From the
Recent studies showed that the Alzheimer amyloid precursor (APP)
occurs as the core protein of a chondroitin sulfate proteoglycan
(appican) in C6 glioma cells. In the present study we show that appican
is present in both human and rat brain tissue. Cortical rat brain cell
cultures were used to identify appican-producing cells. Soluble
secreted and cell-associated appican was produced by mixed glial
cultures but not by primary neuronal cultures. Among the three major
glial cell types, astrocytes produced high levels of appican, while
oligodendrocytes failed to produce any. Only low levels of this
molecule were occasionally detected in microglial cultures. Expression
of appican in astrocyte cultures was regulated by the composition of
the growth media. N2a neuroblastoma cells also produced appican;
however, treatment with dibutyryl cAMP which promotes neuronal
differentiation in these cells inhibited its production without
inhibiting synthesis of APP. In contrast to the restricted expression
of appican, APP was present in all cultures, and its production was
independent of appican synthesis. Neuronal cultures produced mainly
APP
The amyloid peptide (A
Recent studies have shown that certain mutations of the APP
gene are tightly linked to familial Alzheimer disease, implicating them
as causative agents of AD in these families (for review, see Ref. 8).
Production of A
Recently, we have
shown that in C6 glioma cells both truncated and full-length forms of
the KPI-containing APP occur as the core protein of a secreted or
cell-associated chondroitin sulfate proteoglycan (CSPG),
respectively
(16, 17, 18) . CSPGs consist of a
core protein to which one or more chondroitin sulfate (CS)
glycosaminoglycan (GAG) chains are covalently attached. The CS GAG
chains consist of a highly sulfated repeating disaccharide backbone
attached via a carbohydrate linkage region to a serine residue of the
core protein
(19) . Due to the large size of the GAG chains,
proteoglycans (PGs) can be considerably larger than their core
proteins. CSPGs are important constituents of the cell and the
extracellular matrix of mammalian brain. They are involved in many
biological functions including cell adhesion, neurite outgrowth, axonal
guidance, wound healing, and
neuroprotection
(19, 20, 21, 22, 23, 24, 25) .
Identification of the cell types expressing the APP-PG
(appican)
Since our results suggested that
cells with neuronal phenotype do not synthesize appican, we examined
the effects of dibutyryl cAMP on appican production in mouse Neuro-2a
neuroblastoma cells. This treatment is known to induce a neuronal
phenotype in these cells
(46) . Upon dibutyryl cAMP treatment,
N2a cultures adapted a more neuronal phenotype as evidenced by the
elaboration of neuronal processes by the cells (data not shown). It can
be seen in Fig. 5that cAMP treatment of N2a cultures decreased
the amount of appican secreted, even though the amount of APP in
treated and untreated cultures was approximately the same. These
results are in agreement with our observations that primary neuronal
cultures do not synthesize appican and raise the possibility that upon
acquiring the neuronal phenotype cells may shut off production of
appican.
We noticed significant variations in the
production of appican by astrocyte cultures growing in different media.
Serum-containing DMEM media consistently produced the highest levels of
appican. However, Waymouth's medium plus chemically defined
supplements
(31) also induced high levels of appican synthesis.
In contrast, astrocytes growing in DMEM-based defined medium (see
``Materials and Methods'') produced significantly lower
levels of appican, although production of APP was not affected. Since
serum-free media are chemically defined, it should be possible to
isolate the factor(s) responsible for the marked difference in appican
production between astrocytes grown in Waymouth's and DMEM-based
media. It is not clear why, among the three major glial cell types
examined, only astrocytes produced high amounts of appican. Since
production of this molecule in astrocytes is regulated by culture
conditions, we cannot exclude the possibility that conditions may be
found where oligodendrocytes or microglia will also produce appican.
