New headway in battle against neurodegenerative diseases

Conditions which may accelerate the spread of Parkinson’s disease, and a potential means of enhancing naturally-occurring defences against neurodegenerative disorders, have been identified in two new studies.

Two significant breakthroughs which could inform future treatments for neurodegenerative diseases such as Alzheimer’s and Parkinson’s, have been announced by scientists.

The research, published in two separate studies this week, advances understanding of the early development of such disorders and how they might be prevented – in particular by identifying the biological areas and processes that could be pinpointed by future drugs.

Both sets of results have emerged from collaborations between the research groups led by Chris Dobson, Tuomas Knowles and Michele Vendruscolo at the University of Cambridge, who focus on understanding protein “misfolding” diseases. These include Alzheimer’s and Parkinson’s diseases, as well as numerous others.

The first study provides evidence that the early spread of the protein aggregates associated with Parkinson’s appears to happen at an accelerated rate in mildly acidic conditions. This suggests that particular compartments within brain cells, which are slightly more acidic than others, may turn out to be appropriate targets for future treatments fighting the disease.

Meanwhile, researchers behind the second study appear to have identified a way in which the effectiveness of so-called molecular “chaperones”, responsible for limiting the damage caused by misfolded proteins, can be significantly enhanced.

The papers appear in the latest issue of Proceedings of the National Academy of Sciences of the USA.

As the term suggests, protein misfolding diseases stem from the fact that proteins, which need to fold into a particular shape to carry out their assigned function in the body, can sometimes misfold. In certain cases these misfolded proteins then clump together into fibre-like threads, called amyloid fibrils, potentially becoming toxic to other cells.

How this formation begins at a molecular level is still not completely understood, but comprehending the process will be fundamental to the development of future therapies and is the subject of extensive current research.

The first of the new studies builds on research published in 2013, which showed that in Alzheimer’s sufferers, the initial “nucleation” between proteins, which leads to amyloid formation, is followed by an amplification process called secondary nucleation. In these secondary events, the existing amyloid structures facilitate the formation of new aggregates, leading to their exponential increase. This process is likely to be at the heart of the development and spread of the disease in affected brains.

Using the same techniques, the researchers behind the latest study identified a similar process that is relevant in the early stage development of Parkinson’s Disease. Their work focused on a protein called α-synuclein, which is associated with the disorder, and simulated different conditions in which this protein might misfold and form clumps.

As with the previous study on Alzheimer’s, the research identified that Parkinson’s could spread through a series of secondary nucleation events. In addition, however, it showed that in the case of α-synuclein, this happens at a highly accelerated rate only in solutions which are mildly acidic, with a pH below 5.8. The finding is important because certain sub-compartments within cells are more acidic than others, meaning that these may be particularly productive areas for future treatments to target.

Dr Tuomas Knowles, from the Department of Chemistry and a Fellow of St John’s College, Cambridge, said: “This tells us much more about the molecular mechanisms underlying protein aggregation in Parkinson’s and suggests that mildly acidic microenvironments within cells may enhance that process by several orders of magnitude. Not every sub-cellular compartment offers these conditions, so it takes us much closer to understanding how the disease might spread.”

The second study meanwhile suggests a potential route to improving the effectiveness of a particular molecular “chaperone” – a loose classification for proteins which assist in the folding of others, thereby preventing them from causing damage when they misfold.

The researchers focused on a chaperone called α2-macroglobulin (α2M), which is found outside cells themselves. This is important because neurodegenerative diseases often stem from a process which begins with extracellular misfolding. The α2M was tested on a substrate of the amyloid-beta peptide associated with Alzheimer’s Disease.

Typically, the potency of α2M is limited. The new study, however, found that when it comes into contact with the oxidant hypochlorite – the same chemical found in household bleach, which also naturally occurs in our immune systems – its structure is modified in a manner that makes it into a much more dynamic defence.

In their report, the researchers suggest that this increased effectiveness stems from the fact that α2M, which is usually found in a four-part, “tetrameric” form, breaks down into “dimeric”, two-part forms when it comes into contact with hypochlorite.

The chaperone usually plays its role by preventing a misfolded protein from interacting with the membranes that surround and protect cells. Once in its dimeric form, however, receptor binding sites within the α2M are exposed, leading to specific interactions with receptors on the cell itself. If the α2M has already interacted with misfolded proteins, this connection triggers the cell to break the potentially harmful protein down.

“It’s almost like a warning flag for the cell, telling it that something is wrong,” Dr Janet Kumita, from the Department of Chemistry, explained. “It triggers the cell to react in a way that subjects the cargo of misfolded protein to a degradation pathway.”

“Increasing its potency in this way is an exciting prospect. If we could find a way of developing a drug that introduces the same structural alterations, we would have a therapeutic intervention capable of increasing this protective activity in patients with Alzheimer’s Disease.”

Professor Christopher Dobson, from the University’s Department of Chemistry and Master of St John’s College, said: “These studies add very substantially to our detailed understanding of the molecular origins of neurodegenerative diseases, which are now becoming one of the greatest threats to healthcare in the modern world.”

