Transport and regulation of presynaptic release machinery

突触前释放机制的运输和调节

• 批准号: 7969574
• 负责人: Zu-hang Sheng
• 金额: $ 116.99万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 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/7969574
• 关键词: Action Potentials; Arts; Attention; Axon; Axonal Transport; Binding; Biochemistry; Brain; Calcium; Carrier Proteins; Cell Adhesion Molecules; Cell membrane; Cellular biology; Chromaffin Cells; Code; Complex; Coupling; Defect; Dense Core Vesicle; Electrophysiology (science); Event; Exhibits; Exocytosis; Frequencies; Generations; Genes; Genetically Engineered Mouse; Goals; Insulin-Like Growth-Factor Binding Protein 1; Intracellular Membranes; Journals; Kinesin; Kinetics; Knockout Mice; Life; Mediating; Membrane Protein Traffic; Microfilaments; Microtubules; Molecular; Molecular Biology; Motor; Mus; Mutant Strains Mice; Names; Nature; Nerve; Neurodegenerative Disorders; Neurons; Neurosciences; Pan Genus; Paper; Pathway interactions; Phenotype; Physiological; Play; Presynaptic Terminals; Probability; Process; Protein Biosynthesis; Proteins; Publishing; Regulation; Research; Role; Route; SNAP receptor; SNAPIN gene; Shapes; Specificity; Structure; Synapses; Synaptic Transmission; Synaptic Vesicles; Synaptic plasticity; Systems Analysis; Time; Transcriptional Activation; Vesicle; anterograde transport; base; cellular imaging; density; genetic regulatory protein; in vivo; loss of function; mutant; neuron development; neuronal cell body; neuronal transport; neurotransmission; neurotransmitter release; novel; postsynaptic; presynaptic; response; sensor; synaptic function; synaptogenesis; synaptotagmin I; syntaxin; syntaxin 1; trafficking; vesicle-associated membrane protein

Specific Aim 1: Discovery of A New Pathway for the Activity-Dependent Plasticity through Axonal Transport Neuronal transport includes the intracellular trafficking route for membranous protein carriers from the soma to nerve terminals where they deliver cargos for synapse formation. The contents of these transport packets include protein components of SV and AZs, exocytotic machinery, channels, and adhesion molecules. Cargos must be attached to their transport motors with a high degree of specificity to preserve cargo identity and targeted trafficking. However, the mechanism underlying motor-cargo interactions remains unresolved. The SNARE protein syntaxin-1, a key component of SV fusion machinery, is transported to the plasma membrane in cargos. We identified a novel syntaxin-binding and kinesin-1 motor (KIF5)-associated protein named syntabulin (1, 2). Our studies with loss-of-function analysis established that syntabulin is an adaptor capable of conjoining syntaxin-1 and KIF5 motors, thereby mediating transport of syntaxin-1 to neuronal processes. Remodeling of pre-existing synapses and the formation of new synapses play an important role in the various forms of synaptic plasticity of complex neuronal networks. Previously identified mechanisms underlying activity-dependent synaptic plasticity include activation of transcriptional factors, new protein synthesis, and reorganization of the actin filaments at synapses. Thus, efficient and targeted axonal transport of newly synthesized synaptic components to presynaptic boutons would be critical in response to neuronal activity. However, the contribution of the microtubule-based axonal transport to the activity-induced formation of new synapses are unknown. Since syntaxin-1 is a component of the AZ precursor cargos, further characterization of syntabulins role in neuronal trafficking will contribute to understanding the molecular mechanisms of the axonal delivery of presynaptic components. Our ongoing research reveals that syntaxin-1, syntabulin, and KIF5 comprise the transport machinery critical for anterograde axonal transport of the AZ precursors and contributes to presynaptic assembly (3). Knockdown of syntabulin or disruption of the syntaxin-1-syntabulin-KIF5B complex impairs the anterograde transport of AZ components out of the soma and reduces the axonal densities of SV clusters and FM4-64 loading. Furthermore, syntabulin loss-of-function results in a reduction in both the amplitude of postsynaptic currents and the frequency of asynchronous quantal events, and abolishes the activity-induced recruitment of new AZ components into the axons and subsequent co-clustering with SVs. Consequently, syntabulin loss-of-function blocks the formation of new presynaptic boutons during activity-dependent synaptic plasticity. These