Lysophosphatidic acid induces process retraction in CG-4 line oligodendrocytes and oligodendrocyte...

Lysophosphatidic acid induces process retraction in CG-4 line

oligodendrocytes and oligodendrocyte precursor cells but not in

differentiated oligodendrocytes

John Dawson,* Neil Hotchin,� Sian Lax� and Martin Rumsby*

*Department of Biology, University of York, York, UK

�School of Biosciences, University of Birmingham, Birmingham, UK

Abstract

Lysophosphatidic acid is a growth factor-like signalling

phospholipid. We demonstrate here that lysophosphatidic acid

induces process retraction in central glia-4 cells and oligo-

dendrocyte precursors. This lysophosphatidic acid effect is

rapid and concentration-dependent and results in cell round-

ing. It is inhibited by pre-treatment of cells with C3 exoen-

zyme, a specific inhibitor of Rho, or with Y-27632, a specific

inhibitor of ROCK, a downstream kinase of Rho. Processes of

differentiated central glia-4 oligodendrocytes were insensitive

to lysophosphatidic acid treatment but cell bodies became

phase dark, indicating cell spreading on the poly-L-lysine

substratum. RT-PCR and Western blot analyses indicate that

oligodendrocyte precursors and mature oligodendrocytes

express mRNA and protein for LPA1, one of several LPA

receptors. Thus lysophosphatidic acid may be signalling to

Rho and stimulating actomyosin contraction in precursor

oligodendrocytes by this family of receptors. The results show

that lysophosphatidic acid signalling pathways influence

retraction of processes in oligodendrocyte precursors but that

this effect changes as oligodendrocytes differentiate.

Keywords: CG-4 cells, LPA1 receptor, lysophosphatidic

acid, oligodendrocytes, process retraction, S1P.

J. Neurochem. (2003) 87, 947–957.

Oligodendrocyte (OLs) precursor cells arise in subventricular

zones within the embryonic CNS and then undergo prolif-

eration and extensive migration throughout the developing

CNS. These cells then differentiate and produce many more

processes becoming multipolar to myelinate axons (Bau-

mann and Pham-Dinh 2001; Levine et al. 2001; Miller

2002). Process formation therefore is of major importance in

OL development, initially for migration as the precursor and

later for myelination as the mature cell.

An imbalance of motor protein-mediated forces is pro-

posed to account for neurite extension (Hirose et al. 1998;

Ahmad et al. 2000; Baass and Ahmad 2001). This can be

demonstrated by inhibition of actomyosin contraction using

inhibitors of the small GTPase Rho, a known regulator of

myosin activation in addition to other signalling pathways

(Dickson 2001; Ridley 2001). Inhibition of Rho by C3

exoenzyme (Morii and Narumiya 1995) blocks neurite

retraction normally induced by lysophosphatidic acid (LPA)

(Jalink et al. 1994; Tigyi et al. 1996a, 1996b). Inhibition of

ROCK, a Rho-associated kinase, by Y-27632 (Uehata et al.

1997; Narumiya et al. 2000) also blocks LPA-induced

neurite retraction. Sphingosine-1-phosphate (S1P) also

induces process retraction of neurites, like LPA (Sato et al.

1997; van Brocklyn et al. 1999). Such results indicate that

neurite retraction is dependent on Rho and its effectors

(Hirose et al. 1998). In addition, studies with acidic calponin

(Ferhat et al. 2001), which inhibits actin–myosin interaction,

implicate myosin motor proteins in process elaboration.

Studies on central glia-4 (CG-4) line OLs (Louis et al. 1992),

a model precursor OL line, using the microfilament and

microtubule depolymerizing drugs cytochalasin D and

nocodazole, respectively, have demonstrated that motor

Received December 20, 2002; revised manuscript received July 29,

2003; accepted August 4, 2003.

Address correspondence and reprint requests to John Dawson. E-mail:

[email protected]

Abbreviations used: CG-4, central glia-4; DMEM, Dulbecco’s modi-

fied Eagle’s medium; DMSO, dimethyl sulphoxide; DTT, dithiothreitol;

FBS, fetal bovine serum; LPA, lysophosphatidic acid; MAP, mitogen-

activate protein; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-

zonium bromide; OLs, oligodendrocytes; PAGE, polyacrylamide gel

electrophoresis; PBS, phosphate-buffered saline; PLL, poly-L-lysine;

SDS, sodium dodecyl sulphate; S1P, sphingosine-1-phosphate; TBS,

Tris-buffered saline.

Journal of Neurochemistry, 2003, 87, 947–957 doi:10.1046/j.1471-4159.2003.02056.x

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 87, 947–957 947

protein-mediated forces are important for OL process

extension/retraction. Treatment with nocodazole leads to

process retraction but this effect is blocked if microfilaments

are initially depolymerized (Rumsby et al. 2003). This

suggests that there are forces acting on the microtubule and

microfilament networks and that process extension/retraction

in OLs may occur by a similar mechanism to neurites.

