Astrocyte RNA degradation and cognitive function

项目总结 尽管它被认为与许多精神疾病有关，但无稽之谈介导的mRNA衰退(NMD)代表了一种 在脑功能中调节信使核糖核酸稳定性的机制相对未知。NMD在组织中发挥作用-- 细胞类型和细胞状态特异的方式和调节选择性mRNAs的稳定性以微调转录本 富足。缺乏关于这种NMD靶向RNA的识别的知识，特别是在细胞中 在他们正常的活体环境中。该领域的一个特别大的差距是细胞特定的功能和目标 在活体内的NMD。我们最近的工作已经证实，神经元NMD调节GLUR1信号和IS 树突中适当的突触可塑性、认知和局部蛋白质合成所必需的，为 洞察NMD在大脑中的神经元特异性功能。到目前为止，还没有研究报告特定的 NMD的功能，也不确定脑内神经胶质细胞内的NMD底物。突触的星形胶质细胞控制 活动转化为认知调节使星形胶质细胞成为治疗认知障碍的新靶点 功能障碍。然而，星形胶质细胞调节神经元功能的机制并不是很好。 明白了。目前尚不清楚星形胶质细胞中的信使核糖核酸降解是否参与了对 突触可塑性和行为。这项应用的目标是确定星形细胞的贡献 NMD对突触可塑性和认知功能的影响。几个预测的“典型”和“非典型”NMD 靶点在星形胶质细胞中表达。我们对这些预测的NMD目标的基因本体论分析确定了 钙信号的分子功能丰富。与此一致，我们发现国家导弹防御系统在 星形胶质细胞在体外可引起细胞内钙离子活性升高。星形胶质细胞的动态钙瞬变 建议控制适当的基础突触传递，调节海马LTP。我们还发现了 有条件地消融星形胶质细胞的NMD会损害成年小鼠的记忆。根据已发布的 文献和我们的初步研究，我们假设NMD调节星形胶质细胞的钙活性，并且 星形细胞NMD是成人大脑正常认知功能和行为所必需的。为了测试这一点 假设，我们建议确定1)学习和记忆的不同方面是否需要NMD 2)星形细胞NMD消融对神经元的影响(例如，评估神经元网络连通性和 突触可塑性)和3)NMD缺陷星形胶质细胞的功能缺陷(即通过评估体内的钙活性)。 和体内星形胶质细胞的NMD靶点。我们将使用一系列技术，包括诱导性基因 小鼠模型，行为分析，电生理学，双光子显微镜活体动物钙成像， 立体定位病毒标记、多电极阵列分析、体内RNAseq/生物信息学和体内HITS-CLIP。 这项研究的成功完成将为细胞特异性mRNA的降解提供一个连贯的观点 是哺乳动物大脑中高度调节的突触和认知功能的基础，可能是有价值的 为突触功能障碍和神经认知疾病的星形细胞机制提供新的见解。

PROJECT SUMMARY Despite its putative link to many mental illnesses, Nonsense-Mediated mRNA Decay (NMD) represents a relatively unexplored mechanism for regulating mRNA stability in brain function. NMD functions in a tissue-, cell type- and cell-state specific manner and modulates stability of selective mRNAs to fine-tune transcript abundance. There is dearth of knowledge regarding the identity of such NMD target RNAs, particularly in cells in their normal in vivo context. A particularly large gap in the field is the cell-specific function and targets of NMD in vivo. Our recent work has established that neuronal NMD regulates GLUR1 signaling and is required for proper synaptic plasticity, cognition, and local protein synthesis in dendrites, providing fundamental insight into the neuron-specific function of NMD within the brain. To date, no study has reported a specific function for NMD nor identified NMD substrates within glial cells in the brain. Astroglial control of synaptic activity translates into regulation of cognition making astrocytes a novel therapeutic target to treat cognitive dysfunctions. However, the mechanisms through which astrocytes regulate neuronal function are not well understood. Currently, it is not known whether mRNA degradation in astrocytes contribute to the regulation of synaptic plasticity and behavior. The goal of this application is to determine the contribution of astrocytic NMD to synaptic plasticity and cognitive performance. Several predicted ‘canonical’ and ‘atypical’ NMD targets are expressed in astrocytes. Our gene ontology analysis of these predicted NMD targets identified molecular function enrichment for Ca2+ signaling. Consistent with this, we have found that disruption of NMD in astrocytes resulted in elevated Ca2+ activity in vitro. Dynamic Ca2+ transients in astrocytes have been suggested to control proper basal synaptic transmission and modulate hippocampal LTP. We have also found that conditional ablation of NMD in astrocytes impaired memory in the adult mice. Based on the published literature and our preliminary studies, we hypothesize that NMD regulates Ca2+ activity in astrocytes, and astrocytic NMD is required for proper cognitive function and behavior in the adult brain. To test this hypothesis, we propose to determine 1) whether NMD is required for different aspects of learning and memory 2) the effects of astrocytic NMD ablation on neurons (e.g., assessing neuronal network connectivity and synaptic plasticity) and 3) functional deficits of NMD-deficient astrocytes (i.e., by assessing Ca2+ activity in vivo) and in vivo NMD targets in astrocytes. We will use a combination of techniques including an inducible-genetic mouse model, behavioral assays, electrophysiology, live-animal Ca2+ imaging by two-photon microscopy, stereotaxic viral labeling, Multielectrode Array Assay, in vivo RNAseq/bioinformatics, and in vivo HITS-CLIP. The successful completion of this research will provide a coherent view of how cell-specific mRNA degradation underlies the highly regulated synaptic and cognitive function in the mammalian brain and might be valuable for providing new insights into the astrocytic mechanisms of synaptic dysfunction and neurocognitive diseases.