All-D-Enantiomers of beta -Amyloid Exhibit Similar Biological Properties to All-L-beta -Amyloids*

(Received for publication, August 6, 1996, and in revised form, December 6, 1996)

David H. Cribbs Dagger §, Christian J. Pike , Shari L. Weinstein , Peter Velazquez and Carl W. Cotman Dagger

From the Institute for Brain Aging and Dementia, Departments of Psychobiology and Dagger  Neurology, University of California Irvine, Irvine, California 92697-4540

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

The amyloidogenic peptide beta -amyloid has previously been shown to bind to neurons in the form of fibrillar clusters on the cell surface, which induces neurodegeneration and activates a program of cell death characteristic of apoptosis. To further investigate the mechanism of Abeta neurotoxicity, we synthesized the all-D- and all-L-stereoisomers of the neurotoxic truncated form of Abeta (Abeta 25-35) and the full-length peptide (Abeta 1-42) and compared their physical and biological properties. We report that the purified peptides exhibit nearly identical structural and assembly characteristics as assessed by high performance liquid chromatography, electron microscopy, circular dichroism, and sedimentation analysis. In addition, both enantiomers induce similar levels of toxicity in cultured hippocampal neurons. These data suggest that the neurotoxic actions of Abeta result not from stereoisomer-specific ligand-receptor interactions but rather from Abeta cellular interactions in which fibril features of the amyloidogenic peptide are a critical feature. The promiscuous nature of these beta -sheet-containing fibrils suggests that the accumulation of amyloidogenic peptides in vivo as extracellular deposits represents a site of bioactive peptides with the ability to provide inappropriate signals to cells leading to cellular degeneration and disease.


INTRODUCTION

Alzheimer's disease (AD),1 vascular dementia, and hereditary cerebral hemorrhage with the Dutch type are diseases that share an invariant pathological feature, the accumulation of an amyloidogenic peptide into insoluble fibrillar extracellular deposits. In all three cases, the major component of the extracellular debris is the beta -amyloid peptide (Abeta ) that is derived from the proteolytic processing of the large membrane-anchored amyloid precursor protein (APP) encoded by a single gene located on chromosome 21 (1). However, the biological significance of these amyloid deposits has been extensively debated as to whether they are a causative factor of each disease or merely a metabolically inert end product lacking in biological activity. Evidence in support of a causative role for Abeta in neuropathology comes from genetic analysis of the APP gene where several autosomal dominant mutations have been linked with AD and hereditary cerebral hemorrhage with the Dutch type (2, 3). In a recent in vitro study, the beta -APP717 mutation consistently caused a significant increase in the percentage of the longer and more amyloidogenic Abeta 1-42 over the shorter Abeta 1-40 (4). Incorporation of this same mutation into a transgenic mouse model yields Abeta deposition and neuropathology that closely parallels that observed in AD (5). Additional in vivo evidence suggesting that the Abeta peptide itself may be biologically active comes from a transgenic model overexpressing Abeta 1-42, in which Abeta transgene expression was detected in a variety of peripheral tissues but histopathological changes were restricted to the brain. Moreover, the neurodegeneration was largely limited to the cerebral cortex, hippocampus and amygdala, all areas affected in AD, and was essentially undetectable in the cerebellum, which is typically not affected in AD (6). Finally, the amount of beta -amyloid that accumulates in the brain appears to correlate well with the decline of brain function (7).

Insights into the inherent biological activity associated with Abeta have come from in vitro studies that show synthetic Abeta can spontaneously assemble into beta -sheet-containing fibrils (8, 9), that this fibrillar-Abeta can induce neuritic dystrophy in neuronal cultures similar to that seen in the AD brain, and that the mechanism of Abeta -triggered degeneration is via programmed cell death (PCD) (10). These observations have led to the general hypothesis that the biological activity of Abeta is dependent on its transformation into a highly stable protease-resistant antiparallel beta -sheet conformation and higher order quaternary assemblies (11) similar to those found in senile plaques. This is of fundamental importance because it suggests that the biological activity of Abeta is dependent on protein conformation and the transition into this conformation. The consequences of such a relationship between biological activity and protein conformation are critical to understanding the role of Abeta and other beta -pleated sheet protein assemblies such as prion protein in disease.

