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.