Activation Of ARE Pathway Protects Motor Neurons

Amyotrophic lateral sclerosis (ALS) is a common progressive neurodegenerative disease in central nervous system. It is characterized by selective degeneration of motor neurons in brain ,brain stem and spinal cord, which clinical features are delayed onset, chronic progression, weakness of limbs'muscles. Most patients died of respiratory failure 3-5 years later. ALS can be divided into familial amyotrophic lateral sclerosis (FALS) and sporadic amyotrophic lateral sclerosis (SALS). FALS is less than 10% of the total ALS patients, while SALS is more than 90%.FALS is mainly caused by the mutation of copper/zinc superoxide dismutase (Cu/Zn SOD) gene, whose site is at 21q22.1～22.2. More than 100 different mutations of the SOD1 gene have been linked to familial ALS. For many of these mutations, SOD1 enzyme activity is actually normal or elevated, and SOD1 knockout mice have no disease phenotype. These findings indicate that SOD1-associated ALS is not caused by a loss of function, but rather a toxic gain of function.Several mechanisms are implicated in the pathogenesis of motor neuron degeneration, including excitotoxicity, immune activation, mitochondrial dysfunction and oxidative stress, protein aggregation, altered proteosomal function and apoptosis. Although disturbances in each of these pathways may contribute to amplification or even initiation of motor neuron injury, the temporal relation of these pathways and their primacy in dictating disease onset and progression are unclear. These mechanisms are not mutually exclusive but are activated as a communal response that may be coordinated by oxidative stress. Increased oxidative stress appears to be an early and sustained event in association with motor neuron death in ALS, although the specific mechanism leading to oxidative damage on motor neurons remains to be defined. Oxidative stress has a prominent role in the initiation of ALS and is capable of activating pathways that elicit additional oxidative stress and propagate disease.GSH is the most abundant and effective scavenger against ROS directly in mammalian cells. In addition, GSH is also a key substrate for antioxidant enzymes that detoxify hydrogen peroxide and lipid peroxide products. CHI.Lå’ŒKE.Y show that depletion of reduced glutathione enhances motor neuron degeneration in vitro and vivo.The synthesis of GSH involves the actions of two ATP-dependent enzymes,γ-glutamylcysteine ligase (GCL) and GSH synthetase. GCL, the rate-controlling enzyme in the overall pathway, is a heterodimer composed of a catalytic (GCLC) and a modulatory (GCLM).There is a down-regulation of GCLM gene expression in rat CNS tissue during aging, accompany by reduced activity of GCL and GSH level. This means, GCL and GSH play an important role in age-related neuron degeneration disease.Glutathione S-transferase (GST) is a multigene family of more than 100 isoenzymes that catalyze the conjugation of GSH to a variety of electrophilic compounds。Glutathione transferases (GSTs; EC, 2.5.1.18) are phase II enzymes of defense that catalyze the conjugation of reduced glutathione to a wide range of electrophiles, carcinogens and other xenobiotics with genotoxic and cytotoxic activities。GSTs play an important role in protecting neurons against oxidative stress damage. Many study suggest induction of endogenous phase II enzymes,e g,GST may be a strategy for PD and AD.Based on the chronic excitotoxicity pathogenesis, incubation of organotypic spinal cord cultures in presence of threo-hydroxyaspartate (THA), the inhibitor of astrocyte glutamate transporter, causes death of motor neurons. We established the model of chronic motor neuron degeneration.Isummary, the organotypic spinal cord model of chronic motor neuron degeneration promises preclinical feasibility testing of potential neuroprotectants with enhanced relevance for clinical trials in human motor neuron disease. This model has already successfully predicted the efficacy of motor neuron protection by riluzole and neurontin, which are being used for human motor neuron disease. In this model, we want to study neuroprotective effects by inducing endogenous antioxidant agents.The intraneuronal accumulation of reactive oxygen species has been implicated in the pathogenesis of many neurodegenerative diseases .The ability of a cell to neutralize reactive intermediates is, in part, dependent on the activation of a cis-acting regulatory element termed the antioxidant response element (ARE). The ARE is located in the 