In order to understand the degenerative processes induced by Abeta , it is essential to define the characteristics of Abeta salient to its function as a neurotoxic stimulus. In a previous study, we used a series of synthetic Abeta peptides with progressively truncated C-termini to demonstrate that the length of this hydrophobic region is a crucial determinant of peptide ability to both aggregate and induce neurotoxicity in vitro (12). We have also synthesized a series of truncated Abeta peptides to examine the effects of N-terminal heterogeneity, which occurs in vivo on the assembly and biological activity of Abeta . The N-terminal truncated isoforms produced enhanced aggregation into neurotoxic beta -sheet fibrils, which suggests that these truncated peptides may initiate the pathological neurodegeneration in AD by acting as a nucleation site for Abeta deposition (13). Thus far, we have observed that assembled, bioactive Abeta peptides exhibit beta -sheet structure and that amino acid substitutions that disrupt Abeta assembly also prevent beta -sheet structure and abolish toxicity (14). Further analysis of peptides will be useful in elucidating the specific requirements for both the assembly and bioactivity of Abeta .

Since numerous ligand-target interactions are stereospecific, one means to both examine the nature of the Abeta -cellular interactions and assess the validity of several proposed mechanisms of Abeta -induced cell death is to determine whether Abeta bioactivity exhibits stereospecificity. Similar issues of ligand stereospecificity have been investigated in several recent studies by comparing the binding and or activities of D- and L-enantiomers of small peptide ligands (15, 16). In the current study, we have utilized a comparable paradigm, synthesizing the all-D- and all-L-amino acid stereoisomers of the truncated biologically active Abeta 25-35 and the full-length Abeta 1-42 peptide, and compared their physical and biological properties to determine whether the interaction of Abeta with biologically relevant cells is stereospecific.


EXPERIMENTAL PROCEDURES

Peptide Synthesis

Abeta 1-42, Abeta 25-35 (GSNKGAIIGLM), and scrambled sequence Abeta 25-35 (12) were synthesized from either all-D- or all-L- amino acids using solid phase Fmoc (N-(9-fluorenyl)methoxycarbonyl) amino acid chemistry and purified by reverse phase HPLC, as described previously (9). The purified peptides were then routinely analyzed by electrospray mass spectroscopy (9). The all-D-Abeta 25-35 and -Abeta 1-42 enantiomers gave mass values of 1060.4 and 4513.9, respectively. HPLC analysis of the all-D-enantiomers produced identical elution times to the all-L-Abeta peptides, and mixtures of all-D-Abeta 1-42 and all-L-Abeta 25-35 produced a single peak by HPLC. Peptides were solubilized in sterile double deionized water as 2.5 mM stock solutions and allowed to aggregate at room temperature for at least 1 h before using. Aliquots of the peptide stocks were then diluted with an equal volume of 2× Dulbecco's modified Eagle's medium (DMEM) and then with DMEM supplemented with N-2 (17) just prior to treating the cultures.

Circular Dichroism

The mean residue ellipticity of Abeta 25-35 peptides (25 µM in 5 mM potassium phosphate, pH 7.3) was determined using a Jasco J-720 spectropolarimeter equipped with a computerized data processor, as described previously (14). Samples were loaded into a 1.0-cm path length quartz cell and measured over a 190-250-nm wavelength range at 0.5 nm increments. Data from eight scans were averaged and subtracted from base-line values but otherwise are unsmoothed. The instrument was calibrated with a 0.06% (w/v) solution of d-camphorsulfonate.

Electron Microscopy

For ultrastructural analysis, 25 µM samples of Abeta peptides (20 mM MOPS buffer, pH 7.4) were adsorbed onto 200 mesh formvar grids and stained with 2% uranyl acetate prior to viewing with a Zeiss 10CR transmission electron microscope at 80 kV transmission (14).

Peptide Aggregation

Levels of peptide aggregation were quantitatively determined using a sedimentation assay previously described (14). Briefly, Abeta 25-35 peptides (25 µM in 20 mM MOPS, pH 7.3) were ultracentrifuged for 1 h at 100,000 × g. Supernatant peptide concentrations between centrifuged and non-centrifuged samples were compared by fluorescamine assay. Decreased supernatant peptide concentration in centrifuged samples was used as a measure of the aggregated peptide fraction. Analyses were conducted in quadruplicate samples.

Tissue Culture

Cultures of hippocampal neurons from gestational day 18 rat pups were prepared as described previously (12). Cultures were plated at 2.5 × 104 cells/cm2 on poly-L-lysine-treated multiwell plates and maintained in serum-free DMEM supplemented with N-2 components. After two days in vitro, cultures were exposed to the various Abeta peptides for 24 h, after which cell viability was determined on the basis of trypan blue exclusion (12, 18). Raw data were statistically compared by analysis of variance followed by Scheffé f-test.


