Human Molecular Genetics Advance Access originally published online on December 8, 2007
Human Molecular Genetics 2008 17(7):949-962; doi:10.1093/hmg/ddm367
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Selective vulnerability of motor neurons and dissociation of pre- and post-synaptic pathology at the neuromuscular junction in mouse models of spinal muscular atrophy
1 Centre for Integrative Physiology 2 Centre for Neuroscience Research, University of Edinburgh Medical School, Edinburgh EH8 9XD, UK 3 MRC Functional Genetics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, UK
* To whom correspondence should be addressed. Tel: +44 1316503724; Email: t.gillingwater{at}ed.ac.uk
Received October 30, 2007; Accepted December 5, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALFUNDINGREFERENCES |
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Proximal spinal muscular atrophy (SMA) is a common autosomal recessive childhood form of motor neuron disease. Previous studies have highlighted nerve- and muscle-specific events in SMA, including atrophy of muscle fibres and post-synaptic motor endplates, loss of lower motor neuron cell bodies and denervation of neuromuscular junctions caused by loss of pre-synaptic inputs. Here we have undertaken a detailed morphological investigation of neuromuscular synaptic pathology in the Smn–/–;SMN2 and Smn–/–;SMN2;
7 mouse models of SMA. We show that neuromuscular junctions in the transversus abdominis (TVA), levator auris longus (LAL) and lumbrical muscles were disrupted in both mouse models. Pre-synaptic inputs were lost and abnormal accumulations of neurofilament were present, even in early/mid-symptomatic animals in the most severely affected muscle groups. Neuromuscular pathology was more extensive in the postural TVA muscle compared with the fast-twitch LAL and lumbrical muscles. Pre-synaptic pathology in Smn–/–;SMN2;7 mice was reduced compared with Smn–/–;SMN2 mice at late-symptomatic time-points, although post-synaptic pathology was equally severe. We demonstrate that shrinkage of motor endplates does not correlate with loss of motor nerve terminals, signifying that one can occur in the absence of the other. We also demonstrate selective vulnerability of a subpopulation of motor neurons in the caudal muscle band of the LAL. Paralysis with botulinum toxin resulted in less terminal sprouting and ectopic synapse formation in the caudal band compared with the rostral band, suggesting that motor units conforming to a Fast Synapsing (FaSyn) phenotype are likely to be more vulnerable than those with a Delayed Synapsing (DeSyn) phenotype. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALFUNDINGREFERENCES |
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Proximal autosomal recessive spinal muscular atrophy (SMA) is the most common genetic cause of infant mortality in humans, with an incidence of around 1:6000–10 000 and carrier frequency of around 1:35 (1–2). This autosomal recessive neuromuscular disease targets lower (alpha) motor neurons in the ventral horn of the spinal cord, resulting in denervation and atrophy of muscles in the limbs and trunk (3). SMA is divided up into three main subtypes (Type I, II and III), defined by the age of onset and motor milestones reached. The most severe form of SMA, Type I (also known as Werdnig–Hoffmann disease), has a disease onset within the first 6 months of life, is characterized by generalized muscle weakness and hypotonia (presenting as a ‘floppy infant’) and a failure to achieve sitting unaided, with death usually occurring within the first 2 years of life (4).
SMA is caused by low levels of a ubiquitous protein product expressed by a single gene: the survival motor neuron (SMN) gene, which exists in two near identical copies (3,5). Reduced protein levels result from loss or disruption of the telomeric SMN gene (SMN1) with retention of the centromeric SMN gene (SMN2). Copy number of SMN2 determines disease severity. Despite a clear understanding of the genetic cause of SMA, our understanding of the cellular and molecular mechanisms through which mutations in SMN lead to selective motor neuron loss and clinical symptoms remain controversial (2). The lack of availability of suitable human material, either from biopsy or post-mortem, has led many groups to make use of mouse models in order to investigate disease mechanisms. Although several different mouse models of SMA exist (6–9), one common pathological feature shared by most is an unambiguous denervation of skeletal muscle fibres (7–9). As a result, peripheral axons and synapses appear to be important pathological targets of SMN mutations in SMA, with significant disruption of neuromuscular synapses (characterized by a loss of pre-synaptic nerve terminals) occurring alongside muscle atrophy (7–10).
The presence of neuromuscular abnormalities focused on neuromuscular junctions and skeletal muscle fibres suggests that a more detailed understanding of pathological events occurring in these locations will provide important insights into disease mechanisms underlying SMA. For example, despite evidence suggesting that both neuron-specific and muscle-specific events can occur during SMA (11–15), no studies to date have addressed whether muscle atrophy occurs as a direct result of synaptic denervation at the mammalian neuromuscular junction. Teasing apart nerve- and/or muscle-specific roles of SMN will be important for developing therapeutic strategies that target all pathological sites in SMA. Similarly, it is not known whether certain intrinsic characteristics of motor neurons [e.g. fast-fatiguable, fast fatigue-resistant and slow phenotypes (16–18)] can lead to modifications in their susceptibility to SMA-induced disease stimuli. The identification of intrinsic factors that make subpopulations of motor neurons particularly vulnerable, or resistant, to SMA is likely to highlight critical molecular and cellular targets for the development of new therapies and treatments.
