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&p.1:Abstract Recent studies have revealed that proteins
such as growth-associated protein 43 (GAP-43) and neu-
ron-specific enolase (NSE), believed for many years to
be expressed exclusively in neurons, are also present in
glial cells under some circumstances. Here we present an
overview of these observations. GAP-43 is expressed
both in vitro and in vivo transiently in immature rat oli-
godendroglial cells of the central nervous system, in
Schwann cell precursors, and in non-myelin-forming
Schwann cells of the peripheral nervous system. GAP-43
mRNA is also present in oligodendroglial cells and
Schwann cells, indicating that GAP-43 is synthesized in
these cells. GAP-43 is also expressed in type 2 astrocytes
(stellate-shaped astrocytes) and in some reactive astro-
cytes but not in type 1 astrocytes (flat protoplasmic as-
trocytes). These results suggest that GAP-43 plays a
more general role in neural plasticity during develop-
ment of the central and peripheral nervous systems. NSE
enzymatic activity and protein and mRNA have been de-
tected in rat cultured oligodendrocytes at levels compara-
ble to those of cultured neurons. NSE expression in-
creases during the differentiation of oligodendrocyte pre-
cursors into oligodendrocytes. In vivo, NSE protein is
expressed in differentiating oligodendrocytes and is re-
pressed in fully mature adult cells. The upregulation of
NSE in differentiating oligodendrocytes coincides with
the formation of large amounts of membrane structures
and of protoplasmic processes. Similarly, NSE becomes
detectable in glial neoplasms and reactive glial cells at
the time when these cells undergo morphological chang-
es. The expression of the glycolytic isozyme NSE in
these cells, which do not normally contain it, could re-
flect a response to higher energy demands. This expres-
sion may also be related to the neurotrophic and neuro-
protective properties demonstrated for this enolase iso-
form. NSE activity and protein and mRNA have also
been found in cultured rat type 1-like astrocytes but at
much lower levels than in neurons and oligodendrocytes.
Thus GAP-43 and NSE should be used with caution as
neuron-specific markers in studies of normal and patho-
logical neural development.
&kwd:Key words Growth-associated protein 43 · Neuron-
specific enolase · Central nervous system · Peripheral
nervous system · Glia
Abbreviations bFGF Basic fibroblast growth factor ·
CNS Central nervous system · GAP-43 Growth-
associated protein 43 · GC Galactocerebroside ·
GFAP Glial fibrillary acidic protein · MAP microtubule-
associated protein · MBP Myelin basic protein ·
NSE Neuron-specific enolase · PNS Peripheral nervous
system&bdy:
Introduction
During the development of the nervous system changes
in the expression of neuron-specific markers are indica-
Monique Sensenbrenner (
✉
) · Jean-Christophe Deloulme
Laboratoire de Neurobiologie Ontogénique, CNRS ERS 110,
Centre de Neurochimie, 5 rue Blaise Pascal,
F-67084 Strasbourg Cedex, France
Marguerite Lucas
Biochimie Cellulaire, CNRS UPR 9065, Collège de France,
Paris, France.
J Mol Med (1997) 75:653–663 © Springer-Verlag 1997
REVIEW
&roles:Monique Sensenbrenner · Marguerite Lucas
Jean-Christophe Deloulme
Expression of two neuronal markers, growth-associated protein 43
and neuron-specific enolase, in rat glial cells
&misc:Received: 27 January 1997 / Accepted: 9 May 1997
MONIQUE SENSENBRENNER
received the degree of Docteur
és Sciences from the Universi-
ty of Strasbourg, France. She
is presently Directeur de Re-
cherche at CNRS and Chief of
a CNRS Unit (Laboratoire de
Neurobiologie Ontogénique)
at the Centre de Neurochimie
du CNRS in Strasbourg. She
has been a pioneer in her stud-
ies on neural cell cultures, and
her major research interests
concern the effects and mech-
anisms of action of growth
factors on neural cell develop-
ment.&/fn-block:
tive of neuronal cell differentiation and maturation.
However, recent findings from several laboratories, in-
cluding our own, have demonstrated that neuron-specific
proteins such as the growth-associated protein 43 (GAP-
43) and the neuron-specific enolase (NSE) are not con-
fined to neurons but may occur also in glial cells.
