APLP2, a member of the Alzheimer precursor protein family, is required for correct genomic segregation in dividing mouse cells
1998; Springer Nature; Volume: 17; Issue: 16 Linguagem: Inglês
10.1093/emboj/17.16.4647
ISSN1460-2075
Autores Tópico(s)Alzheimer's disease research and treatments
ResumoArticle17 August 1998free access APLP2, a member of the Alzheimer precursor protein family, is required for correct genomic segregation in dividing mouse cells Minoo Rassoulzadegan Minoo Rassoulzadegan Unité 470 de l'Institut National de la Santé et de la Recherche Médicale, Université de Nice, 06108 Nice, France Search for more papers by this author Yinhua Yang Yinhua Yang Unité 470 de l'Institut National de la Santé et de la Recherche Médicale, Université de Nice, 06108 Nice, France Search for more papers by this author François Cuzin Corresponding Author François Cuzin Unité 470 de l'Institut National de la Santé et de la Recherche Médicale, Université de Nice, 06108 Nice, France Search for more papers by this author Minoo Rassoulzadegan Minoo Rassoulzadegan Unité 470 de l'Institut National de la Santé et de la Recherche Médicale, Université de Nice, 06108 Nice, France Search for more papers by this author Yinhua Yang Yinhua Yang Unité 470 de l'Institut National de la Santé et de la Recherche Médicale, Université de Nice, 06108 Nice, France Search for more papers by this author François Cuzin Corresponding Author François Cuzin Unité 470 de l'Institut National de la Santé et de la Recherche Médicale, Université de Nice, 06108 Nice, France Search for more papers by this author Author Information Minoo Rassoulzadegan1, Yinhua Yang1 and François Cuzin 1 1Unité 470 de l'Institut National de la Santé et de la Recherche Médicale, Université de Nice, 06108 Nice, France *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:4647-4656https://doi.org/10.1093/emboj/17.16.4647 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The mouse amyloid precursor-like protein 2 (APLP2) belongs to the Alzheimer peptide precursor family. A possible role in pre-implantation development had been suggested previously, and was investigated further by creating a large deletion in the genomic locus. While heterozygous mice developed normally, homozygous embryos were arrested before reaching the blastocyst stage. One-cell embryos which contained protein of maternal origin underwent a limited number of cleavages. The progressive disappearance of the protein at stages 4 and beyond correlated with the appearance of extensive cytopathological effects. Nuclear DNA contents of the arrested embryos departed widely from the normal 2–4C value, thus suggesting a role for the protein in replication and/or segregation of the embryonic genome. Embryonic mortality was not due to the untimely initiation of programmed cell death, and it occurred before the stage at which apoptotic cells normally appear. The same abnormal distribution of DNA contents was seen in primary cultures of Aplp2 +/− embryonic fibroblasts following transfection of an expression vector for Aplp2 antisense RNA with green fluorescent protein (GFP) expressed from a co-transfected construct. Daughter cells derived from a GFP-positive cell showed abnormal DNA contents both >4C and <2C, thus indicating a role for the protein in the mitotic segregation of the genome and establishment of the proper nuclear structure. Introduction The amyloid precursor-like protein 2 (APLP2) belongs to the Alzheimer peptide precursor (APP) family (Vidal et al., 1992; von der Kammer et al., 1994a, 1994b; von Koch et al., 1995; Yang et al., 1996). After the discovery of APP, the precursor of the βA4 peptide accumulated in the brain of Alzheimer patients (reviewed by Selkoe, 1989), a group of related genes was identified, all evolutionarily well-conserved and, therefore, potentially important. The list includes the mouse Aplp1 (Wasco et al., 1992), the rat Aplp2 (Sandbrink et al., 1994a), the human Aplp2 (also termed Apph) (Sprecher et al., 1993; Wasco et al., 1993), apl-1 of Caenorhabditis elegans (Daigle and Li, 1993) and appl in Drosophila (Rosen et al., 1989), as well as the murine Aplp2 gene. Their products all share with APP three domains of similarity interspersed with completely divergent regions (Figure 1), and their respective functions have remained a matter of speculation. A functional role in the central nervous system was indicated for APP by the phenotype of homozygous negative mutant mice (Müller et al., 1994; Zheng et al., 1995). Development was only slightly impaired, but the mutants exhibited behavioural abnormalities. No such evidence has been obtained for the other genes of the family. The situation is made even more complex by the occurrence for each protein of isoforms generated by alternative splices. Four such forms