Mouse Model Of Dystrophy Research Articles

Generation of mouse model of TGFBI-R124C corneal dystrophy using CRISPR/Cas9-mediated homology-directed repair

Mutations in transforming growth factor-beta-induced (TGFBI) gene cause clinically distinct types of corneal dystrophies. To delineate the mechanisms driving these dystrophies, we focused on the R124C mutation in TGFBI that causes lattice corneal dystrophy type1 (LCD1) and generated novel transgenic mice harbouring a single amino acid substitution of arginine 124 with cysteine in TGFBI via ssODN-mediated base-pair substitution using CRISPR/Cas9 technology. Eighty percent of homozygous and 9.1% of heterozygous TGFBI-R124C mice developed a corneal opacity at 40 weeks of age. Hematoxylin and eosin and Masson trichrome staining showed eosinophilic deposits in subepithelial corneal stroma that stained negative for Congo-red. Although amyloid deposition was not observed in TGFBI-R124C mice, irregular amorphous deposits were clearly observed via transmission electron microscopy near the basement membrane. Interestingly, we found that the corneal deposition of TGFBI protein (TGFBIp) was significantly increased in homozygous TGFBI-R124C mice, suggesting a pathogenic role for the mutant protein accumulation. Furthermore, as observed in the LCD1 patients, corneal epithelial wound healing was significantly delayed in TGFBI-R124C mice. In conclusion, our novel mouse model of TGFBI-R124C corneal dystrophy reproduces features of the human disease. This mouse model will help delineate the pathogenic mechanisms of human corneal dystrophy.

Mouse models for muscular dystrophies: an overview.

ABSTRACTMuscular dystrophies (MDs) encompass a wide variety of inherited disorders that are characterized by loss of muscle tissue associated with a progressive reduction in muscle function. With a cure lacking for MDs, preclinical developments of therapeutic approaches depend on well-characterized animal models that recapitulate the specific pathology in patients. The mouse is the most widely and extensively used model for MDs, and it has played a key role in our understanding of the molecular mechanisms underlying MD pathogenesis. This has enabled the development of therapeutic strategies. Owing to advancements in genetic engineering, a wide variety of mouse models are available for the majority of MDs. Here, we summarize the characteristics of the most commonly used mouse models for a subset of highly studied MDs, collated into a table. Together with references to key publications describing these models, this brief but detailed overview would be useful for those interested in, or working with, mouse models of MD.

Splenic Ly6Chi monocytes are critical players in dystrophic muscle injury and repair.

Dystrophic muscle is characterized by chronic injury and a steady recruitment of inflammatory Ly6Chi monocytes. Recent studies have identified the spleen as the dominant reservoir of these cells during chronic inflammation. Here, we investigated the contribution of splenic Ly6Chi monocytes to dystrophic muscle pathology. Using the mdx mouse model of muscular dystrophy, we show that Ly6Chi monocytes accumulate in great numbers in the spleen over the course of the disease. The chemokine receptor CCR2 was upregulated on Ly6Chi monocytes in mdx spleen before disease onset, thereby enabling their recruitment to dystrophic muscle. Splenectomy performed before disease onset significantly reduced the number of Ly6Chi monocytes infiltrating dystrophic limb muscle. Moreover, in the absence of splenic Ly6Chi monocytes there was a significant reduction in dystrophic muscle inflammation and necrosis, along with improved regeneration during early disease. However, during late disease, a lack of splenic Ly6Chi monocytes adversely affected muscle fiber repair, due to a delay in the phenotypic shift of proinflammatory F4/80+Ly6ChiCD206lo to antiinflammatory F4/80+Ly6CloCD206+ macrophages. Overall, we show that the spleen is an indispensable source of Ly6Chi monocytes in muscular dystrophy and that splenic monocytes are critical players in both muscle fiber injury and repair.

