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1.
Medicina (B Aires) ; 79(Spec 6/1): 582-586, 2019.
Artigo em Espanhol | MEDLINE | ID: mdl-31864230

RESUMO

Alternative splicing of the messenger RNA plays a fundamental role in the flow of genetic information from DNA to proteins by expanding the coding capacity of the genome. The regulation of alternative splicing is as important as the regulation of transcription to determine the specific characteristics of cells and tissues, the normal functioning of cells and the responses of eukaryotic cells to external signals. Basic knowledge of the pre-mRNA sequences and splicing factors that recognize them has allowed scientists to design a therapeutic synthetic oligonucleotide for spinal muscular atrophy. This is an autosomal recessive inherited disease in which the SMN1 gene is mutated and affects one in 10,000 births. By blocking the binding of a negative splicing factor to the mRNA of a paralogue of the SMN1 gene, called SMN2, the Spinraza oligonucleotide corrects an abnormal alternative splicing event of the SMN2 gene and allows the synthesis of high levels of the SMN protein, constituting the first successful case of cure of a neurodegenerative disease.


Assuntos
Processamento Alternativo/genética , Atrofia Muscular Espinal/terapia , Processamento de RNA/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Transcrição Genética/genética , Humanos , Atrofia Muscular Espinal/genética , RNA Mensageiro/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo
2.
Gene Ther ; 26(7-8): 287-295, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31243392

RESUMO

Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, is characterized by the deterioration of alpha motor neurons in the brainstem and spinal cord. Currently, there is no cure for SMA, which calls for an urgent need to explore affordable and effective therapies and to maximize patients' independence and quality of life. Adeno-associated virus (AAV) vector, one of the most promising and well-investigated vehicles for delivering transgenes, is a compelling candidate for gene therapy. Some of the hallmarks of AAVs are their nonpathogenicity, inability to incur an immune response, potential to achieve robust transgene expression, and varied tropism for several tissues of the body. Recently, these features were harnessed in a clinical trial conducted by AveXis in SMA patients, where AAV9 was employed as a vehicle for one-time administration of the SMN gene, the causative gene in SMA. The trial demonstrated remarkable improvements in motor milestones and rates of survival in the patients. This review focuses on the advent of SMA gene therapy and summarizes different preclinical studies that were conducted leading up to the AAV9-SMA trial in SMA patients.


Assuntos
Dependovirus/genética , Terapia Genética/métodos , Atrofia Muscular Espinal/terapia , Animais , Dependovirus/metabolismo , Terapia Genética/efeitos adversos , Humanos , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo
3.
Nucleic Acids Res ; 47(14): 7618-7632, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31127278

RESUMO

Spinal Muscular Atrophy results from loss-of-function mutations in SMN1 but correcting aberrant splicing of SMN2 offers hope of a cure. However, current splice therapy requires repeated infusions and is expensive. We previously rescued SMA mice by promoting the inclusion of a defective exon in SMN2 with germline expression of Exon-Specific U1 snRNAs (ExspeU1). Here we tested viral delivery of SMN2 ExspeU1s encoded by adeno-associated virus AAV9. Strikingly the virus increased SMN2 exon 7 inclusion and SMN protein levels and rescued the phenotype of mild and severe SMA mice. In the severe mouse, the treatment improved the neuromuscular function and increased the life span from 10 to 219 days. ExspeU1 expression persisted for 1 month and was effective at around one five-hundredth of the concentration of the endogenous U1snRNA. RNA-seq analysis revealed our potential drug rescues aberrant SMA expression and splicing profiles, which are mostly related to DNA damage, cell-cycle control and acute phase response. Vastly overexpressing ExspeU1 more than 100-fold above the therapeutic level in human cells did not significantly alter global gene expression or splicing. These results indicate that AAV-mediated delivery of a modified U1snRNP particle may be a novel therapeutic option against SMA.


