Cell type-specific profiling of mTOR-dependent translation

mTOR 依赖性翻译的细胞类型特异性分析

• 批准号: 9316901
• 负责人: Helen S. Bateup
• 金额: $ 19.63万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-15 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9316901
• 关键词: Address; Adverse effects; Affect; Affinity Chromatography; Alpha Cell; Autistic Disorder; Basal Ganglia; Biological Assay; Biometry; Brain; Brain Diseases; Cell physiology; Cells; Cellular Morphology; Complex; Corpus striatum structure; Disadvantaged; Disease; Drug Targeting; Epilepsy; Event; FRAP1 gene; Functional disorder; Generations; Genetic; Genetic Translation; Hippocampus (Brain); Huntington Disease; Lead; Mediating; Mediator of activation protein; Mental disorders; Messenger RNA; Midbrain structure; Molecular; Mus; Neurodegenerative Disorders; Neurologic; Neurons; Neurophysiology - biologic function; Outcome; Parkinson Disease; Pathway interactions; Pharmacology; Population; Positioning Attribute; Process; Protein Biosynthesis; Proteins; Pyramidal Cells; RNA analysis; Reverse Transcriptase Polymerase Chain Reaction; Ribosomal Proteins; Ribosomes; Signal Pathway; Signal Transduction; Specificity; Structure; Synaptic plasticity; Testing; Translating; Translations; Viral; Virus; Western Blotting; Work; cell type; design; dopaminergic neuron; experimental study; genetic approach; hippocampal pyramidal neuron; improved; in vivo; innovation; mTOR Signaling Pathway; nervous system disorder; next generation sequencing; novel; novel strategies; synaptic function; transcriptome sequencing; treatment strategy; virus genetics

PROJECT SUMMARY Elucidating the molecular basis of neurological and psychiatric disease is of critical importance for the design of improved therapies. One signaling pathway that has been identified as a hub of dysregulation in several brain disorders is the mTOR pathway. A large body of work has established that altered mTOR signaling affects key aspects of neuronal structure and function including cell morphology, connectivity, synaptic plasticity, and excitability. However, very little is known about the molecules downstream of mTOR responsible for regulating these processes. This information is essential as the effectors of mTOR signaling represent potential drug targets that could provide improved specificity compared with systemic mTOR blockade; a current treatment strategy that is associated with problematic side-effects. A central cellular process regulated by mTOR complex 1 (mTORC1) is protein synthesis. Therefore, identifying the translational targets of mTORC1 is a key first step towards understanding how this pathway controls neuronal function in both normal and disease states. Here we will generate a comprehensive and unbiased assessment of mTORC1's influence on protein synthesis in distinct disease-relevant cell types in the mouse brain. To do this we will genetically and pharmacologically manipulate mTORC1 signaling in vivo and perform translational profiling of defined classes of neurons. We will use a novel approach, called FLEX-TRAP, to express a tagged-ribosomal protein selectively in sub-populations of neurons and use translating ribosome affinity purification (TRAP) to isolate mRNAs that are being actively translated. These mRNAs will be analyzed using next generation sequencing and translational profiles will be compared across conditions and cell types. For this exploratory project we will examine the impact of mTORC1 signaling on the translational profile of four disease-relevant populations of neurons: hippocampal pyramidal cells, direct and indirect pathway striatal projection neurons, and midbrain dopamine neurons. This analysis will provide the first comprehensive and cell type-specific description of mTORC1's impact on mRNA translation in the brain and identify the molecules downstream of mTORC1 responsible for mediating changes in cellular and synaptic function.

项目摘要 阐明神经和精神疾病的分子基础对于治疗精神疾病至关重要。 设计改进的疗法。一个信号通路已被确定为在细胞内调节异常的枢纽， mTOR通路是几种脑部疾病的关键。大量的工作已经证实，改变mTOR 信号传导影响神经元结构和功能的关键方面，包括细胞形态，连接性， 突触可塑性和兴奋性。然而，对mTOR下游的分子知之甚少 负责管理这些过程。该信息是mTOR信号传导的效应物 代表与系统性mTOR相比可以提供改善的特异性的潜在药物靶点 封锁;目前的治疗策略，与有问题的副作用。 由mTOR复合物1（mTORC 1）调节的中心细胞过程是蛋白质合成。因此，我们认为， 确定mTORC 1的翻译靶点是理解这一途径如何 控制正常和疾病状态下的神经元功能。在这里，我们将生成一个全面的， 无偏评估mTORC 1对不同疾病相关细胞类型中蛋白质合成的影响， 老鼠的大脑为了做到这一点，我们将在体内遗传和生物学上操纵mTORC 1信号传导， 执行定义的神经元类别的平移分析。我们将使用一种称为FLEX-TRAP的新方法， 在神经元亚群中选择性表达标记核糖体蛋白，并使用翻译核糖体 亲和纯化（TRAP）以分离被主动翻译的mRNA。这些mRNA将被分析 使用下一代测序和翻译谱将在不同条件和细胞类型之间进行比较。 对于这个探索性的项目，我们将研究mTORC 1信号转导对四个转录因子的翻译谱的影响。 疾病相关的神经元群体：海马锥体细胞，直接和间接通路纹状体 投射神经元和中脑多巴胺神经元。这项分析将提供第一个全面和细胞 mTORC 1对大脑中mRNA翻译的影响的类型特异性描述，并识别分子 在mTORC 1下游，负责介导细胞和突触功能的变化。