Highly specific σ2R/TMEM97 ligand alleviates neuropathic pain and inhibits the integrated stress response

Abstract

The Sigma 2 receptor (σ₂R) was described pharmacologically more than three decades ago, but its molecular identity remained obscure until recently when it was identified as transmembrane protein 97 (TMEM97). We and others have shown that σ₂R/TMEM97 ligands alleviate mechanical hypersensitivity in mouse neuropathic pain models with a time course wherein maximal anti-nociceptive effect is approximately 24 hours following dosing. We sought to understand this unique anti-neuropathic pain effect by addressing two key questions: do these σ₂R/TMEM97 compounds act selectively via the receptor, and what is their downstream mechanism on nociceptive neurons? Using male and female conventional knockout (KO) mice for Tmem97, we find that a new σ₂R/TMEM97 binding compound, FEM-1689, requires the presence of the gene to produce anti-nociception in the spared nerve injury model in mice. Using primary mouse dorsal root ganglion (DRG) neurons, we demonstrate that FEM-1689 inhibits the integrated stress response (ISR) and promotes neurite outgrowth via a σ₂R/TMEM97-specific action. We extend the clinical translational value of these findings by showing that FEM-1689 reduces ISR and p-eIF2α levels in human sensory neurons and that it alleviates the pathogenic engagement of ISR by methylglyoxal. We also demonstrate that σ₂R/TMEM97 is expressed in human nociceptors and satellite glial cells. These results validate σ₂R/TMEM97 as a promising target for further development for the treatment of neuropathic pain.

Keywords: ISR; Sigma 2 receptor; TMEM97; dorsal root ganglion; drug discovery; pain; σ2 receptor.

Figure 1.

TMEM97 gene is expressed in human dorsal root ganglia (DRG). (A) RNAScope in situ hybridization experiments using lumbar DRGs obtained from organ donors. (B-C) Across 3 donors (1 male and 2 females), we discovered that nearly all DRG neurons (>99%) expressed TMEM97 and notably, all SCN10A-positive nociceptors expressed TMEM97. (D) TMEM97-positive neurons were distributed across all cell sizes. (E) Upon further investigation, we also identified TMEM97 transcripts in FABP7-positive satellite glial cells. (F) Our previously published (36) analysis of near single-cell RNA sequencing of human DRGs showed that TMEM97 is expressed across all neuronal cell types in the ganglia including nociceptors, low-threshold mechanoreceptors (LTMRs), and proprioceptors. TMEM97 transcripts were notably enriched in proenkephalin (PENK)+ nociceptors and Aδ LTMRs.

Figure 2.

Structure, binding profiles, physicochemical properties, and pharmacokinetic parameters for FEM-1689. Values are reported as averages ± standard deviation.

Figure 3.

Mechanical pain hypersensitivity following spared nerve injury in wild-type and global TMEM97-knockout (KO) mice. (A) Experimental paradigm. Mechanical hypersensitivity was assessed using the von Frey filaments test at baseline prior to spared nerve injury (SNI) and for 14 days post-surgery. Mice were treated with FEM-1689 (10 mg/kg) intravenously thirty (30) days after SNI surgery and assessed for mechanical hypersensitivity for the following 7 days. Another intravenous injection of FEM-1689 (20 mg/kg) was given 2 weeks later and mechanical hypersensitivity assessed for 7 days. (B, C) Following SNI, no significant difference in mechanical hypersensitivity between wild-type and TMEM97KO littermates of both sexes was observed, suggesting that TMEM97 did not contribute to the development of neuropathic pain following nerve injury. Wild-type SNI (male n=6, female n=5), wild-type sham (male n=6, female n=5), TMEM97KO SNI (male n=5, female n=5), TMEM97KO sham (male n=5, female n=5). (D, E) A single 20 mg/kg intravenous injection of FEM-1689 reversed mechanical hypersensitivity in wild-type (male n=6, female n=5) but not TMEM97KO (male n=5, female n=5) mice. A lower dose of 10 mg/kg was not sufficient to reduce mechanical hypersensitivity in these animals. Repeated measures two-way ANOVA with Holm-Sidak’s multiple comparison test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (F, G) Effect size analysis demonstrates that a higher dose of 20 mg/kg of FEM-1689 provides maximal anti-nociceptive effect. Two-way ANOVA with Sidak’s multiple comparison test, **p<0.01, ***p<0.001. Blue and red asterisks indicate wild-type and TMEM97KO groups compared to sham controls in (B, C) and wild-type vs. TMEM97KO groups in (D, E).

Figure 4.

