Recruitment of bone-marrow-derived cells by skeletal and cardiac muscle in adult dystrophic mdx mice

 It is commonly accepted, that regenerative capacity of striated muscle is confined to skeletal muscle by activation of satellite cells that normally reside quiescent between the plasmalemma and the basement membrane of muscle fibers. Muscular dystrophies are characterized by repetitive cycles of de- and regeneration of skeletal muscle fibers and by the frequent involvement of the cardiac muscle. Since during the longstanding course of muscular dystrophies there is a permanent demand of myogenic progenitors we hypothesized that this may necessitate a recruitment of additional myogenic precursors from an undifferentiated, permanently renewed cell pool, such as bone marrow (BM) cells. To this end normal and dystrophic (mdx) female mice received bone marrow transplantation (BMT) from normal congenic male donor mice. After 70 days, histological sections of skeletal and cardiac muscle from BMT mice were probed for the donor-derived Y chromosomes. In normal BMT recipients, no Y chromosome-containing myonuclei were detected, either in skeletal or in cardiac muscle. However, in all samples from dystrophic mdx skeletal muscles Y chromosome-specific signals were detected within muscle fiber nuclei, which additionally were found to express the myoregulatory proteins myogenin and myf-5. Moreover, in the hearts of BMT-mdx mice single cardiomyocytes with donor derived nuclei were identified, indicating, that even cardiac muscle cells are able to regenerate by recruitment of circulating BM-derived progenitors. Our findings suggest that further characterization and identification of the BM cells capable of undergoing myogenic differentiation may have an outstanding impact on therapeutic strategies for diseases of skeletal and cardiac muscle. Accepted: 27 October 1998     

A sandwich ELISA kit reveals marked elevation of titin N-terminal fragment levels in the urine of mdx mice

The mdx mouse is a model of Duchenne muscular dystrophy (DMD), a fatal progres-sive muscle wasting disease caused by dystrophin deficiency, and is used most widely in preclinical studies. Mice with dystrophin deficiency, however, show milder muscle strength phenotypes than humans. In human, the introduction of a sandwich enzyme- linked immunosorbent assay (ELISA) kit revealed a more than 700- fold increase in titin N- terminal fragment levels in the urine of pediatric patients with DMD. Notably, the urinary titin level declines with aging, reflecting progression of muscle wast-ing. In mouse, development of a highly sensitive ELISA kit has been awaited. Here, a sandwich ELISA kit to measure titin N- terminal fragment levels in mouse urine was developed. The developed kit showed good linearity, recovery, and repeatability in measuring recombinant or natural mouse titin N- terminal fragment levels. The titin N- terminal fragment concentration in the urine of mdx mice was more than 500- fold higher than that of normal mice. Urinary titin was further analyzed by extending the collection of urine samples to both young (3– 11 weeks old) and aged (56– 58 weeks old) mdx mice. The concentration in the young group was significantly higher than that in the aged group. It was concluded that muscle protein breakdown is active and persistent in mdx mice even though the muscle phenotype is mild. Our results pro-vide an opportunity to develop DMD treatments that aim to alleviate muscle protein breakdown by monitoring urinary titin levels.     

Three-dimensional cytoarchitecture of complex branched fibers in soleus muscle from mdx mutant mice

Three-dimensional cytoarchitecture and types and features of muscle fibers were examined in soleus muscles from mdx mutant mice at different stages of development. In the 2-week-old mice, no abnormal muscle fibers were observed light microscopically, whereas in the 4-week-old animals, disrupted fibers were frequent in light microscopy and scanning electron microscopy. Muscle fibers fused with several short fiber branches appeared at the sixth week after birth and increased in number until the tenth week. In the 1-year-old mice, approximately ten or more muscle fibers were seen fused together. They had many complex branches forming an “anastomosing syncytial reticulum.” Muscle fibers with irregular diameters and aggregations of the same type fibers were also observed. Our results demonstrated that these complex branched fibers might be formed by long term repetition of the degeneration and regeneration cycle during the development of soleus muscles, indicating that the characteristic features of muscle fibers with irregular diameters and aggregations of the same type fibers are certainly dependent on the existence of the complex branched fibers. © 1993 Wiley-Liss, Inc.     

