L\ubfATTIVAZIONE DI TSPO E DI LXR STIMOLA LA NEUROSTEROIDOGENESI ED E\ubf PROTETTIVA IN UN MODELLO DI NEUROPATIA PERIFERICA DI RATTO DIABETICO

Diabetes mellitus (DM) is one of the first leading cause of death in most high-income countries and there is evidence that it is epidemic in many economically developing and newly industrialized nations. Beside these considerations, there are epidemiologic evaluations that show a dramatic increase of the number of diabetic and with impaired glucose tollerance patients in the next years. It is also important to underline that complications from diabetes, such as coronary artery and peripheral vascular disease, stroke, diabetic neuropathy, renal failure and blindness result in increasing disability, reduced life expectancy and enormous health costs for virtually every society. In particular, diabetic peripheral neuropathy can account for the 50-75% of non traumatic amputations; diabetes is therefore undoubtedly one of the most challenging health problems of our century. Recently, in an animal model of diabetic neuropathy (i.e. the rat raised diabetic with the injection of streptozotocin - STZ), it has been demonstrated that, in plasma and in sciatic nerve, the levels of neuroactive steroids are dramatically decreased. Promising results have been obtained in the same experimental model after treatments with progesterone, testosterone and their derivatives. The use of molecules able to increase the neuroactive steroid levels directly in the nervous system could be an interesting therapeutic approach because it may avoid the endocrine side effects usually observed after systemic treatment with steroids. In order to evaluate the possible neuroprotective effects mediated by an increase of neuroactive steroid levels in the peripheral nervous system, using the model of the STZ rat we here analyzed the effects of two different pharmacological approaches. In the first part of this thesis, we analyzed the effect of Ro5-4864, a ligand of the translocator protein of 18 kDa (TSPO). TSPO is a key regulator of neuroactive steroid synthesis and it is involved in the rate limitig step of this process (i.e. the entrance of cholesterol into the mitochondria). Indeed, it has been recently demonstrated that injection of TSPO ligands, like for instance Ro5-4864, was able to increase the capability of steroidogenic machinery in adrenal cortex, placenta, testis, ovary and glial cells. In the second part of this thesis, the effect of GW3965, a ligand of the Liver X Receptor (LXR), was analyzed. The rationale of this approach was based on the finding that cholesterol homeostasis is partially controlled by the regulation of different genes activated by LXR. Moreover, LXR activation is able to increase the steroidogenesis in the adrenal glands. At 2 months after STZ injection, two different protocols of TSPO ligand administration were assessed; in particular, animals were injected every 2 days or once a week whit the ligand (i.e., they received 16 or 4 injections). In the case of GW3965 treatment, at 2 months, diabetic animals were treated once a week (i.e., they received 4 injections). Functional and behavioural parameters, like for instance hot plate test and the recording of nerve conduction velocity, as well as biochemical parameters, like Na+,K+-ATPase activity and myelin protein gene expression, were analyzed in order to assess the neuroprotective effects of treatments. Moreover, in the case of Ro5-4864 treatment, the intra-epidermal nerve fiber density was quantified in the skin of the hindpaw footpad. Furthermore, in the experiment with GW3965 treatment, the levels of gene expression of LXR targets and of steroidogenic enzimes were determined in the sciatic nerve of diabetic animals. Data obtained showed that activation of TSPO and of LXR using their ligands once a week resulted in an increased levels of neuroactive steroids and protection of the sciatic nerve from neuropathy induced by diabetes. Conversely, 16 treatments with Ro5-4864 did not produced any protective effects. The increase of neuroactive steroid levels observed in the sciatic nerve after the treatment with the two molecules was in agreement with the improve of functional and biochimical parameters observed in diabetic rats, like for istance the thermal nociceptive activity, nerve conduction velocity, and Na+,K+-ATPase activity. Moreover, a recover of the intra-epidermal nerve fiber density after Ro5-4864 injection and a significant increase of the gene expression of some enzymes of steroidogenesis after GW3965 administration were detected. In contrast to what observed after TSPO activation, treatment with GW3965 did not consistently countered the diabetes-induced decrease in expression of the myelin protein zero observed in the sciatic nerve of STZ-treated animals. This observation suggests somewhat different mechanisms of action for LXR and TSPO ligands, an issue that it should be explored in the future. In conclusion the data here reported show that Ro5-4864 and GW3965 are neuroprotective in an animal model of diabetic peripheral neuropathy. Since a pharmacological treatment for this pathology is not yet available, data here provide suggest that these two molecules may represent promising candidates for the treatment of diabetic neuropathy

