Neurotransmitter release in motor nerve terminals of amouse model of mild spinal muscular atrophyRoc�ıo Ruiz and Luc�ıa Tabares
Department of Medical Physiology and Biophysics School of Medicine, University of Seville, Seville, Spain
Abstract
Spinal muscular atrophy is a genetic disease which severity depends on the amount of SMN protein, the
product of the genes SMN1 and SMN2. Symptomatology goes from severe neuromuscular impairment leading
to early death in infants to slow progressing motor deficits during adulthood. Much of the knowledge about
the pathophysiology of SMA comes from studies using genetically engineered animal models of the disease.
Here we investigated one of the milder models, the homozygous A2G SMA mice, in which the level of the
protein is restored to almost normal levels by the addition of a mutated transgene to the severe SMN-deficient
background. We examined neuromuscular function and found that calcium-dependent neurotransmitter
release was significantly decreased. In addition, the amplitude of spontaneous endplate potentials was
decreased, the morphology of NMJ altered, and slight changes in short-term synaptic plasticity were found. In
spite of these defects, excitation contraction coupling was well preserved, possibly due to the safety factor of
this synapse. These data further support that the quasi-normal restoration of SMN levels in severe cases
preserves neuromuscular function, even when neurotransmitter release is significantly decreased at motor nerve
terminals. Nevertheless, this deficit could represent a greater risk of motor impairment during aging or after
injuries.
Key words: motor neuron; neurodegeneration; neuromuscular junction; spinal muscular atrophy; synapse.
Introduction
SMN (survival motor neuron) is an essential and ubiquitous
protein which best known functions are its participation in
the assembly of small nuclear ribonucleoproteins and in
mRNA splicing (Fischer et al. 1997; Liu et al. 1997; Meister
et al. 2001; Pellizzoni et al. 2002). A deficiency in SMN
expression produces spinal muscular atrophy (SMA), one of
the most severe diseases of genetic origin in childhood
(Crawford & Pardo, 1996). The disease manifests by deteri-
oration in motor function due to the selective loss of sub-
sets of a-motor neurons in the spinal cord and reduction in
excitatory inputs onto motor neurons (Ling et al. 2010;
Park et al. 2010; Mentis et al. 2011; Martinez et al. 2012) .
There are several clinic forms of SMA which classification is
based on the age of onset and the severity of the symp-
toms (Munsat & Davies, 1992). In SMA type I, by far the
most frequent form of SMA, the disease is diagnosed
within 6 months of birth and is characterized by
profound generalized muscular weakness that usually
results in death before 2 years of age. In SMA type II,
onset of the disease occurs between 6 and 18 months of
age and infants are unable to walk. SMA type III and
type IV are less severe and symptoms start after
18 months of age (type III), or during adulthood (type
IV). Although patients of type III and IV are able to walk
without aid, at least for a time, their motor function
progressively deteriorates.
The different clinic forms of SMA are directly related to
the amount of SMN expressed in patients (McAndrew et al.
1997; Feldkotter et al. 2002; Harada et al. 2002). SMN is
coded by two genes, SMN1 and SMN2 (Lefebvre et al.
1995), and whereas SMN1 produces full length SMN (SMN-
FL), SMN2 produces only 10% of SMN-FL and 90% of a trun-
cated form of the protein (SMND7) (McAndrew et al. 1997).
As the number of copies of SMN2 varies in the population,
the pathology in patients with deleted or mutated SMN1
will depend greatly on the number of SMN2 copies in each
individual (Coovert et al. 1997; Lefebvre et al. 1997). SMA
type I or type II patients have one or two copies of SMN2,
whereas type III or type IV patients have three or more
copies of SMN2.
The generation of animal models of SMA has contributed
enormously to the understanding of the pathophysiology
Correspondence
Luc�ıa Tabares, Department of Medical Physiology and Biophysics,
School of Medicine, University of Seville, Avenida S�anchez Pizju�an, 4,
41009 Seville, Spain. E: [email protected]
Accepted for publication 20 February 2013
© 2013 Anatomical Society
J. Anat. (2013) doi: 10.1111/joa.12038
Journal of Anatomy
of the disease. For example, it has been demonstrated that
the homozygous absence of Smn, the homologous SMN
gene in animals, is embryonically lethal in mice (Schrank et
al. 1997; Hsieh-Li et al. 2000). This early lethality can be pre-
vented by transgenic expression of the human gene SMN2
(Hsieh-Li et al. 2000; Monani et al. 2000b). Remarkably, mice
expressing one or two SMN2 copies have pathologic charac-
teristics similar to those of SMA type I patients (Hsieh-Li
et al. 2000; Monani et al. 2000b), whereas mice expressing
four copies of SMN2 have a normal life span and present
minor alterations such as peripheral necrosis in ears and tail.
On the other hand, expression of 8–16 SMN2 copies in null-
Smn mice results in almost a normal phenotype (Hsieh-Li
et al. 2000; Monani et al. 2000b). Therefore, as in humans,
in mice the disease severity is inversely correlated with the
SMN dosage.
Several SMA type I mouse models have been generated
so far. One of the most used is the so-called SMND7 (Le
et al. 2005). Against a severe background (Smn�/�, plus 1–
2 copies of SMN2), these mice express an additional gene
(SMN1 SMND7) which produces only truncated SMN pro-
tein. As a result, there is a slight increase in maximal survival
(from 5 to 6 days when SMN1 SMND7 is not incorporated
up to 2 weeks after the transgene is included). Importantly,
this provides a wider time window for studying the patho-
logical changes that low levels of SMN produces. For exam-
ple, it has been demonstrated that postnatal maturation
of the NMJ does not take place in most affected muscles
(Kariya et al. 2008; Murray et al. 2008; Kong et al. 2009; Ruiz
et al. 2010), as evidenced at the presynaptic side by the
anomalous distribution and significant decrease in synaptic
vesicles and active zones, reduction in mitochondria, imma-
ture organization of neurofilaments (NF) and microtubules
(Torres-Benito et al. 2011), and up to a ~ 50% reduction in
neurotransmitter release (Kong et al. 2009; Ling et al. 2010;
Ruiz et al. 2010; Torres-Benito et al. 2011, 2012). At the
postsynaptic side, small and immature end-plates are
observed (Kariya et al. 2008; Murray et al. 2008; Kong et al.