Here we have undertaken a detailed analysis of synaptic pathology at the neuromuscular junction in two different mouse models of SMA: the Smn–/–;SMN2 mouse model of type I SMA (6) and the Smn–/–;SMN2;7 mouse model with a modified, less severe phenotype (9). We have used immunocytochemistry, quantitative fluorescence/confocal microscopy and electron microscopy to demonstrate that neuromuscular synapses are grossly disrupted in late-symptomatic (P5–P6) Smn–/–;SMN2 mice, with postural muscles in the trunk being more severely affected than the fast-twitch muscles in both the trunk and lower limbs. Synaptic abnormalities were also present in severely affected muscles at pre-symptomatic time-points (P2). Smn–/–;SMN2;7 mice also showed signs of synaptic disruption at early/mid-symptomatic time-points (P7), but in late-symptomatic mice (P14) post-synaptic pathology had progressed significantly but pre-synaptic pathology had not extended much beyond levels observed at P7. These findings support the hypothesis that synapses are vulnerable in SMA (19). We also show that shrinkage of post-synaptic motor endplates does not correlate with pre-synaptic motor nerve terminal loss, suggesting that pre- (neuronal) and post- (muscular) synaptic pathology can occur independently of one another. Finally, our investigations of neuromuscular pathology in the levator auris longus (LAL) muscle identified selectivity vulnerable subpopulations of motor neurons, with those conforming to a Fast Synapsing (FaSyn) phenotype (20) likely to be more susceptible in SMA than those with a Delayed Synapsing (DeSyn) phenotype.
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RESULTS |
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Motor nerve terminal loss, neurofilament accumulation and muscle fibre shrinkage in Smn–/–;SMN2 mice
We chose to investigate neuromuscular pathology in a range of muscles from the trunk and hind limbs of the mouse, each allowing assessment of the entire synaptic innervation of the muscle in whole-mount preparations. We examined synaptic pathology at the neuromuscular junction in the transversus abdominis [TVA; a postural muscle from the anterior abdominal wall innervated by lower intercostal nerves, known to be affected in other mouse models of motor neuron degeneration (21)], LAL [a pure fast-twitch muscle from the dorsal surface of the head innervated by the facial nerve (22,23)] and deep lumbrical muscles (fast-twitch muscles from the hind-paw innervated by terminal branches of the tibial nerve). These preparations allowed us to quantify and correlate pre- and post-synaptic pathology in muscle groups taken from several anatomical regions of the mouse, whilst also providing the ability to compare pathology in predominantly slow-twitch (TVA) versus fast-twitch (LAL and lumbricals) muscles as well as muscles innervated by nerves with long (lumbricals) and short (TVA/LAL) nerve stumps.
Neuromuscular junctions were labelled in whole-mount TVA, LAL and lumbrical preparations from late-symptomatic (P5–P6) Smn–/–;SMN2 mice with a severe SMA (type I) phenotype. Care was taken during dissection to make sure that the muscle and its nerve supply were removed in their entirety, especially for the LAL which has two constituent muscle bands (rostral and caudal; discussed later). Pre-synaptic axons and motor nerve terminals were labelled using antibodies directed against 150 kDa neurofilament proteins (green; Fig. 1) and post-synaptic acetylcholine receptors were labelled using rhodamine-conjugated 
-bungarotoxin (BTX) (red; Fig. 1). The vast majority of neuromuscular junctions in control preparations from wild-type littermates were, as expected, fully occupied, with a motor nerve terminal branching over the whole post-synaptic motor endplate. As the muscles were taken from animals at P5–P6, most endplates in control preparations were also contacted by more than one incoming motor axon (polyneuronally innervated), as the process of developmental synapse elimination (establishing the adult mononeuronal innervation pattern of one axon innervating one muscle fibre) is not complete until 
2 weeks after birth.
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In contrast, numerous examples of partially occupied and vacant endplates (Fig. 1A) were present in all muscle preparations from Smn–/–;SMN2 mice, indicative of denervation events occurring due to a loss of pre-synaptic motor nerve terminals. The numbers of fully occupied endplates were significantly reduced in all muscles compared with wild-type littermates, albeit with the slow-twitch, postural TVA being more affected than the fast-twitch LAL and lumbricals (Fig. 1C–E; P < 0.01 TVA; P < 0.05 LAL/lumbricals; Mann–Whitney test, two-tailed; n = 4 per muscle wild-type, n = 6 per muscle Smn–/–;SMN2). The extent of nerve terminal loss was similar in the fast-twitch LAL and lumbrical muscles, despite the difference in nerve stump lengths, suggesting that stump length does not modulate the extent of pathology seen. Concurrent to nerve terminal loss, we also found numerous examples of pre-synaptic nerve terminals with abnormal accumulations of neurofilament proteins (Fig. 1B). This data supports similar findings from a previous study of neuromuscular junctions in a different mouse model of SMA, suggesting that neurofilament accumulation in motor nerve terminals is a pathological feature of SMA (7). Measurements of muscle fibre diameter revealed significant shrinkage in the TVA (Fig. 1D), with the LAL much less affected. These data confirm that neuromuscular junctions are primary pathological targets in SMA (7–9), but suggest that significant levels of inter-muscular variability exist within affected individuals, with slow-twitch postural muscles more affected than fast-twitch phasic muscles.
