Ca2+ cellular compartment-specific deficits in iPSC-derived neuronal models of Parkinson's disease

Parkinson’s disease is a neurogenerative disease characterised by the degeneration of dopaminergic neurons of the substantia nigra. I employ induced pluripotent stem cell-derived dopaminergic neurons from patients and controls to study cytotoxic mechanisms in Parkinson’s disease.
Calcium is crucial in the regulation of many neuronal cellular processes, and even more so, in dopaminergic neurons where continuous calcium oscillations occur. This suggests that even small alterations in calcium homeostasis might be deleterious to dopaminergic neurons.
Therefore, I focused my attention on the role of intracellular calcium in the pathogenesis of Parkinson’s disease. To this end, I employed compounds to stimulate intracellular calcium release in conjunction with Fura-2 AM to monitor gross changes in calcium dynamics, and organelle-entrapped genetically encoded calcium indicators to trace back the subcellular source of the defect in dopaminergic neurons derived from Parkinson's patients harbouring mutations in the SNCA and GBA genes respectively encoding for the protein alpha-synuclein and the enzyme beta-glucocerebrosidase. From these experiments, it appeared that SNCA-Trp and GBA-N370S DAns display decreased intracellular calcium release upon ionomycin stimulation in a Fura-2 AM-based assay as well as decreased endoplasmic reticulum calcium release upon ionomycin stimulation using the ER calcium sensor GCEPIAer. SNCA-Trp and GBA-N370S DAns also display decreased mitochondrial calcium uptake upon ionomycin stimulation using the mitochondrial calcium sensor RGECOmt. Further investigation of the source of the deficit was pursued in a functional genetics experiment which employs CRISPRi knock-down of calcium channels and calcium regulators.
I also optimised a proteomic analysis of the endoplasmic reticulum membrane and the outer mitochondrial membrane using proximity biotinylation. In APEX2-catalised proximity labelling subcellular proteomics, spatial resolution is obtained through genetic targeting of the peroxidase APEX2. The experimental design followed the one described by Prof Alice Ting laboratory and involved the use of an endoplasmic reticulum membrane-targeted APEX2 (ERM-APEX2) and an outer mitochondrial membrane-targeted APEX2 (APEX2-OMM) to label the two compartments as well as a cytoplasmic APEX2 (APEX-NES) as a control. I cloned these constructs into an all-in-one doxycycline-inducible PiggyBac transposon construct which I delivered to 3 control iPSC lines and 3 SNCA iPSC lines via nucleofection. We evaluated APEX2 activity in the cells using streptavidin blot analysis and optimised APEX2-biotinylated protein enrichment.
In collaboration with AstraZeneca, Janine Brandes and Dr Brent Ryan in the lab have recently screened more than 4000 compounds for their ability to recover the calcium response to ionomycin in GBA-N370S DAns using a Fura-2 AM-based assay. This screening highlighted a novel potential druggable target for Parkinson’s disease. In this thesis, I evaluated the ability of inhibitors of this target to revert other phenotypes in GBA-N370S DAns. In particular, I focused on calcium handling defects in the endoplasmic reticulum using GCEPIAer. I also explored their efficacy against endoplasmic reticulum stress and autophagy defects.

Keywords: Parkinson's
calcium
iPSC
Advisor: Wade-Martins, R
Lang, C