Oligomeric Properties of Survival Motor Neuron·Gemin2 Complexes

The survival motor neuron (SMN) protein forms the oligomeric core of a multiprotein complex required for the assembly of spliceosomal small nuclear ribonucleoproteins. Deletions and mutations in the SMN1 gene are associated with spinal muscular atrophy (SMA), a devastating neurodegenerative disease that is the leading heritable cause of infant mortality. Oligomerization of SMN is required for its function, and some SMA patient mutations disrupt the ability of SMN to self-associate. Here, we investigate the oligomeric nature of the SMN·Gemin2 complexes from humans and fission yeast (hSMN·Gemin2 and ySMN·Gemin2). We find that hSMN·Gemin2 forms oligomers spanning the dimer to octamer range. The YG box oligomerization domain of SMN is both necessary and sufficient to form these oligomers. ySMN·Gemin2 exists as a dimer-tetramer equilibrium with K_d = 1.0 ± 0.9 μm. A 1.9 Å crystal structure of the ySMN YG box confirms a high level of structural conservation with the human ortholog in this important region of SMN. Disulfide cross-linking experiments indicate that SMN tetramers are formed by self-association of stable, non-dissociating dimers. Thus, SMN tetramers do not form symmetric helical bundles such as those found in glycine zipper transmembrane oligomers. The dimer-tetramer nature of SMN complexes and the dimer of dimers organization of the SMN tetramer provide an important foundation for ongoing studies to understand the mechanism of SMN-assisted small nuclear ribonucleoprotein assembly and the underlying causes of SMA.     

Assembly of higher-order SMN oligomers is essential for metazoan viability and requires an exposed structural motif present in the YG zipper dimer

Protein oligomerization is one mechanism by which homogenous solutions can separate into distinct liquid phases, enabling assembly of membraneless organelles. Survival Motor Neuron (SMN) is the eponymous component of a large macromolecular complex that chaperones biogenesis of eukaryotic ribonucleoproteins and localizes to distinct membraneless organelles in both the nucleus and cytoplasm. SMN forms the oligomeric core of this complex, and missense mutations within its YG box domain are known to cause Spinal Muscular Atrophy (SMA). The SMN YG box utilizes a unique variant of the glycine zipper motif to form dimers, but the mechanism of higher-order oligomerization remains unknown. Here, we use a combination of molecular genetic, phylogenetic, biophysical, biochemical and computational approaches to show that formation of higher-order SMN oligomers depends on a set of YG box residues that are not involved in dimerization. Mutation of key residues within this new structural motif restricts assembly of SMN to dimers and causes locomotor dysfunction and viability defects in animal models.     

A Drosophila model of spinal muscular atrophy uncouples snRNP biogenesis functions of survival motor neuron from locomotion and viability defects

The spinal muscular atrophy (SMA) protein, survival motor neuron (SMN), functions in the biogenesis of small nuclear ribonucleoproteins (snRNPs). SMN has also been implicated in tissue-specific functions; however, it remains unclear which of these is important for the etiology of SMA. Smn null mutants display larval lethality and show significant locomotion defects as well as reductions in minor-class spliceosomal snRNAs. Despite these reductions, we found no appreciable defects in the splicing of mRNAs containing minor-class introns. Transgenic expression of low levels of either wild-type or an SMA patient-derived form of SMN rescued the larval lethality and locomotor defects; however, snRNA levels were not restored. Thus, the snRNP biogenesis function of SMN is not a major contributor to the phenotype of Smn null mutants. These findings have major implications for SMA etiology because they show that SMN's role in snRNP biogenesis can be uncoupled from the organismal viability and locomotor defects.     

VPAC2 receptor agonist BAY 55-9837 increases SMN protein levels and moderates disease phenotype in severe spinal muscular atrophy mouse models

Background

Spinal Muscular Atrophy (SMA) is one of the most common inherited causes of infant death and is caused by the loss of functional survival motor neuron (SMN) protein due to mutations or deletion in the SMN1 gene. One of the treatment strategies for SMA is to induce the expression of the protein from the homologous SMN2 gene, a rescuing paralog for SMA.

Methods and results

Here we demonstrate the promise of pharmacological modulation of SMN2 gene by BAY 55-9837, an agonist of the vasoactive intestinal peptide receptor 2 (VPAC2), a member of G protein coupled receptor family. Treatment with BAY 55-9837 lead to induction of SMN protein levels via activation of MAPK14 or p38 pathway in vitro. Importantly, BAY 55-9837 also ameliorated disease phenotype in severe SMA mouse models.

