The Role of Lysosomal Glucocerebrosidase in Synucleinopathies

Project: Research project

Project Details

Description

Lysosomal dysfunction has been increasingly implicated in the development of neurodege-nerative diseases including Parkinson's disease (PD). Mutations in GBA1 encoding -gluco-cerebrosidase (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 α-syn levels reduced, suggesting that both proteins are functionally linked in the regulation of lysosomal function and α-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 α-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 α-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.
StatusActive
Effective start/end date8/15/177/31/22

Funding

  • National Institute of Neurological Disorders and Stroke (5R01NS076054-09)

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