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R E V I E W A R T I C L E

Spinal muscular atrophy: a motor neuron disorder or a

multi-organ disease?Monir Shababi,1,2, Christian L. Lorson1,2 and Sabine S. Rudnik-Schoneborn3

1Department of Veterinary Pathobiology, Life Sciences Center, University of Missouri, Columbia, MO, USA2Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, MO, USA3Institute of Human Genetics, Medical Faculty, University Hospital Aachen, Aachen, Germany

Abstract

Spinal muscular atrophy (SMA) is an autosomal recessive disorder that is the leading genetic cause of infantile

death. SMA is characterized by loss of motor neurons in the ventral horn of the spinal cord, leading to

weakness and muscle atrophy. SMA occurs as a result of homozygous deletion or mutations in Survival MotorNeuron-1 (SMN1). Loss of SMN1 leads to a dramatic reduction in SMN protein, which is essential for motor

neuron survival. SMA disease severity ranges from extremely severe to a relatively mild adult onset form of

proximal muscle atrophy. Severe SMA patients typically die mostly within months or a few years as a

consequence of respiratory insufficiency and bulbar paralysis. SMA is widely known as a motor neuron disease;

however, there are numerous clinical reports indicating the involvement of additional peripheral organs

contributing to the complete picture of the disease in severe cases. In this review, we have compiled clinical

and experimental reports that demonstrate the association between the loss of SMN and peripheral organ

deficiency and malfunction. Whether defective peripheral organs are a consequence of neuronal damage/

muscle atrophy or a direct result of SMN loss will be discussed.

Key words: Autonomic Nervous System; Cardiac Defects; Peripheral Organs; SMA.

Introduction

Clinical classification of SMA

Spinal muscular atrophy (SMA) is an autosomal recessive dis-

order that is the most common inherited motor neuron dis-

ease and the leading genetic cause of newborn mortality.

SMA occurs in approximately 1 : 10 000 live births with a

carrier frequency of 1 : 50 (Sugarman et al. 2012). SMA

Type I was identified more than a century ago by Werdnig

and Hoffmann as a disorder starting with progressive weak-

ness at infancy and resulting in death at a very early age.

They also characterized the pathology of the disease as loss

of anterior horn cells. Following international consensus,

four types (IIV) of SMA are classified based on the

clinical criteria including physical milestones, the onset of

symptoms, and life span (Munsat & Davies, 1992). The most

common types are acute infantile (SMA Type I, or Werdnig

Hoffmann disease), chronic infantile (SMA Type II), chronic

juvenile (SMA Type III, or KugelbergWelander disease),

and adult onset (SMA Type IV). Infants with the most severe

form of SMA can have decreased movement at prenatal

stage and rarely present with fetal akinesia sequence, con-

genital contractures and postnatal respiratory insufficiency.

The majority of SMA Type I patients have normal strength

at birth but exhibit progressive weakness within a few

weeks or months. Death usually ensues within 12 months

(Burnett et al. 2009; Rudnik-Schoneborn et al. 2009),

although this typical life span can be extended with

improved nutritional and respiratory care (Oskoui et al.

2007). Types II and III SMA present with weakness during

childhood and usually exhibit variable degrees of physical

impairment (Zerres et al. 1997; Bosboom et al. 2009). Type

IV occurs at adulthood and is the mildest form of the dis-

ease; approximately 70% of Type II patients reach adult-

hood but the life expectancy of Types IIIIV is normal

(Zerres et al. 1997; Lewelt et al. 2012).

Genetic determinants of SMA

The cause of SMA was identified as the loss of a gene called

Survival Motor Neuron-1 (SMN1) located on chromosome

Correspondence

Monir Shababi, Department of Veterinary Pathobiology, Life Sciences

Center, University of Missouri, Room 404, Columbia, MO 65211, USA.

T: + 1 573 8842680; F: + 1 573 8849395; E: [email protected]

Accepted for publication26 June 2013

2013 Anatomical Society

J. Anat. (2013) doi: 10.1111/joa.12083

Journal of Anatomy


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5q (Brzustowicz et al. 1990; Lefebvre et al. 1995). A human-

specific copy gene,SMN2, exists on the same region and dif-

fers from SMN1 by only a few silent nucleotides (Lefebvre

et al. 1995; Rochette et al. 2001). Therefore, SMN2encodes

an identical protein to SMN1, although it cannot fullyrestore the function of SMN1. The critical difference

between SMN1 and SMN2 is a C to T transition at the 6th

position of exon 7, leading to an alternatively spliced mRNA

isoform that lacks exon 7 (SMND7) (Lorson et al. 1999;

Monani et al. 1999). While SMN1-derived transcripts

produce full-length and functional SMN protein, nearly

90% ofSMN2-derived transcripts generate a truncated and

unstable protein (SMND7), which lacks the 16 amino acids

encoded by exon 7 (Lorson et al. 1998, 1999; Monani et al.

1999). Importantly, however,SMN2produces a low amount

of full-length SMN, comparable to ~ 10% of SMN1 levels

(Lorson & Androphy, 2000).SMN2is paradoxically a double-

edged sword: it provides only a sufficient level of SMN pro-

tein to allow survival and subsequent development of SMA;

it is also the most potent disease modifier and a bona fide

therapeutic target (Mailman et al. 2002; Wirth et al. 2006).

The number of SMN2copies, and therefore the level of the

full-length SMN created by SMN2 transcripts, directly

impacts disease severity. Type I individuals typically have

one to two copies of SMN2, whereas milder forms of SMA

are mostly associated with three to four copies of SMN2

(Feldkotter et al. 2002). SMA Type IV is only exceptionally

caused by SMN deficiency. In addition, clinically unaffected

individuals have been identified who lack SMN1, pointing

towards further modifiers for the disease phenotype in rareinstances.

