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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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Astrogliotic changes in the anterior horns of spinal cords from mSOD1-Tg mice. (A) GFAP immunostaining reveals normal-shaped astrocytic cytoplasm and fine processes in the anterior horns of spinal cords from non-Tg mice, whereas (B) numerous gemistocytes are visible in the anterior horns of spinal cord from mSOD1-Tg mice at 18 weeks of age. (C,D) Immunoreactivities for Cx43 and AQP4 are increased in the anterior horns of the spinal cords of mSOD1-Tg mice. (E,F) By contrast, expressions of EAAT2 are downregulated in the anterior horns of mSOD1-Tg mice compared with non-Tg mice (arrows). (G,H) At higher magnification, immunoreactivity for EAAT2 is markedly diminished in the anterior horns of mSOD1-Tg mice compared with non-Tg mice. Scale bar; 20 μm (A-D,G,H), 200 μm (E,F).
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Figure 2: Astrogliotic changes in the anterior horns of spinal cords from mSOD1-Tg mice. (A) GFAP immunostaining reveals normal-shaped astrocytic cytoplasm and fine processes in the anterior horns of spinal cords from non-Tg mice, whereas (B) numerous gemistocytes are visible in the anterior horns of spinal cord from mSOD1-Tg mice at 18 weeks of age. (C,D) Immunoreactivities for Cx43 and AQP4 are increased in the anterior horns of the spinal cords of mSOD1-Tg mice. (E,F) By contrast, expressions of EAAT2 are downregulated in the anterior horns of mSOD1-Tg mice compared with non-Tg mice (arrows). (G,H) At higher magnification, immunoreactivity for EAAT2 is markedly diminished in the anterior horns of mSOD1-Tg mice compared with non-Tg mice. Scale bar; 20 μm (A-D,G,H), 200 μm (E,F).

Mentions: There was no difference in the morphology of astrocytes in the anterior horns of spinal cords between non-Tg and mSOD1-Tg mice at 12 weeks of age. However, in the mSOD1-Tg mice at 18 and 20 weeks of age, immunoreactivity for GFAP was stage-dependently upregulated and numerous hypertrophic astrocytes existed in the anterior horns compared with non-Tg mice (Figure 2A,B and Additional file 2: Figure S2D–F). Levels of Cx43 and AQP4 were also upregulated in the anterior horns of the spinal cords of all mSOD1-Tg mice examined (Figure 2C,D). Immunoreactivity for Cx30 was preserved in the anterior horns of mSOD1-Tg mice (data not shown). By contrast, immunoreactivity for EAAT2 was diminished in the anterior horns of mSOD1-Tg mice compared with non-Tg mice (Figure 2E-H). Downregulation of EAAT2 in the anterior horns was observed in three of seven mSOD1-Tg mice (42.9%) at 18 weeks of age and two of five mSOD1-Tg mice (40.0%) at 20 weeks of age. There was no significant alteration of any astrocytic or oligodendrocytic markers in mSOD1-Tg mice compared with non-Tg mice at 12 weeks of age.


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)

Astrogliotic changes in the anterior horns of spinal cords from mSOD1-Tg mice. (A) GFAP immunostaining reveals normal-shaped astrocytic cytoplasm and fine processes in the anterior horns of spinal cords from non-Tg mice, whereas (B) numerous gemistocytes are visible in the anterior horns of spinal cord from mSOD1-Tg mice at 18 weeks of age. (C,D) Immunoreactivities for Cx43 and AQP4 are increased in the anterior horns of the spinal cords of mSOD1-Tg mice. (E,F) By contrast, expressions of EAAT2 are downregulated in the anterior horns of mSOD1-Tg mice compared with non-Tg mice (arrows). (G,H) At higher magnification, immunoreactivity for EAAT2 is markedly diminished in the anterior horns of mSOD1-Tg mice compared with non-Tg mice. Scale bar; 20 μm (A-D,G,H), 200 μm (E,F).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4016493&req=5

Figure 2: Astrogliotic changes in the anterior horns of spinal cords from mSOD1-Tg mice. (A) GFAP immunostaining reveals normal-shaped astrocytic cytoplasm and fine processes in the anterior horns of spinal cords from non-Tg mice, whereas (B) numerous gemistocytes are visible in the anterior horns of spinal cord from mSOD1-Tg mice at 18 weeks of age. (C,D) Immunoreactivities for Cx43 and AQP4 are increased in the anterior horns of the spinal cords of mSOD1-Tg mice. (E,F) By contrast, expressions of EAAT2 are downregulated in the anterior horns of mSOD1-Tg mice compared with non-Tg mice (arrows). (G,H) At higher magnification, immunoreactivity for EAAT2 is markedly diminished in the anterior horns of mSOD1-Tg mice compared with non-Tg mice. Scale bar; 20 μm (A-D,G,H), 200 μm (E,F).
Mentions: There was no difference in the morphology of astrocytes in the anterior horns of spinal cords between non-Tg and mSOD1-Tg mice at 12 weeks of age. However, in the mSOD1-Tg mice at 18 and 20 weeks of age, immunoreactivity for GFAP was stage-dependently upregulated and numerous hypertrophic astrocytes existed in the anterior horns compared with non-Tg mice (Figure 2A,B and Additional file 2: Figure S2D–F). Levels of Cx43 and AQP4 were also upregulated in the anterior horns of the spinal cords of all mSOD1-Tg mice examined (Figure 2C,D). Immunoreactivity for Cx30 was preserved in the anterior horns of mSOD1-Tg mice (data not shown). By contrast, immunoreactivity for EAAT2 was diminished in the anterior horns of mSOD1-Tg mice compared with non-Tg mice (Figure 2E-H). Downregulation of EAAT2 in the anterior horns was observed in three of seven mSOD1-Tg mice (42.9%) at 18 weeks of age and two of five mSOD1-Tg mice (40.0%) at 20 weeks of age. There was no significant alteration of any astrocytic or oligodendrocytic markers in mSOD1-Tg mice compared with non-Tg mice at 12 weeks of age.

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