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During neuropathological states associated with inflammation, the levels of cytokines such as interleukin-1beta (IL-1beta) are increased. Several studies have suggested that the neuronal damage observed in pathogenesis implicating IL-1beta are caused by an alteration in the neurochemical interactions between neurons and astrocytes. We report here that treating striatal astrocytes in primary culture with IL-1beta for 22-24 hr enhances the ATP-evoked release of arachidonic acid (AA) with no effect on the ATP-induced accumulation of inositol phosphates. The molecular mechanism responsible for this effect involves the expression of P2Y2 receptors (a subtype of purinoceptor activated by ATP) and cytosolic phospholipase A2 (cPLA2, an enzyme that mediates AA release). Indeed, P2Y2 antisense oligonucleotides reduce the ATP-evoked release of AA only from IL-1beta-treated astrocytes. Further, both the amount of cPLA2 (as assessed by Western blotting) and the release of AA resulting from direct activation of cPLA2 increased fourfold in cells treated with IL-1beta. We also report evidence indicating that the coupling of newly expressed P2Y2 receptors to cPLA2 is dependent on PKC activity. These results suggest that during inflammatory conditions, IL-1beta reveals a functional P2Y2 signaling pathway in astrocytes that results in a dramatic increase in the levels of free AA. This pathway may thus contribute to the neuronal loss associated with cerebral ischemia or traumatic brain injury.

Neuronal apoptosis induced by survival factor deprivation is strongly regulated at the transcriptional level. Notably, the nuclear factor of activated T cell (NFAT) transcription factors have an important role in the control of the survival/death fate of neurons. However, the mechanisms that regulate NFAT activity in response to apoptotic stimuli and the target genes that mediate their effect on neuronal apoptosis are mostly unknown. In a previous study, we identified Trim17 as a crucial E3 ubiquitin ligase that is necessary and sufficient for neuronal apoptosis. Here, we show that Trim17 binds preferentially SUMOylated forms of NFATc3. Nonetheless, Trim17 does not promote the ubiquitination/degradation of NFATc3. NFAT transcription factors are regulated by calcium/calcineurin-dependent nuclear-cytoplasmic shuttling. Interestingly, Trim17 reduced by twofold the calcium-mediated nuclear localization of NFATc3 and, consistent with this, halved NFATc3 activity, as estimated by luciferase assays and by measurement of target gene expression. Trim17 also inhibited NFATc4 nuclear translocation and activity. NFATc4 is known to induce the expression of survival factors and, as expected, overexpression of NFATc4 protected cerebellar granule neurons from serum/KCl deprivation-induced apoptosis. Inhibition of NFATc4 by Trim17 may thus partially mediate the proapoptotic effect of Trim17. In contrast, overexpression of NFATc3 aggravated neuronal death, whereas knockdown of NFATc3 protected neurons from apoptosis. This proapoptotic effect of NFATc3 might be due to a feedback loop in which NFATc3, but not NFATc4, induces the transcription of the proapoptotic gene Trim17. Indeed, we found that overexpression or silencing of NFATc3, respectively, increased or decreased Trim17 levels, whereas NFATc4 had no significant effect on Trim17 expression. Moreover, we showed that NFATc3 binds to the promoter of the Trim17 gene together with c-Jun. Therefore, our results describe a novel mechanism regulating NFAT transcription factors beyond the calcium/calcineurin-dependent pathway and provide a possible explanation for the opposite effects of NFATc3 and NFATc4 on neuronal apoptosis. [ABSTRACT FROM AUTHOR]