GAP-43, a membrane-associated neuron-specific phos-
phoprotein, was first identified as a protein whose ex-
pression is associated with neuronal development and
neurite outgrowth in the nervous system. Proteins similar
to GAP-43 were later discovered in several laboratories,
and molecular cloning has confirmed that GAP-43 is
identical to neuromodulin, protein P-57, B-50, pp46, and
F1 [1, 2] Expression of GAP-43 has been described for
many years to occur exclusively in neurons both in vivo
[1] and in vitro [3, 4]. Moreover, it has been shown that
this protein is synthesized in the neuronal somata, rapid-
ly transported throughout axons, and accumulates in
growth cones and presynaptic terminals. It has also been
demonstrated that GAP-43 levels remain high during
neuronal development; its expression then declines
markedly following the establishment of final synapto-
genesis [1]. Some mature neurons, however, continue to
express high amounts of GAP-43 throughout adult life
[5]. Moreover, GAP-43 is reexpressed in adult neurons
following nervous system damage and during regenera-
tion processes [6]. GAP-43 was therefore thought to be
involved in the initiation of neurite outgrowth, axonal
elongation, axonal regeneration, synaptic plasticity and
somehow in neurotransmitter release [7, 8].
The notion of GAP-43 as a neuron-specific protein
has been questionable since recent findings from our lab-
oratory and from others have shown that GAP-43 expres-
sion also occurs in nonneuronal cells under some cir-
cumstances. The presence of GAP-43 has been demon-
strated in various glial cells in vitro and in vivo (see
“GAP-43 and glial cell development”). More recent find-
ings indicate that GAP-43 may even be expressed tran-
siently in nonneural cells, such as myoblasts [9, 10].
In 1972, it was demonstrated that the accumulation of
the 14-3-2 protein is a regionally selective process which
can be related to the differential maturation of the CNS
[11]. Later it was shown that the 14-3-2 protein is identi-
cal to the γ subunit of the enolase enzyme [12].
Enolase (2-phospho-
D-glycerate hydrolase, EC
4.2.1.11) is a glycolytic enzyme active as a dimer. In
higher vertebrates these dimers are composed of three
subunits α, β, and γ encoded by different genes. The αα
homodimer is found predominantly in immature cells
and remains expressed in most adult tissues, including
brain. During ontogenesis a switch towards specific iso-
forms occurs, from αα to αβ and ββ in the striated mus-
cle, and from αα to αγ and γγ in the nervous system,
specifically in the neuronal lineage [13]. The functional
significance of this switch towards specific isoforms in
two cell types with high energy demands is not clear.
Two nonexclusive hypothesis can be proposed. First, the
regulation of expression of each enolase subunit can be
specifically controlled at the transcriptional and/or post-
transcriptional levels and thereby adapted to the specific
energy demands of different cell types. Second, the pro-
teins themselves may have specific characteristics lead-
ing them to optimal functioning in their respective envi-
ronmental conditions. It has been shown, for example,
that γγ enolase is more resistant than αα enolase to dena-
turation by a high concentration of chloride ions as can
be found in active neurons. The appearance and expres-
sion of NSE (αγ + γγ enolases) are correlated directly
with structural and functional differentiation processes of
the central nervous system (CNS). Moreover, a net in-
crease in γ enolase gene product is related to the differ-
entiation and the synaptic activity of neuronal cells [14].
In adult tissues high levels of γ enolase are present
only in neurons and neuroendocrine cells. However, ex-
pression of γ enolase is less restricted than previously re-
ported since sensitive immunoassays have demonstrated
significant levels of γ enolase in differentiated tissues
other than nervous tissues [15, 16]. It has been shown
that early rat embryos contain a nonnegligible amount of
γ enolase before the formation of neural tissues [17], and
it has been suggested that γ enolase is rather common in
immature cells [18]. Numerous tumors of cells originat-
ing from various tissues express the γ enolase gene. Con-
cerning tumors of neural origin, glioblastoma, astrocyto-
ma, oligodendroglioma, and schwannoma contain NSE.
This enzyme has also been found in reactive astrocytes.
Recently the presence of NSE has been described in nor-
mal glial cells in vitro and in vivo (see “NSE and glial
cell development”). Neurons in culture express αα, αγ,
and γγ enolases, and the neuron specific isoforms accu-
mulate as differentiation proceeds with time in culture
[19–22].
This review summarizes the recent findings that re-
port on regulation of the expression of GAP-43 and NSE
in various glial populations from the central (CNS) and
peripheral (PNS) nervous systems during in vitro and in
vivo development and discusses the functional signifi-
cance of these observations. The review is divided into
three sections. First, we provide some information on the
various types of vertebrate glial cells in vivo and in cul-
ture. We then present results concerning the expression
of GAP-43 in glial cells. Finally, we address the issue of
the presence of NSE in glial cells.
Glial cells in vivo and in culture
The two major macroglial cell types in the vertebrate
CNS are astrocytes and oligodendrocytes [23]. Fibrous
astrocytes have long, relatively straight processes with
few branches. They have numerous gliofilaments distrib-
uted throughout their cytoplasm. Protoplasmic astrocytes
have thick, extensively branched processes. Their cyto-
plasm contains relatively few gliofilaments. Fibrous as-
trocytes predominate among the myelinated axons in the
white matter, while protoplasmic astrocytes are com-
monly found among the neuronal cell bodies in the gray
matter.
