QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

HOME  |   SEARCH  |   ARCHIVE  |   SUBSCRIBE  |   CONTACT  |   HELP

This Article Abstract Full Text (PDF) Submit an eLetter Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (46) Citing Articles via Google Scholar Google Scholar Articles by Shin, R.-W. Articles by Tateishi, J. Search for Related Content PubMed PubMed Citation Articles by Shin, R.-W. Articles by Tateishi, J. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Medline Plus Health Information Alzheimer's Disease Hazardous Substances DB C.I. DIRECT RED 28

 Previous Article  |  Next Article 

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.

---

REFERENCES

• Buscioglio J, Lorenzo A, Yeh J, Yankner BA (1995) -amyloid fibrils induce tau phosphorylation and loss of microtubule binding. Neuron 14:879-888[Web of Science][Medline].
• Bush AI, Pettingell WH, Multhaup G, Paradis MD, Vonsattel JP, Gusella JF, Beyreuther K, Masters CL, Tanzi RE (1994) Rapid induction of Alzheimer A amyloid formation by zinc. Science 265:1464-1467[Abstract/Free Full Text].
• Davies CA, Mann DM (1993) Is the "preamyloid" of diffuse plaques in Alzheimer's disease really nonfibrillar? Am J Pathol 143:1594-1605[Abstract].
• Dovey HF, Suomesaari-Chrisler S, Lieberburg I, Sinha S, Kiem PS (1993) Cells with a familial Alzheimer's disease mutation produce authentic -peptide. NeuroReport 4:1039-1042[Web of Science][Medline].
• Fraser PE, Nguyen JT, Inouey H, Surewicz WK, Selkoe DJ, Podlisny MB, Kirschner DA (1992) Fibril formation by primate, rodent, and Dutch-hemorrhagic analogues of Alzheimer amyloid -protein. Biochemistry 31:10716-10723[Medline].
• Gowing E, Roher AE, Woods AS, Cotter RJ, Chaney M, Little SP, Ball MJ (1994) Chemical characterization of Ab17-42 peptide, a component of diffuse amyloid deposits of Alzheimer disease. J Biol Chem 269:10987-10990[Abstract/Free Full Text].
• Haas C, Schlossmacher MG, Hung AY, Vigo-Pelfrey C, Mellon A, Ostaszewski BL, Lieberburg I, Koo EH, Schenk D, Teplow DB, Selkoe DJ (1992) Amyloid -peptide is produced by cultured cells during normal metabolism. Nature 359:322-325[Medline].
• Hardy J (1997) Amyloid, the presenilins and Alzheimer's disease. Trends Neurosci 20:154-159[Web of Science][Medline].
• Iwatsubo T, Odaka A, Suzuki N, Mizusawa H, Nukina N, Ihara Y (1994) Visualization of A42(43) and A40 in senile plaques with end-specific A monoclonals: evidence that an initially deposited species is A42(43). Neuron 13:45-53[Web of Science][Medline].
• Iwatsubo T, Mann DMA, Odaka A, Suzuki N, Ihara Y (1995) Amyloid  protein (A) deposition: A42 (43) precedes A40 in Down syndrome. Ann Neurol 37:294-299[Web of Science][Medline].
• Jarret JT, Lansbury Jr PT (1993) Seeding "one-dimensional crystallization" of amyloid: a pathogenetic mechanism in Alzheimer's disease and scrapie? Cell 73:1055-1058[Web of Science][Medline].
• Jarret JT, Berger EP, Lansbury Jr PT (1993) The carboxy terminus of the  amyloid protein is critical for the seeding of amyloid formation: implications for the pathogenesis of Alzheimer's disease. Biochemistry 32:4693-4697[Medline].
• Kang J, Lemaire H-G, Multhaup G, Beyreuther K, Muller-Hill B (1987) The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor. Nature 325:733-736[Medline].
• Khoury JEI, Hickman SE, Christian AT, Cao L, Silverstein SC, Loike JD (1996) Scavenger receptor-mediated adhesion of microglia to -amyloid fibrils. Nature 382:716-719[Medline].
• Kim KS, Wen GY, Bancher C, Chen CMJ, Sapienza VJ, Hong H, Wisniewski HM (1990) Detection and quantitation of amyloid -peptide with two monoclonal antibodies. Neurosci Res Commun 7:113-122.
• Kondo A, Baba S, Iwaki T, Harai H, Koga H, Kimura T, Takamatsu J (1996) Hyperbaric oxygenation prevents delayed neuronal death following transient ischemia in the gerbil hippocampus. Neuropathol Appl Neurobiol 22:350-360[Web of Science][Medline].
• Lorenzo A, Yankner BA (1994) -Amyloid neurotoxicity requires fibril formation and is inhibited by Congo red. Proc Natl Acad Sci USA 91:12243-12247[Abstract/Free Full Text].
