Tau gene (MAPT) sequence variation among primates

Gene 341 (2004) 313 – 322 www.elsevier.com/locate/gene Tau gene (MAPT) sequence variation among primates Max Holzera,b,*, Molly Craxtona, Ross Jakesa, Thomas Arendtb, Michel Goederta a MRC Laboratory of Molecular Biology, Cambridge, UK Department of Neuroanatomy, Paul Flechsig Institute of Brain Research, University of Leipzig, Leipzig, Germany b Received 28 March 2004; received in revised form 16 June 2004; accepted 19 July 2004 Available online 18 September 2004 Received by F. Salvatore Abstract Filamentous tau deposits are a defining feature of a number of human neurodegenerative diseases. Apes and monkeys have been reported to be differentially susceptible to developing tau pathology. Despite this, only little is known about the organisation and sequence of Tau from nonhuman primates. Here we have sequenced Tau exons 1–13, including flanking intronic regions, and the region in intron 9 that contains Saitohin in chimpanzees, gorillas, and gibbons. Partial sequences were obtained for cynomolgus macaque and green monkey. Chimpanzee brain tau was 100% identical to human tau. Identities were 99.5% for gorilla tau and 99.0% for gibbon tau. Chimpanzee DNA was polymorphic for a repeat in intron 9, which was present in human and gorilla tau, and for the nucleotide at position +29 of the intron that follows exon 10. As was the case of the other nonhuman primates examined, chimpanzee DNA was homozygous for nucleotides used to define the H2 haplotype in human Tau. These differences between human and chimpanzee Tau may contribute to the apparent resistance of chimpanzee brain to developing tau pathology. Sequencing of Saitohin revealed an intact open reading frame in chimpanzee and gorilla, but not in gibbon or macaque. D 2004 Elsevier B.V. All rights reserved. Keywords: Chimpanzee; Gibbon; Gorilla; Saitohin; Haplotype; Splicing 1. Introduction The tauopathies constitute a group of neurodegenerative diseases characterized by the presence of amyloid-like, filamentous deposits made of hyperphosphorylated Tau protein in nerve cells or in both nerve cells and glial cells Abbreviations: Ah, beta amyloid peptide; bp, base pair(s); chi, chimpanzee; cDNA, DNA complementary to RNA; CNS, central nervous system; DTT, dithiothreitol; E, exon; EDTA, ethylenediaminetetraacetic acid; gib, gibbon; gor, gorilla; hum, human; kDa, kilodalton(s); mac, macaque; OD, optical density; PCR, polymerase chain reaction; PMSF, phenylmethylsulfonyl fluoride; RT-PCR, reverse transcription polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate polyacrylamide electrophoresis; Tris, tris(hydroxymethyl)aminomethane; UTR, untranslated region. * Corresponding author. Department of Neuroanatomy, Paul Flechsig Institute of Brain Research, University of Leipzig, Jahnallee 59, D-04109 Leipzig, Germany. Tel.: +49 341 9725759; fax: +49 341 9725729. E-mail address: [email protected] (M. Holzer). 0378-1119/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2004.07.013 (Lee et al., 2001; Berriman et al., 2003). They comprise Alzheimer’s disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick’s disease (PiD), argyrophilic grain disease (AGD), and frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). In these diseases, the presence of filamentous tau deposits correlates with the loss of nerve cells. Tau is a neuronally enriched, microtubule-associated protein, which is involved in the nucleation, elongation, and stabilization of microtubules (Lee et al., 2001). In the adult human brain, there are six isoforms of tau, produced from a single gene by alternative mRNA splicing (Goedert et al., 1989; Andreadis et al., 1992). They differ from one another by the presence or absence of a 29- or 58-amino acid insert (encoded by exon 2 and 3, respectively) in the aminoterminal half of the protein and by the inclusion, or not, of a 31-amino acid repeat, encoded by exon 10 of Tau, in the carboxy-terminal