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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

View Article: PubMed Central - PubMed

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 Ca2+ 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 Cells2016;34:2063–2078

No MeSH data available.


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.
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stem2388-fig-0003: 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.

Mentions: In the MNs derived from C9orf72 patients, we identified a significant increase in intranuclear GGGGCC‐repeat containing RNA foci in all lines (Fig. 3A, 3B). The specificity of the probe was confirmed by treatment with RNase, which reduced the number of foci substantially (Supporting Information Fig. S4A), and with DNase I, after which the number of foci was maintained. To investigate evidence for the RAN translation hypothesis, we performed a dot blot analysis using anti‐GA, GP, GR, and PR antibodies, and we could detect traces of all the dipeptides in the MNs from C9orf72 patients (Fig. 3C).


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
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.
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stem2388-fig-0003: 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.
Mentions: In the MNs derived from C9orf72 patients, we identified a significant increase in intranuclear GGGGCC‐repeat containing RNA foci in all lines (Fig. 3A, 3B). The specificity of the probe was confirmed by treatment with RNase, which reduced the number of foci substantially (Supporting Information Fig. S4A), and with DNase I, after which the number of foci was maintained. To investigate evidence for the RAN translation hypothesis, we performed a dot blot analysis using anti‐GA, GP, GR, and PR antibodies, and we could detect traces of all the dipeptides in the MNs from C9orf72 patients (Fig. 3C).

View Article: PubMed Central - PubMed

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&#8208;derived motor neurons, decreased cell survival is correlated with dysfunction in Ca2+ homeostasis, reduced levels of the antiapoptotic protein Bcl&#8208;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&#8208;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 Cells2016;34:2063&ndash;2078

No MeSH data available.