To gain insight to the cellular and molecular mechanisms involved abnormal neuronal migration induced by irradiation, we investigated expression of neuronal cell adhesion molecule L 1 and neuronal migration in the brains through comparison between rats prenatally exposed to X-ray and controls. To observe the pattern of neuronal migration, bromodeoxyuridine (BrdU) was chosen as a marker to label migrating cells. The results showed some of the labeled cells remained in the lower of the cortical plate in the irradiated rats, suggesting that neuronal migration was disrupted by X-ray. To study change of expressing neural cell molecule L1, rat brains were analyzed by SDS-PAGE after isolation of L1 by immunoaffinity chromatography. In the all brain membrane fraction, immunoaffinity purified L1 had bands at 200, 180, 140 and 80 kDa. However, the bands in the irradiated group were very weak when compared with the control. Taking these results into account, abnormal neuronal migration and reduction of expression L1 found in their radiated brain indicated that migration of neural cells may be largely dependent on radial glial fiber as well as neural cell molecules like L1. A decrease in L1 expressionmay be one of reasons of abnormal neuronal migration

Cui, Chun

Fukui, Yoshihiro

Kubota, Yoshihisa

Sawada, Kazuhiko

Sun, Xue-Zhi

Takahashi, Sentaro

Zhang, Rui

Tokushima University Institutional Repository

INTRODUCTIONThe developing brain is one of the fetal structuresmostsusceptible to ionizing radiation. The high vulnerabilityof the fetal brain is a distinctive teratological characteristiccommonly recognized inallmammalianspecies includinghumans (1, 2). Studies of survivors exposed to atomicbombing in Hiroshima and Nagasaki (3) or to X-raysduring medical procedures (4) indicate thatmicrocephalywith typicalheterotopicgraymatter in the lateral ventriclesand mental retardationareprominent findings.Althoughthese biological effects of radiation on embryos havebeen considered to be responsible for cell death andabnormal migration (5), themechanisms involved in celldeath and abnormal migration have remained unclear.Development of the mammalian brain requires theintegration ofmany cellular processes.Neuronmigrationis the critical cellular processwhich initiates histogenesisof neocortex, because the neurons that are born pro-gressively later in gestation must travel increasinglylong distances to reach their correct location. It isnow accepted that glial radial fibers guidance providesa primary mechanism for the positioning of youngneurons in the developing brain (6, 7). To study cell-cellinteractions, neural cell adhesion molecule L1, thatappears to mediate cell-cell adhesion, has been foundto be implicated in several morphogenic processes,especially inneuronalmigration (8 -10). The relationshipbetween neuronal migration and neural cell adhesionORIGINALExpression of neural cell adhesion molecule L1 in the brainof rats exposed to X-irradiation in uteroXue-Zhi Sun, Rui Zhang＊, ChunCui＊, Sentaro Takahashi, Yoshihisa Kubota, KazuhikoSawada＊, and Yoshihiro Fukui＊Environmental and Toxicological Sciences Research Group, National Institute of Radiological Sciences,Chiba, Japan; ＊Department of Anatomy and Developmental Neurobiology, The University of TokushimaSchool of Medicine, Tokushima, JapanAbstract : Togain insight tothecellularandmolecularmechanismsinvolvedabnormalneuronalmigration inducedbyirradiation,weinvestigatedexpressionofneuronalcelladhesionmoleculeL 1 andneuronalmigration in thebrains throughcomparisonbetweenratsprenatallyexposedto X-rayandcontrols. Toobserve thepatternofneuronalmigration,bromodeoxyuridine (BrdU)was chosenasamarkertolabelmigratingcells.Theresultsshowedsomeofthelabeledcellsremainedin the lowerof the cortical plate in the irradiated rats, suggesting thatneuronalmigrationwasdisrupted byX-ray.TostudychangeofexpressingneuralcellmoleculeL1,ratbrainswereanalyzedbySDS-PAGEafter isolationofL1byimmunoaffinitychromatography. Intheallbrainmembranefraction, immunoaffinitypurifiedL1hadbands at 200, 180, 140 and80kDa.However, thebandsin theirradiatedgroupwereveryweakwhencomparedwiththecontrol.Takingtheseresultsintoaccount, abnormalneuronalmigrationandreductionofexpressionL1foundintheirradiatedbrainindicated thatmigrationofneural cellsmaybe largelydependentonradialglial fiberaswellasneural cell molecules like L1. A decrease in L1expressionmay be one of reasons of abnormalneuronal migration. J. Med. Invest. 