The Protective Effect And Underlying Mechanism Of Fibroblast Growth Factor 9 On Cellular Model Of Amyotrophic Lateral Sclerosis

Objective:Amyotrophic lateral sclerosis(ALS) is an adult-onset and progressively fatal neurodegenerative disorder that affects motor neurons in the brain and spinal cord.The loss of neurons leads to muscle atrophy, spasticity and weakness, and eventual death from respiratory failure. Although the clinical disease duration is highly variable, most patients with ALS die within 3 to 5 years after symptom onset. Several pathogenic mechanisms underlying ALS have been reported, including oxidative stress, glutamate-mediated excitotoxicity, abnormal protein aggregation, mitochondrial dysfunction, toxic effects of mutant genes and neurons apoptosis.Recently, TAR DNA binding protein of 43 k Da(TDP-43), as the major protein constituent of ubiquitinated inclusions, was identified in brains of ALS and frontotemporal lobar dementia with ubiquitin-positive inclusions(FTLD-U) and gained more and more attention. TDP-43, a constitutively and ubiquitously expressed DNA-RNA-binding protein of 43-k Da molecular mass with 414 amino acids, is encoded by the gene TARDBP on human chromosome 1. It may be involved in several cellular processes, including specific pre-m RNA splicing and transcription, m RNA stability and the biosynthesis of micro RNAs. To date, over 40 dominant mutations in the TDP-43 gene have been identified and linked to sporadic and familial ALS, providing strong evidence for a direct link between TDP-43 dysfunction and neurodegeneration. TDP-43 C-terminal fragments, TDP-25, is generated by abnormally ubiquitinated TDP-43 and localized in cytoplasmic insoluble aggregates. The fragment is toxic to neurons and induced cell death through a toxic gain-of-function, which plays an important role in disease pathogenesis.Fibroblast growth factor 9(FGF9), a member of the FGF family, is widely distributed in the body. It is essential for cell proliferation 、differentiation、growth and survival and has an important role in many cellular processes in both embryonic development period and adulthood. Recently, more and more attention is paid to the role of FGF9 in promoting cell proliferation and protection in central nervous system. Large studies indicate that FGF9 can promote proliferation and survival in various cell models, regulate the cellular antioxidant defense, reduce the neurons death induced by oxidative insult, upregulate the choline acetyltransferase(Ch AT) activity and has neurotrophic activity. Previous studies show that FGF9 is synthesized and expressed by motoneurons, but very little is known about its role in ALS. Herein, we explored whether FGF9 protect the cellular toxicity induced by TDP-25 and the possible mechanisms in an ALS cellular model stably expressing TDP-25, the C-terminal fragments-25 of TDP-43.Methods:A cellular model stably expressing TDP-25 is used as an ALS cellular model. First, we demonstrated the protein expression level of TDP-25 in the stable cell line by western blot. Then, the stable cell line was exposed to different concentrations of FGF9, and cell viability was measured by the CCK-8 assay and cell damage was measured by LDH assay. In addition, we examined protein levels of antioxidase such as HO-1 and GSS and a biomarker of apoptosis, caspase-3 after the treatment of FGF9 in the stable cell line by western blot.Results:1 The protein expression level of TDP-25 in the stable line. Human TDP-25 plasmid was successfully expressed in the stable cell line. Western blot data showed the expression levels of TDP-25 were approximately 2-fold of that endogenous TDP-43, but there was no expression of TDP-25 in the ordinary NSC-34 cells. The expression levels of endogenous TDP-43 in the stable cell line and ordinary NSC-34 cells were similar and had no markedly difference. The effect of FGF9 on cell viability of the stable cell line. There was no markedly difference in the toxicity in the different concentrations from 0 to 1μM and 10 to 200μM for 24 h. However, cell viability measured by the CCK-8 assay was significantly increased in 2 and 5μM FGF9 in the stable cell line in a dose-dependent manner(114 ± 2.54 VS 100 ± 0.91, p < 0.05;122.3 ± 2.79 VS 100 ± 0.91, p < 0.05). 3 The influence of FGF9 on cell damage of the stable cell line. The level of LDH had no significant difference in the concentration of FGF9 from 0 to 1μM and 10 to 200μM for 24 h, while it had a markedly decrease in 2 and 5μM FGF9 in the stable cell line in a dose-dependent manner(269.4 ± 18.7 VS 357.2 ± 8.59, p < 0.05;210.9 ± 34.54 VS 357.2 ± 8.59, p < 0.05). 4 The protein levels of HO-1 and GSS in the stable cell line with the treatment of FGF9. The stable cell line was exposed to FGF9 in the concentrations of 2 and 5 μM for 24 h, and western blotting analysis revealed a significantly decreasing quantity of HO-1(1.25 ± 0.09 VS 1.95 ±0.45, p < 0.05; 0.62 ± 0.04 VS 1.95 ± 0.45, p < 0.05)and increasing quantity of the GSS(0.75 ± 0.21 VS 0.41 ± 0.14, p﹤0.05; 0.92 ± 0.06 VS 0.41 ± 0.14, p﹤0.05)in the concentrations of 2 and 5 μM FGF9 compared to the control group, but there was no significantly difference of the protein levels of HO-1 and GSS between 2 and 5 μM FGF9. 5 The protein levels of caspase-3 and c-caspase-3 in the stable cell line with the treatment of FGF9. The stable cell line was exposed to FGF9 in the concentrations of 2 and 5 μM for 24 h, and western blotting analysis revealed a markedly increasing quantity of caspase-3(1.46 ± 0.12 VS 1.17 ± 0.02, p﹤0.05; 1.64 ± 0.11 VS 1.17 ± 0.02, p﹤0.05) and decreasing quantity of the c-caspase-3(0.77 ± 0.01 VS 0.91 ± 0.06, p﹤0.05; 0.70 ± 0.03 VS 0.91 ± 0.06, p﹤0.05) in the concentrations of 2 and 5 μM FGF9 compared to the control group, but there was no significant difference of the protein levels of caspase-3 and c-caspase-3 between 2 and 5 μM FGF9.Conclusions:1 In the stable cell line, low-dose of FGF9 could upregulate cell viability and promote cell proliferation. 2 In the stable cell line, low-dose of FGF9 could reduce the cellular level of LDH, indicating a protective effect of FGF9 on the stable cell line. 3 Low-dose of FGF9 could reduce the level of oxidative strss and inhibit apoptosis to protect the stable cell line from TDP-25-induced cell toxicity.