Axonal transport regulates neurotransmission and autophagy-lysosomal function

轴突运输调节神经传递和自噬溶酶体功能

• 批准号: 8342217
• 负责人: Zu-hang Sheng
• 金额: $ 152.07万
• 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/8342217
• 关键词: Action Potentials; Address; Affect; Autophagocytosis; Axon; Axonal Transport; Brain; Cellular biology; Code; Complex; Cytosol; Defect; Disease; Disease model; Distant; Docking; Dynein ATPase; Ensure; Exhibits; Family; Frequencies; Functional disorder; Future; Generations; Genes; Genetic; Genetic Variation; Genetically Engineered Mouse; Goals; Hippocampus (Brain); Homeostasis; Human; Image; Impaired cognition; Intracellular Transport; Investigation; Journals; Kinesin; Kinetics; Knockout Mice; Knowledge; Lead; Life; Link; Lysosomes; Maintenance; Mediating; Membrane; Membrane Protein Traffic; Microtubule-Associated Proteins; Mission; Mitochondria; Modality; Molecular; Molecular Target; Motor; Mus; National Institute of Neurological Disorders and Stroke; Nature; Nerve Degeneration; Neurodegenerative Disorders; Neuroglia; Neurons; Neurosciences; Organelles; Pathogenesis; Pathway interactions; Phenotype; Play; Presynaptic Terminals; Process; Proteins; Proteolysis; Publications; Quality Control; Regulation; Reporting; Research; Role; SNAPIN gene; Schizophrenia; Shapes; Signal Transduction; Site; Sorting - Cell Movement; Specificity; Structure; Susceptibility Gene; Synapses; Synaptic Transmission; Synaptic Vesicles; Synaptic plasticity; System; Systems Analysis; Therapeutic; Time; Transgenes; Vesicle; base; cellular imaging; density; insight; interest; late endosome; loss of function; multidisciplinary; mutant; nervous system disorder; neuronal cell body; neurotransmission; neurotransmitter release; novel therapeutic intervention; postsynaptic; presynaptic; protein misfolding; research study; response; retrograde transport; synaptogenesis; syntaxin; syntaxin 1; targeted delivery; time use; tool; trafficking; trans-Golgi Network; Action Potentials; Address; Affect; Autophagocytosis; Axon; Axonal Transport; Brain; Cellular biology; Code; Complex; Cytosol; Defect; Disease; Disease model; Distant; Docking; Dynein ATPase; Ensure; Exhibits; Family; Frequencies; Functional disorder; Future; Generations; Genes; Genetic; Genetic Variation; Genetically Engineered Mouse; Goals; Hippocampus (Brain); Homeostasis; Human; Image; Impaired cognition; Intracellular Transport; Investigation; Journals; Kinesin; Kinetics; Knockout Mice; Knowledge; Lead; Life; Link; Lysosomes; Maintenance; Mediating; Membrane; Membrane Protein Traffic; Microtubule-Associated Proteins; Mission; Mitochondria; Modality; Molecular; Molecular Target; Motor; Mus; National Institute of Neurological Disorders and Stroke; Nature; Nerve Degeneration; Neurodegenerative Disorders; Neuroglia; Neurons; Neurosciences; Organelles; Pathogenesis; Pathway interactions; Phenotype; Play; Presynaptic Terminals; Process; Proteins; Proteolysis; Publications; Quality Control; Regulation; Reporting; Research; Role; SNAPIN gene; Schizophrenia; Shapes; Signal Transduction; Site; Sorting - Cell Movement; Specificity; Structure; Susceptibility Gene; Synapses; Synaptic Transmission; Synaptic Vesicles; Synaptic plasticity; System; Systems Analysis; Therapeutic; Time; Transgenes; Vesicle; base; cellular imaging; density; insight; interest; late endosome; loss of function; multidisciplinary; mutant; nervous system disorder; neuronal cell body; neurotransmission; neurotransmitter release; novel therapeutic intervention; postsynaptic; presynaptic; protein misfolding; research study; response; retrograde transport; synaptogenesis; syntaxin; syntaxin 1; targeted delivery; time use; tool; trafficking; trans-Golgi Network

