HSP90 Inhibitor, NVP-AUY922, Improves Myelination in Vitro and Supports the Maintenance of Myelinated Axons in Neuropathic Mice

Abstract

Hereditary demyelinating neuropathies linked to peripheral myelin protein 22 (PMP22) involve the disruption of normal protein trafficking and are therefore relevant targets for chaperone therapy. Using a small molecule HSP90 inhibitor, EC137, in cell culture models, we previously validated the chaperone pathway as a viable target for therapy development. Here, we tested five commercially available inhibitors of HSP90 and identified BIIB021 and AUY922 to support Schwann cell viability and enhance chaperone expression. AUY922 showed higher efficacy, compared to BIIB021, in enhancing myelin synthesis in dorsal root ganglion explant cultures from neuropathic mice. For in vivo testing, we randomly assigned 2-3 month old C22 and 6 week old Trembler J (TrJ) mice to receive two weekly injections of either vehicle or AUY922 (2 mg/kg). By the intraperitoneal (i.p.) route, the drug was well-tolerated by all mice over the 5 month long study, without influence on body weight or general grooming behavior. AUY922 improved the maintenance of myelinated nerves of both neuropathic models and attenuated the decline in rotarod performance and peak muscle force production in C22 mice. These studies highlight the significance of proteostasis in neuromuscular function and further validate the HSP90 pathway as a therapeutic target for hereditary neuropathies.

Keywords: C22 mice; Charcot-Marie-Tooth disease; Neuropathy; chaperones; myelin; neuromuscular disease.

Figure 1

Effects of HSP90 inhibitors on Schwann cells. (A) Cell viability after treatment (24 h) with DMSO, GA (50 nM), or the indicated five HSP90 inhibitors (50 and 500 nM) was calculated and graphed, as a percentage of DMSO (vehicle). (B) HSP70 mRNA levels were quantified after 24 h of treatment with the indicated compounds (100 nM). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal control. (A, B) GA (50 nM) served as a positive control. A.U.: arbitrary units. Graphs are plotted as means ± SEM; ***P < 0.001; **P < 0.01; *P < 0.05; n.s., nonsignificant; two-tailed unpaired Student’s t-test.

Figure 2

Treatment with BIIB021 and AUY922 increases chaperone expression in a dose- and time-dependent manner. (A) Steady-state levels of HSP70 and HSP27 in whole Schwann cell lysates (15 μg/lane) were analyzed after 24 h of treatment with DMSO, BIIB021, or AUY922, at the specified doses. GA (50 nM) served as a positive control. (B) HSP70 and HSP27 levels were observed after treatment with 100 nM BIIB021 or AUY922 for the indicated times. (C) Chaperone pathway activation by BIIB021 or AUY922 (100 nM) was studied after 2 or 4 h (treatment), followed by 4, 24, 32, and 48 h chase time points. (A, B) GAPDH and (C) tubulin served as protein loading controls. Molecular mass on left, in kDa. Data shown are representative of n = 3 independent experiments.

Figure 3

Improved myelin production in DRG explant cultures from C22 mice after treatment with AUY922. (A) Steady-state levels of HSP70 and P0 were analyzed in vehicle (Veh)-, AUY922-(A), or BIIB021 (B)-treated explant lysates (35 μg/lane). (B) Wt DRG cultures, with (Neuron + Schwann cell) and without (depleted) Schwann cells, were treated with 100 nM AUY922 and analyzed for the indicated chaperones. (A, B) Tubulin served as a protein loading control. Molecular mass on left, in kDa. MBP-positive myelin internode lengths in explant cultures from (C) Wt and (D) C22 mice treated with vehicle, AUY922, or BIIB021 were measured (n = 100–120 segments per group) and graphed as whisker plots with median (center line), quartiles (box), and extremes (whiskers); ***P < 0.001; *P < 0.05; n.s., nonsignificant; two-tailed unpaired Student’s t-test. (E) Cultures from Wt (top panel) and C22 (lower panel) mice, treated with the indicated compounds were stained for MBP (green). Nuclei were visualized with Hoechst dye (blue). The scale bar is as shown. Data shown are representative of n = 3–4 independent experiments.

