RESUMEN

Hepatitis E virus (HEV) is well-known to cause endemic outbreaks of hepatitis in tropical countries, mostly caused by HEV genotypes 1 or 2 and transmitted from humans to humans via the fecal-oral route. In contrast, HEV genotypes 3 or 4 are commonly encountered as sporadic cases in a non-endemic setting; these autochthonous cases are transmitted from animals to humans and commonly affect elderly male subjects. We report a five-month-old caucasian girl presenting with diarrhea, emesis, and elevated ALT. Surprisingly, acute infection with Hepatitis E virus (HEV) genotype 3 was laboratory-confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) and sequencing. Thirteen months later, RT-PCR for HEV from stool tested negative whereas anti-HEV IgG in serum tested positive. Neither HEV RNA nor anti-HEV antibodies could be detected in stool or serum of the parents. To our knowledge, this is the first pediatric case of a HEV infection in Germany. Thus, HEV should be included into the differential diagnosis of pediatric infectious liver and bowel disease.

Asunto(s)

Virus de la Hepatitis E/aislamiento & purificación , Hepatitis E/diagnóstico , ARN Viral/sangre , Secuencia de Bases , Heces/virología , Femenino , Genotipo , Alemania , Anticuerpos Antihepatitis/sangre , Hepatitis E/virología , Virus de la Hepatitis E/inmunología , Humanos , Inmunoglobulina G/sangre , Lactante , ARN Viral/análisis , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

BACKGROUND: Respiratory infection and failure is a commonly encountered problem in intensive care unit (ICU) patients. However, despite the accumulating body of evidence to suggest that herpes simplex virus type 1 (HSV-1) is associated with pneumonia, the exact role played by this virus in this process is still not fully understood. Therefore, to identify patients at risk, we have conducted a case-control study to characterize patients with HSV-1-positive pneumonia. PATIENTS AND METHODS: Between 2007 and 2009, all patients with suspected viral pneumonia were tested for the presence of herpes viruses using a PCR assay approach with respiratory specimens. To identify possible associations, risk factors, and impact of HSV, HSV-1-positive ICU patients (n = 51) were compared to age-, gender-, and department- and season-matched HSV-negative patients (n = 52). RESULTS: HSV-positive patients differed significantly from the HSV-negative ones only in terms of time of mechanical ventilation (13 vs. 6 days, respectively; p = 0.002). Subgroup analysis in the patients aged >60 years and in those without bacterial detection revealed a similar trend (p = 0.01 and p = 0.004, respectively). Mortality did not differ between the groups or between the HSV-1-positive patients treated with aciclovir and those who were not. A viral load >10E+05 geq/ml was associated with mechanical ventilation (20/21 vs. 17/29; p = 0.004), acute respiratory distress syndrome (ARDS; 19/21 vs. 18/29; p = 0.005), sepsis (18/21 vs. 14/29; p = 0.008), detection of a bacterial pathogen in the same specimen (10/21 vs. 4/29; p = 0.01) and longer ICU stay (25 vs. 30 days; p = 0.04). CONCLUSION: Despite several associations with high viral load, the clinical outcome of HSV-1-positive ICU patients did not differ significantly from the clinical outcome of HSV-negative patients. This finding indicates that HSV-1 viral loads in respiratory specimens are a symptom of a clinically poor condition rather than a cause of it. Longitudinal and therapy studies are therefore needed to distinguish between HSV-1 as a causative pathogen and HSV-1 as a bystander of pneumonia/ARDS.

Asunto(s)

Neumonía Viral/virología , Sistema Respiratorio/virología , Simplexvirus/aislamiento & purificación , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Humanos , Unidades de Cuidados Intensivos , Masculino , Persona de Mediana Edad , Neumonía Viral/mortalidad , Respiración Artificial/efectos adversos , Simplexvirus/genética , Simplexvirus/fisiología , Carga Viral

Asunto(s)

Debilidad Muscular/genética , Mutación Puntual , Proteínas de Transporte Vesicular/genética , Femenino , Humanos , Masculino , Linaje

