©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Amino-terminal Deletions Enhance Aggregation of -Amyloid Peptides in Vitro(*)

(Received for publication, April 17, 1995; and in revised form, August 17, 1995)

Christian J. Pike (§) Michael J. Overman Carl W. Cotman

From the Department of Psychobiology, Institute for Brain Aging and Dementia, University of California, Irvine, California 92717-4550

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

beta-Amyloid protein, which assembles into pathological aggregates deposited in Alzheimer's disease brain tissue, exhibits N-terminal heterogeneity both in vitro and in vivo. To investigate the effects of this N-terminal heterogeneity on the assembly characteristics and biophysical properties of beta-amyloid, we synthesized a series of peptides with progressively shortened N termini (initial residues at positions beta1, beta4, beta8, beta12, and beta17) and C termini extending to residue beta40 or beta42. We report that peptides with N-terminal deletions exhibit enhanced peptide aggregation relative to full-length species, as quantitatively assessed by sedimentation analyses. Overall, sedimentation levels were greater for peptides terminating at residue beta42 than for those terminating at residue beta40. To determine if established biophysical features of the full-length protein were maintained in the truncated peptides, structural and bioactive properties of these peptides were examined and compared. Full-length and truncated peptides exhibiting aggregation showed circular dichroism spectra consistent with predominant beta-sheet conformation, fibrillar morphology under transmission electron microscopy, and significant toxicity in cultures of rat hippocampal neurons. These data demonstrate that N-terminal deletions enhance aggregation of beta-amyloid into neurotoxic, beta-sheet fibrils and suggest that such peptides may initiate and/or nucleate the pathological deposition of beta-amyloid.


INTRODUCTION

beta-Amyloid (Abeta) (^1)is a normal, soluble protein 40-42 amino acid residues in length that, in neuropathological conditions such as Alzheimer's disease (AD), self-assembles into insoluble fibrils, forming characteristic extracellular deposits termed senile plaques(1) . Since a variety of histological, molecular genetic, and in vitro and in vivo studies provide evidence consistent with the possibility that Abeta significantly contributes to the initiation and/or progression of neurodegenerative changes in AD(2, 3, 4) , investigation of the production, assembly, and bioactivity of Abeta is crucial for the successful understanding of and therapeutic intervention in AD and related disorders.

Abeta is derived from proteolytic processing of its precursor, Abeta precursor protein (AbetaPP), by least two distinct and incompletely defined pathways (see (5) for review). In the secretory pathway, transmembrane AbetaPP is cleaved by alpha-secretase between beta16 and beta17(6) , thus precluding the formation of full-length Abeta (beta1-40/42) but generating a 3-kDa beta17-40/42 fragment(7, 8, 9, 10) . In the endosomal/lysosomal pathway, AbetaPP is degraded into several C-terminal fragments containing the complete Abeta sequence(11, 12) that require additional cleavage at the Abeta amino (beta-secretase) and carboxyl (-secretase) termini to generate Abeta. Although the predominant form of Abeta contains the beta1-40 sequence, significant N- and C-terminal heterogeneity has been reported recently in studies of both cell culture (7, 9, 13, 14, 15) and human fluids and tissues(13, 16, 17, 18, 19, 20, 21, 22, 23) , suggesting multiple forms of Abeta that vary in primary structure by a few to several amino acids.

Previous studies suggest that C-terminal heterogeneity of Abeta has profound effects upon the initiation and progression of AD. Specifically, increased length of the hydrophobic C terminus both enhances in vitro aggregation of Abeta (24, 25, 26) and appears to promote early deposition of plaque Abeta in AD brains(27) . In addition, an increased relative production of longer C-terminal forms of Abeta, demonstrated both in vitro(14) and in AD brain (28) , appears to underlie the pathologic action of AbetaPP mutations linked to early onset familial AD.

The effects of N-terminal heterogeneity of Abeta on its assembly and bioactivity characteristics are not well defined. The significance of this issue is underscored by recent data suggesting that early stage plaques may be composed primarily of Abeta peptides with truncated N termini(23, 29) . The possibility of enhanced amyloidogenicity of Abeta peptides with truncated N termini is consistent with a previous study that reported decreased in vitro solubilities of synthetic Abeta peptides beta8-, beta9-, and beta10-43 relative to beta1-, beta2-, and beta4-43(30) .

