The inhalation anesthetic desflurane induces caspase activation and increases amyloid beta-protein levels under hypoxic conditions

Abstract

Perioperative factors including hypoxia, hypocapnia, and certain anesthetics have been suggested to contribute to Alzheimer disease (AD) neuropathogenesis. Desflurane is one of the most commonly used inhalation anesthetics. However, the effects of desflurane on AD neuropathogenesis have not been previously determined. Here, we set out to assess the effects of desflurane and hypoxia on caspase activation, amyloid precursor protein (APP) processing, and amyloid beta-protein (Abeta) generation in H4 human neuroglioma cells (H4 naïve cells) as well as those overexpressing APP (H4-APP cells). Neither 12% desflurane nor hypoxia (18% O(2)) alone affected caspase-3 activation, APP processing, and Abeta generation. However, treatment with a combination of 12% desflurane and hypoxia (18% O(2)) (desflurane/hypoxia) for 6 h induced caspase-3 activation, altered APP processing, and increased Abeta generation in H4-APP cells. Desflurane/hypoxia also increased levels of beta-site APP-cleaving enzyme in H4-APP cells. In addition, desflurane/hypoxia-induced Abeta generation could be reduced by the broad caspase inhibitor benzyloxycarbonyl-VAD. Finally, the Abeta aggregation inhibitor clioquinol and gamma-secretase inhibitor L-685,458 attenuated caspase-3 activation induced by desflurane/hypoxia. In summary, desflurane can induce Abeta production and caspase activation, but only in the presence of hypoxia. Pending in vivo confirmation, these data may have profound implications for anesthesia care in elderly patients, and especially those with AD.

FIGURE 1.

Desflurane does not affect caspase-3 activation, APP processing, nor Aβ levels in H4-APP cells. A, treatment with 12% desflurane for 6 h (lanes 4–6) does not induce caspase-3 cleavage (activation) as compared with control conditions (lanes 1–3) in H4-APP cells. There is no significant difference in the amounts of β-actin in control conditions or desflurane-treated H4-APP cells. B, caspase-3 activation assessed by quantifying ratio of caspase-3 fragment to FL-caspase-3 in the Western blots. Quantification of the Western blot shows that desflurane treatment (black bar) does not increase caspase-3 activation compared with control conditions (white bar)(p = 0.14, NS), normalized to β-actin levels. C, desflurane (lanes 3 and 4) does not affect APP processing as compared with control conditions (lanes 1 and 2) in H4-APP cells. There is no significant difference in amounts of β-actin in control conditions or desflurane-treated H4-APP cells. D, APP processing assessed by quantifying levels of FL-APP in the Western blots. Quantification of the Western blot shows that desflurane treatment (black bar) does not alter levels of FL-APP as compared with control conditions (white bar) (p = 0.11, NS), normalized to β-actin levels. E, APP processing assessed by quantifying levels of APP-CTFs in the Western blots. Quantification of the Western blot shows that desflurane treatment (black bar) does not alter levels of APP-CTFs as compared with control conditions (white bar)(p = 0.09, NS), normalized to β-actin levels. F, desflurane (black bar) does not increase generation of Aβ40 (p = 0.10, NS) and Aβ42 (p = 0.56, NS) as compared with control conditions (white bar).

FIGURE 2.

Desflurane/hypoxia induces caspase-3 activation, affects APP processing, and increases Aβ levels in H4-APP cells. A, treatment with 12% desflurane/hypoxia (18%) for 6 h (lanes 4–6) induces caspase-3 cleavage (activation) as compared with control conditions (lanes 1–3) in H4-APP cells. There is no significant difference in the amounts of β-actin in control conditions or desflurane/hypoxia-treated H4-APP cells. B, quantification of the Western blot shows that desflurane/hypoxia treatment (black bar) (**, p = 0.002) increases caspase-3 activation compared with control conditions (white bar), normalized to β-actin levels. C, desflurane/hypoxia (lanes 4 and 5) reduces levels of APP-CTFs, but not FL-APP, as compared with control conditions (lanes 1–3) in H4-APP cells. There is no significant difference in amounts of β-actin in control conditions or desflurane/hypoxia-treated H4-APP cells. D, quantification of the Western blot shows that desflurane/hypoxia (black bar) does not alter levels of FL-APP as compared with control conditions (white bar)(p = 0.789, NS), normalized to β-actin levels. E, quantification of the Western blot shows that desflurane/hypoxia (black bar) decreases levels of APP-CTFs as compared with control conditions (white bar) (*, p = 0.019), normalized to β-actin levels. F, desflurane/hypoxia (black bar) increases generation of both Aβ40 (**, p = 0.002) and Aβ42 (*, p = 0.018) as compared with control conditions (white bar).

