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Regular Articles |
From the Department of Neuropathology,*
Faculty of
Medicine, University of Tokyo, Tokyo; the Department of Forensic
Medicine,
Kyoto Prefectural University of
Medicine, Kyoto; the Department of Neurology,
Tokyo Women's Medical College, Tokyo; Athena Neurosciences
Inc.,§
South San Francisco, California; the
Department of Pathology,¶
Gunma Cancer Center,
Ohta, Japan; Gunma University School of Health
Sciences,**
Maebashi, Japan; and Core Research
for Evolutional Science and Technology,||
Japan Science
and Technology Corporation, Kawaguchi, Japan
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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Aß42, although a minor Aß species, has received particular attention because (i) it has a higher aggregation potential than Aß40, a major secreted species,8 (ii) immunocytochemistry and two-site enzyme immunoassay (EIA) have revealed that Aß42 is the initially deposited species in the brain,9,10 and (iii) all APP mutations, and presenilin 1 and 2 mutations linked with familial AD (FAD), accompany increased secretion of Aß42.11-13 In fact, plasma from FAD pedigrees14,15 and Down syndrome patients,16 who invariably develop AD pathology in middle age, contains significantly higher levels of Aß42. In addition, the proportion of Aß42 in the Aß deposited in FAD brains is significantly higher than that in sporadic AD brains.17 Thus, several lines of transgenic mice incorporating mutant APP and/or presenilin genes may be excellent models of FAD.1-3
However, sporadic AD, which is far more prevalent than FAD and
is believed to be a polygenic disease, is not associated with increased
levels of Aß42 in plasma.14
It is of note that
the ApoE4 allele (
4), a strong risk factor for AD, is associated
with neither an increased number of Aß42-positive plaques nor
increased deposition of Aß42 in the brain.18,19
Nevertheless, sporadic AD patients and a substantial proportion of
elderly people exhibit extensive deposition of Aß42 in the
brain.7,20
Thus, it is reasonable to speculate
that some unidentified factors other than increased secretion of Aß42
are involved in Aß deposition in sporadic AD patients and among the
general aged population. Consequently, it is of particular importance
to investigate autopsied human brains despite potentially confounding
postmortem artifacts.
We previously quantitated the Aß levels in the cortex and subcortical regions during aging.7,20 There was a strong tendency toward Aß42 accumulation between the ages of 50 and 70 years in T4, putamen, and mamillary body, and a little later in CA1.7,20 Even in cases in which no senile plaques were immunocytochemically detected, EIA clearly showed that significant amounts of Aß42 had already accumulated.7 In contrast to Aß42, Aß40 showed no apparent age-dependent accumulation, and high levels of Aß40 were found to be associated with AD.7 In the course of this work, we noted that Aß dimer at 6~8 kd, but not Aß monomer at ~4 kd, is often prominent on the Western blot of specimens showing negligible levels of Aß42 by EIA.20 Further investigation has clarified that (i) BAN50- or BNT77-based EIA quantitates sodium dodecyl sulfate (SDS)-dissociable Aß at ~4 kd, but not SDS-stable Aß dimer at 6~8 kd, and (ii) specimens containing negligible amounts of Aß as determined by EIA often contain detectable levels of SDS-stable Aß dimer on the Western blot. Although we currently do not know the exact significance of the Aß dimer, it is possible that the SDS-stable Aß dimer accumulates very slowly and plays an important role in the initial stages of ß-amyloidogenesis in human brain.
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Materials and Methods |
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The present study is based on autopsies performed
(n = 74; 56 men, 18 women) during the period
1995–97 at the Tokyo Medical Examiner's Office (Otsuka, Tokyo), as
described previously.7,20
The ages at death of
the 74 subjects ranged from 24 to 92 years (3 at 20–29 years, 4 at
30–39 years, 17 at 40–49 years, 18 at 50–59 years, 13 at 60–69
years, 10 at 70–79 years, 8 at 80–89 years, and 1 at 92 years).
Postmortem delay ranged from 2 to 24 hours. The other source of autopsy
cases (n = 40; 28 men, 12 women) was the Gunma
Cancer Center (Ohta, Gunma); all of these cases had malignant
neoplasms. Their ages at death ranged from 40 to 81 years (5 at 40–49
years, 12 at 50–59 years, 9 at 60–69 years, 13 at 70–79 years, and 1
at 81 years) and postmortem delay ranged from 1 to 13 hours (see Table 2
).