RESUMEN

VAPB is a highly conserved integral membrane protein that is ubiquitously expressed in all eukaryotic organisms and located within the membranes of the endoplasmic reticulum (ER). The P56S missense mutation of the VAPB protein is linked to a hereditary form of amyotrophic lateral sclerosis (ALS8), and the pathogenesis of ALS8 has remained enigmatic. We report the cloning of five novel splice variants of the human VAPB gene, all of which are expressed at the mRNA level in the human nervous system. When transfected into human HEK293 or SH-SY5Y cells, two of these variants (VAPB-2 and VAPB-4,5) were readily detectable by immunoblotting whereas two variants (VAPB-3 and VAPB-3,4) became detectable after proteasomal inhibition, a condition commonly found in neurodegenerative diseases. Interestingly, one of these novel VAPB variants, VAPB-2, co-immunoprecipitated with wt-VAPB. However, so far none of these splice variants could be detected by immunoblotting of lysates from selected human tissues, suggesting that in vivo, the proteins translated from the variant VAPB mRNAs are quickly degraded or, alternatively, the expressed proteins are below detection limit of the available antibodies. We speculate that under conditions of proteasomal inhibition, as encountered in many neurodegenerative diseases including ALS, variant VAPB proteins might accumulate in affected cells and contribute to ALS pathogenesis.

Asunto(s)

Empalme Alternativo , Esclerosis Amiotrófica Lateral/genética , Proteínas de Transporte Vesicular/genética , Esclerosis Amiotrófica Lateral/metabolismo , Línea Celular , Clonación Molecular , Humanos , Inmunoprecipitación , Proteínas de Transporte Vesicular/metabolismo

RESUMEN

Mutations of the neurotrophin receptor tyrosine kinase TrkA (NTRK1) cause congenital sensory neuropathy with insensitivity to pain and anhydrosis (CIPA), also called hereditary sensory and autonomous neuropathy type IV (HSAN IV). The neuronal splice variant of TrkA, TrkAII, binds two neurotrophin ligands, nerve growth factor (NGF) and neurotrophin-3 (NT3). Several studies have demonstrated NGF signaling defects in CIPA-associated TrkA mutants. To date, however, no study has examined NT3/TrkA signaling of CIPA mutants. As the interaction of NT3 and TrkA temporally and spatially precedes the interaction of NGF with TrkA, we examined the signaling of NT3 in a CIPA-associated TrkA mutant. Intriguingly, we revealed remarkable defects in NT3-induced ERK1/2 phosphorylation and neurite outgrowth. The impact of our findings is twofold. First, our data call for a re-examination of previously described TrkAII CIPA mutants regarding their NT3 signaling capability. Second, we envision that CIPA/HSAN IV polyneuropathies might fall into two different subgroups: one with diminished NT3/TrkAII signaling, in which axons actually do not reach their targets, and a second group with sufficient NT3/TrkAII signaling but diminished NGF/TrkAII signaling, in which axons do reach their targets, yet degenerate after successful target engagement.

Asunto(s)

Neurotrofina 3/genética , Receptor trkA/genética , Transducción de Señal/genética , Western Blotting , Células Cultivadas , Humanos , Mutación , Neurotrofina 3/metabolismo , Fosforilación , Receptor trkA/metabolismo

RESUMEN

Neurotrophin receptors of the Trk family promote neuronal survival. The signal transduction of Trk receptors is regulated by endosomal trafficking. Monoubiquitination of receptor tyrosine kinases is an established signal for sorting of internalized receptors to late endosomes. The NGF receptor TrkA is sorted to late endosomes and undergoes ubiquitination, indicating a so far undefined regulatory role of proteasomal activity in the trafficking of TrkA. Surprisingly, we found that proteasomal inhibition alters the trafficking of TrkA from the late endosomal sorting pathway to the recycling pathway. Many neurodegenerative diseases are associated with impaired proteasomal activity. Thus, our study suggests that missorting of neurotrophic receptors might contribute to neuronal death in those neurodegenerative diseases that are known to be associated with impaired proteasomal function.

