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: 10605249
• 负责人: Darshan Sapkota
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF TEXAS DALLAS
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-15 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10605249
• 关键词: 3' Untranslated Regions; APP-PS1; Abeta clearance; Alzheimer' s Disease; Alzheimer' s disease brain; Amyloid beta-Protein; Antibodies; Astrocytes; Basic Science; Behavioral; Binding; Biological; Biology; Blood - brain barrier anatomy; Blood Vessels; Brain; C-terminal; CRISPR interference; Chemicals; Clinical; Clinical Treatment; Collaborations; Data; Defect; Disease; Disease Outcome; Enzyme-Linked Immunosorbent Assay; Ependymal Cell; Epitopes; Etiology; Excision; Exhibits; Fluorescence; Functional disorder; Future; Gene Expression; Gene Expression Regulation; Genetic; Goals; Half-Life; Hippocampus; Impaired cognition; Impairment; Intercellular Fluid; Knockout Mice; Learning; Link; Long-Term Potentiation; Luciferases; Maps; Measures; Membrane; Memory; Mentors; Messenger RNA; Molecular; Morphology; Mus; Neurobiology; Neurons; Outcome; Pathogenesis; Pathway interactions; Phase; Play; Process; Protein Isoforms; Proteins; Regulation; Reporter; Research Personnel; Ribosomes; Role; Sleep; Synapses; Synaptic plasticity; Techniques; Technology; Terminator Codon; Testing; Therapeutic; Time; Transgenic Mice; Translating; Viral; Viral Vector; abeta accumulation; abeta oligomer; aquaporin 4; cell type; deep sequencing; disorder risk; drug use screening; experimental study; genetic manipulation; improved; improved outcome; in vivo; insight; model organism; mouse genetics; mouse model; neurofibrillary tangle formation; neuron loss; new therapeutic target; novel; novel therapeutics; overexpression; preclinical study; prevent; screening; skill acquisition; tau Proteins; viral rescue; water channel; β-amyloid burden

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.

项目摘要/摘要 可溶性淀粉样β蛋白(Aβ)寡聚体可引发tau缠结形成、神经细胞丢失、突触功能障碍和 阿尔茨海默病(AD)患者的认知能力下降。水通道蛋白Aqp4(Aqp4)是一个关键成分 在Aqp4-/-小鼠中，Aβ的清除减少了约55%。 具体地说，星形胶质细胞末端足部集中的Aqp4被证明是在睡眠中移除Aβ所必需的 并在AD中受到干扰，这表明将Aqp4恢复到末端脚可以改善AD的结果。我发现这一点 本地化的Aqp4是Aqp4的终止密码子通读版本。我做了核糖体足迹(RF)， 对小鼠脑中核糖体保护的mRNA片段进行深度测序，并检测到映射到 Aqp4的3‘非翻译区，表明核糖体读过了终止密码子，形成了一个C- Aqp4的终端扩展版本(以下简称Aqp4X)。使用针对通读表位的抗体，我 结果表明，Aqp4X仅限于血管周围的终足，而正常的未延伸的Aqp4是受限的 在星形胶质细胞膜上的其他地方。因此，这个项目的目标是确定Aqp4 通读可增强Aβ清除，从而改善AD结局。 为了实现这一目标，我提出了三个目标。在目标1中，我将确定Aqp4X是否改变了消除 与Aqp4相比，β。我将通过病毒转导在小鼠海马区表达Aqp4X或Aqp4 APP/PS1+/-转基因小鼠，并用新型微免疫电极技术检测A-β率 从活体小鼠的间质液体中清除。接下来，在我获得的Aqp4-/-NICE和Aqp4No_X上 我产生的小鼠，我将使用ELISA来测量它们的大脑总Aβ水平，无论有没有病毒 救援队。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 利用药物筛选和CRISPRi筛选Aqp4的化学和遗传调节因子， 分别进行了分析。最后，在Aim 3中，作为一名独立调查员，我将确定与AD相关的 末端足Aqp4缺失引起的病理生理后果。为此，我将检查Aqp4No_X 小鼠血脑屏障可能存在的结构和功能缺陷以及神经元活性依赖的基因调控 在海马体中。我还会将这些小鼠与APP/PS1小鼠杂交，测试Aβ负荷和行为 当AD小鼠失去末端足部Aqp4时，赤字就会升级。因此，目标1将测试以下各项的必要性和充分性 β许可中的两个Aqp4版本，Aim 2将允许未来对潜在的治疗和生物学研究 和Aim 3将进一步阐明Aqp4X在AD中所起的作用。