Molecular Mechanisms of Synapse Development and Plasticity

1.鉴定突触小泡的一种新的自噬形式。突触小泡是神经元突触前终末内的一种球形小细胞器。它们含有神经递质，并在突触前膜被动作电位去极化时释放神经递质。胞吐后，囊泡被内吞作用回收，并循环形成新的突触囊泡。突触上突触小泡的数量是决定突触强度的重要因素，突触强度在发育过程中受到经验的动态调节。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。关于囊泡胞吐和囊泡内吞的机制和调控已经有了很多的了解。相比之下，内吞作用后囊泡运输的途径不太清楚。多条证据表明，至少有一小部分内细胞性突触小泡通过内体系统。突触内小体的活动是突触小泡内吞和胞吐所必需的。然而，关于突触内小体在突触小泡周期中的作用，人们知之甚少。虽然突触小泡已经被深入研究，但它们的数量是如何维持和调节的，在很大程度上仍然不清楚。 内吞体可以与自噬小体融合，形成两亲体。自噬是在自噬过程中形成的双膜结构，通过这个过程细胞器和聚集的蛋白质被运送到溶酶体进行降解。自噬作为一种促进生存的机制，在由饥饿或生长因子停用等引起的应激期间。各种细胞器可以作为自噬的货物。细胞器的选择性自噬已被发现(如线粒体的有丝分裂吞噬、核糖体的核糖体吞噬、过氧化物体的穿孔吞噬和内质网的网状吞噬)。然而，在神经元中，突触小泡自噬的生理意义仍然难以捉摸。 在这个项目中，我们发现自噬的活性对于海马神经元中突触小泡的动态平衡和活性依赖的循环是必不可少的。突触小泡通过早期和晚期的内噬小体被招募到自噬小体中，突触小泡的自噬受到突触兴奋的调节。因此，我们的研究阐明了突触小泡的一种新型自噬，并揭示了突触小泡循环的新机制。 2.精神分裂症危险基因dybindin在精神分裂症突触病理学中的作用机制。Dybindin是一个含有卷曲结构域的蛋白质，最初被发现是一种抗肌营养不良蛋白结合蛋白，后来被发现是溶酶体相关细胞器复合体1(BLOC-1)生物发生的八个亚基之一。Dybindin基因(Dtnbp1)的单核苷酸多态性与精神分裂症的高风险相关，精神分裂症患者死后大脑中一直显示出低水平的dybindin蛋白和mRNAs。我们早期的工作表明，dybindin通过调节树突的发育来促进神经元连通性的建立，树突包括树突(形成兴奋性突触的微小树突)和丝状足突(在年轻神经元中占主导地位的细长突起)。因此，结合障碍可能通过调节树突棘的发育而增加患精神分裂症的风险。为了确定异常结合蛋白如何在本报告期间调节脊柱发育，我们研究了小鼠大脑P2突触小体部分中异常结合蛋白的相关蛋白质组。我们的数据表明，在小鼠大脑的P2部分，有三种异构体与不同的复合体相关联。为了促进免疫纯化，我们产生了表达标记的dybindin的BAC转基因小鼠，并使用转基因小鼠通过质谱分析鉴定了47个假定的debindin相关蛋白，包括BLOC-1的所有成分。我们用免疫共沉淀法证实了异型结合蛋白与WDR11、FAM91A1、Snapin、muted、pallidin以及两个蛋白酶体亚基PSMD9和PSMA4的相互作用。我们还发现，来自结合障碍缺失突变小鼠(SADY)小鼠大脑的P2部分的蛋白酶体活性显著降低。我们的数据表明，dybindin在功能上与泛素-蛋白酶体系统相关，并为未来研究脑中dybindin功能网络提供了一个分子谱系。

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.