A Multiomic Single Nucleus RNA-seq and ATAC-seq Examination of Key Brainstem Respiratory Control Regions from Fentanyl Overdose Decedents with and without Xylazine Present, and Matched Controls

About This Grant

Over 81,000 individuals died of an opioid overdose (OD) in 2023, and >92% of those cases involved fentanyl. Fentanyl has very potent opioid-induced respiratory depression (OIRD) effects, the primary cause of opioid OD death. Key brainstem regions, the preBötzinger complex and ventral respiratory group (preBot/VRG) and parabrachial and pontine Kölliker-Fuse nuclei (PB/KFn), play critical roles in respiration, and have been implicated in OIRD. Therefore, it is likely that the cellular etiology of opioid OD, notably the sites of action of fentanyl, involves these understudied brain regions. Despite evidence for individual differences including heritable variation in opioid OD (e.g., our GWAS of opioid OD death), most biologically mechanistic studies have focused on addiction-centric brain regions. Consistent with the likelihood of differential cellular signatures across these brain regions, our preliminary data suggest pervasive bulk tissue expression differences in the preBot (i.e., genes distinct from those implicated in regions relevant to addiction). In addition, prior studies have primarily relied on bulk tissue or single omics approaches to characterize the cellular etiology of opioid OD death. New multiome methods allow us to deeply characterize how the partnership between chromatin accessibility and gene expression in these brain regions drives opioid OD involving fentanyl, thus offering insights into biology and implicating genes that could be therapeutically targeted. Against the backdrop of unabated fentanyl-driven OD deaths, our proposal will: (A) generate the first single-nucleus (sn) paired RNAseq and snATACseq (i.e., multiome) data from the preBot/VRG and PB/KFn, from diverse human fentanyl OD decedents (N=40) and matched controls (N=40). (B) Characterize cell populations, states and cell-type specific relationships between differential gene expression and chromatin accessibility in each brain region across groups. (C) Estimate enrichment of opioid GWAS variants and genes, yielding mechanistic insights to large-scale population-scale genetic discovery. (D) Combine existing opioid-related expression differences in the same and other brain regions to delineate a brain-wide network of transcriptional variability associated with OUD and opioid OD, and produce in-silico prioritization of gene-drug pairs that are viable for preclinical follow-up.