FEM-1689 reduces p-eIF2α levels and promotes neurite outgrowth in vitro. (A, B) Cultured mouse DRG neurons obtained from wild-type and TMEM97KO animals were treated with FEM-1689 over 16 hours. (C) Basal levels of p-eIF2α were lower in TMEM97KO neurons. ISRIB (200 nM) treatment reduced p-eIF2α levels in wild-type neurons but failed to change p-eIF2α immunoreactivity in TMEM97KO neurons. (D, E) Immunoreactivity of p-eIF2α was assessed across 5 doses (10 nM, 30 nM, 100 nM, 300 nM, and 1 µM) and a dose-response curve was generated. An IC₅₀ of 30 nM was determined with maximal effect at 100 nM which was comparable to ISRIB (200 nM) treatment. TMEM97KO DRG neurons did not respond to FEM-1689 treatment. (F, G) Wild-type mouse DRG neurons were treated with FEM-1689 (100nM) at 0.5, 1, 3, 6, 12, and 16 hours. We calculated a half-life (t_1/2) for FEM-1689 of 5.0 hours at reducing p-eIF2α levels in vitro. Maximal effect of FEM-1689 was observed at 16 hours of treatment. (H-M) Sholl analysis of mouse DRG neurons following 100 nM of FEM-1689 treatment showed an increase in the number and complexity of neurites in wild-type neurons but not in TMEM97KO neurons. Area under the curve of Sholl analysis was used to statistically demonstrate this effect. Immunoreactivity against β3-tubulin was used to identify neuronal cell bodies and neurites. Arrows indicate neuronal cell bodies. ***p<0.001, ****p<0.0001 two-tailed Student’s t-test. UND=undetermined. &=average mean gray intensity of the primary antibody omission control.

Figure 5.

Norbenzomorphans such as SAS-0132 (A-C) and DKR-1677 (D-F) stimulate the ISR by promoting the phosphorylation of eIF2α in cultured wild-type mouse DRG neurons. Cells were treated for 16 hours with either SAS-0132 or DKR-1677 at 10, 30, 100, or 300 nM concentrations. Data is presented as fold-change compared to the fluorescence measured in the vehicle treated neurons. *p<0.05, ***p<0.001, ****p<0.0001 One-way ANOVA with Tukey’s post hoc test.

Figure 6.

(A, B) HEK293T cells were treated for 2 and 16 hours with FEM-1689 for across a range of 9 concentrations (0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000 nM) in 3–5 replicates. P-eIF2α levels were measured using ICC and spectrophotometry. IC₅₀ was calculated to be 5.89 nM for the 2 hour treatment and 0.74 nM for the 16h treatment. FEM-1689 demonstrated time-dependent effect in reducing p-eIF2α levels in HEK293T cells. (C-K) In a separate experiment, HEK293T cells treated with FEM-1689 (0.1, 1, 10, 100 nM) overnight were used for Western blot analysis. Western blots showed a significant reduction in p-eIF2α, eIF2A, and p-PERK levels following FEM-1689 treatment suggesting the involvement of the PERK arm of ISR. BiP levels remained unchanged. One-way ANOVA with Tukey’s post hoc analysis *p<0.05, **p<0.01.

Figure 7.

(A) Methylglyoxal (MGO, 20 ng) injection in the hind paw induces ISR-dependent mechanical pain hypersensitivity in wild-type mice over a course of 6 days. Mice were treated with FEM-1689 (20 mg/kg, IV) or ISRIB (2.5 mg/kg, IP) 24 hours following MGO administration. (B) FEM-1689 and ISRIB reversed mechanical hypersensitivity in MGO-treated mice. ISRIB’s anti-allodynic effects were observed 3 hours after drug administration and reverted to a hypersensitive state 24 hours after injection. FEM-1689 alleviated mechanical hypersensitivity for the duration of the pain state. *p<0.05, **p<0.01, ***p<0.001 repeated measures two-way ANOVA with Tukey’s post hoc test. Asterisk (*), dollar sign ($), and ampersand (&) denote post-hoc comparison with vehicle (*), MGO+ISRIB ($), and MGO+FEM (&), respectively.

Figure 8.

(A, B) FEM-1689 treatment (10 and 100nM) of cultured human DRG neurons significantly reduced p-eIF2α levels. (C, D) Methylglyoxal (MGO) is known to induce the ISR. Co-treatment of human neurons with MGO (1 µM) and FEM-1689 (100 nM) prevented an increase in p-eIF2α suggesting that FEM-1689 limits the effect of MGO. Peripherin and β3-tubulin were used to identify DRG neurons. One-way ANOVA followed by Tukey’s post-hoc test **p<0.01, ***p<0.001, ****p<0.0001.