RESUMO
BACKGROUND: Hexanucleotide repeat expansion of C9orf72 is a common genetic cause of amyotrophic lateral sclerosis (ALS). No C9orf72-targeted treatments are available. BIIB078 is an investigational antisense oligonucleotide targeting C9orf72 sense RNA. We aimed to assess the safety, tolerability, and pharmacokinetics of BIIB078 in participants with C9orf72-associated ALS. METHODS: This phase 1, randomised controlled trial was done at 22 sites in six countries (Canada, Ireland, Netherlands, Switzerland, UK, and USA). Adults with ALS and a pathogenic repeat expansion in C9orf72 were randomly assigned within six cohorts, via Interactive Response Technology in a 3:1 ratio per cohort, to receive BIIB078 (5 mg, 10 mg, 20 mg, 35 mg, 60 mg, or 90 mg in cohorts 1-6, respectively) or placebo, via an intrathecal bolus injection. The treatment period consisted of three loading doses of study treatment, administered approximately once every 2 weeks, followed by monthly maintenance doses during a treatment period of about 3 months for cohorts 1-3 and about 6 months for cohorts 4-6. Patients and investigators were masked to treatment assignment. The primary endpoint was the incidence of adverse events and serious adverse events. This trial was registered with ClinicalTrials.gov (NCT03626012) and is completed. FINDINGS: Between Sept 10, 2018, and Nov 17, 2021, 124 patients were screened for inclusion in the study. 18 patients were excluded and 106 participants were enrolled and randomly assigned to receive 5 mg (n=6), 10 mg (n=9), 20 mg (n=9), 35 mg (n=19), 60 mg (n=18), or 90 mg (n=18) of BIIB078, or placebo (n=27). 58 (55%) of 106 patients were female. All patients received at least one dose of study treatment and were included in all analyses. All participants had at least one adverse event; most adverse events were mild or moderate in severity and did not lead to treatment discontinuation. The most common adverse events in BIIB078-treated participants were falls, procedural pain, headache, and post lumbar puncture syndrome. 14 (18%) of 79 patients who received any dose of BIIB078 reported serious adverse events, compared with nine (33%) of 27 patients who received placebo. Five participants who received BIIB078 and three participants who received placebo had fatal adverse events: respiratory failure in a participant who received 10 mg BIIB078, ALS worsening in two participants who received 35 mg BIIB078, traumatic intracerebral haemorrhage in one participant who received 35 mg BIIB078, pulmonary embolism in one participant who received 60 mg BIIB078, and respiratory failure in three participants who received placebo. All deaths were assessed as not related to the study treatment by the reporting investigator. INTERPRETATION: On the basis of these phase 1 study results, including secondary and exploratory findings showing no reduction in neurofilament levels and no benefit on clinical outcomes relative to the placebo cohort, BIIB078 clinical development has been discontinued. However, these results will be informative in furthering our understanding of the complex pathobiology of C9orf72-associated ALS. FUNDING: Biogen.
Assuntos
Esclerose Lateral Amiotrófica , Proteína C9orf72 , Oligonucleotídeos Antissenso , Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/genética , Método Duplo-Cego , Proteína C9orf72/genética , Oligonucleotídeos Antissenso/farmacocinética , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/efeitos adversos , Oligonucleotídeos Antissenso/farmacologia , Idoso , Adulto , Relação Dose-Resposta a DrogaRESUMO
BACKGROUND: The intrathecally administered antisense oligonucleotide tofersen reduces synthesis of the superoxide dismutase 1 (SOD1) protein and is being studied in patients with amyotrophic lateral sclerosis (ALS) associated with mutations in SOD1 (SOD1 ALS). METHODS: In this phase 3 trial, we randomly assigned adults with SOD1 ALS in a 2:1 ratio to receive eight doses of tofersen (100 mg) or placebo over a period of 24 weeks. The primary end point was the change from baseline to week 28 in the total score on the ALS Functional Rating Scale-Revised (ALSFRS-R; range, 0 to 48, with higher scores indicating better function) among participants predicted to have faster-progressing disease. Secondary end points included changes in the total concentration of SOD1 protein in cerebrospinal fluid (CSF), in the concentration of neurofilament light chains in plasma, in slow vital capacity, and in handheld dynamometry in 16 muscles. A combined analysis of the randomized component of the trial and its open-label extension at 52 weeks compared the results in participants who started tofersen at trial entry (early-start cohort) with those in participants who switched from placebo to the drug at week 28 (delayed-start cohort). RESULTS: A total of 72 participants received tofersen (39 predicted to have faster progression), and 36 received placebo (21 predicted to have faster progression). Tofersen led