RESUMEN

Nerve growth factor (NGF) was the first neurotrophin described. This neurotrophin contributes to organogenesis by promoting sensory innervation and angiogenesis in the endocrine and immune systems. Neuronal and non-neuronal cells produce and secrete NGF, and several cell types throughout the body express the high-affinity neurotrophin receptor TrkA and the low-affinity receptor p75NTR. NGF is essential for glucose-stimulated insulin secretion and the complete development of pancreatic islets. Plus, this factor is involved in regulating lipolysis and thermogenesis in adipose tissue. Immune cells produce and respond to NGF, modulating their inflammatory phenotype and the secretion of cytokines, contributing to insulin resistance and metabolic homeostasis. This neurotrophin regulates the synthesis of gonadal steroid hormones, which ultimately participate in the metabolic homeostasis of other tissues. Therefore, we propose that this neurotrophin's imbalance in concentrations and signaling during metabolic syndrome contribute to its pathophysiology. In the present work, we describe the multiple roles of NGF in immunoendocrine organs that are important in metabolic homeostasis and related to the pathophysiology of metabolic syndrome.

Asunto(s)

Síndrome Metabólico , Factor de Crecimiento Nervioso , Humanos , Síndrome Metabólico/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Neuronas/metabolismo , Receptor de Factor de Crecimiento Nervioso/metabolismo , Receptor trkA/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo

RESUMEN

OBJECTIVE: The 4-nitroquinoline 1-oxide (4-NQO) model for carcinogenesis has been used to investigate cancer stem cells (CSC), but no study has addressed the role of the p75 neurotrophin receptor (p75NTR) in 4-NQO-induced oral dysplasia and oral squamous cell carcinoma (OSCC). The aim of this study was to evaluate the immunohistochemistry profile of the p75NTR during 4-NQO-induced oral carcinogenesis in rats and to verify whether this profile has an association with proliferating cell nuclear antigen (PCNA) immunolabeling. DESIGN: For 28 weeks, rats were exposed to 4-NQO, which was diluted in the drinking water. After 3, 5, 7, 16, and 28 weeks, the animals were euthanized and their tongues were histologically analyzed using p75NTR and PCNA immunolabeling. RESULTS: In animals without 4-NQO exposure, the p75NTR and PCNA were expressed only in the basal epithelial layer and in a clustered manner. The oral epithelium showed dysplasia and a significant increase in the number of p75NTR- and PCNA-positive cells, which were localized mainly in the basal and suprabasal epithelial layers during weeks 5-16 of 4-NQO exposure. When the epithelium invaded the lamina propria and well-differentiated OSCC began, the p75NTR-positive cell frequency drastically decreased in epithelial cords and nests, showing a negative correlation with PCNA expression. p75NTR immunolabeling during 4-NQO-induced carcinogenesis was similar to that described for human head and neck dysplasia and neoplasia. CONCLUSIONS: p75NTR immunolabeling observed in 4-NQO-induced oral dysplastic and OSCC lesions were related to the early phases of oral carcinogenesis and may help predict cell dysplasia and malignant transformation.

Asunto(s)

Biomarcadores de Tumor , Carcinoma de Células Escamosas , Neoplasias de la Boca , Lesiones Precancerosas , Receptor de Factor de Crecimiento Nervioso , Animales , Masculino , Ratas , 4-Nitroquinolina-1-Óxido , Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/metabolismo , Modelos Animales de Enfermedad , Inmunohistoquímica , Neoplasias de la Boca/metabolismo , Lesiones Precancerosas/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Distribución Aleatoria , Ratas Wistar , Receptor de Factor de Crecimiento Nervioso/metabolismo

