Amyloid fibrils, crystal-like fibrillar aggregates of denatured proteins, are formed linked with the breakdown of supersaturation, causing a series of amyloidosis including Alzheimer’s and Parkinson’s diseases. Although varying in vitro factors are known, in vivo factors breaking supersaturation are unclear. We found that flowing by a peristaltic pump effectively triggers amyloid formation of hen egg white lysozyme, a model amyloidogenic protein, and, moreover, amyloidosis-associated proteins (i.e. α-synuclein, amyloid β 1-40, and β2-microglobulin). The peristaltic pump-dependent amyloid formation was visualized by a fluorescence microscope with looped flow system, revealing dynamic motions under flow. Among them, amyloid fibrils of amyloid β 1-40 were stickier than others, self-associating, absorbing to loop surfaces, and surging upon flicking the loop, implying early stages of cerebral amyloid angiopathy. On the other hand, β2-microglobulin at a neutral pH showed unique two-step amyloid formation with an oligomeric trapped intermediate, which might mimic amyloid formation in patients. Peristalsis-caused strong shear stresses were considered to mechanically break supersaturation. Shearing stresses occur in vivo at varying levels, suggesting that they break otherwise persistent supersaturation, thus triggering amyloid formation and ultimately leading to amyloidosis. (182 words <200 words)
Introduction
Amyloid fibrils, crystal-like fibrillar aggregates of denatured proteins1–6 associated with a series of amyloidosis including Alzheimer’s and Parkinson’s diseases7,8, are formed linked with the breakdown of supersaturation9,10. Amyloid fibrils have been reproduced in vitro even in the absence of seeds under a variety of conditions by distinct mechanisms9,10. These include: (i) a counter ion-binding mechanism observed under acidic conditions in the presence of moderate concentrations of salts, (ii) a salting-out mechanism observed under high salt conditions independent of pH, (iii) a hydrophobic additive-binding mechanism observed in the presence of moderate concentrations of alcohols, detergents like sodium dodecyl sulfate (SDS), or membrane surfaces, and (iv) pI-precipitation under low salt conditions. Common to these mechanisms is the establishment of a supersaturated state of responsible proteins and subsequent breakdown10–15.
Analogous to crystallization, spontaneous amyloid formation occurs in proportion to the degree of supersaturation (S)16:
where [C] and [C]C are the initial solute concentration and thermodynamic solubility (i.e., critical concentration), respectively. Varying conditions as described above increase S and, thus, the risk of amyloid formation10,17. However, under a metastable condition where supersaturation persists in the absence of seeding, mechanical triggers are required to break supersaturation18. Thus, although stirring or ultrasonication is often employed as a mechanical trigger of amyloid formation, we do not know triggers in vivo.
Fluid flow stresses have been increasingly focused on as mechanical triggers initiating amyloid nucleation in vivo19–25. Fluid flows in blood, cerebrospinal fluid, intramural periarterial drainage or interstitial systems are torrents at the microscopic scale, exerting various types of flow stresses including laminar-flow-dependent shear stress and extensional flow stress. The laminar-flow shear stress is governed by the shear rate (s−1), a gradient in velocity perpendicular to the direction of flow. Beyond a critical shear rate, a transition from diffusion-limited flow to advection dominated flow occurs, enabling shorter collision times between solutes and resulting in accelerated aggregation25. On the other hand, an extensional flow field is generated by a gradient in velocity in the direction of flow and is characterized by the strain rate (s−1). The extensional flow is enhanced when the channel diameter decreases or two channels merge. There are an increasing number of reports that extensional flows result in protein aggregation including amyloid fibrils20,26,27. Both laminar flow and extensional flow cause mechanical stresses in liquid, which are believed to trigger protein aggregation. However previous studies did not consider the role of supersaturation in fluid flow stresses. It is likely that the direct role of flow stresses is to break otherwise persistent supersaturation.
Here, during our challenge combining ultrasonication and microchannels, we found that peristaltic pump flow of hen egg white lysozyme (HEWL) resulted in amyloid formation. Peristaltic pump-dependent amyloid formation is common to varying disease-associated amyloidogenic proteins: α-synuclein (αSN), amyloid β 1-40 (Aβ40), and β2-microglobulin (β2m), suggesting that shear stress in vivo triggers amyloid nucleation. We performed a numerical simulation to evaluate the stress in the liquid during the operation of the peristaltic pump and found that significantly large shear stress can be created in the liquid by the peristaltic motion of rotors in contact with the tubes, suggesting that shear stress is the key mechanical factor of amyloid nucleation in vivo. We designed a peristaltic pump-dependent amyloid inducer with fluorescence detection and imaging, which will be useful for assessing amyloidogenic risks10,28 and analyzing the kinetics of shear stress-dependent amyloid formation.