Mercedes Garcia-Gil

Cytosolic 5′-nucleotidase hyperactivity in erythrocytes of Lesch–nyhan syndrome patients

Lesch–Nyhan syndrome is a metabolic–neurological syndrome caused by the X-linked deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Metabolic consequences of HGPRT deficiency have been clarified, but the connection with the neurological manifestations is still unknown. Much effort has been directed to finding other alterations in purine nucleotides in different cells of Lesch–Nyhan patients. A peculiar finding was the measure of appreciable amount of Z-nucleotides in red cells. We found significantly higher IMP-GMP-specific 5′-nucleotidase activity in the erythrocytes of seven patients with Lesch–Nyhan syndrome than in healthy controls. The same alteration was found in one individual with partial HGPRT deficiency displaying a severe neurological syndrome, and in two slightly hyperuricemic patients with a psychomotor delay. Since ZMP was a good substrate of 5′-nucleotidase producing Z-riboside, we incubated murine and human cultured neuronal cells with this nucleoside and found that it is toxic for our models, promoting apoptosis. This finding suggests an involvement of the toxicity of the Z-riboside in the pathogenesis of neurological disorders in Lesch–Nyhan syndrome and possibly in other pediatric neurological syndromes of uncertain origin.

Effect of 1α,25‐dihydroxyvitamin D3 in embryonic hippocampal cells

Although the role of 1α,25‐dihydroxyvitamin D3 in calcium homeostasis of bone tissue is clear, evidence of the involvement of vitamin D3 in the central nervous system functions is increasing. In fact, vitamin D3 regulates vitamin D receptor and nerve growth factor expression, modulates brain development, and reverses experimental autoimmune encephalomyelitis. Only few studies, however, address vitamin D3 effect on embryonic hippocampal cell differentiation. In this investigation, the HN9.10e cell line was used as experimental model; these cells, that are a somatic fusion product of hippocampal cells from embryonic day‐18 C57BL/6 mice and N18TG2 neuroblastoma cells, show morphological and cytoskeletal features similar to their neuronal precursors. By this model, we have studied the time course of vitamin D3 localization in the nucleus and its effect on proteins involved in proliferation and/or differentiation. We found that the translocation of vitamin D3 from cytoplasm to the nucleus is transient, as the maximal nuclear concentration is reached after 10 h of incubation with 3H‐vitamin D3 and decreases to control values by 12 h. The appearance of differentiation markers such as Bcl2, NGF, STAT3, and the decrease of proliferation markers such as cyclin‐1 and PCNA are late events. Moreover, physiological concentrations of vitamin D3 delay cell proliferation and induce cell differentiation of embryonic cells characterized by modification of soma lengthening and formation of axons and dendrites.

Serum deprivation increases ceramide levels and induces apoptosis in undifferentiated HN9.10e cells

Sphingolipid metabolites have been involved in the regulation of proliferation, differentiation and apoptosis. While cellular mechanisms of these processes have been extensively analysed in the post-mitotic neurons, little is known about proliferating neuronal precursors. We have taken as a model of neuroblasts the embryonic hippocampal cell line HN9.10e. Apoptosis was induced by serum deprivation and by treatment with N-acetylsphingosine (C2-Cer), a membrane-permeant analogue of the second messenger ceramide. Following C2-Cer addition, cytochrome c was released from mitochondria, [Ca(2+)](i) and caspase-3-like activity increased. Both cytochrome c release and rise of [Ca(2+)](i) occurred before caspase-3 activation and nuclear condensation. The intracellular levels of ceramide peaked at 1h following the serum deprivation. These results indicate that the serum deprivation induces a rise in the intracellular ceramide level, and that increased ceramide concentration leads to calcium dysregulation and release of cytochrome c followed by caspase-3 activation. We show that cytochrome c is released without a loss of mitochondrial transmembrane potential.

Serum-withdrawal-dependent apoptosis of hippocampal neuroblasts involves Ca++ release by endoplasmic reticulum and caspase-12 activation.