Single SERCA2a Therapy Ameliorated Dilated Cardiomyopathy for 18 Months in a Mouse Model of Duchenne Muscular Dystrophy

Loss of dystrophin leads to Duchenne muscular dystrophy (DMD). A pathogenic feature of DMD is the significant elevation of cytosolic calcium. Supraphysiological calcium triggers protein degradation, membrane damage, and eventually muscle death and dysfunction. Sarcoplasmic/endoplasmic reticulum (SR) calcium ATPase (SERCA) is a calcium pump that transports cytosolic calcium to the SR during excitation-contraction coupling. We hypothesize that a single systemic delivery of SERCA2a with adeno-associated virus (AAV) may improve calcium recycling and provide long-lasting benefits in DMD. To test this, we injected an AAV9 human SERCA2a vector (6× 1012 viral genome particles/mouse) intravenously to 3-month-old mdx mice, the most commonly used DMD model. Immunostaining and western blot showed robust human SERCA2a expression in the heart and skeletal muscle for 18months. Concomitantly, SR calcium uptake was significantly improved in these tissues. SERCA2a therapy significantly enhanced grip force and treadmill performance, completely prevented myocardial fibrosis, and normalized electrocardiograms (ECGs). Cardiac catheterization showed normalization of multiple systolic and diastolic hemodynamic parameters in treated mice. Importantly, chamber dilation was completely prevented, and ejection fraction was restored to the wild-type level. Our results suggest that a single systemic AAV9 SERCA2a therapy has the potential to provide long-lasting benefits for DMD.

Investigation of the effect of taurine supplementation on muscle taurine content in the mdx mouse model of Duchenne muscular dystrophy using chemically specific synchrotron imaging.

Duchenne muscular dystrophy (DMD) is a lethal genetic muscle wasting disorder, which currently has no cure. Supplementation with the drug taurine has been shown to offer therapeutic benefit in the mdx model for DMD, however the mechanism by which taurine protects dystrophic muscle is not fully understood. Mdx muscle is deficient in taurine, however it is not known if this deficiency occurs in the extracellular space, in other cells present in the tissue (such as immune cells) or in the myofibre itself. Likewise, the tissue location of taurine enrichment in taurine treated mdx muscle is not known. In this study we applied X-ray absorption near edge spectroscopy (XANES) at the sulfur K-edge in an imaging format to determine taurine distribution in muscle tissue. XANES is the only technique currently capable of imaging taurine directly in muscle tissue, at a spatial resolution approaching myocyte cell size (20-50 μm). Using a multi-modal approach of XANES imaging and histology on the same tissue sections, we show that in mdx muscle, it is the myofibres that are deficient in taurine, and taurine supplementation ameliorates this deficiency. Increasing the taurine content of mdx myofibres was associated with a decrease in myofibre damage (as shown by the percentage of intact myofibres) and inflammation. These data will help drive future studies to better elucidate the molecular mechanisms through which taurine protects dystrophic muscle; they also support the continued investigation of taurine as a therapeutic intervention for DMD.

Inhibition of FLT1 ameliorates muscular dystrophy phenotype by increased vasculature in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disease in which the dystrophin coding for a membrane stabilizing protein is mutated. Recently, the vasculature has also shown to be perturbed in DMD and DMD model mdx mice. Recent DMD transcriptomics revealed the defects were correlated to a vascular endothelial growth factor (VEGF) signaling pathway. To reveal the relationship between DMD and VEGF signaling, mdx mice were crossed with constitutive (CAGCreERTM:Flt1LoxP/LoxP) and endothelial cell-specific conditional gene knockout mice (Cdh5CreERT2:Flt1LoxP/LoxP) for Flt1 (VEGFR1) which is a decoy receptor for VEGF. Here, we showed that while constitutive deletion of Flt1 is detrimental to the skeletal muscle function, endothelial cell-specific Flt1 deletion resulted in increased vascular density, increased satellite cell number and improvement in the DMD-associated phenotype in the mdx mice. These decreases in pathology, including improved muscle histology and function, were recapitulated in mdx mice given anti-FLT1 peptides or monoclonal antibodies, which blocked VEGF-FLT1 binding. The histological and functional improvement of dystrophic muscle by FLT1 blockade provides a novel pharmacological strategy for the potential treatment of DMD.

