RESUMEN
Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive neurodegenerative disease affecting upper and lower motoneurons (MNs). Although the motor phenotype is a hallmark for ALS, there is increasing evidence that systems other than the efferent MN system can be involved. Mutations of superoxide dismutase 1 (SOD1) gene cause a proportion of familial forms of this disease. Misfolding and aggregation of mutant SOD1 exert neurotoxicity in a noncell autonomous manner, as evidenced in studies using transgenic mouse models. Here, we used the SOD1(G93A) mouse model for ALS to detect, by means of conformational-specific anti-SOD1 antibodies, whether misfolded SOD1-mediated neurotoxicity extended to neuronal types other than MNs. We report that large dorsal root ganglion (DRG) proprioceptive neurons accumulate misfolded SOD1 and suffer a degenerative process involving the inflammatory recruitment of macrophagic cells. Degenerating sensory axons were also detected in association with activated microglial cells in the spinal cord dorsal horn of diseased animals. As large proprioceptive DRG neurons project monosynaptically to ventral horn MNs, we hypothesise that a prion-like mechanism may be responsible for the transsynaptic propagation of SOD1 misfolding from ventral horn MNs to DRG sensory neurons.
Asunto(s)
Esclerosis Amiotrófica Lateral/enzimología , Esclerosis Amiotrófica Lateral/patología , Ganglios Espinales/enzimología , Mutación Missense , Pliegue de Proteína , Células Receptoras Sensoriales/enzimología , Superóxido Dismutasa/metabolismo , Sustitución de Aminoácidos , Esclerosis Amiotrófica Lateral/genética , Animales , Ganglios Espinales/patología , Humanos , Ratones , Ratones Transgénicos , Células Receptoras Sensoriales/patología , Superóxido Dismutasa/genética , Superóxido Dismutasa-1RESUMEN
C boutons are large, cholinergic, synaptic terminals that arise from local interneurons and specifically contact spinal α-motoneurons (MNs). C boutons characteristically display a postsynaptic specialization consisting of an endoplasmic reticulum-related subsurface cistern (SSC) of unknown function. In the present work, by using confocal microscopy and ultrastructural immunolabeling, we demonstrate that neuregulin-1 (NRG1) accumulates in the SSC of mouse spinal MNs. We also show that the NRG1 receptors erbB2 and erbB4 are presynaptically localized within C boutons, suggesting that NRG1-based retrograde signaling may occur in this type of synapse. In most of the cranial nuclei, MNs display the same pattern of NRG1 distribution as that observed in spinal cord MNs. Conversely, MNs in oculomotor nuclei, which are spared in amyotrophic lateral sclerosis (ALS), lack both C boutons and SSC-associated NRG1. NRG1 in spinal MNs is developmentally regulated and depends on the maintenance of nerve-muscle interactions, as we show after nerve transection experiments. Changes in NRG1 in C boutons were also investigated in mouse models of MN diseases: i.e., spinal muscular atrophy (SMNΔ7) and ALS (SOD1(G93A)). In both models, a transient increase in NRG1 in C boutons occurs during disease progression. These data increase our understanding of the role of C boutons in MN physiology and pathology.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Proteínas Aviares/fisiología , Neuronas Motoras/metabolismo , Atrofia Muscular Espinal/metabolismo , Neurregulina-1/fisiología , Orgánulos/química , Densidad Postsináptica/química , Terminales Presinápticos/química , Esclerosis Amiotrófica Lateral/patología , Animales , Proteínas Aviares/análisis , Embrión de Pollo , Pollos , Receptores ErbB/análisis , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas Motoras/patología , Atrofia Muscular Espinal/patología , Neurregulina-1/análisis , Neurregulina-1/biosíntesis , Neurregulina-1/genética , Densidad Postsináptica/ultraestructura , Terminales Presinápticos/ultraestructura , Receptor ErbB-2/análisis , Receptor ErbB-4 , Nervio Ciático/lesiones , Nervio Ciático/ultraestructura , Médula Espinal/citología , Médula Espinal/embriología , Médula Espinal/crecimiento & desarrolloRESUMEN
