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The Journal of Neuroscience, January 1, 1998, 18(1):307-318
Platelet-Activating Factor Receptor Stimulation Disrupts Neuronal
Migration In Vitro
Gregory J.
Bix1 and
Gary D.
Clark1, 2
Departments of
1 Pediatrics, Neurology, and
2 Neuroscience, The Cain Foundation Laboratories, Baylor College of Medicine, Houston,
Texas 77030
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ABSTRACT |
LIS-1 is a gene whose hemi-deletion causes the
human neuronal migration disorder Miller-Dieker lissencephaly. It
encodes a subunit of a brain platelet-activating factor (PAF)
acetylhydrolase, an enzyme that inactivates PAF by hydrolyzing the
acetyl moiety in the sn2 position of this phospholipid.
Because PAF receptor activation has been shown to affect the developing
neuronal cytoskeleton, we have hypothesized that a role for PAF in
neurodevelopment is that of a modulator of neuroblast movement (a
cytoskeletal function) and that an aberrant regulation of PAF could
lead to an early arrest in migration. This report examines the effects
of the nonhydrolyzable PAF receptor agonist methyl carbamyl PAF
(mc-PAF) on the unidirectional in vitro migration of
granule cells from cerebellar cell reaggregates on a laminin substrate.
Bath treatment with mc-PAF yields a dose-dependent decrease in granule
cell migration compared with controls. This effect can be blocked by
the simultaneous bath application of BN 52021 and
trans-BTD, PAF receptor-specific antagonists. Although mc-PAF minimally inhibited neurite growth, its primary effect was on
somal movement along preextended neurites. These experiments suggest
that the stimulation of neuronal PAF receptors could be one crucial
step for the regulation of neuroblast migration and that disturbed PAF
catabolism during neurodevelopment could contribute to the neuronal
migration defects observed in Miller-Dieker lissencephaly.
Key words:
methyl carbamyl platelet-activating factor (mc-PAF); BN
52021; platelet-activating factor (PAF); neuronal migration; Miller-Dieker lissencephaly; LIS-1; PAF acetylhydrolase; PAF
receptor
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INTRODUCTION |
Platelet-activating factor
1-0-alkyl-2-acetyl-sn-glyceryl-3
phosphorylcholine (PAF) is an alkyl-ether phospholipid with probable important roles in the CNS (Kornecki et al., 1984 ; Birkle et al., 1988;
Kornecki and Ehrlich, 1988; Kumar et al., 1988; Del-Cerro et al., 1990;
Arai and Lynch, 1992; Clark et al., 1992; Kato et al., 1994). Brain
contains enzymes that produce (Bazan et al., 1983; Yue et al., 1990;
Baker and Chang, 1993, 1997; Bonoventre and Koroshetz, 1993) and
degrade PAF (Hattori et al., 1993). One brain PAF receptor has been
characterized (Bito et al., 1992, 1993).
PAF has been implicated in the human neuronal migration disorder
Miller-Dieker lissencephaly, a disorder in which neurons are arrested
in the migration that forms the cerebral cortical plate and the
cerebellar internal granule cell layer (IGL) (Stewart et al., 1975;
Jellinger and Rett, 1976; Barth, 1987). In this disorder, the brain has
a smooth cortical surface (lissencephaly) caused by a lack of
complexity of the outer cortex. Additionally, the brains of these
patients manifest a disruption of migration of cerebellar granule cells
(Miller, 1963; Stewart et al., 1975). This brain malformation results
from a haploinsufficiency of the gene LIS-1 (Ledbetter et
al., 1992; Reiner et al., 1993; Mizuguchi et al., 1995; Chong et al.,
1997; Ho et al., 1997; Lo Nigro et al., 1997), which encodes a 45 kDa
subunit of a brain PAF acetylhydrolase (PAF-AH 1b), an enzyme that
converts PAF to the inactive lyso-PAF by removing the acetyl group on
the sn2 position of the PAF molecule (Hattori et al., 1993;
Hattori et al., 1994). The LIS-1 gene haploinsufficiency in
Miller-Dieker lissencephaly could result in defects in PAF catabolism.
Because PAF receptor activation has been shown previously to evoke
changes in the neuronal cytoskeleton leading to growth cone collapse
and neurite withdrawal, it has been suggested that similar PAF
receptor-mediated changes in the neuroblast cytoskeleton could lead to
a disruption of neuronal migration (Clark et al., 1995). This possible
mechanism was probed using a well established in vitro model
of cerebellar granule cell migration from reaggregate cell clusters
(Asou et al., 1992). The migration of cerebellar granule cells is well
characterized (Hatten and Mason, 1990) and often used for the study of
neuronal migration. The neuronal migration from reaggregate clusters,
although similar to migration on glia, minimizes the influence of glia
on migration because the processes of glial cells contained within the
reaggregate clusters do not serve as a migration substrate for the
moving granule cells. Furthermore, unlike the in vitro
migration on glia, granule cell migration from reaggregate clusters was
unidirectional radially away from the cluster margins. Initially, some
neurons extended processes out from the cluster margin on a supportive
extracellular matrix. Then granule cells migrated solely away from the
margin of the reaggregate cluster along the preextended neuronal
processes. They did so by first extending a leading process and then
undergoing somal translocation through this leading process. This
simple cell reaggregate system allowed for the testing of the effects of PAF receptor agonists and antagonists on large numbers of migrating granule cells and allowed for the quantification of the unidirectional movement of these cells (Edmondson et al., 1987).
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MATERIALS AND METHODS |
Materials. Minimal essential medium (MEM) and
Leibovitz's L-15 medium for cell cultures were obtained from
Life Technologies (Grand Island, NY). Albumin, papain, trypan blue,
methanol, and dimethyl sulfoxide (DMSO) were obtained from Sigma (St.
Louis, MO). Methyl carbamyl platelet-activating factor
1-0-hexadecyl-2-0-(methyl carbamyl)-sn-glycero-3-phosphorylcholine (mc-PAF) and
lyso-PAF (1-0-hexadecyl-sn-glycero-3
phosphorylcholine) were obtained from Cayman Chemical Company (Ann
Arbor, MI). BN 52021, trans-2,5-bis-{3,4,5-trimethoxyphenyl}-1,3-dioxolane (trans-BTD), and
cis-2,5-bis-{3,4,5-trimethoxyphenyl}-1,3-dioxolane (cis-BTD) were obtained from Biomol (Plymouth Meeting, PA).
