RESUMEN

Gene mutations and gene copy number variants are associated with autism spectrum disorders (ASDs). Affected gene products are often part of signaling networks implicated in synapse formation and/or function leading to alterations in the excitation/inhibition (E/I) balance. Although the network of parvalbumin (PV)-expressing interneurons has gained particular attention in ASD, little is known on PV's putative role with respect to ASD. Genetic mouse models represent powerful translational tools for studying the role of genetic and neurobiological factors underlying ASD. Here, we report that PV knockout mice (PV(-/-)) display behavioral phenotypes with relevance to all three core symptoms present in human ASD patients: abnormal reciprocal social interactions, impairments in communication and repetitive and stereotyped patterns of behavior. PV-depleted mice also showed several signs of ASD-associated comorbidities, such as reduced pain sensitivity and startle responses yet increased seizure susceptibility, whereas no evidence for behavioral phenotypes with relevance to anxiety, depression and schizophrenia was obtained. Reduced social interactions and communication were also observed in heterozygous (PV(+/-)) mice characterized by lower PV expression levels, indicating that merely a decrease in PV levels might be sufficient to elicit core ASD-like deficits. Structural magnetic resonance imaging measurements in PV(-/-) and PV(+/-) mice further revealed ASD-associated developmental neuroanatomical changes, including transient cortical hypertrophy and cerebellar hypoplasia. Electrophysiological experiments finally demonstrated that the E/I balance in these mice is altered by modification of both inhibitory and excitatory synaptic transmission. On the basis of the reported changes in PV expression patterns in several, mostly genetic rodent models of ASD, we propose that in these models downregulation of PV might represent one of the points of convergence, thus providing a common link between apparently unrelated ASD-associated synapse structure/function phenotypes.

Asunto(s)

Trastorno Autístico/patología , Trastorno Autístico/psicología , Conducta Animal/fisiología , Encéfalo/patología , Neuronas/fisiología , Parvalbúminas , Análisis de Varianza , Animales , Trastorno Autístico/fisiopatología , Encéfalo/fisiopatología , Modelos Animales de Enfermedad , Humanos , Hipertrofia , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Tamaño de los Órganos , Vocalización Animal/fisiología

RESUMEN

In mice, 249 putative members of the superfamily of EF-hand domain Ca(2+)-binding proteins, manifesting great diversity in structure, cellular localization and functions have been identified. Three members in particular, namely, calbindin-D28K, calretinin and parvalbumin, are widely used as markers for specific neuronal subpopulations in different regions of the brain. The aim of the present study was to compile a comprehensive atlas of the gene-expression profiles of the entire EF-hand gene superfamily in the murine brain. This was achieved by a meticulous examination of the in-situ hybridization images in the Allen Brain Atlas database. Topographically, our analysis focused on the olfactory bulb, cerebral cortex (barrel cortex in the primary somatosensory area), basal ganglia, hippocampus, amygdala, thalamus, hypothalamus, cerebellum, midbrain, pons and medulla, and on clearly identifiable sub-structures within each of these areas. The expression profiles of four family-members, namely hippocalcin-like 4, neurocalcin-δ, plastin 3 and tescalcin, that have not been hitherto reported, at either the mRNA (in-situ-hybridization) or the protein (immunohistochemical) levels, are now presented for the first time. The fruit of our analysis is a document in which the gene-expression profiles of all members of the EF-hand family genes are compared, and in which future possible neuronal markers for specific cells/brain areas are identified. The assembled information could afford functional clues to investigators, conducive to further experimental pursuit.

Asunto(s)

Encéfalo/metabolismo , Proteínas de Unión al Calcio/genética , Expresión Génica/genética , Estudio de Asociación del Genoma Completo , Neuronas/metabolismo , Envejecimiento , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Estudio de Asociación del Genoma Completo/métodos , Ratones , ARN Mensajero/metabolismo

RESUMEN

The calcium-binding proteins parvalbumin, calbindin D-28k, calretinin and calcineurin are present in subsets of GABAergic gigantic calyciform presynaptic terminals of the reticular thalamic nucleus (RTN). Previously it was hypothesized that GABA and calcium-binding proteins including parvalbumin are not only colocalized in the same neuron subpopulation, but that GABA synthesis and parvalbumin expression could be also genetically regulated by a common mechanism. Moreover, parvalbumin expression levels could influence GABA synthesis. For this, we analyzed GABA immunoreactivity in RTN gigantic calyciform presynaptic terminals of parvalbumin-deficient (PV-/-) mice. With respect to GABA immunoreactivity we found no differences compared to wild-type animals. However, using a polyclonal parvalbumin antibody raised against full-length rat muscle parvalbumin on brain sections of PV-/- mice, we observed paradoxical parvalbumin immunoreactivity in partly varicose axons in the diencephalon, mainly in the lamina medullaris externa surrounding the thalamus. A detailed immunohistochemical, biochemical and molecular biological analysis revealed this immunoreactivity to be the result of an upregulation of oncomodulin (OM), the mammalian beta isoform of parvalbumin in PV-/- mice. In addition, OM was present in a sparse subpopulation of neurons in the thalamus and in the dentate gyrus. OM expression has not been observed before in neurons of the mammalian brain; its expression was restricted to outer hair cells in the organ of Corti. Our results indicate that the absence of parvalbumin has no major effect on the GABA-synthesizing system in RTN presynaptic terminals excluding a direct effect of parvalbumin on this regulation. However, a likely homeostatic mechanism is induced resulting in the upregulation of OM in selected axons and neuronal perikarya. Our results warrant further detailed investigations on the putative role of OM in the brain.

