The psychoactive drug of abuse mephedrone differentially disrupts blood-brain barrier properties

Abstract

Background: Synthetic cathinones are a category of psychostimulants belonging to the growing number of designer drugs also known as "Novel Psychoactive Substances" (NPS). In recent years, NPS have gained popularity in the recreational drug market due to their amphetamine-like stimulant effects, low cost, ease of availability, and lack of detection by conventional toxicology screening. All these factors have led to an increase in NPS substance abuse among the young adults, followed by spike of overdose-related fatalities and adverse effects, severe neurotoxicity, and cerebral vascular complications. Much remains unknown about how synthetic cathinones negatively affect the CNS and the status of the blood-brain barrier (BBB).

Methods: We used in vitro models of the BBB and primary human brain microvascular endothelial cells (hBMVEC) to investigate the effects of the synthetic cathinone, 4-methyl methcathinone (mephedrone), on BBB properties.

Results: We showed that mephedrone exposure resulted in the loss of barrier properties and endothelial dysfunction of primary hBMVEC. Increased permeability and decreased transendothelial electrical resistance of the endothelial barrier were attributed to changes in key proteins involved in the tight junction formation. Elevated expression of matrix metalloproteinases, angiogenic growth factors, and inflammatory cytokines can be explained by TLR-4-dependent activation of NF-κB signaling.

Conclusions: In this first characterization of the effects of a synthetic cathinone on human brain endothelial cells, it appears clear that mephedrone-induced damage of the BBB is not limited by the disruption of the barrier properties but also include endothelial activation and inflammation. This may especially be important in comorbid situations of mephedrone abuse and HIV-1 infections. In this context, mephedrone could negatively affect HIV-1 neuroinvasion and NeuroAIDS progression.

Keywords: BBB; Cathinones; Mephedrone; Neuroinflammation; Substance abuse.

Fig. 1

Mephedrone does not affect cell growth and viability of hBMVEC. a Treatments (96 h) with 10 μM or 100 μM mephedrone did not cause significant toxicity or cell death compared with untreated controls. Saponin (sap) was used as a positive control. Data is presented as the mean number of live cells ± SEM. Experiments were independently performed three times. Within each individual experimental set, primary cells from 3 different donors were used (n = 9). b Curves represent cell proliferation at 0 h through 120 h. Persistent exposure to 10 μM mephedrone did not change rate of cell growth. Average number of live cells was determined using Cell Viability Assay (Promega). Data is presented as the mean number of live cells ± SEM. Experiments were independently performed three times. Within each individual experimental set, primary cells from 3 different donors were used (n = 9)

Fig. 2

Mephedrone compromises barrier properties of hBMVEC. a Barrier electrical resistance was modelled based on continuous cell-substrate impedance readings recorded at 6 frequencies (400 Hz–48 kHz) every 6 min for the duration of the time shown. Endothelial monolayers were treated with 1 μM, 5 μM, or 10 μM mephedrone or left untreated to serve as a baseline. Treatments were initiated at 0 timepoint. Each data point is represented as the percentage of the mean value ± SEM. Experiments were independently performed three times. Within each individual experimental set, primary cells from four different donors were used (n = 12). b–d Barrier permeability of small molecular tracer was modelled using FITC-conjugated dextrans (3 kDa) of various charges: neutral (b), anionic (c), and cationic (d). Endothelial monolayers were treated with 10 μM mephedrone, 100 ng/mL TNFα, or left untreated to serve as a baseline. Each data point is represented as the apparent permeability coefficient (P_app, mean value ± SEM). Experiments were independently performed three times. Within each individual experimental set, primary cells from 6 different donors were used (n = 18)

Fig. 3

Mephedrone affects tight junction protein expression and localization in hBMVEC. a Representative immunoblots of occludin, claudin-1, and claudin-5 from cytosolic and membrane fractions of endothelial cells treated with 10 μM mephedrone for 24 h. b The plots represent densitometry analysis of the target band intensity normalized to the GAPDH (cytosolic fraction) or NaK-ATPase (membrane fraction) band intensities and relative to the untreated control. The results are shown as the percentage of the mean value ± SEM. Experiments were independently performed three times. Within each individual experimental set, primary cells from four different donors were used (n = 12)

Fig. 4

Mephedrone induces inflammatory activation of hBMVEC. a–c hBMVECs were treated with 10 μM mephedrone for 2 h and analyzed by flow cytometry. Histograms show the expression of adhesion molecules PECAM-1, ICAM-1, and VCAM-1 in response to mephedrone, which was statistically significant only for PECAM-1 at 2 h. Experiments were independently performed three times. Within each individual experimental set, primary cells from three different donors were used (n = 9). d hBMVEC were treated with 10 μM mephedrone for 24 h, and levels of cytokines were analyzed by V-PLEX assay. Levels of IL-1β, IL-2, IL-8, IL-13, and TNFα were significantly upregulated by the treatment. Data shown as the mean concentration ± SEM expressed in pg/mL. Experiments were independently performed three times. Within each individual experimental set, primary cells from three different donors were used (n = 9). e TIMP-1, MMP3, MMP9, and MMP12 mRNA expression in hBMVEC treated with 10 μM mephedrone for 4, 6, or 24 h were determined using qRT-PCR. Data shown as fold change relative to untreated control and normalized to the housekeeping gene (18S). Experiments were independently performed three times. Within each individual experimental set, primary cells from three different donors were used (n = 9). f MMP9 enzyme activity in the culture medium of endothelial cells treated with 10 μM mephedrone for 24 h were determined using Human Active MMP9 Fluorokine assay. Data shown as mean ± SEM and expressed in pg/mL of active enzyme. Experiments were independently performed three times. Within each individual experimental set, primary cells from three different donors were used (n = 9)

Fig. 5

Mephedrone induces angiogenic mechanisms. ELISA was used to measure the concentrations of secreted angiogenic growth factors: PDGF-BB (a) and VEGF-A (b) in cell culture medium of hBMVEC treated with 10 μM mephedrone for 24 h. Data shown as the mean concentration ± SEM expressed in pg/mL. Experiments were independently performed three times. Within each individual experimental set, primary cells from three different donors were used (n = 9)

Fig. 6

Mephedrone activates NF-κB signaling pathway. a TransAM Transcription Factor Activation Assay was used to measure concentrations of activated NF-κB subunits in the nuclear fractions of endothelial cells treated with 10 μM mephedrone for 24 h. Mephedrone significantly upregulates the level of nuclear p65 subunit. Data shown as mean ± SEM. Experiments were independently performed three times. Within each individual experimental set, primary cells from three different donors were used (n = 9). b Representative image of p65 nuclear translocation in endothelial cells. Scale bar is 10 μm. Representative immunoblot (c) and densitometry analysis (d) of TLR4 expression in cytosolic and membrane fractions of hBMVEC. Data shown as mean ± SEM. Experiments were independently performed three times. Within each individual experimental set, primary cells from three different donors were used (n = 9). e TransAM NF-κB p65 Activation Assay was used to measure concentrations of activated p65 in the nuclear fraction of endothelial cells treated with 10 μM mephedrone alone or pre-treated with 100 nM TAK242 for 3 h. TAK242 significantly downregulates the amount of mephedrone-activated p65 subunit. Data shown as mean ± SEM. Experiments were independently performed three times. Within each individual experimental set, primary cells from three different donors were used (n = 9)