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

项目摘要/摘要 这项提议的目标是了解tau-1传播的上游分子机制。 高整合胆碱能基底核内介导的神经纤维缠绕病理-默认模式 无认知障碍(NCI)向轻度认知障碍(NCI)过渡过程中的网络(Ch4-DMN)连接体 损害(MCI)。我们最近发现，有毒的tau寡聚体在Ch4亚区的积累遵循以下规律 在疾病进展过程中，尾端到吻端的梯度。我们新的中试数据显示，tau齐聚物堆积 在DMN内，中枢始于楔前(PREC)，它由尾部Ch4亚场支配，在 扩散到前额叶皮质(Fc)，它由嘴侧Ch4亚区支配。因此，时空 DMN内的缠结演变模式可能反映了在神经支配中观察到的tau病理的地形图 CH4亚区，提示CH4-DMN环内神经变性的病理性扩散。AIM 1将测试 这一假设通过询问DMN组织分类为低病理(LP)-NCI，高病理(HP)-NCI， MCI，或阿尔茨海默病(AD)，具有部位特异性tau椒盐抗体，无偏见的体视学和光学 密度法。在CH4-DMN连接体中驱动tau病理的分子机制是 未知，并可能提供对新的疾病修改目标的关键见解。我们之前的基因表达 Ch4神经元的图谱显示MCI中tau代谢途径的变化，包括表达增加 Tau蛋白激酶活性降低，tau磷酸酶表达减少，3-重复与4-重复tau的比例发生扭曲。 异构体。导致这些变化的一个潜在调控途径可能涉及到小的非编码microRNAs (MiRNAs)，它控制着mRNA的稳定性。试点miRNA测序显示多个miRNAs在 靶向tau代谢的MCI和AD。特别是，miR-298在AD中显著上调，而miR- 298在人类混合脑培养物中的过度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的假设 激光靶向tau代谢的mRNAs在HP-NCI和MCI的DMN连接体中的失调 捕获含椒盐的DMN皮质神经元的显微解剖，随后进行miRNA测序， 生物信息学、靶向信使核糖核酸分析和验证研究。然后，AIM 3将使用一系列严格的miRNA- 信使核糖核酸相互作用和表达分析、tau生化、神经元形态计量学和功能分析 为了验证目标2中确定的候选miRNAs将与tau和tau机械相互作用的假设 代谢途径mRNAs对人类混合脑培养中tau病理的影响。我们还将探索miRNAs 与项目合作涉及胆碱能、RNA剪接、突触、淀粉样蛋白和炎症途径 3和4.总而言之，这些项目的目标将确定选定的miRNA途径作为tau的关键上游调节因子 与Ch4-Tau病理性聚集和基于回路的扩散有关的代谢 DMN连接体先于MCI，从而揭示了治疗的新的潜在靶点。

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.