SMN protein and function

SMN protein is a 38-kD protein localized in the cytoplasm

and nucleus. It is ubiquitously expressed and intimately

involved in a well-defined biochemical pathway that relates

to global gene expression: snRNP biogenesis (Fischer et al.

1997; Liu et al. 1997), as well as a less defined role in RNP

trafficking in neurons (Zhang et al. 2003). Currently, it is

unclear which SMN-associated function underlies SMA

development. snRNPs are the building blocks for the

general splicing machinery and are essential in all tissues.

Initially, it was difficult to envision how a general cellular

defect such as snRNP assembly could account for the motor

neuron pathology associated with SMA, but recent reports

have demonstrated that not all snRNP complexes are

equally affected by reduced SMN levels (Lotti et al. 2012).

The recent identification of specific gene targets that are

dysregulated in SMN-deficient contexts including Drosoph-

ilaand mice has supported the hypothesis that SMA devel-

ops due to splicing abnormalities (Imlach et al. 2012; Lotti

et al. 2012). However, there is still considerable debate over

the specific function(s) that leads to SMA development.

Several theories have been suggested to explain the specific

role of SMN in the survival of motor neurons including:

(i) SMN functions in splicing of the transcripts essential for

integrity and differentiation of motor neurons (Winkler

et al. 2005); (ii) there is a requirement for higher levels of

SMN in motor neurons than other tissues (Battaglia et al.1997); (iii) SMN functions specifically in axonal transport

(Pagliardini et al. 2000) and neuronal development such as

growth cones and neurites (Bechade et al. 1999; Jablonka

et al. 2000; Zhang et al. 2003). Certainly, the snRNP assem-

bly activity is the best characterized function of SMN, but a

conclusive link between SMA and snRNP dysreglation has

not been established.

SMN expression

SMN expression is developmentally regulated (Battaglia

et al. 1997) and there is a high level of SMN expression in

most tissues during embryogenesis (Novelli et al. 1997), fol-

lowed by a significant reduction after birth in all tissues

except in the brain and spinal cord, which remains relatively

high until 2 weeks post birth (La Bella et al. 1998). Based

on this pattern of expression, it is likely that most tissues

require SMN for normal development and that extremely

low SMN levels at prenatal stage are detrimental for nearly

all tissues. The necessity of SMN during embryogenesis for

development of axons and motoneuron dendrites in zebra-

fish (Hao et al. 2013), neuromuscular junction in Drosophila

(Chan et al. 2003), cranial nerves as well as lumbar spinal

nerves and innervations in mice (McGovern et al. 2008; Liu

et al. 2010) is established.Interestingly, prenatal immunoblot studies on SMA Type I

fetuses showed that SMN protein was greatly reduced in all

tissues examined, i.e. skeletal muscle, heart, brain, kidney,

thymus, pancreas and lung (Burlet et al. 1998). However,

when SMN1 and SMN2 expression was analyzed separately

in SMA fetuses and controls at 15 weeks gestation, the

contribution of the SMN2 expression to the amount of full-

length SMN protein was greater in unaffected tissues such

as muscle and kidney compared with the spinal cord (Soler-

Botija et al. 2005). These results suggest the importance of

SMN2 as a potential contributor to the disease mechanism

in SMA and its possible role in compensating pathology in

tissues clinically unaffected by the disease.

Morphological studies of motor neurons in SMA Type I

fetuses gave evidence that programmed motoneuron death

is prolonged in comparison with controls (Fidzianska &

Rafalowska, 2002; Soler-Botija et al. 2002). Nuclear abnor-

malities were found as early as 16 weeks gestation in

affected fetuses (Fidzianska & Rafalowska, 2002). More

recent neuro-pathological analyses of affected fetuses and

patients suggest a fetal developmental maturation error

and a postnatal retrograde dying-back degeneration of

lower motor neurons in SMA (Ito et al. 2011).

This review will summarize the results of studies in SMA

patients and SMA mouse models to highlight the possible


SMA: a motor neuron or multi-organ disease?, M. Shababi et al.2


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role of SMN in development and function of the nervous

system and internal organs. We will also examine the stud-

ies focusing on restoration of SMN and the impact of their

results on understanding the disease pathology.

Clinical reports of cardiac/autonomic defectsin SMA patients

Type I patients

Cardiac defects

Less than 10% of SMA Type I patients retain only one copy

of SMN2 gene and generally have a congenitally lethal

course (also sometimes denoted as SMA Type 0), whereas

80% of patients have two copies of SMN2. Most of the car-

diac defects have been reported in SMA Type I with one

SMN2 copy number, occasionally identified at prenatal

stages (Rijhsinghani et al. 1997; Sarnat & Trevenen, 2007;

Parra et al. 2012). However, the SMN2 copy numbers of

patients showing cardiac defects was not always reported

or determined due to lack of genetic information. Mean-

while, numerous cases of congenital heart defects were

reported in genetically confirmed SMA patients. These

reports indicated a series of heart malformation features

including atrial septal defects, atrioventricular septal

defects, valvular aortic stenosis, hypoplastic aortic arch,

severe coarctation of the aorta, partial atrioventricular

canal, tricuspid atresia, univentricular heart, and several

characteristics of hypoplastic left heart syndrome (Moller

et al. 1990; Burglen et al. 1995; Mulleners et al. 1996;El-Matary et al. 2004; Cook et al. 2006; Bach, 2007; Vaidla

et al. 2007; Menke et al. 2008; Rudnik-Schoneborn et al.

2008). Some of these cases were associated with bradycar-

dia (Bach, 2007), digital necrosis and vascular thrombosis

(Rudnik-Schoneborn et al. 2010). Autonomic nervous sys-

tem (ANS) dysfunction was considered to be the major

cause of distal necrosis in SMA patients.