Asunto(s)

Endosomas/enzimología , Factores de Crecimiento Nervioso/metabolismo , Neuronas/enzimología , Complejo de la Endopetidasa Proteasomal/metabolismo , Receptor trkA/metabolismo , Animales , Apoptosis , Enfermedades Neurodegenerativas/enzimología , Células PC12 , Fosforilación , Inhibidores de Proteasoma , Transporte de Proteínas , Ratas , Transducción de Señal , Ubiquitinación

RESUMEN

Neuroblastoma cell lines are commonly used as models to study neuronal differentiation, as they retain the capacity to differentiate into a neuronal-like phenotype. Receptor tyrosine kinase (RTK) signaling is essential for neuronal differentiation during development, and cholesterol-containing lipid-rafts are important for RTK signaling. Hydroxymethylglutaryl-coenzyme A reductase inhibitors of the statin family impair cholesterol biosynthesis and are in widespread clinical use for the treatment of cardiovascular diseases. It is of great clinical interest that statin treatment also correlates with a lower incidence of malignancies. We found that mevastatin triggered neurite outgrowth of neuroblastoma cells and examined the responsible signaling pathways. Treatment of Neuro2a cells with mevastatin for 24 hr induced neurite outgrowth associated with up-regulation of the neuronal marker protein NeuN. Interestingly, we found that mevastatin triggered phosphorylation of the key kinases epidermal growth factor receptor (EGFR), ERK1/2, and Akt/protein kinase B. Inhibition of EGFR, PI3K, and the mitogen-activated protein kinase cascade blocked mevastatin-induced neurite outgrowth. Moreover, add-back experiments of cell-permeable cholesterol precursors indicated that farnesylated and geranylgeranylated proteins play a major role in statin-induced neurite outgrowth. Taken together, our results provide the first mechanistic insight into statin-triggered signaling pathways that lead to neurite outgrowth in neuroblastoma cells. Surprisingly, we revealed that mevastatin triggered the phosphorylation of the EGFR and that this was because of the inhibition of farnesylated and geranylgeranylated proteins. We propose that members of the large family of farnesylated or geranylgeranylated small GTPases (such as Rabs or Rap1) regulating the trafficking and signaling of EGFR might be responsible for the statin-induced effects on EGFR signaling.

Asunto(s)

Diferenciación Celular/efectos de los fármacos , Receptores ErbB/efectos de los fármacos , Lovastatina/análogos & derivados , Factores de Crecimiento Nervioso/farmacología , Neuritas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Diferenciación Celular/fisiología , Línea Celular Tumoral , Proteínas de Unión al ADN , Receptores ErbB/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Lovastatina/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Proteína Quinasa 3 Activada por Mitógenos/efectos de los fármacos , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas de Unión al GTP Monoméricas/efectos de los fármacos , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Neuritas/metabolismo , Neurogénesis/efectos de los fármacos , Neurogénesis/fisiología , Proteínas Nucleares/efectos de los fármacos , Proteínas Nucleares/metabolismo , Fosfatidilinositol 3-Quinasas/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Prenilación de Proteína/efectos de los fármacos , Prenilación de Proteína/fisiología , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiología

RESUMEN

Growth factors such as the neurotrophins promote neuronal survival and shape neuronal morphology. Neurotrophin receptors are located on the surface of axons and dendrites and must convey their signal retrogradely to the nucleus to influence transcription of target genes. The distance between the site of receptor activation and the nucleus is tremendous. How is the retrograde transmission of survival signals being achieved? Recent work showed that signaling endosomes containing neurotrophin receptors and associated downstream kinases undergo retrograde vesicular transport along microtubules, propelled by the molecular motor dynein. The next objective in the "neurotrophin receptor trafficking meets signal transduction field" will be to elucidate the traffic control mechanisms governing the directed movement of signaling endosomes. Much is already known on the trafficking of the receptor for epidermal growth factor, EGFR. We will summarize the known traffic control mechanisms for EGFR and hypothesize whether EGFR-relevant traffic control mechanisms might also be relevant for neurotrophin receptor traffic control. Moreover, we speculate about potential implications of neurotrophin receptor traffic jams for neurodegenerative diseases.

