alpha-Synuclein in filamentous inclusions of Lewy bodies from Parkinson's disease and dementia with lewy bodies

Abstract

Lewy bodies and Lewy neurites are the defining neuropathological characteristics of Parkinson's disease and dementia with Lewy bodies. They are made of abnormal filamentous assemblies of unknown composition. We show here that Lewy bodies and Lewy neurites from Parkinson's disease and dementia with Lewy bodies are stained strongly by antibodies directed against amino-terminal and carboxyl-terminal sequences of alpha-synuclein, showing the presence of full-length or close to full-length alpha-synuclein. The number of alpha-synuclein-stained structures exceeded that immunoreactive for ubiquitin, which is currently the most sensitive marker of Lewy bodies and Lewy neurites. Staining for alpha-synuclein thus will replace staining for ubiquitin as the preferred method for detecting Lewy bodies and Lewy neurites. We have isolated Lewy body filaments by a method used for the extraction of paired helical filaments from Alzheimer's disease brain. By immunoelectron microscopy, extracted filaments were labeled strongly by anti-alpha-synuclein antibodies. The morphologies of the 5- to 10-nm filaments and their staining characteristics suggest that extended alpha-synuclein molecules run parallel to the filament axis and that the filaments are polar structures. These findings indicate that alpha-synuclein forms the major filamentous component of Lewy bodies and Lewy neurites.

Figure 1

Specificity of anti-synuclein sera PER1, PER4, and PER5 for recombinant synucleins. Gels were stained with Coomassie brilliant blue (A) or immunoblotted with PER1 (B), PER4 (C), and PER5 (D). Lanes: 1, recombinant α-synuclein; 2, recombinant β-synuclein; 3, recombinant γ-synuclein. Molecular mass is shown to the left in M_r × 10⁻³.

Figure 2

Tissue from patients with dementia with Lewy bodies immunostained for α-synuclein (antibodies PER1 and PER2). (A and B) α-Synuclein-positive Lewy bodies and Lewy neurites in substantia nigra stained with PER1 (A) or PER2 (B). [Bar = 100 μm (in B for A and B).] (C and D) α-Synuclein-positive Lewy neurites in hippocampus stained with antibodies PER1 (C) or PER2 (D). [Bar = 80 μm (in D for C and D).] (E) α-Synuclein-positive intraneuritic Lewy body in a Lewy neurite in substantia nigra stained with PER2. (Bar = 40 μm.)

Figure 3

Substantia nigra from patients with Parkinson’s disease (A–F) and cingulate cortex from a patient with dementia with Lewy bodies (G) immunostained for α-synuclein (antibodies PER1 and PER2, in brown) and ubiquitin (in blue). Where only α-synuclein is stained, the color appears as pale reddish-brown, but where ubiquitin also is stained, the superposition of color gives a dark black and brown appearance. The blue staining is not seen on its own, because all the ubiquitin-immunoreactive structures are also α-synuclein-positive. (A) Nerve cell with four Lewy bodies, three of which are double-stained for α-synuclein (antibody PER2) and ubiquitin, whereas one is immunoreactive only for α-synuclein (arrow). A Lewy neurite is stained only for α-synuclein (arrow). (Bar = 30 μm.) (B) Nerve cell with three Lewy bodies that are double-stained for α-synuclein (antibody PER2) and ubiquitin. The halo of each Lewy body is strongly immunoreactive for ubiquitin, whereas both the core and the halo of each Lewy body are immunoreactive for α-synuclein. (Bar = 10 μm.) (C) Nerve cell with two Lewy bodies, one of which is double-stained for α-synuclein (antibody PER1) and ubiquitin, whereas one is immunoreactive only for α-synuclein (arrow). (Bar = 13 μm.) (D) Lewy neurite double-stained for α-synuclein and ubiquitin. (Bar = 90 μm.) (E and F) Lewy neurites single-stained for α-synuclein by using antibodies PER1 (E) and PER2 (F). (Bar = 100 μm.) (G) Intraneuronal and intraneuritic Lewy bodies and Lewy neurites double-stained for α-synuclein (antibody PER1) and ubiquitin. (Bar = 18 μm.)

Figure 4

Filaments from cingulate cortex of patients with dementia with Lewy bodies labeled with anti-α-synuclein antibody PER4. The gold particles conjugated to the second antibody appear as black dots. (A and B) Small clumps of labeled α-synuclein filaments. (C) A labeled α-synuclein filament and an unlabeled paired helical filament (arrow). The labeled filaments have various morphologies, including 5-nm filament (D); 10-nm filament with dark stain-penetrated center line (E); twisted filament showing alternating width (F); 10-nm filament with slender 5-nm extensions at ends (G, also C). [Bar = 100 nm (C).]

Figure 5

Filaments from cingulate cortex of patients with dementia with Lewy bodies labeled with anti-α-synuclein antibody PER1. The gold particles conjugated to the second antibody appear as black dots. In this case one or sometimes two gold particles are associated with one but never two ends of the filament. (Bar = 100 nm.)