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Human Molecular Genetics Advance Access originally published online on February 9, 2005
Human Molecular Genetics 2005 14(6):845-857; doi:10.1093/hmg/ddi078
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Human Molecular Genetics, Vol. 14, No. 6 © Oxford University Press 2005; all rights reserved

SMN{Delta}7, the major product of the centromeric survival motor neuron (SMN2) gene, extends survival in mice with spinal muscular atrophy and associates with full-length SMN

Thanh T. Le1, Lan T. Pham1, Matthew E.R. Butchbach1,{ddagger}, Honglai L. Zhang4,{ddagger}, Umrao R. Monani1,2,{dagger}, Daniel D. Coovert1, Tatiana O. Gavrilina1, Lei Xing4, Gary J. Bassell4 and Arthur H.M. Burghes1,2,3,*

1Department of Molecular and Cellular Biochemistry, 2Department of Neurology, College of Medicine and Public Health, 3Department of Molecular Genetics, College of Biological Sciences, Ohio State University, Columbus, OH, USA and 4Department of Neuroscience, Rose F. Kennedy Center for Mental Retardation, Albert Einstein College of Medicine, Bronx, NY, USA

* To whom correspondence should be addressed at: Department of Molecular and Cellular Biochemistry, 363 Hamilton Hall, 1645 Neil Avenue, Columbus, OH, 43210 USA. Tel: +1 6146884759; Fax: +1 6142924118; Email: burghes.1{at}osu.edu

Received October 29, 2004; Revised January 13, 2005; Accepted January 27, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Spinal muscular atrophy (SMA) is an autosomal recessive disorder in humans which results in the loss of motor neurons. It is caused by reduced levels of the survival motor neuron (SMN) protein as a result of loss or mutation of the SMN1 gene. SMN is encoded by two genes, SMN1 and SMN2, which essentially differ by a single nucleotide in exon 7. As a result, the majority of the transcript from SMN2 lacks exon 7 (SMN{Delta}7). SMN{Delta}7 may be toxic and detrimental in SMA, which, if true, could lead to adverse effects with drugs that stimulate expression of SMN2. To determine the role of SMN{Delta}7 in SMA, we created transgenic mice expressing SMN{Delta}7 and crossed them onto a severe SMA background. We found that the SMN{Delta}7 is not detrimental in that it extends survival of SMA mice from 5.2 to 13.3 days. Unlike mice with selective deletion of SMN exon 7 in muscle, these mice with a small amount of full-length SMN (FL-SMN) did not show a dystrophic phenotype. This indicates that low levels of FL-SMN as found in SMA patients and absence of FL-SMN in muscle tissue have different effects and raises the question of the importance of high SMN levels in muscle in the presentation of SMA. SMN and SMN{Delta}7 can associate with each other and we suggest that this association stabilizes SMN{Delta}7 protein turnover and ameliorates the SMA phenotype by increasing the amount of oligomeric SMN. The increased survival of the SMN{Delta}7 SMA mice we report will facilitate testing of therapies and indicates the importance of considering co-complexes of SMN and SMN{Delta}7 when analyzing SMN function.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by loss of {alpha} motor neurons in the spinal cord (1Go). It is the leading hereditary cause of infant mortality in humans (2Go). SMA is caused by low levels of the survival motor neuron (SMN) protein which is insufficient to protect motor neurons (3Go–7Go). The reduced SMN levels result from loss or mutation of the telomeric survival motor neuron (SMN1) gene and retention of the centromeric survival motor neuron (SMN2) gene (8Go–12Go). The critical difference between these two genes is a C to T transition in exon 7 of SMN2 which affects splicing of this exon (13Go–15Go).

Thus, SMN1 produces full-length transcript whereas SMN2 generates mostly a transcript lacking exon 7 (SMN{Delta}7) (9Go,11Go,16Go). The protein product of SMN{Delta}7 oligomerizes less efficiently (17Go) and appears to be unstable and rapidly degrades (18Go,19Go). The SMN2 gene does, however, produce some full-length transcript and thus full-length SMN (FL-SMN) protein. Increased copy number of SMN2 modulates the severity of SMA (3Go,20Go–25Go).

The 38 kDa SMN protein is ubiquitously expressed and localizes to both cytoplasm and nucleus (3Go,4Go,26Go). In the nucleus, SMN localizes in dot-like structures termed as ‘gems’ which overlap or are in close proximity to coiled bodies (26Go,27Go). SMN has been termed the master RNA assembler and, in particular, has been shown to be important in assembly of the snRNP particles (28Go–31Go). In addition, SMN appears to be important for the assembly of snoRNAs (28Go) and pre-mRNA splicing (32Go) and binds to hnRNP-R and -Q (33Go). Clearly, SMN is a multifunctional protein that binds to a number of proteins, but it is unclear what critical function is disrupted by low levels of SMN (6Go).

In mice, there is one survival motor neuron (Smn) gene which is equivalent to SMN1 (34Go,35Go). Complete loss of this gene results in an embryonic lethal phenotype as would be expected given the housekeeping functions of SMN (36Go). Introduction of one or two copies of SMN2 rescues the embryonic lethal phenotype resulting in mice with severe SMA (7Go,37Go) whereas 8–16 copies of SMN2 results in rescue (7Go). This indicates that SMN2 can produce the SMN required to correct the SMA phenotype although it is not currently clear as to when during development or in which specific tissue(s) high levels of SMN are needed. Expression of a known mild SMA missense mutation (A2G) (38Go) on the severe SMA mouse background results in a mild phenotype (39Go). SMN(A2G) alone does not correct embryonic lethality of the Smn null mouse indicating that mutated SMN needs to be expressed in the presence of small amounts of full-length SMN to get phenotypic modification (39Go). In addition, loss of SMN exon 7 but retention of the ability to produce truncated SMN results in embryonic lethality indicating the importance of retaining at least some full-length SMN in order to function (40Go).

Inducing SMN2 to produce higher levels of SMN protein seems to be an attractive option in developing treatments for SMA. At this time, compounds that stimulate SMN2 to produce more full-length SMN mRNA and SMN protein have been identified (41Go–50Go). These compounds have two modes of action: (1) increase expression of SMN and (2) change the splicing pattern of SMN2 such that more full-length SMN protein is produced. For compounds that stimulate the SMN promoter, the obvious question that arises is whether the concomitant increase in SMN{Delta}7 with that of FL-SMN would be detrimental. In some cases, it appears that some compounds can cause a greater increase in SMN{Delta}7 than in FL-SMN (41Go).

This would seem to be of particular concern in light of recent reports that over-expression of SMN{Delta}7 in neurons or in PC12 cells appears to be pro-apoptotic whereas that of FL-SMN is anti-apoptotic (51Go,52Go) suggesting that SMN{Delta}7 is detrimental in SMA. To test whether SMN{Delta}7 is beneficial or detrimental in SMA, we created transgenes expressing this SMN isoform and introduced them into the severe SMA genetic background. Here, we demonstrate that an increase in SMN{Delta}7 to levels likely to occur in vivo has a positive effect on survival of SMA mice by extending lifespan from 5.2±0.2 to 13.3±0.3 days. We show that SMN and SMN{Delta}7 form heterotypic complexes which stabilize SMN{Delta}7 in transfected cultured cells. We suggest that SMN{Delta}7 results in more oligomeric SMN complex and a reduced phenotypic severity. In these mice, we also show that low SMN levels do not cause disruption of the dystrophin complex as opposed to what was observed in mice with a muscle-specific deletion of SMN exon 7 (53Go). This indicates that low levels of SMN and complete absence of functional SMN in a specific tissue (53Go) have different effects and that it is not possible to draw conclusions about the importance of SMN in SMA by its removal from a tissue.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Generation of SMN{Delta}7 transgenic mice
An SMN cDNA lacking exon 7 was placed under the control of a 3.4 kb SMN promoter fragment (transgene referred to as SMN{Delta}7, Fig. 1A) and microinjected into fertilized mouse oocytes (FVB/N). Three founder lines, 4299, 4352 and 4353, were obtained. The copy number of the transgene was determined by quantitative Southern blot and real-time PCR (Fig. 1B). Real-time measurements were further validated by the use of a transgene of known copy number (SMN2) with the results being in complete concordance. Line 4299 is estimated to have six copies of SMN{Delta}7 (6.12±0.89), line 4352 has 17 copies of SMN{Delta}7 (17.12±1.87) and line 4353 has two copies of SMN{Delta}7 (2.15±0.41). The integration of the transgene for all lines was a single tandem integration and followed Mendelian inheritance consistent with a single locus. The number of SMN{Delta}7 copies was stable in each line with homozygous animals having the expected doubling of copy number. The SMN{Delta}7 transgenic mice were crossed with mice containing SMN2 and a mouse Smn knockout allele (36Go) to obtain double transgenic mice SMN{Delta}7;SMN2;Smn+/– on a FVB/N background. These double transgenic mice were interbred to obtain Smn–/– mice with SMN2 and SMN{Delta}7.



