Humanin Protects Neurons Against NMDA-induced Neurotoxicity By Down Regulation NR1 Expression And Supprestion NR2B Tyrosine Phosphorylation

Excitatory neurotoxicity is triggered by the overactivation of N-methyl-D-aspartate receptors(NMDARs) that induced by overloading of glutamate,which is a critical mechanism contributing to neurodegeneration,as well as neuronal injury.It is a significance target for prevention and therapy of various kinds of neurodegenerative disorders by inhibition of NMDAinduced excitatory neurotoxicity.The NMDARs are prominent ligand-gated ion channels which are preferely permeable to Ca²⁺ and sensitive to voltage-dependt Mg²⁺ block.Three families of genes(NR1,NR2 and NR3) have been identified to encode the NMDA receptor subunits. Functional NMDARs are tetramers which composed of two essential NR1 subunits assembling with two NR2 subunits or in some cases,an NR2 and an NR3 subunit.Furthermore,NMDA receptor subunits interact with various intracellular scaffolding,anchoring and signaling molecules associated with the postsynaptic density.The physiological function of the NMDA receptors could be regulated by its composition of NR1 and NR2 subunit.In particular, selectived expresstion,specific composition and phosphorylation of NMDARs subunits significantly influence the function of NMDA receptors which is related with excitotoxic mechaninsms.Humanin(HN),a 24-amino-acid neuroprotective peptide,was originally found in the occipital lobe of an autopsied Alzheimer's disease(AD) patient.HN suppressed neuronal cell death that is caused by Alzheimer's disease(AD)-specific insults,which including both amyloid-beta peptides and familial AD-causative genes.HN was therefore originally identified as a neuroprotective factor against AD-related neuronal cell death in a process requiring secretion,dimerization and the binding to an as yet unidentified receptor.Recently,HN was reported to protect neurons against cell death by inhibiting the pro-apoptotic Bcl-2 family proteins Bid,Bim,and Bax.We have investigated that HN is a protective factor for neuronal injury induced by the ischemia and hypoxia or excitatory neurotoxicity model induced by NMDA.The results demonstrated that HN is a neuroprotective peptide with a wide spectrum of neuroprotection.The mechanisms of HN inhibited NMDA receptors,however,is uncleared. Based on the damage model of NMDA-induced excitatory neurotoxicity,we assessed the expression of specific NR1 subunits and phosphorylation of NR2B subunits to explore the mechanisms underlying the neuroprotection of HN.Methods:1.Primary culture of rat cerebral cortical neurons and experimental groups Cortical neurons derived from newborn rat(1-3d),the non-neuronal cell division was halted by exposure to cytosine Arabinoside for one day.Subsequently,partial medium replacement was carried out every two days.Only mature cells(8-9d) were used for the experiment.Routinely, cerebral cortical neurons cultured for 9 day in vitro were divided into 4 experimental groups:(1) Control:Locke's(2) NMDA group:NMDA(100μmol/L)+glycine(10μmol/L)(3) MK-801 group:NMDA(100μmol/L)+glycine(10μmol/L)+MK-801(10μmol/L)(4) HN group:NMDA(100μmol/L)+glycine(10μmol/L)+HN(1μmol/L)2.Immunocytochemistry of NR1 subunits Cerebral cortical neurons grown on 6-well plates were fixed with acetonum at -20℃for 5 min,then cells were incubated with antibodies against NR1(1:200) overnight at 4℃.After washed with PBS,cells were incubated with secondary antibodies(FITC) for 1h at room temperature in the dark followed by washed with PBS,and then cellular nucleus were stained with propidium iodide(PI).After extensive washing, cells were observed under a confocal laser scanning microscope.3.Flow cytometry Measurement of immunofluorescence of NR1 subunits which were stained with secondary antibodies(FITC)4.Western-blot Cerebral cortical neurons grown in culture flask were detached by scraping and collected.After adding the sample buffer to cell lysates,the mixtures were boiled for 5 min.Samples containing 40μg of protein were loaded and separated by 10%SDS polyacry-lamide gel electrophoresis,transferred to nitrocellulose membrane.After blocked for 1h in TBST containing 5%fat-free dry milk,protein blots were incubated overnight at 4℃with antibodies against NR2B and NR2BpTyr1472,incubated at room temperature for 1 h with HRP. Immunoreactivities of the protein bands were detected by enhanced chemiluminescence(ECL). Intensities of the bands were measured by using image analysis software and the results expressed as percentage control.Results:1.Cerebral cortical neurons were observed under an inverted phase contrast microscope:NMDA(100μmol/L) triggers obvious neuronal loss and is considered as an appropriate concentration and duration to cause excitatory neurotoxicity.HN(1μmol/L) attenuated NMDA-induced excitatory neurotoxicity.2.The expression level of NMDA receptors NR1 subuints induced by NMDA significantly increased compared to the control group,while pretreatment of HN(1μmol/L) reduced the expression level of NMDA receptors NR1 subuints in primary culture of rat cerebral cortical neurons.3.The level of phosphorylated NR2B induced by NMDA significantly increased compared to the control group,while pretreatment of HN(1 μmol/L) attenuated the phosphorylated of NR2B in primary culture of rat cerebral cortical neurons.Conelusioas:1.NMDA induced the up-regulate of expression of NR1 subunit and increase of phosphorylated of NR2B subunit which might be one of the major causes of excitatory neurotoxicity.2.Pretreatment of HN resulted in down-regulate of NR1 and reduction of phosphorylated of NR2B which might contribute to the neuroprotection against excitotoxicity.