CTCF Is Essential for Up-Regulating Expression ... - Wiley Online Library

Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 1999 International Society for Neurochemistry

CTCF Is Essential for Up-Regulating Expression from the Amyloid Precursor Protein Promoter During Differentiation of Primary Hippocampal Neurons Yaxiong Yang, *Wolfgang W. Quitschke, *Alexander A. Vostrov, and Gregory J. Brewer Department of Medical Microbiology and Immunology, Southern Illinois University School of Medicine, Springfield, Illinois; and *Department of Psychiatry and Behavioral Science, State University of New York, Stony Brook, New York, U.S.A.

Abstract: The transcriptional mechanism underlying amyloid precursor protein (APP) regulation in primary neurons during development was investigated. We observed an approximately threefold elevation of APP mRNA levels in differentiating rat hippocampal neurons between day 1 and day 7 in culture and in rat brain hippocampi between embryonic day 18 and postnatal day 3. When an APP promoter construct extending to position 22,832 upstream from the main transcriptional start site was transfected into primary rat hippocampal neurons, promoter activity increased from day 1 until reaching a maximum on day 7 in culture. This increase in APP promoter activity was correlated more closely with the time course of expression of the synaptic vesicle protein synaptophysin, an indicator of synaptogenesis, than with neurofilament accumulation, an indicator of neuritogenesis. Transfection of 59 APP promoter deletions and internal block mutations indicated that the CTCF binding domain designated APBb was the primary contributor to the increase in APP promoter activity. Furthermore, the binding of transcription factor CTCF to the APBb element increased approximately fivefold between day 1 and day 7, whereas the binding of USF to the APBa sequence increased only twofold. These results suggest that CTCF is pivotal for the up-regulation of APP expression during synaptogenesis in primary neurons. Key Words: Amyloid precursor protein promoter—Rat hippocampal neuron —CTCF—USF—Synaptogenesis—Transcription. J. Neurochem. 73, 2286 –2298 (1999).

sion of APP is developmentally regulated and correlates with the stages of synaptogenesis in the hamster visual system (Moya et al., 1994). A recent study demonstrated the involvement of APP in functional synapse formation in cultured hippocampal neurons (Morimoto et al., 1998). APP also modulates neurite outgrowth in primary neurons (Mattson, 1994; Small et al., 1994; Ishida et al., 1997). These observations indicate that APP expression and metabolism might be linked to brain synaptic plasticity (Beyreuther et al., 1993), and they illustrate the significance of regulation of APP gene expression in neuronal development and synaptogenesis. The human APP gene is located on chromosome 21, which encodes multiple APP transcripts by alternative splicing (Kitaguchi et al., 1988; Tanzi et al., 1988; De Sauvage and Octave, 1989; Jacobsen et al., 1991). APP is differentially expressed in all major tissues (Schmechel et al., 1988). In general, the APP751/770 isoforms predominate in peripheral nonneuronal tissues, whereas in neurons the predominant isoform is APP695 (Kang et al., 1987). APP gene expression is regulated in a developmental and tissue-specific manner (Adler et al., 1991). In culture, APP levels progressively increase during differentiation of both primary neurons (Hung et al., 1992) and neuroblastoma cells (Ko¨nig et al., 1990). Nevertheless, the mechanism by which APP is up-regulated in primary neurons during development has not been determined. As neurons are the primary source of cerebral APP (Schmechel et al., 1988), the relevance of APP to synaptic remodeling and to the neuropathology of AD (Small, 1998) prompted us to inves-

The amyloid precursor protein (APP) contributes to the pathogenesis of Alzheimer’s disease (AD) by generating the neurotoxic b-amyloid protein (Ab) fragment (Selkoe, 1994, 1997). The biological function of APP has yet to be defined. However, evidence is accumulating that it is involved in neuronal development and synaptogenesis. APP is abundant at peripheral and central synaptic sites (Schubert et al., 1991), and it undergoes fast axonal transport to these sites in rat cortical and hippocampal neurons (Koo et al., 1994). In primary cerebellar macroneurons, APP is present in presynaptic clathrin-coated vesicles and participates in synaptic vesicle recycling (Marquez-Sterling et al., 1997). Expres-

Received May 17, 1999; revised manuscript received July 19, 1999; accepted July 29, 1999. Address correspondence and reprint requests to Dr. G. J. Brewer at Department of Medical Microbiology and Immunology, Southern Illinois University School of Medicine, Springfield, IL 62794-9626, U.S.A. Abbreviations used: Ab, b-amyloid protein; AD, Alzheimer’s disease; AdMLP, adenovirus major late promoter; APP, amyloid precursor protein; CAT, chloramphenicol acetyltransferase; DAB, 3,39-diaminobenzidine; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline.

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FIG. 1. Sequence of the proximal human APP promoter from position 2130 to 120. Underlined are the recognition sequences for the three nuclear factor binding sites APBa (CTCF), APBb (USF), and SP1. The transverse block mutations introduced into APBb and APBa are indicated as Mb2 and Ma3, respectively. Numbers show the positions of nucleotides relative to the main transcription start site (11) (Quitschke et al., 1996). Arrows depict the 59 terminal nucleotide of deletions within the proximal promoter.

tigate the transcriptional regulatory mechanism for APP expression in primary neurons. The APP gene promoter lacks typical TATA and CCAAT boxes and has a high GC content and multiple putative regulatory consensus sequences (Salbaum et al., 1988). Of these, sequences in close proximity to the transcriptional start site were shown to confer basal APP expression in nonneuronal cells (Pollwein et al., 1992; Quitschke and Goldgaber, 1992; Bourbonnie`re and Narbantoglu, 1993; Hoffman and Chernak, 1994; Quitschke, 1994). In the human APP gene promoter, two nuclear factor binding domains designated as APBa and APBb (Fig. 1) were found to be functional activating elements for promoter activity in HeLa and PC12 cells (Quitschke, 1994). Transcription factor USF binds to the APBa domain, which is located between positions 242 and 253 upstream from the primary transcriptional start site (Hoffman and Chernak, 1995; Kovacs et al., 1995; Vostrov et al., 1995; Bourbonniere and Nalbantoglu, 1996) (Fig. 1). The factor that binds to the APBb element at position 282 to 293 was revealed recently to be CTCF (Vostrov and Quitschke, 1997), which was identified originally as a regulator of chicken c-myc and lysozyme gene expression (Burcin et al., 1997). In addition, SP1 also binds to the proximal promoter region located at 256 and 263, which contains the recognition sequence GGGGTGGG (position 256 to 263) (Pollwein, 1993). However, much of the APP promoter activity is dependent on APBb, which contributes 70 –90% of the total activity in HeLa and PC12 cells (Quitschke, 1994). We here provide evidence that primarily transcription factor CTCF, and to a lesser extent USF, contribute to most of the increased APP expression in primary neurons during synaptogenesis in culture. MATERIALS AND METHODS Plasmids The APP promoter constructs used in this study include successive deletions designated as APP[22,832], APP[21,359], APP[2488], APP[2303], APP[2204], APP[2121], APP[294], APP[277], and APP[246]. The numbers indicate the 59 extension of the promoter upstream from the main transcriptional start site

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(11) (Fig. 1). In addition, constructs APP[294Ma3], APP[277Ma3], and APP[2488Mb2] contain internal block mutations in the APBa and APBb binding domains that eliminate factor binding to the corresponding sequences (Quitschke, 1994). All promoter fragments were cloned into the polycloning site of vector pCAT2bGAL (Quitschke and Goldgaber, 1992). This plasmid contains the reporter gene chloramphenicol acetyltransferase (CAT) transcribed from the APP promoter, and the b-galactosidase gene activated by the chicken b-actin promoter, which serves as an internal control for transfection efficiency and other experimental variables.

Oligonucleotides and PCR primers Oligonucleotides were synthesized by Life Technologies or Genosys Biotechnologies Inc. Complementary oligonucleotides were annealed prior to 59 end labeling. The sequence of all double-stranded oligonucleotides used as probes are depicted in Fig. 2. These probes include APBb[2125] containing the APP promoter sequence from position 264 to 2125, APBb[2109] (position 264 to 2109), and APBb[294] (position 264 to 294). The wild-type APP promoter sequence in oligonucleotides APBb[2109] and APBb[2125] are flanked by polycloning sequences, whereas in APBb[294], the 59 flanking domain exactly reproduces the vector sequence as it exists in plasmid APP[294]. In addition, mutation Mb2 was introduced into oligonucleotide APBb[2109] (Fig. 2). This transverse mutation converts the wild-type sequence GCCGCT to TAATAG (Fig. 1) and eliminates binding of CTCF to APBb (Quitschke, 1994). Oligonucleotides containing USF binding sites include a sequence from the adenovirus major late promoter (AdMLP) and the APP promoter sequence from position 258 to 230 that encompasses the APBa binding site (Fig. 1). A 3-bp transverse mutation in the APBa sequence, designated Ma3, abolishes the binding of USF to that site (Figs. 1 and 2). A 42-mer oligonucleotide containing the CCAAT domain from the chicken b-actin promoter was used as a binding site for nuclear factor NF-Y (Quitschke et al., 1989; Danilition et al., 1991). The sequence of the APP695 forward primer for PCR, 59-TACCACTGAGTCTGTGGAGG-39, corresponds to bases 849 – 868 of the APP695 cDNA sequence (Rohan de Silva et al., 1997), whereas the reverse primer, 59-GGGGGTCTCCAGGTACTTGT-39, is complementary to bases 936 –917.

Cell culture and transfection Dissection and isolation of primary embryonic hippocampal neurons from rat brain were performed as described (Brewer et al., 1993). Neurons were grown in serum-free Neurobasal medium supplemented with B27, glutamate (25 mM), glutamine (0.5 mM) in 95% O2 and 5% CO2 at 37°C for 5 days. These cultures contain .99% neurons. The cells were plated in 24well plates at a density of 100,000 cells/cm2 (200,000 cells/ well). In brief, 1 mg of plasmid DNA was mixed with 6 mg of Lipofectamine (Life Technologies) in a 20-ml 0.15 M NaCl solution at room temperature for 45 min before being added to the 400-ml culture medium. Immediately before transfection, the medium was shifted to fresh medium without glutamate. The transfection efficiency was estimated to be ;1.5% as determined by in situ b-galactosidase staining (data not shown). Twenty-four hours after transfection, the cells were lysed in lysis buffer (Promega), scraped, and stored at 220°C for subsequent CAT and b-galactosidase assays. Transfections were performed in triplicate for statistical analysis.

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FIG. 2. Double-stranded oligonucleotides used in mobility shift electrophoresis. The sequences directly derived from the APP promoter are underlined. Numbers above the sequences denote positions of nucleotides in the APP promoter. The recognition sequence domains with relevance to CTCF, USF, and NF-Y are double-underlined. These include APBb and APBa in the APP promoter, CACGTGAC in the AdMLP, and GCCAATCAG in the chicken b-actin promoter. Mutated sequences Mb2 and Ma3 in the APP promoter are indicated by brackets. Sequence elements derived from polycloning sites are depicted in italics, and the vector-derived sequence in oligonucleotide APBb[294] is boxed.

Determination of neurofilament and synaptophysin levels Neurofilament and synaptophysin levels were measured in cultured embryonic rat hippocampal neurons by immunochemiluminescence of cell populations in 2-cm2 culture wells (105 cells). For neurofilament measurements, cells were fixed with 4% formaldehyde in phosphate-buffered saline (PBS) for 10 min, permeabilized with 0.5% Triton X-100 for 15 min, and blocked by 5% normal goat serum for 5 min with two washes in PBS between each step. The cells were then incubated with mouse anti-neurofilament 200 IgG (Sigma N5389, 1:1,000 dilution in blocking solution) overnight at 4°C. For synaptophysin measurements, neurons were fixed with 4% formaldehyde and 0.1% glutaraldehyde in PBS for 30 min, incubated with 0.3% H2O2 in PBS for 5 min, permeabilized with 0.5% Triton X-100 for 15 min, and blocked with 1% normal goat serum for 5 min with two washes in PBS between each step. Cells were then incubated with mouse anti-synaptophysin (Sigma S5786, 1:50 dilution) overnight at 4°C. Neurons were then rinsed four times in PBS and incubated with goat anti-mouse IgG conjugated with horseradish peroxidase (Life Technologies, 1:25,000 dilution) overnight at 4°C or for 60 min at room temperature. The signal was detected by a photomultiplier tube using supersignal chemiluminescence substrates (Pierce, Rockford, IL, U.S.A.) and a photomultiplier tube (EMI, Gencom, Plainview, NY, U.S.A.). The immunoreactivity of both neurofilaments and synaptophysin is presented as mean photon counts per 10 s from each of eight cultures. Synaptophysin puncta were analyzed in neurons immunostained with anti-synaptophysin as described above. The substrate consisted of 0.5 mg/ml 3,39-diaminobenzidine (DAB), 0.01% H2O2, and 0.6% NiCl2. Stained neurons were examined under the phase light of a Nikon microscope with a 1003/1.3 oil immersion objective. The images were amplified and relayed by an Ikegami camera to a computer for analysis of the synaptophysin puncta (Global Lab Image software, Data Translation Inc.). The digital pixel size was 0.122 mm2. Cells were selected for digital analysis from eight consecutive images of 7,500 mm2 each. The puncta were counted by the computer from each image at different stages of neuronal differentiation. The numbers of puncta were averaged for statistical analysis.

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As peroxidase-catalyzed deposits of DAB increased with time of incubation, most of the puncta measured were larger than the 0.1– 0.3-mm2 actual sizes of synapses determined by electron microscope (Chang et al., 1993; Hawrylak et al., 1993). Therefore, we set the size criterion to identify puncta between 0.122 and 4 mm2. The 0.122-mm2 setting was the single pixel limit of resolution. The results are presented as number of puncta per cell determined from total number of puncta in each image divided by number of cell bodies in the same area. The same setting was used for calculation of puncta areas.

Determination of APP mRNA levels by RT-PCR

Total RNA was isolated from 6 3 106 neurons of day 1, day 4, and day 7 cultures or from hippocampal tissue of eight rat brains at embryonic day 18 and 22 and postnatal day 3. RTPCR was performed with a kit from Promega (A2150) according to the protocol. In brief, the reaction mixture included 10 mM dNTP, 50 pmol each of forward and reverse APP695 primers (for sequence, see above), 1.5 mM MgSO4, 5 units each of AMV reverse transcriptase and Tfl DNA polymerase, 1.5 mg of total RNA, and 13 reaction buffer (Promega) in a total volume of 50 ml. The reaction tube was placed in the heat block of a thermal cycler, Amplitron-II (Thermolyne, Dubuque, IA, U.S.A.) for 45 min at 48°C for the synthesis of first-strand cDNA. The reaction proceeded to 25 thermal cycles for the synthesis of second-strand cDNA and PCR amplification. Each cycle included 95°C for 1 min, 58°C for 1 min, and 72°C for 1 min. The PCR products were separated by gel electrophoresis in 4% Nusieve-agarose, visualized with 1.5 mg/ml ethidium bromide under UV illumination, and quantified by Image Pro Plus software (Media Cybernetics Inc). The quantification of the APP PCR products was normalized to PCR products from the rat glyceraldehyde-3-phosphate dehydrogenase (G3PDH) control amplimer (Clontech, Palo Alto, CA, U.S.A.).

Nuclear extracts, antibodies, and mobility shift gel electrophoresis Nuclear extracts were prepared from cultured neurons as described elsewhere (Andrews and Faller, 1991) with some modifications. Specifically, harvested cultured neurons (1.5 3 107 cells) were resuspended in 600 ml of buffer A (10 mM

SYNAPTOGENESIS AND APP PROMOTER REGULATION HEPES, pH 7.6, 15 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 20 mM ZnSO4, 1 mM phenylmethylsulfonyl fluoride) and homogenized with 20 strokes of a Dounce tissue grinder. Buffer A (60 ml) containing 1 M KCl was added to the homogenate and mixed immediately. The mixture was centrifuged at 3,000 g for 15 min. Pellets containing nuclei were weighed and resuspended with a pellet pestle device (Kontes) in 5 volumes of buffer E (40 mM HEPES, pH 7.6, 2 mM MgCl2, 0.1 mM EDTA, 20 mM ZnSO4, 1 mM phenylmethylsulfonyl fluoride, 10% glycerol) containing 100 mM KCl. To this suspension was added 0.13 volume of 3 M KCl bringing the total KCl concentration to 0.5 M. The sample was mixed immediately using a wide-orifice pipette tip to prevent DNA shearing and left on ice for 30 min with additional mixing repeated every 5 min. The viscous solution was centrifuged at 18,000 g for 1 h. The supernatant containing the nuclear extract was collected, aliquoted, and stored at 280°C. Protein concentrations were determined with the Bio-Rad protein assay (Bradford) reagent. HeLa cell nuclear extract was prepared as described elsewhere (Quitschke et al., 1996). The his-tagged N-terminal part of the CTCF protein corresponding to amino acids 1–569 was expressed in E. coli and purified by cation exchange and Ni21 affinity chromatography. The purified material was used to raise polyclonal antibodies in rabbits by Pocono Rabbit Farm and Lab (Canadensis, PA, U.S.A.) according to a standard 70-day protocol. Double-stranded oligonucleotides were 59 end-labeled with [g-32P]ATP (Maniatis et al., 1983). Labeled oligonucleotide (10 ng; 50,000 –500,000 cpm) was incubated for 30 min at room temperature with up to 6 ml of nuclear extract in a total reaction volume of 32 ml. When smaller amounts of extract were used, extraction buffer was added to a total volume of 6 ml to maintain uniform salt conditions. Salt concentration in the binding reaction mixture was adjusted with buffer E and 3 M KCl to a final concentration of 150 mM KCl for the CTCF binding assay and to 100 mM KCl for the USF and NF-Y binding assays, respectively. The reaction mixture also contained 1 mg of poly(dI-dC), 3 mg of yeast tRNA, and 2.5% CHAPS. In addition, 1.7 ml of fetal calf serum was added for CTCF and USF binding assays. For CTCF supershift assays, nuclear extracts were preincubated for 1 h at room temperature with 1 ml of either preimmune or immune rabbit serum. To prevent CTCF degradation by serum proteases, the reaction was supplemented with 2 mg/ml aprotinin. The incubation mixture was electrophoresed in 6% polyacrylamide gels containing 0.53 Tris– borate–EDTA buffer (Maniatis et al., 1983) at 180 V for 45 min. Gels were dried and quantified with a phosphorimager (Bio-Rad).

RESULTS Increased accumulation of neurofilaments, synaptophysin, and synaptic puncta during neuronal differentiation Assembly of neurofilaments and expression of synaptophysin in cell culture indicates neuritogenesis and synaptogenesis of developing neurons (Fletcher et al., 1991). We monitored three aspects of synaptogenesis reported by synaptophysin reactivity: (a) total level of synaptophysin per culture, the expression of which must begin before a synapse can be assembled; (b) microscopic size (area) of synaptophysin puncta; synaptophysin must be assembled with synaptic vesicles, which accumulate at

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FIG. 3. A: Total levels of neurofilaments (E) and synaptophysin (F) per culture from day 1 to day 8 determined by chemiluminescence. B: Numbers of puncta per cell (F) and average area (mm2) per puncta (E), representing two independent experiments each from eight cultures that were analyzed individually.

small synapses before reaching the size of mature synapses; and (c) the number of synaptophysin puncta, a measure of the number of synapses. The measurement of the immunoreactivity against neurofilaments and synaptophysin is presented here as chemiluminescent photon counts from a photomultiplier. The abundance of neurofilaments increased about fivefold between day 1 and day 4 cultures, and then slowed to a plateau between day 4 and day 8 (Fig. 3A, open circles). In contrast, the level of synaptophysin continued to increase up to sixfold over the entire 8-day period (Fig. 3A, filled circles). As synaptophysin is a membrane protein of synaptic vesicles, the measurements of synaptophysin puncta reflect the accumulation of immunoreactive vesicles in synapses during neuronal differentiation. Both the number and area of synaptophysin puncta per cell increased in primary neurons from day 1 through day 8 cultures (Fig. 3B). The number of puncta increased more than 35-fold and showed no plateau, whereas the area of the puncta increased 1.4-fold between day 1 and day 4 and then slowed to a maximum size between day 4 and day 8 as expected for a maturing synapse. These measurements are likely to represent only the larger synapses, because synaptic profiles appear to range from 0.1 to 0.3 mm2 as determined by electron microscopy (Chang et al., 1993; Hawrylak et al., 1993). These results were also depicted in microscopic images, comparing neuronal cultures on day 1, day 4, and day 7 with their corresponding synaptophysin puncta (Fig. 4). Although hippocampal neurons express synaptophysin from the beginning of culture, synaptogenesis seems to begin more abruptly after 3 or 4 days. J. Neurochem., Vol. 73, No. 6, 1999

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FIG. 4. Hippocampal neurons in culture on day 1 (A), day 4 (B), and day 7 (C) are displayed with phase-contrast optics. Synaptophysin puncta of neurons in culture on day 1 (D), day 4 (E), and day 7 (F) are displayed with bright-field optics. Neurons were immunostained using anti-synaptophysin and developed with DAB as described. Scale bar for A–C represents 100 mm. Scale bar for D–F represents 20 mm.

Increased levels of APP mRNA and APP promoter activity in hippocampal neurons during synaptogenesis APP expression has been shown to be up-regulated in neuroblastoma cells (Ko¨nig et al., 1990) and primary neurons (Hung et al., 1992) during differentiation in culture. APP expression could be related to neuritogenesis or more directly to synaptogenesis. To study this in rat hippocampal neurons, we measured the level of APP mRNA by RT-PCR on day 1, day 4, and day 7 in culture, and in rat brain hippocampi at different stages of embryonic development using endogenous G3PDH mRNA as an internal standard. However, amplification efficiency J. Neurochem., Vol. 73, No. 6, 1999

for APP and G3PDH cDNA can differ under the selected PCR conditions. In addition, although G3PDH is used routinely as a control for RNA quantification, its developmental regulation in differentiating hippocampal neurons has not been examined systematically. Thus, the results normalized to G3PDH should therefore be considered semiquantitative. To achieve a quantitative PCR, a systematic study of the amplification conditions using several normalizing controls would be necessary. Although APP751 and APP770 transcripts were detected, their levels were so low that their contribution to the total APP mRNA level was negligible. The primary transcript observed was APP695 mRNA, which in-

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FIG. 5. A: APP695 mRNA from rat brain hippocampi on embryonic day 18 (E18), embryonic day 22 (E22), and postnatal day 3 (P3). The increase in APP mRNA is expressed relative to the level at E18, which was assigned the value of 1. B: APP 695 mRNA from embryonic rat hippocampal neurons on day 1 (D1), day 4 (D4), and day 7 (D7) in culture. The increase in APP mRNA is expressed relative to the level at D1, which was assigned the value of 1. C: Relative CAT activity from APP[22,832] transfected neurons at day 1, day 2, day 4, and day 7 in culture. The CAT activity on day 1 was assigned the value of 1, and all other activities are expressed as a ratio thereof. The values represent averages and standard deviations from at least three independent experiments. D: Correlation between APP[22,832] CAT activity and expression of neurofilament and synaptophysin plotted as fold increase relative to levels on day 1. Dashed lines depict linear regression analyses with the resulting equations.

creased approximately threefold both in rat hippocampi between embryonic day 18 and postnatal day 3 (Fig. 5A) and in cultured neurons between day 1 and day 7 (Fig. 5B). Therefore, increased APP transcript levels coincide with intense synaptogenesis during development not only in hippocampal neurons in brain, but also during differentiation in culture. To establish whether the observed increase in APP transcript was attributable to an elevated promoter activity, an APP promoter construct terminating at position 22,832 upstream from the primary transcriptional start site (APP[22,832]) was transfected into primary hippocampal neurons. A dramatic increase in APP promoter activity was observed that reached a 33-fold maximum at day 7 in culture (Fig. 5C). In the linear regression model, the increase in APP promoter activity over a period of 7 days in culture more closely correlates with the increase in the levels of the synaptic vesicle protein synaptophysin (R2 5 0.957) than with the accumulation of neurofilaments (R2 5 0.762) (Fig. 5D). APP expression was also highly correlated with the levels of synaptophysin puncta, both in duration and in magnitude (R2 5 0.9, data not shown). Thus, these results indicate an association of APP expression and neuronal differentiation, and they suggest that APP may play a role in synaptogenesis of primary neurons.

An APP promoter fragment terminating at position 2121 is sufficient for the observed increase in activity during neuronal differentiation To identify which upstream promoter domains are essential for the observed increase in APP promoter activity, plasmids containing APP promoter fragments with sequential 59 deletions were transfected into primary hippocampal neurons. CAT and b-galactosidase activities were determined on day 1, day 2, day 4, and day 7 in culture. All CAT activities were normalized to identical b-galactosidase activities. The normalized CAT activity of the longest promoter fragment (APP[22,832]) on day 1 was assigned the value of 1, and all other activities were expressed as a ratio thereof. After the first day in culture, all promoter constructs displayed similar activities that varied by ,30%. However, over a period of 7 days in culture, promoter fragments APP[22,832], APP[21,359], APP[2488], APP[2303], APP[2202], and APP[2121] all conferred continuous increases in APP promoter activity that culminated in a 30 –35-fold rise on day 7 (Fig. 6). By comparison, although construct APP[294] displayed a similar pattern of up-regulation, the relative activity on day 2, day 4, and day 7 was consistently only ;60 –70% of the levels observed with the longer constructs. This is reflected in a 22-fold maximum increase in activity on day 7 in culture. Further J. Neurochem., Vol. 73, No. 6, 1999

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FIG. 6. Relative CAT activities from neurons transfected with APP promoter constructs consisting of 59 sequential deletions extending to positions 22,832, 21,359, 2488, 2302, 2202, 2121, 294, 277, and 246 on day 1, day 2, day 4, and day 7 in culture. The CAT activities were normalized to identical b-galactosidase activity. All CAT activities are presented relative to the CAT activity of construct APP[22,832] on day 1, which was assigned the value of 1. The data represent averages and standard deviations from at least three independent experiments.

deletions to positions 277 and 246 resulted in additional declines in promoter activity producing only seven- and fourfold increases, respectively (Fig. 6). Moreover, the highest level of activity from constructs APP[277] and APP[246] was attained on day 4 in culture, whereas all other constructs reached their maximum on day 7. These results suggest that 59 sequences between positions 277 and 2121 are essential for the effective up-regulation of the APP promoter activity during neuronal differentiation. Developmental APP up-regulation is controlled predominantly by the APBb element Previous studies on PC12 and HeLa cells (Quitschke, 1994) have shown that the proximal APP promoter sequence terminating at position 294 contains two functional nuclear factor binding sites, designated as APBb (position 282 to 293) and APBa (position 242 to 253) (Fig. 1). APBb was found to contribute most of the constitutive APP promoter activity, whereas much of the remaining activity was attributable to the APBa element. To determine how these elements affect APP expression during development of primary neurons, plasmids containing block mutations that eliminate factor binding to these two domains were transfected into neurons at day 1, day 2, day 4, and day 7 in culture. Construct APP[2488]Mb2, which carries a block mutation that abolishes CTCF binding to the APBb sequence (Fig. 1), reduced promoter activity to ;10 –20% of the level observed with the wild-type construct APP[2488], and this relative decrease persisted throughout the observed time span between days 1 and 7. The results for this mutation are consistent with those reported on HeLa and PC12 cells (Quitschke, 1994). J. Neurochem., Vol. 73, No. 6, 1999

To determine if the residual promoter activity observed with the APP[277] construct might be attributable to the USF binding site, the block mutation Ma3 that eliminates USF binding was analyzed within the promoter fragment terminating at position 277. In this construct, the APBb core recognition sequence from position 282 to 293 has been deleted, making the relatively low contribution of the USF binding site easier to detect. The results show that construct APP[277] is also subject to a low degree of up-regulation despite the absence of the CTCF binding site, in particular on day 4 and day 7 in culture (Fig. 7B). A mutation in APBa that prevents USF binding to this site further reduced promoter activity by about one third, suggesting a contribution of USF to the activity of construct APP[277]. Nevertheless, the activity of construct APP[277]Ma3 remains higher than the activity of APP[246], which is devoid of both the CTCF and USF binding sites. The reason for this is unclear; however, it may be due to the contribution of an SP1 binding site in APP[277] that is unaffected by the Ma3 mutation (Fig. 1). Furthermore, the activity from construct APP[246] is also up-regulated, particularly on day 4, although the level of upregulation is exceedingly low compared with that of promoter constructs containing the CTCF binding site. This indicates that APBb is an essential element in the up-regulation of APP expression during synaptogenesis in primary neurons. However, the reduced expression from the APP[294] fragment suggests that CTCF plays a diminished role in this deletion despite containing the

FIG. 7. Relative CAT activities from neurons transfected with APP constructs on day 1, day 2, day 4, and day 7. A: The block mutation Mb2 in the APBb element was introduced into construct APP[2488] (2488Mb2). The activity of the mutation was compared with those of the wild-type construct APP[2488] and the deletions terminating at positions 277 and 246. B: Relative CAT activities from mutation Ma3 in the APBa element introduced into construct APP[277] (277Ma3). The activity of the mutation was compared to the wild-type deletions extending to positions 277 and 246. CAT activities were normalized to identical b-galactosidase activities and expressed relative to the activity of construct APP[22,832] on day 1 in culture (compare Fig. 6). The values represent averages and standard deviations from at least three independent experiments.

SYNAPTOGENESIS AND APP PROMOTER REGULATION FIG. 8. Binding of neuronal transcription factors CTCF and USF to the APBb and APBa elements, respectively. A and B: Lane 1: mobility shift electrophoresis of 32P-labeled oligonucleotide APBb[2109] with nuclear extract from HeLa cells (A) or hippocampal neurons (B); lane 2: competition with a 100fold molar excess of unlabeled APBb[2109]; lane 3: competition with a 100-fold excess of Mb2; lane 4: nuclear extract incubated with preimmune serum prior to mobility shift electrophoresis; lane 5: nuclear extract preincubated with polyclonal anti-CTCF. C: Lane 1: gel mobility shift of 32P-labeled AdMLP oligonucleotide incubated with nuclear extract from hippocampal neurons; lanes 2– 4: competition with a 100-fold molar excess of unlabeled AdMLP (lane 2), APBa (lane 3), and Ma3 (lane 4). Bound (b) and free (f) oligonucleotides as well as supershift complexes (s) are indicated by brackets.

core binding motif. A functional role for the APBa element is also detectable, however, at a much lower level and is observed primarily when the contribution of APBb has been eliminated (Fig. 7B). CTCF and USF from cultured hippocampal neurons bind to the APP promoter In primary neurons, the APBb element prominently contributes to APP up-regulation during synaptogenesis (Fig. 7A). The nuclear factor that binds to the APBb domain has been isolated from HeLa cell nuclear extract and identified as CTCF (Vostrov and Quitschke, 1997). Transcription factor CTCF contains 11 zinc finger motifs and binds to a range of diverse DNA target sequences, apparently by using different combinations of zinc fingers (Klenova et al., 1993; Filippova et al., 1996). However, it is unknown whether CTCF regulates APP expression in neurons through binding to APBb. Therefore, we performed mobility shift gel electrophoresis with the 80-mer oligonucleotide probe APBb[2109], which contains the APP promoter sequence from position 264 to 2109, including the APBb domain from position 282 to 293 (Figs. 1 and 2). The binding assay was performed with extracts from HeLa cells as well as from rat hippocampal neurons to achieve a precise comparison with previously reported results and to characterize a newly generated antibody against CTCF. Incubation of this oligonucleotide with nuclear extracts from HeLa cells (Fig. 8A, lane 1) and hippocampal neurons (Fig. 8B, lane 1) resulted in the formation of binding complexes with identical electrophoretic mobilities. These complexes were competed with a 100-fold molar excess of unlabeled APBb[2109] (Fig. 8A and B, lane 2), but they were unaffected by the addition of unlabeled Mb2, which carries a deleterious block mutation in the APBb recognition sequence (Fig. 8A and

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B, lane 3). This indicates that factor binding to the probe is APBb-specific. Moreover, with both HeLa and neuronal nuclear extracts, a supershift complex was formed when a polyclonal antibody against CTCF was applied to the binding complex (Fig. 8A and B, lane 5), whereas no supershift complex was detected with preimmune serum (Fig. 8A and B, lane 4). Therefore, in contrast to previously described anti-CTCF antibodies (Vostrov and Quitschke, 1997), these newly generated antibodies recognize nondenatured forms of CTCF. This provides conclusive evidence that the factor that binds to APBb in cultured rat hippocampal neurons is CTCF. Binding of the transcription factor USF to the APBa site in the APP gene promoter was characterized extensively in a variety of tissues and cell lines of human (Kovacs et al., 1995), rat (Hoffman and Chernak, 1995), bovine (Vostrov et al., 1995), and mouse (Bourbonnie`re and Nalbantoglu, 1996) origin. Based on those observations, we inferred that USF most probably binds to the APBa element in embryonic cultured neurons as well. The originally described USF binding sequence from the AdMLP (Sawadogo et al., 1988) has an ;20-fold higher affinity for USF than the APBa sequence (Vostrov et al., 1995). Therefore, we used radiolabeled oligonucleotide AdMLP to assess the USF binding activity in cultured rat neurons (Fig. 8C, lanes 1 and 2). USF binding was completely competed with a 100-fold excess of unlabeled AdMLP oligonucleotide, whereas the wild-type APBa sequence showed partial competition (Fig. 8C, lane 3). In contrast, the oligonucleotide containing the Ma3 mutation in the USF binding site (Figs. 1 and 2) did not compete (Fig. 8C, lane 4). These results are consistent with the binding properties of USF to both the AdMLP and APBa sequences (Vostrov et al., 1995), and they further suggest that USF binds to the APBa domain in cultured primary rat neurons. CTCF binding to the APP promoter increases during neuronal differentiation The binding activity of CTCF and USF to their respective sites in the APP promoter was assessed as a function of neuronal differentiation to account for the up-regulation of promoter expression. To quantify factor binding from different extracts, it was essential to establish a reference point. Total protein concentration would not serve this purpose because the microscale of the extract preparation provided a very crude separation of nuclei from other cellular fragments and organelles. Although approximately the same number of cells was used for all nuclear extract preparations, the protein concentration varied from 2 mg/ml for day 1 cultures to 6 mg/ml for day 7 cultures. Thus, an internal standard corresponding to a uniformly expressed nuclear protein had to be established. For that purpose, we have chosen here the factor NF-Y/CP1/CBF that binds to the CCAAT element of the b-actin and many other promoters (Danilition et al., 1991; Sinha et al., 1995; Stewart and Crabb, 1996). This factor, which will here be referred to as NF-Y, is J. Neurochem., Vol. 73, No. 6, 1999

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FIG. 9. Binding of NF-Y, CTCF, and USF to their respective target sequences during neuronal differentiation on day 1, day 4, and day 7 in culture. Mobility shift electrophoresis with 32P-labeled CCAAT (A), APBb[2109] (B), or APBa (C) oligonucleotides incubated with neuronal nuclear extracts from day 1 (lane 1), day 4 (lane 2), and day 7 (lane 3) cultures is shown. The binding complexes are indicated by brackets. D: Relative CTCF and USF binding activity in nuclear extracts from day 1, day 4, and day 7 cultures. The binding activities of CTCF and USF were normalized to the NF-Y binding activity for each time point. The binding activities of both CTCF and USF in day 1 cultures were assigned the value of 1. The results represent averages and standard deviations from three independent experiments.

exceptionally conserved in phylogeny and was reported to be expressed uniformly and ubiquitously during the mouse embryonic development (Sinha et al., 1996; Stewart and Crabb, 1996). Moreover, we normalized the activity from the APP promoter to the b-actin promoter in expression studies. Therefore, we used NF-Y, a major b-actin promoter activator (Quitschke et al., 1989; Danilition et al., 1991), as a reference point and internal standard. Nuclear extracts were obtained from neurons on day 1, day 4, and day 7 in culture. Binding reactions were assembled with oligonucleotides containing the NF-Y, CTCF, and USF binding sites (Fig. 2), and nuclear extracts were added to the binding mixture in amounts proportional to the number of cells in each culture. Under these conditions, NF-Y binding activity was similar for all three cultures, whereas CTCF and USF binding increased between day 1 and day 7 (Fig. 9A–C). Specifically, when the binding complexes were quantified and normalized to the binding activity of NF-Y, CTCF binding in nuclear extracts progressively increased more than fivefold in nuclear extracts from day 1 to day 7 neuronal cultures (Fig. 9D). In contrast, the level of USF binding increased only twofold during the same period, and the maximal binding activity was reached on day 4 in culture. These results indicate that the rise in promoter activity observed between day 1 and day 7 cultures can be accounted for by an increase in binding of CTCF to the APBb element. CTCF binding is also more closely correlated to synaptophysin than to neurofilament accumulation during development. J. Neurochem., Vol. 73, No. 6, 1999

Replacing the native APP promoter sequence upstream from position 294 with vector sequences reduces the binding affinity of CTCF to the APBb site The results from the promoter deletion studies reported here suggest that the level of up-regulation is reduced significantly in the APP promoter construct terminating at position 294. Although deletion APP[294] indeed contains the core recognition sequence of the CTCF binding site between positions 282 and 293 and block mutations in that sequence largely eliminate upregulation, previous studies have shown that CTCF needs extra flanking sequences outside its recognition motif for optimal binding to the target (Klenova et al., 1993; Vostrov and Quitschke, 1997). It is therefore conceivable that sequence elements immediately upstream from position 294 contribute to the stability of the CTCF binding complex, and modifying these sequences may reduce binding affinity. We therefore compared the binding affinity of CTCF to three oligonucleotides containing different sequences upstream from the APBb site. Oligonucleotide APBb[2125] contains the endogenous wild-type promoter sequence from position 269 to 2125, whereas in oligonucleotide APBb[294], the sequence upstream from position 294 was replaced with vector sequences exactly as they exist in plasmid APP[294]. A third oligonucleotide APBb[2109] contains polycloning sequences upstream from position 2109 as described elsewhere (Vostrov and Quitschke, 1997). APBb[2125] was 59 end-labeled and competed with a three-, 10-, and 30-fold excess of either unlabeled APBb[2125] (Fig. 10A, lanes 2– 4) or oligonucleotides APBb[294] (Fig. 10A, lanes 5–7) and APBb[2109] (Fig. 10A, lanes 8 –10). Complete competition for binding was achieved with a 30-fold excess of oligonucleotides APBb[2125] and APBb[2109]. In contrast, the level of competition observed with oligonucleotide

FIG. 10. Electrophoretic mobility shift competition between oligonucleotides APBb[2125], APBb[294], and APBb[2109] with neuronal nuclear extracts. A: Mobility shift electrophoresis of 32 P-labeled APBb[2125] without competitor (lane 1) and in the presence of a threefold (lane 2), 10-fold (lane 3), and 30-fold (lane 4) molar excess of unlabeled APBb[2125]; a threefold (lane 5), 10-fold (lane 6), and 30-fold (lane 7) excess of APBb[294]; and a threefold (lane 8), 10-fold (lane 9), and 30-fold (lane 10) excess of APBb[2109]. B: Competition of 32P-labeled APBb[294] with the same unlabeled probes as in A. Labeled probes are indicated by asterisks. The binding to the radiolabeled probes without competitor was assigned the value of 100, and all other activities are expressed as a fraction thereof.

SYNAPTOGENESIS AND APP PROMOTER REGULATION APBb[294] was consistently approximately fourfold lower. The same results were observed in reciprocal competitions, in which oligonucleotide APBb[294] was 59 end-labeled (Fig. 10B). These results indicate that the endogenous APP promoter sequence between position 294 and 2109 contributes to the binding affinity of CTCF to APBb. Although replacing this sequence with heterologous vector elements does not abolish CTCF binding, as is the case with mutations in the core recognition sequence, there is nevertheless a significant reduction in binding affinity. It should be noted that occasionally the CTCF binding complex is observed as an apparent doublet on mobility shift gels (Fig. 10), suggesting a possible dimeric binding of CTCF to the target sequence. However, the appearance of this double band occurred in a random manner and was not reproducible from gel to gel. In addition, there is consistent evidence that CTCF binds to the APP promoter as a monomer (manuscript in preparation). It is therefore probable that the apparent doublet is an artifact of the mobility shift electrophoresis caused by a large excess of nonspecific protein used to supplement binding reactions to increase the sensitivity of the assay. DISCUSSION According to a current model, neuronal degeneration in AD could be the consequence of a pathological cascade triggered by genetic or environmental factors to induce toxic levels of Ab, which in turn elicits repair responses in the AD brain (Cotman et al., 1993; Small, 1998). These reactions could result in an increased expression of APP and subsequently produce more toxic Ab, thereby exacerbating the degenerative conditions in the AD brain. Notably, among the events in this pathological loop are neuritic sprouting and synaptic remodeling associated with increased amyloid depositions that occur most frequently in brain regions with high plasticity, such as hippocampus and entorhinal cortex (Byrne, 1987; Masliah et al., 1991; Dawirs et al., 1992). Evidence is also accumulating that implicates a role for APP in synaptogenesis and neuronal development (Schubert et al., 1991; Koo et al., 1994; Marquez-Sterling et al., 1997; Morimoto et al., 1998). Although APP regulation in different species has been the subject of extensive studies (Izumi et al., 1992; Pollwein et al., 1992; Quitschke and Goldgaber, 1992; Pollwein, 1993; Hoffman and Chernak, 1994, 1995; Quitschke, 1994; Song and Lahiri, 1998), few have been conducted in primary neurons that undergo differentiation and synaptogenesis. As APP is abundantly expressed in the brain, and neurons are the major source of cerebral APP (Schmechel et al., 1988), we investigated the transcriptional regulatory mechanism for APP expression in neurons during synaptogenesis in culture. To determine the relationship of APP expression to synaptogenesis and the regulatory factors that contribute to the increased levels of APP during neuronal development, we transfected neurons with plasmids containing the upstream

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regulatory region of the human APP gene. First, we demonstrated that as rat embryonic hippocampal neurons differentiate in culture, they produce increased immunoreactivity for neurofilament and synaptophysin (Figs. 3 and 4). Total immunoreactivity of synaptophysin and the number of puncta continued to increase over an 8-day period in culture, whereas neurofilament reactivity displayed a plateau starting at day 4. By transfecting hippocampal neurons with the APP[22,832] promoter construct, we showed that APP promoter activity steadily increased during neuronal differentiation to a 33-fold maximum from day 1 until day 7 (Fig. 5C). These results are consistent with the increased levels of endogenous APP transcripts from both neuronal culture and rat brain hippocampi (Fig. 5A and B), as well as with previous reports that APP protein levels increase in both neurons and neuroblastoma cells during differentiation in culture (Ko¨nig et al., 1990; Hung et al., 1992). Therefore, a close correlation is established between APP promoter activity and the accumulation of the synaptic vesicle protein synaptophysin in neurons (Fig. 5D). This suggests that APP may be involved in synaptogenesis during neuronal differentiation and that APP expression may be linked more closely to synaptogenesis than to neuritogenesis. However, more direct evidence would colocalize APP and synaptophysin immunoreactivity or block synaptogenesis with antisense oligonucleotides to APP. The observed 33-fold increase in promoter activity compared with an approximately threefold elevation of APP mRNA levels suggests that the APP transcripts may be subject to increased turnover in cells (Zaidi and Malter, 1994). Alternatively, the transfected human promoter may be subject to a higher level of up-regulation than the endogenous chromosomal rat promoter due to the differential utilization of transcription factors. In addition, a region spanning the first exon and part of the first intron was found to act as a negative regulator in the rhesus monkey APP gene (Song and Lahiri, 1998). Such sequence elements are absent in the transfected promoter constructs, and this may account for the lower level of up-regulation of endogenous APP mRNA. To investigate the transcription factors responsible for the observed APP up-regulation in primary neurons, we transfected neurons with APP promoter constructs containing sequential 59 deletions. We found that a promoter sequence extending to position 2121 was as efficient in conferring the observed increase in reporter activity as promoter fragments extending further upstream (Fig. 6). However, a deletion to position 294 resulted in an ;40% relative decline in reporter activity, suggesting that sequence elements between positions 294 and 2121 contribute to full promoter up-regulation in rat hippocampal neurons. Further transfections with promoter constructs containing transverse block mutations confirmed that the APBb core recognition sequence from position 282 to 293 is an essential element for efficient APP promoter up-regulation. In contrast, the contribution of the APBa domain between position 242 and 253 is considerably less pronounced and reaches a maximum level of up-regulation in day 4 cultures (Fig. 7). J. Neurochem., Vol. 73, No. 6, 1999

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This dependence on APBb in the control APP expression in neurons is consistent with results from HeLa and PC12 cells (Quitschke, 1994). Transcription factors USF and CTCF contribute to increased APP expression in neurons by specifically interacting with APBa (USF) and APBb (CTCF) (Fig. 8), and both factors display an increased binding to the two regulatory elements during neuronal differentiation (Fig. 9). However, USF binding only increases until day 4 in culture and levels off thereafter. Incidentally, this binding pattern correlates well with the expression pattern of construct APP[277] (Fig. 7). In contrast, CTCF showed a more substantial and continuous increase in binding activity during differentiation of neurons, a pattern that is reminiscent of the expression from APP promoter constructs containing the APBb binding site (Figs. 6 and 9). This suggests that both CTCF and USF are subject to developmental regulation, which depends on the differentiation stages of neurons. Indeed, a recent study has suggested that CTCF transcription is controlled by a highly conserved nuclear factor YY1 (Klenova et al., 1998). The YY1 activity itself is affected by cell differentiation and aging (Lee et al., 1992) and serves as a nuclear signal transducer during glutamate toxicity (Korhonen et al., 1997). Therefore, it is conceivable that in AD brains, the YY1 activity could be elevated as a result of the pathological responses, which in turn might activate CTCF to promote APP expression. Further investigation to untangle these intertwined regulatory pathways could provide new clues for the understanding of AD development. In contrast to previous reports on APP promoter activity in cell lines (Quitschke and Goldgaber, 1992; Quitschke, 1994), the 294-bp upstream fragment fails to confer full activity in transfected neurons (Fig. 6). Although the APBb domain between positions 282 and 293 is fully included in the APP[294] construct, it has been shown that CTCF needs extra flanking sequences outside its recognition motif for optimal binding to the target (Klenova et al., 1993). For example, in the APP promoter, it requires an oligonucleotide at least 45 bp long for effective DNA binding despite the fact that the essential recognition sequence only extends over 12 bp (Vostrov and Quitschke, 1997). Furthermore, CTCF contains 11 potential zinc finger motifs, and different combinations of zinc fingers may be used to bind apparently divergent DNA sequences (Klenova et al., 1993; Filippova et al., 1996). It is therefore possible that sequence domains outside the APBb core recognition sequence may also contribute to binding affinity, although those sequences are not essential for binding activity per se. To investigate this possibility, we performed binding and competition experiments with three 80-mer oligonucleotide probes containing different sequences 59 to the core recognition domain. Oligonucleotides APBb[294], APBb[2109], and APBb[2125] all contain the native APP promoter sequence starting at position 264 and ending at positions 294, 2109, and 2125, respectively. The 59 flanking sequence in oligonucleotide APBb[294] J. Neurochem., Vol. 73, No. 6, 1999

is identical to the one existing in plasmid APP[294], which is a portion of the pCAT2bGAL cloning site and additional plasmid sequences. The data demonstrate that oligonucleotide APBb[294] binds CTCF with an approximately fourfold lower affinity than either oligonucleotide APBb[2109] or APBb[2125] (Fig. 10). This indicates that the lack of additional native upstream APP sequences beyond position 294 accounts for the reduced CTCF binding. As CTCF is a multivalent transcription factor that binds to a variety of known promoters (Klenova et al., 1998) and presumably to others not yet identified, it is likely that conditions exist where the nuclear level of CTCF available for binding is limiting. Under such circumstances, the binding affinity for a target sequence could become increasingly important for effective interactions. It is conceivable that such a situation exists in primary rat hippocampal neurons, and it may explain why the activity from the APP[294] construct is lower than from those containing additional upstream promoter sequences. In summary, this study has established an association between APP up-regulation and synaptogenesis in primary neurons by presenting a close correlation between increased APP promoter activity and expression of synaptophysin in culture. It demonstrates for the first time the involvement of transcription factors CTCF and USF in the regulation of APP expression during differentiation of primary neurons, especially CTCF in relation to synaptogenesis. This is of physiological significance, because amyloid deposition in AD brain could be the consequence of a pathological cycle involving degeneration and regeneration of synapses in hippocampal and cortical regions. The study also suggests that transcriptional regulation of human APP in rat neurons may be distinct from that in established cell lines in the sense that the level of available endogenous CTCF limits the level of up-regulation. Acknowledgment: We thank John Torricelli, Dr. Adriana B. Marcuzzi, Dr. Kounosuke Watabe, and Dr. Dennis Q. McManus for technical assistance and helpful discussions. This work was supported by Illinois Department of Public Health, Alzheimer’s Disease Research Fund (G.J.B.), and National Institutes of Health grants AG13435 (G.J.B.) and NS30994 (W.W.Q.).

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