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, 113 (51), 14829-14834

Unexpected Central Role of the Androgen Receptor in the Spontaneous Regeneration of Myelin

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Unexpected Central Role of the Androgen Receptor in the Spontaneous Regeneration of Myelin

Bartosz Bielecki et al. Proc Natl Acad Sci U S A.

Abstract

Lost myelin can be replaced after injury or during demyelinating diseases in a regenerative process called remyelination. In the central nervous system (CNS), the myelin sheaths, which protect axons and allow the fast propagation of electrical impulses, are produced by oligodendrocytes. The abundance and widespread distribution of oligodendrocyte progenitors (OPs) within the adult CNS account for this remarkable regenerative potential. Here, we report a key role for the male gonad, testosterone, and androgen receptor (AR) in CNS remyelination. After lysolecithin-induced demyelination of the male mouse ventral spinal cord white matter, the recruitment of glial fibrillary acidic protein-expressing astrocytes was compromised in the absence of testes and testosterone signaling via AR. Concomitantly, the differentiation of OPs into oligodendrocytes forming myelin basic protein (MBP)+ and proteolipid protein-positive myelin was impaired. Instead, in the absence of astrocytes, axons were remyelinated by protein zero (P0)+ and peripheral myelin protein 22-kDa (PMP22)+ myelin, normally only produced by Schwann cells in the peripheral nervous system. Thus, testosterone favors astrocyte recruitment and spontaneous oligodendrocyte-mediated remyelination. This finding may have important implications for demyelinating diseases, psychiatric disorders, and cognitive aging. The testosterone dependency of CNS oligodendrocyte remyelination may have roots in the evolutionary history of the AR, because the receptor has evolved from an ancestral 3-ketosteroid receptor through gene duplication at the time when myelin appeared in jawed vertebrates.

