Mutations in LRRK2 are thus far the most common known cause of late-onset Parkinson’s disease (PD). The most prevalent LRRK2 mutation G2019S has been shown to stimulate LRRK2 kinase activity in vitro and to provoke neurotoxicity in cultured neurons. The challenge is to understand how LRRK2 mutations lead to impairment of key cellular functions through LRRK2-mediated signaling pathways.
The LRRK2 signaling pathways and substrates for LRRK2 are largely unknown. Recent studies have suggested a few candidates such as moesin, but it is unclear if they represent true LRRk2 substrates. Obviously, a more vigorous search for authentic substrates of LRRK2 is urgently needed. A confounding issue in hunting for kinase substrates is the fact that there are about 500 protein kinases in cells that all utilize ATP for phosphorylation. Through a strong collaboration between multiple investigators with diverse expertise relevant to LRRK2 pathobiology, we will undertake an innovative approach to the identification of bona fide LRRK2 substrates using the chemical genetics approach. In the first aim, we propose to identify direct substrates of LRRK2 in mammalian cells using a genetically engineered LRRK2 and chemically modified ATP. In the second aim, we will validate potential LRRK2 substrates by a combination of in vitro kinase assay, intact cell phosphorylation, and phenotyping in cellular and animal models.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
Our proposed studies will likely have important implications for the development of novel therapeutic agents for PD as the identified LRRK2 substrates may represent new targets for pharmacological intervention.
We anticipate that our research will lead to the identification of novel LRRK2 substrates. Scientific knowledge about LRRK2 substrates and signaling partners will likely open a new field of research within the arena of LRRK2 signaling cascades relevant to disease pathogenesis, and may ultimately provide a missing link between the LRRK2 signaling and neuronal dysfunction, leading to a much-needed breakthrough in PD research.
We have made important progress in developing chemical genetics approach to LRRK2 substrate identification. We have generated key reagents and have developed appropriate assays for LRRK2-specific kinase reactions. We have detected cellular proteins that are likely the candidate LRRK2 substrates in cultured cells. Using an invertebrate model, we have identified two neuroprotective genes that counteract mutant LRRK2-induced dopaminergic dysfunction and neurodegeneration. Finally, we have demonstrated the utility of nematode models in the study of LRRK2 kinase inhibitors that have the potential to rescue mutant LRRK2-linked neurodegeneration.
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