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1.
Hum Mol Genet ; 28(3): 396-406, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30281092

RESUMO

Duchenne muscular dystrophy (DMD) is caused by loss of dystrophin protein, leading to progressive muscle weakness and premature death due to respiratory and/or cardiac complications. Cardiac involvement is characterized by progressive dilated cardiomyopathy, decreased fractional shortening and metabolic dysfunction involving reduced metabolism of fatty acids-the major cardiac metabolic substrate. Several mouse models have been developed to study molecular and pathological consequences of dystrophin deficiency, but do not recapitulate all aspects of human disease pathology and exhibit a mild cardiac phenotype. Here we demonstrate that Cmah (cytidine monophosphate-sialic acid hydroxylase)-deficient mdx mice (Cmah-/-;mdx) have an accelerated cardiac phenotype compared to the established mdx model. Cmah-/-;mdx mice display earlier functional deterioration, specifically a reduction in right ventricle (RV) ejection fraction and stroke volume (SV) at 12 weeks of age and decreased left ventricle diastolic volume with subsequent reduced SV compared to mdx mice by 24 weeks. They further show earlier elevation of cardiac damage markers for fibrosis (Ctgf), oxidative damage (Nox4) and haemodynamic load (Nppa). Cardiac metabolic substrate requirement was assessed using hyperpolarized magnetic resonance spectroscopy indicating increased in vivo glycolytic flux in Cmah-/-;mdx mice. Early upregulation of mitochondrial genes (Ucp3 and Cpt1) and downregulation of key glycolytic genes (Pdk1, Pdk4, Ppara), also denote disturbed cardiac metabolism and shift towards glucose utilization in Cmah-/-;mdx mice. Moreover, we show long-term treatment with peptide-conjugated exon skipping antisense oligonucleotides (20-week regimen), resulted in 20% cardiac dystrophin protein restoration and significantly improved RV cardiac function. Therefore, Cmah-/-;mdx mice represent an appropriate model for evaluating cardiac benefit of novel DMD therapeutics.


Assuntos
Monofosfato de Citidina/genética , Distrofina/deficiência , Morfolinos/uso terapêutico , Animais , Cardiomiopatia Dilatada/genética , Carnitina O-Palmitoiltransferase/genética , Fator de Crescimento do Tecido Conjuntivo/análise , Monofosfato de Citidina/fisiologia , Modelos Animais de Doenças , Distrofina/genética , Distrofina/metabolismo , Éxons , Terapia Genética/métodos , Coração/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos mdx , Oxigenases de Função Mista/metabolismo , Distrofia Muscular de Duchenne/genética , Miocárdio/metabolismo , NADPH Oxidase 4/análise , Oligonucleotídeos Antissenso/genética , Peptídeos/genética , Fenótipo , Volume Sistólico , Proteína Desacopladora 3/genética , Função Ventricular Direita
2.
J Muscle Res Cell Motil ; 40(2): 141-150, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31289969

RESUMO

Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle wasting disease. The disease is due to mutations in the DMD gene that encodes for a large intracellular protein called dystrophin. Dystrophin plays a critical role in linking the internal cytoskeleton of the striated muscle cell with the extracellular matrix as well as having cell signalling functions. In its absence muscle contraction is associated with cycles of damage, repair, inflammation and fibrosis with eventual loss of muscle and replacement with fat. Experiments in animal models of DMD have generated a number of different approaches to the induction of dystrophin including viral vector mediated delivery of a recombinant dystrophin gene, antisense oligonucleotide mediated exon-skipping to restore the open reading frame in the dystrophin mRNA, read-through of premature stop mutations, genome modification using CRISPR-Cas9 or cell based transfer of a functional dystrophin gene. In all cases, it will be important to understand how much dystrophin expression is required for a clinically effective therapy and this review examines the data from humans and animal models to estimate the percentage of endogenous dystrophin that is likely to have significant clinical benefit. While there are a number of important caveats to consider, including the appropriate outcome measures, this review suggests that approximately 20% of endogenous levels uniformly distributed within the skeletal muscles and the heart may be sufficient to largely prevent disease progression.


