Virginie Carmignac

Autophagy is increased in laminin α2 chain-deficient muscle and its inhibition improves muscle morphology in a mouse model of MDC1A

Congenital muscular dystrophy caused by laminin α2 chain deficiency (also known as MDC1A) is a severe and incapacitating disease, characterized by massive muscle wasting. The ubiquitin-proteasome system plays a major role in muscle wasting and we recently demonstrated that increased proteasomal activity is a feature of MDC1A. The autophagy-lysosome pathway is the other major system involved in degradation of proteins and organelles within the muscle cell. However, it remains to be determined if the autophagy-lysosome pathway is dysregulated in muscular dystrophies, including MDC1A. Using the dy(3K)/dy(3K) mouse model of laminin α2 chain deficiency and MDC1A patient muscle, we show here that expression of autophagy-related genes is upregulated in laminin α2 chain-deficient muscle. Moreover, we found that autophagy inhibition significantly improves the dystrophic dy(3K)/dy(3K) phenotype. In particular, we show that systemic injection of 3-methyladenine (3-MA) reduces muscle fibrosis, atrophy, apoptosis and increases muscle regeneration and muscle mass. Importantly, lifespan and locomotive behavior were also greatly improved. These findings indicate that enhanced autophagic activity is pathogenic and that autophagy inhibition holds a promising therapeutic potential in the treatment of MDC1A.

PIK3R1 Mutations Cause Syndromic Insulin Resistance with Lipoatrophy

Short stature, hyperextensibility of joints and/or inguinal hernia, ocular depression, Rieger anomaly, and teething delay (SHORT) syndrome is a developmental disorder with an unknown genetic cause and hallmarks that include insulin resistance and lack of subcutaneous fat. We ascertained two unrelated individuals with SHORT syndrome, hypothesized that the observed phenotype was most likely due to de novo mutations in the same gene, and performed whole-exome sequencing in the two probands and their unaffected parents. We then confirmed our initial observations in four other subjects with SHORT syndrome from three families, as well as 14 unrelated subjects presenting with syndromic insulin resistance and/or generalized lipoatrophy associated with dysmorphic features and growth retardation. Overall, we identified in nine affected individuals from eight families de novo or inherited PIK3R1 mutations, including a mutational hotspot (c.1945C>T [p.Arg649Trp]) present in four families. PIK3R1 encodes the p85α, p55α, and p50α regulatory subunits of class IA phosphatidylinositol 3 kinases (PI3Ks), which are known to play a key role in insulin signaling. Functional data from fibroblasts derived from individuals with PIK3R1 mutations showed severe insulin resistance for both proximal and distal PI3K-dependent signaling. Our findings extend the genetic causes of severe insulin-resistance syndromes and provide important information with respect to the function of PIK3R1 in normal development and its role in human diseases, including growth delay, Rieger anomaly and other ocular affections, insulin resistance, diabetes, paucity of fat, and ovarian cysts.

Cell–matrix interactions in muscle disease†

The extracellular matrix (ECM) provides a solid scaffold and signals to cells through ECM receptors. The cell–matrix interactions are crucial for normal biological processes and when disrupted they may lead to pathological processes. In particular, the biological importance of ECM–cell membrane–cytoskeleton interactions in skeletal muscle is accentuated by the number of inherited muscle diseases caused by mutations in proteins conferring these interactions. In this review we introduce laminins, collagens, dystroglycan, integrins, dystrophin and sarcoglycans. Mutations in corresponding genes cause various forms of muscular dystrophy. The muscle disorders are presented as well as advances toward the development of treatment. Copyright

Proteasome inhibition improves the muscle of laminin α2 chain-deficient mice

Muscle atrophy, a significant characteristic of congenital muscular dystrophy with laminin α2 chain deficiency (also known as MDC1A), occurs by a change in the normal balance between protein synthesis and protein degradation. The ubiquitin-proteasome system (UPS) plays a key role in protein degradation in skeletal muscle cells. In order to identify new targets for drug therapy against MDC1A, we have investigated whether increased proteasomal degradation is a feature of MDC1A. Using the generated dy(3K)/dy(3K) mutant mouse model of MDC1A, we studied the expression of members of the ubiquitin-proteasome pathway in laminin α2 chain-deficient muscle, and we treated dy(3K)/dy(3K) mice with the proteasome inhibitor MG-132. We show that members of the UPS are upregulated and that the global ubiquitination of proteins is raised in dystrophic limb muscles. Also, phosphorylation of Akt is diminished in diseased muscles. Importantly, proteasome inhibition significantly improves the dystrophic dy(3K)/dy(3K) phenotype. Specifically, treatment with MG-132 increases lifespan, enhances locomotive activity, enlarges muscle fiber diameter, reduces fibrosis, restores Akt phosphorylation and decreases apoptosis. These studies promote better understanding of the disease process in mice and could lead to a drug therapy for MDC1A patients.

