β-Dystroglycan Restoration and Pathology Progression in the Dystrophic mdx Mouse: Outcome and Implication of a Clinically Oriented Study with a Novel Oral Dasatinib Formulation

ROS-activated cSrc tyrosine kinase (TK) promotes the degradation of β-dystroglycan (β-DG), a dystrophin-glycoprotein complex component, which may reinforce damaging signals in Duchenne muscular dystrophy (DMD). Therefore, cSrc-TK represents a promising therapeutic target. In mdx mice, a 4-week subcutaneous treatment with dasatinib (DAS), a pan-Src-TKs inhibitor approved as anti-leukemic agent, increased muscle β-DG, with minimal amelioration of morphofunctional indices. To address possible dose/pharmacokinetic (PK) issues, a new oral DAS/hydroxypropyl(HP)-β-cyclodextrin(CD) complex was developed and chronically administered to mdx mice. The aim was to better assess the role of β-DG in pathology progression, meanwhile confirming DAS mechanism of action over the long-term, along with its efficacy and tolerability. The 4-week old mdx mice underwent a 12-week treatment with DAS/HP-β-CD10% dissolved in drinking water, at 10 or 20 mg/kg/day. The outcome was evaluated via in vivo/ex vivo disease-relevant readouts. Oral DAS/HP-β-CD efficiently distributed in mdx mice plasma and tissues in a dose-related fashion. The new DAS formulation confirmed its main upstream mechanism of action, by reducing β-DG phosphorylation and restoring its levels dose-dependently in both diaphragm and gastrocnemius muscle. However, it modestly improved in vivo neuromuscular function, ex vivo muscle force, and histopathology, although the partial recovery of muscle elasticity and the decrease of CK and LDH plasma levels suggest an increased sarcolemmal stability of dystrophic muscles. Our clinically oriented study supports the interest in this new, pediatric-suitable DAS formulation for proper exposure and safety and for enhancing β-DG expression. This latter mechanism is, however, not sufficient by itself to impact on pathology progression. In-depth analyses will be dedicated to elucidating the mechanism limiting DAS effectiveness in dystrophic settings, meanwhile assessing its potential synergy with dystrophin-based molecular therapies.

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Several dystrophin-glycoprotein complex members are present in crude surface membranes but they are sodium dodecyl sulphate invisible in KCl-washed microsomes from mdx mouse muscle

Cellular & Molecular Biology Letters ◽

10.2478/s11658-009-0039-8 ◽

2010 ◽

Vol 15 (1) ◽

Cited By ~ 2

Author(s):

Stéphanie Daval ◽

Chantal Rocher ◽

Yan Cherel ◽

Elisabeth Rumeur

Keyword(s):

Muscular Dystrophy ◽

Core Protein ◽

Surface Membrane ◽

Sodium Dodecyl ◽

Mdx Mouse ◽

Mdx Mice ◽

Mouse Muscle ◽

Glycoprotein Complex ◽

Dystrophin Glycoprotein Complex ◽

Dystrophin Glycoprotein

AbstractThe dystrophin-glycoprotein complex (DGC) is a large trans-sarcolemmal complex that provides a linkage between the subsarcolemmal cytoskeleton and the extracellular matrix. In skeletal muscle, it consists of the dystroglycan, sarcoglycan and cytoplasmic complexes, with dystrophin forming the core protein. The DGC has been described as being absent or greatly reduced in dystrophin-deficient muscles, and this lack is considered to be involved in the dystrophic phenotype. Such a decrease in the DGC content was observed in dystrophin-deficient muscle from humans with muscular dystrophy and in mice with X-linked muscular dystrophy (mdx mice). These deficits were observed in total muscle homogenates and in partially membrane-purified muscle fractions, the so-called KCl-washed microsomes. Here, we report that most of the proteins of the DGC are actually present at normal levels in the mdx mouse muscle plasma membrane. The proteins are detected in dystrophic animal muscles when the immunoblot assay is performed with crude surface membrane fractions instead of the usually employed KCl-washed microsomes. We propose that these proteins form SDS-insoluble membrane complexes when dystrophin is absent.

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Investigating the Potential for Sulforaphane to Attenuate Gastrointestinal Dysfunction in mdx Dystrophic Mice

Nutrients ◽

10.3390/nu13124559 ◽

2021 ◽

Vol 13 (12) ◽

pp. 4559

Author(s):

Kristy Swiderski ◽

Suzannah J. Read ◽

Audrey S. Chan ◽

Jin D. Chung ◽

Jennifer Trieu ◽

...

