Polysialic Acid Directs Tumor Cell Growth by Controlling Heterophilic Neural Cell Adhesion Molecule Interactions

Institut für Zoologie (220), Universität Hohenheim, 70593 Stuttgart, Germany.
Molecular and Cellular Biology (Impact Factor: 4.78). 09/2003; 23(16):5908-18. DOI: 10.1128/MCB.23.16.5908-5918.2003
Source: PubMed


Polysialic acid (PSA), a carbohydrate polymer attached to the neural cell adhesion molecule (NCAM), promotes neural plasticity
and tumor malignancy, but its mode of action is controversial. Here we establish that PSA controls tumor cell growth and differentiation
by interfering with NCAM signaling at cell-cell contacts. Interactions between cells with different PSA and NCAM expression
profiles were initiated by enzymatic removal of PSA and by ectopic expression of NCAM or PSA-NCAM. Removal of PSA from the
cell surface led to reduced proliferation and activated extracellular signal-regulated kinase (ERK), inducing enhanced survival
and neuronal differentiation of neuroblastoma cells. Blocking with an NCAM-specific peptide prevented these effects. Combinatorial
transinteraction studies with cells and membranes with different PSA and NCAM phenotypes revealed that heterophilic NCAM binding
mimics the cellular responses to PSA removal. In conclusion, our data demonstrate that PSA masks heterophilic NCAM signals,
having a direct impact on tumor cell growth. This provides a mechanism for how PSA may promote the genesis and progression
of highly aggressive PSA-NCAM-positive tumors.


Available from: Rita Gerardy-Schahn
MOLECULAR AND CELLULAR BIOLOGY, Aug. 2003, p. 5908–5918 Vol. 23, No. 16
0270-7306/03/$08.000 DOI: 10.1128/MCB.23.16.5908–5918.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Polysialic Acid Directs Tumor Cell Growth by Controlling Heterophilic
Neural Cell Adhesion Molecule Interactions
Ralph Seidenfaden,
Andrea Krauter,
Frank Schertzinger,
Rita Gerardy-Schahn,
and Herbert Hildebrandt
Institut fu¨r Zoologie (220), Universita¨t Hohenheim, 70593 Stuttgart,
and Abteilung Zellula¨re Chemie,
Medizinische Hochschule Hannover, 30625 Hannover,
Received 27 December 2002/Returned for modification 3 March 2003/Accepted 22 May 2003
Polysialic acid (PSA), a carbohydrate polymer attached to the neural cell adhesion molecule (NCAM), pro-
motes neural plasticity and tumor malignancy, but its mode of action is controversial. Here we establish that
PSA controls tumor cell growth and differentiation by interfering with NCAM signaling at cell-cell contacts.
Interactions between cells with different PSA and NCAM expression profiles were initiated by enzymatic re-
moval of PSA and by ectopic expression of NCAM or PSA-NCAM. Removal of PSA from the cell surface led
to reduced proliferation and activated extracellular signal-regulated kinase (ERK), inducing enhanced survival
and neuronal differentiation of neuroblastoma cells. Blocking with an NCAM-specific peptide prevented these
effects. Combinatorial transinteraction studies with cells and membranes with different PSA and NCAM phe-
notypes revealed that heterophilic NCAM binding mimics the cellular responses to PSA removal. In conclu-
sion, our data demonstrate that PSA masks heterophilic NCAM signals, having a direct impact on tumor cell
growth. This provides a mechanism for how PSA may promote the genesis and progression of highly aggressive
PSA-NCAM-positive tumors.
The neural cell adhesion molecule (NCAM) exhibits high
structural diversity due to alternative splicing and dynamically
regulated posttranslational modifications (5, 6). Initially iden-
tified as a cell adhesion molecule expressing homophilic bind-
ing properties (20), NCAM was later shown to exert hetero-
philic cis and trans binding interactions with molecules of
different classes, such as cell adhesion molecule L1 (21), mem-
bers of the fibroblast growth factor receptor family (3, 42),
extracellular matrix components (4), and perhaps others. In
accordance with the protein’s complexity, it has been impli-
cated in a multitude of cellular functions, mainly during neural
development and plasticity (reviewed in reference 5) but also
in oncogenesis (3, 36).
The most prominent and unique posttranslational modi-
fication of NCAM is polysialic acid (PSA), a homopolymer
of -2,8-linked sialic acid residues which is added to specific
N-glycan attachment sites in the fifth immunoglobulinlike do-
main of NCAM (30). PSA is abundantly expressed during
embryonic development and downregulated in the course of
maturation and differentiation (41). In the developing nervous
system, PSA-NCAM has been shown to promote plasticity of
cell-cell interactions during cell migration and neurite out-
growth (9, 41). In the adult mammalian brain, PSA appears to
be involved in persistent neurogenesis and some forms of syn-
aptic plasticity (9), and its expression under different patho-
logical conditions implies a role in neural regeneration and
repair (2, 32). As an oncodevelopmental antigen, PSA is reex-
pressed during progression of several malignant human tu-
mors, including small cell lung carcinoma, Wilms’ tumor, neu-
roblastoma, and rhabdomyosarcoma (11, 14, 17). In these
tumors, polysialylation of NCAM appears to increase the met-
astatic potential and has been correlated with tumor progres-
sion and a poor prognosis (7, 8, 14, 16, 17, 48).
Despite the abundant evidence that polysialic acid is criti-
cally involved in neural development and tumor malignancy, its
mode of action on the cellular level is still unclear. According
to the prevailing model, steric inhibition of membrane-mem-
brane apposition by PSA causes a general attenuation of cell
adhesion (41), and this antiadhesive effect of PSA appears to
be independent of NCAM-mediated interactions (13). Besides
modulating cell adhesion, PSA may act as a receptor of se-
creted factors (22), and only recently it was shown that PSA is
needed for the adequate sensitivity of neurons to the brain-
derived neurotrophic factor (BDNF) (49). In contrast to the
antiadhesive properties and the possible receptor functions of
PSA, its potential impact on NCAM signals is unresolved. As
demonstrated previously, enzymatic removal of PSA induces
marked inhibition of cell growth in human SH-SY5Y neuro-
blastoma cells (19).
Using a panel of PSA-NCAM expressing neuroblastoma and
rhabdomyosarcoma cell lines, the present study identifies PSA
as a negative regulator of heterophilic NCAM interactions at
cell-cell contacts. Removal of PSA releases NCAM signals,
inducing growth inhibition as well as mitogen-activated protein
kinase (MAPK)-dependent survival and differentiation. The
present data demonstrate for the first time that expression and
downregulation of PSA are decisive for NCAM-mediated reg-
ulation of tumor cell growth.
Antibodies, reagents and expression vectors. The following monoclonal (MAb)
and polyclonal (PAb) antibodies were used: extracellular signal-regulated kinase
(ERK) 1- and 2-specific rabbit PAb; dually phosphorylated ERK1/2-specific
mouse MAb E10 (9106) or rabbit PAb (9101; New England Biolabs); neurofila-
ment-specific mouse MAb 2F11, recognizing the phosphorylated form of the
* Corresponding author. Mailing address: Institut fu¨r Zoologie (220),
Universita¨t Hohenheim, Garbenstr. 30, 70593 Stuttgart, Germany. Phone:
49 711 459 3763. Fax: 49 711 459 3450. E-mail: hildebra@uni-hohenheim
Page 1
200-kDa and 68-kDa subunits (Neomarkers); mouse MAb 14G2a, specic for
ganglioside GD
(kindly provided by R. Handgretinger); bromodeoxyuridine
(BrdU)-specic rabbit immunoglobulin G (IgG) (Chemicon); normal mouse or
rabbit IgG; alkaline phosphatase- and peroxidase-conjugated goat anti-mouse
IgG; peroxidase-conjugated goat anti-rabbit IgG; 10 nm gold-conjugated goat
anti-mouse IgG (Sigma); Texas Red-conjugated rabbit anti-mouse IgG1; and
uorescein isothiocyanate-conjugated rabbit anti-mouse IgG2 (all from Bio-
trend), and CY2-conjugated goat anti-rabbit IgG and CY3-conjugated goat anti-
mouse IgG (Dianova).