Assuntos
Terapia Genética/métodos , Atrofia Muscular Espinal/terapia , Distrofia Muscular Animal/terapia , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Animais , Dependovirus/genética , Modelos Animais de Doenças , Éxons/genética , Células HEK293 , Humanos , Camundongos Knockout , Atrofia Muscular Espinal/genética , Distrofia Muscular Animal/genética , Mutação , Processamento de RNA , Ribonucleoproteína Nuclear Pequena U1/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo
4.
Mol Neurobiol ; 56(6): 4414-4427, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30327977

RESUMO

Spinal muscular atrophy (SMA), a leading genetic cause of infant death, is caused by the loss of survival motor neuron 1 (SMN1) gene. SMA is characterized by the degeneration and loss of spinal cord motoneurons (MNs), muscular atrophy, and weakness. SMN2 is the centromeric duplication of the SMN gene, whose numbers of copies determine the intracellular levels of SMN protein and define the disease onset and severity. It has been demonstrated that elevating SMN levels can be an important strategy in treating SMA and can be achieved by several mechanisms, including promotion of protein stability. SMN protein is a direct target of the calcium-dependent protease calpain and induces its proteolytic cleavage in muscle cells. In this study, we examined the involvement of calpain in SMN regulation on MNs. In vitro experiments showed that calpain activation induces SMN cleavage in CD1 and SMA mouse spinal cord MNs. Additionally, calpain 1 knockdown or inhibition increased SMN level and prevent neurite degeneration in these cells. We examined the effects of calpain inhibition on the phenotype of two severe SMA mouse models. Treatment with the calpain inhibitor, calpeptin, significantly improved the lifespan and motor function of these mice. Our observations show that calpain regulates SMN level in MNs and calpeptin administration improves SMA phenotype demonstrating the potential utility of calpain inhibitors in SMA therapy.


Assuntos
Calpaína/antagonistas & inibidores , Neurônios Motores/patologia , Atrofia Muscular Espinal/patologia , Medula Espinal/patologia , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Animais , Calpaína/metabolismo , Células Cultivadas , Dipeptídeos/administração & dosagem , Dipeptídeos/farmacologia , Técnicas de Silenciamento de Genes , Glicoproteínas/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/complicações , Atrofia Muscular Espinal/fisiopatologia , Mutação/genética , Degeneração Neural/complicações , Degeneração Neural/patologia , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Fenótipo , Potássio/farmacologia
5.
Pharmacol Res Perspect ; 6(6): e00447, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30519476

RESUMO

Spinal muscular atrophy (SMA) is a rare, inherited neuromuscular disease caused by deletion and/or mutation of the Survival of Motor Neuron 1 (SMN1) gene. A second gene, SMN2, produces low levels of functional SMN protein that are insufficient to fully compensate for the lack of SMN1. Risdiplam (RG7916; RO7034067) is an orally administered, small-molecule SMN2 pre-mRNA splicing modifier that distributes into the central nervous system (CNS) and peripheral tissues. To further explore risdiplam distribution, we assessed in vitro characteristics and in vivo drug levels and effect of risdiplam on SMN protein expression in different tissues in animal models. Total drug levels were similar in plasma, muscle, and brain of mice (n = 90), rats (n = 148), and monkeys (n = 24). As expected mechanistically based on its high passive permeability and not being a human multidrug resistance protein 1 substrate, risdiplam CSF levels reflected free compound concentration in plasma in monkeys. Tissue distribution remained unchanged when monkeys received risdiplam once daily for 39 weeks. A parallel dose-dependent increase in SMN protein levels was seen in CNS and peripheral tissues in two SMA mouse models dosed with risdiplam. These in vitro and in vivo preclinical data strongly suggest that functional SMN protein increases seen in patients' blood following risdiplam treatment should reflect similar increases in functional SMN protein in the CNS, muscle, and other peripheral tissues.


Assuntos
Compostos Azo/farmacocinética , Atrofia Muscular Espinal/tratamento farmacológico , Fármacos Neuromusculares/farmacocinética , Pirimidinas/farmacocinética , Processamento de RNA/efeitos dos fármacos , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo , Animais , Compostos Azo/líquido cefalorraquidiano , Compostos Azo/farmacologia , Compostos Azo/uso terapêutico , Encéfalo/metabolismo , Encéfalo/patologia , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Cães , Avaliação Pré-Clínica de Medicamentos , Éxons/efeitos dos fármacos , Éxons/genética , Feminino , Humanos , Macaca fascicularis , Células Madin Darby de Rim Canino , Masculino , Camundongos , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patologia , Fármacos Neuromusculares/líquido cefalorraquidiano , Fármacos Neuromusculares/farmacologia , Fármacos Neuromusculares/uso terapêutico , Pirimidinas/líquido cefalorraquidiano , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Ratos , Ratos Wistar , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Suínos , Distribuição Tecidual
6.
J Med Chem ; 61(24): 11021-11036, 2018 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-30407821

