RESUMEN

miRNAs are master regulators of gene expression in diverse biological processes, including the modulation of neuronal cytoarchitecture. The identification of their physiological target genes remains one of the outstanding challenges. Recently, it has been demonstrated that the activation of serotonin receptor 7 (5-HT7R) plays a key role in regulating the neuronal structure, synaptogenesis, and synaptic plasticity during embryonic and early postnatal development of the central nervous system (CNS). In order to identify putative miRNAs targeting the 3'UTR of 5-HT7R mouse transcript, we used a computational prediction tool and detected the miR-29 family members as the only candidates. Thus, since miR-29a is more expressed than other members in the brain, we investigated its possible involvement in the regulation of neuronal morphology mediated by 5-HT7R. By luciferase assay, we show that miR-29a can act as a post-transcriptional regulator of 5-HT7R mRNA. Indeed, it downregulates 5-HT7R gene expression in cultured hippocampal neurons, while the expression of other serotonin receptors is not affected. From a functional point of view, miR-29a overexpression in hippocampal primary cultures impairs the 5HT7R-dependent neurite elongation and remodeling through the inhibition of the ERK intracellular signaling pathway. In vivo, the upregulation of miR-29a in the developing hippocampus parallels with the downregulation of 5-HT7R expression, supporting the hypothesis that this miRNA is a physiological modulator of 5-HT7R expression in the CNS.

Asunto(s)

Hipocampo/metabolismo , MicroARNs/metabolismo , Neuronas/citología , Neuronas/metabolismo , Receptores de Serotonina/genética , Regiones no Traducidas 3'/genética , Animales , Secuencia de Bases , Células Cultivadas , Regulación hacia Abajo/genética , Células HEK293 , Células HeLa , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , MicroARNs/genética , Neuritas/metabolismo , Fosforilación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Serotonina/metabolismo , Regulación hacia Arriba/genética

RESUMEN

Spine motility analysis has become the mainstay for investigating synaptic plasticity but is limited in its versatility requiring complex, non automatized instrumentations. We describe an entropy-based method for determining the spatial distribution of dendritic spines that allows successful estimation of spine motility from still images. This method has the potential to extend the applicability of spine motility analysis to ex vivo preparations.

Asunto(s)

Espinas Dendríticas , Modelos Biológicos , Plasticidad Neuronal , Neuronas/citología , Neuronas/fisiología , Algoritmos , Animales , Dendritas , Ratones , Ratones Noqueados

RESUMEN

Serotonergic neurotransmission is mediated by at least 14 subtypes of 5-HT receptors. Among these, the CNS serotonin receptor 7 (5-HTR7) is involved in diverse physiological processes. Here we show that treatment of murine striatal and cortical neuronal cultures with 5-HTR7 agonists (8-OH-DPAT and LP-211) significantly enhances neurite outgrowth. This effect is abolished by the selective 5-HTR7 antagonist SB-269970, by the ERK inhibitor U0126, by the cyclin-dependent kinase 5 (Cdk5) inhibitor roscovitine, as well as by cycloheximide, an inhibitor of protein synthesis. These data indicate that 5-HTR7 activation stimulates extensive neurite elongation in CNS primary cultures, subserved by ERK and Cdk5 activation, and de novo protein synthesis. Two-dimensional (2D) gel electrophoresis coupled to Western blot analyses reveals both qualitative and quantitative expression changes in selected cytoskeletal proteins, following treatment of striatal primary cultures with LP-211. In particular, the 34 kDa isoform of MAP1B is selectively expressed in stimulated cultures, consistent with a role of this protein in tubulin polymerization and neurite elongation. In summary, our results show that agonist-dependent activation of the endogenous 5-HTR7 in CNS neuronal primary cultures stimulates ERK- and Cdk5-dependent neurite outgrowth, sustained by modifications of cytoskeletal proteins. These data support the hypothesis that the 5-HTR7 might play a crucial role in shaping neuronal morphology and behaviorally relevant neuronal networks, paving the way to new approaches able to modulate CNS connectivity.

