RIPK3-mediated cell death is involved in DUX4-mediated toxicity in facioscapulohumeral dystrophy.

BACKGROUND: Facioscapulohumeral dystrophy (FSHD) is caused by mutations leading to the aberrant expression of the DUX4 transcription factor in muscles. DUX4 was proposed to induce cell death, but the involvement of different death pathways is still discussed. A possible pro-apoptotic role of DUX4 was proposed, but as FSHD muscles are characterized by necrosis and inflammatory infiltrates, non-apoptotic pathways may be also involved. METHODS: We explored DUX4-mediated cell death by focusing on the role of one regulated necrosis pathway called necroptosis, which is regulated by RIPK3. We investigated the effect of necroptosis on cell death in vitro and in vivo experiments using RIPK3 inhibitors and a RIPK3-deficient transgenic mouse model. RESULTS: We showed in vitro that DUX4 expression causes a caspase-independent and RIPK3-mediated cell death in both myoblasts and myotubes. In vivo, RIPK3-deficient animals present improved body and muscle weights, a reduction of the aberrant activation of the DUX4 network genes, and an improvement of muscle histology. CONCLUSIONS: These results provide evidence for a role of RIPK3 in DUX4-mediated cell death and open new avenues of research.

Irregular Astigmatism Management Using SPOT Scleral Lenses in the Treatment of Corneal Ectasia and Penetrating Keratoplasty.

PURPOSE: To evaluate the outcome of SPOT scleral lenses in the management of irregular astigmatism in patients with corneal ectasia and penetrating keratoplasty. Second, we analyzed patients' characteristics and tolerance, comfort, and geometries of fitted lenses. METHOD: Over a 5-year period, we included patients experiencing irregular astigmatism fitted with SPOT scleral contact lenses, from the University Hospital of Clermont-Ferrand, France. Data collected included corneal diseases, refractive error, best-corrected visual acuity (VA) with SPOT lenses, geometry of the lens, number of adjustment consultations, and the duration of follow-up. Comfort, quality of vision, less handling, and satisfaction were evaluated using visual analog scales after a 6-month follow-up period. RESULTS: Sixty-five patients were included, analyzing 107 eyes. Eighty percent of patients still daily wore lenses after a follow-up of 22.3±13.8 months. Visual acuity improved by 0.47±0.51 logarithm of the minimum angle of resolution (average increase of 5 lines) (P<0.001) after wearing scleral lenses. Comfort, quality of vision, less handling, and satisfaction of contact lenses were excellent (>75/100). Contact lenses were daily worn 10.0±4.1 hr/day. Most patients wore size M (17 mm) lenses (53.3% of patients), with an average sagittal height of 5.2±1.2 mm. Internal toricity was used in 30% of cases. Best geometry was found after 2.69±0.87 consultations. CONCLUSION: SPOT scleral contact lenses are an effective and well tolerated method to improve the VA of patients with irregular astigmatism.

A Deoxyribonucleic Acid Decoy Trapping DUX4 for the Treatment of Facioscapulohumeral Muscular Dystrophy.

Facioscapulohumeral dystrophy (FSHD) is characterized by a loss of repressive epigenetic marks leading to the aberrant expression of the DUX4 transcription factor. In muscle, DUX4 acts as a poison protein though the induction of multiple downstream genes. So far, there is no therapeutic solution for FSHD. Because DUX4 is a transcription factor, we developed an original therapeutic approach, based on a DNA decoy trapping the DUX4 protein, preventing its binding to genomic DNA and thereby blocking the aberrant activation of DUX4's transcriptional network. In vitro, transfection of a DUX4 decoy into FSHD myotubes reduced the expression of the DUX4 network genes. In vivo, both double-stand DNA DUX4 decoys and adeno-associated viruses (AAVs) carrying DUX4 binding sites reduced transcriptional activation of genes downstream of DUX4 in a DUX4-expressing mouse model. Our study demonstrates, both in vitro and in vivo, the feasibility of the decoy strategy and opens new avenues of research.

