RESUMEN
Traumatic Brain injury affects at least 1.7 million people in the United States alone each year. The majority of injuries are categorized as mild but these still produce lasting symptoms that plague the patient and the medical field. Currently treatments are aimed at reducing a patient's symptoms, but there is no effective method to combat the source of the problem, neuronal loss. We tested a mild, closed head traumatic brain injury model for the effects of modulation of the antioxidant transcription factor Nrf2 by the chemical activator, tert-butylhydroquinone (tBHQ). We found that post-injury visual memory was improved by a 7 day course of treatment and that the level of activated caspase-3 in the hippocampus was reduced. The injury-induced memory loss was also reversed by a single injection at 30 min after injury. Since the protective stress response molecule, HSP70, can be upregulated by Nrf2, we examined protein levels in the hippocampus, and found that HSP70 was elevated by the injury and then further increased by the treatment. To test the possible role of HSP70, model neurons in culture exposed to a mild injury and treated with the Nrf2 activator displayed improved survival that was blocked by the HSP70 inhibitor, VER155008. Following mild traumatic brain injury, there may be a partial protective response and patients could benefit from directed enhancement of regulatory pathways such as Nrf2 for neuroprotection.
Asunto(s)
Antioxidantes/uso terapéutico , Lesiones Encefálicas/complicaciones , Trastornos del Conocimiento/tratamiento farmacológico , Trastornos del Conocimiento/etiología , Hidroquinonas/uso terapéutico , Análisis de Varianza , Animales , Reacción de Prevención/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Lesiones Encefálicas/tratamiento farmacológico , Caspasa 3/genética , Caspasa 3/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Trastornos del Conocimiento/patología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Ratones Endogámicos ICR , Actividad Motora/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Neuroblastoma/patología , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Nucleósidos de Purina/farmacología , ARN Mensajero/metabolismo , Reconocimiento en Psicología/efectos de los fármacos , Prueba de Desempeño de Rotación con Aceleración Constante , Factores de TiempoRESUMEN
Wobbler mice model motor neuron disease with a substantial decline in motor neurons. TDP-43 is a nucleic acid binding protein that accumulates, along with ubiquitin, in the cytoplasm of amyotrophic lateral sclerosis (ALS) motor neurons. Recently, it was reported that Cu/Zn superoxide dismutase type 1 (SOD1) familial amyotrophic lateral sclerosis (fALS) model mice do not mimic the TDP-43 changes seen in sporadic ALS, although they share a large number of other properties with the human disorder. We examined ubiquitin inclusions and TDP-43 expression in wobbler mice. TDP-43 mRNA, measured by quantitative reverse transcription-coupled PCR, was elevated in the wobbler spinal cord. Immunohistochemistry revealed intracellular ubiquitin inclusions and abnormal distribution of TDP-43 into the cytoplasm in wobblers similar to the staining reported in ALS. Finally, nuclear and cytoplasmic fractions, examined by Western immunoblotting, confirmed a delocalization of TDP-43 in the neurodegenerative wobbler. These observations indicate that wobbler mice, which suffer motor neuron loss at 21 days, undergo TDP-43 and ubiquitin changes characteristic of sporadic ALS.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Enfermedad de la Neurona Motora/metabolismo , ARN Mensajero/metabolismo , Animales , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/genética , Ratones , Ratones Mutantes Neurológicos , Enfermedad de la Neurona Motora/patología , Médula Espinal/patología , Ubiquitina/metabolismoRESUMEN
Thrombin is well known in its function as the ultimate serine protease in the coagulation cascade. Emerging evidence indicates that thrombin also functions as a potent signaling molecule that regulates physiologic and pathogenic responses alike in a large variety of cell populations and tissues. Accompanying CNS injury and other cerebral vascular damages, prothrombin activation and leakage of active thrombin into CNS parenchyma has been documented. Due to the irreplaceable feature of neurons, over-reactive inflammatory reactions in the CNS often cause irreversible neuronal damage. Therefore, particular attention is required to develop strategies that restrict CNS inflammatory responses to beneficial, in contrast to neurotoxic ones. In this regard, thrombin not only activates endothelial cells and induces leukocyte infiltration and edema but also activates astrocytes, and particularly microglia, as recently demonstrated, to propagate the focal inflammation and produce potential neurotoxic effects. Recently revealed molecular mechanisms underlying these thrombin effects appear to involve proteolytic activation of two different thrombin-responsive, protease-activated receptors (PARs), PAR1 and PAR4, possibly in concert. Potential therapeutic strategies based on appreciation of the current understanding of molecular mechanisms underlying thrombin-induced CNS inflammation are also discussed.
