Arrayed functional genomic screening of Parkinson’s disease candidate risk genes identifies modifiers of α-synuclein aggregation and mitophagy in human neurons

Thesis

Parkinson’s disease is an age-related neurodegenerative disease, characterised by the death of neurons in the substantia nigra and other brains regions such as the cortex in late-stage disease. Lewy bodies and Lewy neurites composed of the aggregated pre-synaptic protein α-synuclein are a diagnostic finding in affected brain regions at autopsy. This aggregation process is thought to cause neuronal dysfunction, contributing to cell death in substantial part through impairing the function of organelles such as the mitochondria. Environmental toxins targeting mitochondria or mutations in the mitophagy-associated proteins PINK1 and PRKN can also cause PD. 

Treatment of human iPSC-derived cortical neurons with in vitro aggregated α-synuclein pre-formed fibrils modelled α-synuclein aggregation, triggering the aggregation of endogenous monomer. Using up-to-date methodology significantly reduced batch-to-batch variability and increased the robustness of PFF-induced α-synuclein aggregation in cell models, though these improvements were only weakly correlated with measured parameters such as fibril length, suggesting unknown factors influence a preparation’s aggregation efficacy. CRISPRi-mediated knockdown of SNCA abolished PFF-seeded aggregation in human iPSC-derived cortical neurons, confirming that endogenous monomer is required for the process of aggregation. In addition, co-localisation of the mitochondrial mitophagy marker phospho-serine 65 ubiquitin (pS65Ub) and pathological α-synuclein aggregates marked by phosphorylation at serine 129 (pS129) was found in PFF-treated neurons. This association was influenced by aggregate size and time post-seeding, suggesting a pathological cascade where αsynuclein aggregation results in accumulation of pS65Ub.

Recent studies have demonstrated a contribution of 134 risk loci to the development of PD. The mechanism by which these genes increase disease risk is undefined and we hypothesised that these genes might regulate the rate of α-synuclein aggregation or impede the process of mitochondrial autophagy. A library of 71 candidate risk and 4 monogenic PD genes was screened in iPSC-derived cortical neurons and validated in iPSC-derived dopaminergic neurons, identifying several novel and previously-published genes which may modulate the levels of α-synuclein aggregation or of the mitophagy marker phospho-serine 65 ubiquitin. Understanding the underlying mechanisms of risk gene contribution to disease aetiology is expected to enable idiopathic patient stratification and personalised medicine.

Authors: Heon-Roberts, R

• DOI: 10.5287/ora-5jbadddgw
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: mitophagy; high-throughput microscopy; alpha-synuclein aggregation; parkinson's disease; high-content screening
• Subjects: Neurosciences; Molecular biology