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  		Figure 1. Generation of the SMN{Delta}7 transgenic mice. (A) Schematic representation of the SMN promoter driven SMN{Delta}7 construct to generate the transgenic mice. (B) Southern blot of SMN{Delta}7 transgenic lines 4299, 4352 and 4353 in Smn+/– mice. The copy number of the transgene was determined by real-time PCR relative to the ß-globin gene. Similar results were also obtained using the mouse Smn gene or SMN2 as relative standards for copy number.

 
SMN2 and SMN{Delta}7 expression in SMA mice
Mice derived from the same breeding pairs used for survival analysis were analyzed for expression of the SMN{Delta}7 transgene. Semi-quantitative RT–PCR analysis showed that all three lines expressed SMN{Delta}7 with line 4299 showing the highest level of SMN expression (Fig. 2A). Western blot analysis showed that all the transgenic lines produced SMN{Delta}7; furthermore, the amount of SMN{Delta}7 is not more than the FL-SMN produced by SMN2 so the levels are not extremely high. RT–PCR and western blot analyses were performed using the same breeding pairs that were used for survival analysis. The data were consistent between litters. The transgenic line 4299 produced the highest levels of the SMN{Delta}7 protein in the brain (Fig. 2B) as well as in the spinal cord (Fig. 2C). Line 4352 produced slightly lower levels of the protein whereas line 4353 expressed the least amount of protein. SMA mice from line 4299 express relatively high levels of the SMN{Delta}7 RNA. However, despite the high RNA levels, SMN{Delta}7 protein amount was at most only equivocal to the amount of FL-SMN protein from two copies of SMN2 (Fig. 2B). In our experience, it is not uncommon for high copy number transgenes (>~12 copies; i.e. SMN{Delta}7 in line 4352) to show lower transgene expression. When comparing the different transgenic lines, the ratio of SMN{Delta}7 to FL-SMN in spinal cord extracts was highest in line 4299 SMA mice (Fig. 2C). Additionally, the amount of SMN{Delta}7 relative to FL-SMN that is expressed in line 4299 mice is similar in multiple tissues (Fig. 2D). The expression data shown here are consistent with the suggestion that SMN{Delta}7 protein is unstable and rapidly degraded (18Go,19Go). This contrasts with transient transfection and viral infection data where it appears that higher levels of SMN{Delta}7 protein can be reached (19Go,51Go,52Go). In these situations, the level of SMN{Delta}7 is well above what would be expected by induction of SMN2 and represents an artificial situation which is unlikely to occur in vivo.



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  		Figure 2. Expression analysis of transgenic SMN{Delta}7 in mice. (A) Semi-quantitative RT–PCR analysis of SMN RNA present in the transgenic mice. RNA was isolated from the brains of the indicated mice and RT–PCR performed with primers lying in SMN exons 4 and 8 so as to distinguish the various SMN mRNAs. HeLa RNA is used as a marker for the SMN isoforms. All RNA samples were treated with DNase and tested by RT–PCR to ensure no genomic contamination (no amplification when lacking reverse transcriptase). The SMN isoforms are indicated on the right side of the figure and the reactions are all performed with mouse HPRT primers to ensure equal loading of cDNA in each lane. Nor, normal FVB/N mouse lacking any transgene; SMN2, SMA mouse lacking the SMN{Delta}7 transgene (SMN2+/+;Smn–/–); {Delta}7 alone, SMN{Delta}7 transgenic mice that do not contain SMN2; {Delta}7+SMN2, SMA mice containing the SMN{Delta}7 transgene (Smn{Delta}7+/+;SMN2+/+;Smn–/–). All SMA mice used for RT–PCR are from the same breeding pairs as those used for survival experiments. The results are similar between siblings and different litters. (B) Western blot analysis of SMN protein present in brain extracts from SMA mice. The blot was probed with a SMN monoclonal antibody (bottom) and stripped and reprobed with ß-actin to control for loading (top). The SMN antibody cross-reacts with mouse SMN making it not feasible to look at SMN{Delta}7 in a Smn+/– background. 4299, Smn{Delta}7+/+; SMN2+/+; Smn–/– line 4299 mice [postnatal day (PND) 12]; 4352, Smn{Delta}7+/+; SMN2+/+; Smn–/– line 4352 mice (PND11); 4353, Smn{Delta}7+/?; SMN2+/+; Smn–/– line 4353 mice (PND8; it is not known whether the SMN{Delta}7 transgene is heterozygous or homozygous); low, low copy (two copies) SMN2+/+; Smn–/– mice (PND5); high, high copy (8–16 copies) SMN2; Smn–/– mice (PND8); Nor, Smn+/+ mice (PND8). (C) Western blot analysis of spinal cord tissue from PND8 SMN{Delta}7 SMA mice from the three lines 4299, 4352 and 4353. The SMA mice are Smn{Delta}7+/+; SMN2+/+; Smn–/–. The SMA mice used for western blot analysis were from the same breeding pairs as those used for RT–PCR and survival analyses. The results are similar between siblings and different litters. The ratio of SMN{Delta}7 to FL-SMN was determined for each line on independent blots and is shown below the protein lanes. These ratios correlate with the phenotype of the mice and indicates a similar phenotypic modification in lines 4299 and 4352. (D) Western blot analysis of SMN protein in different tissues from line 4299 SMN{Delta}7 SMA mice. SMN{Delta}7 is expressed in a similar manner in different tissues of line 4299 SMN{Delta}7 SMA mice. Br, brain; Sp, spinal cord; Li, liver; Lu, lung; Mu, muscle; Kid, kidney.

 
Phenotype of SMA mice carrying the SMN{Delta}7 transgene
To determine the effect of the SMN{Delta}7 on the phenotype of SMA mice, we interbred SMN2;SMN{Delta}7;Smn+/– mice to obtain SMA mice that carry both SMN2 and SMN{Delta}7 transgenes. Figure 3A shows the survival curves for these mice. The breeding pairs used for survival analysis were the same as those used for expression analysis. Between the three transgenic lines obtained, line 4299 survived the longest with a maximum survival of 16 days and a mean survival of 10.2±0.7 days, 4352 was very similar in its response surviving up to 15 days with a mean survival of 9.4±0.6 days and 4353 survived a maximum 14 days with a mean survival of 7.8±0.6 days. This compares with SMA mice with one copy of SMN2 (SMN2; Smn–/–) and without SMN{Delta}7 transgene, which survive up to 2 days with a mean of 1.4±0.1 days and with SMA mice with two copies of SMN2 which survive up to 8 days with a mean of 5.2±0.2 days. These SMA mice are on a FVB/N background and have been backcrossed for six generations; the original knockout line was generated on a C57bl/6J background (36Go). Backcrossing with C57bl/6J produces a more severe phenotype for the (SMN2; Smn–/–) mice with earlier death (unpublished data). The mice in the earlier crosses that lacked the SMN{Delta}7 transgene but were SMN2 positive and Smn–/– never lived longer than 5 days. This indicates that modification of the phenotype is not caused by the genetic background of the mouse. However, we would predict that SMN{Delta}7; SMN2;Smn–/– mice would be more severe on a C57bl/6J background. SMN{Delta}7 line 4299 mice gave the most improvement in lifespan of SMA mice which is consistent with the fact that this line produced the highest levels of SMN{Delta}7 mRNA and protein (Fig. 2).



