Spinal muscular atrophy: a motor neuron disorder or a multi-organ disease?

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 Motor Neuron-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.

Introduction

Clinical classification of SMA

Spinal muscular atrophy (SMA) is an autosomal recessive disorder that is the most common inherited motor neuron disease 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 weakness 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 (I–IV) 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 Kugelberg–Welander 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, congenital 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-Schöneborn 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 disease; approximately 70% of Type II patients reach adulthood but the life expectancy of Types III–IV 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 5q (Brzustowicz et al. 1990; Lefebvre et al. 1995). A human-specific copy gene, SMN2, exists on the same region and differs from SMN1 by only a few silent nucleotides (Lefebvre et al. 1995; Rochette et al. 2001). Therefore, SMN2 encodes an identical protein to SMN1, although it cannot fully restore 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 (SMNΔ7) (Lorson et al. 1999; Monani et al. 1999). While SMN1-derived transcripts produce full-length and functional SMN protein, nearly 90% of SMN2-derived transcripts generate a truncated and unstable protein (SMNΔ7), which lacks the 16 amino acids encoded by exon 7 (Lorson et al. 1998, 1999; Monani et al. 1999). Importantly, however, SMN2 produces a low amount of full-length SMN, comparable to ∼ 10% of SMN1 levels (Lorson & Androphy, 2000). SMN2 is paradoxically a double-edged sword: it provides only a sufficient level of SMN protein 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 SMN2 copies, 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 rare instances.

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 Drosophila and mice has supported the hypothesis that SMA develops 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 assembly 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), followed 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 zebrafish (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 abnormalities 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 role of SMN in development and function of the nervous system and internal organs. We will also examine the studies focusing on restoration of SMN and the impact of their results on understanding the disease pathology.

Clinical reports of cardiac/autonomic defects in 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 cardiac 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. Meanwhile, 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-Schöneborn et al. 2008). Some of these cases were associated with bradycardia (Bach, 2007), digital necrosis and vascular thrombosis (Rudnik-Schöneborn et al. 2010). Autonomic nervous system (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 SMN2 copy 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 hemodynamically 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-Schöneborn et al. 2008).

Despite these findings there is only little evidence for cardiac rhythm abnormalities in SMA Type I. Electrocardiographic abnormalities were found in 12 of 13 patients with an average age of ∼ 37 months (Coletta et al. 1989). Echocardiography did not reveal cardiac structural defects; therefore, the abnormal ECG was considered to be caused by muscle fasciculations. Similar reports of ECG abnormalities 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 the cause could be pulmonary hypertension due to respiration anomalies (Distefano et al. 1994). An additional study documented 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 muscle weakness, but new clinical features were observed in patients who survived many years under assisted ventilation. 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 pressure, 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 ventricular hypertrophy (Araujo Ade et al. 2009; Rudnik-Schöneborn et al. 2010). In some cases, the distal necrosis occurs with normal cardiac function (Araujo Ade et al. 2009), demonstrating 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 distinction between the two, it would be remarkable to identify 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 common 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 dysfunction, mostly AV conduction and other rhythm disturbances and dilative cardiomyopathy, in SMA Type III patients have to be interpreted with caution as none of 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 in a 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 metabolic diseases (e.g. acid maltase deficiency) or muscular dystrophies. In patients with cardiac arrhythmias and AV conduction disturbances, Emery–Dreifuss muscular dystrophy 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-Schöneborn et al. 2007).

One study performed a standard ECG and a routine echocardiography for 37 genetically confirmed Type II/III SMA patients, aged 6–65 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 considered to be caused by hypertension and/or coronary artery disease (Palladino et al. 2011). These authors concluded that heart dysfunction is not associated with SMA Types 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 the SMN gene. Murine Smn encodes a ‘C’ at the 6th position of exon 7 and is therefore analogous to human SMN1. Two widely used severe SMA mouse models (‘SMN2’ and SMNΔ7) lack murine Smn and contain two copies of the human SMN2 transgene. The SMN2 model lives on average 4–5 days (Monani et al. 2000) and the SMNΔ7 model which contains the cDNA of the Δ7 transcript lives slightly longer, achieving 13–17 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 SMNΔ7 model preceding 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 pronounced 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 skeletal muscle of SMA mice (Somers et al. 2012). Additional structural defects are marked disorganization and degeneration of myofibers with swollen mitochondria in SMNΔ7 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 SMNΔ7 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 compared with 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 properties of the rescued SMNΔ7 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/biochemical defects (Shababi et al. 2012). However, the heart function including the heart rate was only partially restored compared with the age-matched wildtype, further confirming that ANS deficiency leads to inadequate heart function. Similar to the features seen in few SMA Type I patients, distal 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 SMNΔ7 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 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 phosphotransferase reactions) can reduce ATP supply in failing hearts (Ingwall, 2009). Consistent with this, abnormal cardiac 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 balances 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 patients and 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 and die 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 animals models. The peripheral organ defects of SMA mice models are summarized in Table 1.