to greater reductions in concentrations of SOD1 in CSF and of neurofilament light chains in plasma than placebo. In the faster-progression subgroup (primary analysis), the change to week 28 in the ALSFRS-R score was -6.98 with tofersen and -8.14 with placebo (difference, 1.2 points; 95% confidence interval [CI], -3.2 to 5.5; P = 0.97). Results for secondary clinical end points did not differ significantly between the two groups. A total of 95 participants (88%) entered the open-label extension. At 52 weeks, the change in the ALSFRS-R score was -6.0 in the early-start cohort and -9.5 in the delayed-start cohort (difference, 3.5 points; 95% CI, 0.4 to 6.7); non-multiplicity-adjusted differences favoring early-start tofersen were seen for other end points. Lumbar puncture-related adverse events were common. Neurologic serious adverse events occurred in 7% of tofersen recipients. CONCLUSIONS: In persons with SOD1 ALS, tofersen reduced concentrations of SOD1 in CSF and of neurofilament light chains in plasma over 28 weeks but did not improve clinical end points and was associated with adverse events. The potential effects of earlier as compared with delayed initiation of tofersen are being further evaluated in the extension phase. (Funded by Biogen; VALOR and OLE ClinicalTrials.gov numbers, NCT02623699 and NCT03070119; EudraCT numbers, 2015-004098-33 and 2016-003225-41.).
Assuntos
Esclerose Lateral Amiotrófica , Oligonucleotídeos Antissenso , Superóxido Dismutase-1 , Adulto , Esclerose Lateral Amiotrófica/sangue , Esclerose Lateral Amiotrófica/líquido cefalorraquidiano , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/genética , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Método Duplo-Cego , Humanos , Injeções Espinhais , Proteínas de Neurofilamentos/sangue , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/uso terapêutico , Recuperação de Função Fisiológica/efeitos dos fármacos , Superóxido Dismutase-1/líquido cefalorraquidiano , Superóxido Dismutase-1/genéticaRESUMO
Diagnostic criteria for amyotrophic lateral sclerosis (ALS) are complex, incorporating multiple levels of certainty from possible through to definite, and are thereby prone to error. Specifically, interrater variability was previously established to be poor, thereby limiting utility as diagnostic enrollment criteria for clinical trials. In addition, the different levels of diagnostic certainty do not necessarily reflect disease progression, adding confusion to the diagnostic algorithm. Realizing these inherent limitations, the World Federation of Neurology, the International Federation of Clinical Neurophysiology, the International Alliance of ALS/MND Associations, the ALS Association (United States), and the Motor Neuron Disease Association convened a consensus meeting (Gold Coast, Australia, 2019) to consider the development of simpler criteria that better reflect clinical practice, and that could merge diagnostic categories into a single entity. The diagnostic accuracy of the novel Gold Coast criteria was subsequently interrogated through a large cross-sectional study, which established an increased sensitivity for ALS diagnosis when compared with previous criteria. Diagnostic accuracy was maintained irrespective of disease duration, functional status, or site of disease onset. Importantly, the Gold Coast criteria differentiated atypical phenotypes, such as primary lateral sclerosis, from the more typical ALS phenotype. It is proposed that the Gold Coast criteria should be incorporated into routine practice and clinical trial settings.
Assuntos
Esclerose Lateral Amiotrófica , Doença dos Neurônios Motores , Humanos , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/terapia , Austrália , Estudos Transversais , Doença dos Neurônios Motores/diagnósticoRESUMO
Amyotrophic lateral sclerosis (ALS) is a neurological disease that starts at a focal point and gradually spreads to other parts of the nervous system. One of the main clinical symptoms of ALS is muscle weakness. To study spreading patterns of muscle weakness, we analyze spatiotemporal binary muscle strength data, which indicates whether observed muscle strengths are impaired or healthy. We propose a hidden Markov model-based approach that assumes the observed disease status depends on two latent disease states. The model enables us to estimate the incidence rate of ALS disease and the probability of disease state transition. Specifically, the latter is modeled by a logistic autoregression in that the spatial network of susceptible muscles follows a Markov process. The proposed model is flexible to allow both historical muscle conditions and their spatial relationships to be included in the analysis. To estimate the model parameters, we provide an iterative algorithm to maximize sparse-penalized likelihood with bias correction, and use the Viterbi algorithm to label hidden disease states. We apply the proposed approach to analyze the ALS patients' data from EMPOWER Study.
