Lipid microdomains contribute to apoptosis-associated modifications of mitochondria in T cells.

Plasma membrane lipid microdomains have been considered as a sort of 'closed chamber', where several subcellular activities, including CD95/Fas-mediated proapoptotic signaling, take place. In this work we detected GD3 and GM3 gangliosides in isolated mitochondria from lymphoblastoid CEM cells. Moreover, we demonstrated the presence of microdomains in mitochondria by immunogold transmission electron microscopy. We also showed that GD3, the voltage-dependent anion channel-1 (VDAC-1) and the fission protein hFis1 are structural components of a multimolecular signaling complex, in which Bcl-2 family proteins (t-Bid and Bax) are recruited. The disruption of lipid microdomains in isolated mitochondria by methyl-beta-cyclodextrin prevented mitochondria depolarization induced by GD3 or t-Bid. Thus, mitochondrion appears as a subcompartmentalized organelle, in which microdomains may act as controllers of their apoptogenic programs, including fission-associated morphogenetic changes, megapore formation and function. These results disclose a new scenario in which mitochondria-associated lipid microdomains can act as regulators and catalysts of cell fate.

Cytotoxic necrotizing factor 1 hinders skeletal muscle differentiation in vitro by perturbing the activation/deactivation balance of Rho GTPases.

The current knowledge assigns a crucial role to the Rho GTPases family (Rho, Rac, Cdc42) in the complex transductive pathway leading to skeletal muscle cell differentiation. Their exact function in myogenesis, however, remains largely undefined. The protein toxin CNF1 was herein employed as a tool to activate Rho, Rac and Cdc42 in the myogenic cell line C2C12. We demonstrated that CNF1 impaired myogenesis by affecting the muscle regulatory factors MyoD and myogenin and the structural protein MHC expressions. This was principally driven by Rac/Cdc42 activation whereas Rho apparently controlled only the fusion process. More importantly, we proved that a controlled balance between Rho and Rac/Cdc42 activation/deactivation state was crucial for the correct execution of the differentiation program, thus providing a novel view for the role of Rho GTPases in muscle cell differentiation. Also, the use of Rho hijacking toxins can represent a new strategy to pharmacologically influence the differentiative process.

The HIV-1 Nef protein: how an AIDS pathogenetic factor turns to a tool for combating AIDS.

Nef is one of the six regulatory proteins coded by the Human Immunodeficiency Virus (HIV)-1 and -2, and by the Simian Immunodeficiency Virus (SIV). Accumulating experimental evidences indicate that Nef is required for the optimal infectivity of HIV viral particles, and that it plays a critical role in the AIDS pathogenesis progressing. We previously cloned and sequenced a functionally defective HIV-1 genome (F12HIV-1) whose nef gene showed a rather unusual feature, i.e. its expression blocks the HIV-1 release by interfering with the viral assembling/release. Such a striking phenotype appeared to be the result of three amino acid substitutions, and coupled with the loss of the most part of the Nef functions described for the wild type counterpart. The F12Nef properties encouraged the designing of new strategies of anti HIV-1 gene therapy we afforded by recovering an inducible lentivirus vector expressing F12Nef as the cytoplasmic domain of a transmembrane fusion protein including a selectable marker (i.e. the Nerve Growth Factor receptor) as the ecto- and transmembrane domains. As expected, the expression of such a chimeric protein resulted in a potent protection of transduced cells from the HIV-1 spread. In sum, and surprisingly enough, we generated a reagent effectively counteracting the HIV-1 replication through the combination of a slightly mutated AIDS pathogenetic factor together with a lentivirus vector, i.e. the result of artifactual modifications of the HIV-1 genome.

CD95/phosphorylated ezrin association underlies HIV-1 GP120/IL-2-induced susceptibility to CD95(APO-1/Fas)-mediated apoptosis of human resting CD4(+)T lymphocytes.

