Isoflurane increases neuronal cell death vulnerability by downregulating miR-214

Abstract

Since accumulating evidence suggests the application of anesthetics may increase the risk of Alzheimer's disease (AD), we investigated the cytotoxicity of inhaled general anesthesia in neurons and its underlying mechanism. Using primary cultured rat hippocampal neurons as the study model, here we show that isoflurane increases vulnerability to intracellular or extracellular amyloid β with or without serum deprivation. This isoflurane-induced effect is mediated by the downregulation of miR-214 level that lead to an elevated expression of Bax, a prominent target for miR-214. We conclude that isoflurane increases cell death in the presence of amyloid β by increasing Bax level through downregulating miR-214. Our data provide a new insight for inhaled anesthetics toxicity and indicate a possible mechanistic link between anesthetic application and neurodegenration in AD.

Figure 1. Isoflurane increased Aβ and serum deprivation cytotoxicity.

(A) Isoflurane enhanced microinjected intracellular Aβ (iAβ)_1-42, iAβ_1-40 and iAβ_42-1 peptide toxicity in primary rat neurons. Cell death levels were measured by TUNEL staining. (B) Isoflurane did not enhance extracellular Aβ (eAβ)_1-42, eAβ_1-40 and eAβ_42-1 peptide toxicity in primary rat neurons. (C) Pre-treatment of isoflurane for 3 hours increased iAβ_1-42 peptide toxicity. (D) Pre-treatment of isoflurane for 3 hours increased eAβ_1-42 peptide toxicity. (E) Isoflurane enhanced the toxicity of the combination of iAβ_1-42 peptide and serum deprivation (-S). (F) Isoflurane enhanced the toxicity of the combination of eAβ_1-42 peptide and serum deprivation (-S). Data represent Means+SE (n = 200 for each group). **: p<0.01 compared with the control; ##: p<0.01 compared with the Aβ group.

Figure 2. Bax was upregulated by isoflurane treatment.

(A) Left panel: An example of western blot for total Bax (indicated by 2D2 antibody) suggested that total Bax levels were increased by isoflurane in the absence or presence of eAβ_1-42 peptide. Right panel: Quantitative data showed that isoflurane significantly enhanced total Bax levels. Data represent Means±SE (n = 3 repeats for each group). (B) Left panel: Representative images showed that activated Bax (indicated by 6A7 antibody) was upregulated by isoflurane. Scale bar: 100 µm. Right panel: Quantitative data showed that isoflurane significantly enhanced total bax levels. (C) Representative images showed microinjected neurons (indicated by injection marker dye Alex488) with iAβ_1-42 peptide and activated Bax (6A7) staining. Scale bar: 50 µm. (D) Quantitative data showed that isoflurane significantly enhanced total bax levels. (E) Quantitative RT-PCR results showed that there was no significant difference between isoflurane treatment groups on Bax mRNA. (F) Quantitative data showed that isoflurane did not alter exogenous Bax levels at 12 hours after the treatment. Data represent Means±SE (n = 200 cells for each group). **: p<0.01 compared with the control. ##: p<0.01 compared with the Aβ group.

Figure 3. Isoflurane decreased miR-214.

(A) Sequence alignment of human and rat miR-214 and Bax 3′UTR. (B) Levels of relative miR-214 in isoflurane-treated primary rat neurons. (C) Representative images showed that in SH-Sy5y cells, the levels of reporter Bax 3′UTR regulated EGFP (green fluorescent) were regulated by miR-214 and its variants. RFP (red fluorescent) indicated the transfected cells. Scale bar: 50 µm. (D) Quantitative data showed that miR-214 decreased Bax expression. M-miR-214: mimic miR-214; I-miR-214: miR-214 inhibitor. (E) miR-214 decreased Bax expression in a dose-dependent manner. (F) The time course of miR-214 in suppression of Bax expression. Data represent Means±SE (n = 200 cells for each group). **: p<0.01 compared with the control.

Figure 4. Isoflurane regulated cell death through miR-214.

(A) miR-214 specifically acted on Bax 3′UTR. (B) In SH-Sy5y cells, miR-214 inhibited total Bax (indicated by 2D2 antibody) expression. (C) miR-214 did not alter Bax mRNA levels. (D) In the rats treated with inhaled isoflurane, miR-214 levels were decreased in the brain tissues and CSF. (E) The expression level of miR-214 in matched Alzheimer’s disease and control pairs. RT-PCR results for hippocampal tissues from 8 matched control-AD pairs with miR-214 showed that there was no discernible difference detected between two diagnostic groups (p = 0.38281, two-tailed Student’s t-test). (F) Left panel: in cultured primary rat neurons, microinjected miR-214 or mimic miR-214 (M-miR-214) reversed cell death induced by isoflurane in the presence of eAβ_1-42. Right panel: Bax neutralizing antibodies (2D2 and 6A7) and Bax siRNAs (RNAi-1, RNAi-2) blocked cell death induced by isoflurane in the presence of eAβ_1-42. (G) Left panel: microinjected miR-214 or mimic miR-214 (M-miR-214) reversed cell death induced by isoflurane in the presence of iAβ_1-42. Right panel: Bax neutralizing antibodies (2D2 and 6A7) and Bax siRNAs (RNAi-1, RNAi-2) blocked cell death induced by isoflurane in the presence of iAβ_1-42. (H) Left panel: microinjected miR-214 or mimic miR-214 (M-miR-214) reversed cell death induced by isoflurane in the presence of eAβ_1-42 and serum deprivation (-S). Right panel: Bax neutralizing antibodies (2D2 and 6A7) and Bax siRNAs (RNAi-1, RNAi-2) blocked cell death induced by isoflurane in the presence of eAβ_1-42 and serum deprivation (-S). (I) Left panel: microinjected miR-214 or mimic miR-214 (M-miR-214) reversed cell death induced by isoflurane in the presence of iAβ_1-42 and serum deprivation (-S). Right panel: Bax neutralizing antibodies (2D2 and 6A7) and Bax siRNAs (RNAi-1, RNAi-2) blocked cell death induced by isoflurane in the presence of iAβ_1-42 and serum deprivation (-S). Data represent Means±SE (n = 200 cells for each group). **: p<0.01 compared with the control.

Figure 5. Schematic drawing for isoflurane cytotoxicity mechanism.