Spinal muscular atrophies (SMA) are characterized by degeneration of lower motor neurons

Transcription

1 Spinal Muscular Atrophy 175 CHAPTER 14 Spinal Muscular Atrophy Robert Olaso, Jérémie Vitte, Nouzha Salah and Judith Melki Abstract Spinal muscular atrophies (SMA) are characterized by degeneration of lower motor neurons and occasionally bulbar motor neurons leading to progressive limb and trunk paralysis associated with muscular atrophy. SMA is a recessive autosomal disorder with an incidence of 1 out of 5000 newborns and represents one of the most frequent genetic causes of death in childhood. Identification of the survival of motor neuron gene (SMN1) and mutations found in SMA patients greatly improved the clinical management and family-planning options of SMA patients and their parents. The last years have seen major advances in the biochemistry of SMN although the molecular pathway linking SMN defect to the SMA phenotype remains to be elucidated. Animal models of SMA have been generated providing valuable tools to clarify SMA pathogenesis and for designing therapeutic approaches of this devastating neurodegenerative disease for which no curative treatment is known so far. Clinical Aspects Based on age of onset of symptoms, achievement of motor milestones and age at death, childhood SMA has been subdivided into three clinical types. 1 The acute form or Werdnig-Hoffmann disease (type I) is characterized by severe, generalized muscle weakness at birth or within the first 6 months. Feeding and breathing difficulties are usually responsible for death within two years of age. Type II SMA is a more slowly progressive generalized disease with a variable prognosis. Infants are able to sit unsupported but not able to stand or walk unaided. Clinical progression is slow or appears even to arrest. All children will develop, if untreated, severe scoliosis and respiratory ventilation defect. Life expectancy is highly variable, ranging up to adult life in some cases. In juvenile SMA or type III or Kugelberg-Welander disease, symptoms start from 18 months of life to the age of 20 years. All patients are able to walk and proximal muscle weakness is progressive. Subsequently, the patients show difficulties in climbing stairs, gait become waddling. About one fourth of SMA type III patients exhibit a hypertrophy of the calves, a feature similar to that observed in Duchenne or Becker muscular dystrophies. Finally, adult SMA is defined by an age of onset of symptoms beyond 20 years of age, the diagnostic criteria being identical to the other forms. Whatever the age of onset, the clinical features consist of symmetrical muscle weakness (more proximal than distal) associated with muscle atrophy, absence or marked decrease of deep tendon reflexes, fasciculations of the tongue, and tremor of hands. There is no evidence of sensory or upper motor neuron involvement. Electromyographic studies show a pattern of denervation with neither sensory involvement nor marked decrease of motor nerve conduction velocities. Finally, muscle biopsy, which has been recently replaced by genetic testing for diagnosis, provides evidence of skeletal muscle denervation with groups of atrophic and hypertrophic fibers or fiber type grouping most often found in chronic cases (Types II and III). The most striking neuropathological feature found in autopsy material of SMA patients is a loss of the large anterior horn cells of the spinal cord. In the remaining surviving motor Molecular Mechanisms of Muscular Dystrophies, edited by Steve J. Winder Eurekah.com.

2 176 Molecular Mechanisms of Muscular Dystrophies Figure 1. Genomic organization of the SMA locus in human control and SMA individuals. Transcripts derived from SMN1 or SMN2 genes are indicated in boxes. Full-length or truncated transcripts are indicated as ( ) or ( ), respectively. Deletion or gene conversion events of SMN1 gene are responsible for severe (type I) or mild (type III) SMA, respectively. Only one allele is presented on each panel. Truncated transcripts are translated into unstable protein indicated as (0). The abundance of transcripts or protein is indicated as (0, +, ++ or ++++). neurons, severe degree of central chromatolysis is visible. These cells appear as large ballooned cells without stored substances. Other anterior cells are pyknotic. In addition, there are occasional figures of neuronophagia associated with astrogliosis and the anterior roots are small. Genetic Basis of SMA The SMA locus on chromosome 5q13 is characterized by an inverted duplication, each element (about 500 kb) containing several genes. Deletions or conversion events including exon 7 of the telomeric copy of the survival of motor neuron gene (SMN, renamed SMN1) are found in 93% of SMA patients. 2 In the remaining patients, intragenic mutations of SMN1 including missense, nonsense or splice site mutations have identified SMN1 as the SMA disease gene. 2,3 SMN1 is duplicated with a highly homologous copy called SMN2 and both genes are transcribed (Fig. 1). The SMN2 gene is present in all patients but is not able to compensate for the SMN1 gene defects. At the genomic level, the presence of a gene dosage effect in type I but not in type III SMA has suggested that type I SMA was caused by deletion of SMN1 whereas type III was associated with conversion event of SMN1 into SMN2 gene leading to an increased number of SMN2 genes (Fig. 1). 2,4 This is in agreement with the presence of a tight inverted correlation between the amount of the protein encoded by the SMN2 gene and the clinical severity of human SMA disease. 5 In patients, the SMN protein level depends on SMN2 copy number making SMN2 as a modifying gene in SMA. These data have ascribed SMA to a dose effect of SMN. Mutations of SMN1 and SMN2 on both chromosomes have not been reported. Such a genotype would be likely responsible for an extremely severe form of SMA or non viable fetus.