654
During development of the rat and mouse brain astro-
glial precursor cells are generated primarily from the
subventricular zone in the early embryonic and postnatal
periods. Some astrocytes also develop from radial glial
cells, which arise very early in development from the
ventricular zone but are transitory and act as a guiding
substrate for the migration of neurons. Oligodendroglial
precursor cells also arise prenatally from the subventric-
ular zone and proliferate actively during the first 2 weeks
after birth. Both types of precursors then migrate into the
neighboring gray and white matter where they differenti-
ate into astrocytes and oligodendrocytes. In the white
matter oligodendrocytes are closely associated with mye-
linated fibers. Astroglial and oligodendroglial cells are
able to proliferate during the whole postnatal life and
even in aged animals.
Schwann cells are the predominant glial cell type of
the vertebrate PNS and derive from neural crest cells
during the embryonic period [23]. Two distinct subtypes
of Schwann cells have been characterized, myelinating
and nonmyelinating. However, the phenotype of these
cells depends on their association with neurites and un-
der certain experimental conditions can be changed; non-
myelin-forming Schwann cells can become myelin-form-
ing Schwann cells and vice versa.
Many functions have been attributed to astroglial cells
[23]. They are considered to be the supporting cells in
the nervous tissue. They interact with other neural cells
and respond to chemical signals from neurons by releas-
ing molecules such as neuroactive and neurotrophic sub-
stances. Astrocytes also play a role in neural develop-
ment and regeneration processes by promoting neuronal
migration and neurite outgrowth. An important role of
astroglial cells is contributing to the homeostasis of the
extracellular environment. Finally, astrocytes respond to
brain injury, become activated, change their shape, and
transform into reactive fibrous astrocytes. Such reactive
gliosis occurs also under several neuropathological con-
ditions.
For oligodendrocytes and myelinating Schwann cells
a functional role is clearly defined, consisting in myelin-
ation. During this process the cells produce high quanti-
ties of plasma membrane and insert into the lipid matrix
the myelin-specific proteins that participate in the orga-
nization and maintenance of the myelin sheath. Schwann
cells maintain the ability to dedifferentiate after nerve in-
jury and reenter the cell cycle, providing new Schwann
cells for the regeneration process [23].
In culture two populations of astrocytes, the epithelio-
id flat type 1 astrocyte and the stellate type 2 astrocyte,
can be distinguished on the basis of morphological, anti-
genic, and functional criteria [24]. These type 1 and type
2 astroglia have been suggested to reflect protoplasmic
and fibrous astrocytes of the brain, respectively. More-
over, in vitro observations indicate that bipotential oligo-
dendrocyte-type 2 astrocyte precursor cells, termed O-
2A progenitors (characterized by the monoclonal anti-
body A2B5 that binds to polysialogangliosides) differen-
tiate into oligodendrocytes when cultured in a serum-
free, chemically defined medium [24]. On the other
hand, these glial progenitor cells develop into type 2 as-
trocytes when maintained in the presence of serum. The
flat, polygonal-shaped type 1 astrocytes appear to derive
from a different lineage than the O-2A lineage and can
be cultured in either the presence or the absence of se-
rum [25, 26].
Astroglial and oligodendroglial cell cultures are ob-
tained from newborn rat cerebral hemispheres [25–27].
The astrocytes (type 1 and type 2 astrocytes) are charac-
terized by glial fibrillary acidic protein (GFAP) immuno-
reactivity. The progressive developmental stages of oli-
godendroglial cells can be followed by immunostaining
against specific surface markers, such as A2B5, a marker
of 0-2A precursors; O4, a marker of immature oligoden-
drocytes binding to surface sulfatide; and galactocere-
broside (GC) and myelin basic protein (MBP), markers
of mature oligodendrocytes.
Schwann cells, in contact with sensory neurons, are
present in cultures of dissociated adult rat dorsal root
ganglia [28]. Pure cultures of Schwann cells deprived of
axonal contact are obtained from 1- to 5-day-old rat pe-
ripheral sciatic nerves [29, 30]. The Schwann cells can
be characterized by S100 protein immunoreactivity.
GAP-43 and glial cell development
CNS macroglial cells and GAP-43
The expression of a protein with a similar molecular
weight and isoelectric point as GAP-43 in macroglial
cells was first mentioned by the group of Vitkovic in
1987 [31]. This protein was found to be associated with
the plasma membranes of both the somata and cellular
processes of cultured rat cortical type 1 astrocytes. Fur-
ther in vitro studies of this group confirmed the presence
of GAP-43 in cultured astrocytes [32, 33]. In addition,
these cells also express GAP-43 mRNA, indicating that
GAP-43 protein is synthesized rather than taken up by
the astrocytes [34]. These authors point out that GAP-43
expression decreases in type 1 astrocytes with time in
culture. After 2 weeks these glial cell type contains little
if any GAP-43 protein and mRNA.
