Human Molecular Genetics, 2002, Vol. 11, No. 4 465-475
© 2002 Oxford University Press
A candidate molecular mechanism for the association of an intronic polymorphism of FE65 with resistance to very late onset dementia of the Alzheimer type
1Department of Pathology and 2Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA and 3Molecular Pharmacology Research Center, New England Medical Center, Boston, MA 02111, USA
Received November 7, 2001; Revised and Accepted December 15, 2001.
DDBJ/EMBL/GenBank accession nos AF394214 and AF394215.
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ABSTRACT |
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Late onset dementias of the Alzheimer type may be coupled to intrinsic aging processes. Their major pathological hallmarks are the deposition of aggregates of beta amyloid (Aß) peptides, proteolytic products from internal portions of the Aß precursor protein, ßPP. Susceptibility appears to be modulated by polymorphic alleles at multiple loci. Most of these putative assignments, however, have been controversial. It is therefore essential to provide evidence of a plausible biological basis for each such association. Here, we show such evidence for the case of a biallelic polymorphism of the FE65 intron 13. FE65 is an adaptor protein that tightly binds to the cytoplasmic tail of ßPP. Increasing evidence indicates that this binding plays a critical role in a signaling pathway. Our results reveal that a protective (minor) allele alters the splicing of the terminal exon by selection of an alternative acceptor site, resulting in an isoform, FE65a2, with an altered C-terminal region lacking part of a ßPP binding site. Pull down assays confirmed that the FE65a2 isoform binds to ßPP less efficiently, suggesting that an attenuated binding of FE65 with ßPP is, in part, responsible for resistance to the very late onset disease. Sequence analysis of the FE65 of mice, non-human primates and man revealed that the susceptibility allele, which codes for strong binding of the FE65 protein with ßPP, was favored by natural selection leading to our lineage. That allele may contribute to very late onset form of Alzheimer disease when we are aged.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Dementia of the Alzheimer type (DAT) is a complex group of diseases that includes rare familial forms caused by autosomal dominant mutations, generally included under the rubric of an ‘early onset’ category, as affected subjects frequently exhibit cognitive abnormalities before the age of 60. The overall prevalence of DAT, however, rises exponentially with age in our species (
10% >65 years and 47% >85 years) (1). Cases occurring at >65 years are often referred to as late onset DAT (LODAT). Some authors have also differentiated a subset of patients with very late onset DAT (VLODAT) (2). In contrast to the early onset familial forms, the genetic component of susceptibility to late onset forms appears to be polygenic. Moreover, the published data are consistent with the view that these genetic influences may predominate at particular periods of the late portions of human life spans (2,3). However, with the exception of APOE, these assignments have been controversial, in part because of the failure to compare results using similar ranges of age onsets and similar ethnicities. It is therefore essential to provide evidence for a biologically plausible basis for claims that a particular allele provides susceptibility to or protection from LODAT. We previously reported that elderly carriers (average age of onset 75 years) of an intron 13 polymorphic allele (allele 2) of FE65 were relatively resistant to VLODAT (4). The association has been confirmed, but only in populations of equal or more advanced ages (5). The failure of others to replicate that observation may relate, in part, to an inadequate representation of these very old subjects, the use of controls younger than those of VLODAT counterparts or the inclusion of an excessively wide range of ages (Table 1). We are aware that genome-wide screens for LODAT have not disclosed a locus at or near FE65. In some cases, this might have resulted from a predominance of samples from individuals with LODAT, as opposed to VLODAT. It may also reflect differential sensitivities of the two methodological approaches to the discovery of susceptibility alleles in different regions of the genome.
