Using zebrafish to assess the role of inflammation-linked Parkinson’s disease risk genes in neurodegeneration

Parkinson’s disease (PD), a neurodegenerative movement disorder characterised by the loss of dopaminergic neurons in the substantia nigra, has been unequivocally linked to neuroinflammation. Polymorphisms in or near over 90 PD risk genes have been linked with altering the likelihood of developing PD and a number of these genes can also be linked to inflammation. This thesis primarily explores the role of two inflammation-linked risk genes (leucine-rich repeat kinase 2 (LRRK2) and α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD)) using the zebrafish as a model system. acmsd and lrrk2 were shown to be expressed in larval and adult zebrafish, and zebrafish carrying loss-of-function mutations in both genes were generated using CRISPR-Cas9 technology. Homozygous mutants were viable, fertile and exhibited no abnormalities in movement, dopaminergic neurons or microglia. They also demonstrated no change in susceptibility to hepatotoxic, neurotoxic or pro-inflammatory compounds. Metabolomics analysis identified an increase in kynurenine aminotransferase activity in acmsd mutant zebrafish, offering an attractive hypothesis to explain the lack of phenotype in this line; kynurenic acid, the product of kynurenine aminotransferase, can antagonise quinolinic acid, which may reduce the toxic effects of the increase in quinolinic acid detected in both larval and adult acmsd mutants. Additionally, several new lrrk2-mutant zebrafish lines were successfully created using a novel genetic editing technique. This site-directed mutagenesis enabled the induction of specific amino acid changes that recapitulate pathogenic mutations of LRRK2 in PD. This methodology could also now be applied to other genes and research areas, increasing the relevance of zebrafish as a model for human disease. To conclude, whilst both lrrk2 and acmsd are expressed throughout life in zebrafish, a loss of their expression did not result in a PD-like phenotype. A possible explanation for this was identified for acmsd and new stable mutants carrying distinct point mutations were generated for lrrk2.