From the a Garvan Institute of Medical Research, Darlinghurst, Sydney 2010, Australia, b Prince of Wales Medical Research Institute, Randwick 2031, Australia, c University of New South Wales, Sydney 2052, Australia, d Centre for Education and Research on Ageing, University of Sydney and Concord Repatriation General Hospital, Concord 2139, Australia, e Department of Human Genetics, Mount Sinai School of Medicine, New York, New York 10029, f Neurotec, Section of Experimental Geriatrics, Karolinska Institute, Stockholm S-141 86, Sweden, g Laboratory for Alzheimer's Disease, Brain Science Institute, Riken, Saitama 350-01, Japan, h Department of Pathology, University of Tasmania, Hobart, 7001, Australia, and i Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Received for publication, November 20, 2002
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ABSTRACT |
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The mutation L271V in exon 8 of the presenilin-1 (PS-1) gene was detected in an Alzheimer's
disease pedigree. Neuropathological examination of affected
individuals identified variant, large, non-cored plaques without
neuritic dystrophy, reminiscent of cotton wool plaques. Biochemical
analysis of L271V mutation showed that it increased secretion of the
42-amino acid amyloid- The presence of senile plaques is one of the key
neuropathological features of familial as well as sporadic forms of
Alzheimer's disease (AD).1
Senile plaques are extracellular deposits composed mainly of the
amyloid- The range of functions of the wild-type PS-1 protein are still
being defined, although a primary function may be to serve as the
Clinical Description--
The Tas-1 family includes 13 affected
individuals over three generations in a pattern consistent with
autosomal dominant inheritance (Fig. 1A). The clinical
features and disease progression in the five affected
individuals2 were consistent
with AD, with initial difficulties in activities of daily living
followed by progressive memory, language, and visuospatial deficits
leading to severe dementia over a span of several years. All developed
myoclonus late in their illness. In contrast to some other families
with variant plaques (5, 6, 8), none of the members examined had
spastic paraparesis, although individual III.30 had pathologically
brisk reflexes. The onset of symptoms ranged from 43 years of age to
the early sixties (mean, 49 years); the age at death ranged from 52 to
65 years, with one affected individual (III.22) still living at 68 years.
Neuropathology--
The brains of two affected pedigree members
were obtained at autopsy for neuropathological examination (17).
Standardized neuropathological criteria were used for diagnosis (18,
19).
Genetic Analyses--
Intronic polymerase chain reaction
(PCR) primers were used for amplification of PS-1 gene (20). PCR
products were sequenced using Big Dye chemistry on an ABI377 sequencer
(Applied Biosystems). Linkage analysis of the Tas-1 pedigree was
performed using the MLINK program (21). A mutant allele frequency of
0.01 was used in the analysis.
RT-PCR Analysis of PS-1 mRNA--
Total RNA was extracted
from cell lines or frozen brain tissue using the SV Total RNA Isolation
System (Promega). 2 µg of RNA was reverse-transcribed using the
Superscript II RT enzyme (Invitrogen) and a poly(dT) primer
(Invitrogen) followed by PCR amplification using the primers
PS1exon7F( Exon Trapping Analysis--
PCR products, which contained either
the wild type or the L271V mutation in exon 8 along with 62 and 99 bp
of 5' and 3' flanking intronic sequence, were subcloned into the exon
trap vector pSPL3 (Invitrogen). HEK293 and COS-7 cells were transfected
with the exon trap constructs using the Fugene 6 reagent (Roche
Molecular Biochemicals). Cells were collected 48 h
post-transfection, and total RNA was isolated for exon trap analysis
(17).
Construction of PS-1 Expression Plasmids--
The full-length
PS-1 cDNA with the alternatively spliced VRSQ motif was expressed
in the mammalian expression vector, pRc/CMV (Invitrogen). The missense
mutation L271V was introduced into the PS-1 cDNA by
oligonucleotide-directed mutagenesis. The PS-1 Detection of A Metabolic Labeling and Immunoprecipitation of PS-1 Isoforms in
Transfected COS-7 Cells--
COS-7 cells in six-well plates were
transfected with PS-1 cDNAs as described above. After 48 h,
cells were starved for 2 h in methionine- and serum-free COS
medium. Cells were metabolically labeled with 200 µCi of
[35S]methionine for 30 min and then chased with COS
medium with excess unlabeled methionine (0.8 mM) for 4 or
6 h (25). Cells were lysed in 1× lysis buffer (50 mM
Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM
phenylmethylsulfonyl fluoride, 1× complete mixture protease inhibitor
(Roche Molecular Biochemicals)) and 0.01% Tween 20 and immunoprecipitated as described (11) using a rat monoclonal antibody
against the amino terminus of PS-1 (Chemicon). Protein lysates were
heated to 50 °C for 10 min prior to electrophoresis on a 10%
SDS-PAGE and transfer to a nitrocellulose membrane (Trans-blot transfer
medium, Bio-Rad).
Western blot Analysis of PS-1 Isoforms--
COS-7 cells were
transfected with PS-1 cDNAs as described above. After 48 h,
cells were pelleted and lysed in 1× lysis buffer and 0.1% Triton
X-100. Crude membrane extracts of brain tissue were also made (11).
Protein lysates were heated to 50 °C for 10 min prior to
electrophoresis on a 7.5% SDS-PAGE and transfer to a nitrocellulose
membrane. A rat monoclonal antibody (Chemicon), a mouse monoclonal
antibody (NT1) raised against residues 41-49 of PS-1 (26), and a
rabbit polyclonal antibody (Ab14) raised against residues 1-25 of PS-1
(27) were used to detect PS-1 protein. Co-immunoprecipitation
experiments to detect interactions between different PS-1 isoforms and
Tau or GSK-3 PS-1 Mutation (L271V) in Tas-1 Pedigree--
An affected
individual (III:28) was examined for nucleotide substitutions in the
PS-1 gene. As shown in Fig.
