RESUMO
BACKGROUND: Data on combining molecular therapies that increase survival motor neuron protein for spinal muscular atrophy type 1 (SMA1) is lacking. METHODS: This was a retrospective study describing our centers' experiences in treating SMA1 patients with combination therapy. RESULTS: Five children received nusinersen and onasemnogene abeparvovec-xioi (onasemnogene). Four were receiving nusinersen prior to onasemnogene. Nusinersen was continued in three. Marked liver enzyme elevations resulted in prolonged corticosteroid treatment in two patients with hospitalization and liver biopsy in one; milder liver enzyme elevations were noted in the other two. One patient received onasemnogene first, and then nusinersen. No adverse effects were noted. All patients improved. CONCLUSIONS: Combination molecular therapy is tolerated in SMA1 patients. Further studies are needed to determine whether there are circumstances in which combination therapy would be more efficacious than either monotherapy. Prolonged corticosteroid use and liver toxicity monitoring may be necessary with onasemnogene therapy.
Assuntos
Produtos Biológicos/uso terapêutico , Oligonucleotídeos/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Atrofias Musculares Espinais da Infância/tratamento farmacológico , Pré-Escolar , Quimioterapia Combinada , Feminino , Humanos , Lactente , Masculino , Estudos Retrospectivos , Resultado do TratamentoRESUMO
Spinal muscular atrophy (SMA) is caused by insufficient levels of the survival motor neuron (SMN) protein due to the functional loss of the SMN1 gene and the inability of its paralog, SMN2, to fully compensate due to reduced exon 7 splicing efficiency. Since SMA patients have at least one copy of SMN2, drug discovery campaigns have sought to identify SMN2 inducers. C5-substituted quinazolines increase SMN2 promoter activity in cell-based assays and a derivative, RG3039, has progressed to clinical testing. It is orally bioavailable, brain-penetrant and has been shown to be an inhibitor of the mRNA decapping enzyme, DcpS. Our pharmacological characterization of RG3039, reported here, demonstrates that RG3039 can extend survival and improve function in two SMA mouse models of varying disease severity (Taiwanese 5058 Hemi and 2B/- SMA mice), and positively impacts neuromuscular pathologies. In 2B/- SMA mice, RG3039 provided a >600% survival benefit (median 18 days to >112 days) when dosing began at P4, highlighting the importance of early intervention. We determined the minimum effective dose and the associated pharmacokinetic (PK) and exposure relationship of RG3039 and DcpS inhibition ex vivo. These data support the long PK half-life with extended pharmacodynamic outcome of RG3039 in 2B/- SMA mice. In motor neurons, RG3039 significantly increased both the average number of cells with gems and average number of gems per cell, which is used as an indirect measure of SMN levels. These studies contribute to dose selection and exposure estimates for the first studies with RG3039 in human subjects.
Assuntos
Endorribonucleases/antagonistas & inibidores , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Atrofia Muscular Espinal/fisiopatologia , Quinazolinas/farmacologia , Quinazolinas/farmacocinética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo , Administração Oral , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Endorribonucleases/metabolismo , Feminino , Humanos , Camundongos , Camundongos Transgênicos , Atrofia Muscular Espinal/tratamento farmacológico , Quinazolinas/uso terapêutico , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Sinapses/efeitos dos fármacos , Sinapses/fisiologiaRESUMO
Liver steatosis in nonalcoholic fatty liver disease is affected by genetics and diet. It is associated with insulin resistance (IR) in hepatic and peripheral tissues. Here, we aimed to characterize the severity of diet-induced steatosis, obesity, and IR in two phylogenetically distant mouse strains, C57BL/6J and DBA/2J. To this end, mice (male, 8 wk old) were fed a high-fat and high-carbohydrate (HFHC) or control diet for 16 wk followed by the application of a combination of classic physiological, biochemical, and pathological studies to determine obesity and hepatic steatosis. Peripheral IR was characterized by measuring blood glucose level, serum insulin level, homeostasis model assessment of IR, glucose intolerance, insulin intolerance, and AKT phosphorylation in adipose tissues, whereas the level of hepatic IR was determined by measuring insulin-triggered hepatic AKT phosphorylation. We discovered that both C57BL/6J and DBA/2J mice developed obesity to a similar degree without the feature of liver inflammation after being fed an HFHC diet for 16 wk. C57BL/6J mice in the HFHC diet group exhibited severe pan-lobular steatosis, a marked increase in hepatic triglyceride levels, and profound peripheral IR. In contrast, DBA/2J mice in the HFHC diet group developed only a mild degree of pericentrilobular hepatic steatosis that was associated with moderate changes in peripheral IR. Interestingly, both C57BL/6J and DBA/2J developed severe hepatic IR after HFHC diet treatment. Collectively, these data suggest that the severity of diet-induced hepatic steatosis is correlated to the level of peripheral IR, not with the severity of obesity and hepatic IR. Peripheral rather than hepatic IR is a dominant factor of pathophysiology in nonalcoholic fatty liver disease.
