Abstract
The anesthetic isoflurane has been reported to induce apoptosis and increase Aβ generation and aggregation. However, the molecular mechanism underlying these effects remains unknown. We therefore set out to assess whether the effects of isoflurane on apoptosis are linked to amyloid β-protein (Aβ) generation and aggregation. For this purpose, we assessed the effects of isoflurane on β-site amyloid β precursor protein (APP)-cleaving enzyme (BACE) and γ-secretase, the proteases responsible for Aβ generation. We also tested the effects of inhibitors of Aβ aggregation (iAβ5, a β-sheet breaker peptide; clioquinol, a copper–zinc chelator) on the ability of isoflurane to induce apoptosis. All of these studies were performed on naive human H4 neuroglioma cells as well as those overexpressing APP (H4-APP cells). Isoflurane increased the levels of BACE and γ-secretase and secreted Aβ in the H4-APP cells. Isoflurane-induced Aβ generation could be blocked by the broad-based caspase inhibitor Z-VAD. The Aβ aggregation inhibitors, iAβ5 and clioquinol, selectively attenuated caspase-3 activation induced by isoflurane. However, isoflurane was able to induce caspase-3 activation in the absence of any detectable alterations of Aβ generation in naive H4 cells. Finally, Aβ potentiated the isoflurane-induced caspase-3 activation in naive H4 cells. Collectively, these findings suggest that isoflurane can induce apoptosis, which, in turn, increases BACE and γ-secretase levels and Aβ secretion. Isoflurane also promotes Aβ aggregation. Accumulation of aggregated Aβ in the media can then promote apoptosis. The result is a vicious cycle of isoflurane-induced apoptosis, Aβ generation and aggregation, and additional rounds of apoptosis, leading to cell death.
Introduction
Alzheimer's disease (AD), the most common form of age-related dementia, is a rapidly growing health problem. Amyloid β-protein (Aβ) production and/or accumulation are major pathological hallmarks of AD (Glenner and Wong, 1984) (for review, see Tanzi and Bertram, 2005). Aβ is produced via serial proteolysis of the amyloid β precursor protein (APP) by aspartyl protease β-site APP-cleaving enzyme (BACE) or β-secretase and γ-secretase. BACE cleaves APP to generate a 99-residue membrane-associated C-terminal fragment (CTF) (APP-C99). APP-C99 is further cleaved by γ-secretase to release 4 kDa Aβ and AICD (β-amyloid precursor protein intracellular domain) (Gu et al., 2001; Sastre et al., 2001; Yu et al., 2001). Presenilin (PS) and γ-secretase cofractionate as a detergent-sensitive, high-molecular-weight complex (Li et al., 2000) that includes at least three other proteins, nicastrin/anterior pharynx defective protein 2 (APH-2), APH-1, and presenilin enhancer protein 2 (PEN-2), all of which are necessary and sufficient for γ-secretase activity (Yu et al., 2000; Francis et al., 2002; Steiner et al., 2002). Increasing evidence suggests a role for caspase activation and apoptotic cell death in AD, as well as in a large number of other neurodegenerative disorders, such as Huntington's disease, amyotrophic lateral sclerosis, and spinocerebellar ataxia (Holtzman and Deshmukh, 1997; Lunkes et al., 1998; Namura et al., 1998). Aβ has been shown to cause caspase activation and apoptosis, which can in turn potentiate Aβ generation (LeBlanc, 1995; Guo et al., 1997; Galli et al., 1998; Gervais et al., 1999; LeBlanc et al., 1999; Pillot et al., 1999; Sponne et al., 2003; Tesco et al., 2003; Sodhi et al., 2004; Kriem et al., 2005; Florent et al., 2006). Finally, fibrillar aggregates of Aβ and oligomeric species of Aβ are more neurotoxic (Pike et al., 1993; Lorenzo and Yankner, 1994; Lambert et al., 1998; Grace et al., 2002; Walsh et al., 2002; Wang et al., 2002; Kayed et al., 2003; Kim et al., 2003; De Felice et al., 2004).
Perioperative factors, including hypoxia (Kokmen et al., 1996; Jendroska et al., 1997; Nagy et al., 1997; Snowdon et al., 1997; Kalaria, 2000), hypocapnia (Xie et al., 2004), and anesthetics (Eckenhoff et al., 2004; Xie et al., 2006a), have been reported to potentially contribute to AD neuropathogenesis. These perioperative factors may also cause postoperative cognitive dysfunction, a dementia associated with surgery and anesthesia, by trigging AD neuropathogenesis. Treatment with a commonly used inhalation anesthetic isoflurane (1.2–2.5% for 6 h) has been shown to enhance Aβ aggregation and cytotoxicity in pheochromocytoma cells (Eckenhoff et al., 2004). We recently showed that a clinically relevant concentration (2%) of isoflurane can induce apoptosis, alter APP processing, and increase production of Aβ in H4 human neuroglioma cells (Xie et al., 2006a).