“We are beginning to understand exactly how a single, aberrant event can lead to the proliferation and spreading of toxic species throughout the brain, and the manner in which our sophisticated defence mechanisms do their best to suppress such phenomena. It will undoubtedly provide vital clues to the development in due course of new and effective drugs to combat these debilitating and increasingly common disorders.”

Toward unraveling the Alzheimer’s mystery

Getting to the bottom of Alzheimer’s disease has been a rapidly evolving pursuit with many twists, turns and controversies. In the latest crook in the research road, scientists have found a new insight into the interaction between proteins associated with the disease. The report, which appears in the journal ACS Chemical Neuroscience, could have important implications for developing novel treatments.

Witold K. Surewicz, Krzysztof Nieznanski and colleagues explain that for years, research has suggested a link between protein clumps, known as amyloid-beta plaques, in the brain and the development of Alzheimer’s, a devastating condition expected to affect more than 10 million Americans by 2050. But how they inflict their characteristic damage to nerve cells and memory is not fully understood. Recent studies have found that a so-called prion protein binds strongly to small aggregates of amyloid-beta peptides. But the details of how this attachment might contribute to disease — and approaches to treat it — are still up for debate. To resolve at least part of this controversy, Surewicz’s team decided to take a closer look.

Contrary to previous studies, they found that the prion protein also attaches to large fibrillar clumps of amyloid-beta and do not break them down into smaller, more harmful pieces, as once thought. This finding bodes well for researchers investigating a novel approach to treating Alzheimer’s — using prion-protein-based compounds to stop these smaller, toxic amyloid-beta pieces from forming, the authors conclude.

Scientists ID compounds that target amyloid fibrils in Alzheimer’s, other brain diseases

UCLA chemists and molecular biologists have for the first time used a “structure-based” approach to drug design to identify compounds with the potential to delay or treat Alzheimer’s disease, and possibly Parkinson’s, Lou Gehrig’s disease and other degenerative disorders.

All of these diseases are marked by harmful, elongated, rope-like structures known as amyloid fibrils, linked protein molecules that form in the brains of patients.

Structure-based drug design, in which the physical structure of a targeted protein is used to help identify compounds that will interact with it, has already been used to generate therapeutic agents for a number of infectious and metabolic diseases.

The UCLA researchers, led by David Eisenberg, director of the UCLA–Department of Energy Institute of Genomics and Proteomics and a Howard Hughes Medical Institute investigator, report the first application of this technique in the search for molecular compounds that bind to and inhibit the activity of the amyloid-beta protein responsible for forming dangerous plaques in the brain of patients with Alzheimer’s and other degenerative diseases.

In addition to Eisenberg, who is also a professor of chemistry, biochemistry and biological chemistry and a member of UCLA’s California NanoSystems Institute, the team included lead author Lin Jiang, a UCLA postdoctoral scholar in Eisenberg’s laboratory and Howard Hughes Medical Institute researcher, and other UCLA faculty.

The research was published July 16 in eLife, a new open-access science journal backed by the Howard Hughes Medical Institute, the Max Planck Society and the Wellcome Trust.

A number of non-structure-based screening attempts have been made to identify natural and synthetic compounds that might prevent the aggregation and toxicity of amyloid fibrils. Such studies have revealed that polyphenols, naturally occurring compounds found in green tea and in the spice turmeric, can inhibit the formation of amyloid fibrils. In addition, several dyes have been found to reduce amyloid’s toxic effects, although significant side effects prevent them from being used as drugs.

Armed with a precise knowledge of the atomic structure of the amyloid-beta protein, Jiang, Eisenberg and colleagues conducted a computational screening of 18,000 compounds in search of those most likely to bind tightly and effectively to the protein.

Those compounds that showed the strongest potential for binding were then tested for their efficacy in blocking the aggregation of amyloid-beta and for their ability to protect mammalian cells grown in culture from the protein’s toxic effects, which in the past has proved very difficult. Ultimately, the researchers identified eight compounds and three compound derivatives that had a significant effect.

While these compounds did not reduce the amount of protein aggregates, they were found to reduce the protein’s toxicity and to increase the stability of amyloid fibrils — a finding that lends further evidence to the theory that smaller assemblies of amyloid-beta known as oligomers, and not the fibrils themselves, are the toxic agents responsible for Alzheimer’s symptoms.

The researchers hypothesize that by binding snugly to the protein, the compounds they identified may be preventing these smaller oligomers from breaking free of the amyloid-beta fibrils, thus keeping toxicity in check.

An estimated 5 million patients in the U.S. suffer from Alzheimer’s disease, the most common form of dementia. Alzheimer’s health care costs in have been estimated at $178 billion per year, including the value of unpaid care for patients provided by nearly 10 million family members and friends.

In addition to uncovering compounds with therapeutic potential for Alzheimer’s disease, this research presents a new approach for identifying proteins that bind to amyloid fibrils — an approach that could have broad applications for treating many diseases.

