RESUMEN
Human doublecortin (DCX) mutations are associated with severe brain malformations leading to aberrant neuron positioning (heterotopia), intellectual disability and epilepsy. DCX is a microtubule-associated protein which plays a key role during neurodevelopment in neuronal migration and differentiation. Dcx knockout (KO) mice show disorganized hippocampal pyramidal neurons. The CA2/CA3 pyramidal cell layer is present as two abnormal layers and disorganized CA3 KO pyramidal neurons are also more excitable than wild-type (WT) cells. To further identify abnormalities, we characterized Dcx KO hippocampal neurons at subcellular, molecular and ultrastructural levels. Severe defects were observed in mitochondria, affecting number and distribution. Also, the Golgi apparatus was visibly abnormal, increased in volume and abnormally organized. Transcriptome analyses from laser microdissected hippocampal tissue at postnatal day 60 (P60) highlighted organelle abnormalities. Ultrastructural studies of CA3 cells performed in P60 (young adult) and > 9 months (mature) tissue showed that organelle defects are persistent throughout life. Locomotor activity and fear memory of young and mature adults were also abnormal: Dcx KO mice consistently performed less well than WT littermates, with defects becoming more severe with age. Thus, we show that disruption of a neurodevelopmentally-regulated gene can lead to permanent organelle anomalies contributing to abnormal adult behavior.
Asunto(s)
Proteína Doblecortina/genética , Neuropéptidos , Animales , Proteínas de Dominio Doblecortina , Aparato de Golgi , Hipocampo/metabolismo , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/metabolismo , Mutación , Neuropéptidos/genética , Neuropéptidos/metabolismo , Células Piramidales/metabolismoRESUMEN
Prevention of protein misfolding is ensured by chaperone proteins, including the heat shock proteins (HSP) of the DNAJ/HSP40 family. Detection of abnormal protein aggregates in various neurodegenerative diseases has led to the proposal that altered chaperone activity contributes to neurodegeneration. Msj-1, a DNAJ/HSP40 protein located around the spermatozoa acrosome, was recently found to be down-regulated in the testis of wobbler mutant mice. Wobbler is an unidentified recessive mutation which triggers progressive motoneuron degeneration with abnormal intracellular protein accumulations, and defective spermatozoa maturation. Here, we examined Msj-1 expression in the spinal cord of the mutants and their controls. Msj-1 transcripts were amplified by reverse transcription-polymerase chain reaction from mutant and wild-type spinal cord RNA. Sequencing of Msj-1 coding region revealed no change in the mutant. In contrast, decreased Msj-1 mRNA levels were observed in five to six-week-old wobbler mice spinal cord, when motoneuron degeneration is at its apex, as compared to controls. A similar decrease was observed in two-week-old wobbler spinal cord, when the number of motoneurons is still unaltered, indicating that the decreased mRNA content is intrinsic to the mutant and not simply related to the loss of cells expressing Msj-1. Assays of Msj-1 protein levels yielded similar results. Immunofluorescent labeling revealed numerous Msj-1-ir motoneurons in five-week-old control spinal cord while no signal was observed in age-matched wobbler. Our results show, therefore, that Msj-1 expression is down-regulated in both organs affected by the wobbler mutation, the CNS and the testis, and that this defect precedes the first histological signs of motoneuron degeneration. These results provide the first example of an association between transcriptional repression of a chaperone protein and a neurodegenerative process.
