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Lewy pathology in Parkinson’s disease consists of crowded organelles and lipid membranes

Abstract

Parkinson’s disease, the most common age-related movement disorder, is a progressive neurodegenerative disease with unclear etiology. Key neuropathological hallmarks are Lewy bodies and Lewy neurites: neuronal inclusions immunopositive for the protein α-synuclein. In-depth ultrastructural analysis of Lewy pathology is crucial to understanding pathogenesis of this disease. Using correlative light and electron microscopy and tomography on postmortem human brain tissue from Parkinson’s disease brain donors, we identified α-synuclein immunopositive Lewy pathology and show a crowded environment of membranes therein, including vesicular structures and dysmorphic organelles. Filaments interspersed between the membranes and organelles were identifiable in many but not all α-synuclein inclusions. Crowding of organellar components was confirmed by stimulated emission depletion (STED)-based super-resolution microscopy, and high lipid content within α-synuclein immunopositive inclusions was corroborated by confocal imaging, Fourier-transform coherent anti-Stokes Raman scattering infrared imaging and lipidomics. Applying such correlative high-resolution imaging and biophysical approaches, we discovered an aggregated protein–lipid compartmentalization not previously described in the Parkinsons’ disease brain.

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Fig. 1: Lewy pathology shows abundant membranous structures, abnormal organelles and vesicles.
Fig. 2: Electron tomography and subtomogram averaging reveal membranous nature of Lewy pathology.
Fig. 3: Nigral Lewy neurite reveals disrupted cytoskeletal elements, dysmorphic mitochondria and autophagosome-like structures.
Fig. 4: Nigral Lewy neurite revealing disrupted cytoskeletal elements, tubulovesicular, lysosome- and autophagosome-like structures.
Fig. 5: Lewy pathology consisting of abundant tubulovesicular structures.
Fig. 6: Lewy pathology consisting of abundant vesicular structures interspersed with filaments.
Fig. 7: Subcellular features of Lewy pathology reveal the organelle distribution.
Fig. 8: Inner architecture of Lewy pathology shows membrane fragments and organelles.

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Data availability

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

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Acknowledgements

We are grateful to the individuals who participated in the brain donation program and their families, making this study possible. We thank S. Ipsen from the University Hospital Basel and S. Bichet from the Friedrich Miescher Institute for training and assisting with the immunohistochemistry, A. Fecteau-LeFebvre for electron microscopy maintenance, A. Jonker for help with preparing cryostat-cut tissue sections, Prothena for providing the pSer129 11A5 antibody, Advanced Optical Microscopy Core O|2 (www.ao2m.amsterdam) for support with STED imaging, D. Mona for help with labeling antibodies, P. Baumgartner and K. Bergmann for administrative help, and S. Müller for carefully proof-reading and editing the manuscript. S.H.S. was supported by the Roche Postdoctoral Fellowship (RPF) program; this work was in part supported by the Swiss National Science Foundation (SNF Grants no. CRSII3_154461 and CRSII5_177195), the Synapsis Foundation Switzerland, the foundation Heidi Seiler-Stiftung, and the Stichting Parkinson Fonds, the Netherlands.

