Cholinergic effects mediated by M2 muscarinic receptor in human Schwann-like cells induced from adipose mesenchymal stem cells

Cholinergic effects mediated by M2 muscarinic receptor in human Schwann-like cells induced from adipose mesenchymal stem cells Piovesana R.1,2, Faroni A.2, Tata AM1, Reid A.2 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; Schwann cells (SCs) have an important role in peripheral nerve regeneration but there are several restrictions on their clinical application. Adipose derived stem cells (ASCs) present good properties for cell therapies. When exposed to selective growth factors, they can acquire a SC-like phenotype (dASCs), expressing key SCs markers. Our group has demonstrated in rat model that M2 muscarinic receptor causes in vitro, a reversible arrest of cell proliferation, increasing SCs myelinating phenotype. Human dASCs, as rat dASCs, express muscarinic receptors. In the present work we evaluate if M2 muscarinic receptor activation may contribute to human dASCs proliferation and phenotype. M2 selective activation by selective agonist APE, causes a decreased cell proliferation, modulating the expression of gene involved in the proliferative state (i.e. c-jun and egr2) and neurotrophic factors. Although further analyses are needed to best characterise the role of M2 receptor, these data are the first evidence that its selective activation may have effects also on human dASCs proliferation and may favourite a neuroprotective environment relevant for nerve regeneration

Cholinergic effects mediated by M2 muscarinic receptor in human Schwann-like cells induced from adipose mesenchymal stem cells

Schwann cells (SCs) have an important role in peripheral nerve regeneration but there are several restrictions hindering their clinical application. Adipose derived stem cells (ASCs) feature good properties for cell therapies. When exposed to specific growth factors in vitro, they can acquire a SC-like phenotype (dASCs), expressing key SCs markers. Our group has demonstrated that M2 muscarinic receptor in rat dASCs causes a reversible arrest of cell proliferation, increasing SCs myelinating phenotype. Human dASCs, as rat dASCs, express muscarinic receptors. In the present work we evaluate if M2 muscarinic receptor activation may contribute to human dASCs proliferation, migration and phenotype. M2 selective activation by selective agonist Arecaidine Propargyl Ester (APE) causes a decreased cell proliferation, modulating the expression of genes involved in the proliferation/differentiation (i.e. c-jun and egr2) and several neurotrophic factors. Moreover, M2 selective activation is able to decrease cell migration. Although further analyses are needed to best characterise the role of M2 receptor, these results are the first evidence that its selective activation may have effects also on human dASCs proliferation and migration. This may improve our knowledge of these promising therapeutic cells for potential use in nerve regeneration

Anti-proliferative and anti-migratory effects are mediated by M2 muscarinic receptor in Schwann-like cells induced from adipose mesenchymal stem cells: implication in nerve regeneration.

Anti-proliferative and anti-migratory effects are mediated by M2 muscarinic receptor in Schwann-like cells induced from adipose mesenchymal stem cells: implication in nerve regeneration. Piovesana R.1,2, Faroni A.2, Magnaghi V.3, Reid A.2, Tata AM1. 1Dept. Biol and Biotech. C. Darwin, University of Rome “Sapienza”, Rome, Italy; 2Blond McIndoe Laboratories, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK; 3Dept.of Pharmacol and Biomol Sciences, University of Milan, Milan, Italy Patients affected by peripheral nerve injury show great functional morbidity, ranging from sensory and motor loss, chronic pain, or a combination of both. The peripheral nervous system has an intrinsic regeneration capability; nevertheless, nerve regeneration outcomes are rarely satisfactory and full functional recovery is often limited. Schwann Cells (SCs) play a central role in the response of the axon injury. The capacity of SCs to proliferate, to secrete growth factors, to modulate immune response, to migrate and to re-myelinate regenerating nerves have been reported. However, SCs show some drawbacks for tissue engineering, such as the difficulty in collection and culture and the slow rate of in vitro expansion. For this reason, the attention of researchers has moved towards other cell types, such as stem cells, presenting best properties for regenerative medicine. The adipose tissue contains mesenchymal stem cells (ASC). ASC can be differentiated in vitro in Schwann-like cells (dASC) following exposure to suitable culture media. SCs express receptors for different neurotrasmitters (i.e. GABA, acetylcholine). In particular, rat SCs express different muscarinic receptor subtypes with higher levels of M2 subtype. M2 receptor 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 undifferentiated dASC, like SCs, express functional receptors for different neurotrasmitters, including all muscarinic receptor subtypes. In present work, we characterised the effects mediated by M2 receptors in rat dASC. In dASC, similarly to what observed in SCs, M2 receptor activation caused a reversible reduction of cell proliferation and the inhibition of cell migration without affecting cell survival. After 24h of M2 agonist treatment, we have also observed a significant decrease of neurotrophic factors expression (i.e. NGF, BDNF and GDNF) and an increase of P0 transcript level. Finally the stimulation of M2 receptors enhances a pronounced spindle shaped morphology in dASC with similarities to native SCs. Our data demonstrate that muscarinic receptor activity contributes to modulate dASC proliferation/differentiation. Although further analyses are needed to fully characterise the role of the other muscarinic receptor subtypes, these novel data suggest that cholinergic agonists may support the role of dASC in nerve regeneration by differentially modulating neurotrophic potential

Muscarinic receptor in Schwann-like adipose derived stem cells: implications in nerve regeneration.

