Single Cell Gene Expression Profiling in hTau Mice

该提案的目标是了解在htau小鼠前脑内tau沉积之前和之后发生的机制，以帮助描述控制人类中枢神经系统神经变性的细胞事件。目的是评估易感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，我们将在hTau小鼠生命周期的几个关键时间点，包括病理前（2月龄）、早期病理（6-8月龄）、中度病理（12月龄）和重度病理（16-17月龄），对海马CA1锥体神经元、齿状回颗粒细胞和第二层/第二层新皮质锥体神经元的表达谱进行分析。实验设计包括通过激光捕获显微解剖对已鉴定的神经元群体进行微吸，然后是在首席研究员实验室开发的新型单细胞RNA扩增方法，结合定制设计的cDNA阵列分析。这种实验设计允许同时定量分析来自单个神经元的数百个转录本。目的1包括在4个确定的时间点对hTau小鼠的单个神经元群体进行时间过程分析。目的2包括对双转基因htau/APP小鼠进行类似的时间过程分析，以评估积累阿尔茨海默病大脑中两种标志蛋白的小鼠的单细胞表达谱。目的3包括实验性病变模式（穿孔路径横断），以评估中枢神经系统轴切反应对htau和htau/APP小鼠的影响。这些研究被假设为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志物。

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.