Amyloid plaque in a mouse brain with a disease similar to Alzheimer’s, stained by molecules developed by researchers at LiU. The different colours correspond to different structures of amyloid beta. Photo credit: Sofie Nyström
The images remind us of galaxies or clouds of gas tinged with colour far out in the universe. But these images do not show remote objects in space: they show microscopic small lesions in the brain that have been tagged with tracer molecules developed by researchers at Linköping University, LiU.
In several diseases of the brain, long fibres of protein form, and eventually become tangled to form dense bodies known as “plaque”. In Alzheimer’s disease, which causes dementia, the plaque usually consists of faulty variants of two proteins: amyloid beta and tau. The proteins can fold in different ways to form several different 3-dimensional structures, and this can happen even though the protein molecules are built from exactly the same amino acids in the same order.Sofie Nyström, Per Hammarström, Peter Nilsson, Marcus Bäck and Hamid Shirani. Photo credit: LiU
“The structure and function of a protein are normally very closely related. One amino acid sequence gives one structure and one function. When wrongly folded proteins form, however, they have several different structures and we believe that they have different toxic functions. These wrongly folded proteins can convert other molecule to copies of themselves, and they may be very difficult for the body to break down. We believe that some structures are more harmful than others,” says Per Hammarström, professor of protein chemistry in the Department of Physics, Chemistry and Biology, IFM.
The tracer molecules developed by the research group in Linköping have now been used for the first time in investigations of human brain tissue. The results of the international collaboration, which has been led by researchers at the University of Tübingen in German, have been published in the prestigious scientific journal PNAS. The researchers have studied brain tissue from 40 people who have died with various variants of Alzheimer’s disease. The symptoms of the disease differ from one individual to another, and the researchers wanted to investigate whether the large variation is associated with different forms of erroneous folding.
The molecules that are currently used to reveal amyloid plaques in the brain stain the whole of the amyloid aggregate, and can tell us whether amyloid plaque is present or not. The molecules developed in Linköping are more sensitive than the traditional staining methods, and can give information about whether different types of aggregate are present in which the protein is folded into different structures. The researchers investigated wrongly folded proteins in the core of the plaque.
“We discovered that they differ a great deal. An individual does not have a single wrongly folded variant of amyloid beta: there are many different types,” says Per Hammarström.
It is still unclear what the significance of the differences between the folding variants is. More research will be necessary to determine whether the differences in structure are associated with other properties of the plaque and the course of the disease in the patient.
Colour-shifting chameleon molecules
The tracer molecules developed at Linköping University can emit light when light (of a different wavelength) shines on them. We say that they “fluoresce”. What makes these molecules unique, however, is that they have a flexible backbone that adapts to the structure of the protein to which they are bound. When the molecule twists, the colour of the fluorescence emitted changes. This means that the same sort of molecule will emit light of different colours when bound to different structures of amyloid beta, in the same way that a chameleon adapts its colour to the surroundings. This study is the first in which researchers have used a combination of two tracer molecules simultaneously in specimens from patients.Amyloid plaque in a mouse blood vessel with a disease similar to Alzheimer’s, stained by molecules developed by researchers at LiU. The different colours correspond to different structures of amyloid beta. Photo credit: Sofie Nyström
“The two molecules bind with different strengths to different types of amyloid aggregate. One of them emits blue light and the other one red, so it’s easy to see the difference in a microscope,” says Sofie Nyström, principal research engineer in the Department of Physics, Chemistry and Biology.
It is possible to see the different colours visually, but the researchers have carried out a detailed analysis in which the intensity of fluorescence from a sample is measured at several wavelengths. The researchers can calculate how much of each colour is present, and in this way compare samples.
In the clinic, if Alzheimers disease is suspected the presence of plaque in the brain can be studied by positron emission tomography (PET).
“For one patient in the study, the PET examination didn’t reveal any amyloid beta at all, whereas we discovered a large amount of the aggregate by staining with our molecules. This shows why there is a pressing need for further diagnostic tools,” says Peter Nilsson, professor of organic chemistry at the Department of Physics, Chemistry and Biology.
More accurate diagnosis of brain disease
The LiU researchers are now developing the tracer molecules to make it possible to use them in a PET camera. If they succeed, it would improve the accuracy of PET examinations and provide more information about the wrongly folded proteins in a particular patient.
Substances that can be used in PET investigations are needed that can not only detect plaque in the brain, but also distinguish between amyloid beta and tau. This would improve the diagnosis of brain disease. This is because deposits of the tau protein are seen not only in Alzheimer’s disease, but also in a group of less common conditions known as “tau pathologies”.
One such tau pathology is chronic traumatic encephalopathy (CTE), which can occur in individuals who have received blows to the head on repeated occasions, such as may occur in contact sports such as boxing, ice hockey and American football. The only way currently available to make this diagnosis is by autopsy.
Peter Nilsson and his colleagues hope that it will be possible in the long term to use their tracer molecules to detect tau deposits using a PET camera in the clinic. This is why they have developed a variant of the molecular chameleons that can bind to tau plaque and signal its presence. This study has been published in Chemistry-A European Journal. The researchers started with a tracer molecule that recognises only amyloid beta aggregates, and then adapted it so that it became more similar to molecules that bind to tau, developed by other researchers.Brain tissue with Alzheimer’s pathology, stained with one of the new tau-specific molecules (orange) and one of the molecules previously developed by the group at LiU (blue). The image has been taken in a fluorescence microscope. Photo credit: Peter R Nilsson
“We see that extremely small changes, just moving a couple of atoms, can cause the tracer molecule to bind preferentially to another type of aggregate. By making very small changes, we can tailor molecules to recognise different aggregates, based on which protein is present,” says Peter Nilsson.
They hope that it will be possible to discover disease earlier than it is today. In the case of Alzheimer’s disease, we know that plaque starts to form as long as 15-20 years before the first symptoms, such as memory loss and language difficulties, appear.
“This opens the possibility of developing a more advanced diagnostic method in which it is possible to distinguish between amyloid beta and tau. We could then investigate which aggregate forms first, and how they interact,” says Peter Nilsson.
With improved methods to investigate individual plaque deposits, it would also be possible to determine whether different forms of treatment help against one type but not the other, and it would be possible in clinical trials to evaluate continually the effectiveness of drug candidates in living patients.
The researchers have patented the tau tracer molecules in collaboration with a company.
Amyloid polymorphisms constitute distinct clouds of conformational variants in different etiological subtypes of Alzheimer’s disease, Jay Rasmussen, Jasmin Mahler, Natalie Beschorner, Stephan Kaeser, Lisa Häsler, Frank Baumann, Sofie Nyström, Erik Portelius, Kaj Blennow, Tammaryn Lashley, Nick Fox, Diego Sepulveda-Falla, Markus Glatzel, Adrian L. Oblak, Bernardino Ghetti, Peter R. Nilsson, Per Hammarström, Matthias Staufenbiel, Lary Walker and Mathias Jucker, (2017) PNAS, published online November 20 2017, doi: 10.1073/pnas.1713215114
Synthesis of Thiophene-Based Optical Ligands That Selectively Detect Tau Pathology in Alzheimer's Disease, Hamid Shirani, Hanna Appelqvist, Marcus Bäck, Therése Klingstedt, Nigel J. Cairns, Peter R. Nilsson, (2017) Chemistry – A European Journal, published online November 8 2017, doi:10.1002/chem.201703846
Video article about the technique: Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging. Nyström S, Bäck M, Nilsson KPR, Hammarström P. J Vis Exp. 2017 Oct 20;(128). doi: 10.3791/56279