Molecular Mechanisms Of Synapse Development And Plasticity

突触发育和可塑性的分子机制

• 批准号: 7594150
• 负责人: Bai Lu
• 金额: $ 143.66万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7594150
• 关键词: Acute; Adult; Affect; Antidepressive Agents; Anxiety; Appearance; BDNF gene; Behavior; Behavioral; Binding; Biochemical; Biological; Brain; Brain region; Brain-Derived Neurotrophic Factor; Bromodeoxyuridine; CREB1 gene; Calmodulin; Cell model; Cell physiology; Cell surface; Cells; Cellular biology; Chemosensitization; Cholinergic Receptors; Chromosome Pairing; Cognition; Cognitive; Communication; Complication; Cyclic AMP; Cyclic AMP-Responsive DNA-Binding Protein; Cytoplasmic Granules; Defect; Dendrites; Development; Disruption; Electroporation; Embryo; Embryonic Development; Endocytosis; Endopeptidases; Exhibits; FGFR1 gene; Fibroblast Growth Factor 2; Fibroblast Growth Factor Receptor 2; Financial compensation; Fingers; Generations; Genes; Genetic; Genetic Transcription; Germ Lines; Goals; Growth; Hippocampus (Brain); Human; Hypertrophy; Image; Immigration; Impairment; In Vitro; Individual; Interneurons; Knockout Mice; Label; Learning; Link; Medial; Mediating; Mediator of activation protein; Memory; Mental Depression; Mental disorders; Mitogen-Activated Protein Kinases; Modeling; Modification; Molecular; Monomeric GTP-Binding Proteins; Mood Disorders; Moods; Muscle; Muscle Fibers; Mutant Strains Mice; NGFR Protein; Neurites; Neuromuscular Junction; Neurons; Neurotrophic Tyrosine Kinase Receptor Type 2; Neurotrophin 3; Newborn Infant; PC12 Cells; Pathway interactions; Peptide Hydrolases; Perforant Pathway; Phosphotransferases; Physiological; Play; Positioning Attribute; Presynaptic Terminals; Process; Protein Biosynthesis; Protein Overexpression; Protein Tyrosine Kinase; Proteins; RNA Interference; Receptor Protein-Tyrosine Kinases; Regulation; Reporting; Research; Rewards; Role; Schizophrenia; Signal Pathway; Signal Transduction; Skeletal Muscle; Stress; Structure; Surface; Susceptibility Gene; Synapses; Synaptic plasticity; System; Techniques; Thinking; Transcriptional Activation; Transgenic Mice; Ubiquitin; Ubiquitination; Universities; Up-Regulation; Varicosity; Work; Yin-Yang; base; cognitive function; dentate gyrus; depressive symptoms; extracellular; granule cell; in utero; in vivo; insight; interest; knockout animal; long term memory; loss of function; migration; multicatalytic endopeptidase complex; nerve stem cell; neurogenesis; neuronal cell body; neuronal survival; neurotrophic factor; olfactory bulb; postsynaptic; prevent; promoter; protein degradation; receptor; research study; synaptic depression; synaptic function; synaptogenesis; trafficking; ubiquitin ligase; ubiquitin-protein ligase

This year we have made a significant progress in revealing molecular mechanisms underlying long-term synaptic modulation by neurotrophins. 