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Zebrafish models for nemaline myopathy reveal a spectrum of nemaline bodies contributing to reduced muscle function.

Sztal TE, Zhao M, Williams C, Oorschot V, Parslow AC, Giousoh A, Yuen M, Hall TE, Costin A, Ramm G, Bird PI, Busch-Nentwich EM, Stemple DL, Currie PD, Cooper ST, Laing NG, Nowak KJ, Bryson-Richardson RJ - Acta Neuropathol. (2015)

Bottom Line: Another subtype results from a reduction of actin and forms a more stable cytoplasmic body.In contrast, the final type originates at the Z-disk and is associated with myofibrillar disorganization.In addition, we show that the ACTA1(D286G) mutation causes impaired actin incorporation and localization in the sarcomere.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Monash University, Melbourne, VIC, Australia.

ABSTRACT
Nemaline myopathy is characterized by muscle weakness and the presence of rod-like (nemaline) bodies. The genetic etiology of nemaline myopathy is becoming increasingly understood with mutations in ten genes now known to cause the disease. Despite this, the mechanism by which skeletal muscle weakness occurs remains elusive, with previous studies showing no correlation between the frequency of nemaline bodies and disease severity. To investigate the formation of nemaline bodies and their role in pathogenesis, we generated overexpression and loss-of-function zebrafish models for skeletal muscle α-actin (ACTA1) and nebulin (NEB). We identify three distinct types of nemaline bodies and visualize their formation in vivo, demonstrating these nemaline bodies not only exhibit different subcellular origins, but also have distinct pathological consequences within the skeletal muscle. One subtype is highly dynamic and upon breakdown leads to the accumulation of cytoplasmic actin contributing to muscle weakness. Examination of a Neb-deficient model suggests this mechanism may be common in nemaline myopathy. Another subtype results from a reduction of actin and forms a more stable cytoplasmic body. In contrast, the final type originates at the Z-disk and is associated with myofibrillar disorganization. Analysis of zebrafish and muscle biopsies from ACTA1 nemaline myopathy patients demonstrates that nemaline bodies also possess a different protein signature. In addition, we show that the ACTA1(D286G) mutation causes impaired actin incorporation and localization in the sarcomere. Together these data provide a novel examination of nemaline body origins and dynamics in vivo and identifies pathological changes that correlate with muscle weakness.

No MeSH data available.


Related in: MedlinePlus

a Maximum projection confocal microscopy images of Tg(ACTA1wildtype-eGFP)low and Tg(ACTA1wildtype-eGFP)high zebrafish strains injected with two different Nebulin (Neb) morpholinos compared to control uninjected embryos at 2 dpf. There is an increased prevalence of eGFP-positive globular aggregates at the myosepta (arrowheads) in Tg(ACTA1wildtype-eGFP)high Neb morphants compared to controls. Knockdown of Neb produces globular aggregates at the myosepta (arrowheads) in Tg(ACTA1wildtype-eGFP)low Neb morphants that are absent in control uninjected embryos. b Maximum projection confocal microscopy images of wild-type embryos injected with Neb morpholinos at 2 dpf and stained with phalloidin shows an increase in actin-positive aggregates at the myosepta (arrowheads), which are absent in control uninjected embryos
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Fig8: a Maximum projection confocal microscopy images of Tg(ACTA1wildtype-eGFP)low and Tg(ACTA1wildtype-eGFP)high zebrafish strains injected with two different Nebulin (Neb) morpholinos compared to control uninjected embryos at 2 dpf. There is an increased prevalence of eGFP-positive globular aggregates at the myosepta (arrowheads) in Tg(ACTA1wildtype-eGFP)high Neb morphants compared to controls. Knockdown of Neb produces globular aggregates at the myosepta (arrowheads) in Tg(ACTA1wildtype-eGFP)low Neb morphants that are absent in control uninjected embryos. b Maximum projection confocal microscopy images of wild-type embryos injected with Neb morpholinos at 2 dpf and stained with phalloidin shows an increase in actin-positive aggregates at the myosepta (arrowheads), which are absent in control uninjected embryos

Mentions: We next examined a model of nemaline myopathy resulting from a loss of Neb to determine whether actin aggregates were a common feature associated with nemaline myopathy. We injected two neb splice-site-targeting morpholinos (both singularly and in combination) into wild-type zebrafish. The efficacy of the morpholinos was confirmed by qRT-PCR showing that neb mRNA levels are reduced by approximately 80 % by each morpholino and approximately 90 % when both morpholinos are used in combination (Fig. 7b, c). The effect on Neb was further confirmed by examining sarcomere length, with Neb morphants (Fig. 8a) displaying significantly shorter sarcomere lengths compared to controls (Fig. 7d, e).Fig. 7


Zebrafish models for nemaline myopathy reveal a spectrum of nemaline bodies contributing to reduced muscle function.

