Researchers watch amyloid plaques form

Researchers at the University of Toronto Scarborough (UTSC) and Osaka University applied a new approach to take a close look at amyloid plaque formation, a process that plays important roles in Alzheimer's disease. The technique would greatly aid the development and screening for novel therapeutics that can manipulate the formation of the toxic amyloid aggregates.

Anthony Veloso, Prof. Kagan Kerman's PhD student in Chemistry, used a laser to trap amyloid-beta peptides and examined them under a fluorescence microscope as they aggregate, giving them an exceptionally detailed view of the process. The work appears on the cover of the current issue of Analyst, a journal of the Royal Society of Chemistry.

"This technique could accelerate the drug discovery process. It gives us a new way to examine the early phase of plaque formation, when the most toxic species of oligomers are formed," says Prof. Kerman, a faculty with the Department of Physical and Environmental Sciences at UTSC and the corresponding author on the paper.

Amyloid plaques are protein deposits that form around neurons and interfere with their function. The major constituent of these deposits are amyloid-beta, a peptide that clumps together to form harmful plaques in Alzheimer's patients, but is otherwise harmless in normal individuals.

To get a look at the early stages of the process, the Canadian researchers and their Japanese collaborators used a technique called optical trapping. A laser is focused into a very thin beam and aimed at solution containing amyloid-beta particles. The beam creates a small magnetic field, which attracts and holds the particles in place. Amyloid aggregates stained by a dye then glows under the laser light, and the image can be captured by fluorescence microscope.

By using this technique, A. Veloso and Prof. Kerman hope to explore how the aggregates are formed, and to eventually discover the role of amyloid aggregates in Alzheimer's disease. Utilizing the versatility of this technique, Prof. Kerman's research team can extend their studies to understand aggregate formation in other neurodegenerative diseases.

The technique will also become a novel strategy to test therapeutic compounds that could halt the formation of plaques. Prof. Kerman and A. Veloso are working towards the automation of the technique, allowing for many compounds to be tested efficiently.

Provided by University of Toronto Scarborough

Molecules 'light up' Alzheimer's roots: Light-switching complex attaches itself to amyloid proteins

 A breakthrough in sensing at Rice University could make finding signs of Alzheimer's disease nearly as simple as switching on a light.

The technique reported in the Journal of the American Chemical Society should help researchers design better medications to treat the devastating disease.

The lab of Rice bioengineer Angel Martí is testing metallic molecules that naturally attach themselves to a collection of beta amyloid proteins called fibrils, which form plaques in the brains of Alzheimer's sufferers. When the molecules, complexes of dipyridophenazine ruthenium, latch onto amyloid fibrils, their photoluminescence increases 50-fold.

The large increase in fluorescence may be an alternative to molecules currently used to study amyloid fibrils, which researchers believe form when misfolded proteins begin to aggregate. Researchers use changes in fluorescence to characterize the protein transition from disordered monomers to aggregated structures.

Nathan Cook, a former Houston high school teacher and now a Rice graduate student and lead author of the new paper, began studying beta amyloids when he joined Martí's lab after taking a Nanotechnology for Teachers course taught by Rice Dean of Undergraduates and Professor of Chemistry John Hutchinson. Cook's goal was to find a way to dissolve amyloid fibrils in Alzheimer's patients.

But the Colorado native's research led him down a different path when he realized the ruthenium complexes, the subject of much study in Martí's group, had a distinctive ability to luminesce when combined in a solution with amyloid fibrils.

Such fibrils are simple to make in the lab, he said. Molecules of beta amyloid naturally aggregate in a solution, as they appear to do in the brain. Ruthenium-based molecules added to the amyloid monomers do not fluoresce, Cook said. But once the amyloids begin to aggregate into fibrils that resemble "microscopic strands of spaghetti," hydrophobic parts of the metal complex are naturally drawn to them. "The microenvironment around the aggregated peptide changes and flips the switch" that allows the metallic complexes to light up when excited by a spectroscope, he said.

Thioflavin T (ThT) dyes are the standard sensors for detecting amyloid fibrils and work much the same way, Marti said. But ThT has a disadvantage because it fluoresces when excited at 440 nanometers and emits light at 480 nanometers -- a 40-nanometer window.

That gap between excitation and emission wavelengths is known as the Stokes shift. "In the case of our metal complexes, the Stokes is 180 nanometers," said Martí, an assistant professor of chemistry and bioengineering. "We excite at 440 and detect in almost the near-infrared range, at 620 nanometers.

"That's an advantage when we want to screen drugs to retard the growth of amyloid fibrils," he said. "Some of these drugs are also fluorescent and can obscure the fluorescence of ThT, making assays unreliable."

Cook also exploited the metallic's long-lived fluorescence by "time gating" spectroscopic assays. "We specifically took the values only from 300 to 700 nanoseconds after excitation," he said. "At that point, all of the fluorescent media have pretty much disappeared, except for ours. The exciting part of this experiment is that traditional probes primarily measure fluorescence in two dimensions: intensity and wavelength. We have demonstrated that we can add a third dimension -- time -- to enhance the resolution of a fluorescent assay."

