RESUMEN
Mutations in the gene encoding ubiquilin2 (UBQLN2) cause amyotrophic lateral sclerosis (ALS), frontotemporal type of dementia, or both. However, the molecular mechanisms are unknown. Here, we show that ALS/dementia-linked UBQLN2(P497H) transgenic mice develop neuronal pathology with ubiquilin2/ubiquitin/p62-positive inclusions in the brain, especially in the hippocampus, recapitulating several key pathological features of dementia observed in human patients with UBQLN2 mutations. A major feature of the ubiquilin2-related pathology in these mice, and reminiscent of human disease, is a dendritic spinopathy with protein aggregation in the dendritic spines and an associated decrease in dendritic spine density and synaptic dysfunction. Finally, we show that the protein inclusions in the dendritic spines are composed of several components of the proteasome machinery, including Ub(G76V)-GFP, a representative ubiquitinated protein substrate that is accumulated in the transgenic mice. Our data, therefore, directly link impaired protein degradation to inclusion formation that is associated with synaptic dysfunction and cognitive deficits. These data imply a convergent molecular pathway involving synaptic protein recycling that may also be involved in other neurodegenerative disorders, with implications for development of widely applicable rational therapeutics.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Proteínas de Ciclo Celular/genética , Demencia/genética , Mutación , Ubiquitinas/genética , Proteínas Adaptadoras Transductoras de Señales , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Proteínas Relacionadas con la Autofagia , Encéfalo/metabolismo , Encéfalo/patología , Proteínas de Ciclo Celular/metabolismo , Trastornos del Conocimiento/genética , Trastornos del Conocimiento/fisiopatología , Demencia/metabolismo , Demencia/fisiopatología , Espinas Dendríticas/genética , Espinas Dendríticas/metabolismo , Espinas Dendríticas/patología , Espinas Dendríticas/ultraestructura , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Inmunohistoquímica , Cuerpos de Inclusión/metabolismo , Aprendizaje por Laberinto/fisiología , Ratones Endogámicos , Ratones Transgénicos , Microscopía Confocal , Microscopía Electrónica , Actividad Motora/genética , Actividad Motora/fisiología , Complejo de la Endopetidasa Proteasomal/metabolismo , Médula Espinal/metabolismo , Médula Espinal/patología , Médula Espinal/fisiopatología , Transmisión Sináptica/genética , Transmisión Sináptica/fisiología , Ubiquitinas/metabolismoRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disorder characterized by degeneration of motor neurons and atrophy of skeletal muscle. Mutations in the superoxide dismutase (SOD1) gene are linked to 20% cases of inherited ALS. Mitochondrial dysfunction has been implicated in the pathogenic process, but how it contributes to muscle degeneration of ALS is not known. Here we identify a specific deficit in the cellular physiology of skeletal muscle derived from an ALS mouse model (G93A) with transgenic overexpression of the human SOD1(G93A) mutant. The G93A skeletal muscle fibers display localized loss of mitochondrial inner membrane potential in fiber segments near the neuromuscular junction. These defects occur in young G93A mice prior to disease onset. Fiber segments with depolarized mitochondria show greater osmotic stress-induced Ca(2+) release activity, which can include propagating Ca(2+) waves. These Ca(2+) waves are confined to regions of depolarized mitochondria and stop propagating shortly upon entering the regions of normal, polarized mitochondria. Uncoupling of mitochondrial membrane potential with FCCP or inhibition of mitochondrial Ca(2+) uptake by Ru360 lead to cell-wide propagation of such Ca(2+) release events. Our data reveal that mitochondria regulate Ca(2+) signaling in skeletal muscle, and loss of this capacity may contribute to the progression of muscle atrophy in ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Señalización del Calcio , Espacio Intracelular/metabolismo , Mitocondrias/patología , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Animales , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Modelos Animales de Enfermedad , Espacio Intracelular/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Modelos Biológicos , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Fibras Musculares Esqueléticas/ultraestructura , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/ultraestructura , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/metabolismo , Unión Neuromuscular/patología , Unión Neuromuscular/ultraestructura , Compuestos de Rutenio/farmacología , Estrés Fisiológico/efectos de los fármacosRESUMEN
Amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of motoneurons. One potential mechanism is excitotoxicity. We studied the behaviors of spinal neurons using an in vitro preparation of the sacral cord from the G93A SOD1 mouse model of ALS. Measurements were conducted at presymptomatic [approximately postnatal day 50 (approximately P50)], early (approximately P90), and late (>P120) stages of the disease. Short-latency reflexes (SRs) in ventral roots, presumably monosynaptic, were evoked by electrical stimulation of a dorsal root. The fraction of motoneurons capable of responding to this activation was evaluated by measuring the compound action potential [total motor activity (TMA)] evoked by antidromic stimulation of the distal ventral root. In mutant SOD1 (mSOD1) mice, both the SR and the TMA decreased with age compared with nontransgenic littermates, ruling out the SR as a source of increasing excitotoxicity. Spinal interneuron activity was assessed using the synchronized ventral root bursts generated by both bath application of blockers of inhibitory neurotransmitters (glycine, GABA(A)) and agonists of glutamate receptors (especially NMDA receptors). After symptom onset, a higher percentage of preparations from mSOD1 mice exhibited bursting, and these bursts exhibited more sub-bursts and a more disorganized pattern. In mSOD1 mice with clear muscle tremor, the ventral roots exhibited spontaneous synchronized bursts, which were highly sensitive to the blockade of NMDA receptors. These data suggest that although short-latency sensory input does not increase as symptoms develop, interneuron activity does increase and may contribute to excitotoxicity.
Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Neuronas Motoras/patología , Neuronas Motoras/fisiología , Médula Espinal/patología , Sinapsis/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Factores de Edad , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Biofisica/métodos , Distribución de Chi-Cuadrado , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Estimulación Eléctrica , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Humanos , Interneuronas/fisiología , Ratones , Ratones Transgénicos , Mutación , N-Metilaspartato/farmacología , Técnicas de Placa-Clamp/métodos , Desempeño Psicomotor/efectos de los fármacos , Desempeño Psicomotor/fisiología , Quinoxalinas/farmacología , Tiempo de Reacción/genética , Reflejo/efectos de los fármacos , Reflejo/genética , Reflejo/fisiología , Superóxido Dismutasa/genética , Superóxido Dismutasa-1 , Sinapsis/patologíaRESUMEN
Mutations in Cu,Zn superoxide dismutase (SOD1) are associated with amyotrophic lateral sclerosis (ALS). Among more than 100 ALS-associated SOD1 mutations, premature termination codon (PTC) mutations exclusively occur in exon 5, the last exon of SOD1. The molecular basis of ALS-associated toxicity of the mutant SOD1 is not fully understood. Here, we show that nonsense-mediated mRNA decay (NMD) underlies clearance of mutant mRNA with a PTC in the non-terminal exons. To further define the crucial ALS-associated SOD1 fragments, we designed and tested an exon-fusion approach using an artificial transgene SOD1(T116X) that harbors a PTC in exon 4. We found that the SOD1(T116X) transgene with a fused exon could escape NMD in cellular models. We generated a transgenic mouse model that overexpresses SOD1(T116X). This mouse model developed ALS-like phenotype and pathology. Thus, our data have demonstrated that a 'mini-SOD1' of only 115 amino acids is sufficient to cause ALS. This is the smallest ALS-causing SOD1 molecule currently defined. This proof of principle result suggests that the exon-fusion approach may have potential not only to further define a shorter ALS-associated SOD1 fragment, thus providing a molecular target for designing rational therapy, but also to dissect toxicities of other proteins encoded by genes of multiple exons through a 'gain of function' mechanism.
Asunto(s)
Esclerosis Amiotrófica Lateral/enzimología , Esclerosis Amiotrófica Lateral/genética , Fusión Artificial Génica/métodos , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Secuencia de Aminoácidos/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Codón sin Sentido , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Exones , Humanos , Ratones , Ratones Transgénicos , Estabilidad del ARN , ARN Mensajero/metabolismo , Eliminación de Secuencia , Superóxido Dismutasa-1RESUMEN
Mutations in Alsin are associated with chronic juvenile amyotrophic lateral sclerosis (ALS2), juvenile primary lateral sclerosis and infantile-onset ascending spastic paralysis. The primary pathology and pathogenic mechanism of the disease remain largely unknown. Here we show that alsin-deficient mice have motor impairment and degenerative pathology in the distal corticospinal tracts without apparent motor neuron pathology. Our data suggest that ALS2 is predominantly a distal axonopathy, rather than a neuronopathy in the central nervous system of the mouse model, implying that alsin plays an important role in maintaining the integrity of the corticospinal axons.
