1

Ana Villa, Maria Jesús Latasa, and Angel Pascual.

Instituto de Investigaciones Biomédicas. Consejo Superior de Investigaciones

Científicas. Madrid. Spain.

Nerve growth factor modulates the expression and secretion of -

amyloid precursor protein through different mechanisms in PC12 cells.

Address correspondence:

Dr. Angel Pascual

Instituto de Investigaciones Biomédicas (C.S.I.C.)

Arturo Duperier,4

28029 Madrid. Spain.

Tel. 34-91-585 4649 Fax 34-91-585 4587

e-Mail: apascual@iib.uam.es

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ABSTRACT

The -amyloid protein, component of the senile plaques found in Alzheimer

brains is proteolytically derived from the -amyloid precursor protein (APP), a

larger membrane-associated protein that is expressed in both neural and

nonneural cells. Overexpression of APP might be one of the mechanisms that

more directly contributes to the development of Alzheimer disease. APP gene

expression is regulated by a number of cellular mediators including nerve growth

factor (NGF) and other ligands of tyrosine kinase receptors. We have previously

described that NGF increases APP mRNA levels In PC12 cells. However, the

molecular mechanisms and the precise signaling patways that mediate its

regulation are not yet well understood. In the present study we present evidence

that NGF, and to a lesser extent fibroblast growth factor (FGF) and epidermal

growth factor (EGF), stimulate APP promoter activity in PC12 cells. This

induction is mediated by DNA sequences located between the nucleotides -307

and -15, and involves activation of the Ras-MAP kinase signaling pathway. In

contrast, we have also found that NGF-induced secretion of soluble fragments of

APP into the culture medium is mediated by a Ras independent mechanism.

Running title: NGF regulate synthesis and secretion of APP by different

mechanisms.

Key words: Amyloid precursor protein (APP) - Synthesis and secretion - PC12

cells - Nerve growth factor - Promoter - Ras.

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INTRODUCTION

One of the characteristic features of Alzheimer's disease is the presence

of senile plaques in which the -amyloid protein, a 40-42 aminoacids peptide, is

the major component. This 4 kDa peptide is derived by proteolytic cleavage from

a set of alternatively spliced -amyloid precursor proteins (APP), which are

encoded by a single gene located on chromosome 21 (Selkoe, 1994). The APP

gene is ubiquitously expressed in mammalian tissues, and gives rise to three

major APP messenger RNAs that encode for the isoforms APP695, APP751 and

APP770. The precursor protein APP plays a central role in the Alzheimer's

disease, and significant alterations of APP expression might contribute to its

development (Zhong et al., 1994). The appearance of an Alzheimer-like

pathology in Down's syndrome, probably due to the trisomy 21-associated

duplication of the APP gene (Neve et al., 1988), the degeneration of neurons

overexpressing APP (Yoshikawa et al., 1992) or the appearance of -amyloid-

immunoreactive deposits in transgenic mice carrying the human APP cDNA

(Games et al., 1995; Quon et al., 1991), strongly suggest a positive correlation

between the overexpression of APP and the formation of amyloid deposits.

Overexpression of the APP gene might result in an aberrant processing of

the amyloid precursor, which leads to a higher concentration of amyloidogenic

fragments, and is associated with the formation of -amyloid deposits in the

brain (Fukuchi et al., 1992). APP gene expression is regulated in different cell

types by a variety of stimuli, including phorbol esters (Trejo et al., 1994; Yoshikai

et al., 1990), thyroid hormones (Belandia et al., 1998; Latasa et al., 1998), or

retinoic acid (Konig et al., 1990). In addition, and since NGF is considered to be

of benefit in Alzheimer´s and other neurodegenerative diseases, it results of

special interest the effects induced by NGF and other neurotrophins on APP.

Nerve growth factor (NGF), the best characterized neurotrophic factor, has been

found to increase APP mRNA in SH-SY5Y human neuroblastoma cells (Konig et

al., 1990), in mouse brain primary cultures (Ohyagi and Tabira, 1993) and in

developing hamster brain (Mobley et al., 1988). In PC12 cells, a rat

pheochromocytoma cell line which constitutively expresses APP, NGF has been

reported to influence transcript levels (Cosgaya et al., 1996), splicing of APP

mRNA isoforms (Fukuyama et al., 1993; Smith et al., 1991), localization

(Fukuyama et al., 1993), catabolism and secretory processing of APP (Refolo et

al., 1989; Rossner et al., 1998). Moreover, it has been also suggested that APP

4

might act to mediate the effects induced by NGF on neurite outgrowth (Milward

et al., 1992). However, up to date the precise mechanisms involved in this

regulation remain largely unclear.

