![]() ![]() A CMA-specific activator, AR7, induced neuronal LAMP2A transcription and lysosomal activity in MEFs. LAMP2A-knockdown reduced total lysosomal activity and clearance of 'KFERQ'-substrate in WT but not in mutant MEFs, indicating impaired CMA in the latter. LRRK2 mutant MEFs exhibited lower lysosomal degradation than WT indicating lysosomal dysfunction. However, such difference was not observed after the 'KFERQ'-motif was mutated. Using our new reporter protein clearance assay, mutant mouse embryonic fibroblasts (MEFs) expressing either SNCA or CMA recognition 'KFERQ'-like motif conjugated with photoactivated-PAmCherry showed slower cellular clearance compared to WT by 28% and 34%, respectively. Lysosomal clustering and accumulation of CMA-specific LAMP2A and HSPA8/HSC70 proteins were observed in aged mutant striatum along with increased GAPDH (CMA substrate) by immunohistochemistry of dorsal striatum and flow cytometry of ventral midbrain cells. We showed greater age-dependent accumulation of oligomeric SNCA in striatum and cortex of aged LRRK2 R1441G knockin (KI) mice, compared to age-matched wildtype (WT) by 53% and 31%, respectively. Mutant LRRK2 perturbs chaperone-mediated-autophagy (CMA) to degrade SNCA. LRRK2 (leucine-rich repeat kinase-2) mutations predispose to familial and sporadic PD. Impaired catabolism of SNCA potentiates formation of its toxic oligomers. Parkinson disease (PD) is an age-related neurodegenerative disorder associated with misfolded SNCA/α-synuclein accumulation in brain. ![]()
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