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

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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.