FUNCTIONAL ANALYSES OF NEUROGLIAN/L1 IN SYNAPTOGENESIS

描述（由申请人提供）：Drosophila Neuroglian （Nrg）是脊椎动物L1的同源物，是具有多种结合伙伴的多功能细胞粘附分子的主要例子。人类L1不同位点的几种类型的单点突变已被证明可引起多种神经系统疾病（CRASH综合征），包括智力迟钝、脑积水和痉挛。Nrg/L1参与轴突寻路、神经突延伸和细胞迁移。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表征，但对突触形成过程中的潜在功能知之甚少。我们最近发现Nrg在突触发生中确实起着重要的作用。我们发现nrg849等位基因胞外结构域的单个错义突变会破坏巨纤维系统（GFS）神经元回路中中枢突触的组装和功能。我们的数据表明细胞内锚蛋白结合基序Nrg/L1的磷酸化对巨突触的形成至关重要。有趣的是，人类L1能够完全挽救nrg849突变体的表型，而被测试的类似物Neurofascin和NrCAM却不能，尽管它们具有相同的整体结构域结构，包括高度保守的锚蛋白结合基序。这表明GFS是研究l1特异性函数的有效模型系统。我们的初步研究表明，在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。尽管神经突生长、引导或突触形成的缺陷在功能上都可能导致相同的可识别表型，但神经元之间的连接中断，受影响的生物学过程是完全不同的。我们的数据还表明，一些突变不会导致功能表型的丧失，但也可能具有功能增益和主要的负面后果。关于特定生物过程被破坏以及蛋白质功能受损的信息对于未来找到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，该基金旨在进一步探索Nrg和L1在突触形成中的作用，并在单细胞水平上研究已确定的人类L1突变在体内的影响。我们将结合已鉴定的人类L1突变的巨大资源和果蝇遗传和分子工具的力量，确定细胞外L1/Nrg相互作用和细胞内信号通路在突触形成中发挥作用。我们将从电生理和解剖学角度确定野生型nrg849中各种L1/Nrg结构的功能以及功能背景的时间损失。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理解突触发生的机制以及l1相关神经疾病的病理细胞基础。

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.