Impaired Stress-induced Mitophagy In Parkinsonian LRRK2(R1441G) Knockin Mutant Mice

Abstract

Objective: 1. Morphological changes and accumulation of ubiquitinated mitochondria in the striatum of aged leucine-rich-repeat kinase 2 (LRRK2) R1441G knockin mutant mice (Liu et al., 2014, 2016); 2. Mechanism of LRRK2 mutation in defective mitochondria turnover in mutant mouse embryonic fibroblasts (MEFs). Background: Autophagy and ubiquitin-proteasome system maintain mitochondria homeostasis and turnover. Autophagic stress, a key pathological feature of Parkinson’s disease (PD), perturbs mitochondrial quality and energy homeostasis in neurons. LRRK2 mutation is the most common genetic risk of PD. Studies suggested a putative role for LRRK2 in macroautophagy. We hypothesize that LRRK2 mutation disrupts mitophagy process causing defective mitochondria accumulation in aged mutant brains. Methods: For mitochondria morphology, aged mouse striatal sections were fixed and examined under transmission electron microscopy. Total number of mitochondria were quantified and compared with wildtype (WT) controls (20 randomized photomicrographs x 3 animals). Freshly isolated striatal mitochondria were immuno-labeled by antiubiquitin antibody and MitoTracker™. The degree of ubiquitination in total mitochondria pool was determined by flow cytometry. MEF cultures isolated from LRRK2 mutant mice or their WT littermates were treated with rotenone (250uM) or FCCP (10uM) for 0, 30 and 120 min. Autophagic response was determined by levels of autophagic markers (LC3B-I/II and Lamp-1) and immunocytochemistry. Results: Smaller but higher total number of mitochondria was seen in aged LRRK2 mutant mouse striatum as compared to their age-matched WT controls. The relative proportion of ubiquitinated mitochondria was higher in the mutant mice. Abnormal perinuclear clustering of enlarged lysosomes were observed in mutant but not WT MEFs under normal culture condition. Activation of autophagy was observed in both WT and mutant MEFs after exposure to rotenone or FCCP. However, mutant MEFs have significantly lower levels of total LC3B (LC3B-I/II) than the WT controls suggesting autophagosome depletion. Conclusions: Accumulation of ubiquitinated mitochondria in aged LRRK2 mutant mice indicated impaired mitochondria turnover, possibly due to depletion of LC3B for autophagosome maturation and abnormal clustering of lysosomes.