Asunto(s)
Mediadores de Inflamación/fisiología , Enfermedades Neurodegenerativas/fisiopatología , Trombina/fisiología , Traumatismos del Sistema Nervioso/fisiopatología , Animales , Antiinflamatorios/farmacología , Sistema Nervioso Central/patología , Sistema Nervioso Central/fisiopatología , Humanos , Inflamación/patología , Inflamación/fisiopatología , Sistema Nervioso Periférico/patología , Sistema Nervioso Periférico/fisiopatología , Trombina/antagonistas & inhibidoresRESUMEN
The first association of the synapse as a potential site of neurodegenerative disease burden was suggested for Alzheimer's disease (AD) almost 30 years ago. Since then protease:protease inhibitor (P:PI) systems were first linked to functional regulation of synaptogenesis and synapse withdrawal at the neuromuscular junction (NMJ) more than 20 years ago. Confirmatory evidence for the involvement of the synapse, the rate-limiting or key unit in neural function, in AD did not become clear until the beginning of the 1990s. However, over the past 15 years evidence for participation of thrombin, related serine proteases and neural PIs, homologous and even identical to those of the plasma clot cascade, has been mounting. Throughout development a balance between stabilization forces, on the one hand, and breakdown influences, on the other, becomes established at synaptic junctions, just as it does in plasma clot proteins. The formation of protease-resistant cross-links by the transglutaminase (TGase) family of enzymes may add to the stability for this balance. The TGase family includes coagulation factor XIIIA and 8 other different genes, some of which may also influence the persistence of neural connections. Synaptic location of protease-activated, G-protein-coupled receptors (PARs) for thrombin and related proteases, their serpin and Kunitz-type PIs such as protease nexin I (PNI), alpha1-antichymotrypsin (alpha-ACT), and the Kunitz protease inhibitor (KPI)-containing secreted forms of beta-amyloid protein precursor (beta-APP), along with the TGases and their putative substrates, have all been amply documented. These findings strongly add to the conclusion that these molecules participate in the eventual structural stability of synaptic connections, as they do in coagulation cascades, and focus trophic activity on surviving terminals during periods of selective contact elimination. In disease states, this imbalance is likely to be shifted in favor of destabilizing forces: increased and/or altered protease activity, enhanced PAR influence, decreased and/or altered protease inhibitor function, reduction and/or alteration in tTG expression and activity, and alteration in its substrate profile. This imbalance further initiates a cascade of events leading to inappropriate programmed cell death and may well be considered evidence of synaptic apoptosis.
Asunto(s)
Unión Neuromuscular/fisiología , Plasticidad Neuronal/fisiología , Transducción de Señal/fisiología , Sinapsis/fisiología , Trombina/metabolismo , Transglutaminasas/metabolismo , Secuencia de Aminoácidos , Animales , Extensiones de la Superficie Celular/metabolismo , Factor XIII/metabolismo , Humanos , Datos de Secuencia Molecular , Unión Neuromuscular/citología , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismoRESUMEN
Thrombin, the ultimate protease in the blood coagulation cascade, mediates its known cellular effects by unique proteolytic activation of G-protein-coupled protease-activated receptors (PARs), such as PAR1, PAR3, and PAR4, and a "tethered ligand" mechanism. PAR1 is variably expressed in subpopulations of neurons and largely determines thrombin's effects on morphology, calcium mobilization, and caspase-mediated apoptosis. In spinal cord motoneurons, PAR1 expression correlates with transient thrombin-mediated [Ca(2+)](i) flux, receptor cleavage, and elevation of rest [Ca(2+)](i) activating intracellular proteases. At nanomolar concentrations, thrombin retracts neurites via PAR1 activation of the monomeric, 21 kDa Ras G-protein RhoA, which is also involved in neuroprotection at lower thrombin concentrations. Such results suggest potential downstream targets for thrombin's injurious effects. Consequently, we employed several G-protein-specific modulators prior to thrombin exposure in an attempt to uncouple both heterotrimeric and monomeric G-proteins from motoneuronal PAR1. Cholera toxin, stimulating Gs, and lovastatin, which blocks isoprenylation of Rho, reduced thrombin-induced calcium mobilization. In contrast, pertussis toxin and mastoparan, inhibiting or stimulating G(o)/G(i), were found to exacerbate thrombin action. Effects on neuronal rounding and apoptosis were also detected, suggesting therapeutic utility may result from interference with downstream components of thrombin signaling pathways in human motor neuron disorders, and possibly other neurodegenerative diseases. Published 2001 John Wiley & Sons, Inc.
