In vivo non-invasive monitoring of dystrophin correction in a new Duchenne muscular dystrophy reporter mouse.

Duchenne muscular dystrophy (DMD) is a fatal genetic disorder caused by mutations in the dystrophin gene. To enable the non-invasive analysis of DMD gene correction strategies in vivo, we introduced a luciferase reporter in-frame with the C-terminus of the dystrophin gene in mice. Expression of this reporter mimics endogenous dystrophin expression and DMD mutations that disrupt the dystrophin open reading frame extinguish luciferase expression. We evaluated the correction of the dystrophin reading frame coupled to luciferase in mice lacking exon 50, a common mutational hotspot, after delivery of CRISPR/Cas9 gene editing machinery with adeno-associated virus. Bioluminescence monitoring revealed efficient and rapid restoration of dystrophin protein expression in affected skeletal muscles and the heart. Our results provide a sensitive non-invasive means of monitoring dystrophin correction in mouse models of DMD and offer a platform for testing different strategies for amelioration of DMD pathogenesis.

NURR1 activation in skeletal muscle controls systemic energy homeostasis.

Skeletal muscle plays a central role in the control of metabolism and exercise tolerance. Analysis of muscle enhancers activated after exercise in mice revealed the orphan nuclear receptor NURR1/NR4A2 as a prominent component of exercise-responsive enhancers. We show that exercise enhances the expression of NURR1, and transgenic overexpression of NURR1 in skeletal muscle enhances physical performance in mice. NURR1 expression in skeletal muscle is also sufficient to prevent hyperglycemia and hepatic steatosis, by enhancing muscle glucose uptake and storage as glycogen. Furthermore, treatment of obese mice with putative NURR1 agonists increases energy expenditure, improves glucose tolerance, and confers a lean phenotype, mimicking the effects of exercise. These findings identify a key role for NURR1 in governance of skeletal muscle glucose metabolism, and reveal a transcriptional link between exercise and metabolism. Our findings also identify NURR1 agonists as possible exercise mimetics with the potential to ameliorate obesity and other metabolic abnormalities.

CRISPR-Cas9 corrects Duchenne muscular dystrophy exon 44 deletion mutations in mice and human cells.

Mutations in the dystrophin gene cause Duchenne muscular dystrophy (DMD), which is characterized by lethal degeneration of cardiac and skeletal muscles. Mutations that delete exon 44 of the dystrophin gene represent one of the most common causes of DMD and can be corrected in ~12% of patients by editing surrounding exons, which restores the dystrophin open reading frame. Here, we present a simple and efficient strategy for correction of exon 44 deletion mutations by CRISPR-Cas9 gene editing in cardiomyocytes obtained from patient-derived induced pluripotent stem cells and in a new mouse model harboring the same deletion mutation. Using AAV9 encoding Cas9 and single guide RNAs, we also demonstrate the importance of the dosages of these gene editing components for optimal gene correction in vivo. Our findings represent a significant step toward possible clinical application of gene editing for correction of DMD.

Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy.

Mutations in the gene encoding dystrophin, a protein that maintains muscle integrity and function, cause Duchenne muscular dystrophy (DMD). The deltaE50-MD dog model of DMD harbors a mutation corresponding to a mutational "hotspot" in the human DMD gene. We used adeno-associated viruses to deliver CRISPR gene editing components to four dogs and examined dystrophin protein expression 6 weeks after intramuscular delivery (n = 2) or 8 weeks after systemic delivery (n = 2). After systemic delivery in skeletal muscle, dystrophin was restored to levels ranging from 3 to 90% of normal, depending on muscle type. In cardiac muscle, dystrophin levels in the dog receiving the highest dose reached 92% of normal. The treated dogs also showed improved muscle histology. These large-animal data support the concept that, with further development, gene editing approaches may prove clinically useful for the treatment of DMD.

Control of Muscle Metabolism by the Mediator Complex.

