Lysosomal dysfunction has been increasingly implicated in the development of neurodegenerative diseases including Parkinson's disease (PD). Mutations in GBA1 encoding ?-glucocerebrosidase (GCase) cause Gaucher disease (GD), the most prevalent lysosomal storage disorder and represent an important genetic risk factor for synucleinopathies including PD and dementia with Lewy Bodies (DLB). A significant reduction in GCase activity has been also reported in brains of sporadic PD patients, suggesting that reduced GCase activity may represent a common feature of PD pathogenesis. Genetic linkage with PD and DLB has been also demonstrated for lysosomal membrane protein LIMP-2, the lysosomal trafficking receptor for GCase and GD modifier. Mutations in SCARB2 encoding LIMP-2 itself are disease-causing for a rare form of progressive myoclonic epilepsy associated with renal failure (AMRF). Our previous data showed that loss of function of LIMP-2 results in mistrafficking and lysosomal depletion of GCase and is also associated with a PD-like pathology in mice. We have also demonstrated that by elevating the levels of LIMP-2, lysosomal GCase activity was enhanced and ?-syn levels reduced, suggesting that both proteins are functionally linked in the regulation of lysosomal function and ?-syn metabolism. Therefore, we hypothesize that it will be critically important to consider LIMP-2-GCase interaction in the development of activators of GCase as potential therapeutics for PD and related synucleinopathies. To test this hypothesis, we propose to further examine the contribution of LIMP-2 in the pathogenesis of synucleinopathies by providing molecular insights into the formation and trafficking of the LIMP-2/GCase complex, and its role in normal and diseased human neurons.
Aim 1 will examine the assembly and stoichiometry of the wild type LIMP-2/GCase complex in live cells by using photoactivatable amino acids, pulse chase and immunoprecipitations experiments. These studies will be extended to GCase and LIMP-2 patient-linked mutations to determine their impact on the formation of the LIMP-2/GCase complex.
Aim 2 will examine time-dependent phenotypes in LIMP-2- and GCase-deficient human midbrain neurons. We will generate iPSC-derived midbrain neurons from LIMP-2 patient fibroblasts to examine subcellular localization of GCase and GCase/LIMP-2 complex, lysosomal proteolysis and morphology, accumulation of lipid substrates and ?-synuclein in a time-dependent manner.
In Aim 3 we will perform neuropathological characterization of a mouse model expressing LIMP-2 that is deficient in binding to GCase. To directly examine if the neurological phenotypes we previously described in LIMP-2 knockout mice result from depletion of lysosomal GCase, we generated a mouse model expressing LIMP-2-Y163D mutant that cannot bind GCase. These mice will be analyzed for ?-syn and lipid accumulation, lysosomal dysfunction, inflammation and neurotoxicity. These experiments will also establish whether the neuropathology mediated by LIMP-2 deficiency may be at least in part independent of GCase.

Public Health Relevance

Neurodegenerative disorders affect the lives of millions of people worldwide. Recently, enormous strides have been made in identifying disease mechanisms and with these advances, the chances of finding cures are increasing rapidly. We propose a research project that is carried out using new and creative collaborations to accelerate the pre-clinical validation lysosomal proteins as therapeutic targets for PD. If successful, this approach will have high impact for PD as well as other neurodegenerative disorders.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
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Cellular and Molecular Biology of Neurodegeneration Study Section (CMND)
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Sutherland, Margaret L
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Northwestern University at Chicago
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