Mathieu Rederstorff

Selenoprotein function and muscle disease.

The crucial role of the trace element selenium in livestock and human health, in particular in striated muscle function, has been well established but the underlying molecular mechanisms remain poorly understood. Over the last decade, identification of the full repertoire of selenium-containing proteins has opened the way towards a better characterization of these processes. Two selenoproteins have mainly been investigated in muscle, namely SelW and SelN. Here we address their involvement in muscle development and maintenance, through the characterization of various cellular or animal models. In particular, mutations in the SEPN1 gene encoding selenoprotein N (SelN) cause a group of neuromuscular disorders now referred to as SEPN1-related myopathy. Recent findings on the functional consequences of these mutations suggest an important contribution of SelN to the regulation of oxidative stress and calcium homeostasis. Importantly, the conclusions of these experiments have opened new avenues of investigations that provide grounds for the development of therapeutic approaches.

Satellite cell loss and impaired muscle regeneration in selenoprotein N deficiency

Selenoprotein N (SelN) deficiency causes a group of inherited neuromuscular disorders termed SEPN1-related myopathies (SEPN1-RM). Although the function of SelN remains unknown, recent data demonstrated that it is dispensable for mouse embryogenesis and suggested its involvement in the regulation of ryanodine receptors and/or cellular redox homeostasis. Here, we investigate the role of SelN in satellite cell (SC) function and muscle regeneration, using the Sepn1(-/-) mouse model. Following cardiotoxin-induced injury, SelN expression was strongly up-regulated in wild-type muscles and, for the first time, we detected its endogenous expression in a subset of mononucleated cells by immunohistochemistry. We show that SelN deficiency results in a reduced basal SC pool in adult skeletal muscles and in an imperfect muscle restoration following a single injury. A dramatic depletion of the SC pool was detected after the first round of degeneration and regeneration that totally prevented subsequent regeneration of Sepn1(-/-) muscles. We demonstrate that SelN deficiency affects SC dynamics on isolated single fibres and increases the proliferation of Sepn1(-/-) muscle precursors in vivo and in vitro. Most importantly, exhaustion of the SC population was specifically identified in muscle biopsies from patients with mutations in the SEPN1 gene. In conclusion, we describe for the first time a major physiological function of SelN in skeletal muscles, as a key regulator of SC function, which likely plays a central role in the pathophysiological mechanism leading to SEPN1-RM.

Increased Muscle Stress-Sensitivity Induced by Selenoprotein N Inactivation in Mouse: A Mammalian Model for SEPN1-Related Myopathy

Selenium is an essential trace element and selenoprotein N (SelN) was the first selenium-containing protein shown to be directly involved in human inherited diseases. Mutations in the SEPN1 gene, encoding SelN, cause a group of muscular disorders characterized by predominant affection of axial muscles. SelN has been shown to participate in calcium and redox homeostasis, but its pathophysiological role in skeletal muscle remains largely unknown. To address SelN function in vivo, we generated a Sepn1-null mouse model by gene targeting. The Sepn1−/− mice had normal growth and lifespan, and were macroscopically indistinguishable from wild-type littermates. Only minor defects were observed in muscle morphology and contractile properties in SelN-deficient mice in basal conditions. However, when subjected to challenging physical exercise and stress conditions (forced swimming test), Sepn1−/− mice developed an obvious phenotype, characterized by limited motility and body rigidity during the swimming session, as well as a progressive curvature of the spine and predominant alteration of paravertebral muscles. This induced phenotype recapitulates the distribution of muscle involvement in patients with SEPN1-Related Myopathy, hence positioning this new animal model as a valuable tool to dissect the role of SelN in muscle function and to characterize the pathophysiological process.

Identification of differentially expressed non-coding RNAs in embryonic stem cell neural differentiation

Protein-coding genes, guiding differentiation of ES cells into neural cells, have extensively been studied in the past. However, for the class of ncRNAs only the involvement of some specific microRNAs (miRNAs) has been described. Thus, to characterize the entire small non-coding RNA (ncRNA) transcriptome, involved in the differentiation of mouse ES cells into neural cells, we have generated three specialized ribonucleo-protein particle (RNP)-derived cDNA libraries, i.e. from pluripotent ES cells, neural progenitors and differentiated neural cells, respectively. By high-throughput sequencing and transcriptional profiling we identified several novel miRNAs to be involved in ES cell differentiation, as well as seven small nucleolar RNAs. In addition, expression of 7SL, 7SK and vault-2 RNAs was significantly up-regulated during ES cell differentiation. About half of ncRNA sequences from the three cDNA libraries mapped to intergenic or intragenic regions, designated as interRNAs and intraRNAs, respectively. Thereby, novel ncRNA candidates exhibited a predominant size of 18–30 nt, thus resembling miRNA species, but, with few exceptions, lacking canonical miRNA features. Additionally, these novel intraRNAs and interRNAs were not only found to be differentially expressed in stem-cell derivatives, but also in primary cultures of hippocampal neurons and astrocytes, strengthening their potential function in neural ES cell differentiation.