These clinical reports clearly indicate a connection

between cardiac defects and the most severe forms of SMA,

making the previous assumption that this association is

coincidental truly doubtful. Consistent with this, one study

estimated the ratio of SMN2copy numbers and occurrence

of cardiac defects in 65 SMA Type I patients diagnosed

genetically within the first 6 months of age. Three of four

(75%) patients with one copy of SMN2 demonstrated he-

modynamically relevant atrial or ventricular septal defects.

The authors suggested that the probability of simultaneous

occurrence of both conditions is < 1 out of 50 million

patients, suggesting a role for SMN in cardiogenesis in

severe SMA cases (Rudnik-Schoneborn et al. 2008).

Despite these findings there is only little evidence for car-

diac rhythm abnormalities in SMA Type I. Electrocardio-

graphic abnormalities were found in 12 of 13 patients with

an average age of ~37 months (Coletta et al. 1989). Echo-

cardiography did not reveal cardiac structural defects;

therefore, the abnormal ECG was considered to be caused

by muscle fasciculations. Similar reports of ECG abnormali-

ties such as right ventricle overload were documented in

37% of the examined patients who were mostly SMA Type I

(Distefano et al. 1994). The authors speculated that thecause could be pulmonary hypertension due to respiration

anomalies (Distefano et al. 1994). An additional study docu-

mented that 80% of 47 SMA cases (mean age ~40 months,

including all three types) exhibited isoelectric line tremors

in electrocardiogram (ECG) tracings (Huang et al. 1996)

which did not have consequences for heart rhythm and

function.

Autonomic nervous system defects

Patients with SMA Type I normally do not live long enough

to exhibit other organ dysfunction despite progressive mus-

cle weakness, but new clinical features were observed in

patients who survived many years under assisted ventila-

tion. A large retrospective study of long-term ventilated

SMA Type I patients demonstrated that 15 of 63 patients

experienced severe, symptomatic bradycardia (Bach, 2007).

A series of autonomic tests on Type I SMA patients revealed

a sympathetic-vagal imbalance, fluctuation of blood pres-

sure, and irregular skin responses to temperature changes

(Hachiya et al. 2005). Additionally, vascular abnormalities

such as distal necrosis have been reported in SMA Type I

patients, occasionally with ASD and asymmetric left ventric-

ular hypertrophy (Araujo Ade et al. 2009; Rudnik-Schone-

born et al. 2010). In some cases, the distal necrosis occurs

with normal cardiac function (Araujo Ade et al. 2009), dem-onstrating that the autonomic nervous system dysfunction

may lead to impaired regulation of vascular tone. However,

the structural defects of the heart tissue at prenatal stages

cannot be explained by the ANS deficiency. To make a dis-

tinction between the two, it would be remarkable to iden-

tify the intrinsic heart defects independent of ANS and

analyze the ANS defects in relation to the function of other

organs including the heart. Further experiments should

address the question as to whether SMN interacts with

other genes involved in cardio- and neurogenesis. A com-

mon pathway might be provided by functional defects of

neural crest cells which migrate into the developing heart

and the large vessels and also differentiate into autonomic

and sensory nerves (Crane & Trainor, 2006).

SMA type II-III patients

Although the literature supports the conclusion that SMA

Type I patients are more likely to develop cardiac disease,

cardiac function and rhythm remain remarkably stable even

in severely handicapped SMA Type II and III patients in end

stages of the disease. Previous case reports of cardiac dys-

function, mostly AV conduction and other rhythm distur-

bances and dilative cardiomyopathy, in SMA Type III

patients have to be interpreted with caution as none of


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these patients had been genetically confirmed for SMN

deficiency. It has to be taken into account that patients

with SMA have a normal population risk to develop cardiac

insufficiency at an advanced age for unrelated reasons, e.g.

on the basis of a coronary heart disease, as this was seen ina patient with SMA Type III who was diagnosed with

enlargement and diffuse hypokinesia of cardiac ventricles

at age 65 and died at age 67 from respiratory arrest (Kuru

et al. 2009). The situation is also different in patients with

long-standing respiratory dysfunction who are prone to

right heart overloading (Distefano et al. 1994). Nonetheless,

if early-onset cardiomyopathy is seen in patients with mild

SMA, it is most likely that this patient has a different

genetic condition. Phenocopies of SMA Type III include met-

abolic diseases (e.g. acid maltase deficiency) or muscular

dystrophies. In patients with cardiac arrhythmias and AV

conduction disturbances, EmeryDreifuss muscular dystro-

phy or myotonic dystrophy type 2 have to be considered.

Lamin A/C gene mutations were seen in 10% of patients

with the diagnosis of autosomal dominant proximal SMA

(Rudnik-Schoneborn et al. 2007).

One study performed a standard ECG and a routine echo-

cardiography for 37 genetically confirmed Type II/III SMA

patients, aged 665 years, to investigate the possibility of

cardiac involvement (Palladino et al. 2011). Elevated heart

rate and left ventricular dilation were detected in only two

patients, aged 63 and 65, whose heart problems were con-

sidered to be caused by hypertension and/or coronary

artery disease (Palladino et al. 2011). These authors con-

cluded that heart dysfunction is not associated with SMATypes II-III, an observation that is in line with longer natural

history studies and clinical practice.

Cardiac defects in SMA mouse models

In contrast to humans, rodents and all other vertebrates

have a single copy of theSMNgene. MurineSmnencodes a

C at the 6th position of exon 7 and is therefore analogous

to humanSMN1. Two widely used severe SMA mouse mod-

els (SMN2 and SMND7) lack murineSmnand contain two

copies of the human SMN2 transgene. The SMN2 model

lives on average 45 days (Monani et al. 2000) and the

SMND7 model which contains the cDNA of the D7 transcript

lives slightly longer, achieving 1317 days (Le et al. 2005).

