Overexpression of IGF-1 in Muscle
Attenuates Disease in a Mouse Model
of Spinal and Bulbar Muscular Atrophy
J. Paul Taylor,7Charlotte J. Sumner,3Kenneth H. Fischbeck,1and Maria Pennuto8,9,*
1Neurogenetics Branch, NINDS, NIH, Bethesda, MD 20892, USA
2Istituto di Endocrinologia, Universita ` degli Studi di Milano, Milan 20133, Italy
3Department of Neurology, Johns Hopkins School of Medicine, Baltimore, MD 21287, USA
4Institute Pasteur Cenci-Bolognetti, Department of Histology and Medical Embryology, IIM, Sapienza University of Rome, Italy 00161
5Department of Neurology, Nagoya University Graduate School of Medicine, Nagoya, Japan 466-8550
6Institute for Advanced Research, Nagoya University, Nagoya, Japan 466-8550
7Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
8Department of Neurology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
9Department of Neuroscience and Brain Technologies, Italian Institute of Technology, Genoa, Italy 16163
Expansion of a polyglutamine tract in the androgen
receptor (AR) causes spinal and bulbar muscular
atrophy (SBMA). We previously showed that Akt-
mediated phosphorylation of AR reduces ligand
binding and attenuates the mutant AR toxicity. Here,
we show that in culture insulin-like growth factor 1
(IGF-1) reduces AR aggregation and increases AR
clearance via the ubiquitin-proteasome system
through phosphorylation of AR by Akt. In vivo,
SBMA transgenic mice overexpressing a muscle-
show evidence of increased Akt activation and AR
phosphorylation and decreased AR aggregation.
Augmentation of IGF-1/Akt signaling rescues behav-
ioral and histopathological abnormalities, extends
the life span, and reduces both muscle and spinal
IGF-1/Akt-mediated inactivation of mutant AR as
a strategy to counteract disease in vivo and demon-
strates that skeletal muscle is a viable target tissue
for therapeutic intervention in SBMA.
Spinal and bulbar muscular atrophy (SBMA), or Kennedy’s
disease, is an X-linked neurodegenerative disease caused by
expansion of a CAG repeat encoding polyglutamine in the first
exon of the androgen receptor (AR) gene (La Spada et al.,
1991). The repeat is polymorphic in length, and individuals with
an expansion over 36 residues develop disease. Expansion of
polyglutamine is the cause of at least eight other neurodegener-
ative disorders, including Huntington’s disease, dentatorubral
and pallidoluysian atrophy, and six types of spinocerebellar
ataxia (Orr and Zoghbi, 2007). A hallmark of the polyglutamine
diseases is the accumulation of mutant protein into aggregates
and inclusions, which can be detected using biochemical and
histopathological techniques, respectively (Li et al., 2007; Ross
are shared by the polyglutamine diseases, different populations
of neurons are vulnerable to each of the mutant proteins, result-
ing in clinically distinct disease manifestations.
Expansion of polyglutamine in AR causes loss of lower motor
neurons in the brainstem and spinal cord, together with weak-
ness, fasciculations, and muscle atrophy (Katsuno et al., 2006).
SBMA is a gender-specific disease, with only males fully
affected. Females, even if homozygous for the mutation, have
few if any symptoms (Schmidt et al., 2002). In both transgenic
and knockin mouse models of SBMA, male but not female
mice expressing mutant AR develop full disease manifestations
(Katsuno et al., 2002; Yu et al., 2006). Importantly, reduction of
testosterone levels in male mice ameliorates disease manifesta-
tions, suggesting a potential therapy for SBMA (Katsuno et al.,
2003). Indeed, a phase 2 clinical trial shows the benefits of
androgen deprivation by leuprorelin acetate (Banno et al., 2009).
However, the use of anti-androgens as therapy may have unde-
Emerging evidence suggests a role for muscle in SBMA path-
ogenesis. Histological and molecular signs of muscle pathology
are detectable before the appearance of pathological abnormal-
ities in the spinal cord in a knockin mouse model of SBMA (Yu
et al., 2006), suggesting that mutant AR may exert a direct toxic
effect on skeletal muscle. In support of this notion is the obser-
vation that overexpression of normal AR in the skeletal muscle
induces a phenotype similar to SBMA (Monks et al., 2007). Anal-
ysis of muscle biopsy samples derived from SBMA patients
suggests a mixed pathology with both myopathic and neuro-
genic features (Soraru et al., 2008). Although the extent to which
weakness in SBMA is a consequence of motor neuron
316 Neuron 63, 316–328, August 13, 2009 ª2009 Elsevier Inc.
primary muscle degeneration with secondary effects on the
motor neurons, is unknown, these observations suggest that
has been shown to have an anabolic effect on skeletal muscle
(reviewed by Sandri, 2008). Transgenic mice that overexpress
a muscle isoform of IGF-1 (mIGF-1) selectively in skeletal muscle
develop extensive muscle hypertrophy (Musaro et al., 2001).
IGF-1 induces muscle regeneration by stimulating the prolifera-
tionof satellite cellsinnormal(Musaro etal.,2001)andpatholog-
ical conditions, such as amyotrophic lateral sclerosis (Dobro-
wolny et al., 2005). At the molecular level, IGF-1 promotes
muscle hypertrophy through activation of the phosphatidyl-
inositol 3-kinase (PI3K)/Akt pathway (Rommel et al., 2001).
Beyond the general potential benefit for IGF-1 in myopathic
conditions, there is a specific rationale for IGF-1 in the treatment
of SBMA based on its ability to inactivate the AR through an
Akt-dependent mechanism (Palazzolo et al., 2007). We have
binding, thus reducing ligand-induced nuclear translocation and
transactivation of AR in cell culture. Moreover, we have shown
that IGF-1 reduces mutant AR toxicity in cultured cells through
phosphorylation of AR at the Akt consensus sites. These obser-
vations suggest IGF-1 and Akt-mediated inactivation of AR as
potential therapy for SBMA.