RESUMEN

BACKGROUND: Peripheral nerve injury results in retrograde cell body-related changes in the spinal motoneurons that will contribute to the regenerative response of their axons. Successful functional recovery also depends on molecular events mediated by innate immune response during Wallerian degeneration in the nerve microenvironment. A previous study in our lab demonstrated that TLR 2 and 4 develop opposite effects on synaptic stability in the spinal cord after peripheral nerve injury. Therefore, we suggested that the better preservation of spinal cord microenvironment would positively influence distal axonal regrowth. In this context, the present work aimed to investigate the influence of TLR2 and TLR4 on regeneration and functional recovery after peripheral nerve injury. METHODS: Eighty-eight mice were anesthetized and subjected to unilateral sciatic nerve crush (C3H/HeJ, n = 22, C3H/HePas, n = 22; C57Bl6/J, n = 22 and TLR2(-/-), n = 22). After the appropriate survival times (3, 7, 14 days, and 5 weeks), all mice were killed and the sciatic nerves and tibialis cranialis muscles were processed for immunohistochemistry and transmission electron microscopy (TEM). Gait analysis, after sciatic nerve crushing, was performed in another set of mice (minimum of n = 8 per group), by using the walking track test (CatWalk system). RESULTS: TLR4 mutant mice presented greater functional recovery as well as an enhanced p75(NTR) and neurofilament protein expression as compared to the wild-type strain. Moreover, the better functional recovery in mutant mice was correlated to a greater number of nerve terminal sprouts. Knockout mice for TLR2 exhibited 30 % greater number of degenerated axons in the distal stump of the sciatic nerve and a decreased p75(NTR) and neurofilament protein expression compared to the wild type. However, the absence of TLR2 receptor did not influence the overall functional recovery. End-point equivalent functional recovery in transgenic mice may be a result of enhanced axonal diameter found at 2 weeks after lesion. CONCLUSIONS: Altogether, the present results indicate that the lack of TLR2 or the absence of functional TLR4 does affect the nerve regeneration process; however, such changes are minimized through different compensatory mechanisms, resulting in similar motor function recovery, as compared to wild-type mice. These findings contribute to the concept that innate immune-related molecules influence peripheral nerve regeneration by concurrently participating in processes taking place both at the CNS and PNS.

Asunto(s)

Neuropatía Ciática/metabolismo , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Axotomía/efectos adversos , Regulación de la Expresión Génica/genética , Filamentos Intermedios/metabolismo , Filamentos Intermedios/ultraestructura , Cojera Animal/etiología , Locomoción/fisiología , Macrófagos/patología , Macrófagos/ultraestructura , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Unión Neuromuscular/patología , Unión Neuromuscular/ultraestructura , Receptor de Factor de Crecimiento Nervioso/metabolismo , Nervio Ciático/metabolismo , Nervio Ciático/patología , Nervio Ciático/ultraestructura , Neuropatía Ciática/complicaciones , Neuropatía Ciática/etiología , Especificidad de la Especie , Factores de Tiempo , Receptor Toll-Like 2/genética , Receptor Toll-Like 4/genética

RESUMEN

The olfactory and vomeronasal systems of vertebrates are characterised by neurogenesis occurring throughout life. The regenerative ability of olfactory receptor neurons relies on specific glial cells, the olfactory and vomeronasal axon-surrounding cells. Numerous studies have examined mammalian olfactory ensheathing cells which are considered potential candidates for spinal cord injury repair using cell-based therapy. With regard to non-mammalian vertebrates, limited information is available on these glial cells in fish, and there is no information on them in terrestrial anamniotes, the amphibians. In the present research, we studied the immunocytochemical characteristics of axon-surrounding cells in Ambystoma mexicanum. Urodeles have relatively simple olfactory and vomeronasal systems, and represent a good model for studying ensheathing cells in extant representatives of basal tetrapods. Sections from the decalcified heads of A. mexicanum were immunocytochemically processed for the detection of proteins used in research on mammalian olfactory-ensheathing cells. S100, GFAP and NCAM were clearly observed. p75NTR, Gal-1 and PSA-NCAM showed weak staining. No vimentin immunopositivity was observed. The corresponding areas of the olfactory and vomeronasal pathways displayed the same staining characteristics, with the exception of Gal-1, p75NTR and PSA-NCAM in the mucosae. The degree of marker expression was not uniform throughout the sensory pathways. In contrast to fish, both olfactory and vomeronasal nerves displayed uniform staining intensity. This study showed that some markers for mammalian and fish-ensheathing glia are also applicable in urodeles. The olfactory systems of vertebrates show similarities, and also clear dissimilarities. Further investigations are required to ascertain the functional significance of these regional and interspecific differences.

Asunto(s)

Ambystoma mexicanum/metabolismo , Neuroglía/metabolismo , Animales , Axones/metabolismo , Vías Olfatorias/metabolismo , Receptor de Factor de Crecimiento Nervioso/metabolismo , Olfato/fisiología , Traumatismos de la Médula Espinal/metabolismo , Órgano Vomeronasal/fisiología

