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Increased apoptosis of myoblasts in Drosophila model for the Walker-Warburg syndrome.

Ueyama M, Akimoto Y, Ichimiya T, Ueda R, Kawakami H, Aigaki T, Nishihara S - PLoS ONE (2010)

Bottom Line: We demonstrated that expression of RNA interference (RNAi) for the rt gene and the tw mutant was almost completely lethal and semi-lethal, respectively.Flies expressing RNAi had reduced lifespans.We then observed a high density of myoblasts with an enhanced degree of apoptosis in the tw mutant, which completely lost enzymatic activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioinformatics, Soka University, Hachioji, Tokyo, Japan.

ABSTRACT
Walker-Warburg syndrome, a progressive muscular dystrophy, is a severe disease with various kinds of symptoms such as muscle weakness and occasional seizures. The genes of protein O-mannosyltransferases 1 and 2 (POMT1 and POMT2), fukutin, and fukutin-related protein are responsible for this syndrome. In our previous study, we cloned Drosophila orthologs of human POMT1 and POMT2 and identified their activity. However, the mechanism of onset of this syndrome is not well understood. Furthermore, little is known about the behavioral properties of the Drosophila POMT1 and POMT2 mutants, which are called rotated abdomen (rt) and twisted (tw), respectively. First, we performed various kinds of behavioral tests and described in detail the muscle structures by using these mutants. The mutant flies exhibited abnormalities in heavy exercises such as climbing or flight but not in light movements such as locomotion. Defective motor function in mutants appeared immediately after eclosion and was exaggerated with aging. Along with motor function, muscle ultrastructure in the tw mutant was altered, as seen in human patients. We demonstrated that expression of RNA interference (RNAi) for the rt gene and the tw mutant was almost completely lethal and semi-lethal, respectively. Flies expressing RNAi had reduced lifespans. These findings clearly demonstrate that Drosophila POMT mutants are models for human muscular dystrophy. We then observed a high density of myoblasts with an enhanced degree of apoptosis in the tw mutant, which completely lost enzymatic activity. In this paper, we propose a novel mechanism for the development of muscular dystrophy: POMT mutation causes high myoblast density and position derangement, which result in apoptosis, muscle disorganization, and muscle cell defects.

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Representative electron micrographs of thoracic muscles in aged wild-type and tw mutant flies.(A, C, G, and I) Thirty-five-day-old wild-type fly muscles. (B, D, E, F, H, and J) Thirty-five-day-old tw mutant fly muscles. (A and B) Low-magnification images of muscles. (C and D) High-magnification view of the area bordered by the rectangle in Figs. 4A and B. (C) Normal sarcomere with regular Z-lines (arrowheads). (D) Z-lines (arrowheads) are irregular and often streaming. Nemaline bodies (arrows) in the muscle fiber. (E) Actin and myosin filaments are disorganized. (F) Glycogen granules (arrows) are accumulated. (G) Normal sarcoplasmic reticulum (SR). (H) SR is swollen. (I) Normal basement membrane. (J) The basement membrane (arrowheads) is duplicated and multilayered. MT: mitochondria. Bars: (A and B) 2 µm, (C, D, F, G, and H) 1 µm, and (E, I, and J) 500 nm.
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pone-0011557-g004: Representative electron micrographs of thoracic muscles in aged wild-type and tw mutant flies.(A, C, G, and I) Thirty-five-day-old wild-type fly muscles. (B, D, E, F, H, and J) Thirty-five-day-old tw mutant fly muscles. (A and B) Low-magnification images of muscles. (C and D) High-magnification view of the area bordered by the rectangle in Figs. 4A and B. (C) Normal sarcomere with regular Z-lines (arrowheads). (D) Z-lines (arrowheads) are irregular and often streaming. Nemaline bodies (arrows) in the muscle fiber. (E) Actin and myosin filaments are disorganized. (F) Glycogen granules (arrows) are accumulated. (G) Normal sarcoplasmic reticulum (SR). (H) SR is swollen. (I) Normal basement membrane. (J) The basement membrane (arrowheads) is duplicated and multilayered. MT: mitochondria. Bars: (A and B) 2 µm, (C, D, F, G, and H) 1 µm, and (E, I, and J) 500 nm.

