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Volume 17, Number 21,
Issue of November 1, 1997
pp. 8187-8193
Copyright ©1997 Society for Neuroscience
Amyloid  -Protein (A) 1-40 But Not
A1-42 Contributes to the Experimental Formation of
Alzheimer Disease Amyloid Fibrils in Rat Brain
Ryong-Woon Shin1,
Koichi Ogino2,
Akira Kondo3,
Takaomi C. Saido4,
John Q. Trojanowski5,
Tetsuyuki Kitamoto1, and
Jun Tateishi6
1 Department of Neurological Science, Tohoku University
School of Medicine, Sendai 980, Japan, 2 Cellular
Technology Institute, Otsuka Pharmaceutical Co., Tokushima 771-01,
Japan, 3 Department of Neurology, Koga General Hospital,
Miyazaki 880, Japan, 4 Department of Molecular Biology,
Tokyo Metropolitan Institute of Medical Science, Tokyo 113, Japan,
5 Department of Pathology and Laboratory Medicine,
University of Pennsylvania School of Medicine, Philadelphia,
Pennsylvania 19104-4283, and 6 Department of
Neuropathology, Neurological Institute, Kyushu University School of
Medicine, Fukuoka 812-82, Japan
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Two major C-terminal variants ending at Val40 and Ala42 constitute
the majority of amyloid  -protein (A), which undergoes postsecretory aggregation and deposition in the Alzheimer disease (AD)
brain. To probe the differential pathobiology of the two A variants,
we used an in vivo paradigm in which freshly solubilized A1-40 or A1-42 was injected into rat brains, followed by
examination using Congo red birefringence, A immunohistochemistry,
and electron microscopy. In the rat brain, soluble A 1-40 and
A1-42 formed aggregates, and the A1-40 but not the A1-42
aggregates showed Congo red birefringence. Electron microscopy revealed
that the A1-40 aggregates contained fibrillar structures similar to
the amyloid fibrils of AD, whereas the A1-42 aggregates contained nonfibrillar amorphous material. Preincubation of A1-42 solution in vitro led to the formation of birefringent
aggregates, and after injection of the preincubated A1-42, the
aggregates remained birefringent in the rat brain. Thus, a factor or
factors might exist in the rat brain that inhibit the fibrillar
assembly of soluble A1-42. To analyze the postsecretory processing
of A, we used the same in vivo paradigm and showed
that A1-40 and A1-42 were processed at their N termini to yield
variants starting at pyroglutamate, and at their C termini to yield
variants ending at Val40 and at Val39. Thus the normal rat brain could
produce enzymes that mediate the conversion of A 1-40/1-42 into
processed variants similar to those in AD. This experimental paradigm
may facilitate efforts to elucidate mechanisms of A deposition
evolving into amyloid plaques in AD.
Key words:
Alzheimer disease;
senile plaques;
amyloid -protein;
amyloid fibrils;
in vivo;
in vitro
INTRODUCTION
Deposition of amyloid -protein
(A) as amyloid fibrils or nonfibrillar amorphous aggregates in
senile plaques characterizes the Alzheimer disease (AD) brain. There
are two types of senile plaques, i.e., diffuse and dense-cored plaques.
The diffuse plaques are composed of nonfibrillar amorphous A
aggregates that are not associated with degenerative changes, whereas
the cored plaques contain abundant A fibrils that are associated
with pathological changes in the surrounding brain parenchyma (for
review, see Selkoe, 1994 ).
A peptides that are composed of 39-43 amino acids derived from the
amyloid -protein precursor (APP) (Kang et al., 1987) are produced as
soluble metabolic products of the APP, and they are constitutively
secreted into culture medium and cerebrospinal fluids (Haas et al.,
1992; Shoji et al., 1992). The majority of the secreted soluble A
species includes A1-40 and A1-42, which start at Asp1 and end
at either Val40 or Ala42, respectively (Vigo-Pelfrey et al., 1993;
Suzuki et al., 1994).