Laminin, purified from mouse EHS sarcoma, was obtained from Sigma.
Glial fibrillary acidic protein (GFAP) antibody was obtained from Dako Antibody (Carpinteria, CA). TUJ-1 antibody [highly reactive with neuron-specific class III  tubulin but not with tubulin in glia
(Lee et al., 1990; Moody et al., 1996)] was obtained from Babco
Antibody (Berkeley, CA).
Rat cerebellar granule cell reaggregate cultures. Cerebella
were obtained from 8-d-old rats (Harlan Sprague Dawley, Indianapolis, IN) anesthetized with methoxyflurane (Metofane, Pitman-Moore, Mundelein, IL). The cerebellar tissue was digested with papain and then
triturated as described previously (Clark et al., 1995). The
dissociated cells from a single rat cerebellum were placed in a sterile
50 ml polystyrene centrifuge tube and incubated overnight (without
rotation) at 37°C in an atmosphere of 5% C02 in 20 ml of
MEM containing 10% horse serum, 10% fetal calf serum, 6 mM glucose, 200 µM L-glutamine,
50 U/ml penicillin, 50 µg/ml streptomycin, 10 µg/ml human
transferrin, 10 µg/ml insulin, and 10 ng/ml selenium. The cap on the
cell-containing tube was loosened to allow the cells to aerate. This
overnight incubation allowed cellular reaggregates to form (Kobayashi
et al., 1995). On the next day, 2 ml aliquots of this cell reaggregate
suspension were added to 35 × 10 mm tissue culture dishes
(Falcon) coated with laminin for 24 hr at 37°C (25 µg/ml, 2 ml per
dish). The laminin solution was removed, and the dishes were rinsed
once with deionized water immediately before cell plating. The plated
cells were returned to the 37°C incubator for 1 hr to allow for the
reaggregate cell clusters to adhere to the laminin substrate but not to
allow sufficient time for neurite outgrowth, which was slowed by the
presence of serum in the media (Liesi, 1992).
Experimental design. Specific concentrations of mc-PAF
(0.10, 0.25, 0.50, and 1.00 µM), BN 52021 (50 and 100 µM), cis-BTD (50 µM),
trans-BTD (50 µM), lyso-PAF (1 µM), DMSO, and methanol were prepared in serum-free media
(identical to the media described above without the horse serum and
fetal calf serum). The serum-containing media present in the tissue
culture dishes (containing the reaggregate cell clusters) was removed
and replaced with 2 ml aliquots of serum-free media containing the
experimental compounds (one aliquot of one experimental compound per
dish, or a combination of compounds, i.e., mc-PAF and BN 52021, mc-PAF
and cis-BTD, and mc-PAF and trans-BTD). Some
cells (controls) were treated with serum-free media containing no
experimental compound. The cells were returned to the 37°C incubator
and removed for observation after 1, 6, 12, and 24 hr. No detectable
decrease in the bioactivity of these compounds [as measured by rabbit
platelet aggregation (for details, see Clark et al., 1995)] was noted
after 24 hr of cell incubation (data not shown).
Measurements of neurite properties and granule cell
migration. Reaggregate cell clusters were observed and
photographed under a phase contrast microscope (Olympus). After 1 hr of
drug incubation, 12 cell clusters (typically each dish contained ~30
clusters) of similar size (100-120 µm in diameter) were randomly
selected from each dish to make measurements. Clusters of similar size (containing approximately the same number of cells) were selected to
minimize the possibility that any calculated change in the number of
neurites or migrating neurons might be caused by differences in the
number of cells in each cluster. Selected cell clusters were also
required to be farther than 600 µm away from other clusters or the
neurites of other clusters so that the single cell cluster source of
neurons and neurites could be clearly identified. After 24 hr of drug
incubation, the cells were fixed with 4% paraformaldehyde, and the
distribution of granule cells (migrated granule cells) and the lengths
of neurite extension beyond the cluster margin were measured. Some
experiments were conducted with the examiner blinded to the drug
treatment conditions for the cell clusters. All data were statistically
analyzed (two-tailed t test) for significance with SigmaStat
(version 1.0) for Windows (Jandel Scientific, San Rafael, CA). In other
experiments, the number of neurites elaborated from each selected cell
cluster (in the various drug conditions), as identified by indirect
immunofluorescence with TUJ-1 antibody, was recorded at 1, 3, 6, and 12 hr. Time lapse photography was performed for individual cell clusters
using a Bioptechs  T culture dish system to maintain a constant
temperature of 36°C. Video time lapse images were recorded with a CCD
video camera (DAGE-MTI, Michigan City, IN) and Metamorph imaging
software version 2.0 for Windows (Universal Imaging Corporation, West
Chester, PA). Cell clusters for these experiments were plated (in a
similar serum-free media containing a HEPES buffer instead of
HCO3) on laminin-coated (in the same manner as
described above), 0.5-mm-thick glass-bottomed T dishes that were
sealed with a coverslip.
GFAP and TUJ-1 indirect immunofluorescence. Reaggregate cell
clusters were cultured on laminin-coated glass coverslips placed in
35 × 10 mm culture dishes. The cells were washed once with PBS
and then fixed for 30 min with 4% paraformaldehyde. The cells were
blocked and permeabilized for 30 min with 10% goat serum and 0.1%
Triton X-100 in PBS. The primary antibody was applied [1:1000 GFAP
antibody (polyclonal), 1:500 TUJ-1 antibody (monoclonal)] to the cells
for 2 hr followed by three washes with PBS. The cells were again
permeabilized for 1 hr with 0.1% Triton X-100 in PBS. The appropriate
secondary antibody (fluorescein or rhodamine conjugated, 1:100) was
next applied for 2 hr followed by three washes with PBS. The coverslips
were then mounted on glass slides with Vectashield (Vector
Laboratories, Burlingame, CA) and observed for fluorescence with a
Nikon Microphot-FXA microscope equipped with a UV light source.
Trypan blue cell exclusion assay. A 0.2% trypan blue
solution was prepared in a saline solution containing (in
mM) 150 NaCl, 5 CaCl2, 2.5 KCl, 1 MgCl2, 10 glucose, and 10 HEPES, pH 7.3, 290-310 mOsm. The trypan blue solution was warmed to 37°C and then gently added to the cultured cells. After 30 min of cell incubation at 37°C,
the trypan blue solution was removed from the cells, which were
subsequently fixed with 4% paraformaldehyde and observed the same day
for the incorporation of trypan blue into the cells (indicating cell
death).