The morphological correlates of nerve terminal loss appeared distinct from those occurring during more Wallerian-like degenerative processes (24,25), suggesting that a retraction process is more likely to be responsible for removing nerve terminals. In order to confirm this, we examined intramuscular nerves and neuromuscular junctions in deep lumbrical muscles from late-symptomatic (P5–P6) Smn–/–;SMN2 mice using transmission electron microscopy. All intramuscular nerves examined were devoid of classical markers of Wallerian degeneration [e.g. myelin debris and plasma membrane breakdown (24,25)], with intact myelin sheaths, neurofilaments and microtubules (Fig. 2A). Modest accumulations of neurofilament in some axons confirmed the occasional neurofilament accumulations observed using immunocytochemistry (Fig. 2A). At the qualitative level, intact myofibrils were present in all skeletal muscle fibres examined (Fig. 2B). Immature neuromuscular junctions could be identified (devoid of the adult ultrastructural characteristics such as large post-synaptic folds), and many contained large accumulations of neurofilaments, presenting as neurofilament whorls within the body of the motor nerve terminal (Fig. 2C and D). These whorls are likely to be the ultrastructural equivalents of neurofilament accumulations observed using immunocytochemistry (cf. Fig. 1B) and are similar to those previously reported in other mouse models of SMA (7). No evidence of membrane breakdown, organelle fragmentation or phagocytosis by terminal Schwann cells was present, suggesting the involvement of a mechanism distinct from Wallerian or Wallerian-like degeneration (26).
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Occupancy counts underestimate levels of synaptic pathology at the neuromuscular junction in Smn–/–;SMN2 mice
Despite clear evidence of synaptic pathology provided by counts of fully occupied, partially occupied and denervated endplates (Fig. 1), we investigated whether or not the presence of multiple axonal inputs converging on individual endplates – due to ongoing developmental synapse elimination (discussed earlier) – resulted in underestimation of pre-synaptic nerve terminal loss. We quantified the numbers of individual axonal inputs converging on fully occupied endplates (i.e. classified as ‘normal’ in our previous analysis) in the TVA of late-symptomatic Smn–/–;SMN2 mice and wild-type littermates (Fig. 3). The mean number of inputs converging on endplates were significantly reduced (P < 0.01; Mann–Whitney test, two-tailed; n = 4 muscles wild-type, n = 6 muscles Smn–/–;SMN2). Thus, more pre-synaptic inputs were being lost than previously estimated from denervation counts alone.
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It is possible, however, that reduced numbers of synaptic inputs could also result from defective synapse formation, leading to hypo-innervation of neuromuscular junctions before the onset of pathology. To rule out this possibility, and to investigate pre-symptomatic neuromuscular development in SMA in more detail, we undertook a time-course analysis of synaptic pathology in the TVA and LAL of Smn–/–;SMN2 mice (Fig. 4). From a qualitative perspective, we could not identify any discernable differences between neuromuscular innervation in the LAL muscle from P2 (i.e. pre-symptomatic) Smn–/–;SMN2 mice versus wild-type littermates (Fig. 4A and B). Quantification of innervation (Fig. 4C) supported qualitative observations that the LAL was devoid of denervation pathology at P2. Similarly, quantification of the average number of inputs and muscle fibre diameters in the LAL confirmed that synapses were being formed in a similar manner to wild-type littermates (Fig. 4E). By P4, synaptic pathology was evident in the LAL, although not to the extent observed in late-symptomatic P6 mice (Fig. 4C). A similar analysis of TVA from P2 Smn–/–;SMN2 mice showed evidence of low levels of pre-symptomatic synaptic pathology. The average number of inputs per synapse was also decreased at P2, although post-synaptic muscle fibre shrinkage was not yet evident (Fig. 4D). As in the LAL, synaptic pathology in the TVA progressively worsened with time and was always more severe than in LAL at any given time-point (Fig. 4C).
