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

Modeling Alexander disease with patient iPSCs reveals cellular and molecular pathology of astrocytes 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. Keywords: Alexander disease (AxD), Glial fibrillary acidic protein (GFAP), Induced pluripotent stem cells (iPSCs), Disease modeling, Astrocytes, Rosenthal fibers, Heat-shock protein, Alpha-crystallin, Cytokine, Inflammatory response, Inherited astrocytopathy Introduction mutations opened the way to the development of model Alexander disease (AxD) was first described by W. S. systems using tissue culture cells and transgenic mice for Alexander [1]. The clinical phenotypes of AxD are the study of AxD. Transgenic models recapitulated GFAP macrocephaly, frontal leukodystrophy and a variety of aggregations. However, it remained unclear how AxD developmental delays with epileptic seizures, dysphagia, mutations lead to protein aggregation in patient astrocytes or bulbar/pseudobulbar signs. However, the severity of as well as how mutant GFAP-expressing astrocytes these clinical features differs among patients, being contribute to neuronal degeneration [6]. mostly dependent on the age of onset [2]. In 2007, the discovery of a combination of transcription The common neuropathological feature of AxD is the factors that could reprogram somatic cells into cells exhi- presence of Rosenthal fibers, a unique cytoplasmic inclu- biting pluripotency, called induced pluripotent stem cells sion within astrocytes. Rosenthal fibers contain glial (iPSCs), has provided researchers with a revolutionary tool fibrillary acidic protein (GFAP), major astrocytic inter- to study human biology and diseases [7]. iPSCs can be de- mediate filament protein and molecular chaperones, rived from many somatic cell types, including easily access- including alpha-B-crystallin and other heat shock pro- ible dermal fibroblasts and peripheral blood mononuclear teins [3, 4]. After extensive neuropathological investiga- cells [8, 9]. Similar to human embryonic stem cells tions, missense mutations in GFAP have been identified as (hESCs), iPSCs can self-renew and expand indefinitely in a genetic basis for AxD [5]. The discovery of the GFAP culture [7]. More importantly, they share the capacity to generate any cell types in the body, a property that is par- ticularly useful for the study of neurological diseases. The * Correspondence: haruhisa@cira.kyoto-u.ac.jp Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 pluripotency of iPSCs enables the production of astrocytes Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan for disease modeling [10–12]. This remarkable feature of Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kondo et al. Acta Neuropathologica Communications (2016) 4:69 Page 2 of 12 iPSCs facilitates the study of brain cell types that are diffi- Thermo Fisher Scientific), 0.1 M 2-mercaptoethanol cult to obtain from living individuals, including human as- (Thermo Fisher Scientific), and 0.5 % penicillin/strepto- trocytes. To directly examine AxD patient astrocytes, we mycin. The medium was replaced every other day, and the established iPSCs from three AxD patients, with different EB after 8 days was cultured for another 8 days in DMEM GFAP mutations, respectively, and three healthy individ- comprising 10 % FBS on a gelatin-coated coverslip. uals without GFAP mutation. In a recent review of more than 215 cases of AxD with GFAP mutation, AxD was di- Differentiation and enrichment of astrocytes vided into 2 groups: type I was characterized by early on- Human iPSCs were dissociated to single cells and quickly set, seizures, megalencephaly, and typical leukodystrophy reaggregated in U-bottom 96-well plates for suspension cul- as MRI features, and type II with a later age at onset ture (Greiner Bio-One, Frickenhausen, Germany), pre- characterized by brainstem features and atypical MRI find- coated with 2 % Pluronic F-127 (Sigma-Aldrich, St. Louis, ings [13]. Two patients in this study showed type I clinical MO) in 100 % ethanol. Cell aggregates, called embryoid phenotype and one patient showed type II clinical pheno- bodies (EBs), were cultured in ‘DFK5% medium’ (DFK5%; type. In this study, AxD iPSC-derived astrocytes showed DMEM/F12 (Thermo Fisher Scientific) supplemented with GFAP-positive aggregates, like Rosenthal fibers, and also 5 % v/v KSR, 1x NEAA, 1x Glutamax (Thermo Fisher exhibited altered cytokine release. The strategy of this Scientific), 0.1 M 2-mercaptoethanol (Thermo Fisher studywas to providean effectiveand versatilewayof Scientific)) with 2 μM dorsomorphin (Sigma-Aldrich) and pathogenic investigation and drug screening for AxD and 10 μM SB431542 (Cayman Chemical, Ann Arbor, MI) in a other astrocyte-relevant diseases. neural inductive stage (day 0 to 8). After neural induction, EBs were transferred onto Matrigel (Corning, Tewksbury, Materials and methods MA)-coated 6-well culture plates and cultured in DFK5% Human subjects supplemented with 1x N2 supplement (Thermo Fisher Skin or blood samples were obtained from healthy Scientific) and 2 μM dorsomorphin in the patterning stage controls or patients with Alexander disease. The study (day 8 to 24). A large number of neural stem cells was approved by the Institutional Review Board and (NESTIN-positive) were observed to migrate from the EB Ethics Committees of the University of Kyoto and core. After the patterning stage, migrated neural stem cells Kumamoto University and written informed consent was were separated from the plate bottom using Accutase obtained from all participants in this study. (Innovative Cell Technologies, Inc., San Diego, CA) and cultured in Neurobasal medium FULL, Neurobasal Medium (Thermo Fisher Scientific) supplemented with 1x Generation of human iPSCs In this application study, we used dermal fibroblasts N2 supplement, 1x Glutamax, 10 ng/ml BDNF (Peprotech, Rocky Hill, NJ), 10 ng/ml GDNF (Peprotech) and 10 ng/ml or blood cells as patient somatic cells to prepare iPSCs [8, 12, 14]. For the iPSC clones of HC1, HC2, HC3, NT-3 (Peprotech) on Matrigel-coated 6-well culture plates or cover-slips (day 24 to 60). At day 60, iPS-derived neural Alex1, and Alex3, episomal vectors were used to introduce a reprogramming factor (SOX2, KLF4, OCT4, L-MYC, cells were plated at 400,000-2,000,000 cells per 90-mm dish without any coating in DMEM/F12 Glutamax (Thermo LIN28, siRNA for p53) to the somatic cells, which were seeded onto SNL feeder cells. The next day, the medium Fisher Scientific) supplemented with 1x N2 supplement, 10 ng/ml EGF (Peprotech), 12 ng/ml basic FGF (Peprotech) was changed from a dermal fibroblast medium to a human ES cell medium (ReproCell, Yokohama, Japan) and 2 μg/ml heparin (Nacalai Tesque, Kyoto, Japan). After passage, neurons could not attach to a non-coated polystyr- comprising 4 ng/mL of bFGF (Wako Chemicals, Osaka, Japan); the medium was replaced every other day, and ene dish surface or they died by anoikis. On the other hand, astrocytes and a limited number of oligodendrocyte precu- after 30 days, about 20 iPSC colonies were picked up. Later, the presence or absence of residual plasmid was sors could attach and proliferate. By repeated passage in the same manner at days 90, 120, 150 and 180, astrocytes confirmed by PCR, and clones without residual plasmid were selected. Selected clones were run through karyotype increased their own abundance ratio and showed positive GFAP immunostaining. analysis, and normal karyotype clones were analyzed. For the iPSC clones of Alex2, human iPSCs were generated by using Sendai virus vector as described previously [14]. Immunofluorescent study The iPSCs or differentiated astrocytes were immobilized In vitro differentiation into three germ layers at room temperature for 30 min in 4 % paraformal- CTK was used to harvest the iPSCs, and an embryoid dehyde (pH 7.4), and washed with PBS. The cells were body (EB) was formed [12]. Cell masses were cultured in then permeabilized for 10 min with PBS comprising DMEM/F12 (Thermo Fisher Scientific, Waltham, MA) 0.2 % Triton X-100. A non-specific reaction was also comprising 20 % knockout serum replacement (KSR, run for 60 min at room temperature in PBS comprising Thermo Fisher Scientific), 2 mM L-glutamine (Thermo 10 % donkey serum; primary antibodies were reacted Fisher Scientific), 0.1 M nonessential amino acids (NEAA, overnight, and fluorescently labeled secondary antibodies Kondo et al. Acta Neuropathologica Communications (2016) 4:69 Page 3 of 12 were reacted and observed. DAPI (Thermo Fisher Scientific) activation Z-score ≥ 1.2 (or ≤−1.2). The detailed descrip- was used for nuclear staining. The following primary anti- tions of IPA analysis are available under “Upstream bodies were used: NANOG (R&D Systems, Minneapolis, Regulator Analysis”, “Biological Functions Analysis”, and MN, 1:50), TRA1-60 (Millipore, Darmstadt, Germany, “Ingenuity Canonical Pathways Analysis” on the IPA 1:1,000), SOX-17 (R&D Systems, 1:50), αSMA (Dako, website (http://www.ingenuity.com). Glostrup, Denmark, 1:3,000), Tuj1 (Covance, 1:3,000), S100β (Abcam, Cambridge, UK, 1:400), GFAP (DAKO, Immunoblots 1:2,000 or Santa Cruz Biotechnology, Dallas, TX, 1:400), Cells were lysed in RIPA buffer (50 mM Tris-HCl alpha-B crystallin (Millipore, 1:400), and N-cadherin (Santa buffer, pH 8.0, 150 mM NaCl, 1 % NP-40, 0.5 % Cruz Biotechnology, 1:50). Rhodamine phalloidin (Thermo deoxycholate, 0.1 % SDS, protease inhibitor cocktail Fisher Scientific, 1:1,000) were used for F-actin staining. (Roche