Fiber type distribution of <i>m. soleus</i> of NT-1654 and vehicle treated SARCO mice.

<p><b>A:</b> Consecutive muscle cross sections of <i>soleus</i> stained with myosine heavy chain (MHC) specific antibodies or cytochrome C oxidase (COX) staining as indicated. Control and treated SARCO mice show clearly separated type I and II fibers. SARCO mice have a significantly increased amount of hybrid fibers (indicated with asterisks). Cytochrome C staining of <i>soleus</i> sections shows a massive reduction of reactivity in SARCO mice which is reverted back to WT levels in treated SARCO mice. <b>B:</b> Quantitative analysis of muscle fibers of <i>soleus.</i> Bars represent mean values ± standard deviation. Control and treated SARCO mice have almost no hybrid fibers and are indistinguishable from each other. Compared to treated ones, SARCO mice have significantly increased hybrid fibers. Type I fibers are significantly decreased. The total fiber number in SARCO mice is also significantly decreased compared to treated animals. Number of animals: wt  =  3; SARCO treated  =  5, SARCO  =  3. * p<0.05; ** p<0.01. Scale bar: 100 µm.</p

NMJ morphology of <i>m. soleus</i> of NT-1654 and vehicle treated SARCO mice.

<p><b>A:</b> Confocal images of the NMJs in <i>soleus</i> muscle of P30 Control or SARCO mice, treated or not treated with NT-1654 as indicated. The postsynaptic AChRs were stained with Alexa-555 conjugate α-bungarotoxin (red) and the presynapse was stained with anti-neurofilament and synaptophysin antibodies (NF-Syn; green). The postsynapses were highly fragmented, partially or completely lost in <i>soleus</i> muscle of SARCO mice. The postsynapses of NT-1654 treated SARCO mice resembled those in Control mice, showing pretzel like structures and much less fragmentation. Scale bar: 50 µm. <b>B:</b> The postsynapses in Control mice showed fragmentation class 0 (no fragmentation, for illustration see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088739#pone.0088739.s003" target="_blank">figure S3</a>). The postsynapses of NT-1654 treated SARCO mice were mostly classified into class 1, whereas the postsynapse of SARCO mice showed class 1-4 with a peak at class 3. <b>C:</b> Many NMJs of SARCO mice have terminal nerve sprouting, which was dramatically reduced in the NT1654 treated SARCO mice. Data present mean ± standard deviation, n  =  2 mice, 100 NMJs were counted in each mouse.</p

NMJ recovery after sciatic nerve crush.

<p><b>A:</b> Confocal images of the neuromuscular junctions (NMJs) in <i>soleus</i> muscle of Thy1-YFP mice after 14 days sciatic nerve crushing, treated with NT-1654 or PBS (Control) as indicated. The postsynaptic AChR was stained with Alexa-555 conjugate α-bungarotoxin (red) and the presynapse was visualized by the transgenic expression of YFP in motor neurons (green). The NMJs in <i>soleus</i> muscle are fully re-innervated after 14 days sciatic nerve crushing. The NMJs of NT-1654 treated mice showed significantly less nerve sprouting than those treated with PBS (Control). The number of nerve sprouting in each NMJ was counted and shown in <b>B.</b> NMJs in the NT-1654 treated mice have significantly fewer events of nerve sprouting and significantly fewer number of nerve sprouts than those of Control mice. Data present mean ± standard error, *: p<0.05 (two ways t-test), 100 NMJs were counted in each mouse, n  =  3 mice, scale bar: 50 µm.</p