C9orf72 Hexanucleotide Expansions Are Associated with Altered Endoplasmic Reticulum Calcium Homeostasis and Stress Granule Formation in Induced Pluripotent Stem Cell-Derived Neurons from Patients with Amyotrophic Lateral Sclerosis and Frontotemporal Dementia

Abstract

An expanded hexanucleotide repeat in a noncoding region of the C9orf72 gene is a major cause of amyotrophic lateral sclerosis (ALS), accounting for up to 40% of familial cases and 7% of sporadic ALS in European populations. We have generated induced pluripotent stem cells (iPSCs) from fibroblasts of patients carrying C9orf72 hexanucleotide expansions, differentiated these to functional motor and cortical neurons, and performed an extensive phenotypic characterization. In C9orf72 iPSC-derived motor neurons, decreased cell survival is correlated with dysfunction in Ca(2+) homeostasis, reduced levels of the antiapoptotic protein Bcl-2, increased endoplasmic reticulum (ER) stress, and reduced mitochondrial membrane potential. Furthermore, C9orf72 motor neurons, and also cortical neurons, show evidence of abnormal protein aggregation and stress granule formation. This study is an extensive characterization of iPSC-derived motor neurons as cellular models of ALS carrying C9orf72 hexanucleotide repeats, which describes a novel pathogenic link between C9orf72 mutations, dysregulation of calcium signaling, and altered proteostasis and provides a potential pharmacological target for the treatment of ALS and the related neurodegenerative disease frontotemporal dementia. Stem Cells 2016;34:2063-2078.

Keywords: Amyotrophic lateral sclerosis; C9orf72; Calcium dysregulation; Frontotemporal dementia; Induced pluripotent stem cells; Motor neurons.

Figure 1

iPSC line characterization. Characterization data for line iPS C9 T2 7. (A): Karyogram showing genome integrity of iPSC (Illumina SNP array data analyzed using Karyostudio). Autosomal detected regions deviating from reference data are annotated with green (amplification) or orange bands (deletion); the left hand panel shows the parental fibroblasts as reference. (B): FACS analysis of iPSC for pluripotency markers Tra‐1‐60 and Nanog (black line); grey filled plot, isotype control. (C): Human iPSC show pluripotent stem cell‐like morphology (cell‐cell‐contact‐dependent clusters, with high nucleus to cytoplasm ratio) by phase microscopy; scale bar = 100 µm. (D): PluriTest analysis of Illumina HT12v4 transcriptome array data shows that all the iPSC lines used cluster with pluripotent stem cells (red cloud) and not with partly‐ or differentiated cells (blue clouds). Each circle represents one iPSC line, with previously published lines OX1‐19 and NHDF‐1 included as reference points [1 = AH017‐13; 2 = OX3‐9; 3 = OX1‐61; 4 = C902‐10; 5 = AH017‐3; 6 = OX1‐19; 7 = NHDF‐1; 8 = 7245‐1; 9 = 7245‐3; 10 = T2‐7; 11 = C902‐02; 12 = T2‐6]; y axis pluripotency score, x axis novelty score. (E): Assessment of Sendai clearance from iPSC lines reprogrammed with SeVdp(KOSM)302L, by qRT‐PCR, relative to positive control. Abbreviations: iPSC, induced pluripotent stem cell; qPCR, quantitative polymerase chain reaction