Specific Aim 1. Regulation of Synaptic Vesicle Dynamics and 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 the SV fusion process. Our recent studies using the snapin-deficient cortical neurons combined with gene rescue experiments revealed a crucial role for Snapin in enhancing the efficacy of SV priming and in fine-tuning the precision of synchronous release (Pan et al., Neuron 2009). Snapin mutant neurons exhibit EPSCs with multiple peaks and fail to follow sustained firing under high-frequency stimulation. Re-introducing snapin into the mutant presynaptic neurons effectively accelerates EPSC kinetics by boosting the synchronicity of SV fusion. Thus, our studies reveal the role of Snapin as a unique synchronizer of SV fusion at central synapses. Several groups have independently reported an interaction between Snapin and dysbindin (BTNBP1)the product of a susceptibility gene found among the common genetic variations associated with schizophrenia. Our recent study also identified that Snapin acts as dynein motor adaptor for late endosomes (Cai et al., Neuron 2010). This raises an interesting question whether Snapin regulates synaptic vesicle transport and dynamics at release sites, thus contributing to regulation of synaptic transmission. Our current studies aim at (1) elucidating mechanisms by which Snapin regulates synchronized synaptic transmission; (2) determining whether Snapin-dynein transport complex regulates synaptic vesicle density and dynamics at presynaptic terminals; and (3) evaluating Snapins role in the cognitive impairment prominent in schizophrenia. Specific Aim 2. Regulation of Presynaptic Cargo Transport and its Impact on Synaptic Plasticity. The formation of new synapses and remodeling of existing synapses play an important role in the various forms of synaptic plasticity and require the targeted delivery of newly synthesized synaptic components from the trans-Golgi network (TGN) in the soma to the synaptic terminals. Thus, efficient axonal transport of these newly synthesized components to nascent presynaptic boutons is critical in response to neuronal activity. Substantial evidence suggests that AZ precursor carriers are generated from TGN and traverse the developing axon to nascent synapses. Cargo vesicles must attach to their transport motors with a high degree of specificity to preserve cargo identity and targeted trafficking. However, the molecular identities of the motor-adaptor complex essential for assembling presynaptic terminals in developing neurons and in remodeling synapses of mature neurons in response to neuronal activity remain unknown. Our previous studies established that syntabulin is a motor adaptor capable of joining KIF5B and syntaxin-1 and enables syntaxin-1 transport to neuronal processes (Su et al., Nature Cell Biology, 2004). Using time-lapse imaging in live hippocampal neurons, we further demonstrate that the transport complex of syntaxin-1-syntabulin-KIF5B mediates axonal transport of the AZ components essential for presynaptic assembly. Syntabulin loss-of-function blocks formation of new presynaptic boutons during activity-dependent