Assuntos
Distrofina , Regulação da Expressão Gênica/genética , Terapia Genética , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne , Miocárdio/metabolismo , Animais , Modelos Animais de Doenças , Distrofina/biossíntese , Distrofina/genética , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/terapia
3.
Hum Mol Genet ; 24(15): 4225-37, 2015 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-25935000

RESUMO

Splice modulation therapy has shown great clinical promise in Duchenne muscular dystrophy, resulting in the production of dystrophin protein. Despite this, the relationship between restoring dystrophin to established dystrophic muscle and its ability to induce clinically relevant changes in muscle function is poorly understood. In order to robustly evaluate functional improvement, we used in situ protocols in the mdx mouse to measure muscle strength and resistance to eccentric contraction-induced damage. Here, we modelled the treatment of muscle with pre-existing dystrophic pathology using antisense oligonucleotides conjugated to a cell-penetrating peptide. We reveal that 15% homogeneous dystrophin expression is sufficient to protect against eccentric contraction-induced injury. In addition, we demonstrate a >40% increase in specific isometric force following repeated administrations. Strikingly, we show that changes in muscle strength are proportional to dystrophin expression levels. These data define the dystrophin restoration levels required to slow down or prevent disease progression and improve overall muscle function once a dystrophic environment has been established in the mdx mouse model.


Assuntos
Peptídeos Penetradores de Células/genética , Distrofina/biossíntese , Distrofia Muscular Animal/genética , Distrofia Muscular de Duchenne/genética , Oligonucleotídeos Antissenso/genética , Animais , Peptídeos Penetradores de Células/administração & dosagem , Modelos Animais de Doenças , Distrofina/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Terapia Genética , Humanos , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Animal/patologia , Distrofia Muscular Animal/terapia , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/terapia , Oligonucleotídeos Antissenso/administração & dosagem
4.
Hum Mol Genet ; 23(7): 1842-55, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24234655

RESUMO

Mutations in fukutin-related protein (FKRP) underlie a group of muscular dystrophies associated with the hypoglycosylation of α-dystroglycan (α-DG), a proportion of which show central nervous system involvement. Our original FKRP knock-down mouse (FKRP(KD)) replicated many of the characteristics seen in patients at the severe end of the dystroglycanopathy spectrum but died perinatally precluding its full phenotyping and use in testing potential therapies. We have now overcome this by crossing FKRP(KD) mice with those expressing Cre recombinase under the Sox1 promoter. Owing to our original targeting strategy, this has resulted in the restoration of Fkrp levels in the central nervous system but not the muscle, thereby generating a new model (FKRP(MD)) which develops a progressive muscular dystrophy resembling what is observed in limb girdle muscular dystrophy. Like-acetylglucosaminyltransferase (LARGE) is a bifunctional glycosyltransferase previously shown to hyperglycosylate α-DG. To investigate the therapeutic potential of LARGE up-regulation, we have now crossed the FKRP(MD) line with one overexpressing LARGE and show that, contrary to expectation, this results in a worsening of the muscle pathology implying that any future strategies based upon LARGE up-regulation require careful management.


Assuntos
Distroglicanas/metabolismo , N-Acetilglucosaminiltransferases/biossíntese , N-Acetilglucosaminiltransferases/genética , Proteínas/genética , Síndrome de Walker-Warburg/genética , Animais , Membrana Basal/metabolismo , Membrana Basal/patologia , Sistema Nervoso Central/metabolismo , Modelos Animais de Doenças , Glicosilação , Laminina/biossíntese , Camundongos , Camundongos Knockout , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Mutação , Pentosiltransferases , Transferases , Regulação para Cima , Síndrome de Walker-Warburg/mortalidade
5.
J Anat ; 229(4): 514-35, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27173448

