FUNCTIONAL ANALYSES OF NEUROGLIAN/L1 IN SYNAPTOGENESIS

NEUROGLIAN/L1 在突触发生中的功能分析

• 批准号: 7589798
• 负责人: TANJA ANGELA GODENSCHWEGE
• 金额: $ 26.94万
• 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/7589798
• 关键词: Address; Affect; Affinity; Alleles; Ankyrins; Axon; Binding; Biological Models; Biological Process; Brain; Cell Adhesion Molecules; Cells; Clathrin; Clinical Pathology; Clinical Treatment; Collection; Cytoskeleton; Data; Defect; Development; Developmental Process; Disease; 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; Phenotype; Phosphorylation; Physiological; Play; Point Mutation; Process; Protein Isoforms; 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; abstracting; adduct; axonal pathfinding; base; cell motility; design; extracellular; ezrin; fly; follow-up; gain of function; in vivo; knock-down; loss of function; mutant; nervous system disorder; neurofascin; neuroglian; novel; overexpression; 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（由申请人提供）：果蝇神经胶质（NRG），一种脊椎动物L1的同源物，是具有多种结合伴侣的多功能细胞粘附分子的主要例子。人类L1中不同部位的几种单点突变已显示出引起多种神经系统疾病（崩溃综合征），包括智力低下，脑积水和痉挛。 NRG/L1已被证明与轴突探路，神经突延伸和细胞迁移有关。 NRG/L1在这些发育过程中的作用已经在脊椎动物和无脊椎动物中得到了很好的特征，但对突触形成过程中的潜在功能知之甚少。我们最近表明，NRG确实在突触发生中具有重要功能。我们发现，NRG849等位基因的细胞外域中的单个错义突变破坏了中央突触在良好的神经元电路中的中央突触的组装和功能，即巨型纤维系统（GFS）。我们的数据表明，NRG/L1的细胞内踝结合基序的磷酸化对于巨型突触形成至关重要。有趣的是，尽管具有相同的总体域结构，其中包括高度保守的Ankyrin结合基序，但人类L1能够完全营救NRG849突变体中的表型，而NRGASCAN和NRCAM却不能。这表明GFS是用于研究L1特异性功能的有效模型系统。我们的初步研究表明，在L1中鉴定出的一些病理错义突变会影响突触形成，而不是早期的发育过程。尽管神经突生长的缺陷，但引导或突触的形成可能在功能上都在相同的可识别表型中导致，而神经元之间的连接破坏了，但受影响的生物过程完全不同。我们的数据还表明，某些突变不会导致功能表型的丧失，但也可以获得功能和主导的负面后果。有关被破坏的特定生物学过程以及遭受损害的蛋白质功能的信息对于未来与不同类型突变相关的临床病理的适当治疗计划至关重要。因此，该赠款旨在进一步探索NRG和L1在突触形成中的作用，并研究单个细胞水平的L1 IN VIVO中鉴定的人类突变的影响。我们将结合果蝇中确定的人L1突变的巨大资源以及遗传和分子工具的功能，以确定哪些细胞外L1/NRG相互作用和细胞内信号通路在突触形成中起作用。我们将在野生型，NRG849中确定各种L1/NRG构建体的功能，以及功能背景在电生理学上和解剖学上的时间丧失。从本提案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与L1相关神经系统疾病的病理学的细胞基础。 已经鉴定出细胞粘附分子L1中有170多个不同的突变，从而导致各种与智力低下的人神经系统疾病有关，这些疾病与智力低下，脑力（由于大脑上的液体收集）和痉挛（肌肉的非自愿收缩）有关。我们打算研究独特模型系统中单细胞水平上体内这些各种病理突变的影响，这将使我们能够识别受影响的特定生物学过程，以及被突变破坏的蛋白质功能。这些信息对于找到这些突变的临床表现的适当治疗至关重要，因此将来将使L1相关疾病患者的健康受益。