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Extensive dysregulations of oligodendrocytic and astrocytic connexins are associated with disease progression in an amyotrophic lateral sclerosis mouse model.

Cui Y, Masaki K, Yamasaki R, Imamura S, Suzuki SO, Hayashi S, Sato S, Nagara Y, Kawamura MF, Kira J - J Neuroinflammation (2014)

Bottom Line: Non-cell-autonomous motor neuronal death is suggested in a mutant Cu/Zn superoxide dismutase 1 (mSOD1)-mediated amyotrophic lateral sclerosis (ALS) model, in which glial cells play significant roles in disease progression.The levels of Cx47 and Cx32 mRNAs were also decreased at these stages.Our findings indicate that oligodendrocytic and astrocytic GJ proteins in the anterior horns of spinal cord in mSOD1-Tg mice are profoundly affected at the disease-progressive and end stages, where disruption of GJs among glial cells may exacerbate motor neuronal death.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurology, Neurological Institute, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. kira@neuro.med.kyushu-u.ac.jp.

ABSTRACT

Background: Non-cell-autonomous motor neuronal death is suggested in a mutant Cu/Zn superoxide dismutase 1 (mSOD1)-mediated amyotrophic lateral sclerosis (ALS) model, in which glial cells play significant roles in disease progression. Connexins (Cxs) form homotypic or heterotypic gap junctions (GJs) and allow direct intercellular communications among nervous tissue cells. The role of Cxs in motor neuron disease has never been investigated; therefore, we aimed to evaluate alterations of Cxs in mSOD1-transgenic (mSOD1-Tg) mice in comparison with their non-transgenic (non-Tg) littermates at the same ages.

Methods: We pathologically evaluated temporal changes to astrocytic Cx43/Cx30 and oligodendrocytic Cx47/Cx32 immunoreactivities at presymptomatic, disease-progressive, and end stages, relative to aquaporin-4 (AQP4), glial fibrillary acidic protein (GFAP), excitatory amino acid transporter-2 (EAAT2), myelin-oligodendrocyte glycoprotein (MOG), and Nogo-A immunoreactivities, and observed neuronal loss by NeuN and neurofilament immunostaining, and microglial response by Iba-1 immunostaining. We also performed quantitative immunoblotting and real-time PCR analyses for Cxs.

Results: The mSOD1-Tg mice showed neuronal and axonal loss in the anterior horns of the lumbar spinal cord accompanied by increased activation of microglia compared with non-Tg mice at the disease-progressive and end stages. Expression patterns of Cxs were not different between mSOD1-Tg and non-Tg mice at the presymptomatic stage, but immunoreactivities for GFAP, Cx43, Cx30 and AQP4 were increased in the anterior horns of mSOD1-Tg mice at the disease-progressive and end stages. By contrast, Cx47 and Cx32 immunoreactivities were markedly diminished in Nogo-A-positive oligodendrocytes in the anterior horns of mSOD1-Tg mice at the disease-progressive and end stages, especially in oligodendrocytes showing SOD1 accumulation. EAAT2 immunoreactivity was also diminished in the anterior horns of mSOD1-Tg mice at the disease-progressive and end stages. Quantitative immunoblotting revealed a significant reduction in Cx47 and Cx32 protein levels in mSOD1-Tg mice at the disease-progressive and end stages. The levels of Cx47 and Cx32 mRNAs were also decreased at these stages.

Conclusions: Our findings indicate that oligodendrocytic and astrocytic GJ proteins in the anterior horns of spinal cord in mSOD1-Tg mice are profoundly affected at the disease-progressive and end stages, where disruption of GJs among glial cells may exacerbate motor neuronal death.

