Growth Rate of Disease-Causing Amyloid Fibers Now Measurable

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Scientists from the U.K.’s University of Bath and the ISIS Neutron and Muon Source report the invention of a method that can reliably measure the speed of amyloid fibril growth. The formation of amyloid fibers has been linked to a number of serious human diseases, including Alzheimer’s, Parkinson’s, and type 2 diabetes.

The team published its study “Elongation rate and average length of amyloid fibrils in solution using isotope-labelled small-angle neutron scattering,” in RSC Chemical Biology.

“We demonstrate a solution method that allows both elongation rate and average fibril length of assembling amyloid fibrils to be estimated. The approach involves acquisition of real-time neutron scattering data during the initial stages of seeded growth, using contrast matched buffer to make the seeds effectively invisible to neutrons. As deuterated monomers add on to the seeds, the labelled growing ends give rise to scattering patterns that we model as cylinders whose increase in length with time gives an elongation rate,” write the investigators.

“In addition, the absolute intensity of the signal can be used to determine the number of growing ends per unit volume, which in turn provides an estimate of seed length. The number of ends did not change significantly during elongation, demonstrating that any spontaneous or secondary nucleation was not significant compared with growth on the ends of pre-existing fibrils, and in addition providing a method of internal validation for the technique.

“Our experiments on initial growth of alpha synuclein fibrils using 1.2 mg/ml seeds in 2.5 mg/ml deuterated monomer at room temperature gave an elongation rate of 6.3 ± 0.5 Å min -1, and an average seed length estimate of 4.2 ± 1.3 µm.”

“This is an important breakthrough, as information on fiber growth is key to understanding the diseases associated with amyloid fibrils,” said Adam Squires, PhD, from the department of chemistry at Bath, and study co-author. “Knowing what makes these fibers grow faster or slower, or whether they break and what makes them break. In other words, understanding these fibers at a molecular level could eventually have implications for researchers looking for treatments for these serious diseases.

“This new technique will also help scientists investigating non-medical roles of protein folding and self-assembly, as for instance, in biological processes such as inheritance in yeast, or for research into new nanomaterials.”

Most experimental techniques for measuring fibril growth in solution only measure how fast proteins transform into fibril material overall, not how long each fibril is or how fast it is growing, according to the Bath group. Other techniques measure just one fibril attached to a surface such as glass or mica. These conditions do not reflect the real biological process, which occurs in solution.

Researchers for the new study used Small Angle Neutron Scattering (SANS) to study the growth rate and length of amyloid fibrils as they assembled in solution. By using the unique ways neutrons interact with hydrogen and its isotope deuterium, the researchers were able to use “contrast matching” to make all of the fibrils invisible to neutrons apart from the growing tips. Using the SANS2D instrument at the ISIS neutron facility, they watched these tips become longer in real time. This gave a direct measurement of the growth rate, which had never been done before.

The results of growth rate from this study align with values estimated from other methods, indicating that SANS is a suitable tool for measuring amyloid fibril growth.

The technique also allowed the researchers to measure the number of fibril ends present in a given sample. This information told them how many separate fibers were growing, and the length of each one. The fragility of fibrils from different proteins, and how often they break into shorter fragments exposing more growing ends, is a key part of the puzzle to understand fibril disease propagation.

Lead researcher Ben Eves, PhD, carried out the experiments at Bath as part of his ISIS Facility Development studentship.

“I’m thrilled with the success of this method,” he said. “Developing this technique was a truly amazing experience. Understanding the growth of amyloid fibrils is fundamental to understanding their pathogenic, biological and technological properties.

“In the future, I believe this technique could be used to investigate the effect of different factors that affect the growth rate of amyloid fibrils, as well as to measure the impact of therapeutic molecules (the building blocks of medicines) designed to slow down or prevent the growth of amyloid fibrils.”

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