For all that researchers know about Aβ, the peptide so strongly linked to Alzheimer’s disease, they have scant knowledge of its normal function in or out of the cell. A new paper strengthens the idea that Aβ helps fights infection as part of the innate immune system. In the May 25 Science Translational Medicine, scientists led by Robert Moir and Rudolph Tanzi of Massachusetts General Hospital in Charlestown, Massachusetts, report that Aβ helps human cells, worms, and mice ward off invasions by pathogenic yeast and bacteria. The sticky peptide oligomerizes, binds to the surface of these microbes, then fibrillizes to ensnare them in sticky tendrils until other defense mechanisms move in for the kill. Featured in The New York Times, the findings do not directly address the hypothesis that microbes might precipitate AD; rather, they assign Aβ a physiological role. However, they do hint that microbes might seed plaques, which could indirectly explain why different pathogens have been tied to AD.
“This is a superb piece of work,” wrote Guillaume van Niel, Institut Curie, Paris, to Alzforum. “One of the most famous pathological amyloids may finally belong to the emerging class of functional amyloids.” He added that this paper challenges the notion that amyloidogenesis results from misprocessing or mistrafficking, and instead hints that it may be tightly regulated and induced by microbial infection.
A Sticky Web. Aβ oligomers (green, left) bind to yeast to prevent them from attaching to host cells. Those oligomers elongate into fibrils (right) and trap the yeast in sticky clumps. [Courtesy of Science Translational Medicine/AAAS.]
Moir and Tanzi previously reported that synthetic Aβ inhibited the growth of eight pathogens in culture, including the yeast Candida albicans, and the bacteria Escherichia coli and Staphylococcus aureus (Soscia et al., 2010). It performed as well as or better than LL-37, a human antimicrobial peptide (AMP). AMPs are 15 to 20 amino acid peptides that act in a variety of ways throughout the body to combat fungi, bacteria, and viruses as part of the innate immune system. LL-37 typifies AMPs that oligomerize and bind to the surface of microbes, preventing their attachment to host cells, then fibrillizing around them until immune cells kill them off. The authors set out to determine if Aβ oligomerization and fibrillization—usually thought of as pathological—actually served functional roles akin to LL-37.
Co-first authors Deepak Kumar, Se Hoon Choi, and Kevin Washicosky first tested whether Aβ helped three different organisms fend off pathogens. Alzforum covered these results when they were presented at the Zilkha conference last month (May 2016 conference news). In a nutshell, overexpressing Aβ doubled survival of human brain neuroglioma (H4) cells and C. elegans in the face of a Candida infection. Likewise, 5xFAD mice, which overexpress Aβ, survived infection with Salmonella for up to 96 hours, compared to 60-72 hours for wild-type and 54 hours for APP knockouts.
Does Aβ work like LL-37? The scientists compared control H4 cells to H4 cells overexpressing Aβ42 or Aβ40. The Aβ cells bound fewer yeast cells, suggesting that the peptide somehow interferes with yeast attachment, which is required for infection.
Kumar and colleagues next looked to see if Aβ binds the microbes. LL-37 attaches to microbial surface sugars by way of a heparin-binding motif. This six-amino-acid sequence alternates hydrophobic with positively charged amino acids and remains hidden until LL-37 oligomerizes. Aβ also sports a heparin-binding domain between residues 12-17. A binding immunoassay revealed that Aβ bound to Candida cell walls, but only in oligomeric form. However, it failed to bind if incubated with soluble mannan and glucan, two sugars yeast release to gum up the heparin-binding domain on AMPs.
After binding to a microbe’s surface, AMPs such as LL-37 fibrillize to form meshes that ensnare microbes. Can Aβ fibrillization do that, too? The researchers incubated yeast with control H4 cells or cells that overexpressed Aβ. Scanning electron microscope images of the culture medium revealed fibrous material around the yeast in the H4-Aβ42 culture (see image at right). Transmission electron microscopy revealed Aβ-fibrils stuck to the yeast surface.