5'-flanking region of many genes essential for both detoxification and antioxidant proteins. In many tissues of human, rat, mice and primary culture neurons and astrocytes, there are many ARE-drived gene including DCLC, GCLM and GSTs.The organotypic spinal cord model of chronic motor neuron degeneration were used to study the effect of ARE activator on motor neuron cell death and mechanism. The first part: we show that ARE activator completely inhibit glutamate-induced motor neuron death in these explants. The second part: we show that ARE activator inhibits glutamate-induced intracellular Ca²⁺ rise and decrease of tissue glutathione, but don't affect P75NTR pathway.The third part: the rate-controlling enzyme of GSH synthesis, GCL is abnormal in the model, but ARE activator correct the abnormal, which is corresponding to GSH alteration. The forth part: we investigate GSH related enzyme-GSTM protein and mRNA level in the model, ARE activator can change its level, which is accompanied by motor neuron protection.Partâ… ARE activator protects motor neuron against glutamate excitotoxity-induced motor neuron death in organotypic spinal cord model of chronic motor neuron degeneration ,Objective: In organotypic spinal cord model of chronic motor neuron degeneration , to investigate if ARE activator could protect motor neurons against glutamate excitotoxicity.We use three ARE activator :CPDT ,D3T and tBHQ at different concentration,at different time points. To explore new neuroprotective treatment. Methods: Organotypic spinal cord cultures were prepared using lumbar spinal cord slices from 8-day-old rat. Various concentrations of CPDT ,D3T and tBHQ(5μmol/L,15μmol/L,30μmol/L) were continuously added into the culture medium after cultured seven days. Spinal cord slices were treated with THA, a combination of THA and ARE activator , or treatment with the ARE activator without THA for 48 hours .Then we use monoclonal antibody SMI-32, a nonphosphorylated neurofilament marker, immunohistochemistry staining compared with different group.Results: The results showed that the spinal cord explants in control group could maintain excellent organotypic cellular organization and a stable population of ventralα-motor neurons. THA could produce a slow loss ofα-motor neurons. The complete neuron protection was achieved when the explants were pretreated by ARE activator: CPDT, D3T, tBHQ for 48 hour prior to initiating the combination treatment. Whereas ARE activator was only able to offer very limited neuron protection against THA-induced motor neuron death when the two agents were always added together to the culture medium,and same as 5μmol/L ARE activator.Conclusions: It is possibility that ARE activator may block glutamate toxicity to protects motor neuron against glutamate excitotoxity-induced motor neuron death. This subject could provide an effective studying pathogenesis and neuroprotection of ALS.Partâ…¡Tissue glutathione (GSH) contents, intracellular Ca2+ levels and P75NTR protein in organotypic spinal cord model of chronic motor neuron degeneration and alternation by ARE activatorObjective: To study if CPDT stimulates GSH content in rat spinal cord explants Proctention motor neuron survival in CPDT-treated spinal cord explants is accompanied by GSH, intracellular Ca2+ levels and P75NTR protein alteration.Methods: Organotypic spinal cord cultures were prepared using lumbar spinal cord slices from 8-day-old rat. Various concentrations of CPDT, D3T were continuously added into the culture medium after cultured seven days. GSH in tissue homogenates was derivatized by monochlorobimane, then the derivatives (GSH-monochlorobimane) were measured using a fluorescence plate reader. Measurement of intracellular Ca²⁺ level with 8μM Flou3-AM, then them were immediately analyzed by flow cytometry to determine fluorescence intensity.Tissue P75NTR protein was measured by Western blot.Results: After on week recover, the spinal cord tissues were treated with 15μM , 30μMCPDT for 48hours, then collected , measured ,GSH level increase only in 30CPDT group, but continue culture for 96 hours,GSH level was increase eather in 15CPDT group or in 30CPDT group.In the present experiment, the spinal cord tissues were first treated with CPDT at 15 and 30μM for 48 h before combined treatment of CPDT and THA for 3 weeks. We show that THA treatment might deplete tissue GSH. Indeed, 3-week THA treatment markedly reduced GSH level. Significantly, CPDT not only prevented THA-induced GSH depletion but actually elevated tissue GSH level 3-4 fold