Chelation of cytoplasmic Ca2+ increases plasma membrane permeability in murine macrophages.

Cytoplasmic free Ca2+ (Ca2+i) was chelated to 10-20 nM in the macrophage cell line J774 either by incubation with quin2 acetoxymethyl ester in the absence of external Ca2+ (Di Virgilio, F., Lew, P.D., and Pozzan, T. (1984) Nature 310, 691-693) or by loading [ethyl-enebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) into the cytoplasm via reversible permeabilization of the plasma membrane with extracellular ATP (Steinberg, T.H., Newman, A.S., Swanson, J.A., and Silverstein, SS.C. (1987) J. Biol. Chem. 262, 8884-8888; Di Virgilio, F., Meyer, B.C., Greenberg, S., and Silverstein, S.C. (1988) J. Cell Biol. 106, 657-666). After removal of ATP from the incubation medium, ATP-permeabilized Ca2+i-depleted macrophages recovered a near-normal plasma membrane potential which slowly depolarized over a 2-4 h incubation at low [Ca2+]i. In both ATP-treated and quin2-loaded cells, depolarization of plasma membrane potential was paralleled by an increase in plasma membrane permeability to low molecular weight aqueous solutes such as eosin yellowish (Mr 692), ethidium bromide (Mr 394), and lucifer yellow (Mr 463). This increased plasma membrane permeability was not accompanied by release of the cytoplasmic marker lactic dehydrogenase for incubations up to 4 h and was likely a specific effect of Ca2+i depletion since it was not caused by: (i) the mere incubation of macrophages with extracellular EGTA, i.e. at near-normal [Ca2+]i; and (ii) loading into the cytoplasm of diethylenetriaminepentaacetic acid, a specific chelator of heavy metals with low affinity for Ca2+. Treatment of Ca2+i-depleted cells with direct (phorbol 12-myristate 13-acetate) or indirect (platelet-activating factor) activators of protein kinase C prevented the increase in plasma membrane permeability. Down-regulation of protein kinase C rendered Ca2+i-depleted macrophages refractory to the protective effect of phorbol 12-myristate 13-acetate. This report suggests a role for Ca2+i and possibly protein kinase C in the regulation of plasma membrane permeability to low molecular weight aqueous solutes

Familial Alzheimer's disease-linked presenilin mutants and intracellular Ca2+ handling: A single-organelle, FRET-based analysis

Abstract An imbalance in Ca2+ homeostasis represents an early event in the pathogenesis of Alzheimer's disease (AD). Presenilin-1 and -2 (PS1 and PS2) mutations, the major cause of familial AD (FAD), have been extensively associated with alterations in different Ca2+ signaling pathways, in particular those handled by storage compartments. However, FAD-PSs effect on organelles Ca2+ content is still debated and the mechanism of action of mutant proteins is unclear. To fulfil the need of a direct investigation of intracellular stores Ca2+ dynamics, we here present a detailed and quantitative single-cell analysis of FAD-PSs effects on organelle Ca2+ handling using specifically targeted, FRET (Fluorescence/Forster Resonance Energy Transfer)-based Ca2+ indicators. In SH-SY5Y human neuroblastoma cells and in patient-derived fibroblasts expressing different FAD-PSs mutations, we directly measured Ca2+ concentration within the main intracellular Ca2+ stores, e.g., Endoplasmic Reticulum (ER) and Golgi Apparatus (GA) medial- and trans-compartment. We unambiguously demonstrate that the expression of FAD-PS2 mutants, but not FAD-PS1, in either SH-SY5Y cells or FAD patient-derived fibroblasts, is able to alter Ca2+ handling of ER and medial-GA, but not trans-GA, reducing, compared to control cells, the Ca2+ content within these organelles by partially blocking SERCA (Sarco/Endoplasmic Reticulum Ca2+-ATPase) activity. Moreover, by using a cytosolic Ca2+ probe, we show that the expression of both FAD-PS1 and -PS2 reduces the Ca2+ influx activated by stores depletion (Store-Operated Ca2+ Entry; SOCE), by decreasing the expression levels of one of the key molecules, STIM1 (STromal Interaction Molecule 1), controlling this pathway. Our data indicate that FAD-linked PSs mutants differentially modulate the Ca2+ content of intracellular stores yet leading to a complex dysregulation of Ca2+ homeostasis, which represents a common disease phenotype of AD

Experimental Measurement and Numerical Validation of the Flow Ripple in Internal Gear Pumps

