Generation and trans-neuronal seeding of phosphorylated T217-Tau in aging macaque cortical circuits

翻译后摘要：有很大的兴趣，在最近发现的一种新的，血液生物标志物阿尔茨海默氏病 (AD)其中血浆中苏氨酸217磷酸化的tau（pT 217-tau）水平预示着随后的疾病 在认知障碍之前几十年。然而，关于pT 217-tau如何、何时以及在何处表达， 在老化的大脑中产生。拟议的研究将利用一种新的恒河猴模型， 发生tau病理学以检查老化皮质中pT 217-tau表达的模式和序列，和 其与钙调节异常和自噬变性的关系。钙调节异常激活钙蛋白酶， 其切割GSK 3 β以在T217处过度磷酸化tau，并切割热休克蛋白70.1以驱动 自噬性变性，AD中神经元死亡的过程。这些事件可以在 使用灌注固定组织的猴模型，其保留了早期磷酸化事件和细胞内 通常在死后人体组织中降解的细节，例如，检测pT 217-tau运输能力 在神经元之间“播种”高级皮层网络。这项拟议的研究将研究pT 217-tau 出现在脆弱的皮层区域（内嗅皮层和背外侧前额叶皮层）， 在中年（7-13岁）、“年轻”-年龄（17-23岁）和老年（27-33岁）中，初级视觉皮层更具弹性 岁）恒河猴。本研究将采用2种互补技术：1）多标记免疫荧光 （MLIF）与超分辨率STED显微镜检查分子相互作用，和2）免疫电子 显微镜（免疫EM）观察超微结构定位，包括神经元之间的运输。结果将 与Braak/delTredici和Hansson实验室在1999年进行的人类大脑平行研究相比， 欧洲我们假设pT 217-tau是AD的一个成功的生物标志物，因为它预示着AD的发生。 启动tau蛋白过度磷酸化和神经元变性，并且因为其活性的神经元间 神经元之间的运输提供了进入细胞外空间的通道，在那里它可以被捕获， 运输到CSF/血液中。目的1将研究pT 217-tau蛋白在衰老过程中的表达模式和序列 猕猴皮层，以及pT 217-tau表达是否特异性地出现在具有以下迹象的多巴胺能神经元中： 钙调节异常由激活的钙蛋白酶-2表达和低钙结合蛋白证实。ImmunoEM将检测 最早的表达迹象，例如，聚集在远端树突的微管上。目标2将决定 pT 217-tau是否可以在兴奋性神经元之间运输，以种子介导 认知.初步数据表明，pT 217-tau通过欧米茄体跨突触传播。 谷氨酸突触目的3：我们将确定pT 217-tau表达是否与 树突的自噬性变性。阐明pT 217-tau病理学的时间顺序在脆弱的 vs.有弹性的大脑区域和驱动神经变性的机制将有助于解释为什么这种tau蛋白 物种是这样一个适当的先驱，随后的AD，以指导治疗干预策略。

Abstract: There is great interest in the recent discovery of a novel, blood biomarker for Alzheimer’s Disease (AD), where levels of tau phosphorylated at threonine 217 (pT217-tau) in plasma herald ensuing disease decades before cognitive impairment. However, there is little known about how, when and where pT217-tau arises in the aging brain. The proposed research will utilize a novel rhesus macaque model with naturally occurring tau pathology to examine the pattern and sequence of pT217-tau expression in the aging cortex, and its relationship to calcium dysregulation and autophagic degeneration. Calcium dysregulation activates calpain, which cleaves GSK3β to hyperphosphorylate tau at T217, and cleaves heatshock protein 70.1 to drive autophagic degeneration, the process by which neurons die in AD. These events can be detected in the monkey model using perfusion-fixed tissue, which preserves early phosphorylation events and intracellular details that are often degraded in post-mortem human tissue, e.g., the ability to detect pT217-tau trafficking between neurons to “seed” higher cortical networks. The proposed research will examine how pT217-tau arises in vulnerable cortical regions (the entorhinal cortex and dorsolateral prefrontal cortex) compared with the more resilient primary visual cortex, in middle-aged (7-13 yrs), “young”-aged (17-23 yrs), and old aged (27-33 yrs) rhesus monkeys. The research will utilize 2 complementary techniques: 1) multi-label immunofluorescence (MLIF) with super-resolution STED microscopy to examine molecular interactions, and 2) immunoelectron microscopy (immunoEM) to view ultrastructural localization, including trafficking between neurons. Results will be compared to parallel studies of human brain being conducted by the Braak/delTredici and Hansson labs in Europe. We hypothesize that pT217-tau is a successful biomarker of ensuing AD, because it heralds the initiation of tau hyperphosphorylation and neuronal degeneration, and because its active inter-neuronal trafficking between neurons provides access to the extracellular space where it can be captured and transported to CSF/blood. Aim 1 will examine the pattern and sequence of pT217-tau expression in aging macaque cortex, and whether pT217-tau expression specifically arises in glutamatergic neurons with signs of calcium dysregulation evidenced by activated calpain-2 expression and low calbindin. ImmunoEM will detect the earliest signs of expression, e.g., aggregating on microtubules in distal dendrites. Aim 2 will determine whether pT217-tau can traffic between excitatory neurons to seed glutamatergic networks subserving cognition. Preliminary data indicate pT217-tau undergoes trans-synaptic spread via omega bodies across glutamate synapses. Aim 3 we will determine whether pT217-tau expression occurs in concordance with autophagic degeneration in dendrites. Elucidating the temporal sequence of pT217-tau pathology in vulnerable vs. resilient brain regions and mechanisms driving neurodegeneration would help to explain why this tau species is such an appropriate herald of ensuing AD to guide therapeutic intervention strategies.