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 \u3b5 (PKC\u3b5), 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 PKC\u3b5 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 PKC\u3b5 gene expression in DRG neurons. Also the membrane expression of PKC\u3b5 phosphorylated form resulted to be modulated in similar way. These findings suggest a possible involvement of PKC\u3b5 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

Schwann Cell Autocrine and Paracrine Regulatory Mechanisms, Mediated by Allopregnanolone and BDNF, Modulate PKCε in Peripheral Sensory Neurons

Protein kinase type C-\u3b5 (PKC\u3b5) plays important roles in the sensitization of primary afferent nociceptors, such as ion channel phosphorylation, that in turn promotes mechanical hyperalgesia and pain chronification. In these neurons, PKC\u3b5 is modulated through the local release of mediators by the surrounding Schwann cells (SCs). The progesterone metabolite allopregnanolone (ALLO) is endogenously synthesized by SCs, whereas it has proven to be a crucial mediator of neuron-glia interaction in peripheral nerve fibers. Biomolecular and pharmacological studies on rat primary SCs and dorsal root ganglia (DRG) neuronal cultures were aimed at investigating the hypothesis that ALLO modulates neuronal PKC\u3b5, playing a role in peripheral nociception. We found that SCs tonically release ALLO, which, in turn, autocrinally upregulated the synthesis of the growth factor brain-derived neurotrophic factor (BDNF). Subsequently, glial BDNF paracrinally activates PKC\u3b5 via trkB in DRG sensory neurons. Herein, we report a novel mechanism of SCs-neuron cross-talk in the peripheral nervous system, highlighting a key role of ALLO and BDNF in nociceptor sensitization. These findings emphasize promising targets for inhibiting the development and chronification of neuropathic pain

Expression of membrane progesterone receptors (mPRs) in rat peripheral glial cell membranes and their potential role in the modulation of cell migration and protein expression

The role played by progestogens in modulating Schwann cell pathophysiology is well established. Progestogens exert their effects in these cells through both classical genomic and non-genomic mechanisms, the latter mediated by the GABA-A receptor. However, there is evidence that other receptors may be involved. Membrane progesterone receptors (mPRs) are novel 7-transmembrane receptors coupled to G proteins that have been characterized in different tissues and cells, including the central nervous system (CNS). The mPRs were shown to mediate some of progestogens' neuroprotective effects in the CNS, and to be upregulated in glial cells after traumatic brain injury. Based on this evidence, this paper investigated the possible involvement of mPRs in mediating progestogen actions in S42 Schwann cells. All five mPR isoforms and progesterone receptor membrane component 1 (PGRMC1) were detected in Schwann cells, and were present on the cell membrane. Progesterone and the mPR-specific agonist, Org-OD-02-0 (02) bound to these membranes, indicating the presence of functional mPRs. The mPR agonist 02 rapidly increased cell migration in an in vitro assay, suggesting a putative role of mPRs in the nerve regeneration process. Treatment with pertussis toxin, and 8-Br-cAMP blocked 02-induced cell migration, suggesting this progestogen action is mediated by activation of an inhibitory G protein leading to a decrease in intracellular cAMP levels. In contrast, long-term mPR activation led to increased expression levels of myelin associated glycoprotein (MAG). Taken together, these findings show that mPRs are present and active in Schwann cells and have a role in modulating their physiological processes

Membrane Progesterone Receptors (mPRs/PAQRs) Differently Regulate Migration, Proliferation, and Differentiation in Rat Schwann Cells