Assuntos
Esclerose Lateral Amiotrófica , Algoritmos , Humanos , Cadeias de MarkovRESUMO
BACKGROUND: Tofersen is an antisense oligonucleotide that mediates the degradation of superoxide dismutase 1 (SOD1) messenger RNA to reduce SOD1 protein synthesis. Intrathecal administration of tofersen is being studied for the treatment of amyotrophic lateral sclerosis (ALS) due to SOD1 mutations. METHODS: We conducted a phase 1-2 ascending-dose trial evaluating tofersen in adults with ALS due to SOD1 mutations. In each dose cohort (20, 40, 60, or 100 mg), participants were randomly assigned in a 3:1 ratio to receive five doses of tofersen or placebo, administered intrathecally for 12 weeks. The primary outcomes were safety and pharmacokinetics. The secondary outcome was the change from baseline in the cerebrospinal fluid (CSF) SOD1 concentration at day 85. Clinical function and vital capacity were measured. RESULTS: A total of 50 participants underwent randomization and were included in the analyses; 48 participants received all five planned doses. Lumbar puncture-related adverse events were observed in most participants. Elevations in CSF white-cell count and protein were reported as adverse events in 4 and 5 participants, respectively, who received tofersen. Among participants who received tofersen, one died from pulmonary embolus on day 137, and one from respiratory failure on day 152; one participant in the placebo group died from respiratory failure on day 52. The difference at day 85 in the change from baseline in the CSF SOD1 concentration between the tofersen groups and the placebo group was 2 percentage points (95% confidence interval [CI], -18 to 27) for the 20-mg dose, -25 percentage points (95% CI, -40 to -5) for the 40-mg dose, -19 percentage points (95% CI, -35 to 2) for the 60-mg dose, and -33 percentage points (95% CI, -47 to -16) for the 100-mg dose. CONCLUSIONS: In adults with ALS due to SOD1 mutations, CSF SOD1 concentrations decreased at the highest concentration of tofersen administered intrathecally over a period of 12 weeks. CSF pleocytosis occurred in some participants receiving tofersen. Lumbar puncture-related adverse events were observed in most participants. (Funded by Biogen; ClinicalTrials.gov number, NCT02623699; EudraCT number, 2015-004098-33.).
Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos/administração & dosagem , Superóxido Dismutase-1/líquido cefalorraquidiano , Adulto , Esclerose Lateral Amiotrófica/líquido cefalorraquidiano , Esclerose Lateral Amiotrófica/genética , Progressão da Doença , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Cefaleia/induzido quimicamente , Humanos , Injeções Espinhais/efeitos adversos , Filamentos Intermediários , Leucocitose/induzido quimicamente , Masculino , Pessoa de Meia-Idade , Mutação , Oligonucleotídeos/efeitos adversos , Oligonucleotídeos/farmacocinética , Oligonucleotídeos Antissenso/efeitos adversos , Oligonucleotídeos Antissenso/farmacocinética , Superóxido Dismutase-1/genética , Capacidade VitalRESUMO
BACKGROUND: Funding and resources for low prevalent neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) are limited, and optimising their use is vital for efficient drug development. In this study, we review the design assumptions for pivotal ALS clinical trials with time-to-event endpoints and provide optimised settings for future trials. METHODS: We extracted design settings from 13 completed placebo-controlled trials. Optimal assumptions were estimated using parametric survival models in individual participant data (n=4991). Designs were compared in terms of sample size, trial duration, drug use and costs. RESULTS: Previous trials overestimated the hazard rate by 18.9% (95% CI 3.4% to 34.5%, p=0.021). The median expected HR was 0.56 (range 0.33-0.66). Additionally, we found evidence for an increasing mean hazard rate over time (Weibull shape parameter of 2.03, 95% CI 1.93 to 2.15, p<0.001), which affects the design and planning of future clinical trials. Incorporating accrual time and assuming an increasing hazard rate at the design stage reduced sample size by 33.2% (95% CI 27.9 to 39.4), trial duration by 17.4% (95% CI 11.6 to 23.3), drug use by 14.3% (95% CI 9.6 to 19.0) and follow-up costs by 21.2% (95% CI 15.6 to 26.8). CONCLUSIONS: Implementing distributional knowledge and incorporating accrual at the design stage could achieve large gains in the efficiency of ALS clinical trials with time-to-event endpoints. We provide an open-source platform that helps investigators to make more accurate sample size calculations and optimise the use of their available resources.
Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Ensaios Clínicos como Assunto/métodos , Determinação de Ponto Final/métodos , Projetos de Pesquisa , Adulto , Feminino , Humanos , Masculino , Qualidade de Vida , Riluzol/uso terapêuticoRESUMO
This paper focuses on analysis of spatiotemporal binary data with absorbing states. The research was motivated by a clinical study on amyotrophic lateral sclerosis (ALS), a neurological disease marked by gradual loss of muscle strength over time in multiple body regions. We propose an autologistic regression model to capture complex spatial and temporal dependencies in muscle strength among different muscles. As it is not clear how the disease spreads from one muscle to another, it may not be reasonable to define a neighborhood structure based on spatial proximity. Relaxing the requirement for prespecification of spatial neighborhoods as in existing models, our method identifies an underlying network structure empirically to describe the pattern of spreading disease. The model also allows the network autoregressive effects to vary depending on the muscles' previous status. Based on the joint distribution derived from this autologistic model, the joint transition probabilities of responses among locations can be estimated and the disease status can be predicted in the next time interval. Model parameters are estimated through maximization of penalized pseudo-likelihood. Postmodel selection inference was conducted via a bias-correction method, for which the asymptotic distributions were derived. Simulation studies were conducted to evaluate the performance of the proposed method. The method was applied to the analysis of muscle strength loss from the ALS clinical study.
Assuntos
Progressão da Doença , Modelos Logísticos , Análise Espaço-Temporal , Esclerose Lateral Amiotrófica , Simulação por Computador , Humanos , Funções Verossimilhança , Força MuscularRESUMO
Increased excitability of motor neurons in patients with amyotrophic lateral sclerosis (ALS) may be a relevant factor leading to motor neuron damage. This randomized, double-blind, three-way crossover, placebo-controlled study evaluated peripheral motor nerve excitability testing as a biomarker of hyperexcitability and assessed the effects of riluzole and retigabine in 18 patients with ALS. We performed excitability testing at baseline, and twice after participants had received a single dose of either 100 mg riluzole, 300 mg retigabine, or placebo. Between- and within-day repeatability was at least acceptable for 14 out of 18 recorded excitability variables. No effects of riluzole on excitability testing were observed, but retigabine significantly decreased strength-duration time-constant (9.2%) and refractoriness at 2 ms (10.2) compared to placebo. Excitability testing was shown to be a reliable biomarker in patients with ALS, and the acute reversal of previously abnormal variables by retigabine justifies long-term studies evaluating the impact on disease progression and survival.
Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Axônios/efeitos dos fármacos , Carbamatos/administração & dosagem , Potencial Evocado Motor/efeitos dos fármacos , Antagonistas de Aminoácidos Excitatórios/administração & dosagem , Neurônios Motores/efeitos dos fármacos , Fármacos Neuroprotetores/administração & dosagem , Fenilenodiaminas/administração & dosagem , Riluzol/administração & dosagem , Adulto , Idoso , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/fisiopatologia , Axônios/patologia , Carbamatos/efeitos adversos , Carbamatos/farmacocinética , Estudos Cross-Over , Método Duplo-Cego , Eletromiografia , Antagonistas de Aminoácidos Excitatórios/efeitos adversos , Antagonistas de Aminoácidos Excitatórios/farmacocinética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neurônios Motores/patologia , Países Baixos , Fármacos Neuroprotetores/efeitos adversos , Fármacos Neuroprotetores/farmacocinética , Fenilenodiaminas/efeitos adversos , Fenilenodiaminas/farmacocinética , Período Refratário Eletrofisiológico/efeitos dos fármacos , Riluzol/efeitos adversos , Riluzol/farmacocinética , Fatores de Tempo , Resultado do TratamentoRESUMO
OBJECTIVES: Clinical trials in neurodegenerative disorders are facing high futility rates and rising development costs. We aim to review and exemplify the value of group sequential trial designs (i.e., designs with one or more prospectively planned interim analyses) within the field of amyotrophic lateral sclerosis. STUDY DESIGN AND SETTING: We reviewed the literature to identify sequentially conducted trials. Subsequently, we reanalyzed the dexpramipexole trial (EMPOWER), a classically designed and conducted trial involving 942 participants, by sequentially monitoring the functional questionnaire and survival endpoint. Finally, we simulated the performance of the sequential methodology under different treatment effects. RESULTS: Only six (12%) randomized, placebo-controlled trials incorporated stopping rules for both futility and superiority. Despite its high enrollment rate, sequential reanalysis of the EMPOWER study reduced the total trial duration with 140 days (23.4%, 95% confidence interval [CI] 13.2-34.4%), the number of follow-ups with 2,688 visits (23.6%, 95% CI 11.3-38.6%), and the total drug exposure time with 73,377 days (20.6%, 95% CI 9.8-35.9%). The functional questionnaire considerably increased the heterogeneity in the test statistics, which may negatively affect sequential monitoring. CONCLUSION: Group sequential trials can result in important reductions in the trial duration, which could make clinical trials more ethical by reducing the patients' exposure to noneffective treatments or by limiting their time on placebo.
Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Término Precoce de Ensaios Clínicos , Futilidade Médica , Ensaios Clínicos Controlados Aleatórios como Assunto/métodos , Esclerose Lateral Amiotrófica/mortalidade , Fármacos do Sistema Nervoso Central/uso terapêutico , Intervalos de Confiança , Estudos de Equivalência como Asunto , Humanos , Doenças Neurodegenerativas/tratamento farmacológico , Placebos/uso terapêutico , Pramipexol/uso terapêutico , Projetos de Pesquisa , Fatores de TempoRESUMO
OBJECTIVES: Plasma creatinine is a predictor of survival in amyotrophic lateral sclerosis (ALS). It remains, however, to be established whether it can monitor disease progression and serve as surrogate endpoint in clinical trials. METHODS: We used clinical trial data from three cohorts of clinical trial participants in the LITRA, EMPOWER and PROACT studies. Longitudinal associations between functional decline, muscle strength and survival with plasma creatinine were assessed. Results were translated to trial design in terms of sample size and power. RESULTS: A total of 13 564 measurements were obtained for 1241 patients. The variability between patients in rate of decline was lower in plasma creatinine than in ALS functional rating scale-Revised (ALSFRS-R; p<0.001). The average rate of decline was faster in the ALSFRS-R, with less between-patient variability at baseline (p<0.001). Plasma creatinine had strong longitudinal correlations with the ALSFRS-R (0.43 (0.39-0.46), p<0.001), muscle strength (0.55 (0.51-0.58), p<0.001) and overall mortality (HR 0.88 (0.86-0.91, p<0.001)). Using plasma creatinine as outcome could reduce the sample size in trials by 21.5% at 18 months. For trials up to 10 months, the ALSFRS-R required a lower sample size. CONCLUSIONS: Plasma creatinine is an inexpensive and easily accessible biomarker that exhibits less variability between patients with ALS over time and is predictive for the patient's functional status, muscle strength and mortality risk. Plasma creatinine may, therefore, increase the power to detect treatment effects and could be incorporated in future ALS clinical trials as potential surrogate outcome.
Assuntos
Esclerose Lateral Amiotrófica/sangue , Creatinina/sangue , Força Muscular , Idoso , Esclerose Lateral Amiotrófica/mortalidade , Esclerose Lateral Amiotrófica/fisiopatologia , Ensaios Clínicos como Assunto , Progressão da Doença , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Taxa de SobrevidaRESUMO
UNLABELLED: Axon injury is an early event in neurodegenerative diseases that often leads to retrograde neuronal cell death and progressive permanent loss of vital neuronal functions. The connection of these two obviously sequential degenerative events, however, is elusive. Deciphering the upstream signals that trigger the neurodegeneration cascades in both neuronal soma and axon would be a key step toward developing the effective neuroprotectants that are greatly needed in the clinic. We showed previously that optic nerve injury-induced neuronal endoplasmic reticulum (ER) stress plays an important role in retinal ganglion cell (RGC) death. Using two in vivo mouse models of optic neuropathies (traumatic optic nerve injury and glaucoma) and adeno-associated virus-mediated RGC-specific gene targeting, we now show that differential manipulation of unfolded protein response pathways in opposite directions-inhibition of eukaryotic translation initiation factor 2α-C/EBP homologous protein and activation of X-box binding protein 1-promotes both RGC axons and somata survival and preserves visual function. Our results indicate that axon injury-induced neuronal ER stress plays an important role in both axon degeneration and neuron soma death. Neuronal ER stress is therefore a promising therapeutic target for glaucoma and potentially other types of neurodegeneration. SIGNIFICANCE STATEMENT: Neuron soma and axon degeneration have distinct molecular mechanisms although they are clearly connected after axon injury. We previously demonstrated that axon injury induces neuronal endoplasmic reticulum (ER) stress and that manipulation of ER stress molecules synergistically promotes neuron cell body survival. Here we investigated the possibility that ER stress also plays a role in axon degeneration and whether ER stress modulation preserves neuronal function in neurodegenerative diseases. Our results suggest that neuronal ER stress is a general mechanism of degeneration for both neuronal cell body and axon, and that therapeutic targeting of ER stress produces significant functional recovery.
