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

Abstract

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 β1-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 β1-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 β1-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.

BDNF Spinal Overexpression after Spinal Cord Injury Partially Protects Soleus Neuromuscular Junction from Disintegration, Increasing VAChT and AChE Transcripts in Soleus but Not Tibialis Anterior Motoneurons

“…The de-differentiated phenotype does not express S100b, as demonstrated in in vitro experiments [ 103 ]. Taking into account the normalized expression of the M2 receptor in the Soleus muscle after BDNF treatment, and in conjunction with recent findings that the M2 receptor is crucial for switching de-differentiated Schwann cells to the myelinating phenotype [ 59 , 104 ], we hypothesize that the BDNF treatment favors differentiation to regenerative instead of myelinating cells. The tightening of the structure and axonal guidance that characterized this SC phenotype partially explains the larger number of junctions that preserved pre-and postsynaptic contact in the BDNF Sol group.…”

“…This result raised the next question on the molecular changes in the subpopulation of myelinating Schwann cells assisting the most distal parts of the nerve, achievable in the muscle, and in the accompanying, non-myelinating terminal Schwann cells. We found that S-100b expression characterizing both cell populations was significantly reduced; and was similar in each spinal group ( Figure 3 D), which suggested a loss of these cells after spinalization or their de-differentiation [ 59 ].…”

Muscarinic Cholinoreceptors in Skeletal Muscle: Localization and Functional Role

“…This regulation is mediated via the alteration of [Ca 2+ ]i in perisynaptic Schwann cells. It is assumed that M2 mAChR in the Schwann cells of warm-blooded animals is involved in the control of the proliferation, differentiation, and myelination of these cells [ 30 , 49 , 50 ]. In neuromuscular preparations of newborn rats, muscarinic autoreceptors of the M1, M2 and M4 subtypes can participate in the differentiation of “strong” and “weak” synapses in the case of polyinnervation of muscle fibers at early stages of synaptogenesis [ 51 , 52 ].…”

Emerging Roles of Cholinergic Receptors in Schwann Cell Development and Plasticity

“…Acetylcholine was the first molecule identified as a neurotransmitter; its detection in unicellular organisms (i.e., bacteria, protozoa) and primitive plants suggested that ACh may be one of the oldest signalling molecules, highlighting that ACh was widely distributed in primitive organisms before its detection in the nervous system [17,58,59], where it acts as a regulator of nervous system development [60,61]. The cholinergic system is also expressed in several non-neuronal tissues where ACh is not necessarily derived from cholinergic innervation, but it can be synthesised by different cell types (e.g., lymphocytes, keratinocytes, stem cells, and lung epithelial cells) and, upon binding cholinergic receptors, may activate autocrine and/or paracrine signals [17,[62][63][64][65], thereby modulating cell growth, survival, and differentiation [66].…”