Here, we report that augmentation of IGF-1 levels decreases
mutant AR aggregation and increases AR clearance through
the ubiquitin-proteasome system and that this effect is depen-
dent on AR phosphorylation by Akt. In a mouse model of
SBMA, muscle-specific overexpression of IGF-1 activates Akt
and increases AR phosphorylation at Akt consensus sites. This
correlates with decreased AR aggregation in both the muscle
and spinal cord. Furthermore, overexpression of IGF-1 rescues
behavioral and histopathological abnormalities, delays disease
onset, and prolongs the life span of SBMA mice. Importantly,
IGF-1 attenuates both the morphological and molecular signs
of myopathic and neurogenic muscle pathology and increases
motor neuron survival. Our results highlight a disease-specific
mechanism of action of IGF-1 in SBMA muscle, which likely
involves phosphorylation and inactivation of AR by Akt and pres-
ents an opportunity to mitigate the disease manifestations
target tissue and highlights IGF-1 as a promising therapy for
IGF-1 Reduces Mutant AR Aggregation in Cell Culture
through Phosphorylation by Akt
Binding of ligand induces aggregation of mutant AR (Katsuno
et al., 2002; Stenoien et al., 1999). Here, we define AR aggre-
gates as the high molecular weight oligomers soluble in RIPA
buffer after high-speed centrifugation and sonication. These
species are detected as a smear in the stacking portion of
SDS-polyacrylamide gels and are retained in the acetate
membrane in filter retardation assay, as previously described
(Li et al., 2007; Rusmini et al., 2007; Taylor et al., 2003; Williams
et al., 2009). We previously showed that phosphorylation of AR
by Akt impairs ligand binding (Palazzolo et al., 2007). Therefore,
we hypothesized that agents which activate Akt such as IGF-1
would attenuate ligand-dependent AR aggregation. To test
this, we used COS1 cells transiently transfected with a vector
expressing a mutant AR with 65 glutamine residues (AR65Q)
(Figure 1A). The cells were treated with the AR ligand dihydrotes-
tosterone (DHT) with or without IGF-1 supplementation. Treat-
ment of the cells with DHT increased the accumulation of high
molecular weight AR species 2.3-fold (Figure 1A, left panel).
Treatment of the cells with IGF-1 increased phosphorylation of
Akt at serine 473, which is critical for Akt activation (Figure 1A,
right panel) (Alessi et al., 1996), and reduced basal and ligand-
induced AR aggregation by 100% and 80%, respectively
(Figure 1A, left panel). Consistent with this finding, treatment of
the cells with IGF-1 decreased the amount of AR retained in filter
IGF-1 onAR aggregation wasnot dueto decreased transcription
of AR since transcript levels were unchanged (data not shown).
Similar results were obtained in transiently transfected C2C12
myoblast cells and stably transfected motor neuron-derived
MN-1 cells (Figure S1), indicating that the effect of IGF-1 on
AR aggregation is independent of cell type.
Based on our previous findings (Palazzolo et al., 2007), we
hypothesized that the ability of IGF-1 to suppress ligand-depen-
dent AR aggregation was dependent on activation of the PI3K/
Akt pathway and phosphorylation of AR by Akt. To test the role
of PI3K, we used the specific PI3K inhibitor LY294002
(Figure 1A) (Vlahos et al., 1994). Treatment of the COS1 cells
with LY294002 decreased the IGF-1-induced phosphorylation
of IGF-1 on AR aggregation as assayed by both western blotting
and filter retardation assay (Figure 1A, left panel). AR has two
serines that are consensus sites for Akt, serine 215 and serine
792 (Lin et al., 2001). We have previously shown that Akt phos-
phorylates mutant AR at serine 215 (Palazzolo et al., 2007). To
determine whether phosphorylation of AR by Akt reduces aggre-
gation, we used AR variants in which serines 215 and 792 were
or aspartate, which mimics constitutive phosphorylation (Palaz-
zolo et al., 2007). In COS1 cells, DHT increased the aggregation
of both the unaltered AR65Q and the alanine-substituted
(S215A,S792A) AR65Q by 1.5- and 1.9-fold, respectively, in
western blotting (Figure 1B, upper panel), and by 5.8- and 4.9-
fold, respectively, in filter retardation assay (Figure 1B, bottom
panel). In contrast, DHT did not change the aggregation of the
phospho-mimetic variant (S215D,S792D), suggesting that AR
phosphorylation at Akt consensus sites prevents aggregation.
To determine whether the effect of IGF-1 requires phosphoryla-
tion of AR at these serines, we transfected HEK293T cells with
either unaltered AR65Q or the alanine-substituted receptor and
treated the cells with DHT and IGF-1 (Figure 1C). IGF-1
decreased AR-positive aggregates by 43% in cells expressing
AR65Q but did not have any effect in cells expressing the
alanine-substituted receptor. Together, these results show that
IGF-1 reduces AR aggregation through activation of PI3K/Akt
and at least in part through phosphorylation of AR by Akt.
IGF-1 Attenuates SBMA
Neuron 63, 316–328, August 13, 2009 ª2009 Elsevier Inc. 317
IGF-1 Induces Mutant AR Clearance through
the Ubiquitin-Proteasome System
Because the effect of IGF-1 on AR aggregation correlated with
a decrease in the levels of the protein (Figure 1, white bars), we
hypothesized that IGF-1/Akt signaling affects the stability of
mutant AR. To test this, we expressed mutant AR in MN-1 cells
and analyzed protein clearance in the presence of the protein
Figure 1. IGF-1 Decreases AR Aggregation
in Cultured Cells through Activation of the
PI3K/Akt Pathway and Phosphorylation of
AR by Akt
(A) Western blotting (top panel) and filter retarda-
tion assay (bottom panel) analyses of AR aggrega-
ing mutant AR with 65 glutamine residues (AR65Q)
and treated with DHT, LY294002 (LY), and IGF-1.
DHT increased the accumulation of both high
molecular weight (HMW) AR species (black bars)
and monomeric AR (white bars). IGF-1 decreased
both the basal and ligand-induced accumulation
of HMW and monomeric AR, and this effect was
reduced by the PI3K inhibitor LY294002. Graphs,
mean ± SEM, n = 4, (top panel) *p = 0.01 relative
to nonstimulated AR65Q-expressing cells (black
bars), and #p = 0.004 relative to DHT-treated cells
(white bars), (bottom panel) *p = 0.003, (post-hoc t
test). AR was detected with antibody to AR (N20).
ular weight. (Right panel) Phosphorylation of Akt
was analyzed by western blotting under the same
experimental conditions with anti-phospho-serine
473 and anti-total Akt antibodies. Phosphorylation
of Akt is represented as the ratio of the signals
detected with anti-phospho-serine and total Akt
antibodies. Graph, mean ± SEM, n = 4, *p = 0.05,
#p = 0.01.
(B) Western analysis (upper panel) and filter retar-
dation assay (bottom panel) showed that DHT
increased the accumulation of both HMW AR
species (black bars) and monomeric AR (white
bars) in COS1 cells expressing AR65Q and
AR65Q with alanine substitutions for serine 215
and serine 792 (S215A,S792A), but not in cells
(S215D,S792D). Graphs, mean ± SEM, n = 3,
(top panel) *p = 0.01, (bottom panel) *p = 0.05;
NS, nonsignificant relative to the nonstimulated
sample expressing AR65Q (post-hoc t test).