Asunto(s)
Apoptosis/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Hemostáticos/farmacología , Neuronas Motoras/metabolismo , Trombina/farmacología , Anticolesterolemiantes/farmacología , Inhibidores de Caspasas , Línea Celular , Toxina del Cólera/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Proteínas de Unión al GTP/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Lovastatina/farmacología , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/ultraestructura , Neuritas/efectos de los fármacos , Neuritas/fisiología , Oligopéptidos/farmacología , Péptidos , Toxina del Pertussis , Receptor PAR-1 , Receptores de Trombina/metabolismo , Factores de Virulencia de Bordetella/farmacología , Venenos de Avispas/farmacologíaRESUMEN
In order to understand the mechanism for insoluble neurotoxic protein polymerization in Alzheimer's disease (AD) brain neurons, we examined protein and gene expression for transglutaminase (TGase 2; tissue transglutaminase (tTG)) in hippocampus and isocortex. We found co-localization of tTG protein and activity with tau-positive neurofibrillary tangles, whereas mRNA and sequence analysis indicated an absolute increase in tTG synthesized. Although apoptosis in AD hippocampus is now an established mode of neuronal cell death, no definite underlying mechanism(s) is known. Since TGase-mediated protein aggregation is implicated in polyglutamine ((CAG)(n)/Q(n) expansion) disorder apoptosis, and expanded Q(n) repeats are excellent TGase substrates, a role for TGase in AD is possible. However, despite such suggestions almost 20 years ago, the molecular mechanism remained elusive. We now present one possible molecular mechanism for tTG-mediated, neurotoxic protein polymerization leading to neuronal apoptosis in AD that involves not its substrates (like Q(n) repeats) but rather the unique presence of alternative transcripts of tTG mRNA. In addition to a full-length (L) isoform in aged non-demented brains, we found a short isoform (S) lacking a binding domain in all AD brains. Our current results identify intron-exon "switching" between L and S isoforms, implicating G-protein-coupled signaling pathways associated with tTG that may help to determine the dual roles of this enzyme in neuronal life and death processes.
Asunto(s)
Enfermedad de Alzheimer/enzimología , Transglutaminasas/genética , Proteínas tau/metabolismo , Empalme Alternativo/genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Encéfalo/enzimología , Encéfalo/metabolismo , ADN Complementario/análisis , Exones , Humanos , Intrones , Datos de Secuencia Molecular , Neuronas/metabolismo , Unión Proteica , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Factores de Tiempo , Transglutaminasas/metabolismoRESUMEN
Although the precise mechanisms explaining loss of, and failure to regain, function after spinal cord injury are unknown, there is increasing interest in the role of "secondary cell death." One prevalent theme in cell loss in other regions of the CNS involves apoptosis executed by the intracellular caspase proteases. A recent study demonstrated that spinal cord injury rapidly increased the activation of caspase-3. Our previous studies demonstrated peak apoptosis in three of four cellular compartments 3 days after controlled contusion in the rat. We have extended these analyses to include enzyme and substrate studies of caspase subfamilies both in rostral and in caudal adjacent segments compared to the lesion site. Although presumed activation of programmed proenzyme is considered the mechanism for enhanced caspases, our novel analyses were designed to detect upregulation of gene expression. We surveyed traumatically injured spinal cord for caspase family messages with a modified differential mRNA display approach and found that the caspase-3 (CASP3) message was present and upregulated severalfold after injury. Our results clearly demonstrate that cell death in the spinal cord occurs after posttranslational activation of caspases that follow, at least for caspase-3, initial upregulation of CASP3 mRNA levels.
Asunto(s)
Apoptosis/fisiología , Caspasas/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/patología , Médula Espinal/enzimología , Médula Espinal/patología , Animales , Biotina , Proteínas Portadoras/metabolismo , Caspasa 3 , Caspasas/genética , Inhibidores de Cisteína Proteinasa , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Hibridación in Situ , Masculino , Proteínas de Microfilamentos/metabolismo , Microglía/metabolismo , Neuronas Motoras/enzimología , Neuronas Motoras/patología , Oligopéptidos , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas , Proteínas/metabolismo , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Especificidad por SustratoRESUMEN
