| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Unité Mixte de Recherche 788, Institut National de la Santé et de la Recherche Médicale, and University Paris-Sud 11, 94276 Kremlin-Bicêtre, France
Correspondence: Address all correspondence and requests for reprints to: Dr. Michael Schumacher, INSERM UMR 788, 80, rue du Général Leclerc, 94276 Kremlin-Bicêtre, France. E-mail: Michael.Schumacher{at}kb.inserm.fr
 |
Abstract |
|---|
|
I. Introduction |
|---|
Their outcome has been extensively commented upon in the recent literature and has brought many fundamental issues of hormone therapies to light, but discussions were mainly limited to the effects of estrogens (1, 2, 3, 4, 5). In fact, progestagens are generally only considered with respect to their usefulness in preventing uterine hyperplasia and malignancy in response to estrogens. Thus, in a recent review on the clinical effects of progestagens, it was stated that "the only indication for the addition of progestins to estrogen-replacement therapy is endometrial protection" (6). There is even a debate about the real usefulness of progestagens in protecting the endometrium, and the possibility of omitting them from HRT has been considered (7). However, this may underestimate the potential therapeutic promises of progestagens, and in particular those of natural progesterone. These have been particularly well documented for the nervous system, where progesterone itself and its metabolites regulate vital neuronal and glial functions and, like estrogens, exert neuroprotective and neurotrophic effects (8, 9, 10, 11, 12). On the contrary, the effects of the natural hormone and its metabolites on peripheral tissues, including blood vessels, bone, and even classical targets such as the mammary glands, are still a matter of controversy and need to be studied further.
There are many excellent recent reviews on the effects of estrogens on the brain and on cognitive functions and their potential usefulness in HRT, but the effects of progestagens are surprisingly underrepresented in the literature. Two recent papers had the merit of at least calling attention to the potential usefulness of progestagens for HRT and of reminding readers that menopause is characterized by the concomitant loss of estradiol and progesterone (13, 14). The major aim of the present review is to discuss the pleiotropic effects of progesterone and its metabolites in the nervous system and their implications for preventing or treating age-dependent changes and dysfunctions of the brain and peripheral nerves.
Before discussing the neurotrophic, neuroprotective, and promyelinating actions of progesterone, a succinct description will be provided of the major recent HRT trials that have stimulated so much debate and also created some confusion. The commonly used nomenclature for progestagens will then be clarified, stressing the differences between natural progesterone and its synthetic analogs. Promising neural targets of progesterone within the aging nervous system will be examined in detail, including neurons, glial cells, and the myelin sheaths, and the question of whether the aging nervous system remains sensitive to its actions will be discussed, as will the question of whether it is meaningful to attempt the treatment of age-related changes with progestagens. As a matter of fact, when addressing such an important and fundamental problem, it is necessary to also refer to the work on estrogens (15, 16, 17, 18, 19, 20, 21, 22, 23). Indeed, progesterone and estradiol often act in a concerted manner within target cells, and both steroids frequently exhibit similar properties. However, there are also opposing effects; whereas estrogens increase the excitability of neurons, progesterone and its reduced metabolites in general reduce their activity, an effect that may significantly contribute to their neuroprotective actions (24). The effects of progesterone in peripheral tissues will be examined before drawing attention to novel perspectives for the use of progestagens in HRT, resulting from the recent discoveries of their multiple signaling mechanisms and of their local synthesis by neurons and glial cells.
|
II. The Recent HRT Trials |
|---|
The WHI trial comprised two very large placebo-controlled arms: combined estrogen plus progestin and estrogen only. The combined estrogen plus progestin WHI trial involved more than 16,000 women with an intact uterus who received either placebo or CEE (0.625 mg/d) plus MPA (2.5 mg/d) (mean age, 63 yr). This arm of the WHI, designed to continue until 2005, was already terminated in 2002 because the overall risks from use of combined HRT outweighed the benefits: there was a slight increase in the risks of breast cancer and of cardiovascular complications, a significant increase in the levels of inflammatory biomarkers, and an increased risk of ischemic stroke (27, 28, 29, 30). Within this arm of the WHI trial, the effects of the combined HRT on cognitive functions were examined in a subgroup of 4532 women aged 65 yr or older. This so-called "Women’s Health Initiative Memory Study" (WHIM) found no improvement of cognitive functions and no protection against mild cognitive impairment. Instead, the study revealed a very small increase in the risk of cognitive decline and dementia, including Alzheimer’s disease (women with dementia: placebo = 22; CEE+MPA = 45 per 10,000 person-years) (31, 32). The explanation for the small increase in dementia is unknown, but it may result from vascular events (33).
The estrogen-alone arm of the WHI trial compared the effects of CEE alone (0.625 mg/d) vs. placebo in 10,739 postmenopausal women with prior hysterectomy. The use of CEE in the absence of MPA had no incidence on breast cancer or on coronary heart disease, but again an increased risk of cerebral stroke was observed (34). Thus, in all the trials, HRT was found to be associated with an increased risk of cerebral accidents. A recent retrospective analysis of 28 trials, involving a total number of 39,769 women, was consistent with this conclusion and revealed that among women who had a stroke, those taking HRT had a worse outcome (35). As with CEE+MPA, estrogen alone was also found to have adverse effects on cognition in a smaller recent study involving 2808 women aged 65 yr or older (36).
