From the a Adolf Butenandt-Institute, Department of Biochemistry, Laboratory for Alzheimer's Disease Research, Ludwig-Maximilians-University, 80336 Munich, Germany, the c Department of Neuropathology, Institute of Neurology, University College London, Queen Square, London, WC1N 3BG, United Kingdom, the d Institute of Neuropathology, Ludwig-Maximilians-University, 81377 Munich, Germany, e Boehringer Ingelheim KG, CNS Research, 55216 Ingelheim, Germany, the f Gene Center, Ludwig-Maximilians-University, 81377 Munich, Germany, the h Mayo Clinic, Jacksonville, Florida 32224, and i Neurogenetics, Institute of Neurology, Queen Square, London WC1N 3BG, United Kingdom
Received for publication, August 8, 2000, and in revised form, November 16, 2000
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ABSTRACT |
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Familial Alzheimer's disease (FAD)
is frequently associated with mutations in the presenilin-1 (PS1) gene.
Almost all PS1-associated FAD mutations reported so far are exchanges
of single conserved amino acids and cause the increased production of
the highly amyloidogenic 42-residue amyloid Alzheimer's disease
(AD)1 is an
age-dependent neurogenerative disorder. Although most AD
cases occur sporadically, autosomal dominant inheritance has been
recorded in numerous families (1). Mutations in four genes have been
mapped to familial AD (FAD). These include the genes encoding the
PS proteins not only affect the PS proteins not only support the intramembraneous endoproteolysis of
FAD-associated PS mutations occur frequently within the PS1 gene and
are associated with the most aggressive AD phenotype (27). Out of the
numerous PS mutations described to date, only three deletions (28-31)
have been observed so far. However, none of the deletions are directly
associated with a pathological function. We have shown previously that
the pathological activity of the PS1 We have now analyzed the function of a novel PS1 deletion (PS1
Antibodies--
Antibody 3926 to synthetic A Histology and Immunohistochemistry--
Brains from the patient
with the PS1 Genetic Analysis and cDNA Encoding PS1
Cell Culture and Cell Lines--
Human embryonic kidney 293 cells (K293) were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin,
200 µg/ml G418 (to select for Analysis of PS by Combined Immunoprecipitation/Western
Blotting--
Cell lysates from stably transfected K293 cells were
prepared and subjected to immunoprecipitation using the polyclonal
antibody 3027 to PS1 or 3711 to PS2 (32). Following gel
electrophoresis, immunoprecipitated PS proteins were identified by
immunoblotting using the monoclonal antibody BI.3D7 (PS1) or BI.HF5c
(PS2) (32). Bound antibodies were detected by enhanced
chemiluminescence (ECL; Amersham Pharmacia Biotech).
Analysis of A Transgenic Lines of C. elegans and Rescue Assays--
To
construct a new sel-12 expression vector, a 3.0-kilobase
pair fragment of cosmid C08A12 was amplified by polymerase chain reaction using primers CCC GGC TGC AGC TCA ATT ATT CTA GTA AGC and GTC
TCC ATG GAT CCG AAT TCT GAA ACG TTC AAA TAA C and cloned into pPD49.26
(37). The resulting plasmid contains only nontranscribed sequences from
the 5' region of the C. elegans sel-12 gene. PS1 derivatives were cloned into this vector as a
BamHI/SalI fragment. Transgenic lines were
established by microinjection of plasmid DNA mixtures into the C. elegans germ line to create extrachromosomal arrays (26). Four
independent lines from the progeny of F2 generation animals were
established. Since the sel-12(ar171) animals never lay eggs
(26), rescue of the sel-12 defect can be quantified by
scoring egg-laying behavior in transgenic animals (26). 50 transgenic
animals of each line were analyzed for their ability to lay eggs. The
numbers of eggs laid by individual transgenic animals were counted and
placed into four categories: Egl+++, robust egg laying, more than 30 eggs laid; Egl++, 15-30 eggs laid; Egl+, 5-15 eggs laid; Egl Genetic Analysis of a Family with Autosomal Dominant Early Onset AD
with Spastic Paraparesis--
Several families were identified by
Houlden et al. (38) with autosomal dominant early onset AD
with spastic paraparesis. Here we present the detailed pathological,
biochemical, and functional analysis of one of these families.
