Title
Transmission of α-synucleinopathy from olfactory structures deep into the temporal lobe
Citation for published article
Mason, D. M., Nouraei, N., Pant, D. B., Miner, K. M., Hutchison, D. F., Luk, K. C., Stolz, J. F., Leak, R. K. (2016). Transmission of α-synucleinopathy from olfactory structures deep into the temporal lobe. Molecular Neurodegeneration, 11(49). doi:10.1186/s13024-016-0113-4
DOI
10.1186/s13024-016-0113-4
Peer Reviewed
1
Document Type
Supplemental Material
School
School of Pharmacy
Abstract
Supplemental files to the publication "Transmission of α-synucleinopathy from olfactory structures deep into the temporal lobe":
- Supplemental information (PDF): Materials and methods, tables, and supplemental figures S1-S8 (all supplemental figures are mentioned in the main text).
- Two mp4 movie files showing perinuclear localization of pSer129 signal (red) around NeuN+ nuclei (green). One movie shows a rotating cell and in the other video, the red pSer129 signal is peeled away to reveal the underlying green NeuN+ nucleus.
- Four high resolution figures (TIFF files).
Rights
© The Author(s). 2016 This article is distributed under the terms of the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver applies to the data made available in this article, unless otherwise stated.
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.
Repository Citation
Mason, D. M., Nouraei, N., Pant, D. B., Miner, K. M., Hutchison, D. F., Luk, K. C., Stolz, J. F., & Leak, R. (2016). Transmission of α-synucleinopathy from olfactory structures deep into the temporal lobe. Molecular Neurodegeneration. https://doi.org/10.1186/s13024-016-0113-4
Supplemental-Information_Methods-Tables-Figs-S1-S8.pdf (55539 kB)
Materials and methods, tables, and supplemental figures S1-S8 (all supplemental figures are mentioned in the main text).
Figure 1 high resolution.tif (75268 kB)
Fig. 1 α-synucleinopathy is transmitted from the OB/AON to deeper rhinencephalic structures. Two month-old CD1 mice were unilaterally infused with α-synuclein fibrils (5 μg) or an equal volume of phosphate-buffered saline (PBS) into the olfactory bulb and adjoining anterior olfactory nucleus (OB/AON). Fibrils were sonicated for 1 h in a waterbath prior to infusion. Three months later, sagittal brain sections were collected and immunostained for pathologically phosphorylated α-synuclein (pSer129; red). Fibril and PBS groups were stained and photographed in parallel. a Large, high-quality microscopic photomontages of pSer129 and nuclear labeling were stitched together and viewed with Adobe Illustrator software on a tablet. Sagittal schematics of only obvious and clearly visible brain cytoarchitectonics (solid lines), myelinated fiber bundles (gray shading), pSer129+ neurites (red flourishes), and pSer129+ somal inclusions (red dots) were then traced with the pencil and paintbrush tools. All abbreviations are listed in Additional file 1: Table S1. b Examples of stitched photomontages of pSer129 immunostaining and DRAQ5 nuclear labeling following fibril infusions in the OB/AON (different sections than drawn in A). Please download the high-resolution supplemental versions of these files at thelink at the end of this article (Additional file 3) or email the corresponding author at leakr@duq.edu for access to the files and zoom in and out of the stitched montages in order to appreciate the density of the pathology, distinguish it from background, and judge the precise anatomical location. c pSer129 and nuclear Hoechst staining in the olfactory peduncle (a-b), the piriform cortex (c-d), and the hippocampus and amygdala (e-f) in PBS and fibril-infused animals. Abbreviations in Additional file 1: Table S1. d Images of pSer129+ inclusions and Hoechst-stained nuclei were captured using a 100× oil objective. The pSer129+ inclusions were perinuclear or found in processes (also see confocal images in Fig. 2b and Additional file 2, which shows two 3D-like videos of pSer129/NeuN labeled cells). The pSer129+ cell in the OB (uppermost row) was in the mitral cell layer, which is known to house large somata. Images of the sparse label in the OB can be viewed in Additional file 1: Figure S1, S2, and the images below Additional file 1: Table S2. e pSer129+ inclusion numbers were counted in ImageJ software by a blinded observer, after the same threshold values were applied across all images. Inclusion counts were 29.8 ± 20.2 (mean ± SEM) for PBS mice and 817.0 ± 242.5 for fibril mice in the olfactory peduncle; 17.2 ± 10.9 for PBS mice and 154.6 ± 35.7 for fibril mice in the piriform cortex; 4.2 ± 2.2 for PBS mice and 140.8 ± 28.3 for fibril mice in the amygdala; 2.0 ± 0.9 for PBS mice and 102.6 ± 45.9 for fibril mice in the hippocampus; 27.4 ± 9.8 for PBS mice and 137.3 ± 50.7 for fibril mice in the entorhinal cortex; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 vs PBS, Student’s t test (n = 4–5 mice/group). f Polyclonal antibodies against pSer129 were preadsorbed with pSer129 blocking peptide or incubated alone for 24 h prior to application to tissue. Shown are adjacent sagittal sections from the same fibril-infused animal (1 h waterbath sonication), captured with equivalent exposures and intensity scaling. Images showing the overlap between monoclonal and polyclonal pSer129 immunostaining can be examined in Additional file 1: Figure S3. Additional preadsorption control data can also be viewed in Additional file 1: Figure S6
