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

海马神经源性神经龛(HNN)在哺乳动物中产生新的神经元，但目前尚不清楚这是否是 发生在人类身上。成年海马区神经发生是维持完整认知和情绪所必需的 由海马体调节的功能。在HNN的神经细胞中检测到了不成熟的标志 但目前仍不清楚它们代表的是成年后出生的神经元，还是保持未成熟状态的神经细胞。 从出生起就是这样。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80年中，但血管生成和神经可塑性 在老年人中有所下降。其他小组支持我们的发现，而一些小组没有察觉到不成熟 人类海马区的神经细胞。此外，在衰老的小鼠中，更多的神经干细胞分化为神经胶质细胞，而不是 神经元，与年轻的动物相比，但我们不知道这种情况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或通过 其他机制。这些知识上的差距需要使用新技术来研究细胞谱系 在人类HNN中，以及神经前体细胞增殖、细胞命运、分化、成熟和 生死存亡。该项目旨在确定人类差异表达蛋白(DEP)和基因(DEG)。 HNN，在区域和单细胞水平，比较NA和AD。我们将使用高分辨率应用我们的管道 蛋白质组学分析的质谱学，以及单核(SN)RNA和ATAC(转座酶分析)。 可及染色质)测序(SEQ)，以识别基因表达和表观遗传变化。在幻灯片上安装 海马区组织中，我们将应用维西姆(10倍基因组学)和我们定制的空间转录 细胞类型特定mRNAs和蛋白质的解剖共定位技术(DBiT-seq)。新颖的计算方式 方法将确定人类HNN中的神经发生调节因子，可以在细胞或动物中进行测试 模特们。使用这些“OMICS”方法获得的结果将使用HighPlex RNAScope®进行验证 (ADCBio)和免疫荧光，以及qPCR、Western blotts和ELISA法，以可视化和定量DEP DEG在单细胞和区域水平的表达。我们严谨的大脑收集方法确保组织 质量、统一的加工、毒理学和神经病理学的使用，以及严格的临床纳入/排除标准。 分组包括：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) (按年龄、性别和死亡与脑部收集之间的死亡间隔)与40例AD病例(来自 哥伦比亚陶布研究所收藏)，Braak阶段1至4。目标：1.确定与以下项目相关的HNN DEP NA和AD。2.在NA和DG的未成熟和成熟神经元和神经胶质细胞群中鉴定DEG。 AD受试者，使用sn-RNA和sn-ATAC-seq(10倍基因组学)。3.确定细胞的解剖定位 表达与NA和AD相关的DEGS和DEPS，使用VISAM和DBiT-seq。4.检验相关性 在DEPS和DEGS之间，NA和AD的神经前体细胞和未成熟神经元和神经胶质细胞的数量。

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.