• Ma J, Yee A, Brewer Jr HB, Das S, Potter H (1994) Amyloid-associated proteins 1-antichymotrypsin and apolipoprotein E promote assembly of Alzheimer -protein into filaments. Nature 372:92-94[Medline].
• Miller DL, Papayannoopoulos IA, Styles J, Bobin SA, Lin YY, Biemann K, Iqbal K (1993) Peptide compositions of the cerebrovascular and senile plaque core amyloid deposits of Alzheimer's disease. Arch Biochem Biophys 301:41-52[Web of Science][Medline].
• Mori H, Takio K, Ogawara M, Selkoe DJ (1992) Mass spectrometry of purified amyloid  protein in Alzheimer's disease. J Biol Chem 267:17082-17086[Abstract/Free Full Text].
• Roher AE, Lowenson JD, Clarke S, Wolkow C, Wang R, Cotter RJ, Reardon IM, Zurcher-Neely HA, Heinrikson RL, Ball MJ, Greenberg BD (1993a) Structural alterations in the peptide backbone of -amyloid core protein may account for its deposition and stability in Alzheimer's disease. J Biol Chem 268:3072-3083[Abstract/Free Full Text].
• Roher AE, Lowenson JD, Clarke S, Wood AS, Cotter RJ, Gowing E, Ball MJ (1993b) -amyloid-(1-42) is a major component of cerebrovascular amyloid deposits: implications for the pathology of Alzheimer disease. Proc Natl Acad Sci USA 90:10836-10840[Abstract/Free Full Text].
• Rush DK, Aschmies S, Merriman MC (1992) Intracerebral -amyloid (25-35) produces tissue damage. Is it neurotoxic? Neurobiol Aging 13:591-594[Web of Science][Medline].
• Saido TC, Iwatsubo T, Mann DMA, Shimada H, Ihara Y, Kawashima S (1995) Dominant and differential deposition of distinct -amyloid peptide species, AN3(pE), in senile plaques. Neuron 14:457-466[Web of Science][Medline].
• Saido TC, Yamao-Harigaya W, Iwatsubo T, Kawashima S (1996) Amino- and carboxyl-terminal heterogeneity of -amyloid peptides deposited in human brain. Neurosci Lett 215:173-176[Web of Science][Medline].
• Selkoe DJ (1994) Normal and abnormal biology of the -amyloid precursor protein. Annu Rev Neurosci 17:489-517[Web of Science][Medline].
• Seubert P, Vigo-Pelfrey C, Esch F, Lee M, Dovey H, Davis D, Sinha S, Schlossmacher M, Whaley J, Swindlehurst C, McCormack R, Wolfert R, Selkoe D, Lieberburg I, Schenk D (1992) Isolation and quantification of soluble Alzheimer's -peptide from biological fluids. Nature 359:325-327[Medline].
• Shin R-W, Bramblett GT, Lee VM-Y, Trojanowski JQ (1993) Alzheimer disease A68 proteins injected into rat brain induce codeposits of -amyloid, ubiquitin, and 1-antichymotrypsin. Proc Natl Acad Sci USA 90:6825-6828[Abstract/Free Full Text].
• Shin R-W, Lee VM-Y, Trojanowski JQ (1994) Aluminum modifies the properties of Alzheimer's disease PHF proteins in vivo and in vitro. J Neurosci 14:7221-7233[Abstract].
• Shoji M, Golde TE, Ghiso J, Cheung TT, Estus S, Schaffer LM, Cai X-D, McKay D, Tintner R, Frangione B, Younkin SG (1992) Production of the Alzheimer amyloid  protein by normal proteolytic processing. Science 258:126-129[Abstract/Free Full Text].
• Snow AD, Sekiguchi R, Nochlin D, Fraser P, Kimata K, Mizutani A, Arai M, Scherier WA, Morgan DG (1994) An important role of heparan sulfate proteoglycan (Perlecan) in a model system for the deposition and persistence of fibrillar A-amyloid in rat brain. Neuron 12:219-234[Web of Science][Medline].
• Suzuki N, Cheung TT, Cai X-D, Odaka A, Otvos Jr L, Eckman C, Golde TE, Younkin SG (1994) An increased percentage of long amyloid  protein secreted by familial amyloid  protein precursor (APP717) mutants. Science 264:1336-1340[Abstract/Free Full Text].
• Vigo-Pelfrey C, Lee D, Keim P, Lieberburg I, Schenk DB (1993) Characterization of -amyloid peptide from human cerebrospinal fluid. J Neurochem 61:1965-1968[Web of Science][Medline].
• Waite J, Cole GM, Frautschy SA, Conner DJ, Thal LJ (1992) Solvent effects of beta protein toxicity in vivo. Neurobiol Aging 13:595-600[Web of Science][Medline].
• Yamaguchi H, Nakazato Y, Hirai S, Shoji M, Harigaya Y (1989) Electron micrograph of diffuse plaques: initial stage of senile plaque formation in the Alzheimer brain. Am J Pathol 135:593-597[Abstract].

