Crosstalk between autophagic intermediaries in the event of neurotoxic effects mediated by Synthetic Cannabinoids in NG108-15 cells

Synthetic Cannabinoids (SCs) are substantially more potent than cannabis and often associated with severe cases of intoxications and deaths. Although the brain is one of the main target organs for these substances, there is scarce information regarding their neuronal toxicological mechanisms. This work explored the neurotoxicity of 14 SCs, with a particular emphasis on the role played by autophagy.

NG108-15 hybridoma x glioma hybrid cells were exposed to each of the 14 SCs (AMB-FUBINACA, AB-PINACA, MDMB-CHMICA, AB-CHMINACA, ADB-FUBINACA, 5F-AMB, AB-FUBINACA, BZ-2201, X-PB-22F, 5F-PB22, SDB-006, JWH-122, THJ-2201, XLR-11) for 24h, at 1nM and 1μM, in the presence or absence of 0.5µM SR141716A, a CB1r antagonist. Several parameters were analysed, including cell viability (MTT reduction assay), mitochondrial membrane potential (MMP; TMRE labelling), activation of caspase-3, and the occurrence of autophagy (autophagic flux through flow cytometry; and expression of ATG5, Beclin-1, Rab7A, LC3, and ubiquitin by Western Blot).

Viability of NG108-15 cells was affected by the studied SCs in the following order of potency: AB-CHMINACA > ADB-FUBINACA > MDMB-CHMICA > AMB-FUBINACA > X-PB-22F > AB-FUBINACA > JWH-122 > AB-PINACA > FUBIMINA > THJ-2201 > 5F-PB22 > XLR-11 (MTT EC50 values varying from 37.33μM to 1.03mM; no cytotoxicity observed for 5F-AMB and SDB-006 up to 2mM). 5F-PB22 activated caspase-3 (p<0.05) and increased MMP (p<0.0001), at 1μM. FUBIMINA (p<0.0001) and XLR-11 (p<0.01) also significantly increased MMP at 1nM (these were CB1r-independent effects). All but ADB-FUBINACA, THJ-2201, and XLR-11 increased the number of autophagosomes. Preliminary data consistently indicated an increase in ATG5 for all tested SCs, and in Beclin-1 for all except AB-CHMINACA (1nM) and 5F-AMB (1nM). Increases in Rab7A were observed for AB-FUBINACA (1 µM), AB-PINACA (1nM), AB-CHMINACA (1nM) and SDB-006 (1 µM); and in LC3 for all SCs except AMB-FUBINACA (1nM), AB-FUBINACA (1µM) and JWH-122 (1µM). The distinct expression patterns of proteins involved in nucleation (as assessed by Beclin-1), elongation/ maturation (ATG5 and LC3) and fusion (Rab7A) steps, indicate different mechanisms of autophagy triggered by the tested SCs. These distinct data may anticipate the heterogeneity of clinical potency observed in SC intoxication cases. Further investigation is required to explore the mechanisms underlying the SC-elicited neurotoxicity.

Disclosure of Interest Statement: This work was supported by FCT – Fundação para a Ciência e a Tecnologia, I.P. in the scope of the project NeuroSCANN (POCI-01-0145-FEDER-029584) and the grants UIDP/04378/2020 and UIDB/04378/2020 of the UCIBIO – Research Unit on Applied Molecular Biosciences; the project LA/P/0140/2020 of the i4HB – Associate Laboratory Institute for Health and Bioeconomy; and and the PhD grant 2020.04493.BD.