Accumulation of β-amyloid protein (Aβ) has a fundamental role in the neuropathogenesis of Alzheimer disease (AD).1,2 Aβ is produced from amyloid precursor protein (APP) through proteolytic processing by the aspartyl protease β-site APP-cleaving enzyme (BACE) and γ-secretase.3 Increasing evidence suggests a role for caspase activation and apoptosis in AD neuropathogenesis.4–6
Several studies have suggested that isoflurane, a frequently used inhaled anesthetic, can induce caspase activation and apoptosis, affect APP processing (e.g., increases in BACE levels), and enhance levels and aggregation of Aβ.7–14 In addition, mechanistic studies have shown that isoflurane may induce caspase activation and apoptosis via increasing cytosolic calcium levels12,15,16 and cause mitochondrial dysfunction (e.g., increases in reactive oxygen species [ROS] levels).17 We have found that isoflurane increases ROS levels, which causes mitochondrial damage, leading to caspase-3 activation and apoptosis.17 The caspase activation and apoptosis then induce an increase in BACE levels.10 Finally, isoflurane may induce caspase-3 activation and apoptosis by increasing cytosolic calcium levels.12 However, the targeted prevention and treatment of the isoflurane-induced neurotoxicity have not been investigated.
2-Deoxy-D-glucose (2-DG), an analog of glucose, has been shown to have neuroprotective effects via decreasing levels of cellular oxidative stress and enhancing calcium homeostasis.18,19 Specifically, 2-DG attenuates glutamate- or Fe2+-induced ROS generation and the glutamate-induced increase of intracellular calcium levels.18 We sought to determine whether 2-DG can reduce caspase-3 activation and attenuate the increase in BACE, ROS, and cytosolic calcium levels induced by isoflurane. We used H4 human neuroglioma cells (H4 naïve cells) in the studies because these cells are more physiologically relevant without the artificial overexpression of human APP.
We used H4 human neuroglioma cells (H4 naïve cells) in the current experiments. The cells were cultured in Dulbecco's modified Eagle medium (high glucose) containing 9% heat-inactivated fetal calf serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine.
The cell treatments were similar to those in our previous studies.9,10,20 Twenty-one percent O2, 5% CO2, and 2% isoflurane (air component: 2% isoflurane, 5% CO2, 21% O2, and balanced nitrogen) were delivered from an anesthesia machine to a sealed plastic box in a 37°C incubator containing 6-well plates seeded with 1 million cells in 1.5 mL cell culture media as described in our previous studies.9,10,20 The control condition included 5% CO2 plus 21% O2 (air component: 5% CO2, 21% O2, and balanced nitrogen), which did not affect caspase-3 activation or BACE levels.9 A Datex infrared gas analyzer (Puritan-Bennett, Tewksbury, MA) was used to continuously monitor the delivered CO2, O2, and isoflurane concentration. We treated the cells with 2% isoflurane for 6 hours, during which time the cells were incubated in serum-free media. In the interaction studies, the cells were treated with 2-DG (5 mM)18 1 hour before the treatment of 2% isoflurane and throughout the time of the isoflurane treatment, i.e., a total of 7 hours of treatment with 2-DG. The pretreatment time of 2-DG was consistent with that of L-685,458, Congo red, Z-VAD, Aβ, iAβ5, clioquinol, 2-aminoethoxydiphenyl borate, and memantine in our previous studies.10,12,21–24 The treatment of 2-DG was not a preconditioning stimulus. In the studies to determine the effects of serum-free cell culture media on caspase-3 activation, and levels of BACE and ROS, the H4 naïve cells were treated with either cell culture media containing serum or serum-free cell culture media for 6 hours.
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, 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 minutes, and quantified for total proteins using a bicinchoninic acid protein assay kit (Pierce Technology Corporation, 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.10 A caspase-3 antibody (1:1000 dilution; Cell Signaling Technology, Inc., Beverly, MA) was used to recognize the caspase-3 fragment (17–20 kDa), which results from cleavage at the aspartate position 175, and full-length (FL) caspase-3 (35–40 kDa). BACE antibody (1:1000 dilution; Abcam, Cambridge, MA) was used to recognize BACE (65 kDa). Antibody to the nontargeted protein β-actin (42 kDa, 1:5000; Sigma, St. Louis, MO) was used to control for loading differences in total protein amounts. Each band in the Western blot represents an independent experiment. We have averaged the results from 3 to 6 independent experiments. The intensity of signals in each Western blot was analyzed using the National Institutes of Health image program (National Institutes of Health Image 1.62, Bethesda, MD). We quantified the Western blots using 2 steps. First, we used levels of β-actin to normalize (e.g., determine the ratio of the amount of FL caspase-3 to the amount of β-actin) the levels of BACE and caspase-3 to control for any loading differences in total protein amounts. Second, we presented changes in the levels of BACE and caspase-3 in the treated cells as percentages of those in cells from the control condition.