We previously showed that some antipurinergic receptor P2X4 antibodies cross react with misfolded forms of amyotrophic lateral sclerosis (ALS)-linked mutant Cu/Zn superoxide dismutase (SOD1). Cross reactivity might be caused by abnormal exposure of an epitope in the inner hydrophobic region of SOD1 that shares structural homology with the P2X4-immunizing peptide. Here, we raised antibodies against the human SOD1 epitope mimicked by the P2X4 immunizing peptide. One of these antibodies, AJ10, is a recognized mutant/misfolded form of ALS-linked mutant SOD1. This was demonstrated in the hybrid motoneuron cell line NSC34 expressing enhanced green fluorescent protein-tagged G943A or A4V mutant SOD1. We also found AJ10 immunoreactivity to be selectively associated with degenerating neurons but not with glial cells in mice overexpressing either SOD1 or SOD1 mutants. Neurons with strongly positive AJ10 immunostaining were often associated with activated microglia displaying neuronophagic activity. AJ10-immunopositive SOD1 aggregates were also found in spinal cord tissue from a patient with a SOD1-linked familial ALS. AJ10-immunoreactive mutant SOD1 conformers were localized in large intracellular protein aggregates with a filamentous amyloid-like organization by ultrastructural immunolabeling and were also detected in neuronal organelles. These data are consistent with the ability of the AJ10 antibody to recognize misfolded conformations of SOD1 shared by different ALS-linked SOD1 mutations but not with the native protein. The neuronal mutant SOD1 conformers detected with AJ10 may promote neuroinflammation and may define a new epitope in SOD1 for ALS research.
Asunto(s)
Esclerosis Amiotrófica Lateral/complicaciones , Mutación/genética , Degeneración Nerviosa/diagnóstico , Degeneración Nerviosa/enzimología , Superóxido Dismutasa/genética , Anciano , Esclerosis Amiotrófica Lateral/genética , Animales , Especificidad de Anticuerpos , Línea Celular , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Neuronas/patología , Neuronas/ultraestructura , Médula Espinal/metabolismo , Médula Espinal/patología , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1RESUMEN
Childhood spinal muscular atrophy is an autosomal recessive neuromuscular disorder caused by alterations in the Survival Motor Neuron 1 gene that triggers degeneration of motor neurons within the spinal cord. Spinal muscular atrophy is the second most common severe hereditary disease of infancy and early childhood. In the most severe cases (type I), the disease appears in the first months of life, suggesting defects in fetal development. However, it is not yet known how motor neurons, neuromuscular junctions, and muscle interact in the neuropathology of the disease. We report the structure of presynaptic and postsynaptic apparatus of the neuromuscular junctions in control and spinal muscular atrophy prenatal and postnatal human samples. Qualitative and quantitative data from confocal and electron microscopy studies revealed changes in acetylcholine receptor clustering, abnormal preterminal accumulation of vesicles, and aberrant ultrastructure of nerve terminals in the motor endplates of prenatal type I spinal muscular atrophy samples. Fetuses predicted to develop milder type II disease had a similar appearance to controls. Postnatal muscle of type I spinal muscular atrophy patients showed persistence of the fetal subunit of acetylcholine receptors, suggesting a delay in maturation of neuromuscular junctions. We observed that pathology in the severe form of the disease starts in fetal development and that a defect in maintaining the initial innervation is an early finding of neuromuscular dysfunction. These results will improve our understanding of the spinal muscular atrophy pathogenesis and help to define targets for possible presymptomatic therapy for this disease.