BACKGROUND: Mechanisms underlying neurodegeneration are actively sought for new therapeutic strategies. Transgenic, knockout and genetic mouse models greatly aid our understanding of the mechanisms for neuronal cell death. A naturally occurring, autosomal recessive mutant, known as wobbler, and mice transgenic for familial amyotrophic lateral sclerosis (FALS) superoxide dismutase (SOD)1 mutations are available, but the molecular mechanisms remain equally unknown. Both phenotypes are detectable after birth. Wobbler is detectable in the third week of life, when homozygotes (wr/wr) exhibit prominent gliosis and significant motor neuron loss in the cervical, but not in lumbar, spinal cord segments. To address molecular mechanisms, we evaluated "death signals" associated with the multifunctional serine protease, thrombin, which leads to apoptotic motor neuronal cell death in culture by cleavage of a G-protein coupled, protease-activated receptor 1 (PAR-1). MATERIALS AND METHODS: Thrombin activities were determined with chromogenic substrate assays, Western immunoblots and immunohistochemistry were performed with anti-PAR-1 to observe localizations of the receptor and anti-GFAP staining was used to monitor astrocytosis. PAR-1 mRNA levels and locations were determined by reverse transcription polymerase chain reaction (qRT-PCR) and in situ hybridizations. Cell death was monitored with in situ DNA fragmentation assays. RESULTS: In preliminary studies we found a 5-fold increase in PAR-1 mRNA in cervical spinal cords from wr/wr, compared with wild-type (wt) littermates. Our current studies suggested that reactive astrocytosis and motor neuron cell death were causally linked with alterations in thrombin signaling. PAR-1 protein expression was increased, as demonstrated by immunocytochemistry and confirmed with in situ hybridization, in phenotypic wr/wr motor neurons, compared with wt, but not in astrocytes. This increase was much greater in cervical, compared with lumbar, segments, paralleling motor neuron degeneration. We also found, using reverse transcription polymerase chain reaction (qRT-PCR) with RNA from genotyped embryos, that PAR-1 was already increased in wr/wr cords at E12, the earliest time examined. CONCLUSIONS: Thus, motor neuron degeneration and death follows PAR-1 expression both temporally and topographically in wobbler mice. Since our culture studies show that thrombin mobilized [Ca2+]i by activating PAR-1, eventually leading to motor neuron apoptosis, up-regulation of PAR-1 during development may contribute both to "appropriate" as well as "inappropriate" neuronal death in wobbler.
Asunto(s)
Neuronas Motoras/fisiología , Receptores de Trombina/genética , Médula Espinal/patología , Transcripción Genética , Animales , Muerte Celular , Cruzamientos Genéticos , Sondas de ADN , Femenino , Genotipo , Proteína Ácida Fibrilar de la Glía/análisis , Gliosis , Homocigoto , Humanos , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Enfermedad de la Neurona Motora/genética , Enfermedad de la Neurona Motora/fisiopatología , Neuronas Motoras/citología , Neuronas Motoras/patología , ARN Mensajero/análisis , Receptor PAR-1 , Médula Espinal/fisiopatología , Superóxido Dismutasa/genética , Trombina/metabolismoRESUMEN
STUDY DESIGN: Prospective, randomized experimental study in mice. STUDY OBJECTIVE: To determine whether insulin-like growth factor binding proteins (IGFBPs) are present in mouse spinal cord and, if so, what role they play in its development. SUMMARY OF BACKGROUND DATA: Insulin-like growth factors are well recognized hormonal effectors of growth hormone and are expressed in the mammalian spinal cord. The IGFBPs are a group of six genetically distinct proteins that bind IGFs and modulate their bioactivity. They appear in the brain during development, localize to the neuromuscular junction, and promote motor neuron survival. The benefit of IGF-I in amyotrophic lateral sclerosis ALS and its potential use in preventing motor neuron apoptosis in spinal cord injury dictates that studies of the presence and response of IGFBPs in that tissue be performed. METHODS: The IGFBPs in mouse spinal cord were analyzed by Western ligand blot, Western immunoblot, and reverse transcription-polymerase chain reaction at various time points from embryonic day 14 to postnatal day 30. RESULTS: Three IGFBPs with molecular masses of 24, 28, and 32 kDa were found, the latter two being the most prominent. The data indicate that these are IGFBP-4, -5, and -2. CONCLUSION: Both IGFBP-2 and BP-5 are developmentally regulated in mouse spinal cord, with higher levels of those at early embryonic stages indicating their potential role in development of the mouse spinal cord.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Médula Espinal/embriología , Médula Espinal/crecimiento & desarrollo , Animales , Animales Recién Nacidos/metabolismo , Western Blotting , Cartilla de ADN/química , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario y Fetal , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Ligandos , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Médula Espinal/metabolismoRESUMEN
Coagulation Factor XIII (F. VIII), a member of the transglutaminase (TGase) superfamily, is activated by thrombin, cross-links fibrin and stabilizes clots. Another member of this family, tissue TGase (tTG), having similar enzymatic activity, is implicated in neural development and synapse stabilization. Our previous studies indicated that synapse formation and maintenance at the neuromuscular junction (NMJ) involved components of the coagulation cascade in development. Others then showed that either F. XIII or tTG were localized at NMJs in a developmentally-regulated fashion. In the current studies, we addressed the temporal course of skeletal muscle tTG gene expression and found maximal expression at birth and continuing into the immediate postnatal period. Subcellular fractionation revealed a relatively constant particulate isoform of TGase activity which predominated in early embryonic muscle