RESUMO

Musculoskeletal modelling has become a valuable tool with which to understand how neural, muscular, skeletal and other tissues are integrated to produce movement. Most musculoskeletal modelling work has to date focused on humans or their close relatives, with few examples of quadrupedal animal limb models. A musculoskeletal model of the mouse hindlimb could have broad utility for questions in medicine, genetics, locomotion and neuroscience. This is due to this species' position as a premier model of human disease, having an array of genetic tools for manipulation of the animal in vivo, and being a small quadruped, a category for which few models exist. Here, the methods used to develop the first three-dimensional (3D) model of a mouse hindlimb and pelvis are described. The model, which represents bones, joints and 39 musculotendon units, was created through a combination of previously gathered muscle architecture data from microdissections, contrast-enhanced micro-computed tomography (CT) scanning and digital segmentation. The model allowed muscle moment arms as well as muscle forces to be estimated for each musculotendon unit throughout a range of joint rotations. Moment arm analysis supported the reliability of musculotendon unit placement within the model, and comparison to a previously published rat hindlimb model further supported the model's reliability. A sensitivity analysis performed on both the force-generating parameters and muscle's attachment points of the model indicated that the maximal isometric muscle moment is generally most sensitive to changes in either tendon slack length or the coordinates of insertion, although the degree to which the moment is affected depends on several factors. This model represents the first step in the creation of a fully dynamic 3D computer model of the mouse hindlimb and pelvis that has application to neuromuscular disease, comparative biomechanics and the neuromechanical basis of movement. Capturing the morphology and dynamics of the limb, it enables future dissection of the complex interactions between the nervous and musculoskeletal systems as well as the environment.


Assuntos
Osso e Ossos/anatomia & histologia , Osso e Ossos/fisiologia , Simulação por Computador , Modelos Animais , Músculo Esquelético/anatomia & histologia , Músculo Esquelético/fisiologia , Animais , Fenômenos Biomecânicos , Feminino , Membro Posterior , Locomoção/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Microtomografia por Raio-X
6.
Hum Mol Genet ; 21(20): 4508-20, 2012 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-22810924

RESUMO

Loss of dystrophin protein due to mutations in the DMD gene causes Duchenne muscular dystrophy. Dystrophin loss also leads to the loss of the dystrophin glycoprotein complex (DGC) from the sarcolemma which contributes to the dystrophic phenotype. Tyrosine phosphorylation of dystroglycan has been identified as a possible signal to promote the proteasomal degradation of the DGC. In order to test the role of tyrosine phosphorylation of dystroglycan in the aetiology of DMD, we generated a knock-in mouse with a phenylalanine substitution at a key tyrosine phosphorylation site in dystroglycan, Y890. Dystroglycan knock-in mice (Dag1(Y890F/Y890F)) had no overt phenotype. In order to examine the consequence of blocking dystroglycan phosphorylation on the aetiology of dystrophin-deficient muscular dystrophy, the Y890F mice were crossed with mdx mice an established model of muscular dystrophy. Dag1(Y890F/Y890F)/mdx mice showed a significant improvement in several parameters of muscle pathophysiology associated with muscular dystrophy, including a reduction in centrally nucleated fibres, less Evans blue dye infiltration and lower serum creatine kinase levels. With the exception of dystrophin, other DGC components were restored to the sarcolemma including α-sarcoglycan, α-/ß-dystroglycan and sarcospan. Furthermore, Dag1(Y890F/Y890F)/mdx showed a significant resistance to muscle damage and force loss following repeated eccentric contractions when compared with mdx mice. While the Y890F substitution may prevent dystroglycan from proteasomal degradation, an increase in sarcolemmal plectin appeared to confer protection on Dag1(Y890F/Y890F)/mdx mouse muscle. This new model confirms dystroglycan phosphorylation as an important pathway in the aetiology of DMD and provides novel targets for therapeutic intervention.