Asunto(s)

Axones/metabolismo , Proteínas de Unión al Calcio/metabolismo , Diencéfalo/metabolismo , Neuronas/metabolismo , Parvalbúminas/metabolismo , Animales , Giro Dentado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Parvalbúminas/deficiencia , Parvalbúminas/genética , Terminales Presinápticos/metabolismo , Isoformas de Proteínas , Ratas , Ratas Wistar , Núcleos Talámicos/metabolismo , Tálamo/metabolismo , Regulación hacia Arriba , Ácido gamma-Aminobutírico/metabolismo

RESUMEN

AIMS: Parenchymal microcalcification in the brain coincides with neurodegenerative diseases, but is also frequently found in neurologically normal individuals. The origin and role of this process are still under debate. Parvalbumin (PV) is a protein acting as a Ca(2+) buffer and Ca(2+) shuttle towards intracellular Ca(2+) sinks, like mitochondria and the endoplasmic reticulum. Constitutively, it is present in a subset of inhibitory neurones. In transgenic mice expressing pan-neuronal PV, the mitochondrial volume is reduced. We tested whether elevated levels of intraneuronal [Ca(2+)] and reduced mitochondrial volume in the neurone interfere with the generation of parenchymal microcalcification. METHODS: The striatum of wild type and transgenic mice was injected with the glutamate receptor agonist ibotenic acid (IBO), which is known to induce not only excitotoxic neurodegeneration, but also parenchymal calcification. Sections were studied by light and electron microscopy at various time points after IBO application. RESULTS: Morphometric analysis 2, 4 and 20 weeks after IBO application revealed microcalcification in transgenic and wild type mice; the calcification process, however, was enhanced and accelerated in the transgenic animals. Ultrastructural analyses suggest neuronal mitochondria as the nucleators of the deposits which consist of hydroxyapatite. The time-dependent changes in size and surface structure of the deposits indicate the presence of biological mechanisms in the brain promoting regression of bioapatites. CONCLUSIONS: The overload of intraneuronal [Ca(2+)] in combination with impaired mitochondrial function activates neuronal microcalcification. It is hypothesized that this process is an alternative/adaptive mechanism of the neurone to reduce further brain damage.

Asunto(s)

Calcinosis/patología , Mitocondrias/ultraestructura , Neuronas/ultraestructura , Parvalbúminas/metabolismo , Animales , Calcinosis/fisiopatología , Calcio/metabolismo , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/fisiopatología , Cuerpo Estriado/ultraestructura , Durapatita/metabolismo , Agonistas de Aminoácidos Excitadores/administración & dosificación , Ácido Iboténico/administración & dosificación , Inmunohistoquímica , Ratones , Ratones Transgénicos , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Mitocondrias/fisiología , Neuronas/fisiología

RESUMEN

Advances in the understanding of a class of Ca(2+)-binding proteins usually referred to as "Ca(2+) buffers" are reported. Proteins historically embraced within this group include parvalbumins (alpha and beta), calbindin-D9k, calbindin-D28k and calretinin. Within the last few years a wealth of data has accumulated that allow a better understanding of the functions of particular family members of the >240 identified EF-hand Ca(2+)-binding proteins encoded by the human genome. Studies often involving transgenic animal models have revealed that they exert their specific functions within an intricate network consisting of many proteins and cellular mechanisms involved in Ca(2+) signaling and Ca(2+) homeostasis, and are thus an essential part of the Ca(2+) homeostasome. Recent results indicate that calbindin-D28k, possibly also calretinin and oncomodulin, the mammalian beta parvalbumin, might have additional Ca(2+) sensor functions, leaving parvalbumin and calbindin-D9k as the only "pure" Ca(2+) buffers.

Asunto(s)

Tampones (Química) , Calcio/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Calbindina 1 , Calbindinas , Señalización del Calcio/fisiología , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Quelantes/metabolismo , Motivos EF Hand , Homeostasis , Humanos , Riñón/fisiología , Magnesio/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Plasticidad Neuronal , Parvalbúminas/genética , Parvalbúminas/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína G de Unión al Calcio S100/química , Proteína G de Unión al Calcio S100/genética , Proteína G de Unión al Calcio S100/metabolismo

