Environmental and Toxicological Sciences Research Group,
National Institute of Radiological Sciences, Chiba, Japan;*Department
of Anatomy and Developmental Neurobiology, The University
of Tokushima School of Medicine, Tokushima, Japan
Abstract: To gain insight to the cellular and molecular
mechanisms involved abnormal neuronal migration induced
by irradiation, we investigated expression of neuronal cell
adhesion molecule L1 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 L 1 by immunoaffinity chromatography. In the
all brain membrane fraction, immunoaffinity purified L1had
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 the irradiated
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 L1expression may be one of reasons
of abnormal neuronal migration. J. Med. Invest. 50 : 187-191,
2003
Keywords:cell adhesion molecular, irradiation, migration,
neocortex
INTRODUCTION
The developing brain is one of the fetal structures most
susceptible to ionizing radiation. The high vulnerability
of the fetal brain is a distinctive teratological characteristic
commonly recognized in all mammalian species including humans
(1, 2). Studies of survivors exposed to atomic bombing in
Hiroshima and Nagasaki (3) or to X-rays during medical procedures
(4) indicate that microcephaly with typical heterotopic
gray matter in the lateral ventricles and mental retardation
are prominent findings. Although these biological effects
of radiation on embryos have been considered to be responsible
for cell death and abnormal migration (5), the mechanisms
involved in cell death and abnormal migration have remained
unclear.
Development of the mammalian brain requires the integration
of many cellular processes. Neuron migration is the critical
cellular process which initiates histogenesis of neocortex,
because the neurons that are born progressively later in
gestation must travel increasingly long distances to reach
their correct location. It is now accepted that glial radial
fibers guidance provides a primary mechanism for the positioning
of young neurons in the developing brain (6, 7). To study
cell-cell interactions, neural cell adhesion molecule L1,
that appears to mediate cell-cell adhesion, has been found
to be implicated in several morphogenic processes, especially
in neuronal migration (8 -10). The relationship between
neuronal migration and neural cell adhesion molecule L1
has been tested by two different in vitro assay system (microwell
and explant culture systems)(11-14), but few study concerning
such relationship has reported in vivo assay system. To
gain insight to the cellular and molecular mechanisms involved
abnormal neuronal migration induced by X-ray, we investigated
changes of L1 expression and neuronal migration in the brains
through comparison between rats prenatally exposed to X-irradiation
and controls.
MATERIALS AND METHODS
Animals
The animals used were commercially supplied SLc :Wistar
rats (Hamamatsu, Shizuoka, Japan). They were housed in an
are-conditioned room (23±2°C) with a
relative humidity of 55±5% under an alternating
12h light/dark schedule (7:00A.M.-7:00P.M.). Food and water
were provided ad libitum. Nulliparous females about10 weeks
old were caged with potent males in pairs overnight. Pregnancies
were dated as embryonic day 0(E0) when copulation plugs
were found following overnight mating. Rat with positive
plugs were housed in individual cages.
Three dams were randomly selected for study in each experimental
period, and 6 offspring were examined in each group.
Treatment
Pregnant females were exposed to a single whole-body X-irradiation
at a dose of 1.5 Gy On E15. The physical 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
of migrating neurons, bromodeoxyuridine (BrdU) was chosen
as a marker to label migrating cells. Animals of both groups
were injected intraperitoneally with 30mg/Kg BrdU (Becton
Dickinson, San Jose, Calif., USA) on E17.