Keyword(s):

Muscular Dystrophy ◽

Corn Oil ◽

Therapeutic Potential ◽

Ex Vivo ◽

In Vivo Studies ◽

Muscle Pathology ◽

Mdx Mouse ◽

Mdx Mice ◽

Dystrophic Mice

Gastrointestinal (GI) dysfunction is an important, yet understudied condition associated with Duchenne muscular dystrophy (DMD), with patients reporting bloating, diarrhea, and general discomfort, contributing to a reduced quality of life. In the mdx mouse, the most commonly used mouse model of DMD, studies have confirmed GI dysfunction (reported as altered contractility and GI transit through the small and large intestine), associated with increased local and systemic inflammation. Sulforaphane (SFN) is a natural isothiocyanate with anti-inflammatory and anti-oxidative properties via its activation of Nrf2 signalling that has been shown to improve aspects of the skeletal muscle pathology in dystrophic mice. Whether SFN can similarly improve GI function in muscular dystrophy was unknown. Video imaging and spatiotemporal mapping to assess gastrointestinal contractions in isolated colon preparations from mdx and C57BL/10 mice revealed that SFN reduced contraction frequency when administered ex vivo, demonstrating its therapeutic potential to improve GI function in DMD. To confirm this in vivo, four-week-old male C57BL/10 and mdx mice received vehicle (2% DMSO/corn oil) or SFN (2 mg/kg in 2% DMSO/corn oil) via daily oral gavage five days/week for 4 weeks. SFN administration reduced fibrosis in the diaphragm of mdx mice but did not affect other pathological markers. Gene and protein analysis revealed no change in Nrf2 protein expression or activation of Nrf2 signalling after SFN administration and oral SFN supplementation did not improve GI function in mdx mice. Although ex vivo studies demonstrate SFN’s therapeutic potential for reducing colon contractions, in vivo studies should investigate higher doses and/or alternate routes of administration to confirm SFN’s potential to improve GI function in DMD.

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Effect of ethanol and cocaine on [11C]MPC-6827 uptake in SH-SY5Y cells

Molecular Biology Reports ◽

10.1007/s11033-021-06336-7 ◽

2021 ◽

Author(s):

Naresh Damuka ◽

Miranda Orr ◽

Paul W. Czoty ◽

Jeffrey L. Weiner ◽

Thomas J. Martin ◽

...

Keyword(s):

Mechanism Of Action ◽

Ex Vivo ◽

Neuroblastoma Cells ◽

Proper Function ◽

Brain Functions ◽

Biodistribution Studies ◽

Positron Emission ◽

Vitro Binding

AbstractMicrotubules (MTs) are structural units in the cytoskeleton. In brain cells they are responsible for axonal transport, information processing, and signaling mechanisms. Proper function of these processes is critical for healthy brain functions. Alcohol and substance use disorders (AUD/SUDs) affects the function and organization of MTs in the brain, making them a potential neuroimaging marker to study the resulting impairment of overall neurobehavioral and cognitive processes. Our lab reported the first brain-penetrant MT-tracking Positron Emission Tomography (PET) ligand [11C]MPC-6827 and demonstrated its in vivo utility in rodents and non-human primates. To further explore the in vivo imaging potential of [11C]MPC-6827, we need to investigate its mechanism of action. Here, we report preliminary in vitro binding results in SH-SY5Y neuroblastoma cells exposed to ethanol (EtOH) or cocaine in combination with multiple agents that alter MT stability. EtOH and cocaine treatments increased MT stability and decreased free tubulin monomers. Our initial cell-binding assay demonstrated that [11C]MPC-6827 may have high affinity to free/unbound tubulin units. Consistent with this mechanism of action, we observed lower [11C]MPC-6827 uptake in SH-SY5Y cells after EtOH and cocaine treatments (e.g., fewer free tubulin units). We are currently performing in vivo PET imaging and ex vivo biodistribution studies in rodent and nonhuman primate models of AUD and SUDs and Alzheimer's disease.

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Inspiratory loading does not accelerate dystrophy inmdx mouse diaphragm: implications for regenerative therapy

Journal of Applied Physiology ◽

10.1152/japplphysiol.00689.2002 ◽

2003 ◽

Vol 94 (2) ◽

pp. 411-419 ◽

Cited By ~ 6

Author(s):

Alexander S. Krupnick ◽

Jianliang Zhu ◽

Taitan Nguyen ◽

Daniel Kreisel ◽

Keki R. Balsara ◽

...

