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.     

A cell system with targeted disruption of the SMN gene: functional conservation of the SMN protein and dependence of Gemin2 on SMN

The motor neuron degenerative disease spinal muscular atrophy is caused by reduced expression of the survival motor neuron (SMN) protein. Here we report a genetic system developed in the chicken pre-B cell line DT40, in which the endogenous SMN gene is disrupted by homologous recombination, and SMN protein is expressed from a chicken SMN cDNA under control of a tetracycline (tet)-repressible promoter. Addition of tet results in depletion of SMN protein and consequent cell death, which directly demonstrates that SMN is required for cell viability. The tet-induced lethality can be rescued by expression of human SMN, indicating that the function of SMN is highly conserved between the two species. Cells expressing low levels of SMN display slow growth proportional to the amount of SMN they contain. Interestingly, the level of the SMN-interacting protein Gemin2 decreases significantly following depletion of SMN, supporting the conclusion that SMN and Gemin2 form a stable complex in vivo. This system provides a powerful setting for studying the function of SMN in vivo and for screening for potential therapeutics for spinal muscular atrophy.     

Conserved genes act as modifiers of invertebrate SMN loss of function defects

Spinal Muscular Atrophy (SMA) is caused by diminished function of the Survival of Motor Neuron (SMN) protein, but the molecular pathways critical for SMA pathology remain elusive. We have used genetic approaches in invertebrate models to identify conserved SMN loss of function modifier genes. Drosophila melanogaster and Caenorhabditis elegans each have a single gene encoding a protein orthologous to human SMN; diminished function of these invertebrate genes causes lethality and neuromuscular defects. To find genes that modulate SMN function defects across species, two approaches were used. First, a genome-wide RNAi screen for C. elegans SMN modifier genes was undertaken, yielding four genes. Second, we tested the conservation of modifier gene function across species; genes identified in one invertebrate model were tested for function in the other invertebrate model. Drosophila orthologs of two genes, which were identified originally in C. elegans, modified Drosophila SMN loss of function defects. C. elegans orthologs of twelve genes, which were originally identified in a previous Drosophila screen, modified C. elegans SMN loss of function defects. Bioinformatic analysis of the conserved, cross-species, modifier genes suggests that conserved cellular pathways, specifically endocytosis and mRNA regulation, act as critical genetic modifiers of SMN loss of function defects across species.     

SMN gene duplication and the emergence of the SMN2 gene occurred in distinct hominids: SMN2 is unique to Homo sapiens

The spinal muscular atrophy (SMA) region on chromosome 5q13 contains an inverted duplication of about 500 kb, and deleterious mutations in the survival motor neuron 1 (SMN1) gene cause SMA, a common lethal childhood neuropathy. We have used a number of approaches to probe the evolutionary history of these genes and show that SMN gene duplication and the appearance of SMN2 occurred at very distinct evolutionary times. Molecular fossil and molecular clock data suggest that this duplication may have occurred as recently as 3 million years ago in that the position and identity repetitive elements are identical for both human SMN genes and overall sequence divergence ranged from 0.15% to 0.34%. However, these approaches ignore the possibility of sequence homogenization by means of gene conversion. Consequently, we have used quantitative polymerase chain rection and analysis of allelic variants to provide physical evidence for or against SMN gene duplication in the chimpanzee, mankind's closest relative. These studies have revealed that chimpanzees have 2-7 copies of the SMN gene per diploid genome; however, the two nucleotides diagnostic for exons 7-8 and the SMNdelta7 mRNA product of the SMN2 gene are absent in non-human primates. In contrast, the SMN2 gene has been detected in all extant human populations studied to date, including representatives from Europe, the Central African Republic, and the Congo. These data provide conclusive evidence that SMN gene duplication occurred more than 5 million years ago, before the separation of human and chimpanzee lineages, but that SMN2 appears for the first time in Homo sapiens.     

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.     

Characterization of functional domains of the SMN protein in vivo

The Survival of Motor Neurons (SMN) is the disease gene of spinal muscular atrophy. We have previously established a genetic system based on the chicken pre-B cell line DT40, in which expression of SMN protein is regulated by tetracycline, to study the function of SMN in vivo. Depletion of SMN protein is lethal to these cells. Here we tested the functionality of mutant SMN proteins by determining their capacity to rescue the cells after depletion of wild-type SMN. Surprisingly, all of the spinal muscular atrophy-associated missense mutations tested were able to support cell viability and proliferation. Deletion of the amino acids encoded by exon 7 of the SMN gene resulted in a partial loss of function. A mutant SMN protein lacking both the tyrosine/glycine repeat (in exon 6) and exon 7 failed to sustain viability, indicating that the C terminus of the protein is critical for SMN activity. Interestingly, the Tudor domain of SMN, encoded by exon 3, does not appear to be essential for SMN function since a mutant deleted of this domain restored cell viability. Unexpectedly, a chicken SMN mutant (DeltaN39) lacking the N-terminal 39 amino acids that encompass the Gemin2-binding domain also rescued the lethal phenotype. Moreover, the level of Gemin2 in DeltaN39-rescued cells was significantly reduced, indicating that Gemin2 is not required for DeltaN39 to perform the essential function of SMN in DT40 cells. These findings suggest that SMN may perform a novel function in DT40 cells.     