Sztal TE, Zhao M, Williams C, Oorschot V, Parslow AC, Giousoh A, Yuen M, Hall TE, Costin A, Ramm G, Bird PI, Busch-Nentwich EM, Stemple DL, Currie PD, Cooper ST, Laing NG, Nowak KJ, Bryson-Richardson RJ - Acta Neuropathol. (2015)

a Maximum projection confocal microscopy images of Tg(ACTA1wildtype-eGFP)low and Tg(ACTA1wildtype-eGFP)high zebrafish strains injected with two different Nebulin (Neb) morpholinos compared to control uninjected embryos at 2 dpf. There is an increased prevalence of eGFP-positive globular aggregates at the myosepta (arrowheads) in Tg(ACTA1wildtype-eGFP)high Neb morphants compared to controls. Knockdown of Neb produces globular aggregates at the myosepta (arrowheads) in Tg(ACTA1wildtype-eGFP)low Neb morphants that are absent in control uninjected embryos. b Maximum projection confocal microscopy images of wild-type embryos injected with Neb morpholinos at 2 dpf and stained with phalloidin shows an increase in actin-positive aggregates at the myosepta (arrowheads), which are absent in control uninjected embryos
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig8: a Maximum projection confocal microscopy images of Tg(ACTA1wildtype-eGFP)low and Tg(ACTA1wildtype-eGFP)high zebrafish strains injected with two different Nebulin (Neb) morpholinos compared to control uninjected embryos at 2 dpf. There is an increased prevalence of eGFP-positive globular aggregates at the myosepta (arrowheads) in Tg(ACTA1wildtype-eGFP)high Neb morphants compared to controls. Knockdown of Neb produces globular aggregates at the myosepta (arrowheads) in Tg(ACTA1wildtype-eGFP)low Neb morphants that are absent in control uninjected embryos. b Maximum projection confocal microscopy images of wild-type embryos injected with Neb morpholinos at 2 dpf and stained with phalloidin shows an increase in actin-positive aggregates at the myosepta (arrowheads), which are absent in control uninjected embryos
Mentions: We next examined a model of nemaline myopathy resulting from a loss of Neb to determine whether actin aggregates were a common feature associated with nemaline myopathy. We injected two neb splice-site-targeting morpholinos (both singularly and in combination) into wild-type zebrafish. The efficacy of the morpholinos was confirmed by qRT-PCR showing that neb mRNA levels are reduced by approximately 80 % by each morpholino and approximately 90 % when both morpholinos are used in combination (Fig. 7b, c). The effect on Neb was further confirmed by examining sarcomere length, with Neb morphants (Fig. 8a) displaying significantly shorter sarcomere lengths compared to controls (Fig. 7d, e).Fig. 7

Bottom Line: Another subtype results from a reduction of actin and forms a more stable cytoplasmic body.In contrast, the final type originates at the Z-disk and is associated with myofibrillar disorganization.In addition, we show that the ACTA1(D286G) mutation causes impaired actin incorporation and localization in the sarcomere.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Monash University, Melbourne, VIC, Australia.

ABSTRACT
Nemaline myopathy is characterized by muscle weakness and the presence of rod-like (nemaline) bodies. The genetic etiology of nemaline myopathy is becoming increasingly understood with mutations in ten genes now known to cause the disease. Despite this, the mechanism by which skeletal muscle weakness occurs remains elusive, with previous studies showing no correlation between the frequency of nemaline bodies and disease severity. To investigate the formation of nemaline bodies and their role in pathogenesis, we generated overexpression and loss-of-function zebrafish models for skeletal muscle α-actin (ACTA1) and nebulin (NEB). We identify three distinct types of nemaline bodies and visualize their formation in vivo, demonstrating these nemaline bodies not only exhibit different subcellular origins, but also have distinct pathological consequences within the skeletal muscle. One subtype is highly dynamic and upon breakdown leads to the accumulation of cytoplasmic actin contributing to muscle weakness. Examination of a Neb-deficient model suggests this mechanism may be common in nemaline myopathy. Another subtype results from a reduction of actin and forms a more stable cytoplasmic body. In contrast, the final type originates at the Z-disk and is associated with myofibrillar disorganization. Analysis of zebrafish and muscle biopsies from ACTA1 nemaline myopathy patients demonstrates that nemaline bodies also possess a different protein signature. In addition, we show that the ACTA1(D286G) mutation causes impaired actin incorporation and localization in the sarcomere. Together these data provide a novel examination of nemaline body origins and dynamics in vivo and identifies pathological changes that correlate with muscle weakness.

No MeSH data available.


Related in: MedlinePlus