Roberto Spezzano

Neuroactive steroid levels and psychiatric and andrological features in post-finasteride patients

Recent reports show that, in patients treated with finasteride for male pattern hair loss, persistent side effects including sexual side effects, depression, anxiety and cognitive complaints may occur. We here explored the psychiatric and andrological features of patients affected by post-finasteride syndrome (PFS) and verified whether the cerebrospinal fluid (CSF) and plasma levels of neuroactive steroids (i.e., important regulators of nervous function) are modified. We found that eight out of sixteen PFS male patients considered suffered from a DSM-IV major depressive disorder (MDD). In addition, all PFS patients showed erectile dysfunction (ED); in particular, ten patients showed a severe and six a mild-moderate ED. We also reported abnormal somatosensory evoked potentials of the pudendal nerve in PFS patients with severe ED, the first objective evidence of a neuropathy involving peripheral neurogenic control of erection. Testicular volume by ultrasonography was normal in PFS patients. Data obtained on neuroactive steroid levels also indicate interesting features. Indeed, decreased levels of pregnenolone, progesterone and its metabolite (i.e., dihydroprogesterone), dihydrotestosterone and 17beta-estradiol and increased levels of dehydroepiandrosterone, testosterone and 5alpha-androstane-3alpha,17beta-diol were observed in CSF of PFS patients. Neuroactive steroid levels were also altered in plasma of PFS patients, however these changes did not reflect exactly what occurs in CSF. Finally, finasteride did not only affect, as expected, the levels of 5alpha-reduced metabolites of progesterone and testosterone, but also the further metabolites and precursors suggesting that this drug has broad consequence on neuroactive steroid levels of PFS patients.

Role of androgens in dhea-induced rack1 expression and cytokine modulation in monocytes

BackgroundOver the past fifteen years, we have demonstrated that cortisol and dehydroepiandrosterone (DHEA) have opposite effects on the regulation of protein kinase C (PKC) activity in the context of the immune system. The anti-glucocorticoid effect of DHEA is also related to the regulation of splicing of the glucocorticoid receptor (GR), promoting the expression of GRβ isoform, which acts as a negative dominant form on GRα activity. Moreover, it is very well known that DHEA can be metabolized to androgens like testosterone, dihydrotestosterone (DHT), and its metabolites 3α-diol and 3β-diol, which exert their function through the binding of the androgen receptor (AR). Based on this knowledge, and on early observation that castrated animals show results similar to those observed in old animals, the purpose of this study is to investigate the role of androgens and the androgen receptor (AR) in DHEA-induced expression of the PKC signaling molecule RACK1 (Receptor for Activated C Kinase 1) and cytokine production in monocytes.ResultsHere we demonstrated the ability of the anti-androgen molecule, flutamide, to counteract the stimulatory effects of DHEA on RACK1 and GRβ expression, and cytokine production. In both THP-1 cells and human peripheral blood mononuclear cells (PBMC), flutamide blocked the effects of DHEA, suggesting a role of the AR in these effects. As DHEA is not considered a direct AR agonist, we investigated the metabolism of DHEA in THP-1 cells. We evaluated the ability of testosterone, DHT, and androstenedione to induce RACK1 expression and cytokine production. In analogy to DHEA, an increase in RACK1 expression and in LPS-induced IL–8 and TNF–α production was observed after treatment with these selected androgens. Finally, the silencing of AR with siRNA completely prevented DHEA-induced RACK1 mRNA expression, supporting the idea that AR is involved in DHEA effects.ConclusionsWe demonstrated that the conversion of DHEA to active androgens, which act via AR, is a key mechanism in the effect of DHEA on RACK1 expression and monocyte activation. This data supports the existence of a complex hormonal balance in the control of immune modulation, which can be further studied in the context of immunosenescence and endocrinosenescence.