Asunto(s)

Quinasa 5 Dependiente de la Ciclina/fisiología , Neuritas/fisiología , Receptores de Serotonina/metabolismo , Transducción de Señal/fisiología , Animales , Butadienos/farmacología , Células Cultivadas , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Femenino , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Ratones Endogámicos C57BL , Red Nerviosa/efectos de los fármacos , Red Nerviosa/metabolismo , Neuritas/efectos de los fármacos , Nitrilos/farmacología , Embarazo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos

RESUMEN

Neuronal ceroid lipofuscinoses (NCLs) constitute a group of progressive neurodegenerative disorders resulting from mutations in at least eight different genes. Mutations in the most recently identified NCL gene, MFSD8/CLN7, underlie a variant of late-infantile NCL (vLINCL). The MFSD8/CLN7 gene encodes a polytopic protein with unknown function, which shares homology with ion-coupled membrane transporters. In this study, we confirmed the lysosomal localization of the native CLN7 protein. This localization of CLN7 is not impaired by the presence of pathogenic missense mutations or after genetic ablation of the N-glycans. Expression of chimeric and full-length constructs showed that lysosomal targeting of CLN7 is mainly determined by an N-terminal dileucine motif, which specifically binds to the heterotetrameric adaptor AP-1 in vitro. We also show that CLN7 mRNA is more abundant in neurons than astrocytes and microglia, and that it is expressed throughout rat brain, with increased levels in the granular layer of cerebellum and hippocampal pyramidal cells. Interestingly, this cellular and regional distribution is in good agreement with the autofluorescent lysosomal storage and cell loss patterns found in brains from CLN7-defective patients. Overall, these data highlight lysosomes as the primary site of action for CLN7, and suggest that the pathophysiology underpinning CLN7-associated vLINCL is a cell-autonomous process.

Asunto(s)

Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Lipofuscinosis Ceroideas Neuronales/genética , Animales , Animales Recién Nacidos , Encéfalo/metabolismo , Células Cultivadas , Células HEK293 , Células HeLa , Homocigoto , Humanos , Lisosomas/genética , Lisosomas/metabolismo , Mutación , Lipofuscinosis Ceroideas Neuronales/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Transfección

RESUMEN

Brain-resident macrophages (microglia) are key cellular elements in the preservation of tissue integrity. On the other hand, they can also contribute to the development of pathological events by causing an extensive and inappropriate inflammatory response. A growing number of reports indicate the involvement of nucleotides in the control of microglial functions. With this study on P2Y receptors in rat microglia, we want to contribute to the definition of their expression profile and to the characterisation of their signalling mechanisms leading to Ca²âº movements. Endogenous nucleotides, when applied at a concentration of 100 muM, elicited robust Ca²âº transients, thanks to a panel of metabotropic receptors comprising mainly P2Y2, P2Y6 and P2Y12 subtypes. The involvement of P2Y12 receptors in Ca²âº responses induced by adenine nucleotides was confirmed by the pharmacological and pertussis toxin sensitivity of the response induced by adenosine diphosphate (ADP). Beside the G protein involved, Gi and Gq respectively, adenine and uracil nucleotides differed also for induction by the latter of a capacitative Ca²âº plateau. Moreover, when applied at low (sub-micromolar) concentrations with a long-lasting challenge, uracil nucleotides elicited oscillatory Ca²âº changes with low frequency of occurrence (

RESUMEN

Before their exocytotic release during stimulation of nerve terminals, nonpeptide neurotransmitters are loaded into synaptic vesicles by specific transporters. Recently, a protein initially identified as brain-specific Na(+)-dependent inorganic phosphate transporter I (BNPI) has been shown to represent a vesicular glutamate transporter (VGLUT1). In this study, we investigated whether a highly homologous "differentiation-associated Na(+)-dependent inorganic phosphate transporter" (DNPI) is involved in glutamatergic transmission. Vesicles isolated from BON cells expressing recombinant DNPI accumulated l-glutamate with bioenergetical and pharmacological characteristics identical to those displayed by VGLUT1 and by brain synaptic vesicles. Moreover, DNPI localized to synaptic vesicles, at synapses exhibiting classical excitatory features. DNPI thus represents a novel vesicular glutamate transporter (VGLUT2). The distributions of each VGLUT transcript in brain were highly complementary, with only a partial regional and cellular overlap. At the protein level, we could only detect either VGLUT1- or VGLUT2-expressing presynaptic boutons. The existence of two VGLUTs thus defines distinct subsets of glutamatergic neurons.