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Cortical pieces of CA1 and T4 at the level of lateral geniculate body, approximately 80–110 mg each, were sampled from fresh brains at autopsy at the Tokyo Medical Examiner's Office and stored at -80°C until use. The attached leptomeninges and vessels were carefully dissected out. At the Gunma Cancer Center, cortical blocks were obtained from the prefrontal cortex (Brodmann 9, 10, and 11) and stored at -80°C until use. Pieces weighing approximately 200 mg were processed for EIA and Western blotting.
PDAPP transgenic mice, aged 9.3–9.7 months,1,4 were used to examine the effects of postmortem delay on the molecular form of Aß. After death, two each of 12 mice were kept at room temperature for 0, 2, 4, 6, 12, or 18 hours, then frozen at -80°C until use. The mouse brains were similarly processed for EIA and Western blotting.
Tissue Extraction
Each of the sampled pieces was homogenized with a Dounce
homogenizer (20 strokes) in 4 volumes of Tris-saline (50 mmol/L
Tris-HCl, pH 7.6, 0.15 mol/L NaCl) containing 1 mmol/L EGTA, 0.5 mmol/L
diisopropyl fluorophosphate, 0.5 mmol/L phenylmethylsulfonyl fluoride,
1 mg/L N
-p-tosyl-L-lysine chloromethyl ketone,
1 mg/L antipain, 0.1 mg/L pepstatin, and 1 mg/L leupeptin. Each
homogenate was further homogenized with a motor-driven Teflon/glass
homogenizer (20 strokes) and centrifuged at 265,000 x
g for 15 minutes on a TL 100.3 rotor in a TLX centrifuge
(Beckman, Palo Alto, CA). For the cortical blocks from the Gunma Cancer
Center, the Dounce homogenization step was omitted. The resultant
pellet, after being washed once, was further extracted with more than
100 volumes (with respect to the initial tissue volume) of 70% formic
acid. The homogenate was centrifuged on a TL 100.3 rotor as mentioned
above. The supernatant was neutralized with NaOH and trizma base and
subjected to the EIA.
Enzyme Immunoassay
The two-site EIA for Aß consisted of a combination of five monoclonal antibodies: BAN50, BNT77, 4G8, BA27, and BC05. BAN50, BNT77, or 4G8 (Senetek PLC, St. Louis, MO; the epitope is located in Aß17–24) was coated as a capture antibody on a multiwell plate (Immunoplate I, Nunc, Roskilde, Denmark). BAN50 (the epitope is located in Aß1–10) presumably captures full-length Aß, whereas BNT77 (the epitope is thought to be located in Aß11–16)21 is considered to capture all Aß species truncated up to position 10, but not p3 which starts at Aß17. Either BA27 specific for Aß40 or BC05 specific for Aß42 was used as a detection antibody following conjugation with horseradish peroxidase.
Aliquots (100 µl) of appropriately diluted formic acid extracts, as well as a synthetic peptide, Aß1–40 or Aß1–42 (Bachem, Torrance, CA), dissolved in dimethylsulfonyloxide, were applied to a BAN50-, BNT77-, or 4G8-coated multiwell plate and the loaded plate was incubated at 4°C overnight. After being rinsed with phosphate-buffered saline, the loaded wells were incubated with horseradish peroxidase-conjugated BA27 or BC05 at room temperature for 6 hours. Bound enzyme activity was measured using the TMB Microwell Peroxidase Sub- strate System (Kirkegaard & Perry Labs, Gaithersburg, MD). For the insoluble Aß42, the detection limit of EIA was 12 pmol/g wet weight.22
Western Blotting
Small aliquots (10 µl) of the formic acid extracts of the insoluble fractions were dried by Speed Vac (Savant Instruments, Farmingdale, NY), and solubilized with sample buffer (50 mmol/L Tris-HCl (pH 6.8), 12% glycerol, 2% SDS, 2.5% mercaptoethanol, 4 mol/L urea). These samples were subjected to Tris/tricine gel electrophoresis and the separated proteins were blotted onto a nitrocellulose membrane (pore size 0.22 µm, Schleicher & Schuell, Dassel, Germany). The blot, after heat treatment,23 was incubated with BAN50, BA27, BC05, or BC65 (specific for Aß43).24 After washing with Tris-saline-based buffer, the blot was further incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Transduction Laboratories, Lexington, KY). Bound antibodies were visualized using the enhanced chemiluminescence system (Amersham, Buckingham, UK). This modified version of Western blotting23 detected as little as 10 pg (2.5 fmol) of Aß1–42 or Aß1–40 per lane.
