MOLECULAR BASIS FOR THE DEVELOPMENT OF INNOVATIVE THERAPIES FOR PERIPHERAL NEUROPATHIES TREATMENT: ROLE AND CROSS-REGULATION OF THE GABAERGIC SYSTEM AND NEUROACTIVE STEROIDS

Peripheral neuropathies are a heterogeneous group of pathologies with a high prevalence worldwide, which are characterized by alterations of peripheral nerves structure and function. Their treatment is currently a challenge for clinicians. Indeed, even if continuous progresses are made in the study of the basic mechanisms underlying these pathologies, etiology is still unknown in a significant number of cases. Different compounds, such as growth factors, adhesion proteins, neurotransmitters, enzymes, peptides and neuroactive steroids, have been proposed to play important roles in the patho-physiology of the peripheral nervous system. Therefore, most of the research is addressed to identify the molecules that might represent the more promising therapy for this set of pathologies. This thesis focuses on some aspects of the patho-physiological role of the GABAergic system and neuroactive steroids in the peripheral nervous system. Several papers in literature strongly support the hypothesis that they are both present and active in the peripheral nervous system, in particular in Schwann cells, the myelinating cells of the peripheral nervous system. These cells are indeed able to synthesize GABA and neuroactive steroids and express both the ionotropic GABA-A and the metabotropic GABA-B receptor. In order to deepen the knowledge on this topic, four research lines were pursued in my PhD program and are described in this thesis. The first line regarded the analysis of the effects of specific GABA-B ligands on nerve regeneration in a model of neuropathic pain caused by nerve ligation. These studies showed that the specific GABA-B antagonist CGP56433 was able to recover some morphological, functional and biochemical parameters in peripheral nerves. Surprisingly, some of these effects were potentiated by the co-treatment with GABA-B specific agonist baclofen, suggesting the co-activation of possible central and peripheral mechanisms. The second research line regarded the analysis of different GABA-A subunits in dorsal root ganglia (DRG) neurons of a model of conditional knockout mice, in which the GABA-B1 receptor is specifically deleted in Schwann cells. The results showed a modulation of different GABA-A subunits, pointing to a down-regulation of GABA-A receptors, mainly regarding the synaptic ones. This evidence may contribute to understand some of the alterations that were previously observed in this conditional knockout mouse model. The third research line dealt with the study of the modulation of protein kinase C-type ε (PKCε), an important neuropathic pain mediator, and its possible cross-talk with the GABA-A receptor and the neuroactive steroid allopregnanolone. The results showed that allopregnanolone down-modulates PKCε expression in Schwann cells, but the direct treatment on DRG neurons did not lead to any significant effect. However, Schwann cells conditioned medium was able to induce a significant up-regulation of PKCε gene expression in DRG neurons. Also the membrane expression of PKCε phosphorylated form resulted to be modulated in similar way. These findings suggest a possible involvement of PKCε in the GABA-A mediated control of pain transmission exerted by allopregnanolone, also pointing out to a Schwann cell-mediated process. Finally, the fourth research line regarded the identification of a novel family of progestogen receptors localized on the cell membrane (mPRs) and PGRMC1 in Schwann cells; moreover, their putative role in the modulation of Schwann cell physiology was also investigated. The data demonstrated the expression of these receptors in Schwann cell plasma membrane. The treatment with a specific mPR agonist proved able to induce cell migration at short time points (2-4 hours) and increased the expression of myelin associated glycoprotein (MAG) at longer time points (24-36 hours), giving a first demonstration of a role for these receptors in Schwann cells. The identification of this new signaling pathway will allow a better understanding of progestogen actions in Schwann cells. In conclusion, the results presented in this thesis shed some light on some basic mechanism controlling the patho-physiology of the peripheral nervous system, whose comprehension may lead to the identification of new more specific drugs for peripheral neuropathies treatment.