Alzheimer’s disease (AD) is characterized by the formation of large, insoluble deposits, or plaques, of amyloid-β peptide (Aβ) in the brain. These plaques are generated by the aggregation of smaller, soluble clumps made up of perhaps a dozen or so Aβ monomers. Recent research indicates that it is these smaller Aβ aggregates, or oligomers, and not the larger deposits, which represent the toxic elements of AD.

A University of Washington (UW)-headed team of scientists has now developed synthetic alpha sheet structured peptides that inhibited the toxicity of these smaller Aβ aggregates in cultured neural cells, and blocked the Aβ oligomers in two different animal models of Alzheimer’s disease. Reporting on their studies in the Proceedings of the National Academy of Sciences, the team suggests that the synthetic alpha sheets could form the basis of future treatments that clear toxic oligomers in humans.

“This is about targeting a specific structure of amyloid beta formed by the toxic oligomers,” said Valerie Daggett PhD, a UW professor of bioengineering and faculty member at the UW Molecular Engineering & Sciences Institute. “What we’ve shown here is that we can design and build synthetic alpha sheets with complementary structures to inhibit aggregation and toxicity of amyloid beta, while leaving the biologically active monomers intact.” Daggett is corresponding author of the team’s paper, which is titled, “α-sheet secondary structure in amyloid β-peptide drives aggregation and toxicity in Alzheimer’s disease.”

The mechanism by which Aβ peptide aggregation plays a causal role in AD has “eluded scientists for over 50 years,” and new technologies and discoveries only seem to confound our understanding of the disease, the authors wrote. Monomers of Aβ do have important biological functions, and are linked with processes including memory, learning, and neuroprotection. Recent research suggests that small, soluble aggregations of Aβ oligomers represent the main toxic agents in AD, while the larger fibrils and plaques that form from these oligomers are relatively benign. “Amyloid beta definitely plays a lead role in Alzheimer’s disease, but while historically attention has been on the plaques, more and more research instead indicates that amyloid beta oligomers are the toxic agents that disrupt neurons,” said Daggett.

The final structure of any protein depends on how it folds and is held into its 3D shape. One formation, known as the alpha sheet, is a nonstandard structure, which Daggett’s team had discovered using computational methods. The researchers’ previous work linked alpha sheet formation with Aβ aggregation, suggesting that the formation of alpha sheets occurs as a result of incorrect protein folding, which leads to aberrant protein interactions that disrupt normal cellular function, and can then ultimately cause protein-folding diseases such as AD.

The UW team’s latest studies using conventional and novel spectroscopic techniques indicated that Aβ oligomers form an alpha sheet structure as they form longer fibrils and plaques. They observed in human neural cell lines the different stages of plaque formation, from the initial clustering of Aβ monomers, to 12-protein oligomers, fibrils, and plaques. Tests in these cell models also demonstrated that the oligomer stages were the most toxic to neurons.

The researchers then constructed their own, synthetic alpha sheet peptides, each comprising just 23 amino acids. The synthetic peptides folded into nontoxic, hairpin-like structures. “Notably, the peptides alone were not toxic although they too contain α-sheet structure, but by design they remain monomeric to avoid toxicity,” the authors wrote. In vitro tests in neural cell cultures showed that the synthetic alpha sheets bound to toxic oligomeric Aβ tightly and blocked regions of the oligomers that are involved in clump formation, which effectively prevented the formation of larger Aβ aggregates. “De novo-designed α-sheet peptides specifically and tightly bind the toxic oligomers over monomeric and fibrillar forms of Aβ, leading to inhibition of aggregation in vitro and neurotoxicity in neuroblastoma cells,” the researchers stated.

Ball-and-stick model of the structure of AP407, one of the synthetic alpha sheet peptides designed by the research team to inhibit toxic oligomers of amyloid beta. [Shea et al., PNAS, 2019]
Tests on samples of brain tissue from a mouse model of AD showed that treatment using the synthetic alpha sheet peptides was associated with up to an 82% reduction in Aβ oligomer levels. In live mice treatment using the synthetic alpha sheets resulted in a 40% drop in Aβ oligomer levels within 24 hours. The researchers also carried out tests in a well-studied Caenorhabditis elegans worm model for AD, which is used extensively to test compounds against Aβ toxicity. Their experiments first showed that treating the C. elegans AD model with the synthetic alpha sheets delayed the onset of Aβ-induced paralysis. Separate tests also showed that while C. elegans worms treated using Aβ-expressing bacteria developed intestinal damage, pretreatment using the synthetic alpha sheets protected the animals from intestinal damage after the bacteria were administered.

Microscopy images of the anterior intestine of four different C. elegans worms. The gastrointestinal tracts of these worms glow green because they have been fed fluorescent bacteria. Top left: A worm fed normal bacteria. Top right: A worm fed bacteria that contain amyloid beta, which has caused intestinal damage to the worms (white arrows). Bottom left: Intestinal damage is still present if the bacteria contain both amyloid beta and a coil protein that has no effect on aggregation. Bottom right: Intestinal damage is reduced in worms fed bacteria that express amyloid beta and AP5, a synthetic alpha sheet that blocks amyloid beta oligomers. Scale bar is 10 micrometers. [Shea et al., PNAS, 2019]
“Here we provide evidence that Aβ soluble oligomers adopt a nonstandard secondary structure: α-sheet,” the investigators wrote. “This structure forms early in aggregation and is strongly correlated with toxicity … This work challenges the prevailing dogma and sheds light on potential new approaches to the problem … These findings open the possibility of novel therapeutic and diagnostic agents for AD and other amyloid diseases.”

For their reported studies the team created a laboratory assay that uses a synthetic alpha sheet to measure levels of Aβ oligomers, and which could lead to the development of clinical tests that can measure toxic oligomers before symptoms of AD develop. “What we’re really after are potential therapeutics against amyloid beta and diagnostic measures to detect toxic oligomers in people,” said Daggett. “Those are the next steps.”

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