Early programmed cell death in human NT2 cell cultures during differentiation induced by all-trans-retinoic acid.

Previous studies have demonstrated that programmed cell death takes place at different stages during the development of the CNS in vivo. Our purpose in this study was to detect early programmed cell death associated with the induction of differentiation by retinoic acid (RA) in the NT2 cell line. By using the annexin V labeling as a marker of apoptosis, a significant apoptotic cell death was quantified during the third and the fourth days of the RA treatment. Double-labeling studies using the staining of the genomic DNA strand breaks with the terminal deoxyribosyl-transferase-mediated dUTP nick end-labeling (TUNEL) assay and either nestin or microtubule-associated protein 2 (MAP2) showed that 1) the early apoptotic cell death affected mostly nestin-positive cells and 2) after 8 days of differentiation, although cells with neuronal phenotypes are present, no colabeled TUNEL/MAP2 cells were detected. With regard to the neuronal protein MAP2, we observed discrete immunolabeling of a few NT2 cells as early as day 3 of the differentiation and a significant emergence of MAP2-immunopositive cells at days 6-8. Thus, our results show that, when as a whole the differentiating NT2 cell population is considered, 1) the apoptotic cell death observed during the third day of differentiation occurs mostly in undifferentiated cells, 2) this process coincides with the first detection of the neuronal phenotype in NT2 cell cultures, and 3) the end of the cell death period in NT2 cell cultures is marked by both the accumulation of MAP2-positive cells and the beginning of expression of the Bcl-2 protein in the cultures.

Differential involvement of the sigma(1) (sigma(1)) receptor in the anti-amnesic effect of neuroactive steroids, as demonstrated using an in vivo antisense strategy in the mouse.

1. The sigma(1) (sigma(1)) receptor cDNA was cloned in several animal species. Molecular tools are now available to identify its endogenous effectors, such as neuroactive steroids, and to establish its precise physiological role. In particular, the sigma(1) receptor is involved in memory processes, as observed in pharmacological and pathological rodent models of amnesia. 2. In order to establish the involvement of sigma(1) receptors in memory, a 16-mer oligodeoxynucleotide antisense to the sigma(1) receptor cDNA (aODN), and its mismatched control (mODN) were prepared and centrally administered into the mouse brain. The anti-amnesic effects induced by the selective sigma(1) agonist PRE-084 and the steroid dehydroepiandrosterone (DHEA) sulphate or pregnenolone sulphate were examined in ODN-treated animals. 3. The aODN treatment failed to affect the dissociation constant (K(d)) but significantly decreased the number of sigma(1) sites (B(max)) labelled with [(3)H]-(+)-SKF-10,047 in the hippocampus and cortex. In these structures, the in vivo binding levels were also diminished, according to the dose and number of injections, as compared with control animals injected with saline or mODN. 4. Cannulation and injections failed to affect the open-field behaviour of the animals. However, the anti-amnesic effects of PRE-084 and DHEA sulphate against the dizocilpine-induced impairments were blocked after aODN treatment in the short- and long-term memory tests. The anti-amnesic effects of pregnenolone sulphate remained unchanged. 5. These observations bring a molecular basis to the modulatory role of sigma(1) receptors in memory, and reveal that the anti-amnesic action of neuroactive steroids may not similarly involve an interaction with sigma(1) receptors.

Human NT2 neurons express a large variety of neurotransmission phenotypes in vitro.

The NT2 cell line, which was derived from a human teratocarcinoma, exhibits properties that are characteristic of a committed neuronal precursor at an early stage of development. NT2 cells can be induced by retinoic acid to differentiate in vitro into postmitotic central nervous system (CNS) neurons (NT2-N cells). The commitment of NT2-N cells to a stable neuronal phenotype is irreversible. Because it may be possible to transplant these human neurons to compensate for neuronal loss after traumatic injuries or neurodegenerative diseases of the CNS, knowledge of their phenotype is essential. This study aimed to characterize in detail the neurotransmission phenotype of NT2-N cells by using immunocytochemical methods. Single peroxidase immunostaining demonstrated that NT2-N cells expressed the gamma-aminobutyric acidergic (GABAergic), catecholaminergic, and cholinergic phenotypes to a large extent and expressed the serotonergic phenotype to a minor extent. NT2-N cells also expressed different neuropeptides, such as neuropeptide Y, oxytocin, vasopressin, calcitonin gene-related peptide, and Met- and Leu-enkephalin. Double fluorescence immunostaining further indicated that a large number of NT2-N cells could express GABA and another neurotransmitter or neuropeptide at the same time. Finally, electron microscopy demonstrated that these NT2 neurons elaborate classical synaptic contacts. The multipotentiality of these neurons, combined with their apparent functionality, suggests that they may represent useful material for a variety of therapeutic approaches aimed at replacing dead neurons after neurodegenerative diseases or lesions of the CNS.

