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

摘要：最近发现了一种新的阿尔茨海默病血液生物标记物，引起了人们的极大兴趣 (AD)，其中tau在血浆中的苏氨酸217(pT217-tau)水平磷酸化，预示着随后的疾病 比认知障碍早了几十年。然而，关于pT217-tau如何、何时和在哪里发生，人们知之甚少。 出现在老化的大脑中。这项拟议的研究将利用一种新的恒河猴模型和自然 发生tau病理以检测pT217-tau在衰老皮质中的表达模式和序列，以及 它与钙调节失调和自噬变性的关系。钙调节失调会激活钙蛋白酶， 它在T217处将gsk3β裂解为过度磷酸化的tau，并裂解热休克蛋白70.1以驱动 自噬变性，神经元在AD中死亡的过程。这些事件可以在 使用灌流固定组织的猴模型，保存了早期的磷酸化事件和细胞内 身体组织中经常退化的细节，例如检测pT217-tau贩运的能力 在神经元之间“播种”高级皮质网络。拟议的研究将研究pT217-tau如何 发生在易受伤害的皮质区域(内嗅皮层和背外侧前额叶皮质) 中年(7-13岁)、青年(17-23岁)和老年(27-33岁)的初级视觉皮质更具弹性 年)恒河猴。这项研究将利用两项互补技术：1)多标记免疫荧光 (MLIF)使用超分辨率STED显微镜检查分子相互作用，以及2)免疫电子 显微镜(免疫电子显微镜)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#年进行的人类大脑平行研究进行比较 欧洲。我们假设pT217-tau是继发性AD的一个成功的生物标志物，因为它预示着 Tau蛋白过度磷酸化和神经元变性的启动，以及它活跃的神经元间 神经元之间的交易提供了进入细胞外空间的途径，在那里它可以被捕获并 输送到脑脊液/血液。目标1将研究pT217-tau在衰老过程中的表达模式和序列 猕猴大脑皮质，以及pT217-tau是否特异地表达于具有以下症状的谷氨酸能神经元 钙调节失调表现为钙蛋白酶-2表达激活和钙结合蛋白降低。免疫电子显微镜将检测到 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 PT217-tau能否在兴奋性神经元之间传递，从而为谷氨酸能神经网络提供种子 认知力。初步数据显示，pT217-tau通过omega小体跨突触扩散 谷氨酸突触。目的3我们将确定pT217-tau的表达是否与 树突中的自食性退化。VULNERABLE患者pT217-tau病理的时间序列 与弹性脑区和驱动神经退行性变的机制相比，将有助于解释为什么这种tau 物种是这样一个合适的先兆，继而AD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.