(a-d) Phenotypic measures of SBMA degeneration (mean body weight
(a), mean rotarod activity (b), mean grip strength
(c), and survival (d) of NTG (black diamonds) and
AR121Q mice (red squares), in addition to sham-operated (green triangles) and
castrated (blue circles) AR121Q mice. n = 5 mice per group,
****P ≤ 0.0001,
***P =0.0007 by two-way
ANOVA and Dunnett’s posthoc test compared to the NTG group
(a-c) or Kaplan-Meier estimation (log-rank test)
(d). Note that the abrupt reduction in survival reflects animal
protocol criteria that mandate euthanasia of animals with loss of 10%
body weight or the development of substantial weakness. (e-f)
Representative spinal cord (e) and skeletal muscle (f)
sections from 7-week-old NTG and AR121Q mice from one independent experiment.
Sections were stained with hematoxylin and eosin (H&E) stain for
assessment of morphology, in addition to AR (N20) and ubiquitin antibodies.
White arrowheads indicate ubiquitin-positive nuclear inclusions in the spinal
cord. Dotted lines trace around representative myofibers, demonstrating
differences in myofiber size. Arrow indicates atrophied myofibers. Arrowheads
indicate myofibers containing centralized nuclei. (g) PolyQ
(5TF1-1C2) and ubiquitin costaining in spinal cord of NTG and AR121Q mice.
Representative images are shown from one independent experiment.
(h-j) Quantification of the mean muscle fiber diameter of
gastrocnemius/soleus myofibers (h), mean type I and type II
hindlimb muscle fiber staining (i), and ChAT-positive motor neuron
count and area in the anterior horn of the thoracic spinal cord (j)
of NTG and AR121Q mice. Data shown in h-j were evaluated by
unpaired t-test, two-tailed. n = 30 muscle fibers per mouse and 2 mice
per genotype, ****P ≤
0.0001 (h), n=2 and 3 mice for NTG and AR121Q (i), n = 3 slides
per mouse (neuron count), n = 6 sections per mouse (neuron area), 2 mice
per genotype were counted, ***P
=0.0005 (j). (k) Immunofluorescence with antibodies against
NCAM and PSA-NCAM in skeletal muscle tissues of NTG and AR121Q mice.
Representative images are shown from one independent experiment. Scale bars
represent 50 μm. All graphs represent mean ± s.e.m.