On the pH-dependence of α-synuclein amyloid polymorphism and the role of secondary nucleation in seed-based amyloid propagation

Abstract

The aggregation of the protein alpha-synuclein is closely associated with several neurodegenerative disorders and as such the structures of the amyloid fibril aggregates have high scientific and medical significance. However, it seems that there are about as many unique atomic-resolution structures of these aggregates as there are publications describing them. Obviously, this highly polymorphic nature of alpha-synuclein fibrils hampers efforts in disease-relevant in vitro studies on alpha-synuclein amyloid aggregation. In order to better understand the factors that affect polymorph selection we studied the structures of alpha-synuclein fibrils in vitro as a function of pH and buffer using cryo-EM helical reconstruction. We find that in the physiological range of pH 5.8-7.4 a pH-dependent selection between Types 1, 2 and 3 polymorphs occurs. Our results indicate that even in the presence of seeds, the polymorph selection during aggregation is highly dependent on the buffer conditions, attributed to the polymorph-unspecific nature of secondary nucleation. We also uncovered two new polymorphs that occur at pH 7.0 in phosphate buffered saline. The first is a monomeric Type 1 which highly resembles the structure of the juvenile-onset synucleinopathy polymorph found in patient-derived material. The second is a new Type 5 polymorph that resembles a polymorph that has been recently reported in a study that used diseased tissues to seed aggregation. Taken together, our results highlight the shallow amyloid energy hypersurface that can be altered by subtle changes in the environment, including the pH which is shown to play a major role in polymorph selection and in many cases appears to be the determining factor in seeded aggregation. The results also confirm the possibility of producing disease relevant structure in vitro.

Modulation of Alzheimer’s Disease Aβ40 Fibril Polymorphism by the Small Heat Shock Protein αB-Crystallin

“…However, recently it became evident that the so-called “fuzzy coat” of the fibrils plays a crucial role in the process of fibril formation (especially elongation and secondary nucleation), and is an important driving force for intermolecular interactions, e.g. with membranes, mRNA and chaperones. ,, The cryo-EM structures of Aβ42 filaments from human brains in familial AD have an extended fuzzy coat (residues 1–11) compared to the filaments found in sporadic AD (residues 1–8) and differ as well in the packing of the protofilament . A detailed comparison of these polymorphs suggests that G33 and G38 are involved in an interaction with the N-terminus fuzzy coat that shields the hydrophobic C-terminus from the solvent .…”

“…with membranes, mRNA and chaperones. 37,59,60 The cryo-EM structures of Aβ42 filaments from human brains in familial AD have an extended fuzzy coat (residues 1−11) compared to the filaments found in sporadic AD (residues 1−8) and differ as well in the packing of the protofilament. 61 A detailed comparison of these polymorphs suggests that G33 and G38 are involved in an interaction with the N-terminus fuzzy coat that shields the hydrophobic Cterminus from the solvent.…”

“…Amyloid fibrils are formed via distinct mechanisms and pathways such as primary nucleation, elongation, secondary nucleation, fragmentation etc. − Secondary nucleation was found to be the major pathway for the generation of new Aβ amyloid fibrils in which nucleation of Aβ peptide happens on the surface of existing Aβ fibrils. ,, The role of secondary nucleation in the structural polymorphism of fibrils is highly debated. It was suggested that secondary nucleation is highly dependent on environmental conditions and can not ensure the preservation of the seed structure in comparison to elongation. − On the other hand, preservation of the polymorph structure has been observed in secondary nucleation dominated Aβ aggregation . In this context, the role of the so-called fibrillar fuzzy coat, i.e.…”