(C) Western analysisofHEK293T cells treatedwith
DHT and IGF-1 revealed that IGF-1 decreases the
accumulation of HMW (black bars) and mono-
meric (white bars) AR in cells expressing AR65Q,
but not the alanine-substituted receptor. Graph,
mean ± SEM, n = 3, *p = 0.01 and NS, nonsignifi-
cant (post-hoc t test) relative to the corresponding
In (A)–(C) the first sample is represented as 100%.
synthesis inhibitor cycloheximide (CHX)
(Figure 2A). Treatment of the cells with
IGF-1 significantly increased the clear-
ance of AR as compared to the vehicle-
treated samples. To determine whether
phosphorylation by Akt influences AR clearance, we analyzed
the stability of the phospho-defective AR-S215A,S792A variant
(Figure 2B). Loss of AR phosphorylation significantly reduced
the rate of AR clearance, suggesting that phosphorylation of
AR at the Akt consensus sites is important for AR degradation.
Next, we sought to determine which pathway is responsible
for the IGF-1 effect on AR clearance. We have previously shown
IGF-1 Attenuates SBMA
318 Neuron 63, 316–328, August 13, 2009 ª2009 Elsevier Inc.
that phosphorylation by Akt induces AR degradation through the
proteasome (Palazzolo et al., 2007). Consistent with this, treat-
ment of the cells with the proteasome inhibitor MG132 blocked
the effect of IGF-1 in a dose-dependent fashion (Figure 2C).
Proteins degraded through the proteasome are ubiquitylated.
Therefore, we asked whether mutant AR ubiquitylation is influ-
enced by phosphorylation. To address this, we expressed either
nonsubstituted or phospho-defective AR in HEK293T cells
together with vector expressing HA-ubiquitin and processed
the cells for immunoprecipitation with anti-AR antibody (Fig-
ure 2D). The ubiquitylation of the phospho-defective AR was
lower than that of the nonsubstituted receptor. To determine
whether IGF-1 induces AR clearance through other pathways,
we surveyed the target of rapamycin TOR, which is activated
by IGF-1 and induces autophagy (Sarbassov et al., 2005). Treat-
ment of the cells with rapamycin reduced the phosphorylation of
the downstream effector of TOR, S6K p70, but had no effect on
AR clearance (Figure S2). Together, these results indicate that
IGF-1 increases mutant AR clearance through the ubiquitin-pro-
teasome system in a phosphorylation-dependent manner.
IGF-1 Activates Akt and Increases AR Phosphorylation
at Serine 215 in SBMA Muscle
To test the effect of IGF-1 in vivo, we crossed mice that express
human full-length AR with 97 glutamine residues (AR97Q) and
that recapitulate the myogenic changes and the neurogenic
signs of muscle pathology observed in SBMA patients (Katsuno
et al., 2002) (Figure 7) with mice that overexpress a rat noncircu-
lating, muscle-specific isoform of IGF-1 (mIGF-1) under the
control of the rat myosin light chain promoter (Musaro et al.,
2001). The mIGF-1 mice show increased muscle mass and
strength, reduced age-related muscle loss, and augmented
regenerative capacity of muscle. To characterize the AR97Q/
mIGF-1 double-transgenic mice, we analyzed the expression
of the mIGF-1 and AR transgenes. The mRNA levels of the rat
mIGF-1 and human AR transgenes were measured by real-
time PCR and normalized to phosphoglycerate kinase 1
(PGK1) mRNA (Figure 3A) and 18S rRNA (data not shown). In
the skeletal muscle, mIGF-1 mRNA was detected in both
mIGF-1 and AR97Q/mIGF-1 mice, but not wild-type and
AR97Q mice (Figure 3A, left panel). No expression of mIGF-1
was detected in the spinal cord (Figure S3A, left panel). The
human AR transgene is expressed at similar levels in AR97Q
and AR97Q/mIGF-1 mice both in muscle (Figure 3A, right panel)
and spinal cord (Figure S3A, right panel), indicating that mIGF-1
does not alter AR expression in this mouse model.
To determine whether the mIGF-1 is biologically active, we
analyzed the phosphorylation of Akt at serine 473 and of AR at
serine 215 in muscle and spinal cord. Overexpression of mIGF-
1 resulted in augmented phosphorylation of Akt (Figure 3B, left
panel) and endogenous mouse AR (Figure 3B, right panel) in
the muscle, but not spinal cord (Figure S3B) of both mIGF-1
and AR97Q/mIGF-1 mice. Under these experimental conditions,
expanded polyglutamine AR. One explanation for this is that
whenphosphorylated byAkt, mutantARistargetedfordegrada-
tion through the proteasome (Figure 2) (Palazzolo et al., 2007).
Thus, we injected the skeletal muscle of AR97Q and AR97Q/
mIGF-1 mice with the proteasome inhibitor velcade for 24 hr to
block mutant AR degradation (Bonuccelli et al., 2007), and
analyzed AR phosphorylation by western blotting (Figure 3C). In
the velcade-treated muscle, phosphorylation of mutant AR was
increased by 4.3-fold in AR97Q/mIGF-1 mice as compared to
AR97Q mice. Together, these data indicate that overexpression
through the Ubiquitin-Proteasome System
(A) Western analysis of MN-1 cells transfected with
AR65Q and incubated with cycloheximide (CHX) for
1, 4, and 8 hr in the presence and absence of IGF-1
showed that IGF-1 increases the rate of AR clearance.
Graph, mean ± SEM, n = 3, *p = 0.03, **p = 0.001
(Student’s t test).
(B) Western analysis of HEK293T cells transfected
as indicated and treated with CHX showed that the
clearanceof thealanine-substitutedreceptor is slower
than AR65Q. Graph, mean ± SEM, n = 5, *p = 0.04,
**p = 0.01.
(C) Western analysis of MN-1 cells expressing AR65Q
and treated with DHT, IGF-1, and the proteasome
inhibitor MG132showed thatthe IGF-1-inducedclear-
dent fashion. Shown is one experiment representative
(D) Immunoprecipitation with anti-AR antibody of
HEK293T cells expressing the indicated AR65Q
versions together with HA-tagged ubiquitin (HA-Ub),
followed by western analysis with anti-HA or AR anti-
alanine-substituted receptor. Shown is one experi-
ment representative of four.
IGF-1 Attenuates SBMA
Neuron 63, 316–328, August 13, 2009 ª2009 Elsevier Inc. 319
of mIGF-1 in AR97Qmice results in Aktactivation and phosphor-
ylation of AR at the Akt consensus site serine 215.