Asunto(s)

Receptor de Factor de Crecimiento Nervioso/metabolismo , Receptor trkA/metabolismo , Transducción de Señal , Animales , Endosomas/metabolismo , Humanos , Enfermedades Neurodegenerativas/enzimología , Transporte de Proteínas

RESUMEN

The neurotrophin brain-derived neurotrophic factor (BDNF) binds to two cell surface receptors: TrkB receptors that promote neuronal survival and differentiation and p75NTR that induces apoptosis or survival. BDNF, as well as the other members of the neurotrophin family, is synthesized as a larger precursor, pro-BDNF, which undergoes posttranslational modifications and proteolytic processing by furin or related proteases. Both mature neurotrophins and uncleaved proneurotrophins are secreted from cells. The bioactivities of proneurotrophins could differ from those of mature, cleaved neurotrophins; therefore, we wanted to test whether pro-BDNF would differ from mature BDNF in its neurotrophin receptor binding and activation. A furin-resistant pro-BDNF, secreted from COS-7 cells, bound to TrkB-Fc and p75NTR-Fc, but not to TrkA-Fc or TrkC-Fc. Likewise, pro-BDNF elicited prototypical TrkB responses in biological assays, such as TrkB tyrosine phosphorylation, activation of ERK1/2, and neurite outgrowth. Moreover, mutation of the R103 residue of pro-BDNF abrogated its binding to TrkB-Fc but not to p75NTR-Fc. Taken together, these data indicate that pro-BDNF binds to and activates TrkB and could be involved in TrkB-mediated neurotrophic activity in vivo.

Asunto(s)

Factor Neurotrófico Derivado del Encéfalo/metabolismo , Precursores de Proteínas/metabolismo , Receptor trkA/metabolismo , Receptor trkB/metabolismo , Receptor trkC/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Animales , Western Blotting , Factor Neurotrófico Derivado del Encéfalo/genética , Células COS , Chlorocebus aethiops , Humanos , Mutagénesis Sitio-Dirigida , Mutación , Células PC12 , Precursores de Proteínas/genética , Ratas , Receptor de Factor de Crecimiento Nervioso

RESUMEN

Neurotrophins are key signalling molecules in the development of the nervous system. They elicit diverse cellular responses such as proliferation, differentiation, survival and apoptosis. Neurotrophins (NTs) bind to two different classes of cell surface receptors, Trk receptor tyrosine kinases and p75NTR, both of which are expressed by neuroblastoma cells. Neurotrophin signalling via Trks was shown to promote both survival and differentiation of neuroblastoma cells in vitro. The expression of certain Trk receptors is considered to be a prognostic indicator. The p75NTR receptor is the founding member of the Fas/TNF-R family, which is best known for its function in the induction of apoptosis. Its function in neuroblastomas is thus far poorly understood. We analysed neurotrophin receptor (NTR) expression of neuroblastoma cells by surface biotinylation assays and applied recombinant nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4/5 to these cell lines assessing their survival and proliferation in long-term assays lasting 6 days. NGF increased proliferation of Neuro 2a cells, which express p75NTR but no TrkA receptors on their surface. On the other hand, SK-N-BE cell proliferation was decreased after NGF treatment, even though these cells also express p75NTR but no TrkA receptors on their surface. Interestingly, neurotrophin-scavenger proteins (TrkB-Fc and TrkC-Fc) as well as chemical blockers of Trk receptor signalling (K252a, Wortmannin, PD98059) slowed down the proliferation of both cell lines in medium containing serum. Taken together, our results indicate that p75NTR activation has diverse effects on neuroblastomas, depending on the specific neuroblastoma clone. In addition, our studies point towards TrkB-Fc or TrkC-Fc receptor bodies as useful tools to influence the survival of neuroblastoma cells.

Asunto(s)

Factores de Crecimiento Nervioso/farmacología , Neuroblastoma/metabolismo , Neuroblastoma/patología , Receptor trkA/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Animales , División Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Ratones , Receptor de Factor de Crecimiento Nervioso , Receptor trkA/genética , Receptor trkB/genética , Receptor trkC/genética , Receptores Fc/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/farmacología

