Activated cAMP-response Element-binding Protein Regulates Neuronal Expression of Presenilin-1*

Noriaki MitsudaDagger §, Nobutaka OhkuboDagger §, Michio TamataniDagger §, Young-Don LeeDagger §, Manabu TaniguchiDagger §, Kazuhiko Namikawa||, Hiroshi Kiyama||, Atsushi YamaguchiDagger §, Naoyuki Sato**, Kazuko SakataDagger §, Toshio Ogihara**, Michael P. VitekDagger Dagger , and Masaya TohyamaDagger §

From the Dagger  Department of Anatomy and Neuroscience and ** Department of Geriatric Medicine, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan, § CREST, Japan Science and Technology, || Department of Anatomy, Asahikawa Medical College, Asahikawa, Hokkaido 078-8510, Japan, and the Dagger Dagger  Division of Neurology, Duke University Medical Center, Durham, North Carolina 27710

Received for publication, July 12, 2000, and in revised form, November 20, 2000


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Upon binding to the cAMP-response element of a gene's promoter, the transcription factor known as cAMP-response element-binding protein (CREB) facilitates transcription of many different neuronal genes including those involved with synaptic function. Based on our previous reports of gene structure (GenBankTM accession number AF029701), we now demonstrate that activated CREB binds to the proximal promoter of the human presenilin-1 (PS-1) gene to activate PS-1 transcription in rat and in human neuronal cells. Specific stimulation of the N-methyl-D-aspartate subtype of neuronal glutamate receptors activates CREB and results in increased PS-1 expression. Similarly, treatment with brain-derived neurotrophic factor activates CREB and increases PS-1 expression in a dose-dependent fashion. By using adenovirus vectors expressing dominant negative forms of CREB, we were able to show that induction of PS-1 expression requires the activation of CREB. Conversely, constitutive expression of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) results in activation of CREB and increased PS-1 expression that can be blocked by the addition of selective MEK inhibitors. Our findings suggest a hypothesis where stimulation of N-methyl-D-aspartate receptors signals CREB activation to enhance PS-1 gene product expression that contributes to normal neuronal functions.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Alzheimer's disease is a devastating neurological disorder characterized by progressive memory loss and cognitive deficits. To date, four genes have been found to be associated with Alzheimer's disease phenotypes including the amyloid precursor protein gene on chromosome 21 (1-4), the apolipoprotein E gene on chromosome 19 (5-7), the presenilin-1 (PS-1)1 gene on chromosome 14 (8), and the presenilin-2 (PS-2) gene on chromosome 1 (9, 10). The majority of familial Alzheimer's disease cases are associated with over 40 independent mutations in the PS-1 genes of unrelated families that all display an early-age-of-onset Alzheimer's phenotype (8, 11-25). Although the current literature suggests that intracellular PS-1 is intimately associated with the gamma -secretase activity that facilitates amyloid-beta peptide release (26), the precise function of PS-1 proteins found in the synaptic membranes remains elusive and is also a matter of active investigation.

Human PS-1 is widely expressed in a variety of organs throughout the body (8). This distribution raises the question as to why mutations in the PS-1 gene and its products appear to confer a disease state that selectively affects the brains of patients with familial Alzheimer's disease without apparent effect on their peripheral organs. In contrast, rat PS-1 is poorly expressed in liver, kidney, lung, and heart (27), and mouse PS-1 is poorly expressed in skeletal muscle and spleen.2 We also reported that the PS-1 promoter was more active in cells differentiated to become neurons than in cells differentiated to a muscle phenotype (28). These differences suggest both cell type-specific and species-specific distributions of PS-1 expression. The brain, however, is the only organ where PS-1 is universally well expressed in human, rat, and mouse species. Using in situ hybridization, PS-1 mRNA is most highly expressed in neurons and below the limit of detection in other brain cells (29, 30). These results strongly suggest the existence of a mechanism where PS-1 transcription is regulated in a neuron-specific manner in the brain.

To understand presenilin-1 function, one of our approaches is to study regulatory mechanisms in the brain. PS-1 expression is higher in the hippocampus and cerebellum than in the cerebral cortex (29-32). Hippocampal pyramidal neurons, dentate granule neurons, cerebellar Purkinje neurons, and primary olfactory cortical neurons show higher PS-1 expression than the neurons in other cortical regions of rat brain (33, 34). Compared with the adult, the level of PS-1 expression is significantly higher in the developing rat brain. Peaking at postnatal day 10, PS-1 expression in the hippocampus and cerebellum is greatest at a time when proliferation, migration, and synapse formation are actively being completed (35).

Like PS-1, N-methyl-D-aspartate (NMDA) receptors are well expressed in hippocampal pyramidal neurons and dentate granule neurons at this time of development (36-39). Activation of NMDA receptors and consequent Ca2+ entry into postsynaptic spines are critical to the developmental maturation of the brain, to establishment of long term memory, and to the plasticity of synaptic transmission that are characteristic of functional brain neurons (40-44). Furthermore, glutamate released from presynaptic terminals can bind to glutamate receptors at the postsynaptic membrane stimulating Ca2+ influx, phosphatase, and kinase activities that serve to modulate several intracellular signaling systems. Of these systems, phosphorylation of cAMP-response element-binding protein (CREB) activates a signal transduction cascade that eventually modulates downstream protein synthesis in postsynaptic neurons (45-48).

An important relationship between these systems emerged when sequencing of the PS-1 promoter showed the existence of a CREB binding site near the major transcription start site. If CREB regulates the expression of PS-1, then control of CREB activation may be one of the keys to understanding the differences in PS-1 expression between neurons, between nonneuronal cells, during the steps of synapse formation, and during signal transmission at synapses. We now report that activation of CREB leads to increased PS-1 expression in a dose-dependent fashion. We also find that PS-1 colocalizes with neurons expressing NMDA receptors. Thus, we now test the ability of NMDA receptors to signal CREB activation and specifically stimulate PS-1 expression. Our findings support a hypothesis where NMDA-mediated CREB activation regulates PS-1 expression to play a potentially critical role in physiological and pathological, neuron-specific functions of presenilin-1.

    EXPERIMENTAL PROCEDURES
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Reagents-- U0126 and brain-derived neurotrophic factor (BDNF) were purchased from Promega (Madison, WI). Nifedipine and NMDA were purchased from Sigma. (+)-MK801 hydrogen malate was purchased from Research Biochemicals International (Natick, MA). Monoclonal antibody to the amino-terminal fragment of PS-1 (anti-PS-1-NTF) was purchased from Chemicon International (Temecula, CA). Anti-CREB monoclonal antibody (CREB-1, 24H4B) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Cell Culture-- SK-N-SH human neuroblastoma cells were purchased from the American Type Culture Collection and were routinely propagated in Dulbecco's modified Eagle's medium (Sigma) with 10% fetal calf serum (Life Technologies, Inc.). Cultures of rat primary hippocampal neurons were prepared from Harlan Sprague-Dawley rat embryos at embryonic day 18 as described previously (49). All experiments were performed in 5-8-day-old cultures.

Isolation of Genomic DNA Clones for the Promoter Region-- The PCR primer pairs 951/952 and 910/911 amplify PS-1 specific sequences (8) and were used with PCR to screen a human genomic library in a PAC vector, kindly provided by Dr. Pannos A. Ioannu (50). Out of the three PAC clones identified, PAC94 was digested with EcoRI, and the restriction enzyme fragments were subcloned into pBluescript-II-KS (+) phagemid vector (Stratagene) using a DNA ligation kit (Stratagene). The oligonucleotide probe (5'-TGGGACAGGCAGCTCCGGGGTCCGCG-3') from exon 1 of the human PS-1 gene, labeled with gamma -32P and T4-polynucleotide kinase, was used to identify plasmid subclones that contain the putative promoter region by hybridization. One of the positively hybridizing plasmids, clone A4, was sequenced using an Applied Biosystems model 373A automated DNA sequencer with a dye terminator cycle sequencing kit and protocols (PE Biosystems) recommended by the manufacturer.

Searching Transcription Factor Binding Sites-- Potential binding sites of vertebrate transcription factors in the human PS-1 promoter were identified using the TFSEARCH (version 1.3) network service available from Genome Net in Japan.

Isolating Nuclear Protein Extracts to Confirm Functional Transcription Factor Binding Sites-- Nuclear extracts were prepared from NMDA-stimulated SK-N-SH cells according to published methods (51-52) with minor modifications. In brief, cells were stimulated with 100 µM NMDA for 30 min, harvested by scraping, and washed with 0.5 volumes of ice-cold phosphate-buffered saline followed by a gentle centrifugation. The cells were then washed in 0.1 volume of cold buffer A (10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol). The washed cell pellets were then suspended in 50 µl of buffer A plus 0.1% Nonidet P-40 supplemented with 1 µg/ml leupeptin and 1 µg/ml aprotinin and incubated on ice for 10 min before briefly vortexing to mix the pellets, followed by centrifugation at 10,000 rpm at 4 °C for 5 min in a microcentrifuge. The supernatant was carefully removed, and the nuclear pellet was resuspended in 20 µl of ice-cold buffer C (20 mM HEPES, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) containing 1 µg/ml leupeptin and 1 µg/ml aprotinin and then incubated on ice for 15 min with intermittent vortexing. The extracts were then centrifuged at 10,000 rpm at 4 °C for 10 min, and the supernatant was removed and divided into aliquots for freezing at -70 °C and used as purified nuclear extract. Protein concentrations were determined using the Bio-Rad protein assay kit.

Electrophoretic Mobility Shift Assay-- Probes corresponding to the CRE-like element of the human PS-1 promoter sequence (PS1-CRE; 5'-GAGCCGGAAATGACGACAACGGTGAGG-3') and to a mutated CRE-like site containing three nucleotide substitutions (PS1-CREm; 5'-GAGCCGGAAATGATAGCAACGGTGAGG-3') were synthesized as complementary double-stranded oligonucleotides. As a control probe, a consensus CRE element contained in a double-strand oligonucleotide probe (5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3') was purchased from Promega. The probes were 5'-end labeled with [gamma -32P]ATP and T4-polynucleotide kinase and purified with Nucleotrap Push Columns (Stratagene). 5 fmol of labeled probes were incubated with 5 µg of SK-N-SH nuclear extract in a volume of 25 µl containing 0.33 M urea, 0.1 M NaCl, 0.33% Nonidet P-40, 25 mM HEPES, 10 mM dithiothreitol, 10% glycerol, 5 µg of bovine serum albumin, and 2 µg of poly(dI-dC) (Amersham Pharmacia Biotech) for 30 min at room temperature. For antibody supershifts, nuclear extracts were incubated with anti-CREB monoclonal antibody overnight at 4 °C prior to the addition of labeled probes. For competition experiments, a 10-fold excess of cold competitor probe was preincubated with nuclear extracts for 15 min at 4 °C prior to the addition of labeled probes. Incubation mixtures were subjected to 6% polyacrylamide gel electrophoresis using high ionic strength buffer (50 mM Tris-HCl, pH 8.5, 400 mM glycine, 2 mM EDTA) as running buffer. Gels were vacuum-dried and autoradiographed.

Construction of PS-1 Promoter-Firefly Luciferase Reporters-- The plasmid clone A4, which contains a human PS-1 genomic DNA fragment inserted into the EcoRI site of pBluescript II KS (+) vector, was double-digested with NotI and one of SalI, HindIII, or KpnI restriction enzymes, respectively. The promoter fragments were purified with Wizard PCRpreps DNA Purification System (Promega), blunted with a DNA blunting kit (Takara Biochemicals), and inserted into the SmaI site of promoterless pGL3-basic vector (Promega) to make LUC5, LUC4, and LUC3. Similarly, clone A4 was digested with HindIII alone or HindIII and KpnI, blunted, and inserted into pGL3 to make LUC2 and LUC1, respectively. Two synthetic oligonucleotides, PS1-CRE-S (5'-TCGAGAGCCGGAAATGACGACAACGGT-3') and PS1-CRE-AS (5'-AGCTACCGTTGTCGTCATTTCCGGCTC-3') were 5'-phosphorylated with ATP and T4-polynucleotide kinase, mixed together, heated to 95 °C to denature, gradually cooled down to room temperature to anneal, and then inserted into the XhoI/HindIII site of pGL3-basic vector to make LUC6. Similarly, two synthetic oligonucleotides, PS1-CREm-S (5'-TCGAGAGCCGGAAATGATAGCAACGGT-3') and PS1-CREm-AS (5'-AGCTACCGTTGCTATCATTTCCGGCTC-3'), which are identical to the PS1-CRE-S and PS1-CRE-AS, respectively, except for the substitution of three nucleotides at the CRE-like site, were inserted into the XhoI/HindIII site of pGL3-basic vector to make LUC6m.

Transient Transfection and Luciferase Assays-- SK-N-SH cells were plated in six-well tissue culture dishes at 9 × 104 cells/well and allowed to recover for 1 day. Cells containing PS-1-promoter/reporter constructs were then cotransfected with 0.3 pmol of one of the promoter-firefly luciferase plasmid constructs, pGL3-basic vector or pGL3-promoter plasmid (which contains an SV40 promoter upstream of the firefly luciferase gene; Promega), and 0.3 pmol of pRL-TK plasmid (which contains a herpes simplex virus thymidine kinase promoter upstream of the Renilla luciferase gene; Promega), using the Lipofectin procedure (Life Technologies, Inc.) as described in the manufacturer's protocol. Transfected cells were cultured for 24 h, washed twice with 2 ml of Ca2+- and Mg2+-free PBS, and lysed with Passive Lysis Buffer (Promega). Firefly luciferase and Renilla (sea pansy) luciferase activities were measured sequentially using the Dual-Luciferase Reporter Assay System (Promega) and a model TD-20E luminometer (Turner Design). After measuring the firefly luciferase signal (FL) and the Renilla (sea pansy) luciferase signal (RL), the relative promoter activity (RPA) was calculated as follows: RPA = FL/RL.

Northern Blot Hybridization Analysis-- Total RNA extracted from SK-N-SH cells after incubation with or without NMDA for 24 h was Northern blotted at 20 µg/lane. A 368-bp human PS-1 cDNA probe sequence was prepared using PCR with sense primer (5'-CGGGGAAGCGTATACCTAATC-3'), antisense primer (5'-TCAGCTCCTCATCTTCTTCCTCATC-3'), and human PS-1 cDNA as template. The probe was 32P-labeled using the Random Primed DNA Labeling Kit (Roche Molecular Biochemicals) and hybridized to Northern blots.

Recombinant Adenovirus-- Constitutively active mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) adenovirus (Ad-caMEK) was constructed as follows. cDNA fragments comprising the entire coding regions for human MEK1 cDNA were isolated from HEK293 cell cDNAs by using PCR with sense (sequence of sense) and antisense (sequence of antisense) primers. Constitutively active MEK, which lacks its nuclear export signal (amino acids 30-51; Ref. 53) and in which two phosphorylation sites, serine 218 and serine 222, are converted to glutamic acids, was prepared by site-directed mutagenesis as described previously (54). Subsequently, a c-Myc tag sequence was fused to its N-terminal by PCR. The fragment was inserted into pAxCAwt cosmid vector (55) and was designated as pAxCAca-MEK.

A dominant negative form of CREB with a mutation in the DNA binding domain (K-CREB adenovirus or Ad-KCREB; Ref. 56) was constructed as follows. A plasmid bearing wild type rat CREB cDNA was kindly provided by Dr. Hagiwara (Tokyo Medical and Dental University, Japan). The coding sequence was amplified, and a c-Myc tag sequence was fused to its N terminus by PCR. Subsequently, leucine was substituted for arginine 301 by site-directed mutagenesis using the QuickChange Site-Directed Mutagenesis Kit (Stratagene). The fragment was subcloned into pAxCALNLw, Cre-inducible expression cassette (16), and was designated as pAxCALNL-KCREB.

The cosmids, pAxCAca-MEK and pAxCALNL-KCREB, were separately transfected into 293 cells together with the EcoT221-digested adenoviral DNA-terminal protein complex (55) using the calcium phosphate precipitation method. The desired recombinant adenoviruses generated through homologous recombination were purified through a CsCl2 gradient followed by dialysis.

The recombinant adenovirus, AxCANCre, which efficiently produces a nuclear localization signal-tagged Cre recombinase under control of the CAG promoter (57), and AxCANLZ, which can express LacZ under control of the CAG promoter (16), were generously provided by Dr. Saito (University of Tokyo, Japan).

CREB-M1 adenovirus, a dominant negative form of CREB in which Ser-133 is converted to an alanine and that cannot be phosphorylated or activated, was a generous gift from Dr. Anthony Zeleznik (University of Pittsburgh School of Medicine; Ref. 58).

All of the viruses were grown in 293 cells and purified by CsCl2 gradient centrifugation. Virus titers were determined by plaque assay, and concentrated virus was stored at -80 °C. Infection was carried out by adding recombinant adenoviruses to serum-containing medium. The cells were incubated at 37 °C for 60 min with constant agitation. The medium was changed, and the cells were incubated at 37 °C for 24 h before use unless otherwise stated.

Metabolic Labeling and Electrophoresis-- Confluent SK-N-SH cells and rat primary cultured hippocampal neurons were prepared in six-well dishes. Medium was changed to the methionine-free Dulbecco's modified Eagle's medium (Life Technologies, Inc.) with 2% dialyzed fetal bovine serum (Life Technologies, Inc.) and incubated for 7 h, followed by pulse labeling with 200 µCi/ml [35S]methionine for 1 h. The cells were then lysed immediately with ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.5 mM Na3VO4, 0.1% (v/v) 2-mercaptoethanol, 1% Triton X-100, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 10 mM sodium glycerophosphate, 0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml pepstatin, 1 µg/ml leupeptin, and 1 µM microcystin). For NMDA stimulation experiments, 10 or 100 µM NMDA was added to the medium 6 h prior to the addition of radioisotope. For inhibition experiments, 100 µM MK801 or 20 µM nifedipine was added to the medium when the medium was changed. For adenovirus transfection experiments, the cells were transfected with 10-50 multiplicity of infection (m.o.i.) adenovirus 24 h prior to the change of medium. For time course experiments, radioisotope was added to the medium at the indicated time after 100 µM NMDA was added, and then the cells were lysed. Immunoprecipitation with polyclonal antibody against the N-terminal fragment of PS-1 was performed as described previously (59). Electrophoresis was performed with 10% Tris-glycine polyacrylamide gel. The gels were vacuum-dried and autoradiographed.

Quantification of PS-1 mRNA and Labeled PS-1 Protein-- The amount of PS-1 mRNA and labeled PS-1 protein was quantified by scanning the density of radiolabeled or immunodetected bands on x-ray film using ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Characterization of the Human PS-1 Promoter Sequence-- We isolated and sequenced a 5571-bp human genomic DNA that contains the human PS-1 promoter. This genomic DNA includes 4353 bp of the 5'-flanking sequence of exon 1 and exon 2 (Fig. 1 and GenBankTM Accession number AF029701). The comparison of our sequence for the human PS-1 promoter with previously reported sequences (Ref. 60 and GenBankTM accession number L76518) indicated general agreement with several minor differences. This region contains four Alu subfamily repeat sequences where the 3'-end point of the last Alu sequences is position number -812. The region spanning from position -811 to the end of exon 1 at position +140 contains several specific sequence motifs that are known to serve as binding sites for transcription factors. These include one Lyf-1 site, two CdxA sites, one AML-1a site, one Nkx-2 site, one Pbx-1 site, one E47 site, one MZF1 site, two Sp1 sites, one Elk-1 site, and one CREB site for transcription factors that may bind to these motifs and regulate expression of the PS-1 gene (Fig. 1b).


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Fig. 1.   Structure of transcripts and promoter of PS-1 gene. A, structure of two different presenilin-1 transcripts from human brain. DNA sequencing of the cloned products of 5'-rapid amplification of cDNA ends on human brain cDNA revealed the presence of two independent transcripts (transcripts A and B), which appear to derive from two unique transcription start sites. A single vertical arrow represents the major transcription start site at position +1, while a double vertical arrow represents the minor transcription start site at position +344. The position of the major start site was certified by the primer extension method (data not shown). Exonic regions are labeled and are separated by unlabeled introns. Splicing patterns are derived from comparison of cDNA sequence with genomic DNA. The bent arrow represents the translation initiation site. B, structure of 4.5-kb human presenilin-1 promoter region. The human 4.5-kb presenilin-1 promoter region contains four Alu-subfamily sequences. The 4.5-kb full sequence appears in GenBankTM (accession number AF029701). Hatched boxes represent Alu subfamily sequences. The first Alu subfamily sequence starts 812 bp upstream from the major transcription start site. Sequence homologies to consensus motifs for transcription factor binding sites are marked. C, putative transcription factor binding sites on PS-1 proximal promoter around the major transcription start sites. We performed primer extension with an antisense primer (horizontal arrow) and total cellular RNA from SK-N-SH cells and found the major transcription start site. Human PS-1 transcription begins with "A" at position +1 of exon 1 as indicated with a vertical arrow. Our transcription start site (bent arrow) is located 26 bp upstream of the reported start site (60). Pastorcic et al. (65) confirmed this new location of the major transcription start site of PS-1. Consensus binding sites for the transcription factors Elk-1, CREB, and Sp1 are underlined and labeled.

Human PS-1 Proximal Promoter Binds to CREB-- To verify that the CRE sequence appearing in the PS-1 proximal promoter functionally binds to the CREB transcription factor, we synthesized the double-stranded oligonucleotide probe with the PS-1 sequence (PS1-CRE) and the probe identical to PS1-CRE except for the substitution of three nucleotides at the CRE-like site (PS1-CREm) and used them for electrophoretic mobility shift assays. A similar, but nonidentical double-stranded oligonucleotide with a consensus sequence that binds CREB (CRE) was used as a positive control. The PS1-CRE sequence contains a TGACGACA element, the consensus CRE sequence contains a TGACGTCA element, and the PS1-CREm sequence contains a TGATAGCA element (Fig. 2A).


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Fig. 2.   Interaction of PS-1 proximal promoters and CREB. Electrophoretic mobility shift assay revealed binding of CREB to the human presenilin-1 proximal promoter containing putative binding sites. A, the nucleotide sequences of the PS-1 CRE probe (PS1-CRE), the control CRE probe (CRE), and the mutant PS-1 CRE probe (PS1-CREm). The consensus CREB-binding site of CRE probe and the putative CREB-binding site of PS1-CRE and PS1-CREm are underlined. The PS1-CRE is identical to the CRE except for the nucleotide substitution of A for T. The PS1-CREm is identical to the PS1-CRE except for the substitution of three nucleotides at the CRE-like site. B, binding of nuclear proteins from NMDA-stimulated SK-N-SH cells to radiolabeled PS1-CRE probe, CRE probe, and PS1-CREm probe in an electrophoretic mobility shift assay. a and b, band of CREB-CRE complex. PS1-CREm probe did not show either a or b, suggesting that the putative CRE-binding sequence in PS1-CRE probe is crucial for binding to CREB. C, binding of nuclear proteins from NMDA-stimulated SK-N-SH cells to radiolabeled control CRE probe in an electrophoretic mobility shift assay. a and b, bands of CREB-CRE complex. ss, band that supershifted with anti-CREB monoclonal antibody. Anti-beta -galactosidase polyclonal antibody was used as a control antibody, and no supershift was observed. Excess cold CRE probe inhibited the formation of CREB-CRE complex. D, binding of nuclear proteins from NMDA-stimulated SK-N-SH cells to radiolabeled PS1-CRE probe in an electrophoretic mobility shift assay. a and b, band of CREB-PS1-CRE complex. ss, band that supershifted with anti-CREB monoclonal antibody. Excess cold PS1-CRE probe inhibited the formation of CREB-CRE complex.

Each of these elements was radiolabeled and used to probe the binding proteins present in nuclear extracts of cells. Radiolabeled CRE probe incubated with a nuclear extract from NMDA-stimulated SK-N-SH cells formed complexes that were observed on nondenaturing polyacrylamide gels as one strong band and one weaker band (Fig. 2B, bands a and b). To show that these complexes contain the CREB protein, anti-CREB monoclonal antibody was added to the reaction, and the observed band was supershifted to an apparently higher molecular weight (Fig. 2C, band ss). To show that the antibody reaction was specific for CREB protein, a control antibody (anti-beta -gal) was added to the reaction, and no supershift band was observed (Fig. 2C, lane 4), indicating that the supershift effect of the anti-CREB antibody is specific for CRE/CREB-containing complexes.

Using this assay, we then tested whether the CRE-like element present in the PS-1 promoter region could function to bind CREB proteins. Incubation of radiolabeled PS1-CRE probe with nuclear extracts revealed the same a and b bands that were observed when a consensus CRE probe was employed (Fig. 2B). When anti-CREB antibody was added to the PS1-CRE reaction, a supershifted band was observed (Fig. 2D, band ss), indicating that CREB is a component of this protein-PS1-CRE-probe complex as well. As another control, the addition of a molar excess of unlabeled PS1-CRE oligonucleotide probe to the nuclear extracts inhibited detection of this protein-PS1-CRE-probe complex (Fig. 2D, lane 4). The specificity of the complex formation between CREB proteins and PS1-CRE-probes was also tested using a mutated PS1-CRE probe. Incubation of radiolabeled PS1-CREm (mutated) probe with nuclear extracts did not reveal the a or b band (Fig. 2B, lane 4), suggesting that this putative CRE element in the PS-1 promoter is crucial for forming CREB-DNA complexes.

CREB-responsive Element in the PS-1 Proximal Promoter Is Functional-- To test whether the PS1-CRE element identified in the proximal promoter could participate in transcriptional activity, we combined this element with a firefly luciferase reporter (Promega; pGL3-basic vector) to look for CREB-mediated activity in human SK-N-SH cells of neuronal origin. As an internal standard, we used the pRL-TK vector (Promega), which contains the Renilla luciferase gene driven by a herpes simplex virus thymidine kinase promoter. Of all of the DNA fragments tested for promoter activity, plasmid LUC-3 with the human PS-1 fragment -297 to +189 showed the greatest amount of firefly luciferase activity relative to the Renilla luciferase internal control. This ratio of LUC-3 activity to Renilla activity was defined as 100% of relative promoter activity (100% RPA) (Fig. 3A). Larger DNA fragments such as LUC-4 (-1191 to +189) and LUC-5 (-4311 to +189) displayed 84% and 25% of the LUC-3 activity (84% and 25% RPA). LUC-1 (-297 to +1258) and LUC-2 (-1191 to +1258) each displayed less than 10% RPA, suggesting that they contained negative elements that apparently reduce overall promoter activity. Although LUC-6 (-18 to +8) was the shortest fragment tested, it contains a putative CREB binding site and a putative Elk-1 site and displayed 23% RPA. LUC6m, which is identical to LUC-6 except for the substitution of three nucleotides at the CRE-like site, displayed only 8.2% RPA, suggesting that the PS1-CRE element accounts for about two-thirds of the functional activity of this PS-1 promoter fragment.


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Fig. 3.   26-bp core element is critical for human presenilin-1 promoter activation. A, human presenilin-1 promoter-reporter constructs and their relative luciferase activity (%RLA) under unstimulated conditions. Of all the DNA fragments tested for promoter activity, plasmid LUC-3 with the human PS-1 fragment -297 to +189 showed the greatest ratio of firefly activity to Renilla internal control, which was defined as 100% of relative luciferase activity (100% RLA). B, phosphorylation of CREB at Ser-133 was induced with 100 µM NMDA in SK-N-SH cells. Western blots of cell lysates from SK-N-SH cells stimulated with 100 µM NMDA for the indicated time were probed with anti-CREB and anti-phospho-CREB (p-CREB) antibodies. C, transcription activity was induced with 100 µM NMDA in SK-N-SH cells. SK-N-SH cells were transiently transfected with CRE-SEAP plasmid (CLONTECH), which contains the SEAP reporter gene downstream of three copies of the CRE-binding sequence fused to a TATA-like promoter region from the herpes simplex virus thymidine kinase promoter. Alkaline phosphatase activities in the media at the indicated times were measured using Great EscApe SEAP Chemiluminescence Detec tion Kit (CLONTECH). *, p < 0.01 compared with unstimulated control using Student's t test. D, NMDA-transactivated PS-1 promoters. SK-N-SH cells were transiently cotransfected with one of the human presenilin-1 promoter-reporter constructs and pRL-TK plasmid. Cells were cultured for 24 h with (NMDA) or without 100 µM NMDA (Unstimulated), FL and RL activities were measured sequentially, and the RPA was calculated as follows: RPA = FL/RL. -Fold inductions are shown. Each value represents the average of at least four independent determinations, and the error bars indicate the S.E. **, p < 0.01 using Student's t test (n = 3). E, MK801 inhibited the activation of PS-1 promoter by NMDA. The cells were preincubated with 100 µM MK801, a selective NMDA antagonist, prior to the addition of 100 µM NMDA. **, p < 0.01 using Student's t test (n = 3)

Since basal levels of activated CREB are rather low in these cells, we asked whether NMDA treatment of these cells would activate CREB by enhancing its phosphorylation at Ser-133, subsequently resulting in increased PS-1 expression. 15 min after adding 100 µM NMDA, CREB was phosphorylated at Ser-133 and remained significantly phosphorylated for another 360 min as shown on Western blots (Fig. 3B). We then tested whether phosphorylation of CREB led to increased transcriptional activity when CRE element-containing promoters were present. As a positive control, a CRE-SEAP reporter plasmid (CLONTECH), which contains three copies of a consensus CRE element fused to a TATA-like promoter from the herpes simplex virus thymidine kinase promoter and a secreted alkaline phosphatase (SEAP) gene, was transfected into SK-N-SH neuroblastoma cells. NMDA treatment of these cells led to a dose- and time-dependent increase of extracellular phosphatase activity (Fig. 3C). These results suggest that NMDA treatment stimulates phosphorylation of CREB, which appears to function and increase CRE-mediated levels of SEAP activity.

To test whether the PS1-CRE element would also respond to NMDA treatments that activate CREB, we returned to our luciferase activity system to measure functional responses. NMDA treatment of SK-N-SH cells displayed 2.3-fold increase in LUC-3 (-297 to +189) activity and 1.6-fold increase in LUC-4 (-1191 to +189) activity compared with nonstimulated conditions (Fig. 3D). When LUC-6 (positions -18 to +8), the shortest PS-1 promoter fragment that contains a CRE element, was tested, NMDA treatment displayed a 3.7-fold increase in its activity compared with nonstimulated conditions. In contrast, the mutated CRE sequence in LUC-6m displayed similar RPA levels with or without NMDA treatment. On the other hand, NMDA treatments had no significant effect in cells transfected with LUC-1 (positions -297 to +1258), LUC-2 (positions -1191 to +1258), or LUC-5 (positions -4311 to +189) (Fig. 3D).

As a control, cells were preincubated with the selective NMDA receptor antagonist, MK-801, and the subsequent effect of NMDA to induce PS-1 promoter-directed luciferase activity was significantly inhibited (Fig. 3E). These results suggest that the CRE element contained in the 26-bp fragment of the PS-1 promoter spanning positions -18 to +8 can bind phosphorylated CREB and activate transcription at the major transcription start site (position +1).

NMDA Treatment Induces PS-1 Gene Expression-- To extend our finding that CREB activates PS-1 reporter gene expression, we also evaluated the ability of activated CREB to increase expression of the endogenous PS-1 gene products. Like the NMDA-dependent phosphorylation of CREB, NMDA dose-dependently increases the level of PS-1 mRNA in SK-N-SH neuroblastoma cells (Fig. 4). With respect to protein expression, we found that treatment of SK-N-SH cells with NMDA for 6 h followed by a 1-h pulse label with [35S]methionine resulted in a dose-dependent increase of labeled PS-1 protein levels (Fig. 5A). In these experiments, full-length PS-1 (PS-1FL) was observed as a strong band at 45 kDa, and the amino-terminal fragment of PS-1 (PS-1NTF) was observed as faint bands at 30 kDa, suggesting that full-length PS-1 was detected before its cleavage into amino-terminal and C-terminal fragments (61, 62). PS-1 protein synthesis appears to start at 4 h after NMDA treatment and lasts until 18 h after the treatment (Fig. 5C). We could also detect several bands that migrated more slowly than the full-length monomeric PS-1 protein, suggesting that they were aggregated forms of PS-1 and/or its fragments (PS-1ag).


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Fig. 4.   Northern blot hybridization analysis. The SK-N-SH cells were cultured with the indicated concentration of NMDA for 6 h, RNA was extracted from the cells, and PS-1 mRNA was quantified with Northern hybridization analysis. *, p < 0.01 using Student's t test (n = 3).


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Fig. 5.   Metabolic labeling with [35S]Methionine Clarified the Induction of PS-1 by NMDA and BDNF in SK-N-SH cells. The horizontal arrows indicate aggregated PS-1 (PS-1ag), full-length PS-1 (PS-1FL), and amino-terminal fragment of PS-1 (PS-1NTF). A, NMDA induced PS-1, and both MK801 and nifedipine inhibited the induction. Confluent SK-N-SH cells were prepared in six-well dishes. Medium was changed to the methionine-free Dulbecco's modified Eagle's medium with 2% dialyzed fetal bovine serum and incubated for 1 h. Then NMDA was added to the medium at the indicated concentration and incubated for 6 h, followed by pulse labeling with 200 µCi/ml [35S]methionine for 1 h. The cells were lysed immediately, and immunoprecipitation with polyclonal antibody against N-terminal fragment of PS-1 was performed. Electrophoresis was performed with 10% Tris-glycine polyacrylamide gel. The gels were vacuum-dried and autoradiographed. For inhibition experiments, 100 µM MK801 or 20 µM nifedipine was added to the medium when the medium was changed. *, p < 0.01 compared with unstimulated control using Student's t test (n = 3); **, p < 0.01 compared with each other using Student's t test (n = 3). B, U0126-inhibited and dominant negative CREBs reduced PS-1 induction by NMDA. 10 µM U0126 was added to the culture medium when the medium was changed to the methionine-free one, or the cells were transfected with 50 m.o.i. adenovirus bearing control LacZ cDNA or one of the dominant negative CREB cDNAs (CREB-M1 and K-CREB) 24 h prior to change of the medium. C, time course experiments. 200 µCi/ml [35S]methionine was added to the medium at the indicated time after 100 µM NMDA was added. D, induction of PS-1 by BDNF. SK-N-SH cells were cultured with the indicated concentration of BDNF for 6 h, followed by pulse labeling with 200 µCi/ml [35S]methionine for 1 h.

In cells preincubated with MK801, a selective antagonist of the NMDA receptor, or with nifedipine, a selective blocker of the L-type voltage dependent Ca2+ channel (LVDCC), the effect of NMDA treatment to increase PS-1 protein levels was inhibited. These inhibitor treatments support a specific and selective role for NMDA and calcium in stimulating PS-1 gene expression (Fig. 5A). Since NMDA receptor activation can lead to signal transduction mediated through the MEK kinase, then inhibition of MEK kinase activity may modulate PS-1 expression. Preincubation with 10 µM U0126, a selective MEK inhibitor (63), reduced PS-1 induction, suggesting that this kinase activity was necessary for PS-1 induction at the protein level (Fig. 5B).

Relationship of NMDA and CREB to PS-1 Induction-- To test whether CREB directly regulates endogenous human PS-1 expression, we used adenovirus vectors to express different dominant negative forms of CREB before NMDA treatments of neuroblastoma cells and then measured PS-1 expression levels. K-CREB has a mutation in the domain that binds to CRE elements in DNA (56), and CREB-M1 contains a serine 133 to alanine mutation (S133A) that cannot be phosphorylated (58), both of which result in nonfunctional CREB proteins that fail to stimulate gene transcription. Overexpression of K-CREB or CREB-M1 dominant negative forms of CREB in SK-N-SH cells followed by NMDA treatment reduced the induction of PS-1 gene products, indicating that CREB activation is essential for NMDA induction of PS-1 gene expression (Fig. 5B). As a control, adenovirus expressing the lacZ gene product, beta -galactosidase, did not affect NMDA-mediated increases in PS-1 gene expression.

BDNF Induces PS-1 Expression-- BDNF was also reported to stimulate phosphorylation at Ser-133, thereby activating CREB (64). Unlike NMDA treatments, BDNF application does not stimulate influx of Ca2+ from outside of the cell, but it does cause a slow intracellular Ca2+ rise that is probably from an internal Ca2+ store (64). Therefore, we used BDNF to activate CREB to understand whether the influx of extracellular Ca2+ from activated NMDA receptors or from L-type voltage-dependent Ca2+ channel is essential for the induction of PS-1. BDNF treatment of neuroblastoma cells for 6 h resulted in a dose-dependent induction of PS-1 expression (Fig. 5D).

Human PS-1 and Rat PS-1 Are Regulated by Phosphorylation of CREB-- Through the action of one or more kinases, phosphorylation at Ser-133 is a key step in CREB activation leading to induction of gene transcription. The mitogen-activated protein kinase/extracellular signal-regulated kinase kinase, MEK, apparently contributes to CREB activation, because preincubation with 10 µM U0126, a selective MEK inhibitor, reduced the induction of PS-1 by NMDA in SK-N-SH cells. On the other side of the coin, adenovirus-mediated overexpression of constitutively active MEK (54) in SK-N-SH cells dose-dependently induced PS-1 gene expression, while control adenovirus expressing beta -galactosidase did not change PS-1 gene expression levels (Fig. 6A). Like endogenous kinase, the MEK-mediated induction of PS-1 protein expression was reduced by treatment with the U0126 kinase inhibitor (Fig. 6B) and was reduced by adenovirus-mediated expression of dominant negative CREBs (Fig. 6C). Adenovirus-mediated expression of MEK leading to increased PS-1 expression was not inhibited by MK801 treatment as might be expected, since NMDA receptor activation is upstream of MEK in this signal transduction pathway (Fig. 6B). In a parallel experiment where rat primary hippocampal neurons were transfected with an adenoviral vector expressing constitutively active MEK, endogenous rat PS-1 expression was also induced (Fig. 6D). The induction of PS-1 in these primary rat neuronal cultures was fully inhibited by adenovirus-mediated expression of dominant negative CREBs and the U0126 MEK inhibitor, since it was also reduced in human SK-N-SH cells, suggesting the conservation of CREB binding sites in the rat PS-1 promoter (Fig. 6, D and E).


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Fig. 6.   Constitutively active MEK induced PS-1 in SK-N-SH cells and rat hippocampal neurons. The horizontal arrows indicate full-length PS-1 (PS-1FL) and amino-terminal fragment of PS-1 (PS-1NTF). A, SK-N-SH cells were transfected with 50 m.o.i. adenovirus bearing control LacZ cDNA or constitutively active MEK cDNA 24 h prior to change of the medium, followed by metabolic labeling with [35S]methionine. *, p < 0.01 compared with unstimulated control using Student's t test (n = 3). B, U0126 inhibited PS-1 induction by constitutively active MEK, but MK801 did not in SK-N-SH cells. SK-N-SH cells were transfected with 50 m.o.i. adenovirus bearing constitutively active MEK 24 h prior to change of the medium. 100 µM MK801 or 10 µM U0126 was added to the culture medium when the medium was changed. The cells were then incubated for 6 h, followed by pulse labeling with 200 µCi/ml [35S]methionine. *, p < 0.01 compared with unstimulated control using Student's t test (n = 3); **, p < 0.01 compared with each other using Student's t test (n = 3). C, dominant negative CREBs reduced PS-1 induction by constitutively active MEK in SK-N-SH cells. SK-N-SH cells were transfected with 50 m.o.i. adenovirus bearing constitutively active MEK, dominant negative CREB cDNAs (CREB-M1 and K-CREB), and/or control LacZ cDNA. D, U0126 inhibited PS-1 induction by constitutively active MEK, but MK801 did not in rat hippocampal neurons. Rat hippocampal neurons were transfected with 50 m.o.i. adenovirus bearing constitutively active MEK, 24 h prior to change of the medium. 100 µM MK801 or 10 µM U0126 was added to the culture medium when the medium was changed. The cells were then incubated for 6 h, followed by the pulse-labeling with 200 µCi/ml [35S]methionine. E, U0126 and dominant negative CREBs in rat hippocampal neurons inhibited PS-1 induction by constitutively active MEK. Rat hippocampal neurons were transfected with 50 m.o.i. adenovirus bearing constitutively active MEK and/or dominant negative CREB cDNAs (CREB-M1 and K-CREB), 24 h prior to change of the medium. Alternatively, 100 µM MK801 or 10 µM U0126 was added to the culture medium when the medium was changed. The cells were then incubated for 6 h, followed by pulse labeling with 200 µCi/ml [35S]methionine.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Like the amyloid peptide precursor gene, PS-1 is associated with Alzheimer's disease. Except for rare families with mutations, the majority of Alzheimer's disease patients express both of these genes and their normal protein products in a wide variety of cells throughout the body (8, 27). This distribution raises the question as to how PS-1 appears to participate in a disease that primarily affects only the brain. Our studies (30) and those of others (29, 34) show that PS-1 is differentially distributed throughout the human brain. PS-1 is highly expressed in hippocampal pyramidal neurons, dentate granule neurons, and cerebellar Purkinje neurons, while other cortical regions display lower immunoreactivity. To understand this distribution, we began by examining the transcriptional regulation of the human PS-1 gene to look for other factors that control PS-1 and other genes of potential interest in Alzheimer's disease.

Human PS-1 has two independent transcription start sites that are distinguished by position and activity (Ref. 60 and this report). Our initial screening of a Marathon-ready human brain cDNA library (CLONTECH) yielded several human PS-1 cDNA clones whose 5' terminus was actually upstream of the major transcription start site reported by Rogaev et al. at that time (60).3 We performed primer extension with an antisense primer (5'-AGCCGCTGTTTTGTTTCC-3', Fig. 1C) and total cellular RNA from SK-N-SH cells and found the major transcription start site that is located 26 bp upstream of the reported start site (60). Pastorcic et al. (65) confirmed this new location of the major transcription start site of PS-1. Similar to our work with the mouse PS-1 gene, we also identified a minor transcription start site some 343 bp downstream of the major transcription start site. Thus, the majority of the transcripts include exon 1, which is spliced to exon 3, and a minority of transcripts lack exon 1 and include exon 2 spliced to exon 3. Although the 5'-ends of the transcripts are different, the encoded PS-1 proteins are the same, since the translation starts from the ATG codon in exon 3 (Fig. 1A).

In the human PS-1 promoter sequence (GenBankTM accession number AF029701), several DNA elements of interest are found that probably control PS-1 gene expression at the transcriptional level. Four Alu repeats, the size of which varies from 178 to 267 bp, were identified, suggesting to us that the majority of the active elements in the PS-1 promoter were located between the Alu repeat ending at -812 and the major transcription start site (Fig. 1). Several putative transcription factor binding sites were identified in this region including one Lyf-1 site, two CdxA sites, one AML-1a site, one Nkx-2 site, one Pbx-1 site, one E47 site, one MZF1 site, two Sp1 sites, one Elk-1 site, and one CREB site. Among these transcription factor binding sites is a CREB binding site, which overlaps with the putative Elk-1 binding site. To prove our hypothesis that CREB activation controls PS-1 gene expression, which may participate in synaptic function, we closely examined the relationship between CREB and PS-1.

The cAMP response element binding protein must be present and must be activated to bind to CRE elements within the promoter and stimulate PS-1 transcription. Using immunocytochemistry, we found that CREB and PS-1 are similarly distributed in the rat hippocampus and dentate gyrus, being particularly abundant in pyramidal neurons of the hippocampus and granule neurons of dentate gyrus (data not shown). CREB that actively binds its DNA element, CRE, is typically phosphorylated on serine at position 133. We found that the distribution of phosphorylated CREB was also similar to PS-1. As others have reported, the presence of phosphorylated CREB is usually associated with the products of genes that respond to CREB, such as c-Fos, Bcl-2, and BDNF (45-48).

Activation of CREB by its phosphorylation at serine residue 133 is modulated by a variety of effectors. NMDA receptors associated with synapses are involved in neuronal functions like long term potentiation (44). Stimulation of NMDA receptors has also been associated with increased calcium influx into cells, potentiation of kinase activities, and CREB activation. Like hippocampal neurons, the SK-N-SH neuronal cell line expresses functional NMDA receptors (66). Using electrophoretic mobility shift assay, we showed that CREB binds to the human PS-1 proximal promoter. When SK-N-SH cells were stimulated with 10 or 100 µM NMDA, transcription from the human PS-1 gene was dose-dependently induced as shown by promoter-luciferase reporter assay (Fig. 3, D and E), Northern blotting (Fig. 4), and metabolic pulse labeling with [35S]methionine to detect PS-1 proteins (Fig. 5A). We detected full-length PS-1, the amino-terminal fragment of PS-1, and aggregated PS-1 proteins, which is consistent with previous reports showing that PS-1 was cleaved with an average half-life of 50 min after pulse labeling (67). In these experiments, most of the labeled PS-1 protein detected was full-length PS-1, so little amino-terminal fragment of PS-1 was observed. This is because we employed 1-h pulse labeling, which appeared to be shorter than the half-life of PS-1 protein, and full-length PS-1 was detected before its cleavage into amino-terminal and C-terminal fragments. Another cause of the lack of PS-1 amino-terminal fragment observed is that we used several kinds of protease inhibitors after lysing the cells as described under "Experimental Procedures," such as phenylmethylsulfonyl fluoride, aprotinin, pepstatin, and leupeptin. Preincubation with MK801, an antagonist of the NMDA receptor, and with nifedipine, an inhibitor of L-type voltage-dependent calcium channels, inhibited the induction of PS-1, suggesting that the Ca2+ influx contributes to PS-1 gene induction (Fig. 5, A and B). Interestingly, BDNF is also known to phosphorylate CREB at Ser-133 without the participation of NMDA receptors (64). Even in the absence of NMDA, BDNF dose-dependently induces the transcription from PS-1 gene (Fig. 5D). As a whole, these data support our hypothesis that activation of CREB results in increased PS-1 gene expression.

To directly elucidate the effect of CREB on PS-1 gene expression, we used two adenoviruses expressing dominant negative CREB cDNAs. As a control, an adenovirus expressing LacZ cDNA was used. CREB-M1, in which Ser-133 is converted to an alanine and cannot be phosphorylated or activated, was expressed in neuroblastoma cells and reduced the expression of PS-1. K-CREB with a mutation in the DNA binding domain was expressed in neuroblastoma cells and reduced the expression of PS-1, while control LacZ did not affect the expression (Fig. 5B). These data directly indicate that phosphorylation of CREB and its binding to the CRE element in the PS-1 promoter are required for induction of PS-1.

MEK has also been reported to activate extracellular signal-regulated kinases that can phosphorylate proteins such as CREB, resulting in PS-1 induction. MEK phosphorylates extracellular signal-regulated kinases, and these activated extracellular signal-regulated kinases phosphorylate CREB (68). To verify whether MEK can stimulate PS-1 expression, we overexpressed a constitutively active MEK by adenoviral transduction and observed the dose-dependent induction of full-length PS-1 protein, while control LacZ adenovirus did not induce PS-1 (Fig. 6A). The MEK inhibitor U0126 not only inhibited MEK activity, as reported previously (63), but also inhibited PS-1 expression induced by the constitutively active MEK (Fig. 6B). It was also decreased by preoverexpression of either of the dominant negative CREBs, making it clearer that CREB phosphorylation is critical for the induction of PS-1 (Fig. 6C). The discrepancy between the effects of the MEK inhibitor and the dominant negative CREBs in blocking PS-1 induction in SK-N-SH cells by the constitutively active MEK could be due to participation of the transcription factor Elk-1, because MEK can stimulate not only CREB but also Elk-1, and Elk-1 can up-regulate PS-1 transcription as previously described (65). While dominant negative CREBs only partially inhibited the induction of PS-1 by constitutively active MEK in SK-N-SH cells, they fully inhibited the induction of PS-1 in rat hippocampal neurons. (Fig. 6, D and E). This could potentially be due to differences in the organization of the PS-1 promoter between rat and human, although we do not know the exact sequence of the rat PS-1 promoter.

CREB activation is associated with acquisition of learning and memory, which can be demonstrated in the hippocampus (8, 22). Among other types, glutaminergic neurons in the hippocampus display abundant numbers of dendritic spines, and their synapses are rich in NMDA receptors. The activation of NMDA receptors leads to CREB phosphorylation and Ca2+ influx into these postsynaptic spines, which is critical for maintenance of synaptic plasticity and ultimately synaptic transmission (69). When glutamate is released from the presynaptic membrane of glutaminergic neurons, NMDA receptors open and cause Ca2+ influx into postsynaptic spines, resulting in depolarization that triggers the opening of LVDCCS. The activation of LVDCCs, which open during strongly depolarizing conditions, permits the entry of a larger amount of Ca2+ along the dendrites and into the cell bodies. This calcium influx can activate kinases such as extracellular signal-regulated kinase and calmodulin kinase II that function to phosphorylate CREB (45), resulting in PS-1 induction. Our finding that preincubation with nifedipine almost completely inhibited the PS-1 induction stimulated by NMDA treatment suggests that the relevant Ca2+ comes through LVDCC, not the NMDA receptor channel. The importance of calcium influx, however, is not clear, since overexpression of constitutively active MEK does stimulate CREB activation and PS-1 expression, and preoverexpression of dominant negative CREBs reduced the PS-1 induction by constitutively active MEK while failing to stimulate calcium influx.

We have shown by molecular and cell biological methods that the activation of the NMDA receptor-CREB pathway regulates PS-1 transcription. Considering that synaptic plasticity of hippocampal neurons is also associated with the activation of the CREB pathway, then the regulation of PS-1 itself may be associated with synaptic plasticity and long term memory.

    ACKNOWLEDGEMENTS

We thank Dr. Anthony Zeleznik for the generous gift of CREB-M1 adenovirus; Izumu Saito for AxCANCre and AxCANLZ recombinant adenovirus; Dr. Masatoshi Hagiwara for wild-type CREB cDNA; and Drs. Takashi Kudo, Hana Dawson, Kazunori Imaizumi, and Taisuke Katayama for helpful and informative discussions.

    FOOTNOTES

* This work was supported by grants from CREST (Japan Science and Technology); the Ministry of Education, Culture, Sports, Science and Technology, Japan (Priority Area (c)-Advanced Brain Science Project); and Norvatis Foundation for Gerontological Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be 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 Physiology, Ehime University School of Medicine, Shitsukawa, Shigenobu-cho, Ehime 791-0295, Japan. Tel.: 81-89-960-5245; Fax: 81-89-960-5246; E-mail: mitsuda@m.ehime-u.ac.jp.

Published, JBC Papers in Press, December 14, 2000, DOI 10.1074/jbc.M006153200

2 N. Mitsuda, N. Ohkubo, M. Tamatani, Y.-D. Lee, M. Taniguchi, K. Namikawa, H. Kiyama, A. Yamaguchi, N. Sato, K. Sakata, T. Ogihara, M. P. Vitek, and M. Tohyama, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: PS-1 and -2, presenilin-1 and -2, respectively; CREB, cAMP-response element-binding protein; BDNF, brain-derived neurotrophic factor; NMDA, N-methyl-D-aspartate; PCR, polymerase chain reaction; FL, firefly luciferase; RL, Renilla luciferase; RPA, relative promoter activity; bp, base pair(s); kb, kilobase pair(s); m.o.i., multiplicity of infection; CRE, cAMP-response element; SEAP, secreted alkaline phosphatase; LVDCC, L-type voltage dependent Ca2+ channel.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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