Chap 16 Neurotrophic Factors神经营养因子HistoryIt was known that extracellular signals can promote the growth and differentiation of nerve cells in more than half a century ago.Nerve growth factor (NGF) was first discovered by Bueker in 1948.Rita Levi-Montalcini and Victor Hamburger demonstrated NGF is necessary for sympathetic and sensory neurons to keep their survival. Neurotrophic factors (NTFs) influence the growth, differentiation and survival of neurons during development.NTFs are also re-activated in adulthood in tissue renewal or regeneration.The signaling failure of NTFs may underlie neurodegenerative disorders such as Alzheimer disease (AD), Parkinson disease (PD), huntington disease (HD) and amyotrophic lateral sclerosis (ALS). Neurite (神经突起)Neuritic branch is described axon and dendrite. Especially, it is available for growing neuritic which did not assured axon or dendrite.The effect of NGF on cultured spinal neurons(Levi-Montalcini R. 1987. Science 237:1157)The term of Neurotrophic factor The term Neurotrophic Factor refers to any molecule that affects the nerves system by influencing the growth, differentiation, or cell cycles of neurons or glia. The difference between of NTFs and Neuropeptides: 1) NTFs are large molecular proteins, but neuropeptides are the small molecule proteins (14kDaNTF is produced in cell body.Small, 14kDa1kDaSynthesisProducted in cell bodyproduced at the terminalStoragelarge densecored vesiclesSynaptic vesiclesReleaseDetermined active-dependent synthesis. Determined depolarization.Determined Low-frequency stimulationDetermined depolarization.breakdownProtease hydrolyzation, Enshroud by incativation-receptorreuptake, re-use,SignalingThe binding to Trk receptor lead to many of the biologic effectsThe binding G-protein couple receptor and the second messenger lead to the biologic effects.ActionRetograde, anterograde, autocrine, paracrine transmission.anterograde, autocrine, paracrine transmission. acting as quick transmitter.分分类类 Neurotrophic FactorsReceptorsNeurotrophins NGF, BDNF, NT-3, NT-4Trk （R-PTKs） TrkA, TrkB, TrkC1 GDNF family GDNF, neurturin, persephin, LIFRCoupled to Ret GFR1,GFR2,nuknownCNTF family CNTF、LIF、IL-6Coupled to Janus kinase（JAK ） GP130、CNTFFREphrins Eph (R-PTKs)EGF family EGF, TGF, neuregulns2ErbB (R-PTKs)Other growth factors Insulin, IGF, FGF, PDGFR-PTKSinterleukins and related cytokines IL-1, IL-2, IL-3, IL-5,TNF, TNF IL-IR coupled to PS/TK, R-PTK Coupled to JAK, Related to p753TGF family: TGFR-PS/TRsOther cytokinesCoupled to JAK, R-PTKsChemokines CC chemokines (IL-8) CXC chemokines (MIP, MCP) CX3C chemokines (neurotactin) G protein-coupled receotors CC1-CC8R CXC1-CXC4R Cx3C1RNeurotrophinsNeurotrophins are small proteins. They play their physiologic roles through Trk receptor.Family members：NGF、BDNF、Neurotrophin-3(NT-3)、NT-4/5.Effectionsneuronal survival To prevent neuronal deathneurogenesisTo promote the growth of axons and dendritesneurite To promote neurite in adultanabolismTo enhance the size of neuronsdifferentiationTo synthesis proteins which are necessary for Neuronal phenotype regulating the transmittalTo increase the synthesis of neurotransmittors, neuropeptides and their synthetaseselectrical excitabilityTo alter the activation and level of ion channelsNGFBDNFNT-3composing of amino acides 1163 disulfide bonds1193 disulfide bonds1193 disulfide bondsMW13kDa13kDa13kDadistributionWidely distribute in CNS, PNS and peripheral tissuesIn CNS: hypothalamus, cortex, hippocampus in developing; In peripheral tissues: BDNF mRNA was highly expressed in muscle tissuesWidely distrubut in CNS, PNSreceptorsTrkATrkBTrkCSignaling pathwayTrk receptor signaling pathwayCharacteristic of neurotrphinsNGF的结构The -NGF dimer (1 subunit light blue and 2 darker blue) is at the center with the subunits in red and the subunits in green. Interactions between the 1 and 1 (and 2 and 2) subunits are mediated by a zinc ion (off-white spheres).7S NGF Neurotrophins receptorTrk-RTransmembrance receptorGlycoproteins whose molecular