RESUMO
The microtubule motor cytoplasmic dynein and its activator dynactin drive vesicular transport and mitotic spindle organization. Dynactin is ubiquitously expressed in eukaryotes, but a G59S mutation in the p150Glued subunit of dynactin results in the specific degeneration of motor neurons. This mutation in the conserved cytoskeleton-associated protein, glycine-rich (CAP-Gly) domain lowers the affinity of p150Glued for microtubules and EB1. Cell lines from patients are morphologically normal but show delayed recovery after nocodazole treatment, consistent with a subtle disruption of dynein/dynactin function. The G59S mutation disrupts the folding of the CAP-Gly domain, resulting in aggregation of the p150Glued protein both in vitro and in vivo, which is accompanied by an increase in cell death in a motor neuron cell line. Overexpression of the chaperone Hsp70 inhibits aggregate formation and prevents cell death. These data support a model in which a point mutation in p150Glued causes both loss of dynein/dynactin function and gain of toxic function, which together lead to motor neuron cell death.
Assuntos
Transtornos Heredodegenerativos do Sistema Nervoso/genética , Transtornos Heredodegenerativos do Sistema Nervoso/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/fisiologia , Neurônios Motores/metabolismo , Animais , Apoptose/genética , Células COS , Células Cultivadas , Chlorocebus aethiops , Complexo Dinactina , Dineínas/metabolismo , Proteínas de Choque Térmico HSP70/biossíntese , Proteínas de Choque Térmico HSP70/genética , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/química , Microtúbulos/genética , Microtúbulos/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mutação PuntualRESUMO
Cytoplasmic dynein and dynactin drive retrograde axonal transport in neurons, and mutations in dynein/dynactin cause motor neuron degeneration. To test whether defects in dynein/dynactin function are involved in the neurodegenerative disease amyotrophic lateral sclerosis, we examined neurotracer transport from muscle to motor neuron in a transgenic mouse model of amyotrophic lateral sclerosis. Significant inhibition was observed, which was temporally correlated with declines in muscle strength. No decrease in dynein/dynactin expression was observed, but immunohistochemistry suggests that dynein associates with aggregates of mutant Cu/Zn superoxide dismutase 1. Expression of mutant Cu/Zn superoxide dismutase 1 in primary motor neurons altered the cellular localization of dynein, suggesting an inhibition of dynein/dynactin function. Thus, inhibition of dynein/dynactin function may have a role in motor neuron degeneration in amyotrophic lateral sclerosis.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/fisiopatologia , Dineínas/metabolismo , Neurônios Motores/metabolismo , Superóxido Dismutase/genética , Animais , Citoplasma/metabolismo , Complexo Dinactina , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Motores Moleculares/metabolismo , Degeneração Neural/metabolismo , Degeneração Neural/fisiopatologia , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1RESUMO
Impaired axonal transport in motor neurons has been proposed as a mechanism for neuronal degeneration in motor neuron disease. Here we show linkage of a lower motor neuron disease to a region of 4 Mb at chromosome 2p13. Mutation analysis of a gene in this interval that encodes the largest subunit of the axonal transport protein dynactin showed a single base-pair change resulting in an amino-acid substitution that is predicted to distort the folding of dynactin's microtubule-binding domain. Binding assays show decreased binding of the mutant protein to microtubules. Our results show that dysfunction of dynactin-mediated transport can lead to human motor neuron disease.
Assuntos
Cromossomos Humanos Par 2 , Proteínas Associadas aos Microtúbulos/genética , Doença dos Neurônios Motores/genética , Motivos de Aminoácidos , Animais , Transporte Biológico , Centrômero/metabolismo , Clonagem Molecular , Drosophila , Complexo Dinactina , Ligação Genética , Humanos , Camundongos , Camundongos Transgênicos , Microtúbulos/metabolismo , Modelos Genéticos , Modelos Moleculares , Mutação , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Recombinação GenéticaRESUMO
Medical records of 27 horses (including 13 ponies) treated with pergolide or cyproheptadine for pituitary pars intermedia dysfunction were reviewed to determine the effect of treatment on plasma ACTH, insulin, and glucose concentrations and clinical signs. Prior to treatment, the most common clinical signs were laminitis, hirsutism, and abnormal body fat distribution. The median pergolide dose was 3.0 microg/kg p.o. q24h (range, 1.7-5.5 microg/kg). All horses treated with cyproheptadine were given 0.25 mg/kg p.o. q24h. After pergolide treatment, ACTH concentrations (n = 20; median = 30.4 pg/ml; range, 4.2-173) were significantly lower (P < .01) than those in horses treated with cyproheptadine (n = 7; median = 141.0 pg/ml: range, 10-1,230). Among horses treated with pergolide, there was a correlation between ACTH concentration after treatment and the duration of treatment (P < .001) and pergolide dose (P = .04). Significantly (P = .02) more owners of horses treated with pergolide (85%, 17/20) reported an improvement in clinical signs compared to owners of horses treated with cyproheptadine (28%, 2/7).
Assuntos
Síndrome de Cushing/tratamento farmacológico , Síndrome de Cushing/veterinária , Ciproeptadina/farmacologia , Agonistas de Dopamina/farmacologia , Doenças dos Cavalos/tratamento farmacológico , Pergolida/farmacologia , Antagonistas da Serotonina/farmacologia , Hormônio Adrenocorticotrópico/sangue , Animais , Glicemia , Ciproeptadina/administração & dosagem , Agonistas de Dopamina/administração & dosagem , Feminino , Doenças dos Cavalos/patologia , Cavalos , Hipoglicemiantes/sangue , Insulina/sangue , Masculino , Pergolida/administração & dosagem , Antagonistas da Serotonina/administração & dosagem , Resultado do TratamentoRESUMO
To test the hypothesis that inhibition of axonal transport is sufficient to cause motor neuron degeneration such as that observed in amyotrophic lateral sclerosis (ALS), we engineered a targeted disruption of the dynein-dynactin complex in postnatal motor neurons of transgenic mice. Dynamitin overexpression was found to disassemble dynactin, a required activator of cytoplasmic dynein, resulting in an inhibition of retrograde axonal transport. Mice overexpressing dynamitin demonstrate a late-onset progressive motor neuron degenerative disease characterized by decreased strength and endurance, motor neuron degeneration and loss, and denervation of muscle. Previous transgenic mouse models of ALS have shown abnormalities in microtubule-based axonal transport. In this report, we describe a mouse model that confirms the critical role of disrupted axonal transport in the pathogenesis of motor neuron degenerative disease.