Thus far, however, our efforts to stimulate appican production in these
cells by changing growth conditions have been unsuccessful. The
astrocyte-specific expression of this molecule suggests an
astrocyte-specific function distinct from that of the ubiquitously
expressed APP. It is not clear yet, what signal(s) determine the
addition of GAG chains on APP, but recent evidence suggests the CS
chain of appican is attached to serine residue 619 of
KLAPP
Our results show that neuronal cells do not produce
appican. In addition, experiments with the neuroblastoma cell line N2a
suggested that factors such as cAMP inhibit expression of this molecule
without affecting APP expression. Presently it is not clear whether
cAMP, a second messenger, directly inhibits expression of appican by
interfering with its synthesis, or inhibition of this molecule is the
result of the neuronal differentiation induced by cAMP
(46) . The
latter possibility is in accord with the absence of this molecule in
neuronal cultures, however, further work will clarify this important
issue. Although our results suggest that appican is produced by
astrocytes, it is important to determine the cell type and brain
regions that express this molecule in vivo. To address this
question, we are currently producing appican-specific antibodies to be
used in immunocytochemistry.
Recently, it was reported that certain
anti-APP antibodies cross-react with amyloid precursor-like protein 2
(APLP2), which may also occur as the core protein of a
CSPG
(18, 48) . However, R7, R47, and GID antisera used
in the present study showed little or no reactivity against APLP2 (18),
in accordance with the low homology of the respective peptides used to
prepare these antisera with the APLP2 sequence and the absence of the
A
It has recently been shown that
brain CSPGs such as neurocan, phosphacan, and the NG2 PG are potent
inhibitors of cell adhesion and neurite outgrowth, suggesting that
these molecules may be used in vivo to guide growing
axons
(23, 24, 25) . Astrocytes have been
implicated in guidance of neuronal migration during development
(50) and in the inhibition of neurite regeneration following
neuronal injury (51). In addition, in the last decade it has become
clear that these cells are also important in the healing processes
following brain injury
(52) and seem to play a prominent role in
the formation of glial scars
(53) . It has been suggested that
these astrocytic functions are mediated, at least in part, by
CSPGs
(53, 54) . Taken together with the
astrocyte-specific expression of appicans, these observations raise the
possibility that these molecules may mediate several of the observed
astrocytic effects on neuronal extension and healing after brain
damage.
CSPGs are present in both senile plaques and neurofibrillary
tangles (55). Furthermore, in AD, astrocytes have been shown to
surround neuritic plaques, a brain area that also exhibits a
substantial neuronal sprouting activity (regeneration)
(56) .
Appican produced by astrocytes in the vicinity of neuritic plaques may
play a role in guiding the growing axons of the regenerating neurite in
AD brains. Involvement of appican in neurite outgrowth or regeneration
may be important in controlling the development of AD neuropathology.
We thank Drs. T. Saitoh, P. Mehta, and V. Friedrich
for GID, R47 antisera, and GFAP monoclonal antibody, respectively, and
Drs. L. Refolo and R. Hardy for helpful discussions.
while glial cultures produced the Kunitz type
protease inhibitor containing APP. The astrocyte-specific expression of
appican suggests a function distinct from the function of APP. Brain
appicans may play a role in the development of Alzheimer disease
neuropathology.
)
(
)
found in
brain amyloid depositions in Alzheimer disease (AD) patients, derives
from the proteolytic processing of the amyloid precursor protein (APP,
1-4). At least three APP proteins have been detected termed
APP
, APP
, and APP
(5) .
