Analysis of the finasteride treatment and its withdrawal in the rat hypothalamus and hippocampus at whole-transcriptome level.

PURPOSE: As reported in patients treated for androgenetic alopecia with finasteride (i.e., a blocker of the enzyme 5 alpha-reductase) and in an animal model, side effects affecting sexual, psychiatric, neurological, and physical domains, may occur during the treatment and persist with drug suspension. The etiopathogenesis of these side effects has been poorly explored. Therefore, we performed a genome-wide analysis of finasteride effects in the brain of adult male rat. METHODS: Animals were treated (i.e., for 20 days) with finasteride (1mg/rat/day). 24 h after the last treatment and 1 month after drug suspension, RNA sequencing analysis was performed in hypothalamus and hippocampus. Data were analyzed by differential expression analysis and Gene-Set Enrichment Analyses (GSEA). RESULTS: Data obtained after finasteride treatment showed that 186 genes (i.e., 171 up- and 15 downregulated) and 19 (i.e., 17 up- and 2 downregulated) were differentially expressed in the hypothalamus and hippocampus, respectively. Differential expression analysis at the drug withdrawal failed to identify dysregulated genes. Several gene-sets were enriched in these brain areas at both time points. CONCLUSION: Some of the genes reported to be differentially expressed (i.e., TTR, DIO2, CLDN1, CLDN2, SLC4A5, KCNE2, CROT, HCRT, MARCKSL1, VGF, IRF2BPL) and GSEA, suggest a potential link with specific side effects previously observed in patients and in the animal model, such as depression, anxiety, disturbance in memory and attention, and sleep disturbance. These data may provide an important background for future experiments aimed at confirming the pathological role of these genes.

Alterations of gut microbiota composition in post-finasteride patients: a pilot study.

PURPOSE: Post-finasteride syndrome (PFS) has been reported in a subset of patients treated with finasteride (an inhibitor of the enzyme 5alpha-reductase) for androgenetic alopecia. These patients showed, despite the suspension of the treatment, a variety of persistent symptoms, like sexual dysfunction and cognitive and psychological disorders, including depression. A growing body of literature highlights the relevance of the gut microbiota-brain axis in human health and disease. For instance, alterations in gut microbiota composition have been reported in patients with major depressive disorder. Therefore, we have here analyzed the gut microbiota composition in PFS patients in comparison with a healthy cohort. METHODS: Fecal microbiota of 23 PFS patients was analyzed by 16S rRNA gene sequencing and compared with that reported in ten healthy male subjects. RESULTS: Sexual dysfunction, psychological and cognitive complaints, muscular problems, and physical alterations symptoms were reported in more than half of the PFS patients at the moment of sample collection. The quality sequence check revealed a low library depth for two fecal samples. Therefore, the gut microbiota analyses were conducted on 21 patients. The α-diversity was significantly lower in PFS group, showing a reduction of richness and diversity of gut microbiota structure. Moreover, when visualizing ß-diversity, a clustering effect was found in the gut microbiota of a subset of PFS subjects, which was also characterized by a reduction in Faecalibacterium spp. and Ruminococcaceae UCG-005, while Alloprevotella and Odoribacter spp were increased compared to healthy control. CONCLUSION: Gut microbiota population is altered in PFS patients, suggesting that it might represent a diagnostic marker and a possible therapeutic target for this syndrome.

Neuroactive steroids and diabetic complications in the nervous system.

Important complications of diabetes mellitus in the nervous system are represented by diabetic peripheral neuropathy and diabetic encephalopathy. In this context, an important link is represented by neuroactive steroids (i.e., steroids coming from peripheral glands and affecting nervous functionality as well as directly synthesized in the nervous system). Indeed, diabetes does not only affect the reproductive axis and consequently the levels of sex steroid hormones, but also those of neuroactive steroids. Indeed, as will be here summarized, the levels of these neuromodulators present in the central and peripheral nervous system are affected by the pathology in a sex-dimorphic way. In addition, some of these neuroactive steroids, such as the metabolites of progesterone or testosterone, as well as pharmacological tools able to increase their levels have been demonstrated, in experimental models, to be promising protective agents against diabetic peripheral neuropathy and diabetic encephalopathy.

Liver X receptors, nervous system, and lipid metabolism.