RESUMEN

AIMS: Prion diseases are generally characterized by pronounced neuronal loss. In particular, a subpopulation of inhibitory neurones, characterized by the expression of the calcium-binding protein parvalbumin (PV), is selectively destroyed early in the course of human and experimental prion diseases. By contrast, nerve cells expressing calbindin D28 k (CB), another calcium-binding protein, as well as PV/CB coexpressing Purkinje cells, are well preserved. METHODS: To evaluate, if PV and CB may directly contribute to neuronal vulnerability or resistance against nerve cell death, respectively, we inoculated PV- and CB-deficient mice, and corresponding controls, with 139A scrapie and compared them with regard to incubation times and histological lesion profiles. RESULTS: While survival times were slightly but significantly diminished in CB-/-, but not PV-/- mice, scrapie lesion profiles did not differ between knockout mice and controls. There was a highly significant and selective loss of isolectin B(4)-decorated perineuronal nets (which specifically demarcate the extracellular matrix surrounding the 'PV-expressing' subpopulation of cortical interneurones) in scrapie inoculated PV+/+, as well as PV-/- mice. Purkinje cell numbers were not different in CB+/+ and CB-/- mice. CONCLUSIONS: Our results suggest that PV expression is a surrogate marker for neurones highly vulnerable in prion diseases, but that the death of these neurones is unrelated to PV expression and thus based on a still unknown pathomechanism. Further studies including the inoculation of mice ectopically (over)expressing CB are necessary to determine whether the shortened survival of CB-/- mice is indeed due to a neuroprotective effect of this molecule.

Asunto(s)

Parvalbúminas/deficiencia , Parvalbúminas/metabolismo , Proteína G de Unión al Calcio S100/genética , Proteína G de Unión al Calcio S100/metabolismo , Scrapie/metabolismo , Animales , Calbindina 1 , Calbindinas , Modelos Animales de Enfermedad , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Red Nerviosa/patología , Scrapie/genética , Scrapie/patología , Especificidad de la Especie , Análisis de Supervivencia , Vacuolas/patología , Vacuolas/ultraestructura

RESUMEN

The Ca(2+)-binding proteins parvalbumin (PV) and calbindin D-28k (CB) are key players in the intracellular Ca(2+)-buffering in specific cells including neurons and have profound effects on spatiotemporal aspects of Ca(2+) transients. The previously observed increase in mitochondrial volume density in fast-twitch muscle of PV-/- mice is viewed as a specific compensation mechanism to maintain Ca(2+) homeostasis. Since cerebellar Purkinje cells (PC) are characterized by high expression levels of the Ca(2+) buffers PV and CB, the question was raised, whether homeostatic mechanisms are induced in PC lacking these buffers. Mitochondrial volume density, i.e. relative mitochondrial mass was increased by 40% in the soma of PV-/- PC. Upregulation of mitochondrial volume density was not homogenous throughout the soma, but was selectively restricted to a peripheral region of 1.5 microm width underneath the plasma membrane. Accompanied was a decreased surface of subplasmalemmal smooth endoplasmic reticulum (sPL-sER) in a shell of 0.5 microm thickness underneath the plasma membrane. These alterations were specific for the absence of the "slow-onset" buffer PV, since in CB-/- mice neither changes in peripheral mitochondria nor in sPL-sER were observed. This implicates that the morphological alterations are aimed to specifically substitute the function of the slow buffer PV. We propose a novel concept that homeostatic mechanisms of components involved in Ca(2+) homeostasis do not always occur at the level of similar or closely related molecules. Rather the cell attempts to restore spatiotemporal aspects of Ca(2+) signals prevailing in the undisturbed (wildtype) situation by subtly fine tuning existing components involved in the regulation of Ca(2+) fluxes.

Asunto(s)

Retículo Endoplásmico/patología , Retículo Endoplásmico/ultraestructura , Mitocondrias/patología , Parvalbúminas/deficiencia , Células de Purkinje/ultraestructura , Animales , Western Blotting/métodos , Calbindinas , Calcio/metabolismo , Corteza Cerebelosa/citología , Electroforesis en Gel Bidimensional/métodos , Técnica del Anticuerpo Fluorescente/métodos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Transmisión/métodos , Mitocondrias/ultraestructura , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Células de Purkinje/patología , Proteína G de Unión al Calcio S100/genética , Estadísticas no Paramétricas

RESUMEN

Calbindin is a fast Ca2+-binding protein expressed by Purkinje cells and involved in their firing regulation. Its deletion produced approximately 160-Hz oscillation sustained by synchronous, rhythmic Purkinje cells in the cerebellar cortex of mice. Parvalbumin is a slow-onset Ca2+-binding protein expressed in Purkinje cells and interneurons. In order to assess its function in Purkinje cell firing regulation, we studied the firing behavior of Purkinje cells in alert mice lacking parvalbumin (PV-/-), calbindin (CB-/-) or both (PV-/- CB-/-) and in wild-type controls. The absence of either protein resulted in Purkinje cell firing alterations (decreased complex spike duration and pause, increased simple spike firing rate) that were more pronounced in CB-/- than in PV-/- mice. Cumulative effects were found in complex spike alterations in PV-/- CB-/- mice. PV-/- and CB-/- mice manifested approximately 160-Hz oscillation that was sustained by Purkinje cells firing rhythmically and synchronously along the parallel fiber axis. This oscillation was dependent on GABA(A), N-methyl-D-aspartate and gap junction transmission. PV-/- CB-/- mice exhibited a dual-frequency (110 and 240 Hz) oscillation. The instantaneous spectral densities of both components were inversely correlated. Simple and complex spikes of Purkinje cells were phase-locked to one of the two oscillation frequencies. Mono- and dual-frequency oscillations presented similar pharmacological properties. These results demonstrate that the absence of the Ca2+ buffers parvalbumin and calbindin disrupts the regulation of Purkinje cell firing rate and rhythmicity in vivo and suggest that precise Ca2+ transient control is required to maintain the normal spontaneous arrhythmic and asynchronous firing pattern of the Purkinje cells.

