TOOLS TO EXPLORE FHF FUNCTION AND SPINOCEREBELLAR ATAXIA 27

描述(由申请人提供)：细胞内成纤维细胞生长因子(成纤维细胞生长因子)同源因子(FHFs)与海马神经元和异种细胞中的电压门控Na+(Nav)通道相互作用并调节其活性。FGF14/FHF4突变(F145S)是人类进行性脊髓小脑性共济失调和智力低下综合征(SCA27)的原因，缺乏FGF14(Fgf14-/-)的小鼠具有与人类常染色体显性遗传FGF14F145S表型相似的运动和认知表型。我们假设FGF14F145S通过与野生型FGF14或FHF家族其他成员的寡聚作用来阻断它们的正常功能，从而在体内作为主要的负性蛋白发挥作用。尽管我们在了解FGF14在体外的功能以及在海马神经元或异源细胞中错误表达时已经付出了大量的努力，但在理解FGF14(和其他细胞内FGFs)的生理作用和确定FGF14中SCA27突变的表型后果的机制方面的进一步进展受到缺乏必要的实验工具的限制，这些工具是在体内探索这些功能所必需的。为了探索FGF14F145S在体内的功能，并为SCA27建立一种模型，以测试脊髓小脑性共济失调患者的潜在治疗干预措施，我们将构建一种将F145S突变插入到小鼠内源性Fgf14基因中的小鼠模型，并产生用于在体内检测和操纵FHFs的单抗。实现这些目标将允许对FHFs的生理作用进行详细的机制研究，这是目前不可能的。具体目标如下：1.制备、鉴定和验证抗FGF14和其他细胞内成纤维细胞生长因子家族成员的单抗。2.通过胚胎干细胞的同源重组，构建F145S突变插入内源性Fgf14基因的人脊髓小脑性共济失调(SCA27)小鼠模型。

DESCRIPTION (provided by applicant): Intracellular Fibroblast Growth Factor (FGF) Homologous Factors (FHFs) interact with and regulate the activity of voltage-gated Na+ (Nav) channels in hippocampal neurons and in heterologous cells. A mutation (F145S) in FGF14/FHF4 is the cause of a progressive spinocerebellar ataxia and mental retardation syndrome in humans (SCA27), and mice lacking FGF14 (Fgf14-/-) have both motor and cognitive phenotypes that resemble the autosomal dominant FGF14F145S phenotype in humans. We hypothesize that FGF14F145S functions as a dominant negative protein in vivo by oligomerizing with wild type FGF14 or with other members of the FHF family to block their normal function. Although we have put substantial effort into understanding the function of FGF14 in vitro and when mis-expressed in hippocampal neurons or heterologous cells, further advances in understanding the physiological roles of FGF14 (and other intracellular FGFs) and defining the mechanisms underlying the phenotypic consequences of the SCA27 mutation in FGF14 are now limited by the lack of experimental tools that are necessary to probe these functions in vivo. To explore the function of FGF14F145S in vivo and to create a model for SCA27 with which to test potential therapeutic interventions for people with spinocerebellar ataxia, we will construct a mouse model in which the F145S mutation is inserted into the endogenous Fgf14 gene in mice and to generate monoclonal antibodies with which to detect and manipulate FHFs in vivo. Achieving these aims will allow detailed mechanistic studies into the physiological roles of the FHFs that are not presently possible. The following two specific aims are proposed: 1. To generate, characterize and validate monoclonal antibodies (Mabs) to FGF14 and other members of the intracellular FGF family. 2. To construct a mouse model of human spinocerebellar ataxia (SCA27) in which the F145S mutation is inserted into the endogenous Fgf14 gene using homologous recombination in embryonic stem cells.