RESULTS

The initial experiments to investigate whether an all-D-enantiomer of an amyloidogenic peptide retains biological activity were performed with all-D-Abeta 25-35. This is the smallest commonly studied fragment of Abeta that retains both the ability to form beta -sheet-containing fibrils and neurotoxicity (19). An additional advantage of Abeta 25-35 is that it can be modeled relatively easily, and information on the alignment of the antiparallel strands as well as the importance of various side chain interactions in formation of the Abeta fibrils and in defining the surface topography necessary for neurotoxicity can be investigated. In the antiparallel beta -sheet conformation, the surface topography is determined by the amino acid sequence and the alignment of adjacent strands in the beta -sheet. In a computer-generated model of Abeta 25-35 in which the two peptides are maximally overlapped, a cluster of positively charged lysine residues is observed on one face of the beta -sheet while the other face of the beta -sheet contains primarily hydrophobic residues. Previous studies with Abeta 25-35 containing single amino acid substitutions have confirmed the importance of the sequence for retaining the properties of Abeta (14).

Two separate lots of the all-D-Abeta 25-35 were synthesized to control for lot to lot variability in the biological activity of the peptides. Comparison of the all-D and all-L forms of Abeta 25-35 by electrospray mass spectroscopy gave essentialy identical mass values, and HPLC analysis showed that the two enantiomers had similar elution times. Mixed samples of both enantiomers eluted as a single peak from the HPLC (Fig. 1A). All-D-Abeta 25-35 was allowed to assemble in parallel with the all-L-Abeta 25-35, and then both peptides were subjected to a series of commonly used assays to monitor the properties of fibrillar Abeta . Both enantiomers rapidly produced visible aggregates in aqueous solution (Fig. 2, A-D), and analysis of negatively stained specimens by electron microscopy showed similar fibrillar structures (Fig. 1, B and C). Sedimentation analysis was then used to compare the extent of peptide assembly for both enantiomers. Three different lots of the all-L-Abeta 25-35 and two lots of the all-D-Abeta 25-35 were used, and both forms gave similar results (data not shown). The above results show that both peptides have similar physical properties and can only be distinguished by CD analysis where mirror image spectra are generated (data not shown).


Fig. 1. Comparison of the physical properties of the all-L- and all-D-Abeta 25-35 enantiomers. A racemic mixture of all-L- and all-D-Abeta 25-35 produces a single peak upon HPLC analysis using a C4 analytical column (A). Negatively stained samples of the all-L-Abeta 25-35 (B) and all-D-Abeta 25-35 (C) were examined by electron microscopy. Magnification for both samples was ×40,000.
[View Larger Version of this Image (56K GIF file)]



Fig. 2. Comparison of the cellular association and neurotoxic activities of the all-L- and all-D-enantiomers of Abeta 25-35. Primary cultures of rat neurons were treated with pre-assembled forms of the enantiomers of Abeta 25-35. Numerous visible fibrillar aggregates (arrows) of the all-L-Abeta 25-35 (A and B) and all-D-Abeta 25-35 (C and D) can been seen associated with the neurites at 6 h post-treatment with 6 µM Abeta 25-35. At higher magnification (B and D), the fuzzy appearing fibrillar aggregates can been seen decorating the swollen degenerating neurites. Dose response curves were generated to compare the relative neurotoxic activities of the two Abeta 25-35 enantiomers (E). The all-L- and all-D-Abeta 25-35 produced similar dose response curves. Scrambled sequences of all-L- and all-D-peptides were not neurotoxic even at 50 µM (data not shown).
[View Larger Version of this Image (59K GIF file)]


The biological properties of the two enantiomers were then tested by applying the enantiomers to primary cultures of rat hippocampal and cortical neurons that have previously been used to assay the neurotoxic activity of Abeta (12, 18). The all-D-Abeta 25-35 produced visible aggregates in the tissue culture wells and appeared to bind to the surface of neurons equally as well as the all-L-Abeta 25-35 (Fig. 2, A-D). Noticeable neuronal degeneration was apparent at 12 h, and extensive cell death was observed at 24 h for both enantiomers. In order to compare the levels of neurotoxicity between the two enantiomers, a dose response curve was generated. As can be seen in Fig. 2E, the all-D-Abeta 25-35 produced similar toxicity to the all-L-Abeta 25-35 over the entire range of concentrations tested. The specificity of the neurotoxicity was determined by analyzing peptides with scrambled sequences of both the all-L and the all-D enantiomers. Neither scrambled sequence produced detectable neurotoxicity over the entire range of concentrations utilized for the dose response curve (data not shown).

