Human Astrocyte-Based Nanovesicles to Target Neuroinflammation in Alzheimer's Disease

项目摘要-摘要 星形胶质细胞是一种特殊的神经胶质细胞，在神经系统中高度丰富，并维持 神经网络的功能自稳。在老年人和阿尔茨海默病患者的大脑中，星形胶质细胞可以 将炎症信号分子贡献给周围的微环境，进而产生负面影响 神经功能。如何有选择地向这些患者提供抗炎药物等治疗药物？ 功能失调的星形胶质细胞，以减少非靶向药物对其他细胞的影响？目前尚无有效的临床治疗方法。 为实现这一目的，已有各种方法。在这里，我们的目标是通过配制脂类来克服这种技术的缺乏 能够增强靶向递送能力的纳米微囊。这项创新技术的开发将成为可能 通过两个协同实验室的联合专业知识，他们将从 将人类多能干细胞来源的星形胶质细胞植入基于脂质的纳米囊泡表面。我们的预赛 研究表明，与膜蛋白结合的纳米囊泡来自独特的细胞来源 保留独特的细胞黏附蛋白，可能导致细胞特异性靶向。这一发现引发了我们的假设 包裹着来自阿尔茨海默病模型星形胶质细胞的黏附分子的纳米胶囊(又名， AstroVesicles(AVs)将与蛋白质相互作用的伙伴结合，特别是在炎症表面 从而增加功能失调的星形胶质细胞对细胞的摄取。在这种情况下，AVS可能是一个潜在的 新的治疗工具，允许早期识别炎症区域以及提供治疗 货物。淀粉样β-寡聚体治疗阿尔茨海默病模型将诱导星形胶质细胞炎症 疾病微环境和反应性将通过功能钙成像和基因检测来证实 表情分析。为了验证这一假设，在目标1中，我们将构建纳米囊泡，并比较其大小， 含有来自幼稚和低聚物处理的炎性细胞膜蛋白的电荷和稳定性 星形胶质细胞以及其他来源(如神经元、小胶质细胞和细胞来源的外切体)。我们将表演 基于蛋白质组学的纳米囊泡的发现，以鉴定具有高细胞潜力的细胞特异性蛋白 基于已知的细胞-细胞蛋白相互作用的靶向。在目标2中，我们将验证AVS的预期能力 优先针对通过淀粉样β寡聚体治疗而发炎的星形胶质细胞，而不是单纯的 星形胶质细胞和小胶质细胞。我们还将通过干扰候选人来测试潜在的定向机制 初步数据中确定的蛋白质。我们的方法将是测量治疗后动静脉血栓的存在。 由星形胶质细胞、神经元和小胶质细胞组成的球体培养，使用三维光学成像和 流式细胞术。值得注意的是，这些研究将开创利用人类神经球进行纳米胶囊测试的先河。 最后，在目标3中，我们将测试AVs是否能够增强抗炎药物的功能递送，重点是 核因子B途径。在优化和验证自动驾驶系统后，我们预计这一创新的系统将被使用 在整个神经科学界寻找阿尔茨海默病的细胞靶向治疗和成像工具。

PROJECT SUMMARY-ABSTRACT Astrocytes are specialized glial cells that are highly abundant in the nervous system and that maintain functional homeostasis of neural networks. In the aged and the Alzheimer’s disease brain, astrocytes can contribute inflammatory signaling molecules to the surrounding micro-environment, in turn, negatively impacting neural function. How can therapeutics, such as anti-inflammatories, be selectively delivered to these dysfunctional astrocytes in order to reduce off-target drug effects on other cells? At present, no effective clinical approaches exist for this purpose. Here, we aim to overcome this lack of technology by formulating lipid nanovesicles capable of enhanced targeted delivery. Development of this innovative technology will be enabled through the combined expertise of two synergistic laboratories who will bioengineer membrane proteins from human pluripotent stem cell-derived astrocytes into the surface of lipid-based nanovesicles. Our preliminary studies have revealed that nanovesicles integrated with membrane proteins derived from unique cell sources retain unique cell adhesion proteins that may lead to cell-specific targeting. This finding provoked our hypothesis that nanovesicles coated with adhesion molecules derived from Alzheimer’s disease-model astrocytes (a.k.a., AstroVesicles (AVs)) will bind to protein-interacting partners, specifically at the surface of inflammatory astrocytes and, thus, increase cellular uptake by dysfunctional astrocytes. In this way, AVs could be a potential new theranostic tool that allows for the early identification of inflamed areas as well as the delivery of therapeutic cargo. Astrocyte inflammation will be induced by amyloid beta oligomer treatment to model the Alzheimer’s disease microenvironment and then reactivity will be confirmed by functional calcium imaging and gene expression profiling. To test the hypothesis, in Aim 1, we will formulate nanovesicles and compare the size, charge, and stability of those containing membrane proteins from naïve and oligomer-treated inflammatory astrocytes as well as from other sources (e.g., neurons, microglia, and cell-derived exosomes). We will perform proteomics-based discovery of the nanovesicles to identify cell-specific proteins with high potential for cell targeting based on known cell-cell protein interactions. In Aim 2, we will validate the expected capability of AVs to preferentially target astrocytes that are inflamed via amyloid beta oligomer treatment, in comparison to naïve astrocytes and microglia. We will also test potential mechanisms of targeting by interfering with candidate proteins identified in preliminary data. Our approach will be to measure the presence of AVs upon treatment of sphere cultures composed of astrocytes, neurons, and microglia, using three-dimensional optical imaging and flow cytometry. Notably, these studies will pioneer the use of human neural spheres for nanovesicle testing. Finally, in Aim 3, we will test whether AVs yield enhanced functional delivery of anti-inflammatories, focusing on the NFB pathway. After optimizing and validating the AVs, we expect this innovative system will be utilized throughout the neuroscience community for cell-targeted therapeutics and imaging tools in Alzheimer’s disease.