All commercially available antibodies were used according to the recommen-
dations given by the supplier. PSA-specic mouse MAb 735 (12) and MAb 123C3
(29) (clone kindly provided by R. Michalides), reactive with all isoforms of
human NCAM, were used after afnity purication on protein G-Sepharose
(Pharmacia) and applied to live cells at 5 g/ml or for immunoblotting, enzyme-
linked immunosorbent assay (ELISA), immunocytochemistry, and immunouo-
rescence as described (19, 45). N-Acetylneuraminic acid and colominic acid were
purchased from Sigma and used at 50 g/ml. C3d, a synthetic dendrimeric un-
decapeptide which binds to the Ig1 module of NCAM and its inactive variant
C3d2Ala (39) were kindly provided by E. Bock and N. Pedersen. The MEK
inhibitor PD98059 was from Alexis, broblast growth factor 2 (basic broblast
growth factor) was from Merck, BDNF was from Calbiochem, and nerve growth
factor was from Biomol.
To generate NCAM- and PSA-NCAM-positive LS cell lines, the vectors
pAM1, encoding full-length human NCAM-140 in pCDM8 (10), and pcDNAI-
PST, kindly provided by M. Fukuda, encoding human full-length ST8Sia IV in
pcDNAI (33), were used. pEGFP-C1 (BD Clontech) was used as the cotrans-
fected selection marker (neomycin/G418) and to obtain enhanced green uo-
rescent protein (EGFP) expression in the cytosol. Recombinant endoneuramini-
dase NE (endo-NE), specically degrading PSA, was isolated as described (15)
and used in the cell culture medium at 6 or 60 ng/ml to remove PSA from the cell
surface (19). For inactivation, endo-NE was heated for 10 min at 60°C.
Cell culture. The human neuroblastoma cell lines SH-SY5Y (subclone of
SK-N-SH, ATCC CRL-2266), Kelly (ECACC 92110411), LS (40), LAN-5 (ICLC
HTL-96022), and rhabdomyosarcoma cell line TE-671 (ATCC CRL-7774) were
used. Cells were cultured at 37°C and 9% CO
in Dulbeccos modied Eagles
medium-Hams F12 medium containing 10% (vol/vol) heat-inactivated fetal bo-
vine serum (Biochrom), 2 mM glutamate, 100 units of penicillin per ml, and 100
g of streptomycin per ml. Media were changed every 2-day, and cells were
replated before conuency. Experiments were conducted with cells at densities
between 2.5 and 5 10
. To avoid effects of serum deprivation, great
care was taken in all experiments that the different experimental groups received
the same treatment with respect to medium changes, i.e., supplied with fresh se-
rum. For short-term incubations with endo-NE in serum-free medium, cells were
kept without serum for 1 h before changing to serum-free medium containing the
Transfection of neuroblastoma cells. For stable transfections, 3.5 10
SY5Y or 1.5 10
LS cells were plated in a 60-mm dish in culture medium
containing 10% fetal calf serum. After 24 h cells were transfected with 20 lof
Effectene corresponding to the manufacturers instructions (Qiagen) in culture
medium containing 5% fetal calf serum. SH-SY5Y cells were transfected with
1 g of pEGFP-C1. LS cells were cotransfected with 1 g of pAM1 and 0.1 g
of pEGFP-C1 for expression of NCAM-140 or with 0.5 g of pAM1, 0.5 gof
pcDNAI-PST, and 0.05 g of pEGFP-C1 for expression of PSA, or with 1 gof
pCDM8 and 0.1 g of pEGFPC1 as a control (mock transfection); 24 h later the
medium was replaced by culture medium, and 48 h later the cells were passaged
in 12-well plates with culture medium complemented by 400 g of potent G418-
sulfate per ml (Calbiochem). Transfected cells were subcloned by limited dilu-
tion to obtain single-cell clonal lines and checked by immunocytochemistry for
clones with a homogenous cell surface immunoreactivity for PSA or NCAM (45).
The EGFP-positive SH-SY5Ycells are designated SH-SY5Y
, and the mock-
transfected, NCAM- or NCAM-PSA-positive LS cells are designated LS
, and LS
, respectively.
Cellular ELISA , protein extraction, and immunoblotting. The relative amounts
of PSA or NCAM on the cell surface were determined by the cellular ELISA
procedure described previously (44, 45). Briey, cells grown in 96-well plates
were xed with 3.8% paraformaldehydephosphate-buffered saline, blocked with
2% (wt/vol) bovine serum albuminphosphate-buffered saline for 2 h, and incu-
bated with PSA-specic MAb 735 (0.3 g/ml) or NCAM-specic MAb 123C3
(1 g/ml), followed by detection with alkaline phosphatase-labeled anti-mouse
IgG and p-nitrophenylphosphate.
For immunoblotting, cells were washed briey with ice-cold phosphate-buff-
ered saline and harvested with a cell scraper in ice-cold lysis buffer consisting of
1% Brij 96, 150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 10 mM NaF, 1 mM EDTA,
1 mM EGTA, 1 mM Na
, 1 mM PMSF, 10 g of leupeptin per ml, and 10 g
of aprotinin per ml. After 10 min of incubation on ice, the lysates were claried
by centrifugation at 20,000 g for 15 min, and the protein concentration was
determined by the Bio-Rad protein assay. If not specied otherwise, 20 gof
protein was separated on sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis (SDS-PAGE) (10% polyacrylamide gels) and transferred to nitrocellu-
lose lters (Roth). Equal loading of proteins was conrmed by Ponceau S
staining, and proteins of interest were visualized with specic antibodies and the
enhanced chemiluminescence detection system (Amersham) with Kodak Biomax
MS lms. The intensity of enhanced chemiluminescence bands was analyzed as
mean grey value by computerized densitometric scanning and ScionImage soft-
ware (Scion Corporation). For reprobing of membranes, antibodies were
stripped by incubation with 100 mM 2-mercaptoethanol70 mM SDS in 62.5 mM
Tris-HCl, pH 6.7, at 70°C for 45 min.
Preparation of crude membrane fractions. Crude membrane fractions from
, and LS
were homogenized in 10 mM NaHCO
(pH 8.0)
with 1 mM CaCl
and 1 mM MgCl
. Cells were passed 10 times through a 20-
gauge needle, centrifuged at 30,000 g for 10 min, and the resulting pellets were
resuspended in serum-supplemented cell culture medium. This suspension was
added to cultured cells in a 1:1 ratio of extracted to living cells.
Immunocytochemistry, immunouorescence, and immunoelectron microscopy.
Cells were xed for 30 min with 3.8% paraformaldehyde in phosphate buffer,
blocked with 2% bovine serum albuminphosphate buffer for 2 h, and incubated
with primary antibodies overnight at 4°C. For immunouorescence detection of
neurolament, cells were solubilized with 0.1% Triton X-100phosphate buffer
for 15 min before blocking and a CY3-conjugated secondary antibody was used.
PSA and NCAM immunostaining with alkaline phosphatase-coupled secondary
antibodies and 5-bromo-4-chloro-3-indolylphosphate-nitoblue tetrazolium as
well as confocal immunouorescence with Texas Red and uorescein isothio-
cyanate-conjugated secondary antibodies were carried out as described (19, 45).
For ultrastructural analysis, pre-embedding immunolabeling of polysialic acid
on the cell surface was performed as for immunocytochemistry with 10-nm-gold-
labeled secondary antibody. As control, primary antibodies were omitted. Cells
were postxed in the culture plate with 1% glutaraldehydephosphate buffer (5
min, room temperature) and 1% OsO
(10 min, room temperature), washed, and
subjected to a graded series of 30, 50, and 70% ethanol (19 min each), counter-
stained in 1% uranyl acetate70% ethanol (15 min), dehydrated three times with
100% ethanol, incubated 30 min in Araldite (Plano)ethanol, 1:1, and embedded
in Araldite. Then 500-nm-thick horizontal sections were prepared with an Ultra-
cut S (Reichert) and mounted on pioloform-coated copper grids. Imaging of
thick horizontal sections with energy-ltering transmission electron microscopy,
allowing the ultrastructural analysis of large areas of the cell surface, was per-
formed as described (26) with the electron microscope CEM902 (Zeiss)
equipped with a Henry Castaign energy lter (prism-mirror-prism spectrometer).