RESUMEN

Chronic ethanol consumption can produce learning and memory deficits. Brain-derived neurotrophic factor (BDNF) and its receptors affect the pathogenesis of alcoholism. In this study, we examined the expression of BDNF, tropomyosin receptor kinase B (TrkB) and p75 neurotrophin receptor (p75NTR) in the hippocampus of a dog model of chronic alcoholism and abstinence. Twenty domestic dogs (9-10 months old, 15-20 kg; 10 males and 10 females) were obtained from Harbin Medical University. A stable alcoholism model was established through ad libitum feeding, and anti-alcohol drug treatment (Zhong Yao Jie Jiu Ling, the main ingredient was the stems of watermelon; developed in our laboratory), at low- and high-doses, was carried out. The Zhong Yao Jie Jiu Ling was effective for the alcoholism in dogs. The morphology of hippocampal neurons was evaluated using hematoxylin-eosin staining. The number and morphological features of BDNF, TrkB and p75NTR-positive neurons in the dentate gyrus (DG), and the CA1, CA3 and CA4 regions of the hippocampus were observed using immunohistochemistry. One-way ANOVA was used to determine differences in BDNF, TrkB and p75NTR expression. BDNF, TrkB and p75NTR-positive cells were mainly localized in the granular cell layer of the DG and in the pyramidal cell layer of the CA1, CA3 and CA4 regions (DG>CA1>CA3>CA4). Expression levels of both BDNF and TrkB were decreased in chronic alcoholism, and increased after abstinence. The CA4 region appeared to show the greatest differences. Changes in p75NTR expression were the opposite of those of BDNF and TrkB, with the greatest differences observed in the DG and CA4 regions.

Asunto(s)

Animales , Masculino , Femenino , Perros , Abstinencia de Alcohol , Alcoholismo/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Hipocampo/química , Receptor de Factor de Crecimiento Nervioso/metabolismo , Receptor trkB/metabolismo , Factor Neurotrófico Derivado del Encéfalo/genética , Enfermedad Crónica , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Hipocampo/metabolismo , Inmunohistoquímica , Receptor de Factor de Crecimiento Nervioso/genética , Receptor trkB/genética

RESUMEN

Chronic ethanol consumption can produce learning and memory deficits. Brain-derived neurotrophic factor (BDNF) and its receptors affect the pathogenesis of alcoholism. In this study, we examined the expression of BDNF, tropomyosin receptor kinase B (TrkB) and p75 neurotrophin receptor (p75NTR) in the hippocampus of a dog model of chronic alcoholism and abstinence. Twenty domestic dogs (9-10 months old, 15-20 kg; 10 males and 10 females) were obtained from Harbin Medical University. A stable alcoholism model was established through ad libitum feeding, and anti-alcohol drug treatment (Zhong Yao Jie Jiu Ling, the main ingredient was the stems of watermelon; developed in our laboratory), at low- and high-doses, was carried out. The Zhong Yao Jie Jiu Ling was effective for the alcoholism in dogs. The morphology of hippocampal neurons was evaluated using hematoxylin-eosin staining. The number and morphological features of BDNF, TrkB and p75NTR-positive neurons in the dentate gyrus (DG), and the CA1, CA3 and CA4 regions of the hippocampus were observed using immunohistochemistry. One-way ANOVA was used to determine differences in BDNF, TrkB and p75NTR expression. BDNF, TrkB and p75NTR-positive cells were mainly localized in the granular cell layer of the DG and in the pyramidal cell layer of the CA1, CA3 and CA4 regions (DG>CA1>CA3>CA4). Expression levels of both BDNF and TrkB were decreased in chronic alcoholism, and increased after abstinence. The CA4 region appeared to show the greatest differences. Changes in p75NTR expression were the opposite of those of BDNF and TrkB, with the greatest differences observed in the DG and CA4 regions.

Asunto(s)

Abstinencia de Alcohol , Alcoholismo/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Hipocampo/química , Receptor de Factor de Crecimiento Nervioso/metabolismo , Receptor trkB/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Enfermedad Crónica , Modelos Animales de Enfermedad , Perros , Femenino , Regulación de la Expresión Génica , Hipocampo/metabolismo , Inmunohistoquímica , Masculino , Receptor de Factor de Crecimiento Nervioso/genética , Receptor trkB/genética

RESUMEN

INTRODUCTION: Severe lesions in the facial nerve may have extensive axonal loss and leave isolated stumps that impose technical difficulties for nerve grafting. METHODS: We evaluated bone marrow stem cells (BMSC) in a silicone conduit for rat facial nerve regeneration from isolated stumps. Group A utilized empty silicone tubes; in groups B-D, the tube was filled with acellular gel; and, in groups C and D, undifferentiated BMSC (uBMSC) or Schwann-like cells differentiated from BMSC (dBMSC) were added, respectively. Compound muscle action potentials (CMAPs) were measured, and histology was evaluated. RESULTS: Groups C and D had the highest CMAP amplitudes. Group C had shorter CMAP durations than groups A, B, and D. Distal axonal number and density were increased in group C compared with groups A and B. CONCLUSIONS: Regeneration of the facial nerve was improved by both uBMSC and dBMSC in rats, yet uBMSC was associated with superior functional results.