Given these observations, we set out to determine the relationship between isoflurane-induced apoptosis and Aβ generation/aggregation. More specifically, we addressed the hypothesis that isoflurane induces a vicious cycle of apoptosis, Aβ generation, Aβ aggregation, and additional rounds of apoptosis and Aβ production.
Materials and Methods
Cell lines.
We used H4 human neuroglioma cells (naive H4 cells) and H4 human neuroglioma cells stably transfected to express full-length (FL) APP (H4-APP cells) in the experiments. All cell lines were cultured in DMEM (high glucose) containing 9% heat-inactivated fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mm l-glutamine. Stably transfected H4 cells were additionally supplemented with 200 μg/ml G418.
Cell treatment.
The cells were treated with 21% O2, 5% CO2, and 2% isoflurane as described by Xie et al. (2006a). In the interaction studies, the cells were treated with Z-VAD (100 μm), Aβ (2.5, 5, and 7.5 μm), iAβ5 (1.5 μg/μl), and clioquinol (1 μm) 1 h before the treatment with 2% isoflurane or 100 nm staurosporine (STS). Control conditions included 5% CO2 plus 21% O2, which did not affect caspase-3 activation, cell viability, APP processing, and Aβ generation (data not shown).
Cell lysis and protein amount quantification.
Cell pellets were detergent extracted on ice using immunoprecipitation buffer (10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 2 mm EDTA, and 0.5% Nonidet P-40) plus protease inhibitors (1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin A). The lysates were collected, centrifuged at 12,000 rpm for 10 min, and quantified for total proteins by the BCA protein assay kit (Pierce, Iselin, NJ).
Western blot analysis.
The cells were harvested at the end of the experiments and were subjected to Western blot analyses as described by Xie et al. (2005a). Antibodies A8717 (1:2000; Sigma, St. Louis, MO) and anti-β-actin (1:5000; Sigma) were used to visualize APP-FL (110 kDa), APP-CTFs (10–12 kDa), and β-actin (42 kDa), respectively. A caspase-3 antibody (1:1000 dilution; Cell Signaling Technology, Beverly, MA) was used to recognize the caspase-3 fragment (17–20 kDa) resulting from cleavage at aspartate position 175 and caspase-3 FL (35–40 kDa). Rabbit polyclonal anti-BACE-1 antibody ab2077 (1:1000; Abcam, Cambridge, MA) was used to detect the protein levels of BACE (65 kDa). Antibody anti-nicastrin (1:1000; Calbiochem, La Jolla, CA) was used to detect nicastrin levels. The quantitation of Western blots was performed as described by Xie et al. (2005a). Briefly, the intensity of signals was analyzed by using an NIH Image program (NIH Image 1.62). We quantified the Western blots using two steps. First, we used the levels of β-actin to normalize (e.g., determining the ratio of APP-FL amount to β-actin amount) the levels of APP-FL, APP-CTFs, FL-caspase-3, caspase-3 fragment, BACE, and nicastrin to control for the loading differences in total protein amounts. Second, we presented the changes in the levels of APP-FL, APP-CTFs, FL-caspase-3, caspase-3 fragment, and nicastrin in the cells treated with isoflurane, Z-VAD, iAβ5, clioquinol, and Aβ as the percentage of those in the cells treated with controls. In this study, 100% caspase-3 activation, APP-FL, APP-CTFs, BACE, and nicastrin refer to control levels for the purpose of comparison with experimental conditions.
Quantitation of Aβ using sandwich ELISA assay.
Secreted Aβ was measured with a sandwich ELISA assay by using an Aβ measurement kit (Invitrogen, Carlsbad, CA) and by the Aβ ELISA Core Facility at the Center for Neurological Diseases, Brigham and Women's Hospital, Harvard Medical School (Boston, MA), as described by Xie et al. (2005b). Specifically, 96-well plates were coated with mouse monoclonal antibodies specific to Aβ40 (β-amyloid [1-40] cleavage site-specific antibody) or Aβ42 (β-amyloid [1-42] cleavage site-specific antibody). After blocking with Block Ace, wells were incubated overnight at 4°C with test samples of conditioned cell culture media, and then an anti-Aβ (α-Aβ-HR1) conjugated to horseradish peroxidase was added. Plates were then developed with tetramethylbenzidine reagent, and well absorbance was measured at 450 nm. Aβ levels in test samples were determined by comparison with the signal from unconditioned media spiked with known quantities of Aβ40 and Aβ42.
Cell viability study.
