Single cell transcriptome profiling of the human alcohol-dependent brain

Abstract

BackgroundAlcoholism remains a prevalent health concern throughout the world. Previous studies have identified transcriptomic patterns in the brain associated with alcohol dependence in both humans and animal models.But none of these studies have systematically investigated expression within the unique cell types present in the brain. ResultsWe utilized single nucleus RNA sequencing (snRNA-seq) to examine the transcriptomes of over 16,000 nuclei isolated from prefrontal cortex of alcoholic and control individuals. Each nucleus was assigned to one of seven major cell types by unsupervised clustering. Cell type enrichment patterns varied greatly among neuroinflammatory-related genes, which are known to play roles in alcohol dependence and neurodegeneration. Differential expression analysis identified cell type-specific genes with altered expression in alcoholics. The largest number of differentially expressed genes (DEGs), including both protein-coding and non-coding, were detected in astrocytes, oligodendrocytes, and microglia. ConclusionsTo our knowledge, this is the first single cell transcriptome analysis of alcohol-associated gene expression in any species, and the first such analysis in humans for any addictive substance. These findings greatly advance understanding of transcriptomic changes in the brain of alcohol-dependent individuals.1 Background Alcohol abuse is involved in over 200 pathologies and health conditions (e.g. alcohol dependence, liver cirrhosis, cancers, and injuries) and creates substantial social and economic burdens (1,2) . To develop more effective therapeutic strategies, we must first understand how alcohol affects the body at the cellular and molecular level. Previous studies of transcriptomic responses in human alcoholics (3-6) relied on RNA extracted from brain regions using tissue homogenates comprised of multiple cell types. This approach likely masks differences in gene expression patterns among specific cells, as well as heterogeneity from cell-to-cell variation within a given cell type.Single cell RNA sequencing (scRNA-seq) has recently gained attention in cell and molecular biology research for its ability to profile novel cell types and measure cell-to-cell variation in gene expression. To our knowledge, transcriptomic responses to chronic alcohol exposure or any other abused drug have not been studied at the cellular level in the human brain. We hypothesized that cell type-specific gene expression patterns associated with alcoholism will identify novel alcohol targets that were previously missed by bulk analysis of tissue homogenates, as has been shown for other neuropathologies (7,8) . Using single nucleus RNA-seq (snRNA-seq), a popular scRNA-seq alternative for analyzing frozen brain tissue (9-18) , we profiled the transcriptomes of 16,305 nuclei extracted from frozen prefrontal cortex (PFC) samples of 4 control and 3 alcohol dependent individuals. The PFC is involved in executive function and is an important substrate in the reward circuitry associated with development of a...

Increased Toll‐like Receptor‐MyD88‐NFκB‐Proinflammatory neuroimmune signaling in the orbitofrontal cortex of humans with alcohol use disorder

“…In the present study, we report TLR9 co-expression with microglia as well as astrocytes and neurons, consistent with previous studies reporting colocalization of TLR2, TLR3, and TLR4 with neurons in the postmortem human brain (Casula et al, 2011;Crews et al, 2013;Maroso et al, 2010;Zurolo et al, 2011). All TLRs, except TLR3, signal through the TLR adaptor protein MyD88, which is expressed across cell types but is particularly enriched in microglia and astrocytes (Brenner et al, 2020;Soreq et al, 2017). Alcohol releases HMGB1 from neurons and microglia (Crews et al, 2013;Lawrimore & Crews, 2017), and we find HMGB1 is positively correlated with all induced TLRs except TLR3.…”

“…To our knowledge, this is the first study to report upregulation of a broad number of frontal cortical genes involved in HMGB1-TLR-MyD88-NFκB cytokine and chemokine neuroimmune cascades in and extend prefrontal cortex AUD transcriptome studies (Brenner et al, 2020) as well as molecular and histochemical assessments of proinflammatory neuroimmune induction in the AUD brain (Crews et al, 2013;Qin & Crews, 2012b). We report that AUD induces gene expression of HMGB1 as well as multiple TLRs (i.e., TLRs 2-9), but not TLR1.…”

“…The observation that numerous proinflammatory neuroimmune genes are unaltered following microglial depletion in vivo suggests that astrocytes and neurons express higher levels of neuroimmune mediators than previously known. Indeed, astrocytes and neurons are emerging as important contributors to neuroimmune responses (Erickson et al, 2021;Lawrimore et al, 2019;Lawrimore & Crews, 2017;Moffat & Ron, 2021) and a recent cell type-specific transcriptome study in the postmortem human brain revealed unique neuroimmune gene signatures across glia and neurons with robust changes in astrocytes (Brenner et al, 2020;Erickson et al, 2019a;Soreq et al, 2017). While HMGB1 is enriched across all cell types, TLR2, TLR7, and TLR9 are particularly enriched in microglia at baseline, whereas TLR3 is enriched in astrocytes and the remaining TLRs are modestly expressed across glia and neurons (Soreq et al, 2017).…”

Confronting false discoveries in single-cell differential expression

“…Brenner et al, 2020 79 . snRNA-seq data from the prefrontal cortex of alcoholic and control individuals was obtained from GEO (GSE141552).…”

Extracellular microvesicles promote microglia‐mediated pro‐inflammatory responses to ethanol

“…Multiple studies have identified a role of immune signaling in AUD pathology. Transcriptomic studies in both human postmortem brain and chronic ethanol treatments in mice find induction and dysregulation of pro-inflammatory signaling pathways (Brenner et al, 2019;McCarthy et al, 2018). This pro-inflammatory activation might contribute to cellular and synaptic pathology as well as behavioral phenotypes associated with AUD (Warden et al, 2020).…”