Nrf2/ARE Signaling Pathway Activator Protects Selective Motor Neuron Injury

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease, selectively involving in the upper and lower motor neurons. The sufferer usually died 3-5 years after the first appearance of symptoms. Several mechanisms for the pathogenesis of ALS have been proposed: 1)mutations of the copper/zinc superoxide dismutase (Cu/Zn SOD) gene;2)glutamate excitotoxicity;3)mitochondrial dysfunction;4)oxidative stress;5) exogenous toxin and virus;6)Immunity and inflammation, et al. Study on the pathogenesis and therapeutic method have been the focus of research field of neurology. Glutamate excitotoxicity and oxidative stress are important mechanisms for the pathogenesis of amyotrophic lateral sclerosis (ALS), and oxidative stress is believed the final common pathway of neurodegenerative diseases which caused by different reasons.At present, there isn't effective therapeutic method for ALS. ALS therapies include some approaches: antiglutamate, antioxidant agents, neurotrophic factors, gene therapies, immunity therapy and stem cell transplant et al. Aim at the hypothesis of oxidant stress, exogenous antioxidant agents are utilized for ALS patients, such as: N-Acetylcysteine, Vitamine E and melatonin et al. But those medicines show restrict degree of therapeutic effect. Another effective strategy against oxidative stress is inducing the expression of a series of endogenous antioxidant enzyme and antioxidant protein, which coordinately combat oxidative stress.Recently, there is reported that the activation of Nrf2/ARE Signalling pathway could induce a series of antioxidant enzyme, antioxidant protein, antiinflammatory and antitoxic protein. The expression of those antioxidant enzyme and antioxidant proteins in nervous system exhibited broad neuroprotection against injury by glutamate, hydrogen peroxide and dopamine. So Nrf2/ARE Signalling pathway is expected a promising drug target to combat oxidative stress in neurodegenerative disorders.It's reported that dithiolethiones could induce expression of these antioxidant enzyme in different tissue by activation of Nrf2/ARE signalling pathway. CPDT and D3T are potent enzyme inducer in these kind compounds. And both of them have the characterization of small molecule and lipophil, so it is possible for them have the potential therapeutic effect on neurodegenerative disease. It is worth to further study and generalization.The model of organotypic spinal cord culture for ALS is based on the technique of organotypic spinal cord culture. The cultures are treated with threohydroxyaspartate(THA) to selectively inhibit glutamate transporters, which raise the extracellular glutamte concentration and mimick the chronic damage to glutamate transporters that have been observed in ALS patients. It is a very good model to be used to screen agents that can effectively protect against motor neurons damage and provide a possibility for these medicines to be used by ALS patients.Based on the mechanism of glutamate excitotoxicity, and using the technique of organotypic spinal cord culture, we made the organotypic spinal cord culture model of ALS in vitro. On the basis of selectiveαmotor neuro injury in ventral horn, tissues were co-treated with different concentrations of Nrf2/ARE pathway activator to be used to study the protection effect on slective motor neuron injury. Our study included four parts. In the first part, based on glutamate excitotoxicity we establish the organotypic spinal cord culture model of ALS in vitro. Then we screen the effective Nrf2/ARE pathway activator on the basis of the model for further studying the mechanism of protection. In the second part, based on the screening result and using technique of transmission electron microscope and flow cytometry, we treated the spinal cord slices with CPDT to observe the protection effect from different angles. In the third part, the classic antioxidant enzyme NQO1 and antioxidant protein ferrintin were selected, on behalf of a series of downstream protein of Nrf2/ARE pathway. We discussed the mechanisms of the protection of CPDT against selective motor neurons injury, using technique of reverse transcription RT-PCR and immunoblotting. In the fourth part, based on transgenic mice model of ALS we discussed the effect of iron metablism on pathogenesis of ALS for further intervention with CPDT. We observed the expression of ferritin and content of iron in lumbar intumescentia of transgenic mice at endstage of disease.Part I To screen the effective Nrf2/ARE pathway activator at the level of organotypic spinal cord cultureObjective:Based on organotypic spinal cord culture model of ALS, to screen the effective