Mentions: We further examined the effect of the tw mutation on sarcomeric structure by performing detailed electron microscopic analysis on the leg and thoracic muscles from wild-type, tw mutant and the rescue flies. Similar changes in ultrastructure were observed in the leg and thoracic muscles of both male and female tw mutant flies (Table 4). The normal sarcomeric structure of the muscles of wild-type flies is shown in Figs. 4A, 4C, and 5A. Sarcomeric disarray was frequently observed in the muscles of tw mutant flies (Figs. 4B, 4D, 5B, and 5C). In the mutant muscles, Z-lines were irregular and often streaming (Figs. 4D, 5B, and 5C), nemaline bodies were observed in the muscle fiber (Fig. 4D), actin and myosin filaments were disorganized (Figs. 4E and 5B), and accumulated glycogen granules were seen (Fig. 4F). Enlarged mitochondria (Fig. 5F) and swollen sarcoplasmic reticulum (SR) were seen in the tw mutant muscles (Figs. 4H and 5D), while normal mitochondria and SR were observed between the muscle fibers (Figs. 4A, 4C, 4G, 5A, and 5E). The basement membrane was duplicated and multilayered in tw mutant muscles (Figs. 4J and 5H), while normal basement membrane was observed continuously along the sarcolemma in wild-type muscles (Figs. 4I and 5G). The abovementioned defective muscle phenotypes were observed both in 15- and 35-day-old tw mutants but were hardly detected in wild-type flies. The number of mutants with defective phenotypes was higher in 35-day-old tw mutants than in 15-day-old tw mutants. Moreover, these defective phenotypes could not be found among the rescue flies (Table 4), indicating that the defective phenotypes in the mutants were fully rescued. These results demonstrated that tw contributed to the maintenance of muscle ultrastructure. Next, we counted the number of sarcomeric disarray, irregular Z-line, and filament disorganization occurrences in a 590-µm2 muscle area per individual and calculated the percentage of these abnormal structures (number of abnormal structures/number of observed). In thoracic muscles, these abnormal structures were observed in 35-day-old mutants but could not be detected in wild-type flies (Fig. 6A). In leg muscles, these abnormal structures were observed in both 15- and 35-day-old mutants but were hardly detected in wild-type flies (Fig. 6B). The percentages of abnormal structures in the tw mutant were significantly higher than those in wild-type flies except for filament disorganization in the leg muscles of 15-day-old flies, when abnormal structures were observed (Fig. 6). Moreover, these abnormal structures were significantly exaggerated with age in tw mutants but not significantly exaggerated with age in wild-type flies except for sarcomeric disarray in the leg muscles of 15-day-old flies (Table 5). These changes, which are reminiscent of the progressive symptoms in WWS patients, were more frequently observed in 35-day aged mutant muscles than in 15-day aged mutant muscles. The abovementioned results clearly demonstrated several kinds of abnormalities in the muscles of tw mutants that become more severe with age.


Increased apoptosis of myoblasts in Drosophila model for the Walker-Warburg syndrome.

Ueyama M, Akimoto Y, Ichimiya T, Ueda R, Kawakami H, Aigaki T, Nishihara S - PLoS ONE (2010)

Representative electron micrographs of thoracic muscles in aged wild-type and tw mutant flies.(A, C, G, and I) Thirty-five-day-old wild-type fly muscles. (B, D, E, F, H, and J) Thirty-five-day-old tw mutant fly muscles. (A and B) Low-magnification images of muscles. (C and D) High-magnification view of the area bordered by the rectangle in Figs. 4A and B. (C) Normal sarcomere with regular Z-lines (arrowheads). (D) Z-lines (arrowheads) are irregular and often streaming. Nemaline bodies (arrows) in the muscle fiber. (E) Actin and myosin filaments are disorganized. (F) Glycogen granules (arrows) are accumulated. (G) Normal sarcoplasmic reticulum (SR). (H) SR is swollen. (I) Normal basement membrane. (J) The basement membrane (arrowheads) is duplicated and multilayered. MT: mitochondria. Bars: (A and B) 2 µm, (C, D, F, G, and H) 1 µm, and (E, I, and J) 500 nm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2903483&req=5

