The neural androgen receptor: a therapeutic target for myelin repair in chronic demyelination

Abstract

Myelin regeneration is a major therapeutic goal in demyelinating diseases, and the failure to remyelinate rapidly has profound consequences for the health of axons and for brain function. However, there is no efficient treatment for stimulating myelin repair, and current therapies are limited to anti-inflammatory agents. Males are less likely to develop multiple sclerosis than females, but often have a more severe disease course and reach disability milestones at an earlier age than females, and these observations have spurred interest in the potential protective effects of androgens. Here, we demonstrate that testosterone treatment efficiently stimulates the formation of new myelin and reverses myelin damage in chronic demyelinated brain lesions, resulting from the long-term administration of cuprizone, which is toxic for oligodendrocytes. In addition to the strong effect of testosterone on myelin repair, the number of activated astrocytes and microglial cells returned to low control levels, indicating a reduction of neuroinflammatory responses. We also identify the neural androgen receptor as a novel therapeutic target for myelin recovery. After the acute demyelination of cerebellar slices in organotypic culture, the remyelinating actions of testosterone could be mimicked by 5α-dihydrotestosterone, a metabolite that is not converted to oestrogens, and blocked by the androgen receptor antagonist flutamide. Testosterone treatment also failed to promote remyelination after chronic cuprizone-induced demyelination in mice with a non-functional androgen receptor. Importantly, testosterone did not stimulate the formation of new myelin sheaths after specific knockout of the androgen receptor in neurons and macroglial cells. Thus, the neural brain androgen receptor is required for the remyelination effect of testosterone, whereas the presence of the receptor in microglia and in peripheral tissues is not sufficient to enhance remyelination. The potent synthetic testosterone analogue 7α-methyl-19-nortestosterone, which has been developed for long-term male contraception and androgen replacement therapy in hypogonadal males and does not stimulate prostate growth, also efficiently promoted myelin repair. These data establish the efficacy of androgens as remyelinating agents and qualify the brain androgen receptor as a promising drug target for remyelination therapy, thus providing the preclinical rationale for a novel therapeutic use of androgens in males with multiple sclerosis.

Figure 1

Testosterone therapy induces the replenishment with CA II⁺ oligodendrocytes and the recovery of MBP⁺ myelin in the chronically demyelinated corpus callosum of both castrated male (M) and ovariectomized female (F) mice. (A and B) The corpus callosum remained depleted of oligodendrocytes after 12 weeks of cuprizone (CUP) feeding followed by a treatment with empty subcutaneous Silastic® implants for 6 weeks (−T). The administration of Silastic® implants filled with testosterone (+T) during 6 weeks after cuprizone withdrawal restored the number oligodendrocytes in both sexes [overall effect F(1, 31) = 576, P ≤ 0.001; effect of testosterone treatment F(2, 31) = 104, P ≤ 0.001; sex difference in response F(2, 31) = 1.55, P = 0.22] (n = 9–17 per group). (C and D) Cuprizone feeding also strongly reduced myelin basic protein immunostaining, which recovered after testosterone treatment (+T) [overall effect F(1, 57) = 1569, P ≤ 0.001; effect of treatment F(2, 57) = 142, P ≤ 0.001; sex difference in response F(2, 57) = 1.39, P = 0.25] (n = 6–14 per group). Results are presented as mean ± SEM (% of control for myelin basic protein immunostaining) and were analysed by two-way ANOVA (treatment × sex) followed by Newman-Keuls post hoc tests. Significance: ***P ≤ 0.001 when compared with control or testosterone-treated mice, *P ≤ 0.05 as indicated.

Figure 2

Testosterone therapy results in the recovery of EGFP⁺ oligodendroglial cells and myelin throughout the brain of Plp-EGFP mice, expressing the EGFP driven by the mouse myelin proteolipid protein gene promoter. (A) Sagittal brain sections of Plp-EGFP male mice. (A and B) The brains were severely depleted of EGFP⁺ oligodendroglial cells and myelin after 12 weeks of cuprizone (CUP) feeding followed by treatment with empty Silastic® implants (−T) for 6 weeks. As much as 80% of the green fluorescence was recovered after treatment with testosterone-filled implants (+T) for 6 weeks [group differences F(2, 9) = 25.5, P ≤ 0.001] (n = 4 per group). Results are presented as means ± SEM (% of control) and were analysed by one-way ANOVA followed by Newman-Keuls post hoc tests. Significance: ***P ≤ 0.001 when compared with control or testosterone-treated mice, ns = not significant.