Exercise represents an energetic challenge to whole-body homeostasis. In skeletal muscle, exercise activates a variety of signaling pathways that culminate in the nucleus to regulate genes involved in metabolism and contractility; however, much remains to be learned about the transcriptional effectors of exercise. Mediator is a multiprotein complex that links signal-dependent transcription factors and other transcriptional regulators with the basal transcriptional machinery, thereby serving as a transcriptional "hub." In this article, we discuss recent studies highlighting the role of Mediator subunits in metabolic regulation and glucose metabolism, as well as exercise responsiveness. Elucidation of the roles of Mediator subunits in metabolic control has revealed new mechanisms and molecular targets for the modulation of metabolism and metabolic disorders.

Single-cut genome editing restores dystrophin expression in a new mouse model of muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe, progressive muscle disease caused by mutations in the dystrophin gene. The majority of DMD mutations are deletions that prematurely terminate the dystrophin protein. Deletions of exon 50 of the dystrophin gene are among the most common single exon deletions causing DMD. Such mutations can be corrected by skipping exon 51, thereby restoring the dystrophin reading frame. Using clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9), we generated a DMD mouse model by deleting exon 50. These ΔEx50 mice displayed severe muscle dysfunction, which was corrected by systemic delivery of adeno-associated virus encoding CRISPR/Cas9 genome editing components. We optimized the method for dystrophin reading frame correction using a single guide RNA that created reframing mutations and allowed skipping of exon 51. In conjunction with muscle-specific expression of Cas9, this approach restored up to 90% of dystrophin protein expression throughout skeletal muscles and the heart of ΔEx50 mice. This method of permanently bypassing DMD mutations using a single cut in genomic DNA represents a step toward clinical correction of DMD mutations and potentially those of other neuromuscular disorders.

Genome Editing of Monogenic Neuromuscular Diseases: A Systematic Review.

IMPORTANCE: Muscle weakness, the most common symptom of neuromuscular disease, may result from muscle dysfunction or may be caused indirectly by neuronal and neuromuscular junction abnormalities. To date, more than 780 monogenic neuromuscular diseases, linked to 417 different genes, have been identified in humans. Genome-editing methods, especially the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) system, hold clinical potential for curing many monogenic disorders, including neuromuscular diseases such as Duchenne muscular dystrophy, spinal muscular atrophy, amyotrophic lateral sclerosis, and myotonic dystrophy type 1. OBJECTIVES: To provide an overview of genome-editing approaches; to summarize published reports on the feasibility, efficacy, and safety of current genome-editing methods as they relate to the potential correction of monogenic neuromuscular diseases; and to highlight scientific and clinical opportunities and obstacles toward permanent correction of disease-causing mutations responsible for monogenic neuromuscular diseases by genome editing. EVIDENCE REVIEW: PubMed and Google Scholar were searched for articles published from June 30, 1989, through June 9, 2016, using the following keywords: genome editing, CRISPR-Cas9, neuromuscular disease, Duchenne muscular dystrophy, spinal muscular atrophy, amyotrophic lateral sclerosis, and myotonic dystrophy type 1. The following sources were reviewed: 341 articles describing different approaches to edit mammalian genomes; 330 articles describing CRISPR-Cas9-mediated genome editing in cell culture lines (in vitro) and animal models (in vivo); 16 websites used to generate single-guide RNA; 4 websites for off-target effects; and 382 articles describing viral and nonviral delivery systems. Articles describing neuromuscular diseases, including Duchenne muscular dystrophy, spinal muscular atrophy, amyotrophic lateral sclerosis, and myotonic dystrophy type 1, were also reviewed. FINDINGS: Multiple proof-of-concept studies reveal the feasibility and efficacy of genome-editing-meditated correction of monogenic neuromuscular diseases in cultured cells and animal models. CONCLUSIONS AND RELEVANCE: Genome editing is a rapidly evolving technology with enormous translational potential once efficacy, delivery, and safety issues are addressed. The clinical impact of this technology is that genome editing can permanently correct disease-causing mutations and circumvent the hurdles of traditional gene- and cell-based therapies.