Figure 4

Treatment with AUY922 improves neuromuscular performance of C22 mice. (A) Mean ± SEM of the body weight of Wt and C22 mice (n = 6–8 mice per group) was plotted over the treatment period. Performances of individual animals on the accelerating rotarod (B) at baseline (7 weeks age) and (C) at the end of the treatment (25 weeks age) are shown. The bars represent the mean for each group. (D) Mean ± SEM of the rotarod performance of all groups, plotted over the treatment period. (E) Muscle force, analyzed using an in situ technique, was recorded and normalized to the animal’s body weight (mN/g = millinewtons/grams). Each point represents the mean ± SEM force. (F) Distribution of individual measures of the cross-sectional area of TA muscle from C22 mice treated with vehicle or AUY (n = 3–5 mice per group). The bars represent the mean for each group. For all graphs, # indicates a significant (#P < 0.05, ##P < 0.01, ###P < 0.001) genotype difference. *P < 0.05, **P < 0.01, and ***P < 0.001 indicate a significant treatment effect for C22 mice.

Figure 5

AUY922 administration supports the maintenance of myelinated axons in sciatic nerves of C22 mice. (A) Cross-sectional views of nerve sections from Wt (top panels) and C22 (lower panels) male mice. Micron bar, 45 μm. (B) The cross-sectional area occupied by nerve fibers in a 40 μm × 40 μm square (n = 20–25 fibers per animal; n = 6–8 mice per group) was measured and graphed as shown. Graph plotted as means ± SEM; ***P < 0.001, across the treatment groups; #P < 0.05, across the genotypes; two-tailed unpaired Student’s t-test. Correlative analyses between axon and fiber diameter measurements were obtained from sciatic nerve cross-sectional areas from (C) Wt and (D) C22 groups. (E) Comparison of trendlines between the cohorts in parts C and D. Scatter plots comparing the g-ratios (axon diameter/fiber diameter) of individual fibers plotted as a function of axon diameters in nerves of (F) Wt and (G) C22 animals. (C–H) n = 950–1100 fibers per group. (H) Trendline comparisons of graphs in parts F and G.

Figure 6

Improved processing of PMP22 in AUY922-treated C22 mice. (A) Sciatic nerve lysates (5 μg/lane) were treated with either EndoH (column H) or PNGaseF (column N) and probed with antihuman PMP22 antibodies. No enzyme samples served as controls (column C). EndoH-resistant (arrows) and EndoH-sensitive (arrowheads) PMP22 fractions are marked. (B) Quantification of EndoH-resistant PMP22 fractions in sciatic nerves. (C) PMP22-positive aggregates per microscopic field (0.1 mm²) were counted in longitudinal sections of sciatic nerves. (D) Representative images of anti-PMP22 antibody stained (red) nerve sections from Wt (insets) and C22 mice are shown. Arrows mark PMP22-positive aggregates. Hoechst dye (blue) was used to visualize the nuclei. The scale bars are as shown. (E) Steady-state levels of HSP70 and HSP27 in vehicle (Veh)- and AUY922 (AUY)-treated nerve lysates (30 μg/lane) were quantified from (F, G) independent Western blots. (H) Whole liver lysates (30 μg/lane) were processed for (I, J) HSP70 and HSP27 quantification. (E–J) GAPDH or tubulin served as a loading control. Molecular mass on left in kDa. (B, C, F, G, I, J) n = 3–8 mice per group and plotted as means ± SEM; ***P < 0.001; ***P < 0.01; *P < 0.05; n.s., nonsignificant; two-tailed unpaired Student’s t-test.

Figure 7

AUY922 promotes the maintenance of myelinated axons in TrJ mice. (A) Cross-sectional views of nerve sections from vehicle- (left) and AUY-treated (right) TrJ mice. Micron bar, 20 μm. (B–E) Morphometric analysis of nerves from vehicle- and AUY-treated TrJ mice (n = 4 mice, 320 fibers, 30–40 areas per group). (B) Percent area occupied by fibers, (C) fiber diameter, (D) myelin sheath thickness, and (E) g-ratios were graphed. (F) Rotarod performance at the baseline and at the end of treatment is shown for vehicle- or AUY-treated TrJ mice. (G) Myofiber cross-sectional area of TA muscle from the indicated groups (n = 5 mice, 695 fibers per group). (H) Sciatic nerve (30 μg/lane) and whole liver lysates (30 μg/lane), with (I, J) quantification, from vehicle (Veh)- and AUY922 (AUY)-treated TrJ mice, were assessed for HSP70. (H–J) n = 6–14 mice per group, with GAPDH serving as a loading control. Molecular mass on left, in kDa. Graphs plotted as means (C, G) ± SEM (B, F, I, J) or as whisker plots with median (center line), quartiles (box), and extremes (whiskers) (D, E); ***P < 0.001; **P < 0.01; *P < 0.05; two-tailed unpaired Student’s t-test.