In the AD brain, these soluble A peptides undergo aggregation and
are deposited as several variants. The major N-terminal A variants
include AN1, which bears the standard N terminus, and AN3(pE),
which bears an N-terminal pyroglutamate (Mori et al., 1992; Saido et
al., 1995, 1996). Deposition of the AN3(pE) prevails at higher
density than the AN1 and precedes that of the AN1 in plaques
(Saido et al., 1995). The major C-terminal variants include AC40 and
AC42, which retain C-terminal amino acids identical to those of the
secreted A1-40 and A1-42, respectively (Mori et al., 1992;
Miller et al., 1993; Roher et al., 1993a,b). In the parenchymal amyloid
deposits, AC42 is deposited in greater density than AC40
(Iwatsubo et al., 1994, 1995), although soluble A1-40 is more
abundantly secreted than soluble A1-42 (Seubert et al., 1992; Dovey
et al., 1993; Vigo-Pelfrey et al., 1993; Suzuki et al., 1994). Thus,
these distinct A species seem to be metabolized differently and may
play different roles in the deposition of A.
Previous in vitro studies have provided important
information on the pathobiology of A1-40 and A1-42 and their
relevance to the pathological processes in AD, but there has been
little effort to extend these studies to the in vivo brain
environment of experimental animals. In view of the fact that
conditions in vitro and in vivo are quite
different, and given the remarkable discrepancies between the apparent
toxicity of A peptides in vitro versus in
vivo, it is important to undertake in vivo studies of
the fate of A peptides. Therefore, we used an in vivo
paradigm in which A1-40 or A1-42 was injected into rat brain to
dissect their differential pathobiology in AD. Herein we demonstrate
that A1-40 and A1-42 differ in their ability to form amyloid
fibrils in vivo as well as in the postinjection processing
at their C termini. Notably A1-40 but not A1-42 was competent
to form amyloid fibrils in vivo. In addition, A1-42 was
subjected to rapid C-terminal proteolysis, whereas the C-terminal
proteolysis of A1-40 was delayed. Thus, this animal model could be
used to understand the fibrillogenesis of and postsecretory processing
of A in AD.
MATERIALS AND METHODS
Human analog peptides corresponding to A1-40 and A1-42
were synthesized by solid phase methods on an Applied Biosystems (Foster City, CA) synthesizer Model 430A according to the
manufacturer's procedures. The synthesized peptides were purified by
C18 reverse-phase HPLC with the corresponding standards obtained from
Bachem (Torrance, CA) for comparison. The synthesized peptide samples
co-eluted with the corresponding standards, indicating identity with
each other. The A samples were verified further by amino acid
sequencing. The A peptides were lyophilized as stock sample and used
throughout the following experiments.
The lyophilized samples were freshly solubilized in 0.15 M
Tris buffer, pH 8.8, filtered (0.22 µm), and adjusted to 10 µg/µl (referred to hereafter as "freshly solubilized" A1-40 and
A1-42) immediately before each injection. Five microliters of these
A1-40 (10 µg/µl) or A1-42 (10 µg/µl) were injected into
the right side of the hippocampus or deep cerebral cortex of female
Sprague Dawley rats (200-300 gm; n = 50). After
different postinjection survival times, the brains removed from the
rats were fixed in 70% ethanol/0.15 M NaCl and embedded in
paraffin as reported previously (Shin et al., 1993, 1994). The
postinjection survival times included 1 d (n = 5),
1 week (n = 5), 3 weeks (n = 5), 5 weeks (n = 5), and 7 weeks (n = 5) for
A1-40 as well as for A1-42. Paraffin sections were examined by
Congo red staining and immunohistochemistry using several antibodies
that specifically recognize different epitopes in A. The antibodies
used here are summarized in Table 1. The anti-C39 was newly produced in
a rabbit against a synthetic peptide CMVGGV to which keyhole limpet
hemocyanin was conjugated, and was affinity-purified as described
previously (Saido et al., 1995). Its specificity was confirmed by dot
and Western analyses using A1-39, A1-40, A1-41, A1-42,
and A1-43 peptides. Immunostaining of AD sections using the
anti-C39 antiserum revealed that AC39 is present in diffuse and
cored plaques of the AD brain. 4G8 was used to recognize authentic
A, regardless of its processing at the N or C terminus. Anti-N1D and
anti-N3(pE) were used to analyze N-terminal processing of A, and
BC05, anti-C42, BA27, anti-C40, and anti-C39 were used to analyze
C-terminal processing of A.