Diagnostics, Basel, Switzerland), phosphatase Immunostained cells were analyzed using In Cell Analyzer inhibitor cocktail (Roche Diagnostics)). Each 10 μg 6000 (GE Healthcare, Chicago, IL) or the super-resolution sample of protein was subjected to SDS-PAGE (5-20 % structured illumination microscopy with 100 x objective gradient SDS-polyacrylamide gels, BIOCRAFT, Tokyo, lens (N-SIM system, Nikon Instruments, Tokyo, Japan). Japan), and separated proteins were transferred to polyvi- nylidene fluoride membrane (HybondTM-P, GE Health- Transmission electron microscopy care). The membranes were incubated with primary Briefly, iPSC-derived astrocytes were cultured on plastic antibodies, followed by appropriate secondary antibodies, coverslip (CellDesk, Sumitomo Bakelite Co., Ltd., Tokyo, and then visualized using ECL prime (GE Healthcare). For Japan) and fixed in 4 % paraformaldehyde/2 % glutaral- dot-blot analysis, cell lysate samples (each 2 or 4 μg/spot) dehyde/0.1 M phosphate buffer at 4 °C, washed in iso- were loaded on a nitrocellulose membrane. The mem- tonic phosphate-buffered sucrose, and then post-fixed in branes were incubated with primary antibodies, followed 1 % osmic acid. Specimens were dehydrated with ethanol by appropriate secondary antibodies, and then visualized and propylene oxide and subsequently embedded in using ECL prime (GE Healthcare). The images were ac- epoxy resin. Ultrathin sections were cut with an ultrami- quired on LAS 4000 (GE Healthcare). The intensity of the crotome, mounted on grids, stained with uranyl acetate protein band was analyzed using Fiji (http://fiji.sc/). The and lead citrate, and examined by using a Hitachi H- following primary antibodies were used: N-cadherin 7650 electron microscope (Hitachi, Tokyo, Japan). (1:1,000, Santa Cruz Biotechnology), GAPDH (1:3,000, Abcam), 4E-BP1 (1:1,000, Cell Signaling Technology Microarray and pathway analysis for differentiated (CST), Danvers, MA), Phospho-4E-BP1 (Ser65) (1:1,000, astrocytes CST), eIF4B (1:1,000, CST), Phospho-eIF4B (Ser406) Total RNA from differentiated neural cells was extracted by RNeasy micro kit (QIAGEN, Hilden, Germany) and (1:1,000, CST), eIF4E (1:1,000, Abcam), and Phospho- altered into ragmented/biotinylated cDNA by GeneChip® eIF4E (Ser209) (1:1,000, Abcam). WT PLUS Reagent Kit (Affymetrix, Santa Clara, CA). Fragmented cDNA samples were hybridized with Gene- Electrochemiluminescence assays for cytokines Chip Human Gene 2.0 ST Array (Affymetrix). Each sam- Differentiated astroglial cells were replated at 4 x10 ple was hybridized once with the one-color protocol. cells per well in 96-well plates coated with 0.1 % gelatin. Arrays were scanned with a GeneChip® Scanner 3000 Three days after replating, all culture medium was 7Gt (Affymetrix). Data were analyzed by GeneSpring replaced with 100 μL of fresh astrocyte medium. To GX7.3.1 software (Agilent Technologies, Santa Clara, assess extracellular cytokine release, conditioned media CA) to create the list of gene sets. The normalized data were harvested for further analysis. As positive control have been deposited at Gene Expression Omnibus of massive cytokines release, 1 μg/mL LPS was added to (GEO, http://www.ncbi.nlm.nih.gov/geo/) with accession NC1 astrocytes. Cytokines in culture media were number GSE83374. For pathway analysis, we adopted measured by human Cytokine Demonstration 10-Plex gene sets for Ingenuity Pathway Analysis (IPA software, tissue Culture Kits (Meso Scale Discovery, Rockville, QIAGEN), and seek altered canonical pathways. To MD). This assay uses each antibody to capture each understand the upstream of pathway changes, the activa- cytokine and SULFO-TAG-labeled different specific anti- tion status of the functions/pathways was predicted bodies for detection by electrochemiluminescence with using the IPA Upstream Regulator Analysis Tool, by cal- Sector® Imager 2400 (Meso Scale Discovery). Ten kinds culating a regulation Z-score and an overlap p-value, of cytokines, including IL-1β, IL-2, IL4, IL-5, IL-6, IL-8, which were based on the number of known target genes IL-10, IL-12p70, GM-CSF and TNFα, were assayed. The of interest pathways/functions, expression changes of concentrations of IL-1β, IL4, IL-5, IL-6, GM-CSF and these target genes and their agreement with literature findings. It was considered significantly activated (or TNFα were quantified by using standard recombinant inhibited) with an overlap p-value ≤ 0.05 and an IPA proteins, but the signals of IL-2, IL-8, IL-10 and IL- Kondo et al. Acta Neuropathologica Communications (2016) 4:69 Page 4 of 12 12p70 were not able to be detected in the dynamic range expressed TUJ1 (neuronal marker) (Fig. 2b). By repea- of the standard curve (0.05-10,000 pg/ml). ting low-density passage, differentiated neurons, without proliferation, failed to attach to the dish and were selec- Statistics tively removed. After five passages and more than 6 Comparisons of the mean among three groups or more months of cultivation, iPSC-derived astrocytes were were performed by one-way or two-way analysis of enriched (Fig. 2b). Differentiated astrocytes abundantly variance (ANOVA)followedbypost-hoctestusing Tukey– expressed S100β (Fig. 2c) and GFAP (Fig. 3a and b), Kramer method (JMP software version 9.0, SAS Institute which are commonly used as astrocytes markers. We did Inc., Cary, NC). P values < 0.05 were considered significant. not observe any obvious difference in astrocytic differen- tiation efficacy among all individuals (Fig. 2d). Results Generation and characterization of AxD-specific iPSCs GFAP aggregates in iPSCs-derived astrocytes from AxD In the present study, we generated iPSCs from three To evaluate the in vitro recapitulation of Rosenthal AxD patients with heterozygous GFAP mutation (Alex1, fibers, we visualized GFAP of iPSC-derived astrocytes by Alex2 and Alex3) and three healthy controls (HC1, HC2, immunofluorescent staining. Nearly all iPSC-derived and HC3) (Table 1). The disease onset of Alex1 and astrocytes showed positive staining of GFAP (Fig. 3a and Alex2 was infantile and that of Alex3 was adult (Table 1). b). GFAP of healthy control astrocytes formed fine Primary cultures of somatic cells from all six individuals filaments distributed throughout the cytoplasm in a were independently reprogrammed to iPSCs, as judged cytoskeletal array (Fig. 3a, panels of HC1, 2, and 3). In by colony morphology, similar to human embryonic contrast, a proportion of GFAP in AxD astrocytes stem cells (ESCs), growth dynamics, and sustained long- formed fibrous aggregates, similar to Rosenthal fibers of term passaging (>20 passages) (Fig. 1a). The established AxD brain, and also small dot-like patterns (Fig. 3a, iPSCs expressed NANOG and TRA1-60, markers of panels of Alex 1, 2, and 3). These fibrous aggregates pluripotency (Fig. 1a). The pluripotency of the iPSCs were formed in 5-10 % of AxD, and were rarely observed was also evaluated in vitro through the formation of in healthy controls. Small dot-like aggregates were EBs. All iPSC lines spontaneously differentiated into cell formed in 15-20 % of AxD and in a few of the healthy types of the three embryonic germ layers as indicated by controls (Fig. 3c). To characterize small dot-like inclu- expression of the specific markers, including TUJ1 sions in detail, we visualized GFAP-positive dots using (ectoderm marker), αSMA (mesoderm marker), and super-resolution structured illumination microscopy SOX17 (endoderm marker) (Fig. 1b). (N-SIM system). GFAP-positive dots, with a diameter of 50-200 nm, showed a cloud-like amorphous struc- Differentiation of iPSCs into astrocytes ture, adjacent to normal GFAP filament, and were co- The astrocytic differentiation protocol for human iPSCs immunostained with alpha-B crystallin particles (Fig. 4a). was modified from our previous method [12] (Fig. 2a). In addition to super-resolution microscopy, we observed In the neural patterning stage, differentiated cells cytosolic aggregates in AxD astrocytes by using electron expressed NESTIN (marker of neural stem cells) or microscopy. In AxD astrocytes, electron-attenuated, GFAP (marker of radial glia in cortical development) granular, or amorphous-appearing structures, surrounded (Fig. 2b). After 2 months, differentiated cells abundantly by filamentous structure, were observed and determined as Rosenthal fiber-like structures (Fig. 4b). Overexpression of GFAP might contribute to astrocyte dysfunction in Table 1 Summary of iPSCs in this study AxD as was shown in initial studies of overexpressing wild clone name clinical GFAP Sex Age at Age at type GFAP in transgenic mice, which resulted in the for- character genotype onset sampling mation of Rosenthal fibers indistinguishable from those HC1 healthy wild female - 36 found in Alexander disease patients. To investigate the HC2 healthy wild female - 67 GFAP dose effects on aggregates formation, we quantified HC3 healthy wild male - 74 the GFAP expression and found increased GFAP in AxD Alex1 Alexander R239C male 2 6 astrocytes (Additional file 1: Figure S1). disease type I (c. 729 C > T) Alex2 Alexander E63K female 3 10 Pathway analysis of alteration in global gene expression disease type I (c.205 G > A) patterns Alex3 Alexander R276L female 33 45 To uncover molecules involved in the AxD astrocyte disease type II (c.827 G > T) pathogenesis, we analyzed global gene expression pro- Abbreviations: GFAP Glial fibrillary acidic protein, HC Healthy control files of iPSC-derived astrocytes (Fig. 5a and b). Among Alex1 was generated from patient fibroblasts (GM16825) from