Asunto(s)

Sistemas de Transporte de Aminoácidos , Proteínas Portadoras/metabolismo , Ácido Glutámico/metabolismo , Proteínas de Transporte de Membrana , Neuronas/metabolismo , Proteínas de Transporte de Fosfato/metabolismo , Proteínas de Transporte Vesicular , Animales , Transporte Biológico , Encéfalo/citología , Encéfalo/metabolismo , Proteínas Portadoras/genética , Diferenciación Celular , Línea Celular , Hibridación in Situ , Neuronas/clasificación , Especificidad de Órganos , Terminales Presinápticos/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Vesículas Sinápticas/metabolismo , Transfección , Proteína 1 de Transporte Vesicular de Glutamato , Proteína 2 de Transporte Vesicular de Glutamato , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores

RESUMEN

The monomer-dimer equilibrium and the oxygen binding properties of ferrous recombinant Vitreoscilla hemoglobin (Vitreoscilla Hb) have been investigated. Sedimentation equilibrium data indicate that the ferrous deoxygenated and carbonylated derivatives display low values of equilibrium dimerization constants, 6 x 10(2) and 1 x 10(2) M(-1), respectively, at pH 7.0 and 10 degrees C. The behavior of the oxygenated species, as measured in sedimentation velocity experiments, is superimposable to that of the carbonylated derivative. The kinetics of O(2) combination, measured by laser photolysis at pH 7.0 and 20 degrees C, is characterized by a second-order rate constant of 2 x 10(8) M(-1) s(-1) whereas the kinetics of O(2) release at pH 7.0 is biphasic between 10 and 40 degrees C, becoming essentially monophasic below 10 degrees C. Values of the first-order rate constants (at 20 degrees C) and of the activation energies for the fast and slow phases of the Vitreoscilla Hb deoxygenation process are 4.2 s(-1) and 19.2 kcal mol(-1) and 0.15 s(-1) and 24.8 kcal mol(-1), respectively. Thus the biphasic kinetics of Vitreoscilla Hb deoxygenation is unrelated to the association state of the protein. The observed biphasic oxygen release may be accounted for by the presence of two different conformers in thermal equilibrium within the monomer. The two conformers may be assigned to a structure in which the heme-iron-bound ligand is stabilized by direct hydrogen bonding to TyrB10 and a structure in which such interaction is absent. The slow interconversion between the two conformers may reflect a very large conformational rearrangement in the disordered distal pocket segment connecting helices C and E.

Asunto(s)

Proteínas Bacterianas/metabolismo , Compuestos Ferrosos/metabolismo , Hemoglobinas/metabolismo , Oxígeno/metabolismo , Vitreoscilla/química , Proteínas Bacterianas/química , Sitios de Unión , Dimerización , Compuestos Férricos/química , Compuestos Ferrosos/química , Hemoglobinas/química , Cinética , Sonicación , Espectrofotometría , Temperatura , Hemoglobinas Truncadas , Ultracentrifugación

RESUMEN

A fully active recombinant human ceruloplasmin was obtained, and it was mutated to produce a ceruloplasmin stable to proteolysis. The stable ceruloplasmin was further mutated to perturb the environment of copper at the type 1 copper sites in two different domains. The wild type and the mutated ceruloplasmin were produced in the yeast Pichia pastoris and characterized. The mutations R481A, R701A, and K887A were at the proteolytic sites, did not alter the enzymatic activity, and were all necessary to protect ceruloplasmin from degradation. The mutation L329M was at the tricoordinate type 1 site of the domain 2 and was ineffective to induce modifications of the spectroscopic and catalytic properties of ceruloplasmin, supporting the hypothesis that this site is reduced and locked in a rigid frame. In contrast the mutation C1021S at the type 1 site of domain 6 substantially altered the molecular properties of the protein, leaving a small fraction endowed with oxidase activity. This result, while indicating the importance of this site in stabilizing the overall protein structure, suggests that another type 1 site is competent for dioxygen reduction. During the expression of ceruloplasmin, the yeast maintained a high level of Fet3 that was released from membranes of yeast not harboring the ceruloplasmin gene. This indicates that expression of ceruloplasmin induces a state of iron deficiency in yeast because the ferric iron produced in the medium by its ferroxidase activity is not available for the uptake.

Asunto(s)

Ceruloplasmina/metabolismo , Cobre , Proteínas Recombinantes/metabolismo , Secuencia de Aminoácidos , Ceruloplasmina/química , Ceruloplasmina/genética , Endopeptidasas/metabolismo , Estabilidad de Enzimas , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Pichia/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas de Saccharomyces cerevisiae

RESUMEN

A soluble derivative of Fet3 has been obtained from the methylotrophic yeast Pichia pastoris by limited proteolysis of membrane suspensions with trypsin. The soluble protein and the membrane-bound parent Fet3 have been purified to apparent homogeneity. Soluble Fet3 had molecular mass 100 kDa, while the full-length protein had molecular mass 110 kDa, in line with the expected decrease for cleavage and loss of a single transmembrane helix and a small cytoplasmic domain. The optical and EPR spectra of Fet3 were typical of the multicopper oxidases, indicating the presence of one type 1 blue copper site and a type 2/type 3 copper trinuclear cluster. V(max) values for iron oxidation by P. pastoris Fet3 were obtained similar to human ceruloplasmin and much higher than those reported for Saccharomyces cerevisiae Fet3.

