The Protective Effect Of IGF-1 On Cellular Stable Model Of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis(ALS) is a progressive, fatal neurodegenerative disease, that principally affects brain and spinal cord, upper and lower motor neurons, finally leading to paralysis and dead. However,the exact etiology pathogenesis is still unknown and there is no effective therapy.Recently,the abnormal ubiquitin protein aggregates are presented in the motor neurons and glial cells in ALS patients and frontotemporal lobar degeneration with ubiquitin-positive inclusions(FTLD-U) patients.Their main components are TARDNA binding proteins(TDP-43) of 43 KDa,which are encoded by TARDBP gene. Normally TDP-43 mainly localizes within the nucleus,which is involved in splicing and transcriptional regulation.TDP-43 is a 414-amino-acid protein with two RNA recognition motifs and a carboxy-terminal glycine-rich domain.Recent studies have shown that mutations in TARDBP are supposed to be one of the most common mutations in ALS patients,and nearly all of the described ALS-associated TDP-43 mutations are dominant missense mutations within the glycrine-rich domain. The fundamental question of whether mutant TDP43 mediates neurodegeneration through gain of toxic function or a loss of normal function remains unanswered. TDP-43 can be cleaved into a 25 KDa C-terminal fragment(TDP25) which is more prone to misfolding and aggregation.TDP25 plays an important role in the pathological process of ALS.It is reported that stable expressed TDP25 exhibits cytotoxicity in cells and could induce significant oxidative stress and apoptosis.Neuronal development and survival depend on a balanced and tightly regulated support from trophic factors.Such factors are capable of regulating several important physiological processes,such as cell proliferation,neuronal differentiation, synapses formation, and axonal outgrowth.Insulin-like growth factor 1(IGF-1) is a neurotrophic factor which belongs to the insulin family. IGF-1 is used therapeutically in disorders ranging from growth deficiency to diabetes due to its role in growth and metabolism.Now interest in its potential use in neurodegenetative disorders continues to grow.Recent studies have shown that IGF-1 has neuroprotective properties in the central and peripheral nervous systems. IGF-1 can activate the PI3K/Akt and p44/p42 MAPK signaling pathways.The signaling pathways activated by IGF-1 result in a wide range of cellular effects including cell proliferation,differentiation and inhibition of apoptosis.Additional pathways linked to IGF-I signaling include the JNK, p38 MAPK,and m TOR signaling pathways.However,the IGF-1 signaling system is complex and regulated by many factors like IGF-1 receptor(IGF-1R) and insulin-like growth factor binding proteins(IGFBPs).A deep understanding of the IGF-1 system may offer a new therapy for ALS.Objective:To investigate the effects of IGF-1 on NSC34 cells stably expressing TDP25 proteinMethods:The NSC34 cells stably expressing TDP25 were used in this study as a cellular model of amyotrophic lateral sclerosis.After treatment with different concentrations(0ng/ml, 0.1ng/ml, 1ng/ml, 10ng/ml, 100ng/ml, 1000ng/ml) of IGF-1 in NSC34 cells for 24 hours, its effect on the cell morphology was observed by optical microscope,and the cell viability was measured by the CCK-8 assay,finally the optimal drug concentration was decided.After NSC34 cells were exposed to appropriate concentrations for 24 hours,we evaluated the cytotoxicity of TDP25 through LDH and MDA assay. After NSC34 cells were exposed to appropriate concentrations for 24 hours, we examined caspase3(marker of apoptosis) and HO-1(antioxidative protein) level by Western blot.Results:(1)Western blot showed that there was expression of exogenous TDP25 protein in NSC34 cells which was stably transfected by TDP25 plasmid,so the cell model of ALS was established successfully.(2)After treatment with different concentrations(0-1000ng/ml) of IGF-1,we found that 100ng/ml IGF-1 could significantly promote axonal growth,and maintain normal cells form.CCK-8 assay showed that compared with control group, the cell viability was all increased in different drug groups except 0.1ng/ml IGF-1, and the increasing was more remarkable in 10ng/ml and 100ng/ml IGF-1 groups.So finally we chose 10ng/ml and 100ng/ml as the optimal drug concentrations.After NSC34 cells were treated with IGF-1 [0ng/ml, 10ng/ml(low does),100ng/ml(high does)] for 24 hours,LDH and MDA assay showed that both 10ng/ml and 100ng/ml IGF-1 could reduce TDP25 toxicity and there was no marked statistical difference between low does group and high does group.(3)After NSC34 cells were treated with IGF-1[0ng/ml, 10ng/ml(low does),100ng/ml(high does)] for 24 hours,Western blot showed that the two drug groups could inhibit caspase3 expression and up-regulate HO-1 protein level compared with control group,and the regulations is more remarkable in high does group.Conclusions: IGF-1 could reduce cytotoxicity and inhibit apoptosis and oxidative stress in cellular model of ALS stably expressing TDP25.IGF-1 of high does could promote axon growth which exerts a neuroprotective effect.