We have also undertaken in vitro investigations to an-
alyze in more detail the presence and the regulation of
GAP-43 expression in both astrocytes and oligodendro-
glial cells derived from rat brain during their differentia-
tion in culture. Immunocytochemical observations indi-
cate that GAP-43 is present in oligodendroglial cells and
type 2 astrocytes but is absent in type 1 astrocytes [35].
The expression of GAP-43 protein and mRNA in oligo-
dendroglial cells and their absence in protoplasmic astro-
cytes have been confirmed by western and northern blot
analysis [35]. In addition, we have demonstrated that
principally the O-2A bipotential glial precursors (mor-
phologically immature A2B5 positive cells) express
GAP-43 protein, that GAP-43 is also expressed in O4
(sulfatide) positive cells, corresponding to a later devel-
655
opmental stage of the oligodendroglial precursor cells
(Fig. 1), and that cells which acquire a typical oligoden-
drocyte morphology and begin to express the GC antigen
become progressively GAP-43 negative. The precursor
glial cell cultures also show the presence of GAP-43
mRNA, indicating that this protein is synthesized early
in the oligodendrocyte lineage [35]. In the presence of
basic fibroblast growth factor (bFGF), a growth factor
known to enhance the multiplication of oligodendroglial
cells, the proliferation of the GAP-43 positive glial pre-
cursors is stimulated, and the levels of GAP-43 protein
and mRNA are increased [36].
The group of Vitkovic [37] have discussed a constitu-
tive expression of GAP-43 in cultured oligodendrocytes.
Other authors, however, have observed that GAP-43 is
expressed in cultured O-2A progenitor cells but seldom
in GC positive and never in MBP positive mature cul-
tured oligodendrocytes [38]. Moreover, an in vivo immu-
nohistochemical study performed by the latter group on
sections of rat brain demontrated a similar regulation of
GAP-43 in oligodendroglial cells during development.
Indeed, GAP-43 immunoreactivity was seen to be locat-
ed only in the membranes of immature oligodendroglial
cells, and no GAP-43 was found in mature oligodendro-
cytes [38].
Taken together these in vitro and in vivo data indicate
that GAP-43 is associated principally with glial precur-
sor cells, and that its expression characterizes an early
differentiation stage of the oligodendroglial cell lineage
and is lost when these cells differentiate into more ma-
ture oligodendrocytes. Such a transient expression of
GAP-43 has also been described for other nonneuronal
cells belonging to the muscle cell lineage [9].
Biochemical characterization of the GAP-43 has re-
vealed both similarities and differences between the
GAP-43 protein of cultured oligodendroglial and cul-
tured neuronal cells [35]. While three distinct protein
spots are resolved for neurons in two-dimensional gel
electrophoresis, the oligodendroglial GAP-43 migrates
as a single protein spot, with an isoelectric point identi-
cal to that of the more basic form of the neuronal pro-
tein. Moreover, the GAP-43 form of oligodendroglial
cells shows a lower electrophoretic mobility than the
neuronal GAP-43 forms, which may arise from varia-
tions of protein conformation and/or posttranslational
modifications.
Finally, we have also found that GAP-43 immunore-
activity remains in O-2A progenitor cells which differen-
tiate into type 2 astrocytes in serum-containing medium
(Fig. 2) [36], in agreement with the in vitro observations
of Vitkovic’s group [34, 37] and Wilkin’s group [38].
The identification of type 2 astrocytes in tissue sections
has been difficult and controversial. However, it has been
suggested that reactive astrocytes in the damaged hippo-
campus have the phenotypic features of type 2 astrocytes
and express GAP-43 [39]. By contrast, another group has
reported that normal and reactive astrocytes in brain sec-
tions never show GAP-43 immunoreactivity [38]. These
in vivo results should be taken with caution, and further
experiments are required to clarify such contradictory
data.
Schwann cells and GAP-43
Schwann cells, which are the functional counterparts of
oligodendrocytes in the PNS also express GAP-43 both
in vitro and in vivo. The presence of GAP-43 in
Schwann cells, however, has raised controversy. An in
vitro study reported that Schwann/satellite cells grown
together with sensory neurons in dorsal root ganglion
cell cultures express low levels of GAP-43 during the
first 8 days of culture [28]. In this mixed neuronal-glial
culture system the possibility could not be eliminated
that the glial cells might take up the protein from the
neurons. Further in vitro studies have demonstrated that
about 25–40% of Schwann cells cultured in the absence
of neurons can synthesize GAP-43, which is identical to
the neuronal protein [29, 30]. Moreover, cultured
656
Fig. 1a, b Double-immunofluorescence staining of oligodendro-
glial cells grown for 4 days under serum-free condition in second-
ary culture. (The same microscopic fields are shown in a and b.) a
GAP-43 TRITC immunofluorescence. b O4 FITC immunofluores-
cence. Note that all O4 positive oligodendroglial cells are also
GAP-43 positive. Arrows, two GAP-43 positive glial precursor
cells which are O4 negative. Bar=10 µm&/fig.c:
Schwann cells have also been shown to express GAP-43
mRNA [30, 40].