Analysis of cell growth, apoptosis, and neuronal differentiation. Cell growth
or survival was assessed by a colorimetric tetrazolium-formazan assay (Cell Pro-
liferation Assay; Promega), and rates of apoptosis were assessed by the quanti-
tative determination of intracellular mono- and oligonucleosomes with a sand-
wich-ELISA procedure with monoclonal antibodies directed against DNA and
histones (Cell Death Detection ELISA
; Roche). Detached cells were col
lected by centrifugation of the cell culture supernatant (200 g) and lysed together
with the adherent cells. The specic enrichment of intracellular mono- and
oligonucleosomes is given as the apoptotic index, calculated from the absorbance
of the sample relative to untreated controls (see manufacturers instructions for
details). Both assays were applied to cells seeded in parallel on 96-well plates.
For each treatment, phase contrast images of three wells were captured with
an Axiovert 135 microscope (Zeiss) and a charge-coupled device camera, and
cells were counted to compare cell numbers with the results of the metabolic
assay. Alternatively, cells were xed as described for immunocytochemistry,
washed with phosphate buffer, and mounted in Vectashield containing 4,6-di-
amidino-2-phenylindole (DAPI; Linaris) to analyze the morphology of the nuclei
with DAPI uorescence. In some of the experiments, the rate of proliferation
was addressed by incorporation of bromodeoxyuridine (BrdU; Boehringer).
Cells were incubated for 16 h with 10 M BrdU prior to xation. After incubat-
ing with 2 N HCl for 15 min at 37°C and then 0.1 M borate, pH 8.5, for 10 min,
BrdU was detected with an anti-BrdU antibody diluted 1:100 (Chemicon).
Neurite outgrowth of SH-SY5Y
neuroblastoma cells was addressed in
12-well plates. For treatment with endo-NE, dimethyl sulfoxide, or PD98059,
30,000 SH-SY5Y
cells were seeded in the presence of the reagent. For
incubation with crude membrane fractions, SH-SY5Y
cells were grown for
24 h before the membrane suspension was added. After 48 h of treatment, living
cells were imaged by EGFP uorescence with an Axiovert 100 M
microscope equipped with LSM (5 Pa), a helium-neon laser, and a Plan-Neouar
Page 2
10 objective (Zeiss). From each well, ve randomly selected frames were
scanned, SH-SY5Y
cells were counted, and the length of the longest process
per cell was measured by computer-assisted image analysis with the software
package Axio Vision 3.0 (Zeiss). Per frame, the percentage of cells with pro-
cesses longer than 20 m was calculated relative to the total number of cells.
Statistics. Differences between two groups were evaluated with Students t
test. With more than two groups to compare, one-way analysis of variance
(ANOVA) or ANOVA with repeated measures was applied with Prism (Graph-
pad Software). Pearsons
test was used to compare BrdU incorporation.
Removal of PSA from tumor cells inhibits proliferation.
Treatment of living cells with endoneuraminidase NE (endo-
NE) (15) degrades PSA with high specicity and leads to a bet-
ter accessibility of NCAM on the cell surface (19). In NCAM-
and PSA-positive neuroblastoma (SH-SY5Y, Kelly, and LAN-5)
and rhabdomyosarcoma (TE671) cells (44), endo-NE induced
a similar inhibition of cell growth, while growth of the NCAM-
and PSA-negative neuroblastoma cell line LS was unaffected
(Fig. 1a). Control incubations with heat-inactivated endo-NE,
colominic acid (i.e., soluble PSA from bacteria), or N-acetyl-
neuraminic acid had no effect on cell growth. The endo-NE-
induced reduction of cell growth was conrmed by counting
the cells in some of the experiments. Detached, dead, or dam-
aged cells were never observed, and DAPI staining revealed no
abnormal condensation or fragmentation of nuclei. For SH-
SY5Y cells, the absence of apoptosis after endo-NE treatment
was conrmed by a cell death ELISA (see Fig. 6), and the
analysis of BrdU incorporation revealed that the endo-NE-
induced reduction of cell growth was associated with a sig-
nificant decline in proliferation (Fig. 1b). Thus, the observed
growth inhibition was due to reduced rates of proliferation.
In primary cultures of cortical neurons, the expression of
PSA has been shown to improve the sensitivity to BDNF, and
in contrast to other neurotrophins, application of exogenous
BDNF was able to rescue neurons from endo-NE-induced
death (49). Because SH-SY5Y cells express low levels of
BDNF and TrkB (23), we compared the inuence of endo-NE
on the mitogenic activities of BDNF, nerve growth factor, and
broblast growth factor-2. The proliferative response to all
three growth factors was drastically reduced in the absence of
PSA (Fig. 2a). This pleiotropic effect argues against a BDNF-
specic interaction with PSA but suggests that growth inhibi-
tion is a direct result of PSA failure.
We then analyzed whether changes in NCAM binding abil-
ities may be responsible for the effect of endo-NE. In a rst
experiment, the MAb 123C3 (29) was used to interfere with
NCAM binding. The PSA-specic MAb 735 and MAb 14G2a
(31), directed against the ganglioside GD
, served as controls.
All three reagents produced an 80% growth reduction, indi-
cating a strong but nonspecic cytotoxic effect. Incubation with
normal mouse IgG as a nonbinding control antibody had no
effect on cell growth (data not shown). However, the endo-
NE-mediated growth inhibition was completely prevented if
cells were treated with the dendrimeric C3d peptide (39), a
synthetic ligand of the rst immunoglobulinlike domain of
NCAM (Fig. 2b). In contrast, an inactive variant of C3d,
C3d2Ala (39), was unable to protect the cells from the endo-
NE-induced growth inhibition (not shown).
As controlled by cellular ELISA, incubation with the C3d
peptide neither interfered with PSA degradation nor led to
reduced cell surface expression of NCAM (cell surface immu-
noreactivities of untreated controls and of cultures treated with
endo-NE in the presence and absence of C3d [arbitrary units
standard error of the mean] were PSA, 0.77 0.03, 0.02
0.005, and 0.02 0.002, respectively; NCAM, 0.54 0.08, 0.76
0.02, and 0.62 0.05, respectively; n 6). Also, C3d had no
FIG. 1. PSA removal induces growth inhibition in PSA-NCAM-
positive tumor cells. (a) LS, SH-SY5Y, Kelly, and LAN-5 neuroblas-
toma and TE671 rhabdomyosarcoma cells were grown for 2 days in
control medium (ctrl., white bars) or in the presence of 6 ng/ml (80
pM) endo-NE (Endo, grey bars). Growth rates were determined by
metabolic assays performed at day 0 and day 2, and for each cell line,
the rate of the untreated controls was set to 100%. Values represent
means ( standard error of the mean) of a minimum of eight assays for
each treatment. *, P 0.05; ***, P 0.001; t test. Micrographs show
immunostaining of the cell lines with the NCAM- and PSA-specic
antibodies 123C3 and 735 performed with untreated controls and after
2 days of endo-NE treatment (Endo). Scale bar, 50 m. (b) Incor-
poration of BrdU by SH-SY5Y cells grown for 2 days in control
medium (ctrl.) or in the presence of endo-NE (Endo). BrdU was
added 16 h prior to immunouorescence analysis; 400 to 600 cells were
evaluated, and the percentage of BrdU-positive cells is shown. Error
bars indicate the 95% condence intervals. ***, P 0.001,
Page 3
autonomous effect on proliferation (Fig. 2b). The protective
effect of C3d therefore strongly suggests that the inhibition of
proliferation involves interactions of NCAM. Moreover, be-
cause the C3d peptide binds to the rst immunoglobulinlike
domain of NCAM, this module appears to be involved in
mediating this interaction.
