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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

High resolution imaging of aggregates in AxD astrocytes with 3D-SIM or electron microscopy. a Super resolution imaging of GFAP aggregates with dot-like pattern showed accumulated particles of both GFAP and alpha-B crystalline (CRYAB). Scale bar = 200 nm. b Electron microscopy of AxD astrocytes. Electron-dense amorphous-appearing structures (open arrow head) or granular (closed arrow head) structures, surrounded by filamentous structure (*). Scale bars = 100 nm
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Fig4: High resolution imaging of aggregates in AxD astrocytes with 3D-SIM or electron microscopy. a Super resolution imaging of GFAP aggregates with dot-like pattern showed accumulated particles of both GFAP and alpha-B crystalline (CRYAB). Scale bar = 200 nm. b Electron microscopy of AxD astrocytes. Electron-dense amorphous-appearing structures (open arrow head) or granular (closed arrow head) structures, surrounded by filamentous structure (*). Scale bars = 100 nm

Mentions: To evaluate the in vitro recapitulation of Rosenthal fibers, we visualized GFAP of iPSC-derived astrocytes by immunofluorescent staining. Nearly all iPSC-derived astrocytes showed positive staining of GFAP (Fig. 3a and b). GFAP of healthy control astrocytes formed fine filaments distributed throughout the cytoplasm in a cytoskeletal array (Fig. 3a, panels of HC1, 2, and 3). In contrast, a proportion of GFAP in AxD astrocytes formed fibrous aggregates, similar to Rosenthal fibers of AxD brain, and also small dot-like patterns (Fig. 3a, panels of Alex 1, 2, and 3). These fibrous aggregates were formed in 5-10 % of AxD, and were rarely observed in healthy controls. Small dot-like aggregates were formed in 15-20 % of AxD and in a few of the healthy controls (Fig. 3c). To characterize small dot-like inclusions in detail, we visualized GFAP-positive dots using super-resolution structured illumination microscopy (N-SIM system). GFAP-positive dots, with a diameter of 50-200 nm, showed a cloud-like amorphous structure, adjacent to normal GFAP filament, and were co-immunostained with alpha-B crystallin particles (Fig. 4a). In addition to super-resolution microscopy, we observed cytosolic aggregates in AxD astrocytes by using electron microscopy. In AxD astrocytes, electron-attenuated, granular, or amorphous-appearing structures, surrounded by filamentous structure, were observed and determined as Rosenthal fiber-like structures (Fig. 4b). Overexpression of GFAP might contribute to astrocyte dysfunction in AxD as was shown in initial studies of overexpressing wild type GFAP in transgenic mice, which resulted in the formation of Rosenthal fibers indistinguishable from those found in Alexander disease patients. To investigate the GFAP dose effects on aggregates formation, we quantified the GFAP expression and found increased GFAP in AxD astrocytes (Additional file 1: Figure S1).Fig. 4


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)

High resolution imaging of aggregates in AxD astrocytes with 3D-SIM or electron microscopy. a Super resolution imaging of GFAP aggregates with dot-like pattern showed accumulated particles of both GFAP and alpha-B crystalline (CRYAB). Scale bar = 200 nm. b Electron microscopy of AxD astrocytes. Electron-dense amorphous-appearing structures (open arrow head) or granular (closed arrow head) structures, surrounded by filamentous structure (*). Scale bars = 100 nm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4940830&req=5

Fig4: High resolution imaging of aggregates in AxD astrocytes with 3D-SIM or electron microscopy. a Super resolution imaging of GFAP aggregates with dot-like pattern showed accumulated particles of both GFAP and alpha-B crystalline (CRYAB). Scale bar = 200 nm. b Electron microscopy of AxD astrocytes. Electron-dense amorphous-appearing structures (open arrow head) or granular (closed arrow head) structures, surrounded by filamentous structure (*). Scale bars = 100 nm
Mentions: To evaluate the in vitro recapitulation of Rosenthal fibers, we visualized GFAP of iPSC-derived astrocytes by immunofluorescent staining. Nearly all iPSC-derived astrocytes showed positive staining of GFAP (Fig. 3a and b). GFAP of healthy control astrocytes formed fine filaments distributed throughout the cytoplasm in a cytoskeletal array (Fig. 3a, panels of HC1, 2, and 3). In contrast, a proportion of GFAP in AxD astrocytes formed fibrous aggregates, similar to Rosenthal fibers of AxD brain, and also small dot-like patterns (Fig. 3a, panels of Alex 1, 2, and 3). These fibrous aggregates were formed in 5-10 % of AxD, and were rarely observed in healthy controls. Small dot-like aggregates were formed in 15-20 % of AxD and in a few of the healthy controls (Fig. 3c). To characterize small dot-like inclusions in detail, we visualized GFAP-positive dots using super-resolution structured illumination microscopy (N-SIM system). GFAP-positive dots, with a diameter of 50-200 nm, showed a cloud-like amorphous structure, adjacent to normal GFAP filament, and were co-immunostained with alpha-B crystallin particles (Fig. 4a). In addition to super-resolution microscopy, we observed cytosolic aggregates in AxD astrocytes by using electron microscopy. In AxD astrocytes, electron-attenuated, granular, or amorphous-appearing structures, surrounded by filamentous structure, were observed and determined as Rosenthal fiber-like structures (Fig. 4b). Overexpression of GFAP might contribute to astrocyte dysfunction in AxD as was shown in initial studies of overexpressing wild type GFAP in transgenic mice, which resulted in the formation of Rosenthal fibers indistinguishable from those found in Alexander disease patients. To investigate the GFAP dose effects on aggregates formation, we quantified the GFAP expression and found increased GFAP in AxD astrocytes (Additional file 1: Figure S1).Fig. 4

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