Conclusion

Our findings suggest the VPAC2 pathway is a potential SMA therapeutic target.     

The Wallerian degeneration slow (Wld(s)) gene does not attenuate disease in a mouse model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a severe neuromuscular disease characterized by loss of spinal α-motor neurons, resulting in the paralysis of skeletal muscle. SMA is caused by deficiency of survival motor neuron (SMN) protein levels. Recent evidence has highlighted an axon-specific role for SMN protein, raising the possibility that axon degeneration may be an early event in SMA pathogenesis. The Wallerian degeneration slow (Wld^s) gene is a spontaneous dominant mutation in mice that delays axon degeneration by approximately 2-3 weeks. We set out to examine the effect of Wld^s on the phenotype of a mouse model of SMA. We found that Wld^s does not alter the SMA phenotype, indicating that Wallerian degeneration does not directly contribute to the pathogenesis of SMA development.     

Generation and Characterization of a genetic zebrafish model of SMA carrying the human SMN2 gene

Background

Animal models of human diseases are essential as they allow analysis of the disease process at the cellular level and can advance therapeutics by serving as a tool for drug screening and target validation. Here we report the development of a complete genetic model of spinal muscular atrophy (SMA) in the vertebrate zebrafish to complement existing zebrafish, mouse, and invertebrate models and show its utility for testing compounds that alter SMN2 splicing.

Results

The human motoneuron disease SMA is caused by low levels, as opposed to a complete absence, of the survival motor neuron protein (SMN). To generate a true model of SMA in zebrafish, we have generated a transgenic zebrafish expressing the human SMN2 gene (hSMN2), which produces only a low amount of full-length SMN, and crossed this onto the smn ^-/- background. We show that human SMN2 is spliced in zebrafish as it is in humans and makes low levels of SMN protein. Moreover, we show that an antisense oligonucleotide that enhances correct hSMN2 splicing increases full-length hSMN RNA in this model. When we placed this transgene on the smn mutant background it rescued the neuromuscular presynaptic SV2 defect that occurs in smn mutants and increased their survival.

Conclusions

We have generated a transgenic fish carrying the human hSMN2 gene. This gene is spliced in fish as it is in humans and mice suggesting a conserved splicing mechanism in these vertebrates. Moreover, antisense targeting of an intronic splicing silencer site increased the amount of full length SMN generated from this transgene. Having this transgene on the smn mutant fish rescued the presynaptic defect and increased survival. This model of zebrafish SMA has all of the components of human SMA and can thus be used to understand motoneuron dysfunction in SMA, can be used as an vivo test for drugs or antisense approaches that increase full-length SMN, and can be developed for drug screening.     

Molecular analysis of SMN1, SMN2, NAIP, GTF2H2, and H4F5 genes in 157 Chinese patients with spinal muscular atrophy

Spinal muscular atrophy (SMA) is a common and lethal autosomal recessive neurodegenerative disorder, which is caused by mutations of the survival motor neuron 1 (SMN1) gene. Additionally, the phenotype is modified by several genes nearby SMN1 in the 5q13 region. In this study, we analyzed mutations in SMN1 and quantified the modifying genes, including SMN2, NAIP, GTF2H2, and H4F5 by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP), multiplex ligation-dependent probe amplification (MLPA), TA cloning, allele-specific long-range PCR, and Sanger sequencing in 157 SMA patients. Most SMA patients (94.90%) possessed a homozygous SMN1 deletion, while 10 patients demonstrated only the absence of exon 7, but the presence of exon 8. Two missense mutations (c.689 C > T and c.844 C > T) were identified in 2 patients who both carried a single copy of SMN1. We found inverse correlations between SMN2, the NAIP copy number, and the clinical severity of the disease. Furthermore, 7 severe type I patients possessed large-scale deletions, including SMN1, NAIP, and GTF2H2. We conclude that SMN1 gene conversion, SMN1 subtle mutations, SMN2 copy number, and the extent of deletion in the 5q13 region should all be considered in the genotype–phenotype analysis of SMA.     