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Quantitative immunoblot analysis of Cxs in mSOD1-Tg and non-Tg mice at 20 weeks of age. (A) Representative images of GFAP, EAAT2, Cx43, Cx30, Cx32, Cx47, and MOG immunoblots obtained from mSOD1-Tg mice and non-Tg mice (n = 5 per group) at 20 weeks of age. GAPDH blots for loading controls are shown under each protein blot. (B) Results of quantitative analysis for each astrocyte or oligodendrocyte marker. The expression levels of Cx47, Cx32, and EAAT2 are significantly decreased in mSOD1-Tg mice than in non-Tg mice. By contrast, the expression levels of GFAP are significantly increased in mSOD1-Tg mice as compared with non-Tg mice. Cx43, Cx30, and MOG expressions are not significantly different between mSOD1-Tg and non-Tg mice.
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Figure 5: Quantitative immunoblot analysis of Cxs in mSOD1-Tg and non-Tg mice at 20 weeks of age. (A) Representative images of GFAP, EAAT2, Cx43, Cx30, Cx32, Cx47, and MOG immunoblots obtained from mSOD1-Tg mice and non-Tg mice (n = 5 per group) at 20 weeks of age. GAPDH blots for loading controls are shown under each protein blot. (B) Results of quantitative analysis for each astrocyte or oligodendrocyte marker. The expression levels of Cx47, Cx32, and EAAT2 are significantly decreased in mSOD1-Tg mice than in non-Tg mice. By contrast, the expression levels of GFAP are significantly increased in mSOD1-Tg mice as compared with non-Tg mice. Cx43, Cx30, and MOG expressions are not significantly different between mSOD1-Tg and non-Tg mice.

Mentions: We performed immunoblot analyses of Cx proteins at different time points in non-Tg and mSOD1-Tg mice. There was no significant change in any markers of astrocytes or oligodendrocytes in mSOD1-Tg mice compared with non-Tg mice at 12 weeks of age (Additional file 5: Figure S5A,B). At 18 weeks of age, levels of EAAT2 and Cx32 were significantly decreased in mSOD1-Tg mice compared with non-Tg mice (for EAAT2; P = 0.011, for Cx32; P = 0.048), whereas the level of GFAP was increased in mSOD1-Tg mice compared with non-Tg mice (P = 0.025). No statistically significant differences in the levels of Cx43, Cx30, or Cx47 were observed between the two groups (Figure 4A,B). At 20 weeks of age, the levels of Cx47, Cx32, and EAAT2 were significantly reduced in mSOD1-Tg mice compared with those in non-Tg mice (for Cx47, P = 0.009; for Cx32, P = 0.005; for EAAT2, P = 0.024) whereas the levels of Cx43, Cx30, and MOG were not significantly altered between mSOD1-Tg and non-Tg mice. The level of GFAP was increased in mSOD1-Tg mice compared with non-Tg mice at 20 weeks of age (P < 0.001) (Figure 5A,B).


Extensive dysregulations of oligodendrocytic and astrocytic connexins are associated with disease progression in an amyotrophic lateral sclerosis mouse model.

Cui Y, Masaki K, Yamasaki R, Imamura S, Suzuki SO, Hayashi S, Sato S, Nagara Y, Kawamura MF, Kira J - J Neuroinflammation (2014)

Quantitative immunoblot analysis of Cxs in mSOD1-Tg and non-Tg mice at 20 weeks of age. (A) Representative images of GFAP, EAAT2, Cx43, Cx30, Cx32, Cx47, and MOG immunoblots obtained from mSOD1-Tg mice and non-Tg mice (n = 5 per group) at 20 weeks of age. GAPDH blots for loading controls are shown under each protein blot. (B) Results of quantitative analysis for each astrocyte or oligodendrocyte marker. The expression levels of Cx47, Cx32, and EAAT2 are significantly decreased in mSOD1-Tg mice than in non-Tg mice. By contrast, the expression levels of GFAP are significantly increased in mSOD1-Tg mice as compared with non-Tg mice. Cx43, Cx30, and MOG expressions are not significantly different between mSOD1-Tg and non-Tg mice.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 5: Quantitative immunoblot analysis of Cxs in mSOD1-Tg and non-Tg mice at 20 weeks of age. (A) Representative images of GFAP, EAAT2, Cx43, Cx30, Cx32, Cx47, and MOG immunoblots obtained from mSOD1-Tg mice and non-Tg mice (n = 5 per group) at 20 weeks of age. GAPDH blots for loading controls are shown under each protein blot. (B) Results of quantitative analysis for each astrocyte or oligodendrocyte marker. The expression levels of Cx47, Cx32, and EAAT2 are significantly decreased in mSOD1-Tg mice than in non-Tg mice. By contrast, the expression levels of GFAP are significantly increased in mSOD1-Tg mice as compared with non-Tg mice. Cx43, Cx30, and MOG expressions are not significantly different between mSOD1-Tg and non-Tg mice.
Mentions: We performed immunoblot analyses of Cx proteins at different time points in non-Tg and mSOD1-Tg mice. There was no significant change in any markers of astrocytes or oligodendrocytes in mSOD1-Tg mice compared with non-Tg mice at 12 weeks of age (Additional file 5: Figure S5A,B). At 18 weeks of age, levels of EAAT2 and Cx32 were significantly decreased in mSOD1-Tg mice compared with non-Tg mice (for EAAT2; P = 0.011, for Cx32; P = 0.048), whereas the level of GFAP was increased in mSOD1-Tg mice compared with non-Tg mice (P = 0.025). No statistically significant differences in the levels of Cx43, Cx30, or Cx47 were observed between the two groups (Figure 4A,B). At 20 weeks of age, the levels of Cx47, Cx32, and EAAT2 were significantly reduced in mSOD1-Tg mice compared with those in non-Tg mice (for Cx47, P = 0.009; for Cx32, P = 0.005; for EAAT2, P = 0.024) whereas the levels of Cx43, Cx30, and MOG were not significantly altered between mSOD1-Tg and non-Tg mice. The level of GFAP was increased in mSOD1-Tg mice compared with non-Tg mice at 20 weeks of age (P < 0.001) (Figure 5A,B).

