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The Journal of Neuroscience, November 1, 2000, 20(21):7951-7963
Mice with Combined Gene Knock-Outs Reveal Essential and Partially
Redundant Functions of Amyloid Precursor Protein Family
Members
Sabine
Heber1,
Jochen
Herms2,
Vladan
Gajic7,
Johannes
Hainfellner3,
Adriano
Aguzzi3,
Thomas
Rülicke4,
Hans
Kretzschmar2,
Cornelia
von
Koch5,
Sangram
Sisodia5,
Phillippe
Tremml6,
Hans-Peter
Lipp6,
David P.
Wolfer6, and
Ulrike
Müller1, 7
1 Department of Neurochemistry, Max-Planck-Institute
for Brain Research, D-60528 Frankfurt, Germany,
2 Department of Neuropathology, University of
Göttingen, Göttingen, Germany, 3 Institute of
Neuropathology, and 4 Biologisches Zentrallabor, University
Hospital, 8091 Zürich, 5 Department of Neurobiology,
Pharmacology and Physiology, University of Chicago, Chicago, Illinois
60637, and Institutes for 6 Anatomy, and
7 Molecular Biology, University of Zürich, 8057 Zürich, Switzerland
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ABSTRACT |
The amyloid precursor protein (APP) involved in Alzheimer's
disease is a member of a larger gene family including amyloid precursor-like proteins APLP1 and APLP2. We generated and examined the
phenotypes of mice lacking individual or all possible combinations of
APP family members to assess potential functional redundancies within
the gene family. Mice deficient for the nervous system-specific APLP1
protein showed a postnatal growth deficit as the only obvious abnormality. In contrast to this minor phenotype,
APLP2 //APLP1/
and
APLP2//APP/
mice proved lethal early postnatally. Surprisingly,
APLP1//APP/
mice were viable, apparently normal, and showed no
compensatory upregulation of APLP2 expression. These data indicate
redundancy between APLP2 and both other family members and corroborate
a key physiological role for APLP2. This view gains further support by
the observation that
APLP1//APP//APLP2+/
mice display postnatal lethality. In addition, they provide genetic evidence for at least some distinct physiological roles of APP and
APLP2 by demonstrating that combinations of single knock-outs with the
APLP1 mutation resulted in double mutants of clearly different
phenotypes, being either lethal, or viable. None of the lethal double
mutants displayed, however, obvious histopathological abnormalities in
the brain or any other organ examined. Moreover, cortical neurons from
single or combined mutant mice showed unaltered survival rates under
basal culture conditions and unaltered susceptibility to glutamate
excitotoxicity in vitro.
Key words:
amyloid precursor protein; amyloid precursor-like
protein; knock-out mice; functional redundancy; excitotoxicity; cortical neurons; Alzheimer's disease
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INTRODUCTION |
Neurofibrillary tangles and senile
neuritic plaques are the major pathological features of Alzheimer's
disease. The predominant constituent of neuritic plaques is the
 -amyloid peptide (A4), proteolytically derived from the larger
-amyloid precursor protein (APP). APP is a member of a larger gene
family including the two amyloid precursor-like proteins APLP1 and
APLP2 from mammals (Wasco et al., 1992 , 1993; Sprecher et al., 1993;
Sandbrink et al., 1994; Slunt et al., 1994). Both APLPs are highly
homologous to APP and are proteolytically processed in a similar way,
leading to the secretion of the large ectodomains (sAPP and sAPLP)
(Slunt et al., 1994; Paliga et al., 1997). Using in situ
hybridization and RT-PCR analysis, we and others have demonstrated that
APP and APLP2 are expressed ubiquitously in largely overlapping
patterns during embryonic development and in adult tissue (Slunt et
al., 1994; Lorent et al., 1995). In contrast, APLP1 is found primarily in the nervous system (Lorent et al., 1995; Thinakaran et al., 1995c).
APP is axonally transported, has been localized to synapses, and
undergoes retrograde and transcytotic transport in neurons (Simons et
al., 1995; Yamazaki et al., 1995). APLP2 has been detected in
postsynaptic compartments throughout the cortex and in axonal termini
of olfactory sensory neurons (Thinakaran et al., 1995a,b). In summary,
APP/APLP proteins are highly related, are similarly processed, share
overlapping domains of expression, and may therefore also be
functionally conserved.
Multiple functions have been proposed for APP, mainly based on in
vitro experiments (for review, see Mattson, 1997). To address the
physiological functions of APP directly, we and others have generated
mice carrying a hypomorphic mutation of APP (APP ; Müller et
al., 1994) and APP-deficient null mutants (Zheng et al., 1995; Li et
al., 1996). The phenotypes of these mutants suggested that APP may play
a role in neurite outgrowth and the formation of forebrain commissures,
postnatal somatic growth and neurobehavioral development, locomotor
activity and grip strength, copper homeostasis, and the susceptibility
to epileptic seizures and excitotoxic agents (Zheng et al., 1995; Li et
al., 1996; Perez et al., 1997; Steinbach et al., 1998; Tremml et al.,
1998; Magara et al., 1999; White et al., 1999a,b).
APLP2/
mice showed no apparent abnormalities, but double mutants obtained by
crossing
APP/ mice
(obtained from H. Zheng, H. Chen, M. Trumbauer, and L. H. T. van der
Ploeg) to
APLP2/
mice were perinatally lethal, suggesting functional redundancy (von
Koch et al., 1997).
This study investigates the phenotype of
APLP1/
mice and addresses the question of functional complementation within
the gene family by analyzing all possible combinations of double
mutants. We show that APP family members serve essential, at least
partially redundant functions by demonstrating early postnatal
lethality for both
APLP2//APLP1/and
APLP2//APP/double
mutants. In addition, we provide genetic evidence for distinct physiological roles of APP and APLP2, by showing that crosses of the
respective single knock-outs with APLP1 knock-out mice result in double
mutants of clearly different phenotypes, being either lethal
(APLP2//APLP1/),
or viable
(APP//APLP1/).
Moreover, we investigated the presumed neuroprotective role of
endogenous APP family members.
