Analysis of the tumor suppressor gene Tsc1 in motor neuron patterning

抑癌基因Tsc1在运动神经元模式中的分析

• 批准号: 8568581
• 负责人: ONANONG CHIVATAKARN
• 金额: $ 8.63万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-16 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8568581
• 关键词: Affect; Alleles; Amyotrophic Lateral Sclerosis; Autistic Disorder; Axon; Benign; Biochemistry; Biological Assay; Brain Diseases; Cell Nucleus; Cells; Coiled-Coil Domain; Complex; Cranial Nerves; Cues; Data; Defect; Development; Disease; Documentation; Electroporation; Embryo; Ephrins; Epilepsy; Ethylnitrosourea; Exhibits; Exons; Functional disorder; Genes; Genetic Screening; Goals; Growth; Growth Cones; Human; In Vitro; K-Series Research Career Programs; Knowledge; Label; Laboratories; Lateral; Locomotion; Maps; Mediating; Mentors; Missense Mutation; Mission; Molecular; Motor; Motor Neurons; Mus; Muscle; Muscular Atrophy; Mutagenesis; Mutant Strains Mice; Mutation; Natural regeneration; Nervous System Trauma; Nervous system structure; Neurologic; Neurologic Manifestations; Neurons; Outcome; Pathway interactions; Patients; Pattern; Peripheral Nervous System; Phase; Phenotype; Play; Positioning Attribute; Process; Proteins; Public Health; Regulation; Reporter; Research; Respiration; Retinal Ganglion Cells; Role; Sensory; Sensory Ganglia; Signal Pathway; Signal Transduction; Specificity; Spinal; Spinal Cord; Spinal nerve structure; Symptoms; System; TSC2 gene; Testing; Therapeutic Intervention; Tomatoes; Translating; Tuberous Sclerosis; Tuberous sclerosis protein complex; Tumor Suppression; Tumor Suppressor Genes; Vertebrates; Visual system structure; Work; autism spectrum disorder; axon guidance; axonal pathfinding; base; cell type; disability; gain of function; genetic manipulation; hindbrain; human FRAP1 protein; human disease; improved; in vivo; innovation; insight; loss of function; mTOR protein; motor neuron development; mutant; nerve supply; nervous system development; neuronal cell body; neuronal growth; neuronal guidance; novel; overexpression; protein complex; public health relevance; relating to nervous system; repaired; research study; response; transcriptome sequencing; tumor; tumor growth

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.

描述（由申请人提供）：运动神经元（MN）能够控制运动、呼吸和自主反应，并且受到诸如脊髓性肌萎缩（SMA）和肌萎缩性侧索硬化（ALS）等疾病的深刻影响。在脊椎动物中，MN在腹侧脊髓和后脑中发育，它们的细胞体沿着CNS的中-外侧和背-腹轴迁移到特定位置，并且它们的轴突离开CNS以建立精确和刻板的肌肉靶神经支配。虽然已经确定了许多指导分子在MN轴突导航中发挥重要作用，但我们对在特定选择点控制MN轴突转向的信号通路的机械理解仍然是零碎的。这项工作的长期目标是确定体内控制MN轴突导航的新机制。作为一个公正的方法来确定这样的基因，进行了正向遗传筛选，使用报告小鼠与GFP标记的MN轴突和td-番茄标记的MN核。从ENU诱变筛选中，鉴定了9个独立的突变体，并克隆了它们相应的基因。有趣的是，这些突变体中的几个显示轴突靶向缺陷，其中腹侧投射的MN轴突异常地向感觉神经节背侧投射。在一种叫做绿光（GrL）的小鼠突变体中，MN寻路缺陷是由结节性硬化症复合体1（TSC 1）基因中的错义突变引起的。本申请的目的是了解所鉴定的GrL/TSC 1突变如何导致MN寻路缺陷。人类TSC 1的突变会导致一系列神经系统表型，包括良性肿瘤、癫痫和自闭症;然而，这些症状的病理生理学尚不清楚。中心假设是TSC 1与TSC 2一起负调节发育中MN中肝配蛋白/Eph下游的mTOR通路。这一假设是基于我筛选出的GrL/TSC 1突变，以及最近观察到的抑制性轴突引导线索负调节mTOR通路而提出的。拟议研究的基本原理是，对TSC 1功能的详细了解有可能转化为结节性硬化症患者的治疗干预措施。在强有力的初步数据的指导下，该假设将通过追求两个具体目标进行测试：1）研究GrL/TSC 1中鉴定的突变如何影响TSC 1/TSC 2复合物的形成，并详细分析GrL/TSC 1突变如何影响MN和发展中的PNS中的感觉轴突模式。2)研究肝配蛋白/EphA是否在TSC 1上游起作用，以及TSC 1下游TOR复合物1的调节改变是否导致GrL MN寻路缺陷。所提出的实验是创新的，因为它们是基于GrL/TSC 1等位基因作为一种新的突变，导致MN寻路缺陷的鉴定。最终，所获得的知识有可能告知肿瘤抑制基因TSC 1如何在神经系统发育中发挥作用，以及该途径的干扰如何导致通常在TSC患者中观察到的神经学表现。