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Motor nerve terminal loss, neurofilament accumulation and muscle fibre shrinkage in Smn–/–;SMN2;
7 miceIn order to confirm that the synaptic events detailed above were not specific to Smn–/–;SMN2 mice, we quantified neuromuscular junction morphology in early/mid symptomatic (P7) and late-symptomatic (P14) Smn–/–;SMN2;
7 mice. As in the Smn–/–;SMN2 mice, we found synaptic pathology in all muscle groups examined at both P7 and P14, evidenced by partially occupied endplates, vacant endplates and motor nerve terminals with abnormal accumulations of neurofilaments (Fig. 5A and B). Interestingly, neurofilament accumulation appeared to be even more severe in Smn–/–;SMN2;7 mice than previously observed in Smn–/–;SMN2 mice, particularly in the TVA.
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The numbers of fully occupied endplates were significantly reduced in the TVA compared with wild-type littermates in early/mid symptomatic (P7) mice (Fig. 5C; P < 0.05; Mann–Whitney test, two-tailed; n = 6 muscles wild-type, n = 10 muscles Smn–/–;SMN2;
7). However, there was only a subtle decrease in the numbers of fully occupied endplates in the LAL compared with wild-type littermates (Fig. 5C; P > 0.05; Mann–Whitney test, two-tailed; n = 2 muscles wild-type, n = 10 muscles Smn–/–;SMN2;7). This result confirms our finding in Smn–/–;SMN2 mice showing that the LAL is less affected in SMA than the TVA. Endplates of 92 and 45% of endplates in TVA and LAL, respectively showed evidence for neurofilament accumulation, versus 17 and 11% in wild-type littermate controls (P < 0.001 for both muscles, Mann–Whitney test, two-tailed). In line with the data on pre-synaptic pathology, muscle fibre shrinkage was also pronounced in TVA but not LAL (Fig. 5C).
Surprisingly, the numbers of fully occupied endplates in late-symptomatic (P14) mice were not significantly lower than in P7 mice in TVA or LAL (Fig. 5D). However, post-synaptic muscle fibre shrinkage was more pronounced in both the TVA and LAL at P14 (Fig. 5D) suggesting that the attenuation of pre- and post-synaptic pathology may be differently modulated by the SMN7 transgene.
Post-synaptic endplate shrinkage and pre-synaptic nerve terminal loss can occur independently at the neuromuscular junction
As previous studies have identified shrinkage of post-synaptic motor endplates alongside muscle fibre atrophy in SMA (9), we next examined motor endplate size in the TVA of Smn–/–;SMN2 mice (Fig. 6). Quantitative analysis confirmed that motor endplate areas were significantly reduced in Smn–/–;SMN2 mice (Fig. 6C). In order to examine whether post-synaptic changes occurred as a direct result of the loss of innervation at individual neuromuscular junctions, we analysed and compared the percentage occupancy of individual endplates (Fig. 6A and B) with their area. There was no correlation between endplate area and occupancy (Fig. 6D; P = 0.1802, r2 = 0.006018, N = 6 muscles, n = 300 endplates). As a result, endplates with any given area were just as likely to be fully occupied as partially occupied or vacant. The data suggest that post-synaptic changes at motor endplates are not simply a reaction to pre-synaptic pathology.
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Selective vulnerability of a subpopulation of motor neurons in the levator auris longus muscle
We made the observation, whilst quantifying synaptic pathology in LAL, that there appeared to be higher levels of nerve terminal loss in the thinner, caudal band of the muscle compared with the neighbouring rostral band. This was the case in both strains of mouse and at all time-points examined (Fig. 7A and B). Quantification of synaptic pathology (using both occupancy counts and assessment of the average number of inputs) confirmed that neuromuscular pathology was indeed widespread in the caudal band of LAL, but was almost absent in the rostral band of the muscle in both Smn–/–;SMN2 (Fig. 7C and D) and Smn–/–;SMN2;
7 mice (Fig. 7E and F). Assessment of post-synaptic endplate areas and pre-synaptic occupancy in both the caudal and rostral bands of LAL confirmed that pre- and post-synaptic pathology were not correlated (Fig. 7G; caudal band, P = 0.4457, r2 = 0.004314, N = 5 muscles, n = 137 endplates; rostral band, P = 0.1406, r2 = 0.01433, N = 5 muscles, n = 153 endplates). Comparisons of endplate area and muscle fibre diameter showed no significant difference between the rostral and caudal bands of LAL (Supplementary Material, Fig. S1). This data further supports the hypothesis (Fig. 6) that pre-synaptic nerve terminal loss (which is widespread in the caudal band of LAL but not the rostral band) is not correlated with post-synaptic endplate shrinkage at any given neuromuscular junction (which shows no difference between the two muscle bands).
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Previous studies have demonstrated that both rostral and caudal portions of LAL are composed of a homogenous population of fast-twitch muscle fibres, with predominance of MyHC-2b expressing fibres and almost complete absence of slow-twitch fibres (23). We confirmed these findings immunocytochemically in wild-type mice using antibodies raised against slow skeletal muscle myosin (a marker for slow-twitch fibres), and found no more than six slow-twitch fibres in a single LAL from more than eight muscles examined (data not shown). Staining of TVA muscle preparations revealed large numbers of slow-twitch fibres (data not shown), confirming the efficacy of the antibodies used.