LPA and S1P are phospholipids with growth factor-like

signalling properties that can activate a variety of cellular

responses including proliferation, increased Ca2+ levels,

mitogen-activate protein (MAP) kinase activation and actin

cytoskeleton rearrangement (Contos et al. 2000b; Panetti

et al. 2001). LPA- and S1P-signalling pathways are mediated

through G protein-linked receptors (reviewed in Chun et al.

2002). LPA and S1P can also act as intracellular second

messengers (Yatomi et al. 2001; McIntyre et al. 2003). LPA

is implicated in promoting survival and adhesion of Schwann

cells (Weiner and Chun 1999; Weiner et al. 2001). For

example, deletion of LPA1 in mice results in increased

Schwann cell apoptosis, but abnormal movement is not

observed (Contos et al. 2000a), suggesting that LPA1 does

not play a major role in peripheral nervous system (PNS)

myelination, or that other LPA receptors or signalling

molecules, such as S1P, can compensate for the lack of

LPA signalling. However, such results suggest that LPA1 is

required for Schwann cell development. LPA1 is also

expressed by mature OLs during myelination in the CNS

(Allard et al. 1998; Weiner et al. 1998; Beer et al. 2000;

Handford et al. 2001). The jimpy mouse, which has a

mutation in the PLP gene and increased OL apoptosis, shows

a 40% reduction in LPA1 expression (Weiner et al. 1998).

Furthermore, the LPA1 receptor gene in the extinct mouse

mutation vacillens, which suffered from problems with motor

control and shaking, has been mapped to the region of the

mutation (Contos et al. 2000b) further implicating LPA1 in

OL development (Chun et al. 2000; Contos et al. 2000b).

Here we have examined the effect of LPA on process

dynamics in OL precursors and OLs using primary cells and

the CG-4 OL line. We show that processes of OL precursors

and OLs differ in their response to LPA indicating that there

are differences in the signalling pathways regulating process

dynamics in OL precursors and OLs.

Materials and methods

Materials

LPA (1-Oleoyl-sn-glycerol 3-phosphate) and S1P (sphingosine

1-phosphate) were from Sigma (Dorset, UK). LPA was dissolved

in sterile water by sonication to give a 10 mM stock. S1P was

dissolved in methanol similarly. C3 exoenzyme was prepared as

described (Morii and Narumiya 1995). Y-27632 was a gift from

Welfide Corporation (Osaka, Japan). Anti-LPA1 (Edg-2) antibody

was purchased from Calbiochem (Merck Biosciences Ltd, Beeston,

UK). All other reagents were from Sigma, unless otherwise stated.

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum

(FBS), trypsin/EDTA were from Gibco–BRL (Paisley, UK). Tissue

culture plastic was from Nunc (Life Technologies, Paisley, UK).

Cell culture

CG-4 cells, passages 40–50, were cultured in 70% DMEM with N1

supplement and 1.25 mg/mL bovine serum albumin/30% B104-

conditioned medium (Louis et al. 1992). These cells were differ-

entiated to multipolar OLs by culturing for at least 2 days in

DMEM/0.5% FBS with N1 supplement before use. Primary OL

precursor cells were prepared by either the ‘shake off’ method of

McCarthy and de Vellis (1980) or by Percoll density gradient

(Lubetzki et al. 1991) from 1-day dissociated rat pup cerebra. For

the ‘shake off’ method, mixed glial cultures, grown for 7–10 days,

were pre-shaken for 30 min to remove contaminating cells and were

then shaken overnight to release OL precursors. Isolated OL

precursors were purified by two rounds of differential adherence,

which removed microglia, neurones and most astrocytes. Cells

remaining in suspension were pelleted by centrifugation, resus-

pended (70% DMEM with N1 supplement and 1.25 mg/mL bovine

serum albumin/30% B104-conditioned medium) and cultured in

tissue culture flasks and/or 24-well plates coated with 10 lg/mL

poly-L-lysine (PLL; MW 70–150 000) overnight. OL precursor cells

were differentiated to mature OLs as for CG-4 cells.

LPA and S1P treatment

For LPA and S1P experiments cells were grown in serum-free

medium. CG-4 cells and OL precursor cells were seeded at a density

of 2 · 104 cells per well in 10 lg/mL PLL-coated 24-well plates.

LPA/S1P were diluted from 10 mM stock solutions in culture media

and vortexed. Cells were either cultured overnight and then treated

with LPA/S1P for 20 min or LPA was added immediately after

passage and cells cultured for 3 h depending on the experiment.