Short-term proteasome inhibition has been shown to prevent neuronal apoptosis. However, the key pro-survival proteins that must be degraded for triggering neuronal death are mostly unknown. Here, we show that Mcl-1, an anti-apoptotic Bcl-2 family member, is degraded by the proteasome during neuronal apoptosis. Using primary cultures of cerebellar granule neurons deprived of serum and KCl, we found that ubiquitination and proteasomal degradation of Mcl-1 depended on its prior phosphorylation by GSK3, providing the first insight into post-translational regulation of Mcl-1 in neurons. In a previous study, we have reported that the E3 ubiquitin-ligase Trim17 is both necessary and sufficient for neuronal apoptosis. Here, we identified Trim17 as a novel E3 ubiquitin-ligase for Mcl-1. Indeed, Trim17 co-immunoprecipitated with Mcl-1. Trim17 ubiquitinated Mcl-1 in vitro. Overexpression of Trim17 decreased the protein level of Mcl-1 in a phosphorylation- and proteasome-dependent manner. Finally, knock down of Trim17 expression reduced both ubiquitination and degradation of Mcl-1 in neurons. Moreover, impairment of Mcl-1 phosphorylation, by kinase inhibition or point mutations, not only decreased ubiquitination and degradation of Mcl-1, but also blocked the physical interaction between Trim17 and Mcl-1. As this stabilization of Mcl-1 increased its neuroprotective effect, our data strongly suggest that Trim17-mediated ubiquitination and degradation of Mcl-1 is necessary for initiating neuronal death. [ABSTRACT FROM AUTHOR]

Neuronal apoptosis has a major role during development and aberrant apoptosis contributes to the pathology of certain neurological conditions. Studies with nerve growth factor (NGF)-dependent sympathetic neurons have provided important insights into the molecular mechanisms of neuronal apoptosis and the signalling pathways that regulate the cell death programme in neurons. The BH3-only protein Bim is a critical mediator of apoptosis in many cell types and in sympathetic neurons is required for NGF withdrawal-induced death. However, regulation of bim expression is complex and remains incompletely understood. We report that a conserved inverted CCAAT box (ICB) in the rat bim promoter is bound by the heterotrimeric transcription factor NF-Y. Interestingly, NF-Y is required for bim promoter activity and its induction following NGF withdrawal. We demonstrate that NF-Y activity is essential for endogenous Bim expression and contributes to NGF withdrawal-induced death. Furthermore, we find that the transcriptional coactivators CBP and p300 interact with NF-Y and FOXO3a and bind to this region of the bim promoter. The amount of CBP/p300 bound to bim increases after NGF deprivation and inhibition of CBP/p300 activity reduces bim induction. Our results indicate that NF-Y cooperates with FOXO3a to recruit CBP/p300 to the bim promoter to form a stable multi-protein/DNA complex that activates bim transcription after survival factor withdrawal. [ABSTRACT FROM AUTHOR]

Viral neuroinfections represent a major health burden for which the development of antivirals is needed. Antiviral compounds that target the consequences of a brain infection (symptomatic treatment) rather than the cause (direct-acting antivirals) constitute a promising mitigation strategy that requires to be investigated in relevant models. However, physiological surrogates mimicking an adult human cortex are lacking, limiting our understanding of the mechanisms associated with viro-induced neurological disorders. Here, we optimized the Organotypic culture of Post-mortem Adult human cortical Brain explants (OPAB) as a preclinical platform for Artificial Intelligence (AI)-driven antiviral studies. OPAB shows robust viability over weeks, well-preserved 3D cytoarchitecture, viral permissiveness, and spontaneous local field potential (LFP). Using LFP as a surrogate for neurohealth, we developed a machine learning framework to predict with high confidence the infection status of OPAB. As a proof-of-concept, we showed that antiviral-treated OPAB could partially restore LFP-based electrical activity of infected OPAB in a donor-dependent manner. Together, we propose OPAB as a physiologically relevant and versatile model to study neuroinfections and beyond, providing a platform for preclinical drug discovery., (© 2024. The Author(s).)