weight range from 140 to145kDa.Different types of Trk-RNeurotrophins and receptorsDistribution of TrkR in nerval cellsThe most of TrkAs were found in sensory and sympathetic neurons, a few in brain neurons.TrkB and TrkC express in the most of neurons.Trk truncation was existed in astrocytes and oligodendrocytes.P75 receptora 75-kDa proteinas the low-affinity neurotrophin receptor. also appears to modulate Trk signaling; the increases phosphorylation of Trk and then enhances the activity of Trk.may allow Trk-R to response to the low concentration of NGF and the other neurotrophinsis believed to play a key role in cell death pathways.The signaling pathway of Neurotrophins Trk was activated by autophosphorylation of Try (Y490、Y785) in the intracellular domain and subsequently forms to dimer, and then enables phosphorylation the intracellular signaling protein by interacting with its SH2 domain, e.g. Shc and PLC .Three signaling pathways:Ras/MAPK cascadesPI3K/AKTPLC/IP3-dependent Ca2+ releaseIP3DAGCa2+释放释放 The signaling pathway of NeurotrophinsRas/MAPK Ras protein： . 21kD phosphorylating protein; a small G-protein; switch between inactivation of GDP-bound formation and activation of GTP-bound formation; GTP-bound formation keeps the activation of cell growth and activates the cells to overproliferationa cascade of TrkA activates RasShcSosRasGrb2A cascade of Ras/Raf/MEK/ERK ERKRafThe effect of Ras/Raf/MEK cascade in neurite growthPC12 originates from pheochromocytoma TrkA and p75 are expressed in membrane of PC12NGF treatment may stimulate the neurite growth on PC12 cells. The effect of Ras/Raf/MEK cascade in neurite growth Disease of Ras signaling pathwayDefects of that NTFs activated the signaling pathways in the intracellular can lead to the overproliferation of cells, and in turn can give rise to various disorders.Neurofibromatosis is an genetic disease caused by mutation of the Ras-modulatory protein.Oncogenic Ras gene is a mutation form that cause Ras to remain in its active state by two Ras forms: 1) reduced the intrinsic GTPase activity of Ras; 2) inhibited the sensitivity of Ras to GTPase activating proteins (GAPs).Neurotrophins and Trk-R knockout mice Mice, in which the genes encoding NGF or TrkA have been inactivated, exhibited a loss of sympathetic neurons as well as sensory neurons in dorsal root ganglia of the trigeminal system.NGF or TrkA KO mice also exhibited a partial loss of the cholinergic neurons in the basal forebrain that project to the hippocampus and cerebral cortex.TrkB KO mice exhibited the death of cranial motor neurons and trigeminal ganglion neurons.BDNF KO mice exhibited no a loss of facial or spinal motor neurons. In contrast, TrkB KO mice showed significant motor neurons loss.ALS was characterized the degeneration of cortical and spinal motor neurons.The effects of NeurotrophinsNGF activated neurite growth.BDNF maintains survival of motor neurons. NT-4 is important for supporting survival of motor neurons in development. The effect of NT-3, maintaining survival of motor neurons, is less than BDNF and NT-4. The effects of NGFFast effect Regulating or participating in the release of neurotransmitter：altering the activation of ion channels；enhancing the synthesis of transmitters, peptides and its synthetase.Slow effect: Preventing against death of neurons； maintaining development of sympathetic neurons; maintaining neuronal function and plasticity in adult. Regulating or participating in the release of neurotransmitter:To promote the differentiation of embryonic neurons into the functional neurons；The critical affect for maintenance of neuronal survival in maturate.NGF induces the chemotactic direction for nerve fibre growth. Neuritic directions are along the NGF concentrational gradsNGF ensures survival of neuronal synaptic afferent in sensory and sympathetic neuronsTo promote survival of cholinergic neurons:To promote survival of neuronsAs the same of NGF, BDNF also can promote survival of cholinergic neurons and differentiation