Table 1.

Summary of peripheral organ defects in SMA mouse models

	SMA mouse models
Affected organs/systems	SMN2(1)	Δ7(2)	2B/−(3)	Taiwanese (severe)(4)	Transgenic and knockout
Brain/sensory neurons	defective hypocampus development, ↓ neurogenesis(5) ↓ neurite outgrowth, smaller growth cones(6)	Loss of central synapse and sensory neurons(7,8) ↓ motor circuit function(9)	Not Determined	Not Determined	Loss of sensory neurons is due to motor neuron loss(10)
Muscle/NMJ	↓ presynaptic input and post-synaptic pathology ↑ neurofilaments(11), Abnormal differentiation of satellite cells and myotube formation(12)	Denervation in vulnerable muscles(8), abnormal synapses, ↓ Neurotransmission(9,11,13,14,15) small muscle fiber size(2, 16)	↓ paralysis-induced remodeling ↓ nerve-directed re-organization of AChR(17) ↑ Denervation of TVA and LALr ↑neurofilaments(3)	Change in muscle molecular composition(18)	Muscle necrosis/paralysis and death in mice lacking murine Smn in skeletal muscle(19)
Cardio-vascular	IVS remodeling and thin ventricular wall(20)	IVS/Vascular remodeling, interstitial fibrosis(20) ↓ Capillaries smaller cardio-myocytes(21) ↓ Heart function(21,22) and irregular mitochondria(22)	Not Determined	IVS remodeling(23)	Not Determined
Autonomic nervous system	Not Determined	Bradycardia(20,21,22,24), Distal necrosis in the scAAV9-rescued(25,26)	Not Determined	Cataracts and distal necrosis in the rescued(23), distal necrosis in SAHA- treated(4)	Not Determined
Lung	Not Determined	Not Determined	Not Determined	Dark red spots in the lungs, ruptured alveolar septum, and emphysema(27)	Not Determined
Intestine	Not determined	Not Determined	Not Determined	Diarrhea, ↓ numbers of villi, intramural edema in lamina propria, rectal prolapsed(27)	Impacted bowel and pockets of fluid and gas in SMN-induced mice(28)
Pancreas	Not Determined	Not Determined	↓ β cells/↑α cells in pancreatic islets(29)	Not Determined	Not Determined
Liver and metabolic	Not Determined	Hypoglycemia (related to small body mass)(30)	Fasting hyperglycemia glucose intolerance, hypersensitivity to insulin, and hyper-glucagonemia(29)	Reduced level of IGFALS in the liver(23)	Embryonic lethality in transgenic mice lacking murine Smn in the liver(31)

AChR, acetylcholine receptors; IGFALS, IGF-binding-protein acid labile subunit; IVS, interventricular septum; LALr, levator auris longus rostral band; TVA, trans vs. abdominis.

↑: Increase; ↓: Decrease.

¹Monani et al. (2000); ²Le et al. (2005); ³Bowerman et al. (2012a); ⁴Riessland et al. (2010); ⁵Wishart et al. (2010); ⁶Jablonka et al. (2006); ⁷Ling et al. (2010); ⁸Ling et al. (2012); ⁹Mentis et al. (2011); ¹⁰Gogliotti et al. (2012); ¹¹Murray et al. (2008); ¹²Hayhurst et al. (2012); ¹³Kariya et al. (2008); ¹⁴Torres-Benito et al. (2011); ¹⁵Ruiz et al. (2010); ¹⁶Lee et al. (2011); ¹⁷Murray et al. (2012); ¹⁸Mutsaers et al. (2011); ¹⁹Cifuentes-Diaz et al. (2001); ²⁰Shababi et al. (2010a); ²¹Shababi et al. (2012); ²²Bevan et al. (2010); ²³Hua et al. (2011); ²⁴Heier et al. (2010); ²⁵Foust et al. (2010); ²⁶Glascock et al. (2012b); ²⁷Schreml et al. (2012); ²⁸Le et al. (2011); ²⁹Bowerman et al. (2012c); ³⁰Butchbach et al. (2009); ³¹Vitte et al. (2004).