Two different OL precursor preparations were used. For inhibitor

studies, cells were pre-incubated with C3 exoenzyme for 18 h, or

with Y-27632 for 30 min prior to LPA treatment. At least four

replicates of each experiment were made and individual experiments

were repeated at least three times.

Data analysis

LPA effects were recorded on a TE200 Nikon inverted microscope

with heated stage at 37�C using a DXM 1200 Nikon digital camera.

Three random fields of view per well were photographed. Total

number of cells, process length and number of processes per cell

were measured using a Lucia G DXM analysis system (Nikon,

Kingston upon Thames, UK). Process length was defined as the

distance from the end foot to the start of the phase bright cell body.

Process length per cell was determined for each field of view and

then the average of all fields of view per treatment group calculated.

This was termed the mean process length per cell. Standard error,

ANOVA and Chi-squared tests were calculated in Microsoft Excel.

RT-PCR

Total RNA was extracted using TRIzol reagent (Gibco–BRL) and

was treated with DNA-free (Ambion, Austin, TX, USA) to degrade

genomic DNA. Total RNA concentration was determined by

absorbance at 260 nm. 4 lg of total RNA was reverse transcribed

948 J. Dawson et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 87, 947–957

with Superscript II reverse transcriptase using random primers

according to the suppliers, (Promega, Southampton, UK) protocol

and cDNA was stored at )20�C until required. PCRs were

performed in a final reaction volume of 50 lL, containing 2 lL of

cDNA, 1 · PCR buffer, 0.2 mM of each dNTP, 1.5 mM MgCl2,

0.025 units Taq polymerase, and 1 pmol of each primer. PCR

reagents were from Promega. All PCR reactions were heated to

94�C for 4 min then cycled 35 times at 95�C 30 s, 56�C 30 s and

72�C 2 min followed by a final 72�C 10 min step. Primers used to

detect LPA1 transcripts were: forward 5¢-TCTTCTGGGCCATTTT-CAAC-3¢; reverse 5¢-TCGCTRAAGGTGGCGCTCAT-3¢ (Moller

et al. 2001) to give an amplified fragment of 349 bp. Glyceralde-

hyde-3-phosphate dehydrogenase primers were: forward 5¢-CTCCTGGAAGATGGTGATGG-3¢; reverse 5¢-AGACAGCCGCATCTTCTTGTGC-3¢ to give an amplified fragment of 237 base pairs.

Primer specificity was checked by BLAST searching primer

sequences and sequencing PCR products.

Western blotting

Cells were grown to 70–80% confluency, rinsed in Tris-buffered

saline (TBS), drained on ice and scraped out in homogenization

buffer [50 mM Tris–HCL pH 8.8, 0.5 M dithiothreitol (DTT), 2 mM

EGTA, 10 lg/mL aprotinin, 10 lg/mL leupeptin and 2 mM

AEBSF]. Ten percent sodium dodecyl sulphate (SDS) was added

and solubilized protein extracts were mixed by passage through

21/23 gauge needles. Aliquots were analysed for protein by the BCA

assay (Pierce, Chester, UK). Sample buffer was then added and

extracts were boiled for 10 min at 100�C. Proteins were resolved by

SDS–PAGE (polyacrylamide gel electrophoresis) on 12.5% gels and

transferred to Hybond ECL nitrocellulose membrane (Amersham

Pharmacia Biotech, Amersham, UK). Blots were blocked in 5%

dried non-fat milk powder (Marvel, Kwik-Save, York, UK) in TBS/

0.2% Tween before probing overnight at 4�C with antibody to LPA1

(1 : 1000 in 1% Marvel TBS/0.2% Tween). Membranes were rinsed

well and reprobed with anti-rabbit IgG conjugated with horseradish

peroxidase followed by rinsing and ECL detection.

Immunofluorescence

Acid etched 13 mm diameter coverslips were coated in 10 lg/mL

PLL over night. Primary OL precursors were seeded at 1 · 104 cells

per coverslip and allowed to adhere for 3 h. Coverslips were then

rinsed in warm PBS and incubated with an anti-A2B5 antibody

(hybridoma cell line supernatant) or anti-NG2 antibody (gift of

J. Levine, SUNY, Stony Brook, USA) diluted 1 : 1000 in phosphate-

buffered saline (PBS) for 30 min at 37�C. Cells were then fixed in

3% paraformaldehyde, blocked for 30 min in 3% BSA/2% goat

serum, and incubated with antirabbit or antimouse cy3 conjugated

antibody (Stratech Scientific Ltd, Cambridge, UK), diluted 1 : 500

in blocking solution. Coverslips were mounted on glass slides in

DAKO fluorescent mounting medium (Dako Corporation, Ely, UK)

and viewed using a Nikon Optiphot II microscope.

Cell viability

CG-4 cells were seeded into 10 lg/mL PLL-coated wells in 48-well

plates at 1 · 104 cells per well and allowed to settle for 20 min.

