Myelin repair stimulated by CNS-selective thyroid hormone action

Abstract

Oligodendrocyte processes wrap axons to form neuroprotective myelin sheaths, and damage to myelin in disorders, such as multiple sclerosis (MS), leads to neurodegeneration and disability. There are currently no approved treatments for MS that stimulate myelin repair. During development, thyroid hormone (TH) promotes myelination through enhancing oligodendrocyte differentiation; however, TH itself is unsuitable as a remyelination therapy due to adverse systemic effects. This problem is overcome with selective TH agonists, sobetirome and a CNS-selective prodrug of sobetirome called Sob-AM2. We show here that TH and sobetirome stimulated remyelination in standard gliotoxin models of demyelination. We then utilized a genetic mouse model of demyelination and remyelination, in which we employed motor function tests, histology, and MRI to demonstrate that chronic treatment with sobetirome or Sob-AM2 leads to significant improvement in both clinical signs and remyelination. In contrast, chronic treatment with TH in this model inhibited the endogenous myelin repair and exacerbated disease. These results support the clinical investigation of selective CNS-penetrating TH agonists, but not TH, for myelin repair.

Keywords: Endocrinology; Mouse models; Multiple sclerosis; Neuroscience.

Figure 1. Sobetirome mimics thyroid hormone action in oligodendrocyte progenitor cells.

(A) Oligodendrocyte progenitor cells (OPCs) were incubated with vehicle (DMSO), 50 nM T3, or 50 nM sobetirome for 96 hours in the presence of PDGF. Representative images of OPCs stained with antibodies against NG2 for OPCs (green) and MBP for oligodendrocytes (red). Scale bars: 200 μm. (B) Quantification of MBP-positive oligodendrocytes demonstrated that T3 or sobetirome increased OPC differentiation. (C–E) OPCs isolated from rats were incubated with or without PDGF in the presence of DMSO (Veh), T3 (50 nM), or sobetirome (50 nM) for 24 hours. Transcript levels of Mbp, Klf9, and Hr were measured using qPCR. OPC isolation was performed in triplicate with technical qPCR duplicates. Statistical significance was determined by 1-way ANOVA across all groups (P value in figure) followed by a 2-tailed, unpaired t test for comparisons between groups denoted with asterisks (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001). All graphs show mean ± SEM.

Figure 2. T3 and sobetirome treatment promotes myelin repair in lysolecithin and cuprizone models.

(A) BlackGold staining of demyelinated lesion after PBS or lysolecithin injection (lesion outlined in yellow). Scale bars: 200 μm. (B and C) Mice were injected with lysolecithin on day 0, and daily i.p. injections with vehicle, T3 (1 mg/kg), or sobetirome (1 or 5 mg/kg) began on day 5. (D–H) Mice were treated with cuprizone for 12 weeks followed by 3 weeks of normal chow and daily i.p. injections with vehicle, T3 (1 mg/kg), or sobetirome (1 mg/kg). (D) Representative immunofluorescence images in the hippocampus. Brain sections were stained with antibodies for ASPA (red) and MBP (green). Scale bars: 200 μm. (E and F) ASPA-positive cells were quantified, and MBP intensity was quantified by threshold analysis. Data represent the following groups (for ASPA, Veh [n = 6], T3 [n = 5], and Sob [n = 6]; for MBP, Veh [n = 6], T3 [n = 4], and Sob [n = 5]). Two images were quantified for each animal. (G) Quantification of myelinated axons in the corpus callosum. Data represent n = 3, and 3 images were quantified for each animal. Naive mice that did not receive cuprizone (No cup) were not included in the statistical analysis. (H) Representative electron microscopy images from the medial caudal corpus callosum. Scale bars: 1 μm. Statistical significance was determined by 1-way ANOVA across all groups (P value in figure) followed by a 2-tailed, unpaired t test for comparisons between vehicle and treatment denoted with asterisks (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001). All graphs show mean ± SEM.

Figure 3. After Myrf ablation, iCKO-Myrf mice experience clinical signs of demyelination and remyelination consistent with histology findings.

(A) Myrf ablation was induced at 8 weeks of age in iCKO-Myrf mice with daily i.p. doses of tamoxifen over 5 days. Mice performed the rotarod test weekly for 32 weeks after tamoxifen injection. (B) Representative BlackGold images of Cre-positive brains at week 10 and week 24 compared with Cre-negative controls. Scale bars: 1 mm. (C) Serial, semiquantitative magnetization transfer ratio (MTR) maps at week 10 and week 24 (right) averaged from 3 mice. Averaged MTR map from wild-type C57BL6 mice (left, n = 5) was adapted with permission from PLoS ONE (34). Note the hyperintense myelinated white matter tracts in the wild-type mice, compared with isointense and slightly hyperintense corresponding areas in the week 10 and week 24 Cre-positive mice, respectively. Scale bars: 1 mm.

Figure 4. Treatment with sobetirome improves motor performance in iCKO-Myrf mice.

iCKO-Myrf mice were induced with 5 days of once-daily tamoxifen i.p. injections, and starting 2 weeks later, a cohort was fed ad lib chow compounded with sobetirome (80 μg/kg/d nominal dose). The control cohort was provided normal chow. (A) Clinical scores were assigned weekly (0, no disability; 1, wide gait or limp tail; 2, hindlimb weakness; 3, hindlimb paralysis). (B) Rotarod testing was performed weekly. (C) Summary analysis of rotarod data was performed by determining the week at which the mice crossed above a 60-second threshold for the remainder of the experiment. (D) Individual recovery was measured by dividing the average latency during the recovery phase (weeks 18–24) by the latency before decline (weeks 0–4). Statistical significance was determined by a 2-tailed, unpaired t test comparing control to sobetirome (*P ≤ 0.05, **P ≤ 0.01). In A and B, each t test was performed independently. All graphs show mean ± SEM.