1B, the electropherogram traces indicate a C to T mutation that substitutes a valine for a
leucine at codon 271 (L271V). This mutation is situated within exon 8 of the PS-1 gene. An additional 17 pedigree members, including four
affected individuals, were examined for the presence of the L271V
mutation. The affected individuals were found to carry the mutation,
consistent with L271V being the pathogenic mutation within this
pedigree. Linkage analysis of the co-segregation of the L271V mutation
and the disease phenotype revealed a positive logarithm of
odds ratio; score (LOD = 1.8, Variant Neuropathology--
Two affected members of the
pedigree were examined neuropathologically. Subject III:28 died of
pulmonary emboli at the age of 60 years, whereas III:32 died of
bronchopneumonia aged 57. Macroscopically, both brains had considerable
atrophy of the temporal and posterior white matter with enlargement of
the lateral ventricles and narrowing of the corpus callosum. Subject
III:28 had additional atrophy of the frontal and superior temporal gyri
with some loss of white matter in the centrum semiovale. The locus
coeruleus was depigmented in both cases. Microscopically, the most
dramatic pathology was the large number of neocortical plaques,
which resulted in a CERAD (Consortium to Establish a Registry
for Alzheimer's Disease) pathology grading of severe. These plaques
were particularly prominent on the sections immunohistochemically
stained for A Biochemical Analysis of the L271V Mutation--
To determine the
effects of the PS-1 (L271V) mutation on the processing of APP,
wild-type and mutant forms of the PS-1 cDNA were transiently
co-transfected with the APP(Swedish) cDNA into COS-7 cells, and the
amount of secreted A
Specific mutations in the APP gene have been shown to alter the
processing of APP to preferentially generate P3, the peptide cleaved at
position 17 (A L271V Mutation Increases Splicing Out of PS-1 Exon 8--
Exonic
mutations have previously been shown to alter the level of splicing of
their cognate exons (31, 32). We examined RNA transcripts extracted
from the brains of two mutation carriers (III:28 and III:32) for the
presence of PS-1 splice isoforms by RT-PCR using primers that flanked
exon 8. The level of the splice isoform was determined
semiquantitatively (Fig. 4A).
PS-1 RNA transcripts from the two affected mutation carriers were found to have ~17-50% increased levels of PS-1
Exon trapping analysis (17) was used to examine the effects
of the L271V mutation on the efficiency of splicing of PS-1 exon 8. Reverse transcription-PCR of exon trap products yielded two PCR
products, which correspond to either the splicing in or the deletion of
exon 8 (
To confirm that PS-1 Functional Analyses of PS-1
The PS-1
Measurement of total A
Deletion mutants of PS-1 have defined a region spanning amino acids 250 to 298 that is thought to bind to both Tau and GSK-3 We describe an EOFAD pedigree with a novel missense (L271V)
mutation in the PS-1 gene that is associated with a variant
neuropathology. Bielschowsky silver stain revealed that the majority of
plaques in two affected individuals (III:28 and III:32) were large
spherical deposits without defined cores or neuritic dystrophy (Fig. 2, C and D). The morphology of the plaques is
reminiscent of the cotton wool plaques described in other studies
(5-8). Two lines of evidence demonstrate that the PS-1 L271V mutation
is the pathogenic variant within the Tas-1 pedigree. First, the
mutation segregates with the EOFAD phenotype within the pedigree.
Second, in vitro measurements of A The correlation between specific mutations in the APP and PS-1 genes
and the distinctive neuropathology in affected individuals has led to
the definition of two possible molecular mechanisms in the formation of
cotton wool plaques. First, the APP T714I was postulated to play an
integral role in the formation of the variant cotton wool form
of senile plaques, through the generation of P3 and the amino-terminal
truncated A There has been debate concerning whether PS-1 is the actual
A key feature of the cotton wool plaques is the lack of
neuritic dystrophy. Neuritic degeneration may depend on the level of
Tau hyperphosphorylation (40), and GSK-3 Cotton wool plaques have been associated in some AD pedigrees
with the neurological disorder, spastic paraparesis (5, 6). However, it
has been suggested that both cotton wool plaques and spastic
paraparesis are variably expressed in these pedigrees as the outcome of
phenotypic modifiers (6). Accordingly, some PS-1 The EOFAD family, Tas-1, offers a unique opportunity to study the
secondary processes after the initial overproduction of A peptide, suggesting a pathogenic mutation.
Analysis of PS-1 transcripts from the brains of two mutation carriers
revealed a 17-50% increase in PS-1 transcripts with deletion of exon
8 (PS-1
exon8) compared with unrelated Alzheimer's disease brains.
Exon trapping analysis confirmed that L271V mutation enhanced the
deletion of exon 8. Western blots of brain lysates indicated that
PS-1
exon8 was overexpressed in an affected individual. Biochemical
analysis of PS-1
exon8 in COS and BD8 cells indicate the splice
isoform is not intrinsically active but interacts with wild-type PS-1
to generate amyloid-
. Western blots of cell lysates
immunoprecipitated with anti-Tau or anti-GSK-3
antibodies indicated
that PS-1
exon8, unlike wild-type PS-1, does not interact directly
with Tau or GSK-3
, potential modifiers of neuritic dystrophy. We
postulate that variant plaques observed in this family are due in part
to the effects of PS-1
exon8 and that interaction between PS-1 and
various protein complexes are necessary for neuritic plaque formation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
peptide (A
), which is cleaved by a series of secretases from the amyloid precursor protein (APP) (1). Mutations in any of three
genes, APP, presenilin-1 (PS-1), or presenilin-2 (PS-2), give rise to
early onset familial AD (EOFAD). Mutations in the PS-1 gene are
associated with severe neuropathology. Typically, the brains of
affected individuals have a large number of diffuse as well as cored
neuritic plaques that are deposited in the cerebral cortex and in
regions not normally involved in AD, such as the cerebellum (2).
Moreover, there is intense Tau pathology with neuritic dystrophy around
the plaques and neurofibrillary tangles (3, 4). However, four mutations
in PS-1, an in-frame deletion of the exon 9 sequence (PS-1
exon9) (5,
6), a two-amino acid deletion (
I83/
M84) (7), a P436Q (8),
and a E280G (9), have been shown to be associated with a variant
"cotton wool" plaque pathology. Brains from individuals carrying
these specific mutations have extensive deposition of large spherical plaques that lack distinctive cores and neuritic pathology. Biochemical analysis of cells transfected with PS-1 cDNAs, carrying any of these mutations, secrete exceptionally high levels of A
1
42 (8). This suggests that PS-1 has a role not only in determining the levels
of different A
species but also in the morphology of the plaques and
neuritic dystrophy.
-secretase, which cleaves the APP molecule to generate A
1-40 and
A
1-42 (10). PS-1 has also been shown to interact with various
proteins, including Tau (10), GSK-3
(11, 12), and nicastrin (13).