Coriell Institute (Camden, NJ) 40,716 probe sets, we created a gene set with altered Kondo et al. Acta Neuropathologica Communications (2016) 4:69 Page 5 of 12 Alexander disease healthy control HC1 HC2 HC3 Alex1 Alex2 Alex3 Alexander disease healthy control HC1 HC2 HC3 Alex1 Alex2 Alex3 Fig. 1 Generation of iPSCs from Alexander disease patients and healthy controls. a Morphology and expression of human embryonic stem cell markers. iPSCs from both controls and patients with Alexander disease showed ESC-like morphology (phase image) and expressed pluripotent stem cell markers, NANOG and TRA1-60. Scale bars = 200 μm. b In vitro differentiation of established iPSCs to representative three-germ layer: TUJ1 (ectoderm), αSMA (mesoderm), and SOX17 (endoderm). Scale bars = 50 μm expression in AxD astrocytes versus control astrocytes From the results of cellular adherence pathway ana- (fold-change ≥ 2 fold). By adapting this gene set to the lysis, the expression of cell adhesion molecules (CAM), pathway analysis software, we investigated the back- including the cadherin family, was altered in AxD astro- ground pathway of the AxD pathomechanism. Pathway cytes. In iPSC-derived astrocytes of AxD, gene expres- analysis revealed altered function of cellular adherence sion and protein level of N-cadherin were increased, and (Additional file 2: Figure S2) and transcription change those of E-cadherin were decreased (Additional file 2: via mTORC1/mTORC2 (Additional file 3: Figure S3). Figure S2 and Fig. 6b). Cadherin is known to play an Ectoderm human iPSCs Endoderm Mesoderm SOX17 DAPI αSMA DAPI TUJ1 DAPI TRA1-60 DAPI NANOG DAPI phase Kondo et al. Acta Neuropathologica Communications (2016) 4:69 Page 6 of 12 Fig. 2 iPSCs from AxD patients and healthy controls could differentiate into astrocytes with high purity. a Schematic procedures for astroglial differentiation. b Differentiated iPSCs at day 24 expressed neural stem cell markers NESTIN and GFAP. Neural cells at day 60 expressed neuronal or astrocytic marker TUJ1 or GFAP. Most enriched astrocytes expressed GFAP. Scale bars = 20 μm. c Estimation of astroglial differentiation from control and AxD iPSCs. After 180 days of differentiation, astrocytes were immunostained with an antibody against S100β (red color). Scale bars = 20 μm. d Calculated purity of astrocytic differentiation Data represent mean ± SD (biological replicates, n = 3 from randomly picked fields per clone). Two-way analysis of variance (ANOVA) did not show significant variation. F (5, 12) =0.2432; p = 0.935 Kondo et al. Acta Neuropathologica Communications (2016) 4:69 Page 7 of 12 healthy control Alexander disease HC1 Alex1 HC2 Alex2 HC3 Alex3 small dot-like aggregates b c not significant fibrous aggregates 60 15 40 10 20 5 healthy Alexander HC1 HC2 HC3 Alex1 Alex2 Alex3 control disease Fig. 3 Astrocytes of Alexander disease showed GFAP-positive aggregates. a Most iPSC-derived astrocytes showed GFAP-positive staining (green color). In healthy control, GFAP showed filamentous structure. In Alexander disease, GFAP mainly showed filamentous structure, but also fibrous/amorphous (arrows)or dot-like (arrowheads) aggregates. Scale bars = 5 μm. b Calculated purity of astrocytic differentiation data represent mean ± SD (biological replicates, n = 3 from randomly picked fields per clone). c Calculated positivity of GFAP aggregates. Data represent mean ± SD (biological replicates, n = 3 from randomly picked fields per clone) important role in the interactions between cells or their AxD astrocytes. To investigate the detailed structural surrounding matrix, and also to affect the cell mor- changes in AxD astrocytes, we performed an immun- phology [15]. The majority of iPSC-derived astrocytes fluorescence study of N-cadherin and F-actin. The signal showed polygonal shape, and less than 20 % showed stel- intensity of N-cadherin was increased in AxD astrocytes, late or star-like shape. However, we could not find any but the distribution of N-cadherin or F-actin was similar distinct difference in cell shape between control and between control and AxD (Fig. 6c). GFAP positivity GFAP DAPI (% per DAPI) HC1 HC2 HC3 Alex1 Alex2 Alex3 Aggregates positivity (% per DAPI) Decreased top 20 genes Kondo et al. Acta Neuropathologica Communications (2016) 4:69 Page 8 of 12 a Alexander disease super resolution Alex1 Alex2 Alex3 microscope Nuc dot-like aggregates filamentous aggregates * * x50,000 x15,000 x50,000 Fig. 4 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 Additionally, the altered pattern of global gene expres- by western blotting with antibodies, specific to phos- sion in AxD astrocytes suggested activation of the mTOR phorylated 4E-BP, eIF4E, eIF4G, and eIF4B (Fig. 6d). As pathway. Active mTOR promotes protein synthesis by well as the prediction of pathway analysis, the phos- phosphorylating 4E-BPs on several sites that relieve their phorylation statuses of 4E-BP, eIF4E, eIF4G, and eiF4B binding to eIF4E, eIF4G or eIF4B. eIF4E mediates binding were upregulated in AxD astrocytes (Fig. 6d), indicating of eIF4 large protein complex to the 5’ cap structure of activated mTOR pathway. mRNAs. On the other hand, 4E-BPs in their hypopho- To understand the upstream of these two pathway sphorylated state bind to eIF4E competitively, inhibiting changes, the activation status of the functions/pathways the association of eIF4E and eIF4G and leading to a block was predicted. We can predict that altered gene expression in translation [16]. Thus, we evaluated mTOR activation and pathways in AxD is regulated by inflammatory a b (fold change) (fold change) 0 -5 -10 -15 15 10 5 0 Decreased genes LIPH ODZ2 RNU5E-1 LMCD1 FOSL1 GOPC PALMD FNDC1 MID1 ASS1 UIMC1 ARRDC4 PTGIS MMP14 LOC100287562 PSAT1 RN5S150 TPM2 PEG10 THBS2 GPNMB TIMP3 CCL2 SLC7A5 ABI3BP MTHFD2 LOC100506455 NRG1 LOC100505829 MYL9 IFITM2 COL4A1 Increased genes CPA4 ATP10A SEMA5A COL11A1 -4 -2 024 MIR4461 GXYLT2 ND6 PLOD2 Alexander disease (fold change) Fig. 5 Gene expression comparison between healthy control and Alexander’s disease astrocytes. a Scatter plot showing the 2-fold upregulated and downregulated genes (red and blue dots, respectively) in the astrocytes of Alexander disease. b List of increased and decreased top-20 genes (red and blue columns, respectively) Healthy control (fold change) -4 -2 024 Electron microscopy 3D-SIM MIP GFAP CRYAB Increased top 20 genes Kondo et al. Acta Neuropathologica Communications (2016) 4:69 Page 9 of 12 iPSC-derived astrocytes Prediction of upstream regulator Healthy control Altered cytokine release Pathway Prediction of altered pathway analysis Alexander disease Dysfunction in cell-adherence Activation of mTOR signaling M.W. bd M.W. (kDa) (kDa) N-Cadherin 4E-BP GAPDH Phosphorylated 4E-BP (S65) Phosphorylation 1.0 1.1 1.1 1.1 1.2 2.5 ratio of 4E-BP HC1 HC2 HC3 eIF4E Phosphorylated eIF4E (S209) Phosphorylation 1.0 0.5 0.4 1.2 1.4 1.2 ratio of eIF4E eIF4B Alex1 Alex2 Alex3 Phosphorylated eIF4B (S406) Phosphorylation 1.0 1.6 1.8 1.8 1.4 5.6 ratio of eIF4B GAPDH GM-CSF IL-6 N.S. * * 50 25 2500 40 20 2000 30 15 1500 20 10 1000 10 5 500 0 0 0 IL-4 IL-5 N.S. * * 10 2.5 25 8 2 20 6 1.5 15 4 1 10 2 0.5 5 0 0 0 Fig. 6 (See legend on next page.) N-Cadherin F-Actin DAPI (pg/mL) (pg/mL) HC1 HC1 HC2 HC2 HC3 HC3 HC1 Alex1 Alex1 HC2 Alex2 Alex2 Alex3 Alex3 HC3 HC1 HC1 +LPS +LPS Alex1 Alex2 (pg/mL) (pg/mL) Alex3 HC1 HC1 HC2 HC2 HC3 HC3 Alex1 Alex1 Alex2 Alex2 Alex3 Alex3 HC1 HC1 +LPS +LPS (pg/mL) (pg/mL) HC1 HC2 HC1 HC1 HC3 HC2 HC2 HC3 HC3 Alex1 Alex1 Alex1 Alex2 Alex2 Alex2 Alex3 Alex3 Alex3 HC1 HC1 +LPS +LPS Kondo et al. Acta Neuropathologica Communications (2016) 4:69 Page 10 of 12 (See figure on previous page.) Fig. 6 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) cytokines (Fig. 5c). To test the status of cytokines from fibrous aggregates and transformed into fibrous aggre- iPSC-derived astrocytes, cytokines secreted in culture gates after a period of time and/or accumulation of medium were quantified (Fig. 5d). The secretion of GM- cellular stress. We also could observe electron-dense CSF, IL6, IL5 and TNFα was significantly increased in AxD amorphous-appearing structures or granular structures, astrocytes (Fig. 5d). In contrast, the secretion of IL1β and surrounded by filamentous cytoskeleton. These structures IL4 was not altered. Additionally, immunostaining study are similar to Rosenthal fiber in AxD patients brain, but showed intracellular dots of each cytokine in iPSC-derived electron-density of structures in iPSC-derived astrocytes astrocytes (Additional file 4: Figure S4). These results indi- were not as high as that of AxD brain. Relatively low cated that AxD astrocytes secrete more inflammatory cyto- electron-density might reflect the early stage of Rosenthal kines and affect the neural circumstances of AxD patient fiber, which can be an advantage in drug development to brain. modify early pathomechanisms of AxD. Secondly, we investigated how GFAP aggregates elicit Discussion the neurodegenerative process. By comparing global- We generated iPSCs from AxD patients and differen- gene expression between control and AxD astrocytes, we tiated them into astrocytes exhibiting GFAP-positive focused on cell-adhesion pathway and mTOR pathway. aggregates in cytosol. A part of the cytosolic aggregates GFAP is an important cytoskeleton protein, but cell was fibrous and had high immunoreactivity to GFAP shape and cell proliferation are similar between control antibody, resembling Rosenthal fibers of AxD brain. On and AxD astrocytes. In our study, the