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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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Lifespans of flies expressing RNAi for the rt gene.The lifespan of whole-body knockdown flies using Act5C-Gal4 (A), that of neuron-specific knockdown flies using elav-Gal4 (B), and that of glial cell-specific knockdown flies using repo-Gal4 (C). The results of statistical analyses in (A), (B), and (C) are shown in Table 9. The median lifespan for each genotype is shown in Table 10. Flies with ubiquitous expression of RNAi for the rt gene driven by Act5C-Gal4 (Act5C-Gal4/rt-IR) had a shorter lifespan than that of the UAS-rt-IR/+ and Act5C-Gal4/+ control groups (both p<0.001, log-rank test, Table 9). The median lifespan of Act5C-Gal4/rt-IR flies was 23 days, which was −51.1% of that of UAS-rt-IR/+ flies (47 days) and −66.7% of that of Act5C-Gal4/+ flies (69 days) (Tables 9 and 10). On the other hand, the lifespan of [elav-Gal4/+; UAS-rt-IR/+] flies and [UAS-rt-IR/+; repo-Gal4/+] flies was longer than that of [UAS-rt-IR/+; +/+] flies, one control group, and was shorter than that of [elav-Gal4/+; +/+] flies and [+/+; repo-Gal4/+] flies, the other control groups. The median lifespans of [elav-Gal4/+; UAS-rt-IR/+] flies and [UAS-rt-IR/+; repo-Gal4/+] flies were 57 and 59 days, respectively (Table 10). Both lifespans were longer than that of [UAS-rt-IR/+; +/+] flies and shorter than that of [elav-Gal4/+; +/+] flies and [+/+; repo-Gal4/+] flies (Table 9).
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pone-0011557-g011: Lifespans of flies expressing RNAi for the rt gene.The lifespan of whole-body knockdown flies using Act5C-Gal4 (A), that of neuron-specific knockdown flies using elav-Gal4 (B), and that of glial cell-specific knockdown flies using repo-Gal4 (C). The results of statistical analyses in (A), (B), and (C) are shown in Table 9. The median lifespan for each genotype is shown in Table 10. Flies with ubiquitous expression of RNAi for the rt gene driven by Act5C-Gal4 (Act5C-Gal4/rt-IR) had a shorter lifespan than that of the UAS-rt-IR/+ and Act5C-Gal4/+ control groups (both p<0.001, log-rank test, Table 9). The median lifespan of Act5C-Gal4/rt-IR flies was 23 days, which was −51.1% of that of UAS-rt-IR/+ flies (47 days) and −66.7% of that of Act5C-Gal4/+ flies (69 days) (Tables 9 and 10). On the other hand, the lifespan of [elav-Gal4/+; UAS-rt-IR/+] flies and [UAS-rt-IR/+; repo-Gal4/+] flies was longer than that of [UAS-rt-IR/+; +/+] flies, one control group, and was shorter than that of [elav-Gal4/+; +/+] flies and [+/+; repo-Gal4/+] flies, the other control groups. The median lifespans of [elav-Gal4/+; UAS-rt-IR/+] flies and [UAS-rt-IR/+; repo-Gal4/+] flies were 57 and 59 days, respectively (Table 10). Both lifespans were longer than that of [UAS-rt-IR/+; +/+] flies and shorter than that of [elav-Gal4/+; +/+] flies and [+/+; repo-Gal4/+] flies (Table 9).

Mentions: Patients with WWS rarely survive to adulthood [10]. Therefore, we investigated whether rt knockdown mutants had shortened lifespans. Flies with ubiquitous expression of RNAi for the rt gene driven by Act5C-Gal4 had shorter lifespans than those of the control groups (both p<0.001, log-rank test, Table 9) (Fig. 11A). The median lifespan of Act5C-Gal4/rt-IR flies was 23 days, which was −51.1% of that of UAS-rt-IR/+ flies (47 days) and −66.7% of that of Act5C-Gal4/+ flies (69 days) (Tables 9 and 10). On the other hand, the lifespans of flies expressing RNAi for the rt gene in neurons and glial cells driven by elav-Gal4 and repo-Gal4, respectively, were not affected (Figs. 11B and C, Tables 9 and 10). These results indicated that knockdown of the rt gene in all tissues reduced the lifespan, while knockdown in neurons or glial cells did not influence lifespan. Together with the results of the muscle phenotype in Drosophila POMT mutants, these results suggest that age-related weakness of the muscles in the heart and/or gastrointestinal tract may lead to early death in flies.


