Human Molecular Genetics, 2000, Vol. 9, No. 18 2589-2598
© 2000 Oxford University Press
Nonfibrillar diffuse amyloid deposition due to a 
42-secretase site mutation points to an essential role for N-truncated Aß42 in Alzheimer’s disease
Laboratory of Molecular Genetics, Flanders Interuniversity Institute for Biotechnology, Born-Bunge Foundation, University of Antwerp, Universiteitsplein 1, B-2610, Antwerpen, Belgium, 1Laboratory of Neuropathology, Institute of Pathology and 6Laboratory of Human Genetics, Institute for Medical Biology and Human Genetics, University of Graz, Austria, 2Laboratory for Mass Spectrometry, The Rockefeller University, NY, USA, 3Janssen Research Foundation, Beerse, Belgium, 4Laboratory of Neuronal Cell Biology and Gene Transfer, University of Leuven and 5Innogenetics, Zwijnaarde, Belgium
Received 15 June 2000; Revised and Accepted 24 August 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTS DISCUSSIONMATERIALS AND METHODSREFERENCES |
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Amyloidogenic processing of the amyloid precursor protein (APP) with deposition in brain of the 42 amino acid long amyloid ß-peptide (Aß42) is considered central to Alzheimer’s disease (AD) pathology. However, it is generally believed that nonfibrillar pre-amyloid Aß42 deposits have to mature in the presence of Aß40 into fibrillar amyloid plaques to cause neurodegeneration. Here, we describe an aggressive form of AD caused by a novel missense mutation in APP (T714I) directly involving
-secretase cleavages of APP. The mutation had the most drastic effect
on Aß42/Aß40 ratio in vitro of
11-fold, simultaneously increasing Aß42 and decreasing
Aß40 secretion, as measured by matrix-assisted laser
disorption ionization time-of-flight mass spectrometry. This coincided in
brain with deposition of abundant and predominant nonfibrillar pre-amyloid
plaques composed primarily of N-truncated Aß42 in complete
absence of Aß40. These data indicate that N-truncated
Aß42 as diffuse nonfibrillar plaques has an essential but
undermined role in AD pathology. Importantly, inhibiting secretion of
full-length Aß42 by therapeutic targeting of APP processing
should not result in secretion of an equally toxic N-truncated
Aß42. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTS DISCUSSIONMATERIALS AND METHODSREFERENCES |
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The predominant protein component of the cortical and cerebrovascular amyloid deposits of Alzheimer’s disease (AD) is the amyloid ß-peptide (Aß). Accumulating evidence suggests that Aß production from amyloid precursor protein (APP) (1), its aggregation into fibrils and its deposition are key etiological events in AD (2). An understanding of these critical steps will be crucial in devising therapeutic targets.
APP is processed by
ß-secretase (BACE) (3) and a yet unidentified 
-secretase
leading to soluble N-terminal APP fragments (APPs and
APPsß) and membrane bound C-terminal fragments ( and ß CTFs)
(2). Whereas cleavages
by ß- and -secretases release 40–42 amino acids Aß
peptides (Aß1–40 and Aß1–42), the
major secretory pathway utilizes -secretase that cleaves the Aß
sequence between amino acids 16 and 17 of Aß. Further processing of the
CTF by -secretase releases N-terminally cleaved
Aß17–40 or Aß17–42 peptides
(p3). In addition to p3 (Aß17-X), two other major peptides
starting from amino acids 5 (Aß5-X) and 11
(Aß11-X) are secreted by transfected cells (4–6) although their significance for the
pathogenesis of amyloid plaque formation is unclear (2). In contrast, the importance of Aß
C-terminal heterogeneity is more apparent. Immunohistochemistry showed
that, while Aß42 deposits first in diffuse plaques in AD and
in Down’s syndrome (DS) patients (7,8), Aß40 contributes to further
growth of the amyloid plaques to form dense compact plaques (i.e. cored and
neuritic plaques) (8,9). Aß40 is also the
predominant constituent of the amyloid deposits in blood vessel walls
(congophilic amyloid angiopathy and dyshoric angiopathy) (8). Congophilic cored plaques or
fibrillar amyloid deposits, composed chiefly of full-length Aß, are
proposed to be pathogenic in AD and DS patients (10–12). Recently, deposition of p3 in
non-congophilic diffuse plaques has also been recognized (7,13); however, the precise significance of
these amyloid deposits to disease pathogenesis and cognitive decline in AD
and DS is poorly understood. p3 is considered to be relatively benign as it
is nonfibrillar (13)
and is proposed to lack domains involved in APOE binding (14) and microglial and complement
activation (15,16).
