Loss Of SMN1 Research Articles

A rapid and easy-to-use spinal muscular atrophy screening tool based on primers with high specificity and amplification efficiency for SMN1 combined with single-stranded tag hybridization assay.

Spinal muscular atrophy (SMA) is an intractable neuromuscular disorder primarily caused by homozygous deletions in exon 7 of the SMN1 gene. Early diagnosis and prompt treatment of patients with SMA have a significant impact on prognosis, and several therapies have recently been developed. Current SMA screening tests require a significant turnaround time to identify patients with suspected SMA, due both to the interval between the birth of a newborn and the collection of blood for newborn mass screening and the difficulty in distinguishing between SMN1 and SMN2, a paralog gene that requires testing in specialized laboratories. The aim of this study was therefore to develop a novel SMA screening assay that can be rapidly performed in ordinary hospitals and clinics to overcome these issues. We designed over 100 combinations of forward and reverse primers with 3' ends targeting SMN1-specific sites around exon 7, and evaluated their specificity and amplification efficiency by quantitative PCR to identify the best primer pair. Furthermore, we performed a single-stranded tag hybridization assay after PCR. To evaluate the accuracy and practicality of the newly developed assay, we analyzed saliva specimens from five patients with SMA and two SMA carriers collected in an outpatient clinic and DNA specimens from three patients with SMA and four SMA carriers from a biobank, together with those from healthy individuals. DNA and raw saliva specimens from all patients with SMA demonstrated a biallelic loss of SMN1, whereas those from carriers and healthy individuals did not. The results of 50 independent experiments were consistent for all samples. The assay could be completed within one hour. This simple and convenient new screening tool has the potential to allow patients with SMA to receive disease-modifying therapies within a shorter timeframe.

Structure and function analysis of Sam68 and hnRNP A1 synergy in the exclusion of exon 7 from SMN2 transcripts.

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by the absence of a functional copy of the Survival of Motor Neuron 1 gene (SMN1). The nearly identical paralog, SMN2, cannot compensate for the loss of SMN1 because exon 7 is aberrantly skipped from most SMN2 transcripts, a process mediated by synergistic activities of Src-associated during mitosis, 68 kDa (Sam68/KHDRBS1) and heterogeneous nuclear ribonucleoprotein (hnRNP) A1. This results in the production of a truncated, nonfunctional protein that is rapidly degraded. Here, we present several crystal structures of Sam68 RNA-binding domain (RBD). Sam68-RBD forms stable symmetric homodimers by antiparallel association of helices α3 from two monomers. However, the details of domain organization and the dimerization interface differ significantly from previously characterized homologs. We demonstrate that Sam68 and hnRNP A1 can simultaneously bind proximal motifs within the central region of SMN2 (ex7). Furthermore, we show that the RNA-binding pockets of the two proteins are close to each other in their heterodimeric complex and identify contact residues using crosslinking-mass spectrometry. We present a model of the ternary Sam68·SMN2 (ex7)·hnRNP A1 complex that reconciles all available information on SMN1/2 splicing. Our findings have important implications for the etiology of SMA and open new avenues for the design of novel therapeutics to treat splicing diseases.

Characteristics of genetic changes in the <i>SMN1</i> gene in spinal muscular atrophy 5q

