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1 Supplementary Information Title Degeneration and impaired regeneration of gray matter oligodendrocytes in amyotrophic lateral sclerosis Authors Shin H. Kang, Ying Li, Masahiro Fukaya, Ileana Lorenzini, Don W. Cleveland, Lyle W. Ostrow, Jeffrey D. Rothstein, and Dwight E. Bergles
2 Supplementary Fig. 1 Analysis of NG2 + cell proliferation and differentiation in the spinal cord. (a) Schematic showing the three spinal cord regions that were examined for cell proliferation and fate analyses of NG2 + cells and oligodendrocytes. (b) Confocal images of spinal cord sections from Pdgfra-creER;Z/EG mice 4 days after 4HT administration at P30 (P30+4) showing that EGFP expression was restricted to NG2 + cells (PDGF R + Olig2 + ) (c) Confocal images of spinal cord sections from Pdgfra-creER;Z/EG mice two months after administration of 4HT at P30 (P30+60), showing that many EGFP + cells were now CC1 + and PDGF R (white arrowheads), indicating that they had differentiated into oligodendrocytes; other cells remained in the progenitor state (PDGF R + ) (yellow arrows). Scale bars: 20 m. (d - f) Graphs showing the density of EGFP + cells in the ventral gray matter (d), ventral white matter (e), and dorsal gray matter (f) of the spinal cord from PdgfracreER;Z/EG ± SOD1 (G93A) mice. 4HT was administered either at P30 or P60, and mice were examined at P90 or P120. Data are presented as mean + s.e.m. (n = 9 sections from 3 mice per group). * P < 0.05, ** P < , *** P < , unpaired Student s t test.
3 Supplementary Fig. 2 Genetic labeling of spinal cord oligodendrocytes. (a) Fluorescence image of the ventral horn of the spinal cord from a Plp1-creER;ROSA26-EYFP mouse ten days after administration of 4HT at P30 (P30+10). (b) High magnification confocal image of the region highlighted by the yellow box in (a) showing EYFP + cells that were PDGF R and NG2 (white arrowheads). (c) Confocal images of spinal cord showing that EYFP + cells were CC1 + and Olig2 + (yellow arrows). (d) Confocal images showing the presence of EYFP + astrocytes that were GFAP + (asterisk). Astrocytes could be clearly distinguished from oligodendrocytes by their morphology and lack of CC1 immunoreactivity (yellow arrows). Scale bars: 20 m (b - d).
4 Supplementary Fig. 3 Oligodendrocyte-specific expression of EGFP in Mobp-EGFP mice. (a,b) Fluorescence images of spinal cord from an adult Mobp-EGFP mouse (P70) immunostained for EGFP (a) and NG2 (b). (c) High magnification confocal images of spinal cord from a Mobp-EGFP mouse showing co-localization between EGFP, CC1, and Olig2 (yellow arrows), indicating that EGFP is expressed by oligodendrocytes. (d) Confocal images of spinal cord sections immunostained for EGFP, NG2 and PDGF R, indicating that EGFP is not expressed by NG2 + cells in these mice. Scale bars: 20 m.
5 Supplementary Fig. 4 Ablation of spinal motor neurons does not induce reactive changes in NG2 + cells. (a) Transverse section of lumbar spinal cord from a mouse injected with ricin showing NG2 immunoreactivity (brown) and cresyl violet (purple). Scale bar: 200 μm. (b) Magnified views of boxed areas in (a). Boxed areas in upper panel are magnified in lower panels. Note that most motor neurons (arrowheads, blue cell bodies) were lost from the ipsilateral side, but preserved on the contralateral side. NG2 immunoreactivity was similar between the contralateral and ipsilateral sides (black arrows). Red arrows highlight thin NG2 + cell processes. Scale bar: 50 μm. (c) Quantification of motor neurons in the lumbar spinal cord. Data are presented as mean + s.e.m. (n = 14 sections from 3 mice per group) P = , paired Student s t test. (d) Quantification of Ki67 + NG2 + cells. Data are presented as mean + s.e.m. (n = 4 sections from 3 mice per group) P > 0.05, paired Student s t test.
