Store-Operated Calcium Entry Inhibition and Plasma Membrane Calcium Pump Upregulation Contribute to the Maintenance of Resting Cytosolic Calcium Concentration in A1-like Astrocytes.

Highly neurotoxic A1-reactive astrocytes have been associated with several human neurodegenerative diseases. Complement protein C3 expression is strongly upregulated in A1 astrocytes, and this protein has been shown to be a specific biomarker of these astrocytes. Several cytokines released in neurodegenerative diseases have been shown to upregulate the production of amyloid ß protein precursor (APP) and neurotoxic amyloid ß (Aß) peptides in reactive astrocytes. Also, aberrant Ca2+ signals have been proposed as a hallmark of astrocyte functional remodeling in Alzheimer's disease mouse models. In this work, we induced the generation of A1-like reactive astrocytes after the co-treatment of U251 human astroglioma cells with a cocktail of the cytokines TNF-α, IL1-α and C1q. These A1-like astrocytes show increased production of APP and Aß peptides compared to untreated U251 cells. Additionally, A1-like astrocytes show a (75 ± 10)% decrease in the Ca2+ stored in the endoplasmic reticulum (ER), (85 ± 10)% attenuation of Ca2+ entry after complete Ca2+ depletion of the ER, and three-fold upregulation of plasma membrane calcium pump expression, with respect to non-treated Control astrocytes. These altered intracellular Ca2+ dynamics allow A1-like astrocytes to efficiently counterbalance the enhanced release of Ca2+ from the ER, preventing a rise in the resting cytosolic Ca2+ concentration.

The Plasma Membrane Ca2+-ATPase, a Molecular Target for Tau-induced Cytosolic Calcium Dysregulation.

Disruption of calcium (Ca2+) homeostasis is emerging as a prevalent feature of aging and aging-associated neurodegenerative diseases, including Alzheimer's disease (AD), the most common type of tauopathy. This disease is characterized by the combined presence of extracellular neuritic plaques composed by amyloid ß-peptides (Aß) and neurofibrillary tangles of tau. The association of calcium dyshomeostasis with Aß has been extensively studied, however its link with tau has been less investigated. Thus, this review will concentrate on the functional link between tau and the plasma membrane Ca2+ pump (PMCA) and other membrane proteins involved in the regulation of intracellular calcium and/or its association with neurodegeneration.

Inhibition of SERCA and PMCA Ca2+-ATPase activities by polyoxotungstates.

Plasma membrane calcium ATPases (PMCA) and sarco(endo) reticulum calcium ATPases (SERCA) are key proteins in the maintenance of calcium homeostasis. Herein, we compare for the first time the inhibition of SERCA and PMCA calcium pumps by several polyoxotungstates (POTs), namely by Wells-Dawson phosphotungstate anions [P2W18O62]6- (intact, {P2W18}), [P2W17O61]10- (monolacunary, {P2W17}), [P2W15O56]12- (trilacunary, {P2W15}), [H2P2W12O48]12- (hexalacunary, {P2W12}), [H3P2W15V3O62]6- (trivanadium-substituted, {P2W15V3}) and by Preyssler-type anion [NaP5W30O110]14- ({P5W30}). The speciation in the solutions of tested POTs was investigated by 31P and 51V NMR spectroscopy. The tested POTs inhibited SERCA Ca2+-ATPase activity, whereby the Preyssler POT showed the strongest effect, with an IC50 value of 0.37 µM. For {P2W17} and {P2W15V3} higher IC50 values were determined: 0.72 and 0.95 µM, respectively. The studied POTs showed to be more potent inhibitors of PMCA Ca2+-ATPase activity, with lower IC50 values for {P2W17}, {P5W30} and {P2W15V3}.

Sorcin Activates the Brain PMCA and Blocks the Inhibitory Effects of Molecular Markers of Alzheimer's Disease on the Pump Activity.