Background. Proximal spinal muscular atrophy 5q (5q‑SMA) is one of the most common neuromuscular diseases, which is caused by mutations of the SMN1 gene. Despite the fact that most studies consider SMN1 “deletion” as the most common cause of 5q‑SMA, gene loss is in fact associated with both classical deletions and conversions of SMN1 and SMN2, as well as with formation of chimeric structures. Up to now, far too little attention has been paid to the prevalence of types of SMN1 loss. However, different types of mutations can have different influence on the clinical findings and the effectiveness of therapy. A deeper study of the structure of these genes will allow us to determine the predictors of response to treatment and bring us closer to understanding the reasons for the instability of the SMN region.Aim. To study genetic changes in the SMN1 gene, as well as the number of copies of the SMN1 and SMN2 genes in 5q‑SMA.Materials and methods. The study involved 703 patients, for whom the analysis of the number of copies of SMN1 and SMN2 was performed in the center of molecular medicine of I.P. Pavlov First Saint Petersburg State Medical University for 2018–2021. Gene copy number analysis was performed by multiplex ligation‑dependent probe amplification (MLPA) using the SALSA MLPA P021 SMA kit (MRC Holland).Results. Among 703 participants, a homozygous SMN1 deletion was found in 167 (24 %), 76 (11 %) were carriers of the deletion and no aberrations were found in the remaining 460 (65 %). Among patients with a homozygous deletion, 41 (24 %) cases of a true deletion were identified. Also, 11 (7 %) cases of partial deletion with a homozygous loss of the 7th exon and heterozygous of the 8th exon of SMN1 were identified. The most common type of aberration was the conversion of SMN1 to SMN2 – 94 (56 %) cases, which is characterized by a homozygous loss of SMN1 and a reciprocal increase in SMN2 copies. In addition, 21 (13 %) cases of the formation of hybrid genes were found.Conclusion. Types of genetic aberrations in 5q‑SMA have not been studied enough yet. A search of the literature revealed very few studies which results are comparable with ours. However, further research of genetic changes in SMN1 and SMN2 might bring more clarity on the causes and mechanisms of this disease, and get us closer to finding the most effective method of treatment.

Structural Context of a Critical Exon of Spinal Muscular Atrophy Gene.

Humans contain two nearly identical copies of Survival Motor Neuron genes, SMN1 and SMN2. Deletion or mutation of SMN1 causes spinal muscular atrophy (SMA), one of the leading genetic diseases associated with infant mortality. SMN2 is unable to compensate for the loss of SMN1 due to predominant exon 7 skipping, leading to the production of a truncated protein. Antisense oligonucleotide and small molecule-based strategies aimed at the restoration of SMN2 exon 7 inclusion are approved therapies of SMA. Many cis-elements and transacting factors have been implicated in regulation of SMN exon 7 splicing. Also, several structural elements, including those formed by a long-distance interaction, have been implicated in the modulation of SMN exon 7 splicing. Several of these structures have been confirmed by enzymatic and chemical structure-probing methods. Additional structures formed by inter-intronic interactions have been predicted by computational algorithms. SMN genes generate a vast repertoire of circular RNAs through inter-intronic secondary structures formed by inverted Alu repeats present in large number in SMN genes. Here, we review the structural context of the exonic and intronic cis-elements that promote or prevent exon 7 recognition. We discuss how structural rearrangements triggered by single nucleotide substitutions could bring drastic changes in SMN2 exon 7 splicing. We also propose potential mechanisms by which inter-intronic structures might impact the splicing outcomes.

Restoring SMN Expression: An Overview of the Therapeutic Developments for the Treatment of Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disorder and one of the most common genetic causes of infant death. It is characterized by progressive weakness of the muscles, loss of ambulation, and death from respiratory complications. SMA is caused by the homozygous deletion or mutations in the survival of the motor neuron 1 (SMN1) gene. Humans, however, have a nearly identical copy of SMN1 known as the SMN2 gene. The severity of the disease correlates inversely with the number of SMN2 copies present. SMN2 cannot completely compensate for the loss of SMN1 in SMA patients because it can produce only a fraction of functional SMN protein. SMN protein is ubiquitously expressed in the body and has a variety of roles ranging from assembling the spliceosomal machinery, autophagy, RNA metabolism, signal transduction, cellular homeostasis, DNA repair, and recombination. Motor neurons in the anterior horn of the spinal cord are extremely susceptible to the loss of SMN protein, with the reason still being unclear. Due to the ability of the SMN2 gene to produce small amounts of functional SMN, two FDA-approved treatment strategies, including an antisense oligonucleotide (AON) nusinersen and small-molecule risdiplam, target SMN2 to produce more functional SMN. On the other hand, Onasemnogene abeparvovec (brand name Zolgensma) is an FDA-approved adeno-associated vector 9-mediated gene replacement therapy that can deliver a copy of the human SMN1. In this review, we summarize the SMA etiology, the role of SMN, and discuss the challenges of the therapies that are approved for SMA treatment.

