Presence of revertant fibers in hindlimb muscles of <i>P448Lneo-</i> mutant mouse.

<p>Tibialis anterior (TA), extensor digitorum longus (EDL), gastrocnemius (Gast.), and semitendinosus (Semiten.). Strong IIH6 staining shown in all muscle of C57, but almost absent in both 6 week and 6 month old P448Lneo-. Varying numbers of revertant fibers seen at the different ages of <i>P448Lneo-</i>. Images displayed at 20X magnification. Yellow bar (shown in 6 month EDL) represents 200 micron.</p

Distinct expression of functionally glycosylated alpha-dystroglycan in muscle and non-muscle tissues of FKRP mutant mice

<div><p>The glycosylation of alpha-dystroglycan (α-DG) is crucial in maintaining muscle cell membrane integrity. Dystroglycanopathies are identified by the loss of this glycosylation leading to a breakdown of muscle cell membrane integrity and eventual degeneration. However, a small portion of fibers expressing functionally glycosylated α-DG (F-α-DG) (revertant fibers, RF) have been identified. These fibers are generally small in size, centrally nucleated and linked to regenerating fibers. Examination of different muscles have shown various levels of RFs but it is unclear the extent of which they are present. Here we do a body-wide examination of muscles from the FKRP-P448L mutant mouse for the prevalence of RFs. We have identified great variation in the distribution of RF in different muscles and tissues. Triceps shows a large increase in RFs and together with centrally nucleated fibers whereas the pectoralis shows a reduction in revertant but increase in centrally nucleated fibers from 6 weeks to 6 months of age. We have also identified that the sciatic nerve with near normal levels of F-α-DG in the P448Lneo- mouse with reduced levels in the P448Lneo+ and absent in LARGE^myd. The salivary gland of LARGE^myd mice expresses high levels of F-α-DG. Interestingly the same glands in the P448Lneo-and to a lesser degree in P448Lneo+ also maintain considerable amount of F-α-DG, indicating the non-proliferating epithelial cells have a molecular setting permitting glycosylation.</p></div

Percentage of centranucleation (CN) and revertant fibers (RF) in 6 week (6WK) and 6 month (6MO) old <i>P448Lneo-</i> mice.

<p>Percentage of centranucleation (CN) and revertant fibers (RF) in 6 week (6WK) and 6 month (6MO) old <i>P448Lneo-</i> mice.</p

Increase in fiber size of hindlimb muscles of P448Lneo- mutant mouse.

<p>A clear increase in fiber size of the TA, EDL and gastrocnemius is seen from 6 weeks to 6 months of age with only a slight increase in the semitendinosus. However, a larger population of small fibers, <20 micron diameter, is seen in the EDL and gastrocnemius. Significant difference between 6 weeks and 6 months noted with *. Significance determined as p<0.05.</p

Western blot detection of functionally glycosylated α-DG in cecum.

<p>A. Although lower than C57 control tissue, the expression of IIH6 is clearly visible in the P448Lneo- and P448Lneo+ as well as LARGE^myd. However, these levels appear to be higher in the LARGE^myd compared to both P448L mutant mice with lower expression in the P448Lneo+. B. Functional binding of the α-DG to laminin is demonstrated in the cecum of all animals although in reduced levels in the mutant mice compared to C57 controls.</p

Quantitative real time-PCR of salivary gland.

<p><i>FKRP</i> expression levels are normal in LARGE^myd compared to C57 controls while significantly reduced levels are seen in the P448Lneo- and almost absent in the P448Lneo+. <i>LARGE</i> expression levels are high in the C57 as well as both P448Lneo- and neo+, although a decrease is noted in the P448Lneo-. However, as expected, LARGE expression levels are absent in the LARGE^myd mouse. <i>LARGE-2</i> expression shows near normal levels for all three mutant mouse models with no significant difference for any. Significant differences are noted with the same letter as determined by t-test with a p<0.05.</p

F-α-DG in the salivary gland.

<p>Strong IIH6 staining is seen around the striated ducts and acini of C57, P448L- (both 6 weeks and 6 months of age) as well as the LARGE^myd. However, detectable IIH6 staining is seen around the striated ducts of P448L+ and not acini. Images are 20X magnification.</p

Western blot detection of functionally glycosylated α-DG in sciatic nerve.

<p>A. Strong expression is seen in both the 6 week and 6 month old P448Lneo- mouse comparable to C57 control tissue. A decreased expression is seen in the Neo+ and absence in the LARGE^myd. B. Laminin overlay demonstrated functional binding of α-DG to laminin in the sciatic nerve. There is minimal binding in the P448Lneo+ with absence in the LARGE^myd.</p

Fiber size of forelimb, trunk and head muscles of P448Lneo- mutant mouse.

<p>An increase in muscle fiber diameter is clear in both triceps and biceps with little to no increase in smaller diameter fibers. However, pectoralis, deltoid and masseter show an increase in fibers with small diameter, <20 micron. Significant difference between 6 weeks and 6 months noted with *. Significance determined as p<0.05.</p

Percentage of centranucleation (CN) and revertant fibers (RF) in 6 week (6WK) and 6 month (6MO) old <i>P448Lneo-</i> mice.

<p>Percentage of centranucleation (CN) and revertant fibers (RF) in 6 week (6WK) and 6 month (6MO) old <i>P448Lneo-</i> mice.</p