Single Cell Gene Expression Profiling in hTau Mice

这项提议的一个目标是了解tau在htau小鼠前脑内沉积之前和之后发生的机制，以帮助描绘管理人类中枢神经系统内神经退化的细胞事件。一个目标是评估脆弱人群中基因表达的病理变化，同时避免来自备用神经细胞类型和非神经细胞群体的潜在污染。在本文提出的一系列研究中，将在hTau小鼠一生中的几个关键时间点进行海马CA1区锥体神经元、齿状回颗粒细胞和L1/L11层新皮质锥体神经元的表达谱分析，包括病理前期(2个月龄)、早期病理(6-8个月)、中度病理(12个月)和严重病理(16-17个月)。实验设计包括通过激光捕获显微切割对已识别的神经元群体进行微抽吸，然后在首席调查员的实验室中开发出一种新的单细胞RNA扩增方法，并结合定制设计的cDNA阵列分析。这一实验设计允许同时对单个神经元的数百个转录本进行定量分析。目标1包括hTau小鼠在4个确定的时间点的单个神经元群体内的时程分析。AIM 2对双转基因htau/app小鼠进行了类似的时间进程分析，以评估积聚了阿尔茨海默病大脑中两种标志性蛋白的小鼠的单细胞表达谱。目的3包括一种实验性的损毁范式(穿透通路横断)，以评估htau和htau/app小鼠中枢神经系统轴突切断反应的影响。这些研究是为了阐明早期细胞特异性突触和神经退行性疾病的早期生物标记物。

A goal of this proposal is to understand mechanisms that occur prior to and following tau deposition within the forebrain of htau mice to help delineate cellular events that govern neurodegeneration within the human central nervous system. A goal is to assess pathological changes in gene expression within vulnerable populations while avoiding potential contamination from spared neuronal cell types and noneuronal populations. In the series of studies proposed herein, expression profile analysis of hippocampal CA1 pyramidal neurons, dentate gyrus granule cells, and layer ll/lll neocortical pyramidal neurons will be performed on hTau mice at several key time points during the lifespan, including pre-pathology (2 months of age), early pathology (6-8 months), moderate pathology (12 months), and severe pathology (16-17 months). The experimental design consists of microaspiration of identified neuronal populations via laser capture rnicrodissection followed by a novel single cell RNA amplification methodology developed in the laboratory of the Principal Investigator combined with custom-designed cDNA array analysis. This experimental design allows for simultaneous quantitative analysis of hundreds of transcripts from individual neurons. Aim 1 consists of a time course analysis within individual neuronal populations in hTau mice at the 4 defined time points. Aim 2 comprises a similar time course analysis on double transgenic htau/APP mice to assess single cell expression profiles in mice that accumulate both hallmark proteins seen in the Alzheimer's disease brain. Aim 3 consists of an experimental lesion paradigm (perforant path transection) to assess the effects of a central nervous system axotomy response in htau and htau/APP mice. These studies are hypothesized to elucidate early biomarkers for early cell-specific synaptic and neurodegenerative disorders.