Neuropathological examination of a large Scottish family (Fig.
1A) revealed the presence of large cotton wool plaques (see below) similar to those seen in the
Finnish family (30, 39). Sequencing revealed the presence of an exon 4 deletion (ATC-ATG; isoleucine-methionine) of codons 83 and 84 of the
PS1 gene. The mutation was not present in 100 controls. The PS1
Deposition of A
Deposition of A Stable Expression of PS1
As described above, cotton wool plaques were so far found to be
associated with two independent FAD mutations (
Stable expression of PS derivatives results in the displacement of
endogenous presenilins (44) and is a prerequisite for functional
expression of exogenous PS, since PS derivatives not displacing
endogenously expressed fragments are unstable and are rapidly degraded
(36, 45, 46). To prove if PS1 PS1 Reduced Facilitation of Notch Signaling--
PS1 and PS2 are both
required for Notch signaling (19) and functionally replace the
defective C. elegans PS homolog sel-12 (10, 25,
26). We now expressed PS1 The PS1 Although the novel PS1 Our findings may also indicate that therapeutic strategies exclusively
based on the reduction of the amyloid plaque burden (62) may not always
be sufficient to prevent AD symptoms.
-peptide A
42. Here we
report the identification and pathological function of an unusual
FAD-associated PS1 deletion (PS1
I83/
M84). This FAD mutation is
associated with spastic paraparesis clinically and causes accumulation
of noncongophilic A
-positive "cotton wool" plaques in brain
parenchyma. Cerebral amyloid angiopathy due to A
deposition was
widespread as were neurofibrillary tangles and neuropil threads,
although tau-positive neurites were sparse. Although significant
deposition of A
42 was observed, no neuritic pathology was associated
with these unusual lesions. Overexpressing PS1
I83/
M84 in
cultured cells results in a significantly elevated level of the highly
amyloidogenic 42-amino acid amyloid
-peptide A
42. Moreover,
functional analysis in Caenorhabditis elegans reveals
reduced activity of PS1
I83/
M84 in Notch signaling. Our data
therefore demonstrate that a small deletion of PS proteins can
pathologically affect PS function in endoproteolysis of
-amyloid
precursor protein and in Notch signaling. Therefore, the PS1
I83/
M84 deletion shows a very similar biochemical/functional
phenotype like all other FAD-associated PS1 or PS2 point mutations.
Since increased A
42 production is not associated with classical
senile plaque formation, these data demonstrate that amyloid plaque
formation is not a prerequisite for dementia and neurodegeneration.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amyloid precursor protein (
APP), presenilin 1 (PS1), PS2 (1),
and
2-macroglobulin (2). Functional analysis revealed
that
APP and PS mutations affect endoproteolytic processing of
APP in a very similar manner. In the amyloidogenic pathway,
APP
is first cleaved at the N terminus of the A
domain by the recently
identified
-secretase (3). This generates a membrane-retained
C-terminal fragment, which is the substrate for the
-secretase.
-Secretase cleaves its substrate within the membrane, which results
in the physiological secretion of A
(1). About 90% of secreted A
terminates at amino acid 40 (A
40), while most of the remaining A
peptides are elongated by two amino acids (A
42). The rare A
42
appears to aggregate much faster than A
40 (4, 5) and is
therefore the major constituent of senile plaques (6, 7).
FAD-associated mutations found within
APP, PS1, and PS2 all cause
the increased production of this highly amyloidogenic A
variant and
therefore increase the kinetics of A
aggregation and of its
deposition in congophilic senile plaques (1).
-secretase cleavage in FAD cases but
are also required for physiological A
generation, since a PS1
ablation results in a dramatically reduced A
production (8).