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FE65 is an adaptor protein that consists of three protein–protein interaction domains (PIDs)—a WW domain at its N-terminus and two tandem PIDs at its C-terminal half (6). The more C-terminal PID (PID2) binds to a YENPTY motif of the cytoplasmic tail of ßPP (7–13). This interaction may involve regulation of gene expression (14), redistributions of FE65 and the cleaved ßPP tail between cytoplasm and nucleus (14–17), modulation of cell mobility (18), recruitment of other proteins to the cytoplasmic tail of ßPP (19), translocation of ßPP to the cell surface (20) and increases of the proteolytic processing of ßPP (20,21). Formation of the FE65–ßPP complex therefore appears to be a key element for modulations of the functions and metabolism of ßPP. Aberrant processing and functioning of ßPP are thought to be the major causes of all types of Alzheimer’s disease (22–24). The predominant expression of FE65 in neurons of the central nervous system (25,26) is consistent with an important role in the pathogenesis of VLODAT.
Part of the high affinity ßPP binding region in FE65 involves amino acid residues that are encoded by the 3' end of exon 13 and the 5' end of exon 14 (a terminal exon), interrupted by the 84 bp intron 13 (4). The VLODAT-associated biallelic polymorphism in this intron results from two variations usually in linkage disequilibrium: allele 1 has two tandem CTA6–11 sequences (at positions 6–11 from the donor site) and an A57 (at position 57); allele 2 has only one of the two CTA6–11 sequences and a G57. We previously proposed that the resistance to VLODAT in subjects bearing the minor allele (allele 2) was most likely via an influence on the efficiency of the nearby splice site utilization. We suggested that this in turn would produce alternatively spliced isoforms with differential ßPP-binding affinities. In this study, we provide evidence that is completely consistent with that hypothesis. We show that the protective allele 2 does in fact expedite alternative splicing, with the production of an isoform with only weak binding to ßPP. However, the susceptibility allele 1 appears to have been favored by natural selection. Its contribution to VLODAT may therefore be considered to be an example of antagonistic pleiotropy, a type of gene action thought to contribute to the senescent phenotype (27).
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RESULTS |
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Detection of an alternative splicing site within FE65 exon 14 in mini genes
To test the hypothesis that the intron 13 polymorphic locus influences splicing of exon 14, two strategies were chosen. A 3' terminal exon-trapping vector was first used for construction of a series of mini genes containing FE65 intron 13 variants and flanking exons (Fig. 1A and B). In Figure 1B, mini genes 1 (two tandem CTA6–11 and A57) and 4 (a single CTA6–11 and G57) resemble allele 1 and allele 2 in vivo, respectively; mini genes 2 (two tandem CTA6–11 and G57) and 3 (a single CTA6–11 and A57) contain only one variation of the biallelic polymorphism; these are rarely found in vivo (4). Splicing of these mini gene products was evaluated by transfection of the plasmid DNAs into COS-7 cells, an African green monkey kidney carcinoma cell line, followed by a Southern blot analysis of the products of a reverse transcription–polymerase chain reaction (RT–PCR-Southern). The results revealed two major splicing products (Fig. 1C). The sizes of the larger bands matched the expected products when the entire exon 14 was included. The smaller ones, however, were novel, presumably representing an alternatively spliced isoform. The novel bands were much more abundant in cell extracts transfected with mini gene 4 (Fig. 1C, lane 4) than those transfected with any other mini genes, although either G57 or a single CTA6–11 alone (mini gene 2 or 3) also increased the production of the novel bands (Fig. 1C, lanes 2 and 3) when compared with mini gene 1, indicating that both variations at this locus may contribute to the alternative splicing in the case of the mini genes.
