Mechanism Of AQP4 Regulating BDNF Secretion In Astrocytes

Aquaporins(AQPs)are a group of membrane-integrating proteins that are widely involved in water transport in animal and plant cell membranes.They are involved in the transmembrane of water molecules and regulate the homeostasis of the intracellular environment.Currently,13 water channels have been identified in mammals 11,VllAmong them,AQP4 is considered to play an important role in regulating cell homeostasis,potassium ion clearance,and water balance.Recent years,AQP4 has been found to have different effects in garbage cleaning?cell volume dynamics?k+scavenging?neuronal excitability?synaptic plasticity and behavior,and sensing systems:the retina and the inner ear.As a membrane integrin,in addition to providing a basic water balance[3,it has been discovered since 2003 that inhibition of AQP4 expression can lead to NMDA receptor dysregulation and damage to LTP,which is induced by 0 burst,specific dependent on BDNF-TRKB receptorU4],which suggested that there is a correlation between AQP4 and neurotrophic factors.Up to 2019,the specific mechanism of AQP4 involved in the regulation of BDNF has not been studied,mainly related to the cognitive impairment caused by AQP4 and BDNF[5].From the“Yin and Yang Balance Hypothesis"of BDNF in 2009 to the "Continuous Sorting Hypothesis" proposed in 2018,suggesting that we need to evaluate the role of neurotrophic factors as a whole[6].Moreover,the role of brain-derived neurotrophic factor(BDNF)is not limited to mBDNF-TRKB signal coupling,and proBDNF plays an important role in the regulation of BDNF production?secretion?scission and overall cell survival and apoptosis.At the same time,it suggests that new targets and treatment methods will be selected in the future for anti-depression and related neurodegenerative diseases.However,there are few studies on proBDNF and mBDNF at present,and the corresponding mechanism research is also lacking.Two articles on BDNF signal transduction and signal integration published in PNAS and Nature in 2018 suggest that BDNF signal transduction is accompanied by the integration of multiple proteins.and BDNF has a lot of research prospects,many fields are still unclear[7,8].In 2005,Chen et al.found that there is specific binding of the precursor domain of BDNF and sortilin[9],and such complex formation is sufficient to affect the secretion mode of BDNF in cells.In 2019,Jason et al.found that changing the splicing of sorting proteins could change the secretion of activity-dependent BDNF,impair synaptic plasticity and cognitive behavior[10],which were closely related to the precursor domain of proBDNF.This suggests that the BDNF precursor domain plays a significant role in synaptic plasticity and its own secretion.In this work,it was found for the first time that AQP4 knockout can change the expression of BDNF in the hippocampal and cortex.In the depression model,the BDNF expression of AQP4 knockout mice showed specific differences compared with the wild type,suggesting that the regulation of BDNF by AQP4 can affect the expression of BDNF in basic and disease models.Based on the distribution and localization of AQP4 in the brain,we focused on the mechanism of AQP4 regulating the expression of mature BDNF cells in astrocytes,in order to illustrate the mechanism of AQP4 involved in BDNF regulation in the brain.OBJECT:To investigate the specific mechanism of AQP4 regulating the expression of BDNF precursors and matures in astrocytes during the process of astrocyte production and secretion of BDNF.METHOD:Eight-week-old male CD1/AQP4+/+ and CD1/AQP4-/-mice were randomly divided into six groups:AQP4+/+normal control group,AQP4+/+chronic mild stress(CMS)group,AQP4+/+CMS model group+Fluoxetine treatment group,AQP4-/-normal control group,AQP4-/-CMS model group,AQP4-/"CMS model group+fluoxetine treatment group.Each single cage was kept,and the control group was routinely kept.The sucrose preference test(SPT)was used to observe whether the model was successful.1)Western blotting was used to detect the expression of BDNF precursor(proBDNF)and mature(mBDNF)in hippocampus and cortex brain tissue proteins;2)AQP4+/+ and AQP4-/-primary astrocytes were cultured,and the expression of proBDNF and mBDNF in cells was detected by western blotting,and the expression of proBDNF in secreted proteins was detected;3)ELISA was used to detect the intracellular and extracellular BDNF content of astrocytes;4)Detection of neurotrophin mRNA expression in hippocampus?cortex and astrocytes by QPCR;5)Wesrtern blotting was used to detect the expression of BDNF upstream