Catherine Astarie-Dequeker

Nuclear gene OPA1 , encoding a mitochondrial dynamin-related protein, is mutated in dominant optic atrophy

Optic atrophy type 1 (OPA1, MIM 165500) is a dominantly inherited optic neuropathy occurring in 1 in 50,000 individuals that features progressive loss in visual acuity leading, in many cases, to legal blindness. Phenotypic variations and loss of retinal ganglion cells, as found in Leber hereditary optic neuropathy (LHON), have suggested possible mitochondrial impairment. The OPA1 gene has been localized to 3q28–q29 (refs 13–19). We describe here a nuclear gene, OPA1, that maps within the candidate region and encodes a dynamin-related protein localized to mitochondria. We found four different OPA1 mutations, including frameshift and missense mutations, to segregate with the disease, demonstrating a role for mitochondria in retinal ganglion cell pathophysiology.

Activation of the lysosome-associated p61Hck isoform triggers the biogenesis of podosomes.

Haematopoietic cell kinase (Hck) is a protein tyrosine kinase of the Src family specifically expressed in phagocytes as two isoforms, p59Hck and p61Hck, present at the plasma membrane and lysosomes, respectively. We report that ectopic expression of a constitutively active mutant of p61Hck (p61Hckca) triggered the de novo formation of actin‐rich rings at the ventral face of the cells that we characterized as bona fide podosome rosettes, structures involved in cell migration. Their formation required the adaptor domains and the kinase activity of p61Hck, the integrity of microfilament and microtubule networks and concerted action of Cdc42, Rac and Rho. Podosome rosette formation was either abolished when p61Hckca was readdressed from lysosomes to the cytosol or triggered when p59Hckca was relocalized to lysosomes. Lysosomal markers were present at podosome rosettes. By stimulating exocytosis of p61Hckca lysosomes with a calcium ionophore, the formation of podosome rosettes was enhanced. Interestingly, we confirm that, in human macrophages, Hck and lysosomal markers were present at podosomes which were spatially reorganized as clusters, a foregoing step to form rosettes, upon expression of p61Hckca. We propose that lysosomes, under the control of p61Hck, are involved in the biogenesis of podosomes, a key phenomenon in the migration of phagocytes.

Surface-exposed glycopeptidolipids of mycobacterium smegmatis specifically inhibit the phagocytosis of mycobacteria by human macrophages. Identification of a novel family of glycopeptidolipids

Phagocytosis by macrophages represents the early step of the mycobacterial infection. It is governed both by the nature of the host receptors used and the ligands exposed on the bacteria. The outermost molecules of the nonpathogenic Mycobacterium smegmatis were extracted by a mechanical treatment and found to specifically and dose dependently inhibit the phagocytosis of both M. smegmatis and the opportunistic pathogen M. kansasii by human macrophages derived from monocytes. The inhibitory activity was attributed to surface lipids because it is extracted by chloroform and reduced by alkaline hydrolysis but not by protease treatment. Fractionation of surface lipids by adsorption chromatography indicated that the major inhibitory compounds consisted of phospholipids and glycopeptidolipids (GPLs). Mass spectrometry and nuclear magnetic resonance spectroscopy analyses, combined with chemical degradation methods, demonstrated the existence of a novel family of GPLs that consists of a core composed of the long-chain tripeptidyl amino-alcohol with a di-O-acetyl-6-deoxytalosyl unit substituting the allo-threoninyl residue and a 2-succinyl-3,4-di-O-CH3-rhamnosyl unit linked to the alaninol end of the molecules. These compounds, as well as diglycosylated GPLs at the alaninol end and de-O-acylated GPLs, but not the non-serovar-specific di-O-acetylated GPLs, inhibited the phagocytosis of M. smegmatis and M. avium by human macrophages at a few nanomolar concentration without affecting the rate of zymosan internalization. At micromolar concentrations, the native GPLs also inhibit the uptake of both M. tuberculosis and M. kansasii. De-O-acylation experiments established the critical roles of both the succinyl and acetyl substituents. Collectively, these data provide evidence that surface-exposed mycobacterial glycoconjugates are efficient competitors of the interaction between macrophages and mycobacteria and, as such, could represent pharmacological tools for the control of mycobacterial infections.