Linker Protein Repair of LAMA2 Dystrophic Neuromuscular Basement Membranes

An understanding of basement membrane (BM) assembly at a molecular level provides a foundation with which to develop repair strategies for diseases with defects of BM structure. As currently understood, laminins become anchored to cell surfaces through receptor-mediated interactions and polymerize. This provisional matrix binds to proteoglycans, nidogens and type IV collagen to form a mature BM. Identification of BM binding domains and their binding targets has enabled investigators to engineer proteins that link BM components to modify and improve their functions. This approach is illustrated by the development of two linker proteins to repair the LAMA2-deficient muscular dystrophy (LAMA2-MD). Dystrophy-causing mutations of the LAMA2 gene product (Lmα2) disrupt the BM molecular architecture, destabilizing it. In a mild ambulatory type of the dystrophy, α2LN mutations in laminin-211 prevents polymerization. In the more common and severe non-ambulatory type (MDC1A), an absent Lmα2 subunit is replaced by the naturally occurring Lmα4 subunit that is normally largely confined to the microvasculature. The compensatory laminin, however, is a poor substitute because it neither polymerizes nor binds adequately to the anchoring receptor α-dystroglycan. A chimeric laminin-binding protein called αLNNd enables laminins with defective or absent αLN domains to polymerize while another engineered protein, miniagrin (mag), promotes efficient α-dystroglycan receptor-binding in otherwise weakly adhesive laminins. Alone, αLNNd enables Lm211 with a self-assembly defect to polymerize and was used to ameliorate a mouse model of the ambulatory dystrophy. Together, these linker proteins alter Lm411 such that it both polymerizes and binds αDG such that it properly assembles. This combination was used to ameliorate a mouse model of the non-ambulatory dystrophy in which Lm411 replaced Lm211 as seen in the human disease. Collectively, these studies pave the way for the development of somatic gene delivery of repair proteins for treatment of LAMA2-MD. The studies further suggest a more general approach of linker-protein mediated repair in which a variety of existing BM protein domains can be combined together to stabilize BMs in other diseases.

First person – Trace Stay

ABSTRACTFirst Person is a series of interviews with the first authors of a selection of papers published in Disease Models & Mechanisms, helping early-career researchers promote themselves alongside their papers. Trace Stay is first author on ‘In vivo cerebellar circuit function is disrupted in an mdx mouse model of Duchenne muscular dystrophy’, published in DMM. Trace conducted the research described in this article while a PhD student in Roy V. Sillitoe's lab at the Baylor College of Medicine, Houston, TX, USA. He is now a postdoctoral scholar in the lab of Jennifer L. Raymond at Stanford University, Stanford, CA, USA, investigating how the cerebellum interacts with sensory input to influence motor control and more cognitive processing.

Multiple poloxamers increase plasma membrane repair capacity in muscle and nonmuscle cells.

Various previous studies established that the amphiphilic tri-block copolymer known as poloxamer 188 (P188) or Pluronic-F68 can stabilize the plasma membrane following a variety of injuries to multiple mammalian cell types. This characteristic led to proposals for the use of P188 as a therapeutic treatment for various disease states, including muscular dystrophy. Previous studies suggest that P188 increases plasma membrane integrity by resealing plasma membrane disruptions through its affinity for the hydrophobic lipid chains on the lipid bilayer. P188 is one of a large family of copolymers that share the same basic tri-block structure consisting of a middle hydrophobic propylene oxide segment flanked by two hydrophilic ethylene oxide moieties [poly(ethylene oxide)80-poly(propylene oxide)27-poly(ethylene oxide)80]. Despite the similarities of P188 to the other poloxamers in this chemical family, there has been little investigation into the membrane-resealing properties of these other poloxamers. In this study we assessed the resealing properties of poloxamers P181, P124, P182, P234, P108, P407, and P338 on human embryonic kidney 293 (HEK293) cells and isolated muscle from the mdx mouse model of Duchenne muscular dystrophy. Cell membrane injuries from glass bead wounding and multiphoton laser injury show that the majority of poloxamers in our panel improved the plasma membrane resealing of both HEK293 cells and dystrophic muscle fibers. These findings indicate that many tri-block copolymers share characteristics that can increase plasma membrane resealing and that identification of these shared characteristics could help guide design of future therapeutic approaches.