Direct interaction of the spinal muscular atrophy disease protein SMN with the small nucleolar RNA-associated protein fibrillarin

Disruption of the survival motor neuron (SMN) gene leads to selective loss of spinal motor neurons, resulting in the fatal human neurodegenerative disorder spinal muscular atrophy (SMA). SMN has been shown to function in spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis and pre-mRNA splicing. We have demonstrated that SMN also interacts with fibrillarin, a highly conserved nucleolar protein that is associated with all Box C/D small nucleolar RNAs and functions in processing and modification of rRNA. Fibrillarin and SMN co-immunoprecipitate from HeLa cell extracts indicating that the proteins exist as a complex in vivo. Furthermore, in vitro binding studies indicate that the interaction between SMN and fibrillarin is direct and salt-stable. We show that the glycine/arginine-rich domain of fibrillarin is necessary and sufficient for SMN binding and that the region of SMN encoded by exon 3, including the Tudor domain, mediates the binding of fibrillarin. Tudor domain missense mutations, including one found in an SMA patient, impair the interaction between SMN and fibrillarin (as well as the common snRNP protein SmB). Our results suggest a function for SMN in small nucleolar RNP biogenesis (akin to its known role as an snRNP assembly factor) and reveal a potential link between small nucleolar RNP biogenesis and SMA.     

The catalytically inactive tyrosine phosphatase HD-PTP/PTPN23 is a novel regulator of SMN complex localization

The survival motor neuron (SMN) complex fulfils essential functions in the assembly of snRNPs, which are key components in the splicing of pre-mRNAs. Little is known about the regulation of SMN complex activity by posttranslational modification despite its complicated phosphorylation pattern. Several phosphatases had been implicated in the regulation of SMN, including the nuclear phosphatases PPM1G and PP1γ. Here we systematically screened all human phosphatase gene products for a regulatory role in the SMN complex. We used the accumulation of SMN in Cajal bodies of intact proliferating cells, which actively assemble snRNPs, as a readout for unperturbed SMN complex function. Knockdown of 29 protein phosphatases interfered with SMN accumulation in Cajal bodies, suggesting impaired SMN complex function, among those the catalytically inactive, non–receptor-type tyrosine phosphatase PTPN23/HD-PTP. Knockdown of PTPN23 also led to changes in the phosphorylation pattern of SMN without affecting the assembly of the SMN complex. We further show interaction between SMN and PTPN23 and document that PTPN23, like SMN, shuttles between nucleus and cytoplasm. Our data provide the first comprehensive screen for SMN complex regulators and establish a novel regulatory function of PTPN23 in maintaining a highly phosphorylated state of SMN, which is important for its proper function in snRNP assembly.     

PPP4R2 regulates neuronal cell differentiation and survival, functionally cooperating with SMN

Spinal muscular atrophy (SMA) is a human disease caused by reduced levels of the Survival of Motor Neuron (SMN) protein, leading to progressive loss of motor neurons and muscular paralysis. However, it is still not very clear why these cells are specifically sensitive to SMN levels. Therefore, understanding which proteins may functionally interact with SMN in a neuronal context is a very important issue. PPP4R2, a regulatory subunit of the protein phosphatase 4 (PPP4C), was previously identified as a functional interactor of the SMN complex, but has never been studied in neuronal cells. In this report, we show that PPP4R2 displays a very dynamic intracellular localization in mouse and rat neuronal cell lines and in rat primary hippocampal neurons, strongly correlating with differentiation. More importantly, we found that PPP4R2 loss of function impairs the differentiation of the mouse motor-neuronal cell line NSC-34, an effect that can be counteracted by SMN overexpression. In addition, we show that PPP4R2 may specifically protect NSC-34 cells from DNA damage-induced apoptosis and that it is capable to functionally cooperate with SMN in this activity. Our data indicate that PPP4R2 is a SMN partner that may modulate the differentiation and survival of neuronal cells.     

Molecular evolution of the moonlighting protein SMN in metazoans

The spinal muscular atrophy (SMA) associated protein survival of motor neuron (SMN) is known to be a moonlighting protein: having one primary, ancestral function (presumed to be involvement in U snRNP assembly) along with one or more secondary functions. One hypothesis for the evolution of moonlighting proteins is that regions of a structure under relatively weak negative selection could gain new functions without interfering with the primary function. To test this hypothesis, we investigated sequence conservation and dN/dS, which reflects the selection acting on a coding sequence, in SMN and a related protein, splicing factor 30 (SPF30), which is not currently known to be multifunctional. We found very different patterns of evolution in the two genes, with SPF30 characterized by strong sequence conservation and negative selection in most animal taxa investigated, and SMN with much lower sequence conservation, and much weaker negative selection at many sites. Evidence was found of positive selection acting on some sites in primate genes for SMN. SMN was also found to have been duplicated in a number of species, and with patterns that indicate reduced negative selection following some of these duplications. There were also several animal species lacking an SMN gene.     

