The role of linear ubiquitination in memory formation

项目摘要/摘要 这项提议的主要目标是了解记忆是如何在大脑中形成和存储的。 巴甫洛夫恐惧条件反射已被证明是阐明分子机制的有用范例。 基本的记忆形成和存储在细胞中。事实上，现在有很好的证据表明 使用这种范式形成的记忆需要对神经元中的蛋白质进行动态的翻译后修饰。 最近，有证据表明，蛋白质泛素化与记忆密切相关。 在大脑中形成，主要是通过靶向蛋白质，使其被蛋白酶体降解。然而，很多 目前尚不清楚其他独立于蛋白酶体的泛素标记是如何参与的 内存存储过程。在我们的初步研究中，我们发现在杏仁核中，恐惧学习增加了 线性泛素化蛋白的水平，这是一种不以降解为目标的非典型泛素修饰 通过蛋白酶体复合体。重要的是，这些增加选择性地发生在细胞核中，而不是 细胞质或突触，并以转录因子p65为靶点，这表明线性泛素化可能 参与记忆形成过程中的转录控制。这项提案中的工作旨在回答 关于线性泛素化是否参与对恐惧记忆至关重要的基因转录的重要问题 在杏仁核中形成。使用蛋白质纯化方法和质谱学的组合，目标1 将在学习后确定线性泛素化针对哪些蛋白质。此外，这一目标将 将这些鉴定出的蛋白质与siRNA介导的下一代rna-seq数据进行关联。 扁桃体内线性泛素化的减少，这将允许对 这个泛素标记是如何在记忆形成过程中参与转录过程的。目标2 直接测试线性泛素化在记忆形成中的功能作用，通过减少或增强这一点 分别通过体内siRNA或CRISPR-dCas9操作和测试在杏仁核中的泛素标记 情境恐惧条件反射任务的记忆保持。总的来说，这项研究将提供关键的 关于线性泛素化如何参与记忆形成过程中的转录调控的信息 以及它是否对依赖学习的突触可塑性至关重要。这可能会提供潜在有用的 关于记忆是如何储存在大脑中的信息，这可能对治疗有重要意义 与各种精神疾病相关的记忆障碍。

Project Summary/Abstract The broad goal of this proposal is to understand how memories are formed and stored in the brain. Pavlovian fear conditioning has proven to be a useful paradigm in elucidating the molecular mechanisms underlying memory formation and storage in cells. Indeed, good evidence now exists suggesting that memories formed using this paradigm require dynamic post-translational modification of proteins in neurons. Recently, evidence has emerged demonstrating that protein ubiquitination is critically involved in memory formation in the brain, primarily through targeting proteins for degradation by the proteasome. However, much remains unknown about how other ubiquitin marks which are independent of the proteasome are involved in the memory storage process. In our preliminary studies, we found that in the amygdala fear learning increased the levels of linear ubiquitinated proteins, an atypical ubiquitin modification that is not targeted for degradation by the proteasome complex. Importantly, these increases occurred selectively in the nucleus, but not the cytoplasm or at synapses, and targeted the transcription factor p65, suggesting that linear ubiquitination may be involved in transcriptional control during memory formation. The work in this proposal is designed to answer important questions about whether linear ubiquitination is involved in gene transcription critical for fear memory formation in the amygdala. Using a combination of protein purification methods and mass spectrometry, Aim 1 will identify what proteins are targeted by linear ubiquitination following learning. Additionally, this aim will correlate these identified proteins with next generation RNA-seq data obtained following siRNA-mediated reductions in linear ubiquitination within the amygdala, which will allow an unbiased, whole genome analysis of how this ubiquitin mark is involved in transcriptional processes during the process of memory formation. Aim 2 directly tests the functional role of linear ubiquitination in memory formation by reducing or enhancing this ubiquitin mark in the amygdala via in vivo siRNA or CRISPR-dCas9 manipulations, respectively, and testing memory retention for a contextual fear conditioning task. Collectively, this research will provide critical information regarding how linear ubiquitination is involved in transcriptional regulation during memory formation and whether it is critical for learning-dependent synaptic plasticity. This could provide potentially useful information on how memories are stored in the brain which could have important implications for the treatment of memory impairments associated with a variety of psychiatric disorders.