IRE1α-XBP1 Affects the Mitochondrial Function of Aβ25-35-Treated SH-SY5Y Cells by Regulating Mitochondria-Associated Endoplasmic Reticulum Membranes

Abstract

Background: Neurotoxicity induced by the amyloid beta (Aβ) peptide is one of the most important pathological mechanisms of Alzheimer's disease (AD). Activation of the adaptive IRE1α-XBP1 pathway contributes to the pathogenesis of AD, making it a potential target for AD therapeutics. However, the mechanism of IRE1α-XBP1 pathway involvement in AD is unclear. We, therefore, investigated the effect of the IRE1α-XBP1 axis in an in vitro AD model and explored its potential mechanism. Methods: The human neuroblastoma cell line, SH-SY5Y, was used. Cells were treated with Aβ25-35, with or without 4μ8c, an inhibitor of IRE1α. Cells were collected and analyzed by Western blotting, quantitative real-time PCR, electron microscopy, fluorescence microscopy, calcium imaging, and other biochemical assays. Results: Aβ-exposed SH-SY5Y cells showed an increased expression of XBP1s and p-IRE1α. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and calcium imaging analysis showed that the IRE1α inhibitor, 4μ8c, reduced Aβ-induced cytotoxicity. Increased levels of ATP, restoration of mitochondrial membrane potential, and decreased production of mitochondrial reactive oxygen species after Aβ treatment in the presence of 4μ8c showed that inhibiting the IRE1α-XBP1 axis effectively mitigated Aβ-induced mitochondrial dysfunction in SH-SY5Y cells. Furthermore, Aβ treatment increased the expression and interaction of IP3R, Grp75, and vdac1 and led to an increased endoplasmic reticulum (ER)-mitochondria association, malfunction of mitochondria-associated ER-membranes (MAMs), and mitochondrial dysfunction. These deficits were rescued by inhibiting the IRE1α-XBP1 axis. Conclusion: These findings demonstrate that Aβ peptide induces the activation of the IRE1α-XBP1 axis, which may aggravate cytotoxicity and mitochondrial impairment in SH-SY5Y cells by targeting MAMs. Inhibition of the IRE1α-XBP1 axis provides the protection against Aβ-induced injury in SH-SY5Y cells and may, therefore, be a new treatment strategy.

Keywords: Alzheheimer's disease; IRE1α-XBP1; amyloid-beta-protein; cytotoxicity; mitochondria associated ER membranes; mitochondria impairment.

Figure 1

Activation of the IRE1 signaling pathway in Aβ-treated SH-SY5Y cells. Cells were treated with 20 μM Aβ25–35 for 0, 3, 6, 12, and 24 h. (A) Quantitative real-time PCR (qRT-PCR) analysis of spliced-XBP1 mRNA levels. Data were expressed as the fold-change compared with β-actin mRNA levels. (B) Western blot analysis of pIRE1α, IRE1α, and XBP1s proteins. (C–E) Densitometric analysis of pIRE1α, IRE1α, and XBP1s protein levels normalized to the β-actin level. The data are presented as mean ± SD (n = 3), *P < 0.05 compared with the control. **P < 0.01 compared with the control. ****P < 0.0001 compared with the control.

Figure 2

The effect of the IRE1α-XBP1 activation on the viability of Aβ-treated cells. Cells were pretreated with or without 20 μM 4μ8c for 6 h, followed by treatment with 20 μM Aβ25–35 for 24 h. (A) Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (B) Representative trace of Ca²⁺ upon 1 μM bradykinin (BK) stimulation. (C) The amplitude of 1 μM BK-evoked Ca²⁺ increase was calculated as the difference between the peak and the baseline. (D) The number of cells that responded to BK was quantitatively analyzed and is expressed as a percentage of the response. The data are presented aes mean ± SD, *P < 0.05 compared with the control. ****P < 0.0001 compared with the control. ^#P < 0.05 compared with the Aβ25–35-alone group. ^###P < 0.001 compared with the Aβ25–35-alone group.