iPSC-derived as- the other hand, the filamentous structure of GFAP, trocytes were cultivated in the absence of neurons or which is the major intermediate filament of astrocytes, other extracellular matrix and might require years of was almost absent in astrocytes with fibrous aggregates. observation after transplantation into in vivo brain, to In spite of this, however, we could not find any distinct recapitulate morphological phenotypes, such as the morphological alteration in the shape of astrocytes with increased ratio of reactive astrocytes. However, N- fibrous aggregates. We speculate that other species of cadherin protein was increased in AxD astrocytes. Among intermediate filament proteins, including vimentin, may the cadherin family, N-cadherin has been classified as have compensated for the diminished GFAP-filament to “nerve-derived”, and is known as a key CAM in the brain. maintain the cellular shape [17]. In addition to the N-cadherin has also been reported to be upregulated via fibrous aggregates, AxD astrocytes displayed small, cellular-stress signaling after brain injury by using the round shape of GFAP-positive aggregates, described as a N-cadherin knockout model [20]. We speculated that “small dot-like pattern” in the results. These small dots GFAP aggregates in AxD astrocytes can evoke cellular were similar to those of previous reports using cancer stress and upregulated N-cadherin as a stress re- cell line models with overexpression of GFAP (overex- sponse. In the case of in vivo, altered cell-adhesion pression models: OE models) [5, 18, 19]. These small via N-cadherin also might affect the cell-to-cell inter- dots in OE models were observed as irregular dots with action among neurons, oligodendrocytes, microglia or without sand-like diffuse staining patterns. The OE and astrocytes, and consequently could lead to clinical models with mutant GFAP-aggregates did not show any phenotypes of AxD. normal filamentous pattern of GFAP. However, anti- Furthermore, we also focused on activation of the GFAP staining of iPSC-derived astrocytes of AxD mTOR pathway. Activation of the mTOR cascade is showed both normal filamentous structure and small- known as a characteristic feature of the initial stress dot aggregates in the same cells. Furthermore, the response, and is related to reactive astrocytes in brain frequency of the appearance of small-dot aggregates pathologies [21]. According to a previous study by using was greater than that of fibrous aggregates. We GFAP Tg; Gfap+/R236H model mice, the activation speculate that small-dot aggregates, co-existing with through the mTOR pathway appears to be an early wild filamentous GFAP, are a premature form of change, while the later, more severe pathology is Kondo et al. Acta Neuropathologica Communications (2016) 4:69 Page 11 of 12 accompanied by mTOR inactivation [22]. Considering Ethics approval and consent to participate these pieces of evidence, our iPSC-derived astrocytes re- The study was approved by the Institutional Review Board flect the early phase of the AxD pathomechanism, and and Ethics Committees at the University of Kyoto and should be applicable to the development of drugs for an Kumamoto University, and written informed consent was initial insult of GFAP aggregates. obtained from all participants in this study. Previous neuropathological investigations have described that lymphocytic infiltration or microglial activation was Additional files not massive in AxD brain [2, 23]. Olanbarria et al.investi- gated detailed alteration of the cytokine network in AxD Additional file 1: Figure S1. GFAP expression of iPSC-derived astrocytes. Gene expression of GFAP was quantitatively analyzed with model mice with both OE of human wild-type GFAP and RT-qPCR. Two-way analysis of variance (ANOVA) showed significant heterozygous knock-in of mice Gfap R236H, and detected variation. F (5, 12) =9.2490; p = 0.0008. Post hoc analysis revealed inflammatory response [24]. Consistent with this report, in significant increases in GFAP expression in Alex1 and Alex3 (*, p < 0.05). Data represent mean ± SD (biological replicates, n = 3). (PDF 343 kb) our study, AxD astrocytes from patient iPSCs exhibited in- Additional file 2: Figure S2. Alteration in cellular adherence pathway. creased amounts of secreted GM-CSF, IL5, IL6, and TNFα. Gene expression changes were described within epithelial adherens GM-CSF, IL6, and TNFα are well known as proinflamma- junction signaling. Red to orange color = increased in Alexander disease. tory cytokines and are upregulated in various kinds of Blue to green color = decreased in Alexander disease, Grey color = not altered. (PDF 3.50 MB) white matter diseases, including multiple sclerosis (MS) Additional file 3: Figure S3. Alteration in mTORC1/mTORC2 pathway. and neuromyelitis optica (NMO) [25–28]. IL-5 is a Th2 Gene expression changes were described within mTOR signaling. cell-type cytokine that is secreted by astrocytes and micro- Red to orange color = increased in Alexander disease. Blue to green glia [29], turning on the switch of inflammatory response color = decreased in Alexander disease, Grey color = not altered. (PDF 3.63 MB) by activating microglia to upregulate inflammatory re- Additional file 4: Figure S4. Immunofluorescent study of cytokines in sponse in the brain, cooperating with GM-CSF [30, 31]. iPSC-derived astrocytes. iPSC-derived astrocytes showed positive staining IL-5 is also upregulated not only in inflammatory condi- of IL-1β, IL-6, IL-5 (green color) and , GM-CSF, TNFα, IL-4 (red color). Scale tions with parasitic infections but also in neurodegener- bar = 5 μm. (PDF 3.32 MB) ative disorders, including Parkinson’sdisease [32].In addition, clinically, AxD, especially type 2, shows step- Competing interests The authors declare that they have no competing interests. wise or stroke-like progression, which is similar to the typical progression of other white matter diseases Authors’ contributions involving oligodendrocytes, MS and NMO. These find- H.I. conceived the project. T.K. and H.I. designed the experiment. T.K., M.F., M.M., K.T., and H.I. performed the experiments and analyzed the data. T.E. ings suggest that the neuroinflammatory process pro- provided the materials. H.O., T.A., and R.T. provided patient samples and moted by proinflammatory astrocytes may be involved information. T.K. and H.I. wrote the manuscript. in the pathogenesis of AxD, and that immunomodula- tion approaches [33] targeting astrocytopathy would be Acknowledgements We would like to express our sincere gratitude to all our coworkers and a candidate therapy for AxD. Recent studies showed collaborators, to Takako Enami and Ran Shibukawa for their technical that astrocytes themselves can secrete cytokines and support, and to Noriko Endo, Rumi Ueno, and Rie Okuyama for their have responsibility for extrinsic factors, including LPS administrative support. We wish to thank Keiko Furuta and Haruyasu Kohda (Division of Electron Microscopic Study, Center for Anatomical Studies, [34–36]. The cell population of iPSC-derived astrocytes Graduate School of Medicine, Kyoto University) for technical assistance with did not show microglial markers. So, astrocyte-derived electron microscopy. We wish to thank Hiroshi Gomi (Department of cytokines and chemokines might play both neuropro- Veterinary Anatomy, College of Bioresource Sciences, Nihon University) for critical comments on electron microscopy findings. This work was supported tective and neurotoxic roles in AxD, and could be a by the Program for Intractable Diseases Research utilizing disease-specific iPS phenotypic target of future drug development by the cells from the Japan Agency for Medical Research and Development (AMED) use of an iPSC-derived astrocyte platform. to H.I., Research Project for Practical Applications of Regenerative Medicine from AMED to H.I., the grant for Core Center for iPS Cell Research of Research Center Network for Realization of Regenerative Medicine from Conclusions AMED to H.I., the Mochida Memorial Foundation for Medical and iPSCs from AxD patients were used to clarify disease phe- Pharmaceutical Research to H.I., the Daiichi Sankyo Foundation of Life Science to H.I., and Intramural Research Grant (24-9) for Neurological and notypes of astrocytes, which are the target cells of AxD. Psychiatry Disorders of NCNP to H.I.. iPSC-derived astrocytes from AxD patients showed GFAP-aggregates resembling Rothental fibers and altered Author details Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 release of cytokines such as in white matter disease. Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Department of Patient-specific iPSCs of AxD would provide a feasible Cell Modulation, Institute of Molecular Embryology and Genetics (iMEG), platform for the study of inherited astrocytopathies, and Kumamoto University, 2-2-1 Honjo, Tyuou-ku, Kumamoto 860-0811, Japan. Department of Pediatrics, Jichi Medical School, 3311-1 Yakushiji, further studies focusing on pathological crosstalk between Shimotsuke-shi, Tochigi 329-0498, Japan. Department of Neurology, astrocytes and other types of cells in the brain might lead Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, to novel therapies for AxD. Sakyo-ku, Kyoto 606-8507, Japan. 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Modeling Alexander disease with patient iPSCs reveals cellular and molecular pathology of astrocytes

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Springer Journals
Copyright
Copyright © 2016 by The Author(s).