NFATc3 is the predominant member of the NFAT family of transcription factors in neurons, where it plays a pro-apoptotic role. Mechanisms controlling NFAT protein stability are poorly understood. Here we identify Trim39 as an E3 ubiquitin-ligase of NFATc3. Indeed, Trim39 binds and ubiquitinates NFATc3 in vitro and in cells where it reduces NFATc3 protein level and transcriptional activity. In contrast, silencing of endogenous Trim39 decreases NFATc3 ubiquitination and increases its activity, thereby resulting in enhanced neuronal apoptosis. We also show that Trim17 inhibits Trim39-mediated ubiquitination of NFATc3 by reducing both the E3 ubiquitin-ligase activity of Trim39 and the NFATc3/Trim39 interaction. Moreover, we identify Trim39 as a new SUMO-targeted E3 ubiquitin-ligase (STUbL). Indeed, mutation of SUMOylation sites in NFATc3 or SUMO-interacting motifs in Trim39 reduces NFATc3/Trim39 interaction and Trim39-induced ubiquitination of NFATc3. In addition, Trim39 preferentially ubiquitinates SUMOylated forms of NFATc3 in vitro. As a consequence, a SUMOylation-deficient mutant of NFATc3 exhibits increased stability and pro-apoptotic activity in neurons. Taken together, these data indicate that Trim39 modulates neuronal apoptosis by acting as a STUbL for NFATc3., (© 2022. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

Since the Nobel Prize award more than twenty years ago for discovering the core apoptotic pathway in C. elegans , apoptosis and various other forms of regulated cell death have been thoroughly characterized by researchers around the world. Although many aspects of regulated cell death still remain to be elucidated in specific cell subtypes and disease conditions, many predicted that research into cell death was inexorably reaching a plateau. However, this was not the case since the last decade saw a multitude of cell death modalities being described, while harnessing their therapeutic potential reached clinical use in certain cases. In line with keeping research into cell death alive, francophone researchers from several institutions in France and Belgium established the French Cell Death Research Network (FCDRN). The research conducted by FCDRN is at the leading edge of emerging topics such as non-apoptotic functions of apoptotic effectors, paracrine effects of cell death, novel canonical and non-canonical mechanisms to induce apoptosis in cell death-resistant cancer cells or regulated forms of necrosis and the associated immunogenic response. Collectively, these various lines of research all emerged from the study of apoptosis and in the next few years will increase the mechanistic knowledge into regulated cell death and how to harness it for therapy.

The field of the Tripartite Motif (TRIM) family has progressively attracted increasing interest during the last two decades [...].

TRIM17 is a member of the TRIM family, a large class of RING-containing E3 ubiquitin-ligases. It is expressed at low levels in adult tissues, except in testis and in some brain regions. However, it can be highly induced in stress conditions which makes it a putative stress sensor required for the triggering of key cellular responses. As most TRIM members, TRIM17 can act as an E3 ubiquitin-ligase and promote the degradation by the proteasome of substrates such as the antiapoptotic protein MCL1. Intriguingly, TRIM17 can also prevent the ubiquitination of other proteins and stabilize them, by binding to other TRIM proteins and inhibiting their E3 ubiquitin-ligase activity. This duality of action confers several pivotal roles to TRIM17 in crucial cellular processes such as apoptosis, autophagy or cell division, but also in pathological conditions as diverse as Parkinson's disease or cancer. Here, in addition to recent data that endorse this duality, we review what is currently known from public databases and the literature about TRIM17 gene regulation and expression, TRIM17 protein structure and interactions, as well as its involvement in cell physiology and human disorders.

BCL2A1 is an anti-apoptotic member of the BCL-2 family that contributes to chemoresistance in a subset of tumors. BCL2A1 has a short half-life due to its constitutive processing by the ubiquitin-proteasome system. This constitutes a major tumor-suppressor mechanism regulating BCL2A1 function. However, the enzymes involved in the regulation of BCL2A1 protein stability are currently unknown. Here, we provide the first insight into the regulation of BCL2A1 ubiquitination. We present evidence that TRIM28 is an E3 ubiquitin-ligase for BCL2A1. Indeed, endogenous TRIM28 and BCL2A1 bind to each other at the mitochondria and TRIM28 knock-down decreases BCL2A1 ubiquitination. We also show that TRIM17 stabilizes BCL2A1 by blocking TRIM28 from binding and ubiquitinating BCL2A1, and that GSK3 is involved in the phosphorylation-mediated inhibition of BCL2A1 degradation. BCL2A1 and its close relative MCL1 are thus regulated by common factors but with opposite outcome. Finally, overexpression of TRIM28 or knock-out of TRIM17 reduced BCLA1 protein levels and restored sensitivity of melanoma cells to BRAF-targeted therapy. Therefore, our data describe a molecular rheostat in which two proteins of the TRIM family antagonistically regulate BCL2A1 stability and modulate cell death.

