Objective: To evaluate the difference in substrate (Rab8A & Rab10) phosphorylation by LRRK2 variants G2019S & I1371V on membrane fluidity & pathogenicity.

Background: LRRK2 is a multidomain protein reported to have several pathogenic point mutations in Parkinson’s Disease; showing ethnicity bias. The difference between the LRRK2 variants is evident through the reports showing differences with respect to disease risk, disease severity, age of onset, disease progression, brain pathology, clinical symptoms such as RBD, & drug response. PD-causing LRRK2 mutations represent a “gain of function” through a direct activation of kinase activity or by prolonging kinase activity for GTPase domain mutations. Differences among kinase & GTPase domain mutations has been reported in its downstream substrate (Rab) phosphorylation pattern. These may have differential effects on membrane trafficking of proteins & sterols, in turn affecting membrane fluidity & pathogenicity.

Method: SH-SY5Y cells were transfected with WT, LRRK2 G2019S & LRRK2 I1371V variants. Fluorescence polarization was used to study membrane anisotropy. Membrane function such as neurite outgrowth was measured using fluorescent microscopy & level of Caveolin1 was checked using Immunophenotyping. Cholesterol content was measured using ELISA. Substrate phosphorylation (phosphorylated Rab8A, Rab10) & auto-phosphorylation (phosphorylated LRRK2) were measured using Immunophenotyping.

Results: I1371V showed decreased auto-phosphorylation at S935 & increased auto-phosphorylation at S1292 residue while, G2019S showed similar auto-phosphorylation at S935 & decreased auto-phosphorylation at S1292, as compared to the WT. Lesser S1292 phosphorylation at LRRK2 corresponds to lesser neurite length; observed here too in G2019S than I1371V. In contrast, I1371V showed higher Rab8A & Rab10 (substrate) phosphorylation than G2019S. This corresponded to the increased membrane fluidity & decreased Lipid raft (Caveolin1) expression in I1371V than G2019S.

Conclusion: Distinct differences in auto & substrate phosphorylation between variants was manifested through the difference in membrane fluidity, lipid raft expression & neurite length. Importance of featuring the heterogeneity among LRRK2 mutant variants at the cellular level is essential for target-based drug discovery & define predictable population for drug trials targeting LRRK2 protein.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/differential-rab10-rab8a-phosphorylation-by-lrrk2-variants-g2019s-i1371v-manifests-differences-in-membrane-fluidity-pathogenicity/