Moreover, mutagenesis of two critical aspartate residues located within
transmembrane domains 6 and 7 (TM6 and -7) also results in an
inhibition of A
generation (9). Similar mutations in human PS2 also
reduce A
generation (10, 11), and the critical aspartate residues
are functionally conserved during evolution (12). In all cases,
inhibition of PS function not only reduced A
generation but also
concomitantly increased the corresponding membrane-retained
APP
C-terminal fragments, which are the immediate precursors for A
generation. Since two critical aspartate residues are required within
the catalytic center of aspartyl proteases and since
-secretase
function can be blocked by aspartyl protease inhibitors (13), it was
recently claimed that PS proteins may be identical with the
-secretase (14).
APP but are also required for the similar cleavage of Notch (10,
15-17). The endoproteolytic cleavage of Notch appears to be required
for the generation of the Notch intracellular cytoplasmic domain (18),
which translocates to the nucleus, where it is involved in
transcriptional regulation (19). A function of PS in Notch signaling is
also supported by the phenotypes observed in various PS1/PS2 deletions
in mice (20-23), which resemble that observed upon the deletion of the
Notch gene. Moreover, several mutant alleles of the
Caenorhabditis elegans PS homolog sel-12 cause an
egg-laying phenotype, which is due to a functional deficit in Notch
signaling (24). The failure in Notch signaling in worms can be
functionally rescued by transgenic expression of human PS1 or PS2 (10,
25, 26).
exon9 splicing mutation (28) is
independent of the large deletion and rather due to a single amino acid
exchange at the aberrant splice junction at codon 290 (32). A genomic
deletion of the exon 9-encoded domain (
exon9 Finn; see below) was
reported as well (30). However, due to the aberrant splicing of exon 8 with exon 10, the same amino acid exchange is introduced at codon 290 as observed in the original PS1
exon9 splicing mutation. Therefore,
the amino acid sequence of PS1
exon9 Finn is identical to the PS1
exon9 splicing mutation. In analogy to the PS1
exon9 splicing
mutation (32), it would therefore be expected that this mutation (PS1
exon9 Finn) produces A
42 independent of the exon 9 deletion. The
unusual genomic exon 9 deletion of PS1 in the Finnish pedigree is
associated with Alzheimer's disease and spastic paraparesis. In
contrast to all other AD cases, these patients as well as the patients
with the PS1
exon9 splicing mutation develop "cotton wool"
plaques, which lack a congophilic dense core and plaque-related
neuritic pathology (30).2
Finally, the deletion produced by the intron 4 mutation of PS1 could
not be associated with an increased A
42 production (29). It rather
turned out that a single amino acid insertion, which is generated by
aberrant splicing, is responsible for the pathological activity of this
mutation (29). Therefore, no PS1 deletion has so far been associated
with increased A
42 generation.
I83/
M84; Fig. 1), which is also associated with early onset AD
and spastic paraparesis. A potentially pathological function in A
generation and Notch signaling was specifically investigated. We found
that PS1
I83/
M84 causes increased A
42 production like all
other FAD-associated PS1/PS2 mutations. The PS1
I83/
M84 mutation
is associated with A
deposition in noncongophilic cotton wool
plaques, widespread cerebral amyloid angiopathy, neurofibrillary tangles, and neuropil threads, although tau-positive abnormal neurites
are rare.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was
described before (33). The polyclonal and monoclonal antibodies against
amino acids 263-407 of PS1 (3027; BI.3D7) and against amino acids
297-356 of PS2 (3711; BI.HF5c) were described previously (32). For tau
immunohistochemistry, the AT8 antibody (Innogenetics, Belgium) and
mouse monoclonal antibody PHF1 (a gift from Peter Davies) were used. An
anti-GFAP antibody (Dako, UK) was used for the detection of
astrocytosis. The presence of microglia was detected with an antibody
against major histocompatibility complex class II proteins (clone
CR3/43), which was obtained from Dako, UK. For A
immunohistochemistry, N-terminal antibody 6F/3D to A
8-17 (Dako, UK)
or 6E10 to A
1-17 (Senetek) as well as C-terminal specific antisera
recognizing A
ending at Ala42 (antibody 44-344;
Immunogenetics, Belgium) or Val40 (antibody 44-348;
Immunogenetics, Belgium) were used.