2009; Ruiz et al. 2010). In addition to the NMJ changes, loss
of proprioceptive sensory synaptic inputs on motor neurons
is also found (Ling et al. 2010; Mentis et al. 2011). Following
these changes, neurodegenerative signs such as NF accumu-
lation in motor nerve terminals, muscle fiber denervation,
and loss of spinal motor neurons are found (Michaud et al.;
Hsieh-Li et al. 2000; Jablonka et al. 2000; Monani et al.
2000a, 2003; Le et al. 2005; Avila et al. 2007; Kariya et al.
2008; Bowerman et al. 2009; Park et al. 2010; Riessland et
al. 2010; Torres-Benito et al. 2011; Ling et al. 2012).
Much less is known about the pathophysiology of the
milder forms of SMA. However, several mouse models
that resemble SMA type III have already been generated
and are starting to provide valuable information (Jablonka
et al. 2000; Monani et al. 2003; Kariya et al. 2008;
Gladman et al. 2010; Park et al. 2010; Simon et al. 2010;
Osborne et al. 2012). One of these models results in the
expression of the SMN1 A2G mutated transgene on the
severe Smn�/�;SMN2 background (Monani et al. 2003).
A2G is a common missense mutation in the SMN1 gene
in SMA patients (Parsons et al. 1998). Remarkably, the
hemizygous expression of the SMN1 A2G in mouse ame-
liorates the SMA phenotype (from type I towards type
III), significantly improving motor function and extend-
ing mean survival from 5 to 225 days. Even so, these
mice still suffer from motor neuron loss, muscle atrophy,
axonal NF accumulation and immature NMJs (Monani
et al. 2003). In the present study, we investigated a
milder SMA model which expresses the A2G transgene
in homozygosis on the severe Smn�/�;SMN2 back-
ground (Monani et al. 2003). We found that at 1 year
of age, homozygous A2G SMA mice, although smaller,
did not present alterations in the maturation of the
NMJ nor NF accumulation but did have a significant
reduction in neurotransmitter release at motor nerve
terminals of the transversus abdominis (TVA) muscle. In
addition, NMJ morphology appeared slightly altered.
However, assessment of motor function by electromyog-
raphy showed no apparent impairment of hind limbs.
These results allow a better understanding of the alter-
ations present in very mild SMA models in which motor
performance is not apparently affected initially but in
which reduction in the neuromuscular transmission
safety factor could limit motor function in case of
injury, disease or aging.
Methods
Animal model
The A2G mouse line was kindly provided by Dr. A. Burghes
(Department of Molecular Genetics, College of Biological
Sciences, The Ohio State University, Columbus, OH, USA).
Double transgenic Smn+/�;SMN2+/+;SMN A2G+/+ mice on a
FVB/N background were interbred. Genotyping was done
by PCR using tail DNA as previously described (Monani et al.
2003). Control mice were Smn+/+;SMN2+/+;SMN A2G+/+. Smn-
deficient mice were Smn�/�;SMN2+/+;SMN A2G+/+ (referred
to henceforth as homozygous A2G SMA mice). Only male
mice older 1 year or older were used for experiments and
controls were age-matched littermates of homozygous A2G
SMA mice. All experiments were performed according to
the guidelines of the European Council Directive for the
Care of Laboratory Animals.
Electromyography
Compound muscular action potentials (CMAPs) were
recorded as described (Ruiz et al. 2005; Simon et al. 2010) in
anesthetized mice (tribromoethanol 2%, 0.15 mL per 10 g
body weight, i.p.). Stimulating needle electrodes were
placed at the sciatic notch and the head of the fibula. The
© 2013 Anatomical Society
Neurotransmission in mild SMA, R. Ruiz and L. Tabares2
active recording electrode (a circumferential surface elec-
trode) was placed at midthigh. The reference electrode was
inserted at the base of the fifth foot phalanx. A ground
electrode was placed at the base of the tail. Supramaximal
responses were first recorded, followed by responses to
incremental currents, in very small steps, from subthreshold
levels until the progressive recruitment of 10–15 responses.
Each incremental current pulse was applied three times,
and responses were considered stable and therefore accept-
able if they were identical. The mean single motor unit
action potential (SMUAP) was calculated averaging the size
increments of the first 10 responses.
Muscle preparation
Mice were anesthetized with tribromoethanol (2%,
0.15 mL per 10 g body weight, i.p.) and killed by exsangui-
nation. The TVA muscles were dissected with their nerve
branches intact and pinned to the bottom of a 2-mL cham-
ber, over a bed of cured silicone rubber (Sylgard, Dow Corn-
ing). Preparations were continuously perfused with a
solution of the following composition (in mM): 125 NaCl, 5
KCl, 2 CaCl2, 1 MgCl2, 25 NaHCO3 and 15 glucose. The solu-
tion was continuously gassed with 95% O2 and 5% CO2,
which maintained the pH at 7.35. The muscles were used
either for intracellular electrical recordings or were fixed
for performing immunohistochemistry.
Intracellular electrical recording and analysis
The nerve was stimulated by means of a suction elec-
trode. The stimulation consisted of square-wave pulses of
0.5 ms duration and 2–40 V amplitude, at variable fre-
quencies (0.5–100 Hz). A glass microelectrode (10–20 MO)filled with 3 M KCl was connected to an intracellular
recording amplifier (Neuro Data IR283; Cygnus Technol-
ogy) and used to impale single muscle fibers near the
motor nerve endings. Evoked endplate potentials (EPPs)
and miniature EPPs (mEPPs) were recorded from different
NMJs within the muscle as described previously (Ruiz et
al. 2010). Muscular contraction was prevented by includ-
ing in the bath 3–4 lM l-conotoxin GIIIB (Alomone Labo-
ratories), a specific blocker of muscular voltage gated
sodium channels. Recording was performed at room tem-
perature (22–23 °C). The mean amplitudes of the EPP and
mEPPs recorded at each NMJ were linearly normalized to
�70 mV resting membrane potential. EPP amplitudes
were corrected for nonlinear summation (Martin, 1955).
The kinetics of EPP and mEPP were characterized by their
rise time (10–90%) and decay time constant (calculated
from the exponential fit of the decay phase). Quantal
content (QC) was estimated by the direct method, which
consists of recording mEPPs and EPPs (nerve stimulation
0.5 Hz) simultaneously and then calculating the ratio:
QC = average peak EPP/average peak mEPP.
