R.C. Melcangi

Steroid hormones and neurosteroids in normal and pathological aging of the nervous system

Without medical progress, dementing diseases such as Alzheimers disease will become one of the main causes of disability. Preventing or delaying them has thus become a real challenge for biomedical research. Steroids offer interesting therapeutical opportunities for promoting successful aging because of their pleiotropic effects in the nervous system: they regulate main neurotransmitter systems, promote the viability of neurons, play an important role in myelination and influence cognitive processes, in particular learning and memory. Preclinical research has provided evidence that the normally aging nervous system maintains some capacity for regeneration and that age-dependent changes in the nervous system and cognitive dysfunctions can be reversed to some extent by the administration of steroids. The aging nervous system also remains sensitive to the neuroprotective effects of steroids. In contrast to the large number of studies documenting beneficial effects of steroids on the nervous system in young and aged animals, the results from hormone replacement studies in the elderly are so far not conclusive. There is also little information concerning changes of steroid levels in the aging human brain. As steroids present in nervous tissues originate from the endocrine glands (steroid hormones) and from local synthesis (neurosteroids), changes in blood levels of steroids with age do not necessarily reflect changes in their brain levels. There is indeed strong evidence that neurosteroids are also synthesized in human brain and peripheral nerves. The development of a very sensitive and precise method for the analysis of steroids by gas chromatography/mass spectrometry (GC/MS) offers new possibilities for the study of neurosteroids. The concentrations of a range of neurosteroids have recently been measured in various brain regions of aged Alzheimers disease patients and aged non-demented controls by GC/MS, providing reference values. In Alzheimers patients, there was a general trend toward lower levels of neurosteroids in different brain regions, and neurosteroid levels were negatively correlated with two biochemical markers of Alzheimers disease, the phosphorylated tau protein and the beta-amyloid peptides. The metabolism of dehydroepiandrosterone has also been analyzed for the first time in the aging brain from Alzheimer patients and non-demented controls. The conversion of dehydroepiandrosterone to Delta5-androstene-3beta,17beta-diol and to 7alpha-OH-dehydroepiandrosterone occurred in frontal cortex, hippocampus, amygdala, cerebellum and striatum of both Alzheimers patients and controls. The formation of these metabolites within distinct brain regions negatively correlated with the density of beta-amyloid deposits.

Progesterone and its derivatives are neuroprotective agents in experimental diabetic neuropathy: A multimodal analysis

One important complication of diabetes is damage to the peripheral nervous system. However, in spite of the number of studies on human and experimental diabetic neuropathy, the current therapeutic arsenal is meagre. Consequently, the search for substances to protect the nervous system from the degenerative effects of diabetes has high priority in biomedical research. Neuroactive steroids might be interesting since they have been recently identified as promising neuroprotective agents in several models of neurodegeneration. We have assessed whether chronic treatment with progesterone (P), dihydroprogesterone (DHP) or tetrahydroprogesterone (THP) had neuroprotective effects against streptozotocin (STZ)-induced diabetic neuropathy at the neurophysiological, functional, biochemical and neuropathological levels. Using gas chromatography coupled to mass-spectrometry, we found that three months of diabetes markedly lowered P plasma levels in male rats, and chronic treatment with P restored them, with protective effects on peripheral nerves. In the model of STZ-induced of diabetic neuropathy, chronic treatment for 1 month with P, or with its derivatives, DHP and THP, counteracted the impairment of nerve conduction velocity (NCV) and thermal threshold, restored skin innervation density, and improved Na(+),K(+)-ATPase activity and mRNA levels of myelin proteins, such as glycoprotein zero and peripheral myelin protein 22, suggesting that these neuroactive steroids, might be useful protective agents in diabetic neuropathy. Interestingly, different receptors seem to be involved in these effects. Thus, while the expression of myelin proteins and Na(+),K(+)-ATPase activity are only stimulated by P and DHP (i.e. two neuroactive steroids interacting with P receptor, PR), NCV, thermal nociceptive threshold and intra-epidermal nerve fiber (IENF) density are also affected by THP, which interacts with GABA-A receptor. Because, a therapeutic approach with specific synthetic receptor ligands could avoid the typical side effects of steroids, future experiments will be devoted to evaluating the role of PR and GABA-A receptor in these protective effects.

Neuroactive steroids: State of the art and new perspectives

Abstract.Neuroactive steroids include synthetic steroidal compounds and endogenous steroids, produced by endocrine glands (hormonal steroids) or the nervous tissue (neurosteroids), which regulate neural functions. These steroids bind to nuclear receptors or act through the activation of membrane-associated signaling pathways to modulate various important processes including the development of the nervous system, neural plasticity and the adaptive responses of neurons and glial cells under pathological conditions. Reviewed and updated in the present paper are the pleiotropic and protective abilities of neuroactive steroids. The fundamental evidence and knowledge gained constitute a profound background that offers interesting possibilities for developing effective strategies against several disorders of the nervous system.