Besides specimens, synthetic Aß1–40 or 1–42 (10, 20, 50, and 100 pg) was loaded onto each gel for Western blot quantitation of Aß. SDS-stable Aß dimer was quantitated using a standard curve for SDS-dissociable Aß (synthetic Aß) and the concentration was expressed as the Aß monomer equivalent. Thus it was postulated that the blotting efficiency and BA27 or BC05 reactivity of SDS-stable dimer are the same as those of SDS-dissociable Aß. Quantitation of enhanced chemiluminescence bands of interest was performed with a model GS-700 imaging densitometer on Molecular Analyst software (Bio-Rad Laboratories, Hercules, CA).
Aß Immunocytochemistry
The formalin-fixed cortical blocks from the Tokyo Medical Examiner's Office were dehydrated and embedded in paraffin in a routine manner and cut into 6-µm-thick sections. Sections were immunostained with 4G8 (Senetek PLC; specific for Aß17–24) by the avidin-biotin method (Vectastain Elite, Vector Laboratories, Burlingame, CA), after formic acid treatment.7
The cortical blocks from the Gunma Cancer Center were sliced to ~5 mm in thickness and fixed in 4% paraformaldehyde or 10% formalin in phosphate buffer for 24–48 hours at 4°C. Sections, 6 µm thick, were similarly immunostained with Aß polyclonal antibodies.25
Apolipoprotein E Genotyping
Typing of the apolipoprotein E genotype was performed using the polymerase chain reaction (PCR) as described previously.26
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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When Aß is extracted with formic acid from the insoluble
fraction of aged or AD brains, three major molecular forms of Aß40 or
Aß42 were observed on the Western blot: Aß monomer at ~4 kd,
dimer at ~6–8 kd, and larger oligomers and a smear (Figure 1)
. The latter two cannot be dissociated
into 4-kd monomer with SDS or other harsh denaturants including
guanidine hydrochloride (see Figure 2
).
Although these two Aß species are not yet fully characterized, our
data suggest their interrelationship: when Aß monomer is present on
the Western blot of a given brain homogenate, Aß dimer can be also
detected. In the insoluble fraction of human brain homogenate, the
amount of SDS-stable Aß dimer usually exceeds that of the dissociable
Aß form (Figure 1)
.
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.
BAN50- or BNT77-based EIA was found to specifically capture dissociable
Aß40 and Aß42 species, but not SDS-stable dimer or oligomer (Figure 2)
. Thus, the BNT77-based EIA values most likely represent the
levels of SDS-dissociable Aß species, but not of SDS-stable Aß
dimer or oligomer. In contrast, 4G8 captured both dissociable Aß and
SDS-stable Aß dimer. This is consistent with our previous observation
that 4G8 preferentially labeled SDS-stable dimer on the blot (Shinkai
Y, Morishima-Kawashima M, Ihara Y, unpublished observation). SDS-Stable Aß Dimer in Specimens in which Aß is Undetectable by EIA
We examined whether specimens containing negligible levels (below
the detection limit of 12 pmol/g wet weight) of Aß42 by EIA contain
SDS-stable Aß dimer according to the sensitive Western
blotting.23
All these cases from two facilities
showed no immunocytochemically detectable senile plaques in adjacent
sections, as partly described before.7
In a
number of such specimens very prominent bands of Aß dimer at 6~8 kd
were seen (Figure 3, A and B)
, but Aß
monomer at ~4 kd was absent or scarcely detectable, an observation
consistent with the above-described characteristics of the EIA. Because
of the presence of a trace amount of the Aß dimer in the sample
(Table 1
, A and B
, and
Table 2
), we were unable to microsequence
the molecule. However, we consider that the 6~8-kd band represents
Aß dimer because (i) BAN50, 4G8, and BC05 labeled the band on the
blot (see below), (ii) truly end-specific BA27 (Morishima-Kawashima M
and Ihara Y, unpublished observations) often labeled a band at the same
position, (iii) occasionally, above the 6~8-kd band, a 12-kd band was
observed, suggesting the presence of Aß trimer (Figure 1
, Figure 3, A and B
), and thus strengthening the argument that the band at 6~8 kd
represents Aß dimer, and (iv) Western blots of many specimens showed
that there is a transition from 6~8-kd band alone, to appearance of
an additional band at ~4 kd, and finally to the mixture most commonly
seen in aged brain in which the 6~8-kd band is considered to
represent SDS-stable Aß dimer (data not shown; Figure
1).