6 Supplementary Fig. 5 Progressive alterations in the morphology of oligodendrocytes in the spinal cord of ALS mice. (a) Fluorescence images from Mobp-EGFP (top panels) and Mobp- EGFP;SOD1 (G93A) mice (bottom panels). In P120 control mice, EGFP was primarily restricted to the somata of oligodendrocytes, whereas in end stage SOD1 (G93A) mice, numerous large EGFP + structures were visible that lacked DAPI + nuclei and were Olig2 (yellow arrows). (b) Images of EGFP + structures from these mice at P90 and end stage SOD1 (G93A) mice. (c) Selected images of EGFP + structures from cells highlighted by yellow squares in (b), showing that EGFP + Olig2 structures (oligodendrocyte somata and putative apoptotic bodies) are present by P90 in SOD1 (G93A) mice (middle panels). Scale bars: 20 m (a) and 5 m (c). (d) Quantification of oligodendrocyte-derived apoptotic bodies. Schematic at right shows how images were processed and left panels show representative images from the ventral gray matter of Mobp-EGFP (P120, control) and end stage Mobp-EGFP;SOD1 (G93A) mice. Sections of spinal cord were immunostained for EGFP, Olig2 and CC1 and imaged using a confocal fluorescence microscope. Surface rendering of EGFP + structures was performed using Imaris, and Olig2 + EGFP + structures (oligodendrocyte somata) were digitally subtracted. The volume of the remaining EGFP + Olig2 structures was determined. In this example, the volume of these putative apoptotic bodies was 89 m 3 in control mice and 1705 m 3 in SOD1 (G93A) mice. Scale bars: 20 m.
7 Supplementary Fig. 6 Degradation of myelinated axons and altered myelin thickness in the spinal cord gray matter of end stage ALS mice. (a) Electron micrograph showing degenerating axons with swollen myelin (white arrowheads) in the spinal cord ventral gray matter of end stage SOD1 (G93A) mice. Scale bar: 1 m. (b) Cumulative plot showing the distribution of g ratios for compact myelin surrounding non-degenerating axons in control mice at P120 and SOD1 (G93A) mice at end stage. (Control, n = 119 axons from 3 mice; SOD1 (G93A), n = 103 axons from 3 mice) (c,d) Scatter plot of g-ratio vs. axon diameter. The g ratio of end stage SOD1 (G93A) mice was significantly smaller than that of P120 control mice. P = , Kolmogorov-Smirnov test.
8 Supplementary Fig. 7 Decrease in myelin protein abundance in the spinal cord of ALS mice. (a) Western blotting of myelin proteins (MBP, MOG, and CNPase) and PDGF R in wild type and SOD1 (G93A) mice and different stages of disease. (b) Graphs showing the protein expression levels in ALS mice relative to control, after normalization to GAPDH. Data are presented as mean ± s.e.m. (n = 3) * P < 0.05, ** P < 0.01, *** P < Student s t test. (c) Luxol fast blue staining of lumbar spinal cord from control (P120) and end stage SOD1 (G93A) mice. Lower panels are magnified images of boxed areas in upper panels. GM, gray matter, WM, white matter.
9 Supplementary Fig. 8 Reactive NG2 + cells and gray matter demyelination in motor cortex gray matter in ALS patients. (a) Sections of motor cortex gray matter from control and ALS patients showing immunoreactivity to NG2 (brown) and Iba1 (blue). NG2 + cells are highlighted by thick arrows, and Iba1 + microglia/macrophages are highlighted by thin arrows if they were NG2 + or arrowheads if they were NG2. Scale bar: 50 m. (b) Quantification of NG2 immunostaining intensity. NG2 + /Iba1 + or NG2 + blood vessel-associated pericytes were excluded from the analysis. NG2 immunostaining intensity in motor cortex gray matter was approximately 2-fold higher than controls (Mean + s.e.m., Control, n = 51 cells from 3 non-als patients; ALS, n = 69 cells from 4 ALS patients, P < , unpaired Student s t test), while no significant difference was found between occipital cortices of control and ALS patients (Mean + s.e.m. Control, n = 38 cells; ALS, n = 33 cells, P > 0.05, unpaired Student s t test). (c) Western blot analysis for MBP and CNPase in four randomly sampled regions from motor (MOT) cortex and occipital (OCC) cortex. This control subject had diffuse Lewy body disease. (d) Quantification of MBP and CNPase expression measured by western blot, normalized to the average of control. Data are presented as mean ± s.e.m. with individual values. * P < 0.05, ** P < 0.01, *** P < One-way ANOVA and post hoc Tukey test.