Assuntos
Distroglicanas/metabolismo , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Fenótipo , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos mdx , Distrofia Muscular Animal/fisiopatologia , Fosforilação
7.
Ann N Y Acad Sci ; 1534(1): 130-144, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38517756

RESUMO

Myogenesis is essential for skeletal muscle formation, growth, and regeneration and can be altered in Duchenne muscular dystrophy (DMD), an X-linked disorder due to the absence of the cytoskeletal protein dystrophin. Ion channels play a pivotal role in muscle differentiation and interact with the dystrophin complex. To investigate ion channel involvement in myogenesis in dystrophic settings, we performed electrophysiological characterization of two immortalized mouse cell lines, wild-type (WT) H2K-2B4 and the dystrophic (DYS) H2K-SF1, and measured gene expression of differentiation markers and ion channels. Inward and outward currents/density increased as differentiation progressed in both WT and DYS cells. However, day-11 DYS cells showed higher (27%) inward current density with an increased expression ratio of Scn5a/Scn4a and decreased (48%) barium-sensitive outward current compared to WT. Furthermore, day-11 DYS cells showed more positive resting membrane potential (+10 mV) and lower membrane capacitance (50%) compared to WT. DYS cells also had reduced Myog and Myf5 expression at days 6 and 11. Overall, ion channel profile and myogenesis appeared altered in DYS cells. These results are a first step in validating ion channels as potential drug targets to ameliorate muscle degeneration in DMD settings and as differentiation biomarkers in innovative platforms.


Assuntos
Distrofia Muscular de Duchenne , Animais , Camundongos , Distrofia Muscular de Duchenne/metabolismo , Distrofina/metabolismo , Músculo Esquelético/metabolismo , Biomarcadores/metabolismo , Canais Iônicos/metabolismo , Desenvolvimento Muscular
9.
Dis Model Mech ; 16(12)2023 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-38050706

RESUMO

Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin (DMD) gene, is associated with fatal muscle degeneration and atrophy. Patients with DMD have progressive reductions in skeletal muscle strength and resistance to eccentric muscle stretch. Using the DE50-MD dog model of DMD, we assessed tibiotarsal joint (TTJ) flexor and extensor force dynamics, and the resistance of dystrophic muscle to eccentric stretch. Male DE50-MD and wild-type (WT) dogs were analysed every 3 months until 18 months of age. There was an age-associated decline in eccentric contraction resistance in DE50-MD TTJ flexors that discriminated, with high statistical power, WT from DE50-MD individuals. For isometric contraction, at the majority of timepoints, DE50-MD dogs had lower maximum absolute and relative TTJ flexor force, reduced TTJ muscle contraction times and prolonged relaxation compared to those in WT dogs. Cranial tibial muscles, the primary TTJ flexor, of 18-month-old DE50-MD dogs had significant numbers of regenerating fibres as expected, but also fewer type I fibres and more hybrid fibres than those in WT dogs. We conclude that these parameters, in particular, the eccentric contraction decrement, could be used as objective outcome measures for pre-clinical assessment in DE50-MD dogs.


Assuntos
Distrofia Muscular de Duchenne , Humanos , Cães , Masculino , Animais , Lactente , Distrofia Muscular de Duchenne/genética , Músculo Esquelético , Distrofina/genética , Contração Muscular/fisiologia , Força Muscular/fisiologia , Mutação
10.
Lancet ; 378(9791): 595-605, 2011 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-21784508