FIGURE 3.

Caspase inhibitor Z-VAD attenuates caspase-3 activation induced by desflurane/hypoxia in H4-APP cells. A, treatment with desflurane (12%) plus hypoxia (18%) for 6 h (lanes 5 and 6) induces caspase-3 cleavage (activation) as compared with control conditions (lanes 1 and 2) or Z-VAD (50 μm) treatment (lanes 3 and 4). Z-VAD treatment (lane 7 and 8) attenuates caspase-3 cleavage induced by desflurane/hypoxia. There is no significant difference in amounts of β-actin in H4-APP cells with above treatments. B, quantification of the Western blot shows that desflurane/hypoxia (black bar) increases caspase-3 activation as compared with control conditions (white bar) (*, p = 0.016), normalized to β-actin levels. The desflurane/hypoxia-induced caspase-3 activation is reduced by Z-VAD (50 μm) treatment (net bar; ##, p = 0.009). C, desflurane/hypoxia (black bar) increases secreted Aβ levels in H4-APP cells as compared with control conditions (white bar) (**, p = 0.0032). Z-VAD (net bar) reduces the desflurane/hypoxia-induced increases in secreted Aβ levels in H4-APP cells (##, p = 0.00038). DMSO, dimethyl sulfoxide.

FIGURE 4.

Desflurane/hypoxia treatment induces caspase-3 activation independent of APP processing in H4 naïve cells. A, treatment with desflurane (12%) plus hypoxia (18%) for 6 h (lanes 3 and 4) induces caspase-3 cleavage (activation) as compared with control conditions (lanes 1 and 2) in H4 naïve cells. The same treatment (lanes 7 and 8) also induces caspase-3 activation in H4-APP cells as compared with control conditions (lanes 5 and 6). B, quantification of the Western blot shows that desflurane/hypoxia (black bar) increases caspase-3 activation as compared with control conditions (white bar) (*, p = 0.025), normalized to β-actin levels, in H4 naïve cells. C, quantification of the Western blot shows that desflurane/hypoxia (black bar) (**, p = 0.007) increases caspase-3 activation as compared with control conditions (white bar), normalized to β-actin levels, in H4-APP cells. D, desflurane/hypoxia (lane 2) does not alter levels of APP-CTFs as compared with control conditions (lane 1) in H4 naïve cells. Desflurane/hypoxia (lane 4) reduces levels of APP-CTFs as compared with control conditions (lane 3) in H4-APP cells. E, quantification of the Western blot shows that desflurane/hypoxia (black bar) does not alter ratio of APP-CTFs to FL-APP as compared with control conditions (white bar) in H4 naïve cells (p = 0.35, NS). F, quantification of the Western blot shows that desflurane/hypoxia (black bar) decreases ratio of APP-CTFs to FL-APP in H4-APP cells (*, p = 0.03).

FIGURE 5.

Desflurane/hypoxia increases levels of BACE in H4-APP cells. A, desflurane/hypoxia (lanes 4 and 5) increases levels of BACE (65 kDa) as compared with control conditions (lanes 1–3). There is no significant difference in amounts of β-actin in control conditions or desflurane/hypoxia-treated H4-APP cells. B, quantification of the Western blot shows that desflurane/hypoxia (black bar) increases BACE levels as compared with control conditions (white bar) (**, p = 0.006), normalized to β-actin levels.

FIGURE 6.