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and asterisk indicate the positions of the CTA6–11 sequences and the A57/G57 substitution. (C) Splicing of the mini genes. Total RNA isolated from the transfected COS-7 cells was reverse-transcribed with a primer containing a poly (T)17 and an adapter sequence (A). sscDNA was amplified by PCR with a pair of primers indicated by arrows in (A). PCR products were then separated on an agarose gel, transferred to a membrane and hybridized with a [32P]-labeled probe specific to the second exon derived from the vector as indicated in (A). The number of each lane corresponds to the construct number in (B). V, a vector only (control). Ratios of the 606 bp to the 1029 bp bands are indicated at the bottom of the gel. (D) Two alternative pathways for splicing exon 14. Alternatively spliced pathways are indicated by diagonal lines. The pathway on top results in a classical 2.6 kb transcript containing the entire exon 14. The bottom pathway uses a conserved cryptic acceptor site within exon 14 and generates a novel 2.2 kb transcript, named FE65a2. Lower and upper cases represent nucleotide and amino acid sequences, respectively. The subscript numbers indicate nucleotide positions in intron 13.DNA sequence analysis revealed that the production of the novel bands was due to the presence of a cryptic splicing acceptor site within exon 14 at nucleotide 424, downstream from the usual site (Fig. 1D). The cryptic acceptor sites were conserved among human, mouse and rat. Thus, the alternative splicing produces a new mRNA isoform containing a 423 bp deletion from the 5' of exon 14 resulting in a full-length transcript of only 2.2 kb. This short isoform is hereby named FE65a2 in order to distinguish it from the classical 2.6 kb isoform, in which the entire exon 14 is included.
The VLODAT-protective allele causes the alternative splicing in vivo
In order to find in vivo evidence that the presence or absence of the two endogenous FE65 isoforms is actually associated with the intron 13 locus, we applied a second strategy. We examined expression of the two isoforms in various cell cultures and tissues from individuals with different FE65 intron 13 genotypes. RT–PCR-Southern analysis from fibroblast cultures of 13 individuals revealed the existence of endogenous FE65a2 isoforms associated with the presence of the VLODAT-protective allele 2 (Fig. 2). Using three probes that are specific to each isoform or both isoforms (Fig. 2A), we observed that the FE65a2 mRNA was only present in the fibroblast cultures of individuals bearing at least one copy of allele 2 (Fig. 2B, lanes 7–13). After a long exposure (24 h, data not shown), only faint bands could be seen at positions of the FE65a2 in homozygotes for allele 1. However, their intensities were 1% of those with at least one copy of allele 2 (lanes 7–13), suggesting that the intron 13 structure of allele 2 is primarily responsible for the alternative splicing of endogenous FE65 mRNA. Similar results were also observed in lymphoblastoid cultures (data not shown). In contrast to the results of the mini gene splicing assays (Fig. 1C, lane 1), the results of the endogenous assays indicated that homozygotes for allele 1 do not usually produce significant amounts of FE65a2 mRNA in vivo, suggesting that there is a unique cis-element(s) contributing to the selection of the ‘correct’ acceptor site for inclusion of the entire exon 14. This element(s) is very likely located somewhere upstream of exon 13, since more than 9 kb of that part of this gene was missing in the mini gene constructs.
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To further evaluate the regulation of alternative splicing, relative levels of the FE65a2 mRNA isoform were determined in the middle temporal cortex (MTC), cerebellar cortex (CC), and middle frontal cortex (MFC) of six postmortem brains by RT–PCR-Southern analysis (Fig. 2C). Since smaller DNA fragments are more readily amplified than larger ones in PCR reactions, the actual percentages of the FE65a2 isoform in total FE65 mRNAs were determined by normalizing to a series of standards in parallel reactions (Materials and Methods). Once again, we observed that expression of the FE65a2 isoform was associated with allele 2 (Fig. 2C, individuals D–F). Homozygotes for allele 1 do not usually express this isoform (Fig. 2C, individuals B and C), with the exception of individual A, in whom FE65a2 could be detected at levels of 2.2–3.0% of total FE65 mRNAs in the three brain regions. In the heterozygotes, amounts of the FE65a2 isoform varied from 5.3–33.0% in these specimens. Average proportions (means ± SD) of the FE65a2 mRNA in the three brain regions of each individual were 22.87 ± 4.48% (individual D), 21.53 ± 9.59% (individual E) and 10.97 ± 5.60% (individual F). The average proportions of this mRNA in the three individuals of the same brain region were 16.33 ± 10.54% (MTC), 16.77 ± 6.93% (CC) and 22.27 ± 8.89% (MFC). The SD among individuals were relatively larger than those among brain regions. These observations, along with the low but constant levels of the FE65a2 mRNA expressed in all examined regions of individual A, suggest that other genetic variations could also modify the splicing phenotype. Local environments might also influence the splicing of this mRNA.
