Effects Of ProBDNF On Cognition Function In Aged Mice And Its Mechanism

BackgroundAlzheimer'disease(AD) is the most common form of dementia in later life,and a major cause of disability and death in the elderly.Currently there are more than 12 million individuals affected with AD worldwide,and it is projected that the prevalence will nearly quadruple in the next 50 years,by which time approximately 45 million will be afflicted with the disease.It is a huge burden for the families and the society.China has been facing a very serious and irreversible situation of population aging during 21st century.Since 1999 China's population has become aged with the most amount of old people in the world.According to calculation based on the current prevalence data,it is estimated that there will be 8 to 12 million AD patients in China by 2050.Alzheimer's disease is associated with the loss of neurons in areas of the brain that are vital to memory and other mental abilities,like the hippocampus.In elderly humans, imaging studies have shown hippocampal atrophy.It is of great importance and interest to develop effective approaches which are able to prevent the incidence of neuron loss. Neurogenesis in adult mammals occurs in several regions,including the forebrain subventricular zone(SVZ) and the dentate gyrus of the hippocampal formation. Hippocampal neurogenesis occurs throughout the adulthood and is important for learning and memory,but it declines with age.Reduced neurogenesis in the aged hippocampus has been shown to be associated with cognitive deficits.Neurogenesis maybe a potential therapeutic strategy for AD.Brain-derived neurothrophic factor(BDNF) can promote the proliferation, differentiation and survival of neuron.BDNF can enhance the activity-dependent synaptic plasticity.It is recently confirmed that the precursor of BDNF(proBDNF) is the main expressing product of BDNFmRNA outside the cell.proBDNF distributes widely in the adult animal,especially in the central nervous system.The uncleaved precursor may play a different role from that of mature BDNF.It has been shown that a single amino acid mutation(Val66Met) in the proregion of BDNF affects the sorting of the molecule into the nerve terminals,retards activity-dependent secretion and reduces learning functions of the hippocampus.Under pathological conditions,such as aging and early stage of AD,the proBDNF and P75^NTR expression are upregulated.All of these indicate that proBDNF is not merely precursor of BDNF and may have important roles in the development of cognition impairment.It is important for the prevention of AD to study the roles of proBDNF in hippocampal neurogenesis and synaptic plasticity.Thus in the present study,there are main contents including expression,purification of cleave -resistant recombinant proBDNF protein,and study on its functional characterization.Effects of proBDNF on the cognition function in aged mice were observed.By in vivo and in vitro study,we evaluated the effects of proBDNF on hippocampal neurogenesis and synaptic plasticity,and investigated the mechanism of the effects of proBDNF on cognition function.Method1.Expression,purification of recombinant proBDNF protein in E.coli.and its functional characterizationThe cDNA fragment from proBDNF was obtained by standard PCR method with a full-length rat mutated proBDNF cDNA as template,and was inserted into plasmid pET-28a by means of gene rearrangement.The recombinant plasmid pET-proBDNF was identified by restriction endonuclease analysis and DNA sequencing.The cloned pET-proBDNF plasmid was transformed into E.coli host strain,BL21.Following induction by IPTG,the recombinant protein was expressed and then purified by Ni-NAT affinity chromatography under denaturing conditions.The interest protein was viewed by SDS-PAGE,further characterized by western blot.The apoptosis of PC12 cells was evaluated by DAPI assay.2.Effects of proBDNF on cognition function in aged miceThirty aged C57BL/6J mice(18months old)were randomly divided in to three groups: proBDNF group,anti-proBDNF group,or bovine serum albumin(BSA) group(n=10).A Alzet osmotic minipump was connected to right hippocampus of the mouse.Pumps were filled with proBDNF(1μg/μl),sheep antibody to proBDNF(1μg/μl),or BSA(1μg/μl), respectively.Mice received the injection for 6 days at the speed of 0.2μl/h.21days after surgery,the animals were subjected for Morris Water Maze to test mean escape latency, swimming speed and percentage of time spent in the target quardrant.3.Effects of proBDNF on hippocampal neurogenesis in aged mice.Thirty 18-month-old C57BL/6J mice were used in this experiment.Animals received proBDNF,sheep antibody to proBDNF or BSA as described previously