Figure 2 high resolution.tif (69682 kB)
Fig. 2 α-synucleinopathy is found within neuronal somata and some of it colocalizes with the ubiquitin marker of protein aggregates. a Two month-old mice were unilaterally infused with α-synuclein fibrils (5 μg) or an equal volume of phosphate-buffered saline (PBS) into the olfactory bulb and adjoining anterior olfactory nucleus (OB/AON). Fibrils were sonicated for 1 h in a waterbath prior to infusion. Three months later, sagittal brain sections were collected and stained with antibodies against pSer129 and the neuronal nuclear marker NeuN. Hoechst-labeled nuclei are shown in blue. Arrows point to some examples of the many triple-labeled cells in the AON. b Confocal microscopy showing the perinuclear localization of pSer129+ structures in the AON. Arrows: examples of Hoechst+/NeuN+/pSer129+ profiles. Arrowhead: Hoechst+/NeuN−/pSer129+ profile. For three-dimensional movies of pSer129+ and NeuN+ cells, see Additional file 2. c Sagittal sections through the OB/AON were stained with the Thioflavin amyloid stain. Nuclei were labeled blue with Hoechst. An additional sagittal level can be viewed in Additional file 1: Figure S4. d Colocalization of ubiquitin, pSer129, and NeuN in five different brain regions in mice that were sacrificed 3 months after fibril infusions in the OB/AON (1 h waterbath sonication). Some, but not all pSer129+ structures were ubiquitin+. The pSer129 and ubiquitin photos were captured from fibril and PBS-treated animals at the same exposures and intensity scaling. To view the results of the second ubiquitin antibody, please see Additional file 1: Figure S5. e Mice were infused in the OB/AON with 5 μg α-synuclein fibrils (1 h waterbath-sonicated) or PBS and perfused 1.5 h later. Brain sections were stained with rabbit antibodies against phosphorylated or total α-synuclein (red). The needle track is still apparent in the caudal OB at this early timepoint (white arrows) and reveals variability in the precise site of infusion, which would result in differences in inclusion counts in those afferent neurons terminating in the OB/AON in highly topographical manners. Images from PBS and fibril groups were captured at the same intensity scaling and exposure times. Differences in background staining are not the result of differential image processing.
Figure 3 high resolution.tif (103108 kB)
Fig. 3 Areas harboring α-synucleinopathy send first-order projections to the site of infusion in the OB/AON. a The retrograde tracer FluoroGold (blue) was infused at the same stereotaxic coordinates as the α-synuclein fibrils. Animals were perfused after 7 days and sagittal sections were stained with the nuclear marker DRAQ5 (purple). Sections were imaged under UV and CY5 illumination. Dual-stained brain regions appear red (not purple or blue) in panel a a, such as the AON and the horizontal limb of the diagonal band (HDB). Dense FluoroGold label at multiple afferent sites is illustrated at higher magnification in panels c, e, g, i, k, and m. Nuclear DRAQ5 label on the same sections is shown in panels b, d, f, h, j, and l. Note the dense FluoroGold labeling in the horizontal limb of the diagonal band of Broca and the absence of FluoroGold label in the olfactory tubercle (Tu; f-g). Neither of these regions developed α-synucleinopathy. The olfactory tubercle is densely innervated by OB/AON projection neurons but does not project reciprocally back to the OB/AON. Abbreviations in Additional file 1: Table S1. b-c The transmission of α-synucleinopathy after infusions of waterbath-sonicated fibrils can be generalized to other neuroanatomical circuits. Two month-old mice were unilaterally infused with α-synuclein fibrils (5 μg) or an equal volume of phosphate-buffered saline (PBS) into CA2/CA3 of the hippocampus. Fibrils were sonicated for 1 h in a waterbath prior to infusion. Three months later, coronal brain sections were collected. Shown are stitched montages of pSer129 and Hoechst staining in the hippocampus and entorhinal cortex in PBS and fibril-infused animals—both groups were stained in parallel and captured at the same exposure and intensity scaling. The fibril injection in this animal extended from CA2/CA3 into CA1. Note the dense label in the entorhinal cortex, suggesting transmission of pathology through the perforant path, and the absence of label in the thalamus and most of the overlying cortex, demonstrating the lack of nonspecific uptake by neighboring sites from diffusion through the cerebrospinal fluid and interstitial space. For the antibody preadsorption control on coronal hippocampal tissue, see Additional file 1: Figure S6. To view the hippocampus at the same level after infusions of fibrils that were sonicated for 24 h, please examine Additional file 1: Figure S7. Abbreviations in Additional file 1: Table S1. d Electron micrographs of α-synuclein fibrils before and after sonication (1 min probe sonication and 1 h or 24 h waterbath sonication). Most sonicated fibrils were positively, not negatively stained with uranyl acetate. To view the pSer129 pathology in the entorhinal cortex and transentorhinal region in panel c, please zoom in and out of this high resolution version of the file.
