Impact of mutant VPS35 on human dopaminergic neurons

在这一应用中的总体假设是，细胞运输受损在帕金森病(PD)的发病机制中发挥关键作用；这种全球性的侮辱将对更脆弱的神经元产生更明显的影响。帕金森病是第二常见的神经退行性疾病，在病理上与中脑DA神经元的死亡有关。这些神经元具有独特的解剖复杂性，再加上激烈的运输过程对生物能量的要求很高，可能使DA神经元特别容易受到压力的影响。最近，基因空泡蛋白分类35(VPS35)的突变被认为与显性遗传性帕金森病有关，这是一种罕见的单基因形式，临床上与特发性帕金森病无法区分。VPS35已被证明可以调节囊泡的分类和降解。在申请者和选定的主办机构的实验室之间的一项合作研究中，我们的团队发现VPS35结合和运输兴奋性神经递质受体，并控制它们的表面水平和功能命运。这项应用的目的是扩展我们的初步研究，并使用患者来源的含有VPS35突变的诱导多能干细胞(IPSCs)来建立和表征人类DA神经元模型，以检查这种突变的影响。将解决以下三个具体目标：(I)建立从IPSC系(患者和同基因对照)获得的人DA神经元的细胞培养，适用于活细胞分析。这些培养将包含成熟的、可识别的DA神经元，与兴奋性皮质神经元共同培养。(Ii)评估这些神经元的形态和生理表型。鉴定的DA神经元的轴突分枝和突起生长将通过共聚焦显微镜进行评估，固有兴奋性、通道功能和动作电位放电将通过全细胞膜片钳技术进行评估。(Iii)基础和药物刺激的DA释放量将用高效液相色谱法测定。除了对帕金森病的新的病理生理学见解外，该项目的最终价值将是一种被广泛描述的独特的人类疾病模型，该模型高度服从实验操作，并可用于筛选新的治疗化合物。

The overall hypothesis in this application is that impaired cellular trafficking plays a key role in the pathogenesis of Parkinson disease (PD); and that this global insult will have more pronounced effects in more vulnerable neurons. PD is the second most common neurodegenerative disorder and pathologically associated with the death of midbrain DA neurons. These neurons are characterized by a unique anatomical complexity, which is combined with high bioenergetic demands due to intense trafficking processes and may make DA neurons particularly vulnerable to stress. Mutations in the gene vacuolar protein sorting 35 (VPS35) were recently linked to dominantly inherited PD, a rare monogenetic form that is clinically indistinguishable from idiopathic PD. VPS35 has been shown to regulate vesicle sorting and degradation. In a collaborative study between the applicant and the laboratory of the selected host institute, our teams showed that VPS35 binds and traffics excitatory neurotransmitter receptors and governs their surface levels and functional fate. The goal of this application is to extend our preliminary study and to establish and characterize a human DA neuronal model using patient-derived induced pluripotent stem cells (iPSCs) harboring a VPS35 mutation to examine the impact of this mutation. The following three specific aims will be addressed: (i) Establish a cell culture of human DA neurons derived from iPSC lines (patient and isogenic control) amenable to live-cell assays. These cultures will contain mature, identifiable DA neurons in co-culture with excitatory cortical neurons. (ii) Assess these neurons for morphological and physiological phenotypes. Axonal arborisation and neurite outgrowth of identified DA neurons will be assessed by confocal microscopy, and intrinsic excitability, channel function, and action potential firing will be assessed by whole cell patch-clamp technique. (iii) Basal and pharmacologically stimulated DA release will be measured by HPLC. In additional to novel pathophysiological insights into PD, the ultimate value of this project will be an extensively characterized unique model of human disease that is highly amenable to experimental manipulations and available for the screening of new therapeutic compounds.