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

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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).