As yet, eight missense
APP mutations have been identified in families with autosomal
dominant early-onset AD (17,18). All these mutations clustering in close
proximity to the secretase cleavage sites, affect APP metabolism in two
distinct ways. The Swedish double mutation (K670N/M671L) located near
the ß-secretase cleavage site (19), increases the production of both
Aß40 and Aß42 (20,21). In contrast, mutations located near the
-secretase cleavage sites (17,18) result in an increased production of
Aß42, in a similar manner to that described for presenilin
gene (PS1 and PS2) mutations (22,23). Here we report a novel mutation that
involves amino acid 43 of Aß located directly at the
42-secretase cleavage site and leads to a severe AD
pathology with unusual plaque composition and morphology. Whereas
Aß40 was completely absent from amyloid deposits in brain
and formation of typical cored plaques was retarded, abundant diffuse
non-congophilic amyloid plaques in association with dystrophic
neurites and reactive gliosis were predominantly
deposited.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTS DISCUSSIONMATERIALS AND METHODSREFERENCES |
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APP T714I mutation
Family AD156 (Fig. 1A), an Austrian family consistent with autosomal dominant inheritance of early-onset AD, was referred for DNA diagnosis. The proband, her sister and their mother were diagnosed as probable AD according to NINCDS–ADRDA criteria at age 38, 38 and 44 years respectively. However, signs of cognitive impairment and behaviour disturbances were apparent several years earlier in all three patients suggestive of a mean onset age of
34 years in the family. Genomic DNA
of the proband was examined for mutations in APP, PS1 and
PS2. In exon 17 of APP (Fig. 1B), a heterozygous C
T transition was
identified at position 2208 substituting Thr (T) at codon 714 by Ile (I)
(T714I, numbering according to APP770 isoform). The mutation abolishes a
TspRI restriction site that was used to confirm the presence of
the mutation in the proband (156.1) and her sister (156.2)
(Fig. 1C). The
mutation was absent in the father as well as in 50 healthy Austrian
individuals. No other mutations were detected.
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, age at death; arrow, the proband where autopsy was
performed. (B) Sequence analysis for 156.1 and 156.2 showing a
heterozygous CThe Austrian T714I mutation is the first APP mutation reported to date that involves amino acid 43 of Aß located directly at the
42 secretase
cleavage site. The early onset age, as well as rapid progression of the
disease and early death, is comparable with AD caused by mutations in PS1
(18).
Drastically altered
APP processing in vitro
To understand the effect of the
T714I mutation on the cleavage specificity of 42-secretase,
we studied APP processing in transfected cells. Human embryonic kidney
(HEK) 293T cells were transiently transfected with the T714I APP cDNA and
secreted Aß1–42 and Aß1–40 levels
were measured in conditioned medium by enzyme-linked immunosorbent assay
(ELISA) (24,25). Cells overexpressing
wild-type and London V717I (26) APP cDNA were used as controls. The T714I
mutation increased Aß42 and at the same time decreased
Aß40, resulting in a significantly increased
Aß1–42/Aß1–40 ratio (P< 0.001), which was four times higher than in wild-type. In the
same experiment, V717I resulted in a 1.8-fold increased
Aß1–42/Aß1–40 ratio, results
that are comparable with previously published data by Suzuki et
al. (22). We also
measured by ELISA Aß1–42 and
Aß1–40 in plasma of the patient (156.2), her
unaffected father (156.3) and five unrelated age-matched controls. The
Aß1–42/Aß1–40 ratio was
2.5-fold increased compared with the unaffected father and 1.7-fold
compared with the controls.