RESUMO

BACKGROUND: We report clinical safety and biochemical efficacy from a dose-ranging study of intravenously administered AVI-4658 phosphorodiamidate morpholino oligomer (PMO) in patients with Duchenne muscular dystrophy. METHOD: We undertook an open-label, phase 2, dose-escalation study (0·5, 1·0, 2·0, 4·0, 10·0, and 20·0 mg/kg bodyweight) in ambulant patients with Duchenne muscular dystrophy aged 5-15 years with amenable deletions in DMD. Participants had a muscle biopsy before starting treatment and after 12 weekly intravenous infusions of AVI-4658. The primary study objective was to assess safety and tolerability of AVI-4658. The secondary objectives were pharmacokinetic properties and the ability of AVI-4658 to induce exon 51 skipping and dystrophin restoration by RT-PCR, immunohistochemistry, and immunoblotting. The study is registered, number NCT00844597. FINDINGS: 19 patients took part in the study. AVI-4658 was well tolerated with no drug-related serious adverse events. AVI-4658 induced exon 51 skipping in all cohorts and new dystrophin protein expression in a significant dose-dependent (p=0·0203), but variable, manner in boys from cohort 3 (dose 2 mg/kg) onwards. Seven patients responded to treatment, in whom mean dystrophin fluorescence intensity increased from 8·9% (95% CI 7·1-10·6) to 16·4% (10·8-22·0) of normal control after treatment (p=0·0287). The three patients with the greatest responses to treatment had 21%, 15%, and 55% dystrophin-positive fibres after treatment and these findings were confirmed with western blot, which showed an increase after treatment of protein levels from 2% to 18%, from 0·9% to 17%, and from 0% to 7·7% of normal muscle, respectively. The dystrophin-associated proteins α-sarcoglycan and neuronal nitric oxide synthase were also restored at the sarcolemma. Analysis of the inflammatory infiltrate indicated a reduction of cytotoxic T cells in the post-treatment muscle biopsies in the two high-dose cohorts. INTERPRETATION: The safety and biochemical efficacy that we present show the potential of AVI-4658 to become a disease-modifying drug for Duchenne muscular dystrophy. FUNDING: UK Medical Research Council; AVI BioPharma.


Assuntos
Distrofina/metabolismo , Éxons/genética , Morfolinas/administração & dosagem , Distrofia Muscular de Duchenne/tratamento farmacológico , Oligonucleotídeos/administração & dosagem , Adolescente , Processamento Alternativo , Criança , Relação Dose-Resposta a Droga , Distrofina/genética , Humanos , Infusões Intravenosas , Masculino , Morfolinas/farmacocinética , Morfolinos , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Oligonucleotídeos/farmacocinética
12.
Mol Ther ; 19(1): 165-71, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20924363

RESUMO

Antisense-induced exon skipping can restore the open reading frame, and thus correct the dystrophin deficiency that causes Duchenne muscular dystrophy (DMD), a lethal muscle wasting condition. Successful proof-of-principle in preclinical models has led to human clinical trials. However, it is still not known what percentage of dystrophin-positive fibers and what level of expression is necessary for functional improvement. This study directly address these key questions in the mdx mouse model of DMD. To achieve a significant variation in dystrophin expression, we locally administered into tibialis anterior muscles various doses of a phosphorodiamidate morpholino oligomer (PMO) designed to skip the mutated exon 23 from the mRNA of murine dystrophin. We found a highly significant correlation between the number of dystrophin-positive fibers and resistance to contraction-induced injury, with a minimum of 20% of dystrophin-positive fibers required for meaningful improvement. Furthermore, our results also indicate that a relatively low level of dystrophin expression in muscle fibers may have significant clinical benefits. In contrast, improvements in muscle force were not correlated with either the number of positive fibers or total dystrophin levels, which highlight the need to conduct appropriate functional assessments in preclinical testing using the mdx mouse.


Assuntos
Distrofina/biossíntese , Morfolinas/farmacologia , Força Muscular/fisiologia , Distrofia Muscular de Duchenne/fisiopatologia , Distrofia Muscular de Duchenne/terapia , Oligonucleotídeos Antissenso/farmacologia , Animais , Distrofina/genética , Distrofina/metabolismo , Éxons , Feminino , Terapia Genética/métodos , Camundongos , Camundongos Endogâmicos mdx , Modelos Animais , Morfolinas/metabolismo , Morfolinos , Contração Muscular , Fibras Musculares Esqueléticas/metabolismo , Força Muscular/efeitos dos fármacos , Força Muscular/genética , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Fases de Leitura Aberta , RNA Mensageiro/biossíntese , RNA Mensageiro/genética
13.
Mol Ther ; 19(2): 345-54, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21102560

