TRF2 controls telomeric nucleosome organization in a cell cycle phase-dependent manner

Mammalian telomeres stabilize chromosome ends as a result of their assembly into a peculiar form of chromatin comprising a complex of non-histone proteins named shelterin. TRF2, one of the shelterin components, binds to the duplex part of telomeric DNA and is essential to fold the telomeric chromatin into a protective cap. Although most of the human telomeric DNA is organized into tightly spaced nucleosomes, their role in telomere protection and how they interplay with telomere-specific factors in telomere organization is still unclear. In this study we investigated whether TRF2 can regulate nucleosome assembly at telomeres.By means of chromatin immunoprecipitation (ChIP) and Micrococcal Nuclease (MNase) mapping assay, we found that the density of telomeric nucleosomes in human cells was inversely proportional to the dosage of TRF2 at telomeres. This effect was not observed in the G1 phase of the cell cycle but appeared coincident of late or post-replicative events. Moreover, we showed that TRF2 overexpression altered nucleosome spacing at telomeres increasing internucleosomal distance. By means of an in vitro nucleosome assembly system containing purified histones and remodeling factors, we reproduced the short nucleosome spacing found in telomeric chromatin. Importantly, when in vitro assembly was performed in the presence of purified TRF2, nucleosome spacing on a telomeric DNA template increased, in agreement with in vivo MNase mapping.Our results demonstrate that TRF2 negatively regulates the number of nucleosomes at human telomeres by a cell cycle-dependent mechanism that alters internucleosomal distance. These findings raise the intriguing possibility that telomere protection is mediated, at least in part, by the TRF2-dependent regulation of nucleosome organization

Notch Signal Mediates the Cross-Interaction between M2 Muscarinic Acetylcholine Receptor and Neuregulin/ErbB Pathway: Effects on Schwann Cell Proliferation

The cross-talk between axon and glial cells during development and in adulthood is mediated by several molecules. Among them are neurotransmitters and their receptors, which are involved in the control of myelinating and non-myelinating glial cell development and physiology. Our previous studies largely demonstrate the functional expression of cholinergic muscarinic receptors in Schwann cells. In particular, the M2 muscarinic receptor subtype, the most abundant cholinergic receptor expressed in Schwann cells, inhibits cell proliferation downregulating proteins expressed in the immature phenotype and triggers promyelinating differentiation genes. In this study, we analysed the in vitro modulation of the Neuregulin-1 (NRG1)/erbB pathway, mediated by the M2 receptor activation, through the selective agonist arecaidine propargyl ester (APE). M2 agonist treatment significantly downregulates NRG1 and erbB receptors expression, both at transcriptional and protein level, and causes the internalization and intracellular accumulation of the erbB2 receptor. Additionally, starting from our previous results concerning the negative modulation of Notch-active fragment NICD by M2 receptor activation, in this work, we clearly demonstrate that the M2 receptor subtype inhibits erbB2 receptors by Notch-1/NICD downregulation. Our data, together with our previous results, demonstrate the existence of a cross-interaction between the M2 receptor and NRG1/erbB pathway-Notch1 mediated, and that it is responsible for the modulation of Schwann cell proliferation/differentiation

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

Cultures of schwalla cells froill ncoaatal rat sciatic aenes ivere treated with acety1cholilic agonists tvid the cjj cts oil cell Prolificratiou cniluatcd. 3[HI-thyaiidiac incorporation shows that accty1choliac (ACh) receptor agoiiists ilillibit cell proliftratioti, aiid FACS analysis deinoustratcs ccll-cyclc arrest and accunlulatioii of cells ill the Gi phase. The use of arecaiditie, a selective agouist of niuscariiiic M2 receptors reveals that this effect depends nictitily oil jW2 receptor acth,atioii. The arecaidine i1cpetialci i t- block ill Gi is rci,ersible becausc reinoval of arecaidiue froai the culture iiieditaii mduccs progrcssioil to the S phase. The block of the Gj-S transitioil is also characterized by inodulatiou of the expressioti ofscwrol cell-cycic inarkcrs. Morcoi,cr, trcatnicat ivith ACh receptor agmist causes both a decreasc ill the PCNA prolcin lci,cls ill Schivaiiii cell iniclci aad all iiicreasc ill P27 Olld P53 proteins. Filially, iinnnuio-elcctrou inicroscopy dcalonStl-atCS that M2 receptors arc expressed by Schivanii cells ill i,ivo. These results inclicate that ACh, by aiodulatiiig Schwaiin cell prolificratioii through M2 I-CCCPtOl- aclivatioa,)iiight cowributc to their progression to a niore dif rewiated phenotype

