RESUMEN
Bone morphogenetic protein 9 (BMP9) promotes the acquisition of the cholinergic phenotype in basal forebrain cholinergic neurons (BFCN) during development and protects these neurons from cholinergic dedifferentiation following axotomy when administered in vivo. A decline in BFCN function occurs in patients with Alzheimer's disease (AD) and contributes to the AD-associated memory deficits. We infused BMP9 intracerebroventricularly for 7 d in transgenic AD model mice expressing green fluorescent protein specifically in cholinergic neurons (APP.PS1/CHGFP) and in wild-type littermate controls (WT/CHGFP). We used 5-mo-old mice, an age when the AD transgenics display early amyloid deposition and few cholinergic defects, and 10-mo-old mice, by which time these mice exhibit established disease. BMP9 infusion reduced the number of Aß42-positive amyloid plaques in the hippocampus and cerebral cortex of 5- and 10-mo-old APP.PS1/CHGFP mice and reversed the reductions in choline acetyltransferase protein levels in the hippocampus of 10-mo-old APP.PS1/CHGFP mice. The treatment increased cholinergic fiber density in the hippocampus of both WT/CHGFP and APP.PS1/CHGFP mice at both ages. BMP9 infusion also increased hippocampal levels of neurotrophin 3, insulin-like growth factor 1, and nerve growth factor and of the nerve growth factor receptors, tyrosine kinase receptor A and p75/NGFR, irrespective of the genotype of the mice. These data show that BMP9 administration is effective in reducing the Aß42 amyloid plaque burden, reversing cholinergic neuron abnormalities, and generating a neurotrophic milieu for BFCN in a mouse model of AD and provide evidence that the BMP9-signaling pathway may constitute a therapeutic target for AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Amiloidosis/metabolismo , Neuronas Colinérgicas/metabolismo , Factor 2 de Diferenciación de Crecimiento/farmacología , Análisis de Varianza , Animales , Neuronas Colinérgicas/efectos de los fármacos , Femenino , Factor 2 de Diferenciación de Crecimiento/administración & dosificación , Factor 2 de Diferenciación de Crecimiento/metabolismo , Inmunoensayo , Immunoblotting , Inmunohistoquímica , Masculino , Ratones , Microscopía FluorescenteRESUMEN
Numerous studies have demonstrated defects in multiple metabolic pathways in Alzheimer's disease (AD), detected in autopsy brains and in the cerebrospinal fluid in vivo. However, until the advent of techniques capable of measuring thousands of metabolites in a single sample, it has not been possible to rank the relative magnitude of these abnormalities. A recent study provides evidence that the abnormal turnover of the brain's most abundant phospholipids: phosphatidylcholine and phosphatidylethanolamine, constitutes a major metabolic pathology in AD. We place this observation in a historical context and discuss the implications of a central role for phospholipid metabolism in AD pathogenesis.
Asunto(s)
Enfermedad de Alzheimer , Humanos , Enfermedad de Alzheimer/patología , Fosfatidiletanolaminas/metabolismo , Fosfatidilcolinas/metabolismo , Fosfolípidos/metabolismo , Encéfalo/patologíaRESUMEN
The α-secretases A disintegrin and metalloprotease 10 (ADAM10) and ADAM17 trigger constitutive and regulated processing of the cellular prion protein (PrP(c)) yielding N1 fragment. The latter depends on protein kinase C (PKC)-coupled M1/M3 muscarinic receptor activation and subsequent phosphorylation of ADAM17 on its intracytoplasmic threonine 735. Here we show that regulated PrP(c) processing and ADAM17 phosphorylation and activation are controlled by the extracellular-regulated kinase-1/MAP-ERK kinase (ERK1/MEK) cascade. Thus, reductions of ERK1 or MEK activities by dominant-negative analogs, pharmacological inhibition, or genetic ablation all impair N1 secretion, whereas constitutively active proteins increase N1 recovery in the conditioned medium. Interestingly, we also observed an ERK1-mediated enhanced expression of PrP(c). We demonstrate that the ERK1-associated increase in PrP(c) promoter transactivation and mRNA levels involve transcription factor AP-1 as a downstream effector. Altogether, our data identify ERK1 as an important regulator of PrP(c) cellular homeostasis and indicate that this kinase exerts a dual control of PrP(c) levels through transcriptional and post-transcriptional mechanisms.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas PrPC/metabolismo , Regiones Promotoras Genéticas/fisiología , Activación Transcripcional/fisiología , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM10 , Proteína ADAM17 , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Células HEK293 , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Proteína Quinasa 3 Activada por Mitógenos/genética , Fosforilación/fisiología , Proteínas PrPC/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptor Muscarínico M1/genética , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismoRESUMEN
BACKGROUND: The amyloid precursor protein (APP) is cleaved by ß- and γ-secretases to generate toxic amyloid ß (Aß) peptides. Alternatively, α-secretases cleave APP within the Aß domain, precluding Aß formation and releasing the soluble ectodomain, sAPPα. We previously showed that inhibition of the GTPase dynamin reduced APP internalization and increased release of sAPPα, apparently by prolonging the interaction between APP and α-secretases at the plasma membrane. This was accompanied by a reduction in Aß generation. In the present study, we investigated whether surface expression of the α-secretase ADAM (a disintegrin and metalloprotease)10 is also regulated by dynamin-dependent endocytosis. RESULTS: Transfection of human embryonic kidney (HEK) cells stably expressing M3 muscarinic receptors with a dominant negative dynamin I mutant (dyn I K44A), increased surface expression of both immature, and mature, catalytically active forms of co-expressed ADAM10. Surface levels of ADAM10 were unaffected by activation of protein kinase C (PKC) or M3 receptors, indicating that receptor-coupled shedding of the ADAM substrate APP is unlikely to be mediated by inhibition of ADAM10 endocytosis in this cell line. Dyn I K44A strongly increased the formation of a C-terminal fragment of ADAM10, consistent with earlier reports that the ADAM10 ectodomain is itself a target for sheddases. The abundance of this fragment was increased in the presence of a γ-secretase inhibitor, but was not affected by M3 receptor activation. The dynamin mutant did not affect the distribution of ADAM10 and its C-terminal fragment between raft and non-raft membrane compartments. CONCLUSIONS: Surface expression and limited proteolysis of ADAM10 are regulated by dynamin-dependent endocytosis, but are unaffected by activation of signaling pathways that upregulate shedding of ADAM substrates such as APP. Modulation of ADAM10 internalization could affect cellular behavior in two ways: by altering the putative signaling activity of the ADAM10 C-terminal fragment, and by regulating the biological function of ADAM10 substrates such as APP and N-cadherin.
Asunto(s)
Proteínas ADAM/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Dinamina I/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Proteína ADAM10 , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Precursor de Proteína beta-Amiloide/metabolismo , Carbacol/farmacología , Carbamatos/farmacología , Línea Celular , Dipéptidos/farmacología , Dinamina I/genética , Dinamina I/metabolismo , Endocitosis , Humanos , Proteína Quinasa C/metabolismo , Receptor Muscarínico M3/metabolismoRESUMEN
Understanding the molecular mechanisms of aging in vertebrates is a major challenge of modern biology and biomedical science. This is due, in part, to the complexity of the aging process and its multifactorial nature, the paucity of animal models that lend themselves to unbiased high-throughput screening for aging phenotypes, and the difficulty of predicting such phenotypes at an early age. We suggest that the zebrafish genetic model offers a unique opportunity to fill in this gap and contributes to advances in biological and behavioral gerontology. Our recent studies demonstrated that this diurnal vertebrate with gradual senescence is an excellent model in which to study age-dependent changes in musculoskeletal and eye morphology, endocrine factors, gene expression, circadian clock, sleep and cognitive functions. Importantly, we have also found that the presence of a senescence-associated biomarker ('senescence-associated beta-galactosidase') can be documented during early zebrafish development and is predictive of premature aging phenotypes later in adult life. The availability of mutant 'genotypes' with identified aging 'phenotypes' in zebrafish, in combination with a wealth of information about zebrafish development and genetics, and the existence of multiple mutant and transgenic lines, should significantly facilitate the use of this outstanding vertebrate model in deciphering the mechanisms of aging, and in developing preventive and therapeutic strategies to prolong the productive life span ('health span') in humans.
