Project 1: Aberrant neuronal activity and tau distribution in FTD

摘要-项目1 FTLD-tau表现出不平衡的tau蛋白质稳态（细胞内和细胞间）和神经元功能障碍。不过小 已知致病性tau蛋白的积累和传播是如何发生的，以及它如何影响 神经元和突触功能。作为中心的一个组成部分，项目1（P1）的目标是剖析 FTD中异常神经元活性和tau分布的潜在机制。我们将联合收割机 和假设驱动的方法，它们非常强大，但如果没有结构就无法实现 的中心。我们提出三个具体目标。在目标1中，我们将定义FTD突变诱导的异常tau蛋白 翻译后修饰（PTM），并剖析乙酰化如何刺激tau蛋白与P3的释放。目标1将 还通过P2中FTD突变效应的转录组分析进行补充。在目标2中，我们将 确定FTLD-tau如何改变神经元活性和活性依赖性tau释放。我们将扩大我们的 V337 M人类神经元表现出过度兴奋性， 轴突起始段（AIS）。我们将系统地比较同基因FTD中神经元和突触的活动， 通过全细胞记录对突变株和对照株进行了比较。与质谱（MS）核心工作，我们还将剖析 通过解剖tau相互作用体来响应高浓度的 KCL（MS核心）。然后将进行CRISPRi/a以检查如何调节来自相互作用组研究的命中 会影响依赖活性的tau蛋白释放（CRISPR核心）。P1中的目标2将由P2补充， 将神经元活动与转录组和P3相关联，P3检查神经元之间的串扰。 活性和tau降解途径。在目标3中，我们将确定FTD突变如何影响致病性tau蛋白 播种致病性播种是tau蛋白病的一个关键特征，但对其机制知之甚少， 功能性成果。我们将比较在接受人类神经元阳性或阳性的神经元和突触活动， 对于由tau寡聚化产生的FRET信号为阴性。为了剖析介导致病的机制， 接种，我们将比较人类神经元中与内化突变体或WT tau种子相关的蛋白质组 (MS核心）。与突变tau蛋白选择性连接的蛋白质将优先作为潜在的命中物。与CRISPR合作 核心，我们将抑制/激活从种子蛋白质组中选择的基因，并通过FRET测量结果 信号和全细胞记录。目标3中的蛋白质组学方法将得到全基因组蛋白质组学方法的补充。 使用无偏方法（P2和CRISPR核心）筛选以鉴定内化途径。一起 与P2和P3一起，P1将有助于我们解剖tau蛋白质稳态之间的串扰的整体愿景， 失衡和神经元功能障碍。

ABSTRACT — PROJECT 1 FTLD-tau exhibits imbalanced tau proteostasis (intra- and inter-cellar) and neuronal dysfunction. However, little is known about how the accumulation and spread of pathogenic tau could occur, and how it could affect neuronal and synaptic functions. As an integral part of the Center, project 1 (P1)'s objective is to dissect mechanisms underlying aberrant neuronal activity and tau distribution in FTD. We combine unbiased and hypothesis-driven approaches, which are extremely powerful but cannot be achieved without the structure of the Center. We propose three Specific Aims. In Aim 1, we will define FTD mutations-induced aberrant tau post-translational modifications (PTMs) and dissect how acetylation stimulates tau release with P3. Aim 1 will also be complemented by transcriptome analyses of the FTD mutation effects in P2. In Aim 2, we will determine how FTLD-tau alters neuronal activity and activity-dependent tau release. We will expand on our preliminary study that V337M human neurons exhibit hyperexcitability with impaired homeostatic regulation of axon initial segment (AIS). We will systematically compare neuronal and synaptic activity in isogenic FTD mutant and control lines by whole-cell recordings. Working with mass-spec (MS) core, we will also dissect the mechanism underlying the activity-dependent tau release by dissecting tau interactome in response to high KCL (MS core). CRISPRi/a will then be performed to examine how modulating hits from interactome study would affect activity-dependent tau release (CRISPR core). Aim 2 in P1 will be complemented by P2, which correlates neuronal activity with transcriptome, and P3, which examines the crosstalk between neuronal activity and tau degradation pathways. In Aim 3, we will determine how FTD mutations affect pathogenic tau seeding. Pathogenic seeding is a key feature in tauopathy, but little is known about its mechanisms and functional outcome. We will compare neuronal and synaptic activity in receiving human neurons positive or negative for FRET signal resulting from tau oligomerization. To dissect the mechanisms mediating pathogenic seeding, we will compare the proteome associated with internalized mutant or WT tau seeds in human neurons (MS core). Proteins selectively linked with mutant tau will be prioritized as potential hits. Working with CRISPR core, we will inhibit/activate selected genes from the seeding proteome, and measure the outcome by FRET signal and whole-cell recordings. The proteomic approach in Aim 3 will be complemented by the genome-wide screening to identify pathways in internalization using unbiased approach (P2 and CRISPR core). Together with P2 and P3, P1 will contribute to our overall vision of dissecting the crosstalk between tau proteostasis imbalance and neuronal dysfunction.