Phenotypes of SMA patients retaining SMN1 with intragenic mutation

BackgroundSpinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by homozygous deletion or intragenic mutation of the SMN1 gene. It is well-known that high copy number of its homologous gene, SMN2, modifies the phenotype of SMN1-deleted patients. However, in the patients with intragenic SMN1 mutation, the relationship between phenotype and SMN2 copy number remains unclear. MethodsWe have analyzed a total of 515 Japanese patients with SMA-like symptoms (delayed developmental milestones, respiratory failures, muscle weakness etc.) from 1996 to 2019. SMN1 and SMN2 copy numbers were determined by quantitative polymerase chain reaction (PCR) method and/or multiplex ligation-dependent probe amplification (MLPA) method. Intragenic SMN1 mutations were identified through DNA and RNA analysis of the fresh blood samples. ResultsA total of 241 patients were diagnosed as having SMA. The majority of SMA patients showed complete loss of SMN1 (n = 228, 95%), but some patients retained SMN1 and carried an intragenic mutation in the retaining SMN1 (n = 13, 5%). Ten different mutations were identified in these 13 patients, consisting of missense, nonsense, frameshift and splicing defect-causing mutations. The ten mutations were c.275G > C (p.Trp92Ser), c.819_820insT (p.Thr274Tyrfs*32), c.830A > G (p.Tyr277Cys), c.5C > T (p.Ala2Val), c.826 T > C (p.Tyr276His), c.79C > T (p.Gln27*), c.188C > A (p.Ser63*), c.422 T > C (p.Leu141Pro), c.835-2A > G (exon 7 skipping) and c.835-3C > A (exon 7 skipping). It should be noted here that some patients with milder phenotype carried only a single SMN2 copy (n = 3), while other patients with severe phenotype carried 3 SMN2 copies (n = 4). ConclusionIntragenic mutations in SMN1 may contribute more significantly to clinical severity than SMN2 copy numbers.

Intragenic complementation of amino and carboxy terminal SMN missense mutations can rescue Smn null mice.

Spinal muscular atrophy is caused by reduced levels of SMN resulting from the loss of SMN1 and reliance on SMN2 for the production of SMN. Loss of SMN entirely is embryonic lethal in mammals. There are several SMN missense mutations found in humans. These alleles do not show partial function in the absence of wild-type SMN and cannot rescue a null Smn allele in mice. However, these human SMN missense allele transgenes can rescue a null Smn allele when SMN2 is present. We find that the N- and C-terminal regions constitute two independent domains of SMN that can be separated genetically and undergo intragenic complementation. These SMN protein heteromers restore snRNP assembly of Sm proteins onto snRNA and completely rescue both survival of Smn null mice and motor neuron electrophysiology demonstrating that the essential functional unit of SMN is the oligomer.

SMA: REGISTRIES, BIOMARKERS & OUTCOME MEASURES: P.180 Identification of functional SMNs and suppressors of SMA causing mutations to define the critical function of SMN that causes SMA

Spinal muscular atrophy (SMA) is caused by loss of SMN1 and retention of SMN2 leading to SMN deficiency. SMN functions in assembly of spliceosomal snRNPs and U7 snRNP, however many other functions have been proposed such as transport of RNA down axons. It is not understood which of these functions is critical to the development of SMA or whether they act in concert. We have developed a cell line which can be conditionally deleted for SMN via Cre expression. In these lines, removal of SMN always results in lethality, thus they can be used to determine which SMN alleles are functional as well as identify suppressors.

Combinatorial treatment for spinal muscular atrophy: An Editorial for 'Combined treatment with the histone deacetylase inhibitor LBH589 and a splice-switch antisense oligonucleotide enhances SMN2 splicing and SMN expression in Spinal Muscular Atrophy cells' on page 264.