Subsequent to these trials, many medical organizations have recommended that HRT should not be used for the prevention of age-related diseases and, when used for treating acute climacteric symptoms, only at the lowest dose and for the shortest time. These recommendations have recently been renewed by the French Agency for Health Product Safety. Accordingly, estrogen alone or combined estrogen-progestagen HRT has been relegated to strictly short-term treatment of symptoms such as hot flushes at the beginning of the menopause (37, 38, 39, 40, 41).
|
III. Progesterone, Progestagens, and Progestins |
|---|
,5-tetrahydroprogesterone), which is a potent positive modulator of 
-aminobutyric acid (GABA) type A (GABAA) receptors and has been qualified as "neuroactive" (42). The multiple functions of allopregnanolone and its interactions with GABAA receptors will be discussed in detail later.
Progesterone is indeed unidirectionally converted by steroid 5-reductases to 5-dihydroprogesterone, which also activates gene transcription via the intracellular PR (Fig. 1
). These nicotinamide adenine dinucleotide phosphate (reduced form)-dependent enzymes convert a number of 
4–3-ketosteroids, including progestagens, glucocorticoids, mineralocorticoids, and androgens, into their 5-reduced metabolites. Two 5-reductase isozymes are encoded by distinct genes. The type 1 isoform is expressed throughout the rat brain at all stages of development, whereas the type 2 isoform shows a more restricted distribution: it is expressed in the brain almost exclusively around birth, and it is present in the adult spinal cord mainly within gray matter (43, 44).
|
-dihydroprogesterone is catalyzed by two types of enzymes: the cytosolic nicotinamide adenine dinucleotide phosphate-dependent aldo-keto reductases (ARKs) and a subgroup of the membrane-bound nicotinamide adenine dinucleotide-dependent short-chain dehydrogenases/reductases (SDRs), the so-called retinol/sterol dehydrogenase (RODH)-like group of SDRs (45, 46) (Fig. 1
). The four ARK1C1-ARK1C4 isoforms are frequently designated as hydroxysteroid dehydrogenases (HSDs), but also as hydroxysteroid oxidoreductases to insist on the supposedly bidirectional character of the enzyme reactions. However, although bidirectional in vitro, the ARKs may only function in the reductive direction in living cells, and they may thus be mainly responsible for the reduction of 3-ketosteroids to 3-hydroxysteroids, and more specifically of 5-dihydroprogesterone to allopregnanolone (47). On the other hand, the oxidation of 3-hydroxysteroids to 3-ketosteroids, and more specifically of allopregnanolone to 5-dihydroprogesterone, is thought to be catalyzed by the RODH-like SDRs (46). In humans, the RODH-like SDRs comprise four enzymes with 3-HSD activity. Interestingly, two of them also exhibit 3
3ß-hydrosteroid epimerase activity, as shown both in vitro and in living cells, and they may thus play a crucial role in the control of the local concentrations of biologically active allopregnanolone (48, 49). Indeed, as described in detail in Section X.B, some of the neuromodulatory and protective effects of progesterone are mediated by allopregnanolone, a very potent modulator of GABAA receptor activity. On the contrary, the 3ß-epimer of allopregnanolone, iso-allopregnanolone (3ß,5-tetrahydroprogesterone), is not only inactive at GABAA receptors but is also known to antagonize the effects of allopregnanolone (50, 51, 52).
The term "progestin" is not used in a consistent manner. It designates both natural and synthetic progestational molecules, including natural progesterone, or exclusively synthetic ones. In the present review, the term progestin will only be used to designate synthetically produced progestagens, including both C19 testosterone derivatives (19-nortestosterone derivatives) and progesterone derivatives (17-hydroxyprogesterone derivatives and 19-norprogesterone derivatives) (Fig. 2 and Table 1). The pleonasms "natural progesterone" and "synthetic progestins" will be sometimes used to insist on the difference. The 19-norprogesterone derivatives, such as 19-norprogesterone, promegestone (R5020), and nomegestrol acetate, are among the most selective agonists of the PR, and they are sometimes referred to as "pure" progestagens because as they do not in principle possess androgenic, estrogenic, or glucocorticoid activities (53, 54, 55). However, the other progestins bind to several steroid receptors and sometimes exhibit a wide range of nonprogestagenic biological effects. Thus, the 17-hydroxyprogesterone derivative MPA, the most commonly prescribed replacement progestin in the United States and the one used in the recent large HRT trials, also has androgenic and glucocorticoid properties (56). The synthetic 19-nortestosterone-derived progestins, such as norethisterone acetate, a progestin commonly used in Europe, retain varying degrees of androgenic activity despite the removal of carbon 19 (57, 58).