The conditioned medium of the T714I
and wild-type APP transfected HEK293T cells was also analyzed by
matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass
spectrometry (4)
(Fig. 2). This method
allowed us to assess both full-length and N-truncated Aß. Compared with
wild-type, T714I showed a significant elevation of
Aß1–42 (3.5-fold) (P < 0.001), whereas at
the same time Aß1–40 decreased significantly by
68% (P <0.001). This brought down the secreted levels of
Aß1–40 to equal the amounts of secreted
Aß1–42 resulting in an increased
Aß1–42/Aß1–40 ratio by
10.8-fold over wild-type. Similar effects were seen on p3 with nearly equal
levels of secreted Aß17–40 and Aß17–42increasing Aß17–42/Aß17–40ratio by 10.7-fold over wild-type. One other pronounced effect of the
T714I mutation was the significant increase of Aß peptides ending at
V39 and G38 irrespective of the N-terminal residue. Smaller effects were
also seen for full-length Aß ending at G37. These peptides were not
artificially produced by proteolysis of full-length Aß as synthetic
Aß1–40 and Aß1–42 peptides were
not degraded when added to the medium of non-transfected cells (4). Compared with other
APP mutations located close to the 42-secretase
cleavage site (22,26,27), T714I had the highest increase in
Aß42/Aß40 ratio. Interestingly an in
vitro decrease in Aß40 was also reported recently for
the French V715M mutation (27), one amino acid downstream of T714I. The
mechanisms by which these mutations affect Aß40 and
Aß42 secretion or lead to alternative cleavage to
Aß38 or Aß39, is not yet understood. The
transmembrane region of APP is in -helical conformation (28) and its interaction with PS1,
the proposed -secretase (29,30), results in intramembranous
proteolysis. It is plausible that PS1 might have different binding
affinities or cleavage efficiencies to these mutated CTFs and a close
spatial proximity of amino acids three or four positions apart in this
-helical conformation, might explain the effect of T714I or V715M on
40-secretase activity as well.
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Prompted by a description of altered soluble APP for V715M mutation (27), perhaps due to increased trafficking of the mutated APP into cellular compartments where
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and ß-secretases reside, we quantified APPs and APPsßfor T714I. However, no significant changes over wild-type were noted
(Fig. 3).
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Unusual plaque morphology and composition
Neuropathological examination of the proband (156.1) showed extensive neuronal loss accompanied by diffuse gliosis, amyloid plaques and neurofibrillary tangles confirming the diagnosis of AD. We performed an in-depth analysis by histochemistry and immunohistochemistry on serial sections from three select brain areas: hippocampus including entorhinal cortex (ERC) and temporal and frontal cortices. Immunostaining with an antibody recognizing both full-length Aß and p3 (mAb 4G8), remarkably stained a huge plaque load predominantly as ‘cloudy’ diffuse plaques that sometimes enclosed a central lacuna. In the molecular layer of the dentate gyrus, the amyloid plaques had the same non-neuritic ‘cotton wool’ appearance as described for PS1
9 patients (31–33). We determined the fibrillar nature of
the amyloid plaques by congo-red and thioflavin staining. Cored plaques
were congophilic and fluorescent with thioflavin, whereas the majority of
the diffuse plaques were non-congophilic (Fig. 4A and B). In the few diffuse plaques faintly
positive for thioflavin, the fibrillar amyloid occupied only a small
proportion of total amyloid recognized immunohistochemically. In the layer
III of ERC, the amyloid plaques were congophilic whereas in its superficial
and deep layers, the diffuse plaques were again nonfibrillar. A strong
neuritic pathology was evident in CA1 and subicular fields of hippocampus
and ERC. Neurites accumulated hyperphosphorylated tau (AT8),
ubiquitin and APP, irrespective of plaque pathology (Fig. 4C and D). Using endothelial cell
markers (CD31 and CD34) with amyloid staining, core plaques but not the
diffuse plaques were closely associated with blood vessels. This feature
was most remarkable in the endplate region where all fibrillar amyloid
deposits were associated with blood vessels (Fig. 4E and F).