Asunto(s)
Axones/patología , Factores de Intercambio de Guanina Nucleótido/deficiencia , Secuencia de Aminoácidos , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Axones/fisiología , Encéfalo/patología , Modelos Animales de Enfermedad , Exones , Femenino , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/fisiología , Humanos , Masculino , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Neuronas Motoras/patología , Neuronas Motoras/fisiología , Mutación , Embarazo , Médula Espinal/patología , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1RESUMEN
Point mutations in Cu, Zn-superoxide dismutase (SOD1) cause a familial form of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Aggregates of mutant SOD1 proteins are observed in histopathology and are invoked in several proposed mechanisms for motor neuronal death; however, the significant stability and activity of the mature mutant proteins are not readily explained in such models. Recent biochemical studies suggest that it is the immature disulfide-reduced forms of the familial ALS mutant SOD1 proteins that play a critical role; these forms tend to misfold, oligomerize, and readily undergo incorrect disulfide formation upon mild oxidative stress in vitro. Here we provide physiological support for this mechanism of aggregate formation and show that a significant fraction of the insoluble SOD1 aggregates in spinal cord of the ALS-model transgenic mice contain multimers cross-linked via intermolecular disulfide bonds. These insoluble disulfide-linked SOD1 multimers are found only in the spinal cord of symptomatic transgenic animals, are not observed in unafflicted tissue such as brain cortex and liver, and can incorporate WT SOD1 protein. The findings provide a biochemical basis for a pathological hallmark of this disease; namely, incorrect disulfide cross-linking of the immature, misfolded mutant proteins leads to insoluble aggregates.
Asunto(s)
Esclerosis Amiotrófica Lateral/enzimología , Reactivos de Enlaces Cruzados/química , Disulfuros/química , Médula Espinal/enzimología , Superóxido Dismutasa/química , Superóxido Dismutasa/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Cisteína/metabolismo , Humanos , Ratones , Ratones Transgénicos , Modelos Moleculares , Oxidación-Reducción , Estrés Oxidativo , Conformación Proteica , Pliegue de Proteína , Médula Espinal/citología , Superóxido Dismutasa/genéticaRESUMEN
Twenty percent of the familial form of amyotrophic lateral sclerosis (ALS) is caused by mutations in the Cu, Zn-superoxide dismutase gene (SOD1) through the gain of a toxic function. The nature of this toxic function of mutant SOD1 has remained largely unknown. Here we show that WT SOD1 not only hastens onset of the ALS phenotype but can also convert an unaffected phenotype to an ALS phenotype in mutant SOD1 transgenic mouse models. Further analyses of the single- and double-transgenic mice revealed that conversion of mutant SOD1 from a soluble form to an aggregated and detergent-insoluble form was associated with development of the ALS phenotype in transgenic mice. Conversion of WT SOD1 from a soluble form to an aggregated and insoluble form also correlates with exacerbation of the disease or conversion to a disease phenotype in double-transgenic mice. This conversion, observed in the mitochondrial fraction of the spinal cord, involved formation of insoluble SOD1 dimers and multimers that are crosslinked through intermolecular disulfide bonds via oxidation of cysteine residues in SOD1. Our data thus show a molecular mechanism by which SOD1, an important protein in cellular defense against free radicals, is converted to aggregated and apparently ALS-associated toxic dimers and multimers by redox processes. These findings provide evidence of direct links among oxidation, protein aggregation, mitochondrial damage, and SOD1-mediated ALS, with possible applications to the aging process and other late-onset neurodegenerative disorders. Importantly, rational therapy based on these observations can now be developed and tested.
Asunto(s)
Esclerosis Amiotrófica Lateral/enzimología , Mitocondrias/enzimología , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Humanos , Ratones , Ratones Transgénicos , Mitocondrias/ultraestructura , Mutación , Fenotipo , Conformación Proteica , Médula Espinal/citología , Superóxido Dismutasa/química , Superóxido Dismutasa-1 , Tasa de SupervivenciaRESUMEN
ALS (amyotrophic lateral sclerosis) is an adult-onset and deadly neurodegenerative disease characterized by a progressive and selective loss of motoneurons. Transgenic mice overexpressing a mutated human gene (G93A) coding for the enzyme SOD1 (Cu/Zn superoxide dismutase) develop a motoneuron disease resembling ALS in humans. In this generally accepted ALS model, we tested the electrophysiological properties of individual embryonic and neonatal spinal motoneurons in culture by measuring a wide range of electrical properties influencing motoneuron excitability during current clamp. There were no differences in the motoneuron resting potential, input conductance, action potential shape, or afterhyperpolarization between G93A and control motoneurons. The relationship between the motoneuron's firing frequency and injected current (f-I relation) was altered. The slope of the f-I relation and the maximal firing rate of the G93A motoneurons were much greater than in the control motoneurons. Differences in spontaneous synaptic input were excluded as a cause of increased excitability. This finding identifies a markedly elevated intrinsic electrical excitability in cultured embryonic and neonatal mutant G93A spinal motoneurons. We conclude that the observed intrinsic motoneuron hyperexcitability is induced by the SOD1 toxic gain-of-function through an aberration in the process of action potential generation. This hyperexcitability may play a crucial role in the pathogenesis of ALS as the motoneurons were cultured from presymptomatic mice.