Figure 2

Functional characterization of C9orf72 induced pluripotent stem cell (iPSC)‐derived MNs. (A): Immunostaining for Islet1 and Tuj1 in control lines (OX1‐19, OX3‐9, AH017) and six patient lines (C9‐T2‐6, C9‐T2‐7, C9‐7245‐1, C9‐7245‐3, C902‐2, C902‐10). (B): Quantification of Tuj1 positive cells shows 80%–95% of cells are neuronal and (C) quantification of Islet1 positive cells shows 23%–40% of cells are motor neurons, with nonsignificant differences between the genotypes (*, p > .05, one‐way ANOVA). (D): Whole cell recording reveals that trains of action potentials can be evoked reliably in both control MNs and (E) C9orf72 MNs. Representative voltage clamp data showing inward and outward currents recorded during step membrane depolarization of control OX3‐9 (F) and C9‐7245‐3 (G) neurons. (H): Spontaneous calcium oscillations were recorded from neurons after incubation with Fluo‐4 AM, and typical elevations in intracellular calcium were detected on stimulation with (I) KCl and (H) kainate (control neurons are shown in this image). n = 3 independent differentiations; m =10–15 neurons recorded per line; 5 neurons per line for ionic current measurements. Data are represented as average ± SEM. Scale bar = 20 µm. Abbreviations: DAPI, 4′,6‐Diamidino‐2‐Phenylindole; MNs, motor neurons.

Figure 3

RNA foci and repeat‐associated non‐ATG dipeptides are detected in C9orf72 patient‐derived motor neurons (MNs). (A): [CCCCGG]₄ probe was used to detect sense RNA foci from the C9orf72 hexanucleotide expansions. (B): RNA foci were detected in up to 65% of neurons from C9orf72 induced pluripotent stem cell (iPSC)‐derived MNs (121–157 neurons counted), while no foci were detected in the controls (**, p < .01, one‐way ANOVA). (C): Dot blot analysis of GR, PR, GA, and GP dipeptides in controls (OX3‐9 and AH017) and patient lines (C902‐2, C9‐7245‐3 and C902‐3) at baseline and after treatment with MG‐132 for 24 hours. Scale bar = 5 µm. Data are represented as average ± SEM. Abbreviation: DAPI, 4′,6‐Diamidino‐2‐Phenylindole.

Figure 4

Increased ER Ca²⁺ content is detected in C9orf72 induced pluripotent stem cell (iPSC)‐derived MNs along with mitochondrial alterations and Bcl‐2 protein imbalance. (A): Images representing the iPSC‐derived neurons before and after TG stimulation and (B) their representative fluorescent traces. (C): Measurement of the TG‐evoked ER Ca²⁺ release shows significantly higher ER Ca²⁺ levels in C9‐T2 and C9‐7245 lines compared to OX3‐9 (***, p < .001; *, p < .05; one‐way ANOVA with Dunnet's post hoc test); n = 3 independent differentiations, m = 150–200 neurons recorded per genotype. (D): Immunoblotting shows elevated levels of GRP78/BiP in all patient lines and significantly upregulated in C9‐7245‐1 and C9‐7245‐3 (*, p < .05, one‐way ANOVA with Dunnet's post hoc test) (E). n = 3 independent differentiations, m = 142–200 neurons recorded per genotype per differentiation. (F): Immunoblotting for the antiapoptotic Bcl‐2 protein shows downregulation of levels in patient lines and (G) significant reduction in C9‐7245‐1 and C9‐7245‐3 and C902‐2 compared to control OX3‐9, AH017‐13, and OX1‐61 (*, p < .05; **, p < .01, one‐way ANOVA with Dunnett's post hoc test); n = 3 independent differentiations (n = 2 independent differentiations for AH017‐13 and OX1‐61), m = 3–4 technical replicates. (H): Quantitative polymerase chain reaction confirms downregulation of Bcl‐X_L and upregulation of (I) BAK, specifically in the C9‐7245 and C902‐2 MN lines (*, p < .05; **, p < .01; ***, p < .001, one‐way ANOVA with Dunnett's post hoc test); n = 2 independent differentiations; m = 3 technical replicates. (J): Representative image of OX3‐9 loaded with MitoTracker Red CMXRos and immunostained with Islet1 and Tuj1. (K): Quantification of the mean fluorescence intensity of MitoTracker in Islet1⁺/Tuj1⁺ neurons shows reduced mitochondrial membrane potential in all patient lines (***, p < .001, one‐way ANOVA with Dunnett's post hoc test); n = 2 independent differentiations; m = 17–31 neurons analyzed per genotype. (L): Electron micrographs (EM) show enlarged mitochondria and christae in the patient lines C902‐2 and C9‐7245‐3 compared to a healthy control (OX3‐9). Arrows point to mitochondria. (M): Quantification of EM images for mitochondrial alterations show 88% of C9orf72 neurons with swollen mitochondria (**, p < .01, ANOVA with Dunnet's post hoc test). (N): Cytochrome c levels are elevated in all patient lines and significantly higher than healthy controls in C9‐7245‐1 (*, p < .05, one‐way ANOVA with Dunnett's post hoc test). n = 2 independent differentiations, m = 2 technical replicates each. (O): CNs show elevated levels of the apoptotic marker cytochrome c in C902‐10 (*, p < .05; **, p < .01, one‐way ANOVA with Dunnett's post hoc test). n = 2 independent differentiations. Scale bars = 20 µm (J), 1 µm (L). Data are represented as average ± SEM. Abbreviations: CNs, cortical neurons; ER, endoplasmic reticulum; MNs, motor neurons; TG, thapsigargin.

