Effects Of ASC-J9 On NSC34 Cells Stable Trasfected With TDP-43

Neuroblastoma-spinal cord (NSC) was fused the aminopterin-sensitive neuroblastoma with motor neuronenriched embryonic day 12-14 spinal cord cells. NSC hybrids displaying a multipolar neuron-like phenotype: their subclones express additional properties expected of motor neurons, including generation of action potentials,expression of neurofilament tripl- et proteins, and acetylcholine synthesis, storage, and release. In addition, NSC-34 cells induce acetylcholine receptor clusters on cocultured myotub- es, and undergo a vimentin-neurofilament switch with maturation in culture, similar to that occurring in neuronal development. This will not only overc- ome the shortage of primary motor neurons in culture and time-consuming, laborious, spending high enough, but also retaining the original features of primary motor neuron. NSC-34 is commonly used cell line to study amyot- rophic lateral sclerosis (ALS).TDP-43 (43-kDa TAR DNA-binding domain protein) is a major const- ituent of ubiquitin-positive cytoplasmic aggregates present in neurons of patients with fronto-temporal lobular dementia and ALS. The pathologic significance of TDP-43 aggregation is not known. TDP-43 is normally localized to the nucleus, however, in CNS tissue from patients who died of ALS, TDP-43 is redistributed from the nucleus to the cytoplasm,where it appears to be distributed diffusely, or to aggregate as a component of ubiquitinated inclusions (UBIs). Although studies of TDP-43 in human ALS cases are generally consistent, some aspects of the TDP-43 pathology remain controversial.For instance,Mackenzie et al suggested that abnormal localization of TDP-43 is present in most sALS and fALS cases but is absent in fALS caused by SOD1 mutations. In contrast, Robertson et al showed, in two fALS cases carrying SOD1 mutations,that there is misloca- lization of TDP-43 to the cytoplasm as well as association with UBIs. This may suggest that the pathology between abnormal of TDP-43 and SOD1 mutation is different.Recently,30 kinds of TDP-43 mutations have been reported in both sALS and fALS cases. Evidence show that TARDBP genes, is likely to be a high ALS mutations gene following the SOD1 gene. TARDBP gene is on human chromosome 1p36.2, it is highly conserved and ubiquitously expressed in a variety of tissues including the brain. TAR-DNA binding protein is coded by six exon of TARDBP gene. It was identified as part of a complex involved in splicing the cystic fibrosis transmembrane conductan- ce regulator gene.The exon skipping and splicing inhibitory activity requir- es the glycine-rich C-terminal domain that binds to several members of the hnRNP family. TDP-43 has also been shown to act as a scaffold for nuclear bodies through an interaction with survival motor neuron protein. It may also be involved in mRNA stability,microRNA biogenesis,apoptosis and cell division,act as a neuronal activity-response factor involved in the regulation of neuronal plasticity.PartⅠThe culture of NSC34 cells stable trasfected with TDP-43 and the change of MDAObjective:To explore the culturing method of NSC34 cells stable trasfected with TDP-43 and the change of MDA.Thus,the stable cell lines serve as an available model to study ALS.Methods:According to the protocol of culture of motor neuron-like cell line ,to recovery,passage and culture the NSC34 cells stable trasfected with TDP-43 owned by our laboratory.The situation of growth and prolifer- ation were detected. Furthermore,we observed maleic dialdehyde (MDA) changes in the four cell lines.Results:The four different cell lines stable trasfected with empty vcet- or, wild-type (WT), Q331K and M337V mutant human TDP-43 cDNAs were culured. The level of MDA in the cell line expressed wild-type and mutant TDP-43 was higher than that in the cell line bearing empty. Conclusion:The four stable trasfected cell lines could be an ideal in vitro model to study TDP-43 in ALS.The cell lines trasfected with wild- type (WT), Q331K and M337V human TDP-43 cDNAs showed significant lipid peroxidation compared with Empty vector cell line.PartⅡEffects of ASC-J9 on NSC34 cells stable trasfect with TDP-43Objective:To research the protective effects of ASC-J9 on stable transfected with different TDP-43 on NSC34 cell lines. It will create the conditions of ASC-J9 for further study on the effect of ASC-J9 on ALS.Methods:Based on the culture of NSC34 cell lines stable transfected with TDP-43,we changed the medium. After 6-12 hours the cells were randomly divided into different groups: (1) control group:the normal medium;(2) ASC-J9 group:the cells were added with three different concentrations of ASC-J9 containing 0.1,1,and 5μmol/L.After 24 hour further culture, the cells and medium were collected.We measured the cell culture lactate dehydrogenase (LDH) content by LDH assay. The levels of MDA in the cells were measured by TBA assay.Results:After 24 hours treated with ASC-J9 in different concentratio- ns, the morphological changes of cells were not observed with invert micr- oscope. Given ASC-J9 0.1μmol/L for 24 hours, MDA and LDH did not show obvious changes. LDH enzyme activity in culture medium and MDA enzyme activity in cells decreased (P<0.01) in groups treated by ASC-J9 1,5μmol/L compared with normal control group.Conclusion:These data indicate that ASC-J9 can significantly reduce the cell MDA and LDH level and play a role in resisting cell damage of lipid peroxidation ASC-J9 may be an effective agent for clinical neurodeg- enration. ASC-J9 may have certain application such as the prevention and treatment in ALSand other neurodegenerative diseases.