During the comparison of computer-generated models of Abeta 25-35 in antiparallel beta -sheet conformation, we discovered, that with perfect alignment of the individual strands of peptide, that a pseudo-axis of symmetry was generated do to the planar nature of the beta -sheet such that the distribution of the surface groups produced topochemically similar enantiomers. Other cases of topochemically similar peptides that bind to stereoselective receptors and posses similar activities have been reported (20, 21). Based on the high level of bioactivity associated with the all-D-Abeta 25-35, we next determined whether the all-D-enantiomer of the full-length Abeta 1-42 peptide would also bind to cells and produce similar neurotoxicity to the all-L-Abeta 1-42. Although the beta -sheet-containing fibrils of the Abeta 1-42 are predicted to form planar sheets, the longer length of the Abeta 1-42 peptide would reduce the probability of formation of topochemically similar enantiomers since the surface topography of Abeta 1-42 would be far more complex than with Abeta 25-35.

Highly purified all-D-Abeta 1-42 was subjected to CD, and the spectra were compared with the all-L-Abeta 1-42 (Fig. 3A). The all-D-enantiomer produced the expected mirror image spectra, indicating similar secondary structure for the enantiomers. The peptides were then examined by electron microscopy, and, while the filamentous structures were different from those observed with Abeta 25-35, the Abeta 1-42 enantiomers produced fibrils that were indistinguishable from each other (Fig. 3, B and C). The ability to bind certain dyes, such as Congo red and thioflavine T, is a characteristic property of amyloidogenic peptides (22-24) and can be used to measure the amount of peptide in beta -sheet-containing fibrils (25). Analysis of assembled peptides of both enantiomers indicates that the all-D-Abeta 1-42 binds thioflavine with intensity equal to that of the all-L-Abeta 1-42 (data not shown).


Fig. 3. Comparison of the physical properties of the all-L-Abeta 1-42 with the all-D-Abeta 1-42. Circular dichroism analysis of the Abeta 1-42 enantiomers (A) for all-L- and all-D-Abeta 1-42. Negatively stained samples of the all-L-Abeta 1-42 (B) and all-D-Abeta 1-42 (C) were examined by electron microscopy. Magnification for both samples was ×40,000.
[View Larger Version of this Image (48K GIF file)]


The biological response to the full-length all-D-Abeta 1-42 by neurons was assayed as described above for Abeta 25-35. The all-D-enantiomer clearly binds to the neurons since clusters of the fibrillar Abeta 1-42 can be seen over much of the cell surface within 6 h of adding the peptide to the cultures (Fig. 4, C and D). Perhaps more importantly, the fibrillar clusters induce extensive neurodegeneration over a 24-h time course. It should be noted that not all neurons respond equally to fibrillar forms of Abeta , as previous studies have shown that neurons that are immunopositive for GABA are resistant to the neurotoxic activity of Abeta (26). A comparison of the relative neurotoxicity of the two Abeta 1-42 enantiomers is shown in the dose response curves in Fig. 4E. Both enantiomers produce noticeable neurotoxic activity at 5 µM, and at 25 µM, approximately half of the neurons are dead within 24 h.


Fig. 4. Comparison of the cellular association and neurotoxic activities of the all-L- and the all-D-enantiomers of Abeta 1-42. Primary cultures of rat neurons were treated with pre-assembled forms of the enantiomers of Abeta 1-42. Numerous visible fibrillar aggregates (arrows) of the all-L-Abeta 1-42 (A and B) and all-D-Abeta 1-42 (C and D) can been seen associated with the neurites (arrowheads) at 6 h post treatment with 6 µM Abeta 1-42. At higher magnification (B and D), swollen degenerating neurites and fibrillar Abeta aggregates are closely associated with both enantiomers. Dose response curves were generated to compare the relative neurotoxic activities of the two Abeta 1-42 enantiomers (E).
[View Larger Version of this Image (67K GIF file)]



DISCUSSION

This study was designed to probe the stereospecificity of the interaction between Abeta and the plasma membrane of cultured neurons that in vitro leads to programmed cell death (10, 27, 28). According to classic receptor pharmacology, a D-stereoisomer of an amino acid or peptide would not be predicted to exhibit bioactivity comparable with the native L-peptide. For example, glutamate receptors readily discriminate L- versus D-antagonistic agents (29). We have analyzed both the physical and biological properties of the all-D-enantiomers of Abeta 25-35 and Abeta 1-42 and have compared them with their corresponding all-L-enantiomers. With the exception of the CD spectropolarimetry study, which produced mirror image spectrums, both all-D-enantiomers exhibited essentially identical physical and biological properties to their all-L-enantiomers.