Cytosolic Calcium Measurement
Cytosolic calcium levels were determined as described by Zhang et al.24 Specifically, H4 naïve cells were loaded with fura-2 (Invitrogen, Carlsbad, CA), perfused with tyrode buffer, and [Ca2+]i transients were recorded as changes in fura-2 ratio (340/380 nm) using a spectrofluoroscope system (IonOptix, Milton, MA). The cells were exposed to isoflurane (0.57 mM or 2%) or inomycin (10 μM) with or without 2-DG (5 mM) pretreatment.
ROS levels were measured as described by Zhang et al.17 An OxiSelect ™ ROS assay kit (Cell Biolabs, Inc., San Diego, CA) was used in the experiments. The experiment was performed according to the protocol provided by the company. Briefly, cultured H4 naïve cells were placed in a clear 96-well cell culture plate (5 × 104 cells per well) overnight in the incubator. We then added the 2′,7′-dichlorofluorescein diacetate/media solution to the cells for 30 minutes, then washed the cells twice with Dulbecco's phosphate-buffered saline. The H4 naïve cells loaded with 2′,7′-dichlorofluorescein diacetate were then exposed to 2% isoflurane for 6 hours. The treated cells were lysed by adding 100 μL cell lysis buffer and were mixed thoroughly and incubated for 5 minutes at room temperature. We then transferred 150 μL of the mixture to each well of a 96-well plate that was suitable for fluorescence measurement. Finally, the fluorescence was read with a fluorometric plate reader at 480/530 nm.
Given the presence of background caspase-3 activation, BACE, and ROS levels in the cells cultured in serum-free media, we did not use absolute values to describe changes in caspase-3 activation, BACE, and ROS levels. Instead, these changes were presented as a percentage of those of the control group. One hundred percent of caspase-3 activation, BACE, or ROS refers to control levels for the purpose of comparison to experimental conditions. Data were expressed as mean ± SD. The number of samples varied from 3 to 6, and the samples were normally distributed (tested by normality test, data not shown). We used 2-way analysis of variance (ANOVA) and post hoc (Tukey) test to determine the difference among groups and the interaction of 2-DG and isoflurane on caspase-3 activation, and levels of BACE and ROS. This was a type III sum of squares. We used a 2-tailed t test (assuming equal variances) to determine the effects of serum-free cell culture media on ROS levels in the H4 naïve cells. * or ** indicated the difference between the control group and treatment group(s); # or ## indicated the interaction of 2-DG and isoflurane.
2-DG Attenuates the Isoflurane-Induced Caspase-3 Activation
Caspase-3 immunoblotting revealed that isoflurane treatment induced caspase-3 activation (Fig. 1A) as evidenced by increased ratios of cleaved (activated) caspase-3 fragment (17 kDa) to FL (35–40 kDa) caspase-3. Treatment with 2-DG for 6 hours alone did not induce caspase-3 activation, but the 2-DG treatment attenuated the isoflurane-induced caspase-3 activation (Fig. 1A). Quantification of the Western blots, based on the ratio of caspase-3 fragment to FL caspase-3, revealed that the isoflurane treatment led to caspase-3 activation as compared with the control condition: 100% vs 477%, F = 3.94; P = 0.0090. Two-way ANOVA showed that there was an interaction of 2-DG and isoflurane on caspase-3 activation and that 2-DG attenuated the isoflurane-induced caspase-3 activation: 477% vs 186%, F = 8.68; #P = 0.019. These findings suggest that 2-DG mitigates isoflurane-induced caspase-3 activation.
