A novel Toxoplasma gondii calcium-dependent protein kinase.

Toxoplasma gondii is an obligate intracellular parasite that infects all types of cells in humans. A family of calcium-dependent protein kinases (CDPKs), previously identified as important in the development of plants and protists, was recently shown to play a role in the infectivity of apicomplexans, and in motility and host cell invasion in particular. We report here the isolation of a new calcium-dependent protein kinase gene from the human toxoplasmosis parasite, Toxoplasma gondii. The gene consists of 12 exons. The encoded protein, TgCDPK4, consists of the four characteristic domains of members of the CDPK family and is most similar to PfCDPK2 from Plasmodium falciparum. We measured TgCDPK4 activity, induced by calcium influx, using a kinase assay. A calcium chelator (EGTA) inhibited this activity. These findings provide evidence of signal transduction involving members of the CDPK family in T. gondii.

Detection of tyrosine phosphorylated proteins in Trichinella spiralis L1 larvae.

Western-blotting analysis showed the presence of tyrosine phosphorylated proteins in crude extracts of T. spiralis larvae and these phosphorylated proteins were located by immunofluorescence on the striations of the larval cuticle. The patterns of phosphorylated proteins were modified when larvae were incubated with bile.

Tumor necrosis factor alpha activates the phosphorylation of ERK, SAPK/JNK, and P38 kinase in primary cultures of neurons.

Emerging data indicate that the inflammatory cytokine TNFalpha exerts a neuroprotective effect against brain injury. To better understand the mechanism of action of TNFalpha on neurons we have investigated the possible activation of various MAP kinases. Exposure of neurons to TNFalpha triggered the rapid phosphorylation of three members of the MAP kinase family, i.e., extracellular signal-regulated kinase (ERK1/2), stress-activated protein kinase/JUN N-terminal kinase (SAPK/JNK) and the p38 kinase; this activation occured with the same time course and was transient. The TNFalpha-induced activation of ERK1/2 was specifically prevented by compound PD 98059 a specific inhibitor of the MAP kinase kinase MEK1/2. Activation of ERK1/2 was also specifically inhibited by the xanthogenic derivative D609, a specific inhibitor of phosphoinositide phospholipase C suggesting that TNFalpha signaling in neurons involved the acidic sphingomyelinase.

Evidence for the existence of a proteasome in Toxoplasma gondii: intracellular localization and specific peptidase activities.

The proteasome is a large intracellular protein complex whose main function is proteolytic removal of damaged proteins. It has recently been shown that the proteasome has a crucial role in the pathogenesis of protozoan parasites. We attempted to characterize the proteasome of T. gondii (RH strain). In immunoblot experiments, we showed that MCP231 monoclonal antibody, directed against the human 20S proteasome, labelled homologous proteins in T. gondii with a pattern similar to that observed in mammalian cells. The study of in vitro proteolytic activities showed that chymotrypsin-like activity (the only activity obtained with archaebacteria) was present in Toxoplasma, with Km and specific activity values close to those observed with eukaryotic cells. Immunofluorescence studies showed that the Toxoplasma proteasome predominated in the cytosol.

[Mitogen activated protein kinases (MAPK) and Toxoplasma gondii host cell invasion]. / Rôle des mitogen-activated protein (MAP) kinases dans l'invasion de la cellule hôte par Toxoplasma gondii.

Toxoplasmosis is still a big concern in Public Health in France, regarding two particular aspects: congenital toxoplasmosis and reactivated toxoplasmosis in immunodeficient patients. Toxoplasma gondii is an obligate intracellular parasite, that can invade almost all nucleated cells. The invasion step has been widely studied, but its accurate mechanism and the cell receptor remain largely unknown. In this work, we have attempted to investigate the role of kinases and signal transduction in the host cell penetration. We characterized mitogen-activated protein kinases (MAPK). in the parasite itself by immunoblotting, immunofluorescence, and determination of enzymatic activity. In particular, we identified two proteins of 43 and 47 kDa, that could be homologues of extracellular signal-regulated kinases (ERK), i.e. ERK2 and ERK1, respectively. These parasite MAPK are activated by calcium and phorbolmyristyl acetate and inhibited by RO 31-8220, suggesting an activation through protein kinases C (PKC). Indeed the MAPK kinase (= MEK) inhibitor PD98059 inhibits the activation of parasite MAPK, suggesting that a MEK homologue could be responsible for the dual phosphorylation of MAPK on tyrosine and threonine residues, necessary for their activation. Finally, we demonstrated that activation or inhibition of parasite MAPK by preincubation of parasites with various activators or inhibitors led to an increased or reduced host cell invasion in vitro by parasites, respectively. All these results are in favour of a role of ERK-type parasite kinases in T. gondii infectivity in vitro.