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, 94367 in Table 1B
, and
21 and 40 in Table 2
) in which Aß was undetectable by EIA,
Aß42 monomer was detected on the blot in addition to the SDS-stable
dimer. This may be partly due to the difference in sensitivity of the
two assay methods: for quantitation of insoluble Aß Western blotting
is more sensitive than EIA, which requires a neutralization
step22
that results in extensive dilution
of the formic acid extract.
In T4, 26 cases showed negligible Aß levels by EIA. Six cases had
only Aß40 dimer, 2 cases only Aß42 dimer, and 5 cases had both;
thus, there were 13 cases with Aß40 or Aß42 dimer (50%; Table 1A
).
CA1 shows somewhat different characteristics in the Aß accumulation.
As assessed by EIA quantitation, as compared to T4, Aß accumulation
starts a little later.7
There were 32 cases in
which Aß in CA1 fell below the detection limit by EIA. Among them, 2
cases had only Aß40 dimer, 7 cases only Aß42 dimer, 6 cases had
both; thus, 15 cases had Aß40 or Aß42 dimer (47%; Table 1B
).
To exclude the possibility that the dimer is generated postmortem,
cases from a local cancer hospital were similarly examined (Table 2)
.
These cases were generally autopsied shortly after death (see Table 2
).
Even the specimens frozen very shortly after death contained Aß
dimer, strongly suggesting that the Aß dimer is not an artifact
generated postmortem. In 22 cases, insoluble Aß42 was below the
detection limit. One of these cases had only Aß40 dimer, 8 cases had
only Aß42 dimer, and 6 cases had both; thus, there were 15 cases with
Aß dimer (68%; Table 2
).
We also examined PDAPP transgenic mice aged 9.3–9.7 months at
death, an age at which amyloid deposition is apparent (Table 3)
.4
The transgenic
mice were kept at room temperature up to 18 hours after death. Up to 12
hours, Aß dimer was barely observed in the transgenic mice (Figure 3C)
. One of the mice kept for 18 hours was found to contain larger
amounts of Aß dimer than of Aß monomer at 4 kd (data not shown).
Most interestingly, in these transgenic mice, with the one exception
mentioned above, only a trace amount of SDS-stable Aß dimer was
observed, whereas there was a prominent band at ~4 kd representing
SDS-dissociable Aß on the blot (Figure 3C)
. This result can also
exclude the possibility that the SDS-stable dimers are generated during
evaporation of formic acid, a step required for SDS polyacrylamide gel
electrophoresis.
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We examined whether the appearance of SDS-stable dimer is age-dependent in the present two autopsy series. In T4, from the autopsy series at Tokyo Medical Examiner's Office, for ages at death < 50 years, SDS-stable Aß40 and Aß42 dimers were detected in 6 (46%) and 2 (15%) of 13 cases, respectively. For ages 50–59, SDS-stable Aß40 and Aß42 dimers were detected in 4 (44%) and 3 (33%) of 9 cases, respectively. For ages 60–69, SDS-stable Aß40 and Aß42 dimers were detected in 2 (67%) and 2 (67%) of 3 cases, respectively. The incidence of Aß42 dimer, but not Aß40 dimer, tended to increase with age but was not statistically significant (Mantel extension test, P < 0.10).
In CA1 from the same series, for ages at death < 50 years, SDS-stable Aß40 and Aß42 dimers were detected in 2 (15%) and 2 (15%) of 13 cases, respectively. For ages 50–59 years, SDS-stable Aß40 and Aß42 dimers were detected in 5 (42%) and 7 (58%) of 12 cases, respectively. For ages 60–69 years, SDS-stable Aß40 and Aß42 dimer were detected in 1 (17%) and 4 (67%) of 6 cases, respectively. This age-dependent increase in the incidence of Aß42 dimer but not of Aß40 dimer in CA1 was statistically significant (Mantel extension test, P < 0.02).