RESUMEN

Neurotrophins (NTs) play an important role in the modulation of synaptic transmission and in morphological changes in synaptic structures. Although there is agreement that brain-derived neurotrophic factor (BDNF) is sorted to large dense-core vesicles (LDCVs) and released via the regulated secretory pathway, there has been some dispute regarding the mode of secretion of nerve growth factor (NGF) and neurotrophin-3 (NT-3), two structurally related members of the NT family. In this study, we examined the subcellular localization and release characteristics of NGF, BDNF, and NT-3 in adenovirus-infected primary cortical neurons. We found that all members of the NT family colocalized with markers for the endoplasmic reticulum and Golgi within cell bodies and in a punctate manner with a marker for LDCVs within processes. Moreover, their release was triggered by depolarization, indicating that NGF, BDNF, and NT-3 are released via the regulated secretory pathway. When neurons were coinfected with two separate adenoviruses coding for NGF or BDNF, both NTs showed almost complete vesicular colocalization within single cells, suggesting that different NTs might be packaged into shared vesicles. We also examined whether the two splice variants of NGF, the short and long precursors, differ in their release characteristics. We found that neurons infected with viruses coding for either splice variant released NGF in a regulated way. Overall, our study supports the notion that all members of the NT family undergo activity-dependent regulated release from neurons, enabling them to act as "synaptotrophins" on electrically active neurons.

Asunto(s)

Factor Neurotrófico Derivado del Encéfalo/metabolismo , Corteza Cerebral/citología , Factor de Crecimiento Nervioso/metabolismo , Neuronas/metabolismo , Neurotrofina 3/metabolismo , Vesículas Secretoras/metabolismo , Animales , Autoantígenos/metabolismo , Proteínas de Unión a Calmodulina/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos , Retículo Endoplásmico/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Aparato de Golgi/metabolismo , Inmunohistoquímica/métodos , Microscopía Confocal/métodos , Factor de Crecimiento Nervioso/farmacología , Neuronas/efectos de los fármacos , Oligopéptidos/metabolismo , Señales de Clasificación de Proteína , Ratas , Ratas Sprague-Dawley , Vesículas Secretoras/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Transfección/métodos

RESUMEN

The short- and long-term effects of nerve growth factor (NGF) were studied on fibroblast cell lines stably expressing both TrkA and either wild-type p75 or a mutant that lacks the palmitoylation site of p75. The lack of palmitoylation had no effect on the ability of p75 to enhance the short-term NGF-induced tyrosine phosphorylation of TrkA over a wide range of NGF concentrations. Long-term treatment of the cell lines with NGF led to loss of cell adhesion to the culture dishes that increased with increasing concentrations of NGF and increased expression of TrkA. Treatment of the cell lines with mutant NGFs that bound selectively to TrkA or p75 alone revealed that cell detachment was mediated solely through TrkA. Increased cell detachment correlated with a decrease in the expression levels of fibronectin and cadherin, cell surface molecules involved in cell adhesion. The loss of cell adhesion with the cell line expressing the palmitoylation-deficient p75 were identical to those expressing wild type, as was anticipated from the lack of involvement of p75 in this process.

Asunto(s)

Antígenos CD/metabolismo , Moléculas de Adhesión Celular/metabolismo , Fibroblastos/metabolismo , Receptor trkA/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Animales , Antígenos CD/efectos de los fármacos , Moléculas de Adhesión Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Regulación hacia Abajo , Fibroblastos/efectos de los fármacos , Ratones , Factor de Crecimiento Nervioso/farmacología , Receptor trkA/efectos de los fármacos , Receptores del Factor de Necrosis Tumoral/efectos de los fármacos , Receptores Tipo II del Factor de Necrosis Tumoral

RESUMEN

Neurotrophins mediate their signals through two different receptors: the family of receptor tyrosine kinases, Trks, and the low affinity pan-neurotrophin receptor p75. Trk receptors show more restricted ligand specificity, whereas all neurotrophins are able to bind to p75. One important function of p75 is the enhancement of nerve growth factor signaling via TrkA by increasing TrkA tyrosine autophosphorylation. Here, we have examined the importance of p75 on TrkB- and TrkC-mediated neurotrophin signaling in an MG87 fibroblast cell line stably transfected with either p75 and TrkB or p75 and TrkC, as well as in PC12 cells stably transfected with TrkB. In contrast to TrkA signaling, p75 had a negative effect on TrkB tyrosine autophosphorylation in response to its cognate neurotrophins, brain-derived neurotrophic factor and neurotrophin 4/5. On the other hand, p75 had no effect on TrkB or TrkC activation in neurotrophin 3 treatment. p75 did not effect extracellular signal-regulated kinase 2 tyrosine phosphorylation in response to brain-derived neurotrophic factor, neurotrophin 3, or neurotrophin 4/5. These results suggest that the observed reduction in TrkB tyrosine autophosphorylation caused by p75 does not influence Ras/mitogen-activated protein kinase signaling pathway in neurotrophin treatments.

