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. 2013 Mar 19;110(12):4697-702.
doi: 10.1073/pnas.1300398110. Epub 2013 Feb 11.

Astrocyte pathology and the absence of non-cell autonomy in an induced pluripotent stem cell model of TDP-43 proteinopathy

Andrea Serio  1 , Bilada Bilican, Sami J Barmada, Dale Michael Ando, Chen Zhao, Rick Siller, Karen Burr, Ghazal Haghi, David Story, Agnes Lumi Nishimura, Monica A Carrasco, Hemali P Phatnani, Carole Shum, Ian Wilmut, Tom Maniatis, Christopher E Shaw, Steven Finkbeiner, Siddharthan Chandran
Affiliations

Astrocyte pathology and the absence of non-cell autonomy in an induced pluripotent stem cell model of TDP-43 proteinopathy

Andrea Serio et al. Proc Natl Acad Sci U S A. .

Abstract

Glial proliferation and activation are associated with disease progression in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia. In this study, we describe a unique platform to address the question of cell autonomy in transactive response DNA-binding protein (TDP-43) proteinopathies. We generated functional astroglia from human induced pluripotent stem cells carrying an ALS-causing TDP-43 mutation and show that mutant astrocytes exhibit increased levels of TDP-43, subcellular mislocalization of TDP-43, and decreased cell survival. We then performed coculture experiments to evaluate the effects of M337V astrocytes on the survival of wild-type and M337V TDP-43 motor neurons, showing that mutant TDP-43 astrocytes do not adversely affect survival of cocultured neurons. These observations reveal a significant and previously unrecognized glial cell-autonomous pathological phenotype associated with a pathogenic mutation in TDP-43 and show that TDP-43 proteinopathies do not display an astrocyte non-cell-autonomous component in cell culture, as previously described for SOD1 ALS. This study highlights the utility of induced pluripotent stem cell-based in vitro disease models to investigate mechanisms of disease in ALS and other TDP-43 proteinopathies.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Generation of astrocytes from iPSC lines. (A) iPSC-derived neurospheres were enriched for APCs by culturing in EGF/LIF-containing medium for 2–4 wk and then expanded in EGF/FGF2-containing medium. To obtain monolayer cultures, enriched neurospheres were dissociated by using papain and differentiated with CNTF. (B) APCs in monolayer cultures maintained a homogenous identity and morphology, featuring a high percentage of cells positive for the glial progenitors markers Vimentin (85.6 ± 2.9% SEM) and NFIA (79.8 ± 1.9% SEM; scale bar: 100 µm.) (C) APC presented 24.6 ± 2.6% SEM cells positive for GFAP during proliferation in EGF and FGF2; this percentage increased rapidly during the first 7 d of CNTF differentiation (84.9 ± 0.5% SEM), reaching a peak after 14 d (90.8 ± 1.1% SEM; scale bar: 100 μm). (D) Differentiation of monolayer APCs for 14 d in CNTF resulted in a population of astrocytes positive for GFAP (90.6 ± 0.7% SEM) and S100β (91.9 ± 1.9% SEM) (images represent the same field; scale bar: 25 µm.) A few neurons were found in the differentiated cultures (1.7 ± 0.4% SEM). (E) Representative field for a differentiated iPSC-derived astrocytes after 14 d of CNTF differentiation, labeled for GFAP (red) and TuJ1 (green), showing that the protocol gives rise to highly enriched cultures of astrocytes. (Scale bar: 100 μm). (F) M337V and CTRL iPSC lines showed comparable numbers of GFAP-positive cells postdifferentiation (M337V, 92.3 ± 1.0%; WT, 91.36 ± 1.0%; SEM).
Fig. 2.
Fig. 2.
Functional characterization of iPSC-derived astrocytes. (A) l-glutamate uptake assay on iPSC-derived astrocytes. iPSC-derived astrocytes cleared l-glutamate in a time-dependent fashion. ***P < 0.01 (one-way ANOVA). The uptake was abolished by either the absence of sodium or the presence of 2 mM L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC; both negative CTRLs were run for 120 min). (B) M337V iPSC-derived astrocytes did not show a significant difference in glutamate clearance rate, compared with CTRL astrocytes (one-way ANOVA; P > 0.05 for all times considered). (C) Glutamate uptake fold increase. M337V and CTRL astrocytes were able to clear >75% of l-glutamate from the medium in 120 min, and uptake was inhibited equally by PDC. No significant difference was observed between the groups. (D) Representative immunofluorescence panels from synaptogenesis assay. Image shows representative neurite segment used for analysis, stained with TuJ1 (blue), PSY (red), and PSD-95 (green; scale bar: 15 µm). (E) iPSC-derived astrocytes significantly promoted synaptogenesis when cocultured with differentiating neurons. ***P < 0.01 (one-way ANOVA). The increase in synaptogenesis was measured by scoring the number of PSY+/PSD-95+ puncta in 30-µm neurite segments (coculture M337V, 2.4 ± 0.2, SEM; coculture WT, 2.6 ± 0.1, SEM; neurons, 1.00 ± 0.08, SEM). No significant difference was observed in