Analysis of Signal Transduction Pathways Downstream M2 Receptor Activation: Effects on Schwann Cell Migration and Morphology.

BACKGROUND: Schwann cells (SCs) express cholinergic receptors, suggesting a role of cholinergic signaling in the control of SC proliferation, differentiation and/or myelination. Our previous studies largely demonstrated that the pharmacological activation of the M2 muscarinic receptor subtype caused an inhibition of cell proliferation and promoted the expression of pro-myelinating differentiation genes. In order to elucidate the molecular signaling activated downstream the M2 receptor activation, in the present study we investigated the signal transduction pathways activated by the M2 orthosteric agonist arecaidine propargyl ester (APE) in SCs. METHODS: Using Western blot we analyzed some components of the noncanonical pathways involving ß1-arrestin and PI3K/AKT/mTORC1 signaling. A wound healing assay was used to evaluate SC migration. RESULTS: Our results demonstrated that M2 receptor activation negatively modulated the PI3K/Akt/mTORC1 axis, possibly through ß1-arrestin downregulation. The involvement of the mTORC1 complex was also supported by the decreased expression of its specific target p-p70 S6KThr389. Then, we also analyzed the expression of p-AMPKαthr172, a negative regulator of myelination that resulted in reduced levels after M2 agonist treatment. The analysis of cell migration and morphology allowed us to demonstrate that M2 receptor activation caused an arrest of SC migration and modified cell morphology probably by the modulation of ß1-arrestin/cofilin-1 and PKCα expression, respectively. CONCLUSIONS: The data obtained demonstrated that M2 receptor activation in addition to the canonical Gi protein-coupled pathway modulates noncanonical pathways involving the mTORC1 complex and other kinases whose activation may contribute to the inhibition of SC proliferation and migration and address SC differentiation.

The Mechanisms Mediated by α7 Acetylcholine Nicotinic Receptors May Contribute to Peripheral Nerve Regeneration.

Due to the microenvironment created by Schwann cell (SC) activity, peripheral nerve fibers are able to regenerate. Inflammation is the first response to nerve damage and the removal of cellular and myelin debris is essential in preventing the persistence of the local inflammation that may negatively affect nerve regeneration. Acetylcholine (ACh) is one of the neurotransmitters involved in the modulation of inflammation through the activity of its receptors, belonging to both the muscarinic and nicotinic classes. In this report, we evaluated the expression of α7 nicotinic acetylcholine receptors (nAChRs) in rat sciatic nerve, particularly in SCs, after peripheral nerve injury. α7 nAChRs are absent in sciatic nerve immediately after dissection, but their expression is significantly enhanced in SCs after 24 h in cultured sciatic nerve segments or in the presence of the proinflammatory neuropeptide Bradykinin (BK). Moreover, we found that activation of α7 nAChRs with the selective partial agonist ICH3 causes a decreased expression of c-Jun and an upregulation of uPA, MMP2 and MMP9 activity. In addition, ICH3 treatment inhibits IL-6 transcript level expression as well as the cytokine release. These results suggest that ACh, probably released from regenerating axons or by SC themselves, may actively promote through α7 nAChRs activation an anti-inflammatory microenvironment that contributes to better improving the peripheral nerve regeneration.

Cholinergic Modulation of Neuroinflammation: Focus on α7 Nicotinic Receptor.

All nervous system pathologies (e.g., neurodegenerative/demyelinating diseases and brain tumours) develop neuroinflammation, a beneficial process during pathological events, aimed at removing damaged cells, toxic agents, and/or pathogens. Unfortunately, excessive inflammation frequently occurs during nervous system disorders, becoming a detrimental event capable of enhancing neurons and myelinating glial cell impairment, rather than improving their survival and activity. Consequently, targeting the neuroinflammation could be relevant for reducing brain injury and rescuing neuronal and glial cell functions. Several studies have highlighted the role of acetylcholine and its receptors in the regulation of central and peripheral inflammation. In particular, α7 nicotinic receptor has been described as one of the main regulators of the "brain cholinergic anti-inflammatory pathway". Its expression in astrocytes and microglial cells and the ability to modulate anti-inflammatory cytokines make this receptor a new interesting therapeutic target for neuroinflammation regulation. In this review, we summarize the distribution and physiological functions of the α7 nicotinic receptor in glial cells (astrocytes and microglia) and its role in the modulation of neuroinflammation. Moreover, we explore how its altered expression and function contribute to the development of different neurological pathologies and exacerbate neuroinflammatory processes.

Expression of Cholinergic Markers and Characterization of Splice Variants during Ontogenesis of Rat Dorsal Root Ganglia Neurons.

Dorsal root ganglia (DRG) neurons synthesize acetylcholine (ACh), in addition to their peptidergic nature. They also release ACh and are cholinoceptive, as they express cholinergic receptors. During gangliogenesis, ACh plays an important role in neuronal differentiation, modulating neuritic outgrowth and neurospecific gene expression. Starting from these data, we studied the expression of choline acetyltransferase (ChAT) and vesicular ACh transporter (VAChT) expression in rat DRG neurons. ChAT and VAChT genes are arranged in a "cholinergic locus", and several splice variants have been described. Using selective primers, we characterized splice variants of these cholinergic markers, demonstrating that rat DRGs express R1, R2, M, and N variants for ChAT and V1, V2, R1, and R2 splice variants for VAChT. Moreover, by RT-PCR analysis, we observed a progressive decrease in ChAT and VAChT transcripts from the late embryonic developmental stage (E18) to postnatal P2 and P15 and in the adult DRG. Interestingly, Western blot analyses and activity assays demonstrated that ChAT levels significantly increased during DRG ontogenesis. The modulated expression of different ChAT and VAChT splice variants during development suggests a possible differential regulation of cholinergic marker expression in sensory neurons and confirms multiple roles for ACh in DRG neurons, both in the embryo stage and postnatally.