Both APP
and APP
contain a 56-amino-acid
insert with high homology to the Kunitz-type serine protease inhibitors
(KPI). Full-length APPs are transmembrane glycoproteins containing a
large extracytoplasmic domain, a transmembrane region, and a small
cytoplasmic sequence (for review, see Ref. 6). Secreted truncated APP
is produced after full-length cellular APP is cleaved by
``secretases'' close to the junction of the extracytoplasmic
and transmembrane regions. Most of the secreted truncated APP is
non-amyloidogenic because it contains only part of the A
sequence
(7).
and the familial Alzheimer disease mutations
illustrate the importance of APP in the development of AD. Although it
is still not clear how the APP mutations precipitate the AD phenotype,
one possibility is that they increase A
production which may be
neurotoxic
(9) . However, other studies have failed to show a
correlation between amyloid depositions and degree of dementia,
neuronal loss, or synaptic loss (for review, see Ref. 10). Another
possibility is that these mutations precipitate the AD phenotype by
altering the biological function of APP
(11, 12) . These
uncertainties on the pathogenic role of APP in AD emphasize the need to
elucidate both the biological function(s) and metabolism of this family
of proteins. The specific biological functions of the APPs are still
not well defined, but recent studies suggest that they may have cell
and neurite growth promoting activities and may play a role in cell
adhesion
(13, 14, 15) .
(
)
and elucidation of the factors
affecting its production are important for defining the physiological
function of this molecule. In this report, we show that appican is
expressed in both human and rat brain. In addition, using rat brain
primary cultures we show that this molecule is produced mostly by
astrocytes. No appican was produced by neuronal or oligodendrocyte
cultures although all cultures produced high levels of APP. We also
observed that in astrocytes, the expression of this molecule was
regulated by the composition of the growth media, independently of APP
expression.
Preparation of Rat Primary Cell
Cultures
Pregnant Sprague-Dawley rats were purchased (Charles
River) and primary neuronal cultures were prepared from rat embryos on
embryonic day 16 (E16) as described by Park and Mytilineou
(26) .
Cells were plated on polyornithine-coated dishes and maintained in
chemically defined media. Cultures were examined for appican production
after 7-10 days in vitro. Mixed glial cultures were
prepared from brain cortex of newborn rats (P1-2) as
described
(27) . Briefly, cortical tissue was dissected,
dissociated with trypsin, and plated in minimal essential medium (MEM)
supplemented with 10% fetal calf serum (Hyclone). Under these
conditions, neurons did not survive for more than 5 days in
vitro, and the resulting mixed glial cell cultures were examined
for appican production after cells reached confluence (10-30 days
in vitro). Astrocyte, microglia, or oligodendrocyte cultures
were prepared from mixed glial cultures as
described
(27, 28) . Briefly, after 7 days of incubation,
glial cultures were put in Dulbecco's modified Eagle's
medium (DMEM) plus 10% fetal calf serum and then placed on a rotary
shaker to detach non-astrocytic cells from the astrocyte monolayer.
Detached cells were harvested and used to prepare microglia and
oligodendrocyte cultures (see below). Attached astrocytes were detached
by exposure to trypsin/EDTA and then plated on Falcon dishes in the
presence of serum-containing DMEM. After 2 days, medium was replaced by
either serum-containing DMEM or various defined media as
described
(29, 30, 31, 32) . Detached
cells from above were plated on bacteriology dishes, and after 30 min
of incubation, the oligodendrocytes-containing medium was removed, and
attached microglia were grown either in defined or serum-containing
media
(28, 33) . The unattached cells were collected and
plated on poly-L-lysine-coated plates to select
oligodendrocytes. After 2 days, serum-containing medium was replaced
with chemically defined medium
(34) . Additional defined media
known to support oligodendrocyte growth were also
employed
(27, 35, 36) . All culture media and
chemically defined supplements were obtained from Life Technologies,
Inc. or Sigma.
PG Purification from Brain
Human brain CSPGs were
isolated from a phosphate-buffered saline extract of cerebral cortex
according to Kiang et al.(37) . Rat brain CSPGs were
purified by DEAE-cellulose anion-exchange chromatography from a
deoxycholate extract as described for the isolation of heparan sulfate
PGs
(38) . The CSPGs which did not bind to lipoprotein lipase
affinity column were then further purified by gel filtration as
described
(37) .