RESUMO

Spinal muscular atrophy (SMA), a rare neuromuscular disorder, is the leading genetic cause of death in infants and toddlers. SMA is caused by the deletion or a loss of function mutation of the survival motor neuron 1 (SMN1) gene. In humans, a second closely related gene SMN2 exists; however it codes for a less stable SMN protein. In recent years, significant progress has been made toward disease modifying treatments for SMA by modulating SMN2 pre-mRNA splicing. Herein, we describe the discovery of LMI070/branaplam, a small molecule that stabilizes the interaction between the spliceosome and SMN2 pre-mRNA. Branaplam (1) originated from a high-throughput phenotypic screening hit, pyridazine 2, and evolved via multiparameter lead optimization. In a severe mouse SMA model, branaplam treatment increased full-length SMN RNA and protein levels, and extended survival. Currently, branaplam is in clinical studies for SMA.


Assuntos
Encéfalo/efeitos dos fármacos , Canal de Potássio ERG1/metabolismo , Atrofia Muscular Espinal/tratamento farmacológico , Piridazinas/química , Administração Oral , Animais , Encéfalo/metabolismo , Linhagem Celular , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Canal de Potássio ERG1/antagonistas & inibidores , Humanos , Camundongos Endogâmicos C57BL , Neurônios Motores/efeitos dos fármacos , Atrofia Muscular Espinal/genética , Piridazinas/farmacologia , Relação Quantitativa Estrutura-Atividade , Processamento de RNA , Ratos Sprague-Dawley , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/genética
7.
Cell Death Dis ; 9(11): 1100, 2018 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-30368521

RESUMO

Spinal Muscular Atrophy (SMA) is caused by genetic mutations in the SMN1 gene, resulting in drastically reduced levels of Survival of Motor Neuron (SMN) protein. Although SMN is ubiquitously expressed, spinal motor neurons are one of the most affected cell types. Previous studies have identified pathways uniquely activated in SMA motor neurons, including a hyperactivated ER stress pathway, neuronal hyperexcitability, and defective spliceosomes. To investigate why motor neurons are more affected than other neural types, we developed a spinal organoid model of SMA. We demonstrate overt motor neuron degeneration in SMA spinal organoids, and this degeneration can be prevented using a small molecule inhibitor of CDK4/6, indicating that spinal organoids are an ideal platform for therapeutic discovery.


Assuntos
Quinase 4 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/genética , Neurônios Motores/efeitos dos fármacos , Organoides/efeitos dos fármacos , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Diferenciação Celular , Linhagem Celular , Colágeno/química , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/metabolismo , Combinação de Medicamentos , Corpos Embrioides/efeitos dos fármacos , Corpos Embrioides/metabolismo , Corpos Embrioides/patologia , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Laminina/química , Modelos Biológicos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patologia , Organoides/metabolismo , Organoides/patologia , Cultura Primária de Células , Proteoglicanas/química , Transdução de Sinais , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo
8.
PLoS One ; 13(10): e0205589, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30304024

RESUMO

Spinal muscular atrophy (SMA) is a severe genetic disorder that manifests in progressive neuromuscular degeneration. SMA originates from loss-of-function mutations of the SMN1 (Survival of Motor Neuron 1) gene. Recent evidence has implicated peripheral deficits, especially in skeletal muscle, as key contributors to disease progression in SMA. In this study we generated myogenic cells from two SMA-affected human embryonic stem cell (hESC) lines with deletion of SMN1 bearing two copies of the SMN2 gene and recapitulating the molecular phenotype of Type 1 SMA. We characterized myoblasts and myotubes by comparing them to two unaffected, control hESC lines and demonstrate that SMA myoblasts and myotubes showed altered expression of various myogenic markers, which translated into an impaired in vitro myogenic maturation and development process. Additionally, we provide evidence that these SMN1 deficient cells display functional deficits in cholinergic calcium signaling response, glycolysis and oxidative phosphorylation. Our data describe a novel human myogenic SMA model that might be used for interrogating the effect of SMN depletion during skeletal muscle development, and as model to investigate biological mechanisms targeting myogenic differentiation, mitochondrial respiration and calcium signaling processes in SMA muscle cells.