Differential expression of Bcl-2-related proteins in differentiating NT2 cells.

Although the role of Bcl-2-related proteins as regulators of the apoptotic process has been well documented, recent studies suggest that they might also be implicated in neuronal differentiation. We have studied by immunocytochemistry, Western blotting and RT-PCR the expression pattern of Bcl-xL, Bcl-2 and BAX in the in vitro model of neuronal differentiation constituted by retinoic acid (RA)-treated NTera-2/D1 (NT2/D1) cells. Whereas BAX level did not change significantly during the RA treatment, Bcl-xL level increased markedly during the first week, before returning to basal level during the second week. Bcl-2 expression, undetectable in undifferentiated cells, increased progressively from the first week. From our results, we suggest that, at least in our model, Bcl-2-related proteins might be involved in neuronal differentiation.

Immunocytochemical localization of the sigma(1) receptor in the adult rat central nervous system.

In order to characterize the localization of the sigma(1) receptor in the adult rat central nervous system, a polyclonal antibody was raised against a 20 amino acid peptide, corresponding to the fragment 143-162 of the cloned sigma(1) receptor protein. Throughout the rostrocaudal regions of the central nervous system extending from the olfactory bulb to the spinal cord, intense to moderate immunostaining was found to be associated with: (i) ependymocytes bordering the entire ventricular system, and (ii) neuron-like structures located within the parenchyma. Double fluorescence studies confirmed that, throughout the parenchyma, sigma(1) receptor-immunostaining was essentially associated with neuronal structures immunostained for the neuronal marker betaIII-tubulin. In all rats examined, high levels of immunostaining were always associated with neurons located within specific regions including the granular layer of the olfactory bulb, various hypothalamic nuclei, the septum, the central gray, motor nuclei of the hindbrain and the dorsal horn of the spinal cord. In contrast, only faint immunostaining was associated with neurons located in the caudate-putamen and the cerebellum. Electron microscope studies indicated that sigma(1) receptor immunostaining was mostly associated with neuronal perikarya and dendrites, where it was localized to the limiting plasma membrane, the membrane of mitochondria and of some cisternae of the endoplasmic reticulum. At the level of synaptic contacts, intense immunostaining was associated with postsynaptic structures including the postsynaptic thickening and some polymorphous vesicles, whereas the presynaptic axons were devoid of immunostaining. These data indicate that the sigma(1) receptor antibody prepared here, represents a promising tool for further investigating the role of sigma(1) receptors.

Role of rho proteins in agonist regulation of phospholipase D in HL-60 cells.

Rho family GTP-binding proteins have been demonstrated to play a role in the regulation of phospholipase D (PLD) activity. In the present study, we examined the role of Rho proteins in PLD activation in differentiated HL-60 cells using C3 exoenzyme from Clostridium botulinum, which ADP-ribosylates and inactivates Rho proteins. Introduction of C3 exoenzyme into differentiated HL-60 cells by electroporation resulted in complete inhibition of PLD activity stimulated by formyl methionine-leucine-phenylalanine (fMLP) and ATP, two receptor agonists. Phorbol myristate acetate-induced PLD activation was also inhibited in C3 exoenzyme-treated cells, but the inhibition was only partial. GTPgammaS-dependent activation of PLD, measured in the absence or presence of ATP in permeabilized cells, was also partially affected by C3 exoenzyme treatment. Thus, these results indicate that Rho proteins play a key role in receptor-mediated PLD regulation in differentiated HL-60 cells, but play a partial role in the in vivo action of PMA and in vitro action of GTPgammaS on PLD. ATP produced a significant enhancement of the in vitro effect of GTPgammaS on PLD activity, but the effect of ATP was not altered by inhibitors of serine/threonine and tyrosine kinases. However, it was markedly reduced by neomycin and accompanied by an increase in phosphatidylinositol 4,5-bisphosphate (PtdInsP2) synthesis. These data indicate that in permeabilized HL-60 cells, the stimulatory effect of ATP on PLD does not involve protein phosphorylation but is due to an increase in PtdInsP2.

Effects of brefeldin A on phosphatidylcholine phospholipase D and inositolphospholipid metabolism in HL-60 cells.