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MATERIALS AND METHODS |
Generation of APLP1/
embryonic stem cells
Using as a probe a 0.4 kb PstI fragment derived from
the 5'-end of the APLP1 cDNA (Wasco et al., 1992) two overlapping  clones encompassing ~19 kb of genomic sequence were isolated from an isogenic Gem11 genomic library constructed from AB-1 [129/Sv(ev)] embryonic stem (ES) cells. As judged from hybridization
experiments with various oligonucleotides derived from different
regions of the APLP1 cDNA and partial sequencing the genomic clones
contained the whole coding region including the putative first exon
(cDNA positions 9-229) harboring the ATG start codon.
Apart from exon1 and exon2 the precise location of the other exons was
not mapped. The targeting vector pAPLP1targ was constructed by
inserting a blunted 0.9 kb SacI/XhoI fragment
lying 2.5 kb upstream of putative exon1 into the NotI site
of pTKNEOUMSPSA, a slightly modified vector (containing additional
restriction sites) derived from the previously described plasmid
pTKNEOUMS (Ruffner et al., 1993). Subsequently a blunted 4.7 kb
XhoI/KpnI fragment was inserted as the "long
arm" of homology into the unique ClaI site of the vector.
In this construct the neoR gene under the
control of the phosphoglycerate kinase (PGK) promoter was in
antisense orientation to the transcriptional direction of the APLP1
gene and was followed by the 360 bp UMS sequence reported to
mediate transcription termination. A herpes simplex virus thymidine
kinase (HSV-TK) cassette was added at the 3' end to allow for
counterselection. Homologous recombination leads to a ~8 kb genomic
deletion containing ~2.5 kb of the promoter. To determine how much of
the coding region would be deleted by gene targeting, hybridization
experiments were performed showing that the deleted region contained
coding sequences up to approximately position 1000 of the 2.36 kb cDNA,
as judged by hybridization to oligo UM14 (cDNA position
1014-1034).
The targeting vector was subsequently linearized with SacII
at the 5' border of the APLP1 genomic region, electroporated into GS1
ES cells [established by Gerlinde Stark from 129 Sv(ev) mice] grown
on irradiated mouse embryonic fibroblasts in DMEM supplemented with
20% fetal calf serum (D20). Colonies resistant to neomycin and
1-(2-deoxy-2-flouro--D-arabinosifuranosyl)-5-ioduracil
(FIAU) were selected in D20 containing 400 µg of G-418 and 0.2 µM FIAU and screened as described (Müller
et al., 1994) by PCR using a primer derived from the 3' end of the
neo-cassette (P3: 5'-ATTCGCAGCGCATCGCCTTCTATCGCC-3') and a primer
corresponding to a genomic sequence 5' from the targeting vector (UM20:
5'-GGATTTCAGCCCTGGTTCCCATTCTAACCC-3'). The frequency of homologous
recombination was ~1 in 500 G-418/FIAU-resistant colonies.
Generation of
APLP1/ mice
ES cells heterozygous for the mutant APLP1 gene were injected
into 3.5-d-old C57BL/6 blastocysts that were transferred into the uteri
of pseudopregnant NMRI foster mothers. One of four clones investigated (clone GS-34.4) gave rise to a chimeric male that was
mated to C57BL/6 females [and 129 Sv(ev) females for pure genetic
background, respectively] and transmitted the mutant allele in the
germline as revealed by PCR and Southern blot analysis. Heterozygous
offspring were intercrossed and generated 20% homozygous APLP1/ animals (65 animals analyzed).
Animals and PCR genotyping
APLP1 knock-out mice [of either pure 129Sv(ev) or mixed 129 Sv(ev)xC57BL/6 genetic background] were initially screened with primers UM20/P3 as described for ES cells. Later, a simpler 3-primer PCR was used amplifying for the wild-type (wt) allele a 600 bp fragment with primers UM30 (5'-GCTTTCTGCCTTCATGCCTATCTCTAG-3') and UM31
(5'-ACTTTGGCTGAACTGAGTGTACACC-3') derived from the short arm of the
targeting vector or from the region replaced by neo, respectively. For
the mutant allele a 450 bp product was obtained with primers UM30
and P4-neo (5'-ATGCGGTGGGCTCTATGGCTTCTGA-3') derived from the
PGK-Neo-cassette.
APLP2 knock-out mice [129Sv(ev)xC57BL/6 genetic background] were
generated and genotyped as described (von Koch et al., 1997).
APP knock-out animals (129OLAxC57BL/6 genetic background) harbor a 200 kb genomic deletion within the APP locus encompassing exon 2-17 of the
APP gene and were generated as described (Li et al., 1996; Magara et
al., 1999). For genotyping, a 3-primer PCR was set up amplifying for
the wt allele a 650 bp fragment with primers UM44 (5'-GAGACGAGG
ACGCTCAGTCCTAGGG-3) and UM42 (5'-ATCACCT-GGTTCTAATCAGAGGCCC-3') flanking exon 17 and for the mutant allele a 430 bp fragment with primers UM42 located in intron 17 and P3-hygro
(5'-CGAGATCAGCAGCCTCTGTTCCACA-3') derived from the PGK-Hygro-cassette,
respectively. PCR was done on DNA obtained from tail biopsies as
previously described (Müller et al., 1994).
Animals were housed in a special pathogen-free unit kept under optimal
hygiene conditions. Timed matings were set up in the late afternoon
followed by plug check on the next morning. The time point of a
detected plug was considered as embryonic day 0.5 (E0.5).
Generation of combined mutants
Double mutants were generated by three consecutive crosses.
Single mutants of APP family members (e.g.,
APLP1/ × APLP2/)
were intercrossed, and animals heterozygous for both loci were backcrossed to single mutants (e.g.,
APLP1+/APLP2+/ × APLP2/)
to obtain 25% offspring homozygous knock-out for one gene and heterozygous for the other (e.g.,
APLP1+/APLP2/). These animals were
further intercrossed (APLP1+/APLP2/ × APLP1+/APLP2/) to obtain double
knock-outs in the next generation (e.g., 25% APLP1/APLP2/,
25%
APLP1+/+APLP2/,
and 50%
APLP1+/APLP2/).