The next question was if trans-interacting NCAM is also able
to cause growth inhibition. In order to mimic the situation in
the cell culture system, experiments were carried out with
NCAM-positive and NCAM-negative cell membranes isolated
from differentially transfected LS cells. The phenotypes of
transfected LS cells are shown in Fig. 2c. Parental LS are
NCAM and PSA negative (Fig. 1). The same is true for cells
transfected with an empty vector (NCAM negative, PSA neg-
ative; Fig. 2c, LS
). Cells after transfection with a vector
driving the expression of NCAM-140 stained positive in im-
munohistochemistry for NCAM but not for PSA (NCAM pos-
itive, PSA negative; Fig. 2c, LS
), while double transfectants
containing vectors driving NCAM-140 and polysialyltrans-
ferase ST8SiaIV expression, stained positive for both epitopes
(PSA positive, NCAM positive; Fig. 2c, LS
). Mem
branes from PSA-positive, NCAM-positive double transfec-
tants were isolated before and after endo-NE digestion (com-
pare Fig. 2c, LS
, untreated versus LS
In the experiment, SH-SY5Y (PSA positive, NCAM posi-
tive) and the control cells (LS, PSA negative, NCAM negative)
were overlaid with membrane preparations of all phenotypes.
Microscopic control revealed that the membrane particles set-
tled rapidly and dispersed uniformly over cells and cell-free
areas (Fig. 2c, lower panels for an example). After a coculture
period of 2 days, the growth rates were compared (Fig. 2d).
Membranes isolated from mock (PSA negative, NCAM nega-
tive) or double transfected LS cells (AM1PST; PSA positive,
NCAM positive) did not noticeably affect cell growth in either
of the cell systems. In contrast, the membrane fractions derived
from LS
or LS
after endo-NE treatment (both are
FIG. 2. Growth inhibition after removal of PSA is mediated by
NCAM interactions. (a) SH-SY5Y cells were treated for 2-day with 50
ng/ml BDNF, nerve growth factor or broblast growth factor-2 in the
absence (ctrl., white bars) or presence of 6 ng of endo-NE per ml
(Endo, grey bars). Metabolic rates were determined as in Fig. 1 and
expressed as percent increase over the level of untreated controls. (b)
Metabolic rates of SH-SY5Y (white bars) or TE671 cells (grey bars)
treated for 2-day with C3d (1 M), endo-NE (6 ng/ml), or both, as
indicated. (c) Differentially transfected and endo-NE-treated LS cells
(see text for details) were characterized by immunocytochemistry with
PSA- or NCAM-specic antibodies as indicated (upper panels, scale bar,
50 m). SH-SY5Y or parental LS cells were incubated with crude
membrane fractions, prepared from all phenotypes. Shown are repre-
sentative phase contrast images of SH-SY5Y cells without or with
membrane fractions (MF, lower panels) illustrating the variable size
and the density of the membrane particles (white arrowheads; scale
bar, 10 m). (d) Metabolic rates of SH-SY5Y (white bars) or parental
LS cells (gray bars), exposed for 2-day to membrane fractions obtained
from PSA- and NCAM-negative (mock), NCAM-positive (AM1,
AM1PSTEndo) or PSA-NCAM-positive membrane fractions
(AM1PST). Values in panels a, b, and d represent means ( standard
error of the mean) of a minimum of 12 assays for each treatment. *, P
0.05; **, P 0.01; ***, P 0.001; t test against the untreated
controls or between the indicated values.
Page 4
PSA negative, NCAM positive) signicantly inhibited the
growth of SH-SY5Y cells (which are PSA positive, NCAM
positive) and of LS cells (which are PSA negative, NCAM
Although unexpected, this observation indicates that the
growth inhibition induced by endo-NE or by NCAM itself
results from trans binding of nonpolysialylated NCAM to het-
erophilic target structures. These ndings received further
support by the observation that growth of LS
cells and
plus endo-NE (both PSA negative, NCAM positive,
metabolic rates [day 2/day 0 standard error of the mean]:
1.62 0.06 and 1.76 0.09) was signicantly reduced com-
pared to nontransfected LS cells, LS
(both PSA negative,
NCAM negative), or LS
(PSA positive, NCAM positive;
metabolic rates [day 2/day 0 standard error of the mean]:
2.21 0.07, 2.19 0.07, and 2.22 0.08; n 10 to 12; t test,
P 0.01).
The above data imply that PSA regulates NCAM-dependent
cell-cell interactions, which requires its localization at cellular
contact sites. To check this, the cell surface distribution of PSA
and NCAM was studied. As visualized by immunogold detec-
tion with energy-ltering transmission electron microscopy
(26), PSA appears in clusters on SH-SY5Y cells and is prefer-
ably localized at sites of tight cell-cell contacts (Fig. 3a). Im-
munouorescence staining and confocal microscopy clearly
demonstrate that NCAM and PSA are mainly colocalized at
cellular contact sites (Fig. 3b, left panel). Removal of PSA did
not change the strong NCAM-immunoreactivity at contact
sites, however, the number of NCAM-positive contact zones
increased signicantly after 2-day of endo-NE treatment (Fig.
3b, right panel). Even if seeded as single cell suspension at low
densities, the tumor cells used in this study never grew without
any contacts between each other. Time lapse observations per-
formed with SH-SY5Y cells stably transfected to express the
enhanced green uorescent protein (EGFP) in the cytosol
) indicate that, besides the broad and rather sta
ble contacts shown in Fig. 3b, even those cells that have no contact
at a given time point, readily will form new contacts or just broke
up existing contacts due to extensive cellular motility (Fig. 3c).
Taken together, these analyses of cell growth indicate that
downregulation of PSA enables heterophilic NCAM interac-
tions at contact sites between tumor cells leading to reduced
proliferation in the recipient cell.
Endo-NE-induced NCAM signaling involves MAPK activa-
tion. Because previous studies of NCAM signaling demon-
strated the involvement of the p44/p42 MAPK ERK1/2 path-
way (24, 27, 43), this system was surveyed during endo-NE
treatment of SH-SY5Y cells. Western blotting with anti-NCAM
MAb 123C3 was used to monitor the conversion of the hardly
detectable high-molecular weight smear typical for highly sia-
lylated NCAM into PSA-free isoforms seen as sharp bands of
140 and 180-kDa (Fig. 4a). Already after 10 min of endo-NE
digest, signicant amounts of nonpolysialylated NCAM were
generated, but only after 1 h the reaction was completed.
Endo-NE was constantly present in the cultures and no PSA
reappearance was detected during the 2 days of the experi-
ment. Changes in ERK activity were examined by Western
blotting with antibodies directed against dually phosphorylated
ERK1/2, indicative for the activated MAP kinase (28), and
compared to the total amount of ERK protein (Fig. 4b).
FIG. 3. PSA-NCAM is localized at cell-cell contact sites. (a) Im-
munoelectron microscopic localization of cell-surface PSA by pre-
embedding immunogold labeling and energy-ltering transmission
electron microscopy of 500 nm thick sections. Antibodies were applied
to xed cells to achieve cell-surface staining and the technique used
allows the analysis of large cell surface areas (26). Partial view of two
adjacent cells with tight cell-cell contact (black arrow). Clustered la-
beling occurred scattered over the cell surface (black arrowheads) and
was enriched at the contact site (white arrowheads). Scale bar, 0.5 m.
(b) Double immunouorescence staining of PSA and NCAM was
performed with untreated SH-SY5Y cells (control) and after endo-NE
treatment for 2 days (Endo) as indicated. Scale bar, 25 m. Numbers
of NCAM-positive cell-cell contact sites were counted. Values repre-
sent means ( standard error of the mean) of six evaluated frames,
with a total of 312 and 303 cells, respectively. **, P 0.01; t test. (c)
Time lapse uorescence images of EGFP-transfected SH-SY5Y cells
illustrate the high motility of some of the cells. Rapid changes of cell
shape and cell-cell contacts can be observed. Notably, one cell that has
no contact to other cells at 0 min is forming a broad cell-cell contact
zone within 20 min (white arrows). Scale bar, 25 m.
