FUNCTIONAL ANALYSES OF NEUROGLIAN/L1 IN SYNAPTOGENESIS

NEUROGLIAN/L1 在突触发生中的功能分析

• 批准号: 7362957
• 负责人: TANJA ANGELA GODENSCHWEGE
• 金额: $ 29.2万
• 依托单位: FLORIDA ATLANTIC UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-20 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7362957
• 关键词: Address; Affect; Affinity; Alleles; Ankyrins; Axon; Binding; Biological Models; Biological Process; Brain; Cell Adhesion Molecules; Cells; Chromosome Pairing; Clathrin; Clinical Pathology; Clinical Treatment; Collection; Cytoskeleton; Data; Defect; Development; Developmental Process; Disease; Disruption; Dominant-Negative Mutation; Drosophila genus; Endocytosis; Equilibrium; Extracellular Domain; Fiber; Future; Gene Expression; Grant; Head; Health Benefit; Homologous Gene; Human; Hybrids; Hydrocephalus; Invertebrates; Knowledge; Left; Liquid substance; MAP Kinase Signaling Pathways; Masks; Mental Retardation; Missense Mutation; Molecular Genetics; Muscle Contraction; Mutation; Neural Cell Adhesion Molecule L1; Neurites; Neurons; Pathology; Patients; Personal Satisfaction; Phenotype; Phosphorylation; Physiological; Play; Point Mutation; Process; Protein Isoforms; Protein Overexpression; Proteins; RNA; RNA Interference; Reporting; Research; Residual state; Resources; Role; Signal Pathway; Site; Specimen; Staging; Structure; Synapses; Syndrome; System; Temperature; Testing; Thumb structure; Transgenic Organisms; Variant; Vertebrates; adduct; axonal pathfinding; base; cell motility; design; extracellular; ezrin; fly; follow-up; gain of function; gallium alloy GF; in vivo; knock-down; loss of function; mutant; nervous system disorder; neurofascin; neuroglian; novel; paralogous gene; protein function; research study; synaptogenesis; tool; trafficking; treatment planning

DESCRIPTION (provided by applicant): Drosophila Neuroglian (Nrg), a homolog of vertebrate L1, is a prime example of a multifunctional cell adhesion molecule with a multiplicity of binding partners. Several types of single point mutations at different sites in human L1 have been shown to cause a variety of neurological disorders (CRASH syndrome) including mental retardation, hydrocephalus and spasticity. Nrg/L1 has been shown to be involved in axon pathfinding, neurite extension and cell migration. The role of Nrg/L1 in these developmental processes has been well-characterized in vertebrates and invertebrates but much less is known about potential functions during synapse formation. We have recently shown that Nrg does indeed have an essential function in synaptogenesis. We found that a single missense mutation in the extracellular domain of the nrg849 allele disrupts the assembly and functionality of a central synapse in a well- characterized neuronal circuit, the Giant Fiber System (GFS). Our data suggests that phosphorylation of the intracellular ankyrin binding motif of Nrg/L1 is crucial for giant synapse formation. Interestingly, human L1 is able to completely rescue the phenotype in nrg849 mutants while tested paralogs Neurofascin and NrCAM can not, despite having the same overall domain structure inclusive of the highly conserved ankyrin binding motif. This shows that the GFS is a valid model system for studying L1-specific function. Our preliminary studies indicate that some of the pathological missense mutations identified in L1 affect synapse formation rather than earlier developmental processes. Though a defect in neurite outgrowth, guidance or synapse formation may all result functionally in the same discernable phenotype, a disrupted connection between neurons, the biological process affected is completely different. Our data also suggests that some mutations do not result in a loss of a function phenotype but can also have gain of function and dominant negative consequences as well. Information about the particular biological process being disrupted as well as the protein function being compromised is crucial to find appropriate treatment plans for clinical pathologies associated with different types of mutations in the future. Hence, this grant is designed to further explore Nrg and L1's role in synapse formation as well as to study the effects of identified human mutations in L1 in vivo at a single cell level. We will combine the enormous resource of identified human L1 mutations and the power of genetic and molecular tools in Drosophila to determine which extracellular L1/Nrg interactions and intracellular signaling pathways play a role in synapse formation. We will determine the function of various L1/Nrg constructs in wild type, nrg849 and a temporal loss of function background electrophysiologically and anatomically. The knowledge gained from studies listed in this proposal will enable us to have a better understanding of the mechanisms involved in synaptogenesis as well as the cellular basis of the pathologies underlying L1-related neurological disorders. More than 170 different mutations in the cell adhesion molecule L1 have been identified to result in a variety of human neurological disorders which are associated with mental retardation, hydrocephalus (enlarged head due to collection of fluid on the brain) and spasticity (involuntary contraction of muscles). We intend to study the effects of these various pathological mutations in vivo at a single cell level in a unique model system, which will allow us to identify the particular biological process being affected, as well as the protein function being disrupted by the mutation. This information is essential to find appropriate treatment for the clinical manifestations of these mutations and hence will benefit the health of patients with L1-related disorders in the future.

