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

Progesterone derivatives increase expression of Krox-20 and Sox-10 in rat Schwann cells

Neuroactive steroids, like progesterone (P) and its 5alpha-reduced derivatives dihydroprogesterone (DHP) and tetrahydroprogesterone (THP), are involved in the control of Schwann cell proliferation and in the myelinating program of these cells. Here, we demonstrate that in culture of rat Schwann cells, P and its derivatives also increase expression of Sox-10 and Krox-20 (i.e., two transcription factors with a key role in Schwann cell physiology and in their myelinating program). Data obtained by quantitative RT-PCR analysis show that treatment with P, DHP, or THP increases mRNA levels of Krox-20. This stimulatory effect anticipates that exerted by P and DHP on Sox-10 gene expression. Thus, although the effect on Krox-20 occurs after 1 h, that on Sox-10 reaches a peak after 2 h. A similar pattern of effect is also evident on their protein levels. As evaluated by Western blot analysis, Krox-20 is increased after 3 h of treatment with P, DHP, or THP, whereas P or DHP stimulates the expression of Sox-10 after 6 h of exposure. A computer analysis performed on rat and human promoters of these two transcription factors shows that putative P-responsive elements are present in Krox-20 but not in Sox-10. Interestingly, many putative binding sites for Krox-20 are present in the Sox-10 promoter. The observations reported here, together with the concept that P and its derivatives are able to influence directly the expression of myelin proteins, suggest that these neuroactive steroids might coordinate the Schwann cell-myelinating program utilizing different intracellular pathways

Neuroactive Steroid Levels in a transgenic rat model of CMT1A Neuropathy.

Charcot-Marie-Tooth type 1A (CMT1A) represents 80% of all the demyelinating hereditary motor and sensory neuropathies. As recently suggested, neuroactive steroids may have a role in a therapeutic strategy for peripheral neuropathies, including CMT1A. To this aim, an accurate qualitative and quantitative analysis of neuroactive steroid levels in this disease could be extremely important to define effective pharmacological strategies. We here analyzed by liquid chromatography-tandem mass spectrometry the levels of neuroactive steroids present in the sciatic nerve of male and female peripheral myelin protein 22 transgenic rats (PMP22(tg) rats; i.e., an experimental model of CMT1A) and of the corresponding wild-type littermates. We observed that, both in PMP22(tg) rats and in the wild types, the levels of neuroactive steroids, such as progesterone, tetrahydroprogesterone (THP), isopregnanolone (3 beta,5 alpha-THP), testosterone, dihydrotestosterone, and 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) are sexually dimorphic. It is interesting to note that the levels of 3 beta,5 alpha-THP and of 3 alpha-diol, which are exclusively detectable in sciatic nerve of female and male rats, respectively, are strongly decreased in PMP22(tg) rats. 3 beta,5 alpha-THP and 3 alpha-diol are modulators of gamma-amino butyric acid A receptor. Thus, the present findings may be considered an interesting background for experiments aimed to evaluate the possible therapeutic effects of modulators of this neurotransmitter receptor in male and female PMP22(tg) rats

Neuroactive steroids: a therapeutic approach to maintain peripheral nerve integrity during neurodegenerative events

It is now well known that peripheral nerves are a target for the action of neuroactive steroids. This review summarizes observations obtained so far, indicating that through the interaction with classical and nonclassical steroid receptors, neuroactive steroids (e.g., progesterone, testosterone and their derivatives, estrogens, etc.) are able to influence several parameters of the peripheral nervous system, particularly its glial compartment (i.e., Schwann cells). Interestingly, some of these neuroactive steroids might be considered as promising neuroprotective agents. They are able to counteract neurodegenerative events of rat peripheral nerves occurring after experimental physical trauma, during the aging process, or in hereditary demyelinating diseases. On this basis, the hypothesis that neuroactive steroids might represent a new therapeutic strategy for peripheral neuropathy is proposed

Docetaxel-induced peripheral neuropathy: protective effects of dihydroprogesterone and progesterone in an experimental model

Peripheral neurotoxicity is a frequent complication limiting docetaxel chemotherapy in patients with cancer. We developed an experimental model that closely mimics the course of neuropathy in patients, aiming to investigate both the mechanisms of neurotoxicity at biochemical, functional and morphological levels and the potential neuroprotective role of neuroactive steroids. We demonstrated that treatment with dihydroprogesterone (DHP) or progesterone (P) counteracts docetaxel-induced neuropathy, preventing nerve conduction and thermal threshold changes, and degeneration of skin nerves in the foodpad. Neuroactive steroids also counteract the changes in gene expression of several myelin proteins and calcitonin gene-related peptide induced by docetaxel in sciatic nerve and lumbar spinal cord, respectively. Most nerve abnormalities observed during the treatment with docetaxel spontaneously recovered after drug withdrawal, similarly to what occurs in patients. However, results of midterm follow-up experiments indicated that animals cotreated with DHP or P have a faster recovery of the neuropathy compared with docetaxel-treated rats. Our study confirmed that neuroactive steroids exert a protective effect on peripheral nerves at different levels, suggesting that they might represent a new therapeutic frontier for patients with chemotherapy-induced neuropathy