Overexpression of mIGF-1 Reduces Mutant AR
Aggregation in SBMA Muscle
Because we have found that IGF-1/Akt signaling reduces poly-
glutamine AR aggregation in cultured cells (Figure 1), we asked
whether mIGF-1 decreases the accumulation of AR-positive
aggregates in vivo. To test this, we analyzed protein lysates
from skeletal muscle of AR97Q and AR97Q/mIGF-1 mice by
western blotting (Figure 4A) and filter retardation assay
(Figure 4B). In AR97Q mice, high molecular weight AR species
were not detected at 6 weeks of age (Figure S4A), but they
were detected starting at 12 weeks of age (Figure 4), indicating
that appearance of these species correlates with disease
progression (Figures 5 and 6). By contrary, in AR97Q/mIGF-1
mice these species were not detected before 24 weeks of age
(Figure S4A). To further characterize the high molecular weight
AR species, we electrophoresed the muscle lysates on a 1.5%
SDS-agarose gel, which resolves large protein complexes
(Figure 4C) (Bagriantsev et al., 2006; Williams et al., 2009). High
molecular weight AR species were detected in 12-week-old
AR97Q mice, and to a lesser extent in AR97Q/mIGF-1 mice. To
determine the size of these species, we analyzed the muscle
lysates by size-exclusion chromatography followed by SDS-
Figure 3. mIGF-1 Stimulates Phosphorylation of Akt
and AR in SBMA Muscle
(A) Rat mIGF-1 (left panel) and human AR (right panel) mRNA
levels weremeasured by quantitative real-time PCR inskeletal
muscle of 12-week-old mice. mIGF-1 is expressed in both
AR97Q/mIGF-1 and mIGF-1 mice. AR97Q is expressed in
both AR97Q and AR97Q/mIGF-1 mice. Data were normalized
to phosphoglycerate kinase (PGK1) mRNA. Data are repre-
sented relative to mIGF-1 mice = 100% (left) and to AR97Q
mice = 100% (right). Graphs, mean ± SEM; n = 3.
(B) Western analysis to detect phosphorylation at serine 473
of Akt (left panel) and serine 215 of AR (right panel) was
performed using specific phospho-serine antibodies in the
skeletal muscle of 6- (left) and 12- (right) week-old mice. Total
Akt and AR were measured as loading control. Graphs, mean
± SEM, n = 5 right panel, n = 3 left panel, *p = 0.04.
(C) Western analysis of skeletal muscle from AR97Q and
AR97Q/mIGF-1 mice injected with the proteasome inhibitor
expanded polyglutamine AR in mice overexpressing mIGF-1.
Graphs, mean ± SEM, n = 3, *p = 0.01.
In (B) and (C), the first sample is represented as 100%.
polyacrylamide gel electrophoresis (Figure 4D).
Although the molecular weight of monomeric
mutant AR is about 130 kDa and eluted in fraction
9 (Figure 4D, asterisk), in AR97Q mice AR eluted
mostly as high molecular weight species with
a peak in between 440 and 2000 kDa. Importantly,
IGF-1 reduced by 3-fold the amount of AR eluting
off as high molecular weight species.
nuclear accumulation of diffuse AR and nuclear
riceps muscle of AR97Q and AR97Q/mIGF-1 mice were stained
with 1C2 antibody (Figure 4E). Diffuse nuclear staining and
AR-positive nuclear inclusions were evident in AR97Q mice.
Overexpression of mIGF-1 in AR97Q muscle resulted in signifi-
cantly less accumulation of mutant AR in nuclear inclusions.
The effect of mIGF-1 on AR aggregation and nuclear accumula-
mIGF-1 Delays Disease Onset and Extends Disease
Duration and Survival of SBMA Mice
We monitored the effect of overexpression of mIGF-1 on disease
onset (Figure 5A), disease duration (Figure 5B), and survival
(Figure 5C) of SBMA mice. Disease onset was defined as the
week in which the mouse loses 5% body weight for at least two
consecutive weeks, while disease duration was calculated as
and extended the median disease duration by 20 weeks (c2LR =
10.7, p = 0.001). To assess the effect of mIGF-1 on survival, we
monitored the life span of AR97Q and AR97Q/mIGF-1 mice for
64 weeks. Analysis of the survival curves indicated that mIGF-1
extended the median life span by 30 weeks (c2LR = 10.2, p =
0.001). These data show that mIGF-1 delays disease onset and
extends disease duration and life span in SBMA mice. Similar
results were obtained without censoring of the data (Figure S5).
IGF-1 Attenuates SBMA
320 Neuron 63, 316–328, August 13, 2009 ª2009 Elsevier Inc.
Overexpression of mIGF-1 Attenuates Disease
Manifestations in SBMA Mice
SBMA pathogenesis, we analyzed body weight and motor func-
tion. AR97Q/mIGF-1 mice did not show the severe muscle
wasting and reduced body size observed in AR97Q littermates
icantly later than AR97Q mice (p = 0.001; n = 15) (Figure 6B). In
addition, we assessed muscle strength by hanging wire test
(Figure 6C) and motor performance by rotarod (Figure 6D) and
footprinting (Figure 6E) tasks. By hanging wire test, AR97Q mice
showed progressive deterioration in performance starting from
12 weeks of age when compared to wild-type mice (p = 0.001,
(p = 0.001; n = 15). By rotarod task, AR97Q mice showed signifi-
cant performancedeficitscomparedtowild-type mice startingat
16 weeks of age (p = 0.01, n = 7). These deficits were rescued by
overexpression of mIGF-1. On footprint analysis, AR97Q mice
dragged their hind legs, whereas AR97Q/mIGF-1 mice did not
show gait abnormality. Moreover, AR97Q/mIGF-1 mice showed
increased activity, interest in exploration of the surrounding
Figure 4. mIGF-1 Reduces AR Aggregation in SBMA
(A) Western blotting and (B) filter retardation assay analyses
showed a reduction in the accumulation of high molecular
weight AR species in the skeletal muscle of AR97Q/mIGF-1
mice at 12 weeks of age as compared to AR97Q mice. Endog-
enous and expanded polyglutamine AR were detected with
N20 antibody, and expanded polyglutamine AR with 1C2
antibody. Actin was used as a loading control. Shown is one
experiment representative of four in (A) and of three in (B).
Graphs, mean ± SEM, n = 3, *p = 0.001.
(C) 1.5% SDS-agarose gel analysis revealed that the amount
of AR aggregates in the muscle of AR97Q mice was reduced
in AR97Q/mIGF-1 mice.
(D) Size-exclusion chromatography (SEC) and western blot-
ting analysis of muscle lysates showed that IGF-1 reduces
the amount of AR eluting off as high molecular weight species
(fractions 1–4) in AR97Q/mIGF-1 mice compared to AR97Q
mice. The horizontal MW markers (kDa) were obtained by
SEC analysis of protein standards. V, void volume. The graph
and monomeric AR, which eluted off as fraction 9 (asterisk).
Shown is one experiment, which was representative of three.
