代基因、生化和功能分析来研究衰老过程中的神经功能。这些研究的最终目的是确定WDR81在确定进行性神经功能障碍中的作用的新见解。我们将提供广泛的现代遗传、生化和生理技术方面的培训，并提供关于一种独特蛋白质在决定进行性神经功能障碍中的作用的新见解。

A complete understanding of the physiology underlying progressive forms of neurological dysfunction is a key challenge to society. This PhD project aims to understand the role of the WD40-BEACH domain containing neuronal protein WDR81 in these conditions. We have performed a forward genetic suppressor screen to identify novel gene mutations that bypass the requirement of an essential protein in neurotransmitter release. From this screen, we identified that novel mutations in WDR81 could bypass this essential protein and restore wild-type levels of exocytosis. Although WDR81 function has been largely uninvestigated, specific mutations in the protein have been linked with severe pathophysiological defects in humans cerebellar ataxia, cerebro-cerebellar hypoplasia, mental retardation and quadrupedal locomotion (1). In agreement with these symptoms, mice with WDR81 mutations display tremors, abnormal gait as well as progressive neurodegeneration. The mechanism for WDR81 function in these conditions is currently unknown although accumulating evidence points towards a role for WDR81 in intracellular trafficking and autophagy (2). This studentship will allow us to pinpoint the exact function of this novel protein in membrane fusion and determine mechanistically how mutations in WDR81 underlie complex neurological defects in humans. The student undertaking this project will receive multidisciplinary training in a broad combination of genetic, cellular and molecular approaches as well as in vitro biochemical techniques and electrophysiological recordings. A targeted genetic approach will be undertaken in the Barclay lab at the University of Liverpool using Cas9-CRISPR gene editing. The gene edited strains will be analysed for progressive deterioration in neurological function using functional assays for chemosensory and motor neuron output as the animal ages. In conjunction with these experiments, biochemical approaches will be adopted to identify binding partners for WDR81 using proximity-dependent biotin labelling. Finally the student will continue the work in the Seward lab at the University of Sheffield to determine the molecular mechanism of WDR81 function on exocytosis using patch clamp electrophysiology, electrochemical and TIRF (total internal reflection fluorescence) techniques to quantify vesicle docking and fusion at a precise level. From this project, the student will become familiar with modern genetic, biochemical and functional assays to study neurological function during ageing. The ultimate aim of these studies will be to identify novel insights into the role of WDR81 in determining progressive neurological dysfunction. We will provide training in a wide array of modern genetic, biochemical and physiological techniques and deliver novel insights into the role of a unique protein in determining progressive neurological dysfunction.