Assuntos
Carboidratos da Dieta/administração & dosagem , Gorduras na Dieta/administração & dosagem , Fígado Gorduroso/fisiopatologia , Resistência à Insulina/fisiologia , Fígado/fisiologia , Animais , Fígado Gorduroso/etiologia , Glucose/metabolismo , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Hepatopatia Gordurosa não Alcoólica , Obesidade/etiologiaRESUMO
The loss of motor neurons (MNs) is a hallmark of the neuromuscular disease spinal muscular atrophy (SMA); however, it is unclear whether this phenotype autonomously originates within the MN. To address this question, we developed an inducible mouse model of severe SMA that has perinatal lethality, decreased motor function, motor unit pathology, and hyperexcitable MNs. Using an Hb9-Cre allele, we increased Smn levels autonomously within MNs and demonstrate that MN rescue significantly improves all phenotypes and pathologies commonly described in SMA mice. MN rescue also corrects hyperexcitability in SMA motor neurons and prevents sensory-motor synaptic stripping. Survival in MN-rescued SMA mice is extended by only 5 d, due in part to failed autonomic innervation of the heart. Collectively, this work demonstrates that the SMA phenotype autonomously originates in MNs and that sensory-motor synapse loss is a consequence, not a cause, of MN dysfunction.
Assuntos
Neurônios Motores/patologia , Atrofia Muscular Espinal/patologia , Células Receptoras Sensoriais/patologia , Animais , Animais Recém-Nascidos , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Neurônios Motores/fisiologia , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/fisiopatologia , Junção Neuromuscular/patologia , Junção Neuromuscular/fisiologia , Células Receptoras Sensoriais/fisiologiaRESUMO
BACKGROUND: Ambulatory individuals with spinal muscular atrophy experience weakness and impairments of speed and endurance. This leads to decreased motor skill performance required for daily living including transitioning from floor to stand, climbing stairs, and traversing short and community distances. Motor function improvements have been reported in individuals receiving nusinersen, but changes in timed functional tests (TFTs) which assess shorter distance walking and transitions have not been well documented. OBJECTIVE: To evaluate changes in TFT performance over the course of nusinersen treatment in ambulatory individuals with SMA and identify potential factors [age, SMN2 copy number, BMI, Hammersmith Functional Motor Scale Expanded (HFMSE score), Peroneal Compound Motor Action Potential (CMAP) amplitude] associated with TFT performance. METHODS: Nineteen ambulatory participants receiving nusinersen were followed from 2017 through 2019 (range: 0-900 days, mean 624.7 days, median 780 days); thirteen of 19 (mean ageâ=â11.5 years) completed TFTs. The 10-meter walk/run test, time-to-rise from supine, time-to-rise from sitting, 4-stair climb, 6-minute walk test (6MWT), Hammersmith Expanded and peroneal CMAP were assessed at each visit. Linear mixed-effects models were used to evaluate unadjusted and adjusted changes in these outcomes over time. RESULTS: Apart from time to rise from sitting and from supine, all TFTs were found to improve over the course of treatment after adjusting for baseline age and BMI. CONCLUSIONS: Improvement in TFTs over time in patients with SMA treated with nusinersen suggests that shorter TFTs may have value to assess individuals with SMA who have or later gain ambulatory function during treatment.