Age-induced decrease of glycoprotein Po and myelin basic protein gene expression in the rat sciatic nerve. Repair by steroid derivatives

The data here reported show that the gene expression of the glycoprotein Po and of the myelin basic protein, the major components of myelin in the peripheral nervous system, dramatically decreases with ageing in the sciatic nerve of normal male rats. A one-month treatment with dihydroprogesterone, the 5alpha-reduced derivative of progesterone, is able to partially restore the fall in Po gene expression occurring in the sciatic nerve of aged male rats, without significantly modifying the gene expression of the myelin basic protein. In cultures of neonatal Schwann cells (the peripheral nervous system elements involved in the synthesis of myelin), the addition of progesterone and of dihydroprogesterone significantly increases Po gene expression; the 3alpha-reduced metabolite of dihydroprogesterone, tetrahydroprogesterone proved to be even more effective. These data suggest that the effect of progesterone is linked to its conversion into dihydroprogesterone and especially into tetrahydroprogesterone, since Schwann cells possess the 5alpha-reductase-3alpha-hydroxysteroid dehydrogenase system. The data provide the first demonstration that ageing decreases the gene expression of two major components of the peripheral myelin in the sciatic nerve; they also show that this phenomenon may be partially reversed by progesterone derivatives, which might act by stimulating Po gene expression in the Schwann cells.

Aging in peripheral nerves: regulation of myelin protein genes by steroid hormones.

The process of aging deeply influences morphological and functional parameters of the peripheral nerves. Interestingly, recent observations performed in our laboratory on the rat sciatic nerves have indicated that the deterioration of myelin occurring in the peripheral nerves during aging may be explained by the fall of the messenger levels of the major peripheral myelin proteins (glycoprotein Po, myelin basic protein and peripheral myelin protein 22). At least in the case of the Po, the low levels of its messengers and of the protein itself found in aged animals are increased by the treatment with a physiological progesterone derivative like dihydroprogesterone. It has also been found that in normal adult male rats the levels of the messengers for Po in the sciatic nerve are increased by progesterone, dihydroprogesterone and tetrahydroprogesterone; surprisingly, the gene expression of peripheral myelin protein 22 is stimulated only by tetrahydroprogesterone. These observations have been confirmed in parallel studies performed on Schwann cell cultures. Since tetrahydroprogesterone does not bind to the progesterone receptor but is a ligand for the GABAA receptor, the hypothesis has been put forward that part of the steroidal effects reported might occur not through the classical progesterone receptor, but rather via an interaction with the GABAA receptor. In other experiments it has been found that the gene expression of Po may be decreased by orchidectomy and restored by treatment with the androgen dihydrotestosterone. Altogether, these observations suggest the future use of physiological and/ or synthetic steroid hormones as a possible therapeutic approach for some pathological situations occurring in peripheral nerves during aging and demyelinating diseases.

Androgen metabolism in the brain.

The paper summarizes the most recent views on androgen metabolism in the brain. In particular it will be shown that: (1) the enzyme 5 alpha-reductase is particularly concentrated in the white matter; (2) 5 alpha-reductase is also present in the myelin; 5 alpha-reductase is present in higher concentrations in neurons (isolated or cultured) that in glial cells (astrocytes and oligodendrocytes); (4) only neurons possess the capability of aromatizing androgens to estrogens; and (5) a possible role of steroid metabolism in the control of the process of myelinogenesis is suggested.

Neuroprotective effects of a ligand of translocator protein-18kDa (Ro5-4864) in experimental diabetic neuropathy

Peripheral neuropathy represents an important complication of diabetes involving a spectrum of structural, functional and biochemical alterations in peripheral nerves. Recent observations obtained in our laboratory have shown that the levels of neuroactive steroids present in the sciatic nerve of rat raised diabetic by a single injection of streptozotocin (STZ) are reduced and that, in the same experimental model, treatment with neuroactive steroids, such as progesterone, testosterone and their derivatives show neuroprotective effects. On this basis, an interesting therapeutic strategy could be to increase the levels of neuroactive steroids directly in the nervous system. With this perspective, ligands of translocator protein-18 kDa (TSPO) may represent an interesting option. TSPO is mainly present in the mitochondrial outer membrane, where it promotes the translocation of cholesterol to the inner mitochondrial membrane, and, as demonstrated in other cellular systems, it allows the transformation of cholesterol into pregnenolone and the increase of steroid levels. In the diabetic model of STZ rat, we have here assessed whether treatment with Ro5-4864 (i.e., a ligand of TSPO) could increase the low levels of neuroactive steroids in sciatic nerve and consequently to be protective in this experimental model. Data obtained by liquid chromatography-tandem mass spectrometry show that treatment with Ro5-4864 was able to significantly stimulate the low levels of pregnenolone, progesterone and dihydrotestosterone observed in the sciatic nerves of diabetic rats. The treatment with Ro5-4864 also counteracted the impairment of NCV and thermal threshold, restored skin innervation density and P0 mRNA levels, and improved Na+,K+-ATPase activity. In conclusion, data here reported show for the first time that a TSPO ligand, such as Ro5-4864, is effective in reducing the severity of diabetic neuropathy through a local increase of neuroactive steroid levels.