Asunto(s)

Potenciales de Acción/fisiología , Cerebelo/citología , Parvalbúminas/deficiencia , Periodicidad , Células de Purkinje/fisiología , Proteína G de Unión al Calcio S100/genética , 2-Amino-5-fosfonovalerato/farmacología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/efectos de la radiación , Análisis de Varianza , Animales , Calbindina 2 , Calbindinas , Carbenoxolona/farmacología , Potenciales Evocados/efectos de los fármacos , Potenciales Evocados/fisiología , Potenciales Evocados/efectos de la radiación , Antagonistas de Aminoácidos Excitadores/farmacología , Análisis de Fourier , Antagonistas del GABA/farmacología , Inmunohistoquímica/métodos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Inhibición Neural/efectos de la radiación , Piridazinas/farmacología , Proteína G de Unión al Calcio S100/metabolismo , Factores de Tiempo

RESUMEN

Networks of GABAergic interneurons are of utmost importance in generating and promoting synchronous activity and are involved in producing coherent oscillations. These neurons are characterized by their fast-spiking rate and by the expression of the Ca(2+)-binding protein parvalbumin (PV). Alteration of their inhibitory activity has been proposed as a major mechanism leading to epileptic seizures and thus the role of PV in maintaining the stability of neuronal networks was assessed in knockout (PV-/-) mice. Pentylenetetrazole induced generalized tonic-clonic seizures in all genotypes, but the severity of seizures was significantly greater in PV-/- than in PV+/+ animals. Extracellular single-unit activity recorded from over 1000 neurons in vivo in the temporal cortex revealed an increase of units firing regularly and a decrease of cells firing in bursts. In the hippocampus, PV deficiency facilitated the GABA(A)ergic current reversal induced by high-frequency stimulation, a mechanism implied in the generation of epileptic activity. We postulate that PV plays a key role in the regulation of local inhibitory effects exerted by GABAergic interneurons on pyramidal neurons. Through an increase in inhibition, the absence of PV facilitates synchronous activity in the cortex and facilitates hypersynchrony through the depolarizing action of GABA in the hippocampus.

Asunto(s)

Encéfalo/fisiopatología , Epilepsia/fisiopatología , Predisposición Genética a la Enfermedad/genética , Red Nerviosa/fisiopatología , Parvalbúminas/deficiencia , Ácido gamma-Aminobutírico/metabolismo , Potenciales de Acción/fisiología , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Epilepsia/inducido químicamente , Epilepsia/metabolismo , Hipocampo/metabolismo , Hipocampo/fisiopatología , Interneuronas/fisiología , Ratones , Ratones Noqueados , Red Nerviosa/metabolismo , Inhibición Neural/genética , Parvalbúminas/genética , Pentilenotetrazol , Células Piramidales/fisiología , Receptores de GABA-A/metabolismo , Transmisión Sináptica/fisiología

RESUMEN

Following nerve injury in neonatal rats, a large proportion of motoneurons die, possibly as a consequence of an increase in vulnerability to the excitotoxic effects of glutamate. Calcium-dependent glutamate excitotoxicity is thought to play a significant role not only in injury-induced motoneuron death, but also in motoneuron degeneration in diseases such as amyotrophic lateral sclerosis (ALS). Motoneurons are particularly vulnerable to calcium influx following glutamate receptor activation, as they lack a number of calcium binding proteins, such as calbindin-D(28k) and parvalbumin. Therefore, it is possible that increasing the ability of motoneurons to buffer intracellular calcium may protect them from cell death and prevent the decline in motor function that usually occurs as a consequence of motoneuron loss. In this study we have tested this possibility by examining the effect of neonatal axotomy on motoneuron survival and muscle force production in normal and transgenic mice that over-express parvalbumin in their motoneurons.The sciatic nerve was crushed in one hindlimb of new-born transgenic and wildtype mice. The effect on motoneuron survival was assessed 8 weeks later by retrograde labelling of motoneurons innervating the tibialis anterior muscle. Following nerve injury in wildtype mice, only 20.2% (+/-2.2, S.E.M.; n=4) of injured motoneurons survive long term compared with 47.2% (+/-4.4, S.E.M.; n=4) in parvalbumin over-expressing mice. Surprisingly, this dramatic increase in motoneuron survival was not reflected in a significant improvement in muscle function, since 8 weeks after injury there was no improvement in either maximal twitch and tetanic force, or muscle weights.Thus, inducing spinal motoneurons to express parvalbumin protects a large proportion of motoneurons from injury-induced cell death, but this is not sufficient to restore muscle function.

