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Journal of Cerebral Blood Flow & Metabolism (2005) 25, 348–357 & 2005 ISCBFM All rights reserved 0271-678X/05 $30.00 www.jcbfm.com

Nitric oxide inhibits caspase activation and apoptotic morphology but does not rescue neuronal death Ping Zhou, Liping Qian and Costantino Iadecola Division of Neurobiology, Weill Medical College of Cornell University, New York, NY, USA

Nitric oxide (NO) has been shown to inhibit apoptotic cell death by S-nitrosylation of the catalyticsite cysteine residue of caspases. However, it is not clear whether in neurons NO-mediated caspase inactivation leads to improved cell survival. To address this issue, we studied the effect of NO donors on caspase activity and cell survival in cortical neuronal culture treated with the apoptosis inducer staurosporine (STS) and camptothecin. In parallel, cell viability was assessed by the MTS assay and MAP2 staining. We found that NO donors ((7)-S-nitroso-N-acetylpenicillamine, S-nitrosoglutathione, and NONOates) dose-dependently inhibited caspase-3 and -9 activity induced by STS and camptothecin. The reduction in caspase-3 activity was, in large part, because of the blockage of the proteolytic conversion of pro-caspase-3 to active caspase-3. NO donors also inhibited the appearance of the classical apoptotic nuclear morphology. However, inhibition of both caspase activity and apoptotic morphology was not associated with enhancement of cell viability. Thus, inhibition of caspase and apoptotic morphology by NO donors does not improve neuronal survival. The data suggest that inhibition of caspase by NO unmasks a caspase-independent form of cell death. A better understanding of this form of cell death may provide new strategies for neuroprotection in neuropathologies, such as ischemic brain injury, associated with apoptosis. Journal of Cerebral Blood Flow & Metabolism (2005) 25, 348–357. doi:10.1038/sj.jcbfm.9600036 Published online 19 January 2005 Keywords: nitric oxide; S-nitrosylation; caspases; apoptosis; staurosporine

Introduction Apoptotic cell death is a carefully regulated cellular process that is essential for organ development and homeostasis (Vaux and Korsmeyer, 1999). The basic molecular machinery underlying this process is well conserved in all eukaryotic organisms (Danial and Korsmeyer, 2004). One such conserved component in the apoptotic pathway is the caspase family of enzymes (Earnshaw et al, 1999). Caspases are synthesized as inactive zymogens, which become active after proteolytic cleavage on apoptotic stimuli. Activated caspases cleave and destroy key intracellular proteins (Budihardjo et al, 1999), resulting in the morphologic changes that characterize apoptotic cell death.

Correspondence: Dr Ping Zhou, Division of Neurobiology, Weill Medical College of Cornell University, 411 East 69th Street, Kb410, New York, NY 10021, USA. E-mail: [email protected] This work was supported by grants NS34179 and NS 35806. CI is the recipient of a Javits Award from NINDS. Received 15 June 2004; revised 14 September 2004; accepted 15 October 2004; published online 19 January 2005

These include membrane blebbing, nuclear condensation, and appearance of apoptotic bodies (Kerr et al, 1972). Because caspases play such a critical role in apoptotic pathway, their activity is tightly controlled by posttranslational mechanisms (Salvesen and Abrams, 2004), such as inhibitory binding by the inhibitors of the apoptosis (IAPs) family of proteins (Deveraux et al, 1997) and S-nitrosylation by nitric oxide (NO) (Kim et al, 1997; Li et al, 1997; Mannick et al, 1997). Nitric oxide exerts a wide range of biologic effects through activation of soluble guanylyl cyclase (Alderton et al, 2001; Keynes and Garthwaite, 2004), but it can also interact directly with thiol group (SH) on certain cysteine residues, resulting in protein nitrosylation (Stamler, 1994). Because cysteine residues have regulatory roles in enzymes and other proteins, cysteine nitrosylation modulates enzyme activity and alters protein functions. Caspases are among the proteins whose functions are modulated by nitrosylation, and S-nitrosylation of the active cysteine residues of caspase-3 and -9 results in inactivation of the enzymes (Dimmeler et al, 1997; Mannick et al, 1999, 2001).