Subject
Biomedicine; Neurosciences; Pathology; Neurology
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2051-5960
DOI
10.1186/s40478-016-0337-0
pmid
27402089
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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. Keywords: Alexander disease (AxD), Glial fibrillary acidic protein (GFAP), Induced pluripotent stem cells (iPSCs), Disease modeling, Astrocytes, Rosenthal fibers, Heat-shock protein, Alpha-crystallin, Cytokine, Inflammatory response, Inherited astrocytopathy Introduction mutations opened the way to the development of model Alexander disease (AxD) was first described by W. S. systems using tissue culture cells and transgenic mice for Alexander [1]. The clinical phenotypes of AxD are the study of AxD. Transgenic models recapitulated GFAP macrocephaly, frontal leukodystrophy and a variety of aggregations. However, it remained unclear how AxD developmental delays with epileptic seizures, dysphagia, mutations lead to protein aggregation in patient astrocytes or bulbar/pseudobulbar signs. However, the severity of as well as how mutant GFAP-expressing astrocytes these clinical features differs among patients, being contribute to neuronal degeneration [6]. mostly dependent on the age of onset [2]. In 2007, the discovery of a combination of transcription The common neuropathological feature of AxD is the factors that could reprogram somatic cells into cells exhi- presence of Rosenthal fibers, a unique cytoplasmic inclu- biting pluripotency, called induced pluripotent stem cells sion within astrocytes. Rosenthal fibers contain glial (iPSCs), has provided researchers with a revolutionary tool fibrillary acidic protein (GFAP), major astrocytic inter- to study human biology and diseases [7]. iPSCs can be de- mediate filament protein and molecular chaperones, rived from many somatic cell types, including easily access- including alpha-B-crystallin and other heat shock pro- ible dermal fibroblasts and peripheral blood mononuclear teins [3, 4]. After extensive neuropathological investiga- cells [8, 9]. Similar to human embryonic stem cells tions, missense mutations in GFAP have been identified as (hESCs), iPSCs can self-renew and expand indefinitely in a genetic basis for AxD [5]. The discovery of the GFAP culture [7]. More importantly, they share the capacity to generate any cell types in the body, a property that is par- ticularly useful for the study of neurological diseases. The * Correspondence: haruhisa@cira.kyoto-u.ac.jp Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 pluripotency of iPSCs enables the production of astrocytes Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan for disease modeling [10–12]. This remarkable feature of Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kondo et al. Acta Neuropathologica Communications (2016) 4:69 Page 2 of 12 iPSCs facilitates the study of brain cell types that are diffi- Thermo Fisher Scientific), 0.1 M 2-mercaptoethanol cult to obtain from living individuals, including human as- (Thermo Fisher Scientific), and 0.5 % penicillin/strepto- trocytes. To directly examine AxD patient astrocytes, we mycin. The medium was replaced every other day, and the established iPSCs from three AxD patients, with different EB after 8 days was cultured for another 8 days in DMEM GFAP mutations, respectively, and three healthy individ- comprising 10 % FBS on a gelatin-coated coverslip. uals without GFAP mutation. In a recent review of more than 215 cases of AxD with GFAP mutation, AxD was di- Differentiation and enrichment of astrocytes vided into 2 groups: type I was characterized by early on- Human iPSCs were dissociated to single cells and quickly set, seizures, megalencephaly, and typical leukodystrophy reaggregated in U-bottom 96-well plates for suspension cul- as MRI features, and type II with a later age at onset ture (Greiner Bio-One, Frickenhausen, Germany), pre- characterized by brainstem features and atypical MRI find- coated with 2 % Pluronic F-127 (Sigma-Aldrich, St. Louis, ings [13]. Two patients in this study showed type I clinical MO) in 100 % ethanol. Cell aggregates, called embryoid phenotype and one patient showed type II clinical pheno- bodies (EBs), were cultured in ‘DFK5% medium’ (DFK5%; type. In this study, AxD iPSC-derived astrocytes showed DMEM/F12 (Thermo Fisher Scientific) supplemented with GFAP-positive aggregates, like Rosenthal fibers, and also 5 % v/v KSR, 1x NEAA, 1x Glutamax (Thermo Fisher exhibited altered cytokine release. The strategy of this Scientific), 0.1 M 2-mercaptoethanol (Thermo Fisher studywas to providean effectiveand versatilewayof Scientific)) with 2 μM dorsomorphin (Sigma-Aldrich) and pathogenic investigation and drug screening for AxD and 10 μM SB431542 (Cayman Chemical, Ann Arbor, MI) in a other astrocyte-relevant diseases. neural inductive stage (day 0 to 8). After neural induction, EBs were transferred onto Matrigel (Corning, Tewksbury, Materials and methods MA)-coated 6-well culture plates and cultured in DFK5% Human subjects supplemented with 1x N2 supplement (Thermo Fisher Skin or blood samples were obtained from healthy Scientific) and 2 μM dorsomorphin in the patterning stage controls or patients with Alexander disease. The study (day 8 to 24). A large number of neural stem cells was approved by the Institutional Review Board and (NESTIN-positive) were observed to migrate from the EB Ethics Committees of the University of Kyoto and core. After the patterning stage, migrated neural stem cells Kumamoto University and written informed consent was were separated from the plate bottom using Accutase obtained from all participants in this study. (Innovative Cell Technologies, Inc., San Diego, CA) and cultured in Neurobasal medium FULL, Neurobasal Medium (Thermo Fisher Scientific) supplemented with 1x Generation of human iPSCs In this application study, we used dermal fibroblasts N2 supplement, 1x Glutamax, 10 ng/ml BDNF (Peprotech, Rocky Hill, NJ), 10 ng/ml GDNF (Peprotech) and 10 ng/ml or blood cells as patient somatic cells to prepare iPSCs [8, 12, 14]. For the iPSC clones of HC1, HC2, HC3, NT-3 (Peprotech) on Matrigel-coated 6-well culture plates or cover-slips (day 24 to 60). At day 60, iPS-derived neural Alex1, and Alex3, episomal vectors were used to introduce a reprogramming factor (SOX2, KLF4, OCT4, L-MYC, cells were plated at 400,000-2,000,000 cells per 90-mm dish without any coating in DMEM/F12 Glutamax (Thermo LIN28, siRNA for p53) to the somatic cells, which were seeded onto SNL feeder cells. The next day, the medium Fisher Scientific) supplemented with 1x N2 supplement, 10 ng/ml EGF (Peprotech), 12 ng/ml basic FGF (Peprotech) was changed from a dermal fibroblast medium to a human ES cell medium (ReproCell, Yokohama, Japan) and 2 μg/ml heparin (Nacalai Tesque, Kyoto, Japan). After passage, neurons could not attach to a non-coated polystyr- comprising 4 ng/mL of bFGF (Wako Chemicals, Osaka, Japan); the medium was replaced every other day, and ene dish surface or they died by anoikis. On the other hand, astrocytes and a limited number of oligodendrocyte precu- after 30 days, about 20 iPSC colonies were picked up. Later, the presence or absence of residual plasmid was sors could attach and proliferate. By repeated passage in the same manner at days 90, 120, 150 and 180, astrocytes confirmed by PCR, and clones without residual plasmid were selected. Selected clones were run through karyotype increased their own abundance ratio and showed positive GFAP immunostaining. analysis, and normal karyotype clones were analyzed. For the iPSC clones of Alex2, human iPSCs were generated by using Sendai virus vector as described previously [14]. Immunofluorescent study The iPSCs or differentiated astrocytes were immobilized In vitro differentiation into three germ layers at room temperature for 30 min in 4 % paraformal- CTK was used to harvest the iPSCs, and an embryoid dehyde (pH 7.4), and washed with PBS. The cells were body (EB) was formed [12]. Cell masses were cultured in then permeabilized for 10 min with PBS comprising DMEM/F12 (Thermo Fisher Scientific, Waltham, MA) 0.2 % Triton X-100. A non-specific reaction was also comprising 20 % knockout serum replacement (KSR, run for 60 min at room temperature in PBS comprising Thermo Fisher Scientific), 2 mM L-glutamine (Thermo 10 % donkey serum; primary antibodies were reacted Fisher Scientific), 0.1 M nonessential amino acids (NEAA, overnight, and fluorescently labeled secondary antibodies Kondo et al. Acta Neuropathologica Communications (2016) 4:69 Page 3 of 12 were reacted and observed. DAPI (Thermo Fisher Scientific) activation Z-score ≥ 1.2 (or ≤−1.2). The detailed descrip- was used for nuclear staining. The following primary anti- tions of IPA analysis are available under “Upstream bodies were used: NANOG (R&D Systems, Minneapolis, Regulator Analysis”, “Biological Functions Analysis”, and MN, 1:50), TRA1-60 (Millipore, Darmstadt, Germany, “Ingenuity Canonical Pathways Analysis” on the IPA 1:1,000), SOX-17 (R&D Systems, 1:50), αSMA (Dako, website (http://www.ingenuity.com). Glostrup, Denmark, 1:3,000), Tuj1 (Covance, 1:3,000), S100β (Abcam, Cambridge, UK, 1:400), GFAP (DAKO, Immunoblots 1:2,000 or Santa Cruz Biotechnology, Dallas, TX, 1:400), Cells were lysed in RIPA buffer (50 mM Tris-HCl alpha-B crystallin (Millipore, 1:400), and N-cadherin (Santa buffer, pH 8.0, 150 mM NaCl, 1 % NP-40, 0.5 % Cruz Biotechnology, 1:50). Rhodamine phalloidin (Thermo deoxycholate, 0.1 % SDS, protease inhibitor cocktail Fisher