Defective ubiquitinated mitochondria accumulation in aged parkinsonian LRRK2 R1441G knockin mice

Abstract

Objective: (1) To determine potential accumulation of defective ubiquitinated mitochondria in the brains of aged LRRK2R1441G knockin (KI) mice and their age­matched wildtype controls; and (2) to compare the function of mitochondria isolated from these aged knockin mice and their wildtype controls. Background: LRRK2 mutation is the commonest genetic risk of Parkinson's disease (PD). Genetic susceptibility and aging are two major factors contributing to mitochondrial dysfunction in PD. LRRK2 mutations were linked to dysregulation of mitochondrial autophagy. Defective mitochondria are ubiquitinated via Parkin/PINK1 pathway for degradation via mitophagy. We hypothesize that LRRK2 mutation may contribute to accumulation of defective mitochondria in aged brain leading to mitochondria dysfunction. Methods: Twenty­four­month­old LRRK2R1441G knockin mice and their age­matched wildtype controls were cardiac perfused and fixed for examination of mitochondria morphology in dorsal striatum using electron microscopy. Total number of mitochondria and those under fission were quantified and compared with wildtype controls (20 randomized photomicrographs x 3 animals). Degree of ubiquitination in total mitochondria pool was determined by flow cytometry after immuno­labeling. The respiratory chain reactions in isolated mitochondria were assayed using Clark­type oxygen electrodes. Results: Total number of mitochondria in aged LRRK2R1441G knockin mouse striata was higher, whereas the average cross­section area of each mitochondrion was smaller as compared with the wildtype controls. The number of mitochondria under fission was significantly higher in these knockin mice. The amount of mitochondria, which were simultaneously labeled by ubiquitin antibody and MitoTracker, was significantly higher in knockin mice. Oxygen consumption assay showed a trend of decrease in State­IV respiration in isolated mitochondria extracted from knockin mice. Conclusions: Our study showed higher level of ubiquitinated mitochondria accumulation in aged LRRK2R1441G knockin mice, indicating that LRRK2 mutation adversely affected normal mitochondrial function and recycling. These findings also suggested that LRRK2R1441G knockin mouse is a useful experimental model to explore genetic­aging interactions in the pathogenesis of PD.