Humanin Rescues Cortical Neurons From NMDA-Induced Excitatory Neurotoxicity In Rat

Humanin(HN)is a 24-aa peptide encoded by a newly identified gene cloned from an apparently normal brain region from patients with Alzhermer's disease(AD)in 2001.Studies showed that HN protected neurons from insults of various familial Alzheimer's disease mutations(APP,PS-1 and PS-2),anti-APP(amyloid precussor protein,APP)antibodies, amyloid peptide(Aβ)(Aβ1-43 or Aβ1-42)and its segments(Aβ25-35,Aβ31-35)with efficiency.It seemed that HN didn't inhibit other toxic insults to neurons,such as Fas,etoposide, glutamate and NMDA triggered neurotoxicity.HN was,therefore,considered as a selectively neuroprotective factor rescuing neurons from Alzheimer's disease-related insults.Although HN was firstly identified from human brain with Alzheimer's disease,more and more its homologues have been discovered in other species of animals and its location is also far beyond brain,suggesting that the role of HN be versatile other than just attenuating Alzheimer's disease-related insults.In support of this notion,other investigators including our lab have done some works to decipher the neuroprotective mechanism of HN at celluar and molecular level:1)to elevate ATP level,which is necessary for any cells to survive stresses;2) to block apoptosis mediated by extracelullar signals as well as intrinsic signals;3)to interfere with intracellular Ca²⁺accumulation induced by amyloid beta peptide(Aβ).The mechanisms underling the protective activity of HN mentioned above imply that it may play versatile protective roles against various insults other than as an AD- selective neuroprotective peptide.To verify the hypothesis,we employed NMDA induced neurotoxicity, an in vitro paradigm of neurodegeneration representative of pathologically neurotoxic insults of excitotoxicity which brings heavy damage to neurons by complicated mechanisms and is implicated in most of the neurodegenerative processes,to confirm the versatile neuroprotective role of HN in this in vitro insult model.The program of this research are as followings:1) Build an excitatory neurotoxic model in cultured cortical neurons of rat by measurement MTT, LDH in the medium and Ca²⁺concentration in the cytoplasm,which reflect general NMDA-induced neurotoxicity;evaluate the mitochondrial damage in the model by detecting ROS and NO;and assay the neuronal apoptosis by morphological observation of neurons with electron microscopy,TUNEL staining,and the measurement of the activation of caspase-3,and analysis the apoptotic pathways involved in by the measurement of the activities of caspase-8 and caspase-9;2)Evaluate the neuroprotective effects of HN on NMDA induced neurotoxicity by detecting Ca²⁺concentration in cytoplasm,MTT,Calcein staining and LDH release in the medium;3)Evaluate the protective effect of HN on NMDA-induced mitochondrial damage by determining the activity of fumaric reductase(MTT),the level of ROS and NO,and mitochondrial membrane potential;4)Evaluate the protective effects of HN on NMDA-induced neuronal apoptosis by morphological observation of neurons with electron microscopy,TUNEL staining,the measurement of the activity of caspase-3,and sub-summit of PI,analysis the potential protective pathways of HN against apoptosis by the measurement of the activities of caspase-8 and caspase-9,and evaluate the contribution of HN in attenuating NMDA-induced neurotoxicity by blocking neuronal apoptosis with the aid of caspase inhibitors.Our results showed that:1)HN blocked NMDA induced neurotoxicity by attenuating LDH release and Ca²⁺overloading,and rescue cortical neurons from excitatory neurotoxicity with the increase of MTT and Calcein staining cells;2)HN antagonized mitochondrial dysfunction by suppressing ROS and NO with the increase of mitochondrial membrane potential and the activity of fumaric reductase;3)HN inhibited neuronal apoptosis by interfering extrinsic apoptotic signals involved in caspase-dependent pathway as well as neurotoxicity mediated by NO.Part One:NMDA-Induced Neurotoxicity in Cortical Neurons and Potential Mechanisms UnderlyingTo evaluate the neuroprotective effect of HN on excitatory neurotoxicity,an appropriate cellular model must be employed which have definite and moderate neuronal damage and loss.The degree of neuronal loss triggered by NMDA largely depends on the concentration of NMDA in the medium and the time it works.But the