Figure 3

The effect of IRE1α-XBP1 on mitochondrial function in Aβ-treated cells. Cells were pretreated with or without 20 μM 4μ8c for 6 h, followed by treatment with 20 μM Aβ25–35 for 24 h. (A) Cells were lysed on ice with an ATP kit to measure the ATP levels. (B) Mitochondrial membrane potential (MMP) was examined using the fluorescent probe, tetramethylrhodamine (TMRM). (C) Mitochondrial reactive oxygen species (ROS) levels were determined using MitoSoX. Scale bar = 50 μm. (D) The mean fluorescence intensity of each group was stained by TMRM. (E) Mean fluorescence intensity of each group was stained by MitoSoX. The data are presented as mean ± SD (n = 3), *P < 0.05 compared with the control. **P < 0.01 compared with the control. ****P < 0.0001 compared with the control. ^#P < 0.05 compared with the Aβ25–35-alone group. ^##P < 0.01 compared with the Aβ25–35-alone group.

Figure 4

The effect of IRE1α-XBP1 on the length of endoplasmic reticulum (ER)–mitochondria contact sites in Aβ-treated cells. Cells were pretreated with or without 20 μM 4μ8c for 2 h, followed by treatment with 20 μM Aβ25–35 for 4 h. (A–D) Representative electron micrographs of ER–mitochondria contact sites in (A) the control group, (B) the 4μ8c group, (C) the Aβ group, and (D) the Aβ+4μ8c group cells. The white triangles indicate ER–mitochondrial contact sites. Scale bar =0.5 μm. (E) Quantitative analysis of average length of the ER–mitochondria association. (F) Bar graphs show the percentage of mitochondria in contact with ER. (control-72 mitochondria, 4μ8c-79 mitochondria, Aβ-83 mitochondria, Aβ+4μ8c-75 mitochondria). The data are presented as mean ± SD (n = 3), **P < 0.01 compared with the control. ***P < 0.001 compared with the control. ^#P < 0.05 compared with the Aβ25–35-alone group.

Figure 5

The effect of IRE1α-XBP1 on calcium transfer between the ER and mitochondria in Aβ-treated cells. Cells were pretreated with or without 20 μM 4μ8c for 6 h, followed by treatment with 20 μM Aβ25–35 for 24 h. (A) Basal calcium levels in cells were measured using Fura-2 ratios. (B) Quantification of Tg-induced Ca²⁺ release (reflecting ER Ca²⁺ content). ****p < 0.0001 compared with the control group. ^###P < 0.001 compared with the Aβ25-35-alone group. ^####P < 0.0001 compared with the Aβ25–35-alone group. (C) Cytoplasmic and mitochondrial Ca²⁺ content in cells measured by Fura-2/AM and Rhod-2/AM, respectively. Scale bar = 50 μm. (D) The quantification of cytoplasmic calcium and mitochondrial calcium fluorescence intensity. Red * indicates the comparison of the control group with the Aβ25–35 alone group. Green * indicates the comparison of the Aβ25–35 alone group with the Aβ+4μ8c group. Black * indicates the comparison of the Aβ25–35 alone group with the 2APB+Aβ25–35 group. The data are presented as mean ± SD (n = 3).

Figure 6

The effect of IRE1α-XBP1 on the IP3R-Grp75-VDAC1 complex in Aβ-treated cells. Cells were pretreated with or without 20 μM 4μ8c for 6 h, followed by treatment with 20 μM Aβ25–35 for 24 h. (A) Western blot analysis of IP3R, Grp75, and VDAC1 proteins. (B–D) Densitometric analysis of the protein levels of IP3R, Grp75, and VDAC1 normalized to the level of β-actin. (E) Co-immunoprecipitation (Co-IP) was performed to analyze the IP3R-Grp75-VDAC1 interaction. Normal rabbit IgG without antigenicity was used as a negative control. Lysates from cells in each group without immunoprecipitation were used as a positive control (input). The proteins pulled down by anti-IP3Rantibodies were analyzed by Western blotting. The data are presented as mean ± SD (n = 3), *p < 0.05 compared with the control. **p < 0.01 compared with the control. ^#P < 0.05 compared with the Aβ25–35-alone group.

Figure 7

Schematic diagram for Aβ-induced mitochondrial dysfunction via the IRE1α-XBP1 pathway. Based on our experimental data, Aβ-induced activation of the IRE1α-XBP1 pathway facilitated the expression and interaction of IP3R, Grp75, and VDAC1, which further led to the increased the level of ER–mitochondria association and malfunction of mitochondria-associated ER-membranes (MAMs). Then, the increase of calcium ions transferred from the ER to the mitochondria via MAM leads to mitochondrial dysfunction. Inhibition of the IRE1α-XBP1 pathway ameliorated these Aβ25–35-induced changes via the regulation of MAM, which fostered neuroprotection.