Spinal muscular atrophy (SMA) is caused by mutations in the Survival Motor Neuron 1 (SMN1) gene, leading to reduced SMN protein levels and widespread disruption in RNA metabolism. Despite of disease-modifying therapies, which remarkably improve patient outcomes, the long-term effects remain unknown. Dysregulated small non-coding RNAs, including microRNAs, have been reported in SMA, but their contribution to the disease remains unclear. We previously showed that flunarizine could improve the phenotype of an SMA mouse model; however its mode of action is incomplete. Here, we showed that flunarizine modulates the expression of numerous microRNAs. Using small-RNA sequencing of a flunarizine-treated SMA patient fibroblast cell line, we identified several microRNAs, which are also dysregulated in the brains and/or spinal cords of SMA mouse models at early disease stages and corrected with flunarizine. Transfection of the miR-128-3p inhibitor interferes with the flunarizine-induced neurite outgrowth in the murine neuronal NSC34 cells. Among the mRNAs modulated by flunarizine, homeodomain interacting protein kinase 2 (Hipk2) transcripts were revealed as novel miR-128-3p targets using either the mimic or its inhibitor. Our findings suggest that an early microRNA accumulation in spinal cords of SMA models can contribute to molecular dysfunctions and may represent an initiating event in pathogenesis.

Dysregulated RNA metabolism caused by SMN deficiency leads to motor neuron disease spinal muscular atrophy (SMA). Current therapies improve patient outcomes but achieve no definite cure, prompting renewed efforts to better understand disease mechanisms. The calcium channel blocker flunarizine improves motor function in Smn-deficient mice and can help uncover neuroprotective pathways. Murine motor neuron-like NSC34 cells were used to study the molecular cell-autonomous mechanism. Following RNA and protein extraction, RT-qPCR and immunodetection experiments were performed. The relationship between flunarizine mRNA targets and RNA-binding protein GEMIN5 was explored by RNA-immunoprecipitation. Flunarizine increases demethylase Kdm6b transcripts across cell cultures and mouse models. It causes, in NSC34 cells, a temporal expression of GEMIN5 and KDM6B. GEMIN5 binds to flunarizine-modulated mRNAs, including Kdm6b transcripts. Gemin5 depletion reduces Kdm6b mRNA and protein levels and hampers responses to flunarizine, including neurite extension in NSC34 cells. Moreover, flunarizine increases the axonal extension of motor neurons derived from SMA patient-induced pluripotent stem cells. Finally, immunofluorescence studies of spinal cord motor neurons in Smn-deficient mice reveal that flunarizine modulates the expression of KDM6B and its target, the motor neuron-specific transcription factor HB9, driving motor neuron maturation. Our study reveals GEMIN5 regulates Kdm6b expression with implications for motor neuron diseases and therapy.

SMN protein deficiency causes motoneuron disease spinal muscular atrophy (SMA). SMN-based therapies improve patient motor symptoms to variable degrees. An early hallmark of SMA is the perturbation of the neuromuscular junction (NMJ), a synapse between a motoneuron and muscle cell. NMJ formation depends on acetylcholine receptor (AChR) clustering triggered by agrin and its co-receptors lipoprotein receptor-related protein 4 (LRP4) and transmembrane muscle-specific kinase (MuSK) signalling pathway. We have previously shown that flunarizine improves NMJs in SMA model mice, but the mechanisms remain elusive. We show here that flunarizine promotes AChR clustering in cell-autonomous, dose- and agrin-dependent manners in C2C12 myotubes. This is associated with an increase in protein levels of LRP4, integrin-beta-1 and alpha-dystroglycan, three agrin co-receptors. Furthermore, flunarizine enhances MuSK interaction with integrin-beta-1 and phosphotyrosines. Moreover, the drug acts on the expression and splicing of Agrn and Cacna1h genes in a muscle-specific manner. We reveal that the Cacna1h encoded protein Cav3.2 closely associates in vitro with the agrin co-receptor LRP4 . In vivo, it is enriched nearby NMJs during neonatal development and the drug increases this immunolabelling in SMA muscles. Thus, flunarizine modulates key players of the NMJ and identifies Ca v 3.2 as a new protein involved in the NMJ biology.

