Federico Abbiati

Comparison of plasma and cerebrospinal fluid levels of neuroactive steroids with their brain, spinal cord and peripheral nerve levels in male and female rats.

Physiological changes and pathological alterations in the nervous system of rodents are associated with modifications in the levels of neuroactive steroids in the brain, spinal cord and/or peripheral nerves. Measures of tissue levels of steroids in the nervous system present serious limitations for human studies and for longitudinal studies in animals. In this study we have explored whether levels of neuroactive steroids in plasma and the cerebrospinal fluid reflect their levels in neural tissues. To this aim, we have evaluated by liquid chromatography-tandem mass spectrometry the levels of several neuroactive steroids in plasma, cerebrospinal fluid, cerebral cortex, cerebellum, hippocampus, spinal cord and sciatic nerve of male and female rats. Data indicate that plasma and cerebrospinal fluid levels of steroids do not fully reflect their tissue levels. However, the interindividual variations in the levels of all the steroids assessed, with the exception of dehydroepiandrosterone, showed a positive correlation in plasma and cerebral cortex. Most steroids also showed a positive correlation in plasma and the cerebellum, the spinal cord and the sciatic nerve. In the hippocampus, the levels of tetrahydroprogesterone, testosterone and testosterone metabolites showed a significant positive correlation with their respective levels in plasma. The cerebrospinal fluid levels of some steroids, such as testosterone and dihydrotestosterone, showed a full correlation with tissue levels. In addition, cerebrospinal fluid levels of pregnenolone, progesterone, and 17β-estradiol showed a positive correlation with their corresponding levels in the majority of the neural structures analyzed. These findings suggest that the levels of some neuroactive steroids in cerebrospinal fluid as well as in plasma may be valuable to predict their levels in the nervous system.

Activation of the Liver X Receptor Increases Neuroactive Steroid Levels and Protects from Diabetes-Induced Peripheral Neuropathy

Neuroactive steroids act in the peripheral nervous system as physiological regulators and as protective agents for acquired or inherited peripheral neuropathy. In recent years, modulation of neuroactive steroids levels has been studied as a potential therapeutic approach to protect peripheral nerves from damage induced by diabetes. Nuclear receptors of the liver X receptor (LXR) family regulate adrenal steroidogenesis via their ability to control cholesterol homeostasis. Here we show that rat sciatic nerve expresses both LRXα and β isoforms and that these receptors are functional. Activation of liver X receptors using a synthetic ligand results in increased levels of neurosteroids and protection of the sciatic nerve from neuropathy induced by diabetes. LXR ligand treatment of streptozotocin-treated rats increases expression in the sciatic nerve of steroidogenic acute regulatory protein (a molecule involved in the transfer of cholesterol into mitochondria), of the enzyme P450scc (responsible for conversion of cholesterol into pregnenolone), of 5α-reductase (an enzyme involved in the generation of neuroactive steroids) and of classical LXR targets involved in cholesterol efflux, such as ABCA1 and ABCG1. These effects were associated with increased levels of neuroactive steroids (e.g., pregnenolone, progesterone, dihydroprogesterone and 3α-diol) in the sciatic nerve, and with neuroprotective effects on thermal nociceptive activity, nerve conduction velocity, and Na+, K+-ATPase activity. These results suggest that LXR activation may represent a new pharmacological avenue to increase local neuroactive steroid levels that exert neuroprotective effects in diabetic neuropathy.

Neuroactive Steroid Levels are Modified in Cerebrospinal Fluid and Plasma of Post‐Finasteride Patients Showing Persistent Sexual Side Effects and Anxious/Depressive Symptomatology