Although accumulating data indicate that increased α-synuclein expression is crucial for Parkinson disease (PD), mechanisms regulating the transcription of its gene, SNCA, are largely unknown. Here, we describe a pathway regulating α-synuclein expression. Our data show that ZSCAN21 stimulates SNCA transcription in neuronal cells and that TRIM41 is an E3 ubiquitin ligase for ZSCAN21. In contrast, TRIM17 decreases the TRIM41-mediated degradation of ZSCAN21. Silencing of ZSCAN21 and TRIM17 consistently reduces SNCA expression, whereas TRIM41 knockdown increases it. The mRNA levels of TRIM17, ZSCAN21, and SNCA are simultaneously increased in the midbrains of mice following MPTP treatment. In addition, rare genetic variants in ZSCAN21, TRIM17, and TRIM41 genes occur in patients with familial forms of PD. Expression of variants in ZSCAN21 and TRIM41 genes results in the stabilization of the ZSCAN21 protein. Our data thus suggest that deregulation of the TRIM17/TRIM41/ZSCAN21 pathway may be involved in the pathogenesis of PD., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Glycogenin-interacting protein 1 (GNIP1) is a tripartite motif (TRIM) protein with E3 ubiquitin ligase activity that interacts with glycogenin. These data suggest that GNIP1 could play a major role in the control of glycogen metabolism. However, direct evidence based on functional analysis remains to be obtained., Objectives: The aim of this study was 1) to define the expression pattern of glycogenin-interacting protein/Tripartite motif containing protein 7 (GNIP/TRIM7) isoforms in humans, 2) to test their ubiquitin E3 ligase activity, and 3) to analyze the functional effects of GNIP1 on muscle glucose/glycogen metabolism both in human cultured cells and in vivo in mice., Results: We show that GNIP1 was the most abundant GNIP/TRIM7 isoform in human skeletal muscle, whereas in cardiac muscle only TRIM7 was expressed. GNIP1 and TRIM7 had autoubiquitination activity in vitro and were localized in the Golgi apparatus and cytosol respectively in LHCN-M2 myoblasts. GNIP1 overexpression increased glucose uptake in LHCN-M2 myotubes. Overexpression of GNIP1 in mouse muscle in vivo increased glycogen content, glycogen synthase (GS) activity and phospho-GSK-3α/β (Ser21/9) and phospho-Akt (Ser473) content, whereas decreased GS phosphorylation in Ser640. These modifications led to decreased blood glucose levels, lactate levels and body weight, without changing whole-body insulin or glucose tolerance in mouse., Conclusion: GNIP1 is an ubiquitin ligase with a markedly glycogenic effect in skeletal muscle., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Mcl-1 is an anti-apoptotic protein of the Bcl-2 family that is essential for the survival of multiple cell lineages and that is highly amplified in human cancer. Under physiological conditions, Mcl-1 expression is tightly regulated at multiple levels, involving transcriptional, post-transcriptional and post-translational processes. Ubiquitination of Mcl-1, that targets it for proteasomal degradation, allows for rapid elimination of the protein and triggering of cell death, in response to various cellular events. In the last decade, a number of studies have elucidated different pathways controlling Mcl-1 ubiquitination and degradation. Four different E3 ubiquitin-ligases (e.g., Mule, SCFβ-TrCP, SCFFbw7 and Trim17) and one deubiquitinase (e.g., USP9X), that respectively mediate and oppose Mcl-1 ubiquitination, have been formerly identified. The interaction between Mule and Mcl-1 can be modulated by other Bcl-2 family proteins, while recognition of Mcl-1 by the other E3 ubiquitin-ligases and deubiquitinase is influenced by phosphorylation of specific residues in Mcl-1. The protein kinases and E3 ubiquitin-ligases that are involved in the regulation of Mcl-1 stability vary depending on the cellular context, highlighting the complexity and pivotal role of Mcl-1 regulation. In this review, we attempt to recapitulate progress in understanding Mcl-1 regulation by the ubiquitin-proteasome system.