Assuntos
Atrofia Muscular Espinal , Atrofias Musculares Espinais da Infância , Humanos , Criança , Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular Espinal/complicações , Oligonucleotídeos/uso terapêutico , Destreza MotoraRESUMO
Proximal spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality. Traditionally, SMA has been described as a motor neuron disease; however, there is a growing body of evidence that arrhythmia and/or cardiomyopathy may present in SMA patients at an increased frequency. Here, we ask whether SMA model mice possess such phenotypes. We find SMA mice suffer from severe bradyarrhythmia characterized by progressive heart block and impaired ventricular depolarization. Echocardiography further confirms functional cardiac deficits in SMA mice. Additional investigations show evidence of both sympathetic innervation defects and dilated cardiomyopathy at late stages of disease. Based upon these data, we propose a model in which decreased sympathetic innervation causes autonomic imbalance. Such imbalance would be characterized by a relative increase in the level of vagal tone controlling heart rate, which is consistent with bradyarrhythmia and progressive heart block. Finally, treatment with the histone deacetylase inhibitor trichostatin A, a drug known to benefit phenotypes of SMA model mice, produces prolonged maturation of the SMA heartbeat and an increase in cardiac size. Treated mice maintain measures of motor function throughout extended survival though they ultimately reach death endpoints in association with a progression of bradyarrhythmia. These data represent the novel identification of cardiac arrhythmia as an early and progressive feature of murine SMA while providing several new, quantitative indices of mouse health. Together with clinical cases that report similar symptoms, this reveals a new area of investigation that will be important to address as we move SMA therapeutics towards clinical success.
Assuntos
Bradicardia , Atrofia Muscular Espinal , Animais , Bradicardia/tratamento farmacológico , Bradicardia/fisiopatologia , Modelos Animais de Doenças , Ecocardiografia , Eletrocardiografia , Coração/inervação , Bloqueio Cardíaco/etiologia , Bloqueio Cardíaco/fisiopatologia , Frequência Cardíaca/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Atividade Motora , Atrofia Muscular Espinal/complicações , Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/fisiopatologia , Miocárdio/patologia , Sistema Nervoso Simpático/patologia , Sistema Nervoso Simpático/fisiopatologiaRESUMO
The SMN2 transgenic mouse, Tg(SMN2)89Ahmb, has emerged as the most widely used in spinal muscular atrophy (SMA) research. Here we clone the genomic integration site of the transgene and demonstrate it to be in intron 4 of the metabotropic glutamate receptor 7 (mGluR7) gene. We found that the integration of this transgene significantly reduced both mGluR7 mRNA and protein levels (24% and 9%, respectively). To determine if phenotypes associated with mGluR7 knockout mice were present in Tg(SMN2)89Ahmb containing mice, we subjected mice homozygous for the transgene to open field and seizure susceptibility tests. When compared to wild type FVB/N mice, Tg(SMN2)89Ahmb(tg/tg) mice exhibited significantly longer times in finding a safe wall-adjacent square (+54s if Smn(+/+), +90s if Smn(+/-)), as well as a significantly higher frequency of generalized seizure in response to a subthreshold dose of pentylenetrazol (0.11 vs 0.45). These findings aid in explaining the sudden unexpected death that occurs within SMA mouse colonies that contain a homozygous Tg(SMN2)89Ahmb transgene. This should be taken into account in pre-clinical studies that utilize this transgene, especially in therapy-treated SMA mice that have extended survival.
Assuntos
Epilepsia/genética , Medo , Atrofia Muscular Espinal/genética , Receptores de Glutamato Metabotrópico/genética , Animais , Modelos Animais de Doenças , Epilepsia/etiologia , Epilepsia/psicologia , Medo/psicologia , Predisposição Genética para Doença/genética , Genoma/genética , Fatores Hospedeiros de Integração/genética , Íntrons/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atrofia Muscular Espinal/etiologia , Atrofia Muscular Espinal/psicologia , Análise de Sobrevida , Transgenes/genéticaRESUMO
Proximal spinal muscular atrophy (SMA) is a neuromuscular disorder for which there is no available therapy. SMA is caused by loss or mutation of the survival motor neuron 1 gene, SMN1, with retention of a nearly identical copy gene, SMN2. In contrast to SMN1, most SMN2 transcripts lack exon 7. This alternatively spliced transcript, Delta7-SMN, encodes a truncated protein that is rapidly degraded. Inhibiting this degradation may be of therapeutic value for the treatment of SMA. Recently aminoglycosides, which decrease translational fidelity to promote readthrough of termination codons, were shown to increase SMN levels in patient cell lines. Amid uncertainty concerning the role of SMN's C-terminus, the potential of translational readthrough as a therapeutic mechanism for SMA is unclear. Here, we used stable cell lines to demonstrate the SMN C-terminus modulates protein stability in a sequence-independent manner that is reproducible by translational readthrough. Geneticin (G418) was then identified as a potent inducer of the Delta7-SMN target sequence in vitro through a novel quantitative assay amenable to high throughput screens. Subsequent treatment of patient cell lines demonstrated that G418 increases SMN levels and is a potential lead compound. Furthermore, treatment of SMA mice with G418 increased both SMN protein and mouse motor function. Chronic administration, however, was associated with toxicity that may have prevented the detection of a survival benefit. Collectively, these results substantiate a sequence independent role of SMN's C-terminus in protein stability and provide the first in vivo evidence supporting translational readthrough as a therapeutic strategy for the treatment of SMA.
