Acetylcholine inhibits cell cycle progression in rat Schwann cells by activation of the M2 receptor subtype

Cultures of Schwann cells from neonatal rat sciatic nerves were treated with acetylcholine agonists and the effects on cell proliferation evaluated. (3)[H]-thymidine incorporation shows that acetylcholine (ACh) receptor agonists inhibit cell proliferation, and FACS analysis demonstrates cell-cycle arrest and accumulation of cells in the G1 phase. The use of arecaidine, a selective agonist of muscarinic M2 receptors reveals that this effect depends mainly on M2 receptor activation. The arecaidine dependent-block in G1 is reversible because removal of arecaidine from the culture medium induces progression to the S phase. The block of the G1-S transition is also characterized by modulation of the expression of several cell-cycle markers. Moreover, treatment with ACh receptor agonist causes both a decrease in the PCNA protein levels in Schwann cell nuclei and an increase in p27 and p53 proteins. Finally, immuno-electron microscopy demonstrates that M2 receptors are expressed by Schwann cells in vivo. These results indicate that ACh, by modulating Schwann cell proliferation through M2 receptor activation, might contribute to their progression to a more differentiated phenotype.     

Axonal Neuregulin Signals Cells of the Oligodendrocyte Lineage through Activation of HER4 and Schwann Cells through HER2 and HER3

We are interested in the signaling between axons and glia that leads to myelination and maintenance of the myelin internode, and we have focused on the role of neuregulins and their receptors. Neuregulins are a family of ligands that includes heregulin, neu differentiation factor, glial growth factor, and the acetylcholine receptor–inducing activity. Three signal transducing transmembrane receptors for neuregulins, which bear significant homology to the EGF receptor, are currently known: HER2 (erbB2), HER3 (erbB3), and HER4 (erbB4). We have found that oligodendrocite–type II astrocyte (O2A) progenitor cells and mature oligodendrocytes express HER2 and HER4 but no HER3. Schwann cells express HER2 and HER3 but little HER4. In O2A progenitor cells and oligodendrocytes, recombinant neuregulin induces the rapid tyrosine phosphorylation of only HER4. HER2 is not phosphorylated in cells of the oligodendrocyte lineage, but a physical interaction between HER2 and HER4 was detected in coimmunoprecipitation experiments. In Schwann cells, neuregulin induces the phosphorylation of both HER2 and HER3. Coimmunoprecipitation experiments indicate that receptor activation in Schwann cells results in the formation of HER2:HER3 heterodimers. Neuregulin localized immunocytochemically was present on neurites of cultured dorsal root ganglion neurons, and it was released into the medium in a form that promoted receptor tyrosine phosphorylation. Neuregulins therefore meet important criteria expected of molecules involved in axonal-glial signaling. The use of unique neuregulin receptor combinations in oligodendrocytes and Schwann cells likely results in recruitment of different signaling pathways and thus provides a basis for different biological responses.     

In vivo knockdown of ErbB3 in mice inhibits Schwann cell precursor migration

The myelin sheath insulates neuronal axons and markedly increases the nerve conduction velocity. In the peripheral nervous system (PNS), Schwann cell precursors migrate along embryonic neuronal axons to their final destinations, where they eventually wrap around individual axons to form the myelin sheath after birth. ErbB2 and ErbB3 tyrosine kinase receptors form a heterodimer and are extensively expressed in Schwann lineage cells. ErbB2/3 is thought to be one of the primary regulators controlling the entire Schwann cell development. ErbB3 is the bona fide Schwann cell receptor for the neuronal ligand neuregulin-1. Although ErbB2/3 is well known to regulate both Schwann cell precursor migration and myelination by Schwann cells in fishes, it still remains unclear whether in mammals, ErbB2/3 actually regulates Schwann cell precursor migration. Here, we show that knockdown of ErbB3 using a Schwann cell-specific promoter in mice causes delayed migration of Schwann cell precursors. In contrast, littermate control mice display normal migration. Similar results are seen in an in vitro migration assay using reaggregated Schwann cell precursors. Also, ErbB3 knockdown in mice reduces myelin thickness in sciatic nerves, consistent with the established role of ErbB3 in myelination. Thus, ErbB3 plays a key role in migration, as well as in myelination, in mouse Schwann lineage cells, presenting a genetically conservative role of ErbB3 in Schwann cell precursor migration.     

