Regulation of Survival Motor Neuron Gene Expression by Calcium Signaling

Spinal muscular atrophy (SMA) is caused by homozygous survival of motor neurons 1 (SMN1) gene deletion, leaving a duplicate gene, SMN2, as the sole source of SMN protein. However, a defect in SMN2 splicing, involving exon 7 skipping, results in a low level of functional SMN protein. Therefore, the upregulation of SMN protein expression from the SMN2 gene is generally considered to be one of the best therapeutic strategies to treat SMA. Most of the SMA drug discovery is based on synthetic compounds, and very few natural compounds have been explored thus far. Here, we performed an unbiased mechanism-independent and image-based screen of a library of microbial metabolites in SMA fibroblasts using an SMN-specific immunoassay. In doing so, we identified brefeldin A (BFA), a well-known inhibitor of ER-Golgi protein trafficking, as a strong inducer of SMN protein. The profound increase in SMN protein was attributed to, in part, the rescue of the SMN2 pre-mRNA splicing defect. Intriguingly, BFA increased the intracellular calcium concentration, and the BFA-induced exon 7 inclusion of SMN2 splicing, was abrogated by the depletion of intracellular calcium and by the pharmacological inhibition of calcium/calmodulin-dependent kinases (CaMKs). Moreover, BFA considerably reduced the expression of Tra2-β and SRSF9 proteins in SMA fibroblasts and enhanced the binding of PSF and hnRNP M to an exonic splicing enhancer (ESE) of exon 7. Together, our results demonstrate a significant role for calcium and its signaling on the regulation of SMN splicing, probably through modulating the expression/activity of splicing factors.

Download Full-text

Mitochondrial defects in the respiratory complex I contribute to impaired translational initiation via ROS and energy homeostasis in SMA motor neurons

Acta Neuropathologica Communications ◽

10.1186/s40478-020-01101-6 ◽

2020 ◽

Vol 8 (1) ◽

Author(s):

Maximilian Paul Thelen ◽

Brunhilde Wirth ◽

Min Jeong Kye

Keyword(s):

Protein Synthesis ◽

Motor Neuron ◽

Motor Neurons ◽

Complex I ◽

Energy Homeostasis ◽

Muscular Atrophy ◽

Survival Motor Neuron ◽

Translational Initiation ◽

Protein Levels ◽

Smn Protein

AbstractSpinal muscular atrophy (SMA) is a neuromuscular disease characterized by loss of lower motor neurons, which leads to proximal muscle weakness and atrophy. SMA is caused by reduced survival motor neuron (SMN) protein levels due to biallelic deletions or mutations in the SMN1 gene. When SMN levels fall under a certain threshold, a plethora of cellular pathways are disturbed, including RNA processing, protein synthesis, metabolic defects, and mitochondrial function. Dysfunctional mitochondria can harm cells by decreased ATP production and increased oxidative stress due to elevated cellular levels of reactive oxygen species (ROS). Since neurons mainly produce energy via mitochondrial oxidative phosphorylation, restoring metabolic/oxidative homeostasis might rescue SMA pathology. Here, we report, based on proteome analysis, that SMA motor neurons show disturbed energy homeostasis due to dysfunction of mitochondrial complex I. This results in a lower basal ATP concentration and higher ROS production that causes an increase of protein carbonylation and impaired protein synthesis in SMA motor neurons. Counteracting these cellular impairments with pyruvate reduces elevated ROS levels, increases ATP and SMN protein levels in SMA motor neurons. Furthermore, we found that pyruvate-mediated SMN protein synthesis is mTOR-dependent. Most importantly, we showed that ROS regulates protein synthesis at the translational initiation step, which is impaired in SMA. As many neuropathies share pathological phenotypes such as dysfunctional mitochondria, excessive ROS, and impaired protein synthesis, our findings suggest new molecular interactions among these pathways. Additionally, counteracting these impairments by reducing ROS and increasing ATP might be beneficial for motor neuron survival in SMA patients.