Table 1.
Summary of antibodies to A
| Antibody |
Specificity |
Type |
Reference
|
|
| 4G8 |
A17-24 |
M |
Kim et
al., 1990 |
| Anti-N1D |
Standard A bearing the first N-terminal
residue, AN1D |
P |
Saido et al., 1995 |
| Anti-N3
(pE) |
Modified A in which the first and second N-terminal residues
are deleted and the third Glu is converted to pyroGlu, AN3
(pE) |
P |
Saido et al., 1995 |
| BC05 |
A ending at the
forty-second C-terminal residue, AC42 |
M |
Suzuki et al., 1994
|
| Anti-C42 |
A ending at the forty-second C-terminal residue,
AC42 |
P |
Saido et al., 1995 |
| BA27 |
A ending at the
fortieth C-terminal residue, AC40 |
M |
Suzuki et al., 1994
|
| Anti-C40 |
A ending at the fortieth C-terminal residue,
AC40 |
P |
Saido et al., 1995 |
| Anti-C39 |
A ending at the
thirty-ninth C-terminal residue, AC39 |
P |
This study |
|
|
M, Monoclonal antibody; P, polyclonal antibody.
|
|
For electron microscopic study, the same amounts of the freshly
solubilized A1-40 (10 µg/µl) and A1-42 (10 µg/µl) were
injected into the right and left sides of the same rat brain
(n = 18), respectively. After different postinjection
survival times, the rats were perfused with PBS, followed by perfusion
with 4% glutaraldehyde/0.1 M cachodylate buffer, pH 7.4, as described (Shin et al., 1993). The postinjection survival times
included 1 week (n = 8), 2 weeks (n = 5), and 3 weeks (n = 5). The brains were removed, cut
coronally, and dissected into small blocks that contained the injection
sites. The blocks were further fixed in 1% osmium tetroxide and then dehydrated through a graded series of ethanol, immersed in propylene oxide, and embedded in Epoxy resin. One micrometer semithin sections were cut and stained with toluidine blue. Selected areas were thin-sectioned with a diamond knife, stained with uranyl acetate and
lead citrate, and examined with a Hitachi H-7000 electron microscope at
75 kV as reported previously (Kondo et al., 1996).
The A peptides were tested to determine whether they were competent
to assemble into fibrils in vitro under conditions that are
known to favor A fibrillogenesis as described previously (Lorenzo
and Yankner, 1994), with minor modification. Briefly, 10 mg of the A
stock samples was dissolved in double-distilled water, filtered (0.22 µm), and diluted with the same volume of PBS to 350 µM.
The same amount of A1-42 was dissolved in double-distilled water,
filtered (0.22 µm), and adjusted to 350 µM. The A
solutions were incubated at 37°C for 5 d, followed by
centrifugation at 30,000 × g for 20 min. The resultant
pellet was resuspended in PBS to 10 µg/µl (referred to hereafter as
"in vitro preincubated" A1-40 and A1-42).