Asunto(s)
Envejecimiento/genética , Pez Cebra/genética , Envejecimiento/patología , Envejecimiento/fisiología , Envejecimiento/psicología , Animales , Conducta Animal , Ritmo Circadiano/genética , Ojo/anatomía & histología , Marcadores Genéticos , Hígado/anatomía & histología , Modelos Animales , Modelos Genéticos , Mutación , Regeneración/genética , Pez Cebra/crecimiento & desarrollo , Pez Cebra/fisiologíaRESUMEN
The cellular prion protein (PrP(c)) undergoes a physiological cleavage between amino acids 111 and 112, thereby leading to the secretion of an amino-terminal fragment referred to as N1. This proteolytic event is either constitutive or regulated by protein kinase C (PKC) and is operated by the disintegrins ADAM9/ADAM10 or ADAM17 respectively. We recently showed that the stimulation of the M1/M3 muscarinic receptors potentiates this cleavage via the phosphorylation and activation of ADAM17. We have examined the contribution of various PKC isoforms in the regulated processing of PrP(c). First we show that the PDBu- and carbachol-stimulated N1 secretions are blocked by the general PKC inhibitor GF109203X. We establish that HEK293 and human-derived rhabdhomyosarcoma cells over-expressing constitutively active PKCalpha, PKCdelta or PKCepsilon, but not PKCzeta, produce increased amounts of N1 and harbor enhanced ability to hydrolyze the fluorimetric substrate of ADAM17, JMV2770. Conversely, over-expression of the corresponding dominant negative proteins abolishes PDBU-stimulated N1 secretion and restores N1 to levels comparable to constitutive production. Moreover, deletion of PKCalpha lowers N1 recovery in primary cultured fibroblasts. Importantly, mutation of threonine 735 of ADAM17 significantly lowers the PDBu-induced N1 formation while transient over-expression of constitutively active PKCalpha, PKCdelta or PKCepsilon, but not PKCzeta, induced both the phosphorylation of ADAM17 on its threonine residues and N1 secretion. As a corollary, T735A mutation concomitantly reversed PKCalpha-, PKCdelta- and PKCepsilon-induced ADAM17 phosphorylation and N1 recovery. Finally, we established that PKCepsilon-dependent N1 production is fully prevented by ADAM17 deficiency. Altogether, the present results provide strong evidence that the activation of PKCalpha, delta and epsilon, but not zeta, isoforms leads to increased N1 secretion via the phosphorylation and activation of ADAM17, a process that likely accounts for M1/M3 muscarinic receptors-mediated control of N1 production.
Asunto(s)
Isoenzimas/metabolismo , Proteínas PrPC/metabolismo , Proteína Quinasa C/metabolismo , Proteínas ADAM/metabolismo , Proteína ADAM10 , Proteína ADAM17 , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Carbacol/metabolismo , Línea Celular , Agonistas Colinérgicos/metabolismo , Activación Enzimática , Inducción Enzimática , Inhibidores Enzimáticos/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Indoles/metabolismo , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Maleimidas/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Ésteres del Forbol/metabolismo , Proteínas PrPC/genética , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/genéticaRESUMEN
The cellular prion protein (PrP(c)) undergoes a physiological processing yielding the N-terminal fragment referred to as N1, the production of which can be constitutive or protein kinase C regulated. We show that activation of endogenous muscarinic receptors by carbachol and by the M1-selective agonist AF267B increases N1 recovery in an atropine-sensitive manner, in mouse embryonic primary neurons. To identify the muscarinic receptor subtype involved, we used human embryonic kidney HEK293 (HEK) cells stably overexpressing M1, M2, M3, or M4 receptor subtype. Carbachol and the selective M1 agonist AF267B dose dependently increased N1 release by HEK-M3 and HEK-M1 cells, respectively, whereas carbachol did not modify N1 production by HEK-M2 or HEK-M4 cells. We demonstrate that the increase of N1 was not attributable to modified trafficking to the membrane of either PrP(c) or the disintegrin metalloproteases ADAM10 or ADAM17. Furthermore, we establish that carbachol affects the overall phosphorylation of ADAM17 on its threonine and tyrosine but not serine residues, whereas levels of phosphorylated ADAM9 were not affected. Interestingly, carbachol also increases the hydrolysis of the fluorimetric substrate JMV2770, which mimicked the sequence encompassing the N1 site cleavage and was shown previously to behave as an ADAM protease substrate. Mutations of threonine 735 but not of tyrosine 702 of the ADAM17 cytoplasmic tail abolishes the carbachol-induced increase of N1, ADAM17 phosphorylation, and JMV2770-hydrolyzing activity in M1- and M3-expressing HEK293 cells. Thus, our data provide strong evidence that muscarinic receptor activation increases the physiological processing of PrP(c) by upregulating the phosphorylation state and activity of ADAM17 protease.
