Amyloid research enters a new era thanks to breakthrough technologies

Parkinson’s, Alzheimer’s, Diabetes mellitus type 2, Huntington’s, Fatal familial insomnia, prion’s, among many others, are diseases with a common contributor: Amyloids. These are aggregates of proteins that become folded into the wrong shape, allowing many copies of that protein to stick together and form large fibrils. These fibrils disrupt the healthy physiological function of nearby tissues and organs.

Over the last 150 years, technical developments have repeatedly created new horizons in amyloid research, including biophysical and chemical analyses, which have retrieved lots of information related to the mechanistic aspects of fibril formation and possible links with pathogenesis. But in the last couple of years, technologies such as Ion Mobility coupled to Electrospray Ionization Mass Spectrometry (ESI-IM-MS) and Microscale Thermophoresis (MST) have appeared to revolutionize amyloids research, particularly in the characterization of the aggregation process and the screening for amyloid ligands and binding partners.

2015: For the first time, a study challenged the scientific principle about Alzheimer protein Amyloid beta

A comprehensive knowledge of the number of molecules and shape that a protein has when it begins to aggregate is crucial for the design of drugs capable of breaking them up or preventing their formation and the corresponding disease.

In Alzheimer’s disease, the protein that aggregates is called Amyloid beta (Aβ). Two of the most common variants of Aβ are Aβ40 and Aβ42, with the latter being the variant most closely associated with Alzheimer's disease. The literature reports that while Aβ40 self-aggregates to sequentially form dimers, trimers and tetramers, Aβ42 self-aggregates to form pentamers and hexamers.

Among the paradigms in the Alzheimer’s disease field, pentamers and hexamers of Aβ42 have been established as the building blocks for Aβ aggregation. These findings have been cited more than 1000 times and consequently numerous studies have been based on this premise. However, a new study has shown that Aβ40 and Aβ42, actually goes through exactly the same aggregation states. The question then is, what accounts for these differences?  And the answer lies on the technologies that have been used so far and artifacts given by previously used ones.

Results published to date are biased by the technique most widely used to study Aβ aggregates: SDS-PAGE. Most groups rely on this technique because it is characterized by the need for a small amount of sample and therefore is used for more straightforward studies. But the aforementioned study demonstrates that pentamers and hexamers form artifactually from dimers and trimers in the presence of SDS. Thus, SDS-PAGE provided flawed information on the order and distribution of Aβ42 oligomers.

Using a new approach though, based on mass spectrometry, namely ESI-IM-MS, it was shownthat the oligomer distribution for Aβ40 and Aβ42 was the same, comprisingmainly dimers and trimers.

ESI-IM-MS not only has the potential for the characterization of oligomers and to identify chemical modifications, but also to provide dynamic and structural information on the oligomers present in the sample. Thanks to these features, yet another paradigm in the field was challenged, which states that higher order Aβ oligomers adopt a β-sheet structure. This study also demonstrated that Aβ dimers and trimers are globular and lack defined secondary structure, indicating that major structural rearrangements occur during fibril formation. It should be noted that up to now, drug design has been based on the premise of interfering with the beta-sheet structure, so the authors believe that this strategy should be reconsidered and recommend caution when using SDS-PAGE to study Aβ oligomers.

These huge changes on the way scientist used to look at Aβ amyloids have been made thanks to new technologies as well as critical thinking. They highlight the importance of defining the structures and properties of the species involved in Aβ aggregation; it is only through this level of molecular detail that it will be possible to interfere with Aβ aggregation in a rational manner. Specifically, by identifying Aβ40 and Aβ42 dimers and trimers as the earliest oligomers formed during Aβ aggregation, they now constitute the earliest forms to be considered in the design of therapeutic strategies targeting Aβ oligomerization.

2015: Protein-ligand binding affinity measurements can finally be analyzed on supramolecular aggregates such as amyloid fibrils

In Parkinson’s disease, intracellular deposits of the intrinsically disordered protein α-synuclein, form a major characteristic of the pathology. To date, no cure for this disease exists, a consequence at least in part, of the lack of fundamental understanding of the mechanism of aggregation and its associated toxicity, as well as the incomplete characterization of the interactions between aggregates of α-synuclein and other compounds, including small molecules and proteins. But why studying these interactions? Well, they are important for both diagnostic (e.g. for positron emission tomography) and therapeutic purposes (e.g. for targeted aggregation inhibitors). In this context, there is an urgent need for experimental techniques that can be used for high throughput screening to identify such compounds.

Protein aggregates are situated though, at the boundary between soluble and insoluble structures and thus are challenging to study, because classical biophysical techniques, such as scattering, spectroscopic and calorimetric methods, are not well adapted for their study. Standard techniques, such as isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR) can provide important information, but suffer from a number of limitations, including high levels of sample consumption (ITC), potential surface artifacts (SPR) and high sensitivity to solution conditions (both ITC and SPR).

A recent study demonstrated for the first time that Microscale Thermophoresis (MST) is a valuable method for screening for ligands and partners of even such highly challenging samples as supramolecular protein aggregates, such as amyloid fibrils. Due to their polymeric nature, these types of samples are substantially more difficult to handle than most soluble monomeric proteins and this study presents a comprehensive protocol for the use of MST for amyloid-binding assays.

Binding affinities were obtained for a nanobody that binds to monomeric α-synuclein in excellent agreement with a previously determined value obtained from ITC measurements. Its binding affinity to the oligomers was also obtained, which had not previously been reported, partly due to the challenge of obtaining sufficient quantities of pure oligomers. This issue was been able to overcome by exploiting the low sample requirements of MST.

The binding of the small molecule epigallocatechin gallate (EGCG), one of the main constituents of green tea, to α-synuclein aggregates was also investigated by MST. This molecule has been reported to bind to various species on the aggregation pathway of α-synuclein and even to remodel mature amyloid fibrils and other protein aggregates, but its affinity for oligomers had not been previously measured. With this technique it was possible to obtain the binding constants of EGCG to α-synuclein amyloid fibrils and oligomers as well.

The possibility of characterizing novel types of ligands that bind to protein aggregates, both small and large molecules, at high throughput and using minute sample quantities is a highly valuable addition to the experimental toolbox available for the development of diagnostic and therapeutic strategies against protein missfolding diseases. In particular, it should be emphasized that oligomeric structures, which have been highlighted as the most toxic species on the aggregation pathway, often occur only at low concentration and with a short lifetimes and hence experimental methods that are rapid and require only small amounts of sample are vital for their study.