Autosomal-recessive spinal muscular atrophy (SMA) is characterized by the loss of specific motor neurons of the spinal cord and skeletal muscle atrophy. SMA is caused by mutations or deletions of the survival motor neuron 1 (SMN1) gene, and disease severity correlates with the expression levels of the nearly identical copy gene, SMN2. Both genes ubiquitously express SMN protein, but SMN2 generates only low levels of protein that do not fully compensate for the loss-of-function of SMN1. SMN protein forms a multiprotein complex essential for the cellular assembly of ribonucleoprotein particles involved in diverse aspects of RNA metabolism. Other studies using animal models revealed a spatio-temporal requirement of SMN that is high during the development of the neuromuscular system and later, in the general maintenance of cellular and tissues homeostasis. These observations define a period for maximum therapeutic efficiency of SMN restoration, and suggest that cells outside the central nervous system may also participate in the pathogenesis of SMA. Finally, recent innovative therapies have been shown to mitigate SMN deficiency and have been approved to treat SMA patients. We briefly review major findings from the past twenty-five years of SMA research. © 2020 French Society of Pediatrics. Published by Elsevier Masson SAS. All rights reserved.

The motor neurodegenerative disease spinal muscular atrophy (SMA) is caused by alterations of the survival motor neuron 1 (SMN1) gene involved in RNA metabolism. Although the disease mechanisms are not completely elucidated, SMN protein deficiency leads to abnormal small nuclear ribonucleoproteins (snRNPs) assembly responsible for widespread splicing defects. SMN protein localizes in nuclear bodies that are lost in SMA and adult onset amyotrophic lateral sclerosis (ALS) patient cells harboring TDP-43 or FUS/TLS mutations. We previously reported that flunarizine recruits SMN into nuclear bodies and improves the phenotype of an SMA mouse model. However, the precise mode of action remains elusive. Here, a marked reduction of the integral components of the SMN complex is observed in severe SMA patient fibroblast cells. We show that flunarizine increases the protein levels of a subset of components of the SMN-Gemins complex, Gemins2-4, and markedly reduces the RNA and protein levels of the pro-oxydant thioredoxin-interacting protein (TXNIP) encoded by an mRNA target of Gemin5. We further show that SMN deficiency causes a dissociation of the localization of the SMN complex components from the same nuclear bodies. The accumulation of TDP-43 in SMN-positive nuclear bodies is also perturbed in SMA cells. Notably, TDP-43 is found to co-localize with SMN in nuclear bodies of flunarizine-treated SMA cells. Our findings indicate that flunarizine reverses cellular changes caused by SMN deficiency in SMA cells and further support the view of a common pathway in RNA metabolism underlying infantile and adult motor neuron diseases.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The hereditary neurodegenerative disorder spinal muscular atrophy (SMA) is characterized by the loss of spinal cord motor neurons and skeletal muscle atrophy. SMA is caused by mutations of the survival motor neuron (SMN) gene leading to a decrease in SMN protein levels. The SMN deficiency alters nuclear body formation and whether it can contribute to the disease remains unclear. Here we screen a series of small-molecules on SMA patient fibroblasts and identify flunarizine that accumulates SMN into Cajal bodies, the nuclear bodies important for the spliceosomal small nuclear RNA (snRNA)-ribonucleoprotein biogenesis. Using histochemistry, real-time RT-PCR and behavioural analyses in a mouse model of SMA, we show that along with the accumulation of SMN into Cajal bodies of spinal cord motor neurons, flunarizine treatment modulates the relative abundance of specific spliceosomal snRNAs in a tissue-dependent manner and can improve the synaptic connections and survival of spinal cord motor neurons. The treatment also protects skeletal muscles from cell death and atrophy, raises the neuromuscular junction maturation and prolongs life span by as much as 40 percent (p < 0.001). Our findings provide a functional link between flunarizine and SMA pathology, highlighting the potential benefits of flunarizine in a novel therapeutic perspective against neurodegenerative diseases.

The calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway is involved in the modulation of the adult muscle fiber type, but its role in the establishment of the muscle phenotype remains elusive. Here, we show that the NFAT member NFATc2 cooperates with the basic helix-loop-helix transcription factor MyoD to induce the expression of a specific myosin heavy chain (MHC) isoform, the neonatal one, during embryogenesis. We found this cooperation to be crucial, as Myod/Nfatc2 double-null mice die at birth, with a dramatic reduction of the major neonatal MHC isoform normally expressed at birth in skeletal muscles, such as limb and intercostal muscles, whereas its expression is unaffected in myofibers mutated for either factor alone. Using gel shift and chromatin immunoprecipitation assays, we identified NFATc2 bound to the neonatal Mhc gene, whereas NFATc1 and NFATc3 would preferentially bind the embryonic Mhc gene. We provide evidence that MyoD synergistically cooperates with NFATc2 at the neonatal Mhc promoter. Altogether, our findings demonstrate that the calcineurin/NFAT pathway plays a new role in establishing the early muscle fiber type in immature myofibers during embryogenesis.

Spinal muscular atrophy is a severe motor neuron disease caused by reduced levels of the ubiquitous Survival of MotoNeurons (SMN) protein. SMN is part of a complex that is essential for spliceosomal UsnRNP biogenesis. Signal recognition particle (SRP) is a ribonucleoprotein particle crucial for co-translational targeting of secretory and membrane proteins to the endoplasmic reticulum. SRP biogenesis is a nucleo-cytoplasmic multistep process in which the protein components, except SRP54, assemble with 7S RNA in the nucleolus. Then, SRP54 is incorporated after export of the pre-particle into the cytoplasm. The assembly factors necessary for SRP biogenesis remain to be identified. Here, we show that 7S RNA binds to purified SMN complexes in vitro and that SMN complexes associate with SRP in cellular extracts. We identified the RNA determinants required. Moreover, we report a specific reduction of 7S RNA levels in the spinal cord of SMN-deficient mice, and in a Schizosaccharomyces pombe strain carrying a temperature-degron allele of SMN. Additionally, microinjected antibodies directed against SMN or Gemin2 interfere with the association of SRP54 with 7S RNA in Xenopus laevis oocytes. Our data show that reduced levels of the SMN protein lead to defect in SRP steady-state level and describe the SMN complex as the first identified cellular factor required for SRP biogenesis.

The spinal muscular atrophy (SMA) gene product SMN forms with Gemin2 to 8 and unrip the ubiquitous SMN complex that is required for the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs), their nuclear import and localization to subnuclear domain Cajal bodies (CBs). The concentration of the SMN complex and snRNPs in CBs are reduced upon SMN deficiency in SMA cells. Subcellular localization of the SMN complex is regulated in a phosphorylation-dependent manner and the precise mechanisms remain poorly understood. Using coimmunoprecipitation in HeLa cell extracts and in vitro protein binding assays, we show here that the SMN complex and its component Gemin8 interact directly with protein phosphatase PP1γ. Overexpression of Gemin8 in cells increases the number of CBs and results in targeting of PP1γ to CBs. Moreover, depletion of PP1γ by RNA interference enhances the localization of the SMN complex and snRNPs to CBs. Consequently, the interaction between SMN and Gemin8 increases in cytoplasmic and nuclear extracts of PP1γ-depleted cells. Two-dimensional protein gel electrophoresis reveals that SMN is hyperphosphorylated in nuclear extracts of PP1γ-depleted cells and expression of PP1γ restores these isoforms. Notably, SMN deficiency in SMA leads to the aberrant subcellular localization of Gemin8 and PP1γ in the atrophic skeletal muscles, suggesting that the function of PP1γ is likely to be affected in disease. Our findings reveal a role of PP1γ in the formation of the SMN complex and the maintenance of CB integrity. Finally, we propose Gemin8 interaction with PP1γ as a target for therapeutic intervention in SMA.

