Functional specialization of compartmentalized beta2-adrenergic signaling in neurons

神经元中β2-肾上腺素能信号传导的功能特化

• 批准号: 9769131
• 负责人: Nikoleta Georgieva Tsvetanova
• 金额: $ 24.86万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-10 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9769131
• 关键词: Address; Adrenergic Agents; Adrenergic Receptor; Agonist; Alzheimer' s Disease; Amino Acids; Award; Biochemical; Brain; CRISPR interference; Cell Differentiation process; Cell Nucleus; Cell membrane; Cell model; Cell physiology; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Communication; Critical Pathways; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dependence; Down Syndrome; Endocytosis; Endosomes; Foundations; Future; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits, Gs; Gene Expression; Gene Silencing; Genes; Genetic Transcription; Goals; Grant; Hippocampus (Brain); Ion Channel; Learning; Ligands; Link; Long-Term Potentiation; Mass Spectrum Analysis; Mediating; Membrane; Memory; Memory Disorders; Mental disorders; Mentors; Methods; Modeling; Molecular; Neurons; Neurosciences; Norepinephrine; Output; Parkinson Disease; Pathway interactions; Phase; Phosphorylation; Physiological; Post-Traumatic Stress Disorders; Process; Production; Proteins; Regulation; Research Personnel; Role; Signal Pathway; Signal Transduction; Specificity; Stable Isotope Labeling; Synaptic plasticity; System; Testing; Therapeutic; Training; Undifferentiated; Vesicle; Writing; axon growth; base; career; cell motility; experimental study; gene interaction; genetic analysis; genetic regulatory protein; genome-wide; high throughput analysis; memory acquisition; neurotransmitter release; novel; phosphoproteomics; public health relevance; receptor; response; skills; spatiotemporal; trafficking; transcriptome; transcriptome sequencing

 DESCRIPTION (provided by applicant): The beta2-adrenoceptor (β2-AR) is a G protein-coupled receptor (GPCR) that mediates physiological responses to noradrenaline in the brain and critically modulates long-term potentiation, synaptic plasticity and memory acquisition. Disturbances in this pathway have been implicated in mental illnesses such as Alzheimer's disease, Parkinson's disease, Down's syndrome, and post-traumatic stress disorder. Therefore, elucidating the molecular mechanisms that control β2-AR signaling and mediate its functional consequences in neurons will illuminate how cells execute critical brain functions and will enhance our ability to develop more specific and efficient therapies for memory disorders and mental illnesses. It was previously thought that β2- AR signaling occurred via cyclic AMP (cAMP) production strictly at the plasma membrane, but this canonical view has recently been challenged by evidence that ligand-dependent stimulation of β2-ARs also occurs on endosomes following agonist-induced endocytosis. I have demonstrated in HEK293 cells that endosomal β2- AR/cAMP signaling is selectively linked to the receptor-dependent transcriptional response, whereas the plasma membrane signal is effectively uncoupled from this response. My immediate goal is to investigate the functional consequences of compartmentalized β2-AR/cAMP signaling in primary neurons. My long-term goal is to understand how the spatiotemporal dynamics of β2-AR signaling contribute to higher-level neuronal functions. This K99/R00 award will allow me to achieve the following career goals: 1) gain training in neuroscience, 2) obtain additional training in high-throughput genetic analysis using CRISPR interference, 3) develop grant-writing, mentoring, and communication skills I will need in order to become a successful independent researcher. During the mentored phase of the award, I will determine if the transcriptional response to β2-AR activation in primary hippocampal neurons is governed by spatial encoding of cAMP signals (Aim 1). Next, I will systematically define the phosphoproteomic response to β2-AR signaling from distinct cellular compartments, and test the hypothesis that signaling from the plasma membrane (which does not efficiently activate transcription) preferentially stimulates phosphorylation of distinct neuronal target proteins (Aim 2). During the independent phase of the award, I will identify genes that regulate the spatial specificity of β2-AR signaling by conducting a large-scale CRISPR-based genetic analysis (Aim 3). The biochemical and systems-level analysis of β2-AR signaling accomplished with this award will help illuminate how the spatial regulation and molecular consequences of this pathway contribute to critical neuronal functions at the cellular level. Future studies based on these results will elucidate the precise mechanisms by which spatially biased signal transduction is achieved and investigate how the compartment-specific β2-AR/cAMP targets and the mechanisms that regulate them ultimately contribute to higher-level neuronal functions.

 描述（由申请人提供）：β 2-肾上腺素受体（β2-AR）是一种G蛋白偶联受体（GPCR），介导大脑中对去甲肾上腺素的生理反应，并严重调节长时程增强、突触可塑性和记忆获得。这一途径的紊乱与精神疾病有关，如阿尔茨海默病、帕金森病、唐氏综合症和创伤后应激障碍。因此，阐明控制β2-AR信号传导并介导其在神经元中的功能后果的分子机制将阐明细胞如何执行关键的大脑功能，并将增强我们开发更特异和有效的记忆障碍和精神疾病治疗方法的能力。以前认为β2- AR信号传导通过严格在质膜上产生环AMP（cAMP）发生，但最近这一经典观点受到以下证据的挑战：激动剂诱导的内吞作用后，β2-AR的配体依赖性刺激也发生在内体上。我已经在HEK 293细胞中证明，内体β2- AR/cAMP信号选择性地与受体依赖性转录反应相关，而质膜信号与该反应有效地解耦。我的近期目标是研究初级神经元中β2-AR/cAMP信号区室化的功能后果。我的长期目标是了解β2-AR信号的时空动力学如何有助于更高层次的神经元功能。这个K99/R 00奖项将使我能够实现以下职业目标：1）获得神经科学方面的培训，2）获得使用CRISPR干扰进行高通量遗传分析的额外培训，3）发展我需要的赠款写作，指导和沟通技能，以便 成为一名成功的独立研究员。在奖励的指导阶段，我将确定原代海马神经元中对β2-AR激活的转录反应是否受cAMP信号的空间编码控制（目标1）。接下来，我将系统地定义磷酸化蛋白质组学对来自不同细胞区室的β2-AR信号的反应，并测试来自质膜的信号（不能有效地激活转录）优先刺激不同神经元靶蛋白磷酸化的假设（目的2）。在该奖项的独立阶段，我将通过进行大规模的基于CRISPR的遗传分析来确定调节β2-AR信号传导的空间特异性的基因（目标3）。该奖项完成的β2-AR信号的生化和系统水平分析将有助于阐明该途径的空间调节和分子后果如何在细胞水平上促进关键神经元功能。基于这些结果的未来研究将阐明实现空间偏向信号转导的精确机制，并研究隔室特异性β2-AR/cAMP靶点以及调节它们的机制如何最终促进更高水平的神经元功能。