(E) Immunohistochemical analysis using 1C2 antibody on
longitudinal and cross sections of AR97Q and AR97Q/mIGF-
1 muscle showed a reduction of diffuse nuclear staining and
nuclear inclusions in AR97Q/mIGF-1 compared to AR97Q
mice. Graphs, mean ± SEM; n = 5, *p = 0.04. Scale bar, 2 mm.
space, and ability to turn their bodies when sus-
pended by the tail compared to age-matched
AR97Q mice (data not shown). Altogether, these
results show that mIGF-1 reduces muscle weak-
ness and improves motor function in SBMA mice.
mIGF-1 Ameliorates SBMA Muscle
To address whether overexpression of mIGF-1 has
an effect on muscle denervation and myopathy in
SBMA mice, we performed histological analysis of transverse
sections of quadriceps muscles using hematoxylin and eosin
(Figure 7A) and nicotinamide adenine dinucleotide (Figure 7B)
staining. At 12 weeks of age, AR97Q mice showed signs of
muscle denervation, such as grouped and angulated atrophic
fibers (Figure 7A, asterisk), target fibers (Figure 7B, arrow) and
such as the presence of large fibers with central nuclei
(Figure 7A, arrow), which indicate muscle degeneration and
regeneration (Musaro et al., 2001). In contrast, mIGF-1 expres-
sion counteracted muscle atrophy and degeneration and
preserved myofibers architecture (Figures 7A and 7B). This
amelioration of muscle pathology was sustained even at 24
weeks of age (Figure S6).
To further characterize the effect of mIGF-1 on SBMA muscle,
we analyzed by real-time PCR the expression of genes upregu-
lated during denervation, such as runx 1 (AML1), myogenin,
embryonal and perinatal myosin heavy chain, and the alpha
subunit of the acetylcholine receptor (Figure 7C) (Kostrominova
et al., 2005; Zhu et al., 1994). Consistent with the pathological
findings, these genes were all upregulated in AR97Q mice at
IGF-1 Attenuates SBMA
Neuron 63, 316–328, August 13, 2009 ª2009 Elsevier Inc. 321
with disease progression (Figure S7). Importantly, expression of
these genes was also increased in skeletal muscle autopsy
specimens of four SBMA patients compared to unaffected
controls (Figure 7C, black bars). Overexpression of mIGF-1 in
the muscle of SBMA mice significantly attenuated the induction
of these markers. Altogether, these results indicate that mIGF-1
prevents muscle denervation and degeneration in SBMA mice.
Overexpression of mIGF-1 in SBMA Muscle Reduces
Motor Neuron Loss
IGF-1 has a trophic effect on motor neurons (reviewed by Cary
and La Spada, 2008), and increased IGF-1/Akt signaling in the
muscle has previously been shown to improve motor neuron
survivalinanamyotrophic lateralsclerosismouse model(Dobro-
wolny et al., 2005; Kaspar et al., 2003). Therefore, we hypothe-
sized that overexpression of mIGF-1 in muscle protects motor
neurons from damage caused by mutant AR. To test this, we
analyzed ventral spinal cord pathology by Nissl staining (Fig-
ure 8A). Histological analysis revealed that in AR97Q mice the
number of motor neurons/section was reduced by 27% ± 9%
compared to wild-type mice (8.4 ± 0.3 and 11.6 ± 1 motor
neurons/section, respectively), although this difference was not
statistically significant (p = 0.07, n = 3 animals). mIGF-1 restored
the number of motor neurons to levels comparable to wild-type
mice (11.5 ± 2). This result was validated by the analysis of the
that ChAT levels were increased in AR97Q/mIGF-1 mice by1.5 ±
1 promotes survival of motor neurons in SBMA mice (Figure 8B).
Another sign of motor neuron degeneration in SBMA mice is
the reduction of the cross-sectional area of motor neurons (Kat-
suno et al., 2002). Therefore, we measured the area of anterior
horn motor neurons in Nissl-stained spinal cord sections
(Figure 8C). The median area of motor neurons in AR97Q mice
(253.5 mm2) was significantly (W = 6097, p = 0.01, n motor
neurons = 100) smaller than that of wild-type mice (277 mm2),
but rescued in AR97Q/mIGF-1 mice (290 mm2, W = 4676.5,
p = 0.43).
Polyglutamine AR forms aggregates in the spinal cord (Kat-
suno et al., 2002). We asked whether the beneficial effect of
mIGF-1 on motor neurons correlated with a decrease in AR
aggregation. Western analysis of spinal cord lysates showed
that overexpression of mIGF-1 in the muscle reduced AR aggre-
gation in 12- and 24-week-old mice (Figures 8D and S4B).
AR-positive aggregates were detected in AR97Q/mIGF-1 mice
starting from 40 weeks of age. These results indicate that AR
aggregates appear in the spinal cord of AR97Q/mIGF-1 mice
at later stages of disease as compared to muscle.
To determine whether overexpression of mIGF-1 reduces
mutant AR-positive inclusions in the nuclei of motor neurons,
we analyzed 1C2-positive staining in cross sections of ventral
spinal cord of AR97Q and AR97Q/mIGF-1 mice (Figure 8E).
Nuclear 1C2-positive inclusions were decreased in AR97Q/
mIGF-1 mice compared to AR97Q mice. Altogether, these
results indicate that overexpression of mIGF-1 in the skeletal
muscle attenuates spinal cord pathology in SBMA mice.
In conclusion, the data presented here show that IGF-1/Akt
signaling promotes AR phosphorylation, reduces AR aggrega-
tion by stimulating protein clearance, and ameliorates disease
manifestations in SBMA mice. Moreover, our results indicate
that IGF-1-mediated inactivation of mutant AR is a potential
We describe the effect of mIGF-1 on SBMA pathogenesis. We
previously showed that IGF-1 protects cultured cells from the
toxicity of expanded polyglutamine AR through activation of
Akt and induction of AR phosphorylation (Palazzolo et al.,
2007). In this study, we extended these findings by showing
that phosphorylation of AR by IGF-1/Akt signaling increases
protein clearance through the proteasome and reduces mutant
Figure 5. mIGF-1 Delays Disease Onset and Extends Disease Duration and Survival of SBMA Mice
Kaplan-Meier analysis of disease onset (A), disease duration (B), and survival (C) in AR97Q and AR97Q/mIGF-1 mice.
(A) The median disease onset—the week in which the mouse starts to lose 5% body weight—was delayed by 10.5 weeks (arrow) in AR97Q/mIGF-1 mice relative
to AR97Q mice.
(B)The median disease duration—the time intervalfrom diseaseonset todeath—wasextended by 20weeks (arrow) inAR97Q/mIGF-1mice compared toAR97Q
(C) Survival analysis showed that the median lifespan of AR97Q mice was extended by 30 weeks (arrow) in AR97Q/mIGF-1 mice. AR97Q n = 18, AR97Q/mIGF-1
n = 19. Censored data are indicated by the symbol ‘‘:’’ (see Experimental Procedures).
IGF-1 Attenuates SBMA
322 Neuron 63, 316–328, August 13, 2009 ª2009 Elsevier Inc.
AR aggregation in cultured cells. Muscle-specific overexpres-
sion of mIGF-1 in SBMA mice induced phosphorylation of AR
at the Akt consensus site serine 215 and reduced mutant AR
aggregation in the muscle. mIGF-1 significantly improved motor
performance and body weight, counteracted muscle and spinal
cord pathology, and extended survival. We propose that mIGF-1
inactivates mutant AR through phosphorylation by Akt, and that
this results in reduced muscle denervation and degeneration.