Biochemical characterization of mitogen-activated protein (MAP) kinase activity in Toxoplasma gondii.

Mitogen-activated protein (MAP) kinase or extracellular signal-regulated kinase (ERK) are activated by many extracellular stimuli. In this study, we investigated whether MAP kinase and tyrosine kinases were involved in transducing signals in Toxoplasma gondii. Using anti-phosphotyrosine and anti-active ERK antibodies, we identified several phosphorylated proteins in Toxoplasma. In particular, phosphorylation of a 47 kDa and a 43 kDa protein increased strongly after calcium influx. MAP kinase activity, caused by calcium influx, was determined using either a specific synthetic peptide, or an in gel kinase assay. Conversely, calcium chelators (BAPTA and EGTA) and a calcium channel blocker (nifedipine) inhibited this activation. Also, a specific inhibitor of MAP kinase kinase (PD 098059) blocked MAP kinase activity. Three specific anti-MAP kinase antibodies recognized the 47 kDa and 43 kDa proteins, which were putatively identified as ERK1- and ERK2-homologs, respectively. These findings provide early evidence of signal transduction involving members of the MAP kinase family in T. gondii.

Involvement of the mitogen-activated protein (MAP) kinase signalling pathway in host cell invasion by Toxoplasma gondii.

Little is known about signalling in Toxoplasma gondii, but it is likely that protein kinases might play a key role in the parasite proliferation, differentiation and probably invasion. We previously characterized Mitogen-Activated Protein (MAP) kinases in T. gondii lysates. In this study, cultured cells were tested for their susceptibility to Toxoplasma gondii infection after tachyzoite pretreatment with drugs interfering with MAP kinase activation pathways. Protein kinases inhibitors, i.e. genistein, RO31-8220 and PD098059, reduced tachyzoite infectivity by 38 +/- 4.5%, 85.5 +/- 9% and 56 +/- 10%, respectively. Conversely, protein kinases activators, i.e. bombesin and PMA, markedly increased infectivity (by 202 +/- 37% and 258 +/- 14%, respectively). These results suggest that signalling pathways involving PKC and MAP kinases play a role in host cell invasion by Toxoplasma.

Dopamine receptors and groups I and II mGluRs cooperate for long-term depression induction in rat prefrontal cortex through converging postsynaptic activation of MAP kinases.

Tetanic stimuli to layer I-II afferents in rat prefrontal cortex induced long-term depression (LTD) of layer I-II to layer V pyramidal neuron glutamatergic synapses when tetani were coupled to bath application of dopamine. This LTD was blocked by the following metabotropic glutamate receptor (mGluR) antagonists coapplied with dopamine: (S)-alpha-methyl-4-carboxyphenylglycine (MCPG; group I and II antagonist), (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA; group I antagonist), or (RS)-alpha-methylserine-O-phosphate monophenyl ester (MSOPPE; group II antagonist). This suggests that the dopamine-facilitated LTD requires synaptic activation of groups I and II mGluRs during tetanus. LTD could also be induced by coupling tetani to bath application of groups I and II mGluR agonist (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD). In the next series of experiments, coapplication of dopamine and 1S,3R-ACPD, but not application of either drug alone, consistently induced LTD without tetani or even single test stimuli during drug application, suggesting that coactivation of dopamine receptors and the mGluRs is sufficient for LTD induction. Immunoblot analyses with anti-active mitogen-activated protein kinases (MAP-Ks) revealed that D1 receptors, D2 receptors, group I mGluRs, and group II mGluRs all contribute to MAP-K activation in prefrontal cortex, and that combined activation of dopamine receptors and mGluRs synergistically or additively activate MAP-Ks. Consistently, LTD by dopamine + 1S, 3R-ACPD coapplication, as well as the two other forms of LTD (LTD by dopamine + tetani and LTD by 1S,3R-ACPD + tetani), was blocked by bath application of MAP-K kinase inhibitor PD98059. LTD by dopamine + 1S,3R-ACPD coapplication was also blocked by postsynaptic injection of synthetic MAP-K substrate peptide. Our results suggest that dopamine receptors and groups I and II mGluRs cooperate to induce LTD through converging postsynaptic activation of MAP-Ks.

