This grant builds upon the research from a prior grant: Development of a Sensitive Assay to Correlate LRRK2 Kinase Activity and Conformational State
Study Rationale:
The protein LRRK2 has a strong tendency to self-associate in pairs, which plays an important role in how it functions in cells. Mutations in LRRK2 that cause a type of Parkinson's Disease (PD) increase this interaction but it is not clear what regulates this behavior or what the precise effect this has on the different functions of LRRK2. We propose to use a new technique we have developed to "trap" LRRK2 pairs to detect its activity and determine whether other proteins are present that may alter its behavior.
Hypothesis:
We believe that the organization of LRRK2 into pairs plays a key role in determining its behavior in many different cell types, including neurons and cells of the immune system. This organization may indicate a response to a state of activation to many kinds of stress, even in other forms of PD.
Study Design:
We will use our approach to "trap" only LRRK2 that occurs in pairs or binds to another LRRK2 proteins. We will then measure the amount of LRRK2 that is present in this pair versus the total amount of LRRK2, and will compare the effects of different mutations on LRRK2 or different types of cell stress on the amount of LRRK2 present in this form and determine if it has been further modified. We will also compare kinase and GTPase activity in paired-LRRK2 vs single LRRK2. Additionally, we will apply different forms of another protein that is associated with PD, alpha-synuclein, to compare the amount of LRRK2 present in pairs.
Impact on Diagnosis/Treatment of Parkinson's disease:
This study aims to provide answers to important questions on how the function of LRRK2 is regulated and how it responds to various forms of cell stress. This greater understanding will hopefully lead to the development of therapeutic alternatives targeting the toxic properties of LRRK2.
Next Steps for Development:
As inhibitors of LRRK2 function are being tested for potential use in humans, it is important to understand how LRRK2 becomes activated. We need to understand if the target should be certain "shapes" of LRRK2 or when it exists in pairs of proteins to select the best treatment approach.