Combining Mass Spectrometry with Genetic and Pharmacological Approaches to Discover and Validate LRRK2 Substrates

Study Rationale:
The kinase (protein regulator) LRRK2 is thought to play an important role in both familial (inherited) and sporadic (no known cause) Parkinson's disease (PD). Unfortunately, a lack of selective pharmacologic inhibitors has slowed our ability to understand the functional role of LRRK2. The recent development of brain-penetrable inhibitors, combined with state-of-the-art phosphoproteomic (large scale study of proteins) approaches, will enable identification of LRRK2 substrates. These data are a critical first step in the development of biomarkers (markers of disease activity) for use in point-of-care assays for early detection and prognosis, as well as pharmacodynamic studies.

Hypothesis:
Our objective is to identify and validate new LRRK2 protein phosphorylation (modification) substrates.

Study Design:
We will treat pre-clinical models harboring mutations in LRRK2 with small molecule inhibitors. As an important control, we will include in our study design models that express a form of LRRK2 that is resistant to inhibitor treatment. Next, we will use mass spectrometry (test that sorts chemicals) to measure changes in cellular phosphorylation observed across the proteome when these different models are treated with LRRK2 inhibitors. We will focus on those sites which are decreased in one model and active in LRRK2 mutants but are specifically unchanged in the inhibitor-resistant LRRK2 mutant. These proteome-wide assays (tests) will reveal a set of LRRK2 substrates which can be put forth for biochemical and functional validation studies.

Impact on Diagnosis/Treatment of Parkinson's Disease:
There are currently no reliable laboratory tests for Parkinson's. Data from our study may lead to validation and development of markers suitable for use in early detection clinical assays.

Next Steps for Development:
Future work will evaluate the presence and specificity of LRRK2 substrates validated in our study, within a group of individuals with Parkinson's. The goal would be to develop a subset of these LRRK2 substrates for use in early detection assays or as pharmacodynamic markers in drug development and clinical trials.