Getting a clearer picture of amyloid aggregation

Researchers from the Adolphe Merkle Institute’s BioPhysics group have applied atomic force microscopy to improve imaging of protein aggregates and their precursors responsible for the development of brain disorders such as Alzheimer’s Disease.

Amyloids are aggregates of proteins that are linked to a number of human pathologies, among them neurodegenerative disorders such as Parkinson’s or Huntington’s diseases. The presence of aggregated amyloid β (Aβ), in the form of senile plaques and neurofibrillary tangles of microtubule-binding protein tau in the brain, is considered the histopathological hallmark of another neurodegenerative illness, Alzheimer’s dementia (AD). Its two most common forms, Aβ-40 and Aβ-42, are both potential targets to treat AD. Aβ-42 is the form most frequently found in patients’ brains, and the emerging scientific consensus is that its earlier, small and soluble aggregates are generally more neurotoxic than insoluble mature fibrils and dense fibril meshes, otherwise known as senile plaques. To visualize Aβ aggregates and to understand how quickly they form, researchers rely on a variety of imaging and analytical techniques. For the most part though, these techniques do not provide a comprehensive picture, because they cannot visualize the size and shape changes that especially the smallest Aβ aggregates undergo inside physiological solutions.

Atomic force microscopy (AFM) can overcome this hurdle. An atomic force microscope operates by scanning a small cantilever over the surface of a sample, creating a topographical map of it on the nanoscale. This procedure provides images with a resolution in the nanometerrange, and is suitable for imaging of individual proteins and their aggregates. For the best results, the imaging should be carried out on an uncontaminated and artifact-free interface. This requirement is crucial because of the small dimensions of the Aβ peptides: the presence of any contaminants could be mistaken as amyloid aggregates, unlike with extended biomolecules such as DNA, which can be recognized in the image.

To obtain morphological information on amyloid nanostructures with an AF microscope, the Aβ peptides must be adsorbed on a solid surface and maintained in a hydrated state. The surface must also be free from any contaminants, even under ambient conditions. To meet this challenge, the AMI researchers investigated an interface with a graphene surface and pure water. This approach resolved both the soluble forms (monomers and oligomeric aggregates) and insoluble forms (protofibrils with nodular morphology, mature single fibrils, and fibril networks) of Aβ-40 and Aβ-42 with single-particle resolution.

“What was important is that we were able to develop a technique that does not require special labelling or staining, and which allows us to follow the complete amyloid aggregation pathway from start to finish without unwanted artifacts,” says AMI alumnus Peter Nirmalraj, who led the research.

While monitoring changes in oligomer diameter up to 150 hours, the researchers noted faster aggregation rates for peptide Aβ-42 compared to peptide Aβ-40. The resulting mature fibrillar networks showed that those made up of Aβ-42 contained longer, more densely packed and aligned fibrils than networks from Aβ-40. This effect has also been shown on surfaces other than graphene, confirming that the alignment was not influenced by the surface used.

“This hierarchical assembly of the fibrils may be a useful physical property for engineering functional bionanomaterials, because the elongated fibrils with high aspect ratio remain stable under ambient conditions”, adds Nirmalraj.

Reference: Nirmalraj, P. N.; List, J.; Battacharya, S.; Howe, G.; Xu, L.; Thompson, D.; Mayer, M. Complete Aggregation Pathway of Amyloid β (1–40) and (1–42) Resolved on an Atomically Clean Interface. Sci. Adv., 2020, 6 (15), eaaz6014.