half of the protein. The exclusion of exon 10 leads to the production of three isoforms, each containing 314 M. Holzer et al. / Gene 341 (2004) 313–322 three repeats, and its inclusion leads to a further three isoforms, each containing four repeats. The repeats constitute the microtubule-binding region of tau protein. In normal adult human cerebral cortex, there are similar levels of threerepeat and four-repeat tau isoforms (Goedert and Jakes, 1990). In the developing human brain, only the shortest isoform (three repeats and no amino-terminal inserts) is expressed. The longest human tau isoform comprises exons 1–5, 7, 9, and 10–13. In the peripheral nervous system, additional tau isoforms are expressed, which contain a long N-terminal insert encoded by exon 4A (Goedert et al., 1992; Andreadis et al., 1992). Mutations in Tau cause the autosomal dominantly inherited FTDP-17 (Poorkaj et al., 1998; Hutton et al., 1998; Spillantini et al., 1998). Functionally, many mutations reduce the ability of tau to promote microtubule assembly (Hasegawa et al., 1998). Moreover, an imbalance in the ratio of three-repeat to four-repeat tau that results in the relative overproduction of tau with four repeats causes FTDP-17 and leads to the formation of filaments consisting of four-repeat tau (Spillantini et al., 1997, 1998; Hutton et al., 1998). This indicates that in the human brain, a tight regulation of tau isoform ratios is essential for preventing neurodegeneration and dementia. The sporadic tauopathies PSP and CBD are also characterized by the presence of four-repeat taucontaining filaments (Lee et al., 2001). An association between PSP and CBD, and a dinucleotide repeat polymorphism in the intron between exons 9 and 10 of Tau has been reported (Conrad et al., 1997; Di Maria et al., 2000). Moreover, two common Tau haplotypes that differ only at the nucleotide level have been described (Baker et al., 1999). Homozygosity of the more common H1 allele predisposes to PSP and CBD. The intron between exons 9 and 10 of Tau also comprises the putative intronless gene Saitohin (Conrad et al., 2002). It encodes a predicted protein of 128 amino acids of unknown function, which is polymorphic at codon 7 (Q or R). Little is known about the organisation and sequence of the Tau gene in the great apes and other nonhuman primates. It has been reported that the chimpanzee is relatively resistant to developing the neuropathological characteristics of AD (Gearing et al., 1994), whereas baboons, rhesus monkeys, and lemurs have been found to develop tau inclusions, as well as beta amyloid peptide (Ah) deposits, as a function of age (Wisniewski et al., 1973; Giannakopoulos et al., 1997; Härtig et al., 2000; Schultz et al., 2000). Filamentous tau inclusions have been documented in the baboon (Schultz et al., 2000). It remains to be established whether this differential susceptibility to developing tau inclusions is reflected in sequence differences in Tau between humans and nonhuman primates. Here we have addressed this question by sequencing exons 1–13 of Tau and their flanking intronic regions, as well as a region of intron 9 comprising Saitohin, in chimpanzees, gorillas, and gibbons. Partial sequence information was obtained from cynomolgus macaques and green monkeys. 2. Materials and methods 2.1. Materials Genomic DNA, RNA, and proteins were extracted from frozen tissue of the primate species and the cell line listed in Table 1. The tissues were kindly provided by D.C. Gajdusek (National Institutes of Health, Bethesda, MD, USA; for chimp-1, gorilla, and gibbon-1) and P. Khaitovich (Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany). The COS-7 cell line was obtained from the bDeutsche Sammlung von Mikroorganismen und ZellkulturenQ (Braunschweig, Germany). Importation of primate tissue to the UK followed CITES regulations. 