Human brain multi-omics to decipher major depression pathophysiology - Major depressive disorder (MDD) is a common disease with high risk of suicide, great social and economic costs, and poor treatment response. Hippocampus neuroplasticity is altered in MDD, susceptible to stress exposure and critical for mood regulation and the establishment of emotional memories in MDD. At the cellular level, our group and others reported fewer granule neurons, fewer astrocytes, and smaller dentate gyrus in MDD. There are conflicting findings about the extent to which new neurons are generated in adult humans, and we showed this happens into the eighth decade of life, while other groups were unable to confirm these findings. Adult neurogenesis is linked to both the pathogenesis of depressive phenotypes and the action of antidepressants in mouse studies. Pathogenic mechanisms underlying cellular and anatomical changes in MDD remain largely unknown. A meta-analysis from the three largest genome-wide association studies identified 102 independent variants, 269 genes, and 15 gene-sets associated with MDD. There is an urgent need for using new technologies to investigate cell lineages in human hippocampus. This project will identify differentially expressed proteins (DEPs) and genes (DEGs) between MDD and non- psychiatric control (CTRL) hippocampus, at a regional and single cell level. In homogenized hippocampus tissue, we will implement our proteomics pipeline using high resolution mass spectrometry (HRMS), single nuclei (sn)RNA-seq and snATAC (Assay for Transposase-Accessible Chromatin) sequencing. In slide-mounted tissue we will apply Visium (10X Genomics) and our custom-made slide-seq technology, using deterministic barcoding for spatial omics sequencing, co-mapping mRNAs, ATAC and proteins (DBiT-seq). Our pilot proteomics studies showed that DEPs lower in uMDD vs. CTRL promote mitosis, differentiation, and prevent apoptosis. Pilot snRNA-seq and snATAC-seq identified all types of neurons and glia, vasculature, and immature cell clusters, which mapped with Visium onto the expected hippocampus subfields. DBiT-seq pilot studies showed higher variability of RNA splicing in MDD vs. CTRL, RNA velocity revealed high transcriptomic instability in MDD leading to high inter-patient heterogeneity in the pyramidal neuron cluster, and epigenetic ATAC profiling identified markers potentially related to MDD pathogenesis. Studying 80 MDD and 80 CTRL, age 14-90 yrs., 60% males and 40% females, with sudden death, short agonal state, clear toxicology, postmortem interval<24 hrs., RNA integrity number (RIN)>8, tissue pH>6, we will: 1. Identify hippocampus DEPs between MDD and CTRL applying HRMS. 2. Identify cell clusters’ DEGs in MDD vs. CTRL hippocampus applying snRNA- seq and snATAC-seq. 3. Deliver the first hippocampus spatial transcriptomic atlas and epigenomic atlas for chromatin accessibility, identify region-specific RNA dynamics, and determine MDD vs. CTRL multiome differences applying DBiT-seq. Identified molecular regulators of hippocampus cell viability and function will provide potential druggable targets for new MDD treatments, to be later tested in cellular and animal models.
