Molecular mechanisms defining penetrance of LRRK2-associated Parkinson’s disease

帕金森病(PD)基因富含亮氨酸的重复蛋白激酶2(LRRK2)的突变主要是遗传的，外显性降低。到目前为止，对于触发LRRK2-PD发病的分子机制知之甚少。在ProtectMove I中，为了更好地监测进展，并最终识别与外显性相关的细胞通路，我们研究了LRRK2-PD表现的标志物。在G2019S突变显性携带者(LRRK2+/PD+；n=10)和非显性携带者(LRRK2+/PD-；n=21)成纤维细胞中，我们首先证实了PD发病与LRRK2磷酸化之间的联系。其次，受一篇将LRRK2激酶活性与线粒体DNA(MtDNA)损伤联系起来的报道的启发，我们评估了LRRK2+/Pd-和LRRK2+/Pd+成纤维细胞的mtDNA完整性和线粒体功能，并观察到后者mtDNA缺失的积累和复合体I活性的降低。根据一项研究显示，在LRRK2+/Pd+个体中尿酸减少，我们探索了尿酸诱导的Nrf2-ARE抗氧化信号。有趣的是，作为mtDNA转录和包装因子的Nrf2-ARE靶标TFAM在LRRK2+/Pd+神经元中减少，增加了mtDNA对活性氧的暴露。我们假设，环境、线粒体功能和LRRK2激酶活性之间的恶性循环，其中线粒体DNA作为中央界面，定义了LRRK2-PD外显率。为了评估导致外显的环境因素并确定相关毒素(目标1)，我们将从LIPAD队列中收集环境暴露、饮食和药物信息(LRRK2+/PD+，n=1,500；LRRK2+/PD-，n=500；对照，n=500)。此外，在选定的人中，我们将进行无针对性的毒理学检查。为了研究外显性相关(epi-)遗传mtDNA改变(目标2)，我们将在LIPAD的血液样本中进行mtDNA测序和定量分析。为了确定参与抗氧化信号和线粒体功能的核基因的罕见变异对外显率的贡献(目标3)，将对LIPAD患者进行全基因组测序和多基因风险评分分析。最后，为了测试毒素暴露、线粒体DNA解体、线粒体功能障碍、LRRK2激酶活性和抗氧化信号之间的因果联系(目标4)，我们将在暴露于氧化应激源、激酶抑制剂和抗氧化剂前后，对LRRK2+/PD+和LRRK2+/PD-神经元进行RNA-SEQ和功能研究。我们的研究结果将有助于指导旨在延缓LRRK2-PD发病的个体化药物治疗方法。项目P1由精通流行病学、临床、遗传学和细胞学研究的首席调查人员领导。此外，它们还受益于允许将P1扩展到表观遗传学和毒理学分析的(国家间)合作。P1嵌入了ProtectMove II内部紧密的交互网络中。这包括项目P2-P4、P8-P10、INF、Z2和所有核心。

Mutations in the Parkinson’s disease (PD) gene Leucine-rich repeat kinase 2 (LRRK2) are dominantly inherited with reduced penetrance. To date, little is known about the molecular mechanisms that trigger the onset of LRRK2-PD. To allow for better monitoring of progression and, ultimately, for the identification of penetrance-associated cellular pathways, in ProtectMove I, we investigated markers of LRRK2-PD manifestation. In fibroblasts from manifesting (LRRK2+/PD+; n=10) and non-manifesting carriers (LRRK2+/PD-; n=21) of the G2019S mutation, first, we confirmed a link between PD onset and LRRK2 phosphorylation. Second, inspired by a report connecting LRRK2 kinase activity with mitochondrial DNA (mtDNA) lesions, we assessed mtDNA integrity and mitochondrial function in LRRK2+/PD- and LRRK2+/PD+ fibroblasts and observed an accumulation of mtDNA deletions and decreased complex I activity in the latter. In light of a study showing reduced urate in LRRK2+/PD+ individuals, we explored urate-induced Nrf2-ARE antioxidant signaling. Interestingly, the Nrf2-ARE target TFAM, which acts as mtDNA transcription and packaging factor was reduced in LRRK2+/PD+ neurons, increasing mtDNA exposure to reactive oxygen species. We hypothesize that a vicious cycle between environment, mitochondrial function and LRRK2 kinase activity, where the mtDNA acts as central interface, defines LRRK2-PD penetrance. To assess environmental factors contributing to penetrance and to identify relevant toxins (Objective 1), we will collect environmental exposure, diet and medication information from the LIPAD cohort (LRRK2+/PD+, n=1,500; LRRK2+/PD-, n=500; controls, n=500). Moreover, in selected persons, we will perform untargeted toxicology. To investigate penetrance-associated (epi-)genetic mtDNA alterations (Objective 2), we will perform sequencing and quantitative mtDNA analyses in blood samples from LIPAD. To determine the contribution of rare variants in nuclear genes involved in antioxidant signaling and mitochondrial function to penetrance (Objective 3), whole genome sequencing and polygenic risk score analyses will be applied to individuals from LIPAD. Finally, to test the causal link between toxin exposure, mtDNA disintegration, mitochondrial dysfunction, LRRK2 kinase activity, and antioxidant signaling (Objective 4), we will conduct RNA- Seq and functional studies in LRRK2+/PD+ and LRRK2+/PD- neurons before and after exposure to oxidative stressors, kinase inhibitors and antioxidants. The results from our study will help to guide personalized medicine approaches, which aim at delaying the onset of LRRK2-PD. Project P1 is led by Principal Investigators well-versed in epidemiological, clinical, genetic and cellular studies. Furthermore, they benefit from (inter-)national collaborations allowing to expand P1 to epigenetic and toxicology analyses. P1 is embedded in a close network of interactions within ProtectMove II. This includes projects P2-P4, P8-P10, INF, Z2 and all Cores.