Objective: Using a unique fluorescence-based flow cytometry assay on LRRK2(R1441G) mutant mouse embryonic fibroblasts (MEFs) to elucidate whether pathogenic LRRK2 mutation affects cellular lysosomal degradation of a-synuclein.

Background: Accumulation of misfolded a-synuclein aggregates in the brain is integral to the pathogenesis of Parkinson’s disease (PD).Endogenous a-synuclein protein is mainly catabolized via both the proteasome and autophagic pathways. However, when a-synuclein aggregates to form higher molecular weight species, lysosomes play a more dominant role in their degradation. Leucine-rich repeat kinase 2 (LRRK2) mutations form a common genetic risk of familial and sporadic PD. How LRRK2 mutations cause synucleinopathy is unclear. We hypothesize that LRRK2 mutation impairs lysosomal degradation of a-synuclein leading to formation of toxic a-synuclein aggregates in PD brain. We generated knockin mice carrying the homozygous LRRK2(R1441G) mutation as an in vivo model of PD [1,2].

Method: MEFs were derived from LRRK2(R1441G) knockin mice and their wildtype (WT) littermates. Cells were transduced by lentivirus to express full-length a-synuclein conjugated with photoactivated-PAmCherry.  Total cellular clearance of a-synuclein conjugates was measured at different time points by flow cytometry after photoactivation by UV-A (405 nm). Contribution of lysosomes in total cellular degradation was determined by decrease in percentage substrate clearance after treatment of lysosomal inhibitors. Total lysosomal activity in WT and mutant MEFs was also determined based on degradation of a self-quenched lysosomal-specific substrate.

Results: Abnormal perinuclear clustering of lysosomes was observed in LRRK2(R1441G) mutant MEF(s) as compared to WT controls suggesting perturbed lysosomal degradation. Total lysosomal activity and specific clearance of a-synuclein in mutant cells were significantly lower than WT. Lower contribution of lysosomal degradation to overall protein degradation was found in mutant than WT MEFs after treatment with bafilomycin A1 and chloroquine.

Conclusion: Comparing to WT, cells carrying LRRK2(R1441G) mutation degrade a-synuclein more slowly partly because of impaired lysosomal degradation.  Agents to induce lysosome activity to degrade a-synuclein in neurons may be a viable strategy to address synucleinopathies in PD.

References: [1] Liu HF, Ho PWL, Leung GC, Lam CS, Pang SY, Li LF, Kung MHW, Ramsden DB, Ho SL*. Combined LRRK2 mutation, aging and chronic low dose oral rotenone as a model of Parkinson’s disease. Sci Rep. 2017; 7:40887. doi:10.1038/srep40887 [2] Liu HF, Lu S, Ho PWL, Tse ZHM, Pang S, Kung MHW, Ho JWM, Ramsden DB, Zhou ZJ, Ho SL*. LRRK2 R1441G mice are more liable to dopamine depletion and locomotor inactivity. Ann Clin Transl Neurol. 2014; 1(3):199-208.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/impaired-lysosomal-catabolism-of-alpha-synuclein-in-parkinsonian-lrrk2r1441g-mutant-mouse-embryonic-fibroblasts/