Immunohistochemistry
Dissected TVA muscles from control and A2G SMA mice
were fixed with 4% paraformaldehyde (PFA) for 2 h. Mus-
cles were bathed with 0.1 M glycine in phosphate-bufferd
saline (PBS) for 20 min, then permeabilized with 1% (v/v)
Triton X-100 in PBS for 30 min, and incubated then in a
blocking solution containing 5% (w/v) bovine serum albu-
min (BSA), 1% Triton X-100 in PBS for 2 h. Samples were
incubated overnight at 4 °C with a mouse primary antibody
against 160 kDa neurofilament (NF; 1 : 750, Millipore). Next
day, muscles were rinsed for 1 h in PBS containing 1%
Triton X-100, incubated for 1 h both with 4 lg mL–1 Alexa
488-conjugated goat anti-mouse secondary antibody (Invi-
trogen) and 10 ng mL–1 rhodamine-bungarotoxin (Sigma),
and rinsed again with PBS for 90 min. Finally, muscles were
mounted in glycerol containing DABCO and imaged with
an upright Olympus FV1000 confocal microscope. Z-stack
projections were made from serial scanning every 0.5 lm to
reconstruct the NMJ.
Statistics
Results are reported as means � SEM, with n being the
number of muscle fibers per group and N the number of
mice per group. Student’s t-test was performed as unpaired,
two-tailed sets when the distribution was normal and the
Mann–Whitney rank sum test was used when the distribu-
tion was not normal. Results were considered statistically
different when P < 0.05.
Results
Homozygous expression of SMN1 A2G on the SMA severe
mouse background extended survival for more than 1 year,
as previously described (Monani et al. 2003). Body weight
was, however, reduced (~ 20%) in comparison with their
control littermates (Fig. 1A) and mice presented shorter
tails. No other external alterations, such as ear or tail necro-
sis (Hsieh-Li et al. 2000; Osborne et al. 2012), were apparent.
As motor performance was also normal and no signs of
limb muscle wasting or strength loss were observed in
homozygous A2G SMA mice (data not shown), we explored
the morphological characteristics of the NMJ in TVA, one of
the most affected muscles in the severe SMA mouse model
SMND7 (Murray et al. 2008; Ruiz et al. 2010; Torres-Benito
et al. 2011). In the severe model, important defects in post-
natal maturation of the pre- and postsynaptic motor termi-
nals, together with axonal and terminal NF accumulation
and denervation have been reported. In addition, in
8-month-old hemizygous A2G SMA mice poor maturation
of end-plates has been also described at the oblique
abdominal muscle (Kariya et al. 2008).
We stained postsynaptic receptors with bungarotoxin-
rhodamine, and axons and intraterminal axonal branches
© 2013 Anatomical Society
Neurotransmission in mild SMA, R. Ruiz and L. Tabares 3
with an antibody anti-NF, revealed by an Alexa488 second-
ary antibody. Figure 1B shows representative examples of
NMJs from a 1-year-old control (left panel) and a littermate
homozygous A2G SMA mouse. Contrary to what it was
found in most severe mouse models, NMJs from homozy-
gous SMA mice typically presented mature characteristics
[i.e. acetylcholine receptors (AChRs) grouped in bands, and
perforated end-plates], and no signs of denervation. How-
ever, ~ 84% of NMJs showed a fragmented appearance and
separated preterminal axonal branches (Fig. 1B, right),
contrary to the pretzel-like form found in ~ 63% of control
littermates (Fig. 1B, left). In contrast, no difference in NF
accumulation was observed between homozygous A2G
SMA and control mice.
Synaptic transmission reduction in motor nerve
terminals
To investigate whether synaptic transmission was affected
in motor nerve terminals from the TVA in homozygous
A2G SMA mice, we studied the properties of miniature
(mEPP) and evoked (EPP) end-plate potentials in 1-year-
old mice and compared them with those of their control
littermates.
Smaller miniature end-plate potentials in A2G SMA mice
Spontaneous neurotransmitter release was investigated by
recording the miniature end-plate potentials (mEPPs). In
A2G SMA mice, the mEPPs were smaller than in controls, as
can be observed directly from the recording traces (Fig. 2A)
and from their different mEPP size distributions (Fig. 2B).
This was further confirmed in the cumulative frequency dis-
tribution plot, which showed a significant shift to the left
of the mutant curve with respect to that of controls
(Fig. 2C), with median values of 0.39 mV in mutants and
0.57 mV in controls (P < 0.001; Mann–Whitney test). The
origin of this difference was not clear but among the possi-
ble explanations are a decrease in the amount of neuro-
transmitter content (quantal size), a decrease in the
number of postsynaptic acetylcholine receptors, and a
decrease in the sensitivity of the postsynaptic receptors.
On the other hand, when the mean frequency of sponta-
neous neurotransmitter release per fiber was compared, no
statistical difference between controls (1.11 � 0.19 events/s;
n = 18, N = 3) and A2G SMA mice (0.97 � 0.21 events/s;
n = 18,, N = 3; P = 0.56) was found (Fig. 2D).
Decrease in calcium-dependent neurotransmission in
A2G SMA mice
Next, we investigated the postsynaptic electrical response
(end-plate potential, EPP) following presynaptic single
action potential elicited by the electrical stimulation of the
nerve (Fig. 3A). The mean size of the EPP in each fiber was
obtained from recordings lasting 180–200 s during which
the nerve was stimulated repetitively at 0.5 Hz. In A2G
SMA mice, the mean amplitude of the EPP was signifi-
cantly less (P = 0.004; two tailed t-test) than in controls
(23.28 mV � 0.39; n = 18, N = 3, vs. 46.70 mV � 0.48;
n = 18, N = 3, respectively; Fig. 3B). We also compared
quantal content (the amount of synaptic vesicles that fuse
with the plasma membrane in response to an action
potential) and found that it was reduced ~ 21% in A2G
SMA mice (P = 0.03) in comparison with controls
(54.16 � 4.58; n = 18, N = 3, vs. 68.89 � 6.21; n = 18,
N = 3, respectively; Fig. 3C).
The analysis of the kinetics of the EPP, quantified by mea-
suring the rise time between the 10 and 90% of the rising
phase, and the decay time constant of the falling phase,
showed an small, but significant, slower rising phase in A2G
SMA mice than in controls (1.09 ms � 0.05; n = 18, N = 3,
vs. 0.96 ms � 0.05; n = 18, N = 3, respectively (P = 0.02;
Fig. 3A, left graph), suggesting a slight impairment
between pre- and postsynaptic coupling. No significant dif-
ference was found in the decay time constant (A2G SMA:
2.88 ms � 0.24; n = 18, N = 3; controls: 2.58 ms � 0.18; n
= 18, N = 3; P = 0.22; Fig. 3A, right graph).