Expression of Azurophil and specific granule proteins during differentiation of NB4 cells in neutrophils

Neutrophils contain several populations of secretory granules with characteristic sets of proteins. Granule proteins are sorted into their respective granule types by temporal regulation of their expression during cell differentiation and/or by specific targeting signals. We investigated the expression of some granule proteins in human promyelocytic NB4 cells. Like other myeloid cell lines which can be differentiated into neutrophils, NB4 cells lack the specific‐granule population. We report here that, nevertheless, they express the specific‐granule matrix protein lactoferrin, when differentiated with retinoic acid. Lactoferrin and the azurophil‐granule protein β‐glucuronidase were simultaneously expressed, whereas myeloperoxidase expression had stopped, showing that azurophil‐granule proteins are not all produced concomitantly. Cell fractionation by Percoll gradient revealed that while β‐glucuronidase co‐fractionated with myeloperoxidase, lactoferrin was mostly contained in a vesicular compartment free of markers for azurophil granules, plasma membrane, and Golgi. This vesicular compartment was not implicated in regulated exocytosis since it was not mobilized by secretagogues, which, in parallel, induced the release of myeloperoxidase. Furthermore, the specific granule‐membrane protein cytochrome b558 also became expressed during NB4‐cell differentiation. However, it did not co‐localize with lactoferrin but was present in the plasma‐membrane fraction. Therefore, differentiation of NB4 cells with retinoic acid leads to the expression of specific‐ and azurophil‐granule proteins and provides a unique cell line model to study the mechanisms involved in the sorting of azurophil‐ and specific‐granule proteins. J. Cell. Physiol. 175:203–210, 1998.

Playing hide-and-seek with host macrophages through the use of mycobacterial cell envelope phthiocerol dimycocerosates and phenolic glycolipids

Mycobacterial pathogens, including Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), have evolved a remarkable ability to evade the immune system in order to survive and to colonize the host. Among the most important evasion strategies is the capacity of these bacilli to parasitize host macrophages, since these are major effector cells against intracellular pathogens that can be used as long-term cellular reservoirs. Mycobacterial pathogens employ an array of virulence factors that manipulate macrophage function to survive and establish infection. Until recently, however, the role of mycobacterial cell envelope lipids as virulence factors in macrophage subversion has remained elusive. Here, we will address exclusively the proposed role for phthiocerol dimycocerosates (DIM) in the modulation of the resident macrophage response and that of phenolic glycolipids (PGL) in the regulation of the recruitment and phenotype of incoming macrophage precursors to the site of infection. We will provide a unique perspective of potential additional functions for these lipids, and highlight obstacles and opportunities to further understand their role in the pathogenesis of TB and other mycobacterial diseases.

Inhibitory effect of trimetazidine on thrombin-induced aggregation and calcium entry into human platelets

The antiaggregatory properties of trimetazidine were investigated further by analyzing its effects on cytosolic calcium and proton concentrations, well-known regulators of platelet reactivity. Aggregatory responses of washed platelets were assessed by turbidometry, and cytosolic Ca2+ concentration ([Ca2+]i) and pH (pHi) were determined by their respective fluorescent probes: Fura-2 and BCECF. Preincubation with trimetazidine dose-dependently inhibited platelet aggregation induced by 0.05 U/ml thrombin (p < 0.001). At concentrations < or = 1 mM, trimetazidine did not affect the resting [Ca2+]i value but slightly alkalinized the cytosol by 0.05 +/- 0.03 pH units (p < 0.02, n = 11). In platelets stimulated by 0.05 U/ml thrombin, 0.1 mM trimetazidine did not modify pHi variations but decreased [Ca2+]i variations (p < 0.003, n = 16), blunting by 28 +/- 6% the transient peak of [Ca2+]i (p < 0.006) and decreasing by 6 +/- 2% the equilibrium value (p < 0.005). These inhibitory effects were inversely dependent on thrombin concentrations (p < 0.004, n = 21) and were abolished in the virtual absence of external Ca2+. Trimetazidine therefore attenuates the Ca2+ influx evoked by thrombin, thereby limiting Ca2+ accumulation in stimulated platelets. Such a protective effect may participate in the antiaggregatory properties of trimetazidine.