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Next we examined whether in vivo the mutation had similar effects on APP processing. Reactivity to a C-terminal Aß40- specific monoclonal antibody, JRF/cAb40/10, was completely absent in both blood vessel walls and amyloid plaques in the isocortex (Fig. 5A), while a faint immunoreactivity was occasionally present in the endplate region of the hippocampus (2–3 amyloid deposits per 10 fields of 0.7 mm2 each) (Figures 4A and B and 5D). In contrast, a C-terminal Aß42-specific monoclonal antibody, JRF/cAb42/12, imparted a strong reactivity to all amyloid deposits including diffuse plaques, blood vessel walls and ‘infrequent’ cored plaques (Fig. 5B). Sections were stained with 4G8 and compared with immunoreactivity for antibodies specific for Aß40 and Aß42 by image analysis (Fig. 5A–C). In the hippocampus, Aß40 constituted only 1–7% of amyloid deposits while in neocortex Aß40 was completely absent (Fig. 5D). Multi-spectral analysis by confocal laser scanning microscope (CLSM) also demonstrated the absence of Aß40 (Fig. 5E and F) and a full histochemical overlap for 4G8 and Aß42-specific antibody. These observations were confirmed using other Aß40-specific [FCA3340 (34), R209 (35)] and Aß42-specific [21F12 (36), FCA3542 (34), R226 (35)] antibodies (data not shown).
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We studied the N-truncation of Aß in these amyloid deposits and confirmed whether the diffuse plaques were capable of inciting any glial reaction. Reactivity for a panel of antibodies against N-terminal Aß (6E10, 6F/3D and JRF/AßN/11), when compared with 4G8 reactivity, indicated that diffuse plaques consisted of N-truncated Aß while full-length Aß was confined to blood vessel walls, dense amyloid cores and amyloid plaques in ERC layer III and remarkably negative for deep cortical layers (Fig. 6A and B). Abundant glial and inflammatory pathology was noted in association with diffuse but mostly compact plaques using astroglial (GFAP), microglial (CD68, HLA–DR) and complement (C1q) markers. An intense glial activation was noted in many regions without plaque pathology as in white matter (Fig. 6C–F).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTS DISCUSSIONMATERIALS AND METHODSREFERENCES |
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The unusual effect of T714I on Aß40 secretion and deposition in brain allowed for the first time the study of the role of Aß40 in plaque formation and clears many tenets about AD pathophysiology. It is noteworthy that in contrast to in vitro studies where Aß42 was demonstrated to be more fibrillogenic and aggregated faster than Aß40 (37), in vivo studies in rodents point otherwise. Synthetic soluble Aß1–40, but not Aß1–42, when injected in rat brain, form fibrillar congophilic amyloid deposits (38) and transgenic mice overexpressing mutant APP and PS1, deposit predominantly Aß40 and not Aß42 in parenchymal congophilic compact plaques (39). Similarly in AD and DS patients, in cored plaques and especially in the congophilic amyloid cores, Aß40 is the predominant constituent (8,9). Although Aß40 deposition is later in the disease process (8,9), it is unlikely that a rapid disease progression in T714I patients prevented its deposition. The irrefutable argument to support this is that Aß40 was absent from vessels, where it is the predominant constituent at all disease stages (8,40–42). This is the first neuropathological proof in human of the role of Aß40 in plaque maturation where a reduced cored plaque load suggests a role for Aß40in formation of amyloid cores but certainly not in delaying disease progression.
Instead, the severity of the disease paralleled a
massive deposition of diffuse, ‘cloudy’ amyloid plaques,
composed chiefly of N-truncated Aß42. Only amyloid in dense
amyloid cores, congophilic amyloid angiopathy and amyloid plaques in ERC
layer III stained for full-length Aß. Release of p3 by APP expressing
cells during normal metabolism is long known but only recently the
contribution of N-truncated Aß deposition in diffuse plaques is being
appreciated (7,13). It has been recognized that p3 is
relatively insoluble and highly aggregatable (43,44). Furthermore, analysis of T714I clearly
demonstrates that the activity of -secretase might preclude the
formation of full-length Aß, it does not preclude the formation of
amyloid as p3. Developments of drugs targeting secretases have to consider
that partially blocking BACE (3) might switch to increased utilization of
the -secretase pathway, producing p3 which might be as neurotoxic. A
partial co-blockade of -secretase should be seriously
considered.
