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.     

Mouse survival motor neuron alleles that mimic SMN2 splicing and are inducible rescue embryonic lethality early in development but not late

Spinal muscular atrophy (SMA) is caused by low survival motor neuron (SMN) levels and patients represent a clinical spectrum due primarily to varying copies of the survival motor neuron-2 (SMN2) gene. Patient and animals studies show that disease severity is abrogated as SMN levels increase. Since therapies currently being pursued target the induction of SMN, it will be important to understand the dosage, timing and cellular requirements of SMN for disease etiology and potential therapeutic intervention. This requires new mouse models that can induce SMN temporally and/or spatially. Here we describe the generation of two hypomorphic Smn alleles, Smn(C-T-Neo) and Smn(2B-Neo). These alleles mimic SMN2 exon 7 splicing, titre Smn levels and are inducible. They were specifically designed so that up to three independent lines of mice could be generated, herein we describe two. In a homozygous state each allele results in embryonic lethality. Analysis of these mutants indicates that greater than 5% of Smn protein is required for normal development. The severe hypomorphic nature of these alleles is caused by inclusion of a loxP-flanked neomycin gene selection cassette in Smn intron 7, which can be removed with Cre recombinase. In vitro and in vivo experiments demonstrate these as inducible Smn alleles. When combined with an inducible Cre mouse, embryonic lethality caused by low Smn levels can be rescued early in gestation but not late. This provides direct genetic evidence that a therapeutic window for SMN inductive therapies may exist. Importantly, these lines fill a void for inducible Smn alleles. They also provide a base from which to generate a large repertoire of SMA models of varying disease severities when combined with other Smn alleles or SMN2-containing mice.     

Motor defects in a Drosophila model for spinal muscular atrophy result from SMN depletion during early neurogenesis

Spinal muscular atrophy (SMA) is the most common autosomal recessive neurodegenerative disease, and is characterised by spinal motor neuron loss, impaired motor function and, often, premature death. Mutations and deletions in the widely expressed survival motor neuron 1 (SMN1) gene cause SMA; however, the mechanisms underlying the selectivity of motor neuron degeneration are not well understood. Although SMA is degenerative in nature, SMN function during embryonic and early postnatal development appears to be essential for motor neuron survival in animal models and humans. Notwithstanding, how developmental defects contribute to the subversion of postnatal and adult motor function remains elusive. Here, in a Drosophila SMA model, we show that neurodevelopmental defects precede gross locomotor dysfunction in larvae. Furthermore, to specifically address the relevance of SMN during neurogenesis and in neurogenic cell types, we show that SMN knockdown using neuroblast-specific and pan-neuronal drivers, but not differentiated neuron or glial cell drivers, impairs adult motor function. Using targeted knockdown, we further restricted SMN manipulation in neuroblasts to a defined time window. Our aim was to express specifically in the neuronal progenitor cell types that have not formed synapses, and thus a time that precedes neuromuscular junction formation and maturation. By restoring SMN levels in these distinct neuronal population, we partially rescue the larval locomotor defects of Smn mutants. Finally, combinatorial SMN knockdown in immature and mature neurons synergistically enhances the locomotor and survival phenotypes. Our in-vivo study is the first to directly rescue the motor defects of an SMA model by expressing Smn in an identifiable population of Drosophila neuroblasts and developing neurons, highlighting that neuronal sensitivity to SMN loss may arise before synapse establishment and nerve cell maturation.     

Presynaptic Localization of Smn and hnRNP R in Axon Terminals of Embryonic and Postnatal Mouse Motoneurons

