Dissecting molecular consequences of opioid use disorder using single-cell RNA sequencing
Embargo Date
2027-02-07
OA Version
Citation
Abstract
The opioid epidemic is a global public health threat that affects countless individuals worldwide. These issues were precipitated by the significant, unchecked explosion of opioid prescriptions starting in the late 1990’s, leading to a drastic increase in opioid overdose deaths. While prescription rates have been curbed in recent years, the overdose consequences of over-prescription have continued to have consequences to this day. Opioid drugs exert their strongest effect in the central nervous system (CNS), which can be considered the “point of action” for these compounds, but research has also shown that these drugs have an impact on the behavior of cells in the peripheral immune system, which is referred to as the “point of entry”. Given the personal and social consequences of this classification of drugs, it is imperative to characterize the molecular consequences of said drugs; not only to describe the effects these drugs have on the body, but also to identify potential molecular targets for therapeutic intervention. With this goal in mind, the impact opioid drugs have on both the immune system and neurological reward system of opioid-dependent individuals was examined, using both humans and rodents. Peripheral blood mononuclear cells (PBMC) and tissue from the Nucleus Accumbens (NAc) of humans experiencing opioid use disorder (OUD) were examined, comparing them to individuals who were opioid-drug naïve. Rodent models of opioid addiction were also examined both to validate results from our human-participants, and to explore the temporal dynamics of these data. Our results indicate that opioid exposure leads to significant alterations to gene expression in both immune cells and the NAc, with significant consequences for the individuals with substance use disorder.
In the peripheral immune system, disruptions to the ability of the body to defend itself from viruses were observed, likely driven by opioid crosstalk with the Toll-like receptor (TLR) pathway. By utilizing an in-vitro opioid exposure paradigm, it was further found that these alterations are fast-acting, with our previously identified perturbations occurring as quickly as 3 hours post-drug exposure, as assessed by both RT-qPCR and single cell RNA sequencing.
In neurons of the NA of people with OUD differential alterations were found for synaptic activity, with interneurons gaining pro-synaptic gene expression and medium spiny neurons (MSN) losing expression of these genes, while increasing expression of genes associated with the primary cilium. In glia a consistent elevation to immunoreactive gene programs was observed, with a particular association with genes that have previously been shown to be connected to neurodegeneration. A cell-type-agnostic increase in expression of genes connected to energy metabolism was also found, presenting in parallel with an endothelial-cell-associated perturbation to gene expression programs controlling the integrity of the blood-brain barrier. These results were recapitulated using immunofluorescence (IF) in an in-vitro model of the neuronal microenvironment at the blood-brain barrier, indicating that our transcriptional results are borne out at the protein level.
Examination of rodent samples subjected to an opioid addiction simulating protocol, with timepoints associated with drug intoxication, drug withdrawal, and long-term abstinence from drug, implicated both drug withdrawal and abstinence as being predominantly tied to the phenotypes identified in our experimentation with human tissue rather than active intoxication, suggesting removal of drug as the predominant driver of biologically relevant changes to gene expression.
Given the overall societal consequences of opioid drug exposure, these experiments may lead to a greater understanding of the molecular underpinnings of OUD, while lending themselves to future therapeutic interventions to prevent the propagation of OUD-related morbidities.
Description
2024