Assuntos
Aminoglicosídeos/farmacologia , Gentamicinas/farmacologia , Atividade Motora/efeitos dos fármacos , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/fisiopatologia , Biossíntese de Proteínas/efeitos dos fármacos , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Aminoglicosídeos/administração & dosagem , Aminoglicosídeos/uso terapêutico , Animais , Sequência de Bases , Linhagem Celular , Códon de Terminação , Éxons/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Gentamicinas/administração & dosagem , Gentamicinas/uso terapêutico , Humanos , Camundongos , Dados de Sequência Molecular , Atrofia Muscular Espinal/tratamento farmacológico , Estabilidade Proteica/efeitos dos fármacos , Proteína 1 de Sobrevivência do Neurônio Motor/genéticaRESUMO
Purpose The aim of this study was to provide clinicians with an overview of literature relating to dysphagia in spinal muscular atrophy (SMA) to guide assessment and treatment. Method In this clinical focus article, we review literature published in Scopus and PubMed between 1990 and 2020 pertaining to dysphagia in SMA across the life span. Original research articles that were published in English were included. Searches were conducted within four themes of inquiry: (a) etiology and phenotypes, (b) respiratory systemic deficits and management, (c) characteristics of natural history dysphagia and its treatment, and (d) dysphagia outcomes with disease-modifying therapies. Articles for the first two themes were selected by content experts who identified the most salient articles that would provide clinicians foundational background knowledge about SMA. Articles for the third theme were identified using search terms, including spinal muscular atrophy, swallow, dysphagia, bulbar, nutrition, g-tube, alternative nutrition, jaw, mouth, palate, OR mandible. Search terms for the fourth theme included spinal muscular atrophy AND nusinersen OR AVXS-101/onasemnogene abeparvovec-xioi. Review of Pertinent Literature Twenty-nine articles were identified. Findings across identified articles support the fact that patients with SMA who do not receive disease-modifying therapy exhibit clinically significant deficits in oropharyngeal swallow function. Few investigations provided systematic information regarding the underlying physiological deficits responsible for this loss in function, the timing of the degradation, or how disease-modifying therapies change these outcomes. Conclusion Future research outlining the physiological and functional oropharyngeal swallowing deficits among patients with SMA who receive disease-modifying therapy is critical in developing standards of dysphagia care to guide clinicians.
Assuntos
Transtornos de Deglutição , Atrofia Muscular Espinal , Produtos Biológicos , Transtornos de Deglutição/diagnóstico , Transtornos de Deglutição/etiologia , Transtornos de Deglutição/terapia , Humanos , Fenótipo , Proteínas Recombinantes de Fusão , Projetos de PesquisaRESUMO
Until the recent development of disease-modifying therapeutics, spinal muscular atrophy (SMA) was considered a devastating neuromuscular disease with a poor prognosis for most affected individuals. Symptoms generally present during early childhood and manifest as muscle weakness and progressive paralysis, severely compromising the affected individual's quality of life, independence, and lifespan. SMA is most commonly caused by the inheritance of homozygously deleted SMN1 alleles with retention of one or more copies of a paralog gene, SMN2, which inversely correlates with disease severity. The recent advent and use of genetically targeted therapies have transformed SMA into a prototype for monogenic disease treatment in the era of genetic medicine. Many SMA-affected individuals receiving these therapies achieve traditionally unobtainable motor milestones and survival rates as medicines drastically alter the natural progression of this disease. This review discusses historical SMA progression and underlying disease mechanisms, highlights advances made in therapeutic research, clinical trials, and FDA-approved medicines, and discusses possible second-generation and complementary medicines as well as optimal temporal intervention windows in order to optimize motor function and improve quality of life for all SMA-affected individuals.