Spinal muscular atrophy (SMA) is caused by deficiency of the ubiquitously expressed survival motoneuron (SMN) protein. SMN is crucial component of a complex for the assembly of spliceosomal small nuclear ribonucleoprotein (snRNP) particles. Other cellular functions of SMN are less characterized so far. SMA predominantly affects lower motoneurons, but the cellular basis for this relative specificity is still unknown. In contrast to nonneuronal cells where the protein is mainly localized in perinuclear regions and the nucleus, Smn is also present in dendrites, axons and axonal growth cones of isolated motoneurons in vitro. However, this distribution has not been shown in vivo and it is not clear whether Smn and hnRNP R are also present in presynaptic axon terminals of motoneurons in postnatal mice. Smn also associates with components not included in the classical SMN complex like RNA-binding proteins FUS, TDP43, HuD and hnRNP R which are involved in RNA processing, subcellular localization and translation. We show here that Smn and hnRNP R are present in presynaptic compartments at neuromuscular endplates of embryonic and postnatal mice. Smn and hnRNP R are localized in close proximity to each other in axons and axon terminals both in vitro and in vivo. We also provide new evidence for a direct interaction of Smn and hnRNP R in vitro and in vivo, particularly in the cytosol of motoneurons. These data point to functions of SMN beyond snRNP assembly which could be crucial for recruitment and transport of RNA particles into axons and axon terminals, a mechanism which may contribute to SMA pathogenesis.     

Restoration of SMN to Emx-1 expressing cortical neurons is not sufficient to provide benefit to a severe mouse model of Spinal Muscular Atrophy

Spinal Muscular Atrophy (SMA), an autosomal recessive neuromuscular disorder, is a leading genetic cause of infant mortality. SMA is caused by the homozygous loss of Survival Motor Neuron-1 (SMN1). However, low, but essential, levels of SMN protein are produced by a nearly identical copy gene called SMN2. Detailed analysis of neuromuscular junctions in SMA mice has revealed a selective vulnerability in a subset of muscle targets, suggesting that while SMN is reduced uniformly, the functional deficits manifest sporadically. Additionally, in severe SMA models, it is becoming increasing apparent that SMA is not restricted solely to motor neurons. Rather, additional tissues including the heart, vasculature, and the pancreas contribute to the complete SMA-associated pathology. Recently, transgenic models have been utilized to examine the tissue-specific requirements of SMN, including selective depletion and restoration of SMN in motor neurons. To determine whether the cortical neuronal populations expressing the Emx-1 promoter are involved in SMA pathology, we generated a novel SMA mouse model in which SMN expression was specifically induced in Emx-1 expressing cortical neurons utilizing an Emx-1-Cre transgene. While SMN expression was robust in the central nervous system as expected, SMA mice did not live longer. Weight and time-to-right motor function were not significantly improved.     

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.     

A cell-autonomous defect in skeletal muscle satellite cells expressing low levels of survival of motor neuron protein

Mutations in the Survival of Motor Neuron (SMN) gene underlie the development of spinal muscular atrophy (SMA), which currently represents the leading genetic cause of mortality in infants and toddlers. SMA is characterized by degeneration of spinal cord motor neurons and muscle atrophy. Although SMA is often considered to be a motor neuron disease, accumulating evidence suggests that muscle cells themselves may be affected by low levels of SMN. Here, we examine satellite cells, tissue-resident stem cells that play an essential role in the growth and repair of skeletal muscle, isolated from a severe SMA mouse model (Smn(-/-); SMN2(+/+)). We found similar numbers of satellite cells in the muscles of SMA and wild-type (Smn(+/+); SMN2(+/+)) mice at postnatal day 2 (P2), and, when isolated from skeletal muscle using cell surface marker expression, these cells showed comparable survival and proliferative potential. However, SMA satellite cells differentiate abnormally, revealed by the premature expression of muscle differentiation markers, and, especially, by a reduced efficiency in forming myotubes. These phenotypes suggest a critical role of SMN protein in the intrinsic regulation of muscle differentiation and suggest that abnormal muscle development contributes to the manifestation of SMA symptoms.     

SMA-Causing Missense Mutations in Survival motor neuron (Smn) Display a Wide Range of Phenotypes When Modeled in Drosophila