Immunohistological examination of sporadic and familial AD brains
reveals that PS-1 protein is associated with the amyloid cores of the
senile plaques and dystrophic neurites (14, 15), as well as
neurofibrillary tangles, the other neuropathological feature of AD (15,
16). Whether protein interactions with PS-1 are actively required for
plaque formation, or neuritic dystrophy, has not been demonstrated. We
report the genetic and functional analysis of a novel mutation (L271V)
in the PS-1 gene in an EOFAD pedigree with a variant neuropathology.
The missense mutation also results in the production of PS-1 that lacks
exon 8. We show that exon 8 sequences of the PS-1 molecule interact
with Tau and GSK-3
, a candidate protein kinase that can catalyze the
phosphorylation and polymerization of Tau and perhaps initiate neuritic
dystrophy. These results suggest that the presence of mutant PS-1 plays
a major role in the development of neuritic senile plaques.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
exon8) (5'-TCCCTGAATGGACTGCGTGGCTC-3') and
PS1exon9R(
exon8) (5'-CTTTGAGCTTCCGGGTCTCCTTC-3'). The relative ratio
of PCR products with and without exon 8 sequences was determined semiquantitatively by PCR amplification using 0.2 µg of cDNA
template and 33P end-labeled PS1exon7F(
exon8) primer
(17).
exon8 construct was
made by RT-PCR amplification using RNA isolated from an affected
pedigree member as was the PS-1
exon 9 construct (6).
Species--
PS-1 cDNA was
co-transfected with a second construct, which expresses the 751-amino
acid isoform of APP carrying the Swedish mutation, pCMV-APP(Swedish)
(22) into COS-7 cells using the Fugene 6 reagent. Cells were left to
recover for 48 h. For ELISA of full-length A
species, medium
was removed and replaced with 700 µl of fresh growth medium per well
(six-well plate), and conditioned medium was collected after 24 h.
200 µl of each conditioned medium sample was used to assay for
secreted A
1-40 and A
1-42 using the SignalSelect
-amyloid
ELISA kits (BIOSOURCE Int.). For the assay of
truncated A
species, transfection medium was removed and replaced
with 2 ml of serum-reduced growth medium (0.2% fetal calf serum)/10-cm
plate, and conditioned medium was collected after 24 h. 500 µl
of conditioned medium was assayed for the presence of A
species by
immunoprecipitation/mass spectrometry (IP-MS) A
assay (23). For
the luciferase-based assay of
-secretase activity in BD8 (PS-1/PS-2
knockout) cells, PS-1 cDNAs were transiently transfected into cells
and assayed for activity after 24 h as described (24).
were performed as described previously (11).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
= 0).
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Fig. 1.
A, pedigree of Tas-1 family showing the
autosomal dominant inheritance of EOFAD. Affected individuals are
indicated by filled symbols. Genotyped individuals are
indicated by asterisks. B, electropherogram
traces of nucleotide sequence from exon 8 of PS-1 gene. Affected
individuals III:28 and III:32 have a C to G nucleotide substitution,
which is absent in an unaffected individual, III:21.
(Fig. 2, A
and B) but were also visible as large diffuse plaques without neuritic dystrophy in the silver-stained sections (Fig. 2,
C and D). These were reminiscent of the cotton
wool plaques described in the Finnish EOFAD pedigree (5). In III:28,
all plaques in all regions were diffuse and without neuritic dystrophy or cores (Fig. 2, A and C), whereas neuritic and
cored plaques were observed in III:32 in the hippocampus (Fig.
2F). However, the large diffuse cotton wool plaques were the
most numerous plaque type in both cases (Fig. 2, A and
B). Despite the lack of neurites within the plaques, both
cases had significant numbers of neurofibrillary tangles using either a
silver stain or Tau immunohistochemistry (Fig. 2, E-H).
These cases reached a neocortical stage for tangle formation (19),
substantiating their clinical diagnosis as AD. Amyloid angiopathy was
also prominent in both cases.
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Fig. 2.
Photomicrographs of cortical sections from
the right hemisphere of III:28 (A, C,
E, G) and the left hemisphere of III:32
(B, D, F, H). The
scale shown in B is equivalent for A and
B, the scale in D is equivalent for C
and D, and the scale in H is equivalent for
E-H. A and B, section of the temporal
neocortex immunohistochemically stained with A antibodies. Large
numbers of A
deposits were seen throughout the neocortex.
C and D, higher magnification of the temporal
neocortex stained using the Bielschowsky silver technique. The majority
of neocortical plaque deposits in both cases was of the diffuse cotton
wool type without cores or neuritic dystrophy. E and
F, silver-stained section through the hippocampal CA1 region
showing neurofibrillary tangles in both cases. Note the neuritic
dystrophy within the plaques in this region in III:32 and the classical
cored plaque at the top of the section (F). This
case (F) appeared to have more substantial neuronal loss as
compared with III:28 (E). G and H,
section through the CA1 region of the hippocampus immunohistochemically
stained with Tau II antibodies. A large proportion of neurons,
in both cases, contained fibrillar Tau deposits. Note also the
substantial numbers of Tau-positive neurites within the hippocampus in
case III:32 (H).
was measured by ELISA. The L271V mutation
resulted in a significant 1.3-fold increase (p < 0.05, Student's t test) in the secretion of A
1-42 compared with the wild-type sequence (Table I), in
the same manner as another pathogenic missense mutation, L286V (28),
which also showed a 1.3-fold increase in A
1-42 levels. Our assay
also demonstrated that the PS-1
exon 9 mutation results in an
exceptionally high, 2-fold increase in production of A
1
42
compared with other PS-1 mutations (8) (Table I).
A secretion induced expression of by PS-1 cDNAs
17-40 and A
17-42), as well as a series of
amino-terminally truncated A
species (29, 30). To determine whether
the PS-1 L271V mutation altered processing of APP in a similar manner,
the conditioned media from triplicate transfections were analyzed for
the presence of the major P3 species or any truncated A
species by
IP-MS (23). As shown in Fig. 3, PS-1 L271V mutation did not result in the generation of P3. Similarly, P3
was undetectable in the conditioned media of cells transfected with the
PS-1
exon 9 cDNA. Our assays were capable of detecting the
full-length A
species corresponding to A
1-40 and A
1-42 in
all of the PS-1 transfections. Moreover, the ratios of
A
1-42/A
1-40 were consistent with the results obtained using an
ELISA, with all mutant PS-1 cDNAs increasing the secretion of
A
1-42 compared with the wild-type PS-1 (Table I).