Accordingly, to obtain APP/APLP2 and APP/APLP mutants the respective analogous crosses were set up, and double knock-outs were
generated in the third round of crossing from the following matings:
(APP+/APLP2/ × APP+/APLP2/)
and
(APP+/APLP1/ × APP+/APLP1/).
APP//APLP1//APLP2+/
mice were generated by crossing
APP//APLP1/
mice with
APP//APLP1+//APLP2+/ mice.
Southern and Northern blot analysis of APLP/
mice
Southern blots were prepared from APLP1 ES cell or tail DNA
digested with HindIII or EcoRI, cutting outside
of the targeting vector and separated by conventional or pulsed-field
gel electrophoresis [1% agarose in 45 mM Tris-borate, 1 mM EDTA run for 15 hr at 6 V/cm, 120° angle, switch time
being from 0.5-2.0 sec in a Bio-Rad (Hercules, CA) DRIII apparatus].
Hybridization was performed with a random primer
32P-labeled (PrimeIt; Stratagene, La
Jolla, CA) genomic 0.5 kb PstI fragment (Fig.
1, probe B) and a 0.6 kb
PstI/XbaI-fragment of pGKneo.
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Figure 1.
Disruption of the APLP1 gene by gene targeting in
ES cells. Our targeting strategy was aimed at abolishing transcription
and translation by generating an ~8 kb genomic deletion comprising
2.5 kb of the putative promoter, the first exon containing the ATG
translational start codon and genomic sequences containing ~50% of
the APLP1 coding region. Top, Genomic segment
containing the APLP1 locus. Apart from the first and second exon the
precise location of the other exons (Exn) was not
mapped. The stippled box represents an arbitrary
positioned exon corresponding to coding sequences around cDNA position
1000. Parentheses indicate a restriction site derived
from one of two overlapping phages. Middle,
Targeting vector pAPLP1-targ. Horizontal arrows indicate
the direction of transcription. The hatched box
represents a HSV-TK gene. Bottom, The disrupted
allele after homologous recombination. E,
EcoRI; H, HindIII;
K, KpnI; P,
PstI; S, SacI;
X, XhoI; neo-probe, 0.6 kb
PstI/XbaI fragment of pGKneo; probe B,
genomic 0.5 kb PstI fragment. Brackets
represent restriction sites that were destroyed during cloning.
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For Northern blots, total RNA (prepared as described in Müller et
al., 1994) or polyA-RNA [prepared using Oligotex columns from Qiagen
(Hilden, Germany)] was separated on 1% denaturing agarose gels and
transferred by capillarity to HybondN+ (Amersham, Arlington Heights,
IL) membrane. A 32P-labeled RNA probe
containing 570 nt from the 3' end of the APLP1 cDNA was prepared by
in vitro transcription with T7 polymerase using
EcoRV linearized pBSKAPLP1 as the template (Wasco et al., 1992). To check for APP expression, a genomic 0.7 kb
EcoRI/ScaI fragment subcloned into pBluescript
and comprising exon 2 was used as the template for in vitro transcription.
Northern blots were hybridized with riboprobes overnight at 65°C in
50% formamide solution containing 5× SSC, 5× Denhardt's solution,
1% SDS, 0.1% Na Pyrrophosphate, 5 mM EDTA, and 300 µg/ml yeast tRNA. Washing of blots was at 65°C for 20 min in 0.2×
SSC and 0.5% SDS. After autoradiography blots were
stripped by boiling for 10 min in 20 mM Tris-HCl, pH 8.0, 2 mM EDTA, and 0.5% SDS and rehybridized with a random
primer 32P-labeled 490 bp XhoII
fragment of the rat glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) cDNA.
Western blot analysis
Mouse organs were homogenized in ~10 vol of homogenization
buffer (0.5% SDS and 50 mM Tris-HCl, pH 6.8, 5 mM EDTA, 50 µg/ml pepstatin, and 0.25 mM
PMSF) per gram of tissue, using a Ultra-Turrax T25 (IKA Labortechnik)
blender. The homogenates were boiled for 10 min, centrifuged for 10 min
at 12,000 × g, and the supernatants were recovered.
Protein concentrations were determined by the BCA method (Pierce,
Rockford, IL). Proteins were separated on Standard (Lämmli) 12%
SDS-polyacrylamide gels using the protean II system (Bio-Rad) and
electrotransferred to nitrocellulose membrane in low glycine buffer at
10 mA for 17 hr with wet transfer equipment (Bio-Rad). Filters were
blocked for 1 hr in TBST (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, and 0.1% Tween 20) containing 5%
nonfat dry milk. The blot was incubated overnight with the primary
antibody (e.g., rabbit antiserum CT-11 at a dilution of 1:2000 in TBST and 1% nonfat dry milk) followed by an 1 hr incubation with
HRP-coupled secondary antibody [swine anti-rabbit IgG from Dako
(Carpinteria, CA) at a dilution of 1:2500 in TBST and 1% nonfat dry
milk] and developed by the ECL reaction (Pierce).
Antibodies used for Western blotting: CT-11 is a rabbit polyclonal
serum raised against a synthetic peptide comprising the 11 C-terminal
amino acids of murine APLP1 and is not cross-reacting with either APP
or APLP2 (Thinakaran et al., 1995c). APLP2 was specifically detected
with rabbit serum D2II (dilution 1:2000) as described (Thinakaran and
Sisodia, 1994), and APP-specific staining was obtained with antibody
22C11 (dilution 1:1000; Chemicon). anti-Actin staining was performed
with antibody C4 (dilution 1:10,000; ICN Biomedicals, Cleveland, OH).