Proteomic and cell biological profiling of the renal phenotype of the mdx-4cv mouse model of Duchenne muscular dystrophy

The X-linked inherited muscle wasting disease Duchenne muscular dystrophy, which is caused by primary abnormalities in the membrane cytoskeletal protein dystrophin, is a multi-system disorder. Highly progressive forms of dystrophinopathy are associated with a complex secondary pathophysiology, including renal dysfunction. It was therefore of interest to carry out a systematic survey of potential proteome-wide changes in the kidney of the established mdx-4cv mouse model of dystrophinopathy. Of 5878 mass spectrometrically identified kidney proteins, 82 versus 142 proteins were shown to be decreased or increased, respectively, in association with muscular dystrophy. The most decreased versus increased protein species are the ACSM3 isoform of mitochondrial acyl-coenzyme A synthetase and the FABP1 isoform of fatty acid binding protein, respectively. Both proteomic findings were verified by immunofluorescence microscopy and immunoblot analysis. Interestingly, haematoxylin/eosin staining indicated diffuse whitish deposits in the mdx-4cv kidney, and an increased intensity of Sudan Black labelling of kidney cells revealed ectopic fat deposition. Although the proteomic results and cell biological findings do not demonstrate a direct functional link between increased FABP1 and fat accumulation, the results suggest that the up-regulation of FABP1 may be related to abnormal fat metabolism. This makes FABP1 potentially a novel pathobiochemical indicator for studying kidney abnormalities in the mdx-4cv model of dystrophinopathy.

Alpha-Dystroglycan Supports Platelet Aggregation and Thrombus Formation

No relevant conflicts of interest to declare.

Shape Therapeutics raises funds to edit RNA

Seattle-based Shape Therapeutics has raised $35.5 million in series A financing to develop RNA-editing therapies. Unlike DNA, RNA is made continually in the cell, so some scientists view RNA editing as potentially safer than gene editing. Shape was cofounded by University of California San Diego scientist Prashant Mali. His lab has designed synthetic RNA molecules that direct enzymes called ADAR to make precise, single-letter changes to RNA in human cells. Mali’s lab has already tested the technique in a mouse model of muscular dystrophy.

Isolation and Characterization of Muscle-Derived Stem Cells from Dystrophic Mouse Models.

The study of the population of muscle satellite cells (SC) is important to understand muscle regeneration and its involvement in the different dystrophic processes. We studied two dystrophic mouse models, Largemyd and Lama2dy2j/J, that show an intense and very similar pattern of muscle degeneration, but with differences in the expression of genes involved in the regeneration cascade. They are, therefore, interesting models to study possible differences in the mechanism of activation and action of satellite cells in the dystrophic muscle. The main objectives of this chapter are to describe the isolation and characterization of SC populations, evaluating the presence of myogenic and pluripotent stem cells markers in normal and dystrophic muscles.

Macrophages fine tune satellite cell fate in dystrophic skeletal muscle of mdx mice.

