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.

帕金森病（PD）是第二常见的神经退行性疾病。其病理特征包括错误折叠的α-突触核蛋白（αSyn）聚集体沉积和线粒体功能障碍，这些因素共同导致黑质纹状体多巴胺能神经退行性变。尽管大多数PD病例为散发病例，但LRRK2突变是家族性和散发PD中最常见的遗传风险之一，其发病机制尚不明确。本论文阐明了LRRK2R1441G突变与PD中两种常见事件之间的新型分子联系，即应激诱导的线粒体钙（Ca2+）信号传导和脑部突触核蛋白病。我提出了LRRK2抑制（使用GNE-7915）的可行治疗方案，以减轻PD突变型LRRK2敲入（KI）小鼠模型中αSyn寡聚体的积累。携带LRRK2R1441G突变的胚胎成纤维细胞（MEFs）表现出功能障碍的线粒体积累和ATP缺乏，这与受损的线粒体自噬和Drp1激活（一种与线粒体分裂相关的蛋白）有关。我探讨了LRRK2作为线粒体Ca2+信号传导的介质及其与线粒体应激反应中细胞外信号调节激酶（ERK）/Drp1信号轴的相关性。与野生型（WT）MEFs相比，KI MEFs显示出较慢的基底线粒体清除、较低的ATP:ADP比率、线粒体膜电位（MMP）和线粒体Ca2+水平。这些缺陷在LRRK2敲除（KO）MEFs中并未观察到，这表明LRRK2本身并不直接参与。由羰基氰化物4-（三氟甲氧基）苯基脒（FCCP，一种特定的线粒体解偶联剂）诱导的线粒体去极化导致WT和KO MEFs中细胞质Ca2+激增，而在KI MEFs中未观察到。这种FCCP诱导的Ca2+激增被发现是通过线粒体Ca2+外流通道（NCLX）介导的，而不是通过线粒体通透性转换孔（mPTP）。在KI MEFs中缺乏线粒体Ca2+反应和CaMKII、MEK、ERK和Drp1激活受损的情况并未被LRRK2激酶抑制剂（MLi-2）所挽救。这些发现表明，由LRRK2突变引起的固有线粒体缺陷可能使细胞易受环境应激的影响，而环境应激是PD的另一主要风险因素。LRRK2突变导致突触核蛋白病。在本研究的第二部分，我测试了慢性抑制LRRK2激酶活性是否是一种可行的治疗方法，以减轻老年LRRK2R1441G突变小鼠（KI小鼠）大脑中有毒αSyn寡聚体的积累。因此，我为WT和KI小鼠设计了18周的方案，每周两次皮下注射GNE-7915。在KI小鼠中，这种治疗方法显著降低了纹状体αSyn寡聚体和皮质Ser129-αSyn磷酸化水平至相应的WT水平，但通过形态学和免疫组化评估以及功能试验未观察到对肺、肾和肝脏的副作用。磷酸化的Rab12（一个真正的LRRK2磷酸化靶点）的减少证实了GNE-7915在脑部和周围组织中的疗效。这种治疗方案表明，轻度、慢性抑制突变型LRRK2是PD中一种安全且可行的治疗策略。总之，我的当前研究阐明了LRRK2R1441G突变与1）线粒体功能障碍和受损的线粒体自噬，以及2）与PD相关的突触核蛋白病之间的新型分子联系。这些发现为关于线粒体Ca2+信号传导的作用和慢性LRRK2抑制在LRRK2相关PD治疗中的可行性的进一步研究提供了启示。