The cell viability was determined by using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT; Sigma). The experiments were performed according to the manufacturer's protocol. Briefly, we added 150 μl of MTT solution to each well, containing 1.5 ml of cell culture media, of a six-well plate. We then returned the cell culture to the incubator for 2 h. Finally, we removed the cell culture fluid and added 1.5 ml of isopropanol into the wells. We spectrophotometrically measured the absorbance at a wavelength of 570 nm. We present the changes in the absorbance, as a measure of cell viability, in the cells treated with isoflurane as the percentage of those in the cells treated with control conditions.
Statistics.
Given the presence of background caspase-3 activation and cell death in cells cultured in serum-free media, we did not use absolute values to describe changes in caspase-3 activation and cell viability. Instead, changes in caspase-3 activation and cell viability were presented as a percentage of those of the control group. One-hundred percent caspase-3 activation or cell viability refers to control levels for purposes of comparison with experimental conditions. Data were expressed as mean ± SD. The number of samples varied from three to 10, and the samples were normally distributed. We used a two-tailed t test to compare the difference between the experimental groups. p values <0.05 and 0.01 were considered statistically significant.
Results
Isoflurane can induce caspase-3 activation in naive H4 cells
We previously reported that isoflurane can induce apoptosis and potentiate Aβ levels in the conditioned media of H4-APP cells (Xie et al., 2006a). To test whether isoflurane can induce apoptosis in cells with low (barely detectable) basal levels of secreted Αβ, naive H4 human neuroglioma cells were treated with 2% isoflurane for 6 h. Because caspase-3 activation is one of the final steps of cellular apoptosis (Thornberry, 1998), we assessed the effects of isoflurane on caspase-3 activation by quantitative Western blot analyses. Isoflurane treatment led to caspase-3 activation (Fig. 1 A,B), as evidenced by increased ratios of cleaved (activated) caspase-3 fragment (17–19 kDa) to FL caspase-3 (35–40 kDa). Quantitation of the Western blots, based on the ratio of caspase-3 fragment to FL caspase-3, revealed that the 2% isoflurane treatment (Fig. 1 B, black bar) led to a 240% increase in caspase-3 activation compared with control cells (Fig. 1 B, white bar, **p < 0.01). Treatment with 2% isoflurane (Fig. 1 C, black bar) also decreased cell viability by 17% compared with the control cells (Fig. 1 C, white bar, *p < 0.05). APP immunoblotting revealed no significant differences in the levels of APP-CTFs and APP-FL between 2% isoflurane-treated (Fig. 1 D, lanes 3, 4; E,F, black bar) and control (Fig. 1 D, lanes 1, 2; E,F, white bar) naive H4 cells. Treatment with 2% isoflurane (black bar) did not increase levels of Aβ40 (27 vs 30 pg/ml) or Aβ42 (7 vs 9 pg/ml) compared with the control cells (white bar) (Fig. 1 G). These results suggest that the isoflurane can induce apoptosis independently of changes in APP processing and Aβ generation in naive H4 cells. Collectively, these findings suggest that isoflurane can induce apoptosis/caspase activation in the absence of any significant alterations in APP processing and Aβ generation.
Treatment with 2% isoflurane induces caspase-3 activation and decreases cell viability without detectable changes in APP processing and Aβ generation in naive H4 cells. A , The 2% isoflurane treatment (lanes 1–3) induces caspase-3 cleavage (activation) compared with control conditions (lanes 4–6) in naive H4 cells. There is no significant difference in the amounts of β-actin between the control- or 2% isoflurane-treated naive H4 cells. B , Caspase-3 activation assessed by quantifying the ratio of caspase-3 fragment to caspase-3-FL in the Western blots. Quantification of the Western blot shows that the 2% isoflurane treatment (black bar) increases caspase-3 activation compared with control conditions (white bar), normalized to β-actin levels. C , Treatment with 2% isoflurane (black bar) decreases cell viability compared with control conditions (white bar) in naive H4 cells. D , Treatment with 2% isoflurane (lanes 3, 4) does not alter the levels of APP-FL and APP-CTFs compared with control conditions (lanes 1, 2). There is no significant difference in the amounts of β-actin in the control- or 2% isoflurane-treated naive H4 cells. E , Quantification of the Western blot shows that the 2% isoflurane treatment (black bar) does not alter the protein levels of APP-FL compared with control conditions (white bar) in H4 naive cells, normalized to β-actin levels. F , Quantification of the Western blot shows that the 2% isoflurane treatment (black bar) does not alter the protein levels of APP-CTFs compared with control conditions (white bar) in H4 naive cells, normalized to β-actin levels. G , Treatment with 2% isoflurane (black bar) does not increase the generation of Aβ40 and Aβ42 compared with control conditions (white bar). Data are means ± SD; n = 9–10 for each experimental group. t test was used to compare the difference between control condition and the 2% isoflurane treatment condition (*p < 0.05; **p < 0.01).