Immunoprecipitation of Appican and APP
Primary
neuronal or glial cultures were incubated in sulfate-free medium with
sodium [S]sulfate (carrier-free, ICN; at 400
µCi/5 ml/10-cm dish). Conditioned medium was collected, and cells
were washed and scraped from the dish in the presence of 0.2% SDS.
Lysed cells were further homogenized by passing through syringe needles
of 21 and 23 gauges. To compare relative production of appican in
astrocytes, microglia, and oligodendrocytes, 0.5 mg of cell extract
protein was used for immoprecipitation. Chondroitinase digestion,
immunoprecipitation, and Western blotting were performed as described
(16-18). Appican and APP were detected using the following
antisera (amino acid numbering according to APP
):
anti-GID which is specific for amino acids 179-185 and recognizes
all APP isoforms (39); anti-R7 which is specific for amino acids
296-315 and recognizes the KPI containing APP
(40) ;
anti-R47 which is specific for amino acids 652-667 and also
recognizes A
sequence 1-16
(16) ; and anti-R1 which is
specific for amino acids 729-751 of the cytoplasmic sequence and
recognizes all full-length APP
(17) . In addition, monoclonal
antibody 22C11 (Boehringer Mannheim) specific for amino acids
46-61 of all APP isoforms was used.
Immunocytochemistry
Cells were fixed on a chamber
slide (Nunc) with 2% paraformaldehyde and labeled with antibodies
against a variety of cell marker proteins as shown in the figure
legends. Rat monoclonal anti-GFAP (clone 2.2B10) was provided by Dr.
Victor Friedrich (Mount Sinai Medical Center). Mouse monoclonal
anti-Mac 1 (anti-rat CD11b, clone MRC OX-42), rabbit polyclonal
anti-galactocerebroside, and mouse monoclonal anti-A2B5 (clone
A2B5-105) were obtained from SEROTEC or Boehringer Mannheim. For
GFAP staining, cells were permeabilized with either acid/ethanol or
Triton X-100. Alkaline phosphatase-linked or fluorescence dye-labeled
secondary antibodies were used. Fluorescein isothiocyanate or Texas-red
linked species-specific secondary antibodies were obtained from Jackson
ImmunoResearch Laboratories or Vector Laboratories. Nuclear DNA
staining fluorescent dye DAPI (Sigma), at 2 µg/ml, was used to
detect cell nuclei.
RESULTS
Recent protein purification and sequencing studies with rat
glioblastoma cell line C6 have shown that a significant fraction of APP
occurs as the core protein of a chondroitin sulfate proteoglycan,
termed appican
(16) . Anti-APP antibodies used for appican
detection included R47, R7, GID, and
R1
(16, 17, 18) . In addition, antibodies
specific for the stub disaccharide remaining on the core protein
following enzymatic digestion of the CS chains reacted with the core
APP produced after chondroitinase treatment of appican, clearly
demonstrating the proteoglycan nature of APP
(16) . To determine
whether appican is present in brain tissue, we examined highly purified
PG preparations from rat brain (see ``Materials and
Methods''). It can be seen in Fig. 1A that R7
antiserum specific to the KPI domain of APP detected the characteristic
heterogeneous staining of appican at about 200 kDa
(16) .
Furthermore, digestion of the CS chains with chondroitinase eliminated
the PG staining and resulted in the production of a core protein which
migrated on SDS gels similar to the KPI-containing secreted APP nexin
II
(41) , purified from PC12 cell culture medium
(Fig. 1A, lanes 2 and 3). These
results indicate that most of the core protein of the detected brain
appican derives from the KPI-containing APP. The core protein of the C6
cell appican was also derived from the KPI-containing APP and displayed
the same mobility on SDS-PAGE as nexin II
(16) . These
observations indicate that brain and C6 cell appicans have the same APP
core protein. A crude soluble fraction of human brain homogenate
enriched in proteoglycans (see ``Materials and Methods'') was
also examined for the presence of appican. It can be seen in
Fig. 1B that R7 antiserum detected the diffuse staining
of appican, which was eliminated after chondroitinase digestion.