of their phenotype in vitro. But both effect in different growth phases of cholingergic neurons, BDNF in forepart but NGF in anaphase.BDNF、NT-3 and NT-4 can promote survival of some motorial cortical neurons and hippocampal neurons. Neurotrophins can promote survival of the neurons which were activated by NE, DA, 5-HT in brain stem. To regulate synaptic plasticityStudies in the formation of ocular dominance columns in the developing visual cortex have be confirmed that neurotrophins mediate forms of synaptic plasticity.Neurotrophins also are involved in regulating synaptic plasticity in the fully differentiated adult brain.Neurotrophins can modulate synaptic transmission and regulate the formation and strengthening of synapses.Neurotrophins also regulate synaptic transmission in the hippocampus. But this still controversial.GDNF FamilyGDNF was first isolated from culturing glial cell line that maintains survival of mesencephalic dopaminergic neurons. Glial cell line-Derived Neurotrophic FactorGDNF, a glycosylated protein of 18 kDa. GDNF family includes Neurturin、persphinGDNF receptors and its signaling pathwayGDNF plays a role via activation of protein tyrosine kinase. But such activation is achieved indirectly through the receptor protein inserted.A GDNF dimer binds to a specific receptor, termed GFR1, a protein of 40 kDa, which is anchored to the plasma membrane by GPI.GDNF bind triggers GFR1 to combine with Ret.Ret is a transmembrane protein tyrosinekinase of about 150 kDa.This binding results in activation of Ret, which is believed that lead to many biologic effects of GDNF via triggering the phosphorylation of specific substrates.The autophosphorylated Ret in turn activates several intracellular signaling proteins that regulate cell survival, differentiation, proliferation, migration, neurite growth and synaptic modulation. These signaling proteins, belonging to protein and lipid kinases and phospholipases, act on several signal transduction pathways. PLC-Regulate Ca2+iMAP kinaseneurite growthneuronal survivalPI3Kneuronal survival neurite growthSeveral signal transduction pathways:Ret signaling by GDNF mediated can interact with and activate the Src-family kinases eliciting optimal neurite outgrowth and neuronal survival. In Ret deficient cell lines and primary neurons, GDNF triggers Src-family kinase activation and phosphorylation of ERK/MAP kinase, PLC- and the transcription factor CREB, and production of FosGDNF family receptors complexThe effects of GDNF family1. Dopamine Neurons GDNF selectively enhanced the survival and development of dopaminergic neurons.In vitro, GDNF protected mesencephalic dopaminergic cells against the effects of the neurotoxin MPP+. GDNF improved the survival of cells, prevented further cell death, and stimulated the re-growth of dopaminergic fibers with damaged by MPP+. In vivo, in PD model induced by the administration of the 6-OHDA into the medial forebrain bundle, GDNF has been shown to reverse apomorphine-induced rotation and rescue dopaminergic neurons lesion.GDNF may be a valuable therapeutic agent in treating Parkinsons disease.Neurturin is also a factor for treatment of PD.The effects of GDNF family2. Motor neurons GDNF was shown to protect facial motoneurons from injury-induced cell death.GDNF can affect motoneuron development and motoneuron survival after axotomy, novel approaches for motor neuron disease are suggested.GDNF can rescue some spinal motoneuron from programmed cell death in development and promote its survival. GDNF can prevent the neuronal degeneration which was caused by axon broken. The effects of GDNF family3.PNS neurons GDNF promotes the survival and neurite outgrowth of cultured sympathetic neurons, parasympathetic ciliary ganglion neurons and sensory dorsal root ganglion neurons.GDNF is important for the developing of enteric neurons and renal neurons. Ret ( loss-of-function) mutation has the same symptoms as human Hirschsprung disease. Ret (gain-of-function) mutation has the same