Isoflurane-induced alterations in APP processing and Aβ generation can be attenuated by the caspase inhibitor Z-VAD in H4-APP cells
We reported previously that isoflurane can alter APP processing and increase Aβ generation in H4-APP cells (Xie et al., 2006a). We next asked whether these effects are dependent on caspase activation. For this purpose, we incubated H4-APP cells with Z-VAD (100 μm), a caspase inhibitor, for 1 h, followed by treatment with 2% isoflurane for 6 h. Isoflurane induced caspase-3 activation, which was blocked by treatment with Z-VAD (Fig. 2 A). Quantification of the Western blots, based on the ratio of caspase-3 fragment to FL caspase-3, revealed that treatment with 2% isoflurane induced caspase-3 activation by 350% over that of control cells (Fig. 2 B, **p < 0.01). Treatment with isoflurane and Z-VAD reduced caspase-3 activation from 350 to 151% (Fig. 2 B, ## p < 0.01). Treatment with Z-VAD also attenuated isoflurane-induced alterations in APP processing and Aβ generation. As can be seen in Figure 2 C, APP immunoblotting revealed that isoflurane treatment (lanes 5, 6) decreased protein levels of APP-FL and APP-CTFs, compared with the control cells (lanes 1, 2). Z-VAD (lanes 7, 8) suppressed these decreases in levels of APP-FL and APP-CTFs induced by isoflurane, whereas Z-VAD treatment alone (lanes 3, 4) did not alter APP-FL and APP-CTF protein levels (Fig. 2 C). Quantification of the Western blots showed that treatment with isoflurane (black bar) led to a 35% reduction in levels of APP-FL (Fig. 2 D) and 51% reduction in levels of APP-CTFs (Fig. 2 E). Treatment with isoflurane plus Z-VAD (striped bar) resulted in only a 10% decrease in protein levels of APP-FL (Fig. 2 D) and a 12% decrease in protein levels of APP-CTFs (Fig. 2 E). Isoflurane treatments, but not Z-VAD treatment, significantly increased Aβ levels in the conditioned media, whereas treatment with isoflurane plus Z-VAD (striped bar) led to smaller increases in Aβ levels (157%) versus treatment with isoflurane alone (black bar; 204%) (Fig. 2 F) compared with control conditions (white bar). These results indicate that isoflurane-induced alterations in APP processing and Aβ generation are dependent on the ability of isoflurane to induce caspase-3 activation and apoptosis.
The caspase inhibitor Z-VAD inhibits the caspase-3 activation and the increases in Aβ generation induced by 2% isoflurane in H4-APP cells. A , Treatment with 2% isoflurane (lanes 5, 6) induces caspase-3 cleavage (activation) compared with control conditions (lanes 1, 2) or Z-VAD (100 μm) treatment (lanes 3, 4). The Z-VAD treatment inhibits the caspase-3 cleavage induced by 2% isoflurane (lane 7, 8). There is no significant difference in the amounts of β-actin in the H4-APP cells with the above treatments. B , Quantitation of the Western blot shows that the 2% isoflurane treatment (black bar) increases caspase-3 activation compared with control conditions (white bar) or the Z-VAD (100 μm) treatment (gray bar), normalized to β-actin levels. The isoflurane-induced caspase-3 activation is inhibited by the Z-VAD treatment (striped bar). C , Z-VAD inhibits the isoflurane-induced changes in APP processing in H4-APP cells. Treatment with 2% isoflurane (lanes 5, 6) decreases the protein levels of APP-FL and APP-CTFs compared with control conditions (lanes 1, 2) or Z-VAD (100 μm) treatment (lanes 3, 4). The Z-VAD treatment (lanes 7, 8) inhibits the isoflurane-induced decreases in the protein levels of APP-FL and APP-CTFs. There is no significant difference in the amounts of β-actin in the H4-APP cells with all of the above treatments. D , Quantification of the Western blot shows that 2% isoflurane treatment (black bar) decreases the protein levels of APP-FL compared with the control condition (white bar) or Z-VAD treatment (gray bar), normalized to β-actin levels. The isoflurane-induced decrease in the protein levels of APP-FL is inhibited by the Z-VAD treatment (striped bar). E , Quantification of the Western blot also shows that 2% isoflurane treatment (black bar) decreases the protein levels of APP-CTFs compared with the control condition (white bar) or Z-VAD treatment (gray bar), normalized to β-actin levels. The isoflurane-induced decrease in the protein levels of APP-CTFs is also inhibited by the Z-VAD treatment (striped bar). F , Z-VAD inhibits the isoflurane-induced increases in Aβ generation. Treatment with 2% isoflurane (black bar) increases the levels of Aβ40 compared with the control condition (white bar). Z-VAD treatment alone (gray bar) does not change the levels of Aβ40; however, Z-VAD treatment inhibits the isoflurane-induced increases in the levels of Aβ40. Data are means ± SD; n = 6 for each experimental group. t test is used to compare the difference between control conditions and 2% isoflurane treatment (*p < 0.05; **p < 0.01) and the difference between 2% isoflurane plus DMSO treatment and 2% isoflurane plus Z-VAD treatment (# p < 0.05; ## p < 0.01).