50 : 187-191, 2003Keywords : cell adhesion molecular, irradiation, migration, neocortexReceived for publication June 23, 2003 ; accepted July 23, 2003.Address correspondence and reprint requests to Dr. Xue-ZhiSun, Environmental and Toxicological Sciences Research Group,National Institute of Radiological Sciences, Anagawa4-9-1,Inage-ku, Chiba 263-8555, Japan and Fax : +81-43-251-4853.The Journal of Medical Investigation Vol. 50 2003１８７molecule L1 has been tested by two different in vitroassay system (microwell and explant culture systems)(11-14), but few study concerning such relationshiphas reported in vivo assay system. To gain insight to thecellular and molecular mechanisms involved abnormalneuronal migration induced by X-ray, we investigatedchanges of L1 expression and neuronalmigration in thebrains through comparison between rats prenatallyexposed to X-irradiation and controls.MATERIALS AND METHODSAnimalsThe animals used were commercially supplied SLc :Wistar rats (Hamamatsu, Shizuoka, Japan). They werehoused in an are-conditioned room (23±2℃) with arelative humidity of 55±5% under an alternating 12hlight/dark schedule (7 : 00 A.M.-7 : 00 P.M.). Foodand water were provided ad libitum.Nulliparous femalesabout 10 weeks old were caged with potent males inpairs overnight. Pregnancies were dated as embryonicday 0(E0) when copulation plugswere found followingovernight mating. Rat with positive plugswere housedin individual cages.Three damswere randomly selected for study in eachexperimental period, and 6 offspring were examinedin each group.TreatmentPregnant females were exposed to a single whole-body X-irradiation at a dose of 1.5 GyOnE15. The physi-cal factors of the X-rays used were 200kVp, 15mA, 0.5mm Cu＋0.5mm Al filter, 90cm distance, and 0.45Gy/minute exposure rate. Control pregnant rats were sham-treated. To observe the pattern ofmigrating neurons,bromodeoxyuridine (BrdU) was chosen as amarkerto label migrating cells. Animals of both groups wereinjected intraperitoneally with 30mg/KgBrdU(BectonDickinson, San Jose, Calif., USA) on E 17.HE-staining and Immunohistochemical stainingThe brains of the fetuses were obtained at 6h (E15)of exposure, E17 and postnatal day 4 (P4).The sampleswere fixed in Bouin’s solution, dehydrated, embeddedin paraffin, and serially sectioned in frontal plane at5 µm. The sections were stainedwith hematoxylin andeosin, or by immunohistochemical staining usingmono-clonal antibody specific for BrdU (Becton Dickinson,San Jose, Calif., USA). The visualization of BrdU inbrainsections was performed basically according to themethod ofRoberts et al. (15). Sectionsweredenatures in4NHCl for 20min at room temperature, neutralizedwith0.1 N sodiumborate buffer (pH8.5),washed inphosphate-buffered saline (PBS), and reachedwith anti-BrdUmon-oclonal antibody (1:100 in PBS with 1mg/ml bovineserum albumin) for 60 min at room temperature. Thesections were then sequentially incubatedwith goat anti-rat immunoglobulin (Ig) biotinylated second antibody,horseradish peroxidase (HRP)-conjugated streptavidin,and coloredwith 3, 3-diaminobenzidine (DAB), resultingin the formation of a brown precipitate in the nucleiof BrdU-labeled cells.Isolation of L1molecule by immunoaffinity chromatographyThe purification of the L1molecule from rat brainwas performed according to the method of Rathjenand Schachner (16). The crude membrane fractionwere prepared from rat brains of E17 and solubilizedin a buffer containing 0.5% NP-40, 20mMTris, 150mMNaCl, 1mMEDTA and 4U aprotinin/ml, pH 7.2. Proteinconcentrations were adjusted