Role of Fragile X Mental Retardation Protein on Gene Expression in Auditory Brainstem Development

脆性X智力低下蛋白对听觉脑干发育中基因表达的作用

• 批准号: 10190073
• 负责人: HITOMI SAKANO
• 金额: $ 18.41万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10190073
• 关键词: Affect; Age; Antibodies; Auditory; Auditory system; Binding; Binding Proteins; Biology; Brain; Brain Stem; Brain region; Cochlear nucleus; Collaborations; Data; Development; Diphtheria Toxin; Disease; Down-Regulation; Exhibits; FMR1; Fibroblasts; Fragile X Syndrome; Gene Expression; General Population; Genes; Genetic Translation; Goals; Heritability; Hyperacusis; Hypersensitivity; Inherited; Knock-out; Knockout Mice; Loudness; Measures; Mediating; Mentors; Messenger RNA; Methods; Modeling; Molecular Biology; Mus; Neuronal Plasticity; Neurons; Noise; Pathway interactions; Patients; Phenocopy; Phenotype; Population; Process; Proteins; Proxy; Publishing; RNA; RNA immunoprecipitation sequencing; Research; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Role; Seizures; Stains; Synapses; Techniques; Technology; Testing; Therapeutic; Tissues; Translational Repression; Translations; Western Blotting; Wild Type Mouse; Work; auditory deprivation; auditory pathway; autism spectrum disorder; base; deafness; experience; glutamatergic signaling; induced pluripotent stem cell; inhibitor/antagonist; mRNA Decay; mRNA Precursor; mouse model; novel; postnatal; protein function; response; small molecule; sound; therapeutic target; transcriptome; transcriptome sequencing; translation factor

Fragile X Syndrome (FXS) is the most common heritable autism spectrum disorder and is associated with auditory features such as hypersensitivity to sound (hyperacusis). FXS is caused by the absence of Fragile X mental retardation protein (FMRP), which is known to bind specific mRNAs and repress their translation. Little is known about how FMRP impacts the central auditory pathway. We will study FMRP effects on gene expression in the auditory brainstem, using the well-established fmr1-knockout (KO) mouse model, which exhibits auditory hypersensitivity and seizures in response to loud noise. Although the well-established role of FMRP is translational repression, it has been shown recently in neurons that FMRP can also change the level of many mRNAs. Whether this occurs in the auditory brainstem is unknown. We have novel transcriptome data (unpublished) showing that the levels of many mRNAs that are known to be bound by FMRP, and to function in synaptic pathways, are decreased in the fmr1-KO cochlear nucleus. How this occurs is not known. Aim 1 will test the hypothesis that direct FMRP binding stabilizes the bound mRNA, but in the absence of FMRP, these mRNAs have decreased stability (and, therefore, decreased level). We will also determine if decreased stabilization of mRNA leads to decreased protein level or if it is offset by the loss of FMRP-mediated translational repression so as to manifest as increased protein level. Another possibility is that FMRP acts indirectly through translational repression of factors, such nonsense-mediated mRNA decay (NMD) factor, UPF1. Unpublished data from my research mentor’s lab has shown that induced pluripotent stem cells derived from FXS-patient fibroblasts manifest an abnormally high level of UPF1 (whose mRNA is bound by FMRP), resulting in hyperactivated NMD and, as a consequence, reduced levels of cellular NMD target mRNAs. Based on these data, Aim 2 will test the hypothesis that NMD is hyperactivated in fmr1-KO cochlear nucleus, leading to gene downregulation. Lastly, it is known that FMRP is involved in activity dependent processes. For example, dendritic localization of FMRP is increased with glutamatergic signaling and loss of afferent activity can blunt translational repression by FMRP. Aim 3 will test the hypothesis that the FMRP effects on gene expression are dependent on afferent activity. We will examine an inducible deafness mouse model to determine if it can phenocopy the fmr1-KO, indicating that afferent activity is required for FMRP function. The PI has extensive molecular biology experience, and with the guidance of a primary mentor who is a respected RNA biologist. The PI will master current RNA techniques and work towards becoming an independent investigator. Results will reveal how FMRP regulates genes important for auditory development and plasticity. The ultimate goal is to reveal potential therapeutic targets to treat auditory hypersensitivity and processing disorders. The mechanism leading to hyperacusis is poorly understood, and because it is not limited to FXS and affects up to 15% of the population, the study is relevant to the general population.

脆性X综合征(FXS)是最常见的遗传性自闭症谱系障碍，与 听觉特征，如对声音敏感(听觉过敏症)。FXS是由脆性X的缺失引起的 智力低下蛋白(FMRP)，已知能结合特定的mRNAs并抑制其翻译。一点儿 已知FMRP如何影响中枢听觉通路。我们将研究FMRP对基因的影响 利用已建立的fmr1基因敲除(KO)小鼠模型，在听觉脑干中表达 表现出对大噪音的听觉过敏和癫痫发作。尽管已经确立的角色是 FMRP是翻译抑制，最近在神经元中显示FMRP也可以改变水平 在许多mRNA中。这是否发生在听觉脑干中还不得而知。我们有新的转录组数据 (未发表)表明许多已知受FMRP结合的mRNAs的水平，并在 突触通路，在fmr1-kO耳蜗核减少。目前尚不清楚这种情况是如何发生的。目标1将 测试直接FMRP结合稳定结合的mRNA的假设，但在没有FMRP的情况下，这些 MRNAs的稳定性降低(因此，水平降低)。我们还将确定是否减少 MRNA的稳定导致蛋白质水平的降低，或者如果它被FMRP介导的缺失所抵消 翻译抑制，从而表现为蛋白质水平的增加。另一种可能性是FMRP采取行动 间接通过翻译抑制因子，如无意义介导的mRNA衰变(NMD)因子， UPF1。来自我的研究导师的实验室的未发表的数据表明，诱导的多能干细胞来源于 来自FXS患者的成纤维细胞显示异常高水平的UPF1(其mRNA被FMRP结合)， 导致NMD过度激活，从而导致细胞内NMD靶向mRNAs水平降低。基座 根据这些数据，目标2将检验NMD在fmr1-kO耳蜗核过度激活的假设，导致 到基因下调。最后，众所周知，FMRP参与了依赖活动的过程。为 例如，随着谷氨酸能信号转导和传入活性的丧失，FMRP的树突定位增加 可以钝化FMRP对翻译的抑制。目标3将检验FMRP对基因影响的假设 表达依赖于传入活动。我们将研究一种诱导性耳聋小鼠模型 确定它是否能复制fmr1-kO，表明FMRP功能需要传入活动。这个 PI拥有丰富的分子生物学经验，并在一位受人尊敬的主要导师的指导下 核糖核酸生物学家。PI将掌握当前的RNA技术，并努力成为一名独立的 调查员。结果将揭示FMRP如何调节对听觉发育和可塑性重要的基因。 最终目标是揭示治疗听觉过敏症和加工的潜在治疗靶点。 精神错乱。导致听力亢进的机制尚不清楚，因为它并不局限于FXS 并影响高达15%的人口，这项研究与普通人群相关。