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Fig. 3.
Representative mass spectrometry traces
of A species from conditioned media
of COS-7 cells transfected with PS-1 cDNAs. A
species are
indicated by amino acid size. Significant peaks corresponding to
carboxyl-terminal truncated A
species are indicated by
vertical arrows.
exon8 compared with RNAs isolated from two unrelated EOFAD brains (which do not have mutations in the PS-1 gene) and the neuroblastoma cell line SK-N-MC.
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Fig. 4.
A, semiquantification of
RT-PCR-amplified PS-1 transcripts with and without exon 8. RNAs used
for each reaction were isolated from the SK-N-MC cell line and the
brain cortex of two unrelated EOFAD patients without PS-1 mutations
(EOFAD1 and EOFAD2), III:28, and III:32. Mean
values (±S.E.) were obtained from triplicate RT-PCR reactions.
B, upper panel, electrophoresis of exon trap
products on a 2% agarose gel. Exon trapping was performed in two cell
lines, HEK293 and COS-7, transfected with the parental pSPL3 vector
(vec) or constructs carrying either genomic fragment with
wild-type PS-1 exon 8 sequence (wt) or with the L271V
mutation. The 177-bp product corresponds to the deletion of PS-1 exon 8 sequence, and the 275-bp product corresponds to the inclusion of exon
8. Lower panel, semiquantitative analysis of exon trap
products isolated from cells transfected with either the pSPL3 vector
carrying the wild-type (open bars) or L271V nucleotide
substitution (hatched bars) genomic fragment. Mean values
(±S.E.) were obtained from three separate transfections. Pairwise
Student's t test comparisons were performed between
wild-type and L271V exon trap products. Statistical significance is
indicated (* = p < 0.05). C, Western blot
analysis of PS-1 in transfected COS-7 cell and human brain lysates
using the NT1 antibody. Approximately 100 µg of total protein was
loaded in each lane. PS-1 exon8 (arrow) is detected as a
47-kDa molecule as compared with the 50-kDa wild-type full-length
(FL) and amino-terminal fragment (NTF).
Nonspecific immunoreactive bands are indicated (*).
exon8) (Fig. 4B). In both the HEK293 and COS-7
cell lines, the presence of the L271V mutation resulted in
significantly more of the
exon8 product compared with wild-type sequence (Fig. 4B). The increase in exon trap
exon8 PCR
products for the L271V mutation was determined semiquantitatively in
both the HEK293 and COS-7 cells and was shown to be significantly
increased by 35 and 70% (p < 0.05, Student's
t test), respectively, compared with wild-type sequence.
exon8 was overexpressed in the brain of III:32,
we performed Western blot analysis on the membrane fractions of
proteins extracted from frontal cortical brain tissue in
addition to COS-7 cells transfected with PS-1 cDNAs. As shown in
Fig. 4C, a distinct 47-kDa polypeptide corresponding to
PS-1
exon8 was detected in the COS-7 cell lysate (transfected with
the PS-1
exon8 cDNA) and in the brain extracts of EOFAD2 and
III:32 as detected by Western blot analysis using the NT1 antibody. The
47-kDa band can be distinguished from a 50-kDa band, which corresponds
to the full-length wild-type PS-1 molecule. The same size bands were also detected when either the rat monoclonal PS-1 antibody or rabbit
polyclonal PS-1 antibody (Ab14) were used for Western blotting (data
not shown). Nonspecific bands (Fig. 4C) were detected in the
brain extracts when the NT1 antibody was used but were not detected
with the other two PS-1 antibodies.
exon8--
We examined the stability
of the PS-1
exon8 isoform in metabolically labeled COS-7 cells
transfected with PS-1 cDNAs. The PS-1
exon9 molecule is normally
rapidly degraded via the proteosome pathway (25). As shown in Fig.
5, A and B, newly
synthesized full-length PS-1 molecules, uncleaved by the presenilinase,
are rapidly degraded over a span of several hours. However,
PS-1
exon8 appeared to be the most stable of the PS-1 isoforms. Six
hours after the cells were pulse-labeled, there was a 1.2-1.6-fold
increase in the PS-1
exon8 isoform compared with wild-type
(p = 0.32, Student's t test) and
PS-1
exon9 isoforms (p < 0.05, Student's
t test), respectively.
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Fig. 5.
A, autoradiograph of pulse-chased PS-1
protein immunoprecipitated with a rat monoclonal antibody against PS-1
and electrophoresed on a 10% SDS-PAGE. Cells were harvested
after 4 h. B, quantification of the degradation of
pulse-labeled wild-type (diamond), PS-1 exon8
(squares), and PS-1
exon9 (triangles) isoforms.
Values were normalized to total PS-1 protein at the 0 h time point
by Western blotting using the Ab14 antibody. Error bars were
derived from five independent experiments for the 4-h time point and
three independent experiments for the 6-h time point. ELISA
quantification of total A
in COS-7 cells transfected with PS-1
cDNAs is shown. Error bars are derived from eight
separate transfections. D, a luciferase-based quantification
of
-secretase activity in BD8 cells transfected with PS-1 cDNAs.
Error bars are derived from three independent
experiments with triplicates of each transfection.
exon8 splice isoform did not have an effect on the secretion
of A
1-42 compared with wild-type cDNA when measured using an
ELISA of transiently transfected cells (Table I). This finding is in
agreement with previous studies of the effect of PS-1
exon8 in stably
transfected cell lines (33, 34). However, when we examined all of the
A
species in the conditioned media using IP-MS, we found a small
increase in the ratio of A
1
42/1-40 in the cells transfected with
PS-1
exon8 (0.19) compared with the wild-type cDNA (0.14) (Table
I). Moreover, two new peaks were detected in the mass spectra that
correspond to carboxyl-terminally truncated A
species, A
1-19 and
A
1-28. These A
species were not detected in the conditioned
media of cells transfected with wild-type, L271V, or PS-1
exon9
cDNAs (Fig. 3).
in COS cells transfected with either a
lacZ control vector or PS-1 cDNAs was estimated
by a combined ELISA of A
1-40 and A
1-42. As shown in Fig.