Asunto(s)

Muñones de Amputación/cirugía , Trasplante de Médula Ósea/métodos , Nervio Facial/citología , Células Madre Mesenquimatosas/fisiología , Músculo Esquelético/fisiopatología , Regeneración Nerviosa/fisiología , Potenciales de Acción/fisiología , Animales , Axones/patología , Células Cultivadas , Electromiografía , Estudios de Seguimiento , Masculino , Factor 6 de Transcripción de Unión a Octámeros/metabolismo , Técnicas de Cultivo de Órganos , Ratas , Ratas Wistar , Receptor de Factor de Crecimiento Nervioso/metabolismo , Proteínas S100/metabolismo , Estadísticas no Paramétricas , Transducción Genética , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismo

RESUMEN

Beta-amyloid (Abeta) deposition is one important pathological hallmark in Alzheimer's disease (AD). However, low levels of Abeta may modify critical endogenous protection systems before neurodegeneration occurs. We examined the time-course effect of sublethal concentrations of Abeta on total BDNF (panBDNF), BDNF transcripts (I, II, IV and VI), trkB.FL, trkB.T1 and p75(NGFR) mRNA expression in cultured cortical neurons. We have shown that Abeta exhibited a dual response on BDNF mRNA, i.e. an increase at short times (3-5 h) and a dramatic decrease at longer times (24 or 48 h). The early increase in BDNF expression seems to be driven by increased expression of transcripts I and IV. The BDNF drop was specific since did not occur for other mRNAs examined. The BDNF protein content showed a similar profile but did not follow the dramatic reduction as its encoding mRNA. These observations may help to explain cognitive deficits observed at initial stages of AD.

Asunto(s)

Péptidos beta-Amiloides/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Corteza Cerebral/fisiología , Neuronas/fisiología , Fragmentos de Péptidos/metabolismo , Animales , Western Blotting , Supervivencia Celular/fisiología , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Inmunoensayo , Hibridación in Situ , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor de Factor de Crecimiento Nervioso/metabolismo , Receptor trkB/metabolismo , Factores de Tiempo

RESUMEN

In neuronal cultures it has been demonstrated that neurotrophins can elicit neuronal death through the p75 neurotrophic receptor (p75ntr) in the absence of concomitant Trk signaling. However, it was suggested that p75ntr induces neuronal death after status epilepticus (SE) in neuronal populations that express relatively high quantities of tropomyosin receptor kinase B (TrkB). Here, using Western blot and immunohistochemistry analyses in the hippocampus, we found that 3-h SE caused a remarkable decrease in TrkB expression and phosphorylation, and a significant increase in p75ntr. TrkB modification occurs before the overexpression of the tumor suppressor protein p53, accompanies the cell damage taking place in the dentate gyrus, and precedes the CA1 neuronal injury as assessed by Fluoro-Jade B and Nissl staining. Co-immunoprecipitation of brain-derived neurotrophic factor (BDNF) or its immature form proBDNF showed increased interaction with p75ntr after its binding to TrkB was reduced. Interestingly, proBDNF also increases its binding with p75ntr after seizures that do not cause neuronal death (animals injected with pilocarpine that fail to enter SE). However, in those animals, TrkB protein levels remained unchanged and its phosphorylation increased. Our results indicate an intrinsic capacity of neurons in vivo to modify final neurotrophin output by changing the proportion of their receptors' expression and the receptors' interaction with their ligands. These early events support the idea that neurotrophins may be involved in the induction of neuronal death in vivo under pathological conditions.

Asunto(s)

Factor Neurotrófico Derivado del Encéfalo/metabolismo , Neuronas/patología , Receptor de Factor de Crecimiento Nervioso/metabolismo , Receptor trkB/metabolismo , Estado Epiléptico/metabolismo , Estado Epiléptico/patología , Animales , Benzoxazinas , Western Blotting , Colorantes , Electrochoque , Fluoresceínas , Inmunohistoquímica , Inmunoprecipitación , Etiquetado Corte-Fin in Situ , Masculino , Agonistas Muscarínicos , Compuestos Orgánicos , Oxazinas , Pilocarpina , Ratas , Ratas Wistar , Estado Epiléptico/inducido químicamente