Cells were then treated with LPA and basic fibroblast growth factor

for 24 h or 48 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazo-

nium bromide (MTT) dissolved in distilled water at 5 mg/mL, was

diluted 1 : 10 in culture media 24 or 48 h after cell seeding. Cells

were incubated with MTT for 90 min at 37�C. Reduction of MTT

by mitochondrial dehydrogenase of viable cells forms a blue

formazan precipitate, which is proportional to the number of viable

cells. The precipitate was solubilized in dimethyl sulphoxide

(DMSO) and the colour intensity measured using a 490-nm filter.

Cell viability was also assessed with the nucleic acid stain SYTOX

green (Molecular Probes, Leiden, Netherlands). CG-4 cells grown in

10 lg/mL PLL-coated 24-well plates were treated with LPA. Cells

were stained for 5 min with 100 nM SYTOX green diluted in PBS.

Excess SYTOX green was then rinsed off and cells examined using

a TE200 Nikon inverted fluorescence microscope.

Results

Oligodendrocyte characterization

The characteristics of the CG-4 cells used in this work are as

reported previously (Rumsby et al. 1999). Freshly isolated

primary OL precursors were 84.5 ± 18.5% A2B5-positive

and 77.2 ± 18.1% NG2-positive. After two rounds of

differential adherence to remove contaminating cells OL

precursor cell cultures were greater than 95% pure. These

cells were NG2+ and were bipolar in appearance (see Fig. 1).

LPA causes a rapid, concentration-dependent retraction

of processes of CG-4 line OLs and OL precursor cells

Treatment of CG-4 cells and OL precursors with 1 lM LPA

caused rapid process retraction within 5 min: by 20 min the

majority of cells had rounded up and were phase-bright

+ LPA

CG-4

OPC

Fig. 1 Effect of LPA on undifferentiated OLs. CG-4 cells or OL pre-

cursors (OPC) plated on 10 lg/mL PLL for 24 h retracted their pro-

cesses and rounded up in response to a 20-min treatment with 1 lM

LPA. Scale bar is 30 lM.

LPA-induced oligodendrocyte process retraction 949


(Fig. 1). Time-lapse recordings revealed that after retraction,

random short protrusions about 2 lm in length, formed

transiently and repeatedly from the cell body (results not

shown but see Fig. 8, arrows). The LPA effect was quantified

by treating bipolar CG-4 cells and OL precursors with

increasing concentrations of LPA up to 1 lM (Figs 2a–c). LPA

treatment resulted in a concentration-dependent decrease in

the number of processes per cell. For CG-4 cells (Fig. 2a),

82.2% of cells had no processes after treatment with 1 lMLPA

compared with 11.4% in no LPA controls (zero LPA).

Similar results were obtained with OL precursors (Fig. 2b).

Furthermore, LPA reduced the mean process length per cell

(Fig. 2c): untreated CG-4 cells had processes of 35.2 ±

1.2 lm, which was reduced to 2.7 ± 0.4 lm by 1 lM LPA.

LPA is not toxic to oligodendrocytes

CG-4 cells treated with increasing concentrations of LPA up to

1 lM for 24 or 48 h showed no signs of toxicity comparedwith

untreated cells (Fig. 3). Only at a higher concentration of LPA

(10 lM), was a slight reduction in cell numbers observed in the

MTTassay but this was not significant. Treatment of cells with

10 ng/mL basic fibroblast growth factor significantly

Fig. 2 Quantification of LPA-induced pro-

cess retraction in CG-4 cells and OL pre-

cursors. Treatment of CG-4 cells (a) and OL

precursor cells (OPC; b) with increasing

concentrations of LPA up to 1 lM for

20 min, decreased the number of proces-

ses per cell. (c) Increasing concentrations

of LPA also reduced the mean process

length per cell (see Methods) of CG-4 cells

and OL precursor cells. For (a) and (b)

percentage of cells is shown and statistical

significance was calculated compared to

untreated cells using Chi-squared

(***p < 0.001). (c) Mean process length per

cell ± SEM shown (n ¼ 12). Statistical sig-

nificance was calculated compared to

untreated cells using ANOVA followed by a

Tukey’s test (*p < 0.05). All graphs

represent one experimental data set.

Repeat experiments gave similar results.



increased the MTT response compared to untreated controls,

indicative of increased cell numbers, as would be expected

(Engel andWolswijk 1996). In addition, to demonstrate if LPA

had any short-term membranolytic effects, CG-4 cells were

treated with 1 lM LPA for up to 4 h and then incubated with

the nucleic acid stain SYTOX green. Cells with damaged

membranes will take up this stain. LPA treatment did not

increase the number of cells taking up SYTOX green

compared with untreated controls (data not shown).