In order to examine the effects of Abeta N-terminal heterogeneity on both peptide assembly and bioactivity, we have synthesized two series of Abeta peptides with progressive N-terminal deletions (beginning at positions beta1, beta4, beta8, beta12, and beta17), one of which terminates at residue beta40 (betan-40 series) and the other at beta42 (betan-42 series). These Abeta peptides have been examined for their rates and levels of peptide assembly, fibrillar ultrastructures under electron microscope, secondary structures by circular dichroism, and bioactivities with cultured neurons.


MATERIALS AND METHODS

Synthetic Peptides

Peptides were synthesized by solid-phase Fmoc (N-(9-fluorenyl)methoxycarbonyl) amino acid chemistry using a continuous flow semiautomatic instrument, as described previously(25) . All peptides were synthesized within a single lot, purified by reverse-phase high performance liquid chromatography, and solubilized at 250 µM in sterile double deionized H(2)O (ddH(2)O); the purity of this lot was estimated by electrospray mass spectrometry and amino acid sequencing to be approximately 70% expected product. Due to their solubility characteristics, only trace amounts of purified beta17-40 and beta17-42 were recovered in the initial synthesis; thus, these peptides were synthesized a second time, partially purified by ether precipitation, and initially solubilized at 25 mM in 1,1,1,3,3,3-hexafluoro-2-propanol prior to dilution to 250 µM in ddH(2)O. It is unlikely that these differences in synthesis lot, purification, and solubilization introduced a significant source of variability between the peptides, since fractions of beta17-40 recovered from the initial lot behaved in a manner nearly indistinguishable from that in the second lot. Based upon findings from our previous studies(31, 32) , we examined the peptides at three time points: (i) immediately following solubilization; (ii) 2 days after solubilization; (iii) 7 days following solubilization.

Sedimentation Assay

The proportion of pelletable peptide was determined using our previously described technique(32) . Briefly, peptide stock solutions were diluted to 25 µM samples in 20 mM MOPS buffer (pH 7.3) and then ultracentrifuged for 1 h at 1 times 10^5g. The ratio of protein concentrations, as determined by fluorescamine assay, of the supernatant relative to non-centrifuged peptide was used to calculate the levels of sedimentable peptide. Data represent the mean of triplicate samples statistically compared by analysis of variance.

Electron Microscopy

For electron microscope analysis(32) , Abeta peptides were diluted from stock solutions to 25 µM in 20 mM MOPS buffer (pH 7.3) and equilibrated for 1 h. Peptide samples were then adsorbed onto carbon-stabilized, Formvar-coated grids, rinsed with ddH(2)O, and stained with 2% (w/v) uranyl acetate. Samples were viewed under a Ziess 10CR transmission electron microscope at times80,000 (80 kV).

Circular Dichroism

To study secondary structure of Abeta peptides in solution, circular dichroism (CD) spectra were examined using a Jasco J-720 spectropolarimeter, as described previously(32) . Briefly, stock solutions of Abeta peptides were diluted to 25 µM in 5 mM potassium phosphate (pH 7.3) and then loaded into a 1-cm pathlength quartz cell. Measurements were taken at 0.5-nm steps over a 195-250-nm wavelength range, averaged over eight scans, and subtracted from base-line (buffer only) values. Data are presented as mean residue ellipticity (degreesbulletcm^2/dmol).

Cell Culture

Primary cultures of hippocampal neurons were established from embryonic day 18 Sprague-Dawley rat pups, as described previously(31) . Cultures were maintained in serum-free Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with N2 components. After 2 days in vitro, cultures were exposed to 25 µM Abeta peptides; cell survival was determined 48 h later based on counts of viable cells using the ethidium homodimer/calcein AM (Molecular Probes, Eugene, OR) combination of vital dyes(31, 33, 34) . Experiments were performed in triplicate wells and repeated in at least three independent experiments. Data were statistically examined with analysis of variance followed by pairwise comparisons using Scheffé F-test.


RESULTS AND DISCUSSION

Given the N- and C-terminal heterogeneity of Abeta isolated from AD brain tissue(16, 17, 18, 19, 20, 21, 22, 23) as well as the demonstrated profound influence of C-terminal heterogeneity on Abeta assembly in vitro(24, 25, 26) and apparently in vivo(27) , the potential influence of N-terminal heterogeneity on Abeta assembly is an intriguing possibility that may significantly contribute to the pathological deposition of Abeta. In this study, we sought to determine (i) how the length of the N terminus influences the overall levels and kinetics of Abeta aggregation and (ii) whether aggregates of N-terminal truncated Abeta retain the biophysical properties of full-length Abeta.

