Address correspondence and reprint requests to Professor Antonio Contestabile, Department of Biology, University of Bologna, via Selmi 3, 40126 Bologna, Italy. E-mail: email@example.com
Nitric oxide (NO) is a small, diffusible, highly reactive molecule with a dichotomous regulatory role in the brain: an intra- and intercellular messenger under physiological conditions and a neurodegenerative agent under pathological conditions. We have recently demonstrated that long-lasting exposure to an neuronal nitric oxide synthase (nNOS) inhibitor down-regulated serine/threonine kinase (Akt) survival pathway and caused apoptosis in cerebellar granule cell cultures. The present study further substantiates the role of NO in neuronal survival by demonstrating that blocking its production down-regulates the activity of cAMP-responsive element binding protein (CREB), a transcription factor involved in cell survival and synaptic plasticity. Pharmacological dissection of the pathway linking NO to CREB shows that cGMP and its kinase are intermediate effectors. We also identify Bcl-2 as one of the anti-apoptotic genes down-regulated by NO shortage and decreased CREB phosphorylation. These results not only confirm the role of CREB in neuronal survival but also provide circumstantial evidence for a novel link among NO, CREB activation and survival.
Some evidence has been recently obtained that nitric oxide (NO) signaling may be functionally coupled to CREB activation in nerve cells. This diffusible messenger has been implicated in developmental neural plasticity (Gally et al. 1990; Edelman and Gally 1992; Contestabile 2000) as well as in hippocampal long-term potentiation (LTP), a long-lasting form of synaptic plasticity related to some types of learning and memory (Bliss and Collingridge 1993; Hawkins et al. 1998). A recent paper (Lu et al. 1999) has demonstrated that the cGMP-dependent protein kinase (PKG) activation by NO, contributes to the increased phosphorylation of CREB occurring during the late phase of hippocampal LTP. This NO-dependent pathway seems also involved in the induction of another well known form of synaptic plasticity, the long-term sensitization in sensory neurons of Aplysia (Lewin and Walters 1999). In other cellular systems, NO-induced CREB phosphorylation through the guanylate cyclase/PKG pathway has also been demonstrated (Ohki et al. 1995; Gudi et al. 2000). Evidence for a similar functional link has also been obtained in vivo where light stimulation during the dark phase is able to elicit a NO-dependent CREB phosphorylation which contributes to reset circadian rhythms (Ding et al. 1997). NO has been defined asa ‘Janus-faced’ molecule (Snyder 1993) due to its dual involvement in neuron survival, development and synaptic plasticity on one side, or in neurodegeneration on the other side, depending on the efficacy of the control upon its production and diffusion (Dawson and Dawson 1995; Estevez et al. 1995; Peunova and Enikolopov 1995; Farinelli et al. 1996; Yun et al. 1997; Contestabile 2000; Chung et al. 2001). The previously reviewed data suggest that CREB activation is one of the ways by which NO exerts its action on neuronal survival and synaptic plasticity. However, while there is evidence for a NO-dependent involvement of CREB in synaptic plasticity, no experimental evidence for this functional link has been so far obtained concerning neuronal survival.
As stated above, CREB activity is regulated through phosphorylation that can be mediated by various kinases. Among these, the signaling pathway involving phosphoinositide 3′ kinase (PI3′ kinase) and the serine/threonine protein kinase Akt (also known as protein kinase B, PKB) plays a central role in the survival action mediated by downstream CREB activation (Dudek et al. 1997; Kulik et al. 1997; Du and Montminy 1998; Walton and Dragunow 2000). A novel link between the NO-mediated survival effect and Akt activation has been recently disclosed using primary cultures of cerebellar granule cells (CGC) (Ciani et al. 2002). We have, indeed, demonstrated that chronic inhibition of NO production by differentiated granule neurons resulted in their progressive apoptotic death, accompanied by down-regulation of the Akt pathway. Both these effects were reverted by slow-releasing NO donors or by an analog of the NO-related second messenger, cGMP (Ciani et al. 2002), thus demonstrating the specificity of the effects and the validity of the functional link among NO, survival and Akt activation.