In order to try and identify the nature of the selective vulnerability of motor neurons and neuromuscular synapses in the caudal band of LAL, we mapped the innervation patterns of immunocytochemically labelled LAL muscles in wild-type mice (Fig. 8A–C). Every muscle of over 40 examined conformed to a consistent pattern of innervation as shown in Figure 8C. The thin caudal band consistently had two clusters of neuromuscular junctions (C1 and C2), located at medial and lateral ends of the muscle. The thicker rostral band consistently had five clusters of neuromuscular junctions (R1–R5), which were arranged in two groups (R1 and R2 together and R3–R5 together).
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In order to investigate whether the rostral and caudal bands shared pools of innervating motor neurons, or whether they were composed of distinct and separate motor units, we traced individual, immunocytochemically labelled motor neuron axons from the point at which they entered the muscle (via a single muscle entry point; arrow in Fig. 8C) through to their ultimate termination in one of the neuromuscular junction clusters in LAL (Fig. 8D). In every muscle examined motor units exclusively supplied either the rostral or the caudal band. We found no motor units that had synaptic terminals in both bands. To confirm these observations, we examined 16 LAL muscles from YFP-H mice in which only a small number of motor neurons (usually one or two) expressed endogenous yellow fluorescent protein (27). In each case, individually labelled motor units innervated either the caudal band or the rostral band, but never both (Fig. 8E). These experiments show that distinct populations of motor neurons supply the rostral and caudal bands of LAL. The preferential loss of innervation in the caudal band of Smn–/–;SMN2 and Smn–/–;SMN2;
7 mice is therefore likely to represent a selectively vulnerable population of motor neurons.
Selectively vulnerable motor neurons conform to a FaSyn phenotype
One possible explanation for the selective vulnerability of synapses in the caudal band of LAL was that these motor units were larger (and hence had a more severe demand on intrinsic resources required to maintain viability following a degenerative stimulus) than motor units innervating the rostral band. However, quantitative analysis of motor unit sizes in the rostral and caudal bands of the LAL, using YFP-H mice to label the entire synaptic cohort of individual motor units, showed no difference between the two muscle bands (Supplementary Material, Fig. S2). Similarly, a previous study of motor neuron vulnerability in SOD1 mouse models of amyotrophic lateral sclerosis demonstrated that axons of fast-fatiguable motor neurons are more vulnerable than slow motor neurons (18). However, as the LAL muscle is composed of an entirely homogenous population of fast-twitch muscle fibres (discussed earlier), it is unlikely that the selective vulnerability of motor neurons innervating the caudal band is due to a difference in the fast/slow status of motor neurons between the two bands of the muscle.
We therefore examined whether motor neurons innervating the caudal band differed from those innervating the rostral band with respect to their FaSyn/DeSyn characteristics. Intrinsic distinctions between neuromuscular junctions with respect to their development and stability have previously been reported between different skeletal muscles (20). In particular, neuromuscular junctions conforming to a DeSyn phenotype have been shown to undergo dramatic collateral sprouting and formation of ectopic endplates following prolonged paralysis with botulinum toxin type A (BotA), whilst FaSyn neuromuscular junctions remain largely unaffected.
To test whether neuromuscular synapses in the caudal band of LAL differed from those in the rostral band with respect to their FaSyn/DeSyn characteristics, we paralysed the LAL muscle in wild-type mice for 1 month with repeated subcutaneous injections of BotA (see Materials and Methods). Subsequent quantitative analysis of collateral sprouting from nerve terminals and ectopic endplate formation in immunocytochemically stained LAL muscles revealed striking differences between the two bands (Fig. 9A and B). Very few motor nerve terminals (<5%) showed any signs of collateral sprouting in the caudal band of the muscle (Fig. 9C). In contrast, more than 20% of endplates in the rostral band showed clear evidence of terminal collateral sprouting (Fig. 9C; P < 0.01, Mann–Whitney test, two tailed; n = 10 muscles BotA, n = 6 muscles vehicle control). Similarly, there were significantly fewer ectopic endplates formed in the caudal band compared with the rostral band (Fig. 9D). In addition, the differential disruption of post-synaptic acetylcholine receptor clusters, previously reported in FaSyn/DeSyn muscles in response to BotA (20), was observed in the rostral and caudal bands, with the former undergoing significant post-synaptic disassembly and the latter less affected (data not shown). These experiments reveal that motor neurons supplying the caudal band of the LAL are more FaSyn-like than their counterparts in the rostral band. Motor neurons with FaSyn-like characteristics may therefore be more likely to be vulnerable to SMA-induced synapse loss than those with DeSyn characteristics.