Scientific, 1:1,000) were used for F-actin staining. (Roche Diagnostics, Basel, Switzerland), phosphatase Immunostained cells were analyzed using In Cell Analyzer inhibitor cocktail (Roche Diagnostics)). Each 10 μg 6000 (GE Healthcare, Chicago, IL) or the super-resolution sample of protein was subjected to SDS-PAGE (5-20 % structured illumination microscopy with 100 x objective gradient SDS-polyacrylamide gels, BIOCRAFT, Tokyo, lens (N-SIM system, Nikon Instruments, Tokyo, Japan). Japan), and separated proteins were transferred to polyvi- nylidene fluoride membrane (HybondTM-P, GE Health- Transmission electron microscopy care). The membranes were incubated with primary Briefly, iPSC-derived astrocytes were cultured on plastic antibodies, followed by appropriate secondary antibodies, coverslip (CellDesk, Sumitomo Bakelite Co., Ltd., Tokyo, and then visualized using ECL prime (GE Healthcare). For Japan) and fixed in 4 % paraformaldehyde/2 % glutaral- dot-blot analysis, cell lysate samples (each 2 or 4 μg/spot) dehyde/0.1 M phosphate buffer at 4 °C, washed in iso- were loaded on a nitrocellulose membrane. The mem- tonic phosphate-buffered sucrose, and then post-fixed in branes were incubated with primary antibodies, followed 1 % osmic acid. Specimens were dehydrated with ethanol by appropriate secondary antibodies, and then visualized and propylene oxide and subsequently embedded in using ECL prime (GE Healthcare). The images were ac- epoxy resin. Ultrathin sections were cut with an ultrami- quired on LAS 4000 (GE Healthcare). The intensity of the crotome, mounted on grids, stained with uranyl acetate protein band was analyzed using Fiji (http://fiji.sc/). The and lead citrate, and examined by using a Hitachi H- following primary antibodies were used: N-cadherin 7650 electron microscope (Hitachi, Tokyo, Japan). (1:1,000, Santa Cruz Biotechnology), GAPDH (1:3,000, Abcam), 4E-BP1 (1:1,000, Cell Signaling Technology Microarray and pathway analysis for differentiated (CST), Danvers, MA), Phospho-4E-BP1 (Ser65) (1:1,000, astrocytes CST), eIF4B (1:1,000, CST), Phospho-eIF4B (Ser406) Total RNA from differentiated neural cells was extracted by RNeasy micro kit (QIAGEN, Hilden, Germany) and (1:1,000, CST), eIF4E (1:1,000, Abcam), and Phospho- altered into ragmented/biotinylated cDNA by GeneChip® eIF4E (Ser209) (1:1,000, Abcam). WT PLUS Reagent Kit (Affymetrix, Santa Clara, CA). Fragmented cDNA samples were hybridized with Gene- Electrochemiluminescence assays for cytokines Chip Human Gene 2.0 ST Array (Affymetrix). Each sam- Differentiated astroglial cells were replated at 4 x10 ple was hybridized once with the one-color protocol. cells per well in 96-well plates coated with 0.1 % gelatin. Arrays were scanned with a GeneChip® Scanner 3000 Three days after replating, all culture medium was 7Gt (Affymetrix). Data were analyzed by GeneSpring replaced with 100 μL of fresh astrocyte medium. To GX7.3.1 software (Agilent Technologies, Santa Clara, assess extracellular cytokine release, conditioned media CA) to create the list of gene sets. The normalized data were harvested for further analysis. As positive control have been deposited at Gene Expression Omnibus of massive cytokines release, 1 μg/mL LPS was added to (GEO, http://www.ncbi.nlm.nih.gov/geo/) with accession NC1 astrocytes. Cytokines in culture media were number GSE83374. For pathway analysis, we adopted measured by human Cytokine Demonstration 10-Plex gene sets for Ingenuity Pathway Analysis (IPA software, tissue Culture Kits (Meso Scale Discovery, Rockville, QIAGEN), and seek altered canonical pathways. To MD). This assay uses each antibody to capture each understand the upstream of pathway changes, the activa- cytokine and SULFO-TAG-labeled different specific anti- tion status of the functions/pathways was predicted bodies for detection by electrochemiluminescence with using the IPA Upstream Regulator Analysis Tool, by cal- Sector® Imager 2400 (Meso Scale Discovery). Ten kinds culating a regulation Z-score and an overlap p-value, of cytokines, including IL-1β, IL-2, IL4, IL-5, IL-6, IL-8, which were based on the number of known target genes IL-10, IL-12p70, GM-CSF and TNFα, were assayed. The of interest pathways/functions, expression changes of concentrations of IL-1β, IL4, IL-5, IL-6, GM-CSF and these target genes and their agreement with literature findings. It was considered significantly activated (or TNFα were quantified by using standard recombinant inhibited) with an overlap p-value ≤ 0.05 and an IPA proteins, but the signals of IL-2, IL-8, IL-10 and IL- Kondo et al. Acta Neuropathologica Communications (2016) 4:69 Page 4 of 12 12p70 were not able to be detected in the dynamic range expressed TUJ1 (neuronal marker) (Fig. 2b). By repea- of the standard curve (0.05-10,000 pg/ml). ting low-density passage, differentiated neurons, without proliferation, failed to attach to the dish and were selec- Statistics tively removed. After five passages and more than 6 Comparisons of the mean among three groups or more months of cultivation, iPSC-derived astrocytes were were performed by one-way or two-way analysis of enriched (Fig. 2b). Differentiated astrocytes abundantly variance (ANOVA)followedbypost-hoctestusing Tukey– expressed S100β (Fig. 2c) and GFAP (Fig. 3a and b), Kramer method (JMP software version 9.0, SAS Institute which are commonly used as astrocytes markers. We did Inc., Cary, NC). P values < 0.05 were considered significant. not observe any obvious difference in astrocytic differen- tiation efficacy among all individuals (Fig. 2d). Results Generation and characterization of AxD-specific iPSCs GFAP aggregates in iPSCs-derived astrocytes from AxD In the present study, we generated iPSCs from three To evaluate the in vitro recapitulation of Rosenthal AxD patients with heterozygous GFAP mutation (Alex1, fibers, we visualized GFAP of iPSC-derived astrocytes by Alex2 and Alex3) and three healthy controls (HC1, HC2, immunofluorescent staining. Nearly all iPSC-derived and HC3) (Table 1). The disease onset of Alex1 and astrocytes showed positive staining of GFAP (Fig. 3a and Alex2 was infantile and that of Alex3 was adult (Table 1). b). GFAP of healthy control astrocytes formed fine Primary cultures of somatic cells from all six individuals filaments distributed throughout the cytoplasm in a were independently reprogrammed to iPSCs, as judged cytoskeletal array (Fig. 3a, panels of HC1, 2, and 3). In by colony morphology, similar to human embryonic contrast, a proportion of GFAP in AxD astrocytes stem cells (ESCs), growth dynamics, and sustained long- formed fibrous aggregates, similar to Rosenthal fibers of term passaging (>20 passages) (Fig. 1a). The established AxD brain, and also small dot-like patterns (Fig. 3a, iPSCs expressed NANOG and TRA1-60, markers of panels of Alex 1, 2, and 3). These fibrous aggregates pluripotency (Fig. 1a). The pluripotency of the iPSCs were formed in 5-10 % of AxD, and were rarely observed was also evaluated in vitro through the formation of in healthy controls. Small dot-like aggregates were EBs. All iPSC lines spontaneously differentiated into cell formed in 15-20 % of AxD and in a few of the healthy types of the three embryonic germ layers as indicated by controls (Fig. 3c). To characterize small dot-like inclu- expression of the specific markers, including TUJ1 sions in detail, we visualized GFAP-positive dots using (ectoderm marker), αSMA (mesoderm marker), and super-resolution structured illumination microscopy SOX17 (endoderm marker) (Fig. 1b). (N-SIM system). GFAP-positive dots, with a diameter of 50-200 nm, showed a cloud-like amorphous struc- Differentiation of iPSCs into astrocytes ture, adjacent to normal GFAP filament, and were co- The astrocytic differentiation protocol for human iPSCs immunostained with alpha-B crystallin particles (Fig. 4a). was modified from our previous method [12] (Fig. 2a). In addition to super-resolution microscopy, we observed In the neural patterning stage, differentiated cells cytosolic aggregates in AxD astrocytes by using electron expressed NESTIN (marker of neural stem cells) or microscopy. In AxD astrocytes, electron-attenuated, GFAP (marker of radial glia in cortical development) granular, or amorphous-appearing structures, surrounded (Fig. 2b). After 2 months, differentiated cells abundantly by filamentous structure, were observed and determined as Rosenthal fiber-like structures (Fig. 4b). Overexpression of GFAP might contribute to astrocyte dysfunction in Table 1 Summary of iPSCs in this study AxD as was shown in initial studies of overexpressing wild clone name clinical GFAP Sex Age at Age at type GFAP in transgenic mice, which resulted in the for- character genotype onset sampling mation of Rosenthal fibers indistinguishable from those HC1 healthy wild female - 36 found in Alexander disease patients. To investigate the HC2 healthy wild female - 67 GFAP dose effects on aggregates formation, we quantified HC3 healthy wild male - 74 the GFAP expression and found increased GFAP in AxD Alex1 Alexander R239C male 2 6 astrocytes (Additional file 1: Figure S1). disease type I (c. 729 C > T) Alex2 Alexander E63K female 3 10 Pathway analysis of alteration in global gene expression disease type I (c.205 G > A) patterns Alex3 Alexander R276L female 33 45 To uncover molecules involved in the AxD astrocyte disease type II (c.827 G > T) pathogenesis, we analyzed global gene expression pro- Abbreviations: GFAP Glial fibrillary acidic protein, HC Healthy control files of iPSC-derived astrocytes (Fig. 5a