Clearance and In Vivo Detection of Tau Pathology

神经元Ca 2+稳态失调多年来一直与阿尔茨海默病有关 (AD)增加的Ca 2+内流已被证明与tau病理学有密切联系，但这种联系 在动物模型中没有得到很好的检验。一些先前的研究已经检查了Tau病中的Ca 2+信号传导 小鼠模型的双光子成像，具有可变的结果，这可能与不同的实验 参数我们最近报道了tau蛋白病小鼠中更明显的神经元功能障碍， 在增强的神经元活动期间通过双光子成像来观察它们。这一赤字可以在很大程度上通过 急性tau抗体治疗和益处与可溶性磷酸化tau蛋白的清除有关。 这些先前的研究是在通过脑内AAV感染表达神经元钙指示剂的小鼠中进行的。为 一种更好的方法，我们最近通过培育一种转基因模型， 一个钙指示剂在皮质神经元（Tg-Thy-1-GCAMP 6）与PS19 tau蛋白病小鼠，并显示在这里， 神经元功能障碍在年轻的时候，支持其在这个建议中的用途。 除了神经元功能障碍外，小胶质细胞活化与AD发病机制相关。可是 关于小胶质细胞如何与积累tau的细胞相互作用以及它们如何响应tau抗体， 配合物虽然这可以在一定程度上在培养中探索，但众所周知，离体小胶质细胞表现为 与在体内的情况非常不同。通过将在小胶质细胞中表达GFP或dTomato的小鼠与PS19 tau蛋白病杂交， 小鼠，我们可以第一次可视化tau病理进展期间小胶质细胞的结构动力学， 真实的时间监测tau-抗体复合物的小胶质细胞介导的吞噬清除。 关于诊断成像，小分子PET配体可以在体内对tau病变进行成像，但是因为 这些探针都是β折叠结合剂，它们可以识别各种淀粉样蛋白聚集体。相比之下，整个 抗体对它们的靶蛋白非常特异，但是太大而不能以足够的量进入大脑进行PET 侦测然而，单结构域抗体（sdAb）保留了这种特异性，同时小于1/10的大小 因此进入大脑的数量要大得多。 为了解决这些与tau蛋白病高度相关的重要问题， 正在进行的tau抗体治疗的临床试验，并得到初步和已发表数据的充分支持，我们 提出以下独立的目的：1）确定tau蛋白病理诱导的神经元凋亡的时程， 已建立tau蛋白病和神经元钙在清醒的新型转基因小鼠杂交中的体内功能障碍 2）确定小胶质细胞形态是否受清醒小鼠中tau病理学的影响，以及如何影响 它对具有或不具有效应子功能的tau抗体的治疗有反应; 3）检查 tau蛋白病理学进展过程中的神经元功能障碍和小胶质细胞反应及其治疗 清除，和; 4）针对tau蛋白的sdAb的诊断成像潜力的细化。

Neuronal Ca2+ dyshomeostasis has for many years been implicated in Alzheimer's disease (AD), and increased Ca2+ influx has been shown to have a close link to tau pathology, but this connection has not been well examined in animal models. A few prior studies have examined Ca2+ signaling in tauopathy mouse models by two-photon imaging, with variable results, which may relate to different experimental parameters. We recently reported more pronounced neuronal dysfunction in tauopathy mice by examining them by two-photon imaging during enhanced neuronal activity. This deficit could be largely corrected with an acute tau antibody treatment and the benefits were associated with clearance of soluble phospho-tau protein. Those prior studies were in mice expressing neuronal calcium indicator via an intracerebral AAV infection. For a better approach, we recently generated a novel mouse cross by breeding a transgenic model that expresses a calcium indicator in cortical neurons (Tg-Thy-1-GCAMP6) with PS19 tauopathy mice, and show here its neuronal dysfunction at a young age, supporting its use in this proposal. Beside neuronal dysfunction, microglial activation is associated with AD pathogenesis. However, very little is known about how microglia interact with cells accumulating tau and how they respond to tau-antibody complexes. While this can be explored to some extent in culture, it is well known that ex vivo microglia behave very differently from in vivo. By crossing mice expressing GFP or dTomato in microglia with PS19 tauopathy mice, we can for the first time visualize structural dynamics of microglia during tau pathology progression and monitor in real time microglia-mediated phagocytic clearance of tau-antibody complexes. Regarding diagnostic imaging, small molecule PET ligands can image tau lesions in vivo but because these probes are all β-sheet binders, they can recognize various amyloid aggregates. In contrast, whole antibodies are very specific for their target protein but too large to enter the brain in sufficient quantities for PET detection. However, single domain antibodies (sdAbs) retain this specificity while being less than 1/10 the size of whole antibodies, and therefore get into the brain in much larger quantities. To address these important issues, which are of a very high relevance to tauopathies, including several ongoing clinical trials on tau antibody therapies, and well supported by preliminary and published data, we propose the following independent aims: 1) To determine the time course of tau pathology-induced neuronal dysfunction in vivo in awake novel transgenic mouse cross of established tauopathy and neuronal calcium indicator lines; 2) To determine if microglial morphology is influenced by tau pathology in awake mice, and how it responds to therapy with tau antibodies with or without effector function; 3) To examine the interplay between neuronal dysfunction and microglial response during the progression of tau pathology and its therapeutic clearance, and; 4) Refinement of the diagnostic imaging potential of sdAbs against the tau protein.