Muscarinic receptor activation modulates proliferation and neurotrophic factors production in rat Schwann-like cells: implications in nerve regeneration.

Muscarinic receptor activation modulates proliferation and neurotrophic factors production in rat Schwann-like cells: implications in nerve regeneration. Theme B: Neural Excitability, Synapses, and Glia: Cellular Mechanisms *Roberta Piovesana1,2, Alessandro Faroni2, Marzia Soligo3, Alessandro Matera1, Luigi Manni3, Valerio Magnaghi4, Adam J Reid2 & Ada Maria Tata1 1Dept. Biol and Biotech. C. Darwin, University of Rome “Sapienza”, Rome, Italy; 2Blond McIndoe Lab, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK; 3Institute of Translational Pharmacology-CNR, Rome, Italy; 4Dept.of Pharmacol and Biomol Sciences, Univ of Milan, Milan, Italy Introduction: Peripheral nerve injury is caused by direct mechanical trauma or surgical resection secondary to tumor excision. Patients show sensory and motor loss and chronic pain. The peripheral nervous system has an intrinsic regeneration capability; nevertheless, full functional recovery is limited. Schwann Cells (SCs) play a pivotal role in the response of the axon injury, thus, SCs are an attractive therapeutic target. In fact SCs enhance nerve regeneration, producing neurotrophic factors (NFs). Unfortunately, SCs have some clinical limitations, such as the difficulty in collection and culture and a slow proliferation rate. The ideal transplantable cells should be easily accessible, proliferate rapidly in culture and successfully integrate into host tissue. Methods: Adipose-derived stem cells (ASCs) can be differentiated in SC phenotype (Schwann-like, dASCs) following exposure to a growth factor cocktail. Gene expression was analysed by Real Time PCR; 25 kDa proNGF isoform was detected by Western Blotting analysis. By custom made Elisa Assay, the production of two different NGF forms, precursor (proNGF) and mature NGF (mNGF), have been detected. The cell migration has been analysed by wound healing assay. Results: SCs express receptors for different neurotransmitters. Rat SCs express different muscarinic receptor subtypes. In particular, the most expressed is M2 receptor. Its activation causes a negative effect on SC proliferation, upregulating transcription factors involved in the promyelinating phase (e.g., Sox10 and Krox20) and downregulating proteins involved in the maintenance of the proliferative state (c-jun, Notch-1). dASCs, like SCs, express functional receptors for different neurotrasmitters, including all muscarinic receptor subtypes. In present work, we characterized the effects mediated by M2 receptors in rat dASCs. As for SCs, M2 receptor activation caused a reversible decrease of cell proliferation and the inhibition of cell migration without affecting cell survival. After 24hs of M2 agonist treatment (Arecaidine propargyl ester, APE), it has been observed a significant decrease of NF expression (i.e. NGF, BDNF and GDNF) and an increase of P0 transcript level. APE treatment induced a decreased release of both NGF forms, whereas the non-selective agonist muscarine stimulated an increased release of mNGF. Both agonists caused a significant decreased expression of the 25 kDa proNGF isoform, which is involved in the modulation of apoptotic processes. Furthermore, the selective activation of M2 receptors enhanced a pronounced spindle shaped morphology in dASCs similarly to native SCs. Conclusions: Our data demonstrate that M2 receptor activity inhibits dASCs proliferation and migration and it could improve their differentiation. Conversely muscarinic receptors activation positively modulates NFs production. These data suggest that cholinergic agonists may differently contribute to the dASC functions during nerve regeneration.