The potentially deleterious effects of CBD on the immature neural cells have not been deeply studied nor well reproduced by in vitro assays. In this study, we analyzed the exposure of NPCs to CBD in different concentrations and evaluated the effects on cell morphology, viability, proliferation, differentiation, death, migration, and gene expression. Our data indicated that high CBD concentrations showed increased cytotoxicity and reduced cell viability in NPCs compared to the control condition in both 2D and 3D cell culture methods.
Our data corroborated the study by Brvar´s group in 2022, as they also observed cytotoxicity in primary cortical neurons and astrocyte cultures exposed to increased concentrations of CBD (0.1, 0.5, 1, and 5 µM)(Jurič et al. 2022). The authors observed a reduction in metabolic activity in neurons exposed to 0.1 µM CBD, and in astrocytes exposed to 0.5 µM CBD (Jurič et al. 2022), while in our neurospheres cell viability began to decrease at 5 µM dose of CBD. In another study, primary cerebellar granule neurons obtained from newborn rats were exposed to CBD in different doses. A high dose of CBD (10 µM) evoked a reduction in cell viability, although, in lower doses, no significant effects on cell viability was observed (Echeverry et al. 2021).
High concentration of CBD decreases cell viability in NPC not only in cells from rodents. Miranda e collaborators observed a massive cell death when CBD was added at 10 µM to neuronal cells differentiated from human induced pluripotent stem cells (hiPSC) lines. In addition, the authors reported that CBD at 1 µM concentration does not promote or inhibit the neuronal differentiation (Miranda et al. 2020). Likewise, our study also found no differences in cell viability and neural differentiation when 1 µM CBD was added in NPC cultures; however, previous exposure to CBD at 5 µM affected neural differentiation into GFAP+ cells and decreased the growth of neurites in βtubulin-III + cells. Interestingly, in one in vivo experiment in mice, CBD showed a dose-dependent effect. Schiavon and collaborators showed that repeated chronic higher doses of CBD (30 mg/kg) in male mice decreased proliferation and neurogenesis, whereas lower dose (3 mg/kg) showed opposite effects (Schiavon et al. 2016). We can suggest that the effect of CBD on cell viability is dose-dependent, and the effective dose range for CBD action should be narrow.
Similarly, a possible mechanism behind the increased toxicity of high CBD concentrations in our NPC seems to be the rise of intracellular Ca2+ in the mitochondria. The presence of high calcium levels directly impacts the ATP synthase and the activity of Krebs enzymes (Mato, Sánchez-Gómez, and Matute 2010; Kafkova et al. 2023); resulting in inhibited mitochondria consuming more ATP and compromising the cell energetic supply, which may finally lead to autophagy and cell death (Tsujimoto 1997; Dando et al. 2013). We hypothesize that the raised intramitochondrial Ca2+ establishes the formation of the mitochondrial permeability transition pore, that leads to severe oxidative stress, mitochondrial dysfunction, depolarization of the inner mitochondrial membrane, and the release of Cyt-c and Ca2+ into the cytosol (Olivas-Aguirre et al. 2019).
A dynamic metabolic shift in the NPCs was also observed in our data in which, at higher doses of CBD (5 µM), a decrease in glycolysis and an increase in β-oxidation were favored, mainly from the early to the final stages of metabolic pathways. Enzymes such as hexokinase and pyruvate kinase play an essential role in the glycolytic pathway as both enzymes are allosterically regulated, which means they can regulate glycolysis (Ishfaq et al. 2022). Therefore, the decrease of HK2 and PKM2 expression observed in our study at higher concentrations of CBD can be interpreted as the allosteric regulation of both enzymes due to the lower presence of glucose. Some studies have shown that the downregulation of PKM2 and HK2 can inhibit proliferation and promote apoptosis (Li et al. 2018; Zhang et al. 2019). Additionally, the downregulation of PKM2 decreases the production of lactic acid, implying that the flow of glucose is shifter to the pentose phosphate pathway to ensure cell survival (Li et al. 2018). Likewise, Zang and collaborators observed that the downregulation of HK2 can inhibit cell proliferation and colony formation and promote apoptosis (Zhang et al. 2019).
