Treating small fiber neuropathy by topical application of a small molecule modulator of ligand-induced GFRα/RET receptor signaling

Abstract

Small-fiber neuropathy (SFN) is a disorder of peripheral nerves commonly found in patients with diabetes mellitus, HIV infection, and those receiving chemotherapy. The complexity of disease etiology has led to a scarcity of effective treatments. Using two models of progressive SFN, we show that overexpression of glial cell line-derived neurotrophic factor (GDNF) in skin keratinocytes or topical application of XIB4035, a reported nonpeptidyl agonist of GDNF receptor α1 (GFRα1), are effective treatments for SFN. We also demonstrate that XIB4035 is not a GFRα1 agonist, but rather it enhances GFRα family receptor signaling in conjunction with ligand stimulation. Taken together, our results indicate that topical application of GFRα/RET receptor signaling modulators may be a unique therapy for SFN, and we have identified XIB4035 as a candidate therapeutic agent.

Keywords: pain; sensory; thermonociception; topical drug; trophic factor.

Fig. 1.

Overexpression of GDNF in the skin or topically applied XIB4035 curtails progression of SFN in two animal models. (A) Hot-plate (54 °C) test shows that the loss of thermal nociception in line-D mice is prevented by GDNF overexpression in keratinocytes (K14-GDNF). Only responses of line-D differ from those observed in the other three genotypes (one-way ANOVA with Newman–Keuls post hoc test, n ≥ 3; ***P < 0.001; error bars indicate SEM). (B) Hot-plate (54 °C) test shows that XIB4035 treatment starting at P21 prevents loss of thermal nociception in line-D mice. Wild-type (WT) and line-D mice were randomly divided into two groups at P21, a stage at which line-D mice had a small but significant increase in paw withdrawal latency compared with WT animals (Student t test, P = 0.0002). No difference existed within the groups of each genotype (Student t test, n ≥ 6; WT +vehicle vs. WT +XIB4035, P = 0.3067; line-D +vehicle vs. line-D +XIB4035, P = 0.285). Line-D mice treated with vehicle had progressively longer withdrawal latencies over the duration of the treatment. In contrast, WT mice treated with either vehicle or XIB4035, and XIB4035-treated line-D mice had similar latencies throughout the duration of the treatment. For P28, P35, P42, and P49: one-way ANOVA with Newman–Keuls post hoc test, n ≥ 6; WT +vehicle vs. line-D +vehicle (P < 0.001), WT +vehicle vs. line-D +XIB4035, n.s., line-D +vehicle vs. line-D +XIB4035 (P < 0.001); error bars indicate SEM. (C) Electron micrographs of transverse sections from the sciatic nerve of line-D mice treated for 4 wk (P49) show that Remak bundle structure is lost in mutants treated with vehicle but preserved in line-D mice treated with XIB4035. Remak bundle structure in wild types is not changed by the treatment. (Scale bar: 4 μm.) (D) Modified Hargreaves test show that XIB4035 treatment improves thermal nociception in diabetic mice. STZ-injected mice were treated with either XIB4035-containing cream or vehicle from onset of hyperglycemia. Hindpaw thermal response latency was measured every other week from week 8 posthyperglycemia onward. At week 8, vehicle-treated diabetic (diabetic +vehicle) mice showed thermal hypoalgesia (P < 0.01 vs. control +vehicle by ANOVA with Newman–Keuls post hoc test) that was attenuated by XIB4035 treatment (*P < 0.05 for diabetic +vehicle vs. diabetic +XIB4035; n ≥ 7; error bars indicate SEM) but not completely normalized (P < 0.05 for control +vehicle vs. diabetic +XIB4035). The attenuation of paw thermal hypoalgesia persisted throughout the study (*P < 0.05, **P < 0.01, ***P < 0.001, for diabetic +vehicle vs. diabetic +XIB4035 mice: one-way ANOVA with Newman–Keuls post hoc test; error bars indicate SEM).

Fig. 2.