3 Spinal Muscular Atrophy 177 Why is SMN2, which remains present in SMA patients, not able to compensate for the SMN1 defect? Five nucleotides distinguish SMN2 from SMN1 genes without any effect on the amino acid sequence. 2 One of these nucleotide substitutions located in exon 7 is responsible for the alternative splicing of exon 7 which is specific to the SMN2 transcripts. 2,6 Full-length transcripts are almost exclusively produced by SMN1, whereas the predominant form encoded by SMN2 is lacking exon 7. Full length transcript is also encoded by SMN2 and translated into functional protein but its abundance is much lower than that encoded by SMN1. The truncated transcript lacking exon 7 encodes a protein lacking the last C-terminal 16 residues (SMN 7 ). In vitro data strongly suggested a dominant negative effect of SMN 7 on the full length SMN protein. 7-9 However, such effect was not observed in vivo in both mice and humans carrying heterozygous deletion of SMN exon Dominant negative effect SMN 7 could be ascribed to over-expression of SMN 7 induced by in vitro but not in vivo systems. Therefore, mutations of SMN1 are responsible for a loss of SMN function. SMN: A Multifunctional Protein SMN is an ubiquitously expressed protein of 294 amino acids with a molecular weight of 38 kda. Sequence analysis showed that SMN contains a phylogenetically conserved sequence called the Tudor domain which has been found in many proteins involved in RNA metabolism. 11 The SMN complex is found both in the cytoplasm and in the nucleus where it is concentrated in a structure called gems (for gemini of coiled bodies ) most often associated with or identical to Cajal bodies (coiled bodies) depending on the cell type or tissue analyzed. 12 Cajal bodies are nuclear structures enriched in U small nuclear ribonucleoprotein (snrnp) and U small nucleolar RNP proteins (snornp). SMN forms a large complex of approximately 1 MDa and most of components of this complex have been identified. SMN interacting proteins include Gemin 2 (formerly SIP1 for SMN interacting protein 1 ), Gemin 3 (a DEAD box putative RNA helicase previously known as dp103), Gemin 5, the spliceosomal snrnp Sm and Lsm protein, the snornp including fibrillarin and GAR1, heterogeneous nuclear RNP-Q and coilin. 13 The SMN complex is also composed of Gemin 4 and 6, two novel proteins of unknown function. 13 The interaction of SMN with components of this large complex is mediated directly by the Tudor domain and is enhanced by a SMN oligomerization domain corresponding to exon 6. The identification of SMN interacting proteins of known function strongly supports the view that SMN is involved and facilitates cytoplasmic assembly of snrnp into the spliceosome, a large RNA-protein complex that catalyzes the splicing reaction. In the nucleus, SMN appears to be directly involved in pre-mrna splicing, transcription and metabolism of ribosomal RNA. SMN can be regarded as an assembly factor that mediates formation of the Sm core domain. SMN has also been shown to interact directly with other proteins including Bcl-2 and p53, two proteins involved in apoptotic processes, although these interactions and the involvement of an apoptotic process in SMA remain to be clarified in vivo. Other direct or indirect partners of SMN include the FUSE binding protein, profilin II, a zinc-finger protein called ZRP1, RNA helicase A, RNA polymerase II, and RNA. 3 Therefore, SMN appears to be a multifunctional protein. How does partial deficiency of SMN, but not complete absence, lead to the SMA phenotype? Among the various putative functions of SMN, which deficiency is responsible for the SMA phenotype? SMN is indeed ubiquitously expressed and the selective involvement of the neuromuscular system in SMA suggest that cells of different types do not require the same amount of SMN for survival unless SMN has an as yet unknown function specific to neurons or skeletal muscle. To answer these questions, animal models of SMA have been generated. Animal Models of SMA In various species including M. musculus, D. melanogaster, C. elegans, SMN orthologue is not duplicated and its deletion leads to early embryonic lethality These data hampered our ability to clarify the function of SMN and the pathway involved in SMA pathogenesis in