Anatomical and histological analysis
Immunohistochemistry. Immunohistochemistry was done
as described previously (Steinbach et al., 1998). Formaldehyde-fixed, paraffin-embedded, rehydrated, 2-µm-thick sections were incubated with monoclonal mouse and polyclonal rabbit antibodies (MAP II; 1:400,
Boehringer, Mannheim, Germany), anti-synaptophysin (1:50, Dako), glial
fibrillary acid protein (GFAP; 1:50, Dako), and -III tubulin
(1:2000, Promega) diluted in PBS for 2 hr at room temperature. This was
followed by incubation with the secondary antibody (rabbit-anti mouse
IgG, Dako) diluted 1:50 in PBS for 45 min at room
temperature. Bound secondary antibody was detected by using the
alkaline phosphatase-anti-alkaline phosphatase complex (APAAP; mouse
monoclonal, Dako) diluted 1:40 in PBS and incubated for 45 min at room
temperature. The alkaline phosphatase activity was visualized by using
Astranenfuchsin (Aldrich, Milwaukee, WI). The sections were
counterstained with hemalaun. The immunohistochemical methods used have
previously been validated by us as a sensitive assay to detect lesions
induced by systemic injection of sublethal, low doses of kainate
(Steinbach et al., 1998).
In situ end-labeling assay. Terminal deoxynucleotidyl
transferase-mediated biotinylated UTP nick end labeling (TUNEL)
assays were performed on formalin-fixed sections: staining was
performed incubating sections under coverslips with 50 µl of labeling
mix [25 U/µl terminal deoxynucleotidyl transferase (TdT)
(Boehringer), Dig-DNA labeling mix 10× concentration (Boehringer), and
2 mM CaCl2 in reaction buffer for
terminal transferase (Boehringer) containing 0.2 M
potassium cacodylate and 25 mM Tris-HCl, pH 7.5] for 60 min at 37°C. After rinsing in Tris-buffered saline (TBS), sections
were blocked with 10% fetal calf serum (FCS) (Seromed). Sections were
then treated for 60 min with alkaline phosphatase-labeled anti-dioxigenin antibody Fab-fragment (Boehringer) at a dilution of
1:250 in 10% FCS. After washing in TBS, the color reaction was
visualized by incubating the sections in reaction buffer (in mM: Tris 100, NaCl 100, and MgCl2 50, pH 9.5) containing 100 mg/ml 4-nitroblue tetrazolium salt and 50 mg/ml
5-bromo-4-chloro-3-indolyl phosphate (Boehringer) for 5-15 min. The
reaction was stopped with TBS, and sections were counterstained with
nuclear fast red (Merck).
Electron microscopy
Brains from newborn mice obtained from wt matings or by
intercrosses of
ALPL1+/APLP2/
mice, or
APP+/APLP2/
mice, respectively, were dissected and fixed by immersion in 2%
glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, containing 2% polyvinyl-pyrrolidone (PVP) for 2 hr. After
several rinses in cacodylate buffer with 2% PVP, the brainstems were
post-fixed in 1% OsO4 in cacodylate buffer
followed by dehydration in ethanol and embedding in Epon 812 substitute
(Fluka, Neu-Ulm, Germany). Ultrathin 75 nm sections were cut with an
ultramicrotome (Reichert Ultracut E). Sections were stained with uranyl
acetate/lead citrate and examined under the electron microscope (Zeiss
EM 10C). Images were recorded with a BioScan Camera (Gatan).
Primary cultures from mouse cortex
Cultures of cortical neurons were prepared with some minor
modifications in analogy to the procedure described by Banker and Goslin (1998) for hippocampal neurons. Embryos for cortical cultures were obtained from intercrosses of
APLP1+/APLP2/
mice or
APP+/APLP2/
mice, respectively. Genotyping was done by PCR on tail tissue. Wild-type control mice were obtained from 129 Sv(ev) × C57B6
matings and processed in parallel. Cortices from single E14.5 mouse
embryos were collected in Ca 2+- and Mg
2+-free HBSS/10 mM
HEPES, pH 7.2 (Life Technologies, Gaithersburg, MD). After addition of
trypsin (final concentration 0.05%) neuronal tissue was incubated for
8 min at 37°C. Trypsin was removed, the suspension was washed with 15 ml of HBSS/HEPES, and cortices were triturated with 5 ml glass
pipettes in plating medium (serum-free Neurobasal medium supplemented
with B27, 0.5 mM glutamine, and 50 U of
penicillin-streptomycin; Life Technologies). Cells were seeded in 500 µl of plating medium in 24-well plastic dishes (coated overnight with
10 µg/ml poly-L-lysine hydrobromide in 0.1 M
borate buffer, pH 8.5) at a density of 130,000 cells/well (~73,000
cells/cm2) and maintained at 37°C in 5%
CO2. Neuronal cultures were treated with
cytosine--D-arabinofuranoside (Ara-C; final
concentration 3 µM, Sigma) dissolved in 250 µl of
plating medium on day 3 in vitro (DIV 3) to prevent
glial proliferation. Neuronal purity was >95% as determined by
immunohistochemical staining against GFAP, performed on DIV 10. Cultures were kept without further medium change until DIV 15 when
survival experiments were done.
Survival assays and glutamate treatment
To assess spontaneous survival rates, neurons from individual
embryos were cultured on gridded cellocate coverslips (Eppendorf, Hamburg). Live neurons, as judged by their morphological integrity (with an extended neurite network, smooth membrane appearance, and
noncondensed soma) were counted within the same area (six nonoverlapping gridded fields, each covering a total area of 350 × 350 µm, containing ~40-60 neurons per field, on average
240-360 neurons per sample) once on DIV 1 and again on DIV 7. Neuron
counts were performed for two or three independent embryos for each
genotype. Values represent averages ± SEM. The rate of
spontaneous survival on DIV 7 was calculated relative to neuron counts
obtained on DIV 1.
Cell survival after glutamate treatment was monitored by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromide (MTT) assays: yellow MTT (Sigma) dissolved in PBS was added to the neuronal cultures to a final concentration of 0.5 mg/ml and incubated for 1 hr
at 37°C. A violet formazan product is produced by viable cells
because of the metabolic activity of mitochondrial enzymes. The
formazan product is dissolved in 250 µl of DMSO and detected by its optical density
(OD) 540-690. The
OD540-690 is
taken as a measure of the number of living cells ( 95%
neurons) in the culture (Mosmann, 1983).
