Molecular Mechanisms of Synapse Development and Plasticity

突触发育和可塑性的分子机制

• 批准号: 9152116
• 负责人: Zheng Li
• 金额: $ 127.98万
• 依托单位: NATIONAL INSTITUTE OF MENTAL HEALTH
• 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/9152116
• 关键词: Action Potentials; Adverse event; Anxiety Disorders; Autistic Disorder; Autophagocytosis; Autophagosome; Autopsy; Behavioral; Binding Proteins; Biogenesis; Brain; Co-Immunoprecipitations; Cognitive; Coiled-Coil Domain; Complex; Data; Dendritic Spines; Development; Dystrophin; Early Endosome; Emotional; Endocytosis; Endoplasmic Reticulum; Endosomes; Ensure; Excitatory Synapse; Exhibits; Exocytosis; Filopodia; Future; Genes; Goals; Growth Factor; Hippocampus (Brain); Homeostasis; Lysosomes; Mass Spectrum Analysis; Membrane; Mental Depression; Mental disorders; Messenger RNA; Mitochondria; Molecular; Mus; Neurons; Neurotransmitters; Organelles; Pathology; Patients; Physiological; Presynaptic Terminals; Prevention strategy; Process; Protein Isoforms; Proteins; Proteome; Recruitment Activity; Recycling; Regulation; Reporting; Ribosomes; Risk; Role; Route; SNAPIN gene; Schizophrenia; Single Nucleotide Polymorphism; Site; Starvation; Stress; Structure; Synapses; Synaptic Vesicles; Synaptic plasticity; Synaptosomes; System; Transgenic Mice; Ubiquitin; Vertebral column; Vesicle; Withdrawal; Work; experience; flexibility; high risk; insight; late endosome; multicatalytic endopeptidase complex; mutant; neural circuit; neurotransmitter release; peroxisome; postsynaptic neurons; presynaptic; presynaptic neurons; protein aggregate; risk variant; stem; synaptic function; trafficking; treatment strategy

1. Identification of a new form of autophagy for synaptic vesicles. Synaptic vesicles are small spherical organelles in the presynaptic terminals of neurons. They contain neurotransmitter and release neurotransmitter when the presynaptic membrane is depolarized by action potentials. After exocytosis, vesicles are retrieved by endocytosis and recycled to form new synaptic vesicles. The number of synaptic vesicles at a synapse is an important factor determining synaptic strength, which is dynamically regulated during development and by experience. A stable, yet flexible, pool of synaptic vesicles is therefore critical to ensure the reliability and adaptability of neural circuits. Much has been known about the machinery and regulation of vesicle exocytosis and endocytosis. The route of vesicle trafficking post endocytosis, by contrast, is less clear. Multiple lines of evidence indicate that at least a fraction of endocytic synaptic vesicles go through the endosomal system. The activity of synaptic endosomes is necessary for endocytosis and exocytosis of synaptic vesicles. However, little is known about the role of synaptic endosomes in the synaptic vesicle cycle. Although synaptic vesicles have been intensively studied, how their number is maintained and regulated remains largely unclear. Endosomes can fuse with autophagosomes to form amphisomes. Autophagosomes are doublemembrane structures formed during autophagy, a process by which organelles and aggregated proteins are delivered to lysosomes for degradation. Autophagy serves as a pro-survival mechanism during stress induced by, for instance, starvation or growth factor withdrawal. Various organelles can be cargos of autophagy. Selective autophagy for organelles (such as mitophagy for mitochondria, ribophagy for ribosomes, pexophagy for peroxisomes, and reticulophagy for endoplasmic reticulum) has been uncovered. In neurons, however, the physiological significance of autophagy for synaptic vesicles remains elusive. In this project, we found that the activity of autophagy is essential for the homeostasis and activity-dependent cycling of synaptic vesicles in hippocampal neurons. Synaptic vesicles are recruited to autophagosomes via early and late endosomes, and autophagy of synaptic vesicles is regulated by synaptic excitation. Our study therefore elucidates a new type of autophagy for synaptic vesicles and reveals a new mechanism underlying the cycling of synaptic vesicles. 2. The mechanism by which the schizophrenia risk gene dysbindin contributes to synaptopathology in schizophrenia. Dysbindin is a coiled-coil domain containing protein, initially discovered as a dystrophin-binding protein and later found to be one of eight subunits of biogenesis of lysosome-related organelles complex 1 (BLOC-1). Single-nucleotide polymorphisms of the dysbindin gene (Dtnbp1) have been associated with higher risk for schizophrenia, and the postmortem brains of schizophrenia patients consistently exhibit low levels of dysbindin proteins and mRNAs. Our earlier work shows that dysbindin contributes to the establishment of neuronal connectivity by regulating the development of dendritic protrusions, including dendritic spines (tiny dendritic protrusions where excitatory synapses are formed) and filopodia (long, thin protrusions that predominant in young neurons). Dysbindin, therefore, may confer the risk for schizophrenia by regulating the development of dendritic spines. To determine how dysbindins regulates spine development in this reporting period, we investigated the associated proteome of dysbindin in the P2 synaptosome fraction of mouse brain. Our data suggest that dysbindin has three isoforms associating with different complexes in the P2 fraction of mouse brain. To facilitate immunopurification, we generated BAC transgenic mice expressing a tagged dysbindin and using the transgenic mice identified 47 putative dysbindin-associated proteins, including all components of BLOC-1, by mass spectrometry. We confirmed the interaction of dysbindin with several identified proteins, including WDR11, FAM91A1, snapin, muted, pallidin, and two proteasome subunits, PSMD9 and PSMA4 by co-immunoprecipitation. We also found that proteasomal activity is significantly reduced in the P2 fraction from the brains of dysbindin-null mutant (sandy) mice. Our data suggest that dysbindin is functionally interrelated to the ubiquitin-proteasome system and offer a molecular repertoire for future study of dysbindin functional networks in brain.