Therefore, both models likely represent very severe SMA

disease contexts. Recently, three individual labs identified

functional and structural heart defects in murine SMA,

demonstrating a link between extremely low levels of SMN

and cardiac defects (Bevan et al. 2010; Heier et al. 2010;

Shababi et al. 2010a).

Pathological/structural defects

Cardiac structural defects include inter-ventricular septum

(IVS) remodeling, under-developed left ventricular wall,

and dilated ventricles occurring at embryonic stage in

SMN2 model and shortly after birth in SMND7 model pre-

ceding motor neuron degeneration (Shababi et al. 2010a).

Analysis of the vasculature in the SMA heart also revealed a

significant reduction in the width of arterial wall and a pro-nounced decrease in the density of capillary bed (Shababi

et al. 2010a, 2012), which can certainly hinder the ability of

the heart to pump blood into other tissues/organs. A

reduced number of capillaries was also shown in the skele-

tal muscle of SMA mice (Somers et al. 2012). Additional

structural defects are marked disorganization and degener-

ation of myofibers with swollen mitochondria in SMND7

mice at postnatal day 14 (P14) (Bevan et al. 2010).

Cardiac functional defects and autonomic nervous

system

Cardiac functional analysis indicated significantly reduced

heart rate, stroke volume (SV), cardiac output, fractional

shortening (FS), and LV mass in the SMND7 heart at P7 and

14 (Bevan et al. 2010). Interestingly, systemic injection of

self-complementary (sc) AAV9 expressing the full-length

SMN cDNA which rescues the SMA phenotype (Foust et al.

2010) could not fully rescue the functional defects except

the heart rate. Therefore, the possibility of SMN functioning

in cardiogenesis and/or ANS abnormalities that contribute

to defective heart function was suggested (Bevan et al.

2010). Consistent with this, whole-mount heart immuno-

staining indicated a lack of prominent sympathetic nerves

and fewer, thinner branches in the SMA heart comparedwith age-matched heterozygous littermates (Heier et al.

2010). To distinguish the intrinsic cardiac defects from ANS

defects in SMA mice, we examined cardiac function using

cine-MRI in parallel with the structural/biochemical proper-

ties of the rescued SMND7 heart following systemic delivery

of scAAV9-SMN. Rescued mice demonstrated a significant

repair of motor function, a high level of SMN protein in the

heart, and a great improvement in cardiac structural/bio-

chemical defects (Shababi et al. 2012). However, the heart

function including the heart rate was only partially restored

compared with the age-matched wildtype, further confirm-

ing that ANS deficiency leads to inadequate heart function.

Similar to the features seen in few SMA Type I patients, dis-

tal necrosis has also been reported in intermediate SMA

mice models and therapeutically rescued severe SMA mice

(Avila et al. 2007; Gavrilina et al. 2008; Passini et al. 2010).

Extension of life span has led to increased recognition of

necrosis in the tail and ear tips of these mice. It is likely that

the necrosis is a result of impaired vascularization due to

autonomic nervous system dysfunction, which is consistent

with vascular defects detected in the SMND7 heart (Shababi

et al. 2012).

The reduced contractility in the SMA heart is attributed

to decreased sympathetic stimulation and bradycardia.

However, restoring the heart rate in the rescued SMA mice




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or increasing the heart rate in SMA mice by using drugs

cannot restore the contractile dysfunction, suggesting a

very low contractile reserve in the SMA heart (Bevan et al.

2010). Abnormalities in ATP-generating pathways (e.g.

mitochondrial function, glycolysis, glycogenolysis and phos-photransferase reactions) can reduce ATP supply in failing

hearts (Ingwall, 2009). Consistent with this, abnormal car-

diac mitochondria and hypoglycemia in SMA mice and

patients have been reported (Bruce et al. 1995; Butchbach

et al. 2009; Bevan et al. 2010). Measurement of contractility

in isolated cardiomyocytes of the SMA heart will clarify

whether the contractile dysfunction is intrinsic to the heart

or depends on ANS function. To detect ANS defects related

to cardiac function, tail-cuff and telemetric measurements

will be suitable to reveal differences in sympatho-vagal bal-

ances between the rescued SMA and wildtype mice. The

activity of autonomic nervous system regarding cardiac

function can be measured more directly during telemetry

using pharmacological drugs and examining significant

alterations in the heart rate under the inhibition of

parasympathetic and/or sympathetic nervous activity in the

rescued SMA mice.

Additional organ deficiencies in SMA patientsand mice

In contrast to numerous reports of cardiac defects in SMA

patients, the number of instances showing defects in other

peripheral organs is limited. This may be due to the fact

that severe SMA patients have various complications anddie prematurely before the damage to other organs is fully

recognized. In the following section, we examine additional

organs with reported deficiencies in SMA patients and ani-

mals models. The peripheral organ defects of SMA mice

models are summarized in Table 1.

Brain and sensory neurons

Earlier studies of SMN expression in rat, monkey and human

revealed a widespread but uneven SMN expression within

several areas of the brain. Intense expression was observed

in specific neuronal cell populations, such as in layer V pyra-

midal neurons of the neocortex, the pallidal neurons in the

basal ganglia, and the neurons of the deep cerebellar nuclei

as well as the motor neurons of the brainstem and spinal

cord (Battaglia et al. 1997). High SMN expression in spinal

preganglionic sympathetic neurons and the neocortical

pyramidal neurons, which was considered to have no effect

in SMA development at the time, may possibly be responsi-

ble for peripheral organ function and normal cognitive abil-

ity. Cognitive function is well preserved in chronic SMA,

and the overall experience in clinical practice is that infants

with SMA Type II have an early speech development in com-

parison to healthy (normally moving) toddlers. However,

formal assessments of cognitive functions are hampered by

the fact that these are standardized only for children

beyond 5 years of age. Therefore, only few data from natu-

ral history studies are available for SMA Type I patients.