Satellite cells (SCs) are muscle stem cells that remain quiescent during homeostasis and are activated in response to acute muscle damage or in chronic degenerative conditions such as Duchenne Muscular Dystrophy. The activity of SCs is supported by specialized cells which either reside in the muscle or are recruited in regenerating skeletal muscles, such as for instance macrophages (MΦs). By using a dystrophic mouse model of transient MΦ depletion, we describe a shift in identity of muscle stem cells dependent on the crosstalk between MΦs and SCs. Indeed MΦ depletion determines adipogenic conversion of SCs and exhaustion of the SC pool leading to an exacerbated dystrophic phenotype. The reported data could also provide new insights into therapeutic approaches targeting inflammation in dystrophic muscles.

Plasmid-Mediated Gene Therapy in Mouse Models of Limb Girdle Muscular Dystrophy

We delivered plasmid DNA encoding therapeutic genes to the muscles of mouse models of limb girdle muscular dystrophy (LGMD) 2A, 2B, and 2D, deficient in calpain3, dysferlin, and alpha-sarcoglycan, respectively. We also delivered the human follistatin gene, which has the potential to increase therapeutic benefit. After intramuscular injection of DNA, electroporation was applied to enhance delivery to muscle fibers. When plasmids encoding the human calpain3 or dysferlin cDNA sequences were injected into quadriceps muscles of LGMD2A and LGMD2B mouse models, respectively, in 3-month studies, robust levels of calpain3 and dysferlin proteins were detected. We observed a statistically significant decrease in Evans blue dye penetration in LGMD2B mouse muscles after delivery of the dysferlin gene, consistent with repair of the muscle membrane defect in these mice. The therapeutic value of delivery of the genes for alpha-sarcoglycan and follistatin was documented by significant drops in Evans blue dye penetration in gastrocnemius muscles of LGMD2D mice. These results indicated for the first time that a combined gene therapy involving both alpha-sarcoglycan and follistatin would be valuable for LGMD2D patients. We suggest that this non-viral gene delivery method should be explored for its translational potential in patients.

Early skeletal muscle pathology and disease progress in the dy3K/dy3K mouse model of congenital muscular dystrophy with laminin \u03b12 chain-deficiency

Deficiency of laminin α2 chain leads to a severe form of congenital muscular dystrophy (LAMA2-CMD), and dystrophic symptoms progress rapidly in early childhood. Currently, there is no treatment for this detrimental disorder. Development of therapies is largely hindered by lack of understanding of mechanisms involved in the disease initiation and progress, both in patients but also in mouse models that are commonly used in the preclinical setup. Here, we unveil the first pathogenic events and characterise the disease development in a mouse model for LAMA2-CMD (dy3K/dy3K), by analysing muscles at perinatal, neonatal and postnatal stages. We found that apoptotic muscle fibres were present as early as postnatal day 1. Other typical dystrophic hallmarks (muscle degeneration, inflammation, and extensive production of the extracellular matrix proteins) were clearly evident already at postnatal day 4, and the highest degree of muscle deterioration was reached by day 7. Interestingly, the severe phenotype of limb muscles partially recovered on days 14 and 21, despite worsening of the general condition of the dy3K/dy3K mouse by that age. We found that masticatory muscles were severely affected in dy3K/dy3K mice and this may be an underlying cause of their malnutrition, which contributes to death around day 21. We also showed that several signalling pathways were affected already in 1-day-old dy3K/dy3K muscle. Therapeutic tests in the dy3K/dy3K mouse model should therefore be initiated shortly after birth, but should also take into account timing and correlation between regenerative and pathogenic events.

Better living through peptide-conjugated chemistry: next-generation antisense oligonucleotides.

Two different antisense oligonucleotide-based (ASO-based) therapies are currently in clinical use to treat neuromuscular diseases. This success, for Duchenne muscular dystrophy and spinal muscular atrophy, offers hope not only for additional neuromuscular diseases, but also for other disorders that could benefit from RNA-targeted therapies. A major limitation for more widespread application of ASOs relates to relatively poor tissue penetration. In this issue of the JCI, Klein et al. showed that conjugating an ASO with an arginine-rich cell-penetrating peptide, Pip6a, enhanced delivery, resulting in corrective outcome for a mouse model of myotonic dystrophy. Linking ASOs to cell-penetrating peptides, or even other moieties, is an approach currently under development with treatment potential to expand to other disorders.

