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Endogenously regulated Dab2 worsens inflammatory injury in experimental autoimmune encephalomyelitis

Jokubaitis, Vilija G, Gresle, Melissa M, Kemper, Dennis A, Doherty, William, Perreau, Victoria M, Cipriani, Tania L, Jonas, Anna, Shaw, Gerry, Kuhlmann, Tanja, Kilpatrick, Trevor J, Butzkueven, Helmut·2013·Acta Neuropathologica Communications

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Figure 1 Dab2 expression is up-regulated in the murine C57B/6 MOG-EAE model relative to health. a: Realtime PCR quantitation of dab2 gene expression in the spinal cords of MOG EAE-immunised mice was expressed relative to healthy controls. Analysis reveals that dab2 gene expression is positively correlated with disease severity (HC: Healthy Control; Spearmann’s rank correlation r2=0.921, p<0.0001). b: Western blot analysis of Dab2 expression in healthy control mice and those subjected to EAE (grade 3.0). 1. Healthy spinal cord 2. EAE spinal cord 3. Healthy forebrain 4. EAE forebrain 5. Healthy hindbrain 6. EAE hindbrain 7. Healthy spleen 8. EAE spleen. Shown is a marked up-regulation of both the p96 and p67 Dab2 isoforms in the EAE spinal cord and hindbrain. c/d/e: Scale bar represents 200μm c: Dab2 staining is undetectable in the healthy spinal cord. d: A region of EAE spinal cord spared from inflammation shows no Dab2 staining. e: A region of EAE spinal cord containing inflammatory infiltrates (DAPI-positive nuclear accumulation) shows Dab2 staining throughout the lesions.
Figure 1 Dab2 expression is up-regulated in the murine C57B/6 MOG-EAE model relative to health. a: Realtime PCR quantitation of dab2 gene expression in the spinal cords of MOG EAE-immunised mice was expressed relative to healthy controls. Analysis reveals that dab2 gene expression is positively correlated with disease severity (HC: Healthy Control; Spearmann’s rank correlation r2=0.921, p<0.0001). b: Western blot analysis of Dab2 expression in healthy control mice and those subjected to EAE (grade 3.0). 1. Healthy spinal cord 2. EAE spinal cord 3. Healthy forebrain 4. EAE forebrain 5. Healthy hindbrain 6. EAE hindbrain 7. Healthy spleen 8. EAE spleen. Shown is a marked up-regulation of both the p96 and p67 Dab2 isoforms in the EAE spinal cord and hindbrain. c/d/e: Scale bar represents 200μm c: Dab2 staining is undetectable in the healthy spinal cord. d: A region of EAE spinal cord spared from inflammation shows no Dab2 staining. e: A region of EAE spinal cord containing inflammatory infiltrates (DAPI-positive nuclear accumulation) shows Dab2 staining throughout the lesions.CC BY · PubMed Central
Figure 2 Characterization of Dab2 expression within the EAE spinal cord. Immunoflourescent staining for Dab2 in the lumbar expansion in an EAE grade 3.0 C57B/6 wild-type mouse spinal cord. Representative confocal images show a: Dual epifluorescence image showing that the vast majority of CD11b-positive macrophages/microglia also stain positively for Dab2. Dab2-negative microglia (arrowhead). b: Dual epifluoresence image showing that a large proportion of GFAP-positive astrocytes also stain positively for Dab2 (arrows). c: Dual epifluoresence image showing that CD3-positive T-lymphocytes do not express Dab2 within inflammatory infiltrates. Scale bars represent 50 μm.
