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.

High affinity binding of amyloid ß-peptide to calmodulin: Structural and functional implications.

Amyloid ß-peptides (Aß) are a major hallmark of Alzheimer's disease (AD) and their neurotoxicity develop with cytosolic calcium dysregulation. On the other hand, calmodulin (CaM), a protein which plays a major multifunctional role in neuronal calcium signaling, has been shown to be involved in the regulation of non-amyloidogenic processing of amyloid ß precursor protein (APP). Using fluorescent 6-bromoacetyl-2-dimethylaminonaphthalene derivatives of CaM, Badan-CaM, and human amyloid ß(1-42) HiLyte™-Fluor555, we show in this work that Aß binds with high affinity to CaM through the neurotoxic Aß25-35 domain. In addition, the affinity of Aß for calcium-saturated CaM conformation is approximately 20-fold higher than for CaM conformation in the absence of calcium (apo-CaM). Moreover, the value of Kd of 0.98 ± 0.11 nM obtained for Aß1-42 dissociation from CaM saturated by calcium points out that CaM is one of the cellular targets with highest affinity for neurotoxic Aß peptides. A major functional consequence of Aß-CaM interaction is that it slowdowns Aß fibrillation. The novel and high affinity interaction between calmodulin and Aß shown in this work opens a yet-unexplored gateway to further understand the neurotoxic effect of Aß in different neural cells and also to address the potential of calmodulin and calmodulin-derived peptides as therapeutic agents in 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.

Dependence of Saccharomyces cerevisiae Golgi functions on V-ATPase activity.

The V-ATPase of Saccharomyces cerevisiae is an ATP-dependent proton pump responsible for acidification of the vacuole and other internal compartments including the whole secretory pathway. We have studied the behavior of several glycoprotein processing reactions occurring in different Golgi compartments of representative vmaΔ mutants. We found that outer chain initiation is not altered in the mutants while mannosylphosphate transfer, α(1,3)-linked mannoses addition, and α factor maturation seem to be affected. The results suggest a gradation in the dependence of Golgi functions on V-ATPase activity, from early Golgi (unaffected) to late Golgi (significantly reduced). These findings are in agreement with the internal pH of Golgi cisternae measured in mammalian cells, which is more acidic in the late region. The mutant defects can be partially restored by buffering the external medium to pH 6.0, which supports the existence of a mechanism that, in the absence of a functional V-ATPase, could contribute to pH regulation at least in the late Golgi.

Standard YPD, even supplemented with extra nutrients, does not always compensate growth defects of Saccharomyces cerevisiae auxotrophic strains.

Conventional complex media are routinely used to grow auxotrophic strains under the assumption that they can compensate the latter's nutritional deficiencies. We here demonstrate that this is not always true. This study compares the growth parameters of Saccharomyces cerevisiae (S288C) and its derived auxotrophic strains FY1679-14C and BY4741 in synthetic minimal medium (SD), standard YPD medium from two of the most commonly used suppliers, or modified YPD medium. Maximum specific growth rates of auxotrophic strains were slightly lower than the prototrophic case in all growth conditions tested. Also, the biomass production of auxotrophic strains in synthetic medium was slightly less than the prototrophic case. However in both of the two standard YPD media used, the biomass production of both auxotrophic strains was markedly lower than that of the prototrophic one. The extent of the differences depended on the medium used. Indeed in one of the two YPD media, the lower biomass production of auxotrophic strains was evident even at the diauxic shift. Uracil seems to be the main limiting growth factor for both auxotrophic strains growing in the two standard YPD medium tested. No YPD media or specific supplement was able to compensate for the effect of the auxotrophic mutations in the multiple auxotrophic marker strain BY4741. The fact that auxotrophic strains grew poorly on YPD when compared to their prototrophic counterpart indicates that standard YPD medium is not sufficient to overcome the effect of auxotrophic mutations.

Identification of the MNN3 gene of Saccharomyces cerevisiae.