Figure 4 high resolution.tif (88891 kB)
Fig. 4 α-synucleinopathy transmission from olfactory structures in older mice. Mice (17 months old) were unilaterally infused in the OB/AON with α-synuclein fibrils (1 h waterbath sonication; see study on older animals in main text and in Additional file 1). Three months later, sagittal brain sections were collected and stained for DRAQ5 (a, c, e, g, i) and pSer129 (b, d, f, h, j). In order to fully appreciate the density of the inclusions and the neuroanatomical sublocalization, the reader must zoom in and out of the stitched pSer129 montages in the high-resolution versions of these files at the link at the end of this article (Additional file 3) or email the corresponding author at leakr@duq.edu for access to the files and examine panels k-m. The extent and density of the label in these fibril-infused animals should be contrasted with the lack of inclusions in large stitched montages of PBS-infused animals in Additional file 1: Figure S8. Abbreviations in Additional file 1: Table S1. k-m Higher magnification views of PBS versus fibril-infused cases. Shown are the olfactory peduncle (k), the hippocampus (l), and the piriform cortex (m). The entire brain section of these PBS cases can be fully examined for background staining in Additional file 1: Figure S8. The scale bar for a-j is in panel A and the scale bar for k-m is in panel m
pSer129red NeuNgreen AON.mp4 (984 kB)
Video file showing perinuclear localization of pSer129 signal (red) around NeuN+ nuclei (green). This file shows a rotating cell.
pSer129red NeuNgreen Hoechstblue AON.mp4 (1283 kB)
Video file showing perinuclear localization of pSer129 signal (red) around NeuN+ nuclei (green). In this file, the red pSer129 signal is peeled away to reveal the underlying green NeuN+nucleus.
Materials and methods, tables, and supplemental figures S1-S8 (all supplemental figures are mentioned in the main text).
Figure 1 high resolution.tif (75268 kB)
Fig. 1 α-synucleinopathy is transmitted from the OB/AON to deeper rhinencephalic structures. Two month-old CD1 mice were unilaterally infused with α-synuclein fibrils (5 μg) or an equal volume of phosphate-buffered saline (PBS) into the olfactory bulb and adjoining anterior olfactory nucleus (OB/AON). Fibrils were sonicated for 1 h in a waterbath prior to infusion. Three months later, sagittal brain sections were collected and immunostained for pathologically phosphorylated α-synuclein (pSer129; red). Fibril and PBS groups were stained and photographed in parallel. a Large, high-quality microscopic photomontages of pSer129 and nuclear labeling were stitched together and viewed with Adobe Illustrator software on a tablet. Sagittal schematics of only obvious and clearly visible brain cytoarchitectonics (solid lines), myelinated fiber bundles (gray shading), pSer129+ neurites (red flourishes), and pSer129+ somal inclusions (red dots) were then traced with the pencil and paintbrush tools. All abbreviations are listed in Additional file 1: Table S1. b Examples of stitched photomontages of pSer129 immunostaining and DRAQ5 nuclear labeling following fibril infusions in the OB/AON (different sections than drawn in A). Please download the high-resolution supplemental versions of these files at thelink at the end of this article (Additional file 3) or email the corresponding author at leakr@duq.edu for access to the files and zoom in and out of the stitched montages in order to appreciate the density of the pathology, distinguish it from background, and judge the precise anatomical location. c pSer129 and nuclear Hoechst staining in the olfactory peduncle (a-b), the piriform cortex (c-d), and the hippocampus and amygdala (e-f) in PBS and fibril-infused animals. Abbreviations in Additional file 1: Table S1. d Images of pSer129+ inclusions and Hoechst-stained nuclei were captured using a 100× oil objective. The pSer129+ inclusions were perinuclear or