Page 5
To apply equal amounts of endo-NE, the enzyme was di-
luted in culture medium and medium were changed in all
experimental groups at the beginning of the experiment. In the
control group, the supply with fresh serum resulted in a mod-
erate increase of ERK phosphorylation, relapsing to baseline
within 16 h (Fig. 4b and c). In endo-NE treated cultures a
strong increase of ERK phosphorylation was observed (Fig. 4b
and c), while the total amount of ERK1/2 protein remained
constant throughout the experiment (Fig. 4b, lower panel).
ERK phosphorylation was maximal as soon as 10 min after the
beginning of the treatment and the peak level was maintained
up to 1 h before it slowly returned to the level of the time-
matched controls (Fig. 4b and c). In contrast to the effect of
medium replacement, the strong MAPK activation during PSA
digest was not caused by serum factors, because in the absence
of serum endo-NE treatment carried out for 10, 30, or 60 min
resulted in the same activation of MAPK as in serum-supple-
mented cultures (Table 1). However, in contrast to the MAPK
activation after supply with fresh serum, ERK phosphorylation
was not increased by medium replacement and these experi-
ments were limited to 1 h, because longer periods of serum
withdrawal led to a marked increase of cell death. The parallel
analysis of TE671 rhabdomyosarcoma cells produced an iden-
tical time course of ERK phosphorylation after endo-NE treat-
ment (Table 1).
If the endo-NE-induced MAPK activation relates to signal-
ing via the NCAM molecule, factors that prevented growth
retardation as shown in Fig. 2b, should be able to interfere with
this induction. In fact, a complete inhibition of ERK phosphor-
ylation was observed, if PSA removal was carried out in the
presence of 1 M C3d peptide (Fig. 5a). As shown in Fig. 5b,
similar results were obtained by antibody inhibition. Here it is
important to note that, in contrast to the long-term incubations
described above, short periods of antibody application had no
apparent cytotoxic effect. After endo-NE treatment for 1 h,
SH-SY5Y cells were incubated for 10 min with antibodies
directed against NCAM (MAb 123C3), ganglioside GD
14G2a) or PSA (MAb 735). Although no complete reversal
could be achieved by this specic protocol, application of MAb
123C3, but not 14G2a or 735, clearly reduced the ERK phos-
phorylation induced by endo-NE (Fig. 5b, endo-NE-induced
pERK intensities relative to the untreated control: 2.29 with-
out additional antibody application, 1.38 after MAb 123C3,
1.96 and 2.53 after MAb 14G2a and 735, respectively). The
same experiment performed with TE671 cells yielded identical
results (endo-NE-induced pERK intensities relative to the un-
treated control: 1.77 without antibody, 1.30 after MAb 123C3,
2.23 and 1.95 after MAb 14G2a and 735, respectively).
Next, NCAM-positive membranes were tested for their ca-
pability to activate MAPK. No MAP kinase activity could be
detected in the membrane fractions themselves (not shown).
NCAM-positive membranes (AM1, AM1PST Endo) were
able to induce ERK phosphorylation in PSA- and NCAM-
positive SH-SY5Y cells, but membranes containing polysialy-
lated NCAM (AM1PST) were ineffective (Fig. 5c). Likewise,
ERK phosphorylation in TE671 cells was stimulated only by
NCAM-positive, PSA-negative membranes (data not shown).
Exposure of the PSA- and NCAM-negative LS cells to NCAM-
positive membranes resulted in an ERK activation with a time-
course similar to that, seen after removing PSA from the sur-
face of the PSA-NCAM-positive neuroblastoma cells (Fig. 5d).
As for the endo-NE treatment of the PSA- and NCAM-posi-
tive SH-SY5Y cells, this induction of MAPK signaling was
clearly inhibited by concomitant application of C3d (Fig. 5e,
left panel) and completely blocked in the presence of PSA
FIG. 4. Removal of PSA leads to ERK activation. SH-SY5Y cells
were supplied with fresh cell culture medium containing 60 ng of endo-
NE per ml (Endo, Endo) or solvent (ctrl., Endo), incubated for the
times indicated and analyzed by immunoblots with (a) NCAM-specic
MAb 123C3 or (b) MAb E10, specic for dually phosphorylated
ERK1/2 (pERK). Loading and transfer of proteins was controlled by
Ponceau S staining of the blot membrane and only lanes with equal
amounts of protein were used. (b) Antibodies were stripped off and
membranes were reprobed with ERK1/2-specic antibodies to control
for changes in ERK protein levels (ERK). Due to saturation of the
ERK bands, a higher dilution of the ERK 1/2 specic antibody was
used in all further experiments. Changes in the amount of ERK pro-
tein were never detected. (c) Densitometric evaluation of pERK rel-
ative to the mean value of control cultures analyzed 48 h after medium
change. Since ERK1 and ERK2 were not always separated unambig-
uously, the pERK bands were evaluated together. Values are means
( standard error of the mean) of three to six independent experiments.
*, P 0.05; **, P 0.01; t test against the time-matched controls.
TABLE 1. Effect of endo-NE treatment on ERK phosphorylation
Cell type
Relative pERK intensity
(endo-NE treated/untreated control)
10 min 30 min 1 h 4 h 16 h
FCS 2.10 2.02 1.75 1.79 0.78
FCS 1.75 2.52 1.86
FCS 1.67 1.93 1.51 1.43 0.92
pERK intensities were determined as described for Fig. 4, and ratios of endo-
NE treated samples relative to time-matched controls are quoted. Values for
SH-SY5Y cells with fetal calf serum (FCS) were calculated from values in Fig. 4.
All other values represent means of at least two independent experiments with
similar results.
Page 6
(Fig. 5e, right panel, AM1PST). In accordance with the growth
inhibition experiment (see Fig. 2d), these data are most con-
sistent with the assumption that the MAPK activation after
removal of PSA is induced by heterophilic NCAM interactions.
PSA removal promotes MAPK-dependent survival. To elu-
cidate the contribution of MAPK activity to changes of cell
growth induced by endo-NE, the effects of MAPK inhibition
were compared to those of PSA removal. In serum-supple-
mented cultures of SH-SY5Y cells, the inhibition of ERK
phosphorylation by the MEK inhibitor PD98059 (Fig. 6a) re-
duced cell growth, and growth inhibition induced by endo-NE
was signicantly enhanced in the presence of PD98059 (Fig.
6b, upper graph). The semiquantitative evaluation of intracel-
lular mono- and oligonucleosomes by ELISA revealed that
EndoNE treatment did not induce apoptotic cell death, where-
as the inhibition of MAPK led to a small but clear-cut increase
of apoptosis, which occurred independent of endo-NE treat-
ment (Fig. 6b, lower graph). As evident from the intracellular
fragmentation of nuclei, a strong induction of apoptosis was
observed after 2-day of serum withdrawal (Fig. 6c). Only 40%
of the cells survived the 2-day starvation in otherwise untreated
or in dimethyl sulfoxide-treated controls (Fig. 6d, upper graph),
and the high rate of apoptosis was conrmed by the semiquan-
titative evaluation of intracellular mono- and oligonucleo-
somes (Fig. 6d, lower graph).
By endo-NE treatment, the survival rate of the serum-
starved cells was signicantly improved (Fig. 6d, upper graph),
while apoptosis was reduced (Fig. 6d, lower graph). In the
presence and in the absence of endo-NE, MAPK inhibition
with PD98059 reduced the survival and increased apoptosis
(Fig. 6d). In a second set of experiments, a clear increase of
apoptosis was detected as soon as 4 h after the onset of serum
withdrawal. In accordance with the results shown in Fig. 6d for
the 2-day incubation period, the incidence of apoptosis was
enhanced by the inhibition of MAPK with PD98059, reduced
by PSA removal with endo-NE and the anti-apoptotic effect of
endo-NE was reversed by MAPK inhibition (apoptotic indices
for the 4 h treatments: solvent-control [dimethyl sulfoxide]:
0.291, PD98059: 0.334, endo-NE: 0.213, endo-NEPD98059:
0.302). Despite the signicantly higher number of cells result-
FIG. 5. ERK activation by heterophilic NCAM interactions. ERK
phosphorylation (pERK) and total ERK protein (ERK) were analyzed
as in Fig. 4, with the ERK1/2-specic antibody at a dilution of 1:2,000.