The data suggest that in cell culture, overexpressing Aβ helps cells resist microbes via a mechanism similar to that of LL-37. To test whether Aβ works the same way in animals, the researchers infected Aβ-overexpressing C. elegans with yeast. In just two hours, the surface of yeast in the worm’s gut tested positive for Aβ by immunogold labelling. Later, clumps of yeast stained positive for thioflavin S, implying Aβ had formed plaques around them. In four-week-old 5xFAD mice, the authors saw something similar. At this age, these transgenic mice typically have no plaques but, two days after infection with Salmonella, Aβ had deposited throughout their brain. The bacteria and plaques were in the same place; indeed transmission electron microscopy revealed the microbes embedded inside plaques (see video below).
Plaques Trap Bacteria. Confocal fluorescence microscopy shows Salmonella (green) sheathed in b-amyloid (red) in 5XFAD mouse brain. [Courtesy of Science Translational Medicine/AAAS.]
What happens to the microbes inside these plaques? Moir said it appears these extracellular traps generate hydrogen peroxide, which both kills the microbe and cross-links the Aβ fibrils, making them resistant to proteases. That’s good for sealing in the bug, but makes plaques hard to clear, he said.
The data support the idea that Aβ functions as an AMP, and counter the prevailing view that it is intrinsically pathological, Moir said. “Its activities start to make more sense when you view Aβ as an AMP,” he told Alzforum, “It’s provocative to see this peptide so quickly form amyloids that are protective.” Moir noted that Aβ has been conserved in vertebrates for 400 million years, suggesting it has a function.
He believes his findings explain why many microbes have been tied to Alzheimer’s disease. “This is a general response,” said Moir. “Aβ will do the same thing with whatever you throw at it.” Of course, Aβ itself is known to be toxic to cells, especially neurons, suppressing synaptic activity and causing cell death. It could be that this innate immune process becomes dysregulated in Alzheimer’s disease and becomes pathological, said Moir.
Moir emphasized that the study does not claim infection causes Alzheimer’s disease. It only assigns Aβ a function. However, the microbial hypothesis deserves to be looked at more seriously, he said. He plans to systematically characterize microbes lurking in the brains of AD patients, and examine other amyloidogenic proteins implicated in disease—such as amylin in diabetes—to test whether they function as AMPs.
“Moir and colleagues did a marvelous job using these different models to demonstrate that Aβ oligomers and fibrils protect against infection,” said Brian Balin, Philadelphia College of Osteopathic Medicine. While this mechanism may help neutralize invaders that remain outside of cells, pathogens that enter cells—such as viruses—may trigger β-amyloid production without being killed, hiding safe inside their hosts, he noted. They may persist in the brain and keep generating an amyloid response, Balin said. This could explain the links between intracellular pathogens such as herpes and chlamydia and AD, which have been proposed by Balin and others, (Wozniak et al., 2009; Balin et al., 2008).
“It’s a provocative hypothesis that addresses the long-standing question of why Aβ aggregates,” said Douglas Fowler, University of Washington, Seattle, who called the idea that these aggregates could be functional compelling and interesting. “If it indeed turns out to be true, then they will have resolved one of the fundamental mysteries of how Aβ aggregation relates to its native function in the brain and possibly to Alzheimer’s disease,” he said.
Other scientists remain skeptical. For example, Bruce Kagan at the University of California, Los Angles, cautioned that this mechanism has still only been examined in animal models. Few of many known amyloid proteins have a known antimicrobial function, Kagan noted, though he and colleagues reported that the amyloid-forming serum amyloid A has potent antimicrobial activity (Hirakura et al., 2002). For his part, Kagan believes that Aβ’s destructive effect on pathogens comes from amyloid proteins damaging membranes and killing cells due to their β-sheet structure. —Gwyneth Dickey Zakaib
Research Models Citations
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