Since dysregulation of intracellular calcium (Ca2+) levels is a common occurrence in neurodegenerative diseases, including Alzheimer's disease (AD), the study of proteins that can correct neuronal Ca2+ dysregulation is of great interest. In previous work, we have shown that plasma membrane Ca2+-ATPase (PMCA), a high-affinity Ca2+ pump, is functionally impaired in AD and is inhibited by amyloid-ß peptide (Aß) and tau, two key components of pathological AD hallmarks. On the other hand, sorcin is a Ca2+-binding protein highly expressed in the brain, although its mechanism of action is far from being clear. Sorcin has been shown to interact with the intracellular sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), and other modulators of intracellular Ca2+ signaling, such as the ryanodine receptor or presenilin 2, which is closely associated with AD. The present work focuses on sorcin in search of new regulators of PMCA and antagonists of Aß and tau toxicity. Results show sorcin as an activator of PMCA, which also prevents the inhibitory effects of Aß and tau on the pump, and counteracts the neurotoxicity of Aß and tau by interacting with them.

Methylene Blue Blocks and Reverses the Inhibitory Effect of Tau on PMCA Function.

Methylene blue (MB) is a synthetic phenothiazine dye that, in the last years, has generated much debate about whether it could be a useful therapeutic drug for tau-related pathologies, such as Alzheimer's disease (AD). However, the molecular mechanism of action is far from clear. Recently we reported that MB activates the plasma membrane Ca2+-ATPase (PMCA) in membranes from human and pig tissues and from cells cultures, and that it could protect against inactivation of PMCA by amyloid ß-peptide (Aß). The purpose of the present study is to further examine whether the MB could also modulate the inhibitory effect of tau, another key molecular marker of AD, on PMCA activity. By using kinetic assays in membranes from several tissues and cell cultures, we found that this phenothiazine was able to block and even to completely reverse the inhibitory effect of tau on PMCA. The results of this work point out that MB could mediate the toxic effect of tau related to the deregulation of calcium homeostasis by blocking the impairment of PMCA activity by tau. We then could conclude that MB could interfere with the toxic effects of tau by restoring the function of PMCA pump as a fine tuner of calcium homeostasis.

Overview of bioremediation with technology assessment and emphasis on fungal bioremediation of oil contaminated soils.

Environmental contamination is a problem that requires sustainable solutions. Bioremediation technologies have been developed in the last decades and are increasingly used to mitigate environmental accidents and systematic contaminations. A review of bioremediation technologies, based on published article and patent documents, is presented for different types of contaminated matrices, bioremediation agents and contaminants. The worldwide database of the European Patent Office was searched using radicals of keyword as well as the International Patent Classification (IPC) to identify patents in our areas of concern. Technological domains, annual filing volume, legal status, assignee countries and development collaborations are presented and examples are discussed. The total number of patents is compared with the total number of articles. A SWOT analysis for bioremediation technologies is presented. The technologies for water (53%), soils (36%), and sludges (11%) are growing yearly at nearly constant rates. The bioremediation agents are predominantly bacteria (57%), enzymes (19%), fungi (13%), algae (6%), plants (4%) and protozoa. The major contaminants are oils (38%), followed by metals (21%), organic waste (21%), polymers (10%), food (5%), cellulose (5%) and biodiesel. Most of the patents are generally originated from China and United States of America. The soils bioremediation technology of oil is centered on bacteria usage (about two thirds of the articles and patents), being fungi a technology with critical mass and high growth potential. A recent trend in oil bioremediation of soils is the combination of bioremediation agents (fungi and bacteria) in the same process, thus making the process more robust to environment changes.

STIM1 deficiency is linked to Alzheimer's disease and triggers cell death in SH-SY5Y cells by upregulation of L-type voltage-operated Ca2+ entry.