Physiopathological role of the enzymatic complex 5α-reductase and 3α/β-hydroxysteroid oxidoreductase in the generation of progesterone and testosterone neuroactive metabolites

The enzymatic complex 5\u3b1-reductase (5\u3b1-R) and 3\u3b1/3\u3b2-hydroxysteroid oxidoreductase (HSOR) is expressed in the nervous system, where it transforms progesterone (PROG) and testosterone (T) into neuroactive metabolites. These metabolites regulate myelination, brain maturation, neurotransmission, reproductive behavior and the stress response. The expression of 5\u3b1-R and 3\u3b1-HSOR and the levels of PROG and T reduced metabolites show regional and sex differences in the nervous system and are affected by changing physiological conditions as well as by neurodegenerative and psychiatric disorders. A decrease in their nervous tissue levels may negatively impact the course and outcome of some pathological events. However, in other pathological conditions their increased levels may have a negative impact. Thus, the use of synthetic analogues of these steroids or 5\u3b1-R modulation have been proposed as therapeutic approaches for several nervous system pathologies. However, further research is needed to fully understand the consequences of these manipulations, in particular with 5\u3b1-R inhibitors

Sex differences in steroid levels and steroidogenesis in the nervous system : Physiopathological role

The nervous system, in addition to be a target for steroid hormones, is the source of a variety of neuroactive steroids, which are synthesized and metabolized by neurons and glial cells. Recent evidence indicates that the expression of neurosteroidogenic proteins and enzymes and the levels of neuroactive steroids are different in the nervous system of males and females. We here summarized the state of the art of neuroactive steroids, particularly taking in consideration sex differences occurring in the synthesis and levels of these molecules. In addition, we discuss the consequences of sex differences in neurosteroidogenesis for the function of the nervous system under healthy and pathological conditions and the implications of neuroactive steroids and neurosteroidogenesis for the development of sex-specific therapeutic interventions

Altered methylation pattern of the SRD5A2 gene in the cerebrospinal fluid of post-finasteride patients: A pilot study

Context: Post-finasteride syndrome (PFS) occurs in patients with androgenic alopecia after suspension of the finasteride treatment, leading to a large variety of persistent side effects. Despite the severity of the clinical picture, the mechanism underlying the PFS symptoms onset and persistence is still unclear. Objective: To study whether epigenetic modifications occur in PFS patients. Methods: Retrospective analysis of a multicentric, prospective, longitudinal, case\u2013control clinical trial, enrolling 16 PFS patients, compared to 20 age-matched healthy men. Main outcomes were methylation pattern of SRD5A1 and SRD5A2 promoters and concentration of 11 neuroactive steroids, measured by liquid chromatography-tandem mass spectrometry, in blood and cerebrospinal fluid (CSF) samples. Results: SRD5A1 and SRD5A2 methylation analysis was performed in all blood samples (n = 16 PFS patients and n = 20 controls), in 16 CSF samples from PFS patients and in 13 CSF samples from controls. The SRD5A2 promoter was more frequently methylated in CSF of PFS patients compared to controls (56.3 vs 7.7%). No promoter methylation was detected in blood samples in both groups. No methylation occurred in the SRD5A1 promoter of both groups. Unmethylated controls compared to unmethylated SRD5A2 patients showed higher pregnenolone, dihydrotestosterone and dihydroprogesterone, together with lower testosterone CSF levels. Andrological and neurological assessments did not differ between methylated and unmethylated subjects. Conclusions: For the first time, we demonstrate a tissue-specific methylation pattern of SRD5A2 promoter in PFS patients. Although we cannot conclude whether this pattern is prenatally established or induced by finasteride treatment, it could represent an important mechanism of neuroactive steroid levels and behavioural disturbances previously described in PFS

Evaluation of neuroactive steroid levels by liquid chromatography-tandem mass spectrometry in central and peripheral nervous system : effect of diabetes