Neurosteroid allopregnanolone regulates EAAC1-mediated glutamate uptake and triggers actin changes in Schwann cells

Recent evidence shows that neurotransmitters (e.g., GABA, Ach, adenosine, glutamate) are active on Schwann cells, which form myelin sheaths in the peripheral nervous system under different pathophysiologic conditions. Glutamate, the most important excitatory neurotransmitter, has been recently involved in peripheral neuropathies, thus prevention of its toxic effect is desirable to preserve the integrity of peripheral nervous system and Schwann cells physiology. Removal of glutamate from the extracellular space is accomplished by the high affinity glutamate transporters, so we address our studies to analyze their functional presence in Schwann cells. We first demonstrate that Schwann cells express the EAAC1 transporter in the plasma membrane and in intracellular vesicular compartments of the endocytic recycling pathways. Uptake experiments confirm its presence and functional activity in Schwann cells. Secondly, we demonstrate that the EAAC1 activity can be modulated by exposure to the neurosteroid allopregnanolone 10 nM (a progesterone metabolite proved to support Schwann cells). Transporter up-regulation by allopregnanolone is rapid, does not involve protein neo-synthesis and is prevented by actin depolymerization. Allopregnanolone modulation involves GABA-A receptor and PKC activation, promotes the exocytosis of the EAAC1 transporter from intracellular stores to the Schwann cell membrane, in actin-rich cell tips, and modifies the morphology of cell processes. Finally, we provide evidence that glutamate transporters control the allopregnanolone-mediated effects on cell proliferation. Our findings are the first to demonstrate the presence of a functional glutamate uptake system, which can be dynamically modulated by allopregnanolone in Schwann cells. Glutamate transporters may represent a potential therapeutic target to control Schwann cell physiology.     

N-WASP is required for membrane wrapping and myelination by Schwann cells

During peripheral nerve myelination, Schwann cells sort larger axons, ensheath them, and eventually wrap their membrane to form the myelin sheath. These processes involve extensive changes in cell shape, but the exact mechanisms involved are still unknown. Neural Wiskott-Aldrich syndrome protein (N-WASP) integrates various extracellular signals to control actin dynamics and cytoskeletal reorganization through activation of the Arp2/3 complex. By generating mice lacking N-WASP in myelinating Schwann cells, we show that N-WASP is crucial for myelination. In N-WASP-deficient nerves, Schwann cells sort and ensheath axons, but most of them fail to myelinate and arrest at the promyelinating stage. Yet, a limited number of Schwann cells form unusually short internodes, containing thin myelin sheaths, with the occasional appearance of myelin misfoldings. These data suggest that regulation of actin filament nucleation in Schwann cells by N-WASP is crucial for membrane wrapping, longitudinal extension, and myelination.     

Gpr126 is essential for peripheral nerve development and myelination in mammals

In peripheral nerves, Schwann cells form the myelin sheath that insulates axons and allows rapid propagation of action potentials. Although a number of regulators of Schwann cell development are known, the signaling pathways that control myelination are incompletely understood. In this study, we show that Gpr126 is essential for myelination and other aspects of peripheral nerve development in mammals. A mutation in Gpr126 causes a severe congenital hypomyelinating peripheral neuropathy in mice, and expression of differentiated Schwann cell markers, including Pou3f1, Egr2, myelin protein zero and myelin basic protein, is reduced. Ultrastructural studies of Gpr126-/- mice showed that axonal sorting by Schwann cells is delayed, Remak bundles (non-myelinating Schwann cells associated with small caliber axons) are not observed, and Schwann cells are ultimately arrested at the promyelinating stage. Additionally, ectopic perineurial fibroblasts form aberrant fascicles throughout the endoneurium of the mutant sciatic nerve. This analysis shows that Gpr126 is required for Schwann cell myelination in mammals, and defines new roles for Gpr126 in axonal sorting, formation of mature non-myelinating Schwann cells and organization of the perineurium.     

M2 receptor activation inhibits cell cycle progression and survival in human glioblastoma cells

Muscarinic receptors, expressed in several primary and metastatic tumours, appear to be implicated in their growth and propagation. In this work we have demonstrated that M2 muscarinic receptors are expressed in glioblastoma human specimens and in glioblastoma cell lines. Moreover, we have characterized the effects of the M2 agonist arecaidine on cell growth and survival both in two different glioblastoma cell lines (U251MG and U87MG) and in primary cultures obtained from different human biopsies. Cell growth analysis has demonstrated that the M2 agonist arecaidine strongly decreased cell proliferation in both glioma cell lines and primary cultures. This effect was dose and time dependent. FACS analysis has confirmed cell cycle arrest at G1/S and at G2/M phase in U87 cells and U251 respectively. Cell viability analysis has also shown that arecaidine induced severe apoptosis, especially in U251 cells. Chemosensitivity assays have, moreover, shown arecaidine and temozolomide similar effects on glioma cell lines, although IC50 value for arecaidine was significantly lower than temozolomide. In conclusion, we report for the first time that M2 receptor activation has a relevant role in the inhibition of glioma cell growth and survival, suggesting that M2 may be a new interesting therapeutic target to investigate for glioblastoma therapy.     