Both the 2.6 kb and 2.2 kb FE65 transcripts had previously been observed on northern blots (with a probe to exons 11–13) of a single sample of an adult human brain, and brain and testis from mice tissues, in which FE65 mRNAs are predominantly expressed (8). However, the identity of the 2.2 kb transcript was not determined in that study. Retrospective analysis of the previous northern blot revealed that the percentages of the 2.2 kb isoform among the total FE65 mRNA species (the 2.6 kb isoform plus the 2.2 kb isoform) were 11%, 28% and 43% in human brain, mouse brain and mouse testis, respectively. These numbers closely approximated the quantitative results (RT–PCR-Southern) in this study.
The VLODAT-susceptible allele may be favored by natural selection
To investigate the genetic origin of the intron 13 polymorphism, we compared FE65 intron 13 sequences among several mammalian species including human, common chimpanzee (Pan troglodytes), African green monkey (Cercopithecus aethiops) and the laboratory mouse (Fig. 3). The nucleotide sequences of chimpanzee and African green monkey are 96.3 and 92.6% identical to human allele 2, and 94.0 and 90.5% identical to human allele 1, respectively, while the mouse sequence is much less well conserved. Interestingly, the number of CTA6–11 sequences is increased during evolution: no CTA in mouse and African green monkey, one CTA in chimpanzee and human allele 2, and two CTAs in human allele 1. In contrast, the A57 is conserved in all species examined except in human allele 2. These comparative results suggest that the two tandem CTA6–11 sequences in human allele 1 probably resulted from a meiotic replicative or recombinant event leading to an extra CTA in the pre-existing allele 2, while G57 was due to a point mutation. Natural selection towards our lineage apparently favored two tandem CTA6–11 sequences because it has become the predominant allele in humans, at least among populations of predominantly northern European extraction.
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Expressions of the FE65a2 mRNA in the tissues from these lower species are equivalent to or more abundant than those of the human heterozygotes. The proportions of FE65a2 mRNA in total FE65 were 33% in cultures of COS-7 cells (African green monkey) (Fig. 2C), and 25–45% in various mouse tissues (data not shown) as determined by RT–PCR-Southern assays, and 28% and 43% in mouse brain and testis tissues as determined previously by northern analysis (8). Both African green monkey and mouse have the conserved A57, but no CTA6–11 (Fig. 3), suggesting that the possession of two tandem CTA6–11 sequences may be the primary factor for successful inclusion of the entire exon 14. The mechanism may involve the formation of a new secondary structure that favors the selection of the first over the second acceptor site. On the other hand, the evidence that the inclusion of the 5' region of exon 14 is favored by natural selection suggests that the amino acids encoded by this region may be very important for human brain development and its sophisticated functions, given the predominant expression of FE65 in neurons of the central nervous system (26).
FE65a2 inefficiently binds to the C-terminal region of ßPP
We compared the predicted amino acid sequences of FE65a2 with the sequences of the classical FE65. The results indicate that FE65a2 is identical to FE65 through amino acid 655 (Fig. 4A). However, the 55 amino acids present at the C-terminus of the FE65, including the partial ßPP binding site and the stop codons, are deleted in FE65a2. Because of the deletion of the stop codons, the open reading frame is extended for 53 additional amino acids before reaching the terminal polyadenylation cleavage site. The open reading frame of FE65a2 could be extended further into the poly(A) tail resulting in a polylysyl-C-terminus. It has been reported that mutant transcripts [those lacking part of coding sequences and stop codons and extending the open reading frame into the poly(A) tail] of a growth hormone receptor gene from a dwarf strain of chickens were successfully translated in vivo (28). This altered gene action was thought to have been the cause of the dwarf phenotype. The novel C-terminal region of FE65a2 shows no homology with any other known proteins, but the sequences of the regions are 58% identical in a total of 53 amino acids, or 85% identical at the N-terminal halves of human versus mouse (Fig. 4A). These highly conserved sequences suggest that the region may have unique functions when expressed.
