RESUMEN
BACKGROUND: The two major isoforms of the human amyloid precursor protein (APP) are APP695 and APP751. They differ by the insertion of a Kunitz-type protease inhibitor (KPI) sequence in the extracellular domain of APP751. APP-KPI isoforms are increased in Alzheimer's disease brains, and they could be associated with disease progression. Recent studies have shown that APP processing to Aß is regulated by homodimerization, which involves both extracellular and juxtamembrane/transmembrane (JM/TM) regions. OBJECTIVE: Our aim is to understand the mechanisms controlling APP dimerization and the contribution of the ectodomain and JM/TM regions to this process. METHODS: We used bimolecular fluorescence complementation approaches coupled to fluorescence-activated cell sorting analysis to measure the dimerization level of different APP isoforms and APP C-terminal fragments (C99) mutated in their JM/TM region. RESULTS: APP751 was found to form significantly more homodimers than APP695. Mutation of dimerization motifs in the TM domain of APP or C99 did not significantly affect fluorescence complementation. CONCLUSION: These findings indicate that the KPI domain plays a major role in APP dimerization. They set the basis for further investigation of the relation between dimerization, metabolism and function of APP.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Dimerización , Inhibidores de Proteasas/metabolismo , Multimerización de Proteína/fisiología , Precursor de Proteína beta-Amiloide/genética , Animales , Proteínas Bacterianas/genética , Células COS , Chlorocebus aethiops , Citometría de Flujo , Humanos , Proteínas Luminiscentes/genética , Mutación/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerización de Proteína/genética , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
Nucleotide concentrations were normal in adenylosuccinate lyase-deficient fibroblasts, and the succinylpurines were not toxic for cultured neuronal cells.
Asunto(s)
Adenilosuccinato Liasa/deficiencia , Adenilosuccinato Liasa/genética , Purinas/metabolismo , Animales , Calcio/metabolismo , Células Cultivadas , Fibroblastos/enzimología , Fibroblastos/metabolismo , Humanos , Neuronas/metabolismo , Errores Innatos del Metabolismo de la Purina-Pirimidina/diagnóstico , Errores Innatos del Metabolismo de la Purina-Pirimidina/genética , Purinas/química , Ratas , Ratas Wistar , Piel/metabolismo , Sales de Tetrazolio/farmacología , Tiazoles/farmacología , Factores de TiempoAsunto(s)
Envejecimiento/patología , Geriatría , Envejecimiento/inmunología , Envejecimiento/fisiología , Enfermedad de Alzheimer/etiología , Animales , Bélgica , Encéfalo/patología , Encéfalo/fisiopatología , Caenorhabditis elegans , Senescencia Celular , Drosophila melanogaster , Humanos , Longevidad , Ratones , Ratones Transgénicos , Modelos Animales , Enfermedades Neurodegenerativas/etiología , Estrés Oxidativo , Ratas , Proyectos de Investigación , Envejecimiento de la PielRESUMEN
One of the major neuropathological hallmarks of Alzheimer's disease is the presence of senile plaques in vulnerable regions of CNS. These plaques are formed of aggregated amyloid peptide. Amyloid peptide is released by the cleavage of its precursor (APP). The establishment of cell lines expressing human APP allowed to characterize both amyloidogenic and non-amyloidogneic pathways of APP catabolism and to identify some of the proteins involved in this processing (known as secretases). This led to a better comprehension of amyloid peptide production, which needs to be further characterized since gamma-secretase is as yet not identified; moreover, we still lack a clear overview of the interactions between APP and other proteins promoting Alzheimer's disease (tau, presinilinsellipsis). An important limitation of these cell lines for studying the mechanisms involved in Alzheimer's disease is supported by the observation that human APP expression does not modify transfected cells survival. The infection of primary neuronal cultures with full-length human APP indicates that APP expression induces neuronal apoptosis by itself; this neurotoxicity does not rely on extracellular production of APP derivatives (secreted APP, amyloid peptide). It is now essential to understand, in neuronal models, the production, localization and involvement of amyloid peptide in neurodegenerative processes.
