Lipid droplet biogenesis induced by stress involves triacylglycerol synthesis that depends on group VIA phospholipase A2

This work investigates the metabolic origin of triacylglycerol (TAG) formed during lipid droplet (LD) biogenesis induced by stress. Cytotoxic inhibitors of fatty acid synthase induced TAG synthesis and LD biogenesis in CHO-K1 cells, in the absence of external sources of fatty acids. TAG synthesis was required for LD biogenesis and was sensitive to inhibition and down-regulation of the expression of group VIA phospholipase A2 (iPLA2-VIA). Induction of stress with acidic pH, C2-ceramide, tunicamycin, or deprivation of glucose also stimulated TAG synthesis and LD formation in a manner dependent on iPLA2-VIA. Overexpression of the enzyme enhanced TAG synthesis from endogenous fatty acids and LD occurrence. During stress, LD biogenesis but not TAG synthesis required phosphorylation and activation of group IVA PLA2 (cPLA2α). The results demonstrate that iPLA2-VIA provides fatty acids for TAG synthesis while cPLA2α allows LD biogenesis. LD biogenesis during stress may be a survival strategy, recycling structural phospholipids into energy-generating substrates

Group IVA phospholipase A2 is necessary for the biogenesis of lipid droplets

Lipid droplets (LD) are organelles present in all cell types, consisting of a hydrophobic core of triacylglycerols and cholesteryl esters, surrounded by a monolayer of phospholipids and cholesterol. This work shows that LD biogenesis induced by serum, by long-chain fatty acids, or the combination of both in CHO-K1 cells was prevented by phospholipase A2 inhibitors with a pharmacological profile consistent with the implication of group IVA cytosolic phospholipase A2 (cPLA2α). Knocking down cPLA2α expression with short interfering RNA was similar to pharmacological inhibition in terms of enzyme activity and LD biogenesis. A Chinese hamster ovary cell clone stably expressing an enhanced green fluorescent protein-cPLA2α fusion protein (EGFP-cPLA2) displayed higher LD occurrence under basal conditions and upon LD induction. Induction of LD took place with concurrent phosphorylation of cPLA2α at Ser505. Transfection of a S505A mutant cPLA2α showed that phosphorylation at Ser505 is key for enzyme activity and LD formation. cPLA2α contribution to LD biogenesis was not because of the generation of arachidonic acid, nor was it related to neutral lipid synthesis. cPLA2α inhibition in cells induced to form LD resulted in the appearance of tubulo-vesicular profiles of the smooth endoplasmic reticulum, compatible with a role of cPLA2α in the formation of nascent LD from the endoplasmic reticulum

Neuroimaging analyses from a randomized, controlled study to evaluate plasma exchange with albumin replacement in mild-to-moderate Alzheimer's disease : additional results from the AMBAR study

This study was designed to detect structural and functional brain changes in Alzheimer's disease (AD) patients treated with therapeutic plasma exchange (PE) with albumin replacement, as part of the recent AMBAR phase 2b/3 clinical trial. Mild-to-moderate AD patients were randomized into four arms: three arms receiving PE with albumin (one with low-dose albumin, and two with low/high doses of albumin alternated with IVIG), and a placebo (sham PE) arm. All arms underwent 6 weeks of weekly conventional PE followed by 12 months of monthly low-volume PE. Magnetic resonance imaging (MRI) volumetric analyses and regional and statistical parametric mapping (SPM) analysis on 18 F-fluorodeoxyglucose positron emission tomography (18 FDG-PET) were performed. MRI analyses (n = 198 patients) of selected subcortical structures showed fewer volume changes from baseline to final visit in the high albumin + IVIG treatment group (p < 0.05 in 3 structures vs. 4 to 9 in other groups). The high albumin + IVIG group showed no statistically significant reduction of right hippocampus. SPM 18 FDG-PET analyses (n = 213 patients) showed a worsening of metabolic activity in the specific areas affected in AD (posterior cingulate, precuneus, and parieto-temporal regions). The high-albumin + IVIG treatment group showed the greatest metabolic stability over the course of the study, i.e., the smallest percent decline in metabolism (MaskAD), and least progression of defect compared to placebo. PE with albumin replacement was associated with fewer deleterious changes in subcortical structures and less metabolic decline compared to the typical of the progression of AD. This effect was more marked in the group treated with high albumin + IVIG. (AMBAR trial registration: EudraCT#: 2011-001,598-25; ClinicalTrials.gov ID: NCT01561053). The online version contains supplementary material available at 10.1007/s00259-022-05915-

