Neurotoxicity Of The Prodomain Peptide Of BDNF On Neurons And Its Effects On Learning And Memory In AD Mice

BackgroundBrain-derived neurotrophic factor(BDNF)is a prominent member of the neurotrophic factor family,which helps to enhance the proliferation,differentiation and survival of neural stem cells,and facilitates synaptic formation,transmission and synaptic plasticity,and promotes long-term potentiation(LTP).The precursor form of BDNF(proBDNF)is cleaved by several kinds of enzymes such as furin,plasmin,tissue plasminogen activator(tPA)and selective matrix metalloproteinases(MMPs)to generate mature BDNF(mBDNF)and its prodomain(pBDNF).It has been proved that both mBDNF and pBDNF were stored in presynaptic dense core vesicles in brain neurons and also attended in roughly equimolar ratio which was 10-fold more abundant than proBDNF in the hippocampus.While BDNF is neuroprotective,proBDNF has an opposite effect to that of BDNF.proBDNF can bind to the p75 neurotrophin receptor(p75NTR),attenuate hippocampal neurogenesis,and induce learning and memory deficits in aged mice.Moreover,proBDNF accelerates amyloid-β(Aβ)deposition in the brain,and suppresses the migration of cerebellar granule cells and neural stem cells.The N-terminal fragment cleaved from proBDNF is an 120 amino acid long prodomain peptide(pBDNF).In the past,pBDNF was considered only as a molecular chaperone which assists folding of BDNF.However,recent evidence demonstrated that pBDNF also has important biological functions.pBDNF is localized in dense core vesicles in excitatory presynaptic terminals,and is secreted in an activity-dependent manner from cultured hippocampal neurons.pBDNF induces neuronal growth cone retraction by coupling with the p75NTR and the sortilin family member Vps10p-domain sorting receptor 2(SorCS2).Furthermore,pBDNF was reported to reduce the number of dendritic spines,and facilitate hippocampal long-term depression(LTD).The hippocampal pBDNF level of AD patients was 16-fold higher than that of the healthy individuals,and was 30-fold higher than the mBDNF level.Collectively,it is likely that pBDNF exerts pathogenic role.In the present study,we investigated the effects of pBDNF on neuronal viability and neurite growth in vitro.Materials and MethodsCCK-8 viability assay.Primary hippocampal neurons were plated in replicates of five in 96-well plates at a density of 1×10~5 cells per well in 100μL,and treated with human BDNF prodomain at various concentrations(0,10,50,100,200,300 ng/mL,respectively)and anti-p75NTR extracellular domain antibody(140 nM)with pBDNF(300 ng/mL)for 72 h.Hippocampal neuron survival was measured using the Cell Counting Kit-8 following the manufacturer’s protocol.The amount of CCK-8 reagent reduced to formazan by cellular dehydrogenase indicated cell viability and was measured by reading the absorbance at 450 nm in a 96-well plate reader.Immunofluorescent assay.Neurons on coverslips were washed with PBS,fixed with 4%paraformaldehyde,blocked with 3%BSA,incubated with antibody at 4°C overnight followed with 488-and/or Cy3-labeled secondary antibody.Nuclei were labeled with DAPI.To determine the identity of cultured cells,cultured cells were double labeled with mouse anti-DCX antibody to stain immature neurons and rabbit anti-GFAP antibody to stain astrocytes when cultured for 48 h.DCX-positive cells of at least 8 random viewfields which were counted to calculate the average purity.A high proportion of DCX-positive cells indicates that hippocampal neurons had high purity.In order to characterise NSCs,mouse anti-Nestin antibody was used.More than90%cells were found Nestin-positive.In order to learn whether primary hippocampal neurons and NSCs are p75NTR positive,rabbit anti-p75NTR antibody were used to examine the presence of p75NTR.For neurite growth measurement,after 72 h incubation with PBS control or pBDNF(300 ng/mL)or together with anti-p75NTR antibody(140 nM),hippocampal neurons were fixed and incubated with rabbit anti-MAP2 antibody.Neurites were viewed using the fluorescence microscope.The coverslips were scanned from left to right,and 8 fields were randomly selected.TUNEL assay.Hippocampal neurons were plated(1×10~5/well)on PDL-coated coverslips and were treated with PBS control or pBDNF(300 ng/mL)with or without anti-p75NTR antibody(140 nM)for 72 h.Neurons were then processed for TUNEL analysis and counterstained with DAPI to visualize nuclei.TUNEL-positive cells of at least 8 random view fields were counted for each culture condition.The ratio of apoptotic nuclei to the total number of nuclei was then calculated.EDU incorporation assay.NSCs were dissociated in 0.125%trypsin at 37℃for 3-5min and plated in 24-well plates with PDL-coated coverslips.After incubation with PBS control or pBDNF(300 ng/mL)or together with anti-p75NTR antibody(140 nM)for 72h,EDU Kit was used to detect the cells which were proliferating and counterstained with DAPI to visualize nuclei according to the manufacturer’s protocol.EDU-positive cells of at least 8 random view fields were counted for each culture condition.The cell proliferation rate was represented by the percentage of EDU-positive cells.Western blot.Protein samples from cells were extracted using RIPA buffer supplemented with a phosphatase inhibitor and a protease inhibitor.Equal amounts of extracts from different preparations were separated on a 10-15%SDS-PAGE