Law, Bernard Man Hin; (2011) Characterisation of LRRK2 - tubulin interactions in Parkinson's disease. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

Mutations in PARK8 encoding the cytosolic protein leucine-rich repeat kinase 2 (LRRK2) are the most common known cause for Parkinson's disease (PD). LRRK2 belongs to the ROCO family of proteins which are characterised by a Roc (Ras in complex proteins) domain with intrinsic GTPase activity and a COR (C-terminal o f Roc) domain. The modification of LRRK2 GTPase and kinase activity by PARK8 mutations is believed to lead to neuronal cell death but the pathways involved remain elusive. To further our understanding of LRRK2 signalling pathways leading to cell death, several previously identified LRRK2 interacting proteins were characterised. As evidence of an involvement of axonal transport defects in the pathogenesis of PD is accumulating, I decided to focus on the LRRK2 interaction with β-tubulin. Tubulin is the main component of microtubules, a part of the cytoskeleton. Changes in phosphorylation of tubulin and microtubule-associated proteins lead to changes in the dynamic instability of microtubules with detrimental effects for axonal transport, ultimately leading to synaptic defects and axonal degeneration. Using the yeast two- hybrid (YTH) system I found that the LRRK2 Roc-COR domain interacts specifically with some neuronally expressed β-tubulin isoforms. This interaction was further characterised using the YTH system, biochemical methods and mammalian cellular models. The binding domains on LRRK2 and β-tubulin isoforms mediating the interaction were determined and LRRK2 association with tubulin was shown in a differentiated dopaminergic human neuroblastoma cell model. Importantly, 1 found that PARK8 mutations segregating with PD and the phosphorylation state of LRRK2 and β-tubulin might influence the interaction between LRRK2 and microtubules. Finally, genetic screening for PARK8 mutations using LightScanner technology of postmortem samples from the Imperial Parkinson's Disease Society Brain Bank revealed a novel mutation in the LRRK2 GTPase domain. The modulation of the interaction between β-tubulin isoforms and LRRK2 by PARKS mutations and phosphorylation as well as the expression of these proteins in human substantia nigra indicate the patho-physiological relevance of these interactions and might lead us towards a better understanding of the disruption of signalling pathways resulting in neurodegeneration.

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