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: 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 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10409864
• 关键词: 3' Untranslated Regions; APP-PS1; Abeta clearance; Alzheimer' s Disease; Alzheimer' s disease brain; Amyloid beta-Protein; Animal Model; Antibodies; Astrocytes; Basic Science; Behavioral; Biological; Biology; Blood - brain barrier anatomy; Brain; C-terminal; CRISPR interference; Clinical; Clinical Treatment; 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 (Brain); Impaired cognition; Impairment; Intercellular Fluid; Knockout Mice; Learning; Link; Long-Term Potentiation; Luciferases; 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; Structure; Synapses; Synaptic plasticity; Techniques; Technology; Terminator Codon; Testing; Therapeutic; Time; Transgenic Mice; Translating; Viral; Viral Vector; abeta accumulation; abeta oligomer; aquaporin 4; cell type; chemical genetics; deep sequencing; disorder risk; drug use screening; experimental study; genetic manipulation; improved; improved outcome; in vivo; insight; mouse genetics; mouse model; neurofibrillary tangle formation; neuron loss; new therapeutic target; novel; novel therapeutics; overexpression; preclinical study; prevent; screening; skills; 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.

项目概要/摘要 可溶性 β 淀粉样蛋白 (Aβ) 寡聚物会引发 tau 缠结形成、神经元细胞损失、突触功能障碍和 阿尔茨海默病 (AD) 中出现的认知能力下降水通道蛋白 4 (Aqp4) 是一个关键成分。 Aqp4-/- 小鼠中 Aβ 去除减少约 55% 就证明了这一点。 具体而言，星形胶质细胞末端浓缩的 Aqp4 被证明是睡眠期间去除 Aβ 所必需的 并在 AD 中受到干扰，表明将 Aqp4 恢复到 endfeet 可以改善 AD 的结果。 endfeet-localized Aqp4 是 Aqp4 的终止密码子通读版本，我进行了核糖体足迹 (RF)， 对小鼠大脑中核糖体保护的 mRNA 片段进行深度测序，并检测到映射到的读数 Aqp4 的 3' 非翻译区域，表明核糖体读过终止密码子并生成 C- Aqp4 的末端延伸版本（以下简称 Aqp4X）。 表明 Aqp4X 只存在于血管周围终足，而正常未延伸的 Aqp4 则受到限制 因此，该项目的目标是确定 Aqp4 是否存在。 通读可增强 Aβ 清除率，从而改善 AD 结局。 我提出了 3 个目标来实现这一目标，在目标 1 中，我将确定 Aqp4X 是否改变了消除功效。 Aβ 与 Aqp4 相比，我将使用病毒转导在海马中表达 Aqp4X 或 Aqp4。 APP/PS1+/-转基因小鼠，并采用新型微免疫电极技术测量Aβ率 接下来，在我获得的 Aqp4-/- 和 Aqp4No_X 上进行去除。 对于我培育的小鼠，我将使用 ELISA 来测量它们的大脑总 Aβ 水平，无论是否含有病毒 我还将检查 Aqp4No_X 小鼠的记忆和其他行为缺陷。在目标 2 中，我将确定哪些缺陷。 使用药物筛选和 CRISPRi 筛选对 Aqp4 的化学和遗传调节因子进行通读， 最后，在目标 3 中，作为独立研究者，我将确定 AD 相关的问题。 端足 Aqp4 缺失引起的病理生理后果 为此，我将检查 Aqp4No_X。 小鼠 BBB 可能的结构和功能缺陷以及神经元活动依赖性基因调控 我还将这些小鼠与 APP/PS1 小鼠进行杂交，测试 Aβ 负荷和行为。 当 AD 小鼠失去末端 Aqp4 时，缺陷会加剧。因此，目标 1 将测试 Aqp4 的必要性和充分性。 Aβ 清除中的两个 Aqp4 版本，目标 2 将允许未来对潜在的治疗和生物学研究 目标 3 将进一步阐明 Aqp4X 在 AD 中的作用。