Objective: To determine the molecular link of LRRK2^R1441G mutation to impaired CaMKII/ERK signaling under mitochondrial depolarization stress using mutant mouse embryonic fibroblast (MEF) model of Parkinson’s disease (PD).

Background: Cellular stress response to preserve mitochondrial quality promotes neuronal survival, but the underlying mechanism is unclear. ERK/DRP1 activation and mitophagy were impaired in mutant LRRK2^R1441G knockin mice which showed accumulation of dysfunctional mitochondria in brains [1,2]. LRRK2 mutation may perturb mitochondrial Ca²⁺ handling and Ca²⁺-dependent stress response pathways that mediate mitophagy.

Method: LRRK2^R1441G knockin (KI) and wildtype control (WT) MEFs were used to assess mitochondrial quality under depolarization stress with FCCP. Mitochondrial membrane potential (MMP) and mitochondrial Ca²⁺ levels were determined from staining intensity of TMRM and Cal-520, respectively, in flow cytometry. Cytosolic Ca²⁺ response and activation of CaMKII, ERK and DRP1 were determined by imaging and immunoblotting. Mitochondrial specificity of Ca²⁺ signal was assessed by inhibiting mitochondrial Ca²⁺ efflux channel (NCLX) with CGP-37157. The effect of mutant LRRK2 hyperkinase activity was tested using kinase inhibitor MLi-2.

Results: LRRK2 KI MEFs showed lower basal MMP and mitochondrial Ca²⁺ level compared to WT, indicating inherent mitochondrial defects caused by gene mutation. FCCP induced cytosolic Ca²⁺ surge in WT mediated via NCLX, but not mitochondrial permeability transition pore. Such Ca²⁺ surge and CaMKII/ERK/DRP1 activation in WT was not observed in KI MEFs with FCCP, which explained impaired mitophagy and accumulation of dysfunctional mitochondria in the mutant mice [1]. LRRK2 inhibitor did not rescue ERK activation defect in KI MEFs, indicating LRRK2 activity was not involved.

Conclusion: LRRK2^R1441G mutation caused inherent mitochondrial defects, increasing susceptibility to accumulate dysfunctional mitochondria and energy crisis in PD brains. This study shows a novel link between LRRK2 mutation and dysregulated mitochondrial Ca²⁺ signaling, which may impair downstream CaMKII/ERK/DRP1 activation to mediate mitochondrial fission and mitophagy for mitochondrial quality control. Our findings shed light on novel neuroprotective approach to improve mitochondrial quality and energy supply in LRRK2-PD.

References: [1] Liu H, Ho PW, Leung CT, Pang SY, Chang EES, Choi ZY, Kung MH, Ramsden DB, Ho SL. Aberrant mitochondrial morphology and function associated with impaired mitophagy and DNM1L-MAPK/ERK signaling are found in aged mutant Parkinsonian LRRK2R1441G mice. Autophagy. 2021 Oct;17(10):3196-3220. doi: 10.1080/15548627.2020.1850008. Epub 2020 Dec 10. PMID: 33300446; PMCID: PMC8526027.

[2] Ho PW, Leung CT, Liu H, Pang SY, Lam CS, Xian J, Li L, Kung MH, Ramsden DB, Ho SL. Age-dependent accumulation of oligomeric SNCA/α-synuclein from impaired degradation in mutant LRRK2 knockin mouse model of Parkinson disease: role for therapeutic activation of chaperone-mediated autophagy (CMA). Autophagy. 2020 Feb;16(2):347-370. doi: 10.1080/15548627.2019.1603545. Epub 2019 Apr 14. PMID: 30983487; PMCID: PMC6984454.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/impaired-camkii-erk-activation-in-mouse-mutant-lrrk2-r1441g-fibroblasts-was-associated-with-reduced-mitochondrial-calcium-store-and-efflux-in-response-to-depolarization-stress/