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Abstract

Testosterone acts on the brain and spinal cord as an important regulator of neural functions, either by binding to the androgen receptor (AR) or via its aromatization to estradiol. Neurons have long been considered as its primary cellular target, but testosterone also plays a key role in the formation of myelin within the central nervous system (CNS). Remarkable features of CNS myelin are its plasticity and great capacity of regeneration. After a demyelinating lesion, new myelin sheaths are formed by oligodendrocytes recently generated from oligodendrocyte progenitor cells or by spared mature oligodendrocytes. Testosterone has allowed to demonstrate strong remyelinating effects, mediated by the AR receptor, in mouse models of toxin-induced demyelination. Enhancing these regenerative responses has become an objective for the treatment of demyelinating diseases, possibly including testosterone or AR ligands.

Introduction

Testosterone is well known as the male reproductive hormone and an anabolic steroid.¹ However, it is now well documented that this hormone exerts major influences on the nervous system that go far beyond the control of reproductive functions. Testosterone, indeed, plays major roles in the viability, functions, and plasticity of neurons, and it is also a key regulator of the myelination of axons, as presented in this review.

Neurons have long been considered as the primary cellular targets of testosterone in the central nervous system (CNS). The motoneurons of the rat lumbar spinal nucleus of the bulbocavernosus are one of the neuronal populations on which the neurotrophic effects of testosterone have been extensively explored. These motoneurons innervate striated muscles of the penis and are more numerous and larger in males, mainly resulting from the effects of testosterone on target muscles during development and on both neurons and muscles during adulthood.^2,3

Other neurons on which neurotrophic effects of testosterone have been documented are the Syrian hamster facial motoneurons.^4,5 Notably, testosterone affects cognitive processes and executive functions by exerting direct influences on the mesocorticolimbic system,⁶ and testosterone modulates synaptic plasticity and dendritic spine density in the rat and non-human primate hippocampus and prefrontal cortex.^7–9

Neuroprotective effects of testosterone have been documented, mainly in cultures of neuronal cells: protection of cultured rat cerebellar neurons against oxidative stress,¹⁰ of rat hippocampal neurons against β-amyloid toxicity,¹¹ and of human neurons isolated from fetal brain tissue against serum-deprived apoptosis.¹² Importantly, the neurotrophic and neuroprotective effects of testosterone in the CNS mainly involve direct signaling of testosterone via the androgen receptor (AR).

There are also examples of protective effects involving both androgens and estrogens, as in a transgenic mouse model of Alzheimer's disease, where both testosterone and estradiol (E2) delayed the brain accumulation of β-amyloid.¹³ A remarkable implication of testosterone and AR in determining the caliber of axons within white matter tracts has been observed by magnetic resonance imaging in adolescent boys.^14,15

There is now accumulating evidence that testosterone also plays a major role in myelin formation, both during development and during myelin regeneration in adulthood. The aim of this review is to discuss the implication of testosterone in the regeneration of myelin. In the first part of the review, particular attention is paid to our present understanding of the signaling mechanisms and metabolism of testosterone in the rodent and human CNS. In the second part, we will discuss the effects of testosterone in remyelination mechanisms that are associated with myelin repair in demyelinating diseases.

Testosterone Actions on the Brain

Sources of testosterone: the endocrine glands and local synthesis

Extra-cerebral sources: circulating testosterone

In men, reference morning levels of testosterone determined in three cohorts by liquid chromatography-tandem mass spectrometry (LC-MS/MS) have provided a basis for categorizing testosterone levels as normal (about 20 nM) or low (about 10 nM).¹⁶ The study also revealed that the age-related decrease in mean testosterone levels is rather modest, but that individual testosterone concentrations show great variability between individuals. It is more precisely the prevalence of men with low testosterone levels that markedly increases with age, with about 4% in young and 20% in aged men.¹⁶

Another LC-MS/MS study found mean levels of testosterone of 17 nM in young men and of 10 nM in men older than 80.¹⁷ An earlier study using a radioimmunoassay also reported a very progressive decline in testosterone levels between the ages of 30 and 80, and an age-dependent increase in the proportion of men with testosterone values in the hypogonadal range, reaching about 10% between the ages 40 and 50, and about 20% in men older than 60.¹⁸

Importantly, testosterone is not only a male hormone. Circulating levels of testosterone in fertile women determined by LC-MS/MS are around 1 nM, with a slight elevation at mid-cycle.¹⁹ These results are consistent with those of another study comparing testosterone levels measured by LC-MS/MS between men and women.¹⁷ Contrasting with the drop of the main ovarian steroids, E2 and progesterone, testosterone levels do not significantly decrease at the time of menopause, with about half of the testosterone provided by the ovaries and half by the adrenal glands.²⁰

Brain synthesis of testosterone

Brain levels of testosterone do not necessarily reflect their circulating levels, as testosterone may be synthesized locally de novo or from circulating adrenal precursor steroids by neural cells. Evidence is mainly based on the expression and activity of steroidogenic enzymes and the persistence of low amounts of testosterone and its metabolites in the brain after removal of the steroidogenic endocrine glands, in particular within the hippocampus and mesocorticolimbic system.^6,21–23

The de novo synthesis of steroids from cholesterol within the brain has been first reported by Baulieu, and he named them “neurosteroids” to refer to their site of synthesis.²⁴ At first, it was thought that the synthesis of neurosteroids was limited to pregnenolone, progesterone and their immediate metabolites, whereas testosterone and E2 were considered to be brought to the brain from the steroidogenic endocrine glands.²⁵

This view was consistent with the striking effects of gonadectomy on brain functions and structure and with reports failing to detect in the rat brain cytochrome P450c17, which catalyzes the conversion of pregnenolone to dehydroepiandrosterone (DHEA) or of progesterone to androstenedione, two obligatory precursors of androgens²⁶ (Fig. 1A). However, one study reported P450c17 mRNA expression in different regions of the rat and mouse brain.²⁷ In any case, P450c17 was shown to be expressed in the embryonic rat brain and in cultures of neurons and astrocytes, but not of oligodendrocytes, purified from neonatal rat brains.^28,29

FIG. 1.

The biosynthesis and metabolism of testosterone. (A) Cholesterol is converted to pregnenolone, the precursor of all steroid hormones, by cytochrome P450scc. Androstenedione, the direct precursor of testosterone, results from the conversion of DHEA by the 3β-HSD or of progesterone by P450c17. Cytochrome P450c17 catalyzes both 17α-hydroxylase and 17,20-lyase reactions. The reduction of androstenedione to testosterone is catalyzed by the 17β-HSD3. The conversion of DHEA to androstenediol corresponds to an alternative testosterone biosynthetic pathway. The transcriptional activity of testosterone mediated by AR is amplified via its 5α-reduction to 5α-DHT. Here again exists an additional pathway via the 5α-reduction of androstenedione to 5α-androstanedione, which is then converted to 5α-DHT by the 17β-HSD3. Two metabolites of 5α-DHT, 3α,5α-diol and 3β,5α-diol, are, respectively, activators of GABA_A receptors and of the ERβ. (B) As an alternative pathway to its 5α-reduction, testosterone is converted to E2 by the aromatase. Androstenedione is aromatized to estrone by the enzyme. (C) A third metabolic pathway of testosterone and androstenedione is their conversion to 11β-hydroxylated metabolites via an 11β-hydroxylase. The 11β-hydroxylated androgens are further metabolized to 11-ketoandrostenedione and 11-ketotestosterone, with the latter also being a substrate of the 5α-reductases. Both 11-ketotestosterone and 11-ketodihydrotestosterone are AR agonists. Potent activators of AR are indicated in red. 3α,5α-diol, 3α,5α-androstanediol; 5α-DHT, 5α-dihydrotestosterone; 3β,5α-diol, 3β,5α-androstanediol; 17β-HSD3, 17β-hydroxysteroid dehydrogenase type 3; 3β-HSD, 3β-hydroxysteroid dehydrogenase; AR, androgen receptors; DHEA, dehydroepiandrosterone; E2, estradiol; ERβ, estrogen receptor beta.

