Who is to blame? Investigating Genes within the 22q11.2 Deletion Region as potential Risk Factors for Parkinson's Disease using Human iPS Cells and CRISPR Perturbation

帕金森氏病(PD)是全球第二常见的神经退行性疾病。尽管如此，帕金森病的发病机制尚不完全清楚，治疗帕金森病仅限于对症治疗，这可能会导致不良的长期副作用。对帕金森病的认识和治疗的主要局限是其病因与遗传和环境危险因素的异质性，以及目前的帕金森病模型不能完全概括该疾病的所有临床和神经病理学相关方面的挑战。新的体外人源性干细胞模型的发展为帕金森病的发病机制提供了新的线索，并证实了神经发育与帕金森病神经变性的联系。发现和研究导致神经发育缺陷和帕金森病发病风险增加的新的遗传危险因素可能为更好地理解帕金森病的发病机制提供一个切入点。最近，最常见的缺失综合征之一(约1：2,000-4,000活产)22q11.2DS被发现是发生早发性帕金森病的重要遗传危险因素。由于22q11.2DS的帕金森病患者可出现与散发性帕金森病患者相似的症状，这表明22q11缺失区受影响基因的失调引发了常见的疾病机制和途径。我们假设，识别22q.11.2缺失区域内导致22q.11.2DS发育缺陷的基因将有助于发现与PD病理相关的新的靶基因，并可能揭示对制定未来治疗策略至关重要的机制。本研究的总体目标是增加我们对22q11.2缺失区域内的基因及其在22q11.2DS和22q11.2DS PD中的潜在作用的理解。[目的1]我将用22q11.2DS患者的人干细胞来源的神经元建立与22q11.2DS相关的体外神经元网络，并利用一种新型的高含量微电极阵列芯片，通过高含量的成像、分子生物学读数和电生理记录，探索与疾病相关的表型。[目的2]我将通过基因克隆进一步产生表达簇状规则间隔短回文重复激活(CRISPRa)或干扰(CRISPRi)机制的IPSCs，以调节22q11DS内源基因的表达。[目标3]这将使我能够通过在已开发的神经元网络中使用CRISPR修饰符筛选来识别22q11.2缺失内的特定基因，这些基因涉及22q11.2DS PD的表型检测(在目标1中)。

Parkinson’s disease (PD) is the second most common neurodegenerative disorder worldwide. Still, the pathogenesis of PD is incompletely understood, and treating PD is limited to symptomatic therapies, which can lead to undesirable long-term side effects. Major limitations in understanding and treating PD is its heterogeneous etiology with genetic as well as environmental risk factors and the challenge that current models of PD do not fully recapitulate all clinical and neuropathological relevant aspects of the disease. The development of new human-derived stem cell models in vitro shed new light on PD pathogenesis and confirmed a neurodevelopmental link to neurodegeneration of PD. Discovery and investigation of new genetic risk factors contributing to neurodevelopmental defects and increased risk for the development of PD might provide an entry point towards better understanding PD pathogenesis.Only recently one of the most common deletion syndromes (~1:2,000-4,000 live births) the 22q11.2DS was found to constitute a significant genetic risk factor for developing early-onset PD. Since PD patients with 22q11.2DS can develop similar symptoms as compared to patients with sporadic PD, this indicates common disease mechanisms and pathways triggered by the dysregulation of affected genes in the 22q11 deletion region. We hypothesize that identifying genes within the 22q.11.2 deletion that are responsible for developmental defects described for 22q.11.2DS will enable the discovery of new target genes involved in PD pathology and might bear the potential to uncover mechanisms that are essential in developing future treatment strategies.The overall goal of this fellowship is to increase our understanding of genes within the 22q11.2 deletion region and their potential role in 22q11.2DS and 22q11.2DS PD. [Objective 1] I will build an in vitro neuronal network relevant for 22q11.2DS with neurons derived from human stem cells from 22q11.2DS patients and probe for well-described disease-associated phenotypes by using high-content imaging, molecular biological readouts, and electrophysiological recordings with a novel high-content microelectrode array chip. [Objective 2] I will further generate iPSCs expressing Clustered Regularly Interspaced Short Palindromic Repeats activation (CRISPRa) or interference (CRISPRi) machinery by genetic cloning to regulate gene expression from endogenous loci within the 22q11DS. [Objective 3] This will allow me to identify specific genes inside the 22q11.2 deletion involved in phenotypes detected (in Objective 1) for 22q11.2DS PD by employing a CRISPR-modifier screen in the developed neuronal networks.