The Changes Of Nrf2-ARE Signaling Path In The Aging And Mutant SOD1G93A Astrocytes

Astrocytes constitute the major class of cells in the central nervous system (CNS). They are no longer considered to be passive structural and trophic supports for neurons, but rather play important and integral roles in the physiological functioning of the CNS in both health and disease. Astrocytes participate in ionic homeostasis by siphoning away potentially excitotoxic factors such as excess extracellular glutamate and K+, and are involved in CNS communication and information processing through interactions with neurons and other astrocytes. Glia in general, and astrocytes in particular, have also been directly and indirectly implicated in the pathophysiology of various traumatic and neurodegenerative diseases. Of particular clinical interest, they have demonstrated properties which may be targeted in novel therapeutic initiatives based on the neuroprotective properties they display following neuronal injury in vitro and in brain and spinal cord injuries. It is critical to have well characterized pure astrocyte in vitro systems which allow the isolation and study of specific components of astrocyte physiology and biochemistry by well established electrophysiological, molecular, and immunohistochemical techniques. Typically, methods which aim at establishing highly pure astrocyte cultures have been time- and resource consuming, and technically complex, while simpler methods yielded cultures that, depending on the procedure, are contaminated with fibroblasts, microglia, oligodendrocytes, endothelial cells, ependymal cells and (occasionally) even neurons. In an attempt to address these issues, we have developed a simple non-enzymatic technique for culturing astrocytes which takes advantage of the ease and minimal time required for culturing offered by mechanical membrane dissociation methods, and which produce astrocyte cultures which are 99% pure. The brain is particularly vulnerable to oxidative stress because of its high metabolic rate, high level of polyunsaturated fatty acids and low antioxidant defences. Free radicals are constantly generated in the brain in vivo. Moreover, excessive production of the superoxide radical and hydrogen peroxide (H2O2) in the presence of iron or copper ions can result in the generation of the highly reactive hydroxyl radical. Changes in the function of astrocytes may reduce their neuroprotective capacity, contributing to the neuronal aging development. Oxidative stress is believed to neuronal aging development. Oxidative stress is believed to play a key role in brain aging progression, although the molecular mechanisms involved are not clear. Numerous evidences on alterations in astrocyte function during aging have been related to changes in redox homeostasis. Astrocyte activation increases progressively during aging in most brain regions, as estimated by an increased expression of glial fibrillary acidic protein (GFAP) and S100b protein; and GFAP and S100b mRNA. Both proteins also increase with age in culture in mouse and rat cortical astrocytes. Part 1 The establishment of model of aged astrocytesObjective: To explore the characteristics of aged astrocytes.Material and Methods: Primary cultures enriched in astrocytes were established from spinal cord tissue of 1-day-old ICR mouse. The cell suspension was then spun at 300g for 5 min, the supernatant removed, and the pellet resuspended in glial medium: DMEM (Invitrogen) containing 10% FBS, 100 U ml–1 penicillin(Amrisco) and 100 mg ml–1 streptomycin sulfate (Sigma). The resuspended cells were plated onto glass flasks. After 2 weeks, glial cultures contained 95-98% GFAP+ astrocytes. To eliminate residual microglia, 2-week-old cultures were agitated at 200 r.p.m. for 6 h. Experiments were performed at 14 days (14 DIV) and 30 days (30 DIV) and 60 days (60 DIV) after seeding. Fresh medium was added twice a week. Astrocytes were rinsed twice with PBS and then incubated with 0.25% trypsin for 5 min, and centrifuged at 500g for 5 min at 4°C. Protein was extracted using total protein extraction kit and nuclear-cytosol extraction kit. Protein extracts were quantified using the Bradford method. Ten ug of the total protein extracts, 10ug of nuclear extracts or 40ug cytocol extraction were denatured at 95°C for 5 min, loaded onto a 10% SDS– polyacrylamide gel and electrophoresed, respectively. After electrophoresis, the proteins in gel were transferred onto a polyvinylidene difluoride membrane (Millipore). The membrane was then incubated overnight at 4°C with the following primary antibodies: mouse anti-GFAP (diluted 1: 500). Membranes were then incubated for 1 h at 22–24°C with a fluorescence conjugated secondary antibody (1:3000). The levels of protein immunoreactivity were normalized to that ofβ-actin. Mitochondrial transmembrane potential (?Ψm) was measured using flow cytometry. The cell suspensions were centrifuged at 800 g for 3 min and the cells were resuspended in PBS. The entire procedure was performed at room temperature. For measurement of ?