Resolution of skeletal muscle inflammation in mdx dystrophic mouse is accompanied by increased immunoglobulin and interferon-gamma production

Mdx mouse, the animal model of Duchenne muscular dystrophy, develops an X-linked recessive inflammatory myopathy with an apparent sustained capacity for muscle regeneration. We analysed whether changes in the skeletal muscle during myonecrosis and regeneration would correlate with functional alterations in peripheral lymphoid tissues. Here we show that during the height of myonecrosis, mdx mice display marked atrophy of peripheral lymph nodes and extensive muscle inflammation. In contrast, enlargement of draining lymph nodes with accumulation of CD4+ CD44+, CD4+ CD25+, CD8+ CD44+ T lymphocytes and type-2 B cells was consistently observed during amelioration of the muscle lesion. In addition, regeneration of the muscular tissue was accompanied by concomitant increase of immunoglobulin-secreting cells in regional lymph nodes and bone marrow. Double immunolabelling analysis revealed intense B cell proliferation and formation of germinal centre in the follicles of dystrophic regional lymph nodes. Furthermore, lymph node cells produced large amounts of IFN-gamma but not IL-4, IL-6 or IL-10 after in vitro mitogen stimulation with Concanavalin A. As these alterations occurred mainly during the recovery period, we suggested that local activation of the immune system could be an influence which mitigates the myonecrosis of muscular tissue in the mdx dystrophic mouse.     

Effects of muscle damage on 31phosphorus magnetic resonance spectroscopy indices of energetic status and sarcolemma integrity in young mdx mice

³¹Phosphorus magnetic resonance spectroscopy (³¹P-MRS) has been shown to detect altered energetic status (e.g. the ratio of inorganic phosphate to phosphocreatine: Pi/PCr), intracellular acid–base status, and free intracellular magnesium ([Mg²⁺]) in dystrophic muscle compared with unaffected muscle; however, the causes of these differences are not well understood. The purposes of this study were to examine ³¹P-MRS indices of energetic status and sarcolemma integrity in young mdx mice compared with wild-type and to evaluate the effects of downhill running to induce muscle damage on ³¹P-MRS indices in dystrophic muscle. In vivo ³¹P-MRS spectra were acquired from the posterior hindlimb muscles in young (4–10 weeks of age) mdx (C57BL/10ScSn-DMDmdx) and wild-type (C57BL/10ScSnJ) mice using an 11.1-T MR system. The flux of phosphate from PCr to ATP was estimated by ³¹P-MRS saturation transfer experiments. Relative concentrations of high-energy phosphates were measured, and intracellular pH and [Mg²⁺] were calculated. ¹H₂O-T₂ was measured using single-voxel ¹H-MRS from the gastrocnemius and soleus using a 4.7-T MR system. Downhill treadmill running was performed in a subset of mice. Young mdx mice were characterized by elevated ¹H₂O-T₂ (p < 0.01)_, Pi/PCr (p = 0.02), PCr to ATP flux (p = 0.04) and histological inflammatory markers (p < 0.05) and reduced (p < 0.01) [Mg²⁺] compared with wild-type. Furthermore, 24 h after downhill running, an increase (p = 0.02) in Pi/PCr was observed in mdx and wild-type mice compared with baseline, and a decrease (p < 0.001) in [Mg²⁺] and a lower (p = 0.048) intracellular [H⁺] in damaged muscle regions of mdx mice were observed, consistent with impaired sarcolemma integrity. Overall, our findings demonstrate that ³¹P-MRS markers of energetic status and sarcolemma integrity are altered in young mdx compared with wild-type mice, and these indices are exacerbated following downhill running.     

Ixazomib,an oral proteasome inhibitor,exhibits potential effect in dystrophin‐deficient mdx mice

Dystrophin deficiency makes the sarcolemma fragile and susceptible to degeneration in Duchenne muscular dystrophy. The proteasome is a multimeric protease complex and is central to the regulation of cellular proteins. Previous studies have shown that proteasome inhibition improved pathological changes in mdx mice. Ixazomib is the first oral proteasome inhibitor used as a therapy in multiple myeloma. This study investigated the effects of ixazomib on the dystrophic muscle of mdx mice. MDX mice were treated with ixazomib (7.5 mg/kg/wk by gavage) or 0.2 mL of saline for 12 weeks. The Kondziela test was performed to measure muscle strength. The tibialis anterior (TA) and diaphragm (DIA) muscles were used for morphological analysis, and blood samples were collected for biochemical measurement. We observed maintenance of the muscle strength in the animals treated with ixazomib. Treatment with ixazomib had no toxic effect on the mdx mouse. The morphological analysis showed a reduction in the inflammatory area and fibres with central nuclei in the TA and DIA muscles and an increase in the number of fibres with a diameter of 20 µm² in the DIA muscle after treatment with ixazomib. There was an increase in the expression of dystrophin and utrophin in the TA and DIA muscles and a reduction in the expression of osteopontin and TGF‐β in the DIA muscle of mdx mice treated with ixazomib. Ixazomib was thus shown to increase the expression of dystrophin and utrophin associated with improved pathological and functional changes in the dystrophic muscles of mdx mice.     

