Ana M. Mata

Reversible inactivation of Saccharomyces cerevisiae glutathione reductase under reducing conditions

Glutathione reductase from Saccharomyces cerevisiae was rapidly inactivated following aerobic incubation with NADPH, NADH, and several other reductants, in a time- and temperature-dependent process. The inactivation had already reached 50% when the NADPH concentration reached that of the glutathione reductase subunit. The inactivation was very marked at pH values below 5.5 and over 7, while only a slight activity decrease was noticed at pH values between these two values. After elimination of excess NADPH the enzyme remained inactive for at least 4 h. The enzyme was protected against redox inactivation by low concentrations of GSSG, ferricyanide, GSH, or dithiothreitol, and high concentrations of NAD(P)+; oxidized glutathione effectively protected the enzyme at concentrations even lower than GSH. The inactive enzyme was efficiently reactivated after incubation with GSSG, ferricyanide, GSH, or dithiothreitol, whether NADPH was present or not. The reactivation with GSH was rapid even at 0 degree C, whereas the optimum temperature for reactivation with GSSG was 30 degrees C. A tentative model for the redox interconversion, involving an erroneous intramolecular disulfide bridge, is put forward.

The Plasma Membrane Ca2+-ATPase Isoform 4 Is Localized in Lipid Rafts of Cerebellum Synaptic Plasma Membranes

Here we describe the association of the synaptosomal plasma membrane Ca2+-ATPase (PMCA) from pig cerebellum with cholesterol/sphingomyelin-rich membrane domains (rafts). The PMCA4 was localized exclusively in rafts prepared by floatation in Nycodenz density gradients of ice-cold Brij 96 extracts. This was corroborated by its colocalization with the raft markers cholesterol, ganglioside GM1, and PrPC. The remaining PMCA isoforms were found in the detergent-soluble fractions, with the majority of the membrane proteins. Activity assays confirmed the bimodal distribution of the PMCA isoforms in the density gradient, with a lower activity for PMCA4 and greater stimulation by calmodulin than for the other isoforms. By providing an ordered membrane microenvironment, lipid rafts may contribute to the interaction of PMCA4 with proteins involved in Ca2+ signaling at discrete functional positions on the synaptic nerve terminals.

Altered Ca2+ dependence of synaptosomal plasma membrane Ca2+-ATPase in human brain affected by Alzheimer's disease.

High‐affinity Ca2+ transport ATPases play a crucial role in controlling cytosolic Ca2+. The amyloid β‐peptide (Aβ) is a neurotoxic agent found in affected neurons in Alzheimers disease (AD) that has been implicated in dysregulation of Ca2+ homeostasis. Using kinetic assays, we have shown that the Ca2+ dependencies of intracellular Ca2+‐ATPase (SERCA and SPCA) activity are the same in human AD and normal brain but that of plasma membrane Ca2+‐ATPase (PMCA) is different. The addition of Aβ to normal brain decreases the PMCA activity measured at pCa 5.5, resulting in the same Ca2+ dependency as that seen in AD brain, whereas the addition of Aβ to AD brain has no effect on PMCA activity. Aβ also decreases the activity of PMCA purified from pig cerebrum, the effect being isoform specific. The level of inhibition of purified PMCA caused by Aβ is reduced by cholesterol, and the level of inhibition of PMCA activity by Aβ in the raft fraction of pig synaptosomal membranes is lower than for the nonraft fraction. We conclude that the effect of Aβ on PMCA activity could be important in amyloid toxicity, resulting in cytoplasmic Ca2+ dysregulation and could explain the different Ca2+ dependencies of PMCA activity observed in normal and AD brain.—Berrocal, M.,Marcos, D., Sepulveda, M.R., Perez, M., Avila, J., Mata, A.M. Altered Ca2+ dependence of synaptosomal plasma membrane Ca2+‐ATPase in human brain affected by Alzheimers disease. FASEB J. 23, 1826–1834 (2009)

Silencing the SPCA1 (Secretory Pathway Ca2+-ATPase Isoform 1) Impairs Ca2+ Homeostasis in the Golgi and Disturbs Neural Polarity