A number of studies have been done utilizing D-enantiomers of various ligands to investigate the stereospecific requirements for binding to their respective receptor proteins. In the cases of three peptide hormones, bradykinin (30), oxytocin (31), and angiotensin (32), that must interact with chiral receptors on the plasma membrane, the all-D-forms of the ligands were inactive. However, different results were obtained in the case of a synthetic beta -endorphin analog that contained 18 D-amino acid residues in the C-terminal portion of the peptide but 5 L-residues within the actual binding site. The all-D-containing region was designed to form a left-handed amphiphilic helical segment that was topochemically similar to the native right-handed amphiphilic helix. The D/L chimeric peptide retained equal ability to bind and to activate the opiate receptor (21). In some cases, the all-D-peptide enantiomers can still resemble the parent compound, both in the overall spatial arrangements and with respect to the electronic nature of the functional groups. In the case of the antibiotic enniatin B, the topochemically similar enantio-enniatin B possessed similar antimicrobial activity (20). In two recent studies for example, the all-D-peptide analogs were found to bind with similar affinities to their respective receptors. In the first one, two all-D-amphiphilic helical peptides were shown to interact with calmodulin in a sterically malleable fashion (16, 33), and the second example reported that the laminin segment containing the IKVAV amino acid sequence, which is responsible for cell attachment and tumor-promoting activities, was retained in the all-D-peptide. Peptide analogs with either alternating D-L-substitutions or randomized IKVAV sequence were inactive, indicating that the sequence and conformational status of the domain contribute to the biological activity but that no stereospecific requirement exists (15).

Although bilayer lipids and membranes are also chiral and contain numerous asymmetric centers, the partitioning of chiral channel-forming antibiotic peptides into membranes does not require a specific chirality. The all-D-analogs of cecropin, magainin II amide, and melittin were equally effective when tested on achiral synthetic planar bilayers and as antibiotics against bacteria containing chiral membranes (34). The reports that Abeta 1-40 forms giant multivalent cation channels when incorporated into synthetic bilayers (35, 36) and our findings that the neurotoxic activity associated with both Abeta 25-35 and Abeta 1-42 are not chirally dependent are consistent with the results obtained with the channel-forming antibiotic peptides. Unfortunately, after many years of extensive study on Abeta , there is no definitive evidence that Abeta forms channels in neurons. Another more likely possibility is that Abeta is active as a membrane perturbant, which may alter the microenvironment between the bilayer and membrane-bound enzymes or receptors.

We currently favor a mechanism dependent on the interaction of Abeta with membrane receptor proteins on the surface of neurons, and other cell types such as astrocytes, because assembled fibrillar forms of the amyloidogenic peptides are required for activity (12, 14, 37). It is possible, for example, that Abeta acts as a ligand to cross-link receptors at the cell surface and activates cell death pathways via activation-induced cell death similar to Fas (38). Consistent with a mechanism involving membrane receptors, we have shown that the lectin ConA, which forms clusters of membrane glycoproteins on the cell surface, also causes neurodegeneration and apoptotic death in cultured neurons similar to that observed with Abeta while succinyl ConA, which binds but does not cross-link, is inactive (39). Recently, Burdick, et al. (40) have shown that a substantial portion of the Abeta that binds to cells can be removed by treatment with trypsin and several receptors that appear to bind Abeta peptides have been identifed (41-46). In the case of the receptor for advanced glycation end products (RAGE), some evidence has been presented that it may be directly involved in Abeta -induced neurotoxicity (45). Experiments with all-D-Abeta and these putative Abeta receptors are in progress and should provide information on the specificity of these receptors for Abeta . Thus, we suggest that extracellular macromolecular assemblies such as Abeta can serve as stimuli or agonists that trigger a particular sequence of cellular reactions in neurons that initiate an apoptotic program of cell death. These PCD agonists are characterized in part by beta -sheet fibrillar structure but, in addition, have the common ability to access critical signal transduction and downstream mechanisms that drive PCD.