Mutations in the human survival motor neuron 1 (SMN) gene are the primary cause of spinal muscular atrophy (SMA), a devastating neuromuscular disorder. SMN protein has a well-characterized role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), core components of the spliceosome. Additional tissue-specific and global functions have been ascribed to SMN; however, their relevance to SMA pathology is poorly understood and controversial. Using Drosophila as a model system, we created an allelic series of twelve Smn missense mutations, originally identified in human SMA patients. We show that animals expressing these SMA-causing mutations display a broad range of phenotypic severities, similar to the human disease. Furthermore, specific interactions with other proteins known to be important for SMN''s role in RNP assembly are conserved. Intragenic complementation analyses revealed that the three most severe mutations, all of which map to the YG box self-oligomerization domain of SMN, display a stronger phenotype than the null allele and behave in a dominant fashion. In support of this finding, the severe YG box mutants are defective in self-interaction assays, yet maintain their ability to heterodimerize with wild-type SMN. When expressed at high levels, wild-type SMN is able to suppress the activity of the mutant protein. These results suggest that certain SMN mutants can sequester the wild-type protein into inactive complexes. Molecular modeling of the SMN YG box dimer provides a structural basis for this dominant phenotype. These data demonstrate that important structural and functional features of the SMN YG box are conserved between vertebrates and invertebrates, emphasizing the importance of self-interaction to the proper functioning of SMN.     

Smn deficiency causes neuritogenesis and neurogenesis defects in the retinal neurons of a mouse model of spinal muscular atrophy

The eye is an excellent model for the study of neuronal development and pathogenesis of central nervous system disorders because of its relative ease of accessibility and the well‐characterized cellular makeup. We have used this model to study spinal muscular atrophy (SMA), an autosomal recessive neuromuscular disease caused by deletions or mutations in the survival of motor neuron 1 gene (SMN1). We have investigated the expression pattern of mouse Smn mRNA and protein in the neural retina and the optic nerve of wild type mice. Smn protein is present in retinal ganglion cells and amacrine cells within the neural retina as well as in glial cells in the optic nerve. Histopathological analysis in phenotype stage SMA mice revealed that Smn deficiency is associated with a reduction in ganglion cell axon and glial cell number in the optic nerve, as well as compromised cellular processes and altered organization of neurofilaments in the neural retina. Whole mount preparation and retinal neuron primary culture provided further evidence of abnormal synaptogenesis and neurofilament accumulation in the neurites of Smn‐deficient retinal neurons. A subset of amacrine cells is absent, in a cell‐autonomous fashion, in the retina of SMA mice. Finally, the retinas of SMA mice have altered electroretinograms. Altogether, our study has demonstrated defects in axodendritic outgrowth and cellular composition in Smn‐depleted retinal neurons, indicating a role for Smn in neuritogenesis and neurogenesis, and providing us with an insight into pathogenesis of SMA. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 71: 153‐169, 2011     

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 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.     

High Expression Level of Tra2-β1 Is Responsible for Increased SMN2 Exon 7 Inclusion in the Testis of SMA Mice

Spinal muscular atrophy (SMA) is an inherited neuromuscular disease caused by deletion or mutation of SMN1 gene. All SMA patients carry a nearly identical SMN2 gene, which produces low level of SMN protein due to mRNA exon 7 exclusion. Previously, we found that the testis of SMA mice (smn^−/− SMN2) expresses high level of SMN2 full-length mRNA, indicating a testis-specific mechanism for SMN2 exon 7 inclusion. To elucidate the underlying mechanism, we established primary cultures of testis cells from SMA mice and analyzed them for SMN2 exon 7 splicing. We found that primary testis cells after a 2-hour culture still expressed high level of SMN2 full-length mRNA, but the level decreased after longer cultures. We then compared the protein levels of relevant splicing factors, and found that the level of Tra2-β1 also decreased during testis cell culture, correlated with SMN2 full-length mRNA downregulation. In addition, the testis of SMA mice expressed the highest level of Tra2-β1 among the many tissues examined. Furthermore, overexpression of Tra2-β1, but not ASF/SF2, increased SMN2 minigene exon 7 inclusion in primary testis cells and spinal cord neurons, whereas knockdown of Tra2-β1 decreased SMN2 exon 7 inclusion in primary testis cells of SMA mice. Therefore, our results indicate that high expression level of Tra2-β1 is responsible for increased SMN2 exon 7 inclusion in the testis of SMA mice. This study also suggests that the expression level of Tra2-β1 may be a modifying factor of SMA disease and a potential target for SMA treatment.     