Assuntos
Glaucoma/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/metabolismo , Degeneração Retiniana/metabolismo , Resposta a Proteínas não Dobradas , Animais , Estresse do Retículo Endoplasmático , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Ganglionares da Retina/metabolismoRESUMO
Dual leucine-zipper kinase (DLK) is critical for axon-to-soma retrograde signaling following nerve injury. However, it is unknown how DLK, a predicted soluble kinase, conveys long-distance signals and why homologous kinases cannot compensate for loss of DLK. Here, we report that DLK, but not homologous kinases, is palmitoylated at a conserved site adjacent to its kinase domain. Using short-hairpin RNA knockdown/rescue, we find that palmitoylation is critical for DLK-dependent retrograde signaling in sensory axons. This functional importance is because of three novel cellular and molecular roles of palmitoylation, which targets DLK to trafficking vesicles, is required to assemble DLK signaling complexes and, unexpectedly, is essential for DLK's kinase activity. By simultaneously controlling DLK localization, interactions, and activity, palmitoylation ensures that only vesicle-bound DLK is active in neurons. These findings explain how DLK specifically mediates nerve injury responses and reveal a novel cellular mechanism that ensures the specificity of neuronal kinase signaling.
Assuntos
Axônios/metabolismo , Axônios/patologia , Proteínas de Caenorhabditis elegans/metabolismo , Lipoilação , MAP Quinase Quinase Quinases/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Sequência Conservada , Evolução Molecular , Corantes Fluorescentes/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , MAP Quinase Quinase Quinases/química , Microfluídica , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Fosforilação , Ligação Proteica , Multimerização Proteica , Transporte Proteico , RNA Interferente Pequeno/metabolismo , Ratos , Células Receptoras Sensoriais/metabolismo , Transfecção , Vesículas Transportadoras/metabolismoRESUMO
IMPORTANCE: New genomic strategies can now be applied to identify a diagnosis in patients and families with previously undiagnosed rare genetic conditions. The large family evaluated in the present study was described in 1966 and now expands the phenotype of a known neuromuscular gene. OBJECTIVE: To determine the genetic cause of a slowly progressive, autosomal dominant, scapuloperoneal neuromuscular disorder by using linkage and exome sequencing. DESIGN, SETTING, AND PARTICIPANTS: Fourteen affected individuals in a 6-generation family with a progressive scapuloperoneal disorder were evaluated. Participants were examined at pediatric, neuromuscular, and research clinics from March 1, 2005, to May 31, 2014. Exome and linkage were performed in genetics laboratories of research institutions. MAIN OUTCOMES AND MEASURES: Examination and evaluation by magnetic resonance imaging, ultrasonography, electrodiagnostic studies, and muscle biopsies (n = 3). Genetic analysis included linkage analysis (n = 17) with exome sequencing (n = 7). RESULTS: Clinical findings included progressive muscle weakness in an initially scapuloperoneal and distal distribution, including wrist extensor weakness, finger and foot drop, scapular winging, mild facial weakness, Achilles tendon contractures, and diminished or absent deep tendon reflexes. Both age at onset and progression of the disease showed clinical variability within the family. Muscle biopsy specimens demonstrated type I fiber atrophy and trabeculated fibers without nemaline rods. Analysis of exome sequences within the linkage region (4.8 megabases) revealed missense mutation c.591C>A p.Glu197Asp in a highly conserved residue in exon 4 of ACTA1. The mutation cosegregated with disease in all tested individuals and was not present in unaffected individuals. CONCLUSIONS AND RELEVANCE: This family defines a new scapuloperoneal phenotype associated with an ACTA1 mutation. A highly conserved protein, ACTA1 is implicated in multiple muscle diseases, including nemaline myopathy, actin aggregate myopathy, fiber-type disproportion, and rod-core myopathy. To our knowledge, mutations in Glu197 have not been reported previously. This residue is highly conserved and located in an exposed position in the protein; the mutation affects the intermolecular and intramolecular electrostatic interactions as shown by structural modeling. The mutation in this residue does not appear to lead to rod formation or actin accumulation in vitro or in vivo, suggesting a different molecular mechanism from that of other ACTA1 diseases.
