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.

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.

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.

Neuroprotective effects of progesterone in chronic experimental autoimmune encephalomyelitis.

Observations so far obtained in experimental autoimmune encephalomyelitis (EAE) have revealed the promising neuroprotective effects exerted by progesterone (PROG). The findings suggest that this neuroactive steroid may potentially represent a therapeutic tool for multiple sclerosis (MS). However, up to now, the efficacy of PROG has been only tested in the acute phase of the disease, whereas it is well known that MS expresses different features depending on the phase of the disease. Accordingly, we have evaluated the effect of PROG treatment in EAE induced in Dark Agouti rats (i.e. an experimental model showing a protracted relapsing EAE). Data obtained 45 days after EAE induction show that PROG treatment exerts a beneficial effect on clinical score, confirming surrogate parameters of spinal cord damage in chronic EAE (i.e. reactive microglia, cytokine levels, activity of the Na(+) ,K(+) -ATPase pump and myelin basic protein expression). An increase of the levels of dihydroprogesterone and isopregnanolone (i.e. two PROG metabolites) was also observed in the spinal cord after PROG treatment. Taken together, these results indicate that PROG is effective in reducing the severity of chronic EAE and, consequently, may have potential with respect to MS treatment.

Sex-dimorphic effects of dehydroepiandrosterone in diabetic neuropathy.

Our recent observations have demonstrated that gonadectomy in female, but not in male diabetic animals, exert protection in the peripheral nervous system and that these effects were associated with an increase in the levels of dehydroepiandrosterone (DHEA) in the female sciatic nerve [Pesaresi M, Giatti S, Cavaletti G, Abbiati F, Calabrese D, Bianchi R, Caruso D, Garcia-Segura LM, Melcangi RC (2011) Exp Neurol 228:215-221]. That is interesting because the neuroprotective effects of this neuroactive steroid have so far only been analyzed in male diabetic animals. Using the experimental model of streptozotocin-induced diabetic neuropathy, we have here compared the effect of DHEA treatment in male and in female animals. Data obtained indicate that DHEA treatment is able to counteract the decrease in nerve conduction velocity (NCV) induced by diabetes in both sexes. However, it was only in females that this neuroactive steroid was able to reestablish NCV to control levels. In addition, it was only in females that DHEA exerted neuroprotective actions on functional (i.e., thermal sensitivity) or molecular parameters, such as gene expression of myelin proteins. Sex-depending neuroprotective effects of DHEA were also confirmed by the finding that it was only in females that this neuroactive steroid fully restored the intra-epidermal nerve fiber density, which was decreased by diabetes. Interestingly, the metabolic fate of DHEA is also different in males and females. Thus, analysis of the neuroactive steroid levels after the treatment with DHEA indicates that in the sciatic nerve of male diabetic animals 17α-estradiol levels decrease in association with an increase of its isomer 17ß-estradiol and with a decrease in the levels of α-androstane-3α, 17ß-diol. These changes were not observed in the sciatic nerve of females. Altogether, these results suggest that DHEA could be considered as a candidate for a sex-specific therapy based on neuroactive steroids.

Effect of short-and long-term gonadectomy on neuroactive steroid levels in the central and peripheral nervous system of male and female rats.

Significant levels of neuroactive steroids are still detected in the nervous system of rodents after the removal of peripheral steroidogenic glands. However, the influence of the plasma levels of gonadal steroids on the levels of neuroactive steroids in the nervous system has not so far been clarified in detail. Accordingly, by liquid chromatography tandem mass spectrometry, we have analysed the levels of neuroactive steroids in the sciatic nerve, in three central nervous system (CNS) regions (i.e. cerebellum, cerebral cortex and spinal cord) and in the plasma of male and female animals. The levels present in gonadally intact animals were compared with those present in short- and long-term gonadectomised animals. We observed that: (i) changes in neuroactive steroid levels in the nervous system after gonadectomy do not necessarily reflect the changes in plasma levels; (ii) long-term gonadectomy induces changes in the levels of neuroactive steroids in the peripheral nervous system (PNS) and the CNS that, in some cases, are different to those induced by short-term gonadectomy; (iii) the effect of gonadectomy on neuroactive steroid levels is different between the PNS and the CNS and within different CNS regions; and (iv) the effects of gonadectomy on neuroactive steroid levels in the nervous system show sex differences. Altogether, these observations indicate that the nervous system adapts its local levels of neuroactive steroids in response to changes in gonadal hormones with sex and regional specificity and depending on the duration of the peripheral modifications.

Neuroprotective effects of a ligand of translocator protein-18 kDa (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.

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.

Testosterone derivatives are neuroprotective agents in experimental diabetic neuropathy.

In this study we have assessed the effect of testosterone (T), dihydrotestosterone (DHT) and 5alphaandrostan-3alpha, 17beta-diol (3alpha-diol) therapies on diabetic neuropathy. Diabetes was induced in adult male rats by the injection of streptozotocin and resulted in decreased T and increased 3alpha-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 5alpha-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 3alpha-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, 3alpha-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.