Aliquots of these samples were plated onto poly-L-lysine-coated glass plates, dried, stained with
Congo red, and viewed under polarized microscopy. Other aliquots of the
in vitro preincubated A1-40 and A1-42 peptides were
also used for injection into rat brains (n = 40) and
examined as described above, with postinjection survival times of
1 d (n = 5), 1 week (n = 5), 3 weeks (n = 5), and 5 weeks (n = 5) for
A1-40 as well as for A1-42. In addition, the A1-40 and
A1-42 peptides were freshly solubilized in the same buffer and in
the same concentration as those that were used for injection into rat
brain to test further the ability of these peptides to undergo
fibrillogenesis in vitro. After incubation at 37°C for
5 d, the A samples were plated onto poly-L-lysine-coated glass plates and examined as described
above.
RESULTS
The near-serial sections through the injection sites in the rat
brains were immunostained with 4G8 (Figs.
1A,D, 3A,D,
4A,E) to document the location of the injected
material. The A1-40 and A1-42 samples that were soluble before
injection formed 4G8-positive aggregates in the rat brain at the
earliest postinjection survival time (1 d) (Figs. 1D,
3A,D, 4A,E). Although these aggregates
persisted for a prolonged period, they were cleared from the rat brains by ~5-7 weeks postinjection. Thus, in the experimental system used
here, the temporal profiles of aggregation, persistence, and clearance
of the injected A peptides were the same for A1-40 and
A1-42, except that the abundance of the A1-40 aggregates was
greater than the A1-42 aggregates at the temporal stage between injection and final clearance.
Fig. 1.
Photomicrographs of rat brain sections showing
aggregates formed after intracerebral injection of freshly solubilized
A1-40 (A, B) and A1-42 (D, E) at
3 weeks (A, B) and 1 d (D, E)
postinjection survival times. The sections were probed by
immunohistochemistry with 4G8 (A, D) and by Congo red
staining (CR) (B, E). The preparations of
A1-40 (C) and A1-42
(F) were preincubated in vitro to
form fibrils, followed by staining with Congo red. Photomicrographs (B, C, E, F) were taken under illumination with
polarized light.
[View Larger Version of this Image (68K GIF file)]
Fig. 3.
Photomicrographs of rat brain sections showing
aggregates formed after injection of freshly solubilized A1-40
(A-C) and A1-42 (D-F)
at 1 d postinjection survival time. The sections were
immunostained with 4G8 (A, D), anti-N1D (B,
E), and anti-N3(pE) (C, F).
[View Larger Version of this Image (83K GIF file)]
Fig. 4.
Photomicrographs of rat brain sections showing
aggregates formed after injection of freshly solubilized A1-40
(A-D) and A1-42 (E-H)
at 1 d (A-C, E-H) and 3 weeks
(D) postinjection survival times. The sections
were immunostained with 4G8 (A, E), BA27
(B), anti-C39 (C, D, H),
BC05 (F), and anti-C40
(G).
[View Larger Version of this Image (59K GIF file)]
Although it is plausible that endogenous rodent A species contribute
to the A aggregates detected here, there were significant differences in the immunohistochemical and histochemical profile of the
aggregates induced by injections of A1-40 versus A1-42. Thus,
for simplicity, we consider the immunohistochemical and other data
described here to be a consequence of the injected human A.
Freshly solubilized A1-40 but not A1-42 is assembled into
amyloid fibrils in rat brain
The in vivo paradigm of extracellular injection of
freshly solubilized As in rat brain was analyzed for the ability of
these peptides to assemble into amyloid fibrils. The rat brain sections including the A aggregates were stained with Congo red. The
A1-40 aggregates demonstrated intense birefringence under polarized microscopy (Fig. 1B), indicating the formation of
amyloid fibrils by the A1-40 peptides. The birefringence in the
A1-40 aggregates appeared at 1 d postinjection survival time,
and this birefringence persisted until the aggregates were cleared away
by 5-7 weeks postinjection survival times (not shown). In contrast,
the aggregates formed by the A1-42 peptides were not birefringent
after Congo red staining at any of the postinjection survival times
(Fig. 1E), indicating that these peptides did not
form amyloid fibrils.
