Homer1a Protects Brain From NMDA Induced Brain Injury And Correlation With NNOS

Traumatic brain injury (TBI) is one of the main causes for disability in human. Thebrain tissue damage caused by traumatic brain injury can be divided into the primary braininjury and secondary brain injury. Secondary brain injury is the important factor to affectthe clinical outcomes of traumatic brain injury. After brain damage, amount of glutamatereleases at early stage, and glutamate can keep different kinds of Ca2+channels open andcontribute to the excitatory neurotoxicity, which play an important role in the secondaryinjury. The NMDARs opened after combination with glutamate, and amount of Ca2+couldget through from NMDA receptors. Moreover, the slow inactivation of NMDAR keepsvery high Ca2+concentration in neurons after injury and leads to a heavier brain damage.Therefore, the study to reduce the neuronal damage caused by the NMDA receptor has aremarkable clinical significance.Homer1a belongs to the Homer protein family, which is an IEG (Immediate EarlyGene). Recent studies have found that Homer1a regulates a variety of cellular andmolecular functions in brain diseases. As member of the PSD (postsynaptic density),homer1a could regulate the signal transduction between the PSD. Moreover,Homer1a canwork as an important calmodulin, and Homer1a play negative regulatory roles by physicalreplacement of some proteins. According to previous studies, Homer1a could interact withShank, NMDA, nNOS and other proteins. However, the relationship between homer1a andNMDA induced neuron injury has not been studied yet, and how homer1a acts onNMDAR and NMDAR’s downstream pathways was still not known, so it is extremelymeaningful to investigate the effects of homer1a on NMDA induced neuron injury.Part I Establish of Mouse NMDA injury modelObjective: To establish a simple and reliable NMDA brain injury model at the animal level, for the further investment of the pathophysiological changes and the mechanisms ofbrain injury. Methods: To clarify the effect of NMDA injury model in mice, mice wasdivided into two groups randomly according to the treatment. We detected the changes ofNSS, serum NSE, and the toluidine blue staining was carried out after brain damage.Results: Toluidine blue staining showed that: after the cortical injection of NMDA in mice,the cortical neurons and nissl bodies were clear in sham group with no damage to cortex;while in NMDA injury group, there is a significantly reduce of neurons, the integrity ofcortex was broken at24h. For the behavior of mice, NMDA injury greatly increased theNSS at24h. In addition, NMDA injection increased the serum NSE at12h and24h.Conclusion: The mouse NMDA injury model induced by NMDA cortex injection is asuccessful and simple model.Part II NMDA injury model in vitroObjective: In order to study NMDA injury in vitro, we established an effective andreliable NMDA injury model in vitro. Methods: Purity of culture neurons wasidentificated at7d after cortex neurons was cultured. In accordance with the method ofKoh et al, we built similar model with addition of NMDA in cultured cortex neurons. Thenthe lactate dehydrogenase (LDH) release, ROS generation, Hoechst stain experiments wascarried out to value the injury effects of NMDA to neurons. Results: The NF200stainingshows a perfect purity of culture neurons. After NMDA injury, the LDH concentrationincreased from6h to24h compared with the control group. At24h, the apoptosis rate andROS fluorescence intensity significantly increased compared to the control group.Conclusion: By detection of LDH, ROS, as well as the change of Hoechst staining, weconfirmed that the NMDA injury model of cortical neurons is a simple and effective toolfor the study of NMDA neuron injury.Part III Homer1a expression after NMDA brain injury in vitro and in vivoObjective: To investigate the expression of Homer1a after NMDA brain injury invivo and in vitro. Methods: Base on NMDA brain injury model in vitro and in vivo, westudied the changes of Homer1a protein expression using western blotting andimmunohistochemistry, while the expression of Homer1a mRNA by RT-PCR. In addition,mice was randomly divide into NDMA injury group and sham group. Results: In vivomodel, we observed the changes of Homer1a protein expression in the surrounding braintissue, and found that the rate of Homer1a positive neurons in NMDA injury group is higher than that in the control group at24h. In vitro model, we find the expression ofHomer1a protein significantly increased at6h to24h; moreover, Homer1a mRNA greatlyincreased at1h and3h using RT-PCR compared to the control group. Conclusion: Theexpression of protein levels and mRNA levels of Homer1a increased after NMDA injury,suggesting that the Homer1a may play important roles in NMDA injury, and it may beinvolved in the mechanisms of neuron’s self-protection.Part IV Study of NMDA injury effects to Homer1a K/O MouseObjective: To study of the NMDA injury effects to Homer1a K/O mouse. Methods:We identified