Bottom Line: Non-cell-autonomous motor neuronal death is suggested in a mutant Cu/Zn superoxide dismutase 1 (mSOD1)-mediated amyotrophic lateral sclerosis (ALS) model, in which glial cells play significant roles in disease progression.The levels of Cx47 and Cx32 mRNAs were also decreased at these stages.Our findings indicate that oligodendrocytic and astrocytic GJ proteins in the anterior horns of spinal cord in mSOD1-Tg mice are profoundly affected at the disease-progressive and end stages, where disruption of GJs among glial cells may exacerbate motor neuronal death.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurology, Neurological Institute, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. kira@neuro.med.kyushu-u.ac.jp.

ABSTRACT

Background: Non-cell-autonomous motor neuronal death is suggested in a mutant Cu/Zn superoxide dismutase 1 (mSOD1)-mediated amyotrophic lateral sclerosis (ALS) model, in which glial cells play significant roles in disease progression. Connexins (Cxs) form homotypic or heterotypic gap junctions (GJs) and allow direct intercellular communications among nervous tissue cells. The role of Cxs in motor neuron disease has never been investigated; therefore, we aimed to evaluate alterations of Cxs in mSOD1-transgenic (mSOD1-Tg) mice in comparison with their non-transgenic (non-Tg) littermates at the same ages.

Methods: We pathologically evaluated temporal changes to astrocytic Cx43/Cx30 and oligodendrocytic Cx47/Cx32 immunoreactivities at presymptomatic, disease-progressive, and end stages, relative to aquaporin-4 (AQP4), glial fibrillary acidic protein (GFAP), excitatory amino acid transporter-2 (EAAT2), myelin-oligodendrocyte glycoprotein (MOG), and Nogo-A immunoreactivities, and observed neuronal loss by NeuN and neurofilament immunostaining, and microglial response by Iba-1 immunostaining. We also performed quantitative immunoblotting and real-time PCR analyses for Cxs.

Results: The mSOD1-Tg mice showed neuronal and axonal loss in the anterior horns of the lumbar spinal cord accompanied by increased activation of microglia compared with non-Tg mice at the disease-progressive and end stages. Expression patterns of Cxs were not different between mSOD1-Tg and non-Tg mice at the presymptomatic stage, but immunoreactivities for GFAP, Cx43, Cx30 and AQP4 were increased in the anterior horns of mSOD1-Tg mice at the disease-progressive and end stages. By contrast, Cx47 and Cx32 immunoreactivities were markedly diminished in Nogo-A-positive oligodendrocytes in the anterior horns of mSOD1-Tg mice at the disease-progressive and end stages, especially in oligodendrocytes showing SOD1 accumulation. EAAT2 immunoreactivity was also diminished in the anterior horns of mSOD1-Tg mice at the disease-progressive and end stages. Quantitative immunoblotting revealed a significant reduction in Cx47 and Cx32 protein levels in mSOD1-Tg mice at the disease-progressive and end stages. The levels of Cx47 and Cx32 mRNAs were also decreased at these stages.

Conclusions: Our findings indicate that oligodendrocytic and astrocytic GJ proteins in the anterior horns of spinal cord in mSOD1-Tg mice are profoundly affected at the disease-progressive and end stages, where disruption of GJs among glial cells may exacerbate motor neuronal death.

Show MeSH
Related in: MedlinePlus