Spinal muscular atrophy (SMA) is a severe autosomal recessive motor neuron disease caused by the loss of SMN1, which encodes a protein essential for motor neuron survival. SMA patients have one or more copies of an alternate SMN gene, SMN2, which is nearly identical to SMN1. SMN2 differs at a single nucleotide from SMN1 which results in the skipping of exon 7 in the mRNA and produces an unstable protein (SMNΔ7). Therapeutic approaches that have been undertaken include (1) replacement of SMN1 by gene delivery mediated by adeno-associated virus serotype 9 (AAV9) (Zolgensma), (2) correction of the aberrant SMN2 splicing using an antisense oligonucleotide (ASO) or small molecule (nusinersin, risdiplam), and (3) increased expression of SMN2 mediated by histone deacetylase (HDAC) inhibitors. Two of these three approaches have given rise to successful treatments for SMA, but they are very expensive, and their long-term safety is not well known. In addition, the ability of ASOs and viral vectors to reach their targets in the CNS with peripheral administration is limited. Small molecules may cross the brain-blood barrier when orally delivered and can be discontinued if needed to mitigate adverse effects. This Editorial highlights this study by Pagliarni et al. in which they used combined treatment of cell models of SMA with an ASO and an orally delivered HDAC inhibitor (panobinostat) to overcome the limitations of a single-therapeutic approach. Panobinostat enhanced the expression of SMN2, increasing the amount of SMN2 mRNA available for splicing correction mediated by the ASO. In addition, panobinostat increased exon 7 retention in the SMN2 mRNA. This combinatorial treatment might allow lower or less frequent ASO doses, reducing the need for repeated intrathecal administration. The combined effects of panobinostat and nusinersen can now be tested in SMA animal models to determine whether this approach will be translatable to patients.

Amyotrophie spinale infantile

Spinal muscular atrophy (SMA) is the most common genetic disease leading to infant mortality. This neuro-muscular disorder is caused by the loss or mutation of the telomeric copy of the 'survival of motor neuron' (Smn) gene, termed SMN1. Loss of SMN1 leads to reduced SMN protein levels, inducing degeneration of motor neurons (MN) and progressive muscle weakness and atrophy. Gene therapy, consisting of reintroducing SMN1 in the MNs, is an attractive approach for SMA. We showed the most efficient rescue of SMA mice to date after a single intravenous injection of an AAV9 expressing SMN1, highlighting the considerable potential of this method for the treatment of human SMA. Recently, a startup led by the Dr Kaspar decided to test this experimental approach in children with SMA type 1. Dr Mendell, in charge of this clinical project, showed a very significant increase of the lifespan and motor function of the patients (until 4 years) after a single injection of AAV9-SMN1 (named ZolgenSMA®) into an arm or leg vein. This gene therapy treatment obtained a marketing authorization by the FDA in May 24 and is now the first efficient therapy for neuromuscular disease.

The First Orally Deliverable Small Molecule for the Treatment of Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is 1 of the leading causes of infant mortality. SMA is mostly caused by low levels of Survival Motor Neuron (SMN) protein due to deletion of or mutation in the SMN1 gene. Its nearly identical copy, SMN2, fails to compensate for the loss of SMN1 due to predominant skipping of exon 7. Correction of SMN2 exon 7 splicing by an antisense oligonucleotide (ASO), nusinersen (Spinraza™), that targets the intronic splicing silencer N1 (ISS-N1) became the first approved therapy for SMA. Restoration of SMN levels using gene therapy was the next. Very recently, an orally deliverable small molecule, risdiplam (Evrysdi™), became the third approved therapy for SMA. Here we discuss how these therapies are positioned to meet the needs of the broad phenotypic spectrum of SMA patients.