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  		Figure 3. Survival analysis and phenotype of SMA mice expressing the SMN{Delta}7 transgene. (A) Kaplan–Meier survival curves of SMN{Delta}7 SMA mice from lines 4299, 4352 and 4353. The mice used to generate these curves may be either heterozygous or homozygous for the SMN{Delta}7 transgene. The mice used for survival analysis were from the same breeding pairs as those used for expression analysis. Line 4299 mice survived a maximum of 16 days with a mean survival of 10.2±0.7 days (n=40), line 4352 mice survived a maximum 15 days with a mean survival of 9.4±0.6 days (n=25) and line 4353 survived a maximum 14 days with a mean survival of 7.8±0.6 days (n=25). SMA mice with one copy of SMN2 (SMN2+/–; Smn–/–) and without the SMN{Delta}7 transgene, on the other hand, survive up to 2 days with a mean of 1.4±0.1 days (n=13) and SMA mice with two copies of SMN2 (SMN2+/+; Smn–/–) survive up to 8 days with a mean of 5.2±0.2 days (n=57). (B) Kaplan–Meier survival analysis of homozygous SMA mice with and without the SMN{Delta}7 transgene. Line 4299 SMA mice (SMN2+/+; Smn{Delta}7+/+; Smn–/–) survived for a maximum of 17 days with a mean of 13.3±0.3 days (n=95). This compares with SMA mice lacking the SMN{Delta}7 transgene but with two copies of SMN2 (SMN2+/+; Smn–/–), which survived up to 8 days with a mean of 5.2±0.2 days (n=57). The log-rank test shows that the difference in lifespan between SMA mice with and without the SMN{Delta}7 transgene is highly significant (P<0.00001). (C) Weight curves of SMA (SMN2+/+; Smn–/–, closed diamonds), SMN{Delta}7 SMA (SMN2+/+; Smn{Delta}7+/+; Smn–/–, open circles) and non-SMA mice (SMN2+/+; Smn{Delta}7+/+; Smn+/– or Smn+/+, closed triangles). All plots are shown as means of weight at each day with error bars representing standard deviation. (D) A micrograph showing a non-SMA mouse (SMN2+/+; Smn{Delta}7+/+; Smn+/– or Smn+/+) and two SMN{Delta}7 SMA mice (SMN2+/+; Smn{Delta}7+/+; Smn–/–) at PND13. The SMA mice ({Delta}7) are smaller in size and have difficulty in righting themselves when compared with non-SMA mice (Nor).

 
Because the mice in the initial crosses could carry one or two copies of SMN2 and could be either heterozygous or homozygous for the SMN{Delta}7 transgene, lines 4352 and 4299 were bred to homozygosity for both the SMN2 transgene and the SMN{Delta}7 transgene. Survival curves for mice from line 4299 homozygous for both transgenes and homozygous for SMN2, but lacking SMN{Delta}7, are compared in Figure 3B. In line 4299, SMN{Delta}7 in the homozygous state extends the lifespan of SMA mice with two copies of SMN2 to a maximum of 17 days with a mean of 13.3±0.3 days (P>0.00001 using the log-rank test). Similar results were obtained for the line 4352, indicating a similar effect for two independent transgenic insertions (data not shown). It is clear from this data that expression of SMN{Delta}7 extends the life of SMA mice and is not detrimental. The levels of SMN{Delta}7 reported here are closer to what could occur in vivo by induction of SMN2 expression; this level of SMN{Delta}7 is not toxic to the mice and appears beneficial to SMA mice. The first sign of a phenotypic difference between SMN{Delta}7;SMN2;Smn–/– mice homozygous for the two transgenes and non-SMA littermates (Smn+/+ and Smn+/– with the transgenes) is in body weight. SMA mice with or without the SMN{Delta}7 transgene tend to be smaller at postnatal day (PND) 1: SMN2;Smn–/– mice were 1.13±0.05 g, SMN{Delta}7; SMN2;Smn–/– mice were 1.12±0.08 g and non-SMA mice (Smn+/+ or +/– with the transgenes) were 1.50±0.07 g (Fig. 3C). However, the ranges in weight do overlap and we have seen SMA mice with normal weights on PND1–2. The weight difference became readily apparent at 5 days (Fig. 3C; 2.22±0.19 g compared with 3.52±0.18 g). SMA mice tend to be smaller at PND1–2. By 5 days of age, this is true for all SMA mice. The difference in size between SMN{Delta}7 SMA mice and non-SMA littermates remains throughout life (Fig. 3C). At 5 days of age, SMN{Delta}7 mice also have considerable difficulty righting themselves when placed on their backs. The weakness becomes progressively more pronounced over the next week of life. By 10 days of age, it is clear that SMA mice homozygous for the SMN{Delta}7 and SMN2 transgenes have difficult ambulating and often fall over while walking (Fig. 3D) (Supplementary Material). When they do walk, they display an abnormal gait and shakiness (fibrillation) of the hind limbs.

Spinal motor neurons were counted in the lumbar region (L3–L6) of non-SMA mice (siblings containing mouse Smn;n=5) and SMA mice (SMN2+/+;SMN{Delta}7+/+; Smn–/–; n=5). At PND9, non-SMA mice had 707±16 motor neurons, whereas SMN{Delta}7 SMA mice had 574±20 motor neurons (P=0.004 as determined by one-way ANOVA analysis). No significant difference in the population of motor neurons was detected at PND4. These mice were from parents that were homozygous for the SMN2 and SMN{Delta}7 transgenes and the comparison was conducted between siblings containing mouse Smn. We have previously reported that motor neuron loss in SMN2+/+;Smn–/– mice occurs at PND3 (these SMA mice survive at most 8 days) (7Go). Motor neuron loss in SMN(A2G) mild SMA mice occurred later at ~3 months (39Go). Thus, the time of motor neuron loss appears to be related to disease severity.

Effect of low SMN levels on muscle morphology and dystrophy
A conditional mutant which results in loss of FL-SMN from mature skeletal muscle results in a clear dystrophic phenotype with major disruption of the dystrophin complex (53Go). In this situation, the muscle fiber becomes completely depleted of functional FL-SMN. However, functional FL-SMN is present at low levels in SMA patients or in SMA mice described here. We determined whether low levels of SMN resulted in a dystrophic phenotype or any obvious disruption of the dystrophin complex as reported in mice which lack FL-SMN in their muscle. We first stained gastrocnemius muscle from PND14 SMN{Delta}7 SMA mice and non-SMA littermates with dystrophin antibodies. All of the muscle fibers in non-SMA mice (Fig. 4A) and SMN{Delta}7 SMA animals (Fig. 4B) were positive for dystrophin. Higher magnification shows that there were no discontinuous gaps in dystrophin staining in non-SMA (Fig. 4D) or SMN{Delta}7 SMA (Fig. 4C) muscle fibers. Thus, there is no obvious disruption in the dystrophin complex in our SMA mice. Hematoxylin and eosin staining also showed no dystrophic features in SMN{Delta}7 SMA gastrocnemius fibers (Fig. 4F) relative to non-SMA mice (Fig. 4E). The only striking feature was the small size of fibers due to atrophy in the SMA mouse (Fig. 4G). There was no increase in the proportion of muscle fibers with centrally localized nuclei in SMN{Delta}7 SMA mice when compared to non-SMA mice (data not shown). Additionally, we found that the serum CK levels were not elevated in SMN{Delta}7 SMA mice when compared with SMN{Delta}7 non-SMA mice (data not shown). Motor endplates, as visualized with a fluorescently-conjugated {alpha}-bungarotoxin, are present along the sarcolemma (stained with dystrophin) in both SMN{Delta}7 SMA (Fig. 4I) and non-SMA (Fig. 4H) mice. It is, therefore, clear from these data that reduction of SMN levels, unlike loss of SMN, does not cause major disruption of the dystrophin complex or result in muscular dystrophy.