Mode of regulation of the extracellular signal-regulated kinases in the pancreatic beta-cell line MIN6 and their implication in the regulation of insulin gene transcription.

Physiological concentrations of glucose that lead to Ca2+ entry and insulin secretion activate extracellular signal-regulated protein kinases (ERK1 and ERK2) in the MIN6 pancreatic beta-cell line. Here we show that this activation is inhibited by the down-regulation of protein kinase C (PKC) and by genistein, an inhibitor of protein tyrosine kinases. In contrast with results obtained in other cell types, neither the epidermal growth factor activity nor the Src family protein tyrosine kinases seem to be involved in the Ca2+-dependent activation of ERKs. inhibition of tyrosine phosphatases by vanadate leads to the activation of ERKs. As observed in the response to glucose, this activation is dependent on Ca2+ entry through L-type voltage-dependent Ca2+ channels. Thus the activation of ERKs in response to glucose depends on PKC and possibly on a tyrosine kinase/tyrosine phosphatase couple. To define the role of ERK activation by glucose we studied the regulation of transcription of the insulin gene. We found that this transcription is regulated in the MIN6 cells in the same range of glucose concentration as in primary islets, and that specific inhibition of mitogen-activated protein kinase kinase, the direct activator of ERK, impaired the response of the insulin gene to glucose. This was observed by analysis of the transfected rat insulin I gene promoter activity and a Northern blot of endogenous insulin mRNA.

Rapid activation and nuclear translocation of mitogen-activated protein kinases in response to physiological concentration of glucose in the MIN6 pancreatic beta cell line.

MIN6 is one of the few pancreatic beta cell lines that respond to physiological concentrations of glucose by secreting insulin, and little is known about the triggered molecular mechanisms. We report below that the response to glucose in the MIN6 cells includes an activation of the p42 and p44 mitogen-activated protein (MAP) kinases (ERK2 and ERK1). This activation also occurred with the antidiabetic sulfonylurea glibenclamide and kainate, a specific agonist of a subtype of the ionotropic glutamate receptors, which depolarize the cytoplasmic membrane. The requirement for a calcium entry through the L-type voltage-gated channels and other characteristics of the regulation of the MAP kinase activity, such as the effect of the elevation of the cAMP concentration by forskolin, were similar to those of the secretion of insulin. However, the activation of the MAP kinases is not required for the secretion of insulin, inasmuch as this effect of glucose was not abolished when the MAP kinases were prevented from activation by PD098059, an inhibitor of the MAP kinase kinase. However, as the MAP kinases were translocated into the nucleus, they might be implicated in the calcium-dependent transcriptional response of the cells to glucose and thus regulate the expression of the insulin gene.

Neurons in primary culture are less efficiently infected by Toxoplasma gondii than glial cells.

Infection by Toxoplasma gondii is asymptomatic, leading to an immune response that controls the disease. In immune-compromised patients, however, quiescent cysts can reactivate, leading to toxoplasmic encephalitis. We studied the infection of cells of the central nervous system in an attempt to understand the process leading to this complication. Primary cultures of rat hippocampal glial cells and neurons were infected with the virulent strain RH and examined by immunofluorescent microscopy after fixation of cells and staining with antibodies specific to the different cell types. After 24 h of infection, glial cells were highly infected and showed active division of the parasite. Neurons, on the other hand, were much less efficiently infected than glial cells, but actual penetration of the parasites was demonstrated by confocal microscopy. Whereas glial cells contained vacuoles with several parasites, the vacuoles observed in neurons usually contained one parasite or, rarely, two, indicating that the parasites inside neurons did not undergo active division. This was corroborated by determination of the incorporation of [3H]-uracil. Little is known about the mechanism of neuronal infection by Toxoplasma. The experimental setup used in this study should help to improve our understanding of neuronal infection and bring insight into the physiopathology of toxoplasmic encephalitis.

Differential expression of PKC isoforms in hippocampal neuronal cultures: modifications after basic FGF treatment.