Nitric oxide inhibits caspase but not cell death P Zhou et al 349

However, it is not known whether the inhibition of caspase activity in neurons produced by Snitrosylation results in improved cell viability. There is evidence that caspase inhibition does not always enhance neuronal survival, but it may promote a caspase-independent form of cell death (Stefanis et al, 1999). Therefore, in this study, we investigated the effect of NO on staurosporine (STS) and camptothecin (Camp)-induced caspase-3 activation, apoptotic morphology, and cell viability in neuronal cultures. We found that NO inhibits STSinduced caspase-9 and -3 activation and eliminates the classical apoptotic nuclear morphology. However, such inhibition of caspase and apoptotic features did not rescue neurons from STS-induced cell death. The data suggest that caspase inhibition by NO drives cells towards a caspase-independent form of neuronal death that counteracts the potentially beneficial effects of caspase inhibition.

Methods Primary Culture of Cortical Neurons Cortical neurons were prepared from the mouse brain at embryonic day 16 to 18 from timed pregnant mouse (C57BL/6J, Jackson laboratory, Bar Harbor, ME) using a method described by Brewer (1995). The cerebral cortex was dissected into 2 mL Neurobasal Medium supplemented with B27 (Invitrogen) at room temperature and triturated through a fire-polished Pasteur pipet. After counting cell viability with trypan blue dye exclusion, the cells were seeded into poly-D-lysine-coated plates at a density of 8 105 /mL and incubated at 371C. The composition of the neuronal culture assessed immunocytochemically at day 5 in culture with specific markers was: neurons 495% (MAP2), for astroglial cells 3% to 5% (GFAP), and microglial cells o1% (CD11).

In Vitro Mouse Brain Slice Culture and Apoptosis Induction The procedure for culturing mouse brain slices and induction of apoptosis in slice by STS was described previously (Zhou et al, 2001). Briefly, the brains from adult mice were sliced coronally into 300-mm sections using a McIlwain tissue chopper. The slices were then plated onto 30 mm Millicell pore filters (Millipore, Bradford, MA) inside a six-well plate with 1.1 mL medium (25% horse serum (Sigma, St Louis, MO), 50% Eagle’s Basal Medium (Sigma), 25% HBSS (Invitrogen), 5 mg/mL glucose (Sigma), 50 U/mL penicillin, 50 mg/mL streptomycin) in each well. Apoptosis was induced with 8 mmol/L STS (Sigma, 5 mmol/L stock in dimethyl sulfoxide). The DMSO concentration in culture medium is 0.16% in volume.

Total RNA Isolation from Cultured Mouse Brain Slice and RT-PCR Detection for iNOS Expression Total RNA from the brain slices was isolated using Trizol reagent (Invitrogen). mRNA was reverse transcribed to

cDNA using reagents from TheremoScript systems (Invitrogen). PCR detection for iNOS expression in the slices after STS incubation was performed as previously described using identical primer sequences (forward: 50 ACAACGTGAAGAAAACCCCTTGTG-30 ; reverse: 50 -ACA GTTCCGAGCGTCAAAGACC-30 ) (Iadecola et al, 1995). PCR parameters were: 951C for 2 mins followed by 40 cycles at 941C for 30 secs, 601C for 30 secs, 721C for 30 secs. The reaction produces a band of 556 base pairs representing mouse iNOS. Beta-actin was used as a control for reaction condition and gel loading. PCR products were loaded on to a 1% agarose gel and imaged with a Kodak F440 Image Station.