The presence of neuritic plaques containing
abundant Aß-derived amyloid fibrils in AD brain tissues supports
the concept that fibril accumulation per se underlies neuronal
dysfunction in AD (11,12). However, recent observations have
challenged this assumption by suggesting that earlier Aß assemblies
formed during the process of fibrillogenesis may also play a role in AD
pathogenesis (45,46). Protofibrils, metastable intermediates
in fibril formation, were shown to be toxic to cultured neurons (47). Although full
electron-microscopical data are lacking but based on Congo-red and
thioslavin-S binding, we present here novel pathological findings that
diffuse, non-congophilic nonfibrillar Aß plaques are associated
with neuritic pathology. The ‘cloudy’ diffuse plaques as a
predominant plaque type in AD partially resemble those reported for the PS1
9 patients (31–33) that present with AD and spastic
paraparesis. The prevalent, large, nonfibrillar, but non-neuritic
‘cotton wool’ plaques described in these patients are also
composed primarily of N-truncated Aß42 (33 and unpublished data),
suggesting that a heavily increased load of truncated Aß42
changes the plaque morphology. We also show here in T714I patients, with a
primarily APP etiology, a completely plaque-independent development of
dystrophic neurites and neurofibrillary pathology in few brain
regions. This suggests that while nonfibrillar pre-amyloid deposits might
have the potential to cause neuronal toxicity, development of neuritic and
amyloid pathology might be dissociated at some stage of the disease.
In conclusion, our study demonstrates the possible role of Aß40 in formation of dense cored plaques in AD and presents for the first time evidence that N-truncated Aß42 even of pre-amyloid nature might play a necessary and sufficient role to cause neuronal toxicity and cognitive decline in AD patients.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTS DISCUSSIONMATERIALS AND METHODSREFERENCES |
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AD diagnosis
Patients in family AD156 were diagnozed with AD based on neurological examination, neuropsychological testing, neuroimaging and neuropathology (R. Kleinert et al., manuscript in preparation). The mother was diagnosed at 44 years old. She had progressive memory problems and was disoriented in time. EEG showed moderate but generalized unspecific changes while CT showed brain atrophy. The proband as well as her sister had a neurological examination at 38 years old. They both suffered from severe depression. Mini metal state examination (MMSE) confirmed the presence of dementia. Single photon emission tomography showed clear hypoactivity while CT confirmed the presence of brain atrophy. In both sisters, the dementia was rapidly progressing as measured repeatedly by MMSE. For example at the age of 39 the proband scored 18/30 and her sister 11/30, at the age of 40 the scores had already dropped to 10/30 and 3/30, respectively. The actual age of onset of the symptoms was several years earlier according to the neurologists who treated the patients. Onset age was estimated 5–7 years earlier for the mother and 4–5 years earlier for the daughters. Therefore mean onset age in family AD156 was estimated to be
34 years. The
APOE genotype of the proband was E3E3, that of the sister
E2E3. The APOE genotype of the mother E2E3 was
inferred from that of the father and siblings. The proband died at 41 years
of age and had brain autopsy. The sister is still alive and is 42
years. Macroscopic examination of the brain showed gross atrophy weighing
1000 g. Sections derived from the fore-, mid- and hindbrain were
stained with haematoxylin and eosin (H&E), Nissl, Congo-red and
modified Bielshowsky. A definite diagnosis of presenile AD was
made.
Genetic analysis
Exons 16 and 17 of
APP were PCR amplified from genomic DNA of patient 156.1 using
published primer sets and PCR conditions (48) and PCR fragments were sequenced using
the Ready Reaction Rhodamine Dye Terminator Cycle Sequencing kit (Applied
Biosystems, Foster City, CA). The products were analyzed on an ABI310
capillary DNA sequencer (Applied Biosystems). The APP T714I
mutation was analyzed by TspRI digestion of PCR amplified
APP exon 17. Wild-type fragments of 354 bp were cut into two
fragments of 232 and 122 bp, respectively, whereas the T714I mutant
fragments were not cut (Fig. 1). All coding exons of PS1 and
PS2 were PCR amplified from genomic DNA using published primer
sets and screened for mutations by single strand conformational
polymorphism analysis as described by Cruts et al. (49). APOE genotype was
determined as described (50,51).