Isoflurane enhances levels of BACE and γ-secretase in H4-APP cells
Given that 2% isoflurane can induce apoptosis and increase Aβ generation, we next asked whether isoflurane can increase the levels of the amyloidogenic secretases, BACE and γ-secretase, in H4-APP cells. Treatment with 2% isoflurane for 6 h increased caspase-3 activation and simultaneously increased the levels of BACE. The level of APP-FL was also decreased by isoflurane-induced caspase activation (Fig. 3 A,B). Next, we assessed the effects of isoflurane on the levels of nicastrin, a γ-secretase complex component, in H4-APP cells. Treatment with 2% isoflurane increased protein levels of mature (121%) and immature (156%) nicastrin compared with the control cells (Fig. 3 C,D).
Isoflurane increases levels of BACE and nicastrin in H4-APP cells. A , Treatment with 2% isoflurane (lane 1) induces caspase-3 cleavage (17 kDa; activation), increases the level of BACE (65 kDa), and decreases the APP-FL level (110 kDa) compared with the control condition (lane 2). There is no significant difference in the amounts of β-actin between the control condition- or 2% isoflurane-treated H4-APP cells. B , Quantification of the Western blot shows that 2% isoflurane treatment (black bar) induces caspase-3 activation, increases BACE levels, and decreases APP-FL levels, normalized to β-actin levels. C , Treatment with 2% isoflurane (lanes 4–6) increases the levels of immature and mature nicastrin compared with the control condition (lanes 1–3). There is no significant difference in the amounts of β-actin between the control- or 2% isoflurane-treated H4-APP cells. D , Quantification of the Western blot shows that 2% isoflurane treatment (black bar) increases the levels of both immature and mature nicastrin, normalized to β-actin levels. Data are means ± SD; n = 3 for each experimental group. t test was used to compare the difference between control condition and 2% isoflurane treatment (*p < 0.05; **p < 0.01).
Aβ aggregation inhibitors, iAβ5 and clioquinol, attenuate isoflurane-induced caspase-3 activation
Isoflurane has previously been shown to enhance Aβ aggregation (Eckenhoff et al., 2004). Thus, we next tested whether Aβ aggregation can potentiate the effects of isoflurane on caspase-3 activation and apoptosis. For this purpose, we set out to assess the effects of two known Aβ aggregation inhibitors, the β-sheet breaker peptide iAβ5 (Soto et al., 1998) and the metal protein attenuation compound (MPAC), clioquinol (Cherny et al., 2001), on isoflurane-induced caspase-3 activation. H4-APP cells were incubated with iAβ5 (1.5 μg/μl) or clioquinol (1 μm) for 1 h, followed by treatment with 2% isoflurane or 100 nm STS for 6 h. Treatment with 2% isoflurane alone induced caspase-3 activation in H4-APP cells (Fig. 4 A,B). iAβ5 plus 2% isoflurane treatment reduced caspase-3 activation relative to the treatment with isoflurane alone (170 vs 247%) (Fig. 4 A,B). In contrast, iAβ5 treatment did not attenuate STS-induced caspase-3 activation in H4-APP cells. iAβ5 alone did not induce caspase-3 activation; treatment with either STS or STS plus iAβ5 led to similar increases in caspase-3 activation (1200 vs 1250%) (Fig. 4 C,D). These results suggest that iAβ5 selectively attenuates isoflurane-induced, but not STS-induced, caspase-3 activation.
