The role of endoplasmic reticulum-mitochondria contacts in neurodegeneration

背景。神经退行性疾病是一种复杂的疾病，也是一个重大的公共卫生问题。神经元功能障碍有多种分子机制，但其中许多机制集中在线粒体和/或内质网（ER）功能的改变上。内质网和线粒体具有相互依赖的功能，并在特定的接触部位相互作用。调节内质网线粒体接触的基因突变导致遗传形式的神经变性，但是内质网线粒体通讯缺陷导致神经变性的确切机制仍然知之甚少。在这里，我们建议研究er -线粒体接触在charco - marie - tooth病2A型（CMT2A）中的作用，CMT2A是一种由线粒体融合因子mitofusin 2 （MFN2）突变引起的运动和感觉神经病变。MNF2最近被证明是一种可能的系链蛋白，调节内质网线粒体的接触。我们的假设是，MFN2中的CMT2A突变影响er -线粒体接触，导致线粒体动力学和功能破坏，从而导致神经元死亡。我们将使用一种新的人类多能干细胞（hPSC） CMT2A模型，即在第一导师实验室开发的MFN2R94Q/+运动和感觉神经元，以及在第二导师实验室建立的细胞和分子er线粒体和神经生物学分析来验证这一假设。1：确定改变er -线粒体接触对线粒体动力学和运动神经元和感觉神经元功能行为的影响。我们将操纵er -线粒体接触（例如，通过敲除MNF2或PTPIP51/VAPB系链或通过合成系链的过表达），并分析此类操作对线粒体形态、动力学和神经元功能的影响。2：表征来自MFN2R94Q/+ hPSCs及其等基因野生型对应物的运动和感觉神经元中的er线粒体接触。我们将使用接近结扎法和电子显微镜定量er -线粒体接触，并通过钙成像分析功能接触。3：确定er -线粒体接触在MFN2R94Q/+表型中的作用。我们将评估操纵MFN2R94Q/+神经元中er -线粒体接触对这些细胞线粒体运输缺陷特征及其行为（电生理、存活/死亡）的影响。新奇和时效性。内质网线粒体接触最近才与CMT和其他几种神经退行性疾病有关。对er -线粒体接触机制的理解将直接为CMT治疗提供新方法，并可能与其他神经退行性疾病相关。实验方法。该项目使用干细胞技术、超分辨率显微镜、电生理学、生物化学和分子生物学方法，包括使用CRISPR/Cas9。

Background. Neurodegenerative disorders are complex diseases and a significant public health concern. Multiple molecular mechanisms underpin neuronal dysfunction, but many of them converge on altered function of mitochondria and/or endoplasmic reticulum (ER). ER and mitochondria have interdependent functions and physically interact with each other at specialised contact sites. Mutations in genes that regulate ER-mitochondria contact cause genetic forms of neurodegeneration, but the exact mechanisms by which defects in ER-mitochondria communication underpin neurodegeneration remain poorly understood. Here, we propose to investigate the role of ER-mitochondria contacts in Charcot-Marie-Tooth disease type 2A (CMT2A), a motor and sensory neuropathy caused by mutations in a mitochondrial fusion factor, mitofusin 2 (MFN2). MNF2 was recently shown to act as a possible tether protein regulating ER-mitochondria contacts. Our hypothesis is that CMT2A mutations in MFN2 affect ER-mitochondria contacts causing disruption of mitochondrial dynamics and function, and consequently neuronal death. We will test this hypothesis using a novel human pluripotent stem cell (hPSC) model of CMT2A, namely MFN2R94Q/+ motor and sensory neurons developed in the laboratory of the first supervisor and the cellular and molecular ER-mitochondria and neurobiology assays established in the laboratory of the second supervisor.Objectives.1: Determine the impact of altering ER-mitochondria contacts on mitochondrial dynamics and the functional behaviour of motor and sensory neurons.We will manipulate ER-mitochondria contacts (e.g. by knocking down MNF2 or the PTPIP51/VAPB tether or by overexpression of synthetic tethers) and analyse the impact of such manipulations on mitochondrial morphology and dynamics and neuronal function.2: Characterise ER-mitochondria contacts in motor and sensory neurons derived from MFN2R94Q/+ hPSCs and their isogenic wild type counterparts. We will quantify ER-mitochondria contacts using proximity-ligation assays and electron microscopy, and assay the contact functionally by calcium imaging.3: Determine the role of ER-mitochondria contacts in the phenotype of from MFN2R94Q/+. We will assess the impact of manipulating ER-mitochondria contacts in MFN2R94Q/+ neurons on the mitochondrial trafficking defects characteristic of these cells and on their behaviour (electrophysiology, survival/death).Novelty and timeliness. ER-mitochondria contacts have been implicated in CMT and several other neurodegenerative diseases only recently. The mechanistic understanding of ER-mitochondria contacts uncovered here will directly inform novel approaches for CMT treatment and may be relevant to other neurodegenerative diseases.Experimental approach. This project uses stem cell technology, super-resolution microscopy, electrophysiology, biochemistry and molecular biology approaches, including the use of CRISPR/Cas9.