STIM1 is an endoplasmic reticulum protein with a role in Ca2+ mobilization and signaling. As a sensor of intraluminal Ca2+ levels, STIM1 modulates plasma membrane Ca2+ channels to regulate Ca2+ entry. In neuroblastoma SH-SY5Y cells and in familial Alzheimer's disease patient skin fibroblasts, STIM1 is cleaved at the transmembrane domain by the presenilin-1-associated γ-secretase, leading to dysregulation of Ca2+ homeostasis. In this report, we investigated expression levels of STIM1 in brain tissues (medium frontal gyrus) of pathologically confirmed Alzheimer's disease patients, and observed that STIM1 protein expression level decreased with the progression of neurodegeneration. To study the role of STIM1 in neurodegeneration, a strategy was designed to knock-out the expression of STIM1 gene in the SH-SY5Y neuroblastoma cell line by CRISPR/Cas9-mediated genome editing, as an in vitro model to examine the phenotype of STIM1-deficient neuronal cells. It was proved that, while STIM1 is not required for the differentiation of SH-SY5Y cells, it is absolutely essential for cell survival in differentiating cells. Differentiated STIM1-KO cells showed a significant decrease of mitochondrial respiratory chain complex I activity, mitochondrial inner membrane depolarization, reduced mitochondrial free Ca2+ concentration, and higher levels of senescence as compared with wild-type cells. In parallel, STIM1-KO cells showed a potentiated Ca2+ entry in response to depolarization, which was sensitive to nifedipine, pointing to L-type voltage-operated Ca2+ channels as mediators of the upregulated Ca2+ entry. The stable knocking-down of CACNA1C transcripts restored mitochondrial function, increased mitochondrial Ca2+ levels, and dropped senescence to basal levels, demonstrating the essential role of the upregulation of voltage-operated Ca2+ entry through Cav1.2 channels in STIM1-deficient SH-SY5Y cell death. KEY MESSAGES: STIM1 protein expression decreases with the progression of neurodegeneration in Alzheimer's disease. STIM1 is essential for cell viability in differentiated SH-SY5Y cells. STIM1 deficiency triggers voltage-regulated Ca2+ entry-dependent cell death. Mitochondrial dysfunction and senescence are features of STIM1-deficient differentiated cells.

Methylene blue activates the PMCA activity and cross-interacts with amyloid ß-peptide, blocking Aß-mediated PMCA inhibition.

The phenothiazine methylene blue (MB) is attracting increasing attention because it seems to have beneficial effects in the pathogenesis of Alzheimer's disease (AD). Among other factors, the presence of neuritic plaques of amyloid-ß peptide (Aß) aggregates, neurofibrilar tangles of tau and perturbation of cytosolic Ca2+ are important players of the disease. It has been proposed that MB decreases the formation of neuritic plaques due to Aß aggregation. However, the molecular mechanism underlying this effect is far from clear. In this work, we show that MB stimulates the Ca2+-ATPase activity of the plasma membrane Ca2+-ATPase (PMCA) in human tissues from AD-affected brain and age-matched controls and also from pig brain and cell cultures. In addition, MB prevents and even blocks the inhibitory effect of Aß on PMCA activity. Functional analysis with mutants and fluorescence experiments strongly suggest that MB binds to PMCA, at the C-terminal tail, in a site located close to the last transmembrane helix and also that MB binds to the peptide. Besides, Aß increases PMCA affinity for MB. These results point out a novel molecular basis of MB action on Aß and PMCA as mediator of its beneficial effect on AD.

Functional interplay between plasma membrane Ca2+-ATPase, amyloid ß-peptide and tau.

It is well known that dysregulation of Ca2+ homeostasis is involved in Alzheimers disease (AD), a neurodegenerative disorder characterized by the presence of toxic aggregates of amyloid ß-peptide (Aß) and neurofibrillary tangles of tau. Alteration of calcium signaling has been linked to Aß and tau pathologies, although the understanding of underlying molecular and cellular mechanisms is far from clear. This review summarizes the functional inhibition of plasma membrane Ca2+-ATPase (PMCA) by Aß and tau, and its modulation by calmodulin and the ionic nature of phospholipids. The data obtained until now in our laboratory suggest that PMCA injury linked to Aß and tau can be significantly involved in the cascade of events leading to intracellular calcium overload associated to AD.

Phospholipids and calmodulin modulate the inhibition of PMCA activity by tau.