Short-term effects of diabetes on neurosteroidogenesis in the rat hippocampus

Diabetes may induce neurophysiological and structural changes in the central nervous system (i.e., diabetic encephalopathy). We here explored whether the levels of neuroactive steroids (i.e., neuroprotective agents) in the hippocampus may be altered by short-term diabetes (i.e., one month). To this aim, by liquid chromatography-tandem mass spectrometry we observed that in the experimental model of the rat raised diabetic by streptozotocin injection, one month of pathology induced changes in the levels of several neuroactive steroids, such as pregnenolone, progesterone and its metabolites (i.e., tetrahydroprogesterone and isopregnanolone) and testosterone and its metabolites (i.e., dihydrotestosterone and 3α-diol). Interestingly these brain changes were not fully reflected by the plasma level changes, suggesting that early phase of diabetes directly affects steroidogenesis and/or steroid metabolism in the hippocampus. These concepts are also supported by the findings that crucial steps of steroidogenic machinery, such as the gene expression of steroidogenic acute regulatory protein (i.e., molecule involved in the translocation of cholesterol into mitochondria) and cytochrome P450 side chain cleavage (i.e., enzyme converting cholesterol into pregnenolone) and 5α-reductase (enzyme converting progesterone and testosterone into their metabolites) are also affected in the hippocampus. In addition, cholesterol homeostasis as well as the functionality of mitochondria, a key organelle in which the limiting step of neuroactive steroid synthesis takes place, are also affected. Data obtained indicate that short-term diabetes alters hippocampal steroidogenic machinery and that these changes are associated with impaired cholesterol homeostasis and mitochondrial dysfunction in the hippocampus, suggesting them as relevant factors for the development of diabetic encephalopathy.

Diabetes alters myelin lipid profile in rat cerebral cortex: Protective effects of dihydroprogesterone

Due to the emerging association of diabetes with several psychiatric and neurodegenerative events, the evaluation of the effects of this pathology on the brain function has now a high priority in biomedical research. In particular, the effects of diabetes on myelin compartment have been poorly taken into consideration. To this purpose, we performed a deep lipidomic analysis of cortical myelin in the streptozotocin-induced diabetic rat model. In male rats three months of diabetes induced an extensive alterations in levels of phosphatidylcholines and phosphatidylethanolamines (the main species present in myelin membranes), plasmalogens as well as phosphatidylinositols and phosphatidylserines. In addition, the levels of cholesterol and myelin basic protein were also decreased. Because these lipids exert important functional and structural roles in the myelin compartment, our data indicate that cerebral cortex myelin is severely compromised in diabetic status. Treatment for one-month with a metabolite of progesterone, dihydroprogesterone, restored the lipid and protein myelin profiles to the levels observed in non-diabetic animals. These data suggest the potential of therapeutic efficacy of DHP to restore myelin in the diabetic brain.

Axonal transport in a peripheral diabetic neuropathy model : sex-dimorphic features

BackgroundDisruption of axonal transport plays a pivotal role in diabetic neuropathy. A sex-dimorphism exists in the incidence and symptomatology of diabetic neuropathy; however, no studies so far have addressed sex differences in axonal motor proteins expression in early diabetes as well as the possible involvement of neuroactive steroids. Interestingly, recent data point to a role for mitochondria in the sexual dimorphism of neurodegenerative diseases. Mitochondria have a fundamental role in axonal transport by producing the motors’ energy source, ATP. Moreover, neuroactive steroids can also regulate mitochondrial function.MethodsHere, we investigated the impact of short-term diabetes in the peripheral nervous system of male and female rats on key motor proteins important for axonal transport, mitochondrial function, and neuroactive steroids levels.ResultsWe show that short-term diabetes alters mRNA levels and axoplasm protein contents of kinesin family member KIF1A, KIF5B, KIF5A and Myosin Va in male but not in female rats. Similarly, the expression of peroxisome proliferator-activated receptor γ co-activator-1α, a subunit of the respiratory chain complex IV, ATP levels and the key regulators of mitochondrial dynamics were affected in males but not in females. Concomitant analysis of neuroactive steroid levels in sciatic nerve showed an alteration of testosterone, dihydrotestosterone, and allopregnanolone in diabetic males, whereas no changes were observed in female rats.ConclusionsThese findings suggest that sex-specific decrease in neuroactive steroid levels in male diabetic animals may cause an alteration in their mitochondrial function that in turn might impact in axonal transport, contributing to the sex difference observed in diabetic neuropathy.