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One of the best known functions of the C-terminal region of the classical FE65 isoform is its high binding ability to the cytoplasmic C-terminus of ßPP. The amino acid sequences of FE65a2 suggest that this isoform might not bind to ßPP efficiently. To test this hypothesis, the ßPP-binding abilities of the two isoforms were compared by fusion protein pull-down assays. A fusion protein (GST-C48) containing glutathione S-transferase (GST) and the 48 C-terminal amino acids of the human ßPP was used as a ‘bait’ to entrap FE65 isoforms from the lysates of COS-7 cells transfected with full-length FE65 or FE65a2 cDNAs (Fig. 4B). The identities and expression levels of these recombinant proteins were first evaluated by western analysis with isoform-specific antibodies (Fig. 4B, top). As shown in Figure 4B (bottom), the FE65 isoform showed strong binding to the C-terminus of ßPP; in contrast, the FE65a2 isoform showed dramatically reduced binding to that region. Since the FE65a2 isoform is encoded by the VLODAT-protective allele 2, these results are consistent with the hypothesis that an attenuated interaction of FE65 with ßPP may be partially responsible for the association of that allele with resistance to VLODAT.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We present here compelling evidence that the VLODAT-associated polymorphic variation in the structure of the FE65 intron 13 influences alternative splicing of the last exon that encodes part of the ßPP binding site. Altered splicing of pre-mRNAs has recently been implicated in the pathogenesis of several neurological diseases (29). VLODAT may now be added to that list, given the association with preferential selection of acceptor sites for splicing of exon 14, and the consequent synthesis of protein isoforms with differential ßPP binding affinities. This mechanism is in agreement with the current hypothesis that intracellular signaling resulting from the metabolism of ßPP may be involved in the development of DAT as binding of FE65 to the ßPP tail might be one of the early steps in a signaling pathway (14,23,24).
We previously found increased expression of FE65 mRNA in the cerebellar cortex of postmortem brains of VLODAT subjects. That region exhibits only minimal evidence of DAT-related pathology; mainly variable amounts of diffuse deposits of ßPP-derived polypeptides. FE65 may therefore play an early role in VLODAT pathogenesis (30). Recent evidence also suggests that increased FE65 immunoreactivity in hippocampal area CA4 of VLODAT brains might be associated with the severity of the disease (31). Finally, overexpression of FE65 in cell cultures has been shown to increase the proteolytic processing of ßPP (20,21). These observations, including the present results, are consistent with the hypothesis that the downstream actions that follow strong FE65–ßPP interactions, as mediated by allele 1, may accelerate VLODAT pathogenesis late in the life span. Conversely, the attenuated interaction mediated by allele 2 may play a protective role (Fig. 5).
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?, the influential trends are not clear. Thick arrows suggest that the pathways might be enhanced. Asterisks indicate the genes in which DAT-related mutations and/or polymorphisms have been reported.In addition to binding to ßPP, FE65 interacts with several other proteins via its two other binding domains. These proteins include the WW domain-bound Mena (mammalian enabled, involved in cytoskeletal dynamics and neural development) (18,32) and Abl (a non-receptor tyrosine kinase) (19), and the PID1-bound histone acetyltransferase Tip60 (14), the LDL receptor-related protein (LRP, a neuronal ApoE receptor) (33) and the CP2/LSF/LBP1 transcription factor (34). With this protein network, FE65 appears to play a critical role in escorting FE65–ßPP/derivative complexes, via its interactions with the other proteins, to alternative destinations. One such example is that the release and transport of the FE65–ßPP tail complex into the nucleus for subsequent binding to histone acetyltransferase Tip60 (14). By this action, signals could be transduced from extracellular environments into the nucleus for regulation of gene expression. Another example is the observation that FE65–ßPP complex selectively localizes with Mena in integrin-based focal complexes in a mobile membrane compartment, thereby modulating cell movement (18). Formation of the FE65–ßPP complex can also influence the local environments around the ßPP tail because when FE65 binds to ßPP, active Abl is recruited to this location, followed by phosphorylation of ßPP tail (19).
