Investigation of molecular pathways associated with Parkin

Parkinson’s disease (PD) is an incurable neurodegenerative disease. Although the majority of PD cases are sporadic, 5-10% of cases are inherited. Studies of sporadic and genetic forms of PD suggest shared pathogenesis such as mitochondrial dysfunction. Mutations in the gene encoding Parkin are the most common cause of autosomal recessive, young-onset PD. Parkin has been shown to regulate mitochondrial quality control (mitophagy), however the molecular pathways that regulate Parkin activity remain poorly characterised. MEKK3/p38, MAPK/ERK, and PI3K/Akt signalling pathways have been described in association with Parkin regulation. In this thesis, I have investigated whether activation of any of these pathways could lead to Parkin phosphorylation by utilising inducible cell lines overexpressing MEKK3-ER, Raf¬ER or Akt-ER genes. I found that Parkin was not phosphorylated following the activation of the p38, ERK and Akt pathways. In an attempt to depolarise mitochondria in neuroblastoma SH-SY5Y cells lines by mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP), I found that Parkin was phosphorylated at serine 101 (S101). In order to investigate the role of phosphorylation of Parkin S101 in mitochondrial quality control, I established SH-SY5Y clones stably expressing wild type (WT), non-phosphorylatable (S101A), or phosphomimetic (S101D) FLAG-Parkin. I found that this phosphorylation is associated with increased Parkin’s E3 ligase activity. S101A cells showed deficiencies in translocation of Parkin to depolarised mitochondria, ubiquitination of outer mitochondrial membrane proteins, p62 (an autophagy adaptor) recruitment, perinuclear mitochondrial clustering and mitophagy. Overall the work presented in this thesis demonstrates that Parkin is activated during mitochondrial depolarisation, and that the regulation of Parkin function via phosphorylation at S101 plays an important role in mitochondrial quality control associated with PD pathophysiology