Ψm, 1 ml cell suspension (106 cells) was incubated with 26.3 uM rhodamin 123 at 37oC for 30 min. The cells were then washed twice with PBS and were immediately analyzed by flow cytometry to determine fluorescence intensity (Excitation/emission wavelengths: 505nm/534nm). Fixed astrocytes cultures were treated with 10% horse serum for half an hour at room temperature, and then, incubated with an anti-GFAP antibody (1:500) overnight at 4 oC. The cultures were washed three-time with TBS-T and incubated with a biotinylated secondary antibody for 1 h. The cultures were further washed and then incubated with an HRP-conjugated ABC staining solution.Result: (1) Changes of GFAP in the primary spinal cord astrocyte cultures. Densitometric analysis of the immunoblots showed that Glial fibrillary acidic protein (GFAP) levels in 30 DIV and 60 DIV cultures were 1.6- and 1.7-fold higher, respectively, than that in the 14 DIV cultures, although In GFAP level at 60 DIV was similar to that at 30 DIV. (2) Mitochondrial transmembrane potential decreased with aging. Mitochondria are key regulators of cell survival and death and play a central role in aging. In the 60 DIV, the astrocytes had a lower mitochondrial transmembrane potential than that in the 14 DIV (Fig. 1I-J), which reflected a damage of function of the mitochondria. (3) GFAP-positive astrocytes exhibited different morphologies, such as stellate, pancake, or elongation. In the aged astrocytes, the expression of GFAP increased and the volume of aged astrocytes appeared larger than that of the 14 DIV astrocytes.Conclusions: In our aging model, the expression of GFAP and mitochondral membrane potentials reflected the function of aged spinal cord astrocytes.PartⅡThe Nrf2 activity lost in the spinal cord and its astrocytes in the aged animalsObjective: To study the antioxidant ability in the aged spinal cord and its astrocytes.Materal and Methods: We established the vitro and vivo aging models. The expression of Nrf2 and HO1 and NQO1 induced by Nrf2 were analized by the Western Blot and Immunochemistry. The expression EAAT2 and ferritin was also checked by the Western Blot. Furthermore, the effect of EGCG on aged astrocytes was analyzed by the Western Blot, LDH and Cytoflow.Results: Glial fibrillary acidic protein (GFAP) expression increased and glutamate transporter 1(GLT1), known as EAAT2 decreased in cultured cells and animals during aging. The mitochondrial transmembrane potential decreased in the 60 DIV cultures. Ferritin was up-regulated, while transferrin receptor 1(TfR) was down-regulated in aged astrocytes and animals. Given the relationship between aging and loss of antioxidant tolerance capacity, we examined the expression of heme oxygenase 1(HO1) and NAD-(P)-H: quinone oxidoreductase 1 (NQO1) in the old astrocytes and animals. Indeed, both antioxidant enzymes decreased there. Furthermore, we found that total Nrf2 which governed basal and inducible HO1 and NQO1 expression decreased significantly in the old astrocytes and animals. Furthermore, whether the age-related decrease of Nrf2 transcriptional activity can be restored and whether elevating the Nrf2 activity by EGCG can protect the aged cells, aged astrocytes were treated with EGCG (5uM for 30 days), which is known to activate Nrf2. EGCG induced Nrf2 nuclear translocation, and higher expression of NQO1. Astrocyte activation decreased after EGCG treatment.Conclusions: Older astrocytes have a decreased level of HO1, NQO1 and Nrf2 and antioxidants, like EGCG, protect the aged astrocytes.PartⅢThe activity of Nrf2 was also lost in the astrocytes of ALS-like animalsObjective: SOD1G93A astrocytes have a deadly effect on the survival of motor neurons. But, the mechanisms are poorly understood. So, we wanted to know whether SOD1G93A astrocytes had a lower antioxidant and detoxifying capacity than normal astrocytes.Material and Methods: The cell toxicity of astrocytes deporized serum was measured by MTT. The level of ROS was analysized by the fluence of DCF by using Confocal Microscope. The expression of Nrf2, HO1 and NQO1 in the different cells was detected by the Western blot.Results: In deed, the level of expression of Nrf2, HO1 and NQO1 decreased 44 percent, 43 percent and 40 percent respectively in the mutant astrocytes. Epigallocatechin gallate (EGCG) has been known to have anti-neurodegenerative effects and activates Nrf2. Furthermore, we studied the effect of EGCG on the astrocytes bearing mutant SOD1. EGCG up-regulated the expression of Nrf2 and NQO1 and caused the Nrf2 translocation from the cytoplasm to the nucleus. So, the transcriptional activity of Nrf2 was increased by using of EGCG. In addition, the expression of EAAT2 which was down-regulated by mutant SOD1G93A also was up-regulated by using EGCG. Serum withdrawal induces oxidative stress with the generation of more than one free radical species in cell. It was interesting to note that the astrocytes bearing mutant SOD1 were subjected to oxidative stress in the form of serum withdrawal. Moreover, we tested the effect of the serum deprivation on the mutant astrocytes and mutant astrocytes pretreated by EGCG.Conclusion: Our results suggested that EGCG protected mutant SOD1 astrocytes from oxidative damage.PartⅣThe culture of NSC-34 Objective: To study the characteristics of the growth and histocytochemistry of NSC-34.Material and Methods: According to the protocols of culture of NSC34, to passage and culture the cell line. The proper growth of NSC-34 was detected by the different of density of NSC-34. The expression of SMI32 was struded by histocytochemistry.Results: The NSC-34 showed versatile morphology. The dencity of 8000cells/cm2 was fitted to transfection. SMI32 was expressed in the NSC-34.Conclusion: We find out the proper densicty of NSC-34 to transfection and the expression of SMI32 may be the marker of cytoplasm.