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病，其特征是线粒体功能和蛋白质代谢的损害随着年龄的增长而发生。有证据表明，改变控制线粒体移除和溶酶体功能的过程可能会引起或促成这些人类神经退行性疾病的病因学。光遗传学允许膜对光的响应去极化，这项技术已被广泛用于神经元功能的研究。通过设计视紫红质2和古紫质的特异性定位标签，强大的光遗传学技术现在也可以用来研究特定细胞群体和单个细胞器水平上的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调节邻近星形胶质细胞的有丝分裂吞噬(线粒体的移除)，这一过程被称为传递吞噬。因此，除了研究有丝分裂吞噬和溶血作用外，我们还将使用慢病毒载体在细胞特异性启动子的控制下调查吞噬传播性，以评估其在中枢神经系统中的广泛分布程度，并评估其与帕金森氏病的特殊相关性。实验将在哺乳动物细胞系(包括EGFP-Parkin或LC3-eGFPCHE过表达细胞)中进行，使用功能分析和高含量显微镜。这个项目为学生提供了一个独特的机会，学习分子(例如慢病毒的构建、克隆)、光遗传学/成像(例如共聚焦和高含量荧光显微镜)、生化(细胞生物能量学测量、蛋白质聚集、线粒体功能分析)和干细胞生物学(培养来自人类IPS细胞的神经元，这些神经元携带神经退行性疾病相关基因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).