The disruption of Ca2+ signaling in neurons, together with a failure to keep optimal intracellular Ca2+ concentrations, have been proposed as significant factors for neuronal dysfunction in the Ca2+ hypothesis of Alzheimer's disease (AD). Tau is a protein that plays an essential role in axonal transport and can form abnormal structures such as neurofibrillary tangles that constitute one of the hallmarks of AD. We have recently shown that plasma membrane Ca2+-ATPase (PMCA), a key enzyme in the maintenance of optimal cytosolic Ca2+ levels in cells, is inhibited by tau in membrane vesicles. In the present study we show that tau inhibits synaptosomal PMCA purified from pig cerebrum, and reconstituted in phosphatidylserine-containing lipid bilayers, with a Ki value of 1.5±0.2nM tau. Noteworthy, the inhibitory effect of tau is dependent on the charge of the phospholipid used for PMCA reconstitution. In addition, nanomolar concentrations of calmodulin, the major endogenous activator of PMCA, protects against inhibition of the Ca2+-ATPase activity by tau. Our results in a cellular model such as SH-SY5Y human neuroblastoma cells yielded an inhibition of PMCA by nanomolar tau concentrations and protection by calmodulin against this inhibition similar to those obtained with purified synaptosomal PMCA. Functional studies were also performed with native and truncated versions of human cerebral PMCA4b, an isoform that has been showed to be functionally regulated by amyloid peptides, whose aggregates constitutes another hallmark of AD. Kinetic assays point out that tau binds to the C-terminal tail of PMCA, at a site distinct but close to the calmodulin binding domain. In conclusion, PMCA can be seen as a molecular target for tau-induced cytosolic calcium dysregulation in synaptic terminals. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

An improved method for expression and purification of functional human Ca(2+) transporter PMCA4b in Saccharomyces cerevisiae.

Human plasma membrane calcium ATPases (PMCAs) are highly regulated transporters responsible for the extrusion of calcium out of the cell. Since calcium homeostasis is implicated in several diseases and neurodegenerative disorders, understanding PMCAs activity is crucial. One of the major hindrances is the availability of these proteins for functional and structural analysis. Here, using the yeast Saccharomyces cerevisiae system, we show a new and enhanced method for the expression of the full-length human PMCA isoform 4b (hPMCA4b) and a truncated form lacking its auto-inhibitory domain. We have also improved a method for the purification of the native isoform by calmodulin-agarose affinity chromatography, and developed a new method to purify the truncated isoform by glutathione-Sepharose affinity chromatography. One of the most relevant features of this work is that, when compared to PMCAs purification from pig brain, our method provides a pure single isoform instead of a mixture of isoforms, essential for fine-tuning the activity of PMCA4b. Another relevant feature is that the method described in this work has a superior yield of protein than previously established methods to purify PMCA proteins expressed in yeasts.

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 Alzheimer's disease (AD). This disease is characterized by the accumulation of ß-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.

Presynaptic control of glycine transporter 2 (GlyT2) by physical and functional association with plasma membrane Ca2+-ATPase (PMCA) and Na+-Ca2+ exchanger (NCX).

Fast inhibitory glycinergic transmission occurs in spinal cord, brainstem, and retina to modulate the processing of motor and sensory information. After synaptic vesicle fusion, glycine is recovered back to the presynaptic terminal by the neuronal glycine transporter 2 (GlyT2) to maintain quantal glycine content in synaptic vesicles. The loss of presynaptic GlyT2 drastically impairs the refilling of glycinergic synaptic vesicles and severely disrupts neurotransmission. Indeed, mutations in the gene encoding GlyT2 are the main presynaptic cause of hyperekplexia in humans. Here, we show a novel endogenous regulatory mechanism that can modulate GlyT2 activity based on a compartmentalized interaction between GlyT2, neuronal plasma membrane Ca(2+)-ATPase (PMCA) isoforms 2 and 3, and Na(+)/Ca(2+)-exchanger 1 (NCX1). This GlyT2·PMCA2,3·NCX1 complex is found in lipid raft subdomains where GlyT2 has been previously found to be fully active. We show that endogenous PMCA and NCX activities are necessary for GlyT2 activity and that this modulation depends on lipid raft integrity. Besides, we propose a model in which GlyT2·PMCA2-3·NCX complex would help Na(+)/K(+)-ATPase in controlling local Na(+) increases derived from GlyT2 activity after neurotransmitter release.

High levels of Mn²âº inhibit secretory pathway Ca²âº/Mn²âº-ATPase (SPCA) activity and cause Golgi fragmentation in neurons and glia.