RESUMEN

Signaling by the p75 neurotrophin receptor (p75) has been implicated in diverse neuronal responses, including the control of neuronal survival versus death and axonal regeneration and growth cone collapse, involving p75 in different neuropathological conditions. There are different levels of complexity regulating p75-mediated signaling. First, p75 can interact with different ligands and co-receptors in the plasma membrane, forming tripartite complexes, whose activation result in different cellular outcomes. Moreover, it was recently described that trafficking capacities of p75 in neurons are regulating, in addition to p75 downstream interactions, also the sequential cleavage of p75. The proteolytical processing of p75 involves, first, a shedding event that releases a membrane-bound carboxiterminal fragment (p75-CTF), followed by a gamma-secretase mediated cleavage, generating a soluble intracellular domain (p75-ICD) with signaling capabilities. The first shedding event, generating a p75-CTF, is the key step to regulating the production of p75-ICD, and although the generation of p75-ICD is important for both p75-mediated control of neuronal survival and the control of neurite outgrowth, little is known how both cleavage events are regulated. In this review, we argue that both sheddases and gamma-secretase are key membrane components regulating p75-mediated signaling transduction; therefore, further attention should be paid to their roles as p75 signaling regulators.

Asunto(s)

Secretasas de la Proteína Precursora del Amiloide/fisiología , Metaloproteasas/fisiología , Receptor de Factor de Crecimiento Nervioso/metabolismo , Transducción de Señal/fisiología , Animales , Modelos Biológicos

RESUMEN

Nerve growth factor (NGF) can induce apoptosis by signaling through the p75 neurotrophin receptor (p75(NTR)) in several nerve cell populations. Cultured embryonic motor neurons expressing p75(NTR) are not vulnerable to NGF unless they are exposed to an exogenous flux of nitric oxide (*NO). In the present study, we show that p75(NTR)-mediated apoptosis in motor neurons involved neutral sphingomyelinase activation, increased mitochondrial superoxide production, and cytochrome c release to the cytosol. The mitochondria-targeted antioxidants mitoQ and mitoCP prevented neuronal loss, further evidencing the role of mitochondria in NGF-induced apoptosis. In motor neurons overexpressing the amyotrophic lateral sclerosis (ALS)-linked superoxide dismutase 1(G93A) (SOD1(G93A)) mutation, NGF induced apoptosis even in the absence of an external source of *NO. The increased susceptibility of SOD1(G93A) motor neurons to NGF was associated to decreased nuclear factor erythroid 2-related factor 2 (Nrf2) expression and downregulation of the enzymes involved in glutathione biosynthesis. In agreement, depletion of glutathione in nontransgenic motor neurons reproduced the effect of SOD1(G93A) expression, increasing their sensitivity to NGF. In contrast, rising antioxidant defenses by Nrf2 activation prevented NGF-induced apoptosis. Together, our data indicate that p75(NTR)-mediated motor neuron apoptosis involves ceramide-dependent increased mitochondrial superoxide production. This apoptotic pathway is facilitated by the expression of ALS-linked SOD1 mutations and critically modulated by Nrf2 activity.

Asunto(s)

Apoptosis/fisiología , Mitocondrias/metabolismo , Neuronas Motoras/fisiología , Factor 2 Relacionado con NF-E2/metabolismo , Receptor de Factor de Crecimiento Nervioso/metabolismo , Médula Espinal/citología , Animales , Animales Modificados Genéticamente , Apoptosis/efectos de los fármacos , Células Cultivadas , Citocromos c/metabolismo , Embrión de Mamíferos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Mitocondrias/efectos de los fármacos , Neuronas Motoras/efectos de los fármacos , Factor de Crecimiento Nervioso/farmacología , Donantes de Óxido Nítrico/farmacología , Compuestos Nitrosos/farmacología , Oligodesoxirribonucleótidos Antisentido/farmacología , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/farmacología

RESUMEN

The retrograde transport of nerve growth factor (NGF) in neurite-like processes of living differentiated PC12 cells was studied using streptavidin-quantum dots (QDs) coupled to monobiotin-NGF. These reagents were active in differentiation, binding, internalization, and transport. Ten-35% of the QD-NGF-receptor complexes were mobile. Quantitative single particle tracking revealed a bidirectional step-like motion, requiring intact microtubules, with a net retrograde velocity of 0.054+/-0.020 microm/s. Individual runs had a mean velocity of approximately 0.15 microm/s at room temperature, and the run times were exponentially distributed. The photostability and brightness of QDs permit extended real-time analysis of individual QDbNGF- receptor complexes trafficking within neurites.