RESUMO

The administration of antisense oligonucleotides (AOs) to skip one or more exons in mutated forms of the DMD gene and so restore the reading frame of the transcript is one of the most promising approaches to treat Duchenne muscular dystrophy (DMD). At present, preclinical studies demonstrating the efficacy and safety of long-term AO administration have not been conducted. Furthermore, it is essential to determine the minimal effective dose and frequency of administration. In this study, two different low doses (LDs) of phosphorodiamidate morpholino oligomer (PMO) designed to skip the mutated exon 23 in the mdx dystrophic mouse were administered for up to 12 months. Mice treated for 50 weeks showed a substantial dose-related amelioration of the pathology, particularly in the diaphragm. Moreover, the generalized physical activity was profoundly enhanced compared to untreated mdx mice showing that widespread, albeit partial, dystrophin expression restores the normal activity in mdx mice. Our results show for the first time that a chronic long-term administration of LDs of unmodified PMO, equivalent to doses in use in DMD boys, is safe, significantly ameliorates the muscular dystrophic phenotype and improves the activity of dystrophin-deficient mice, thus encouraging the further clinical translation of this approach in humans.


Assuntos
Morfolinas/uso terapêutico , Distrofia Muscular de Duchenne/tratamento farmacológico , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Western Blotting , Creatina Quinase/sangue , Creatinina/sangue , Eletrofisiologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos mdx , Morfolinos , Distrofia Muscular de Duchenne/sangue , gama-Glutamiltransferase
14.
Dis Model Mech ; 15(3)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35019137

RESUMO

Duchenne muscular dystrophy (DMD), a fatal musculoskeletal disease, is associated with neurodevelopmental disorders and cognitive impairment caused by brain dystrophin deficiency. Dog models of DMD represent key translational tools to study dystrophin biology and to develop novel therapeutics. However, characterisation of dystrophin expression and function in the canine brain is lacking. We studied the DE50-MD canine model of DMD that has a missense mutation in the donor splice site of exon 50. Using a battery of cognitive tests, we detected a neurocognitive phenotype in DE50-MD dogs, including reduced attention, problem solving and exploration of novel objects. Through a combination of capillary immunoelectrophoresis, immunolabelling, quantitative PCR and RNAScope in situ hybridisation, we show that regional dystrophin expression in the adult canine brain reflects that of humans, and that the DE50-MD dog lacks full-length dystrophin (Dp427) protein expression but retains expression of the two shorter brain-expressed isoforms, Dp140 and Dp71. Thus, the DE50-MD dog is a translationally relevant pre-clinical model to study the consequences of Dp427 deficiency in the brain and to develop therapeutic strategies for the neurological sequelae of DMD.


Assuntos
Distrofina , Distrofia Muscular de Duchenne , Animais , Encéfalo/metabolismo , Cães , Distrofina/genética , Distrofina/metabolismo , Éxons/genética , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Fenótipo
15.
Dis Model Mech ; 15(12)2022 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-36444978

RESUMO

Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disease, caused by mutations in the dystrophin gene, characterised by cycles of muscle degeneration, inflammation and regeneration. Recently, there has been renewed interest specifically in drugs that ameliorate muscle inflammation in DMD patients. The DE50-MD dog is a model of DMD that closely mimics the human DMD phenotype. We quantified inflammatory proteins in serum from wild-type (WT) and DE50-MD dogs aged 3-18 months to identify biomarkers for future pre-clinical trials. Significantly higher concentrations of C-C motif chemokine ligand 2 (CCL2), granulocyte-macrophage colony-stimulating factor (GM-CSF or CSF2), keratinocyte chemotactic-like (KC-like, homologous to mouse CXCL1), TNFα (or TNF), and interleukins IL2, IL6, IL7, IL8 (CXCL8), IL10, IL15 and IL18 were detected in DE50-MD serum compared to WT serum. Of these, CCL2 best differentiated the two genotypes. The relative level of CCL2 mRNA was greater in the vastus lateralis muscle of DE50-MD dogs than in that of WT dogs, and CCL2 was expressed both within and at the periphery of damaged myofibres. Serum CCL2 concentration was significantly associated with acid phosphatase staining in vastus lateralis biopsy samples in DE50-MD dogs. In conclusion, the serum cytokine profile suggests that inflammation is a feature of the DE50-MD phenotype. Quantification of serum CCL2 in particular is a useful non-invasive biomarker of the DE50-MD phenotype.