Keywords: Schwann cells; androgen receptor; myelin; oligodendrocytes; testosterone.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Testes, testosterone, and AR are required for the recruitment of astrocytes into a demyelinating lesion, thus determining the balance of central versus peripheral types of remyelination. (AE, Left) Visualization of astrocytes stained with an antibody to GFAP (red). (AE, Right) Immunohistochemical staining of MBP+ CNS myelin (red) and P0+ PNS myelin (green) 4 wk after lysolecithin-induced demyelination. Cell nuclei were blue-counterstained with DAPI. Dotted lines delineate the boundary between the ventral white matter (VWM) and top gray matter. (A) Recruitment of astrocytes and recovery of MBP+ CNS myelin in the lesion area of gonadally intact males. Wt, wild type. (Scale bars: 100 μm.) (B) After testes removal and treatment with an empty implant (+ V), astrocytes remained sparse within the remyelinated area and remyelination was mediated by Schwann cells (P0+ myelin). (C) Recruitment of GFAP+ astrocytes and regeneration of MBP+ myelin in castrated males treated with testosterone (+ T). (D and E) Despite testosterone treatment, astrocytes were almost absent within the remyelinated area in ARTfm or ARNesCre mice, and axons were remyelinated by Schwann cells. The thin arrows show the areas depleted in GFAP+ labeling and the thick arrows point out the areas displaying a limited expression of P0. (F, Left) Mean area of GFAP immunolabeling (±SEM) (F5,28 = 5.67, P ≤ 0.001). (F, Right) Mean area of P0 immunolabeling (±SEM) (F5,46 = 3.78, P ≤ 0.01). Letters on top of columns indicate least significant differences (LSDs) [P ≤ 0.001 (Left), P ≤ 0.01 (Right), post hoc LSD tests; n = 5–6].
Fig. S1.
Fig. S1.
Perfect matching of the immunostaining of CNS myelin with an antibody against PLP (red) or MBP (green). The bottom panel indicates the colocalization of PLP and MBP immunoreactivity.
Fig. S2.
Fig. S2.
In the absence of testes and testosterone, GFAP+ astrocytes are sparse and axons are remyelinated by Schwann cells within the demyelinated lesion. (Top) Myelin formed by oligodendrocytes within the area demyelinated by LPC was stained on sagittal sections with an antibody to MBP (red). Cell nuclei were stained with DAPI in blue. (Bottom) Myelin formed by Schwann cells was stained with an antibody to P0 (green), and astrocytes were stained with an antibody to GFAP (white). (A) When castrated male mice were treated during 4 wk after LPC-induced demyelination with an empty s.c. Silastic implant (+ V), few GFAP+ astrocytes were observed within the lesion area. Axons were preferentially remyelinated by Schwann cells, as indicated by the high expression of P0+ PNS myelin. (B) When castrated males received a testosterone-filled implant (+ T), GFAP+ astrocytes were widely detected within the area of remyelination and axons were preferentially remyelinated by oligodendrocytes, as shown by the high expression of MBP+ CNS myelin. The arrows point out the wide or limited areas expressing the P0 marker in each condition. Representative images from five to six animals are shown. (Scale bar: 100 μm.)
Fig. S3.
Fig. S3.
Large-caliber axons are largely preserved after lysolecithin-induced demyelination. Immunohistochemical staining of neurofilament 200 kDa (NF-200; green) and MBP (red) in sagittal spinal cord sections. (Left) Cell nuclei were blue-counterstained with DAPI. All male mice were castrated. (Top) Control (CTRL) mice remained unlesioned. Dotted lines delineate the boundary between the ventral white matter (VWM, Bottom) and gray matter (Top). (Scale bars: 100 μm.) (Middle and Bottom) In the other mice, a demyelinating lesion was induced by LPC, and they were treated during 4 wk with an empty (+ V) or testosterone-filled (+ T) s.c. implant. Results confirm the failure of MBP-immunoreactive myelin regeneration in the absence of testosterone and show that NF-200+ axons are largely preserved (as indicated by the arrow). With testosterone treatment, NF-200+ axons are again ensheathed by MBP-immunoreactive myelin. Representative images from five to six animals per group are shown.
Fig. S4.
Fig. S4.
Metabolism and signaling mechanisms of testosterone in the nervous system. In the CNS, testosterone is converted by the aromatase enzyme to estradiol, which activates gene transcription via the binding of estrogen receptors (ERs; comprising ERα and ERβ) to estrogen response element (ERE). The conversion of testosterone to estradiol has a key role in the viability and plasticity of neurons and in cognitive functions (54, 55). In addition, testosterone is converted by the 5α-reductases to its potent metabolite 5α-dihydrotestosterone, which also acts via AR binding to androgen response element (ARE) (56). The 5α-dihydrotestosterone can be further metabolized by hydroxysteroid dehydrogenase (HSD) to 3β-androstanediol or 3α-androstanediol. Whereas 3β-androstanediol is a ligand of ERs, 3α-androstanediol is a positive modulator of γ-aminobutyric acid type A (GABAA) receptors, the main receptors mediating neuronal inhibition in the brain (–59).
Fig. 2.
Fig. 2.