Assuntos
Actinas/genética , Distrofia Muscular de Emery-Dreifuss/genética , Distrofia Muscular de Emery-Dreifuss/fisiopatologia , Adulto , Idade de Início , Criança , Progressão da Doença , Exoma/genética , Ligação Genética , Humanos , Masculino , Distrofia Muscular de Emery-Dreifuss/patologia , Mutação de Sentido Incorreto/genética , Miopatias da Nemalina , Linhagem , FenótipoRESUMO
In both the central nervous system (CNS) and peripheral nervous system (PNS), transected axons undergo Wallerian degeneration. Even though Augustus Waller first described this process after transection of axons in 1850, the molecular mechanisms may be shared, at least in part, by many human diseases. Early pathology includes failure of synaptic transmission, target denervation, and granular disintegration of the axonal cytoskeleton (GDC). The Ca(2+)-dependent protease calpains have been implicated in GDC but causality has not been established. To test the hypothesis that calpains play a causal role in axonal and synaptic degeneration in vivo, we studied transgenic mice that express human calpastatin (hCAST), the endogenous calpain inhibitor, in optic and sciatic nerve axons. Five days after optic nerve transection and 48 h after sciatic nerve transection, robust neurofilament proteolysis observed in wild-type controls was reduced in hCAST transgenic mice. Protection of the axonal cytoskeleton in sciatic nerves of hCAST mice was nearly complete 48 h post-transection. In addition, hCAST expression preserved the morphological integrity of neuromuscular junctions. However, compound muscle action potential amplitudes after nerve transection were similar in wild-type and hCAST mice. These results, in total, provide direct evidence that calpains are responsible for the morphological degeneration of the axon and synapse during Wallerian degeneration.
Assuntos
Axônios/patologia , Calpaína/fisiologia , Citoesqueleto/patologia , Degeneração Walleriana/patologia , Animais , Proteínas de Ligação ao Cálcio/fisiologia , Córtex Cerebral/patologia , Eletromiografia , Fenômenos Eletrofisiológicos/fisiologia , Feminino , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Proteínas de Neurofilamentos/metabolismo , Junção Neuromuscular/patologia , Nervo Óptico/patologia , Retina/patologia , Nervo Isquiático/patologiaRESUMO
We examined the neurite outgrowth of sensory neurons on astrocytes following the genetic deletion of N-cadherin (NCAD). Deletion abolished immunostaining for NCAD and the other classical cadherins, indicating that NCAD is likely the only classical cadherin expressed by astrocytes. Only 38% of neurons grown on NCAD-deficient astrocytes for 24 h produced neurites, as compared to 74% of neurons grown on NCAD-expressing astrocytes. Of the neurons that produced neurites, those grown on NCAD-deficient astrocytes had a mean total length of 378 µm, as compared to 1093 µm for neurons grown on NCAD-expressing astrocytes. Thus, the loss of NCAD greatly impairs the formation and extension neurites on astrocytes.
Assuntos
Astrócitos/fisiologia , Caderinas/fisiologia , Neuritos/fisiologia , Células Receptoras Sensoriais/fisiologia , Animais , Animais Recém-Nascidos , Astrócitos/ultraestrutura , Caderinas/genética , Células Cultivadas , Dependovirus/genética , Gânglios Espinais/citologia , Camundongos , Células Receptoras Sensoriais/ultraestruturaRESUMO
Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration(1). Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents(2). Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal(3,4). This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae(4). Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions(5,4). Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration(6, 7). Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps(8,9,10,11,12). Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads(13,14,1). We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.
Assuntos
Músculo Esquelético/inervação , Compressão Nervosa/métodos , Regeneração Nervosa/fisiologia , Nervo Isquiático/fisiologia , Nervo Isquiático/cirurgia , Animais , Modelos Animais de Doenças , Membro Posterior/cirurgia , Camundongos , Camundongos Endogâmicos C57BL , Reprodutibilidade dos Testes , Nervo Isquiático/lesõesRESUMO
After central nervous system (CNS) injury axons fail to regenerate often leading to persistent neurologic deficit although injured peripheral nervous system (PNS) axons mount a robust regenerative response that may lead to functional recovery. Some of the failures of CNS regeneration arise from the many glial-based inhibitory molecules found in the injured CNS, whereas the intrinsic regenerative potential of some CNS neurons is actively curtailed during CNS maturation and limited after injury. In this review, the molecular basis for extrinsic and intrinsic modulation of axon regeneration within the nervous system is evaluated. A more complete understanding of the factors limiting axonal regeneration will provide a rational basis, which is used to develop improved treatments for nervous system injury.