---

Copyright ©1997 Society for Neuroscience   0270-6474/1997/178187-07$05.00/0

This article has been cited by other articles:

				J. D. Best, M. T. Jay, F. Otu, J. Ma, A. Nadin, S. Ellis, H. D. Lewis, C. Pattison, M. Reilly, T. Harrison, et al. Quantitative Measurement of Changes in Amyloid-{beta}(40) in the Rat Brain and Cerebrospinal Fluid following Treatment with the {gamma}-Secretase Inhibitor LY-411575 [N2-[(2S)-2-(3,5-Difluorophenyl)-2-hydroxyethanoyl]-N1-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-L-alaninamide] J. Pharmacol. Exp. Ther., May 1, 2005; 313(2): 902 - 908. [Abstract] [Full Text] [PDF]

				K. Murakami, K. Irie, A. Morimoto, H. Ohigashi, M. Shindo, M. Nagao, T. Shimizu, and T. Shirasawa Neurotoxicity and Physicochemical Properties of A{beta} Mutant Peptides from Cerebral Amyloid Angiopathy: IMPLICATION FOR THE PATHOGENESIS OF CEREBRAL AMYLOID ANGIOPATHY AND ALZHEIMER'S DISEASE J. Biol. Chem., November 14, 2003; 278(46): 46179 - 46187. [Abstract] [Full Text] [PDF]

				J. P. Melchor, R. Pawlak, and S. Strickland The Tissue Plasminogen Activator-Plasminogen Proteolytic Cascade Accelerates Amyloid-{beta} (A{beta}) Degradation and Inhibits A{beta}-Induced Neurodegeneration J. Neurosci., October 1, 2003; 23(26): 8867 - 8871. [Abstract] [Full Text] [PDF]

				S. Kumar-Singh, C. De Jonghe, M. Cruts, R. Kleinert, R. Wang, M. Mercken, B. De Strooper, H. Vanderstichele, A. Lofgren, I. Vanderhoeven, et al. Nonfibrillar diffuse amyloid deposition due to a {gamma}42-secretase site mutation points to an essential role for N-truncated A{beta}42 in Alzheimer's disease Hum. Mol. Genet., November 1, 2000; 9(18): 2589 - 2598. [Abstract] [Full Text] [PDF]

				R. P. Mason, R. F. Jacob, M. F. Walter, P. E. Mason, N. A. Avdulov, S. V. Chochina, U. Igbavboa, and W. G. Wood Distribution and Fluidizing Action of Soluble and Aggregated Amyloid beta -Peptide in Rat Synaptic Plasma Membranes J. Biol. Chem., June 25, 1999; 274(26): 18801 - 18807. [Abstract] [Full Text] [PDF]

				M. Enya, M. Morishima-Kawashima, M. Yoshimura, Y. Shinkai, K. Kusui, K. Khan, D. Games, D. Schenk, S. Sugihara, H. Yamaguchi, et al. Appearance of Sodium Dodecyl Sulfate-Stable Amyloid ß-Protein (Aß) Dimer in the Cortex During Aging Am. J. Pathol., January 1, 1999; 154(1): 271 - 279. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit an eLetter Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (46) Citing Articles via Google Scholar Google Scholar Articles by Shin, R.-W. Articles by Tateishi, J. Search for Related Content PubMed PubMed Citation Articles by Shin, R.-W. Articles by Tateishi, J. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Medline Plus Health Information Alzheimer's Disease Hazardous Substances DB C.I. DIRECT RED 28

Home  |   Search  |   Archive  |   Subscribe  |   Contact  |   Help