Academic literature on the topic 'Human neuroblast'

The rostral migratory stream (RMS) is viewed as a glia-enriched conduit of forward-migrating neuroblasts in which chemorepulsive signals control the pace of forward migration. Here we demonstrate the existence of a scaffold of neurons that receive synaptic inputs within the rat, mouse, and human fetal RMS equivalents. These neurons express secretagogin, a Ca2+-sensor protein, to execute an annexin V-dependent externalization of matrix metalloprotease-2 (MMP-2) for reconfiguring the extracellular matrix locally. Mouse genetics combined with pharmacological probing in vivo and in vitro demonstrate that MMP-2 externalization occurs on demand and that its loss slows neuroblast migration. Loss of function is particularly remarkable upon injury to the olfactory bulb. Cumulatively, we identify a signaling cascade that provokes structural remodeling of the RMS through recruitment of MMP-2 by a previously unrecognized neuronal constituent. Given the life-long presence of secretagogin-containing neurons in human, this mechanism might be exploited for therapeutic benefit in rescue strategies.

SH-SY5Y human neuroblastoma cells express IGF receptors, IGFs and IGF binding proteins (IGFBPs), and provide a model for studying the role of the IGF system in human neuronal development. We investigated the effect of IGF-I and des(1-3)IGF-I on the motility of SH-SY5Y cells by a cell migration assay based on the assessment of the number of cells which migrated across 8 microm pore size membranes and around an agarose drop. IGF-I and des(1-3)IGF-I stimulated neuroblast chemotaxis in a dose-dependent manner. Treatment of cells with these agents for 24 h resulted in a significant increase (IGF-I by 70% and des(1-3)IGF-I by 90%; P<0. 0001) in cell motility relative to control conditions. Addition of monoclonal antibody against type 1 IGF receptor (alpha-IR3), significantly (P<0.05) reduced the cell motility induced by IGF-I (by 30%) and des(1-3)IGF-I (by 70%). Wortmannin, a specific inhibitor of phosphatidylinositol (PI)-3 kinase intracellular signalling, also reduced the IGF-stimulated cell migration (by over 40%, P<0.01), indicating a key role of the PI-3 kinase pathway in mediating the IGF effect on neuroblast migration. Finally, cell treatment with plasminogen (PLG) markedly enhanced neuroblast migration (by over 200%, P<0.01), whereas incubation with the PLG inhibitor 4-(2-aminoethyl)-benzenesulphonyl fluoride reduced cell motility (by 80%, P<0.01), thus suggesting an involvement of PLG-dependent IGFBP proteolysis in the regulation of neuroblast motility. In conclusion, IGF-I is a potent stimulator of neuroblast migration through the activation of type 1 IGF receptor and the PI-3 kinase intracellular pathway. IGFBPs and the plasmin system seem to play a role in cell motility, although the nature and the extent of their involvement has yet to be elucidated.

AbstractHuman MYCN is an oncogene amplified in neuroblastoma and many other tumors. Both human MYCN and mouse Mycn genes are important in embryonic brain development, but their functions in adult healthy nerve system are completely unknown. Here, with Mycn-eGFP mice and quantitative RT-PCR, we found that Mycn was expressed in specific brain regions of young adult mice, including subventricular zone (SVZ), subgranular zone (SGZ), olfactory bulb (OB), subcallosal zone (SCZ), and corpus callosum (CC). With immunohistochemistry (IHC), we found that many Mycn-expressing cells expressed neuroblast marker doublecortin (DCX) and proliferation marker Ki67. With Dcx-creER and Mki67-creER mouse lines, we fate mapped Dcx-expressing neuroblasts and Mki67-expressing proliferation cells, along with deleting Mycn from these cells in adult mice. We found that knocking out Mycn from adult neuroblasts or proliferating cells significantly reduced cells in proliferation in SVZ, SGZ, OB, SCZ, and CC. We also demonstrated that the Mycn-deficient neuroblasts in SGZ matured quicker than wild-type neuroblasts, and that Mycn-deficient proliferating cells were more likely to survive in SVZ, SGZ, OB, SCZ, and CC compared to wild type. Thus, our results demonstrate that, in addition to causing tumors in the nervous system, oncogene Mycn has a crucial function in neurogenesis and oligodendrogenesis in adult healthy brain.