At least three other proteins bind, via their PID domains, to the YENPTY motif of the ßPP tail, potentially competing with FE65. They include X11 and its family members (neural adaptor proteins) (9,10,35,36), mDabl (mammalian Disabled, a neuronal adaptor protein interacting with non-receptor tyrosine kinases) (33,37,38), and mouse c-Jun N-terminal kinase (JNK)-interacting protein-1b (JIP-1b) and its human homolog IB1 (scaffold proteins of the JNK signaling pathway) (39). In fact, the binding of FE65 or X11 to ßPP has been shown to have opposing effects on ßPP processing (20,21,40,41). Among these, X11, mDabl and JIP-1b bind to the YENPTY motif with comparable affinities, whereas the affinity of FE65 for ßPP appears to be at least 3-fold greater than that of the other proteins (38,39,42). This suggests that the FE65–ßPP complex may play a predominant regulating role. Thus, the FE65–ßPP complex could modulate the strengths of other signaling pathways mediated by the YENPTY motif (Fig. 5).
The mechanism for resistance to VLODAT mediated by the FE65 polymorphism might not be limited to a reduced binding of FE65a2 to ßPP. The last 55 amino acids at the C-terminus of the classical FE65 isoform, which are deleted in FE65a2, are 53% identical to the C-terminal region of FE65L1, a member of the FE65 family; that region contains apoptosis-inhibitory activity (43). On the other hand, the newly acquired but highly conserved sequences at the C-terminus of the FE65a2 isoform may possess yet-to-be-defined functions.
Remarkably, allele 1, which codes for strong ßPP binding and enhanced susceptibility to VLODAT, was favored by natural selection leading to our lineage. This allele is present in humans but not in any of the other species we have examined. This observation suggests that the inclusion of the entire exon 14 and an intact ßPP-binding site into FE65 protein of humans may have evolved together with specializations associated with the development of the human brain. This speculation is consistent with observations that expression of FE65 mRNA is restricted to neural tissues in mouse embryos (25) and is predominant in adult brain tissues (26,44). During neuronal differentiation, expression of FE65 mRNA is dramatically up-regulated (30-fold) and is switched from a non-neuronal isoform to a neuronal isoform (26). Likewise, ßPP is also highly expressed in neurons; its proteolytic products are constitutively released in cell cultures, and can be found in normal brains (22). Extensive studies suggest that ßPP and its derivatives are important factors involved in the viability, morphologic differentiation and functional plasticity of nervous cells, and appear to play a role in memory processes of mice (45). However, during the aging of many humans, Aß peptides, released after ß- and 
-cleavages from an internal portion of ßPP, are deposited in brain regions that are important for cognitive functions (22). The extent of these depositions is coupled to the availability of the C-terminal fragments of ßPP. The latter are barely detectable in tissues because that fragment is either unstable or is rapidly degraded, as has been shown by cell culture experiments (16). This suggests that the C-terminus-mediated signaling pathways need to be highly regulated in vivo. Intrinsic aging processes might perturb such regulation. Alternatively, chronological time, rather than an intrinsic biological aging process, may lead to the acceleration of stochastic alterations in such regulation. This notion is consistent with the fact that DAT is mainly found in the species that have long life span and high cognitive functions (46). Thus, a strong FE65–ßPP interaction, beneficial during the early phase of our life history, may become a liability during aging.
Since only a small and variable portion (5–33%) of the pre-FE65 mRNA would be expected to undergo alternative splicing in heterozygotes (25% in our population), it would take many decades for phenotypic expression of differential susceptibility, consistent with the nature of the association that is modest and could only be detected in a very old population [Table 1; note particularly the data of Lambert et al. (5)]. As we have discussed, other functional variants in this region, such as polymorphisms in the cis-element(s) upstream of intron 13, might also have modulating effects. One can imagine a complex pattern of allelic variations that could contribute to such functional modulation since these polymorphisms are likely to be in linkage disequilibrium, at least in populations derived from northern Europe (47). It will therefore be important to pursue haplotype analysis at such loci.