To confirm that the birefringent A aggregates contained amyloid
fibrils, we performed electron microscopy on sections of rat brains
injected with freshly solubilized A peptides. In the right side of
the rat brains injected with A1-40 peptides, 5-10 nm fibrillar
structures were observed (Fig.
2A,B) that appeared similar to the fibrils seen in amyloid plaques of the AD brain. However, in the left side of the rat brains injected with A1-42 peptides, only amorphous material was found (Fig. 2C,D).
Thus, these electron microscopic studies demonstrate that freshly
solubilized A1-40, but not A1-42, is assembled into amyloid
fibrils after injection into the rat brain, consistent with the results
obtained by the examination using Congo red birefringence.
Fig. 2.
Electron micrographs of rat brain injected with
freshly solubilized A1-40 (A, B) and A1-42
(C, D) at 1 week postinjection survival time. Injected
A1-40 consists of 5-10 nm fibrils, and injected A1-42 consists
of nonfibrillar amorphous material (indicated as *). Note that
A1-42 amorphous aggregates are intimately intermingled with
macrophages (C) and astrocytes (C,
D). Scale bars: A, 1 µm; B, 250 nm; C, 1 µm; D, 250 nm.
[View Larger Version of this Image (150K GIF file)]
Because previous in vitro experiments showed that both
A1-40 and A1-42 peptides self-assemble into amyloid fibrils
(Fraser et al., 1992; Bush et al., 1994; Lorenzo and Yankner, 1994; Ma et al., 1994), and biophysical experiments indicated that A1-42 forms amyloid fibrils even more readily than A1-40 (Jarret and Lansbury, 1993; Jarret et al., 1993), we examined the ability of each
of the species of A peptides to form amyloid fibrils in
vitro under two different conditions, as described in Materials and Methods. After incubation under each condition, we observed that
both A1-40 and A1-42 peptides formed Congo red-stained birefringent aggregates (Fig. 1C,F), indicating that
they assembled into amyloid fibrils. Thus, A1-40 is competent to
form amyloid fibrils in vitro and in vivo,
whereas A1-42 is competent to form amyloid fibrils in
vitro but not in vivo.
To examine the in vivo properties of these A1-40 and
A1-42 fibrils generated in vitro, we injected aliquots
of these fibrillar A samples into rat brains, and we observed
aggregates of these A fibrils that persisted in abundance much more
than aggregates of A that formed after injection of freshly
solubilized A samples at all postinjection survival times examined
(1 d to 5 weeks). The aggregates of in vitro preincubated
A1-40 and A1-42 were intensely birefringent after Congo red
staining (not shown), and they appeared similar to those produced with
injections of freshly solubilized A1-40 (Fig.
1B).
In vivo N- and C-terminal processing of freshly
solubilized A1-40 and A1-42
To gain insights into the postsecretory processing of A1-40
and A1-42 in the in vivo brain, we next examined whether
freshly solubilized As injected into the rat neocortex and
hippocampus were modified as were A deposits in amyloid plaques of
the AD brain. For this purpose, we performed immunohistochemistry using antibodies that specifically recognize different N-terminal variants of
A, including A1N and AN3(pE), as well as C-terminal variants of A, including AC42, AC40, and AC39, on rat brain sections containing aggregates of the A peptides (see Table
1 for a summary of the specificities of
these antibodies). The A aggregates were positively immunostained
with the anti-N1 as well as with the anti-N3(pE) antibodies as early as
1 d postinjection (Fig.