Asunto(s)
Proteínas ADAM/metabolismo , Priones/fisiología , Receptor Muscarínico M1/fisiología , Receptor Muscarínico M3/fisiología , Proteínas ADAM/antagonistas & inhibidores , Proteína ADAM17 , Animales , Carbacol/farmacología , Línea Celular , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Humanos , Ratones , Fosforilación/efectos de los fármacos , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M3/agonistasRESUMEN
Muscarinic receptors subserve many functions in both peripheral and central nervous systems. Some of these processes depend on increases in protein synthesis, which may be achieved by activation of mammalian target of rapamycin (mTOR), a kinase that regulates protein translation capacity. Here, we examined the regulation of mTOR-dependent signaling pathways by muscarinic receptors in SK-N-SH human neuroblastoma cells, and in human embryonic kidney (HEK) cell lines transfected with individual muscarinic receptor subtypes. In SK-N-SH cells, the acetylcholine analog carbachol stimulated phosphorylation of the ribosomal S6 protein, a downstream target of mTOR. The sensitivity of the response to subtype-selective muscarinic receptor antagonists indicated that it was mediated by M3 receptors. Carbachol-evoked S6 phosphorylation was blocked by the mTOR inhibitor rapamycin, but was independent of phosphoinositide 3-kinase activation. The response was significantly reduced by the mitogen-activated protein kinase kinase (MEK) inhibitor U0126, which also inhibited carbachol-evoked S6 phosphorylation in HEK cells expressing M2 receptors, but was ineffective in M3 receptor-expressing HEK cells, although carbachol activated MAPK in both transfected lines. The p90 ribosomal S6 kinase has been implicated in mTOR regulation by phorbol esters, but was not activated by carbachol in any of the cell lines tested. The protein kinase C inhibitor bisindolylmaleimide I reduced carbachol-stimulated S6 phosphorylation in SK-N-SH cells, and in HEK cells expressing M3 receptors, but not in HEK cells expressing M2 receptors. The results demonstrate that multiple muscarinic receptor subtypes regulate mTOR, and that both MAPK-dependent and -independent mechanisms may mediate the response in a cell context-specific manner.
Asunto(s)
Proteínas Quinasas/metabolismo , Receptores Muscarínicos/metabolismo , Proteína S6 Ribosómica/metabolismo , Carbacol/farmacología , Línea Celular Tumoral , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Proteína Quinasa C/metabolismo , Receptor Muscarínico M2/metabolismo , Receptor Muscarínico M3/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Serina-Treonina Quinasas TOR , Acetato de Tetradecanoilforbol/farmacología , TransfecciónRESUMEN
Choline is an essential nutrient for humans. It is a precursor of membrane phospholipids (e.g., phosphatidylcholine (PC)), the neurotransmitter acetylcholine, and via betaine, the methyl group donor S-adenosylmethionine. High choline intake during gestation and early postnatal development in rat and mouse models improves cognitive function in adulthood, prevents age-related memory decline, and protects the brain from the neuropathological changes associated with Alzheimer's disease (AD), and neurological damage associated with epilepsy, fetal alcohol syndrome, and inherited conditions such as Down and Rett syndromes. These effects of choline are correlated with modifications in histone and DNA methylation in brain, and with alterations in the expression of genes that encode proteins important for learning and memory processing, suggesting a possible epigenomic mechanism of action. Dietary choline intake in the adult may also influence cognitive function via an effect on PC containing eicosapentaenoic and docosahexaenoic acids; polyunsaturated species of PC whose levels are reduced in brains from AD patients, and is associated with higher memory performance, and resistance to cognitive decline.
Asunto(s)
Colina/farmacología , Fármacos Neuroprotectores/farmacología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Colina/administración & dosificación , Cognición/efectos de los fármacos , Dieta , Humanos , Fármacos Neuroprotectores/administración & dosificaciónRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0170450.].