concentration of NMDA and the time it works varied dramatically in reported models currently.To find a reasonable concentration of NMDA and the time it works is therefore a key factor in this part.Methods:Cortical neurons from neonatal rat(P1-3)were primarily cultured.An appropriate concentration of NMDA and the time it works were explored by monitoring morphological changes under contrast phase microscope,MTT,LDH release in the medium,and Ca²⁺ concentration in the cytoplasm so as to build an appropriate cellular model in cortical neurons in rat.ROS and NO were detected to evaluate the damage of mitochondria in the model.Electron microscopy,TUNEL staining,and the measurement of the activation of caspase-3,caspase-8 and caspase-9 were employed to evaluate neuronal apoptosis and analysis the pathways involved in the model.Data coming from 5-8 separate experiments were subjected to SPSS10.0 and were expressed as mean±S.D.Means of different groups were compared by ANOVA,followed by the Dunnett's test.Significance level was 0.05.Results:The administration of NMDA in the cultures caused pathological changes of morphology and cell viability decrease dose-dependently and time-dependently.NMDA (100μmol/L,2h)caused cell viability decrease by about 47%and LDH release increase by about 66%compared with control.It triggered the rise of Ca²⁺in cytoplasm and the maintenance of the highest level during the observation.Excessive ROS and NO were detected in the model.NMDA also caused the appearance of neuronal apoptosis suggested by electron microscopy,TUNEL staining.Caspase-3,caspase-8 and caspase-9 were activated in the model.Conclusions:1)NMDA(100μmol/L,2h)triggers obvious neuronal loss and is considered as an appropriate concentration and duration to cause excitatory neurotoxicity;2)Mitochondria are damaged in the model;3)NMDA(100μmol/L,2h)triggers neuronal apoptosis mediated both by extrinsic and intrinsic apoptotic signal pathways.Part Two:Humanin Attenuates NMDA-Induced Excitatory NeurotoxicityAlthough HN was considered as a selectively neuroprotective factor against AD-related insults, the mechanism underlying its neuroprotective functions at molecular and cellular level strongly suggests that HN plays protective roles for neurons at various stresses other than AD-related insults.The current research was to investigate the protective role of HN against excitatory neurotoxicity.Methods:Cortical neurons from neonatal rat(P1-3)were primarily cultured.For the experiment, cortical neurons were grouped as following:1)control,cultured neurons;2)HN,neurons incubated with HN(10μmol/L);3)NMDA,neurons coincubated with NMDA(100μmol/L) and Gly 10μmol/L)for 2h;4)NMDA + MK-801(10μmol/L),additional MK-801(10μmol/L) was needed compared with NMDA group;5)NMDA + HN(various concentration),having extra HN compared with NMDA group.Primary cultures were treated with chemicals as grouped.HN was administrated 16h in advance.2h later,neurons were collected,labeled with Fluo-3 and subjected to laser scanning confocl microscopy.Dynamic changes of cytoplasmic Ca²⁺were captured and recorded every 660ms and 300 records(for about 198s)were needed for each neuron.24h after NMDA treatment,the medium was collected for the measurement of LDH. Neurons were subjected to MTT detection and Calcein staining.Data coming from 5-8 separate experiments were subjected to SPSS10.0 and were expressed as mean±S.D.Means of different groups were compared by ANOVA,followed by the Dunnett's test.Significance level was 0.05.Results:1)HN inhibits the increase of cytoplasmic Ca²⁺concentration induced by NMDA. NMDA triggered the rapid increase of cytoplasmic Ca²⁺concentration and maintenance of the highest level during the observation.From the control level to the highest one,it took only 2s. HN(0.1μmol/L)has no effect on interfering with NMDA-induced increase of cytoplasmic Ca²⁺ concentration.Although HN(1μmol/L)didn't block the increase of cytoplasmic Ca²⁺ concentration induced by NMDA,it caused dropping of the Ca²⁺concentration;2)HN inhibited the loss of living neurons caused by NMDA.NMDA triggered the loss of neurons by 51.3%compared with control showed by Calcein staining.Pretreated with HN(100μmol/L), the loss of neurons was almost blocked completely.The rate of living cells reached 98.6%of control;3)HN blocked the decrease of cell viability caused by NMDA.NMDA caused cell viability dropping by 47%showed by MTT.HN blocked the toxic effect of