Several in vivo observations support the results of the
in vitro studies. GAP-43 is detectable in all Schwann cell
precursors in developing peripheral nerves [29]. More-
over, this protein is expressed in most mature non-my-
elin-forming Schwann cells [29, 30]. By contrast, adult
myelin-forming Schwann cells are not normally immu-
noreactive for GAP-43 but begin to synthesize GAP-43
only several weeks following peripheral nerve injury [29,
30]. Furthermore, expression of GAP-43 mRNA is high
in Schwann cells of degenerating peripheral nerves [30,
40]. Other authors have shown that non-myelin-forming
Schwann cells present on adult skeletal muscle motor
endplates are not GAP-43 immunoreactive [41]. These
cells, however, become positive for a GAP-43 mRNA in
situ hybridization signal and are GAP-43 immunoreac-
tive shortly after denervation of the muscles by either a
cut or crush lesion of the sciatic nerve [41]. A parallel
has been noted between GAP-43 expression and the
elaboration by denervated Schwann cells of long fine
processes and branches. After reinnervation occurs, the
Schwann cell processes disappear, and the cells loose
GAP-43 immunoreactivity. This indicates that GAP-43
expression in Schwann cells is state dependent, and in
this respect it resembles neuronal expression of the pro-
tein.
The developmental expression of GAP-43 in rat glial
cells of the CNS and PNS is summarized in Table 1.
657
Fig. 2a, b Double-immunoflu-
orescence staining of type 2 as-
trocytes grown for 1 week in
the presence of serum in sec-
ondary culture. (The same mi-
croscopic fields are shown in a
and b.) a GAP-43 FITC immu-
nofluorescence. b GFAP
TRITC-immunofluorescence.
Note that the GFAP-positive
astrocytes are also GAP-43
positive. Bar=10 µm&/fig.c:
Table 1 Rat glial cells: expres-
sion of GAP-43: comparison
of in vitro and in vivo results.
(M myelinating Schwann cells,
Non-M nonmyelinating
Schwann cells, R reactive
Schwann cells; n.d. not deter-
mined)&/tbl.c:&tbl.b:
Develop- Glial cell type
mental stage
Astrocytes Oligodendrocytes Schwann cells
Type 1 Type 2 (in vitro)
or reactive (in vivo)
Precursors Culture ± + + +
In vivo n.d. n.d. n.d. +
Immature Culture ± + + +
cells In vivo n.d. n.d. + +
Mature Culture – + – +
cells In vivo – +/− – M Non-M R
− +/− +
&/tbl.b:
NSE and glial cell development
NSE in neoplastic glial cells and reactive astrocytes
Studies performed a number of years ago revealed that
many types of CNS and PNS tumors of glial origin such
as glioblastoma, astrocytoma, oligodendroglioma, and
schwannoma contain the γ subunit of the glycolytic en-
zyme enolase [42–45]. However, no relationship between
the presence of γ enolase and cellular anaplasia has been
found. The putative cell type from which these tumors
originate was found at that time not to contain demon-
strable NSE. All these findings which indicate that γ
enolase is detected not only in neuronal-derived tumors
but also in glial neoplasms demonstrate that this protein
can no longer be regarded as a neuron-specific marker in
neuropathology. Indeed, clinical interest has focused on
this enolase isoform over the years as a possible marker
antigen of some neuronal diseases. However, NSE im-
munohistochemistry is still a valuable method in neuro-
nal and glial tumor diagnosis and for evaluating tumor
metabolic activity.
In addition, nonneoplastic reactive astrocytes adjacent
to and within CNS tumors have been demonstrated often
to be NSE positive [42]. Moreover, reactive astrocytes in
the adult brain after injury also become NSE positive
[46]. It has been hypothesized that for cells normally
containing only ubiquitous enolase (αα enolase) the
change to a neoplastic or a reactive process necessitates
an increase in glycolytic activity, and therefore that NSE
must additionally be synthesized in the altered glial cell
to adapt to the new metabolic demands [47]. Further-
more, it has been suggested that glial cells in response to
injury may reexpress fetal characteristics of progenitors.
This response may represent a reversion to a common or-
igin of glial cells/neurons. This putative conversion to
early developmental stages may be favorable for repair
and regeneration processes through the neurotrophic ef-
fect of γ enolase (see “Discussion”).
