Objective/Rationale:
Mutations in LRRK2 are the most common genetic cause of Parkinson’s disease. The LRRK2 gene encodes a large multi-domain protein with kinase activity. LRRK2 protein is normally located both in the cytosol of cells and bound to cellular membranes. The membrane-bound form of LRRK2 has greater kinase activity, which is implicated in LRRK2-mediated neurotoxicity. We propose to investigate potential cellular mechanisms that regulate LRRK2 membrane association because this may directly or indirectly regulate LRRK2 kinase activity and LRRK2-mediated neurotoxicity.
Project Description:
We will attempt to address major unanswered questions including what causes LRRK2 protein to associate with cellular membranes and can preventing LRRK2 membrane association mitigate the effects of LRRK2 mutations that cause Parkinson’s disease. We hypothesize that LRRK2 cycles between cytosolic and membrane-bound states according to various post-translational modifications. We will use an array of biochemical and cell biological methods to identify the cellular mechanisms that regulate LRRK2 membrane association. We will also determine if these mechanisms are affected by disease-linked LRRK2 mutations. We will attempt to generate variants of LRRK2 with reduced ability to associate with cellular membranes. These LRRK2 variants may be useful research tools to discover novel therapies for Parkinson’s disease based on inhibiting LRRK2-mediated neurotoxicity.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
Because the membrane-bound form of LRRK2 has greater kinase activity compared to the non-membrane-bound form of LRRK2 and because the kinase activity of LRRK2 is required for LRRK2-mediated neurotoxicity, disruption of LRRK2 membrane association may serve as a potential therapeutic target to treat or prevent Parkinson’s disease or parkinsonism caused by LRRK2 mutations.
Anticipated Outcome:
We anticipate developing research tools and advancing basic scientific understanding of LRRK2 biology that will enable more detailed studies of how mutations in LRRK2 cause Parkinson’s disease. Completion of these studies may lead to the development of therapeutic strategies to treat or prevent Parkinson’s disease based on manipulating the ability of LRRK2 to associate with cellular membranes.
Final Outcome
We have identified methods to increase or decrease the ratio of cytosolic LRRK2 compared to membrane-localized LRRK2 as well as methods to increase or decrease the ratio of LRRK2 dimer compared to LRRK2 monomer. These findings are functionally significant and likely relevant to Parkinson’s disease because we found that the same treatments that decrease LRRK2 membrane localization and LRRK2 dimerization also decrease LRRK2-mediated cell toxicity and mitigate the effects of LRRK2 mutations causally linked to Parkinson’s disease. We have determined that the N-terminal half of LRRK2 localizes to the membrane fraction of cell lysates more than the C-terminal half of LRRK2. Our ongoing analyses are aimed at further defining the sites within the LRRK2 protein that are most required for LRRK2 membrane localization. These studies are important for guiding the development of novel therapies based on minimizing LRRK2-mediated toxicity and for understanding the biophysical properties of LRRK2, which associates with membranes despite the absence of any transmembrane sequences or known membrane association motifs.