HE-staining and Immunohistochemical staining
The brains of the fetuses were obtained at 6h (E15) of exposure,
E17 and postnatal day 4 (P4). The samples were fixed in
Bouin's solution, dehydrated, embedded in paraffin, and
serially sectioned in frontal plane at5µm. The
sections were stained with hematoxylin and eosin, or by
immunohistochemical staining using monoclonal antibody specific
for BrdU (Becton Dickinson, San Jose, Calif., USA). The
visualization of BrdU in brain sections was performed basically
according to the method of Roberts et al. (15). Sections
were denatures in 4N HCl for 20 min at room temperature,
neutralized with0.1N sodium borate buffer (pH 8.5), washed
in phosphate-buffered saline (PBS), and reached with anti-BrdU
monoclonal antibody (1:100 in PBS with 1mg/ml bovine serum
albumin) for 60 min at room temperature. The sections were
then sequentially incubated with goat anti-rat immunoglobulin
(Ig) biotinylated second antibody, horseradish peroxidase
(HRP)-conjugated streptavidin, and colored with 3, 3 -diaminobenzidine
(DAB), resulting in the formation of a brown precipitate
in the nuclei of BrdU-labeled cells.
Isolation of L1molecule by immunoaffinity chromatography
The purification of the L1molecule from rat brain was performed
according to the method of Rathjen and Schachner (16). The
crude membrane fraction were prepared from rat brains of
E17 and solubilized in a buffer containing 0.5% NP- 40,
20mM Tris, 150mM NaCl, 1mM EDTA and 4U aprotinin/ml, pH
7.2. Protein concentrations were adjusted to1mg/ml. Non-solubilized
material was removed by centrifugation at 100000g and4°C
for 1h. The supernatant was added first to a column containing
immunobilized non-specific rat-IgG antiserum. The flow-through
of this column was connected directly to the L1antibody-containing
colum(5mg IgG/ml CNBr sepharose, Pharmacia). The bound protein
from anti-L1antibody column was eluted with an alkaline
buffer containing 0.1M diethylamine, 0.5% deoxycholate (DOC),
pH 11.5. Collecting volumes were neutralized by the addition
of 0.5 M Tris-HCl, pH 6.8. The protein was then dialyzed
against 20mM Tris,0.1%DOC, pH8.5.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) analysis
After dialysis, the protein was concentrated by microdialyzer
system (Pierce Chemical Company) for the SDS-PAGE analysis.
SDS -PAGE analysis was carried out on7% polyacrylamide gels
according to Laemmli (17).Staining of gels was performed
with Coomassie brilliant blue (CBB).
RESULTS
Pattern of neuronal migration:distribution of BrdU-labeled
cells
Histological examination showed that extensive cell death
(pyknotic cells), which were extremely dark and often had
fragmented nuclei, occurred 6h after X-irradiation, and
dead cells disappeared by 48h (E17)after exposure. On E17,
at6h after BrdU injection, BrdU-labeled cells (yellowish-brown
color) were observed only in the bottom of the ventricular
zone in the control brain. In the irradiated brain, most
labeled cells were observed in the ventricular zone, while
some labeled cells had appeared in the middle of the brain
mantle. By P4, the labeled cells has further migrated from
the ventricular zone to the cortical plate, almost labeled
cells were reached in the upper part of the cerebral mantel
in the control rat (Fig.1a). In the irradiated group, a
high proportion of the labeled cells were located in the
superficial layers of the cortical plate, but some of the
labeled cells were remained in the lower of the cortical
plate (Fig.1b).
Expression of neural cell adhesion molecule L1
To study change of expressing neural cell molecule L1 both
in control and irradiated brain, rat brains were analyzed
by SDS -PAGE after isolation of L1 by immunoaffinity chromatography.
In the all brain membrane fraction, immunoaffinity purified
L1had bands at 200,180, 140 and 80kDa. However, the bands
in irradiated the group were very weak when compared with
the control, even at the place of 80kDa protein marker,
the band had disappeared (Fig.2).
DISCUSSION
According to a schedule of brain development, E15for rats
is critical stage for histogenesis of the cerebral cortex,
which corresponds to a time when an apparent dramatic surge
normally results in neurogenesis along with the establishment
of architectonic stratification of the cerebral wall (18).
Therefore, X-irradiation resulted in extensive cell death
in the rat cerebral cortex on E15. That no dead cells were
observed 48h after exposure indicated developing brain had
a high recovery capacity. Thus, BrdU injection on E17 may
be the proper time to label actively migrating cells.