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RESULTS |
Within the first hour of plating, cerebellar reaggregate clusters
appeared as a sphere of similar cells that adhered to the laminin
substrate coating the dish bottom. Minutes after the serum-containing media in the dish was replaced with serum-free media (serum-free media
control), cell process extension was observed. In the ensuing 5 hr,
more processes extended beyond the margin of the clusters. Many of
these processes formed bundles, whereas a few remained as individual,
discrete processes. TUJ-1 immunofluorescence confirmed that they were
exclusively neurites (Fig. 1). Time-lapse
observation of the clusters under the microscope with strict
temperature control (36°C) revealed that small bipolar cells migrated
unidirectionally away from the cell clusters along these preextended
neurites and neurite bundles (Figs. 1, 2;
see Fig. 7). The neuronal identity of these cells was confirmed with
TUJ-1 staining (Fig. 1). These cells were deemed to be cerebellar
granule cells, rather than stellate or basket cells, on the basis of
size [granule cells were ~5-7 µm in diameter (15 µm polarized
ends), a well characterized size for granule cells in vivo
(Altman, 1972) and in vitro (Gregory et al., 1988) compared
with stellate and basket cells, which were 7-12 µm], and shape
[granule cells were bipolar, with two long small neurites elaborated
in opposite directions, a well characterized feature of granule cells
in vitro (Selak et al., 1985; Rivas and Hatten, 1995) (Figs.
1, 2, 3; see Fig. 7) and in
vivo (Altman, 1972), whereas stellate and basket cells were
elliptical].
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Figure 1.
Characterization of cellular composition of and
granule cell migration from cerebellar reaggregate clusters.
A shows an image (40× magnification) of a cerebellar
reaggregate cluster immunolabeled with the neuronal cell marker
anti-TUJ-1 (neuronal class III tubulin-specific marker) antibody
(green) and with a glial cell marker anti-GFAP
antibody (red/yellow; curved arrow
indicates a glial cell). Note that the glia present are localized
almost exclusively to the reaggregate core, and their processes do not contribute to the processes elaborated from the cluster
(green, neuritic processes, straight
arrows). Scale bar, 50 µm. B shows three
granule cells (small arrows, 100× magnification,
immunolabeled in the same manner as in A) migrating away
from a reaggregate cluster along a neurite bundle. Note the nearby
red/yellow glial cell (large arrow) that
does not contribute any processes as migration substrate for the
granule cells. Granule cell migration from the reaggregate clusters
occurs along preextended neurites (homotypic migration). Scale bar, 10 µm.
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Figure 2.
Time-lapse characterization of granule cell
migration from a cerebellar reaggregate cell cluster. A
and B are images of the same reaggregate cell cluster
(in serum-free media at 36°C plated on laminin) taken 1 hr apart.
Scale bar, 50 µm. C-E are magnified images taken
every 20 min of the reaggregate cell cluster area outlined in
A and B. The arrow in
C indicates the barely visible soma of a granule cell
that is extending a process (indicated by arrowheads)
out along another preextended neurite to migrate. This cell can be seen
to migrate away from the cluster in D and more so in
E. Immediately after images B and
E were taken, the serum-free bathing solution was
bath-exchanged with one containing 1 µM mc-PAF, and image
F was taken 20 min later. Note the slight rounding of
the granule cell. The granule cell has not moved appreciably from its
position in E. Other experiments that were performed (Fig. 7) determined that the mc-PAF-treated granule cells migrated again after removal of mc-PAF. Scale bar, 15 µm.
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Figure 3.
Effects of mc-PAF and BN 52021 on the migration of
granule cells from reaggregate cell clusters. The above images show
typical cerebellar reaggregate cell clusters plated on a laminin
substrate 24 hr after drug incubation. These and all cell clusters used for data collection were selected for similar initial cell number contained in the cluster and for distance away from other clusters or
their processes (at least 600 µm) to clearly identify the single cell
cluster source of neurons and neurites. A shows a
cluster treated with serum-free media alone (control). A substantial
number of granule cells have elaborated processes and migrated away
from the cluster. B shows a cluster treated with 1 µM mc-PAF in serum-free media. Many neurites can be seen
extending from the cluster, but very few cells have migrated.
C shows a cluster treated with 1 µM mc-PAF
and 50 µM BN 52021 in serum-free media. The disruptive effect of mc-PAF on migration is inhibited. D shows a
cluster treated with 100 µM BN 52021 in serum-free media.
Many more cells have migrated than in control conditions shown in
A. Scale bar, 50 µm.
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Migration of granule cells from reaggregate clusters involved the
extension of processes along other preextended neurites (TUJ-1
positive, GFAP negative) and then over time, the translocation of their
cell bodies outside of the reaggregate cell cluster margin. Higher
magnification, time lapse, infrared differential-interference contrast
(IR-DIC) video microscopy of these migrating neurons revealed that they
migrated by somal translocation through a leading process (see Fig. 7).
In addition to granule cells, a small number of basket and stellate
cerebellar interneurons (<10% of the total number of cells that
migrated) could be seen (Fig. 3A) to have migrated. These
cells were not GFAP positive and were not used for granule cell
migration calculations.
The reaggregate cluster method used in this study involved neuronal
migration along preextended neurites (TUJ-1 positive) elaborated on a
defined laminin substrate. GFAP immunofluorescence labeled some cells
within the reaggregate cluster (average number of GFAP-positive cells
per cluster = 15.1 ± 0.8; n = 12), but none
of the processes elaborated from the cluster that served as a neuronal
migration substrate (Fig. 1A). Therefore, the
homotypic migration of granule cells in this system was confirmed to be similar to that described in other in vitro cerebellar
systems such as cerebellar microexplant cultures (Nagata et al.,
1990).