2.2. Tissue culture COS-7 cells were maintained in Dulbecco’s modified Eagle’s medium+10% foetal calf serum, supplemented with penicillin and streptomycin. The medium was changed every 2 days and the cell culture stopped after 14 days by the addition of 6 ml of Trizol per dish. 2.3. Polymerase chain reaction (PCR) and DNA sequencing Genomic DNA was extracted using the DNeasy tissue kit (Qiagen) and included a mock-extracted sample, where water was substituted for the tissue sample. PCR primers were designed to amplify tau exons 1, 2, 3, 4, 4a, 5, 6, 7, 8, 9, 10, 11, 12, and 13, including 100–300 bp of flanking intronic regions. A DNA stretch in exon 9 comprising Saitohin was also amplified. A list of PCR and sequencing primers is given as Supplementary Table 1. PCR was performed in a final volume of 50 Al using TaqPCR mastermix (Qiagen), supplemented with 0.7 U of Pfu-Turbo polymerase (Stratagene), 30 ng of genomic DNA, and 50 pmol of each primer. Most reactions used a touchdown temperature profile of 5 cycles (94 8C, 5 s; 72 8C, 2 min), 5 cycles (94 8C, 5 s; 70 8C, 2 min), and 25 cycles (94 8C, 5 s; 68 8C, 2 min), with a final 10-min Table 1 List of primate tissues and cell line Individual Designation Species Tissue Chimpanzee-1 Chimpanzee-2 C8 Herman Pan troglodytes Pan troglodytes Chimpanzee-3 Gorilla Gibbon-1 Gibbon-2 13306 15/08/1973 939 Falco Pan troglodytes Gorilla gorilla Hylobates lar Hylobates concolor Macaca fascicularis Chlorocebus aethiops Parietal cortex Frontal cortex and cerebellar cortex Muscle Temporal cortex Temporal cortex Temporal cortex Cynomolgus macaque Green monkey COS-7 Sclera SV40-transformed kidney cell line 315 M. Holzer et al. / Gene 341 (2004) 313–322 extension at 72 8C. Less stringent conditions were used for some reactions using gibbon and macaque DNA samples. They consisted of 5 cycles (94 8C, 5 s; 70 8C, 2 min), 5 cycles (94 8C, 5 s; 68 8C, 2 min), and 25 cycles (94 8C, 5 s; 66 8C, 15 s; 72 8C, 2 min), with a final 10-min extension at 72 8C. The PCR products were purified from 1% agarose gels using QIAquick spin columns (Qiagen) and eluted with 50 Al of water. For DNA sequencing, 10 Al of eluate was mixed with 6 Al of big dye terminator mastermix (v 3.0; PE Biosystems), 3.5 pmol of sequencing primer, 0.3 Al of 25 mM MgCl2, and 3 Al of water. Cycle sequencing used a total of 27 cycles (96 8C, 30 s; 45 8C, 15 s; 60 8C, 4 min). The reactions were run on an ABI 377 DNA sequencer. Nucleotide sequences were deposited in the database with accession numbers AY574122–AY574186. Sequence alignments made use of the multiple alignment general interface (MAGI, http://www.hgmp.mrc.ac.uk) and the clustal algorithm. The human Tau gene sequence (accession number AC091628) was used for sequence comparison. 2.4. Reverse transcription polymerase chain reaction (RT-PCR) RNA was extracted from the temporal cortex of chimpanzee, gorilla, gibbon, and human using the Trizol reagent (Invitrogen) and reverse-transcribed with Superscript II enzyme (Invitrogen). Alternative splicing of exons 2, 3, 6, 8, and 10 of Tau was analyzed using primers T5Vf/ TE5r, TE5f/TE7r, T5f/T9r, and THF1/THR1, respectively (see Supplementary Table 1). Full-length tau transcripts were amplified using primers T5Vf/T3Vr, cloned into vector pZero2.0 (Invitrogen), and sequenced. The PCR conditions were identical to those used for genomic DNA, with the exception of the reactions using primers THF1/THR1 and Esaif/Esair, for which we used a total of 35 cycles (94 8C, 20 s; 55 8C, 20 s; 72 8C, 45 s), with a final 5-min extension at 72 8C. For DNase I treatment, 15 Ag of RNA was incubated with RNase-free DNase I (190 U/ml; Qiagen) for 20 min at room temperature. RNA was purified using RNAeasy minElute cleanup columns (Qiagen) and brought to 0.5 mg/ml. Table 2 Rate of base substitutions in different primates compared to the human MAPT sequence Species Nocod/ 1000 bp Syncod/ 1000 bp Nosyncod/ 1000 bp chi-1 Gor gib-1 Mac Cos 10.6 13.2 34.0 56.7 56.7 4.0 3.7 9.2 21.1 18.5 2.2 3.7 11.4 14.1 7.1 Nocod = substitution in noncoding