Along with the observation of ANS dysfunction arising in

ventilated SMA Type I patients, it would be important tostudy cognitive development and function in long-term sur-

vivors. One larger cognitive and intelligence study of SMA

Types I-III, aged 6.018 years, was undertaken in Germany

and demonstrated no difference between SMA patients

and healthy controls (von Gontard et al. 2002).

Defective hippocampus development due to abnormali-

ties in cellular proliferation and neurogenesis is reported in

severe SMN2 mice model (Wishart et al. 2010). Additionally,

several cases of defective sensory neurons and thalamic

lesions have been detected in genetically confirmed SMA

Type I patients (Rudnik-Schoneborn et al. 2003; Ito et al.

2004), although reports of sensory neuron disorder are

more common in SMA mice (Jablonka et al. 2006; Ling

et al. 2010; Mentis et al. 2011; Gogliotti et al. 2012; Marti-

nez et al. 2012). The role of SMN in development and main-

tenance of sensory neurons is supported by high SMN levels

in dorsal root ganglia and posterior horn of the spinal cord

in human fetus, albeit with less intensity than anterior horn

(Tizzano et al. 1998). There is a considerable debate

whether the loss of synapse in sensory neurons results in

motor neuron cell death or is a consequence of SMN loss in

motor neurons. Studies in zebrafish andDrosophilasuggest

that abnormalities in sensory-motor circuit create the motor

system defects and repair of motor neural network activity

is essential to ameliorate the disease phenotype (Imlachet al. 2012; Lotti et al. 2012). On the other hand, restora-

tion of SMN in motor neurons of SMA mice repairs the NMJ

defects and also restores synapses in the sensory neurons

(Gogliotti et al. 2012; Martinez et al. 2012), suggesting that

the sensory motor circuit function is dependent on SMN lev-

els in motor neurons.

Muscle and neuromuscular junction (NMJ)

A direct effect of the SMN loss on NMJ dysfunction and

SMA progression is fully established and reviewed in great

detail (Murray et al. 2010b; Bottai & Adami, 2013; Goulet

et al. 2013); therefore, we briefly mention the reported

defects in NMJ and muscle.

In human muscle, SMN protein is localized at NMJ (Fan &

Simard, 2002) and its role in NMJ development was verified

by the failure of the cultured muscle cells derived from SMA

patients to cluster acetylcholine receptors (AChRs) at the

junction (Arnold et al. 2004). Delayed maturation of myotu-

bes in SMA fetuses has been identified (Martinez-Hernandez

et al. 2009). In addition, neurofilament accumulation along

with poor terminal arborization in postnatal diaphragm

samples of SMA Type I have been reported (Kariya et al.

2008). Further evidence for the role of NMJ in human

SMA pathology comes from a recent study that provided a




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detailed structural characterization of NMJ defects in SMA

fetuses (Martinez-Hernandez et al. 2013). The main prena-

tal defects were abnormal modification of acetylcholine

receptor clustering, irregular accumulation and positioning

of synaptic vesicles, and atypical nerve terminals in motorendplates of SMA Type I samples, whereas SMA Type II

fetuses were similar to controls.

In SMA mice, disruption of skeletal muscle molecular

composition with increased activity of cell death pathways

(Mutsaers et al. 2011), abnormal differentiation in muscle

satellite cells, deficient formation of myotubes, and

decreased muscle fiber size are reported (Le et al. 2005; Lee

et al. 2011; Hayhurst et al. 2012). Interestingly, loss of mur-

ine Smn specifically in the skeletal muscle causes muscle

necrosis, paralysis, and death (Cifuentes-Diaz et al. 2001).

Yet, restoration of SMN in the muscle or increasing the mus-

cle mass by using compounds has limited therapeutic bene-

fits in treated mice (Gavrilina et al. 2008; Rose et al. 2009;

Bosch-Marce et al. 2011), further confirming that NMJ mat-

uration defects and abnormal synapses are the hallmark of

SMA pathology (Kariya et al. 2008).

Interestingly, there are no detectable pre-symptomatic

neurodevelopmental defects in SMA mice models (Kariya

et al. 2008; McGovern et al. 2008; Murray et al. 2010a). Yet,

rapid degeneration of motor neurons at disease onset cause

several post-natal NMJ defects in SMA mice which clearly

resemble human defects. NMJ pathology in SMA mice

includes neurofilament buildup and poor axonal sprouting,

denervation, abnormal calcium homeostasis leading to

defects in motor neuron excitability, reduced terminalarborization, disruption in synaptic vesicle release, aberrant

expression of synaptic proteins, delayed post-synaptic matu-

ration, loss of Schwann cells leading to defects in compensa-

tory endplate remodeling and nerve-directed maturation of

acetylcholine receptor (AChR) clusters (Cifuentes-Diaz et al.

2002; Jablonka et al. 2007; Kariya et al. 2008; Murray

et al. 2008, 2012; Kong et al. 2009; Ling et al. 2010; Ruiz

et al. 2010; Torres-Benito et al. 2011).

A recent report demonstrates that not all the muscles in

SMA mice are uniformly affected (Ling et al. 2012). The sen-

sitive muscles in SMND7 mice which have normal NMJ for-

mation at P1, become highly denervated by P4 as the

disease progresses (Ling et al. 2012). Severely denervated

muscles in SMA mice are among axial and appendicular

muscles which are clinically related to SMA patients. This

study shows that the NMJ denervation in these muscles is

likely due to the loss in preservation of the synapse which is

consistent with other studies (Kariya et al. 2008; Murray

et al. 2012).

