Abstract:
Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra. While environmental and genetic factors both contribute to disease risk, pathogenic mutations in PINK1 are among the most well-established causes of autosomal recessive early-onset PD. Despite two decades of research, the precise physiological cellular mechanisms through which PINK1 regulates neuronal health remain incompletely understood.
This thesis explores the biology of PINK1 in human dopaminergic neurons using stem cell derived neuronal models, proteomics, and functional assays. First, a novel pathogenic mutation, PINK1 p.F385S, was characterized in an Indian family with early-onset PD. Functional studies revealed that this mutation destabilizes the kinase domain, abolishes phosphorylation of ubiquitin at Ser65, prevents Parkin recruitment, and thereby disrupts PINK1/Parkin mitophagy.
Second, spatio-temporal proteomics using microfluidic chambers was applied to investigate protein turnover, axonal enriched and synthesized proteins and protein trafficking between the soma and axonal part of human dopaminergic neurons. These studies provide a quantitative resource of proteome dynamics and highlight neuronal compartment specific regulation of proteostasis.
Third, a previously unrecognized role of PINK1 in regulating cholesterol metabolism was identified in human dopaminergic neurons. Using phosphoproteomics, we showed altered phosphorylation of a sterol regulatory protein, SCAP. Loss of PINK1 stabilizes SCAP, increases cholesterol biosynthesis and leads to cholesterol accumulation in plasma membranes and lipid rafts. This dysregulation disrupts dopamine transporter localization and impairs neurotransmitter uptake, identifying cholesterol imbalance as an early phenotype in the pathogenesis of PINK1 associated PD.
Collectively, this work expands the mechanistic repertoire of PINK1 biology beyond mitochondrial quality control and identifies a previously unreported mechanism on how PINK1 dysfunction contributes to PD. Additionally, we also provide a proteomic framework using human dopaminergic neurons for future studies into the cellular pathways driving PD pathogenesis.
PINK1