RESUMO
Spinal muscular atrophy (SMA) is a pediatric neuromuscular disease caused by genetic deficiency of the survival motor neuron (SMN) protein. Pathological hallmarks of SMA are spinal motor neuron loss and skeletal muscle atrophy. The molecular mechanisms that elicit and drive preferential motor neuron degeneration and death in SMA remain unclear. Transcriptomic studies consistently report p53 pathway activation in motor neurons and spinal cord tissue of SMA mice. Recent work has identified p53 as an inducer of spinal motor neuron loss in severe Δ7 SMA mice. Additionally, the cyclin-dependent kinase inhibitor P21 (Cdkn1a), an inducer of cell cycle arrest and mediator of skeletal muscle atrophy, is consistently increased in motor neurons, spinal cords, and other tissues of various SMA models. p21 is a p53 transcriptional target but can be independently induced by cellular stressors. To ascertain whether p53 and p21 signaling pathways mediate spinal motor neuron death in milder SMA mice, and how they affect the overall SMA phenotype, we introduced Trp53 and P21 null alleles onto the Smn2B/- background. We found that p53 and p21 depletion did not modulate the timing or degree of Smn2B/- motor neuron loss as evaluated using electrophysiological and immunohistochemical methods. Moreover, we determined that Trp53 and P21 knockout differentially affected Smn2B/- mouse lifespan: p53 ablation impaired survival while p21 ablation extended survival through Smn-independent mechanisms. These results demonstrate that p53 and p21 are not primary drivers of spinal motor neuron death in Smn2B/- mice, a milder SMA mouse model, as motor neuron loss is not alleviated by their ablation.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/genética , Neurônios Motores/patologia , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patologia , Medula Espinal/patologia , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína Supressora de Tumor p53/genética , Animais , Morte Celular , Sobrevivência Celular , Modelos Animais de Doenças , Feminino , Imuno-Histoquímica , Expectativa de Vida , Masculino , Camundongos , Camundongos Knockout , Transdução de Sinais , Análise de SobrevidaRESUMO
Spinal muscular atrophy (SMA) is caused by loss of the survival motor neuron gene (SMN1) and retention of the SMN2 gene. The copy number of SMN2 affects the amount of SMN protein produced and the severity of the SMA phenotype. While loss of mouse Smn is embryonic lethal, two copies of SMN2 prevents this embryonic lethality resulting in a mouse with severe SMA that dies 5 days after birth. Here we show that expression of full-length SMN under the prion promoter (PrP) rescues severe SMA mice. The PrP results in high levels of SMN in neurons at embryonic day 15. Mice homozygous for PrP-SMN with two copies of SMN2 and lacking mouse Smn survive for an average of 210 days and lumbar motor neuron root counts in these mice were normal. Expression of SMN solely in skeletal muscle using the human skeletal actin (HSA) promoter resulted in no improvement of the SMA phenotype or extension of survival. One HSA line displaying nerve expression of SMN did affect the SMA phenotype with mice living for an average of 160 days. Thus, we conclude that expression of full-length SMN in neurons can correct the severe SMA phenotype in mice. Furthermore, a small increase of SMN in neurons has a substantial impact on survival of SMA mice while high SMN levels in mature skeletal muscle alone has no impact.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Atrofia Muscular Espinal/genética , Proteínas do Tecido Nervoso/genética , Proteínas de Ligação a RNA/genética , Animais , Embrião de Mamíferos/metabolismo , Dosagem de Genes , Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular Espinal/patologia , Atrofia Muscular Espinal/fisiopatologia , Neurônios , Regiões Promotoras Genéticas , Proteínas do Complexo SMN , Análise de Sobrevida , Proteína 1 de Sobrevivência do Neurônio Motor , Proteína 2 de Sobrevivência do Neurônio MotorRESUMO