and b). Among Alex1 was generated from patient fibroblasts (GM16825) from Coriell Institute (Camden, NJ) 40,716 probe sets, we created a gene set with altered Kondo et al. Acta Neuropathologica Communications (2016) 4:69 Page 5 of 12 Alexander disease healthy control HC1 HC2 HC3 Alex1 Alex2 Alex3 Alexander disease healthy control HC1 HC2 HC3 Alex1 Alex2 Alex3 Fig. 1 Generation of iPSCs from Alexander disease patients and healthy controls. a Morphology and expression of human embryonic stem cell markers. iPSCs from both controls and patients with Alexander disease showed ESC-like morphology (phase image) and expressed pluripotent stem cell markers, NANOG and TRA1-60. Scale bars = 200 μm. b In vitro differentiation of established iPSCs to representative three-germ layer: TUJ1 (ectoderm), αSMA (mesoderm), and SOX17 (endoderm). Scale bars = 50 μm expression in AxD astrocytes versus control astrocytes From the results of cellular adherence pathway ana- (fold-change ≥ 2 fold). By adapting this gene set to the lysis, the expression of cell adhesion molecules (CAM), pathway analysis software, we investigated the back- including the cadherin family, was altered in AxD astro- ground pathway of the AxD pathomechanism. Pathway cytes. In iPSC-derived astrocytes of AxD, gene expres- analysis revealed altered function of cellular adherence sion and protein level of N-cadherin were increased, and (Additional file 2: Figure S2) and transcription change those of E-cadherin were decreased (Additional file 2: via mTORC1/mTORC2 (Additional file 3: Figure S3). Figure S2 and Fig. 6b). Cadherin is known to play an Ectoderm human iPSCs Endoderm Mesoderm SOX17 DAPI αSMA DAPI TUJ1 DAPI TRA1-60 DAPI NANOG DAPI phase Kondo et al. Acta Neuropathologica Communications (2016) 4:69 Page 6 of 12 Fig. 2 iPSCs from AxD patients and healthy controls could differentiate into astrocytes with high purity. a Schematic procedures for astroglial differentiation. b Differentiated iPSCs at day 24 expressed neural stem cell markers NESTIN and GFAP. Neural cells at day 60 expressed neuronal or astrocytic marker TUJ1 or GFAP. Most enriched astrocytes expressed GFAP. Scale bars = 20 μm. c Estimation of astroglial differentiation from control and AxD iPSCs. After 180 days of differentiation, astrocytes were immunostained with an antibody against S100β (red color). Scale bars = 20 μm. d Calculated purity of astrocytic differentiation Data represent mean ± SD (biological replicates, n = 3 from randomly picked fields per clone). Two-way analysis of variance (ANOVA) did not show significant variation. F (5, 12) =0.2432; p = 0.935 Kondo et al. Acta Neuropathologica Communications (2016) 4:69 Page 7 of 12 healthy control Alexander disease HC1 Alex1 HC2 Alex2 HC3 Alex3 small dot-like aggregates b c not significant fibrous aggregates 60 15 40 10 20 5 healthy Alexander HC1 HC2 HC3 Alex1 Alex2 Alex3 control disease Fig. 3 Astrocytes of Alexander disease showed GFAP-positive aggregates. a Most iPSC-derived astrocytes showed GFAP-positive staining (green color). In healthy control, GFAP showed filamentous structure. In Alexander disease, GFAP mainly showed filamentous structure, but also fibrous/amorphous (arrows)or dot-like (arrowheads) aggregates. Scale bars = 5 μm. b Calculated purity of astrocytic differentiation data represent mean ± SD (biological replicates, n = 3 from randomly picked fields per clone). c Calculated positivity of GFAP aggregates. Data represent mean ± SD (biological replicates, n = 3 from randomly picked fields per clone) important role in the interactions between cells or their AxD astrocytes. To investigate the detailed structural surrounding matrix, and also to affect the cell mor- changes in AxD astrocytes, we performed an immun- phology [15]. The majority of iPSC-derived astrocytes fluorescence study of N-cadherin and F-actin. The signal showed polygonal shape, and less than 20 % showed stel- intensity of N-cadherin was increased in AxD astrocytes, late or star-like shape. However, we could not find any but the distribution of N-cadherin or F-actin was similar distinct difference in cell shape between control and between control and AxD (Fig. 6c). GFAP positivity GFAP DAPI (% per DAPI) HC1 HC2 HC3 Alex1 Alex2 Alex3 Aggregates positivity (% per DAPI) Decreased top 20 genes Kondo et al. Acta Neuropathologica Communications (2016) 4:69 Page 8 of 12 a Alexander disease super resolution Alex1 Alex2 Alex3 microscope Nuc dot-like aggregates filamentous aggregates * * x50,000 x15,000 x50,000 Fig. 4 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 Additionally, the altered pattern of global gene expres- by western blotting with antibodies, specific to phos- sion in AxD astrocytes suggested activation of the mTOR phorylated 4E-BP, eIF4E, eIF4G, and eIF4B (Fig. 6d). As pathway. Active mTOR promotes protein synthesis by well as the prediction of pathway analysis, the phos- phosphorylating 4E-BPs on several sites that relieve their phorylation statuses of 4E-BP, eIF4E, eIF4G, and eiF4B binding to eIF4E, eIF4G or eIF4B. eIF4E mediates binding were upregulated in AxD astrocytes (Fig. 6d), indicating of eIF4 large protein complex to the 5’ cap structure of activated mTOR pathway. mRNAs. On the other hand, 4E-BPs in their hypopho- To understand the upstream of these two pathway sphorylated state bind to eIF4E competitively, inhibiting changes, the activation status of the functions/pathways the association of eIF4E and eIF4G and leading to a block was predicted. We can predict that altered gene expression in translation [16]. Thus, we evaluated mTOR activation and pathways in AxD is regulated by inflammatory a b (fold change) (fold change) 0 -5 -10 -15 15 10 5 0 Decreased genes LIPH ODZ2 RNU5E-1 LMCD1 FOSL1 GOPC PALMD FNDC1 MID1 ASS1 UIMC1 ARRDC4 PTGIS MMP14 LOC100287562 PSAT1 RN5S150 TPM2 PEG10 THBS2 GPNMB TIMP3 CCL2 SLC7A5 ABI3BP MTHFD2 LOC100506455 NRG1 LOC100505829 MYL9 IFITM2 COL4A1 Increased genes CPA4 ATP10A SEMA5A COL11A1 -4 -2 024 MIR4461 GXYLT2 ND6 PLOD2 Alexander disease (fold change) Fig. 5 Gene expression comparison between healthy control and Alexander’s disease astrocytes. a Scatter plot showing the 2-fold upregulated and downregulated genes (red and blue dots, respectively) in the astrocytes of Alexander disease. b List of increased and decreased top-20 genes (red and blue columns, respectively) Healthy control (fold change) -4 -2 024 Electron microscopy 3D-SIM MIP GFAP CRYAB Increased top 20 genes Kondo et al. Acta Neuropathologica Communications (2016) 4:69 Page 9 of 12 iPSC-derived astrocytes Prediction of upstream regulator Healthy control Altered cytokine release Pathway Prediction of altered pathway analysis Alexander disease Dysfunction in cell-adherence Activation of mTOR signaling M.W. bd M.W. (kDa) (kDa) N-Cadherin 4E-BP GAPDH Phosphorylated 4E-BP (S65) Phosphorylation 1.0 1.1 1.1 1.1 1.2 2.5 ratio of 4E-BP HC1 HC2 HC3 eIF4E Phosphorylated eIF4E (S209) Phosphorylation 1.0 0.5 0.4 1.2 1.4 1.2 ratio of eIF4E eIF4B Alex1 Alex2 Alex3 Phosphorylated eIF4B (S406) Phosphorylation 1.0 1.6 1.8 1.8 1.4 5.6 ratio of eIF4B GAPDH GM-CSF IL-6 N.S. * * 50 25 2500 40 20 2000 30 15 1500 20 10 1000 10 5 500 0 0 0 IL-4 IL-5 N.S. * * 10 2.5 25 8 2 20 6 1.5 15 4 1 10 2 0.5 5 0 0 0 Fig. 6 (See legend on next page.) N-Cadherin F-Actin DAPI (pg/mL) (pg/mL) HC1 HC1 HC2 HC2 HC3 HC3 HC1 Alex1 Alex1 HC2 Alex2 Alex2 Alex3 Alex3 HC3 HC1 HC1 +LPS +LPS Alex1 Alex2 (pg/mL) (pg/mL) Alex3 HC1 HC1 HC2 HC2 HC3 HC3 Alex1 Alex1 Alex2 Alex2 Alex3 Alex3 HC1 HC1 +LPS +LPS (pg/mL) (pg/mL) HC1 HC2 HC1 HC1 HC3 HC2 HC2 HC3 HC3 Alex1 Alex1 Alex1 Alex2 Alex2 Alex2 Alex3 Alex3 Alex3 HC1 HC1 +LPS +LPS Kondo et al. Acta Neuropathologica Communications (2016) 4:69 Page 10 of 12 (See figure on previous page.) Fig. 6 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) cytokines (Fig. 5c). To test the status of cytokines from fibrous aggregates and transformed into fibrous aggre- iPSC-derived astrocytes, cytokines secreted in culture gates after a period of time and/or accumulation of medium were quantified (Fig. 5d). The secretion of GM- cellular stress. We also could observe electron-dense CSF, IL6, IL5 and TNFα was significantly increased in AxD amorphous-appearing structures or granular structures, astrocytes (Fig. 5d). In contrast, the secretion of IL1β and surrounded by filamentous cytoskeleton. These structures IL4 was not altered. Additionally, immunostaining study are similar to Rosenthal fiber in AxD patients brain, but showed intracellular dots of each cytokine in iPSC-derived electron-density of structures in iPSC-derived astrocytes astrocytes (Additional file 4: Figure S4). These results indi- were not as high as that of AxD brain. Relatively low cated that AxD astrocytes secrete more inflammatory cyto- electron-density might reflect the early stage of Rosenthal kines and affect the neural circumstances of AxD patient fiber, which can be an advantage in drug development to brain. modify early pathomechanisms of AxD. Secondly, we investigated how GFAP aggregates elicit Discussion the neurodegenerative process. By comparing global- We generated iPSCs from AxD patients and differen- gene expression between control and AxD astrocytes, we tiated them into astrocytes exhibiting GFAP-positive focused on cell-adhesion pathway and mTOR pathway. aggregates in cytosol. A part of the cytosolic aggregates GFAP is an important cytoskeleton protein, but cell was fibrous and had high immunoreactivity to GFAP shape and cell proliferation are similar between control antibody, resembling