Download Full-text

Molecular Mechanisms of Neurodegeneration in Spinal Muscular Atrophy

Journal of Experimental Neuroscience ◽

10.4137/jen.s33122 ◽

2016 ◽

Vol 10 ◽

pp. JEN.S33122 ◽

Cited By ~ 36

Author(s):

Saif Ahmad ◽

Kanchan Bhatia ◽

Annapoorna Kannan ◽

Laxman Gangwani

Keyword(s):

Spinal Muscular Atrophy ◽

Motor Neuron ◽

Motor Neurons ◽

Molecular Mechanisms ◽

Muscular Atrophy ◽

Survival Motor Neuron ◽

Skeletal Muscle Atrophy ◽

Spinal Motor Neurons ◽

Low Levels ◽

Smn Protein

Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease with a high incidence and is the most common genetic cause of infant mortality. SMA is primarily characterized by degeneration of the spinal motor neurons that leads to skeletal muscle atrophy followed by symmetric limb paralysis, respiratory failure, and death. In humans, mutation of the Survival Motor Neuron 1 (SMN1) gene shifts the load of expression of SMN protein to the SMN2 gene that produces low levels of full-length SMN protein because of alternative splicing, which are sufficient for embryonic development and survival but result in SMA. The molecular mechanisms of the (a) regulation of SMN gene expression and (b) degeneration of motor neurons caused by low levels of SMN are unclear. However, some progress has been made in recent years that have provided new insights into understanding of the cellular and molecular basis of SMA pathogenesis. In this review, we have briefly summarized recent advances toward understanding of the molecular mechanisms of regulation of SMN levels and signaling mechanisms that mediate neurodegeneration in SMA.

Download Full-text

Deletion of Murine SMN Exon 7 Directed to Skeletal Muscle Leads to Severe Muscular Dystrophy

The Journal of Cell Biology ◽

10.1083/jcb.152.5.1107 ◽

2001 ◽

Vol 152 (5) ◽

pp. 1107-1114 ◽

Cited By ~ 150

Author(s):

Carmen Cifuentes-Diaz ◽

Tony Frugier ◽

Francesco D. Tiziano ◽

Emmanuelle Lacène ◽

Natacha Roblot ◽

...

Keyword(s):

Skeletal Muscle ◽

Motor Neuron ◽

Motor Neurons ◽

Muscular Atrophy ◽

Creatine Kinase Activity ◽

Survival Motor Neuron ◽

Mutant Mice ◽

Blue Dye ◽

Muscle Paralysis ◽

Survival Motor Neuron Gene

Spinal muscular atrophy (SMA) is characterized by degeneration of motor neurons of the spinal cord associated with muscle paralysis and caused by mutations of the survival motor neuron gene (SMN). To determine whether SMN gene defect in skeletal muscle might have a role in SMA pathogenesis, deletion of murine SMN exon 7, the most frequent mutation found in SMA, has been restricted to skeletal muscle by using the Cre-loxP system. Mutant mice display ongoing muscle necrosis with a dystrophic phenotype leading to muscle paralysis and death. The dystrophic phenotype is associated with elevated levels of creatine kinase activity, Evans blue dye uptake into muscle fibers, reduced amount of dystrophin and upregulation of utrophin expression suggesting a destabilization of the sarcolemma components. The mutant mice will be a valuable model for elucidating the underlying mechanism. Moreover, our results suggest a primary involvement of skeletal muscle in human SMA, which may contribute to motor defect in addition to muscle denervation caused by the motor neuron degeneration. These data may have important implications for the development of therapeutic strategies in SMA.

Download Full-text

Circulating microRNAs as potential biomarkers and therapeutic targets in spinal muscular atrophy

Therapeutic Advances in Neurological Disorders ◽

10.1177/1756286420979954 ◽

2020 ◽

Vol 13 ◽

pp. 175628642097995

Author(s):

Tai-Heng Chen

Keyword(s):

