Researchers at Washington University School of Medicine in St. Louis have identified a druggable pathway that potentially could be used to help prevent Alzheimer’s disease (AD). The scientists found a way to increase clearance of waste products from the brains of mice by ramping up a genetic quirk known as readthrough, which boosted production of a variant of the astrocytic water channel protein Aquaporin 4. Initial experiments confirmed that small molecule compounds could boost this readthrough mechanism, and ultimately enhance clearance of amyloid-β (Aβ) in live mice.
The team suggests that their approach may also be effective for other neurodegenerative disorders, such as Parkinson’s disease, which are characterized by the build-up of toxic proteins. The study was led by Darshan Sapkota, PhD, at the time a postdoctoral researcher at Washington University. Sapkota, now an assistant professor of biological sciences at the University of Texas, Dallas, and colleagues reported on their findings in Brain, in a paper titled “Aqp4 stop codon readthrough facilitates amyloid-β clearance from the brain.”
Amyloid-β accumulation in the brain is the first step in the development of Alzheimer’s dementia. Scientists have poured countless hours and millions of dollars into finding ways to clear amyloid away before cognitive symptoms arise, with largely disappointing results. “Immunotherapeutics aimed at reducing amyloid beta are in clinical trials but with very limited success to date,” the team wrote. “Identification of orthogonal approaches for clearing amyloid-β may complement these approaches for treating Alzheimer’s disease.”
Scientists already know that a cell’s protein-building machinery occasionally fails to stop where it should. When the machinery doesn’t stop — a phenomenon known as readthrough — it creates extended forms of proteins that sometimes function differently than the regular forms. “A skipped termination, and hence the addition of extra amino acids to the growing polypeptide chain, can result in a protein product that becomes rapidly degraded or acquires untoward functions,” they explained. The astrocyte-specific water channel protein Aquaporin 4 (AQP4) is a key mediator of the glymphatic system, which clears amyloid-β and other waste products from the brain. And every once in a while, readthrough results in the synthesis of a C-terminally extended aquaporin 4, known as AQP4X, which effectively has an extra little tail on the end.
At first, Sapkota thought this tail represented nothing more than an occasional failure of quality control in the protein-manufacturing process. “We were studying this very wonky basic science question—‘How do proteins get made?’—and we noticed this funny thing,” said senior author Joseph D. Dougherty, PhD, a Washington University professor of genetics and of psychiatry, and Sapkota’s former mentor. “Sometimes the protein-synthesizing machinery blew right through the stop sign at the end and made this extra bit on the end of aquaporin 4. At first, we thought it couldn’t possibly be relevant. But then we looked at the gene sequence, and it was conserved across species. And it had this really striking pattern in the brain: It was only in structures that are important for waste clearance. So that’s when we got excited.”
Sapkota and Dougherty created tools to see whether the long AQPX4 form of aquaporin 4 behaved differently in the brain than the regular form. They found the long form — but not the short one — in the endfeet of astrocytes. “… we and others have shown that the readthrough-extended version (AQP4X) becomes exclusively perivascular within astrocytic endfeet in the brain whereas the normal-length AQP4 is localized to parenchyma away from blood vessels,” they wrote.
Astrocytes are a kind of support cell that help maintain the barrier between the brain and the rest of the body. Their endfeet wrap around tiny blood vessels in the brain and help regulate blood flow. Astrocytic endfeet are the perfect place to be if your job is to keep the brain free of unwanted proteins by flushing waste out of the brain and into the bloodstream, where it can be carried away and disposed of.
Interestingly, the investigators noted, the fraction of normal length AQP4 found perivascularly is decreased in AD, “ … with the extent of reduction being proportional to Aβ levels.” However, they continued, “… it has not yet been established whether it is the perivascular AQP4 that specifically clears endogenous Aβ, and/or whether the AQP4X isoform mediates this function.”
Reasoning that increasing the amount of long aquaporin 4 might, in fact, increase waste clearance, Sapkota screened 2,560 compounds for the ability to increase readthrough of the aquaporin 4 gene. The screen highlighted two candidates: apigenin, a dietary flavone found in chamomile, parsley, onions and other edible plants; and sulphaquinoxaline, a veterinary antibiotic used in the meat and poultry industries.
Sapkota and Dougherty teamed up with Alzheimer’s researchers and co-authors John Cirrito, PhD, an associate professor of neurology, and Carla Yuede, PhD, an associate professor of psychiatry, of neurology and of neuroscience, to figure out the relationship between long aquaporin 4 and amyloid-β clearance.
The researchers studied mice genetically engineered to have high levels of amyloid in their brains. They treated the mice with one of either apigenin, sulphaquinoxaline, an inert liquid, or a placebo compound that has no effect on readthrough. The results confirmed that mice treated using either apigenin or sulphaquinoxaline cleared amyloid beta significantly faster than those treated with either of the two inactive substances. Further experiments confirmed that “Mice treated with apigenin or sulphaquinoxaline had a significantly faster elimination half-life of Aβ than mice treated with vehicle, suggesting the drugs indeed worked by increasing clearance,” they stated. Conversely, in mice genetically engineered such that they could not produce the long, AQP4X variant, the drugs completely lost their effects, “indicating the ability of the drugs to alter Aβ levels required AQP4 readthrough in vivo.”
Neither compound used in these studies would be suitable as a clinical drug candidate. Sulphaquinoxaline is not safe for use in people, and while apigenin is available as a dietary supplement, it’s not known how much gets into the brain, and Cirrito cautions against consuming large amounts of apigenin in an attempt to stave off Alzheimer’s. “Although these specific compounds may or may not be suitable for therapeutics (sulphaquinoxaline, an antiparasitic in chickens, can crystallize in primate kidney), they indicate this is a modulatable pathway in vivo,” the team commented. The researchers are working on finding better drugs that influence the production of the long form of aquaporin 4, testing several derivatives of sulphaquinoxaline and additional compounds.
“We’re looking for something that could be quickly translated into the clinic,” Sapkota said. “Just knowing that it’s targetable at all by a drug is a helpful hint that there’s going to be something out there we can use.”
Cirrito noted, “There’s a lot of data that says reducing amyloid levels by just 20% to 25% stops amyloid build-up, at least in mice, and the effects we saw were in that ballpark. That tells me that this could be a novel approach to treating Alzheimer’s and other neurodegenerative diseases that involve protein aggregation in the brain. There’s nothing that says this process is specific for amyloid beta. It may be enhancing, say, alpha-synuclein clearance, too, which could benefit people with Parkinson’s disease.”
The investigators concluded, “… while we have focused here on Aβ clearance, it is possible that enhancing readthrough might equally promote clearance of other proteins involved in neurodegenerative diseases (e.g. tau, synuclein), thus providing a singular broadly applicable therapeutic strategy for diseases involving aggregation in the brain.”