In vitro
mutagenesis
Site-directed mutagenesis was performed on wild-type
APP695 cDNA cloned in pCDNA3 (52) using the QuikChange site-directed
mutagenesis system (Stratagene, La Jolla, CA). Primers app714s,
5'-CGGTGTTGTCATAGCGATAGTGATCGTCATCACC-3',
and app714as, 5'-GGTGATGACGATCACTATCGCTATGACAACACCG-3'
were used to insert the APP T714I mutation into the construct. The
sequence of the constructs was confirmed by direct PCR sequencing of the
insertion fragment using the Taq dye terminator sequenase II
sequencing kit (Applied Biosystems). The products were analyzed on an
ABI373 automated DNA sequencer (Applied Biosystems). Mutant APP V717I were
constructed as described previously by Hendriks et al. (52).
cDNA
transfection
HEK293T cells were transiently transfected with pCDNA3
vector containing the T714I, V717I or wild-type APP695 cDNA
constructs using Fugene (Roche Diagnostics, Nutley, NJ) according to the
manufacturer’s instructions. The presence of the constructs in the
cells was confirmed by western blotting. To normalize for APP expression,
cells were lysed in 300 µl RIPA buffer (50 mM Tris, pH 8.0, 150 mM
NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS
plus complete protease inhibitors). A dilution series of a 5 µl
aliquot was separated on a 4–12% NuPAGE polyacrylamide
gel. Proteins were blotted on a PVDF membrane and immunodetection was
performed with antibody B10/4 using the Western Star Chemiluminescence
system (Tropix, Bedford, MA). The full-length APP immunoreactive band was
quantified using self written softwares for the VIDAS image analysis system
(Kontron, München, Germany) (53).
AßELISA
HEK293T cells were transfected in triplicate with
wild-type or T714I APP cDNA in a six-well plate. One day after
transfection, 1 ml OPTIMEM medium without additives was added to the
HEK293T cells and conditioned for 24 h. Medium was collected and pooled
from six transfections. A 1 ml aliquot was used for each
ELISA. Aß42 concentrations were measured by a prototype
version of the INNOTEST ß-amyloid1–42 HS ELISA
detecting Aß42 peptide (24). Aß40 was measured by
ELISA using rabbit antiserum R209 (35) as capturing antibody and biotinylated
3D6 (36) as detector
antibody, as described (24,25). Each experiment was performed in
triplicate and the results were averaged. A two-tailed unpaired
t-test was used to compare the mean level of Aß produced by
the wild-type and mutant transfectants.
Mass
spectrometric Aß analysis
In a second aliquot of
supernatant, collected as described above, proteinase inhibitors (2 mM
EDTA–Na, 10 µM leupeptin, 1 µM pepstatin A, 1 mM PMSF,
0.1 mM TLCK, 0.2 mM TPCK) were added. Aß peptides were analyzed by
immunoprecipitation/mass spectrometric Aß assay (IP/MS) as
described previously (4). The Aß peptides were
immunoprecipitated from 1.0 ml of conditioned media using mAb 4G8 (Senetek,
Maryland Heights, MO) and protein G Plus/Protein A-agarose beads
(Oncogene Science, Cambridge, MA) and analyzed using a MALDI-TOF mass
spectrometer (Voyager-DE STR BioSpectrometry Workstation, PE/PerSeptive
Biosystem, Foster City, CA). Each mass spectrum was averaged from 256
measurements and calibrated by using bovine insulin as internal mass
calibrant. For comparing the peptide levels in the conditioned media,
synthetic Aß (12–28) peptide (10 nM) was used as internal
standard and the relative peak intensity was used. Both ELISA and MALDI-TOF
mass spectrometric analysis were performed by experimenters blinded to
sample identity.
Quantification of APPs and
APPsß
Conditioned medium (5 µl) was separated on a
4–12% NuPage gel (Novex). Proteins were transferred to a PVDF
membrane, the membrane was blocked in PBS + 0.2% I-block +
0.1% Tween-20, incubated overnight at 4°C with primary
antibodies 6E10 diluted 1:2000 (for APPs) or 53/4 diluted 1:500
(for APPsß), incubated with alkaline phosphatase labeled secondary
antibody (1:4000 diluted), and detected with either Western Star
chemiluminescent reagent (Tropix) or peroxidase labeled secondary antibody
(Amersham Pharmacia, Little Chalfont,
UK).
Immunohistochemistry
Monoclonal antibody
JRF/cAb40/10 and JRF/cAb42/12 specific for the C-terminus
of Aß40 and Aß42, respectively, were raised by immunizing mice with
synthetic peptides corresponding to Aß residues 36–40 (VGGVV) or
residues 33–42 (GLMVGGVVIA) (54). Specificity of the Aß40
and Aß42 mAbs was validated by ELISA and western blotting
showing no cross reactivity. Similarly mAb JRF/AßN/11 specific
for N-terminus of Aß was raised against Aß residues 1–7
(DAEFRHD) and recognizes full-length Aß (54).
