Proteomic Profile of Circulating Extracellular Vesicles in the Brain after Δ9-Tetrahydrocannabinol Inhalation

Abstract

Given the increasing use of cannabis in the US, there is an urgent need to better understand the drug's effects on central signaling mechanisms. Extracellular vesicles (EVs) have been identified as intercellular signaling mediators that contain a variety of cargo, including proteins. Here, we examined whether the main psychoactive component in cannabis, Δ9-tetrahydrocannabinol (THC), alters EV protein signaling dynamics in the brain. We first conducted in vitro studies, which found that THC activates signaling in choroid plexus epithelial cells, resulting in transcriptional upregulation of the cannabinoid 1 receptor and immediate early gene c-fos, in addition to the release of EVs containing RNA cargo. Next, male and female rats were examined for the effects of either acute or chronic exposure to aerosolized ('vaped') THC on circulating brain EVs. Cerebrospinal fluid was extracted from the brain, and EVs were isolated and processed with label-free quantitative proteomic analyses via high-resolution tandem mass spectrometry. Interestingly, circulating EV-localized proteins were differentially expressed based on acute or chronic THC exposure in a sex-specific manner. Taken together, these findings reveal that THC acts in the brain to modulate circulating EV signaling, thereby providing a novel understanding of how exogenous factors can regulate intercellular communication in the brain.

Keywords: THC; cannabis; cerebrospinal fluid; extracellular vesicles; proteomic.

Figure 1

THC-induced changes in c-fos and CB1 receptor mRNA in primary choroid plexus cells and mir-204 in released EVs. Primary epithelial cell culture from the choroid plexus was examined following THC exposure (n = 6 rats). (A) THC induced a significant upregulation of cannabinoid receptor (CB1) mRNA. (B) Cellular activation was evidenced with THC exposure based on increased expression of c-fos mRNA. (C) From the cell culture medium, extracellular vesicles (EVs) were extracted, and THC treatment was found to induce a significant increase in the expression of EV-localized mir-204 as compared to control. ** p < 0.01. Data were normalized based on the expression of β-actin (Cnr1 gene for CB1 and Fos gene for C-fos) or U6 (mir-204). Data represent mean normalized values ± SEM.

Figure 2

Acute THC-induced changes in the proteomic profile of CSF EVs from males. Male rats (n = 12/group) were permitted to inhale aerosolized THC or vehicle across a 1 h session. CSF was collected and pooled from 3 subjects for EV extraction (resulting in 4 samples per treatment group), and then proteomic analysis was performed. (A) The heatmap displays differentially expressed proteins for each sample analyzed per group (p < 0.05). (B) The volcano plot depicts the proteomic alterations within EVs isolated from the CSF based on significance set at p < 0.05. (C) Ingenuity Pathway Analysis reveals the networks governing the biological systems implicated in EV-localized proteins differentially regulated by acute THC vape exposure in male rats.

Figure 3

Chronic THC-induced changes in the proteomic profile of CSF EVs from males. Male rats (n = 12/group) were permitted to inhale aerosolized THC or vehicle across 1 h sessions for 14 consecutive days. CSF was collected and pooled from 3 subjects for EV extraction (resulting in 4 samples per treatment group), and proteomic analysis was performed. (A) The heatmap displays differentially expressed proteins for each sample analyzed per group (p < 0.05). (B) The volcano plot depicts the proteomic alterations within EVs isolated from the CSF based on significance set at p < 0.05. (C) Ingenuity Pathway Analysis reveals the networks governing the biological systems implicated in EV-localized proteins differentially regulated by chronic THC vape exposure in male rats.

Figure 4

Acute THC-induced changes in the proteomic profile of brain EVs from females. Female rats (n = 9–12/group) were permitted to inhale aerosolized THC or vehicle across a 1 h session. CSF was collected and pooled from 3 subjects for EV extraction (resulting in 3–4 samples per treatment group), and then proteomic analysis was performed. (A) The heatmap displays differentially expressed proteins for each sample analyzed per group (p < 0.05). (B) The volcano plot depicts the proteomic alterations within EVs isolated from the CSF based on significance set at p < 0.05. (C) Ingenuity Pathway Analysis reveals the networks governing the biological systems implicated in EV-localized proteins differentially regulated by acute THC vape exposure in female rats.

Figure 5

Chronic THC-induced changes in the proteomic profile of brain EVs from females. Female rats (n = 12/group) were permitted to inhale aerosolized THC or vehicle across 1 h sessions for 14 consecutive days. CSF was collected and pooled from 3 subjects for EV extraction (resulting in 4 samples per treatment group), and proteomic analysis was performed. (A) The heatmap displays differentially expressed proteins for each sample analyzed per group (p < 0.05). (B) The volcano plot depicts the proteomic alterations within EVs isolated from the CSF based on significance set at p < 0.05. (C) Ingenuity Pathway Analysis reveals the networks governing the biological systems implicated in EV-localized proteins differentially regulated by chronic THC vape exposure in female rats.

Figure 6

Pathway analysis for upstream regulators and downstream functions of proteins localized in circulating brain EVs with THC exposure. (A–D) Pathway analysis highlights upstream regulators of select identified proteins localized in EVs within the CSF following either acute THC exposure (males, (A); females, (C)) or chronic THC exposure (males, (B); females, (D)). The symbol and color legend are adapted from https://qiagen.my.salesforce-sites.com/KnowledgeBase/articles/Knowledge/Legend (accessed on 1 July 2024).