Jean de Barry

Two chromogranin a-derived peptides induce calcium entry in human neutrophils by calmodulin-regulated calcium independent phospholipase A2

Background Antimicrobial peptides derived from the natural processing of chromogranin A (CgA) are co-secreted with catecholamines upon stimulation of chromaffin cells. Since PMNs play a central role in innate immunity, we examine responses by PMNs following stimulation by two antimicrobial CgA-derived peptides. Methodology/Principal Findings PMNs were treated with different concentrations of CgA-derived peptides in presence of several drugs. Calcium mobilization was observed by using flow cytometry and calcium imaging experiments. Immunocytochemistry and confocal microscopy have shown the intracellular localization of the peptides. The calmodulin-binding and iPLA2 activating properties of the peptides were shown by Surface Plasmon Resonance and iPLA2 activity assays. Finally, a proteomic analysis of the material released after PMNs treatment with CgA-derived peptides was performed by using HPLC and Nano-LC MS-MS. By using flow cytometry we first observed that after 15 s, in presence of extracellular calcium, Chromofungin (CHR) or Catestatin (CAT) induce a concentration-dependent transient increase of intracellular calcium. In contrast, in absence of extra cellular calcium the peptides are unable to induce calcium depletion from the stores after 10 minutes exposure. Treatment with 2-APB (2-aminoethoxydiphenyl borate), a store operated channels (SOCs) blocker, inhibits completely the calcium entry, as shown by calcium imaging. We also showed that they activate iPLA2 as the two CaM-binding factors (W7 and CMZ) and that the two sequences can be aligned with the two CaM-binding domains reported for iPLA2. We finally analyzed by HPLC and Nano-LC MS-MS the material released by PMNs following stimulation by CHR and CAT. We characterized several factors important for inflammation and innate immunity. Conclusions/Significance For the first time, we demonstrate that CHR and CAT, penetrate into PMNs, inducing extracellular calcium entry by a CaM-regulated iPLA2 pathway. Our study highlights the role of two CgA-derived peptides in the active communication between neuroendocrine and immune systems.

The expression of presynaptic t-ACPD receptor in rat cerebellum

The expression of a receptor subtype for one type of excitatory amino acid agonist, t-ACPD, was examined in developing Purkinje cells of cerebellar slices. The t-ACPD-induced responses were compared with those induced by QA in current response, single cell Ca2+ imaging and changes in the miniature currents in the same preparation. It was found that t-ACPD induced a single component of inward current, and an increase in the frequency of miniature currents associated with the presence of external Ca2+, but was ineffective at mobilizing intracellular Ca2+ even in the presence of external Ca2+. The present study suggests the expression of at least two types of metabotropic receptors in the Purkinje cell region, one of which, expressed in the Purkinje cell dendrites, is highly sensitive to QA, but relatively insensitive to t-ACPD, and the other of which is a t-ACPD-sensitive receptor expressed on the presynaptic terminals of the neurons making synapses onto Purkinje cells.

ARF6 Regulates the Synthesis of Fusogenic Lipids for Calcium-regulated Exocytosis in Neuroendocrine Cells

An important role for specific lipids in membrane fusion has recently emerged, but regulation of their biosynthesis remains poorly understood. Among fusogenic lipids, phosphatidic acid and phosphoinositol 4,5-bisphosphate (PIP2) have been proposed to act at various steps of neurotransmitter and hormone exocytosis. Using real time FRET (fluorescence resonance energy transfer) measurements, we show here that the GTPase ARF6, potentially involved in the synthesis of these lipids, is activated at the exocytotic sites in PC12 cells stimulated for secretion. Depletion of endogenous ARF6 by siRNA dramatically inhibited secretagogue-evoked exocytosis. ARF6-siRNA greatly reduced secretagogue-evoked phospholipase D (PLD) activation and phosphatidic acid formation at the plasma membrane and moderately reduced constitutive levels of PIP2 present at the plasma membrane in resting cells. Expression of an ARF6 insensitive to short interference RNA (siRNA) fully rescued secretion in ARF6-depleted cells. However, a mutated ARF6 protein specifically impaired in its ability to stimulate PLD had no effect. Finally, we show that the ARF6-siRNA-mediated inhibition of exocytosis could be rescued by an exogenous addition of lysophosphatidylcholine, a lipid that favors negative curvature on the inner leaflet of the plasma membrane. Altogether these data indicate that ARF6 is a critical upstream signaling element in the activation of PLD necessary to produce the fusogenic lipids required for exocytosis.