4 178 Molecular Mechanisms of Muscular Dystrophies vivo. To circumvent embryonic lethality of mice knocked out for the Smn gene, several transgenic approaches were undertaken to create mouse models. One strategy was based on the generation of mice carrying genomic organization similar to that of human SMA. 18,19 It consisted of the creation of two mouse lines, one carrying a deletion of Smn through homologous recombination and the other line carrying a transgene expressing the human SMN2 gene. Mice carrying both homozygous deletion of Smn and the human SMN2 gene developed a phenotype depending on the number of SMN2 transgenes. Mice carrying low copy number of SMN2 and harbouring a homozygous null Smn allele developed a severe phenotype leading to death either in utero or in the first days after birth. Mutant mice displayed abnormal motor defects associated with a moderate loss of motor neurons of the spinal cord (up to 35%) at the latest stage of disease. The absence of abnormal phenotype in mutant mice carrying a high copy number of SMN2 transgene indicated that SMN2 was able to prevent the embryonic lethality of the Smn knock out mice and confirmed that an increased copy number of SMN2 reduced the severity of the phenotype, in agreement with data observed in human SMA. 18,19 Another approach has been carried out by using the Cre-loxP recombination system. A mouse line carrying two loxp sequences flanking Smn exon 7 (SmnF7) has been established through homologous recombination. Cre-mediated deletion of Smn exon 7, the most frequent mutation found in SMA patients, has been directed to neurons by crossing SmnF7 mice to transgenic mice expressing Cre recombinase in neurons ( neuronal mutant). 17,20 Neuronal mutant mice in which full length Smn transcripts are lacking, displayed severe motor defect leading to complete paralysis and death at a mean age of 4 weeks. Analysis of skeletal muscle revealed a severe muscle denervation process, a dramatic and progressive loss of motor axons (up to 73% reduction) contrasting with mild reduction of motor neuron cell bodies (29%). In addition, abnormal synaptic terminals of neuromuscular junctions filled with neurofilaments, including phosphorylated forms, were observed in mutant mice associated with defects in axonal sprouting. 20 These findings are likely responsible for motor neuron dysfunction and suggest that loss of motor neurons occurs through a dying back axonopathy. These data demonstrated that motor neurons are a primary target of the Smn gene defect in SMA. Consistently, antisense morpholinos to reduce SMN levels in zebrafish causes defects in motor axon pathfinding suggesting that SMN has a role in motor axon development. 21 Moreover, point mutations in SMN similar to those found in SMA patients have been found in Drosophila resulting in defects of the neuromuscular junctions including disorganization of synaptic motor neuron boutons and reduction of post synaptic receptor subunits. 22 Therefore, defects in SMN in various organisms highlighted an essential role of SMN in motor axon and neuromuscular junction development or maintenance. Is there an effect of SMN gene defect in other tissues? Deletion of Smn exon 7 has been directed to murine skeletal muscle using the same strategy than that described above ( muscular mutant ). 10 Unexpectedly, mutant mice displayed a dystrophic phenotype leading to muscle paralysis and death at a mean age of 1 month. The dystrophic phenotype was associated with destabilization of the sarcolemma. Although sarcolemma destabilization is not sufficient to explain death of mutant mice, this study revealed that Smn gene defect leads to a degenerative process of skeletal muscle fibers. The severity of muscle phenotype suggested an impairment of muscle regeneration resulting from SMN defect in both skeletal muscle fibers and progenitors in SMN muscular mutant. This was supported by Smn targeting directed to myotubes but not muscle progenitor cells. Mutant mice develop similar myopathic process but exhibit mild phenotype with median survival of 8 months (instead of 1 month), motor performance similar to that of controls within the first six months of age and high proportion of regenerating myofibers. These data suggest that SMN has an important role in skeletal muscle differentiation and/or regeneration. 23 To know whether SMN defects lead to deleterious effect in all mammalian cell types or is restricted to the neuromuscular system, which might suggest functions of SMN specific to this system, Smn mutation has been directed to liver, a tissue non affected in human SMA. Using a transgenic line that expressed the Cre recombinase in liver only, deletion of Smn exon 7 was