Polyglutamylation is a posttranslational modification that generates glutamate side chains on tubulins and other proteins. Although this modification has been shown to be reversible, little is known about the enzymes catalyzing deglutamylation. Here we describe the enzymatic mechanism of protein deglutamylation by members of the cytosolic carboxypeptidase (CCP) family. Three enzymes (CCP1, CCP4, and CCP6) catalyze the shortening of polyglutamate chains and a fourth (CCP5) specifically removes the branching point glutamates. In addition, CCP1, CCP4, and CCP6 also remove gene-encoded glutamates from the carboxyl termini of proteins. Accordingly, we show that these enzymes convert detyrosinated tubulin into Δ2-tubulin and also modify other substrates, including myosin light chain kinase 1. We further analyze Purkinje cell degeneration (pcd) mice that lack functional CCP1 and show that microtubule hyperglutamylation is directly linked to neurodegeneration. Taken together, our results reveal that controlling the length of the polyglutamate side chains on tubulin is critical for neuronal survival., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Unlabelled: An unresolved question regarding the physiopathology of hepatitis C virus (HCV) infection is the remarkable efficiency with which host defenses are neutralized to establish chronic infection. Modulation of an apoptotic response is one strategy used by viruses to escape immune surveillance. We previously showed that HCV proteins down-regulate expression of BH3-only Bcl2 interacting domain (Bid) in hepatocytes of HCV transgenic mice. As a consequence, cells acquire resistance to Fas-mediated apoptosis, which in turn leads to increased persistence of experimental viral infections in vivo. This mechanism might participate in the establishment of chronic infections and the resulting pathologies, including hepatocellular carcinoma. We now report that Bid is also down-regulated in patients in the context of noncirrhotic HCV-linked tumorigenesis and in the HCV RNA replicon system. We show that the nonstructural HCV viral protein NS5A is sufficient to activate a calpain cysteine protease, leading to degradation of Bid. Moreover, pharmacological inhibitors of calpains restore both the physiological levels of Bid and the sensitivity of cells toward a death receptor-mediated apoptotic signal. Finally, human HCV-related tumors and hepatocytes from HCV transgenic mice that display low Bid expression contain activated calpains., Conclusion: Calpains activated by HCV proteins degrade Bid and thus dampen apoptotic signaling. These results suggest that inhibiting calpains could lead to an improved efficiency of immune-mediated elimination of HCV-infected cells.

Neuronal apoptosis has been shown to require de novo RNA/protein synthesis. However, very few genes whose expression is necessary for inducing apoptosis have been identified so far. To systematically identify such genes, we have used genome-scale, long oligonucleotide microarrays and characterized the gene expression profile of cerebellar granule neurons in the early phase of apoptosis elicited by KCl deprivation. We identified 368 significantly differentially expressed genes, including most of the genes previously reported to be transcriptionally regulated in this paradigm. In addition, we identified several hundreds of genes whose transcriptional regulation has never been associated with neuronal apoptosis. We used automated Gene Ontology annotation, analysis of promoter sequences, and statistical tools to characterize these regulations. Although differentially expressed genes included some components of the apoptotic machinery, this functional category was not significantly over-represented among regulated genes. On the other hand, categories related to signal transduction were the most significantly over-represented group. This indicates that the apoptotic machinery is mainly constitutive, whereas molecular pathways that lead to the activation of apoptotic components are transcriptionally regulated. In particular, we show for the first time that signaling pathways known to be involved in the control of neuronal survival are regulated at the transcriptional level and not only by post-translational mechanisms. Moreover, our approach provides insights into novel transcription factors and novel mechanisms, such as the unfolded protein response and cell adhesion, that may contribute to the induction of neuronal apoptosis.

