Imaging of protein synthesis and ubiquitination in fragile x syndrome

 描述(由申请人提供)：脆性X综合征(FXS)是由脆性X智力低下蛋白(FMRP)遗传缺失引起的，是最常见的遗传性智力残疾形式，也是导致自闭症的主要单基因原因。FMRP是一种mRNA结合蛋白，可以结合大量的mRNAs，并经常抑制它们的翻译。FMRP在树突棘内的突触后部位被检测到，在那里它被认为在参与突触发育和突触可塑性的局部蛋白质合成中发挥作用，这些蛋白质参与了学习和记忆。我们的长期目标是表征神经元局部蛋白质合成的机制，阐明它们在突触发育中的功能，并利用这些知识开发策略，在脆性X综合征和其他神经发育障碍中恢复突触蛋白质的动态平衡。一个关键的差距是缺乏对FMRP介导的局部蛋白质合成调节的潜在机制的了解，以及翻译异常如何可能有助于脆性x综合征患者突触蛋白稳态受损和树突棘突发育。我们推测，FMRP对树突和棘中mRNA翻译的活性依赖性调节是必需的，FXS中FMRP的缺失导致翻译失调和突触后蛋白稳态改变，导致突触发育受损。为了实现这些目标，我们将开发和使用新的荧光报告和成像分析来可视化和表征FMRP介导的活体培养海马神经元局部蛋白质合成的新机制。目的1将验证在FXS小鼠模型中FMRP的缺失导致树突状细胞和棘细胞中的mRNA翻译失调的假设。目的2将验证一种假说，即参与局部蛋白质合成的FMRP或mRNA靶序列的丢失会导致树突和脊柱中蛋白质泛素化和内稳态的失调。脆性X综合征突触蛋白稳态失调的特征对于阐明自闭症谱系障碍突触病变的共同神经生物学具有更广泛的意义。这项研究将为评估其他自闭症疾病模型和测试恢复突触蛋白稳态的治疗策略提供方法和理论基础。

 DESCRIPTION (provided by applicant): Fragile X syndrome (FXS), caused by the inherited loss of the Fragile X Mental Retardation Protein (FMRP), is the most common form of inherited intellectual disability and the leading monogenetic cause of autism. FMRP is an mRNA binding protein that binds numerous mRNAs and often represses their translation. FMRP is detected at postsynaptic sites within dendritic spines where it is believed to play a role in local protein synthesis involved in synapse development and synaptic plasticity underlying learning and memory. Our long-term objectives are to characterize mechanisms of local protein synthesis in neurons, elucidate their functions in synapse development, and use this knowledge to develop strategies to restore synaptic protein homeostasis in fragile x syndrome and other neurodevelopmental disorders. A critical gap is lack of understanding of the underlying mechanisms of FMRP mediated regulation of local protein synthesis and how dysregulated translation may contribute to impaired synaptic protein homeostasis and dendritic spine development in fragile x syndrome. We hypothesize that FMRP is necessary for the activity dependent regulation of mRNA translation in dendrites and spines, and that loss of FMRP in FXS results in dysregulated translation and altered postsynaptic protein homeostasis leading to impaired synaptic development. To accomplish these goals, we will develop and employ novel fluorescent reporters and imaging assays to visualize and characterize novel mechanisms of FMRP mediated local protein synthesis in live cultured hippocampal neurons. Aim 1 will test the hypothesis that loss of FMRP in a mouse model of FXS results in dysregulation of mRNA translation in dendrites and spines. Aim 2 will test the hypothesis that loss of either FMRP, or mRNA target sequences involved in local protein synthesis, results in dysregulation of protein ubiquitination and homeostasis in dendrites and spines. The characterization of dysregulated protein homeostasis at synapses in fragile x syndrome has broader significance toward elucidation of the shared neurobiology of synaptopathies in autism spectrum disorders. This research will provide methods and rationale to assess other autism disease models and test therapeutic strategies that restore synaptic protein homeostasis.