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Neurobiological significance of Aqp4 stop codon readthrough

Neurobiological significance of Aqp4 stop codon readthrough
Aqp4 终止密码子通读的神经生物学意义
批准号:
10409864
负责人:
Darshan Sapkota
金额:
$24.9万
依托单位:
依托单位国家:
美国
项目类别:
财政年份:
2021
资助国家:
美国
项目状态:
已结题
起止时间:
2021-07-15 至 2024-04-30

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项目成果

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中文摘要
翻译
项目概要/摘要 可溶性淀粉样蛋白β(Aβ)寡聚体触发tau缠结形成、神经元细胞损失、突触功能障碍和神经元凋亡。 阿尔茨海默病(AD)中出现的认知能力下降。水通道水通道蛋白4(Aqp 4)是一个关键组成部分, Aqp 4-/-小鼠中Aβ清除减少约55%证明了这一点。 特别是,星形胶质细胞足端浓缩的Aqp 4被证明是在睡眠期间清除Aβ所必需的。 并且在AD中受到干扰,这表明将Aqp 4恢复到末端足可以改善AD的结果。我发现这 末端定位的Aqp 4是Aqp 4的终止密码子通读形式。我进行了核糖体足迹法(RF), 核糖体保护的mRNA片段的深度测序,在小鼠大脑中,检测到的读数映射到 Aqp 4的3'非翻译区,这表明核糖体阅读通过终止密码子,并使C- Aqp 4的末端延伸形式(下文称为Aqp 4X)。使用针对通读表位的抗体, 表明Aqp 4X仅限于血管周围终足,而正常的未延伸的Aqp 4被限制在血管周围终足, 沿着星形胶质细胞膜。因此,本项目的目标是确定Aqp 4是否 通读增强Aβ清除,从而改善AD结局。 我提出了三个目标来实现这一目标。在目标1中,我将确定Aqp 4X是否改变了消除 Aβ与Aqp 4相比。我将使用病毒转导在大肠杆菌中表达Aqp 4X或Aqp 4。 APP/PS1+/-转基因小鼠,并采用新型微量免疫电极技术测定Aβ 从活小鼠的间质液中去除。接下来,在我获得的Aqp 4-/- nice和Aqp4No_X上, 我将使用ELISA来测量它们的总脑Aβ水平,无论是否有病毒感染, 救援我还将检查Aqp4No_X小鼠的记忆和其他行为缺陷。在目标2中,我将确定 使用药物筛选和CRISPRi筛选的Aqp 4通读的化学和遗传调节剂, 分别最后,在目标3中,作为一名独立调查员,我将确定与AD相关的 终足缺失引起的病理生理学后果Aqp 4。为此,我将研究Aqp4No_X 小鼠的BBB和神经元活性依赖性基因调控中可能的结构和功能缺陷 在海马区。我还将这些小鼠与APP/PS1小鼠杂交,并测试Aβ负荷和行为 当AD小鼠失去末端足Aqp 4时,缺陷升级。因此,目标1将检验以下方面的必要性和充分性: Aqp 4在Aβ清除中的两个版本,aim 2将允许未来对潜在的治疗和生物学的研究。 调节因子,目的3将进一步阐明Aqp 4X在AD中发挥的作用。
英文摘要
Project Summary/Abstract Soluble amyloid beta (Aβ) oligomers trigger tau tangle formation, neuronal cell loss, synaptic dysfunction and cognitive decline seen in Alzheimer's disease (AD). The water channel Aquaporin 4 (Aqp4) is a key component of the Aβ removal machinery in the brain, as evidenced by ~ 55% reduction in Aβ removal in Aqp4-/- mice. Specifically, astrocyte endfeet-concentrated Aqp4 is shown to be both required for removing Aβ during sleep and perturbed in AD, suggesting that restoring Aqp4 to endfeet can improve the outcome of AD. I find that this endfeet-localized Aqp4 is a stop codon readthrough version of Aqp4. I performed ribosome footprinting (RF), deep-sequencing of ribosome-protected mRNA fragments, in the mouse brain and detected reads mapping to the 3' untranslated region of Aqp4, suggesting that ribosomes read past the stop codon and make a C- terminally extended version of Aqp4 (Aqp4X hereafter). Using an antibody against the readthrough epitope, I show that Aqp4X is exclusive to the perivascular endfeet, whereas the normal un-extended Aqp4 is confined elsewhere along the astrocyte membrane. Therefore, the objective of this project is to determine if Aqp4 readthrough enhances Aβ clearance and thus improve AD outcome. I propose 3 aims to meet this objective. In aim 1, I will determine if Aqp4X has altered efficacy in eliminating Aβ compared to Aqp4. I will express either Aqp4X or Aqp4 using viral transduction in the hippocampi of APP/PS1+/- transgenic mice, and use a novel micro-immunoelectrode technology to measure the rate of Aβ removal from the interstitial fluid in live mice. Next, on the Aqp4-/- nice that I have acquired and Aqp4No_X mice that I have generated, I will use ELISA to measure their total brain Aβ levels, with or without viral rescue. I will also examine Aqp4No_X mice for memory and other behavioral deficits. In aim 2, I will identify the chemical and genetic regulators of Aqp4 readthrough using drug screening and CRISPRi screening, respectively. Finally, in aim 3, as an independent investigator, I will determine the AD-related pathophysiological consequences arising from the loss of endfeet Aqp4. To this end, I will examine Aqp4No_X mice for possible structural and functional defects in the BBB and neuronal-activity dependent gene regulation in the hippocampus. I will also cross these mice with APP/PS1 mice and test if Aβ burden and behavioral deficits escalate when an AD mouse loses endfeet Aqp4. Thus, aim 1 will test the necessity and sufficiency of the two Aqp4 versions in Aβ clearance, aim 2 will allow future studies on potential therapeutics and biological regulators, and aim 3 will further elucidate the role Aqp4X plays in AD.
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Neurobiological significance of Aqp4 stop codon readthrough
  • 批准号:
    10605249
  • 项目类别:
  • 资助金额:
    $24.9万
  • 财政年份:
    2021
  • 负责人:
    Darshan Sapkota
  • 依托单位:
Neurobiological significance of Aqp4 stop codon readthrough
  • 批准号:
    10451765
  • 项目类别:
  • 资助金额:
    $24.9万
  • 财政年份:
    2021
  • 负责人:
    Darshan Sapkota
  • 依托单位:
海外基金