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DISCUSSION |
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The findings of this study provide novel insights into neuromuscular pathology occurring during SMA. First, we demonstrated that neuromuscular pathology was prominent in muscle groups taken from different locations throughout the body of two SMA mouse models. Consistently, the postural TVA was more severely affected than fast-twitch LAL and lumbrical muscles, suggesting that muscle fibre-type and body location are likely to be important determining factors in regulating synaptic vulnerability during SMA. The morphological and ultrastructural correlates of nerve terminal loss support the hypothesis that SMA demonstrates characteristics consistent with a dying back neuropathy (7). Second, we showed that synapses form normally in SMA mouse models, but that in the most severely affected muscles (e.g. TVA) pre-synaptic pathology occurs early on in the disease course. Third, we identified differences in the time-course and severity of neuromuscular pathology between Smn–/–;SMN2 and Smn–/–;SMN2;
7 mice. Severe pre-synaptic pathology in late-symptomatic Smn–/–;SMN2 mice was not mirrored in late-symptomatic Smn–/–;SMN2;7 mice. Post-synaptic pathology, however, appeared to be much more severe in late-symptomatic Smn–/–;SMN2;7 mice, suggesting that the 7 transgene does not simply delay the neuropathology observed in Smn–/–;SMN2 mice. Rather, it appears that the 7 transgene has an affect in attenuating pre-synaptic pathology. Fourth, we demonstrated that post-synaptic changes resulting in shrinkage of motor endplates do not correlate with pre-synaptic pathology (loss of nerve terminals). Thus, pre- and post-synaptic changes can occur independently of one another at any given neuromuscular junction. And finally, we have identified a selectively vulnerable population of motor neurons innervating the caudal band of the LAL muscle. These motor neurons conform to a FaSyn phenotype when challenged with paralysis (20), suggesting that FaSyn motor neurons may be particularly vulnerable in SMA. The finding that synaptic pathology at the neuromuscular junction was a significant event in mouse models of SMA is in agreement with previous studies on human tissue and animal models of SMA (7–9,12,28,29), as well as human patients and mouse models of other motor neuron diseases, including amyotrophic lateral sclerosis (16,19,21,30). The data therefore support the hypothesis that synaptic compartments of neurons are particularly vulnerable to a wide variety of neurodegenerative stimuli, ranging from Alzheimer's disease and prion disease through to stroke and motor neuron disease (19,31). The future development of synaptoprotective treatments is therefore likely to be of critical importance in order to delay and/or prevent the onset and progression of conditions such as SMA (32–34). Furthermore, the presence of synaptic abnormalities in early/mid-symptomatic SMA mice suggests that early intervention will be required to adequately treat the disease and block the development of neuronal-specific pathologies. The current study suggests that whole-mount TVA and LAL muscles are good experimental systems in which to study such synaptic pathology and its amelioration through therapeutic intervention in SMA and other motor neuron diseases, allowing quantification of pathology in the majority of synapses present in both muscles without requiring sectioning or teasing of muscle fibres. However, all future investigations of synaptic pathology occurring in muscles still undergoing synapse elimination need to consider quantifying the number of inputs at individual neuromuscular junctions as well as simply the presence or absence of complete denervation.
The data showed a clear dissociation of pre- and post-synaptic pathology in all muscle groups examined, and in both different mouse models of SMA. These findings provide strong support for the hypothesis that pre- and post-synaptic pathology can occur independently of one another in SMA. One possible explanation for this, which the current study has not been able to directly address, is that SMN gene activity may be required in both neuronal and muscle tissue in order to alleviate the pathological phenotype (12). Neuronal-specific functions of SMN are well documented, and supported by data in the current study, with SMN being shown to be important for events including axon outgrowth, pathfinding and neuronal maintenance (11,13,35). The muscle-specific role of SMN is rather less clear, although deletion of SMN exon 7 in mouse muscle is known to cause severe muscular dystrophy (36) and a recent study utilizing another Drosophila model of SMA has identified an important role for SMN protein in the form and function of muscle sarcomeres (15). One way to address this question would be to repeat the current set of experiments in mouse models in which SMN levels have been reduced, but not completely abolished, in a tissue specific fashion in muscle or nerve individually. Another possible explanation for the apparent dissociation of pre- and post-synaptic pathology could be that muscle fibre pathology was a consequence of pre-synaptic inactivity – resulting in deficient neuromuscular transmission – occurring in the absence of pre-synaptic pathology. However, our finding that some nerve terminals were removed from corresponding muscle fibres devoid of any pathology suggests that pre-synaptic inactivity is likely to play, at most, a minor role in regulating post-synaptic pathology.