Asunto(s)
Precursor de Proteína beta-Amiloide/fisiología , Enfermedad de Alzheimer/etiología , Animales , Células CHO , Cricetinae , Humanos , Neuronas/metabolismo , RatasRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal, paralytic disorder that primarily affects motoneurons. By combining physiological and morphological approaches, we examined the effect of a murine superoxide dismutase 1 (SOD1) mutation (G86R), which induces neurological disorders resembling human familial ALS (FALS), on the arginine vasopressin (AVP) hypothalamo-neurohypophysial axis, an unmyelinated tract poor in neurofilaments. First, we observed that G86R mice progressively consumed more water than wild-type littermates. Furthermore, levels of plasma AVP and neurohypophysial AVP content were decreased in the SOD1 mutant mice, whereas the amount of hypothalamic AVP increased in an age-dependent manner. However, hypothalamic AVP mRNA levels were not significantly modified in these animals. At the ultrastructural level, we found that the neurohypophysis of G86R mice had a decreased number of neurosecretory axons. Conversely, the presence of large axon swellings was more pronounced in the SOD1 mutant mice. In addition, the size of neurosecretory granules was higher in G86R than in wild-type animals. All these findings strongly suggest that the FALS-associated SOD1 mutation injures the hypothalamo-neurohypophysial axis by provoking early, progressive disturbances in the axonal transport of neurosecretory products from neuronal perikarya to nerve terminals. This blockade could ultimately result in degeneration of the tract, as proposed for the myelinated, neurofilament-enriched motor axons affected by ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/fisiopatología , Arginina Vasopresina/metabolismo , Sistema Hipotálamo-Hipofisario/metabolismo , Superóxido Dismutasa/genética , Factores de Edad , Animales , Arginina Vasopresina/genética , Transporte Axonal/genética , Axones/clasificación , Axones/metabolismo , Axones/ultraestructura , Agua Corporal/metabolismo , Gránulos Citoplasmáticos/ultraestructura , Modelos Animales de Enfermedad , Femenino , Sistema Hipotálamo-Hipofisario/patología , Masculino , Ratones , Ratones Transgénicos , Mutación Missense , Neurosecreción , Hipófisis/metabolismo , Hipófisis/patología , Hipófisis/ultraestructura , ARN Mensajero/metabolismo , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1RESUMEN
Depolarization and subsequent calcium entry exert essential neuroprotective effects but the ultimate effector by which calcium blocks apoptosis is not known. Here we show that inhibition of calcium entry into cerebellar neurons by switching from high to low extracellular K+ concentrations (30-5 mM) induces apoptosis, that correlates with a rapid accumulation of cyclin D1 (CD1), an early marker of the G1/S transition of the cell cycle. These effects on apoptosis and cyclin D1 are mimicked either by blocking calcium entry into neurons (LaCl3, 100 microM or nifedipine, 10(-6) M) or by inhibiting the calcium/calmodulin pathway (calmidazolium, 10(-7) M). The increased CD1 protein levels do not result from a transcriptional upregulation of the CD1 gene by the Ca2+/calmodulin pathway but rather reflect an accumulation due to the lack of degradation by the proteasome-dependent pathway. Specific proteasome antagonists: carbobenzoxyl-leucinyl-leucinyl-norvalinal-H (MG-115), carbobenzoxyl-leucinyl-leucinyl-leucinal-H (MG-132) and clastolactacystin beta-lactone, induce neuronal apoptosis by themselves. Finally, this pathway is functional only at neuroprotective concentrations of K+ (30 mM), suggesting that calcium/CamK signalling pathway may regulate neuronal death by regulating the proteasome-mediated degradation activity of rapidly turning-over proteins (constitutively expressed genes or pre-existing pools of mRNA).