5C, PS-1
exon8, in the same manner as the other PS-1
cDNAs, significantly increased A
secretion compared with cells
expressing only endogenous PS-1 (which correspond to the
lacZ control). This is similar to the results reported for
stably transfected cell lines (34) and suggests that PS-1
exon8 was
capable of supporting the generation of A
. To determine whether
PS-1
exon8 has intrinsic
-secretase activity, the splice
isoform was transfected into BD8 (PS-1/PS-2 knockout) cells
(24). In contrast to COS cells, which contain endogenous PS-1, the BD8
cells were unable to support the generation of A
in the presence of
PS-1
exon8, although the wild-type and mutant PS-1 cDNAs restored
this activity to levels similar to that observed in COS cells with
endogenous presenilins (Fig. 5D). Moreover, we noted, for
the mutant PS-1 cDNAs, that an increase in the production of
A
1-42 (Table I) was associated with an overall decrease in the
secretion of total A
(Fig. 5, C and D).
(11) and
includes the sequence encoded by exon 8 (amino acids 257-289). Thus,
the splice isoform PS-1
exon8 would not be expected to interact with
either Tau or GSK-3
. We examined whether this was the case for
GSK-3
by co-immunoprecipitation of lysates from cells transfected
with PS-1 cDNAs. As shown in Fig.
6A, wild-type PS-1 was
detected by Western blot analysis in lysates immunoprecipitated with an
anti-GSK-3
antibody. In contrast, no immunoprecipitated PS-1
exon8
protein was detected. In the same manner, the PS-1 deletion mutant,
which lacks amino acids 251-467 (PS-1 N250), also failed to be
immunoprecipitated by the anti-GSK-3
antibody. A similar result was
obtained for the co-immunoprecipitation experiment between PS-1 and Tau
(Fig. 6B). Finally, the co-immunoprecipitation experiment
was repeated to determine whether PS-1
exon9, which lacks amino acids
residues 290-319, had the same effect as the PS-1
exon8 isoform. As
shown in Fig. 6C, PS-1
exon9 did co-immunoprecipitate with
GSK-3
. The binding affinity between the PS-1 protein and GSK-3
can be estimated semiquantitatively by the difference in levels of
protein detected in the crude lysate and the immunoprecipitate fractions. Thus, PS-1
exon9, like PS-1
exon8, appears to bind with
lesser affinity to GSK-3
than wild-type PS-1 (Fig.
6C).
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Fig. 6.
A, co-immunoprecipitation of PS-1 and
GSK-3 in lysates from COS-7 cells transfected with PS-1 cDNAs.
Western blot analysis was used to detect PS-1 protein present in 5% of
crude lysate and the GSK-3
immunoprecipitate (IP).
Full-length uncleaved PS-1 protein was detected in the lysate, and
immunoprecipitate was obtained from cells transfected with the
wild-type (wt) PS-1 cDNA (arrow). A smaller,
uncleaved PS-1 protein is observed in the lysate of cells transfected
with the PS-1
exon8 cDNA but not in the corresponding
immunoprecipitate. vec, lacZ control
vector. B, uncleaved PS-1 (arrow) is also
detected in lysates of cells transfected with wild-type, but not
PS-1
exon8 cDNA, when immunoprecipitated with a monoclonal
antibody against Tau. C, decreased detection of PS-1
exon9
protein in the anti-GSK-3
immunoprecipitate compared with the amount
present in the lysate (arrow). Nonspecific immunoglobulin
bands are indicated (*).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
levels using either
an ELISA or by IP-MS indicate that the L271V mutation increases
A
1-42 secretion to comparable levels to other characterized PS-1
mutations. However, the simple elevation of A
1-42 secretion does
not explain the presence of the variant plaques that occur in this pedigree.
species (29). However, we did not detect the presence of
P3 in conditioned media from cells transfected with the L271V cDNA.
Furthermore, we were also unable to detect the presence of the
truncated A
species in cells transfected with the PS-1
exon9
cDNA, another PS-1 mutation associated with cotton wool plaques (5,
6). This suggests that, unlike the APP T714I mutation, cotton wool
plaques found in PS-1 L271V mutation carriers are not attributable to
the production of P3. The second mechanism, proposed by Houlden
et al. (8), for the formation of the cotton wool plaques is
a high level of A
1-42 production such as observed for the
PS-1
exon9 mutation. However, levels of A
1-42 secretion induced
by the PS-1 L271V mutation were not as high as those seen with the
PS-1
exon9 mutation (Fig. 3). This suggests that although the L271V
mutation is necessary for the generation of the cytotoxic A
1-42
species, biochemical analyses indicate that the L271V mutation was not
sufficient to explain the presence of the cotton wool plaques in our
pedigree, at least by the two mechanisms proposed above. Exonic
mutations can have the unexpected consequence of altering the splicing
of their cognate exons. For example, the exonic deletion
K280
mutation in the tau gene, MAPT
(microtubule-associated protein
Tau) have been shown to decrease the splicing in of exon 10 of the gene due to the disruption of a purine-rich exon splicing
enhancer (32). We demonstrate that the L271V mutation has a similar
effect on the alternative splicing of exon 8 of the PS-1 gene, possibly by a similar mechanism. RT-PCR and Western blot analysis of PS-1 expression in the frontal cortex of the two mutation carriers (III:28
and III:32) revealed a 17-50% increase in levels of PS-1
exon 8 splice isoform compared with unrelated EOFAD brains and a neuroblastoma cell line (Fig. 4, A and C). The
exon8 isoform
is not proteolytically cleaved like the wild-type molecule (33, 34),
which may effect its stability in vivo. This
hypothesis was supported by our pulse-chase study of PS-1 isoforms,
which demonstrated that PS-1
exon8 was not as rapidly degraded as
either the wild-type or PS-1
exon9 isoform (Fig. 5B).
Thus, the modest increase in the level of PS-1
exon8 transcripts
would lead to accumulation of higher levels of the
exon8
isoform than the full-length wild-type molecule (Fig. 4C), and have functional consequences.