These previous results prompted us to exploit the same experimental model to confirm the link between NO and CREB activation in these primary neuronal cultures and, additionally to seek evidence for the involvement of this cellular pathway in survival. We report here that NO shortage down-regulates CREB phosphorylation in CGC, decreases the expression of the CREB-regulated anti-apoptotic gene, Bcl-2, and that similar effects are obtained by interfering at different levels with the main cellular pathway linking NO to CREB, namely the one related to guanylate cyclase and PKG activation.
Basal modified Eagle's medium (BME), fetal calf serum (FCS), and l-glutamine were purchased from Life Technologies (Gaithersburg, MD, USA). Polyclonal phospo-CREB Ser133, CREB and Bcl-2 antibodies were obtained from Transduction Laboratories (Lexingston, KY, USA), and anti-mouse β-actin monoclonal antibody from Sigma (St Louis, MO, USA). Quinoxaline-1-one (ODQ) and KT5823 were obtained from Calbiochem (San Diego, CA, USA) and Glyco-SNAP-2 from Alexis Biochemicals (San Diego, CA, USA). Other reagents were purchased from Sigma unless indicated otherwise.
Cell cultures and treatments
Primary cultures of cerebellar granule cells were prepared from the cerebella of 8-day-old rat pups (Wistar) and maintained in BME containing 10% heat-inactivated FCS, as previously described (Gallo et al. 1982). Briefly, cerebella were removed and dissected from their meninges in Krebs' buffer containing 0.3% BSA. Tissue was dissociated with trypsin at 37°C for 15 min and triturated 15 times using a Pasteur pipette in a DNAase/soybean trypsin inhibitor solution. The cells were plated at a density of 2.4 × 106/35 mm dish previously coated with poly l-lysine, and maintained in BME medium containing 10% FCS, 2 mm glutamine, 100 µg/mL gentamicin, and 25 mm KCl. Cytosine β-d-arabinofuranoside (10 µm) was added 16 h after plating to arrest the growth of non-neuronal cells.
Cultures were treated with l-nitro arginine methyl ester (l-NAME) (1 mm) after 7 days in vitro for different periods and with ODQ, KT5823, Glyco-SNAP-2 or 8Br-cGMP at the concentrations and for the times specified in the figure legends. 8Br-cGMP was replaced after 48 h from the beginning of the treatment. Fresh NO donor (Glyco-SNAP-2) was added every 24 h.
A sandwich ELISA method was used to assess apoptosis (Cell Death ELISA; Roche Molecular Biochemicals, Indianapolis, IN, USA). The assay measures the enrichment of histone-associated DNA fragments in the cytoplasm of apoptotic cells. Detection of bound nucleosomes from the samples is made using a monoclonal anti-DNA antibody with a peroxidase label. Bound anti-DNA-peroxidase is quantified using the peroxidase substrate 2,2′-azino-di-(3-ethylbenzthiazoline sulfonate), whose product is measured by absorbance at 405 nm. Cell cultures, which had been treated with l-NAME for different periods, were assayed according to the protocol supplied with the kit.
Cells were washed twice with phosphate-buffered saline and were lysed with 500 µL of lysis buffer (20 mm Tris, pH 7.5, 150 mm NaCl, 2 mm EDTA, 0.2% NP-40, 1 mm sodium orthovanadate, 100 µm phenylmethanesulfonyl fluoride, 1 nm okadaic acid, and 10 µg/mL each of leupeptin, aprotinin and pepstatin). The lysates were collected in microcentrifuge tubes, sonicated for 10 s, and centrifuged at 14 000 g for 15 min. Protein concentration in the supernatants was determined using the folin method (Lowry et al. 1951). The lysates were stored at − 80°C until used for immunoblotting. Cell lysates were mixed with Laemmli sample buffer containing 2% sodium dodecyl sulfate (SDS) and placed in a boiling water bath for 10 min. Proteins (50 µg per lane) were separated using 10% SDS–polyacrylamide gel electrophoresis (PAGE), and transferred to nitrocellulose membranes. Blots were probed with antibodies to CREB, phospho-serine-133-CREB, Bcl-2 and β-actin (1 : 1000). Immunoblots were developed using horseradish peroxidase-conjugated secondary antibodies (1 : 2000), detected with enhanced chemiluminescence (Amersham-Pharmacia, Piscataway, NJ, USA), and the optical density was quantified by using the image processing and analysis program Scion Image (Ederick, MA, USA).
