Analysis of chromatin remodeling and transcriptional alterations in heroin brain in single cell resolution

Opioid Use Disorder (OUD) has escalated into a global epidemic, marked by a notable surge in opioid overdose deaths. U.S. military veterans have been particularly affected by this crisis, with a more than 50% increase in drug overdose mortality over the last decade. Deepening our understanding of the biological mechanisms behind OUD and identifying its molecular markers are essential for grasping addiction vulnerability and discovering new treatment targets. In our ongoing research, we used fluorescence-activated nuclear sorting (FANS) to isolate nuclei from major brain cell types in postmortem brains. This method enabled us to identify numerous gene expression and epigenetic changes associated with OUD in these cells. However, although recent single-cell analyses have uncovered various subpopulations within major brain cell types, each with unique transcriptional and epigenetic profiles that distinguish between healthy and diseased brains in disorders like depression, Alzheimer's disease, and Parkinson's disease, no studies to date have applied these single-cell techniques to explore gene expression and epigenetic changes in the brains of individuals with heroin addiction. We hypothesize that certain neuronal and/or glial subpopulations may exhibit heightened vulnerability to OUD. To expedite the discovery of genes and regulatory drivers relevant to the pathophysiology of OUD, in Aim 1 we propose to provide the first comprehensive characterization of molecular alterations in the brains of heroin users at a single-cell resolution. To strengthen our investigation, we will employ a 10x Genomics multiome assay that enables simultaneous profiling of chromatin accessibility and gene expression from the same nucleus, allowing the direct coordination between expression and regulation. Using these data, we will harness cutting-edge bioinformatic tools to infer cell- and subpopulation-specific Gene Regulatory Networks (GRNs) and pivotal regulators—transcription factors (TFs) driving the transcriptional changes linked to OUD. While postmortem studies offer valuable insights, they offers a static snapshot of disorders like OUD at the time of death, without clarity on whether molecular profiles result from chronic drug use, environmental factors, or genetic background. To overcome this, Aim 2 will utilize induced pluripotent stem cells (iPSC)-derived glutamatergic and GABAergic neurons. This approach allows for the dynamic study of opioid-induced gene expression and epigenetic changes. It also offers a platform for evaluating potential pharmacological treatments. We plan to test the VGF-derived TLQP-62 peptide, suggested in our preliminary research, for its therapeutic potential. Additionally, we aim to validate the computationally inferred GRNs, which encompass key TFs along with their target genes using iPSC-derived neurons. This will be achieved by integrating CRISPR-based screening with snRNA-seq. These studies should identify strong candidates for future animal studies, which, while essential, are beyond the immediate scope of this grant. Whereas distinct in their objectives, the collective impact of investigations in postmortem brain and iPSC- derived neurons is anticipated to synergistically uncover the cell-type-specific molecular mechanisms underpinning OUD. Additionally, this concerted effort aims to offer validation of potential novel targets for OUD treatment, nominating strong candidates for future animal studies. Project Number: 1I01BX006642-01A1 | Fiscal Year: 2025 | NIH Institute/Center: Veterans Affairs (VA) | Principal Investigator: STELLA DRACHEVA | Institution: JAMES J PETERS VA MEDICAL CENTER, BRONX, NY | Activity Code: I01 | Study Section: Special Emphasis Panel[ZRD1 NURA-U (01)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11052076