Asunto(s)

Membrana Celular/metabolismo , Ceruloplasmina/metabolismo , Endopeptidasas/metabolismo , Pichia/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Catálisis , Ceruloplasmina/química , Ceruloplasmina/aislamiento & purificación , Cobre/metabolismo , Cobre/farmacología , Espectroscopía de Resonancia por Spin del Electrón , Proteínas Fúngicas/química , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Glicosilación , Humanos , Hierro/metabolismo , Hierro/farmacología , Quelantes del Hierro/farmacología , Cinética , Proteínas de la Membrana/química , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Pichia/citología , Pichia/efectos de los fármacos , Pichia/enzimología , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae , Solubilidad

RESUMEN

The effect of multiple binding of azide, N3-, on the structural and functional properties of ceruloplasmin (CP) has been reinvestigated by means of both spectroscopic and enzymatic techniques. High affinity binding of the anion to human CP resulted in a dramatic increase of the absorbance at 610 nm and in a concomitant decrease of the optical density at 330 nm. The oxidase activity toward Fe(II) was essentially unaffected, while turnover parameters versus nonferrous substrates dramatically changed, with an approximately 100-fold enhancement of the kcat/Km parameter. Chloride at physiological concentration proved to behave very similarly to N3- bound with high affinity, in that it not only induced the spectroscopic changes previously interpreted in terms of an intramolecular electron transfer from reduced type 1 to type 3 copper ions [Musci, G., Bonaccorsi di Patti, M.C. & Calabrese, L. (1995) J. Protein Chem. 14, 611-617], but it also enhanced some 60-fold the kcat/Km value. A different behavior was observed with chicken CP, where a decrease at 330 nm occurred without a concomitant modification at 603 nm. The chicken enzyme was less sensitive also in terms of enzymatic activity, which was nearly unchanged in the presence of either high affinity N3- or Cl-. At higher N3- concentrations, optical changes of both human and chicken CP were mainly focussed on the appearance of ligand-to-metal charge transfer bands below 500 nm, and the anion behaved as an inhibitor of the oxidase activity versus Fe(II) as well as noniron substrates. The well known bleaching of the blue chromophore could be observed, at neutral pH, only at very high N3-/CP ratios. The data presented in this paper are consistent with a mechanism of structural and functional modulation of CP by anions, that would be able to dictate the substrate specificity of the cuproprotein, and suggest the possibility that CP may act in vivo as a multifunctional oxidase.

Asunto(s)

Ceruloplasmina/química , Oxidorreductasas/metabolismo , Animales , Aniones/química , Azidas/química , Pollos , Cloruros/química , Cobre/química , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón , Humanos , Hierro/química , Cinética , Unión Proteica , Espectrofotometría

RESUMEN

The copper-glutathione complex (Cu(I)-GSH) efficiently acted in vitro as the source of Cu(I) in the reconstitution of apoceruloplasmin. Copper was found to reinstate in the various sites in a multistep process, with metal entry into the protein in a first phase, and a second step involving conformational changes of the protein leading to the recovery of the native structural and functional properties. This latter phase was found to be strongly facilitated by Mg2+ or Ca2+ and by ATP. Both Mg2+ and ATP had to be present for optimal reconstitution. These results may shed some light on the mechanisms governing the biosynthesis of ceruloplasmin in vivo. Cu(I)-GSH was the only complex able to reconstitute ceruloplasmin at neutral pH. Glutathione may thus function to shuttle the metal from the membrane copper pump, as the Wilson disease ATPase, and ceruloplasmin in the secretory compartments of the cell. The finding that ceruloplasmin acquires the native conformation after metal entry through a complex pathway triggered by Mg2+ and ATP suggests that they may act as physiological modulators of this process in vivo.

Asunto(s)

Ceruloplasmina/química , Cobre/química , Adenosina Trifosfato/química , Animales , Apoproteínas/química , Espectroscopía de Resonancia por Spin del Electrón , Electroforesis en Gel de Poliacrilamida , Glutatión/química , Humanos , Cinética , Magnesio/química , Ovinos , Análisis Espectral