Spinal Muscular Atrophy ◽

Motor Neurons ◽

Molecular Mechanisms ◽

Muscular Atrophy ◽

Therapeutic Targets ◽

Homozygous Deletion ◽

Survival Motor Neuron ◽

Novel Mirna ◽

Potential Applications ◽

Smn Protein

Spinal muscular atrophy (SMA), a leading genetic cause of infant death, is a neurodegenerative disease characterized by the selective loss of particular groups of motor neurons (MNs) in the anterior horn of the spinal cord with progressive muscle wasting. SMA is caused by a deficiency of the survival motor neuron (SMN) protein due to a homozygous deletion or mutation of the SMN1 gene. However, the molecular mechanisms whereby the SMN complex regulates MN functions are not fully elucidated. Emerging studies on SMA pathogenesis have turned the attention of researchers to RNA metabolism, given that increasingly identified SMN-associated modifiers are involved in both coding and non-coding RNA (ncRNA) processing. Among various ncRNAs, microRNAs (miRNAs) are the most studied in terms of regulation of posttranscriptional gene expression. Recently, the discovery that miRNAs are critical to MN function and survival led to the study of dysregulated miRNAs in SMA pathogenesis. Circulating miRNAs have drawn attention as a readily available biomarker due to their property of being clinically detectable in numerous human biofluids through non-invasive approaches. As there are recent promising findings from novel miRNA-based medicines, this article presents an extensive review of the most up-to-date studies connecting specific miRNAs to SMA pathogenesis and the potential applications of miRNAs as biomarkers and therapeutic targets for SMA.

Download Full-text

Utilizing gene therapy methods to probe the genetic requirements to prevent spinal muscular atrophy.

10.32469/10355/57243 ◽

2016 ◽

Author(s):

◽

Madeline R. Miller

Keyword(s):

Neuromuscular Junction ◽

Spinal Muscular Atrophy ◽

Motor Neuron ◽

Motor Neurons ◽

Muscular Atrophy ◽

Survival Motor Neuron ◽

Downstream Effects ◽

Smn Protein ◽

Progressive Weakness

Spinal Muscular Atrophy is clinically recognized as a progressive weakness within the trunk and proximal limbs that will lead to breathing failure and death within infants. As a neurodegenerative genetic disease, SMA is caused by loss of motor neurons, which in turn is caused by low levels of the Survival Motor Neuron (SMN) protein. The mechanism by which a ubiquitously expressed protein such as SMN is able to cause the specific death of motor neurons is highly debated and of great interest. Work presented here focuses on understanding the biological requirements of SMN and its downstream effects on the neuromuscular junction. To this end we utilize viral based gene delivery as a powerful tool to assess the effects of genes of interest in vivo. Our findings contribute to the conversation regarding whether SMA is truly a "motor neuron" disease, suggesting that astrocytes play a meaningful role in staving off SMA. Further, we investigate the domains within SMN needed to maintain its function in a mammalian system. We take a novel and challenging approach to identify a minimal domain capable of maintaining function. Finally, we demonstrate the practical use of morophological analysis of the neuromuscular junction as a means to characterize SMA pathology.

Download Full-text

Assessment of motor neuron pathology and potential compensatory mechanisms in phenotypically normal mice with reduced Survival Motor Neuron levels.

Journal of Student Science and Technology ◽

10.13034/jsst.v8i1.42 ◽

2015 ◽

Vol 8 (1) ◽

Author(s):

Rebecca Xu Xu ◽

Lyndsay M. Murray M. Murray Murray ◽

Yves De Repentigny De Repentigny ◽

Rashmi Kothary Kothary

Keyword(s):

Motor Neuron ◽

Mouse Models ◽

Motor Neurons ◽

Neuromuscular Junctions ◽

Muscular Atrophy ◽

Neuromuscular Disorder ◽

Survival Motor Neuron ◽

Compensatory Mechanisms ◽

Protein Levels ◽

Smn Protein

Spinal muscular atrophy (SMA) is a destructive pediatric neuromuscular disorder caused by low survival motor neuron (Smn) protein levels due to mutations and deletions within the survival motor neuron 1 (SMN1) gene. Motor neurons are the main pathological targets, and along with neuromuscular junctions (NMJs), they play an early significant role in the pathogenesis of SMA. Previous studies demonstrate that a pathological reduction in Smn levels can lead to significant remodeling defects in both the outgrowth of axonal sprouts and in the nerve-directed clustering of AChRs in mouse models. However, whether this pathological reduction in Smn leads to ubclinical features has not been investigated. Here, we have employed the Smn2B/2B and Smn+/- mouse models to study whether similar SMA pathology is present sub-clinically, and if so whether there is any compensation present. We show a decrease in the motor neuron number in the mouse models, no change in myelin thickness and modest NMJ pathology in both mouse models. Additionally, compensation through the expansion of the motor unit size is suggested.L’amyotrophie spinale (AMS) est un trouble neuromusculaire pédiatrique destructif causé par le niveau bas de protéine du neurone de moteur de survie (NMS) en raison des mutations et des effacements dans le neurone de moteur de survie 1 gène (NMS1). Des neurones du moteur sont les cibles pathologiques principales, et ce, avec des jonctions neuromusculaires (JNMs), ils jouent, en avance, un rôle significatif dans la pathogénie de AMS. Des études précédentes démontrent qu’une réduction pathologique de niveaux de NMS peut mener aux défauts importants de réorganisation tant dans l’excroissance axonale que dans l’agrégation du récepteur de l’acétylcholine (AChR) sous la terminaison nerveuse dans des modèles de souris. Cependant, si cette reduction pathologique de NMS mène aux caractéristiques infracliniques n’a pas été à l’étude. Ici, nous avons employé le NMS2B/2B et NMS +/- des modèles de souris afin de déterminer si une pathologie semblable à l’AMS est présente infracliniquement, ainsi s’il y a présence de quelconque compensation. Nous montrons une diminution dans le nombre des neurones du moteur dans les modèles de souris, aucun changement de l’épaisseur du myelin et une pathologie modeste de JNM dans les deux modèles de souris. De plus, une compensation par l’expansion de la taille d’unité du moteur est suggérée.

