Glycolaldehyde induces endoplasmic reticulum stress and apoptosis in Schwann cells

Abstract

Schwann cell injury is caused by diabetic neuropathy. The apoptosis of Schwann cells plays a pivotal role in diabetic nerve dysfunction. Glycolaldehyde is a precursor of advanced glycation end products that contribute to the pathogenesis of diabetic neuropathy. In this study, we examined whether glycolaldehyde induces endoplasmic reticulum (ER) stress and apoptosis in rat Schwann cells. Schwann cells treated with 500 μM glycolaldehyde showed morphological changes characteristic of apoptosis. Glycolaldehyde activated apoptotic signals, such as caspase-3 and caspase-8. Furthermore, it induced ER stress response involving RNA-dependent protein kinase-like ER kinase (PERK), inositol-requiring ER-to-nucleus signal kinase 1α (IRE1α), and eukaryotic initiation factor 2α (eIF2α). In addition, glycolaldehyde activated CCAAT/enhancer-binding homologous protein (CHOP), an ER stress response factor crucial to executing apoptosis. Knockdown of nuclear factor E2-related factor 2 (Nrf2), which is involved in the promotion of cell survival following ER stress, enhanced glycolaldehyde-induced cytotoxicity, indicating that Nrf2 plays a protective role in the cytotoxicity caused by glycolaldehyde. Taken together, these findings indicate that glycolaldehyde is capable of inducing apoptosis and ER stress in Schwann cells. The ER stress induced by glycolaldehyde may trigger the glycolaldehyde-induced apoptosis in Schwann cells. This study demonstrated for the first time that glycolaldehyde induced ER stress.

Keywords: AGEs, advanced glycation end products; ATF6, activating transcription factor 6; Apoptosis; CHOP, CCAAT/enhancer-binding homologous protein; ER, endoplasmic reticulum; Endoplasmic reticulum stress; GA, glycolaldehyde; Glycolaldehyde; HO-1, heme oxygenase-1; IRE1, inositol-requiring ER-to-nucleus signal kinase 1; MG, methylglyoxal; Nrf2, nuclear factor E2-related factor 2; Nuclear factor E2-related factor 2; PERK, RNA-dependent protein kinase-like ER kinase; Schwann cell; eIF2, eukaryotic initiation factor 2.

Fig. 1

Effect of GA on morphology of apoptotic cells. (A) Schwann cells were treated with GA (100, 250 or 500 μM) for 24 h. Cells were analyzed under a laser scanning microscope using the annexin-V/PI double staining method. Magnification, 40ÿ. Scale bar, 20 μm. (B) Schwann cells were treated with 500 μM GA (0⿿24 h). Cells were analyzed by flow cytometry using the annexin-V/PI double staining method. A, viable or undamaged cells (annexin-V⿿, PI⿿); B, early apoptotic cells (annexin-V+, PI⿿); and C, late apoptotic cells (annexin-V+, PI+).

Fig. 2

Effect of GA on caspase-8 and -3 activities. Schwann cells were treated with 500 μM GA for 24 h. Subsequently, caspase-8 (A) and caspase-3 (C and E) activation was analyzed by flow cytometry and by western blot. (B) and (D) Relative fluorescence of untreated cells was set to 1.0. (F) Relative band intensity was normalized for β-actin. Data are means ± S.D. of three independent experiments. *Significant difference from the value of control (p < 0.05).

Fig. 3

Effect of GA on ER stress sensors. Schwann cells were treated with 500 μM GA for 24 h. (A) PERK phosphorylation was analyzed under a laser scanning microscope. Scale bar, 20 μm. (B) PERK mRNA expression was analyzed by real-time RT-PCR. (C) IRE1α phosphorylation was analyzed by Western blot. (D) IRE1α mRNA expression was analyzed by real-time RT-PCR. (E) eIF2α phosphorylation was analyzed by Western blot. (F) CHOP protein expression was analyzed under a laser scanning microscope. Scale bar, 20 μm. (G) CHOP mRNA expression was analyzed by RT-PCR. Values are means ± S.D. of three experiments. *Significant difference from the value of control (p < 0.05).

Fig. 4

Effect of Nrf2 on GA-induced cytotoxicity. Schwann cells were treated with 500 μM GA for 24 h. (A) HO-1 mRNA expression was analyzed by real-time RT-PCR. (B) HO-1 protein expression was analyzed under a laser scanning microscope. Scale bar, 20 μm. (C) Schwann cells were transfected with control siRNA (ctrl siRNA) or Nrf2 siRNA and were treated or not treated with GA. Data are means ± S.D. of three independent experiments. *Significant difference from the value of control (p < 0.05). **Significant difference from the value of control treated with GA (p < 0.05).