3B,C,E,F). Because
aggregates of injected A1-40 and A1-42 were promptly processed
at their N termini to yield the AN3(pE) variant similar to A
deposited in amyloid plaques of the AD brain (Mori et al., 1992; Saido
et al., 1995, 1996), both A1-40 and A1-42 appear to be equally
susceptible to this modification. Furthermore, the A1-42 aggregates
were positively immunostained with the BA27, the anti-C40, and the
anti-C39 antibodies at the earliest postinjection survival time (1 d)
(Fig. 4F-H),
suggesting that injected A1-42 peptides underwent rapid C-terminal
proteolysis to yield AC40 and AC39. In contrast, the aggregates
of injected A1-40 were not immunostained with the anti-C39 at
1 d postinjection survival time (Fig. 4C), but were
positively immunostained with this antibody at 1-3 weeks postinjection
survival times (Fig. 4D). This suggests that injected
A1-40 yielded AC39 after a more attenuated process of C-terminal
proteolysis. Thus, A1-40 and A1-42 are processed at their C
termini, but this processing occurs more rapidly for A1-42 than for
A1-40.
DISCUSSION
In 1987, Kang et al. first reported biochemical evidence that
A1-42/43 consists of the neuritic plaques (Kang et al., 1987). In
1993, Jarret and Lansbury suggested the "seeding" hypothesis wherein A1-42 serves as a seed for plaque formation and A1-40 is incorporated later as A progressively deposits in the AD brain (Jarret and Lansbury, 1993; Jarret et al., 1993). Since then, considerable attention has focused on the differential amyloidogenic capabilities of A species with variable C termini, especially the
AC40 and AC42 variants. Indeed, support for this concept has come
from immunohistochemical studies (Iwatsubo et al., 1994, 1995)
conducted with antibodies that specifically recognize AC40 versus
AC42 (Suzuki et al., 1994). For example, in the brains of patients
with AD or Down's syndrome, AC42 is deposited before AC40, and
AC42 is the predominant species of A in amyloid plaques at all
stages of these diseases. Furthermore, prominent accumulations of
AC42 are seen in the diffuse plaques that are thought to represent an early stage in the formation of amyloid plaques, whereas
accumulations of AC40 are more characteristic of the cored plaques
that are believed to form later in the process of amyloidogenesis.
A17-42 is a unique proteolytic fragment of A in that it is
biochemically extractable from the diffuse plaques but not from the
cored plaques (Gowing et al., 1994). Taken together, these observations
suggest that AC40 and AC42 may play distinct roles in the
progressive deposition of A in the AD brain.
The studies described here were designed to gain insight into the
differential pathobiology of A1-40 versus A1-42 in the brains
of living mammals. To this end, we studied synthetic A1-40 or
A1-42 peptides that were injected into the neocortex and
hippocampus of rats, and we showed here that these A variants
exhibited striking differences in their ability to form amyloid
fibrils. Specifically, fibrils were generated consistently from
A1-40 but not from A1-42. Although previous in vivo
studies (Rush et al., 1992; Snow et al., 1994) reported the formation
of amyloid fibrils in the rat brain after injections of A1-40, and
other in vivo studies (Waite et al., 1992) of injections of
A1-42 failed to produce amyloid fibrils, our study is novel because
it directly assessed the differential amyloidogenic capabilities of
these two important species of A in vivo, and this has
enabled us to reconcile a critical discrepancy in the literature on
amyloidogenesis in the AD brain. Indeed, our data are consistent with
studies showing that AC42 dominates in diffuse plaques with few
amyloid fibrils (Yamaguchi et al., 1989; Davies and Mann, 1993;
Iwatsubo et al., 1994, 1995), whereas AC40 is most prominent in
cored plaques with abundant amyloid fibrils (Iwatsubo et al., 1994,
1995). Thus our observations on the preferential contribution of
A1-40 to the formation of A fibrils in the rat brain suggest the
these findings may reflect authentic molecular events underlying the
fibrillogenesis of A in the AD brain. It should be emphasized,
however, that these findings do not contradict the putative role of
A1-42 in the pathogenesis of AD; this A variant is shown to be
the earliest and most abundant species of A deposited in the
amyloid-rich senile plaques in AD brains, and the production of more
A1-42 has been linked to the onset of familial AD attributable to
mutations in the presenilin and APP genes (Hardy, 1997). Therefore,
initial deposition of A1-42 is a necessary early pathological
process, but it is not sufficient to develop mature amyloid plaques
unless succeeded by further deposition of A1-40.