CD95(APO-1/Fas)-mediated apoptosis of bystander uninfected T cells exerts a major role in the HIV-1-mediated CD4+ T-cell depletion. HIV-1 gp120 has a key role in the induction of sensitivity of human lymphocytes to CD95-mediated apoptosis through its interaction with the CD4 receptor. Recently, we have shown the importance of CD95/ezrin/actin association in CD95-mediated apoptosis. In this study, we explored the hypothesis that the gp120-mediated CD4 engagement could be involved in the induction of susceptibility of primary human T lymphocytes to CD95-mediated apoptosis through ezrin phosphorylation and ezrin-to-CD95 association. Here, we show that gp120/IL-2 combined stimuli, as well as the direct CD4 triggering, on human primary CD4(+)T lymphocytes induced an early and stable ezrin activation through phosphorylation, consistent with the induction of ezrin/CD95 association and susceptibility to CD95-mediated apoptosis. Our results provide a new mechanism through which HIV-1-gp120 may predispose resting CD4(+)T cell to bystander CD95-mediated apoptosis and support the key role of ezrin/CD95 linkage in regulating susceptibility to CD95-mediated apoptosis.

Expression of CCR-7, MIP-3beta, and Th-1 chemokines in type I IFN-induced monocyte-derived dendritic cells: importance for the rapid acquisition of potent migratory and functional activities.

The migration capability of dendritic cells (DCs) is regulated by their response to factors, namely chemokines, that characterize maturation stage and shape their functional activities. This study examines the morphology, expression of chemokines/chemokine receptors, and migration properties of DCs generated after treatment of monocytes with type I interferon (IFN) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (IFN-DCs). IFN-DCs showed phenotypical and morphologic features undetectable in DCs generated in the presence of interleukin 4 (IL-4) and GM-CSF, such as expression of CD83 and CD25 and the presence of CD44+, highly polarized, thin, and long dendrites. IFN-DCs markedly migrated in response to beta-chemokines (especially MIP-1beta) and expressed the Th-1 chemokine IP-10. Notably, IFN-DCs showed an up-regulation of CCR7 as well as of its natural ligand MIP-3beta, characteristics typical of mature DCs. Of interest, IFN-DCs exhibited a marked chemotactic response to MIP-3beta in vitro and strong migratory behavior in severe combined immunodeficient (SCID) mice. In SCID mice reconstituted with human peripheral blood leukocytes, IFN-DCs induced a potent primary human antibody response and IFN-gamma production, indicative of a Th-1 immune response. These results define the highly specialized maturation state of IFN-DCs and point out the existence of a "natural alliance" between type I IFN and monocyte/DC development, instrumental for ensuring an efficient connection between innate and adaptive immunity.

GD3 glycosphingolipid contributes to Fas-mediated apoptosis via association with ezrin cytoskeletal protein.

Efficiency of Fas-mediated apoptosis of lymphoid cells is regulated, among other means, by a mechanism involving its association with ezrin, a cytoskeletal protein belonging to the 4.1 family of proteins. In the present work, we provide evidence for a further molecule that associates to ezrin in Fas-triggered apoptosis, the disialoganglioside GD3. In fact, as an early event, GD3 redistributed in membrane-associated domains in uropods and co-localized with ezrin. Co-immunoprecipitation analyses confirmed this result, indicating a GD3-ezrin association. Altogether, these results are suggestive for a role of GD3 in Fas/ezrin-mediated apoptosis, supporting the view that uropods contain a multimolecular signaling complex involved in Fas-mediated apoptosis.

The A3 adenosine receptor induces cytoskeleton rearrangement in human astrocytoma cells via a specific action on Rho proteins.

In previous studies, we have demonstrated that exposure of astroglial cells to A3 adenosine receptor agonists results in dual actions on cell survival, with "trophic" and antiapoptotic effects at nanomolar concentrations and induction of cell death at micromolar agonist concentrations. The protective actions of A3 agonists have been associated with a reinforcement of the actin cytoskeleton, which likely results in increased resistance of cells to cytotoxic stimuli. The molecular mechanisms at the basis of this effect and the signalling pathway(s) linking the A3 receptor to the actin cytoskeleton have never been elucidated. Based on previous literature data suggesting that the actin cytoskeleton is controlled by small GTP-binding proteins of the Rho family, in the study reported here we investigated the involvement of these proteins in the effects induced by A3 agonists on human astrocytoma ADF cells. The presence of the A3 adenosine receptor in these cells has been confirmed by immunoblotting analysis. As expected, exposure of human astrocytoma ADF cells to nanomolar concentrations of the selective A3 agonist 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (CI-IB-MECA) resulted in formation of thick actin positive stress fibers. Preexposure of cells to the C3B toxin that inactivates Rho-proteins completely prevented the actin changes induced by CI-IB-MECA. Exposure to the A3 agonist also resulted in significant reduction of Rho-GDI, an inhibitory protein known to maintain Rho proteins in their inactive state, suggesting a potentiation of Rho-mediated effects. This effect was fully counteracted by the concomitant exposure to the selective A3 receptor antagonist MRS1191. These results suggest that the reinforcement of the actin cytoskeleton induced by A3 receptor agonists is mediated by an interference with the activation/inactivation cycle of Rho proteins, which may, therefore, represent a biological target for the identification of novel neuroprotective strategies.