Inhibition of granule cell migration by mc-PAF
(Fig. 3B)
We examined the long-term effects of PAF receptor activation on
neuronal migration using a nonhydrolyzable PAF receptor agonist, mc-PAF, which is not subject to the ubiquitous PAF acetylhydrolases present in CNS tissue in vitro (O'Flaherty et al., 1987;
Clark et al., 1995). mc-PAF exposure altered the expected migration of
granule cells from the reaggregate cell clusters. Figure 3B illustrates a typical cell cluster treated with 1 µM
mc-PAF for 24 hr. This figure reveals a dramatic decrease in the number
of granule cells that migrated from the cell cluster compared with the
cluster shown in Figure 3A (serum-free media control after 24 hr). mc-PAF caused a significant dose-dependent decrease in the mean
total number of granule cells that migrated (78.1 ± 2.7 cells in
500 nM mc-PAF vs 256.6 ± 1.9 cells in control
conditions) (Table 1). In addition, the
mean distance of migration from the cluster margin measured after 24 hr
of mc-PAF incubation also demonstrated a dose-dependent decrease at
three mc-PAF concentrations (119.3 ± 0.6 µm in control
conditions vs 72.8 ± 0.4 µm in 500 nM mc-PAF). No
significant effect was noted at 100 nM mc-PAF (Table 1).
Experiments in which the investigator was blinded to the drug
conditions yielded a mean total number of migrated cells (46.8 ± 1.9) and a mean migration distance (55.5 ± 0.8 µm) for 1 µM mc-PAF-treated cell clusters (n = 12)
that were significantly different from blinded serum-free media
controls (256.6 ± 1.9 cells and 119.3 ± 0.6 µm mean
migration distance) (Table 1) but not from unblinded 1 µM
mc-PAF experiments (45.0 ± 1.5 cells migrated and 60.1 ± 0.5 mean migration distance).
Granule cell migration from the cell clusters was not
significantly affected by treatment with methanol alone, indicating that the drug vehicle used for mc-PAF was not responsible for the
changes in migration. Also, lyso-PAF, which is structurally similar to
PAF and mc-PAF but with little PAF receptor activity, did not have any
effects on neuronal migration at a concentration of 1 µM
(Fig. 4, Table 1). This suggests that
mc-PAF effects were receptor-specific. Trypan blue cell exclusion
assays revealed no significant differences in the number of dead
neurons after 24 hr of 1 µM mc-PAF treatment (2.3 ± 0.1 trypan blue-labeled cells per cell cluster; n = 48)
and 24 hr of serum-free media treatment (2.2 ± 0.2 trypan
blue-labeled cells per cell cluster; n = 48).
Therefore, mc-PAF did not affect neuronal migration by killing cells.
Finally, few cells were detached from the laminin substrate in any of
the drug conditions used, and no differences could be seen between 1 µM mc-PAF-treated dishes and serum-free media
controls.
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Figure 4.
mc-PAF inhibits cerebellar granule cell migration
in vitro. The graph illustrates the
number of cerebellar granule cells that have migrated and the distance
of migration from their reaggregate cell cluster source 24 hr after
incubation in serum-free media with 250 nM, 500 nM, and 1 µM mc-PAF, methanol (the drug
vehicle for mc-PAF, at the final concentration for 1 µM
mc-PAF), or serum-free media control (n = 48 for
each condition) at 37°C and 5% CO2. Error bars indicate
SE and were removed from several points above for clarity. SEs from
left to right: serum-free media 2.3, 3.0, 2.6, 1.0, 2.8, 3.1, 3.3, 0.0, 0.0; methanol drug vehicle control 1.4, 1.2, 2.3, 2.0, 4.0, 1.4, 1.3, 0.0, 0.0; 250 nM mc-PAF 3.5, 3.3, 3.2, 2.7, 0.0, 0.0, 0.0, 0.0, 0.0; 500 nM mc-PAF 3.5, 3.3, 3.2, 2.7, 0.0, 0.0, 0.0, 0.0, 0.0; 1 µM mc-PAF, all shown.
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Effects of mc-PAF on the migration substrate
Significant effects of mc-PAF on neurites have been reported
(Clark et al., 1995). Therefore, an effect of mc-PAF on the neurite migration substrate in this study could have contributed to the observed inhibition of cell movement. mc-PAF decreased the overall mean
number of neurites elaborated (TUJ-1 positive) in a dose-dependent manner during the first 12 hr of cell cluster incubation (Fig. 5). The mean number of neurites
elaborated from 1 µM mc-PAF-treated clusters was
significantly lower than in serum-free media controls (p < 0.001) at all times measured, whereas the
mean number of neurites elaborated from 500 nM
mc-PAF-treated clusters (Fig. 5) was only significantly different from
serum-free media controls after 6 and 12 hr of incubation; 250 nM mc-PAF treatment had no significant effect on neurite
number at any time. Measurements of neurite number were not attempted
after 12 hr of drug incubation, because after that time larger numbers
of migrating neurons in non-mc-PAF conditions tended to obscure
individual neurites. However, mc-PAF-treated clusters appeared to have
fewer neurites at this time compared with serum-free media-treated
controls (Fig. 3A,B).
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Figure 5.
PAF receptor activation causes a dose-dependent
decrease in mean neurite number, whereas PAF receptor blockade causes a
dose-dependent increase in mean neurite number. The
graph illustrates the mean number of neurites elaborated
from reaggregate granule cell clusters measured 1, 3, 6, and 12 hr
after incubation with 500 nM mc-PAF, 1 µM
mc-PAF, 50 µM BN 52021, 100 µM BN 52021, 1 µM mc-PAF, and 50 µM BN 52021, or
serum-free media control (n = 48 for each
condition) at 37°C and 5% CO2. Mean neurite counts were
significantly lower than serum-free media control for 500 nM mc-PAF at t = 6 and 12 hr
(p < 0.0001). All mean neurite counts for 1 µM mc-PAF were significantly lower than controls
(p < 0.0001). Mean neurite counts for 50 and 100 µM BN 52021 were significantly higher than
controls (p < 0.0001) at
t = 3, 6, and 12 hr.
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It is also possible that mc-PAF could have altered migration by
affecting the fasciculation of neurites. It has been shown in
cerebellar microexplants that the disruption of neurite fasciculation with anti-L1 or anti-NCAM antibodies increased the percentage of
neurons that migrated away from the explant core (Fischer et al.,
1986). In the present study, there was no significant difference in the
number of fasciculated TUJ-1-positive neurites of 1 µM mc-PAF or serum-free media treatment (67.5 ± 0.7% of the total number of neurites in mc-PAF clusters were fasciculated,
n = 12 clusters, all neurites counted,
p = NS; 68.9 ± 0.8% fasciculated neurites in
control clusters, n = 12 clusters, all neurites
counted). Additionally, no significant differences were found in the
mean number of neurites per neurite bundle after 12 hr (3.4 ± 0.7 for 1 µM mc-PAF treated, 3.5 ± 0.2 for serum-free
media controls, p = NS; one similarly positioned
neurite bundle analyzed per cluster, n = 48 per drug
condition). Therefore, no significant effects were observed on the
characteristics of the neurite bundles.
