Proteomic Analysis of Synaptic Vesicle Pools

描述(申请人提供)：突触传递领域的经典研究假设神经递质的释放来自生物化学上相同的突触小泡群体，但许多实验系统的大量工作表明，突触小泡属于不同的池，它们对刺激的反应不同。这些观察结果引发了两个相互矛盾的假设，一个是这两个池在生化上是相同的，行为上的差异严格地是随机的，或者是外在的，反映了与细胞骨架或既往病史的不同联系，而不是组成上的任何内在差异。或者，分子组成的差异是不同突触囊泡池行为的基础，最近的研究表明，这些池在循环后可能保持其身份。尽管有争议，但我们最近发现不同的突触囊泡蛋白对刺激的反应不同，这提供了一些最初的证据，表明突触囊泡池的组成不同。然而，这些实验涉及单个报告结构的光学成像，理解膜蛋白组成如何决定突触小泡的属性需要更系统的方法。因此，我们将严格根据磁性纳米颗粒对活性的反应来标记特定的突触囊泡池，并通过定量蛋白质组学分析来确定它们的组成：目的1：优化从高纯度突触神经体中回收突触囊泡。标准程序无法恢复与质膜相关的突触小泡，因此我们将使用物理和化学相结合的方法优化从突触神经小体中恢复突触小泡。目的2：优化刺激过程中磁性纳米颗粒标记突触小泡的分离。我们将合成小的磁性纳米颗粒，并根据不同的刺激模式优化不同突触囊泡池的标记。目的3：利用相对和绝对定量等压标签(ITRAQ)或哺乳动物稳定同位素标记(SILAM)，通过定量蛋白质组学方法确定重复和静止突触囊泡池的组成。确定不同突触囊泡池的分子组成将为进一步探索已识别的成分在递质释放、突触发育和可塑性中的作用奠定基础。

DESCRIPTION (provided by applicant): Classical studies in the field of synaptic transmission have assumed that neurotransmitter release occurs from a biochemically homogeneous population of synaptic vesicles, but considerable work from many experimental systems has shown that synaptic vesicles belong to pools that differ in their response to stimulation. These observations have given rise to two competing hypotheses, one that the pools are biochemically the same, with differences in behavior strictly stochastic, or extrinsic, reflecting differential association with the cytoskeleton or prior history rather than any intrinsic differences in composition. Alternatively, differences in molecular composition underlie the behavior of different synaptic vesicle pools, and recent work has suggested that the pools may retain their identity after recycling. Although controversial, we have recently shown that different synaptic vesicle proteins respond differently to stimulation, providing some of the first evidence that synaptic vesicle pools differ in composition. However, these experiments involved optical imaging of individual reporter constructs, and understanding how membrane protein composition determines the properties of synaptic vesicles requires a more systematic approach. We will thus label specific synaptic vesicle pools with magnetic nanoparticles strictly on the basis of their response to activity, and determine their composition by quantitative proteomic analysis: Aim 1: Optimize synaptic vesicle recovery from highly purified synaptoneurosomes. Standard procedures fail to recover synaptic vesicles associated with the plasma membrane, so we will optimize synaptic vesicle recovery from synaptoneurosomes using a combination of physical and chemical approaches. Aim 2: Optimize isolation of synaptic vesicles labeled with magnetic nanoparticles during stimulation. We will synthesize small magnetic nanoparticles, and optimize the labeling of different synaptic vesicle pools by different patterns of stimulation. Aim 3: Determine the composition of recycling and resting synaptic vesicle pools by quantitative proteomics using isobaric tag for relative and absolute quantitation (iTRAQ) or stable isotope labeling in mammals (SILAM). Identifying the molecular composition of different synaptic vesicle pools will provide a foundation for future work to explore the functin of the identified components in transmitter release, synapse development and plasticity.