One-hour universal protocol for mouse genotyping.

BACKGROUND: Transgenic animals are widely used for research and for most of them, genotyping is unavoidable. Published protocols may be powerful but may also present disadvantages such as their cost or the requirement of additional steps/equipment. Moreover, if more than one strain must be genotyped, several protocols may need to be developed. METHODS: we adapted the existing amplification-resistant mutation protocol to develop the 1-h universal genotyping protocol (1-HUG), which allows the robust genotyping of genetically modified mice in 1 h from sample isolation to polymerase chain reaction gel running. RESULTS: This protocol allows the genotyping of different mouse models including mdx mouse, and FLExDUX4 and HSA-MerCreMer alone or in combination. It can be applied to different types of genomic modifications and to sexing. CONCLUSIONS: The 1-HUG protocol can be used routinely in any laboratory using mouse models for neuromuscular diseases.

Targeting the Polyadenylation Signal of Pre-mRNA: A New Gene Silencing Approach for Facioscapulohumeral Dystrophy.

Facioscapulohumeral dystrophy (FSHD) is characterized by the contraction of the D4Z4 array located in the sub-telomeric region of the chromosome 4, leading to the aberrant expression of the DUX4 transcription factor and the mis-regulation of hundreds of genes. Several therapeutic strategies have been proposed among which the possibility to target the polyadenylation signal to silence the causative gene of the disease. Indeed, defects in mRNA polyadenylation leads to an alteration of the transcription termination, a disruption of mRNA transport from the nucleus to the cytoplasm decreasing the mRNA stability and translation efficiency. This review discusses the polyadenylation mechanisms, why alternative polyadenylation impacts gene expression, and how targeting polyadenylation signal may be a potential therapeutic approach for FSHD.

Downregulation of myostatin pathway in neuromuscular diseases may explain challenges of anti-myostatin therapeutic approaches.

Muscular dystrophies are characterized by weakness and wasting of skeletal muscle tissues. Several drugs targeting the myostatin pathway have been used in clinical trials to increase muscle mass and function but most showed limited efficacy. Here we show that the expression of components of the myostatin signaling pathway is downregulated in muscle wasting or atrophying diseases, with a decrease of myostatin and activin receptor, and an increase of the myostatin antagonist, follistatin. We also provide in vivo evidence in the congenital myotubular myopathy mouse model (knock-out for the myotubularin coding gene Mtm1) that a down-regulated myostatin pathway can be reactivated by correcting the underlying gene defect. Our data may explain the poor clinical efficacy of anti-myostatin approaches in several of the clinical studies and the apparent contradictory results in mice regarding the efficacy of anti-myostatin approaches and may inform patient selection and stratification for future trials.

Retinoic Acid Engineered Amniotic Membrane Used as Graft or Homogenate: Positive Effects on Corneal Alkali Burns.

Purpose: Alkali burns are the most common, severe chemical ocular injuries, their functional prognosis depending on corneal wound healing efficiency. The purpose of our study was to compare the benefits of amniotic membrane (AM) grafts and homogenates for wound healing in the presence or absence of previous all-trans retinoic acid (atRA) treatment. Methods: Fifty male CD1 mice with reproducible corneal chemical burn were divided into five groups, as follows: group 1 was treated with saline solution; groups 2 and 3 received untreated AM grafts or grafts treated with atRA, respectively; and groups 4 and 5 received untreated AM homogenates or homogenates treated with atRA, respectively. After 7 days of treatment, ulcer area and depth were measured, and vascular endothelial growth factor (VEGF) and matrix metalloproteinase 9 (MMP-9) were quantified. Results: AM induction by atRA was confirmed via quantification of retinoic acid receptor ß (RARß), a well-established retinoic acid-induced gene. Significant improvements of corneal wound healing in terms of ulcer area and depth were obtained with both strategies. No major differences were found between the efficiency of AM homogenates and grafts. This positive action was increased when AM was pretreated with atRA. Furthermore, AM induced a decrease in VEGF and MMP-9 levels during the wound healing process. The atRA treatment led to an even greater decrease in the expression of both proteins. Conclusions: Amnion homogenate is as effective as AM grafts in promoting corneal wound healing in a mouse model. A higher positive effect was obtained with atRA treatment.