iAβ5 and clioquinol specifically attenuate isoflurane-induced caspase-3 activation in H4-APP cells. A , iAβ5 plus 2% isoflurane treatment (lane 2) results in a lower degree of caspase-3 cleavage than 2% isoflurane treatment alone (lane 1). There is no significant difference in the amounts of β-actin between the 2% isoflurane- and iAβ5 plus 2% isoflurane-treated H4-APP cells. B , Quantification of the Western blot, based on the ratio of caspase-3 fragment to caspase-3 FL, shows that iAβ5 treatment (black bar) reduces the isoflurane-induced caspase-3 activation (white bar), normalized to β-actin levels. C , STS treatment (lane 3), but not iAβ5 treatment (lane 2), causes caspase-3 activation compared with control condition (lane 1) in H4-APP cells. iAβ5 plus STS treatment (lane 4) leads to a degree of caspase-3 activation similar to STS treatment alone (lane 3). There is no significant difference in the amounts of β-actin in the control-, iAβ5-, STS-, or iAβ5 plus STS-treated H4-APP cells. D , Quantification of the Western blot shows that iAβ5 (striped bar) does not reduce the STS-induced (black bar) caspase-3 activation, normalized to β-actin levels. E , Two percent isoflurane (lane 3) or STS (lane 5) treatment causes caspase-3 activation compared with control condition (lane 1) or clioquinol treatment (lane 2) in H4-APP cells. Clioquinol plus 2% isoflurane treatment (lane 4) leads to a lower degree of caspase-3 activation than 2% isoflurane treatment alone (lane 3). Clioquinol plus STS treatment (lane 6) leads to a degree of caspase-3 activation similar to STS treatment alone (lane 5). There is no significant difference in the amounts of β-actin in all of the above treatments in H4-APP cells. F , Quantification of the Western blot shows that clioquinol reduces the isoflurane-induced (gray bar vs black bar), but not STS-induced (dotted bar vs striped bar), caspase-3 activation, normalized to β-actin levels. Data are means ± SD; n = 3 for each experimental group. t test was used to compare the difference of caspase-3 activation between control condition and STS or 2% isoflurane treatment (*p < 0.05; **p < 0.01) and between saline treatment and iAβ5 or clioquinol treatment (# p < 0.05).
We next tested the ability of clioquinol to attenuate isoflurane-induced caspase activation in the H4-APP cells. Treatment with clioquinol alone did not increase caspase-3 cleavage (Fig. 4 E,F). Both 2% isoflurane and STS induced caspase-3 activation. Treatment with 2% isoflurane plus clioquinol reduced caspase-3 activation relative to treatment with 2% isoflurane alone (174 vs 257%) (Fig. 4 E,F). In contrast, treatment with STS alone or STS plus clioquinol led to similar increases in caspase-3 activation (487 vs 501%) (Fig. 4 E,F). Thus, both Aβ aggregation inhibitors, iAβ5 and clioquinol, selectively attenuated isoflurane-induced, but not STS-induced, caspase-3 activation. These findings suggest that Aβ aggregation can potentiate the ability of isoflurane to induce caspase-3 activation.
Exogenously added Aβ can potentiate isoflurane-induced caspase-3 activation
Given that that Aβ aggregation potentiates isoflurane-induced caspase-3 activation, we next asked whether exogenously added Aβ can potentiate isoflurane-induced caspase activation. Naive H4 cells were incubated with Aβ (2.5, 5, and 7.5 μm both Aβ40 and Aβ42) for 1 h, followed by treatment with 2% isoflurane for 6 h. Both 2% isoflurane and Aβ alone induced caspase-3 activation in naive H4 cells (Fig. 5 A,B). However, treatment with Aβ plus 2% isoflurane resulted in a greater degree of caspase-3 cleavage than either treatment alone, in a dose-dependent manner (469, 403, 612, and 1223%) (Fig. 5 A,B). These results suggest that exogenously added Aβ can potentiate the isoflurane-induced caspase-3 activation in naive H4 cells.
Aβ potentiates the isoflurane-induced caspase-3 activation in H4 naive cells. A , Treatments with 2% isoflurane (lanes 3, 7, 11) cause caspase-3 cleavage (activation) compared with control conditions (lanes 1, 5, 9) in H4 naive cells. Aβ plus 2% isoflurane treatment (lanes 4, 8, 12) leads to a greater degree of caspase-3 cleavage than 2% isoflurane treatment alone (lanes 3, 7, 11) in a dose-dependent manner. Aβ treatments alone (lanes 2, 6, 10) also cause caspase-3 activation compared with saline treatments (lanes 1, 5, 9) in a dose-dependent manner. There is no significant difference in the amounts of β-actin between the control- or 2% isoflurane-treated H4-APP cells. B , Quantification of the Western blot shows that Aβ [0 (white bar), 2.5 (gray bar), 5 (black bar), and 7.5 μm (striped bar)] induces caspase-3 activation and potentiates the isoflurane-induced caspase-3 activation in a dose-dependent manner, normalized to β-actin levels. Data are means ± SD; n = 3 for each experimental group. t test was used to compare the difference of caspase-3 activation between saline and Aβ treatment in 2% isoflurane-treated cells (*p < 0.05; **p < 0.01) and in cells with the control condition (# p < 0.05; ## p < 0.01).