Epineurial adipocytes are dispensable for Schwann cell myelination

Previous clinical observations and data from mouse models with defects in lipid metabolism suggested that epineurial adipocytes may play a role in peripheral nervous system myelination. We have used adipocyte‐specific Lpin1 knockout mice to characterize the consequences of the presence of impaired epineurial adipocytes on the myelinating peripheral nerve. Our data revealed that the capacity of Schwann cells to establish myelin, and the functional properties of peripheral nerves, were not affected by compromised epineurial adipocytes in adipocyte‐specific Lpin1 knockout mice. To evaluate the possibility that Lpin1‐negative adipocytes are still able to support endoneurial Schwann cells, we also characterized sciatic nerves from mice carrying epiblast‐specific deletion of peroxisome proliferator‐activated receptor gamma, which develop general lipoatrophy. Interestingly, even the complete loss of adipocytes in the epineurium of peroxisome proliferator‐activated receptor gamma knockout mice did not lead to detectable defects in Schwann cell myelination. However, probably as a consequence of their hyperglycemia, these mice have reduced nerve conduction velocity, thus mimicking the phenotype observed under diabetic condition. Together, our data indicate that while adipocytes, as regulators of lipid and glucose homeostasis, play a role in nerve function, their presence in epineurium is not essential for establishment or maintenance of proper myelin.     

Immunoelectron microscopic localization of neural cell adhesion molecules (L1, N-CAM, and MAG) and their shared carbohydrate epitope and myelin basic protein in developing sciatic nerve

The cellular and subcellular localization of the neural cell adhesion molecules L1, N-CAM, and myelin-associated glycoprotein (MAG), their shared carbohydrate epitope L2/HNK-1, and the myelin basic protein (MBP) were studied by pre- and post-embedding immunoelectron microscopic labeling procedures in developing mouse sciatic nerve. L1 and N-CAM showed a similar staining pattern. Both were localized on small, non-myelinated, fasciculating axons and axons ensheathed by non- myelinating Schwann cells. Schwann cells were also positive for L1 and N-CAM in their non-myelinating state and at the onset of myelination, when the Schwann cell processes had turned approximately 1.5 loops. Thereafter, neither axon nor Schwann cell could be detected to express the L1 antigen, whereas N-CAM was found in the periaxonal area and, more weakly, in compact myelin of myelinated fibers. Compact myelin, Schmidt-Lanterman incisures, paranodal loops, and finger-like processes of Schwann cells at nodes of Ranvier were L1-negative. At the nodes of Ranvier, the axolemma was also always L1- and N-CAM-negative. The L2/HNK-1 carbohydrate epitope coincided in its cellular and subcellular localization most closely to that observed for L1. MAG appeared on Schwann cells at the time L1 expression ceased. MAG was then expressed at sites of axon-myelinating Schwann cell apposition and non-compacted loops of developing myelin. When compaction of myelin occurred, MAG remained present only at the axon-Schwann cell interface; Schmidt- Lanterman incisures, inner and outer mesaxons, and paranodal loops, but not at finger-like processes of Schwann cells at nodes of Ranvier or compacted myelin. All three adhesion molecules and the L2/HNK-1 epitope could be detected in a non-uniform staining pattern in basement membrane of Schwann cells and collagen fibrils of the endoneurium. MBP was detectable in compacted myelin, but not in Schmidt-Lanterman incisures, inner and outer mesaxon, paranodal loops, and finger-like processes at nodes of Ranvier, nor in the periaxonal regions of myelinated fibers, thus showing a complementary distribution to MAG. These studies show that axon-Schwann cell interactions are characterized by the sequential appearance of cell adhesion molecules and MBP apparently coordinated in time and space. From this sequence it may be deduced that L1 and N-CAM are involved in fasciculation, initial axon-Schwann cell interaction, and onset of myelination, with MAG to follow and MBP to appear only in compacted myelin. In contrast to L1, N- CAM may be further involved in the maintenance of compact myelin and axon-myelin apposition of larger diameter axons.     

A dual role of erbB2 in myelination and in expansion of the schwann cell precursor pool

Neuregulin-1 provides an important axonally derived signal for the survival and growth of developing Schwann cells, which is transmitted by the ErbB2/ErbB3 receptor tyrosine kinases. Null mutations of the neuregulin-1, erbB2, or erbB3 mouse genes cause severe deficits in early Schwann cell development. Here, we employ Cre-loxP technology to introduce erbB2 mutations late in Schwann cell development, using a Krox20-cre allele. Cre-mediated erbB2 ablation occurs perinatally in peripheral nerves, but already at E11 within spinal roots. The mutant mice exhibit a widespread peripheral neuropathy characterized by abnormally thin myelin sheaths, containing fewer myelin wraps. In addition, in spinal roots the Schwann cell precursor pool is not correctly established. Thus, the Neuregulin signaling system functions during multiple stages of Schwann cell development and is essential for correct myelination. The thickness of the myelin sheath is determined by the axon diameter, and we suggest that trophic signals provided by the nerve determine the number of times a Schwann cell wraps an axon.     