Selenoprotein N is dynamically expressed during mouse development and detected early in muscle precursors

BackgroundIn humans, mutations in the SEPN1 gene, encoding selenoprotein N (SelN), are involved in early onset recessive neuromuscular disorders, referred to as SEPN1-related-myopathies. The mechanisms behind these pathologies are poorly understood since the function of SelN remains elusive. However, previous results obtained in humans and more recently in zebrafish pointed to a potential role for SelN during embryogenesis. Using qRT-PCR, Western blot and whole mount in situ hybridization, we characterized in detail the spatio-temporal expression pattern of the murine Sepn1 gene during development, focusing particularly on skeletal muscles.ResultsIn whole embryos, Sepn1 transcripts were detected as early as E5.5, with expression levels peaking at E12.5, and then strongly decreasing until birth. In isolated tissues, only mild transcriptional variations were observed during development, whereas a striking reduction of the protein expression was detected during the perinatal period. Furthermore, we demonstrated that Sepn1 is expressed early in somites and restricted to the myotome, the sub-ectodermal mesenchyme and the dorsal root ganglia at mid-gestation stages. Interestingly, Sepn1 deficiency did not alter somitogenesis in embryos, suggesting that SelN is dispensable for these processes in mouse.ConclusionWe characterized for the first time the expression pattern of Sepn1 during mammalian embryogenesis and we demonstrated that its differential expression is most likely dependent on major post-transcriptional regulations. Overall, our data strongly suggest a potential role for selenoprotein N from mid-gestation stages to the perinatal period. Interestingly, its specific expression pattern could be related to the current hypothesis that selenoprotein N may regulate the activity of the ryanodine receptors.

Compartmentalization and Regulation of Mitochondrial Function by Methionine Sulfoxide Reductases in Yeast

Elevated levels of reactive oxygen species can damage proteins. Sulfur-containing amino acid residues, cysteine and methionine, are particularly susceptible to such damage. Various enzymes evolved to protect proteins or repair oxidized residues, including methionine sulfoxide reductases MsrA and MsrB, which reduce methionine (S)-sulfoxide (Met-SO) and methionine (R)-sulfoxide (Met-RO) residues, respectively, back to methionine. Here, we show that MsrA and MsrB are involved in the regulation of mitochondrial function. Saccharomyces cerevisiae mutant cells lacking MsrA, MsrB, or both proteins had normal levels of mitochondria but lower levels of cytochrome c and fewer respiration-competent mitochondria. The growth of single MsrA or MsrB mutants on respiratory carbon sources was inhibited, and that of the double mutant was severely compromised, indicating impairment of mitochondrial function. Although MsrA and MsrB are thought to have similar roles in oxidative protein repair each targeting a diastereomer of methionine sulfoxide, their deletion resulted in different phenotypes. GFP fusions of MsrA and MsrB showed different localization patterns and primarily localized to cytoplasm and mitochondria, respectively. This finding agreed with compartment-specific enrichment of MsrA and MsrB activities. These results show that oxidative stress contributes to mitochondrial dysfunction through oxidation of methionine residues in proteins located in different cellular compartments.