regions; syncod = synonymous base substitutions in coding regions; nosyncod = nonsynonymous base substitutions in coding regions. Table 3 Comparison of base substitutions in coding and noncoding sequences in constitutively and nonconstitutively spliced exons Base substitutions Constitutive exon substitutions/ 1000 bp Nonconstitutive exon substitutions/ 1000 bp Coding sequence Intronic flanking sequence 18.8 58.3 40.2 53.5 2.5. Exon trapping Exon 10, together with 5 V and 3V flanking intronic sequences, was amplified from human, chimpanzee, gorilla, and macaque DNA using primers etr10f/etr10r and cloned into the vector pSPL3 (Gibco-BRL) via the SacI/BamHI sites. The 3V intronic sequence included 113 bp for all constructs, and the 5V intronic sequence was 343 bp for human and gorilla, and 283 bp (lacking a 60-bp repeat) for chimpanzee and macaque. All constructs were verified by DNA sequencing. COS-7 cells in six-well plates were transfected with 1.2 Ag of each construct. RNA was prepared 24 h after transfection and SA2L-primed cDNA produced by reverse transcription using Superscript II following the manufacturer’s recommendations. Amplification was carried out for 25 cycles (94 8C, 30 s; 60 8C, 30 s; 72 8C, 60 s). Secondary PCR was performed with nested primers SD2/ SA4 for 25 cycles (94 8C, 30 s; 60 8C, 30 s; 72 8C, 60 s), with a final 10-min extension at 72 8C. The relative proportions of exon 10-containing/exon 10-lacking transcripts were obtained by densitometric image analysis of ethidium bromide-stained 2% agarose gels. 2.6. Tau protein extraction and analysis Proteins were precipitated from the phenol phase of the Trizol reagent with isopropyl alcohol, and the pellets were dissolved in 6 M guanidine hydrochloride and dialyzed against 20 mM Tris/HCl, pH 8.0, 0.1 mM EDTA, 0.1 mM DTT, and 1 mM PMSF. Dephosphorylation was done with Escherichia coli alkaline phosphatase for 3 h at 67 8C, following adjustment of the MgCl2 concentration to 0.5 mM. For immunoblotting, samples were run using 10% sodium dodecyl sulfate polyacrylamide electrophoresis Table 4 List of human-specific nucleotides Location on MAPT nt change Location on MAPT nt change E1 E4 E6 E6 E7 E7 E8 C to T A to G G to A C to T T to C C to T G to C E10 185 E10 205 E11 170 E12 +16 Saitohin 29 Cys10Arg Saitohin +39 G to A G to A G to A C to T T to C C to T 185 76 +67 +105 +11 +94 201 Nucleotide (nt) change describes the conversion of the conserved bases in the nonhuman primates to the human-specific nucleotides. 316 M. Holzer et al. / Gene 341 (2004) 313–322 Fig. 1. Amino acid alignment of human (hum), chimpanzee (chi), gorilla (gor) and gibbon-1 (gib) exons found in central nervous system tau isoforms (A) and of exon 4a present in bigtau from the peripheral nervous system (B). The first amino acid of each exon is marked in bold. M. Holzer et al. / Gene 341 (2004) 313–322 (SDS-PAGE) and transferred to polyvinylidene difluoride membranes. After soaking in blocking solution, the membranes were incubated with the Tau-1 antibody (1:5000; Chemicon), which recognizes an unphosphorylated epitope spanning amino acids 198–205 in tau. Detection was carried out with goat anti mouse/rabbit horseradish peroxidase conjugate, prior to visualization with Supersignal ECL substrate (Pierce, Rockford). 3. Results 3.1. MAPT sequence variation at the nucleotide and protein levels We sequenced 14 Tau exons (1, 2, 3, 4, 4a, 5, 6, 7, 8, 9, 10, 11, 12, and 13) with flanking intronic regions and the 317 putative Saitohin gene in two chimpanzees, one gorilla, and one gibbon. We obtained partial genomic sequence from chimpanzee-3, a second gibbon, a cynomolgus macaque, and a green monkey cell line. The sequences of tau transcripts from the chimpanzee obtained by RT-PCR were identical to the assembled exonic sequences. Table 2 summarizes the nucleotide variation in the coding and noncoding regions of Tau. The chimpanzee sequence was most similar to the human sequence. The