Nrf2/ARE pathway activator by morphological method and count the number ofαmotor neurons in the ventral horn for further study the protection effect.Methods:Organotypic spinal cord cultures were prepared using lumber spinal cord slices from 8-day-old rat pups. Lumber spinal cords were collected under sterile condition and sectioned transversely at 350-μm intervals with a Mcllwain tissue chopper. Slices were carefully placed on the surface of Millipore Millicell-CM insert(five slices per membrane).Cultures were incubated at 37℃in a 5% CO2/ 95% humidified enviroment. To establish the organotypic spinal cord culture model of ALS, the culture medium were continuously added with 100μmol/L THA, an inhibitor of glutamate transporter. On the basis of the model, the spinal cord slices were treated with Nrf2/ARE pathway activator CPDT or D3T. The cultures were randomly divided into five groups:①normal control group;②THA model group.③CPDT or D3T pretreatment group(CPDT,D3T pretreated 48h+THA)④CPDT or D3T simultaneous treatment group(CPDT,D3T+THA)⑤solvent DMSO control group. To observe the growth condition of cultures with inverted microscope every day. After 4 weeks culture in vitro, the spinal cord cultures of each group were taken out andαmotor neurons in the ventral horn were identified and evaluated with SMI-32 immuohistochemical staining, a neuron specific marker.Results:The spinal cord cultures of normal control group had excellent organotyopic cellular organization and became bigger. There are about 15-20 αmotor neurons in the ventral horn in this group. Whileαmotor neurons in the ventral horn in THA model group significant decrease while the inter-neurons in the dorsal horn were less affected. Pretreated with CPDT or D3T, the condition of spinal cord slices in both groups is similar to normal control group. Compared with model group,αmotor neurons in the ventral horn in these group increased significantly(P<0.05). Compared with 15,30μmol/L D3T pretreatment group,αmotor neurons in 15,30μmol/L CPDT pretreatment group increased slightly(P>0.05). In contrast there aren't increase ofαmotor neurons in 5μmol/LCPDT,D3T pretreatment group and CPDT,D3T simultaneous treatment group(P>0.05).Conclusions:In this study, based on organotypic spinal cord culture model of ALS, we founded that the selective motor neuron loss by THA was inhibited completely after CPDT(15, 30μmol/L) pretreatment. The effect of CPDT is slight better than D3T. The activation of Nrf2/ARE signal pathway may be the new strategy against glutamate exicitotoxicity. CPDT and D3T were promising neuroprotective agents.PartⅡA study on protection effect of CPDT against selective motor neuron injuryObjective: Based on organotypic spinal cord culture model of ALS, the culture medium were added with different concentration of Nrf2/ARE pathway activator CPDT. Using technique of transmission electron microscope and flow cytometry, to evaluate the protection effect onαmotor neuron by CPDT from different angles.Methods: Organotypic spinal cord cultures were prepared using lumbar spinal cord slices from 8-day-old rat pups. The cultures were randomly divided into 4 groups:①normal control group;②THA model group.③15μmol/L CPDT pretreatment group(CPDT pretreated 48h+THA)④30μmol/L CPDT pretreatment group(CPDT pretreated 48h+THA). The culture medium were changed twice a week, and collected at different time point for further study. After 4 weeks culture in vitro, the spinal cord cultures of each group were taken out. The ultrastructural change of cultures and mitochondrial membrane potential were observed with transmission electron microscope and flow cytometry. The level of lactate dehydrogenase (LDH) and malondialdehyde (MDA) in culture medium was assayed and compared with model group.Results: The ventralα-motor neurons in control group showed normal appearance under TEM; While motor neurons in THA model group showed midrange or severe degree motor neuron degeneration with mitochondrial vacuolar degeneration. In contrast motor neurons in 15, 30μmol/L CPDT pretreatment group showed roughly normal appearance. THA could also produce a loss of mitochondrial membrane potential. CPDT(15, 30μmol/L) pretreatment could inhibit significantly the decrease of mitochondrial membrane potential(P<0.05).Compared with normal control group, THA model group could result in an increase of LDH enzyme activity and content of MDA in culture medium. CPDT(15, 30μmol/L) pretreatment caused the level of LDH and MDA in culture medium decreased, the difference is significant(P<0.01).Conclusions: It is verified that pretreatment with CPDT, a Nrf2/ARE pathway activator, could obviously protect selectiveαmotor neurons damage by protection of