development. In contrast, cytosolic TGase specific activity became the major isoform in the postnatal period. The timing of muscle TGase activity correlated well with expression of tTG mRNA and we now present novel data of Tgm 2 gene expression for tTG in skeletal muscle. Confirming and extending the previous studies, TGase becomes localized at NMJs in the early, further ramifying in the late, neonatal period. These data suggest that the early pulse of particulate activity could coincide with the period of myoblast cell death in embryonic muscle. On the other hand, the peak cytosolic TGase activity occurs in the neonatal period, correlating temporally with muscle prothrombin expression during activity-dependent synapse elimination and possibly the source of the enzyme localized to the NMJ extracellular matrix resulting in synaptic stabilization.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Isoenzimas/genética , Unión Neuromuscular/enzimología , Unión Neuromuscular/crecimiento & desarrollo , Transglutaminasas/genética , Envejecimiento , Animales , Técnicas de Cultivo , Citosol/enzimología , Femenino , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Peso Molecular , Músculo Esquelético/enzimología , Unión Neuromuscular/embriología , Embarazo , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Transglutaminasas/análisisRESUMEN
Apoptosis, well-established in development and now also in degenerative disease, occurs with regularity in several cell compartments early after controlled contusion spinal cord injury (SCI). Cell death in astrocytic, microglial, and neuronal populations peaks at 3 days, while oligodendroglial apoptosis is found 10-14 days later. In this regard, the executioners of apoptosis, the caspase proteases, are also activated within 3 days of SCI. On the other hand, serine proteases, which have been shown to initiate apoptosis and activate caspases in culture models, have not been extensively studied in regards to nervous system trauma. As part of an ongoing effort to examine the spectrum of genes that are up- and downregulated in the injured rat spinal cord, we synthesized serine protease family specific primers to take advantage of conserved residues in the charge relay system and the codon preferences of these mammalian genes. These primers were then employed in a modified, family-specific differential mRNA display technique. One specific serine protease gene we found that was upregulated after injury was prothrombin. Qualitative and quantitative RT-PCR techniques indicated that this increase occurred early, already evident at 8 h after injury, and reached a maximum level fourfold above baseline at 24 h. Peak expression for prothrombin mRNA occurred prior to peak levels of apoptosis in astrocytic, microglial and neuronal compartments at 72 h. Of additional interest, gene database mining revealed that prothrombin shared approximately 48% similarity with myelencephalon-specific protease (MSP), a neurotoxic serine protease previously found to be increased two- to threefold at 3 days after excitotoxic SCI. Since thrombin induces apoptosis in murine and chick motor and rat hippocampal neurons by activating a member of the novel protease-activated receptor (PAR) gene family known as PAR-1, we also analyzed PAR-1 by similar techniques and found that it, too, was upregulated after SCI with the same kinetics as prothrombin. We confirmed these results with gene array analyses that revealed more than one trypsin subfamily serine protease was activated by SCI. They imply the possibility of using specific, tissue-directed serine protease inhibition at translational or transcriptional levels, and offer a potential paradigm shift in drug discovery for SCI to limit the extent of apoptosis, and consequent functional loss, in the human spinal cord.
Asunto(s)
Neurotoxinas/metabolismo , Protrombina/metabolismo , Receptores de Trombina/metabolismo , Serina Endopeptidasas/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Secuencias de Aminoácidos , Animales , Muerte Celular , Femenino , Expresión Génica , Familia de Multigenes , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor PAR-1 , Receptores de Trombina/genética , Lugares Marcados de Secuencia , Serina Endopeptidasas/genética , Transducción de Señal , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatología , Trombina/fisiología , Factores de Tiempo , Regulación hacia ArribaRESUMEN
We analyzed samples of insulin-like growth factor binding proteins (IGFBPs) in human cerebrospinal fluid (CSF) in neurologically normal patients from one day after birth to age 76 years. CSF samples were separated on SDS-PAGE and then transferred to nitrocellulose membranes where IGFBPs were detected by Western ligand blot using [(125)I]-IGF-II, confirming other reports where we found the presence of IGFBP-2, 3, 4, 5. The 34 kDa IGFBP-2 was present in all samples, and progressively decreased with age. A broad 28- to 30-kDa IGFBP band, having the appearance of IGFBP-5, was triphasic: faint during infancy, barely detectable at 6 months, but intense in adult and aged individuals. The 24-kDa IGFBP-4 band was only seen in neonatal CSF samples, while the IGFBP-3 doublet gradually increased during aging. Thus, these present results show that IGFBP-2, 3, 4 and 5 in CSF are developmentally regulated, suggesting roles for these molecules in the development of the nervous system.