Asunto(s)

Complejos Multiproteicos/metabolismo , Receptor de Factor de Crecimiento Nervioso/metabolismo , Coloración y Etiquetado/métodos , Animales , Compuestos Cromogénicos/farmacocinética , Endocitosis , Microtúbulos/fisiología , Neuritas/metabolismo , Neuritas/ultraestructura , Células PC12 , Unión Proteica , Transporte de Proteínas , Puntos Cuánticos , Ratas , Sensibilidad y Especificidad , Especificidad por Sustrato

RESUMEN

Neurotrophins are trophic factors that regulate important neuronal functions. They bind two unrelated receptors, the Trk family of receptor-tyrosine kinases and the p75 neurotrophin receptor (p75). p75 was recently identified as a new substrate for gamma-secretase-mediated intramembrane proteolysis, generating a p75-derived intracellular domain (p75-ICD) with signaling capabilities. Using PC12 cells as a model, we studied how neurotrophins activate p75 processing and where these events occur in the cell. We demonstrate that activation of the TrkA receptor upon binding of nerve growth factor (NGF) regulates the metalloprotease-mediated shedding of p75 leaving a membrane-bound p75 C-terminal fragment (p75-CTF). Using subcellular fractionation to isolate a highly purified endosomal fraction, we demonstrate that p75-CTF ends up in endosomes where gamma-secretase-mediated p75-CTF cleavage occurs, resulting in the release of a p75-ICD. Moreover, we show similar structural requirements for gamma-secretase processing of p75 and amyloid precursor protein-derived CTFs. Thus, NGF-induced endocytosis regulates both signaling and proteolytic processing of p75.

Asunto(s)

Secretasas de la Proteína Precursora del Amiloide/fisiología , Endosomas/metabolismo , Factor de Crecimiento Nervioso/farmacología , Receptor de Factor de Crecimiento Nervioso/metabolismo , Receptor trkA/fisiología , Animales , Factor Neurotrófico Derivado del Encéfalo/farmacología , Endocitosis , Células PC12 , Estructura Terciaria de Proteína , Ratas , Receptor de Factor de Crecimiento Nervioso/química , Transducción de Señal , Acetato de Tetradecanoilforbol/farmacología

RESUMEN

Nerve growth factor (NGF) overexpression and increased production of peroxynitrite occur in several neurodegenerative diseases. We investigated whether NGF could undergo posttranslational oxidative or nitrative modifications that would modulate its biological activity. Compared to native NGF, peroxynitrite-treated NGF showed an exceptional ability to induce p75(NTR)-dependent motor neuron apoptosis at physiologically relevant concentrations. Whereas native NGF requires an external source of nitric oxide (NO) to induce motor neuron death, peroxynitrite-treated NGF induced motor neuron apoptosis in the absence of exogenous NO. Nevertheless, NO potentiated the apoptotic activity of peroxynitrite-modified NGF. Blocking antibodies to p75(NTR) or downregulation of p75(NTR) expression by antisense treatment prevented motor neuron apoptosis induced by peroxynitrite-treated NGF. We investigated what oxidative modifications were responsible for inducing a toxic gain of function and found that peroxynitrite induced tyrosine nitration in a dose-dependent manner. Moreover, peroxynitrite triggered the formation of stable high-molecular-weight oligomers of NGF. Preventing tyrosine nitration by urate abolished the effect of peroxynitrite on NGF apoptotic activity. These results indicate that the oxidation of NGF by peroxynitrite enhances NGF apoptotic activity through p75(NTR) 10,000-fold. To our knowledge, this is the first known posttranslational modification that transforms a neurotrophin into an apoptotic agent.

Asunto(s)

Apoptosis/fisiología , Neuronas Motoras/metabolismo , Factor de Crecimiento Nervioso/química , Ácido Peroxinitroso/farmacología , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Células Cultivadas , Ensayo de Cambio de Movilidad Electroforética , Espectrometría de Masas , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/patología , Factor de Crecimiento Nervioso/metabolismo , Oligonucleótidos Antisentido/farmacología , Ratas , Receptor de Factor de Crecimiento Nervioso/antagonistas & inhibidores , Receptor de Factor de Crecimiento Nervioso/efectos de los fármacos , Receptor de Factor de Crecimiento Nervioso/metabolismo , Tirosina/metabolismo