XIB4035 treats line-D mice with advanced SFN symptoms. (A) Hot-plate (54 °C) test shows XIB4035 improves the SFN symptoms of line-D mice when treatment is initiated after severe symptoms are apparent (P28). Mutant mice showed significant improvement in thermal nociception after 1 wk of treatment (P35), and this was maintained as long as the treatment continued [red line indicates treatment duration; one-way ANOVA Newman–Keuls post hoc test, n ≥ 7; line-D +vehicle vs. line-D +XIB4035; n.s., not statistically significant; **P < 0.01, ***P < 0.001; WT +vehicle vs. line-D +vehicle, P < 0.001, WT +vehicle vs. line-D +XIB4035, P < 0.001 (P28, P35, P42), P < 0.01 (P49), P > 0.05 (P56); error bars indicate SEM]. (B) Chronic XIB4035 treatment is necessary to maintain gains. If mice were treated beginning at P28 (as in A), but treatment was interrupted at P35, neuropathic symptoms returned [red line indicates treatment duration; one-way ANOVA Newman–Keuls post hoc test, n ≥ 7; line-D +vehicle vs. line-D +XIB4035; n.s., not statistically significant; *P < 0.05, ***P < 0.001; WT +vehicle vs. line-D +vehicle, P < 0.001; WT +vehicle vs. line-D +XIB4035, P < 0.001 (P28, P35, P42, and P49), P < 0.01 (P56); error bars indicate SEM]. (C) If XIB4035 treatment is initiated at P28, the sensory recovery at P35 is not accompanied by recovery of IENF density. Representative images of PGP9.5-positive IENF staining at P35 in line-D hindpaw skin. (Scale bar: 100 μm.) (D) XIB4035 treatment initiated at P28 results in improved IB4 staining in the dorsal spinal cord. (Left) Representative images of spinal cord sections from wild-type mice treated with vehicle from P28 to P35 show the normal appearance of TrpV1+ (red) and IB4+ (green) nerve terminals. (Center) Images of line-D mice treated with vehicle show complete absence of IB4 staining. (Right) Images from line-D mice treated with XIB4035-containing cream show clear presence of IB4+ fibers. No obvious difference in TrpV1 labeling was observed between wild-type and mutant animals regardless of treatment. (Scale bar: 50 μm.)

Fig. 3.

XIB4035 potentiates ligand-induced RET signaling but does not act as a GFRα/RET receptor agonist. (A) XIB4035 does not activate a TH-Luciferase reporter in SH-SY5Y cells. SH-SY5Y cells transfected with the reporter construct were treated with either 2 nM GDNF or increasing concentrations of XIB4035 either for 10 min and then incubated overnight in growth media or exposed to treatments overnight. Measurements of luciferase activity after the treatments show that GDNF treatment increases TH promoter activity in both conditions, but XIB4035 has no effect (one-way ANOVA Newman–Keuls post hoc test, n = 3; vs. control, ***P < 0.001; error bars indicate SEM). (B) XIB4035 does not induce RET phosphorylation. SH-SY5Y cells were treated with either 2 nM GDNF or various concentrations of XIB4035 for 2 or 10 min, and cells were lysed immediately. Anti-phosphotyrosine Western blot analysis shows that RET phosphorylation (arrow) is induced by GDNF but not by XIB4035. (C) XIB4035 enhances GFL-induced RET signaling. SH-SY5Y-THpGL3 stable cells were treated with increasing concentrations of GDNF or ARTN with or without 20 µM XIB4035 for 10 min. Treatments were replaced with growth media, and cells were maintained overnight before being assayed for luciferase activity. For both ligands, XIB4035 cotreatment caused a shift in the nonlinear regression of the dose–response curve (F test: GDNF vs. GDNF + 20 µM XIB4035, P = 0.00006; ARTN vs. ARTN + 20 µM XIB4035, P = 0.000005), reduced minimal ligand dose necessary to induce luciferase activity above control [Student t test vs. control: GDNF = 75 pM (P = 0.0063), GDNF + 20 µM XIB4035 = 2.7 pM (P = 0.038), ARTN = 75 pM (P = 0.0271), ARTN + 20 µM XIB4035 = 2.7 pM (P = 0.0124)], and increased maximal effect (Student t test: fold over control: 18 nM GDNF = 2.76 ± 0.32 vs. 18 nM GDNF + 20 µM XIB4035 = 3.41 ± 0.41, P = 0.0189; 18 nM ARTN = 2.78 ± 0.44 vs. 18 nM ARTN + 20 µM XIB4035 = 3.61 ± 0.47, P = 0.0241). (D) XIB4035 prolongs GFL-induced RET phosphorylation. SH-SY5Y cells were treated with 2 nM GDNF or 1 nM ARTN with or without 10 or 20 µM XIB4035 for 10 min. Cell lysates were either collected immediately or treatment was washed out and replaced with growth media for 30 or 60 min. Cell lysates were subjected to phosphotyrosine Western blot. RET phosphorylation (arrow) in the GDNF- or ARTN-treated samples returns to baseline by 60 min but not with addition of XIB4035.