Figure 2 Characterization of Dab2 expression within the EAE spinal cord. Immunoflourescent staining for Dab2 in the lumbar expansion in an EAE grade 3.0 C57B/6 wild-type mouse spinal cord. Representative confocal images show a: Dual epifluorescence image showing that the vast majority of CD11b-positive macrophages/microglia also stain positively for Dab2. Dab2-negative microglia (arrowhead). b: Dual epifluoresence image showing that a large proportion of GFAP-positive astrocytes also stain positively for Dab2 (arrows). c: Dual epifluoresence image showing that CD3-positive T-lymphocytes do not express Dab2 within inflammatory infiltrates. Scale bars represent 50 μm.CC BY · PubMed Central
Figure 3 Dab2 deletion ameliorates EAE disease severity. a/b: EAE clinical scores of MOG-induced C57B/6 wild-type, Dab2 heterozygote and Dab2 knockout mice. a: Mice heterozygous for Dab2 have significantly repressed EAE disease scores from 14 days post-EAE induction (Mann–Whitney rank sum test; *p<0.05, **p<0.01, ***p<0.001) b: Dab2 knockout mice have significantly repressed EAE disease scores from 12 days post-EAE induction (Mann–Whitney rank sum test; *p<0.05, **p<0.01) c/d: Kaplan-Meier survival curves for wild-type, Dab2 heterozygote and Dab2 knockout mice. c: Figure shows that one hundred percent Dab2 heterozygote mice survived the EAE insult relative to eighty-one percent of wild-type mice (Chi-Square test, p<0.01). d: Figure shows that ninety-six percent of Dab2 knockout mice survived the EAE insult realtive to sixty-seven percent of wild-type mice (Chi-Square test, p<0.05). e/f: Serum phosphorylated neurofilament heavy chain (pNF-H) ELISA measure of axonal injury. e: Figure shows that mice heterozygous for Dab2 have a significantly lower concentration of pNF-H in their serum than wild-type mice (Two-tailed Student’s t-test p<0.01). f: Figure shows that Dab2 knockout mice have significantly less pNF-H in their serum than wild-type mice (Two-tailed Student’s t-test, p<0.01).
Figure 3 Dab2 deletion ameliorates EAE disease severity. a/b: EAE clinical scores of MOG-induced C57B/6 wild-type, Dab2 heterozygote and Dab2 knockout mice. a: Mice heterozygous for Dab2 have significantly repressed EAE disease scores from 14 days post-EAE induction (Mann–Whitney rank sum test; *p<0.05, **p<0.01, ***p<0.001) b: Dab2 knockout mice have significantly repressed EAE disease scores from 12 days post-EAE induction (Mann–Whitney rank sum test; *p<0.05, **p<0.01) c/d: Kaplan-Meier survival curves for wild-type, Dab2 heterozygote and Dab2 knockout mice. c: Figure shows that one hundred percent Dab2 heterozygote mice survived the EAE insult relative to eighty-one percent of wild-type mice (Chi-Square test, p<0.01). d: Figure shows that ninety-six percent of Dab2 knockout mice survived the EAE insult realtive to sixty-seven percent of wild-type mice (Chi-Square test, p<0.05). e/f: Serum phosphorylated neurofilament heavy chain (pNF-H) ELISA measure of axonal injury. e: Figure shows that mice heterozygous for Dab2 have a significantly lower concentration of pNF-H in their serum than wild-type mice (Two-tailed Student’s t-test p<0.01). f: Figure shows that Dab2 knockout mice have significantly less pNF-H in their serum than wild-type mice (Two-tailed Student’s t-test, p<0.01).CC BY · PubMed Central
Figure 4 Histological analysis of the lumbar expansion in Dab2wt/wt, Dab2wt/ko and Dab2ko/ko mice subjected to EAE. Representative sections of the lumbar expansion of the spinal cord of a: a wild-type mouse subjected to EAE b: a Dab2 heterozygote mouse subjected to EAE c: a Dab2 knockout mouse subjected to EAE. a/b/c: Orange lines outline immune infiltrates. Scale bar represents 500μm d: Analysis of average number of immune lesions in the spinal cords of Dab2 heterozygous and knockout mice relative to wild-type controls reveals that mice heterozygous for Dab2 and knockout for Dab2 have a similar number of lesions to wild-type mice (p>0.05) e: Analysis of average infiltrate area reveals that the size of immune infiltrates found in the lumbar expansion of Dab2 heterozygous mice and Dab2 knockout mice is significantly smaller than those found in the lumbar expansion of wild-type mice (***p<0.001) Similarly, the average size of immune infiltrates in the spinal cords of Dab2 knockout mice is significantly smaller those found in Dab2 heterozygous mice (*p<0.05). f: Cell densitometry analysis within the core of EAE lesions reveals that there are no significant differences between the proportional composition of various glial cell types within lesions in wild-type and Dab2 heterozygous mice, however, total cell numbers per lesion are significantly less in Dab2 heterozygous and knockout lesions (**p<0.01). Average ± SEM; One-Way ANOVA, Tukey’s post-hoc test; n ≥ 4 mice/genotype for all above analyses.
