Conference Agenda

Beclin-1 is a core component of the class III phosphatidylinositol 3-kinase/vacuolar protein sorting 34 (PI3K-III/Vps34) complex, essential for autophagy initiation and for the recruitment of subsequent effectors. Beclin-1 takes part in different protein networks, thus switching its role from supporting autophagy induction to regulating autophagosomal maturation and endosomal trafficking. This protein is critical for embryonic development and is involved in several disorders, including cancer and neurodegenerative diseases. Concerning the nervous system, while assessed in neuronal populations, astrocytes and microglia, its role was far less investigated in myelinating glia, including Schwann cells (SCs), responsible for myelin production in peripheral nerves. Of note, autophagic flux disruption and endosomal trafficking dysregulation are emerging as potential pathophysiological mechanisms underlying Charcot-Marie-Tooth peripheral neuropathies.

To investigate the role of Beclin-1 in SCs, we generated a new mouse line with Beclin-1 ablation specifically in SCs (Becn1 cKO). Becn1 cKO mice displayed a severe demyelination, detectable since early post-natal days and worsening over time, becoming almost complete in adults, and accompanied by progressive involuntary tremors, body weight loss and premature death. Ultrastructural analysis revealed the presence of enlarged SC cytoplasm, with progressive accumulation of intracellular vesicles. To gain better insight in the processes elicited by Beclin-1 ablation we performed transcriptomic analysis from sciatic nerves of pups and 2-month-old mice. This analysis revealed a pro-mitotic alteration of cell cycle at P10, sustained by immunofluorescence experiments indicating increased SC proliferation. Moreover, transcriptomic analysis also pointed to impaired nervous system development and increased immune infiltration at 2 months of age, underlying a neuropathic degeneration that well reflects the progressive motor and sensory functional impairment characterizing Becn1 cKO mice, compared to controls. Our study establishes a further step in understanding key mechanisms in SC development and points to Beclin-1 and its binding partners as novel candidate genes for demyelinating CMT forms.

Immature neurons as well as mature neurons of the peripheral nervous system can repair their axons upon injury, while axon regeneration failure in the central nervous system leads to permanent motor, cognitive and/or sensory disabilities representing a health, social and economic burden. The growing number of nervous system disorders worldwide makes axon regeneration and functional recovery major challenges of public health. The lecture will depict the permessive niche that governs regeneration of the neuromuscular synapse, which arises from the orchestrated contribution of all its cell components. Thus, the NMJ represents a privileged point of observation, whose injury response provides a valuable source of pro-regenerative candidates to be tested in different forms of nerve damage.

Schwann cells (SCs) involvement in neurotransmission has been understudied for a long time. In the early 2000s, the general term of gliotransmission has been proposed, even still controversial. In the last decade, anyway, some observations stated the participation of SCs in purinergic, cholinergic, glutamatergic as well as GABAergic peripheral neurotransmission. In particular, it was found that SCs in vivo and in vitro express the GABA receptors, i.e. GABA-A and GABA-B, and the GABA synthetic machinery. The GABA receptors mediate some autocrine actions in SCs, where participate in the regulation of proliferation, differentiation, axonal sorting, and myelination, likely during the peripheral nervous system development. Undoubtedly, the SCs are a source of endogenous GABA, which takes an important role also in peripheral glia-to-neuron cross-talk. In this regard, we found that the axons of DRG neurons, especially those forming the nociceptive C-fibres, are depolarized by GABA-mediated currents, thus shaping the nociceptor computation process. The axonal responses to GABA are coupled to the Na+/K+/Cl- cotransporter (NKCC1) and secondary to the Cl- conductance. The regulation of axonal GABA-A receptor and Cl- concentration, therefore, might be a promising strategy to control C-fiber hyperexcitability. Furthermore, the molecular mechanism and the potential triggers underlying the tonic GABA release by SCs have been investigated. We found GABA transaminase (GABA-T) and GABA transporters (GAT-1 and GAT-3) expressed in SCs, suggesting that they may be involved in GABA release, although the involvement of other anion channels cannot be excluded. In conclusion, our findings shed light on a new functional role of GABA in peripheral SC-axon cross-talk, with relevance to the mechanisms modulating the nociception in physio-pathological conditions.

Schwann cells (SCs) play a strategic role after peripheral nerve injury, driving axons regeneration and regulating local inflammation Recently we demonstrated the presence of α7 nicotinic acetylcholine receptors (nAChRs) in rat SCs. α7 nAChR is expressed in SCs after peripheral nerve axotomy and its expression is significantly enhanced after 24 h when sciatic nerve segments are cultured alone or in presence of the proinflammatory neuropeptide Bradykinin (BK). To clarify the role that α7 nAChRs play after peripheral axon damage, we investigated the signal transduction pathways triggered by receptor activation and the effects produced by their activation. Both ionotropic and metabotropic signaling pathway were analyzed by calcium imaging and Western blot analysis, respectively, following α7 nAChR activation by partial agonist ICH3. In addition, the expression of c-Jun was evaluated by immunocytochemistry and Western blot analysis. Finally, the cell migration was studied by a wound healing assay. Activation of α7 nAChRs by the selective partial agonist ICH3, did not induce calcium mobilization in SCs but positively modulated the metabotropic pathway involving PI3K/AKT/mTORC1 axis. Activation of the mTORC1 complex was confirmed by the up-regulated expression of its specific p-p70 S6K^Thr389 target. Moreover, the up-regulation of p-AMPK^Thr172, a negative regulator of myelination, was also observed concomitantly to an increased nuclear accumulation of the transcription factor c-Jun. Cell migration and morphology analyses demonstrate that α7 nAChR activation also promotes SCs migration. Moreover α7 nAChRs caused an upregulation of uPA, MMP2 and MMP9 activity and decrease the IL-6 production and release. These results demonstrate that ACh, probably released from regenerating axons or by SC themselves, may actively promote, through α7 nAChRs activation the Repair Schwann Cell phenotype and contribute to favour an anti-inflammatory microenvironment. These conditions are relevant to support the peripheral nerve regeneration.