Glioma induced alterations in neuronal transcription and translation

描述(申请人提供)：弥漫性浸润性胶质瘤是最常见的原发脑肿瘤类型之一。胶质瘤细胞广泛侵入大脑，在那里它们与肿瘤微环境中的神经元混合并相互作用。这会导致神经生理学的病理变化，可能会产生毁灭性的临床后果，如无法控制的癫痫发作。以前的研究表明，神经元翻译的失调是癫痫的一个原因。然而，人们对胶质瘤患者神经元功能障碍背后的分子变化知之甚少。作为解决这一重要问题的第一步，我们建议表征在弥漫性浸润性胶质瘤小鼠模型中发生的神经元特异性转录和翻译变化。我们会 通过将表达PDGF的逆转录病毒注射到CaMKII-cre/RiboTag小鼠的皮质下白质中诱导胶质瘤的形成，这种逆转录病毒被设计成在神经元中选择性地表达HA标记的核糖体蛋白(Rpl22)。我们将免疫沉淀小鼠脑瘤匀浆中神经元来源的翻译核糖体，并对信使核糖核酸足迹进行测序，以量化与每个基因相关的核糖体数量。我们还将对总组织匀浆中的mRNA进行RNA-SEQ，并应用计算方法将复合表达数据去卷积到细胞类型特定的图谱中。总之，这些分析将使我们能够计算每个基因的翻译效率(每个转录本的核糖体密度)，这为翻译调控提供了一个敏感的衡量标准。该提案中开发的实验系统将为未来的研究提供一个强大的工具，以探索胶质瘤-神经元相互作用如何导致癫痫发作的机制问题，以及测试旨在抑制胶质瘤相关癫痫的靶向治疗的效果的临床前研究。

DESCRIPTION (provided by applicant): Diffusely infiltrating gliomas are among the most common type of primary brain tumors. The glioma cells widely invade the brain, where they intermingle with, and interact with, neurons in the tumor microenvironment. This leads to pathological alterations in neuronal physiology that can have devastating clinical consequences, such as uncontrollable seizures. Previous studies have implicated dysregulation of neuronal translation as a cause of epilepsy. However, little is known about the molecular alterations underlying the neuronal dysfunctions seen in glioma patients. As a first step towards addressing this important question, we propose to characterize the neuron-specific alterations in transcription and translation that occur in a mouse model of diffusely infiltrating glioma. We will induce gliomas formation by injecting a PDGF expressing retrovirus into the subcortical white matter of CamKII-cre/RiboTag mice, which are engineered to express HA-tagged ribosomal protein (Rpl22) selectively in neurons. We will immunoprecipitate neuron-derived translating ribosomes from mouse brain tumor homogenate and sequence the mRNA footprints to quantify the number of ribosomes associated with each gene. We will also perform RNA-seq on mRNA from total tissue homogenate and apply a computational approach to deconvolve the composite expression data into cell-type specific profiles. Together, these analyses will enable us to calculate translation efficiency for every gene (ribosome density per transcript), which provides a sensitive measure of translational regulation. The experimental system developed in this proposal will provide a powerful tool for future studies to explore mechanistic questions about how glioma-neuron interactions cause seizures, as well as preclinical studies to test the effects target therapies designed to inhibit glioma-associated epilepsy.