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，是一个重要的公共卫生问题。多种分子机制支持神经元功能障碍，但其中许多都集中在线粒体和/或内质网（ER）的功能改变。ER和线粒体具有相互依赖的功能，并在专门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，但内质网-线粒体通讯缺陷支持神经变性的确切机制仍然知之甚少。在这里，我们建议调查ER-线粒体接触在Charcot-Marie-Tooth病2A型（CMT 2A）中的作用，这是一种由线粒体融合因子mitofusin 2（MFN 2）突变引起的运动和感觉神经病。MNF 2最近被证明是一种可能的系绳蛋白调节ER-线粒体接触。我们的假设是MFN 2中的CMT 2A突变影响ER-线粒体接触，导致线粒体动力学和功能的破坏，并因此导致神经元死亡。我们将使用CMT 2A的新的人多能干细胞（hPSC）模型（即在第一监督者的实验室中开发的MFN 2 R94 Q/+运动和感觉神经元）和在第二监督者的实验室中建立的细胞和分子ER-线粒体和神经生物学测定来测试该假设。确定改变内质网-线粒体接触对线粒体动力学以及运动和感觉神经元功能行为的影响。（例如通过敲低MNF 2或PTPIP 51/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和动力学以及神经元功能的影响。2：表征源自MFN 2 R94 Q/+ hPSC及其同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。我们将使用邻近连接测定和电子显微镜定量ER-线粒体接触，并通过钙成像功能测定接触。3：确定ER-线粒体接触在MFN 2 R94 Q/+表型中的作用。我们将评估在MFN 2 R94 Q/+神经元中操纵ER-线粒体接触对这些细胞的线粒体运输缺陷特征及其行为（电生理学，存活/死亡）的影响。新奇和及时性。ER-线粒体接触最近才与CMT和其他几种神经退行性疾病有关。在这里发现的ER-线粒体接触的机制理解将直接为CMT治疗提供新的方法，并可能与其他神经退行性疾病相关。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.