The inhibitory glycine receptor (GlyR) is a ligand‐gated ion channel which mediates post‐synaptic inhibition in spinal cord and other regions of the vertebrate central nervous system. Previous biochemical and molecular cloning studies have indicated heterogeneity of GlyRs during development. Here, the distribution of GlyR subunit transcripts in rat brain and spinal cord was investigated by in situ hybridization using sequence‐specific oligonucleotide probes. In adult animals, GlyR alpha 1 subunit mRNA was abundant in spinal cord, but was also seen in a few brain areas, e.g. superior and inferior colliculi, whereas alpha 2 transcripts were found in several brain regions including layer VI of the cerebral cortex and hippocampus. GlyR alpha 3 subunit mRNA was expressed at low levels in cerebellum, olfactory bulb and hippocampus, while high amounts of beta subunit transcripts were widely distributed throughout spinal cord and brain. During development, alpha 2 mRNA accumulated already prenatally and decreased after birth, whereas alpha 1 and alpha 3 subunit transcripts appeared only in postnatal brain structures. Hybridization signals of beta subunit mRNA were seen already at early embryonic stages and continuously increased to high levels in adult rats. These data reveal unexpected differences in the regional and developmental expression of GlyR subunit mRNAs and point to novel functions of GlyR proteins in the mammalian central nervous system.

IntroductionBone marrow (BM) is a complex tissue containing hematopoietic progenitor cells and a connective-tissue network of stromal cells. Marrow stroma includes a subpopulation of undifferentiated cells that are capable of becoming one of a number of phenotypes, including bone and cartilage, tendon, muscle, fat, and marrow stromal connective tissue that supports hematopoietic cell differentiation. 1,2 These cells are referred to as mesenchymal stem cells (MSCs), since they are known to have capacity of proliferation and differentiation into the mesenchymal lineage. Due to their potential for differentiation into different tissues, MSCs have emerged as a promising tool for clinical applications such as tissue engineering and cell and gene therapy. [3][4][5] Several reports underline the ability of MSCs to migrate. [6][7][8][9][10][11][12][13] MSCs are thought to migrate in the bloodstream to seed new sites of hematopoiesis and to various tissues during embryonic and fetal development. 14,15 MSCs are present in large numbers in human blood from at least 7 weeks' gestation and they persist until approximately 12 weeks' gestation. 14 Although circulating MSCs decrease after 12 weeks, there is evidence that a very lowfrequency population of circulating multipotent nonhematopoietic cells resembling the classical MSCs persist through to adult life. [16][17][18] MSCs migrate efficiently to hematopoietic tissues (BM and spleen) after transplantation in some experimental animal models, 19,20 whereas reports of BM homing in humans are inconsistent. [21][22][23][24][25][26] Of particular interest for tissue remodeling, intravenous delivery of MSCs results in their specific migration to a site of injury. [6][7][8]10,27 This ability of implanted MSCs to seek out the site of tissue damage has been demonstrated in bone or cartilage fracture, 28 myocardial infarction, 8,29 and ischemic cerebral injury. 6,10,11 Because MSCs have been shown to give rise to many tissues (such as bone, cartilage, fat, endothelia, muscle, brain, and pancreatic islet cells 30,31 ), migrating MSCs may represent a source of pluripotent cells that are constantly available for the repair of damaged organs. The mechanisms that guide homing of implanted cells are unclear. In this study, we examined the role of chemokines and their receptors in the migration of human MSCs. Moreover the interaction between human pancreatic islets and MSCs was investigated as a model of tissue cross talk. Material and methods Human bone marrow mesenchymal stem cell cultureHuman bone marrow mesenchymal stem cells (BM-MSCs) were obtained from Cambrex (Baltimore, MD). There were 3 different batches used for the study. Before use, the cells were analyzed for morphology, marker For personal use only. on May 11, 2018. by guest www.bloodjournal.org From expression, and osteogenic differentiation. All batches used had a fibroblastlike morphology in culture, were homogeneously CD73 ϩ , CD105 ϩ , HLA I ϩ , ␣V␤3 ϩ , ␣V␤5 ϩ , CD34 Ϫ , CD45 Ϫ , CD117 Ϫ , CD31 Ϫ , HLAII Ϫ , CD18 Ϫ , CD80...

