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.

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.

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.