In addition, other antibodies used in this study were the following: mAb 6E10 (Senetek, Maryland Heights, MO; raised against Aß 1–17, recognizes Aß 5–11), mAb 4G8 (Senetek; Aß residues 17–24), 6F3D (Dako, Glostrup, Denmark; raised against Aß residues 7–17), mAb 21F12 (for Aß42, Innogenetics, Gent, Belgium), rabbit antisera FCA3542 and FCA3340 (34), rabbit antisera R209 (35) (Aß40) and R226 (35) (Aß42), rabbit anti-Aß1–40 (Sigma, St Louis, MO), mAb 22C11 (N-terminus APP; Roche), mAb AT8 (abnormally phosphorylated tau; Innogenetics) and anti-glial fibrillary acidic protein (GFAP; Dako), CD68 (macrophage; Dako), rabbit anti-ubiquitin (Dako), C1q complement (Dako), HLA–DR [HLA–DP,DQ,DR (Dako)], CD31 (Dako) and CD34 [QBEnd (Dako)].
Antigen retrieval for Aß immunohistochemistry was performed on sections treated with 98% formic acid for 10 min and for other antibodies as recommended by the supplier. Staining for single antigen was performed using streptavidin–biotin–horse radish peroxidase (ABC/HRP) or peroxidase–anti-peroxidase, utilizing 3'3'diaminobenzidine as a chromogen as described by Kumar-Singh et al. (55). Immunohistochemistry involving detection of more than one antigen was performed using species-specific or IgG subtype-specific secondary antibodies conjugated directly with biotin, HRP, alkaline phosphatase or galactosidase (Southern Biotechnology, Birmingham, AL). This was followed by colour development using one of the following chromogens (Roche): DAB, 3-amino-9-ethylcarbazole (AEC), Fast-red, 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium soluton (BCIP/NBT) or 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X–gal). For Aß40 immunohistochemistry, a sensitive tyramide amplification system (DuPont NEN, Boston, MA) was utilized.
Densitometric analysis
Densitometric
analysis was performed for 5 µm thick serial sections stained with
21F12, JRF/cAb42/12 and FCA3542 (for Aß42),
JRF/cAb40/10, R209 and FCA3340 (for Aß40) and 4G8,
was performed using the VIDAS image analysis system. The obtained results
were compared with staining of similar brain regions of patients with
sporadic AD cases and PS1 (I143T) related familial AD. Pixels
representing the immunocytochemical stain were counted to calculate the
size of each plaque. Also, the relative area occupied by Aß staining in
five fields from hippocampus or ERC with most intense staining and
envisaging an area of 0.7 mm2, was determined as described by
Kumar-Singh et al. (53).
Fluorescent
microscopy
For multiple labeling on a CLSM, 10 µm sections
were incubated overnight with mAb 21F12 and a JRF/cAb42/12, washed
and labeled with an anti-mouse TRITC-conjugated and an anti-rabbit
FITC-conjugated antibody (Molecular Probes, Eugene, OR). Images were
acquired with a Zeiss CLM-410 using either 488 nm line of argon single
laser or 632 nm helium–neon double laser for excitation. One
micrometer thick consecutive optical slices were captured for both
fluorochromes separately and the relative
Aß42/Aß40 content and ratios in amyloid
plaques were
determined.
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ACKNOWLEDGEMENTS |
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The authors are grateful to F. Checler for FCA3340 and FCA3542 antisera, P. Mehta for R209 and R226 antisera, M. Savage for providing 53/4, E. Van Marck for the use of the image analysis system, J.-P. Timmermans for the use of CLSM and B. Dermaut for translating the patients’ medical records. This work was supported by the Fund for Scientific Research-Flanders (FWO-F), the Interuniversity Attraction Poles (IUAP P4/17), the International Alzheimer Research Foundation (IARF), Belgium, the FWF project S7403-MOB, Austria, and Alzheimer’s Association Grant (RG1-96–070 to R.W.), USA. C.D.J. and M.C. are postdoctoral fellows of the FWO-F.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +32 3 820 2601; Fax:+32 3 820 2541; Email: cvbroeck@uia.ua.ac.be
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTS DISCUSSIONMATERIALS AND METHODSREFERENCES |
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