2-Chloro-adenosine induces a glutamate-dependent calcium response in C2C12 myotubes.

Adenosine and its derivatives may induce acute changes, i.e., injury and death, in muscle cells. In the present work, we evaluated the intracellular calcium concentration in C2C12 myogenic cells differentiated in vitro to form myotubes and exposed to a metabolically stable analogue of adenosine, 2-chloro-adenosine. The compound was able to significantly modify ionic homeostasis by sensitizing muscle cells to the excitatory amino acid glutamate. A single exposure to glutamate led to a marked increase in intracellular calcium level. This is the first demonstration that adenosine analogues can regulate muscle cell integrity and function via an indirect increase of intracellular calcium ions.

CD95 (APO-1/Fas) linkage to the actin cytoskeleton through ezrin in human T lymphocytes: a novel regulatory mechanism of the CD95 apoptotic pathway.

CD95 (APO-1/Fas) is a member of the tumor necrosis factor receptor family, which can trigger apoptosis in a variety of cell types. However, little is known of the mechanisms underlying cell susceptibility to CD95-mediated apoptosis. Here we show that human T cells that are susceptible to CD95-mediated apoptosis, exhibit a constitutive polarized morphology, and that CD95 colocalizes with ezrin at the site of cellular polarization. In fact, CD95 co-immunoprecipitates with ezrin exclusively in lymphoblastoid CD4(+) T cells and primary long-term activated T lymphocytes, which are prone to CD95-mediated apoptosis, but not in short-term activated T lymphocytes, which are refractory to the same stimuli, even expressing equal levels of CD95 on the cell membrane. Pre-treatment with ezrin antisense oligonucleotides specifically protected from the CD95-mediated apoptosis. Moreover, we show that the actin cytoskeleton integrity is essential for this function. These findings strongly suggest that the CD95 cell membrane polarization, through an ezrin-mediated association with the actin cytoskeleton, is a key intracellular mechanism in rendering human T lymphocytes susceptible to the CD95-mediated apoptosis.

Overexpression of lymphocytic GD3 ganglioside and presence of anti-GD3 antibodies in patients with HIV infection.

This study was undertaken to analyze the role of disialoganglioside GD3 in HIV infection and disease progression. We report here the results obtained by both ex vivo and in vitro experiments on (1) surface and cytoplasmic expression and distribution of GD3 in HIV-infected cells, (2) the presence of anti-GD3 antibodies in sera of patients with HIV infection in various stages of the disease, and (3) the association of GD3 expression with HIV-related apoptotic events. GD3 expression was determined by high-performance thin-layer chromatography (HPTLC) and lipid-bound sialic acid and by static and flow cytometric analyses in peripheral blood lymphocytes from 22 AIDS patients, 20 anti-HIV Ab(+) asymptomatic subjects, and 25 healthy donors. Results obtained clearly indicated a significantly higher expression of plasma membrane GD3 content in lymphocytes from HIV-infected patients with respect to healthy controls. These HIV-induced perturbations of glycosphingolipid metabolism could be detected in all stages of the disease, including asymptomatic individuals. In addition, a significant percentage of patients showing disease progression displayed in serum samples an increased presence of anti-GD3 antibodies. Interestingly, ex vivo studies of lymphocytes from patients with HIV infection also indicated that GD3 expression is strictly associated with annexin V binding, an early marker of apoptosis. Moreover, cytofluorimetric analysis showed that virtually all anti-p24 Ab-positive cells were also immunolabeled with anti-GD3 antibodies. Accordingly, in vitro studies showed a significant redistribution and increase in GD3 expression in cultured U937 cells chronically infected with HIV-1 with respect to uninfected counterparts. In conclusion, our data clearly indicate that a significant increase in GD3 content in HIV-infected lymphocytes can occur and that this GD3 overexpression is paralleled by the presence of anti-GD3 antibodies in the plasma of patients. This is the first demonstration that disialoganglioside GD3, independent of the therapeutic schedule employed, can be considered as one of the early markers of HIV infection and can contribute to the early events leading to T cell depletion by apoptosis.