Liver and metabolic disorders

Reye-like syndrome of fulminant liver failure is reported in

only one case of SMA type II patient following a spinal sur-

gery, which was suggested to be caused by a combination

of different factors such as pre-operative fasting, prolonged

anesthesia, post-operative stress, and reduced fatty acid oxi-

dation (Zolkipli et al. 2012). Abnormal fatty acid metabo-

lism is the most common metabolic defect reported in

severe patients and some younger SMA Type II patients,which may return to normal with aging (Tein et al. 1995;

Crawford et al. 1999). Mild to moderate dicarboxylic acidu-

ria is consistently found in SMA Types I and II patients which

is very similar to mitochondrial b oxidation abnormalities

(Tein et al. 1995; Crawford et al. 1999). In addition, one

study reported an increased level of esterified carnitine, a

factor that performs a role in fatty acid transport from the

cytosol to the mitochondria in plasma of severe SMA

patients, concomitant with abnormalities in mitochondrial

multifunctional enzyme complex (Tein et al. 1995). How-

ever, it is not certain whether data of patients with other

entities, e.g. mitochondrial disease, were included in this

study, as it was published before SMN gene testing was

available. The level of ketone bodies (secondary products of

mitochondrial fatty acid b-oxidation in liver) in SMA

patients with metabolic disorder was normal or not signifi-

cantly reduced (Crawford et al. 1999), suggesting a normal

fatty acid utilization by the liver and, therefore, a muscle

specific defect in fatty acid metabolism. To investigate

whether fatty acid metabolism defect is a consequence of

denervation and muscle atrophy, fatty acid levels in the

plasma of 33 infants with severe SMA were measured and

compared with that of normal infants and six diseased con-

trol infants affected with equally severe denervating non-

SMA (Crawford et al. 1999). They found a significantlyincreased ratio of dodecanoic acid (C12) to tetradecanoic

acid (C14) in the plasma of all SMA patients compared with

control patients. The authors suggested that the fatty acid

metabolism disorder in SMA is directly related to the loss of

SMN1 or possibly other genes in the chromosome 5q and is

not the consequence of muscle denervation and/or atrophy

(Crawford et al. 1999). Further evidence of fatty acid oxida-

tion disorder is based on the autopsy samples of some

infants with severe SMA who showed fatty vacuolization of

the liver (Crawford et al. 1999).

The role of SMN in the development and function of liver

in mice was demonstrated by a study in which a mutation

in the exon 7 of murine Smn directed to liver led to liver

failure and late embryonic lethality of transgenic mice (Vitte

et al. 2004). Importantly, however, this animal model may

produce SMN levels that are comparable to a null-state

which is not SMA, but would be predicted to be lethal for

any tissue. The importance of liver in SMA pathology was

indicated by the experiments in which restoration of SMN

and subsequent increase in insulin-like growth factor (IGF)1

levels in the liver of SMA mice, through subcutaneous injec-

tion of therapeutic oligonucleotide (ASO), was an impor-

tant factor for high degree of rescue (Hua et al. 2011).

Premature death of SMA severe patients in infancy is a

significant hurdle for continuous analysis of the urine and




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plasma to investigate the exact role that SMN may play in

metabolic defects. While controversial in the community, a

specific diet has not been scientifically evaluated. On the

other hand, extreme muscle wasting in severe SMA patients

could be explained by defects in fatty acid transport/oxida-tion, which plays a major role in energy production during

prolonged fasting, resulting in a detrimental effect on

working cardiac and skeletal muscle. A thorough investiga-

tion of lipid content in the liver and possibly muscle of SMA

mice models, before and after the disease onset, can pro-

vide valuable information regarding the direct or indirect

effect of SMN deficiency on metabolic abnormalities.

Pancreas

While single case reports do not provide sufficient evidence

for an association of diabetes mellitus and SMA, consider-

ing the frequency of both conditions, there are occasional

cases reporting diabetes and abnormalities in the glucose

metabolism in a few genetically confirmed SMA Type II and

III patients (Bowerman et al. 2012c; Lamarca et al. 2012). In

addition, three cases of acute pancreatitis among long-term

survivors of ventilated SMA Type I patients (Bach, 2007) and

pathological pancreatic defects in autopsied pancreas speci-

mens from infants and an intermediate SMA mouse model

(SMA2B/) have been documented (Bowerman et al. 2012c).

The metabolic defects in SMA2B/ mice were characterized

by fasting hyperglycemia, glucose intolerance, hypersensi-

tivity to insulin, and hyperglucagonemia. Pathological

defects were identified by loss of insulin-producing b cellsand a corresponding increase in the number of the gluca-

gon-producing a cells in pancreatic islets of mice and

human specimens. Based on the observation that the pan-

creatic pathology and fasting hyperglycemia occurred

before the onset of SMA symptoms, the authors suggested

that the pancreatic phenotype is independent of the neuro-

nal SMA phenotype and is rather a direct consequence of

SMN deficiency within the pancreas (Bowerman et al.

2012c).

Careful observation of SMA patients regarding their glu-

cose homeostasis is essential to shed light on the role of

SMN in glucose metabolism and pancreas function. In the

study of Bach (2007), one patient who had acute pancreati-

tis was taking valproic acid in a research protocol. Valproic

acid is well known to be associated with an increased pan-

creatitis risk (Asconape et al. 1993). Therefore, considering

a possible disposition to pancreatic dysfunction in severe

SMA, clinicians have to be alerted about corresponding side

effects of medications used in clinical trials or clinical care.

Intestine

Thus far, the existence of intestinal issues in SMA patients is

considered to be the result of muscle weakness. Intestinal

problems have been recorded in SMA mice (Le et al. 2011;

Schreml et al. 2012) and two SMA patients (Type II and Type

IV) represented by severe constipation (Ionasescu et al.

1994; Khawaja et al. 2004). In addition, a micro-dissection

study revealed significantly low values for fractional area of

neural tissue in small intestine and colon of severe SMApatients (Galvis et al. 1992), suggesting that the inadequacy

of the autonomic nervous system contributes to functional

impairments.

