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Neurobiological significance of Aqp4 stop codon readthrough

Aqp4 终止密码子通读的神经生物学意义

基本信息

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
项目类别：
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&apos Untranslated Regions APP-PS1 Abeta clearance Alzheimer&apos s Disease Alzheimer&apos 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

项目摘要

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.

项目总结/文摘