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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session D

Poster U14 - Spatial Omic Cellular Taxonomy of Human Hippocampus Reveals Region-Specific Heterogeneity in Proteome, Transcriptome and Epigenome

Friday, October 25, 2024
3:30 PM – 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction: The human hippocampus is essential for memory formation and spatial navigation, exhibiting significant cellular and molecular diversity (Bird and Burgess, 2008). It contains structurally distinct subfields, including the dentate gyrus (DG) and cornu Ammonis regions (CA1, CA2, CA3) (Duvernoy et al., 2013), and plays a critical role in the limbic–cortical–striatal–pallidal–thalamic (LCSPT) circuits that regulate mood, emotions, and learning. This makes the hippocampus a key focus in neuroscience and psychiatric research, where a comprehensive understanding of its cellular and molecular landscape is crucial for studying normal brain functions and the mechanisms underlying various neurological disorders.

The cellular taxonomy of the human brain has primarily been investigated through two methodologies: single-cell analysis and spatial transcriptomics. Previous single-cell studies have provided a cell type-specific RNA database of the human hippocampus (Ayhan et al., 2021; Darmanis et al., 2015; Franjic et al., 2022; Tran et al., 2021; Zhong et al., 2020), providing valuable insights but at the cost of losing spatial context due to the dissociation involved in single-cell extraction from postmortem brain tissues. This process complicates the accurate mapping of cellular subtypes and states back to their original microenvironments.

Furthermore, the dissociation of postmortem human brain into single nuclei disrupts cell-cell contacts and cell integrity, leading to the loss of neuronal and glial processes. Single-nucleus RNA (snRNA) sequencing fails to capture transcriptomic information from the cytosol and neuropil, which constitute the majority of neuronal volume and are vital for synaptic activity. These components are crucial in the pathogenesis of neuropsychiatric disorders.

Materials and

Methods: We used 25-multiplexed protein imaging to generate high-resolution cellular maps comprising 1.6 million cells, allowing for a systematic quantitative analysis of cell-cell distances and spatial neighborhood contexts. We performed spatial mRNA sequencing by DBiT-seq and constructed a multi-omic view of the tissue. The integration of spatial transcriptomic atlas with reference single-nuclei sequencing data has defined the relative abundance of each cell type in hippocampal subfields and revealed surprisingly variable RNA synthesis or degradation activities in Major Depressive Disorder (MDD) versus control samples. RNA velocity analysis suggested that cell-type and gene-specific transcriptomic instability may be characteristic of MDD, showing high inter-patient heterogeneity particularly in cornu Ammonis pyramidal neurons, which was not observed in controls.

Moreover, we introduced the first epigenetic gene regulatory maps of the human hippocampus using Spatial-ATAC-seq, which profiles genome-wide chromatin accessibility pixel by pixel at 50 µm resolution (Deng et al., 2022). Subfield-specific transcription factors were identified based on open chromatin read peaks and motif sequence. This approach has allowed us to decode the non-coding regions in the genome and reveal the epigenetic regulation of different cell types in the hippocampus, particularly showing that the chromatin states in granule neurons are more readily available for cFOS binding, indicating a primed state for memory formation.

Results, Conclusions, and Discussions: In this study, we present the first spatial multi-omic atlas of the adult human hippocampus based on proteomic, transcriptomic, and epigenomic sequencing of 17 hippocampi from 9 neurotypical donors and 8 major depressive disorder (MDD) patients (age, 44 ± 12 years; 15 males and 2 females; RNA integrity number (RIN), 7.58 ± 0.88). We deconvoluted the cellular composition in each pixel to build a gene expression landscape of cell types and cell states. Psychiatrically healthy brains were compared with MDD brains to showcase how spatial multi-omics identify differentially expressed genes and altered biological pathways in a region-specific manner. High variability of RNA synthesis rates was observed in MDD compared with controls. RNA velocity analysis further suggested a model for how transcriptomic instability in MDD contributes to a high inter-patient heterogeneity particularly in the pyramidal neuron cluster. Spatially ATAC-seq further identified the differential levels of open chromatin in the subfields. The motif binding readiness of certain transcription factors explained the differential neuronal excitability in memory formation. In this work, not only did we construct the first spatial atlas of adult human hippocampus, but also generated new insights into molecular underpinnings of hippocampal neuropathology in MDD, in a subregion-specific manner.

Acknowledgements (Optional):