Figure 3

Testosterone therapy promotes the remyelination of corpus callosum. (A) Representative semi-thin plastic-embedded tissue sections stained with toluidine blue (top) and ultra-thin sections examined by electron microscopy (EM) (bottom) from corpus callosum of castrated control male mice (Cont), cuprizone-fed mice for 12 weeks (CUP 12 W), cuprizone-fed mice for 12 weeks followed by a treatment of 6 weeks with empty implants (6 W−T) and cuprizone-fed mice treated testosterone-filled Silastic® implants (6 W+T). Dramatic loss of myelin was observed after 12 weeks of cuprizone treatment (CUP-12 W) and the following weeks (CUP12+W6−T). We noticed a significant reduction of axonal diameter as a consequence of myelin loss. Testosterone promoted the remyelination of axons CUP12W+(6 W+T), and the the axonal diameter of remyelinated axons retruned to normal size. (B) The number of myelinated axons counted from electron microscopy micrographs [group differences F(3, 15) = 144, P ≤ 0.001] (n = 4–5 per group). (C) The average of axonal diameters measured from electron microscopy graphs [group differences F(3, 15) = 78, P ≤ 0.001] (n = 4–5 per group). (D) Density of SMI-31⁺ axons quantified by light microscopy on 50 µm vibratome sections [group differences F(3, 17) = 2.9, P = 0.063] (n = 5–6 per group). Values represent means ± SEM and were analysed by one-way ANOVA followed by Newman-Keuls post hoc tests. Significance: ***P ≤ 0.001 when compared with control or testosterone-treated mice or as indicated.

Figure 4

Testosterone treatment significantly increased the number of Olig2⁺ cells and NG2⁺ oligodendrocyte precursor cells in the chronically demyelinated corpus callosum. Following cuprizone-induced chronic demyelination, castrated male mice were treated for 6 weeks with empty (−T) or testosterone-filled (+T) subcutaneous Silastic® implants. (A and C) Following the feeding of cuprizone (CUP), the density of Olig2⁺ cells was markedly decreased within the corpus callosum, and testosterone (T) treatment stimulated their recruitment [group differences F(2, 20) = 34.3, P ≤ 0.001] (n = 6–9 per group). (B and D) Testosterone treatment also caused a 5-fold increase in the number of NG2⁺ oligodendrocyte precursor cells [group differences F(2, 9) = 32.4, P ≤ 0.001] (n = 4 per group). Values presented in C and D correspond to means ± SEM and were analysed by one-way ANOVA followed by Newman-Keuls post hoc tests. Significance: ***P ≤ 0.001 when compared with controls or testosterone-treated mice (C), or when compared with controls or mice receiving an empty implant (D). **P ≤ 0.01 and *P ≤ 0.05 as indicated. (E–J) The identification of cells induced by testosterone within the cuprizone-demyelinated corpus callosum. (E) Cells double-labelled for Olig2 (yellow-green stained nuclei) and NG2 (black cell processes). (F) Peroxidase immunostaining of Olig2⁺ nuclei. (G) Double-labeling of the peroxidase immunostained Olig2⁺ nuclei (black) and CA II⁺ oligodendrocytes (green) is indicated by arrows. (H) Immunodetection of BrdU⁺ nuclei of dividing cells. (I) Immunodetection of CA II⁺ oligodendroglial cells. (J) Co-localization of BrdU and CA II (arrows), visualized by merging H and I.

Figure 5

The activation of astrocytes and microglial cells in response to cuprizone-induced demyelination is markedly attenuated by prolonged testosterone treatment. (A and C) Cuprizone-induced demyelination induced strong astrogliosis in the corpus callosum of castrated male mice. Administration of testosterone (T) for 6 weeks following cuprizone (CUP) withdrawal did not significantly affect glial fibrillary acidic protein staining intensity. However, after 9 weeks of testosterone therapy, glial fibrillary acidic protein staining was downregulated to control (C) levels [group differences F(4, 19) = 54.5, P ≤ 0.001] (n = 4–6 per group). (B and D) Cuprizone-induced demyelination also strongly increased the number of activated Iba1⁺ microglial cells within the corpus callosum. Similarly to astrogliosis, microglial activation was reduced to control levels after 9 weeks of testosterone treatment, but not after 6 weeks [group differences F(4, 23) = 27.3, P ≤ 0.001] (n = 4–8 per group). Values represent means ± SEM and were analysed by one-way ANOVA followed by Newman-Keuls post hoc tests. Significance: ***P ≤ 0.001 when compared with control mice or as indicated.