studies establish for the first time that a kinesin motor-adaptor complex is critical for the anterograde axonal transport of AZ components, thus contributing to activity-dependent presynaptic assembly during neuronal development. To explore the function of syntabulin in vivo, we will generate the conditional KO mice with functional disruption of the syntabulin gene. Phenotype analysis of the KO mice and the mutant neurons will clarify the molecular details of how this protein mediates the trafficking of the AZ precursor vesicles essential for presynapse assembly and synaptic plasticity. Specific Aim 2: Identification of An Essential Role of Snapin in Synchronizing Fast Neurotransmitter Release Information coding in the brain depends on the timing of action potentials, which is influenced by integration of unitary excitatory inputs. The size and shape of excitatory postsynaptic currents (EPSCs) are two decisive factors in tuning the temporal and spatial precision of spiking and can be modulated by synaptic vesicle (SV) fusion process. Ca2+-triggered neurotransmitter release depends on the presence of a pool of primed release-ready SVs, which determines the release probability of a synapse. The priming step corresponds to assembly of the SNARE complex in which synaptobrevin interacts with SNAP-25 and syntaxin-1 to form a metastable structure before fusion. Maturation of SVs into a release-ready state requires synaptotagmin I (Syt I), a Ca2+ sensor of fast neurotransmission. Accurate assembly of Syt I-SNARE fusion machineries is critical for the precise timing of fast release. Ca2+-dependent and independent interactions between Syt I and SNAREs suggest that before the Ca2+ trigger, a loose pre-fusion Syt I-SNARE complex is assembled during the priming process. Ca2+ influx sensitizes the Ca2+ sensor Syt I and induces its subsequent tight coupling to the SNARE complex. While much attention in the past decade has been given to the SNARE-regulatory proteins in studying SV release probability and short-term plasticity, our understanding of the molecular mechanisms that govern the tuning of EPSC shape is largely lacking. We initially identified Snapin as a SNAP-25-binding protein that enhances the association of Syt I with the SNAREs (4-6). Using snapin knockout mice, we demonstrated that Snapin modulates fast exocytosis of large dense-core vesicles in chromaffin cells (7). Deletion of snapin leads to a reduced amount of Syt I-SNARE complex in mouse brain. We recently characterized the function of Snapin in synchronizing SV fusion at central synapses (8). By recording synaptic transmission between cultured cortical neurons from snapin-deficient mice, we found that snapin mutant neurons exhibit EPSCs with multiple peaks and fail to follow sustained firing under high-frequency stimulations. Re-introducing snapin into the mutant presynaptic neurons effectively accelerates EPSC kinetics to the greater extent found in (+/+) neurons by boosting the synchronicity of SV fusion. The marked increase in rise/decay time and synaptic delay time observed in snapin-deficient neurons changes the shape of the EPSC and impairs both synaptic efficacy and precision. At snapin-deficient nerve terminals, SVs are likely heterogeneously primed due to the unfavorable or unstable association of Syt I with the metastable SNARE complex before the Ca2+ sensing. It leads to two defects: (1) fewer fusion competent vesicles, and hence decreased size of EPSCs; and (2) fewer vesicles undergoing synchronized fusion within a narrow time window during excitation-secretion coupling. Thus, our studies reveal the role of Snapin as a unique synchronizer of calcium-triggered SV fusion at central synapses. Papers published from the lab related to the projects: 1. Qingning Su*, Qian Cai*, Claudia Gerwin, Carolyn L. Smith, Zu-Hang Sheng (2004). Nature Cell Biology 6, 941-953. 2. Qian Cai, Claudia Gerwin, and Zu-Hang Sheng. (2005). Journal of Cell Biology 170, 959-969. 3. Qian Cai, Pingyue Pan, and Zu-Hang Sheng. (2007). Journal of Neuroscience 27, 7284-7296. 4. Jeffrey M. Ilardi, Sumiko Mochida, and Zu-Hang Sheng (1999). Nature Neuroscience 2, 119-124. 5. Milan G. Chheda, Uri Ashery, Pratima Thakur, Jens Rettig, and Zu-Hang Sheng (2001). Nature Cell Biology 3, 331-338. 6. Pratima Thakur, David R. Stevens, Zu-Hang Sheng and Jens Rettig (2004), Journal of Neuroscience 24, 6476-6481. 7. Jin-Hua Tian, et al (2005). Journal of Neuroscience 25, 10546-10555. 8. Ping-Yue Pan, Jin-Hua Tian and Zu-Hang Sheng (2009). Neuron 61, 412-424.