Assuntos
Células-Tronco Embrionárias Humanas/metabolismo , Desenvolvimento Muscular/fisiologia , Fibras Musculares Esqueléticas/metabolismo , Atrofia Muscular Espinal/metabolismo , Mioblastos/metabolismo , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Linhagem Celular , Expressão Gênica , Células-Tronco Embrionárias Humanas/patologia , Humanos , Fibras Musculares Esqueléticas/patologia , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patologia , Mioblastos/patologia , Receptores Colinérgicos/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo
9.
Nucleic Acids Res ; 46(20): 10983-11001, 2018 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-30165668

RESUMO

The Survival Motor Neuron (SMN) protein is essential for survival of all animal cells. SMN harbors a nucleic acid-binding domain and plays an important role in RNA metabolism. However, the RNA-binding property of SMN is poorly understood. Here we employ iterative in vitro selection and chemical structure probing to identify sequence and structural motif(s) critical for RNA-SMN interactions. Our results reveal that motifs that drive RNA-SMN interactions are diverse and suggest that tight RNA-SMN interaction requires presence of multiple contact sites on the RNA molecule. We performed UV crosslinking and immunoprecipitation coupled with high-throughput sequencing (HITS-CLIP) to identify cellular RNA targets of SMN in neuronal SH-SY5Y cells. Results of HITS-CLIP identified a wide variety of targets, including mRNAs coding for ribosome biogenesis and cytoskeleton dynamics. We show critical determinants of ANXA2 mRNA for a direct SMN interaction in vitro. Our data confirms the ability of SMN to discriminate among close RNA sequences, and represent the first validation of a direct interaction of SMN with a cellular RNA target. Our findings suggest direct RNA-SMN interaction as a novel mechanism to initiate the cascade of events leading to the execution of SMN-specific functions.


Assuntos
Motivos de Nucleotídeos , Domínios Proteicos , RNA/química , Proteína 1 de Sobrevivência do Neurônio Motor/química , Animais , Sequência de Bases , Sítios de Ligação/genética , Ligação Competitiva , Linhagem Celular Tumoral , Humanos , Neurônios/metabolismo , Ligação Proteica , RNA/genética , RNA/metabolismo , Homologia de Sequência do Ácido Nucleico , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/química , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo
10.
J Clin Invest ; 128(8): 3219-3227, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29985170

RESUMO

The motor neuron disease spinal muscular atrophy (SMA) is caused by recessive, loss-of-function mutations of the survival motor neuron 1 gene (SMN1). Alone, such mutations are embryonically lethal, but SMA patients retain a paralog gene, SMN2, that undergoes alternative pre-mRNA splicing, producing low levels of SMN protein. By mechanisms that are not well understood, reduced expression of the ubiquitously expressed SMN protein causes an early-onset motor neuron disease that often results in infantile or childhood mortality. Recently, striking clinical improvements have resulted from two novel treatment strategies to increase SMN protein by (a) modulating the splicing of existing SMN2 pre-mRNAs using antisense oligonucleotides, and (b) transducing motor neurons with self-complementary adeno-associated virus 9 (scAAV9) expressing exogenous SMN1 cDNA. We review the recently published clinical trial results and discuss the differing administration, tissue targeting, and potential toxicities of these two therapies. We also focus on the challenges that remain, emphasizing the many clinical and biologic questions that remain open. Answers to these questions will enable further optimization of these remarkable SMA treatments as well as provide insights that may well be useful in application of these therapeutic platforms to other diseases.


Assuntos
Marcação de Genes/métodos , Atrofia Muscular Espinal , Oligonucleotídeos Antissenso/uso terapêutico , Proteína 1 de Sobrevivência do Neurônio Motor , Dependovirus , Humanos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/terapia , Oligonucleotídeos Antissenso/genética , Processamento de RNA/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo
11.
Nat Commun ; 9(1): 2794, 2018 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-30022074

RESUMO

Mutations in proteins like FUS which cause Amyotrophic Lateral Sclerosis (ALS) result in the aberrant formation of stress granules while ALS-linked mutations in other proteins impede elimination of stress granules. Repeat expansions in C9ORF72, the major cause of ALS, reduce C9ORF72 levels but how this impacts stress granules is uncertain. Here, we demonstrate that C9ORF72 associates with the autophagy receptor p62 and controls elimination of stress granules by autophagy. This requires p62 to associate via the Tudor protein SMN with proteins, including FUS, that are symmetrically methylated on arginines. Mice lacking p62 accumulate arginine-methylated proteins and alterations in FUS-dependent splicing. Patients with C9ORF72 repeat expansions accumulate symmetric arginine dimethylated proteins which co-localize with p62. This suggests that C9ORF72 initiates a cascade of ALS-linked proteins (C9ORF72, p62, SMN, FUS) to recognize stress granules for degradation by autophagy and hallmarks of a defect in this process are observable in ALS patients.