MED12 Regulates HSC-Specific Enhancers Independently of Mediator Kinase Activity to Control Hematopoiesis.

Hematopoietic-specific transcription factors require coactivators to communicate with the general transcription machinery and establish transcriptional programs that maintain hematopoietic stem cell (HSC) self-renewal, promote differentiation, and prevent malignant transformation. Mediator is a large coactivator complex that bridges enhancer-localized transcription factors with promoters, but little is known about Mediator function in adult stem cell self-renewal and differentiation. We show that MED12, a member of the Mediator kinase module, is an essential regulator of HSC homeostasis, as in vivo deletion of Med12 causes rapid bone marrow aplasia leading to acute lethality. Deleting other members of the Mediator kinase module does not affect HSC function, suggesting kinase-independent roles of MED12. MED12 deletion destabilizes P300 binding at lineage-specific enhancers, resulting in H3K27Ac depletion, enhancer de-activation, and consequent loss of HSC stemness signatures. As MED12 mutations have been described recently in blood malignancies, alterations in MED12-dependent enhancer regulation may control both physiological and malignant hematopoiesis.

A MED13-dependent skeletal muscle gene program controls systemic glucose homeostasis and hepatic metabolism.

The Mediator complex governs gene expression by linking upstream signaling pathways with the basal transcriptional machinery. However, how individual Mediator subunits may function in different tissues remains to be investigated. Through skeletal muscle-specific deletion of the Mediator subunit MED13 in mice, we discovered a gene regulatory mechanism by which skeletal muscle modulates the response of the liver to a high-fat diet. Skeletal muscle-specific deletion of MED13 in mice conferred resistance to hepatic steatosis by activating a metabolic gene program that enhances muscle glucose uptake and storage as glycogen. The consequent insulin-sensitizing effect within skeletal muscle lowered systemic glucose and insulin levels independently of weight gain and adiposity and prevented hepatic lipid accumulation. MED13 suppressed the expression of genes involved in glucose uptake and metabolism in skeletal muscle by inhibiting the nuclear receptor NURR1 and the MEF2 transcription factor. These findings reveal a fundamental molecular mechanism for the governance of glucose metabolism and the control of hepatic lipid accumulation by skeletal muscle. Intriguingly, MED13 exerts opposing metabolic actions in skeletal muscle and the heart, highlighting the customized, tissue-specific functions of the Mediator complex.

Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy.

CRISPR/Cas9-mediated genome editing holds clinical potential for treating genetic diseases, such as Duchenne muscular dystrophy (DMD), which is caused by mutations in the dystrophin gene. To correct DMD by skipping mutant dystrophin exons in postnatal muscle tissue in vivo, we used adeno-associated virus-9 (AAV9) to deliver gene-editing components to postnatal mdx mice, a model of DMD. Different modes of AAV9 delivery were systematically tested, including intraperitoneal at postnatal day 1 (P1), intramuscular at P12, and retro-orbital at P18. Each of these methods restored dystrophin protein expression in cardiac and skeletal muscle to varying degrees, and expression increased from 3 to 12 weeks after injection. Postnatal gene editing also enhanced skeletal muscle function, as measured by grip strength tests 4 weeks after injection. This method provides a potential means of correcting mutations responsible for DMD and other monogenic disorders after birth.

Loss of catalytically inactive lipid phosphatase myotubularin-related protein 12 impairs myotubularin stability and promotes centronuclear myopathy in zebrafish.