This difficulty of detecting P450c17 mRNA and protein in the adult rat brain has encouraged the search for alternative synthesis pathways. Thus, a P450c17-independent alternative non-enzymatic pathway has been reported for the conversion of progesterone to androstenedione by rat brain cortex microsomes and cultured oligodendrocytes, astrocytes, and C6 rat glioma tumor cells.^30,31

More recently, studies have provided evidence that the de novo synthesis of testosterone and E2 occurs in neurons of the rodent hippocampus.^23,32,33 Moreover, androgens and estrogens are also synthesized within the rat medial prefrontal cortex, nucleus accumbens, and ventral tegmental area, where they may be involved in the regulation of executive functions.^6,34 Although castration eliminated circulating testosterone, the hormone remained present within these brain regions for as long as 6 weeks, although at lower levels, providing further support for its localized brain synthesis.³⁵ However, as shown later on, the brain synthesis of androgens does not compensate for the effects of castration on myelin formation in mice.³⁶

In the human brain, although data remain limited, the presence of steroidogenic enzymes has been documented by immunocytochemistry, RT-PCR and their activity has been assessed by the conversion of ³H- or¹⁴ C-labeled precursors. The presence of the cytochrome P450 cholesterol side-chain cleavage, which converts cholesterol to pregnenolone, a rate-limiting step in steroid synthesis, was first detected in the human brain by immunocytochemistry (Fig. 1A).³⁷

P450scc mRNA levels were then quantified by RT-PCR in hippocampal tissue obtained at neurosurgery from patients with temporal lobe epilepsy. Interestingly, hippocampal P450scc mRNA concentrations increased during childhood, reaching adult levels around puberty, and they were higher in adult women than in men.³⁸

Cytochrome P450c17 mRNA could not be detected in biopsies of human temporal lobe or hippocampus.³⁹ However, the enzyme has been localized by immunocytochemistry in the nervous system of the fetal nervous system at mid-gestation, pointing again to its possible role in developmental processes.⁴⁰ In the search for alternative pathways to P450c17 for the synthesis of DHEA, human astroglial and oligodendroglial cell lines, as described earlier for rat neural cells, made DHEA via a non-enzymatic mechanism.⁴¹

Investigation of mRNA expression by RT-PCR showed that 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3), which converts androstenedione to testosterone, 5α-androstanedione to 5α-dihydrotestosterone (5α-DHT) and estrone to E2, is expressed in biopsies of the human temporal lobe, interestingly of both men and women (Fig. 1A,B).⁴² Of note, metabolic conversions of labeled androstenedione, testosterone and also of estrogens were higher in subcortical white matter than in the cerebral cortex.⁴³

In the neurosurgically removed cerebral cortex, hippocampus, and subcortical white matter, 5α-reductase and 3α-HSD activities colocalized, suggesting the important roles of 5α-reduced metabolites of testosterone and other 5α-reducible neurosteroids in these brain regions.^44,45 Aromatase expression and activity were also demonstrated in the cerebral cortex and subcortical white matter.⁴⁶ Surprisingly, no differences between sexes were observed for the brain 5α-reductase and aromatase pathways.^45,47 Thus, all enzymes necessary for androgen and estrogen biosynthesis from cholesterol have been identified in the adult human brain, with the exception of P450c17.

Adrenal androgens as an important source of brain testosterone

DHEA and its sulfated conjugate dehydroepiandrosterone sulfate (DHEAS), both produced by the adrenal glands, are substrates for the local synthesis of androgens and estrogens within hormone-sensitive tissues, including the nervous system (Fig. 1A).^48,49 In humans, they are the most abundant steroid hormones, with DHEA reaching high nanomolar concentrations and DHEAS even micromolar levels in both sexes.⁵⁰ The DHEAS concentrations peak in men (around 10 μM) and in women (around 8 μM) between 20 and 30 years of age and then continually decline until 60 years, followed by a more moderate decrease and stabilizing around 20% of their maximal levels.^51,52

It is important to realize that peak DHEAS levels are about 1000-fold higher than those of testosterone in men and about 10,000-fold higher in women.⁵² This implies that DHEAS and, to a lesser extent, DHEA may represent throughout life, and importantly in both sexes, a great potential reservoir for the local formation of bioactive androgens and estrogens within target tissues, including the CNS.

It is important to be reminded here that elevated levels of DHEA and DHEAS are limited to humans and nonhuman primates and are not observed in laboratory rodents. Early reports of elevated levels of DHEAS and pregnenolone sulfate in rats were analytical artefacts resulting from the autoxidation of cholesterol present in millimolar amounts in tissue samples.⁵³ We have, thus, to be aware of important differences between species when discussing the significance of steroid hormones and neurosteroids in neural functions.^53–55

Another important adrenal androgen is androstenedione, which is converted to testosterone (and estrone) by the 17β-HSD3 present in the rodent and human brain. Androstenedione results from the conversion of DHEA by the 3β-hydroxysteroid dehydrogenase, the privileged pathway in humans, or of progesterone by P450c17, the privileged pathway in rodents (Fig. 1A). Androstenedione is also produced by the gonads, but in women the adrenal glands are considered to be its main source.⁵⁶ In both sexes, levels of androstenedione are in the low nanomolar range as determined by LC-MS/MS, with slightly higher levels in women, and they also gradually decrease with age.¹⁷

The advent of precise and sensitive mass spectrometric methods, allowing the extensive and precise analysis and profiling of steroids in biological samples, has enabled the emergence of an increasingly complex picture of adrenal androgens. The second most abundant, but still under-recognized androgens produced by the human and primate adrenal glands are 11-oxygenated androgens, also named 11-oxyandrogens (Fig. 1C). They are synthesized from androstenedione (11-hydroxyandrostenedione) or testosterone (11β-hydroxytestosterone) via the adrenal cytochrome P450 11β-hydroxylase.⁵⁷

These adrenal androgens can then be respectively converted within tissues to 11-ketoandrostenedione and 11-ketotestosterone and their respective 5α-reduced metabolites. Importantly, both 11-ketotestosterone and 11-ketodihydrotestosterone are bioactive androgens, with potencies equivalent to those of testosterone (Fig. 1C).^58,59