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  		Figure 4. Morphology of skeletal muscles from SMN{Delta}7 SMA mice. (A and B) Dystrophin staining of gastrocnemius muscle from PND14 SMN{Delta}7 SMA (SMN2+/+; Smn{Delta}7+/+; Smn–/–) and non-SMA (SMN2+/+; Smn{Delta}7+/+; Smn+/?) mice. These images show that all muscle fibers stain for dystrophin in both SMA (B) and non-SMA (A) mice. Scale bar for (A and B) is 5 µm. (C and D) High magnification confocal images of dystrophin-staining myofibers. Dystrophin staining is continuous along the sarcolemma in both SMA (D) and non-SMA (C) mice. Scale bar for (C and D) is 5 µm. (E and F) Hematoxylin and eosin staining of gastrocnemius muscle sections from PND14 SMN{Delta}7 SMA (F) and non-SMA (E) mice. There are no indications of dystrophy in these muscles. Scale bar for (E and F) is 5 µm. (G) Distribution of muscle fiber areas in PND14 SMN{Delta}7 SMA and non-SMA mouse gastrocnemii. SMN{Delta}7 SMA gastrocnemii tend to have smaller myofibers than age-matched non-SMA mice. (H and I) Confocal images showing the presence of motor endplates (green nicotinic acetylcholine receptors) along the sarcolemma (red, dystrophin) in both SMN{Delta}7 SMA (I) and non-SMA (H) mice. Scale bar for (H and I) is 50 µm.

 
Effect of low SMN levels on innervation of skeletal muscle
We investigated the consequences of low SMN levels on the morphology and innervation state of neuromuscular junctions (NMJs) within the gastrocnemius of PND14 mice. Nicotinic acetylcholine receptors (AChRs) which are present on the NMJs were labeled with {alpha}-bungarotoxin whereas the motor neuron axons were labeled with antibodies directed against neurofilament heavy chains and presynaptic vesicles (SV2). In a non-SMA transgenic mouse, most of the NMJs within the gastrocnemius are completely innervated (closed arrow) at PND14 (Fig. 5A and C). NMJs which are either partially innervated (open arrow) or not innervated (arrowhead) are not abundant in non-SMA transgenic mice. There is, however, a higher proportion of NMJs that are either partially innervated or not innervated in SMN{Delta}7 SMA mice at PND14 (Fig. 5B and C). Closer examination of the gastrocnemius NMJs revealed that the diameters of those NMJs from SMN{Delta}7 SMA mice (Fig. 5E) tended to be smaller than those from SMN{Delta}7 non-SMA mice (Fig. 5D). The diameters of the gastrocnemius NMJs from SMN{Delta}7 non-SMA and SMA mice are graphically shown in Figure 5F. We also examined the integrity of postsynaptic AChR clusters in the gastrocnemius of SMN{Delta}7 non-SMA and SMA mice at PND14, using a scoring system developed by Pun et al. (54Go). A higher proportion of NMJs from SMN{Delta}7 SMA displayed disassembled AChR clusters (low AChR cluster score) than those from SMN{Delta}7 non-SMA mice (Fig. 5G). This type of disassembly of postsynaptic receptors has also been observed in NMJs following denervation injury (54Go).



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  		Figure 5. Innervation and morphologies of NMJs in SMN{Delta}7 SMA mice. (A and B) Immunofluorescent micrographs showing the innervation of NMJs within the gastrocnemii of SMN{Delta}7 SMA (B, SMN2+/+; Smn{Delta}7+/+; Smn–/–) and non-SMA (A, SMN2+/+; Smn{Delta}7+/+; Smn+/–) mice. NMJs were labeled with AlexaFluor 488-conjugated {alpha}-bungarotoxin (green) and motor neuron axons were labeled with antibodies directed against the heavy chain of neurofilament (NF-H) and synaptic vesicles (SV2) which were visualized with AlexaFluor 594-conjugated secondary antibodies (red). The closed arrow points to an innervated NMJ, the open arrow to a partially innervated NMJ and the closed arrowhead to a noninnervated NMJ. Scale bar, 50 µm. (C) Distribution of innervated (black), partially innervated (light gray) and non-innervated (dark gray) NMJs in SMN{Delta}7 SMA and non-SMA gastrocnemii. There are fewer innervated NMJs in SMN{Delta}7 SMA samples than those in non-SMA mice. (D and E) Micrographs showing the morphologies of NMJs within the gastrocnemius muscle of SMN{Delta}7 SMA (E) and non-SMA (D) mice at PND14. NMJs are shown in green and the corresponding motor neuron axons are in red. Scale bar, 10 µm. (F) Histogram showing the mean NMJ diameter in SMN{Delta}7 SMA and non-SMA gastrocnemii at PND14. (G) Histogram showing the distribution of postsynaptic AChR cluster scores for SMN{Delta}7 SMA (gray bars) and non-SMA mice (black bars). The AChR cluster scores represent the NMJ postsynaptic integrity which is compromised in denervated muscles (54Go).

 
Association of SMN and SMN{Delta}7
It has been previously shown with in vitro binding assays that SMN and SMN{Delta}7 can associate with each other, albeit at a reduced affinity (55Go). To demonstrate that SMN and SMN{Delta}7 associated to form heterotypic complexes in a cell, we transfected SMN constructs that are tagged at their N-terminal with either the Flag epitope or the enhanced green fluorescent protein (EGFP). As shown in Figure 6A, epitope–tagged SMN{Delta}7 co-immunoprecipitates tagged SMN, indicating that they form an oligomeric complex in cultured cells. The oligomeric complex is important in efficiently binding SMN associated proteins (56Go). In addition, the incorporation of SMN{Delta}7 into a SMN complex stabilizes SMN{Delta}7 expression levels as shown in Figure 6B. Co-transfection of FL-SMN with SMN{Delta}7 stabilized SMN{Delta}7 such that more SMN{Delta}7 protein was present at 36 and 48 h when compared with those transfections with only SMN{Delta}7 (Fig. 6B). Not only does co-transfection of SMN{Delta}7 and FL-SMN stabilize the SMN{Delta}7 protein but also it is localized in a pattern that is indistinguishable from FL-SMN (55Go) (Fig. 6E). This contrasts with the situation that occurs when SMN{Delta}7 is transfected by itself despite the presence of endogenous FL-SMN (Fig. 6C). Thus, when SMN{Delta}7 is in excess, the SMN complex is retained in the nucleus; whereas when FL-SMN and SMN{Delta}7 are expressed in a 1 : 1 ratio, the SMN complex is correctly localized. We suggest that this is similar to the situation that occurs in the SMN{Delta}7 transgene reported here with FL-SMN and SMN{Delta}7 protein being in an ~1 : 1 ratio and the heterotypic complex being the required location. Thus, the transgene and co-transfection data are consistent with the idea that SMN{Delta}7 simply increases the amount of functional oligomeric SMN complex present in motor neurons and thus improves the phenotype of SMA animals.