The trophic effects of NGF are mediated by the specific tyrosine kinase

receptor TrkA, and also by the low-affinity neurotrophin receptor p75NTR

(Barbacid, 1994; Segal and Greenberg, 1996). NGF binds to the receptor, and

activates various intracellular signaling pathways that mediate the

phosphorylation of specific transcription factors, the activation of target genes,

and finally the biological responses induced by the neurotrophin. Accumulating

evidence indicates that binding of NGF to its receptors, p75NTR and TrkA,

mediates the responses of the neurotrophin on APP mRNA expression, splicing,

and protein secretion in PC12 cells (Rossner et al., 1998). In addition, TrkA

receptor gene expression is decreased in nucleus basalis (Higgins and Mufson,

1989), and parietal cortex (Hock et al., 1998) of patients with Alzheimer's

disease, suggesting that this receptor could play a role in the neurodegenerative

process associated with this pathology. We have previously reported that in

PC12 cells NGF, as well as epidermal (EGF) and basic fibroblast (bFGF) growth

factors, increase APP mRNA levels by a mechanism that very likely involves

activation of ras (Cosgaya et al., 1996), and is probably mediated by specific

sequences of the APP promoter (Lahiri and Nall, 1995; Lahiri et al., 1999).

However, the exact mechanisms, and the precise signaling pathways that

mediate these effects of NGF remain elusive.

In this work we have examined the role of the ras oncogene in the

response induced by NGF on APP in PC12 cells, and also in two different

subclones of PC12 stably transfected with a dominant negative mutant of the ras

oncogene (M-M17-26), or with an activated ras oncogene under control of the

MMTV promoter (UR61). We have determined the promoter activity induced by

NGF or dexamethasone in those cell lines, and partially analyzed the sequences

of DNA that mediate the response of the APP promoter to NGF. We have also

examined whether the mitogen-activated protein kinase cascade is involved in

this response by using several dominant negative mutants, and finally analyzed

the specific pattern of intracellular and secreted isoforms of APP induced by

NGF in these cells. NGF induces the APP promoter activity, and according to our

results this activation is mediated by Ras throughout the MAP kinase signaling

pathway. NGF-induced activation of the promoter and the specific increase of

the intracellular content of APP. In contrast, the release of APP isoforms to the

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culture medium appeared to be quite Ras-independent. On the other hand, the

response to NGF was significantly reduced in a small fragment (-15/+102) of the

promoter that, however, maintains a residual response likely involving direct

effects of NGF on different components of the basal transcriptional machinery.

MATERIALS AND METHODS

Cell cultures - PC12 cells were cultured in RPMI medium containing 10%

donor horse serum (Quality Biological Inc.) and 5% fetal calf serum (GIBCO) in

collagen-treated plates. The subclones of PC12 cells, UR61 and M-M17-26 cells,

were grown in a similar medium containing 10% horse serum - 5% fetal calf

serum. UR61 cells were derived from PC12 cells following transfection with a

plasmid containing the transformant mouse N-ras oncogene under the

transcriptional control of the dexamethasone-inducible mouse mammary tumor

virus promoter (Guerrero et al., 1988). The PC12 subline M-M17-26, was

obtained by Szeberényi et al. (Szeberenyi et al., 1990) after transfection with the

dominant negative mutant Ha-ras (Asn-17) gene transcribed from the promoter

of the mouse methallothionein-1 gene. This subclone constitutively express high

levels of mutant Ras (Val-12) protein which could not be further induced by zinc.

In our culture conditions both subclones, as well as parental PC12 cells, exhibit

more than 95% viability as determined by trypan blue exclusion. In addition,

proliferative ability of M-M71-26 cells is not affected by constitutive expression of

RasAsn17 (Szeberényi et al., 1990). In UR-61 cells, RasVal12 induces

differentiation; however, differentiation is not accompanied by a decrease in the

protein content per culture. The same occurs in parental PC12 cells treated with

NGF, whereas proliferative ability of M-M17-26 cells is not significantly affected

by the neurotrophin.

The PC12 subclones were incubated with the different factors at the

concentrations and for the times indicated in the figures. NGF and

dexamethasone were obtained from Sigma, and bFGF and EGF were obtained

from Austral Biologicals.