1) Regulation of the development of the neuromuscular junction Neurotrophins elicit both structural and functional changes of synapses. Whether these changes are mediated by the same or different mechanisms are not known. We report the mechanistic separation of functional and structural synaptic regulation by neurotrophin 3 (NT-3), using the neuromuscular junction (NMJ) as a model. Inhibition of cAMP response element (CRE)-binding protein (CREB)-mediated transcription blocks the enhancement of transmitter release elicited by NT-3, without affecting the synaptic varicosity of the presynaptic terminals. Further analysis indicates that CREB is activated through Ca(2)calmodulin-dependent kinase IV (CaMKIV) pathway, rather than the mitogen-activated protein kinase (MAPK) or cAMP pathway. In contrast, inhibition of MAPK prevents the NT-3-induced structural, but not functional, changes. Genetic and imaging experiments indicate that the small GTPase Rap1, but not Ras, acts upstream of MAPK activation by NT-3. Thus, NT-3 initiates parallel structural and functional modifications of synapses through the Rap1-MAPK and CaMKIV-CREB pathways, respectively. These findings may have implications in the general mechanisms of long-term synaptic modulation by neurotrophins. Long-term synaptic modulation by neurotrophins requires protein synthesis, but the role of protein degradation has not been studies. In this study, we investigated whether ubiquitin-proteasome pathway is involved in the development of NMJ. We have identified a PDZ domain containing RING finger 3 (PDZRN3) as a synapse-associated E3 ubiquitin ligase and have demonstrated that it regulates the surface expression of muscle-specific receptor tyrosine kinase (MuSK), the key organizer of postsynaptic development at the NMJ. PDZRN3 binds to MuSK and promotes its ubiquitination. Regulation of cell surface levels of MuSK by PDZRN3 requires the ubiquitin ligase domain and is mediated by accelerated endocytosis. Gain- and loss-of-function studies in cultured myotubes show that regulation of MuSK by PDZRN3 plays an important role in MuSK-mediated ACh receptor clustering. Furthermore, overexpression of PDZRN3 in skeletal muscle of transgenic mice perturbs the growth and maturation of the NMJ. These results identify a synapse-associated E3 ubiquitin ligase as an important regulator of synapse development. 2) Neurotrophic regulation of adult neurogenesis, memory and mood disorders While adult neurogenesis is thought to play a role in learning and memory as well as in depression, how newly generated neurons contribute to the cognitive process remains unknown. Fibroblast growth factor 2 (FGF-2) is known to stimulate the proliferation of neuronal progenitor cells (NPCs) in adult brain. Using conditional knockout mice that lack brain expression of FGFR1, a major receptor for FGF-2, we have investigated the role of adult neurogenesis in hippocampal synaptic plasticity and learning and memory. Bromodeoxyuridine labeling experiments demonstrate that FGFR1 is required for the proliferation of NPCs as well as generation of new neurons in the adult dentate gyrus (DG). Moreover, deficits in neurogenesis in Fgfr1 mutant mice are accompanied by a severe impairment of LTP at the medial perforant path (MPP)-granule neuron synapses in the hippocampal dentate. Finally, the Fgfr1 mutant mice exhibit significant deficits in memory consolidation but not spatial learning. Our study suggests a critical role of FGFR1 in adult neurogenesis in vivo, provides a potential link between proliferative neurogenesis