Intestinal problems in mice were recorded as impacted

bowel and pockets of fluid and gas (pneumoperitoneum) in

SMN-induced transgenic mice following extended survival

(Le et al. 2011). Pathological defects in intestine of severe

Taiwanese SMA mice are characterized by string-like intes-

tine, reduced numbers of villi, intracytoplasmic vacuoles

predominantly at the tips of the villi, severe intramural

edema in the lamina propria, severe diarrhea, and opaque

fluid in the abdomen of most late-stage SMA animals

(Schreml et al. 2012). Similar to other organs, the pathologi-

cal impact of SMN deficiency on the intestine may not be

fully recognized due to the premature death of severe SMA

patients and mice.

Lung

Pulmonary complications are the most reported condition

and by far the most devastating aspect of the disease

leading to the death of severe SMA patients at infancy.

In contrast to infantile SMA with respiratory distress

Type 1 (SMARD1), where diaphragmatic palsy is a hallmark

(Rudnik-Schoneborn et al. 2004), SMA with SMN deficiencyresults in intercostal muscle weakness and thoracic cage

deformation, thereby reducing pulmonary volume and ven-

tilatory capacity. SMA Type I patients frequently show para-

doxical breathing caused by preserved diaphragmatic

movements. Complications include airway obstruction, air-

way inflammation, increased mucus production, aspiration

causing pneumonia, lung damage, diminished ability to

clear secretions, weakened pulmonary defenses, restrictive

lung disease, and respiratory arrest. Non-invasive mechani-

cal ventilation and tracheostomy can both extend survival

of SMA Type I patients, but tracheostomy results in perma-

nent ventilator reliance and prevents speech development

(Bach et al. 2007).

Since respiratory complications mostly arise from hypo-

ventilation and subsequent respiratory infections, there is

little data focusing upon the structural lung damage in

SMA autopsies. Several cases of aletectasis (one was associ-

ated with bradycardia) are reported (Collado-Ortiz et al.

2007; Modi et al. 2010; Henrichsen et al. 2012). Most

recently, lung structural defects were indicated in the severe

Taiwanese mice (Schreml et al. 2012). These defects were

described as discolorations of the lungs compatible with

atelectasis or pulmonary infarctions, ruptured alveolar sep-

tum, and emphysema. Whether the pathological defects in

the lungs of SMA mice are relevant to SMA patients is an




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open question. Extension of survival through mechanical

means or any other therapeutic strategies may reveal more

potential pathological defects in the lungs of SMA patients

than presently known.

Restoration of SMN only in motor neuronsdoes not fully rescue severe SMA mice

Many researchers have focused on different methods to

restore SMN protein in motor neurons of SMA mice. The

outcome of these experiments demonstrate that increasing

SMN levels exclusively in motor neurons has a surprisingly

small impact upon survival and weight gain of severe SMA

mice. As therapeutics move forward towards the clinic, it

is essential to fully understand the pathology of SMA

and determine the temporal requirements for SMN restora-

tion within these tissues. Different approaches designed

to increase the SMN levels in motor neurons and their

outcomes are summarized.

Transgenic mouse models

Transgenic models have been created to examine the con-

tribution of specific cell types, most notably, skeletal muscle

and neurons (Gavrilina et al. 2008). When full-length SMN

is expressed under the control of the HSA promoter (which

should be highly expressed in skeletal muscle), the severe

SMA phenotype was only marginally improved. In contrast,

SMNexpression driven by the PrP promoter (which should

be highly expressed in several populations within the cen-tral nervous system, including neuronal and glial popula-

tions) significantly extended survival (Gavrilina et al. 2008).

Interestingly, although average life span was extended

from ~5 days to 150210 days, some mice died prematurely

for unknown reasons (Gavrilina et al. 2008). This could be

due to the nature of transgenic animals or may suggest that

additional tissues are required for a complete rescue. In a

recent study, doxycycline-dependent induction of SMN lev-

els in a transgenic SMA model at P1 rescued the SMA phe-

notype (Le et al. 2011). However, 70% of the animals died

within a month once SMN induction was stopped, even

though the physiology and morphology of the neuromus-

cular junctions were largely normal at the time of death (Le

et al. 2011). Lutz et al. (2011) have used an inducible SMN

rescue allele that was induced at different time points to

investigate the temporal requirement for SMN in order to

achieve rescue. They observed a significant extension of life

span in approximately 50% of the rescued mice when the

SMN allele was induced at symptomatic stage of PND46.

They concluded that the beneficial effect of SMN induction

in their rescued mice was due to SMN restoration to all tis-

sues, and not exclusively to motor neurons. Their justifica-

tion for the lack of rescue in 50% of their SMA mice relies

on the possibility of the heart abnormalities. Another

report described using an inducible allele (Hb9-Cre) that

restored SMN solely in motor neurons and repaired the

motor neuron phenotype but resulted in a very modest

increase in survival (Gogliotti et al. 2012). The authors spec-

ulated that the minimal extension of life span may be due

to lack of autonomic innervations of the heart. In a comple-mentary study, SMN was depleted specifically in motor neu-

rons and resulted in only a mild cellular phenotype rather

than the predicted severe SMA phenotype (Park et al.

2010). These studies suggest that the composite SMA phe-

notype and disease progression is the result of SMN short-

age in additional tissues and the full phenotypic rescue

requires SMN restoration in all tissues.

Therapeutics and delivery methods

The delivery method of the therapeutics to restore the SMN

in the CNS as well as the peripheral organs may have a

great impact on the rate of the rescue. One of the most

effective RNA therapeutics is a short, antisense oligonucleo-

tide (ASO) that blocks the inhibitory activity of ISS-N1

located within intron 7 and increases the inclusion of exon 7

(Singh et al. 2009, 2010; Hua et al. 2010, 2011; Passini et al.

