Diroximel fumarate acts through Nrf2 to attenuate methylglyoxal-induced nociception in mice and decreases ISR activation in DRG neurons

Abstract

Diabetic neuropathic pain is associated with elevated plasma levels of methylglyoxal (MGO). MGO is a metabolite of glycolysis that causes mechanical hypersensitivity in mice by inducing the integrated stress response (ISR), which is characterized by phosphorylation of eukaryotic initiation factor 2α (p-eIF2α). Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that regulates the expression of antioxidant proteins that neutralize MGO. We hypothesized that activating Nrf2 using diroximel fumarate (DRF) would alleviate MGO-induced pain hypersensitivity. We pretreated male and female C57BL/6 mice daily with oral DRF prior to intraplantar injection of MGO (20 ng). DRF (100 mg/kg) treated animals were protected from developing MGO-induced mechanical and cold hypersensitivity. Using Nrf2 knockout mice we demonstrate that Nrf2 is necessary for the anti-nociceptive effects of DRF. In cultured mouse and human dorsal root ganglion (DRG) sensory neurons, we found that MGO induced elevated levels of p-eIF2α. Co-treatment of MGO (1 μM) with monomethyl fumarate (MMF, 10, 20, 50 μM), the active metabolite of DRF, reduced p-eIF2α levels and prevented aberrant neurite outgrowth in human DRG neurons. Our data show that targeting the Nrf2 antioxidant system with DRF is a strategy to potentially alleviate pain associated with elevated MGO levels.

Keywords: Neuroscience; Pharmacology; diroximel fumarate; integrated stress response; methylglyoxal; nrf2; pain.

Figure 1.. Diroximel fumarate (DRF) pretreatment protects against methylglyoxal (MGO)-induced pain hypersensitvity.

(A) MGO stimulates the integrated stress response (ISR) and promotes the phosphorylation of eIF2α. DRF is a prodrug and is metabolized to MMF which activates the Nrf2 antioxidant response neutralizing MGO. (B) Male (n=8 per condition) and female (n=8 per condition) mice were treated with DRF (60 or 100 mg/kg) for five days. MGO (20 ng) was administered in an intraplantar injection on the third day, 1-hour after oral DRF administration for the day. Mechanical and cold sensitivity was assessed using von Frey and acetone tests, respectively. Acetone test was performed an hour after von Frey testing was complete. We observed that male (C) and female (D) mice had reduced von Frey thresholds in response to MGO injection on day 3. Daily treatment with DRF prevented MGO-induced mechanical hypersensitivity following MGO injection on day 3. (E, F) Using area under the curve of von Frey thresholds, we determined that DRF at a dose of 100 mg/kg were effective at preventing MGO-induced tactile hypersensitivity in males while both 60 mg/kg and 100 mg/kg doses were effective in females. MGO produced cold hypersensitivity in male (G) and female (H) mice which was abrogated in DRF treated mice at both doses. For (C, D, G, H) significance was calculated with repeated measures two-way ANOVA with Tukey’s multiple comparison test. *, &, # p<0.05; **, ## p<0.01; ***, &&& p<0.001; **** p<0.0001. * indicates comparison to veh-only, & indicates comparison to MGO+DRF-60, # indicates comparison to MGO+DRF-100. For (E, F) signficance was caluclated with a one-way ANOVA with Brown-Forsythe correction. * p<0.05; ** p<0.01.

Figure 2.. MGO induces mechanical pain hypersensitivity in Nrf2KO mice.

Wild-type (A) and Nrf2KO (B) animals were injected MGO (20ng) or vehicle (saline) in the left paw. Mechanical sensitivity was assessed using the von Frey test for up to 20 days after injection. MGO (20ng) injection produced mechanical hypersensitivity in both wild-type and Nrf2KO mice. (C) Area under the curve of von Frey thresholds was used to directly compare the response of wild-type and Nrf2KO animal to MGO injection. Each condition included 4 male and 4 female mice for a total of 8 animals. For (A, B) statistical analysis was performed with repeated measures two-way ANOVA followed by Sidak multiple comparison test. * p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001. In (C), a two-way ANOVA with Tukey’s test was used. ** p<0.01, ***p<0.001.

Figure 3.. DRF treatment fails to prevent mechanical hypersensitivity in Nrf2KO mice.

Wild-type and Nrf2KO animals were treated with DRF at 100 mg/kg based on the paradigm outlined in Fig 1A. MGO (20 ng) was injected into the paw on day 3 since the start of oral DRF treatment in wild-type (n=8, 4 males and 4 females) and Nrf2KO (n=8, 4 males and 4 females) littermates. Nrf2KO animals developed mechanical hypersensitivity compared to the wild-type animals suggesting that DRF treatment was unable to prevent MGO-induced hypersensitivity in Nrf2KO animals. A repeated measures two-way ANOVA was used followed by Sidak’s post hoc test. *p<0.05, **p<0.01, ***p<0.001.

Figure 4.. Monomethyl fumarate (MMF) prevents MGO-evoked increases in p-eIF2α levels in mouse DRG neurons.

(A-C) Representative images of immunohistochemistry of mouse DRG neurons cultured for 24 hours and subsequently treated with vehicle-only, MGO (1μM), or MGO (1μM) plus MMF (10, 20, 50 μM). (D) We observed a significant increase in the levels of p-eIF2α when cells were treated with MGO as compared to vehicle-treated cells. MMF treatment with MGO prevented elevation of p-eIF2a particularly at 20 μM and 50 μM concentrations. Neurons were identified by their expression of beta3 tubulin. Vehicle-only (n=18), MGO (n=37), MGO+10μM MMF (n=20), MGO+20μM MMF (n=16), MGO+50μM MMF (n=32). A one-way ANOVA was used to calculate significance. *p<0.05, **p<0.01.

Figure 5.. Monomethyl fumarate (MMF) prevents MGO-evoked increases in p-eIF2α levels in human DRG neurons.

(A-C) Representative images of human DRG neurons obtained from organ donors treated with vehicle, MGO (1μM), and MGO plus MMF. (D) MGO (1μM) increases p-eIF2α in human DRG neurons which is prevented with 20 μM and 50 μM MMF cotreatment. Vehicle-only (n=170), MGO (n=152), MGO+10μM MMF (n=125), MGO+20μM MMF (n=176), MGO+50μM MMF (n=111). A one-way ANOVA was used to calculate statistical significance. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 6.. Neurite outgrowth induced by MGO is prevented with MMF treatment.

(A-C) Example images of human DRG neurons immunolabeled with β3 tubulin (green) to visualize neuronal cell body and nerites. Neurons were treated with vehicle, MGO (1μM), or MGO (1μM) plus MMF (10, 20, 50 μM) for 24 hours prior to fixation and immunostaining. (D) Sholl analysis was performed to quantify neurite outgrowth and complexity. (E) Area under the curve of Sholl analysis shows that MMF treatment prevents MGO-induced neurite outgrowth, particularly at 20 μM and 50 μM concentrations. Vehicle-only (n=24), MGO (n=25), MGO+10μM MMF (n=27), MGO+20μM MMF (n=27), MGO+50μM MMF (n=23). A one-way ANOVA was used to calculate statistical significance. *p<0.05, ***p<0.001, ****p<0.0001.