Molecular control of synaptobrevin retrieval and its biological function by synaptophysin

脑细胞(神经元)通过释放化学神经递质进行交流。神经递质储存在神经元内称为突触小泡(SVS)的小球状隔间中。当神经元交流时，SVS与神经元的外表面融合，导致神经递质释放。在神经递质释放后，这些SVS通过一种称为内吞作用的过程进行改造。内吞过程中SVS的正确形成对于维持神经递质的释放是至关重要的，因为组成错误的SVS将不利于后续的融合。SV生成的关键部分是将正确数量的正确蛋白质包装到SVS中。这通常是由称为接头蛋白的特定分子完成的，但在某些情况下需要额外的分子。我们最近确定了突触素在协调SybII包装进入SVS的内吞过程中的重要作用。虽然已知这些蛋白质相互粘连，但没有人知道这些蛋白质的哪些区域对此很重要。还建议需要其他分子来控制sybII和突触素包装到SVS中。它们是AP180和AP-2。我们提出了一个工作模型，即所有四种蛋白质以协调的方式粘连在一起，以控制sybII和突触素的包装进入SVS。该模型将在此应用程序中进行测试。最后，在SVS上发现了各种不同类型的Syb，它们都在神经递质释放的控制中发挥着特定的作用。因此，突触素也可能控制它们的包装进入SVS，从而控制它们的生物学作用。我们认为，突触素是在内吞作用中将不同的Syb分子包装成SVS的中心组织者。我们将通过许多方法来测试这一点。我们将研究当编码突触素的基因从玻璃片上生长的神经元中移除时，sb运动有何不同。我们还将研究改变形式的Syb和突触素的同时运动，它们彼此不粘着，看看它们的相互作用如何控制它们的功能。我们还将监测在没有AP180或AP-2的情况下Syb和突触素的运动。最后，我们将通过间接检测邻近神经元的电变化来确定干扰突触素在SYB恢复过程中的正常功能如何改变神经递质的释放。本申请中概述的实验组合将系统地剖析突触素在控制不同SyB包装到SVS中的作用以及它在神经递质释放中的下游功能。这一点非常重要，因为将syb包装到SVS中的效率下降被认为是一系列神经退行性和神经发育障碍的基础，如阿尔茨海默病、帕金森病和X连锁智力残疾。它还将提供这一领域以外的重要线索，因为在这一应用中创建的突变体可用于检测突触素的其他可能的神经元功能，如大脑中神经元之间连接的形成。

Brain cells (neurones) communicate by releasing chemical neurotransmitters. Neurotransmitters are stored in small spherical compartments within neurones called synaptic vesicles (SVs). When neurones communicate, SVs fuse with the outer surface of the neurone causing neurotransmitter release. After neurotransmitter release these SVs are reformed by a process called endocytosis. The correct formation of SVs during endocytosis is essential for the maintenance of neurotransmitter release, since SVs with the wrong composition will be faulty for subsequent fusion. A critical part of SV generation is the packaging the correct proteins in the correct amounts into SVs. This is usually done by specific molecules called adaptor proteins, however in some cases additional molecules are required. We have recently identified an essential role for the protein synaptophysin in coordinating the packaging of sybII into SVs during endocytosis. While these proteins are known to stick to each other, no-one knows which regions of the proteins are important for this. Other molecules are also suggested to be required to control sybII and synaptophysin packaging into SVs. These are AP180 and AP-2. We have proposed a working model whereby all four proteins stick together in a coordinated manner to control packaging of sybII and synaptophysin into SVs. This model will be tested in this application. Finally various different types of syb are found on SVs, all of which have specific jobs in the control of neurotransmitter release. Therefore synaptophysin may also control their packaging into SVs and subsequently their biological role. We propose that synaptophysin is the central organiser in the packaging of different syb molecules into SVs during endocytosis. We will test this by a number of approaches. We will examine how syb movements differ when the gene encoding synaptophysin is removed from neurones grown on glass coverslips. We will also examine the simultaneous movement of altered forms of sybs and synaptophysin which do not stick to each other to see how their interaction controls their function. We will also monitor syb and synaptophysin movement in the absence of AP180 or AP-2. Finally we will determine how interfering with the normal function of synaptophysin in syb retrieval alters neurotransmitter release by detecting it indirectly via electrical changes in neighbouring neurones. The combination of experiments outlined in this application will systematically dissect the role of synaptophysin in both the control of the packaging of different sybs into SVs but also its downstream functions in neurotransmitter release. This is very important, since a decrease in the efficiency of syb packaging into SVs is proposed to underlie a series of neurodegenerative and neurodevelopmental disorders such as Alzheimer's Disease, Parkinson's Disease and X-linked intellectual disability. It will also provide important leads outside this field, since the mutants created in this application can be used to examine other possible neuronal functions of synaptophysin such as the formation of connections between neurones in brain.