Author information

Authors and Affiliations

Authors

Contributions

S.H.S. performed CLEM/TEM and tomography, SBFSEM imaging and 2D/3D color segmentations, analyzed data and wrote the manuscript. A.J.L. performed CLEM/TEM and tomography, and contributed to analyzing and interpreting the data and writing the manuscript. C.G. and A.G.M. trained and supported S.H.S. and A.J.L. with SBFSEM and CLEM tissue preparation and imaging. J. Hench screened light microscopy slides and analyzed light microscopy data of CLEM for localizing Lewy pathology. J. Hench, G.S. and A.J.L. designed and optimized the staining for CLEM and localization of Lewy pathology in light microscopy data with S.H.S. W.D.J.v.d.B., T.M. and E.H. performed STED imaging. P.P.N. trained and supported A.J.L. in sample preparation, data collection and image processing for TEM tomography. K.N.G. and J.W. assisted with TEM tomography. R.S. and S.H.S. optimized and performed lipid and αSyn co-staining and confocal imaging. D.C.-D. performed subtomogram analysis. A.I. processed tissue samples collected at the autopsy room, prepared cryostat tissue and sectioned paraffin embedded tissue. Y.d.G. prepared cryostat tissue for CARS and FTIR imaging. A.J.M.R. and W.D.J.v.d.B. performed rapid autopsies of PD brain donors and controls, collected brain tissue and preformed neuropathological assessment. W.D.J.v.d.B. performed neuroanatomical dissections and performed laser-capture micro dissection with S.H.S. A.D.P. performed Raman imaging tests. J.E., A.S. and J. Hoernschemeyer performed LC–MS analysis. D.N., S.F.E.M. and K.N.G. performed and analyzed CARS imaging. F.G. performed FTIR imaging. M.Q., W.F.J.v.I.J. and V.B. provided whole exome sequencing and genetic analysis of PD brain donors. B.B. provided technical input to electron microscopy analysis of brain tissue in neurodegeneration. S.F. provided expertise in neuropathology, differentiation of Lewy pathology versus corpora amylacea, and provided optical microscopy data of corpora amylacea and Lewy pathology in same tissues. M.B., H.S., W.D.J.v.d.B. and M.E.L. designed research, analyzed and interpreted the data, and contributed to writing the manuscript.

Corresponding authors

Correspondence to Henning Stahlberg, Wilma D. J. Van de Berg or Matthias E. Lauer.

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Competing interests

A.d.P., J.E., A.S., J. Hoernschemeyer, B.B., M.B. and M.E.L. are full-time employees of Roche/F. Hoffmann–La Roche Ltd, and they may additionally hold Roche stock and/or stock options.

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Integrated supplementary information

Supplementary Figure 1 Histopathalogical analysis of PD brain donors.

(a–d) Immunohistochemical analysis of aSyn pathology. aSyn (KM-51) immunostaining in the CA2 region of the hippocampus of Donor A-PD. (e–h) aSyn (KM-51) immunostaining of the substantia nigra of Donor B-PD. Images shown are from tissues that were taken from the same region of the same brain donors used for the other methods employed in this study, including CLEM and SBFSEM. Scale bars = 50 µm. (i–l) Conventional histopathological aspect of FFPE sample of the substantia nigra obtained from a PD brain donor shows Lewy pathology and corpora amylacea side by side. H&E stained tissue sections; yellow arrowheads indicate CA; black arrowheads indicate Lewy bodies. Note the similar size of the two structures and that they can occur in close proximity to one another. Scale bars: i-k = 10 µm; l = 20 µm.

Supplementary Figure 2 Correlative light and electron microscopy (CLEM) to identify Lewy pathology.

aSyn-immunopositive inclusion from Donor D-PD is shown as an example. The same one inclusion serially sectioned is shown in each white circle in a-g. The same CLEM procedure shown here was applied to identify all Lewy structures in this study. (a) EM montage of 100–200 nm-thick tissue sections collected on an EM grid. (b) Light microscopy montage of aSyn-immunostained adjacent tissue sections (also 100–200nm-thick), overlaid onto the EM montage at 100% opacity. (c) Light microscopy montage overlay at 80% opacity. (d–f) Higher magnification area of the white box depicted in ‘a-c’; black arrowhead indicates blood vessel and white arrowhead indicates nucleus of nearby cell. Dotted white circle shows aSyn-immunopositive inclusion. (e, f) Colored feature represents inclusion, immunostained for aSyn; bound antibody complex detected by Permanent HRP Green Kit (Zytomed Systems), slides were counterstained with hematoxylin. (g) Higher magnification area of the subregion shown in ‘d’ containing the inclusion (dotted circle) and neighboring nucleus. (h) Higher magnification of inclusion. Scale bars a-c = 200 µm, d-f = 20 µm, g = 5 µm, h = 1 µm.

Supplementary Figure 3 CLEM and CLSM to identify Lewy pathology.