C3 exoenzyme or Y-27632 block LPA-induced process

retraction

Process retraction induced by LPA was blocked by pre-

incubation of CG-4 cells with either 10 lg/mL C3 exoenzyme

or 10 lMY-27632. In addition, we noted that cells treated with

C3 exoenzyme alone had an increased number of minor

branches along processes (data not shown). Preincubation of

CG-4 cells with C3 exoenzyme inhibited the effect of 1 lM

LPA in a concentration-dependent manner, as shown in

Fig. 4(a). Furthermore, C3 exoenzyme treatment inhibited the

LPA-induced reduction in the number of processes per cell

(data not shown). Pre-incubation of CG-4 cells with increas-

ing concentrations of Y-27632 up to 10 lM also inhibited

LPA-induced process retraction (Fig. 4b). As with C3 exoen-

zyme, Y-27632 inhibited the reduction of the number of

processes per cell induced by LPA treatment (data not shown).

OLs metabolize extracellular LPA

Cell culture medium sampled 18 h after LPA treatment did

not induce process retraction when added to fresh CG-4 cells,

suggesting that the LPA had been metabolised or inactivated.

LPA does not cause process retraction

in differentiated CG-4 OLs

Treatment of 3-day differentiated CG-4 OLs with 1 lM LPA

did not induce process retraction (Fig. 5), even after 60 min

(data not shown). However, on LPA treatment, the cell body

of differentiated CG-4 cells rapidly (within 5 min) became

phase-dark, indicating flattening and spreading onto the poly-

L-lysine substratum (Fig. 5). In untreated control CG-4 cells,

9% of cells were phase-dark, while in 1 lM LPA-treated cells

this figure increased to 94%. This spreading effect was

Fig. 3 Effect of LPA on CG-4 cell viability. Cell viability was assessed

using the MTT assay on CG-4 cells grown in the presence of

increasing concentrations of LPA up to 10 lM for 24 or 48 h. Basic

fibroblast growth factor was used as a positive control. Mean optical

density (OD) ± SEM shown (n ¼ 6). Statistical significance was cal-

culated compared to untreated cells using ANOVA followed by a Tukey’s

test (*p < 0.05). One experiment shown. Repeat experiments gave

similar results.

Fig. 4 Effect of C3 exoenzyme on LPA-induced process retraction in

CG-4 cells. (a) Treatment of CG-4 cells with increasing concentrations

of the Rho inhibitor, C3 exoenzyme, up to 10 lg/mL for 18 h before

addition of 1 lM LPA, inhibited the LPA-induced decrease in mean

process length per cell. (b) Treatment of CG-4 cells with increasing

concentrations of the ROCK inhibitor, Y-27632, up to 10 lM for 30 min

before addition of 1 lM LPA, inhibited the LPA-induced decrease in the

mean process length. Mean process length per cell ± SEM shown

(n ¼ 6). Statistical significance was calculated compared to untreated

cells using ANOVA followed by a Tukey’s test (*p < 0.05). One result for

each inhibitor is shown. Repeat experiments gave similar results.

+ LPA

Fig. 5 Effect of LPA on differentiated CG-4 cells. CG-4 cells plated on

10 lg/mL PLL and differentiated for 3 days by withdrawal of growth

factors (see Materials and methods) treated with 1 lM LPA for 20 min

did not retract processes. The only observable effect on morphology,

viewed under phase optics, was a rapid darkening of the cell bodies

suggesting the cells were spreading on the PLL substratum. Scale bar

is 30 lm.



observed at even the lowest LPA concentration (0.0625 lM),

where 76% of cells were phase dark.

LPA blocks process formation by freshly passaged

CG-4 cells, but not differentiated CG-4 OLs

One micromolar LPA added to freshly passaged CG-4 cells

blocked process formation so that after 3 h cells were still

rounded up compared with untreated controls (Fig. 6). By

18 h after passage, CG-4 cells treated with LPA had formed

processes (Fig. 6) and appeared identical to untreated

controls. In contrast, 1 lM LPA did not block process

extension by freshly passaged differentiated CG-4 cells

(Fig. 6), but did cause phase-darkening of the cell body as

described above (Fig. 5). Three hours after passage and LPA

treatment, 100% of differentiated CG-4 cells were phase-dark,

while approximately 30% were phase-dark in untreated

controls. After 18 h, LPA-treated differentiated CG-4 cells

had regained a phase-bright cell body, presumably because

LPA had been metabolized.

S1P also causes process retraction in CG-4 cells

Treatment of CG-4 cells with 1 lM S1P resulted in rapid

process retraction (Fig. 7). As with LPA, S1P-induced

retraction also caused membrane ruffling at the cell body

(Fig. 8, arrows). Unlike LPA, however, S1P also caused

occasional membrane ruffling at the tips of longer processes

as they were retracting (Fig. 8, arrowheads), after shorter

processes had retracted.

