Assessment of a novel tau propagation pathway from layer II medial entorhinal cortical neurons to CA1 pyramidal neurons as an early Braak stage mouse model

异常磷酸化的微管相关蛋白tau（p-tau）是AD的诊断标志之一 病理学，这与AD的突触丧失和认知能力下降密切相关。Tau病理学第一 出现在经内嗅皮层和内嗅皮层II层（EC II），然后扩散到氨角 1（CA1）领域的海马区在前驱阶段的AD（Braak阶段I-II）。然而，没有动物 模型曾经成功地显示了tau从EC II特异性传播到CA 1，如早期典型的 布拉克准备。最近的一项研究发现，EC II中的Wolfram综合征-1（Wfs1）阳性细胞投射到 CA 1通过沿着颞氨（TA）通路的分子陷窝层（stratum lacunosum moleculare）。我们假设 错误折叠的tau蛋白通过TA途径从EC II中的Wfs1+细胞传播到CA 1，并且TA途径是一种新的途径。 新的治疗靶点，以抑制前驱AD阶段的tau蛋白传播。我们令人兴奋的初步数据 结果显示，将表达Cre诱导的P301L tau的腺相关病毒立体定位注射到EC II中， 诱导Wfs 1-Cre小鼠：1）人类tau从EC II稳健转移到CA 1锥体神经元，2）直接tau Wfs 1 + EC II神经元的轴突终末和CA 1锥体神经元的树突之间的转移，3） 抑制CA1锥体神经元的兴奋性，和4）受损的联想工作记忆。因此， 新的小鼠模型可以重现从Braak I到II的tau病理学进展，并发展成 神经生理功能障碍和海马学习障碍。本申请的目的是 是充分表征这种EC II-CA1 tau传播小鼠模型的病理学，并描绘出 从EC II到CA 1的tau传递的连接性和模式。我们的首要目标是发明治疗方法 for prodromal前驱AD using运用this mouse小鼠model模型.在目标1中，我们将i）表征以下的翻译后修饰： 使用多种p-tau抗体和神经元特异性标记物在EC II-CA 1小鼠中检测tau和细胞类型。（ii） 使用早期Braak分期和年龄/性别匹配的人AD脑组织中结果的可翻译性 iii）研究EC、CA1和前额叶皮质区的基因表达谱， 雄性和雌性小鼠，以了解行为结果中两性异形的分子基础。在 目标2，我们将i）采用免疫电子显微镜和超分辨率共聚焦显微成像， 捕获EC II轴突终末和CA 1锥体神经元的放射状树突之间的tau转移; ii）探索 利用Cre依赖性互补在CA 1锥体细胞和EC II神经元之间的单突触追踪 和iii）确定修饰的狂犬病病毒和WGA-GFP报告系统的激活/抑制作用， 神经元放电对tau蛋白传播的影响，神经元细胞外囊泡释放的作用，以及LDL受体相关 蛋白质1作为预测的突触末梢分泌的游离tau蛋白受体。拟议研究 该项目将对早期Braak阶段的tau传播机制产生新的理解， 为认知表型的性别二态性提供了新的认识。

Abnormally phosphorylated microtubule-associated protein tau (p-tau) is one of the diagnostic hallmarks of AD pathologies, which strongly correlates with synaptic loss and cognitive decline in AD. Tau pathology first appears in the transentorhinal cortex and entorhinal cortex layer II (EC II), then spreads to the Cornu Ammonis 1 (CA1) field of the hippocampal region at the prodromal stage of AD (Braak stage I-II). However, no animal model has ever succeeded in showing tau propagation from EC II specific to CA1 as typically seen in the early Braak staging. A recent study has discovered that Wolfram syndrome-1 (Wfs1) positive cells in EC II project to CA1 via the stratum lacunosum moleculare along the temporammonic (TA) pathway. We hypothesize that misfolded tau propagates from Wfs1+ cells in EC II to CA1 via the TA pathway, and that the TA pathway is a novel therapeutic target to suppress tau propagation in the prodromal AD stage. Our exciting preliminary data showed that the stereotaxic injection of adeno-associated virus expressing Cre-inducible P301L tau into EC II of Wfs1-Cre mice induced: 1) robust human tau transfer from EC II to CA1 pyramidal neurons, 2) direct tau transfer between axonal terminals of Wfs1+ EC II neurons and dendrites of CA1 pyramidal neurons, 3) suppression of excitability of CA1 pyramidal neurons, and 4) impaired associative working memory. Thus, this new mouse model may recapitulate tau pathology progression from Braak I to II, and develop neurophysiological dysfunction and hippocampal learning impairment. The object of this current application is to fully characterize the pathology of this EC II-CA1 tau propagation mouse model and delineate the connectivity and mode of tau transmission from EC II to CA1. Our overarching goal is to invent therapeutics for prodromal AD using this mouse model. In Aim 1, we will i) characterize the post-translational modification of tau and cell types in EC II-CA1 mice using multiple p-tau antibodies and neuronal specific markers. ii) Validate the translatability of the findings in human AD brain tissues using early Braak stage and age/sex matched control specimens.iii) Investigate the gene expression profiles in EC, CA1, and prefrontal cortical regions of male and female mice to understand the molecular basis of sexual dimorphism in the behavioral outcome. In Aim 2, we will i) employ immuno-electron microscopy and super-resolution confocal microscopic imaging to capture tau transfer between EC II axonal terminals and radial dendrites of CA1 pyramidal neurons;ii) explore monosynaptic tracing between CA1 pyramidal cells and EC II neurons using Cre-dependent complementation of a modified rabies virus and WGA-GFP reporter system, and iii) determine the effect of activating/inhibiting neuronal firing on tau propagation, the role of neuronal extracellular vesicle release, and LDL Receptor Related Protein 1 as the predicted receptor for free tau secreted from the synaptic terminals. The proposed research project will develop a new understanding of tau propagation mechanism seen in the early Braak stages and provide a new insight for the sexual dimorphism in cognitive phenotype.