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. 1999 Oct 12;96(21):11872-7.
doi: 10.1073/pnas.96.21.11872.

Presenilin 2 deficiency causes a mild pulmonary phenotype and no changes in amyloid precursor protein processing but enhances the embryonic lethal phenotype of presenilin 1 deficiency

A Herreman  1 , D Hartmann, W Annaert, P Saftig, K Craessaerts, L Serneels, L Umans, V Schrijvers, F Checler, H Vanderstichele, V Baekelandt, R Dressel, P Cupers, D Huylebroeck, A Zwijsen, F Van Leuven, B De Strooper
Affiliations

Presenilin 2 deficiency causes a mild pulmonary phenotype and no changes in amyloid precursor protein processing but enhances the embryonic lethal phenotype of presenilin 1 deficiency

A Herreman et al. Proc Natl Acad Sci U S A. .

Abstract

Mutations in the homologous presenilin 1 (PS1) and presenilin 2 (PS2) genes cause the most common and aggressive form of familial Alzheimer's disease. Although PS1 function and dysfunction have been extensively studied, little is known about the function of PS2 in vivo. To delineate the relationships of PS2 and PS1 activities and whether PS2 mutations involve gain or loss of function, we generated PS2 homozygous deficient (-/-) and PS1/PS2 double homozygous deficient mice. In contrast to PS1(-/-) mice, PS2(-/-) mice are viable and fertile and develop only mild pulmonary fibrosis and hemorrhage with age. Absence of PS2 does not detectably alter processing of amyloid precursor protein and has little or no effect on physiologically important apoptotic processes, indicating that Alzheimer's disease-causing mutations in PS2, as in PS1, result in gain of function. Although PS1(+/-) PS2( -/-) mice survive in relatively good health, complete deletion of both PS2 and PS1 genes causes a phenotype closely resembling full Notch-1 deficiency. These results demonstrate in vivo that PS1 and PS2 have partially overlapping functions and that PS1 is essential and PS2 is redundant for normal Notch signaling during mammalian embryological development.

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Figures

Figure 1
Figure 1
Generation of PS2-deficient (PS2−/−) mice. (A) Targeting strategy to replace coding exon 5 and flanking sequences of the mouse PS2 gene by a hygromycin cassette. Boxes represent PS2 exons 4, 5, and 6 in the wild-type PS2 gene (upper panel). The lower panel displays the gene locus after targeting. The direction of transcription of the inserted hygromycin cassette is opposite to PS2 transcription (arrow). The predicted lengths of the restriction fragments obtained from the wild-type allele (top) or the targeted allele (bottom) after digestion with EcoRV or combined XbaI and SpeI are shown. (B) Genomic DNA from different ES clones was digested with XbaI and SpeI and was analyzed by Southern blotting using the external 3′probe. (C) Tail genomic DNA from littermate embryos obtained after heterozygote crossings, digested with EcoRV and analyzed by Southern blotting using the external 5′ probe. (D) PCR diagnosis of the PS2 deficient gene in tail genomic DNA.
Figure 2
Figure 2
Complete absence of PS2 in homozygote PS2−/− mice. (A) Northern blot analysis of PS2 expression. Polyadenylated RNA was isolated from brain from littermate E14 embryos and was hybridized with the EcoRI-digested PS2 cDNA probe. An actin probe was used as positive control. (B) Western blot analysis of PS2 synthesis. Membrane-enriched fractions were prepared from kidney, lung, brain, heart, and liver of 1-week-old animals, and 50 μg of protein was applied on 4–20% SDS/PAGE. Ponceau S staining of the blot confirmed that equal amounts of protein were applied in every lane. The blot was developed by using amino-terminal (NTF) and carboxy-terminal (CTF) specific PS2 antisera. (C) Semiquantitative Western blot analysis of PS2 expression in PS2−/− animals aged 3 months. Membrane material was probed with PS2CTF antiserum. No signal is observed when 100 μg of membrane-enriched material from lung or from brain of PS2−/− animals is applied whereas a signal remains visible in the lanes containing 0.3 μg (lung) or 1 μg (brain) of material derived from wild-type littermates.
Figure 3
Figure 3
Expression of Alg-3 in adult liver. (A) Western blot analysis of material of 3-month-old animals. The blot was stained first with PS2CTF antiserum and afterward with anti-PS1NTF. Notice that PS2CTF are expressed only in liver in the PS2−/− mice. PS1NTF are present at equal levels in PS2 wild-type and knock-out animals. (B) Membrane-enriched material from liver was prepared from E13.5 embryos and 1-week- and 3-month-old animals and was probed with antibodies specific for the carboxyterminus of PS1 (PS1CTF) or PS2 (PS2CTF). Notice that only in 3-month-old PS2−/− animals are PS2CTF fragments expressed.
Figure 4
Figure 4
Phenotypical analysis of PS2 knock-out mice. (A and B) Hippocampal formation of control (A) and PS2-deficient (B) mice at age 6 months as seen in routine histological preparation. No differences in tissue architecture can be demonstrated. (C and D) Lung parenchyma of control (C) and PS2-deficient (D) mice at age 3 months. Note the increase in cell mass and the reduction of alveolar space as compared with the control. (E and F) Lung of a PS2-deficient mouse at 6 months exhibiting profuse hemorrhages. Both alveolar spaces and lower conductive airways are virtually filled with extravasated erythrocytes. (F and G) Lung specimens of 6-month-old PS2−/− mice stained for single-stranded DNA after thermal denaturation (F) and tissue transglutaminase type II (G). Both markers for cells undergoing apoptosis label scattered cells within the lung parenchyma as well as in the corresponding blood vessels (BV) and bronchioli (BR). (H and I) Similar experiments carried out on wild-type mice of the same age did not result in any staining either for tissue type transglutaminase (H) or single-stranded DNA (I).
Figure 5
Figure 5
APP processing in neurons derived from E14 PS2−/− embryos. (A) Analysis of APP expression and generation of α- and β-secretase-generated APP carboxyterminal stubs (23, 38). Neurons were infected with recombinant Semliki Forest Virus driving expression of human wild-type APP (APPWt) or APP containing the London (APPLo) or the Swedish (APPSw) type of AD-causing mutations. After metabolic labeling, antibody B12/4 recognizing the 20 carboxyterminal amino acid residues of APP was used to immunoprecipitate holo-APP and α- and β-secretase-generated carboxyterminal stubs from the cell extracts. The material was analyzed on a Tris⋅tricine 10–20% gel. The lower part of the figure shows a stronger exposure of the bottom part of the gel to better reveal the APP carboxyterminal fragments. (B) Analysis of Aβ secretion into the medium (23, 38). The conditioned medium of the same neurons examined in A was immunoprecipitated with the Aβ antibody B7/6. This antibody recognizes an epitope between the α- and the β-secretase sites and therefore does not precipitate the p3 fragment. Material was analyzed on a Tris⋅tricine 10–20% gel.
Figure 6
Figure 6
PS1−/−PS2−/− embryos at E9.5 display severe growth retardation. The two upper panels display the yolk sac of E9.5 PS1+/−PS2−/− (A) and PS1−/−PS2−/− (B) littermates (identical magnification). The arrowhead points to the vascular plexus. The four lower panels display E9.5 embryos (identical magnification). See text for description of the phenotype.
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