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

Human neurodegenerative disorders such as Parkinson's, and Huntington's diseases are characterised
by the impairment of mitochondrial function and protein metabolism occurring with age. Evidence
suggests that altering the processes governing the removal of mitochondria and function of lysosomes
may 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 been
used extensively in studies of neuronal function. By engineering specific localization tags to
Chanelrhodopsin2 and Archaerhodopsin powerful optogenetic techniques can now also be used to
study mitochondrial and lysosomal function with spatiotemporal control in specific cell populations
and at the level of individual organelles. Recent evidence also suggests that certain neuronal
populations can mediate mitophagy (removal of mitochondria) in neighbouring astrocytes, a process
referred to as transmitophagy. Hence as well as studying mitophagy and lysophagy we will also
investigate transmitophagy using lentiviral vectors under the control of cell specific promoters to
evaluate how widespread it is in the CNS and to evaluate its particular relevance to Parkinson's
disease. Experiments will be performed in mammalian cell lines (including eGFP-Parkin or LC3-eGFPCHE
over-expressing cells) using functional assays and high content-microscopy. Studies will also be
performed in cultures of astrocytes and neurones derived from stem cells and human induced
pluripotent 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) and
stem cell biology (culturing of neurons derived from human IPS cells carrying autosomal dominant
mutations in genes implicated in neurodegenerative disease).