Rosenthal fibers of AxD brain. On and AxD astrocytes. In our study, the iPSC-derived as- the other hand, the filamentous structure of GFAP, trocytes were cultivated in the absence of neurons or which is the major intermediate filament of astrocytes, other extracellular matrix and might require years of was almost absent in astrocytes with fibrous aggregates. observation after transplantation into in vivo brain, to In spite of this, however, we could not find any distinct recapitulate morphological phenotypes, such as the morphological alteration in the shape of astrocytes with increased ratio of reactive astrocytes. However, N- fibrous aggregates. We speculate that other species of cadherin protein was increased in AxD astrocytes. Among intermediate filament proteins, including vimentin, may the cadherin family, N-cadherin has been classified as have compensated for the diminished GFAP-filament to “nerve-derived”, and is known as a key CAM in the brain. maintain the cellular shape [17]. In addition to the N-cadherin has also been reported to be upregulated via fibrous aggregates, AxD astrocytes displayed small, cellular-stress signaling after brain injury by using the round shape of GFAP-positive aggregates, described as a N-cadherin knockout model [20]. We speculated that “small dot-like pattern” in the results. These small dots GFAP aggregates in AxD astrocytes can evoke cellular were similar to those of previous reports using cancer stress and upregulated N-cadherin as a stress re- cell line models with overexpression of GFAP (overex- sponse. In the case of in vivo, altered cell-adhesion pression models: OE models) [5, 18, 19]. These small via N-cadherin also might affect the cell-to-cell inter- dots in OE models were observed as irregular dots with action among neurons, oligodendrocytes, microglia or without sand-like diffuse staining patterns. The OE and astrocytes, and consequently could lead to clinical models with mutant GFAP-aggregates did not show any phenotypes of AxD. normal filamentous pattern of GFAP. However, anti- Furthermore, we also focused on activation of the GFAP staining of iPSC-derived astrocytes of AxD mTOR pathway. Activation of the mTOR cascade is showed both normal filamentous structure and small- known as a characteristic feature of the initial stress dot aggregates in the same cells. Furthermore, the response, and is related to reactive astrocytes in brain frequency of the appearance of small-dot aggregates pathologies [21]. According to a previous study by using was greater than that of fibrous aggregates. We GFAP Tg; Gfap+/R236H model mice, the activation speculate that small-dot aggregates, co-existing with through the mTOR pathway appears to be an early wild filamentous GFAP, are a premature form of change, while the later, more severe pathology is Kondo et al. Acta Neuropathologica Communications (2016) 4:69 Page 11 of 12 accompanied by mTOR inactivation [22]. Considering Ethics approval and consent to participate these pieces of evidence, our iPSC-derived astrocytes re- The study was approved by the Institutional Review Board flect the early phase of the AxD pathomechanism, and and Ethics Committees at the University of Kyoto and should be applicable to the development of drugs for an Kumamoto University, and written informed consent was initial insult of GFAP aggregates. obtained from all participants in this study. Previous neuropathological investigations have described that lymphocytic infiltration or microglial activation was Additional files not massive in AxD brain [2, 23]. Olanbarria et al.investi- gated detailed alteration of the cytokine network in AxD Additional file 1: Figure S1. GFAP expression of iPSC-derived astrocytes. Gene expression of GFAP was quantitatively analyzed with model mice with both OE of human wild-type GFAP and RT-qPCR. Two-way analysis of variance (ANOVA) showed significant heterozygous knock-in of mice Gfap R236H, and detected variation. F (5, 12) =9.2490; p = 0.0008. Post hoc analysis revealed inflammatory response [24]. Consistent with this report, in significant increases in GFAP expression in Alex1 and Alex3 (*, p < 0.05). Data represent mean ± SD (biological replicates, n = 3). (PDF 343 kb) our study, AxD astrocytes from patient iPSCs exhibited in- Additional file 2: Figure S2. Alteration in cellular adherence pathway. creased amounts of secreted GM-CSF, IL5, IL6, and TNFα. Gene expression changes were described within epithelial adherens GM-CSF, IL6, and TNFα are well known as proinflamma- junction signaling. Red to orange color = increased in Alexander disease. tory cytokines and are upregulated in various kinds of Blue to green color = decreased in Alexander disease, Grey color = not altered. (PDF 3.50 MB) white matter diseases, including multiple sclerosis (MS) Additional file 3: Figure S3. Alteration in mTORC1/mTORC2 pathway. and neuromyelitis optica (NMO) [25–28]. IL-5 is a Th2 Gene expression changes were described within mTOR signaling. cell-type cytokine that is secreted by astrocytes and micro- Red to orange color = increased in Alexander disease. Blue to green glia [29], turning on the switch of inflammatory response color = decreased in Alexander disease, Grey color = not altered. (PDF 3.63 MB) by activating microglia to upregulate inflammatory re- Additional file 4: Figure S4. Immunofluorescent study of cytokines in sponse in the brain, cooperating with GM-CSF [30, 31]. iPSC-derived astrocytes. iPSC-derived astrocytes showed positive staining IL-5 is also upregulated not only in inflammatory condi- of IL-1β, IL-6, IL-5 (green color) and , GM-CSF, TNFα, IL-4 (red color). Scale tions with parasitic infections but also in neurodegener- bar = 5 μm. (PDF 3.32 MB) ative disorders, including Parkinson’sdisease [32].In addition, clinically, AxD, especially type 2, shows step- Competing interests The authors declare that they have no competing interests. wise or stroke-like progression, which is similar to the typical progression of other white matter diseases Authors’ contributions involving oligodendrocytes, MS and NMO. These find- H.I. conceived the project. T.K. and H.I. designed the experiment. T.K., M.F., M.M., K.T., and H.I. performed the experiments and analyzed the data. T.E. ings suggest that the neuroinflammatory process pro- provided the materials. H.O., T.A., and R.T. provided patient samples and moted by proinflammatory astrocytes may be involved information. T.K. and H.I. wrote the manuscript. in the pathogenesis of AxD, and that immunomodula- tion approaches [33] targeting astrocytopathy would be Acknowledgements We would like to express our sincere gratitude to all our coworkers and a candidate therapy for AxD. Recent studies showed collaborators, to Takako Enami and Ran Shibukawa for their technical that astrocytes themselves can secrete cytokines and support, and to Noriko Endo, Rumi Ueno, and Rie Okuyama for their have responsibility for extrinsic factors, including LPS administrative support. We wish to thank Keiko Furuta and Haruyasu Kohda (Division of Electron Microscopic Study, Center for Anatomical Studies, [34–36]. The cell population of iPSC-derived astrocytes Graduate School of Medicine, Kyoto University) for technical assistance with did not show microglial markers. So, astrocyte-derived electron microscopy. We wish to thank Hiroshi Gomi (Department of cytokines and chemokines might play both neuropro- Veterinary Anatomy, College of Bioresource Sciences, Nihon University) for critical comments on electron microscopy findings. This work was supported tective and neurotoxic roles in AxD, and could be a by the Program for Intractable Diseases Research utilizing disease-specific iPS phenotypic target of future drug development by the cells from the Japan Agency for Medical Research and Development (AMED) use of an iPSC-derived astrocyte platform. to H.I., Research Project for Practical Applications of Regenerative Medicine from AMED to H.I., the grant for Core Center for iPS Cell Research of Research Center Network for Realization of Regenerative Medicine from Conclusions AMED to H.I., the Mochida Memorial Foundation for Medical and iPSCs from AxD patients were used to clarify disease phe- Pharmaceutical Research to H.I., the Daiichi Sankyo Foundation of Life Science to H.I., and Intramural Research Grant (24-9) for Neurological and notypes of astrocytes, which are the target cells of AxD. Psychiatry Disorders of NCNP to H.I.. iPSC-derived astrocytes from AxD patients showed GFAP-aggregates resembling Rothental fibers and altered Author details Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 release of cytokines such as in white matter disease. Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Department of Patient-specific iPSCs of AxD would provide a feasible Cell Modulation, Institute of Molecular Embryology and Genetics (iMEG), platform for the study of inherited astrocytopathies, and Kumamoto University, 2-2-1 Honjo, Tyuou-ku, Kumamoto 860-0811, Japan. Department of Pediatrics, Jichi Medical School, 3311-1 Yakushiji, further studies focusing on pathological crosstalk between Shimotsuke-shi, Tochigi 329-0498, Japan. Department of Neurology, astrocytes and other types of cells in the brain might lead Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, to novel therapies for AxD. Sakyo-ku, Kyoto 606-8507, Japan. 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Acta Neuropathologica CommunicationsSpringer Journals

Published: Jul 11, 2016

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