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Modeling Alexander disease with patient iPSCs reveals cellular and molecular pathology of astrocytes.

Kondo T, Funayama M, Miyake M, Tsukita K, Era T, Osaka H, Ayaki T, Takahashi R, Inoue H - Acta Neuropathol Commun (2016)

Bottom Line: Alexander disease is a fatal neurological illness characterized by white-matter degeneration and formation of Rosenthal fibers, which contain glial fibrillary acidic protein as astrocytic inclusion.We established induced pluripotent stem cells from Alexander disease patients, and differentiated induced pluripotent stem cells into astrocytes.Alexander disease patient astrocytes exhibited Rosenthal fiber-like structures, a key Alexander disease pathology, and increased inflammatory cytokine release compared to healthy control.

View Article: PubMed Central - PubMed

Affiliation: Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.

ABSTRACT
Alexander disease is a fatal neurological illness characterized by white-matter degeneration and formation of Rosenthal fibers, which contain glial fibrillary acidic protein as astrocytic inclusion. Alexander disease is mainly caused by a gene mutation encoding glial fibrillary acidic protein, although the underlying pathomechanism remains unclear. We established induced pluripotent stem cells from Alexander disease patients, and differentiated induced pluripotent stem cells into astrocytes. Alexander disease patient astrocytes exhibited Rosenthal fiber-like structures, a key Alexander disease pathology, and increased inflammatory cytokine release compared to healthy control. These results suggested that Alexander disease astrocytes contribute to leukodystrophy and a variety of symptoms as an inflammatory source in the Alexander disease patient brain. Astrocytes, differentiated from induced pluripotent stem cells of Alexander disease, could be a cellular model for future translational medicine.

No MeSH data available.


Related in: MedlinePlus

Pathway analysis revealed altered status of cell-adhesion, mTOR, and cytokine release in Alexander disease astrocytes. a Schema of pathway analysis and upstream prediction analysis. b Whole-cell lysates of iPSC-derived astrocytes were prepared and equivalent amounts of total protein were loaded per lane on a polyacrylamide gel for Western blot (WB) analysis using N-cadherin or GAPDH antibodies shown on the left. c Astrocytes were immunostained with an antibody against N-cadherin (green color) and F-actin (red color) was also visualized by using Rhodamine phalloidin. Scale bar = 10 μm. d Whole-cell lysates were prepared and equivalent amounts of total protein were loaded per lane on a polyacrylamide gel for WB analysis using the panel of antibodies shown on the left. The values shown below each blot represent the ratio of phosphorylated/total band densities, calculated and normalized to the ratio in “HC1” astrocytes. e Quantification of cytokine release from iPSC-derived astrocytes. Gray/pink-colored columns indicate astrocytes of healthy controls/Alexander disease. Black-colored column indicates healthy astrocytes with addition of LPS as positive control of cytokine release. LPS: lipopolysaccharide. (*, p < 0.05, N.S.: not significant) Data represent mean ± SD (biological replicates, n = 3)
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Fig6: Pathway analysis revealed altered status of cell-adhesion, mTOR, and cytokine release in Alexander disease astrocytes. a Schema of pathway analysis and upstream prediction analysis. b Whole-cell lysates of iPSC-derived astrocytes were prepared and equivalent amounts of total protein were loaded per lane on a polyacrylamide gel for Western blot (WB) analysis using N-cadherin or GAPDH antibodies shown on the left. c Astrocytes were immunostained with an antibody against N-cadherin (green color) and F-actin (red color) was also visualized by using Rhodamine phalloidin. Scale bar = 10 μm. d Whole-cell lysates were prepared and equivalent amounts of total protein were loaded per lane on a polyacrylamide gel for WB analysis using the panel of antibodies shown on the left. The values shown below each blot represent the ratio of phosphorylated/total band densities, calculated and normalized to the ratio in “HC1” astrocytes. e Quantification of cytokine release from iPSC-derived astrocytes. Gray/pink-colored columns indicate astrocytes of healthy controls/Alexander disease. Black-colored column indicates healthy astrocytes with addition of LPS as positive control of cytokine release. LPS: lipopolysaccharide. (*, p < 0.05, N.S.: not significant) Data represent mean ± SD (biological replicates, n = 3)

Mentions: From the results of cellular adherence pathway analysis, the expression of cell adhesion molecules (CAM), including the cadherin family, was altered in AxD astrocytes. In iPSC-derived astrocytes of AxD, gene expression and protein level of N-cadherin were increased, and those of E-cadherin were decreased (Additional file 2: Figure S2 and Fig. 6b). Cadherin is known to play an important role in the interactions between cells or their surrounding matrix, and also to affect the cell morphology [15]. The majority of iPSC-derived astrocytes showed polygonal shape, and less than 20 % showed stellate or star-like shape. However, we could not find any distinct difference in cell shape between control and AxD astrocytes. To investigate the detailed structural changes in AxD astrocytes, we performed an immunfluorescence study of N-cadherin and F-actin. The signal intensity of N-cadherin was increased in AxD astrocytes, but the distribution of N-cadherin or F-actin was similar between control and AxD (Fig. 6c).Fig. 6


Modeling Alexander disease with patient iPSCs reveals cellular and molecular pathology of astrocytes.