Assuntos
Citocinas , Distrofia Muscular de Duchenne , Humanos , Cães , Camundongos , Animais , Atrofia Muscular
16.
Wellcome Open Res ; 7: 238, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36865375

RESUMO

Background: Animal models of Duchenne muscular dystrophy (DMD) are essential to study disease progression and assess efficacy of therapeutic intervention, however dystrophic mice fail to display a clinically relevant phenotype, limiting translational utility. Dystrophin-deficient dogs exhibit disease similar to humans, making them increasingly important for late-stage preclinical evaluation of candidate therapeutics. The DE50-MD canine model of DMD carries a mutation within a human 'hotspot' region of the dystrophin gene, amenable to exon-skipping and gene editing strategies. As part of a large natural history study of disease progression, we have characterised the DE50-MD skeletal muscle phenotype to identify parameters that could serve as efficacy biomarkers in future preclinical trials. Methods: Vastus lateralis muscles were biopsied from a large cohort of DE50-MD dogs and healthy male littermates at 3-monthly intervals (3-18 months) for longitudinal analysis, with multiple muscles collected post-mortem to evaluate body-wide changes. Pathology was characterised quantitatively using histology and measurement of gene expression to determine statistical power and sample sizes appropriate for future work. Results: DE50-MD skeletal muscle exhibits widespread degeneration/regeneration, fibrosis, atrophy and inflammation. Degenerative/inflammatory changes peak during the first year of life, while fibrotic remodelling appears more gradual. Pathology is similar in most skeletal muscles, but in the diaphragm, fibrosis is more prominent, associated with fibre splitting and pathological hypertrophy. Picrosirius red and acid phosphatase staining represent useful quantitative histological biomarkers for fibrosis and inflammation respectively, while qPCR can be used to measure regeneration ( MYH3, MYH8), fibrosis ( COL1A1), inflammation ( SPP1), and stability of DE50-MD dp427 transcripts. Conclusion: The DE50-MD dog is a valuable model of DMD, with pathological features similar to young, ambulant human patients. Sample size and power calculations show that our panel of muscle biomarkers are of strong pre-clinical value, able to detect therapeutic improvements of even 25%, using trials with only six animals per group.

17.
Curr Opin Neurol ; 24(5): 437-42, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21825985

RESUMO

PURPOSE OF REVIEW: Dystroglycanopathies are a common group of diseases characterized by a reduction in α-dystroglycan glycosylation. This review discusses the recent novel discovery of additional dystroglycanopathy variants and progress in dystroglycanopathy animal models. RECENT FINDINGS: Several novel glycosyltransferase genes have been found to be responsible for a dystroglycanopathy phenotype, and in addition recessive mutations in DAG1 have been identified for the first time in a primary dystroglycanopathy. Studies in dystroglycanopathy mouse models have clarified some aspects of the structural defects observed in the central nervous system and in the eye, whereas a study in zebrafish implicates unfolded protein response in the pathogenesis of two of the secondary dystroglycanopathies. SUMMARY: Improved understanding of the molecular bases of dystroglycanopathies will lead to more precise diagnosis and genetic counseling; therapeutic strategies are being developed and tested in the preclinical models and it is hoped that these observations will pave the way to therapeutic interventions in humans.


Assuntos
Distroglicanas/metabolismo , Distrofias Musculares/metabolismo , Síndrome de Walker-Warburg/metabolismo , Animais , Modelos Animais de Doenças , Distroglicanas/genética , Glicosilação , Humanos , Camundongos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Distrofias Musculares/genética , Distrofias Musculares/terapia , Síndrome de Walker-Warburg/genética , Síndrome de Walker-Warburg/terapia , Peixe-Zebra
18.
Appl Opt ; 50(36): 6583-90, 2011 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-22193187

RESUMO

We describe a new light transport model, which was applied to three-dimensional lifetime imaging of Förster resonance energy transfer in mice in vivo. The model is an approximation to the radiative transfer equation and combines light diffusion and ray optics. This approximation is well adopted to wide-field time-gated intensity-based data acquisition. Reconstructed image data are presented and compared with results obtained by using the telegraph equation approximation. The new approach provides improved recovery of absorption and scattering parameters while returning similar values for the fluorescence parameters.