Spinal Muscular Atrophy Type B Awareness and Symptoms Prediction Using 3D Segmentation Process in MRI Images

Spinal muscular atrophy (SMA) describes a group of disorders associated with spinal motor neuron loss. In this review we provide an update regarding the most common form of SMA, proximal or 5q SMA, and discuss the contemporary approach to diagnosis and treatment. Electromyography and muscle biopsy features of denervation were once the basis for diagnosis, but molecular testing for homozygous deletion or mutation of the SMN1 gene allows efficient and specific diagnosis. In combination with loss of SMN1, patients retain variable numbers of copies of a second similar gene, SMN2, which produce reduced levels of the survival motor neuron (SMN) protein that are insufficient for normal motor neuron function. Despite the fact that the understanding of how ubiquitous reduction of SMN protein leads to motor neuron loss remains incomplete, several promising therapeutics are now being tested in early phase clinical trials. This proposed model investigates the symptoms and scans readings from the initial MRI scan images of babies with mutation progress and SMN proteins formation benchmark values for this particular disorder SMA and further this segmented parameters are acquitted into the K-means clustering technique that predict the report with the disorder symptoms with MSE (mean square error) values that helps the babies in future to take prevention measures to overcome this problem.

Evaluation of Exon Inclusion Induced by Splice Switching Antisense Oligonucleotides in SMA Patient Fibroblasts.

Spinal muscular atrophy (SMA), a lethal neurological disease caused by the loss of SMN1, presents a unique case in the field of antisense oligonucleotide (AON)-mediated therapy. While SMN1 mutations are responsible for the disease, AONs targeting intronic splice silencer (ISS) sites in SMN2, including FDA-approved nusinersen, have been shown to restore SMN expression and ameliorate the symptoms. Currently, many studies involving AON therapy for SMA focus on investigating novel AON chemistries targeting SMN2 that may be more effective and less toxic than nusinersen. Here, we describe a protocol for in vitro evaluation of exon inclusion using lipotransfection of AONs followed by reverse transcription polymerase chain reaction (RT-PCR), quantitative polymerase chain reaction (qPCR), and Western blotting. This method can be employed for various types of AON chemistries. Using this method, we demonstrate that AONs composed of alternating locked nucleic acids (LNAs) and DNA nucleotides (LNA/DNA mixmers) lead to efficient SMN2 exon inclusion and restoration of SMN protein at a very low concentration, and therefore, LNA/DNA mixmer-based antisense oligonucleotides may be an attractive therapeutic strategy to treat splicing defects caused by genetic diseases. The in vitro evaluation method described here is fast, easy, and sensitive enough for the testing of various novel AONs.

Antisense Oligonucleotide-Mediated Terminal Intron Retention of the SMN2 Transcript

The severe childhood disease spinal muscular atrophy (SMA) arises from the homozygous loss of the survival motor neuron 1 gene (SMN1). A homologous gene potentially encoding an identical protein, SMN2 can partially compensate for the loss of SMN1; however, the exclusion of a critical exon in the coding region during mRNA maturation results in insufficient levels of functional protein. The rate of transcription is known to influence the alternative splicing of gene transcripts, with a fast transcription rate correlating to an increase in alternative splicing. Conversely, a slower transcription rate is more likely to result in the inclusion of all exons in the transcript. Targeting SMN2 with antisense oligonucleotides to influence the processing of terminal exon 8 could be a way to slow transcription and induce the inclusion of exon 7. Interestingly, following oligomer treatment of SMA patient fibroblasts, we observed the inclusion of exon 7, as well as intron 7, in the transcript. Because the normal termination codon is located in exon 7, this exon/intron 7-SMN2 transcript should encode the normal protein and only carry a longer 3′ UTR. Further studies showed the extra 3′ UTR length contained a number of regulatory motifs that modify transcript and protein regulation, leading to translational repression of SMN. Although unlikely to provide therapeutic benefit for SMA patients, this novel technique for gene regulation could provide another avenue for the repression of undesirable gene expression in a variety of other diseases.