The flow ripple in an internal gear pump was measured by means of a new instantaneous high-pressure flowmeter. The flowmeter consists of two pressure sensors mounted on a piece of the straight steel pump delivery line, and a variable-diameter orifice was installed along such a line, downstream of the flowmeter, to generate a variable load. Three distinct configurations of the high-pressure flowmeter, characterized by a different distance between the pressure transducers, were analyzed. Furthermore, a comprehensive fluid dynamic 3D model of the pump and of its high-pressure delivery line was developed and validated in terms of both the delivery pressure and the flow ripple for different pump working conditions. For the three examined configurations of the flowmeter, the measured flowrate time histories matched the corresponding numerical distributions at the various operating points. Finally, the validated 3D model was applied to predict the incomplete filling working of the interteeth chambers, and the obtained numerical pressure time histories along the delivery line were used, as input data, to assess the reliability of the flowmeter algorithm even in these severe operating conditions

Presenilin 2 Modulates Endoplasmic Reticulum-Mitochondria Coupling by Tuning the Antagonistic Effect of Mitofusin 2

Communication between organelles plays key roles in cell biology. In particular, physical and functional coupling of the endoplasmic reticulum (ER) and mitochondria is crucial for regulation of various physiological and pathophysiological processes. Here, we demonstrate that Presenilin 2 (PS2), mutations in which underlie familial Alzheimer’s disease (FAD), promotes ER-mitochondria coupling only in the presence of mitofusin 2 (Mfn2). PS2 is not necessary for the antagonistic effect of Mfn2 on organelle coupling, although its abundance can tune it. The two proteins physically interact, whereas their homologues Mfn1 and PS1 are dispensable for this interplay. Moreover, PS2 mutants associated with FAD are more effective than the wild-type form in modulating ER-mitochondria tethering because their binding to Mfn2 in mitochondrial-associated membranes is favored. We propose a revised model for ER-mitochondria interaction to account for these findings and discuss possible implications for FAD pathogenesis

Presenilin 2 Modulates Endoplasmic Reticulum-Mitochondria Coupling by Tuning the Antagonistic Effect of Mitofusin 2

Communication between organelles plays key roles in cell biology. In particular, physical and functional coupling of the endoplasmic reticulum (ER) and mitochondria is crucial for regulation of various physiological and pathophysiological processes. Here, we demonstrate that Presenilin 2 (PS2), mutations in which underlie familial Alzheimer's disease (FAD), promotes ER-mitochondria coupling only in the presence of mitofusin 2 (Mfn2). PS2 is not necessary for the antagonistic effect of Mfn2 on organelle coupling, although its abundance can tune it. The two proteins physically interact, whereas their homologues Mfn1 and PS1 are dispensable for this interplay. Moreover, PS2 mutants associated with FAD are more effective than the wild-type form in modulating ER-mitochondria tethering because their binding to Mfn2 in mitochondria-associated membranes is favored. We propose a revised model for ER-mitochondria interaction to account for these findings and discuss possible implications for FAD pathogenesis

Defective Mitochondrial Pyruvate Flux Affects Cell Bioenergetics in Alzheimer's Disease-Related Models

Summary: Mitochondria are key organelles for brain health. Mitochondrial alterations have been reported in several neurodegenerative disorders, including Alzheimer's disease (AD), and the comprehension of the underlying mechanisms appears crucial to understand their relationship with the pathology. Using multiple genetic, pharmacological, imaging, and biochemical approaches, we demonstrate that, in different familial AD cell models, mitochondrial ATP synthesis is affected. The defect depends on reduced mitochondrial pyruvate oxidation, due to both lower Ca2+-mediated stimulation of the Krebs cycle and dampened mitochondrial pyruvate uptake. Importantly, this latter event is linked to glycogen-synthase-kinase-3β (GSK-3β) hyper-activation, leading, in turn, to impaired recruitment of hexokinase 1 (HK1) to mitochondria, destabilization of mitochondrial-pyruvate-carrier (MPC) complexes, and decreased MPC2 protein levels. Remarkably, pharmacological GSK-3β inhibition in AD cells rescues MPC2 expression and improves mitochondrial ATP synthesis and respiration. The defective mitochondrial bioenergetics influences glutamate-induced neuronal excitotoxicity, thus representing a possible target for future therapeutic interventions. : Mitochondria are key organelles for brain health. Rossi et al. show that, in different Alzheimer's disease cell models, lower mitochondrial Ca2+ signal and pyruvate uptake reduce ATP synthesis. GSK-3β hyper-activation contributes to the defect by impairing HK1-mitochondria association, decreasing MPC2 levels and destabilizing MPC complexes. Defective bioenergetics affects neuronal functionality. Keywords: Alzheimer's disease, presenilin, mitochondrial metabolism, bioenergetics, calcium homeostasis, pyruvate, mitochondrial pyruvate carrier, hexokinase 1, GSK-3

Paediatric Sleep Questionnaire for Obstructive Sleep Apnoea Syndrome Screening: Is Sleep Quality Worthy of Note?