Plasmids - The chloramphenicol acetyl transferase (CAT) reporter plasmid

containing the -1099/+105 fragment of the human APP gene has been

previously described (Belandia et al., 1998). Progressive 5' deletions to -487, -

307, -15 +55 and +75 bp were prepared by polymerase chain reaction from the

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original -1099/+105 bp fragment, kindly provided by Dr. Lahiri's laboratory (Lahiri

and Robakis, 1991), and subcloned into the BamH1 site of pBL-CAT8, a plasmid

which lacks the AP-1 binding site present in the pUC backbone. Expression

vectors for the dominant negative mutants of Ras, Raf and MAPK have been

previously described. These vectors contain the inhibitory Ha-rasAsn17 mutant

(Feig and Cooper, 1988), a dominant negative raf (Raf-C4) (Bruder et al., 1992),

or the Chinese hamster p44MAPK mutated in the kinase domain (Meloche et al.,

1992).

DNA transfection and determination of CAT activity - The PC12 cells were

transfected in Dulbecco´s modified Eagle´s medium (DMEM) containing 10%

fetal calf serum. The RPMI growing culture medium was replaced by DMEM

four-six hours before transfection, and cells transfected by the calcium

phosphate coprecipitation method with 1g of reporter plasmids and carrier DNA.

One hundred nanograms of a luciferase reference vector was simultaneously

used as an internal control of the transfection efficiency. In cotransfection

experiments, 1g of reporter plasmid and 10 g of the corresponding dominant

negative expression vector were used. After 16h of incubation in the presence of

calcium phosphate, the medium was discarded and washed with 5 ml of

phosphate-buffered saline. A new RPMI medium containing 0,5% serum was

added and the cells were then incubated for an additional period of 48 hours in

the presence or absence of NGF (50 ng/ml). Each treatment was performed in

duplicate cultures that normally showed less than 5-15% variation in CAT activity

which was determined by incubation of [14C]-chloramphenicol with the same

amount of cell lysate protein. After autoradiography, the non-acetylated and

acetylated [14C]-chloramphenicol were quantified. Each experiment was

repeated at least 2-3 times with similar relative differences in regulated

expression. All data are expressed as the mean ± standard deviation.

Western blot analysis. - Cellular proteins were extracted by lysis with a

buffer (150 mM NaCl, 50 mM Tris pH8, 2 mM EDTA, 1% Triton, 0.1% SDS)

containing the protease inhibitors PMSF (1 mM) and leupeptin (10 g/ml). The

protein content of cells was determined by using the BCA assay (Pierce. Illinois),

according to the manufacturer's instructions. Identical amounts, 40 g, of cell

extracts were then electrophoresed in a 8% SDS-polyacrylamide gel and

transferred to an immobilon polyvinylidine difluoride membrane. Non specific

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binding was blocked with 5% nonfat dried milk in TBS-T (Tris buffered saline,

0.1% Tween 20) for 2-3 h at room temperature and the cellular APP detected

with a 1/1,500 dilution of the rabbit polyclonal antibody 369A, raised against the

carboxi-terminal domain of human APP. After 1 hour incubation at room

temperature the membrane was washed and incubated with a second

biotinylated anti-rabbit antibody (1/2,000) for an additional hour, washed again

and finally incubated for 1 hour with 1/2,000 peroxidase-conjugated

streptavidine. All incubations took place at room temperature, and detection by

enhanced chemiluminiscence (ECL, Amersham International plc. England) was

carried out according to manufacturer's indications.

Secreted full-length APP isoforms were detected by the same method

from 50 l (1:100 from total) of conditioned medium using the monoclonal

antibody 22C11 at a final concentration of 10 g/ml. The apparent molecular

mass (kDa) of the detected bands was always determined by using a wide range

protein standard (Mark 12 from Novex.-S. Diego. CA).

RESULTS

Induction of APP promoter activity by NGF and other neurotrophins.