Figure 5. Sobetirome hastens myelin recovery and oligodendrocyte maturation in iCKO-Myrf mice.

(A and B) Representative BlackGold images from Cre-positive mice treated with control or sobetirome chow. Scale bars: 1 mm (full-brain images); 0.25 mm (magnified inset, blue box). (C and D) Quantification of BlackGold images was performed by threshold analysis for white matter tracts (n = 4 for control and n = 6 for Sob, 2 images per animal). (E) Representative spinal cord EM images from control- and sobetirome-treated Cre-positive mice and Cre-negative mice at 24 weeks. Scale bars: 2 μm. (F) Myelinated axons were quantified in the ventrolateral spinal cord (n = 4 for control, n = 5 for Sob, 2 images per animal). (G) G-ratio analysis of spinal cord axons representing 100–200 axons per group. (H) Representative images from the caudal corpus callosum at week 10 stained for PDGFRα (white), ASPA (red), and EdU (green) from mice treated with control or sobetirome chow from weeks 2–10 and coadministered EdU from weeks 4–10. Scale bars: 20 μm. (I and J) Quantification of the PDGFRα/EdU colabeled oligodendrocyte precursor cells (OPCs) and ASPA/EdU-colabeled oligodendrocytes (n = 4 for all groups, 2 images per animal). Statistical significance was determined by a 2-tailed, unpaired t test comparing control to sobetirome (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). All graphs show mean ± SEM.

Figure 6. Hyper- and hypothyroidism produce different outcomes in the iCKO-Myrf model.

(A) Mice were administered T3/T4 chow starting 2 weeks after tamoxifen. The mice experienced a more severe disease course with no recovery and were euthanized at 14 weeks. (B) Mice were administered hypothyroid-inducing water starting 2 weeks after tamoxifen and were followed weekly by rotarod analysis. (C) Individual recovery was measured by dividing the average latency during the recovery phase (weeks 18–24) by the latency before decline (weeks 0–4). (D and F) Representative BlackGold brain images from control and hypothyroid groups. Scale bars: 1 mm (full-brain images); 0.25 mm (magnified insets, blue box). (E and G) Quantification of BlackGold images was performed by threshold analysis for white matter tracts (n = 4, 2 images per animal). Statistical significance was determined by a 2-tailed, unpaired t test comparing control to treatment (*P ≤ 0.05). In A and B, each t test was performed independently (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). No differences were significant for C (*P = 0.42), E (*P = 0.56), or G (*P = 0.31). All graphs show mean ± SEM.

Figure 7. Hyperthyroid treatment in iCKO-Myrf mice reduces oligodendrocyte progenitor cell and oligodendrocyte populations.

(A) Representative images from the caudal corpus callosum at week 10 stained for PDGFRα (white), ASPA (red), and EdU (green) from mice treated with control chow, T3/T4 chow, or hypothyroid water from weeks 2–10 and coadministered EdU from weeks 4–10. Scale bars: 20 μm. (B and C) Quantification of the PDGFRα/EdU colabeled oligodendrocyte progenitor cells (OPCs) and ASPA/EdU colabeled oligodendrocytes (n = 4, 2 images per animal). Statistical significance was determined by ANOVA analysis with Dunnett’s multiple comparisons test comparing control to treatment groups (**P ≤ 0.01, ***P ≤ 0.001). All graphs show mean ± SEM.

Figure 8. Sob-AM2 treatment in iCKO-Myrf mice improves motor performance and increases myelin recovery, as observed by MRI analysis.

Ablation of Myrf was induced in iCKO-Myrf mice with 5 days of once-daily tamoxifen i.p. injections, and starting 2 weeks later, mice were fed ad lib chow compounded with Sob-AM2 (84 μg/kg/d) or control chow. (A) Rotarod testing was performed weekly. (B) Summary analysis of rotarod data was performed by determining the week at which the mice crossed above a 60-second threshold for the remainder of the experiment. (C) Recovery was measured by dividing the average latency during the recovery phase (weeks 18–24) by the latency before decline (weeks 0–4). (D and E) Quantification of BlackGold images was performed by threshold analysis for white matter tracts (n = 7, 2 images per animal). Data points marked with orange indicate mice imaged by MRI (n = 3, 2 images per animal). (F) Group-average magnetization transfer ratio (MTR) maps from control and Sob-AM2 mice (n = 3) at week 24. Arrows indicate the body/isthmus of the corpus callosum, and arrowheads indicate lateral corpus callosum (orange for weak remyelination and white for strong remyelination). Scale bars: 1 mm. (G) BlackGold caudal brain images from representative control and Sob-AM2 mice imaged by MRI. Scale bars: 1 mm. Statistical significance was determined by a 2-tailed, unpaired t test comparing control to Sob-AM2 (*P ≤ 0.05, **P ≤ 0.01). In A, each t test was performed independently; green asterisks indicate weeks in which Sob-AM2 mice were significantly different from control Cre-positive mice (*P ≤ 0.05), and orange asterisks indicate weeks in which the Sob-AM2–treated mice were not significantly different from unaffected Cre-negative mice (*P > 0.10). All graphs show mean ± SEM.