Asunto(s)

Neuronas Motoras/patología , Músculo Esquelético/fisiopatología , Parvalbúminas/biosíntesis , Animales , Animales Recién Nacidos , Muerte Celular , Femenino , Inmunohistoquímica , Masculino , Ratones , Ratones Transgénicos , Neuronas Motoras/metabolismo , Músculo Esquelético/inervación , Compresión Nerviosa , Degeneración Nerviosa , Regeneración Nerviosa , Parvalbúminas/genética , Reacción en Cadena de la Polimerasa , Recuperación de la Función , Nervio Ciático/fisiología , Médula Espinal/metabolismo , Médula Espinal/patología

RESUMEN

We tested the hypothesis whether the mild dystrophy in mdx mice could result from the contribution of the cytosolic calcium buffer parvalbumin in maintaining a normal cytosolic [Ca2+]i, in spite of an increased passive Ca2+ influx. By crossing mdx mice with parvalbumin-deficient mice, double mutant mice, lacking both dystrophin and parvalbumin, were obtained. Though resting cytosolic [Ca2+]i and total calcium content were similar to that of mdx muscles, this new animal model presented a slightly more severe phenotype than the mdx mouse. Muscle pseudo-hypertrophy, the density of myotubes and of centronucleated fibres as well as the loss of IIB fibres were all increased in parvalbumin-deficient mdx mice. Many of these deficits were overcome in late adulthood, albeit fibrosis was clearly more pronounced than in mdx muscles. At 90 days, parvalbumin-deficient mdx mice showed higher levels of creatine phosphokinase and lower muscle strength, in vivo, than mdx mice. Isometric tension of isolated muscle was reduced, but the susceptibility to eccentric contraction was not increased. The slight aggravation of muscle dystrophy observed in mdx mice deprived of parvalbumin cannot explain the severity of the affection observed in xmd dogs and Duchenne dystrophy patients where parvalbumin is constitutively not expressed.

Asunto(s)

Calcio/metabolismo , Distrofina/deficiencia , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Mutación , Parvalbúminas/deficiencia , Fenotipo , Factores de Edad , Animales , Creatina Quinasa/sangre , Citosol/metabolismo , Fibrosis/fisiopatología , Contracción Isométrica , Ratones , Ratones Endogámicos mdx , Fibras Musculares de Contracción Rápida/patología , Fibras Musculares Esqueléticas/patología , Distrofias Musculares/metabolismo , Distrofias Musculares/patología , Distrofias Musculares/fisiopatología , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patología , Distrofia Muscular de Duchenne/fisiopatología , Cadenas Pesadas de Miosina , Factores de Tiempo

RESUMEN

Excitotoxic effects leading to neuronal cell degeneration are often accompanied by a prolonged increase in the intracellular level of Ca(2+) ions and L-glutamate-induced toxicity is assumed to be mediated via a Ca(2+)-dependent mechanism. Due to their buffering properties, EF-hand Ca(2+)-binding proteins (CaBPs) can affect intracellular Ca(2+) homeostasis and a neuroprotective role has been attributed to some of the family members including calretinin, calbindin D-28k and parvalbumin. We have stably transfected N18-RE 105 neuroblastoma-retina hybrid cells with the cDNAs for the three CaBPs and investigated the effect of these proteins on the L-glutamate-induced, Ca(2+)-dependent cytotoxicity. Several clones for each CaBP were selected according to immunocytochemical staining and characterization of the overexpressed proteins by Western blot analysis. In calretinin- and parvalbumin-expressing clones, expression levels were quantitatively determined by ELISA techniques. Cytotoxicity of transfected clones was quantified by measurement of the activity of lactate dehydrogenase (LDH) that was released into the medium after L-glutamate (10 mM) exposure as a result of necrotic cell death. In untransfected and parvalbumin-transfected cells, LDH released into the medium progressively increased (starting from the 20th hour) reaching maximum levels after 28-30 h of glutamate application. In contrast, LDH release in both, calretinin and calbindin D-28k-transfected clones, was not significantly different from unstimulated transfected or untransfected cells over the same period of time. The results indicate that the 'fast' Ca(2+)-buffers calretinin and calbindin D-28k, but not the 'slow' buffer parvalbumin can protect N18-RE 105 cells from this type of Ca(2+)-dependent L-glutamate-induced delayed cytotoxicity.

Asunto(s)

Ácido Glutámico/toxicidad , Parvalbúminas/genética , Parvalbúminas/fisiología , Retina/patología , Proteína G de Unión al Calcio S100/genética , Proteína G de Unión al Calcio S100/fisiología , Animales , Western Blotting , Neoplasias Encefálicas/patología , Calbindina 2 , Calbindinas , División Celular/efectos de los fármacos , Línea Celular , Células Clonales , Colorantes , Ensayo de Inmunoadsorción Enzimática , Células Híbridas , Inmunohistoquímica , Ratones , Neuroblastoma/patología , Plásmidos/genética , Ratas , Sales de Tetrazolio , Tiazoles , Transfección