The involvement of the small GTP-binding protein ADP-ribosylation factor (ARF) in guanosine 5'-[gamma-thio]triphosphate (GTP[S])-dependent activation of phospholipase D (PLD) in HL-60 cells has been well established in vitro. In this study, we tested the effect of brefeldin A, which prevents ARF activation by inhibiting guanine-nucleotide-exchange activity, on PLD stimulation by receptor agonists (formyl-Met-Leu-Phe and ATP) and by the phorbol ester phorbol 12-myristate 13-acetate (PMA) in differentiated HL-60 cells. However, brefeldin A did not affect the activation of PLD at a time (1 h) when it eliminated the activity of the trans-Golgi enzyme galactosyltransferase. It also did not inhibit PLD activity in Golgi-enriched membranes treated with GTP[S] with or without ARF in vitro. However, longer times of brefeldin A treatment (>6 h), progressively and completely inhibited the activation of PLD by formyl-Met-Leu-Phe and partly inhibited (approximately 50%) the activation by PMA. In contrast, long-term brefeldin A treatment did not inhibit the effect of GTP[S] on PLD in permeabilized HL-60 cells. Long-term brefeldin A treatment completely inhibited inositol phosphate production in response to formyl-Met-Leu-Phe and ATP, indicating that it affected inositolphospholipid-specific phospholipase C activity. These data indicate that the rapid inhibitory effect of brefeldin A on Golgi function is not associated with inhibition of receptor-mediated or PMA-mediated PLD activation in HL-60 cells. However, longer-term effects, presumably arising from the disruption of the Golgi, lead to a total inhibition of agonist activation of PLD and inositolphospholipid-specific phospholipase C. In summary, these results do not support a role for brefeldin-A-sensitive ARF in agonist regulation of PLD in HL-60 cells.

Immunolocalization of Bcl-xL/S in the central nervous system of neonatal and adult rats.

A polyclonal antibody raised against a peptide corresponding to the (2-19) amino-terminal sequence of the Bcl-xL/S protein was used to localize Bcl-x immunostaining in the central nervous system of rats at various postnatal ages. Whereas Bcl-x immunostaining was present in virtually all neurons of young animals (4 days postnatal), this staining became progressively restricted during the course of postnatal development. In adults, Bcl-x immunostaining was particularly strong in certain neurons present in a few hypothalamic nuclei, such as the supraoptic or the arcuate nuclei. Moderate staining was observed in some discrete brain regions, such as the olfactory bulb, the hippocampus, some catecholaminergic nuclei of the brainstem, and the cerebellum. Strong Bcl-x immunostaining was also exhibited in axon-like fibers located in the pyriform cortex, the median eminence, the dorsal medulla oblongata, and spinal cord. Bcl-x immunostaining was also present in astrocytes scattered throughout the white matter in the brain and the spinal cord, but was absent from those located in gray matter. Staining was particularly strongly expressed in reactive astrocytes densely packed along the borders of a central lesion or surrounding them, and in a large number of reactive astrocytes detected at a distance from the lesion. Our data suggest that, in addition to the possible stimulating effects on cell survival generally ascribed to Bcl-x, its maintained expression throughout adulthood or its re-expression following injury characterizes those neuronal or non-neuronal cells of the adult central nervous system that synthesize a range of molecules enabling them to adapt rapidly and successfully to a changing environment.

Protein kinase C and phospholipase D activation in rat parotid glands.

We have previously demonstrated that muscarinic and alpha-adrenergic receptors regulated a phospholipase D (PLD) activity in parotid glands. Since phorbol 12-myristate, 13-acetate (PMA) induced production of phosphatidylethanol (PEt), a stable metabolite widely accepted as marker of PLD activation, we have investigated the role of protein kinase C (PKC) in PLD stimulation in parotid acini. We tested PKC inhibitors on PEt formation elicited by PMA, by muscarinic and adrenergic agents. Staurosporine and chelerythrine, which act on the catalytic domain of PKC, did not allow the attribution of a role for PKC in PLD activation. Indeed, staurosporine did not affect PMA-mediated PLD activity and chelerythrine showed an important non-specific effect, independent of PKC inhibition. On the other hand, calphostin C, which acts on the regulatory domain of PKC, affected PMA- and receptor-mediated PLD stimulation. We attributed this effect to PKC inhibition and we suggested PKC involvement in PLD regulation in parotid gland. Since only PKC inhibitor acting on the regulatory part of the enzyme affected PLD activity, we also suggested that PKC could be involved in PLD activation through a pathway independent of the phosphorylation mechanism.

Receptor- and phorbol ester-mediated phospholipase D activation in rat parotid involves two different pathways.