2-DG Attenuates the Isoflurane-Induced Increases in BACE Levels
As can be seen in Figure 2A, BACE immunoblotting illustrated that isoflurane increased BACE levels as compared with the control. 2-DG alone did not change BACE levels as compared with controls. However, 2-DG attenuated the isoflurane-induced increases in the BACE levels (Fig. 2A). Western blot quantification showed that isoflurane increased BACE levels as compared with controls: 100% vs 345%, F = 1.013; P = 0.001. Two-way ANOVA showed that there was an interaction of 2-DG and isoflurane on BACE, and that 2-DG attenuated the isoflurane-induced increase in BACE levels: 345% vs 123%, F = 42.24; ##P = 0.0002. These findings suggest that 2-DG mitigates isoflurane-induced increases in BACE.
2-DG Decreases Cytosolic Calcium Levels
Two percent isoflurane increased cytosolic calcium (Fig. 3B, upper panel) in the H4 naïve cells. 2-DG treatment inhibited the isoflurane-induced increase in cytosolic calcium (Fig. 3B, lower panel). 2-DG attenuated the increase in cytosolic calcium induced by inomycin, which can increase intracellular calcium25 (Fig. 3A). Thus, H4 naïve cells released calcium, and 2-DG inhibited both the inomycin and isoflurane-induced increase in cytosolic calcium. Finally, 2-DG alone did not increase cytosolic calcium (data not shown). Taken together, these findings suggest that 2-DG may attenuate an isoflurane-induced increase in cytosolic calcium.
2-DG Decreases ROS Levels
Isoflurane increased the levels of ROS (gray bar, F = 1.78; P = 0.006) and 2-DG treatment attenuated ROS levels (black bar, F = 1.94; P = 0.014) (Fig. 4). Two-way ANOVA showed that there was no interaction of isoflurane and 2-DG on ROS (F = 0.69; P = 0.400, not significant). These results suggest that 2-DG may not specifically attenuate an isoflurane-induced increase in ROS, but 2-DG decreased ROS, mitigating the isoflurane-induced increase in ROS.
Effects of Serum-Free Cell Culture Media on Caspase-3 Activation, BACE Levels, and ROS Levels
Treatment with serum-free cell culture media for 6 hours induced a slight caspase-3 activation (Fig. 5A), slight increases in BACE (Fig. 5B), and significant increases in ROS (Fig. 5C) as compared with the cell culture media containing serum in the H4 naïve cells. These results suggest that the serum deprivation could induce caspase-3 activation and increase BACE and ROS, and that these effects can be attenuated by 2-DG (Figs. 1, 2, and 4, black bars).
AD is one of the most common forms of dementia, currently affects 5.4 million Americans, and costs $183 billion a year in the United States.a An estimated 200 million patients worldwide undergo anesthesia and surgery each year. Even though anesthesia and surgery may not increase the incidence of AD,26 it has been reported that age of onset of AD is inversely related to cumulative exposure to anesthesia and surgery before age 50 years.27 Several studies have suggested an association between anesthesia exposure and AD risk.28,29 However, other studies have suggested that there is little or no relationship between anesthesia and AD.30–32 More population studies, defining the role of anesthesia and surgery in AD, are necessary.14,33 Because we cannot clinically prove or disprove the relationship of anesthesia and AD and performing such studies may take many years, it is equally important to perform animal studies and in vitro studies, which will establish mechanistic hypotheses, vulnerable windows, less provocative anesthetics, and potential interventions. The results from these in vitro and animal studies would help to design more targeted and focused clinical research.14
We found that 2-DG attenuated isoflurane-induced caspase-3 activation in H4 naïve cells. Our previous studies have shown that isoflurane-induced caspase-3 activation can increase the levels of BACE, which will promote APP processing to generate Aβ.10 Thus, we determined the effects of 2-DG on the BACE levels and have found that 2-DG can also attenuate the isoflurane-induced increase in BACE levels. Given that many studies have shown that isoflurane can promote AD neuropathogenesis,13,14,17 the results from the current studies may suggest that 2-DG could mitigate isoflurane-induced promotion of AD neuropathogenesis.
We chose to use H4 naïve cells because these cells are more physiologically relevant without the artificial overexpression of human APP. However, it is technically difficult to detect APP processing and Aβ levels in these human cells without APP overexpressions, as described in our previous studies.10 Therefore, one limitation in the current studies is that we were unable to directly assess the effects of 2-DG on isoflurane-induced APP processing and Aβ generation. However, increases in BACE levels strongly suggest the promotion of APP processing and Aβ generation.10,34 Our previous studies have shown that isoflurane can promote APP processing and increase Aβ generation by enhancing BACE levels.10 Thus, the measurement of BACE could serve as an indirect assay for Aβ in the current studies. Taken together, our findings that 2-DG mitigates the effects of isoflurane on increasing BACE levels highly suggest that 2-DG is able to mitigate the effects of isoflurane on promoting APP processing and Aβ generation. Our future experiments will include the in vivo relevance studies in primary (cortex/hippocampus) cultured neurons, wild-type mice, and AD transgenic mice (which have higher Aβ levels for a relatively easy measurement of Aβ) to further assess the potential protective effects of 2-DG on isoflurane or other anesthetic (e.g., sevoflurane) induced neurotoxicity.
