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

组织工程黑质纹状体通路作为评估帕金森病轴突病理生理学的试验台。

• 批准号: 10005614
• 负责人: JOHN Eric DUDA
• 金额: --
• 依托单位: PHILADELPHIA VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10005614
• 关键词: 3-Dimensional; Acute; Address; Affect; American; Anatomy; Animal Model; Architecture; Axon; Beds; Brain; Cell Proliferation; Cell Survival; Cell model; Cells; Clinical; Complex; Corpus striatum structure; Diagnosis; Disease; Disease Progression; Disease model; Dopamine; Elements; Endowment; Ensure; FRAP1 gene; Feedback; Functional disorder; Genetic; Growth; Human; In Vitro; Intervention; Label; Lead; Length; Life; Mediating; Metabolic; Microscopy; Modeling; Motor; Nerve Degeneration; Nervous system structure; Neurodegenerative Disorders; Neuroglia; Neurons; Output; Parkinson Disease; Parkinsonian Disorders; Pathogenesis; Pathologic; Pathology; Pathway interactions; Patients; Periodicity; Pharmacology; Phenotype; Play; Population; Pre-Clinical Model; Predisposition; Presynaptic Terminals; Prevention; Process; Proteins; Research; Research Personnel; Resolution; Rodent; Rodent Model; Role; Scanning; Signal Transduction; Sirolimus; Source; Specialist; Structure; Substantia nigra structure; Symptoms; Synapses; System; Testing; Therapeutic; Time; Tissue Engineering; Translations; Tyrosine 3-Monooxygenase; Validation; Work; alpha synuclein; axonal degeneration; axonopathy; base; brain pathway; cell type; density; disability; dopaminergic neuron; foot; human disease; human stem cells; immunocytochemistry; in vitro testing; in vivo Model; induced pluripotent stem cell; mTOR inhibition; motor control; motor symptom; multidisciplinary; neural circuit; neuroimaging; nigrostriatal pathway; novel; overexpression; pars compacta; postsynaptic; presynaptic; prevent; response; stem cells; synaptogenesis; synuclein; synucleinopathy; targeted treatment; therapeutic development; three dimensional structure; translational approach; translational impact; transmission process; uptake

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.

项目概要 帕金森病 (PD) 是一种进行性神经退行性疾病，每年诊断人数为 50,000-60,000 例 总共有超过 100 万美国人受到影响。 PD 相关运动症状是由于选择性丧失 黑质致密部 (SNpc) 中的多巴胺能神经元。因为 SNpc 神经元发送长 将轴突投射到纹状体，这种刻板的神经变性剥夺了纹状体重要的多巴胺能 输入，从而使重要的运动反馈通路无效。 α突触核蛋白是已知的 作为PD病理学的病理标志。 α突触核蛋白在健康大脑中的功能并不 完全被理解，但是它大量地沿着轴突运输，在突触前高度丰富 终端，并被认为负责 PD 病理学的传播和进展 神经系统的不同区域。尽管研究人员对 PD 有了很多了解 通过细胞和动物模型进行病理生理学研究，由于以下原因，这些发现的转化影响有限 概括人类大脑结构和功能与疾病最相关的属性面临的挑战。在 特别是，目前的体外和体内模型在概括人类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相关的疾病。例如，与以下相关的一个关键功能 早期 SNpc 的弱点是每个多巴胺能神经元都有一个长突出的轴突，轴突具有复杂的结构。 纹状体内总长度可达 15 英尺的树状结构，产生独特的运输和代谢需求 这些神经元。来自人类的多巴胺能神经元的长轴突投射的这一特征—— 人力资源确保能够发展和应对突触核蛋白病的遗传天赋 – PD 临床前模型中不存在投射到纹状体神经元源的信号，因此代表性不足 轴突病变和代谢易感性在 PD 发病机制中的作用。为了解决这个需求，我们有 开发了第一个组织工程黑质纹状体通路（TE-NSP），概括了天然的关键要素 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-连接 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 激发微组织，然后应用这个新平台来研究 PD 轴突病变、机制 突触核蛋白传播和药物干预以阻止轴突介导的病理传播 α-突触核蛋白跨越离散的大脑结构。我们将首先证明 TE-NSP 适当 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表征突触形成，并展示输入-输出 基于诱发多巴胺释放 (AIM 1)。然后我们将通过添加外源 alpha 来模拟 PD 突触核蛋白原纤维并表征轴突长度和密度、酪氨酸的急性轴突病理生理变化 羟化酶表达，α突触核蛋白从多巴胺能神经元转移到中等多刺神经元，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测试平台 旨在通过 MTOR 抑制来抑制 α 突触核蛋白扩散 (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种新颖的体外平台。该平台的成功示范将 显着推进转化方法，最终使用多巴胺能构建个性化 TE-NSP 来自帕金森病患者的神经元用于评估药物治疗的神经保护功效 旨在防止 α 突触核蛋白传输，以延迟和/或防止轴突/神经元变性 患者特定的方式。