Apoptotic death caused by diseases or toxic insults is preceded and determined by endoplasmic reticulum dysfunction and altered intraluminar calcium homeostasis in many different cell types. With the present study we have explored the possibility that the ER stress could be involved also in apoptotic death induced by serum deprivation in neuronal cells. We have chosen as a model of study the cell line HN9.10e, constituted by immortalized hippocampal neuroblasts. The Ca(++) concentration in the lumen of the ER has been evaluated by using the low affinity Ca(++) probe Mag-fluo-4. We show that serum deprivation lowers the ER Ca(++) concentration with a time course closely related to the increase of apoptosis incidence. Serum deprivation also enhances the expression of a well-known marker of ER stress, the glucose-regulated protein-78 (GRP-78), a member of the heat shock/stress response protein family. Moreover, in serum-deprived neuroblasts, following GRP-78 up-regulation, the ER-associated procaspase-12 is cleaved with a time course which parallels the ER calcium loss while activation of caspase-3 is a later event. Depletion of ER Ca(++) by thapsigargin, a specific inhibitor of the ER-associated Ca(++) ATPase, also produces caspase-12 processing and apoptotic cell death, whereas agents capable of reducing the ER calcium loss protect the cells from serum-deprivation-induced apoptosis. These findings indicate that, in hippocampal neuroblasts, Ca(++) mobilization from ER and caspase-12 activation are components of the molecular pathway that leads to apoptosis triggered by serum deprivation and may constitute an amplifying loop of the mitochondrial pathway.

Nuclear sphingomyelin pathway in serum deprivation-induced apoptosis of embryonic hippocampal cells

Sphingomyelin (SM) cycle has been involved in the regulation of proliferation, differentiation, and apoptosis. Increases in ceramide have been found after a larger number of apoptotic stimuli including cytokines, cytotoxic drugs, and environmental stresses. Accumulating evidence suggest that the subcellular localization of ceramide generation is a critical factor in determining the cellular behavior. Since recently enzymes involved in ceramide metabolism such as sphingomyelinase, SM synthase, sphingosine kinase and ceramidase have been found in the nucleus of hepatocyte cells, we have studied first the presence and the physicochemical characteristics of SM metabolism enzymes in nuclei isolated from embryonic hippocampal cells (cell line HN9.10e). The activities of sphingomyelinase and SM‐synthase have been assayed and the ceramide production evaluated at different times after serum deprivation in these neurones cultivated in serum‐deficient medium. We report that both enzymes are present in the nucleus of embryonic hippocampal cells and differ from those present in the homogenate in optimum pH. After serum deprivation, that induces a time‐dependent decrease in cell viability and increase of the cell percentage in G1 phase of the cell cycle, a nuclear sphingomyelinase activation together with SM‐synthase inhibition and a consequent increase of nuclear ceramide pool have been demonstrated. No similar enzyme activity modifications in homogenate have been identified. The possible role of nuclear sphingomyelinase/sphingomyelin‐synthase balance in serum deprivation‐induced apoptosis in the embryonic hippocampal cell is discussed.

Sphingomyelinase metabolites control survival and apoptotic death in SH-SY5Y neuroblastoma cells

There is increasing evidence that sphingolipids are involved in cell survival, differentiation or commitment to death. The effect of different sphingolipids and inhibitors of mitogen-activated protein kinase (MAPK) cascade on SH-SY5Y neuroblastoma cell death has been studied. Permeant ceramide analogues C2-Cer, C8-Cer, and C8-Cer-1-phosphate, but not dihydro C2-Cer induce apoptosis, as shown by Hoechst staining. Inhibition of ceramidase and sphingosine kinase, as well as incubation with sphingosine, decreases cell viability, measured as 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide reduction, whereas addition of sphingosine-1-phosphate increases proliferation. Both PD98059 (MAPKK inhibitor) and SB202190 (p38 MAPK inhibitor) decreased viability, but only SB202190 abolished the effect of ceramide. These results suggest that in SH-SY5Y neuroblastoma cells, death is signalled by increases in ceramide, ceramide-phosphate or sphingosine content through p38 MAPK pathway while survival requires MAPK and high sphingosine-1-phosphate/ceramide ratio.

Cytotoxic effects and apoptotic signalling mechanisms of the sesquiterpenoid euplotin C, a secondary metabolite of the marine ciliate Euplotes crassus , in tumour cells

Most antitumour agents with cytotoxic properties induce apoptosis. The lipophilic compound euplotin C, isolated from the ciliate Euplotes crassus, is toxic to a number of different opportunistic or pathogenic microorganisms, although its mechanism of action is currently unknown. We report here that euplotin C is a powerful cytotoxic and pro-apoptotic agent in mouse AtT-20 and rat PC12 tumour-derived cell lines. In addition, we provide evidence that euplotin C treatment results in rapid activation of ryanodine receptors, depletion of Ca2+ stores in the endoplasmic reticulum (ER), the release of cytochrome c from the mitochondria, activation of caspase-12, and activation of caspase-3, leading to apoptosis. Intracellular Ca2+ overload is an early event which induces apoptosis and is parallelled by ER stress and the release of cytochrome c, whereas caspase-12 may be activated by euplotin C at a later stage in the apoptosis pathway. These events, either independently or concomitantly, lead to the activation of the caspase-3 and its downstream effectors, triggering the cell to undergo apoptosis. These results demonstrate that euplotin C may be considered for the design of cytotoxic and pro-apoptotic new drugs.