I83/
M84 mutation and from a patient with a PS1 T115C
mutation were collected at postmortem and fixed in 10% formalin in
phosphate-buffered saline. Blocks from the major anatomical areas,
including the hippocampal formation, were processed in paraffin wax.
Tissue sections were stained with hematoxylin and eosin and
Bielschowsky's silver impregnation methods. Congo red and
thioflavine S methods were used to detect A
deposits in
-sheet conformation. For immunohistochemistry, 4-, 7-, or 20-µm
sections were deparaffinized in xylene and rehydrated using graded
alcohols. For PHF1, AT8, and CR3/43 immunohistochemistry, sections were
pretreated in a microwave oven in sodium citrate buffer for 20 min, for
GFAP immunohistochemistry in trypsin for 10 min, and for A
, A
40,
and A
42 immunohistochemistry in formic acid for 10 min followed by
treatment in a pressure cooker in citrate buffer for 10 min. After
washes in phosphate-buffered saline and 10% milk, sections were
incubated with the PHF1 antibody at 4 °C or with the GFAP, AT8,
CR3/43, A
, A
40, and A
42 antibodies at room temperature.
Detection of antibody binding was either performed with the ABC or the
alkaline phosphatase anti-alkaline phosphatase system (DAKO) according
to the manufacturer's instructions. Either
diaminobenzidine/H2O2 or neufuchsin was used as chromogen.
I83/
M84--
Genomic DNA and mRNA were extracted from blood
and frozen brain, respectively. All exons of the PS1 gene were analyzed
by polymerase chain reaction amplification of genomic DNA and Big Dye
sequencing. Sequence analyses of PS1 exon 4 revealed a heterozygous deletion of ATCATG at codons 83 and 84 (isoleucine-methionine) of the
gene. The corresponding cDNA encoding PS1
I83/
M84 was cloned
into pcDNA3.1-zeo(+) expression vector (Invitrogen).
APP expression), and 200 µg/ml
zeocin (to select for presenilin expression). K293 cells stably
expressing PS1
I83/
M84 were generated by transfection of K293
cells stably expressing
APP containing the Swedish mutation (34).
K293 cells stably coexpressing Swedish
APP695 and wt PS1 or PS1
exon9 were described previously (35, 36).
by ELISA--
Conditioned media (2 ml) were
collected from confluent K293 cells in six-well dishes for 24 h.
The media were assayed for A
40 and A
42 according to a previously
described enzyme-linked immunosorbent assay (36).
, 0-5
eggs laid.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
I83/
M84 deletion occurs within TM1 of PS1 (Fig. 1B).
TM1 may be functionally important, since other mutations were
previously located in that region. Interestingly, the PS1
I83/
M84
deletion is located immediately C-terminal to the V82L mutation (40).
Moreover, a third mutation has been observed in TM1, which results in
the exchange of valine at position 96 to phenylalanine (41).
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Fig. 1.
A FAD-associated PS1 deletion.
A, three generation family with five affected members. All
affected individuals had signs of AD with spastic paraplegia; this was
progressive over 6-10 years, and all cases were wheelchair bound in
the later stages of the disease. Pedigree is disguised for family
protection, and the numbers to the right of
individual symbols refer to the age at death or
current age. Arrow, sequenced cDNA of affected patient.
B, schematic representation of PS1 I83/
M84. The
deletion of amino acids Ile83 and Met84 in TM1
is shown as well as neighboring point mutations associated with FAD
(asterisks). The black box represents
the cleavage site domain of PS1.