In all experiments shown, dual phosphorylation of ERK was analyzed
with MAb E10, except for panel a, where a rabbit PAb with a higher
afnity towards pERK1 (p44) was used. (a to c) SH-SY5Y cells were
treated as indicated: (a) with C3d (1 M), endo-NE (60 ng/ml), or both
for 2 h; (b) without or with endo-NE (60 ng/ml) for 1 h, followed by a
10-min exposure to control medium or medium containing NCAM-
specic MAb 123C3, PSA-specic MAb 735 or ganglioside GD2-spe-
cic MAb 14G2a (5 g/ml each); (c) with membrane fractions pre-
pared from LS
(PSA negative, NCAM negative), LS
negative, NCAM positive), LS
(PSA positive, NCAM positive), or
(PSA negative, NCAM positive) for 2 h. (d and e) As
indicated, parental LS cells (PSA negative, NCAM negative) were
treated with the different membrane fractions specied in panel c in
the presence or absence of C3d (1 M). The experiments shown in
panels b and and c were repeated at least once with identical outcome
and equal results were obtained with TE671 cells (see text). Densito-
metric evaluation of the pERK signal in panels a and d represents
means ( standard error of the mean) of three to ve independent
experiments each. Values in panel a are normalized to the untreated
controls, and ** indicates a signicant difference to all other groups
shown (P 0.01, repeated measure ANOVA). Due to the moderate
activation of MAPK by the supply with fresh serum (see text related to
Fig. 4), data in panel d were expressed relative to control cultures
analyzed 48 h after medium change. *, P 0.05; **, P 0.01; paired
t tests against the time-matched controls.
Page 7
ing from the endo-NE treatment, the percentage of BrdU-
positive cells was decreased (Fig. 6e). Thus, removal of PSA
from serum-deprived cells induced a similar inhibition of pro-
liferation as under serum-supplemented conditions (see Fig.
1b). Together, these data indicate that the activation of MAPK
after PSA removal or NCAM application exerts a survival
promoting, anti-apoptotic effect but did not cause the growth
inhibition observed after these treatments.
PSA removal induces neuronal differentiation. The activa-
tion of MAPK observed in our experiments is highly reminis-
cent to the time course of ERK activity underlying the induc-
tion of neuronal differentiation in PC12 cells (28). The
induction of neuronal differentiation was therefore investi-
gated by counting neuritic extensions in NCAM- and endo-NE
treated SH-SY5Y cells. SH-SY5Y
were used in these ex
periments because all processes can be reliably detected by
uorescence microscopy of living cells (Fig. 7a to d). In line
with the well-characterized potential of SH-SY5Y cells to dif-
ferentiate into a neuron-like phenotype (35), process-bearing
cells can be stained with an antibody against
phosphorylated neurolament, a marker of neuronal differen-
tiation (46) (Fig. 7a and b, insets). Accordingly, long processes
were referred to as neurites and cells with neurites longer than
20 m were evaluated to assess the degree of neuronal differ-
entiation. In untreated cultures grown for 48 h, an average of
21% of the SH-SY5Y
cells developed processes longer
than 20 m, ranging up to 120 m (mean length standard
error of the mean, 36 0.8 m, n 401).
Incubation with solvent (dimethyl sulfoxide) or MAPK in-
hibition by PD98059 caused no signicant changes in neurite
lengths or in the amount of neurite-bearing cells, while in the
presence of endo-NE the neurites grew slightly longer and the
number of neurite-bearing cells was clearly increased (Fig. 7e
and f). This increase was completely prevented by the concom-
itant application of PD98059 (Fig. 7f). Similar to the effect of
endo-NE, coculture of SH-SY5Y
with NCAM-positive
membrane fractions from LS
or endo-NE treated
induced a strong increase in the relative amounts of
neurite-bearing cells together with a slight enhancement of
neurite length (Fig. 7g and h). Consistent with the results on
FIG. 6. PSA removal modulates MAPK-dependent survival and
apoptosis. Serum-supplemented or serum-deprived SH-SY5Y cells
were treated with the MEK inhibitor PD98059 (50 M in dimethyl
sulfoxide) or solvent (1 l of dimethyl sulfoxide/ml) in the presence or
absence of endo-NE (6 ng/ml). (a) PD98059 inhibits ERK phosphor-
ylation in SH-SY5Y cells. Cells were treated for2hinserum-supple-
mented medium as indicated and ERK phosphorylation was analyzed
(see Fig. 4 and 5). (b to d) SH-SY5Y cells were grown for 2-day in
(b) serum-supplemented or (c and d) serum-deprived medium in the
presence of dimethyl sulfoxide, PD98059 or endo-NE as indicated.
Cell growth or survival was determined by metabolic assays (b and d,
upper graphs). To detect apoptosis, the morphology of nuclei was
analyzed by DAPI immunouorescence and the occurrence of apo-
ptotic nuclei after serum withdrawal is shown (c, white arrowheads,
scale bar 10 m). ELISA detection of intracellular mono- and oligo-
nucleosomes was used to calculate an arbitrary apoptotic index (b and
d, lower graphs). Values in panels b and d represent means ( stan-
dard error of the mean) of a minimum of eight independent metabolic
assays or three to ve determinations of apoptotic indices. Different
letters denote signicant differences between groups (P 0.05, one-
way ANOVA). (e) Effect of endo-NE treatment on cell number and
BrdU incorporation of serum-deprived SH-SY5Y cells. Phase contrast
images of control (ctrl.) or endo-NE-treated cells (Endo) were com-
bined with immunouorescent labeling for BrdU (white arrowheads,
scale bar, 100 m). Total cell numbers and BrdU-labeled nuclei were
counted from six frames each. Means of cell counts are shown
standard error of the mean, *, P 0.05, t test. The percentage of
BrdU-positive cells is given 95% condence intervals, **, P 0.01,
Page 8
MAPK activation (see Fig. 5c), NCAM negative (mock) or
PSA-NCAM-positive (AM1PST) membrane fractions had no
effect (Fig. 7g, h). Despite the prolonged activation of MAPK
after NCAM-exposure of untransfected LS cells (see Fig. 5d),
no neurite outgrowth could be induced in this cell line (data
not shown). Since protocols such as retinoic acid or growth
factor treatment, which induce neuronal differentiation in
other neuroblastoma cell lines, were also ineffective with LS
cells (Seidenfaden, unpublished observation), these cells ap-
pear unable to differentiate morphologically. Thus, downregu-
lation of PSA or exposure to nonpolysialylated NCAM induces
a MAPK-dependent change towards a neuron-like phenotype
in cells that exhibit the ability for such a differentiation.
Numerous studies indicate a major contribution of PSA to
cellular plasticity in neural development, remodeling, and re-
pair (9, 41), and PSA is increasingly recognized as a positive
modulator of tumor malignancy (7, 8, 14). The present study
establishes for the rst time that expression of PSA affects
NCAM-dependent signaling implicated in the regulation of
tumor cell proliferation, survival and differentiation. This func-
tion of PSA appears clearly distinct from its role as a positive
regulator of chain migration and axon fasciculation (9) and
from the anti-adhesive properties of PSA, which seems to be
dominated by the modulation of NCAM-independent cell ad-
hesion (13).