Functional Implication of Neuronal Calcium Sensor-1 and Phosphoinositol 4-Kinase-β Interaction in Regulated Exocytosis of PC12 Cells

Several studies have shown that the neuronal calcium sensor (NCS-1) and phosphoinositol 4-kinase-β (PI4K-β) regulate the exocytotic process of nerve and neuroendocrine cells. The aim of our study was to investigate their possible interaction at rest and during stimulation in living cells and to decipher the role of this interaction in the secretory process. In PC12 cells, we observed a stimulation-induced recruitment of NCS-1 and PI4K-β from the intracellular compartment toward the plasma membrane. This recruitment was highly correlated to the intracellular Ca2+ rise induced by secretagogues. Using fluorescence resonance energy transfer between PI4K-β-ECFP and NCS-1-EYFP, we show that both proteins are interacting in resting cells and that this interaction increases with stimulation. It appears that the membrane insertion of NCS-1 is necessary for the interaction with PI4K-β, since a mutation that prevented the membrane insertion of NCS-1 abolished NCS-1-PI4K-β interaction, as revealed by fluorescence resonance energy transfer analysis. Additionally, the overexpression of mutated NCS-1 prevents the stimulatory effect on secretion induced by PI4K-β, suggesting that the interaction of the two proteins on a membrane compartment is necessary for the secretory function. Moreover, extinction of endogenous PI4K-β by small interfering RNA inhibits secretion and completely prevents the stimulatory effect of NCS-1 on calcium-evoked exocytosis from permeabilized PC12 cells, showing directly for the first time the functional implication of a NCS-1·PI4K-β complex in regulated exocytosis.

Clostridium perfringens Epsilon Toxin Targets Granule Cells in the Mouse Cerebellum and Stimulates Glutamate Release

Epsilon toxin (ET) produced by C. perfringens types B and D is a highly potent pore-forming toxin. ET-intoxicated animals express severe neurological disorders that are thought to result from the formation of vasogenic brain edemas and indirect neuronal excitotoxicity. The cerebellum is a predilection site for ET damage. ET has been proposed to bind to glial cells such as astrocytes and oligodendrocytes. However, the possibility that ET binds and attacks the neurons remains an open question. Using specific anti-ET mouse polyclonal antibodies and mouse brain slices preincubated with ET, we found that several brain structures were labeled, the cerebellum being a prominent one. In cerebellar slices, we analyzed the co-staining of ET with specific cell markers, and found that ET binds to the cell body of granule cells, oligodendrocytes, but not astrocytes or nerve endings. Identification of granule cells as neuronal ET targets was confirmed by the observation that ET induced intracellular Ca2+ rises and glutamate release in primary cultures of granule cells. In cultured cerebellar slices, whole cell patch-clamp recordings of synaptic currents in Purkinje cells revealed that ET greatly stimulates both spontaneous excitatory and inhibitory activities. However, pharmacological dissection of these effects indicated that they were only a result of an increased granule cell firing activity and did not involve a direct action of the toxin on glutamatergic nerve terminals or inhibitory interneurons. Patch-clamp recordings of granule cell somata showed that ET causes a decrease in neuronal membrane resistance associated with pore-opening and depolarization of the neuronal membrane, which subsequently lead to the firing of the neuronal network and stimulation of glutamate release. This work demonstrates that a subset of neurons can be directly targeted by ET, suggesting that part of ET-induced neuronal damage observed in neuronal tissue is due to a direct effect of ET on neurons.

Protein kinase C as a peripheral biomarker for Alzheimer’s disease

Numerous studies have been performed, which assess an important role of protein kinase C (PKC) in the physiopathology of Alzheimer disease (AD). The alteration of PKC activity stimulates amyloid-beta peptides production and protein tau hyperphosphorylation. This recently led to consider PKC as a potential therapeutic target for disease modifying drugs. Moreover PKC alterations were also observed in peripheral cells including blood cells. This short review recalls the main findings on the role of PKC in the disease process and focuses on its use as an AD biomarker in blood cells. Using fluorescent probes specific for PKC, it is possible to detect the conformational changes of the enzyme in living cells. Such probes can be used to detect PKC alterations in red blood cells and thus to distinguish AD patients from healthy controls with unmatched specificity and sensitivity.