RESUMO
Distal hereditary motor neuropathies comprise a clinically and genetically heterogeneous group of disorders. We recently mapped an X-linked form of this condition to chromosome Xq13.1-q21 in two large unrelated families. The region of genetic linkage included ATP7A, which encodes a copper-transporting P-type ATPase mutated in patients with Menkes disease, a severe infantile-onset neurodegenerative condition. We identified two unique ATP7A missense mutations (p.P1386S and p.T994I) in males with distal motor neuropathy in two families. These molecular alterations impact highly conserved amino acids in the carboxyl half of ATP7A and do not directly involve the copper transporter's known critical functional domains. Studies of p.P1386S revealed normal ATP7A mRNA and protein levels, a defect in ATP7A trafficking, and partial rescue of a S. cerevisiae copper transport knockout. Although ATP7A mutations are typically associated with severe Menkes disease or its milder allelic variant, occipital horn syndrome, we demonstrate here that certain missense mutations at this locus can cause a syndrome restricted to progressive distal motor neuropathy without overt signs of systemic copper deficiency. This previously unrecognized genotype-phenotype correlation suggests an important role of the ATP7A copper transporter in motor-neuron maintenance and function.
Assuntos
Adenosina Trifosfatases/genética , Proteínas de Transporte de Cátions/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doença dos Neurônios Motores/genética , Mutação de Sentido Incorreto , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Adolescente , Adulto , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Células Cultivadas , Pré-Escolar , Cobre/metabolismo , ATPases Transportadoras de Cobre , Primers do DNA/genética , Feminino , Estudos de Associação Genética , Teste de Complementação Genética , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Síndrome dos Cabelos Torcidos/genética , Síndrome dos Cabelos Torcidos/metabolismo , Pessoa de Meia-Idade , Modelos Moleculares , Dados de Sequência Molecular , Doença dos Neurônios Motores/metabolismo , Linhagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Síndrome , Adulto JovemRESUMO
Progressive loss of motor neurons causes Amyotrophic Lateral Sclerosis. Patients complain, most often, of progressive weakness in the distal limbs. However, weakness may manifest in any body segment (bulbar, cervical, thoracic, or lumbosacral). The diagnosis of ALS is suggested by clinical examination that reveals both upper and lower motor neuron failure. Formal diagnostic criteria have been developed and validated. Nerve conduction and electromyography studies improve diagnostic sensitivity and exclude some alternate, treatable diagnoses. Likewise, conventional imaging studies and laboratory evaluation refute other diseases that may masquerade as ALS. Experimental imaging and laboratory evaluations may improve ALS diagnosis in the future. The cause of motor neuron death is not known but inherited forms of motor neuron disease may suggest mechanisms. The goal of ALS treatment is control of the symptoms of progressive weakness, especially respiratory insufficiency and dysphagia and is best managed in an integrated clinic.
Assuntos
Esclerose Lateral Amiotrófica/diagnóstico , Esclerose Lateral Amiotrófica/etiologia , Esclerose Lateral Amiotrófica/fisiopatologia , Diagnóstico Diferencial , Humanos , Condução Nervosa/fisiologia , Superóxido Dismutase/genética , Superóxido Dismutase-1RESUMO
Our past work indicates that growth-inhibiting chondroitin sulfate proteoglycan (CSPG) is abundant in the peripheral nerve sheaths and interstitium. In this study we tested if degradation of CSPG by chondroitinase enhances axonal regeneration through the site of injury after (a) nerve crush and (b) nerve transection and coaptation. Adult rats received the same injury bilaterally to the sciatic nerves and then chondroitinase ABC was injected near the injury site on one side, and the contralateral nerve was injected with vehicle alone. Nerves were examined 2 days after injury in the nerve crush model and 4 days after injury in the nerve transection model. Chondroitinase-dependent neoepitope immunolabeling showed that CSPG was thoroughly degraded around the injury site in the chondroitinase-treated nerves. Axonal regeneration through the injury site and into the distal nerve was assessed by GAP-43 immunolabeling. Axonal regeneration after crush injury was similar in chondroitinase-treated and control nerves. In contrast, axonal regrowth through the coaptation of transected nerves was markedly accelerated and the ingress of axons into the distal segment was increased severalfold in nerves injected with chondroitinase. On the basis of these results we concluded that growth inhibition by CSPG contributes critically to the poor regenerative growth of axons in nerve transection repair. In addition, degradation of CSPG by injection of chondroitinase ABC at the site of nerve repair increased the ingress of axonal sprouts into basal laminae of the distal nerve segment, presumably by enabling more latitude in growth at the interface of coapted nerve. This suggests that chondroitinase application may be used clinically to improve the outcome of primary peripheral nerve repair.