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 pyramidal 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 responsible for peripheral organ function and normal cognitive ability. 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 comparison 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 natural 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 to study cognitive development and function in long-term survivors. One larger cognitive and intelligence study of SMA Types I-III, aged 6.0–18 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 abnormalities 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-Schöneborn 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; Martinez et al. 2012). The role of SMN in development and maintenance 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 and Drosophila suggest 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 (Imlach et al. 2012; Lotti et al. 2012). On the other hand, restoration 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 levels 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 myotubes 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 detailed structural characterization of NMJ defects in SMA fetuses (Martinez-Hernandez et al. 2013). The main prenatal defects were abnormal modification of acetylcholine receptor clustering, irregular accumulation and positioning of synaptic vesicles, and atypical nerve terminals in motor endplates 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 murine 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 muscle mass by using compounds has limited therapeutic benefits in treated mice (Gavrilina et al. 2008; Rose et al. 2009; Bosch-Marce et al. 2011), further confirming that NMJ maturation 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 terminal arborization, disruption in synaptic vesicle release, aberrant expression of synaptic proteins, delayed post-synaptic maturation, loss of Schwann cells leading to defects in compensatory 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 sensitive muscles in SMNΔ7 mice which have normal NMJ formation 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 surgery, 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 oxidation (Zolkipli et al. 2012). Abnormal fatty acid metabolism 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 aciduria is consistently found in SMA Types I and II patients which is very similar to mitochondrial β 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). However, 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 β-oxidation in liver) in SMA patients with metabolic disorder was normal or not significantly 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 control infants affected with equally severe denervating non-SMA (Crawford et al. 1999). They found a significantly increased 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 oxidation 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 injection of therapeutic oligonucleotide (ASO), was an important 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 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/oxidation, which plays a major role in energy production during prolonged fasting, resulting in a detrimental effect on working cardiac and skeletal muscle. A thorough investigation of lipid content in the liver and possibly muscle of SMA mice models, before and after the disease onset, can provide 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, considering 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 specimens from infants and an intermediate SMA mouse model (SMA^2B/−) have been documented (Bowerman et al. 2012c). The metabolic defects in SMA^2B/− mice were characterized by fasting hyperglycemia, glucose intolerance, hypersensitivity to insulin, and hyperglucagonemia. Pathological defects were identified by loss of insulin-producing β cells and a corresponding increase in the number of the glucagon-producing α cells in pancreatic islets of mice and human specimens. Based on the observation that the pancreatic pathology and fasting hyperglycemia occurred before the onset of SMA symptoms, the authors suggested that the pancreatic phenotype is independent of the neuronal 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 glucose 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 pancreatitis was taking valproic acid in a research protocol. Valproic acid is well known to be associated with an increased pancreatitis 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 SMA patients (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 intestine, 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 pathological 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-Schöneborn et al. 2004), SMA with SMN deficiency results in intercostal muscle weakness and thoracic cage deformation, thereby reducing pulmonary volume and ventilatory capacity. SMA Type I patients frequently show paradoxical breathing caused by preserved diaphragmatic movements. Complications include airway obstruction, airway 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 mechanical ventilation and tracheostomy can both extend survival of SMA Type I patients, but tracheostomy results in permanent ventilator reliance and prevents speech development (Bach et al. 2007).

Since respiratory complications mostly arise from hypoventilation and subsequent respiratory infections, there is little data focusing upon the structural lung damage in SMA autopsies. Several cases of aletectasis (one was associated 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 septum, and emphysema. Whether the pathological defects in the lungs of SMA mice are relevant to SMA patients is an 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 neurons does 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 restoration 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 contribution 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, SMN expression driven by the PrP promoter (which should be highly expressed in several populations within the central nervous system, including neuronal and glial populations) significantly extended survival (Gavrilina et al. 2008). Interestingly, although average life span was extended from ∼ 5 days to 150–210 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 levels in a transgenic SMA model at P1 rescued the SMA phenotype (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 neuromuscular 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 PND4–6. They concluded that the beneficial effect of SMN induction in their rescued mice was due to SMN restoration to all tissues, and not exclusively to motor neurons. Their justification 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 speculated that the minimal extension of life span may be due to lack of autonomic innervations of the heart. In a complementary study, SMN was depleted specifically in motor neurons 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 phenotype and disease progression is the result of SMN shortage 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 oligonucleotide (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 SMNΔ7 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 peripheral 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 SMNΔ7 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 phenotype. Additional therapeutic intervention is gene replacement 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). However, sudden deaths, cardiac/respiratory complications, and average life spans of 65–70 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 alternative 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 SMNΔ7 mice had a modest extension 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 clinical 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 dysfunction, and metabolic deficiencies. In SMA mouse models, further features are observed, such as cardiac structural and functional defects along with rhythm disturbances, defective development of specific brain areas, and more extended necrosis of tail and ears. However, mice are not men, and there remain significant differences in the phenotypic 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 tissue. Therefore, to develop the most efficient therapeutic approach and also prevent further complications that may arise with extended survival following therapeutic interventions, 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 autopsied 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 atypical complications which may arise due to the strain on peripheral organs as a result of increased survival.