Endo-NE treatment of PSA-NCAM-positive neuroblastoma
or rhabdomyosarcoma cells removed PSA without altering
NCAM expression and induced growth inhibition, MAPK ac-
tivation, as well as MAPK-dependent survival and differentia-
tion. Our data provide substantial evidence that these effects
are due to the release of heterophilic NCAM interactions at
cell-cell contact sites and neither due to endo-NE effects other
than PSA removal, nor due to interactions of PSA itself. (i)
Unspecic responses to endo-NE can be excluded, since heat-
inactivated endo-NE or endo-NE treatment of PSA-negative
cells had no effect. (ii) In contrast to the experiments suggest-
ing a role of PSA in BDNF signaling (49), the effects of endo-
NE on tumor cells could not be mimicked by soluble PSA. The
PSA-dependent growth of SH-SY5Y cells cannot be assigned
to a specic interaction with BDNF, since the mitogenic re-
sponses to different growth factors were uniformly reduced by
endo-NE treatment. (iii) Together with the appearance of non-
polysialylated NCAM, ERK phosphorylation was increased
after 10 min of endo-NE treatment. As the complete removal
of PSA takes 1 h, the occurrence of some nonpolysialylated
NCAM appears sufcient to stimulate MAPK. Interactions of
PSA or nonpolysialylated NCAM with serum factors cannot
FIG. 7. Effect of PSA removal, trans-interacting NCAM and MAPK
on neuronal differentiation. (a to d) EGFP uorescence of SH-SY5Y
cells grown for 48 h (a) in the absence or (b) in the presence of endo-
NE (6 ng/ml) or together with crude membrane fractions of (c) NCAM-
negative LS
or (d) NCAM-positive LS
. Scale bar, 100 m.
(Insets in panels a and b) With or without endo-NE treatment, neurite-
bearing SH-SY5Y
cells were positive for neurolament immuno
uorescence. Scale bar, 50 m. (e to h) Length of neurites (e and g)
and percentage of neurite-bearing SH-SY5Y
cells (f and h) rela
tive to untreated controls (see text for details). (e and f) Neurite
formation of SH-SY5Y
cells grown in the presence of solvent (1
l of dimethyl sulfoxide/ml), the MEK-inhibitor PD 98059 (50 Min
dimethyl sulfoxide), or endo-NE (6 ng/ml) as indicated. (g and h)
Neurite formation of SH-SY5Y
cells cocultured with crude mem
brane fractions prepared from NCAM-negative LS
NCAM-positive LS
(AM1), PSA-NCAM-positive LS
(AM1PST), or LS
treated with endo-NE to remove PSA
(AM1PSTEndo). In each of the three experimental series, three to
six independent experiments were evaluated and between 300 and 600
cells per experiment were analyzed for each experimental group. Due
to some variability between the different experiments, the values of
each experiment were standardized relative to the untreated control
(100%). Means ( standard error of the mean) are shown, and dif-
ferent letters denote signicant differences between groups (one-way
ANOVA, P 0.05 in panels e, g, and h; P 0.01 in panel f).
Page 9
account for this effect, since MAPK is activated in the presence
and absence of serum. Similarly, the survival promoting activity
of PSA removal under conditions of serum-withdrawal appears
not compatible with the idea that growth inhibition could be due
to a depletion of soluble factors by nonpolysialylated NCAM.
With or without PSA, NCAM is highly concentrated at cell-cell
contact sites, and the number of NCAM-positive cell-cell con-
tacts increased after PSA removal. Even at low densities, the
tumor cells never grow without any contact between each
other. This supports the view that removal of PSA enables
NCAM interactions at cell-cell contact sites. (v) Growth inhi-
bition and MAPK activation after PSA removal were abolished
by the NCAM-specic ligand C3d, which previously has been
shown to prevent the neuritogenic effect of trans-interacting
NCAM (39). Despite the nonspecic cytotoxicity of antibodies
that bind to the cell surface, short-term incubations with an
antibody against NCAM specically interfered with the MAPK
activation induced by endo-NE. Thus, two distinct NCAM-spe-
cic ligands were able to inhibit effects induced by endo-NE.
(vi) Incubations with membrane fractions containing PSA-
NCAM, nonpolysialylated NCAM or no NCAM at all, was
chosen as an approach to imitate cell-cell contacts as close to
the in vivo situation as possible. For a functional assay of
PSA-NCAM versus nonpolysialylated NCAM, the membrane
association appears particularly important, as differences in
polysialic acid content do not alter the binding properties of
solubilized NCAM (18). In line with the assumption of a con-
tact-dependent effect of NCAM, growth inhibition, MAPK
activation and MAPK-dependent differentiation were induced
by NCAM-positive but not by PSA-NCAM-positive or NCAM-
negative membranes. (vii) Membranes from PSA-NCAM-pos-
itive cells treated with endo-NE induced the same effects as
membranes derived from NCAM-positive, PSA-negative cells.
Thus, nonpolysialylated NCAM synthesized de novo or pro-
duced by endo-NE treatment was equally effective, i.e., PSA
expression either has no effect or a reversible effect on the
membrane representation of NCAM. (viii) NCAM-positive
membrane fractions induced growth inhibition and MAPK ac-
tivation of the PSA- and NCAM-negative LS cells. PSA-
NCAM positive membranes never induced any effect, indicat-
ing that PSA prevents the NCAM interactions in question.
Nevertheless, the NCAM-positive membranes were effective, if
applied on PSA-NCAM-positive cells and were able to mimic
all the effects observed after endo-NE treatment. The data
therefore provide direct evidence for heterophilic NCAM in-
teractions and strongly imply that heterophilic NCAM interac-
tions underlie the changes of cell growth and differentiation
induced by PSA removal.
Although the nature of these heterophilic NCAM interac-
tions remains to be resolved, the known cell surface binding
partners of NCAM can either be excluded or appear highly
unlikely to account for the effects of PSA removal. Interactions
of NCAM with L1 (21) cannot underlie the effects of endo-NE
treatment on TE671, as these cells are negative for L1 (Hilde-
brandt, unpublished data). Substantial evidence points towards
activating cis-interactions of NCAM with broblast growth fac-
tor receptors (3, 42). However, broblast growth factor, such as
BDNF and nerve growth factor, was mitogenic for SH-SY5Y
cells and thus induced the opposite effect of PSA removal or
NCAM exposure. NCAM binds to heparan sulfate proteogly-
cans (4) but this binding is promoted by the presence of PSA
(47), i.e., in contrast to the results of the current study removal
of PSA and NCAM exposure should have contrary effects. In
addition, NCAM binding to heparan sulfate proteoglycans is
engaged in cell-substrate rather than cell-cell interactions (3,
37) affecting the NCAM-bearing cell and not an NCAM-neg-
ative recipient cell as in the current study.
The rst indication for a potent heterophilic effect of NCAM
came from mutant mice, which produce only secreted forms of
NCAM and die during embryonic development (38). Trans-
interacting NCAM has been repeatedly reported to induce
growth inhibition, but the signicance of PSA for this process
has never been addressed (5). Notably, heterophilic interactions
of NCAM inhibit proliferation and promote differentiation of
hippocampal progenitor cells (1). Thus, the effects of PSA re-
moval or trans-interacting NCAM on neuroblastoma cells
were highly reminiscent to the stimulation of neural progeni-
tors with NCAM. In contrast to these trans-interactions with a
heterophilic NCAM binding partner on the recipient cell, the
neurite elongation and MAPK activation of PC12 cells and
cultured neurons after NCAM activation were assigned to ho-
mophilic NCAM-NCAM binding with NCAM as the neurito-
genic receptor (24, 25, 34) and the NCAM-dependent formation
of a signaling complex in pancreatic tumor cells is thought to
induce neurite outgrowth independent of cell-cell contacts (3).