Restoration of SMN expression in mesenchymal stem cells derived from gene-targeted patient-specific iPSCs

Spinal muscular atrophy (SMA) is primarily a neurodegenerative disease caused by the homozygous deletion of the survival motor neuron 1 (SMN1) gene, thereby reducing SMN protein expression. Mesenchymal stem cells (MSCs) have been implicated in the treatment of SMA. In the present study, we overexpressed exogenous SMN1 at the ribosomal DNA (rDNA) locus of induced pluripotent stem cells (iPSCs) generated from a SMA patient using an rDNA-targeting vector. The gene-targeted patient iPSCs differentiated into MSCs (SMN1-MSCs). A 2.1-fold higher expression level of SMN protein was detected in SMN1-MSCs than that detected in MSCs derived from patient iPSCs, and the results of the immunofluorescence analysis showed no difference in the quantity of SMN nuclear structures (gems) between SMN1-MSCs and MSCs derived from normal human iPSCs (h-MSCs). These findings provide a novel strategy for obtaining gene-targeted MSCs for potential clinical applications in autologous cell-based therapy.     

Molecular determinants of survival motor neuron (SMN) protein cleavage by the calcium-activated protease, calpain

Spinal muscular atrophy (SMA) is a leading genetic cause of childhood mortality, caused by reduced levels of survival motor neuron (SMN) protein. SMN functions as part of a large complex in the biogenesis of small nuclear ribonucleoproteins (snRNPs). It is not clear if defects in snRNP biogenesis cause SMA or if loss of some tissue-specific function causes disease. We recently demonstrated that the SMN complex localizes to the Z-discs of skeletal and cardiac muscle sarcomeres, and that SMN is a proteolytic target of calpain. Calpains are implicated in muscle and neurodegenerative disorders, although their relationship to SMA is unclear. Using mass spectrometry, we identified two adjacent calpain cleavage sites in SMN, S192 and F193. Deletion of small motifs in the region surrounding these sites inhibited cleavage. Patient-derived SMA mutations within SMN reduced calpain cleavage. SMN(D44V), reported to impair Gemin2 binding and amino-terminal SMN association, drastically inhibited cleavage, suggesting a role for these interactions in regulating calpain cleavage. Deletion of A188, a residue mutated in SMA type I (A188S), abrogated calpain cleavage, highlighting the importance of this region. Conversely, SMA mutations that interfere with self-oligomerization of SMN, Y272C and SMNΔ7, had no effect on cleavage. Removal of the recently-identified SMN degron (Δ268-294) resulted in increased calpain sensitivity, suggesting that the C-terminus of SMN is important in dictating availability of the cleavage site. Investigation into the spatial determinants of SMN cleavage revealed that endogenous calpains can cleave cytosolic, but not nuclear, SMN. Collectively, the results provide insight into a novel aspect of the post-translation regulation of SMN.     

Construction of a plasmid containing human SMN, the SMA determining gene, coupled to EGFP

We describe here the construction of plasmid pEGFP-C3/SMN, bearing the human SMN gene coupled to the green fluorescent protein (GFP) sequence. The mutation of the SMN gene is responsible for spinal muscular atrophy (SMA), a frequent human infantile genetic disease. We introduced the SMN cDNA into the multiple cloning site of pEGFP-C3. This plasmid bears the neomycin-resistance sequence and the enhanced green fluorescent protein (EGFP). It results in the expression of a fusion protein bearing SMN coupled to a carboxy-terminal GFP tag, used for fluorescence localization studies. Transfection of primary human myoblasts with pEGFP-C3 or pEGFP-C3/SMN revealed that EGFP is intracellularly localized within the cytosol as well as in the nucleus, while the fusion protein EGFP-SMN localized within the nucleus in prominent dot-like structures termed "gems." These data demonstrate that human primary muscle cells can be efficiently transfected and may have important implications for the development of therapeutic strategies in SMA.     

SMN and the Gemin proteins form sub-complexes that localise to both stationary and dynamic neurite granules

Childhood spinal muscular atrophy (SMA) is caused by a reduction in survival motor neuron (SMN) protein. SMN is expressed in every cell type, but it is predominantly the lower motor neurones of the spinal cord that degenerate in SMA. SMN has been linked to the axonal transport of β-actin mRNA, a breakdown in which could trigger disease onset. It is known that SMN is present in transport ribonucleoproteins (RNPs) granules that also contain Gemin2 and Gemin3. To further characterise these granules we have performed live cell imaging of GFP-tagged SMN, GFP-Gemin2, GFP-Gemin3, GFP-Gemin6 and GFP-Gemin7. In all, we have made two important observations: (1) SMN granules appear metamorphic; and (2) the SMN-Gemin complex(es) appears to localise to two distinct subsets of bodies in neurites; stationary bodies and smaller dynamic bodies. This study provides an insight into the neuronal function of the SMN complex.