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MATERIALS AND METHODS |
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Cell cultures and transfection
Cells were cultured as previously described (26). For DNA transfection, COS-7 cells were seeded at 3–5 x 105 cells/60 mm dish for 16 h and were transiently transfected with 2.5 µg of plasmid DNAs and 12.5 µl of SuperfectTM (Qiagen, Valencia, CA) per dish as described in the manufacturer’s instructions. Cells were assayed
48 h after transfection.
Generation of mini genes and recombinant protein constructs
The initial mini gene, pTAG-FE65, was generated by PCR amplification of a human P1 clone (4). Forward and reverse primers were tagged with EcoRI and BamHI sites. Their PCR product (1277 bp) overlaps with the sequences of nucleotide –74 from the acceptor site of intron 12 to nucleotide +379 from the polyadenylation signal. After digestions, the purified DNA fragment was cloned into a pTAG4 vector (48) at the corresponding sites by standard cloning procedures. The constructs with A
G mutations and CTA deletions were derived from the pTAG4-FE65 construct with a Quick Change mutagenesis kit (Stratagene, La Jolla, CA). The constructs, pcDNA3.1FE65 and pcDNAFE65a2, containing full-length cDNAs of human FE65 and FE65a2, were generated by RT–PCR with RNA isolated from human brain tissues of a homozygote for allele 1 and from fibroblasts of a homozygote for allele 2, respectively. The resulting 2.2 kb cDNA sequences contained the entire open reading frames from the first Met in exon 2 to the first stop codon in exon 14 for the FE65 isoform or to the beginning of the polylysine chain for the FE65a2 isoform. cDNAs were then cloned into the BamHI and NotI sites of pcDNA3.1(+)B (Clontech, Palo Alto, CA). GST expression vector pGEX-4T-2 (Amersham Pharmacia Biotech, Piscataway, NJ) and a fusion protein between GST and the 48 C-terminal amino acids of the human ßPP protein (GST-C48, amino acids 590–695, of ßPP695) were described in the previous study (8). The DNA sequences of all the constructs were verified by sequencing.
3' terminal exon trapping and RT–PCR-Southern analysis
The pTAG4-FE65 and its mutants were transiently transfected into COS-7 cells. Total RNA was isolated 24 h after transfection. Reverse transcription was performed with a primer AP containing poly-T plus an adapter sequence (5'-AAGGATCCGTCGACATCGATAATACGACTTTTT(T)12-3') under the conditions described previously (26). Single strand cDNA (sscDNA) was then amplified by PCR with a forward primer SV40P (5'-AGCTATTCCAGAAGTAGTGA-3') corresponding to the DNA sequences of the first internal exon derived from the pTAG vector (Fig. 1A) and a reverse primer UAP (the underlined sequences of the primer AP). PCR products were separated on an agarose gel, transferred to a nylon membrane and hybridized with a probe corresponding to the sequences of the second internal exon (Fig. 1A). For detection of the endogenous FE65a2 mRNA, total RNA was isolated from cell cultures of 13 human fibroblast strains from GRC longitudinal study/NIA aging cell repository at Coriell Institute. RNA from postmortem human brains of six individuals (specimens A–F; 80–92 years old; A, C and E are male, the others are female) was obtained from a previous study (30). Genotyping of these samples for the intron 13 polymorphism was performed as described previously (4). Total RNA (5 µg) was reverse transcribed with primer AP for fibroblasts RNA or random hexamers for brain RNA. sscDNA was amplified by PCR with a forward primer, 230–19F (5'-TGGACCCCAAGTCATGTCAG-3') in exon 12 and reverse primers, 230–37R (5'-CAGCAGAGGTGCCCATGGAG-3' for the sscDNA from fibroblasts) or 230–167R (5'-CTGGCCTGGACCAGTTCCTC-3' for the sscDNA from brain tissues and COS-7 cells) in exon 14. PCR products were separated on agarose gels, transferred and hybridized with isoform-specific probes as indicated in Figure 2A. DNA fragments (800 bp) used as standards for determination of the percentages of the FE65a2 mRNA isoform were amplified by PCR from pcDNA3.1FE65 and pcDNAFE65a2 constructs, and purified by gel electroelution. The copy numbers of the standards were determined as described previously (26). The standards for the FE65 and FE65a2 cDNAs at 5 x 103 copies/µl were mixed at ratios of 0, 5, 10, 20, 30, 40 and 100% [FE65a2/(FE65a2 + FE65) x 100%]. Each mixture at 2 µl was used for PCR reactions. Intensities of DNA bands were quantified as described previously (26).
