Astrocytic exocytosis of ATP in amyloid pathology and Alzheimer's disease

项目摘要/摘要： 阿尔茨海默病(AD)是一种无法治愈的破坏性神经退行性疾病。它的特点是β- 大脑中含有淀粉样蛋白(Aβ)的老年斑和含有tau的神经原纤维缠结。在众多的 在阿尔茨海默病的发病机制中，毒性Aβ多肽的过度沉积被认为起着关键作用。 最近的研究表明，β在大脑中的沉积会触发强大的形态和 星形胶质细胞的转录改变。这些星形胶质细胞功能的改变如何有助于 AD的发展仍不清楚。PI研究小组的初步研究表明，Aβ42 诱导培养的星形胶质细胞释放三磷酸腺苷。此外，囊泡核苷酸转运体(Vnut)的丢失，是 负责将胞浆三磷酸腺苷装载到星形胶质细胞的排泄性溶酶体中，显著降低Aβ 使6月龄雌性5xFAD小鼠的斑块减少约50%，并改善认知功能。这些数据强烈地 提示消除星形胶质细胞中的ATP胞吐作用阻止淀粉样斑块的积累和 缓解认知功能衰退。在这些令人振奋的初步数据的大力支持下，拟议的 研究假设β沉积增强星形细胞对三磷酸腺苷的胞吐作用，并慢性升高 星形胶质细胞衍生的三磷酸腺苷进一步诱导神经炎症，损害Aβ的产生和清除，以及 会加剧认知能力的下降。拟议的研究将在具体目标上解决这一中心假设：Aim 1.探讨星形细胞胞吐与Aβ沉积之间的动态相互作用。简单地说， 微透析将被用来评估当Vnut被删除时大脑中细胞外ATP水平是如何改变的 星形胶质细胞对Aβ沉积的反应。Aβ沉降量的减少可能是由于产量减少 或者增加净空。为此，APP产品的相关内容，包括可溶性APPα、Aβ40、 Aβ42将被量化以评估Aβ生产的偏好。此外，Aβ相关星形胶质细胞和 小胶质细胞将被量化为神经胶质依赖的Aβ清除的指标。目标2：评估捐款 三磷酸腺苷的星形细胞胞吐作用对阿尔茨海默病小鼠神经炎症的影响。神经性炎症 用免疫印迹、免疫荧光、定量聚合酶链式反应和免疫印迹等方法对5xFAD小鼠进行检测。 ELISA法，并与相应的对照组小鼠进行比较。此外，为了探索更多的贡献机制， 这些小鼠的星形胶质细胞的转录本将使用RNA序列和生物信息学进行分析。贯穿始终 这个项目，所有关于神经炎症、细胞外三磷酸腺苷水平和Aβ沉积的数据都将是相关的 与同样的小鼠在6个月和12个月大时的学习和记忆能力相同。通过一种新的遗传小鼠模型， 这项拟议的研究将为星形胶质细胞和嘌呤能信号在AD中的作用提供有价值的见解。 这将有助于生成关键的初步数据，以支持在未来的R01赠款中进行更深入的调查。在……里面 从长远来看，这一系列研究可能会揭示治疗阿尔茨海默病和其他疾病的新方法 神经退行性疾病，通过靶向嘌呤能信号。

Project Summary/Abstract: Alzheimer’s disease (AD) is a devastating neurodegenerative disease with no cure. It is characterized by β- amyloid (Aβ)-containing senile plaques and tau-containing neurofibrillary tangles in the brain. Among many mechanisms contributing to AD, excessive deposition of toxic Aβ peptides is considered to play a key role. Recent studies have demonstrated that Aβ deposition in the brain triggers robust morphological and transcriptomic changes in astrocytes. How these alterations of astrocyte functions may contribute to the development of AD remains unclear. The preliminary studies of the PI’s research group show that Aβ42 induces ATP release in cultured astrocytes. Further, loss of vesicular nucleotide transporter (Vnut), which is responsible of loading cytosolic ATP into the excretory lysosomes, in astrocytes dramatically reduces Aβ plaques by ~50% and improved cognitive function in 6-month-old female 5xFAD mice. These data strongly suggest that elimination of ATP exocytosis in astrocytes blocks the accumulation of amyloid plaques and alleviates the cognitive decline. With the strong support of these exciting preliminary data, the proposed research hypothesize that Aβ deposition enhances astrocytic exocytosis of ATP, and chronic elevation of astrocyte-derived ATP further induces neuroinflammation, impairs Aβ production and clearance, and exacerbates cognitive decline. The proposed research will address this central hypothesis in specific aims: Aim 1: To examine the dynamic interaction between astrocytic exocytosis of ATP and Aβ deposition. Briefly, microdialysis will be used to assess how extracellular ATP levels in the brain are altered when Vnut is deleted in astrocytes in response to Aβ deposition. The reduced Aβ deposition could be due to decreased production or increased clearance. To this end, the relative contents of APP products, including soluble APPα, Aβ40, and Aβ42 will be quantified to assess the preference of Aβ production. In addition, Aβ-associated astrocytes and microglia will be quantified as an indicator of glial-dependent Aβ clearance. Aim 2: To assess the contributions of the astrocytic exocytosis of ATP on neuroinflammation in mouse models of AD. Neuroinflammation in 5xFAD mice without astrocytic Vnut will be examined using immunoblotting, immunofluorescence, qPCR and ELISA, and compared to appropriate control mice. In addition, to explore additional contributing mechanisms, the transcriptomes of astrocytes of these mice will be analyzed using RNA Seq and bioinformatics. Throughout the project, all the data on neuroinflammation, the extracellular ATP levels, and Aβ deposition will be correlated with the learning and memory of the same mice at 6 and 12 months of age. With a novel genetic mouse model, the proposed research will provide valuable insight into the roles of astrocytes and purinergic signaling in AD. This will help generate key preliminary data to support a more in-depth investigation in a future R01 grant. In the long term, this line of research may reveal new therapeutic approaches to treat AD and possibly other neurodegenerative diseases, by targeting purinergic signaling.