RESUMEN

The p75 neurotrophin receptor (p75NTR) is expressed by degenerating spinal motor neurons in amyotrophic lateral sclerosis (ALS). The mature and pro-form of nerve growth factor (NGF) activate p75NTR to trigger motor neuron apoptosis. However, attempts to modulate p75NTR-mediated neuronal death in ALS models by downregulating or antagonizing p75NTR with synthetic peptides have led to only modest results. Recently, a novel ligand of p75NTR, compound LM11A-24, has been identified. It is a non-peptidyl mimetic of the neurotrophin loop 1 domain that promotes hippocampal neuron survival through p75NTR and exerts protection against p75NTR-mediated apoptosis of oligodendrocytes induced by proNGF. Thus, LM11A-24 appears to activate p75NTR-linked survival but not death mechanisms, and may interfere with the ability of neurotrophins to induce apoptosis. Given these findings, we hypothesized that LM11A-24 might be a particularly potent inhibitor of motor neuron degeneration. We examined the effects of LM11A-24 on apoptosis of cultured rat embryonic motor neurons. Interestingly, in contrast to the effects observed in hippocampal cultures, LM11A-24 was unable to prevent motor neuron apoptosis induced by trophic factor deprivation. However, picomolar concentrations of LM11A-24 prevented p75NTR-dependent motor neuron death induced by either exogenous addition of NGF or spinal cord extracts from symptomatic superoxide dismutase-1G93A mice, in the presence of low steady-state concentrations of nitric oxide. LM11A-24 also inhibited motor neuron death induced by NGF-producing reactive astrocytes in co-culture conditions. These studies suggest that modulation of p75NTR by small molecule ligands targeting this receptor might constitute a novel strategy for preventing motor neuron degeneration.

Asunto(s)

Neuronas Motoras/efectos de los fármacos , Factores de Crecimiento Nervioso/química , Factores de Crecimiento Nervioso/farmacología , Receptor de Factor de Crecimiento Nervioso/metabolismo , Análisis de Varianza , Animales , Animales Recién Nacidos , Astrocitos , Cafeína/análogos & derivados , Cafeína/farmacología , Recuento de Células/métodos , Muerte Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Embrión de Mamíferos , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Lipopolisacáridos/farmacología , Neuronas Motoras/fisiología , Factor de Crecimiento Nervioso/farmacología , Estructura Terciaria de Proteína , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Médula Espinal/citología

RESUMEN

Chronic stress affects brain areas involved in learning and emotional responses. Although most studies have concentrated on the effect of stress on limbic-related brain structures, in this study we investigated whether chronic stress might induce impairments in diencephalic structures associated with limbic components of the stress response. Specifically, we analyzed the effect of chronic immobilization stress on the expression of sympathetic markers in the rat epithalamic pineal gland by immunohistochemistry and western blot, whereas the plasma melatonin concentration was determined by radioimmunoassay. We found that chronic stress decreased the expression of three sympathetic markers in the pineal gland, tyrosine hydroxylase, the p75 neurotrophin receptor and alpha-tubulin, while the same treatment did not affect the expression of the non-specific sympathetic markers Erk1 and Erk2, and glyceraldehyde-3-phosphate dehydrogenase. Furthermore, these results were correlated with a significant increase in plasma melatonin concentration in stressed rats when compared with control animals. Our findings indicate that stress may impair pineal sympathetic inputs, leading to an abnormal melatonin release that may contribute to environmental maladaptation. In addition, we propose that the pineal gland is a target of glucocorticoid damage during stress.

Asunto(s)

Melatonina/sangre , Glándula Pineal/metabolismo , Estrés Psicológico/sangre , Sistema Nervioso Simpático/metabolismo , Animales , Biomarcadores/metabolismo , Enfermedad Crónica , Regulación hacia Abajo/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Gliceraldehído 3-Fosfato/metabolismo , Inmunohistoquímica , Masculino , Melatonina/metabolismo , Actividad Motora/fisiología , Glándula Pineal/inervación , Ratas , Ratas Sprague-Dawley , Receptor de Factor de Crecimiento Nervioso/metabolismo , Restricción Física , Estrés Psicológico/fisiopatología , Fibras Simpáticas Posganglionares/metabolismo , Fibras Simpáticas Posganglionares/fisiopatología , Sistema Nervioso Simpático/fisiopatología , Tubulina (Proteína)/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Regulación hacia Arriba/fisiología