Asunto(s)
Neuronas Motoras/patología , Músculo Esquelético/patología , Unión Neuromuscular/patología , Atrofias Musculares Espinales de la Infancia/patología , Estudios de Casos y Controles , Predisposición Genética a la Enfermedad , Edad Gestacional , Humanos , Lactante , Recién Nacido , Microscopía Confocal , Microscopía Electrónica , Morfogénesis , Placa Motora/patología , Neuronas Motoras/química , Neuronas Motoras/ultraestructura , Músculo Esquelético/embriología , Músculo Esquelético/inervación , Músculo Esquelético/ultraestructura , Unión Neuromuscular/embriología , Unión Neuromuscular/ultraestructura , Fenotipo , Terminales Presinápticos/química , Terminales Presinápticos/patología , Receptores Colinérgicos/análisis , Índice de Severidad de la Enfermedad , Atrofias Musculares Espinales de la Infancia/embriología , Atrofias Musculares Espinales de la Infancia/genética , Atrofias Musculares Espinales de la Infancia/metabolismoRESUMEN
A detailed pathologic analysis was performed on Smn(-/-);SMN2 mice as a mouse model for human type I spinal muscular atrophy (SMA). We provide new data concerning changes in the spinal cord, neuromuscular junctions and muscle cells, and in the organs of the immune system. The expression of 10 synaptic proteins was analyzed in 3-dimensionally reconstructed neuromuscular junctions by confocal microscopy. In addition to defects in postsynaptic occupancy, there was a marked reduction in calcitonin gene-related peptide and Rab3A in the presynaptic motor terminals of some, but not all, of the skeletal muscles analyzed. Defects in the organization of presynaptic nerve terminals were also detected by electron microscopy. Moreover, degenerative changes in muscle cells, defective postnatal muscle growth, and prominent muscle satellite cell apoptosis were also observed. All of these changes occurred in the absence of massive loss of spinal cord motoneurons. On the other hand, astroglia, but not microglia, increased in the ventral horn of newborn SMA mice. In skeletal muscles, the density of interstitial macrophages was significantly reduced, and monocyte chemotactic protein-1 was downregulated. These findings raise questions regarding the primary contribution of a muscle cell defect to the SMA phenotype.
Asunto(s)
Desarrollo de Músculos/fisiología , Atrofia Muscular Espinal/patología , Enfermedades de la Unión Neuromuscular/patología , Unión Neuromuscular/patología , Animales , Animales Recién Nacidos , Apoptosis/genética , Péptido Relacionado con Gen de Calcitonina/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo/genética , Embrión de Mamíferos , Humanos , Etiquetado Corte-Fin in Situ/métodos , Ratones , Ratones Transgénicos , Desarrollo de Músculos/genética , Músculo Esquelético/patología , Músculo Esquelético/ultraestructura , Atrofia Muscular Espinal/complicaciones , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/mortalidad , Unión Neuromuscular/genética , Unión Neuromuscular/crecimiento & desarrollo , Unión Neuromuscular/ultraestructura , Enfermedades de la Unión Neuromuscular/etiología , Enfermedades de la Unión Neuromuscular/metabolismo , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Proteína 2 para la Supervivencia de la Neurona Motora/genética , Proteínas de Unión al GTP rab3/metabolismoRESUMEN
The role of microglia during normal development of the nervous system is still not well understood. In the present study, a chick embryo model was used to examine the development of microglia in the spinal cord and characterize their changes in response to naturally occurring and pathological death of motoneurons (MNs). The microglial response to MN axotomy and the effects of microglial activation on MN survival were also studied. We found that: 1) macrophages/microglial cells were present in the spinal cord at early developmental stages (E3) and that they were recruited after normal and induced MN apoptosis; 2) although many microglial cells were seen phagocytosing apoptotic bodies, a proportion of dying cells were devoid of engulfing microglia; 3) axotomy of mature MNs was accompanied by microglial activation in the absence of MN death; 4) excitotoxic (necrotic) MN death provoked a rapid and massive microglial recruitment with phagocytic activity; 5) lipopolysaccharide-induced microglial activation in vivo resulted in the death of immature, but not mature, microglia; and 6) overactivation of microglia modulated the survival of mature MNs, either by killing them or by enhancing their vulnerability to die in response to a mild injury. Taken together, these observations indicate that normal microglia do not play an active role in triggering apoptosis of developing MNs. Rather, they act as phagocytes for the removal of dying cells during the process of programmed cell death.