INTRODUCTION Observations performed in a subset of subjects treated with finasteride (an inhibitor of the enzyme 5α-reductase) for male pattern hair loss seem to indicate that sexual dysfunction as well as anxious/depressive symptomatology may occur at the end of the treatment and continue after discontinuation. AIM A possible hypothesis to explain depression symptoms after finasteride treatment might be impairment in the levels of neuroactive steroids. Therefore, neuroactive steroid levels were evaluated in paired plasma and cerebrospinal fluid samples obtained from male patients who received finasteride for the treatment of androgenic alopecia and who, after drug discontinuation, still show long-term sexual side effects as well as anxious/depressive symptomatology. METHODS The levels of neuroactive steroids were evaluated by liquid chromatography-tandem mass spectrometry in three postfinasteride patients and compared to those of five healthy controls. MAIN OUTCOME MEASURES Neuroactive steroid levels in plasma and cerebrospinal fluid of postfinasteride patients and healthy controls. RESULTS At the examination, the three postfinasteride patients reported muscular stiffness, cramps, tremors, and chronic fatigue in the absence of clinical evidence of any muscular disorder or strength reduction. Severity and frequency of the anxious/depressive symptoms were quite variable; overall, all the subjects had a fairly complex and constant neuropsychiatric pattern. Assessment of neuroactive steroid levels in patients showed some interindividual differences. However, the most important finding was the comparison of their neuroactive steroid levels with those of healthy controls. Indeed, decreased levels of tetrahydroprogesterone, isopregnanolone and dihydrotestosterone and increased levels of testosterone and 17β-estradiol were reported in cerebrospinal fluid of postfinasteride patients. Moreover, decreased levels of dihydroprogesterone and increased levels of 5α-androstane-3α,17β-diol and 17β-estradiol were observed in plasma. CONCLUSION The present observations confirm that an impairment of neuroactive steroid levels, associated with depression symptoms, is still present in androgenic alopecia patients treated with finasteride despite the discontinuation of the treatment.

Age-related changes in neuroactive steroid levels in 3xTg-AD mice.

Although neuroactive steroids exert neuroprotective actions in different experimental models of neurodegenerative diseases, including those of Alzheimers disease (AD), their relationships with aged related physiologic and pathologic brain changes remain to be clarified. In this study the levels of pregnenolone, dehydroepiandrosterone, progesterone, dihydroprogesterone, tetrahydroprogesterone, isopregnanolone, testosterone, dihydrotestosterone, 5α-androstane-3α,17β-diol, 5α-androstane-3β,17β-diol, 17α-estradiol, and 17β-estradiol were assessed in the limbic region of young adult (7 months) and aged (24 months) male wild type and triple transgenic AD mice. Age related neuropathological changes in AD brains, such as β-amyloid accumulation and gliosis, were associated with modified levels of specific neuroactive steroids and particularly with changes in the levels of progesterone and testosterone metabolites. The altered levels of neuroactive steroids in aged AD brains might impact on the activation of neuroprotective signaling mediated by classic and nonclassic steroid receptors, like the gamma-aminobuttyric acid (GABA)-A receptor.

Diabetes-induced myelin abnormalities are associated with an altered lipid pattern: protective effects of LXR activation

Diabetic peripheral neuropathy (DPN) is characterized by myelin abnormalities; however, the molecular mechanisms underlying such deficits remain obscure. To uncover the effects of diabetes on myelin alterations, we have analyzed myelin composition. In a streptozotocin-treated rat model of diabetic neuropathy, analysis of sciatic nerve myelin lipids revealed that diabetes alters myelins phospholipid, FA, and cholesterol content in a pattern that can modify membrane fluidity. Reduced expression of relevant genes in the FA biosynthetic pathway and decreased levels of the transcriptionally active form of the lipogenic factor sterol-regulatory element binding factor-1c (SREBF-1c) were found in diabetic sciatic nerve. Expression of myelins major protein, myelin protein zero (P0), was also suppressed by diabetes. In addition, we confirmed that diabetes induces sciatic nerve myelin abnormalities, primarily infoldings that have previously been associated with altered membrane fluidity. In a diabetic setting, synthetic activator of the nuclear receptor liver X receptor (LXR) increased SREBF-1c function and restored myelin lipid species and P0 expression levels to normal. These LXR-modulated improvements were associated with restored myelin structure in sciatic nerve and enhanced performance in functional tests such as thermal nociceptive threshold and nerve conduction velocity. These findings demonstrate an important role for the LXR-SREBF-1c axis in protection from diabetes-induced myelin abnormalities.

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.