|
|
-, 3,5- and 3ß,5-reduced metabolites (59, 60). Interestingly, whereas the 5-reduction significantly increases the androgenic potency of testosterone, the 5-reduction of norethisterone results in a significant diminution of androgenicity (61). The 3ß,5-reduced metabolites of norethisterone, levonorgestrel, and gestodene bind to ER, although with a lower affinity than estradiol, and activate gene transcription via this receptor (60, 62, 63). Whether A-ring-reduced metabolites of progestins act on GABAA receptors needs to be clarified. Norethisterone acetate and MPA were shown to produce some anxiolytic-like effects when rats were tested in the "elevated plus maze" and the "shock-probe burying test". In contrast, the norprogesterone derivative trimegestone only had little effect (64). However, these behavioral effects of the progestin metabolites do not necessarily result from the direct modulation of GABAA receptors. Indeed, some observations suggest that the administration of progestins affects the concentrations of endogenous allopregnanolone in the brain and influences the activity of enzymes involved in the metabolism of progestagens (65, 66). For example, MPA (Provera), which does not directly act on GABAA receptors, enhances GABAA receptor-mediated inhibitory neurotransmission in the rat hippocampus by inhibiting the metabolism of allopregnanolone (67). Another recent study has shown that 2-wk oral treatment with MPA increases allopregnanolone levels within the hippocampus, cerebral cortex, and hypothalamus of ovariectomized female rats (68). The effect of MPA on the endogenous levels of brain allopregnanolone may explain why the progestin is therapeutically beneficial for catamenial epilepsy (69) and sometimes improves anxiety and mood in postmenopausal women (70, 71).
Only a few progestins have been tested for their effects on the nervous system, but concerns are particularly serious about the negative effects of MPA. Thus, MPA has been shown to antagonize the neuroprotective and promnesic effects of estrogen. Whereas progesterone and 19-norprogesterone, alone or in combination with estradiol, protected cultured hippocampal neurons against glutamate toxicity, MPA not only failed to be effective but also attenuated the estrogen-induced neuroprotection. At the molecular levels, MPA blocked estrogen-induced expression of the antiapoptotic protein Bcl-2 and antagonized estradiol-induced attenuation of the glutamate-induced rise in intracellular calcium (72, 73). Thus, one of the most prescribed progestins for HRT and contraception opposes some of the beneficial effects of estradiol in the brain and may even exacerbate the excitotoxic death of neurons (74). In vivo, MPA has recently been reported to diminish the ability of CEE to reduce stroke damage in subcortical regions of the rat brain (75). In female monkeys, treatment with MPA reduced the increase in sexual initiation induced by estradiol treatment and increased aggressive behavior, which may represent a serious behavioral side effect (76). MPA has also been shown to directly inhibit the activity of steroidogenic enzymes, in particular of the human type II 3ß-HSD, an enzyme that converts pregnenolone (PREG) to progesterone, and the progestin thus interferes with steroid biosynthetic pathways (77).
It is important to draw attention to differences in HRT regimens between countries. In the United States, the most commonly used progestin is MPA, generally combined with CEE, an association of more than 10 different estrogens. Most of them are sulfated and distinct from the predominant endogenous estrogens in women, that is, estradiol before and estrone after menopause (78). Nevertheless, estrogen components of CEE have recently been shown to have potent antioxidant and neuroprotective effects and also to reduce the cortical infarction volume in a rodent model of stroke (75, 79, 80, 81). In the United Kingdom, the progestins mainly used are 19-nortestosterone derivatives (norethisterone acetate, norgestrel, and levonorgestrel). In central and southern Europe, both 19-nortestosterone derivatives and a range of progesterone derivatives are used. In France, micronized progesterone and 19-norprogesterone derivatives are commonly prescribed in combination with oral or transdermal estradiol (82, 83, 84). It would certainly be worthwhile to attempt retrospective comparisons of the different HRT formulations.
Oral micronized progesterone has been widely used in Europe, and in particular in France, since 1980. Micronized progesterone is natural progesterone, whose average particle size has been reduced, leading to decreased destruction in the gastrointestinal tract, a longer half-life, and enhanced bioavailability. Before, the discovery of the micronization process, progesterone could not be taken orally because it is poorly absorbed and rapidly metabolized. The use of micronized progesterone is well tolerated, with mild and transient sedation as a side effect that can be minimized by taking the hormone at bedtime (85). Moreover, the elevation of circulating levels of progesterone by oral administration of the micronized hormone has been shown to be as effective as the administration of progestins for the control of endometrial growth (86, 87). Earlier studies have also reported that micronized progesterone may improve mood in patients with premenstrual mood disturbances and in postmenopausal women (88, 89). When compared with the MPA-containing regimen, micronized progesterone was found to improve significantly vasomotor symptoms, somatic complaints, anxiety, and depressive symptoms in postmenopausal women (90). However, work by Bäckström and collaborators (91, 92) has shown that treatment with progesterone can also result in adverse mood changes (tension, irritability, depression), and that the metabolite allopregnanolone may be the mediator of these effects. Thus, in two studies of postmenopausal women with climacteric symptoms, negative mood effects during treatment with vaginal progesterone implants were related to the blood concentrations of allopregnanolone. During the progesterone treatment period, women had increased negative mood symptoms when compared with the estradiol-only period, but only when serum concentrations of allopregnanolone were increased to those seen during the midluteal phase of the menstrual cycle, not when they were either higher or lower (91, 92). These observations suggest a bimodal association between allopregnanolone and adverse mood, and they point to the importance of a well-dosed HRT.
