Tissue Engineered Nigrostriatal Pathway for Anatomical Tract Reconstruction in Parkinson's Disease

摘要 帕金森病（PD）是一种进行性神经退行性疾病，影响全球1000万人。 其运动症状是由于黑质部多巴胺能神经元选择性变性所致 这导致了它们对纹状体的长投射轴突输入的损失。常规细胞治疗 包括将多巴胺能神经元植入纹状体;然而，这种策略忽视了重要的 原生神经解剖学的系统级含义。路径重建战略旨在解决这一问题 通过以恢复解剖结构的方式替换神经元和轴突纤维来限制-因此 电路功能-丢失的路径。我们开发了一种重建策略， 黑质纹状体通路（TE-NSP）是在体外预制的，其特征在于人干细胞来源的细胞群。 多巴胺能神经元和它们的长突出轴突束包裹在生物相容性管状水凝胶内。 TE-NSPs可以被植入以直接替代该通路，向黑质供应多巴胺能神经元和多巴胺能神经元。 并向纹状体提供轴突输入，从而恢复基底神经节的重要互连性。在 这个建议，我们将回答一个基本的和被忽视的问题，在细胞治疗PD的特点， 使用TE-NSP的通路重建是否能够改善运动功能的恢复， PD大鼠模型中的常规纹状体移植物。我们的总体假设是，TE-NSP将导致更多 与纹状体移植物相比，通过一种涉及生理重建的机制， 神经支配和纹状体多巴胺调节模式更接近于天然基底神经节。这 本研究将从三个方面对这一假设进行检验：（1）建立TE-NSP重建基底神经节的能力 （2）证明TE-NSP在恢复神经元功能方面的实时功效; 黑质纹状体功能;（3）评估TE-NSP活性对运动恢复的影响。TE-NSP机制 和疗效将进行比较，水凝胶包裹黑质或纹状体移植物，脱细胞水凝胶植入物，以及 作为非植入和非损伤动物，直至植入后24周。将评估运动功能 通过旋转、前肢不对称和粘合剂去除测试。神经和连接模式将是 用免疫组织化学和单突触狂犬病示踪法评估，而离体和体内伏安法 和[18F]F-DOPA正电子发射断层扫描将用于分析实时多巴胺释放， 纹状体中的摄取。我们还将采用化学遗传学来沉默TE-NSP中的神经活动，以测试TE-NSP的神经活性。 对运动功能的影响总的来说，TE-NSP通过提供一种手段， 直接取代黑质纹状体途径，这可能会产生显着的好处比其他方法，提供 纹状体中多巴胺的适当调节是整合基底神经节回路的特征。这些 研究将进一步推进TE-NSPs作为组织工程医疗产品的长期目标， 减轻帕金森病患者运动症状的神经轴突损失。

ABSTRACT Parkinson’s disease (PD) is a progressive neurodegenerative disease that affects 10 million people worldwide. Its motor symptoms result from selective degeneration of dopaminergic neurons in the substantia nigra pars compacta, leading to a loss of their long-projecting axonal inputs to the striatum. Conventional cell therapy involves implanting dopaminergic neurons into the striatum; however, this strategy disregards the important systems-level implications of the native neuroanatomy. Pathway reconstruction strategies aim to address this limitation by replacing both neurons and axonal fibers in a manner that restores the anatomy – and hence circuit function – of the lost pathway. We have developed a reconstruction strategy whereby tissue-engineered nigrostriatal pathways (TE-NSPs) are pre-fabricated in vitro featuring a population of human stem cell-derived dopaminergic neurons and their long-projecting axonal tracts encased within a biocompatible tubular hydrogel. TE-NSPs may be implanted to directly replace the pathway, supplying both dopaminergic neurons to the nigra and providing axonal inputs to the striatum, thereby restoring crucial interconnectivity of the basal ganglia. In this proposal, we will answer a fundamental and neglected question in cell therapy for PD by characterizing whether pathway reconstruction with the TE-NSPs enables improved restoration of motor function compared to conventional striatal grafts in a rat model of PD. Our overarching hypothesis is that TE-NSPs will lead to more robust motor recovery than striatal grafts through a mechanism involving the reestablishment of physiological innervation and striatal dopamine regulation patterns more closely matching those of native basal ganglia. This hypothesis will be tested over three Aims: (1) Establish the ability of TE-NSPs to reconstruct basal ganglia circuitry via axonal-dendritic synaptic integration; (2) Demonstrate real-time efficacy of TE-NSPs in restoring nigrostriatal functionality; (3) Assess the influence of TE-NSP activity on motor recovery. TE-NSP mechanisms and efficacy will be compared to hydrogel-encased nigral or striatal grafts, acellular hydrogel implants, as well as non-implant and non-lesioned animals out to 24 weeks post-implantation. Motor function will be evaluated with rotational, forelimb asymmetry and adhesive removal tests. Innervation and connectivity patterns will be assessed with immunohistochemistry and monosynaptic rabies tracing, while ex vivo and in vivo voltammetry and [18F]F-DOPA positron emission tomography will be used to analyze real-time dopamine release and uptake in the striatum. We will also employ chemogenetics to silence neural activity in TE-NSPs to test the effects on motor function. Overall, TE-NSPs address a crucial gap in clinical treatment by providing a means to directly replace the nigrostriatal pathway, which may yield significant benefits over other methods by providing properly-regulated dopamine in the striatum as characteristic of integrated basal ganglia circuitry. These studies will further the long-term goal of advancing TE-NSPs as a Tissue Engineered Medical Product to mitigate the neuronal-axonal loss underlying the motor symptoms in patients afflicted by PD.