Assuntos
Esclerose Amiotrófica Lateral/genética , Autofagia/genética , Proteína C9orf72/genética , Proteína FUS de Ligação a RNA/genética , Proteína Sequestossoma-1/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Arginina/metabolismo , Proteína C9orf72/metabolismo , Linhagem Celular Tumoral , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/patologia , Embrião de Mamíferos , Células HeLa , Humanos , Metilação , Camundongos , Camundongos Knockout , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Cultura Primária de Células , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Proteína Sequestossoma-1/metabolismo , Estresse Fisiológico , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo
12.
Neurology ; 91(7): e620-e624, 2018 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-30006410

RESUMO

OBJECTIVE: To report our experience delivering intrathecal nusinersen through cervical puncture in patients with spinal muscular atrophy (SMA) with no lumbar access. BACKGROUND: SMA is a neuromuscular disorder characterized by profound muscle weakness, atrophy, and paralysis due to degeneration of the anterior horn cells. Nusinersen, the first Food and Drug Administration-approved treatment for SMA, is administered intrathecally via lumbar puncture; however, many patients with SMA have scoliosis or solid spinal fusion with hardware that makes lumbar access impossible. Studies in primates have demonstrated better spinal cord tissue concentration with intrathecal injections than with intracerebral ventricular injections. Therefore we have used C1/C2 puncture as an alternative to administer nusinersen. METHOD: Retrospective chart review. RESULTS: Intrathecal nusinersen via cervical puncture was given to 3 patients who had thoracic and lumbosacral spinal fusion: a 12-year-old girl with type 1 SMA and 2 17-year-old girls with type 2 SMA. Cervical puncture was performed without deep sedation under fluoroscopic guidance using a 25-G or a 24-G Whitacre needle in the posterior aspect of C1-C2 interspace and full dose of nusinersen (12 mg/5 mL) was injected after visualizing free CSF flow. Patients completed their 4 loading doses and first maintenance dose of nusinersen, and 15 procedures were successful and well-tolerated. CONCLUSION: Cervical puncture is a feasible alternative delivery route to administer intrathecal nusinersen in patients with longstanding SMA and spine anatomy precluding lumbar access when done by providers with expertise in this procedure.


Assuntos
Atrofia Muscular Espinal/tratamento farmacológico , Oligonucleotídeos/uso terapêutico , Punção Espinal/métodos , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Adolescente , Criança , Feminino , Humanos , Região Lombossacral , Masculino , Atrofia Muscular Espinal/diagnóstico por imagem , RNA Mensageiro/metabolismo , Estudos Retrospectivos , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Resultado do Tratamento , Raios X
13.
Adv Neurobiol ; 20: 31-61, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29916015

RESUMO

Spinal muscular atrophy (SMA) is one of the major genetic disorders associated with infant mortality. More than 90% cases of SMA result from deletions or mutations of Survival Motor Neuron 1 (SMN1) gene. SMN2, a nearly identical copy of SMN1, does not compensate for the loss of SMN1 due to predominant skipping of exon 7. However, correction of SMN2 exon 7 splicing has proven to confer therapeutic benefits in SMA patients. The only approved drug for SMA is an antisense oligonucleotide (Spinraza™/Nusinersen), which corrects SMN2 exon 7 splicing by blocking intronic splicing silencer N1 (ISS-N1) located immediately downstream of exon 7. ISS-N1 is a complex regulatory element encompassing overlapping negative motifs and sequestering a cryptic splice site. More than 40 protein factors have been implicated in the regulation of SMN exon 7 splicing. There is evidence to support that multiple exons of SMN are alternatively spliced during oxidative stress, which is associated with a growing number of pathological conditions. Here, we provide the most up to date account of the mechanism of splicing regulation of the SMN genes.