Asunto(s)

Factor Neurotrófico Derivado del Encéfalo/farmacología , Factores de Crecimiento Nervioso/farmacología , Receptor trkB/fisiología , Receptores de Factor de Crecimiento Nervioso/fisiología , Animales , Línea Celular , Cinética , Ratones , Fármacos Neuroprotectores/farmacología , Células PC12 , Fosforilación , Ratas , Receptor trkA/genética , Receptor trkA/fisiología , Receptor trkB/química , Receptor trkB/genética , Receptor trkC/química , Receptor trkC/fisiología , Receptores de Factor de Crecimiento Nervioso/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Transfección

RESUMEN

Particle-mediated gene transfer and two-photon microscopy were used to monitor the behavior of dendrites of individual cortical pyramidal neurons coexpressing green fluorescent protein (GFP) and brain-derived neurotrophic factor (BDNF). While the dendrites and spines of neurons expressing GFP alone grew modestly over 24-48 hr, coexpressing BDNF elicited dramatic sprouting of basal dendrites, accompanied by a regression of dendritic spines. Compared to GFP-transfected controls, the newly formed dendrites and spines were highly unstable. Experiments utilizing Trk receptor bodies, K252a, and overexpression of nerve growth factor (NGF) demonstrated that these effects were mediated by secreted BDNF interacting with extracellular TrkB receptors. Thus, BDNF induces structural instability in dendrites and spines, which, when restricted to particular portions of a dendritic arbor, may help translate activity patterns into specific morphological changes.

Asunto(s)

Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Dendritas/fisiología , Células Piramidales/fisiología , Animales , Factor Neurotrófico Derivado del Encéfalo/fisiología , Carbazoles/metabolismo , Dendritas/metabolismo , Dendritas/ultraestructura , Hurones , Proteínas Fluorescentes Verdes , Inmunohistoquímica , Técnicas In Vitro , Alcaloides Indólicos , Proteínas Luminiscentes/biosíntesis , Factor de Crecimiento Nervioso/biosíntesis , Células Piramidales/metabolismo , Células Piramidales/ultraestructura , Receptor de Factor Neurotrófico Ciliar/biosíntesis , Transfección , Corteza Visual/citología , Corteza Visual/metabolismo

RESUMEN

Trophic factor gene therapy may provide a rational treatment strategy for neurodegenerative disease. Recombinant adeno-associated virus vectors, incorporating a neuron-specific promoter driving bicistronic expression of green fluorescent protein and either nerve growth factor or brain-derived neurotrophic factor, transduced 10,000-15,000 neurons in the medial septum for periods of at least six months. Both cholinergic and non-cholinergic neurons expressed green fluorescent protein. Nerve growth factor and brain-derived neurotrophic factor vectors produced up to 50% increases in immunohistochemical detection of the acetylcholine-synthesizing enzyme in septal neurons ipsilateral to the injection. Increased levels of this enzyme, choline acetyltransferase, persisted for six months with the brain-derived neurotrophic factor vector. The nerve growth factor vector increased Trk receptor immunoreactivity in a volume of brain exceeding that of the transduced cells. Counterstaining for the neuronal marker, NeuN, or Nissl substance did not reveal any vector toxicity at any time-point. It therefore appears that the lasting effects of vector-mediated trophic factor gene transfer will offer a new approach for modulating septal cholinergic transmission and Trk receptor activity.