|
IV. Trophic and Protective Effects of Progesterone in the Nervous System |
|---|
Beneficial effects of progesterone have also been demonstrated in experimental models of traumatic brain injury (TBI). A much-studied system corresponds to bilateral contusion lesion of the rat medial prefrontal cortex, which produces cognitive deficits typically observed after human frontal lobe injury (99, 100). The medial prefrontal cortex receives glutamatergic and cholinergic afferents, respectively, from the mediodorsal thalamic nucleus (MDN) and from the nucleus basalis magnocellularis (NBM). TBI leads to edema, to secondary excitotoxic neuronal death in the vicinity of the lesion, and subsequently to retrograde neuronal degeneration in both MDN and NBM (101). Edema is a very important negative factor for the outcome of TBI. Therefore, the observation that progesterone treatment reduced both edema and secondary neuronal losses and improved behavioral recovery after TBI in male rats was particularly encouraging. Females are protected by their high endogenous levels of progesterone, and their brains have much less water content after TBI when compared with males (102, 103). Following these important observations, a phase II, randomized, double-blind, placebo-controlled trial, named "ProTECT", has been conducted in Atlanta to test the usefulness of progesterone as a treatment for moderate to severe TBI. In this study, which included 100 trauma patients, stable progesterone levels were rapidly achieved after TBI by its iv infusion (104). The very promising outcome of the trial has now been published. Progesterone-treated patients had a lower 30-d mortality rate than controls, and survivors of moderate TBI who received progesterone had better outcomes. However, the administration of progesterone had no effect on the disability of severe TBI survivors. It is important to note that no adverse events could be attributed to progesterone in this trial (105).
What makes progesterone a particularly attractive neuroprotective agent for the treatment of brain lesions is its surprisingly large therapeutic window. Even when administered as late as 2 h after the onset of MCAO, progesterone still provided therapeutic benefit (106), and the steroid was effective in reducing edema and in protecting neurons after TBI when treatment was delayed as much as 24 h after injury (107). Pretreating ovariectomized female rats with low physiological concentrations of progesterone also allowed hippocampal neuron loss in response to TBI to be reduced (108). With respect to the duration of the progesterone treatment and its mode of administration, available experimental data show that both prolonged and continuous administration of the hormone leads to more complete behavioral recovery after TBI (109, 110).
An important finding was that administration of the enantiomer of progesterone (ent-progesterone) also decreased cerebral edema, neuron death, inflammatory cytokines, and reactive gliosis (111). Enantiomers of steroids indeed have a therapeutic potential for treating lesions and age-dependent dysfunctions of the nervous system (112, 113). An enantiomer is a mirror-symmetric, non-superimposable image of a molecule, with identical physical properties, except for the different rotation of polarized light, but with different biological actions (114). The protective effects of ent-progesterone were not mediated by the intracellular PR because the compound did not activate PR-mediated gene transcription, and its mechanisms of action, which may involve membrane receptors, need to be clarified. A previous study had shown that ent-progesterone is a potent competitive inhibitor of human enzymes involved in steroid metabolism, namely, the cytochromes P450c17 and P450c21 (115).
Neuroprotective effects of progesterone have also been demonstrated for midbrain dopaminergic neurons. Both progesterone and estradiol were found to protect dopaminergic neurons against degeneration induced by 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine, a finding consistent with a possible role of these hormones in the deterioration of dopaminergic functions with age and in the development of Parkinson’s disease (116). There is indeed a higher incidence of Parkinson’s disease in men when compared with women, and the risk of its occurrence is increased in women with an early onset of menopause (117).
In the spinal cord of male rats, chronic treatment with progesterone for 5 d reduced the size of the lesion and prevented secondary neuronal loss after contusion injury (118). Progesterone is indeed an important neuroprotective factor for spinal motoneurons, as has been shown after spinal cord lesion and in a genetic model of motoneuron disease, the Wobbler mouse (11, 119, 120). After spinal cord transection, progesterone treatment preserved the Nissl bodies of the ventral horn motoneurons, restored choline acetyltransferase levels, normalized the expression of the Na,K-ATPase, and increased growth-associated protein of 43 kDa (GAP-43) and brain-derived neurotrophic factor (BDNF) message and protein (121, 122).
The Wobbler mouse is a particularly useful model for the study of motoneuron diseases, including amyotropic lateral sclerosis. The Wobbler phenotype is due to a missense mutation in the gene encoding the vacuolar-vesicular protein sorting factor Vps54 (123). The first manifestations of the disease are already observed at 2–3 wk of age (124, 125). When 2-month-old, symptomatic Wobbler mice presenting tremor, ambulatory difficulty, and diminished muscle strength received sc implants of progesterone that produced constant high physiological levels of the hormone for only 2 wk, the neuropathological changes of spinal motoneurons were less severe, motoneuron vacuolation was reduced, and there was better preservation of the endoplasmic reticulum and of the mitochondria. Most importantly, progesterone treatment also had beneficial effects on muscle strength of the animals (126, 127, 128). The link between the mutant Vps54 and the severe impairment of Wobbler motoneurons needs to be explored, but it is likely that the mutation results in altered axonal transport. It was thus a significant finding that retrograde axonal transport is indeed impaired in Wobbler motoneurons, as shown by the injection of fluorogold into the limb muscles and its retrograde tracing, and that it can be restored by treating the animals for 8 wk with progesterone (119).