Download Full-text

Self-oligomerization regulates stability of Survival Motor Neuron (SMN) protein isoforms by sequestering an SCFSlmb degron

10.1101/078337 ◽

2016 ◽

Author(s):

Kelsey M. Gray ◽

Kevin A. Kaifer ◽

David Baillat ◽

Ying Wen ◽

Thomas R. Bonacci ◽

...

Keyword(s):

Motor Neuron ◽

Muscular Atrophy ◽

Exon Skipping ◽

Duplicate Gene ◽

Survival Motor Neuron ◽

Protein Isoforms ◽

Missense Mutations ◽

Label Free ◽

Ubiquitin E3 Ligase ◽

Smn Protein

AbstractSpinal muscular atrophy (SMA) is caused by homozygous mutations in human SMN1. Expression of a duplicate gene (SMN2) primarily results in skipping of exon 7 and production of an unstable protein isoform, SMNΔ7. Although SMN2 exon skipping is the principal contributor to SMA severity, mechanisms governing stability of SMN isoforms are poorly understood. We used a Drosophila model system and label-free proteomics to identify the SCFSlmb ubiquitin E3 ligase complex as a novel SMN binding partner. SCFSlmb interacts with a phospho-degron embedded within the human and fruitfly SMN YG-box oligomerization domains. Substitution of a conserved serine (S270A) interferes with SCFSlmb binding and stabilizes SMNΔ7. SMA-causing missense mutations that block multimerization of full-length SMN are also stabilized in the degron mutant background. Overexpression of SMNΔ7S270A, but not wild-type SMNΔ7, provides a protective effect in SMA model mice and human motor neuron cell culture systems. Our findings support a model wherein the degron is exposed when SMN is monomeric, and sequestered when SMN forms higher-order multimers.

Download Full-text

AAV9-Stathmin1 gene delivery improves disease phenotype in an intermediate mouse model of spinal muscular atrophy

Human Molecular Genetics ◽

10.1093/hmg/ddz188 ◽

2019 ◽

Vol 28 (22) ◽

pp. 3742-3754 ◽

Cited By ~ 7

Author(s):

E Villalón ◽

R A Kline ◽

C E Smith ◽

Z C Lorson ◽

E Y Osman ◽

...

Keyword(s):