At high concentrations, mc-PAF had a small but significant effect on
the mean neurite length beyond the cluster margin (Table 1) compared
with the mean neurite length in serum-free media controls after 24 hr.
Blinded experiments yielded a mean neurite length beyond the cluster
margin in 1 µM mc-PAF-treated clusters that was also
significantly different from that of serum-free media controls (Table
1). However, 100, 250, and 500 nM mc-PAF had no significant
effect on the mean neurite length beyond the cluster margin (Table 1)
compared with serum-free media controls, although significant effects
on migration were noted at 250 and 500 nM mc-PAF.
Therefore, mc-PAF effects (at concentrations below 1 µM)
on the mean distance of granule cell migration cannot be attributed to
shorter neurites.
The decrease in neurite number and to a lesser extent the decrease in
mean neurite length (for 1 µM mc-PAF only) limited the amount of migration substrate for the granule cells and therefore could
account for some of the decrease in the number of cells that migrated
from mc-PAF-treated cell clusters after 24 hr (Figs. 3B, 4,
Table 1). However, such a mechanism is insufficient to account for the
difference calculated in the mean number of migrating neurons found per
neurite bundle after 24 hr (3.1 ± 0.7 for 1 µM
mc-PAF treated vs 10.6 ± 0.5 for serum-free media controls; p < 0.001; n = 48 per drug condition).
Stated differently, when presented with similar neurite migration
substrate, fewer granule cells migrated on this substrate in 1 µM mc-PAF conditions than in control conditions.
To determine whether the difference in mean migration distance
induced with 1 µM mc-PAF treatment could be entirely
accounted for by effects on neurite length, it was hypothesized that
the potential range of somal migration was limited by the length of the
preextended neurites elaborated from the cell cluster that the granule
cells migrated on. Therefore, a percentage of migration potential
fulfilled by each cluster after 24 hr in serum-free media and 1 µM mc-PAF treatment was calculated with the following equation: % of migration potential fulfilled at 24 hr = (mean cell migration distance at 24 hr)/(mean neurite length beyond the
cluster margin at 24 hr) × 100. If 1 µM mc-PAF did not
affect migration but only decreased the mean neurite length, then a
larger percentage of migration fulfillment would be expected. If 1 µM mc-PAF had affected primarily migration along the
neurite, then a smaller percentage of migration potential fulfillment
was expected. This calculation was independent of the number of
neurites elaborated from a cell cluster and the total number of cells
that migrated. It revealed very significant differences between the
mean percentage for 1 µM mc-PAF-treated clusters
(18.5 ± 0.1% fulfillment) and serum-free media-treated control
clusters (35.8 ± 0.2% fulfillment; p < 0.0001)
(Table 1). Thus, there was a twofold decrease in mean migration
distance (Table 1), whereas 1 µM mc-PAF reduced mean
neurite length beyond the cluster margin only 2.5%. Therefore, reduced
neurite length was not likely to be the main mechanism for 1 µM mc-PAF disruption of migration. Furthermore, it seemed unlikely that under 1 µM mc-PAF conditions a decrease in
mean neurite length of 8.9 µm could explain a decrease in mean
distance of migration of 59.2 µm.
Dynamic studies of mc-PAF effects on granule cell motility
To determine whether mc-PAF had more direct effects on the
somal movement of migration, two different sets of experiments were
performed. In the first set of experiments, cell clusters were treated
with 1 µM mc-PAF 6 hr after process extension and cell
migration had begun. Before mc-PAF addition, some neurons were observed
to have migrated 70-80 µm from the cell cluster edge. Eighteen hours
after mc-PAF addition (24 hr after serum-free media exchange), the mean
length of neurite extension beyond the cluster margin for
mc-PAF-treated clusters (330.3 ± 1.6 µm; p = NS) was not significantly different from the serum-free media control
(332.9 ± 1.5 µm) (Table 1). Under 1 µM mc-PAF
conditions, however, few neurons were found to have migrated beyond 80 µm from the cluster, and the mean distance of migration for these cells (29.8 ± 0.6 µm; p < 0.0001) was
significantly different from that of serum-free media controls
(119.3 ± 0.6 µm) (Table 1). The percentage of migration
potential fulfilled after 24 hr was also substantially different from
serum-free media controls (8.0 ± 0.1% for mc-PAF vs 35.8 ± 0.2% for serum-free media controls; p = 0.00001)
(Table 1). These values were also significantly different from those
determined for 24 hr of 1 µM mc-PAF exposure (Table 1).
These differences may have resulted from the obscuring of the cluster
margin in the delayed mc-PAF experiments by the relatively large number
of cells that began to migrate before mc-PAF addition, or they may have
resulted from a retraction of the neurite substrate (Clark et al.,
1995). These factors would result in an underestimate of migration
parameters. Although the results from the delayed addition of mc-PAF
suggested that mc-PAF blocked the further migration of neurons along
preextended neurites (Fig. 6), these
experiments were not conclusive.
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Figure 6.
mc-PAF disrupts previously initiated granule cell
migration. The images of cerebellar granule cells and their processes
elaborated from reaggregate clusters plated on laminin demonstrate the
effect of adding 1 µM mc-PAF to the clusters 6 hr after
plating them in serum-free media. A shows some processes
and migrating granule cells from a cell cluster (to illustrate the
distribution of migrated cells, the cluster itself is located just
below the image) after 24 hr of serum-free media
incubation at 37°C and 5% CO2. Cells have migrated as
far as 300 µm from the cluster source. B shows some
processes and migrating granule cells from a similarly positioned cell
cluster that was incubated in serum-free media at 37°C and 5%
CO2 for 6 hr and then with 1 µM mc-PAF in
serum-free media for an additional 18 hr. Cells elaborated neurites and
migrated 80 µm from the cell cluster before mc-PAF addition and then
no farther after mc-PAF addition. Scale bar, 50 µm.