Neural cell differentiation involves a complex regulatory signal transduction network in which Ca2+ ions and the secretory pathway play pivotal roles. The secretory pathway Ca2+-ATPase isoform 1 (SPCA1) is found in the Golgi apparatus where it is actively involved in the transport of Ca2+ or Mn2+ from the cytosol to the Golgi lumen. Its expression during brain development in different types of neurons has been documented recently, which raises the possibility that SPCA1 contributes to neuronal differentiation. In the present study, we investigated the potential impact of SPCA1 on neuronal polarization both in a cell line and in primary neuronal culture. In N2a neuroblastoma cells, SPCA1 was immunocytochemically localized in the juxtanuclear Golgi. Knockdown of SPCA1 by RNA interference markedly delayed the differentiation in these cells. The cells retarded in differentiation showed increased numbers of neurites of reduced length compared with control cells. Ca2+ imaging assays showed that the lack of SPCA1 impaired Golgi Ca2+ homeostasis and resulted in disturbed trafficking of different classes of proteins including normally Golgi-localized cameleon GT-YC3.3, bearing a Golgi-specific galactosyltransferase N terminus, and a normally plasma membrane-targeted, glycosyl phosphatidyl inositol-anchored cyan fluorescent protein construct. Also in hippocampal primary neurons, which showed a differential distribution of SPCA1 expression in Golgi stacks depending on differentiation stage, partial silencing of SPCA1 resulted in delayed differentiation, whereas total suppression drastically affected the cell survival. The disturbed overall cellular Ca2+ homeostasis and/or the altered targeting of organellar proteins under conditions of SPCA1 knockdown highlight the importance of SPCA1 function for normal neural differentiation.

Activity and localization of the secretory pathway Ca2+-ATPase isoform 1 (SPCA1) in different areas of the mouse brain during postnatal development.

Ca2+ and Mn2+ play an important role in many events in the nervous system, ranging from neural morphogenesis to neurodegeneration. As part of the homeostatic control of these ions, the Secretory Pathway Ca2+-ATPase isoform 1 (SPCA1) mediates the accumulation of Ca2+ or Mn2+ with high affinity into Golgi reservoirs. This SPCA1 represents a relatively recently characterized P-type pump that is highly expressed in nervous tissue, but information on its involvement in neural maturation is currently lacking. In this study, we have analyzed the expression and distribution of the SPCA1 pump in mouse brain during postnatal development. RT-PCR and Western blot assays showed that SPCA1 is particularly highly expressed at nearly constant levels during this entire period of development in cortex, hippocampus, and cerebellum. In spite of the apparently unchanged expression levels, functional assays showed that SPCA-associated Ca2+-ATPase activity increased with the stage of development in these areas. Immunohistochemical studies pointed to SPCA1 localization in Golgi stacks of the soma and the initial part of primary dendritic trunk in main cortical, hippocampal and cerebellar neurons from the earliest postnatal stages. This suggests a potential role in intracellular signaling and in Golgi secretory processes involved in dendritic growth and in functional maturation of the mouse nervous system.

Localization of endoplasmic reticulum and plasma membrane Ca2+-ATPases in subcellular fractions and sections of pig cerebellum.

Subcellular fractions and sections of the cerebellum were analysed to evaluate the relative activity and distribution of organellar and plasma membrane Ca2+‐ATPases (SERCA and PMCA). Western blot analysis of the fractions with IID8 or Y/1F4 SERCA‐specific antibodies or else with 5F10 or pbPMCA antibodies, specific to PMCA pump, revealed a major content of SERCA protein in microsomes and of PMCA protein in plasma membrane vesicles. The Ca2+‐ATPase activity of microsomes was more sensitive to thapsigargin, a SERCA‐specific inhibitor, whereas the activity of the plasma membrane vesicle fraction was inhibited more by vanadate, a blocker of PMCA activity. The SERCA and PMCA distribution analysed in cerebellar sections revealed IID8 antibody reactions in Purkinje cell cytoplasm, granule cells and cerebellar glomeruli. Y/1F4 gave immunostaining in Purkinje cells, molecular layer interneurons (basket and stellate cells) and glomeruli, but granule cells were not labelled. The 5F10 antibody reacted with Purkinje cells, including their dendritic spines, as well as cerebellar glomeruli, whereas the pbPMCA antibody labelled several processes in all three layers and some synaptic interaction sites. The differential content and localization of the two types of Ca2+ pumps in specific neuronal areas of pig cerebellum indicate precise Ca2+ requirements of specific cellular regions.

Evaluation of manganese uptake and toxicity in mouse brain during continuous MnCl2 administration using osmotic pumps.