RESUMEN

Astrocytes may modulate the survival of motor neurons in amyotrophic lateral sclerosis (ALS). We have previously shown that fibroblast growth factor-1 (FGF-1) activates astrocytes to increase secretion of nerve growth factor (NGF). NGF in turn induces apoptosis in co-cultured motor neurons expressing the p75 neurotrophin receptor (p75NTR) by a mechanism involving nitric oxide (NO) and peroxynitrite formation. We show here that FGF-1 increased the expression of inducible nitric oxide synthase and NO production in astrocytes, making adjacent motor neurons vulnerable to NGF-induced apoptosis. Spinal cord astrocytes isolated from transgenic SOD1G93A rats displayed increased NO production and spontaneously induced apoptosis of co-cultured motor neurons. FGF-1 also activates the redox-sensitive transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) in astrocytes. Because Nrf2 increases glutathione (GSH) biosynthesis, we investigated the role of GSH production by astrocytes on p75NTR-dependent motor neuron apoptosis. The combined treatment of astrocytes with FGF-1 and t-butylhydroquinone (tBHQ) increased GSH production and secretion, preventing motor neuron apoptosis. Moreover, Nrf2 activation in SOD1G93A astrocytes abolished their apoptotic activity. The protection exerted by increased Nrf2 activity was overcome by adding the NO donor DETA-NONOate to the co-cultures or by inhibiting GSH synthesis and release from astrocytes. These results suggest that activation of Nrf2 in astrocytes can reduce NO-dependent toxicity to motor neurons by increasing GSH biosynthesis.

Asunto(s)

Astrocitos/metabolismo , Glutatión/biosíntesis , Neuronas Motoras/fisiología , Factor 2 Relacionado con NF-E2/metabolismo , Receptor de Factor de Crecimiento Nervioso/metabolismo , Animales , Animales Modificados Genéticamente , Animales Recién Nacidos , Antioxidantes/farmacología , Astrocitos/efectos de los fármacos , Western Blotting/métodos , Recuento de Células/métodos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo/métodos , Interacciones Farmacológicas , Embrión de Mamíferos , Activación Enzimática/efectos de los fármacos , Factor 1 de Crecimiento de Fibroblastos/farmacología , Proteína Ácida Fibrilar de la Glía/metabolismo , Glutatión/metabolismo , Hidroquinonas/farmacología , Metionina/análogos & derivados , Metionina/farmacología , Factor de Crecimiento Nervioso/farmacología , Nitratos/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Nitritos/metabolismo , Propionatos/farmacología , Pirimidinas/farmacología , Quinolinas/farmacología , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Médula Espinal/citología , Superóxido Dismutasa , Transfección/métodos , Urea/análogos & derivados , Urea/farmacología

RESUMEN

Oxidative stress mediated by nitric oxide (NO) and its toxic metabolite peroxynitrite has previously been associated with motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Degenerating spinal motor neurons in familial and sporadic ALS are typically surrounded by reactive astrocytes expressing the inducible form of NO synthase (iNOS), suggesting that astroglia may have a pathogenic role in ALS. We report here that a brief exposure of spinal cord astrocyte monolayers to peroxynitrite (0.25-1 mM) provoked long-lasting reactive morphological changes characterized by process-bearing cells displaying intense glial fibrillary acidic protein and iNOS immunoreactivity. Furthermore, peroxynitrite caused astrocytes to promote apoptosis of embryonic motor neurons subsequently plated on the monolayers. Neuronal death occurred within 24 hr after plating, as evidenced by the presence of degenerating motor neurons positively stained for activated caspase-3 and nitrotyrosine. Motor neuron death was largely prevented by NOS inhibitors and peroxynitrite scavengers but not by trophic factors that otherwise will support motor neuron survival in the absence of astrocytes. The bacterial lipopolysaccharide, a well-known inflammatory stimulus that induces iNOS expression in astrocytes, provoked the same effects on astrocytes as peroxynitrite. Thus, spinal cord astrocytes respond to extracellular peroxynitrite by adopting a phenotype that is cytotoxic to motor neurons through peroxynitrite-dependent mechanisms.

Asunto(s)

Esclerosis Amiotrófica Lateral/metabolismo , Apoptosis/fisiología , Astrocitos/metabolismo , Neuronas Motoras/metabolismo , Óxido Nítrico/metabolismo , Ácido Peroxinitroso/metabolismo , Médula Espinal/metabolismo , Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Astrocitos/efectos de los fármacos , Astrocitos/patología , Comunicación Celular/efectos de los fármacos , Comunicación Celular/fisiología , Tamaño de la Célula/efectos de los fármacos , Tamaño de la Célula/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Técnica del Anticuerpo Fluorescente , Depuradores de Radicales Libres/farmacología , Gliosis/metabolismo , Gliosis/patología , Gliosis/fisiopatología , Lipopolisacáridos/farmacología , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/patología , Óxido Nítrico Sintasa/metabolismo , Ácido Peroxinitroso/toxicidad , Fenotipo , Ratas , Receptor de Factor de Crecimiento Nervioso/efectos de los fármacos , Receptor de Factor de Crecimiento Nervioso/metabolismo , Médula Espinal/efectos de los fármacos , Médula Espinal/fisiopatología