Implicació dels lisosomes en la mort cel·lular per inhibició de la síntesi de fosfatidilcolina i en la senyalització de calci per NAADP

La fosfatidilcolina (PtdCho) és el fosfolípid majoritari de les membranes cel·lulars. La inhibició de la seva síntesi ha estat relacionada amb la mort cel·lular apoptòtica, que ha estat estudiada principalment mitjançant inhibidors farmacòlogics. La manca d'especificitat dels inhibidors utilitzats ha posat de relleu la necessitat d'utilitzar un model cel·lular on només la via de síntesi de PtdCho estigui afectada. La línia cel·lular CHO-MT58 presenta una mutació termosensible en l'enzim CTP:fosfocolina citi¬dililtransferasa (CCT), enzim regulador de la síntesi de PtdCho.Quan es cultiven les cèl·lules CHO-MT58 a 40ºC, observem una ràpida inhibició de la síntesi de PtdCho, un descens del contingut cel·lular de PtdCho i la pèrdua de viabilitat cel·lular en una seqüència temporal definida. La mort cel·lular produïda per la inhibició de la síntesi de PtdCho en cèl·lules CHO-MT58 genera poca condensació de la cromatina i fragmentació del DNA i no hi ha activació de caspasa-3 si es compara amb la mort apoptòtica induïda pel tractament amb Actinomicina D (ActD). A més, en la inhibició de la síntesi de PtdCho en cèl·lules CHO-MT58 es produeix en paral·lel un increment de la catepsina D i l'aparició de vesícules autofàgiques identificades en el microscopi electrònic de transmissió. Quan seguim el procés de l'autofàgia amb el marcador LC3 podem observar que l'aparició del fragment LC3-II, indicatiu de la presència d'autofagosomes, no disminueix al llarg dels temps a 40ºC suggerint un bloqueig de l'autofàgia causat per la inhibició de la síntesi de PtdCho en cèl·lules CHO-MT58.Per tant, la inhibició de la síntesi de PtdCho en cèl·lules CHO-MT58 a 40ºC provoca mort cel·lular no apoptòtica tot aturant l'autofàgia que actuaria com a mecanisme constitutiu de supervivència. Addicionalment, les catepsines actuarien com a possible mecanisme alternatiu de mort cel·lular.D'altra banda, actualment es sap que els astròcits tenen un paper importantíssim en la regulació de la transmissió sinàptica i en la plasticitat neuronal. Aquestes noves funcions dels astròcits són possibles perquè són cèl·lules excitables, tot i que la seva excitabilitat no es basa en potencials d'acció sinó en ones de Ca2+ intra- i intercel·lulars. Els mecanismes que controlen la senyalització via Ca2+ en astròcits, però, no es coneixen del tot. El nostre projecte ha demostrat que les vesícules acídiques de tipus lisosomals (VAL) són reservoris intracel·lulars de Ca2+ i el NAADP és un segon missatger capaç d'alliberar-ne el Ca2+ en astròcits corticals. Així hem determinat nivells endògens de NAADP i que aquests s'incrementen després d'estimulació amb diferents agonistes. Per altra banda, el NAADP-AM, un anàleg del NAADP permeable a la membrana plasmàtica, és capaç d'induir respostes de Ca2+ a diferents concentracions a través de l'alliberament de Ca2+ de les VAL en forma de campana (inhibició a altes concentracions de NAADP-AM). A més, hem demostrat l'expressió del receptor de NAADP en vesícules acídiques de tipus lisosomal. Per altra banda, la inhibició dels receptors de NAADP amb Ned-19 i la destrucció de les VAL amb GPN inhibeixen en part les respostes de calci induïdes per ATP, Endotelina-1 (ET-1) i Acetilcolina (Ach) però no per Bradiquinina. Finalment, el NAADP i les VAL també tenen un paper en l'excitabilitat astrocitària en la modulació de la magnitud de les ones de Ca2+ entre astròcits induïdes per estrès mecànic. Per tant, podem concloure com l'excitabilitat i senyalització