10 Supplementary Fig. 9 Excision of SOD1 (G37R) from NG2 + cells reduced gliosis. (a) Delayed gliosis at normal age of disease onset after SOD1 (G37R) deletion in oligodendroglia. Images show sections of lumbar spinal cord ventral horn from Pdgfra-creER;loxSOD1 (G37R) mice ± 4HT that were immunostained for GFAP and Iba1. White arrowheads in upper left panel highlight region of increased GFAP immunoreactivity in the ventral horn gray matter. Note the reduced gliosis in sections from 4HT treated mice, as indicated by the lower GFAP and Iba1immunoreactivity. Dashed lines indicate the border between gray and white matter. Scale bar: 100 μm. (b) Immunofluorescence staining showing preservation of mutant SOD1 expression (green) in lumbar spinal cord neurons (SMI32 +, red) in Pdgfra-creER;loxSOD1 (G37R) mice with and without 4HT treatment. Scale bar: 50 μm. (c, d) Western blot showing that SOD1 expression was slightly lower (P = ) in the lumbar spinal cord in 4HT treated mice. Data are presented as mean + s.e.m. ( 4HT, n = 3; +4HT, n = 4), unpaired Student s t test.
11 Supplementary Fig. 10 Abnormal dynamics of gray matter oligodendrocytes and their progenitors in ALS. (a) Schematic representation of the change in oligodendrocyte number in the normal and ALS spinal cord. Black line represents the total number of oligodendrocytes, the red line shows the fate of oligodendrocytes born early in postnatal life, and the blue line show the generation of new oligodendrocytes in adult life (note this shows only net accumulation). The progressive degeneration of early-born oligodendrocytes in ALS enhances oligodendrogenesis to maintain their density. (b) Model derived from genetic fate tracing of both NG2 + cells (green, middle) and oligodendrocytes showing the relationship between NG2 + cell proliferation and oligodendrocyte generation in the normal spinal cord, and oligodendrocyte degeneration and enhanced proliferation and differentiation of NG2 + cells in the ALS spinal cord. The death of early-born oligodendrocytes (red) triggers an increase in differentiation of NG2 + cells to maintain the oligodendrocyte population. NG2 + cell proliferation is enhanced to replace cells that have differentiated (blue), thereby maintaining their density.
12 Supplementary Fig. 11 Full-length pictures of the blots presented in the main figures.
13 Supplementary Table 1. Clinical and demographic information on control and ALS human cases studied, use of tissue, and descriptive analysis of demyelination for each case. Cause of Death Age (at DOD) Sex PMD (hour) Disease duration (year) ALS Mutation Analyzed regions and methods MOT OCC SC Gray Matter demyelination in MOT or SC Control 1 Cardiac failure 74 M 4 NA NA WB WB NA No 2 Cardiac failure 91 F 8 NA NA WB WB NA No Metastatic NA 3 68 F 12 NA WB WB NA No adenocarcinoma 4 Respiratory failure 82 M 50 NA NA LFB; IHC LFB; IHC LFB; IHC No 5 Respiratory failure 81 F 40 NA NA LFB LFB LFB;IHC No 6 Myocardial infarction 56 M 11 NA NA LFB; IHC LFB;IHC LFB No NA 7 Respiratory failure 62 M 8 NA LFB LFB LFB;IHC No 8 Respiratory failure 68 M 12 NA NA LFB; IHC LFB;IHC LFB;IHC No 9 Cardiac failure 42 M 19 NA NA WB WB NA No 10 Cardiac failure 60 M 16 NA NA WB WB NA No 11 Cardiac failure 56 M 28 NA NA WB WB NA No 12 AD 48 F 13 