Mechanisms Of Neurotrophic Factors In Neurodevelopment And Neurodegenerative Disease

BackgroundNeurotrophins are a group of proteins which are critical to maintaining the neuron survival and normal physiological functions.In 1952,Italian neuroscientist Dr.Rita Levi-Montaicini first discovered nerve growth factor（NGF）.Currently,the neurotrophic factor family mainly includes NGF,BDNF,NT-3 and NT-4.They play physiological roles mainly in promoting nerve growth and maintaining neuron survival by binding to their corresponding high-affinity tyrosine kinase receptors and activating downstream signaling pathways.Neurotrophic factors are critical in neurodevelopment,neuro-maturation,maintenance of physiological functions and post-injury repair.However,the molecular mechanisms of neurotrophic factors in development,degeneration and repair is not clear.And the neurotrophic factors applications in the clinical treatment of neurodegenerative diseases and nervous system injury is very limited,which remains an unexplored area in it.Therefore,an in-depth study of the neurotrophic factors’physiological molecular mechanisms in neurodevelopment and pathological process of neurological diseases can not only further explore the neurotrophic factors’applications in the clinic but also provide a novel way to understand the pathological mechanism of neurological diseases.Aims1.To investigate how sphingosine 1-phosphate（S1P）affects neuronal differentiation and nerve growth by regulating NGF-TrkA pathways in trigeminal ophthalmic nerve innervation development model.2.To explore the pathologic molecular mechanism of AD（Alzheimer’s disease）-like pathological changes caused by the association between Tau protein and the BDNF receptor after BDNF decreasing in AD.Methods1.Sphingosine 1-phosphate effects on the development of trigeminal ophthalmic nerve fibers.In ophthalmic nerve innervation models,immunofluorescence staining was used to observe cornea nerve innervation of sphingosine 1-phosphate transporter（Spns2）mutant rat at different embryonic days.2.The mechanisms of S1P regulating NGF and its receptor TrkA signaling pathways and the resultant effects on trigeminal ganglion（TG）development.Immunofluorescence staining was used to detect Trk expression in Spns2 mutant TG neurons at different embryonic days.S1P or NGF was added to rescue abnormal TG nerve growth in primary TG neuron culture and observe its effects on neuron morphologies.S1P receptor s1pr3siRNA was transfected into primary TG cells to knock down S1PR3 expression,live-cell imaging was used to observe cell conditions and nerve growth.These experiments were performed to investigate the effects of S1P-S1PR3 pathways on TrkA expression and NGF-TrkA signaling activation.3.The mechanisms of BDNF deprivation on asparagine endopeptidase（AEP）cleaved Tau protein and resultant effects on TrkB signaling pathways.Tau mutant mice P301S mice,AD transgenic mice 3xTg and AD patients’brain tissue samples were used to detect AEP activation,cleaved Tau N368 expression and Tau N368-TrkB binding.We cultured HEK293 and SH-SY5Y cells and transfected TrkB and Tau N368 plasmids,observed TrkB-Tau N368 binding and TrkB signals by using GST-pull down,immunoprecipitation and immunofluorescence staining.BDNF deprivation in vitro model was built to investigate TrkB and Tau N368 association and AD-like pathologic change in primary cultured neurons.4.In vivo BDNF deprivation model building and rescue interventions.BDNF deprivation in vivo model was established by injecting Cre virus into the hippocampal area of BDNF^2lox mice,and rescue experiments were conducted as followed:1,one week following the Cre viral injection,treated the mice with R1 peptide from Tau Repeat domain1（R1）via i.p.administration（10 mg/kg）once every 5 days for 6 weeks;2,injected the Cre virus into the hippocampus of BDNF^2lox mice together with a vector expressing the delta-secretase-uncleavable form of Tau（N255A/N368A）.Then detected TrkB-Tau N368binding,TrkB signaling changing and AD-related pathological changing after BDNF deprivation by using immunofluorescence staining,Western Blot,immunoprecipitation,etc.We observed the synaptic structure and spine morphologies by using Golgi staining and electron microscope analysis.The electrophysiological analysis was to validate the effects on synaptic functions.Cognitive behavior tests in Morris water maze,fear conditioning and novel object recognition were conducted to investigate AD-related learning and memory change.Results and Conclusions1.Spns2 mutant causes the delayed ophthalmic nerve innervation into the cornea and myelinating malformation.E22.5 Spns2 mutant rat cornea nerve showed nerve density decreased in cornea stromal layer,leading to disruption of normal cornea nerve structures.The myelination on Spns2 mutant rat is delayed compared to WT rat,and malformation of myelin was observed.From E16.5 to E18.5,the TG nerve innervation to the cornea is delayed.These results showed Spns2 mutant resulting in S1P extracellular decreasing contributes to TG ophthalmic nerve innervation disruption during development.2.TrkA expression was decreased and NGF-TrkA signaling pathways deactivated in Spns2 mutant rat TG neurons.Addition S1P to Spns2 mutant primary TG neurons can rescue delayed nerve growth.Knocked down WT TG neuron S1PR3 by transfecting s1pr3siRNA resulted in abnormal nerve growth and cannot be rescued by adding NGF or S1P.These results showed S1P signaling pathways acting as upstream of NGF-TrkA signaling and participate in TG development by regulating TrkA signaling pathway.3.After BDNF deprivation,phosphorylated Akt decreased and AEP is activated,subsequently cleaves Tau N368.This truncated Tau can bind with TrkB and inhibit TrkB signals,finally induce neuron apoptosis and trigger AD-like pathologies.Our data demonstrated that BDNF deprivation triggered TrkB-Tau N368 binding is the important mechanism of AD pathogenesis.4.Based on former in vitro data to build in vivo BDNF deprivation animal model.Injecting Cre virus into the hippocampal area of BDNF^2lox mice to trigger BDNF deprivation in vivo,we observed AEP activation in the injection region,Tau N368expression increasing,and TrkB-Tau N368 binding.The rescue interventions,i.p.injection TAT-R1-FITC peptides to interrupt TrkB-Tau N368 binding and over-expression uncleavable Tau N255A/N368A to inhibit Tau N368 production,can restore the AD-like pathologies triggered by BDNF deprivation.These experiments showed that BDNF deprivation might be a critical factor for AD pathogenesis and development.