synaptic plasticity in developing neurons (Cai et al., J Neuroscience 2007). Our studies establish that kinesin-mediated and MT-based anterograde axonal transport is another critical factor in the cellular mechanism underlying activity-dependent presynaptic plasticity. Our recent study further demonstrated the critical role of syntabulin-mediated axonal transport in the maintenance of presynaptic function and regulation of synaptic plasticity in well-matured sympathetic SCG neurons in culture (Ma et al., J Neuroscience 2009). Our findings provide a molecular basis for future studies. Conditional syntabulin knockout mice are in generation and we will use this genetic mouse line to (1) determine whether deficiency in syntabulin/KIF5-mediated anterograde axonal transport has any impact on synapse formation and maintenance, and synaptic plasticity; (2) determine whether the motor-adaptor complex regulates the transport rate in response to synaptic activity; (3) identify the sorting signals for the axon-targeted delivery of the AZ cargo. Specific Aim 3. Retrograde Transport and Impact on Neuronal Autophagy-Lysosomal Function. Maintaining cellular homeostasis in neurons depends on efficient intracellular transport. Late endocytic trafficking, which delivers target materials into lysosomes, is critical for maintaining efficient degradation capacities via autophagy-lysosomal pathways. Autophagy-lysosomal system is essential for quality control of intracellular components and mitochondria, and maintenance of cellular homeostasis. An impaired autophagy-lysosomal system has been associated with the pathogenesis of several major neurodegenerative diseases. However, the mechanisms regulating the autophagy-lysosomal system in neurons remain incompletely understood. Dynein-mediated retrograde transport can enhance late endocytic trafficking to some, where lysosomes are predominantly localized, and drive late endosomes and lysosomes close enough to fuse with higher efficiency, thus ensuring proper autophagy-lysosomal function. In addition to its association with SVs, Snapin is present in cytosol and membrane-associated fractions in neuronal and non-neuronal cells and is co-purified with late endocytic organelles. Our recent study uncovered a critical role for Snapin in regulating late endocytic transport and membrane trafficking (Cai et al., Neuron 2010). Snapin acts as a motor adaptor by attaching dynein to late endosomes. Snapin (-/-) neurons exhibit aberrant accumulation of immature lysosomes, impaired retrograde transport of late endosomes along processes, reduced lysosomal proteolysis, and impaired clearance of autolysosomes, combined with reduced neuron viability and neurodegeneration. The phenotypes are rescued by expressing the snapin transgene. Thus, our study highlights new mechanistic insights into how Snapin-dynein coordinates retrograde transport and late endosomal-lysosomal trafficking critical for autophagy-lysosomal function. Our research goal is to identify the cellular pathways for clearance of aggregation-prone proteins by regulating the autophagy-lysosomal system. The snapin KO mouse provides us with a unique genetic tool for characterizing the role of late endocytic transport in neurodegeneration. The conditional snapin KO mice are generated and are being crossing with several disease mouse lines including mutant SOD1-linked ALS disease model. These studies will