Objective/Rationale:
One of the most exciting recent advances in PD research is that mutations within the gene coding for a protein of unknown function, LRRK2, predispose people to PD. Since a number of these mutations increase the protein kinase activity of LRRK2 the goal of this project is to investigate three recently identified potential targets of LRRK2’s kinase activity. Such information will enable the development LRRK2 inhibitors, efforts currently hampered by the lack of validated substrates and cellular assays and significantly increase our understanding of critical pathways underlying the disorder.
Project Description:
The project will initially seek to validate that the three putative substrates identified are phosphorylated by LRRK2 in a cellular environment. To perform this objective the three potential substrates will be individually transfected into two different recombinant LRRK2 cell lines that we have developed. Using a combination of mass spectroscopy, commercial and in-house developed phospho-specific antibodies specific consensus phosphorylation sites will be investigated. To extend this characterization into neurons, cultured primary neurons and brains from LRRK2 BAC transgenic mice will be used. under normal and ‘stressed’ conditions. The most compelling substrate from these studies will be studied in vivo using LRRK2 BAC transgenic mice and viral overexpression of LRRK2 to elucidate the pathological implications of its phosphorylation by LRRK2.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
The overall relevance of this application is that it seeks to develop disease-modifying treatments for PD patients. The recent advances linking human mutations in genes such as LRRK2 to Parkinson’s disease, has opened the way to investigate why certain genes can lead to this pathology, and will also allow the development of therapeutics to slow or halt the underlying progression of this devastating disease. Understanding the phosphorylation and modulation of LRRK2 targets within neurons and animals should validate LRRK2 kinase activity, and may also validate one or more intracellular pathways that can be targeted to yield disease modifying therapies.
Anticipated Outcome:
At the conclusion of this study we will have determined the cellular, neuronal, and in vivo relevance of three target proteins identified as putative substrates of LRRK2 kinase activity. By examining putative LRRK2 substrates in multiple cell types, as well as in vivo using PD model transgenic animals, the relative importance of these substrates in the context of normal and stressed cells should emerge. These data will provide critical insights into the mechanistic basis of PD progression, and therefore the identity of targets for pharmacological intervention in the treatment of PD.