Although A1-42 failed to form fibrils in vivo as we
observed in the present experimental animal system, it spontaneously assembled into fibrils after preincubation in vitro, and
this is consistent in turn with previous in vitro studies
(Lorenzo and Yankner, 1994; Ma et al., 1994; Buscioglio et al., 1995). The mechanism that accounts for this differential assembly of A1-42
into fibrils in vitro but not in vivo is
currently unknown, but it is plausible that a factor or factors exist
in the rat brain that inhibit the assembly of A1-42 into amyloid
fibrils. However, such factors do not induce A1-42 fibrils formed
in vitro to disassemble after injection into the rat brain,
because these A1-42 fibrils retained their Congo red birefringence
for prolonged intervals in vivo. On the basis of the data
reported here, we speculate that our model system could be used to
identify factors that prevent the assembly of soluble A1-42 into
amyloid fibrils in the mammalian brain. For example, cells of the
macrophage/microglia lineage and glial cells in the brain could secrete
such factors into the extracellular space because these cells
accumulate around plaques in the AD brain and appear to intermingle
preferably with the A1-42 aggregates in the rat brain (our
unpublished results). Lending support for this hypothesis, a recent
in vitro study showed that microglia adhere to fibrillar
A1-42 via their scavenger receptors, followed by secretion of
reactive oxygen species from the microglia, leading to clearance of the
fibrillar A1-42 (Khoury et al., 1996). Because in vitro
experiments (Lorenzo et al., 1994) suggest that the neurotoxicity of
A is mediated by the fibrillar rather than the amorphous forms of
the peptides, and fibril-rich amyloid plaques induce the most reactive
changes in the AD brain, the assembly of A into fibrils rather than
the mere accumulation of A as amorphous deposits in the
extracellular space may be a critical event that leads to the
degeneration of neurons in AD. Thus, the model system described here
may enable the elucidation of the mechanisms that regulate the
acquisition of A neurotoxicity via fibrillogenesis in the AD
brain.
In view of current uncertainties about the biological consequences of
A deposition in the AD brain, it is important to elucidate the
mechanisms that regulate production, aggregation, and proteolysis of
A. The model system described here could be used to unravel the
molecular basis of the postsecretory processing of A. In the rat
brain, injected A1-40 and A1-42 were similarly processed at
their N termini to yield AN3(pE), whereas they were processed differently at their C termini where A1-42 was rapidly processed to
yield AC40 and AC39, but the proteolysis of A1-40 to yield AC39 occurred more slowly. Taken together, these data suggest that
the normal rat brain secretes enzymes that differentially process
A1-40 and A1-42 to yield AN3(pE), AC40, and AC39. Thus, the N- and C-terminal processing of A occurs in the rat brain,
and this processing is not a pathological process unique to the AD
brain. Additional studies of the processing and fate of human A
peptides injected into the rodent brain may clarify the mechanisms
responsible for fibrillogenesis and neurodegenerative processes in the
AD brain.
FOOTNOTES
Received June 26, 1997; revised Aug. 13, 1997; accepted Aug. 20, 1997.
This research was supported by Grants-in-Aid from the Japanese Ministry
of Education (R.-W.S, T.K.) and by a Grant from the Japan Brain
Foundation (R.-W.S, T.K.). This study was conducted in accordance with
the Guide for Animal Experimentation, Tohoku University and Tohoku
University School of Medicine. We thank Drs. K. S. Kim and H. M. Wisniewski, New York State Institute for Basic Research in
Developmental Disabilities, for providing 4G8, and Drs. M. Umemiya, S. Shibuya, and J. Higuchi, and Ms. H. Kudo, Tohoku University, for
comments and technical assistance.
Correspondence should be addressed to Ryong-Woon Shin, Department of
Neurological Science, Tohoku University School of Medicine, Seiryo-machi 2-1, Sendai 980, Japan.
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