Diabetes induces mitochondrial dysfunction and alters cholesterol homeostasis and neurosteroidogenesis in the rat cerebral cortex

The nervous system synthesizes and metabolizes steroids (i.e., neurosteroidogenesis). Recent observations indicate that neurosteroidogenesis is affected by different nervous pathologies. Among these, long-term type 1 diabetes, together with other functional and biochemical changes, has been shown to alter neuroactive steroid levels in the nervous system. Using an experimental model of type 1 diabetes (i.e., streptozotocin injection) we here show that the levels of these molecules are already decreased in the rat cerebral cortex after one month of the initiation of the pathology. Moreover, decreased levels of free cholesterol, together with alterations in the expression of molecules involved in cholesterol biosynthesis, bioavailability, trafficking and metabolism were detected in the rat cerebral cortex after one month of diabetes. Furthermore, mitochondrial functionality was also affected in the cerebral cortex and consequently may also contribute to the decrease in neuroactive steroid levels. Altogether, these results indicate that neurosteroidogenesis is an early target for the effect of type 1 diabetes in the cerebral cortex.

Sterol regulatory element binding protein-1c Knockout mice show altered neuroactive steroid levels in sciatic nerve

Neuroactive steroid levels are altered in several experimental models of peripheral neuropathy, and on this basis, they have been proposed as protective agents. For the first time, the levels of these molecules were assessed here in sterol regulatory element binding protein ‐1c knock‐out male mice (i.e., an experimental model of peripheral neuropathy) and compared with observations in wild type animals. The levels of neuroactive steroids have been evaluated by liquid chromatography–tandem mass spectrometry in plasma and sciatic nerve at 2 and 10 months of age and these analyses were implemented analyzing the gene expression of crucial steroidogenic enzymes in sciatic nerve. Data obtained at 2 months of age showed high levels of pregnenolone in sciatic nerve, associated with low levels of its first metabolite, progesterone, and further metabolites (i.e., 5α‐pregnane‐3,20‐dione and 5α‐pregnan‐3β‐ol‐20‐one). High levels of testosterone and 17β‐estradiol were also observed. At 10 months of age, the neuroactive steroid profile showed some differences. Indeed, low levels of pregnenolone and high levels of 5α‐pregnan‐3α‐ol‐20‐one and 5α‐pregnan‐3β‐ol‐20‐one were observed. The analysis of the gene expression of steroidogenic enzymes considered here generally followed these changes. Interestingly, the levels of pregnenolone and progesterone were unmodified in plasma suggesting a specific effect of sterol regulatory element binding protein‐1c on neurosteroidogenesis. Because this peripheral neuropathy is due to altered fatty acid biosynthesis, data reported here support the belief that the cross‐talk between this biosynthetic pathway and neuroactive steroids may represent a possible therapeutic strategy for peripheral neuropathy.

Valorizing coffee pulp by-products as anti-inflammatory ingredient of food supplements acting on IL-8 release

Coffee is the second traded food commodity in the world. Beyond roasted seeds, the most part of the original fruit -and in particular pulp- is discarded as waste, with severe environmental and economic consequences in many developing countries. Our research focused on developing an eco-friendly extraction protocol of phytocomplexes from coffee pulp and evaluating their bioactivity and beneficial effects to human health as food supplements. Antioxidant activity assays (Folin-Ciocalteu and DPPH assays) were adopted to select the most effective extraction technique and results show antioxidant activity of coffee pulp extracts. After analysis of cytotoxicity on human epithelial gastric cells, measurements of IL-8 release of treated or pre-treated cells were performed. Results showed that the use of soft technical equipment and sustainable solvents (i.e. maceration process, aqueous extraction) can extract phytocomplexes with antioxidant properties. Moreover, IL-8 measurements showed impairment of this chemokine release at concentrations that may be reached in vivo in the gastrointestinal tract, following consumption of reasonable amount of extract. Pre-treatments analysis demonstrated the ability of coffee pulp extracts to prevent IL-8 release by gastric epithelial cells. Chemical evaluation performed by liquid chromatography mass spectrometry showed that quinic acid derivatives are abundant in coffee pulp extract together with procyanidins derivatives: those compounds might be responsible for the high biological activity. This evidence supports future applications of coffee pulp extracts as food supplement with high added value, starting from a waste that can be valorized through simple yet efficient extraction methods.