The lethal spotted (ls) mouse has been used as a model for the human disorder Hirschsprung's disease, because as in the latter condition, ls/ls homozygotes are born without ganglion cells in their terminal colons and, without surgical intervention, die early as a consequence of intestinal obstruction. Previous studies have led to the conclusion that hereditary aganglionosis in ls/ls mice occurs because neural crest-derived enteric neuroblasts fail to colonize the distal large intestine during embryogenesis, perhaps due to a primary defect in non-neuroblastic mesenchyme rather than migrating neuroblasts themselves. In this investigation, the latter issue was addressed directly, in vivo, by comparing the distributions of ls/ls and wild-type neurons in aggregation chimeras. Expression of a transgene, D beta H-nlacZ, in enteric neurons derived from the vagal neural crest, was used as a marker for ls/ls enteric neurons in chimeric mice. In these animals, when greater than 20% of the cells were wild-type, the ls/ls phenotype was rescued; such mice were neither spotted nor aganglionic. In addition, these ‘rescued’ mice had mixtures of ls/ls and wild-type neurons throughout their gastrointestinal systems including distal rectum. In contrast, mice with smaller relative numbers of wild-type cells exhibited the classic ls/ls phenotype. The aganglionic terminal bowel of the latter mice contained neither ls/ls nor wild-type neurons. These results confirm that the primary defect in ls/ls embryos is not autonomous to enteric neuroblasts, but instead exists in the non-neuroblastic mesenchyme of the large intestine.

A hallmark of the aging brain is the robust inflammation mediated by microglial activation. Pathophysiology of common neurodegenerative diseases involves oxidative stress and neuroinflammation. Chronic treatment of aging rats by ladostigil, a compound with antioxidant and anti-inflammatory function, prevented microglial activation and learning deficits. In this study, we further investigate the effect of ladostigil on undifferentiated SH-SY5Y cells. We show that SH-SY5Y cells exposed to acute (by H2O2) or chronic oxidative stress (by Sin1, 3-morpholinosydnonimine) induced apoptotic cell death. However, in the presence of ladostigil, the decline in cell viability and the increase of oxidative levels were partially reversed. RNA-seq analysis showed that prolonged oxidation by Sin1 resulted in a simultaneous reduction of the expression level of endoplasmic reticulum (ER) genes that participate in proteostasis. By comparing the differential gene expression profile of Sin1 treated cells to cells incubated with ladostigil before being exposed to Sin1, we observed an over-expression of Clk1 (Cdc2-like kinase 1) which was implicated in psychophysiological stress in mice and Alzheimer’s disease. Ladostigil also suppressed the expression of Ccpg1 (Cell cycle progression 1) and Synj1 (Synaptojanin 1) that are involved in ER-autophagy and endocytic pathways. We postulate that ladostigil alleviated cell damage induced by oxidation. Therefore, under conditions of chronic stress that are observed in the aging brain, ladostigil may block oxidative stress processes and consequently reduce neurotoxicity.

Stimulation of postnatal neurogenesis in the subventricular zone (SVZ) and robust migration of neuroblasts to the lesion site in response to traumatic brain injury (TBI) is well established in rodent species; however, it is not yet known whether postnatal neurogenesis plays a role in repair after TBI in gyrencephalic species. Here we describe the anatomy of the SVZ in the piglet for the first time and initiate an investigation into the effect of TBI on the SVZ architecture and the number of neuroblasts in the white matter. Among all ages of immaturity examined the SVZ contained a dense mesh network of neurogenic precursor cells (doublecortin+) positioned directly adjacent to the ependymal cells (ventricular SVZ, Vsvz) and neuroblasts organized into chains that were distinct from the Vsvz (abventricular SVZ, Asvz). Though the architecture of the SVZ was similar among ages, the areas of Vsvz and Asvz neuroblast chains declined with age. At postnatal day (PND) 14 the white matter tracts have a tremendous number of individual neuroblasts. In our scaled cortical impact model, lesion size increased with age. Similarly, the response of the SVZ to injury was also age dependent. The younger age groups that sustained the proportionately smallest lesions had the largest SVZ areas, which further increased in response to injury. In piglets that were injured at 4 months of age and had the largest lesions, the SVZ did not increase in response to injury. Similar to humans, swine have abundant gyri and gyral white matter, providing a unique platform to study neuroblasts potentially migrating from the SVZ to the lesioned cortex along these white matter tracts. In piglets injured at PND 7, TBI did not increase the total number of neuroblasts in the white matter compared to uninjured piglets, but redistribution occurred with a greater number of neuroblasts in the white matter of the hemisphere ipsilateral to the injury compared to the contralateral hemisphere. At 7 days after injury, less than 1% of neuroblasts in the white matter were born in the 2 days following injury. These data show that the SVZ in the piglet shares many anatomical similarities with the SVZ in the human infant, and that TBI had only modest effects on the SVZ and the number of neuroblasts in the white matter. Piglets at an equivalent developmental stage to human infants were equipped with the largest SVZ and a tremendous number of neuroblasts in the white matter, which may be sufficient in lesion repair without the dramatic stimulation of neurogenic machinery. It has yet to be determined whether neurogenesis and migrating neuroblasts play a role in repair after TBI and/or whether an alteration of normal migration during active postnatal population of brain regions is beneficial in species with gyrencephalic brains.