Lipids in the nervous system are represented by cholesterol and phospholipids as constituents of cell membranes and, in particular, of myelin. Therefore, lipids are finely regulated to guarantee physiological functions. In the central nervous system, cholesterol is locally synthesized due to the presence of the blood brain barrier. In the peripheral nervous system cholesterol is either up-taken by lipoproteins and/or produced by de novo biosynthesis. Defects in lipid homeostasis in these tissues lead to structural and functional changes that often result in different pathological conditions depending on the affected pathways (i.e. cholesterol biosynthesis, cholesterol efflux, fatty acid biosynthesis etc.). Alterations in cholesterol metabolism in the central nervous system are linked to several disorders such as Alzheimer's disease, Huntington disease, Parkinson disease, Multiple sclerosis, Smith-Lemli-Opitz syndrome, Niemann-Pick type C disease, and glioblastoma. In the peripheral nervous system changes in lipid metabolism are associated with the development of peripheral neuropathy that may be caused by metabolic disorders, injuries, therapeutics, and autoimmune diseases. Transcription factors, such as the Liver X receptors (LXR), regulate both cholesterol and fatty acid metabolism in several tissues including the nervous system. In the last few years several studies elucidated the biology of LXR in the nervous system due to the availability of knock-out mice and the development of synthetic ligands. Here, we review a survey of the literature focused on the central and peripheral nervous system and in physiological and pathological settings with particular attention to the roles played by LXR in both districts.

Steroidogenic machinery in the adult rat colon.

Gastrointestinal function is known to be regulated by steroid molecules produced by the gonads, the adrenal glands and the gut microbiota. However, we have a limited knowledge on the functional significance of local steroid production by gastrointestinal tract tissue. On this basis, we have here evaluated, as a first methodological approach, the expression of steroidogenic molecules and the local levels of key steroids in the male rat colon. Our findings indicate that the colon tissue expresses molecules involved in the early steps of steroidogenesis and in the consecutive synthesis and metabolism of steroid hormones, such as progesterone, testosterone and 17ß-estradiol. In addition, the levels of the steroid hormone precursor pregnenolone and the levels of active metabolites of progesterone and testosterone, such as dihydroprogesterone, tetrahydroprogesterone, dihydrotestosterone and 17ß-estradiol, were higher in colon than in plasma. Higher levels of the androgen metabolite 3α-diol were detected in the colon in comparison with another non-classical steroidogenic tissue, such as the cerebral cortex. These findings suggest the existence of local steroid synthesis and metabolism in the colon, with the production of active steroid metabolites that may impact on the activity of the enteric nervous system and on the composition of the gut microbiota.

Sex dimorphism in an animal model of multiple sclerosis: Focus on pregnenolone synthesis.

Neuroactive steroids, molecules produced from cholesterol in steroidogenic cells (i.e., peripheral glands and nervous system) are physiological modulators and protective agents of nervous function. A possible role for neuroactive steroids in the sex-dimorphic clinical manifestation, onset and progression of Multiple Sclerosis (MS) has been recently suggested. To explore this possibility, we assessed the synthesis of the first steroidogenic product (pregnenolone; PREG) in the spinal cord of experimental autoimmune encephalomyelitis rats, a MS model. Data obtained indicate that the synthesis of PREG in the spinal cord is altered by the pathology in a sex-dimorphic way and depending on the pathological progression. Indeed, in male spinal cord the synthesis was already decreased at the acute phase of the disease (i.e., 14 days post induction - dpi) and maintained low during the chronic phase (i.e., 45 dpi), while in females this effect was observed only at the chronic phase. Substrate availability had also a role in the sex-dimorphic kinetics. Indeed, at the chronic phase, male animals showed a reduction in the levels of free cholesterol coupled to alteration of cholesterol metabolism into oxysterols; these effects were not observed in female animals. These findings suggest that the comprehension of the neurosteroidogenic processes could be relevant to better understand the sexual dimorphism of MS and to possibly design sex-oriented therapeutic strategies based on neuroactive steroids.

The endocrine nervous system: source and target for neuroactive steroids.

For a long time the endocrine brain was considered to the hypothalamus and to its special relationships with the hypophysis. The discovery of the wide distribution of steroid hormone receptors, as well as that of the possibility of metabolizing or synthesizing steroids by neural cells (neuroactive steroids), suggest, on the contrary, that interactions among steroids and nervous system are key points of the regulatory processes in the central and peripheral nervous system in normal conditions as well as in pathological conditions. In this brief overview we illustrate a few examples of these relationships with major emphasis on papers collected in this special issue.

Neuroprotective effects of dihydroprogesterone and progesterone in an experimental model of nerve crush injury.