Other amyloidogenic proteins, which do not share sequence homology with Abeta (e.g.. prion and amylin), do form structurally similar extracellular deposits and have been found to have similar neurotoxic activity in vitro (47, 48). One possible mechanism that could explain the common biological activity seen with different amyloidogenic peptides is beta -sheet augmentation, whereby a peptide forms a "peptide-surface association" (49) either by inserting itself into a beta -sheet-containing domain (50), which has been proposed for other diseases involving protein conformational changes (51-54), or by adding to the edge of an anti-parallel beta -strand, as has been implicated in regulating protein associations governing signal transduction pathways and assembly interactions in certain viral capsids (49). The beta -sheet augmentation mechanism provides much greater flexibility than classical domain-domain association because the peptide is not constrained by a rigidly folded domain. The specificity in this model is dependent on the ability of the peptide to augment an appropriate beta -strand on a protein. Consistent with this model, Abeta has shown a pronounced ability to bind to other proteins, such as alpha -1-antichymotrypsin, and transthyretin which are rich in beta -sheet. Finally, the cell surface contains numerous proteins with Ig superfamily homology with extensive beta -sheet content, which include receptors (55) and cell adhesion molecules (i.e. NCAM and N-cadherin) (56), and in several reports, the cell surface appears to be able to actually nucleate Abeta assembly (40, 57, 58), which is also consistent with the model (49).

The fact that the all-D analogs of Abeta retain bioactivity may present new avenues for therapeutic intervention by allowing the D-enantiomers of inhibitory peptides to be utilized. An approach similar to this has recently been used to identify an all-D-amino acid opioid peptide with analgesic activity capable of crossing the blood brain barrier using a synthetic combinatorial library made up of D-amino acid hexapeptides (59). In addition, the identification of D-peptide ligands through mirror image phage display using genetically encoded libraries (60) offers the promise of rapidly screening for D-peptide ligands that can block assembly and or neurotoxicity of the Abeta peptide. All-D-ligands are generally resistant to proteolysis and D-amino acid proteins are reported to have low immunogenicity, thus making them useful for pharmacological applications (61).

The results obtained in this study suggest that the neurotoxic activity of Abeta is independent of the classical stereoisomer-specific ligand-receptor interaction. Rather, Abeta -induced neurotoxicity is dependent on the primary sequence of the peptide that regulates both the ability of the peptides to assemble into active conformations and bind to cellular surfaces. While mechanisms dependent on the perturbation of cellular membranes or the formation of calcium ion channels cannot be excluded, the requirement for higher order protein assemblies by amyloidogenic peptides for biological activity does not readily support these mechanisms. Further investigation of the mechanism of Abeta -induced toxicity will likely benefit attempts to understand the neurodegeneration that occurs in AD and perhaps other amyloid-related disorders.


FOOTNOTES

*   This work was supported by NIA, National Institutes of Health Grants AG 13007 (to C. W. C.) and AG 07918 (to D. H. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed. Tel.: 714-824-3482; Fax: 714-824-2071; E-mail: dhcribbs{at}uci.edu.
1   The abbreviations used are: AD, Alzheimer's disease; Abeta , beta -amyloid protein; APP, amyloid precursor protein; CD, circular dichroism; PCD, programmed cell death; DMEM, Dulbecco's modified Eagle's medium; HPLC, high performance liquid chromatography; MOPS, 4-morpholinepropanesulfonic acid; ConA, concanavalin A; GABA, gamma-aminobutyric acid.

Acknowledgments

We thank Virany Kreng for excellent technical assistance and Dr. Charles Glabe for synthesis and characterization of the amyloid peptides.