The current studies are also limited by the use of serum-free cell culture media, which suggest that the changes in caspase-3 activation and levels of BACE and ROS could result from the combination of isoflurane treatment and serum deprivation. We used serum-free cell culture media to more easily demonstrate the effects of isoflurane on caspase-3 activation, and increases in BACE and ROS levels, because serum could protect the cells from injuries, which may cover the potential cytotoxic effects of isoflurane. The findings that the anesthetics isoflurane and sevoflurane induce caspase activation, apoptosis, cell death, increase in BACE and Aβ levels in the brain tissues of mice11,12,21,22 and in the cells incubated in the cell culture media containing serum7,8,15,35 suggest that the isoflurane-induced cytotoxic effects may not be dependent on serum deprivation. Moreover, we have found that serum deprivation increases the levels of ROS but not caspase-3 activation and levels of BACE in the H4 naïve cells (Fig. 5). Nevertheless, given the fact that serum-free cell culture media were used in the current experiments, it is conceivable that 2-DG may attenuate caspase-3 activation and increases in the levels of BACE and ROS induced by the combination of isoflurane and serum deprivation. As such, conclusions about the effects of 2-DG on isoflurane-induced cytotoxicity must be made cautiously and will require independent confirmation both in vitro and in vivo.
Several studies have shown that 2-DG has cytoprotective effects through decreasing ROS generation and cytosolic calcium increase.18,19 Isoflurane has been shown to induce caspase-3 activation by increasing cytosolic calcium levels24 and increasing ROS generation.17 We therefore investigated the underlying mechanism by which 2-DG inhibits the isoflurane-induced caspase-3 activation by assessing the effects of 2-DG on the isoflurane-induced increase of cytosolic calcium levels and ROS accumulation in H4 naïve cells. The findings that 2-DG can attenuate the inomycin-induced increase of cytosolic calcium (Fig. 3A) have validated the calcium study system in the H4 naïve cells used in our experiments. We then found that the effects of 2-DG on attenuating the levels of cytosolic calcium and ROS mitigate the effects of isoflurane on increasing the levels of cytosolic calcium and ROS. Collectively, these studies have further demonstrated that isoflurane may induce neurotoxicity via the mitochondria pathway of apoptosis17 and have suggested that 2-DG may ameliorate isoflurane-induced neurotoxicity by inhibiting the mitochondrial pathway of apoptosis. Specifically, we have postulated that 2-DG inhibits the isoflurane-induced increase of cytosolic calcium levels, leading to attenuation of the isoflurane-induced increase of ROS. The reduced ROS levels will prohibit isoflurane-induced caspase activation and apoptosis as suggested by Zhang et al.17 Finally, the inhibition of caspase activation and apoptosis will lead to attenuation of isoflurane-induced increases in BACE levels.10 Alternatively, 2-DG may attenuate the isoflurane-induced increase of BACE levels via affecting oxidative stress and hypoxia-inducible factor 1 (HIF1)-α because BACE levels can be enhanced by oxidative stress and HIF1-α.36 Finally, p75 neurotrophin receptor (p75NTR) can regulate BACE levels and this regulation can be prevented by caloric restriction (e.g., 2-DG treatments).37 Thus, 2-DG may mitigate isoflurane-induced increases in BACE levels through affecting p75NTR. Future studies should include an assessment of the interaction of 2-DG and isoflurane on the levels of HIF1-α and p75NTR to further understand the underlying mechanisms by which 2-DG attenuates isoflurane-induced cytotoxicity.
It is interesting that 2-DG can reduce an increase of cytosolic calcium levels induced by both isoflurane and inomycin. Given that isoflurane and inomycin may increase the levels of cytosolic calcium via different mechanisms, it is likely that the mitigation effects of 2-DG on the isoflurane-induced increase of cytosolic calcium are not specific to isoflurane. Moreover, these findings may suggest that the effects of isoflurane and 2-DG on cytosolic calcium levels, ROS generation, caspase-3 activation, and BACE could be through independent mechanistic pathways.