RESUMEN
Prevention of Alzheimer's disease (AD) is a major goal of biomedical sciences. In previous studies we showed that high intake of the essential nutrient, choline, during gestation prevented age-related memory decline in a rat model. In this study we investigated the effects of a similar treatment on AD-related phenotypes in a mouse model of AD. We crossed wild type (WT) female mice with hemizygous APPswe/PS1dE9 (APP.PS1) AD model male mice and maintained the pregnant and lactating dams on a control AIN76A diet containing 1.1 g/kg of choline or a choline-supplemented (5 g/kg) diet. After weaning all offspring consumed the control diet. As compared to APP.PS1 mice reared on the control diet, the hippocampus of the perinatally choline-supplemented APP.PS1 mice exhibited: 1) altered levels of amyloid precursor protein (APP) metabolites-specifically elevated amounts of ß-C-terminal fragment (ß-CTF) and reduced levels of solubilized amyloid Aß40 and Aß42 peptides; 2) reduced number and total area of amyloid plaques; 3) preserved levels of choline acetyltransferase protein (CHAT) and insulin-like growth factor II (IGF2) and 4) absence of astrogliosis. The data suggest that dietary supplementation of choline during fetal development and early postnatal life may constitute a preventive strategy for AD.
Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/prevención & control , Precursor de Proteína beta-Amiloide/genética , Amiloidosis/prevención & control , Colina O-Acetiltransferasa/metabolismo , Colina/administración & dosificación , Suplementos Dietéticos , Hipocampo/metabolismo , Presenilina-1/genética , Enfermedad de Alzheimer/dietoterapia , Precursor de Proteína beta-Amiloide/metabolismo , Amiloidosis/patología , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/patología , Masculino , Ratones , Ratones Mutantes , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Neurogénesis/efectos de los fármacos , Embarazo , Presenilina-1/metabolismoRESUMEN
Basal forebrain cholinergic neurons play critical roles in the organization of brain cortical structures and in processes such as learning and memory. We have previously shown that bone morphogenetic protein (BMP) 9, a member of the transforming growth factor (TGF) beta superfamily of cytokines, is a differentiating factor for cholinergic central nervous system neurons. However, whereas the basic signal transduction pathways for most known members of the TGF-beta superfamily have been well characterized in brain and other organs, nothing is known about the signal transduction pathway of BMP9 in the brain. Here, we describe the pattern of expression of BMP receptors, including Bmpr-Ia, Bmpr-Ib, Bmpr-II, Actr-I. Actr-Ib, Actr-II and Actr-IIb, Alk-1, and Smad proteins (Smads 1-5 and Smad8) in the septal region of the basal forebrain during mouse development. Using cultured basal forebrain cells derived from embryonic day (E) 14 mice, we show that BMP9 causes phosphorylation of Smad1 and Smad5, formation of a complex of Smad4 with Samd1 and/or Smad5, and translocation of these proteins into the nucleus. These data show that BMP9 activates the canonical BMP signaling pathway and suggest that this could be one of the mechanisms responsible for the induction of the cholinergic phenotype by BMP9 in the basal forebrain.
Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Prosencéfalo/fisiología , Proteínas Smad/metabolismo , Animales , Animales Recién Nacidos , Western Blotting/métodos , Receptores de Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/farmacología , Células Cultivadas , Embrión de Mamíferos , Activación Enzimática/efectos de los fármacos , Factor 2 de Diferenciación de Crecimiento , Ratones , Prosencéfalo/citología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Proteínas Smad/genéticaRESUMEN
BACKGROUND: The amyloid precursor protein (APP) is transported via the secretory pathway to the cell surface, where it may be cleaved within its ectodomain by alpha-secretase, or internalized within clathrin-coated vesicles. An alternative proteolytic pathway occurs within the endocytic compartment, where the sequential action of beta- and gamma-secretases generates the amyloid beta protein (Abeta). In this study, we investigated the effects of modulators of endocytosis on APP processing. RESULTS: Human embryonic kidney cells were transfected with a dominant negative mutant of dynamin I, an important mediator of clathrin-dependent endocytosis, and APP proteolysis was analyzed. Overexpression of the mutant dynamin (dyn I K44A) resulted in increased shedding of the APP ectodomain (sAPPalpha), accumulation of the C-terminal alpha-secretase product C83, and a reduction in the release of Abeta. Levels of mature APP on the cell surface were increased in cells expressing dyn I K44A, and internalization of surface-immunolabeled APP, assessed by fluorescence microscopy, was inhibited. Dynamin is a substrate for protein kinase C (PKC), and it was hypothesized that activators of PKC, which are known to stimulate alpha-secretase-mediated cleavage of APP, might exert their effects by inhibiting dynamin-dependent endocytosis. However, the internalization of surface-biotinylated APP was unaffected by treatment of cells with phorbol 12-myristate 13-acetate in the presence of the alpha-secretase inhibitor TAPI-1. CONCLUSION: The results indicate that APP is internalized by a dynamin-dependent process, and suggest that alterations in the activity of proteins that mediate endocytosis might lead to significant changes in Abeta production.