Nitric Oxide Donors (7)-S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione (GSNO) were from Sigma. Spermine NONOate and dipropylenetriamine NONOate (DPTA NONOate) were from the Cayman Chemical Company. All NO donor stock solutions were made fresh right before the experiment and diluted into cell culture together with apoptosis-inducing agents STS or Camp, except for DPTA NONOate, which was diluted into culture 30 mins before the addition of STS or Camp. The concentrations of NO donors used were determined based on dose–response experiments and were in the range of those used by others (Badorff et al, 2000; Chen et al, 2001; Kim et al, 2000; Reynaert et al, 2004). However, in cell cultures, the concentration of NO to which the cells are exposed is much smaller than that of the NO donor added to the culture and is likely to be even smaller in the cytoplasm when NO reaches its intracellular targets (Beltran et al, 2000; Kindler et al, 2003).

Caspase-3 and -9 Activity and Cell Viability Assays After 5 days in culture, neurons were treated with STS, Camp, or the NO donors. Caspase-3 and -9 activity was assayed as described (Zhou et al, 2001). Briefly, cells in a 12-well plate were lysed in 200 mL of lysis buffer (25 mmol/L Hepes, pH 7.4, 0.1% Triton X-100, 5 mmol/L MgCl2, 2 mmol/L DTT, 1.5 mmol/L EDTA, 1 mmol/L EGTA, 74 mmol/L antipain, 0.15 mmol/L aprotinin, 15 mmol/L pepstin, 20 mmol/L leupeptin). After a 10-mins incubation on ice, the cells were scraped off the plate and transferred to a tube. Lysates were cleared by centrifugation at 13,000g, 41C for 5 mins. The lysates were incubated with the fluorescent substrate (ac-DEVD-afc for caspase-3 and Ac-LEHD-afc for caspase-9) in freshly made caspase assay buffer (20 mmol/L Hepes, pH 7.4, 100 mmol/L NaCl, 1 mmol/L EDTA, 0.1% Chaps, 10% sucrose, 10 mmol/L DTT) for 60 mins at 37oC. Protein concentration in each sample was measured using a detergent compatible (DC) protein assay kit (Bio-Rad, Hercules, CA). The fluorescence intensity generated by the caspase-3 and -9 activity was measured using a fluorescence plate reader (Perkin-Elmer), with a 400 nmol/L excitation filter and a 505 nmol/L emission filter. The fluorescence reading of each sample was normalized with its protein content and Journal of Cerebral Blood Flow & Metabolism (2005) 25, 348–357


expressed as caspase-3 or -9 activity in relative fluorescence intensity (RFI). Caspase-3 activity in mouse brain slices was assayed essentially the same way as described above. Cell viability was assessed by morphologic criteria using nuclear staining (DAPI) (Zhou et al, 1997) and neuronal cytoskeletal staining (MAP2). For nuclear morphology assessment, cells on the coverslip were fixed in methanol for 5 mins and incubated with DAPI (1 mg/mL in PBS) for 5 mins at room temperature. The cells were then mounted and viewed under a fluorescence microscope equipped with a DAPI filter (Nikon, Garden City, NY). For MAP2 staining, cells were fixed by 4% paraformaldehyde followed by incubation in blocking buffer (3% BSA in Tris-buffered saline þ 0.1% Triton X-100). The cells were then incubated with diluted (1:200) primary antibody (Mouse monoclonal MAP2 from Sigma) in blocking buffer. After washing, the cells were incubated with fluorescein (FITC)-conjugated secondary antibody (goat anti-mouse, Southern Biotech, Birmingham, AL). Labeled cells were viewed using a Nikon fluorescence microscope equipped with a digital camera. Cell viability was also assayed by MTS cellular metabolism measurement. MTS viability assay (Promega) is based on the conversion of a tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS)) in live cells into a formazan product that is soluble in tissue culture medium.

Statistical Analysis Data are presented as mean7s.d. Statistical differences among groups were evaluated by the one-way ANOVA and Turkey’s test. Differences were considered significant for Po0.05.