-secretase catalytic enzyme or whether PS-1 forms part of a larger heteromeric complex that facilitates the positioning of
-secretase and its substrates (35). Our analysis of
-secretase activity associated with transfected PS-1 cDNAs using ELISA or the
luciferase-based reporter system (Fig. 5, C and
D) may provide some insight into this debate. First, the
ELISA measurements of
-secretase activity of the L271V and
PS-1
exon9 mutants indicated that an increase in the secretion of
A
1-42 was associated with a concomitant decrease in the production
of total A
(which consists mostly of A
1-40) (Fig. 5,
C and D). This suggests that the mutations
resulted in an alteration of PS-1 activity, such that the mutant PS-1
was more efficient in cleavage of A
1-42 than A
1-40. This is
supported by the nonsteroidal anti-inflammatory drug, sulindac sulfide, which has been shown to inhibit selectively the production of A
1-42
while increasing the carboxyl-terminal truncated A
1-38 species
(36). The ELISA indicated that PS-1
exon8 might be capable of
supporting the generation of A
(Fig. 5C), consistent with a similar study using stably transfected HEK cell lines (34). To remove
the effects of endogenous presenilins on the ability of the PS-1
cDNAs to generate A
, this experiment was repeated in PS-1/PS-2
knockout cells (24). As shown in Fig. 5D, the PS-1
exon8 isoform was unable to support the generation of A
. We inferred that
in the COS-7 cells, PS-1
exon8 must form oligomers with endogenous PS-1 to give rise to the modified
-secretase activity. This
hypothesis is supported by studies showing that oligomeric PS-1 is part
of a high molecular weight complex in vivo (37) and that
PS-1 molecules exist as homodimers with direct interactions between
adjacent PS-1 amino-terminal fragments (38). The mechanism by which the unusual carboxyl-terminally truncated A
species (A
1-19 and
A
1-28) are generated when PS-1
exon8 is transfected into
wild-type cells (Fig. 3) remains unknown. However, a recent IP-MS study
using a series of specific
-secretase inhibitors demonstrated that there was a wide number of apparently authentic cleavage sites for the
enzyme, ranging from position 19 to 42 (39). The issues outlined above
are unlikely to be resolved until the
-secretase activity can be
reconstituted in vitro.
appears to play a crucial
role in this process, as GSK-3
conditional transgenic mice displayed
increased Tau hyperphosphorylation (41). PS-1 has been proposed to
interact with GSK-3
(11, 12) and Tau (11). In addition, PS-1 is
co-localized to neuritic processes in AD brains (14-16). These studies
consistently suggest a vital role for PS-1 in influencing the neuritic
activity surrounding the senile plaques by bringing together GSK-3
and its substrate Tau during the formation of the plaques. Our
co-immunoprecipitation experiments demonstrate that the PS-1
exon8
splice isoform did not interact with GSK-3
or Tau (Fig. 6). This
finding is supported by the observation that PS-1
exon9,
another mutation associated with plaques that lack neuritic dystrophy
(5), also appears to have a lower affinity for GSK-3
as assessed by
co-immunoprecipitation (Fig. 6C). Thus, we propose that the
lack of neuritic dystrophy observed in the senile plaques of affected
individuals within this pedigree (III:28 and III:32) is due to the
increased levels of the PS-1
exon8 molecule. It would be of interest
to determine whether the variable levels of neuritic dystrophy
displayed in other pedigrees (6) might correlate with levels of
PS-1
exon8.
exon9 pedigrees have
been reported to lack cotton wool plaques or spastic paraparesis (42,
43). The Tas-1 family is of interest because there is no indication of
spastic paraparesis in the mutation carriers. The relationship of the
phenotype to the specific nature of the mutation remains to be
determined. The presentation of the neuropathology also differs from
other pedigrees with cotton wool plaques. In several pedigrees with
PS-1
exon9 mutations, the distribution of cored neuritic plaques and
cotton wool plaques was fairly even (5, 6). In the Tas-1 pedigree,
cored neuritic plaques were found only in the hippocampus of one
patient. This distribution of cored and cotton wool plaques is similar
to that described in a patient with an APP T714I missense mutation
(27). However, the biological significance of this distribution
of neuritic dystrophy remains unknown and may simply reflect the usual
relatively high intensity of Tau pathology of the hippocampus in AD brains.
species
by mutant PS-1. Our data provides the first example of a pathogenic
PS-1 mutation that generates a molecule that cannot support A
production on its own but causes aberrant carboxyl truncated A
species to be generated when endogenous wild-type presenilins are
present. Our data suggest a central role for PS-1 not only in
determining the overall level of A
but also in specifying the
morphology of plaques and neuritic dystrophy. Moreover, identification of factors that affect the splicing of PS-1 may also be of importance in understanding the pathogenesis of AD, as the PS-1
exon8 isoform has biochemical properties that differ from those of wild-type, full-length PS-1. Correlation of the specific neuropathology of AD
cases with genetic mutations will lead to a better understanding of the
role that PS-1 plays in the disease process.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Heather McCann and Heidi Cartwright for laboratory assistance. We are grateful to Dorit Donoviel for providing the BD8 cells. We thank Sam Sisodia, Jeff Nye, Peter St. George-Hyslop, Christian Haass, and Mike Wolfe for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by Department of Veteran Affairs (Australia) Grant 9937441 (to J. B. J. K., W. S. B., and P. R. S.), National Health and Medical Research Council (Australia) grants, Project Fellowship Grant 113804 (to G. M. H.), Block Grant 993050 (to P. R. S.), Network Grant 983302 (to J. B. J. K., W. S. B., and P. R. S.), and National Institutes of Health Grant NIA AG10491 (to S. E. G. and R. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
j To whom correspondence should be addressed: Garvan Institute of Medical Research, 384 Victoria St., Darlinghurst, Sydney, New South Wales 2010, Australia. Tel.: 61-2-9295-8285; Fax: 61-2-9295-8281; E-mail: p.schofield@garvan.org.au.
Published, JBC Papers in Press, December 19, 2002, DOI 10.1074/jbc.M211827200
2 Subjects were examined by W. S. Brooks.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
AD, Alzheimer's
disease;
A, amyloid-
protein;
APP, amyloid precursor protein;
EOFAD, early onset familial AD;
ELISA, enzyme-linked immunosorbent
assay;
GSK, glycogen synthase
kinase;
PS-1 and -2, presenilin-1 and -2;
IP-MS, immunoprecipitation-mass spectrometry;
RT-PCR, reverse
transcription-PCR.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Ray, W. J., Ashall, F., and Goate, A. M. (1998) Mol. Med. Today 4, 151-157[CrossRef][Medline] [Order article via Infotrieve] |
2. | Lemere, C. A., Lopera, F., Kosik, K., Lendon, C. L., Ossa, J., Saido, T. C., Yamaguchi, H., Ruiz, A., Martinez, A., Madrigal, L., Hincapie, L., Arango, J. C., Anthony, D. C., Koo, E. H., Goate, A. M., and Selkoe, D. J. (1996) Nat. Med. 2, 1146-1150[Medline] [Order article via Infotrieve] |
3. | Smith, M. J., Gardner, R. J. M., Knight, M. A., Forrest, S. M., Beyreuther, K., Storey, E., McLean, C. A., Cotton, R. G. H., Cappai, R., and Masters, C. L. (1999) NeuroReport 10, 503-507[Medline] [Order article via Infotrieve] |
4. |
Singleton, A. B.,
Hall, R.,
Ballard, C. G.,
Perry, R. H.,
Xuereb, J. H.,
Rubinztein, D. C.,
Tysoe, C.,
Mathews, P.,
Cordell, B.,
Kumar-Singh, S., De,
Jonghe, C.,
Cruts, M.,
van Broeckhoven, C.,
and Morris, C. M.