have been described for APLP2, corresponding to the different combinations generated by two alternatively spliced exons (Sandbrink et al., 1994b). Some of these isoforms undergo post-translational modifications, in the case of APLP2 by chondroitin sulfate glycosaminoglycan addition (Thinakaran and Sisodia, 1994). A possible function in axogenesis was proposed on the basis of the preferential accumulation of one of the APLP2 isoforms in the olfactory tract of the mouse (Thinakaran et al., 1995). Figure 1.The three domains of similarity between the APP and APLP2 proteins. Diagram (not to scale) of the respective exon–intron structures of the genomic loci encoding the APP and APLP2 proteins (from Yang et al., 1996). The exons indicated by grey boxes encode protein domains with 68–70% identical residues. No significant similarity is found in the other exons, shown as open boxes. Closed boxes correspond to the non-translated 5′ and 3′ mRNA sequences. The open double-headed arrow shows the position of the deletion in the Aplp2− mutant. Download figure Download PowerPoint Our previous results led us to suggest a different function for an APP family protein. The same gene which was designated Aplp2 on the basis of the sequence similarities between APP and the encoded protein (von der Kammer et al., 1994b; von Koch et al., 1995) initially had been described under the name Cdebp as that of a DNA-binding protein which recognizes the sequence [A/G]TCAC[G/A]TG, identical to the CDEI element of the yeast centromere (Vidal et al., 1992; Hanes et al., 1993). Immunocytochemical analysis localized the protein at discrete spots in the interphase nucleus (Blangy et al., 1995), and a series of convergent observations then suggested a role in the replication and/or segregation of genomic DNA. Protein binding to a CDEI motif in the genome of bovine papillomavirus type 1 was found to be required for maximal efficiency of replication of the viral DNA in transfected cells and for its subsequent episomal maintenance in stable transformants (Pierrefite and Cuzin, 1995; Pierrefite et al., 1996). A strong inhibitory effect on early development had been observed upon microinjection into fertilized mouse eggs of double-stranded oligonucleotides containing the CDEI sequence, and after treatment of one-cell embryos with antisense oligonucleotides. In both instances, the development was arrested before the blastocyst stage, with the characteristic accumulation of abnormal nuclear structures and DNA contents (Blangy et al., 1991, 1995), suggestive of a possible role for the protein in DNA replication and/or segregation. To investigate further the biological function(s) of the APLP2 protein, we first isolated the entire genomic structure, mapped it to mouse chromosome 9 and elucidated its exon–intron organization (Yang et al., 1996). We then generated an intragenic 11.35 kb deletion, which abolishes the expression of all the known isoforms. No obvious developmental defect was noted in the heterozygotes, which consistently were produced with normal Mendelian ratios. In sharp contrast, homozygotes failed to reach the blastocyst stage. Results Generation of the Aplp2-deleted mice The replacement targeting vector pYY-V8 (Figure 2) was designed to create a null mutation in the Aplp2 (Cdebp) gene. It contains a 3 kb fragment covering exons 5 and 6, with the adjacent and intervening intron sequences linked to a neo cassette and to 2.9 kb of sequences from intron 14. The size and location of the expected deletion (11.35 kb corresponding to eight exons and seven introns) were chosen in such a way that none of the isoform mRNAs could possibly be generated from the mutated allele. WW6 embryonic stem (ES) cells were electroporated with linearized pYY-V8 DNA. Positive/negative selection was applied in medium containing both G418 and ganciclovir. A first screen by Southern blot hybridization after BamHI cleavage (see below) detected 18 homologous recombinants among 78 survivors analysed. They exhibited identical genomic structures, with the neo cassette inserted between exon 4 on the 5′ side and exon 15 on the 3′ side. Long-distance PCR amplification (primers p44 and p45; Figure 2B) yielded the expected 8.5 kb fragment hybridizing with the neo probe. Results of PCR analysis were confirmed by subsequent Southern blot analysis of tail DNA after germline transmission of the mutant allele (see below). Figure 2.Homologous recombination at the Aplp2 (Cdebp) locus of the mouse. (A) Map of the genomic locus (Yang et al., 1996), of the targeting vector pYY-V8 and predicted structure of the targeted locus. Restriction sites: Ba, BamHI; Bc, BclI; Bg, BglII; S, SspI. The targeting vector includes a total of 5.9 kb of sequences homologous to the genomic locus, with 3 kb covering exons 5 and 6 