γ-Secretase inhibitor L-685,458 and Aβ aggregation inhibitor CQ reduce the desflurane/hypoxia-induced caspase-3 activation, but Aβ potentiates the desflurane/hypoxia-induced caspase-3 activation in H4-APP cells. A, treatment with desflurane (12%) plus hypoxia (18%) for 6 h (lanes 5 and 6) induces caspase-3 cleavage (activation) as compared with control conditions (lanes 1 and 2) or L-685,458 (0.5 μm) treatment (lanes 3 and 4). L-685,458 (0.5 μm) treatment (lane 7 and 8) attenuates caspase-3 cleavage induced by desflurane/hypoxia. There is no significant difference in amounts of β-actin in H4-APP cells with above treatments. B, quantification of the Western blot shows that desflurane/hypoxia (black bar) increases caspase-3 activation as compared with control conditions (white bar) (**, p = 0.0001), normalized to β-actin levels. The desflurane/hypoxia-induced caspase-3 activation is reduced by L-685,458 (0.5 μm) (net bar) (#, p = 0.018). C, L-685,458 (0.5 μm) reduces the desflurane/hypoxia-induced increases in secreted Aβ40 (**, p = 0.00003) and Aβ42 (**, p = 0.0009) levels in H4-APP cells. D, desflurane/hypoxia (lanes 5 and 6) induces caspase-3 cleavage (activation) as compared with control conditions (lanes 1 and 2) or CQ (1 μm) treatment (lanes 3 and 4). CQ (1 μm)(lanes 7 and 8) treatment attenuates caspase-3 cleavage induced by desflurane/hypoxia. There is no significant difference in amounts of β-actin in H4-APP cells with above treatments. E, quantification of the Western blot shows that desflurane/hypoxia (black bar) increases caspase-3 activation as compared with control conditions (white bar) (*, p = 0.035), normalized to β-actin levels. The desflurane/hypoxia-induced caspase-3 activation is attenuated by treatment of CQ (1 μm) (net bar) (#, p = 0.046). F, desflurane/hypoxia (lane 3) induces caspase-3 cleavage (activation) as compared with control conditions (lane 1). Aβ40 (7.5 μm) plus Aβ42 (7.5 μm) treatment potentiates caspase-3 activation induced by desflurane/hypoxia (lane 4). There is no significant difference in amounts of β-actin in H4-APP cells with the above treatments. G, quantification of the Western blot shows that desflurane/hypoxia (black bar; *, p = 0.024) increases caspase-3 activation as compared with control conditions (white bar), normalized to β-actin levels. The desflurane/hypoxia-induced caspase-3 activation is potentiated by treatment of Aβ (net bar) (##, p = 0.0012). DMSO, dimethyl sulfoxide.

FIGURE 7.

Hypoxia (18%) does not affect caspase-3 activation, APP processing, nor Aβ generation in H4-APP cells. A, hypoxia treatment alone (lanes 4–6) does not induce caspase-3 cleavage (activation) as compared with control conditions (lanes 1–3) in H4-APP cells. There is no significant difference in amounts of β-actin in control conditions or hypoxia-treated H4-APP cells. B, quantification of the Western blot shows that hypoxia treatment (black bar) does not increase caspase-3 activation compared with control conditions (white bar)(p = 0.16, NS), normalized to β-actin levels. C, hypoxia (lanes 4–6) does not affect APP processing as compared with control conditions (lanes 1–3) in H4-APP cells. There is no significant difference in amounts of β-actin in control conditions or hypoxia-treated H4-APP cells. D, quantification of the Western blot shows that hypoxia treatment (black bar) does not alter levels of FL-APP as compared with control conditions (white bar) (p = 0.85, NS), normalized to β-actin levels. E, quantification of the Western blot shows that hypoxia treatment (black bar) does not alter levels of APP-CTFs as compared with control conditions (white bar)(p = 0.24, NS), normalized to β-actin levels. F, hypoxia (black bar) does not increase generation of Aβ40 as compared with control conditions (white bar)(p = 0.45, NS).

FIGURE 8.

Hypothetical pathway by which desflurane/hypoxia induces a vicious cycle of apoptosis and Aβ generation/aggregation. Desflurane/hypoxia induces caspase-3 activation/apoptosis. Caspase activation, in turn, increases BACE levels, which serves to increase Aβ generation. Desflurane/hypoxia also enhances Aβ aggregation, which further induces caspase-3 activation and apoptosis. Elevated Aβ generation and Aβ aggregation then further induces apoptosis leading to a vicious cycle of desflurane/hypoxia-induced apoptosis and Aβ generation/aggregation.