P.129Latent myostatin specific elimination by sweeping antibody® is a novel therapeutic approach to improve muscle strength

Myostatin, or GDF8, is predominantly secreted from the skeletal muscles as a latent form, which is an inactive form. After it is cleaved by BMP1/tolloid proteases, the then active C-terminal mature form binds to receptor ActRIIB, inducing muscle atrophy related signaling. Monoclonal antibodies that target myostatin have been shown to increase muscle mass and strength in many mouse models of muscle atrophy, but results in some clinical trials did not indicate sufficient improvement of muscle strength and physical function. We generated an antibody that binds specifically to the latent form of myostatin, blocking its activation. We engineered it with a recently developed antibody engineering technology–the sweeping technology–which results in the very effective elimination of the specific antigen from circulation by forcing the internalization of antibody-antigen complex into cells. The effect of the latent myostatin-sweeping antibodies in increasing muscle strength and mass was tested in a muscle atrophy and muscular dystrophy mouse models. Our results showed that, by eliminating the myostatin from both skeletal muscle and from plasma, this novel and potent antibody greatly increased muscle strength and mass at about 10-fold lower doses compared to other mature myostatin antibodies or non-sweeping latent myostatin antibodies. We selected the final candidate for clinical development among them, and called it GYM329/RG6237. Our data suggest that the elimination of latent myostatin with sweeping technology could lead to a more efficient increase of muscle mass and strength in patients with degenerative muscle disorders.

P.311BIO101 demonstrates combined beneficial effects on skeletal muscle and respiratory functions in a mouse model of Duchenne muscular dystrophy

P.311BIO101 demonstrates combined beneficial effects on skeletal muscle and respiratory functions in a mouse model of Duchenne muscular dystrophy

P.321Diaphragm echodensity and function in mdx mice by non-invasive ultrasonography: validation and correlation with in vivo and ex vivo functional readouts

In the mdx mouse model of Duchenne muscular dystrophy (DMD), diaphragm (DIA) alterations occur at early age, with a progression which closely resembles patients' condition. High frequency ultrasound has been recently proposed as a longitudinal, non-invasive technique to monitor the progression of mdx DIA dysfunction. At present, data available are still limited, particularly in mdx mice at early disease stages. Moreover, no evidence has ever been provided about the usefulness of ultrasonography to measure echodensity, as an index of contractile tissue loss in mdx DIA. In light of this, and considering the importance assumed by an independent validation of new experimental procedures in preclinical studies on DMD, our study was aimed to perform an ultrasonography evaluation of DIA amplitude and echodensity, in 3- and 6-month-old mdx mice (the age window typically used for pharmacological studies), compared to wild type (wt). In parallel, in vivo (forelimb force, fatigability) and ex vivo (DIA contractile recordings, TGF-β1 levels) dystrophy-relevant outcomes were assessed. Ultrasonography showed a significant decrease of DIA amplitude in 3- and 6-month-old mdx mice compared to wt, which correlated with the isometric force reduction observed ex vivo. An age-dependent increase of mdx DIA echodensity was disclosed; this paralleled the high levels of muscular pro-fibrotic TGF-β1. DIA from 6-month-old mdx mice also showed a consistent force drop after eccentric contraction and a change in stiffness compared to wt, likely in relation to an increased fibrosis. Notably, no alteration of left ventricular function was found in mdx hearts, supporting the view that respiratory complications occur independently from cardiac ones. Our findings corroborate the usefulness of DIA ultrasonography as a tool to evaluate mdx mouse pathology progression since early stages, and this possibility opens important new perspectives to improve translational research on DMD (Supported by PRIN-MIUR n. 20108YB5W3_004).