Keyword(s):

Therapeutic Strategy ◽

Mdx Mouse ◽

Mdx Mice ◽

Blue Dye ◽

Regenerative Therapy ◽

Regenerative Capacity ◽

Progressive Degeneration ◽

Mouse Diaphragm ◽

The Relationship

Since the finding that the mdx mouse diaphragm, in contrast to limb muscles, undergoes progressive degeneration analogous to that seen in Duchenne muscular dystrophy, the relationship between the workload on a muscle and the pathogenesis of dystrophy has remained controversial. We increased the work performed by the mdx mouse diaphragm in vivo by tracheal banding and evaluated the progression of dystrophic changes in that muscle. Despite the establishment of dramatically increased respiratory workload and accelerated myofiber damage documented by Evans blue dye, no change in the pace of progression of dystrophy was seen in banded animals vs. unbanded, sham-operated controls. At the completion of the study, more centrally nucleated fibers were evident in the diaphragms of banded mdx mice than in sham-operated mdx controls, indicating that myofiber regeneration increases to meet the demands of the work-induced damage. These data suggest that there is untapped regenerative capacity in dystrophin-deficient muscle and validates experimental efforts aimed at augmenting regeneration within skeletal muscle as a therapeutic strategy in the treatment of dystrophinopathies.

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Talin, vinculin and DRP (utrophin) concentrations are increased at mdx myotendinous junctions following onset of necrosis

Journal of Cell Science ◽

10.1242/jcs.107.6.1477 ◽

1994 ◽

Vol 107 (6) ◽

pp. 1477-1483 ◽

Cited By ~ 1

Author(s):

D.J. Law ◽

D.L. Allen ◽

J.G. Tidball

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Cytoskeletal Proteins ◽

Mdx Mouse ◽

Mdx Mice ◽

Myotendinous Junction ◽

Glycoprotein Complex ◽

Transmembrane Glycoprotein ◽

Muscle Necrosis ◽

Myotendinous Junctions

Duchenne muscular dystrophy (DMD) and the myopathy seen in the mdx mouse both result from absence of the protein dystrophin. Structural similarities between dystrophin and other cytoskeletal proteins, its enrichment at myotendinous junctions, and its indirect association with laminin mediated by a transmembrane glycoprotein complex suggest that one of dystrophin's functions in normal muscle is to form one of the links between the actin cytoskeleton and the extracellular matrix. Unlike Duchenne muscular dystrophy patients, mdx mice suffer only transient muscle necrosis, and are able to regenerate damaged muscle tissue. The present study tests the hypothesis that mdx mice partially compensate for dystrophin's absence by upregulating one or more dystrophin-independent mechanisms of cytoskeleton-membrane association. Quantitative analysis of immunoblots of adult mdx muscle samples showed an increase of approximately 200% for vinculin and talin, cytoskeletal proteins that mediate thin filament-membrane interactions at myotendinous junctions. Blots also showed an increase (143%) in the dystrophin-related protein called utrophin, another myotendinous junction constituent, which may be able to substitute for dystrophin directly. Muscle samples from 2-week-old animals, a period immediately preceding the onset of muscle necrosis, showed no significant differences in protein concentration between mdx and controls. Quantitative analyses of confocal images of myotendinous junctions from mdx and control muscles show significantly higher concentrations of talin and vinculin at the myotendinous junctions of mdx muscle. These findings indicate that mdx mice may compensate in part for the absence of dystrophin by increased expression of other molecules that subsume dystrophin's mechanical function.

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

Neuromuscular Disorders ◽

10.1016/j.nmd.2019.06.435 ◽

2019 ◽

Vol 29 ◽

pp. S160-S161

Author(s):

P. Mantuano ◽

A. Mele ◽

O. Cappellari ◽

A. Fonzino ◽

F. Sanarica ◽

...