symptoms as neural crest malignancies, e. g. mult-adenomatosis, medullar thyroid cancer. CNTF family(Ciliary neurotriphic factor)CNTF familyReceptors (JAK-coupled receptors)CNTF Gp130 LIFRCNTFRLIF (leukemia inhibitory factor)IL-6，prolactin, growth hormone, leptinIL-11、OSM(oncostatin M ) CNTF familyCNTF, a protein approximately 24 kDa in size, was studied as a survival factor for chick ciliary genglion neurons. CNTF is one of cytokine that plays a role among several processes within the human body from endogenous neuroprotection to regulatethe energy expenditure.CNTF is a pluripotent neurochtokine expressed by glial cells in the peropheral nerves and in the CNS. The family members include LIF, IL-6, OSM, IL-1, prolactin, growth hormone, leptin and interferons. CNTF receptor complex Consists of three components: CNTF family, CNTFR, and signal transducing receptor component（LIFR, gp130）The effects of CNTFCNTF has been proven to regulate the survival or differentiation of many other neuronal cells, including sympathetic neurons, sensory neurons, motor neurons, cultured hippocampal neurons and mesencephalic dopaminergic neurons. CNTF on motor neurons plays the more significant role：CNTF not only supports the neuronal survival in vitro, but also prevents nerve degeneration after axotomy. CNTF improves some motor against motor neuron defects in murine.LIF and IL-6 may regulate the survival or differentiation of neurons.IL-6 promotes the survival of septal cholinergic, mesencephalic catecholaminergic, and hypothalamic neurons in vitro.CNTF and CNTFRCNTF KO nice develop normally, exhibiting only mild motor neuron defects during adulthood.Approximately 2.5% of the Japanese population are homozygous for inactivation mutations of CNTF and thus they are human CNTF “knockouts” , like CNTF KO mice.In contrast, mice lacking the CNTFR lost almost all of their motor neurons and died within 24h after birth. It suggested the existence of another endogenous ligand for CNTFR, but that has not been discovered yet. Therapeutic agents directed at NTFTreatment with BDNF, CNTF, or combination of these factors has been shown to slow or attest the progression of motor neuron degengration in the wobbler mouse Wobbler.Due to BDNF, GDNG, and other factors have enhanced the survival of dopaminergic neurons in animal models, the use of NTFs in the treatment of PD is a top research priority.Investigators are exploring the use of NGF as a treatment for AD. Indeed, after treatment with NGF, aged rats have exhibited improvement in their ability to perform memory tasks.The difficulty of therapy at NTFThe need to deliver such therapeutic factors directly to targeted neurons of the CNS represents a significant challenge.Humans have not been able to tolerate the highest doses of NGF, although such doses are 10,000 times lower than effective doses used in animal modes.Patients treated with this neurotrophic factor can develop neutralizing antibodies to CNTF that limit its bioavailability.小小 结结1. 神经营养因子是一种支持神经元的生长、分化及成活的多肽。2.与多肽的神经递质相比，神经营养因子是通过酪氨酸激酶的活化产生作用的，酪氨酸激酶也可能是神经营养因子实质上的受体或者是分离的蛋白质。3.在适当神经营养因子缺乏的情况下，神经元通过程序性死亡（成为凋亡）而死。小 结4.靶源性生长因子如NGF通过靶组织以有限定量分泌，因而，只有神经元可产生适当的突触间隙的连接存活。 5.神经元自己本身、神经胶质细胞体也可释放神经营养因子，并且以autocrine和paracrine的方式。6.神经营养因子的某些功能与神经递质交迭；例如，脑源性的神经营养因子（BDNF）可用来调节突触间隙传递。小 结7.神经营养素家族由NGF、BDNF、NTF-3以及NTF-4组成。神经营养素家族通过结合到酪氨酸激酶活性受体上而起作用，这些受体称为Trk受体(TrkA、TrkB 、TrkC)8. P75蛋白也与神经营养素结合，但其亲和力较Trk受体低；在某种环境下，p75被包含在程序性死亡的起始中。9. GDNF家 族 除 GDNF外 ， 还 包 括 Neurturin、Persphin和Enovin/artemin三个新发现的成员。GDNF是 通 过 与 GDNF受 体 亚 基 -1（GFR1）结合后引起Ret酪氨酸激酶受体的磷酸化介导细胞内信号转导通路。 小结10. CNTF受体复合物中CNTFR依靠GPI锚定在浆膜上，CNTF与CNTFR结合，触发gp130连接并且随后连接LIFR。三元受体复合物的形成触发JAK和相关的蛋白酪氨酸激酶（如Trk激酶）的活化，引起信号转导级联反应，介导生物效应如转录因子STAT家族的活化。 11. CNTF可调节交感神经元、感觉神经元、运动神经元、海马神经元及中脑多巴胺能神经元的存活和分化。12.营养因子是治疗神经退化疾病的重要候选者；例如，神经胶质细胞分泌的神经营养因子在某种模型系统中支持着多巴胺神经元的存活，因而对Parkinson病的治疗有兴趣。基本概念：神经营养因子Trk受体CNTF受体复合物GFR1P75思考题试述神经营养因子的作用方式。简述NGF如何通过Ret启动胞内Ras/Raf/MEK级联反应。简述神经营养素在调节突触可塑性中的作用。试述GDNF受体及其信号转导通路。试述GDNF对多巴氨能神经元的作用。试述CNTF家成员及其功能。试述神经营养因子在治疗神经退行性疾病中的应用前景。谢 谢！