Discussion
We have shown previously that the commonly used inhalation anesthetic isoflurane can induce cellular apoptosis and increase Aβ generation in H4-APP cells (Xie et al., 2006a). Here, we set out to assess whether the effects of isoflurane on apoptosis are linked to Aβ generation and aggregation. First, we found that isoflurane can induce caspase-3 activation in naive H4 cells without significantly affecting APP processing and Aβ generation (Fig. 1). This indicates that isoflurane-induced apoptosis can occur independently of alterations in APP processing and Aβ generation. However, it is still possible that isoflurane may lead to undetectable changes in APP processing and Aβ generation in naive H4 cells.
Next, we examined whether isoflurane-induced alterations in APP processing and Aβ generation are dependent on isoflurane-induced apoptosis. Using the broad caspase activation inhibitor, Z-VAD, we were able to show that inhibition of isoflurane-induced caspase-3 activation was coupled to inhibition of the effects of isoflurane on APP processing and Aβ generation (Fig. 2). These findings revealed that the isoflurane-induced alterations in APP processing and Aβ generation are largely dependent on the ability of isoflurane to induce apoptosis. Collectively, these findings suggest that isoflurane can induce caspase-3 activation and apoptosis, which, in turn, alter APP processing and increase Aβ generation. In contrast, isoflurane was able to induce apoptosis in the absence of any detectable effects on APP processing and Aβ generation.
In exploring the mechanism by which isoflurane increases Aβ generation, we tested the effects of isoflurane on the amyloidogenic secretases, BACE and γ-secretase. We showed that treatment of H4-APP cells with 2% isoflurane for 6 h was able to enhance protein levels of the BACE and γ-secretase complex component nicastrin (Fig. 3). These findings suggest that isoflurane-induced apoptosis is linked to increases in the levels of BACE and nicastrin, leading to elevated BACE and γ-secretase activity, as evidenced by enhanced levels of Aβ. In contrast, treatment with 2% isoflurane for 6 h did not alter the protein levels of the three other components of γ-secretase, PS-1, PEN-2, and APH-1 (data not shown). It is possible that longer treatment time may be required to affect the levels of the other components of the γ-secretase complex.
Isoflurane has previously been shown to enhance Aβ aggregation and potentiate the cytotoxicity of Aβ (Eckenhoff et al., 2004). It has been reported that oligomeric and fibrillar species of Aβ are more neurotoxic (Pike et al., 1993; Lorenzo and Yankner, 1994; Lambert et al., 1998; Grace et al., 2002; Walsh et al., 2002; Wang et al., 2002; Kayed et al., 2003; Kim et al., 2003; De Felice et al., 2004). We have shown previously that an amyloid fibril-binding dye, Congo red, which has been reported to inhibit Aβ fibrillar aggregation and to prevent neurotoxicity (Lorenzo and Yankner, 1994), can attenuate 2% isoflurane-induced apoptosis. Thus, Aβ aggregation induced by isoflurane may be able to further potentiate the proapoptotic effects of isoflurane (Xie et al., 2006b). To further explore this effect with more specific Aβ aggregation inhibitors, we tested the ability of iAβ5, a β-sheet breaker peptide (Soto et al., 1998), and clioquinol, an MPAC (Cherny et al., 2001), to attenuate isoflurane-induced apoptosis. We found that both compounds were able to attenuate the 2% isoflurane-induced apoptosis. The effects were also specific for isoflurane, because neither compound attenuated STS-induced apoptosis. Given that both iAβ5 and clioquinol have been reported to inhibit Aβ aggregation (Soto et al., 1998; Cherny et al., 2001), these findings suggest that isoflurane-induced apoptosis can be potentiated by Aβ aggregation. Finally, testing for the possibility of a vicious cycle of isoflurane-induced apoptosis and Aβ accumulation, we found that exogenously added Aβ potentiated isoflurane-induced caspase-3 activation in a dose-dependent manner in naive H4 cells. These findings suggest that the increased Aβ generation induced by isoflurane can further potentiate the isoflurane-induced apoptosis, leading to a vicious cycle.
Collectively, our studies defined the molecular pathways by which isoflurane induces apoptosis, alters APP processing, increases Aβ levels, and enhances Aβ aggregation. As can be seen in Figure 6, our studies illustrate that isoflurane can induce caspase activation and apoptosis, which then increase the activities of BACE and γ-secretase. The enhanced BACE and γ-secretase will be able to facilitate the APP processing to increase Aβ generation/accumulation. Finally, the increased Aβ generation/accumulation will potentiate the isoflurane-induced caspase activation and apoptosis.