NSE in normal glial cells
An in vivo study performed by Vinores et al. in 1984
[48] reported that fibrillary astrocytes in the white mat-
ter of rat and mouse cerebrum and cerebellum contain
NSE, while no other nonneuronal cells in the brain ex-
hibit positive immunostaining for NSE. Another investi-
gation later demonstrated that Müller cells in the retina
of Anura are immunolabeled with an antihuman NSE se-
rum [49].
We recently undertook studies to examine the pres-
ence of NSE in rat brain glial elements and to analyze
the regulation of its expression at various steps of their
differentiation/maturation stages [50, 51]. We showed
that NSE, both αγ- and γγ dimer forms, is expressed in
rat cultured oligodendrocytes. Both NSE enzymatic ac-
tivity and γ enolase protein and mRNA are detected in
these glial cells at levels similar to those of cultured neu-
rons (Figs. 3–5). We have also demonstrated that NSE is
expressed in oligodendroglial cells only at a certain stage
of differentiation both in vitro and in vivo [50]. Indeed,
NSE immunoreactivity is never seen in the cultured O-
2A precursors, which are A2B5 immunopositive. NSE is
expressed only in developing oligodendrocytes that pres-
ent GC and MBP immunostaining (Fig. 6). The immuno-
reactivity has been found to be localized mostly in the
cytoplasm, as expected for a glycolytic enzyme. The lev-
els of NSE protein and mRNA are high in oligodendro-
cytes but low in O-2A progenitor cells (Fig. 5).
We have also shown that in the presence of bFGF,
when the proliferation of O-2A precursor cells is stimu-
lated and their differentiation into oligodendrocytes re-
tarded [27], the amounts of γ enolase protein and mRNA
remain low in these oligodendroglial cell cultures. By
658
Fig. 3 Specific activities of NSE (γγ enolase) in cultured neurons,
astrocytes, and oligodendrocytes at various times of culture. d,
Days; w, weeks. Values are mean±SEM of 7–10 determinations&/fig.c:
Fig. 4 Western blot analysis of NSE in protein extracts from adult
rat brain (Br; 150 µg protein), 6-day-old oligodendrocyte cultures
(OL; 400 µg protein), 3-week-old astrocyte cultures (As; 400 µg
protein), and 4-day-old neuronal cell cultures (N; 80 µg protein).
Last lane, sample of purified γγ enolase (NSE; 0.5 µg protein).
The γ enolase subunit was detected by a rabbit anti-rat NSE poly-
clonal antibody (1:5000)&/fig.c:
contrast, treatment with bFGF has no effects on α eno-
lase protein or mRNA levels [50]. Thus bFGF prevents
specifically γ gene expression in cultured oligodendro-
glial cells, which is probably due to the fact that this fac-
tor delays the differentiation of these cells into oligoden-
drocytes. The cultures have been used only over a short
period of time (4 days), and therefore the possibility
cannot be excluded of a transient expression of NSE in
these oligodendroglial cells in vitro, as has been found
for oligodendroglial cells in vivo. Indeed, we found NSE
highly expressed in oligodendroglial cells of 8-day-old
rat brain, while it is no longer detected in adult rats
by day 60 [50]. All these data indicate that γ enolase
gene expression is associated with differentiation of the
oligodendrocytes and is repressed in fully mature adult
cells.
Biochemical analysis by two-dimensional gel electro-
phoresis has revealed a heterogeneity of γ enolase sub-
unit, consisting in the identification of six distinct pro-
tein spots, which is strictly the same in cultured oligo-
dendrocytes and in adult rat brain [50]. This indicates
that the same posttranscriptional modifications of γ sub-
unit occur in neurons in vivo and in oligodendroglial
cells in vitro.
We have also shown that NSE activity, γ enolase pro-
tein, and mRNA are present in cultured rat brain type
1 astrocytes [51]. However, NSE levels in this glial
cell type are much lower than those in cultured neurons
and oligodendrocytes (Figs. 4, 5). One of us (M.L. in
collaboration with Y. Berwald-Netter’s group) also ob-
served the presence of NSE mRNA in cultured mouse
type 1 astrocytes. On the other hand, type 2 astrocytes,
which develop in culture from the O-2A progenitor cells
in the presence of serum, have not been found to express
NSE.
659
Fig. 5 NSE mRNA expression studied by northern blot analysis
of total RNA (15 µg per sample) in cultures of neurons (N; 4-day-
old), O-2A precursors (P; 2-day-old secondary culture), oligoden-
drocytes (O; 6-day-old secondary culture), and astrocytes (A; 3-
week-old). The same blot was rehybridized with an oligonucle-
otide probe for 18S ribosomal RNA, confirming that equal
amounts of total RNAs were used in all samples (not shown)&/fig.c:
Fig. 6a, b Double-immunoflu-
orescence staining of rat brain
oligodendrocytes grown under
serum-free conditions for 4
days in secondary culture. (The
same microscopic fields are
shown in a and b). a NSE
DTAF-immunofluorescence. b
MBP TRITC-immunofluores-
cence. Note that all MBP posi-
tive oligodendrocytes are also
NSE positive. Bar=10 µm.