Gene therapy prolongs survival and restores function in murine and canine models of myotubular myopathy.

Loss-of-function mutations in the myotubularin gene (MTM1) cause X-linked myotubular myopathy (XLMTM), a fatal, congenital pediatric disease that affects the entire skeletal musculature. Systemic administration of a single dose of a recombinant serotype 8 adeno-associated virus (AAV8) vector expressing murine myotubularin to Mtm1-deficient knockout mice at the onset or at late stages of the disease resulted in robust improvement in motor activity and contractile force, corrected muscle pathology, and prolonged survival throughout a 6-month study. Similarly, single-dose intravascular delivery of a canine AAV8-MTM1 vector in XLMTM dogs markedly improved severe muscle weakness and respiratory impairment, and prolonged life span to more than 1 year in the absence of toxicity or a humoral or cell-mediated immune response. These results demonstrate the therapeutic efficacy of AAV-mediated gene therapy for myotubular myopathy in small- and large-animal models, and provide proof of concept for future clinical trials in XLMTM patients.

Site-specific Mtm1 mutagenesis by an AAV-Cre vector reveals that myotubularin is essential in adult muscle.

Manipulation of the mouse genome by site-specific mutagenesis has been extensively used to study gene function and model human disorders. Mouse models of myotubular myopathy (XLMTM), a severe congenital muscular disorder due to loss-of-function mutations in the MTM1 gene, have been generated by homologous recombination and shown that myotubularin is essential for skeletal muscle. However, since the Mtm1 deletion occurred constitutively or shortly after birth in these mice, it is not known whether myotubularin is required during adulthood, an important issue in the context of not only muscle biology but also therapies. To delete the Mtm1 gene in adult muscle fibers, we constructed a recombinant adeno-associated vector (AAV) that expresses the Cre recombinase under the muscle-specific desmin promoter. We report that a single injection of this vector into muscles of 3-month-old Mtm1 conditional mice leads to a myotubular myopathy phenotype with myofiber atrophy, disorganization of organelle positioning, such as mitochondria and nuclei, T-tubule defects and severe muscle weakness. In addition, our results show that MTM1-related atrophy and dysfunction correlate with abnormalities in satellite cell number and markers of autophagy, protein synthesis and neuromuscular junction transmission. The expression level of atrogenes was also analyzed. Therefore, we provide a valuable tissue model that recapitulates the main features of the disease, and it is useful to study pathogenesis and evaluate therapeutic strategies. We establish the proof-of-concept that myotubularin is required for the proper function of skeletal muscle during adulthood, suggesting that therapies will be required for the entire life of XLMTM patients.

Myotubularin-deficient myoblasts display increased apoptosis, delayed proliferation, and poor cell engraftment.