Asunto(s)
Apoptosis/fisiología , Ciclina D1/metabolismo , Cisteína Endopeptidasas/metabolismo , Complejos Multienzimáticos/metabolismo , Neuronas/citología , Sulfonamidas , Ubiquitinas/metabolismo , Animales , Calcio/fisiología , Calmodulina/fisiología , Cerebelo/citología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ciclina D1/genética , Inhibidores de Cisteína Proteinasa/farmacología , Cartilla de ADN , Inhibidores Enzimáticos/farmacología , Expresión Génica/fisiología , Imidazoles/farmacología , Etiquetado Corte-Fin in Situ , Isoquinolinas/farmacología , Leupeptinas/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos , Neuronas/enzimología , Cloruro de Potasio/farmacología , Inhibidores de Proteasas/farmacología , Complejo de la Endopetidasa Proteasomal , ARN Mensajero/análisis , Transducción de Señal/fisiologíaRESUMEN
Melatonin secretion from the mammalian pineal gland is strongly stimulated by noradrenaline and also by vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP). Three types of receptors for VIP and PACAP have been characterized so far: VIP1/PACAP receptors and VIP2/PACAP receptors, which possess similar high affinities for VIP and PACAP, and PACAP1 receptors which exhibit a 100-1000-fold higher affinity for PACAP. The aim of the present study was to characterize the receptor subtype(s) mediating the stimulatory effects of VIP and PACAP on melatonin synthesis in the rat pineal gland. Autoradiographic studies showed that PACAP and VIP were equally potent in displacing binding of radioiodinated PACAP27 from pineal sections. Amplification of pineal complementary DNAs by polymerase chain reaction using specific primers for the different receptor subtypes revealed that all three receptor messenger RNAs are expressed and that VIP1/PACAP receptor messenger RNA was predominant over VIP2/PACAP receptor messenger RNA. In vitro, VIP and PACAP stimulated melatonin synthesis with similar high potency and the effect of the two peptides were not additive. The selective VIP1/PACAP receptor agonists [R16]chicken secretin (1-25) and [K15, R16, L27]VIP(1-7)/growth hormone releasing factor(8-27) were significantly more potent than the selective VIP2/PACAP receptor agonist RO 25-1553 in stimulating melatonin secretion. The stimulatory effects of VIP and PACAP were similarly inhibited by the VIP1/PACAP antagonist [acetyl-His1, D-Phe2, K15, R16, L27]VIP(3-7)/growth hormone releasing factor(8-27). These data strongly suggest that VIP and PACAP exert a stimulatory effect on melatonin synthesis mainly through activation of a pineal VIP1/PACAP receptor subtype.
Asunto(s)
Adenilil Ciclasas/metabolismo , Glándula Pineal/química , Glándula Pineal/enzimología , Receptores de la Hormona Hipofisaria/análisis , Receptores de Péptido Intestinal Vasoactivo/análisis , Agonistas Adrenérgicos beta/farmacología , Animales , Autorradiografía , Broncodilatadores/farmacología , Activación Enzimática/efectos de los fármacos , Expresión Génica/fisiología , Radioisótopos de Yodo , Isoproterenol/farmacología , Masculino , Melatonina/metabolismo , Neuropéptidos/farmacología , Neurotransmisores/farmacología , Fragmentos de Péptidos/farmacología , Péptidos Cíclicos/farmacología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , ARN Mensajero/análisis , Ratas , Ratas Wistar , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores de la Hormona Hipofisaria/genética , Receptores de la Hormona Hipofisaria/metabolismo , Receptores de Péptido Intestinal Vasoactivo/genética , Receptores de Péptido Intestinal Vasoactivo/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Péptido Intestinal Vasoactivo/análogos & derivados , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
Continuous exposure of cells to agonists develops a process that determines the extent to which the cells eventually respond to further stimuli. Here we used CATH.a cells (a catecholaminergic neuron-like cell line), which express pituitary adenylate cyclase-activating polypeptide (PACAP) receptors linked to both adenylyl cyclase and phospholipase C-beta pathways, to investigate the influence of prolonged hormonal treatment on dual signaling and gene transcription. Prolonged incubation of cells with PACAP failed to down-regulate the density and affinity of membrane binding sites and caused opposite changes in messenger systems: PACAP-stimulated cyclic AMP accumulation was attenuated in a time- and dose-dependent fashion (t(1/2) = 6.7 h and IC50 = 0.1 nM), whereas phosphoinositide turnover was overstimulated. Both effects were insensitive to pertussis toxin, whereas the drop in cyclic AMP concentration was also unchanged in the presence of 3-isobutyl-1-methylxanthine, indicating that neither Gi-like proteins nor cyclic nucleotide phosphodiesterases play a critical role in these processes. Blockade of protein synthesis with cycloheximide, as well as inhibition by H89 of cyclic AMP-dependent protein kinase (but not by bisindolylmaleimide of protein kinase C) antagonized the influences exerted by PACAP on adenylyl cyclase activity and inositol phosphate formation. Transcription of the chimeric GAL4-CREB construct, transiently transfected into CATH.a cells, was stimulated by PACAP, and this effect was potentiated as a result of chronic PACAP treatment. The results of the present investigation provide new insight into the possible differential regulation and cross-talks of transduction signals of receptors linked to multiplex signaling. They demonstrate that prolonged exposure of CATH.a cells to PACAP results in the desensitization of the cyclic AMP pathway and superinduction of the inositol phosphate signal, through protein neosynthesis and cyclic AMP-dependent protein kinase activation. At the same time, they show that desensitization of cyclic AMP signaling not only fails to hamper, but actually amplifies PACAP-stimulated CREB-regulated transcription.