In addition, the NO donors spermine NONOate and DPTA NONOate dose-dependently reduced the STS-induced caspase-3 activity, while the donors alone did not increase the caspase-3 activity (Figure 1E). Active caspase-3 is a cleavage product of inactive precursor zymogen by initiator caspase-8 or -9 (Earnshaw et al, 1999). To determine whether the reduced caspase-9 activity attenuates procaspase-3 cleavage, we examined the effect of NO donors on the levels of cleaved caspase-3. As expected, STS induced a dramatic rise in active caspase-3 levels (see Figure 1E, lanes 1 and 2), an effect markedly attenuated by NO donors (Figure 1E, lanes 3 and 4). Therefore, NO donors affect the cleavage of procaspase-3. Nitric Oxide Donors Reduce Camptothecin-Induced Caspase-3 Activity

Camptothecin (Camp) is an inhibitor of DNA topoisomerase I and causes DNA damage and apoptosis (Liu et al, 2000; Morris and Geller, 1996). To test if NO donors could also reduce the caspase-3 activity induced by DNA damage, we in duced neuronal apoptosis with Camp (10 mmol/L) and measured caspase-3 activity after adding NO donors. (7)-S-nitroso-N-acetylpenicillamine (50 mmol/ L) reduced Camp-induced caspase-3 activity by approximately 50% (Figure 2). Spermine NONOate and DPTA NONOate (40 mmol/L) also reduced the Camp-induced caspase-3 activity (Figure 2). Staurosporine-Induced Caspase-3 Activity is Increased in Brain Slice of iNOS Null Mice

Results Nitric Oxide Donors Reduce Staurosporine-Induced Caspase-3 and -9 Activation in Primary Neuronal Cultures

We first evaluated the induction of apoptosis in neurons using STS, a protein kinase inhibitor known to induce apoptosis in many cell lines (Bertrand et al, 1994; Jacobsen et al, 1996). Staurosporine induced a robust caspase-3 and -9 activation in primary neuronal cultures (Figure 1A). Since caspase-3 is predominantly activated by caspase-9 in STS-induced apoptosis, we also measured caspase-9 activity. Although total caspase-9 activity was lower than caspase-3, a similar pattern of activity induction by STS was seen (Figure 1B). Incubation with the NO donor SNAP reduced STS-induced caspase-3 activity in a dose-dependent manner (Figure 1C). (7)-S-nitroso-N-acetylpenicillamine alone (600 mmol/L) did not increase caspase3 activity (data not shown). Similarly, STS-induced caspase-9 activity was blocked by SNAP or GSNO. (7)-S-nitroso-N-acetylpenicillamine or GSNO alone did not induce caspase-9 activation (Figure 1D). Journal of Cerebral Blood Flow & Metabolism (2005) 25, 348–357

We then used a brain slice system to determine whether endogenous NO could also affect caspase activity. Staurosporine leads to iNOS induction in some cells (Hecker et al, 1997). Therefore, we studied caspase-3 activity in brain slices of iNOS null mice and wild-type littermates. In wild-type mice, STS induced iNOS mRNA expression (Figure 3A, lane 3) and increased caspase-3 activity (Figure 3B, right panel). In iNOS null mice, STS failed to increase iNOS expression (Figure 3A, lane 4), and caspase-3 activity was significantly higher than in wild-type mice (Figure 3B). The effect of iNOS induction on caspase-3 activity is relatively small (30%), probably reflecting inadequate cell death induction in thick brain slices because of limited penetration of STS (Zhou et al, 2001). Nitric Oxide Donors Prevent Staurosporine-Induced Apoptotic Nuclear Morphology but do not Improve Neuronal Survival

Staurosporine induced robust caspase-3 activation in neurons, which resulted in the characteristic