A study across species has revealed that 11-oxyandrogens are present in nonhuman primates and humans, but like DHEA and DHEAS neither in rats nor in mice, calling again attention to important species differences in steroid biosynthetic pathways.⁶⁰ In nonhuman primates and humans, circulating levels of bioactive 11-ketotestosterone are similar in males and females (around 1 nM), and these are thus comparable to testosterone levels in women but 20 times lower than testosterone levels in men.^60,61 Unlike DHEA, DHEAS, and androstenedione, the 11-oxyandrogens do not decline in aging women and remain elevated after menopause.^17,62

The presence of comparable levels of DHEA, DHEAS, androstenedione, and 11-ketotestosterone in both sexes implies that circulating testosterone levels are not the only indicator of the androgen status, especially in women. It also requires to reconsider the role and importance of androgens in women. Androgens, indeed, play a significant role in women's health, not only for reproductive and sexual functions, but they also exert beneficial effects on the skeletal, metabolic, cardiovascular, and nervous systems.⁶³ For this reason, testosterone therapy at a low dosing may be beneficial in women.^64,65

The brain metabolism of testosterone: the aromatase and 5α-reductase pathways

The aromatase pathway

The aromatase converts testosterone to E2 and androstenedione to estrone (Fig. 1B).⁶⁶ The first evidence for the aromatization of testosterone to E2 by neural cells was provided by Naftolin et al. in the 1970s.⁶⁷ Thus, testosterone serves as a local source of estrogens within the CNS. The aromatase is widely expressed in the brain of both male and female rodents, mainly in neurons, and also throughout the human brain.⁶⁸

A comparative study of the subcellular location of aromatase in neurons of different species by light and electron microscopy revealed the presence of the enzyme in perikarya, dendrites, and axons and at the level of synapses.⁶⁹ This suggests that the bioavailability of E2 may be rapidly modulated at the synaptic levels via local aromatase activity.⁷⁰ It is of importance that the brain aromatase is a highly regulated enzyme, which is upregulated by its substrate testosterone and induced in astrocytes in response to injury or neurodegenerative conditions.^66,70

Numerous effects of testosterone in the brain are mediated by E2, which acts through the estrogen receptors (estrogen receptor alpha and estrogen receptor beta [ERβ]).⁶⁶ E2 is involved in the regulation of a variety of neural processes such as synaptic plasticity, neurotransmission, and the proliferation of neural progenitor cells.^71–76 As a consequence, besides its reproductive functions, E2 influences memory and cognitive functions in rodents, nonhuman primates, and humans.⁷⁷ Moreover, estrogens exert protective effects on neurons and glial cells exposed to degenerative conditions or injury.^66,78 However, the most important for our purpose are the effects of estrogens in myelin formation, as discussed later in the text.

The 5α-reductase pathway

The conversion of 5α-DHT by two 5α-reductase enzymes is the other major metabolic pathway of testosterone in the CNS (Fig. 1A). As 5α-DHT binds more tightly to AR than testosterone and is more potent in the transactivation of AR target genes, this enzymatic reaction corresponds to an amplification of the testosterone signal at the pre-receptor level.⁷⁹

The 5α-DHT cannot be converted to E2 by the aromatase, and for this reason it is commonly used in experimental designs to ensure that AR-mediated signaling is involved in the effects of testosterone. However, one should be aware that 5α-DHT can be converted in a reversible manner to 3β,5α-androstanediol (3β,5α-diol), which acts on ERβ and is involved in the regulation of the hypothalamo-pituitary-adrenal axis.⁸⁰

Two 5α-reductase isoforms, named the 5α-reductase type 1 (5α-R1) and the 5α-reductase type 2 (5α-R2), have been identified in rodents, nonhuman primates, and humans. Although both isozymes catalyze the same reaction, they share only a limited degree of homology and each possesses distinctive biochemical properties.⁸¹ In addition to testosterone, the two enzymes catalyze the conversion of all steroids with a carbon 3 ketone group and a C4–C5 double bond to their corresponding 5α-reduced metabolites, including androgens, glucocorticoids, progestins, and mineralocorticoids.⁸²

Immunocytochemical studies have shown that of the two 5α-reductase isoforms, 5α-R1 is widely expressed in the brain of both sexes and in all neural cell types. In one study, 5α-R1 immunoreactivity was found to be present in glial cells throughout the brain of male and female rats.⁸³ Interestingly, within the brain, 5α-R1 immunopositive cells were preferentially located in white matter, consistent with the high 5α-reductase activity in purified myelin.^84–86

Thus, testosterone signaling via AR may be preferentially amplified via its conversion to 5α-DHT in areas of dense myelin. A role of the 5α-reduction of testosterone in myelination was suggested by the observations that the formation of 5α-DHT is highest in the rat hypothalamus and cerebral cortex during the first 2 weeks of life, when the myelination process starts, and also takes place in cultured oligodendrocytes and their progenitor cells.^87,88

The 5α-R2 is the major isoform of the urogenital system, including the prostate, epididymis, testicles, and seminal vesicles. In the rat brain, the presence of 5α-R2 was initially considered to be limited to the late fetal and early postnatal period.⁸⁹ More recently, the presence of the isoform has been detected in most regions of the adult brain, but in contrast to 5α-R1, it was found to be exclusively localized to neurons.⁹⁰ However, when considering the cellular distribution of steroid metabolizing enzymes, we have to be aware of possible methodological differences between studies, in particular those using immunohistological or mRNA amplification methods, and also of possible species differences. Thus, in the mouse brain, 5α-R1 was only observed in neurons.⁹¹ In contrast, in humans, 5α-R1 was found to be present in neurons and glial cells and 5α-R2 only in neurons of both men and women.^92,93

We have already mentioned the conversion of 5α-DHT to 3β,5α-diol, which modulates ERβ gene transcription in neuronal cells.⁹⁴ In addition, 5α-DHT can be metabolized to 3α,5α-androstanediol (3α,5α-diol), which in the brain is not just an elimination product, but also a positive modulator of GABA_A receptors.⁹⁵ This is a property that 3α,5α-diol shares with the other 3α,5α-reduced neurosteroids.^96–98

AR in the CNS

Testosterone and 5α-DHT are the principal ligands of AR, with the conversion of testosterone to 5α-DHT corresponding to a signal amplification step as discussed earlier. The AR is encoded by a single gene located on chromosomeX.⁹⁹ Unliganded AR are predominantly cytoplasmic and undergo nucleocytoplasmic shuttling via mechanisms involving nuclear important and export signals as well as association with heat-shock proteins.^100,101 After ligand binding, the receptor translocates to the nucleus, where it interacts with coregulators and mediates the transcriptional regulation of androgen-sensitive target genes.^102–105

Properties that distinguish AR from the other intracellular steroid receptors are that transcriptionally activated nuclear receptors are recycled into the cytoplasm, where they become again available for ligand binding, and that ligand binding protects the receptors from degradation by the proteasome.^106,107

Similar to the intracellular movements of other steroid receptors, AR can also translocate to the cellular membrane and associate with membrane-associated proteins such as caveolins, filamins, and Src tyrosine kinases after palmitoylation of a conserved 9 amino acid localization sequence.^108–110 Thus, besides its genomic actions, testosterone can exert direct effects on cell membranes via its intracellular receptor.^111,112 Membrane actions of AR may be particularly important in the CNS, as suggested by receptor localization studies.