5 Spinal Muscular Atrophy 179 restricted to that tissue. Homozygous deletion of Smn exon 7 leads to late embryonic lethality. Mutant phenotype was characterized by marked atrophy of liver associated with severe liver dysfunction and absence of regeneration (J.V. and J.M., unpublished data). Altogether, these data support the hypothesis that SMN plays an ubiquitous and essential role in all cells including motor neurons. How to explain the difference of effect in response to SMN defect between human and mouse? Murine Smn gene is present as a single copy while in human, SMN1 is duplicated in a highly homologous copy SMN2. The residual amount of SMN produced by full length transcripts of the SMN2 gene, which remains present in patients, is likely sufficient to ensure normal functions of various organs including liver but not motor neurons. Why are motor neurons more sensitive to reduced level of SMN? Motor axons can reach more than 1 m long, making motor neuron one of the largest cells in the body by both volume and surface area. Such large cellular unit requires molecular machinery able to produce and regulate molecules, including proteins and RNA, from the cell body to the neuromuscular junction through motor axons. Mild impairment in production or stability of molecules involved in these processes could have detrimental effect on axonal growth. Consistently, defects of axonal growth and neuromuscular junctions are the main features found in animal models. However, other neurons including sensory neurons are also very large in size. Recently, involvement of these neurons has been described in severe cases of SMA in agreement with such hypothesis. 24 Skeletal muscle has also been recently shown to be affected by severe SMN defect in mice, 10 features found in clinical reports suggesting a primary involvement of skeletal muscle, in addition to motor neurons, in human SMA The involvement of skeletal muscle which has a critical role in motor neuron maintenance could modulate the expression or progression of SMA disease. Altogether, these data suggest that involvement of other tissues may occur during prolonged SMA disease course or in very severe forms of SMA. Ubiquitous expression of SMN and the deleterious effect of lack of SMN in various mammalian cell types strongly suggest an ubiquitous role of SMN rather than a function specific to the neuromuscular system. Recently, SMN has been localized in neurites and growth cones in which SMN appeared to be transported linked to components of the cytoskeleton. 29 Moreover, RNA and RNA binding proteins were shown to interact with microtubules in neurons or microfilaments in fibroblasts. 30 Therefore, it is tempting to hypothesize that defects in transport of mrna complexes resulting from reduced levels of SMN could have detrimental effect in large cells, such as motor neurons. Alternatively, neurons express high numbers of genes with respect to other cell types and alternative splicing or stability of mrna are important post-transcriptional mechanisms to create or regulate protein diversity. 31 Although a recent report did not reveal abnormal splicing pattern of a single gene in a heterozygous SMA mouse mutant, 32 we cannot however exclude the hypothesis of a defect in metabolism of some RNA sub-classes involved in the structural specificities of motor neurons. Search for transcript defects through microarray analysis should allow to test this hypothesis. Therapeutic Strategies in SMA From a better knowledge of genetic basis of SMA, upregulation of SMN2 gene expression, preventing exon 7 skipping of SMN2 transcripts or stabilizing SMN 7 should represent attractive therapeutic strategies in SMA (Fig. 2). Several groups have generated cell systems to identify compounds able to induce these modifications. Molecules including interferon, sodium butyrate, valproic acid or aclarubicin have been recently found to increase the amount of SMN protein encoded by the SMN2 gene either by activating the SMN2 gene promoter and/or by preventing the alternative splicing of exon 7 in vitro Their effects should be validated in vivo on animal models carrying the human SMN2 gene and could represent candidate molecules for therapeutic trials in human SMA. Identifying molecules involved in or associated with the SMA degenerative process should help in designing targeted therapeutics in SMA. Cellular or animal models should contribute