The Analgesic Effect on Neuropathic Pain of Retrogradely Transported botulinum Neurotoxin A Involves Schwann Cells and Astrocytes

In recent years a growing debate is about whether botulinum neurotoxins are retrogradely transported from the site of injection. Immunodetection of cleaved SNAP-25 (cl-SNAP-25), the protein of the SNARE complex targeted by botulinum neurotoxin serotype A (BoNT/A), could represent an excellent approach to investigate the mechanism of action on the nociceptive pathways at peripheral and/or central level. After peripheral administration of BoNT/A, we analyzed the expression of cl-SNAP-25, from the hindpaw's nerve endings to the spinal cord, together with the behavioral effects on neuropathic pain. We used the chronic constriction injury of the sciatic nerve in CD1 mice as animal model of neuropathic pain. We evaluated immunostaining of cl-SNAP-25 in the peripheral nerve endings, along the sciatic nerve, in dorsal root ganglia and in spinal dorsal horns after intraplantar injection of saline or BoNT/A, alone or colocalized with either glial fibrillar acidic protein, GFAP, or complement receptor 3/cluster of differentiation 11b, CD11b, or neuronal nuclei, NeuN, depending on the area investigated. Immunofluorescence analysis shows the presence of the cl-SNAP-25 in all tissues examined, from the peripheral endings to the spinal cord, suggesting a retrograde transport of BoNT/A. Moreover, we performed in vitro experiments to ascertain if BoNT/A was able to interact with the proliferative state of Schwann cells (SC). We found that BoNT/A modulates the proliferation of SC and inhibits the acetylcholine release from SC, evidencing a new biological effect of the toxin and further supporting the retrograde transport of the toxin along the nerve and its ability to influence regenerative processes. The present results strongly sustain a combinatorial action at peripheral and central neural levels and encourage the use of BoNT/A for the pathological pain conditions difficult to treat in clinical practice and dramatically impairing patients' quality of life. © 2012 Marinelli et al

Immunofluorescence detection of cl-SNAP-25 in DRG.

<p>Representative examples of high magnification confocal images taken from section of DRG in naïve mice injected with saline (<i>panels </i><b>A-C</b>) or BoNT/A (<i>panels </i><b>D-F</b>; 15 pg/paw). In this tissue NeuN (green) is a protein marker expressed by neuronal cells. Scale bar: 10 µM.</p

Immunofluorescence detection of cl-SNAP-25 in dorsal horns from mice ipl-injected with saline. A)

<p>Representative examples of low and high magnification confocal images taken from lumbar (L4/L5) sections of spinal cord in naïve mice, not subjected to CCI, injected (<i>panel b</i>) or not with saline (<i>panel a</i>). Dorsal horn region considered for high magnification images is shown by IDH square in the low magnification image. Scale bar: 20 µM. <b>B)</b> Representative examples of high magnification images taken form lumbar (L4/L5) sections of spinal cord in CCI mice injected with saline. In these images, GFAP (<i>panel a</i>) and CD11b (<i>panel d</i>), are protein markers of astrocytes and microglia respectively. <i>Panels b and e</i> show immunofluorescence detection of cl-SNAP-25, while <i>panels c</i> and <i>f</i> show colocalization of cl-SNAP-25 with astrocytes and microglia, and also staining of nuclei with DAPI. Scale bar: 20 µM.</p