via Ca2+ dels astròcits és un fenomen complex en el qual participen rutes de senyalització clàssiques (IP3 i ER; cADPR i RyR) però també nous segons missatgers i reservoris intracel·lulars de calci com ara el NAADP i les VAL.Phosphatidylcholine (PtdCho) is the major phospholipid of the cell membrane. Inhibition of their synthesis has been linked to apoptotic cell death, when it has been studied primarily using pharmacological inhibitors. The lack of specificity of these inhibitors has highlighted the need to use a cell line where the only PtdCho synthesis is affected. The cell line CHO-MT58 has a termosensitive mutation in the enzyme Thermal CTP:phosphocholine cytidyltransferase (CCT), the regulatory enzyme of PtdCho synthesis.When CHO MT58 cells are grown at 40°C, we observed a rapid inhibition of PtdCho synthesis, a decrease in cellular mass of PtdCho and loss of cell viability in a defined temporal sequence. The cell death caused by inhibition of PtdCho synthesis in CHO-MT58 cells generates little chromatin condensation and late DNA fragmentation without activation of caspase-3 when compared with the apoptotic death induced by treatment with Actinomycin D (ActD). In addition, CHO-MT58 cells at 40ºC induce an increase of cathepsin D and the emergence of autophagic vesicles identified in transmission electron microscopy. When we follow the autophagic process using LC3 as marker, we show the appearance of LC3-II fragment, indicative of the presence of autofagosomes and LC3-II form do not decrease over time at 40ºC, suggesting a blockade of autophagy caused by inhibition of PtdCho synthesis in CHO-MT58 cells. Therefore, inhibition of PtdCho synthesis in CHO MT58 cells at 40ºC causes non apoptotic cell death stopping the autophagy that acts as a constitutive survival mechanism. In addition, cathepsin acts as a possible alternative mechanism of cell death.Moreover, it is currently known that astrocytes play a huge role in the regulation of synaptic transmission and neuronal plasticity. These new features are possible because astrocytes are excitable cells, although their excitability is not based on action potentials, but in Ca2+ waves intra- and intercellularly. The mechanisms that control Ca2+ signalling in astrocytes are not completely known. Our project has shown that the lysosome-related vesicles are intracellular Ca2+ stores and NAADP is a second messenger able to release their Ca2+ in cortical astrocytes. Thus we determined endogenous NAADP levels and how they increase after stimulation with different agonists. Furthermore, the NAADP-AM, a cell permeable analog of NAADP, is able to induce Ca2+ responses at different concentrations by releasing Ca2+ from lysosome-related vesicles in a bell-shape manner (inhibition at high concentrations of NAADP-AM). In addition, we have demonstrated the expression of the NAADP receptor in lysosome-related vesicles. Furthermore, inhibition of NAADP receptors by Ned-19 and the destruction of lysosome-related vesicles with GPN partly inhibit Ca2+ responses induced by ATP, Endothelin-1 (ET-1) and Acetylcholine (Ach) but not Bradykinin. Finally, the Ca2+ signalling by NAADP and lysosome-related vesicles also play a role in the astrocitic excitability with a modulation of the magnitude of the Ca2+ waves between astrocytes induced by mechanical stress. Therefore, we concluded that the excitability and Ca2+ signalling of astroyctes is a complex phenomenon in which classical signalling pathways (ER and IP3, cADPR and RyR) as well as new Ca2+ stores and intracellular second Ca2+ messengers such as NAADP and lysosome-related vesicles