Total RNA, free from chromosomal DNA contamination, was isolated using TRIZOL reagent according to the supplier's instructions and reverse transcribed with SUPERSCRIPT II RNase H-reverse transcriptase (Life Technologies, Inc.) using customer-synthesized (Life Technologies) oligo-dT12-18 primers. RT-PCR primers for glyceraldehyde-3-phosphate dehydrogenase (GADPH) and Bcl-2 are listed: GADPH (451 bp) sense: 5′-accacagtccatgccatcac-3′, antisense: 5′-tccaccaccctgttgctgta-3′; Bcl-2 (349 bp) sense: 5′-cacccctggcatcttctcct-3′, antisense 5′-gttgacgctccccacacaca-3′.
Briefly, 2 µg of total RNA was incubated for 45 min at 42°C in 20 µL containing 25 µm of the primer, 10 mm dithiothreitol, 0.5 mm dATP, dCTP. dGTP and dTTP, 200 units of reverse transcriptase, and 1X First Strand Buffer (Life Technologies). A 1 µL aliquot of reverse transcribed cDNA was subjected to PCR in a 50-µL mixture containing 3′ and 5′ primers (2 µm each). PCR amplification was conducted for 21 cycles (GADPH), or 27 cycles (Bcl-2) using the following conditions: denaturation 40 s at 94°C, annealing 55°C (Bcl-2) or 56°C (GADPH), and primer extension 2 min at 72°C. PCR products were electrophoresed on agarose gels containing ethidium bromide and visualized under UV light. Band intensities (arbitrary optical density units) were evaluated using a Bio-Imaging Analyzer System (Amersham) combined with densitometry. Intensities for amplified Bcl-2 were normalized against those obtained for GADPH in the same samples. The linearity of PCR amplification was verified by amplifying several dilutions of RT-cDNA.
Transfection and analysis of reporter gene activity
Cells were seeded in six-well plates at the density of 106/mL and were transfected the second day in culture with 2 µg of plasmids using polethylenimine PEI (25K) as DNA carrier according to a previously described method (Sée et al. 2001). Starting from the seventh day in vitro (DIV) cells were exposed to l-NAME for different times. Plasmids containing a tandem repeat of a CREB/ATF-1 or AP-1 or nuclear factor-κB (NF-κB) consensus response element placed upstream to a luciferase cDNA (CRE-Luc, AP-1-Luc, NF-κB-Luc) or a control plasmid (Tk-Luc) were used as reporter genes. After l-NAME treatment, cells were washed twice with ice-cold phosphate-buffered saline and lysed by incubation in 50 mm Tris-MES (pH 7.8), 1 mm dithiothreitol, and 1% Triton X-100 for 5 min on ice. The lysate was cleared of cellular debris by centrifugation at 11 000 g(de Wet et al. 1987). Luciferase assay was performed with a TD-20/20 luminometer (Promega, Madison, WI, USA).
The results are expressed as means ± SEM of four or more experiments. The data were analyzed by one-way anova followed by Bonferroni's test. The differences between means were considered statistically significant when p < 0.05.
l-NAME-induced apoptosis is preceded by inhibition of phosphorylation and transcriptional activity of CREB
As previously reported (Ciani et al. 2002) inhibition of NO production through l-NAME resulted in progressive apoptotic death of granule neurons (Fig. 1a). By using a new sensitive method to reveal cell death, we can demonstrate here that the process of cell death begins after 48 h of pharmacological NOS blockade and becomes highly significant after 72 h (Fig. 1a). CREB phosphorylation at Ser133 activation site was examined in cerebellar granule cells treated with l-NAME for 24–72 h starting from the seventh DIV. A progressive decrease of CREB phosphorylation was found under these experimental conditions (Fig. 1b). The observed decrease of P-CREB was not accounted for by the lower expression of CREB protein, since western blot analysis with an antibody that recognizes native CREB showed that the total amount of CREB was essentially stable during the first 24 h of treatment and decreased much more slowly that P-CREB thereafter (Fig. 1b).