Asunto(s)
Envejecimiento/líquido cefalorraquídeo , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/líquido cefalorraquídeo , Adolescente , Adulto , Anciano , Niño , Preescolar , Electroforesis en Gel de Poliacrilamida , Humanos , Lactante , Recién Nacido , Persona de Mediana EdadRESUMEN
Fibrinogen has been reported to interact with phospholipid; however, the properties of this binding interaction have not been characterized. Purified preparations of human fibrinogen bound to small unilamellar vesicles containing phosphatidylserine (PS) as measured by light scattering and radioisotope filtration. Binding to 100% PS was saturable (apparent Kd=5 microM, Bmax=1.9 g protein/g lipid), reversible, and involved a minor subfraction of the fibrinogen preparation (3-6% of total protein). Fibrinogen interacted minimally with phosphatidylinositol, and not at all with pure phosphatidylcholine (PC) or PC vesicles containing 5% glycosphingolipid (lactosylceramide, ganglioside GM3, ganglioside GD3). Binding efficiency decreased as the PS content of vesicles was diluted with PC. Calcium chloride (2 mM) enhanced protein binding to PS, which was reversed by EDTA. Fibrin clot formation almost quantitatively precipitated the PS binding activity. PS, but not PC, increased the final turbidity of fibrin clots. Computerized sequence analysis of fibrinogen revealed three candidate acidic phospholipid binding motifs located at position 143-210 in the alpha chain, and positions 59-77 and 101-139 in the beta chain. Further study of the PS binding activity of fibrinogen may lead to new insights about fibrinogen function.
Asunto(s)
Fibrinógeno/química , Fosfolípidos/química , Fibrinógeno/metabolismo , Humanos , Fosfolípidos/metabolismo , Unión ProteicaRESUMEN
Thrombin acts on cells through the surface protease-activated receptor 1 (PAR-1), a G-protein-coupled member of the seven-transmembrane domain superfamily. On neural cells, thrombin has deleterious effects, killing neurons through apoptosis. Consequently, knowledge of PAR-1 expression in the nervous system may help to elucidate the role of thrombin in neurodegenerative disease. We developed a mimic construct to facilitate the highly sensitive technique of quantitative reverse transcriptase to PCR (qRT-PCR) to measure the differential expression of low copy number PAR-1 mRNA in neurodegenerative model systems. In this article, we report our results comparing homozygous wobbler (wr/wr) mice and normal littermates. By optimizing the transcription and quantitative PCR procedures to facilitate rapid copy number determination in small RNA samples, we documented a fivefold greater level of PAR-1 mRNA in the cervical spinal cord of wr/wr.
Asunto(s)
Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/análisis , Receptores de Trombina/genética , Animales , Femenino , Masculino , Ratones , Ratones Mutantes , Imitación Molecular , Receptor PAR-1 , Mapeo Restrictivo , Médula Espinal/químicaRESUMEN
Apoptosis, often also termed "programmed cell death", occurs in normal development in the brain and spinal cord. Important to concepts of disease and potential intervention is the exciting finding that apoptosis is also found after neurotrauma and in a number of neurodegenerative diseases. Although the precise mechanism of neuronal cell loss remains unknown, much emphasis has been placed recently on the activation of cell death protease cascades within the cell. How these cascades may be activated, especially from extracellular influences, is currently poorly understood. Thrombin, the multifunctional coagulation protease, is an early phase modulator at sites of tissue injury and has been shown to induce cell death in neurons by an apoptotic mechanism by activating its receptor, PAR-1. Using a model motor neuronal cell line, NSC19, which we have shown undergoes apoptosis after treatment with classic apoptosis inducers such as the topoisomerase inhibitors camptothecin and etoposide, we unambiguously found that nanomolar thrombin induced characteristic signs of apoptosis. Strikingly, endonucleolysis was accompanied by an increase in caspase-3-like activity in cellular extracts, which correlated with both detection of caspase-induced signature cleavage of the cortical cytoskeleton component nonerythroid spectrin (alpha-fodrin) and identification of increased accessibility of a caspase cleavage domain, using an antibody (Ab127) made against a synthetic peptide KGDEVD. Demonstrating that thrombin activation of death proteases was linked to cell death, we were able to inhibit thrombin-induced apoptosis by using a caspase family inhibitor, benzyloxycarbonyl-Asp-(oMe)-fluoromethyl ketone (Boc-D-FMK). These novel results demonstrate that thrombin serves as an extracellular "death signal" to activate intracellular protease pathways. These pathways lead to apoptotic cell death and can be modulated by inhibiting caspase activity downstream to PAR-1.
