Establishing FoxO pathway function in neuronal morphogenesis

描述（由申请人提供）：我们发现转录因子FoxO控制果蝇神经元微管组织和神经元形态发生。FoxO同系物最近也被认为与蠕虫和小鼠的神经元形态发生有关，这是进化守恒的观点。在这些系统中，foxo调节极化、生长和形态——所有这些过程都需要适当的微管组织。我们的研究表明，果蝇FoxO在NMJ限制微管稳定性（或者促进微管不稳定）。我们还发现FoxO蛋白水平在几种细胞骨架应激范式中显著降低，认为FoxO动态调节微管网络。我们认为FoxO水平的降低是抵消微管扰动的细胞策略的一部分。这一假设是基于FoxO突变体的表型分析所揭示的FoxO的微管不稳定功能。我们确定FoxO既是神经元微管网络的调节剂，也是神经元应激调节的转录因子，这表明FoxO有助于神经元对细胞骨架破坏的反应。在本提案中，我们将定义FoxO信号系统，以深入了解调节神经元形态稳定性和可塑性的机制。此外，神经元微管功能障碍与衰弱的神经元病理有关，包括运动神经元和神经退行性疾病，以及年龄和疾病相关的神经病变。因此，分析微管组织的动态调控将揭示对人类健康和衰老的重要机制。我们提出了一套系统的实验来建立foxo依赖通路。我们将确定脂质磷酸酶PTEN在NMJ中促进FoxO活性的作用，以及E3泛素连接酶Nedd4在细胞骨架扰动后FoxO降解中的作用。此外，我们提出的表型分析和表达数据表明，微管相关蛋白CRMP（塌陷反应中介蛋白）受到FoxO的抑制。因此，我们将确定CRMP在调节运动神经元微管稳定性中的作用，并确定其与FoxO的关系。最后，我们将建立FoxO在树突发生和老化神经元中的功能，定义其神经元表型的范围，并阐明FoxO在这些不同神经元环境中的功能。

DESCRIPTION (provided by applicant): We found that the transcription factor FoxO controls neuronal microtubule organization and neuronal morphogenesis in Drosophila. Arguing for evolutionary conservation, FoxO homologs have also recently been implicated in neuronal morphogenesis in worms and mice. In these systems, FoxOs regulate polarization, outgrowth, and morphology-all processes demanding appropriate microtubule organization. Our work demonstrated that Drosophila FoxO limits microtubule stability (or alternatively, promotes microtubule destabilization) at the NMJ. We also found that FoxO protein levels are strikingly reduced in several cytoskeletal stress paradigms, arguing that FoxO dynamically regulates the microtubule network. We propose that FoxO levels are reduced as part of a cellular strategy to counteract microtubule perturbation. This hypothesis is based on FoxO's microtubule- destabilizing function as revealed by phenotypic analysis of foxO mutants. Our identification of FoxO as both a regulator of the neuronal microtubule network, and a neuronal stress-regulated transcription factor, argues that FoxO contributes to the neuronal response to cytoskeletal disruption. In this proposal we will define FoxO signaling systems to gain insight into mechanisms that regulate stability and plasticity of neuronal morphology. Moreover, neuronal microtubule dysfunction is linked to debilitating neuronal pathologies, including motorneuron and neurodegenerative diseases, as well as age and disease-related neuropathies. Thus, analyzing the dynamic regulation of microtubule organization will shed light on mechanisms important to human health and aging. We present a systematic set of experiments to establish FoxO-dependent pathways. We will determine the role of the lipid phosphatase PTEN in promoting FoxO activity at the NMJ, and the role of the E3 ubiquitin ligase Nedd4 in FoxO degradation following cytoskeletal perturbation. Furthermore, we present phenotypic analysis and expression data arguing that the microtubule - associated protein CRMP (Collapsin Response Mediator Protein) is repressed by FoxO. Thus, we will establish the role of CRMP in regulating microtubule stability in motorneurons, and define its relationship with FoxO. Finally, we will establish the FoxO's function in dendritogenesis and in aging neurons, to define the scope of its neuronal phenotypes, and to illuminate FoxO function in these diverse neuronal contexts.