Small alterations in short-term plasticity
To explore the ability of A2G SMA motor terminals to
change their synaptic strength, trains of repetitive stimuli of
05
10152025303540
0 100 200 300 400
Bod
y w
eigh
t (g)
Age (days)
ControlA2G SMA
A2G SMAControl
NF
BTX-Rho
Merge
A
B
Fig. 1 Body weight and NMJ in homozygous A2G SMA mice. (A)
Body weight in control (open symbols) and homozygous A2G SMA
(closed symbols) mice. (B) Representative NMJ confocal images from
control and homozygous A2G SMA mice. Neurofilaments have been
labeled by specific antibodies (green) and AChR have been labeled
with BTX-Rho (red). Scale bar: 15 lm.
© 2013 Anatomical Society
Neurotransmission in mild SMA, R. Ruiz and L. Tabares4
different frequencies and durations were applied to the
nerve and the amplitude of the EPP measured in control
and A2G SMA fibers. Figure 4A shows representative exam-
ples of postsynaptic responses and appears to illustrate no
large differences between genotypes. This was confirmed
by statistical analysis in a number of terminals as shown in
the graph in Fig. 4B, representing the mean size of the EPPs
along the train at 50 Hz in control and A2G SMA mice
fibers, normalized to the size of the first response. At this
frequency, the degree of facilitation and depression along
the train was not different between genotypes.
We also measured the paired-pulse facilitation (EPP2/
EPP1), which gives an indication of the initial vesicular
release probability. No differences between controls and
A2G SMA mice were found with 1-s stimulations at differ-
ent frequencies (10–100 Hz; Fig. 4C). Finally, when the train
index was calculated (see figure legend), which is an indica-
tion of the amount of short-term depression, a trend
toward less depression in mutant in comparison with con-
trol terminals was observed, although this only reached sta-
tistical significance at 20 Hz (0.97 � 0.02; n = 18, N = 3, vs.
0.91 � 0.02; n = 23, N = 3; P = 0.04; Fig. 4D). These results
show that short-term plasticity in A2G SMAmouse terminals
was, in general, well preserved in spite of the decrease in
quantal content.
No failures
In contrast to frequent failures in neurotransmission
reported in 9-month-old hemizygous A2G SMA mice (Kariya
et al. 2008), we found no signs of failures in 1-year-old
homozygous mice at any of the stimulation frequencies
explored (0.5–100 Hz; data not shown).
Electromyography
Although no signs of limb muscle weakness were appar-
ent in homozygous A2G SMA mice, EMG recordings were
performed to test whether a reduction in compound mus-
cular action potential (CMAP) amplitude or alteration in
the size of the motor units was detectable, as occurs in
SMA patients (Swoboda et al. 2005) and in other mild
SMA mouse models (Kariya et al. 2008; Simon et al.
2010).
Recordings were done by means of a surface ring elec-
trode placed at the middle of the thigh in 1-year-old male
mice homozygous for A2G and wild-type littermates. Maxi-
mum compound muscle action potential (CMAP) and motor
unit (MU) recruitment were recorded after sciatic nerve
stimulation.
As previously reported (Monani et al. 2003), no spontane-
ous activity (fibrillation or fasciculation potentials) in resting
0
20
40
60
80
100
0 0.5 1 1.5 2 2.5 3
Control (946 events; 18.3)A2G SMA (910 events; 18.3) 0.2
0.6
1
1.4
0.5 mV
mEPPs amplitude (mV)
Cum
. fre
quen
cy %
mE
PP
s pe
r sec
.(18.3) (18.3)
Control A2G SMA
100
80
60
40
20
02.01.51.00.50.0
mEPP size (mV)
200
150
100
50
02.01.51.00.50.0
mEPP size (mV)
# of
obs
erva
tions
# of
obs
erva
tions
500 ms
ControlA2G SMA
A
B
C DFig. 2 Spontaneous end-plate potentials in
homozygous A2G SMA mice.
(A) Representative recordings from the TVA
muscle in control (black traces) and in
homozygous A2G SMA mice (gray traces).
(B) Frequency histograms of mEPP sizes in
representative examples in both genotypes.
(C) Cumulative mEPP size curves in both
genotypes. (D) Frequency of mEPP
occurrence. The numbers between
parentheses are the number of fibers and the
number of mice studied, respectively.
© 2013 Anatomical Society
Neurotransmission in mild SMA, R. Ruiz and L. Tabares 5
(18.3) (18.3)EP
Ps
ampl
itude
(mV
)
0
10
20
30
40
50
**
(18.3) (18.3)
Qua
ntal
Con
tent
0
20
40
60
80
*
*
(18.3) (18.3) (18.3) (18.3)
Ris
e tim
e (m
s)
Dec
ay τ
(m
s)
5 ms 0
0.2
0.4
0.6
0.8
1
1.2
1
2
3
ControlA2G SMAA
B C
Fig. 3 Decreased calcium-dependent neurotransmitter release in motor nerve terminals of homozygous A2G SMA mice. (A) EPP traces from
control (black trace) and homozygous A2G SMA TVA muscles (gray trace). Recordings were normalized to the same peak amplitude for compari-
son. Rising (left graph) and decay (right graph) times of evoked EPPs in control and homozygous A2G mice. (B,C) Mean EPP size (B) and quantal
content (C) in control and homozygous A2G mice. *P < 0.05; ** P < 0.005.
0.5 mV
Control
0
0.2
0.4
0.6
Trai
n in
dex
10 20 50 100Frequency (Hz)
*
PP
F
10 20 50 100Frequency (Hz)
1 mV
50 ms
0.8
1
1.2
0 2 4 6 8 10EP
Ps
ampl
itude
(N)
Stimulus number
Control (17.3)
A2G SMA (13.3)
50 Hz
50 ms
A2G SMA
0.8
1
00.20.40.60.8
11.2
A
B C D
Fig. 4 Short-term plasticity in homozygous A2G SMA mice. (A) Representative EPP recordings from the TVA muscle in control (black trace) and in
homozygous A2G SMA mice (gray trace) during a train of stimuli at 50 Hz. (B) For each fiber, we constructed the mean curve observed for three
sequential stimulus trains normalized to the first EPP, separated by 20-s intervals. (C) PPF quantification (EPP2/EPP1) at different frequencies
(10–100 Hz) in control (white bars) and in homozygous A2G SMA mice (gray bars). (D) Normalized depression measured as train index,
[(EPP9 + EPP10)/2]/EPP1, at different frequencies (10–100 Hz) in control (white bars) and in homozygous A2G SMA mice (gray bars) terminals.