Examination with immunohistochemistry for anti-BrdU antibody
showed a clear course of histogenesis of the neocortex.
Neuronal cells in the neocortex were born in a region of
proliferating cells as seeing BrdU-labeled cells in the
ventricular zone at 6h after BrdU injection, and later these
cells migrated to the superficial region of the cortical
plate to establish neuronal lamina and settle onto the outmost
layer. However, that some of the labeled cells were remained
in the lower of the cortical plate in the irradiated rats
suggested that neuronal migration was disrupted by X-ray.
Neuronal migration is a remarkable complex process. Normal
neuronal migrating needs a guidance of radial glial fiber
(6, 7) and also needs proper receptors, ligands, neuronal
cell adhesion molecules (NCAM, L1, etc.)(19). Studies on
neuronal cell adhesion molecules has identified that L1
is a200kDa integral membrane glycoprotein and it plays various
important roles in the dynamic phases of neural development,
neuronal cell migration, neurite elongation and fasciculation
of axons(14, 16, 20). It has been believed that the important
roles of L1are related to its biological characterization.
Expression of L1is one of the first signs of neuronal differentiation.
Following L1expression, other NCAMs are always coexpressed
on the L1-positive neuron. It has been demonstrated that
NCAM enhances L1-dependent cell-cell interaction (21). Moreover,
L1 first appears in the subplate (the first neurons in the
cortex) coexpressed with NCAM during the development of
the neocortex, and it is predominantly expressed on axons
and scarcely on cell bodies or dendrites. Antibodies to
L1efficiently perturb neurite elongation and fasciculation
(22, 23). Furthermore, L1 is restrictedly expressed in particular
to post-mitotic neurons in the central nervous system, but
no on glia or several non-neural tissues tested. If L1 functions
as a ligand in cell-cell adhesion and if the corresponding
receptors are also localized on neurons, the L1may be responsible
for adhesion between neuronal cells only, and not between
neurons and glia. It is conceivable that L1 mediates adhesion
among neurons in the form of a "self-aggregation"(24).
Alternatively, L1 may mediate adhesion of neurons to other
cell types carrying different receptors. This mode of interaction
may lead to an association between post-mitotic pre-migratory
neurons with radial glial fibers which have been postulated
to guide migrating neurons (25).
As mentioned above, neuronal migration is a complex process
and also highly sensitive to various physical including
to irradiation. Any disturbance of the normal process may
result in neuronal migration disorder. Previous study has
confirmed that prenatal irradiation caused a disturbance
of radial glial fibers (26). By immunohistochemistry for
anti-BrdU antibody and the SDS -PAGE analysis, we have presented
here the first direct evidence that relationship between
neuronal migration disorder and expression of L1 molecule
in the brain following prenatal exposure to irradiation.
Neuronal migration disorder and reduction of expression
L1 were found in the irradiated brain when compared with
the control, suggesting 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 expression should
be one of reasons of disruption of neuronal migration. As
to mechanisms caused reduction of L1 expression by prenatal
exposed to irradiation, neuron death and/or inhibition of
any step of L1 expressing has been considered. Further studies
are needed to clarify these points.
REFFERENCES
1.Schardein JL : Chemically induced birth defects.Marcel
Dekker, Inc, New York, 1985
2.Shepard TH : Catalog of teratogenic agents. 5th ed. Johns
Hopkins Univ Press, Baltimore, 1986
3.Blot WJ, Miller RW : Mental retardation following in uteri
exposure to the atomic bombs of Hiroshima and Nagasaki.
Radiology106:617-619, 1973
4.Dekaban AS : Abnormal lities in children exposed to X-radiation
during various stags of gestation : Tentative timetable
of radiation injury to the human fetus. J Nucl Med9:471-477,
1968
5.Schull WJ, Norton S, Jensh RP : Ionizing radiation and
the developing brain. Neurotoxicol and Teratol12:249 - 260,
1990
6.Rakic P : Neuronal-glial interaction during brain development.