Spinal muscular atrophy (SMA) is a common autosomal recessive neurodegenerative disease caused by reduced survival motor neuron (SMN) levels. The assembly machinery containing SMN is implicated in the biogenesis of the spliceosomal small nuclear ribonucleoproteins (snRNPs). SMN is present in both the cytoplasm and nucleus, where it transiently accumulates in subnuclear domains named Cajal bodies (CBs) and functions in the maturation of snRNPs and small nucleolar (sno)RNPs. The impact of lowering SMN levels on the composition of CBs in SMA cells is still not completely understood. Here, we analyse the CB composition in immortalized and primary fibroblasts from SMA patients. We show that the U snRNA export factors PHAX and chromosome region maintenance 1 and the box C/D snoRNP core protein fibrillarin concentrate in CBs from SMA cells, whereas the box H/ACA core proteins GAR1 and NAP57/dyskerin show reduced CB localization. Remarkably, the functional deficiency in SMA cells is associated with decreased localization of the snoRNP chaperone Nopp140 in CBs that correlates with disease severity. Indeed, RNA interference knockdown experiments in control fibroblasts demonstrate that SMN is required for accumulation of Nopp140 in CBs. Conversely, overexpression of SMN in SMA cells restores the CB localization of Nopp140, whereas SMN mutants found in SMA patients are defective in promoting the association of Nopp140 with CBs. Taken together, we demonstrate that only a subset of CB functions (as indicated by the association of representative factors) are impaired in SMA cells and, importantly, we identify the decrease of Nopp140 localization in CBs as a phenotypic marker for SMA.

Mutations of the survival motor neuron gene SMN1 cause the inherited disease spinal muscular atrophy (SMA). The ubiquitous SMN protein facilitates the biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs). The protein is detected in the cytoplasm, nucleoplasm and enriched with snRNPs in nuclear Cajal bodies. It is structurally divided into at least an amino-terminal region rich in basic amino acid residues, a central Tudor domain, a self-association tyrosine-glycine-box and an exon7-encoded C-terminus. To examine the domains required for the intranuclear localization of SMN, we have used fluorescently tagged protein mutants transiently overexpressed in mammalian cells. The basic amino acid residues direct nucleolar localization of SMN mutants. The Tudor domain promotes localization of proteins in the nucleus and it cooperates with the basic amino acid residues and the tyrosine-glycine-box for protein localization in Cajal bodies. Moreover, the most frequent disease-linked mutant SMNΔex7 reduces accumulation of snRNPs in Cajal bodies, suggesting that the C-terminus of SMN participates in targeting to Cajal bodies. A reduced number of Cajal bodies in patient fibroblasts associates with the absence of snRNPs in Cajal bodies, revealing that intranuclear snRNA organization is modified in disease. These results indicate that direct and indirect mechanisms regulate localization of SMN in Cajal bodies.

Objective: To describe the clinical features of a novel variant of autosomal recessive lower motor neuron disease (LMND) with childhood onset and to map the disease-causing gene. Methods: The authors performed a clinical study in a large consanguineous African family. After linkage exclusion to SMN1 and SOD1 loci, they performed a genome-wide linkage analysis to map the underlying genetic defect. Results: This novel variant of LMND with childhood onset and autosomal recessive mode of inheritance is characterized by a progressive symmetric and generalized involvement of the musculature. Four of the five affected patients had muscle weakness since age 3, strongly worsening during childhood and leading to generalized tetraplegia in adulthood. Genetic analyses using homozygosity mapping strategy assigned this progressive generalized LMND locus to an interval of 3.9 cM (or 1.5 megabases) on chromosome 1p36, between loci D1S508 and D1S2633 (Z(max) = 3.79 at theta = 0.00 at locus D1S253). This region encloses 27 candidate genes. Conclusion: Genetic mapping of a novel rare phenotype of lower motor neuron disease opens the way toward the identification of a new gene involved in motor neuron degeneration, located in the 1p36 chromosomal region.

