AbstractAutophagy is an intracellular lysosomal degradation pathway by which cytoplasmic cargoes are removed to maintain cellular homeostasis. This process is essential in neurons that need to cope with prolonged and sustained operational stress, and thus autophagy is known to protect against neurodegenerative diseases including Parkinson’s disease (PD). In PD, a specific subset of regional neurons – midbrain dopaminergic neurons (mDANs) in the substantia nigra pars compacta – are lost, so it is essential to understand the selective vulnerability of these cells. In this context, we have described an improved induced pluripotent stem cell (iPSC) protocol to obtain large numbers of mDANs in culture. This rapid and tractable monolayer-type iPSC differentiation protocol generates >70% verified mDANs within 25 days, and is optimised for fixed and live cell imaging-based studies of autophagy. Overall, this information will provide a better understanding of the regulation of autophagy specifically in the neurons that are lost in PD. The LIM homeobox transcription factors LMX1A and LMX1B cooperatively regulate the specification and proliferation of mDANs during development, and are also needed for their functional maintenance and survival in the adult midbrain. Here we show that LMX1A and LMX1B are autophagy transcription factors in iPSC-derived human mDANs. Their suppression reduces basal autophagy, lowers mitochondrial respiration, and elevates mitochondrial ROS levels; while their inducible overexpression protects against rotenone toxicity in mDANs in vitro. Significantly, we show that LMX1A and LMX1B bind to multiple members of the ATG8 family of core autophagy proteins via LIR-type interaction, contingent on subcellular localisation and nutrient status: under basal conditions, LMX1B interacts with LC3B in the nucleus, but binds to cytosolic LC3B and is degraded by autophagy during nutrient starvation. Crucially, ATG8 binding stimulates LMX1B-mediated transcription, whereas LIR mutant LMX1B is unable to protect mDANs against rotenone. This establishes a novel LMX1B-autophagy regulatory axis that contributes to mDAN maintenance in the adult midbrain with implications for mDAN protection/decline in PD.
|Date of Award||23 Jun 2020|
|Supervisor||Jon D Lane (Supervisor), Pete J Cullen (Supervisor) & Paul B Martin (Supervisor)|
Reciprocal Autophagy Control by the LIM Homeodomain Transcription Factors LMX1A and LMX1B Safeguards Human Midbrain Dopaminergic Neurons
Jimenez Moreno, N. (Author). 23 Jun 2020
Student thesis: Doctoral Thesis › Doctor of Philosophy (PhD)