Assuntos
Éxons , Atrofia Muscular Espinal/genética , Processamento de RNA , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Humanos , Atrofia Muscular Espinal/metabolismo , Sítios de Splice de RNA , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo
14.
Adv Neurobiol ; 20: 143-171, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29916019

RESUMO

Spinal muscular atrophy (SMA) is a motor neuron disease caused by mutations/deletions within the survival of motor neuron 1 (SMN1) gene that lead to a pathological reduction of SMN protein levels. SMN is part of a multiprotein complex, functioning as a molecular chaperone that facilitates the assembly of spliceosomal small nuclear ribonucleoproteins (snRNP). In addition to its role in spliceosome formation, SMN has also been found to interact with mRNA-binding proteins (mRBPs), and facilitate their assembly into mRNP transport granules. The association of protein and RNA in RNP complexes plays an important role in an extensive and diverse set of cellular processes that regulate neuronal growth, differentiation, and the maturation and plasticity of synapses. This review discusses the role of SMN in RNP assembly and localization, focusing on molecular defects that affect mRNA processing and may contribute to SMA pathology.


Assuntos
Atrofia Muscular Espinal/metabolismo , Ribonucleoproteínas/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo
15.
J Mol Neurosci ; 65(2): 196-202, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29799103

RESUMO

Spinal muscular atrophy (SMA) is an autosomal recessive genetic disorder caused by survival motor neuron (SMN) protein deficiency leading the loss of motor neurons in the anterior horns of the spinal cord and brainstem. More than 95% of SMA patients are attributed to the homozygous deletion of survival motor neuron 1 (SMN1) gene, and approximately 5% are caused by compound heterozygous with a SMN1 deletion and a subtle mutation. Here, we identified a rare variant c.835-5T>G in intron 6 of SMN1 in a patient affected with type I SMA. We analyzed the functional consequences of this mutation on mRNA splicing in vitro. After transfecting pCI-SMN1, pCI-SMN2, and pCI-SMN1 c.835-5T>G minigenes into HEK293, Neuro-2a, and SHSY5Y cells, reverse transcription polymerase chain reaction (RT-PCR) was performed to compare the splicing effects of these minigenes. Finally, we found that this mutation resulted in the skipping of exon 7 in SMN1, which confirmed the genetic diagnosis of SMA.


Assuntos
Atrofia Muscular Espinal/genética , Mutação , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Linhagem Celular Tumoral , Células HEK293 , Humanos , Lactente , Masculino , Atrofia Muscular Espinal/patologia , Processamento de RNA , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo
16.
J Clin Invest ; 128(7): 3008-3023, 2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29672276

RESUMO

Spinal muscular atrophy (SMA), a degenerative motor neuron (MN) disease, caused by loss of functional survival of motor neuron (SMN) protein due to SMN1 gene mutations, is a leading cause of infant mortality. Increasing SMN levels ameliorates the disease phenotype and is unanimously accepted as a therapeutic approach for patients with SMA. The ubiquitin/proteasome system is known to regulate SMN protein levels; however, whether autophagy controls SMN levels remains poorly explored. Here, we show that SMN protein is degraded by autophagy. Pharmacological and genetic inhibition of autophagy increases SMN levels, while induction of autophagy decreases these levels. SMN degradation occurs via its interaction with the autophagy adapter p62 (also known as SQSTM1). We also show that SMA neurons display reduced autophagosome clearance, increased p62 and ubiquitinated proteins levels, and hyperactivated mTORC1 signaling. Importantly, reducing p62 levels markedly increases SMN and its binding partner gemin2, promotes MN survival, and extends lifespan in fly and mouse SMA models, revealing p62 as a potential new therapeutic target for the treatment of SMA.


Assuntos
Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular Espinal/metabolismo , Proteínas do Complexo SMN/metabolismo , Proteína Sequestossoma-1/antagonistas & inibidores , Animais , Autofagia , Células Cultivadas , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/patologia , Mutação , Fenótipo , Proteólise , RNA Interferente Pequeno/genética , Proteínas do Complexo SMN/deficiência , Proteínas do Complexo SMN/genética , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/antagonistas & inibidores , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Serina-Treonina Quinases TOR/metabolismo
17.
FEBS Lett ; 592(8): 1400-1411, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29537490

RESUMO

Gemin5 acts as a U1 small nuclear RNA (snRNA)-binding protein in U1 small nuclear ribonucleic protein (snRNP) biogenesis. Here, we report a role for Gemin5 in unassembled U1 snRNP disposal under survival of motor neuron (SMN) protein-deficient conditions. We demonstrate that non-Sm protein-associated U1 snRNA and U1A are enriched in cytoplasmic granules and colocalize to P bodies in SMN-deficient cells. Immunoprecipitation assays show increased associations of the U1 snRNP component U1A with P body components and Gemin5 in SMN-deficient cells. More importantly, Gemin5 knockdown eliminates the unassembled U1 snRNP granules and rescues U1 snRNA levels in SMN-deficient cells. Taken together, our study provides direct evidence that Gemin5 is involved in unassembled-U1 snRNA disposal under conditions of SMN deficiency.