Schwann Cells (SCs) play a central role in the physiology and in the response of the axon injury. The capacity of SCs to proliferate, to secrete growth factors, to modulate immune response, to migrate and to re-myelinate regenerating nerves have been reported (Jessen et al, 2016). However, SCs present limited clinical application, such as the difficulty in collection and culture and the slow rate of in vitro expansion. Some papers describe that Adipose-derived stem cells (ASCs) have the ability to differentiate towards SCs phenotype (Schwann-like, dASC) following exposure to suitable culture media (Kingham et al, 2007). dASC, like SCs, express functional receptors for different neurotrasmitters, including muscarinic receptor subtypes (M1-M4) that regulate some physiological events. In the present work, we have characterised the effects mediated by muscarinic receptors on proliferation and neurotrophic factors (NFs) expression and production

Muscarinic receptor activation modulates neurotrophic factors production in rat Schwann-like cells derived from adipose mesenchymal stem cells.

Muscarinic receptor activation modulates neurotrophic factors production in rat Schwann-like cells derived from adipose mesenchymal stem cells Piovesana R1, Faroni A2, Soligo M3, Manni L3, Reid AJ2 & Tata AM1 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 Peripheral nerve injury is commonly caused by direct mechanical trauma. Regeneration depends on the ability of Schwann cells (SCs) to create a favourable environment, by producing neurotrophic factors. Although SCs are effective in promoting nerve regeneration, they are not a convenient source of transplantable cells to improve outcomes after injury. Mesenchymal Stem Cells derived from adipose tissue (ASCs) seem to be a promising alternative source considering their ability to differentiate towards SC phenotype (Schwann-like). SCs express different receptors for neurotransmitters. In particular cholinergic stimulation of M2 muscarinic receptor decreases SCs proliferation whilst upregulating myelination. Previously, we demonstrated that Schwann-like cells express muscarinic receptors; in particular the M2 receptor activation resulted in decreased proliferation and reduced migration. In present work, we have characterised the effects mediated by muscarinic receptors on neurotrophic factors (NFs) expression and production. The selective activation of M2 receptors by arecaidine propargyl ester (APE) caused a significant decrease of the transcript levels for NFs (NGF, BDNF and GDNF), while the non-selective agonist muscarine did not influence NFs mRNA expression. By custom made Elisa Assay, we analysed the production of two different NGF forms, precursor (proNGF) and mature NGF (mNGF). APE treatment induced a decreased release of both NGF forms, whereas muscarine treatment stimulated an increased release of mNGF. Western blot analysis indicated that both agonists caused a significant decrease in the expression of the proNGF isoform at 25 kDa, which is likely involved in the modulation of apoptotic processes. The data obtained suggest a relevant role of muscarinic receptors in the modulation of NFs production in Schwann-like cells. In particular the ability of both muscarinic agonists to negatively modulate the proNGF isoform, thereby suggesting a neuroprotective role of muscarinic receptors towards regenerating axons

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

M2 receptors activation affects proliferation, migration and neurotrophic factors production in rat Schwann-like cells.

Introduction: Schwann cells (SCs) are key regulators of peripheral nerve regeneration and axonal myelination. Although SCs are an attractive therapeutic option for peripheral nerve injuries, there are several restrictions on their clinical application. Adipose derived stem cells (ASCs) represent an attractive source for cell therapies. When exposed to selective growth factors, they can acquire a SC-like phenotype (dASCs) expressing key SCs markers and adopting a spindle-shape morphology. Our group has demonstrated that Acetylcholine, via M2 muscarinic receptors, causes a reversible arrest of cell proliferation in SCs, increasing myelin proteins expression. dASCs also express muscarinic receptors. In the present work we evaluate if M2 muscarinic receptor activation may contribute to dASCs proliferation and phenotype. Methods: Cell growth was analysed by MTS assay. By ELISA and western blot analysis, two different NGF isoforms were detected and their release measured. Cell migration was analysed by wound healing assay. Results: M2 receptor activation causes a reversible arrest of the cell growth, without affecting cell survival. Moreover, they inhibit dASCs migration. M2 receptor stimulation also causes a significant decrease in neurotrophins expression, accompanied by a decreased release of proNGF and mNGF forms. In particular, the apoptotic proNGF isoform (25kDa) is strongly reduced after M2 receptor stimulation. Conclusions: Our data clearly demonstrates that M2 receptor activation inhibits dASCs proliferation and migration, negatively modulating neurotrophins expression and maturation. This indicates that M2 receptors may be relevant for dASCs maturation and myelination, perhaps in a similar way to that observed in SCs