描述(申请人提供)：果蝇神经胶质(NRG)，脊椎动物L1的同源物，是具有多种结合伙伴的多功能细胞黏附分子的主要例子。人类L1不同部位的几种类型的单点突变已被证明会导致各种神经疾病(撞车综合征)，包括智力低下、脑积水和痉挛。NRG/L1参与轴突的寻路、突起延伸和细胞迁移。NRG/L1在这些发育过程中的作用在脊椎动物和无脊椎动物中已经得到了很好的描述，但对突触形成过程中的潜在功能知之甚少。我们最近的研究表明，NRG在突触发生中确实具有重要的功能。我们发现，nrg849等位基因胞外域的单个错义突变破坏了巨型纤维系统(GFS)中中央突触的组装和功能。我们的数据表明，NRG/L1细胞内Anyrin结合基序的磷酸化对巨型突触的形成至关重要。有趣的是，人的L1能够完全挽救nrg849突变体的表型，而测试的近亲动物NeuroFascin和NrCAM则不能，尽管它们具有相同的结构域结构，包括高度保守的锚蛋白结合基序。这表明GFS是研究L1特有功能的有效模式系统。我们的初步研究表明，L1中发现的一些病理性错义突变影响突触的形成，而不是早期的发育过程。尽管轴突生长、引导或突触形成的缺陷在功能上都可能导致相同的可辨别表型，即神经元之间的连接中断，但受影响的生物过程完全不同。我们的数据还表明，一些突变不会导致功能表型的丧失，但也可能产生功能获得和显性负面后果。关于被破坏的特定生物过程以及被损害的蛋白质功能的信息对于在未来为与不同类型的突变相关的临床病理找到适当的治疗计划至关重要。因此，这项拨款旨在进一步探索NRG和L1‘S在突触形成中的作用，以及在单细胞水平上研究体内L1中已发现的人类突变的影响。我们将结合已识别的人类L1突变的巨大资源以及果蝇遗传和分子工具的力量来确定哪些细胞外L1/NRG相互作用和细胞内信号通路在突触形成中发挥作用。我们将从电生理学和解剖学的角度确定野生型nrg849中各种L1/NRG结构的功能和暂时功能丧失的背景。从这项建议中列出的研究中获得的知识将使我们能够更好地了解突触发生的机制以及L1相关神经疾病的病理基础。 细胞黏附分子L1的170多种不同突变已被确定可导致各种人类神经疾病，这些疾病与智力低下、脑积水(由于脑内积液而导致的头部增大)和痉挛(肌肉的不自主收缩)有关。我们打算在一个独特的模型系统中，在体内的单个细胞水平上研究这些不同的病理突变的影响，这将使我们能够识别特定的生物过程受到影响，以及这些突变破坏了蛋白质功能。这些信息对于为这些突变的临床表现找到适当的治疗至关重要，因此将有益于未来L1相关疾病患者的健康。