Our results represent the first evidence that muscle is a thera-
peutic target in SBMA and provide the rationale for the use of
mIGF-1 as a novel therapy for SBMA.
Phosphorylation of AR by mIGF-1/Akt Signaling
Overexpression of mIGF-1 attenuates disease manifestations in
SBMA mice. One possible mechanism involves a direct effect of
mIGF-1 on polyglutamine AR. AR has two serines, serine 215
and serine 792, that conform to the Akt consensus site (Lawlor
and Alessi, 2001) and are conserved through evolution (Palaz-
Figure 6. Overexpression of mIGF-1 Atten-
uates Behavioral Abnormalities in SBMA
(A and B) Body weight analysis showed that
AR97Q mice progressively lose body weight start-
ing from 8 weeks of age. AR97Q/mIGF-1 mice
have normal body weight up to 12 weeks of age
and do not show body weight loss until 16 weeks
of age. Scale bars, SEM, n = 15.
(C) Hanging wire (n = 15) and (D) rotarod (n = 7)
analysis showed progressive deterioration in
performance of AR97Q mice, which was delayed
and improved in AR97Q/mIGF-1 mice. Scale
(E) Footprints of 12-week-old mice showed that
AR97Q mice dragged
AR97Q/mIGF-1 mice did not show defects in
coordination of steps. Red, front paws; blue,
hind paws. Shown is one experiment representa-
tive of three.
hind legs, whereas
zolo et al., 2007). We have previously
shown that phosphorylation of mutant
AR by Akt decreases ligand binding,
thus inhibiting ligand-dependent protein
stabilization, nuclear translocation, tran-
scription activation, and mostimportantly
toxicity in cultured cells (Palazzolo et al.,
2007). Akt phosphorylates other polyglut-
amine disease proteins. For example,
phosphorylation of mutant huntingtin at
serine 421 is protective in cultured striatal
neurons (Humbert et al., 2002). Here, we
show that phosphorylation of mutant AR
by Akt results in decreased aggregation
both in vitro and in vivo, and this corre-
lates with reduced toxicity in a mouse
model of SBMA. Importantly, we show
that the mechanism by which IGF-1
reduces AR aggregation involves degradation of mutant AR
through the proteasome in a process that is dependent on AR
phosphorylation by Akt. These results are consistent with
previous observations from Chang’s group, which show that
phosphorylation of nonexpanded AR by Akt induces the degra-
dation of the receptor through the proteasome (Lin et al.,
involves inactivation of mutant AR through phosphorylation by
Akt and decreased toxicity.
IGF-1 Promotes Muscle Hypertrophy and Inhibits
IGF-1 can have additional effects that are independent of AR
modifications. IGF-1 has long been established as an anabolic
factor for the skeletal muscle (Florini et al., 1996). Overexpres-
sion of IGF-1 in muscle induces muscle hypertrophy and actively
counteracts muscle atrophy (Coleman et al., 1995; Musaro et al.,
2001; Sandri et al., 2004), with a mechanism that involves activa-
tion of PI3K/Akt signaling (Bodine et al., 2001; Rommel et al.,
IGF-1 Attenuates SBMA
Neuron 63, 316–328, August 13, 2009 ª2009 Elsevier Inc. 323
2001). Several downstream effectors of Akt have been shown to
play a role in muscle atrophy. For instance, Akt inhibits FOXO,
which in muscle promotes protein degradation via the ubiqui-
tin-proteasome system (Sandri et al., 2004) and autophagy
(Zhao et al., 2007). FOXO activates transcription of the ubiquitin
ligases, atrogin1/MAFbx and MuRF1, and this process is
inhibited by IGF-1 (Stitt et al., 2004). Because we did not find
any change in expression of these genes in the mouse model
of SBMA used in this study, this pathway is not likely to be rele-
vant to SBMA (I.P. and M.P., unpublished data). Instead, it is
possible that IGF-1 restores SBMA muscle via inhibition of
FOXO and FOXO-induced autophagy. IGF-1/Akt signaling
promotes muscle hypertrophy by inducing novel protein
synthesis through inhibition of the glycogen synthase kinase 3b
and activation of the mammalian target of rapamycin (mTOR)
(Bodine et al., 2001; Rommel et al., 2001). The contribution of
these pathways to SBMA pathogenesis remains to be eluci-
IGF-1 is a Trophic Factor for Motor Neurons
The rescue of morphological abnormalities that we observe in
the spinal cord of SBMA mice suggests a trophic effect for
mIGF-1 on motor neurons. IGF-1 promotes sprouting, axonal
growth, and survival of embryonic motor neurons (Caroni and
Grandes, 1990), and after nerve injury (Hughes et al., 1993;
Neff et al., 1993). We have previously shown that IGF-1 is a pro-
survival factor for motor neuron-derived cells that express
expanded polyglutamine AR (Palazzolo et al., 2007). Therefore,
it is possible that overexpression of mIGF-1 in the muscle of
SBMA mice has direct beneficial effect on motor neurons.
Also, muscle isitself a source of neurotrophic and growthfactors
that may have protective effect on motor neurons (reviewed by
Cary and La Spada, 2008). Muscle-released brain-derived neu-
rotrophic factor is retrogradely transported by motor neurons
(DiStefano et al., 1992). Muscle-specific overexpression of
trophins from muscle, which in turn have beneficial effect on
motor neurons. The decreased AR aggregation that we found
in the spinal cord of AR97Q/mIGF-1 mice suggests a direct
effect of mIGF-1 on AR. However, we did not observe a change
in AR phosphorylation in the spinal cord of AR97Q/mIGF-1 mice
compared to AR97Q mice.
The results that we describe in this study suggest mIGF-I as
a good candidate for therapy in SBMA. Akt activation is pre-
dicted to result in a loss of AR function (Palazzolo et al., 2007),
which in turn may alter fertility. However, we have found that
overexpression of mIGF-1 selectively in muscle does not affect
fertility of SBMA mice (Figure S8). Subcutaneous injection of
human recombinant IGF-1 has not been effective in ALS clinical
trials (Borasio et al., 1998; Lai et al., 1997; Sorenson et al., 2008).
However, the muscle-specific IGF-1 isoform used in this study
may be more beneficial. Moreover, we provide evidence that,
in contrast to ALS, IGF-1/Akt signaling has a direct inhibitory
effect on the mutant protein, which helps to prevent motor
neuron degeneration in SBMA.
All experiments were carried out in accordance with the National Institutes of
Health Guide for the Care and Use of Laboratory Animals, and were approved
by the NINDS Animal Care Committee. Mice were observed daily and sacri-
ficed when they showed signs of muribundity (see below). AR97Q and
AR97Q/mIGF-1 mice, as well as mIGF-1and wild-typemice used in the exper-
iments described here were derived by crossing C57Bl6 mIGF-1 mice with
BDF1 AR97Q mice. All the experiments were performed in male mice coming
from the F1 generation of the cross described above. Similar to AR97Q mice,
we crossed mice that express normal AR (AR24Q) (Katsuno et al., 2002) with
Figure 7. mIGF-1 Ameliorates SBMA Muscle Pathology
(A) Hematoxylin and eosin and (B) nicotinamide adenine dinucleotide staining
of transverse sections of quadriceps of 12-week-old mice. (A) Arrows = fibers
with central nuclei; asterisks = angulated and grouped fibers. (B) Arrows =
target fibers; asterisks = moth-eaten fibers. Shown is one experiment repre-
sentative of three. Bar, 50 mm.