Bid plays an essential role in Fas-mediated apoptosis of the so-called type II cells. In these cells, following cleavage by caspase 8, the C-terminal fragment of Bid translocates to mitochondria and triggers the release of apoptogenic factors, thereby inducing cell death. Here we report that Bid is phosphorylated by casein kinase I (CKI) and casein kinase II (CKII). Inhibition of CKI and CKII accelerated Fas-mediated apoptosis and Bid cleavage, whereas hyperactivity of the kinases delayed apoptosis. When phosphorylated, Bid was insensitive to caspase 8 cleavage in vitro. Moreover, a mutant of Bid that cannot be phosphorylated was found to be more toxic than wild-type Bid. Together, these data indicate that phosphorylation of Bid represents a new mechanism whereby cells control apoptosis.

In many types of apoptosis, the proapoptotic protein Bax undergoes a change in conformation at the level of the mitochondria. This event always precedes the release of mitochondrial cytochrome c, which, in the cytosol, activates caspases through binding to Apaf-1. The mechanisms by which Bax triggers cytochrome c release are unknown. Here we show that following binding to the BH3-domain-only proapoptotic protein Bid, Bax oligomerizes and then integrates in the outer mitochondrial membrane, where it triggers cytochrome c release. Bax mitochondrial membrane insertion triggered by Bid may represent a key step in pathways leading to apoptosis.

Here we report that in staurosporine-induced apoptosis of HeLa cells, Bid, a BH3 domain containing protein, translocates from the cytosol to mitochondria. This event is associated with a change in conformation of Bax which leads to the unmasking of its NH2-terminal domain and is accompanied by the release of cytochrome c from mitochondria. A similar finding is reported for cerebellar granule cells undergoing apoptosis induced by serum and potassium deprivation. The Bax-conformational change is prevented by Bcl-2 and Bcl-xL but not by caspase inhibitors. Using isolated mitochondria and various BH3 mutants of Bid, we demonstrate that direct binding of Bid to Bax is a prerequisite for Bax structural change and cytochrome c release. Bcl-xL can inhibit the effect of Bid by interacting directly with Bax. Moreover, using mitochondria from Bax-deficient tumor cell lines, we show that Bid- induced release of cytochrome c is negligible when Bid is added alone, but dramatically increased when Bid and Bax are added together. Taken together, our results suggest that, during certain types of apoptosis, Bid translocates to mitochondria and binds to Bax, leading to a change in conformation of Bax and to cytochrome c release from mitochondria.

During apoptosis induced by various stimuli, cytochrome c is released from mitochondria into the cytosol where it participates in caspase activation. This process has been proposed to be an irreversible consequence of mitochondrial permeability transition pore opening, which leads to mitochondrial swelling and rupture of the outer mitochondrial membrane. Here we present data demonstrating that NGF-deprived sympathetic neurons protected from apoptosis by caspase inhibitors possess mitochondria which, though depleted of cytochrome c and reduced in size, remained structurally intact as viewed by electron microscopy. After re-exposure of neurons to NGF, mitochondria recovered their normal size and their cytochrome c content, by a process requiring de novo protein synthesis. Altogether, these data suggest that depletion of cytochrome c from mitochondria is a controlled process compatible with function recovery. The ability of sympathetic neurons to recover fully from trophic factor deprivation provided irreversible caspase inhibitors have been present during the insult period, has therapeutical implications for a number of acute neuropathologies.

Hydrogen peroxide (H2O2) is suspected to be involved in numerous brain pathologies such as neurodegenerative diseases or in acute injury such as ischemia or trauma. In this study, we examined the ability of pyruvate to improve the survival of cultured striatal neurons exposed for 30 min to H2O2, as estimated 24 hr later by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide assay. Pyruvate strongly protected neurons against both H2O2 added to the external medium and H2O2 endogenously produced through the redox cycling of the experimental quinone menadione. The neuroprotective effect of pyruvate appeared to result rather from the ability of alpha-ketoacids to undergo nonenzymatic decarboxylation in the presence of H2O2 than from an improvement of energy metabolism. Indeed, several other alpha-ketoacids, including alpha-ketobutyrate, which is not an energy substrate, reproduced the neuroprotective effect of pyruvate. In contrast, lactate, a neuronal energy substrate, did not protect neurons from H2O2. Optimal neuroprotection was achieved with relatively low concentrations of pyruvate (