to1mg/ml.Non-solubilizedmaterial was removed by centrifugationat 100000gand4℃ for 1h. The supernatant was added first to a columncontaining immunobilizednon-specific rat-IgGantiserum.The flow-through of this columnwas connected directlyto the L 1 antibody-containing colum(5 mg IgG/mlCNBr sepharose, Pharmacia). The bound protein fromanti-L 1 antibody column was eluted with an alkalinebuffer containing 0.1Mdiethylamine, 0.5% deoxycholate(DOC), pH 11.5. Collecting volumeswere neutralizedby the additionof 0.5MTris-HCl, pH6.8.Theproteinwasthen dialyzed against 20mM Tris,0.1%DOC, pH8.5.Sodium dodecyl sulfate-polyacrylamide gel electropho-resis (SDS-PAGE) analysisAfter dialysis, the proteinwasconcentratedbymicrodia-lyzer system (Pierce Chemical Company) for the SDS-PAGE analysis. SDS-PAGE analysis was carried outon 7% polyacrylamide gels according to Laemmli (17).Staining of gels was performedwithCoomassie brilliantblue (CBB).RESULTSPattern of neuronal migration:distribution of BrdU-labeled cellsHistological examination showed that extensive celldeath (pyknotic cells), which were extremely darkand often had fragmented nuclei, occurred 6h afterX-irradiation, and dead cells disappeared by 48h (E17)X. Sun et al. L 1 in brain exposed to X-irradiation１８８after exposure. On E17, at 6 h after BrdU injection,BrdU-labeled cells (yellowish-browncolor)wereobservedonly in the bottom of the ventricular zone in the controlbrain. In the irradiated brain, most labeled cells wereobserved in the ventricular zone, while some labeledcells had appeared in the middle of the brain mantle.By P4, the labeled cells has furthermigrated from theventricular zone to the cortical plate, almost labeledcells were reached in the upper part of the cerebralmantel in the control rat (Fig.1a). In the irradiatedgroup,a high proportion of the labeled cells were located inthe superficial layers of the cortical plate, but someof the labeled cells were remained in the lower ofthe cortical plate (Fig.1 b).Expression of neural cell adhesion molecule L1To study change of expressing neural cell moleculeL1 both in control and irradiated brain, rat brains wereanalyzed by SDS-PAGE after isolation of L1 by immu-noaffinity chromatography. In the all brain membranefraction, immunoaffinity purified L1had bands at 200,180, 140 and 80kDa. However, the bands in irradiatedthe group were very weak when compared with thecontrol, even at the place of 80kDa protein marker,the band had disappeared (Fig.2).DISCUSSIONAccording to a schedule of brain development, E15for rats is critical stage for histogenesis of the cerebralcortex, which corresponds to a timewhen an apparentdramatic surge normally results in neurogenesis alongwith the establishment of architectonic stratificationof the cerebralwall (18). Therefore,X-irradiation resultedin extensive cell death in the rat cerebral cortex onE15.That no dead cells were observed 48h after exposureindicated developing brain had a high recovery capacity.Thus, BrdU injection on E17 may be the proper timeto label actively migrating cells.Examination with immunohistochemistry for anti-BrdU antibody showed a clear course of histogenesisof the neocortex. Neuronal cells in the neocortex wereborn in a region of proliferating cells as seeing BrdU-labeled cells in the ventricular zone at 6h after BrdUinjection, and later these cellsmigrated to the superficialregion of the cortical plate to establish neuronal laminaand settle onto the outmost layer. However, that someofthe labeledcellswere remained in the lowerof the corticalplate in the irradiated rats suggested that neuronalmigration was disrupted by X-ray.Neuronalmigration is a remarkable complex process.Normal neuronalmigrating needs a guidance of radialglial fiber (6, 7) andalsoneedsproper receptors, ligands,neuronal cell adhesion molecules (NCAM, L1, etc.)(19). Studies on neuronal cell adhesionmolecules hasidentified that L1 is a 200kDa integral membraneFig.1.Micrographs of the ratbrainmantle(parietal region)atpostnatalday 4 showing distribution of BrdU-labeled cells.