Asunto(s)
AMP Cíclico/fisiología , Fosfatidilinositoles/fisiología , Receptores de la Hormona Hipofisaria/fisiología , Transducción de Señal/fisiología , Línea Celular , Membrana Celular/metabolismo , AMP Cíclico/biosíntesis , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Cicloheximida/farmacología , Inhibidores Enzimáticos/farmacología , Hidrólisis/efectos de los fármacos , Neuronas/metabolismo , Neuropéptidos/farmacología , Fosfatidilinositoles/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Biosíntesis de Proteínas , Proteína Quinasa C/antagonistas & inhibidores , Proteínas Quinasas/fisiología , Inhibidores de la Síntesis de la Proteína/farmacología , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores de la Hormona Hipofisaria/metabolismo , Transducción de Señal/efectos de los fármacos , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
The molecular nature, transduction pathways, and neurotrophic functions of pituitary adenylate cyclase activating peptide (PACAP) receptors were studied in primary culture of rat cerebellar granule cells. We show that cerebellar neurons express several PACAP type I receptor (PVR I) isoforms, including the short (PVR Is) and the Hop (PVR I-Hop) splice variants, the latter being restricted to neurons and not found in cerebellar glial cell cultures. In vitro, cerebellar granule cells die rapidly in the absence of a high concentration of K+ (25 mM), as demonstrated by TUNEL histochemistry, which shows that K+ deprivation induces massive neuronal apoptosis within 12 hr. This effect was reversed by PACAP 27 and 38. Both forms of PACAP prevent DNA fragmentation and allow long-term neuronal survival in the absence of high K+ (as shown by MAP2 immunostaining) and stimulate a reporter gene driven by the full-length c-fos promoter. These effects of PACAP are fully abolished upon transient transfection of cells with a dominant inhibitory mutant of the cAMP-dependent protein kinase (PKA). Taken together, these results show that in cerebellar granule neurons, PACAP type I receptors regulate gene expression and promote neuronal survival through the cAMP/PKA pathway.
Asunto(s)
Cerebelo/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Neuronas/metabolismo , Neuropéptidos/metabolismo , Receptores de la Hormona Hipofisaria/metabolismo , Transducción de Señal , Animales , Muerte Celular , Cerebelo/patología , Genes fos , Neuronas/patología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Ratas , Ratas Wistar , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , TransfecciónRESUMEN
GABAB receptors affect short-term signalling in various cell types. However, nothing is known about possible long-term effects on transcription. To analyse such effects in the CNS, we studied GABAB receptor-mediated gene regulation in primary cultures of cerebellar granule neurons. Transcription was followed using a chloramphenicol acetyl transferase reporter gene driven by the minimal cyclic AMP-responsive element (TGACGTCA). Transcription was stimulated by activation of both the cyclic AMP (forskolin: 5 x 10(-6) M) and the Ca2+ dependent (KCl: 30 mM) pathways (-)-Baclofen (10(-6) M to 10(-4) M), a specific GABAB receptor agonist, reduced by 50-70% the transcriptional stimulation evoked by both forskolin and KCl, whereas isoguvacine, a GABAA receptor agonist, was without effect. Moreover, the GABAB antagonist CGP 35348 abrogated the inhibitory effects of both GABA and baclofen, indicating that GABAB receptors were specifically implicated in this response. Measurements of cyclic AMP levels suggested that (-) baclofen inhibits forskolin-initiated transcription by reducing cyclic AMP production. Direct transcriptional activation, via the cyclic AMP pathway, by overexpression of the catalytic subunit of the cyclic AMP-dependent protein kinase, was not significantly altered by (-) baclofen. This indicates again that (-) baclofen-dependent inhibitory mechanisms operate upstream of cyclic AMP-dependent protein kinase at the level of second messenger formation. Further, we used a yeast transcriptional activator GAL4-cyclic AMP-responsive element binding protein to analyse whether GABAB receptor-mediated inhibition of cyclic AMP-responsive element transcription implicated the transacting factor cyclic AMP-responsive element binding protein. We show that the negative effects of (-) baclofen implicate this transcription factor and this holds good for both the forskolin and KCl-stimulated pathways. The results indicate that GABAB receptors negatively regulate cyclic AMP-responsive element binding protein-mediated transcription in the CNS.