α-Synuclein & Parkinson’s Disease (PD)

Parkinson’s disease (PD) is a neurodegenerative movement disorder that is characterized by the loss of dopaminergic neurons from the substantia nigra, and the formation of fibrillar intraneuronal inclusions (called Lewy bodies). Several lines of evidence point towards a central role for the process of α-synuclein fibrillization in the etiology of PD. First, α-synuclein is the primary component of Lewy bodies in all PD patients. Second, two different α-synuclein missense mutations (A30P and A53T) are associated with rare, autosomal dominant, early-onset PD and have been shown to form fibrils. Third, transgenic mice and Drosophila expressing human wild-type (WT) α-synuclein or, in the flies, the mutants, are characterized by α-synuclein inclusions that resemble Lewy bodies. In both models, the formation of these inclusions is correlated to the onset of disease phenotype. Finally, the PD-linked mutations (A30P and A53T), promote in vitro α-synuclein oligomerization, suggesting that the process of α-synuclein fibrillization may initiate neurodegeneration. α-synuclein amyloid fibril formation proceeds through a series of discrete oligomeric intermediates, referred to as protofibrils, that disappear upon fibril formation. Although both PD-linked mutations accelerate the formation of α-synuclein protofibrils, the A30P mutation was shown to delay the formation of amyloid fibrils relative to WT, suggesting that α-synuclein protofibrils, rather than fibrils, may be the pathogenic species. This hypothesis is supported by the observation that α-synuclein deposits in the brains of the “symptomatic” transgenic mice are non-fibrillar, and the fact that dopaminergic neurons that contain Lewy bodies appear to be healthier than neighboring neurons. Atomic force microscopy analysis of α-synuclein oligomerization demonstrated that α-synuclein protofibrils exist in spherical, chainlike and annular morphologies. In collaboration with Peter Lansbury (Brigham and Women’s Hospital), we investigated the effect of the familial PD mutations on the structural properties of α-synuclein protofibrils. By extending the biophysical studies to electron microscopy, analytical ultracentrifugation and scanning transmission electron microscopy, we showed that protofibrils of defined molecular size distribution and morphology are formed. In addition, we performed a detailed analysis of the morphological types of protofibrils using electron microscopy and single-particle averaging of negatively stained specimens.

To gain insight into the structural features of protofibrillar intermediates, the species in the α-synuclein protofibril peak were partially purified by size exclusion chromatography fractionation of the void peak. Three fractions corresponding to the early, middle and late fractions of the void peak were collected (top left panel). The protofibril morphology in these fractions were analyzed by single particle electron microscopy. The early fraction of A53T contained predominantly large spherical and tubular species with an average diameter of 24 nm and 19 nm, respectively (top right panel). Occasionally, annular protofibrils with an average diameter of 11 nm could be seen in this fraction. The amount of annular protofibrils increased significantly in the fraction corresponding to the middle of the A53T protofibril peak (bottom left panel), whereas the majority of the species in the late fraction were 10–12 nm annular protofibrils (bottom right panel). The late fraction contained a significant amount of tubular species, which had an average diameter of 11 nm and an average length of 24 nm.

Electron micrographs of the late and middle fractions of the void peak for A53T and A30P showed annular and tubular structures, which were classified into 100 classes. In the case of A53T (top), the first group consisted of what appears to be incomplete rings, where one end of the protofibril seemed to lie above the other end, suggesting a helical symmetry for these structures (panels 1–3). The second group showed annular structures (panels 4–6). The third group showed rectangular particles (panels 7–9). The presence of rectangular particles with similar diameter but closely spaced lengths is likely to reflect different stages of protofibrillar growth. The fourth group showed large aggregates, some of which appeared to be formed by two rectangular particles joint at a 90° angle (panels 10–12). In the case of A30P (bottom), at least three groups of protofibrillar structures were evident. The first group contained chain-like structures that formed rings with slightly displaced ends, which are likely to represent the precursors for the helical fibrils (panels 1–3). The second group represents the majority of the structures for A30P that consist of annular structures (panels 4–6). The third group contains large spherical particles (panels 7–9). Unlike A53T, tubular particles were not observed for A30P.


» Lashuel et al. (2002) J. Mol. Biol. 322: 1089-1102.