Figure 2.

Methylglyoxal induces the ISR in cultured mouse DRG neurons. (A) Schematic representation of ISR signaling in DRG neurons under normal and pathophysiological MGO concentrations. (B) Incubation of DRG neurons at day 6 in vitro with MGO (1 μM) up-regulates the expression of the immunoglobulin heavy-chain-binding protein (BiP) and phosphorylates the PKR-like ER-localized eIF2α kinase (PERK^Thr981). For BiP: 1-way ANOVA, F_(5,26) = 4.339, P = 0.0053; post-hoc Dunnett: vehicle (veh) + MGO at 24 hours: *P = 0.0472 and at 48 hours: **P = 0.0050. n = 5 to 6. For p-PERK^Thr981: 1-way ANOVA, F_(5,30) = 2.161, P = 0.0851; post-hoc Dunnett: vehicle + MGO at 24 hours: *P = 0.0427. n = 6. (C and D) BiP activation and PERK^Thr981 phosphorylation converge on the subsequent phosphorylation of the eukaryotic initiation factor 2α at serine 51 (eIF2α^Ser51). For Western blot: 1-way ANOVA, F_{(5, 30)} = 4.52, P = 0.0034; post-hoc Dunnett: vehicle + MGO at 24 hours: *P = 0.0475, at 48 hours: 0.0144. n = 6. For immunofluorescence: Unpaired t test: t = 2.396; *P = 0.0224, vehicle (n = 10) vs MGO (n = 25). (E) In the surface sensing of translation (SUnSET) method, cultured DRG neurons are incubated with MGO (1 μM) for 24 hours after addition of puromycin (1 μM) for an additional 15 minutes. Incubation with either MGO (1 μM for 24 hours) or the elongation inhibitor homoharringtonine (HHT) (50 μM for 1 hour), but not vehicle, significantly reduces nascent protein synthesis in DRG neurons. Staining is shown from top to bottom for puromycin (green), peripherin (red), or a merge. 1-way ANOVA, F_{(3, 21)} = 28.83, P < 0.0001; post-hoc Tukey: ****P < 0.0001, veh-puro vs vehicle + puro; ^&P = 0.0445, vehicle + puro vs MGO + puro. (F) Treatment with MGO (1 μM) represses the phosphorylation of proteins mainly associated with cap-dependent translation such as the extracellular signal-regulated kinases 1 and 2 (ERK1/2) and the ribosomal protein S6. (G) Methylglyoxal (2 ng, i.p.) administration produces mechanical hypersensitivity in mice lacking phosphorylation of the cap-binding protein eIF4E (eIF4E^S209A) and (H) precipitates mechanical hypersensitivity after an intraplantar injection of prostaglandin E₂ (PGE₂) at day 9 after i.p. MGO. (I) Activation of the ISR by MGO (1 μM, for 24 hours), but not vehicle, drives neuronal hyperexcitability in cultured DRG neurons revealed by an increase in Ca²⁺ signaling when they are stimulated with 50 mM KCl. Unpaired t test: = 3.13; *P = 0.0022, vehicle (n = 57) vs MGO (n = 76). Scale bar, 50 μm. ANOVA, analysis of variance; ISR, integrated stress response.