M. Rosario Sepúlveda

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)

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.

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.

Calmodulin antagonizes amyloid-β peptides-mediated inhibition of brain plasma membrane Ca2+-ATPase

The synaptosomal plasma membrane Ca(2+)-ATPase (PMCA) plays an essential role in regulating intracellular Ca(2+) concentration in brain. We have recently found that PMCA is the only Ca(2+) pump in brain which is inhibited by amyloid-β peptide (Aβ), a neurotoxic peptide implicated in the pathology of Alzheimers disease (AD) [1], but the mechanism of inhibition is lacking. In the present study we have characterized the inhibition of PMCA by Aβ. Results from kinetic assays indicate that Aβ aggregates are more potent inhibitors of PMCA activity than monomers. The inhibitory effect of Aβ could be blocked by pretreating the purified protein with Ca(2+)-calmodulin, the main endogenous activator of PMCA, and the activity of truncated PMCA lacking the calmodulin binding domain was not affected by Aβ. Dot-overlay experiments indicated a physical association of Aβ with PMCA and also with calmodulin. Thus, calmodulin could protect PMCA from inhibition by Aβ by burying exposed sites on PMCA, making them inaccessible to Aβ, and also by direct binding to the peptide. These results suggest a protective role of calmodulin against neuronal Ca(2+) dysregulation by PMCA inhibition induced by Aβ.

Inhibition of PMCA activity by tau as a function of aging and Alzheimer's neuropathology

Ca2+-ATPases are plasma membrane and intracellular membrane transporters that use the energy of ATP hydrolysis to pump cytosolic Ca2+ out of the cell (PMCA) or into internal stores. These pumps are the main high-affinity Ca2+ systems involved in the maintenance of intracellular free Ca2+ at the properly low level in eukaryotic cells. The failure of neurons to keep optimal intracellular Ca2+ concentrations is a common feature of neurodegeneration by aging and aging-linked neuropathologies, such as Alzheimers disease (AD). This disease is characterized by the accumulation of β-amyloid senile plaques and neurofibrillary tangles of tau, a protein that plays a key role in axonal transport. Here we show a novel inhibition of PMCA activity by tau which is concentration-dependent. The extent of inhibition significantly decreases with aging in mice and control human brain membranes, but inhibition profiles were similar in AD-affected brain membrane preparations, independently of age. No significant changes in PMCA expression and localization with aging or neuropathology were found. These results point out a link between Ca2+-transporters, aging and neurodegeneration mediated by tau protein.

Impairment of the activity of the plasma membrane Ca2+-ATPase in Alzheimer's disease

AD (Alzheimers disease) is an age-associated neurodegenerative disorder where the accumulation of neurotoxic Aβ (amyloid β-peptide) in senile plaques is a typical feature. Recent studies point out a relationship between Aβ neurotoxicity and Ca2+ dyshomoeostasis, but the molecular mechanisms involved are still under discussion. The PMCAs (plasma membrane Ca2+-ATPases) are a multi-isoform family of proteins highly expressed in brain that is implicated in the maintenance of low intraneural Ca2+ concentration. Therefore the malfunction of this pump may also be responsible for Ca2+ homoeostasis failure in AD. We have found that the Ca2+-dependence of PMCA activity is affected in human brains diagnosed with AD, being related to the enrichment of Aβ. The peptide produces an inhibitory effect on the activity of PMCA which is isoform-specific, with the greatest inhibition of PMCA4. Besides, cholesterol blocked the inhibitory effect of Aβ, which is consistent with the lack of any Aβ effect on PMCA4 found in cholesterol-enriched lipid rafts isolated from pig brain. These observations suggest that PMCAs are a functional component of the machinery that leads to Ca2+ dysregulation in AD and propose cholesterol enrichment in rafts as a protector of the Aβ-mediated inhibition on PMCA.

Effect of spermine on the activity of synaptosomal plasma membrane Ca2+-ATPase reconstituted in neutral or acidic phospholipids

The activity of purified plasma membrane Ca(2+)-ATPase (PMCA) from pig brain was inhibited by spermine (a naturally occurring and highly abundant polycation in brain). The level of inhibition was dependent on the phospholipid used for reconstitution as well as on the intact or truncated state of the enzyme. An IC(50) value of 12.5 mM spermine was obtained for both, the intact protein plus calmodulin and the trypsin-digested protein, reconstituted in phosphatidylcholine (PC). In the absence of calmodulin the intact Ca(2+)-ATPase gave an IC(50) of 27 mM. This form was more sensitive to spermine inhibition when it was reconstituted with phosphatidylserine (PS), showing an IC(50) value of 2.5 mM spermine. However, the truncated form was less responsive to spermine inhibition, having an IC(50) value of 12.5 mM. Spermine has no effect on the affinity of the PMCA for Ca(2+) or ATP, but its effect on the protein is pH-dependent. It is suggested that spermine could bind to negatively charged residues on the ATPase with different accessibility, depending on the structural rearrangement of the protein. Further, when the protein is reconstituted in PS, spermine also binds to the lipid.