To investigate the influence of N-terminal deletions on Abeta assembly properties, we solubilized the betan-40 and betan-42 peptides and quantitatively determined the levels of peptide aggregates at various time points by sedimentation assay. As illustrated in Fig. 1A, the betan-40 peptides exhibited initially low levels of sedimentation, which gradually increased over 7 days. This progressive increase in peptide aggregation is consistent with previous observations of the time-dependent nature of Abeta aggregation(25, 26, 31) .


Figure 1: Abeta peptides with N-terminal deletions exhibit enhanced sedimentation. A, betan-40 peptides show negligible levels of sedimentation at day 0 that gradually increase by day 7. B, betan-42 peptides exhibit relatively high sedimentation levels, which reach maintained, maximal values by day 2. Means (±S.E.) of triplicate values are shown. * denotes significant (p < 0.05) peptide sedimentation relative to non-centrifuged peptide samples.



In comparison to the betan-40 series, the betan-42 peptides exhibited more rapid and extensive assembly as evidenced by high initial sedimentation and early attainment of maintained peak values; maximal levels were reached within 2 days for beta1-, beta4-, and beta8-42 and by the initial time point (i.e. within minutes of solubilization) for beta12- and beta17-42 (Fig. 1B). The significantly greater amounts of sedimentable aggregates and more rapid kinetics observed in the betan-42 relative to the betan-40 peptides are consistent with the conclusions of previous studies that the length of the C terminus is a critical variable in Abeta assembly(24, 25, 26, 31) . More importantly, we observed a general trend in the betan-42 series of increasing levels of sedimentation with decreasing length of N terminus, with the exception of increased sedimentation levels for beta8-42 at the 2- and 7-day time points; a similar relationship was also apparent in the betan-40 peptides. Note that in both series of peptides, the greatest levels of sedimentation were observed in those peptides beginning at either residue beta8 or beta17.

The increased relative aggregation of Abeta peptides with N-terminal deletions shown by the above data suggests that these and/or related peptides may be the initial Abeta species deposited in senile plaques; recent observations in AD brain support this possibility(27, 29) . Interestingly, Abeta deposited in early stage or diffuse plaques generally lacks the beta-sheet fibrillar structure and associated degenerating neurites characteristic of classic senile plaques. Thus, we sought to determine whether N-terminal deletions might affect previously defined bioactive and structural properties of full-length Abeta, including neurotoxicity, beta-sheet conformation, and fibrillar structure.

Our previous studies have demonstrated that the presence of peptide aggregates in solutions of full-length Abeta accurately predicts significant neurotoxic activity(31, 35, 36) . To determine whether this assembly/toxicity relationship also characterizes Abeta peptides with N-terminal deletions, we examined potential neurotoxic activities of the betan-40 and betan-42 peptides in cultured hippocampal neurons. Within the betan-40 peptide series, neurotoxicity induced by the truncated peptides was greater than that induced by beta1-40 (Fig. 2A). Neurotoxicity generally increased within the peptide series between the 0- and 7-day time points, a trend that parallels the sedimentation data.


Figure 2: Abeta peptides with N-terminal deletions induce significant neurotoxicity in cultures of hippocampal neurons. A, betan-40 peptides induce the greatest cell loss at day 7, the time point associated with maximal sedimentation (Fig. 1A). Note the enhanced toxicity of betan-40 deleted peptides relative to beta1-40. B, betan-42 peptides cause the greatest cell loss at day 0, a time point associated with maximal or near maximal sedimentation values (Fig. 1B). At day 7, all betan-42 peptides induce comparable but diminished levels of toxicity. * denotes significant (p < 0.05) differences in cell viability relative to untreated controls;** denotes significant (p < 0.05) differences relative to beta1-42 at the day 0 time point.