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  		Figure 6. Association of FL-SMN and SMN{Delta}7 in cultured cells. Formation of a heterotypic complex between FL-SMN and SMN{Delta}7 can increase the stability of SMN{Delta}7 and is co-localized in neuritic granules. (A) HEK293 cells were co-transfected with indicated EGFP and Flag-tagged SMN constructs. Co-immunoprecipitation of Flag–SMN and EGFP–SMN reveal formation of both homotypic (full length/full length; {Delta}7/{Delta}7) and heterotypic (full length/{Delta}7) complexes. Flag-tagged SMN was first immunoprecipitated and then western blot was performed on both supernatants and pellets by co-incubation with monoclonal antibodies to EGFP and Flag. The top lane depicts the EGFP–SMN fusion proteins; bottom lane shows Flag– SMN fusion proteins. (B) Western blot analysis of total protein extracts from co-transfected cells. Expression of EGFP–SMN and Flag–SMN after 24 h. (lane 1). Expression of EGFP–SMN{Delta}7 and Flag–SMN at 24, 36 and 48 h. (lanes 2–4). Expression of EGFP–SMN{Delta}7 and Flag–SMN{Delta}7 at 24, 36 and 48 h. (lanes 5–7). Note that EGFP–SMN{Delta}7 levels were significantly lower when co-expressed with Flag–SMN{Delta}7 compared with Flag–SMN. These results suggest formation of a heterotypic complex which can stabilize the truncated SMN{Delta}7 form. Bottom panel: ß-actin was detected with a monoclonal antibody as a loading control. (C) Transfection of primary neurons with YFP–SMN{Delta}7 (red) and CFP–SMN{Delta}7 (green) showed co-localization in nuclear foci in the nucleus (yellow, arrow). (D) Phase optic indicates a nuclear position (arrowhead). (E) In contrast, cytoplasmic localization of CFP–SMN{Delta}7 (green) was restored by the co-transfection with FL-SMN (YFP–SMN). Note co-localized granules of CFP–SMN{Delta}7 (green) and YFP–SMN (red) in the processes (yellow, arrows). (F) The position of nucleus shown with phase optics (arrowhead).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Spinal muscular atrophy is caused by loss of the SMN1 gene and retention of the SMN2 gene (9Go). A single copy of the SMN2 gene does not produce sufficient SMN protein to compensate for the loss of SMN1 but multiple copies of SMN2 alter the severity of phenotype (7Go,22Go,23Go). The difference between the SMN1 and SMN2 genes is a single nucleotide in exon 7 which alters the splicing pattern of the two genes (6Go,13Go,48Go). The majority of the transcript from SMN2 lacks exon 7 (SMN{Delta}7) although it does produce some FL-SMN (9Go,11Go,16Go). SMN2 copy number is known to modify phenotypic severity, although there are exceptions where the SMN2 copy number does not correlate with the phenotype in that particular patient (22Go–25Go). Detection of SMN2 and determination of its copy number rely on the sequence difference in exon 7 and thus only indicates the presence of the 3' end of the SMN2 gene but not necessarily an intact gene. The analysis of the polymorphic marker Ag1-CA (C272), which lies at the 5'end of the SMN genes, indicates a correlation of Ag1-CA copy number to SMN2 copy number in SMA patients which demonstrates that the majority of SMN2 genes are intact (20Go,24Go,57Go). The lack of complete correlation, however, could indicate that non-intact SMN2 genes do exist and explain some of those exception cases where SMN2 copy number does not correlate with severity. In addition, the genome region that contains the SMN genes is complex and not all individuals contain the same size or arrangement of genes as indicated by the different size fragments obtained on pulsed field gels (21Go). These arrangements, as well as genes outside the SMA locus, could influence the splicing pattern of the SMN2 gene and thus alter the amount of FL-SMN and SMN{Delta}7 from any particular SMN2 gene (58Go). Thus, the major modifier of SMA phenotype is SMN2 and the critical parameter is the amount of SMN it can produce. In this paper, we show that the amount of SMN{Delta}7 as well as FL-SMN influences the severity of the SMA phenotype indicating that the critical parameter is the amount of total SMN protein that is produced.

A number of studies have investigated SMN mRNA and the spliced isoforms present in SMA patients (11Go,16Go,59Go–62Go). It is clear from these studies that there is an increase in the amount of SMN{Delta}7 relative to FL-SMN in SMA patients. Attempts at quantifying the amounts of the different SMN isoforms in SMA are inconsistent. Various tissues and, in particular, cultured cells derived from SMA patients have been examined. Control of growth conditions is particularly important in assessing SMN levels in culture. Furthermore, transforming cells using agents such as the SV40 large T antigen does alter SMN levels (3Go,4Go,42Go). In a number of the studies indicated earlier, high cycle numbers during amplification and the use of non-denaturing gels which will not separate heteroduplex bands formed between various isoforms further complicates the quantitative analysis of the SMN isoforms in different SMA types. In one study (60Go), it was suggested that the level of SMN{Delta}7 was increased in types I and II individuals when compared with levels in type III individuals. The authors concluded that SMN{Delta}7 was or could produce a product that had a negative impact on severity in SMA. In these cases SMN is always expressed from SMN2 so there is some FL-SMN present this is similar to the situation in the transgenes in this paper. Others (51Go,52Go) have shown that SMN{Delta}7 is pro-apoptotic when produced at very high levels in a cell and also suggest that SMN{Delta}7 has a deleterious effect in SMA. This is in striking contrast to the transgenic data we present in this paper. We find that increased amounts of SMN{Delta}7 are not only not detrimental but also beneficial to the SMA phenotype. The survival of the mice increases from 5.2 to 13.3 days. We have also observed a trend towards more total SMN mRNA when moving from severe to mild SMA using mRNA isolated from patient fibroblasts (63Go). It is certainly possible that some type I patients have a deletion of exon 7 and still produce SMN{Delta}7 from this locus whereas gene conversion to SMN2 is more frequent in mild SMA patients, thus altering the ratio of FL-SMN-to-SMN{Delta}7. The critical correlation seems to be the amount of SMN protein that is produced and is stable. The transgenic data obtained in this paper is consistent with the data obtained by Wang and Dreyfuss (64Go) who showed that SMN{Delta}7 does rescue a cell line expressing very low levels of SMN thus indicating that it can function in the presence of low levels of full length SMN.

Taken together, these reports and our data argue strongly that increased levels of SMN{Delta}7 positively modify the SMA phenotype and exert no negative effects, at least at levels that could reasonably be expected in vivo. It is possible that SMN{Delta}7 acts in a dominant negative manner by capturing FL-SMN when expressed at extremely high levels, using viral vectors or transient transfections; however, it is unlikely that drug treatments could achieve these high levels. The loss of SMN exon 7 but retention of the ability to produce SMN{Delta}7 results in early embryonic death (40Go). It could be argued that the Smn{Delta}7/Smn{Delta}7 embryos live longer than in a complete knockout (36Go), but this could well be due to the contribution of maternal SMN and SMN{Delta}7 as opposed to function of SMN{Delta}7 on its own. We have found that the mild mutant SMNA2G protein does not rescue embryonic lethality of Smn–/– mice (39Go). Thus, both SMN{Delta}7 and mild SMA mutation appear to have minimal, if any function without full length SMN being present. This would indicate that FL-SMN and SMN{Delta}7 interact to modify the phenotype. Indeed, transfection data present in this paper shows that FL-SMN and SMN{Delta}7 interact and that FL-SMN can stabilize the SMN{Delta}7 protein expression. It is most likely that the functional unit for SMN is the oligomer, which is composed of both FL-SMN and SMN{Delta}7 in the transgenes presented here. In the case of transfection of SMN{Delta}7 alone into a cell line, the high expression levels of SMN{Delta}7 will result in the vast majority of oligomer containing only SMN{Delta}7 or very little endogenous FL-SMN, hence its accumulation in the nucleus (55Go) and its degradation. The localization of heterotypic FL-SMN/SMN{Delta}7 complexes is the same as FL-SMN; thus, expression of SMN{Delta}7 at these levels simply adds to the total amount of oligomeric SMN complex, thereby enhancing SMN function. We would suggest that SMN2 modifies the SMA phenotype through the total amount of SMN protein that the gene can produce in neurons with both full-length and SMN{Delta}7 contributing SMN protein to different extents. This is consistent with the correlation of SMN protein level with severity of SMA (3Go,4Go).