In primary cultures of mouse striatal astrocytes prelabeled with [3H]myristic acid, endothelin (ET)-1 induced a time-dependent formation of [3H]phosphatidic acid and [3H]diacylglycerol. In the presence of ethanol, a production of [3H]phosphatidylethanol was observed, indicating the activation of a phospholipase D (PLD). ET-1 and ET-3 were equipotent in stimulating PLD activity (EC50 = 2-5 nM). Pretreatment of the cells with pertussis toxin partially abolished the effect of ET-1, indicating the involvement of a Gi/G(o) protein. Inhibition of protein kinase C by Ro 31-8220 or down-regulation of the kinase by a long-time treatment with phorbol 12-myristate 13-acetate (PMA) totally abolished the ET-1-induced stimulation of PLD. In contrast, a cyclic AMP-dependent process is not involved in the activation of PLD, because the ET-1-evoked formation of [3H]phosphatidylethanol was not affected when cells were coincubated with either isoproterenol, 8-bromo-cyclic AMP, or forskolin. Acute treatment with PMA also stimulated PLD through a protein kinase C-dependent process. However, the ET-1 and PMA responses were additive. Furthermore, the ET-1-evoked response, contrary to that of PMA, totally dependent on the presence of extracellular calcium. These results suggest that at least two distinct mechanisms are involved in the control of PLD activity in striatal astrocytes. Finally, ET-1, ET-3, and PMA also stimulated PLD in astrocytes from the mesencephalon, the cerebral cortex, and the hippocampus.

The role of cholinergic mechanisms in N-methyl-D-aspartate (NMDA)-mediated neuronal death was investigated using mouse striatal neurones in primary culture. A 30 min exposure of striatal neurones to increasing concentrations of NMDA resulted 24 h later in dramatic neuronal degeneration as assessed by MTT staining, crystal violet incorporation and determination of microtubule-associated protein 2. The NMDA-induced neurodegeneration was strongly inhibited by the co-application of two non-selective cholinergic agonists, acetylcholine or carbachol. This protective effect appears to be mediated by nicotinic receptors since it was insensitive to the muscarinic antagonist atropine but mimicked by nicotine, nornicotine and 1,1-dimethyl-4-phenyl-piperazinium. Moreover, the nicotine-evoked neuroprotection was inhibited by the central nicotinic antagonist hexamethonium. Therefore, this study suggests that cholinergic interneurones play an important role in neuronal survival in the striatum.

We studied the effects of nitric oxide (NO)-producing agents on N-methyl-D-aspartate (NMDA) receptor activation in cultured neurons. 3-Morpholino-sydnonimine (SIN-1) blocked both NMDA-induced currents and the associated increase in intracellular Ca2+. The actions of SIN-1 were reversible and suppressed by hemoglobin. A degraded SIN-1 solution that did not release NO was unable to block NMDA receptors. This showed that the SIN-1 effects were due to NO and not to another breakdown product. Similar results were obtained with 1-nitrosopyrrolidine (an NO-containing drug) and with NO released from NaNO2. Pretreatment with hemoglobin potentiated NMDA-induced effects, demonstrating that endogenous NO modulates NMDA receptors. Since NMDA receptor activation induces NO synthesis, these results suggest a feedback inhibition of NMDA receptors by NO under physiological condition.

The effects of a nitric oxide (NO) donor, sodium nitroprusside (SNP), on N-methyl-D-aspartate (NMDA) receptors were assessed by optical measurements of intracellular calcium concentration ([Ca2+]i) and patch-clamp techniques in cultured central neurons. SNP selectively blocked NMDA-mediated currents and increases in [Ca2+]i. SNP inhibited the binding of [3H]-CGS 19755. The blockade of NMDA responses by SNP was prevented by CPP or APV which are selective competitive NMDA receptor antagonists. These effects were not necessarily mediated by NO, since they were mimicked by ferrocyanide ions, the NO companion photolysis product of SNP.