We have previously reported that NGF, as well as EGF or bFGF,

effectively increases APP-mRNA levels in PC12 cells by a mechanism that

requires activation of Ras (Cosgaya et al., 1996). To examine whether the

specific changes induced by these growth factors on the APP mRNA are induced

at transcriptional levels, we analyzed the effect of NGF, EGF and bFGF on the

APP promoter activity. As shown in figure 1A, treatment with 50 ng/mlNGF for 48

hours, induced by almost 4-fold the CAT activity in PC12 cells transiently

transfected with a chimeric plasmid containing the APP promoter linked to the

CAT reporter gene. EGF and bFGF also stimulated APP promoter activity

although their effect was weaker than that found with NGF. Since the ras

oncogene is involved in different actions of growth factors ligands of tyrosine

kinase receptors in PC12 cells, we examined the effect of activated Ras on APP

promoter activity in the PC12 subline UR61 which contains a transfected N-Ras

oncogene (Rasval12) under control of the glucocorticoid-incucible MMTV

promoter. Figure 1B shows that promoter activity was significantly induced in

UR61 cells treated for 48 hours with 100 nM dexamethasone. Since, as also

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shown in figure 1A, dexamethasone treatment of PC12 cells did not increase

promoter activity, these results indicate that activated ras is responsible for

promoter stimulation by the glucocorticoid in UR61 cells. The induction of of APP

promoter activity by dexamethasone in UR61 was comparable to that produced

by NGF in the parental PC12 cells. In contrast, NGF caused a weaker increase

in UR61 cells, in which the neurotrophin does not elicit neurite outgrouth

(Cosgaya et al., 1997; Guerrero et al., 1988).

To determine whether endogenous Ras is required for growth factor

stimulation of the APP promoter in PC12 cells, we examined the ability of NGF,

EGF and bFGF to increase APP promoter activity in the PC12 subclone M-M17-

26 which as indicated above constitutively expresses the dominant inhibitory

RasAsn17 mutant. As illustrated in figure 1C, neither growth factor was able to

stimulate APP promoter activity in the presence of the dominant negative ras

mutant, showing the requirement of functional Ras for this response.

Characterization of the signaling pathway that mediates the effects of NGF

on the APP promoter.

The results obtained in M-M17-26 cells transfected in a stable manner with

the dominant inhibitory ras mutant were confirmed in parental PC12 cells

transiently transfected with RasAsn17. Figure 2 shows that transfection with this

mutant totally abolished the response to NGF in PC12 cells. Since Raf can act

downstream of Ras in growth factor signal transduction, the influence of transient

transfection with an expression vector for a dominant negative form of Raf was

also analyzed. As shown in figure 2, the mutant Raf had an effect identical to

that caused by RasAsn17, blocking the response of the APP promoter to the

neurotrophin in PC12 cells. The same was found with a MAP kinase mutant,

which also reduced very significantly the promoter response to NGF. In addition,

transfection of these mutants decreased basal CAT levels (data not shown), thus

suggesting that even in the absence of NGF exist a certain activation of the

Ras/MAPK pathway that contributes to basal APP promoter activity.

Identification of DNA regions mediating the regulation of APP

transcriptional activity.

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To map the DNA sequences of the APP gene that mediate the NGF-

induced response, progressively deleted fragments of the promoter (-1099, -487,

-307 and -15) were linked to the CAT reporter gene and transfected into PC12

cells. As illustrated in figure 3, not only the basal activity, but also the response

to NGF was affected along the different fragments of promoter studied. Deletion

from nucleotides -1099 to -487 increased basal promoter activity without

affecting significantly the response to NGF. Deletion to nucleotide -307 caused a

decrease of CAT levels which were similar to those found with the fragment

extending to nucleotide -1099. In addition, induction with NGF was similar,

indicating that sequences between nucleotides -307 and -1099 do not

significantly contribute to the response of the APP promoter to NGF. However,

further deletion of sequences between -307 and -15 caused not only a strong

decrease of basal promoter activity, but also a marked reduction of the response

to NGF, since only a residual response to the neurotrophin was found in PC12

cells transfected with the plasmid containing the -15 t0 +102 fragment.

Effects of NGF on the cell-associated proteins and the accumulation of

secreted forms into the culture medium.

After 48 hours of incubation in the presence or in the absence of NGF

(PC12 and M-M17-26 cells), or dexamethasone (UR61 cells), the content of APP

was analyzed in cells and culture medium by Western blot using the antibodies

369A and 22C11, respectively. The analysis of the intracellular APP isoforms is

illustrated in figure 4A. Based on previous descriptions, the immunoreactive

bands detected by Western blot analysis in PC12 cells contain at least six

protein species corresponding to immature and mature forms of APP (APP695,

APP751 and APP770) (Buxbaum et al., 1990; Weidemann et al., 1989).