(a-f) Light microscopy (LM) image and correlating electron microscopy image (EM) for aSyn inclusions. 150 nm tissue sections collected on LM slides were processed using the LB509 antibody and immunopositive aggregates identified using a peroxidase detection system and green chromogen. Slides were counterstained with hematoxylin in order to identify cellular features for correlation with EM images. The same immunopositive inclusion is indicated (dashed green circle) in an adjacent 150 nm tissue section collected on an EM grid. White arrows indicate tissue features that were used for correlating the LM and EM images. (a) CLEM for Fig. 1d and Supplementary Fig. 8, Donor C-PD, (b-d) CLEM for Supplementary Fig. 6d-f, respectively, Donor D-PDD, (e-f) CLEM for Supplementary Fig. 10 and Fig 6, respectively, Donor E-PD. (g, h) Examples of differential antibody staining for two aSyn-immunopositive inclusions. Adjacent tissue sections were stained with either LB509, phosphorylated aSyn (pSyn; 11a5) antibody or ubiquitin (Ubq). The correlating EM picture for each inclusion is shown. (g) CLEM for Supplementary Fig. 9, Donor D-PDD, (h) CLEM for Fig. 5, Donor D-PDD. All scale bars, a-h = 10 µm. (i) CLSM images from a LB in a neuromelanin-containing neuron in the SN of Donor A-PDD, immunolabeled for alpha-synuclein (LB-509), Serine 129 phosphorylated alpha-synuclein (11A5) and ubiquitin (Ubq).

Supplementary Figure 4 CLEM workflow.

Correlative light and electron microscopy (CLEM) is often used to localize specific molecules of interest within the complex and diverse biological landscape of cells and tissues, typically via genetically encoded fluorescent or enzymatic markers. Light microscopy is first used to visualize wide-field images with limited resolution, essentially providing a map to the labeled structures of interest. Such a map is then used to guide to the structure of interest for higher-resolution visualization by electron microscopy at a smaller imaging window. The general sequence of steps taken to achieve this for PD brain tissue sections is shown. EM = electron microscopy; LM = light microscopy.

Supplementary Figure 5 Correlative light and electron microscopy (CLEM) to identify Lewy neuritis.

(a, b) aSyn-immunopositive inclusion from Donor E-PD is shown as an example. The essential procedure was used to identify all LN in this study. (a) Light microscopy image of aSyn-immunostained adjacent tissue sections (also 100–200 nm-thick); Green colored features represents LN, immunostained for aSyn; bound antibody complex detected by Permanent HRP Green Kit (Zytomed Systems), slides were counterstained with hematoxylin. (b) Corresponding 2D EM image showing the same two regions of LN (circled in yellow) as identified by aSyn immunostaining in ‘a.’ Scale bar = 10 µm. (c) Lewy neurite from Donor B-PD, substantia nigra. 2D EM micrograph indicating the LN (yellow dotted oval) as identified by aSyn immunostaining in adjacent tissue section, and the specific positions where electron tomograms were collected (pink dotted boxes). Higher magnification images of pink dotted boxes represented in Fig. 3. Scale bar = 3 µm.

Supplementary Figure 6 Lewy pathology as identified by CLEM.

Projections of the central 20 slices of each reconstructed 3D tomogram are shown for each aSyn-immunopositive inclusion and surrounding cellular milieu. Feature details (arrowheads) are tabulated in Supplementary Table 1. Additional aSyn-immunopositive Lewy pathological inclusions are shown in Figs. 3, 4 and Supplementary Fig. 512. Donor identities are shown in Table 1. (a) aSyn-immunopositive inclusion in Donor A-PD (Movie 5), (b, c) in Donor B-PD (Supplementary Movies 6, 7), (d–f) in Donor D-PD (Movies 810, CLEM data Supplementary Fig. 3b-d). Scale bars = 1 µm.

Supplementary Figure 7 Filamentous Lewy pathology within neuromelanin-containing organelles.

Identified by CLEM in Donor C-PD. CLEM data shown in Supplementary Fig. 3a 2D electron micrographs showing the ultrastructure of a predominantly filamentous aSyn-immunopositive inclusion (same as shown in Fig. 1d) at (a) low magnification (white dotted circle) in which it can be seen amongst neuromelanin-containing organelles (black high contrast spots), and increasingly higher magnification in (b, c). In addition to filaments and vesicles, distorted mitochondria are also interspersed at the periphery of the inclusion. Scale bars: a = 5 µm; b, c = 1 µm.

Supplementary Figure 8 Membranous Lewy pathology within neuromelanin-containing organelles.

Identified by CLEM in Donor D-PD. CLEM data shown in Supplementary Fig. 3g 2D electron micrographs showing the ultrastructure of a predominantly membranous aSyn-immunopositive inclusion at (a) low magnification (white dotted circle) in which it can be seen amongst neuromelanin-containing organelles (black high contrast spots), and increasingly higher magnification in (b, c). Abundant clustered mitochondria (vesicles with cristae) are interspersed amongst the other notated features. Scale bars: a, b = 5 µm; c = 1 µm.