Protein kinase C (PKC) is present in high concentrations in neuronal tissues and participates in various neuronal functions. Ten isoforms have so far been identified and each PKC isoform may be activated by a variety of stimuli. By immunoblot analysis the presence of PKC isoforms was examined in dissociated cell cultures of the hippocampus and during the development in vivo. As soon as embryonic day 17 the hippocampus contains detectable amounts of PKC epsilon and zeta and low levels of PKC alpha. PKC beta and gamma appear during the first and second post-natal week. All isoforms progressively increase until the adult age. Cultures of hippocampal neurons derived from rat embryos express PKC alpha, epsilon and zeta whereas PKC beta and gamma are undetectable; a distinct pattern is observed in cultures of hippocampal glial cells. The neuronal levels of PKC alpha, epsilon and zeta increase during the period in culture and are enhanced when hippocampal neurons are exposed to the continuous presence of basic fibroblast growth factor. Immunofluorescence of PKC epsilon and zeta occupies all the cytoplasmic neuronal compartment. The early expression of some PKC isoforms in cultures of post-mitotic hippocampal neurons suggest their involvement in morphological events that occur during this period; in particular the neuritic outgrowth.

Implication of protein kinase C in mechanisms of potassium-induced long-term potentiation in rat hippocampal slices.

The involvement of Ca2+/phospholipid-dependent (alpha, beta, gamma, PKCs) and Ca(2+)-independent PKC (epsilon and zeta isoforms) in mechanisms of long-term potentiation was investigated in CA1 hippocampal slices, using a brief high potassium pulse (50 mM, 40 s) to induce long-term potentiation (K+/LTP). The K+ pulse induced first, in 15 s a translocation of PKC activity to the membrane. This was rapidly followed, from 1 to 60 min after the pulse, by a selective activation of PKC in the cytosol. This activation, which could be blocked by the NMDA (N-methyl-D-aspartate) receptor antagonist 2-amino-5-phosphonovalerate (APV), was associated with a significant increase n immunoreactivity for gamma PKC in he cytosol, and also to a less degree for beta PKC. In contrast, application of the phorbol ester PMA (phorbol 12-mirystate 13 acetate) to other slices induced a rapid and persistent translocation to the membrane of alpha, beta, epsilon and zeta PKCs. A major role for the activation role for the activation of cytosolic gamma PKC in the maintenance of LTP is discussed.

Potassium chloride pulse enhances mitogen-activated protein kinase activity in rat hippocampal slices.

Mitogen-activated protein (MAP) kinases have been implicated in multiple responses to extracellular stimuli. In this study we show that MAP kinase activity is enhanced after a KCl pulse. This activation correlates with an increased tyrosine phosphorylation of a 42-kDa protein as determined by antiphosphotyrosine immunoblot. The same band is found in an anti-MAP kinase immunoblot. Activity is enhanced within 1 min, reaches a maximum at 2 min, and returns to basal level after 10 min. A second peak of activity is observed between 12 and 30 min. The activation is completely blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), showing the involvement of the AMPA type of glutamate receptor. Partial inhibition of MAP kinase activation by 2-amino-5-phosphonovalerate (APV) also shows the involvement of the NMDA receptor. Because the KCl pulse used induces long-term potentiation (LTP) in rat hippocampal slice, we conclude that MAP kinase may be involved in neuronal transduction events leading to LTP.

Identification and cellular localization of protein kinase C isoforms in cultures of rat type-1 astrocytes.

We have examined which isoforms of protein kinase C were present in rat brain astrocytes and found that: (1) the total of calcium-independent isoforms was greater than the total of calcium-dependent isoforms; (2) there were differences in the intracellular distribution of different isoforms; and (3) the abundance of total protein kinase C was greater in astrocytes from cortex than astrocytes from diencephalon.

Different GABAB-mediated effects on protein kinase C activity and immunoreactivity in neonatal and adult rat hippocampal slices.

The effects of GABA on protein kinase C (PKC) were investigated in rat hippocampal slices at various postnatal ages [postnatal day (P) 1-P60]. At P4, GABA (300 microM) induced a rapid (in 1-2 min) 40-50% increase of PKC activity in the membrane fraction and a decrease in the cytosol. These effects were mediated by GABAB receptors because (a) they were neither blocked by 10 microM bicuculline nor reproduced by 10 microM isoguvacine and (b) they were mimicked by the GABAB agonist baclofen (3-30 microM), an effect fully antagonized by the GABAB antagonist 2-hydroxysaclofen (10 microM). A baclofen-induced increased PKC activity in the membrane fraction was only present during the early postnatal period (P1-P14); it was associated with a translocation from the cytosol to the membrane of the immunoreactivity of some PKC isoforms (alpha-, beta-, and epsilon-PKCs). In contrast, after P21, PKC activity and alpha-, beta-, epsilon-, and gamma-PKC immunoreactivities were decreased by baclofen in the membrane fraction and increased in the cytosol. These results suggest that the stimulation of GABAB receptors differentially modulates PKC activity via distinct second messenger pathways in developing and mature hippocampi.