To investigate cell survival in response to glutamate, neurons were
prepared from individual embryos (8-12 processed in parallel), and
cell suspensions of two cultures were counted before seeding. To
correct for small differences in initial plating densities between
individual cultures, cell survival was always expressed as relative
values of glutamate-treated cultures normalized to values obtained from
untreated cultures derived from the same embryo. For dose-response
experiments different concentrations of glutamate (10, 25, 50, 100 µM) dissolved in plating medium were added to cortical
neurons for 24 hr followed by MTT assays. Average values from
triplicate cultures were calculated relative to values from nontreated
cultures for which the mean was set as 100% survival. For each
experiment two or three independent embryos were used, and the
experiment was replicated at least three times. In addition, cortical
neurons were treated with 50 µM glutamate for 1 or 3 hr.
For each time point, a triplicate of 24-well cultures were processed in
parallel, and mean values were determined. In addition, triplicate sets
of nontreated cultures were analyzed for each time point. The mean MTT
value obtained from these nontreated cultures (after 0, 1, and 3 hr of
incubation) was set as 100% cell survival for each embryo. Percentage
of survival was calculated relative to these mean values.
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RESULTS |
Generation and characterization of APLP1 knock-out mice
Inactivation of the murine APLP1 gene was achieved by gene
targeting with a strategy depicted in Figure 1. By homologous
recombination in ES cells we generated an ~8 kb genomic deletion
comprising 2.5 kb of the putative promoter, the first exon containing
the ATG translational start codon and genomic sequences containing ~50% of the APLP1 coding region. Deletion of the transcription and
translation start sites should completely abolish APLP1 expression. Mutant ES cells were injected into blastocysts and gave rise to one
chimera that transmitted the mutant allele in the germ line. Correct
homologous recombination was confirmed by Southern blot analysis of
tail DNA that showed the expected pattern for an interrupted APLP1
locus (Fig. 2A,B).
Heterozygous animals were intercrossed and yielded homozygous APLP1
knock-out animals at a normal Mendelian frequency. To determine whether
the APLP1 gene was also functionally inactive, Northern and Southern
blot analysis was performed on brain tissue. An antisense RNA probe
(cDNA position 1788-2360) lying downstream of the targeted deletion
revealed a band of ~2.6 kb on brain of wt animals, whereas no
transcript was detected in
APLP1/
mutants (Fig. 2D). Western blot analysis with the
antibody CT-11, which is directed against the 11 C-terminal amino acids
of APLP1 and discriminates against the related APP and APLP2 proteins, gave rise to a set of APLP-1-specific bands of 85-100 kDa for wt
brain, but not for mutants, indicating that the gene had been completely inactivated. Expression of the other family members APP and
APLP2 was unaltered, as judged from Northern (data not shown) and
Western blot analysis (Fig. 3A,
lane 1, lane 2 ).
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Figure 2.
Molecular validation of the APLP1 knock-out.
Functional inactivation of the APLP1 gene was confirmed by a
combination of Southern (A, B), Western
(C), and Northern (D) blot
analysis. Genomic tail DNA from F1 offspring of the germline chimera
was digested with HindIII and hybridized with genomic
probe B, as depicted in Figure 1. Note that wt animals show a single
band of 18 kb, whereas in heterozygous APLP1 mutants an additional band
of the expected size of 14 kb was detected (A).
Intercrossing of these heterozygous animals lead to homozygous mutants
(B), as judged by analysis of DNA from mouse
embryonic fibroblasts (MEF). The respective
genotype is depicted above the blots. Western blots
(C) of wt and
APLP1/ mice were prepared as
described in Materials and Methods. Forty micrograms of brain extracts
from two APLP1/ mice
(lanes 1, 2), a wt APLP1+/+
littermate (lane 3), and a 129 Sv(ev) wt mouse
(lane 4) were separated on a 12% standard
Lämmli gel. Incubation with APLP1-specific antiserum CT11
followed by anti-rabbit horseradish peroxidase-linked secondary
antibody showed a set of APLP-1-specific bands of 85-100 kDa for wt
brain homogenates. In contrast, brain homogenates from
APLP1/ animals
showed neither APLP1-specific bands of wt size nor any shorter
polypeptides. The blot was developed with chemiluminescence reagents
(ECL; Amersham). Marker proteins of the indicated sizes were from
Bio-Rad (broad range rainbow markers). Total RNA was isolated from
brain of either mutant or wt animals, and poly(A+) RNA was prepared
from 120 µg of total RNA. Northern blot analysis
(D) with an antisense RNA probe (cDNA position
1788-2360) lying downstream of the targeted deletion revealed a band
of 2.4 kb on brain of wt animals, whereas no transcript was detected in
organs from APLP1/ mutant
animals. After autoradiography, filters were stripped and rehybridized
with a GAPDH probe to monitor loading (bottom
panel).
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Figure 3.
Western blot analysis of APP/APLP expression in
single and double mutants. A, Total brains of newborn
APP/ (lane 9),
APLP2/ (lane
7), and wt (lane 8) mice and in addition
brains of newborn mice generated by intercrossing heterozygous
APP+//APLP1/
mice (lanes 1-6) were homogenized, and equal
amounts of protein (20 µg/lane) were resolved on 8% PAA gels. From
each blot the bottom half was cut off and probed with an Actin-specific
antibody to monitor loading (second row of each panel).