Supplementary Figure 9 Membranous Lewy pathology within neuromelanin-containing organelles.

Identified by CLEM in Donor E-PD. CLEM data shown in Supplementary Fig. 3f 2D electron micrographs showing the ultrastructure of a predominantly membranous aSyn-immunopositive inclusion at (a) low magnification (white dotted circle) in which it can be seen amongst neuromelanin-containing organelles (black high contrast spots), and increasingly higher magnification in (b, c). Abundant clustered mitochondria (vesicles with cristae) are interspersed amongst the other notated features. Scale bars: a = 5 µm; b = 2 µm; c = 1 µm.

Supplementary Figure 10 Subcellular distribution of aSyn and organelle markers within Lewy pathology without a p-aSyn positive outer layer.

STED microscopy showing distribution of (a) marker for phosphorylated aSyn (pS129), (b) marker for mitochondria (porin VDAC1), (c) marker for lysosomes (LAMP1), (d) overlay of ‘a-c’, (e) higher magnification view of the edge of the aSyn-immunopositive inclusion shown in ‘d’ (f–i) Same STED microscopy and markers as in ‘a-d’, but a different inclusion, showing empty vacuoles that may represent autophagic vacuolar-like structures reminiscent of CLEM (Supplementary Fig. 12), (j) higher magnification view of center of the inclusion shown in ‘i’. (k–n) Same STED microscopy and markers as in ‘a-d,’ but a LN, (o) higher magnification view of the LN as in ‘n.’. Images are representative across 14 PD donors for Lewy structures without the p-aSyn outer layer. Scale bars: d, i, n = 2 µm; e, j, o = 1 µm.

Supplementary Figure 11 Co-localization of lipids with aSyn in Lewy pathology.

Confocal fluorescence light microscopy projected image stacks of snap-frozen 10µm-thick tissue showing aSyn-immunopositive inclusions in the (a–c) hippocampal CA2 region of Donor A-PD, and (d–f) SN of Donor B-PD. Inclusions immunopositive for aSyn are visualized in green (LB509 antibody), lipid-rich structures are visualized in red by Nile Red staining, and nuclei are visualized in blue by DAPI. Column i = aSyn (green), nuclei (blue); Column ii = lipids (red), nuclei (blue); Column iii = overlay of aSyn (green), lipids (red), and nuclei (blue); Column iv = co-localization of aSyn and lipids (yellow). Scale bars: a, d = 20 µm; b, c, e, f = 5 µm.

Supplementary Figure 12 Lipid and protein distributions in Lewy pathology detected by label-free CARS or FTIR imaging combined with correlative immunofluorescence CLSM for aSyn.

In Donor A-PD, CA2. (a) CARS image of lipids in an aSyn-immunopositive inclusion in PD brain tissue, recorded at 2850 cm-1. (b) Projected confocal immunofluorescence stack showing the same area, after immunostaining for aSyn (LB509). (c) Overlay of the CARS and aSyn immunofluorescence data shown in ‘a’ and ‘b’. (d) CARS intensity distribution profiles for lipids and proteins within the area, showing high peaks in the region of the LB. (e) FTIR image of lipids in an aSyn-immunopositive inclusion in PD brain tissue. (f) Projected confocal immunofluorescence stack showing the same area, immunostained for aSyn (LB509). (g) Overlay of the FTIR and aSyn immunofluorescence data shown in e and f. (h) FTIR intensity distribution profiles of lipids and proteins within the inclusion. Scale bars: 20 µm.

Supplementary Figure 13 Detection of the lipid and protein distribution in neuron of control patient by label-free CARS and FTIR.