Mechanical and morphological aspects of the calcaneal tendon of mdx mice at 21 days of age

A relationship between compromised muscles and other tissues has been demonstrated in mdx mouse, an animal model studied for understanding of Duchenne muscular dystrophy. The hypothesis is that changes in the calcaneal tendon of mdx mice occur previous to the onset of rigorous and most marked episodes of muscle degeneration, which start suddenly after 21 days of life. Thus, this study aimed to identify possible alterations in the calcaneal tendon of mdx mouse at 21 days of age. Control and mdx tendons were submitted to mechanical tensile testing, quantification of hydroxyproline, and staining with toluidine blue and picrosirius red. Hydroxyproline content was similar between mdx and control groups. The control tendon presented higher mechanical strength (load, stress, and elastic modulus) and its morphological analysis showed a larger number of round fibroblasts, nuclei with well‐decondensed chromatin, and slightly metachromatic well‐stained cytoplasmic material, different from that observed in mdx tendons. The results suggest that the absence of dystrophin in mdx mouse can provoke directly or indirectly alterations in the mechanical properties and morphology of the calcaneal tendon. Anat Rec, 296:1546–1551, 2013. © 2013 Wiley Periodicals, Inc.     

Erythropoietin reduces the expression of myostatin in mdx dystrophic mice

Erythropoietin (EPO) has been well characterized as a renal glycoprotein hormone regulating red blood cell production by inhibiting apoptosis of erythrocyte progenitors in hematopoietic tissues. EPO exerts regulatory effects in cardiac and skeletal muscles. Duchenne muscular dystrophy is a lethal degenerative disorder of skeletal and cardiac muscle. In this study, we tested the possible therapeutic beneficial effect of recombinant EPO (rhEPO) in dystrophic muscles in mdx mice. Total strength was measured using a force transducer coupled to a computer. Gene expression for myostatin, transforming growth factor-β1 (TGF-β1), and tumor necrosis factor-α (TNF-α) was determined by quantitative real time polymerase chain reaction. Myostatin expression was significantly decreased in quadriceps from mdx mice treated with rhEPO (rhEPO=0.60±0.11, control=1.07±0.11). On the other hand, rhEPO had no significant effect on the expression of TGF-β1 (rhEPO=0.95±0.14, control=1.05±0.16) and TNF-α (rhEPO=0.73±0.20, control=1.01±0.09). These results may help to clarify some of the direct actions of EPO on skeletal muscle.     

Hereditary muscular dystrophy in mdx mice as a homologous model for introduction of cell technologies in the treatment of progressive muscular dystrophies in humans

Life-time monitoring of the main clinical and laboratory manifestations of hereditary muscular dystrophy in mdx mice confirmed the presence of mutation in exon 23 of dystrophin gene and the absence of this protein in skeletal muscles of mutant animals. Muscular dystrophy in mice was similar to human progressive muscle disorder, which allows the use of this model for the development of cell technologies for the treatment of hereditary muscular diseases in humans.Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 138, No. 10, pp. 477–480, October, 2004     

Cardiac and skeletal muscle changes associated with rosuvastatin therapy in dystrophic mdx mice

Statins are prescribed to prevent and treat atherosclerotic cardiovascular and metabolic diseases but have controversial effects on skeletal muscles. While statins are a reported cause of myopathy, some studies have suggested that statins could potentially ameliorate dystrophy due to their pleiotropic effects on inflammation, myonecrosis, and autophagy. In the present study, we evaluated the potential benefit of rosuvastatin treatment on heart, limb, and diaphragm muscles in dystrophin-deficient mdx mice at an early stage (45 days of age) of disease. Mdx mice received rosuvastatin (10 mg/kg) by gavage for 30 days beginning at 15 days of age. Normal C57BL/10 mice received rosuvastatin by the same route over the same interval. In the mdx group, rosuvastatin significantly increased IgG-positive fibers (myonecrosis) and the inflammatory areas in the biceps brachii and diaphragm muscles and decreased the anterior limb muscle force (grip strength). Molecular markers of inflammation (TNF-α and NF-kB) and fibrosis (fibronectin) were not altered by rosuvastatin in mdx mice skeletal and cardiac muscles. In normal mice, rosuvastatin increased CK, TNF-α (heart), NF-kB (diaphragm), and fibronectin (heart and diaphragm). Inflammatory areas were seen in all normal muscles of rosuvastatin-treated mice. Rosuvastatin did not benefit dystrophy in the mdx mice and was associated with inflammation in normal cardiac and skeletal muscles.     