A fraction enriched in rat hippocampal mossy fibre synaptosomes contains trophic activities.

Subcellular fractions prepared from the rat hippocampus, were assessed for the presence of trophic activities. The cytosol of synaptosomal fractions induced mitotic reinitiation of confluent 3T3 fibroblasts. The synaptosomal fraction, enriched in mossy fibre terminals, contained the highest mitotic activity. The mitogenic activity was heat and trypsin sensitive, suggesting that polypeptides are involved. The cytosol of the mossy fibre synaptosomal fraction promoted neuritic outgrowth of PC 12 cells and embryonic hippocampal neurones in primary cultures. These results suggest that mossy fibres contain both mitogenic and neurotrophic activities. These factors could participate in mossy fibre sprouting that occur following brief seizures or experimental lesions.

Subcellular fractionation on Percoll gradient of mossy fiber synaptosomes: evoked release of glutamate, GABA, aspartate and glutamate decarboxylase activity in control and degranulated rat hippocampus.

Using discontinuous density gradient centrifugation in isotonic Percoll sucrose, we have characterized two subcellular fractions (PII and PIII) enriched in mossy fiber synaptosomes and two others (SII and SIII) enriched in small synaptosomes. These synaptosomal fractions were compared with those obtained from adult hippocampus irradiated at neonatal stage to destroy granule cells and their mossy fibers. Synaptosomes were viable as judged by their ability to release aspartate, glutamate and GABA upon K+ depolarization. After irradiation, compared to the control values, the release of glutamate and GABA was decreased by 57 and 74% in the PIII fraction, but not in the other fractions and the content of glutamate, aspartate and GABA was also decreased in PIII fraction by 62, 44 and 52% respectively. These results suggest that mossy fiber (MF) synaptosomes contain and release glutamate and GABA. Measurement of the GABA synthesizing enzyme, glutamate decarboxylase, exhibited no significant difference after irradiation, suggesting that GABA is not synthesized by this enzyme in mossy fibers.

Subcellular fractionation on Percoll gradient of mossy fiber synaptosomes: morphological and biochemical characterization in control and degranulated rat hippocampus.

A method for preparation of hippocampal mossy fiber synaptosomes directly from the postnuclear pellet is presented. This method represents an adaptation of that previously described for the isolation of synaptosomes by centrifugation through Percoll gradients directly from the supernatant fraction. We have characterized by electron microscopy two fractions, PII and PIII, enriched in mossy fiber synaptosomes; fraction PIII had 75% mossy fiber synaptosomes with well-preserved morphology (large size 3 microns, complex morphology, high synaptic vesicle density, multisynapses), whereas fraction PII contained 12%. These fractions were enriched in lactate dehydrogenase activity indicating that the integrity of synaptosomes was preserved. Compared with the other synaptosomal fractions, these fractions showed greater levels of dynorphin A (1-8) immunoreactivity and endogenous zinc, which are particularly concentrated in hippocampal mossy fiber terminals. Furthermore, we prepared synaptosomes from adult hippocampus after neonatal irradiation, which destroys the majority of granule cells and associated mossy fibers. The levels of dynorphin and zinc decreased by 88 and 70% in fraction PII and by 95 and 90%, respectively, in PIII. These results suggest that the rapid Percoll procedure is convenient for the purification of mossy fiber synaptosomes.

Basic fibroblast growth factor-induced increase in zif/268 and c-fos mRNA levels is Ca2+ dependent in primary cultures of hippocampal neurons.

Basic fibroblast growth factor (bFGF) is present in the developing rat brain and has been shown to provide critical trophic support for hippocampal neurons in culture. The influence of bFGF on the expression of mRNAs encoding the transcription factors zif/268 and c-fos was studied in primary cultures of hippocampal neurons (derived from rat embryos) using reverse transcription-coupled PCR. In these cultures grown for 3 days in the absence of serum, bFGF causes a dramatic and transient increase in the levels of zif/268 and c-fos, within 15 and 30 min, respectively. A similar induction of these two early genes occurs following activation of protein kinase C (PKC). The bFGF-induced activation persists after PKC desensitization but is inhibited by chelation of intracellular Ca2+. These results suggest that in primary cultures of hippocampal neurons, bFGF induces the expression of immediate early genes through a pathway that requires Ca2+ mobilization.