Iron Is A Potential Key Mediator Of Glutamate Excitotoxicity And The Strategies To Protect Motor Neurons

Amyotrophic lateral sclerosis (ALS) is an adult-onset, progressive and lethal neurodegenerative disease, characterizing by the degeneration of motor neurons from cortex, brainstem, and spinal cord. ALS is one of the most common neurodegenerative disorders, occurring both sporadically (sALS) and as a familial disorder (fALS) with inherited cases accounting for about 10% of patients. Using the current standard therapy, the typical survival time for patients after diagnosis is three years, although large deviation has been observed. Present evidence indicates that loss of neurons in ALS results from a complex interplay among oxidative injury, excitotoxic stimulation, aggregation and/or dysfunction of critical proteins and genetic factors. Recent investigations support that these mechanisms are not mutually exclusive but are activated as a communal response that may be coordinated by oxidative stress.Reactive oxygen species (ROS), such as superoxide anion (O2–), hydrogen peroxide (H2O2), and hydroxyl radical (OH?), are continuously generated during oxidative metabolism in biological systems. ROS are prominent toxic intermediates, potentially damaging all types of biological molecules. Although cellular antioxidants act in concert to detoxify these species, an increase in the production of ROS and/or a decrease in the antioxidant capacity of cells can cause oxidative stress.CNS is particularly vulnerable to oxidative damage, due to a high level of oxidative capacity, high concentrations of polyunsaturated fatty acids. In addition, the CNS contains non-haem iron and certain brain structures are particularly rich in iron.Iron is essential for normal physiology, and it is implicated in many pathological processes, including neuron degenerative disorders. Iron is known to play an important role in Alzheimer disease, including accelerating amyloid-βaggregation and promoting oxidative damage. We suspected that iron might play an important role in glutamate excitotoxicity and neuronal death. Iron is known to potentiate the toxic effects of ROS by catalyzing the formation of highly reactive hydroxyl radicals from hydrogen peroxide through the Fenton chemistry.In the present study, we studied effect of iron on glutamate excitotoxicity and the strategies to protect motor neurons. We measured tissue iron levels and tissue levels of transferrin receptor, divalent metal-ion transporter 1 and ferritin in organotypic culture of rat spinal cord with and without THA-induced glutamate excitotoxicity. Moreover, the role of iron in glutamate excitotoxicity was assessed by measuring the protective activity of iron chelator deferoxamine. Moreover, we studied the mechanisms of EGCG and sulforaphane protects motor neurons in organotypic culture of rat spinal cord.We found: 1. THA could increase tissue iron level in the spinal cord tissue, with concomitant modulation of several iron transport and storage proteins, including transferrin receptor, divalent metal-ion transporter 1 and ferritin. More significantly, iron chelator deferoxamine was able to prevent THA-induced motor neuron degeneration completely. 2. EGCG could protect motor neurons via regulating glutamate levels in organotypic culture of rat spinal cord. 3. SF could prevent motor neuron death caused by THA toxicity via inducing the expression of phaseⅡenzymes.Part I Iron is a potential key mediator of glutamate excitotoxicity in spinal cord motor neuronsObjective:To study effect of iron on glutamate excitotoxicity. Methods:Organotypic spinal cord cultures were prepared using lumber spinal cord slices from 7-day-old rat pups. The cultures were divided into three groups at random: control, 100μmol/L THA group, DFO plus THA group. The number of motor neurons was assessed by immunohistochemistry, the expression of transferrin receptor, divalent metal-ion transporter 1 and ferritin were assayed with western blot, the level of lactate dehydrogenase, malondialdehyde and glutamate was assayed with ELIASA, the iron level in the explants was assayed with atomic absorption spectrometry.Results:At the end of the 3-week THA treatment, we found that the expression levels of TfR, DMT-IRE and DMT1+IRE were all increased significantly after THA treatment, whereas the level of ferritin decreased significantly, total iron content in the explants increased 21.4%. DFO prevented THA-induced neuron death, the number of motor neurons per ventral horn was higher in explants treated with 100μmol/L DFO plus 100μmol/L THA than in the control. Moreover, DFO could prevent the increase of lactate dehydrogenase and malondialdehyde induced by THA. But DFO had no effect on the medium glutamate.Conclusion: Iron is a potential key mediator of motor neuron excitotoxicity in organotypic culture of rat spinal cord. Blocking THA-induced iron rise alone may be sufficient for prevention of glutamate excitotoxicity.PartⅡThe mechanisms of green tea polyphenol protecting motor neuronsObjective: To study the mechanisms of green tea polyphenol, EGCG, protecting motor neurons in organotypic culture of rat spinal cord. Methods: The SD rat pups spinal cord organotypic cultures were divided into three groups at random: control, 100μmol/L THA group, EGCG plus THA group. The number of motor neurons was assessed by immunohistochemistry, and the expression of phaseⅡenzymes and EAAT2 were assayed with western blot, the level of malondialdehyde and glutamate was assayed with ELIASA.Results: At the end of 3-week treatment, the motor neurons number in the group treated with THA was less than the control group (P<0.05). However, the motor neurons number in the group treated with 5μmol/L EGCG plus 100μmol/L THA was more than group treated with THA at the end of 3 weeks (P<0.05). 5μmol/L EGCG could prevent the increase of glutamate induced by THA (P<0.05). The level of MDA decreased after treated with 5μmol/L EGCG plus 100μmol/L THA. Interestingly, the phaseⅡenzymes (NQO-1 and HO-1) in the group treated with 5μmol/L EGCG plus 100μmol/L THA expressed less than group treated with THA only.Conclusions: EGCG could prevent motor neuron death caused by THA toxicity via decreasing the synaptic cleft glutamate level in organotypic culture of rat spinal cord.PartⅢSulforaphane protects motor neurons of rat spinal cord via inducing phase II enzymesObjective: To investigate whether sulforaphane (SF) protects motor neurons via inducing phaseⅡenzymes.Methods: The SD rat pups spinal cord organotypic cultures were divided into three groups at random: control, 100μmol/L THA group, SF plus THA group. The number of motor neurons was assessed by immunohistochemistry, the expression of phaseⅡenzymes were assayed with western blot, and he level of glutamate was assayed with ELIASA.Results: At the end of 3-week treatment, the motor neurons number in the group treated with THA was less than the control group (P<0.05). However, the motor neurons number in the group treated with 10μmol/L SF plus 100μmol/L THA was more than group treated with THA at the end of 3 weeks (P<0.05). Meanwhile, the phaseⅡenzymes (NQO-1 and HO-1) in the group treated with 10μmol/L SF plus 100μmol/L THA expressed more than group treated with THA only (P<0.05). But SF had no effect on the medium glutamate.Conclusions: SF could prevent motor neuron death caused by THA toxicity via inducing the expression of phaseⅡenzymes.PartⅣStudy the model of glutamate excitotoxicityObjective: To study the levels of glutamate in medium of THA-induced spinal cord organotypic culture.Methods: The SD rat pups spinal cord organotypic cultures were divided into two groups at random: control and 100μmol/L THA group. The number of motor neurons was assessed by immunohistochemistry and the level of glutamate was assayed with ELIASA.Results: There is glutamate in minimal essential medium and horse serum. At the end of the 3-week THA treatment, we found that the medium glutamate level treated with THA is higher than the control, and the motor neurons number in the group treated with THA was less than the control group.Conclusions: It is important to assay the medium glutamate level in THA-induced spinal cord organotypic culture, and perfect results acquired from this model should be include the effect of treatment on the medium glutamate.