Distal spinal muscular atrophy is a heterogeneous group of neuromuscular disorders caused by progressive anterior horn cell degeneration and characterized by progressive motor weakness and muscular atrophy, predominantly in the distal parts of the limbs. Here we report on chronic autosomal recessive distal spinal muscular atrophy in a large, inbred family with onset at various ages. Because this condition had some of the same clinical features as spinal muscular atrophy with respiratory distress, we tested the disease gene for linkage to chromosome 11q and mapped the disease locus to chromosome 11q13 in the genetic interval that included the spinal muscular atrophy with respiratory distress gene (D11S1889‐D11S1321, Z max = 4.59 at θ = 0 at locus D11S4136). The sequencing of IGHMBP2 , the human homologue of the mouse neuromuscular degeneration gene ( nmd ) that accounts for spinal muscular atrophy with respiratory distress, failed to detect any mutation in our chronic distal spinal muscular atrophy patients, suggesting that spinal muscular atrophy with respiratory distress and chronic distal spinal muscular atrophy are caused by distinct genes located in the same chromosomal region. In addition, the high intrafamilial variability in age at onset raises the question of whether nonallelic modifying genes could be involved in chronic distal spinal muscular atrophy.

Spinal muscular atrophy (SMA) is caused by the loss of functional survival motor neuron 1 (SMN1) protein. This ubiquitously expressed protein is a component of a novel complex immunodetected in both the cytoplasm and the nucleus, which is associated with complexes involved in mRNA splicing, ribosome biogenesis and transcription. Here, we study a mutant protein corresponding to the N-terminal half of the protein that is encoded by the SMA frameshift mutation SMN 472del5. We show by confocal microscopy that the resulting mutant protein exhibits various distribution patterns in different transiently transfected COS cells. The mutant distributes into the nucleoplasm and/or the nucleolus, whereas the normal SMN protein accumulates at discrete nucleocytoplasmic dot-like structures previously named gems/Cajal bodies. The cell population with the nucleolar distribution is enriched upon treatment with mimosine, a synchronizing drug in late G(1) phase. Co-immunoprecipitation studies carried out on nuclear extracts reveal that both the endogenous SMN and mutant proteins are associated with complexes containing two major non-ribosomal nucleolar proteins, namely nucleolin and protein B23, and that the association is mediated, by among other things, RNA moieties. Both the association of the SMN protein with nucleolin-containing complexes and the nucleolin/B23 complex are disrupted in fibroblasts derived from a type I SMA patient harboring a homozygous SMN1 gene deletion. These findings suggest that altered assembly and/or stability of ribonucleoprotein complexes may contribute to the pathophysiological processes in SMA.

Editor—With a prevalence of 1/6000 live births, spinal muscular atrophy (SMA) represents the second most common fatal autosomal recessive disorder after cystic fibrosis.1 2 SMA is characterised by the degeneration of anterior horn cells of the spinal cord, resulting in progressive, symmetrical limb and trunk paralysis associated with muscular atrophy. This condition is clinically heterogeneous and has been subdivided into three types according to age of onset and clinical course3: type I (Werdnig-Hoffmann disease, MIM 253300), type II (intermediate form, MIM 253550), and type III SMA (Kugelberg-Welander disease, MIM 253400). The SMA locus has been mapped to chromosome 5q11.2-q13.3 within a region characterised by the large inverted duplication of a 500 kb element.4-6 The survival motor neurone ( SMN ) gene, which lies within this element, is duplicated and both copies are expressed. The telomeric gene ( SMN1 ) has been shown to be deleted or mutated in all three types of SMA.4 SMN1 encodes almost the full length transcript whereas the centromeric copy ( SMN2 ) generates alternatively spliced variants lacking the C-terminal sequence.5 7 The SMN region contains low copy repeats triggering homologous recombination events. Indeed, approximately 95% of SMA patients lack both SMN1 genes owing to either deletion or gene conversion.4 In SMA patients who lack only one SMN1 gene, allelic intragenic mutations have been identified, confirming the involvement of SMN1 in the pathogenesis of SMA.5 8-10 The heterozygote frequency has been estimated to be 1/40. However, the duplication of the SMA locus makes the detection of SMA carriers in the general population difficult, and this has hampered genetic counselling in affected families. Initial attempts to estimate the SMN copy number were based on the measurement of the SMN1 / SMN2 ratios,11-13 but the broad variability of SMN2 copy number hinders reliable …