(From [50])&/fig.c:
Discussion
For many years GAP-43 and NSE were thought to have a
restricted neuron-specific expression. However, this view
has now been questioned, particularly by in vitro studies
and by a few in vivo reports summarized in this review,
showing the expression of these proteins in various types
of central and peripheral glial cells, such as differentiat-
ing astrocytes, oligodendrocytes, and Schwann cells. We
have performed investigations preferentially on oligo-
dendroglial cells, and a transient expression of both
GAP-43 and NSE has been found during the in vitro and
in vivo development of these cells. Figure 7 presents
schematic representation of the expression of these two
proteins in developing oligodendrocytes. GAP-43 and
NSE gene expressions are regulated differentially. In-
deed GAP-43 is downregulated while NSE is upregulat-
ed during the differentiation of oligodendrocyte precur-
sors into oligodendrocytes. Moreover, it should be point-
ed out that the time courses of expression of each of
these proteins are similar in culture and in vivo.
Some investigators have mentioned other examples of
proteins, such as microtubule-associated protein 2 (MAP
2) and neurofilament (NF) protein M, normally found in
neurons and which are in fact also expressed in glial
cells under certain circumstances. Kelly et al. [52] have
reported that myelin-forming Schwann cells of the rat
sciatic nerve in culture express NF-M at a very early
stage of their development. Other authors have found de-
tectable levels of the high molecular weight isoforms of
MAP 2 in specific populations of normal rat optic nerve
astrocytes [53]. Furthermore, reactive astrocytes after
brain injury have been described to contain the low mo-
lecular weight isoform MAP 2c [54]. More recently a
transient expression of a MAP 2c isoform in cultured oli-
godendrocyte precursors has been described [55]. All
these data demonstrating that neuron-specific proteins
can also be expressed in vitro and in vivo by glial cells in
the CNS and PNS have raised new questions about the
role of these proteins.
In both neuronal and glial cells GAP-43 is preferen-
tially expressed in vitro and in vivo during early develop-
ment, and its level dramatically declines late in develop-
ment [1, 29, 36, 38]. Moreover, in glial cells this protein
is expressed at very restricted developmental stages [29,
36, 38]. It is not known, however, whether the upregula-
tion or the downregulation of the GAP-43 gene within
neuronal and glial cell types uses the same regulatory
mechanisms.
In neurons of the CNS as well as of the PNS, GAP-43
expression increases during periods of growth cone ac-
tivity and axonal outgrowth both in vivo and in vitro [1,
3, 4]. The highest amounts of expression occur in vitro
during axonal growing and in vivo when branching neu-
rites establish stable synaptic formations. Thereafter
GAP-43 expression decreases sharply in the cell body of
cultured neurons and in both the cell bodies and the neu-
rites of most neurons in vivo. However, in several brain
areas which retain the capacity to undergo synaptic reor-
ganization some mature neurons continue to express
GAP-43 at high levels throughout adult life. High ex-
pression of GAP-43 also has been associated with re-
growth of injured neurites as regeneration procedes [6].
Thus GAP-43 may play a key role in regulating mem-
brane dynamics during neurite growth and synaptic mod-
ulation and has been considered as a plasticity protein in-
volved in neuronal shaping and repair [6].
This hypothesis has been confirmed by recent find-
ings. An in vivo study has shown that overexpression of
GAP-43 in adult neurons of transgenic mice induces
both spontanous nerve sprouting and the formation of
new synapses and promotes massive sprouting after inju-
ry [56]. Another in vivo investigation observed that mice
which lack GAP-43 display abnormal axonal pathfinding
during early development [57]. Finally, in vitro experi-
ments show that cultured neurons depleted of GAP-43
by an antisense oligonucleotide approach extend only
thin, unramified neurites with very small growth cones
adhering poorly and displaying unstable lamellar exten-
sions [58].
Other functions have been ascribed to GAP-43 in neu-
ronal cells, such as to modulate neurotransmitter secre-
tion [8].
In oligodendrocytes and Schwann cells, as in neurons,
the expression of both GAP-43 and NF-M intermediate
filaments and MAP-2 coincides with developmental
stages of major morphological changes of these cells,
consisting in the formation of extensive branches and of
elevated motile activity [1, 29, 36, 52, 55]. GAP-43 is
also expressed in cultured type 2 astrocytes [36] and in
hippocampal reactive astrocytes, when large and long
processes are formed, and this protein may contribute to
an increase in the motility of these hypertrophic astro-
cytes [39]. By contrast, type 1 astrocytes in culture lack
long processes and express little if any GAP-43. All
these observations indicate that GAP-43 distribution in
glial cells is correlated with the formation of cytoplasmic
processes and with sites of new membrane synthesis.