X-linked myotubular myopathy (XLMTM) is a congenital disorder caused by mutations of the myotubularin gene, MTM1. Myotubularin belongs to a large family of conserved lipid phosphatases that include both catalytically active and inactive myotubularin-related proteins (i.e., "MTMRs"). Biochemically, catalytically inactive MTMRs have been shown to form heteroligomers with active members within the myotubularin family through protein-protein interactions. However, the pathophysiological significance of catalytically inactive MTMRs remains unknown in muscle. By in vitro as well as in vivo studies, we have identified that catalytically inactive myotubularin-related protein 12 (MTMR12) binds to myotubularin in skeletal muscle. Knockdown of the mtmr12 gene in zebrafish resulted in skeletal muscle defects and impaired motor function. Analysis of mtmr12 morphant fish showed pathological changes with central nucleation, disorganized Triads, myofiber hypotrophy and whorled membrane structures similar to those seen in X-linked myotubular myopathy. Biochemical studies showed that deficiency of MTMR12 results in reduced levels of myotubularin protein in zebrafish and mammalian C2C12 cells. Loss of myotubularin also resulted in reduction of MTMR12 protein in C2C12 cells, mice and humans. Moreover, XLMTM mutations within the myotubularin interaction domain disrupted binding to MTMR12 in cell culture. Analysis of human XLMTM patient myotubes showed that mutations that disrupt the interaction between myotubularin and MTMR12 proteins result in reduction of both myotubularin and MTMR12. These studies strongly support the concept that interactions between myotubularin and MTMR12 are required for the stability of their functional protein complex in normal skeletal muscles. This work highlights an important physiological function of catalytically inactive phosphatases in the pathophysiology of myotubular myopathy and suggests a novel therapeutic approach through identification of drugs that could stabilize the myotubularin-MTMR12 complex and hence ameliorate this disorder.

Lack of myotubularin (MTM1) leads to muscle hypotrophy through unbalanced regulation of the autophagy and ubiquitin-proteasome pathways.

Mutations in the phosphoinositide phosphatase myotubularin (MTM1) results in X-linked myotubular/centronuclear myopathy (XLMTM), characterized by a severe decrease in muscle mass and strength in patients and murine models. However, the molecular mechanism involved in the muscle hypotrophy is unclear. Here we show that the IGF1R/Akt pathway is affected in Mtm1-deficient murine muscles, characterized by an increase in IGF1 receptor and Akt levels in both the presymptomatic and symptomatic phases. Moreover, up-regulation of atrogenes was observed in the presymptomatic phase of the myopathy, supporting overactivation of the ubiquitin-proteasome pathway. In parallel, the autophagy machinery was affected as indicated by the increase in the number of autophagosomes and of autophagy markers, such as LC3 and P62. However, phosphorylation of FOXO3a and mTOR were abnormal at late but not at early stages of the disease, suggesting that myotubularin acts both upstream in the IGF1R/Akt pathway and downstream on the balance between the autophagy and ubiquitin-proteasome pathways in vivo. Adeno-associated virus-mediated delivery of Mtm1 into Mtm1-null muscles rescued muscle mass and normalized the expression levels of IGF1 receptor, the ubiquitin-proteasome pathway, and autophagy markers. These data support the hypothesis that the unbalanced regulation of the ubiquitin proteasome pathway and the autophagy machinery is a primary cause of the XLMTM pathogenesis.

Myotubularin and PtdIns3P remodel the sarcoplasmic reticulum in muscle in vivo.

The sarcoplasmic reticulum (SR) is a specialized form of endoplasmic reticulum (ER) in skeletal muscle and is essential for calcium homeostasis. The mechanisms involved in SR remodeling and maintenance of SR subdomains are elusive. In this study, we identified myotubularin (MTM1), a phosphoinositide phosphatase mutated in X-linked centronuclear myopathy (XLCNM, or myotubular myopathy), as a key regulator of phosphatidylinositol 3-monophosphate (PtdIns3P) levels at the SR. MTM1 is predominantly located at the SR cisternae of the muscle triads, and Mtm1-deficient mouse muscles and myoblasts from XLCNM patients exhibit abnormal SR/ER networks. In vivo modulation of MTM1 enzymatic activity in skeletal muscle using ectopic expression of wild-type or a dead-phosphatase MTM1 protein leads to differential SR remodeling. Active MTM1 is associated with flat membrane stacks, whereas dead-phosphatase MTM1 mutant promotes highly curved cubic membranes originating from the SR and enriched in PtdIns3P. Overexpression of a tandem FYVE domain with high affinity for PtdIns3P alters the shape of the SR cisternae at the triad. Our findings, supported by the parallel analysis of the Mtm1-null mouse and an in vivo study, reveal a direct function of MTM1 enzymatic activity in SR remodeling and a key role for PtdIns3P in promoting SR membrane curvature in skeletal muscle. We propose that alteration in SR remodeling is a primary cause of X-linked centronuclear myopathy. The tight regulation of PtdIns3P on specific membrane subdomains may be a general mechanism to control membrane curvature.