Implicació dels lisosomes en la mort cel·lular per inhibició de la síntesi de fosfatidilcolina i en la senyalització de calci per NAADP

Descripció del recurs: el 02 de novembre de 2010Títol en català i text de la tesi en anglèsLa fosfatidilcolina (PtdCho) és el fosfolípid majoritari de les membranes cel·lulars. La inhibició de la seva síntesi ha estat relacionada amb la mort cel·lular apoptòtica, que ha estat estudiada principalment mitjançant inhibidors farmacòlogics. La manca d'especificitat dels inhibidors utilitzats ha posat de relleu la necessitat d'utilitzar un model cel·lular on només la via de síntesi de PtdCho estigui afectada. La línia cel·lular CHO-MT58 presenta una mutació termosensible en l'enzim CTP:fosfocolina citi¬dililtransferasa (CCT), enzim regulador de la síntesi de PtdCho. Quan es cultiven les cèl·lules CHO-MT58 a 40ºC, observem una ràpida inhibició de la síntesi de PtdCho, un descens del contingut cel·lular de PtdCho i la pèrdua de viabilitat cel·lular en una seqüència temporal definida. La mort cel·lular produïda per la inhibició de la síntesi de PtdCho en cèl·lules CHO-MT58 genera poca condensació de la cromatina i fragmentació del DNA i no hi ha activació de caspasa-3 si es compara amb la mort apoptòtica induïda pel tractament amb Actinomicina D (ActD). A més, en la inhibició de la síntesi de PtdCho en cèl·lules CHO-MT58 es produeix en paral·lel un increment de la catepsina D i l'aparició de vesícules autofàgiques identificades en el microscopi electrònic de transmissió. Quan seguim el procés de l'autofàgia amb el marcador LC3 podem observar que l'aparició del fragment LC3-II, indicatiu de la presència d'autofagosomes, no disminueix al llarg dels temps a 40ºC suggerint un bloqueig de l'autofàgia causat per la inhibició de la síntesi de PtdCho en cèl·lules CHO-MT58. Per tant, la inhibició de la síntesi de PtdCho en cèl·lules CHO-MT58 a 40ºC provoca mort cel·lular no apoptòtica tot aturant l'autofàgia que actuaria com a mecanisme constitutiu de supervivència. Addicionalment, les catepsines actuarien com a possible mecanisme alternatiu de mort cel·lular. D'altra banda, actualment es sap que els astròcits tenen un paper importantíssim en la regulació de la transmissió sinàptica i en la plasticitat neuronal. Aquestes noves funcions dels astròcits són possibles perquè són cèl·lules excitables, tot i que la seva excitabilitat no es basa en potencials d'acció sinó en ones de Ca2+ intra- i intercel·lulars. Els mecanismes que controlen la senyalització via Ca2+ en astròcits, però, no es coneixen del tot. El nostre projecte ha demostrat que les vesícules acídiques de tipus lisosomals (VAL) són reservoris intracel·lulars de Ca2+ i el NAADP és un segon missatger capaç d'alliberar-ne el Ca2+ en astròcits corticals. Així hem determinat nivells endògens de NAADP i que aquests s'incrementen després d'estimulació amb diferents agonistes. Per altra banda, el NAADP-AM, un anàleg del NAADP permeable a la membrana plasmàtica, és capaç d'induir respostes de Ca2+ a diferents concentracions a través de l'alliberament de Ca2+ de les VAL en forma de campana (inhibició a altes concentracions de NAADP-AM). A més, hem demostrat l'expressió del receptor de NAADP en vesícules acídiques de tipus lisosomal. Per altra banda, la inhibició dels receptors de NAADP amb Ned-19 i la destrucció de les VAL amb GPN inhibeixen en part les respostes de calci induïdes per ATP, Endotelina-1 (ET-1) i Acetilcolina (Ach) però no per Bradiquinina. Finalment, el NAADP i les VAL també tenen un paper en l'excitabilitat astrocitària en la modulació de la magnitud de les ones de Ca2+ entre astròcits induïdes per estrès mecànic. Per tant, podem