1.一种新的突触囊泡自噬形式的鉴定。突触囊泡是位于神经元突触前末梢的小的球形细胞器。它们含有神经递质，当突触前膜被动作电位去极化时释放神经递质。胞吐作用后，囊泡通过内吞作用回收并再循环形成新的突触囊泡。突触囊泡的数量是决定突触强度的重要因素，其在发育期间和通过经验被动态调节。因此，一个稳定而灵活的突触囊泡池对于确保神经回路的可靠性和适应性至关重要。关于囊泡胞吐和胞吞作用的机制和调节已经知道很多。相比之下，内吞作用后囊泡运输的途径不太清楚。多种证据表明，至少有一小部分内吞突触囊泡通过内体系统。突触内体的活性是突触囊泡内吞和胞吐所必需的。然而，很少有人知道突触内体在突触囊泡周期中的作用。虽然突触囊泡已被深入研究，但它们的数量如何维持和调节仍不清楚。 内体可以与自噬体融合形成两性体。自噬体是在自噬过程中形成的双膜结构，自噬是细胞器和聚集的蛋白质被递送到溶酶体进行降解的过程。自噬在由例如饥饿或生长因子撤回诱导的应激期间充当促存活机制。各种细胞器都可以作为自噬的载体。细胞器的选择性自噬（如线粒体的自噬，核糖体的自噬，过氧化物酶体的自噬和内质网的自噬）已经被发现。然而，在神经元中，突触囊泡自噬的生理意义仍然难以捉摸。 在本项目中，我们发现自噬活性对于海马神经元突触囊泡的稳态和活性依赖性循环是必不可少的。突触囊泡通过早期和晚期内体被募集到自噬体中，突触囊泡的自噬受突触兴奋的调节。因此，我们的研究阐明了一种新型的突触囊泡自噬，并揭示了一个新的机制，突触囊泡的循环。 2.精神分裂症风险基因dysbindin在精神分裂症突触病理学中的作用机制。Dysbindin是一种含有卷曲螺旋结构域的蛋白质，最初被发现为抗肌萎缩蛋白结合蛋白，后来被发现是溶酶体相关细胞器复合物1（BLOC-1）生物发生的八个亚基之一。dysbindin基因（Dtnbp 1）的单核苷酸多态性与精神分裂症的高风险相关，精神分裂症患者的死后大脑始终表现出低水平的dysbindin蛋白和mRNA。我们早期的工作表明，dysbindin通过调节树突突起的发育来促进神经元连接的建立，包括树突棘（形成兴奋性突触的微小树突突起）和丝状伪足（在年轻神经元中占主导地位的细长突起）。因此，Dysbindin可能通过调节树突棘的发育而赋予精神分裂症的风险。为了确定dysbindins如何调节脊柱发育在本报告期间，我们调查了相关的蛋白质组dysbindins在小鼠大脑的P2突触体部分。我们的数据表明，dysbindin有三个亚型与不同的复合物在小鼠脑的P2部分。为了促进免疫纯化，我们产生了表达标记dysbindin的BAC转基因小鼠，并使用转基因小鼠通过质谱鉴定了47种推定的dysbindin相关蛋白，包括BLOC-1的所有组分。我们通过免疫共沉淀证实了dysbindin与几种鉴定的蛋白质的相互作用，包括WDR 11，FAM 91 A1，snapin，muted，pallidin和两种蛋白酶体亚基PSMD 9和PSMA 4。我们还发现，蛋白酶体活性显着降低P2馏分从dysbindin无效突变（桑迪）小鼠的大脑。我们的数据表明，dysbindin是功能相关的泛素-蛋白酶体系统，并提供了一个分子库，为未来的研究dysbindin功能网络在大脑中。