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），由脆性X智力迟钝蛋白（FMRP）的遗传性缺失引起，是遗传性智力残疾的最常见形式，也是自闭症的主要单基因原因。FMRP是一种mRNA结合蛋白，其结合许多mRNA并通常抑制它们的翻译。FMRP在树突棘内的突触后位点处被检测到，在那里它被认为在参与突触发育的局部蛋白质合成和作为学习和记忆基础的突触可塑性中起作用。我们的长期目标是描述神经元中局部蛋白质合成的机制，阐明它们在突触发育中的功能，并利用这些知识来发展恢复脆性X综合征和其他神经发育障碍中突触蛋白稳态的策略。一个关键的差距是缺乏理解的潜在机制FMRP介导的调节局部蛋白质合成和翻译失调如何可能有助于受损的突触蛋白稳态和树突棘发育脆性X综合征。我们假设，FMRP是必要的树突和棘的mRNA翻译的活性依赖性调节，并在FXS的FMRP的损失导致失调的翻译和改变突触后蛋白质稳态，导致受损的突触发育。为了实现这些目标，我们将开发和采用新的荧光报告和成像分析，以可视化和表征FMRP介导的活培养海马神经元的局部蛋白质合成的新机制。目的1将检验FMRP在FXS小鼠模型中的缺失导致树突和棘中mRNA翻译失调的假设。目的2将测试的假设，无论是FMRP，或mRNA的靶序列参与局部蛋白质合成的损失，导致蛋白质泛素化和树突和棘的稳态失调。脆性x染色体综合征中突触蛋白质稳态失调的特征对于阐明自闭症谱系障碍中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.