AR are, in fact, widely distributed throughout the brain and spinal cord, with elevated expression in the cerebral cortex, strongly suggesting that testosterone may modulate a wide range of neural functions via its intracellular receptor.^113–115 In accordance with the effects of testosterone on executive functions, AR are also abundant within mesocorticolimbic nuclei and associated structures in rodents.⁶

The distribution of AR has been examined in detail in the rodent brain at the cellular level by combining light with immuno-electron microscopy. These studies have demonstrated that in addition to their presence in the cell nucleus, AR are located in axons and dendrites, where they may activate kinase signaling pathways.^116,117 Interestingly, AR-immunoreactive (AR-ir) axons and dendrites were primarily observed in brain regions where nuclear AR staining is low.¹¹⁶

AR-ir has also been observed in glial processes.¹¹⁷ Within the male rat hippocampus, AR-ir was detected in astrocyte processes close to axon terminals,¹¹⁸ consistent with the increase in hippocampal spine density by 5α-DHT.^119,120 Other studies have shown that various populations of astrocytes contain AR.^115,121 When considering the distribution of AR in the CNS, it is important to keep in mind that its expression is regulated. For example, in response to excitotoxic or traumatic injuries to the parietal cortex or hippocampus, AR is induced in microglial cells.¹²²

The brain distribution of AR shows differences between species. However, because of the differential, dynamic, and brain region-specific subcellular distribution of AR, individual studies carried out under precise experimental and methodological conditions require to be interpreted with caution. In contrast to rodents, the prefrontal cortex of adult rhesus monkeys contains, in addition to neurons, a large number of glial cells that bear AR.

Most of the identified AR-ir glial cells were astrocytes, with a small proportion of oligodendrocytes. Conversely, no AR-ir was detected in microglial cells.¹²³ A study of fetal rhesus monkeys showed that by 124 days of gestation (term at 167 days), the distribution of brain AR-ir neurons became similar to that observed in adults, with marked labeling in the hypothalamus, amygdala, and hippocampus.¹²⁴ Similar AR expression patterns have been reported for mouse and human brain.¹²⁵

In the human brain, AR-ir was detected in neurons, astrocytes, oligodendrocytes, and microglia in temporal cortex tissue resected from epileptic patients.¹²⁶ The presence of AR-ir in the human cerebral cortex and hippocampus has been confirmed by another study.¹²⁷

Binding studies of tritiated 5α-DHT within microdissected brain nuclei confirmed the wide distribution of AR and that nuclear receptor levels are higher in male than in female rats.¹¹³ Insertion of reporter genes into the locus of the AR gene has allowed the identification of sexually dimorphic populations AR-expressing neurons within the mouse hypothalamus, bed nucleus of the stria terminalis, and forebrain.^128,129

A study of AR-ir in the basal forebrain of young men and women revealed less staining in women than in men for some nuclei, but similar staining intensities in the bed nucleus of the stria terminalis, the nucleus basalis of Meynert (NBM), and the islands of Calleja.¹³⁰ The marked reduction in AR-ir observed within the cholinergic nuclei of the human basal forebrain with age or in Alzheimer's disease also showed sex-specific patterns.¹³¹

Within the vertical limb of the diagonal band of Broca (VDB) and the NBM of men and women older than the age of 56, cytoplasmic AR immunostaining was decreased compared with young subjects. This decrease was more pronounced in men, resulting in a higher proportion of AR-positive neurons in aged women. The percentage of AR-positive neurons in both VDB and NBM was further decreased in women with Alzheimer's disease, but unexpectedly not in men with Alzheimer's disease.¹³¹

Differences in testosterone levels contribute to the observed differences in brain AR expression between sexes. Indeed, the AR is an autoregulated protein, which means that its expression is positively regulated by its ligand testosterone.^132–134 In mice, males exhibited more intense AR-ir than females in different brain regions. AR-ir was reduced after gonadectomy in both sexes, with a more marked decrease in males. Importantly, testosterone treatment upregulated brain AR expression to a similar extent in both males and females.

Thus, although there is a sexual dimorphism in AR expression within some brain regions, regulatory responses to both gonadectomy and testosterone treatment are essentially identical in both sexes.¹³³

Other hormones and, in particular, estrogens may also be involved in the positive regulation of AR expression. Thus, in hamster facial motoneurons, androgens and estrogens work synergistically to regulate AR expression and ovarian hormones play a role in the regulation of AR expression.^5,135 A role of ovarian hormones in regulating brain AR expression is further supported by the observation that AR-ir in the female rat hindbrain varies throughout the estrus cycle, and that ovariectomy decreases AR expression.¹¹⁷ During the postnatal period, testosterone does not directly induce AR expression via an auto-regulatory mechanism in the male rat forebrain, but it does so via its conversion to E2.¹³⁶

However, it is important to mention here that a downregulation of AR by androgens has also been reported for various cells and tissues.^137,138 The regulation of AR expression by androgens may be dependent on the cellular context as well as on the concentration of testosterone. In cell lines derived from castration-resistant prostate cancers, testosterone was found to downregulate AR expression, even at very low concentrations.^139,140

AR may also be downregulated by high concentrations of testosterone.^141,142 Several Cis-regulatory elements involved in the androgen-mediated regulation of AR expression have been identified. Thus, 4 consensus androgen response elements (ARE) within exons 4 and 5 mediate androgen-dependent upregulation.¹⁴³ Conversely, a regulatory sequence in the second intron is involved in androgen-mediated downregulation of AR mRNA.¹⁴⁴ It is also important to be aware of species differences. Thus, an active non-consensus ARE within the 5′ untranslated region of the human AR gene binds AR and elicits the repression of AR transcription. Multiple-species comparison revealed that this ARE is specific to primates.¹⁴⁵

Testosterone and Demyelinating Diseases

Myelin and its plasticity

In the vertebrate CNS, myelin is formed by oligodendrocytes. The lipid-rich and insulating myelin sheaths, which surround large axons, are required for the rapid saltatory conduction of nerve impulses.¹⁴⁶ In addition, oligodendrocytes also provide trophic support to axons.^147,148

First mainly regarded as an isolating structural component of neuronal circuits, myelin is now considered as a functional dynamic structure contributing to brain plasticity.¹⁴⁹ The structure of the myelin sheaths, in particular their thickness and the lengths of myelinated axon segments (internodes), affects the conduction of neuronal signals. Moreover, myelin is not evenly distributed along axons, another feature that has been proposed to play a role in the modulation of neuronal signal conduction velocity.¹⁵⁰ The conduction properties of axons and entire neuronal networks can, thus, be affected by the structure and organization of the myelin sheaths.^151,152