Like the betan-40 peptides, the betan-42 peptides also exhibited significant neurotoxicity (Fig. 2B). The greater toxic activities of the betan-42 series may reflect in part their higher sedimentation levels. Toxicity of betan-42 peptides was generally greatest at day 0 and showed a significant decrease in intensity with decreasing length of the peptide N termini. At the day 7 time point, neurotoxicity was comparable between the different betan-42 peptides but generally reduced within individual peptides relative to the initial time point. The decreased neurotoxicities, both across the peptide series at day 0 and within individual peptides over the two time points, may reflect higher order assembly (i.e. aggregation of Abeta oligomers) of these Abeta peptide aggregates resulting in a corresponding decrease in aggregate-cell interactions, in agreement with previous observations(37) . Thus, although the property of neurotoxicity for individual Abeta peptides is predicted well by their tendency to form sedimentable peptide aggregates, the relationship between these two factors does not necessarily exhibit a strict quantitative correlation(31) .

In addition to neurotoxicity, aggregating Abeta peptides are also characterized by specific structural features, including beta-sheet secondary structure (24, 37) and fibrillar morphology(25, 38, 39, 40) ; like aggregation measures, these factors have been demonstrated to be associated with Abeta neurotoxicity(32, 37, 41) . The following structural studies focused on the betan-42 series, since these peptides not only exhibited significantly higher levels of sedimentation and neurotoxicity than the betan-40 series but also have been demonstrated to be the initial Abeta species deposited within AD plaques(27, 29) .

Examination of CD spectra was used to provide qualitative information regarding the secondary structure of betan-42 peptides in solution. Predominant beta-sheet structure is recognized by a single negative peak near 218 nm, as opposed to the doublet of negative peaks near 208 and 222 nm observed in alpha-helical structure; both structures exhibit a single positive peak near 200 nm(42, 43) . An absence of ordered structure, random coil, is indicated by a single negative peak near 198 nm. Like beta1-42, the truncated betan-42 peptides exhibit characteristics of predominant beta-sheet structure (Fig. 3). Although the CD spectra of all the betan-42 peptides are similar, beta8-42 and beta17-42 exhibit slightly greater negative peaks than the other betan-42 peptides; such enhanced peak values within predominant beta-sheet solutions suggest higher beta-sheet content resulting from a reduced proportion of random coil(43) .


Figure 3: betan-42 peptides exhibit CD spectra characteristic of predominant beta-sheet conformation. The CD spectrum of each betan-42 peptide shows a positive peak near 200 nm and a single negative peak near 218 nm, defining characteristics of beta-sheet secondary structure.



These CD observations verify that, like full-length beta1-42, the truncated betan-42 peptide assemblies exist in the predominantly beta-sheet conformations characteristic of amyloid fibrils. In addition, the CD spectra are consistent with the increased amyloidogenicity of Abeta peptides with N-terminal deletions suggested by the sedimentation data. In particular, the beta8-42 and beta17-42 peptides exhibited both the greatest sedimentation levels and the highest apparent beta-sheet content. The enhanced relative amyloidogenicity of these peptides likely reflects the influences of altered primary structure in promoting and/or stabilizing the beta-sheet conformation underlying Abeta fibril formation; this conclusion is consistent with recent data demonstrating significant contributions by N-terminal residues to Abeta amyloidogenicity(44) . Further studies are required to determine the relevant contributing factors (e.g. increased hydrophobicity, improved interstrand registration).

In order to compare further the amyloidogenic nature of the truncated betan-42 peptides relative to full-length beta1-42, we examined their aggregate structures under electron microscopy. Previous studies have shown that several synthetic Abeta peptides form 5-10-nm diameter fibrils in vitro(25, 38, 39, 40, 41) , which are morphologically similar to Abeta fibrils from AD plaques(38, 45) . We observed that all truncated betan-42 peptides exhibited negatively stained fibrils with morphological features similar to beta1-42 and consistent with previous observations of Abeta fibrils (Fig. 4). Interestingly, although beta17-42 fibrils usually exhibited morphology comparable with fibrils of the other betan-42 peptides, beta17-42 fibrils in some samples appeared to be relatively shorter in length and narrower in diameter (data not shown). Similar discrete populations of relatively wide and narrow Abeta fibrils have been observed in plaque core preparations (45) . In addition, beta17-42 fibrils were organized into exceptionally large, dense meshworks (Fig. 4), consistent with observations of beta17-40 fibrils recently reported by Näslund et al.(46) . However, these authors also reported (46) that beta17-40 aggregates lack the positive thioflavine staining characteristic of all amyloids. In contrast, using our thioflavine assay(32) , we have observed that aggregates formed by all the betan-40 and betan-42 peptides exhibit positive thioflavine staining. (^2)


Figure 4: betan-42 peptides exhibit fibrillar morphology by transmission electron microscopy. A, negatively stained fibrils formed by all betan-42 peptides exhibit similar morphology under electron microscopy that is typical of amyloid; representative fibrils from beta8-42 are shown here. B, fibrils formed by beta17-42 typically are organized into a relatively dense meshwork. Scale bar, 25 nm.