Adenosine- and 2-chloro-adenosine-induced cytopathic effects on myoblastic cells and myotubes: involvement of different intracellular mechanisms.

We recently suggested that, in muscular dystrophies, the excessive accumulation of adenosine as a result of an altered purine metabolism may contribute to progressive functional deterioration and muscle cell death. To verify this hypothesis, we have taken advantage of C2C12 myoblastic cells, which can be differentiated in vitro into multinucleated cells (myotubes). Exposure of both proliferating myoblasts and differentiated myotubes to adenosine or its metabolically-stable analog, 2-chloro-adenosine, resulted in apoptotic cell death and myotube disruption. Cytotoxicity by either nucleoside did not depend upon extracellular adenosine receptors, but, at least in part, by entry into cells via the membrane nitro-benzyl-thio-inosine-sensitive transporter. The adenosine kinase inhibitor, 5-iodotubercidin, prevented 2-chloro-adenosine-induced (but not adenosine-induced) effects, suggesting that an intracellular phosphorylation/activation reaction plays a key role in 2-chloro-adenosine-mediated cytotoxicity. Conversely, adenosine cytotoxicity was aggravated by the addition of homocysteine, suggesting that adenosine effects may be due to the accumulation of S-adenosyl-homocysteine, which blocks intracellular methylation-dependent reactions. Both nucleosides markedly disrupted the myotube structure via an effect on the actin cytoskeleton; however, also for myotubes, there were marked differences in the morphological alterations induced by these two nucleosides. These results show that adenosine and 2-chloro-adenosine induce apoptosis of myogenic cells via completely different metabolic pathways, and are consistent with the hypothesis that adenosine accumulation in dystrophic muscles may represent a novel pathogenetic pathway in muscle diseases.

Apoptosis induced by 2-chloro-adenosine and 2-chloro-2'-deoxy-adenosine in a human astrocytoma cell line: differential mechanisms and possible clinical relevance.

We have previously demonstrated that 2-chloro-adenosine (2-CA) can induce apoptosis of rat astroglial cells (Abbracchio et al. [1995] Biochem. Biophys. Res. Commun. 213:908-915). In the present study, we have characterized, for the first time, the effects induced on a human astrocytoma cell line (ADF cells) by both 2-CA and its related analog 2-chloro-2'-deoxy-adenosine (2-CdA, that is employed as anti-cancer agent in chronic lymphoid malignancies). Exposure of these cells to either adenosine analog resulted in time- and concentration-dependent apoptosis. Experiments with pharmacological agents known to interfere with adenosine receptors, its membrane transporter, and intracellular nucleoside kinases showed that: (i) cell death induced by either adenosine analog did not depend on extracellular adenosine receptors, but on a direct intracellular action; however, only in the case of 2-CA, was entry into cells mediated by the specific nitrobenzyl-tioinosine-sensitive transporter; (ii) for both adenosine analogs, induction of apoptosis required the phosphorylation/activation by specific intracellular nucleoside kinases, i.e., adenosine kinase for 2-CA, and deoxycytidine kinase for 2-CdA. In addition, only in the case of 2-CdA, was induction of apoptosis preceded by a block of cells at the G2/M phase of the cell cycle. Finally, at concentrations of either analog that killed about 80-90% of astrocytoma cells, a significantly lower effect on the viability of primary cortical neurons was observed. In conclusion, both adenosine analogs can trigger apoptosis of human astrocytoma cells, albeit with different mechanisms. This effect together with the relative sparing of neuronal cells, may have potential clinical implications for the therapy of tumors of glial origin.

Involvement of interleukin-1beta in the mechanism of human immunodeficiency virus type 1 (HIV-1) recombinant protein gp120-induced apoptosis in the neocortex of rat.