Keyword(s):

Ex Vivo ◽

Mdx Mice ◽

Non Invasive ◽

And Function

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Systemic administration of IGF-I enhances oxidative status and reduces contraction-induced injury in skeletal muscles of mdx dystrophic mice

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00101.2006 ◽

2006 ◽

Vol 291 (3) ◽

pp. E499-E505 ◽

Cited By ~ 44

Author(s):

Jonathan D. Schertzer ◽

James G. Ryall ◽

Gordon S. Lynch

Keyword(s):

Tibialis Anterior ◽

Skeletal Muscles ◽

Force Production ◽

Mdx Mice ◽

Cross Sectional ◽

Myosin Heavy Chain Isoform ◽

Glycoprotein Complex ◽

Igf I ◽

Dystrophin Glycoprotein Complex ◽

Endocrine Factor

The absence of dystrophin and resultant disruption of the dystrophin glycoprotein complex renders skeletal muscles of dystrophic patients and dystrophic mdx mice susceptible to contraction-induced injury. Strategies to reduce contraction-induced injury are of critical importance, because this mode of damage contributes to the etiology of myofiber breakdown in the dystrophic pathology. Transgenic overexpression of insulin-like growth factor-I (IGF-I) causes myofiber hypertrophy, increases force production, and can improve the dystrophic pathology in mdx mice. In contrast, the predominant effect of continuous exogenous administration of IGF-I to mdx mice at a low dose (1.0–1.5 mg·kg−1·day−1) is a shift in muscle phenotype from fast glycolytic toward a more oxidative, fatigue-resistant, slow muscle without alterations in myofiber cross-sectional area, muscle mass, or maximum force-producing capacity. We found that exogenous administration of IGF-I to mdx mice increased myofiber succinate dehydrogenase activity, shifted the overall myosin heavy chain isoform composition toward a slower phenotype, and, most importantly, reduced contraction-induced damage in tibialis anterior muscles. The deficit in force-producing capacity after two damaging lengthening contractions was reduced significantly in tibialis anterior muscles of IGF-I-treated (53 ± 4%) compared with untreated mdx mice (70 ± 5%, P < 0.05). The results provide further evidence that IGF-I administration can enhance the functional properties of dystrophic skeletal muscle and, compared with results in transgenic mice or virus-mediated overexpression, highlight the disparities in different models of endocrine factor delivery.

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382. In Utero Intramuscular Delivery of a High-Capacity Adenoviral Vector Carrying a Full-Length Murine Dystrophin cDNA Provides Functional Benefit and Reassembly of the Dystrophin-Glycoprotein Complex in Hind Limb Muscles of mdx Mice

Molecular Therapy ◽

10.1016/j.ymthe.2004.06.919 ◽

2004 ◽

Vol 9 ◽

pp. S146

Keyword(s):

Adenoviral Vector ◽

Hind Limb ◽

High Capacity ◽

In Utero ◽

Mdx Mice ◽

Glycoprotein Complex ◽

Functional Benefit ◽

Dystrophin Glycoprotein Complex ◽

Dystrophin Glycoprotein ◽

Intramuscular Delivery

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TAT-μUtrophin mitigates the pathophysiology of dystrophin and utrophin double-knockout mice

Journal of Applied Physiology ◽

10.1152/japplphysiol.00248.2011 ◽

2011 ◽

Vol 111 (1) ◽

pp. 200-205 ◽

Cited By ~ 15

Author(s):

Jarrod A. Call ◽

James M. Ervasti ◽

Dawn A. Lowe

Keyword(s):

Knockout Mice ◽

Skeletal Muscles ◽

Ex Vivo ◽

Eccentric Contraction ◽

Mdx Mice ◽

Double Knockout ◽

Replacement Treatment ◽

Generating Capacity

Previously, we demonstrated functional substitution of dystrophin by TAT-μUtrophin (TAT-μUtr) in dystrophin-deficient mdx mice. Herein, we addressed whether TAT-μUtr could improve the phenotype of dystrophin and utrophin double-knockout ( mdx:utr−/−) mice. Specifically, we quantitatively compared survival and quality of life assessments in mdx:utr−/− mice receiving TAT-μUtr protein administration against placebo-treated mdx:utr−/− mice (PBS). Additionally, skeletal muscles from TAT-μUtr and PBS mice were tested in vivo and ex vivo for strength and susceptibility to eccentric contraction-induced injury. We found the TAT-μUtr treatment extended life span 45% compared with mice administered PBS. This was attributed to significantly increased food consumption (3.1 vs. 1.8 g/24 h) due to improved ability to search for food as daily cage activities were greater in TAT-μUtr mice (e.g., 364 vs. 201 m ambulation/24 h). The extensor digitorum longus muscles of TAT-μUtr-treated double-knockout mice also displayed increased force-generating capacity ex vivo (8.3 vs. 6.4 N/cm2) and decreased susceptibility to injury ex vivo and in vivo. These data indicate that the functional benefits of TAT-μUtr replacement treatment extend to the mdx:utr−/− double-knockout mouse and support its development as a therapy to mitigate muscle weakness in patients with Duchenne muscular dystrophy.

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