6 180 Molecular Mechanisms of Muscular Dystrophies Figure 2. Schematic representation of the current knowledge on SMA pathophysiology and therapeutic research strategies. For each therapeutic strategy, molecules found to be active on either cell system or animal model are indicated. MN: motor neuron. to elucidate the molecular events leading to SMA. Neuroprotective agents or neurotrophic factors may protect neurons against toxicity or promote axonal sprouting of motor neurons. The availability of animal models allows us to test the efficiency, however non specific, of molecules with such biological properties. Recently, cardiotrophin I and riluzole which exhibit neurotrophic activities have shown therapeutic benefits on neuronal mouse model of SMA indicating for the first time that SMA disease progression might be attenuated (Fig. 2) Finally, several reports have revealed that stem cells derived from either bone marrow or other sources have the capacity to differentiate into or to fuse to various cell types including skeletal muscle or neurons. The fantastic capacity of these cells could serve as shuttle for gene therapy. Mouse models of SMA offer hereafter the opportunity of testing the capacity of cells to migrate to damaged tissue, to divide and to differentiate into functional cells. Conclusion Refined characterization of the degenerative process in SMA and the identification of the defective molecular pathway downstream from the SMN defect will provide further exciting insight into this disease in the near future. They should contribute to clarify the pathophysiology of SMA, the function of SMN and should help in designing potential targeted or non targeted therapeutic molecules. Acknowledgements Work made in our laboratory was supported by INSERM, the Association Française contre les Myopathies, Families of SMA (U.S.A.), the Fondation Bettencourt Schueller and Genopole. We gratefully thank Natacha Roblot, Bénédicte Desforges, Gaelle Millet, Sabrina Courageot and Vandana Joshi for their invaluable assistance.

7 Spinal Muscular Atrophy 181 References 1. Munsat TL. Workshop report: International SMA Collaboration. Neuromuscul Disord 1991; 1: Lefebvre S, Burglen L, Reboullet S et al. Identification and characterization of a spinal muscular atrophy-determining gene. Cell 1995; 80: Frugier T, Nicole S, Cifuentes-Diaz C et al. The molecular bases of spinal muscular atrophy. Curr Opin Genet Dev 2002; 12: Campbell L, Potter A, Ignatius J et al. Genomic variation and gene conversion in spinal muscular atrophy: Implications for disease process and clinical phenotype. Am J Hum Genet 1997; 61: Lefebvre S, Burlet P, Liu Q et al. Correlation between severity and SMN protein level in spinal muscular atrophy. Nat Genet 1997; 16: Lorson CL, Androphy EJ. An exonic enhancer is required for inclusion of an essential exon in the SMA-determining gene SMN. Hum Mol Genet 2000; 9: Pellizzoni L, Kataoka N, Charroux B et al. A novel function for SMN, the spinal muscular atrophy disease gene product, in premrna splicing. Cell 1998; 95: Lorson CL, Strasswimmer J, Yao JM et al. SMN oligomerization defect correlates with spinal muscular atrophy severity. Nat Genet 1998; 19: Vyas S, Bechade C, Riveau B et al. Involvement of survival motor neuron (SMN) protein in cell death. Hum Mol Genet 2002; 11: Cifuentes-Diaz C, Frugier T, Tiziano FD et al. Deletion of murine SMN exon 7 directed to skeletal muscle leads to severe muscular dystrophy. J Cell Biol 2001; 152: Talbot K, Ponting CP, Theodosiou AM et al. Missense mutation clustering in the survival motor neuron gene: A role for a conserved tyrosine and glycine rich region of the protein in RNA metabolism? Hum Mol Genet 1997; 6: Liu Q, Dreyfuss G. A novel nuclear structure containing the survival of motor neurons