Epigenetic modulation of in vitro neurodifferentiation of NG108-15 cells by the synthetic cannabinoid THJ-2201

Background: The increasing use of synthetic cannabinoids (SCs) among women of reproductive age, including breastfeeding and pregnant women, poses a major public health concern, as it may contribute to the onset of neurodevelopmental disorders in their offspring. We previously observed that THJ-2201 (THJ), a frequently used SC, increased neurite outgrowth in NG108-15 neuroblastoma x glioma cells via CB1 receptor activation. As epigenetic signaling critically controls neurodifferentiation, we herein assessed whether THJ-induced changes in neurite outgrowth are linked to changes in global DNA methylation and histone acetylation. 
Methods: NG108-15 cell differentiation was induced in serum-starved (1% FBS) cell culture medium supplemented with 10mM retinoic acid and 30mM forskolin. Global DNA methylation (i.e., % 5-methylCytosine, %5-mC), and total histone H3 and H4 acetylation were assessed using commercially available colorimetric and fluorometric kits (Abcam), respectively, in 3 distinct exposure settings: A) single THJ addition at the start of differentiation (d0), sample collection on day 3 (d3); B) single THJ addition at d3, sample collection on day 6 (d6); C) THJ additions at d0 and d3, sample collection at d6. THJ was tested at 1pM, 1nM, and 1µM. In a set of assays, 500nM SR141716A, a selective CB1 receptor antagonist, was added 20 min before THJ to assess CB1’s involvement in DNA methylation. DNA Methyltransferase (DNMT) and Ten-Eleven Translocation (TET) enzyme activities were measured with commercially available fluorometric (Abcam) and colorimetric (Epigentek) kits, respectively.
Results: THJ decreased global DNA methylation by 48.5% (compared to vehicle control) at 1pM in setting B, and by 43%-60% (compared to control) at all tested concentrations in setting C. These changes seemed to be independent of CB1 activation, as they were not reversed by the CB1 antagonist. Interestingly, such a decrease in global DNA methylation was associated with increased TET activity (suggesting increased demethylation), but not a decreased DNMT activity. Notably, THJ did not seem to alter total histone H3 (n=4), or H4 (n=2) acetylation.
Conclusions: Overall, the stimulation of THJ-induced neurite outgrowth in NG108-15 cells was shown to be accompanied by a reduction in global DNA methylation, triggered by the TET-mediated oxidation of 5-mC. These findings suggest that this SC regulates gene expression, by activating genes whose transcription would be otherwise repressed. However, further investigations are warranted to delve into the specific genes that can be affected by THJ-induced modulation of neurogenesis.
Acknowledgments: This work was supported by Fundação para a Ciência e Tecnologia (FTC) through projects UIDP/04378/2020 and UIDB/04378/2020 (UCIBIO), and LA/P/0140/2020 (I4HB). CPT and JPS also acknowledge FCT for the PhD grant 2022.13857.BD and research contract (under Scientific Employment Stimulus) 2021.01789.CEECIND/CP1662/CT0014, respectively.