in step 2.BrdU(50 mg/kg,i.p.) was injected twice per day for 6 days immediately after surgery.7days and 28 days postsurgery,immunohistochemistry was processed to examine the BrdU positive cells, and immunofluoresent triple labeling was processed to examine the phenotype of the newly generated cells.4.Effects of proBDNF on hippocampal synaptic plasticity in aged mice.Fifteen 18-month-old C57BL/6J mice were used in this experiment.Animals received proBDNF,sheep antibody to proBDNF or BSA as described previously in step 2.28 days postsurgery,western blot were used to observe changes of the synaptic plasticity marker-pCREB and synapsin I.5.Effects of proBDNF on the proliferation and apoptosis of cultured hippocampal neuron.The primary hippocampal cell cultures were prepared from 1-day-old female P75^NTR knocked out mice and their wild-type littermates.Cells were co-cultured with cleaved resistant proBDNF,proBDNF+sortilin-FC,or BSA,respectively.Proliferation was tested by immunohistochemstry on BrdU,and apoptosis of hippocampal neuron was detected by TUNEL assay.Results1.Expression,purification of recombinant proBDNF protein in E.coli.and its functional characterizationThe rat proBDNF cDNA was amplified and cloned into prokaryotic expression vector. The restriction endonuclease analysis and DNA sequencing proved that the plasmid pET-proBDNF was obtained.The recombinant protein was expressed in E.coli system. After Coomassie brilliant blue staining,the recombinant protein showed an exclusive band; western blot analysis with anti-proBDNF antibody supported that the expressed protein was recombinant proBDNF.The recombinant rat proBDNF protein could enhance the apoptosis of PC12 cells.Affinity purified antibody to proBDNF could neutralize the effect of proBDNF2.Effects of proBDNF on cognition function in aged miceCompared with BSA control group,proBDNF group had longer mean escape latency (p＜0.05),while the mean escape latency in antiproBDNF group was shorter(p＜0.05).When it comes to swimming speed,there had no difference between each group.Compared with control group,mice in proBDNF group spent less time in the target quadrant,while mice in anti-proBDNF group spent more time in the target quadrant(p＜0.05).3.Effects of proBDNF on hippocampal neurogenesis in aged mice.7days and 28days postsurgery,proBDNF group had less BrdU positive cells in dentate gyrus(p＜0.01),while antiproBDNF had more BrdU positive cells(p＜0.01),compared with control group,respectively.After 28days postsurgery,the BrdU positive cells decreased in all the groups,but proBDNF decreased the most dramatically.Immunofluoresent triple labeling showed that there had not difference of phenotype of BrdU positive cells between each group.4.Effects of proBDNF on hippocampal synaptic plasticity in aged mice.28days postsurgery,western blot showed that synapsin I and P-CREB in hippocampus were lower in proBDNF group than that of control group(p＜0.01).But anti-proBDNF group had more synapsin I and pCREB,compared with control group(p＜0.01)5.Effects of proBDNF on the proliferation and apoptosis of cultured hippocampal neuron.The number of BrdU positive cells in wild type mice decreased significantly in proBDNF group(p＜0.01,compared with control group),but there had no difference between proBDNF+sortilin-Fc group and contral group.In P75^NTR knockout mice,the BrdU positive cells in proBDNF group was the same as the control group.On the contrary,the number of TUNEL positive cells in wild type mice increased significantly in proBDNF group(p＜0.01,compared with control group),while there had no difference between proBDNF+sortilin-Fc group and control group.In P75^NTR knockout mice,both the control group and proBDNF group had the same percentage of TUNEL positive cell. Conclusions1.The prokaryotic expression vector for rat proBDNF is successfully constructed.The recombinant rat proBDNF protein can be expressed and purified in E.coli.and has a good biological activity.2.proBDNF can suppress the learning and memory ability of aged mice.It indicates that proBDNF plays a different role from that of mature BDNF.3.proBDNF suppressed the proliferation and survival of hippocampal neuron in dentate gyrus in aged mice.4.proBDNF downregulated the expression of pCREB and synapsin I protein in hippocampus of aged mice.5.proBDNF can inhibit the proliferation of neurons in dentate gyrus and initiate cell death.Interaction of proBDNF with both receptors of sortilin and P75^NTR on the cell surface is required for these effects.6.proBDNF can suppress the learning and memory ability of aged mice by inhibiting the hippocampal neurogenesis and downregulating the expression of pCREB and synapsin I protein in hippocampus.