Proximal spinal muscular atrophy (SMA) results from loss of the survival motor neuron 1 (SMN1) gene, with retention of its nearly identical homolog, SMN2. There is a direct correlation between disease severity and SMN2 copy number. Mice do not have a Smn2 gene, and thus cannot naturally replicate the disorder. However, two murine models of SMA have been generated using SMN2-BAC transgenic mice bred onto a mutant Smn background. In these instances mice die shortly after birth, have variable phenotypes within the same litter, or completely correct the SMA phenotype. Both models have been imported to The Jackson Laboratory for distribution to the research community. To ensure that similar results are obtained after importation to The Jackson Laboratory to what was originally reported in the literature, we have begun a molecular and phenotypic evaluation of these mouse models. Here we report our findings for the SMA mouse model that has been deposited by the Li group from Taiwan. These mice, JAX stock number TJL-005058, are homozygous for the SMN2 transgene, Tg(SMN2)2Hung, and a targeted Smn allele that lacks exon 7, Smn1(tm1Hung). Our findings are consistent with those reported originally for this line and clarify some of the original data. In addition, we have cloned and mapped the integration site for Tg(SMN2)2Hung to Chromosome 4, and provide a simple genotyping assay that is specific to the junction fragment. Finally, based upon the survival data from our genetic crosses, we suggest that this underused SMA model may be a useful compliment or alternative to the more commonly used "delta7" SMA mouse. We provide breeding schemes in which two genotypes of mice can be generated so that 50% of the litter will be SMA-like pups while 50% will be controls.
Assuntos
Modelos Animais de Doenças , Camundongos , Atrofia Muscular Espinal/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Animais , Sequência de Bases , Heterozigoto , Humanos , Camundongos Knockout , Camundongos Mutantes Neurológicos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação , Fenótipo , Proteína 2 de Sobrevivência do Neurônio Motor/genéticaRESUMO
BACKGROUND: Electrocardiography remains the best diagnostic tool and therapeutic biomarker for a spectrum of pediatric diseases involving cardiac or autonomic nervous system defects. As genetic links to these disorders are established and transgenic mouse models produced in efforts to understand and treat them, there is a surprising lack of information on electrocardiograms (ECGs) and ECG abnormalities in neonate mice. This is likely due to the trauma and anaesthesia required of many legacy approaches to ECG recording in mice, exacerbated by the fragility of many mutant neonates. Here, we use a non-invasive system to characterize development of the heart rate and electrocardiogram throughout the growth of conscious neonate FVB/N mice. RESULTS: We examine ECG waveforms as early as two days after birth. At this point males and females demonstrate comparable heart rates that are 50% lower than adult mice. Neonatal mice exhibit very low heart rate variability. Within 12 days of birth PR, QRS and QTc interval durations are near adult values while heart rate continues to increase until weaning. Upon weaning FVB/N females quickly develop slower heart rates than males, though PR intervals are comparable between sexes until a later age. This suggests separate developmental events may contribute to these gender differences in electrocardiography. CONCLUSIONS: We provide insight with a new level of detail to the natural course of heart rate establishment in neonate mice. ECG can now be conveniently and repeatedly used in neonatal mice. This should serve to be of broad utility, facilitating further investigations into development of a diverse group of diseases and therapeutics in preclinical mouse studies.
Assuntos
Sistema Nervoso Autônomo/crescimento & desenvolvimento , Eletrocardiografia/métodos , Frequência Cardíaca/fisiologia , Animais , Animais Recém-Nascidos , Feminino , Modelos Logísticos , Masculino , Camundongos , Distribuição Aleatória , Fatores SexuaisRESUMO
Friedreich's ataxia is a neurodegenerative disorder associated with a GAA trinucleotide repeat expansion in intron 1 of the frataxin (FXN) gene. It is the most common autosomal recessive cerebellar ataxia, with a mean age of onset at 16 years. Nearly 95-98% of patients are homozygous for a 90-1300 GAA repeat expansion with only 2-5% demonstrating compound heterozygosity. Compound heterozygous individuals have a repeat expansion in one allele and a point mutation/deletion/insertion in the other. Compound heterozygosity and point mutations are very rare causes of Friedreich's ataxia and nonsense mutations are a further rarity among point mutations. We report a rare compound heterozygous Friedrich's ataxia patient who was found to have one expanded GAA FXN allele and a nonsense point mutation in the other. We summarize the four previously published cases of nonsense mutations and compare the phenotype to that of our patient. We compared clinical information from our patient with other nonsense FXN mutations reported in the literature. This nonsense mutation, to our knowledge, has only been described once previously; interestingly the individual was also of Cuban ancestry. A comparison with previously published cases of nonsense mutations demonstrates some common clinical characteristics.
