Mutations in LRRK2 cause familial Parkinson disease (PD) and, in some populations, may account for up to 40% of all cases3. The LRRK2 gene locus also contains a risk factor for `idiopathic' PD (iPD); however, the role of LRRK2 in typical iPD is not clear. Furthermore, the relationship of LRRK2 to other genes and proteins associated with PD, such as ?-synuclein, is also relatively unexplored. While the mechanism(s) by which mutant LRRK2 causes neurodegeneration are not entirely certain, it is generally believed that disease-causing mutations may be associated with increased kinase activity, at least in situ in intact cells. However, a critical barrier to understanding the role of endogenous, wildtype LRRK2 in iPD is the absence of a practical, high-resolution assay for its activation state. We have developed and validated a pair of novel proximity ligation assays with excellent anatomical resolution that can rapidly provide information regarding activation state, cellular localization and physiological regulators of LRRK2. The assay is based on (i) S1292 phosphorylation and (ii) dissociation of 14-3-3 from LRRK2. Using this and other assays, we have preliminary evidence that (i) LRRK2 is activated in nigrostriatal neurons in iPD; (ii) sublethal concentrations of rotenone activate LRRK2; (iii) overexpression of ?-synuclein in vivo activates LRRK2 in nigrostriatal neurons; (iv) LRRK2 activation is mediated by oxidative mechanisms. We now propose to examine the role of LRRK2 in iPD and its potential physiological interactions with ?-synuclein and mitochondria. We will investigate the following questions: (1) What is the activation state of LRRK2 in human iPD brain tissue? (2) To what extent can endogenous LRRK2 activation be modeled in the rotenone rat? (3) Does!?-synuclein activate endogenous WT LRRK2? (4) Does oxidative stress activate LRRK2?

Public Health Relevance

Mutations in a protein called LRRK2 are the most common cause of familial Parkinson's disease, and there is a possibility that the normal (non-mutated) form of the protein may also be important in contributing to the neurodegeneration in idiopathic (non-familial) Parkinson's disease (iPD). A critical barrier to studying LRRK2 in iPD has been lack of a sensitive, easy biochemical assay. We have now developed and validated such an assay and our preliminary results suggest that normal LRRK2 may play a central role in iPD. If this can be confirmed with the studies proposed here, it will have enormous implications on how we can slow or stop degeneration in iPD.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
1R01NS100744-01A1
Application #
9440529
Study Section
Neural Oxidative Metabolism and Death Study Section (NOMD)
Program Officer
Sieber, Beth-Anne
Project Start
2017-09-01
Project End
2022-05-31
Budget Start
2017-09-01
Budget End
2018-05-31
Support Year
1
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
Neurology
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213