Myotubularin phosphoinositide phosphatases in human diseases.

The level and turnover of phosphoinositides (PIs) are tightly controlled by a large set of PI-specific enzymes (PI kinases and phosphatases). Mammalian PI phosphatases are conserved through evolution and among this large family the dual-specificity phosphatase (PTP/DSP) are metal-independent enzymes displaying the amino acid signature Cys-X5-Arg-Thr/Ser (CX5RT/S) in their active site. Such catalytic site characterizes the myotubularin 3-phosphatases that dephosphorylate PtdIns3P and PtdIns(3,5)P2 and produce PtdIns5P. Substrates of myotubularins have been implicated in endocytosis and membrane trafficking while PtdIns5P may have a role in signal transduction. As a paradox, 6 of the 14 members of the myotubularin family lack enzymatic activity and are considered as dead phosphatases. Several myotubularins have been genetically linked to human diseases: MTM1 is mutated in the congenital myopathy X-linked centronuclear or myotubular myopathy (XLCNM) and MTMR14 (JUMPY) has been linked to an autosomal form of such disease, while MTMR2 and MTMR13 are mutated in Charcot-Marie-Tooth (CMT) neuropathies. Furthermore, recent evidences from genetic association studies revealed that several other myotubularins could be associated to chronic disorders such as cancer and obesity, highlighting their importance for human health. Here, we discuss cellular and physiological roles of myotubularins and their implication in human diseases, and we present potential pathological mechanisms affecting specific tissues in myotubularin-associated diseases.

Phosphatase-dead myotubularin ameliorates X-linked centronuclear myopathy phenotypes in mice.

Myotubularin MTM1 is a phosphoinositide (PPIn) 3-phosphatase mutated in X-linked centronuclear myopathy (XLCNM; myotubular myopathy). We investigated the involvement of MTM1 enzymatic activity on XLCNM phenotypes. Exogenous expression of human MTM1 in yeast resulted in vacuolar enlargement, as a consequence of its phosphatase activity. Expression of mutants from patients with different clinical progression and determination of PtdIns3P and PtdIns5P cellular levels confirmed the link between vacuolar morphology and MTM1 phosphatase activity, and showed that some disease mutants retain phosphatase activity. Viral gene transfer of phosphatase-dead myotubularin mutants (MTM1(C375S) and MTM1(S376N)) significantly improved most histological signs of XLCNM displayed by a Mtm1-null mouse, at similar levels as wild-type MTM1. Moreover, the MTM1(C375S) mutant improved muscle performance and restored the localization of nuclei, triad alignment, and the desmin intermediate filament network, while it did not normalize PtdIns3P levels, supporting phosphatase-independent roles of MTM1 in maintaining normal muscle performance and organelle positioning in skeletal muscle. Among the different XLCNM signs investigated, we identified only triad shape and fiber size distribution as being partially dependent on MTM1 phosphatase activity. In conclusion, this work uncovers MTM1 roles in the structural organization of muscle fibers that are independent of its enzymatic activity. This underlines that removal of enzymes should be used with care to conclude on the physiological importance of their activity.

Increased expression of wild-type or a centronuclear myopathy mutant of dynamin 2 in skeletal muscle of adult mice leads to structural defects and muscle weakness.