Expression of LPA1 in cultured OLs

With primers specific for LPA1 (Moller et al. 2001) a single

amplified fragment of the expected size (349 bp) was

detected indicating the presence of the LPA1 transcript in

CG-4 cells, differentiated CG-4 OLs, OL precursors and

3-day differentiated OLs (Fig. 9). Western blotting with a

commercial anti-LPA1 antibody revealed that CG-4 cells and

OLs express LPA1 protein as a band of approximately

62 kDa as also observed in post-natal day 10 rat brain where

a faster migrating band of approximately 44 kDa was also

observed (Fig. 10). Further investigation of LPA1 expression

in post-myelination CNS tissue (post-natal day 30 and

7 month) demonstrated that the expression of both the

44 kDa and the 62 kDa bands are also detected later in

development (results not shown).

Fig. 6 Effect of LPA on process extension by freshly passaged CG-4

cells. CG-4 cells had not extended processes 3 h after passage in the

presence of 1 lM LPA, whereas untreated CG-4 cells had extended

processes. Differentiated CG-4 OLs extended processes after 3 h

even in the presence of 1 lM LPA. LPA-treated differentiated CG-4

cells after 3 h appeared phase-dark compared to control cells, sug-

gesting they were flattening onto the PLL substratum. By 18 h after

passage and LPA addition, all treated CG-4 cells were similar to

controls. Scale bar is 30 lm.

+ S1P

Fig. 7 Effect of S1P on CG-4 cells. Treatment of CG-4 cells with 1 lM

S1P for 20 min resulted in a rapid retraction of processes, similar to

LPA (see Fig. 1). Scale bar is 30 lM.



Discussion

Formation and extension of processes is a fundamental

characteristic of OLs in the CNS. Initially, OL precursors

require a few dynamic processes for migration throughout

the developing CNS. After differentiation, mature OLs

extend many more stable processes, which myelinate axons

(Baumann and Pham-Dinh 2001; Miller 2002). Understand-

ing how an OL regulates such changes in cellular morphol-

ogy and defining the signalling molecules involved therefore

is of interest and may have relevance for understanding why

remyelination fails in multiple sclerosis (Franklin 2002).

Many soluble factors and extracellular matrix ligands that

influence OL survival and development have been identified

(reviewed in Baumann and Pham-Dinh 2001). Few if any

produce the dramatic effects on OL precursor morphology

that we report here with the growth factor-like signalling

phospholipid LPA.

LPA, synthesized from membrane phospholipids by the

action of phopholipases (Pages et al. 2001), stimulates a

variety of cellular responses (Contos et al. 2000b; Panetti

et al. 2001). These include rearrangement of the actin

cytoskeleton, which is mediated through the small G protein

Rho. The Rho signalling pathway has been implicated in

regulating LPA-induced retraction of neurites and Schwann

cell processes (Fukushima et al. 1998; Hirose et al. 1998;

Weiner and Chun 1999; Weiner et al. 2001; Tigyi et al.

1996a, 1996b) acting through LPA receptors (Fukushima

et al. 1998; Ishii et al. 2000). Das and Hajra (1989) reported

Fig. 10 Expression of LPA1 protein in OLs. Protein extracts of CG-4

cells (1), differentiated CG-4 OLs (2), post-natal day 10 rat brain (3),

OL precursors (4) and 3-day differentiated OLs (5) were resolved by

SDS–PAGE for Western blotting. Blots were probed with a commercial

antibody for LPA1. Protein was detected as a band at approximately

62 kDa in all lanes. In addition, a 42-kDa band was detected in post-

natal day 10 rat brain. Markers in kDa.

19:30.41

19:35.41

19:40.42

19:45.41

19:50.41

19:55.43

20:00.41

Fig. 8 S1P induces ruffling of the cell body and at the tips of long

retracting processes. CG-4 cells were grown on 10 lg/mL PLL.

Treatment with 1 lM S1P for 20 min induced ruffling at the cell body

(arrows) of rounded up cells and at the tips of long retracting pro-

cesses (arrowheads). Scale bar is 10 lM. Pictures taken at five-s

intervals.

1 2 3 4 5

LPA1

GAPDH

Fig. 9 mRNA expression of LPA1. Expression of LPA1 mRNA was

detected by RT–PCR (see methods). LPA1 mRNA is expressed by

CG-4 cells (1), differentiated CG-4 OLs (2), OL precursors (3), 3-day

differentiated OLs (4) and P10 rat brain (5). Results for GAPDH show

that similar amounts of mRNA were used in each PCR reaction. None

of the primer pairs produced bands in negative control reactions where

total RNA was used as the template.



that the highest tissue concentration of LPA

(86.2 ± 4.2 nmol/g tissue) is found in the brain. Post-mitotic

neurones and Schwann cells in culture secrete low nM

concentrations of LPA into the medium (Fukushima et al.