In the prefrontal cortex from the autopsy series at the Gunma Cancer
Center, for ages at death < 50 years, SDS-stable Aß40 and 42
dimer were detected in 0 (0%) and 1 (50%) of 2 cases, respectively.
For ages 50–59 years, the SDS-stable Aß40 and Aß42 dimers were
detected in 2 (40%) and 2 (40%) of 5 cases, respectively. For ages
60–69 years, SDS-stable Aß40 and Aß42 dimers were detected in 2
(33%) and 5 (83%) of 6 cases, respectively. For ages 70–79 years,
SDS-stable Aß40 and Aß42 dimers were detected in 3 (50%) and 6
(100%) of 6 cases, respectively. This age-dependent increase in the
incidence of the Aß dimer in the prefrontal cortex was statistically
significant (Mantel extension test, P < 0.05).
Although the number of cases was small, the presence of Aß dimer is
presumably unrelated to ApoE genotypes (Table 1
, A and B
, and Table 2
).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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The unexpected finding that BAN50 or BNT77 cannot capture the dimer but
4G8 can raises the possibility that the 6~8-kd band represents the
dimer of p3 (Aß17–42). However, this is unlikely because the
6~8-kd band in a number of specimens examined was also labeled with
BAN50 (the epitope is located in Aß1–10; see Figure 1C
). Apparently,
this conflicts with its capturing characteristic in EIA.
SDS-denatured Aß dimers on the Western blot may not take the same
conformation as those in diluted guanidine hydrochloride. Possibly, the
latter solution leads SDS-stable Aß dimers and dissociable Aß to
take more native conformations. Thus, the BAN50 immunoreactivity with
the 6~8-kd band on the blot is presumably created by the use of SDS.
The carboxyl terminus of the SDS-stable Aß dimer is not uniform;
generally, a large proportion of the dimers reacts with BC05 but not
with BA27, whereas a small proportion of the dimers is labeled with
BA27 and not with BC05. Predominance of BC05 or BA27 immunoreactivity
in each case appears to be unrelated to postmortem delay. When the
dimer in a given specimen is reactive with both antibodies, usually a
fast-migrating part of the band was BC05-reactive and a slow-migrating
part was BA27-reactive (see Figures 1 and 2
). Thus, apparently there
are two forms of the SDS-stable Aß dimer: species ending at Aß40 or
Aß42 exist presumably as homodimer. At present we do not have a
proper explanation for the presence of two Aß dimers in EIA-negative
brains. One possible explanation would be that the generated SDS-stable
Aß42 dimers are rapidly converted to Aß40 dimers through cleavage
with a specific carboxyl dipeptidase in some brains, but very slowly in
other brains. The activity of the carboxyl dipeptidase may be
relatively high in the brain because a significant proportion of
synthetic Aß1–42 injected into rat brain is rapidly converted to
Aß40.31
Related to this, one may point to the
possibility that SDS-stable Aß42 dimer is generated from a potential
precursor, Aß43 dimer, by the action of another carboxyl peptidase.
However, we were unable to detect BC65 immunoreactivity on the blot
(data not shown). We are still not certain about whether the formation
of Aß42 dimer is indeed age-dependent, whereas that of Aß40 dimer
is not. To clarify this point, a much larger number of cases must be
carefully studied.
The presence of soluble SDS-stable dimer was previously reported in CSF,32 and we have independently confirmed the presence of SDS-stable Aß40 dimer in CSF by Western blotting in a substantial proportion of aged control subjects and AD patients (Shinkai Y, Morishima-Kawashima M, Arai H, Ihara Y, unpublished observations). This may suggest that only Aß42 dimer, not Aß40 dimer, is pathogenic. It was also reported that the soluble fraction of AD brain contains SDS-stable Aß dimer and ApoE complexes.33 On the other hand, some cultured cells (CHO cells) secrete SDS-stable Aß dimer and trimer34 and transfection of mutant presenilin 1 or 2 to the cells, compared with that of wild-type presenilins, enhances SDS-stable oligomerization of the secreted Aß.35,36 A remarkable characteristic of this in vitro phenomenon is the oligomerization of Aß at nanomolar or subnanomolar concentrations of Aß, very close to physiological concentrations of Aß in the extracellular space.