Manganese is a vital element and cofactor of many key enzymes, but it is toxic at high levels, causing pronounced disturbances in the mammalian brain. Magnetic resonance imaging (MRI) studies using manganese ions as a paramagnetic contrast agent are often limited by the neurotoxicity of Mn(2+) . In this work, we have explored a new in vivo model to study Mn(2+) uptake, distribution and neurotoxicity in mice by subcutaneous implantation of mini-osmotic pumps delivering MnCl(2) continuously for 21 days. Fractionated injections can reduce the toxicity; however, constant administration at very low doses using osmotic pumps caused a substantial effect on the T(1) contrast in MRI while reducing toxicity. Manganese-enhanced MRI documented fast but reversible Mn(2+) deposition largely in glomerular and mitral cell layers of the olfactory bulb, in the CA3 area of the hippocampus, and in the gray matter of the cerebellum. Mn(2+) accumulated as early as the first days after implantation, with a fast dispersal 9 days after stopping a 12-days Mn(2+) exposure. Prominent Mn(2+) accumulation was also seen in salivary glands and in the endocrine thyroid and posterior pituitary gland. These structures with enhanced Mn(2+) accumulation correlated well with those showing high expression of the secretory pathway Ca(2+) /Mn(2+) -ATPase (SPCA1), i.e. a transporter that could take part in Mn(2+) detoxification. Our new experimental model for continuous low-dosage administration of Mn(2+) is an easy alternative for enhancing Mn(2+) -based contrast in MEMRI studies, and might provide insight into the etiology of neuropathologies resulting from chronic Mn(2+) exposure in vivo.

Ontogeny of ATP hydrolysis and isoform expression of the Plasma Membrane Ca2+-ATPase in mouse brain

BackgroundPlasma membrane Ca2+-ATPases (PMCAs) are high affinity Ca2+ transporters actively involved in intracellular Ca2+ homeostasis. Considering the critical role of Ca2+ signalling in neuronal development and plasticity, we have analyzed PMCA-mediated Ca2+-ATPase activity and PMCA-isoform content in membranes from mouse cortex, hippocampus and cerebellum during postnatal development.ResultsPMCA activity was detected from birth, with a faster evolution in cortex than in hippocampus and cerebellum. Western blots revealed the presence of the four isoforms in all regions, with similar increase in their expression patterns as those seen for the activity profile. Immunohistochemistry assays in cortex and hippocampus showed co-expression of all isoforms in the neuropil associated with synapses and in the plasma membrane of pyramidal cells soma, while cerebellum showed a more isoform-specific distribution pattern in Purkinje cells.ConclusionThese results show an upregulation of PMCA activity and PMCA isoforms expression during brain development in mouse, with specific localizations mainly in cerebellum. Overall, our findings support a close relationship between the ontogeny of PMCA isoforms and specific requirements of Ca2+ during development of different brain areas.

A developmental profile of the levels of calcium pumps in chick cerebellum

The functional expression and distribution of intracellular ATPase (sarco(endo)plasmic reticulum Ca2+‐ATPase: SERCA) and plasma membrane Ca2+‐ATPase (PMCA) was analyzed in the developing chick cerebellum. The activity and Ca2+ uptake increase with development for both ATPases. However, the protein content increases with the stage of development only for SERCA, remaining constant for PMCA. Immunohistochemical assays showed that the ontogenesis of these ATPases goes along with definite stages of cerebellum histogenesis, and is complete at hatching. The SERCA is mainly distributed in Purkinje neurons, whereas the PMCA seems to be expressed initially in climbing fibers, shifting to soma and spiny branchlets of Purkinje cells at late embryonic stages. Granule cells express both ATPases according to their degree of maturity, whereas only PMCA is present in cerebellar glomeruli. These pumps are present in deep nuclei and the choroid plexus, although in this latter tissue their expression declines with development. The spatio‐temporal distribution of SERCA and PMCA must be closely related to their association with the development of specific cells and processes of the chick cerebellum.

Developmental distribution of plasma membrane Ca2+-ATPase isoforms in chick cerebellum

The plasma membrane Ca2+‐ATPase (PMCA) is highly expressed in the nervous system, but little information is available about its implication in neuronal development. We have analyzed the expression and localization of different isoforms of PMCA in membrane vesicles and sections of chick cerebellum from embryonic day 10 to hatching. We found that the relative amount of each PMCA isoform and their spatiotemporal distribution in the cerebellum are directly linked to precise cellular types during the cerebellar maturation, even in a non‐neural tissue as choroid plexus. Purkinje cells contain the highest diversity of PMCA isoforms of the cerebellar cortex since the moment of its morphogenesis. From embryonic day 15, the PMCA2 was highly expressed in the whole Purkinje cell, while PMCAs 1 and 3 had a more restricted distribution in the soma and dendritic branches, and these distributions were evolving according with cell maturation. Other cellular types seem to contain a specific combination of isoforms, but with a well‐defined distribution pattern at late moments of development. Thus, PMCAs 1 and 3 were located in the soma of molecular layer interneurons, and only the PMCA2 was observed in granule cells at hatching. Furthermore, PMCA isoforms are also expressed in cellular compartments characterized by a high amount of synapses, suggesting a key role of these proteins in synaptogenesis and in the maturation of neuronal electrophysiological properties. Developmental Dynamics 236:1227–1236, 2007.