Abstract To investigate the involvement of pituitary adenylate cyclase- activating polypeptide (PACAP) and GH-releasing factor (GRF) during early chick brain development, we established neuroblast- enriched primary cell cultures derived from embryonic day 3.5 chick brain. We measured increases in cAMP generated by several species-specific forms of the peptides. Dose-dependent increases up to 5-fold of control values were measured in response to physiological concentrations of human/salmon, chicken, and tunicate PACAP27. Responses to PACAP38 were more variable, ranging from 5-fold for human PACAP38 to 4-fold for chicken PACAP38, to no significant response for salmon PACAP38, compared with control values. The responses to PACAP38 may reflect a greater difference in peptide structure compared with PACAP27 among species. Increases in cAMP generated by human, chicken, and salmon/carp GRF were not statistically significant, whereas increases in response to lower-range doses of tunicate GRF27-like peptide were significant, but small. We also used immunocytochemistry and Western blot to show synthesis of the PACAP38 peptide. RT-PCR was used to demonstrate that messenger RNAs for PACAP and GRF and a PACAP-specific receptor were present in the cells. This is a first report suggesting an autocrine/paracrine system for PACAP in early chick brain development, based on the presence of the ligand, messages for the ligand and receptor, and activation of the receptor in neuroblast-enriched cultures.

Tumeur embryonnaire du tissu sympathique, le neuroblaste (nb) est l'un des cancers pediatriques les plus frequents. Le nb metastatique, au pronostic generalement severe, est traite par chimiotherapie mais manifeste souvent une chimioresistance a laquelle sont associes des taux eleves de transcrit du gene mdr1, gene implique dans le phenomene de resistance multiple aux drogues (mdr). D'autre part, l'oncogene n-myc, qui code pour une proteine nucleaire agissant comme un facteur transcriptionnel, est frequemment active par amplification genique dans ces formes disseminees de la maladie. Cette these etudie la regulation de l'expression du gene mdr1 humain et la compare a celle de l'oncogene n-myc dans des neuroblastes humains differencies, proliferatifs et metastatiques

ABSTRACT I focused on the analysis of a protein involved in the development of the peripheral nervous system: the Wilms tumor1 (WT1). My thesis is divided into two experimental phases: I) spatio-temporal distribution of WT1 during human embryonic development, II) expression and functional roles of WT1 during development of the peripheral sympathetic nervous system and gastrointestinal tract. The Wilms tumor (WT1) gene and its protein product are known to exhibit a dynamic expression profile during development and in the adult organism. Apart from a nuclear expression observed in the urogenital system, its precise localization in other developing human tissues is still largely unknown. Accordingly, the aim of this study was to investigate immunohistochemically the temporal and spatial distribution of WT1 in epithelial and mesenchymal developing human tissues from gestational weeks 7 24. For this purpose we used antibodies against the N-terminal of WT1. As might be expected, WT1 nuclear expression was observed in mesonephric/metanephric glomeruli, metanephric blastema, celomderived membranes (pleura, peritoneum, serosal surfaces) and sex cords. With regard to mesenchymal tissues, a similar nuclear staining was also obtained in the mesenchyme surrounding Müllerian and Wolffian ducts, as well as in the submesothelial mesenchymal cells of all celomatic-derived membranes. The most striking finding was the detection of strong WT1 cytoplasmic immunostaining in developing skeletal and cardiac muscle cells and endothelial cells. The tissue-specific expression of WT1, together with its different nuclear/cytoplasmic localization, both suggest that WT1 protein may have shuttling properties, acting as a protein with complex regulator activity in transcriptional/translation processes during human ontogenesis. The reported cytoplasmic expression of WT1 in human rhabdomyosarcomas and in many vascular tumors strongly suggests an oncofetal expression of this protein. Although not specific, WT1 cytoplasmic expression can be used as a marker of skeletal muscle and endothelial differentiation in an appropriate morphological context. Developmental expression of Wilms tumor gene (WT1) and protein is crucial for cell proliferation, apoptosis, differentiation and cytoskeletal architecture regulation. More recently, it has been suggested a potential role of WT1 in the development of neural tissue and in neurodegenerative disorders. We have investigated immunohistochemically the developmentally regulated expression and distribution of WT1 in human fetal (from the 8th to the 28th week gestational age) peripheral sympathetic nervous system (PSNS) and gastro-enteric nervous system (GENS). Interestingly WT1 expression was restricted to the cytoplasm of sympathetic neuroblasts, while it progressively disappeared with advancing morphologic differentiation of these cells along both ganglionic and chromaffin cell lineages. In adult tissues, both ganglion and chromaffin cells lacked any WT1 expression. These findings show that WT1 is a reliable marker of human sympathetic neuroblasts, which can be used routinely in formalin-fixed, paraffin-embedded tissues. The progressive loss of WT1 in both ganglion and chromaffin cells, suggests its potential repressor role of differentiation in a precise temporal window during human PSNS and GENS development.