Dynamin 2 (DNM2) is a large GTPase implicated in many cellular functions, including cytoskeleton regulation and endocytosis. Although ubiquitously expressed, DNM2 was found mutated in two genetic disorders affecting different tissues: autosomal dominant centronuclear myopathy (ADCNM; skeletal muscle) and peripheral Charcot-Marie-Tooth neuropathy (peripheral nerve). To gain insight into the function of DNM2 in skeletal muscle and the pathological mechanisms leading to ADCNM, we introduced wild-type DNM2 (WT-DNM2) or R465W DNM2 (RW-DNM2), the most common ADCNM mutation, into adult wild-type mouse skeletal muscle by intramuscular adeno-associated virus injections. We detected altered localization of RW-DNM2 in mouse muscle. Several ADCNM features were present in RW-DNM2 mice: fiber atrophy, nuclear mislocalization, and altered mitochondrial staining, with a corresponding reduction in specific maximal muscle force. The sarcomere and triad structures were also altered. We report similar findings in muscle biopsy specimens from an ADCNM patient with the R465W mutation. In addition, expression of wild-type DNM2 induced some muscle defects, albeit to a lesser extent than RW-DNM2, suggesting that the R465W mutation has enhanced activity in vivo. In conclusion, we show the RW-DNM2 mutation acts in a dominant manner to cause ADCNM in adult muscle, and the disease arises from a primary defect in skeletal muscle rather than secondary to peripheral nerve involvement. Therefore, DNM2 plays important roles in the maintenance of adult muscle fibers.

Receptor activator of NF-kappaB (RANK) stimulates the proliferation of epithelial cells of the epidermo-pilosebaceous unit.

Receptor activator of NF-κB (RANK), known for controlling bone mass, has been recognized for its role in epithelial cell activation of the mammary gland. Because bone and the epidermo-pilosebaceous unit of the skin share a lifelong renewal activity where similar molecular players operate, and because mammary glands and hair follicles are both skin appendages, we have addressed the function of RANK in the hair follicle and the epidermis. Here, we show that mice deficient in RANK ligand (RANKL) are unable to initiate a new growth phase of the hair cycle and display arrested epidermal homeostasis. However, transgenic mice overexpressing RANK in the hair follicle or administration of recombinant RANKL both activate the hair cycle and epidermal growth. RANK is expressed by the hair follicle germ and bulge stem cells and the epidermal basal cells, cell types implicated in the renewal of the epidermo-pilosebaceous unit. RANK signaling is dispensable for the formation of the stem cell compartment and the inductive hair follicle mesenchyme, and the hair cycle can be rescued by Rankl knockout skin transplantation onto nude mice. RANKL is actively transcribed by the hair follicle at initiation of its growth phase, providing a mechanism for stem cell RANK engagement and hair-cycle entry. Thus, RANK-RANKL regulates hair renewal and epidermal homeostasis and provides a link between these two activities.

Myotubularin controls desmin intermediate filament architecture and mitochondrial dynamics in human and mouse skeletal muscle.

Muscle contraction relies on a highly organized intracellular network of membrane organelles and cytoskeleton proteins. Among the latter are the intermediate filaments (IFs), a large family of proteins mutated in more than 30 human diseases. For example, mutations in the DES gene, which encodes the IF desmin, lead to desmin-related myopathy and cardiomyopathy. Here, we demonstrate that myotubularin (MTM1), which is mutated in individuals with X-linked centronuclear myopathy (XLCNM; also known as myotubular myopathy), is a desmin-binding protein and provide evidence for direct regulation of desmin by MTM1 in vitro and in vivo. XLCNM-causing mutations in MTM1 disrupted the MTM1-desmin complex, resulting in abnormal IF assembly and architecture in muscle cells and both mouse and human skeletal muscles. Adeno-associated virus-mediated ectopic expression of WT MTM1 in Mtm1-KO muscle reestablished normal desmin expression and localization. In addition, decreased MTM1 expression and XLCNM-causing mutations induced abnormal mitochondrial positioning, shape, dynamics, and function. We therefore conclude that MTM1 is a major regulator of both the desmin cytoskeleton and mitochondria homeostasis, specifically in skeletal muscle. Defects in IF stabilization and mitochondrial dynamics appear as common physiopathological features of centronuclear myopathies and desmin-related myopathies.