The MNN3 gene of Saccharomyces cerevisiae has been identified as a synonym of VPS74. We have compared phenotype characteristics of the original mnn3 mutant, including low dye binding phenotype, size of external invertase, clump formation, and sodium orthovanadate resistance and found these to be identical to those shown by vps74Δ. Mating of both haploid strains resulted in non-complementation of mutant phenotypes. Finally, a vector containing wild-type VPS74 complemented the defects of both vps74Δ and mnn3. This work completes the identification of the entire collection of genes that are defective in mnn mutants. In addition, we have identified the mnn3 mutation by sequencing the VPS74 gene from the original mnn3 strain. We found a single amino acid change of Arg97 to Cys. This unique alteration seems to be sufficient to account for the phenotype of mnn3.

Genome-wide expression profile of the mnn2 Delta mutant of Saccharomyces cerevisiae.

The MNN2 gene of S. cerevisiae encodes an alpha (1,2) mannosyl transferase required for branching the outer chain of N-linked oligosaccharides (Rayner J.C. and Munro S. 1998. J. Biol. Chem. 273: 26836-26843) and it also seems to have some effect on the transfer of mannosyl phosphate groups to the inner core (Olivero I. et al. 2000. FEBS Lett. 475: 111-116). In order to reveal possible interactions of MNN2 expression with other cellular pathways, we analyzed the transcriptome of the deletion mutant S. cerevisiae mnn2 Delta using cDNA microarrays. We found 151 genes that showed an altered expression level of > or =2-fold, 58 of them up-regulated and 93 down-regulated. Quite a high proportion of these genes (29%) encode unclassified proteins. In contrast to other defects affecting the integrity of the cell wall, deletion of MNN2 does not stimulate the expression of any of the genes included in the previously defined 'cell wall compensatory cluster' (Lagorce et al. 2003. J. Biol. Chem. 278: 20345-20357). We also found that 15% of the selected genes are related to central metabolic pathways. In addition, the mnn2 Delta strain seems to have a certain level of stimulation of DNA processing reactions while some genes involved in intracellular transport pathways are under-regulated.

A genome-wide screen for Saccharomyces cerevisiae nonessential genes involved in mannosyl phosphate transfer to mannoprotein-linked oligosaccharides.

A collection of haploid Saccharomyces cerevisiae deletion strains--both MAT a and alpha--was screened for mutants that exhibit low dye binding (ldb) phenotype. This phenotype has previously been associated with reduced incorporation of mannosyl phosphate groups into the mannoprotein-linked oligosaccharides. We identified 199 nonessential genes whose deletion resulted in a detectable ldb phenotype. They fell into diverse functional categories, including those involved in protein glycosylation, vacuolar function, intracellular transport, cytoskeleton organization, transcription, signal transduction, among others. The study extends the number of known genes that affect mannosyl phosphorylation of mannoprotein-linked oligosaccharides, and establishes a link with other relevant pathways in the cell, especially vacuolar function. We have assigned an LDB name to four uncharacterized ORFs identified in this study: YCL005W, LDB16; YDL146W, LDB17; YLL049W, LDB18; and YOR322C, LDB19.

Identification of low-dye-binding (ldb) mutants of Saccharomyces cerevisiae.

We have completed the identification of Saccharomyces cerevisiae genes that are defective in previously isolated ldb (low-dye-binding) mutants. This was done by complementation of the mutant's phenotype with DNA fragments from a genomic library and by running standard tests of allelism with single-gene deletion mutants of similar phenotype. The results were as follows: LDB2 is allelic to ERD1; LDB4 to SPC72; LDB5 to RLR1; LDB6 to GON7/YJL184W; LDB7 to YBL006C; LDB9 to ELM1; LDB10 to CWH36; LDB11 to COG1; LDB12 to OCH1; LDB13 to VAN1; LDB14 to BUD32; and LDB15 to PHO85. Since the precise function of some of the genes is not known, these data may contribute to the functional characterization of the S. cerevisiae genome.

The ldb1 mutant of Saccharomyces cerevisiae is defective in Pmr1p, the yeast secretory pathway/Golgi Ca(2+)/Mn(2+)-ATPase.

The LDB1 gene of Saccharomyces cerevisiae was identified by complementation of the ldb1 mutant phenotype with a genomic library. We found that the ldb1 defect is complemented by PMR1 which codes for the yeast secretory pathway/Golgi Ca(2+)/Mn(2+)-ATPase. Besides that, the analysis of a null mutation of the PMR1 gene revealed a phenotype identical to that of ldb1 mutant. Thus, LDB1 must be considered a synonym of PMR1.