X-linked myotubular myopathy is a severe congenital myopathy caused by deficiency of the lipid phosphatase, myotubularin. Recent studies of human tissue and animal models have discovered structural and physiological abnormalities in myotubularin-deficient muscle, but the impact of myotubularin deficiency on myogenic stem cells within muscles is unclear. In the present study, we evaluated the viability, proliferative capacity, and in vivo engraftment of myogenic cells obtained from severely symptomatic (Mtm1δ4) myotubularin-deficient mice. Mtm1δ4 muscle contains fewer myogenic cells than wild-type (WT) littermates, and the number of myogenic cells decreases with age. The behavior of Mtm1δ4 myoblasts is also abnormal, because they engraft poorly into C57BL/6/Rag1null/mdx5cv mice and display decreased proliferation and increased apoptosis compared with WT myoblasts. Evaluation of Mtm1δ4 animals at 21 and 42 days of life detected fewer satellite cells in Mtm1δ4 muscle compared with WT littermates, and the decrease in satellite cells correlated with progression of disease. In addition, analysis of WT and Mtm1δ4 regeneration after injury detected similar abnormalities of satellite cell function, with fewer satellite cells, fewer dividing cells, and increased apoptotic cells in Mtm1δ4 muscle. These studies demonstrate specific abnormalities in myogenic cell number and behavior that may relate to the progression of disease in myotubularin deficiency, and may also be used to develop in vitro assays by which novel treatment strategies can be assessed.

Myotubular myopathy and the neuromuscular junction: a novel therapeutic approach from mouse models.

Myotubular myopathy (MTM) is a severe congenital muscle disease characterized by profound weakness, early respiratory failure and premature lethality. MTM is defined by muscle biopsy findings that include centralized nuclei and disorganization of perinuclear organelles. No treatments currently exist for MTM. We hypothesized that aberrant neuromuscular junction (NMJ) transmission is an important and potentially treatable aspect of the disease pathogenesis. We tested this hypothesis in two murine models of MTM. In both models we uncovered evidence of a disorder of NMJ transmission: fatigable weakness, improved strength with neostigmine, and electrodecrement with repetitive nerve stimulation. Histopathological analysis revealed abnormalities in the organization, appearance and size of individual NMJs, abnormalities that correlated with changes in acetylcholine receptor gene expression and subcellular localization. We additionally determined the ability of pyridostigmine, an acetylcholinesterase inhibitor, to ameliorate aspects of the behavioral phenotype related to NMJ dysfunction. Pyridostigmine treatment resulted in significant improvement in fatigable weakness and treadmill endurance. In all, these results describe a newly identified pathological abnormality in MTM, and uncover a potential disease-modifying therapy for this devastating disorder.

Regulation of the expression of the avian uncoupling protein 3 by isoproterenol and fatty acids in chick myoblasts: possible involvement of AMPK and PPARalpha?

The avian uncoupling protein 3 (UCP3), mainly expressed in muscle tissue, could be involved in fatty acid (FA) metabolism, limitation of reactive oxygen species production, and/or nonshivering thermogenesis. We recently demonstrated that UCP3 mRNA expression was increased by isoproterenol (Iso), a ß-agonist, in chicken Pectoralis major. This upregulation was associated with changes in FA metabolism and variations in the activation of AMP-activated protein kinase (AMPK) and in the expression of the transcription factors peroxisome proliferator-activated receptor (PPAR)α, PPARß/δ, and PPARγ coactivator-1α (PGC-1α). The aim of the present study was to elucidate the mechanisms involving AMPK and PPARα in UCP3 regulation in primary cultures of chick myoblasts. Avian UCP3 mRNA expression, associated with p38 mitogen-activated protein kinase (p38 MAPK) activation, was increased by Iso and/or FAs. The PKA pathway mediated the effects of Iso on UCP3 expression. FA stimulation also led to AMPK activation. Furthermore, the direct involvement of AMPK on UCP3 regulation was shown by using 5-aminoimidazole-4-carboxyamide ribonucleoside and Compound C. The use of the p38 MAPK inhibitor SB202190, which was associated with AMPK activation, also dramatically enhanced UCP3 mRNA expression. Finally the PPARα agonist WY-14643 strongly increased UCP3 mRNA expression. This study highlights the control of UCP3 expression by the ß-adrenergic system and FA in chick myoblasts and demonstrates that its expression is directly regulated by AMPK and by PPARα. Overexpression of avian UCP3 might modulate energy utilization or limit oxidative stress when mitochondrial metabolism of FA is triggered by catecholamines.