Asunto(s)
Proteínas de Choque Térmico/biosíntesis , Enfermedad de la Neurona Motora/metabolismo , Espermatozoides/metabolismo , Médula Espinal/metabolismo , Animales , Regulación hacia Abajo/fisiología , Proteínas del Choque Térmico HSP40 , Proteínas de Choque Térmico/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NZB , Ratones Mutantes Neurológicos , Enfermedad de la Neurona Motora/genética , Mutación/fisiología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Testículo/metabolismoRESUMEN
Excitotoxic lesions in the gray matter induce profuse demyelination of passage and afferent fibers in areas of neuronal loss, independent of Wallerian degeneration. The time course of this phenomenon, which extends over weeks after the excitotoxin injection, suggests that demyelination is not related only to a direct effect of the toxin. In order to define mechanisms at work, a parallel study of myelin and oligodendrocytes was carried out following kainate injections into the adult rat thalamus. Within the 1st day postlesion, myelin alteration appeared throughout the area exhibiting neuronal loss, while the number of oligodendrocytes fell by 45%. No apoptotic oligodendrocytes were identified at that time. Over the following 2 days, there was no further loss of myelin and oligodendrocytes, but there was an increase in the number of oligodendrocytes displaying typical signs of apoptosis as revealed with TUNEL-end-labeled nuclei, Hoechst-labeled condensed chromatin bodies, or bax immunoreactivity. This resulted in a second, progressive loss of both myelin and oligodendrocytes leading to their almost complete disappearance 2 weeks postlesion. These results demonstrate two temporal stages of oligodendroglial cell death. The excitotoxin injection resulted in the rapid destruction of a first oligodendroglial population, most probably by necrosis. A second population died in a delayed manner from apoptosis. This second wave of death coincided with an activated microglia/macrophage invasion of the lesion, suggesting that delayed oligodendroglial death results from toxic microglia/macrophage effects. In addition, the longest surviving oligodendrocytes were located next to reactive astrocytes, suggesting the existence of trophic interactions between these two glial populations.
Asunto(s)
Encéfalo/patología , Oligodendroglía/patología , Proteínas Proto-Oncogénicas c-bcl-2 , Animales , Apoptosis , Astrocitos/patología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Recuento de Células , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/patología , Progresión de la Enfermedad , Femenino , Etiquetado Corte-Fin in Situ , Ácido Kaínico , Macrófagos/patología , Microglía/patología , Vaina de Mielina/efectos de los fármacos , Vaina de Mielina/patología , Vaina de Mielina/ultraestructura , Necrosis , Oligodendroglía/efectos de los fármacos , Oligodendroglía/metabolismo , Antígeno Nuclear de Célula en Proliferación/biosíntesis , Proteínas Proto-Oncogénicas/biosíntesis , Ratas , Ratas Sprague-Dawley , Tálamo/efectos de los fármacos , Tálamo/patología , Factores de Tiempo , Proteína X Asociada a bcl-2RESUMEN
Expression of transforming growth factor alpha (TGFalpha), a member of the epidermal growth factor (EGF) family, is a general response of adult murine motoneurons to genetic and experimental lesions, TGFalpha appearing as an inducer of astrogliosis in these situations. Here we address the possibility that TGFalpha expression is not specific to pathological situations but may participate to the embryonic development of motoneurons. mRNA of TGFalpha and its receptor, the EGF receptor (EGFR), were detected by ribonuclease protection assay in the ventral part of the cervical spinal cord from embryonic day 12 (E12) until adult ages. Reverse transcription-PCR amplification of their transcripts from immunopurified E15 motoneurons, associated with in situ double-immunohistological assays, identified embryonic motoneurons as cellular sources of the TGFalpha-EGFR couple. In vitro, TGFalpha promoted the survival of immunopurified E15 motoneurons in a dose-dependent manner, with a magnitude similar to BDNF neuroprotective effects at equivalent concentrations. In a transgenic mouse expressing a human TGFalpha transgene under the control of the metallothionein 1 promoter, axotomy of the facial nerve provoked significantly less degeneration in the relevant motor pool of 1-week-old mice than in wild-type animals. No protection was observed in neonates, when the transgene exhibits only weak expression levels in the brainstem. In conclusion, our results point to TGFalpha as a physiologically relevant candidate for a neurotrophic role on developing motoneurons. Its expression by the embryonic motoneurons, which also synthesize its receptor, suggests that this chemokine is endowed with the capability to promote motoneuron survival in an autocrine-paracrine manner.