The researchers said their complexes could be fitting partners in a new technique called fluorescence lifetime imaging microscopy, which discriminates microenvironments based on the length of a particle's fluorescence rather than its wavelength.

Cook's goal remains the same: to treat Alzheimer's -- and possibly such other diseases as Parkinson's -- through the technique. He sees a path forward that may combine the ruthenium complex's ability to target fibrils and other molecules' potential to dissolve them in the brain.

"That's something we are actively trying to target," Martí said.

More information: http://pubs.acs.or … 21/ja204656r

Provided by Rice University (news : web)

Simulating amyloid formation

Many neurodegenerative diseases are characterized by proteins that assume an abnormal configuration, which leads to their aggregation and deposition in and around rve cells, causing cell death. This process, called amyloid formation, is a common pathological feature in diseases such as Alzheimer’s, Parkinson’s and prion diseases, as well as type II diabetes. Charlotte Hauser and co-workers from the A*STAR Institute of Bioengineering and Nanotechnology and Institute of High Performance Computing along with colleagues in Europe have now designed a class of ultrasmall peptides that simulate the self-assembly of abnormally folded proteins in such neurodegenerative conditions.

Hauser and her co-workers designed ultrasmall peptides consisting of three to six amino acid residues, each containing a characteristic motif—a ‘tail’ of uncharged residues with decreasing affinity to water capped by a polar ‘head’ residue. These peptides spontaneously self-assembled in water to form fibril structures (pictured) resembling those that make up the amyloid-ß plaques found in the brains of Alzheimer’s patients.

The researchers hypothesize that fiber assembly is a complex stepwise mechanism involving at least three distinct stages. Individual peptide molecules first bond to each other in an anti-parallel arrangement to form dimers. The pairs then line up to form single ?-helical fibers as intermediate structures, which continue to assemble and then condense into fibrous scaffolds in the form of solid hydrogels.

The team further examined the driving forces for self-assembly and found that gel formation was critically dependent on the length of the tail and the polar nature of the head. Peptides containing six amino acid residues formed gels more readily than the others, and the strongest gels were formed by peptides containing an acidic head residue. A minimum peptide concentration was required for fiber formation, and increasing the temperature was found to accelerate the self-assembly process.

Investigation into the assembly process and experimental results were verified by computer simulations. This helped the research team confirm that the formation of peptide pairs precedes fiber formation, suggesting that the peptides have a strong tendency to aggregate because the sheet-like structures have a lower free energy state than individual fibers, and are therefore more stable.

“Understanding the driving forces that enable these ultrasmall peptides to stably self-assemble into macromolecular structures will shed light on aggregate formation in amyloidogenesis,” says Hauser. “This would facilitate the design of new therapeutics to prevent and control plaque formation in neurodegenerative disorders and a wide range of other debilitating diseases.”

More information: Hauser, C. A. E. et al. Natural tri- to hexapeptides self-assemble in water to amyloid ß-type fiber aggregates by unexpected ?-helical intermediate structures. Proceedings of the National Academy of Sciences 108, 1361–1366 (2011). http://dx.doi.org/ … s.1014796108

Abstract
Many fatal neurodegenerative diseases such as Alzheimer’s, Parkinson, the prion-related diseases, and non-neurodegenerative disorders such as type II diabetes are characterized by abnormal amyloid fiber aggregates, suggesting a common mechanism of pathogenesis. We have discovered that a class of systematically designed natural tri- to hexapeptides with a characteristic sequential motif can simulate the process of fiber assembly and further condensation to amyloid fibrils, probably via unexpected dimeric ?-helical intermediate structures. The characteristic sequence motif of the novel peptide class consists of an aliphatic amino acid tail of decreasing hydrophobicity capped by a polar head. To our knowledge, the investigated aliphatic tripeptides are the shortest ever reported naturally occurring amino acid sequence that can adopt ?-helical structure and promote amyloid formation. We propose the stepwise assembly process to be associated with characteristic conformational changes from random coil to ?-helical intermediates terminating in cross-ß peptide structures. Circular dichroism and X-ray fiber diffraction analyses confirmed the concentration-dependent conformational changes of the peptides in water. Molecular dynamics simulating peptide behavior in water revealed monomer antiparallel pairing to dimer structures by complementary structural alignment that further aggregated and stably condensed into coiled fibers. The ultrasmall size and the dynamic facile assembly process make this novel peptide class an excellent model system for studying the mechanism of amyloidogenesis, its evolution and pathogenicity. The ability to modify the properties of the assembled structures under defined conditions will shed light on strategies to manipulate the pathogenic amyloid aggregates in order to prevent or control aggregate formation.

Provided by Agency for Science, Technology and Research (A*STAR)