RESUMO
Spinal muscular atrophy, a common autosomal recessive motor neuron disorder, is caused by the loss of the survival motor neuron gene (SMN1). SMN2, a nearly identical copy gene, is present in all spinal muscular atrophy patients but differs by a critical nucleotide that alters exon 7 splicing efficiency. This results in low survival motor neuron protein levels, which are not enough to sustain motor neurons. SMN disruption has been undertaken in different organisms (yeast, nematode, fly, zebrafish, and mouse) in an attempt to model this disease and gain fundamental knowledge about the survival motor neuron protein. This review compares the various animal models generated to date and summarizes a research picture that reveals a pleiotropic role for survival motor neuron protein; this summary also points to unique requirements for survival motor neuron protein in motor neurons. It is hoped that these observations will aid in pointing towards complementary paths for therapeutic discovery research.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Modelos Animais de Doenças , Biologia Molecular/tendências , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas de Ligação a RNA/genética , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Evolução Molecular , Humanos , Invertebrados/genética , Invertebrados/metabolismo , Biologia Molecular/métodos , Atrofia Muscular Espinal/fisiopatologia , Mutação/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas do Complexo SMN , Proteína 1 de Sobrevivência do Neurônio Motor , Proteína 2 de Sobrevivência do Neurônio Motor , Vertebrados/genética , Vertebrados/metabolismoRESUMO
Proximal spinal muscular atrophy is caused by deletion or mutation of the survival motor neuron 1 gene, SMN1. Rentention of a nearly identical copy gene, SMN2, enables survival but is unable to fully compensate for the loss of SMN1. The SMN1 and SMN2 genes differ by a single nucleotide that results in alternative splicing of SMN2 exon 7 due to the disruption of a binding site for an essential splicing factor. This alternatively spliced form encodes a partially functional truncated protein. Because SMN2 is present in patients with spinal muscular atrophy, it is an ideal therapeutic target. Some of the current approaches to increase SMN protein levels are aimed at increasing the transcription from SMN2 or at preventing exon 7 skipping. One area that has yet to be investigated is the stability of messenger ribonucleic acid (RNA) transcripts produced from SMN2. We postulated that transcripts derived from SMN2 may be less stable because alternative splicing, recruitment of RNA-binding proteins, and alteration of stop codons have been associated with changes in rates of messenger RNA decay; these features are all characteristic of SMN2. Accordingly, transcript degradation was examined within primary fibroblast cells that exclusively contained SMN1 or SMN2 by treating cultures with a transcriptional inhibitor to observe messenger RNA stability. The results indicate that SMN transcript instability does not play a role in the disease mechanism, suggesting that therapeutic modulation of messenger RNA degradation would not target a molecular defect in patients with spinal muscular atrophy, although it could provide general benefits by increasing total pools of SMN2 transcripts.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Atrofia Muscular Espinal/genética , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Estabilidade de RNA/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/genética , Processamento Alternativo/genética , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Dosagem de Genes/genética , Predisposição Genética para Doença/genética , Humanos , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/terapia , Proteínas do Tecido Nervoso/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas do Complexo SMN , Proteína 1 de Sobrevivência do Neurônio Motor , Proteína 2 de Sobrevivência do Neurônio Motor , Transcrição Gênica/genéticaRESUMO
