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Biomolecules Mar 2024Duchenne muscular dystrophy is caused by loss of the dystrophin protein. This pathology is accompanied by mitochondrial dysfunction contributing to muscle fiber...
Duchenne muscular dystrophy is caused by loss of the dystrophin protein. This pathology is accompanied by mitochondrial dysfunction contributing to muscle fiber instability. It is known that mitochondria-targeted in vivo therapy mitigates pathology and improves the quality of life of model animals. In the present work, we applied mitochondrial transplantation therapy (MTT) to correct the pathology in dystrophin-deficient mice. Intramuscular injections of allogeneic mitochondria obtained from healthy animals into the hind limbs of mice alleviated skeletal muscle injury, reduced calcium deposits in muscles and serum creatine kinase levels, and improved the grip strength of the hind limbs and motor activity of recipient mice. We noted normalization of the mitochondrial ultrastructure and sarcoplasmic reticulum/mitochondria interactions in muscles. At the same time, we revealed a decrease in the efficiency of oxidative phosphorylation in the skeletal muscle mitochondria of recipient mice accompanied by a reduction in lipid peroxidation products (MDA products) and reduced calcium overloading. We found no effect of MTT on the expression of mitochondrial signature genes (, , , , ) and on the level of mtDNA. Our results show that systemic MTT mitigates the development of destructive processes in the quadriceps muscle of mice.
Topics: Animals; Mice; Mice, Inbred mdx; Dystrophin; Calcium; Quality of Life; Muscular Dystrophy, Duchenne; Muscle, Skeletal; Mitochondria
PubMed: 38540736
DOI: 10.3390/biom14030316 -
Biomedicines Mar 2024Duchenne Muscular Dystrophy (DMD) is a lethal disease caused by mutation in the dystrophin gene. Currently there is no cure for DMD. We introduced a novel human...
Amelioration of Morphological Pathology in Cardiac, Respiratory, and Skeletal Muscles Following Intraosseous Administration of Human Dystrophin Expressing Chimeric (DEC) Cells in Duchenne Muscular Dystrophy Model.
Duchenne Muscular Dystrophy (DMD) is a lethal disease caused by mutation in the dystrophin gene. Currently there is no cure for DMD. We introduced a novel human Dystrophin Expressing Chimeric (DEC) cell therapy of myoblast origin and confirmed the safety and efficacy of DEC in the mouse models of DMD. In this study, we assessed histological and morphological changes in the cardiac, diaphragm, and gastrocnemius muscles of the / mice after the transplantation of human DEC therapy via the systemic-intraosseous route. The efficacy of different DEC doses was evaluated at 90 days (0.5 × 10 and 1 × 10 DEC cells) and 180 days (1 × 10 and 5 × 10 DEC cells) after administration. The evaluation of Hematoxylin & Eosin (H&E)-stained sectional slices of cardiac, diaphragm, and gastrocnemius muscles included assessment of muscle fiber size by minimal Feret's diameter method using ImageJ software. The overall improvement in muscle morphology was observed in DMD-affected target muscles in both studies, as evidenced by a shift in fiber size distribution toward the wild type (WT) phenotype and by an increase in the mean Feret's diameter compared to the vehicle-injected controls. These findings confirm the long-term efficacy of human DEC therapy in the improvement of overall morphological pathology in the muscles affected by DMD and introduce DEC as a novel therapeutic approach for DMD patients.
PubMed: 38540201
DOI: 10.3390/biomedicines12030586 -
CPT: Pharmacometrics & Systems... May 2024Duchenne muscular dystrophy (DMD) is a rare X-linked recessive disorder characterized by loss-of-function mutations in the gene encoding dystrophin. These mutations lead...
Duchenne muscular dystrophy (DMD) is a rare X-linked recessive disorder characterized by loss-of-function mutations in the gene encoding dystrophin. These mutations lead to progressive functional deterioration including muscle weakness, respiratory insufficiency, and musculoskeletal deformities. Three-dimensional gait analysis (3DGA) has been used as a tool to analyze gait pathology through the quantification of altered joint kinematics, kinetics, and muscle activity patterns. Among 3DGA indices, the Gait Profile Score (GPS), has been used as a sensitive overall measure to detect clinically relevant changes in gait patterns in children with DMD. To enhance our understanding of the clinical translation of 3DGA, we report here the development of a population nonlinear mixed-effect model that jointly describes the disease progression of the 3DGA index, GPS, and the functional endpoint, North Star Ambulatory Assessment (NSAA). The final model consists of a quadratic structure for GPS progression and a linear structure for GPS-NSAA correlation. Our model was able to capture the improvement in function in GPS and NSAA in younger subjects, as well as the decline of function in older subjects. Furthermore, the model predicted NSAA (CFB) at 1 year reasonably well for DMD subjects ≤7 years old at baseline. The model tended to slightly underpredict the decline in NSAA after 1 year for those >7 years old at baseline, but the prediction summary statistics were well maintained within the standard deviation of observed data. Quantitative models such as this may help answer clinically relevant questions to facilitate the development of novel therapies in DMD.