Membrane abnormalities and Ca homeostasis in muscles of the mdx mouse,an animal model of the Duchenne muscular dystrophy: a review

Muscles of the mdx mouse lack dystrophin, a cytoskeletal protein. Mdx fibres exhibit an increased fragility to hypo-osmotic shock and to forced lengthening, an abnormal opening time of stretch-sensitive calcium channels. The question of a chronic elevated [Ca²⁺]_i value is a matter of controversy. We have analysed Ca homeostasis in smooth and skeletal muscles from the adult mdx mouse. The wall of the vas deferens was loaded with the fluorescent Ca indicator Fura-2-AM (cell-diffusible). Resting [Ca²⁺]_i was measured after changes of the electrochemical potential for Ca²⁺ and after KCl or electrical stimulations. In no instance was a difference observed between these and similar muscles from control mice. Single striated fibres were isolated by collagenase treatment of the flexor digitorum brevis muscle and loaded with Fura-2-AM. The value of resting [Ca²⁺]_i was measured using an in situ calibration procedure which took account of Ca buffering by Fura-2. A chronic increase of cytosolic Ca²⁺ was not confirmed. The expression of the intracellular Ca-binding protein, parvalbumin, was measured. It increased by about threefold in fast mdx muscles (tibialis anterior) but remained undetectable in the soleus. It is hypothesized that parvalbumin helps to maintain [Ca²⁺]_i within normal values. This hypothesis will be discussed in connection with dystrophy phenotypes in mutant dogs and in human patients.     

Restoration of gastrocnemius muscle in mdx mice of different age after injury and implantation of xenogenic muscle tissue

The intensity of regeneration of crossed gastrocnemius muscle was evaluated in two groups of mdx mice of different age 2 weeks after implantation of crushed muscle tissue from newborn rats into the wound defect area. The effect of xenoplasty manifested in increased weight of the damaged muscle. The effect was observed in mice aging 12–16 weeks but not in those aged 40–48-weeks. Structural changes in the skeletal muscle tissue intrinsic of mdx mice and augmenting with age were detected in intact mice before the experiment. Activity of muscle fiber regeneration in intact and injured muscle of 40–48-week-old mice was significantly lower than in 12–16-week-old ones. Myoblasts of the xenogenic transplant retained viability in recipient muscles for at least 2 weeks. Post-traumatic regeneration was stimulated in only 12–16-week animals. Xenoplasty was ineffective in older animals and even somewhat enhanced the destructive processes in the muscle. It seems that age-specific regeneration activity of the recipient skeletal muscle tissue should be taken into consideration in the development of effective strategy of cell therapy for progressive muscular dystrophy. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 8, pp. 216–220, August, 2006     

Therapeutic effects of exon skipping and losartan on skeletal muscle of mdx mice

Various attempts have been made to find treatments for Duchenne muscular dystrophy (DMD) patients. Exon skipping is one of the promising technologies for DMD treatment by restoring dystropin protein, which is one of the muscle components. It is well known that losartan, an angiotensin II type1 receptor blocker, promotes muscle regeneration and differentiation by lowering the level of transforming growth factor–beta1 signaling. In this study, we illustrated the combined effects of exon skipping and losartan on skeletal muscle of mdx mice. We supplied mdx mice with losartan for 2 weeks before exon skipping treatment. The losartan with the exon skipping group showed less expression of myf5 than the losartan treated group. Also the losartan with exon skipping group recovered normal muscle architecture, in contrast to the losartan group which still showed many central nuclei. However, the exon skipping efficiency and the restoration of dystrophin protein were lower in the losartan with exon skipping group compared to the exon skipping group. We reveal that losartan promotes muscle regeneration and shortens the time taken to restore normal muscle structure when combined with exon skipping. However, combined treatment of exon skipping and losartan decreases the restoration of dystrophin protein meaning decrease of exon skipping efficiency.     