Heterogeneous Immunoreactivity of Glial Cells in the Mesencephalon of a Lizard: A Double Labeling Immunohistochemical Study

Astrocytes and radial glia coexist in the adult mesencephalon of the lizard Gallotia galloti. Radial glia and star‐shaped astrocytes express glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS). The same cell markers are also expressed by round or pear‐shaped cells that are therefore astrocytes with unusual morphology.

Reversible protein kinase C activation in PC12 cells: effect of NGF treatment

Although protein kinase C (PKC) is a key enzyme in the signal transduction process, there is little information on the mechanism leading to PKC activation in living cells. Using a new fluorescence imaging method, we studied this mechanism and correlated PKC conformational changes with intracellular Ca2+ concentration. PC12 cells were simultaneously loaded with Fura‐2‐AM and Fim‐1, two fluorescent probes, which recognize Ca2+ and PKC, respectively. KCl and carbachol (an agonist to muscarinic receptors) applications induced dose‐dependent increases of fluorescence for both probes. Both Ca2+ and PKC responses were observed within seconds following KCl or carbachol application, and were reversible upon stimulus withdrawal. PKC activation kinetics was slightly more rapid than the Ca2+ response after KCl application. After nerve growth factor (NGF) treatment of the cells, the amplitude of the KCl‐induced PKC responses was larger indicating an increase in the activated PKC‐pool in these cells. This difference between control and NGF‐treated cells was not observed following carbachol application, suggesting the involvement of different PKC pools. While the Ca2+ response uniformly occurred in the cytosol, the PKC response displayed a patch pattern with higher intensities in the peripheral zone near the plasma membrane. This heterogeneous distribution of PKC activation sites was similar to the immunocytological localization of Ca2+‐dependent and independent PKC isoforms, which suggested that at least several PKC isoforms interacted with intracellular elements. Upon repeated stimulation, the PKC response rapidly desensitized.

Ca2+ Mobilization in Cultured Rat Cerebellar Cells: Astrocytes are Activated by t-ACPD

Using primary rat cerebellar cell cultures we observed that trans‐1‐amino‐cyclopentyl‐1, 3‐dicarboxylic acid (t‐ACPD) was able to induce an increase in intracellular [Ca2+] in different cell types. This response was not abolished by external Ca2+ withdrawal, indicating that t‐ACPD triggered the release of intracellularly stored Ca2+. In neurons the t‐ACPD response was monophasic and inhibited by L‐2‐amino‐4‐phosphonobutyrate (APB). In astrocytes, characterized by their immunoreactivity to antisera to glial fibrillary acidic protein and S‐100 protein, the response was oscillatory and resistant to APB application. These results suggest the presence of glutamate metabotropic receptor subtypes in the mammalian brain.

NMDA antagonist properties of γ-L-glutamyl-L-aspartate demonstrated on chemically induced seizures in mice

In order to determine the gamma-L-glutamyl-L-aspartate (gamma-LGLA) site of action in excitatory amino acids (EAA) systems, we studied the gamma-LGLA anticonvulsant activity against seizures induced in mice by pentylenetetrazol, picrotoxin and EAA agonists. The mice were protected against seizures induced by pentylenetetrazol (80 mg/kg s.c.) and picrotoxin (2.75 mg/kg s.c.) after intraperitoneal administration of gamma-LGLA with two significant peak effects around the doses of 0.25 and 200 mumol/kg as revealed by the dose-response curves obtained in both experiments. Use of an intracerebroventricular co-injection procedure showed that gamma-LGLA dose dependently suppressed the seizures induced by NMDA (1 nmol/mouse) with a maximal effect at 80 nmol/mouse but, at the same dose, it only slightly suppressed seizures induced by kainate (0.3 and 0.8 nmol/mouse) or by quisqualate (18.5 nmol/mouse). The anticonvulsant activity of gamma-LGLA on these chemically induced seizures is consistent with an antagonistic action of gamma-LGLA on NMDA receptor subtypes.