found in processes (also see confocal images in Fig. 2b and Additional file 2, which shows two 3D-like videos of pSer129/NeuN labeled cells). The pSer129+ cell in the OB (uppermost row) was in the mitral cell layer, which is known to house large somata. Images of the sparse label in the OB can be viewed in Additional file 1: Figure S1, S2, and the images below Additional file 1: Table S2. e pSer129+ inclusion numbers were counted in ImageJ software by a blinded observer, after the same threshold values were applied across all images. Inclusion counts were 29.8 ± 20.2 (mean ± SEM) for PBS mice and 817.0 ± 242.5 for fibril mice in the olfactory peduncle; 17.2 ± 10.9 for PBS mice and 154.6 ± 35.7 for fibril mice in the piriform cortex; 4.2 ± 2.2 for PBS mice and 140.8 ± 28.3 for fibril mice in the amygdala; 2.0 ± 0.9 for PBS mice and 102.6 ± 45.9 for fibril mice in the hippocampus; 27.4 ± 9.8 for PBS mice and 137.3 ± 50.7 for fibril mice in the entorhinal cortex; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 vs PBS, Student’s t test (n = 4–5 mice/group). f Polyclonal antibodies against pSer129 were preadsorbed with pSer129 blocking peptide or incubated alone for 24 h prior to application to tissue. Shown are adjacent sagittal sections from the same fibril-infused animal (1 h waterbath sonication), captured with equivalent exposures and intensity scaling. Images showing the overlap between monoclonal and polyclonal pSer129 immunostaining can be examined in Additional file 1: Figure S3. Additional preadsorption control data can also be viewed in Additional file 1: Figure S6
Figure 2 high resolution.tif (69682 kB)
Fig. 2 α-synucleinopathy is found within neuronal somata and some of it colocalizes with the ubiquitin marker of protein aggregates. a Two month-old mice were unilaterally infused with α-synuclein fibrils (5 μg) or an equal volume of phosphate-buffered saline (PBS) into the olfactory bulb and adjoining anterior olfactory nucleus (OB/AON). Fibrils were sonicated for 1 h in a waterbath prior to infusion. Three months later, sagittal brain sections were collected and stained with antibodies against pSer129 and the neuronal nuclear marker NeuN. Hoechst-labeled nuclei are shown in blue. Arrows point to some examples of the many triple-labeled cells in the AON. b Confocal microscopy showing the perinuclear localization of pSer129+ structures in the AON. Arrows: examples of Hoechst+/NeuN+/pSer129+ profiles. Arrowhead: Hoechst+/NeuN−/pSer129+ profile. For three-dimensional movies of pSer129+ and NeuN+ cells, see Additional file 2. c Sagittal sections through the OB/AON were stained with the Thioflavin amyloid stain. Nuclei were labeled blue with Hoechst. An additional sagittal level can be viewed in Additional file 1: Figure S4. d Colocalization of ubiquitin, pSer129, and NeuN in five different brain regions in mice that were sacrificed 3 months after fibril infusions in the OB/AON (1 h waterbath sonication). Some, but not all pSer129+ structures were ubiquitin+. The pSer129 and ubiquitin photos were captured from fibril and PBS-treated animals at the same exposures and intensity scaling. To view the results of the second ubiquitin antibody, please see Additional file 1: Figure S5. e Mice were infused in the OB/AON with 5 μg α-synuclein fibrils (1 h waterbath-sonicated) or PBS and perfused 1.5 h later. Brain sections were stained with rabbit antibodies against phosphorylated or total α-synuclein (red). The needle track is still apparent in the caudal OB at this early timepoint (white arrows) and reveals variability in the precise site of infusion, which would result in differences in inclusion counts in those afferent neurons terminating in the OB/AON in highly topographical manners. Images from PBS and fibril groups were captured at the same intensity scaling and exposure times. Differences in background staining are not the result of differential image processing.
