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An RNAi screen to discover novel modulators of PINK1 related effects on mitochondrial dynamics.

Pogson, J (2012) An RNAi screen to discover novel modulators of PINK1 related effects on mitochondrial dynamics. PhD thesis, University of Sheffield.

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Abstract

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder. It is characterised by the loss of dopaminergic neurons in the substantia nigra. Causes of PD are largely unknown although cases of familial PD have lead to the identification of disease causing genetic factors such as loss of function mutations in Pten-induced Kinase 1 (PINK1). PINK1 is a putative kinase that contains a mitochondrial localisation sequence. PINK1 acts in a common pathway with another PD gene, parkin, that appears to regulate mitophagy but little else is known about the components of this pathway. This study aims to dissect the pathway and find novel modulators of PINK1 loss of function. Loss of PINK1 in Drosophila causes dopaminergic neuron loss, muscle degeneration, mitochondrial defects along with reduced climbing and flight ability. Previous studies have shown that PINK1 genetically interacts with genes that control mitochondrial fusion (OPA1 and Mfn) and fission (Drp1 and Fis1). RNAi mediated knockdown of PINK1 in Drosophila S2R+ cells results in elongation of the mitochondrial network. This cellular phenotype was used to perform a cell based RNAi screen on a kinome and phosphatome library consisting of around 700 genes. Results were compared from two parallel screens; wild type and PINK1 knockdown (KD) background. Two groups of hits were identified from the results 40 rescuers of PINK1 KD and 23 phenocopiers of PINK1 KD. These were rescreened under more stringent conditions resulting in 24 confirmed rescuers and 11 confirmed phenocopiers. In vitro rescuers and phenocopiers were tested for suppression or recapitulation of PINK1 mutant phenotypes in vivo using VDRC transgenic RNAi lines. Several PINK1 phenotypes were tested including thoracic indentations, climbing and flight ability. KD of 4 genes have shown rescue of PINK1 mutant phenotypes. KD of 6 genes has shown to phenocopy PINK1 mutant phenotypes. Follow up studies on human homologues of 2 genes nuf (Rab11 FIP3 and Rab11 FIP4) and fwd (PI4K-β) assayed changes in levels of mitophagy using over- expression and knockdown via RNAi in a CCCP induced HeLa cell model of mitophagy. Rab11 FIP4 and PI4K-β, but not Rab11 FIP3, were found to have significant effects on levels of mitophagy.

Item Type: Thesis (PhD)
Academic Units: The University of Sheffield > Faculty of Science (Sheffield) > Biomedical Science (Sheffield)
The University of Sheffield > Faculty of Medicine, Dentistry and Health (Sheffield) > Biomedical Science (Sheffield)
Depositing User: Dr J Pogson
Date Deposited: 20 Sep 2012 14:08
Last Modified: 08 Aug 2013 08:50
URI: http://etheses.whiterose.ac.uk/id/eprint/2770

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