Distinct regulation of evoked and spontaneous vesicle fusion site distributions

诱发和自发囊泡融合位点分布的独特调节

• 批准号: 9065944
• 负责人: Haiwen Chen
• 金额: $ 3.4万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9065944
• 关键词: Affect; Area; Autistic Disorder; Behavioral; Biological; Biological Assay; Brain; Cognition; Cognitive; Comprehension; Data; Disease; Event; Excitatory Synapse; Exocytosis; Functional disorder; Gene Mutation; Glutamates; Health; Hippocampus (Brain); Individual; Knowledge; Learning; Link; Location; Maps; Measures; Mediating; Memory; Modeling; Molecular; Mutant Strains Mice; Neurons; Noise; PHluorin; Pathology; Pattern; Physiological; Presynaptic Terminals; Prevention approach; Probability; Protein Isoforms; Proteins; Receptor Activation; Regulation; Resolution; Scaffolding Protein; Schizophrenia; Shapes; Site; Spatial Distribution; Sum; Synapses; Synaptic Receptors; Synaptic Transmission; Synaptic plasticity; Techniques; Testing; Vesicle; Work; bassoon protein; biophysical properties; density; design; experience; genetic regulatory protein; improved; insight; knock-down; molecular marker; nanoscale; neural circuit; neuropsychiatric disorder; neurotransmitter release; novel; novel strategies; overexpression; postsynaptic; presynaptic; receptor; receptor density; research study; scaffold; simulation; stem; synaptic function; temporal measurement; trafficking; transmission process; vesicular release

 DESCRIPTION (provided by applicant): Healthy synapse function relies on precise structural alignment between the presynaptic active zone (AZ) and the postsynaptic density (PSD). A central question is whether localization of vesicle fusion sites influences the efficacy of synapti transmission. The question is important because receptor activation at glutamatergic synapses is limited by both biophysical properties of the receptors themselves and their distance from vesicle release sites. Thus, release events can fail to activate all synaptic receptors. Recently, our lab found that PSD scaffolding proteins cluster receptors into nanometer-scale subregions. This organization is expected to increase the impact of fusion site organization: simulations show that EPSC amplitude at synapses with clustered receptor distributions is greatest when vesicle fusion is aligned with postsynaptic clusters. However, it has not been previously possible to precisely localize vesicle fusion sites, and so there is little information about where within te AZ vesicles fuse. The significance of this issue is emphasized by the growing list of diseases in which cognitive and behavioral deficits appear to stem from disruption of synapse function caused by mutations of genes such as RIM1 and Munc13 that encode synaptic proteins. To map vesicle fusion sites, I developed a novel technique to localize single-vesicle fusion with high spatial resolution, which I call "pHluorin uncovering sites of exocytosis" or pHuse. Using this approach, I mapped the pattern of evoked or spontaneous vesicle fusion at individual presynaptic terminals of cultured hippocampal neurons. Spontaneous release of neurotransmitter was previously thought to be biological noise but has recently been linked to distinct physiological functions. Interestingly, there is controversy over whether spontaneous and evoked release utilize different vesicle pools, involve different trafficking and fusion machinery, or activate different groups of receptors. All these factors suggest that spontaneous and evoked fusion could take place at spatially distinct regions of the AZ, but this has not been tested. My preliminary data using pHuse indicate that evoked and spontaneous fusion in fact occur over different subregions of the terminal. I hypothesize that the spatial distributions of evoked and spontaneous vesicle fusion are differentially regulated by specific active zone proteins and activity. Mouse mutants suggest that two key presynaptic proteins may mediate this effect: loss of the dominant RIM isoform RIM1a specifically disrupts evoked but not spontaneous release57 while loss of Munc13 disrupts both58. Thus, I will test whether these proteins maintain the differential distribution of evoked and spontaneous release sites. Furthermore, molecular remodeling of the AZ to alter vesicle release has been proposed to underlie changes in presynaptic function associated with synaptic plasticity. Thus, I will assess whether the spatial distribution of evoked and spontaneous fusion within the AZ is differentially regulated by key paradigms of synaptic plasticity. In sum, these Aims will help elucidate the organization and regulation of a key aspect of synaptic function, and test an unexpected new mechanism of activity-driven synaptic remodeling.

 描述（由申请人提供）：健康的突触功能依赖于突触前活动区（AZ）和突触后密度（PSD）之间的精确结构对齐。一个中心问题是囊泡融合位点的定位是否影响突触传递的效率。这个问题是重要的，因为受体激活在突触受体本身的生物物理性质和它们的距离囊泡释放位点的限制。因此，释放事件可能无法激活所有突触受体。最近，我们的实验室发现PSD支架蛋白将受体聚集成纳米级的亚区。该组织预计将增加融合位点组织的影响：模拟显示，当囊泡融合与突触后簇对齐时，具有簇状受体分布的突触处的EPSC振幅最大。然而，以前不可能精确定位囊泡融合位点，因此关于AZ囊泡融合的位置的信息很少。越来越多的疾病强调了这一问题的重要性，这些疾病中的认知和行为缺陷似乎源于编码突触蛋白的RIM1和Munc13等基因突变引起的突触功能破坏。为了绘制囊泡融合位点，我开发了一种新的技术，以高空间分辨率定位单囊泡融合，我称之为“pHluorin发现胞吐位点”或pHuse。使用这种方法，我映射的模式，诱发或自发囊泡融合在个别突触前末梢培养海马神经元。神经递质的自发释放以前被认为是生物噪音，但最近被认为与不同的生理功能有关。有趣的是，有争议的自发和诱发释放是否利用不同的囊泡池，涉及不同的贩运和融合机制，或激活不同的受体。所有这些因素表明，自发和诱发融合可能发生在AZ的空间不同的区域，但这还没有得到测试。我使用pHuse的初步数据表明，诱发和自发融合实际上发生在终端的不同子区域。我推测，诱发和自发囊泡融合的空间分布差异调节特定的活性区蛋白和活动。小鼠突变体表明，两种关键的突触前蛋白可能介导这种效应：显性RIM同种型RIM1a的缺失特异性地破坏诱发释放，但不破坏自发释放57，而Munc13的缺失破坏两者58。因此，我将测试这些蛋白质是否维持诱发和自发释放位点的差异分布。此外，AZ改变囊泡释放的分子重塑被认为是与突触可塑性相关的突触前功能变化的基础。因此，我将评估是否诱发和自发融合的AZ内的空间分布差异调节的关键范例的突触可塑性。总之，这些目标将有助于阐明突触功能的一个关键方面的组织和调节，并测试活动驱动的突触重塑的一个意想不到的新机制。