NMDA and attenuated the decrease dose-dependently;4)HN neutralized the LDH release triggered by NMDA.Compared with control,additional LDH release(about 66%of control)was detected when neurons were treated with NMDA.HN inhibited the additional release of LDH does-dependently although it didn't stop it completely.Conclusions:1)HN inhibits the increase of cytoplasmic Ca²⁺concentration induced by NMDA; 2)HN inhibits the loss of living nerurons,blocks the decrease of cell viability,and neutralizes the additional release of LDH when neurons confront with NMDA-induced extitatory stress. HN,therefore,rescues cortical neurons from NMDA-triggered excitatory neurotoxicity effectively.Part Three:Humanin Attenuates NMDA-Induced Mitochondria DysfunctionExcitatory neurotoxicity is triggered by the overloading of Ca²⁺for the overactivation of NMDA receptor.Since mitochondria is an important Ca²⁺pool beside producing ATP,it plays an important role in soothing excessive Ca²⁺in the situation.Once more Ca²⁺is uptaken into mitochondria than its capacity,mitochondria itself is damaged and loss normal function of producing ATP.In the part,the protective role of HN against mitochondria dysfunction caused by NMDA were to investigate since we have confirmed its neuroprotective effect on NMDA-induced excitatory neurotoxicity and its inhibitory effect on Ca²⁺increase triggered by NMDA.Methods:Cortical neurons from neonatal rat(P1-3)were primarily cultured.For the experiment, cortical neurons were grouped as following:1)control,cultured neurons;2)HN,neurons incubated with HN(10μmol/L);3)NMDA,neurons coincubated with NMDA(100μmol/L)and Gly(10μmol/L)for 2h;4)NMDA + MK-801(10μmol/L),additional MK-801(10μmol/L)was needed compared with NMDA group;5)NMDA + HN(various concentration),having extra HN compared with NMDA group.Primary cultures were treated with chemicals as grouped.HN was administrated 16h in advance.2h later,neurons were collected and labeled with JC-1 for mitochondria membrane potential or DCFH-DA for ROS.Neurons were subjected to laser scanning confocl microscopy and FCM.The medium was collected for the detection of NO.24h later,neurons were collected for the determination of fumaric reductase by MTT.Data coming from 5-8 separate experiments were subjected to SPSS10.0 and were expressed as mean±S.D. Means of different groups were compared by ANOVA,followed by the Dunnett's test. Significance level was 0.05.Results:1)HN blocked the decrease of the activity of fumaric reductase.NMDA triggered the decrease of the activity of fumaric reductase by about 47%of control.HN inhibited the effect of NMDA dose-dependently showed by MTT;2)HN attenuated the drop of mitochondrial membrane potential caused by NMDA.The red fluorescent intensity of JC-1 labeled on neurons for mitochondrial membrane potential was detected and analyzed both by laser scanning confocal microscopy and FCM.The fluorescent intensity decreases when treated with NMDA. When HN(1μmol/L)and NMDA coincubated with the neurons,the decrease of the fluorescent intensity of JC-1 was inhibited;3)HN inhibited the excessive production of ROS triggered by NMDA.The measurement of fluorescent intensity of DCFH-DA for ROS by laser scanning confocal microscopy and FCM showed that NMDA induces excessive production of ROS and HN(1μmol/L)inhibited the production of ROS caused by NMDA.4)HN neutralized the excessive production of NO.Compared with control,NMDA triggered more NO and HN attenuated the activity of NMDA.Conclusions:1)HN blocks the decrease of the activity of fumaric reductase;2)HN attenuates the drop of mitochondrial membrane potential caused by NMDA.3)HN inhibits the excessive production of ROS triggered by NMDA;4)HN neutralizes the excessive production of NO. HN,therefore,protects cortical neurons from NMDA-induced excitatory neurotoxicity by attenuating mitochondrial dysfunction.Part Four:Humanin Attenuates NMDA-Induced Neuronal ApoptosisExcitatory neurotoxicity causes neuronal loss by necrosis and apoptosis.Apoptosis draws more and more attention nowadays because it is a delayed cell death and can be controlled and regulated to some extent.Mitochondria play a key role in apoptosis.For one reason,it is in the centre of intrinsic apoptotic signal system.For another,it is involved in extrinsic apoptotic signals via Bid.In the part,the protective role of HN against neuronal apoptosis was to explore since it protects cortical neurons from NMDA-induced