Moreover, following chondroitinase treatment the amount of APP
increased showing that a fraction of human brain APP occurred as a
CSPG
(16) .
Figure 1:
Detection of appican in brain tissue.
A, a highly enriched rat brain PG preparation was obtained as
the unbound fraction from a lipoprotein lipase-Sepharose column (see
``Materials and Methods''). The PG-containing fractions were
incubated for 1 h at 37 °C with (lane 2) or without
(lane 1) 0.5 milliunits of chondroitinase ABC/1 µg of
protein. After SDS-PAGE (8% gel) running, anti-APP immunoreactivity was
detected by Western blotting using R7 antiserum. Purified nexin II, the
secreted form of the KPI-containing APP, isolated from PC12 cells (8)
was used as a reference (lane 3). B, cerebral cortex
from human brain was homogenized in phosphate-buffered saline, and the
crude PG fraction was prepared by anion-exchange chromatography.
Aliquots of the fraction were treated without (lane 4) or with
(lane 5) chondroitinase as described above. Closed and open arrows indicate the positions of APP (nexin II)
and appican, respectively. Numbers on the left represent
mobilities of molecular mass markers in kDa.
Rat brain primary cultures were used to determine
the cell type responsible for production of the brain appican. Primary
neuronal cultures were prepared from the cerebral cortex of rat
embryonic brain (see ``Materials and Methods''). Staining
with the neuron-specific antibody anti-tau showed that these cultures
consisted primarily of neurons (data not shown). Cultures were grown in
the presence of [S]sulfate which predominantly
labels the CS chains of CSPG
(17) . APP and appican were then
immunoprecipitated from the culture conditioned medium either with
22C11 antibody which recognizes all APP isoforms or with R7-antiserum.
Cellular appican containing full-length APP as a core protein was
immunoprecipitated with R1 antiserum
(17) . Fig. 2shows
that these cultures produced substantial amounts of APP, which is
tyrosine-sulfated
(39) . The predominant APP species in our
neuronal cultures was APP
, in agreement with previous
in situ studies showing that neurons synthesize predominantly
this APP isoform (42). However, neither culture medium nor cell
extracts of the primary neuronal cultures contained the characteristic
heterogeneous staining of the PG form of APP, suggesting that neurons
produced little or no appican (Fig. 2).
Figure 2:
Appican production in primary neuronal
cultures. Primary neuronal cultures were prepared from cerebral rat
cortices (see ``Materials and Methods''). Cells were labeled
with [S]sulfate overnight. Conditioned medium
was collected and heat treated in the presence of 0.1% SDS. Cells
attached to the dish were washed and lysed in the presence of 0.2% SDS.
Aliquots of conditioned medium or cell extracts containing 1.0
10
(for the immunoprecipitation with polyclonal antibodies)
or 2.0
10
(for monoclonal antibody) trichloroacetic
acid-precipitable disintegrations/min were immunoprecipitated with
anti-APP antibodies as indicated. Immunoprecipitates were treated with
or without chondroitinase ABC (see ``Materials and Methods'')
and then analyzed by SDS-PAGE (6% gel) and fluorography. Panels A and B, immunoprecipitation of conditioned medium and cell
extracts, respectively, using 22C11 antibody (lanes 1 and
2), R7 antiserum (lanes 3 and 4), or R1
antiserum (lanes 5 and 6). Lanes 1,
3, and 5, control (no chondroitinase digestion);
lanes 2, 4, and 6, after chondroitinase
digestion. Positions of secreted (A) or full-length
(B) APP
and APP
are marked by
arrows and arrowheads,
respectively.
