The soluble-to-toxic conversion of amyloid proteins such as Aβ, α-synuclein, mHTT among others is a critical milestone in the onset and pathogenesis of amyloid-specific neurodegeneraive disorders. Efforts to develop an understanding of this biophysical transformation are driven by spectroscopic and immunohistochemical tools. Nevertheless, access to instruments such as solid-state NMR, microscopes (TEM, HR-TEM, SEM, AFM), ATR-IR, DLS instruments and biochemical kits precludes routine studies of the process for many laboratories and investigators.
Even if high-resolution microscopes are accessible, extensive sample preparation protocols, analyses times and availability of very specific technical/instrumentation expertise are barriers that still need to be overcome. Finally, and critically, higher-resolution structural techniques are not amenable to quantification and kinetics measurements. As previously noted, quantification of oligomers and fibrils formed from soluble monomers and, perhaphs more importantly, the reverse process is important for advancing biomedical intervention. The in vitro screening of small-molecules that intervene in amyloidogenesis precedes testing in preclinical models.
Optical methods such as DLS or fluorescence using ThT or Congo red to identify fibrils are frequently confounded by interference from small-molecule fluorescence (26). Others techniques such as solid-state NMR, are not amenable to easy use, lack access, and fail to satisfactorily quantify the interconversion between the monomeric amyloid, its intermediates and the mature fibril. Often, necessary sample preparation conditions do not recapitulate solution conditions.
Through several inroads, the method described here reduce barriers towards the study of amyloidogenesis which has traditionally involved elaborate sample preparation, mounting of “dried” samples, expensive instrumentation and protracted sample analyses times (16, 17). Even though the technique is chemically and structurally “low-resolution” in nature, it provides a rapid, facile and inexpensive mechanism by which to quantify the loss of monomers (via their conversion to dimers, oligomers, proto-fibrils and fibrils), starting from a known concentration of the amyloid nomer. Importantly, by quantifying the intensity of the bands on the gel, it permits the user to build a kinetic profile of the consumption of monomers, formation of dimers, oligomers and finally the transformation of the amyloid protein into mature fibrils. From a biomedical perspective, the use of PAGE to establish a quantitative and dose-dependent profile of small-molecule efficiency in dissolving fibrils and oligomeric aggregates to their monomeric counterparts is highly desired.
In conclusion, we demonstrate that a readily existing method and easily accessible appartatus can be used to obtain rich biophysical (kinetic) data about amyloid forming trajectories and the interplay between intermediates therein. Equally importantly, it can be used to screen small-molecules and also determine, via size analysis, where along the trajectory that the small-molecule intervenes. It provides undergdatuates, graduate students and advanced biomedical researchers in an insittituion with a powerful, affordable, facile method, which is already widely availble, to study an important neurodegeneration-associated process.