In conclusion, our data strongly suggest that PSA acts as a
negative regulator of heterophilic NCAM signals at sites of
cell-cell contact, which after downregulation of PSA trigger the
cell to cease proliferation and to differentiate. The dynamic
regulation of PSA therefore provides the control over an in-
structive signal for tumor cell growth. The PSA-positive neu-
roblastoma and rhabdomyosarcoma cell lines will be important
to unravel the molecular mechanisms underlying the changes
in cell growth and differentiation after downregulation of PSA,
while the PSA- and NCAM-negative LS neuroblastoma cells
appear suited for searching heterophilic NCAM receptors in-
volved. Further unravelling the impact of PSA on NCAM sig-
nals will allow new insights into cell contact dependent growth
control and opens up new therapeutic options for PSA-positive
Ralph Seidenfaden and Andrea Krauter contributed equally to this
We thank U. Paulus for electron microscopy, S. Kustermann,
K. Marquart, and I. Ro¨ckle for cell culture work, K.-H- Herzog and
A. Schulz for BrdU immunostaining, E. Bock, N. Pedersen, V. Ma-
tranga, R. Handgretinger, M. Fukuda, A. Mu¨nster, and R. Michae-
lidis for cells and reagents, and M. Mu¨hlenhoff for critical com-
ments on the manuscript.
This work was supported by grants from the Deutsche Forschungs-
gemeinschaft and Fonds der Chemischen Industrie to H.H. and R.G.-S.
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    • "We report that berberine inhibits EMT process in neuroblastoma as evidenced by decreased MMP2 and MMP9 levels. PSA-NCAM is known to promote neural malignancy and stemness while its removal shows benefits (Seidenfaden et al., 2003M A N U S C R I P T 14 ERK1/2 MAPK promotes EMT by increasing the expression of EMT transcription factors and regulators of cell motility and invasion (Lamouille et al., 2014). Herein, we report berberine inhibits phosphorylation of RAS–RAF–ERK MAPK signalling pathway; thus inhibiting EMT in neuroblastoma. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: Berberine, a plant alkaloid, has been used since many years for treatment of gastrointestinal disorders. It shows promising medicinal use against metabolic disorders, neurodegenerative disorders and cancer; however its efficacy in neuroblastoma (NB) is poorly explored. Hypothesis: EMT is important in cancer stemness and metastasis resulting in failure to differentiate; thus targeting EMT and related pathways can have clinical benefits. Study design: Potential of berberine was investigated for (i) neuronal differentiation and cancer stemness inhibition, (ii) underlying molecular mechanisms regulating cancer-stemness and (iii) EMT reversal. Methods: Using neuro2a (N2a) neuroblastoma cells (NB); we investigated effect of berberine on neuronal differentiation, cancer-stemness, EMT and underlying signaling by immunofluorescence, RT-PCR, Western blot. High glucose-induced TGF-β mediated EMT model was used to test EMT reversal potential by Western blot and RT-PCR. STRING analysis was done to determine and validate functional protein-interaction networks. Results: We demonstrate berberine induces neuronal differentiation accompanying increased neuronal differentiation markers like MAP2, β-III tubulin and NCAM; generated neurons were viable. Berberine attenuated cancer stemness markers CD133, β-catenin, n-myc, sox2, notch2 and nestin. Berberine potentiated G0/G1 cell cycle arrest by inhibiting proliferation, cyclin dependent kinases and cyclins resulting in apoptosis through increased bax/bcl-2 ratio. Restoration of tumor suppressor proteins, p27 and p53, indicate promising anti-cancer property. The induction of NCAM and reduction in its polysialylation, indicates anti-migratory potential which is supported by down regulation of MMP-2/9. It increased epithelial marker laminin and smad and increased Hsp70 levels also suggests its protective role. Molecular insights revealed that berberine regulates EMT via downregulation of PI3/Akt and Ras-Raf-ERK signalling and subsequent upregulation of p38-MAPK. TGF-β secretion from N2a cells was potentiated by high glucose and negatively regulated by berberine through modulation of TGF-β receptors II and III. Berberine reverted mesenchymal markers, vimentin and fibronectin, with restoration of epithelial marker E-cadherin, highlighting the role of berberine in reversal of EMT. Conclusion: Collectively, the study demonstrates prospective use of berberine against neuroblastoma as elucidated through inhibition of fundamental characteristics of cancer stem cells: tumorigenicity and failure to differentiation and instigates reversal in the EMT.
    No preview · Article · Apr 2016 · Phytomedicine
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    • "The PSA modification is also involved in NCAM's effect on tumorigenesis but its role is discussed controversially. Depending on the tumor type, PSA seems either to reduce or to increase the tumorigenic potential [42,54,55]. Soluble NCAM forms are generated by different members of the disintegrin and metalloprotease (ADAM) family cleaving close to the plasma membrane resulting in an approximately 115 kDa fragment [18,19,102,103]. "
    [Show abstract] [Hide abstract] ABSTRACT: Cell adhesion molecules of the immunoglobulin (Ig) superfamily represent the biggest group of cell adhesion molecules. They have been analyzed since approximately 40 years ago and most of them have been shown to play a role in tumor progression and in the nervous system. All members of the Ig superfamily are intensively posttranslationally modified. However, many aspects of their cellular functions are not yet known. Since a few years ago it is known that some of the Ig superfamily members are modified by ubiquitin. Ubiquitination has classically been described as a proteasomal degradation signal but during the last years it became obvious that it can regulate many other processes including internalization of cell surface molecules and lysosomal sorting. The purpose of this review is to summarize the current knowledge about the ubiquitination of cell adhesion molecules of the Ig superfamily and to discuss its potential physiological roles in tumorigenesis and in the nervous system.
    Full-text · Article · Dec 2015 · Biology
    • "This is evident from the aberrant development of brain axon tracts in 2 2/2 4 2/2 mice, which is not seen in Ncam1 2/2 (N 2/2 ) or 2 2/2 4 2/2 N 2/2 triple knockout mice (Weinhold et al., 2005; Hildebrandt et al., 2009). Congruently, in vitro studies indicate that removal of polySia initiates NCAM interactions, which inhibit migration and promote neuron-like differentiation of neuroblastoma cells as well as the maturation of primary cultured precursors of the postnatal mouse SVZ into a calretinin-positive (CR 1 ) phenotype (Seidenfaden et al., 2003; R€ ockle et al., 2008; Eggers et al., 2011). As shown recently, removal of polySia by endosialidase injection into the lateral ventricles of adult mice causes a dispersed pattern of newborn, BrdU labeled CR 1 cells around the RMS indicating aberrant migration of precursors and premature differentiation (Battista and Rutishauser, 2010 ). "
    [Show abstract] [Hide abstract] ABSTRACT: The neurogenic niche of the anterior subventricular zone (SVZ) persistently generates neuroblasts, which migrate along the rostral migratory stream (RMS) into the olfactory bulb (OB), where they differentiate into granule and periglomerular cells. Loss of the neural cell adhesion molecule NCAM or its post-translational modification polysialic acid (polySia) impairs migration causing accumulations of cells in the proximal RMS and decreased OB volume. Polysialylation of NCAM is implemented by two polysialyltransferases, ST8SIA2 and ST8SIA4, with overlapping functions. Here, we used mice with Ncam1 and polysialyltransferase deletions to analyze how partial or complete loss of polySia synthesis or a combined loss of polySia and NCAM affects the RMS and the interneuron composition in the OB. Numerous calretinin-positive cells were detected dispersed around the RMS in Ncam1 knockout, St8sia2, St8sia4 double-knockout, and St8sia2, St8sia4, Ncam1 triple-knockout mice, as well as in St8sia2(-/-) but not in St8sia4(-/-) mice. These changes were not reflected by reductions of calretinin-positive cells in the granule or glomerular layer of the OB. Instead, calbindin-positive periglomerular interneurons were strongly reduced in all polySia-NCAM negative mice and slightly attenuated in St8sia2(-/-) as well as in the St8sia4(-/-) mice which were devoid of ectopic calretinin-positive cells along the RMS. Consistent with the early developmental generation of calbindin- as compared to calretinin-positive OB interneurons, this phenotype was fully developed at postnatal day 5. Together, these results demonstrate that the early development of calbindin-positive periglomerular interneurons depends on the presentation of polySia on NCAM and requires the activity of both polysialyltransferases. This article is protected by copyright. All rights reserved. © 2015 Wiley Periodicals, Inc.
    No preview · Article · Jul 2015 · Developmental Neurobiology
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