Restoring FMRP Phenotypes Frpm Temporal Regional and Splice- Isoforms Varations

项目四：威廉·格林诺 恢复FMRP：来自时间、区域和剪接等形变异的表型 脆性X综合征（FXS）是遗传性智力低下的最常见原因。某些治疗 似乎恢复/拯救fmM敲除小鼠模型中的特定WT表型特征。 基因的重新引入或替换正在成为未来的一个重要选择，研究正在进行中。 然而，FXS表型是异质的。神经系统的一系列特征似乎 影响。虽然影响单一神经递质系统的药物治疗可能会改善 一些症状（例如，Fmr 1 KO小鼠中听源性癫痫发作易感性的MPEP降低）， 越来越多的证据表明脆性X蛋白有多种亚型，脆性X基因在这一过程中的作用 以及病毒载体传递基因的策略。开始工作（目标 1)通过解决FMRP的不同亚型在它们的功能方面的可能不同的作用， 通过评估小鼠中的一系列表型， 同种型。这些研究的结果将为我们首次尝试病毒驱动表达 外源性FMRP（Aim 3），使用在Aim 1中特别有效的那些同种型。最后，AIM 2 调查的稳定性和持久性的症状，在存在的恢复完整的 基因在发育的特定阶段。 我们的方法将包括调查的神经解剖表型的脊柱形状，脊柱密度 和树突状领域的构象，而不仅仅是静态图像，但在形态随着时间的推移而变化， 关于传入活动，除了常规方法外，还使用内源性YFP标记的 神经元和多个双光子显微镜视图随着时间的推移，发展的脊柱和树突 网络此外，我们将采用生物标志物ERK磷酸化作为正常功能的标准， 细胞信号传导级联，以表征具有短暂或永久恢复的组织， FMRP分子的全部或部分阵列。

PROJECT IV: WILLIAM GREENOUGH RESTORING FMRP: PHENOTYPES FROM TEMPORAL, REGIONAL, AND SPLICE-ISOFORM VARIATIONS Fragile X syndrome (FXS) is the most frequent cause of inherited mental retardation. Certain treatments appear to restore/rescue particular WT phenotypic characteristics in the fmM-knockout mouse model. Gene reintroduction or replacement is becoming a serious future alternative, with research well under way. However, the FXS phenotype is heterogeneous. An array of features of the nervous system appear to be affected. While pharmacological treatments that affect a single neurotransmitter system may ameliorate some symptons (e.g. MPEP reduction of audiogenic seizure susceptibility in the Fmr1 KO mouse), the growing evidence of multiple isoforms of the Fragile X protein, the role of the Fragile X gene in the course of brain development, and a strategy for viral-vector delivery of the gene. The proposed work begins (Aim 1) by addressing the presumably varied roles of the different isoforms of the FMRP in terms of their relationship to phenotype by assessing a range of phenotypes in mice able to produce only single isoforms. The outcomes of these studies will inform our first attempts at viral-driven expression of exogenous FMRP (Aim 3), using those isoforms that were particularly effective in Aim 1. Finally, Aim2 investigates the stability and permanence of symptoms in the presence of restoration of the complete gene at selected stages in development. Our methods will include investigations of the neuroanatomical phenotypes of spine shape, spine density and dendritic field conformation, not just as static images but at the morphology changes over time with respect to afferent activity, using, in addition to conventional approaches, intrinsically-YFP-labeled neurons and multiple two-photon microscopic views over time of the developing spine and dendritic network. In addition, we will employ a biomarker, ERK phosphoylation, as a criterion for normallyfunctioning cellular signaling cascades, to characterize tissues with transient or permanent restoration of full or partial arrays of FMRP molecules.