42 in Cotton Wool Plaques--
Neuropathological
investigation of the PS1
I83/
M84 case by hematoxylin/eosin
staining, Bielschowsky's silver staining, and A
immunohistochemistry revealed the presence of widespread cotton wool
plaques (Figs. 2 and
3). These plaques were most frequently found in the neocortex, hippocampus, and striatum. Cotton wool plaques
appeared in the neuropil as round, eosinophilic, and strongly A
-positive structures often larger than 100 µm in diameter. These frequently seemed to displace other elements such as neurons, a finding
readily noticeable in the hippocampus (Fig. 2, A and B). Cotton wool plaques did not generally contain amyloid,
since they were negative or occasionally very weakly stained by Congo red and weakly positive with thioflavine S (Fig. 2,
C-E). Cerebral amyloid angiopathy was
widespread, capillaries having thickened walls, which, together with
the affected arterioles, showed apple green birefringence following
Congo red staining and strong fluorescence with thioflavine S (Fig. 2,
C-E). Bielschowsky silver staining (Fig.
2B) and tau immunohistochemistry (Fig. 2, F and
H) revealed that neurofibrillary tangle pathology was
widespread in the neocortex and hippocampal formation, although the
dentate fascia was spared. Fine neuropil threads were a prominent
feature within the cortices, and AT8 as well as PHF1
immunohistochemistry also revealed that the cotton wool plaques
contained many thread-like processes but were only rarely associated
with abnormal neurites (Fig. 2, F and H). In
contrast, numerous tau (Fig. 2G) and silver-positive abnormal neurites (data not shown) were seen in association with the
classical plaques found in the control case with a PS1 T115C mutation
(42) (Fig. 2G). GFAP immunostaining demonstrated a relatively sparse astrocytic response to the cotton wool plaques (Fig.
2I). Furthermore, no significant microglial activation
(CR3/43 immunostaining) was observed in association with cotton wool
plaques (Fig. 2J), in contrast to widespread activated
microglia that were clustered mostly around the amyloid plaques in the
PS1 T115C case (Fig. 2K).
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Fig. 2.
Neuropathological findings in a case with
PS1 I83/
M84
mutation. A, hippocampal cotton wool plaques
(arrow) displacing neurons of the CA1 hippocampal subregion
(Hematoxylin and eosin, X 115). B, nonneuritic cotton wool
plaques in the inner molecular layer of the dentate fascia
(iML) and CA4 (arrows) (Bielschowsky's silver
impregnation, × 115). C and D, Congo
red-positive blood vessels but negative cotton wool plaques (Congo red
preparation, × 50). E, strongly thioflavine S-positive
blood vessel and weakly positive cotton wool plaques in the temporal
neocortex (thioflavine S, × 150). F, an antibody
recognizing phosphorylated serine 202/threonine 205 epitopes of tau
labels no abnormal neurites in hippocampal cotton wool plaques
(G), although numerous such structures are stained in
classical plaques of a FAD case with the PS1 T115C mutation (AT8
immunohistochemistry, × 200). H, no abnormal neurites but
numerous neurofibrillary tangles and neuropil threads in the temporal
neocortex (PHF1 immunohistochemistry, × 190). I, relatively
sparse astrocytic response to cotton wool plaques in the frontal
neocortex (GFAP immunohistochemistry, × 115). J, activated
microglia in the temporal cortex is found around blood vessels, but not
around cotton wool plaques in the PS1
I83/
M84 case (CR3/43
immunohistochemistry, × 200). K, activated microglia
clusters around classical plaques in the PS1 T115C case (CR3/43
immunohistochemistry, × 200).
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Fig. 3.