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To better characterize the effects of mc-PAF on previously
migrating neurons, time-lapse experiments of migrating granule cells
were conducted. Cell clusters plated on laminin coated T dishes
(see Materials and Methods) in serum-free media were removed from the
incubator after 6 hr, as described above. Granule cell migration was
then observed in a controlled temperature environment for 1 hr in
serum-free media. After 1 hr, the serum-free media in the dish was
bath-exchanged with warmed serum-free media containing 1 µM mc-PAF (n = 9) or no additional drug
compound (n = 3). Within 2 min of 1 µM
mc-PAF addition, the migrating neurons stopped moving and remained
immobile for the entire hour of mc-PAF exposure (n = 9). mc-PAF did not cause any observable morphological change in the
neurite migration substrate (n = 9), although slight
rounding of the soma of the migrating granule cell was observed within 4 min of mc-PAF addition (Figs. 2, 7).
Cell migration usually resumed within 6 min of removal of the mc-PAF
and continued uninterrupted for >30 min (n = 9). To
avoid adding mc-PAF to migrating neurons coincidentally at a time when
they might naturally stop moving (Edmondson et al., 1987; Komuro et
al., 1996), the time of mc-PAF application was varied from 8 min, 1 hr,
and 1.5 hr after the initial observation of the migrating neuron, and
it was determined that the application of 1 µM mc-PAF
stopped the movement of the cell regardless of the time that it was
applied (n = 9; three migrating neurons per migration
time interval).
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Figure 7.
mc-PAF disrupts previously initiated granule cell
migration observed with time-lapse IR-DIC video microscopy.
1a-d, Video time-lapse images taken every 2 min of the
same granule cell (arrow) migrating on a bundle of
neurites at 36°C in vitro. Immediately after image
1d was taken, the bathing solution was exchanged with 1 µM mc-PAF in serum-free media (at 36°C), and image
2a was taken 10 sec later. Note the slight rounding of
the soma of the cell in 2a-d (images taken every 15 min
of the same granule cell in 1 µM mc-PAF conditions) as
compared with before mc-PAF exposure in 1a-d. The cell
appears to remain stationary for the entire hour. The mc-PAF was then
removed with three bath exchanges of serum-free media (at 36°C)
immediately after image 2d was taken, and image
3a was taken 8 min later. 3a-d images
(taken every 8 min) appear to show that the granule cell resumes
migration after mc-PAF removal. An asterisk in each
image denotes the same fixed location. Scale bar (shown in
3d): 5 µm.
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Inhibition of mc-PAF migration effects with BN 52021 and trans-BTD
The specific PAF receptor antagonists BN 52021 and
trans-BTD blocked the decrease in migration brought about by
1 µM mc-PAF (n = 48) (Figs.
3C, 8). The combination of 1 µM mc-PAF and either one of the antagonists resulted in
no significant differences in either the mean total number of cells
that migrated (Table 2) or the mean
distance of migration as compared with serum-free media controls (Table
2). Figure 3C illustrates a typical cell cluster after 24 hr
of incubation with 1 µM mc-PAF and 50 µM BN 52021. The extent of neuronal migration from this cluster closely resembled that seen in the serum-free media control rather than that
observed in the mc-PAF-treated cell clusters. BN 52021 (50 µM) inhibited the effects of 1 µM mc-PAF on
neurite number measured at 1, 3, 6, and 12 hr after drug incubation
(p = not significant from serum-free media
control) (Fig. 3). These inhibitory effects were not reproduced by
adding a combination of DMSO, the drug vehicle used for BN 52021 and
trans-BTD, and 1 µM mc-PAF to cell clusters
(Table 2) nor were they reproduced by the combined addition of DMSO and
methanol (n = 48; data not shown), which indicates that
DMSO was not responsible for blocking the mc-PAF effects. The
inhibition of mc-PAF effects on neuronal migration was not reproduced
with cis-BTD (Table 2), a stereoisomer of
trans-BTD that is not a PAF receptor antagonist. Finally,
blinded experiments with 1 µM mc-PAF and with 50 µM BN 52021 (n = 12) yielded a mean total
number of migrated cells, a mean migration distance, and a mean neurite
length that were all not significantly different from serum-free media
control values (Table 2).
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Figure 8.
PAF receptor antagonists BN 52021 and
trans-BTD, but not cis-BTD (a
stereoisomer of trans-BTD that is not a PAF receptor
antagonist), inhibit mc-PAF disruption of cerebellar granule cell
migration in vitro. The graph illustrates
the number of cerebellar granule cells that have migrated and the
distance of migration from their reaggregate cell cluster source 24 hr
after incubation with serum-free media (control), 1 µM
mc-PAF alone, 1 µM mc-PAF with 50 µM BN 52021, 50 µM trans-BTD, or 50 µM cis-BTD (all in serum-free media; n = 48) at 37°C and 5% CO2. Error
bars indicate SE and were removed from several points above for
clarity. SEs from left to right: serum-free media and 1 µM mc-PAF (Fig. 2); 1 µM mc-PAF and 50 µM BN 52021 3.1, 2.3, 2.5, 2.4, 1.8, 0.8, 0.9, 0.0, 0.0;
1 µM mc-PAF and 50 µM
trans-BTD 2.8, 1.8, 2.6, 1.6, 2.4, 1.0, 0.7, 0.0, 0.0; 1 µM mc-PAF and 50 µM cis-BTD
3.2, 2.8, 2.0, 0.0, 0.0, 0.0, 0.0, 0.0, 0.0.
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Possible enhancement of migration with BN 52021 and trans-BTD
The addition of 50 or 100 µM BN 52021 or 50 µM trans-BTD alone to the reaggregate cell
clusters in some experiments led to a significant increase in the mean
total number of granule cells that migrated (407.4 ± 2.3 cells in
100 µM BN 52021 vs 256.6 ± 1.9 cells in control)
(Fig. 9, Table
3) and their mean distance of migration
(159.6 ± 0.7 µm in 100 µM BN 52021 vs 119.3 µm ± 0.6 in control) (Table 3). Figure 3D
illustrates a typical cluster after 24 hr of 100 µM BN
52021 treatment. This image reveals an apparent increase in the number
of neurons that migrated from the cluster as compared with the
serum-free media control in Figure 3A. The mean neurite
lengths for 50 µM BN 52021, 100 µM BN
52021, or 50 µM trans-BTD-treated clusters
after 24 hr were not significantly different from those of serum-free
media controls (Table 3). It should be noted, however, that blinded
experiments with 100 µM BN 52021 (n = 12)
alone failed to yield any significant difference in the mean distance
of migration after 24 hr of incubation (Table 3) compared with
serum-free media controls, which suggests that BN 52021 may not
significantly enhance granule cell migration after all. If these data
are included in the above comparison, then a significant effect on the
mean distance of migration was still seen (157.6 ± 0.5 µm
compared with 119.3 ± 0.6 µm for serum-free media control;
p < 0.001). The possible migration-promoting effects of BN 52021 and trans-BTD were not sufficient in magnitude
to explain the apparent block of mc-PAF effects in previously described experiments. Finally, 50 µM cis-BTD and DMSO
(vehicle for all antagonists) did not appear to have any enhancing
affect on migration (Fig. 8, Table 3).