Loss of FL-SMN from muscle in a conditional gene knockout leads to a dystrophic phenotype and loss of dystrophin staining on the membrane (53Go). In this study, we show that low SMN levels from SMN2 protect against loss or disruption of the dystrophin complex, and there are the no obvious signs of dystrophy. There is no loss of dystrophin staining in muscle biopsies from SMA patients (65Go), thereby making our results consistent with those observed in human patients.

There are signs of denervation and atrophy as would be expected. We suggest that loss of functional SMN from muscle does not give an equivalent phenotype to low SMN levels and cannot be used to indicate SMN at high levels is critical in the pathogenesis of SMA. Currently, it appears that the critical function of SMN for motor neurons is cell autonomous and involved in motor axonal patterning (66Go). This would predict an early developmental requirement for high SMN levels to ensure correct guidance of motor neurons, at least in severe SMA (63Go). It will become important to determine whether this occurs in SMA mice and how this affects the function and development of synaptic connections with muscle.

Drug compounds that either activate the SMN promoter or increase the amount of FL-SMN have been identified (41Go–47Go,49Go). Some compounds that activate the promoter can increase the amount of SMN{Delta}7 considerably (41Go) (unpublished data). This study indicates that this is not harmful. On the contrary, it would appear that increasing the amount of SMN{Delta}7 alleviates the disease phenotype. The SMN{Delta}7 SMA mice presented here live up to 17 days and with their extended lifespan over our previously reported severe SMA mice are useful for testing of either gene or drug therapy in vivo.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Generation of transgenic mice and breeding strategy
The generation of the SMN construct lacking exon 7 but containing exon 8 has been previously described (19Go). Briefly, RT–PCR was performed from total RNA isolated from a type I SMA patient fibroblast line (number 3813), using eLONGase (Invitrogen, Grand Island, NY, USA). The PCR primers were 5'-CAGGATCCCTATGGCGATGAGCAGC-3' and 5'-CGGAATTCAGTACAATGAACAGCCATGTCCA-3'. The conditions are described in Le et al. (19Go). The amplified 1.37 kb fragments were digested with BamHI and EcoRI (engineered into the primer sequences) and subcloned into pcDNA3. The resulting clones were then amplified with primers in exons 4 and 8, as described previously for RT–PCR of SMN mRNA (38Go,42Go) to distinguish clones lacking exon 7. SMN{Delta}7 clones were sequenced so as to ensure that these clones did not carry additional mutations.

Separately, an ~3.4 kb KpnI–SacII SMN promoter fragment (67Go) was cloned into the pEGFP (BD Biosciences Clontech, San Diego, CA, USA) vector and then excised out using KpnI and BamHI. This fragment was directionally ligated to the SMN{Delta}7 cDNA in pcDNA3 and the CMV promoter removed (39Go). The 5.4 kb promoter–SMN{Delta}7 construct was excised with KpnI and DraIII and injected into fertilized FVB/N oocytes. Potential founders were screened by Southern blot analysis using exons 6–8 SMN cDNA probe or by PCR using the following primers in exons 4 and 8: 5'-AGTGAGAACTCCAGGTCTCCTG-3' and 5'-TTCTCAACTGCCTCACCACCGT-3'. Southern blots were performed as described previously (39Go) except that the fractionated DNA bands were transferred onto a nitrocellulose membrane using the Genie electrophoretic blotter (Idea Scientific, Minneapolis, MN, USA) as per manufacturer's directions. The quantitation of Southern blots was performed by co-hybridizing with a mouse Smn probe followed by densitometric analysis as described previously (39Go). Southern blots were first performed with restriction enzymes that cut once within the construct to determine integrants (all were single) and subsequently with an enzyme that releases an internal fragment for quantitation.

Real-time PCR for copy number determination was performed using the ABI Prism 7700 sequence detection system. The method for determination of copy number is described by Schaeffeler et al. (68Go); however, we used the mouse ß-globin gene as the control gene (two copies). The data were also confirmed using SMN2 intron primers and mouse Smn primers. The following primers were used and were tested to have similar efficiencies using the method described by Liu and Saint (69Go): ß-globin; forward, 5'-AAACAAGAGCAAACTAAGTAAGATGCAT-3' and reverse, 5'-TGAGTGCATGGATGAATCTTCT-3'; transgenic human SMN{Delta}7: forward, 5'-GCGGCGGCAGTGGTGGCGGC-3' and reverse, 5'-AGTAGATCGGACAGATTTTGCT-3'. PCR was performed with SYBR Green Jump Start Tag Ready mix (Sigma-Aldrich, St. Louis, MO, USA) and the following cycling conditions: 2 min initial denaturation at 94°C, 15 s at 94°C, 1 min at 60°C and 1 min at 72°C. The validity of this method was confirmed using the SMN2 transgene of known copy number and the mice that were either heterozygous or homozygouse for the Smn knockout allele.

Founders were bred to SMN2;Smn+/– mice (6Go). SMN{Delta}7;SMN2;Smn+/– mice were interbred resulting in SMN{Delta}7;SMN2;Smn–/– mice. To identify mice that were homozygous for the SMN{Delta}7 and SMN2 transgenes, mice carrying the transgenes were bred to non-transgenic FVB/N mice. Mice that had 100% of their offspring positive for the transgenes for a minimum of 40 offspring were considered homozygous. The SMN2 transgene was genotyped with the primers 5'-GCGATAGAGTGAGACTCCATCT-3' and 5'-GACATAGAGGTCTGATCTTTAGCT-3'. The mouse knockout allele was detected with two primer sets: one set for neomycin resistance (NeoR) gene (5'-GCAGCTGTGCTCGACGTTGTC-3' and 5'-CACCATGATATTCGGCAAGCAGGC-3') and the other set detects the junction fragment between mouse Smn exon 2A and NeoR (5'-CGGCTACCTGCCCATTCGACCACC-3' and 5'-CCTTAAAGGAAGCCACAGCTTTATC-3'). The mouse Smn gene is detected with the primers 5'-TCCAGCTCCGGGATATTGGGATTG-3' and 5'-AGGTCCCACCACCTAAGAAAGCC-3'.

RT–PCR and western analysis of transgenes
Total RNA was isolated from 100 mg of mouse tissue using the TRIzol reagent (Invitrogen), according to the manufacturer's directions. To eliminate DNA contamination, RNA was treated with DNase Inactivation Reagent (DNA-freeTM, Ambion, Austin, TX, USA) for 3 h at 37°C. Elimination of DNA contamination was confirmed by PCR of treated RNA. To produce cDNA, the following protocol was followed: 4–5 µg of treated RNA was mixed with 250 ng of random hexamers (Amersham Biosciences) in a final volume of 17 µl. The mixture was heated to 70°C for 10 min, and then chilled on ice to anneal the primer-template. The first strand cDNA was synthesized in the following buffer: 1 mM dNTPs, 50 mM Tris–HCl, pH 8.3, 8 mM MgCl2, 50 mM NaCl, 1 mM DTT, 10 U RNase inhibitor (RNAguardTM, Amersham Biosciences, Piscataway, NJ, USA) and 15 U AMV reverse transcriptase (US Biologicals, Cleveland, OH, USA) at 42°C for 1 h. We used a multiplex PCR reaction using SMN and mouse hypoxanthine phosphoribosyltransferase (HPRT) primers, as described previously (11Go,38Go,42Go). The PCR was performed with a {gamma}-32P-labeled exon 4 primer (5'-GTGAGAACTCCAGGTCTCCTGG-3') and an unlabeled human specific primer in exon 8 (5'-CTACAACACCCTTCTCACAG-3'). The primers for mouse HPRT were 5'-AGTCCCAGCGTCGTGATTAGC-3' and 5'-TTCACCAGCAAGCTTGCAACC-3'. The forward primer was {gamma}-32P labeled and the HPRT primers were 50 times more dilute than the SMN primers. The PCR conditions were as follows: denaturation for 4 min at 94°C, 20 cycles of 1 min at 60°C, 1 min at 72°C, 30 s at 95°C, followed by a final extension for 4 min at 72°C. The PCR products were run on a 6.7% polyacrylamide denaturing gel and analyzed on a Storm imager (Amersham Biosciences), as described by the manufacturer.