Mouse Model ◽

Spinal Muscular Atrophy ◽

Motor Neuron ◽

Motor Neurons ◽

Muscular Atrophy ◽

Genetic Disorder ◽

Premature Death ◽

Survival Motor Neuron ◽

Motor Neuron Diseases ◽

Smn Protein

Abstract Spinal muscular atrophy (SMA) is a devastating infantile genetic disorder caused by the loss of survival motor neuron (SMN) protein that leads to premature death due to loss of motor neurons and muscle atrophy. The approval of an antisense oligonucleotide therapy for SMA was an important milestone in SMA research; however, effective next-generation therapeutics will likely require combinatorial SMN-dependent therapeutics and SMN-independent disease modifiers. A recent cross-disease transcriptomic analysis identified Stathmin-1 (STMN1), a tubulin-depolymerizing protein, as a potential disease modifier across different motor neuron diseases, including SMA. Here, we investigated whether viral-based delivery of STMN1 decreased disease severity in a well-characterized SMA mouse model. Intracerebroventricular delivery of scAAV9-STMN1 in SMA mice at P2 significantly increased survival and weight gain compared to untreated SMA mice without elevating Smn levels. scAAV9-STMN1 improved important hallmarks of disease, including motor function, NMJ pathology and motor neuron cell preservation. Furthermore, scAAV9-STMN1 treatment restored microtubule networks and tubulin expression without affecting tubulin stability. Our results show that scAAV9-STMN1 treatment improves SMA pathology possibly by increasing microtubule turnover leading to restored levels of stable microtubules. Overall, these data demonstrate that STMN1 can significantly reduce the SMA phenotype independent of restoring SMN protein and highlight the importance of developing SMN-independent therapeutics for the treatment of SMA.

Download Full-text

Genomic Variability in the Survival Motor Neuron Genes (SMN1 and SMN2): Implications for Spinal Muscular Atrophy Phenotype and Therapeutics Development

International Journal of Molecular Sciences ◽

10.3390/ijms22157896 ◽

2021 ◽

Vol 22 (15) ◽

pp. 7896

Author(s):

Matthew E. R. Butchbach

Keyword(s):

Spinal Muscular Atrophy ◽

Motor Neuron ◽

Motor Neurons ◽

Muscular Atrophy ◽

Survival Motor Neuron ◽

Smn1 Gene ◽

Spinal Motor Neurons ◽

Copy Numbers ◽

Smn Protein ◽

High Degree

Spinal muscular atrophy (SMA) is a leading genetic cause of infant death worldwide that is characterized by loss of spinal motor neurons leading to muscle weakness and atrophy. SMA results from the loss of survival motor neuron 1 (SMN1) gene but retention of its paralog SMN2. The copy numbers of SMN1 and SMN2 are variable within the human population with SMN2 copy number inversely correlating with SMA severity. Current therapeutic options for SMA focus on increasing SMN2 expression and alternative splicing so as to increase the amount of SMN protein. Recent work has demonstrated that not all SMN2, or SMN1, genes are equivalent and there is a high degree of genomic heterogeneity with respect to the SMN genes. Because SMA is now an actionable disease with SMN2 being the primary target, it is imperative to have a comprehensive understanding of this genomic heterogeneity with respect to hybrid SMN1–SMN2 genes generated by gene conversion events as well as partial deletions of the SMN genes. This review will describe this genetic heterogeneity in SMA and its impact on disease phenotype as well as therapeutic efficacy.

Download Full-text

Drug screening with human SMN2 reporter identifies SMN protein stabilizers to correct SMA pathology

Life Science Alliance ◽

10.26508/lsa.201800268 ◽

2019 ◽

Vol 2 (2) ◽

pp. e201800268 ◽

Cited By ~ 4

Author(s):

Yiran Wang ◽

Chongchong Xu ◽

Lin Ma ◽

Yongchao Mou ◽

Bowen Zhang ◽

...

Keyword(s):

Protease Inhibitor ◽

Motor Neurons ◽

Cysteine Protease ◽

Muscular Atrophy ◽

Survival Motor Neuron ◽

Cysteine Protease Inhibitor ◽

Therapeutic Effects ◽

Protective Effects ◽

Pass Through ◽

Smn Protein

Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, is caused by reduced levels of functional survival motor neuron (SMN) protein. To identify therapeutic agents for SMA, we established a versatile SMN2-GFP reporter line by targeting the human SMN2 gene. We then screened a compound library and identified Z-FA-FMK as a potent candidate. Z-FA-FMK, a cysteine protease inhibitor, increased functional SMN through inhibiting the protease-mediated degradation of both full-length and exon 7–deleted forms of SMN. Further studies reveal that CAPN1, CAPN7, CTSB, and CTSL mediate the degradation of SMN proteins, providing novel targets for SMA. Notably, Z-FA-FMK mitigated mitochondriopathy and neuropathy in SMA patient–derived motor neurons and showed protective effects in SMA animal model after intracerebroventricular injection. E64d, another cysteine protease inhibitor which can pass through the blood–brain barrier, showed even more potent therapeutic effects after subcutaneous delivery to SMA mice. Taken together, we have successfully established a human SMN2 reporter for future drug discovery and identified the potential therapeutic value of cysteine protease inhibitors in treating SMA via stabilizing SMN proteins.

Download Full-text