Abstract
BACKGROUND: Neuropeptides are a diverse class (>100 members) of endogenous neurotransmitters. They are understood to be strongly influenced by psychological stress, specifically glucocorticoids like cortisol, with evidence of (mal)adaptation across several phenotypes of psychiatric illness. While it is hypothesised that changes in their expression contribute to an altered balance of inhibitory and excitatory signalling in the brain, the mechanistic details - including the relevant spatial context - of these changes are yet to be fully understood. To move forward, the field of psychiatry requires a high-resolution understanding of how stress manifests at the molecular level across the postmortem human brain. AIMS & OBJECTIVES: The mechanistic analysis of neuropeptides is confounded by several of their innate structural and chemical properties. As a result, their study has previously been confined to predominantly bulk analysis methods, many of which are no longer sufficient in allowing us to recapitulate the biological basis for stress regulation in the human brain. Here, we directly target this gap in our understanding by providing a cell-subtype specific analysis of neuropeptide (dys)regulation in the postmortem human orbitofrontal cortex at a single cell resolution using a novel multi-omic experimental pipeline. METHOD: Using genome-wide transcriptomic data from >800,000 individual postmortem nuclei, we used bioinformatics to understand the effects stress exposure has on the cell type-specific expression patterns of neuropeptides in the human orbitofrontal cortex (n=101). Spatial transcriptomic data from the same subjects allowed us to visualise the spatial context of these changes. Finally, we are working to adapt a multimodal approach to tissue analysis by Vicari et al (Nature Biotechnology, 2023) which combines histology, mass spectrometry imaging and spatial transcriptomics to give a holistic overview of mRNA, proteins, lipids and metabolites from within a single fresh frozen postmortem brain tissue sample, to also detect neuropeptides. RESULTS: Preliminary analysis of the snRNAseq data showed that compared to controls, individuals exposed to early life stress have differentially expressed levels of SST (somatostatin) and PENK (proenkephalin) mRNA, with the most pronounced changes seen in excitatory and inhibitory neurons. Using spatial transcriptomics, we found that somatostatin expression appeared consistent throughout the grey matter of the orbitofrontal cortex, which was distinct from somatostatin positive inhibitory neurons that were predominantly located within the deeper grey matter layers of the cortex. We also investigated mRNA expression for several neuropeptide-related synaptic receptors, three of which (SYNPR; synaptoporin, SCGN; secretagogin and HTR2C; serotonin) showed cell type specific changes in early life stress compared to controls. DISCUSSION & CONCLUSIONS: In line with previous evidence, we show that exposure to severe stress, particularly early in life, associates with an altered landscape of neuropeptide expression in the human orbitofrontal cortex. Further, we show that the spatial context over which said changes occur are peptide specific, and therefore, likely unique to disorder phenotype. Given the importance of neuropeptides as facilitators of neuronal transmission, and therefore, upstream regulators of gene and protein expression, understanding these changes moves us closer to developing a system of biological classification for psychopathology. Specifically, identifying treatment options which directly target the underlying molecular changes at a patient-specific level.