Cotton wool plaques associated with the
PS1 I83/
M84 mutation
contain A
42. A and
B, A
positive cotton wool plaques and blood vessels
(arrow) in the neocortex (A, 6F/3D
immunohistochemistry, × 115; B, 6E10 immunohistochemistry, × 120). The cotton wool plaques are weakly positive for A
40 (44-348 immunohistochemistry, × 50) (C) but strongly positive for
A
42 (44-344 immunohistochemistry, × 115) (D).
species ending at position 42 is believed to be
closely associated with neuritic plaque formation in previously reported cases with different PS1 mutations (6, 7). In our PS1
I83/
M84 case, the cotton wool plaques were strongly positive not
only with antibodies raised against epitopes 8-17 (6F/3D
immunostaining) or 1-17 (6E10 immunostaining) of the A
peptide
(Fig. 3, A and B), but also with an end-specific
antiserum to position 42 (Fig. 3D). In contrast, the cotton
wool plaques were only weakly reactive for A
40, which was found to
be the predominant A
species deposited in blood vessels (Fig.
3C). These findings show that the cotton wool plaques are
predominantly composed of A
ending at position 42, and, since they
were also positive with the 6E10 antibody recognizing amino acids 1-17
of A
, at least some full-length A
1-42 is deposited (Fig.
3B).
I83/
M84 in Human Cell Lines--
To
further prove the pathological activity of PS1
I83/
M84 on A
production, we analyzed its function in a tissue culture system, which
has previously been proven to be very sensitive for the detection of
abnormal A
42 generation caused by the expression of FAD mutant
presenilins (see Ref. 43 and references therein).
exon9 and
exon9
Finn), which both affect PS1 endoproteolysis. Since the PS1
I83/
M84 mutation also results in a very similar pathology, we
first investigated endoproteolysis of this mutant PS1 variant. cDNAs encoding PS1
I83/
M84, PS1
exon9, and wt PS1 were
stably transfected into human embryonic kidney 293 cells expressing
Swedish mutant
APP (34). To prove ectopic expression and
endoproteolysis of the transfected PS1 derivatives, cell lysates were
immunoprecipitated with antibody 3027 to the cytoplasmic loop of PS1
(32). Immunoprecipitated PS1 proteins were identified by immunoblotting
using the monoclonal antibody BI.3D7 to the C terminus of PS1 (32).
Consistent with previous results (44), large amounts of uncleaved PS1
holoprotein accumulated in cells expressing PS1
exon9 (Fig.
4A, upper
panel). Robust amounts of PS1 C-terminal fragments (CTFs)
were observed in all other cell lines including those overexpressing
PS1
I83/
M84 (Fig. 4A, upper
panel). This demonstrates that the novel deletion mutation
does not affect endoproteolysis of PS1 like the PS1
exon 9 deletion.
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Fig. 4.
Endoproteolytic cleavage, replacement of
endogenous PS, and elevated A 42 production by
stable expression of PS1
I83/
M84 in K293
cells. A, upper panel, cell lysates from
K293 cells expressing endogenous presenilins or overexpressing the
indicated PS variants were immunoprecipitated with antibodies specific
to the large loop of PS1 (3027). PS1 holoproteins and PS1 CTFs were
detected by immunoblotting using the monoclonal antibody BI.3D7 (to
PS1). Endoproteolytic cleavage occurs in cell lines stably expressing
PS1
I83/
M84 or wt PS1. As observed before, cell lines stably
expressing PS1
exon9 replace endogenous PS1 and accumulate as
uncleaved full-length PS proteins (32, 44). Lower
panel, cell lysates from the cell lines in (A) were
immunoprecipitated with antibodies specific to the large loop of PS2
(3711), and PS2 CTFs were detected by immunoblotting using the
monoclonal antibody BI.HF5c (to PS2). Overexpression of all indicated
PS1 variants results in efficient replacement of endogenous PS2.
B, quantitation of the A
42 and A
40 concentrations in
conditioned media of K293 cells expressing the indicated presenilin
variants using a previously described highly specific enzyme-linked
immunosorbent assay (36). Expression of PS1
I83/
M84 results in a
1.5-1.8 fold increase of A
42 production. Consistent with previous
results, the PS1
exon9 mutation causes the production of higher
A
42 levels than the majority of other FAD mutations (63).
However, the PS1
exon9 also causes the generation of cotton wool
plaques (30).
