Homocysteine and cytosolic GSH depletion induce apoptosis and oxidative toxicity through cytosolic calcium overload in the hippocampus of aged mice: Involvement of TRPM2 and TRPV1 channels

- Oxidative stress and Ca2+ influx play an important role in functions of the hippocampus.
- Effects of homocysteine (Hcy) on Ca2+ and apoptosis were investigated.
- Hcy induced Ca2+ entry through TRPM2 channel in the hippocampus.
- Glutathione reduced Hcy-induced apoptosis and Ca2+ influx in the hippocampus.
- The study is the first to compare effects of Hcy on TRP channels in the hippocampus.

Oxidative stress and apoptosis were induced in neuronal cultures by inhibition of glutathione (GSH) biosynthesis with d,l-buthionine-S,R-sulfoximine (BSO). Transient receptor potential melastatin 2 (TRPM2) and transient receptor potential vanilloid 1 (TRPV1) cation channels are gated by oxidative stress. The oxidant effects of homocysteine (Hcy) may induce activation of TRPV1 and TRPM2 channels in aged mice as a model of Alzheimer's disease (AD). We tested the effects of Hcy, BSO and GSH on oxidative stress, apoptosis and Ca2+ and influx via TRPM2 and TRPV1 channels in the hippocampus of mice.Native mice hippocampal neurons were divided into five groups as follows; control, Hcy, BSO, Hcy + BSO and Hcy + BSO + GSH groups. The neurons in TRPM2 and TRPV1 experiments were stimulated by hydrogen peroxide and capsaicin, respectively. BSO and Hcy incubations increased intracellular free Ca2+ concentrations, reactive oxygen species, apoptosis, mitochondrial depolarization, and levels of caspase 3 and 9. All of these increases were reduced by GSH treatments. Treatment with 2-aminoethoxydiphenyl borate (2-APB) and N-(p-amylcinnamoyl)anthranilic acid (ACA) as potent inhibitors of TRPM2, capsazepine as a potent inhibitor of TRPV1, verapamil + diltiazem (V + D) as inhibitors of the voltage-gated Ca2+ channels (VGCC) and MK-801 as a N-methyl-d-aspartate (NMDA) channel antagonist indicated that GSH depletion and Hcy elevation activated Ca2+ entry into the neurons through TRPM2, TRPV1, VGCC and NMDA channels. Inhibitor roles of 2-APB and capsazepine on the Ca2+ entry higher than in V + D and MK-801 antagonists.In conclusion, these findings support the idea that GSH depletion and Hcy elevation can have damaging effects on hippocampal neurons by perturbing calcium homeostasis, mainly through TRPM2 and TRPV1 channels. GSH treatment can partially reverse these effects.

Possible molecular pathways of cytosolic glutathione (GSH) depletion and homocysteine (Hcy) elevation on oxidative stress, apoptosis and Ca2+ entry through TRPM2 and TRPV1 channels in hippocampal neurons of aged mice. Vitamin B6 functions as a coenzyme, pyridoxal 5′-phosphate (PLP). In the transsulfuration pathway homocysteine condenses with serine to form cystathionine in a reaction that is catalyzed by cystathionine β-synthase (CBS) and requires PLP. Cystathionine is hydrolyzed by a second PLP-containing enzyme, gamma-cystathionase, to form cysteine. Excess cysteine is oxidized to taurine and inorganic sulfates. It is likely that TRPM2 and TRPV1-mediated cytosolic Ca2+ accumulation in the hippocampus via cytosolic GSH depletion and Hcy elevation involves accumulation of extracellular and intracellular ROS and opening of the mitochondrial permeability transition (MPT) pore that consequently leads to mitochondrial dysfunction. As the oxidative damage and Ca2+ entry increase, molecules such as cytochrome c may be released from mitochondria and trigger apoptosis. At the extreme, oxidative stress and Ca2+ entry cause severe MPT or even the rupture of the mitochondrial membrane, substantial swelling of the mitochondria with rupture of the outer membrane and release of apoptosis-inducing factors such as caspase 3 and 9.84