To examine the
expression of appican in glial cells, primary cultures of mixed
neuroglia were prepared from neonatal rat brains (see ``Materials
and Methods''). It can be seen in Fig. 3that
immunoprecipitation of conditioned media with several APP antibodies
resulted in the appearance of the characteristic diffuse staining of
appican between 140-250 kDa
(16) . This staining was
eliminated after digestion with chondroitinase ABC (Fig. 3). The
smear was also sensitive to chondroitinase AC (data not shown)
indicating that the GAG chains of this PG contain chondroitin sulfate
but not dermatan sulfate. The absence of an obvious increase in the APP
protein upon chondroitinase treatment is due to the presence of large
amounts of unmodified APP which is produced by all glial cells and
migrates similar to the appican core APP
(16) . In contrast,
appican is produced only by astrocytes, where only a fraction of the
total astrocytic APP occurs as PG (see below). Immunoprecipitation of
cell extracts with R1 antiserum indicated that, in addition to APP,
glial cultures produced significant amounts of cellular appican
containing full-length APP as a core protein (Fig. 3B).
Immunostaining of the glial cultures with markers specific for
astrocytes, oligodendrocytes, or microglia showed that all three cell
types were present (data not shown). Glial cellular appican also
reacted with R7 antiserum (data not shown). Thus, like brain tissue,
mixed primary glia cultures produced appican containing the KPI-APP as
a core protein.
Figure 3:
Appican production in primary glial
cultures. Primary cultures of neuroglia (11 days in vitro DIV)
were prepared from cerebral cortices of neonatal rat brains (see
``Materials and Methods''). Labeled medium and cell extracts
were prepared and analyzed as described in the legend to Fig. 2. In
some experiments, immunoprecipitations were conducted in the presence
of epitope peptides (10 µg/ml). Panels A and B,
immunoprecipitation of conditioned medium and cell extracts,
respectively, using 22C11 antibody (lanes 1 and 2),
R7 antiserum (lane 3), R7 antiserum plus R7 peptide (lane
4), GID antiserum (lane 5), GID antiserum plus GID
peptide (lane 6), and R1 antiserum (lanes 7 and
8). Lanes 1 and 7, control (no
chondroitinase digestion); lanes 2 and 8, after
chondroitinase digestion. Positions of secreted (A) or
full-length (B) APP and APP
are marked by arrows and arrowheads,
respectively. Large open arrows indicate position of
appican.
To further define the glial cell type responsible
for the synthesis of appican, primary cultures of astrocytes,
oligodendrocytes, or microglia were prepared (see ``Materials and
Methods''). The homogeneity of these cultures was assayed by
fluorescence immunocytochemistry using markers specific for each glial
cell type. High purity of each cell culture was demonstrated in
fluorescent light micrographs where cell nuclei of all cell types were
visualized by DAPI staining (Fig. 4A). It can be seen in
Fig. 4B that although oligodendrocyte and microglia
cultures secreted substantial amounts of APP in the culture medium, no
appican was detected in oligodendrocyte conditioned medium (lane
1), while in microglia cultures very low levels of secreted
appican were occasionally detected (lane 2; see below). In
contrast, the characteristic appican staining was clearly detected in
the conditioned medium of astrocytic cultures (lane 3).
Astrocyte appican was also detected with R47 antiserum, indicating that
the A sequence is present in the core protein
(Fig. 4C). Similar results were obtained with 22C11
antibody (data not shown). Staining of astrocyte cultures with
anti-fibronectin showed no appreciable contamination by fibroblasts.