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Figure 9.
BN 52021 and trans-BTD alone but
not cis-BTD may enhance cerebellar granule cell
migration in vitro. The graph illustrates the number of cerebellar granule cells that have migrated and the
distance of migration from their reaggregate cell cluster source 24 hr
after incubation with 50 µM cis-BTD, 50 µM trans-BTD, 50 and 100 µM
BN 52021, and DMSO (at the final concentration for 50 µM
BN 52021 in serum-free media) all in serum-free media
(n = 48; 4 dishes, 12 clusters measured per dish)
at 37°C and 5% CO2. Error bars indicate SE and were
removed from several points above for clarity. SEs from left to
right: DMSO 1.8, 1.4, 2.0, 1.6, 2.0, 1.4, 2.1, 0.0, 0.0; 50 µM cis-BTD 1.3, 2.3, 1.7, 2.5, 1.3, 1.0, 0.8, 0.0, 0.0; 50 µM BN 52021 3.3, 1.3, 1.8, 2.10, 1.50, 1.6, 2.1, 2.9, 0.0; 50 µM trans-BTD 2.6, 2.3, 1.8, 1.9, 1.3, 2.8, 3.1, 1.2, 0.7; 100 µM BN 52021 2.1, 1.5, 2.7, 1.8, 3.1, 1.0, 1.5, 3.0, 0.3.
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Cultures treated with 50 and 100 µM BN 52021 also
elaborated more neurites per cluster after BN 52021 incubation (Fig. 5) than serum-free media controls at all times measured. This increase in
neurite number represented an increase in migration substrate that
could have accounted for some of the increase in the number of cells
that migrated under these conditions. Because clusters appeared larger
in PAF receptor antagonist conditions, an effect of these compounds on
granule cell proliferation could not be excluded. However, the effects
on migration observed by video microscopy occurred in a short time
frame (6-30 min) during which proliferation could not account for the
apparent changes in cell position.
| |
DISCUSSION |
In the present study, the effects of PAF receptor agonists and
antagonists on homotypic or neuron on neurite migration were examined.
Although in vivo neuronal migration occurs via neuronal attachment to radial glia in the cerebral cortex (radial migration) or
to Bergmann glia in the cerebellum (Fishell and Hatten, 1991; Liesi,
1992), in vivo tangential migration involving the attachment of neurons to other neuronal processes for contact guidance support has
also been proposed (O'Rourke et al., 1995, 1997). Additionally, cerebellar granule cells have been shown to migrate predominantly along
migration pathways of other neuronal fibers instead of Bergmann glia
during the first 8 d of postnatal rat cerebellum development (Hager et al., 1995). In vitro preparations from the
neonatal cerebellum including cerebellar reaggregate clusters (this
study) and cerebellar microexplants (Nagata et al., 1990) demonstrate this neurite-guided granule cell migration.
In the cerebellar reaggregate system, it was observed that migrating
granule cells extended leading processes out along preextended neurites. Soon after this leading process extension, the soma of the
granule cells could be seen to move in the direction of leading process
extension. This mode of migration appeared to be similar to what occurs
in glial-guided migration, in which a migrating cell extends a leading
process along a glial substrate before movement of the soma (Rakic,
1990). Therefore, the observed granule cell migration appears to be a
process of attachment to a migration substrate, extension of neural
processes in the direction of migration, and subsequent movement of the
soma in the direction of the leading process.
The observed inhibition of migration in this reaggregate system by the
PAF receptor agonist mc-PAF could be caused by an effect on the neurite
migration substrate, because this agonist has been shown to produce
significant effects on neurites (Clark et al., 1995). Alternatively,
because modifications of adhesive substrates [both on glia (Stitt et
al., 1990) and on neurites (Linder et al., 1983; Liesi et al., 1992,
1995)] have been shown to alter migration, PAF receptor activation
could have disturbed the ability of the granule cells to bind and
adhere to neuritic processes. Finally, the microtubules and other
cytoskeletal components that individual neurons use to migrate could
have been affected by mc-PAF, perhaps in a manner similar to the
effects of agonist on growth cones and neurites (Clark et al.,
1995).
In this study, neuronal PAF receptor activation by mc-PAF interfered
with the ability of the neurons to migrate on preextended neurites.
Soma of neurons that did attempt to migrate on the neurite substrate in
mc-PAF-treated conditions, or on preextended neurites that formed
before mc-PAF addition, did not migrate as far from the reaggregate
cluster as did controls. Although some effects of mc-PAF on neurite
substrate were noted, it seemed unlikely that these effects could
explain a larger, more significant effect on migration. These
observations are consistent with previous studies, because in those
studies neurites were acutely treated with mc-PAF to bring about a
rapid neurite withdrawal measured as a change in length (Clark et al.,
1995). In the present experiments, neurites were chronically exposed to
mc-PAF, and the total neurite length was measured. These conclusions
were further supported by video time-lapse experiments that
demonstrated that bath application of 1 µM mc-PAF stopped
the movement of neurons previously migrating along preextended
neurites, with no obvious effects on the neuritic migration substrate.
A rapid (~4 min) shape change was noted in previously migrating
granule cells (cell rounding) to accompany the arrest of migration. The
rapidity of these observed morphological effects suggested that a
downregulation of extracellular cell adhesion molecules or of the
recognition sites for these molecules was unlikely. Taken together,
these data suggest that PAF receptor activation interferes with the
cell motility, perhaps through similar cytoskeletal alterations that
lead to growth cone collapse and neurite withdrawal (Clark et al.,
1995).