Survival motor neuron protein reduction deregulates autophagy in spinal cord motoneurons in vitro

Spinal muscular atrophy (SMA) is a genetic disorder characterized by degeneration of spinal cord motoneurons (MNs), resulting in muscular atrophy and weakness. SMA is caused by mutations in the Survival Motor Neuron 1 (SMN1) gene and decreased SMN protein. SMN is ubiquitously expressed and has a general role in the assembly of small nuclear ribonucleoproteins and pre-mRNA splicing requirements. SMN reduction causes neurite degeneration and cell death without classical apoptotic features, but the direct events leading to SMN degeneration in SMA are still unknown. Autophagy is a conserved lysosomal protein degradation pathway whose precise roles in neurodegenerative diseases remain largely unknown. In particular, it is unclear whether autophagosome accumulation is protective or destructive, but the accumulation of autophagosomes in the neuritic beadings observed in several neurite degeneration models suggests a close relationship between the autophagic process and neurite collapse. In the present work, we describe an increase in the levels of the autophagy markers including autophagosomes, Beclin1 and light chain (LC)3-II proteins in cultured mouse spinal cord MNs from two SMA cellular models, suggesting an upregulation of the autophagy process in Smn (murine survival motor neuron protein)-reduced MNs. Overexpression of Bcl-x_L counteracts LC3-II increase, contributing to the hypothesis that the protective role of Bcl-x_L observed in some SMA models may be mediated by its role in autophagy inhibition. Our in vitro experimental data indicate an upregulation in the autophagy process and autophagosome accumulation in the pathogenesis of SMA, thus providing a valuable clue in understanding the mechanisms of axonal degeneration and a possible therapeutic target in the treatment of SMA.     

The splicing regulator Sam68 binds to a novel exonic splicing silencer and functions in SMN2 alternative splicing in spinal muscular atrophy

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss of motor neurons in patients with null mutations in the SMN1 gene. An almost identical SMN2 gene is unable to compensate for this deficiency because a single C‐to‐T transition at position +6 in exon‐7 causes skipping of the exon by a mechanism not yet fully elucidated. We observed that the C‐to‐T transition in SMN2 creates a putative binding site for the RNA‐binding protein Sam68. RNA pull‐down assays and UV‐crosslink experiments showed that Sam68 binds to this sequence. In vivo splicing assays showed that Sam68 triggers SMN2 exon‐7 skipping. Moreover, mutations in the Sam68‐binding site of SMN2 or in the RNA‐binding domain of Sam68 completely abrogated its effect on exon‐7 skipping. Retroviral infection of dominant‐negative mutants of Sam68 that interfere with its RNA‐binding activity, or with its binding to the splicing repressor hnRNP A1, enhanced exon‐7 inclusion in endogenous SMN2 and rescued SMN protein expression in fibroblasts of SMA patients. Our results thus indicate that Sam68 is a novel crucial regulator of SMN2 splicing.     

In vitro and in cellulo evidences for association of the survival of motor neuron complex with the fragile X mental retardation protein

Spinal muscular atrophy (SMA) is caused by reduced levels of the survival of motor neuron (SMN) protein. Although the SMN complex is essential for assembly of spliceosomal U small nuclear RNPs, it is still not understood why reduced levels of the SMN protein specifically cause motor neuron degeneration. SMN was recently proposed to have specific functions in mRNA transport and translation regulation in neuronal processes. The defective protein in Fragile X mental retardation syndrome (FMRP) also plays a role in transport of mRNPs and in their translation. Therefore, we examined possible relationships of SMN with FMRP. We observed granules containing both transiently expressed red fluorescent protein(RFP)-tagged SMN and green fluorescent protein(GFP)-tagged FMRP in cell bodies and processes of rat primary neurons of hypothalamus in culture. By immunoprecipitation experiments, we detected an association of FMRP with the SMN complex in human neuroblastoma SH-SY5Y cells and in murine motor neuron MN-1 cells. Then, by in vitro experiments, we demonstrated that the SMN protein is essential for this association. We showed that the COOH-terminal region of FMRP, as well as the conserved YG box and the region encoded by exon 7 of SMN, are required for the interaction. Our findings suggest a link between the SMN complex and FMRP in neuronal cells.     