Only a few experimental studies have reported an absence of effect or negative actions of progesterone in the injured nervous system (attention has already been drawn in Section III to the disruptive effects of progestins such as MPA). Thus, one study did not find a beneficial effect of progesterone after ischemic insult in senescent female rats (129), and two dose-response studies have raised concerns about the possibility that high doses of progesterone (30–60 mg/d) may exacerbate the outcome of MCAO in ovariectomized female rats or of TBI in male rats (130, 131). The influence of sex hormones may also be dependent on the type and the severity of a brain lesion. Thus, sex differences in the outcome of TBI favoring female rats have been consistently observed after diffuse weight-drop-induced TBI, but these results could not be confirmed after more severe focal impact injury, qualified as "focal TBI" and characterized by a very fast evolution of neurodegeneration (132, 133). One study has reported that progesterone inhibited the neuroprotective effects of estradiol in the rat hippocampus observed after systemic kainate administration, which induces excitotoxic neuron death (134). Although very sparse, these negative results indicate that one should remain cautious and demonstrate the need for more research on the modes of action of progesterone and of its metabolites in the nervous system.
The mechanisms by which progesterone promotes morphological and functional recovery after brain injury are indeed not well understood, and they seem to involve multiple actions, some of which may be particularly relevant to the potential role of progesterone in the aging nervous system. In this regard, the above-mentioned observation that progesterone may improve impaired axonal transport is particularly interesting. There is indeed strong evidence that the slowing of axonal transport may be a key event in the aging process of the brain and of peripheral nerves (135, 136, 137, 138). Reduced axonal transport has also been proposed to play an early and causative role in the development of Alzheimer’s disease. That is, deficits in axonal transport are characteristic of the early stages of the disease in mouse models and in patients, and they may lead to aberrant amyloid-ß peptide formation and subsequently to neurodegeneration (139).
Another important consequence of progesterone treatment is the reduction of lipid peroxidation, but the underlying mechanisms still need to be specified (140). The peroxidation of lipids is indeed a complex phenomenon, involving distinct enzymatic pathways as well as nonenzymatic mechanisms, such as free radical mediated peroxidation, and it is reduced by the actions of different antioxidant enzymes (141). During aging, there is an increase in the concentration of lipid peroxidation products, and the oxidative damage of lipids may play an important role in mediating or even initiating specific aspects of age-dependent changes (142). In addition, the oxidation of neuronal lipids resulting from an oxidative imbalance has been proposed to play a significant role in Alzheimer pathogenesis and, consequently, may represent an interesting therapeutic target at early stages of the disease (143). The increased exposure of aging tissues to oxidative stress partly results from decreased activity of antioxidant enzymes such as the superoxide dismutases. The prolonged treatment of middle-aged and old acyclic female rats (12, 18, and 24 months of age) with low doses of progesterone, estradiol, or a combination of both steroids increased superoxide dismutase activity and reduced lipid peroxidation (144, 145).
The age-dependent accumulation of oxidative damage is also a consequence of a decline in mitochondrial function, another major component of the normal aging process and of neurodegeneration (146, 147, 148, 149, 150). Many of the reactive oxygen species involved in oxidative stress are indeed toxic byproducts of the mitochondrial energy production pathway, and they damage not only lipids, but also proteins and nucleic acids. Over the past few years, attention has indeed focused on the role of the mitochondria in brain aging and neurodegeneration (151, 152, 153), and this cellular organelle is another important target for the actions of progesterone. One of the prevalent neuropathological changes found in spinal motoneurons of Wobbler mice is damaged mitochondria with severe vacuolation, and treatment with progesterone allowed the restoration of a normal appearance of the mitochondria (120). Progesterone can protect neurons against apoptotic cell death by increasing the expression of antiapoptotic proteins residing in the outer mitochondrial membrane, such as Bcl-2, and by down-regulating proapoptotic gene expression (bax and bad) and the caspase-3 enzyme (73, 154, 155). The expression of Bcl-2-family proteins is regulated by nuclear steroid actions, and the newly synthesized proteins are translocated to the outer mitochondrial membrane (156).
Some studies have suggested direct actions of steroids on mitochondria. Thus, estrogen and glucocorticoid receptors have been detected in mitochondria, and the mitochondrial genome contains nucleotide sequences with high similarity to known steroid-responsive elements (157, 158, 159, 160). It has also been proposed that estradiol may protect against the formation of mitochondrial reactive oxygen species by directly acting on mitochondrial estrogen receptors (ERs) (161). Whether progesterone may also exert direct effects on mitochondria remains to be explored. In the low physiological range, progesterone has been shown to completely reverse postinjury alterations in mitochondrial respiration (108). The mitochondria is also a target for other steroids, such as dehydroepiandrosterone (DHEA), and it is the site where the first step in steroid hormone biosynthesis takes place, the conversion of cholesterol to PREG by the cytochrome P450scc, as shall be discussed later. There obviously exist complex relationships between steroids, mitochondrial activity, oxidative stress, the aging of brain cells, and neurodegenerative events. Unfortunately, the available data are still much too fragmentary for an integrated picture.
Progesterone exerts many other actions, which can be related to its neuroprotective effects and may also have implications for the aging brain. Thus, progesterone regulates the expression of aquaporin 4 in the injured brain, a membrane-channel protein involved in water homeostasis, which is largely distributed throughout the brain and may play a significant role during edema formation (162). It is puzzling that some of the actions of progesterone resemble those of the estrogens. For example, like estradiol, progesterone up-regulates the expression of antiapoptotic proteins such as Bcl-2 (163, 164, 165), reduces inflammation by repressing the activation of microglial cells and by inhibiting the production of proinflammatory cytokines (101, 166, 167, 168), up-regulates the expression of neurotrophins such as BDNF (122, 169, 170), and protects neurons against glucose deprivation and the toxicity of glutamate, FeSO4, and ß-amyloid peptides (72, 171, 172).
