Abstract

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and decreased levels of dopamine. 5% of PD cases are linked to genetic mutations. Point mutations in α-synuclein (SNCA), PTEN-inducible serine/threonine kinase 1 (PINK1) and Parkin ubiquitin E3 ligase (PRKN) genes, but also duplications and triplications of the SNCA gene, have been identified as causes of familial PD. Mitochondrial dysfunction and aggregation of α-synuclein into fibrillary structures are characteristics of the disease and are involved in neurodegeneration. The proteins PINK1 and Parkin have been implicated in the targeting of damaged mitochondria for mitophagy, while alpha-synuclein has been shown to have an impact on mitochondrial function and transport.

Our lab has developed novel models of PD by simultaneously using induced pluripotent stem cell (iPSC)-derived dopaminergic or cortical neurons and CRISPR interference (CRISPRi) technology, a genetic technique that allows sequence-specific repression of gene expression by using single guide RNAs (sgRNAs) to target genes such as PINK1 and PRKN. Using CRISPRi, we have been able to recapitulate patient cell phenotypes, such as loss of the mitophagy marker phospho-serine 65 ubiquitin, seen in iPSC-derived dopaminergic neurons obtained from patients carrying PINK1 and PRKN mutations, confirming that CRISPRi is functional and can be used to model genetic PD. This technique can be used in combination with genetic mutations such as alpha-synuclein triplications, in order to analyse the effects of specific gene repressions in an otherwise similar cellular and genetic context.

Our data supports the idea that CRISPRi technology can phenocopy patient-derived neuronal cells. Aligning patient phenotypes with CRISPRi models, we can pinpoint the overall mechanisms by which this disease occurs, focusing on mitochondria, and the basis to identify potential therapeutic approaches that could be used to treat patients.