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Title: Molecular characterisation of autophagy deficits in a LRRK2-BAC transgenic rat model of Parkinson's disease
Author: Wallings, Rebecca
ISNI:       0000 0004 7652 9808
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2018
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Parkinson's disease (PD) is the second most common neurodegenerative disorder worldwide. PD is characterised by the preferential loss of dopaminergic neurons in the Substantia Nigra pars compacta in the midbrain accompanied by progressive motor dysfunction. The precise aetiology of PD is unknown, however a causative role of Leucine-rich repeat kinase 2 (LRRK2) has been proposed. Mutations in the LRRK2 gene are the most frequent cause of familial PD and are also an independent risk factor for sporadic PD. Although the function of LRRK2 is not well characterised, a role of LRRK2 in the autophagy pathway has been suggested. The disruption of the autophagy pathway by LRRK2 pathogenic mutations has been described. However, the literature is often contradictory and the exact underlying mechanisms remain unknown. In this study, primary cortical cultures were generated from three bacterial artificial chromosome (BAC) transgenic (TG) rat models of PD harbouring either the whole human wild-type LRRK2 gene, the G2019S mutant (the most common LRRK2 mutation) or the R1441C mutant (the LRRK2 mutation leading to a more aggressive pathology). After characterising the autophagy pathway it was observed that the presence of either hWT-LRRK2 or LRRK2-G2019S inhibits autophagosome biogenesis in primary cortical cultures. hWT-LRRK2 and LRRK2-G2019S each localise to the Golgi apparatus where autophagy signalling complexes are situated, which may underlie the inhibitory effect on autophagosome biogenesis. The presence of LRRK2-R1441C, however, induces a lysosomal deficit that causes an accumulation in autophagosomes and decreased autolysosome maturation and lysosomal protein degradation. LRRK2-R1441C also increases lysosomal pH levels and causes lysosomal Ca2+ release deficits. Furthermore, hWT-LRRK2 and LRRK2 G2019S were found to bind to the a1 subunit of the v-type ATPase pump (vATPase a1) which is responsible for modulating lysosomal pH. Whereas LRRK2-R1441C showed a loss of binding capacity to this subunit, which was associated with a decrease in a1 subunit protein expression and cellular mislocalisation. Lastly, the Zn2+ ionophore, clioquinol, was able to rescue the LRRK2-R1441C-mediated lysosomal phenotypes through modulating lysosomal zinc levels and increased vATPase a1 expression which re-acidified lysosomes, corrected localised calcium release and increased autolysosome maturation. These data describe a novel functional link between LRRK2 and vATPase a1, and define critical mechanisms underlying the inhibition of autophagy by different pathogenic LRRK2 mutations. These findings demonstrate a novel mode of action by which drugs may rescue lysosomal dysfunction and alleviate blockage in autophagic flux. These results demonstrate the importance of LRRK2 in lysosomal biology, as well as the critical role of the lysosome in PD and the potential of lysosome-targeting compounds as a novel therapeutic for the disease.
Supervisor: Wade-Martins, Richard ; Connor-Robson, Natalie Sponsor: Parkinson's UK
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available