Functional specialization of compartmentalized beta2-adrenergic signaling in neurons

 描述(申请人提供)：β2-肾上腺素受体(β2-AR)是一种G蛋白偶联受体(GPCR2)，在大脑中介导对去甲肾上腺素的生理反应，并关键地调节长时程增强、突触可塑性和记忆获得。这一通路的紊乱与阿尔茨海默病、帕金森氏病、唐氏综合症和创伤后应激障碍等精神疾病有关。因此，阐明控制β2-AR信号并在神经元中介导其功能后果的分子机制将有助于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治疗记忆障碍和精神疾病的能力。以往的观点认为β2-AR信号转导完全是通过在质膜上产生环磷酸腺苷(CAMP)来实现的，但最近有证据表明，激动剂诱导的内吞作用后，对β2-AR的配体依赖性刺激也发生在内体上，这一经典观点受到了挑战。我已经在HEK293细胞中证明了内体β2-AR/cAMP信号选择性地与受体依赖的转录反应相连，而质膜信号则有效地与这种反应解偶联。我的近期目标是研究β2-AR/cAMP信号在初级神经元中的功能后果。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水平的神经元功能。这个K99/R00奖项将使我实现以下职业目标：1)获得神经科学方面的培训，2)获得使用CRISPR干扰的高通量基因分析的额外培训，3)发展我需要的赠款撰写、指导和沟通技能 成为一名成功的独立研究员。在授奖的指导阶段，我将确定初级海马神经元对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，我将系统地定义来自不同细胞间隔的β2-AR信号的磷酸蛋白质组反应，并测试来自质膜的信号(不能有效激活转录)优先刺激不同神经元靶蛋白的磷酸化(Aim 2)的假设。在奖项的独立阶段，我将通过进行基于CRISPR的大规模遗传分析来识别调节β2-AR信号的空间特异性的基因(目标3)。获得该奖项后完成的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有助于在细胞水平上发挥关键的神经元功能。基于这些结果的未来研究将阐明空间偏向信号转导的精确机制，并研究区室特异性β2-AR/cAMP靶点及其最终调节机制如何有助于更高水平的神经元功能。

 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.