Far from being static, the myelin sheaths are constantly remodeled by neuronal activity and impacted by internal as well as environmental influences. This new concept, referred to as “myelin plasticity” or “adaptative myelination,” has been observed throughout the vertebrate lineage.¹⁵³ Adaptative myelination is controlled in adult rodents by the learning of new motor skills, sensory enrichment, and social experience, and it plays an important role in learning and memory processes.^154–159 Human neuroimaging studies indicated that new brain areas are myelinated during adult life, and that the learning of new skills results in white matter changes.^{156,160–162}

The regeneration of myelin

Damaged myelin and lost oligodendrocytes can be replaced in the adult CNS by a process known as remyelination or myelin repair. Animal studies have provided evidence that the formation of new myelin sheaths involves migration to demyelinated axons, proliferation, and differentiation of oligodendrocyte progenitor cells (OPC). These stem-like cells are present and widely distributed in the adult rodent brain and spinal cord.¹⁶³ Although the newly formed myelin sheaths are thin, they resist over time and preserve axonal functions.¹⁶⁴

These findings have furthered interest in stimulating the regenerative capacity of myelin in demyelinating diseases such as MS by promoting the proliferation and differentiation of CNS-resident OPC.¹⁶⁵ Myelin repair may, indeed, also occur in MS patients, as suggested by the initial relapsing-remitting course of MS in most patients, and by the presence of thin myelin sheaths in MS lesions, forming the so called “shadow plaques” and interpreted as areas of remyelination.^166,167 Of note, the number of OPC and maturing oligodendrocytes is increased in early MS lesions, whereas they become rare in chronic lesions, consistent with their participation in myelin repair during the early stages of the disease.¹⁶⁸

Over the past few years, there has been a regained interest in the contribution of preexisting mature oligodendrocytes in regenerating myelin. A recent study showed that in feline and nonhuman primate models of demyelination, not only oligodendrocytes newly generated from OPC, but also spared mature oligodendrocytes participate in myelin repair.¹⁶⁹ This was already suggested by earlier studies in rodents.^170–173 In adult mice, myelin regeneration by preexisting mature oligodendrocytes has been shown to be sustained by the learning of a new motor task after demyelination, pointing to the plasticity of remyelinating oligodendrocytes.¹⁷⁴

In humans, oligodendrocytes surviving demyelination may represent a major source of newly formed myelin. Thus, oligodendrocytes present in remyelinated MS lesions, defined by their thin myelin sheaths, were found to be generated during early life and not at the time of demyelination. These results were based on the integration of test-derived nuclear bombs.^{14 C.175} They also pointed to a very low turnover of oligodendrocytes in the adult brain.¹⁷⁶ Evidence for actively myelinating spared oligodendrocytes in MS came from single-cell RNA sequencing, showing that mature oligodendrocytes can reactivate transcriptional programs involved in myelination.¹⁷⁷

These observations could shift the therapeutic focus from OPC to surviving mature oligodendrocytes. Clarifying the respective contribution of preexisting oligodendrocytes and OPC in myelin repair now represents a challenge for remyelination therapies. A recent review on the question has presented interesting directions for future research.¹⁷⁸ Factors that were proposed to influence the balance of the two modes of remyelination were the types of demyelination, regions of the CNS, the heterogeneity of oligodendrocytes, the effect of age, and differences between species.

Age may be particularly important, as OPC-driven remyelination in rodents markedly decreases with advancing age, and oligodendrocytes that survive demyelination may gradually play an increasingly important role.^178,179 The hormonal environment of OPC and regenerating oligodendrocytes may represent an additional important factor. Indeed, progesterone, estrogens, and androgens, the latter being the focus of the present review, all exert strong influences on CNS myelination and remyelination.^180–187

Beneficial effects of testosterone in experimental autoimmune encephalomyelitis and MS

Beneficial effects of testosterone were first demonstrated in experimental autoimmune encephalomyelitis (EAE), the most commonly used animal model to investigate MS pathology and therapeutic interventions.¹⁸⁸ The EAE can be induced in animals by immunization with whole myelin proteins or peptide epitopes, or by the adoptive transfer of autoreactive T cells. Many of the disease-modifying therapies developed for MS have been tested or validated on the basis of EAE studies.^189,190

Physiologic levels of testosterone are protective in male mice with EAE since their castration worsens disease course. Interestingly, the removal of endogenous testosterone by orchidectomy affected EAE pathology only in those mouse strains showing sex differences in EAE susceptibility.^191,192 However, treatment with exogenous testosterone had myelin-protective and anti-inflammatory effects and ameliorated EAE symptoms in all strains of mice so far tested, and importantly in both males and females.^191–193

Beneficial effects of testosterone on mice with EAE are mediated by the AR, as they could be inhibited by the AR antagonist flutamide, and as they could be mimicked by 5α-DHT, a potent activator of AR.^191,193,194 Treatment with 5α-DHT had also beneficial effects on clinical scores in male rats.¹⁹⁵ Reduced levels of testosterone and 5α-DHT in the brain and spinal cord of male rats with EAE, analyzed by LC-MS/MS, may have contributed to the efficacy of the androgen therapy.¹⁹⁶ However, although animal studies demonstrate a role of direct androgen signaling via AR in improving EAE disease course, it is important to be aware that estrogens also exert protective and anti-inflammatory effects in EAE.¹⁹⁷

The first studies documenting the beneficial effects of testosterone therapy in EAE provided support for a small pilot clinical trial, in which the effects of transdermal testosterone supplementation were studied in 10 men with relapsing-remitting MS during 1 year. Although no effect was found on active lesions, there was a reduction in brain atrophy. Other outcomes were improved cognitive performance, anti-inflammatory effects, and reduced cortical gray matter loss evaluated by voxel-based morphometry.^198–200

The beneficial effects of testosterone treatment and reduced levels of endogenous androgens in experimental EAE as well as the efficacy of testosterone therapy in the small clinical trial of men with MS raise the question of a possible relationship between reduced testosterone levels and the incidence and progression of demyelinating diseases. Several studies reported that low testosterone levels are associated with disability in men with MS.^201–204

Reduced testosterone levels in MS may, of course, be a consequence of the disease. However, the analysis of clinical records revealed a relationship between testicular hypofunction and an increased risk of developing MS.²⁰⁵ Steroid profiling by LC-MS/MS in male patients with relapsing-remitting MS showed that in blood plasma and cerebrospinal fluid (CSF), levels of several neuroactive steroids were modified. Levels of 5α-DHT were decreased in CSF, and levels of 3α,5α-diol were increased. In contrast, levels of 5α-DHT were increased in plasma.

No significant differences in testosterone levels were observed between MS patients and controls in this study.²⁰⁶ The results of another study suggest that reduced levels of testosterone may even play a role in women with MS, as low levels were associated with increased brain lesions and clinical disability.²⁰⁷

Testosterone promotes myelin repair in the adult CNS

Current treatments of MS target the immune system during relapses with immunomodulatory and immunosuppressive drugs and recently introduced immune reconstitution therapies.^208,209 However, these treatments remain mainly disease-modifying. A complementary therapeutic approach, which has gained interest during the past few years, consists of stimulating the remyelination of axons.^210–213 Improving and accelerating this process is important not only for restoring nerve conduction, but also for preventing axonal degeneration at the origin of permanent lesions and disability.