特定目的1：通过轴突传输发现新的依赖活性可塑性的途径 神经元的运输包括从躯体到神经末端的膜蛋白载体的细胞内贩运途径，在那里它们输送cargos以形成突触。这些传输包的内容包括SV和AZS的蛋白质成分，胞外机械，通道和粘附分子。必须具有高度特异性的货物，以保持货物身份和有针对性的贩运。然而，电动货车相互作用的基础机制仍未解决。 SNARE蛋白Syntaxin-1是SV融合机械的关键组成部分，被运输到嘉戈斯的质膜。我们鉴定了一种新型的语法结合和驱动蛋白-1运动（KIF5）相关的蛋白质蛋白（1，2）。我们对功能丧失分析的研究表明，征素蛋白是一种能够连接语法1和KIF5电动机的适配器，从而介导了义taxin-1向神经元过程的转运。 在复杂神经元网络的各种突触可塑性中重塑现有的突触和新突触的形成起着重要作用。先前确定的基础活性依赖性突触可塑性的机制包括转录因子的激活，新的蛋白质合成以及突触处肌动蛋白丝的重组。因此，新合成的突触成分向突触前胸子的有效且有针对性的轴突运输对于响应神经元活性至关重要。但是，基于微管的轴突转运对活性诱导的新突触形成的贡献尚不清楚。由于Syntaxin-1是AZ前体cargos的组成部分，因此在神经元运输中的征素蛋白作用的进一步表征将有助于理解突触前成分的轴突递送的分子机制。我们正在进行的研究表明，Syntaxin-1，syntabulin和KIF5构成了AZ前体轴突运输至关重要的运输机制，并有助于突触前组装（3）。敲低征素蛋白或索法蛋白-1-辅助蛋白-KIF5B复合物的破坏会损害AZ组件从SOMA中的顺行传输，并降低SV簇和FM4-64载荷的轴突密度。此外，征肌丧失的功能丧失导致突触后电流的振幅和异步定量事件的频率都降低，并废除了将新的AZ组件募集到轴突中的活性诱导的募集，并与SVS进行了后续共聚类。因此，征功能丧失阻碍了活动依赖性突触可塑性期间新的突触前胸子的形成。这些研究首次确定了动力蛋白运动适应器复合物对于AZ成分的顺行轴突运输至关重要，因此有助于神经元发育过程中活性依赖性突触前组装。为了探索体内征素蛋白的功能，我们将产生有条件的KO小鼠，并具有征素基因基因的功能破坏。对KO小鼠和突变神经元的表型分析将阐明该蛋白如何介导AZ前体囊泡的运输对于促肿瘤前组装和突触可塑性必不可少的分子细节。 特定目标2：识别Snapin在同步快速神经递质释放中的基本作用 大脑中的信息编码取决于动作电位的时间，这受到单位兴奋性输入的整合的影响。兴奋性突触后电流（EPSC）的大小和形状是调整峰值的时间和空间精度的两个决定性因素，可以通过突触囊泡（SV）融合过程调节。 Ca2+触发的神经递质释放取决于启动释放的SV池的存在，这决定了突触的释放概率。启动步骤对应于SNARE复合物的组装，在该复合物中，突触纤维与SNAP-25和Syntaxin-1相互作用以在融合前形成亚稳态结构。将SVS成熟到释放就绪状态需要SynaptoTagmin I（SYT I），这是快速神经传递的CA2+传感器。 SYT I-SNARE融合机器的准确组装对于快速释放的精确时机至关重要。 CA2+依赖性和独立的相互作用与SYT I和SNARES之间的相互作用表明，在Ca2+触发之前，在启动过程中组装了松散的融合前SYT I-SNARE复合物。 Ca2+涌入使Ca2+传感器SYT I敏感，并诱导其随后的紧密耦合到SNARE复合物。尽管在过去的十年中，在研究SV释放概率和短期可塑性方面已经引起了编调控蛋白的广泛关注，但我们对控制EPSC形状调整的分子机制的理解在很大程度上缺乏。 我们最初将Snapin鉴定为一种SNAP-25结合蛋白，可增强SYT I与SNARES的缔合（4-6）。使用Snapin基因敲除小鼠，我们证明了Snapin调节铬蛋白细胞中大型致密囊泡的快速胞吐作用（7）。 Snapin的缺失导致小鼠大脑中SYT I-SNARE复合物的量减少。我们最近表征了Snapin在中央突触下同步SV融合中的功能（8）。通过记录来自Snapin缺陷型小鼠培养的皮质神经元之间的突触传播，我们发现Snapin突变神经元具有多个峰值EPSC，并且在高频刺激下未能持续持续发射。通过提高（+/+）神经元中发现的更大程度地，将Snapin重新引入突触前神经元中有效加速EPSC动力学，从而提高SV融合的同步性。在缺乏Snapin的神经元中观察到的上升/衰减时间和突触延迟时间的明显增加改变了EPSC的形状，并损害了突触功效和精度。在Snapin缺乏的神经末端，由于SYT I与CA2+传感之前的亚稳态SNARE复合物的不利或不稳定的关联，SV可能是异质启动的。它导致两个缺陷：（1）较少的融合囊泡，因此EPSC的大小降低； （2）在激发分泌耦合期间，在狭窄的时间窗口内进行同步融合的囊泡较少。因此，我们的研究揭示了Snapin作为中央突触中钙触发的SV融合的独特同步器的作用。 从实验室发表的论文与项目有关： 1。自然细胞生物学6，941-953。 2。 （2005）。细胞生物学杂志170，959-969。 3。Qian Cai，Pingyue Pan和Zu-Hang Sheng。 （2007）。神经科学杂志27，7284-7296。 4。JeffreyM. Ilardi，Sumiko Mochida和Zu-Hang Sheng（1999）。自然神经科学2，119-124。 5。MilanG. Chheda，Uri Ashery，Pratima Thakur，Jens Rettig和Zu-Hang Sheng（2001）。 自然细胞生物学3，331-338。 6。PratimaThakur，David R. Stevens，Zu-Hang Sheng和Jens Rettig（2004），《神经科学杂志》 24，6476-6481。 7。Jin-Hua Tian等人（2005）。神经科学杂志25，10546-10555。 8。Ping-Yue Pan，Jin-Hua Tian和Zu-Hang Sheng（2009）。 Neuron 61，412-424。