Three-layered structure shared between Lewy bodies and lewy neurites-three-dimensional reconstruction of triple-labeled sections

Abstract

Lewy bodies (LBs) and Lewy neurites (LNs) are the hallmarks of Parkinson's disease (PD). Although LBs and LNs, frequently coexistent, share some histological properties, their appearances are quite different under conventional two-dimensional observation. In order to clarify how these apparently different structures (LBs and LNs) are related during their formation, we performed three-dimensional observation on post-mortem brainstem tissues with PD. Sixty-microm thick floating sections were multi-immunofluorolabeled for alpha-synuclein (alphaS), ubiquitin (Ub) and neurofilament (NF). Serial confocal images were reconstructed with software. External three-dimensional configuration of LBs, double-labeled for alphaS and NF, exhibited frequent continuity with LNs (70%). Internally, alphaS and Ub formed the three-dimensional concentric inner layers and NF rimmed these inner layers. This layered structure was shared among spherical LBs, rod-shaped LNs and even convoluted forms of LBs/LNs. Furthermore, each layer exhibited continuity without interruption even in the convoluted form and around its junction to spherical LBs. This three-layered structure shared among various Lewy pathologies and their layered continuity on three-dimensional basis favor the hypothesis that LNs evolve into LBs. Besides progression from pale bodies to LBs, structural evolution from LNs into LBs may provide an alternative explanation for the variability of alphaS deposits and their interrelation.

Figure 1

Lewy bodies (LBs) and Lewy neurites (LNs) in the locus ceruleus labeled with anti‐α‐synuclein (αS) antibody. αS immunohistochemical staining of brainstems demonstrated LBs (indicated by the arrow) and LNs (indicated by the arrowhead), which are apparently distinct from each other. Bar = 20 µm.

Figure 2

Serial optical sections of Lewy bodies (LBs) were obtained from thick floating sections double‐immunofluorolabeled for α‐synuclein (red) and neurofilament (green). They were reconstructed to yield external configurations of LBs, frequently in continuity with Lewy neurites. Bar = 20 µm.

Figure 3

Triple immunofluorolabeling of LBs (A) and LNs (B) with anti‐αS (red), anti‐Ub (blue) and anti‐NF (green) antibodies. They are stratified into concentric layers. Ub epitope is in the center of inner layer and αS epitope is surrounding the Ub epitope. NF epitope at the outermost layer rims these internal core structures. This three‐layered structure of spherical LBs was shared with that of rod‐shaped LNs. Bars = 20µm. Abbreviations: LBs = Lewy bodies; LNs = Lewy neurites; αS = α‐synuclein; Ub = ubiquitin; NF = neurofilament.

Figure 4

Stacked images of 300 serial optical sections of triple‐immunofluorolabeled LBs and LNs with an interval of 0.1 µm. LBs (A–D) and LNs (E–H) are immunopositive for α‐synuclein (red), ubiquitin (blue) and neurofilament (green). There is a significant overlap between the epitopes. Bars = 20 µm. Abbreviations: LBs = Lewy bodies; LNs = Lewy neurites.

Figure 5

Cross sections of reconstructed LBs (A–C) and LNs (D–F). A,D. One of the optical sections (X–Y) at the depth indicated with blue lines in B,C and E,F. B,E. Cross‐sectional X–Z image along the blue lines indicated in A,D and C, F. C,F. Cross‐sectional Y–Z image along the blue lines indicated in A,D and B,E. LBs and LNs showed three‐layered structure in whatever optical planes. Red = α‐synuclein; blue = ubiquitin; green = neurofilament. Bars = 20 µm. Abbreviations: LBs = Lewy bodies; LNs = Lewy neurites.

Figure 6

External and internal views of the three‐dimensional reconstructed LBs and LNs. Three‐dimensional reconstruction from serial optical sections with the interval of 0.1 µm along Z‐axis was performed on a software “Delta Viewer,” and the same LBs and LNs were observed from different angles: upper view (A,E); front view (B,F); and side view (C,G). The three‐layered structure was maintained when a part of LBs was removed after a three‐dimensional reconstruction (D). Red = α‐synuclein, blue = ubiquitin; green = neurofilament. Bars =  20 µm. Abbreviations: LBs = Lewy bodies; LNs = Lewy neurites.

Figure 7

Internal (A,B) and external (C,D) views of reconstructed LNs/LBs with convolution. Three‐dimensional analyses demonstrated LNs/LBs with convolution. The same three‐layered structure is maintained in convoluted LNs/LBs reconstructed and shown along XY (A), XZ (B) and YZ (data not shown) planes. Each layer of the internal structure is in continuity (indicated by the arrow) with that of the adjacent spherical LB (C,D). Red = α‐synuclein, blue = ubiquitin; green = neurofilament. Bars = 20 µm (A–D). Abbreviations: LBs = Lewy bodies; LNs = Lewy neurites.