2000; Weiner et al. 2001). One receptor for LPA, LPA1, is

expressed by mature OLs in post-natal rat and mouse brain

(Allard et al. 1998; Weiner et al. 1998; Stankoff et al. 2002);

peak expression of LPA1 coincides with peak expression of

the proteolipid protein gene (Weiner et al. 1998), i.e. with

myelination. Allard et al. (1999) have shown that LPA1

exists as two splice variants, short and long forms, and that

expression of the long variant sharply increases post-natally,

coinciding with the peak of myelination. Immunohistological

studies have revealed that LPA1 expression is confined to

myelinated white matter and that LPA1 is associated

specifically with the ‘myelin-containing region of the OL

rather than the cell body region’ (Beer et al. 2000; Handford

et al. 2001). In contrast, Cervera et al. (2002) have recently

shown that LPA1 is localized to a greater extent in the OL

cell body rather than in myelinated fibres. Localization of

LPA1 in mature OLs needs clarification but such results do

indicate that LPA1 expression may be important for myeli-

nation. Currently, little is known about LPA receptor

expression by OL precursors though freshly isolated oligo-

dendroglial cells from post-natal day 4 and older optic nerves

show evidence of LPA1 expression (Stankoff et al. 2002).

Our results using OL precursors show that such cells express

LPA1 when maintained in culture. In addition, we show that

LPA regulates the process dynamics of OL precursors and

that the action of LPA changes as OLs differentiate. It should

be noted that, throughout this study, experiments have been

undertaken in the absence of serum which is well known to

contain LPA and S1P. Whether B104-conditioned medium,

used to culture CG-4 and OL precursor cells, contains LPA

and S1P is not known but we think this unlikely as CG-4

cells and OL precursor cells rapidly extend processes in

B104-conditioned medium but do not when LPA is added.

Treatment of OL precursors with LPA causes rapid process

retraction in a concentration-dependent manner resulting in

phase-bright rounded cells. This is comparable with obser-

vations on neurite retraction in 3-day differentiated PC12

cells (Tigyi et al. 1996a, 1996b) and Schwann cells (Weiner

et al. 2001) except that in these studies LPA treatment caused

cells to become phase-dark indicating flattening onto the

substratum. Treatment of differentiated OLs with LPA had no

observable effect on processes, in keeping with Stankoff

et al. (2002). However, a rapid darkening of the cell body

under phase optics was observed, suggesting the cells were

spreading onto the PLL substratum, perhaps by a GTP-Rac-

dependent mechanism (van Leeuwen et al. 2002). This

spreading effect is also observed in 8-day cultured PC12 cells

treated with LPA (Tigyi et al. 1996a). By 18 h after LPA

addition, differentiated OLs have adopted a morphology

similar to controls as the LPA has been metabolized. Our

observed difference with respect to the effect of LPA on OL

precursors and mature OLs may arise because (i) mature OLs

may be more firmly attached to the substratum limiting

retraction, (ii) mature OLs have more stable microtubules

(Lunn et al. 1997; Song et al. 1999) perhaps giving more

stable processes, (iii) changes in LPA receptor expression

may have occurred and/or (iv) LPA signalling pathways may

have altered during differentiation. The fact that semaphorin-

3 A-conditioned medium reduces process extension in

mature OLs (Ricard et al. 2001) suggests that the latter

explanation is most likely. Moller et al. (1999) demonstrated

that GalC+ mature OLs but not O4+/GalC– OL precursors,

exhibited a LPA-induced increase in intracellular [Ca2+]

further indicating that LPA effects change as OLs differen-

tiate. Such [Ca2+] effects were not observed when mature

OLs isolated from 4-week rat brain were treated with LPA

(Stankoff et al. 2002). These authors also reported that LPA

had no effect on the survival, maturation, myelination or

cytoskeletal organization of primary mature OLs. However,

before such conclusions on the effect of LPA on mature OLs

can be made, it is essential to be certain that endogenous LPA

is not being secreted into the medium by the cells present.

LPA is secreted by both post-mitotic neurones and Schwann

cells (Fukushima et al. 2000; Weiner et al. 2001) and has not

been examined for OLs.

The actin cytoskeleton is regulated by the small G proteins

Rho, Rac and Cdc42 (Ridley 2001). We have confirmed that

Rho signalling is involved in LPA-induced process retraction

in precursor OLs by using C3 exoenzyme, an inhibitor of

Rho (Morii and Narumiya 1995) and Y-27632, an inhibitor

of ROCK (Uehata et al. 1997; Narumiya et al. 2000). C3

exoenzyme and Y-27632 concentration-dependently inhibit

LPA-induced precursor OL process retraction, consistent

with results obtained with neuronal cells and Schwann cells

(Jalink et al. 1994; Tigyi et al. 1996a, 1996b; Hirose et al.