In-Frame Mutations in Exon 1 of SKI Cause Dominant Shprintzen-Goldberg Syndrome

Shprintzen-Goldberg syndrome (SGS) is characterized by severe marfanoid habitus, intellectual disability, camptodactyly, typical facial dysmorphism, and craniosynostosis. Using family-based exome sequencing, we identified a dominantly inherited heterozygous in-frame deletion in exon 1 of SKI. Direct sequencing of SKI further identified one overlapping heterozygous in-frame deletion and ten heterozygous missense mutations affecting recurrent residues in 18 of the 19 individuals screened for SGS; these individuals included one family affected by somatic mosaicism. All mutations were located in a restricted area of exon 1, within the R-SMAD binding domain of SKI. No mutation was found in a cohort of 11 individuals with other marfanoid-craniosynostosis phenotypes. The interaction between SKI and Smad2/3 and Smad 4 regulates TGF-β signaling, and the pattern of anomalies in Ski-deficient mice corresponds to the clinical manifestations of SGS. These findings define SGS as a member of the family of diseases associated with the TGF-β-signaling pathway.

12p13.33 microdeletion including ELKS/ERC1, a new locus associated with childhood apraxia of speech.

Speech sound disorders are heterogeneous conditions, and sporadic and familial cases have been described. However, monogenic inheritance explains only a small proportion of such disorders, in particular in cases with childhood apraxia of speech (CAS). Deletions of <5 Mb involving the 12p13.33 locus is one of the least commonly deleted subtelomeric regions. Only four patients have been reported with such a deletion diagnosed with fluorescence in situ hybridisation telomere analysis or array CGH. To further delineate this rare microdeletional syndrome, a French collaboration together with a search in the Decipher database allowed us to gather nine new patients with a 12p13.33 subtelomeric or interstitial rearrangement identified by array CGH. Speech delay was found in all patients, which could be defined as CAS when patients had been evaluated by a speech therapist (5/9 patients). Intellectual deficiency was found in 5/9 patients only, and often associated with psychiatric manifestations of various severity. Two such deletions were inherited from an apparently healthy parent, but reevaluation revealed abnormal speech production at least in childhood, suggesting variable expressivity. The ELKS/ERC1 gene, which encodes for a synaptic factor, is found in the smallest region of overlap. These results reinforce the hypothesis that deletions of the 12p13.33 locus may be responsible for variable phenotypes including CAS associated with neurobehavioural troubles and that the presence of CAS justifies a genetic work-up.

Distinct roles for laminin globular domains in laminin alpha1 chain mediated rescue of murine laminin alpha2 chain deficiency.

Background Laminin α2 chain mutations cause congenital muscular dystrophy with dysmyelination neuropathy (MDC1A). Previously, we demonstrated that laminin α1 chain ameliorates the disease in mice. Dystroglycan and integrins are major laminin receptors. Unlike laminin α2 chain, α1 chain binds the receptors by separate domains; laminin globular (LG) domains 4 and LG1-3, respectively. Thus, the laminin α1 chain is an excellent tool to distinguish between the roles of dystroglycan and integrins in the neuromuscular system. Methodology/Principal Findings Here, we provide insights into the functions of laminin α1LG domains and the division of their roles in MDC1A pathogenesis and rescue. Overexpression of laminin α1 chain that lacks the dystroglycan binding LG4-5 domains in α2 chain deficient mice resulted in prolonged lifespan and improved health. Importantly, diaphragm and heart muscles were corrected, whereas limb muscles were dystrophic, indicating that different muscles have different requirements for LG4-5 domains. Furthermore, the regenerative capacity of the skeletal muscle did not depend on laminin α1LG4-5. However, this domain was crucial for preventing apoptosis in limb muscles, essential for myelination in peripheral nerve and important for basement membrane assembly. Conclusions/Significance These results show that laminin α1LG domains and consequently their receptors have disparate functions in the neuromuscular system. Understanding these interactions could contribute to design and optimization of future medical treatment for MDC1A patients.