Figure 1 Nitric oxide donors elicit a dose-dependent reduction in STS-induced caspase-3 activation in primary neuronal culture. Neurons (5 days in culture) were treated with STS with and without the NO donors SNAP, GNSO, spermine NONOate, or DPTA NONOate. After 5 hours’ incubation, the cells were washed and harvested for caspase-3 and -9 activity assay or lysed for immunoblotting analysis of caspase-3 cleavage. Each data point represents three to four independent experiments (*Po0.001; # Po0.05). (A) Induction of caspase-3 activity in STS- (0.25 mmol/L) treated neurons. (B) Induction of caspase-9 activity in STStreated neurons. (C) Dose-dependent reduction in caspase-3 activity by SNAP. (D) Caspase-9 activity inhibition by SNAP (200 mmol/L) and GSNO (100 mmol/L). (E) Dose-dependent reduction in STS- (0.1 mmol/L) induced caspase-3 activity by NONOate-type NO donors. (F) Western blot analysis of caspase-3 cleavage. The protein lysates on membrane from neurons treated with STS and NO donors were probed with an antibody specific for cleaved larger fragment of caspase-3 (17 kDa). Beta actin was used as a loading standard. An equal number of cells (8 105) was loaded on each lane.

apoptotic nuclear morphology. As shown in Figure 4B, nuclei from neurons treated with 0.25 mmol/L STS for 24 hours were condensed and fragmented, typical features of apoptotic nuclei. On average, 86.7%73.5% cells had apoptotic nuclei after STS

treatment (n ¼ 300) compared with 14.5%71.1% in controls (n ¼ 500, Po0.001). However, neurons cotreated with STS and SNAP showed no typical apoptotic nuclear features compared with neurons treated with STS alone. No nuclear fragmentations Journal of Cerebral Blood Flow & Metabolism (2005) 25, 348–357


Figure 2 Nitric oxide donors reduce camptothecin-induced caspase-3 activation. Neurons in culture were cotreated with Camp and SNAP or spermine NONOate, or preincubated with DPTA/NO for 30 mins before the addition of Camp. Caspase-3 activity was assayed after 5 hours additional incubation. Data were derived from three separate experiments. *Po0.001.

and apoptotic bodies were observed (Figure 4C). Compared with the nucleus of normal neurons (Figure 4A), STS and SNAP cotreated neurons showed normal but smaller and denser nuclei (see Figures 4A and 4C). This type of morphology is not caused by SNAP because cells treated with SNAP alone had the same nuclear morphology as untreated neurons (see Figures 4A and 4D). Because NO donors inhibited caspase-9 and -3 activities and abolished the apoptotic nuclear morphology, we further examined whether NO donors improved the viability of STS-treated neurons. At concentrations that inhibited caspase-3 activity, NO donors failed to rescue neurons from STS-induced death assessed either by the MTS assay or MAP2 staining (Figures 5 and 6). Rather, the viability of cells decreased with the addition of NO donors (Figures 5A and 5B), reflecting a synergistic deleterious effect of STS and NO treatments. However, when NO donors were applied alone, there was no significant loss of cell viability even at high concentrations (Figures 5C and 5D).

Discussion

Figure 3 In slice culture, STS induces higher caspase-3 activity in iNOS null mice than in wild-type littermates. Mouse brain slices from either iNOS/ or wild-type littermates (C57BL/6) were incubated with STS (8 mmol/L) and collected for RNA isolation and caspase-3 activity assay at the time points indicated. (A) RT-PCR detection of iNOS expression in slice incubated with STS. Housekeeping gene GAPDH was used here as a loading control. (B) Caspase-3 activity assay in slices from iNOS null and wild-type mice (N ¼ 8 pairs and Po0.05). Journal of Cerebral Blood Flow & Metabolism (2005) 25, 348–357