A satisfactory management to ensure a full restoration of peripheral nerve after trauma is not yet available. Using an experimental protocol, in which crush injury was applied 1 cm above the bifurcation of the rat sciatic nerve for 20 s, we here demonstrate that the levels of neuroactive steroids, such as pregnenolone and progesterone (P) metabolites (i.e. dihydroprogesterone, DHP, and tetrahydroprogesterone, THP) present in injured sciatic nerve were significantly decreased. On this basis, we have focused our attention on DHP and its direct precursor, P, analyzing whether these two neuroactive steroids may have neuroprotective effects on biochemical, functional and morphological alterations occurring during crush-induced degeneration-regeneration. We demonstrate that DHP and/or P counteract biochemical alterations (i.e. myelin proteins and Na(+),K(+)-ATPase pump) and stimulate reelin gene expression. These two neuroactive steroids also counteract nociception impairment, and DHP treatment significantly decreases the up-regulation of myelinated fibers' density occurring in crushed animals. Altogether, these observations suggest that DHP and P (i.e. two neuroactive steroids interacting with progesterone receptor) may be considered protective agents in case of nerve crush injury.

Steroids, stress and the gut microbiome-brain axis.

It is becoming well established that the gut microbiome has a profound impact on human health and disease. In this review, we explore how steroids can influence the gut microbiota and, in turn, how the gut microbiota can influence hormone levels. Within the context of the gut microbiome-brain axis, we discuss how perturbations in the gut microbiota can alter the stress axis and behaviour. In addition, human studies on the possible role of gut microbiota in depression and anxiety are examined. Finally, we present some of the challenges and important questions that need to be addressed by future research in this exciting new area at the intersection of steroids, stress, gut-brain axis and human health.

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: old players in a new game.

It is now clear that the study of the effects exerted by steroids on the nervous system may be considered as one of the most interesting and promising topics for biomedical research. Indeed, new effects, mechanisms of action and targets are becoming more and more evident suggesting that steroids are not only important key regulators of nervous system function but they may also represent a new therapeutic tool to combat certain diseases of the nervous system. The present review summarizes recent observations on this topic indicating that while the concept of the nervous system as a target for steroid hormones has been appreciated for decades, a promising new era for the study of these molecules and their actions in the nervous system has been initiated in the last few years.

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.

Neuroactive steroids influence peripheral myelination: a promising opportunity for preventing or treating age-dependent dysfunctions of peripheral nerves.

The process of aging deeply influences morphological and functional parameters of peripheral nerves. The observations summarized here indicate that the deterioration of myelin occurring in the peripheral nerves during aging may be explained by the fall of the levels of the major peripheral myelin proteins [e.g., glycoprotein Po (Po) and peripheral myelin protein 22 (PMP22)]. Neuroactive steroids, such as progesterone (PROG), dihydroprogesterone (5alpha-DH PROG), and tetrahydroprogesterone (3alpha,5alpha-TH PROG), are able to stimulate the low expression of these two myelin proteins present in the sciatic nerve of aged male rats. Since Po and PMP22 play an important physiological role in the maintenance of the multilamellar structure of PNS myelin, we have evaluated the effect of PROG and its neuroactive derivatives, 5alpha-DH PROG and 3alpha,5alpha-TH PROG, on the morphological alterations of myelinated fibers in the sciatic nerve of 22-24-month-old male rats. Data obtained clearly indicate that neuroactive steroids are able to reduce aging-associated morphological abnormalities of myelin and aging-associated myelin fiber loss in the sciatic nerve.

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

Without medical progress, dementing diseases such as Alzheimer's 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 Alzheimer's disease patients and aged non-demented controls by GC/MS, providing reference values. In Alzheimer's 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 Alzheimer's 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 Alzheimer's patients and controls. The formation of these metabolites within distinct brain regions negatively correlated with the density of beta-amyloid deposits.

Steroids and the reversal of age-associated changes in myelination and remyelination.

The myelin sheaths that surround all but the smallest diameter axons within the mammalian central nervous system (CNS) must maintain their structural integrity for many years. Like many tissues, however, this function is prone to the effects of ageing, and various structural anomalies become apparent in the aged CNS. Similarly, the regenerative process by which myelin sheaths, lost as a consequence of exposure to a demyelinating insult, are restored (remyelination) is also affected by age. As animals grow older, the efficiency of remyelination progressively declines. In this article, we review both phenomena and describe how both can be partially reversed by steroid hormones and their derivatives.

Neurosteroidogenesis Today: Novel Targets for Neuroactive Steroid Synthesis and Action and Their Relevance for Translational Research.

