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

项目概要/摘要 活动依赖的联想赫布可塑性，被认为是一个核心机制的基础学习和 记忆、认知功能和大脑发育。赫布塑性本质上是不稳定的，因为它 积极的反馈性质，并已被平衡的稳态机制，保持稳定的 整体神经元活动。该领域的一个主要差距是对细胞通路的不完全理解 神经元如何维持体内平衡以及这些机制如何与赫布可塑性相互作用， 尤其是在快速的时间尺度上。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）可能参与体内快速稳态机制。NMPs表达于 蝌蚪脑和降解新生蛋白质。NMP活性的抑制导致细胞内的 自发神经元活动和取消学习诱导的行为改善的视觉系统 行为范式活性诱导的蛋白质的从头合成，这些蛋白质对表达 下游塑性机制是赫布塑性的一个标志。我们假设NMP介导的 活性诱导的新生蛋白质的降解作为一种负反馈机制， 神经元活动对可塑性诱导活动的稳态调节。我们将测试这个 非洲爪蟾视觉驱动经验依赖可塑性范式假说 蝌蚪，它允许生物化学，生理学，分子遗传学和行为学的结合， 在一个完整的神经回路与生理相关的感官刺激实验。最关键的是， 实验系统提供了快速的时间分辨率，这是关键的研究快速 NMPs在体内降解新生蛋白质。具体而言，在目标1中，我们将使用体内BONCAT标记 表征不同活性方案下NMP的蛋白水解活性和用途扩展 显微镜下描绘时空的表达谱的核基质蛋白在视顶盖的发展。 在目标2中，我们将联合收割机结合体内Ca++成像和分子遗传学工具来研究NMPs如何调节 自发和视觉诱发的活动，并确定是否NMP介导的调节 是细胞自主的。在Aim 3中，我们将使用延时结构和功能成像， 视觉回避行为，以评估NMPs在经验依赖性可塑性中的功能作用， 在视觉系统的细胞和电路水平。拟议的实验将产生数据， 了解NMP在体内的功能。这些结果将揭示一种新的基于蛋白质稳定的快速 稳态机制，并奠定了基础，为今后的研究，以进一步阐明下游细胞和 活性依赖性蛋白质稳态与新生血管内皮细胞的功能相互作用的分子途径 蛋白质和经验依赖的可塑性机制。

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.