In the CNS, acetylcholine (ACh) is a key neurotransmitter implicated in higher brain functions, including cognitive processes. The Nucleus Basalis of Meynert (NBM) is the major source of cholinergic input to the cerebral cortex and projects to most cortical areas and is implicated in several roles, as memory, attention, and behaviour. It has been demonstrated that the degeneration of NBM is involved in various forms of dementia, as Alzheimer and Parkinson diseases, but also in schizophrenia. The first aim of this thesis was to took advantage from the availability of human foetal nucleus basalis of Meynert (hfNBM) cultures to investigate the electrophysiological properties of immature, non-differentiating, cholinergic neurons from the human developing CNS and their functional responses to cholinergic agonists. For this purpose, we used electrophysiological patch-clamp recordings and selective cholinergic agonist and/or antagonist, to investigate functional metabotropic receptors in hfNBM cultures. Carbachol activated atropine-sensitive muscarinic receptors that enhanced IK and reduced INa currents by activating Gi- or phospholipase C-dependent pathways, respectively. When investigated in the current-clamp mode, cells presented either a single, small amplitude action potential, or high-frequency oscillations in membrane potential. This latter phenomenon, defined by us “voltage waves”, was impaired by intracellular thapsigargin, a potent inhibitor of endoplasmic reticulum Ca++-ATPases, or BAPTA and prevented by extracellular Iberiotoxin, a selective blocker of BK channel, or Ba++, demonstrating the involvement of intracellular Ca++ rise, BK and Kir channels, respectively. This knowledge could be of relevance to understand the mechanisms of cholinergic system development and functions in the human brain, either in health or disease. In addition, it is well recognised that purinergic signalling plays a fundamental role in several biological systems, including both short-term (neurotransmission, endothelial-mediated vasodilatation, platelet aggregation) and long-term (cell proliferation, differentiation, migration and death) phenomenon. In particular, about this field, we studied the role of adenosine in oligodendrogliogenesis and pain. Oligodendrocytes are the only myelinating cells in the brain and differentiate from their progenitors (OPCs) throughout adult life. However, this process fails in demyelinating pathologies. Adenosine is emerging as an important player in OPC differentiation and it is demonstrated that adenosine A2A receptors inhibit cell maturation by reducing voltage-dependent K+ currents. Therefore, no data are available to date about the A2B receptor (A2BR) subtype. On the other hand, the bioactive lipid mediator sphingosine-1-phosphate (S1P) and its receptors (S1P1–5) are also crucial modulators of OPC development. In addition, an interaction between this pathway and the A2BR is reported in peripheral cells. Therefore, the second aim was to study the role of A2BRs in modulating K+ currents and cell differentiation in OPC cultures and we investigated a possible interplay with S1P signalling by electrophysiological recordings and biochemical assays, as real-time quantitative polymerase chain reaction experiments (RT-PCR), western-blot, small interference RNA transfection and immunofluorescence analysis. Our data indicate that the A2BR agonist BAY60-6583 and its new analogue P453 inhibit K+ currents in cultured OPC. This effect was prevented by the A2BR antagonist MRS1706, by K+ channel blockers and was differently modulated by the S1P analogue FTY720-P. An acute (10 min) exposure of OPCs to BAY60-6583 also increased the phosphorylated form of sphingosine kinase 1 (SphK1). A chronic treatment with A2BR agonists decreased OPC differentiation whereas SphK1/2 inhibition exerted the opposite effect. Furthermore, A2BR was overexpressed during OPC differentiation, an effect prevented by the pan SphK1/2 inhibitor VPC69047. Finally, A2BR silenced cells showed increased cell maturation, decreased SphK1 expression and enhanced S1P lyase levels. In conclusion, A2BRs inhibit K+ currents and cell differentiation and positively modulate S1P synthesis in cultured OPCs. Recently, studies have focused on the antihyperalgesic activity of the A3 adenosine receptor (A3R) in several chronic pain models, but the cellular and molecular basis of this effect is still unknown. Therefore, in the last aim, we investigated the expression and functional effects of A3R on the excitability of small- to medium-sized, capsaicin-sensitive, dorsal root ganglion (DRG) neurons isolated from 3- to 4-week-old rats, by using patch-clamp and RT-PCR experiments and immunofluorescence analysis. Patch-clamp experiments demonstrated that two distinct A3R agonists, Cl-IB-MECA and the highly selective MRS5980, inhibited Ca++-activated K+ (KCa) currents evoked by a voltage-ramp protocol. This effect was dependent on a reduction in Ca++ influx via N-type voltage-dependent Ca++ channels, as Cl-IB-MECA–induced inhibition was sensitive to the N-type blocker PD173212 but not to the L-type blocker, lacidipine. The endogenous agonist adenosine also reduced N-type Ca++ currents, and its effect was inhibited by 56% in the presence of A3R antagonist MRS1523, demonstrating that the majority of adenosine’s effect is mediated by this receptor subtype. Furthermore, current-clamp recordings demonstrated that neuronal firing of rat DRG neurons was also significantly reduced by A3R activation in a MRS1523-sensitive but PD173212-insensitive manner. Intracellular Ca++ measurements confirmed the inhibitory role of A3R on DRG neuronal firing. We conclude that pain-relieving effects observed on A3R activation could be mediated through N-type Ca++ channel block and action potential inhibition as independent mechanisms in isolated rat DRG neurons. These findings support A3R-based therapy as a viable approach to alleviate pain in different pathologies.