(2000)
Brain
123,
2467-2474 |
5. | Crook, R., Verkkoniemi, A., Perez-Tur, J., Mehta, N., Baker, M., Houlden, H., Farrer, M., Hutton, M., Lincoln, S., Hardy, J., Gwinn, K., Somer, M., Paetau, A., Kalimo, H., Ylikoski, R., Poyhonen, M., Kucera, S., and Haltia, M. (1998) Nat. Med. 4, 452-455[Medline] [Order article via Infotrieve] |
6. | Smith, M. J., Kwok, J. B. J., McLean, C. A., Kril, J. J., Broe, G. A., Nicholson, G. A., Cappai, R., Hallupp, M., Cotton, R. G. H., Masters, C. L., and Schofield, P. R. (2001) Ann. Neurol. 49, 125-129[CrossRef][Medline] [Order article via Infotrieve] |
7. |
Steiner, H.,
Revesz, T.,
Neumann, M.,
Romig, H.,
Grim, M. G.,
Pesold, B.,
Kretzschmar, H. A.,
Hardy, J.,
Holton, J. L.,
Baumeister, R.,
Houlden, H.,
and Haass, C.
(2001)
J. Biol. Chem.
276,
7233-7239 |
8. | Houlden, H., Baker, M., McGowan, E., Lewis, P., Hutton, M., Crook, R., Wood, N. W., Kumar-Singh, S., Geddes, J., Swash, M., Scaravilli, F., Holton, J. L., Lashley, T., Tomita, T., Hashimoto, T., Verkkoniemi, A., Kalimo, H., Somer, M., Paetau, A., Martin, J.-J., Van Broeckhoven, C., Golde, T., Hardy, J., Haltia, M., and Revesz, T. (2000) Ann. Neurol. 48, 806-808[CrossRef][Medline] [Order article via Infotrieve] |
9. |
O'Riordan, S.,
McMonagle, P.,
Janssen, J. C.,
Fox, N. C.,
Farrell, M.,
Collinge, J.,
Rossor, M. N.,
and Hutchinson, M.
(2002)
Neurology
59,
1108-1110 |
10. | Li, Y.-M., Xu, M., Lai, M.-T., Huang, Q., Castro, J. L., DiMuzio-Mower, J., Harrison, T., Lellis, C., Nadin, A., Neduvelil, J. G., Register, R. B., Sardana, M. H., Shearman, M. S., Smith, A. L., Shi, X.-P., Yin, K.-C., Shafer, J. A., and Gardell, S. J. (2000) Nature 405, 689-694[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Takashima, A.,
Murayama, M.,
Murayama, O.,
Kohno, T.,
Honda, T.,
Yasutake, K.,
Nihonmatsu, N,
Mercken, M.,
Yamaguchi, H.,
Sugihara, S.,
and Wolozin, B.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
9637-9641 |
12. | Gantier, R., Gilbert, D., Dumanchin, C., Campion, D., Davoust, D., Toma, F., and Frebourg, T. (2000) Neurosci Lett. 283, 217-220[CrossRef][Medline] [Order article via Infotrieve] |
13. | Yu, G., Nishimura, M., Arawaka, S., Levitan, D., Zhang, L., Tandon, A., Song, Y.-Q., Rogaeva, E., Chen, F., Kawarai, T., Supala, A., Levesque, L., Yu, H., Yang, D.-S., Holmes, E., Milman, P., Liang, Y., Zhang, D. M., Xu, D. H., Sato, C., Rogaev, E., Smitth, M., Janus, C., Zhang, Y., Aeberold, R., Farrer, L., Sorbi, S., Bruni, A., Fraser, P., and St George-Hyslop, P. (2000) Nature 407, 48-54[CrossRef][Medline] [Order article via Infotrieve] |
14. | Weggen, S., Diehlmann, A., Buslei, R., Beyreuther, K., and Bayer, T. A. (1998) NeuroReport 9, 3279-3283[Medline] [Order article via Infotrieve] |
15. | Chui, D.-H., Shirotani, K., Tanahashi, H., Akiyama, H., Ozawa, K., Kunishita, T., Takahashi, K., Makifuchi, T., and Tabira, T. (1998) J. Neurosci. Res. 53, 99-106[CrossRef][Medline] [Order article via Infotrieve] |
16. | Tomidokoro, Y., Ishiguro, K., Igeta, Y., Matsubara, E., Kanai, M., Shizuka, M., Kawarabayashi, T., Harigaya, Y., Kawakatsu, S., Li, K., Ikeda, M., St, George-Hyslop, P., Hirai, S., Okamoto, K., and Shoji, M. (1999) Biochem. Biophys. Res. Commun. 256, 512-518[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Stanford, P. M.,
Halliday, G. M.,
Brooks, W. S.,
Kwok, J. B. J.,
Storey, C. E.,
Creasey, H.,
Morris, J. G. L.,
Fulham, M. J.,
and Schofield, P. R.