(C) The mRNA levels of runx1, myogenin, myosin heavy chain (MyHC) embry-
onal and perinatal, and acetylcholine receptor alpha were measured by real-
time PCR in the skeletal muscle of 12-week-old mice and SBMA patients.
Data from mice and patients were normalized to PGK1 and beta-glucoroni-
dase mRNA, respectively. Data are represented relative to wild-type mice or
control human samples = 1. Graphs, mean ± SEM, n = 3 mice and n = 4
IGF-1 Attenuates SBMA
324 Neuron 63, 316–328, August 13, 2009 ª2009 Elsevier Inc.
mIGF-1 mice. Overexpression of IGF-1 in the skeletal muscle of AR24Q mice
did not result in any change of behavior and survival (data not shown). Mice
were genotyped by PCR on tail DNA as previously described (Katsuno et al.,
2002; Musaro et al., 2001), using REDExtract-N-Amp Tissue PCR kit (Sigma)
according to manufacturer’s instructions.
Behavioral and Survival Analysis
All the tests were performed weekly. Data were analyzed retrospectively.
Rotarod analysis: Three trials were performed weekly using the Ugo Basile
ROTA-ROD for mice (Ugo Basile). Mice were put on the rod at speed of 16
rpm for a maximum period of 180 s, and the best performance for each mouse
was recorded. Hanging wire test: The mouse was placed on top of a wire cage
lid. The lid was shaken slightly three times to cause the mouse to grip the wires
and then the lid was turned upside down. The latency to fall was recorded for
a maximum time of 60 s (Crawley, 2008). Foot printing analysis: The forepaws
and the hindpawsof the mouse were painted with red and blue colors, respec-
Figure 8. mIGF-1 Reduces Spinal Cord
Pathology in SBMA Mice
(A) Nissl-stained transverse sections of ventral
spinal cords of 12-week-old mice. Quantitative
analysis showed that the number of motor
neurons/section is increased in AR97Q/mIGF-1
mice as compared to AR97Q mice. Graphs,
mean ± SEM, n = 3.
(B) Western analysis showed that expression of
choline acetyltransferase (ChAT) is increased in
the spinal cord of 12-week-old AR97Q/mIGF-1
mice as compared to AR97Q mice. Graphs,
(C) Histograms of the cross-sectional area of
motor neurons (see Experimental Procedures).
The distribution of motor neuron area is altered
in AR97Q mice, but not in AR97Q/mIGF-1 mice
relative to wild-type mice. Graphs, mean ± SEM,
n = 3 animals.
(D) Western analysis revealed that AR aggregation
is decreased in the spinal cord of AR97Q/mIGF-1
mice as compared to AR97Q mice at 12 weeks of
age. AR was detected with N20 antibody. Actin is
shown as loading control. Shown is one experi-
ment representative of three.
(E) Immunohistochemical analysis using 1C2 anti-
body on cross sections of AR97Q and AR97Q/
mIGF-1 ventral spinal cord showed a reduction
of nuclear inclusions in AR97Q/mIGF-1 relative
toAR97Qmice.Graphs, mean± SEM, n=4.Scale
bar, 2 mm.
tively, and the mouse was allowed to walk along
a narrow, paper-covered corridor. Survival anal-
ysis: Moribundity was set as the time in which
the mouse lost more than 30% and up to 35% of
body weight, or showed inability to move, dehy-
dration, and cachexia.
Human Skeletal Muscle Samples
Thisstudywasconducted according tothe Decla-
ration of Helsinki (Hong Kong Amendment).
Autopsy sample collection was approved by the
ethics committee of Nagoya University Graduate
School of Medicine. Written informed consent
was obtained from the family of each patient.
Confidentiality was ensured by assigning a study
code to each patient. All studies conformed to the ethics guidelines for human
genome/gene analysis research and the ethics guidelines for epidemiological
studies endorsed by the Japanese government. Anonymized control biopsy
samples were obtained from Dr A. Lieberman (Ann Arbor, MI, USA) following
the University of Michigan IRB guidelines. A supplemental control Human
Skeletal muscle total RNA has been purchased from Ambion/Applied Biosys-
Taqman Quantitative PCR Analysis
and spinal cord were flash frozen in liquid nitrogen. Total RNA was extracted
with Trizol (Invitrogen) as previously described (Pennuto et al., 2008). One mg
RNA was retro-transcribed using the cDNA Archive kit (Applied Biosystems)
following manufacturer’s instructions. Gene expression was measured by
quantitative real-time PCR using ABI 9900 Sequence Detector System
(Applied Biosystems).Specificassays on demand formyogenin
IGF-1 Attenuates SBMA
Neuron 63, 316–328, August 13, 2009 ª2009 Elsevier Inc. 325
(Mm_00446194-m1 and Hs_00231167-m1), embryonic (Mm_01332452-m1
and Hs_00159463-m1), perinatal (Mm_01329512-m1 and Hs_00267293-m1),
and adult (Mm_01332489-m1 and Hs_00428600-m1) myosin heavy chain,
acetylcholine receptor alpha (Mm_00431629-m1 and Hs_00175578-m1),
runx1 (Mm_00486762-m1 and Hs_00231079-m1), human AR (Hs_00907244-
m1), PGK1 (Mm_00435617-m1), and beta-glucuronidase (Hs_99999908-m1)
were from Applied Biosystems. Rat mIGF1 transgene expression was
measured using the following primers 50-GCACTCTGCTTGCTCACCTTTA
and 50-CCCCGCAAAGGGTCTCT (Assay on Design, Applied Biosystems).