The aim of this research study is to highlight and to compare the behavior of two human neuroblastoma cell lines, representative of two tumor phenotypes, respectively the SHSY5Y less aggressive (not N-MYC amplified), and the IMR-32 more aggressive (N-MYC amplified), after two different hypoxic exposures, that are the first for 4 h or acute hypoxia and the second for 24 h or prolonged hypoxia. The interesting results obtained in this initial study allowed to outline probable scenarios of oxygen deficiency action on neuroblastoma cell cultures. The response of the two neuroblastoma cell cultures to two different hypoxic stimuli showed its greatest differences especially when treatment in hypoxia prolonged over time. The overall view of these first results led to hypothesis that daily hypoxia triggered a series of phenomena that induced SHSY5Y neuroblastoma cells to differentiation towards a neuronal phenotype. These phenomena were precisely the mortality from apoptosis, the cell cycle arrest in G1 phase, the presence of TG2-S involved in differentiation processes, the low presence of metalloproteases involved in tumor invasion and the accumulation of adhesion molecules and in particular ICAM, whose presence seems to be related to neuroblastoma cell differentiation. Instead the IMR-32 cell line, after the same treatment of SHSY5Y cell line, showed good cellular viability and the cell cycle progression. The most interesting result was the unexpected detection of both transglutaminase 2 isoforms in hypoxia, where it was already proven that TG2-L acts to stimulate cell proliferation in contrast to TG2-S. Instead the adhesion molecules ICAM and VCAM and the metalloproteases showed a behavior that must be further verified. These results obtained for the IMR-32 cell line induced the intriguing idea that hypoxia strongly stimulated the proliferation of these neuroblastoma cells, making the tumor even more dangerous and invasive. This first approach to the study of the processes triggered by hypoxia in two different neuroblastoma cell cultures opened interesting horizons on increasingly specific understanding of the behavior of this malignant neoplasm. Furthermore it also offered further insights into the clinic and pediatric surgery on the possibility or the risk of applying techniques such as minimally invasive surgery for biopsies of this tumor, precisely because of the hypoxic environment created by the carbon dioxide insufflation.