Asunto(s)
Apoptosis/fisiología , Endopeptidasas/fisiología , Espacio Extracelular/fisiología , Membranas Intracelulares/enzimología , Neuronas Motoras/fisiología , Transducción de Señal/fisiología , Trombina/fisiología , Clorometilcetonas de Aminoácidos/farmacología , Animales , Línea Celular , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Fragmentación del ADN , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Ratones , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/enzimología , Trombina/farmacología , Inhibidores de Topoisomerasa I , Inhibidores de Topoisomerasa IIRESUMEN
Following a controlled, severe contusion lesion to the lower thoracic spinal cord in adult rats, we found that apoptosis occurred in cells located in both gray and white matter. This suggested that both nonneuronal cells, including astrocytes, oligodendroglia and microglia, as well as neurons, might participate in programmed cell death (PCD) following spinal cord injury (SCI). Determination of which cell populations participate, and the kinetics and extent of their involvement might reveal new paradigms for approaches to therapy. Consequently, we assessed the functional deficit, comparing a comprehensive locomotor rating scale (LRS) with the inclined plane test at various times after injury. Using standard histology, along with cell-specific markers, we assessed PCD in different spinal cord segments using several parameters of apoptosis. Our results indicate that hind limb motor function was lost at day 1, and then only gradually and ineffectively (about 10-15%) recovered over the next month. Evidence for increased cell number was present for astrocytes and microglia beginning at day 1 after injury. Over the postinjury time period, apoptotic cells appeared (from day 1 to 14), and peaked (in terms of apoptotic index) on day 3. About one-third were microglia, whereas neurons, both large and small, also underwent apoptosis, again peaking at day 3. However, neurons continued to die and were not replaced by proliferation, so that at day 7, three times as many neurons (as a percentage) underwent PCD compared with the glial compartment. Oligodendrocytes also underwent apoptosis, with a biphasic curve, both at days 3 and 14 following injury. Thus, in addition to immediate, passive necrosis, delayed and apoptotic PCD also occurred in all cell populations in severely injured spinal cord.
Asunto(s)
Apoptosis/fisiología , Traumatismos de la Médula Espinal/patología , Médula Espinal/patología , Animales , Astrocitos/patología , Modelos Animales de Enfermedad , Femenino , Análisis de los Mínimos Cuadrados , Microglía/patología , Trastornos del Movimiento/fisiopatología , Necrosis , Neuronas/patología , Oligodendroglía/patología , Ratas , Ratas Sprague-Dawley , Médula Espinal/fisiopatología , Médula Espinal/ultraestructura , Traumatismos de la Médula Espinal/fisiopatología , Factores de TiempoRESUMEN
Thrombin, the ultimate enzyme in the blood coagulation cascade, has prominent actions on various cells, including neurons. As in platelets, thrombin increases [Ca2+]i mobilization in neurons, and also retracts neurites. Both these effects are mediated through a G protein-coupled, proteolytically activated receptor for thrombin (PAR-1). Prolonged exposure to thrombin kills neurons via apoptosis, that may also involve PAR-1 activation. Increased [Ca2+]i has been a unifying mechanism proposed for cell death in several neurodegenerative diseases. Thrombin-elevated calcium levels may activate intracellular cascades in neurons leading to cell death. Since thrombin mediates its diverse effects on cells through both heterotrimeric and monomeric G proteins, we also explored what effect altering differential G protein coupling would have on the neuronal response to thrombin. We studied calcium mobilization by thrombin in a model motor neuronal cell line, NSC19, using fluorescence image analysis. Confirming effects in other neuronal types, thrombin caused dramatic increases in [Ca2+]i levels, both transiently and after prolonged exposure, which involved activation and cleavage of the PAR-1 receptor. Using enzyme linked immunosorbent assay (ELISA) and dot-blot analysis, we found that the N-terminal fragment of PAR-1 was released into the medium after exposure to thrombin. We confirmed that PAR-1 protein and mRNA expression occurred in motor neurons. We found that cholera toxin inhibited thrombin-mediated Ca2+ influx, pertussis toxin did not significantly alter thrombin action, and lovastatin, a small 21-kDa Ras GTPase (Rho) modulator, showed a tendency to reduce the thrombin effect. These data indicate that thrombin-increased [Ca2+]i, sufficient to trigger cell death in motor neurons, might be approached in vivo by modulating thrombin signaling through PAR-1.
