Regulation and Function of Spontaneous Mini Release at Synapses

描述（由申请人提供）：长期目标是了解突触信号传导如何调节神经元连接并导致神经系统疾病，我们建议使用果蝇作为模型系统，用于确定自发神经递质释放调节的分子机制以及它如何调节突触连接。突触处神经元通信的表征主要集中在动作电位触发的突触囊泡融合，其中自发微型电位（minis）主要被认为代表背景噪声。然而，我们最近发现，突触的自发释放是由突触特异性SNARE复合物结合蛋白complexin独立于诱发释放来调节的，并且可能调节突触生长。果蝇复合蛋白的突变导致突触自发融合的显著增加，这与细胞外钙离子或动作电位无关，并导致突触结的过度生长。复合蛋白也是PKA依赖性磷酸化的靶点，表明微频率可以在突触处以PKA依赖性方式独立调节。通过复合素融合钳调节自发融合为独立于诱发突触电位的神经元突触信息传递提供了新途径。此外，人类中复合蛋白水平的改变与从精神分裂症到帕金森病和亨廷顿病的许多神经疾病有关，表明复合蛋白功能障碍和自发释放的异常速率可能导致几种人类神经病理学。在这里，我们建议利用果蝇中可用的遗传工具，以确定复合蛋白如何调节自发释放，如果它的功能可以调节神经元活动和PKA通路。这些实验的完成将使我们能够提出未来的研究，以定义minis的生物学作用及其在大脑中的调节模式，可能为我们理解由minis介导的突触和神经元通信的生物学提供根本性的进步-这是R21水平支持的理想高风险/高回报实验类型。 公共卫生相关性：突触通信的表征主要集中在动作电位触发的突触囊泡融合，自发微小电位（minis）主要被认为是代表背景噪声。我们最近的工作表明，minis可以在突触中发挥信号作用，并受到神经元特异性蛋白复合素的调节。我们将探讨复合素陷阱机制如何调节突触的自发释放，并有助于突触的生长和可塑性。确定minis在突触生物学中的作用将是我们理解大脑功能的重要进展，并为进一步理解复杂蛋白失调如何导致神经系统疾病奠定基础。

DESCRIPTION (provided by applicant): With the long-term goal of understanding how synaptic signaling regulates neuronal connectivity and contributes to neurological disease, we propose to use Drosophila as a model system for determining the molecular mechanisms by which spontaneous neurotransmitter release is regulated and how it may regulate synaptic connectivity. Characterization of neuronal communication at synapses has largely focused on action potential-triggered synaptic vesicle fusion, with spontaneous miniature potentials (minis) largely thought to represent background noise. However, we have recently discovered that spontaneous release at synapses is regulated independently of evoked release by complexin, a synapse-specific SNARE complex binding protein, and may modulate synaptic growth. Mutations in Drosophila complexin lead to a dramatic increase in spontaneous fusion at synapses that is independent of extracellular calcium or action potentials, and cause a profound overgrowth of synaptic boutons. Complexin is also a target for PKA-dependent phosphorylation, suggesting that mini frequency can be independently regulated at synapses in a PKA-dependent manner. Regulation of spontaneous fusion by a complexin fusion clamp provides a new avenue for information transfer at neuronal synapses independent of evoked synaptic potentials. In addition, alterations in complexin levels in humans have been implicated in a host of neurological diseases ranging from schizophrenia to Parkinson's and Huntington's Disease, suggesting complexin dysfunction and abnormal rates of spontaneous release may contribute to several human neuropathologies. Here, we propose to take advantage of the genetic tools available in Drosophila to determine how complexin modulates spontaneous release and if its function can be regulated by neuronal activity and the PKA pathway. The completion of these experiments will allow us to propose future studies to define the biological role of minis and their mode of regulation in the brain, potentially providing a fundamental advance in our understanding of the biology of the synapse and neuronal communication mediated by minis - the type of high risk/high reward experiments ideal for R21-level support. PUBLIC HEALTH RELEVANCE: Characterization of synaptic communication has largely focused on action potential-triggered synaptic vesicle fusion, with spontaneous miniature potentials (minis) largely thought to represent background noise. Our recent work suggests that minis can function in a signaling role at the synapse, and are regulated by the neuron specific protein complexin. We will explore how the complexin-SNARE machinery regulates spontaneous release at synapses and contributes to synaptic growth and plasticity. Defining the role of minis in the biology of the synapse would be an important advance in our understanding of brain function and set the stage for further understanding of how complexin dysregulation contributes to neurological disease.