660
Fig. 7 Schematic representation of GAP-43 and NSE expression
during the developmental stages of rat oligodendroglial cells in
culture and in vivo. A2B5, a marker of O-2A precursors; O4, a
marker of immature oligodendrocytes binding to surface sulfa-
tides; galactocerebroside (GC) and myelin basic myelin (MBP),
markers of mature oligodendrocytes&/fig.c:
Similarly, transfection experiments have shown that the
expression of GAP-43 in nonneural cells causes changes
in their shape, associated with the formation of long and
thin processes [59, 60]. Thus GAP-43 appears to be a
protein in glial cells involved in the regulation of their
shape, and which plays a role in their motility at specific
stages of development. This is consistent with the pro-
posed function of GAP-43 in neuronal morphological
changes.
The observations showing the presence of GAP-43 in
neurons and in glial cells of both the CNS and the PNS
and the findings indicating changes in the amounts of
GAP-43 expression during development and regenera-
tion suggest that GAP-43 is probably a multifunctional
protein. It has been proposed that some of its functions
are specific to neurons while some others are associated
with various types of morphological remodeling in both
neurons and glial cells.
It has been demonstrated that levels of NSE are very
low in embryos and at birth and rapidly increase con-
comitantly with morphological and functional matura-
tion of neurons in vivo [14, 61] and in vitro [19, 20, 22,
62, 63]. Similarly, the expression of NSE seems to be
linked to the process of glial cell, astroglia and oligoden-
droglia, differentiation [50, 51]. Moreover, relatively ma-
ture cultured neurons and oligodendrocytes which no
longer proliferate express high amounts of NSE. The ex-
pression of NSE is therefore indicative not only of neu-
ronal maturation but also of the relative degree of oligo-
dendroglial cell differentiation.
In vivo a transient expression of NSE has been found
in oligodendroglial cells at a period corresponding to the
beginning of myelination when these cells must to form
a large amount of membranous structures. It has been
suggested that higher levels of NSE reflect more intense
metabolic activity of neurons [64]. Therefore the expres-
sion of the glycolytic isozyme γ enolase in oligodendro-
cytes which do not normally contain it, could reflect a
response to higher energy demands occurring in these
cells. In culture, the upregulation of NSE coincides with
the formation of the protoplasmic processes, suggesting
again that increased glycolytic activity takes place as an
adjustement to new metabolic demands. The same has
been hypothesized for glial cells that change to neoplas-
tic glial cells or to reactive astrocytes and begin to ex-
press NSE [42, 46, 47].
Concerning the hypothetical physiological signifi-
cance of γ enolase expression in all these cells it is worth
noting that, as it is the case of many other glycolytic en-
zymes [65, 66], γγ enolase has another function in addi-
tion to its enzymatic activity. Indeed, γγ enolase but not
αα enolase has neurotrophic and neuroprotective effects
on a rather broad spectrum of neurons [67, 68]. Increas-
ing levels of γ enolase are found in cerebrospinal fluid
after cerebrovascular accidents, brain tumors, and Creuz-
feld-Jakob disease (see [68]), and this can be helpful in
promoting the survival of injured neurons. Furthermore,
NSE shows neuroprotective action on neurons cultivated
in a low-oxygen atmosphere [68]. The presence of this
protein in reactive astrocytes surrounding tumors or in-
farcts may also support survival of neurons. It is not
known whether γ enolase can be secreted, but it is of in-
terest to mention that it is present in nonnegligible levels
in the intact retinal extracellular matrix [69]. Thus NSE
may play an important role during brain development as
well as during repair of injured neurons in the adult
CNS.
Finally, unraveling how specific brain proteins are ex-
pressed and regulated in neural cells is of great interest
for understanding normal and disturbed brain develop-
ment and function. The data presented in this review un-
derline the difficulty of defining specific neural markers,
since they can be expressed transiently in different cell
types during development. GAP-43 and NSE are clearly
not exclusively neuron-specific proteins, but they proba-
bly play a fundamental role in neuronal development and
regeneration as well as a critical function in normal and
pathological glial development.
&p.2:Acknowledgements The work of the authors was supported by
grants from the French Centre National de la Recherche Scientifi-
que, Institut National de la Santé et de la Recherche Médicale,
Association pour la Recherche sur le Cancer, Fondation pour la
Recherche Médicale, and Association Française contre les Myo-
pathies. The authors acknowledge the collaboration of Drs. J.
Baudier, F. Eclancher, T. Janet, G. Labourdette, P. Laeng, and M.
Ledig; of Mrs. A. Helies and M.F. Knoetgen; and of Mr. P. Bouil-
lon and C. Gaber. We thank Dr. A. Keller for critical reading of
the manuscript, Mrs. C. Orphanides for excellent secretarial work,
and Mr. J.C. Barthe for skillful assistance with the photomicro-
graphs.
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