We have investigated phospholipase D (PLD) activation in rat parotid acini prelabeled with [14C]stearic acid. In the presence of 2% ethanol, muscarinic and alpha-adrenergic agonists stimulated the formation of [14C]phosphatidylethanol as a result of a PLD activity. The calcium ionophore, ionomycin, and the phorbol esters, 4 beta-phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu), also stimulated phosphatidylethanol accumulation, but 1-oleyl-2-acetyl-sn-glycerol (OAG), a permeant analogue of diacylglycerol did not. Chelerythrine and staurosporine, two inhibitors of protein kinase C, failed to affect any response. These results suggest that protein kinase C was not involved in the regulation of PLD activity. A difference between PLD regulation by PMA and receptor-mediated agonists was observed with regard to the extracellular calcium requirement. Our results strongly suggest that PLD activation in parotid acini involved different pathways: a calcium-dependent pathway activated by receptor-mediated agonists and a calcium-independent pathway activated by phorbol esters. Moreover, we observed that PLD activation did not result in any change in phosphatidic acid level. We propose that the phosphatidyl transferase activity of PLD reflected a metabolic pathway which may allow a base-exchange reaction in parotid gland.

Evidence for receptor-linked activation of phospholipase D in rat parotid glands. Stimulation by carbamylcholine, PMA and calcium.

In order to test if phospholipase D (PLD) activity exists in the rat parotid gland, we took advantage of the fact that, in the presence of ethanol, PLD generates phosphatidylethanol (PEth) via a transphosphatidylation reaction. Lipid extracts of parotid acini prelabelled with [3H]myristic acid were analyzed by thin layer chromatography to determine [3H]phosphatidylethanol ([3H]PEth) formation. Carbamylcholine (1 mM) stimulated [3H]PEth formation in the presence of 2% ethanol, this effect was completely inhibited by atropine (10 microM). PMA (0.1-1 microM) and ionomycine (10 microM) also caused [3H]PEth generation. We conclude that a phospholipase D activity is present in the rat parotid gland and is regulated by muscarinic cholinergic receptors. Protein kinase C and calcium could also modulate this activity. This report provides the first evidence for the existence and receptor-linked regulation of phospholipase D in an exocrine gland, the rat parotid gland.

The NK-1 receptor and a calcium-phospholipid pathway: inositol trisphosphate production and calcium movements induced by selective agonists of neurokinin receptors in rat parotid glands.

In the rat parotid gland, substance P has been shown to induce a phosphatidylinositol bisphosphate breakdown resulting in an inositol trisphosphate production. These data suggested that substance P activated a phospholipase C and thus mediated its effects through the calcium-phospholipid pathway. To determine which neurokinin (NK) receptor was involved in the substance P response, we have used selective agonists of the different NK receptors and examined their effects on both inositol trisphosphate production and calcium movements. A selective NK-1 receptor agonist, [Sar9Met(O2)11]-substance P, evoked an [3H]inositol trisphosphate production and a rapid and transient 45Ca2+ efflux. On the other hand, selective NK-2 and NK-3 receptor agonists, [beta-Ala8]-NKA(4-10) and [MePhe7]-NKB, respectively, were without effect. We conclude that, in the rat parotid glands, only the NK-1 receptors are coupled to the calcium-phospholipid pathway. The C-terminal part of substance P appeared to be sufficient to stimulate this route because the C-terminal octapeptide, substance P(4-11), mimicked substance P effects on both inositol trisphosphate production and calcium movements. The NK-2 and NK-3 receptors, if present in the rat parotid glands, are not associated with the calcium-phospholipid pathway.

Involvement of NK1 receptors and importance of the N-terminal sequence of substance P in the stimulation of protein secretion in rat parotid glands.

Recent in vitro studies have shown that the dose-response curve of substance P on [3H]protein secretion from rat parotid glands is biphasic. Such a response could result either from the activation of tachykinin receptors or from the amphiphilic character of substance P, since it has previously been shown that the N-terminal part of substance P may play an important role in the activation of phosphoinositides in rat parotid glands. To investigate these possibilities, we studied the effects of selective NK1, NK2, NK3 receptor agonists and C-terminal fragments of substance P and neurokinin A on protein secretion from rat parotid lobules. The poor activity of NK2 (neurokinin A-(4-10) and [beta-Ala8]neurokinin A-(4-10)) as well as of NK3 ([MePhe7]neurokinin B) selective agonists allowed us to rule out a possible involvement of NK2 and NK3 receptors in the parotid gland secretory process. Conversely, the selective NK1 receptor agonist, [Sar9,Met(O2)11]substance P, reproduced the biphasic dose-response curve for [3H]protein secretion typical of native substance P. However, a biphasic response was not observed with peptides deprived of the N-terminal moiety of substance P, such as substance P-(4-11) or [AcArg6,Sar9,Met(O2)11] substance P-(6-11). Our data therefore indicate that the [3H]protein secretion obtained with substance P results from the activation of NK1 receptors. Moreover, our data suggest that the N-terminal tripeptide of substance P is also active, and could stimulate different phospholipases either by acting through a second functional site on the NK1 receptor or by directly activating G-proteins.