* P < 0.05.
© 2013 Anatomical Society
Neurotransmission in mild SMA, R. Ruiz and L. Tabares6
muscles was observed in homozygous A2G SMA mice
(n = 5), suggesting no denervation or anomalous nerve
excitability.
Recording of the maximal CMAP after supramaximal
nerve stimulation in both mouse groups showed no signifi-
cant differences in CMAP first peak amplitude between
control and A2G SMA mice (37.8 � 11.6 mV; n = 3; vs.
45.7 � 3.4 mV; n = 5; respectively, P = 0.58), suggesting no
differences in the number of functional motor units
between genotypes (Fig. 5A).
To test whether mutant muscles were more predisposed
to fatigue, the nerve was supramaximally stimulated at
20 Hz for 1 s while the successive CMAP were recorded
(Fig. 5B). The amount of fatigue was calculated as the per-
cent of decrease in the CMAP peak amplitude at the end of
the stimulation train relative to the first response. In control
mice the signal depression was 17 � 2% (N = 3) and in
homozygous A2G SMA mice it was 16 � 4% (N = 2); a non-
statistical difference (P = 0.67).
Finally, to estimate the average single motor unit action
potential (SMUAP) we applied 10 ‘successful’ successive
stimuli of increasing strength and recorded the increment
in the responses which represent the successive recruitment
of single motor units. Examples of the sizes of the
increments are shown in Fig. 5C, in control and A2G SMA
mice. In neither of the genotypes were large steps
observed, which is an indication that no giant motor units
were recruited. Moreover, quantification of the average
SMUAP amplitude in control and A2G SMA mice showed
no statistical differences (1.4 � 0.33 mV; N = 3 vs.
1 � 0.14 mV; N = 5, respectively; P = 0.34), further support-
ing that there was no axonal sprouting, similar to what it
has been reported in another mild SMA model (Gladman
et al. 2010; but see Monani et al. 2003). Therefore, these
data demonstrate that the average size of measured motor
units was not different between genotypes and support
that no significant loss of motor units occurs in A2G SMA
mice.
Discussion
SMA is not only a neurodegenerative disease in children
and adults but also a neurodevelopment disease when SMN
levels are low before birth. At early stages of development,
SMN is implicated in the control of neurite length, in the
correct development of axonal growth cone, in motor axon
guidance, and in the maturation and maintenance of
synaptic contacts in motor circuits. Most of this information
has been obtained from in vivo and in vitro SMA models in
which SMN is much decreased. However, less is known
about the pathological changes in mild animal models of
the disease in which SMN is not greatly reduced. The aim of
this work has been to investigate possible alterations in a
very mild SMA mouse model, the homozygous A2G SMA
mouse (Monani et al. 2003), with the aim of better
understanding which properties are preserved and which
are not in the milder forms of the disease.
In mouse models of slowly progressing motor neuron dis-
eases, loss of motor neurons is partially compensated by
axonal sprouting and reinnervation, a process in which
Control
30 mV
2 ms
0
0.4
0.8
1.2
0
0.4
0.8
1.2
0 2 4 6 8 100 2 4 6 8 10
200 ms
30 mV
ΔV (m
V)
Stimulus Stimulus
0
0.8
1.6
SM
UA
P (m
V)
ΔV (m
V)
0
20
40
60
CM
AP
(mV
)
0
0.4
0.8
A20
/A1
ControlA2G SMAA2G SMA
(2) (3)
(3) (5)
(3) (5)
A
B
C
Fig. 5 EMG characteristics of hind limb
muscles in homozygous A2G SMA mice.
(A) Representative recording of CMAP in both
genotypes. Graph represents the
quantification of the absolute first peak
amplitude of CMAP in response to a
supramaximal stimulation in control (white
bars) and in homozygous A2G SMA mice
(gray bars) terminals. (B) Representative
recordings during a train of stimuli at 100 Hz
in control (left trace) and in homozygous A2G
SMA mice (right trace). The bar graph shows
the depression of CMAP amplitudes (A20/A1)
in both genotypes. (C) Amplitudes of
SMUAPs in control and homozygous A2G
mice in response to stimuli of increasing
amplitude. Each number in the x-axis
represents a stimulus that elicited an
increment in the amplitude of the response.
SMUAP mean size is showed in the graph in
both studied groups. The numbers between
parentheses are the number of fibers and the
number of mice studied, respectively.
© 2013 Anatomical Society
Neurotransmission in mild SMA, R. Ruiz and L. Tabares 7
CNTF (ciliary neurotrophic factor) participates (Simon et al.
2010; Selvaraj et al. 2012). This remodeling gives rise to
anomalously large motor units that, in many instances, can
be shown by electromyography (EMG). In addition, other
anomalous features in motor neuron diseases, such as spon-
taneous activity and alterations in the normal size of
CMAPs, are good indications of the degree of affectation
and of the compensation capability of the system. For
example, in contrast to hemizygous A2G SMA mice and to
heterozygous (Smn+/�) mice, which present abnormal EMG
activity (Monani et al. 2003; Simon et al. 2010), homozygous
A2G SMA mice exhibited an apparent absence of EMG
abnormalities in hind limb muscles. This result is not unex-
pected given the higher SMN expression level in A2G SMA
mice in comparison with other type III SMA mouse models
(Monani et al. 2003). The absence of spontaneous muscle
electrical activity at rest in homozygous A2G SMA mice sug-
gests that end-plates were not denervated and that motor
axons were not anomalously excited. In patients, no sponta-
neous activity in the EMG has been reported in very mild
type III SMA forms of the disease (Hausmanowa-Petrusewicz
& Jozwik, 1986). In the same way, the size of the CMAP was
not different between control and homozygous A2G SMA
mice, which is indicative of either no alteration or the pres-
ence of compensatory large motor units resulting from
sprouting and reinnervation, as occurs in Smn+/� mice
(Simon et al. 2010). We favor the first possibility, as the
mean size of motor units was not different between control
and A2G SMA mice, contrary to what has been found in
Smn+/� mice (Simon et al. 2010) and in other mouse models
of motor neuron diseases, such in that of spinal muscular
atrophy with respiratory distress type I (Ruiz et al. 2005) or
in a model of ALS (Hegedus et al. 2009). Nevertheless, the
presence of high threshold anomalously large motor units
in this SMA model cannot be completely excluded, as the
technique used for this estimation is based on the recruit-
ment of 10–12 motor units with the lower thresholds. In
fact, in a mouse model of ALS it has been shown that fast-
twitch and fast-fatigable (FF) motor neurons, which usually
have a higher threshold for activation, are lost first, fol-
lowed by fast-twitch and fatigue-resistant (FR) motor neu-
rons, whereas slow-twitch (S) are less affected (Pun et al.
