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（FGF）同源因子（FHF）与海马神经元和异源细胞中的电压门控Na+（Nav）通道相互作用并调节其活性。FGF 14/FHF 4中的突变（F145 S）是人类进行性脊髓小脑共济失调和智力迟钝综合征（SCA 27）的原因，并且缺乏FGF 14（Fgf 14-/-）的小鼠具有类似于人类中常染色体显性FGF 14 F145 S表型的运动和认知表型。我们假设FGF 14 F145 S在体内通过与野生型FGF 14或FHF家族的其他成员寡聚而作为显性负性蛋白发挥功能，从而阻断它们的正常功能。虽然我们已经投入了大量的努力来了解FGF 14在体外的功能，以及当在海马神经元或异源细胞中错误表达时，进一步了解FGF 14的生理作用（和其他细胞内FGF）目前，由于缺乏必要的实验工具来探测这些结果，在体内发挥作用。为了探索FGF 14 F145 S在体内的功能，并建立SCA 27模型，以测试脊髓小脑共济失调患者的潜在治疗干预措施，我们将构建一个小鼠模型，其中F145 S突变插入小鼠内源性Fgf 14基因，并产生单克隆抗体，用于检测和操纵体内FHF。实现这些目标将允许对FHF的生理作用进行详细的机制研究，这是目前不可能的。提出了以下两个具体目标：1.生成、表征和验证针对FGF 14和细胞内FGF家族其他成员的单克隆抗体（Mab）。2.利用胚胎干细胞同源重组技术，将F145 S突变插入内源性Fgf 14基因，构建人脊髓小脑共济失调（SCA 27）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.