The effect of subchronic intracerebroventricular injection of the human immunodeficiency virus type 1 (HIV-1) recombinant protein gp120 (100 ng, given daily for up to seven consecutive days) on interleukin-1beta expression was studied by immunohistochemistry in the brain of adult rats. In comparison to control, bovine serum albumin (300 ng, given intracerebroventricularly for up to seven days) -treated animals (n=6), interleukin-1beta immunoreactivity increased in the brain cortex and hippocampus of rats (n=6) receiving a single injection of the viral protein 24 h before analysis with more substantial increases being observed in these regions of the brain (n=6) after seven days treatment. Double-labelling immunofluorescence experiments support a neuronal and, possibly, a microglial cell origin for gp120-enhanced interleukin-1beta expression. Transmission electron microscopy analysis of brain tissue sections revealed that combination treatments (given intracerebroventricularly daily for seven days) with gp120 (100 ng) and interleukin-1 receptor antagonist (80 ng) or with the interleukin converting enzyme inhibitor II (100 pmol), but not with leupeptin (100 pmol), prevented apoptotic death of rat (n=6/group) brain cortical cells typically elicited by the viral protein. These data demonstrate that gp120 enhances interleukin-1beta expression in the brain and this may be involved in the mechanism underlying apoptosis induced by gp120 in the brain cortex of rat. Further support to this hypothesis comes from the evidence that intracerebroventricular injection of murine recombinant interleukin-1beta (200 U, given daily for seven consecutive days) produces DNA fragmentation in the brain cortex of rat (n=6). Interestingly, the latter treatment enhanced nerve growth factor level in the hippocampus but not in the cerebral cortex and this coincides with a similar effect recently reported in identical brain areas of rats treated likewise with gp120. In conclusion, the present data demonstrate that treatment with gp120 enhances interleukin-1beta expression and this participates in the mechanism of apoptotic cell death in the brain cortex of rat. By contrast, in the hippocampus, gp120-enhanced interleukin-1beta expression elevates nerve growth factor that may prevent or delay apoptosis in this plastic region of the rat brain.

Inhibition of proteasome function prevents thymocyte apoptosis: involvement of ornithine decarboxylase.

We have previously shown that polyamine levels rapidly decrease in thymocytes undergoing apoptosis, and that ornithine decarboxylase increases early but too transiently to maintain elevated polyamine levels. These data led us to suppose that a precocious ornithine decarboxylase degradation might be responsible for the imbalance of polyamine metabolism. Ornithine decarboxylase is known to be degraded by the cytosolic 26S proteasome that plays an essential role in thymocyte apoptosis. In this paper we demonstrate that the inhibition of proteasome function preserves ornithine decarboxylase activity and prevents thymocytes from undergoing apoptosis after dexamethasone treatment. Since intracellular polyamine levels are also preserved, ornithine decarboxylase seems to be functionally active in maintaining polyamine homeostasis after proteasome inhibition in thymocytes. Our proposed role for the proteasome in quiescent cells upon an apoptotic stimulus is to degrade proteins like ornithine decarboxylase that are involved in the control of the cell cycle and cell survival.

Protection against apoptosis by monoamine oxidase A inhibitors.

Several lines of evidence have been accumulating indicating that an important role may be played by mitochondrial homeostasis in the initiation phase, the first stage of apoptosis. This work describes the results obtained by using different inhibitors of monoamine oxidases (MAO), i.e. pargyline, clorgyline and deprenyl, on mitochondrial integrity and apoptosis. Both pargyline and clorgyline are capable of protecting cells from apoptosis induced by serum starvation while deprenyl is ineffective. These data represent the first demonstration that MAO-A inhibitors may protect cells from apoptosis through a mechanism involving the maintenance of mitochondrial homeostasis.

HIV-1 gp120-induced apoptosis in the rat neocortex involves enhanced expression of cyclo-oxygenase type 2 (COX-2).

The effect of subchronic intracerebroventricular (i.c.v.) injection of the human immunodeficiency virus type 1 (HIV-1) recombinant protein gp120 (100 ng, given daily for up to 7 consecutive days) on cyclooxygenase type 2 (COX-2) expression was studied by immunohistochemistry in the brain of adult rats. In comparison to control, bovine serum albumin (100 ng, given i.c.v. for up to 7 days) treated animals (n = 6), a single daily injection of the viral protein for 7 consecutive days enhanced the number of COX-2 immunoreactive cells in the brain cortex of rats (n = 6 per group) and this was accompanied by a 50% increase over control PGE2 content in whole brain tissue homogenates (n = 6). In another series of experiments, pretreatment of rats (n = 6) with indomethacin (6.0 mg/kg given i.p. 1 h before gp120 injection), an inhibitor COX activity, prevented apoptotic death typically produced by gp120 in the neocortex of rat suggesting that enhancement of COX-2 expression may be involved in the mechanisms of apoptosis yielded by the HIV-1 coat protein.