Recent Advances and Clinical Applications of Exon Inclusion for Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive disorder caused by a mutation in SMN1 that stops production of SMN (survival of motor neuron) protein. Insufficient levels of SMN results in the loss of motor neurons, which causes muscle weakness, respiratory distress, and paralysis. A nearly identical gene (SMN2) contains a C-to-T transition which excludes exon 7 from 90% of the mature mRNA transcripts, leading to unstable proteins which are targeted for degradation. Although SMN2 cannot fully compensate for a loss of SMN1 due to only 10% functional mRNA produced, the discovery of the intronic splicing silencer (ISS-N1) opened a doorway for therapy. By blocking its function with antisense oligonucleotides manipulated for high specificity and efficiency, exon 7 can be included to produce full-length mRNA, which then compensates for the loss of SMN1. Nusinersen (Spinraza), the first FDA-approved antisense oligonucleotide drug targeting SMA, was designed based on this concept and clinical studies have demonstrated a dramatic improvement in patients. Novel chemistries including phosphorodiamidate morpholino oligomers (PMOs) and locked nucleic acids (LNAs), as well as peptide conjugates such as Pip that facilitate accurate targeting to the central nervous system, are explored to increase the efficiency of exon 7 inclusion in the appropriate tissues to ameliorate the SMA phenotype. Due to the rapid advancement of treatments for SMA following the discovery of ISS-N1, the future of SMA treatment is highly promising.

A Multilayered Control of the Human Survival Motor Neuron Gene Expression by Alu Elements.

Humans carry two nearly identical copies of Survival Motor Neuron gene: SMN1 and SMN2. Mutations or deletions of SMN1, which codes for SMN, cause spinal muscular atrophy (SMA), a leading genetic disease associated with infant mortality. Aberrant expression or localization of SMN has been also implicated in other pathological conditions, including male infertility, inclusion body myositis, amyotrophic lateral sclerosis and osteoarthritis. SMN2 fails to compensate for the loss of SMN1 due to skipping of exon 7, leading to the production of SMNΔ7, an unstable protein. In addition, SMNΔ7 is less functional due to the lack of a critical C-terminus of the full-length SMN, a multifunctional protein. Alu elements are specific to primates and are generally found within protein coding genes. About 41% of the human SMN gene including promoter region is occupied by more than 60 Alu-like sequences. Here we discuss how such an abundance of Alu-like sequences may contribute toward SMA pathogenesis. We describe the likely impact of Alu elements on expression of SMN. We have recently identified a novel exon 6B, created by exonization of an Alu-element located within SMN intron 6. Irrespective of the exon 7 inclusion or skipping, transcripts harboring exon 6B code for the same SMN6B protein that has altered C-terminus compared to the full-length SMN. We have demonstrated that SMN6B is more stable than SMNΔ7 and likely functions similarly to the full-length SMN. We discuss the possible mechanism(s) of regulation of SMN exon 6B splicing and potential consequences of the generation of exon 6B-containing transcripts.

How the discovery of ISS-N1 led to the first medical therapy for spinal muscular atrophy.

Spinal muscular atrophy (SMA), a prominent genetic disease of infant mortality, is caused by low levels of survival motor neuron (SMN) protein owing to deletions or mutations of the SMN1 gene. SMN2, a nearly identical copy of SMN1 present in humans, cannot compensate for the loss of SMN1 because of predominant skipping of exon 7 during pre-mRNA splicing. With the recent US Food and Drug Administration approval of nusinersen (Spinraza), the potential for correction of SMN2 exon 7 splicing as an SMA therapy has been affirmed. Nusinersen is an antisense oligonucleotide that targets intronic splicing silencer N1 (ISS-N1) discovered in 2004 at the University of Massachusetts Medical School. ISS-N1 has emerged as the model target for testing the therapeutic efficacy of antisense oligonucleotides using different chemistries as well as different mouse models of SMA. Here, we provide a historical account of events that led to the discovery of ISS-N1 and describe the impact of independent validations that raised the profile of ISS-N1 as one of the most potent antisense targets for the treatment of a genetic disease. Recent approval of nusinersen provides a much-needed boost for antisense technology that is just beginning to realize its potential. Beyond treating SMA, the ISS-N1 target offers myriad potentials for perfecting various aspects of the nucleic-acid-based technology for the amelioration of the countless number of pathological conditions.