For western blot analysis, 100 mg of tissue was homogenized in blending buffer (10% SDS, 62.5 mM Tris, pH 6.8, 5 mM EDTA). The sample was mixed with an equal volume of loading buffer (62.5 mM Tris, pH 6.8, 20% glycerol, 200 mM DTT, 0.2% bromophenol blue) and run on a 12.5% polyacrylamide gel. Samples were transferred to Immobilon-P (Millipore, Bedford, MA, USA), as previously described (3Go). The blot was blocked in 5% milk powder, 0.5% BSA in PBS–Tween for 1 h, and then incubated for 1 h with primary antibody MANSMA2 (27Go). Bound primary antibody was detected by horseradish peroxidase conjugated secondary antibody followed by chemiluminescence (ECLTM Western Blotting Detection Reagents, Amersham Biosciences). The blots were then stripped and re-probed with a ß-actin monoclonal antibody (clone AC-15, Sigma-Aldrich) to control for protein loading.

Association of SMN and SMN{Delta}7 in cultured cells
Full-length cDNA of the human SMN1 was subcloned into either EGFP-C1 or YFP-C1 (BD Biosciences Clontech, Palo Alto, CA, USA) at the sites of HindIII and EcoRI and was termed EGFP–SMN or YFP–SMN, respectively. cDNA of exon-7 deletion mutant (SMN{Delta}7) which includes four amino acids (EMLA) encoded by the exon-8, was generated by PCR using specific primers and then subcloned into vectors of EGFP-C1, YFP-C1 or CFP-C1 at the sites of HindIII and EcoRI, and termed EGFP–SMN{Delta}7, YFP–SMN{Delta}7 or CFP–SMN{Delta}7. For CO-IP experiments, the fluorescence proteins in the constructs were replaced with a Flag-tag (MDYKDDDDK) at the sites of AgeI and HindIII and termed Flag-SMN and Flag-SMN{Delta}7. All of the constructs were purified (Qiagen) and sequenced to ensure that no frame shift had occurred.

HEK293 cells were cultured in Dulbecco's modified eagle's medium (DMEM) containing 10% FBS (Sigma). Cells were briefly washed with prewarmed DMEM before transfection. An equivalent amount of Flag- and EGFP-tagged constructs (2–3 µg total DNA) were diluted to 100 µl in transfection buffer (20 mM HEPES, 150 mM NaCl, pH 7.4) and then incubated with 5 µl of FuGENE 6 transfection reagent (Roche Molecular Biologicals, Indianapolis, IN, USA) for 15 min at room temperature. After incubation with the DNA mixture for 1 h, cells were cultured in DMEM with 10% FBS for 24 h.

Protein extracts were then prepared using a lysis buffer (50 mM Tris–HCl, pH 7.4, 150  mM NaCl, 0.1% NP-40 and 0.1% sodium deoxycholate). The protein extract (1 ml) was incubated with a rabbit antibody to Flag (2 µl; Sigma-Aldrich) and protein-A agarose (60 µl) at 4°C overnight on a rotatory platform. After washing with a high-salt buffer (50 mM Tris–HCl, 300 mM NaCl, 0.1% NP-40), the beads were collected by centrifugation and re-suspended in SDS–PAGE loading buffer (60 µl). The bound proteins from each sample (20 µl), as well as an equivalent volume of supernatant from each transfection, were resolved through a 12% polyacrylamide gel containing SDS. Fractionated proteins were transferred to Hybond ECL nitrocellulose membrane (Amersham Biosciences). Flag fusion proteins for SMN and SMN{Delta}7 were detected with a monoclonal antibody to Flag (1 : 3000; Sigma-Aldrich) diluted in TBS buffer. EGFP–SMN and EGFP–SMN{Delta}7 were detected using a monoclonal antibody to EGFP (1 : 5000; BD Biosciences Clontech). The membrane was washed and incubated with peroxidase-conjugated donkey anti-mouse IgG (Jackson Immunoresearch, West Grove, PA, USA). The signal was developed using ECL detection reagents (Amersham Biosciences).

Histology and immunofluorescence
Motor neuron counts were performed as previously described (7Go,39Go). For histology and low resolution dystrophin staining, SMA mice and non-SMA controls were killed by cervical dislocation and the muscles were dissected. The tissues were then mounted on wooden blocks and flash frozen in liquid nitrogen-cooled isopentane (70Go). Ten to twelve micrometers thick transverse muscle sections were cut, mounted on Superfrost (Fisher Scientific) slides and then air dried before processing. For hematoxylin/eosin staining, sections were fixed in 100% alcohol (30 s), rinsed in tap water and stained in Harris's hematoxylin (Sigma-Aldrich) for 30 s followed by a second rinse in tap water. They were then stained in eosin Y (Sigma-Aldrich) for 20 s, rinsed in tap water, dehydrated in alcohol and cleared in xylene before mounting in Permount (Sigma-Aldrich) for light microscopy. Myofiber area was calculated using a SPOT-RT slider digital camera and SPOT software (Diagnostic Instruments, Inc, Sterling Heights, MI, USA).

For immunocytochemistry of dystrophin, the tissue sections on slides were brought to room temperature and rinsed for 5 min in potassium phosphate buffered saline (KPBS) and blocked for 15 min with KPBS containing 1% gelatin. The sections were rinsed in KPBS containing 0.2% gelatin. A rabbit polyclonal antibody directed against the N-terminal of mouse dystrophin (71Go) was diluted 1 : 500 in KPBS containing 1% gelatin and 1% goat serum and incubated with the sections overnight at room temperature. The sections were then washed in three 5 min changes of KPBS with 1% gelatin and incubated with goat anti-rabbit FITC secondary (Jackson ImmunoResearch) in KPBS with 1% gelatin for 1 h. They were rinsed in three 5 min changes of KPBS with 1% gelatin and mounted with 50 µl of Vectashield (Vector). Images were obtained with an Eclipse E800 fluorescent microscope (Nikon) and MagnaFIRE v2.1C software (Optronics).

Confocal microscopy
Dissected muscle tissues which were used for high resolution dystrophin immunofluorescence were postfixed for 60 min in 1% paraformaldehyde in phosphate buffer followed by cryoprotection with 20% sucrose in PBS. For NMJ and motor neuron axon staining, mice were deeply anesthesized and perfused transcardially first with PBS and then with 4% paraformaldehyde in phosphate buffer. After perfusion, the muscles were dissected and cryoprotected in 20% sucrose solution overnight. The tissues were then mounted on wooden blocks and flash frozen in liquid nitrogen-cooled isopentane. Eight micrometers cross-sections (for high resolution dystrophin immunofluorescence) or 30 µm longitudinal sections (for NMJ and motor neuron axon staining) were cut on a cryostat, mounted onto gelatin-coated slides and air dried. The tissue sections on slides were brought to room temperature and washed for 60 min in 100 mM glycine in PBS at room temperature. The sections were then permeabilized with 0.5% Triton X-100 in PBS for 5 min on ice followed by rinsing with PBS. The slides were then blocked in 1% gelatin dissolved in PBS for 60 min. The sections were incubated in primary antibody solution (0.2% gelatin in PBS) overnight at room temperature. Either a rabbit polyclonal antibody directed against the N-terminal of mouse dystrophin (1 : 200) (71Go) or a cocktail of a chicken anti-neurofilament heavy chain pAb (1 : 500; EnCor Biotechnology, Inc., Alachua, FL, USA) and a mouse anti-synaptic vesicle monoclonal antibody (SV2; 1 : 50; Developmental Studies Hybridoma Bank, Iowa City, IA, USA) were used as primary antibodies. The sections were then washed in three 15 min changes of PBS with 0.2% gelatin and 0.1% Tween-20. Afterwards, the sections were incubated for 3 h at room temperature in a mixture of AlexaFluor 594-conjugated secondary antibodies (1 : 500; Molecular Probes, Eugene, OR, USA) and AlexaFluor 488-conjugated {alpha}-bungarotoxin (50 ng/mL; Molecular Probes) dissolved in 0.2% gelatin in PBS. They were rinsed in three 15 min changes PBS with 0.2% gelatin and 0.1% Tween-20 and mounted with ImmuMount (Shandon Lipshaw). Images were obtained using a Zeiss 510 META laser scanning confocal microscope (Carl Zeiss Inc., Thornwood, NY, USA) using either a 40x (NA=1.3), 63x (NA=1.4) or 100x (NA=1.4) oil objective. A zoom factor of 0.7 was used for the images obtained with the 40x objective, whereas those obtained with the 63x objective were obtained with a zoom factor of 2. A zoom factor of 3 was used to obtain images from the 100x objective. Optical sections were taken through the z-axis of each cell at a thickness of 500 nm per section.