Cib2 binds integrin a7Bb1D and is reduced in laminin a2 chain deficient muscular dystrophy

Mutations in the gene encoding laminin α2 chain cause congenital muscular dystrophy type 1A. In skeletal muscle, laminin α2 chain binds at least two receptor complexes: the dystrophin-glycoprotein complex and integrin α7β1. To gain insight into the molecular mechanisms underlying this disorder, we performed gene expression profiling of laminin α2 chain-deficient mouse limb muscle. One of the down-regulated genes encodes a protein called Cib2 (calcium- and integrin-binding protein 2) whose expression and function is unknown. However, the closely related Cib1 has been reported to bind integrin αIIb and may be involved in outside-in-signaling in platelets. Since Cib2 might be a novel integrin α7β1-binding protein in muscle, we have studied Cib2 expression in the developing and adult mouse. Cib2 mRNA is mainly expressed in the developing central nervous system and in developing and adult skeletal muscle. In skeletal muscle, Cib2 colocalizes with the integrin α7B subunit at the sarcolemma and at the neuromuscular and myotendinous junctions. Finally, we demonstrate that Cib2 is a calcium-binding protein that interacts with integrin α7Bβ1D. Thus, our data suggest a role for Cib2 as a cytoplasmic effector of integrin α7Bβ1D signaling in skeletal muscle.

The placenta: phenotypic and epigenetic modifications induced by Assisted Reproductive Technologies throughout pregnancy

Today, there is growing interest in the potential epigenetic risk related to assisted reproductive technologies (ART). Much evidence in the literature supports the hypothesis that adverse pregnancy outcomes linked to ART are associated with abnormal trophoblastic invasion. The aim of this review is to investigate the relationship between epigenetic dysregulation caused by ART and subsequent placental response. The dialogue between the endometrium and the embryo is a crucial step to achieve successful trophoblastic invasion, thus ensuring a non-complicated pregnancy and healthy offspring. However, as described in this review, ART could impair both actors involved in this dialogue. First, ART may induce epigenetic defects in the conceptus by modifying the embryo environment. Second, as a result of hormone treatments, ART may impair endometrial receptivity. In some cases, it results in embryonic growth arrest but, when the development of the embryo continues, the placenta could bring adaptive responses throughout pregnancy. Amongst the different mechanisms, epigenetics, especially thanks to a finely tuned network of imprinted genes stimulated by foetal signals, may modify nutrient transfer, placental growth and vascularization. If these coping mechanisms are overwhelmed, improper maternal-foetal exchanges occur, potentially leading to adverse pregnancy outcomes such as abortion, preeclampsia or intra-uterine growth restriction. But in most cases, successful placental adaptation enables normal progress of the pregnancy. Nevertheless, the risks induced by these modifications during pregnancy are not fully understood. Metabolic diseases later in life could be exacerbated through the memory of epigenetic adaptation mechanisms established during pregnancy. Thus, more research is still needed to better understand abnormal interactions between the embryo and the milieu in artificial conditions. As trophectoderm cells are in direct contact with the environment, they deserve to be studied in more detail. The ultimate goal of these studies will be to render ART protocols safer. Optimization of the environment will be the key to improving the dialogue between the endometrium and embryo, so as to ensure that placentation after ART is similar to that following natural conception.

Tif1γ regulates the TGF-β1 receptor and promotes physiological aging of hematopoietic stem cells

Significance Hematopoietic stem cell aging has been directly linked to the development of several hematological disorders, including myeloproliferative diseases. Here we show that in elderly mice (20 mo old), physiological aging of the hematopoietic system is linked to a decreased expression of transcription intermediary factor 1γ (Tif1γ) in HSCs. In turn, in young Tif1γ−/− mice (4 mo old), the hematopoiesis aging phenotype is exacerbated. In both sets of mice, Tif1γ level controls the TGF-β receptor 1 (Tgfbr1) turnover and subtly regulates the number of myeloid-biased HSCs in bone marrow. We establish that young Tif1γ−/− mice develop a phenotype of premature hematopoietic aging that may explain their predisposition to myeloproliferative disease. The hematopoietic system declines with age. Myeloid-biased differentiation and increased incidence of myeloid malignancies feature aging of hematopoietic stem cells (HSCs), but the mechanisms involved remain uncertain. Here, we report that 4-mo-old mice deleted for transcription intermediary factor 1γ (Tif1γ) in HSCs developed an accelerated aging phenotype. To reinforce this result, we also show that Tif1γ is down-regulated in HSCs during aging in 20-mo-old wild-type mice. We established that Tif1γ controls TGF-β1 receptor (Tgfbr1) turnover. Compared with young HSCs, Tif1γ−/− and old HSCs are more sensitive to TGF-β signaling. Importantly, we identified two populations of HSCs specifically discriminated by Tgfbr1 expression level and provided evidence of the capture of myeloid-biased (Tgfbr1hi) and myeloid-lymphoid-balanced (Tgfbr1lo) HSCs. In conclusion, our data provide a new paradigm for Tif1γ in regulating the balance between lymphoid- and myeloid-derived HSCs through TGF-β signaling, leading to HSC aging.