gorilla sequence showed an increased rate of nonsynonymous base substitutions in the coding region, whereas the gibbon sequence showed a threefold greater variation in coding and noncoding regions than the chimpanzee sequence. The estimation of sequence variation in macaque and green monkey may have been somewhat skewed, since it only relied on partial sequence information (exons 2, 3, and 8–13 for the macaque; exons 1, 2, 5, and 11–13 for the green monkey). Fig. 2. Sequence alignment of exon 10 with intronic flanking regions. Human (hum), chimpanzee (chi), gorilla (gor), gibbon-1 (gib) and macaque (mac) sequences are shown. r : g/a. The tandem repeat is underlined (first repeat single underlined; second repeat double underlined); grey shading highlights base substitutions. The +29 polymorphism in chimpanzee-1 is shown in bold. For chimpanzee-3, the two alleles are shown separately. 318 M. Holzer et al. / Gene 341 (2004) 313–322 3.2. Analysis of the alternative splicing of exon 10 by exon trapping Previous work has identified a 59- or 60-bp tandem repeat in intron 9 of human Tau, at positions 139 to 21 relative to exon 10 (Poorkaj et al., 2001a). Both copies of the repeat were present in gorilla DNA, whereas two chimpanzees had only one copy of the repeat and chimpanzee-3 was polymorphic for the tandem repeat. Gibbon and macaque DNA carried only one repeat each (Fig. 2). By exon trapping, transfection with chimpanzee and macaque constructs with one repeat gave a smaller proportion of exon 10-containing transcripts than transfection with human and gorilla constructs having two repeats (Fig. 3). Fig. 3. Presence of the tandem repeat affects exon 10 splicing. Boxplot showing the abundance of exon 10-containing exon-trapping PCR products (270 bp) in relation to exon 10-lacking PCR products (177 bp) evaluated by densitometric analysis of ethidium bromide-stained agarose gel. Chimpanzee (chi) and macaque (mac) sequences without the tandem repeat yield less exon 10-containing PCR products than sequences from species with the tandem repeat, such as humans (hum) and gorilla (gor). The constitutively spliced Tau exons 1, 4, 5, 7, 9, 11, 12, and 13 were more highly conserved than exons 2, 3, 4a, 6, 8, and 10, which are not constitutively spliced (Table 3). The flanking intronic regions of all exons had comparable nucleotide substitution rates. We examined the sequences for the presence of nucleotide changes unique to humans (Table 4). Of the seven changes, all but one resided in intronic regions. The exception was a T-to-C transition in the first nucleotide of codon 10 of Saitohin, resulting in a C10R change in nonhuman primates. No human-specific insertions/deletions were observed. The predicted amino acid sequence of the longest chimpanzee brain tau isoform (441 amino acids) was identical to that of the corresponding isoform from human brain (Fig. 1A). The gorilla sequence differed in two amino acids (H32L and K87E) and the gibbon sequence in four amino acids (E9D, H32L, T52A, and G86S) from human tau. Exon 4A was less conserved. Of 251 amino acids, the chimpanzee sequence differed at three positions and the gorilla sequence at four positions from the human sequence, whereas gibbon and human sequences showed 15 differences (Fig. 1B). Fig. 4. The +29 A allele in the chimpanzee sequence exhibits reduced exon 10 splicing. Ethidium bromide-stained agarose gel of exon-trapping PCR products. Fig. 5. RT-PCR analysis of tau isoform and saitohin expression. RNA was extracted from the temporal cortex of gorilla (lane 1), gibbon (lane 2), chimpanzee-1 (lane 3), and human (lane 6); from the cerebellum of chimpanzee-2 (lane 4); and from the frontal cortex of chimpanzee 2 (lane 5). The first panel shows the amplification of a 5Vportion of tau transcripts with primers binding in the 5V untranslated region (UTR) and exon 5 showing alternative splicing of exons 2 and 3. The second panel shows that exon 6-containing tau transcripts