New technologies to identify molecular regulators of the human hippocampus neurogenic niche in healthy aging and Alzheimer's Disease

海马神经原性龛（HNN）在哺乳动物中产生新的神经元，但尚不清楚这是否是 发生在人类身上。成人海马神经发生是维持完整的认知和情感所必需的 由海马体调节的功能。在HNN的神经元细胞中检测到不成熟的标记物 但目前还不清楚它们是否代表了成年出生的神经元，或者是保持不成熟状态的神经元细胞 从出生到现在我们发现神经前体细胞和未成熟神经元的数量是稳定的 在正常衰老（NA）受试者的整个第八个十年的生活中，血管生成和神经可塑性， 老年人减少。其他的研究小组也支持我们的发现， 人类海马区的神经元细胞。此外，在衰老的小鼠中，更多的NPC分化为神经胶质细胞，而不是神经胶质细胞。 神经元，相比年轻的动物，但我们不知道这是否发生在人类身上。成人神经发生是 阿尔茨海默病（AD）的发病率较低，目前尚不清楚这是因为更多的NPC分化为神经胶质细胞还是通过 其他机制。这些知识上的空白保证了使用新技术来研究细胞谱系 在人HNN中，以及NPC增殖、细胞命运、分化、成熟和 生存本项目旨在鉴定人类组织中差异表达的蛋白质（DEPs）和基因（DEGs）， HNN，在区域和单细胞水平上，比较NA和AD。我们将应用我们的管道使用高分辨率 用于蛋白质组学分析的质谱法，以及单核（sn）RNA和ATAC（转座酶- 可重复染色质）测序（seq），以鉴定基因表达和表观遗传变化。在滑动安装中 海马组织，我们将应用Visium（10 X Genomics）和我们定制的空间转录组学 细胞类型特异性mRNA和蛋白质的解剖学共映射技术（DBiT-seq）。新型计算 这种方法将确定人类HNN中的神经发生调节因子，这些调节因子可以在细胞或动物中进行测试。 模型通过这些“OMICS”方法获得的结果将使用HighGlass RNAscope® （ADCBio）和免疫荧光以及qPCR、蛋白质印迹和ELISA测定，以可视化和定量DEP 和DEG在单细胞和区域水平的表达。我们严格的大脑采集方法确保 质量、统一处理、使用毒理学和神经病理学以及严格的临床入选/排除标准。 组包括：NA受试者（N=100），Braak分期0-1，年龄14-99岁，其中40人（60岁及以上） 与40例AD病例（来自 哥伦比亚陶布研究所收藏），布拉克阶段1至4。目的：1.识别与以下各项相关的HNN DEP NA和AD。2.鉴定NA中DG的未成熟和成熟神经元和神经胶质细胞群中的DG， AD受试者，使用sn-RNA和sn-ATAC-seq（10 X Genomics）。3.确定细胞的解剖定位 表达与NA和AD相关的DEG和DEP，使用Visium和DBiT-seq。4.测试相关性 DEPs和DEG之间的差异，以及NA和AD中NPC和未成熟神经元和胶质细胞的数量。

The hippocampus neurogenic niche (HNN) generates new neurons in mammals, but it is unclear if this is happening in humans. Adult hippocampal neurogenesis is necessary to maintain intact cognitive and emotional functions regulated by the hippocampus. Markers of immaturity have been detected in neuronal cells of the HNN but it is still unclear if they represent adult-born neurons, or neuronal cells that have maintained their immaturity since birth. We found that the number of neural progenitor cells (NPCs) and immature neurons was stable throughout the eighth decade of life in normal aging (NA) subjects, but angiogenesis and neuroplasticity were decreased in older people. Other groups have supported our findings, while some could not detect immature neuronal cells in human hippocampus. Moreover, in aging mice, more NPCs differentiate into glia rather than neurons, compared to younger animals, but we do not know if this happens in humans. Adult neurogenesis is lower in Alzheimer’s Disease (AD) and it is unknown if this is because more NPCs differentiate into glia or through other mechanisms. These gaps in knowledge warrant the use of new technologies to investigate cellular lineages in the human HNN, and molecular regulators of NPCs proliferation, cell fate, differentiation, maturation and survival. This project aims to identify differentially expressed proteins (DEPs) and genes (DEGs) in the human HNN, at the regional and single cell level, comparing NA and AD. We will apply our pipeline using high resolution mass spectrometry for proteomics analysis, and single nuclei (sn) RNA and ATAC (Assay for Transposase- Accessible Chromatin) sequencing (seq), to identify gene expression and epigenetic changes. In slide-mounted hippocampus tissue, we will apply Visium (10X Genomics) and our custom-made spatial transcriptomic technology for anatomical co-mapping of cell-type specific mRNAs and proteins (DBiT-seq). Novel computational approaches will identify neurogenesis regulators in the human HNN that can be tested in cellular or animal models. Findings obtained with these “OMICS” approaches will be validated using HighPlex RNAscope® (ADCBio) and immunofluorescence, and qPCR, Western blots, and ELISA assays, to visualize and quantify DEP and DEG expression at the single cell and regional level. Our rigorous brain collection methods assure tissue quality, uniform processing, use of toxicology and neuropathology, and strict clinical inclusion/exclusion criteria. Groups include: NA subjects (N=100), Braak stage 0-1, age 14-99 yrs., 40 of which (60 years of age and older) are matched (by age, sex and postmortem interval between death and brain collection) with 40 AD cases (from the Columbia Taub Institute collection), Braak stage 1 through 4. Aims: 1. Identify HNN DEPs associated with NA and AD. 2. Identify DEGs in immature and mature neuronal and glial cell populations of the DG in NA and AD subjects, using sn-RNA and sn-ATAC-seq (10X Genomics). 3. Determine the anatomical localization of cell expressing DEGs and DEPs associated with NA and AD, using Visium and DBiT-seq. 4. Test correlations between DEPs and DEGs, and numbers of NPCs and immature neurons and glia in NA and AD.