The nervous system is a target for physiological and protective effects of neuroactive steroids. Consequently, the assessment of their levels in nervous structures under physiological and pathological conditions is a top priority. To this aim, identification and quantification of pregnenolone (PREG), progesterone (PROG), dihydroprogesterone (DHP), tetrahydroprogesterone (THP), testosterone (T), dihydrotestosterone (DHT), 5aandrostan- 3a, 17b-diol (3a-diol), 17a- and 17b-estradiol (17a-E and 17b-E) by liquid chromatography and tandem mass spectrometry (LC\u2013MS/ MS) has been set up. After validation, this method was applied to determine the levels of neuroactive steroids in central (i.e., cerebral cortex, cerebellum and spinal cord) and peripheral (i.e., brachial nerve) nervous system of control and diabetic rats. In controls only the brachial nerve had detectable levels of all these neuroactive steroids. In contrast, 17a-E in cerebellum, 17a-E, 17b-E, DHP and THP in cerebral cortex, and 17a-E, 17b-E and DHP in spinal cord were under the detection limit. Diabetes, induced by injection with streptozotocin, strongly affected the levels of some neuroactive steroids. In particular, the levels of PREG, PROG and T in cerebellum, of PROG, T and 3a-diol in cerebral cortex, of PROG, DHTand 3a-diol in spinal cord and of PREG, DHP, THP, T, DHTand 3a-diol in brachial nerve were significantly decreased. In conclusion, the data here reported demonstrate that the LC\u2013MS/MS method allows the assessment of neuroactive steroids in the nervous system with high sensitivity and specificity and that diabetes strongly affects their levels, providing a further basis for new therapeutic tools based on neuroactive steroids aimed at counteracting diabetic neuropathy

Gender effect on neurodegeneration and myelin markers in an animal model for multiple sclerosis

Multiple sclerosis (MS) varies considerably in its incidence and progression in females and males. In spite of clinical evidence, relatively few studies have explored molecular mechanisms possibly involved in gender-related differences. The present study describes possible cellular- and molecular-involved markers which are differentially regulated in male and female rats and result in gender-dependent EAE evolution and progression. Attention was focused on markers of myelination (MBP and PDGF\u3b1R) and neuronal distress and/or damage (GABA synthesis enzymes, GAD65 and GAD67, NGF, BDNF and related receptors), in two CNS areas, i.e. spinal cord and cerebellum, which are respectively severely and mildly affected by inflammation and demyelination. Tissues were sampled during acute, relapse/remission and chronic phases and results were analysed by two-way ANOVA

Serotonin transporter (SERT) and translocator protein (TSPO) expression in the obese ob/ob mouse

Background: An ever growing body of evidences is emerging concerning metabolism hormones, neurotransmitters or stress-related biomarkers as effective modulators of eating behavior and body weight in mammals. The present study sought at examining the density and affinity of two proteins related to neurotransmission and cell metabolism, the serotonin transporter SERT and the cholesterol import-benzodiazepine site TSPO (translocator protein), in a rodent leptin-lacking mutant, the obese ob/ob mouse. Binding studies were thus carried out in brain or peripheral tissues, blood platelets (SERT) and kidneys (TSPO), of ob/ob and WT mice supplied with a standard diet, using the selective radiochemical ligands [(3)H]-paroxetine and [(3)H]-PK11195. Results: We observed comparable SERT number or affinity in brain and platelets of ob/ob and WT mice, whilst a significantly higher [(3)H]-PK11195 density was reported in the brain of ob/ob animals. TSPO binding parameters were similar in the kidneys of all tested mice. By [(3)H]-PK11195 autoradiography of coronal hypothalamic-hippocampal sections, an increased TSPO signal was detected in the dentate gyrus (hippocampus) and choroids plexus of ob/ob mice, without appreciable changes in the cortex or hypothalamic-thalamic regions. Conclusions: These findings show that TSPO expression is up-regulated in cerebral regions of ob/ob leptin-deficient mice, suggesting a role of the translocator protein in leptin-dependent CNS trophism and metabolism. Unchanged SERT in mutant mice is discussed herein in the context of previous literature as the forerunner to a deeper biochemical investigation