Eosinophil-induced release of acetylcholine from differentiated cholinergic nerve cells

One immunological component of asthma is believed to be the interaction of eosinophils with parasympathetic cholinergic nerves and a consequent inhibition of acetylcholine muscarinic M2 receptor activity, leading to enhanced acetylcholine release and bronchoconstriction. Here we have used an in vitro model of cholinergic nerve function, the human IMR32 cell line, to study this interaction. IMR32 cells, differentiated in culture for 7 days, expressed M2 receptors. Cells were radiolabeled with [3H]choline and electrically stimulated. The stimulation-induced release of acetylcholine was prevented by the removal of Ca2+. The muscarinic M1/M2 receptor agonist arecaidine reduced the release of acetylcholine after stimulation (to 82 +/- 2% of control at 10(-7) M), and the M2 receptor antagonist AF-DX 116 increased it (to 175 +/- 23% of control at 10(-5) M), indicating the presence of a functional M2 receptor that modulated acetylcholine release. When human eosinophils were added to IMR32 cells, they enhanced acetylcholine release by 36 +/- 10%. This effect was prevented by inhibitors of adhesion of the eosinophils to the IMR32 cells. Pretreatment of IMR32 cells with 10 mM carbachol, to desensitize acetylcholine receptors, prevented the potentiation of acetylcholine release by eosinophils or AF-DX 116. Acetylcholine release was similarly potentiated (by up to 45 +/- 7%) by degranulation products from eosinophils that had been treated with N-formyl-methionyl-leucyl-phenylalanine or that had been in contact with IMR32 cells. Contact between eosinophils and IMR32 cells led to an initial increase in expression of M2 receptors, whereas prolonged exposure reduced M2 receptor expression.     

P0 promoter directs expression of reporter and toxin genes to Schwann cells of transgenic mice.

We generated transgenic mice that specifically express foreign genes in myelinating Schwann cells. A 1.1 kb segment of 5' flanking sequence from the rat P0 gene was used to drive expression of the genes encoding human growth hormone (hGH) and bacterial diphtheria toxin A chain (DT-A). The P0-hGH mice expressed hGH in myelinating Schwann cells, but not in nonmyelinating Schwann cells, the central nervous system, or any other tissue assayed. This expression was activated on a developmental schedule comparable to that of endogenous myelin gene expression. One line of P0-DT-A mice developed a generalized hypomyelinating peripheral neuropathy, with Schwann cell deficiency apparent in newborn animals. Peripheral nerves from adult mice of this line displayed morphological alterations ranging from completely denuded axons to myelinated Schwann cells undergoing degeneration, although occasional Schwann cells were able to form apparently normal myelin sheaths. Pronounced secondary changes, including proliferation and retraction of processes, occurred in the nonmyelinating Schwann cells of these P0-DT-A mice.     

Muscarinic receptor subtypes mediating central and peripheral antinociception studied with muscarinic receptor knockout mice: a review

To gain new insight into the physiological and pathophysiological roles of the muscarinic cholinergic system, we generated mutant mouse strains deficient in each of the five muscarinic acetylcholine receptor subtypes (M(1)-M(5)). In this chapter, we review a set of recent studies dealing with the identification of the muscarinic receptor subtypes mediating muscarinic agonist-dependent analgesic effects by central and peripheral mechanisms. Most of these studies were carried out with mutant mouse strains lacking M(2) or/and M(4) muscarinic receptors. It is well known that administration of centrally active muscarinic agonists induces pronounced analgesic effects. To identify the muscarinic receptors mediating this activity, wild-type and muscarinic receptor mutant mice were injected with the non-subtype-selective muscarinic agonist, oxotremorine (s.c., i.t., and i.c.v.), and analgesic effects were assessed in the tail-flick and hot-plate tests. These studies showed that M(2) receptors play a key role in mediating the analgesic effects of oxotremorine, both at the spinal and supraspinal level. However, studies with M(2)/M(4) receptor double KO mice indicated that M(4) receptors also contribute to this activity. Recent evidence suggests that activation of muscarinic receptors located in the skin can reduce the sensitivity of peripheral nociceptors. Electrophysiological and neurochemical studies with skin preparations from muscarinic receptor mutant mice indicated that muscarine-induced peripheral antinociception is mediated by M(2) receptors. Since acetylcholine is synthesized and released by different cell types of the skin, it is possible that non-neuronally released acetylcholine plays a role in modulating peripheral nociception. Our results highlight the usefulness of muscarinic receptor mutant mice to shed light on the functional roles of acetylcholine released from both neuronal and non-neuronal cells.