concloure com l'excitabilitat i senyalització via Ca2+ dels astròcits és un fenomen complex en el qual participen rutes de senyalització clàssiques (IP3 i ER; cADPR i RyR) però també nous segons missatgers i reservoris intracel·lulars de calci com ara el NAADP i les VAL.Phosphatidylcholine (PtdCho) is the major phospholipid of the cell membrane. Inhibition of their synthesis has been linked to apoptotic cell death, when it has been studied primarily using pharmacological inhibitors. The lack of specificity of these inhibitors has highlighted the need to use a cell line where the only PtdCho synthesis is affected. The cell line CHO-MT58 has a termosensitive mutation in the enzyme Thermal CTP:phosphocholine cytidyltransferase (CCT), the regulatory enzyme of PtdCho synthesis. When CHO MT58 cells are grown at 40°C, we observed a rapid inhibition of PtdCho synthesis, a decrease in cellular mass of PtdCho and loss of cell viability in a defined temporal sequence. The cell death caused by inhibition of PtdCho synthesis in CHO-MT58 cells generates little chromatin condensation and late DNA fragmentation without activation of caspase-3 when compared with the apoptotic death induced by treatment with Actinomycin D (ActD). In addition, CHO-MT58 cells at 40ºC induce an increase of cathepsin D and the emergence of autophagic vesicles identified in transmission electron microscopy. When we follow the autophagic process using LC3 as marker, we show the appearance of LC3-II fragment, indicative of the presence of autofagosomes and LC3-II form do not decrease over time at 40ºC, suggesting a blockade of autophagy caused by inhibition of PtdCho synthesis in CHO-MT58 cells. Therefore, inhibition of PtdCho synthesis in CHO MT58 cells at 40ºC causes non apoptotic cell death stopping the autophagy that acts as a constitutive survival mechanism. In addition, cathepsin acts as a possible alternative mechanism of cell death. Moreover, it is currently known that astrocytes play a huge role in the regulation of synaptic transmission and neuronal plasticity. These new features are possible because astrocytes are excitable cells, although their excitability is not based on action potentials, but in Ca2+ waves intra- and intercellularly. The mechanisms that control Ca2+ signalling in astrocytes are not completely known. Our project has shown that the lysosome-related vesicles are intracellular Ca2+ stores and NAADP is a second messenger able to release their Ca2+ in cortical astrocytes. Thus we determined endogenous NAADP levels and how they increase after stimulation with different agonists. Furthermore, the NAADP-AM, a cell permeable analog of NAADP, is able to induce Ca2+ responses at different concentrations by releasing Ca2+ from lysosome-related vesicles in a bell-shape manner (inhibition at high concentrations of NAADP-AM). In addition, we have demonstrated the expression of the NAADP receptor in lysosome-related vesicles. Furthermore, inhibition of NAADP receptors by Ned-19 and the destruction of lysosome-related vesicles with GPN partly inhibit Ca2+ responses induced by ATP, Endothelin-1 (ET-1) and Acetylcholine (Ach) but not Bradykinin. Finally, the Ca2+ signalling by NAADP and lysosome-related vesicles also play a role in the astrocitic excitability with a modulation of the magnitude of the Ca2+ waves between astrocytes induced by mechanical stress. Therefore, we concluded that the excitability and Ca2+ signalling of astroyctes is a complex phenomenon in which classical signalling pathways (ER and IP3, cADPR and RyR) as well as new Ca2+ stores and intracellular second Ca2+ messengers such as NAADP and lysosome-related vesicles