over the control. We found that CPDT fully prevented both intracellular Ca²⁺ rise.In the model, we could not find any specific P75NTR protein expression , but we could see it on one day old rat, which is control. After treatment with CPDT, there is no any specific protein expression at 75KD marker can be seen.Conclusions: CPDT could increase GSH level in spinal cord tissues ,which is time-depented and dose-depented.THA treatment reduced GSH level and CPDT not only prevented that but all so elevated GSH level 3-4 fold .CPDT,D3T fully prevented intracellular Ca²⁺ rise. CPDT have no effection on P75NTR pathway.Partâ…¢ARE target gene-GCL expression in organotypic spinal cord model of chronic motor neuron degeneration and alternation by ARE activatorObjective: to investigate one of ARE target gene GCL (include two subunit GCLC and GCLM) expression in organotypic spinal cord model of chronic motor neuron degeneration. If their alteration are accompany by motor neuron protection and ARE activitor can prevented THA-induced alteration.Methods: Various concentrations of CPDT were continuously added into the culture medium after cultured seven days. In the present study, lumber spinal cord explants prepared from 7-day old rats, after one week of culture, were treated with CPDT for 48 h and then harvested for measurement of GCLC, GCLMmRNA.Thus, explants after one week of culture were exposed to either solvent, THA or THA plus CPDT. In the THA plus CPDT group, the explants were first treated with CPDT for 48 h before the combination treatment, since this treatment schedule allowed CPDT to fully protect motor neurons.RT-PCR was used to measure the expression of two genes, including GCLCand GCLMalong withβ-actin gene as a control. The Trizol method was used to extract total RNA from the rat spinal cord explants.Results: At treat for 48hours group, only the rat spinal cord explants treated with 30μmol/LCPDT can increase GCLCmRNA expression, 15μmol/LCPDT could not affect it. On the other hand, two concentration CPDT treatment spinal cord explants show no alteration in GCLMmRNA expression at all.In the THA plus CPDT group,. In THA group, GCLMmRNA expression is lower than others. However, THA seems to have no effect on GCLC expression. Just as above, the combination treatment of THA with CPDT could not cause increase in GCLMmRNA expression. But this way caused a more significant increase in GCLCmRNA expression.Conclusions: Hight concentration CPDT (30μM) could cause increase expression of GCLC, but could not affect GCLM. However THA decrease GCLMmRNA expression, but can not affect GCLC.CPDT could increase GCLCmRNA last for 4 weeks, accompany by protection motor neuron.Partâ…£GSTM protein and gene expression in organotypic spinal cord model of chronic motor neuron degeneration and alternation by ARE activatorObjective: to investigate one of ARE target gene GSTM protein and mRNA expression in organotypic spinal cord model of chronic motor neuron degeneration, If their alteration are accompany by motor neuron protection and ARE activator can prevented THA-induced alteration.Methods: Organotypic spinal cord cultures were prepared using lumbar spinal cord slices from 8-day-old rat. Various concentrations of CPDT were continuously added into the culture medium after cultured seven days. In the present study, lumber spinal cord explants prepared from 7-day old rats, after one week of culture, were treated with CPDT for 48 h and then harvested for measurement of GSTM protein.Thus, explants after one week of culture were exposed to either solvent, THA or THA plus CPDT. In the THA plus CPDT group, the explants were first treated with CPDT for 48 h before the combination treatment, since this treatment schedule allowed CPDT to fully protect motor neurons.Prepare whole tissue extracts for Western Blot analysis. The Trizol method was used to extract total RNA from the rat spinal cord explants.Results: In lumber spinal cord explants treated with CPDT for 48 h shows CPDT and tBHQ CPDT at 15 and 30μM caused significant increase in expression of GSTM protein.In cultured for 4 weeks group, exposure of the explants to CPDT in the presence of THA lead to increased expression of GSTM in mRNA and protein .Interestingly, THA itself also seems to positively modulate some of the genes, GSTM.Conclusions: CPDT and tBHQ could stimulates ARE target genes in rat spinal cord explants. Increased motor neuron survival in CPDT-treated spinal cord explants is accompanied by activation of ARE targen gene GSTM expression and protein level.