Activation of chaperone-mediated autophagy prevents accumulation of oligomeric a-synuclein in LRRK2 R1441G knockin mice of Parkinson's disease

Abstract

OBJECTIVE: Explore effects of LRRK2 mutation and aging, on a-synuclein accumulation and lysosomal chaperone-mediated autophagy (CMA) and disease-modification strategy for Parkinson’s disease (PD). BACKGROUND: Leucine-rich-repeat kinase-2 (LRRK2) mutations are the commonest genetic cause of PD. a-synuclein accumulation with aging is a key pathogenic process. Its accumulation enhances formation of its toxic oligomers. Unwanted protein clearance by CMA is essential for neurons. CMA dysfunction was linked to PD. DESIGN/METHODS: a-synuclein levels in striatal lysates of LRRK2(R1441G) knockin (KI) mice and wild-type (WT) from 3 to 18 months of age were determined by Western and dot-blotting. Primary cortical neurons were incubated with recombinant a-synuclein to determine its clearance rate. Levels of lysosomal lamp2a and hsc70 levels relative to lamp1, and multimeric lamp2a complexes in cerebral cortices of 3 and 18-month-old mice were assayed to determine CMA activity. Primary cortical neurons were treated with AR7 [specific retinoic acid receptor alpha-antagonist and CMA activator] at 10 and 20μM, for 72-hr or 12 days in culture. a-synuclein levels were determined in cell lysates and conditioned medium. RESULTS: Greater age-dependent increase of striatal oligomeric a-synuclein in KI mice compared with WT, was apparent by 15 months, reaching significance at 18 months of age. KI neurons had higher maximal monomeric a-synuclein levels with slower decline indicating impaired a-synuclein clearance. Unlike young mice, aged KI mice had greater reduction of lamp2a and hsc70 levels, with increased multimeric lamp2a complexes compared to WT mice indicating greater age-dependent decrease in CMA activity. AR7 significantly decreased monomeric a-synuclein levels, and prevented oligomeric a-synuclein accumulation in KI neuronal lysates and conditioned medium. AR7 did not show cytotoxic effects by LDH release assay. CONCLUSIONS: Aged related accumulation of striatal oligomeric a-synuclein was due to impaired clearance of a-synuclein in KI mice, associated with impaired CMA activity. CMA activation prevented oligomeric a-synuclein accumulation.