2-DG, a glucose analog, is a competitive inhibitor of hexokinase, which can decrease glucose availability and energy of cells. 2-DG has been shown to significantly reduce seizure-induced spatial memory deficits and hippocampus neuron loss in rats.18 High concentrations of 2-DG (e.g., 10 or 20 mM) may induce ROS release from the mitochondria, leading to cytotoxic effects.18 However, low concentrations of 2-DG (e.g., 5 mM) can protect oxidative injury in primary neurons, neural progenitor cells, or synaptic terminals.38,39 We therefore used a low concentration of 2-DG (5 mM) in our current experiments to determine whether 2-DG can mitigate the isoflurane-induced cytotoxicity. It is unclear why 2-DG decreased the ROS levels as presented in Figure 4. We used serum-free cell culture media in the studies and serum deprivation can increase ROS levels (Fig. 5). It is therefore conceivable that 2-DG may also mitigate the serum deprivation–induced increases in ROS levels (Fig. 4, black bar). It is also possible that 2-DG may affect the autophagic state of cells; however, the results from such studies would only provide additional evidence to demonstrate that 2-DG may mitigate isoflurane-induced cytotoxicity, which will only serve to further support the current conclusion. We therefore will systematically assess the interactive effects of 2-DG with isoflurane and other anesthetics (e.g., sevoflurane) on the autophagic state of cells in our future studies. Impaired glucose use and reduced energy in the brain are associated with AD neuropathogenesis,40,41 and the anesthetic isoflurane has been shown to promote AD neuropathogenesis.13,14 Furthermore, our current findings suggest that 2-DG can attenuate isoflurane-induced neurotoxicity through mitochondria-related mechanisms. Taken together, it is conceivable that isoflurane may induce mitochondrial dysfunction and impair generation of cell energy, leading to caspase activation, apoptosis, and Aβ accumulation. Our future studies should include systematically assessing the effects of isoflurane and other anesthetics on mitochondrial function and cellular energy levels.
In conclusion, we found that 2-DG might protect against isoflurane-induced cytotoxicity in cultured cells. Specifically, 2-DG decreased ROS and cytosolic calcium, mitigating caspase-3 activation and increase in BACE induced by isoflurane. These findings should lead to more studies investigating the neurotoxicity of anesthesia and AD neuropathogenesis, especially the underlying mechanisms and possible interventions.
Name: Jun Zhang, MD, PhD.
Contribution: Data collection and data analysis.
Attestation: Jun Zhang approved the final manuscript.
Name: Yuanlin Dong, MD, MS.
Contribution: Data collection and data analysis.
Attestation: Yuanlin Dong approved the final manuscript.
Name: Zhipeng Xu, MD, PhD.
Contribution: Data collection and data analysis.
Attestation: Zhipeng Xu approved the final manuscript.
Name: Yiying Zhang, MD, MS.
Contribution: Data collection and data analysis.
Attestation: Yiying Zhang approved the final manuscript.
Name: Chuxiong Pan, MD, MS.
Contribution: Data collection and data analysis.
Attestation: Chuxiong Pan approved the final manuscript.
Name: Sayre McAuliffe, BA.
Contribution: Data collection, data analysis, and manuscript preparation.
Attestation: Sayre McAuliffe approved the final manuscript.
Name: Fumito Ichinose, MD.
Contribution: Data collection, data analysis, and manuscript preparation.
Attestation: Fumito Ichinose approved the final manuscript.
Name: Yun Yue, MD, MS.
Contribution: Manuscript preparation.
Attestation: Yun Yue approved the final manuscript.
Name: Weimin Liang, MD, PhD.
Contribution: Manuscript preparation.
Attestation: Weimin Liang approved the final manuscript.
Name: Zhongcong Xie, MD, PhD.
Contribution: Study design and manuscript preparation.
Attestation: Zhongcong Xie approved the final manuscript.
This manuscript was handled by: Steven L. Shafer, MD.
The authors thank Dr. Hui Zheng, Assistant Professor in Medicine and statistician in Massachusetts General Hospital and Harvard Medical School, for the advice regarding statistical analysis of the data in the studies.
a Alzheimer's Association. 2011 Alzheimer's Facts and Figures. Available at: http://www.alz.org/downloads/Facts_Figures_2011.pdf. Accessed July 15, 2011.
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