However, to eliminate the possibility that appican is produced by a
very small population of fibroblasts, we maintained the astrocyte
cultures in medium containing D-valine instead of
L-valine. This substitution inhibits fibroblast
growth
(30) . Under these conditions, production of secreted
appican was not reduced, suggesting that this molecule is not produced
by undetectable contaminating fibroblasts. Immunostaining of astrocyte
cultures with anti-A2B5, a reagent specific for type 2
astrocytes
(43) , showed that astrocyte cultures producing
appican did not contain appreciable levels of this type of astrocytes
(GFAP
A2B5
; data not shown)
indicating that the CSPG isoform is mostly produced by type I
astrocytes (GFAP
A2B5
). In agreement
with this suggestion, type 2 astrocyte cultures prepared by growing
oligodendrocyte precursor cells in serum
(44) did not produce
appican (data not shown).
Figure 4:
Appican production in various primary glia
cultures. Astrocytes, oligodendrocytes, and microglia were isolated or
enriched as described under ``Materials and Methods.''
Oligodendrocytes were grown in DMEM/Ham's F12 mixture (1:1) with
supplements (35), and microglia and astrocytes in DMEM with 10% fetal
calf serum. A, immunocytochemistry of primary neuroglial
cultures. Panels a-c show immunostaining of oligodendrocyte,
microglia, and astrocyte cultures, with anti-GC, anti-Mac1, and
anti-GFAP, respectively. All cell nuclei were stained with DAPI
(bright white color). Cultures of three different cell types
were labeled with [S]sulfate as described in the
legend to Fig. 2. Cell culture conditioned medium containing 1.0
10
trichloroacetic acid-precipitable
disintegrations/min was immunoprecipitated with R7 antiserum
(B) or R47 antiserum (C) and analyzed as described in
the legend to Fig. 2. The weak signal of the appican in lane 4 is due to the low immunoreactivity of this molecule with R47
antiserum (16, 18). Cell extracts (0.5 mg of protein) from different
cell cultures were immunoprecipitated with R1 antiserum (D).
Anti-R1 immunoprecipitates from astrocyte cell extracts were incubated
with or without chondroitinase ABC (E). Lanes 1 and
5, oligodendrocytes; lanes 2 and 6,
microglia; lanes 3, 4, and 7-9,
astrocytes. Positions of secreted (B and C) or
cellular (D and E) APP
are marked
by arrow. Large open arrows indicate position of
appican. Immunoreactive band at 97 kDa in lane 1 probably
shows a degradation product of APP.
To examine the production of cellular
appican containing full-length APP as a core protein, cell extracts
from glial cultures were immunoprecipitated with R1 antiserum. In
agreement with the results obtained from the culture medium, cellular
appican was detected primarily in the astrocytic culture. As expected,
cellular appican also reacted with anti-R7 and anti-22C11 antibodies
(data not shown). The characteristic diffuse band of appican was
eliminated by chondroitinase treatment (Fig. 4E,
lanes 8 and 9). Microglia or oligodendrocytes
contained little or no detectable cellular full-length appican,
respectively, even though these cultures contained significant amounts
of cellular full-length APP. Oligodendrocyte cultures derived from long
term (16-21 days in vitro) mixed glial
cultures
(45) , or grown in several distinct defined media, known
to support growth and differentiation of
oligodendrocytes
(27, 35, 36) , also failed to
induce production of appican (data not shown). Similarly, activation of
microglia with retinoic acid failed to increase production of this
molecule. Since microglial cultures may contain contaminating
astrocytes, the low levels of appican detected in these cultures may
derive from astrocytes rather than microglia. In agreement with this
suggestion, treatment of microglia cultures with L-leucine
methyl ester, an inhibitor of microglial growth
(28) , did not
affect appican production, suggesting that the detected appican may not
be produced by microglia cells.
Figure 5:
Effect of cAMP on the production of
appican. N2a cell cultures of approximately 50% confluence were
incubated without (lane 1) or with (lane 2) 1
mM dibutyryl cAMP for 3 days. Media were analyzed by Western
blotting using R7 antiserum as described under ``Materials and
Methods.'' The APP and appican bands are marked by filled and open arrows, respectively. Analysis of five
independent experiments showed that media from cAMP-treated cultures
contained 28 ± 9% of the appican present in control cultures,
whereas APP secretion was unaffected. Similar results were obtained
using 22C11 monoclonal antibody (data not
shown).