Lipid droplet biogenesis induced by stress involves triacylglycerol synthesis that depends on group VIA phospholipase A2

This work investigates the metabolic origin of triacylglycerol (TAG) formed during lipid droplet (LD) biogenesis induced by stress. Cytotoxic inhibitors of fatty acid synthase induced TAG synthesis and LD biogenesis in CHO-K1 cells, in the absence of external sources of fatty acids. TAG synthesis was required for LD biogenesis and was sensitive to inhibition and down-regulation of the expression of group VIA phospholipase A2 (iPLA2-VIA). Induction of stress with acidic pH, C2-ceramide, tunicamycin, or deprivation of glucose also stimulated TAG synthesis and LD formation in a manner dependent on iPLA2-VIA. Overexpression of the enzyme enhanced TAG synthesis from endogenous fatty acids and LD occurrence. During stress, LD biogenesis but not TAG synthesis required phosphorylation and activation of group IVA PLA2 (cPLA2α). The results demonstrate that iPLA2-VIA provides fatty acids for TAG synthesis while cPLA2α allows LD biogenesis. LD biogenesis during stress may be a survival strategy, recycling structural phospholipids into energy-generating substrates

Introduction

Intracellular Ca2+ signals provide astrocytes with a specific form of excitability that enables them to regulate synaptic transmission. In this study, we demonstrate that NAADP-AM, a membrane-permeant analogue of the new second messenger nicotinic acid-adenine dinucleotide phosphate (NAADP), mobilizes Ca2+ in astrocytes and that the response is blocked by Ned-19, an antagonist of NAADP signalling. We also show that NAADP receptors are expressed in lysosome-related acidic vesicles. Pharmacological disruption of either NAADP or lysosomal signalling reduced Ca2+ responses induced by ATP and endothelin-1, but not by bradykinin. Furthermore, ATP increased endogenous NAADP levels. Overall, our data provide evidence for NAADP being an intracellular messenger for agonist-mediated calcium signalling in astrocytes

NAADP mediates ATP-induced Ca2+ signals in astrocytes

Intracellular Ca2+ signals provide astrocytes with a specific form of excitability that enables them to regulate synaptic transmission. In this study, we demonstrate that NAADP-AM, a membrane-permeant analogue of the new second messenger nicotinic acid-adenine dinucleotide phosphate (NAADP), mobilizes Ca2+ in astrocytes and that the response is blocked by Ned-19, an antagonist of NAADP signalling. We also show that NAADP receptors are expressed in lysosome-related acidic vesicles. Pharmacological disruption of either NAADP or lysosomal signalling reduced Ca2+ responses induced by ATP and endothelin-1, but not by bradykinin. Furthermore, ATP increased endogenous NAADP levels. Overall, our data provide evidence for NAADP being an intracellular messenger for agonist-mediated calcium signalling in astrocytes

Group IVA phospholipase A2 is necessary for the biogenesis of lipid droplets

Lipid droplets (LD) are organelles present in all cell types, consisting of a hydrophobic core of triacylglycerols and cholesteryl esters, surrounded by a monolayer of phospholipids and cholesterol. This work shows that LD biogenesis induced by serum, by long-chain fatty acids, or the combination of both in CHO-K1 cells was prevented by phospholipase A2 inhibitors with a pharmacological profile consistent with the implication of group IVA cytosolic phospholipase A2 (cPLA2α). Knocking down cPLA2α expression with short interfering RNA was similar to pharmacological inhibition in terms of enzyme activity and LD biogenesis. A Chinese hamster ovary cell clone stably expressing an enhanced green fluorescent protein-cPLA2α fusion protein (EGFP-cPLA2) displayed higher LD occurrence under basal conditions and upon LD induction. Induction of LD took place with concurrent phosphorylation of cPLA2α at Ser505. Transfection of a S505A mutant cPLA2α showed that phosphorylation at Ser505 is key for enzyme activity and LD formation. cPLA2α contribution to LD biogenesis was not because of the generation of arachidonic acid, nor was it related to neutral lipid synthesis. cPLA2α inhibition in cells induced to form LD resulted in the appearance of tubulo-vesicular profiles of the smooth endoplasmic reticulum, compatible with a role of cPLA2α in the formation of nascent LD from the endoplasmic reticulum