A Drosophila melanogaster model of spinal muscular atrophy reveals a function for SMN in striated muscle

Mutations in human survival motor neurons 1 (SMN1) cause spinal muscular atrophy (SMA) and are associated with defects in assembly of small nuclear ribonucleoproteins (snRNPs) in vitro. However, the etiological link between snRNPs and SMA is unclear. We have developed a Drosophila melanogaster system to model SMA in vivo. Larval-lethal Smn-null mutations show no detectable snRNP reduction, making it unlikely that these animals die from global snRNP deprivation. Hypomorphic mutations in Smn reduce dSMN protein levels in the adult thorax, causing flightlessness and acute muscular atrophy. Mutant flight muscle motoneurons display pronounced axon routing and arborization defects. Moreover, Smn mutant myofibers fail to form thin filaments and phenocopy null mutations in Act88F, which is the flight muscle-specific actin isoform. In wild-type muscles, dSMN colocalizes with sarcomeric actin and forms a complex with alpha-actinin, the thin filament crosslinker. The sarcomeric localization of Smn is conserved in mouse myofibrils. These observations suggest a muscle-specific function for SMN and underline the importance of this tissue in modulating SMA severity.     

Gemin2 plays an important role in stabilizing the survival of motor neuron complex

The survival of motor neuron (SMN) protein, responsible for the neurodegenerative disease spinal muscular atrophy (SMA), oligomerizes and forms a stable complex with seven other major components, the Gemin proteins. Besides the SMN protein, Gemin2 is a core protein that is essential for the formation of the SMN complex, although the mechanism by which it drives formation is unclear. We have found a novel interaction, a Gemin2 self-association, using the mammalian two-hybrid system and the in vitro pull-down assays. Using in vitro dissociation assays, we also found that the self-interaction of the amino-terminal SMN protein, which was confirmed in this study, became stable in the presence of Gemin2. In addition, Gemin2 knockdown using small interference RNA treatment revealed a drastic decrease in SMN oligomer formation and in the assembly activity of spliceosomal small nuclear ribonucleoprotein (snRNP). Taken together, these results indicate that Gemin2 plays an important role in snRNP assembly through the stabilization of the SMN oligomer/complex via novel self-interaction. Applying the results/techniques to amino-terminal SMN missense mutants that were recently identified from SMA patients, we successfully showed that amino-terminal self-association, Gemin2 binding, the stabilization effect of Gemin2, and snRNP assembly activity were all lowered in the mutant SMN(D44V), suggesting that instability of the amino-terminal SMN self-association may cause SMA in patients carrying this allele.     

Targeting SR Proteins Improves SMN Expression in Spinal Muscular Atrophy Cells

Spinal muscular atrophy (SMA) is one of the most common inherited causes of pediatric mortality. SMA is caused by deletions or mutations in the survival of motor neuron 1 (SMN1) gene, which results in SMN protein deficiency. Humans have a centromeric copy of the survival of motor neuron gene, SMN2, which is nearly identical to SMN1. However, SMN2 cannot compensate for the loss of SMN1 because SMN2 has a single-nucleotide difference in exon 7, which negatively affects splicing of the exon. As a result, most mRNA produced from SMN2 lacks exon 7. SMN2 mRNA lacking exon 7 encodes a truncated protein with reduced functionality. Improving SMN2 exon 7 inclusion is a goal of many SMA therapeutic strategies. The identification of regulators of exon 7 inclusion may provide additional therapeutic targets or improve the design of existing strategies. Although a number of regulators of exon 7 inclusion have been identified, the function of most splicing proteins in exon 7 inclusion is unknown. Here, we test the role of SR proteins and hnRNP proteins in SMN2 exon 7 inclusion. Knockdown and overexpression studies reveal that SRSF1, SRSF2, SRSF3, SRSF4, SRSF5, SRSF6, SRSF7, SRSF11, hnRNPA1/B1 and hnRNP U can inhibit exon 7 inclusion. Depletion of two of the most potent inhibitors of exon 7 inclusion, SRSF2 or SRSF3, in cell lines derived from SMA patients, increased SMN2 exon 7 inclusion and SMN protein. Our results identify novel regulators of SMN2 exon 7 inclusion, revealing potential targets for SMA therapeutics.     

The contribution of mouse models to understanding the pathogenesis of spinal muscular atrophy

