Nascent protein degradation-based fast homeostatic mechanism mediated by neuronal membrane proteasomes

项目摘要/摘要 活动依赖的联想Hebbian可塑性被认为是学习和学习的核心机制 记忆、认知功能和大脑发育。Hebbian塑性本质上是不稳定的，因为它 正反馈性质，必须由维持稳定的动态平衡机制来平衡 整体神经元活动。该领域的一个主要空白涉及对细胞通路的不完全理解。 神经元维持内稳态的机制以及这些机制如何与Hebbian可塑性相互作用， 尤其是在快速时标上。我们的初步数据表明，最近发现的一种神经细胞膜 蛋白酶体(NMP)可能参与体内快速动态平衡机制。NMP表示为 蝌蚪大脑和体内降解新生蛋白质。抑制NMP活性导致血管内皮细胞迅速增加 视觉运动患者的自发神经元活动和取消学习诱导的行为改善 行为范式。活性诱导蛋白质的从头合成，这些蛋白质对表达 下游塑性机制是Hebbian塑性的一个标志。我们假设NMP介导的 活性诱导的新生蛋白降解是FAST的负反馈机制 神经元活动的动态平衡调节，以响应可塑性诱导活动。我们将对此进行测试 非洲爪哇视觉驱动经验依赖性可塑性范式的假说 蝌蚪，它允许生化、生理、分子遗传学和行为的结合 在具有生理相关感官刺激的完整神经回路中进行实验。最关键的是，这 实验系统提供了快速的时间分辨率，这是快速研究的关键 NMPs体内降解新生蛋白质的研究。具体地说，在目标1中，我们将使用体内BONCAT标记 不同活性方案下NMPs的蛋白水解性表征及应用扩展 显微镜下描绘视顶盖发育过程中NMPs的时空表达谱。 在目标2中，我们将结合体内钙成像和分子遗传学工具来研究NMP是如何调节的 顶盖神经元的自发和视觉诱发活动，并确定NMP介导的调节 神经元活动的一部分是细胞自主的。在Aim3中，我们将使用延时结构和功能成像以及 视觉回避行为评估NMP在经验依赖性可塑性中的功能作用 视觉系统中的细胞水平和电路水平。拟议的实验将为深入研究提供数据 了解NMP在体内的功能。这些结果将为一种新的基于蛋白质平衡的FAST提供线索 动态平衡机制，为进一步阐明下游细胞和 新生细胞活性依赖蛋白稳定期之间功能相互作用的分子途径 蛋白质和依赖经验的可塑性机制。

Project Summary/Abstract Activity-dependent associative Hebbian plasticity, is recognized as a core mechanism underlying learning and memory, cognitive function, and brain development. Hebbian plasticity is intrinsically unstable due to its positive feedback nature, and has to be balanced by homeostatic mechanisms, which maintain the stability of overall neuronal activity. A major gap in the field concerns the incomplete understanding of cellular pathways by which neurons maintain homeostasis and how these mechanisms interact with Hebbian plasticity, especially on fast time scales. Our preliminary data suggest that a recently discovered neuronal membrane proteasome (NMP) may be involved in the fast homeostatic mechanism in vivo. NMPs are expressed in the tadpole brain and degrade nascent proteins in vivo. Inhibition of NMP activity led to a rapid increase in spontaneous neuronal activity and abolished learning-induced behavioral improvement in a visuomotor behavior paradigm. Activity-induced de novo synthesis of proteins that are important for the expression of downstream plasticity mechanisms is a hallmark of Hebbian plasticity. We hypothesize that NMP-mediated degradation of activity-induced nascent proteins serves as a negative feedback mechanism for fast homeostatic regulation of neuronal activity in response to plasticity-inducing activities. We will test this hypothesis in a well-established visually driven experience-dependent plasticity paradigm in Xenopus laevis tadpoles, which allows the combination of biochemical, physiological, molecular genetics, and behavioral experiments in an intact neural circuit with physiologically relevant sensory stimulation. Most critically, this experimental system provides the fast temporal resolution that is pivotal for the investigation of the rapid degradation of nascent proteins by NMPs in vivo. Specifically, in Aim 1, we will use in vivo BONCAT labeling to characterize the proteolytic activity of NMPs under different activity regimens and use expansion microscopy to delineate the spatiotemporal expression profile of NMPs in the optic tectum over development. In Aim 2, we will combine in vivo Ca++ imaging with molecular genetic tools to examine how NMPs regulate spontaneous and visually-evoked activity in tectal neurons, and determine whether NMP-mediated regulation of neuronal activity is cell-autonomous. In Aim3, we will use time-lapse structural and functional imaging and the visual avoidance behavior to assess the functional role of NMPs in experience-dependent plasticity at both cellular and circuit levels in the visual system. The proposed experiments will generate data for in-depth understanding of the NMP function in vivo. These results will shed light on a novel proteostasis-based fast homeostatic mechanism and lay the groundwork for future studies to further elucidate downstream cellular and molecular pathways underlying the functional interplay between activity-dependent proteostasis of nascent proteins and experience- dependent plasticity mechanisms.