Top, Note that probing with an antiserum specific for
APLP2 (D2II) showed no major alterations of APLP2 expression in animals
of different APP/APLP1 genotype compared to wt levels (lane
8). Bottom, Probing with an APP-specific
antibody (22C11) showed comparable amounts of total APP expression in
APLP1/ (lanes 1, 2) and wt (lane 8) mice. The seemingly higher
expression of APP in APLP2/
mice (lane 7) is attributable to unequal loading
as evidenced by more intense actin staining. As expected, APP
expression was abolished in
APP/ single mutants
(lane 9) and
APP//APLP1/
double mutants (lanes 5, 6). Note
that in heterozygous
APP+//APLP1/
mice APP expression is reduced to ~50%, arguing against a
compensatory upregulation of APP expression. B, Total
brains of newborn pups obtained from heterozygous
APLP2+//APLP1/
intercrosses were homogenized, and equal amounts of protein
(20 µg/lane) were separated on a 8% PAA gel. Top,
Probing with an APP-specific antibody (22C11) showed similar amounts of
total APP expression in both viable
APLP1+//APLP2/
heterozygotes (lanes 3, 4) or in
lethal
APLP1//APLP2/
double knock-outs (lanes 5, 6), compared to the
amount of APP expression in
APLP2/ single mutants
(lanes 1, 2) or in a wt control
(lane 7). Note that no significant compensatory
upregulation of APP expression was found. Bottom,
Probing with an antiserum specific for APLP1 (CT11) showed similar
APLP1 expression levels in
APLP2/
mice (lanes 1, 2) compared to the wt
control. Note that in heterozygous
APLP1+//APLP2/
animals (lanes 3, 4) expression is
reduced to ~50% and abolished in
APLP1//APLP2/
double mutants (lanes 5, 6). Genotypes of animals
analyzed are as indicated above blot panels.
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APLP1/-animals
were viable, fertile, and showed, apart from a significant body weight
deficit of ~10% (mutant males, 25.37 ± 0.70 gm; wt males,
27.72 ± 0.76 gm; mutant females, 20.01 ± 0.47 gm; wt
females, 22.12 ± 0.55 gm; average age, 9 weeks; n = 59; p < 0.001 by two-way ANOVA), emerging in the
second week postnatally (data not shown), no overt abnormalities until
at least 18 months of age. In contrast to APP-deficient mice that exhibit reduced locomotor activity and reduced forelimb grip strength (Müller et al., 1994; Zheng et al., 1995; Tremml et al., 1997), APLP1/
mice showed, compared to wt controls, normal locomotor activity, and in
adult animals grip strength was unchanged (data not shown). A subtle
retardation of neurobehavioral development [as assessed by tests of
the Fox (1965) battery] was observed early postnatally (Tremml et al.,
1997; data not shown). Histopathological analysis of brain sections
(see Materials and Methods) showed no morphological alterations; in
particular no agenesis of the corpus callosum was observed [even on a
pure 129Sv(ev) genetic background], as opposed to APP-deficient mice
exhibiting commissure defects and a high incidence of callosal agenesis
on a 129-specific background (Magara et al., 1999). Spatial learning
was analyzed by the Morris water maze test, which revealed similar
cognitive abilities of APLP1/
animals as wt controls (as assessed by
their normal performance in reversal learning). Surprisingly, however,
APLP1-deficient mice showed improved acquisition learning, which could
be attributed to altered noncognitive components of the behavior, in
particular reduced thigmotaxis (Tremml et al., 1997; P. Tremml, U. Müller, H.-P. Lipp, and D. Wolfer, manuscript in
preparation). In summary, ablation of the neuron-specific APLP1
gene results in a rather minor phenotype that is clearly distinct from
that of APP-deficient mice, suggesting at least some different
functions of both proteins.
Generation of mutants with combined gene deficiencies
To address the question of whether the minor phenotype of single
mutants is caused by functional compensation by other APP family
members, we set out to generate all three possible combinations of
double mutants
(APP//APLP2/,
APLP1//APLP2/,
and
APP//APLP1/)
by crossing the respective single mutants (see Materials and Methods
for details). To obtain
APP//APLP2/
mice, animals deficient for APLP2 and heterozygous for APP
(APLP2//APP+/)
were intercrossed and expected to yield 25% offspring deficient for
both proteins. When we genotyped the offspring at weaning (~4 weeks
of age) initially none and, after analysis of a larger sample of 355 mice, only a single surviving double mutant was found (Table
1A),
suggesting that a combined deficiency of APP/APLP2 is lethal either
during development or within the first weeks after birth. Analysis of
the genotype distribution of offspring at E19 and shortly after birth
(P0) showed that almost the theoretically expected number of double
mutants (21% of 131 animals screened; Table 1A)
survived until this time point. A smaller sample of 26 mice was
analyzed at postnatal day 1 (P1), however, only two APP//APLP1/
mice were found (one of which died at P2 and the other after 4 weeks)
indicating postnatal lethality, predominantly within the first day
after birth.
Analogously,
APLP1+//APLP2/
mice were intercrossed, and the offspring were genotyped at weaning. As
seen for
APP//APLP2/
mice, a combined APLP1/APLP2 deficiency proved lethal, in this case
with 100% penetrance. Among 326 animals analyzed at weaning, not a
single surviving double mutant was found (Table 1B).
A smaller sample of pups was screened shortly after birth (P0), and
another set on P1 (Table 1B). Whereas
APLP1//APLP2/
mice were born at normal Mendelian frequency (29% of 171 animals), no
surviving double mutants were found at P1, indicating that a combined
APLP1/APLP2 deficiency results in lethality within the first day after
birth. These results demonstrate that APP family members serve
essential but at least partially redundant functions in vivo
and corroborate a key physiological role for APLP2.
To our surprise,
APP//APLP1/
mice generated in a similar manner were viable, fertile, and showed no
apparent abnormalities, apart from a body weight deficit comparable to
that of single mutants, until at least 18 months of age. This
unexpected finding that not all three possible combinations of the
single mutants results in a lethal phenotype has crucial implications
for the specific and redundant functions exerted by APP family members (see Discussion).
The key physiological function or functions of APLP2 are further
supported by recent preliminary data demonstrating postnatal lethality
for
APP//APLP1//APLP2+/
mice, which suggests haploinsufficiency for the remaining single APLP2
allele. In an attempt to ultimately generate triple knock-outs, which
should theoretically be feasible by intercrossing
APP//APLP1//APLP2+/
mice, we set up appropriate matings (see Material and Methods) that were expected to yield 25% offspring of the respective
(APP//APLP1//APLP2+/)
genotype. Among 350 animals screened at weaning, we found only four
APP//APLP1// APLP2+/
mice (1% instead of the expected 25%). As observed for double mutants
(see below)
APP//APLP1//APLP2+/
mice showed (when analyzed on P0) no apparent histopathological abnormalities (data not shown). The surviving animals were severely impaired in breeding, and we have so far been unable to obtain any
litters from these mice. Again, lethality occurred postnatally, because
an
APP//APLP1//APLP2+/
allele frequency of 27% was found in a
smaller sample of mice, analyzed shortly after birth (data not shown).