In vitro inhibition by endothelins of thrombin-induced aggregation and Ca2+ mobilization in human platelets.

1 The in vitro effects of endothelins (ET‐1 and ET‐3) on human platelets were investigated by measurement of the aggregatory responses of washed platelets to thrombin and by the determination of cytosolic pH (pHi) and free Ca2+ concentration ([Ca2+]i) determined with the fluorescent indicators, BCECF and Fura‐2. 2 ET‐1 and ET‐3 at concentrations ranging from 10−10 to 5 × 10−7 m, did not promote platelet aggregation but inhibited in a dose‐dependent manner the aggregation induced by 0.05 u ml−1 thrombin (P < 0.002 and < 0.001, respectively) with maximal effects reached at 10−8 m (17 ± 3 and 15 ± 2%, n = 11, P = 0.002 for each). 3 Even at 5 × 10−7 m, ET‐1 and ET‐3 did not cause a measurable change in basal [Ca2+]i and pHi. When tested in combination with thrombin, 5 × 10−7 m ET‐1 and ET‐3 decreased the transient peak of [Ca2+]i by 17 ± 7 and 28 ± 7% (n = 7 and 11, P = 0.03 and P = 0.002). No effect on pHi variations was detected. In the virtual absence of external Ca2+, 5 × 10−7 m ET‐3 inhibited the peak of [Ca2+]i by 18 ± 6% (n = 6, P = 0.02). 4 The anti‐aggregating agents, prostacyclin (PGI2, 10−8−10−7 m) and nitroprusside (NP, 10 ng‐50 μg l−1) also induced a dose‐dependent inhibition of the thrombin‐induced [Ca2+]i peak (P = 0.001 for each). A combination of 10−9 m PGI2 and 10 ng l−1 NP augmented the inhibitory effect of each drug (PGI2 alone 52 ± 11, plus NP 90 ± 2; NP alone 26 ± 4, plus PGI2 69 ± 5% inhibition of [Ca2+]i peak, n = 6 for each, P < 0.01 and P < 0.001, respectively). Platelet preincubation with 5 × 10−7 m ET‐3 increased by 34 ± 11% (n = 6, P = 0.014) the inhibitory effect of NP 10 ng l−1 without a significant influence on the PGI2 effect. 5 In conclusion, endothelins ET‐1 and ET‐3 can reduce in vitro the aggregating response of human platelets to thrombin by a mechanism that is probably due to decrease Ca2+ mobilization.

Lipoarabinomannans Activate the Protein Tyrosine Kinase Hck in Human Neutrophils

ABSTRACT The mycobacterial lipoarabinomannans (LAMs) are glycosylphosphatidyl-myo-inositol-anchored lipoglycans with diverse biological activities. It has been shown that purified LAMs interact directly, or indirectly, through receptors with the plasma membrane receptors of target cells located in domains rich in glycosylphosphatidylinositol-anchored proteins that contain Src family protein tyrosine kinases. To examine whether LAMs could activate Src-related kinases, human neutrophils were exposed to mannosylated LAMs (ManLAMs) purified from the vaccinal strain Mycobacterium bovis BCG and to phosphoinositol-capped LAMs (AraLAM or PILAM) obtained from the nonpathogenic species Mycobacterium smegmatis. We report first that both ManLAMs and PILAMs activate Hck in a rapid and transient manner and second that complete deacylation of ManLAM abolished its effect on Hck activity, thereby demonstrating that acylation of LAM but not mannosylation is critical for Hck activation. These data indicate that Hck is involved in the signaling pathway of LAMs, molecules known for their ability to trigger several responses in eukaryotic cells.

