Restoring FMRP Phenotypes Frpm Temporal Regional and Splice- Isoforms Varations

项目四：威廉·格雷厄姆 恢复FMRP：来自时间、区域和剪接-异构体变异的表型 脆性X综合征(FXS)是遗传性智力低下最常见的原因。某些治疗方法 在FMM基因敲除小鼠模型中，似乎可以恢复/挽救特定的WT表型特征。 随着研究的顺利进行，基因重新导入或替换正在成为未来一种严肃的替代方案。 然而，FXS的表型是异质性的。神经系统的一系列特征似乎是 受影响。虽然影响单一神经递质系统的药物治疗可能会改善 某些症状(例如，Fmr1KO小鼠听源性惊厥敏感性的MPEP降低)， 越来越多的证据表明脆性X蛋白的多种异构体，脆性X基因在这一过程中的作用 以及通过病毒载体传递基因的策略。提议的工作开始(目标 1)通过阐述FMRP的不同异构体在其 通过评估一系列只能产生单一表型的小鼠的表型与表型的关系 异构体。这些研究的结果将为我们首次尝试通过病毒驱动的表达提供信息 外源FMRP(AIM 3)，使用在AIM 1中特别有效的亚型。最后，AIM2 研究在存在完全修复的情况下症状的稳定性和持久性 基因处于选定的发育阶段。 我们的方法将包括研究脊柱形状、脊柱密度的神经解剖学表型。 和树枝晶场的构象，不仅是静态图像，而且在形态上随着时间的推移而变化 关于传入活动，除了使用常规方法外，还使用固有的YFP标记 发育中的脊髓和树突的神经元和多个双光子显微镜图像 网络。此外，我们将使用一种生物标志物，ERK磷酸化，作为正常功能的标准 细胞信号级联，以表征具有短暂或永久修复的组织 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.