Although EAE is valuable for studying the pathogenesis of MS and for testing new treatments, this is not suitable for investigating the regeneration of myelin. For this reason, experimental models of demyelination based on the use of toxins have been developed to study the cellular and molecular mechanisms involved in the process of myelin repair.²¹⁴ Their major advantage is the spatiotemporal predictability of axon demyelination and remyelination.

A widely used toxin-based model is the induction of a focal demyelinating lesion in white matter tracts of the brain or spinal cord by the stereotaxic infusion of lysophosphatidylcholine (LPC), often referred to as lysolecithin, which destroys myelin and kills oligodendrocytes but spares axons if well dosed. A major asset of this model is the separation between the phases of demyelination and spontaneous remyelination.²¹⁵

Another model of toxin-induced demyelination consists of feeding the copper chelator cuprizone to mice, resulting in widespread demyelination in the brain, mainly caused by oligodendrocyte apoptosis. Remyelination accompanies demyelination if the cuprizone challenge does not exceed 6 weeks (acute demyelination), but it is much delayed after 12 weeks of cuprizone feeding.^216–218

The effects of testosterone on remyelination were first assessed after 12 weeks of cuprizone. To avoid the confusing effects of endogenous gonadal steroid hormones, male and female mice were gonadectomized, reducing brain and plasma testosterone levels to about 100 pM. No spontaneous myelin regeneration was observed as late as 12 weeks after the withdrawal of cuprizone from the diet, confirming long-lasting demyelination.¹⁸⁵

However, when mice received a subcutaneous Silastic implant filled with testosterone, producing male-like plasma and brain levels of the hormone during 6 weeks (8–12 nM), oligodendrocytes were replenished with the recruitment of proliferating OPC and axons were remyelinated within the corpus callosum. Of note, testosterone therapy stimulated myelin regeneration in both male and female mice with similar efficacy. The remyelinating effect of testosterone was remarkably strong, as no spontaneous myelin recovery was observed in the absence of treatment. As an additional therapeutic benefit, testosterone modulated astrocyte and microglial responses.¹⁸⁵

After the demonstration that testosterone promotes myelin repair in chronic demyelination, it became mandatory to also examine the therapeutic effect of testosterone under experimental conditions where spontaneous remyelination takes place. The effects of testosterone on myelin regeneration were tested after the stereotaxic infusion of LPC into the male mouse ventral spinal cord white matter tract (ventral funiculus). In this model, demyelinated axons are fully remyelinated by oligodendrocytes within 4 weeks (Fig. 2).

FIG. 2.

The role of testes, testosterone, and brain AR in spontaneous myelin regeneration within the male mouse spinal cord. (A) A focal demyelinating lesion was induced unilaterally in the ventral white matter tract of the spinal cord by the stereotaxic infusion of LPC. The orange cylinder schematically indicates the extent of demyelination. (B) In gonadally intact males or in CX males treated with testosterone, axons within the demyelinated lesion (delimited by dotted lines) were fully remyelinated as early as 4 weeks after LPC (CNS myelin was stained in green by an antibody against MBP). The remyelinated area was also replenished with mature oligodendrocytes (immunostained in red by an antibody to carbonic anhydrase II). (C) In CX males, in the absence of testosterone therapy, and in transgenic males with conditional inactivation of the AR within the CNS (AR^NesCre), even when treated with testosterone, no remyelination by oligodendrocytes was observed 4 weeks after LPC. Cell nuclei were blue-counterstained with DAPI. The blue staining mainly corresponds to microglial cells. The selected pictures are representative of the different experimental conditions, with spontaneous remyelination in an intact male (B) and in a CX male (C). Scale bars: 100 μm. CNS, central nervous system; CX, castrated; DAPI, 4′,6-diamidino-2-phenylindole; LPC, lysophosphatidylcholine; MBP, myelin basic protein.

Results of this experiment came as a great surprise: Myelin rapidly regenerated as expected in gonadally intact male mice, within only 4 weeks, whereas neither replenishment of the demyelinated lesion by oligodendrocytes, nor spontaneous remyelination of axons by oligodendrocytes was observed in castrated (CX) males.³⁶ However, when CX males received a subcutaneous implant filled with testosterone, producing constant physiological levels of the hormone, axons were fully remyelinated by oligodendrocytes (Fig. 2).

The use of transgenic mice, expressing the enhanced green fluorescent protein selectively in cells of the oligodendrocyte lineage under the control of the mouse proteolipid protein gene promoter,²¹⁹ allowed to demonstrate that testosterone increases both the proliferation and differentiation of OPC.³⁶ Thus, testosterone plays an essential role in this regenerative process. These observations also imply that low levels of testosterone, remaining present in the CNS (about 100 nM) after removal of the testes, whatever their origin, are not sufficient to promote remyelination. As in the model of cuprizone-induced demyelination, testosterone modulated local neuroinflammatory responses.³⁶

However, in the absence of testosterone, axons did not remain demyelinated. They were, instead, remyelinated by cells presenting a Schwann cell-like phenotype³⁶ (Fig. 3). Schwann cells are the myelin-forming glial cells of the peripheral nervous system (PNS), which produce peripheral myelin containing the peripheral myelin proteins P0 (myelin protein zero) and PMP22 (peripheral myelin protein 22). How the presence or absence of testosterone determines whether CNS axon remyelination is carried out by oligodendrocytes or by Schwann-like cells remains to be elucidated.

FIG. 3.

CNS remyelination by oligodendrocytes or Schwann cells. The remyelination of axons by oligodendrocytes after LPC-induced demyelination in the male mouse ventral spinal cord requires signaling of T via AR, and in addition the conversion of T to E2. In the presence of T and AR, the demyelinated lesion was replenished by astrocytes before the remyelination of axons by oligodendrocytes. Conversely, in CX males or after AR ablation (AR^NesCre mice), astrocytes did not penetrate the lesion and axons were remyelinated by Schwann-like cells, derived either from OPC or from invading PNS Schwann cells. OPC, oligodendrocyte progenitor cells; PNS, peripheral nervous system; T, testosterone.

Interestingly, in the presence of testosterone and AR, the demyelinated lesion becomes occupied by astrocytes before the remyelination of axons by oligodendrocytes³⁶ (Fig. 3). Conversely, in the absence of androgen signaling, astrocytes remain absent when axons are remyelinated by Schwann-like cells. These observations point to the possibility that astrocytes may mediate or at least may play a key role in the remyelinating actions of testosterone.

Previously, it has been observed in both experimental animal models and human demyelinating diseases that demyelinated axons in the CNS can be remyelinated by Schwann cells.²²⁰ One possibility is that Schwann cells from PNS sources are recruited to remyelinate the CNS.^221,222 Alternatively, as revealed by genetic fate-mapping, Schwann cells contributing to remyelination in the CNS may also be derived from OPC²²³ (Fig. 3). Such a surprising plasticity of OPC with a neuroepithelial origin is intriguing and requires to be clarified, as Schwann cells arise during development from the neural crest. In that case, testosterone signaling may represent a switch in the fate of OPC.