I83/
M84 also displaces endogenous
presenilins, PS2 was immunoprecipitated using antibody 3711 (32).
Precipitated PS2 derivatives were identified by immunoblotting using
the monoclonal antibody BI.HF5c (32). As demonstrated in Fig.
4A (lower panel), overexpression of
PS1
I83/
M84 led to a significant displacement of endogenous PS2
CTFs. Consistent with previous results (32, 44), expression of wt PS1
and PS1
exon9 also strongly reduced the accumulation of endogenous
PS2 CTFs (Fig. 4B, lower panel).
I83/
M84 Promotes Pathological A
42
Generation--
After demonstrating that overexpressed PS1
I83/
M84 efficiently replaces endogenous PS2 CTFs, we analyzed the
pathological function of the deletion mutation on A
42 production by
using a sensitive and specific enzyme-linked immunosorbent assay (36). Conditioned media were collected from cells stably expressing wt PS1,
PS1
exon9, or PS1
I83/
M84, and A
42/A
total
ratios were determined. This revealed that the PS1
I83/
M84
mutation induces elevated levels of A
42 (Fig. 4B) that
are similar to those produced by other FAD-associated point mutations
(47). In contrast, and consistent with previous results (32, 35), the
PS1
exon9 mutation increases A
42 production to even higher levels.
I83/
M84 in a mutant strain of C. elegans, which lacks a functional PS homolog
(sel-12(ar171)). The sel12(ar171) animals show an
egg-laying defective phenotype, which is due to a defect in
Notch-signaling during vulva differentiation (24). This system can be
used to monitor PS function in the facilitation of Notch signaling in
an in vivo rescue assay by transgenic expression of the
corresponding human cDNA constructs (24, 26). We (10, 26) and
others (25) have previously shown that human wt PS1 and PS2 rescue the
egg laying phenotype of the mutant worm, whereas FAD-associated PS
point mutations showed a reduced rescuing activity. Consistent with
previous results (25, 26), transgenic expression of wt PS1 in the
mutant worm lead to a rescue of the egg laying phenotype (Table
I). In contrast, PS1
I83/
M84 showed
significantly less rescuing activity (Table I). Therefore, similar to
all other PS1-associated FAD mutations investigated so far (25, 32),
the PS1
I83/
M84 deletion also lost activity in Notch signaling in
the in vivo rescuing assay.
Reduced rescuing activity of the sel-12 egg laying defect by PS1
I83/
M84
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
I83/
M84 mutation is to our knowledge the first
FAD-associated deletion. Almost all FAD mutations described within PS1
or PS2 are point mutations exchanging single highly conserved amino
acids (48, 49). Although the recently identified intron 4 mutation can
result in C-terminal truncated PS1 derivatives, these are
pathologically inactive and do not cause increased A
42 generation
(29). This is consistent with the finding that C-terminal deleted
artificial presenilins containing a FAD mutation are unable to induce
A
42 production as well. In that regard, mutant PS fragments have
been overexpressed, which correspond to the proteolytically generated
N-terminal fragment. Such PS fragments consistently failed to induce
A
42 generation (36, 50, 51), and even very minor deletions at the C
terminus of PS1/PS2 inactivate PS function (52). The lack of
pathological activity of truncated presenilins appears to be due to the
failure of such PS derivatives to incorporate into the functionally
required PS complex (36, 45, 50, 51, 53-56). Based on these findings,
it would be unexpected that large deletions in the PS sequence would
indeed be associated with early onset AD. However, one of the
previously identified PS1 mutations results in the deletion of the
complete exon 9-encoded domain due to a splicing error (28). Since exon 9 encodes the cleavage site of PS1 (43, 44, 57), this deletion mutation
is associated with a lack of endoproteolysis and consequently the
accumulation of the PS1
exon9 holoprotein (44). This very drastic
phenotype was believed to be the cause for the pathological activity of
PS1
exon9 in A
42 generation. However, we have shown recently that
the pathological activity of PS1
exon9 splicing mutation in regard
of A
42 generation is solely due to a single amino acid exchange of
the conserved Ser290 to cysteine (32). Therefore, this
demonstrates that deletions of the PS amino acid sequence have so far
not been associated with a pathological function in A
generation.
