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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

Characterization of nemaline bodies in patient muscle biopsies. In ACTA1T66I muscle, nemaline bodies (white arrowheads), darkly stained by Gomori trichome, are positive for actinin2 and phalloidin (labeling F-actin). In ACTA1I136M muscle, nemaline bodies are phalloidin positive and actinin2 positive (yellow arrowheads) or actinin2 negative (gray arrowheads). Control (unaffected) muscle shows normal localization of markers in the absence of nemaline bodies
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Fig6: Characterization of nemaline bodies in patient muscle biopsies. In ACTA1T66I muscle, nemaline bodies (white arrowheads), darkly stained by Gomori trichome, are positive for actinin2 and phalloidin (labeling F-actin). In ACTA1I136M muscle, nemaline bodies are phalloidin positive and actinin2 positive (yellow arrowheads) or actinin2 negative (gray arrowheads). Control (unaffected) muscle shows normal localization of markers in the absence of nemaline bodies

Mentions: To determine if the different aggregates could be distinguished immunologically, we performed antibody and phalloidin (labeling F-actin) staining on zebrafish muscle expressing ACTA1D286G-eGFP. Although nemaline bodies could be detected at 2 dpf using an antibody against eGFP, they were not detected by Actinin2 or Actinin3 antibodies, nor by phalloidin (Fig. 5a, Supplementary Figure S2). In contrast, the globular aggregates at 4 dpf were positive for Actinin2, Actinin3 and phalloidin (Fig. 5a, Supplementary Figure S2). We also co-expressed the cardiac α-actin cDNA fused with mCherry (actc1a-mCherry) together with ACTA1D286G-eGFP in the skeletal muscle and showed that both types of aggregates incorporated cardiac actin (Supplementary Figure S2). To determine if similar α-actinin-negative nemaline bodies were present in patients with mutations in ACTA1, we tested patient skeletal muscle biopsies. As observed in Tg(ACTA1D286G-eGFP)high zebrafish, we found that nemaline bodies can exhibit different protein signatures. In a biopsy from a patient with a T66I mutation in ACTA1 [18], nemaline bodies darkly stained by Gomori trichome are both phalloidin and actinin2 positive (Fig. 6). Whereas for a patient with an I136M mutation in ACTA1 [18], nemaline bodies darkly stained by Gomori trichome are phalloidin positive and either actinin2 positive or negative (Fig. 6).Fig. 5


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)

Characterization of nemaline bodies in patient muscle biopsies. In ACTA1T66I muscle, nemaline bodies (white arrowheads), darkly stained by Gomori trichome, are positive for actinin2 and phalloidin (labeling F-actin). In ACTA1I136M muscle, nemaline bodies are phalloidin positive and actinin2 positive (yellow arrowheads) or actinin2 negative (gray arrowheads). Control (unaffected) muscle shows normal localization of markers in the absence of nemaline bodies
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4541704&req=5

Fig6: Characterization of nemaline bodies in patient muscle biopsies. In ACTA1T66I muscle, nemaline bodies (white arrowheads), darkly stained by Gomori trichome, are positive for actinin2 and phalloidin (labeling F-actin). In ACTA1I136M muscle, nemaline bodies are phalloidin positive and actinin2 positive (yellow arrowheads) or actinin2 negative (gray arrowheads). Control (unaffected) muscle shows normal localization of markers in the absence of nemaline bodies
Mentions: To determine if the different aggregates could be distinguished immunologically, we performed antibody and phalloidin (labeling F-actin) staining on zebrafish muscle expressing ACTA1D286G-eGFP. Although nemaline bodies could be detected at 2 dpf using an antibody against eGFP, they were not detected by Actinin2 or Actinin3 antibodies, nor by phalloidin (Fig. 5a, Supplementary Figure S2). In contrast, the globular aggregates at 4 dpf were positive for Actinin2, Actinin3 and phalloidin (Fig. 5a, Supplementary Figure S2). We also co-expressed the cardiac α-actin cDNA fused with mCherry (actc1a-mCherry) together with ACTA1D286G-eGFP in the skeletal muscle and showed that both types of aggregates incorporated cardiac actin (Supplementary Figure S2). To determine if similar α-actinin-negative nemaline bodies were present in patients with mutations in ACTA1, we tested patient skeletal muscle biopsies. As observed in Tg(ACTA1D286G-eGFP)high zebrafish, we found that nemaline bodies can exhibit different protein signatures. In a biopsy from a patient with a T66I mutation in ACTA1 [18], nemaline bodies darkly stained by Gomori trichome are both phalloidin and actinin2 positive (Fig. 6). Whereas for a patient with an I136M mutation in ACTA1 [18], nemaline bodies darkly stained by Gomori trichome are phalloidin positive and either actinin2 positive or negative (Fig. 6).Fig. 5

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