Loss of mitochondrial Ca response and CaMKII/ERK activation by LRRK2 mutation correlate with impaired depolarization-induced mitophagy.

BACKGROUND

Stress-induced activation of ERK/Drp1 serves as a checkpoint in the segregation of damaged mitochondria for autophagic clearance (mitophagy). Elevated cytosolic calcium (Ca) activates ERK, which is pivotal to mitophagy initiation. This process is altered in Parkinson's disease (PD) with mutations in leucine-rich repeat kinase 2 (LRRK2), potentially contributing to mitochondrial dysfunction. Pathogenic LRRK2 mutation is linked to dysregulated cellular Ca signaling but the mechanism involved remains unclear.

METHODS

Mitochondrial damages lead to membrane depolarization. To investigate how LRRK2 mutation impairs cellular response to mitochondrial damages, mitochondrial depolarization was induced by artificial uncoupler (FCCP) in wild-type (WT) and LRRK2 mutant knockin (KI) mouse embryonic fibroblasts (MEFs). The resultant cytosolic Ca flux was assessed using live-cell Ca imaging. The role of mitochondria in FCCP-induced cytosolic Ca surge was confirmed by co-treatment with the mitochondrial sodium-calcium exchanger (NCLX) inhibitor. Cellular mitochondrial quality and function were evaluated by Seahorse™ real-time cell metabolic analysis, flow cytometry, and confocal imaging. Mitochondrial morphology was visualized using transmission electron microscopy (TEM). Activation (phosphorylation) of stress response pathways were assessed by immunoblotting.

RESULTS

Acute mitochondrial depolarization induced by FCCP resulted in an immediate cytosolic Ca surge in WT MEFs, mediated predominantly via mitochondrial NCLX. However, such cytosolic Ca response was abolished in LRRK2 KI MEFs. This loss of response in KI was associated with impaired activation of Ca/calmodulin-dependent kinase II (CaMKII) and MEK, the two upstream kinases of ERK. Treatment of LRRK2 inhibitor did not rescue this phenotype indicating that it was not caused by mutant LRRK2 kinase hyperactivity. KI MEFs exhibited swollen mitochondria with distorted cristae, depolarized mitochondrial membrane potential, and reduced mitochondrial Ca store and mitochondrial calcium uniporter (MCU) expression. These mutant cells also exhibited lower cellular ATP: ADP ratio albeit higher basal respiration than WT, indicating compensation for mitochondrial dysfunction. These defects may hinder cellular stress response and signals to Drp1-mediated mitophagy, as evident by impaired mitochondrial clearance in the mutant.

CONCLUSIONS

Pathogenic LRRK2 mutation abolished mitochondrial depolarization-induced Ca response and impaired the basal mitochondrial clearance. Inherent defects from LRRK2 mutation have weakened the cellular ability to scavenge damaged mitochondria, which may further aggravate mitochondrial dysfunction and neurodegeneration in PD.