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，FGF 14，细胞内成纤维细胞生长因子（iFGF）亚家族的成员，作为神经元兴奋性的新型调节剂发挥作用。缺乏Fgf 14（Fgf 14-/-）的小鼠中的主要表型是共济失调，并且人中Fgf 14的突变引起进行性脊髓小脑共济失调综合征SCA 27。还已经证明，FGF 14和其他iFGF与电压门控Na+（Nav）通道孔形成（1）亚基的C-末端结构域相互作用，并调节异源表达的Nav通道的性质。此外，利用经验证的（在Fgf 14-/-小鼠中）抗FGF 14特异性抗体，我们发现FGF 14与Nav通道1亚基共定位在小脑浦肯野神经元中富含锚蛋白G的轴突起始段（AIS）处。这些观察结果使我们假设FGF 14的损失产生浦肯野神经元（小脑皮质的唯一输出神经元）的放电特性的缺陷。在直接探索这一假设的初步研究中，我们发现，与野生型浦肯野神经元相比，Fgf 14-/-的自发活动和重复放电显著减少。额外的初步研究显示，在Fgf 14-/-浦肯野神经元中，Nav通道1亚基Nav1.6的表达和AIS定位显著降低，而AIS处的锚蛋白G表达没有受到显著影响。这些发现表明FGF 14- 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。本提案中概述的实验将直接测试这些假设，并探索参与介导FGF 14对小脑浦肯野神经元中Nav通道的表达、定位和功能的影响的分子机制。另外的实验将集中于直接测试以下假设：SCA 27连接的FGF 14突变蛋白FGF 14 F145 S在体内作为显性负性功能破坏野生型FGF 14蛋白和Nav通道1亚基之间的相互作用，从而降低Nav通道表达/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FGF 14的功能作用和FGF 14介导的神经元兴奋性作用的潜在分子机制提供新的和根本性的重要见解。公共卫生相关性：健康相关性声明（两三句话，描述这项研究与公共卫生的相关性）SCA 27是一种显性遗传性脊髓小脑共济失调（SCA）综合征，由FGF 14基因突变引起。SCA 27的特征在于进行性共济失调、小脑变性和认知障碍，并且在表型上与缺乏功能性Fgf 14基因的小鼠非常相似。提出的分子、细胞和生理学研究将为FGF 14在调节神经元兴奋性中的功能作用以及FGF 14突变导致人类疾病的潜在分子机制提供新的和根本性的重要见解。

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.