Figure 3 high resolution.tif (103108 kB)
Fig. 3 Areas harboring α-synucleinopathy send first-order projections to the site of infusion in the OB/AON. a The retrograde tracer FluoroGold (blue) was infused at the same stereotaxic coordinates as the α-synuclein fibrils. Animals were perfused after 7 days and sagittal sections were stained with the nuclear marker DRAQ5 (purple). Sections were imaged under UV and CY5 illumination. Dual-stained brain regions appear red (not purple or blue) in panel a a, such as the AON and the horizontal limb of the diagonal band (HDB). Dense FluoroGold label at multiple afferent sites is illustrated at higher magnification in panels c, e, g, i, k, and m. Nuclear DRAQ5 label on the same sections is shown in panels b, d, f, h, j, and l. Note the dense FluoroGold labeling in the horizontal limb of the diagonal band of Broca and the absence of FluoroGold label in the olfactory tubercle (Tu; f-g). Neither of these regions developed α-synucleinopathy. The olfactory tubercle is densely innervated by OB/AON projection neurons but does not project reciprocally back to the OB/AON. Abbreviations in Additional file 1: Table S1. b-c The transmission of α-synucleinopathy after infusions of waterbath-sonicated fibrils can be generalized to other neuroanatomical circuits. Two month-old mice were unilaterally infused with α-synuclein fibrils (5 μg) or an equal volume of phosphate-buffered saline (PBS) into CA2/CA3 of the hippocampus. Fibrils were sonicated for 1 h in a waterbath prior to infusion. Three months later, coronal brain sections were collected. Shown are stitched montages of pSer129 and Hoechst staining in the hippocampus and entorhinal cortex in PBS and fibril-infused animals—both groups were stained in parallel and captured at the same exposure and intensity scaling. The fibril injection in this animal extended from CA2/CA3 into CA1. Note the dense label in the entorhinal cortex, suggesting transmission of pathology through the perforant path, and the absence of label in the thalamus and most of the overlying cortex, demonstrating the lack of nonspecific uptake by neighboring sites from diffusion through the cerebrospinal fluid and interstitial space. For the antibody preadsorption control on coronal hippocampal tissue, see Additional file 1: Figure S6. To view the hippocampus at the same level after infusions of fibrils that were sonicated for 24 h, please examine Additional file 1: Figure S7. Abbreviations in Additional file 1: Table S1. d Electron micrographs of α-synuclein fibrils before and after sonication (1 min probe sonication and 1 h or 24 h waterbath sonication). Most sonicated fibrils were positively, not negatively stained with uranyl acetate. To view the pSer129 pathology in the entorhinal cortex and transentorhinal region in panel c, please zoom in and out of this high resolution version of the file.
Figure 4 high resolution.tif (88891 kB)
Fig. 4 α-synucleinopathy transmission from olfactory structures in older mice. Mice (17 months old) were unilaterally infused in the OB/AON with α-synuclein fibrils (1 h waterbath sonication; see study on older animals in main text and in Additional file 1). Three months later, sagittal brain sections were collected and stained for DRAQ5 (a, c, e, g, i) and pSer129 (b, d, f, h, j). In order to fully appreciate the density of the inclusions and the neuroanatomical sublocalization, the reader must zoom in and out of the stitched pSer129 montages in the high-resolution versions of these files at the link at the end of this article (Additional file 3) or email the corresponding author at leakr@duq.edu for access to the files and examine panels k-m. The extent and density of the label in these fibril-infused animals should be contrasted with the lack of inclusions in large stitched montages of PBS-infused animals in Additional file 1: Figure S8. Abbreviations in Additional file 1: Table S1. k-m Higher magnification views of PBS versus fibril-infused cases. Shown are the olfactory peduncle (k), the hippocampus (l), and the piriform cortex (m). The entire brain section of these PBS cases can be fully examined for background staining in Additional file 1: Figure S8. The scale bar for a-j is in panel A and the scale bar for k-m is in panel m
pSer129red NeuNgreen AON.mp4 (984 kB)
Video file showing perinuclear localization of pSer129 signal (red) around NeuN+ nuclei (green). This file shows a rotating cell.
pSer129red NeuNgreen Hoechstblue AON.mp4 (1283 kB)
Video file showing perinuclear localization of pSer129 signal (red) around NeuN+ nuclei (green). In this file, the red pSer129 signal is peeled away to reveal the underlying green NeuN+nucleus.
Comments
Daniel M. Mason and Negin Nouraei contributed equally to this publication.
Rehana Leak is the corresponding author at 407 Mellon Hall, 600 Forbes Ave, Duquesne University, Pittsburgh, PA 15282, United States.