C5-substituted 2,4-diaminoquinazoline inhibitors of the decapping scavenger enzyme DcpS (DAQ-DcpSi) have been developed for the treatment of spinal muscular atrophy (SMA), which is caused by genetic deficiency in the Survival Motor Neuron (SMN) protein. These compounds are claimed to act as SMN2 transcriptional activators but data underlying that claim are equivocal. In addition it is unclear whether the claimed effects on SMN2 are a direct consequence of DcpS inhibitor or might be a consequence of lysosomotropism, which is known to be neuroprotective. DAQ-DcpSi effects were characterized in cells in vitro utilizing DcpS knockdown and 7-methyl analogues as probes for DcpS vs non-DcpS-mediated effects. We also performed analysis of Smn transcript levels, RNA-Seq analysis of the transcriptome and SMN protein in order to identify affected pathways underlying the therapeutic effect, and studied lysosomotropic and non-lysosomotropic DAQ-DCpSi effects in 2B/- SMA mice. Treatment of cells caused modest and transient SMN2 mRNA increases with either no change or a decrease in SMNΔ7 and no change in SMN1 transcripts or SMN protein. RNA-Seq analysis of DAQ-DcpSi-treated N2a cells revealed significant changes in expression (both up and down) of approximately 2,000 genes across a broad range of pathways. Treatment of 2B/- SMA mice with both lysomotropic and non-lysosomotropic DAQ-DcpSi compounds had similar effects on disease phenotype indicating that the therapeutic mechanism of action is not a consequence of lysosomotropism. In striking contrast to the findings in vitro, Smn transcripts were robustly changed in tissues but there was no increase in SMN protein levels in spinal cord. We conclude that DAQ-DcpSi have reproducible benefit in SMA mice and a broad spectrum of biological effects in vitro and in vivo, but these are complex, context specific, and not the result of simple SMN2 transcriptional activation.
Assuntos
Endorribonucleases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular Espinal/enzimologia , Quinazolinas/farmacologia , Animais , Linhagem Celular , Modelos Animais de Doenças , Inibidores Enzimáticos/química , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Knockout , Atrofia Muscular Espinal/genética , Regiões Promotoras Genéticas , Quinazolinas/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/deficiência , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismoRESUMO
OBJECTIVE: The aim of this study was to investigate the role of ubiquitin C-terminal hydrolase-L1 (UCHL1) for motor neuron circuitry and especially in spinal motor neuron (SMN) health, function, and connectivity. METHODS: Since mutations in UCHL1 gene leads to motor dysfunction in patients, we investigated the role of UCHL1 on SMN survival, axon health, and connectivity with the muscle, by employing molecular and cellular marker expression analysis and electrophysiological recordings, in healthy wild-type and Uchl1 (nm3419) (UCHL1-/-) mice, which lack all UCHL1 function. RESULTS: There is pure motor neuropathy with selective degeneration of the motor, but not sensory axons in the absence of UCHL1 function. Neuromuscular junctions (NMJ) are impaired in muscle groups that are innervated by slow-twitch or fast-twitch SMN. However, unlike corticospinal motor neurons, SMN cell bodies remain intact with no signs of elevated endoplasmic reticulum (ER) stress. INTERPRETATION: Presence of NMJ defects and progressive retrograde axonal degeneration in the absence of major SMN soma loss suggest that defining pathology as a function of neuron number is misleading and that upper and lower motor neurons utilize UCHL1 function in different cellular events. In line with findings in patients with mutations in UCHL1 gene, our results suggest a unique role of UCHL1, especially for motor neuron circuitry. SMN require UCHL1 to maintain NMJ and motor axon health, and that observed motor dysfunction in the absence of UCHL1 is not due to SMN loss, but mostly due to disintegrated circuitry.
RESUMO
Molecular technologies have produced diverse arrays of animal models for studying genetic diseases and potential therapeutics. Many have neonatal phenotypes. Spinal muscular atrophy (SMA) is a neuromuscular disorder primarily affecting children, and is of great interest in translational medicine. The most widely used SMA mouse models require all phenotyping to be performed in neonates since they do not survive much past weaning. Pre-clinical studies in neonate mice can be hindered by toxicity and a lack of quality phenotyping assays, since many assays are invalid in pups or require subjective scoring with poor inter-rater variability. We find, however, that passive electrocardiography (ECG) recording in conscious 11-day old SMA mice provides sensitive outcome measures, detecting large differences in heart rate, cardiac conduction, and autonomic control resulting from disease. We find significant drug benefits upon treatment with G418, an aminoglycoside targeting the underlying protein deficiency, even in the absence of overt effects on growth and survival. These findings provide several quantitative physiological biomarkers for SMA preclinical studies, and will be of utility to diverse disease models featuring neonatal cardiac arrhythmias.