Hypothetical pathway by which isoflurane induces a vicious cycle of apoptosis and Aβ generation and aggregation. Isoflurane induces caspase-3 activation/apoptosis. Caspase activation, in turn, increases the activities of both BACE and γ-secretase, which serve to increase Aβ generation/accumulation. Isoflurane also enhances Aβ aggregation, which further induces caspase-3 activation and apoptosis. Elevated Aβ generation/accumulation and Aβ aggregation then further induce apoptosis, leading to a vicious cycle of isoflurane-induced apoptosis and Aβ generation/accumulation and aggregation.
Alternatively, isoflurane may induce the cycle of apoptosis and Aβ generation/accumulation by first promoting Aβ aggregation, especially because isoflurane has previously been reported to induce Aβ aggregation (Eckenhoff et al., 2004). Moreover, in the present study, we found that iAβ5 and clioquinol, effective inhibitors of Aβ aggregation, specifically attenuated isoflurane-induced apoptosis. Collectively, our studies have shown that isoflurane can induce a vicious cycle of apoptosis and Aβ generation/accumulation and aggregation, followed by additional rounds of apoptosis, ultimately leading to cell death. These findings suggest that patients with elevated Aβ levels could be more vulnerable to isoflurane-induced cytotoxicity.
It is possible that the elevated Aβ levels are also associated with postoperative cognitive dysfunction, a subtle form of dementia after surgery and anesthesia (Xie and Tanzi, 2006). Additional investigation will be necessary to determine whether occurrences of postoperative cognitive dysfunction are linked to elevated Aβ levels in the human brain, CSF, and plasma.
Isoflurane induces apoptosis, which, in turn, increases BACE levels in both naive H4 cells and H4-APP cells. However, the effects on APP processing and Aβ generation are only detectable in H4-APP cells that contain sufficient levels of APP.
The molecular mechanism by which isoflurane induces apoptosis remains unclear and is an important topic for future studies. Previous studies have shown that isoflurane can induce calcium release from endoplasmic reticulum (ER) in cerebrocortical and hippocampal neurons (Kindler et al., 1999). Wei et al. (2005) reported that dantrolene, a selective ryanodine receptor antagonist that inhibits calcium release from ER, can suppress isoflurane-induced cytotoxicity. These findings suggest that isoflurane may induce cellular apoptosis by facilitating calcium release from ER. Aβ has also been shown to elevate basal intracellular calcium levels and facilitate calcium overload after activation of glutamate receptors (Mattson et al., 1992; Mark et al., 1995) (for review, see Mattson and Chan, 2003). Furthermore, dantrolene has also been shown to inhibit Aβ-induced cytotoxicity (Guo et al., 1997). Thus, these findings suggest that isoflurane can induce apoptosis by facilitating calcium release from ER to elevate intracellular calcium levels. Aggregated Aβ may then potentiate isoflurane-induced apoptosis by further enhancing the intracellular calcium concentration. Future studies will be necessary to assess whether the effects of isoflurane and Aβ on cell death are mediated by cellular calcium homeostasis (Yoo et al., 2000).
Although our findings and the results from other studies suggest that isoflurane may affect AD neuropathogenesis, these experiments were performed only in cultured cells. The determination of the in vivo relevance of isoflurane on AD neuropathogenesis will be necessary before we can conclude that the inhalation anesthetic isoflurane facilitates or exacerbates AD neuropathogenesis in humans.
In conclusion, we found that isoflurane can induce both apoptosis and changes in APP processing, leading to increased generation of Aβ. Increased levels of Aβ generation and subsequent aggregation induced by isoflurane can further potentiate isoflurane-induced apoptosis, forming a vicious cycle of apoptosis and Aβ generation/accumulation and aggregation. These studies should facilitate future strategies for delivering safer anesthesia care to patients, especially senior patients, who are particularly susceptible to the incidence of postoperative cognitive dysfunction and risk for AD.
Footnotes
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This work was supported by National Institutes of Health (NIH) Grants R01AG 014713 and R01MH 60009 (R.E.T.); NIH Grants K12AG 000294, K08 NS048140, and P60 AG008812 and the American Geriatrics Society Jahnigen Award (Z.X.); NIH Grant K08GM077057 (D.J.C.); and NIH Grant R01AG20253 (G.C.). The cost of anesthetic isoflurane and salary support of Yuanlin Dong and Uta Maeda were generously provided by the Department of Anesthesia and Critical Care, Massachusetts General Hospital and Harvard Medical School (Boston, MA).
- Correspondence should be addressed to Dr. Rudolph E. Tanzi, Genetics and Aging Research Unit, MassGeneral Institute for Neurodegenerative Disease, and Department of Neurology, Massachusetts General Hospital and Harvard Medical School, 114 16th Street, C3009, Charlestown, MA 02129-4404. tanzi{at}helix.mgh.harvard.edu
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