Astrocytes grown in chemically defined DMEM produced
substantially lower amounts of appican than astrocytes grown in
serum-containing DMEM (Fig. 6). A number of different chemically
defined media that have been previously used to grow
astrocytes
(29, 31, 32) were examined for their
effects on appican production. Among them, Waymouth's medium
(31) induced almost as much appican synthesis as
serum-containing media (Fig. 6). Astrocytes grown in DMEM
produced substantially lower levels of appican than astrocytes grown in
Waymouth's medium even though both cultures synthesized similar
levels of APP. These results suggest that production of appican is
regulated by growth conditions independently of APP synthesis.
Figure 6:
Effect of growth media on the appican
production in astrocytes. Astrocytes were grown in either
serum-containing or defined medium. Labeled media (A) or cell
extracts (B) were prepared as described in the legend to Fig.
2, and immunoprecipitation was done using either R7 (A) or R1
antiserum (B). Lanes 1 and 4, DMEM
supplemented with 10% serum; lanes 2 and 5, DMEM with
defined supplements (29); lanes 3 and 6,
Waymouth's medium 752/1 with defined supplements
(31).
DISCUSSION
In this report we show that 1) appican is present in human
and rat brains suggesting that this molecule has a physiological
function in brain, 2) in primary brain cell cultures, appican is
primarily produced by astrocytes but not neurons, oligodendrocytes or
microglia, 3) most of the core proteins of both soluble and
cell-associated appicans detected in brain cell cultures derive from
the KPI-containing APP, and 4) production of appican in primary
astrocyte cultures can be regulated by growth conditions. Our results
also show that primary neuronal cells produce mainly non-KPI APP,
whereas glial cells produce predominantly the KPI-containing APP.
Although fibroblasts may be a minor contaminant of glial
cultures
(30) , anti-fibronectin staining of our cultures
revealed no significant fibroblast contamination. In addition,
inhibition of fibroblast growth by D-valine did not affect
appican production, thus excluding the possibility that the detected
appican is produced by contaminating fibroblasts. This conclusion is
further supported by the absence of appican in fibroblast cell lines
including kidney cell line 293 and COS cells
(18) and by the
synthesis of this molecule in C6 cells which display an astrocytic
phenotype
(47) . Estimates of the relative amount of appican
detected in serum-grown astrocytes from 10 independent experiments
showed that approximately 20% of the total APP was in the PG form. Some
microglial cultures, particularly long term cultures, produced low
levels of appican. Examination of these cultures by fluorescence
immunocytochemistry (see ``Materials and Methods''), revealed
that they were sometimes contaminated by astrocytes. Thus, occasional
detection of CSPG in microglial cultures may be due to astrocytes which
proliferate during long term culturing. This hypothesis is further
supported by the finding that leucine methyl ester did not affect
appican expression.
, 16 amino acids upstream of the A
sequence of
APP
(57) .
region from the APLP2
(49) . Furthermore, in contrast to
appican, APLP2 proteoglycan is produced at similar levels in both
primary astrocyte and microglial cultures,
(
)
and
contains a core protein which on SDS-PAGE migrates differently from the
core protein of appican
(18) . Similarly, expression of appican
in transformed cell lines is more restricted than expression of
endogenous APLP2 PG
(18) .
,
/A4 amyloid; AD, Alzheimer
Disease; APLP2, amyloid precursor-like protein 2; APP, amyloid
precursor protein; CS, chondroitin sulfate; CSPG, chondroitin sulfate
proteoglycan; DMEM, Dulbecco's modified Eagle's medium;
GAG, glycosaminoglycan; KPI, Kunitz type protease inhibitor; MEM,
minimal essential medium; PAGE, polyacrylamide gel electrophoresis; PG,
proteoglycan.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.