The reported experiments revealed that the effects of mc-PAF were
blocked by the PAF receptor antagonists BN 52021 and
trans-BTD, indicating that the phenomenon observed is likely
a PAF receptor-mediated one. This conclusion is further supported by
the failure of lyso-PAF, a biologically inactive lipid that is
structurally similar to PAF and mc-PAF, to have any effects on neuronal
migration at the concentrations tested, and the failure of
cis-BTD to block the effects of mc-PAF on migration in the
cell reaggregate cluster system. Experiments to determine the
effectiveness of these compounds in blocking mc-PAF effects on
migration observed in video time-lapse experiments are ongoing.
In some experiments, the application of BN 52021 or
trans-BTD alone to the granule cells appeared to enhance
their migration by increasing the number of neuronal processes that
formed and by increasing the distance of neuronal migration from the
cell clusters. BN 52021 and trans-BTD could have blocked the
effects of PAF produced in vitro by the cerebellar granule
cells; in vitro PAF production by rat cerebellar granule
cells has been described (Yue et al., 1990). Another possibility could
have been that both of these antagonists enhanced neuronal survival or
neuroblast cell divisions (Gao et al., 1991). The former possibility
was deemed unlikely, because trypan blue stains failed to show any differences in the number of dead cells between BN 52021-treated and
serum-free media-treated clusters (data not shown). The later possibility could not be excluded; however, in time-lapse video microscopy experiments, cells were under observation for an
insufficient time for proliferation to occur. Finally, blinded studies
with 100 µM BN 52021 failed to reveal the same
enhancement in neuronal migration, but when the data from the blinded
studies were included with the data from the unblinded studies, a
significant effect on the mean distance of migration was still seen.
These results therefore must be interpreted cautiously but may suggest
that PAF and PAF receptors have de novo roles in the
regulation of migration.
The neuronal PAF receptor mediating the reported effects seems to have
led to neuronal cytoskeletal changes that altered cell motility.
Motility of the neuroblast is accomplished by changes in the internal
cytoskeleton (Gregory et al., 1988). The leading processes of migrating
neurons have a microtubular arrangement such that tubulin is added at
the end of the microtubular polymer directed toward the migratory
trajectory (Rakic et al., 1996). This arrangement makes these processes
subject to profound effects from an alteration of microtubules; it has
been hypothesized that a microtubule-based mechanism could be
responsible for the movement of the soma of migrating neurons (Rakic et
al., 1996). Neurite retraction evoked by PAF receptor activation (Clark
et al., 1995) and the presently observed cell rounding are likely to
involve microtubules.
Although these data support a hypothesis that PAF may normally
serve as a migration stop signal, other possibilities should be
considered. In the present study, a nonhydrolyzable PAF receptor agonist was applied in a homogenous manner to migrating neurons in vitro. It is unlikely that PAF would exist in such a
manner in vivo. Rather, it may be expressed in some gradient
similar to that of many other molecules important in neurodevelopment, such as the lipid retinoic acid (Thaller and Eichele, 1987; Chen et
al., 1994). By presenting a nonhydrolyzable PAF receptor agonist (mc-PAF) in a ubiquitous manner, PAF receptors could have been saturated, and this could have effectively removed an important extracellular or intracellular gradient for PAF in the reaggregate system.
Little is known about the in vivo distribution of PAF
and PAF receptors in the developing cerebellum or cerebral cortex to support or refute a role of PAF as a modulator of neuronal migration. Cells taken from immature rat cerebella (granule cells from 8-d-old rats) (Yue et al., 1990) or human fetal brains (microglia) (Jaranowska et al., 1995) have been shown to synthesize PAF in vitro.
In situ hybridization experiments in the brain for a PAF
receptor originally cloned from guinea pig lung (Honda et al., 1991)
showed a ubiquitous signal in the rat brain but particularly intense
signals in the cerebral cortex, olfactory bulb, pyramidal cell layer of
the hippocampus, medial thalamus, hypothalamus, and cerebellar granule
cell layer, with developmental expression constant from E18 (Mori et
al., 1996). If this PAF receptor were expressed by cerebellar granule cells, one might expect PAF receptor expression in the developing cerebellum to increase substantially, because granule cells undergo dramatic proliferation in the first 2 weeks after birth in the rat.
However, the predominant expression of this PAF receptor was found in
rat brain microglia. Microglia are not found in significant numbers in
our culture system, and recent experiments with Percoll gradient-purified granule cell reaggregate clusters (containing no
glia) appear to yield results that are comparable to the experiments described here (unpublished observations). Taken together, these observations suggest that the mechanism of mc-PAF action on cerebellar granule cells in this study is via a different, as yet uncharacterized, neuronal PAF receptor. One possible function of this neuronal PAF
receptor could be to transduce a PAF "migration stop signal" as
migrating granule cells (from the external granule cell layer) approach
the end point of migration in the IGL.
Although highly speculative, it is intriguing to consider that mc-PAF
could interact with PAF acetylhydrolase 1B (PAF-AH 1B), a novel
G-protein-like complex, to produce changes in the cytoskeleton as
theorized recently (Xiang et al., 1995; Ho et al., 1997). In other
words, this complex could theoretically serve as a recognition site for
PAF that functions as a modulator of the cytoskeleton. The subunits of
this complex, including Lis-encoded proteins, are expressed in a
pattern that supports a role in neuronal migration (Reiner et al.,
1995; Albrecht et al., 1996; Clark et al., 1997).
The data reported here demonstrate that PAF receptor activation
inhibits the migration of neurons on preextended neurites. These
observations support the hypothesis that PAF receptors, presumably
activated in the brain by the lipid messenger PAF, may play a role in
the regulation of neuronal migration in vivo. Furthermore,
these results suggest that overstimulation of PAF receptors via
abnormal levels of PAF in vivo could have dire consequences for the developing brain, consequences that may manifest as neuronal migration disorders such as Miller-Dieker lissencephaly, in which the
potential for defects in PAF catabolism exist.
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FOOTNOTES |
Received Aug. 6, 1997; revised Oct. 16, 1997; accepted Oct. 20, 1997.
This work was supported by National Institutes of Health (NIH) Grants
NS01433 and NS37146 and the Mental Retardation Research Center-NIH
Grant HD24064. We thank Dr. M. Elizabeth Ross for suggestions regarding
this manuscript, and Robert McNeil for assistance with immunofluorescence experiments.
Correspondence should be addressed to Dr. Gary D. Clark, Department of
Pediatrics, Texas Children's Hospital, 6621 Fannin, MC 3-3311, Houston, TX 77030.
| |
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Top
Abstract
Introduction