TINS4:184-187, 1981
7.Rakic P : Principles of neural cell migration. Experientia46:882-891,
1990
8.Miura M, Asou H, Kobayashi M, Uyemura K : Functional expression
of a full-length cDNA coding for rat neuronal cell adhesion
molecule L1 mediates homophilic intercellular adhesion and
migration of cerebellar neurons. J Biol Chem267:10752-10758,
1992
9.Hiavin ML, Lemmon V : Molecular structure and functional
testing for human L1CAM : a interspecies comparison. Genomics
2:416 - 423, 1991
10.Prince JT, Alberti L, Healy PA, Nauman SJ, Stallcup WB
: Molecular cloning of NILF glycoprotein and evidence for
its continued expression in mature rat CNS. J Neurosci Res30:567-581,
1991
11.Hatten ME, Liem RKH, Mason CA : Two forms of cerebellar
glial cells interact differently with neurons in vitro.
J Cell Biol98:193-204, 1984
12.Hatten ME : Riding the glial monorail : a common mechanism
for gllial-guide migration in differentregions of the develping
brain. TINS 13:179 -184, 1990
13.Lindner J, Zinser G, Wertz W, Goridis C, Bizzini B, Schachner
M : Experimental modification of postnatal cerebellar granule
cell migration in vitro. Brain Res377:298-304, 1985
14.Linder J, Rathjen FG, Schachner M : L1 molecular and
polyclonal antibodies modify cell migration in early postnatal
mouse cerebellum. Nature305:427-430, 1983
15.Roberts JS, O'Rourke NA, McConnell SK : Cell migration
in cultured cerebral cortical slices. Dev Biol 155:396 -408,
1993
16.Rathjen FG, Schachner M : Immunocytological and biochemical
characterization of a new neural cell surface component
(L1 antigen) which is involved in cell adhesion. EMBO J3:1-10,
1984
17.Laemmli UK : Cleavage of structural protein during the
assembly of the bacteriophage T4. Nature 227 :680-685, 1970
18.Kameyama Y : High vulnerability of the developing fetal
brain to ionizing radiation and hyperthermia. Environ Med33:1-17,
1989
19.Sun XZ, Takahashi S, Cui C, Zhang R, Sakata-Haga H, Sawada
K, Fukui Y : Normal and abnormal neuronal migration in the
developing cerebral cortex. J Med Invest49:97-110, 2002
20.Faissner A, Teplow DB, Kubler D, Keihauer G, Kinzel V,
Schachner M : Biosynthesis and membrane topography of the
neural cell adhesion molecule L1. EMBO J 12:3105-3113, 1985
21.Kadmon G, Kowitz A, Altevogt P, Schachner M : The neuronal
cell adhesion molecule N-CAM enhances L1-dependent cell-cell
interactions. J Cell Biol110:193-208, 1990
22.Chang S, Rathjen FG, Raper JA : Extension of neuritis
on axons is impaired by antibodies against specific neural
cell surface glycoproteins. J Cell Biol104:355-362, 1987
23.Lemmon V, Farr KL, Lagenaur C : L1-mediated axon outgrowth
occurs via a homophilic binding mechanism. Neuron2:1597-1603,
1989
24.Rutishauser U, Hoffman S, Edelman GM : Binding properties
of a cell adhesion molecule from neural tissue. Proc Natl
Acad Sci USA 79 : 685 - 689, 1982
25.Rakic P, Sidman RL : Weaver mutant mouse cerebellum:
defective neuronal migration secondary to abnormality of
Bergmann glia. Proc Natl Acad Sci USA70:240-244, 1973
26.Sun XZ, Inouye M, Fukui Y, Hisano S, Sawada K, Muramatsu
H, Muramatsu T : An immunohistochemical study of radial
glial cells in the mouse brain prenatally exposed to gamma-irradiation.
J Neuropathol Exp Neurol56:1339-1348, 1997
|
