Protective Effects And Potential Mechanisms Of Metallothionein Against NMDA-Induced Neurotoxicity

Gluta mate, the important excitatory neurotransmitter in ma mma l Central nervoussystem, participates in excitatory synaptic transmission, mediates signa l transduction ,constitute the foundation of remembra nce and cognitive, be relevant with some importantphysiologica l functions , such as synaptic plasticity, neuron growth and survival long-termpotentiation. NMDA-Ca2+-NO pathway is the activation of the neurona l or interneurona lNMDA receptor by Glu or N-methyl-D-aspartate (NMDA), which can induce the increaseof intracellular calcium ion ([Ca2+]i) and nitric oxide (NO), accordingly generate s the toxiceffect or protection of a series of physiology or pathologica l change . Excessive activationof NMDA receptor can cause of ma ny nervous system disease and the nervous lesion ofcerebrum including neuron cellular necrosis and apoptotic. The etiopathogenesis of nervoussystem degenera tive disease such as Alzheimer's disease , has close relationship with theabnormity of NMDA receptor, therefore it is considered that NMDA-med iated the neurona lexcitability death is releva nt with nervous degenera tive disease marked by dysmnesia forclinica l ma nifestation.Metallothioneins (MTs) are a group of low-molecular, cysteine rich intracellular meta lbinding proteins which can be induced by metals. The functions of MTs in CNS has oftenbeen considered as the homeostasis and metabolism regulation of essentia l meta ls, the storeand attenuate of nonessentia l meta ls, as well as antiapoptosis, free radica l scavenging,regulation intercellular oxidation-reduction equilibration. Recently, ma ny researchesdemonstrate that glutamate has an important role in med iating the communication betweenthe neurons and astrocytes, and regulateing the expression of MTs with free radica l andother cytokines. Thus, research on the role of MTs in glumatic acid signa l pathway willprovide new idea and new target for drug action for therapeutics of nervous system diseaseand CNS injury. Our study includes two sections.1 The effect of MT on cortical neuron growth and developmentThe anima l model of MT gene knocked out mice (MT-/-) and the correspond ing wildtypemice (MT+/+) were used for experiment. MT content of brain was measured by cadmium saturation methods to confidence the anima l model. The cellular localiza tion ofMT-â… /â…¡within the neuron-glia l cell culture was detected by immunofluorescence . Glia land neurons of cortex has weak positive of MT-â… /â…¡. Studies demonstrating that MT-â… /â…¡ma inly exist in the cell nucleus of glia l cell, as well as the intracytoplasm of neuron, thecell nucleus of neuron presented weaker fluorescence tha n intracytoplasm. For observationof the effect of the MT-deficient on neuron growth and development, cortical neuroncultured for 2, 4, 24, 72h was fixed and stained with hema toxylin and eosine (HE) formorphometric analysis of the percentage of neurite-bearing neurons, the number of neuritesper cell of the neurite-bearing neurons and neurite length of developing neurites over 3days using the Ima ge-Pro Plus Version 6.0; Cortical neuron cultured for 1, 4, 7, 10d wastreated as above for cell body analysis. Results shows that MT-deficient inhibit the thenumber of neurites per cell of the neurite-bearing neurons , neurite length and cell body.MT-I/II has inhibitory effect of the neuron growth and development.2 The protective effect of MT on nervous excitability injury induced by NMDAN-methyl-D-aspartate receptor agonist was used to establish the cortica l neuronNMDA-ind uce excitability injury model. For study the protective effect of MT on nervousexcitability injury induced by NMDA, we observed and compared the effect of NMDAinducednervous excitability injury on cortica l neuron-glia l cell of MT gene knocked outmice (MT-/-) and that of correspond ing wild-type mice (MT+/+). MTT and LDH resultsshows that cell survival was lower in MT-/- neuron-glia l cells, lacta te dehydrogenaserelease was higher in MT+/+ neuron-glia l cells after NMDA injury. Western blot analysisof MT content shows that NMDA can upregulate MT-â… /â…¡expression. Therefore MT-â… /â…¡has neuroprotective effect to NMDA-ind uced nervous excitability injury.Excessive activation of N-methyl-D-aspartate receptor can lead to the overload ing ofCa2+, which beyond the capability of mitochond rial, interferes with electron transfer andinduces the reactive oxygen species (ROS) production. To investigate whether theprotective mecha nism of MT-â… /â…¡is releva nt with the block of pathologica l changecaused by NMDA, we detected some index releva nt with the pathologica l change describedabove. After NMDA treatment with MT+/+ and MT-/- neuron-glia l cells, the nitriteconcentration in the culture med ium was measured as an indica tor of NO production,according to the Griess reaction. Using the correspond ing fluorescent probe (Hoechst33258R,DCFH-DA,Fluo-3/AM,Rhodamine123) respectivly for detecting theapoptosis, ROS overprod uction, eleva tion of [Ca2+]i, mitochomdria l depolarization, thephotofluorogram was analyzed and showed as mean value of fluorecence using the Ima ge-Pro Plus Version 6.0. The lipid peroxidation concentration was determined using the thiobarbituric acid (TBA) method. DNA damage was evaluated by comet-NE assay. In allcase, MT-â… /â…¡displa y antagonistic effect to NMDA toxicity, indica ted that the protectiveeffect of MT-â… /â…¡to NMDA-ind uced nervous excitability injury ma y act by influencingthe NMDA-Ca2+-NO pathway.In our studies, we also found that the lacta te dehydrogenase release , nitriteconcentration, ROS overprod uction and DNA damage was more serious in MT-/- neuronglial cells than MT+/+. This diference between MT+/+ and MT-/- neuron-glia l cellsindica ted that MT-â… /â…¡deficient ma y lead to the change of base redox state.In conclusion, the present study demonstrated that MT deletion is correla ted with thegrowth and development of neuron, and has an essentia l role in ma inta ining the the norma lredox state.