Considering our results on colony formation with the different CBD concentrations, we may speculate that the reduced size of the colony at high CBD concentrations (5 and 10 µM) compared to the CTRL and CBD-1µM groups may also be related to the downregulation of HK2 and PKM2 enzymes. Lum and collaborators found that when the energy supply is limited, the activation of a cell survival response is related to autophagy; the catabolic pathway digests cellular organelles and macromolecules to generate sufficient energy (Lum et al. 2005).
Our study showed that the metabolic shift was reversed in the N2a cell lineage compared with the NPCs. During the initial stages, the mitochondrial β-oxidation was reduced, whereas the glycolysis increased during the final step of cell metabolism. In the β-oxidation mechanism energy for the cells is generated via acetyl Co-A (Griffin and Ackerman 2020). The first and rate limiting enzyme in peroxisomal β-oxidation of the very-long-chain fatty acid (VLCFAs) is the acyl-CoA oxidase 1 (ACOX1). It has been observed that loss-of-function mutations in ACOX1 lead to neurodegeneration. This is often associated with exaggerated reflexes, developmental regression, visual and hearing loss, and death (Griffin and Ackerman 2020). Additionally, severe peroxisome biogenesis disorders have been linked to the neuronal migration defect and elevated levels of VLCFA that disrupt the physicochemical properties of membranes are indications of these peroxisome disorders (Krysko et al. 2010). Based on these findings, it appears that CBD may interfere with the global metabolic dynamics, as both metabolic shifts are present in different cell types.
Considering the reduced cellular migration observed in the astrocytes at low doses of CBD, cell migration was only affected after higher doses, These data clearly reinforce the Brvar group´s statement that CBD is not only concentration-dependent but also cell-specific (Jurič et al. 2022), showing that astrocytes are more sensitive to CBD effects than neurons. Astrocytes express pattern recognition receptors on their surface to detect signals from other cells and environmental conditions, making them sensible to very subtle changes (Egaña-Huguet, Soria-Gómez, and Grandes 2021), and it seems reasonable that this would be the reason for the reduced migration pattern at low CBD dose.
Regarding the cellular phenotypes of the positive GFAP cells, we observed that the exposure to CBD before differentiation revealed qualitative differences as shortened and thickened extensions were observed in CBD 5 µM and 10 µM groups. In contrast, longer branch points were observed in the control and CBD 1 µM groups. One possible interpretation is that astrocytes respond rapidly to the surrounding stimuli such as osmotic stimulation and stress, leading to neuroprotective or neurotoxic changes in their phenotype and molecular function (Afridi et al. 2020; Zhou, Zuo, and Jiang 2019). Astrocytes and microglia are the primary mediators of “reactive gliosis,” a response of glial cells to injury or other pathological processes (Kozela, Juknat, and Vogel 2017), such as the increased intracellular Ca2+ in our case. Reactive astrocytes are characterized by high-level expression of GFAP, and their alterations vary with the nature and severity of the insult. Inflammatory processes and modest metabolic stimuli induce moderate reactive astrogliosis characterized by changes in the molecular expression of pro-inflammatory cytokines and cellular hypertrophy (Acaz-Fonseca et al. 2019).
In conclusion, we demonstrate that CBD at higher concentrations is toxic to neural primordial cells in two different systems (2D and 3D), inducing significant alteration in function, gene expression of metabolic proteins, and morphological changes in neurons and astrocytes. Higher doses of CBD switch cellular metabolism, affecting multiple cellular functions, which is even more critical during neurodevelopment; differentiation, mobility, cell death, and cell viability are negatively impacted. Thus, the toxicity of CBD at different doses should be further investigated for deleterious effects, and CBD extracts should be recommended with prudence during brain development.