Soluble amyloid beta (A?) 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 A? removal machinery in the brain, as evidenced by ~ 55% reduction in A? removal in Aqp4-/- mice. Specifically, astrocyte endfeet-concentrated Aqp4 is shown to be both required for removing A? 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 A? 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 A? 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 A? 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 A? 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 A? 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 A? 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.

Public Health Relevance

The concentration of amyloid-? (A?) is directly linked to its likelihood to aggregate into toxic species in the Alzheimer's disease brain. This proposal will determine the role of Aquaporin-4 (Aqp4), particularly an elongated readthrough version of the protein that occurs naturally, in regulating A? clearance via astrocytes. Elucidating the mechanisms that regulate A? concentration will provide insight into disease pathogenesis as well as risk of disease, and could suggest a new therapeutic target to enhance A? clearance from the brain.

National Institute of Health (NIH)
National Institute on Aging (NIA)
Career Transition Award (K99)
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Neuroscience of Aging Review Committee (NIA)
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Mackiewicz, Miroslaw
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Washington University
Schools of Medicine
Saint Louis
United States
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