5 NA NA NA WB No 13 AD 66 F NA NA NA WB No 14 AD 74 M 2 14 NA NA NA WB No 15 AD 62 M 6 7 NA NA NA WB No 16 AD 68 M 14 8 NA NA NA WB No 17 ALS Diffuse Lewy body disease 80 M 15 NA C9orf72 neg. WB; ECM; IHC WB; ECM, IHC WB; ECM, IHC 1 FTD/ALS 62 M C9orf72 WB WB NA MOT decreased 2 ALS 59 F 2 2 NK WB WB NA MOT significantly decreased 3 ALS 65 M NK WB WB NA MOT decreased 4 ALS 71 M 17 4 NK WB WB NA MOT significantly decreased 5 ALS 69 F 13 4 NK WB WB NA No 6 ALS 54 M 9 3 NK WB; WB; WB; MOT patchy ECM; IHC ECM; IHC ECM; IHC demyelination 7 ALS 44 M NK WB; WB; WB; MOT patchy ECM; IHC ECM; IHC ECM; IHC demyelination 8 ALS 67 M C9orf72 ECM; IHC ECM; IHC WB No 9 ALS 73 M 54 7 NK LFB; IHC LFB; IHC LFB No 10 ALS 51 F NK LFB; IHC LFB; IHC LFB LSC VH less myelin staining 11 ALS 70 M 48 6 NK LFB; IHC LFB; IHC LFB No 12 ALS 79 M 57 6 NK LFB; IHC LFB; IHC LFB LSC VH less demyelination 13 ALS 67 M NK LFB LFB LFB No 14 ALS 41 M 52 9 NK LFB; IHC LFB; IHC LFB LSC VH less myelin staining 15 ALS 71 M 28 7 NK LFB LFB LFB LSC VH less myelin staining 16 ALS 62 M 8 8 NK LFB; IHC LFB; IHC LFB No 17 ALS 67 M 4 13 NK LFB; IHC LFB; IHC LFB MOT patchy demyelination and VH less myelin staining 18 ALS 77 M 5 6 NK LFB; IHC LFB; IHC LFB indeterminate 19 ALS 63 M 8 NA NK NA NA IHC indeterminate 20 ALS 60 F 9 NA NK NA NA IHC indeterminate 21 ALS 55 M 10 NA NK NA NA IHC Indeterminate 22 ALS 79 F 3 NA NK NA NA IHC indeterminate 23 ALS 47 M 5 NA NK NA NA IHC indeterminate 24 ALS 59 M 5 NA NK LFB; IHC LFB; IHC LFB; IHC LSC VH less myelin staining 25 ALS 68 F NK ECM ECM ECM CSC & TSC VH less myelin staining 26 ALS 63 F C9orf72 LSC VH less myelin ECM ECM ECM neg. staining 27 fals 55 M NA 1.75 A4V CSC & TSC VH less ECM; IHC ECM; IHC ECM SOD1 myelin staining 28 ALS 84 F NK ECM; IHC NA ECM MOT demyelination No
14 29 ALS 65 F NA NA NK ECM; IHC NA NA MOT demyelination 30 ALS 56 F 4 3 C9orf72 repeats + ECM ECM ECM; WB indeterminate 31 ALS 64 F 13 1 NK NA NA ECM indeterminate 32 fals 66 M C9orf72 ECM; IHC ECM; IHC ECM; IHC indeterminate 33 ALS 49 F NK NA NA WB; IHC LSC VH demyelination 34 ALS 71 M NK NA NA WB; IHC No 35 ALS 62 F 5 3 NK NA NA WB; IHC LSC VH demyelination 36 ALS 68 M 11 2 NK NA NA WB; IHC No 37 ALS 61 M NK NA NA WB; IHC No 38 ALS 34 F 5 1 C9orf72 NA NA ECM LSC VH demyelination 39 fals 47 M 5 3 C9orf72 NA NA ECM indeterminate Notes: AD: Alzheimer s disease; fals: familial ALS; C9orf72 neg: C9orf72 repeats negative; CSC: cervical spinal cord; DOD: Date of death; ECM: Erichrome Cyanine R myelin staining; F: female; FTD: frontal-temporal dementia; GM: gray matter; IHC: immunohistochemistry; LFB: Luxol fast blue myelin staining; LSC: Lumbar spinal cord; M: male; MOT: motor cortex; NA: not applicable/available; NK: not known; PMD: postmortem duration; SOD1: superoxide dismutase 1; TSC: thoracic spinal cord; VH: ventral horn; WB: Western blot analysis. c9orf72: c9orf72 repeat expansion and c9orf72 neg: c9orf72 repeat expansion negative. Post mortem delay average (± s.e.m.): 16.2 ± 3 hrs Control (range: 2-50), 15.5 ± 3 hrs ALS (range: 2-57) Median: Control 13 hrs; ALS, 8 hrs (No significant differences between post mortem delay) Mean age (± s.e.m.): Control: 66 ± 3 yrs ALS: 62 ± 2 yrs (No significant difference in ages)
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