Effects of the cannabinoid receptor antagonist hemopressin on amyloid-β induced toxicity in mouse Neuro-2a neuroblastoma cells

S. J. Werner, S. W. Martin, A. W. Paterson, N. G. Milton. School of Clinical & Applied Sciences, Leeds Beckett University, Leeds, United Kingdom.

Introduction: Pepcans are a group of peptide based cannabinoids which are haemoglobin derived. They act as cannabinoid (CB) receptor antagonists/agonists and are found throughout the CNS¹. The pepcan hemopressin (Hp) is a nonapeptide antagonist of the CB₁ receptor¹. Studies have suggested that cannabinoid antagonists, such as the synthetic CB₁ antagonist AM251, have memory enhancing activities following brain trauma². The levels of amyloid-β (Aβ) are elevated during brain trauma and may contribute to tissue damage³. Previous in vitro studies have demonstrated the protective properties of CB₁ receptor ligands against Aβ induced neurotoxicity⁴. The aim of this study was to determine the effects of Hp on Aβ toxicity.

Method: This study employed MTT cell viability assays to investigate the effects of the peptide CB₁ antagonist Hp, the peptide CB₁ agonist RVD, the lipid CB agonist 2-arachidonoylglycerol (2-AG), the synthetic CB agonist O-2545, the synthetic CB₁ antagonist AM251, the synthetic CB₂ antagonist AM630 plus the synthetic GPR-55 antagonist CID16020046 (10μM for each drug), on 10μM Aβ 25-35 induced neurotoxicity in mouse neuroblastoma Neuro-2a (N2a) cells. Data were analyzed by one-way analysis of variance (ANOVA).

Results: Both Hp and 2-AG had a significant protective effect (P<0.05) on Aβ 25-35 induced neurotoxicity in N2a cells. The RVD, O-2545 and AM251 had no effect on Aβ 25-35 induced toxicity in N2a cells (Fig 1A). The RVD, AM251, AM630 and CID16020046 had no effect on Hp protection against Aβ 25-35 induced toxicity in N2a cells (Fig 1B).

Figure 1. (A) Effect of Aβ 25-35 plus Hp, RVD, 2-AG, O-2545 or AM251 on MTT reduction in N2a cells. (B) Effect of Aβ 25-35 with Hp alone or plus RVD, AM251, AM630 or CID16020046 on MTT reduction in N2a cells. Results are mean ± SEM (n=8 for each data point); * = P< 0.05 vs control; † = P<0.05 vs Aβ 25-35 alone; (one-way ANOVA).

Conclusion: In conclusion, the cannabinoid antagonist Hp is protective against Aβ 25-35 induced neurotoxicity in N2a cells. The Hp protection is not antagonised by RVD or the CB₁, CB₂ or GRP-55 antagonists suggesting either an action via a non-cannabinoid receptor or that Hp is acting allosterically at an alternate binding site within a CB receptor.

References:

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(2) Sunada H et al. (2017). J Exp Biol. 220:3026-3038.

(3) Edwards G et al. (2017). Biochem Biophys Res Commun. 483: 1137-1142.

(4) Milton NGN (2002). Neurosci Letts 332: 127-130.