Pathogenic mutant LRRK2R1441G in mitochondrial dysfunction and synucleinopathy in Parkinson's disease

Abstract

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder characterized by selective loss of nigrostriatal dopaminergic neurons. Key neuropathological hallmarks include deposition of α-synuclein (αSyn) aggregates (as Lewy bodies) and mitochondria dysfunction in the brain. Whilst most PD cases are sporadic, LRRK2 mutation is one of the most common genetic risks of both familial and sporadic PD. This thesis explored links of LRRK2R1441G mutation to two pathogenic features in PD, namely stress-induced response of mitochondrial calcium (Ca2+) signaling and brain synucleinopathy. I explored chronic LRRK2 inhibition (using GNE-7915) as a therapeutic strategy to attenuate toxic αSyn oligomer accumulation in a mutant LRRK2 knock-in (KI) mouse model of PD.

Mouse embryonic fibroblasts (MEFs) carrying LRRK2R1441G mutation exhibit accumulation of dysfunctional mitochondria and ATP deficiency, which are associated with impaired mitophagy and Drp1 activation, a mitochondrial fission related protein. Here, I demonstrated a link between LRRK2 and mitochondrial Ca2+ signaling, and extracellular signal-regulated kinase (ERK)/Drp1 axis in response to mitochondrial stress. KI MEFs showed a slower basal mitochondrial clearance, and lower levels of ATP:ADP, mitochondrial membrane potential (MMP), and mitochondrial Ca2+ compared to 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 mitochondrial-specific uncoupler, triggered a rapid cytosolic Ca2+ surge in WT and KO MEFs, which was not observed in KI MEFs. Such Ca2+ surge was found to be mediated predominantly through mitochondrial Na+/Ca2+ exchanger (NCLX), but not via mitochondrial permeability transition pore (mPTP). The lack of mitochondrial Ca2+ response in KI MEFs was associated with impaired activation of CaMKII/MEK/ERK/Drp1 signal axis. This defective cell response was not alleviated by LRRK2 kinase inhibition (using MLi-2), indicating that it was not due to aberrant hyperactive kinase activity of mutant LRRK2.

Pathogenic LRRK2 mutations contribute to synucleinopathy in PD. In the second part of this study, I tested whether chronic therapeutic LRRK2 inhibition was a feasible approach to attenuate the accumulation of toxic αSyn oligomers in aged LRRK2R1441G KI mouse brains. I devised an 18 consecutive weeks of twice-weekly subcutaneous injections of GNE-7915 in WT and KI mice. In KI mice, this treatment protocol significantly reduced striatal αSyn oligomers and cortical Ser129-αSyn phosphorylation without histological abnormalities in lung, kidney, and liver. Circulatory IL-6 (pro-inflammatory cytokine) and alanine aminotransaminase (ALT) assays revealed no systemic inflammation and drug-induced hepatotoxicity after treatment. Reduced phosphorylated-Rab12, a bona fide LRRK2 phosphorylation target, confirmed GNE-7915 efficacy in brain and lung. This treatment regimen indicates that chronic inhibition of mutant LRRK2 hyperactivity to WT levels is a safe and feasible therapeutic approach in PD.

In summary, my study 1) elucidated a novel molecular link of LRRK2R1441G mutation with dysregulated stress-induced mitochondrial Ca2+ response, and 2) demonstrated a novel LRRK2 inhibitor treatment regimen to ameliorate αSyn oligomer accumulation in LRRK2 KI mice. My findings shed light on better understanding of LRRK2 pathogenesis relevant to mitochondrial dysfunction, and the feasibility of chronic LRRK2 inhibition to alleviate the synucleinopathy in PD.