Spinal muscular atrophy (SMA), which is caused by inactivating mutations in the survival motor neuron 1 (SMN1) gene, is characterized by loss of lower motor neurons in the spinal cord. The gene encoding SMN is very highly conserved in evolution, allowing the disease to be modeled in a range of species. The similarities in anatomy and physiology to the human neuromuscular system, coupled with the ease of genetic manipulation, make the mouse the most suitable model for exploring the basic pathogenesis of motor neuron loss and for testing potential treatments. Therapies that increase SMN levels, either through direct viral delivery or by enhancing full-length SMN protein expression from the SMN1 paralog, SMN2, are approaching the translational stage of development. It is therefore timely to consider the role of mouse models in addressing aspects of disease pathogenesis that are most relevant to SMA therapy. Here, we review evidence suggesting that the apparent selective vulnerability of motor neurons to SMN deficiency is relative rather than absolute, signifying that therapies will need to be delivered systemically. We also consider evidence from mouse models suggesting that SMN has its predominant action on the neuromuscular system in early postnatal life, during a discrete phase of development. Data from these experiments suggest that the timing of therapy to increase SMN levels might be crucial. The extent to which SMN is required for the maintenance of motor neurons in later life and whether augmenting its levels could treat degenerative motor neuron diseases, such as amyotrophic lateral sclerosis (ALS), requires further exploration.     

Salbutamol inhibits ubiquitin-mediated survival motor neuron protein degradation in spinal muscular atrophy cells

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder that is currently incurable. SMA is caused by decreased levels of the survival motor neuron protein (SMN), as a result of loss or mutation of SMN1. Although the SMN1 homolog SMN2 also produces some SMN protein, it does not fully compensate for the loss or dysfunction of SMN1. Salbutamol, a β2-adrenergic receptor agonist and well-known bronchodilator used in asthma patients, has recently been shown to ameliorate symptoms in SMA patients. However, the precise mechanism of salbutamol action is unclear. We treated SMA fibroblast cells lacking SMN1 and HeLa cells with salbutamol and analyzed SMN2 mRNA and SMN protein levels in SMA fibroblasts, and changes in SMN protein ubiquitination in HeLa cells. Salbutamol increased SMN protein levels in a dose-dependent manner in SMA fibroblast cells lacking SMN1, though no significant changes in SMN2 mRNA levels were observed. Notably, the salbutamol-induced increase in SMN was blocked by a protein kinase A (PKA) inhibitor and deubiquitinase inhibitor, respectively. Co-immunoprecipitation assay using HeLa cells showed that ubiquitinated SMN levels decreased in the presence of salbutamol, suggesting that salbutamol inhibited ubiquitination. The results of this study suggest that salbutamol may increase SMN protein levels in SMA by inhibiting ubiquitin-mediated SMN degradation via activating β2-adrenergic receptor-PKA pathways.     

Molecular genetic basis of proximal spinal muscular atrophy and experience in its pharmaceutical treatment

The review considers the original and published data on the molecular genetic basis of proximal spinal muscular atrophy (SMA), the most common monogenic neuromuscular disease. The structures of the SMN1 gene and SMN2 pseudogene, mutations distorting the SMN1 function, the structure and functions of the Smn neurotrophic protein, its role in biogenesis of small nuclear ribonucleoproteins (snRNPs), and the principles and problems of molecular diagnosis in SMA are described. Special consideration is given to the current approaches and prospects of gene and cell therapy of SMA, pharmacogenetic methods to correct the SMN2 function, and original results of long-term treatment of SMA patients with valproic acid drugs.     

A Cell System for Phenotypic Screening of Modifiers of SMN2 Gene Expression and Function

Spinal muscular atrophy (SMA) is an inherited neurodegenerative disease caused by homozygous inactivation of the SMN1 gene and reduced levels of the survival motor neuron (SMN) protein. Since higher copy numbers of the nearly identical SMN2 gene reduce disease severity, to date most efforts to develop a therapy for SMA have focused on enhancing SMN expression. Identification of alternative therapeutic approaches has partly been hindered by limited knowledge of potential targets and the lack of cell-based screening assays that serve as readouts of SMN function. Here, we established a cell system in which proliferation of cultured mouse fibroblasts is dependent on functional SMN produced from the SMN2 gene. To do so, we introduced the entire human SMN2 gene into NIH3T3 cell lines in which regulated knockdown of endogenous mouse Smn severely decreases cell proliferation. We found that low SMN2 copy number has modest effects on the cell proliferation phenotype induced by Smn depletion, while high SMN2 copy number is strongly protective. Additionally, cell proliferation correlates with the level of SMN activity in small nuclear ribonucleoprotein assembly. Following miniaturization into a high-throughput format, our cell-based phenotypic assay accurately measures the beneficial effects of both pharmacological and genetic treatments leading to SMN upregulation. This cell model provides a novel platform for phenotypic screening of modifiers of SMN2 gene expression and function that act through multiple mechanisms, and a powerful new tool for studies of SMN biology and SMA therapeutic development.