2006). For the most severe form of SMA, a similar suscepti-
bility may also exist (Kanning et al. 2010). In severe SMA
mouse models it has been shown that large motor neurons,
which usually have a high threshold for activation and
belong to motor units of the FF type (Hashizume et al.
1988), are more vulnerable than smaller ones (Baumer et al.
2009). Accordingly, in severe SMA patients, type II muscle
fibers, which normally are innervated by fast motor neu-
rons, are atrophic, whereas slow type I muscle fibers are
better preserved and even hypertrophic (Dubowitz, 1978).
However, this sole hypothesis is not sufficient to explain
why in severe SMA mouse models, muscles with predomi-
nant oxidative slow-twitch fibers, such as the TVA, are more
affected than glycolytic fast-twitch muscles, such the LAL
(Murray et al. 2008; Ruiz et al. 2010). Other proposed fac-
tors for motor neuron vulnerability have been the location
of motor neurons at the spinal cord, the synapsing pheno-
type or the metabolic demand (Monani et al. 2000b; Kariya
et al. 2008; Murray et al. 2008; Baumer et al. 2009; Ruiz
et al. 2010; Ling et al. 2012). Finally, it is of interest to note
that in SMA and ALS patients, extraocular muscles are very
well preserved during the course of the disease (Kubota
et al. 2000; Mitsumoto et al. 2006). Although these muscles
have fast-twitch muscle fibers, they also have extremely
small motor units. However, it is not clear whether this is
indeed a protective factor.
In homozygous A2G SMA mice, the absence of pathologi-
cal electromyography signs in hind limb muscles contrasts
with the alterations in neuromuscular transmission
observed in the TVA, namely smaller mEPPs and ~ 21%
reduction in the number of vesicles fused with the plasma
membrane per action potential (quantal content). The
reduction in mEPP size was unexpected considering the
smaller size of homozygous mice in comparison with con-
trol littermates. Smaller muscle fibers normally have a
higher input resistance, which implies a larger voltage
change for the same amount of current flow. Nevertheless,
our results are in accordance with a previously reported
reduction in miniature postsynaptic currents in a severe
SMA model (Martinez et al. 2012). Among the reasons that
could explain this reduction are a decrease in synaptic vesi-
cle ACh content, a reduction in the density of ACh postsyn-
aptic receptors, or a combination of both. On the other
hand, the reduction in quantal content in SMA mouse mod-
els is a constant in most muscles in different SMA mouse
models (Torres-Benito et al. 2011; Ling et al. 2012). In the
TVA, it is around 50% at 2 weeks of age in the SMND7
model (Ruiz et al. 2010; Torres-Benito et al. 2011) but
reduced only 21% in 1-year homozygous mice (Fig. 3C).
Given the high safety factor in neuromuscular transmission,
this deficit probably does not compromise function. Fur-
thermore, repetitive stimulation of the nerve terminals did
not result in larger synaptic depression than in control litter-
mates. The lack of alteration in short-term synaptic plastic-
ity, in spite of the decrease in quantum content, is not
surprising given the capability of the neuromuscular system
to adapt to chronic changes (Ferraiuolo et al. 2009). Also, at
the observational level, no postural abnormalities or respi-
ratory distress was observed in mutant mice as would be
expected if the TVA function were deficient. It cannot be
discarded, however, that some functional deficit could
appear at mice older than 1 year, when age-related motor
neuron loss occurs.
Paradoxically, in hemizygous A2G SMA mice at 8–9-
month-old mice, an increase in quantal content was
detected in the semitendinosus muscle in relation to litter-
mate controls, in addition to frequent neurotransmission
failures in a percentage of fibers (Kariya et al. 2008). These
© 2013 Anatomical Society
Neurotransmission in mild SMA, R. Ruiz and L. Tabares8
changes were accompanied by immature end-plates and NF
accumulation in axonal motor branches of the gastrocne-
mius muscle (Kariya et al. 2008). We did not observe these
alterations in the TVA muscle of the homozygous mice,
probably because the homozygous model is a form of the
disease that is even milder than the heterozygous Smn+/�(Jablonka et al. 2000; Simon et al. 2010). In accordance with
the apparent lack of muscle weakness, our morphological
and functional results showed no signs of denervation, con-
trary to what it is found in other mouse SMA models that
present different degrees of impairment, from large neuro-
muscular denervation in severe SMND7 mice (Ling et al.
2012) to reduced remodeling potential of NMJs in Smn2B/�
mice, apparently in relation to the loss of terminal Schwann
cells (Murray et al. 2012). On the other hand, we did not
find any signs of sprouting and reinnervation, contrary to
what it has been reported in SMA intermediate/mild models
such as hemizygous A2G (Monani et al. 2003;) and in Smn+/
� mice (Simon et al. 2010).
The ability of SMN1 A2G to rescue the SMA phenotype is
of great interest. It has been shown previously that A2G
does not efficiently self-associate, but it can form complexes
when some amount of SMN (full-length) exists (Monani
et al. 2003). Therefore, it is critical that SMN reaches a cer-
tain minimal threshold, as the requirement for this is higher
during the embryonic and early postnatal period than in
the adulthood, as demonstrated in the different therapeutic
assays in SMA animal models (Lorson et al. 2010; Sendtner,
2010; Hua et al. 2011; Porensky et al. 2012). If the SMN
level is below its critical threshold at birth and is not rap-
idly restored, the postnatal maturation of motor nerve ter-
minals stops (Kariya et al. 2008; Torres-Benito et al. 2011).
Besides this effect, neurodegeneration changes at the NMJ
and other synapses takes place. Furthermore, when the
SMN level is well below its normal threshold, the degener-
ative changes are so fast that there is no time for
compensatory mechanisms such as sprouting and reinner-
vation. The correlation, if any, between synapse matura-
tion defects and neurodegeneration is not clear as yet. It is
evident that alteration of the NMJ can exist in apparently
mature terminals, as in the SMA model in the present
study. However, it could be also that an inappropriate
maturation process of the NMJ could accelerate the neu-
rodegeneration process.
In summary, we found that homozygous A2G SMA mice,
in spite of having no apparent motor functional deficits
and having mature NMJs, present a significant reduction in
neurotransmitter release and morphological NMJ altera-
tions (fragmentation). However, evaluation of neuromuscu-
lar coupling by electromyography showed no alterations in
hind limb muscles. These results suggest that although in
mild cases of SMA, motor symptoms could be initially
absent or very mild, the safety factor at the NMJ is
decreased by the reduction in neurotransmitter release.