Different pathways of apoptosis revealed by 2-chloro-adenosine and deoxy-D-ribose in mammalian astroglial cells.

Both the adenosine analogue 2-chloro-adenosine (2-CA) and the reducing sugar deoxy-D-ribose (dRib) induce apoptosis of astroglial cells in rat brain primary cultures (Abbracchio et al.: Biochem Biophys Res Commun 213:908-915, 1995). The present study was undertaken to elucidate by both morphological and cytofluorimetric analyses the intracellular mechanism(s) involved in induction of apoptosis by these two agents. The poly(ADP-ribose)polymerase (PARP) inhibitor 3-aminobenzamide did not prevent either 2-CA- or dRib-induced cell death, suggesting that activation of PARP is not critically important for induction of apoptosis in astrocytes. The radical scavenger N-acetyl-cysteine (NAC) strongly inhibited dRib- but not 2-CA-induced cell death, suggesting a differential role for radical formation in apoptosis by these two agents. A time-dependent increase of cells with depolarized mitochondria was observed in dRib-, and to a lesser extent, in 2-CA-treated cultures. NAC also prevented dRib- but not 2-CA-induced mitochondrial changes. We conclude that, in mammalian astrocytes, apoptosis can proceed through diverse and multiple pathways, depending upon the apoptotic stimulus. For dRib, apoptosis likely proceeds through generation of radicals and mitochondrial involvement. An adenosine extracellular receptor linked to an as yet unidentified signaling pathway may instead mediate 2-CA-induced cell death, which may have intriguing implications for both nervous system development and brain response to trauma and ischemia.

Combined effect of 3-aminobenzamide and N-acetylcysteine on HIV replication in chronically infected U937 cells.

The existence of a close relationship between apoptosis associated with oxidative stress and the increase of viral progeny in chronically HIV-infected cells has been previously reported. The possibility of modulating both phenomena by using an antioxidant such as N-acetylcysteine (NAC) has also been demonstrated. The present investigation was designed to study the role of the nuclear enzyme poly-(ADP-ribose)-polymerase (PARP) when HIV- infected cells are treated with tumour necrosis factor alpha (TNFα), a cytokine capable of inducing both apoptosis and intracellular oxygen free radical production. PARP overexpression may result in a rapid drop of intracellular NAD(+) and ATP concentration, thus contributing to cellular redox imbalance. We have used the specific PARP inhibitor 3- aminobenzamide (3-ABA), alone or in a combination with NAC. 3-ABA was only partially capable of inhibiting viral replication and apoptosis induced by TNFα. In contrast, the combination of NAC and 3-ABA led to an inhibition of apoptosis as well as to a marked decrease in viral particle production, with a parallel replenishment of intracellular reduced glutathione content. The results reported here confirm the potential role of antioxidant drug treatment in specific phases of HIV infection.

The A3 adenosine receptor mediates cell spreading, reorganization of actin cytoskeleton, and distribution of Bcl-XL: studies in human astroglioma cells.

The pathophysiological role of the adenosine A3 receptor in the central nervous system is largely unknown. We have investigated the effects of the selective A3 receptor agonist 2-chloro-N6-(3-iodobenzyl)-adenosine, Cl-IB-MECA, in cells of the astroglial lineage (human astrocytoma ADF cells). A marked reorganization of the cytoskeleton, with appearance of stress fibers and numerous cell protrusions, was found following exposure of cells to low (nM) concentrations of Cl-IB-MECA. These "trophic" effects were accompanied by induction of the expression of Rho, a small GTP-binding protein, which was virtually absent in control cells, and by changes of the intracellular distribution of the antiapoptotic protein Bcl-XL, that, in agonist-exposed cells, became specifically associated to cell protrusions. This is the first demonstration that the intracellular organization of Bcl-XL can be modulated by the activation of a G-protein-coupled membrane receptor, such as the A3 adenosine receptor. Moreover, modulation of the astrocytic cytoskeleton by adenosine may have intriguing implications in both nervous system development and in the response of the brain to trauma and ischemia.