Figure 6

Testosterone promotes the remyelination of cerebellar slice cultures after lysophosphatidyl choline (LPC)-induced demyelination. (A and B) Effect of testosterone (T) and lysophosphatidyl choline treatment on the number of myelinated axons. (A) Antibodies against MBP and calbindin protein were used for the immunostaining of myelin (green) and axons (red). Most axons surrounded by myelin sheaths in control slices were remyelinated after 4 days of treatment with testosterone following lysophosphatidyl choline–induced demyelination. The arrowhead indicates a myelinating oligodendrocyte and the arrow denotes a Purkinje cell. Images were analysed using confocal microscopy. (B) While most axonal myelin was lost, the number of axons was not significantly affected by lysophosphatidyl choline treatment [group differences F(2, 13) = 0.86, P > 0.4], but testosterone restored the number of myelinated axons to normal level [group differences F(2, 12) = 45.0, P ≤ 0.001] (n = 5–6 per group). Values represent means ± SEM and were analysed by one-way ANOVA followed by Newman-Keuls post hoc tests. Significance: ***P ≤ 0.001 when compared with sections treated with vehicle (controls) or testosterone (10 µM) after lysophosphatidyl choline. (C and D) The remyelinating effect of testosterone can be blocked by the selective androgen receptor antagonist flutamide and mimicked by its non-aromatizable metabolite 5α-DHT. (C) Representative immunofluorescence of MBP⁺ myelin in control slices, after lysophosphatidyl choline–induced demyelination followed by 4 days of vehicle, testosterone or testosterone+flutamide treatment. (D) The corresponding quantification of myelin basic protein immunofluorescence in the cerebellar slices [group differences F(4, 68) = 39.7, P ≤ 0.001] (n = 9–20 per group). Values represent means ± SEM (% of control) and were analysed by one-way ANOVA followed by Newman-Keuls post hoc tests. Significance: ***P ≤ 0.001 when compared with controls or sections treated with testosterone alone or 5α-DHT.

Figure 7

Testosterone fails to stimulate remyelination in the chronically demyelinated corpus callosum of mice with a non-functional androgen receptor (AR^Tfm mice) or after selective neural androgen receptor ablation (AR^NesCre mice). (A and B) In AR^Tfm mice, the corpus callosum was depleted of CA II⁺ oligodendrocytes and MBP⁺ myelin after 12 weeks of cuprizone (CUP) treatment followed by the administration of empty implants during 6 weeks (CUP−T). Treatment for 6 weeks with testosterone implants (CUP+T) failed to stimulate the recruitment of new oligodendrocytes and myelin repair [group differences for CA II⁺ oligodendrocytes: F(2, 16) = 56.7, P ≤ 0.001, and for myelin basic protein immunofluorescence: F(2, 15) = 21.3, P ≤ 0.001] (n = 6–7 per group). (C–F) As in AR^Tfm mice, testosterone treatment failed to stimulate oligodendrocyte recruitment and remyelination of the cuprizone-demyelinated corpus callosum of AR^NesCre mice. (C and E) CA II⁺ oligodendrocytes in the corpus callosum of control AR^NesCre mice or after feeding cuprizone for 12 weeks followed by treatment for 6 weeks with empty (CUP−T) or testosterone-filled (CUP+T) Silastic® implants [group differences F(2, 12) = 48.6, P ≤ 0.001]. (D and F) Myelin basic protein immunofluorescence staining of the corpus callosum of control, CUP−T and CUP+T AR^NesCre mice. Results are expressed as % of control [group differences F(2, 13) = 76.3, P ≤ 0.001] (n = 4–7 per group). Values represent means ± SEM and were analysed by one-way ANOVA followed by Newman-Keuls post hoc tests. Significance: ***P ≤ 0.001 when compared with the corresponding controls.

Figure 8

After cuprizone-induced chronic demyelination of the corpus callosum, the remyelinating effect of testosterone (T) can be mimicked by its non-aromatizable metabolite 5α-DHT and by the potent androgen receptor ligand 7α-methyl-19-nortestosterone (MENT). (A) Treatment of castrated males after cuprizone withdrawal for 6 weeks with a Silastic® implant filled with testosterone, 5α-DHT or 7α-methyl-19-nortestosterone efficiently replenished the number of CA II⁺ oligodendrocytes. The number of oligodendrocytes remained low in controls (C) receiving an empty implant [group differences F(3, 24) = 62, P ≤ 0.001] (n = 5–7 per group). (B) Treatment with testosterone, 5α-DHT or 7α-methyl-19-nortestosterone also efficiently restored myelin basic protein immunoreactive myelin, whereas myelin basic protein immunofluorescence remained low in controls. Results are expressed as % of myelin basic protein immunofluorescence in normal males [group differences F(3, 25) = 25.2, P ≤ 0.001] (n = 5–13 per group). Values represent means ± SEM and were analysed by one-way ANOVA followed by Newman-Keuls post hoc tests. Significance: ***P ≤ 0.001 when compared with the corresponding controls.