ronidase mRNA for human tissues. Values were normalized to the mean of the
wild-type animals in each group, which was assigned as 1, unless differently
HEK293T, Cos1, and MN-1 cells were cultured and transfected as previously
described (Palazzolo et al., 2007). Cells were treated with DHT (10 nM, 24 hr,
Sigma), IGF-1 (100 ng/ml, 24–48 hr, Calbiochem), MG132 (1–10 mM, 16 hr,
Calbiochem), cycloheximide (25 mM, Calbiochem), and LY294002 (10 mM,
24 hr, Calbiochem), as indicated. Cells were processed for western blotting
as previously described (Palazzolo et al., 2007). Tissues were snap frozen in
liquid nitrogen. Frozen tissue samples (50–100 mg) were pulverized and
homogenized in 500 ml RIPA buffer (150 mM NaCl, 6 mM Na2HPO4, 4 mM
NaH2PO4, 2 mM EDTA pH 8, 1% sodium deoxycholate, 0.5% Triton X-100,
0.1% sodium dodecyl sulfate [SDS]) and protease inhibitor cocktail (Roche),
sonicated for 10 s, and centrifuged 10 min at 15,700 3 g at 4?C. The soluble
fraction was collected and total protein concentrations were determined by
the bicinchoninic acid Protein Assay Kit (Pierce) following manufacturer’s
instructions. For western blotting, protein lysates (100 mg) were separated
on 7.5% SDS-polyacrylamide gels and transferred to PVDF membranes (Milli-
pore). For the filter retardation assay, protein lysates (20 mg) were loaded onto
acetate and nitrocellulose membranes and washed twice with phosphate-
buffered saline. For SDS-agarose gels, protein lysates (100 mg) were diluted
in Laemmli buffer and loaded on 1.5% high melting agarose gels containing
1% SDS, as previously described (Bagriantsev et al., 2006). For size-exclusion
chromatography, muscle lysates (3 mg/500 ml) were resolved on a Super-
dex 200 10/300 GL column (GE Healthcare BioSciences) at a flow rate of
0.3 ml/min in 0.5 M phosphate buffer pH 7.4 using a Pharmacia P-500 series
FPLC system. Fractions (0.6 ml) were concentrated with Amicon-Ultra4 50K
filters (Millipore). To calibrate the column, high molecular weight standards
(GE Healthcare BioSciences) were eluted under the same conditions. The
void volume of the column was determined by eluting dextran blue (2000
kDa).To analyze phosphorylation, muscle lysates were prepared in lysis buffer
(50 mM HEPES, 250 mM NaCl, 5 mM EDTA, 0.1% NP40) with protease and
phosphatase inhibitor cocktails (Pierce). Membranes were probed with the
following antibodies: anti-AR (N20) (sc-816, Santa Cruz), anti a-tubulin
(T6199, Sigma), anti-phospho-serine 473 Akt and total Akt (9271 and 9272,
Cell Signaling), anti-polyglutamine 1C2 (MAB1574, Chemicon), anti-phos-
pho-serine 215 AR (PA137082, Pierce), anti-actin (sc-1616, Santa Cruz),
anti-choline acetyltransferase (AB144P, Chemicon), anti-HA (2367, Cell
Signaling). Relative signal intensities were quantified by NIH Image J software
and are shown in graphs below the corresponding panels.
Quadriceps muscles were snap frozen in isopentane. Sections of unfixed
muscle tissue were cut at 7 mm in a ?28?C cryostat and processed for hema-
toxylin and eosin or nicotinamide adenine dinucleotide (NADH) staining.
Hematoxylin and eosin staining: Slides were incubated in hematoxylin for
3 min, and rinsed in water. They were then stained with eosin, dehydrated
through gradient ethanol, then immersed in xylene for 30 min followed by
mounting in Permount (Electron Microscopy Science, PA). NADH staining:
Slides were incubated in NADH and NBT solution in the oven for 1 hr or until
the sections become dark. Slides were then rinsed and mounted with Shur/
Mount mounting medium (Pella Inc, CA). Motor neuroncount:Deeply anesthe-
tized mice were transcardially perfused with 4% paraformaldehyde (PFA).
Lumbar spinal cords and proximal hind limbs were dissected, post-fixed for
2 hr in 4% PFA and stored in 70% ethanol. Paraffin-embedded lumbar spinal
cord was serially sectioned at 5 mm steps, mounted on slides, and processed
for Nissl staining. Images of ten contiguous sections, 100 mm apart (original
magnification, 310) were analyzed with NIH ImageJ software. Motor neurons
wereidentified as cells positive for Nissl staining, with clear nucleus and nucle-
olus, with a maximum diameter greater than 20 mm. All neurons in the region
below a line drawn horizontally at the level of the spinal canal were measured.
Counting was performed in a blinded fashion. Motor neuron cross-sectional
area: the distribution of motor neurons was obtained by ranking the neurons
by size, and the mean ± SEM of the number of motor neurons of n = 3 animals
was calculated for each size range. For statistical analysis, we used Wilcoxon
rank sum test with continuity correction to compare distribution differences
among genotypes. 1C2 staining: 6 mm-thick tissue sections were deparaffi-
nized and rehydrated through a graded series of alcohol-water solutions,
and pretreated with microwave oven heating for 10 min in 10 mM citrate buffer
at pH 6.0 and then in formic acid for 5 min at room temperature. The tissue
sections were stained with mouse 1C2 antibody (1:10000) using Envision-
plus kit (Dako K2006) according to the manufacturer’s instructions. Sections
were counterstained with Mayer’s hematoxylin. For the ventral horn of the
spinal cord, 50 consecutive transverse sections of the lumbar spinal cord
were prepared from each individual mouse and 1C2-positive cells within the
ventral horn of every fifth section were counted. For muscle, more than 500
fibers were counted in randomly selected areas of individual mice.
A one-way ANOVA was used to evaluate the effect of IGF-1 among treatment
groups. Two-sample t tests were used for post-hoc comparisons. A two-way
mixed design ANOVA was conducted to compare the effects of genotype on
performance at the hanging wire and rotarod tasks as well as on body weight
over time. Genotype was used as a between-subjects factor, and time was
used as a within-subjects factor. A Fisher’s Least Significant Difference
(LSD) post-hoc was used to compare the performance and body weight
across genotypes at each time point. A log rank test was used to compare
Kaplan-Meier survival, disease onset and disease duration curves between
AR97Q and AR97Q/mIGF-1 genotypes. In these curves data were censored
either when a mouse died for a cause independent of SBMA (one mouse
died for prolapsed colon and one during blood collection) or when a mouse
was sacrificed at a fixed time point for biochemical and histopathologicalanal-
yses or at the end of the observation period. Censoring is indicated in Figure 5
by a vertical thick mark at the time of the event.
Supplemental Data include eight figures and can be found with this article
online at http://www.cell.com/neuron/supplemental/S0896-6273(09)00548-0.
We thank G. Harmison for skillful technical assistance, Dr. S. Ranganathan
and the other members of Dr. Fischbeck’s lab for advice and discussion,
Dr. A. Lieberman for providing us with control tissue samples, Dr. J. Crawley
for advice about mouse behavioral studies, Drs. L. Wrabetz and S. Previtali
for kindly providing us with muscle samples from denervated mice, Dr A. Anti-
gnani for instructions for size-exclusion chromatography, and Dr. F. Samba-
taro for statistical analysis. This work was supported by NINDS intramural
funds and by grants from Telethon-Italy (GFP04005, M.P.; A.M.), the Ken-
nedy’s Disease Association (M.P.), Muscular Dystrophy Association (Develop-
ment Grant, M.P.; Research Grant, J.P.T.), and the NIH (NS053825, J.P.T.).
Accepted: July 16, 2009
Published: August 12, 2009
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