Tumor perineural dissemination is a hallmark of human pancreatic ductal adenocarcinoma (PDAC) and represents a major source of local tumor recurrence after surgery. In this study, we provide in vitro and in vivo evidence that the chemokine receptor CX3CR1 may be involved in the neurotropism of PDAC cells to local peripheral nerves. Neoplastic cells from PDAC cell lines and surgical specimens express the chemokine receptor CX3CR1, absent in normal pancreatic ducts. Its unique ligand, the transmembrane chemokine CX3CL1, is expressed by neurons and nerve fibers. CX3CR1 + PDAC cell lines migrated in response to human recombinant CX3CL1 and specifically adhered to CX3CL1-expressing cells of neural origin via mechanisms involving activation of G proteins, B1 integrins, and focal adhesion kinase. In vivo experiments with transplanted PDAC showed that only CX3CR1-transfected tumor cells infiltrated the local peripheral nerves. Immunohistochemistry of CX3CR1 in PDAC specimens revealed that 90% of the samples were positive with a heterogeneous pattern of expression. High receptor score was significantly associated with more prominent tumor perineural infiltration evaluated histologically (P = 0.026). Regression analyses (univariate and multivariate) showed that high CX3CR1 expression and perineural invasion were strongly associated with local and earlier tumor recurrence (P = 0.007). Collectively, this study shows that the CX3CR1 receptor may be involved in PDAC tumor neurotropism and is a relevant and independent risk factor to predict an early local tumor relapse in resected patients. Thus, the CX3CR1-CX3CL1 axis could represent a valuable therapeutic target to prevent tumor perineural dissemination in pancreatic cancer. [Cancer Res 2008;68(21):9060-9]

The neurosecretory process, by which neurons and neurosecretory cells store their neurotransmitters and/or neurohormones within unique organelles and discharge them by regulated exocytosis, is the central event of neurosecretion, acquired by the cells in the course of their progression from pluripotent stem cells to restricted lineages. Extensive studies carried out during the last decade (reviewed by Wu and Xie 2006) have shown that this progression depends on a complex signaling network orchestrated by the repressor element 1-silencing transcription factor (REST, also referred to as NRSF), operating also through the involvement of additional factors, active at the transcriptional and post-transcriptional level (Conaco et al. 2006;Ma 2006;Wu and Xie 2006). Binding of REST to a specific DNA sequence distributed in its numerous target genes, the repressor element 1 (RE-1), entails the assembly, at the N and C terminal domains of the repressor, of two protein complexes, including histone and DNA modifying enzymes. The activity of these complexes consists in the repression of transcription. The differential development of Received December 12, 2007; revised manuscript received January 7, 2008; accepted January 8, 2008.Address correspondence and reprint requests to Jacopo Meldolesi, Vita-Salute San Raffaele University, DIBIT, via Olgettina 58, 20132 Milan, Italy. E-mail: meldolesi.jacopo@hsr.itAbbreviations used: DBD, DNA-binding domain; DBD/REST, dominant negative construct of REST; DCV, dense-core vesicle; FITC, fluorescein isothiocyanate; GC, Golgi complex; hChgA and ChgA, human and rat chromogranin A; hChgB and ChgB, human and rat chromogranin B; HDAC, histone deacetylase; ICA512, Islet cell antigen 512; Q-SNARE and R-SNARE, target and vesicle SNAREs; RE-1, responsive element 1; REST, RE-1 silencing transcription (factor); Scg2, secretogranin 2; SLMV, synaptic-like microvesicles; Stx1a, syntaxin 1a; SynI, synapsin I; Syp1, synaptophysin 1; Syt1, synaptotagmin 1; TH, tyrosine hydroxylase; TRITC, tetramethyl-rhodamine isothiocyanate; TSA, trichostatin A; VAMP, vesicle associated protein; wt, wild-type. AbstractThe neurosecretory process is acquired during differentiation and can be lost en block by differentiated cells. To investigate the role of REST/NRSF, a transcription repressor, in the maintenance of the process we studied two PC12 clones, one wt and one defective, expressing low and high levels of endogenous RE-1 silencing transcription (factor) (REST), respectively. Stable transfection of constructs demonstrated that REST represses 10 genes coding for proteins of neurosecretory vesicles and their exocytosis, eight including and two lacking the REST-binding sequence, RE-1. Of these genes, those of chromogranins were strongly repressed by fewfold increases of REST, those of VAMP2 and syntaxin1a required much higher levels. Moreover, in wt cells transfected with an active construct the dense-core vesicles, still competent for regulated exocytosis, were much smaller, with lighter cores; in defective cells, the...

