Article In Brief
Research in an animal model of Alzheimer’s disease offers insights into the role of aquaporin 4 in the glymphatic system and its implications for treatment or prevention of amyloid buildup in the brain.
A naturally, elongated form of the astrocytic protein 4 (AQP4) promotes clearance of amyloid-beta from the rodent brain, a process that can be enhanced with either of two promising drug candidates, according to a published Sept.14 in Brain.
The results provide new insights into the essential but still somewhat mysterious role AQ4 plays in the brain-cleansing mechanism known as the glymphatic system and may have implications for treatment or prevention of amyloid buildup in the brain.
AQP4 sits on the endfeet of astrocytes, which wrap around blood vessels and play an essential role in maintaining the blood-brain barrier. As its name suggests, AQP4 is a water channel and does not transport amyloid. Instead, in ways still not entirely clear, said lead author Joseph Dougherty, PhD, it is thought that AQP4 creates the flow of interstitial fluid that carries extracellular amyloid from its origin to transporter proteins that flush it from the brain into the vasculature. This is mechanism behind the glymphatic system.
Dr. Dougherty, professor of genetics and psychiatry at Washington University School of Medicine in St. Louis, discovered the extended form of AQP4 (AQP4X) through basic research aimed at understanding transcriptional regulation, looking at the population of ribosomes on RNA transcripts in different cell types in the brain.
“We noticed there were ribosomes on some transcripts where they shouldn’t be,” he said.
In most cases, transcription ends when a ribosome encounters a stop codon, which causes the ribosome to unbind from the RNA and stop adding new amino acids to the polypeptide chain. In rare cases, a ribosome will “read through” a stop codon, elongating the chain. Dr. Dougherty found that in astrocytes, when transcribing the aquaporin 4 messenger RNA, “something like 30 percent of the time, the ribosome just blows past the stop codon and keeps making protein,” extending the polypeptide by an average of 29 amino acids.
The extended form was unknown until very recently, in part because the existing antibodies, used to locate AQP4 on the astrocyte membrane, didn’t distinguish between the shorter and longer forms. So the research team developed an AQP4X-specific antibody and applied it to astrocytes.
“The antibodies just lit up right along the blood vessels. This seemed to suggest that this form of AQP4 was the form that functioned at the endfeet,” Dr. Dougherty said.
The shorter form, in contrast, was dispersed widely over the surface of the astrocyte. While a small amount did localize to the endfeet, that localization was lost when AQP4X was deleted, suggesting that the short form may simply tag along, or may form a complex with, AQP4X.
To understand its role in amyloid clearance, Dr. Dougherty crossed mice that made only the short form of AQP4 with APP/PS1 transgenic mice, which make excess amyloid. Absence of AQP4X reduced amyloid clearance from the mouse brain, increasing its elimination half-life by more than 40 percent, indicating the central importance of AQP4X to the glymphatic disposal of amyloid.
With that in mind, Dr. Dougherty sought compounds that could promote ribosomal read-through of the AQP4 transcript to increase the proportion of the extended form. He tested a collection of 2,560 compounds, a group comprising drug candidates in clinical trials, drugs sold in Asia or Europe, or natural products and their derivatives. Two compounds emerged from the screen: apigenin and sulphaquinoxaline; apigenin, a natural product available as a supplement, performed best in the cell-based system.
Finally, they infused the compounds into the hippocampus of APP/PS1 mice and measured the level of amyloid-beta in the interstitial fluid over time. Both compounds lowered amyloid-beta by about 30 percent or more over the course of 20 hours of treatment. This effect was lost when the drugs were administered to mice in which AQP4X production was blocked, “indicating that the ability of the drugs to alter amyloid-beta levels required AQP4 read-through in vivo,” Dr. Dougherty said.
The team is currently testing whether increasing the level of AQP4X genetically or pharmaceutically has a functional benefit in Alzheimer’s mice and “whether an increase in read-through might slow down the progression of disease,” he said.
Whether or not compound either becomes a clinically useful treatment, Dr. Dougherty said, “What’s exciting is that we have potentially found a way to ‘turn up the knob’ on the waste system by encouraging astrocytes to create more of this long isoform of aquaporin.” That is promising.”
Importantly, he said, studies have shown a loss of AQP4 at the blood vessels over time in the Alzheimer’s brain “exactly at the time you need it the most.” Any treatment to slow that loss may be beneficial, he added.
“This study is exciting,” said Jeffrey Iliff, PhD, Arthur J. and Marcella McCaffray Professor in Alzheimer’s Disease and professor of psychiatry and behavioral sciences and of neurology at the University of Washington School of Medicine in Seattle, who was part of the team that initially featured the glymphatic system.
“Over the past 10 years, as the function of the glymphatic system has been worked out, one of the key findings has been the dependence of the system on this water channel,” he said. “But up to this point, identifying a point for intervention has been elusive.”
The identification of the role of the extended form of aquaporin 4 might provide that point of intervention, he added.
The demonstration that elimination of the elongated form slows amyloid clearance, and that enhances its expression improves clearance, “suggests that this elongated form, which may be the perivascular form of AQ4, may be really important in the development of Alzheimer’s disease.”
Nonetheless, there are hurdles to overcome before the potential benefits of AQP4 read-through enhancement can be fully understood. The amount of aquaporin localized to the endfeet differs in mice and humans, which may affect the importance of AQP4X and thus the translation of the results to people. Further, he said, there likely will be differences in amyloid clearance between species and in different pathological states as well as differences in clearance from different brain regions.
“No one really understands yet how these influences contribute to the sum total of amyloid clearance,” he added.
Finally, Dr. Iliff said, “We still will need to know what the effect, if any, these or other compounds have on read-through in other transcripts and what the consequences of that could be. That will need to be figured out.”
Even more important is determining whether amyloid reduction has a beneficial effect on cognition, said Samuel E. Gandy, MD, PhD, professor of neurology and psychiatry and Mount Sinai Professor of Alzheimer’s Disease Research at the Icahn School of Medicine at Mount Sinai in New York . So far in human trials, he pointed out, the evidence is not there.
A 2021 study in the British Medical Journal by Sarah F. Ackley, PhD, and colleagues pooled results from 14 randomized trials of amyloid-targeting drugs that used neuroimaging to track amyloid reduction. It found “no consistent relationship with clinical cognition,” Dr. Gandy said. “In other words, therapeutic manipulation of AQP4 might modulate amyloid burden, but there is no guarantee that patients would have their risk for dementia reduced even if all amyloid is purged from their brains.”
“The potential significance of the AQP4 amyloid paper is impossible to judge until the observation can be both validated in humans and shown to cause a meaningful clinical benefit for human patients,” he said.
“The work is significant from the perspective of understanding the pathophysiology of Alzheimer’s disease and is “very convincing about the importance of AQP4 in regulating amyloid-beta clearance,” said Benjamin Wolozin, MD, PhD, professor of pharmacology and neurology at Boston University School of Medicine.
However, he said, from a therapeutic standpoint, “we know that once the disease process has started, completely removing amyloid-beta impacts disease only a bit, and that even a 50 percent removal progress would be unlikely to have any demonstrable impact on disease.” progression. The effect is just too small.”
None of the individuals quoted here have relevant disclosures.