B. Promyelinating effects
Progesterone is also known to have a role in myelination and remyelination. By preserving or restoring the integrity of myelin sheaths, which insulate the large axons and are required for the efficient and rapid conduction of electrical impulses along nerve fibers, progesterone may not only play an important role in the efficient communication between neurons but also promote their viability. In fact, myelin has neuroprotective functions, and myelin-associated proteins influence the caliber of axons (173). Moreover, after injury, secondary demyelination of spared axons contributes to neuronal degeneration, the extension of the lesion, and functional deterioration (174).
A role of progesterone in myelination was first demonstrated in the regenerating mouse sciatic nerve after lesion as well as in explant cultures of rat dorsal root ganglia composed of sensory neurons and Schwann cells, which are the myelinating glial cells of the PNS (175). Progesterone also enhanced the rate of myelin formation in dissociated cocultures of neurons and Schwann cells (176). Whether progesterone promotes myelination of peripheral nerves directly by acting on Schwann cells, or indirectly by acting on neurons, needs to be clarified because there are contradictory reports. Whole-cell radioligand binding assays suggested the presence of specific and saturable progesterone binding sites in Schwann cells (177). The PR has also been detected in Schwann cells either grown in culture or within the rat sciatic nerve by immunocytochemistry (178). On the contrary, in cocultures of dorsal root ganglia neurons and Schwann cells, the presence of PR mRNA and protein was only detected in the neurons, not in the Schwann cells (179). Also, in a recent study, purified rat Schwann cells and various Schwann cell lines were found to only express extremely low amounts of PR mRNA (180). In the mouse Schwann cell line MSC80, progesterone did not activate the transcription of a progesterone-sensitive reporter gene. Demonstration that the PR was the limiting factor was provided by the positive transcriptional responses obtained when exogenous receptors were transiently expressed (180). Whether present in neurons or glial cells, the PR in the peripheral nervous system is a potential pharmacological target for the therapy of inherited and acquired peripheral neuropathies. In a transgenic rat model of Charcot-Marie-Tooth disease overexpressing the peripheral myelin protein PMP22, the culprit of the disease, treatment with a progesterone antagonist reduced PMP22 expression and had a beneficial influence on the evolution of the disease (181). In a model of diabetic neuropathy, induced in rats by an injection of streptozotocin, prolonged treatment with progesterone or its reduced metabolites had beneficial effects on peripheral nerves at the neurophysiological, functional, and neuropathological levels (182).
In the CNS, brain, and spinal cord, axons are myelinated by oligodendrocytes (183, 184). That progesterone also promotes myelination by oligodendrocytes has been demonstrated in explant cultures of cerebellar slices taken from 7-d-old rats and mice (185). These organotypic cultures closely reproduce developmental events and provide a unique model for examining neuronal survival and maturation, as well as the myelination of axons (186, 187). In these explants, myelination is very intense during the second postnatal week, exactly at a time when endogenous levels of progesterone are elevated in the cerebellum (188, 189). A stimulatory effect of progesterone on myelination was observed in cerebellar slices of both sexes and involved the classical PR: 1) it could be mimicked by the selective progestin promegestone; 2) it was completely abolished by the PR antagonist mifepristone (RU486); and 3) it was not observed in cerebellar slice cultures from 7-d-old PR knockout mice (185). In these slices, progesterone was shown to stimulate the proliferation and maturation of oligodendrocyte progenitor cells (190). An earlier study had already shown that adding progesterone to cultures of glial cells isolated from neonatal rat brains increased the number of oligodendrocytes (191). More recently, the addition of progesterone to the medium of cultured oligodendrocytes has been shown to increase their branching, whereas estradiol stimulated myelin membrane formation (192). Progesterone also promotes remyelination by oligodendrocytes in vivo. After toxin-induced demyelination, the systematic administration of progesterone promoted the slow endogenous remyelination of axons within the cerebellar peduncle of aging male rats (193). In the lesioned rat spinal cord, treatment with progesterone was found to increase the density of NG2+ oligodendrocyte progenitor cells and the expression of myelin basic protein (194).
The promyelinating effects of progesterone are particularly relevant when discussing the significance of the hormone in the aging nervous system. Indeed, it is less well appreciated that overall loss of myelin and altered integrity of myelin sheaths are among the most reliable markers of the aging nervous system, correlating with chronological age and cognitive decline (195, 196). Age-dependent changes in brain myelin, which have been extensively studied in rhesus monkeys, include alterations in oligodendrocytes, abnormalities and breakdown of the myelin sheaths, and loss of white matter (195, 197, 198, 199). Based on these observations, it has been proposed that myelin changes may significantly contribute to age-related cognitive decline by altering conduction velocities along axons (196). A postmortem study has provided evidence that aging in humans is also accompanied by the loss of myelinated fibers (200). By using the method of diffusion tensor magnetic resonance imaging (MRI), it has been shown that myelin disruption occurs in men even during normal aging. Most importantly, alterations of cortical myelin correlated with declined cognitive ability (201). In parallel with these morphological and functional studies, only a few reports have dealt with biochemical changes in myelin during aging. Increase in water and decrease in cholesterol within white matter have been described (202), and decreased expression of the major peripheral myelin proteins has also been reported in peripheral nerves of aged rodents and humans (203, 204, 205, 206).