Asunto(s)
Péptidos beta-Amiloides/biosíntesis , Precursor de Proteína beta-Amiloide/metabolismo , Dinamina I/fisiología , Endocitosis/fisiología , Secretasas de la Proteína Precursora del Amiloide , Precursor de Proteína beta-Amiloide/química , Ácido Aspártico Endopeptidasas , Línea Celular , Dinamina I/antagonistas & inhibidores , Dinamina I/genética , Endopeptidasas/metabolismo , Humanos , Mutación , Proteína Quinasa C/metabolismo , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , TransfecciónRESUMEN
F-spondin, an extracellular matrix protein, is an important player in embryonic morphogenesis and CNS development, but its presence and role later in life remains largely unknown. We generated a transgenic zebrafish in which GFP is expressed under the control of the F-spondin (spon1b) promoter, and used it in combination with complementary techniques to undertake a detailed characterization of the expression patterns of F-spondin in developing and adult brain and periphery. We found that F-spondin is often associated with structures forming long neuronal tracts, including retinal ganglion cells, the olfactory bulb, the habenula, and the nucleus of the medial longitudinal fasciculus (nMLF). F-spondin expression coincides with zones of adult neurogenesis and is abundant in CSF-contacting secretory neurons, especially those in the hypothalamus. Use of this new transgenic model also revealed F-spondin expression patterns in the peripheral CNS, notably in enteric neurons, and in peripheral tissues involved in active patterning or proliferation in adults, including the endoskeleton of zebrafish fins and the continuously regenerating pharyngeal teeth. Moreover, patterning of the regenerating caudal fin following fin amputation in adult zebrafish was associated with F-spondin expression in the blastema, a proliferative region critical for tissue reconstitution. Together, these findings suggest major roles for F-spondin in the CNS and periphery of the developing and adult vertebrate.
Asunto(s)
Envejecimiento/genética , Proteínas de la Matriz Extracelular/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Pez Cebra/genética , Pez Cebra/crecimiento & desarrollo , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Embrión no Mamífero/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Larva/genética , Larva/crecimiento & desarrollo , Masculino , Neurogénesis/genética , Especificidad de Órganos/genética , Homología de Secuencia de Aminoácido , Pez Cebra/embriología , Proteínas de Pez Cebra/metabolismoRESUMEN
The discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that is highly expressed in breast carcinoma cells. Upon binding to collagen, DDR1 undergoes autophosphorylation followed by limited proteolysis to generate a tyrosine phosphorylated C-terminal fragment (CTF). Although it was postulated that this fragment is formed as a result of shedding of the N-terminal ectodomain, collagen-dependent release of the DDR1 extracellular domain has not been demonstrated. We now report that, in conjunction with CTF formation, collagen type I stimulates concentration-dependent, saturable shedding of the DDR1 ectodomain from two carcinoma cell lines, and from transfected cells. In contrast, collagen did not promote cleavage of other transmembrane proteins including the amyloid precursor protein (APP), ErbB2, and E-cadherin. Collagen-dependent tyrosine phosphorylation and proteolysis of DDR1 in carcinoma cells were reduced by a pharmacologic Src inhibitor. Moreover, expression of a dominant negative Src mutant protein in human embryonic kidney cells inhibited collagen-dependent phosphorylation and shedding of co-transfected DDR1. The hydroxamate-based metalloproteinase inhibitor TAPI-1 (tumor necrosis factor-alpha protease inhibitor-1), and tissue inhibitor of metalloproteinase (TIMP)-3, also blocked collagen-evoked DDR1 shedding, but did not reduce levels of the phosphorylated CTF. Neither shedding nor CTF formation were affected by the gamma-secretase inhibitor, L-685,458. The results demonstrate that collagen-evoked ectodomain cleavage of DDR1 is mediated in part by Src-dependent activation or recruitment of a matrix- or disintegrin metalloproteinase, and that CTF formation can occur independently of ectodomain shedding. Delayed shedding of the DDR1 ectodomain may represent a mechanism that limits DDR1-dependent cell adhesion and migration on collagen matrices.