pone-0011557-g004: Representative electron micrographs of thoracic muscles in aged wild-type and tw mutant flies.(A, C, G, and I) Thirty-five-day-old wild-type fly muscles. (B, D, E, F, H, and J) Thirty-five-day-old tw mutant fly muscles. (A and B) Low-magnification images of muscles. (C and D) High-magnification view of the area bordered by the rectangle in Figs. 4A and B. (C) Normal sarcomere with regular Z-lines (arrowheads). (D) Z-lines (arrowheads) are irregular and often streaming. Nemaline bodies (arrows) in the muscle fiber. (E) Actin and myosin filaments are disorganized. (F) Glycogen granules (arrows) are accumulated. (G) Normal sarcoplasmic reticulum (SR). (H) SR is swollen. (I) Normal basement membrane. (J) The basement membrane (arrowheads) is duplicated and multilayered. MT: mitochondria. Bars: (A and B) 2 µm, (C, D, F, G, and H) 1 µm, and (E, I, and J) 500 nm.
Mentions: We further examined the effect of the tw mutation on sarcomeric structure by performing detailed electron microscopic analysis on the leg and thoracic muscles from wild-type, tw mutant and the rescue flies. Similar changes in ultrastructure were observed in the leg and thoracic muscles of both male and female tw mutant flies (Table 4). The normal sarcomeric structure of the muscles of wild-type flies is shown in Figs. 4A, 4C, and 5A. Sarcomeric disarray was frequently observed in the muscles of tw mutant flies (Figs. 4B, 4D, 5B, and 5C). In the mutant muscles, Z-lines were irregular and often streaming (Figs. 4D, 5B, and 5C), nemaline bodies were observed in the muscle fiber (Fig. 4D), actin and myosin filaments were disorganized (Figs. 4E and 5B), and accumulated glycogen granules were seen (Fig. 4F). Enlarged mitochondria (Fig. 5F) and swollen sarcoplasmic reticulum (SR) were seen in the tw mutant muscles (Figs. 4H and 5D), while normal mitochondria and SR were observed between the muscle fibers (Figs. 4A, 4C, 4G, 5A, and 5E). The basement membrane was duplicated and multilayered in tw mutant muscles (Figs. 4J and 5H), while normal basement membrane was observed continuously along the sarcolemma in wild-type muscles (Figs. 4I and 5G). The abovementioned defective muscle phenotypes were observed both in 15- and 35-day-old tw mutants but were hardly detected in wild-type flies. The number of mutants with defective phenotypes was higher in 35-day-old tw mutants than in 15-day-old tw mutants. Moreover, these defective phenotypes could not be found among the rescue flies (Table 4), indicating that the defective phenotypes in the mutants were fully rescued. These results demonstrated that tw contributed to the maintenance of muscle ultrastructure. Next, we counted the number of sarcomeric disarray, irregular Z-line, and filament disorganization occurrences in a 590-µm2 muscle area per individual and calculated the percentage of these abnormal structures (number of abnormal structures/number of observed). In thoracic muscles, these abnormal structures were observed in 35-day-old mutants but could not be detected in wild-type flies (Fig. 6A). In leg muscles, these abnormal structures were observed in both 15- and 35-day-old mutants but were hardly detected in wild-type flies (Fig. 6B). The percentages of abnormal structures in the tw mutant were significantly higher than those in wild-type flies except for filament disorganization in the leg muscles of 15-day-old flies, when abnormal structures were observed (Fig. 6). Moreover, these abnormal structures were significantly exaggerated with age in tw mutants but not significantly exaggerated with age in wild-type flies except for sarcomeric disarray in the leg muscles of 15-day-old flies (Table 5). These changes, which are reminiscent of the progressive symptoms in WWS patients, were more frequently observed in 35-day aged mutant muscles than in 15-day aged mutant muscles. The abovementioned results clearly demonstrated several kinds of abnormalities in the muscles of tw mutants that become more severe with age.

Bottom Line: We demonstrated that expression of RNA interference (RNAi) for the rt gene and the tw mutant was almost completely lethal and semi-lethal, respectively.Flies expressing RNAi had reduced lifespans.We then observed a high density of myoblasts with an enhanced degree of apoptosis in the tw mutant, which completely lost enzymatic activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioinformatics, Soka University, Hachioji, Tokyo, Japan.

ABSTRACT
Walker-Warburg syndrome, a progressive muscular dystrophy, is a severe disease with various kinds of symptoms such as muscle weakness and occasional seizures. The genes of protein O-mannosyltransferases 1 and 2 (POMT1 and POMT2), fukutin, and fukutin-related protein are responsible for this syndrome. In our previous study, we cloned Drosophila orthologs of human POMT1 and POMT2 and identified their activity. However, the mechanism of onset of this syndrome is not well understood. Furthermore, little is known about the behavioral properties of the Drosophila POMT1 and POMT2 mutants, which are called rotated abdomen (rt) and twisted (tw), respectively. First, we performed various kinds of behavioral tests and described in detail the muscle structures by using these mutants. The mutant flies exhibited abnormalities in heavy exercises such as climbing or flight but not in light movements such as locomotion. Defective motor function in mutants appeared immediately after eclosion and was exaggerated with aging. Along with motor function, muscle ultrastructure in the tw mutant was altered, as seen in human patients. We demonstrated that expression of RNA interference (RNAi) for the rt gene and the tw mutant was almost completely lethal and semi-lethal, respectively. Flies expressing RNAi had reduced lifespans. These findings clearly demonstrate that Drosophila POMT mutants are models for human muscular dystrophy. We then observed a high density of myoblasts with an enhanced degree of apoptosis in the tw mutant, which completely lost enzymatic activity. In this paper, we propose a novel mechanism for the development of muscular dystrophy: POMT mutation causes high myoblast density and position derangement, which result in apoptosis, muscle disorganization, and muscle cell defects.

Show MeSH
Related in: MedlinePlus