In this paper, we have provided experimental evidence demonstrating a significant influence of N-terminal length on Abeta peptide assembly. Specifically, Abeta peptides with N-terminal deletions exhibit enhanced levels of aggregation in comparison with full-length Abeta peptides, as quantitatively assessed by sedimentation assay. Despite the variable absence of N-terminal residues, the truncated peptides retain the neurotoxicity and beta-sheet, fibrillar structure associated with aggregated full-length Abeta. The structure/activity relationship suggested by these data may impact current concepts regarding the pathogenic potential of Abeta peptide fragments. The prevailing doctrine within the literature has been that alpha-secretase cleavage, which cleaves AbetaPP between Abeta residues beta16 and beta17(6) , is the preferred proteolytic pathway in terms of minimizing disease potential since it generates the theoretically benign beta17-40/42 as opposed to the more pathologic beta1-40/42. In contrast to this position, our data predict that beta17-40/42 is actually more likely than beta1-40/42 to assemble into deposits in vivo and that the shortened peptide retains significant neurotoxic activity. Consistent with the idea of enhanced amyloidogenicity, recent data by Gowing et al.(29) suggest that beta17-42 is a primary component of early stage, diffuse plaques. We suggest that beta17-42 and other prevalent betan-42 peptides may initiate and/or accelerate plaque formation, perhaps by acting as nucleating centers that seed the subsequent deposition of relatively less amyloidogenic but apparently more abundant full-length Abeta; similar seeding events have been described previously for Abeta peptides in vitro(26) .

In summary, the present findings predict that N-terminal heterogeneity of Abeta peptides, demonstrated to occur both in vitro(7, 9, 13, 15) and in AD brain(16, 18, 21, 22, 23) , may accelerate Abeta deposition into plaques. Thus, proteolytic events contributing to the cleavage of AbetaPP within the N-terminal domain of Abeta (e.g. activities of alpha- and beta-secretases) may be of considerable significance in the pathogenesis of AD and related disorders.


FOOTNOTES

*
This work was supported by National Institute on Aging Grant AG12663 (to C. W. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 714-824-6071; Fax: 714-824-2071.

(^1)
The abbreviations used are: Abeta, beta-amyloid protein; AbetaPP, beta-amyloid precursor protein; AD, Alzheimer's disease; ddH(2)O, double deionized water; MOPS, 3-(N-morpholino)propanesulfonic acid.

(^2)
C. J. Pike and C. W. Cotman, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. David H. Cribbs for helpful discussions and technical advice, Dr. Charles Glabe and Joseph Kosmoski for synthesis of peptides, and Scott Paladichuk for assistance with electron microscopy.