RESUMEN

Monoclonal antibodies are very helpful tools to investigate the localization and sometimes even the function of specific proteins in cells and tissues. By generating monoclonal antibodies against calretinin-22k (CR-22k), a C-terminally truncated isoform of calretinin (CR) as a result of alternative splicing of the CR mRNA, we envisaged that screening multiple monoclonal antibodies would allow the identification of CR-22k as well as CR. Both proteins share the first 178 amino acids, but have different C-termini. All three antibodies 10C10, 6B3 and 2H4 recognize recombinant CR-22k and the specificity to also recognize CR was demonstrated in brain extracts of different species and human tumour cells, which express CR. All monoclonal antibodies did not crossreact with the closely related protein calbindin D-28k. Antibody binding was depending on the Ca2+-binding status of both forms of calretinin. Generally, the Ca2+-bound form was better recognized than the Ca2+-free form. Carboxy- and amino-terminally truncated CR proteins were expressed in E. coli in order to characterize the epitopes recognized by the three antibodies. Additionally, tryptic and cyanogen bromide fragments were produced to further narrow down the sequences recognized by the three antibodies. 10C10 recognizes an epitope consisting of the linker region between EF-hand domains I and II and the N-terminal part of EF-hand II, while the others (6B3, 2H4) bind to a region including the linker between EF-hand domains III and IV. These antibodies are valuable tools to further investigate the distribution and eventually the specific function of these two proteins in the nervous tissue and under pathological conditions, e.g. in colon tumours and mesotheliomas.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Calcio/metabolismo , Neoplasias del Colon/inmunología , Epítopos/inmunología , Proteína G de Unión al Calcio S100/inmunología , Secuencia de Aminoácidos , Animales , Western Blotting , Calbindina 2 , Calbindinas , Calcio/inmunología , Neoplasias del Colon/metabolismo , Bromuro de Cianógeno , Mapeo Epitopo , Humanos , Inmunohistoquímica , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Ratas , Proteína G de Unión al Calcio S100/metabolismo , Homología de Secuencia , Tripsina , Células Tumorales Cultivadas

RESUMEN

The mechanism responsible for the selective vulnerability of motor neurons in amyotrophic lateral sclerosis (ALS) is poorly understood. Several lines of evidence indicate that susceptibility of motor neurons to Ca(2+) overload induced by excitotoxic stimuli is involved. In this study, we investigated whether the high density of Ca(2+)-permeable AMPA receptors on motor neurons gives rise to higher Ca(2+) transients in motor neurons compared to dorsal horn neurons. Dorsal horn neurons were chosen as controls as these cells do not degenerate in ALS. In cultured spinal motor neurons, the rise of the cytosolic Ca(2+) concentration induced by kainic acid (KA) and mediated by the AMPA receptor was almost twice as high as in spinal neurons from the dorsal horn. Furthermore, we investigated whether increasing the motor neuron's cytosolic Ca(2+)-buffering capacity protects them from excitotoxic death. To obtain motor neurons with increased Ca(2+) buffering capacity, we generated transgenic mice overexpressing parvalbumin (PV). These mice have no apparent phenotype. PV overexpression was present in the central nervous system, kidney, thymus, and spleen. Motor neurons from these transgenic mice expressed PV in culture and were partially protected from KA-induced death as compared to those isolated from nontransgenic littermates. PV overexpression also attenuated KA-induced Ca(2+) transients, but not those induced by depolarization. We conclude that the high density of Ca(2+)-permeable AMPA receptors on the motor neuron's surface results in high Ca(2+) transients upon stimulation and that the low cytosolic Ca(2+)-buffering capacity of motor neurons may contribute to the selective vulnerability of these cells in ALS. Overexpression of a high-affinity Ca(2+) buffer such as PV protects the motor neuron from excitotoxicity and this protective effect depends upon the mode of Ca(2+) entry into the cell.

Asunto(s)

Agonistas de Aminoácidos Excitadores/toxicidad , Neuronas Motoras/efectos de los fármacos , Neurotoxinas/toxicidad , Parvalbúminas/farmacología , Esclerosis Amiotrófica Lateral/etiología , Animales , Western Blotting , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Células Cultivadas , Citoprotección/efectos de los fármacos , Citoprotección/genética , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Ácido Kaínico/toxicidad , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas Motoras/citología , Neuronas Motoras/metabolismo , Especificidad de Órganos , Parvalbúminas/genética , Parvalbúminas/metabolismo , Fenotipo , Células del Asta Posterior/citología , Células del Asta Posterior/efectos de los fármacos , Células del Asta Posterior/metabolismo , Receptores AMPA/metabolismo

RESUMEN

In some neurological diseases, injury to neurones reflects an over-stimulation of their receptors for excitatory amino acids. This response may disturb the Ca(2+)-homeostasis and lead to a pronounced and sustained increase in the intracellular concentration of this ion. On the basis of data derived from correlative studies, calcium-binding proteins have been postulated to play a protective role in these pathologies. We tested, directly, the capacity of the three calcium-binding proteins calretinin (CR), calbindin D-28k (CB) and parvalbumin (PV) to buffer [Ca(2+)], and to protect cells against excitotoxic death. We used P19 murine embryonic carcinoma cells, which can be specifically induced (by retinoic acid) to transform into nerve-like ones. The differentiated cells express functional glutamate-receptors and are susceptible to excitotoxic shock. Undifferentiated P19-cells were stably transfected with the cDNA for CR, CB or PV, induced to differentiate, and then exposed to NMDA, a glutamate-receptor agonist. The survival rates of clones expressing CR, CB or PV were compared with those of untransfected P19-cells using the lactate-dehydrogenase assay. CR- and CB-expressing cells were protected from death during the first 2 h of exposure to NMDA. This protection was, however, transient, and did not suffice to rescue P19-cells after prolonged stimulation. Two of the three PV-transfected clones raised were vulnerable to NMDA-induced excitotoxicity; the third, which expressed the lowest level of PV, was protected to a similar degree as that found for the CR- and CB-transfected clones. Our results indicate that in the P19-cell model, CR and CB can help to delay the onset of cell death after excitotoxic stimulation.

