©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Alzheimer Amyloid Precursor Proteoglycan (Appican) Is Present in Brain and Is Produced by Astrocytes but Not by Neurons in Primary Neural Cultures (*)

Junichi Shioi (1)(§), Menelas N. Pangalos (1), James A. Ripellino (1), Dido Vassilacopoulou (1), Catherine Mytilineou (2), Richard U. Margolis (3), Nikolaos K. Robakis (1)

From the (1) Departments of Psychiatry and Fishberg Research Center for Neurobiology and (2) Neurology, Mount Sinai School of Medicine, New York, New York 10029 and the (3) Department of Pharmacology, New York University Medical Center, New York, New York 10016

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

The amyloid peptide (A)() 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).

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 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) .

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)() 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.


MATERIALS AND METHODS

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.

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.


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.

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, 16 amino acids upstream of the A sequence of APP (57) .

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 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) .

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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants AG08200 and AG05138 (to N. K. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Psychiatry and Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, One Gustave Levy Place, New York, NY 10029. Tel.: 212-241-9380; Fax: 212-831-1947.

The abbreviations used are: A, /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.

In accordance with the nomenclature of other proteoglycans, we propose the term appican (APP-proteoglycan) for the APP-PG.

J. Shioi and N. K. Robakis, unpublished results.


ACKNOWLEDGEMENTS

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.


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