An All Optical Platform for Modeling and Monitoring Early Neurodegeneration in Human Neural Circuits

项目摘要 减缓或延迟疾病发作、减轻症状和改善神经退行性疾病的治疗 病人的生活质量是迫切需要的。神经退行性疾病的一个共同特征， 帕金森病（Parkinson's Disease，PD）是一种神经元突触功能紊乱和兴奋抑制失衡的疾病。虽然传统上 被视为细胞死亡的后果，新兴的研究表明，这些过程可能发生在 广泛的变性，使它们成为开发神经保护疗法的有希望的靶点。 因此，该项目旨在开发新的工具，以更好地了解全球神经化学 动力学、突触生理学和神经变性中的电路结构。退化前模型 在本项目中，将通过内源性α-突触核蛋白的遗传操作建立散发性PD， 疾病的中心标志（目标1）。鉴于啮齿动物PD模型的可翻译性的局限性， 该项目利用人类多能干细胞衍生的神经元组织在功能电路使用微流体 培养装置。该装置不仅允许对α-突触核蛋白进行电路特异性操作，而且允许对α-突触核蛋白进行电路特异性操作。 特殊的成熟和组织不同的细胞类型，以重演基底神经节的输入。高保真 和电路特定的测量电路结构和功能的变化，在这个模型中，创新， 将寻求新的光学工具。在该提案中开发的光学传感器利用混合 化学遗传学策略，包括遗传编码的HaloTag蛋白质支架和明亮的红色- 位移染料远红混合HaloTag为基础的化学遗传谷氨酸传感器将被利用一起， 钙指示剂和光遗传致动器，以允许全光学操纵和读出电路功能， 早期PD（目标2）。将开发一种基于分裂HaloTag的突触传感器， 突触密度和突触谷氨酸传递随细胞类型和输入的变化的测量 特异性（目标3）。这些工具将使我们能够测试中心假设，即特定于电路的操纵， 人皮质-基底神经节回路中的α-突触核蛋白将导致谷氨酸活性的不同调节， 突触功能和电路结构。该项目的成功完成还将导致 建立了一个通用平台，其他神经退行性疾病和治疗 干预是可以理解的。

PROJECT SUMMARY Treatments for neurodegenerative disorders that slow or delay disease onset, mitigate symptoms, and improve patient quality of life are desperately needed. A common feature of neurodegenerative diseases such as Parkinson’s Disease (PD) is synaptic dysfunction and excitatory-inhibitory imbalance. Although traditionally seen as consequences of cell death, emerging research has shown that these processes may occur before widespread degeneration, making them promising targets for the development of neuroprotective therapeutics. Therefore, this project aims to develop new tools to better understand early defects in global neurochemical dynamics, synaptic physiology, and circuit structure in neurodegeneration. A pre-degeneration model of sporadic PD will be established in this project by genetic manipulation of endogenous alpha-synuclein, a central hallmark of the disease (Aim 1). Given the limitations in the translatability of rodent models of PD, this project utilizes human pluripotent stem cell derived neurons organized in functional circuits using a microfluidic culture device. This device allows for not only circuit-specific manipulation of alpha-synuclein, but also the specific maturation and organization of distinct cell types to recapitulate basal ganglia inputs. For high-fidelity and circuit-specific measurements of changes in circuit structure and function in this model, innovations in novel optical tools will be pursued. The optical sensors developed in this proposal utilizes a hybrid chemigenetic strategy that involves a genetically encoded HaloTag based protein scaffold and bright, red- shifted dyes. A far-red hybrid HaloTag-based chemigenetic glutamate sensor will be utilized alongside a calcium indicator and optogenetic actuator to allow for all-optical manipulation and read-out of circuit function in early PD (Aim 2). A split HaloTag-based synaptic sensor will be developed to enable simultaneous measurement of changes in synaptic density and synaptic glutamate transmission with cell type and input specificity (Aim 3). These tools will allow us to test the central hypothesis that circuit-specific manipulation of alpha-synuclein in human cortico-basal ganglia circuitry will result in disparate modulation of glutamate activity, synapse function, and circuit structure. Successful completion of this project will also result in the establishment of a general platform from which other neurodegenerative disorders and therapeutic interventions can be understood.