| The pathogenesis and treatment of spinal cord injury(SCI) have been one of the most difficult and attractive field in the neuroscience. Our preliminary proteomics analysis suggested that expression of the microtubule-associated protein tau is elevated in the spinal cord after injury which seized our attraction.Microtubule-associated protein tau has been focused studied on its relationship with central nervous system disease such as Alzheimer’s disease and overexpression of tau in neuron was closely related to nerve injury. However, there is little research available on tau expression in SCI. Therefore, the first aim of the present study was to examine tau expression in the injured spinal cord, which provide theoretical basis for tau expression in peripheral nervous system desease.Traditional treatments for SCI have many limitations. With great advance in cell biology in recent years, SCI treated by cell transplantation has attracted more and more attention. Neural stem cells(NSCs), which have the potential to differentiate into neurons and glial cells, releasing neurotransmitters and producing neurotrophic factors, are regarded as a promising treatment for SCI. The most important factors for the success of NSC transplantation in the treatment of SCI are the migration and differentiation abilities of the NSCs. Dr. Yang in our research group found microtubule associated protein could affect cell migration. Previous studies have shown that tau plays an important regulatory role in neuronal migration and tau phosphorylation modification may play an important role in this process. However,the relationship between phosphorylation of the microtubule-associated protein tau and NSCs migration has not been examined to date. Here, we used agonist and inhibitor of protein phosphatase 2A(PP2A) to change tau phosphorylation level.Transwell assay and stem cell transplantation was used to explore howpost-translational modification of tau(phosphorylation/dephosphorylation) affects NSC migration in vitro and in vivo. We utilized PI3 K,ERK,p38 MAPK and JNK pathway specific blocker to pretreat NSCs, observing p-tau expression and NSCs migration by western blot and transwell assay. Our present study will provide new perspective on affecting factors and related pathway of NSCs migration, then offer theoretical basis for SCI treated by NSCs transplantation.Objective:The first aim of this study is to examine tau expression in the injured spinal cord.The second aim was to determine whether tau phosphorylation can regulate neural stem cell migration, a critical factor in the successful treatment of spinal cord injury.We also want to investigate the related pathway of NSCs migration.Method:Part 1: Twenty adult Sprague-Dawley rats were equally and randomly assigned to an SCI group and a control group. In the SCI group, the abdominal aorta was blocked with a vascular clamp for 30 minutes;.The control group underwent the same procedure except that the abdominal aorta was exposed for 30 min without obstruction, before the incision was sutured. On the days 1, 3, 5 after surgery, when the rats had regained consciousness, three investigators who had not participated in the model establishment performed hindlimb motor function testing using the modified Tarlov score. HE staining was then performed to observe pathologic changes.Part 2: Fetuses were obtained from mouse uteri. Hippocampi were isolated at embryonic day 14 under a dissecting microscope, cut into pieces, triturated mechanically. 5 × 105 cells were incubated in a culture flask. The NSCs morphologic changes were observed for 7d. Anti-nestin antibody was used to indentify NSCs.Part 3: L3–5 spinal cord segment was obtained from rats of both groups, sectioned and collected for free-floating immunohistochemistry. Immunohistochemistry.and western blot were performed to make qualitative and quantitative analysis for tauexpression in injured spinal cord.Part 4: We used agonist and inhibitor of PP2 A to change tau phosphorylation level.Radioactive counting of 32 p labeled substrate and MTT was used to determine PP2 A and NSCs activity respectively. NSCs was randomly assigned to control group, OA group, C2-ceramide group and unpretreated group. Each group were analysized by western blot and transwell assay to observe tau/p-tau expression and NSCs migration.We transplanted DAPI labeled NSCs,to SCI model, investigating NSCs migration 3days after transplantation.Part 5: We utilized PI3 K, ERK, p38 MAPK and JNK pathway specific blocker to pretreat NSCs, observing p-tau expression and NSCs migration by western blot and transwell assay.Result:Part 1: When the rats regained consciousness, hindlimb motor function was evaluated using the Tarlov scoring system. Hindlimb function was normal in the control group(6.00 ± 0.00 points), but markedly impaired in the SCI group(2-3 points). HE staining showed neuronal swelling and deformation as time went on, indicating success of the SCI model.Part 2: Neurospheres were observed after primary cell culture, proliferation and passage. As time in culture increased, the neurospheres grew, refractive index decreased, and boundaries became clearer. No growth of processes was observed.Nestin immunoreactive neurospheres had been discovered. The cells grew well and met the requirements of subsequent experiments.Part 3: Immunohistochemistry revealed swollen and deformed cells in the SCI group,with dark stained cytoplasm. In the control group, cells had a normal shape, and tau was observed in the cytoplasm and axon. Average optical density value and positive neurons were significantly higher in the SCI group than in the control group. Western blot also showed tau expression levels normalized to b-actin increased gradually after SCI.Part 4: 32 p labeled substrate and western blot revealed together that OA andC2-ceramide intervene PP2 A activity and tau phosphorylation. Tau expression levels normalized to b-actin had no significant difference among three groups on days 1, 3, 5 after cell treatment. P-tau expression levels normalized to b-actin had no significant difference among three groups on days 3 and 5 after cell treatment. Compared with unpretreated group at the first day after cell treatment, p-tau expression levels of OA group was significantly higher while C2-ceramide group showed the opposite. Transwell showed that optical density of crystal violet was significantly higher in the unpretreated NSCs than in the groups exposed to OA and C2-ceramide. Growth curve of NSCs pretreated with OA, C2-ceramide and DAPI were almost normal. DAPI labeled NSCs migrated to injured spinal cord segment, while migrated NSCS in unpretreated group was significantly higher in the OA group and C2-ceramide group.Part 5: The relative densitometry showed expression of p-tau in PI3 K group was significant higher than unpretreated group. P-tau expression in ERK, p38 MAPK and JNK groups was significant lower than unpretreated group. Compared with unpretreated NSCs, NSCs pretreated with PI3 K and MAPK pathway blocker showed weaker migration ablility by transwell assay.Conlusion:1. Tau overexpressed in the inured spinal cord. Tau expression and tau-positive neuron number have positive correlation with the degree of SCI.2. NSCs have targeted migration capability at the site of injury, intracellular tau phosphorylation/dephosphorylation dynamic balance can ensure NSCs normal migration. PI3 K, ERK, p38 MAPK and JNK pathway get involved in NSCs migration.Innovation:1. Our study made qualitative and quantitative analysis for tau expression in injured spinal cord.2. We focused on the relationship between tau phosphorylation and NSCs migration,suggesting tau phosphorylation/dephosphorylation dynamic balance is significant inNSCs migration, which have not any relate reports in the world.3. Our paper also demonstrates that NSCs migration influenced by tau phosphorylation is regulated by some cellular pathways like PI3 K and MAPK.Key words... |
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