gel and transferred with the NC membrane.Then the membrane was blocked by milk for 1 hour and incubated with the primary antibodies against caspase-3,Bcl-2,Bax,p75NTR,p-ERK 1/2,total ERK 1/2,PSD-95,andβ-actin.The membranes were incubated with IRDye 800CW secondary antibodies and scanned using the Odyssey fluorescent scanner.For NSCs proliferation experiment,there was 2 h starvation culture in a growth factor-free medium to synchronize cell cycle,and then NSCs were treated with pBDNF or together with anti-p75NTR.Morris water maze.AD mice were received microinjection into hippocampus with AAV-GFP,AAV-pBDNF,and AAV-pBDNF+anti-p75NTR.4 weeks later,they were tested by morris maze.Statistical analysis.Statistical analysis was carried out with Graph Pad Prism 6software.Data were displayed as a mean±SD of three independent experiments.For experiments with more than two groups,results were compared using one-way ANOVA with post hoc Tukey’s test.For two group-designed experiments,comparisons were performed using unpaired Student’s t-test.A p-value of<0.05 was considered to be statistically significant.ResultspBDNF inhibits the viability and proliferation of neurons.We found that pBDNF inhibited neuronal survival in a concentration-dependent manner.To explore whether the neurotoxicity of pBDNF is dependent on the p75NTR signaling pathway,primary hippocampal neurons were treated with pBDNF(300 ng/mL)together with anti-p75NTR antibody(140 nM)for 72 h.We found that there was a significant recovery in cell viability in the presence of anti-p75NTR antibody,in comparison to the pBDNF group.Furthermore,to investigate whether pBDNF inhibits NSCs proliferation,EDU incorporation assay was performed.The immunostaining demonstrated that the proportion of EDU-positive neurons was significantly decreased when incubated with pBDNF(300 ng/mL).When the NSCs were co-treated with pBNDF and anti-p75NTR antibody,the proportion of EDU-positive neurons was significantly increased compared with the pBDNF group,but were lower than PBS group.In brief,these data confirmed that pBDNF is neurotoxic,which not only inhibited the viability of primary hippocampal neurons,but also the proliferation of NSCs in vitro,and this effects were dependent on the receptor p75NTR signaling pathway.pBDNF induces neuronal apoptosis in vitro.TUNEL assay was performed,which reflected apoptosis-associated DNA strand breaks.The TUNEL-positive cells in pBDNF-treated(300 ng/mL)group were significantly more than the PBS-treated group.To provide further evidence for the involvement of p75NTR in pBDNF-induced apoptosis,anti-p75NTR antibody(140 nM)was applied to hippocampal neurons and NSCs together with pBDNF(300 ng/mL).Exactly,anti-p75NTR antibody antagonized the pro-apoptotic effect of pBDNF on neurons in vitro.pBDNF inhibits neurite growth in primary hippocampal neurons.Primary hippocampal neurons were treated with either PBS or pBDNF(300 ng/mL)or pBDNF together with anti-p75NTR antibody(140 nM)for 72 h.In the pBDNF group there was a significant reduction in the average neurite length compared with the PBS group.However,in the presence of anti-p75NTR antibody,the reduction in neurite length was less than pBDNF alone.pBDNF promotes primary hippocampal neurons apoptosis through the caspase-3pathway.To investigate themolecular mechanism of pBDNF-induced neuronal apoptosis,the pro-apoptotic proteins Bcl-2,Bax,caspase-3 in primary hippocampal neurons were tested.We found that pBDNF(300 ng/mL)increased caspase-3 and Bax expression and decreased the expression of Bcl-2.Furthermore,anti-p75NTR antibody(140 nM)can reverse the inhibitory effect of pBDNF.These results suggested that the caspase-3pathway may be the crucial pathway regulating neuronal apoptosis of pBDNF.pBDNF inhibits ERK 1/2 phosphorylation which affected NSCs proliferation.In order to investigate if MEK/ERK 1/2 pathway is involved in NSCs proliferation,there was a 2 h starvation culture in a growth factor-free medium to synchronize cell cycle.After the starvation culture,NSCs displayed an increase in p-ERK 1/2,with an irritation of EGE and bFGF.Then,NSCs were treated with pBDNF(300 ng/mL)or together with anti-p75NTR(140 nM).There was no significant difference between PBS-and pBDNF-treated group within 30 min.The expression of p-ERK 1/2 was significantly reduced after the NSCs were cultured with pBDNF after 360 min.However,pBDNF treatment did not change the expression of total ERK 1/2.Also,the effect of pBDNF on ERK 1/2 expression can be neutralized by anti-p75NTR antibody.pBDNF impaires learning and memory in AD mice and anti-p75NTR acts as an antagonist.The results of Morris water maze showed that AAV-pBDNF group did not have a shorter latency compared to the first day.But control group and AAV-pBDNF+anti-p75NTR group have a shorter latency.Also,it was found that the annulus crossing times in AAV-pBDNF group were less than other two groups.And the level of PSD-95 in AAV-pBDNF group was less than other two groups,too.ConclusionpBDNF not only inhibits the activity of primary hippocampal neurons,inhibits the growth of neuron processes,but also promotes the apoptosis of primary neurons and inhibits the proliferation of hippocampal neural stem cells,suggesting that pBDNF has toxic effects,which may be mediated by p75NTR receptor.pBDNF can impaire the learning and memory of AD mice,which is involved with p75NTR.