Abstract
Lysosomal dysfunction lies at the centre of the cellular mechanisms underlying Parkinson’s disease although the precise underlying mechanisms remain unknown. We investigated the role of leucine-rich repeat kinase 2 (LRRK2) on lysosome biology and the autophagy pathway in primary neurons expressing the human LRRK2-G2019S or LRKK2-R1441C mutant or the human wild-type (hWT-LRRK2) genomic locus. The expression of LRRK2-G2019S or hWT-LRRK2 inhibited autophagosome production, whereas LRRK2-R1441C induced a decrease in autophagosome/lysosome fusion and increased lysosomal pH. In vivo data from the cortex and substantia nigra pars compacta of aged LRRK2 transgenic animals revealed alterations in autophagosome puncta number reflecting those phenotypes seen in vitro. Using the two selective and potent LRRK2 kinase inhibitors, MLi-2 and PF-06447475, we demonstrated that the LRRK2-R1441C-mediated decrease in autolysosome maturation is not dependent on LRRK2 kinase activity. We showed that hWT-LRRK2 and LRRK2-G2019S bind to the a1 subunit of vATPase, which is abolished by the LRRK2-R1441C mutation, leading to a decrease in a1 protein and cellular mislocalization. Modulation of lysosomal zinc increased vATPase a1 protein levels and rescued the LRRK2-R1441C-mediated cellular phenotypes. Our work defines a novel interaction between the LRRK2 protein and the vATPase a1 subunit and demonstrates a mode of action by which drugs may rescue lysosomal dysfunction. These results demonstrate the importance of LRRK2 in lysosomal biology, as well as the critical role of the lysosome in PD.
Highlights
Parkinson’s disease (PD) is a common progressive neurodegenerative disease, affecting ∼1–2% of the population over the age of 65 years[1]
Co-immunofluorescence staining for the autophagy proteins LC3 and LAMP1 to quantify autophagosomes and autolysosomes demonstrated leucine-rich repeat kinase 2 (LRRK2)-R1441C neurons showed a significant increase in autophagosome number and a significant decrease in autolysosome number (Fig. 1D and E; Supplementary Material, Fig. S1f)
Our work has demonstrated differential effects on the autophagy/lysosomal pathway by the LRRK2-R1441C and hWTLRRK2 and LRRK2-G2019S variants in primary neuronal cultures
Summary
Parkinson’s disease (PD) is a common progressive neurodegenerative disease, affecting ∼1–2% of the population over the age of 65 years[1]. Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of familial PD [2], and cases have an indistinguishable clinico-pathological phenotype from sporadic PD. Pathogenic mutations are found in the enzymatic domains of the LRRK2 protein. The LRRK2-G2019S mutation in the kinase domain leads to an increase in kinase activity [4], a potential pathogenic mechanism of LRRK2 that is further supported by the protective effects of LRRK2 kinase inactivation [5,6]. Located in the GTPase domain of LRRK2, Arg1441 is the second most common site of pathogenic LRRK2 substitutions (LRRK2-R1441C/G), leading to decreased GTP hydrolysis [7,8] and motor deficits and defects in dopaminergic neurotransmission in rodent models [9,10,11,12]
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