the behavior of M337V or CTRL iPSC-derived astrocytes. Data represent the average of three independent experiments, each with two clones of M337V (M337V-1 and -2) iPSC and two independent CTRL lines (CTRL-1 and -2); data from different lines were pooled for graphical presentation. (F) Synaptogenesis assay with M337V or WT patterned neurons revealed that astrocyte promotion of synapse formation was independent from the genotype of cocultured neurons and that M337V astrocytes do not exert a negative effect on synaptic formation, even to mutant neurons. (M337V neurons on WT astrocytes, 0.90 ± 0.04, SEM; M337V neurons on M337V astrocytes, 0.98 ± 0.03 SEM; WT neurons on WT astrocytes, 1.00 ± 0.01 SEM; WT neurons on M337V astrocytes, 1.01 ± 0.02, SEM). (G) iPSC-derived astrocytes propagate calcium waves to adjacent cells upon mechanical stimulation. Red outline represents glass bead used for stimulation (200-µm diameter). Annotation represents time in seconds from stimulation.
Fig. 3.
Fig. 3.
Characterization of TDP-43 in iPSC-derived astrocytes. (A) qRT-PCR expression analysis of iPSC-derived astrocytes shows no significant difference (P > 0.05 for all pairs; unpaired t test) in mRNA levels of TARDBP, HDAC6, S100β, or GFAP. Results are presented as expression fold increase normalized to M337V-1. (B) Representative Western blot from soluble TDP-43 analysis of M337V-1, -2, and CTRL-1 astrocytes. (C) Semiquantitative analyses of band signal intensity was accomplished by using ImageJ on three independent extractions (M337V-1 mean = 2.0 ± 0.4, SEM; M337V-2 mean = 1.9 ± 0.4, SEM; CTRL-1 mean = 1.1 ± 0.3, SEM). **P = 0.003 (repeated measures ANOVA). (D) Representative confocal images of TDP-43/GFAP immunolabeling used for densitometric analysis. (E) Densitometric quantification of TDP-43 localization by confocal microscopy showing a significant increase of cytoplasmic TDP-43 in M337V astrocytes; results are presented as densitometric signal fold increase normalized to the CTRL (n = 4 independent experiments). ***P < 0.001.
Fig. 4.
Fig. 4.
Ectopic TDP-43 expression partially reproduces the iPSC phenotype. (A) Representative images of cytoplasmic and nuclear-only TDP43:mApple-transfected astrocytes showing the criteria for inclusion of astrocytes in either cytoplasmic (Upper) or nuclear (Lower). (B) Analysis of exogenous WT TDP-43 localization on stratified relative expression groups. The localization of WT TDP-43 was significantly related to relative expression levels [nuclear vs. cytoplasmic TDP-43: A, not significant (n.s.); B, n.s.; C, n.s.; D, P < 0.05; E, P < 0.01; F, P < 0.001). Data are pooled from three independent experiments. (C) Analysis of exogenous M337 TDP-43 localization on stratified relative expression groups. M337V TDP-43 remained localized mainly to the cytoplasm regardless of expression level (nuclear vs. cytoplasmic TDP-43: A–F, P < 0.001). Data are pooled from three independent experiments. (D) Comparison of ectopic TDP-43 localization in astrocytes from the lowest relative expression level groups (group A in B and C). At low expression levels, 73.6 ± 3.3% of astrocytes from the TDP43M337V:mApple group presented mainly or exclusively cytoplasmic localization of the protein, compared with 44.5 ± 1.6% in the TDP43WT:mApple. **P < 0.01; ***P < 0.001 (one-way ANOVA).
Fig. 5.
Fig. 5.
Survival analysis on iPSC-derived astrocytes and MN-astrocyte cocultures. (A) Real-time survival analysis of M337V iPSC-derived astrocytes and CTRLs. Mutant astrocytes showed increased cumulative risk of death associated with M337V TDP-43 under basal conditions (HR = 2.5, P = 2 × 10−16; CTRL taken as baseline; ***P < 0.001). (B) Real-time survival analysis of M337V and CTRL iPSC-derived astrocytes in the presence of 10 µM QVD-oph. The inhibition of caspase activation reduced the risk of death in M337V and CTRL astrocytes (with CTRL+vehicle as a reference, in M337V+vehicle, HR = 2.61, P = 8.13 × 10−12; M337V+QVD, HR = 0.72, P = 0.0145; CTRL+QVD, HR = 0.33, P = 5.07 × 10−11; ***P < 0.001). (C) Comparison between cumulative HR of M337V astrocytes in QVD- and vehicle-treated groups. The error bars represent 95% confidence intervals. (D) Real-time survival analysis of WT iPSC-derived MNs plated on either M337V or CTRL iPSC-derived astrocytes. Mutant astrocytes were not toxic to cocultured WT MNs (with WT MNs on WT astrocytes as a reference, in WT MNs on M337V astrocytes, HR 0.98; P = 0.68). (E) Real-time survival analysis of MNs cultured in isolation (n = 3), demonstrating cell-autonomous toxicity of mutant TDP43 (with WT as a reference, in M337V, HR = 2.02; P = 8.91 × 10−7). (F) Longitudinal analysis of M337V MNs plated on either WT or M337V astrocytes. Both WT and M337V astrocytes improved the survival of M337V MNs. M337 astrocytes had no detectable toxicity to cocultured MNs. All graphs represent pooled data from three independent experiments.
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