Effect of head-down hanging on the course of degenerative process in the hind paw muscles of 12-month-old MDX mice

Effect of functional unloading on the course of degenerative process in muscles was studied in dystrophin-deficient mdx mice. Head down-hanging of animals led to a significant decrease in the cross-section area of muscle fibers, increase in the percentage of fibers with centrally located nuclei and of Evans blue-stained fibers. Gravitation unloading of 12-month-old animals with pronounced manifestations of muscular dystrophy did not inhibit this pathological process. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 141, No. 6, pp. 702–705, June, 2006     

DMD模型鼠基因型鉴定及其肌组织病理学与超微结构研究

目的:探讨DMD动物模型dko小鼠的基因型鉴定方法及其肌组织的病理学与超微结构情况。方法:运用序列特异性引物PCR-SSP技术鉴定杂合子种鼠的子代基因型,并对子代dko小鼠的骨骼肌组织冰冻切片进行HE染色和dystrophin/utrophin的SABC-Cy3荧光免疫组织化学检测以及肌组织的透射电镜观察。结果:112只子代鼠中,mdx小鼠28只,dko小鼠26只,杂合子鼠58只,按百分比计算,分别占25.0%、23.2%、51.8%,基因型鉴定结果符合孟德尔遗传规律;dko小鼠肌组织HE染色显示细胞大小形态不均,轮廓变圆,核中移,肌间隙变宽,有炎症细胞浸润与结缔组织增生现象;免疫荧光检测肌膜未见有dystrophin/utrophin表达;电镜观察有肌膜断裂不完整、膜与膜下组织分离并水肿、肌原纤维结构松散、结缔组织增生及炎症细胞浸润等现象。结论:PCR-SSP技术鉴定子代鼠基因型快捷准确;dko小鼠的病理生理学表现与DMD极为相像,是DMD临床治疗研究的理想疾病动物模型。     

Calcium-binding proteins in skeletal muscles of the mdx mice: potential role in the pathogenesis of Duchenne muscular dystrophy

Duchenne muscular dystrophy is one of the most common hereditary diseases. Abnormal ion handling renders dystrophic muscle fibers more susceptible to necrosis and a rise in intracellular calcium is an important initiating event in dystrophic muscle pathogenesis. In the mdx mice, muscles are affected with different intensities and some muscles are spared. We investigated the levels of the calcium-binding proteins calsequestrin and calmodulin in the non-spared axial (sternomastoid and diaphragm), limb (tibialis anterior and soleus), cardiac and in the spared extraocular muscles (EOM) of control and mdx mice. Immunoblotting analysis showed a significant increase of the proteins in the spared mdx EOM and a significant decrease in the most affected diaphragm. Both proteins were comparable to the cardiac muscle controls. In limb and sternomastoid muscles, calmodulin and calsequestrin were affected differently. These results suggest that differential levels of the calcium-handling proteins may be involved in the pathogenesis of myonecrosis in mdx muscles. Understanding the signaling mechanisms involving Ca²⁺-calmodulin activation and calsequestrin expression may be a valuable way to develop new therapeutic approaches to the dystrophinopaties.     

Morpholino-induced exon skipping stimulates cell-mediated and humoral responses to dystrophin in mdx mice