Asunto(s)

Proteínas de Unión al Calcio/genética , Muerte Celular/fisiología , Enfermedades del Sistema Nervioso Central/tratamiento farmacológico , Sistema Nervioso Central/metabolismo , Neuronas/metabolismo , Fármacos Neuroprotectores/metabolismo , Neurotoxinas/metabolismo , Animales , Calbindina 2 , Calbindinas , Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Muerte Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Tamaño de la Célula/efectos de los fármacos , Tamaño de la Célula/fisiología , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/fisiopatología , Enfermedades del Sistema Nervioso Central/metabolismo , Enfermedades del Sistema Nervioso Central/fisiopatología , Agonistas de Aminoácidos Excitadores/farmacología , Aminoácidos Excitadores/metabolismo , Glicina/farmacología , Inmunohistoquímica , L-Lactato Deshidrogenasa/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , N-Metilaspartato/farmacología , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Neuronas/efectos de los fármacos , Neuronas/patología , Parvalbúminas/genética , Parvalbúminas/metabolismo , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Proteína G de Unión al Calcio S100/genética , Proteína G de Unión al Calcio S100/metabolismo , Tretinoina/farmacología

RESUMEN

Calretinin (CR) and the alternatively spliced form calretinin-22k (CR-22k) are members of the EF-hand family of Ca(2+)-binding proteins (CaBPs). CR is expressed in more than 60% of poorly differentiated human colon tumors and both isoforms are present in several colon carcinoma cell lines (e.g., WiDr). They are absent in normal enterocytes and in well-differentiated adenocarcinoma cell lines such as CaCo-2. Calretinins are thought to act as Ca(2+) buffers and to be involved in the regulation of Ca(2+)-dependent processes. Down-regulation of calretinins in WiDr cells by antisense oligonucleotides leads to growth inhibition and treatment with sodium butyrate (NaBt, an inducer of differentiation) leads to a blockage of the cell cycle and, in parallel, to down-regulation of CR. It has been proposed that CR and/or CR-22k may be involved in maintaining the undifferentiated phenotype of WiDr cells and contributing to the transformation of enterocytes. Expression levels and distribution of CR-22k were investigated in WiDr cells. CR-22k was down-regulated in NaBt-treated cells and the normally cytoplasmic protein was preferentially localized in the nucleus either as a result of translocation or selective nuclear maintenance, a process more pronounced than in the case of CR. To compare functional differences of calretinins, CR-negative Caco-2 cells were stably transfected with cDNAs encoding CR or CR-22k. Cell growth of CR-transfected cells was increased, an effect that was not observed in CR-22k-transfected ones. The CR-expressing clones were almost completely resistant to treatment with 0.5 mM NaBt, a concentration significantly reducing cell growth in control cells. The same effect was obtained in the CR-22k-expressing clones, although to a lesser extent. This implicates that expression of CR and/or CR-22k in colon tumor cells may contribute to tumorigenesis by blocking differentiation-promoting signals.

Asunto(s)

Adenocarcinoma/metabolismo , Butiratos/farmacología , Neoplasias del Colon/metabolismo , Proteína G de Unión al Calcio S100/farmacología , Acetamidas/farmacología , Antineoplásicos/farmacología , Células CACO-2 , Calbindina 2 , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Humanos , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Isoformas de Proteínas/farmacología , Proteína G de Unión al Calcio S100/biosíntesis , Proteína G de Unión al Calcio S100/genética , Fracciones Subcelulares/metabolismo , Transfección

RESUMEN

The soluble Ca2+-binding protein parvalbumin (PV) is expressed at high levels in fast-twitch muscles of mice. Deficiency of PV in knockout mice (PV -/-) slows down the speed of twitch relaxation, while maximum force generated during tetanic contraction is unaltered. We observed that PV-deficient fast-twitch muscles were significantly more resistant to fatigue than were the wild type. Thus components involved in Ca2+ homeostasis during the contraction-relaxation cycle were analyzed. No upregulation of another cytosolic Ca2+-binding protein was found. Mitochondria are thought to play a physiological role during muscle relaxation and were thus analyzed. The fractional volume of mitochondria in the fast-twitch muscle extensor digitorum longus (EDL) was almost doubled in PV -/- mice, and this was reflected in an increase of cytochrome c oxidase. A faster removal of intracellular Ca2+ concentration ([Ca2+]i) 200-700 ms after fast-twitch muscle stimulation observed in PV -/- muscles supports the role for mitochondria in late [Ca2+]i removal. The present results also show a significant increase of the density of capillaries in EDL muscles of PV -/- mice. Thus alterations in the dynamics of Ca2+ transients detected in fast-twitch muscles of PV -/- mice might be linked to the increase in mitochondria volume and capillary density, which contribute to the greater fatigue resistance of these muscles.