in the CNS are rare in all species examined. The third panel shows differences in alternative splicing of exon 10. The fourth panel depicts Saitohin cDNA (Sai). M. Holzer et al. / Gene 341 (2004) 313–322 The intronic sequence close to the splice donor site of exon 10 was conserved between humans and nonhuman primates. The only sequence difference was found in one of the chimpanzees, which was heterozygous (g or a) for the nucleotide at position +29. The other species were homozygous for +29 g. By exon trapping, the ratio of exon 10-containing/exon 10-lacking transcripts from the chimpanzee was 0.29 for the +29 g allele and 0.13 for the +29 a allele (Fig. 4). 319 3.3. Analysis of the alternative splicing of exons 2, 3, 6, 8, and 10 by RT-PCR The splicing pattern of exons 2, 3, 6, 8, and 10 in the brains of nonhuman primates was similar to that in human brain (Fig. 5). The nonhuman primates yielded the transcripts +E2+E3, +E2 E3, E2 E3, +E10, and E10, making it likely that they express six tau isoforms in brain. There appeared to be differences in the proportion of exon 10- Fig. 6. Intron 9 nucleotide sequence alignment of the region containing Saitohin. The protein sequence of human saitohin is depicted above the nucleotide sequence alignment. Human (hum), chimpanzee (chi), gorilla (gor), gibbon-1 (gib) and macaque (mac) sequences are shown. Gray shading highlights base substitutions. 320 M. Holzer et al. / Gene 341 (2004) 313–322 containing over exon 10-lacking transcripts, in that chimpanzees and gorillas expressed a higher proportion of transcripts lacking exon 10 than humans (Fig. 5). Transcripts encoding exon 6 were rare or undetectable. Transcripts encoding exon 8 were undetectable (not shown). 3.4. Saitohin is present in chimpanzee, gorilla, and orangutan, but not in gibbon or macaque Saitohin has been described as an intronless gene that encodes a predicted protein of 128 amino acids and is nested in intron 9 of human Tau (Conrad et al., 2002). Codon 7 was found to be polymorphic, coding either for the more common Q or the less common R. We sequenced this putative gene in nonhuman primates and found an open reading frame encoding a predicted 128-amino acid protein in both chimpanzee and gorilla (Fig. 6). Codon 7 was homozygous for R. A C10R change relative to the human sequence was present, with the chimpanzee carrying an additional change at position 83 (S83N). In the gibbon (Hylobates lar), the open reading frame of saitohin was coding for a predicted protein of only amino acids, because of a 2-bp insertion that generated a stop codon. Cynomolgus macaque DNA lacked the putative start codon of saitohin, but was otherwise similar in sequence to up to 9 bp downstream of the stop codon (Fig. 6). In human brain, a saitohin transcript could be readily amplified by RT-PCR. Amplification was still possible following extensive DNase I treatment of human brain RNA, even though it was somewhat reduced compared to untreated RNA (not shown). Analysis of saitohin expression by RT-PCR showed transcripts in each primate species examined. The gibbon, with a dysfunctional open reading frame, showed comparable expression to the gorilla, with an intact open reading frame (Fig. 5). 3.5. Tau haplotype analysis in nonhuman primates Eight single nucleotide polymorphisms, which define the H1 and H2 human Tau haplotypes, were analyzed (Table 5) (Supplementary Table 2). Of these polymor- Fig. 7. Western blot of dephosphorylated brain proteins from chimpanzee (lane 1) and gibbon (lane 2), together with a mix of six recombinant human tau isoforms (lane 3). The membrane was probed with anti-tau antibody Tau-1. phisms, seven had the sequence of the H2 haplotype. No sequence variation was observed between the chimpanzee, gorilla, gibbon, macaque, and green monkey. Supplementary Table 2 lists the sequences in nonhuman primates of additional single nucleotide polymorphisms previously described in humans. In the nonhuman primates studied, these sequences were conserved and nonpolymorphic. 