Neuroactive steroids are endogenous neuromodulators synthesised in the brain that rapidly alter neuronal excitability by binding to membrane receptors, in addition to the regulation of gene expression via intracellular steroid receptors. Neuroactive steroids induce potent anxiolytic, antidepressant, anticonvulsant, sedative, analgesic and amnesic effects, mainly through interaction with the GABAA receptor. They also exert neuroprotective, neurotrophic and antiapoptotic effects in several animal models of neurodegenerative diseases. Neuroactive steroids regulate many physiological functions, such as the stress response, puberty, the ovarian cycle, pregnancy and reward. Their levels are altered in several neuropsychiatric and neurological diseases and both preclinical and clinical studies emphasise a therapeutic potential of neuroactive steroids for these diseases, whereby symptomatology ameliorates upon restoration of neuroactive steroid concentrations. However, direct administration of neuroactive steroids has several challenges, including pharmacokinetics, low bioavailability, addiction potential, safety and tolerability, which limit its therapeutic use. Therefore, modulation of neurosteroidogenesis to restore the altered endogenous neuroactive steroid tone may represent a better therapeutic approach. This review summarises recent approaches that target the neuroactive steroid biosynthetic pathway at different levels aiming to promote neurosteroidogenesis. These include modulation of neurosteroidogenesis through ligands of the translocator protein 18 kDa and the pregnane xenobiotic receptor, as well as targeting of specific neurosteroidogenic enzymes such as 17ß-hydroxysteroid dehydrogenase type 10 or P450 side chain cleavage. Enhanced neurosteroidogenesis through these targets may be beneficial not only for neurodegenerative diseases, such as Alzheimer's disease and age-related dementia, but also for neuropsychiatric diseases, including alcohol use disorders.

Effect of streptozotocin-induced diabetes on the gene expression and biological activity of 3beta-hydroxysteroid dehydrogenase in the rat spinal cord.

Abnormal secretion of steroids by the adrenals and gonads is one of the disturbances occurring in diabetics but the impact of diabetes on steroid formation in the nervous system has never been studied. However, it is well known that numerous actions of peripheral steroids on the nervous system require their conversion into neuroactive metabolites within the neural tissue. As this in situ steroid synthesis/metabolism is crucial for the control of several neurobiological functions, we investigated the effects of streptozotocin-induced diabetes on the gene expression and activity of 3beta-hydroxysteroid dehydrogenase in the spinal cord, a pivotal structure involved in sensorimotor and neurovegetative mechanisms. 3beta-Hydroxysteroid dehydrogenase is a key enzyme which participates to the biosynthesis of all classes of steroids by converting delta5-3beta-hydroxysteroids such as pregnenolone and dehydroepiandrosterone into delta4-3-ketosteroids as progesterone and androstenedione, respectively. Reverse transcription coupled with quantitative real-time polymerase chain reaction revealed that 3beta-hydroxysteroid dehydrogenase gene was over-expressed in the spinal cord of streptozotocin-treated rats compared with controls. Pulse-chase experiments combined with high performance liquid chromatography and continuous flow detection of newly-synthesized steroids showed an increase of 3beta-hydroxysteroid dehydrogenase activity responsible for a hyper-production of progesterone in the spinal cord of diabetic rats. This up-regulation of progesterone biosynthesis was concomitant with a decrease of its transformation into tetrahydroprogesterone, a process which facilitated progesterone accumulation in the spinal cord of streptozotocin-treated rats. Since progesterone is a potent neuroprotective steroid, increase of its production appeared as an endogenous molecular and biochemical mechanism triggered by spinal nerve cells to cope with degenerative effects of streptozotocin-induced diabetes. Our results constitute the first direct evidence showing an impact of diabetes on steroid biosynthetic and metabolic pathways in the nervous system. The data open new perspectives for the modulation of deleterious effects of diabetes by neuroprotective steroids.

Smad proteins are targets of transforming growth factor beta1 in immortalised gonadotrophin-releasing hormone releasing neurones.

Transforming growth factor beta (TGFbeta) is one of the growth factors involved in the neuroendocrine control of the gonadotrophin-releasing hormone (GnRH) neurones. It is produced and released by the astrocytes surrounding GnRH neurones and directly controls their secretory activity. TGFbeta signalling is based on a complex of two receptors that transduces the signal through peculiar intracellular substrates, the Smad proteins, which, upon activation, move into the nucleus, and modify the transcription of TGFbeta responsive genes. The present study aimed to verify whether TGFbeta1 is able to regulate the Smad pathway in GT1-1 cells (i.e. an immortalised neuronal cell line releasing GnRH). We show that: (i) GT1-1 cells express Smad 2, 3, 4, and 7; (ii) TGFbeta1 enhances the phosphorylation of Smad 2 and 3 at short times of exposure (15-30 min); (iii) TGFbeta1 induces the synthesis of the inhibitory Smad 7 at longer times (60-120-240 min); (iv) the conditioned medium of type 1 astrocytes enhances the phosphorylation of Smad 2 and 3 in GT1-1 cells and a TGFbeta1 neutralising antibody counteracts this effect. The results indicate that Smads are targets of TGFbeta1 and that astrocytes are able to modulate Smads proteins in GT1-1 cells through the release of TGFbeta1. Taken together, the data provide new evidence that glial cells are important regulators of the GnRH neuronal activity.