(a). Almost labeledcells have arrived the superficial layer in the control. (b).Somelabeledcells (arrows) are remained in the lower of the cortical plate, althougha high proportion of the labeled cells have reached in thesuperficial layer in the irradiated brain. Scale bar=150µm.Fig.2. SDS -PAGE showing ex-pression of neuronal cell adhesionmolecule L1. L1 was purified byimmunoaffinity chromatographyusing monoclonal antibody frombrains of embryonic day 17. The7% SDS-polyacrylamide gel wasstained byCoomassiebrilliantblue(CBB). In the all brainmembranefraction, immunoaffinity purifiedL 1 hadbands at 200, 180, 140 and80kDa. However, the bands in theirradiated group were veryweakwhen compared with the control.C : Control ; R : Irradiation.The Journal of Medical Investigation Vol. 50 2003 １８９glycoprotein and it plays various important roles in thedynamic phases of neural development, neuronal cellmigration, neurite elongation and fasciculation of axons(14, 16, 20). It has been believed that the importantroles of L1are related to its biological characterization.Expression of L1is one of the first signs of neuronaldifferentiation. Following L1expression, otherNCAMsare always coexpressed on the L 1-positive neuron.It has been demonstrated that NCAM enhances L1-dependent cell-cell interaction (21).Moreover, L1 firstappears in the subplate (the first neurons in the cortex)coexpressed with NCAM during the development ofthe neocortex, and it is predominantly expressed onaxons and scarcely on cell bodiesordendrites. Antibodiesto L1efficiently perturb neurite elongation and fascicu-lation (22, 23). Furthermore, L1 is restrictedly expressedin particular to post-mitotic neurons in thecentral nervoussystem, but no on glia or several non-neural tissuestested. If L1 functions as a ligand in cell-cell adhesionand if the corresponding receptors are also localizedon neurons, the L1may be responsible for adhesionbetween neuronal cells only, and not between neuronsand glia. It is conceivable that L1 mediates adhesionamong neurons in the form of a “self-aggregation”(24). Alternatively, L1maymediate adhesion ofneuronsto other cell types carrying different receptors. Thismode of interactionmay lead to an association betweenpost-mitotic pre-migratory neurons with radial glialfibers which have been postulated to guidemigratingneurons (25).As mentionedabove, neuronalmigration is a complexprocess and also highly sensitive to various physicalincluding to irradiation. Any disturbance of the normalprocess may result in neuronal migration disorder.Previous study has confirmed that prenatal irradiationcaused a disturbance of radial glial fibers (26). Byimmunohistochemistry for anti-BrdU antibody andthe SDS-PAGE analysis, we have presented here thefirst direct evidence that relationship betweenneuronalmigration disorder and expression of L1 molecule inthe brain following prenatal exposure to irradiation.Neuronal migration disorder andreductionof expressionL1 were found in the irradiated brain when comparedwith the control, suggesting that migration of neuralcells may be largely dependent on radial glial fiberas well as neural cell molecules like L1. A decreasein L1 expression should be one of reasons of disruptionof neuronal migration. As to mechanisms causedreduction of L 1 expression by prenatal exposed toirradiation, neuron death and/or inhibition of any stepof L1 expressing has been considered. Further studiesare needed to clarify these points.REFERENCES1. Schardein JL : Chemically induced birth defects.Marcel Dekker, Inc, New York, 19852. Shepard TH : Catalog of teratogenic agents. 5thed. 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Expression of neural cell adhesion molecule L1 in the brain of rats exposed to X-irradiation in utero

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Expression of neural cell adhesion molecule L1 in the brain of rats exposed to X-irradiation in utero

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