Asunto(s)
Calcio/metabolismo , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Receptores de Trombina/metabolismo , Trombina/farmacología , Animales , Línea Celular , Toxina del Cólera/farmacología , Proteínas de Unión al GTP/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Células Híbridas , Interfase/efectos de los fármacos , Líquido Intracelular/efectos de los fármacos , Líquido Intracelular/metabolismo , Lovastatina/farmacología , Ratones , Toxina del Pertussis , Receptor PAR-1 , Receptores de Trombina/efectos de los fármacos , Trombina/antagonistas & inhibidores , Trombina/efectos de los fármacos , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
STUDY DESIGN: Serum withdrawal was introduced to a spinal cord motor neuron cell line to investigate the mode of cell death. OBJECTIVES: To characterize the death of motor neurons in culture, to gain insight into mechanisms that could be important in spinal cord diseases. SUMMARY OF BACKGROUND DATA: Normal reduction of cell number during central nervous system development is brought about by programmed cell death. These same apoptotic processes probably play a role in a variety of central nervous system disorders, including traumatic injury. Although certain proteolytic processes are involved, the molecular details involved in the apoptotic induction have not been fully elucidated. METHODS: To identify apoptosis, several criteria were used, including analysis of chromatin condensation with DNA-specific stains (propidium iodide and Hoechst 33342); in situ end-labeling of DNA fragments in apoptotic nuclei with terminal deoxynucleotidyl transferase; fragmentation of DNA separated on agarose gel electrophoresis; and cleavage of a characteristic substrate for apoptotic proteases, alpha-fodrin, into signature cleavage fragments. RESULTS: The NSC19 cell line exhibited motor neuron characteristics morphologically, with typical cellular structure, and biochemically, by synthesizing choline acetyl transferase. Under various treatments including serum withdrawal (loss of trophic factors), cell loss occurred through an apoptotic cell death pathway. CONCLUSIONS: A murine motor neuron cell line, NSC19, has been used to investigate apoptosis in this in vitro system. Cell death occurs by apoptosis, suggesting that this cell line may provide a useful model for studying apoptotic mechanisms in spinal cord degeneration and injury.
Asunto(s)
Apoptosis/fisiología , Neuronas Motoras/fisiología , Animales , Azacitidina/farmacología , Proteínas Portadoras/metabolismo , Línea Celular , Inhibidores Enzimáticos/farmacología , Ratones , Proteínas de Microfilamentos/metabolismo , Mitosis/efectos de los fármacos , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Inhibidores de Proteínas QuinasasRESUMEN
Precise determination of mRNA levels is an essential element in any investigation of complex regulatory systems. Classical methodologies such as Northern hybridization suffer from requirements for significant samples of material and also a degree of nonspecificity. Recently, quantitative techniques involving PCR amplification have been devised. We have developed and applied such procedures to the determination of prothrombin messages in skeletal muscle cells during development. In addition to its role in the blood coagulation cascade, the serine protease thrombin has been shown to participate in several signaling events in the neuromuscular system. The inactive precursor, prothrombin, primarily produced in the liver, has also been shown to be synthesized and developmentally-regulated in the brain. In skeletal muscle, thrombin is a mediator of activity-dependent polyneuronal synapse elimination (ADPSE) which occurs in early postnatal development. Recent experiments showing that thrombin is released from myotubes in culture under the influence of acetylcholine suggest that locally-synthesized prothrombin may be the source of this Hebbian synaptic interaction. We have determined that prothrombin is expressed in skeletal muscle, as the likely source of thrombin involved in ADPSE, and the current results show the quantitative expression of muscle prothrombin during this time of intense synapse remodeling.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Reacción en Cadena de la Polimerasa , Protrombina/genética , ARN Mensajero/biosíntesis , Animales , Células Cultivadas , Clonación Molecular , Hígado/metabolismo , Ratones , Ratones Endogámicos BALB C , Desarrollo de Músculos , Proteínas Musculares/biosíntesis , Músculo Esquelético/embriología , Músculo Esquelético/crecimiento & desarrollo , Especificidad de Órganos , Protrombina/biosíntesis , ARN Mensajero/análisis , ARN Mensajero/genética , Sinapsis/metabolismoRESUMEN
In order to develop in vitro models of CNS injury, astrocytes have been mechanically injured in culture to study reactive astrocytosis. However, scratch injury models of pure neuronal cultures have not yet been exploited to study programmed cell death (PCD). For this study, we examined model motor neurons (NSC19 cells) in culture and found time-dependent cell death in proximity (within 2.5 mm) to a physical scratch injury. Injury-induced cell death was apoptotic verified by positively-stained nuclei using both the in situ end-labeling (ISEL) procedure and Hoechst 33342. Unexpectedly, cells proximal to the injury site were not affected by the injury until 3 days later suggesting that adjacent motor neuron loss was dependent on a 'death signal' produced by direct injury to sister neurons. 'Executioners' in apoptosis include free radicals, cell cycle kinases and cysteine proteases (caspases). Extracellular serine proteases, such as thrombin and granzyme B, may activate such intracellular pathways and several inhibitors (serpins), such as CrmA, are effective in blocking apoptosis. Since protease nexin I (PNI), a serpin homologous with CrmA, prevents apoptosis of lumbar motor neurons and is increased after nerve injury, we examined mRNA by RT-PCR for PNI expression. Of interest, although we were unable to find significant levels of PNI message in NSC19 cells, we did detect it in the parent neuroblastoma.