Cdk5 activity in triceps surae samples from G93A hSOD1 and Non-Tg littermates were examined at three different ages: 27 days, 47 days, and 67 days. A statistically significant age-dependent change in cdk5 activity was observed in the muscle samples from Non-Tg littermates (One-way ANOVA, F_2,7=6.65, p=0.02). Multiple comparison post-hoc analysis showed higher cdk5 activity in 27-day-old and 47-day-old mice compared to 67-day-old mice (27 days vs. 67 days [a vs. c], p=0.03; 47 days vs. 67 days [b vs. c], p=0.009, least square difference test). Such transient change in cdk5 activity was not detected in the muscle samples from G93A hSOD1 at the ages tested (F_2,7=0.454, p=0.65). The values in graphs are mean±SD.

Cdk5 interaction with either mutant G93A or wildtype (WT) hSOD1 was examined in supernatants from muscle and spinal cord lysate samples from 27-day-old G93A and WT hSOD1 transgenic mice. (A) More G93A hSOD1, compared to WT hSOD1, co-immunoprecipitated with cdk5 in the muscle lysates (two-tailed Student’s t test; p=0.05). No increase in the interaction between mutant G93A hSOD1 and cdk5 was observed in spinal cord samples. (B) The greater interaction between mutant G93A hSOD1 and cdk5 is not a consequence of higher protein expression. In contrast, the hindlimb muscle lysates from WT hSOD1 mice showed higher hSOD1 levels than those from G93A hSOD1 mice, suggesting higher expression level for WT hSOD1. (C) Examination of cdk5 activity in the same samples corresponding to (A) showed decreased cdk5 activity in G93A hSOD1 muscle samples compared to WT hSOD1 muscle samples (two-tailed Student’s t-test; p=0.001). Again, no difference in the cdk5 activities was observed between the spinal cord samples from G93A hSOD1 and WT hSOD1 mice. (D) To determine whether the presence of mutant G93A hSOD1 is responsible for the reduced cdk5 activity, immunoprecipitates of either mutant G93A or wildtype hSOD1 was added to the supernatants of Non-Tg mouse muscle lysates prior to the immunopreciptation kinase assay. Immunoprecipitation kinase assay following the incubation with mutant G93A hSOD1 immunoisolated from brain resulted in a concentration dependent reduction of cdk5 activity (0 μg, n=6; 0.1 μg, n=1; 0.5 μg, n=2; 1 μg, n=2; 3 μg, n=3). Addition of 3 μg of WT hSOD1 (n=3) immunoisolated from brain to the supernatants of Non-Tg mouse muscle lysates did not result in a reduction of the cdk5 activity (One-way ANOVA, F_5,11=6.06, p=0.006, post hoc multiple comparison test, * p<0.05). The values in graphs are mean±SD.

In vitro analysis of muscle samples revealed a mutant hSOD1 interaction-mediated reduction in cdk5 activity. Cdk5 and mutant hSOD1 localization were examined in muscle sections to determine whether the observed interactions are detectable in situ. (A) Nuclear staining of cdk5 is more widespread than that of mutant hSOD1. Only a subpopulation of cdk5 positive nuclei showed mutant hSOD1 immunostaining. Triple immunofluorescence shows distinct co-localization of cdk5 and mutant hSOD1 in the myonuclei. (B) Muscle fibers in G93A hSOD1 mice show aberrant nuclear patterning compared to those from Non-Tg mice. Nuclear patterning in the G93A hSOD1 mice muscle was clustered and disorganized in appearance compared to non-transgenic mice as shown by DAPI staining. Scale bar = 50 μm.

(A) Two-way ANOVA analysis of hindlimb muscle samples from 27- and 47-day-old G93A hSOD1 and Non-Tg mice showed significant reduction in cdk5 activity in samples from G93A hSOD1 mice compared to age-matched samples from Non-Tg mice (F_1,10=10.11, p=0.009). Multiple comparison post hoc analysis showed statistically significant reduction in the cdk5 activity in samples from G93A hSOD1 mice compared to Non-Tg mice at both 27 and 47 days of age (27-day-old animal comparison: G93A hSOD1 vs. Non-Tg: p=0.04; 47-day-old animal comparison: G93A hSOD1 vs. Non-Tg; p=0.04; least-significant-difference test). Cdk5 activity in samples from 27-day-old G93A hSOD1 mice also showed significant decrease compared to samples from 47-day-old Non-Tg mice (p=0.006; least significant difference test). In contrast, no deficits were observed in cdk5 (B) and p35 (C) protein levels. The values in graphs are mean±SD.

Protein levels of a myogenic marker and various cell cycle regulators were examined in the muscle samples of 27-day-old G93A hSOD1 and non-transgenic (Non-Tg) mice. Protein levels for (A) MyoD and (B) cyclin D1, a G1 phase cell-cycle regulator, were significantly lower in the muscle samples from G93A hSOD1 compared to those from Non-Tg mice (two-tailed Student’s t-test, p=0.001 and p=0.01, respectively). We did not detect any differences in the protein levels for (C) cdk4, another G1 phase cell-cycle marker, (D) PCNA, an S phase cell-cycle regulator and (E) cyclin B1, a G2/M phase cell-cycle regulator, in the muscle samples between the G93A hSOD1 and Non-Tg mice. The values in graphs are mean±SD.