Several studies in the last decade demonstrated that progestogens play an important role in the biology of Schwann cells, the main neuroglial cells of the peripheral nervous system. Since a recent study showed that the S42 rat Schwann cell line expressed membrane progesterone receptors (mPRs), members of the PAQR family, in this study, we examined mPR expression in a more physiological model, primary rat Schwann cells. We demonstrated that primary rat Schwann cells show a different pattern of mPR expression compared to the previously studied model; mPR\u3b1 (PAQR7) and \u3b2 (PAQR8) isoforms were the major mPR members identified, with different sub-cellular localizations. Activation of the nuclear progesterone receptor (PR) with the specific agonist R5020 upregulated mPR expression, while activation of mPRs with the specific agonist Org OD 02-0 changed their sub-cellular localization. An in-depth analysis revealed additional effects of mPR activation, such as AKT activation, reduced expression of the myelin-associated glycoprotein (MAG), morphological changes, altered expression of several Schwann cell differentiation markers, and increased Schwann cell migration and proliferation. In conclusion, we identified mPR\u3b1 and mPR\u3b2 in primary rat Schwann cells, and our findings suggest a putative role for mPRs in the regulation of Schwann cell migration, proliferation, and differentiation. Therefore, mPRs are a potential pharmacological target for Schwann cell\u2013related disorders and neurodegenerative diseases, especially those in which Schwann cell\u2013mediated axon remyelination is desirable

Schwann cell development, maturation and regeneration : a focus on classic and emerging intracellular signaling pathways

The development, maturation and regeneration of Schwann cells (SCs), the main glial cells of the peripheral nervous system, require the coordinate and complementary interaction among several factors, signals and intracellular pathways. These regulatory molecules consist of integrins, neuregulins, growth factors, hormones, neurotransmitters, as well as entire intracellular pathways including protein-kinase A, C, Akt, Erk/MAPK, Hippo, mTOR, etc. For instance, Hippo pathway is overall involved in proliferation, apoptosis, regeneration and organ size control, being crucial in cancer proliferation process. In SCs, Hippo is linked to merlin and YAP/TAZ signaling and it seems to respond to mechanic/physical challenges. Recently, among factors regulating SCs, also the signaling intermediates Src tyrosine kinase and focal adhesion kinase (FAK) proved relevant for SC fate, participating in the regulation of adhesion, motility, migration and in vitro myelination. In SCs, the factors Src and FAK are regulated by the neuroactive steroid allopregnanolone, thus corroborating the importance of this steroid in the control of SC maturation. In this review, we illustrate some old and novel signaling pathways modulating SC biology and functions during the different developmental, mature and regenerative states

GABA-B1 Receptor-Null Schwann Cells Exhibit Compromised In Vitro Myelination

GABA-B receptors are important for Schwann cell (SC) commitment to a non-myelinating phenotype during development. However, the P0-GABA-B1fl/fl conditional knockout mice, lacking the GABA-B1 receptor specifically in SCs, also presented axon modifications, suggesting SC non-autonomous effects through the neuronal compartment. In this in vitro study, we evaluated whether the specific deletion of the GABA-B1 receptor in SCs may induce autonomous or non-autonomous cross-changes in sensory dorsal root ganglia (DRG) neurons. To this end, we performed an in vitro biomolecular and transcriptomic analysis of SC and DRG neuron primary cultures from P0-GABA-B1fl/fl mice. We found that cells from conditional P0-GABA-B1fl/fl mice exhibited proliferative, migratory and myelinating alterations. Moreover, we found transcriptomic changes in novel molecules that are involved in peripheral neuron-SC interaction

The biology of platelet-rich plasma and its application in oral surgery: literature review.

Item does not contain fulltextPlatelet-rich plasma (PRP) is a new approach in tissue regeneration and a developing area for clinicians and researchers. It is used in various surgical fields, including oral and maxillofacial surgery. PRP is prepared from the patient's own blood and contains growth factors that influence wound healing. Of these growth factors, platelet-derived growth factor, transforming growth factor, insulin-like growth factor, and epidermal growth factor play a pivotal role in tissue repair mechanisms. Although the growth factors and mechanisms involved are still poorly understood, the easy application of PRP in the clinic and its possible beneficial outcome, including reduction of bleeding, rapid soft tissue healing, and bone regeneration, hold promise for new treatment approaches. However, animal studies and human trials demonstrate conflicting results regarding the application of PRP. Therefore the aim of this literature review is to evaluate the scientific evidence regarding the use of PRP in dentistry, to describe the different bioactive substances included in PRP and their participation in the healing process, to elucidate the different techniques and available technology for PRP preparation, to review animal and human studies, to clarify risks, and to provide guidance for future research