1998; Weiner et al. 2001). Our observation that treating cells

with C3 exoenzyme leads to an increase of branching of

processes on precursor cells suggests that a basal activity of

Rho is required to maintain the OL precursor morphology.

This is in keeping with observations that expression of

dominant negative Rho in primary OLs leads to hyper-

extension of processes, whereas expression of constitutively

active Rho reduced process formation (Wolf et al. 2001).

Our results show that mRNA and protein for LPA1 is

expressed by CG-4 cells and OL precursors in culture. LPA1

protein was detected in post-natal day 10 rat brain at

approximately 44 kDa in good agreement with the supplier’s

data and a report for human white matter (Cervera et al. 2002)

and mouse neuroblast cells (Ishii et al. 2000). In our cultured

OL samples, LPA1 was also detected routinely at approxi-

mately 62 kDa, with a similar band in post-natal day 10 rat

brain. Both 44 kDa and 62 kDa bands were also detected in

post-natal day 30 and mature rat brain samples (7 month).

Comparable results were obtained with another anti-LPA1



antibody (gift from Dr P. Maycox, GlaxoSmithKline, Harlow,

UK, results not shown). Stankoff et al. (2002) report a major

band of approximately 55 kDa for LPA1 in RH7777 cells

transfected with cDNA for LPA1 similar to that reported by

Cervera et al. (2002) in the same cells. LPA1 is known to

occur as short and long splice variants (Allard et al. 1999) but

this is unlikely to account for such differences as the two

splice variants only differ by 18 amino acids. The identity of

the higher band is presently unknown.

LPA signalling through LPA receptors to Rho in neuronal

cells occurs through a Ga12/13 pathway, which may be the

same in OLs (Kranenburg et al. 1999). GTP-Rho can

activate ROCK among other targets (Bishop and Hall

2000), leading to an overall effect increasing myosin light

chain phosphorylation and activation of myosin. This in turn

will increase actomyosin contraction and process retraction

as shown in Fig. 11 (Kimura et al. 1996; Amano et al.

1998). Therefore, LPA-induced process retraction in OLs

appears similar to that in neurites (Jalink et al. 1994; Tigyi

et al. 1996a, 1996b), so process dynamics in OLs may be

regulated in a similar way to neurites as proposed by Baass

and Ahmad (2001). This could explain why LPA does not

cause process retraction in mature OLs. Our observation that

freshly passaged OL precursors do not extend processes in

the presence of LPA is explained by the fact that a net

retractile force overrides any attempt by the cell to extend

processes. LPA-treated cells extend processes after 18 h

because the ligand has been metabolized, perhaps by a lipid

phosphate–phosphatase (Pilquil et al. 2001). After LPA-

induced process retraction and cell rounding, we noted that

random short protrusions were being extended regularly from

the cell body. This suggests that cells were still attempting to

extend processes, but could not do so fully due to stronger

net retractile forces.

S1P, a structurally related phospholipid to LPA, evokes

similar cellular responses to LPA (Takuwa et al. 2001),

which are mediated through S1P receptors (Hla 2001) one of

which, S1P5, is expressed by OLs (Im et al. 2000; Terai

et al. 2003). We find that S1P induces rapid process

retraction in OL precursors comparable to LPA, consistent

with S1P-induced retraction of neurites (Sato et al. 1997; van

Brocklyn et al. 1999). In contrast to LPA, however, S1P

induces membrane ruffling at the tips of long retracting

processes of OL precursors. This suggests that LPA and S1P

signalling pathways may differ.

It is unlikely that phosphorylation of the myosin light

chain is the sole target of LPA signalling leading to process

retraction. In neurites, for example, phosphatidylinositol

4-phosphate 5-kinase (van Horck et al. 2002; Yamazaki

et al. 2002), glycogen synthase kinase-3 (Sayas et al. 1999)

and LIM-kinase (Maekawa et al. 1999) have all been

implicated in LPA-induced process retraction. Further work

to investigate the involvement of these signalling interme-

diates in LPA-induced process retraction in OL precursors is

in progress. The expression pattern of the LPA1 receptor

suggests that LPA may play an important role in myelination.

However, a role for LPA in OL precursor motility is currently

unclear. LPA is clearly able to modulate OL precursor

migration because it influences process retraction, essential

for cell motility (Horwitz and Parsons 1999).

Our results also demonstrate that LPA affects CG-4 cells

and OL precursors similarly suggesting that CG-4 line cells

are a suitable model system for investigating OL process

formation as reviewed by Stariha and Kim (2001).

Acknowledgements

We thank Dr J. M. Levine (SUNY, USA) for the anti-NG2 antibody

and the Welfide Coporation (Japan) for Y-27632. This work was

supported by the BBSRC (research studentship to JCD).

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