To examine whether l-NAME also affected the transcriptional activity of CREB, a reporter gene assay was performed using reporter plasmids containing a tandem repeat of a CREB/ATF-1 or AP-1 or NF-κB consensus response elements, placed upstream to a luciferase cDNA (CRE-Luc, AP-1-Luc, NF-κB-Luc) or a control plasmid (Tk-Luc). Cells were transfected by the second day in culture to obtain the maximum transfection efficiency. Four days after transfection with the reporter plasmids, cells were cultured for additional 24 h in the presence or absence of l-NAME.
While no changes in luciferase activity were observed as a consequence of l-NAME treatment concerning AP-1-Luc, NF-κB-Luc or Tk-Luc transfected cells, this treatment remarkably reduced (up to 50%) the transcriptional activity on CREB/ATF-1 (Fig. 1c).
The effect of l-NAME inhibition for 24 and 48 h on CREB phosphorylation was completely reverted by adding to the medium a slow-releasing NO donor (Glyco-SNAP-2, 10 µm) (Fig. 2), that had no effect by itself for the same exposure periods (Fig. 2). A similar result was also obtained by adding a cGMP analog (8Br-cGMP, 250 µm) to the medium which was completely effective in the 24 h range of exposure and partially but significantly effective in the 48 h range (Fig. 2).
Effect of NOS inhibition at downstream levels of the NO-cGMP pathway
Since the previous experiments indicated that the NO deprivation-dependent decrease of CREB phosphorylation occurred through a cGMP-mediated pathway, we attempted to replicate the results by interfering at two different levels with the cGMP cascade, downstream to NO. To this aim, a specific inhibitor of the NO-dependent soluble guanylate cyclase (ODQ, 100 µm) and a PKG inhibitor (KT5823, 500 nm) were used. As shown by Fig. 3, both inhibitors essentially replicated the effect of l-NAME on CREB phosphorylation, with ODQ appearing more efficient than KT5823 (Fig. 3a). This was confirmed by further time-course experiments that demonstrated a more rapid and drastic decrease of CREB phosphorylation following guanylate cyclase inhibition than following PKG inhibition (Fig. 3b).
Effect of l-NAME on the CREB-regulated anti-apoptotic gene Bcl-2
The Bcl-2 gene has been demonstrated to be one of the main anti-apoptotic genes in many types of cells (Farrow and Brown 1996) and it is known to be transcriptionally regulated by P-CREB (Wilson et al. 1996). Bcl-2 mRNA levels and protein expression were therefore selected as a possible correlate for the survival role of NO in our system.
l-NAME exposure for 24 or 48 h led to a significant decrease of Bcl-2 mRNA level and the effect was reverted by adding to the medium Glyco-SNAP-2 (Fig. 4a). Noticeably, also ODQ and, to a lower extent KT5823, significantly replicated the effect of NO deprivation (Fig. 4a). The negative effects of prolonged NOS inhibition were even clearer when studied on Bcl-2 protein expression, which was progressively down-regulated by exposure to l-NAME (24–72 h) (Fig. 4b). No effect on Bcl-2 protein levels were noted in the presence of Glyco-SNAP-2 alone (data not shown). Also, in the case of Bcl-2 protein, the effect of l-NAME exposure was mimicked by inhibition of guanylate cyclase, while it was significantly reverted by inclusion in the medium of a slowly releasing NO donor (Fig. 4b).
In the present study we have used a previously tested experimental model in which the chronic NO deprivation leads to extensive apoptotic neuronal death in CGC cultures (Ciani et al. 2002), and we have obtained evidence that, under these conditions, a dramatic decrease of CREB phosphorylation does occur. By using a sensitive method to detect apoptotic cell death, we demonstrate here that the drop of CREB phosphorylation temporally preceded the progression of apoptosis. The drop in CREB phosphorylation was not, therefore, a trivial consequence of decreased cell viability because western blotting for the inactive form of CREB showed only a modest and much-delayed decrease in l-NAME-treated cultures. Rather, we propose that the decreased activity of CREB is an early marker for the functional derangement of neurons committed to die. Our experiments further demonstrate that the decrease of CREB activity related to the blockade of NO production, takes place through the cGMP/PKG cascade, the most important and widespread intracellular signaling pathway activated by NO (Mayer 1993; Garthwaite and Boulton 1995; Dawson et al. 1998). Figure 5 summarizes the various steps of the signaling pathway linking NO to CREB activation, targeted by the experiments described in the present report.
