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

项目总结 帕金森病（Parkinson's Disease，PD）是一种进行性神经退行性疾病，每年有5 - 6万例诊断 超过100万的美国人受到感染。PD相关的运动症状是由于选择性丧失了 多巴胺能神经元在黑质pars延髓（SNpc）。因为SNpc神经元发送长- 这种典型的神经退行性病变剥夺了纹状体中至关重要的多巴胺能神经元， 输入，从而使重要的运动反馈通路无效。已知α突触核蛋白 作为帕金森病的病理标志在健康大脑中，阿尔法突触核蛋白的功能不是 完全理解，但是它是大量运输下来的轴突，是高度富集在突触前 终末，并被认为是负责传播和发展的PD病理跨越 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，研究结果具有有限的翻译影响，由于 在概括人类大脑结构和功能的最相关的疾病属性的挑战。在 特别是，目前的体外和体内模型在概括人的基本特征方面存在局限性。 与轴突病理生理学和突触核蛋白传递有关的疾病。例如，一个与以下内容相关的关键功能 早期SNpc的脆弱性是每个多巴胺能神经元都有一个长的投射轴突， 在纹状体内的树枝状结构可以达到15英尺长，引起独特的运输和代谢需求 这些神经元。这种来自人源性多巴胺能神经元的长轴突投射的特征- 人类来源，确保能够发展和应对突触核蛋白病的遗传禀赋- 在PD的临床前模型中不存在向纹状体神经元源的投射， 轴突病变和代谢易感性在PD发病机制中的作用。为了满足这一需求，我们 开发了第一个组织工程黑质纹状体通路（TE-NSP），重现了天然黑质纹状体通路的关键元件。 途径：离散的人类干细胞衍生的，表型控制的神经元群体通过长- 突出的轴突束。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，然后应用这个新的平台研究PD轴突病变， 突触核蛋白传递，以及阻断轴突介导的病理性 α-突触核蛋白穿过离散的大脑结构。我们将首先证明TE-NSP可以适当地 通过识别所有源和目标细胞类型来概括相关的系统级架构， 用突触前和突触后标记物表征突触形成，并证明输入输出 基于诱发多巴胺释放（AIM 1）。然后，我们将通过添加外源性α来模拟PD 突触核蛋白纤维和表征轴突长度和密度、酪氨酸 羟化酶表达，α突触核蛋白从多巴胺能神经元转移到中型棘神经元， 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平台 目的是通过MTOR抑制（AIM 3）来抑制α突触核蛋白扩散。我们已经建立了一个多- 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种新的体外平台。该平台的成功演示将 显着推进了使用多巴胺能最终构建个性化TE-NSP转化方法 来自PD患者的神经元，以评估药物治疗的神经保护功效 靶向预防α突触核蛋白传递以延迟和/或预防神经元变性， 患者的具体方式。

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.