LXR and TSPO as new therapeutic targets to increase the levels of neuroactive steroids in the central nervous system of diabetic animals

Neuroactive steroid levels are decreased in the central nervous system (CNS) of streptozotocin (STZ) diabetic rats. In agreement, they exert protective effects in this experimental model, counteracting degenerative events occurring in the CNS. Therefore, an interesting therapeutic strategy could be to increase their levels directly in the CNS. In this study we have evaluated whether activation of translocator protein-18kDa (TSPO) or liver X receptors (LXRs) may affect the levels of neuroactive steroids present in the CNS of diabetic and non-diabetic animals. We observed that the treatment with either Ro5-4864 (i.e., a ligand of TSPO) or with GW3965 (i.e., a ligand of LXRs) induced an increase of neuroactive steroids in the spinal cord, the cerebellum and the cerebral cortex of STZ-rats, but not in the CNS of non-pathological animals. Interestingly, the pattern of induction was different among the three CNS areas analyzed and between the two pharmacological tools. In particular, the activation of LXRs might represent a promising neuroprotective strategy, because the treatment with GW3965, at variance to Ro5-4864 treatment, did not induce significant changes in the plasma levels of neuroactive steroids. This suggests that activation of LXRs may selectively increase the CNS levels of neuroactive steroids avoiding possible endocrine side effects exerted by the systemic treatment with these molecules. Interestingly GW3965 treatment induced an increase of dihydroprogesterone in the spinal cord of diabetic animals in association with an increase of myelin basic protein expression. Thus we demonstrated that LXR activation was able to rescue CNS symptoms of diabetes.

Modifications of neuroactive steroid levels in an experimental model of nigrostriatal degeneration: potential relevance to the pathophysiology of Parkinson's disease.

An important link between neuroactive steroids and neurodegenerative disorders has recently been suggested. Indeed, in several neurodegenerative experimental models the levels of neuroactive steroids are affected and their administration exerts neuroprotective effects. However, scarce information has so far been obtained on the neuroactive steroid levels present in Parkinson’s disease. To this aim, using an experimental model of loss of nigrostriatal dopaminergic neurons obtained by stereotaxic injection of the neurotoxin 6-hydroxydopamine (6-OHDA), we evaluated by liquid chromatography tandem mass spectrometry the levels of several neuroactive steroids in the striatum and cerebral cortex of 6-OHDA-lesioned male rats. Among the neuroactive steroid levels assessed (i.e., pregnenolone, progesterone, dihydroprogesterone, tetrahydroprogesterone, isopregnanolone, testosterone, dihydrotestosterone, 3α-diol, dehydroepiandrosterone, 17α-estradiol, and 17β-estradiol), we observed a significant decrease of pregnenolone in the striatum. A similar effect was also observed on the levels of dihydroprogesterone present in this cerebral area and also in the cerebral cortex. Interestingly, an increase of isopregnanolone also occurred in the striatum and in the cerebral cortex. Altogether, these results suggesting that progesterone metabolism is affected in an experimental model of Parkinson’s disease further highlight the link between neuroactive steroids and the neurodegenerative diseases.

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.

Sex differences in the manifestation of peripheral diabetic neuropathy in gonadectomized rats: A correlation with the levels of neuroactive steroids in the sciatic nerve

Clinical observations suggest a sex-dimorphism in the incidence and symptomatology of diabetic neuropathy, but this possible gender effect has never been investigated in detail in a well-characterized experimental model such as streptozotocin (STZ)-induced diabetes. Therefore, in this study we have compared with a multimodal set of tests the impact of diabetes on the sciatic nerve in male and female rats. To assess whether sex-dimorphism in peripheral diabetic neuropathy is dependent on gonadal hormones we have also analyzed the effect of ovariectomy and orchidectomy on the sciatic nerve of STZ-diabetic rats. Nerve conduction velocity (NCV), Na(+),K(+)-ATPase activity, expression of myelin proteins, thermal sensitivity and reactive oxygen species production were similarly affected in male and female animals by STZ. However, ovariectomy, but not orchidectomy, significantly counteracted STZ-induced alterations on NCV, Na(+),K(+)-ATPase activity, and expression of myelin proteins. This effect of ovariactomy was associated to an increase in the levels of neuroactive steroids, such as dehydroepiandrosterone, testosterone and dihydrotestosterone, in the sciatic nerve of diabetic rats. These neuroactive steroids have been demonstrated to be protective agents in this experimental model of diabetic neuropathy. However, their efficacy has been so far tested only in male animals. Therefore, the present data might represent an important background to evaluate their efficacy also in female diabetic animals.