Figure 5

Cleaved caspase‐3 is frequently detected in C9orf72 MNs and cortical neurons (CNs) and reduced cell viability is found in C9orf72 MNs and CNs. (A, B): Immunostaining for cleaved caspase‐3 shows increased frequency of apoptotic neurons in C9orf72 MNs compared to control MNs (*, p < .05; **, p < .01, one‐way ANOVA with Dunnett's post hoc test); n = 3 independent differentiations; m = 400 cells per genotype analyzed for each individual experiment. (C): Reduced cell viability was detected in C9orf72 MNs by PI staining (***, p < .001, one‐way ANOVA with Dunnett's post hoc test) n = 2 independent differentiations. (D, E): Immunostaining with cleaved caspase‐3 shows significantly increased frequency of positive cortical neurons in C902‐2 and C902‐10 lines compared to two healthy control lines (OX3‐9 and AH017‐13) (*, p < .05; ***, p < .001, one‐way ANOVA with Dunnett's post hoc test). (F): Reduced viability was detected by PI staining in the C9orf72 patient (***p < .001, one‐way ANOVA with Dunnett's post hoc test). n = 3 independent differentiations. Data are represented as average ± SEM. Scale bar = 20 µm. Abbreviations: MNs, motor neurons; PI, propidium iodide.

Figure 6

p62 is elevated in C9orf72 neurons and PABP immunopositive stress granules form spontaneously in C9orf72 neurons. (A): Immunostaining for p62 in C9 motor neurons (MNs) shows aggregates are formed under baseline conditions. (B, C): Immunoblotting demonstrates high levels of p62 in C9‐T2‐6, C9‐7245‐3, and C902‐2 MNs compared to controls under baseline conditions (*, p < .05; **, p < .01, one‐way ANOVA with Dunnet's post hoc test); n = 2 independent differentiations, m = 3 technical replicates. (D, E): p62 aggregates are detected more frequently in C9 CNs compared to controls (***, p < .001, one‐way ANOVA with Dunnett's post hoc test). (F, G): Immunoblotting shows elevated levels of LC3‐II in both lines from the C9orf72 patient C902 compared to controls (***, p < .001, one‐way ANOVA with Dunnett's post hoc test); (H) PABP⁺ and TIA‐1⁺ stress granules are detected in all five C9 patient lines under baseline conditions and (I) PABP⁺ MNs are significantly more frequent than in controls (*, p < .05; ***, p < .01, one‐way ANOVA with Dunnet's post hoc test); n = 3 independent differentiations; m = 387–918 neurons analyzed per genotype. (J, K): PABP⁺ stress granules form in C9 CNs (***, p < .001, one‐way ANOVA with Dunnett's post hoc test). Scale bars = 20 µm; scale bars in the bottom panels of (H) = 10 µm. Data are represented as average ± SEM. Abbreviations: CNs, cortical neurons; DAPI, 4′,6‐Diamidino‐2‐Phenylindole; PABP, poly‐A‐binding protein.