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Supporting data for "Pathogenic mutant LRRK2R1441G in mitochondrial dysfunction and synucleinopathy in Parkinson’s disease"
Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder. Disease hallmarks include deposition of misfolded α-synuclein (αSyn) aggregates and mitochondrial dysfunction that induce nigrostriatal dopaminergic neurodegeneration. Whilst most PD cases are sporadic, LRRK2 mutation is one of the most common genetic risks of both familial and sporadic PD with unclear pathogenesis. This thesis elucidates novel molecular links of LRRK2R1441G mutation to two common events in PD, namely stress-induced response of mitochondrial calcium (Ca2+) signaling and brain synucleinopathies. I proposed a feasible therapeutic strategy of LRRK2 inhibition (using GNE-7915) to attenuate αSyn oligomer accumulation in a mutant LRRK2 knock-in (KI) mouse model of PD.
Mouse embryonic fibroblasts (MEFs) carrying LRRK2R1441G mutation exhibit the accumulation of dysfunctional mitochondria and ATP deficiency, which are associated with impaired mitophagy and Drp1 activation, a mitochondrial fission related protein. I explored LRRK2 as a mediator of mitochondrial Ca2+ signaling and its correlation with the extracellular signal-regulated kinase (ERK)/Drp1 signaling axis in response to mitochondrial stress. KI MEFs revealed a slower basal mitochondrial clearance, and lower levels of ATP:ADP ratio, mitochondrial membrane potential (MMP) and mitochondrial Ca2+ compared to those in wildtype (WT) MEFs. These defects were not seen in LRRK2 knockout (KO) MEFs, indicating that LRRK2 per se is not directly involved. Mitochondrial depolarization induced by carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), a specific mitochondrial uncoupler, resulted in a cytosolic Ca2+ surge in WT and KO MEFs, which was not seen in KI MEFs. This FCCP-induced Ca2+ surge was found to mediate via the mitochondrial Ca2+ efflux channel (NCLX), but not the mitochondrial permeability transition pore (mPTP). The lack of mitochondrial Ca2+ response in KI MEFs and impaired activation of CaMKII, MEK, ERK and Drp1 were not rescued by LRRK2 kinase inhibitor (MLi-2). These findings suggest that the inherent mitochondrial defects caused by LRRK2 mutation may render cells susceptible to environmental stress, another major risk factor of PD.
LRRK2 mutations contribute to synucleinopathy. In the second part of this study, I tested whether chronic inhibition of LRRK2 kinase hyperactivity was a viable therapeutic approach to attenuate the accumulation of toxic αSyn oligomers in the brain of aged LRRK2R1441G mutant mice (KI mice). Therefore, I devised an 18-week protocol of twice-weekly subcutaneous injections of GNE-7915 in WT and KI mice. In KI mice, this significantly reduced striatal αSyn oligomers and cortical Ser129-αSyn phosphorylation to the corresponding WT levels but showed no adverse effects in lung, kidney and liver by morphological and immunohistochemical assessments and functional assays. Reduced phosphorylated-Rab12, a bona fide LRRK2 phosphorylation target, confirmed GNE-7915 efficacy in both brain and peripheral tissues. This treatment regimen indicates that mild, chronic inhibition of mutant LRRK2 is a safe and feasible therapeutic strategy in PD.
In summary, my current study elucidated a novel molecular link of LRRK2R1441G mutation with 1) mitochondrial dysfunction and impaired mitophagy, and 2) synucleinopathies as implicated in PD. These findings shed light on more investigations regarding the role of mitochondrial Ca2+ signaling and the feasibility of chronic LRRK2 inhibition in LRRK2-associated PD therapies.