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)

Lifespans of flies expressing RNAi for the rt gene.The lifespan of whole-body knockdown flies using Act5C-Gal4 (A), that of neuron-specific knockdown flies using elav-Gal4 (B), and that of glial cell-specific knockdown flies using repo-Gal4 (C). The results of statistical analyses in (A), (B), and (C) are shown in Table 9. The median lifespan for each genotype is shown in Table 10. Flies with ubiquitous expression of RNAi for the rt gene driven by Act5C-Gal4 (Act5C-Gal4/rt-IR) had a shorter lifespan than that of the UAS-rt-IR/+ and Act5C-Gal4/+ control groups (both p<0.001, log-rank test, Table 9). The median lifespan of Act5C-Gal4/rt-IR flies was 23 days, which was −51.1% of that of UAS-rt-IR/+ flies (47 days) and −66.7% of that of Act5C-Gal4/+ flies (69 days) (Tables 9 and 10). On the other hand, the lifespan of [elav-Gal4/+; UAS-rt-IR/+] flies and [UAS-rt-IR/+; repo-Gal4/+] flies was longer than that of [UAS-rt-IR/+; +/+] flies, one control group, and was shorter than that of [elav-Gal4/+; +/+] flies and [+/+; repo-Gal4/+] flies, the other control groups. The median lifespans of [elav-Gal4/+; UAS-rt-IR/+] flies and [UAS-rt-IR/+; repo-Gal4/+] flies were 57 and 59 days, respectively (Table 10). Both lifespans were longer than that of [UAS-rt-IR/+; +/+] flies and shorter than that of [elav-Gal4/+; +/+] flies and [+/+; repo-Gal4/+] flies (Table 9).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2903483&req=5

pone-0011557-g011: Lifespans of flies expressing RNAi for the rt gene.The lifespan of whole-body knockdown flies using Act5C-Gal4 (A), that of neuron-specific knockdown flies using elav-Gal4 (B), and that of glial cell-specific knockdown flies using repo-Gal4 (C). The results of statistical analyses in (A), (B), and (C) are shown in Table 9. The median lifespan for each genotype is shown in Table 10. Flies with ubiquitous expression of RNAi for the rt gene driven by Act5C-Gal4 (Act5C-Gal4/rt-IR) had a shorter lifespan than that of the UAS-rt-IR/+ and Act5C-Gal4/+ control groups (both p<0.001, log-rank test, Table 9). The median lifespan of Act5C-Gal4/rt-IR flies was 23 days, which was −51.1% of that of UAS-rt-IR/+ flies (47 days) and −66.7% of that of Act5C-Gal4/+ flies (69 days) (Tables 9 and 10). On the other hand, the lifespan of [elav-Gal4/+; UAS-rt-IR/+] flies and [UAS-rt-IR/+; repo-Gal4/+] flies was longer than that of [UAS-rt-IR/+; +/+] flies, one control group, and was shorter than that of [elav-Gal4/+; +/+] flies and [+/+; repo-Gal4/+] flies, the other control groups. The median lifespans of [elav-Gal4/+; UAS-rt-IR/+] flies and [UAS-rt-IR/+; repo-Gal4/+] flies were 57 and 59 days, respectively (Table 10). Both lifespans were longer than that of [UAS-rt-IR/+; +/+] flies and shorter than that of [elav-Gal4/+; +/+] flies and [+/+; repo-Gal4/+] flies (Table 9).
Mentions: Patients with WWS rarely survive to adulthood [10]. Therefore, we investigated whether rt knockdown mutants had shortened lifespans. Flies with ubiquitous expression of RNAi for the rt gene driven by Act5C-Gal4 had shorter lifespans than those of the control groups (both p<0.001, log-rank test, Table 9) (Fig. 11A). The median lifespan of Act5C-Gal4/rt-IR flies was 23 days, which was −51.1% of that of UAS-rt-IR/+ flies (47 days) and −66.7% of that of Act5C-Gal4/+ flies (69 days) (Tables 9 and 10). On the other hand, the lifespans of flies expressing RNAi for the rt gene in neurons and glial cells driven by elav-Gal4 and repo-Gal4, respectively, were not affected (Figs. 11B and C, Tables 9 and 10). These results indicated that knockdown of the rt gene in all tissues reduced the lifespan, while knockdown in neurons or glial cells did not influence lifespan. Together with the results of the muscle phenotype in Drosophila POMT mutants, these results suggest that age-related weakness of the muscles in the heart and/or gastrointestinal tract may lead to early death in flies.

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