We investigated whether NO could inhibit caspase activity in neurons and improve their survival in a model of STS-induced apoptosis. We found that NO donors inhibit STS-induced caspase-3 and -9 activities and Camp-induced caspase-3 activity in neuronal cultures. Furthermore, STS induces higher levels of caspase-3 activity in iNOS null mice than in controls. The inhibition in caspase-3 activity is mainly because of the reduction of cleavage of caspase-3 precursor by caspase-9. NO donors also prevented the classic apoptotic nuclear morphology on STS incubation. However, the inhibition of caspase activity and apoptotic nuclear fragmentation failed to rescue neurons from STS-induced cell death, as determined by MAP2 staining and viability (MTS) assay. These findings suggest that NO, while inhibiting STS-induced caspase activity and apoptotic nuclear morphology, does not prevent cell death. Neuronal apoptosis was induced using STS or Camp. Staurosporine is a potent apoptotic trigger for nonneuronal cells (Bertrand et al, 1994) and neurons (Koh et al, 1995). Staurosporine induces classical features of apoptosis, such as mitochondria dysfunction, caspase activation, and nuclear fragmentation (Krohn et al, 1998; Kruman et al, 1998). Camp induces DNA damage (Liu et al, 2000) and has been used to study DNA damage-induced neuronal apoptosis (Hetman et al, 1999; Lang-Rollin et al, 2003; Morris and Geller, 1996; Morris et al, 2001; Stefanis et al, 1999). In our experimental system, the STS-induced apoptotic morphology was eliminated when caspase activity was inhibited with NO donors, but the neurons were not rescued from cell death. These data indicate that caspase inhibition is


Figure 4 Nitric oxide donors prevent the occurrence of apoptotic nuclear morphology in neuronal cultures treated with STS. Five-dayold neuronal cultures were treated with 0.25 mmol/L STS together with NO donors for 24 hours, and the cells were stained with DAPI to observe nuclear morphology. (A) Normal nuclear morphology in neurons without treatment. (B) Apoptotic nuclei in neurons treated with STS. (C) Neurons treated with 0.25 mmol/L STS and 500 mmol/L SNAP. (D) Neurons treated with 500 mmol/L SNAP alone. Note the disappearance of apoptotic nuclear morphology in panel C (compared with B). Bar ¼ 10 mm. (E) Quantitative analysis of condensed or fragmented nuclei in control (A) and STS-treated (B) neuronal cultures. DAPI-stained cells were photographed and apoptotic nuclei were counted according to morphologic criteria. The nuclei counted were 500 in controls and 300 cells in the treatment groups from three separate experiments. *Po0.001.

sufficient for blocking the morphologic features of apoptosis, but not for cell survival. Our findings suggest that, after exposure to STS, the cell fate is determined before the activation of caspase. Caspase

inhibition by NO donors causes the cell to adopt a caspase-independent pathway to death. Caspase-independent cell death has been reported previously. Stefanis et al (1999) showed in rat Journal of Cerebral Blood Flow & Metabolism (2005) 25, 348–357


cortical neurons that a broad-spectrum caspase inhibitor, boc-aspartyl(OMe)-fluoromethylketone (BAF), inhibited camptothecin-induced caspase activation and apoptosis, resulting in caspaseindependent neuronal death. This type of cell death involves energy depletion and free radical generation, but not apoptosis-inducing factor (AIF) trans-

location (Lang-Rollin et al, 2003). In sympathetic neurons, nerve growth factor (NGF) deprivation induces a caspase-dependent apoptotic death (Deshmukh et al, 1996). Caspase activity inhibition by BAF can protect NGF-deprived cells for a period of time. The BAF-saved cells eventually die by a nonapoptotic, caspase-independent mechanism if readdition of NGF is beyond the point of mitochondrial membrane potential collapse (Deshmukh et al, 2000). In the present study, the apoptosis inducer and caspase inhibitor are different from those used in the above studies, but the ultimate outcome for cell fate appears to be the same. Collectively, these observations suggest a common pathway involved in this aborted apoptosis that is caspase-independent. In our study, inhibition of caspase activity was achieved through the use of NO donors or iNOS induction. Two types of NO donors were used in this study: the S-nitrosothiol NO donors, SNAP and GSNO, and the NONOate donors, spermine/NO and DPTA/NO. Both types of NO donors produced similar inhibition of caspase-3 activity, indicating that the effect is likely mediated by NO released from these donors. Nitric oxide has been shown to directly nitrosylate a critical cysteine residue in the active site of caspases and renders the protease inactive (Li et al, 1997). Activation of caspase-3 is the direct result of cleavage of procaspase-3 by upstream initiator caspases such as caspase-9. The caspase-3 activity inhibition by NO donors could either be because of inactivation of caspase-9, which results in lack of cleavage of pro-caspase-3, or direct nitrosylation of active caspase-3. Our results indicate that NO donors inhibit pro-caspase-3 cleavage by caspase-9. Therefore, reduction of active caspase3 contributes to the attenuation in caspase-3 activity produced by NO donors. However, we cannot rule out direct caspase-3 inhibition resulting from cysteine nitrosylation by NO donors. In addition to inhibition of apoptosis, NO has also been shown to induce apoptosis (Brune et al, 1998).