Securinine enhances SMN2 exon 7 inclusion in spinal muscular atrophy cells.

Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by the degeneration of motor neurons in the spinal cord, leading to muscular atrophy. SMA is caused by deletions or mutations in the survival motor neuron gene (SMN1) on chromosome 5q13. A second copy of the SMN gene (SMN2) also exists on chromosome 5, and both genes can produce functional protein. However, due to alternative splicing of the exon 7, the majority of SMN protein produced by SMN2 is truncated and unable to compensate for the loss of SMN1. Increasing full-length SMN protein production by promoting the exon 7 inclusion in SMN2 mRNA or increasing SMN2 gene transcription could be a therapeutic approach for SMA. In this study, we screened for the compounds that enhance SMN2 exon 7 inclusion by using SMN2 minigene-luciferase reporter system. We found that securinine can increase luciferase activity, indicating that securinine promoted SMN2 exon 7 inclusion. In addition, securinine increased full-length SMN2 mRNA and SMN protein expression in SMA patient-derived lymphoid cell lines. To investigate the mechanism of securinine effect on SMN2 splicing, we compared the protein levels of relevant splicing factors between securinine-treated and untreated cells. We found that securinine downregulated hnRNP A1 and Sam68 and upregulated Tra2-β1 expression. However, securinine, unlike HDAC inhibitors, did not enhance tra2-β1 gene transcription, indicating a post-transcriptional mechanism for Tra2-β1 upregulation. Furthermore, we treated SMA-like mice with securinine by i.p. injection and found that securinine treatment increased SMN2 exon 7 inclusion and SMN protein expression in the brain and spinal cord. According to our results, securinine might have the potential to become a therapeutic drug for SMA disease.

The Power of Human Protective Modifiers: PLS3 and CORO1C Unravel Impaired Endocytosis in Spinal Muscular Atrophy and Rescue SMA Phenotype

Homozygous loss of SMN1 causes spinal muscular atrophy (SMA), the most common and devastating childhood genetic motor-neuron disease. The copy gene SMN2 produces only ∼10% functional SMN protein, insufficient to counteract development of SMA. In contrast, the human genetic modifier plastin 3 (PLS3), an actin-binding and -bundling protein, fully protects against SMA in SMN1-deleted individuals carrying 3-4 SMN2 copies. Here, we demonstrate that the combinatorial effect of suboptimal SMN antisense oligonucleotide treatment and PLS3 overexpression-a situation resembling the human condition in asymptomatic SMN1-deleted individuals-rescues survival (from 14 to >250days) and motoric abilities in a severe SMA mouse model. Because PLS3 knockout in yeast impairs endocytosis, we hypothesized that disturbed endocytosis might be a key cellular mechanism underlying impaired neurotransmission and neuromuscular junction maintenance in SMA. Indeed, SMN deficit dramatically reduced endocytosis, which was restored to normal levels by PLS3overexpression. Upon low-frequency electro-stimulation, endocytotic FM1-43 (SynaptoGreen) uptake in the presynaptic terminal ofneuromuscular junctions was restored to control levels in SMA-PLS3 mice. Moreover, proteomics and biochemical analysis revealed CORO1C, another F-actin binding protein, whose direct binding to PLS3 is dependent on calcium. Similar to PLS3 overexpression, CORO1C overexpression restored fluid-phase endocytosis in SMN-knockdown cells by elevating F-actin amounts and rescued the axonal truncation and branching phenotype in Smn-depleted zebrafish. Our findings emphasize the power of genetic modifiers to unravel the cellular pathomechanisms underlying SMA and the power of combinatorial therapy based on splice correction of SMN2 and endocytosis improvement to efficiently treat SMA.

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.