Obstructive sleep apnoea syndrome (OSAS) is the most severe condition on the spectrum of sleep-related breathing disorders (SRBDs). The Paediatric Sleep Questionnaire (PSQ) is one of the most used and validated screening tools, but it lacks the comprehensive assessment of some determinants of OSAS, specifically anamnestic assessment and sleep quality. This study aims to assess the accuracy of some specific items added to the original PSQ, particularly related to the patient’s anamnestic history and to the quality of sleep, for the screening of OSAS in a paediatric population living in Sicily (Italy). Fifteen specific items, divided into “anamnestic” and “related to sleep quality” were added to the original PSQ. The whole questionnaire was administered via a digital form to the parents of children at 4 schools (age range: 3–13 years). For each item, sensitivity and specificity, positive and negative predictive values, and positive and negative likelihood ratios were calculated. The highest sensitivity (80.0, 95% CI: 28.4; 99.5), in combination with the highest specificity (61.1, 95% CI: 35.7; 82.7), was found for the Item 32 (“assumption of bizarre or abnormal positions during sleep”). This item was found statistically significant for predicting the occurrence of OSAS in children (p-value ≤0.003). The study demonstrates the accuracy of specific items related to sleep quality disturbance for the preliminary assessment of the disease. Although these results should be validated on a larger sample of subjects, they suggest that including the factors discriminating sleep quality could further increase the efficiency and accuracy of PSQ

Prion Protein Paralog Doppel Protein Interacts with Alpha-2-Macroglobulin: A Plausible Mechanism for Doppel-Mediated Neurodegeneration

Doppel protein (Dpl) is a paralog of the cellular form of the prion protein (PrPC), together sharing common structural and biochemical properties. Unlike PrPC, which is abundantly expressed throughout the central nervous system (CNS), Dpl protein expression is not detectable in the CNS. Interestingly, its ectopic expression in the brain elicits neurodegeneration in transgenic mice. Here, by combining native isoelectric focusing plus non-denaturing polyacrylamide gel electrophoresis and mass spectrometry analysis, we identified two Dpl binding partners: rat alpha-1-inhibitor-3 (α1I3) and, by sequence homology, alpha-2-macroglobulin (α2M), two known plasma metalloproteinase inhibitors. Biochemical investigations excluded the direct interaction of PrPC with either α1I3 or α2M. Nevertheless, enzyme-linked immunosorbent assays and surface plasmon resonance experiments revealed a high affinity binding occurring between PrPC and Dpl. In light of these findings, we suggest a mechanism for Dpl-induced neurodegeneration in mice expressing Dpl ectopically in the brain, linked to a withdrawal of natural inhibitors of metalloproteinase such as α2M. Interestingly, α2M has been proven to be a susceptibility factor in Alzheimer's disease, and as our findings imply, it may also play a relevant role in other neurodegenerative disorders, including prion diseases

SPLICS: a split green fluorescent protein-based contact site sensor for narrow and wide heterotypic organelle juxtaposition

Contact sites are discrete areas of organelle proximity that coordinate essential physiological processes across membranes, including Ca2+ signaling, lipid biosynthesis, apoptosis, and autophagy. However, tools to easily image inter-organelle proximity over a range of distances in living cells and in vivo are lacking. Here we report a split-GFP-based contact site sensor (SPLICS) engineered to fluoresce when organelles are in proximity. Two SPLICS versions efficiently measured narrow (8\u201310 nm) and wide (40\u201350 nm) juxtapositions between endoplasmic reticulum and mitochondria, documenting the existence of at least two types of contact sites in human cells. Narrow and wide ER\u2013mitochondria contact sites responded differently to starvation, ER stress, mitochondrial shape modifications, and changes in the levels of modulators of ER\u2013mitochondria juxtaposition. SPLICS detected contact sites in soma and axons of D. rerio Rohon Beard (RB) sensory neurons in vivo, extending its use to analyses of organelle juxtaposition in the whole anim