Role of major and brain-specific Sgce isoforms in the pathogenesis of myoclonus-dystonia syndrome.

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/ß-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.

Myoclonus dystonia and muscular dystrophy: ɛ-sarcoglycan is part of the dystrophin-associated protein complex in brain.

BACKGROUND: Myoclonus-dystonia is a neurogenic movement disorder caused by mutations in the gene encoding ɛ-sarcoglycan. By contrast, mutations in the α-, ß-, γ-, and δ-sarcoglycan genes cause limb girdle muscular dystrophies. The sarcoglycans are part of the dystrophin-associated protein complex in muscle that is disrupted in several types of muscular dystrophy. Intriguingly, patients with myoclonus-dystonia have no muscle pathology; conversely, limb-girdle muscular dystrophy patients have not been reported to have dystonia-associated features. To gain further insight into the molecular mechanisms underlying these differences, we searched for evidence of a sarcoglycan complex in the brain. METHODS: Immunoaffinity chromatography and mass spectrometry were used to purify ubiquitous and brain-specific ɛ-sarcoglycan directly from tissue. Cell models were used to determine the effect of mutations on the trafficking and assembly of the brain sarcoglycan complex. RESULTS: Ubiquitous and brain-specific ɛ-sarcoglycan isoforms copurify with ß-, δ-, and ζ-sarcoglycan, ß-dystroglycan, and dystrophin Dp71 from brain. Incorporation of a muscular dystrophy-associated ß-sarcoglycan mutant into the brain sarcoglycan complex impairs the formation of the ßδ-sarcoglycan core but fails to abrogate the association and membrane trafficking of ɛ- and ζ-sarcoglycan. CONCLUSIONS: ɛ-Sarcoglycan is part of the dystrophin-associated protein complex in brain. Partial preservation of ɛ- and ζ-sarcoglycan in brain may explain the absence of myoclonus dystonia-like features in muscular dystrophy patients. © 2016 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

Naive pluripotent stem cell-based models capture FGF-dependent human hypoblast lineage specification.

The hypoblast is an essential extraembryonic tissue set aside within the inner cell mass in the blastocyst. Research with human embryos is challenging. Thus, stem cell models that reproduce hypoblast differentiation provide valuable alternatives. We show here that human naive pluripotent stem cell (PSC) to hypoblast differentiation proceeds via reversion to a transitional ICM-like state from which the hypoblast emerges in concordance with the trajectory in human blastocysts. We identified a window when fibroblast growth factor (FGF) signaling is critical for hypoblast specification. Revisiting FGF signaling in human embryos revealed that inhibition in the early blastocyst suppresses hypoblast formation. In vitro, the induction of hypoblast is synergistically enhanced by limiting trophectoderm and epiblast fates. This finding revises previous reports and establishes a conservation in lineage specification between mice and humans. Overall, this study demonstrates the utility of human naive PSC-based models in elucidating the mechanistic features of early human embryogenesis.

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

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 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 ε-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.

Ryanodine receptors are part of the myospryn complex in cardiac muscle.

The Cardiomyopathy-associated gene 5 (Cmya5) encodes myospryn, a large tripartite motif (TRIM)-related protein found predominantly in cardiac and skeletal muscle. Cmya5 is an expression biomarker for a number of diseases affecting striated muscle and may also be a schizophrenia risk gene. To further understand the function of myospryn in striated muscle, we searched for additional myospryn paralogs. Here we identify a novel muscle-expressed TRIM-related protein minispryn, encoded by Fsd2, that has extensive sequence similarity with the C-terminus of myospryn. Cmya5 and Fsd2 appear to have originated by a chromosomal duplication and are found within evolutionarily-conserved gene clusters on different chromosomes. Using immunoaffinity purification and mass spectrometry we show that minispryn co-purifies with myospryn and the major cardiac ryanodine receptor (RyR2) from heart. Accordingly, myospryn, minispryn and RyR2 co-localise at the junctional sarcoplasmic reticulum of isolated cardiomyocytes. Myospryn redistributes RyR2 into clusters when co-expressed in heterologous cells whereas minispryn lacks this activity. Together these data suggest a novel role for the myospryn complex in the assembly of ryanodine receptor clusters in striated muscle.

S1PR2 variants associated with auditory function in humans and endocochlear potential decline in mouse.

Progressive hearing loss is very common in the population but we still know little about the underlying pathology. A new spontaneous mouse mutation (stonedeaf, stdf ) leading to recessive, early-onset progressive hearing loss was detected and exome sequencing revealed a Thr289Arg substitution in Sphingosine-1-Phosphate Receptor-2 (S1pr2). Mutants aged 2 weeks had normal hearing sensitivity, but at 4 weeks most showed variable degrees of hearing impairment, which became severe or profound in all mutants by 14 weeks. Endocochlear potential (EP) was normal at 2 weeks old but was reduced by 4 and 8 weeks old in mutants, and the stria vascularis, which generates the EP, showed degenerative changes. Three independent mouse knockout alleles of S1pr2 have been described previously, but this is the first time that a reduced EP has been reported. Genomic markers close to the human S1PR2 gene were significantly associated with auditory thresholds in the 1958 British Birth Cohort (n = 6099), suggesting involvement of S1P signalling in human hearing loss. The finding of early onset loss of EP gives new mechanistic insight into the disease process and suggests that therapies for humans with hearing loss due to S1P signalling defects need to target strial function.

Huntingtin Exists as Multiple Splice Forms in Human Brain.

BACKGROUND: A CAG repeat expansion in HTT has been known to cause Huntington's disease for over 20 years. The genomic sequence of the 67 exon HTT is clear but few reports have detailed alternative splicing or alternative transcripts. Most eukaryotic genes with multiple exons show alternative splicing that increases the diversity of the transcriptome and proteome: it would be surprising if a gene with 67 known exons in its two major transcripts did not present some alternative transcripts. OBJECTIVE: To investigate the presence of alternative transcripts directly in human HTT. METHODS: An overlapping RT-PCR based approach was used to determine novel HTT splice variants in human brain from HD patients and controls and 3D protein homology modelling employed to investigate their significance on the function of the HTT protein. RESULTS: Here we show multiple previously unreported novel transcripts of HTT. Of the 22 splice variants found, eight were in-frame with the potential to encode novel HTT protein isoforms. Two splice variants were selected for further study; HTT Δex4,5,6 which results in the skipping of exons 4, 5 and 6 and HTTex41b which includes a novel exon created via partial retention of intron 41. 3D protein homology modelling showed that both splice variants are of potential functional significance leading to the loss of a karyopherin nuclear localisation signal and alterations to sites of posttranslational modification. CONCLUSIONS: The identification of novel HTT transcripts has implications for HTT protein isoform expression and function. Understanding the functional significance of HTT alternative splicing would be critical to guide the design of potential therapeutics in HD that aim to reduce the toxic HTT transcript or protein product including RNA silencing and correction of mis-splicing in disease.

Specific expression of Kcna10, Pxn and Odf2 in the organ of Corti.

The development of the organ of Corti and the highly specialized cells required for hearing involves a multitude of genes, many of which remain unknown. Here we describe the expression pattern of three genes not previously studied in the inner ear in mice at a range of ages both embryonic and early postnatal. Kcna10, a tetrameric Shaker-like potassium channel, is expressed strongly in the hair cells themselves. Odf2, as its centriolar isoform Cenexin, marks the dendrites extending to and contacting hair cells, and Pxn, a focal adhesion scaffold protein, is most strongly expressed in pillar cells during the ages studied. The roles of these genes are yet to be elucidated, but their specific expression patterns imply potential functional significance in the inner ear.