Excess Mn(2+) in humans causes a neurological disorder known as manganism, which shares symptoms with Parkinson's disease. However, the cellular mechanisms underlying Mn(2+) -neurotoxicity and the involvement of Mn(2+) -transporters in cellular homeostasis and repair are poorly understood and require further investigation. In this work, we have analyzed the effect of Mn(2+) on neurons and glia from mice in primary cultures. Mn(2+) overload compromised survival of both cell types, specifically affecting cellular integrity and Golgi organization, where the secretory pathway Ca(2+) /Mn(2+) -ATPase is localized. This ATP-driven Mn(2+) transporter might take part in Mn(2+) accumulation/detoxification at low loads of Mn(2+) , but its ATPase activity is inhibited at high concentration of Mn(2+) . Glial cells appear to be significantly more resistant to this toxicity than neurons and their presence in cocultures provided some protection to neurons against degeneration induced by Mn(2+) . Interestingly, the Mn(2+) toxicity was partially reversed upon Mn(2+) removal by wash out or by the addition of EDTA as a chelating agent, in particular in glial cells. These studies provide data on Mn(2+) neurotoxicity and may contribute to explore new therapeutic approaches for reducing Mn(2+) poisoning.

Calmodulin antagonizes amyloid-ß peptides-mediated inhibition of brain plasma membrane Ca(2+)-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-ß peptide (Aß), a neurotoxic peptide implicated in the pathology of Alzheimer's disease (AD) [1], but the mechanism of inhibition is lacking. In the present study we have characterized the inhibition of PMCA by Aß. Results from kinetic assays indicate that Aß aggregates are more potent inhibitors of PMCA activity than monomers. The inhibitory effect of Aß 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 Aß. Dot-overlay experiments indicated a physical association of Aß with PMCA and also with calmodulin. Thus, calmodulin could protect PMCA from inhibition by Aß by burying exposed sites on PMCA, making them inaccessible to Aß, 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 Aß.

Impairment of the activity of the plasma membrane Ca²âº-ATPase in Alzheimer's disease.

AD (Alzheimer's disease) is an age-associated neurodegenerative disorder where the accumulation of neurotoxic Aß (amyloid ß-peptide) in senile plaques is a typical feature. Recent studies point out a relationship between Aß 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 Aß. 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 Aß, which is consistent with the lack of any Aß 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 Aß-mediated inhibition on PMCA.

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 Ca(2+) ions and the secretory pathway play pivotal roles. The secretory pathway Ca(2+)-ATPase isoform 1 (SPCA1) is found in the Golgi apparatus where it is actively involved in the transport of Ca(2+) or Mn(2+) 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. Ca(2+) imaging assays showed that the lack of SPCA1 impaired Golgi Ca(2+) 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 Ca(2+) homeostasis and/or the altered targeting of organellar proteins under conditions of SPCA1 knockdown highlight the importance of SPCA1 function for normal neural differentiation.

Ontogeny of ATP hydrolysis and isoform expression of the plasma membrane Ca(2+)-ATPase in mouse brain.

BACKGROUND: Plasma 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. RESULTS: PMCA 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. CONCLUSION: These 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.

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

High-affinity Ca(2+) transport ATPases play a crucial role in controlling cytosolic Ca(2+). The amyloid beta-peptide (Abeta) is a neurotoxic agent found in affected neurons in Alzheimer's disease (AD) that has been implicated in dysregulation of Ca(2+) homeostasis. Using kinetic assays, we have shown that the Ca(2+) dependencies of intracellular Ca(2+)-ATPase (SERCA and SPCA) activity are the same in human AD and normal brain but that of plasma membrane Ca(2+)-ATPase (PMCA) is different. The addition of Abeta to normal brain decreases the PMCA activity measured at pCa 5.5, resulting in the same Ca(2+)dependency as that seen in AD brain, whereas the addition of Abeta to AD brain has no effect on PMCA activity. Abeta also decreases the activity of PMCA purified from pig cerebrum, the effect being isoform specific. The level of inhibition of purified PMCA caused by Abeta is reduced by cholesterol, and the level of inhibition of PMCA activity by Abeta in the raft fraction of pig synaptosomal membranes is lower than for the nonraft fraction. We conclude that the effect of Abeta on PMCA activity could be important in amyloid toxicity, resulting in cytoplasmic Ca(2+) dysregulation and could explain the different Ca(2+) dependencies of PMCA activity observed in normal and AD brain.

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.