Asunto(s)

Calcio/metabolismo , Mitocondrias/metabolismo , Fatiga Muscular/fisiología , Fibras Musculares de Contracción Rápida/fisiología , Parvalbúminas/fisiología , Animales , Señalización del Calcio , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fibras Musculares de Contracción Rápida/química , Fibras Musculares de Contracción Rápida/ultraestructura , Parvalbúminas/deficiencia , Parvalbúminas/genética , Unión Proteica

RESUMEN

The molecular components surrounding a neurone serve as recognition cues for the nerve terminals and glial processes that contact them and the constellations formed by these inputs will therefore be determined by the blend of adhesive and repulsive components therein. Using immunohistochemical methods, we observed that the large extracellular matrix-protein, tenascin-R (Restrictin, J1-160-180, Janusin), associates preferentially with the parvalbumin-positive subpopulation of interneurones within the cerebral cortex. In situ-hybridization indicated that tenascin-R-mRNA was expressed in a subpopulation of nerve cells distinct from that containing parvalbumin, suggesting that this protein's association with the latter is receptor mediated. These nerve cells thus modulate at a distance the composition of the extracellular matrix around parvalbuminneurons.

Asunto(s)

Corteza Cerebral/metabolismo , Matriz Extracelular/metabolismo , Neuronas/metabolismo , Parvalbúminas/metabolismo , Tenascina/biosíntesis , Animales , Corteza Cerebral/química , Corteza Cerebral/ultraestructura , Matriz Extracelular/química , Matriz Extracelular/ultraestructura , Inmunoquímica/métodos , Interneuronas/metabolismo , Interneuronas/ultraestructura , Masculino , Neuronas/química , Neuronas/ultraestructura , Ratas , Tenascina/metabolismo , Tenascina/ultraestructura

RESUMEN

GABAergic (GABA = gamma-aminobutyric acid) neurons from different brain regions contain high levels of parvalbumin, both in their soma and in their neurites. Parvalbumin is a slow Ca(2+) buffer that may affect the amplitude and time course of intracellular Ca(2+) transients in terminals after an action potential, and hence may regulate short-term synaptic plasticity. To test this possibility, we have applied paired-pulse stimulations (with 30- to 300-ms intervals) at GABAergic synapses between interneurons and Purkinje cells, both in wild-type (PV+/+) mice and in parvalbumin knockout (PV-/-) mice. We observed paired-pulse depression in PV+/+ mice, but paired-pulse facilitation in PV-/- mice. In paired recordings of connected interneuron-Purkinje cells, dialysis of the presynaptic interneuron with the slow Ca(2+) buffer EGTA (1 mM) rescues paired-pulse depression in PV-/- mice. These data show that parvalbumin potently modulates short-term synaptic plasticity.

Asunto(s)

Cerebelo/fisiología , Interneuronas/fisiología , Plasticidad Neuronal/fisiología , Parvalbúminas/fisiología , Células de Purkinje/fisiología , Sinapsis/fisiología , 2-Amino-5-fosfonovalerato/farmacología , Animales , Cerebelo/efectos de los fármacos , Ácido Egtácico/farmacología , Estimulación Eléctrica , Electrofisiología/métodos , Antagonistas de Aminoácidos Excitadores/farmacología , Técnicas In Vitro , Interneuronas/efectos de los fármacos , Ratones , Ratones Noqueados , Plasticidad Neuronal/efectos de los fármacos , Parvalbúminas/deficiencia , Parvalbúminas/genética , Células de Purkinje/efectos de los fármacos , Quinoxalinas/farmacología , Sinapsis/efectos de los fármacos

RESUMEN

The effects of tetanus duration on the relaxation rate of extensor digitorum longus (EDL) and flexor digitorum brevis (FDB) muscles were studied in normal (wild-type, WT) and parvalbumin-deficient (PVKO) mice, at 20 C. In EDL of PVKO, the relaxation rate was low and unaffected by tetanus duration (< 3.2 s). In contrast, the relaxation rate of WT muscles decreased when tetanus duration increased from 0.2 to 3.2 s. In WT muscles, fast relaxation recovered as the rest interval increased. Specific effect of parvalbumin was asserted by calculating the difference in relaxation rate between WT and PVKO muscles. For EDL, the rate constant of relaxation slowing was 1.10 s-1 of tetanization; the rate constant of relaxation recovery was 0.05 s-1 of rest. In FDB, the effects of tetanus duration on WT and PVKO muscles were qualitatively similar to those observed in EDL. Relaxation slowing as tetanus duration increases, reflects the progressive saturation of parvalbumin by Ca2+, while recovery as rest interval increases reflects the return to Ca2+-free parvalbumin. At all tetanus durations, relaxation rate still remained slightly faster in WT muscles. This suggested that parvalbumin facilitates calcium traffic from myofibrils to the SR. No difference was found between WT and PVKO muscles for: (i) the expression of the fast isoforms of myosin heavy chains, (ii) the force-velocity relationship and maximal shortening velocity and (iii) the Ca2+-activated ATPase activity from isolated preparations of the sarcoplasmic reticulum (SR).

Asunto(s)

Fibras Musculares de Contracción Rápida/fisiología , Parvalbúminas/deficiencia , Parvalbúminas/genética , Animales , Calcio/fisiología , ATPasas Transportadoras de Calcio/metabolismo , Difusión , Estimulación Eléctrica , Ratones , Ratones Noqueados , Fibras Musculares de Contracción Rápida/metabolismo , Relajación Muscular/fisiología , Miofibrillas/metabolismo , Miofibrillas/fisiología , Miofibrillas/ultraestructura , Cadenas Pesadas de Miosina/metabolismo , Parvalbúminas/metabolismo , Fenotipo , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/fisiología , Retículo Sarcoplasmático/ultraestructura