Laminin induced local axonal translation of β-actin mRNA is impaired in SMN-deficient motoneurons

Reduced levels of the SMN (survival of motoneuron) protein cause spinal muscular atrophy, the main form of motoneuron disease in children and young adults. In cultured motoneurons, reduced SMN levels lead to disturbed axon growth that correlates with reduced actin mRNA and protein in growth cones, indicating that anterograde transport and local translation of β-actin mRNA are altered in this disease. However, it is not fully understood how local translation of the β-actin mRNA is regulated in SMN-deficient motoneurons. Here, we established a lentiviral GFP-based reporter construct to monitor local translation of β-actin mRNA. Time-lapse imaging of fluorescence recovery after photobleaching (FRAP) in living motoneurons revealed that β-actin is locally translated in the growth cones of embryonic motoneurons. Interestingly, local translation of the β-actin reporter construct was differentially regulated by various Laminin isoforms, indicating that Laminins provide extracellular cues for the regulation of local translation in growth cones. Notably, local translation of β-actin mRNA was deregulated in motoneurons from a mouse model for the most severe form of SMA (Smn ^?/? ;SMN2). Taken together our findings suggest that local translation of β-actin in growth cones of motoneurons is regulated by Laminin signalling and that this signalling is disturbed in SMA.     

Modeling spinal muscular atrophy in Drosophila links Smn to FGF signaling

Spinal muscular atrophy (SMA), a devastating neurodegenerative disorder characterized by motor neuron loss and muscle atrophy, has been linked to mutations in the Survival Motor Neuron (SMN) gene. Based on an SMA model we developed in Drosophila, which displays features that are analogous to the human pathology and vertebrate SMA models, we functionally linked the fibroblast growth factor (FGF) signaling pathway to the Drosophila homologue of SMN, Smn. Here, we characterize this relationship and demonstrate that Smn activity regulates the expression of FGF signaling components and thus FGF signaling. Furthermore, we show that alterations in FGF signaling activity are able to modify the neuromuscular junction defects caused by loss of Smn function and that muscle-specific activation of FGF is sufficient to rescue Smn-associated abnormalities.     

Generation of a tamoxifen inducible SMN mouse for temporal SMN replacement

Proximal spinal muscular atrophy (SMA) is caused by low levels of the SMN protein, encoded by the Survival Motor Neuron genes (SMN1 and SMN2). Mouse models of SMA can be rescued by increased SMN expression, but the timing of SMN replacement for complete rescue is unknown. Studies in zebrafish predict restoration of SMN function during embryogenesis may be important for axonal pathfinding, while the mouse models and normal human disease progression suggest that post-natal treatment may be sufficient for amelioration of disease. To evaluate the timing for SMN replacement, we have generated a stably integrated Cre-inducible SMN mouse in which expression of full-length SMN2 occurs after tamoxifen administration. Our temporally inducible SMN transgene is able to express SMN in embryonic, neonatal, and weanling mice and as such can be utilized in severe and mild SMA mouse models to identify the therapeutic window for SMN replacement.     

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.     

Oxidative Stress Triggers Body-Wide Skipping of Multiple Exons of the Spinal Muscular Atrophy Gene

Humans carry two nearly identical copies of Survival Motor Neuron gene: SMN1 and SMN2. Loss of SMN1 leads to spinal muscular atrophy (SMA), the most frequent genetic cause of infant mortality. While SMN2 cannot compensate for the loss of SMN1 due to predominant skipping of exon 7, correction of SMN2 exon 7 splicing holds the promise of a cure for SMA. Previously, we used cell-based models coupled with a multi-exon-skipping detection assay (MESDA) to demonstrate the vulnerability of SMN2 exons to aberrant splicing under the conditions of oxidative stress (OS). Here we employ a transgenic mouse model and MESDA to examine the OS-induced splicing regulation of SMN2 exons. We induced OS using paraquat that is known to trigger production of reactive oxygen species and cause mitochondrial dysfunction. We show an overwhelming co-skipping of SMN2 exon 5 and exon 7 under OS in all tissues except testis. We also show that OS increases skipping of SMN2 exon 3 in all tissues except testis. We uncover several new SMN2 splice isoforms expressed at elevated levels under the conditions of OS. We analyze cis-elements and transacting factors to demonstrate the diversity of mechanisms for splicing misregulation under OS. Our results of proteome analysis reveal downregulation of hnRNP H as one of the potential consequences of OS in brain. Our findings suggest SMN2 as a sensor of OS with implications to SMA and other diseases impacted by low levels of SMN protein.