mitochondrial, strengthening the ability of clearance of free radical. It is speculated that protect effect is correlated with that the expression of downstream target gene of Nrf2/ARE pathway, Nrf2/ARE signal pathway activator maybe an effective agent for clinical neurodegenration.PartⅢA study on the mechanisms of the protection of Nrf2/ARE pathway activator on motor neuronsObjective: The classic antioxidant enzyme NQO1 and antioxidant protein ferrintin were on behalf of a series of downstream protein of Nrf2/ARE pathway. To observe whether CPDT could induce functional expression of NQO1 and Ferritin, which locate downstream of Nrf2/ARE pathway. And to further discuss the machnism of this agent.Methods: Based on organotypic spinal cord culture model of ALS, the spinal cord slices were divided into two group of different time point. One group is treated 48h by CPDT. Three subgroups is①control group;②15μmol/L CPDT treated group.③30μμmol/L CPDT treated group. The other group is treated 3 weeks by CPDT. Four subgroups is①normal control group;②THA model group.③15μmol/L CPDT pretreatment group(CPDT pretreated 48h + THA)④30μmol/L CPDT pretreatment group CPDT pretreated 48h+THA). After 4 weeks culture in vitro, the spinal cord cultures of each group were collected. The total RNA and protein were extracted from these two groups. To measure the expression of two genes and protein, NQO1 and Ferritin-H, by the methods of RT-PCR and Western blotting in different subgroups. And to detect the enzyme activity of NQO1 in each subgroup.Results:At the time point of CPDT treated 48h, the expression mRNA and protein of NQO1 and Ferritin-H were significant increased in 15,30μmol/L CPDT treated group, compared with control group. The enzyme activity of NQO1 is increased dose-dependent. The expression of NQO1 and Ferritin-H in 30μmol/L CPDT treated group is higher than those in 15μmol/L CPDT treated group, the difference is significant.At the time point of CPDT treated 3 weeks, the expression of NQO1 and Ferritin-H mRNA and protein were significant increased in 15,30μmol/L CPDT pretreatment group, compared with model group. The enzyme activity of NQO1 is also increased in CPDT pretreatment group. The expression of NQO1 and Ferritin-H were mildly increased in THA group, compared with control group.Conclusions: In spinal cord CPDT could induce functional expression of antioxidant enzyme NQO1 and antioxidant protein Ferritin which locates downstream of Nrf2/ARE pathway. Upregulated expression of NQO1 and Ferritin contribute a certain degree to against selective motor neuron damage. It is speculated that the activation of Nrf2/ARE induces the expression of a series of target gene, which coordinately strengthern the antioxidant ability and protect the selective motor neuron injury. CPDT and riluzole aim at different directions, so it may strengthen therapeutic efficacy of riluzole. The activation of Nrf2/ARE pathway maybe a new strategy against glutamate exicitotoxicity.PartⅣAn elementary study on iron metabolism of transgenic mice model of ALSObjective: To discuss whether there are iron overload and difference of ferritin expression between transgenic mice of B6SJL-TgN (SOD1G93A)1Gur(with mutant human SOD1G93A gene), wild type littermate and B6SJL-TgN (SOD1)2 Gur(with normal human SOD1 gene). It would provide experimental data for further intervention with CPDT.Methods: The animal were divided into 3 groups:①model group: 5 B6SJL-TgN (SOD1G93A)1Gur mouse ;②control group1: 5 wild type littermates;③control group2: 3 B6SJL-TgN (SOD1)2Gur mouse. All the animal were sacrificed at endstage disease of model group (score =5.0). The lumbar intumescentia of all animal were divided into two parts. One part is used to measure the content of iron by atomic absorption spectrometry. The other part is used to extract total protein and detect the expression of Ferrintin-H.Results:There is an increase of iron content in lumbar intumescentia of model group mouse(42.20±3.74μg/g) at endstage, compared with control group 1(33.35±3.67μg/g) and control group 2(30.74±2.82μg/g). The expression of ferritin-H protein also increase in lumbar intumescentia of model group mouse at endstage. While there isn't any difference between control group 1 and control group 2.Conclusion: The results showed that there are iron overload and up-regulation of Ferrintin-H protein in lumbar intumescentia of model group at end-stage disease,compared with control group 1 and control group 2. It is speculated that over accumulated iron contribute to the disease process of transgenic mouse and up-regulation of Ferrintin-H is a protective response of body. It would provide experimental data for further intervention with CPDT.