(2000)
Brain
123,
880-893 |
18. | Mirra, S. S., Heyman, A., McKeel, D., Crain, B. J., Brownlee, L. M., and Vogel, F. S. (1991) Neurology 41, 479-486[Abstract] |
19. | Harding, A. J., Kril, J. J., and Halliday, G. M. (2000) Acta Neuropathol. 99, 199-208[Medline] [Order article via Infotrieve] |
20. | Kwok, J. B. J., Taddei, K., Fisher, C., Hallupp, M., Brooks, W. S., Nicholson, G. A., Hardy, J., Stell, R., St, George-Hyslop, P. H., Fraser, P. E., Kakulas, B., Clarnette, R., Relkin, N., Gandy, S. E., Schofield, P. R., and Martins, R. N. (1997) NeuroReport 8, 1537-1542[Medline] [Order article via Infotrieve] |
21. | Badenhop, R. F., Moses, M. J., Scimone, A., Mitchell, P. B., Ewen, K. R., Rosso, A., Donald, J. A., Adams, L. J., and Schofield, P. R. (2001) Mol. Psychiatry 6, 396-403[CrossRef][Medline] [Order article via Infotrieve] |
22. | Kwok, J. B. J, Li, Q.-X., Hallupp, M., Whyte, S., Ames, D., Beyreuther, K., Masters, C. L., and Schofield, P. R. (2000) Ann. Neurol. 47, 249-253[CrossRef][Medline] [Order article via Infotrieve] |
23. |
Wang, R.,
Sweeney, D.,
Gandy, S. E.,
and Sisodia, S. S.
(1996)
J. Biol. Chem.
271,
31894-31902 |
24. |
Karlstrom, H.,
Bergman, A.,
Lendahl, U.,
Naslund, J.,
and Lundkvist, J.
(2002)
J. Biol. Chem.
277,
6763-6766 |
25. |
Steiner, H.,
Capell, A.,
Pesold, B.,
Citron, M.,
Kloetzel, P. M.,
Selkoe, D. J.,
Romig, H.,
Mendla, K.,
and Haass, C.
(1998)
J. Biol. Chem.
273,
32322-32331 |
26. |
Verdile, G.,
Martins, R. N.,
Duthie, M.,
Holmes, E., St,
George-Hyslop, P. H.,
and Fraser, P. E.
(2000)
J. Biol. Chem.
275,
20794-20798 |
27. | Thinakaran, G., Borchelt, D. R., Lee, M. K., Slunt, H. H., Spitzer, L., Kim, G., Ratovitsky, T., Davenport, F., Nordstedt, C., Seeger, M., Hardy, J., Levey, A. I., Gandy, S. E., Jenkins, N. A., Copeland, N. G., Price, D. L., and Sisodia, S. S. (1996) Neuron 17, 181-190[Medline] [Order article via Infotrieve] |
28. | Murayama, O., Tomita, T., Nihonmatsu, N., Murayama, M., Sun, X.-Y., Honda, T., Iwatsubo, T., and Takashima, A. (1999) Neurosci. Lett. 265, 61-63[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Kumar-Singh, S., De,
Jonghe, C.,
Cruts, M.,
Kleinert, R.,
Wang, R.,
Mercken, M., De,
Strooper, B.,
Vanderstichele, H.,
Lofgren, A.,
Vanderhoeven, I.,
Backhovens, H.,
Vanmechelen, E.,
Kroisel, P. M.,
and Van Broeckhoven, C.
(2000)
Hum. Mol. Genet.
9,
2589-2598 |
30. |
Ancolio, K.,
Dumanchin, C.,
Barelli, H.,
Warter, J. M.,
Brice, A.,
Campion, D.,
Frebourg, T.,
and Checler, F.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
4119-4124 |
31. | Hasegawa, M., Smith, M. J., Iijima, M., Tabira, T., and Goedert, M. (1999) FEBS Lett. 443, 93-96[CrossRef][Medline] [Order article via Infotrieve] |
32. |
D'Souza, I.,
and Schellenberg, G. D.
(2000)
J. Biol. Chem.
275,
17700-17709 |
33. | Morihara, T., Katayama, T., Sato, N., Yoneda, T., Manabe, T., Hitomi, A., Imaizumi, K., and Tohyama, M. (2000) Mol. Brain Res. 85, 85-90[CrossRef][Medline] [Order article via Infotrieve] |
34. | Capell, A., Steiner, H., Romig, H., Keck, S., Baader, M., Grim, M. G., Baumeister, R., and Haass, C. (2000) Nat. Cell Biol. 2, 205-211[CrossRef][Medline] [Order article via Infotrieve] |
35. | Sisodia, S. S., Annaert, W, Kim, S.-H., and De Strooper, B. (2001) Trends Neurosci. 24, S2-S6[CrossRef][Medline] [Order article via Infotrieve] |
36. | Weggen, S., Eriksen, J. L., Das, P., Sagi, S. A., Wang, R., Pietrzik, C. U., Findlay, K. A., Smith, T. E., Murphy, M. P., Bulter, T., Kang, D. E., Marquez-Sterling, N., Golde, T. E., and Koo, E. H. (2001) Nature 414, 212-216[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Seeger, M.,
Nordsted, C.,
Petanceska, S.,
Kovacs, D. M.,
Gouras, G. K.,
Hahne, S.,
Fraser, P.,
Levesque, L.,
Czernik, A. J., St,
George-Hyslop, P.,
Sisodia, S. S.,
Thinakaran, G.,
Tanzi, R. E.,
Greengard, P.,
and Gandy, S.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
5090-5094 |
38. | Cervantes, S., Gonzalez-Duarte, R., and Marfany, G. (2001) FEBS Lett. 505, 81-86[CrossRef][Medline] [Order article via Infotrieve] |
39. | Beher, D., Wrigley, J. D. J., Owens, A. P., and Shearman, M. S. (2002) J. Neurochem. 82, 563-575[CrossRef][Medline] [Order article via Infotrieve] |
40. | Rapoport, M., and Ferreira, A. (2000) J. Neurochem. 74, 125-133[CrossRef][Medline] [Order article via Infotrieve] |
41. |
Lucas, J. J.,
Hernandez, F.,
Gomez-Ramos, P.,
Moran, M. A.,
Hen, R.,
and Avila, J.
(2001)
EMBO J.
20,
27-39 |
42. | Perez-Tur, J., Froelich, S., Prihar, G., Crook, R., Baker, M., Duff, K., Wragg, M., Busfield, F., Lendon, C., and Clark, R. F. (1995) NeuroReport 7, 297-301[Medline] [Order article via Infotrieve] |
43. | Hiltunen, M., Helisalmi, S., Mannermaa, A., Alafuzoff, I., Koivisto, A. M., Lehtovirta, M., Pirskanen, M., Sulkava, R., Verkkoniemi, A., and Soininen, H. (2000) Eur. J. Hum. Genet. 8, 259-266[CrossRef][Medline] [Order article via Infotrieve] |