Consistent with these findings, De Jonghe et al. (29)
reported that the pathological activity of the intron 4 mutation of PS1
was surprisingly associated with an amino acid insertion generated by
the utilization of a cryptic splice site but not with the deleted PS1
derivatives. Therefore, the PS1
I83/
M84 mutation is the first
pathogenic deletion of presenilins, which indeed is directly associated
with a malfunction in A
42 production.
I83/
M84 mutation is the first pathogenic
PS1 deletion, it is associated with a pathological phenotype similar to
that first described in association with the PS1
exon9 Finn
mutation. The cotton wool plaques observed lack congophilia including
an amyloid core and associated abnormal neurites (30). Non-neuritic
parenchymal deposits in association with extensive neurofibrillary
degeneration are, however, not a unique feature of variant AD as
lesions resembling cotton wool plaques, but composed of different
amyloidogenic peptides also occur in the BRI gene related
diseases, in familial British dementia (64-66) and familial Danish
dementia (67). It is of interest that the clinical phenotype of
familial British dementia and familial Danish dementia also resembles
that seen in variant AD with cotton wool plaques and spasticity (38).
Since A
42 has a great propensity to accumulate in classical senile
plaques (6, 7), one would have expected that PS1 mutations associated
with noncongophilic cotton wool plaques might not affect A
42
generation. However, our data demonstrate that A
42 generation is
significantly induced in cultured cells by the PS1
I83/
M84
mutation very similar to all other FAD associated PS mutations
investigated so far. In addition, we showed immunohistochemically that
the cotton wool plaques associated with the PS1
I83/
M84 mutation
are predominantly composed of A
42, which is similar to that seen in
classical plaques in sporadic AD and AD caused by either APP or other
PS1 mutations (6, 58, 59). PS1
I83/
M84 not only behaves in terms
of A
42 generation like a typical PS-associated FAD mutation but also
exhibits a similar loss of function in Notch signaling in C. elegans. Therefore, the question arises whether amyloid plaques
composed of A
42 are the primary cause of AD and whether such amyloid
plaques initiate neurodegeneration. Apparently, the lack of classical
dense core congophilic plaques did not prevent the cotton wool plaque
cases from developing neurological symptoms including dementia,
suggesting that the potential pathological activity of A
42 may be
acting upstream of amyloid deposition. Although this could indicate
that increased A
42 production is an epiphenomenon of FAD-associated
mutations, we think it is much more likely that the previously
characterized protofibrils of A
(4, 5) may be the primary cause for
the observed neurological deficits. Since A
42 can be observed
intracellularly (33, 60, 61), primary pathological consequences may be
induced long before A
finally precipitates into amyloid plaques.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Liane Meyn, Gabi Basset, Tammaryn Lashley, and Irvna Pigur for expert experimental assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the European Community and the Deutsche Forschungsgemeinschaft (to C. H.).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.
b These authors contributed equally to this work.
g Present address: Central Institute of Mental Health, Dept. of Molecular Biology, J5, 68159 Mannheim, Germany.
j Supported by a grant from the Wellcome Trust.
k To whom correspondence should be addressed: Adolf-Butenandt-Institute, Ludwig-Maximilians-University Munich, Dept. of Biochemistry, Schillerstr. 44, 80336 München, Germany. Tel.: 49-89-5996-471/472; Fax: 49-89-5996-415; E-mail: chaass@pbm.med.uni-muenchen.de.
Published, JBC Papers in Press, November 17, 2000, DOI 10.1074/jbc.M007183200
2 H. Houlden and J. Hardy, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
AD, Alzheimer's
disease;
FAD, familial AD;
APP,
-amyloid precursor protein;
TM, transmembrane domain;
wt, wild type.
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