Reduced penetrance in Parkin and PINK1 deficiency: Inflammation as a Parkinson’s disease penetrance modifier

复发性遗传性帕金森病（PD）的致病基因包括帕金蛋白和磷酸酶和张力蛋白同源物诱导的推定激酶1（PINK 1）;这些基因中罕见的双等位基因突变导致明确的疾病表现。另一方面，杂合突变-发生在约2%的人群中-可能以显性方式易患PD，并伴有高度降低的突变率。PD的特征在于黑质中多巴胺能神经元的损失，并且有新的证据表明线粒体功能障碍诱导的炎症在Parkin和PINK 1相关PD的发病机制中起作用：我们最近发现，杂合突变的患者具有较高的异质性线粒体DNA（mtDNA）与健康突变携带者相比，变异负荷表明Parkin和PINK 1突变的突变率受到低水平mtDNA异质性的影响。此外，我们探讨了炎症在PINK 1或Parkin相关PD中的作用，并检测到Parkin突变携带者与健康对照相比白细胞介素6水平升高。P2项目的首要目标是定义细胞因子特征，并进一步探索帕金和PINK 1缺乏症患者的炎症途径。我们将通过对受影响与未受影响的Parkin和PINK 1突变携带者进行细胞因子组分析，确定血清和脑脊液样本中炎症因子的水平（目标1）。此外，我们将通过评估多种细胞外细胞因子分析物和基础条件下小胶质细胞的活细胞成像以及iPSC衍生的神经元/小胶质细胞共培养物中对线粒体和炎症应激源的响应来研究Parkin和PINK 1缺陷模型中的小胶质细胞活化（目标2）。为了查明参与Parkin和PINK 1在神经炎症中的保护作用的途径和因素，并探索可能解释未受影响的杂合突变携带者中不存在临床帕金森症的途径，我们将通过单细胞测序对诱导多能干细胞（iPSC）衍生的神经元/小胶质细胞共培养物进行转录组分析。然后，通过用选定途径的特异性激动剂和拮抗剂处理培养物，在药理学拯救实验中进一步验证这些途径（目的3）。我们将利用最先进的技术，即，iPSC和单细胞RNA测序。该项目建立在PI在运动障碍遗传学以及从iPSC生成和使用神经元模型方面的广泛专业知识的基础上。

Genes causative for recessively inherited Parkinson’s disease (PD) include Parkin and Phosphatase and tensin homolog-induced putative kinase 1 (PINK1); rare biallelic mutations in these genes result in definite disease manifestation. On the other hand, heterozygous mutations – occurring in about 2% of the population – may predispose to PD in a dominant manner with highly reduced penetrance. PD is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta, and there is new evidence that mitochondrial dysfunction-induced inflammation plays a role in the pathogenesis of Parkin- and PINK1-linked PD: We recently found that patients with heterozygous mutations have a higher heteroplasmic mitochondrial DNA (mtDNA) variant load compared to healthy mutation carriers suggesting that penetrance of Parkin and PINK1 mutations are influenced by low-level mtDNA heteroplasmy. Moreover, we explored the role of inflammation in PINK1- or Parkin-associated PD and detected elevated interleukin 6 levels in Parkin mutation carriers compared to healthy controls. The overarching goal of Project P2 is to define cytokine signatures and to further explore inflammatory pathways in patients with Parkin and PINK1 deficiency. We will determine the levels of inflammatory factors in serum and cerebrospinal fluid samples by performing a cytokine panel analysis from affected vs. unaffected Parkin and PINK1 mutation carriers (Objective 1). Further, we will study microglia activation in Parkin and PINK1 deficiency models by assessing multiple extracellular cytokine analytes and live-cell imaging of microglia under basal conditions and in response to mitochondrial and inflammatory stressors in an iPSC-derived neuron/microglia co-culture (Objective 2). To pinpoint the pathways and factors involved in the protective role of Parkin and PINK1 in neuroinflammation and to explore pathways that may explain the absence of clinical parkinsonism in unaffected heterozygous mutation carriers, we will perform transcriptome profiling of induced pluripotent stem cell (iPSC)-derived neuron/microglia co-cultures by single-cell sequencing. These pathways will then be further validated in pharmacological rescue experiments by treating the cultures with specific agonists and antagonists of selected pathways (Objective 3). We will utilize state-of-the-art technologies, i.e., iPSCs and single-cell RNA sequencing. The project builds on the extensive expertise of the PIs in genetics of movement disorders and the generation and use of neuronal models from iPSCs.