MULTI-OMICS PROFILING OF HUMAN HIPPOCAMPUS NEUROGENIC NICHE TO DECIPHER THE NEUROBIOLOGY OF MAJOR DEPRESSION
Authors: Boldrini *Maura , Mariani Madeline B , Sissoko Chieck A , Xiao Yang , Dwork Andrew J , Rosoklija Gorazd B , Mann J John , Fan Ron , Leong Kam , Su Grham 
Published: February 2025

To investigate the presence of a neurogenic niche in human adult hippocampus and the molecular basis of smaller dentate gyrus (DG) in Major Depressive Disorder (MDD), we applied single-nucleus RNA (snRNA- seq) and accessible chromatin sequencing (snATAC-seq), together with spatial RNA sequencing (Visium, 10x Genomics). We assayed the hippocampus proper from 9 neurotypical (age 40 ± 12 yrs.) and 7 untreated MDD (age 48 ± 10 yrs.) males (2-3 biological replicates). A subsample (3 MDDs; 3 neurotypicals) were used for Visium (2 biological replicates, 12 capture areas total). Samples had RIN ³ 7 (7.9 ± 0.4). Libraries were sequenced (NovaSeq 6000 Sequencer, Illumina) to 62,000 reads/cell for RNA libraries, 33,000 reads/cell for ATAC libraries, and 67,000 reads/spot for Visium libraries (total 41,878 spots). RNA and ATAC data were preprocessed using Seurat, Signac, and DoubletFinder, batch effects removed using Harmony. Multimodal data for 293,236 cells were integrated using Weighted Nearest Neighbor analysis in Seurat, cell types annotated using canonical gene expression, enriched genes identified by a Wilcoxon rank-sum test (Bonferroni-adjusted p-value), and prediction scores from integration with spatial data implemented with TransferData function in Seurat. Gene ontology (GO) terms were generated using clusterProfiler (FDR-adjusted q-value). Differentially expressed genes (DEGs) in MDDs vs. neurotypicals, were identified using a mixed linear model implemented in limma with a pseudobulk approach, we aggregated count matrix across sample and cluster, and used a random effect to control for multiple samples/donor and fixed effects for age, PMI, and batch.

We identified hippocampus canonical cell types: granule neurons (GN), non-granule excitatory neurons (ExN), inhibitory neurons (InN), astrocytes, oligodendrocytes, microglia, vasculature, ciliated epithelial cells, choroid plexus, Cajal-Retzius, and oligodendrocyte progenitor cells (OPc). We discovered: a neural progenitor cell cluster (NPC) enriched for EGFR (p = 1.68E-35), BMPR1B (p = 1.25E-31), NOTCH2 (p = 4.43E-11), and DLL3 (p = 4.48E-21), with top GO terms ‘nervous system development’ (q=2.11E-05) and ‘cell fate commitment’ (q = 2.11E-05); and immature granule neurons (ImN) enriched for PROX1, DCX, CALB2 (p &lt;2.23E-308), that expressed GAD1 (p &lt;2.23E-308),with GO terms ‘neuron axonogenesis’ (q = 9.11E-05), ‘excitatory synapse assembly’ (q = 0.004), and ‘ephrin receptor signaling pathway’ (q = 0.024). ATAC showed NPC and ImN had increased chromatin accessibility of ASCL1, involved in neuronal commitment (p = 0.00013) and differentiation (p &lt;2.23E-308). Visium confirmed the expected anatomical location of canonical cell clusters, and mapped NPCs and ImNs to the DG region. In MDD vs. neurotypicals, DEGs included: upregulated interferon inducible genes (IFI44, IFI6, IFIT1, etc.) in GN, ExN, InN, ImN, astrocyte, microglia, choroid plexus, OPC (p &lt;0.01), and downregulated transthyretin (TTR), which transports thyroid hormone, in GN, ExN, InN, astrocyte, microglia, vasculature, and ciliated epithelial cells (p &lt;4.06E-05). This first single cell and spatial multiome study of human adult hippocampus reveals a neurogenic niche and supports a role of neuroinflammation in MDD. GAD1 expression in ImN explains their known inhibition of mature GNs. Molecular landscape of DEGs in MDD cell population points to druggable targets for MDD.

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