Synthetic cannabinoids 5F-PB22 and THJ-2201 promote neuronal differentiation in neureblastoma cells at biologically relevant concentrations

The fast-paced emergence of synthetic cannabinoids (SCBs) - new psychoactive substances (NPS) structurally similar, but substantially more potent than tetrahydrocannabinol (THC) - represents a major challenge for public health and policy-makers in view of the increasing number of SCB-related acute intoxications and deaths. SCB use by pregnant women and women of child-bearing age is especially alarming due to the potential onset of neurodevelopment disorders in the offspring.

Here, we assessed how SCB exposure affects in vitro neuronal development-related processes, like neuronal differentiation and proliferation.

Neuroblastoma (NG108-15) cells were differentiated in serum-starved (1%) cell culture medium supplemented with forskolin and retinoic acid, in the presence/absence of 2 SCBs: 5F-PB-22 and THJ-2201. These were added once (at day 0) or every 24h for 3 days (3 additions), at non-toxic in vivo relevant concentrations (1pM-1nM). Differentiation ratios (number of newly formed neurites per total cell number) and total neurite length were determined. 500 nM SR141716A, a specific CB1 receptor (CB1R) antagonist, was also added prior to SCB exposure in the assays to assess CB1R involvement on SCBs effect on neurodifferentiation. Expression of mature (e.g. MAP-2, synaptophysin) and immature (e.g. ß3-tubulin, p73) neuron markers was evaluated by Western-Blot and/or immunocytochemistry. Cell proliferation was followed up to 72h in the presence of SCBs and measured with the sulforhodamine B (SRB) assay.

NG108-15 cell exposure to 1pM-1nM 5F-PB22 and THJ-2201 resulted in enhanced differentiation ratios (about 2-fold) and total neurite length, compared to cells cultured in standard differentiation medium. Of note, the cells’ metabolic activity (MTT reduction) was not altered as a result of the differentiation process. Interestingly, multiple additions of 5F-PB22 were required to produce similar effects to those obtained by a single dose of THJ-2201. Regulation of such SCB-mediated effects was CB1R activation-dependent, as the selective inhibition of this receptor abrogated the 5F-PB22 and THJ-2201-induced responses. Overexpression of ß3-tubulin and p73, with no significant alteration in the number of MAP-2-positive cells were detected following 5F-PB22 treatment. In contrast, THJ-2201 exposure increased the number of MAP-2-positive cells (at 1pM), with no significant changes in the expression of ß3-Tubulin and decreased p73 expression. Preliminary assessment of synaptophysin expression seem to support this conclusion, as this marker of mature neurons displayed higher expression in THJ-2201-treated cells (especially at 1pM) and no significant changes after 5F-PB22 exposure. These results suggest that neurons differentiated upon THJ-2201 exposure may have reached a more mature state, compared with 5F-PB22-differentiated neurons. In addition, none of the tested SCBs significantly affected neuronal cell proliferation.

Overall, our results evidence the CB1R-mediated enhancement of neuronal cells’ differentiation by 5F-PB22 and THJ-2201 at concentrations below 1nM, with different levels of neuronal maturity attained in response to each SCB. Nevertheless, further research is required to identify the mechanisms involved.