The Neurogenesis Role Of Annexin 2 In Traumatic Brain Injury And Its Related Mechanism

ObjectiveNeuron loss caused by traumatic brain injury?TBI?leading severe disability and neurological dysfunction,which bringing great mental stress and economic burden to patients and their families.However,there are still no effective treatment to restore impaired neurofunction.Previous study demonstrated that there are neural stem cells and neural progenitor cells in the subventricular zone?SVZ?and subgranular zone?SGZ?of the adult mammal's brain,including human being.Interestingly,TBI not only causes brain damage,but also triggered the neural regeneration process,including neural progenitor cells proliferation,migration and etc.It is promising to study the mechanism of neural regeneration after TBI for repairing damaged neurofunctions.Annexin 2 is a pleiotropic protein encoded by ANXA2 gene,closely related to cytoskeletal dynamics.It reported that Annexin 2 involved in survival,proliferation,migration and signal transduction of various cells.Recent study reported that ANXA2 gene deficiency mice have worse long-term neurological deficits after TBI.Whether Annexin 2 participates in nerve regeneration process after TBI has not been reported.To this end,in present study,firstly,we established a mouse TBI model through Controlled Cortical Impact?CCI?to detect the expression and distribution of Annexin 2,and then interfere ANXA2expression in TBI mice to detect its role of Annexin 2 on neural function recovery after TBI.Secondly,we tested the effect of ANXA2 interference or over-expression on the proliferation and migration of neural progenitor cells in neurospheres cultures and SVZ explants.Finally,we apply recombinant annexin 2?rA2?in vivo and in vitro to explore the molecular mechanism of Annexin 2 on promoting neural progenitor cells proliferation and migration.Methods1.Tissue samples near the SVZ of peri-injured side of the adult human TBI brain were collected from the Forensic Science department of Chongqing Medical University.Then paraffin-embed and section.The expression of DCX,a neural progenitor cells marker and the expression of Annexin 2 were detected by immunostaining for the evidence of neurogenesis in adult human TBI brain and its perusable relationship between Annexin 2 and neuroblast.2.Sixty adult,male C57BL/6 mice were randomly divided into sham group?n=12?and TBI group?n=48?,and then a moderate TBI model was established by CCI.Western blotting was used to detect the expression trend of Annexin 2 in peri-injured hemispheres at 6h and 1,3,5,7,14,28days after CCI.Annexin 2 distribution in the mice brain at 7day after TBI were detected by immunostaining.3.Fifty-eight adult,male C57BL/6 mice were randomly divided into4 groups:sham group?n=13?,vehicle group?n=13?;siR-ANXA2 group?n=16?;and siR-scramble group?n=16?.ANXA2 interference adeno-associated virus or control reagent was injected into the lateral ventricle at 15 days before CCI.Wire grip test and Neurological severity scores?NSS?were detected pre and 1,3,5,7,14,21 days after CCI.Morris water maze?MWM?test was performed at 16 day after CCI.4.Neural progenitor cells and SVZ explants were extracted from the SVZ area of C57BL/6 mice and cultured in vitro and ANXA2 interference or overexpression adenovirus were applied to modulate Annexin 2expression.The ratio of BrdU positive cells was detected by immunostaining in neurosphere cultures.The migration distance of neural progenitor cells was measured in SVZ explant.5.Recombinant Annexin 2?rA2?and OSU-03012,a PDK1 inhibitor,was added in neurosphere culture medium,and then proliferation and migration related PDK1-Akt-Girdin pathway were detected by Western blotting.Twenty-four adult,male C57/BL6 mice were randomly divided into sham group,Control group,rA2 group,rA2+OSU-03012 group,6mice in each group.rA2 or equal volume of control medium was injected through the lateral ventricle catheter,and OSU-03012 was injected intraperitoneally at 6h after CCI and once a day for ten consecutive days.And on the third day after CCI,BrdU at a dose of 50 mg/kg was injected intraperitoneally once a day and for seven consecutive days.The DCX,BrdU and NeuN were detected by immunostaining at 14 day after CCI.Results1.DCX-positive neural progenitor cells were found in all samples of three adult human TBI brain,and these cells simultaneously expressed Annexin2.2.The expression of Annexin 2 in the injured hemisphere after CCI was significantly increased begin at the 6th hour?**p<0.01?,and continued to increase until it reached a peak on the 7th day?****p<0.0001?,and then gradually decreased.Immunostaining confirmed that the increased Annexin 2 expression was mainly distributed near the injured side of SVZ,and it was co-expressed in DCX positive cells in the SVZ area.3.Down-regulate the expression of Annexin 2 in the brain of mice after CCI,reduced the performance of NSS and wire grip test?****p<0.0001?,which indicate weakened motor-sensory functional recovery.And it also increased the latency for find the hidden platform?****p<0.0001?,reduced the proportion of time spent in the platform quadrant?*p<0.05?and reduced the times crossed the original hidden platform position?*p<0.05?.in MWM test,that indicate more worsen learning and memory ability.4.Down-regulate the expression of Annexin 2 in neural progenitor cells,reduced the ratio of BrdU positive cells in neurospheres?**P<0.01?and reduced the migration distance of neural progenitor cells in SVZ explants?***P<0.001?;Up-regulate the expression of Annexin 2 in neural progenitor cells,increased the ratio of BrdU positive cells in neurospheres?****P<0.0001?and increased the migration distance of neural progenitor cells in SVZ explants?####P<0.0001?.5.The phosphorylation of PDK1^ser241er241 and downstream molecule Akt^ser473er473 and Girdin^s-1416?****P<0.0001?were increased after rA2treatment in neurosphere in vitro by western blotting.Continuous injection of rA2 in the lateral ventricle in mice after CCI increased the proportion of DCX positive area in the SVZ?####P<0.0001?,and increased the number of DCX/NeuN double-positive cells?****P<0.0001?and BrdU/NeuN double-positive cells?***p<0.001?in peri-injury site.And it also increased the number of DCX positive cells in the SGZ?###p<0.001?and the number of BrdU/NeuN double-positive cells hippocampal?##p<0.01?by immunostaining.OSU-03012 abolished the effect of rA2 on neuroblast proliferation and migration.ConclusionAnnexin 2 promote neural progenitor cell proliferation and migration through activates PDK1-Akt-Girdin signaling pathway after TBI.