New signalling pathway involved in the anti-proliferative action of vitamin D3 and its analogues in human neuroblastoma cells. A role for ceramide kinase

1α,25-Dihydroxyvitamin D3 (1,25(OH)₂D₃), a crucial regulator of calcium/phosphorus homeostasis, has important physiological effects on growth and differentiation in a variety of malignant and non-malignant cells. Synthetic structural hormone analogues, with lower hypercalcemic side effects, are currently under clinical investigation. Sphingolipids appear to be crucial bioactive factors in the control of the cell fate: the phosphorylated forms, sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P), are mitogenic factors, whereas sphingosine and ceramide (Cer) usually act as pro-apoptotic agents. Although many studies correlate S1P function to impaired cell growth, the relevance of C1P/Cer system and its involvement in neuroblastoma cells remain to be clarified. Here, we demonstrated the anti-proliferative effect of 1,25(OH)₂D₃ as well as of its structural analogues, ZK156979 and ZK191784, in human SH-SY5Y cells, as judged by [³H]thymidine incorporation, cell growth and evaluation of active ERK1/2 levels. The inhibition of ceramide kinase (CerK), the enzyme responsible for C1P synthesis, by specific gene silencing or pharmacological inhibition, drastically reduced cell proliferation. 1,25(OH)₂D₃ and ZK191784 treatment induced a significant decrease in CerK expression and C1P content, and an increase of Cer. Notably, the treatment of SH-SY5Y cells with ZK159222, antagonist of 1,25(OH)₂D₃ receptor, trichostatin A, inhibitor of histone deacetylases, and COUP-TFI-siRNA prevented the decrease of CerK expression elicited by 1,25(OH)₂D₃ supporting the involvement of VDR/COUP-TFI/histone deacetylase complex in CerK regulation. Altogether, these findings provide the first evidence that CerK/C1P axis acts as molecular effector of the anti-proliferative action of 1,25(OH)₂D₃ and its analogues, thereby representing a new possible target for anti-cancer therapy of human neuroblastoma.

Molecular mechanisms of euplotin C-induced apoptosis: involvement of mitochondrial dysfunction, oxidative stress and proteases

The metabolite euplotin C (EC), isolated from the marine ciliate Euplotes crassus, is a powerful cytotoxic and pro-apoptotic agent in tumour cell lines. For instance, EC induces the rapid depletion of ryanodine Ca2+ stores, the release of cytochrome c from the mitochondria, and the activation of caspase-3, leading to apoptosis. The purpose of this study was to gain further insight into the mechanisms of EC-induced apoptosis in rat pheochromocytoma PC12 cells. We found that EC increases Bax/Bcl-2 ratio and that Bax is responsible of the EC-induced dissipation of the mitochondrial membrane potential (Δψm). In addition, EC induces the generation of reactive oxygene species (ROS) without involvement of p53. The inhibition of ROS generation prevents, at least in part, the pro-apoptotic effects of EC as well as the effects of EC on Bax, Δψm and intracellular free Ca2+, indicating a cross-talk between different pathways. However, definition of the effector cascade turns out to be more complex than expected and caspase-independent mechanisms, acting in parallel with caspases, should also be considered. Among them, EC increases the expression/activity of calpains downstream of ROS generation, although calpains seem to exert protective effects.

Seasonal variation of serotonin content and nonassociative learning of swim induction in the leech Hirudo medicinalis

SummaryIt is possible to obtain habituation of swim induction by stimulating the leech with repetitive light electrical trains. After obtaining this simple form of nonassociative learning, it is also possible to potentiate its response by a series of nociceptive skin brushings (dishabituation). Serotonin applied to the animal is the only neurotransmitter found to mimick dishabituation. We have observed that in the period April–June most animals did not exhibit potentiation of the swimming response after nociceptive stimulation while injection of serotonin mimicked dishabituation as in the animals treated in the period October–March. We have seen correlation between the changes in nonassociative learning and the seasonal variation of serotonin levels in segmental ganglia. This finding strengthens the hypothesis of serotonin as the neurotransmitter mediating dishabituation in swim induction of the leech.