Importantly, remyelination continues to take place in the brains of aged monkeys, but the newly formed myelin sheaths are thin and have short internodes (207). Age is indeed a negative factor for the capacity to regenerate myelin sheaths, as has been demonstrated in the rodent CNS after demyelination induced by a gliotoxin; in old rats, the process of remyelination takes much longer than in young animals (208, 209, 210). The reasons for the age-associated slowing down of myelin repair are not well understood, but impaired recruitment of progenitor cells and their delayed differentiation into myelinating oligodendrocytes, as well as delayed expression of growth factors, may be responsible (211, 212). In humans, differences in the speed of remyelination could explain the much slower functional recovery in older patients after demyelinating diseases such as optic neuritis (213). In addition, a reduced capacity for myelin repair with age is consistent with the observation that the prognosis of multiple sclerosis is mainly age-dependent (214).
In conclusion, a substantial number of animal studies have documented neuroprotective effects of progesterone or its reduced metabolites in the lesioned or diseased nervous system of young adult rodents. Particularly promising for the treatment of traumatic lesions is the large therapeutic window of progesterone. Progesterone may exert neuroprotective effects and promote neuroregeneration by a dual action: by directly acting on neurons and increasing their survival, and by accelerating the formation of new myelin sheaths. Progesterone and its metabolites may exert similar beneficial effects in the aging brain and peripheral nerves. The significance of progesterone in aging will be further explored when discussing the question of whether the aging nervous system remains sensitive to the beneficial effects of steroids.
|
V. Neurons and Glial Cells in the Aging Nervous System |
|---|
However, brain structures may differ in the involvement of neuron loss, and some populations of neurons may be more affected by the aging process than others. For example, within subregions of the rat hippocampal formation, the number of neurons may significantly decrease at advanced ages, in particular within the subiculum and the hilus of the dentate gyrus (225). Among the most vulnerable cells of the nervous system are the cerebellar Purkinje cells, and there is consistent evidence for their significant loss during normal aging in rodents and humans. The age-dependent loss of Purkinje cells correlates with decreased eye-blink conditioning, a reflex pathway mediated by these neurons, and elderly people with very slow eye-blink conditioning may have an increased risk of becoming demented (226, 227).
B. Aging glial cells
Neurons have long been the main focus of studies on brain aging, and glial changes have been largely neglected. As already point out, there are significant deteriorations of the myelin sheaths with age, which may reflect age-dependent changes in the myelinating glial cells, oligodendrocytes in the CNS and Schwann cells in the PNS. However, the formation and maintenance of myelin sheaths are also dependent on neuronal signals, and there are complex reciprocal interactions between axons and myelinating glial cells in both compartments of the nervous system (228, 229, 230, 231). Consequently, any age-dependent alterations of myelin sheaths may result from impaired neuronal functions, from changes in the myelinating glial cells themselves, or from both events.
Another type of glial cell also plays an essential role in brain aging. The general assumption was that the increased number of astrocytes (astrogliosis) during aging may be a consequence of neuron degeneration. However, there is now strong experimental evidence provided by Finch and collaborators (232) that changes in astrocytes are in fact a very early event in the aging process, and that increased glial fibrillary acidic protein (GFAP) expression by astrocytes may contribute to decreased synaptic functioning and plasticity in the aged brain. Indeed, in cocultures of neurons and old astrocytes, diminishing GFAP levels by RNA interference restored neurite outgrowth, whereas overexpression of GFAP in young astrocytes modeled the effects of aging by reducing neurite outgrowth (233). Consistent with these in vitro findings is the observation that inactivation of the GFAP gene in mice improves both neuronal survival and neurite growth (234, 235).
Important for the present discussion is the observation that astrocytes can mediate some of the effects of progesterone and estradiol on neuronal plasticity, and that steroids are a critical component of the cross-talk between neurons and glial cells (236, 237). Thus, enhanced neuronal sprouting after lesion in response to estradiol is mediated in part by the repression of GFAP expression in astrocytes (238). Astrocytes are also a target for the actions of progesterone; after a penetrating brain injury, treatment with progesterone decreased astrocyte accumulation in both female and male rats (239, 240). Progesterone was also shown to reduce astrocytic hypertrophy after TBI close to the lesion site (154). However, in two other models, progesterone was not found to modify astrocyte accumulation in rats, either after spinal cord transection or after medial frontal cortex contusion (241, 242).
|
VI. Gender Differences and Sensitivity to Progesterone |
|---|
Only a few studies have investigated the possible influence of gender on the response of the adult nervous system to the trophic and protective effects of ovarian steroids, and there are surprisingly few observations of sex differences. A recent study has revealed that the effects of progesterone and its 5-reduced metabolites on the expression of peripheral myelin protein genes differs between males and females. This was shown by using sex-specific cultures of Schwann cells prepared from neonatal rats (259). On the other hand, the differential sensitivity of the male and female rodent brain to injury appears to be largely determined by the presence of different levels of progesterone. Thus, the more favorable outcome after cerebral stroke or TBI in female rats when compared with males mainly results from the presence of high endogenous levels of progesterone in the females. Similarly, treatment with progesterone provides similar neuroprotection in males and in females after TBI (102, 260).