The inhibitory 81yeine r~eptor (GlyR) i~ a liitand.gated chloride channel protein which dkpla),~ devdopmental heteroiLeneiiy in the mammalian central ncrvou~ ~y~tem. H©r~ wo descril~ 2 novel eDNA variants of the rat GlyR ~2 subunit and demonstrate that alternative ~pllcinll Ilenerate~ these 2 isoforms. The d¢dueed protein sequences (~2A and ~2B) exhibit 99~ identity wilh the previousl~¢ characterized human ~2 subunit. In site hy'bridizatt0n revealed expression of botll ",2A and crab raRNAs in the prenatal rat brain, suilgestinll that these variant protcin~l may have a role in synaptoilenesis. Heteroloilous expression in XenOl)US ooeytes showed that the more abtmdantll¢ ~xpre~d a2A subunit forms strychnlne..~asitiv~ ior ehannel~l which resemble human ~2 ~abunit GlyRs in their clectrophysiologieal properties.Glycine receptor; Alternative splicing; Receptor helcro~eneity: Brain development

H 2 O 2 produced by extracellular NADPH oxidases regulates tyrosine kinase signaling inhibiting phosphatases. How does it cross the membrane to reach its cytosolic targets? Silencing aquaporin-8 (AQP8), but not AQP3 or AQP4, inhibited H 2 O 2 entry into HeLa cells. Re-expression of AQP8 with silencing-resistant vectors rescued H 2 O 2 transport, whereas a C173A-AQP8 mutant failed to do so. Lowering AQP8 levels affected H 2 O 2 entry into the endoplasmic reticulum, but not into mitochondria. AQP8 silencing also inhibited the H 2 O 2 spikes and phosphorylation of downstream proteins induced by epidermal growth factor. These observations lead to the hypothesis that H 2 O 2 does not freely diffuse across the plasma membrane and AQP8 and other H 2 O 2 transporters are potential targets for manipulating key signaling pathways in cancer and degenerative diseases.

The peripheral membrane protein gephyrin copurifies with the inhibitory glycine receptor of mammalian spinal cord. It binds with high affinity to polymerized tubulin and has been implicated in the anchoring of the glycine receptor to cytoskeletal elements. Recently, cDNA cloning has identified variants of the gephyrin mRNA, which originate from alternative splicing of four exonic regions (cassettes 1-4). In this study, the expression patterns of gephyrin splice variants were determined in the adult and developing rat brain by in situ hybridization with synthetic oligonucleotide probes. Gephyrin transcripts were detected throughout the brain and spinal cord, with mRNAs containing cassette 2 (C2 transcripts) being predominant in adult animals. C3 and C4 transcripts were seen in cerebellar granule cells and in the dentate gyrus, whereas a C1 probe did not produce detectable hybridization signals. During development, C2 and C3 mRNAs were found in most brain regions. Generally, the spatial and temporal distribution of gephyrin transcripts is similar to that of the glycine receptor beta subunit mRNA reported previously.

Expression of dense-core granules, a typical exocytic organelle, is widely believed to be controlled by coordinate gene expression mechanisms specific to neurones and neurosecretory cells. Recent studies in PC12 cells, however, have suggested the number of granules/cells depends on the levels of only one of their cargo proteins, chromogranin A, regulating the metabolism of the other proteins, and thus the composition of the organelles, by an on/off switch mechanism. In addition, transfection of chromogranin A was reported to induce appearance of dense-core granules in the non-neurosecretory fibroblasts of the CV-1 line. Here the role of chromogranin A has been reinvestigated using not the heterogeneous PC12 line but several clones isolated therefrom. In these clones, investigated as such or after transfection with chromogranin A antisense sequences, the ratio between chromogranin A and its secretory protein mate, chromogranin B, was not constant but highly and apparently randomly variable. Variability of the chromogranin A/chromogranin B ratio was seen by confocal immunofluorescence also among the cells of single clones and subclones and among the granules of single cells. Moreover, stable and transient transfections of chromogranin A in a PC12 clone characterised by a low number of dense-core granules (one fifth of the reference clone) failed to modify significantly the number of the organelles, despite the several-fold increase of the granin. Finally, in three types of non-neurosecretory cells (CV-1, adenocarcinoma TS/A and a clone of PC12 incompetent for secretion) the transfected chromogranin A accumulated mostly in the Golgi/transGolgi area and was released rapidly from resting cells (constitutive secretion) as revealed by both immunofluorescence during cycloheximide treatment and pulse-chase experiments. Only a minor fraction was sorted to discrete organelles that were not dense-core granules, but primarily lysosomes because they contained no chromogranin B, and were largely positive for the late endosomal-lysosomal markers, lamp1 and lamp3. Dense-core granules are therefore true hallmarks of neurones and neurosecretory cells. Their number/cell appears independent of chromogranin A and their composition does not appear to be constant; in particular, they exhibit considerable, and so far unexplained variability in the chromogranin A/chromogranin B ratio.