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Faithful SGCE imprinting in iPSC-derived cortical neurons: an endogenous cellular model of myoclonus-dystonia.

ABSTRACT: In neuropathology research, induced pluripotent stem cell (iPSC)-derived neurons are considered a tool closely resembling the patient brain. Albeit in respect to epigenetics, this concept has been challenged. We generated iPSC-derived cortical neurons from myoclonus-dystonia patients with mutations (W100G and R102X) in the maternally imprinted ?-sarcoglycan (SGCE) gene and analysed properties such as imprinting, mRNA and protein expression. Comparison of the promoter during reprogramming and differentiation showed tissue-independent differential methylation. DNA sequencing with methylation-specific primers and cDNA analysis in patient neurons indicated selective expression of the mutated paternal SGCE allele. While fibroblasts only expressed the ubiquitous mRNA isoform, brain-specific SGCE mRNA and ?-sarcoglycan protein were detected in iPSC-derived control neurons. However, neuronal protein levels were reduced in both mutants. Our phenotypic characterization highlights the suitability of iPSC-derived cortical neurons with SGCE mutations for myoclonus-dystonia research and, in more general terms, prompts the use of iPSC-derived cellular models to study epigenetic mechanisms impacting on health and disease.

SUBMITTER: Grutz K

PROVIDER: S-EPMC5290732 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Faithful SGCE imprinting in iPSC-derived cortical neurons: an endogenous cellular model of myoclonus-dystonia.

Grütz Karen K Seibler Philip P Weissbach Anne A Lohmann Katja K Carlisle Francesca A FA Blake Derek J DJ Westenberger Ana A Klein Christine C Grünewald Anne A

Scientific reports 20170203

In neuropathology research, induced pluripotent stem cell (iPSC)-derived neurons are considered a tool closely resembling the patient brain. Albeit in respect to epigenetics, this concept has been challenged. We generated iPSC-derived cortical neurons from myoclonus-dystonia patients with mutations (W100G and R102X) in the maternally imprinted ε-sarcoglycan (SGCE) gene and analysed properties such as imprinting, mRNA and protein expression. Comparison of the promoter during reprogramming and dif ...[more]

PMID: 28155872

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Project description:Loss-of-function mutations in SGCE, which encodes ε-sarcoglycan (ε-SG), cause myoclonus-dystonia syndrome (OMIM159900, DYT11). A "major" ε-SG protein derived from CCDS5637.1 (NM_003919.2) and a "brain-specific" protein, that includes sequence derived from alternative exon 11b (CCDS47642.1, NM_001099400.1), are reportedly localized in post- and pre-synaptic membrane fractions, respectively. Moreover, deficiency of the "brain-specific" isoform and other isoforms derived from exon 11b may be central to the pathogenesis of DYT11. However, no animal model supports this hypothesis. Gene-trapped ES cells (CMHD-GT_148G1-3, intron 9 of NM_011360) were used to generate a novel Sgce mouse model (C57BL/6J background) with markedly reduced expression of isoforms derived from exons 3' to exon 9 of NM_011360. Among those brain regions analyzed in adult (2month-old) wild-type (WT) mice, cerebellum showed the highest relative expression of isoforms incorporating exon 11b. Homozygotes (SgceGt(148G1)Cmhd/Gt(148G1)Cmhd or SgceGt/Gt) and paternal heterozygotes (Sgcem+/pGt, m-maternal, p-paternal) showed 60 to 70% reductions in expression of total Sgce. Although expression of the major (NM_011360) and brain-specific (NM_001130189) isoforms was markedly reduced, expression of short isoforms was preserved and relatively small amounts of chimeric ε-SG/β-galactosidase fusion protein was produced by the Sgce gene-trap locus. Immunoaffinity purification followed by mass spectrometry assessments of Sgcem+/pGt mouse brain using pan- or brain-specific ε-SG antibodies revealed significant reductions of ε-SG and other interacting sarcoglycans. Genome-wide gene-expression data using RNA derived from adult Sgcem+/pGt mouse cerebellum showed that the top up-regulated genes were involved in cell cycle, cellular development, cell death and survival, while the top down-regulated genes were associated with protein synthesis, cellular development, and cell death and survival. In comparison to WT littermates, Sgcem+/pGt mice exhibited "tiptoe" gait and stimulus-induced appendicular posturing between Postnatal Days 14 to 16. Abnormalities noted in older Sgcem+/pGt mice included reduced body weight, altered gait dynamics, and reduced open-field activity. Overt spontaneous or stimulus-sensitive myoclonus was not apparent on the C57BL/6J background or mixed C57BL/6J-BALB/c and C57BL/6J-129S2 backgrounds. Our data confirm that mouse Sgce is a maternally imprinted gene and suggests that short Sgce isoforms may compensate, in part, for deficiency of major and brain-specific Sgce isoforms.

Dataset Information

Faithful SGCE imprinting in iPSC-derived cortical neurons: an endogenous cellular model of myoclonus-dystonia.

Publications

Faithful SGCE imprinting in iPSC-derived cortical neurons: an endogenous cellular model of myoclonus-dystonia.

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