Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
The list of neurodegenerative disorders associated with misfolded or aggregated proteins is continually growing, and along with it the attempts to understand them. The pathologies of common diseases such as Alzheimer’s disease (AD) and Parkinson’s disease have much in common with the plaques and tangles seen in the classic prion diseases—mad cow, kuru, scrapie, Creutzfeldt-Jakob and their relatives. And other maladies—from Huntington’s disease and amyotrophic lateral sclerosis (ALS, or Lou Gehrig’s disease) to type II diabetes—have been shown to involve amyloids, as well.
Studying these disorders involves a host of overlapping disciplines, each contributing its own tools and methodologies. At the same time, there are unique aspects and challenges to these investigations. Among these, perhaps paradoxically, is the need to distinguish not only the amyloid β (Aβ) aggregates seen in AD from the α-synuclein-composed Lewy bodies seen in Parkinson’s disease, for example, but also to identify different forms of Aβ from each other.
What is an amyloid?
Amyloids are hydrogen-bonded protein aggregates. They tend to be composed of (often misfolded) normal proteins or peptides that have come together to form structures with a preponderance of β-sheet motifs. There are very likely mechanisms to clear these from the cell. But under some circumstances, there is a breakdown in the regulatory pathway that leads to more and more protein monomers, ultimately forming insoluble plaques. It’s believed that the intermediate stages—soluble oligomers and fibrils—are the more biologically active forms of (at least) Aβ, says Harry LeVine III, associate professor of molecular and cellular biochemistry in the Sanders-Brown Center on Aging at the University of Kentucky College of Medicine. The plaques themselves cause problems by taking up space and gluing things together, but they are otherwise relatively inert.
In some ways, an amyloid is an amyloid, whether it’s made up of Aβ or α-synuclein. The actual sequence is hidden in the beta sheet, where half the amino acids are hidden on the other side, says Charles Glabe, professor of molecular biology and biochemistry at the University of California, Irvine. Antibodies used to detect them “see the structures on the surface of the fibril that are identical, regardless of the sequence”—common, generic epitopes found on many amyloids regardless of sequence.
“That’s why a lot of these new protein-misfolding diseases have been discovered—because the antibodies do, in fact, recognize common structures,” he explains. Collaborators using Glabe’s anti-amyloid antibodies have identified “two types of cardiomyopathy and a kidney disease, and we also discovered that pre-eclampsia is an amyloidosis.”
Polymorphic conformations
Yet even an affinity reagent that specifically recognizes an amyloid plaque may only bind a subset of aggregates made up of proteins or peptides of the same sequence. LeVine, for example, has found that the small molecule Pittsburgh Compound B (PiB) sees only about 10% to 15% of human Aβ plaques. And PiB won’t bind the same sequence expressed in other animals, including nonhuman primates, at all.
“The amyloid peptide is like an origami: It can fold up into at least five or six different fibril structures that have been published,” says Glabe. He and others hypothesize that such structural variation may lie behind the heterogeneity of the diseases themselves. Studying that heterogeneity “could hold promise in clarifying why some people can run around and have massive amount of amyloid in their brains, and they’re not demented. One possibility is that not all amyloids are the same.”
“To study this, you’re going to need antibodies that can detect these conformationally distinct variants,” notes Nicola Hodson, neuroscience content marketing executive at Abcam. Abcam and other companies have licensed and are marketing some of Glabe’s conformation-specific Aβ antibodies—and are continuing to develop others. “We also appreciate the importance of structural variation and different isoforms of proteins such as tau [a second misfolded protein associated with AD] and α-synuclein – this is something we’re working on” Hodson says.
The sequence
Of course, recognizing that something is a plaque or an insoluble fibril is one thing, but it’s still important to identify the protein(s) that make up the structure. Rodrigo Morales, assistant professor of neurology at the University of Texas Health Science Center, divides his time between AD and prion-disease research, investigating whether pathology can be exogenously introduced by injecting an animal with aggregated proteins. He generally uses a sequence-specific antibody to a denatured form of a protein, only occasionally relying on conformation-specific reagents as a validation of structure. “We don’t know statistically which conformation we are forming in our animals—this is something we plan to explore later—but right now, we are focused on the amount of protein we have in the brain,” he says.
Researchers may sometimes want to know which particular protein fragments are present. “There are other neo-epitopes that may result from cleavage of a protein to a truncated form, or otherwise cleaving from an inactive to an active form,” notes Peggy Taylor, general manager of Biolegend’s neuroscience division. Similarly, AD researchers are interested in studying the different isoforms and differentially phosphorylated isoforms of tau (τ) protein, for example. “Biolegend makes antibodies that will not only enable customers to ask is the protein present or absent, but in what form is it present? Is it phosphorylated at this epitope? Is it truncated?”
Use multiple approaches
Neurodegenerative-disorder research doesn’t exist in a vacuum. It shares techniques with a host of disciplines: electrophysiology and behavioral tests; ELISA, Western blotting and immunohistochemistry; confocal microscopy and PET scanning; and molecular biology, genetics and genomics. Researchers may precede multicolor fluorescent assays with the introduction of agonists, antagonists, activators or inhibitors involved in transit or recycling pathways. Or they may investigate how altering the immune response alters the pathology. As the number of neurodegenerative disorders linked to misfolded proteins continues to grow, the tools available to researchers continues to evolve and become more comprehensive.