REFERENCES

  1. Kang, J., Lemaire, H.-G., Unterbeck, A., Salbaum, J. M., Masters, C. L., Grzeschik, K.-H., Multhaup, G., Beyruther, K., and Müller-Hill, B. (1987) Nature 325, 733-736 [CrossRef][Medline] [Order article via Infotrieve]
  2. Selkoe, D. J. (1993) Trends Neurosci. 16, 403-409 [CrossRef][Medline] [Order article via Infotrieve]
  3. Schellenberg, G. D. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8552-8559 [Abstract]
  4. Suzuki, N., Cheung, T. T., Cai, X. D., Odaka, A., Otvos, L. J., Eckman, C., Golde, T. E., and Younkin, S. G. (1994) Science 264, 1336-1340 [Medline] [Order article via Infotrieve]
  5. Games, D., Adams, D., Alessandrini, R., Barbour, R., Berthelette, P., Blackwell, C., Carr, T., Clemens, J., Donaldson, T., Gillespie, F., et al. (1995) Nature 373, 523-527 [CrossRef][Medline] [Order article via Infotrieve]
  6. LaFerla, F. M., Tinkle, B. T., Bieberich, C. J., Haudenschild, C. C., and Jay, G. (1995) Nat. Genet. 9, 21-30 [Medline] [Order article via Infotrieve]
  7. Cummings, B. J., and Cotman, C. W. (1995) Lancet 346, 1524-1528 [CrossRef][Medline] [Order article via Infotrieve]
  8. 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]
  9. 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]
  10. Loo, D. T., Copani, A., Pike, C. J., Whittemore, E. R., Walencewicz, A. J., and Cotman, C. W. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7951-7955 [Abstract/Free Full Text]
  11. Cotman, C. W., and Anderson, A. J. (1995) Mol. Neurobiol. 10, 19-45 [Medline] [Order article via Infotrieve]
  12. Pike, C. J., Burdick, D., Walencewicz, A. J., Glabe, C. G., and Cotman, C. W. (1993) J. Neurosci. 13, 1676-1687 [Abstract]
  13. Pike, C. J., Overman, M. J., and Cotman, C. W. (1995) J. Biol. Chem. 270, 23895-23898 [Abstract/Free Full Text]
  14. 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]
  15. Nomizu, M., Utani, A., Shiraishi, N., Kibbey, M. C., Yamada, Y., and Roller, P. P. (1992) J. Biol. Chem. 267, 14118-14121 [Abstract/Free Full Text]
  16. Fisher, P. J., Prendergast, F. G., Ehrhardt, M. R., Urbauer, J. L., Wand, A. J., Sedarous, S. S., McCormick, D. J., and Buckley, P. J. (1994) Nature 368, 651-653 [Medline] [Order article via Infotrieve]
  17. Bottenstein, J. E., and Sato, G. H. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 514-517 [Abstract]
  18. 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]
  19. Yankner, B. A., Duffy, L. K., and Kirschner, D. A. (1990) Science 250, 279-282 [Medline] [Order article via Infotrieve]
  20. Shemyakin, M. M., Ovchinnikov, Yu. A., Ivanov, V. T., and Evstratov, A. V. (1967) Nature 213, 412-413 [Medline] [Order article via Infotrieve]
  21. Blanc, J. P., and Kaiser, E. T. (1984) J. Biol. Chem. 259, 9549-9556 [Abstract/Free Full Text]
  22. Naiki, H., Higuchi, K., Hosokawa, M., and Takeda, T. (1989) Anal. Biochem. 177, 244-249 [Medline] [Order article via Infotrieve]
  23. Naiki, H., Higuchi, K., Matsushima, K., Shimada, A., Chen, W. H., Hosokawa, M., and Takeda, T. (1990) Lab. Invest. 62, 768-773 [Medline] [Order article via Infotrieve]
  24. Naiki, H., Higuchi, K., Nakakuki, K., and Takeda, T. (1991) Lab. Invest. 65, 104-110 [Medline] [Order article via Infotrieve]
  25. Levine, H., III (1995) Neurobiol. Aging 16, 755-764 [CrossRef][Medline] [Order article via Infotrieve]
  26. Pike, C. J., and Cotman, C. W. (1993) Neuroscience 56, 269-274 [Medline] [Order article via Infotrieve]
  27. Forloni, G., Chiesa, R., Smiroldo, S., Verga, L., Salmona, M., Tagliavini, F., and Angeretti, N. (1993) Neuroreport 4, 523-526 [Medline] [Order article via Infotrieve]
  28. Watt, J. A., Pike, C. J., Wancewicz-Wasserman, A. J., and Cotman, C. W. (1994) Brain Res. 661, 147-156 [CrossRef][Medline] [Order article via Infotrieve]
  29. Monaghan, D. T., Bridges, R. J., and Cotman, C. W. (1989) Annu. Rev. Pharmacol. Toxicol. 29, 365-402 [CrossRef][Medline] [Order article via Infotrieve]
  30. Stewart, J. M., and Woolley, D. W. (1965) Nature 206, 619-620
  31. Flouret, G., and Du Vigneaud, V. (1965) J. Am. Chem. Soc. 87, 3775-3776 [Medline] [Order article via Infotrieve]
