Leucine-rich repeat kinase 2 (LRRK2)

Abstract

Mutations in PARK8 associated leucine-rich repeat kinase 2 (LRRK2) have been shown to be the leading cause of autosomal dominant Parkinson's disease (PD). The multidomain LRRK2 is expressed ubiquitously, including the central nervous system and various organs. Previously novel R767H, S885N and reported R1441H were found in Taiwanese PD patients, in addition to R1628P and G2385R risk factors in ethnic Chinese populations. In the first part of this study, EGFP-tagged wild type, R1441H, R1628P and G2385R LRRK2 constructs (with S1647T and M2397T SNPs) were prepared for transient expression in HEK-293T cells. Western blot analysis and fuorescence microscopy examination revealed that neither localization nor processing of LRRK2 was affected by R1441H, R1628P and G2385R variations. Secondly, α-synuclein cDNA was cloned and co-transfected with the EGFP-tagged wild type or mutant (R767H, S885N, R1441H, G2019S) LRRK2 constructs in SK-N-SH cells. Confocal microscopy examination revealed that wild-type LRRK2 was widespread cytoplasmic and partially in association with α-synuclein. The distribution of R767H and S885N proteins were also mainly in cytoplasm. In contrast, both R1441H and G2019S (included as an aggregation control) LRRK2 mutants formed α-synuclein-negative perinuclear aggregates in a smaller, but still appreciable, proportion of cells, in addition to cytoplasmic distribution. Fluorescent microscopy examination and quantitation of the number of cells with LRRK2-EGFP fluorescent aggregates out of the transfected HEK-293T cell population further revealed that R1441H and G2019S both induced significant more inclusions as compared to wild-type LRRK2. Finally, V5-tagged ARHGEF7 cDNA was cloned and co-expressed with the Myc-tagged wild type or mutant (R767H, S885N, R1441H, G2019S) LRRK2 constructs in HEK-293T cells. Co-immunoprecipitation assay revealed that S885N, R1441H and G2019S reduced interaction between LRRK2 and ARHGEF7.