Transcript Flutamide Rescues Transgenic Male Mice from the Toxic Effects of Over-expressing Wildtype Androgen Receptors in Skeletal Muscle Fibers Jamie A.

Flutamide Rescues Transgenic Male Mice from the Toxic Effects of Over-expressing Wildtype Androgen Receptors in Skeletal Muscle Fibers
Jamie A. Johansen 1, S. Marc Breedlove 1,2, Cynthia L. Jordan 1,2
1Neuroscience Program, 2Department of Psychology, Michigan State University
[email protected]
• Spinal Bulbar Muscular Atrophy (SBMA) is a heritable X-linked,
slowly progressive lower motor neuron disease characterized by
adult onset of muscle weakness, atrophy, and loss of motoneurons.
Prenatal flutamide rescues transgenic (tg)
males from death.
Percent alive at weaning
35
• Unexpectedly, tg male mice as adults show a disease phenotype
typical of SBMA in humans.
30
• Tg males weighed less, and developed signs of SBMA as they
reached adulthood.
25
20
15
• This included kyphosis, and a profound loss in muscle strength
indicated by the hang test. Loss of muscle strength is an early
indicator of the disease.
10
5
TG
untreated
FLUT WT
FLUT TG
Figure 1. Prenatal flutamide treatment in tg male mice was successful in
rescuing males from death. Flutamide treated tg males (FLUT TG) had a
higher rate of survival at weaning compared to untreated tg male mice.
Flutamide treated tg males are feminized, but do
develop symptoms of SBMA.
AGD
3.5
30
3
2.5
2
1.5
1
140
10
.5
5
0
0
• Histology: Extensor Digitorum Longus (EDL) muscles were
dissected from transgenic and wildtype males, cryostat sectioned at
10um, and stained for hematoxylin and eosin (H&E). Ventral roots
were embedded in plastic and 1µm cross-sections were stained
with toluidine blue.
Fiber Number
1800
600
400
200
0
FLUT WT
n=5
FLUT TG
n=6
1200
1000
800
600
400
200
FLUT WT
n=5
FLUT TG
n=6
Figure 5. Left) FLUT tg mice show a significant reduction in the number of
muscle fibers in the extensor digitorum longus (EDL) muscle compared to
FLUT wt controls (p=.014). Right) No change in average fiber size was
detected.
1000
900
800
700
600
500
400
300
200
100
0
Significance
0
FLUT WT
n=5
FLUT TG
n=6
FLUT TG
n=6
• Tg males did not show a reduction in the number of L5 ventral
root axons, also suggesting that the disease has not progressed to
late-stage SBMA where motoneurons begin to die.
2
1
FLUT WT
n=5
Axon Number
• Tg males showed a reduction in the number of EDL muscle
fibers, but no change in average fiber size.
3
FLUT TG
n=6
No loss in the number of size of ventral root
axons is observed.
Fiber Size
• However, stride length was not decreased suggesting that the
disease in flutamide treated males had not progressed to latestage SBMA.
• No change in average axon size was seen.
4
Figure 4. Left) FLUT tg males show a profound loss of motor function when
tested with the Hang Test compared to FLUT wt brothers (p=.0003). This is
similar to untreated tg males. Right) FLUT tg males do not show a loss in
stride length, suggesting that the disease has not progressed as far in these
males.
1400
0
*
FLUT WT
n=5
Number of Axons
Number of Fibers
*
800
60
0
FLUT TG
n=6
1600
1000
Average Fiber Area (um2)
1200
80
20
FLUT WT
n=5
Stride Length
5
40
Flutamide treated tg males have fewer muscle fibers,
but no change in fiber size.
• Prenatal Flutamide: 5 mg flutamide/day on gestational days 1520 was given to pregnant dams.
6
100
20
15
Hang Test
120
*
Figure 3. Left) FLUT tg males and FLUT wt males have ano-genital distances
(AGD) similar to females at the age of weaning. Right) FLUT tg males show a
significant reduction in body weight (p=.023) compared to wt brothers (FLUT
WT) at sacrifice.
• Behavioral Testing: Hang Test - Mice were placed on a wire
grid, and then turned upside down, and the time to fall was
measured in seconds. Paw Print Analysis – Front feet were painted
with non-toxic acrylic red paint, and hind feet were painted with
blue. Mice were then guided to walk along a strip of filter paper.
Flutamide treated tg males show a loss
of motor function.
Body Weight
25
FLUT WT FLUT TG FLUT WT FLUT TG
male
male
female
female
• Construct: AR cDNA was subcloned from pCMVAR. cDNAs
were ligated into the NotI site of pBSX-HSA.
Figure 2. Left) FLUT tg males are approximately half the size of their flutamide
treated wildtype (FLUT WT) brothers. Right) FLUT tg males exhibit kyhosis, or
hunched back (arrow).
Stride Length (cm)
WT
untreated
Average Axon Area (um2)
Methods
TG WT
Body Weight (g)
• To examine whether blocking androgen action prenatally would
protect our tg males from death on postnatal day 1, we treated
pregnant dams with 5 mg flutamide/day on gestational days 15-20 .
• Prenatal flutamide treatment was successful in rescuing
transgenic (tg) males from death.
• Tg males and their wildtype brothers were feminized, showing
shorter ano-genital differences than untreated males.
0
Ano-genital distance (mm)
• Given that tg females do not experience a similar mortality, we
suspect that perinatal testosterone may cause many tg males to die
as newborns.
Flutamide treated tg male mice are markedly
smaller and exhibit kyphosis.
Survival
40
• We created a transgenic mouse line in which a rat AR cDNA with
a wildtype (Wt) number of glutamine residues (22) is overexpressed in muscle fibers using an HSA (human skeletal actin)
promoter.
• Very few transgenic males survive to weaning and recent
evidence suggests that many tg males die on the day of birth.
Summary
Results: Flutamide rescues transgenic males.
Time (s)
Introduction
50
45
40
35
30
25
20
15
10
5
0
Axon Size
FLUT WT
n=5
• This work demonstrates that prenatal flutamide can rescue tg
males from death on postnatal day 1 but is not sufficient to prevent
the eventual emergence of SBMA later in life.
• Flutamide treatment may delay the onset of SBMA, and disease
progression may be slower in flutamide treated males.
• These results suggest that the wildtype AR, at sufficiently high
levels, is toxic to muscle fibers.
• Ultimately, loss in muscle fiber function may lead to a loss of
fibers, and eventually to a loss in motor neurons.
• Our results also suggest that SBMA and other motor neuron
diseases may be myogenic in origin, contrary to the prevailing
view.
FLUT TG
n=6
Figure 6. Left) There was no reduction in the number of L5 ventral root axons.
Right) No change in average axon size was detected. Perhaps, if the disease was
allowed to progress farther there would be a detectable difference in axon
numbers, as seen in untreated tg males.
Acknowledgments
Technical support was provided by Cindy Knaff, Chas Jensen, and
Sandy Troxell.
These studies were funded by NIH NS-045195 (CLJ) and the
MSU foundation (CLJ).