Although we cannot exclude the possibility that SDS-stable Aß dimer is produced within the cell and released, it is attractive to postulate that SDS-stable Aß dimer is generated in the extracellular space of the brain from dissociable Aß constitutively secreted from brain cells. It should be noted that Aß40 (and presumably also Aß42) exists as dimer under physiological conditions.37,38 In this context, it is of particular interest that oligomerization of Aß, including dimerization, is enhanced in conditioned culture media, and that the addition of Congo red blocks the oligomerization.36 The conditioned media appear to contain a factor or factors that enhance oligomerization. Perhaps one of the factors is Aß42 itself because the culture media of mutant APP or presenilin 1- or 2-transfected cells that enhance Aß oligomerization are known to contain increased levels of Aß42.36
Thus, it is possible that SDS-stable dimer and oligomer are generated from secreted dissociable Aß in the extracellular space under influences of many factors. At the very initial stage of Aß accumulation the concentrations of soluble SDS-stable Aß dimer and oligomers may increase. This may be because of increased Aß, particularly Aß42, production and/or decreased Aß degradation. As the clearance becomes more defective with age, soluble SDS-stable Aß oligomers reach saturation level and is deposited, a step which makes clearance or elimination more difficult. An alternative but not mutually exclusive possibility is that SDS-stable Aß dimer may have a stronger affinity to extracellular matrix in brain. Thus, the presence of SDS-stable Aß dimer may reflect the unusually slow process in the Aß accumulation in the human brain.
In the above context, it is particularly intriguing that a quite recent
report describes the neurotoxicity of diffusible, nonfibrillar
(SDS-stable) Aß1–42 oligomers.39
These
oligomers are claimed to show potent neurotoxicity at nanomolar
concentrations, through a particular (as yet unidentified) cell-surface
receptor, and the activation of fyn, a protein tyrosine kinase of the
src family.39
Furthermore, it has been reported
that incubation of rat hippocampal slices with these oligomers prevents
long term potentiation before signs of neuronal degeneration
appear.39
Related to this, it is rather
surprising to note that a substantial proportion of CA1 specimens
similar to that of T4 specimens already contain SDS-stable Aß dimers
(Table 1A and B)
. This indicates that, although CA1 is the site least
affected by the deposition of Aß (SDS-dissociable
Aß),7
SDS-stable Aß dimers appear in CA1 as
early as in T4, one of the most affected sites. This may further
suggest that there are two stages for ß-amyloid deposition in humans:
initial deposition of SDS-stable Aß dimers followed by
SDS-dissociable Aß. Presumably, subsequent deposition of
SDS-dissociable Aß is significantly delayed in CA1 for unknown
reasons. If diffusible Aß42 oligomers were indeed toxic, this
might explain why a number of neurofibrillary tangles in CA1 are
already present in the stage showing no accumulation of SDS-dissociable
Aß as judged by EIA (Funato H and Ihara Y, unpublished observations).
Thus, the major issue is whether the very low levels of SDS-stable Aß
dimer in the transgenic mice is related to the absence of neuronal
loss6
that defines the degree of dementia in
humans.40
Taken together, the findings of the
present study suggest the possibility that SDS-stable dimers play
important roles in ß-amyloidogenesis in aged human brain and the
neurodegeneration of AD.
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Note Added in Proof |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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Acknowledgements |
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Footnotes |
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Supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (No.09835003 to MM) and the Ministry of Health and Welfare of Japan.
Accepted for publication September 12, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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4 allele dosage in sporadic Alzheimer's disease. Brain Res 1997, 748:250-252[Medline]
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T. Yamazaki, T.-Y. Chang, C. Haass, and Y. Ihara Accumulation and Aggregation of Amyloid beta -Protein in Late Endosomes of Niemann-Pick Type C Cells J. Biol. Chem., February 2, 2001; 276(6): 4454 - 4460. [Abstract] [Full Text] [PDF]
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S. Scheuermann, B. Hambsch, L. Hesse, J. Stumm, C. Schmidt, D. Beher, T. A. Bayer, K. Beyreuther, and G. Multhaup Homodimerization of Amyloid Precursor Protein and Its Implication in the Amyloidogenic Pathway of Alzheimer's Disease J. Biol. Chem., August 31, 2001; 276(36): 33923 - 33929. [Abstract] [Full Text] [PDF]
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