Assuntos
Algoritmos , Transferência Ressonante de Energia de Fluorescência/métodos , Óptica e Fotônica/métodos , Animais , Fluorescência , Imageamento Tridimensional/métodos , Camundongos , Modelos Teóricos , Espalhamento de Radiação , Tomografia Óptica/métodos
19.
Wellcome Open Res ; 6: 197, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35509373

RESUMO

Background: Progression through mammalian embryogenesis involves many interacting cell types and multiple differentiating cell lineages. Quantitative polymerase chain reaction (qPCR) analysis of gene expression in the developing embryo is a valuable tool for deciphering these processes, but normalisation to stably-expressed reference genes is essential for such analyses. Gene expression patterns change globally and dramatically as embryonic development proceeds, rendering identification of consistently appropriate reference genes challenging. Methods: We have investigated expression stability in mouse embryos from mid to late gestation (E11.5-E18.5), both at the whole-embryo level, and within the head and forelimb specifically, using 15 candidate reference genes ( ACTB, 18S, SDHA, GAPDH, HTATSF1, CDC40, RPL13A, CSNK2A2, AP3D1, HPRT1, CYC1, EIF4A, UBC, B2M and PAK1IP1), and four complementary algorithms (geNorm, Normfinder, Bestkeeper and deltaCt). Results: Unexpectedly, all methods suggest that many genes within our candidate panel are acceptable references, though AP3D1, RPL13A and PAK1IP1 are the strongest performing genes overall (scoring highly in whole embryos, heads or forelimbs alone, and in all samples collectively). HPRT1 and B2M are conversely poor choices, and show strong developmental regulation. We further show that normalisation using our three highest-scoring references can reveal subtle patterns of developmental expression even in genes ostensibly ranked as acceptably stable ( CDC40, HTATSF1). Conclusion: AP3D1, RPL13A and PAK1IP1 represent universally suitable reference genes for expression studies in the E11.5-E18.5 mouse embryo.

20.
Wellcome Open Res ; 6: 84, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-37942409

RESUMO

Background: In addition to progressive, debilitating muscle degeneration, ~50% of patients with Duchenne muscular dystrophy (DMD) have associated cognitive and behavioural disorders secondary to deficiency of dystrophin protein in the brain. The brain expresses a variety of dystrophin isoforms (Dp427, Dp140 and Dp71) whose functions remain to be fully elucidated. Detailed comparative analysis of gene expression in healthy and dystrophin-deficient brain is fundamental to understanding the functions of each isoform, and the consequences of their deficiency, with animal models representing a key tool in this endeavour. Reverse transcription quantitative real-time PCR (RT-qPCR) is a widely used method to study gene expression. However, accurate quantitative assessment requires normalisation of expression data using validated reference genes. The aim of this study was to identify a panel of suitable reference genes that can be used to normalise gene expression in the brain of healthy and dystrophic dogs and mice. Methods: Using the DE50-MD dog and mdx mouse models of DMD we performed RT-qPCR from fresh frozen brain tissue and employed the geNorm, BestKeeper and Normfinder algorithms to determine the stability of expression of a panel of candidate reference genes across healthy and dystrophic animals, and across different brain regions. Results: We show that SDHA, UBC and YWHAZ are suitable reference genes for normalising gene expression in healthy and dystrophic canine brain, and GAPDH, RPL13A and CYC1 in healthy and dystrophic murine brain. Notably, there was no overlap in the highest performing reference genes between the two species. Conclusions: Our findings suggest that gene expression normalisation is possible across six regions of the canine brain, and three regions of the murine brain. Our results should facilitate future work to study gene expression in the brains of normal and dystrophic dogs and mice and thus decipher the transcriptional consequences of dystrophin deficiency in the brain.

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