Asunto(s)

Factor Neurotrófico Derivado del Encéfalo/farmacología , Colina O-Acetiltransferasa/metabolismo , Factores de Crecimiento Nervioso/farmacología , Prosencéfalo/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Dependovirus/genética , Expresión Génica/fisiología , Vectores Genéticos , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Factores de Crecimiento Nervioso/genética , Ratas , Ratas Sprague-Dawley , Receptor de Factor Neurotrófico Ciliar , Recombinación Genética , Factores de Tiempo , Transgenes/genética

RESUMEN

Here, we describe a novel mechanism for the rapid regulation of surface levels of the neurotrophin receptor TrkB. Unlike nodose ganglion neurons, both retinal ganglion cells (RGCs) and spinal motor neurons (SMNs) in culture display only low levels of surface TrkB, though high levels are present intracellularly. Within minutes of depolarization or cAMP elevation, surface TrkB levels increase by nearly 4-fold, and this increase is not blocked by cycloheximide. These findings suggest that activity and cAMP elevation rapidly recruit TrkB to the plasma membrane by translocation from intracellular stores. We propose that a fundamental difference between peripheral nervous system (PNS) and central nervous system (CNS) neurons is the activity dependence of CNS neurons for responsiveness to their peptide trophic factors and that differences in membrane compartmentalization of the receptors underlie this difference.

Asunto(s)

AMP Cíclico/metabolismo , Neuronas Motoras/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Células Ganglionares de la Retina/metabolismo , Médula Espinal/metabolismo , Animales , Transporte Biológico/fisiología , Factor Neurotrófico Derivado del Encéfalo/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Membrana Celular/metabolismo , Supervivencia Celular/fisiología , Células Cultivadas , Electrofisiología , Factores de Crecimiento Nervioso/farmacología , Neuronas/efectos de los fármacos , Nervios Periféricos/citología , Nervios Periféricos/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptor de Factor Neurotrófico Ciliar , Médula Espinal/citología

RESUMEN

Recent studies have established that neurotrophin synthesis and secretion are regulated by activity and that these factors are involved in activity-dependent processes in the nervous system. Neurotrophins also are known to induce increases in intracellular calcium, a trigger for regulated secretion. This finding raises the possibility that neurotrophins themselves may stimulate regulated secretion of neurotrophins. To address this question, we studied the release of neurotrophins from transfected PC12 cells, a widely used model for neuronal secretion and neurotrophin signal transduction. We found that neurotrophins induced the regulated secretion of brain-derived neurotrophic factor, neurotrophin-3 (NT-3), and neurotrophin-4/5. The effect of brain-derived neurotrophic factor on release of NT-3 could be abolished by REX, a p75 blocking antibody, but not by K252a, an inhibitor of neurotrophin tyrosine kinase receptor (Trk) signaling. The nerve growth factor effect on release of NT-3 could be blocked only by simultaneous application of REX and K252a, suggesting that they are mediated by TrkA as well as p75. Our data show that neurotrophins are able to induce the regulated secretion of neurotrophins and suggest a signal-transducing role for both TrkA and p75 in this process. The neurotrophin-induced release of neurotrophins may be relevant for activity-dependent processes such as synaptic plasticity and memory formation.

Asunto(s)

Factores de Crecimiento Nervioso/fisiología , Animales , Células COS , Células Clonales , Genes myc , Factores de Crecimiento Nervioso/metabolismo , Células PC12 , Ratas , Transfección

RESUMEN

A growing body of evidence suggests that neurotrophins (NTs) play a critical role in synaptic plasticity and other activity dependent processes in the CNS. Release of these growth factors by neurons and neuroendocrine cells was recently shown to occur via the regulated secretory pathway, representing a possible mechanism for preferentially supplying NTs locally to active synapses. However, the identity and characteristics of the intracellular storage compartment for NTs undergoing stimulus-coupled secretion remains controversial. As a step towards addressing these issues we have investigated the subcellular localization of epitope-tagged nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) in neuroendocrine cells. Placement of the myc-epitope tag at the neurotrophin carboxy terminus did not affect essential properties of the NTs such as their ability to induce Trk tyrosine phosphorylation or their sorting into the regulated secretory pathway in PC12 and AtT-20 neuroendocrine cells. Epitope-tagged NTs colocalize with dense core vesicle (DCV)-markers at the light microscopic level in both cell lines investigated. Furthermore, at an EM level immunoreactivity (IR) for myc-tagged NGF was found over dense core granules (DCGs) in PC12 cells. These data provide evidence that NTs can be stored in DCVs in neuronal model cell lines and, potentially, in neurons as well.