Homer1a gene knockout mice, and then the mice were divided into twogroups, KO mice group and WT mice group. To study the difference of NMDA impact onthese different group, the mouse NSS, serum NSE, and toluidine blue stain was detectedand recorded. Results: We identified Homer1a knockout mice successfully. There is noHomer1a gene expression, while normal Homer1c gene expression in these K/O mouse.Nissl staining showed enlarged infarct size in Homer1a K/O group compared with WTgroup at24h after the injection of NMDA. Deletion of Homer1a gene increased NSS at12h,24h, and showed a higher serum NSE level in Homer1a K/O group at24h.Conclusion: The data indicate that the deletion of Homer1a gene increased NMDAinduced brain injury, while homer1a may have protective effects against NMDA inducedinjury.Part V The effects of overexpression of Homer1a to the NMDA induced injury invitroObjective: Previous experiments suggested the knockout of Homer1a gene increasedNMDA induced brain injury, so homer1a may have protective effects against NMDAinduced injury in vitro. Using the Lentiviral vector, we overexpressed Homer1a in vitro tostudy the effects of homer1a to NMDA induced neuron injury. Methods: After thetransfection of LV-Homer1a to cultured cortical neurons, we identified the expression ofHomer1a.Neurons was divide into threes groups according to treatment, then we observedthe effect of overexpressed Homer1a on LDH release, apoptosis and p-caspase-3expression after NMDA induced injury. Results: Western blot analysis showed that thetransfection of LV-Homer1a could effectively increase the level of Homer1a in neurons.Compared with the empty vector group, transfection of LV-Homer1a can significantlyreduce the apoptosis rate of neurons, and inhibit the increase of LDH and the expression ofp-Caspase3after NMDA injury. Conclusion: These results suggest that Homer1a has a protective effect on neurons after NMDA injury in vitro.Part VI Effects of Homer1a on NMDA induced injury and nNOS activityObjective: The previous experiments confirmed a direct protective of Homer1a effecton the NMDA induced neuron injury, but the mechanism is not clear yet. This study wasdesigned to explore the effects of Homer1a on NMDAR’s downstream pathways, andwhether nNOS activity was effected was also studied. Methods: After the transfection ofLV-Homer1a to cultured cortical neurons, neurons was divide into different groupsaccording to the treatment. After the establishment of NMDA injury model, we investedthe cellular ROS, Ca2+, and carried out whole-cell patch-clamp recording for the peakvalue of NMDA. Western Blot was used to measure the expression of p-nNOS, p-ERKand p-CREB of each group after NMDA injury. Results: Using the LV-Homer1a, wefound that the overexpression of Homer1a could significantly decrease the generation ofROS, Ca2+influx compared to the empty vector group after NMDA stimulation and thepeak value of NMDAR was decreased, too. Transfection of LV-Homer1a decreased theexpression of p-nNOS, p-ERK and p-CREB compared to empty vector. Conclusion:These results suggested that the transfection of LV-Homer1a changed the properties of theNMDAR and reduced the permeability and the activity of downstream pathwaysdecreased, too. This laid the foundation to further clarify the mechanisms of protection ofHomer1a in NMDA induced injury.Part VII Effects of Homer1a on NMDAR and NMDAR complexObjective: The previous experiments confirmed a direct protective effect ofHomer1a to NMDA-induced neuron injury, and change its properties, as well as theactivity of the downstream pathways, but the specific mechanism is not clear yet. Wespeculated that the protective effect of the Homer1a may rely on the regulation ofNMDAR distribution and the regulation of related NMDAR complex.Methods:HEK293T cells was co-transfected with NR1,NR2B receptor as well as Homer1a,cell wasdivide into different groups according to the different molecular been transfected, thencells were committed with patch clamp recording, for study the direct effects of Homer1ato the NMDA receptor. After transfected with LV-Homer1a in neurons, membrane proteinwas extracted and purified, and the distribution of NMDA receptor subunit NR2B wasstudied. After transfected with LV-Homer1a, immunoprecipitation was laid out to detectthe effects of homer1a to the combination of the NMDA receptor subunit NR2B andnNOS.Results: We successfully expressed NR1, NR2B, Homer1a in HEK293T cells, and found that Homer1a has no direct effects on NR1NR2B complex’s peak value. Aftertransfected with LV-Homer1a in cultured neurons, the membrane NR2B expressiondecreased compared with the empty vector. By immunoprecipitation of NR2B as wellnNOS, we found that the Homer1a reduced the binding of NR2B and nNOS in culturedneurons. Conclusion: These results suggested that, Homer1a can promote NMDARendocytosis and dismiss the combination between NR2B and nNOS, which may the veryimportant mechanisms for the protective effects of homer1a against NMDA inducedneuron injury.