excitatory neurotoxicity by attenuating mitochondrial dysfunction.Methods:Cortical neurons from neonatal rat(P1-3)were primarily cultured.For the experiment, cortical neurons were grouped as following:1)control,cultured neurons;2)HN,neurons incubated with HN(10μmol/L);3)NMDA,neurons coincubated with NMDA(100μmol/L)and Gly(10μmol/L)for 2h;4)NMDA + MK-801(10μmol/L),additional MK-801(10μmol/L)was needed compared with NMDA group;5)NMDA + HN(1μmol/L),having extra HN compared with NMDA group.Primary cultures were treated with chemicals as grouped.HN was administrated 16h in advance.6h later,neurons were collected and subjected to electron microscopy,TUNEL staining,the measurement of the activation of caspase-3,caspase-8 and caspase-9,and the detection of sub-summit of PI by FCM.By electron microscopy,TUNEL staining and the measurement of the activity of caspase-3,NMDA-induced neuronal apoptosis were to confirm.By the quantitative analysis of the activity of caspase-3 and the sub-summit of PI,the protective effect of HN on NMDA-induced neuronal apoptosis were to evaluate.By comparing the activity of caspase-8 and caspase-9 in different groups,the mechanism underlying HN's protective role against neuronal apoptosis were to investigate.By two caspase inhibitors, caspase-8 inhibitor(IETD)and general caspase inhibitor(VAD),and the detection of MTT and LDH release,the share of HN's inhibiting apoptosis in its protective effect against excitatory neurotoxicity were to evaluate.By caspase-8 inhibitor(IETD)and the detection of NO,the effect of extrinsic apoptosis signals on the function of mitochondria was to explore.Data coming from 5-8 separate experiments were subjected to SPSS10.0 and were expressed as mean±S.D.Means of different groups were compared by ANOVA,followed by the Dunnett's test.Significance level was 0.05.Results:1)Electron microscopy,TUNEL staining and the measurement of caspase-3 suggested that NMDA induce neuronal apoptosis.The quantitative analysis of sub-summit of PI and caspase-3 showed that HN rescued cortical neurons from NMDA-induced apoptosis;2)The detection of caspase-8 and caspase-9 indicated that NMDA triggered neuronal apoptosis by both extrinsic and intrinsic apoptosis signals.HN attenuated neuronal apoptosis induced by NMDA via extrinsic apoptotic pathway;3)With caspase-8 inhibitor(IETD)and the detection of MTT and LDH release,it was believed that the contribution of neuronal apoptosis mediated by extrinsic apoptotic signal was limited in NMDA-induced neurotoxicity.The contribution of HN's inhibiting extrinsic apoptotic signal-mediated apoptosis to its protective effect on NMDA-induced nerotoxicity was,therefore,limited;4)With general caspase inhibitor(VAD) and the detection of MTT and LDH release,it was believed that the share of caspase-dependent neuronal apoptosis was limited in NMDA-induced neurotoxicity.The contribution of HN's inhibiting caspase-dependent neuronal apoptosis to its protective effect on NMDA-induced neurotoxicity was,therefore,limited;5)With caspase-8 inhibitor(IETD)and the detection of NO,excessive NO triggered by NMDA was found to be inhibited suggesting that HN attenuates the excessive production of NO induced by NMDA by interfering with extrinsic apoptotic signal.Conclusions:1)HN attenuates NMDA-induced neuronal apoptosis;2)HN attenuates NMDA-induced neuronal apoptosis by interfering with extrinsic apoptotic signal pathway;3) The contribution of HN to inhibition of extrinsic apoptotic signal-mediated apoptosis to its protective effect on NMDA-induced nerotoxicity was limited;4)the contribution of HN to inhibition of caspase-dependent neuronal apoptosis to its protective effect on NMDA-induced neurotoxicity was limited;5)HN attenuates NO mediated neurotoxicity induced by NMDA at least in part via interfering with extrinsic apoptotic signal.The conclusion in all:1)NMDA(100μmol/L,2h)triggers obvious neuronal loss and is considered as an appropriate cortical neuronal model of excitatory neurotoxicity with mitochondrial dysfunction and neuronal apoptosis;2)HN rescues cortical neurons from NMDA induced excitatory neurotoxicity with efficiency;3)HN attenuates NMDA-triggered mitochondrial dysfunction;4)HN inhibits NMDA-induced neuronal apoptosis by interfering with the activity of extrinsic apoptotic signals;5)HN inhibits NO mediated neurotoxicity triggered by NMDA at least in part by interfering with the activity of extrinsic apoptotic signals.