protein. Embo J 1996; 15: Paushkin S, Gubitz AK, Massenet S et al. The SMN complex, an assemblyosome of ribonucleoproteins. Curr Opin Cell Biol 2002; 14: Miguel-Aliaga I, Culetto E, Walker DS et al. The Caenorhabditis elegans orthologue of the human gene responsible for spinal muscular atrophy is a maternal product critical for germline maturation and embryonic viability. Hum Mol Genet 1999; 8: Miguel-Aliaga I, Chan YB, Davies KE et al. Disruption of SMN function by ectopic expression of the human SMN gene in Drosophila. FEBS Lett 2000; 486: Schrank B, Gotz R, Gunnersen JM et al. Inactivation of the survival motor neuron gene, a candidate gene for human spinal muscular atrophy, leads to massive cell death in early mouse embryos. Proc Natl Acad Sci USA 1997; 94: Frugier T, Tiziano FD, Cifuentes-Diaz C et al. Nuclear targeting defect of SMN lacking the C-terminus in a mouse model of spinal muscular atrophy. Hum Mol Genet 2000; 9: Hsieh-Li HM, Chang JG, Jong YJ et al. A mouse model for spinal muscular atrophy. Nat Genet 2000; 24: Monani UR, Sendtner M, Coovert DD et al. The human centromeric survival motor neuron gene (SMN2) rescues embryonic lethality in Smn(-/-) mice and results in a mouse with spinal muscular atrophy. Hum Mol Genet 2000; 9: Cifuentes-Diaz C, Nicole S, Velasco ME et al. Neurofilament accumulation at the motor endplate and lack of axonal sprouting in a spinal muscular atrophy mouse model. Hum Mol Genet 2002; 11: McWhorter ML, Monani UR, Burghes AH et al. Knockdown of the survival motor neuron (Smn) protein in zebrafish causes defects in motor axon outgrowth and pathfinding. J Cell Biol 2003; 162: Chan YB, Miguel-Aliaga I, Franks C et al. Neuromuscular defects in a Drosophila survival motor neuron gene mutant. Hum Mol Genet 2003; 12: Nicole S, Desforges B, Millet G et al. Intact satellite cells lead to remarkable protection against Smn gene defect in differentiated skeletal muscle. J Cell Biol 2003; 161: Rudnik-Schoneborn S, Goebel HH, Schlote W et al. Classical infantile spinal muscular atrophy with SMN deficiency causes sensory neuronopathy. Neurology 2003; 60: Kugelberg E, Welander V. Heredofamilial juvenile muscular atrophy simulating muscular dystrophy. Acta Neurol Psychiatr 1956; 75: Namba T, Aberfeld DC, Grob D. Chronic spinal muscular atrophy. J Neurol Sci 1970; 11: Mastaglia FL, Walton JN. Histological and histochemical changes from cases of chronic juvenile and early adult spinal muscular atrophy (the Kugelberg-Welander syndrome). J Neurol Sci 1971; 12:15-44.

8 182 Molecular Mechanisms of Muscular Dystrophies 28. Bouwsma G, Vanwijngaarden GK. Spinal muscular atrophy and hypertrophy of the calves. J Neurol Sci 1980; 44: Zhang HL, Pan F, Hong D et al. Active transport of the survival motor neuron protein and the role of exon-7 in cytoplasmic localization. J Neurosci 2003; 23: Bassell GJ, Singer RH. Neuronal mrna localization and the cytoskeleton. In: Richter D, ed. Cell polarity and subcellular localization. Springer, Berlin: 2001: Stamm S, Zhang MQ, Marr TG et al. A sequence compilation and comparison of exons that are alternatively spliced in neurons. Nucleic Acids Res 1994; 22: Jablonka S, Schrank B, Kralewski M et al. Reduced survival motor neuron (Smn) gene dose in mice leads to motor neuron degeneration: An animal model for spinal muscular atrophy type III. Hum Mol Genet 2000; 9: Baron-Delage S, Abadie A, Echaniz-Laguna A et al. Interferons and IRF-1 induce expression of the survival motor neuron (SMN) genes. Mol Med 2000; 6: Chang JG, Hsieh-Li HM, Jong YJ et al. Treatment of spinal muscular atrophy by sodium butyrate. Proc Natl Acad Sci USA 2001; 98: Brichta L, Hofmann Y, Hahnen E et al. Valproic acid increases the SMN2 protein level: A well-known drug as a potential therapy for spinal muscular atrophy. Hum Mol Genet 2003; 12: Andreassi C, Jarecki J, Zhou J et al. Aclarubicin treatment restores SMN levels to cells derived from type I spinal muscular atrophy patients. Hum Mol Genet 2001; 10: Lesbordes JC, Cifuentes-Diaz C, Miroglio A et al. Therapeutic benefits of cardiotrophin-1 gene transfer in a mouse model of spinal muscular atrophy. Hum Mol Genet 2003; 12: Haddad H, Cifuentes-Diaz C, Miroglio A et al. Riluzole attenuates spinal muscular atrophy disease progression in a mouse model. Muscle and Nerve 2003; 28: