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Human Molecular Genetics, 2003, Vol. 12, No. 8 869-877
DOI: 10.1093/hmg/ddg098
© 2003 Oxford University Press

Alzheimer-associated C allele of the promoter polymorphism -22C>T causes a critical neuron-specific decrease of presenilin 1 expression

Jessie Theuns1, Jacques Remacle2, Richard Killick3, Ellen Corsmit1, Krist'l Vennekens1, Danny Huylebroeck2, Marc Cruts1 and Christine Van Broeckhoven1,*

1Department of Molecular Genetics, Flanders Interuniversity Institute for Biotechnology (VIB), University of Antwerp (UIA), Antwerpen, Belgium, 2Department of Developmental Biology, Flanders Interuniversity Institute for Biotechnology (VIB) and Laboratory of Molecular Biology (CELGEN), University of Leuven (KUL), Leuven, Belgium and 3The Institute of Psychiatry, Department of Neuroscience, London, UK

Received December 19, 2002; Accepted February 10, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We, amongst others, have shown that CC homozygosity at the -22C>T promoter polymorphism in presenilin 1 (PSEN1) is associated with increased risk for Alzheimer's disease (AD). Also, studies in AD brains suggested that CC homozygosity increased the risk for AD by increasing the Aß load. We characterized the PSEN1 promoter by deletion mapping, and analysed the effect of the -22C and -22T alleles on the transcriptional activity of PSEN1 in a transient transfection system. We showed a neuron-specific 2-fold decrease in promoter activity for the -22C risk allele, which in homozygous individuals would lead to a critical decrease in PSEN1 expression. The deletion mapping suggested that the 13 bp region (-33/-20) spanning the -22C>T polymorphism harbours a binding site for a negative regulatory factor. This factor has a higher affinity for the -22C risk allele and is strongly dependent on downstream sequences for cell-type-specific expression differences. Together, these studies provide evidence that the increased risk for AD associated with PSEN1 may result from genetic variations in the regulatory region, leading to altered expression levels of PSEN1 in neurons.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Alzheimer's disease (AD) is the most common form of senile dementia and is caused by progressive degeneration of the central nervous system. There is substantial evidence that genetic factors play a role in AD. The genetic contribution to AD is particularly strong in cases with the early-onset form of the disease (onset<65 years; EOAD). A positive family history was observed for 60% of EOAD cases and in 10% of the familial cases AD is inherited as an autosomal-dominant trait (1). Three genes have been identified that together explain 18–50% of all autosomal dominant EOAD families (2–6). In addition the E4 allele of the apolipoprotein E gene (APOE4) was shown to explain 12% of AD in the general population, with a 2.3-fold increase of the E4 allele in EOAD patients (7,8).

The most frequently mutated gene in autosomal dominant EOAD is presenilin 1 (PSEN1) with the majority of mutations being missense mutations (http://molgen-www.uia.ac.be/ADMutations/) (2,9). Missense mutations in PSEN1 were shown to lead to autosomal-dominant EOAD by pathways dependent upon the production of increased amounts of the amyloidogenic Aß42 peptide (10). Also, effective inhibition (up to 50% of the normal level) of PSEN1 expression in cultured cells by an anti-sense RNA approach resulted in increased Aß42 production (11). On the other hand, in neuronal cell cultures derived from PSEN1 deficient mice, APP processing into Aß was prevented (12). Together, these observations suggested that variable expression levels of PSEN1 modulate Aß production.

Previously, we reported a potential role for the PSEN1 promoter in increased risk for EOAD in a Dutch population-based study (13). Significant association was found with the promoter polymorphism -22C>T [numbering relative to the major transcription initiation site (TIS) P1 reported by Pastorcic and Das (14); formerly, this polymorphism was reported as -48C>T (2) with numbering relative to the TIS t2 reported by Rogaev et al. (15)]. The most frequent allele -22C was associated with an increased risk for EOAD (OR=2.6) due to significant overrepresentation of the CC genotype in patients, independent of the APOE4 genotype (13). In a systematic screen of the PSEN1 regulatory region we identified two additional polymorphisms (-2128G>A and -2797I/D) associated with increased risk for EOAD (16). All three promoter polymorphisms showed a high degree of linkage disequilibrium allowing the identification of a risk haplotype (-22C/-2128G/-2797D). Homozygosity for the risk haplotype was significantly higher in EOAD patients than in controls (P=0.03). In addition, we observed two heterozygous mutations (-254C>G and -2792A>G) in two patients homozygous for the risk haplotype (16). Each polymorphism and mutation involved consensus sequences of transcriptional regulatory elements and therefore might modulate PSEN1 transcription. Indeed, reporter gene data using luciferase demonstrated that the -254G mutation decreased PSEN1 transcriptional activity by 30% in neuroblastoma cells (17).

Recently, the association between -22CC and AD was replicated in a UK population of EOAD and late-onset AD patients (P=0.04) (18). Moreover, this study showed a phenotypic correlation with Aß40, Aß42 and total Aß load in AD brains, suggesting that the C allele of the -22C>T polymorphism in the PSEN1 promoter increased the risk of AD by altering PSEN1 expression and thereby influencing Aß load. Here, we performed additional studies of the proximal PSEN1 promoter, focusing on the functionality of the -22C variant in relation to AD.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Alternatively transcription initiation of PSEN1 in multiple tissues
Two alternative initiated transcripts of PSEN1 have been reported (15). The major transcript starts at exon 1A and is present in multiple tissues. The transcript starting at exon 1B has been detected only once in a colon adenocarcinoma cell line (15) and has not been studied since. We examined in more detail both transcripts in different tissues and cell types. RT–PCR analysis of the same amount of poly (A) RNA showed that exon 1A transcripts were indeed present at high levels in brain, liver, testis, colon and lymphoblasts (Fig. 1A). Increasing the amount of DNA polymerase enabled us to show that exon 1B transcripts were also present in all these tissues though at lower levels, with a significantly lower expression in brain and almost undetectable expression in liver (Fig. 1B). RT–PCR analysis of comparable levels of total RNA of human neuroblastoma and kidney cells also showed lower expression of exon 1B versus exon 1A transcripts (data not shown). RNA ligase-mediated rapid amplification of cDNA ends (RLM-5'RACE) in different tissues and cell types using an exon 3 primer confirmed these results. Sequence analysis of ~20 RLM-5'RACE cloned transcripts for each of the eight different tissues and cell types (160 clones in total) revealed the presence of multiple TISs for PSEN1 exon1A as reported (14,15). In all tissues and cell types the TIS of the majority (93.4%) of exon 1A transcripts is located at (+1) or downstream of P1 (Table 1) (10). Interestingly, 6.6% of the transcripts started 6–11 bp upstream of P1, suggesting that the true TIS of exon 1A might be located 11 bp upstream of P1. Also interesting was that 7% of the transcripts lacked the last 4 bp of exon 1A. None of the RLM-RACE clones contained exon 1B, however preliminary RLM-RACE experiments using an exon 1B primer identified exon 1B transcripts in different tissues and cell types (data not shown). The integrity of the RLM-RACE products of the different tissues and cell types was verified using ß-actin control primers. Thirty-eight out of 40 clones analysed contained the full-length ß-actin 5' end and only two clones lacked 3 and 6 bp, respectively. Therefore, we can conclude that variation in transcription initiation of PSEN1 is a gene-specific effect and is not due to general degradation of RNA.



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  		Figure 1. RT–PCR analysis of PSEN1 exon 1A and 1B transcripts in different human tissues. Tissues are presented by: B, brain; H, liver; T, testes; C, colon, L, lymphoblast. (0) No RNA present in RT reaction; (-) negative control PCR reaction; (M) 100 bp ladder. The DNA polymerase amount per PCR reaction is shown at the top of the gels. No RT, no reverse transcriptase. PSEN1 cDNA sequences from exons 1A–3 and exons 1B–3, respectively, are presented at the bottom of the graphs. Exon boundaries are marked by vertical lines and primers are presented by arrows. (A) Exon 1A amplification. (B) Exon 1B amplification.

 

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  		Table 1. Transcription initiation sites (TIS) of the human PSEN1 exon 1A transcripts mapped by RLM-RACE in different tissues and cell types. The numbering is relative to the most upstream exon 1A TIS (P1 or +1) previously reported by Pastorcic and Das (14)
 
Deletion mapping of the PSEN1 regulatory region
We constructed a deletion panel of the PSEN1 upstream region encompassing nucleotides -3501 to +1413 [numbering relative to P1 (14); Fig. 2]. Transcriptional activity of the promoter constructs was analysed in transiently transfected HEK293 and N2a cells by measuring firefly luciferase activity.



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  		Figure 2. Deletion mapping of functional PSEN1 promoter elements. The upper horizontal line represents a detailed restriction map of plasmid B22 (27). Exons 1A (Ex 1A, +1 to +139) and 1B (Ex 1B, +373 to +779) of PSEN1 are represented by the respective black boxes. The position of the PSEN1 -22C>T promoter polymorphism is indicated by an arrow. Restriction enzyme recognition sites used for subcloning into the pGL3 basic vector are represented in bold. The deletion constructs in the pGL3 basic vector are represented by horizontal lines. Position of the 5'- and 3'-end of each construct is given based on the exon1A transcription initiations site P1 reported by Pastorcic and Das (14). Bars represent the promoter activity of each construct in N2a (black) and HEK293 (white) cells. Transcriptional activities are expressed as a percentage of the SV40 early promoter activity. Values are the mean±SD of in duplo measurements of at least three transfection experiments of three independent DNA preparations each.

 
The relative changes in transcriptional activity between most PSEN1 promoter constructs were in general similar in both cell lines (Fig. 2). All promoter constructs with 3'-ends downstream of exon1B at +1413 or +1257 showed an activity below background levels. Further 3' deletion of sequences up to +849 increased activity ~10-fold to a maximum of 50% of SV40 promoter activity in HEK293, and 80% in N2a cells. Another 2-fold increase resulted from deletion of the remaining intron 1B sequences from +849 to +752. Of all promoter constructs ending in exon 1B at +752, the -113/+752 fragment showed the highest activity. Additional 5' deletion of this fragment to -33/+752 decreased the activity 5-fold suggesting the presence of a general transcriptional activator in the -113/-33 region. Further deletion of 5' sequences between -33 and -20 increased activity again 2-fold in HEK293 cells, while in N2a cells this deletion resulted in a more dramatic 5-fold increase, leading to a transcriptional activity comparable with the activity of the -113/+752 fragment. These data suggested the presence of a strong silencer in the -33/-20 region with a higher impact on expression in N2a cells than in HEK293 cells. However, upon further deletion of the 3' end, the -33/+80 fragment showed the highest activity detected for both cell lines in this study, while the activity of the -20/+80 fragment did not exceed background levels. In rat primary neurons the -33/+80 fragment exhibited at least 30-fold greater activity than the -20/+80 fragment (Fig. 3). Therefore, it appeared that the -33/-20 region harbours an important regulatory element that is strongly dependent on downstream regulatory elements and which is particularly responsive in neurons.



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  		Figure 3. Transcriptional activity of the PSEN1 -22C/T variants in transient transfection experiments. Bars represent firefly/renilla luciferase ratios for the different constructs (relative luciferase activity, RLA). (A) For the rat primary neurons transcriptional activities are presented as the percentage of the activity of the -20/+80 construct. (B) For N2A and HEK293 cells transcriptional activities are presented as the percentage of the activity of the wild-type construct (-22T, black bars). Values are the mean±SD of in duplo measurements of at least three transfection experiments of three independent DNA preparations each.

 
5' deletion of the major exon 1A TISs (+43/+849, +43/+752) did not completely abolish transcriptional activity, but resulted in a residual activity of about one-third of the activity of the respective exon 1A containing fragments (-20/+849 and -20/+752) in both cell types. Further deletion of the start of exon 1B (+536/+849, +536/+752) caused transcriptional activity to drop again to background levels.

Transcriptional effect of PSEN1 -22C>T polymorphism
We introduced the T-allele of -22C>T in the -292/+1257 fragment. In transiently transfected HEK293 cells no significant differences in expression level were detected between -22T and -22C promoter constructs, whereas in N2a cells a 2-fold decrease was observed for -22C (Fig. 3). We also introduced the -22T allele in the -33/+849 fragment. The transcriptional activity of -33/+849T was comparable with that of -20/+849 lacking the -22 position, while a 2-fold reduction in activity was detected for the C-allele in transiently transfected N2a and HEK293 cells (Fig. 3). MatInspector v2.2 (19) indicated the presence of a high affinity binding site for NF-1, a member of an emerging family of DNA binding proteins, some being involved in neuron-specific regulation of transcription (20).

Electrophoretic mobility shift assays
We examined whether -22C>T affected the specific recognition of the PSEN1 promoter by nuclear factors using electrophoretic mobility shift assays (EMSA). We started with -26/+1 double-stranded oligonucleotide probes containing either the C- or the T-allele. Both HEK293 and human neuroblastoma (SHSY-5Y) nuclear extracts contained nuclear proteins binding specifically to this region of the PSEN1 promoter resulting in the formation of one major complex. Competition with 10–40-fold excess of unlabeled -26/+1 C or T probes resulted in complete inhibition of complex formation (Fig. 4). However, no significant differences in complex mobility or binding affinity were detected between -26/+1C and -26/+1T. Also, the phosphorylation status of the proteins in the nuclear extract had no influence on binding (Fig. 4). Competition with an Ets1/2 consensus probe also inhibited binding of nuclear factors to the PSEN1 -26/+1 probe, suggesting that the -22C>T polymorphism may effect the binding of ETS proteins to its previously reported binding site at position -12 (14). However, pre-incubation with {alpha}-Ets1/2 or {alpha}-Elk1 antibodies did not result in a super shift of the complexes (data not shown). Even when we extended the region of investigation into the 5' direction, using a probe spanning the -43/-6 region, we could not detect any allele-specific differences.



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  		Figure 4. EMSA analysis of allele-specific effect of the -22C>T variation on the interaction of nuclear protein complexes extracted from HEK293 and SHSY-5Y cells with the (-26/+1) region. DIG-labelled double-stranded probes (50 fmol) were incubated with 10 µg nuclear extract from HEK293 and SH-SY5Y cells, respectively. PI, presence of phosphatase inhibitors 1 and 2 (Sigma). Lanes 1, 3–6 and 11–14 include the -22C risk allele. Lanes 2, 7–10 and 15–18 include the wild type -22T allele. In competition experiments, 20-fold excess of unlabeled probe was added prior to the addition of the labelled probe.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Polymorphic variability within a gene promoter can effect gene expression and has previously been associated with age-related neurodegeneration (16,18,21–24). In this study we set out to analyse the PSEN1 promoter region in more detail with respect to the AD-associated -22C>T variation. First we showed that both alternatively initiated exon 1A and 1B transcripts are present in different tissues and cell lines, although the exon 1B transcripts are at significant lower levels. None of the transcripts contained both exon 1A and exon 1B. RLM-RACE analysis of the PSEN1 exon 1A transcripts revealed the presence of multiple TISs and provided evidence that the real start site of PSEN1 exon 1A is located 11 bp upstream of P1. Together our data indicated that the initiation of transcription and splicing of the 5' UTR exons of PSEN1 is complex and that its effect on transcription efficiency warrants further investigation. Next we constructed an extended deletion panel of the PSEN1 upstream regulatory region to allow for identification of sequence determinants required for the PSEN1 transcriptional activity. One interesting result emerging from the 5' deletion analyses is the observation that transcription is not completely abolished when deleting the major exon 1A TISs. The residual activity can be due to transcription from the more downstream located minor TISs (15). Another, more likely, scenario is that transcription initiating from exon 1B is driven by promoter elements located downstream of the exon 1A TISs. The latter hypothesis is favoured since transcriptional activity increases with deletion of all reported exon 1A TISs. MatInspector v2.2 detected in the close proximity of exon 1B, the same combination of an Ets-1/2 binding site spanning the TIS and a number of GC-rich elements including an SP1 binding site, as was shown for exon 1A (14). When removing the start of exon 1B, promoter activity dropped to background levels. Our study is the first to demonstrate that the region downstream of the major exon 1A TISs contains functional promoter elements, most probably driving transcription from exon 1B both in neuronal and non-neuronal cells. To study the effect of the promoter deletions on the alternative usage of the first exons and the activity of the exon 1B promoter in the presence of the exon 1A promoter, we propose to determine the occurrence of exon 1A and 1B transcripts in cells transfected with the PSEN1 promoter deletion constructs.

Further we analysed the effect of the C- and T-alleles of the -22 polymorphism, located in the PSEN1 core promoter, on transcriptional activity by reporter gene expression in HEK293, N2a and rat primary neurons. The -22C>T variation, had an even larger effect on PSEN1 promoter activity than the previously reported EOAD mutation -254C>G (16). It also provided evidence that -22C>T modified transcriptional activity of the PSEN1 promoter in a cell-type specific manner. In contrast to the human neuroblastoma cells (N2a), human embryonic kidney (HEK293) cells showed the 2-fold decrease in transcriptional activity for the risk (C) allele as compared with the wild-type (T) allele only for the -33/+849 promoter fragment. Therefore, we suggest the presence of a cell-type-specific element in the sequence up or down stream of -33/+849 masking the transcriptional effect of -22C>T in HEK293 cells in vivo. This can be either a HEK293 cell-specific inhibitor or a neuron-restrictive silencer element (NRSE) (25,26). Although we did not detect the 21 bp NRSE consensus sequence in the human PSEN1 upstream region, we cannot rule out the presence of other elements with similar function. Additionally, deletion analysis showed that, for constructs ending at positions +849 or +752, the 5' deletion of the -33/-20 region resulted in an at least 2-fold increase of promoter activity, comparable to the presence of the T allele, suggesting that the presence of the -22 C allele results in a negative regulation of transcription. However, further deletion at the 3' end showed that the silencing effect of the -33/-20 region is completely abolished in the absence of downstream elements, especially in neuronal cells. These data suggested that a strong negative regulator binds to the sequence surrounding position -22 with the highest affinity for the AD-associated C-allele and that this binding is strongly dependent on downstream promoter elements. This might be either a real inhibitor or a weak activator competing for a binding site with a stronger activator. In this view, it is interesting that -22C>T is located in the core promoter, which has been shown to contain a functional Ets-1 like element at position -12 (14). Although MatInspector v2.2 predicted the presence of multiple transcription factor binding sites, some of which have been described to be involved in neuron-specific regulation of transcription (20), so far, no functional elements have been mapped to this region of the PSEN1 promoter. However, DNaseI footprint analyses in human neuroblastoma cells revealed the presence of a large protected area from -82 to -21 on the top strand, and even a larger footprint on the bottom strand from +33 to -82 (14). The very large footprints probably result from the binding of several proteins. In contrast, in HepG2 cells, no detectable footprint could be observed between -40 and -20, again suggesting the presence of a cell-type-specific regulatory element.

Although EMSA showed that nuclear proteins extracted from HEK293 and human neuroblastoma cells (SHSY-5Y) specifically recognized the PSEN1 promoter region covering the -22 position, no significant differences in complex mobility or binding affinity were detected between the risk C- and wild-type T-alleles. Competition with an Ets1/2 consensus probe inhibited binding of nuclear factors to the PSEN1 -26/+1 probe suggesting the presence of a protein of the ETS family. However, pre-incubation with {alpha}-Ets1/2 or {alpha}-Elk1 antibodies did not result in a super shift of the complexes, suggesting that these proteins are either not accessible to antibody recognition or not present in the complex binding to this region under these conditions. However, since downstream elements are important for promoter activity it is possible that the differential binding to the -33/-20 region can only be detected when these elements are included in the probes. However, the exact downstream sequences involved in this cooperative action have still to be identified and the analysis of long probes in the presence of crude nuclear extracts poses technical difficulties.

In conclusion, we detected maximal PSEN1 promoter activity for the smallest fragment -33/+80 spanning all exon 1A TISs in both N2A and HEK293 cells and therefore delineated the PSEN1 core promoter to this region. Also, we provided evidence for transcription initiation from exon 1B driven by its own promoter and the presence of exon1B transcripts in different tissues. The previously reported -22C>T polymorphism, associated with AD, is located in the PSEN1 core promoter in the 13 bp (-33/-20) fragment containing a strong negative regulatory element involved in cell-type-specific inhibition of PSEN1 transcription. We demonstrated a 2-fold neuron-specific decrease for the -22C allele associated with AD using reporter gene analysis. Previously, it was shown that a 50% reduction of PSEN1 levels significantly increased Aß42 production (11). Since most AD patients are homozygous for -22C, the corresponding 50% decrease in promoter activity would have a major effect on PSEN1 expression levels, possibly leading to increased Aß42 production. In combination with other promoter variations, such as the -254C>G mutation, it might even lead to enhanced transcriptional effects. Indeed, we have already demonstrated a neuron-specific 30% decrease (16) for this heterozygous EOAD-related mutation. Together, our studies suggested an important role for variability in regulatory elements in the genetic predisposition for developing AD. Further characterization of the factors involved, and their mechanism of action, will no doubt contribute to the unravelling of AD pathology.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
RNA isolation, RT–PCR and 5'RACE
Total RNA from SH-SY5Y and HEK 293 cells was isolated using the SV Total RNA Isolation System (Promega, Madison, WI, USA). Poly(A) RNA from human lymphoblasts was isolated using the Poly(A) Pure Kit (Ambion, Austin, USA). Human brain, testicle, liver and colon poly(A) RNA were purchased from Ambion. First-strand cDNA was generated from 100 ng poly(A) RNA or 5 µg total RNA using random hexamers and oligo dT primers and the Superscript first strand synthesis system (Life Technologies, Paisley, UK) according to the manufacturer's protocol. Exon 1A transcripts were PCR amplified using primers 5'-tcacatcggaaacaaaacagc-3' (exon 1A) and 5'-acgtacagtattgctcaggtggtt-3' (exon 3) and exon 1B using primers 5'-ggagtggagtaggagaaagaggaa-3' (exon 1B) and 5'-acgtacagtattgctcaggtggtt-3' (exon 3) using standard PCR conditions and increasing amounts of HotGoldstar DNA polymerase (Eurogentec, Seraing, Belgium). 5' RLM-RACE was performed on 250 ng mRNA using random hexamers and the GeneRacer Kit (Life Technologies) following the manufacturer's protocol. The 5' cDNA ends were amplified using the GeneRacer 5' Primer in combination with primer 5'-gtaggacaacggtgcaggtaactct-3' located in PSEN1 exon 3. PCR products were cloned using the TOPO TA cloning for sequencing kit (Life Technologies) and sequenced using the Big Dye Terminator sequencing kit (ABI, Foster City, CA, USA).

PSEN1 promoter luciferase reporter constructs
Genomic DNA fragments spanning parts of the putative PSEN1 promoter were subcloned upstream of the firefly luciferase gene (Fig. 2A). Different fragments of plasmid B22, containing exons 1A and 1B of PSEN1 and ~6.5 kb of upstream sequences (27,16), were subcloned into the promoterless pGL3-basic vector (Promega): the 4914 bp BglII/BamHI fragment spanning nucleotides -3501/+1413, the 2447 bp HindIII fragment spanning -1190/+1257, the 1743 bp NheI/HindIII fragment spanning -486/+1257, the 1549 bp KpnI/HindIII fragment spanning -292/+1257, the 1370 bp SacI/HindIII fragment spanning -113/+1257 and the 632 bp XhoI/HindIII fragment spanning +626/+1257 (numbering according to the major exon 1A TIS reported by Pastorcic and Das, (14). Later this deletion panel was refined with PCR-based deletion clones (Fig. 2A). Genomic fragments were obtained by PCR amplification using the proofreading Pfu polymerase (Promega) and primers designed to our published sequence of the human PSEN1 upstream region (AF205592) (16). Primers were designed to incorporate restriction enzyme sites at the end of the amplified products to facilitate subcloning of the fragments in pGL3-basic. The Quick-change in vitro mutagenesis kit (Stratagene, La Jolla, CA, USA) was used to introduce the T allele at position -22 in the 1549 bp KpnI/HindIII fragment using primer 5'-gtgggccggccgccaacgaTgccagagccggaaatgacg-3' and its reverse complement. The upper primer designed to amplify the 882 bp -33/+849 fragment spans position -22, the T allele was introduced in this fragment using an upper primer corresponding to the T allele. -22T clones were selected by HgaI digestion. The integrity of all inserts was confirmed by sequence analysis using the Thermo SequenaseTM II Dye Terminator Cycle Sequencing Kit (Amersham) using vector- and PSEN1-specific primers designed for screening of the PSEN1 5' upstream region (16). Sequences were assembled using the Lasergene software (DNASTAR Inc., Madison, WI, USA).

Eukaryotic cell culture and transient transfection
Mouse Neuro2a neuroblastoma (28) and human SH-SY5Y neuroblastoma cells were propagated in a minimal essential medium with Earle's salt (Life Technologies), 10% foetal bovine serum (Life Technologies), 2 mM L-glutamine (Life Technologies), 200 IU/ml of penicillin, 200 g/ml of streptomycin (Life Technologies) and 0.1 mM nonessential amino acids (Life Technologies). Human embryonic kidney cells (HEK293) were propagated in OptiMem (Life Technologies) with 10% fetal bovine serum, 200 IU/ml of penicillin and 200 g/ml of streptomycin. For transient transfection N2a and HEK293 cells were seeded in six-well tissue culture dishes, at 9x104 and 7x105 cells/well, respectively, and allowed to recover for 24 h. Cells were co-transfected with 20 ng of pRL-TK plasmid containing the herpes simplex virus thymidine kinase promoter upstream of the renilla luciferase gene (Promega) and 1 µg of either one of the PSEN1 promoter constructs or one of the control plasmids, using the Lipofectamine procedure (Life Technologies). Empty pGL3-basic vector was used as a negative control, pGL3-promoter plasmid containing the SV40 early promoter upstream of the firefly luciferase gene (Promega) as a positive control.

Primary neuron culture and transient transfection
Time-mated Sprague–Dawley dams (B&K, UK Ltd) were sacrificed by cervical dislocation. The E18 fetuses were collected and placed in Ca2+–Mg2+-free HBSS. The brains were removed and meningeal tissue dissected away. The cerebral hemispheres were isolated by dissection and transferred to a 15 ml tube and washed three times with fresh HBSS. Ten millilitres of trypsin solution were then added to the tissue and incubated at 37°C for 20 min. The tissue was then washed in B27 supplemented Neurobasal containing 10% FCS and DNAse 1 and then triturated with a fire polished pasture pipette. Dissociated cells were diluted to 1x106 cells/ml in Neurobasal medium +B27 and plated onto poly-D-lysine coated 24 well Falcon plates at 5x105/well for reporter gene assay.

Primary neurones were cultured for 7 days in Neurobasal+B27 supplement (Invitrogen) and were subsequently transfected with 200 ng of each PSEN1-luciferae reporter construct plus 25 ng of Renilla luciferase under a TK promoter (Promega) with 2 µl Lipofectamine 2000 in 300 µl of Optimem according to the manufacturers instructions (Invitrogen).

Luciferase activity
Transfected cells were cultured for 48 h, washed with 1 ml phosphate-buffered saline (PBS, Life Technologies), and lysed with Passive lysis buffer (Promega). Firefly luciferase activities (LAF) and renilla luciferase activities (LAR) were measured sequentially using a Dual-Luciferase reporter assay system (Promega) and a model TD-20E Luminometer (Turner design). To correct for transfection efficiency and DNA uptake, the relative luciferase activity (RLA) was calculated as RLA=LAF/LAR. To compare the RLAs between different cell lines, relative RLA was calculated as a percentage of the RLA of the SV40 promoter construct %RLA=(RLA/RLASV40)*100 or the wild-type construct %RLA=(RLAmt/RLAwt)*100.

Preparation of nuclear extracts
HEK293 and SH-SY5Y cells were grown under normal growth conditions to a density of 0.5–1x106 cells/ml. Approximately 109 cells were harvested in PBS (Life Technologies), washed twice in PBS, homogenized to single cell suspension and pelleted by centrifugation at 950g at 4°C for 15 min. Nuclear extracts were prepared according to a modified Dignam et al. (29) procedure. Cells were resuspended in five packed cell volumes (pcv) of hypotonic buffer H containing 20 mM Hepes pH 7.9, 1.5 mM MgCl2, 10 mM KCl and freshly added 1 mM DTT, 1 mM PMSF, 1xprotease inhibitor Complete (Roche, Basel, Switzerland) and different phosphatase inhibitor cocktails (Sigma St. Louis, USA). After 15 min of swelling on ice the cells were vortexed for 10 s. Nuclei were collected by centrifugation at 6000g for 1 min and the nuclear pellet was resuspended in 5 pcv of buffer D containing 20 mM Hepes pH 7.9, 1.5 mM MgCl2, 0.2 mM EDTA, 20% glycerol, 0.42 M KCl and freshly added 1 mM DTT, 1 mM PMSF, 1xprotease inhibitor complete (Roche) and phosphatase inhibitors (Sigma). The suspension was incubated on ice for 20 min and insoluble debris were pelleted by centrifugation at 10 000g for 2 min at 4°C. Nuclear extracts were divided in aliquots and stored at -80°C. The protein concentration of the extracts was determined using a Bradford assay (Bio-Rad, Munich, Germany).

Electrophoretic mobility shift assays
DIG-labelled single strand oligonucleotides with different lengths were designed spanning the PSEN1 -22 promoter variation: -43-6C, 5'-ccgtgggccggccgccaacgaCgccagagccggaaatg-3'; -43-6T, 5'-ccgtgggccggccgccaacgaTgccagagccggaaatg-3'; -26+1C, 5'-acgaCgccagagccggaaatgacgaca-3' and -26+1T, 5'-acgaTgccagagccggaaatgacgaca-3' and HPLC-purified. Blunt-ended double stranded probes were obtained by annealing of the oligonucleotides with their respective reverse complements and checked on a non-denaturing 15% polyacrylamide gel in 0.25xTBE. For the binding reactions 30–60 fmol DIG-labelled double stranded probe were added to a total reaction volume of 20 µl containing 2–10 µg nuclear extract, 1xHepes binding buffer (12% glycerol, 20 mM HEPES or Tris, 50 mM KCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF) and 1 µg poly (dA–dT) (Boehringer-Mannheim, Germany). For competition assays unlabelled double stranded probes were added to the reaction mixture prior to addition of the labelled probe. In the supershift experiments the extracts were pre-incubated on ice with rabbit polyclonal anti-Ets-1/Ets-2 (SC-112X; Santa Cruz Inc., CA, USA). The binding reactions were incubated at room temperature for 15–25 min. Protein–DNA complexes were analysed by electrophoresis on non-denaturing 6% polyacrylamide gels in 0.25xTBE and visualized by chemiluminescent detection using the DIG gel shift kit (Roche).


   ACKNOWLEDGEMENTS
 
We thank Cold Spring Harbor Laboratories for the technical support in the modified Dignam procedure for preparation of nuclear extracts. We are grateful to Ayodeji Asuni for the supply of the primary neurons. This work was supported by the Fund for Scientific Research-Flanders (Belgium; FWO-V), the Medical Foundation Queen Elisabeth, the Interuniversity Attraction Poles (IUAP) program P5/19 of the Federal Office of Scientific, Technical and Cultural Affairs (OSTC), the International Alzheimer's Research Foundation (IARF), Belgium; and the Welcome Trust, UK. M.C. and J.T. are postdoctoral fellows of the FWO-V.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Department of Molecular Genetics (VIB8), University of Antwerp (UIA), Universiteitsplein 1, B-2610 Antwerpen, Belgium. Tel: +32 38202601; Fax: +32 38202541; Email: christine.vanbroeckhoven{at}ua.ac.be Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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