(a) CARS image of lipids in a neuron of brain tissue, recorded at 2850 cm−1. (b) Confocal fluorescence showing the same area, after staining with Neurotrace (530/615) to stain the Nissl substance. (c) Overlay of the CARS and Neurotrace fluorescence data shown in ‘a’ and ‘b’. (d) CARS intensity distribution profiles for lipids and proteins within the neuron (black circle). These results show decreased lipid and similar protein intensities in neurons compared to neighboring tissue, which is in contrast with the results, that is increased lipids and proteins in Lewy structures (Supplementary Figure 15). (e) FTIR image of lipids in a neuron of brain tissue, recorded at 2850 cm-1. (f) Confocal fluorescence showing the same area, after staining with Neurotrace. (g) Overlay of the CARS and Neurotrace fluorescence data shown in ‘e’ and ‘f’. (h) FTIR intensity distribution profiles for lipids and proteins within the neuron (black circle). These results show decreased lipid and similar protein intensities in neurons compared to neighboring tissue, which is in contrast with the results, that is increased lipids and proteins in Lewy structures (Supplementary Figure. 15).

Supplementary Figure 14 Liquid chromatography (LC) mass spectrometry (MS) and lipidomics reveal lipid content of Lewy pathology.

Predominant peaks in all traces (a–c) represent the presence of phosphatidylcholine (PC) and sphingomyelin (SM) lipids. Mass spectrometric trace of (a) myelin as dissected from corpus callosum of non-neurological control donor, Donor F-Control, (b) laser capture micro-dissected Lewy bodies (~ 2700) from hippocampal CA2 region of Donor A-PD, (c) laser capture micro-dissected Lewy bodies (~ 3,050) from substantia nigra of Donor B-PD. (d) Mass spectrometric traces of controls: dentate gyrus (DG) laser capture micro-dissected, not shown to contain any LB, from hippocampus of Donor A-PD; (e) blank tube, and (f) blank solvent, 5 µl chloroform/MeOH (2:1), as control experiments. Each curve shows MS signal over LC retention time with the m/z window scanning for the ratios indicated at the right end of the curves in ‘a’. These m/z windows select for the lipids indicated above the vertical lines in ‘a’.

Supplementary Figure 15 Hypothetical mechanism for the formation of membranous Lewy pathology in PD.

(a) Organelles including mitochondria as they exist physiologically in the cell, and (b) in the presence of pathologically aggregated or modified aSyn (for example, phosphorylated at Ser129, oxidated, truncated, etc.) together with other protein aggregates. Over time, this may lead to (c) disruption of organellar membranes and (d) further aggregation of organelles and disruption and fragmentation of their lipid membranes. (e) Larger clumps of lipid membrane fragments, aggregated proteins, vesicles and other general membrane bound structures, which compact over time in the restricted cellular environment, would give rise to the ultrastructure of the majority of Lewy pathology (membrane-rich) as observed by the CLEM, SBFSEM and STED methods used in this study.

Supplementary information

Supplementary Figs. 1–15 and Supplementary Tables 1 and 2.

Reporting Summary

Supplementary Video 1

Reconstructed and color-segmented 3D transmission electron tomogram of aSyn-immunopositive inclusion (LB). Corresponds to Fig. 1a, Donor A-PD. Thickness of tissue section imaged ~150 nm.

Supplementary Video 2

Reconstructed and color-segmented 3D transmission electron tomogram of aSyn-immunopositive inclusion (LB). Corresponds to Fig. 1b, Donor B-PD. Thickness of tissue section imaged ~150 nm.

Supplementary Video 3

Reconstructed and color-segmented 3D transmission electron tomogram of aSyn-immunopositive inclusion (LB). Corresponds to Fig. 1c, Donor D-PD. Thickness of tissue section imaged ~150 nm.

Supplementary Video 4

Reconstructed and color-segmented 3D transmission electron tomogram of aSyn-immunopositive inclusion (LB). Corresponds to Fig. 1d, Donor C-PD, S7. Thickness of tissue section imaged ~150 nm.

Supplementary Video 5

Reconstructed and color-segmented 3D transmission electron tomogram of aSyn-immunopositive inclusion (LB). Corresponds to Supplementary Fig. 6a, Donor A-PD. Thickness of tissue section imaged ~150 nm.

Supplementary Video 6

Reconstructed and color-segmented 3D transmission electron tomogram of aSyn-immunopositive inclusion (LB). Corresponds to Supplementary Fig. 6b, Donor B-PD. Thickness of tissue section imaged ~150 nm.

Supplementary Video 7

Reconstructed and color-segmented 3D transmission electron tomogram of aSyn-immunopositive inclusion (LB). Corresponds to Supplementary Fig. 6c, Donor B-PD. Thickness of tissue section imaged ~150 nm.