Assuntos
Grânulos Citoplasmáticos/metabolismo , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Grânulos Citoplasmáticos/genética , Células HeLa , Humanos , RNA Nuclear Pequeno/genética , Ribonucleoproteína Nuclear Pequena U1/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo
18.
Brain Dev ; 40(8): 670-677, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29580671

RESUMO

BACKGROUND: The SMN genes, SMN1 and SMN2, are highly homologous genes which are related to the development or clinical severity of spinal muscular atrophy. Some alternative splicing patterns of the SMN genes have been well documented. In 2007, an SMN1 transcript with a full sequence of intron 3 was reported as the first intron-retained SMN transcript. METHODS: Intron-retained SMN transcripts in various cells and tissues were studied using reverse transcription (RT)-PCR. HeLa cells were used for subcellular localization of the transcripts and protein expression analysis with Western blotting. RESULTS: Two intron-retained SMN transcripts were detected, which contain full sequences of intron 2b or intron 3. These transcripts were produced from SMN1 and SMN2, and ubiquitously expressed in human cells and tissues. Western blotting analysis showed no proteins derived from the intron-retained transcripts. Fractionation analysis showed that these intron-retained transcripts were localized mainly in the nucleus. Contrary to our expectation, the intron-retained transcript levels decreased during the treatment of cycloheximide, an inhibitor of nonsense-mediated decay (NMD), suggesting that they were not targets of NMD. CONCLUSION: Intron 2b-retained SMN transcript and intron3-retained SMN transcript were ubiquitously expressed in human cells and tissues. The intron-retained transcripts were mainly localized in the nucleus and decreased through non-NMD pathway.


Assuntos
Íntrons , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Western Blotting , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Cicloeximida/farmacologia , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Atrofia Muscular Espinal/genética , Degradação do RNAm Mediada por Códon sem Sentido/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo
19.
Sci Rep ; 8(1): 2075, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29391529

RESUMO

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.


Assuntos
Corpos Enovelados/efeitos dos fármacos , Flunarizina/farmacologia , Atrofia Muscular Espinal/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Animais , Linhagem Celular , Corpos Enovelados/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Flunarizina/uso terapêutico , Células HeLa , Humanos , Masculino , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Atrofia Muscular Espinal/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia
20.
Brain Res ; 1693(Pt A): 92-97, 2018 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-29462610

RESUMO

Spinal muscular atrophy is caused by deletions or mutations in the SMN1 gene that result in reduced expression of the SMN protein. The SMN protein is an essential molecular chaperone that is required for the biogenesis of multiple ribonucleoprotein (RNP) complexes including spliceosomal small nuclear RNPs (snRNPs). Reductions in SMN expression result in a reduced abundance of snRNPs and to downstream RNA splicing alterations. SMN is also present in axons and dendrites and appears to have important roles in the formation of neuronal mRNA-protein complexes during development or neuronal repair. Thus, SMA is an exemplar, selective motor neuron disorder that is caused by defects in fundamental RNA processing events. A detailed molecular understanding of how motor neurons fail, and why other neurons do not, in SMA will yield important principals about motor neuron maintenance and neuronal specificity in neurodegenerative diseases.


Assuntos
Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patologia , Ribonucleoproteínas/metabolismo , Animais , Axônios/metabolismo , Humanos , Doença dos Neurônios Motores/genética , Neurônios Motores/metabolismo , Neurônios Motores/fisiologia , Degeneração Neural/metabolismo , Processamento Pós-Transcricional do RNA/genética , Processamento Pós-Transcricional do RNA/fisiologia , Processamento de RNA , Proteínas de Ligação a RNA , Ribonucleoproteínas/fisiologia , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Ribonucleoproteínas Nucleares Pequenas/fisiologia , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo
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