and dentate LTP, and raises the possibility that adult neurogenesis might contribute to memory consolidation. The role of FGFR1 in depression remains to be further investigated. The neurotrophin hypothesis of depression is based largely on correlations between stress or antidepressant treatment and down- or up-regulation, respectively, of BDNF. The current status of research on BDNF and depression is reviewed. Genetic disruption of the signaling pathways involving BDNF and its TrkB receptor does not seem to cause depressive behaviors, but does hamper the effect of antidepressant drugs. Thus, BDNF may be a target of antidepressants, but not the sole mediator of depression or anxiety. Advances in BDNF cell biology, including its transcription through multiple promoters, trafficking and secretion, may provide new insights into its role in mood disorders. Moreover, as the precursor proBDNF and the mature protein mBDNF can elicit opposite effects on cellular functions, the impact of proBDNF and its cleavage on mood should be considered. Opposing influences of mBDNF and proBDNF on LTP and LTD might contribute to the dichotomy of BDNF actions on behaviors mediated by the brain stress and reward systems. 3) Studies of genes involved in schizophrenia A new line of research in this lab is to study genes involved in cognition and schizophrenia. We have recently collaborated with Dr. Hong-Jun Songs group at Johns Hopkins University to investigate the role of Disrupted-In-Schizophrenia 1 (disc1), a susceptibility gene for schizophrenia. DISC1 expression is broad in many brain regions during embryonic development and fairly restricted in the adult brain with particularly high expression in dentate granule cells of the hippocampus and interneurons of the olfactory bulb, two neuronal types that are continuously generated through adult neurogenesis. A role of DISC1 in neuronal development was first suggested by biochemical identification of interacting proteins. For example, DISC1 binds Ndel1 (NUDEL), a molecule involved in embryonic neuronal development including migration. In vitro studies with PC12 cells and primary neurons showed that blocking DISC1 function impairs neurite outgrowth. Furthermore, in utero electroporation-mediated expression of DISC1 shRNAs in embryos leads to retarded migration. The finding that DISC1 promotes migration in the embryonic cortex and neurite outgrowth in vitro, as well as its restricted expression in neurons produced during adult neurogenesis, raise a tantalizing possibility that DISC1 may play an important role in regulating the process of adult neurogenesis. To ascertain the in vivo function of DISC1 in adult neurogenesis, we employed an oncoretrovirus-mediated RNA interference approach to genetically manipulate DISC1 expression within individual cells in specific brain regions. Such in vivo single-cell genetic approach allows characterization of cell-autonomous roles of DISC1 specifically in adult neurogenesis, without the complication of potential developmental defects andor compensations in traditional germ-line knockout animals. We demonstrated that DISC1 regulates almost all essential steps of neuronal integration in adult neurogenesis. In contrast