Supplementary Video 8

Reconstructed and color-segmented 3D transmission electron tomogram of aSyn-immunopositive inclusion (LB). Corresponds to Supplementary Fig. 6d, Donor D-PD. Thickness of tissue section imaged ~150 nm.

Supplementary Video 9

Reconstructed and color-segmented 3D transmission electron tomogram of aSyn-immunopositive inclusion (LB). Corresponds to Supplementary Fig. 6e, Donor D-PD. Thickness of tissue section imaged ~150 nm.

Supplementary Video 10

Reconstructed and color-segmented 3D transmission electron tomogram of aSyn-immunopositive inclusion in neurite (LN). Corresponds to Supplementary Fig. 6f, Donor D-PD. Thickness of tissue section imaged ~150 nm.

Supplementary Video 11

Reconstructed and color-segmented 3D transmission electron tomogram of a region inside aSyn-immunopositive inclusion (LB, Fig. 1a, Donor A-PD) collected at higher magnification. Thickness of tissue section imaged ~150 nm.

Supplementary Video 12

Reconstructed and color-segmented 3D transmission electron tomogram of a region inside aSyn-immunopositive inclusion (LB, Fig. 1a, Donor A-PD) collected at higher magnification. Tailed membrane stacks are clearly visible, as indicated in Fig. 1a (two yellow arrow-heads on right-hand side). Thickness of tissue section imaged ~150 nm.

Supplementary Video 13

Reconstructed and color-segmented 3D transmission electron tomogram of region at the edge of the aSyn-immunopositive inclusion (LB, Fig. 1a, Donor A-PD) collected at higher magnification. A mitochondrion is clearly visible, as indicated in Fig. 2c (white oval). Thickness of tissue section imaged ~150 nm.

Supplementary Video 14

Reconstructed and color-segmented 3D transmission electron tomogram of region at the edge of the aSyn-immunopositive inclusion (LB, Supplementary Fig. 6a, Donor A-PD) collected at higher magnification. Thickness of tissue section imaged ~150 nm.

Supplementary Video 15

Reconstructed and color-segmented 3D transmission electron tomogram of region inside the aSyn-immunopositive inclusion (LB, Supplementary Fig. 6a, Donor A-PD) collected at higher magnification. Cluster of vesicles in separate adjacent compartment to LB is visible as shown in Fig. 2d. Thickness of tissue section imaged ~150 nm.

Supplementary Video 16

Reconstructed and color-segmented 3D transmission electron tomogram of region within an aSyn-immunopositive Lewy neurite (same as shown in Fig. 3a, Donor B-PD) collected at high magnification. Thickness of tissue section imaged ~150 nm.

Supplementary Video 17

Reconstructed and color-segmented 3D transmission electron tomogram of region within an aSyn-immunopositive Lewy neurite (same as shown in Fig. 3b, Donor B-PD) collected at high magnification. Thickness of tissue section imaged ~150 nm.

Supplementary Video 18

Reconstructed and color-segmented 3D transmission electron tomogram of region within a ‘control’ neurite in brain tissue from a non-neurological, age-matched control donor (same as shown in Fig. 3c, Donor F-control) collected at high magnification. Thickness of tissue section imaged ~150 nm.

Supplementary Video 19

Reconstructed and color-segmented 3D transmission electron tomogram of region within a ‘control’ neurite in brain tissue from a non-neurological, age-matched control donor (same as shown in Fig. 3d, Donor F-control) collected at high magnification. Thickness of tissue section imaged ~150 nm.

Supplementary Video 20

Reconstructed serial block-face scanning electron tomograms (SBFSEM) depicting three separate Lewy pathological inclusions within the substantia nigra of Donor B-PD. Scale bar, 5 µm.

Supplementary Video 21

Stimulated emission depletion microscopy showing a Lewy pathological inclusion in the same tissues (Donor B-PD, substantia nigra) as taken from parallel blocks for the SBFSEM ultrastructural analysis (Fig. 5). Thickness of tissue section = 20 µm.

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Shahmoradian, S.H., Lewis, A.J., Genoud, C. et al. Lewy pathology in Parkinson’s disease consists of crowded organelles and lipid membranes. Nat Neurosci 22, 1099–1109 (2019). https://doi.org/10.1038/s41593-019-0423-2

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