FGF14 IN THE REGULATION OF PURKINJE NEURON EXCITABILITY AND SCA27

描述(由申请人提供)：最近的研究表明，FGF14是细胞内成纤维细胞生长因子(IFGF)亚家族的成员，是一种新的神经元兴奋性调节因子。缺乏Fgf14(Fgf14-/-)的小鼠的主要表型是共济失调，而人类FGF14的突变导致进行性脊髓小脑性共济失调综合征SCA27。研究还表明，FGF14和其他iFGFs与电压门控Na+(NAV)通道成孔亚基(1)的C-末端结构域相互作用，并调节异源表达的NAV通道的特性。此外，利用一种有效的(在Fgf14-/-小鼠中)抗FGF14特异性抗体，我们发现FGF14与NAV通道1亚单位共同定位于小脑浦肯野神经元中富含G的轴突起始段(AIS)，这些观察结果导致我们假设，失去FGF14会导致小脑皮质唯一输出神经元-浦肯野神经元的放电特性缺陷。在直接探索这一假说的初步研究中，我们发现与野生型浦肯野神经元相比，Fgf14-/-神经元的自发活动和重复放电显著减少。进一步的初步研究表明，NAV通道1亚单位Nav1.6在Fgf14-/-Purkinje神经元中的表达和AIS定位显著降低，而Ankyrin G在AIS的表达没有明显影响。这些发现表明，FGF14-NAV 1亚基相互作用在调节NAV通道的表达和/或AIS定位以及控制小脑浦肯野神经元的放电(输出)特性中起关键作用。这些实验将直接验证这些假说，并探索FGF14对小脑浦肯野神经元NAV通道表达、定位和功能的影响的分子机制。其他实验将集中在直接测试假设，即SCA27连锁的FGF14突变蛋白FGF14F145S在体内作为显性负向干扰野生型FGF14蛋白和NAV通道1亚单位之间的相互作用，从而减少NAV通道的表达/定位，并改变小脑浦肯野神经元的放电特性。预计这些研究将为FGF14的功能作用以及FGF14介导的神经元兴奋性效应所涉及的潜在分子机制提供新的和根本上重要的见解。公共卫生相关性：健康相关性声明(两到三句话，描述这项研究与公共健康的相关性)SCA27是一种由FGF14基因突变引起的显性遗传性脊髓小脑性共济失调(SCA)综合征。SCA27以进行性共济失调、小脑变性和认知障碍为特征，其表型与缺乏功能性Fgf14基因的小鼠非常相似。所提出的分子、细胞和生理研究将为FGF14在调节神经元兴奋性中的功能作用以及FGF14突变导致人类疾病的潜在分子机制提供新的和根本上重要的见解。

DESCRIPTION (provided by applicant): Recent studies suggest that FGF14, a member of the intracellular fibroblast growth factor (iFGF) subfamily functions as a novel regulator of neuronal excitability. The major phenotype in mice lacking Fgf14 (Fgf14-/-) is ataxia and mutations in FGF14 in humans cause a progressive spinocerebellar ataxia syndrome, SCA27. It has also been demonstrated that FGF14, and other iFGFs interact with the C-terminal domains of voltage-gated Na+ (Nav) channel pore-forming (1) subunits and modulate the properties of heterologously expressed Nav channels. In addition, exploiting a validated (in Fgf14-/-mice) anti-FGF14 specific antibody, we find that FGF14 co- localizes with Nav channel 1 subunits at the ankyrin G-rich axon initial segments (AIS) in cerebellar Purkinje neurons, These observations led us to hypothesize that loss of FGF14 produces a defect in the firing properties of Purkinje neurons, the sole output neurons of the cerebellar cortex. In preliminary studies focused on exploring this hypothesis directly, we found that spontaneous activity and repetitive firing were decreased significantly in Fgf14-/-, compared with wild type, Purkinje neurons. Additional preliminary studies revealed that the expression and AIS localization of the Nav channel 1 subunit, Nav1.6, was reduced markedly in Fgf14-/- Purkinje neurons, whereas Ankyrin G expression at the AIS was not significantly affected. These findings suggest that FGF14- Nav 1 subunit interactions play a critical role in regulating the expression and/or the AIS localization of Nav channels and in controlling the firing (output) properties of cerebellar Purkinje neurons. The experiments outlined in this proposal will test these hypotheses directly and explore the molecular mechanisms involved in mediating the effects of FGF14 on the expression, localization and functioning of Nav channels in cerebellar Purkinje neurons. Additional experiments will be focused on testing directly the hypothesis that the SCA27-linked FGF14 mutant protein, FGF14F145S, functions in vivo as a dominant negative to disrupt the interaction between the wild type FGF14 protein and Nav channel 1 subunits, thereby reducing Nav channel expression/localization and altering the firing properties of cerebellar Purkinje neurons. It is anticipated that these studies will provide new and fundamentally important insights into the functional roles of FGF14 and into the underlying molecular mechanisms involved in FGF14- mediated effects on neuronal excitability. PUBLIC HEALTH RELEVANCE: Health relatedness statement (two or three sentences, describe the relevance of this research to public health) SCA27 is a dominantly inherited spinocerebellar ataxia (SCA) syndrome caused by mutations in the FGF14 gene. SCA27 is characterized by progressive ataxia, cerebellar degeneration and cognitive impairment, and is phenotypically very similar to mice that lack a functional Fgf14 gene. The molecular, cellular and physiological studies proposed will provide new and fundamentally important insights into the functional roles of FGF14 in regulating neuronal excitability and into the underlying molecular mechanisms by which mutations in FGF14 cause disease in humans.