It is worth mentioning here that a few studies have reported that some brain responses to estrogen are sexually dimorphic. In one study, estradiol was found to improve neurological outcome after TBI in male rats, but to exacerbate brain injury in females (261). In another study, although estrogen therapy protected both male and female brains against ischemic insult, the responses differed between sexes: acute exposure to estrogen was sufficient to ameliorate ischemic brain injury in males, whereas females required longer-term replacement (262). As will be discussed later, the response of aged hippocampal synapses to estrogen is also sexually dimorphic (263).
Very few studies have addressed the question of the effects of gender on the outcome of injury in the human nervous system, and in TBI patients the role of gender is still controversial. One clinical study has reported that female TBI patients have a better outcome than male patients (264). More recently, another group has shown that female patients have lower levels of cerebrospinal fluid (CSF) lipid peroxidation and oxidative damage products, consistent with the already discussed antioxidant properties of ovarian hormones (265, 266). However, other studies have not shown a beneficial effect of female gender on TBI outcome (267, 268).
Gender is also an influential factor in the incidence and progression of multiple sclerosis, a demyelinating disease that selectively affects the brain and spinal cord (214, 269, 270, 271). The questions of why more women have multiple sclerosis than men and why it affects women differently from men have been mainly addressed experimentally by examining hormonal influences on autoimmune responses (269, 272, 273). However, the recent observation showing that myelin is sexually dimorphic casts a new light on the role of gender and hormones in the maintenance, alterations, and diseases of myelin (274). In this study, gender differences in myelin components of white matter tracts of young and aged rodents were found to be so dramatic that it was possible to determine the sex of an animal from blind sections immunostained for oligodendrocyte-specific markers. Gonad-derived steroids appeared to be a major contributor to these sex differences because castration of adult males produced a female phenotype (274). Consistent with clinical observations showing that the course of multiple sclerosis is mainly age-dependent, and that women reach disability milestones at older ages than males (214, 275), the extent of oligodendrocyte remyelination after a demyelinating lesion was found to be significantly reduced in aging rats, and middle-aged males and females (12 months of age) differed in their capacity to remyelinate axons. This sex difference was not influenced by castration, suggesting a more stable sex difference (210).
|
VII. The Sensitivity of the Aging Nervous System to Progesterone and Estradiol |
|---|
A. Maintained sensitivity to ovarian steroids
1. Progesterone.
Beneficial effects of progesterone on the aging nervous system have been particularly well demonstrated for myelinated nerve fibers. In Section IV.B, progesterone plays an important role in peripheral nerve myelination, and recent studies have shown that treatment with progesterone or its 5-reduced metabolites allows reversal of age-related myelin abnormalities. Thus, in the sciatic nerves of aged male rats, a significant decrease in myelin-associated activity of the 5-reductase, the enzyme that converts progesterone to 5-dihydroprogesterone (or testosterone to 5-dihydrotestosterone), is associated with a reduction in myelin gene expression. Treatment of the aged rats for 1 month with progesterone, 5-dihydroprogesterone, or allopregnanolone allowed reversal of the age-dependent decline in peripheral myelin protein expression, whereas repeated injections of androgens were without effect (204, 205). The administration of progesterone not only counteracted the drop in myelin protein expression, it also allowed reversal of age-related structural abnormalities of the peripheral myelin sheaths. Indeed, the prolonged treatment of old male rats (22–24 months) with progesterone or its 5-reduced metabolites significantly decreased the percentage of fibers with myelin abnormalities as well as the number of fibers with irregular shapes, and it increased the number of small myelinated fibers. Again, as previously observed for the normalization of myelin gene expression, the effects were specific for progesterone and its metabolites because the administration of androgens was inefficient (276).
As already mentioned, the capacity to repair myelin in the brain decreases with age; spontaneous remyelination after gliotoxin-induced demyelination is very rapid in the brain of young rats, but it is very much delayed in middle-aged rats. Whereas no beneficial effect of progesterone on central myelin repair could be observed in young males (10 wk old), because spontaneous remyelination was too rapid, the implantation of sc progesterone pellets stimulated a slow remyelination of axons in middle-aged animals (9 months old) (193, 277).
It has been proposed that the disappearance of the protection against ischemic brain injury in females after reproductive senescence may be a consequence of ovarian hormone deficiency. However, aging female rats remain responsive to the protective actions of ovarian hormones, at least until a certain age. Thus, in middle-aged female rats (16 months), the administration of either progesterone or estradiol alleviated cerebral stroke (278).
Animal models also support the axiolytic effects of progesterone, which are mediated by its conversion allopregnanolone, a potent positive modulator of GABAA receptors (see Section X.B) (279, 280, 281). In fact, the anxiolytic actions of progesterone do not require the intracellular PR because as they are still observed in PR knockout mice, which even exhibit a greater anxiolytic response than their wild-type littermates (282). However, progesterone does not enhance anxiolytic behavior in mice deficient of the type 5-reductase (283). Most importantly, middle-aged (between 9 and 12 months of age) and old (between 18 and 24 months of age) wild-type and PR knockout mice continue to respond to the anxiety-reducing effects of progesterone (284). That the brain
Top
Abstract
I. Introduction