Figure 5 The inhibition in caspase-3 and -9 activity by NO donors does not result in improved neuronal survival. Neuronal cultures were treated with NO donors (SNAP or GSNO) in the presence or absence of STS. The viability was assessed 24 hours later by the MTS assay and expressed as a percentage of controls. Results shown were from three independent experiments, #Po0.01 compared with controls in (A) and (B). Valves in (C) and (D) are statistically insignificant between controls and experimental groups. (A) Neurons were treated with STS alone or together with SNAP in indicated concentrations. (B) Neurons were treated with STS along or together with GSNO in indicated concentrations. Note the reduction in cell viability after treatment with STS, or combined with SNAP and GSNO. (C) and (D) SNAP (500 mmol/L) or GSNO (200 mmol/L) alone does not affect cell viability. Neurons were treated with different doses of SNAP or GSNO for 24 hours without STS and assayed for viability. Note that there is no significant reduction in viability across the concentration range. Journal of Cerebral Blood Flow & Metabolism (2005) 25, 348–357


Figure 6 The inhibition in caspase-3 activity induced by NO donors does not result in improved neuronal survival, neuronal morphology. Neuronal cultures were treated as indicated, fixed 24 hours later and labeled with MAP2 antibody and counterstained with DAPI to assess the effect of SNAP on morphology. (A) Normal neurons without any treatment. (B) Neurons with 0.25 mmol/L STS for 5 hours, washed and changed to normal medium for 19 more hours. Note the significant reduction in MAP-2 labeling (compare with panel A). (C) Neurons cotreated with 0.25 mmol/L STS and 500 mmol/L SNAP for 5 hours. (D) Dead neurons after 24 hours treatment with 0.25 mmol/L STS. Note the nearly complete loss of MAP2 staining. (E) Neurons cotreated with STS and 500 mmol/L SNAP for 24 hours. Bar ¼ 100 mm.

Interestingly, we did not observe toxicity when NO donors were applied alone in neuronal culture even at high concentrations (see Figures 5C and 5D). However, the toxicity of a high concentration of NO became evident when STS was coincubated with NO donors. This observation suggests a synergistic toxic effect that reduced cell viability in a concentration dependent way. This is likely because of the oxidative stress in neurons exposed to NO and STS simultaneously. Staurosporine is able to induce the

production of superoxide in neurons (Krohn et al, 1998). The presence of abundant NO helps to form the strong oxidant peroxynitrite (Lipton et al, 1993) and contributes to the oxidative stress leading to accelerated cell death. In conclusion, we found that different types of NO donors effectively inhibit caspase-3 and -9 activity in primary neuronal cultures exposed to STS and Camp. However, such caspase inhibition did not enhance neuronal survival. Rather, the cells died in Journal of Cerebral Blood Flow & Metabolism (2005) 25, 348–357


a caspase-independent manner. Our results raise the possibility that in neurons caspase inhibition, while reversing apoptotic morphology, is not sufficient to prevent cell death in the presence of lethal insults. Understanding the mechanism of this type of cell death could provide better strategies for neuroprotection in apoptotic neuronal death.

Acknowledgements We thank Dr Rajiv Ratan for helpful suggestions.

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