The Synthetic Cannabinoids ADB-FUBINACA Modulate Mitochondrial Function and Dynamics at Biologically Relevant Concentrations During In Vitro Neurodifferentiation

Abstract

Synthetic cannabinoids (SCs) pose a significant risk to neurodevelopment, as they may disrupt the proper brain development by interfering with the endocannabinoid system. Previously, we demonstrated that the SC ADB-FUBINACA (ADB) enhances neurodifferentiation of NG108-15 neuroblastoma x glioma hybrid cells via CB1 receptor activation. Interestingly, the influence of mitochondria on cellular homeostatic responses has emerged as a central regulator of neural stem cell fate. Thus, here we aimed to evaluate the effects of this SC on mitochondrial function and dynamics during in vitro neurodifferentiation. NG108-15 cells were differentiated in serum-starved (1% FBS) cell culture medium supplemented with 30µM retinoic acid and 10µM forskolin. ADB was added once at the beginning of differentiation at in vivo relevant concentration (between 1 pM and 1 µM). Mitochondrial membrane potential (assessed by TMRE labelling) and intracellular ATP levels (luciferase-based luminescence assay) were evaluated after 24h and 72h. Specific cell-permeable or cell-impermeable CB1R antagonists/ inverse agonists (SR141716A and hemopressin, respectively) were added 20 minutes prior to ADB exposure to assess the role of CB1R in the observed effects. The expression of the mitochondrial fission marker dynamin-related protein 1 (DRP1), fusion marker mitochondrial dynamin-like GTPase (OPA1), and Voltage-dependent anion channel (VDAC) was analyzed by Western-blot at 24 or 72h. ADB (1pM and 1nM) reduced intracellular ATP levels by approximately 30-35% at 24h, which returned to control levels after 72h. These effects were mediated by CB1R signaling, as they were prevented by both SR141716A and hemopressin. Reduced intracellular TMRE retention by around 1.2-1.3-fold was observed for all concentrations tested at 72h, but this effect was not blocked by CB1R antagonists. Notably, while the higher concentration of ADB (1 µM) increased DRP1 levels around 1.6-fold at 24h, the levels of OPA1 and VDAC, an indirect marker of mitochondrial mass, decreased by 1.6 to 2.1-fold at 72h after exposure to 1nM and 1 µM. Overall, ADB seems to disrupt both mitochondrial function and dynamics during the neurodifferentiation process of NG108-15 cells. Different mechanisms seem to underlie mitochondrial function-related effects, as only the modulation of energy supply was dependent on CB1R activation. However, further research is thus required to better understand the mechanisms underlying cannabinoids’ modulation of mitochondrial activity and their role in the SCs-induced enhancement of neurodifferentiation.