Objective/Rationale:
A major function of LRRK2, a protein kinase, is the modification of other proteins through a process known as phosphorylation. It appears that the kinase activity of LRRK2 is linked to its ability to self-associate. We have developed an assay to separate distinct pools of LRRK2 based on their size, whether it is present alone (as a monomer) or in a large complex with multiple LRRK2 molecules, and then directly measure its activity.
Project Description:
Wild type or mutant LRRK2 expressed in cultured cells or in pre-clinical models will be separated into individual “fractions” based on their size using a technique known as size-exclusion chromatography. We will then assess multiple aspects of LRRK2 kinase activity simultaneously; measuring not only its ability to phosphorylate other natural or artificial substrates, but also its ability to phosphorylate itself. This assay will then be adapted to measure LRRK2 activity in tissue samples from PD patients. It is important to assess these different activities of LRRK2 in parallel, and how they correlate with its ability to self-associate, since changes in the activity of distinct groups of LRRK2 molecules may have unique implications on the function and survival of neurons.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
Since neuronal death caused by mutant LRRK2 is linked to its kinase activity, the development of selective inhibitors of LRRK2 is an extremely active area of research. However, since it is not known if certain pools of LRRK2 possess neurotoxic properties while other remain innocuous, the broad inhibition of all LRRK2 activity may have detrimental effects towards other vital functions of the protein. This project will help clarify the neurotoxic properties of LRRK2 and aid in screening agents that can selectively inhibit the “toxic species” of LRRK2.
Anticipated Outcome:
With the proposed assay, we will be able to directly correlate kinase activity with LRRK2 self-association, to determine whether increases in this form of interaction (e.g. such as that seen in some mutant forms of the protein) lead to increased kinase activity. Furthermore, we hope that this platform can aid in the development of LRRK2 inhibitors that selectively target the activity of LRRK2 responsible for neuronal death in PD.