3.6. Tau protein isoforms in chimpanzee and gibbon brain By immunoblotting of dephosphorylated brain extracts with anti-tau antibody Tau-1, six bands were detected in the chimpanzee and gibbon brains; they aligned with the six recombinant human brain tau isoforms (Fig. 7). Four strong tau bands and two weaker bands were present in the chimpanzee brain, and six bands of similar intensity were found in the gibbon brain. Table 5 Summary of primate Tau gene typing for H1 and H2 haplotypes Position Human H1/H2 Amino acid Chimpanzeea Gorilla Gibbona Macaque COS-7 Nonhuman primates Reference Exon 1 5 Intron 2 +18 Intron 3 +9 Exon 9 125 Exon 9 209 Saitohin 20 Intron 11 +34 Intron 13 +34 A/G C/T A/G A/G T/C A/G G/A T/C na na na Ala/Ala Asn/Asn Gln/Arg na na G C G G C G A C G C G G C G A C G C G G C G A C nd C G G C G A C G C nd nd nd nd A C H2 H1 H2 H2 H2 H2 H2 H2 Baker et Baker et Baker et Baker et Baker et Verpillat Baker et Baker et na=Not applicable; nd=not determined. a Two individuals were analysed per species/genus and were found to have nucleotide assignment. al. al. al. al. al. et al. al. al. M. Holzer et al. / Gene 341 (2004) 313–322 4. Discussion The predicted amino acid sequence of the longest brain isoform of chimpanzee tau was 100% identical to that of the corresponding isoform from human brain. It was identical to recently published partial sequences of chimpanzee tau (Caceres et al., 2003; Clark et al., 2003). Gorilla tau was 99.5% and gibbon tau was 99.0% identical to human tau. Six isoforms that aligned with recombinant human tau were present in chimpanzee and gibbon brain by immunoblotting. Sequence differences with human tau were located in exons 1–3, where they are unlikely to affect the ability of tau to interact with microtubules. It remains to be determined whether they can influence the ability of tau to assemble into filaments. Exon 4A from chimpanzee and gorilla was 98.8% identical to the corresponding human sequence, with 94% identity for exon 4A from gibbon. The lower degree of conservation of exon 4A is compatible with the proposed spacer function of this exon. Previous work has shown that baboon brain tau is 99% (four-repeat isoform lacking exons 2 and 3) and rhesus monkey brain tau is 98% (longest isoform) identical to human tau (Nelson et al., 1996). This contrasts with 88% identity between mouse and human tau (Poorkaj et al., 2001a), and 74% identity between chicken and human tau (Yoshida and Goedert, 2002). From the above, it is clear that the apparent resistance of the chimpanzee to developing a filamentous tau pathology cannot be due to differences in amino acid sequence with human tau. It could result instead from differences in the nucleotide sequences that are located outside the coding region. Upstream of exon 10, human tau possesses two repeats of 59 or 60 bp (Poorkaj et al., 2001a). Gorilla DNA also carries two repeats, chimpanzee DNA can have either one or two repeats, whereas gibbon and macaque DNA have only one repeat each. By exon trapping, constructs with one repeat gave rise to a smaller proportion of exon 10containing transcripts than constructs with both repeats. In humans, intronic sequences located close to the splice donor site of exon 10 regulate the splicing of exon 10 (Lee et al., 2001). Mutations in this region cause FTDP-17, most probably through the destabilization of a stem loop structure (Hutton et al., 1998; Spillantini et al., 1998; Varani et al., 1999). This results in the relative overproduction of fourrepeat tau (Spillantini et al., 1998). The sequences of exon 10 and the stem loop were completely conserved between humans and the nonhuman primates studied. One chimpanzee was heterozygous (g or a) at position +29 of the intron following exon 10. The other species were homozygous for +29 g. By exon trapping, +29 a constructs showed a relative increase in exon 10-lacking transcripts