Kondo T, Funayama M, Miyake M, Tsukita K, Era T, Osaka H, Ayaki T, Takahashi R, Inoue H - Acta Neuropathol Commun (2016)

Pathway analysis revealed altered status of cell-adhesion, mTOR, and cytokine release in Alexander disease astrocytes. a Schema of pathway analysis and upstream prediction analysis. b Whole-cell lysates of iPSC-derived astrocytes were prepared and equivalent amounts of total protein were loaded per lane on a polyacrylamide gel for Western blot (WB) analysis using N-cadherin or GAPDH antibodies shown on the left. c Astrocytes were immunostained with an antibody against N-cadherin (green color) and F-actin (red color) was also visualized by using Rhodamine phalloidin. Scale bar = 10 μm. d Whole-cell lysates were prepared and equivalent amounts of total protein were loaded per lane on a polyacrylamide gel for WB analysis using the panel of antibodies shown on the left. The values shown below each blot represent the ratio of phosphorylated/total band densities, calculated and normalized to the ratio in “HC1” astrocytes. e Quantification of cytokine release from iPSC-derived astrocytes. Gray/pink-colored columns indicate astrocytes of healthy controls/Alexander disease. Black-colored column indicates healthy astrocytes with addition of LPS as positive control of cytokine release. LPS: lipopolysaccharide. (*, p < 0.05, N.S.: not significant) Data represent mean ± SD (biological replicates, n = 3)
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Fig6: Pathway analysis revealed altered status of cell-adhesion, mTOR, and cytokine release in Alexander disease astrocytes. a Schema of pathway analysis and upstream prediction analysis. b Whole-cell lysates of iPSC-derived astrocytes were prepared and equivalent amounts of total protein were loaded per lane on a polyacrylamide gel for Western blot (WB) analysis using N-cadherin or GAPDH antibodies shown on the left. c Astrocytes were immunostained with an antibody against N-cadherin (green color) and F-actin (red color) was also visualized by using Rhodamine phalloidin. Scale bar = 10 μm. d Whole-cell lysates were prepared and equivalent amounts of total protein were loaded per lane on a polyacrylamide gel for WB analysis using the panel of antibodies shown on the left. The values shown below each blot represent the ratio of phosphorylated/total band densities, calculated and normalized to the ratio in “HC1” astrocytes. e Quantification of cytokine release from iPSC-derived astrocytes. Gray/pink-colored columns indicate astrocytes of healthy controls/Alexander disease. Black-colored column indicates healthy astrocytes with addition of LPS as positive control of cytokine release. LPS: lipopolysaccharide. (*, p < 0.05, N.S.: not significant) Data represent mean ± SD (biological replicates, n = 3)
Mentions: From the results of cellular adherence pathway analysis, the expression of cell adhesion molecules (CAM), including the cadherin family, was altered in AxD astrocytes. In iPSC-derived astrocytes of AxD, gene expression and protein level of N-cadherin were increased, and those of E-cadherin were decreased (Additional file 2: Figure S2 and Fig. 6b). Cadherin is known to play an important role in the interactions between cells or their surrounding matrix, and also to affect the cell morphology [15]. The majority of iPSC-derived astrocytes showed polygonal shape, and less than 20 % showed stellate or star-like shape. However, we could not find any distinct difference in cell shape between control and AxD astrocytes. To investigate the detailed structural changes in AxD astrocytes, we performed an immunfluorescence study of N-cadherin and F-actin. The signal intensity of N-cadherin was increased in AxD astrocytes, but the distribution of N-cadherin or F-actin was similar between control and AxD (Fig. 6c).Fig. 6

Bottom Line: Alexander disease is a fatal neurological illness characterized by white-matter degeneration and formation of Rosenthal fibers, which contain glial fibrillary acidic protein as astrocytic inclusion.We established induced pluripotent stem cells from Alexander disease patients, and differentiated induced pluripotent stem cells into astrocytes.Alexander disease patient astrocytes exhibited Rosenthal fiber-like structures, a key Alexander disease pathology, and increased inflammatory cytokine release compared to healthy control.

View Article: PubMed Central - PubMed

Affiliation: Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.

ABSTRACT
Alexander disease is a fatal neurological illness characterized by white-matter degeneration and formation of Rosenthal fibers, which contain glial fibrillary acidic protein as astrocytic inclusion. Alexander disease is mainly caused by a gene mutation encoding glial fibrillary acidic protein, although the underlying pathomechanism remains unclear. We established induced pluripotent stem cells from Alexander disease patients, and differentiated induced pluripotent stem cells into astrocytes. Alexander disease patient astrocytes exhibited Rosenthal fiber-like structures, a key Alexander disease pathology, and increased inflammatory cytokine release compared to healthy control. These results suggested that Alexander disease astrocytes contribute to leukodystrophy and a variety of symptoms as an inflammatory source in the Alexander disease patient brain. Astrocytes, differentiated from induced pluripotent stem cells of Alexander disease, could be a cellular model for future translational medicine.

No MeSH data available.


Related in: MedlinePlus