Asunto(s)
Neuronas Motoras/efectos de los fármacos , Factor de Crecimiento Transformador alfa/farmacología , Animales , Animales Recién Nacidos , Células del Asta Anterior/citología , Células del Asta Anterior/metabolismo , Axotomía , Supervivencia Celular/efectos de los fármacos , Medio de Cultivo Libre de Suero/farmacología , Relación Dosis-Respuesta a Droga , Receptores ErbB/genética , Receptores ErbB/metabolismo , Nervio Facial/fisiología , Regulación del Desarrollo de la Expresión Génica , Humanos , Hibridación in Situ , Metalotioneína/genética , Ratones , Ratones Transgénicos , Neuronas Motoras/citología , Cuello , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Médula Espinal/citología , Médula Espinal/embriología , Médula Espinal/metabolismo , Factor de Crecimiento Transformador alfa/genética , Factor de Crecimiento Transformador alfa/metabolismo , TransgenesRESUMEN
In the adult brain, neurotrophins play a key role in adaptive processes linked to increased neuronal activity. A growing body of evidence suggests that chronic pain results from long-term plasticity of central pathways involved in nociception. We have investigated the involvement of nerve growth factor (NGF) in adaptive responses of primary sensory neurons during the course of a long-lasting inflammatory pain model. The amount and distribution of the NGF receptors p75(NTR) and TrkA were measured in the dorsal horn and dorsal root ganglia (DRG) of animals subjected to Freund's adjuvant-induced arthritis (AIA). We observed an increased immunoreactivity of both receptors in the central terminals of primary sensory neurons in the arthritic state. The increases were seen in the same population of afferent terminals in deep dorsal horn laminae. These changes paralleled the variations of clinical and behavioral parameters that characterize the course of the disease. They occurred in NGF-sensitive, but not GDNF-sensitive, nerve terminals. However, p75(NTR) and TrkA protein levels in the DRG (in the cell body of these neurons) showed different response patterns. An immediate rise of p75(NTR) was seen in parallel with the initial inflammation that developed after administration of Freund's adjuvant in hindpaws. In contrast, increases of the mature (gp140(trk)) form of TrkA occurred later and seemed to be linked to the development of the long-lasting inflammatory response. The changes in receptor expression were observed exclusively at lumbar levels, L3-L5, somatotopically appropriate for the inflammation. Together, these results implicate NGF in long-term mechanisms accompanying chronic inflammatory pain, via the up-regulation of its high affinity receptor, and offer additional evidence for differential processes underlying short- versus long-lasting inflammatory pain.
Asunto(s)
Artritis Experimental/metabolismo , Ganglios Espinales/metabolismo , Células del Asta Posterior/metabolismo , Receptor trkA/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Animales , Péptido Relacionado con Gen de Calcitonina/metabolismo , Masculino , Neuronas Aferentes/metabolismo , Proteínas/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor de Factor de Crecimiento Nervioso , Sustancia P/metabolismoRESUMEN
Repetitive noxious stimulation leads to permanent adaptive changes of central pathways involved in the genesis and integration of nociception. Several classes of neurotrophic factors that affect brain plasticity are also involved in the regulation of sensory functions in adulthood. To investigate a putative role of nerve growth factor (NGF) in central plasticity linked to chronic pain, modifications in immunoreactivity (IR) for the high-affinity NGF receptor, TrkA, were studied at spinal levels in a rat model of inflammatory chronic pain, adjuvant-induced arthritis (AIA). We report a specific increase in the number of TrkA-IR profiles in laminae V-VI at lumbar levels L3 and L4 in arthritic rats. Tract tracing using FluoroGold injections in the ventrobasal complex of the thalamus and in the brainstem showed that these increased TrkA-IR profiles are spinoreticular neurons. Dual labeling with calcitonin gene-related peptide or substance P showed that TrkA-IR neurons were mainly located in projection fields of small- to medium-sized primary afferent fibers, which convey nociceptive inputs. These results suggest that TrkA-containing neurons of the spinal dorsal horn participate in the first central relay of transmission of nociceptive information to supraspinal centers. Enhanced numbers of TrkA-IR neurons during AIA strongly support the hypothesis of a participation of NGF in adaptive mechanisms of central nociceptive pathways observed in chronic pain states.