Tissue engineered Nigrostriatal Pathway as a testbed for evaluating axonal pathophysiology in Parkinson's disease.

项目总结 帕金森病(PD)是一种进行性神经退行性疾病，每年诊断5-6万例 总共有100多万美国人受到影响。帕金森病相关的运动症状是由于选择性地丢失 黑质致密部多巴胺能神经元(SNPC)。因为SNPC神经元发送长时间的 将轴突投射到纹状体，这种刻板的神经退行性变剥夺了纹状体至关重要的多巴胺能 从而使一条重要的运动反馈途径失效。已知α突触核蛋白 作为帕金森病的病理标志。α-突触核蛋白在健康大脑中的功能不是 完全理解，无论它是如何运输下来的大量轴突，是高度丰富的突触前 终末，并被认为是帕金森病的传播和进展的跨 神经系统的不同区域。尽管研究人员已经对帕金森病有了很多了解 通过细胞和动物模型的病理生理学，这些发现对翻译的影响有限，因为 在总结人类大脑结构和功能中与疾病最相关的属性方面面临的挑战。在……里面 特别是，目前的体外和体内模型在概括人类的基本特征方面存在局限性。 与轴突病理生理和突触核蛋白传递相关的疾病。例如，与以下内容相关的关键功能 早期SNPC的弱点是每个多巴胺能神经元都有一个长投射的轴突和复合体 纹状体内总长可达15英尺的树枝状结构，产生独特的运输和代谢需求 这些神经元。来自人源性多巴胺能神经元的长轴突投射的这一特征 人类来源确保了能够发展和应对同核症的遗传天赋- 在帕金森病的临床前模型中，纹状体神经元来源的投射是不存在的，因此不能很好地代表 轴索病变和代谢易感性在帕金森病发病中的作用为了满足这一需求，我们有 开发了第一个组织工程黑质纹状体通路(TE-NSP)，概括了天然的关键成分 途径：离散的人类干细胞来源的、表型受控的神经细胞群体通过长时间的 投射出轴索。该项目将验证TE-NSP作为第一个具有解剖学特征的PD模型 启发微组织，然后应用这一新的平台来研究帕金森病的轴突，机制 突触核蛋白传递和药物干预以阻断轴突介导的病理性传播 α-突触核蛋白横跨离散的大脑结构。我们将首先适当地演示TE-NSP 通过确定所有源和目标小区类型来概括相关的系统级架构， 用突触前和突触后标志物表征突触的形成，并展示输入-输出 基于诱发的多巴胺释放(AIM 1)。然后，我们将通过添加外源阿尔法来模拟PD 突触核蛋白纤维和表征急性轴突长度和密度、酪氨酸的病理生理变化 羟基酶表达，α-突触核蛋白从多巴胺能神经元转移到中等刺神经元，并影响 多巴胺释放(AIM 2)。最后，我们将利用TE-NSP作为评估治疗策略的试验床 旨在通过MTOR抑制来抑制α突触核蛋白的扩散(AIM 3)。我们已经组装了一个多- 由干细胞专家、神经生物学家、组织工程师和 临床医生验证和应用这一新颖的体外平台。该平台的成功演示将 显著推进了最终使用多巴胺能构建个性化TE-NSP的转换方法 帕金森病患者神经来源神经元对药物治疗的神经保护作用 旨在防止α-突触核蛋白传递，以延迟和/或防止轴突/神经元变性 特定于患者的方式。

PROJECT SUMARY Parkinson’s Disease (PD) is a progressive neurodegenerative disease with 50,000-60,000 diagnoses annually and over 1 million Americans afflicted in total. PD-associated motor symptoms arise from the selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Because SNpc neurons send long- projecting axons to the striatum, this stereotypical neurodegeneration robs the striatum of crucial dopaminergic inputs and thereby renders an important motor feedback pathway ineffective. Alpha synuclein protein is known as the pathological hallmark of PD pathology. The function of alpha synuclein in the healthy brain is not completely understood, however it is transported down axons in abundance, is highly enriched in presynaptic terminals, and is believed to be responsible for the transmission and progression of PD pathology across different regions of the nervous system. Although researchers have learned a great deal about PD pathophysiology through cellular and animal models, the findings have had limited translational impact due to challenges in recapitulating the most disease-relevant attributes of human brain structure and function. In particular, there are limitations of current in vitro and in vivo models to recapitulate essential features of human disease related to axon pathophysiology and synuclein transmission. For instance, a key feature related to early SNpc vulnerability is that each dopaminergic neuron features a long-projecting axon with complex arborization that can total 15 feet in length within the striatum, incurring unique transport and metabolic needs of these neurons. This feature of long axonal projections from human derived dopaminergic neurons – the human source ensuring a genetic endowment capable of developing and responding to synucleinopathy – projecting to a striatal neuronal source has been absent in preclinical models of PD thereby underrepresenting the role of axonopathy and metabolic susceptibility in PD pathogenesis. To address this need, we have developed the first tissue engineered nigrostriatal pathway (TE-NSP) recapitulating key elements of the native pathway: discrete human stem cell derived, phenotypically-controlled neuronal populations connected by long- projecting axonal tracts. This project will validate TE-NSPs as the first PD model featuring anatomically inspired microtissue, and then apply this novel platform for the study of PD axonopathy, mechanisms of synuclein transmission, and pharmacological interventions to block axon-mediated spread of pathological alpha-synuclein across discrete brain structures. We will first demonstrate that TE-NSPs appropriately recapitulate the relevant systems-level architecture by identifying all source and target cell types, characterizing synaptic formation with presynaptic and postsynaptic markers, and demonstrating input-output based on evoked dopamine release (AIM 1). We will then model PD via the addition of exogenous alpha synuclein fibrils and characterize acute axonal pathophysiological changes to axon length and density, tyrosine hydroxylase expression, alpha synuclein transfer from dopaminergic to medium spiny neurons, and affects on dopamine release (AIM 2). Finally, we will utilize TE-NSPs as a testbed for evaluating therapeutic strategies aimed at inhibiting alpha synuclein spread through MTOR inhibition (AIM 3). We have assembled a multi- disciplinary team of researchers consisting of stem cell specialists, neurobiologists, tissue engineers, and clinicians to validate and apply this novel in vitro platform. Successful demonstration of this platform will significantly advance a translational approach to ultimately build personalized TE-NSPs using dopaminergic neurons derived from PD patients to evaluate the neuroprotective efficacy of pharmacological therapies targeted at preventing alpha synuclein transmission to delay and/or prevent axonal/neuronal degeneration in a patient-specific manner.