Topics: Humans; Muscular Dystrophy, Duchenne; Child; Disease Progression; Longitudinal Studies; Child, Preschool; Gait; Male; Adolescent; Gait Analysis
PubMed: 38539027
DOI: 10.1002/psp4.13126 -
Fluids and Barriers of the CNS Mar 2024Waste from the brain has been shown to be cleared via the perivascular spaces through the so-called glymphatic system. According to this model the cerebrospinal fluid...
Waste from the brain has been shown to be cleared via the perivascular spaces through the so-called glymphatic system. According to this model the cerebrospinal fluid (CSF) enters the brain in perivascular spaces of arteries, crosses the astrocyte endfoot layer, flows through the parenchyma collecting waste that is subsequently drained along veins. Glymphatic clearance is dependent on astrocytic aquaporin-4 (AQP4) water channels that are highly enriched in the endfeet. Even though the polarized expression of AQP4 in endfeet is thought to be of crucial importance for glymphatic CSF influx, its role in extracellular solute clearance has only been evaluated using non-quantitative fluorescence measurements. Here we have quantitatively evaluated clearance of intrastriatally infused small and large radioactively labeled solutes in mice lacking AQP4 (Aqp4) or lacking the endfoot pool of AQP4 (Snta1). We confirm that Aqp4 mice show reduced clearance of both small and large extracellular solutes. Moreover, we find that the Snta1 mice have reduced clearance only for the 500 kDa [H]dextran, but not 0.18 kDa [H]mannitol suggesting that polarization of AQP4 to the endfeet is primarily important for clearance of large, but not small molecules. Lastly, we observed that clearance of 500 kDa [H]dextran increased with age in adult mice. Based on our quantitative measurements, we confirm that presence of AQP4 is important for clearance of extracellular solutes, while the perivascular AQP4 localization seems to have a greater impact on clearance of large versus small molecules.
Topics: Animals; Mice; Aquaporin 4; Astrocytes; Brain; Dextrans; Glymphatic System
PubMed: 38532513
DOI: 10.1186/s12987-024-00527-7 -
Sleep Advances : a Journal of the Sleep... 2024Brain oscillations of non-rapid eye movement sleep, including slow oscillations (SO, 0.5-1.5 Hz) and spindles (10-16 Hz), mirror underlying brain maturation across...
Brain oscillations of non-rapid eye movement sleep, including slow oscillations (SO, 0.5-1.5 Hz) and spindles (10-16 Hz), mirror underlying brain maturation across development and are associated with cognition. Hence, age-associated emergence and changes in the electrophysiological properties of these rhythms can lend insight into cortical development, specifically in comparisons between pediatric populations and typically developing peers. We previously evaluated age-associated changes in SOs in male patients with Duchenne muscular dystrophy (DMD), finding a significant age-related decline between 4 and 18 years. While primarily a muscle disorder, male patients with DMD can also have sleep, cognitive, and cortical abnormalities, thought to be driven by altered dystrophin expression in the brain. In this follow-up study, we characterized the age-associated changes in sleep spindles. We found that age-dependent spindle characteristics in patients with DMD, including density, frequency, amplitude, and duration, were consistent with age-associated trends reported in the literature for typically developing controls. Combined with our prior finding of age-associated decline in SOs, our results suggest that SOs, but not spindles, are a candidate intervention target to enhance sleep in patients with DMD.
PubMed: 38525359
DOI: 10.1093/sleepadvances/zpae015 -
Cellular and Molecular Life Sciences :... Mar 2024Deposition of the exon junction complex (EJC) upstream of exon-exon junctions helps maintain transcriptome integrity by preventing spurious re-splicing events in already...