REFERENCES

  1. Selkoe, D. J. (1991) Neuron 6,487-498 [Medline] [Order article via Infotrieve]
  2. Mattson, M. P., Barger, S. W., Cheng, B., Lieberburg, I., Smith-Swintosky, V. L., and Rydel, R. E. (1993) Trends Neurosci. 16,409-414 [CrossRef][Medline] [Order article via Infotrieve]
  3. Mullan, M., and Crawford, F. (1993) Trends Neurosci. 16,398-403 [Medline] [Order article via Infotrieve]
  4. Selkoe, D. J. (1993) Trends Neurosci. 16,403-409 [CrossRef][Medline] [Order article via Infotrieve]
  5. Haass, C., and Selkoe, D. J. (1993) Cell 75,1039-1042 [Medline] [Order article via Infotrieve]
  6. Esch, F. S., Keim, P. S., Beattie, E. C., Blacher, R. W., Culwell, A. R., Oltersdorf, T., McClure, D., and Ward, P. J. (1990) Science 248,1122-1124 [Medline] [Order article via Infotrieve]
  7. Haass, C., Schlossmacher, M. G., Hung, A. Y., Vigo-Pelfrey, C., Mellon, A., Ostaszewski, B. L., Lieberburg, I., Koo, E. H., Schenk, D., Teplow, D. B., and Selkoe, D. J. (1992) Nature 359,322-325 [CrossRef][Medline] [Order article via Infotrieve]
  8. Shoji, M., Golde, T. E., Ghiso, J., Cheung, T. T., Estus, S., Shaffer, L. M., Cai, X.-D., McKay, D. M., Tintner, R., Frangione, B., and Younkin, S. G. (1992) Science 258,126-129 [Medline] [Order article via Infotrieve]
  9. Busciglio, J., Gabuzda, D. H., Matsudaira, P., and Yankner, B. A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,2092-2096 [Abstract]
  10. Haass, C., Hung, A. Y., Schlossmacher, M. G., Teplow, D. B., and Selkoe, D. J. (1993) J. Biol. Chem. 268,3021-3024 [Abstract/Free Full Text]
  11. Golde, T. E., Estus, S., Younkin, L. H., Selkoe, D. J., and Younkin, S. G. (1992) Science 255,728-730 [Medline] [Order article via Infotrieve]
  12. Haass, C., Koo, E. H., Mellon, A., Hung, A. Y., and Selkoe, D. J. (1992) Nature 357,500-503 [CrossRef][Medline] [Order article via Infotrieve]
  13. 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., and Schenk, D. (1992) Nature 359,325-327 [CrossRef][Medline] [Order article via Infotrieve]
  14. Suzuki, N., Cheung, T. T., Cai, X.-D., Odaka, A., Otvos, L., Jr., Eckman, C., Golde, T. E., and Younkin, S. G. (1994) Science 264,1336-1340 [Medline] [Order article via Infotrieve]
  15. Zhong, Z., Higaki, J., Murakami, K., Wang, Y., Catalano, R., Quon, D., and Cordell, B. (1994) J. Biol. Chem. 269,627-632 [Abstract/Free Full Text]
  16. Masters, C. L., Simms, G., Weinman, N. A., Multhaup, G., McDonald, B. L., and Beyreuther, K. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,4245-4249 [Abstract]
  17. Prelli, F., Castaño, E., Glenner, G., and Frangione, B. (1988) J. Neurochem. 51,648-651 [Medline] [Order article via Infotrieve]
  18. Miller, D. L., Papayannopoulos, I. A., Styles, J., Bobin, S. A., Lin, Y. Y., Biemann, K., and Iqbal, K. (1993) Arch. Biochem. Biophys. 301,41-52 [CrossRef][Medline] [Order article via Infotrieve]
  19. Roher, A. E., Lowenson, J. D., Clarke, S., Wolkow, C., Wang, R., Cotter, R. J., Reardon, I. M., Zürcher-Neely, H. A., Heinrikson, R. L., Ball, M. J., and Greenberg, B. D. (1993) J. Biol. Chem. 268,3072-3083 [Abstract/Free Full Text]
  20. Roher, A. E., Lowenson, J. D., Clarke, S., Woods, A. S., Cotter, R. J., Gowing, E., and Ball, M. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,10836-10840 [Abstract]
  21. Näslund, J., Schierhorn, A., Hellman, U., Lannfelt, L., Roses, A. D., Tjernberg, L. O., Silberring, J., Gandy, S. E., Winblad, B., Greengard, P., Nordstedt, C., and Terenius, L. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,8378-8382 [Abstract]
  22. Tabaton, M., Nunzi, M. G., Xue, R., Usiak, M., Autilio-Gambetti, L., and Gambetti, P. (1994) Biochem. Biophys. Res. Commun. 200,1598-1603 [CrossRef][Medline] [Order article via Infotrieve]
