Human-relevant concentrations of synthetic cannabinoid THJ-2201 alter mitochondrial dynamics during neurodifferentiation
Background: THJ-2201 is a potent synthetic cannabinoid (SC) linked to acute intoxications and fatalities. Of particular concern is its potential use by women of child-bearing age or those who are pregnant/lactating, as it poses a heightened risk of inducing neurodevelopmental disorders in their offspring. Our previous research has revealed that THJ-2201 enhances neurite outgrowth in NG108-15 neuroblastoma x glioma cells via CB1 receptor activation, concurrently leading to a decrease in intracellular ATP levels and mitochondrial membrane potential. Based on these findings, we postulate that this SC could interfere with cellular mechanisms related to mitochondrial dynamics during neurodifferentiation.
Methods: To address this hypothesis, NG108-15 cells were differentiated in serum-starved (1% fetal bovine serum) cell culture medium, supplemented with 10 µM forskolin and 30 µM retinoic acid. THJ-2201 was then added, at non-toxic, in vivo-relevant concentrations (1 pM – 1 µM). The expression of mitochondrial markers, including mitochondrial mass (e.g., voltage-gated anion channel, VDAC), biogenesis (e.g., PGC-1α), fusion (e.g., OPA1, MNF2), and fission (e.g., DRP1), was assessed following 24 and 72h through Western blot analysis on total cell extracts.
Results: We observed that cells exposed for 72h to 1 µM THJ-2201 did not sustain the increase in VDAC levels observed in differentiating cells between 24 and 72h. Instead, there was a notable nearly 25% reduction in VDAC levels (compared to the control at 72h). PGC-1α expression also decreased under this condition, suggesting a downregulation in the biogenesis pathway and a reduction in mitochondrial turnover. Concurrently, the levels of DRP1, MFN2 and OPA1 decreased by approximately 40-50% following exposure to THJ-2201, indicating a diminished fusion/fission dynamic. Importantly, no significant changes in these endpoints were observed at an earlier stage of NG108-15 cell differentiation (24H).
Conclusion: Our data underscore the disruption of mitochondrial dynamics processes induced by THJ-2201 during the differentiation of NG108-15 cells. Such effects may potentially compromise mitochondrial physiology (e.g. membrane potential) and function efficiency (e.g. ATP production), crucial to neurodifferentiation. Nevertheless, further research is required to comprehensively understand the mechanisms involved.
Acknowledgements: This work was funded by FEDER and by national funds from Fundação para a Ciência e a Tecnologia (FCT) in the scope of the project NeuroSCANN (POCI-01-0145-FEDER-029584) and the grants UIDB/04378/2020 and UIDP/04378/2020 (UCIBIO) and LA/P/0140/2020 (i4HB). RFM and JPS acknowledge FCT for PhD grant 2020.07135.BD and research contract (under Scientific Employment Stimulus) 2021.01789.CEECIND/CP1662/CT0014, respectively.