Therefore, complementary strategies for avoiding age-
related or disease-related impairment of motor function
might be required in patients with the milder form of the
disease. For example, in future studies it might be of inter-
est to test whether caloric restriction or programmed exer-
cise could prevent NMJ alterations.
Acknowledgements
The authors thank A. Burghes for the generous gift of this mouse
model. The financial support of the following research grants is
gratefully acknowledged: Spanish Ministry of Science and Innova-
tion BFU2010-21648, and Junta de Andaluc�ıa P09-CVI-4862.
References
Avila AM, Burnett BG, Taye AA, et al. (2007) Trichostatin A
increases SMN expression and survival in a mouse model of
spinal muscular atrophy. J Clin Invest 117, 659–671.
Baumer D, Lee S, Nicholson G, et al. (2009) Alternative splicing
events are a late feature of pathology in a mouse model of
spinal muscular atrophy. PLoS Genet 5, e1000773.
Bowerman M, Anderson CL, Beauvais A, et al. (2009) SMN,
profilin IIa and plastin 3: a link between the deregulation
of actin dynamics and SMA pathogenesis. Mol Cell Neurosci
42, 66–74.
Coovert DD, Le TT, McAndrew PE, et al. (1997) The survival
motor neuron protein in spinal muscular atrophy. Hum Mol
Genet 6, 1205–1214.
Crawford TO, Pardo CA (1996) The neurobiology of childhood
spinal muscular atrophy. Neurobiol Dis 3, 97–110.
Dubowitz V (1978) Muscle disorders in childhood. Major Probl
Clin Pediatr 16, iii-xiii, 1–282.
Feldkotter M, Schwarzer V, Wirth R, et al. (2002) Quantitative
analyses of SMN1 and SMN2 based on real-time lightCycler PCR:
fast and highly reliable carrier testing and prediction of severity
of spinal muscular atrophy. Am J Hum Genet 70, 358–368.
Ferraiuolo L, De Bono JP, Heath PR, et al. (2009) Transcriptional
response of the neuromuscular system to exercise training and
potential implications for ALS. J Neurochem 109, 1714–1724.
Fischer U, Liu Q, Dreyfuss G (1997) The SMN-SIP1 complex has
an essential role in spliceosomal snRNP biogenesis. Cell 90,
1023–1029.
Gladman JT, Bebee TW, Edwards C, et al. (2010) A humanized
Smn gene containing the SMN2 nucleotide alteration in exon
7 mimics SMN2 splicing and the SMA disease phenotype. Hum
Mol Genet 19, 4239–4252.
Harada Y, Sutomo R, Sadewa AH, et al. (2002) Correlation
between SMN2 copy number and clinical phenotype of
spinal muscular atrophy: three SMN2 copies fail to rescue
some patients from the disease severity. J Neurol 249,
1211–1219.
Hashizume K, Kanda K, Burke RE (1988) Medial gastrocnemius
motor nucleus in the rat: age-related changes in the number
and size of motoneurons. J Comp Neurol 269, 425–430.
Hausmanowa-Petrusewicz I, Jozwik A (1986) The application of
the nearest neighbor decision rule in the evaluation of elec-
tromyogram in spinal muscular atrophy (SMA) of childhood.
Electromyogr Clin Neurophysiol 26, 689–703.
Hegedus J, Putman CT, Gordon T (2009) Progressive motor unit
loss in the G93A mouse model of amyotrophic lateral sclerosis
is unaffected by gender. Muscle Nerve 39, 318–327.
© 2013 Anatomical Society
Neurotransmission in mild SMA, R. Ruiz and L. Tabares 9
Hsieh-Li HM, Chang JG, Jong YJ, et al. (2000) A mouse model
for spinal muscular atrophy. Nat Genet 24, 66–70.
Hua Y, Sahashi K, Rigo F, et al. (2011) Peripheral SMN restora-
tion is essential for long-term rescue of a severe spinal muscu-
lar atrophy mouse model. Nature 478, 123–126.
Jablonka S, Schrank B, Kralewski M, et al. (2000) Reduced sur-
vival motor neuron (Smn) gene dose in mice leads to motor
neuron degeneration: an animal model for spinal muscular
atrophy type III. Hum Mol Genet 9, 341–346.
Kanning KC, Kaplan A, Henderson CE (2010) Motor neuron
diversity in development and disease. Annu Rev Neurosci 33,
409–440.
Kariya S, Park GH, Maeno-Hikichi Y, et al. (2008) Reduced SMN
protein impairs maturation of the neuromuscular junctions in
mouse models of spinal muscular atrophy. Hum Mol Genet 17,
2552–2569.
Kong L, Wang X, Choe DW, et al. (2009) Impaired synaptic vesi-
cle release and immaturity of neuromuscular junctions in
spinal muscular atrophy mice. J Neurosci 29, 842–851.
Kubota M, Sakakihara Y, Uchiyama Y, et al. (2000) New ocular
movement detector system as a communication tool in venti-
lator-assisted Werdnig-Hoffmann disease. Dev Med Child
Neurol 42, 61–64.
Le TT, Pham LT, Butchbach ME, et al. (2005) SMNDelta7, the
major product of the centromeric survival motor neuron
(SMN2) gene, extends survival in mice with spinal muscular
atrophy and associates with full-length SMN. Hum Mol Genet
14, 845–857.
Lefebvre S, Burglen L, Reboullet S, et al. (1995) Identification
and characterization of a spinal muscular atrophy-determining
gene. Cell 80, 155–165.
Lefebvre S, Burlet P, Liu Q, et al. (1997) Correlation between
severity and SMN protein level in spinal muscular atrophy. Nat
Genet 16, 265–269.
Ling KK, Lin MY, Zingg B, et al. (2010) Synaptic defects in the
spinal and neuromuscular circuitry in a mouse model of spinal
muscular atrophy. PLoS ONE 5, e15457.
Ling KK, Gibbs RM, Feng Z, et al. (2012) Severe neuromuscular
denervation of clinically relevant muscles in a mouse model of
spinal muscular atrophy. Hum Mol Genet 21, 185–195.
Liu Q, Fischer U, Wang F, et al. (1997) The spinal muscular atro-
phy disease gene product, SMN, and its associated protein
SIP1 are in a complex with spliceosomal snRNP proteins. Cell
90, 1013–1021.