There is increasing evidence for a key role played by CREB activation in developmental survival of neuronal cells (Finkbeiner et al. 1997; Riccio et al. 1999; Finkbeiner 2000; Walton and Dragunow 2000). In parallel, several results suggest promotion of neuronal survival as one important physiological function of NO (Peunova and Enikolopov 1995; Estevez et al. 1998; Contestabile 2000; Ciani et al. 2002). The present results establish CREB as a novel cellular mediator for the survival-promoting action exerted by NO during brain development. The data reported here, indeed, are the first demonstration linking NO survival effects to a cellular pathway having CREB activation at its center. Previous researches have demonstrated a cGMP/PKG-mediated survival action of NO on serum-deprived PC12 cells, but involvement of CREB activation was not investigated in these experiments (Kim et al. 1999). Other recent results, have established functional links between NO and protection from apoptosis in neural cells deprived of survival factors known to mediate their anti-apoptotic effects through CREB activation (Peunova and Enikolopov 1995; Farinelli et al. 1996; Estevez et al. 1998). In none of these works, however, was the demonstration of a possible link between NO-mediated survival effect and CREB phosphorylation experimentally sought.
Recent reviews (Contestabile 2000; Walton and Dragunow 2000) have stressed the importance of identifying the cellular and molecular correlates of NO and CREB actions on both synaptic plasticity and survival with the dual aim of better characterizing these related aspects of brain function and to devise strategies for neuroprotection and cognitive enhancement (Walton and Dragunow 2000). The present results, by strongly suggesting a functional link between these two molecules in promoting survival, make the previously outlined goals an even more significant target for future research.
CREB activation is involved in the transcriptional control of numerous genes whose identification is crucial to understand the mechanisms and to dissect the molecular steps implicated in the survival effects of the NO-CREB cascade in neurons. The present results point to Bcl-2 as one such genes in granule cells, as both mRNA and protein levels were decreased by l-NAME and ODQ treatments and rescued by the NO donor. It has been shown in several cellular systems that Bcl-2 is one of the most important of the CREB-regulated genes, related to the survival role played by this transcriptional factor (Walton and Dragunow 2000). Our results point to Bcl-2 as an anti-apoptotic gene whose expression, under the transcriptional control of CREB, is regulated, together with other previously determined intracellular pathway (Walton and Dragunow 2000), also by the NO-cGMP cascade. Further studies involving the use of dominant negative mutants of CREB and Bcl-2 could lead to an unequivocal demonstration of the proposed functional links.
The present report may help with solving the paradox of the neurotrophic action of NO that can turn into neurodegeneration (Snyder 1993; Contestabile 2000; Chung et al. 2001). Indeed, it discloses a specific cellular pathway linking NO to CREB and targeted at neuronal survival. Noticeably, this seems to be the same pathway operating in processes of neural plasticity related to learning and memory (Lewin and Walters 1999; Lu et al. 1999). This coincidence clearly puts the NO-CREB pathway at the very center of nervous function because it is involved in the two essential mechanisms for neural circuit development: maintenance, i.e. neuronal survival, and activity-dependent modifications, i.e. synaptic plasticity. The neuropathological action of NO, as evidenced by several authors (Dawson and Dawson 1998; Kaufman 1999), is due to the uncontrolled escape from these very important physiological functions, which might result in activation of other cellular pathways potentially able to lead to cell damage and, eventually, death. A better understanding of these mechanisms is of the utmost relevance in view of the increasing interest in finding drugs able to maintain NO physiological functions in the brain while keeping under control the potential pathological consequences of uncontrolled bursts of NO production. By characterizing a new survival pathway linking NO to CREB and to regulation of an anti-apoptotic gene, the present study provides a further target for research.
This study was supported by a research grant from the Italian National Research Council in the framework of the targeted project ‘Biotechnologies’. We thank Miss Luciana di Pietrangelo for competent technical help.