Statistical analysis
The quantitative data presented here were expressed at the mean±SEM. One-way ANOVA analysis was performed on quantitative data using SPSS v 12.0 (SPSS, Inc., Chicago, IL, USA). Kaplan–Meier survival curves were generated with SPSS v.12 and statistical significance was determined with the log-rank test.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We wish to thank Kristie Schussler for help in editing the manuscript, the Campus Microscopy and Imaging Facility for providing access to the confocal microscope, Dr. Jill Rafael-Fortney for generously providing the dystrophin antibody as well as providing access to the SPOT system and Dr Michael Sendtner and colleagues for training in counting motor neurons. The SV2 hybridoma developed by Dr Kathleen M. Buckley was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences. These studies were funded by NIH grant NS3860 to A.H.M.B., Families of SMA to M.E.R.B. and SMA Foundation to G.J.B. and H.L.Z. U.R.M. was a recipient of a MDA transition award.


   FOOTNOTES
 
{dagger} Present address: Department of Neurology, Columbia University, New York, NY, USA. Back

{ddagger} These authors contributed equally to this manuscript. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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[Abstract] [Full Text] [PDF]

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 Hum Mol GenetHome page
T. O. Gavrilina, V. L. McGovern, E. Workman, T. O. Crawford, R. G. Gogliotti, C. J. DiDonato, U. R. Monani, G. E. Morris, and A. H.M. Burghes
Neuronal SMN expression corrects spinal muscular atrophy in severe SMA mice while muscle-specific SMN expression has no phenotypic effect
Hum. Mol. Genet., April 15, 2008; 17(8): 1063 - 1075.
[Abstract] [Full Text] [PDF]

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 Hum Mol GenetHome page
L. M. Murray, L. H. Comley, D. Thomson, N. Parkinson, K. Talbot, and T. H. Gillingwater
Selective vulnerability of motor neurons and dissociation of pre- and post-synaptic pathology at the neuromuscular junction in mouse models of spinal muscular atrophy
Hum. Mol. Genet., April 1, 2008; 17(7): 949 - 962.
[Abstract] [Full Text] [PDF]

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 J. Neurosci.Home page
O. Biondi, C. Grondard, S. Lecolle, S. Deforges, C. Pariset, P. Lopes, C. Cifuentes-Diaz, H. Li, B. della Gaspera, C. Chanoine, et al.
Exercise-Induced Activation of NMDA Receptor Promotes Motor Unit Development and Survival in a Type 2 Spinal Muscular Atrophy Model Mouse
J. Neurosci., January 23, 2008; 28(4): 953 - 962.
[Abstract] [Full Text] [PDF]

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R. Morse, D. J. Shaw, A. G. Todd, and P. J. Young
Targeting of SMN to Cajal bodies is mediated by self-association
Hum. Mol. Genet., October 1, 2007; 16(19): 2349 - 2358.
[Abstract] [Full Text] [PDF]

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 Am. J. Pathol.Home page
J. Vitte, C. Fassier, F. D. Tiziano, C. Dalard, S. Soave, N. Roblot, C. Brahe, P. Saugier-Veber, J. P. Bonnefont, and J. Melki
Refined Characterization of the Expression and Stability of the SMN Gene Products
Am. J. Pathol., October 1, 2007; 171(4): 1269 - 1280.
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 Proc. Natl. Acad. Sci. USAHome page
A. K. Mishra, L. Gangwani, R. J. Davis, and D. G. Lambright
Structural insights into the interaction of the evolutionarily conserved ZPR1 domain tandem with eukaryotic EF1A, receptors, and SMN complexes
PNAS, August 28, 2007; 104(35): 13930 - 13935.
[Abstract] [Full Text] [PDF]

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 J Child NeurolHome page
S. Shanmugarajan, K. J. Swoboda, S. T. Iannaccone, W. L. Ries, B. L. Maria, and S. V. Reddy
Congenital Bone Fractures in Spinal Muscular Atrophy: Functional Role for SMN Protein in Bone Remodeling
J Child Neurol, August 1, 2007; 22(8): 967 - 973.
[Abstract] [PDF]

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C. J. Sumner
Molecular Mechanisms of Spinal Muscular Atrophy
J Child Neurol, August 1, 2007; 22(8): 979 - 989.
[Abstract] [PDF]

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C. E. Beattie, T. L. Carrel, and M. L. McWhorter
Fishing for a Mechanism: Using Zebrafish to Understand Spinal Muscular Atrophy
J Child Neurol, August 1, 2007; 22(8): 995 - 1003.
[Abstract] [PDF]

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A. Schmid and C. J. DiDonato
Animal Models of Spinal Muscular Atrophy
J Child Neurol, August 1, 2007; 22(8): 1004 - 1012.
[Abstract] [PDF]

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C. R. Heier, R. G. Gogliotti, and C. J. DiDonato
SMN Transcript Stability: Could Modulation of Messenger RNA Degradation Provide a Novel Therapy for Spinal Muscular Atrophy?
J Child Neurol, August 1, 2007; 22(8): 1013 - 1018.
[Abstract] [PDF]

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J. van Bergeijk, K. Rydel-Konecke, C. Grothe, and P. Claus
The spinal muscular atrophy gene product regulates neurite outgrowth: importance of the C terminus
FASEB J, May 1, 2007; 21(7): 1492 - 1502.
[Abstract] [Full Text] [PDF]

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 NeurologyHome page
L. R. Simard, M-C Belanger, S. Morissette, M. Wride, T. W. Prior, and K. J. Swoboda
Preclinical validation of a multiplex real-time assay to quantify SMN mRNA in patients with SMA
Neurology, February 6, 2007; 68(6): 451 - 456.
[Abstract] [Full Text] [PDF]

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 Proc. Natl. Acad. Sci. USAHome page
V. Setola, M. Terao, D. Locatelli, S. Bassanini, E. Garattini, and G. Battaglia
Axonal-SMN (a-SMN), a protein isoform of the survival motor neuron gene, is specifically involved in axonogenesis
PNAS, February 6, 2007; 104(6): 1959 - 1964.
[Abstract] [Full Text] [PDF]

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 NeurologyHome page
C. J. Sumner, S. J. Kolb, G. G. Harmison, N. O. Jeffries, K. Schadt, R. S. Finkel, G. Dreyfuss, and K. H. Fischbeck
SMN mRNA and protein levels in peripheral blood: Biomarkers for SMA clinical trials
Neurology, April 11, 2006; 66(7): 1067 - 1073.
[Abstract] [Full Text] [PDF]

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B. Renvoise, K. Khoobarry, M.-C. Gendron, C. Cibert, L. Viollet, and S. Lefebvre
Distinct domains of the spinal muscular atrophy protein SMN are required for targeting to Cajal bodies in mammalian cells
J. Cell Sci., February 15, 2006; 119(4): 680 - 692.
[Abstract] [Full Text] [PDF]

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