Project 1 (MSU)-Neurofibrillary tangle evolution in mild cognitive impairment

项目总结/摘要 这项提案的目标是了解tau蛋白传播的上游分子机制， 高度整合的胆碱能基底核内介导的神经缠结病理学-默认模式 在从无认知障碍（NCI）到轻度认知障碍的过渡期间， 损害（MCI）。我们最近发现，毒性tau寡聚体在Ch 4亚场中的积累遵循一个新的机制。 在疾病进展期间的尾侧-吻侧梯度。我们新的试验数据表明，tau寡聚体的积累 在DMN枢纽开始于楔前叶（PreC），这是由尾部Ch 4亚场支配， 扩散到额叶皮层（FC），其由吻侧Ch 4子场支配。因此，时空 DMN内缠结演变的模式可能反映了在神经支配中观察到的tau病理学的拓扑结构。 Ch 4子字段，表明Ch 4-DMN电路内神经变性的病理性扩散。目标1将测试 通过询问分类为低病理（LP）-NCI，高病理（HP）-NCI， MCI或阿尔茨海默病（AD）与位点特异性tau蛋白前缠结抗体、无偏体视学和光学显微镜的结合。 密度法在Ch 4-DMN连接体中驱动tau病理的分子机制是 未知的，并可能提供新的疾病修饰靶点的关键见解。我们之前的基因表达 Ch 4神经元的分析揭示了MCI中tau代谢途径的改变，包括表达增加， tau激酶的表达降低，tau磷酸酶的表达降低，以及3-重复与4-重复tau蛋白的比例倾斜 同种型。引起这些变化的一个潜在调控途径可能涉及小的非编码microRNA （miRNAs），其控制mRNA稳定性。中试性miRNA测序显示， 靶向tau代谢的MCI和AD。具体而言，miR-298在AD中显著上调，而miR-298在AD中显著上调。 298在人脑混合培养物中的过表达降低了较高分子量tau单体的水平， 提示miR-298影响tau亚型组成。因此，目标2将检验miRNAs 通过使用激光，靶向tau代谢mRNA在HP-NCI和MCI中的DMN连接体内失调 捕获带有预缠结的DMN皮质神经元的显微切割，然后进行miRNA测序， 生物信息学、靶mRNA分析和验证研究。Aim 3将使用一系列严格的miRNA- mRNA相互作用和表达测定、tau生物化学以及神经元形态和功能分析 为了检验Aim 2中鉴定的候选miRNAs将与tau和tau相互作用的假设， 代谢途径mRNA影响人类混合脑培养物中的tau病理学。我们还将探索miRNAs 与胆碱能、RNA剪接、突触、淀粉样蛋白和炎症通路相关，与项目合作 3和4总之，这些项目的目标将确定选择miRNA途径作为tau蛋白的关键上游调控因子 代谢相关的病理聚集和电路为基础的传播tau病理在Ch 4- DMN连接体先于MCI，从而揭示了新的潜在治疗靶点。

PROJECT SUMMARY/ABSTRACT The goal of this proposal is to understand the upstream molecular mechanisms underlying the spread of tau- mediated neurofibrillary tangle pathology within the highly integrated cholinergic nucleus basalis-default mode network (Ch4-DMN) connectome during the transition from no cognitive impairment (NCI) to mild cognitive impairment (MCI). We recently showed that toxic tau oligomer accumulation within Ch4 subfields follows a caudal-to-rostral gradient during disease progression. Our new pilot data show that tau oligomer accumulation within DMN hubs begins in the precuneus (PreC), which is innervated by the caudal Ch4 subfields, before spreading to the frontal cortex (FC), which is innervated by the rostral Ch4 subfields. Hence, the spatiotemporal pattern of tangle evolution within the DMN may mirror the topography of tau pathology observed in innervating Ch4 subfields, suggesting a pathological spread of neurodegeneration within Ch4-DMN circuits. Aim 1 will test this hypothesis by interrogating DMN tissue categorized as low pathology (LP)-NCI, high pathology (HP)-NCI, MCI, or Alzheimer’s disease (AD) with site-specific tau pretangle antibodies, unbiased stereology, and optical density methods. The molecular mechanisms driving tau pathology within the Ch4-DMN connectome are unknown and may provide key insights into novel disease-modifying targets. Our previous gene expression profiling of Ch4 neurons revealed alterations in tau metabolic pathways in MCI, including increased expression of tau kinases, decreased expression of tau phosphatases, and skewing of the ratio of 3-repeat to 4-repeat tau isoforms. One potential regulatory pathway causing these changes may involve small non-coding microRNAs (miRNAs), which control mRNA stability. Pilot miRNA sequencing revealed multiple miRNAs dysregulated in MCI and AD that target tau metabolism. In particular, miR-298 was significantly upregulated in AD, whereas miR- 298 overexpression in human mixed brain cultures reduced the levels of higher molecular-weight tau monomers, suggesting miR-298 influences tau isoform composition. Therefore, Aim 2 will test the hypothesis that miRNAs targeting tau metabolic mRNAs are dysregulated within the DMN connectome in HP-NCI and MCI by using laser capture microdissection of pretangle-bearing DMN cortical neurons, followed by miRNA sequencing, bioinformatics, target mRNA analysis, and validation studies. Aim 3 will then use a host of stringent miRNA- mRNA interaction and expression assays, tau biochemical, and neuronal morphometric and functional analyses to test the hypothesis that candidate miRNAs identified in Aim 2 will mechanistically interact with tau and tau metabolic pathway mRNAs to impact tau pathology in human mixed brain cultures. We will also explore miRNAs related to cholinergic, RNA splicing, synaptic, amyloid, and inflammatory pathways in collaboration with Projects 3 and 4. Altogether, these project aims will identify select miRNA pathways as critical upstream regulators of tau metabolism related to the pathological aggregation and circuit-based spread of tau pathology within the Ch4- DMN connectome prior to MCI, thus revealing novel potential targets for therapy.