Elsevier

Molecular Brain Research

Volume 134, Issue 2, 4 April 2005, Pages 215-225
Molecular Brain Research

Research report
Protective effects of Δ9-tetrahydrocannabinol against N-methyl-d-aspartate-induced AF5 cell death

https://doi.org/10.1016/j.molbrainres.2004.10.044Get rights and content

Abstract

The neuroprotective effects of Δ9-tetrahydrocannabinol (THC) were examined using an in vitro model in which the AF5 CNS cell line was exposed to toxic levels of N-methyl-d-aspartate (NMDA), an agonist of the NMDA glutamate receptor. NMDA toxicity was reduced by THC, but not by the more specific cannabinoid receptor agonist, WIN55,212-2. Addition of dibutyryl cAMP (dbcAMP) to the culture medium did not alter the neuroprotective effect of THC and did not unmask a neuroprotective effect of WIN55,212-2. The cannabinoid antagonist SR141716A did not inhibit the neuroprotection induced by THC or alter the response to WIN55,212-2, even in the presence of dbcAMP, indicating that the neuroprotective effect of THC was cannabinoid receptor-independent. On the other hand, both THC and WIN55,212-2 produced cellular toxicology at higher dosages, an effect which was blocked in part by SR141716A. Capsaicin, an antioxidant and vanilloid receptor agonist, also produced a protective effect against NMDA toxicology. The protective effect of capsaicin was blocked by co-application of ruthenium red, but was not blocked by the specific vanilloid receptor antagonist capsazepine, and the transient receptor potential vanilloid type 1 (TRPV1) and ANKTM1 transcripts were not detected in AF5 cells. Thus, the neuroprotective effects of THC and capsaicin did not appear to be mediated by TRP ion channel family receptors. The antioxidant α-tocopherol prevented neurotoxicity in a dose-dependent manner. Therefore, THC may function as an antioxidant to increase cell survival in NMDA-induced neurotoxicity in the AF5 cell model, while higher dosages produce toxicity mediated by CB1 receptor stimulation.

Introduction

Cannabinoids, such as the pharmacologically active component of marijuana (−)Δ9-tetrahydrocannabinol (THC), and the endogenous cannabinoid anandamide, exert a variety of biological actions which are mediated by activation of cannabinoid (CB) receptors [15], [23], receptors of the transient receptor potential (TRP) family of ion channels [7], [16], and antioxidant effects which are not mediated by receptors [2], [10], [11].

One of the most promising potential medical applications of cannabinoids involves their ability to protect cells from a variety of toxic events [8]. Cannabinoids have been reported to protect neurons from death caused by glutamatergic overstimulation, ischemia, and oxidative damage [13]. The mechanisms of these cellular protectant effects are not, however, entirely clear. On one hand, there are several studies which suggest that for cell death induced by glutamate, hydrogen peroxide, retinoids, and serum deprivation, the protectant actions of THC are mediated primarily or exclusively by chemical antioxidant effects [2], [10], [19]. For example, Hampson et al. [10] reported that cell death in neuronal cultures induced by stimulation of the AMPA/kainate glutamate receptor was similarly inhibited by THC and by cannabidiol, which does not stimulate CB1 receptors, and that the CB1 antagonist SR141716A did not inhibit the protective effect of either THC or cannabidiol. Marsicano et al. [19] found that alteration of CB1 receptor expression using knockout animals, or gene transfer in the HT22 cell line, did not influence the protectant effect of THC against hydrogen peroxide toxicity in vitro.

On the other hand, THC and the synthetic cannabinoid WIN55,212-2, which does not have an antioxidant effect [2], [10], [19], prevent glutamate- and NMDA-induced neurotoxicity in isolated neurons [26] as well as in the brain [21] via activation of the cannabinoid CB1 receptor [6], [31]. Moreover, El-Remessy et al. [4] investigated the neuroprotective effect of THC in a model of NMDA-induced retinal toxicity. In this model, the effect of THC was partially, although not completely, blocked by the CB1 antagonist SR141716A. Therefore, it appears that in some circumstances, THC exerts neuroprotective effects exclusively through non-receptor-mediated antioxidant properties, while in other models or other cell types, stimulation of CB1 receptors by THC or WIN55212-2 can produce neuroprotection. Differences between effects observed in different models may be related to the cell type or model system, and to differences in the toxic events which have been employed.

