Single-cell transcriptomic and epigenomic analysis of brain cell vulnerabilities to tauopathies in early AD impacted brain regions

项目摘要 阿尔茨海默病（AD）是一种与年龄相关的、进行性的神经退行性疾病，可导致脑细胞的丧失 和他们的关系现有文献提供了AD/ADRD相关分子变化的广泛观点 从异质细胞群体中分离出来。然而，细胞特异性和脑区域特异性AD/ADRD相关的细胞凋亡的丧失， 细胞和连通性尚未解决，特别是在机械水平上。为了响应RFA-AG-23-028，我们 在多个机构组建了一支强大的多研究者团队，在以下方面具有互补的专业知识： 单细胞转录组学和表观基因组学分析，神经回路映射和下一代AD小鼠 模型开发我们将使用多个互补系的tau小鼠模型，结合APOE， 基因调节或致病触发在目标大脑区域。1)我们将使用Tau P301 S转基因 人APOE 4敲入背景（TE 4）或小鼠Apoe敲除背景（TEKO）的小鼠。 TE 4基因敲入显著加剧了tau介导的神经变性，而TEKO小鼠表现出很大程度上减弱了 与P301 S小鼠相比，神经元损失和脑萎缩。2)此外，我们还将使用新开发的 人源化Tau小鼠模型，其用正常的或非正常的小鼠MAPT基因替代内源性小鼠MAPT基因。 整个人MAPT基因的致病性变体（MAPT基因置换，MAPT-GR），其表达所有 在生理水平和比率下的人tau的同种型。我们假设早期AD中的脆弱细胞类型- 受影响脑区（蓝斑、内嗅皮层、海马CA 1和下托）显示出早期 适应不良的基因表达谱和表观基因组特征，定义他们的分子脆弱性， AD/ADRD发病机制。为了验证我们的假设，在目标1中，我们将应用单细胞表观基因组学， 转录组学技术在年龄匹配的对照组和AD小鼠中早期AD影响的大脑区域， 细胞类型分辨的基因表达和染色质可及性的多组学图谱。Tau病进展 因此，我们将在两个月内比较对照和病理性tau模型小鼠， 对于不同的小鼠系，每个小鼠系具有不同的年龄（对于TE和TEKO，4个月、10个月;对于TEKO，6个月、12个月;对于TEKO，6个月、12个月）。 MAPT-GR系）的行为和神经病理学特征。在目标2中，我们将使用 多重错误鲁棒荧光原位杂交（MERFISH）技术，以产生单细胞 早期AD影响的脑区域的分辨率空间转录组学图。MERFISH将扩展单细胞 组学和空间基因组学，以高空间分辨率绘制神经回路和病理。在目标3中，我们 使用执行计算分析来整合单细胞多组学数据、基于图像的解剖学和 从不同年龄的不同小鼠模型中获得的目标1-2的分子基因表达图谱， 在细胞水平上表征神经元回路和AD脆弱性。拟议的研究很好地协调了 RFA的目标，并预计提供新的生物学见解AD/ADRD发病机制， 前所未有的细胞和空间分辨率。

Project Summary Alzheimer's disease (AD) is an age-related, progressive neurogenerative disease that leads to loss of brain cells and their connections. The existing literature provides a broad view of AD/ADRD-related molecular changes from heterogeneous cell populations. However, cell-specific and brain-region specific AD/ADRD-related loss of cells and connectivity is not yet resolved, particularly at a mechanistic level. In response to RFA-AG-23-028, we have assembled a strong multi-investigator team across multiple institutions with complementary expertise in single-cell transcriptomics and epigenomics analysis, neural circuit mapping, and next-generation AD mouse model development. We will use multiple complementary lines of tau mouse models, in conjunction with APOE genetic modulation or pathogenic triggers in targeted brain regions. 1) We will use the Tau P301S transgenic mice on either a human APOE4 knock-in background (TE4) or a mouse Apoe knock-out background (TEKO). TE4 knock-in markedly exacerbates tau-mediated neurodegeneration, while TEKO mice show largely attenuated neuronal loss and brain atrophy compared to P301S mice. 2) We will additionally use recently developed novel humanized Tau mouse models that replace the endogenous mouse MAPT gene with either a normal or pathogenic variant of the entire human MAPT gene (MAPT gene replacement, MAPT-GR), which express all isoforms of human tau at physiologic levels and ratios. We hypothesize that vulnerable cell types in early AD- impact brain regions (locus coeruleus, entorhinal cortex, and hippocampal CA1 and subiculum) show early maladaptive gene expression profiles and epigenomic signatures that define their molecular vulnerability during AD/ADRD pathogenesis. To test our hypothesis, in Aim 1 we will apply single-cell epigenomics and transcriptomics technologies to early AD-impacted brain regions in age-matched control and AD mice, creating cell-type-resolved multi-omic maps of gene expression and chromatin accessibility. Tauopathy progression unfolds in an age-dependent manner, thus we will compare control and pathological tau model mice at two different ages each for different mouse lines (4 months, 10 months for TE and TEKO; 6 months, 12 months for MAPT-GR lines) based upon their behavioral and neuropathological characterization. In Aim 2, we will use the multiplexed error-robust fluorescence in situ hybridization (MERFISH) technology to generate single-cell resolution spatial transcriptomic maps for the early AD-impacted brain regions. MERFISH will extend single-cell omics and spatial genomics to map neural circuits and pathologies at high spatial resolution. In Aim 3, we will use perform computational analysis to integrate single-cell multi-omics data, image-based anatomical and molecular gene expression maps from Aims 1-2, acquired from different mouse models at different ages, to fully characterize the neuronal circuits and AD-vulnerability at cellular level. The proposed research is well aligned to the RFA goals and is expected to provide new biological insights into AD/ADRD pathogenesis at unprecedented cellular and spatial resolution.