Lorson CL, Rindt H, Shababi M (2010) Spinal muscular atrophy:
mechanisms and therapeutic strategies. Hum Mol Genet 19,
R111–118.
Martin AR (1955) A further study of the statistical composition
on the end-plate potential. J Physiol 130, 114–122.
Martinez TL, Kong L, Wang X, et al. (2012) Survival motor neu-
ron protein in motor neurons determines synaptic integrity in
spinal muscular atrophy. J Neurosci 32, 8703–8715.
McAndrew PE, Parsons DW, Simard LR, et al. (1997) Identifica-
tion of proximal spinal muscular atrophy carriers and patients
by analysis of SMNT and SMNC gene copy number. Am J Hum
Genet 60, 1411–1422.
Meister G, Buhler D, Pillai R, et al. (2001) A multiprotein com-
plex mediates the ATP-dependent assembly of spliceosomal U
snRNPs. Nat Cell Biol 3, 945–949.
Mentis GZ, Blivis D, Liu W, et al. (2011) Early functional impair-
ment of sensory-motor connectivity in a mouse model of
spinal muscular atrophy. Neuron 69, 453–467.
Michaud M, Arnoux T, Bielli S, et al. (2010) Neuromuscular
defects and breathing disorders in a new mouse model of
spinal muscular atrophy. Neurobiol Dis 38, 125–135.
Mitsumoto H, Floyd A, Tang MX, et al. (2006) Transcranial mag-
netic stimulation for upper motor neuron involvement in
amyotrophic lateral sclerosis (ALS). Suppl Clin Neurophysiol
59, 327–332.
Monani UR, Coovert DD, Burghes AH (2000a) Animal models of
spinal muscular atrophy. Hum Mol Genet 9, 2451–2457.
Monani UR, Sendtner M, Coovert DD, et al. (2000b) The
human centromeric survival motor neuron gene (SMN2) res-
cues embryonic lethality in Smn(�/�) mice and results in a
mouse with spinal muscular atrophy. Hum Mol Genet 9,
333–339.
Monani UR, Pastore MT, Gavrilina TO, et al. (2003) A transgene
carrying an A2G missense mutation in the SMN gene modu-
lates phenotypic severity in mice with severe (type I) spinal
muscular atrophy. J Cell Biol 160, 41–52.
Munsat TL, Davies KE (1992) International SMA consortium
meeting. 26–28 June 1992, Bonn, Germany. Neuromuscul
Disord 2, 423–428.
Murray LM, Comley LH, Thomson D, et al. (2008) Selective vul-
nerability of motor neurons and dissociation of pre- and post-
synaptic pathology at the neuromuscular junction in mouse
models of spinal muscular atrophy. Hum Mol Genet 17,
949–962.
Murray LM, Beauvais A, Bhanot K, et al. (2012) Defects in neu-
romuscular junction remodelling in the Smn(2B/–) mouse
model of spinal muscular atrophy. Neurobiol Dis 49C, 57–67.
Osborne M, Gomez D, Feng Z, et al. (2012) Characterization of
behavioral and neuromuscular junction phenotypes in a novel
allelic series of SMA mouse models. Hum Mol Genet 21,
4431–4447.
Park GH, Maeno-Hikichi Y, Awano T, et al. (2010) Reduced sur-
vival of motor neuron (SMN) protein in motor neuronal pro-
genitors functions cell autonomously to cause spinal muscular
atrophy in model mice expressing the human centromeric
(SMN2) gene. J Neurosci 30, 12005–12019.
Parsons DW, McAndrew PE, Iannaccone ST, et al. (1998) Intra-
genic telSMN mutations: frequency, distribution, evidence of
a founder effect, and modification of the spinal muscular
atrophy phenotype by cenSMN copy number. Am J Hum
Genet 63, 1712–1723.
Pellizzoni L, Yong J, Dreyfuss G (2002) Essential role for the
SMN complex in the specificity of snRNP assembly. Science
298, 1775–1779.
Porensky PN, Mitrpant C, McGovern VL, et al. (2012) A single
administration of morpholino antisense oligomer rescues
spinal muscular atrophy in mouse. Hum Mol Genet 21,
1625–1638.
Pun S, Santos AF, Saxena S, et al. (2006) Selective vulnerability
and pruning of phasic motoneuron axons in motoneuron
disease alleviated by CNTF. Nat Neurosci 9, 408–419.
Riessland M, Ackermann B, Forster A, et al. (2010) SAHA amelio-
rates the SMA phenotype in two mouse models for spinal
muscular atrophy. Hum Mol Genet 19, 1492–1506.
Ruiz R, Lin J, Forgie A, et al. (2005) Treatment with trkC agonist
antibodies delays disease progression in neuromuscular
degeneration (nmd) mice. Hum Mol Genet 14, 1825–1837.
Ruiz R, Casanas JJ, Torres-Benito L, et al. (2010) Altered intracel-
lular Ca2+ homeostasis in nerve terminals of severe spinal mus-
cular atrophy mice. J Neurosci 30, 849–857.
© 2013 Anatomical Society
Neurotransmission in mild SMA, R. Ruiz and L. Tabares10
Schrank B, Gotz R, Gunnersen JM, et al. (1997) Inactivation of
the survival motor neuron gene, a candidate gene for human
spinal muscular atrophy, leads to massive cell death in early
mouse embryos. Proc Natl Acad Sci U S A 94, 9920–9925.
Selvaraj BT, Frank N, Bender FL, et al. (2012) Local axonal func-
tion of STAT3 rescues axon degeneration in the pmn model of
motoneuron disease. J Cell Biol 199, 437–451.
Sendtner M (2010) Therapy development in spinal muscular
atrophy. Nat Neurosci 13, 795–799.
Simon CM, Jablonka S, Ruiz R, et al. (2010) Ciliary neurotrophic
factor-induced sprouting preserves motor function in a mouse
model of mild spinal muscular atrophy. Hum Mol Genet 19,
973–986.
Swoboda KJ, Prior TW, Scott CB, et al. (2005) Natural history of
denervation in SMA: relation to age, SMN2 copy number, and
function. Ann Neurol 57, 704–712.
Torres-Benito L, Neher MF, Cano R, et al. (2011) SMN require-
ment for synaptic vesicle, active zone and microtubule postna-
tal organization in motor nerve terminals. PLoS ONE 6, e26164.
Torres-Benito L, Ruiz R, Tabares L (2012) Synaptic defects in
SMA animal models. Dev Neurobiol 72, 126–133.
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