Our finding that motor neurons innervating the TVA (a predominantly slow-twitch muscle) appear to be more susceptible in SMA models than the LAL and lumbrical muscles (exclusively fast-twitch muscles) is in contrast to previous studies of adult onset motor neuron diseases and provides a potential explanation for the proximal distribution of muscle weakness in SMA. Studies of SOD1 mouse models of adult-onset motor neuron disease have demonstrated that phasic, fast-twitch motor neurons are selectively vulnerable with slow-twitch motor neurons reported to be resistant and even capable of initiating regenerative responses (18). This contrasting data is likely to reflect the different cellular stimuli and neuronal environments (e.g. age of disease onset) present in SMA and amyotrophic lateral sclerosis, but it also highlights the fact that motor neurons cannot be grouped into those that are generically vulnerable and those that are not. Motor neurons that are particularly vulnerable in one form of motor neuron disease may not be vulnerable in another.
The surprising result that motor neurons innervating the caudal band of the LAL muscle are more susceptible than those innervating the rostral band supports the hypothesis that not all pools of motor neurons are affected in the same way and at the same time in different motor neuron diseases (16,17,35). Whereas differences in vulnerability based around different motor neuron properties (e.g. fast-fatiguable, fast-fatigue resistant) and muscle fibre types are certain to exist, the current study demonstrates that motor neurons within a single muscle consisting of a homogeneous population of muscle fibres can also be differentially vulnerable in motor neuron disease. Our studies have identified a correlation between vulnerable motor units and FaSyn characteristics (20). Whilst these experiments do not directly demonstrate a causal link between FaSyn characteristics and selective vulnerability (genetic tools to experimentally manipulate FaSyn and DeSyn characteristics are unfortunately not yet available), the hypothesis is strongly supported by the selective vulnerability of muscle groups present in human SMA patients. For example, the diaphragm [a DeSyn muscle (20)] is relatively spared whilst the intercostal muscles [FaSyn muscles (20)] are severely affected (37).
We believe that this is the first time that FaSyn and DeSyn characteristics have been identified in subpopulations of neurons innervating the same muscle. The data suggest that further studies providing insights into the mechanisms through which motor neurons and their synapses are specified to be, and develop into, FaSyn or DeSyn motor units may provide important insights into vulnerability of motor neurons in SMA. The ability to identify factors associated with the more resistant DeSyn synapses may provide new targets for therapeutic intervention. The neuroanatomical map of LAL innervation and the identification of its differential neuronal susceptibility provided in the current study is likely to provide a useful model system with which to explore the differences between FaSyn and DeSyn synapses, and their vulnerability in SMA, in more detail.
In summary, we have shown that neuromuscular synapses are important and early pathological targets in SMA and that the levels of pre- and post-synaptic vulnerability can be differentially modulated by the presence of the SMN7 transgene. We demonstrated that the shrinkage of a post-synaptic motor endplate does not necessarily correlated with loss of its pre-synaptic motor nerve terminal, showing that pre- (neuronal) and post- (muscular) synaptic pathology can occur independently of one another in SMA. We have also revealed that motor neurons innervating the TVA (a slow-twitch muscle) are more vulnerable than those innervating fast-twitch muscles (LAL and lumbricals), and that neuromuscular synapses conforming to a FaSyn phenotype may be more susceptible than those with a DeSyn phenotype, regardless of the muscle fibre type with which they synapse.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALFUNDINGREFERENCES |
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Mice
C57Bl/6 and YFP-H mice were maintained as breeding colonies in the animal care facilities in Edinburgh under standard SPF conditions. Smn+/–;SMN2 mice (Jackson labs strain no. 005024) were also maintained as heterozygote breeding pairs in animal care facilities in Edinburgh. Smn+/–;SMN2;SMN
7 (Jackson labs strain no. 005025) breeding pairs were maintained in animal care facilities in Oxford. All animal procedures and breeding were performed in accordance with Home Office and institutional guidelines. Litters produced from SMA colonies were retrospectively genotyped by standard PCR protocols (JAX® Mice Resources).
Muscle preparation
Neonatal Smn–/–;SMN2 (P2, P4 and P5/6) and Smn–/–;SMN2;SMN7 (P7 and P14) mice and wild-type littermates were killed by intra-peritoneal injection of sodium pentabarbitol. C57Bl/6 (6 weeks) and C57Bl/6-YFP-H (3 months) mice were killed by exposure to rising concentrations of CO2. The LAL (from the back of the neck), lumbricals (from the hind-paw) and/or TVA (from the abdominal wall) muscles were dissected in oxygenated mammalian physiological saline (mM: NaCl 120, KCl 5, CaCl2 2, MgCl2 1, NaH2PO4 0.4, NaHCO3 23.8, D-glucose 5.6). Muscles were exposed to -BTX conjugated to tetramethyl-rhodamine isothiocyanate (TRITC--BTX; 5 mg/ml, Molecular Probes) for 10 min and fixed in 0.1M PBS (phosphate-buffered saline) containing 4% Paraformaldehyde (Fischer) for 15 min at 4°C. YFP-H muscles were mounted at this stage (discussed later). All other muscles were subsequently processed for immunocytochemistry.