Testosterone increases the reactivity and number of astrocytes in vivo and in mixed cultures of glial cells. (A, Top) Immunohistochemical staining of reactive GFAP+ astrocytes (green) and MBP+ myelin (red) in the ventrolateral part of the unlesioned male mouse spinal cord [control (CTRL)]. Cell nuclei were blue-counterstained with DAPI. (Scale bar: 50 μm.) (A, Middle and Bottom) GFAP+ astrocytes and MBP+ myelin at the top border of the demyelinated area (dotted line) in castrated male mice that received lysolecithin injection and were treated for 4 d with an empty (+ V) or testosterone filled (+ T) s.c. Silastic implant. (B) Mean area covered by GFAP+ astrocytes (±SEM) (F2,21 = 14.19, P ≤ 0.001). Letters on top of columns indicate LSDs (P ≤ 0.05, post hoc LSD tests; n = 5–6). (C, Top) Immunohistochemical staining of GFAP+ astrocytes (red) in CTRL mixed cultures of glial cells. (C, Middle) Exposing the cultures for 12 h to lysolecithin and treating them for 3 d with vehicle (+ V) increased the density of astrocytes (details are provided in legend of Fig. S5). (C, Bottom) Further increase in the density of astrocytes was observed when cultures were treated for 3 d with testosterone (+ T; 1 μM). (D) Mean area covered by GFAP+ astrocytes (±SEM) (F2,43 = 21.3, P < 0.001). Letters on top of columns indicate LSDs [P ≤ 0.05 (Left), post hoc LSD tests; n = 3–4]. LPC, lysolecithin.
Fig. 3.
Fig. 3.
Testes, testosterone, and AR are required for the spontaneous regeneration of CNS myelin by oligodendrocytes. (AF, Left) MBP-immunoreactive CNS myelin (green) in sagittal sections at 4 wk after lysolecithin microinjection into the right ventrolateral white matter tract of the spinal cord. Cell nuclei were counterstained with DAPI. (AF, Right) Immunohistochemical staining of CA II+ oligodendrocytes (red). (A) Recovery of MBP and mature oligodendrocytes in gonadally intact Wt mice. Dotted lines delineate the boundary between the ventral white matter (VWM) and the top gray matter. (Scale bars: 100 μm.) (B) Absence of recovery of MBP and oligodendrocytes after the removal of testes and treatment with an empty s.c. implant (+ V). Castration was performed at the age of 4–6 wk, 4 wk before lysolecithin-induced demyelination. (C) Regeneration of MBP-immunoreactive myelin and replenishment of oligodendrocytes in castrated males treated with a testosterone-filled implant (+ T). In ARTfm mice with a nonfunctional AR (D) or in ARNesCre mice with CNS-selective ablation of AR (E), testosterone failed to stimulate CNS remyelination and the replenishment of oligodendrocytes. (F, Left) Mean area devoid of MBP immunostaining (F5,30 = 44.8, P ≤ 0.001). Letters on top of columns indicate LSDs [P ≤ 0.01 (Left), P ≤ 0.05 (Right), post hoc LSD tests; n = 5–6]. (F, Right) Mean number of CA II+ oligodendrocytes (±SEM) within the area of demyelination (F5,34 = 9.2, P ≤ 0.001).
Fig. S5.
Fig. S5.
Testosterone increases the density of neonatal OPs in cell culture and of adult OPs in vivo. (A and B) Visualization of a confluent mixed glial cell culture prepared from the postnatal (postnatal days 0–3) brain of Plp-EGFP mice, exposed for 12 h to LPC (0.1 mg/mL culture medium) and then treated during 3 d with vehicle (0.1% ethanol, + V) or testosterone (1 μM, + T). (A) Oligodendroglial cells expressing EGFP were first daily observed. (B) Then, the culture was fixed and NG2+ cells were immunostained. (C) Density of EGFP+ cells in cultures treated for 1, 2, or 3 d with V or T after LPC (F2,28 = 5.53, P ≤ 0.01). (D) Density of NG2+ OPs in cultures treated for 3 d with V or T (F2,15 = 60.2, P ≤ 0.001). (E, Left) Olig2 and proliferating cell nuclear antigen (PCNA) immunostaining performed on sagittal sections of the ventrolateral spinal cord derived from an unlesioned CTRL male (Top) and from animals that received a stereotaxic injection of LPC before being treated for 4 wk with either an empty (+ V) or testosterone-filled (+ T) implant (Middle and Bottom). (E, Right) Histograms indicate the density of Olig2+ cells (Top; F2,12 = 53.5, P ≤ 0.001) and of proliferating Olig2+ cells double-labeled with PCNA (Bottom; F2,12 = 63.7, P ≤ 0.001). Letters on top of columns indicate least significant differences [LSDs; P ≤ 0.01 (Top), P ≤ 0.05 (Bottom), post hoc LSD tests; n = 5–6]. (Scale bars: 50 μm.)
Fig. 4.
Fig. 4.
Coordinated acquisition of a hinged jaw and myelin, and parallel diversification of steroid receptors during vertebrate evolution. The surrounding of axons with myelin was a prerequisite for the emergence of predation and escape behavior in vertebrates. The evolutionary appearance of myelin indeed parallels the acquisition of a hinged jaw (gnathostomata) (31). The oldest contemporary vertebrates with myelin are cartilaginous fishes (rays and sharks), whereas myelin is absent in jawless fishes (cyclostomata: hagfishes and lampreys) (32). Notably, at the time when a hinged jaw and myelin appeared during vertebrate evolution, the six nuclear steroid hormone receptors diverged from three ancestral receptors during a second round of whole-genome duplication, namely, the AR, progesterone (PR), glucocorticosteroid (GR), mineralocorticosteroid (MR), and the two estrogen (ER) receptors (25, 26). In extant cyclostomata, only ancestral progesterone (Anc PR), corticoid (Anc CR), and estrogen (Anc ER) receptors are present, themselves derived from two ancestral cephalochordata 3-ketosteroid (AncSR) and estrogen (Anc ER) receptors (16). Cartilaginous fishes, the earliest group of living jawed vertebrates with myelinated axons, contain the most ancient type of AR activated by testosterone (27).

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