The self-assembly of α-synuclein (αS) into intraneuronal inclusion bodies is a key characteristic of Parkinson’s disease. To define the nature of the species giving rise to neuronal damage, we have investigated the mechanism of action of the main αS populations that have been observed to form progressively during fibril growth. The αS fibrils release soluble prefibrillar oligomeric species with cross-β structure and solvent-exposed hydrophobic clusters. αS prefibrillar oligomers are efficient in crossing and permeabilize neuronal membranes, causing cellular insults. Short fibrils are more neurotoxic than long fibrils due to the higher proportion of fibrillar ends, resulting in a rapid release of oligomers. The kinetics of released αS oligomers match the observed kinetics of toxicity in cellular systems. In addition to previous evidence that αS fibrils can spread in different brain areas, our in vitro results reveal that αS fibrils can also release oligomeric species responsible for an immediate dysfunction of the neurons in the vicinity of these species.

Edited by Paul E. FraserParkinson's disease (PD) is a neurodegenerative disorder characterized by fibrillar neuronal inclusions composed of aggregated ␣-synuclein (␣-syn). These inclusions are associated with behavioral and pathological PD phenotypes. One strategy for therapeutic interventions is to prevent the formation of these inclusions to halt disease progression. ␣-Synuclein exists in multiple structural forms, including disordered, nonamyloid oligomers, ordered amyloid oligomers, and fibrils. It is critical to understand which conformers contribute to specific PD phenotypes. Here, we utilized a mouse model to explore the pathological effects of stable ␤-amyloid-sheet oligomers compared with those of fibrillar ␣-synuclein. We biophysically characterized these species with transmission EM, atomic-force microscopy, CD spectroscopy, FTIR spectroscopy, analytical ultracentrifugation, and thioflavin T assays. We then injected these different ␣-synuclein forms into the mouse striatum to determine their ability to induce PD-related phenotypes. We found that ␤-sheet oligomers produce a small but significant loss of dopamine neurons in the substantia nigra pars compacta (SNc). Injection of small ␤-sheet fibril fragments, however, produced the most robust phenotypes, including reduction of striatal dopamine terminals, SNc loss of dopamine neurons, and motor-behavior defects. We conclude that although the ␤-sheet oligomers cause some toxicity, the potent effects of the short fibrillar fragments can be attributed to their ability to recruit monomeric ␣-synuclein and spread in vivo and hence contribute to the development of PD-like phenotypes. These results suggest that strategies to reduce the formation and propagation of ␤-sheet fibrillar species could be an important route for therapeutic intervention in PD and related disorders. Figure 2. Structural characterization of ␣-synuclein species used in the in vivo mouse studies. A, absorbance unit (a.u.) sedimentation velocity measurement of human (blue dashed line) and mouse oligomers (orange line) shows 10S and 15S species. B, FTIR spectra show that F-L, F-M, and F-S species are primarily composed of parallel ␤-sheets (band at 1620 -1630) and that the oligomeric (mouse and human) species are primarily antiparallel (band at 1620 -1630 cm Ϫ1 and shoulder at 1695 cm Ϫ1 ). The mouse and human oligomers have ␤-sheet structures of about 40 and 65%, respectively. C, CD shows that ␤-sheet content of oligomers (mouse and human) are intermediate between monomer and fibrils. M.R.E., mean residue ellipticity. D, ThioT binding shows that the fibrils adopt an amyloid conformation; the oligomers show limited ThioT binding, and the monomer shows no ThioT binding.

The extent of protein hydration modulates the free energy barrier of both heterogeneous and homogeneous α-synuclein nucleation, leading to the formation of distinct amyloid polymorphs depending on the water activity of the protein microenvironment.