Exon skipping is a promising genetic therapeutic strategy for restoring dystrophin expression in the treatment of Duchenne muscular dystrophy (DMD). The potential for newly synthesized dystrophin to trigger an immune response in DMD patients, however, is not well established. We have evaluated the effect of chronic phosphorodiamidate morpholino oligomer (PMO) treatment on skeletal muscle pathology and asked whether sustained dystrophin expression elicits a dystrophin-specific autoimmune response. Here, two independent cohorts of dystrophic mdx mice were treated chronically with either 800 mg/kg/month PMO for 6 months (n = 8) or 100 mg/kg/week PMO for 12 weeks (n = 11). We found that significant muscle inflammation persisted after exon skipping in skeletal muscle. Evaluation of humoral responses showed serum-circulating antibodies directed against de novo dystrophin in a subset of mice, as assessed both by Western blotting and immunofluorescent staining; however, no dystrophin-specific antibodies were observed in the control saline-treated mdx cohorts (n = 8) or in aged (12-month-old) mdx mice with expanded ‘revertant’ dystrophin-expressing fibers. Reactive antibodies recognized both full-length and truncated exon-skipped dystrophin isoforms in mouse skeletal muscle. We found more antigen-specific T-cell cytokine responses (e.g. IFN-g, IL-2) in dystrophin antibody-positive mice than in dystrophin antibody-negative mice. We also found expression of major histocompatibility complex class I on some of the dystrophin-expressing fibers along with CD8+ and perforin-positive T cells in the vicinity, suggesting an activation of cell-mediated damage had occurred in the muscle. Evaluation of complement membrane attack complex (MAC) deposition on the muscle fibers further revealed lower MAC deposition on muscle fibers of dystrophin antibody-negative mice than on those of dystrophin antibody-positive mice. Our results indicate that de novo dystrophin expression after exon skipping can trigger both cell-mediated and humoral immune responses in mdx mice. Our data highlights the need to further investigate the autoimmune response and its long-term consequences after exon-skipping therapy. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Evaluation of the gastrointestinal tract in mdx mice: an experimental model of Duchenne muscular dystrophy

This study describes the functional and morphological alterations in the intestines of mdx mice (n = 4) compared with the intestinal features of C57BL/10 mice (n = 7) at 2 months of age. The whole gut transit time (carmine red) and the upper gut transit time (activated charcoal) were measured, and light microscopy was utilized to view stained sections (H&E and picrosirius red) for histological analysis. No significant difference in mean evacuation time for the whole gut was observed between the two groups, but a significant delay in activated charcoal passage was observed in the mdx mice. Visually, a higher concentration of collagen fibers in the submucosal region was apparent in the mdx mice. The concentration of collagen fibers in the stomach and small intestine suggests a direct relationship with the decrease in motility of the upper gastrointestinal tract in the mdx mice. Further experimental studies should be conducted to develop therapeutic alternatives to collagen inhibition to control these manifestations.     

Induction of a regenerative microenvironment in skeletal muscle is sufficient to induce embryonal rhabdomyosarcoma in p53-deficient mice

We have previously reported that mice with muscular dystrophy, including mdx mice, develop embryonal rhabdomyosarcoma (eRMS) with a low incidence after 1 year of age and that almost all such tumours contain cancer-associated p53 mutations. To further demonstrate the relevance of p53 inactivation, we created p53-deficient mdx mice. Here we demonstrate that loss of one or both p53 (Trp53) alleles accelerates eRMS incidence in the mdx background, such that almost all Trp53(-/-) mdx animals develop eRMS by 5 months of age. To ascertain whether increased tumour incidence was due to the regenerative microenvironment found in dystrophic skeletal muscles, we induced muscle regeneration in Trp53(+/+) and Trp53(-/-) animals using cardiotoxin (Ctx). Wild-type (Trp53(+/+) ) animals treated with Ctx, either once every 7 days or once every 14 days from 1 month of age onwards, developed no eRMS; however, all similarly Ctx-treated Trp53(-/-) animals developed eRMS by 5 months of age at the site of injection. Most of these tumours displayed markers of human eRMS, including over-expression of Igf2 and phosphorylated Akt. These data demonstrate that the presence of a regenerative microenvironment in skeletal muscle, coupled with Trp53 deficiency, is sufficient to robustly induce eRMS in young mice. These studies further suggest that consideration should be given to the potential of the muscle microenvironment to support tumourigenesis in regenerative therapies for myopathies.     

Huntingtin fragmentation and increased caspase 3, 8 and 9 activities in lymphoblasts with heterozygous and homozygous Huntington's disease mutation

Huntington's disease (HD) is caused by mutated huntingtin (htt), a toxic protein ubiquitously expressed in nervous and non-nervous system tissues. Fragmentation of htt by caspases and further accumulation in cells of protein aggregates contribute to cell dysfunction and death. In the attempt to elucidate whether this mechanism depends on patients' genotype, we analysed the pattern of htt fragmentation, the caspase 3, 8 and 9 activities and their variation in lymphoblasts with heterozygous and homozygous CAG mutation and in controls. Cells homozygous for expanded mutation showed greater amount of mutated fragments than heterozygotes and controls, caspase 3, 8 and 9 activities greater in mutated than control cell lines, after cyanide treatment, the caspase 3 and 8 particularly increased in homozygotes. This data offers a biological explanation to the clinical in-patients evidence of mutation homozygosity associated with more severe phenotype.