linked to 2.9 kb from the intron between exons 14 and 15. The expected recombinant allele has therefore deleted eight exons and seven introns (11.35 kb). The neo and tk genes allow positive and negative selection, respectively, of the homologous recombinants. p45–p50: oligonucleotide primers, those with the same 5′–3′ orientation as the mRNA are indicated by italics. The vector was linearized by SspI cleavage prior to electroporation. (B–E) PCR and Southern blot analysis of wild–type mice (+/+) and of putative heterozygous mutant mice (+/−), initially identified by positive hybridization of tail DNA with neo sequences (probe C) in the F1 progeny obtained from chimeric animals. (B) Long PCR analysis: hybridization with probe C (neo sequences) of the product amplified from primers p44 (exon 4) and p45 (exon 15) in the presence of increasing concentrations of dimethylsulfoxide: 1% (lane 3), 5% (lanes 1 and 4) and 10% (lane 2 and 5). Lanes 1 and 2, wild-type DNA; lanes 3–5, heterozygous mutant DNA. The product expected from the recombined locus is a 8.5 kb fragment hybridizing with probe C. (C) Southern blot hybridization with the neo probe after cleavage with BglII (lanes 1–3) and EcoRI (lanes 4–6) of a wild-type (lanes 1 and 4) and two putative recombinants (lanes 2, 5, 3 and 6). Both enzymes are expected to generate from the recombined locus neo-containing fragments of nearly identical sizes (7.3 kb). (D) Southern blot analysis of one of the wild-type and the two putative recombinants identified in (C) using the 5′ probe A, after cleavage with BclI (1), BglII (2) and BamHI (3). (E) Same as in (D), but hybridization is with the 3′ probe B; cleavage with BclI (1), BglII (2) and EcoRI (3). Download figure Download PowerPoint Two clones, designated 8-W54 and 8-W76, were used to generate chimeric mice by blastocyst microinjection. For each clone, mouse colonies were established from the agouti progeny of two different chimeras, by back-crossing heterozygous animals twice onto either BALB/c or B6/D2 genetic backgrounds. The properties of these four families of mice were identical and will not be described separately. Although a detailed analysis remains to be performed, no obvious developmental or behavioural defect was noted in heterozygous animals. Litters in crosses with wild-type partners were of normal sizes and frequencies. Half of the offspring were heterozygous for the targeted allele, with an equal representation of males and females. Transmission of the targeted allele was checked by Southern blot analysis of tail DNA using two probes, A and B, on both sides of the deletion, and one, C, corresponding to the neo sequences (Figure 2). Probe A detected the expected 2.7 kb recombined BamHI fragment, which also hybridized with the neo probe (not shown), BglII generated a fragment of 7.3 kb reacting with probes A and B, BclI, a 12 kb fragment hybridizing with probes A and B, and EcoRI, a fragment of 7.3 kb detected by probe B. Absence of homozygous mutants in the progeny of heterozygous mating To find evidence for a possible role for Aplp2 in development, crosses between heterozygotes were performed to generate homozygous animals. Litters were significantly smaller, yielding an average of seven pups as compared with nine in crosses with wild-type mice, with one-third of the mice homozygous for the wild-type allele, and two-thirds, heterozygous (Table I). The proportion of heterozygous and wild-type and the lack of the homozygous genotypes are consistent with the notion that homozygous embryos die before birth. If this is the case, death must occur at an early embryonic stage, since the same distorted allelic distribution was found among embryos dissected at about mid-gestation (see below). Table 1. Litter size and hereditary transmission Parent genotypes No. of litters Total progeny Litter size Genotypesa +/+ +/− −/− +/−×+/+ 59 516 8.7 ± 0.12b 279 237 0 +/−×+/− 42 292 6.9 ± 0.18 99 193 0 a Tail DNA was analysed by Southern blot hybridization after BamHI cleavage (see Figure 2). b Average ± SEM; P 4C DNA content, while one or several nuclei exhibited DNA contents 4C (arrows) and <2C (stars) DNA contents. 1a-3a: GFP protein fluorescence. 1b-3b: Hoechst 33258 fluorescence of the same cells. Download figure Download PowerPoint Discussion To elucidate the role of the APLP2, we engineered mutant mice in which the gene is disrupted by a large deletion (11.35 kb). Heterozygous mice were apparently normal, but embryos homozygous for the disrupted locus underwent only a limited number of cleavages, corresponding to the period where they contain protein of maternal origin. From previous experiments performed on early mouse embryos (Blangy et al., 1991) as well as on a model viral replicon (Pierrefi
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