Figure 4 Histological analysis of the lumbar expansion in Dab2wt/wt, Dab2wt/ko and Dab2ko/ko mice subjected to EAE. Representative sections of the lumbar expansion of the spinal cord of a: a wild-type mouse subjected to EAE b: a Dab2 heterozygote mouse subjected to EAE c: a Dab2 knockout mouse subjected to EAE. a/b/c: Orange lines outline immune infiltrates. Scale bar represents 500μm d: Analysis of average number of immune lesions in the spinal cords of Dab2 heterozygous and knockout mice relative to wild-type controls reveals that mice heterozygous for Dab2 and knockout for Dab2 have a similar number of lesions to wild-type mice (p>0.05) e: Analysis of average infiltrate area reveals that the size of immune infiltrates found in the lumbar expansion of Dab2 heterozygous mice and Dab2 knockout mice is significantly smaller than those found in the lumbar expansion of wild-type mice (***p<0.001) Similarly, the average size of immune infiltrates in the spinal cords of Dab2 knockout mice is significantly smaller those found in Dab2 heterozygous mice (*p<0.05). f: Cell densitometry analysis within the core of EAE lesions reveals that there are no significant differences between the proportional composition of various glial cell types within lesions in wild-type and Dab2 heterozygous mice, however, total cell numbers per lesion are significantly less in Dab2 heterozygous and knockout lesions (**p<0.01). Average ± SEM; One-Way ANOVA, Tukey’s post-hoc test; n ≥ 4 mice/genotype for all above analyses.CC BY · PubMed Central
Figure 5 qPCR quantification of gene expression in the lumbar expansion of spinal cords of Dab2wt/wt mice (n=15) in acute EAE. Multivariate linear regression analysis reveals that Dab2 gene expression is positively correlated with a: iNOS gene expression (adjusted r2=0.65, p=0.003) and b: IL-1β gene expression (adjusted r2=0.45, p=0.015) when controlling for disease grade 18 days post-EAE induction.
Figure 5 qPCR quantification of gene expression in the lumbar expansion of spinal cords of Dab2wt/wt mice (n=15) in acute EAE. Multivariate linear regression analysis reveals that Dab2 gene expression is positively correlated with a: iNOS gene expression (adjusted r2=0.65, p=0.003) and b: IL-1β gene expression (adjusted r2=0.45, p=0.015) when controlling for disease grade 18 days post-EAE induction.CC BY · PubMed Central
Figure 6 Characterization of Dab2 expression within MS lesions. Immunohistochemical staining of an early acitve lesion (biopsy) and a chronic active lesion. a/b: Luxol Fast Blue/Periodic Acid Schiff stain showing regions of demyelination (pink). c/d: Staining for the macrophage/microglial marker CD68 (brown) showing large numbers of macrophages throughtout both lesion types. e/f: Dab2 staining in the early acitve and chronic active lesion. e: Dab2 staining can be seen throughout the early active lesion expressed by cells of a macrophage morphology. f: Very little perivenular Dab2 staining (arrows) can be found in the chronic active lesion.
Figure 6 Characterization of Dab2 expression within MS lesions. Immunohistochemical staining of an early acitve lesion (biopsy) and a chronic active lesion. a/b: Luxol Fast Blue/Periodic Acid Schiff stain showing regions of demyelination (pink). c/d: Staining for the macrophage/microglial marker CD68 (brown) showing large numbers of macrophages throughtout both lesion types. e/f: Dab2 staining in the early acitve and chronic active lesion. e: Dab2 staining can be seen throughout the early active lesion expressed by cells of a macrophage morphology. f: Very little perivenular Dab2 staining (arrows) can be found in the chronic active lesion.CC BY · PubMed Central

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Authors
Jokubaitis, V. G., Gresle, M. M., Kemper, D. A., Doherty, W., Perreau, V. M., Cipriani, T. L., Jonas, A., Shaw, G., Kuhlmann, T., Kilpatrick, T. J., & Butzkueven, H.
Venue
Acta Neuropathologica Communications
Published
2013-07-09
Volume
1
Issue
1
ISSN
2051-5960
Cited by
31
References
1
Language
en

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