The purpose of the present study was to further explore the mechanisms of neuroprotection induced by THC, using a neural progenitor cell line model [24], [28]. The AF5 cell line retains its plasticity in culture and possesses some of the characteristics of primary mesencephalic neural progenitors [29]. In the present study, the AF5 cell line was examined for expression of NMDA and CB1 receptors and susceptibility to toxicity mediated by NMDA. NMDA was employed as a toxic agent since most of the studies which have found CB1 receptor-mediated neuroprotection, mentioned above, have employed NMDA-induced toxicity. This model was then employed to assess the neuroprotectant properties of THC and the antioxidants α-tocopherol and capsaicin.

Section snippets

Chemicals and materials

N-methyl-d-aspartate (NMDA), N-methyl-l-aspartate (NMLA), Δ9-THC, Hoechst 33342, Ruthenium red, and α-tocopherol were obtained from Sigma (St. Louis, MO). (R)-(+) WIN55,212-2 mesylate salt, (+)-MK-801, Capsazepine, and (E)-Capsaicin were from Tocris Cookson (Ellisville, MO). SR141716A was obtained from the National Institute on Drug Abuse drug supply system. Dibutyryl cAMP (dbcAMP) was from Calbiochem (San Diego, CA). The MTT cell proliferation assay kit was purchased from ATCC (Manassas, VA).

NMDA receptor expression and NMDA-induced cell death

AF5 cells were found to express the NMDA receptor using immunocytochemistry, which showed that the NMDA1 receptor protein was present in AF5 cell bodies (Fig. 1A).

Exposure of AF5 cells to 5 to 10 mM NMDA reduced cell viability in a dose-dependent manner, as measured by the MTT assay (Fig. 1B). At 7.5 mM NMDA, cell survival was reduced to approximately 40–50% of vehicle-treated cells. Only about 20% of cells survived following exposure to 10 mM NMDA. Co-treatment with 10 μM MK-801, a selective

Discussion

The present study demonstrates that the AF5 cell line can be employed to investigate the effects of neuroprotectants against NMDA-induced neurotoxicity. The AF5 cell line expresses functional NMDA1 receptors and is vulnerable to toxicity induced by exposure to NMDA in high concentrations. NMDA-induced toxicity was blocked by the NMDA antagonist MK-801, and the inactive stereoisomer of NMDA, N-methyl-l-aspartate, was inactive. These results suggest that NMDA-induced cell death in AF5 cells is

References (34)

  • M.E. Truckenmiller et al.

    AF5, a CNS cell line immortalized with an n-terminal fragment of SV40 large T: growth, differentiation, genetic stability and gene expression

    Exp. Neurol.

    (2002)
  • E.L. Watson et al.

    Ca 2+-activated membrane ATPase: selective inhibition by ruthenium red

    Biochim. Biophys. Acta

    (1971)
  • G.C. Chan et al.

    Hippocampal neurotoxicity of Delta9-tetrahydrocannabinol

    J. Neurosci.

    (1998)
  • Y. Chen et al.

    Cannabinoids protect cells from oxidative cell death: a receptor-independent mechanism

    J. Pharmacol. Exp. Ther.

    (2000)
  • J.M. Dubinsky et al.

    Intracellular calcium concentrations during “chemical hypoxia” and excitotoxic neuronal injury

    J. Neurosci.

    (1991)
  • M. Guzman et al.

    Control of the cell survival/death decision by cannabinoids

    J. Mol. Med.

    (2001)
  • M.G. Hamilton et al.

    Effect of ruthenium red on voltage-sensitive Ca++ channels

    J. Pharmacol. Exp. Ther.

    (1995)

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