Deposition of the exon junction complex (EJC) upstream of exon-exon junctions helps maintain transcriptome integrity by preventing spurious re-splicing events in already spliced mRNAs. Here we investigate the importance of EJC for the correct splicing of the 2.2-megabase-long human DMD pre-mRNA, which encodes dystrophin, an essential protein involved in cytoskeletal organization and cell signaling. Using targeted RNA-seq, we show that knock-down of the eIF4A3 and Y14 core components of EJC in a human muscle cell line causes an accumulation of mis-splicing events clustered towards the 3' end of the DMD transcript (Dp427m). This deregulation is conserved in the short Dp71 isoform expressed ubiquitously except in adult skeletal muscle and is rescued with wild-type eIF4A3 and Y14 proteins but not with an EJC assembly-defective mutant eIF4A3. MLN51 protein and EJC-associated ASAP/PSAP complexes independently modulate the inclusion of the regulated exons 71 and 78. Our data confirm the protective role of EJC in maintaining splicing fidelity, which in the DMD gene is necessary to preserve the function of the critical C-terminal protein-protein interaction domain of dystrophin present in all tissue-specific isoforms. Given the role of the EJC in maintaining the integrity of dystrophin, we asked whether the EJC could also be involved in the regulation of a mechanism as complex as skeletal muscle differentiation. We found that eIF4A3 knockdown impairs myogenic differentiation by blocking myotube formation. Collectively, our data provide new insights into the functional roles of EJC in human skeletal muscle.
Topics: Humans; Cell Nucleus; Dystrophin; Exons; RNA Splicing; RNA, Messenger
PubMed: 38512499
DOI: 10.1007/s00018-024-05188-1 -
BMC Genomics Mar 2024Dystrophinopathies are the most common X-linked inherited muscle diseases, and the disease-causing gene is DMD. Exonic duplications are a common type of pathogenic...
BACKGROUND
Dystrophinopathies are the most common X-linked inherited muscle diseases, and the disease-causing gene is DMD. Exonic duplications are a common type of pathogenic variants in the DMD gene, however, 5' end exonic duplications containing exon 1 are less common. When assessing the pathogenicity of exonic duplications in the DMD gene, consideration must be given to their impact on the reading frame. Traditional molecular methods, such as multiplex ligation-dependent probe amplification (MLPA) and next-generation sequencing (NGS), are commonly used in clinics. However, they cannot discriminate the precise physical locations of breakpoints and structural features of genomic rearrangement. Long-read sequencing (LRS) can effectively overcome this limitation.
RESULTS
We used LRS technology to perform whole genome sequencing on three families and analyze the structural variations of the DMD gene, which involves the duplications of exon 1 and/or exon 2. Two distinct variant types encompassing exon 1 in the DMD Dp427m isoform and/or Dp427c isoform are identified, which have been infrequently reported previously. In pedigree 1, the male individuals harboring duplication variant of consecutive exons 1-2 in the DMD canonical transcript (Dp427m) and exon 1 in the Dp427c transcript are normal, indicating the variant is likely benign. In pedigree 3, the patient carries complex SVs involving exon 1 of the DMD Dp427c transcript showing an obvious phenotype. The locations of the breakpoints and the characteristics of structural variants (SVs) are identified by LRS, enabling the classification of the variants' pathogenicity.
CONCLUSIONS
Our research sheds light on the complexity of DMD variants encompassing Dp427c/Dp427m promoter regions and emphasizes the importance of cautious interpretation when assessing the pathogenicity of DMD 5' end exonic duplications, particularly in carrier screening scenarios without an affected proband.
Topics: Humans; Male; Dystrophin; Exons; Genomics; Muscular Dystrophy, Duchenne; Protein Isoforms
PubMed: 38504154
DOI: 10.1186/s12864-024-10224-2 -
Orphanet Journal of Rare Diseases Mar 2024Pathogenic missense variants in the dystrophin (DMD) gene are rarely reported in dystrophinopathies. Most DMD missense variants are of uncertain significance and their...
BACKGROUND
Pathogenic missense variants in the dystrophin (DMD) gene are rarely reported in dystrophinopathies. Most DMD missense variants are of uncertain significance and their pathogenicity interpretation remains complicated. We aimed to investigate whether DMD missense variants would cause aberrant splicing and re-interpret their pathogenicity based on mRNA and protein studies.
METHODS
Nine unrelated patients who had an elevated serum creatine kinase level with or without muscle weakness were enrolled. They underwent a detailed clinical, imaging, and pathological assessment. Routine genetic testing and muscle-derived mRNA and protein studies of dystrophin and sarcoglycan genes were performed in them.