  23. Saido, T. C., Iwatsubo, T., Mann, D. M. A., Shimada, H., Ihara, Y., and Kawashima, S. (1995) Neuron 14,457-466 [Medline] [Order article via Infotrieve]
  24. Barrow, C. J., and Zagorski, M. G. (1991) Science 253,179-182 [Medline] [Order article via Infotrieve]
  25. Burdick, D., Soreghan, B., Kwon, M., Kosmoski, J., Knauer, M., Henschen, A., Yates, J., Cotman, C., and Glabe, C. (1992) J. Biol. Chem. 267,546-554 [Abstract/Free Full Text]
  26. Jarret, J. T., Berger, E. P., and Lansbury, P. T., Jr. (1993) Biochemistry 32,4693-4697 [Medline] [Order article via Infotrieve]
  27. Iwatsubo, T., Odaka, A., Suzuki, N., Mizusawa, H., Nukina, N., and Ihara, Y. (1994) Neuron 13,45-53 [Medline] [Order article via Infotrieve]
  28. Tamaoka, A., Odaka, A., Ishibashi, Y., Usami, M., Sahara, N., Suzuki, N., Nukina, N., Mizusawa, H., Shoji, S., and Kanazawa, I. (1994) J. Biol. Chem. 269,32721-32724 [Abstract/Free Full Text]
  29. Gowing, E., Roher, A. E., Woods, A. S., Cotter, R. J., Chaney, M., Little, S. P., and Ball, M. J. (1994) J. Biol. Chem. 269,10987-10990 [Abstract/Free Full Text]
  30. Hilbich, C., Kisters-Woike, B., Reed, J., Masters, C. L., and Beyreuther, K. (1991) J. Mol. Biol. 218,149-163 [Medline] [Order article via Infotrieve]
  31. Pike, C. J., Burdick, D., Walencewicz, A. J., Glabe, C. G., and Cotman, C. W. (1993) J. Neurosci. 13,1676-1687 [Abstract]
  32. Pike, C. J., Walencewicz-Wasserman, A. J., Kosmoski, J., Cribbs, D. H., Glabe, C. G., and Cotman, C. W. (1995) J. Neurochem. 64,253-265 [Medline] [Order article via Infotrieve]
  33. Pike, C. J., and Cotman, C. W. (1993) Neuroscience 56,269-274 [Medline] [Order article via Infotrieve]
  34. Pike, C. J., and Cotman, C. W. (1995) Brain Res. 671,293-298 [CrossRef][Medline] [Order article via Infotrieve]
  35. Pike, C. J., Walencewicz, A. J., Glabe, C. G., and Cotman, C. W. (1991) Brain Res. 563,311-314 [CrossRef][Medline] [Order article via Infotrieve]
  36. Pike, C. J., Walencewicz, A. J., Glabe, C. G., and Cotman, C. W. (1991) Eur. J. Pharmacol. 207,367-368 [CrossRef][Medline] [Order article via Infotrieve]
  37. Simmons, L. K., May, P. C., Tomaselli, K. J., Rydel, R. E., Fuson, K. S., Brigham, E. F., Wright, S., Lieberburg, I., Becker, G. W., Brems, D. N., and Li, W. Y. (1994) Mol. Pharmacol. 45,373-379 [Abstract]
  38. Castaño, E. M., Ghiso, J., Prelli, F., Gorevic, P. D., Migheli, A., and Frangione, B. (1986) Biochem. Biophys. Res. Commun. 141,782-789 [Medline] [Order article via Infotrieve]
  39. Gorevic, P. D., Castaño, E. M., Sarma, R., and Frangione, B. (1987) Biochem. Biophys. Res. Commun. 147,854-862 [Medline] [Order article via Infotrieve]
  40. Kirschner, D. A., Inouye, H., Duffy, L. K., Sinclair, A., Lind, M., and Selkoe, D. J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,6953-6957 [Abstract]
  41. Lorenzo, A., and Yankner, B. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,12243-12247 [Abstract/Free Full Text]
  42. Sarkar, P. K., and Doty, P. (1966) Proc. Natl. Acad. Sci. U. S. A. 55,981-989 [Medline] [Order article via Infotrieve]
  43. Greenfield, N., and Fasman, G. D. (1969) Biochemistry 8,4108-4116 [Medline] [Order article via Infotrieve]
  44. Soto, C., Castaño, E. M., Frangione, B., and Inestrosa, N. C. (1995) J. Biol. Chem. 270,3063-3067 [Abstract/Free Full Text]
  45. Roher, A., Wolfe, D., Palutke, M., and KuKuruga, D. (1986) Proc. Natl. Acad. Sci. U. S. A. 83,2662-2666 [Abstract]
  46. Näslund, J., Jensen, M., Tjernberg, L. O., Thyberg, J., Terenius, L., and Nordstedt, C. (1994) Biochem. Biophys. Res. Commun. 204,780-787 [CrossRef][Medline] [Order article via Infotrieve]

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