The finding that testosterone plays a key role in myelin regeneration by oligodendrocytes raised the important question of the mechanism of its remyelinating action. This has motivated the search for interactions between testosterone and signaling mechanisms involved in oligodendrocyte differentiation and myelin formation. A recent study has uncovered a cooperation between androgen and sonic hedgehog (Shh) signaling in accelerating myelin regeneration.²²⁴ In the CNS, Shh signaling has been previously shown to be involved in myelin repair.^211,225

The canonical Shh signaling is initiated by binding of the secreted morphogen protein Shh to the transmembrane protein Patched, abrogating its repressive activity on a second transmembrane protein named Smoothened (Smo). The disinhibition of Smo then initiates downstream events, resulting in the translocation of Gli transcription factors into the nucleus and the transcription of Shh target genes.²²⁶

Local administration of the Smoothened (Smo) agonist (SAG) during LPC-induced focal demyelination within the corpus callosum of gonadally intact male mice increased the proliferation of OPC and their differentiation into mature oligodendrocytes. The pro-differentiating effect of SAG on OPC was indirect, involving Smo signaling on microglial cells and their polarization toward a non-inflammatory promyelinating phenotype (Fig. 4).^224,227

FIG. 4.

Cellular mechanisms of androgens mediating remyelination. In CX male mice, both the Smo agonist SAG and T signaling via AR stimulated the proliferation of OPC, but without additive effects. In contrast, SAG and T signaling cooperated in promoting OPC differentiation. The pro-differentiating action of SAG was indirect, involving Smo signaling on microglial cells. On the other hand, the pro-differentiating effects of T took place in the presence of astrocytes and required both AR signaling and the aromatization of testosterone to E2. SAG, Smoothened (Smo) agonist.

In CX males, both SAG and testosterone treatment enhanced the number of proliferating OPC within the demyelinated lesion to a similar extend, but no additive effect was observed after their co-administration. In contrast, the density of mature oligodendrocytes was higher after their combined than after their separate administration, demonstrating that testosterone and Shh signaling cooperates in promoting OPC differentiation.

Of note, the OPC proliferating effect of testosterone was mediated by AR, whereas the differentiating effects of testosterone not only involved AR, but also required its conversion to E2, as they could be inhibited by the selective and competitive aromatase inhibitor fadrozole (Fig. 4). Consistent with an additional role of testosterone aromatization in myelin repair, a strong upregulation of aromatase expression was observed in the LPC-induced demyelinated lesion.²²⁴ These findings hint toward the complexity of the interactions between testosterone, E2 and Shh signaling in myelin repair.

Cooperation between the androgen and Shh pathways was also observed in EAE, induced in CX male mice by a single subcutaneous injection of a myelin oligodendrocyte glycoprotein (MOG) peptide. Immunization of mice with myelin-derived MOG antigen causes the demyelination of axons and neuroinflammation, and this model is widely used for evaluating the potential of MS therapeutics. Treatment with vehicle, testosterone, SAG, or their combination was started at the onset of clinical signs.

Although both testosterone and SAG administered individually significantly reduced disease scores, their combined administration was more efficient. The reduction in neurological disability was accompanied by the preservation of axon integrity, a significant reduction of abnormally myelinated axons, and the modulation of microglial activation.²²⁴ In these experiments, testosterone was administered in female mice via the intranasal route to preferentially target the brain.²²⁸ When CX male mice received intranasal testosterone, its levels measured by gas chromatography-tandem mass spectrometry after 2 hours reached about 7 times higher concentrations in brain (about 70 nM) than in plasma (about 10 nM). These results are, thus, consistent with a privileged delivery of the hormone to the brain.²²⁴

The neural AR as a target for the remyelinating effects of testosterone

Testosterone can exert its effects on target cells via the intracellular AR, either directly or after its signal-amplifying conversion to 5α-DHT, or via the estrogen receptors, after its enzymatic aromatization to E2. Clarifying the signaling mechanisms underlying the remyelinating effects of testosterone was, thus, a major issue, especially as estrogens have been shown to exert beneficial effects on myelin, oligodendrocytes, and axons and have protective effects in EAE mice as well as in women with MS.^229–233 The presence and wide distribution of AR in the male and female CNS was in support of its possible role in the strong remyelinating effects of testosterone after cuprizone- or LPC-induced demyelination.

In both animal models, the remyelinating and anti-inflammatory effects of testosterone were dependent on the presence of AR, as they were no longer observed in male mice carrying the testicular feminization mutation (AR^Tfm mice). In these mice, a frame shift mutation in exon 1 of the AR gene produces a non-functional protein in all androgen target tissues.^234,235

The CNS AR was then identified as target of the remyelinating effects of testosterone by selective genetic deletion of AR in neural cells. In transgenic mice with selective conditional invalidation of AR in neurons and macroglial cells (AR^NesCre mice), testosterone therapy also failed to promote remyelination. As in these mice AR was not inactivated in microglial cells, its presence in this neural cell type is not sufficient to enhance remyelination by testosterone.^36,185

After acute LPC-induced demyelination in the spinal cord of male mice, no spontaneous remyelination by oligodendrocytes was observed in the AR^NesCre mice (Fig. 2C). Instead, as reported earlier for CX wild-type males, axons within the demyelinated lesion were remyelinated by PNS-type myelin.³⁶ Thus, testosterone signaling via AR governs the balance between oligodendrocyte- or Schwann cell-mediated remyelination.

Consistent with a key role of AR in myelin regeneration, treatment of male mice with 5α-DHT or the potent AR agonist 7α-methyl-19-nortestosterone (MENT) also efficiently stimulated corpus callosum remyelination after cuprizone-induced demyelination.¹⁸⁵

Perspectives

The multiple beneficial effects of testosterone treatment in EAE ranged from myelin-protective and anti-inflammatory effects to the improvement of disease symptoms. Recently, the use of mouse models of toxin-induced demyelination has allowed to demonstrate that testosterone also stimulates the remyelination of demyelinated axons. The promyelinating effect of testosterone was a particularly strong one, as its administration allowed the recovery of oligodendrocytes and myelin in a model of chronic demyelination.¹⁸⁵

Remarkably, no spontaneous myelin regeneration took place in the absence of testes-derived testosterone, demonstrating a key role of physiological male levels of testosterone.³⁶ The essential role of testosterone in oligodendrocyte myelination in male mice raises, of course, the question of the role of steroid hormones in females. One possibility is that the ovarian steroids progesterone and E2, which also have promyelination effects, are important for remyelination, but low levels of testosterone in females may also contribute to the regeneration of myelin, as both sexes are sensitive to the remyelinating effects of testosterone.

The two studies demonstrating an essential role of testosterone in myelin repair focused on its effects on OPC, showing that the hormone stimulates their proliferation and differentiation. The recognition that preexisting mature oligodendrocytes surviving demyelination contribute to remyelination, and that they could even represent a major source of newly formed myelin, opens new perspectives for testosterone therapy. Thus, the focus could shift from the effects of testosterone on OPC to the protection of mature oligodendrocytes and the recovery of damaged ones.