Incubation of PC12 cells with NGF (50 ng/ml) for 48 hours, does not affect the

general pattern of intracellular proteins (data not shown), but leads to a

generalized increase of the intracellular APP content, that is not observed in the

M-M17-26 cell line which constitutively expresses the dominant negative Ras

mutant. In contrast, incubation of UR61 cells with 100 nM dexamethasone, which

induces the expression of the stably transfected Ras oncogene, leads to a

similar increase in the intracellular content of APP. In addition, dexamethasone

did not affect the intracellular content of APP in the native PC12 cells or in M-

M17-26 cells (data not shown). Figure 4B shows the effects of NGF and

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dexamethasone on the levels of APP soluble isoforms accumulated into the

culture medium. As expected, NGF treatment leads to an increased sAPP

content in the PC12 conditioned medium. However, and in contrast to that

observed with the intracellular APP content, the amount of secreted isoforms

was not modified by dexamethasone in UR61 cells. Furthermore, the amount of

sAPP accumulated into the culture medium was increased in the NGF-treated M-

M17-26 cells.

DISCUSSION

It has been extensively documented that NGF can modulate the

postranscriptional processing and secretion of APP in PC12 cells (Refolo et al.,

1989; Rossner et al., 1998; Rossner et al., 1999). In addition, we have previously

demonstrated that NGF, as well as the growth factors bFGF and EGF, increases

APP-mRNA levels through a Ras-dependent mechanism (Cosgaya et al., 1996).

In this study we have examined the effects of NGF on APP expression in

three variants of PC12 cells, the parental cell line and two different subclones,

which represent a good model to investigate the role of Ras in NGF-induced

effects in PC12 cells. UR61 cells express oncogenic N-ras and M-M17-26 cells

contain a dominant inhibitory mutant of ras. Transient transfection studies

demonstrate that NGF and to a lesser extent bFGF and EGF stimulate APP

promoter activity in PC12 cells. It has been reported that NGF can stimulate APP

promoter activity when these cells are treated with the growth factor for a

duration of four days prior to and four days after transfection with a plasmid

containing the APP promoter (Lahiri and Nall 1995). We demonstrate here that

48 hours of incubation with NGF after transfection are sufficient to activate the

APP promoter. These results demonstrate that NGF increases APP gene

expression at transcriptional level.

Activation of the protein tyrosine kinase receptors initiates a cascade of

intracellular signalling events leading to regulation of specific genes and finally to

regulation of a wide variety of cellular responses. Numerous proteins and

several second messengers, among them the Ras-MAP kinase cascade, have

been implicated in the signalling pathway that follows binding of neurotrophins to

their Trk receptors. Evidence that activation of endogenous Ras mediates the

effects of NGF on APP promoter activity comes from the experiments with the

PC12 subclone M-M17-26 which constitutively expresses the dominant inhibitory

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mutant of ras Asn-17. This mutant has a reduced affinity for GTP and inhibition

of endogenous Ras function by this mutant has been suggested to occur through

competition with normal Ras for regulatory proteins which promote nucleotide

exchange (Feig and Cooper, 1988). Szerebényi et al., (Szeberenyi et al., 1990),

have shown that the mutant blocks the neurite outgrowth induced by NGF or

FGF, as well as the induction of different genes. Our present results demonstrate

that expression of the Asn-17 mutant also blocks the stimulation of APP

promoter activity induced by NGF in PC12 cells, thus showing the requirement of

functional Ras for this response. The results obtained in dexamethasone-

induced UR61 cells support this hypothesis further. In this subclone of PC12

cells the transfected ras proto-oncogene is under control of a dexamethasone

inducible MMTV promoter. Since dexamethasone does not affect the expression

of APP in PC12 cells, it can be assumed that Ras is the signal transduction

pathway used by dexamethasone to increase the intracellular content of APP in

UR61 cells. Moreover, the results obtained with dominant negative mutants of

Ras, Raf and MAP kinase, strongly suggest that NGF-induced stimulation of the

APP promoter activity not only requires activation of Ras, but also the complete

activation of the Ras-MAP kinase signaling pathway.

Many transcription factors which constitute final targets of specific

transduction pathways (Hunter et al., 1992) bind to specific response elements in

the regulated genes, and mediate the effects induced by neurotrophins. The

APP promoter has the typical structure of a housekeeping gene, lacking the

TATA and CAAT elements. Its sequence is extremely well conserved between

rat, mouse and human (Chernak, 1993), and contains multiple positive and

negative regulatory elements and consensus sequences for the binding of

several transcription factors (La Fauci et al., 1989; Salbaum et al., 1988;

Quertfurth et al., 1999; Wright et al., 1999; Vostrov et al., 1997). In agreement

with previous descriptions (Lahiri et al., 2000), we found that progressive

deletions of promoter sequences led to significant variations of the basal

promoter activity. This activity was maximal in the construct containing the -487

fragment of the APP promoter, suggesting the presence of a silencer element

between this position and the nucleotide -1099, and it was further decreased in

succesive deletions to nucleotides -307 and -15. Deletion to nucleotide -307

caused a significant decrease of basal activity, which could be secondary to the

loss of the "5'-GC element/AP1 site" tandem described in the -383/-358 region of

the promoter (Quertfurth et al., 1999). Further deletion of sequences to

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nucleotide -15 caused an additional reduction which very like represents the loss

of previously described response elements which are essential to maintain the

basal promoter activity (La Fauci et al., 1989; Salbaum et al., 1988; Quertfurth et

al., 1999; Vostrov et al., 1997).

The response to NGF was maintained in the plasmid which contains the

first 307 bp of the 5' upstream sequence, but it was significantly reduced in the

next deletion plasmid extending to nucleotide -15. These results indicate that

stimulation of the APP promoter activity by NGF is mainly mediated through

sequences located between nucleotides -307 and -15. In addition the smallest

promoter fragment used (-15/+102) retains a residual response that probably

involves direct effects of NGF on different components of the basal

transcriptional machinery, or alternatively the existence of regulatory sequences

located downstream of the main transcriptional initiation site.

Our results strongly suggest that transcriptional activation of APP gene

expression is responsible for the NGF-induced increase in APP transcripts

previously described by us in PC12 cells (Cosgaya et al., 1996). We have

reported that NGF increases APP mRNA levels in PC12 cells. Furthermore,

expression of activated ras in UR61 cells also leads to a significant increase in

APP transcripts, whereas the expression of a dominant negative mutant of ras

blocked the induction of APP gene expression by NGF in the M-M17-26 cell line.

Stimulation of transcription should lead also to an increase in the cellular content

of APP proteins. This was indeed observed in PC12 cells, where NGF treatment

resulted in a significant increase in the intracellular levels of the different APP

isoforms. Moreover, our results indicate that this effect was also largely

dependent of Ras. As also occurred with promoter activity (Cosgaya et al.,

1996), the NGF-induced increase of cellular APP observed in PC12 cells was not

reproduced in the M-M17-26 subline, which expresses the inhibitory mutant of

Ras, and was mimicked by dexamethasone-induced expression of Ras in UR61

cells. According to our results NGF appears to induce a generalized increase of

the different APP species detected. However, and since a complete definition of

bands has not been possible, we cannot exclude that, as previously described

(Fukuyama et al., 1993), NGF only affected the synthesis of APP695, thus

increasing specifically the levels of both the immature and mature forms of

APP695 in PC12 cells.

In addition, it has been also reported that NGF, as well as other

neurotrophins, regulate the secretion of sAPP in PC12 cells (Desdouits-Magnen

13

et al., 1998; Fukuyama et al., 1993; Refolo et al., 1989). In agreement with those

descriptions we have found that NGF increases the amount of sAPP

accumulated into the cultured medium in the parental PC12 cells. In contrast,

expression of activated Ras in the UR61 subline was unable to increase the

extracellular sAPP content. In addition, the increased release of sAPP induced

by NGF in PC12 cells was not completely abolished in the M-M17-26 cells which

express a dominant negative mutant of Ras. Therefore, and contrarily to those

effects induced by NGF on promoter activity, levels of mRNA, and cellular

content of APP, the regulation of APP secretion by NGF in PC12 cells appears

to be mainly mediated by a Ras-independent mechanism.