  32. Vogler, K., Lanz, P., Lergier, W., and Haefely, W. (1966) Helv. Chim. Acta 49, 390-403 [Medline] [Order article via Infotrieve]
  33. Seaton, B. A. (1994) Nat. Struct. Biol. 1, 350-351 [Medline] [Order article via Infotrieve]
  34. Wade, D., Boman, A., Wahlin, B., Drain, C. M., Andreu, D., Boman, H. G., and Merrifield, R. B. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4761-4765 [Abstract]
  35. Arispe, N., Rojas, E., and Pollard, H. B. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 567-571 [Abstract]
  36. Arispe, N., Pollard, H. B., and Rojas, E. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10573-10577 [Abstract]
  37. 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]
  38. Cotman, C. W., Cribbs, D. H., and Anderson, A. J. (1997) in The Molecular Mechanisms for Dementia (Tanzi, R., and Wasco, W. M., eds), pp. 73-90, The Humana Press, Totowa, NJ
  39. Cribbs, D. H., Kreng, V. M., Anderson, A. J., and Cotman, C. W. (1996) Neuroscience 75, 173-185 [CrossRef][Medline] [Order article via Infotrieve]
  40. Burdick, D., Kosmoski, J., Knauer, M. F., and Glabe, C. G. (1997) Brain Res., in press
  41. Boland, K., Manias, K., and Perlmutter, D. H. (1995) J. Biol. Chem. 270, 28022-28028 [Abstract/Free Full Text]
  42. Boland, K., Behrens, M., Choi, D., Manias, K., and Perlmutter, D. H. (1996) J. Biol. Chem. 271, 18032-18044 [Abstract/Free Full Text]
  43. Paresce, D. M., Ghosh, R. N., and Maxfield, F. R. (1996) Neuron 17, 553-565 [Medline] [Order article via Infotrieve]
  44. El Khoury, J., Hickman, S. E., Thomas, C. A., Cao, L., Silverstein, S. C., and Loike, J. D. (1996) Nature 382, 716-719 [CrossRef][Medline] [Order article via Infotrieve]
  45. Yan, S. D., Chen, X., Fu, J., Chen, M., Zhu, H., Roher, A., Slattery, T., Zhao, L., Nagashima, M., Morser, J., Migheli, A., Nawroth, P., Stern, D., and Schmidt, A. M. (1996) Nature 382, 685-691 [CrossRef][Medline] [Order article via Infotrieve]
  46. Giulian, D., Haverkamp, L. J., Yu, J. H., Karshin, W., Ton, D., Li, J., Kirkpatrick, J., Kuo, Y.-M., and Roher, A. E. (1996) J. Neurosci. 16, 6021-6037 [Abstract/Free Full Text]
  47. Forloni, G., Angeretti, N., Chiesa, R., Monzani, E., Salmona, M., Bugiani, O., and Tagliavini, F. (1993) Nature 362, 543-546 [CrossRef][Medline] [Order article via Infotrieve]
  48. May, P. C., Boggs, L. N., and Fuson, K. S. (1993) J. Neurochem. 61, 2330-2333 [Medline] [Order article via Infotrieve]
  49. Harrison, S. C. (1996) Cell 86, 341-343 [Medline] [Order article via Infotrieve]
  50. Banzon, J. A., and Kelly, J. W. (1992) Protein Eng. 5, 113-115 [Medline] [Order article via Infotrieve]
  51. Carrell, R. W., Whisstock, J., and Lomas, D. A. (1994) Amer. J. Respir. Crit. Care Med. 150, 171-175
  52. Bruce, D., Perry, D. J., Borg, J. Y., Carrell, R. W., and Wardell, M. R. (1994) J. Clin. Invest. 94, 2265-2274 [Medline] [Order article via Infotrieve]
  53. Sifers, R. N. (1995) Nat. Struct. Biol. 2, 355-357 [Medline] [Order article via Infotrieve]
  54. Kelly, J. W. (1996) Curr. Opin. Struct. Biol. 6, 11-17 [CrossRef][Medline] [Order article via Infotrieve]
  55. Ullrich, A., and Schlessinger, J. (1990) Cell 61, 203-212 [Medline] [Order article via Infotrieve]
  56. Vaughn, D. E., and Bjorkman, P. J. (1996) Neuron 16, 261-273 [Medline] [Order article via Infotrieve]
  57. Busciglio, J., Lorenzo, A., and Yankner, B. A. (1992) Neurobiol. Aging 13, 609-612 [CrossRef][Medline] [Order article via Infotrieve]
  58. Davis-Salinas, J., Saporito-Irwin, S. M., Cotman, C. W., and Van Nostrand, W. E. (1995) J. Neurochem. 65, 931-934 [Medline] [Order article via Infotrieve]
  59. Dooley, C. T., Chung, N. N., Wilkes, B. C., Schiller, P. W., Bidlack, J. M., Pasternak, G. W., and Houghten, R. A. (1994) Science 266, 2019-2022 [Medline] [Order article via Infotrieve]
  60. Schumacher, T. N. M., Mayr, L. M., Minor, D. L., Jr., Milhollen, M. A., Burgess, M. W., and Kim, P. S. (1996) Science 271, 1854-1857 [Abstract]
  61. Dintzis, H. M., Symer, D. E., Dintzis, R. Z., Zawadzke, L. E., and Berg, J. M. (1993) Proteins Struct. Funct. Genet. 16, 306-308 [Medline] [Order article via Infotrieve]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.