promoters(3-catenin?CREB and phosphorylated CREB(pCREB)in hippocampus,cortical brain regions and astrocytes;6)Detection of binding efficiency of intracellular sorting protein sortilin to proBDNF domain in astrocytes by COIP;7)Flow cytometry was used to detect the amount of calcium ions in AQP4+/+and AQP4-/-astrocytes incubated with the calcium probe Fluo-3 am,and to detect the content of calcium ions in astrocytes of AQP4+/+ and AQP4-/-stars after administration.8Using TSA-conjugated multiplex fluorescent staining-cell technique to observe the binding efficiency of sortilin to proBDNF in AQP4+/+and AQP4-/-astrocytes.RESULTS:1)Abnormal expression of BDNF precursor and mature protein in hippocampus and cortical brain tissue of AQP4 knockout mice.Under the basal state,AQP4 knockdown resulted in increased expression of proBDNF in hippocampus and cortex of mice,and decreased expression of mBDNF(p<0.05).After CMS modeling,proBDNF in hippocampus and cortex of AQP4+/+group increased,and mBDNF decreased(P<0.05).In the AQP4-/-group,there were no differences of proBDNF and mBDNF in hippocampus and cortical after CMS modeling,and the therapeutic effect of fluoxetine was abolished.,which suggested that AQP4 regulates the expression of'proBDNF and mBDNF in the brain;2)AQP4 gene knockout leads to abnormal expression of BDNF precursor and mature protein in astrocytes.Under basal conditions,AQP4 knockout resulted in increased expression of intracellular proBDNF in primary astrocytes and decreased expression of mBDNF(p<0.05).AQP4 knockout did not affect BDNF mRNA levels and upstream promoters CREB??-catenin and p-?-catenin in hippocampus and cortex.QPCR detected the mRNA levels of BDNF?NGF?GDNF and CDNF in hippocampus and cortex of AQP4+/+group and AQP4-/-group.It was found that AQP4 knockout did not affect the mRNA levels of various brain trophic factors in the hippocampus and cortex.At the same time,under the basic state,western blotting was used to detect the BDNF?upstream promoter ?-catenin,?CREB and pCREB,which found that AQP4 knockout had no significant effect on ?-catenin,CREB and pCREB levels in the brain.3)AQP4 gene knockout did not affect the formation of BDNF in astrocytes.QPCR detected the expression of BDNF mRNA in astrocytes and found that there was no significant difference in BDNF mRNA between AQP4 knockout and control group.There was no significant difference in the level of P-catenin?CREB and pCREB in BDNF upstream by western blotting.By ELISA kit,we found that there was no significant difference in the total amount of BDNF in astrocytes between AQP4-/-and AQP4+/+group.It indicated that AQP4 did not participate in the formation of BDNF in astrocyte.4)AQP4 gene knockout inhibits astrocyte exocrine BDNF.The ELISA kit was used to detect the extracellular BDNF content at 6h?12h and 24h after stimulation with hunger The direction of AQP4 knockout resulted in a decrease in the amount of BDNF secreted by astrocytes to the extracellular.Significant differences(p<0.05)occurred from 12 h of hunger.Western blotting was used to detect the supernatant protein and found that the secretion of proBDNF was decreased(p<0.01).5)AQP4 gene knockout leads to a decrease in BDNF secretion by astrocytes and is associated with intracellular calcium ion content.AQP4 knockdown can result in decreased expression of calcium channel protein on cells(Laboratory preliminary results);administration of NMD A?AMPA receptor inhibitors can restore abnormal secretion of BDNF in AQP4-/-astrocytes.Calcium channel blockers can cause a decrease in BDNF secretion in AQP4+/+astrocytes.By flow cytometry,the calcium ion content of AQP4 knockout astrocytes in the basal state was higher than that of the control group.The administration of NMD A and AMPA receptor inhibitors can reduce the intracellular calcium content of AQP4-/-astrocytes.6)AQP4 gene knockdown inhibits the binding of sortilin to the BDNF precursor domain.There was no significant difference in the protein expression of sortilin in AQP4-/-and AQP4+/+astrocytes.In the basal state,the binding of sortilin to the BDNF precursor domain in AQP4-/-astrocytes was reduced(p<0.05).This indicates that AQP4 inhibits the binding of sortilin to the BDNF precursor domain.In summary:AQP4 knockout increases intracellular calcium levels and attenuates the binding of sortilin to proBDNF,inhibiting the secretion of BDNF by astrocytes.The decrease in proBDNF that lead to intercellular involvement in splicing ultimately leads to a decrease in the production of mBDNF,which in turn alters the expression of BDNF in cells?hippocampus and cortex precursors.