compared to +29 g constructs, consistent with findings using human DNA (Stanford et al., 2003). The latter study also reported a heterozygous +29 g-to-a change in a family with frontotemporal dementia, but without tau pathology. The authors suggested that a relative increase in three-repeat tau may cause disease through an ill-defined mechanism that does not 321 involve the formation of tau filaments. However, Stanford et al. also found the +29 g-to-a change in 1 of 200 control individuals, in line with earlier works (D’Souza et al., 1999; Roks et al., 1999; Poorkaj et al., 2001b). Together with the present findings in the chimpanzee, it therefore appears likely that the +29 change is a polymorphism in both humans and chimpanzees. Analysis of a larger number of samples will be required to establish the frequency of this polymorphism in the chimpanzee population. It also remains to be seen whether chimpanzee brain expresses a higher relative proportion of three-repeat tau than human brain, as suggested by our RT-PCR experiments. This could be relevant with respect to the resistance of the chimpanzee to developing a filamentous tau pathology. Haplotypes H1 and H2 characterize human Tau (Baker et al., 1999). We analysed eight single nucleotide polymorphisms that distinguish H1 from H2 in the nonhuman primates. At seven positions, nonhuman primates were homozygous for the H2 haplotype sequence. This suggests that H1, the more prevalent of the two haplotypes, may be human-specific and may have evolved as the result of selective pressure. One of these single nucleotide polymorphisms is located in the coding region of saitohin, where codon 7 encodes either Q (H1 haplotype) or R (H2 haplotype) (Conrad et al., 2002; Verpillat et al., 2002). In saitohin from chimpanzee and gorilla, this residue was R, as expected from the haplotype findings. Compared with the human sequence, chimpanzee and gorilla saitohin carried a C10R substitution, with an additional amino acid difference (S83N) in chimpanzee. The functional relevance of saitohin is unknown. It has been reported to be coexpressed with tau in human tissues, leading to the suggestion that both proteins may function in a common pathway (Conrad et al., 2002). The present findings indicate that this is unlikely to be of general relevance, since gibbon and cynomolgus macaque did not encode intact saitohin. This contrasts with tau protein, which is conserved from chickens to humans (Goedert et al., 1989; Yoshida and Goedert, 2002). Transcription of the genomic sequence encompassing Saitohin in the gibbon may be a sign of noise, or may indicate a regulatory role of the noncoding RNA. In conclusion, our results show that humans and great apes have very similar tau protein sequences, and that their differential susceptibility to developing filamentous tau inclusions may be influenced by intronic Tau sequences. Acknowledgements We wish to thank Drs. D.C. Gajdusek, P. Khaitovich, and S. P77bo for providing primate tissues. This work was supported by the UK Medical Research Council, the European Commission (QLK6-1999-51519), and the Deutsche Forschungsgemeinschaft (HO23681-1). We gratefully acknowledge the Interdisziplin7res Zentrum fqr Klinische Forschung (IZKF) at the Faculty of Medicine of the 322 M. Holzer et al. / Gene 341 (2004) 313–322 University of Leipzig (C24) and the IZKF Leipzig core unit Z03 for DNA sequencing. Appendix A. Supplementary information Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.gene.2004. 07.013. References Andreadis, A., Brown, W.M., Kosik, K.S., 1992. Structure and novel exons of the human tau gene. Biochemistry 31, 10626 – 10633. Baker, M., Litvan, I., Houlden, H., Adamson, J., Dickson, D., Perez-Tur, J., Hardy, J., Lynch, T., Bigio, E., Hutton, M., 1999. Association of an extended haplotype in the tau gene with progressive supranuclear palsy. Hum. Mol. Genet. 8, 711 – 715. 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