These results are in apparent contradiction with the description that

activation of the MAP kinase cascade in PC12 results in the rapid secretion of

sAPP isoforms. Nevertheless, it has been also reported that although activation

of MAP kinase promotes APP secretion, the inhibition of this kinase is not

sufficient to reduce APP secretion (Rossner et al., 1999). Taken together, these

results are compatible with the existence of a Ras-independent mechanism

responsible for APP secretion in response to NGF in PC12 cells. A number of

possible mechanisms could explain the specific MAP kinase-dependent / Ras-

independent regulation of APP secretion by NGF. Other TrkA-interacting

proteins, such as PLC- or PI 3-Kinase (Greene and Kaplan, 1995), or docking

proteins such as FRS2, that are phosphorylated in response to NGF stimulation

(Hadari et al., 1998; Kouhara et al., 1997) could be involved in the observed

effect. In addition, activation of protein kinase C (PKC) downstream of PLC-

could contribute to Ras-independent activation of MAP kinase through PKC

phosphorylation of Raf (Sozeri et al., 1992).

Our results demostrate that NGF activates transcription of the APP gene,

thus increasing expression of APP. If this also occurs in humans in vivo, the use

of NGF, proposed as a paliative treatment in Alzheimer, could rather result in a

risk factor for the disease. However, the neurotrophins can also increase the

release of neurotrophic sAPP fragments, which very likely should be

accompanied by a reduced generation and release of the -amyloid peptide. If

this is the case NGF could indeed be useful to define new therapeutic strategies

for the disease. Therefore, it will be of interest to analyze the effect of NGF on

APP gene expression and on the secretion of neurotrophic sAPP in humans.

14

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21

LEGEND TO THE FIGURES

Fig. 1.

Regulation of APP promoter activity by NGF and other neurotrophins in PC12

cells.

Promoter activity was determined in three different subclones of PC12 cells, the

native cell line (left panel), the M-M17-26 cell line which expresses a dominant

negative ras mutant (rigth panel), and UR61 cells which express the N-ras (val-

12) oncogene in response to dexamethasone (middle panel). The cells were

transfected with a CAT reporter gene containing the -1099/+105 fragment of the

APP promoter, and CAT activity was measured after a 48 h period of incubation

in the presence or absence of 100 nM dexamethasone (Dex), 50 ng/ml NGF, 17

ng/ml bFGF or60 ng/ml EGF. CAT activity was normalized for total protein, and

results are expressed as fold induction over the levels obtained in the

corresponding control untreated cells. Data are the mean ± SD from three

independent experiments.

Fig. 2.

Stimulation of APP promoter activity by NGF is mediated by the MAP kinase

signaling pathway in PC12 cells.

The CAT plasmid containing the -1099/+105 fragment of the APP promoter was

transfected alone or in combination with dominant negative forms (dn) of Ras,

Raf and MAPK. CAT activity was determined in untreated cells and in cells

treated with 50 ng/ml NGF for 48 h. CAT activity is expressed in each case as

fold induction above the control values found in NGF-untreated cells, and

represent mean ± SD values of four determinations (duplicates from two

independent experiments).

Fig. 3.

Analysis of DNA regions that mediate the effect of NGF on the APP promoter.

PC12 cells were transiently transfected with pBL-CAT plasmids containing

progressive deletions of the APP promoter and CAT activity was determined in

cell extracts after a 48 h incubation in the absence or presence of NGF. Data are

expressed relative to CAT activity obtained with the construct containing the -

1099 to + 105 bp fragment and are the mean ± SD from two separate

experiments performed with duplicates.

22

Fig. 4.

Western blot analysis of cellular and secreted APP isoforms.

After 48 h of incubation in the absence or presence of NGF (PC12 and M-M17-

26 cells), or dexamethasone (UR61 cells), the intracellular and secreted isoforms

of APP were analyzed by western blot with the antibodies 369 A (A) and 22C11

(B), respectively. The figure include the blots obtained in a representative

experiment. The apparent size (kilodaltons) of the protein bands is indicated at

the rigth by arrows. The relative changes induced by the different treatments are

indicated by a number, at the bottom, that represents the mean ± SD obtained

from three independent experiments

23

ACKNOWLEDGMENTS

This research was funded by grants from the Spanish Comisión Interministerial

de Ciencia y Tecnología (SAF 97-0183) and Dirección General de Investigación

de la Comunidad de Madrid (08.5/0036/1998. Ana Villa was recipient of a

fellowship from Consejo Superior de Investigaciones Científicas - Glaxo-

Wellcome. We thank DRs. G. M. Cooper and A. Pellicer for the PC12 subclones,

D. K. Lahiri and N. K. Robakis for providing the APP promoter and S. E. Gandy

for the polyclonal antibody 369A .We also thank Dr. A. Aranda for critical reading

of the manuscript.