provide genetic evidence as to whether manipulating the late endocytic pathway will ultimately lead to new therapeutic approaches. Pursuing these investigations will advance our knowledge of fundamental processes that may affect human neurological disorders and is thus the very essence of the mission of the National Institute of Neurological Disorders and Stroke. Related publications from the lab: Qingning Su, Qian Cai, Claudia Gerwin, Carolyn L. Smith, Zu-Hang Sheng (2004) Syntabulin: a microtubule-associated protein implicated in syntaxin transport in neurons, Nature Cell Biology 6, 941-953. Qian Cai, Pingyue Pan, and Zu-Hang Sheng. (2007). Syntabulin-kinesin-1 family 5B-mediated axonal transport contributes to activity-dependent presynaptic assembly. Journal of Neuroscience 27, 7284-7296. Ping-Yue Pan, Jin-Hua Tian and Zu-Hang Sheng (2009). Snapin Facilitates the Synchronization of Synaptic Vesicle Fusion. Neuron 61, 412-424.

特定目标1。突触囊泡动力学和释放的调节。 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SV融合过程调节。我们最近使用Snapin缺陷型皮质神经元与基因救援实验相结合的研究表明，Snapin在增强SV启动的功效以及微调同步释放的精确度方面至关重要（Pan等人，Neuron 2009）。 Snapin突变神经元表现出具有多个峰的EPSC，并且在高频刺激下未能持续发射。将Snapin重新引入突触前神经元中，通过提高SV融合的同步性有效地加速了EPSC动力学。因此，我们的研究揭示了Snapin作为中央突触中SV融合的独特同步器的作用。几个组已经独立地报道了Snapin和Dysbindin（BTNBP1）之间的相互作用（BTNBP1）在与精神分裂症相关的常见遗传变异中发现的易感基因的乘积。我们最近的研究还确定，Snapin充当晚期内体的动力蛋白运动适配器（Cai等，Neuron，2010年）。这就提出了一个有趣的问题，Snapin是否调节释放位点的突触囊泡运输和动力学，从而有助于调节突触传播。我们目前的研究旨在（1）阐明Snapin调节同步突触传播的机制； （2）确定Snapin-Dynein转运复合物是否调节突触前末端的突触囊泡密度和动力学； （3）评估Snapins在精神分裂症中显着的认知障碍中的作用。 特定目的2。调节突触前货物运输及其对突触可塑性的影响。 新的突触的形成和现有突触的重塑在各种形式的突触可塑性中起着重要作用，并需要从Soma中的跨加利基网络（TGN）靶向递送新合成的突触组件到突触终端。因此，这些新合成的成分向新生的突触前胸子的有效轴突转运对于响应神经元活性至关重要。大量证据表明，AZ前体载体是由TGN产生的，并遍历了发育中的轴突到新生的突触。货物囊泡必须具有高度特异性，以保持货物身份和有针对性的贩运。然而，在发育神经元中组装突触前末端和成熟神经元的重塑突触中，对于神经元活性的重塑突触而言，运动适应器复合物的分子身份仍然未知。 我们以前的研究表明，征素蛋白是一种能够连接KIF5B和Syntaxin-1的运动适配器，并使Syntaxin-1转运到神经元过程（Su等，自然细胞生物学，2004年）。使用活海马神经元中的延时成像，我们进一步证明了语法1-辅助蛋白-KIF5B的转运复合物介导AZ成分的轴突转运，这对于突触前组件必不可少。在发育神经元中活性依赖性突触可塑性期间，征功能丧失阻碍了新的突触前胸子的形成（Cai等，J Neuroscience 2007）。我们的研究表明，动力学介导的和基于MT的轴突转运是依赖活性依赖性突触前塑性的细胞机制的另一个关键因素。我们最近的研究进一步证明了女介蛋白介导的轴突转运在培养良好成熟的交感神经元中突触前功能和突触可塑性的调节中的关键作用（Ma等，J Neuroscience 2009）。我们的发现为将来的研究提供了分子基础。有条件的征素基因敲除小鼠是生成的，我们将使用该遗传小鼠线来（1）确定征素蛋白/KIF5介导的顺行轴突转运是否对突触的形成和维持和突触可塑性有任何影响； （2）确定运动适应器络合物是否根据突触活动调节运输速率； （3）确定AZ货物的轴突靶向输送的排序信号。 具体目标3。逆行运输和对神经元自噬 - 溶酶体功能的影响。 维持神经元中的细胞稳态取决于有效的细胞内转运。晚期的内吞运输将目标材料传递到溶酶体中，对于通过自噬 - 溶酶体途径保持有效的降解能力至关重要。自噬 - 溶酶体系统对于细胞内组件和线粒体的质量控制以及细胞稳态的维持至关重要。自噬溶质体系统受损与几种主要神经退行性疾病的发病机理有关。然而，调节神经元中自噬溶质体系统的机制尚不完全了解。动力蛋白介导的逆行转运可以增强溶酶体主要位置的某些晚期内吞运输，并使晚期内体和溶酶体足够接近以融合更高的效率，从而确保适当的自噬溶质体功能。 除了与SVS的关联外，Snapin还存在于神经元和非神经元细胞中的细胞质和膜相关馏分中，并与晚期内吞细胞器共纯化。我们最近的研究发现了Snapin在调节晚期转运和膜运输中的关键作用（Cai等，Neuron，2010年）。 Snapin通过将动力蛋白连接到晚期内体来充当运动适配器。 Snapin（ - / - ）神经元表现出异常的未成熟溶酶体积累，内体晚期沿过程的逆行运输受损，溶酶体蛋白水解降低以及自染色体的清除受损，以及降低的神经元通过耐用性和神经脱发。通过表达Snapin转基因来挽救表型。 因此，我们的研究强调了新的机械洞察力对Snapin-Dynein如何坐在逆行转运和晚期内体 - 溶酶体贩运对自噬 - 溶酶体功能至关重要的情况下。我们的研究目标是通过调节自噬溶质体系统来确定可清除聚集蛋白的细胞途径。 Snapin KO小鼠为我们提供了一种独特的遗传工具，用于表征晚期内吞转运在神经变性中的作用。有条件的Snapin KO小鼠是生成的，并且正在与包括突变体SOD1连接ALS疾病模型的几种疾病小鼠系交叉。这些研究将提供有关操纵晚期内吞途径是否最终导致新的治疗方法的遗传证据。进行这些调查将促进我们对可能影响人类神经系统疾病的基本过程的了解，因此是国家神经疾病和中风研究所使命的本质。 实验室的相关出版物： singning su，Qian Cai，Claudia Gerwin，Carolyn L. Smith，Zu-hang Sheng（2004）征素蛋白：与神经元中索道毒素转运有关的微管相关蛋白，自然细胞生物学6，941-953。 Qian Cai，Pingyue Pan和Zu-Hang Sheng。 （2007）。征素 - 运动蛋白-1家族5B介导的轴突转运有助于活动依赖性突触前组装。神经科学杂志27，7284-7296。 Ping-Yue Pan，Jin-Hua Tian和Zu-Hang Sheng（2009）。 Snapin促进突触囊泡融合的同步。 Neuron 61，412-424。