Molecular Basis for the Role of Selenium in Muscle Development and Function

Selenium Deficiencies Leading to Muscular Diseases. – Selenium (Se) is an essential trace element; identification of pathologies due to its dietary restriction or mis-absorption pointed to its vital nutrient function, in animals and in humans. In addition, optimal Se supplementation was shown to be beneficial to many aspects of human health [1]. It is important for cancer and heart disease prevention, inhibition of viral infection, and delaying the progression of AIDS in HIV positive patients. Moreover, Se has been reported to have a role in immune function, male fertility, mammalian development, and retarding the aging process. Characterized defects caused by Se deficiency also include several cardiac and skeletal muscle disorders [2] [3]. Previous observations showed that the lack of Se is the cause for different forms of muscular diseases in both cattle and humans, defining a new syndrome called nutritional muscular dystrophy (for references, see [3]). Animal myopathies – white muscle disease in cows, calves, and sheep, or rigid lamb syndrome – are characterized by the alteration of cardiac and skeletal muscle fibers, with extensive calcification. Emergence of these diseases was linked to Se content in the food and Se supplementation was shown to protect the animals. In humans, Keshan disease is another Se-related disorder occurring in different regions of eastern China with very low Se in the food supply, due to raising crops in Se-poor soils. This disease corresponds to a cardiomyopathy, characterized by necrotic lesions, inflammatory areas, and calcification throughout the myocardium. Experimental approaches in mouse demonstrated a dual ethiology for this disease, caused by both dietary deficiency in the trace element and infection by the enterovirus Coxsackie. Proliferation of an originally nonvirulent strain in Se-deficient individuals introduced mutations in the genome of the virus, turning it into a cardiovirulent one (reviewed in [4]). The pathogenicity and irreversibility of the mutations were demonstrated as the modified viral strain also provoked heart damages in adequate selenium mice. Connection between the selenoenzyme glutathione peroxidase and the oxidative damage occurring at the viral DNA level was demonstrated, since glutathione peroxidase knock-out mice infected with the non-virulent strain recapitulated the symptoms. Vitamin E deficiency and aging were shown to be two other conditions for the cardio-virulent conversion of the viral strain [5]. Prolonged parenteral nutrition in humans is another cause for proximal muscle pain and weakness associated with severe Se deficiency in humans. In these CHEMISTRY & BIODIVERSITY – Vol. 5 (2008) 408

Expression Profiling of a Heterogeneous Population of ncRNAs Employing a Mixed DNA/LNA Microarray

Mammalian transcriptomes mainly consist of non protein coding RNAs. These ncRNAs play various roles in all cells and are involved in multiple regulation pathways. More recently, ncRNAs have also been described as valuable diagnostic tools. While RNA-seq approaches progressively replace microarray-based technologies for high-throughput expression profiling, they are still not routinely used in diagnostic. Microarrays, on the other hand, are more widely used for diagnostic profiling, especially for very small ncRNA (e.g., miRNAs), employing locked nucleic acid (LNA) arrays. However, LNA microarrays are quite expensive for high-throughput studies targeting longer ncRNAs, while DNA arrays do not provide satisfying results for the analysis of small RNAs. Here, we describe a mixed DNA/LNA microarray platform, where directly labeled small and longer ncRNAs are hybridized on LNA probes or custom DNA probes, respectively, enabling sensitive and specific analysis of a complex RNA population on a unique array in one single experiment. The DNA/LNA system, requiring relatively low amounts of total RNA, which complies with diagnostic references, was successfully applied to the analysis of differential ncRNA expression in mouse embryonic stem cells and adult brain cells.

Expression Profiling of ncRNAs Employing RNP Libraries and Custom LNA/DNA Microarray Analysis

Recently, it has been shown by the ENCODE consortium that more than 90% of the human genome might be transcribed. While only about 1.5% of these transcripts correspond to mRNAs, it was proposed that the majority of them (i.e., 88.5%) might correspond to regulatory noncoding RNAs (ncRNAs). Numerous protocols dedicated to the generation of cDNA libraries coupled to next-generation sequencing (NGS) technologies are currently available to identify novel ncRNA species, and we have recently developed a novel procedure for the generation of ribonucleoprotein (RNP) libraries. To validate differential expression of ncRNAs identified using our or any library generation approach, we describe an innovative ncRNA profiling approach based on microarray technology. Employing LNA probes, dedicated to the analysis of small/microRNAs, and DNA probes, dedicated to the study of longer ncRNAs, our platform enables the study of most ncRNAs independently of their length in a single experiment. Detailed methodological solution description includes the automated design of probes to be spotted on the array, optimization of spotting and labeling of probes, as well as hybridization conditions. All the steps have been improved for the analysis of ncRNAs, which are generally difficult to study owing to their peculiarities in terms of secondary structure or abundance.

O.3 Satellite cell loss is the pathomechanism leading to muscle atrophy in selenoprotein N deficiency

zygous for the RT insertion mutation, five of whom carried a novel intronic mutation that activates a pseudoexon between exons 5 and 6 (c.647 + 2084G > T). Compared with individuals that were homozygous for the RT insertion mutation, the seven heterozygotes for the RT insertion mutation, including five patients with the novel pseudoexon mutation, exhibited a more severe clinical phenotype in terms of motor abilities and more extensive brain MRI abnormalities (i.e., a wider distribution of cortical malformation, pons and cerebellar hypoplasia, and more frequent diffuse white matter changes and ventricular dilatation). Conclusions FKTN mutations are the most common genetic cause of CMD with defective a-DG glycosylation in Korea. Compound heterozygosity of the RT insertion and the novel pseudoexon mutation is the most prevalent genotype in Korea and is associated with a more severe clinical phenotype and a wider extent of brain MRI abnormalities compared with homozygosity for the RT insertion mutation.