S100A11 Protects Against Neuronal Cell Apoptosis Induced By Cerebral Ischemia Via Inhibiting The Nuclear Translocation Of Annexin A1

BackgroundAnnexin A1（ANXA1）is a Ca²⁺phospholipid binding protein involved in a series of cellular events,including cell proliferation,differentiation,apoptosis,migration,and repair.Our previous study found that the different subcellular localization of ANXA1 could change the final fate of neurons after ischemia-reperfusion injury.ANXA1 nucleus localization can initiate caspase cascade reaction and induce neuronal apoptosis,while its membrane localization can promote the release of anti-inflammatory medium and neurotrophic factors in microglial cells and protect ischemic injury neurons.Therefore,it will bring dawn to the treatment and prognosis of patients with ischemic reperfusion injury by inhibiting ANXA1 nuclear translocation and increase its membrane translocation and secretion.S100A11 is also a Ca²⁺phospholipid binding protein,which is easy to form a tetramer with ANXA1and is an important chaperone.Is S100A11 one of the key targets for regulating ANXA1 subcellular translocation?Is it possible that ameliorating neurological damage after cerebral ischemia by regulation the expression of S100A11?In response to these scientific questions,we have confirmed that in vivo and in vitro up-regulation of S100A11 expression could reduce neuronal apoptosis,improve neurological function after ischemia by promoting ANXA1 membrane translocation,inhibiting ANXA1 nuclear translocation.Our research initially stated that S100A11 is the important signaling molecules regulating ANXA1 subcellular translocation which may provide a new target for the development of new therapeutic methods and drugs for ischemic stroke.ObjectiveThe aim of this study was to investigate the effect of S100A11 on the subcellular localization of ANXA1,and to find out the molecular mechanism of S100A11regulating the subcellular localization of ANXA1.It is proposed that S100A11 has protective effects on neurons after cerebral ischemia-reperfusion injury.MethodsBased on the model of cerebral ischemia-reperfusion injury in mice by MCAO surgery,the neurological function score,water maze,runner experiment and open field test were used to detect the motor and learning and memory ability of mice after reperfusion injury.TTC staining was used to detect the size of ischemic infarction.N2a cells were treated with OGD/R,TUNEL staining and MTT assay were used to detect apoptosis and viability.Apoptosis was detected by flow cytometry and Annexin V-FITC-PI staining.The expression of Bid mRNA was detected by qPCR.The expression of apoptosis-related molecules was detected by Western blot.Different S100A11 and ANXA1 mutant plasmids were constructed respectively,and Co-IP and FRET assays were used to determine the binding domains between ANXA1 and S100A11.Results（1）After ischemia-reperfusion injury,the expression of S100A11 is decreased and up-regulation of S100A11 expression can play a protective role in the brain.After treatment with MCAO,immunofluorescence staining showed S100A11 significantly decreased compared with the control group.TTC staining showed overexpression of S100A11 obviously decreased the infarct volume and Longa neurological function showed the neurological function score decreased.TUNEL staining showed overexpression of S100A11 decreased neuronal apoptosis in cortex and hippocampus.The water maze,the open field experiment and the runner experiment showed that overexpressing S100A11 significantly improve spatial learning and memory ability and motor coordination ability in mice.（2）S100A11 protects against neurons injury induced by OGD/R relying on ANXA1.N2a cells were treated with OGD/R,Co-IP assay showed OGD/R decreased the interaction of S100A11 and ANXA1.MTT and Annexin V-FITC-PI showed that OGD/R+ANXA1 decreased cell viability and increased apoptosis compared with OGD/R group;however,in OGD/R+S100A11+ANXA1 group compared with OGD/R+ANXA1 group,cell viability was improved.（3）S100A11 is involved in reducing the expression of apoptosis-related molecules caused by ANXA1 nuclear translocation after OGD/R.qPCR results indicated that S100A11 could decrease the level of Bid mRNA involed in ANXA1 in N2a cells after OGD/R.Western Blot results indicated that S100A11 could decrease the level of tBid,cleaved caspase-3,and cleaved PARP protein involed in ANXA1 in N2a cells after OGD/R.Annexin V/FITC results indicated that both S100A11 and ANXA1 regulated neurons survival depending on the caspase-3 apoptosis pathway.（4）Overexpression of S100A11 decrease ANXA1 nuclear translocation and increase membrane translocation following OGD/R.N2a cells were treated with OGD/R,immunofluorescence and Western blot showed that OGD/R increased the distribution of ANXA1 membrane and nucleus and decreased cytoplasmic accumulation.After up-regulating the expression of S100A11,the distribution of ANXA1 cell membrane increased and the nuclear distribution decreased.After down-regulating the expression of S100A11,the distribution of ANXA1 cell membrane decreased and the nuclear distribution increased.（5）S100A11 directly suppresses ANXA1 nuclear translocation after OGD/R.After treatment with OGD/R,Western blot showed that shPEX14 decreased the distribution of ANXA1 cell membrane.The distribution of cytoplasm increased and the distribution of nuclear did not change significantly.On the other hand,in the OGD/R+PEX14 group compared with the OGD/R group,the ANXA1 cell membrane distribution increased,the cytoplasmic distribution decreased,and the nuclear distribution did not change significantly;while the OGD/R+PEX14+shS100A11 group was relative to the OGD/R+shS100A11 group,there was no significant change in the distribution of cell membrane,cytoplasm and nucleus of ANXA1 in N2a cells.（6）S100A11 interacts with NTS of ANXA1.N2a cells were transfected with different ANXA1 mutant plasmids,Co-IP showed that N-terminal and C-terminal of ANXA1 protein could bind to S100A11;while ANXA1-ΔN-ΔNTS could not bind to S100A11.The binding of different ANXA1 mutant plasmids to S100A11 was further verified by fluorescence resonance transfer experiments.The results showed that both ANXA1-ΔN and ANXA1-ΔNTS could bind to S100A11,but ANXA1-ΔN-ΔNTS could not bind to S100A11.（7）S100A11 Leu⁴²-Ile⁹⁸ is involved in the direct inhibition of ANXA1 nuclear translocation and reduces the expression of apoptosis-related molecules in N2a cells.Western blot analysis showed that S100A11-（42-98）could mimic the function of full-length S100A11 to promote ANXA1 membrane translocation and inhibit ANXA1 nuclear translocation after OGD/R.After treatment with OGD/R in N2a cells,qPCR results showed that S100A11-（42-98）decreased Bid mRNA expression caused by overexpression of ANXA1.Similarly,Western Blot showed that S100A11-（42-98）reduced the expression of tBid,cleaved caspase-3,and cleaved PARP protein caused by overexpression of ANXA1.TUNEL staining showed that overexpression of S100A11-（42-98）decreased cells apoptosis induced OGD/R.ConclusionsTaken together,we first discovered that S100A11 is involved in the regulation of subcellular localization of ANXA1 in neurons after cerebral ischemia.S100A11inhibits the nuclear traslocation of ANXA1 by competing with Importinβfor nuclear localization signal（NTS）,reducing the transcription of Bid gene,thereby inhibiting the caspase pathway,and ultimately reducing neuronal apoptosis caused by cerebral ischemia-reperfusion injury.In addition,we also initially explored the key domain of S100A11 binding to ANXA1.Therefore,studies aiming to identify the factors that specifically block the nuclear translocation of ANXA1 may provide promising targeted strategies for the treatment of ischemic stroke.