2011; Osman et al. 2012; Porensky et al. 2012). Extension in

life span and preservation of neuromuscular junctions of

SMA mice has been observed with 2-O-2-methoxyethyl-

modified ASO, referred to as ASO-10-27, when delivered

via a single intracerebroventricular (ICV) injection into

SMND7 mice (Passini et al. 2011). Surprisingly, a recent

report described the positive effect of the ASO-10-27 on

the survival of severe Taiwanese mice by subcutaneous(SC) injection (Hua et al. 2011). Their results indicated that

SC injection of ASO extended the survival much more

effectively than ICV injection, even though the ASO dose

used in ICV injection was lower than that in SC injection.

Interestingly, combination of both injection methods

increased the life span even longer than each individual

injection. They concluded that SMN restoration in periph-

eral tissues in combination with partial restoration in the

CNS can achieve efficient rescue of severe SMA mice. On

the other hand, a recent study reported a life span of

more than 100 days for SMND7 mice following a single

ICV injection of morpholino (MO) oligomer against ISS-N1

(Porensky et al. 2012). This study concluded that there was

no difference in the survival of the ICV-injected animals vs.

those treated with combined ICV and systemic injection.

According to their results, SMN increase was minimal in

the peripheral organs of ICV-injected mice, suggesting that

the rescue was due to early salvage of the motor pheno-

type. Additional therapeutic intervention is gene replace-

ment therapy using scAAV9 expressing full-length SMN

cDNA that has provided the most substantial improvement

in severe SMA mice models (Foust et al., 2010; Valori et al.

2010; Dominguez et al. 2011; Glascock et al. 2012a,b;

Shababi et al. 2012; Benkhelifa-Ziyyat et al. 2013). How-

ever, sudden deaths, cardiac/respiratory complications, and




10/14

average life spans of 6570 days are common following

intravenous delivery of scAAV9-SMN. We have recently

reported a significantly increased level of strength and

weight gain in scAAV9-SMN injected mice through ICV

delivery than IV delivery using a low dose of scAAV9-SMN(Glascock et al. 2012b). Additionally, our survival analysis

demonstrated that ICV-treated mice displayed fewer early

deaths than IV-treated animals. ICV injection at P2 can also

result in dissemination of the virus into the peripheral

organs (due to immature state of the blood brain barrier)

and, therefore, the possibility remains that direct delivery

of SMN into the CNS along with partial restoration in the

peripheral organs leads to an efficient rescue. An alterna-

tive possibility is that ICV delivery of the therapeutic virus

is more suitable than IV delivery for efficient transduction

of the ANS and subsequently leads to more improved

function of the peripheral organs.

Chemical compounds

A variety of chemical compounds have been examined

within the context of SMA models as well as SMA patients.

Recent reviews have focused upon these compounds and

their modes-of-action (Lorson et al., 2010; Shababi et al.

2010b; Cherry & Androphy, 2012; Lewelt et al. 2012; Lorson

& Lorson, 2012). Several therapeutic compounds used in

SMA treatment such as Rock inhibitors and follistatin had a

positive effect on NMJ maturation (Rock inhibitors) and

increased the numbers of motor neurons in the lumbar

spinal cord (Follistatin) (Rose et al. 2009; Bowerman et al.2010, 2012b). However, their effect is SMN-independent,

resulting in a very modest increase in the survival of the

SMA mice.

Additional compounds such as HDACi and prolactin (PLR)

are capable of SMN induction, but their effect on survival

is either insignificant or modest, respectively. The HDACi-

treated severe Taiwanese mice demonstrated normal motor

endplate and NMJ morphology but significant structural

damages to the heart, intestine, and lung at P5 (Schreml

et al. 2012). PLR-treated SMND7 mice had a modest exten-

sion in the survival (~70%), even though SMN protein

induction in the CNS of these mice was higher than that in

wildtype or heterozygous mice (Farooq et al. 2011). One of

the factors considered to contribute to the moderate rescue

was the lack of SMN induction in the peripheral organs,

including cardiac tissue.

Conclusion

Although it is clear that the primary pathology in SMA is

neurodegeneration, there is increasing evidence from clini-

cal reports and animal studies that other tissues are

involved in the overall phenotype, especially in the most

severe forms of the disease. Additional complications in

patients include autonomic nervous system involvement,

congenital heart defects, liver, pancreas and intestinal dys-

function, and metabolic deficiencies. In SMA mouse models,

further features are observed, such as cardiac structural and

functional defects along with rhythm disturbances, defec-

tive development of specific brain areas, and moreextended necrosis of tail and ears. However, mice are not

men, and there remain significant differences in the pheno-

typic features of patients and mouse models which cannot

be explained so far. Taking the impact of respiratory, limbic,

cardiac, and autonomic nervous system dysfunction in

severe SMA patients into account, it will be beneficial to

reveal the potential pathological defects in each organ

before and after the disease onset to distinguish the specific

defects occurring as a direct result of SMN shortage from

those which are the byproduct of disease progression. Since

the total loss of SMN results in embryonic lethality, it is not

surprising that the extremely low levels of SMN in severely

affected patients lead to detrimental damages in every tis-

sue. Therefore, to develop the most efficient therapeutic

approach and also prevent further complications that may

arise with extended survival following therapeutic interven-

tions, it is necessary to investigate the specific damages to

every system independently in detail. The comparison of

the defects in SMA mice models will provide valuable

insights if they are accompanied by similar studies in autop-

sied specimens of SMA patients. One of the most important

issues in regard to treating SMA patients by any therapeutic

means is to maintain a broad and extensive outlook to

ensure the ability of clinicians to predict and contain atypi-

cal complications which may arise due to the strain onperipheral organs as a result of increased survival.

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