RESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal disease of unknown etiology. Mutations in copper/zinc superoxide dismutase (SOD1) are the most commonly associated genetic abnormality. Given that SOD1 is ubiquitously expressed, the exclusive vulnerability of motor neurons is one of the most puzzling issues in ALS research. We here report that wild-type SOD1 mRNA forms ribonucleoprotein (RNP) complexes with protein homogenates of neuronal tissue but not with homogenates of non-neuronal tissues. 3' Untranslated region of SOD1 mRNA-dependent RNP complexes functioned to stabilize SOD1 mRNA. Moreover, SOD1 mRNAs harboring ALS-associated mutations, including silent mutations, were deficient in forming RNP complexes. In contrast, SOD1 mRNAs harboring artificial mutations, not known to be associated with ALS, demonstrated preserved RNP complex formation. This paper reports RNP complex formation on SOD1 mRNA as a neuronal tissue-specific and ALS-associated mutation sensitive feature.
Asunto(s)
Enfermedad de la Neurona Motora/genética , Mutación , ARN Mensajero/genética , Superóxido Dismutasa/genética , Humanos , Enfermedad de la Neurona Motora/enzimología , Neuronas , ARN/genética , ARN/aislamiento & purificación , Sondas ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleoproteínas/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1 , Transcripción GenéticaRESUMEN
Neurofilament (NF) aggregate formation within motor neurons is a pathological hallmark of both the sporadic and familial forms of amyotrophic lateral sclerosis (ALS). The relationship between aggregate formation and both microglial and astrocytic proliferation, as well as additional neuropathological features of ALS, is unknown. To examine this, we have used transgenic mice that develop NF aggregates, through either a lack of the low-molecular-weight NF subunit [NFL (-/-)] or the overexpression of human NFL [hNFL (+/+)]. Transgenic and wild-type C57bl/6 mice were examined from 1 month to 18 months of age, and the temporal pattern of motor neuron degeneration, microglial and astrocytic proliferation, and heat shock protein-70 (HSP-70) expression characterized. We observed three overlapping phases in both transgenic mice, including transient aggregate formation, reactive microgliosis, and progressive motor neuron loss. However, only NFL (-/-) mice demonstrated significant astrogliosis and HSP-70 upregulation in both motor neurons and astrocytes. These in vivo models suggest that the development of NF aggregates in motor neurons leads to motor neuron death, but that the interaction between the degenerating motor neurons and the adjacent non-neuronal cells may differ significantly depending on the etiology of the NF aggregate itself.
Asunto(s)
Esclerosis Amiotrófica Lateral/fisiopatología , Gliosis/fisiopatología , Degeneración Nerviosa/fisiopatología , Proteínas de Neurofilamentos/genética , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Astrocitos/metabolismo , Astrocitos/patología , Caspasa 3 , Caspasas/metabolismo , Muerte Celular/fisiología , Proliferación Celular , Modelos Animales de Enfermedad , Gliosis/genética , Gliosis/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microglía/metabolismo , Microglía/patología , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Degeneración Nerviosa/genética , Degeneración Nerviosa/metabolismo , Factores de TiempoRESUMEN
The significance of copper/zinc superoxide dismutase (SOD1) and neuronal nitric oxide synthase (nNOS) co-localization to neurofilamentous (NF) aggregates in amyotrophic lateral sclerosis (ALS) is unknown. In this study, we have used dissociated motor neurons from either C57BL/6 or mice that over-express the human low molecular weight neurofilament protein (hNFL+/+) to examine the relationship between NF aggregate formation, SOD1 and nNOS co-localization, and the regulation of NMDA-mediated calcium influx in vitro. The intracellular distribution of NF aggregates, SOD1 and nNOS was examined by confocal microscopy and NMDA-induced alterations in intracellular calcium levels using either Oregon green fluorescence or FURA-2 photometric imaging. Cell death was assessed using an antibody to activated caspase-3. C57 Bl/6 motor neurons expressed nNOS in a punctate manner, whereas SOD1 was distributed homogeneously throughout the cytosol. In contrast, hNFL+/+ motor neurons demonstrated co-localization of SOD1 and nNOS by day 9 post-plating, preceding the formation of NF aggregates. Both proteins co-localized to NF aggregates once formed. With NMDA stimulation, aggregate-bearing hNFL+/+ motor neurons demonstrated significant increases in intracellular calcium, whereas only a minimal alteration in intracellular calcium was observed in C57 Bl/6 neurons. Following stimulation with 100 microM NMDA, 75.5+/-5.5% of hNFL+/+ neurons became apoptotic, whereas only 16.3+/-5.3% of C57 Bl/6 were. These observations suggest that the presence of NF aggregates results in a failure of regulation of NMDA-mediated calcium influx, and that this occurs due to the sequestration of nNOS to the NF aggregate, preventing its down-regulation of the NMDA receptor.