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 mechan...