We conclude from these data that the presence of a single APLP2 allele
in the absence of other APP family members is not sufficient for survival.
Analysis of APP/APLP expression in double mutants
We then asked whether the viable phenotype of
APP//APLP1/
mice may be attributed to a compensatory upregulation of APLP2 (Fig. 3). Western blot analysis of brain homogenates prepared from newborn mice showed APLP2 levels comparable to those found in single mutants or
wt animals (Fig. 3A, top) suggesting that basal wt-APLP2
levels are not limiting for survival. More subtle or region-specific alterations in APLP2 expression cannot be ruled out, however. Likewise,
APP protein levels were not upregulated in heterozygous, viable
APP+//APLP1/
littermates, showing an ~50% reduction
of APP as compared to APP+/+/APLP1/
single knock-outs (Fig. 3A,
bottom). We then determined whether in the lethal
APLP2//APLP1/
double knock-out, expression of the remaining third family member, APP,
is altered. Both Northern (data not shown) and Western blot analysis
failed to reveal changes in the expression level of APP in the brains
of both newborn viable
APLP2//APLP1+/
pups and of
APLP2//APLP1/
littermates that would have died within
the next hour (Fig. 3B, top). However, APLP1 expression was
found to be reduced to ~50% of wt level in viable
APLP2//APLP1+/
heterozygous newborns (Fig. 3B,
bottom). Thus, a loss of APP/APLP family members does not cause
compensatory upregulation of related family members in double
knock-outs.
Gross and histopathological analysis of double mutants
Newborn double mutants were initially (for several hours)
indistinguishable from their littermates and were able to suckle. Between ~5 and 15 hr after birth, however, they had no or less milk
in their stomachs, appeared weaker, became pale, and died. From the
observation of several litters of mice of both types of lethal double
mutants, we have no evidence for motor paralysis or overt seizure
activity. To address the cause of the early postnatal lethality
observed in
APLP2//APLP1/
and
APLP2//APP/double
knock-outs, we used various histological approaches to examine newborn
double mutants, as compared to
APLP2/
single mutants and wt controls. No obvious
abnormalities were observed after macroscopic examination, revealing
normal sizes of the head, face, and body postnatally.
Hematoxylin-eosin-stained sections of internal organs also did not
show any abnormalities. In particular, no malformations of the palate,
esophagus, stomach, intestine, colon, and rectum were found that could
result in impaired feeding behavior. Moreover, meconium was present in
the intestine of all mutants. Furthermore, cranial nerves implicated in
feeding, including trigeminal ganglion and facial motor nucleus, were
present and appeared of normal size. The heart and lungs appeared
normal in maturity and size, which argues against an impairment of
cardiopulmonary function as the cause of lethality, however alterations
in the efferent autonomic innervation of the heart cannot be excluded. None of the organs examined (thyroid, thymus, spleen, pancreas, liver,
kidneys, bladder, testis, and ovaries) showed differences in size or
histopathological abnormalities in both types of double mutants, or
single mutants, compared to wt controls (data not shown).
In the brain no signs of increased cell death were found, as evidenced
by pyknotic nuclei in Nissl-stained sections or by TUNEL staining (Fig.
4). The neocortex showed the normal
layered structure, and cortical neurons were of normal appearance. CA1 through CA3 regions and the dentate gyrus of the hippocampus appeared normal, based on Nissl staining pattern and MAP-II and
synaptophysin immunohistochemistry, indicating that lack of APLP2
either in combination with a deficiency for APP, or APLP1,
respectively, did not affect hippocampal architecture and cell/neurite
density (Fig. 4). GFAP immunohistochemistry revealed no signs of
gliosis (data not shown).
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Figure 4.
Characterization of mutants by immunocytochemistry
on brain sections. Histological analysis of the cortex and hippocampus
from age-matched newborn wild-type,
APLP2/, and double knock-out
mice lacking either APLP2/APLP1 or APLP2/APP revealed no apparent
anomalies in any of the mutants examined. Pictures show frontal
sections of parietal cortex (a-d) and hippocampus
(a'-d') from wild-type (a),
APLP2/
(b),
APP//APLP2/
(c), and
APLP1//APLP2/(d)
knock-out mice. Shown are Nissl stains and immunohistochemistry with
antibodies directed against synaptophysin (Syn) and
MAP-II, as well as TUNEL stains. Scale bars: d, d', 50 µm (applies to all panels). The cortical layers are indicated as:
MZ, marginal zone; CP, cortical plate;
SP, subplate; IZ, intermediate zone. The
hippocampal structures are indicated as follows: St.O,
stratum oriens; P, CA1 pyramidal cells;
St.R, stratum radiatum; St.M, stratum
moleculare; DG, dentate gyrus granule cells. The
arrows mark apoptotic nuclei.
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Because APLP2 has been suggested to play a role in axon outgrowth in
the olfactory system, where it is highly expressed in olfactory sensory
neurons and axon terminal glomeruli (Thinakaran et al., 1995a), we
analyzed the olfactory system of both types of double mutants. However,
the distribution and density of neurons in the olfactory bulb, as well
as the number and thickness of neuron-specific tubulin-positive axon
bundles, were not found to be different between the lines studied (data
not shown). No differences were observed in size and nerve cell density
of brainstem nuclei (data not shown).
APP undergoes fast axonal trafficking to presynaptic terminals (Tienari
et al., 1996) and has been detected in rab5-containing (Ikin et al.,
1996) and clathrin-coated vesicles (Nordstedt et al., 1993), organelles
that are likely to be involved in endocytosis. To assess whether a lack
of APP family members affected synaptic architecture, we examined by
electron microscopy coronal sections of the cortex and the brainstem
from newborn double mutants and controls (see Fig.