to what has been found in embryonic cortical development and cultured neuronal cells, DISC1 knockdown in newborn dentate granule cells of the adult hippocampus leads to soma hypertrophy, accelerated dendritic outgrowth with appearance of ectopic dendrites, mis-positioning from over-extended migration, enhanced intrinsic excitability and accelerated synapse formation of new neurons. These findings indicate that DISC1, a schizophrenia susceptibility gene, serves as a key regulator that controls the tempo of neuronal development and therefore keeps the progress of new neuron integration in the adult brain in check.

今年，我们在揭示神经营养蛋白长期突触调节的分子机制方面取得了重大进展。 1）调节神经肌肉连接的发展 神经营养蛋白引起突触的结构变化和功能变化。这些变化是由相同或不同机制介导的。我们使用神经肌肉连接（NMJ）作为模型报告了神经营养蛋白3（NT-3）的功能和结构突触调节的机械分离。抑制营地反应元件（CRE）结合蛋白（CREB）介导的转录阻断了NT-3引起的发射机释放的增强，而不会影响突触前末端的突触静脉曲张。进一步的分析表明，CREB是通过Ca（2）钙调蛋白依赖性激酶IV（CAMKIV）途径激活的，而不是促有丝分裂原激活的蛋白激酶（MAPK）或cAMP途径。相反，对MAPK的抑制可阻止NT-3诱导的结构性变化，但不是功能性的变化。遗传和成像实验表明，小的GTPase RAP1（而不是RAS）作用于NT-3的MAPK激活的上游。因此，NT-3分别通过RAP1-MAPK和CAMKIV-CREB途径启动突触的平行结构和功能修饰。这些发现可能对神经营养蛋白长期突触调节的一般机制有影响。 神经营养蛋白长期的突触调节需要蛋白质合成，但蛋白质降解的作用并不是研究。 在这项研究中，我们研究了泛素 - 蛋白酶体途径是否参与NMJ的发展。我们已经确定了一个含有环手指3（PDZRN3）的PDZ结构域是突触相关的E3泛素连接酶，并证明它调节了NMJ的突触后发育的关键组织者。 PDZRN3与麝香结合并促进其泛素化。 PDZRN3对麝香的细胞表面水平的调节需要泛素连接酶结构域，并由加速内吞作用介导。在培养的肌管中的功能丧失研究表明，PDZRN3对MUSK的调节在Musk介导的ACH受体聚类中起重要作用。此外，在转基因小鼠的骨骼肌中PDZRN3的过表达呈现NMJ的生长和成熟。这些结果确定了与突触相关的E3泛素连接酶是突触发育的重要调节剂。 2）成人神经发生，记忆和情绪障碍的神经营养调节 尽管人们认为成年神经发生在学习和记忆以及抑郁症中发挥作用，但新产生的神经元如何促进认知过程仍然未知。已知成纤维细胞生长因子2（FGF-2）刺激成人脑中神经元细胞（NPC）的增殖。使用缺乏FGFR1的脑表达的条件敲除小鼠，FGFR1是FGF-2的主要受体，我们研究了成年神经发生在海马突触可塑性以及学习以及记忆中的作用。溴脱氧尿苷的标记实验表明，NPC的增殖以及成人齿状回（DG）的新神经元的生成是必需的。此外，FGFR1突变小鼠的神经发生缺陷伴随着内侧穿孔路径（MPP） - 海马齿状颗粒神经元突触的LTP严重损害。最后，FGFR1突变小鼠在记忆巩固中表现出明显的缺陷，但没有空间学习。我们的研究表明，FGFR1在体内成人神经发生中的关键作用，提供了增生性神经发生和齿状LTP之间的潜在联系，并提高了成人神经发生可能有助于记忆巩固的可能性。 FGFR1在抑郁症中的作用仍有待进一步研究。 抑郁症的神经营养蛋白假说主要基于BDNF的压力或抗抑郁药治疗与下调或上调之间的相关性。审查了关于BDNF和抑郁症的研究现状。涉及BDNF及其TRKB受体的信号通路的遗传破坏似乎并没有引起抑郁行为，但会阻碍抗抑郁药的作用。因此，BDNF可能是抗抑郁药的靶标，而不是抑郁或焦虑的唯一介体。 BDNF细胞生物学的进步，包括通过多个启动子，贩运和分泌的转录，可能会为其在情绪障碍中的作用提供新的见解。此外，由于前体ProbDNF和成熟的蛋白质MBDNF可以对细胞功能产生相反的影响，因此应考虑ProbDNF及其裂解对情绪的影响。 MBDNF和ProbDNF对LTP和LTD的相反影响可能有助于BDNF作用对由大脑压力和奖励系统介导的行为的二分法。 3）研究中涉及精神分裂症的基因 该实验室的一项新研究是研究涉及认知和精神分裂症的基因。我们最近与约翰·霍普金斯大学（Johns Hopkins University）的Hong-jun Songs Group合作，调查了雪橇症1（Disc1）的作用，这是精神分裂症的易感基因。在胚胎发育过程中，许多大脑区域的表达广泛，并且在成年大脑中受到相当限制，在海马齿状细胞和嗅球的齿状颗粒细胞中特别高表达，两种神经元类型是通过成人神经生成不断产生的两种神经元类型。首先通过对相互作用蛋白的生化鉴定提出了DISC1在神经元发育中的作用。例如，Disc1结合NDEL1（NUDEL），NDEL1（NUDEL）是一种参与胚胎神经元发育的分子，包括迁移。对PC12细胞和原发性神经元的体外研究表明，阻断Disc1功能会损害神经突生长。此外，在子宫电穿孔介导的胚胎中的表达中，胚胎中会导致较低的迁移。 DISC1促进体外胚胎皮质和神经突生长的迁移的发现，以及其在成人神经发生过程中产生的神经元中的受限表达，提高了Disc1在调节成人神经发生过程中起重要作用的诱人可能性。 为了确定成年神经发生中椎间盘1的体内功能，我们采用了肠内病毒介导的RNA干扰方法来对特定大脑区域中单个细胞内的二氧化激素表达进行遗传操纵。这种体内单细胞遗传学方法允许表征DISC1在成人神经发生中的细胞自主作用，而没有潜在的发育缺陷的并发症Andor补偿在传统的种系敲除动物中。我们证明，DISC1几乎调节成人神经发生中神经元整合的所有基本步骤。 In contrast to what has been found in embryonic cortical development and cultured neuronal cells, DISC1 knockdown in newborn dentate granule cells of the adult hippocampus leads to soma hypertrophy, accelerated dendritic outgrowth with appearance of ectopic dendrites, mis-positioning from over-extended migration, enhanced intrinsic excitability and accelerated synapse formation of new neurons.这些发现表明，精神分裂症易感基因Disc1是控制神经元发育节奏的关键调节剂，因此可以检查成人大脑中新的神经元整合的进步。