Asunto(s)
Colágeno Tipo I/farmacología , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/metabolismo , Familia-src Quinasas/metabolismo , Secretasas de la Proteína Precursora del Amiloide , Ácido Aspártico Endopeptidasas , Células Cultivadas , Receptor con Dominio Discoidina 1 , Endopeptidasas/metabolismo , Humanos , Fosfotirosina/metabolismo , Estructura Terciaria de Proteína/efectos de los fármacos , Factores de Tiempo , Inhibidor Tisular de Metaloproteinasa-3/metabolismoRESUMEN
The mitogen-activated protein kinases (MAPKs) are activated by extracellular signals, and translocate to the nucleus where they modulate transcription. Integrin-mediated cell adhesion to extracellular matrix (ECM) proteins is required for efficient transmission of MAPK-based signals initiated by growth factors. However, the modulation of G protein-coupled receptor (GPCR) signaling by adhesion is less well understood. In the present study, we assessed the impact of cell adhesion on MAPK activation by muscarinic M3 receptors. The muscarinic agonist carbachol more efficiently promoted stress fiber formation and tyrosine phosphorylation of focal adhesion-associated proteins in M3 receptor-expressing cells adherent to fibronectin or collagen type I, as compared to polylysine. Overall MAPK activation was robust in cells adherent to all three substrata. However, total levels of MAPK and mitogen-activated protein kinase kinase (MEK) in the nucleus were significantly greater in cells adherent to ECM proteins for 2.5 h, and levels of activated MAPK and MEK in the nuclei of these cells were higher following carbachol stimulation, relative to levels in cells adherent to polylysine. MEK inhibitors did not prevent adhesion-dependent translocation of MAPK and MEK to the nucleus, and increased nuclear phospho-MEK levels in carbachol-stimulated cells. The results suggest that adhesion of cells to ECM triggers the redistribution of MAPK and MEK to the nucleus, possibly as a result of the cytoskeletal rearrangements that accompany cell spreading. This may represent a mechanism for priming the nucleus with MEK and MAPK, leading to more rapid and pronounced increases in intranuclear phospho-MAPK upon GPCR stimulation.
Asunto(s)
Adhesión Celular , Núcleo Celular/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Receptores Muscarínicos/fisiología , Transducción de Señal/fisiología , Carbacol/farmacología , Línea Celular , Colágeno Tipo I/fisiología , Proteínas de la Matriz Extracelular/fisiología , Fibronectinas/fisiología , Humanos , Microscopía Fluorescente , Fosforilación , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Basal forebrain cholinergic neurons (BFCN) participate in processes of learning, memory, and attention. Little is known about the genes expressed by BFCN and the extracellular signals that control their expression. Previous studies showed that bone morphogenetic protein (BMP) 9 induces and maintains the cholinergic phenotype of embryonic BFCN. We measured gene expression patterns in septal cultures of embryonic day 14 mice and rats grown in the presence or absence of BMP9 by using species-specific microarrays and validated the RNA expression data of selected genes by immunoblot and immunocytochemistry analysis of their protein products. BMP9 enhanced the expression of multiple genes in a time-dependent and, in most cases, reversible manner. The set of BMP9-responsive genes was concordant between mouse and rat and included genes encoding cell-cycle/growth control proteins, transcription factors, signal transduction molecules, extracellular matrix, and adhesion molecules, enzymes, transporters, and chaperonins. BMP9 induced the p75 neurotrophin receptor (NGFR), a marker of BFCN, and Cntf and Serpinf1, two trophic factors for cholinergic neurons, suggesting that BMP9 creates a trophic environment for BFCN. To determine whether the genes induced by BMP9 in culture were constituents of the BFCN transcriptome, we purified BFCN from embryonic day 18 mouse septum by using fluorescence-activated cell sorting of NGFR(+) cells and profiled mRNA expression of these and NGFR(-) cells. Approximately 30% of genes induced by BMP9 in vitro were overexpressed in purified BFCN, indicating that they belong to the BFCN transcriptome in situ and suggesting that BMP signaling contributes to maturation of BFCN in vivo.