5 for representative images). Inspection
of many electron micrographs revealed in both types of lethal double mutants (Fig. 5B,C) mature synapses in the brainstem, at
densities similar to that found in wt controls (Fig. 5A). In
contrast, predominantly immature synapses were detectable in the
developing cortices of these mice (data not shown). Again, no apparent
differences in synapse densities could be detected between sections
from wt and the lethal double mutants (data not shown). Also, no
obvious changes were found in the ultrastructure of the nerve terminals
(Fig. 5). We therefore conclude that, at least with regard to these morphological aspects, synapse formation proceeds apparently normal in
the brainstem of mutant animals. We also consider neuromuscular deficits as an unlikely cause of lethality in double mutants because some of us have addressed this question in a previous study
investigating APP/APLP2 double knock-outs (von Koch et al., 1997).
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Figure 5.
Ultrastructure of brainstem synapses from single
or combined mutants. Comparison of the morphology of synapses in
brainstem ultrathin sections from newborn mice revealed no obvious
changes in the ultrastructure of the nerve terminals in mutant compared
to wt mice. Shown are electron micrographs of representative active
zones obtained from wt (A),
APLP2//APP/
(B), and
APLP2//APLP1/
(C) samples processed as described in
Materials and Methods. At the presynaptic site mutants and control mice
showed comparably sized vesicle clouds, including docked vesicles in
close proximity to the membrane. Electron dense postsynaptic
specializations were clearly detectable in both double mutants.
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Viability of primary neuronal cultures derived from single and
combined mutants
Viability of cortical neurons derived either from
APLP2/
single mutants or from both types of
lethal double mutants
(APP//APLP2/
and
APLP1//APLP2/)
was investigated by counting viable neurons on gridded coverslips after
plating (DIV 1) and again after 7 d in culture (DIV 7; Fig. 6). No significant differences relative
to wt neurons were detectable for any of the mutants analyzed,
indicating that under the culture conditions used, loss of endogenous
APLP2 alone, or in combination with APLP1 (see Fig.
6A for
APLP1//APLP2/)
or with APP (see Fig. 6B for
APP//APLP2/),
respectively, does not affect neuronal survival. Moreover, we saw no
significant differences in plating efficiency of viable neurons between
wt and mutant cultures on DIV 1 (data not shown), indicating that lack
of APP family members did not affect the viability of major neuronal
subpopulations in these cultures.
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Figure 6.
Viability of cortical neurons from single or
combined mutants. Neuronal viability was similar to that of wt neurons
in APLP2/ single
mutants (A, B) or both types of double mutants.
A,
APLP2//APLP1/
double mutants. B,
APLP2//APP/
double mutants. Cortical neurons were obtained from
individual embryos, as described in Materials and Methods. Viable
neurons were counted using gridded coverslips once at DIV 1 (set as
100%) and again on DIV 7. For each embryo ~240-360 neurons were
counted, and two or three embryos were analyzed for each genotype.
Values represent mean neuron counts obtained for mice of the indicated
genotypes ± SEM, normalized to initial values on DIV 1.
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Survival of neuronal cultures derived from single or combined
mutants against excitotoxic stress
Studies by Mattson et al. (1993) and Mattson (1994) have
demonstrated that primary neuronal cultures can be protected from glutamate-induced excitotoxicity by addition of exogenous secreted forms of human APP (sAPP) to the culture medium. We have previously shown that APP-deficient mice are hypersensitive to seizures induced by
the glutamate receptor agonist kainate, although the amount of tissue
damage was similar to that observed for wt animals (Steinbach et al.,
1998). However, using primary neuronal cultures derived from APP mutant
mice, we and others (Steinbach et al., 1998; White et al., 1998) have
been unable to show differences in the glutamate sensitivity of
APP-deficient neurons. To unravel whether endogenous APP plays any role
in the protection of neurons against glutamate excitotoxicity and
whether the related APLPs may compensate for such a protection by APP,
we compared the survival of neurons derived from single APP family
mutants and from lethal mutants deficient for a combination of
APLP2/APP, or APLP2/APLP1, respectively. Two sets of experiments were
performed. We first investigated the response of neurons prepared from
single embryos obtained by intercrossing heterozygous
APP+//APLP2/
mice and as controls, neurons prepared from wt embryos of
129Sv(ev)xC57BL/6 matings (Fig.
7A,C). Second, we analyzed
neurons of embryos derived from intercrosses of
APLP1+//APLP2/
mice in comparison to wt neurons of 129Sv(ev)xC57BL/6 matings (Fig.
7B,D). Cortical neurons were used because sufficient numbers of cells can be obtained from individual cortices and moreover, this
cell type has previously been shown to be protected from glutamate
excitotoxicity by (hu)sAPP (Mattson et al., 1993). Cortical neurons
cultured under serum-free conditions for 15 d in vitro were treated with increasing amounts of glutamate (10-100
µM) for 24 hr, and their survival was assessed
by MTT assays. This resulted in a glutamate concentration-dependent
reduction in cell survival for all cultures examined. Neither
APLP2-deficient neurons nor
APP//APLP2/
double knock-out cultures differed,
however, significantly in their survival rates compared to wt control
neurons at any glutamate concentration used (Fig. 7B).
Likewise,
APLP1//APLP2/
double knock-out cultures showed similar
survival rates as
APLP2/
single knock-outs or wt control cultures
(Fig. 7A). To test for possible differences in the response
kinetics of the various mutants we investigated cell survival at 1, 3, and 24 hr after addition of a constant amount of 50 µM glutamate (Fig. 7C,D for 1 and 3 hr, Fig. 7A,B for 24 hr time point). Again, no significant
differences for both sets of mutants compared to wt neurons were found
at any time point investigated. As expected from these results, we were
also unable to detect significant differences when we tested the whole
panel of single mutants
APP/,
APLP2/,
and
APLP1/
relative to wt controls (data not shown).
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Figure 7.
Susceptibility of neurons from single or combined
mutants to glutamate excitotoxicity. Cortical neurons from single
mutants, heterozygotes, or double mu |
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TOP
ABSTRACT
INTRODUCTION
