Effects Of Resveratrol On Motor Neuron-like Cell Line Transfected By TDP-43

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive and selective death of upper and lower motor neuron, the etiology and pathogenesis are still unclear. Many studies showed that oxidative stress induced neuron death and played an important role in neurodegenerative diseases. So antioxidant therapy became an important strategy for ALS.Abnormal TDP-43(TAR-DNA binding protein) protein was found in nerve tissues in patients with ALS and is redistributed from the nucleus to the cytoplasm. TDP-43 protein is encoded by six exons of TARDBP(transactive response DNA binding protein), which is located on human chromosome 1,1p36.2.The TARDBP gene is highly conserved and widely expressed in various tissues.Many studies showed that TDP-43 played an important role in expression and regulation of genes, including: participating in shear of cystic fibrosis transmembrane conductance regulator (CFTR) gene and apolipoprotein AⅡ(ApoA-Ⅱ) gene; affecting the exon skipping splicing and inhibiting splicing activity through combination of the glycine-rich C- end of the sugar with the heterogeneous nuclear nucleoprotein (hnRNP) family members: providing a scaffold for nuclear bodies through an interaction with survival motor neuron protein; involving in regulating the stability of mRNA , the biosynthesis of microRNA, apoptosis, and cell division; regulating neuronal plasticity in response to neuronal activity and other factors.Since the last two years, nearly 30 kinds of mutations in TARDBP gene in different populations of familial ALS (familial ALS, FALS) and sporadic ALS (sporadic ALS, SALS) patients have been reported, the studies on the function and pathogenesis of the gene are going on, suggesting that TARDBP gene is potentially relevant to ALS.Studies in our laboratory showed that oxidative damage, mitochondrial dysfunction was more serious in motor neuron-like cell line (NSC-34) transfected with TDP-43 Q331K,M337V than that in NSC-34 cells transfected with Empty vector,TDP-43 Wide type.Resveratrol (3,5,4-trihydroxy-trans-stilbene, Res) is a phytoalexin structurally related to stilbenes, which was extracted from the skin and seeds of grapes in 1976. Now it has been reported that the resveratrol has a wide spectrum of biological activity, such as antioxidant, neuroprotective effects, cardioprotective, antiproliferative. It is capable of scavenging lipid hydroperoxyl free radical as well as hydroxyl and superoxide anion radicals. The most important effect is antioxidant activities and neuroprotective properties.In this study, NSC34 cells with stable transfection of wild-type and mutant TDP-43 were used as the cell model of oxidative stress in ALS to further observe the effect of resveratrol on NSC-34 cells transfected with mutant TDP-43.Objective: To explore the method of NSC34 cell lines stably transfected with TDP-43; The NSC-34 cells transfected with mutant TDP-43 were used as a cell model of ALS to investigate the effects of resveratrol on NSC-34 cells transfected with mutant TDP-43.Methods: According to the protocol of culture of motor neuron-like cell line,the NSC34 cells stable trasfected with TDP-43 owned by our laboratory were recovered, passaged and cultured. The situation of growth and proliferation were observed. The NSC34 cell lines stably transfected with TDP-43 were cultured, then the medium was changed after 24 hours of passage. After 6-12 hours the cells were randomly divided into different groups: normal control group and Res groups. In the Res groups, three concentrations of Res by 1, 5, 25μmol/L were added to the cells. 24 hours later, the cells and medium were collected,the proliferation of the cells was measured by MTT and the content of LDH were detected by multifunctional enzyme instrument.Results: (1) The four different cell lines stably transfected with empty vcetor, wild-type (WT), Q331K and M337V mutant human TDP-43 cDNAs were cultured, the cells are in good condition under inverted microscope; (2) After 24 hours treated with Res by 1,5 and 25μmol/L, the morphological changes of cells were observed with invert microscope. (3) The proliferation of cells treated with Res1,5μmol/L for 24 hours increased compared to that in the control and Res 25μmol/Lgroup; and the level of LDH decreased(P<0.05); (4) The cell proliferation decreased significantly in group treated with Res 25μmol/L for 24 hours compared to that in control group and Res1,5μmol/L group, while the level of LDH increased (P<0.05).Conclusions: Four cell lines with stable transfection with TDP-43 were set up to serve as the cell model of the ALS in vitro; Res by low concentration of 1,5μmol/L can increase the cell proliferation and decrease the level of LDH in culture medium . The study indicates that resveratrol by low concentration can protect NSC34 cells from oxidative damage and may be as a promising neuroprotective agent in neurodegenerative diseases.