Another conceptual advance is likely to broaden the influences of testosterone on the brain. Myelin is, indeed, now considered as a dynamic component of brain plasticity, and it has been proposed to play a role in cognitive functions and in the development of neuropsychiatric diseases that were traditionally exclusively attributed to neuronal abnormalities, including depression and schizophrenia.^236–239 The stimulation of myelin formation and the modulation of myelin plasticity by testosterone may, thus, not only be of interest for demyelinating diseases.

They may have consequences for a variety of neuronal functions and diseases of the nervous system. This prospect opens completely new opportunities for our understanding of hormonal influences on the nervous system and for the therapeutic use of androgens.

When considering testosterone therapy for the treatment of demyelinating lesions or for sustaining brain functions, eventually in both men and women at different dosing levels, it is important take into account sources and endogenous levels. There is an age-dependent increase in the proportion of men with testosterone values in the hypogonadal range. Low testosterone levels may have an influence on the incidence of MS and on its evolution toward a progressive state.²⁰⁵

Although testosterone levels are higher in men, they are far from being negligible in women, reaching concentrations around 1 nM. Testosterone therapy may, thus, also be considered in women at lower dosing.

Our present understanding of the key role of androgen signaling via AR in myelin regeneration does not exclude benefits of other steroid hormones, in particular of the estrogens and also of progesterone, mediated by their respective intracellular receptors. In this regard, clarifying the signaling mechanisms underlying the promyelination effects of testosterone is a major question, especially as estrogens have been shown to exert beneficial effects on myelin, oligodendrocytes, and axons and have protective effects in EAE mice, as well as in women with MS.^229–233

It is interesting to mention here that other nuclear receptors are involved in myelin regeneration and have been identified as potential targets for myelin repair therapies. Among them are the thyroid hormone receptors,^240–242 the retinoid X receptor,²⁴³ the liver X receptors,²⁴⁴ and the vitamin D receptor.²⁴⁵ The brain AR has joined this list of nuclear receptors as a potential key pharmacological target for promoting remyelination.²⁴⁶

The identification of AR as a key pharmacological target for remyelination allows one to consider the therapeutic use of AR selective synthetic androgens. Thus, the potent and selective AR agonist MENT was shown to efficiently stimulate myelin regeneration in male mice after cuprizone-induced demyelination.¹⁸⁵ A particular advantage of this synthetic androgen is that it is not a substrate for the 5α-reductase enzymes and does not stimulate prostate growth, but that it can be converted to estrogens and thus does not deprive men of their major physiological effects.²⁴⁷ It is interesting to also mention that MENT has been developed and tested for androgen replacement therapy in hypogonadal men and also for male contraception.²⁴⁸

The choice of the type of androgen used for promoting myelin repair is important. As E2 and other metabolites mediate multiple effects of testosterone within the CNS, the use of bioidentical testosterone may be considered preferable to synthetic analogs. Androgens such as MENT can be converted to weak estrogens, whereas synthetic androgens that cannot undergo local tissue amplification or aromatization, and thus lack the full spectrum of the effects of testosterone, may be of particular concern.

Their administration may not only deprive patients of valuable neuroactive metabolites, but may even block their endogenous biosynthetic pathways via negative regulatory mechanisms. They also shut down testosterone production by the testes via their powerful negative feedback effects on the hypothalamic-pituitary-gonadal axis.^249–251

A therapeutic use of bioidentical testosterone raises the question of its mode of administration since when given orally, the hormone is inactivated in the liver. For this reason, parenteral forms of administration have been developed for hypogonadal and elderly men. Intramuscular injectable testosterone esters and surgically implanted subdermal testosterone pellets are increasingly replaced by transdermal patches and gels.^252–254

Testosterone gel has, indeed, been used in the pilot MS trial reported earlier. Nasal gels are less common, but they allow safe, prolonged, and intermittent administration of testosterone to humans, resulting in serum concentrations of testosterone in the normal physiological range.²⁵⁵ Importantly, intranasal administration of testosterone results in relatively low levels of circulating 5α-DHT, which may present a risk factor for the prostate.^256,257

Testosterone administration via the intranasal route efficiently reduced disease scores in EAE and was preferentially delivered to the brain and spinal cord, indicating the usefulness of this mode of administration for the treatment of demyelinating diseases.²²⁴

Identifying the cellular targets of the promyelinating effects of testosterone remains a major challenge, and it is very likely that testosterone acts on different neural cell types. Oligodendrocytes and their precursors may be direct targets, although this remains to be demonstrated. Testosterone may also stimulate myelin formation by acting on neurons, as the hormone has been shown to be neurotrophic and neuroprotective.

Oligodendrocyte maturation and myelination, indeed, involves reciprocal communication with neurons, and neuronal signals including neurotrophins, electrical activity, and contact-mediated factors regulate myelin formation and myelin plasticity.^258,259 After cuprizone-induced demyelination, testosterone-dependent remyelination was, indeed, accompanied by an increase in the diameter of axons.¹⁸⁵ However, an increase in axon size may also reflect the trophic effects of recovered or regenerated oligodendrocytes on neurons.¹⁴⁸

The myelination of axons also involves interactions of oligodendrocytes and neurons with other neural cells, especially astrocytes and microglial cells.^260,261 Astrocytes have been shown to play a particularly important role in OPC proliferation, differentiation, and myelination.²⁶² They may, indeed, play a key role in the remyelinating actions of testosterone. Thus, after LPC-induced demyelination, testosterone favors astrocyte recruitment, which is a prerequisite for myelin regeneration by oligodendrocytes.³⁶

How astrocytes participate in the remyelinating actions of testosterone remains to be clarified. It is conceivable that an androgen-dependent neuronal signal may attract astrocytes to the demyelinating lesion or, alternatively, testosterone may directly act on astrocytes or oligodendrocytes.

Footnotes

Authors' Contributions

All authors actively contributed to the writing of this review and the accumulation of relevant information. M.S. also prepared the figures.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

Cite this article as: Schumacher M, Ghoumari A, Mattern C, Bougnères P, Traiffort E (2021) Testosterone and myelin regeneration in the central nervous system. Androgens: Clinical Research and Therapeutics 2.1, 231–251, DOI: 10.1089/andro.2021.0023.

Abbreviations Used

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Testosterone and Myelin Regeneration in the Central Nervous System

Abstract

Introduction

Testosterone Actions on the Brain

Sources of testosterone: the endocrine glands and local synthesis

Extra-cerebral sources: circulating testosterone

Brain synthesis of testosterone

Adrenal androgens as an important source of brain testosterone

The brain metabolism of testosterone: the aromatase and 5α-reductase pathways

The aromatase pathway

The 5α-reductase pathway

AR in the CNS

Testosterone and Demyelinating Diseases

Myelin and its plasticity

The regeneration of myelin

Beneficial effects of testosterone in experimental autoimmune encephalomyelitis and MS

Testosterone promotes myelin repair in the adult CNS

The neural AR as a target for the remyelinating effects of testosterone

Perspectives

Footnotes

Authors' Contributions

Author Disclosure Statement

Funding Information

Abbreviations Used

References