Endothelin-3, Ca2+ mobilization and cyclic GMP content in human platelets

As previously described for endothelin-3, platelet exposure to cyclic GMP-elevating agents such as sodium nitroprusside and M&B-22948 (2-o-propoxyphenyl-8-azapurin-6-one), a cGMP phosphodiesterase inhibitor, lowered Ca2+ mobilization in response to thrombin. Interestingly, when cGMP phosphodiesterases were blocked, endothelin-3 produced a dose-dependent cGMP accumulation (P < 0.001). Since endothelin-3 has been proposed to decrease the activity of Ca2+ accumulating pumps, we examined whether this latter effect could be mediated by a rise in cGMP content. Cyclic GMP decreased in a dose-dependent manner the initial rate and plateau value of the ATP-dependent 45Ca2+ uptake in platelet membrane vesicles (P = 0.006 for each). Furthermore, combined treatment with endothelin-3 and M&B-22948 or a moderate concentration of Na(+)-nitroprusside further reduced the thrombin-evoked Ca2+ discharge (P = 0.004 and 0.01, respectively), suggesting that endothelin-3 pre-exposure had reduced the amount of mobilizable Ca2+. We propose that the depletion of platelet Ca2+ stores and the reduction of Ca2+ release evoked by endothelin-3 could be due, at least in part, to the elevation of cGMP content and to a decrease in Ca2+ accumulating pump activity.

Different effects of endothelin‐3 on the Ca2+ discharge induced by agonists and Ca2+‐ATPase inhibitors in human platelets

1 The present study demonstrates that endothelin‐3 (ET‐3), previously shown to attenuate thrombin‐evoked aggregation of human platelets, delayed the dose‐dependent aggregatory response to thapsigargin (Tg). As this Ca2+‐ATPase inhibitor induces platelet activation in part through the depletion of internal Ca2+‐stores, we examined the influence of ET‐3 on Ca2+ discharge from internal pools. 2 Cytosolic Ca2+ concentration was evaluated with Fura‐2 in the absence of Ca2+ influx. Platelet preincubation for 15 min with 5 times 10−7 m ET‐3 decreased the Ca2+ release evoked by thrombin and U46619, a thromboxane‐mimetic. However, ET‐3 did not affect Ca2+ movements induced by 1 μm ADP. Addition of Tg (0.5 to 5 μm) to resting platelets induced a cytosolic [Ca2+] rise with concentration‐dependent increase of the initial rate and decrease of the time to reach the peak. ET‐3 slowed down these dose‐dependent effects with a more marked influence on the responses induced by low concentrations of Tg. 3 ET‐3 did not modify the Ca2+ response to another Ca2+‐ATPase inhibitor, 2,5‐di‐(tert‐butyl)‐1,4‐benzohydroquinone(tBuBHQ). The thromboxane A2 receptor antagonist, SQ 29548, reduced by 53% the calcium signal evoked by 1 μm Tg, which became similar to that induced by 15 μm tBuBHQ. Under these conditions, the ET‐3 effects were suppressed. A subsequent addition of thrombin induced a substantial further Ca2+ increase which was again sensitive to ET‐3. 4 ET‐3 attenuates Ca2+ mobilization from an internal pool dependent on the stimulation of thrombin and thromboxane A2 receptors and insensitive to the direct effect of Ca2+‐ATPase inhibitors. The small but significant inhibitory effect of ET‐3 leads us to propose that endothelin‐3 acts as a modulator of platelet activation.