RESULTS
Three of the 9 patients presented with a Duchenne muscular dystrophy (DMD) phenotype and the remaining 6 patients had a suspected diagnosis of Becker muscular dystrophy (BMD) or sarcoglycanopathy based on their clinical and pathological characteristics. Routine genetic testing detected only 9 predicted DMD missense variants in them, of which 6 were novel and interpreted as uncertain significance. Muscle-derived mRNA studies of sarcoglycan genes didn't reveal any aberrant transcripts in them. Dystrophin mRNA studies confirmed that 3 predicted DMD missense variants (c.2380G > C, c.4977C > G, and c.5444A > G) were in fact splicing and frameshift variants due to aberrant splicing. The 9 DMD variants were re-interpreted as pathogenic or likely pathogenic based on mRNA and protein studies. Therefore, 3 patients with DMD splicing variants and 6 patients with confirmed DMD missense variants were diagnosed with DMD and BMD, respectively.
CONCLUSION
Our study highlights the importance of muscle biopsy and aberrant splicing for clinical and genetic interpretation of uncertain DMD missense variants.
Topics: Humans; Dystrophin; Muscular Dystrophy, Duchenne; Mutation, Missense; RNA, Messenger; Sarcoglycans
PubMed: 38486238
DOI: 10.1186/s13023-024-03128-7 -
Progress in Neurobiology Apr 2024Dystrophin loss due to mutations in the Duchenne muscular dystrophy (DMD) gene is associated with a wide spectrum of neurocognitive comorbidities, including an aberrant... (Review)
Review
Dystrophin loss due to mutations in the Duchenne muscular dystrophy (DMD) gene is associated with a wide spectrum of neurocognitive comorbidities, including an aberrant unconditioned fear response to stressful/threat stimuli. Dystrophin-deficient animal models of DMD demonstrate enhanced stress reactivity that manifests as sustained periods of immobility. When the threat is repetitive or severe in nature, dystrophinopathy phenotypes can be exacerbated and even cause sudden death. Thus, it is apparent that enhanced sensitivity to stressful/threat stimuli in dystrophin-deficient vertebrates is a legitimate cause of concern for patients with DMD that could impact neurocognition and pathophysiology. This review discusses our current understanding of the mechanisms and consequences of the hypersensitive fear response in preclinical models of DMD and the potential challenges facing clinical translatability.
Topics: Animals; Humans; Dystrophin; Fear; Muscular Dystrophy, Duchenne; Mutation; Vertebrates
PubMed: 38484964
DOI: 10.1016/j.pneurobio.2024.102590 -
International Journal of Molecular... Feb 2024Dystrophin () gene mutations are associated with skeletal muscle diseases such as Duchenne and Becker Muscular Dystrophy (BMD) and X-linked dilated cardiomyopathy...
Dystrophin () gene mutations are associated with skeletal muscle diseases such as Duchenne and Becker Muscular Dystrophy (BMD) and X-linked dilated cardiomyopathy (XL-DCM). To investigate the molecular basis of DCM in a 37-year-old woman. Clinical and genetic investigations were performed. Genetic testing was performed with whole exome sequencing (WES) using the Illumina platform. According to the standard protocol, a variant found by WES was confirmed in all available members of the family by bi-directional capillary Sanger resequencing. The effect of the variant was investigated by using an in silico prediction of pathogenicity. The index case was a 37-year-old woman diagnosed with DCM at the age of 33. A germline heterozygous A>G transversion at nucleotide 10103 in the gene, leading to an aspartic acid-glycine substitution at the amino acid 3368 of the DMD protein (c.10103A>G p.Asp3368Gly), was identified and confirmed by PCR-based Sanger sequencing of the exon 70. In silico prediction suggests that this variant could have a deleterious impact on protein structure and functionality (CADD = 30). The genetic analysis was extended to the first-degree relatives of the proband (mother, father, and sister) and because of the absence of the variant in both parents, the p.Asp3368Gly substitution was considered as occurring de novo. Then, the direct sequencing analysis of her 8-year-old son identified as hemizygous for the same variant. The young patient did not present any signs or symptoms attributable to DCM, but reported asthenia and presented with bilateral calf hypertrophy at clinical examination. Laboratory testing revealed increased levels of creatinine kinase (maximum value of 19,000 IU/L). We report an early presentation of dilated cardiomyopathy in a 33-year-old woman due to a de novo pathogenic variant of the dystrophin () gene (p.Asp3368Gly). Genetic identification of this variant allowed an early diagnosis of a skeletal muscle disease in her son.
Topics: Humans; Female; Adult; Child; Dystrophin; Cardiomyopathy, Dilated; Muscular Dystrophy, Duchenne; Mothers
PubMed: 38474032
DOI: 10.3390/ijms25052787