Asunto(s)

Epítopos/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Sistemas Neurosecretores/metabolismo , Animales , Línea Celular , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/ultraestructura , Inmunohistoquímica , Ratones , Factores de Crecimiento Nervioso/inmunología , Factores de Crecimiento Nervioso/ultraestructura , Sistemas Neurosecretores/ultraestructura , Células PC12 , Adenohipófisis , Proteínas Proto-Oncogénicas c-myc/metabolismo , Ratas , Fracciones Subcelulares/metabolismo

RESUMEN

The role of the nerve growth factor (NGF) carboxyl terminus in the function of NGF is not well understood. Previous work showed that deletion of residues 112-120 abolished NGF bioactivity. Several mutagenesis studies, however, have localized the binding sites of the two NGF receptors, p75 and TrkA, to other regions of the NGF molecule. To investigate the role of the NGF COOH terminus, we performed a detailed structure-function analysis of this region by deleting stepwise each of the nine COOH-terminal residues as well as constructing six point mutants. We found that point mutations within the 111-115 region, but not deletion of residues 116-120, significantly decreased NGF bioactivity, as determined by TrkA tyrosine phosphorylation and neurite outgrowth from PC12 cells. Mutation of the absolutely conserved Leu112 led to severely disrupted p75 binding on A875 cells but had only a modest effect on TrkA binding to MG87-TrkA fibroblasts. This suggests that the p75 binding surface is more extended than previously believed and includes not only charged residues within loops 1 and 5 but also spatially discontinuous, uncharged residues in a region where the NH2 and COOH termini are in close proximity. Unexpectedly, deletion of COOH-terminal residues beyond Ala116 led to significantly decreased stability. These results demonstrate that residues 111-115, but not residues 116-120, are important for both the structural stability and biological activity of NGF.

Asunto(s)

Factores de Crecimiento Nervioso/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Ratones , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Factores de Crecimiento Nervioso/química , Factores de Crecimiento Nervioso/genética , Unión Proteica , Conformación Proteica , Ratas , Eliminación de Secuencia

RESUMEN

The varied roles that neurotrophins play in the development and activity-dependent plasticity of the nervous system presumably require that the sites and quantity of neurotrophin release be precisely regulated. As a step toward understanding how different neurotrophins are sorted and secreted by neurons, we expressed nerve growth factor (NGF), brain-derived neurotrophic factor, and neurotrophin-3 in cell lines used as models for neuronal protein sorting. All three neurotrophins were secreted by a regulated pathway in transfected AtT-20 and PC12 neuroendocrine cells, with a 3-6-fold increase in neurotrophin release in response to 8-bromo-cAMP or depolarization, respectively. To determine if the propeptide directs the intracellular sorting of mature NGF, we examined mutants in which regions spanning the propeptide were deleted. These mutants underwent regulated release in every case in which expression could be detected. Similarly, NGF sorting was not significantly altered by mutations which specifically abolished N-glycosylation or proteolytic processing sites within the NGF precursor. Finally, we found that all three neurotrophins were secreted 65-75% basolaterally by polarized Madin-Darby canine kidney epithelial cells. These findings suggest that the determinants of regulated neurotrophin secretion lie within the mature neurotrophin moiety and that NGF, brain-derived neurotrophic factor, and neurotrophin-3 are likely to be sorted similarly and released in a regulated manner by neurons.

Asunto(s)

Factores de Crecimiento Nervioso/biosíntesis , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Células COS , Línea Celular , Chlorocebus aethiops , Cicloheximida/farmacología , Dactinomicina/farmacología , Perros , Epitelio/metabolismo , Glicosilación , Cinética , Ratones , Mutagénesis , Factores de Crecimiento Nervioso/metabolismo , Neurotrofina 3 , Células PC12 , Neoplasias Hipofisarias , Procesamiento Proteico-Postraduccional , Ratas , Proteínas Recombinantes/biosíntesis , Transfección , Células Tumorales Cultivadas