DNA sequencing
Genomic DNA was isolated from a C57BL/6J mouse and COS-7 cells using TRIzolTM (Invitrogen Corporation, Carlsbad, CA) and a protocol for sequential isolation of RNA and DNA as provided by the manufacturer. Chimpanzee DNA from eight individual animals was provided by Dr Samir S.Deeb (University of Washington). DNA sequencing was performed as described previously (4).
Antibodies and western analysis
Two rabbit polyclonal antibodies against FE65 and FE65a2 (FE518), and FE65a2 (hFEa2-663) were made by Research Genetics Inc. (Huntsville, AL). Oligopeptide sequences (amino acids 518–527, LDHSKLVDVP for FE518; amino acids 663–678, QARPHPPGPSRGRRRK for hFEa2-663) were chosen and sent to the company for custom peptide synthesis and antibody production. FE518 recognizes both FE65 and FE65a2; its epitope has no homology to the other known family members, FE65L1 (11) and FE65L2 (12) while hFEa2-633 is unique to human FE65a2. For western analysis, cell lysates or beads from GST pull-down assays were mixed with 2x Laemmli buffer and boiled for 5 min before being loaded on 7.5% SDS polyacrylamide gels (SDS–PAGE). After electrophoresis, samples were transferred to Immobilon-P transfer membranes (Millipore, Bedford, MA) and probed with 1:3000 diluted preimmune sera, FE518 or hFEa2-663 antisera. Rabbit IgG was then detected by a peroxidase-conjugated monoclonal antibody against rabbit IgG (Vector, Burlingame, CA) and enhanced chemiluminescence reagents (Amersham Pharmacia Biotech).
GST pull-down assays
GST and GST-C48 fusion proteins were produced in Escherichia coli BL21 cells and purified on glutathione sepharose 4B beads (Amersham Phamacia Biotech). Concentrations of the purified proteins were determined by SDS–PAGE with bovine serum albumin as standards. COS-7 cells were transfected with pcDNA3.1FE65, pcDNA3.1FE65a2 or a control vector, pcDNA3.1LacZ. Cell lysates (60–120 µl), prepared as described previously (42), were incubated for 3 h with equal amounts of the GST-C48 or GST beads at 4°C. The beads were then washed three times in the same lysis buffer before being subjected to western analysis.
Accession numbers
Nucleotide sequences 3' to human FE65 gene, AF394215; human FE65 cDNA, L77864; human FE65a2 cDNA, AF394214; human FE65 gene, AF029234.
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ACKNOWLEDGEMENTS |
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We thank Drs David B.Krizman, Bryce L.Sopher, Samir S.Deeb and Lee-Way Jin for providing the pTAG4 vector, pGEX-GST-C48 construct, chimpanzee genomic DNA and human brain tissues. We thank Dr Mark Bothwell for his review of the manuscript. This work was supported in part by National Institute on Aging Research Awards: AG19711 (G.M.M.), AG10917 (G.M.M.), AG05136 (G.M.M.) and AG15645 (M.G.H.), and Alzheimer Association Grants: PRG-98-023 (Q.H.) and TLL-99-1885 (G.M.M.).
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 206 616 4226; Fax: +1 206 685 8356; Email: qhu@u.washington.edu
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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