Analysis of the tumor suppressor gene Tsc1 in motor neuron patterning

描述(申请人提供)：运动神经元(MN)能够控制运动、呼吸和自主神经反应，并受到脊髓肌萎缩(SMA)和肌萎缩侧索硬化症(ALS)等疾病的深刻影响。在脊椎动物中，MN在腹侧脊髓和后脑发育，它们的胞体沿着CNS的内侧轴和背腹轴迁移到特定的位置，它们的轴突离开CNS，建立精确的和定型的肌肉靶神经支配。尽管已经发现了许多在MN轴突导航中起重要作用的导向分子，但我们对控制特定选择点MN轴突导向的信号通路的机械性理解仍然不够深入。这项拟议工作的长期目标是确定在体内控制MN轴突导航的新机制。作为一种识别这些基因的无偏见的方法，使用带有GFP标记的MN轴突和TD标记的MN核的报告鼠进行了正向遗传筛选。在ENU诱变筛选中，共鉴定出9个独立突变体，并克隆了相应的基因。有趣的是，其中几个突变体显示出轴突靶向缺陷，即腹侧投射的MN轴突异常地投射到感觉神经节的背侧。名为Greenlight(GRL)的小鼠突变体中的MN寻路缺陷是由结节性硬化症复合体1(TSC1)基因的错义突变引起的。本应用的目的是了解已识别的GRL/TSC1突变如何导致MN通路缺陷。人类TSC1基因突变导致一系列神经表型，包括良性肿瘤、癫痫和自闭症；然而，这些症状的病理生理学机制尚不清楚。中心假说是，在MNS的发生过程中，TSC1与TSC2一起负向调节ePhin/Eph下游的mTOR通路。这一假说是基于在My Screen中发现的GRL/TSC1突变，以及最近观察到的抑制性轴突引导信号对mTOR途径的负面调节而产生的。这项拟议研究的基本原理是，对TSC1功能的详细了解有可能转化为改善结节性硬化症患者的治疗干预措施。在强大的初步数据的指导下，将通过追求两个具体目标来检验这一假说：1)研究已发现的GRL/TSC1突变如何影响TSC1/TSC2复合体的形成，并详细分析GRL/TSC1突变如何影响MN和发育中的PNS中的感觉轴突模式。2)研究EPhin/EphA是否作用于TSC1的上游，以及TSC1下游的TOR复合体1的调节改变是否导致GRL MN通路缺陷。拟议的实验是创新的，因为它们基于GRL/TSC1等位基因的鉴定，这是一种导致MN寻路缺陷的新突变。最终，所获得的知识有可能告知肿瘤抑制基因TSC1如何在神经系统发育中发挥作用，以及该途径的扰动如何导致在TSC患者中典型的神经表现。

DESCRIPTION (provided by applicant): Motor neurons (MNs) enable control over locomotion, respiration and autonomic responses, and are profoundly affected by diseases such as spinal muscle atrophy (SMA) and amyotrophic lateral sclerosis (ALS). In vertebrates, MNs develop in the ventral spinal cord and hindbrain, their cell bodies migrate to specific positions along the medio-lateral and dorso-ventral axes of the CNS and their axons exit the CNS to established precise and stereotypic innervation of muscle targets. Although numerous guidance molecules have been identified that play an essential role in MN axon navigation, our mechanistic understanding of the signaling pathways that control MN axon steering at specific choice points remains fragmentary. The long-term goal of the proposed work is to identify novel mechanisms that control MN axon navigation in vivo. As an unbiased approach to identify such genes, a forward genetic screen was carried out using a reporter mouse with GFP-labeled MN axons and td-tomato-labeled MN nuclei. From the ENU mutagenesis screen, nine independent mutants were identified and their corresponding genes were cloned. Interestingly, several of these mutants display axon targeting defects in which ventral-projecting MN axons aberrantly project dorsally into the sensory ganglia. The MN pathfinding defects in a mouse mutant called Greenlight (GrL) are caused by a missense mutation in the tuberous sclerosis complex 1 (TSC1) gene. The objective in this application is to understand how the identified GrL/TSC1 mutation contributes to MN pathfinding defects. Mutations in human TSC1 cause a spectrum of neurological phenotypes including benign tumors, epilepsy and autism; however the pathophysiology of these symptoms is poorly understood. The central hypothesis is that TSC1, in conjunction with TSC2, negatively regulates the mTOR pathway downstream of ephrin/Eph in developing MNs. This hypothesis has been developed based on the identification of the GrL/TSC1 mutation in my screen, and the recent observation that inhibitory axon guidance cues negatively regulate the mTOR pathway. The rationale for the proposed research is that a detailed understanding of TSC1 function has the potential to translate into improved therapeutic interventions in patients suffering from tuberous sclerosis. Guided by strong preliminary data, the hypothesis will be tested by pursuing two specific aims: 1) to study how the identified mutation in GrL/TSC1 influences TSC1/TSC2 complex formation and to analyze in detail how the GrL/TSC1 mutation affects MN and sensory axon patterning in the developing PNS. 2) to study whether ephrin/EphA operates upstream of TSC1 and whether altered regulation of the TOR complex 1 downstream of TSC1 results in the GrL MN pathfinding defects. The proposed experiments are innovative because they are based on the identification of the GrL/TSC1 allele as a novel mutation that causes MN pathfinding defects. Ultimately, the knowledge gained has the potential to inform how the tumor suppressor gene TSC1 functions in nervous system development and how perturbation of this pathway can cause the neurological manifestations typically observed in TSC patients.