Zebrafish Assays for Identifying Potential Muscular Dystrophy Drug Candidates - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 229

dystroglycan MO knockdown zebrafish (Parsons et al., 2002). Disruption of DGC in
KD zebrafish resulted in loss of sarcomere and sarcoplasmic organization, indicating
that dystroglycan is required to maintain long-term muscle cell survival (Parsons
et al., 2002). Inhibition of dystroglycan protein production in zebrafish has been
confirmed by dystroglycan antibody staining. Recently, overexpression of myostatin-
2, a member of the TGF-b superfamily and a potent negative regulator of skeletal
muscle and growth, was found to decrease dystrophin-associated protein complex
(DAPC) expression, resulting in muscle dystrophy (Amali et al., 2008).
RNA interference, which is primarily used for research applications, has received
increasing attention as a potential therapeutic strategy and both long ds (double
stranded) RNAs ( 100) and short (20-80 nucleotides, nt) ds RNAs have been assessed.

Long ds siRNAs have been shown to cause nonspecific effects and a short 21-nt ds
siRNA has also been used to knockdown the dystrophin gene in zebrafish (Dodd
et al., 2004). A recent research report (Zhao et al., 2008) sheds additional light on
reducing potential nonspecific siRNA effects and coinjection of preprocessed micro-
RNA-430 bases (b) can prevent nonspecific defects associated with siRNA injection.
siRNA injection transiently inhibits protein production until siRNA degrades. In our
zebrafish MD model, using whole mount dystroglycan antibody staining, we observed
elimination of dystroglycan protein

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20 h post fertilization (hpf). In general, siRNA
dystroglycan knockdown zebrafish phenotypes were similar to MO phenotypes in-
cluding developmental delay, truncated bodies, curved tails (data not shown), and
decreased and uncoordinated movement. These results are also consistent with data
reported by Parsons et al. (2002). In humans, DMD progression leads to a variety of
physical symptoms affecting spine, legs, feet, joints, and tendons. Symptoms can
include general muscle weakness, overdeveloped calves (pseudohypertrophy), in-
creased muscle volume due to fat deposits, lordosis and scoliosis, curvature of the
spine, joint and tendon restriction, speech and mental impairment, and respiratory
difficulties. In dystroglycan KD zebrafish, we observed several defects that resemble
human congenital myopathies, including rapid, progressive muscular degeneration,
immobility, muscle and brain deformities, bent spine (notochord), and cardiac defects.

18.1.5 MD Treatment Options

Previous studies have shown that three- to fourfold increase in utrophin expression in
mdx mouse muscle was sufficient to prevent or dramatically reduce muscular
dystrophy pathology (Tinsley et al., 1998). However, for the majority of DMD
patients, steroids, including prednisone, which reduces inflammation, are the only
available therapeutic option (Campbell and Jacob, 2003; Manzur et al., 2004, 2008;
Beenakker et al., 2005). Use of antibodies that block the action of myostatin
(Bogdanovich et al., 2002, 2005), a negative regulator of muscle mass, has resulted
in functional improvement of dystrophic muscle in mice. MYO-029, a neutralizing
antibody, has also been assessed in clinical trials for various forms of muscular
dystrophy. More recently, inhibition of histone deacetylases (HDACs) resulted in
upregulation of regeneration-activated genes and formation of hypernucleated, larger