A Novel Astrocyte Endfoot Protein Arising from Translational Readthrough and Influencing Alzheimer's Disease Pathology

/Abstract Epidemiological data on Alzheimer’s disease (AD) paint a bleak picture for American society, with the death toll from the disease surpassing that from breast cancer and prostate cancer combined. As the most common cause of dementia, AD afflicts about 7 million Americans as of 2024 and will affect 13 million Americans by 2050. The earliest neuropathology of AD is often the extracellular deposits of Amyloid-beta (Aβ) peptides. If not removed efficiently, Aβ causes synaptic dysfunction and starts to aggregate into plaques, which in turn deform neuronal processes, activate glial cells, and induce inflammation. Recent research has established Aquaporin 4 (AQP4), an astrocyte-specific water channel protein, as a key regulator of Aβ. The mechanism is debated: some researchers argue AQP4 facilitates Aβ removal, while others suggest it helps sequester plaques to minimize neuronal harm. Nonetheless, there is widespread agreement on AQP4's critical role in AD. Recently, we detected an isoform of AQP4, termed AQP4X, and showed that it facilitates the clearance of Aβ peptides and the remodeling of plaques. AQP4X arises from a rare phenomenon called ‘translational readthrough’, where about 20% of the translating ribosomes continue beyond the stop codon, generating an extended isoform of the protein. Unlike the normal-length isoforms of AQP4, which are located away from blood vessels, the extended AQP4X is found in specialized astrocytic projections called ‘endfeet’ that surround blood vessels. To investigate AQP4X in AD, we have developed a loss-of-function mouse (Aqp4No_X, extra stops added to abolish readthrough) and a gain-of-function mouse (Aqp4All_X, stop mutated to sense for constitutive readthrough). When crossed to an AD model (APP/PS1), Aqp4No_X shows impaired Aβ clearance and Aqp4All_X shows enhanced clearance, indicating the importance of AQP4X in Aβ removal. AQP4X may function through the glymphatic system, a pathway thought to involve perivascular AQP4 and the cerebrospinal fluid flow in the brain and remove Aβ from the interstitial fluid during sleep. Unfortunately, in AD, the perivascular pool of AQP4 is altered, and the glymphatic system is compromised. In this proposal, we test the hypothesis that AQP4X is a critical modulator of astrocytes’ response in AD pathology. Aim 1 focuses on identifying pathways relevant to AQP4X function, including the glymphatic pathway, meningeal lymphatics, and circadian rhythms, using techniques such as cerebrospinal fluid tracing and MRI. Aim 2 characterizes the effect of AQP4X on Aβ plaque morphology by quantifying changes in Aβ fibrillation, dystrophic neurites, astrocyte hypertrophy, and microglia activation. Finally, Aim 3 seeks to characterize the effect of Aβ plaques on AQP4X and astrocyte endfeet through biochemical, imaging, and endfoot-specific translational profiling. Overall, our proposal will illuminate the role of Aqp4 readthrough in AD pathology and may establish the phenomenon as a potential therapeutic approach to enhance perivascular AQP4 and to mitigate AD pathology. Project Number: 1R01AG093941-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Institute on Aging (NIA) | Principal Investigator: Darshan Sapkota | Institution: UNIVERSITY OF TEXAS DALLAS, RICHARDSON, TX | Award Amount: $347,657 | Activity Code: R01 | Study Section: Special Emphasis Panel[ZRG1 AN-Z (55)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11301725