Immunocytochemistry
Muscles were blocked in 4% bovine serum albumin and 1% Triton X in 0.1M PBS for 30 min before incubation in primary antibodies directed against 150 kDa neurofilament proteins (1:350 dilution; Chemicon International) overnight. After washing for 30 min in 0.1M PBS, muscles were incubated for 4 h in a 1:40 dilution of swine anti-rabbit secondary antibody conjugated to the fluorescent label FITC (Dako). Muscles were then whole-mounted in Mowoil® (Calbiochem) on glass slides and cover-slipped for subsequent imaging.
Paralysis with botulinum toxin type A
BotA (0.5 µl/g of a 5 ng/ml solution; Sigma) or vehicle control (non-sprouting buffer) was repeatedly injected subcutaneously above each LAL at postnatal days 15, 19, 22, 26 and 29 [as previously described (20)]. Mice were sacrificed and muscles analysed at P45. Muscles were quantified for presence/absence of collateral terminal sprouting and percentage of ectopic endplates (as described later).
Imaging and analysis
Muscle preparations were viewed using either a phase contrast microscope (for muscle measurements), a standard epi-fluorescence microscope equipped with a chilled CCD camera (40x objective; 0.8NA; Nikon IX71 microscope; Hammamatsu C4742–95), and/or a laser scanning confocal microscope (40x objective; 0.8NA; BioRad Radiance 2000, Hemel Hempstead, UK). TRITC--BTX-labelled preparations were imaged using 543 nm excitation and 590 nm emission optics and FITC-labelled preparations utilized 488 nm excitation and 520 nm emission optics. For confocal microscopy, 488 nm and 543 nm laser lines were used for excitation and confocal Z-series were merged using Lasersharp (Biorad) software. All images were then assembled using Adobe Photoshop.
Montages and reconstructions
Reconstructions of immunocytochemically labelled and YFP-H labelled muscle preparations were produced in Adobe Photoshop software by layering and combining multiple micrographs covering the entire muscle. Individual motor neurons were traced and pseudo-coloured manually in Adobe Photoshop software.
Quantification and statistics
A minimum of 80 endplates, selected at random, were assessed in each muscle preparation. Muscles with poor staining and/or damage were excluded from further analysis. Wherever possible, all analysis was performed without the operator knowing the status of the material. For basic occupancy counts, the occupancy of individual neuromuscular junctions was evaluated by categorizing endplates as either fully occupied (neurofilament entirely overlies endplate), partially occupied (neurofilament partially covers endplate) or vacant (no neurofilament overlies endplate). For correlated pre- and post-synaptic measurements, calibrated individual images of neuromuscular junctions were imported into ImageJ software and split into red (endplate) and green (axon and motor nerve terminal) channels. Each endplate and corresponding motor nerve terminal was manually outlined in ImageJ (Fig. 3A and B) and the software used to calculate total areas. To evaluate the number of neural inputs, the number of axons converging on a single motor endplate was counted. Collateral terminal sprouts were identified by the presence of nerve terminal projections exceeding 2 µm in length, to exclude the possibility that sprouting was due to ongoing synaptic reorganization. Ectopic endplates were identified by clusters of acetylcholine receptors under 5 µm in diameter. Individual muscle fibre diameters were measured in ImageJ using regions of the muscle with teased fibres present. Only isolated fibres with no overlapping fibres obscuring their profile were included in these analyses. All data were collected and analysed using GraphPad Prism software.
Electron microscopy
Deep lumbrical muscles were dissected in normal mammalian physiological saline (mM: NaCl, 120; KCl, 5; CaCl2, 2; MgCl2, 1; NaH2PO4, 0.4; NaHCO3, 23.8; D-glucose, 5.6) bubbled to equilibrium with a 5% CO2/95% O2 mixture before fixation in ice-cold 0.1M phosphate buffer containing 4% formaldehyde and 2.5% glutaraldehyde for 4 h. Preparations were then washed in 0.1M phosphate buffer before post-fixation in 1% osmium tetroxide for 45 min. Following dehydration through an ascending series of ethanol solutions and propylene oxide, the preparations were embedded in Durcupan resin. Ultra-thin sections (75–90 nm) were cut and collected on formvar-coated grids (Agar Scientific, UK), stained with uranyl acetate and lead citrate in an LKB ‘Ultrostainer’ and then viewed in a Philips CM12 transmission electron microscope.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALFUNDINGREFERENCES |
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Supplementary Material is available at HMG Online.
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FUNDING |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALFUNDINGREFERENCES |
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This work was supported by Medical Research Scotland (THG), The Anatomical Society of Great Britain & Ireland (LM/THG), the BBSRC (THG), The Muscular Dystrophy Campaign (NP/KT) and The SMA Trust (NP/KT).
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ACKNOWLEDGEMENTS |
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We thank Dr T. Wishart, B. Baxter, A. Edwards and P. Geldsetzer for advice and assistance with experiments and Dr S. Parson for helpful comments on the manuscript.
Conflict of Interest statement. The authors have no conflicts of interest to declare.
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