New clues illuminate Alzheimer’s roots

Scientists at Rice University and the University of Miami have figured out how synthetic molecules designed at Rice latch onto the amyloid peptide fibrils thought to be responsible for Alzheimer’s disease. Their discovery could point the way toward therapies to halt or even reverse the insidious disease.

The metallic dipyridophenazine ruthenium molecules strongly bind to pockets created when fibrils form from misfolded proteins that cells fail to destroy. When excited under a spectroscope, the molecules luminesce, which indicates the presence of the fibrils. That much was known by Rice researchers, but until now the process was a mystery.

By combining their talents in biophysics (at Rice) and computer simulation (at Miami), researchers pinpointed four such pockets along the fibril where the hydrophobic (water-averse) molecules can bind. They believe their work will help chemists design molecules to keep the fibrils from forming the plaques found in Alzheimer’s patients.

The teams led by Rice chemist Angel Martí and Miami chemist Rajeev Prabhakar reported their results in the Journal of the American Chemical Society this month.

Two years ago, Martí and Nathan Cook, a graduate student in his lab and lead author of the new paper, combined ruthenium complexes with solutions containing the spaghetti-like amyloid fibrils. The complexes don’t luminesce by themselves, but when they link to an amyloid fibril, they can be triggered by light at one wavelength to glow at another; this helps the researchers “see” the fibrils.

This ability to track amyloids was a great step forward, but left open the question of why the complexes latched onto the fibrils at all, Cook said.

“We had no way to figure it out because our experimental techniques can’t identify binding sites,” he said. “The standard (used to analyze proteins) is to crystallize your material and use X-rays to determine where everything is positioned. The problem with amyloid beta is the fibrils are not uniform, and you can’t crystallize them. All you would get is an amorphous lump.”

But a door opened when Prabhakar, a theoretical and computational chemist who specializes in amyloids, contacted Martí and suggested a collaboration. “We both knew the other was working with amyloid betas,” Martí said. “We were able to figure out how many amyloid beta monomers (molecules that can bind with each other) had to come together to form fibrils, while he modeled the interactions. When we brought all the data together, we had a perfect match.”

“Basically, we learned from the model that we need two monomers to form a binding site,” Marti said. “The cleft where the ruthenium complex binds is completely hydrophobic, the same as the complex. Neither wants to be exposed to water, so when they find each other, they don’t have a choice but to come together. It turns out that’s exactly what needs to happen to turn on the photoluminescent response of the compound.”

Martí said testing various concentrations of monomers with ruthenium complexes helped them determine that a little more than two monomers, on average, was sufficient to get the “light switch” effect. Prabhakar’s analysis found four specific locations along the aggregating monomers where the ruthenium complexes could bind: two at the ends where the monomers tend to bind to each other, and two in the middle.

“It was a complicated system to model and we tried hard, using a variety of computational techniques,” Prabhakar said. “In the end, we were amazed to find our results in perfect agreement with the experiments performed in the Martí lab.”

The researchers called the end locations “A and B,” and the middle clefts “C and D.” The hydrophobic A and B sites exist only at the edges of the fibrils, which limits their exposure to the complexes, Martí said. “But there are lots of C and D sites,” he said. “That explains why the ruthenium complexes don’t inhibit the aggregation of fibrils. It seems the system prefers to bind another monomer, rather than a ruthenium complex, at the ends.

“But now that we understand the mechanism, we can design more hydrophobic complexes that could bind strongly to the ends and prevent further elongation of the fibril,” he said.

“There’s a whole variety of ways to tweak this that could potentially disrupt a binding pocket,” Cook said.

More challenges lie beyond the new discovery, he said. New research indicates toxic oligomers may be catalyzed by the formation of amyloid fibrils. “We might be able to prevent the formation of these oligomeric species by binding ruthenium complexes to the surface, which would completely change the surface chemistry of the fibrils,” Martí said. “These are the things we are really interested in doing right now.”

Yeast Protein Breaks up Amyloid Fibrils and Disordered Protein Clumps In Different Ways

Several fatal brain disorders, including Parkinson’s disease, are connected by the misfolding of specific proteins into disordered clumps and stable, insoluble fibrils called amyloid. Amyloid fibrils are hard to break up due to their stable, ordered structure. For example, a-synuclein forms amyloid fibrils that accumulate in Lewy Bodies in Parkinson’s disease. By contrast, protein clumps that accumulate in response to environmental stress, such as heat shock, possess a less stable, disordered architecture.

Hsp104, an enzyme from yeast, breaks up both amyloid fibrils and disordered clumps. In the most recent issue of Cell, James Shorter, PhD, assistant professor of Biochemistry and Biophysics, and colleagues from the Perelman School of Medicine, University of Pennsylvania, show that Hsp104 switches mechanism to break up amyloid versus disordered clumps. For stable amyloid-type structures, Hsp104 needs all six of its subunits, which together make a hexamer, to pull the clumps apart. By contrast, for the more amorphous, non-amyloid clumps, Hsp104 required only one of its six subunits.