Optogenetic control of subcellular electrochemical gradients for the study of mitophagy and lysosomal function in neural cells

人类神经退行性疾病如帕金森氏病和亨廷顿氏病的特征是随着年龄的增长而发生的线粒体功能和蛋白质代谢的损伤。有证据表明，改变线粒体的去除和溶酶体功能的过程可能导致或有助于这些人类神经退行性疾病的病因。光遗传学允许细胞膜对光的反应去极化，该技术已被广泛用于神经元功能的研究。通过对Chanelrhodopsin 2和Archaerhodopsin的特异性定位标签进行工程改造，强大的光遗传学技术现在也可以用于研究特定细胞群体和单个细胞器水平上时空控制的线粒体和溶酶体功能.最近的证据还表明，某些神经元群体可以介导邻近星形胶质细胞的线粒体自噬（线粒体的去除），这一过程被称为transmantophagy。因此，在研究线粒体自噬和溶血自噬的同时，我们还将使用细胞特异性启动子控制的慢病毒载体研究传递自噬，以评估其在中枢神经系统中的广泛程度，并评估其与帕金森病的特殊相关性。将使用功能测定和高含量显微镜在哺乳动物细胞系（包括eGFP-Parkin或LC 3-eGFPCHE过表达细胞）中进行实验。研究也将在培养星形胶质细胞和神经元来源于干细胞和人类诱导多能干细胞（iPSC）。这个项目提供了一个独特的机会，让学生学习分子生物学。（例如慢病毒构建、克隆）、光遗传学/成像（例如，共聚焦和高含量荧光显微镜），生物化学（测量细胞生物能量学，蛋白质聚集，线粒体功能测定）和干细胞生物学（培养衍生自人IPS细胞的神经元，所述人IPS细胞携带涉及神经变性疾病的基因中的常染色体显性突变）。

Human neurodegenerative disorders such as Parkinson's, and Huntington's diseases are characterisedby the impairment of mitochondrial function and protein metabolism occurring with age. Evidencesuggests that altering the processes governing the removal of mitochondria and function of lysosomesmay cause or contribute to the aetiology of these human neurodegenerative illnesses.Optogenetics allow membranes to be depolarised in response to light and the technique has beenused extensively in studies of neuronal function. By engineering specific localization tags toChanelrhodopsin2 and Archaerhodopsin powerful optogenetic techniques can now also be used tostudy mitochondrial and lysosomal function with spatiotemporal control in specific cell populationsand at the level of individual organelles. Recent evidence also suggests that certain neuronalpopulations can mediate mitophagy (removal of mitochondria) in neighbouring astrocytes, a processreferred to as transmitophagy. Hence as well as studying mitophagy and lysophagy we will alsoinvestigate transmitophagy using lentiviral vectors under the control of cell specific promoters toevaluate how widespread it is in the CNS and to evaluate its particular relevance to Parkinson'sdisease. Experiments will be performed in mammalian cell lines (including eGFP-Parkin or LC3-eGFPCHEover-expressing cells) using functional assays and high content-microscopy. Studies will also beperformed in cultures of astrocytes and neurones derived from stem cells and human inducedpluripotent stem cells (iPSCs).This project offers a unique opportunity for the student to learn molecular (e.g. lentiviral construction,cloning), optogenetics/imaging (e.g. confocal and high content fluorescent microscopy), biochemical(measurements of cellular bioenergetics, protein aggregation, assays of mitochondrial function) andstem cell biology (culturing of neurons derived from human IPS cells carrying autosomal dominantmutations in genes implicated in neurodegenerative disease).