Testosterone 5α-reductase activity in the rat brain is highly concentrated in white matter structures and in purified myelin sheaths of axons

Previous results obtained in this laboratory indicate that in the rat brain the 5 alpha-reductase, the enzymatic activity involved in metabolizing testosterone into 5 alpha-androstan-17 beta-ol-3-one (dihydrotestosterone), is particularly concentrated in the white matter. In the present experiments, this enzymatic activity was studied in the following white matter structures, which were microdissected using the punch technique of Palkovits: anterior commissure (CA), fornix (FX), habenulo-interpeduncular tract (HP), corpus callosum (CC), stria medullaris (SM), optic chiasm (CO), fimbria of the hippocampus (FI), cerebral peduncle (PC), pontine fibers (FP), cerebellar medulla (CMD) and corticospinal tract (TCS). Moreover brain myelin was isolated and purified by sucrose density gradient ultracentrifugation. The results obtained confirm that, in the rat brain, the enzymes involved in testosterone 5 alpha-reduction are preferentially localized in the white matter. However, clearcut differences in the metabolic activity exist between the different structures examined so far. DHT formation increases rostro-caudally, so that the highest activity has been recorded in the white matter structures punched at the level of pons (FP), medulla oblungata (TCS) and cerebellum (CMD). The high metabolic activity associated with the white matter structures appears to be linked to the presence of myelin, since the specific activity of the enzyme is particularly elevated in purified preparations of myelin sheaths.

Effect of progesterone, testosterone and their 5α-reduced metabolites on GFAP gene expression in type 1 astrocytes

Astrocytes possess steroid receptors as well as several enzymes typical of steroid target cells, such as 5 alpha-reductase, which converts testosterone (T) and progesterone (P) into their respective 5 alpha-reduced metabolites, and the 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD). Because of this, it was deemed of interest to analyze whether the original hormones P and T, and their 5 alpha-reduced metabolites dihydrotestosterone (DHT), 5 alpha-androstan-3 alpha, 17 beta-diol (3 alpha-diol), dihydroprogesterone (DHP) and 5 alpha-pregnan-3 alpha-ol-20-one (THP), might exert some effects on the expression of the most typical astrocytic marker, i.e. the glial fibrillary acidic protein (GFAP). Cultures of rat type 1 astrocytes were exposed to the various steroids for 2, 6, and 24 h, and the variations of GFAP mRNA were measured by Northern blot analysis. A significant elevation of GFAP mRNA levels was observed after exposure to either P or DHP; the effect of DHP appeared more promptly (at 2 h) than that of P (at 6 h). This result suggests that the effect of P might be linked to its conversion into DHP; this hypothesis has been confirmed by showing that the addition of finasteride (a specific blocker of the 5 alpha-reductase) is able to completely abolish the effect of P. After exposure to DHP or THP, a decrease of GFAP gene expression was observed at later intervals (24 h). In the case of androgens, T and 3 alpha-diol did not change GFAP expression at any time of exposure, while DHT produced a significant decrease of GFAP mRNA only after 24 h of exposure. Taken together, the data indicate that the 5 alpha-reduced metabolites of P and T may modulate the expression of GFAP in type 1 rat astrocytes.

Testosterone derivatives are neuroprotective agents in experimental diabetic neuropathy.

Abstract.In this study we have assessed the effect of testosterone (T), dihydrotestosterone (DHT) and 5αandrostan-3α, 17β-diol (3α-diol) therapies on diabetic neuropathy. Diabetes was induced in adult male rats by the injection of streptozotocin and resulted in decreased T and increased 3α-diol levels in plasma and in decreased levels of pregnenolone and DHT in the sciatic nerve. Moreover, a reduced expression of the enzyme converting Tinto DHT (i.e., the 5α-reductase) also occurs at the level of sciatic nerve, suggesting that the decrease of DHT levels could be due to an impairment of this enzyme. Chronic treatment for 1 month with DHT or 3α-diol increased tail nerve conduction velocity and partially counteracted the increase of thermal threshold induced by diabetes. Treatment with DHT increased tibial Na+,K+-ATPase activity and the expression of myelin protein P0 in the sciatic nerve.DHT, 3α-diol and T reversed the reduction of intra-epidermal nerve fiber density induced by diabetes. These observations indicate that T metabolites can reverse behavioral, neurophysiological, morphological and biochemical alterations induced by peripheral diabetic neuropathy.