Structural Transitions Associated with the Interaction of Alzheimer beta -Amyloid Peptides with Gangliosides*

JoAnne McLaurinDagger §, Trudy Franklin, Paul E. FraserDagger par , and Avijit ChakrabarttyDagger **

From the Dagger  Department of Medical Biophysics and  Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario M5G 2M9, Canada

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Alzheimer's disease is characterized pathologically by the presence of neurofibrillary tangles and amyloid plaques. The principal component of the plaque is the beta -amyloid peptide (Abeta ), a 39-43-residue peptide. The conformational change required for the conversion of soluble peptide into amyloid fibrils is modulated by pH, Abeta concentration, addition of kinetic and thermodynamic enhancers, and alterations in the primary sequence of Abeta . We report here the ability of gangliosides to induce an alpha -helical structure in Abeta and thereby diminish fibrillogenesis. Circular dichroism and a fluorescence dye release assay data indicate that gangliosides interact with and induce alpha -helix formation in Abeta . We find that the sialic acid moiety of gangliosides is necessary for the induction of alpha -helical structure. Differences in the amount and the position of the sialic acid on the carbohydrate backbone also affect the conformational switch. The Abeta -ganglioside interaction at pH 7.0, monitored by CD, is stable over time and resistant to high concentrations of NaCl. The induction of alpha -helical structure is greater with Abeta 1-40 than Abeta 1-42. The ability of gangliosides to sequester Abeta from fibril formation was also evaluated by electron microscopy.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Alzheimer's disease is characterized by the presence of amyloid plaques surrounded by dead and dying neurons in the brain (1, 2). The principal component of the plaque is the beta -amyloid (Abeta )1 peptide, a 39-43-residue peptide found in normal human tissue and generated as a cleavage product from the larger amyloid precursor protein (3-6). Abeta in the plaque is in the form of an amyloid fibril, approximately 100 Å in diameter and several microns long (7). The conformational changes required for the conversion of soluble Abeta into amyloid fibrils have been demonstrated to be a nucleation-dependent process (8, 9) which is modulated by pH, Abeta -peptide concentrations, and presence of nucleation seeds, such as proteoglycans and apolipoproteins (10-14). Alterations in the primary sequence of Abeta peptides greatly affect amyloid fibril formation (15). The E22Q mutation in Abeta , found in hereditary cerebral hemorrhage with amyloidosis of the dutch type (16), yields a peptide with increased ability to form amyloid fibrils (17). On the other hand, the single mutation of V18A increases the apparent alpha -helical content of Abeta and diminishes fibrillogenesis (18). We have found that the interaction of Abeta 40 and Abeta 42 with mixed ganglioside preparations and GM1 increases the alpha -helical content, and we speculated that this interaction may prevent amyloid fibril formation (19).

It has been well documented that amyloid fibrils are intimately associated with neuronal, microglial, and endothethial membranes. Cell processes are extended out into close proximity of amyloid deposits (20, 21), and cell culture studies have demonstrated that overexpression of Abeta results in cell surface ruffling (22-24). These results, as well as the studies on Abeta -membrane interactions (19, 25-31), suggested that the interaction of Abeta on cellular membranes may be a mechanism leading to cell death. In addition, cell culture experiments have demonstrated that Abeta neurotoxicity is dependent on the formation of beta -structure and fibrils (12, 32, 33). Therefore, it is of interest to investigate possible Abeta interactions that decrease beta -structure transitions, fibril formation, and subsequent neurotoxicity.

We report here the investigation of the ability of various gangliosides to induce an alpha -helical structure in Abeta 40 and Abeta 42. It was our aim to investigate whether a specific ganglioside could reproduce the structural transition seen with mixed gangliosides and whether this was a function of the ceramide backbone or variation in carbohydrate and sialic acid content. We also investigated the effect of mixed gangliosides on Abeta 40 and Abeta 42 fibril formation using electron microscopy.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Abeta Peptides-- Abeta 40 and Abeta 42 were purchased from U.S. Peptide Inc. (Rancho Cucamonga, CA). To minimize the presence of endogenous fibril nucleation seeds, peptides were initially dissolved in 0.5 ml of 100% trifluoroacetic acid (Aldrich), diluted in distilled H2O, and immediately lyophilized. The lyophilized peptide was then dissolved in 40% trifluoroethanol (TFE, Aldrich) in H2O and stored at -20 °C until use.

Vesicle Preparation-- Pure lipids or mixtures of lipids consisting of bovine brain phosphatidylcholine (PC), bovine brain mixed ganglioside preparation, the monosialogangliosides GM1, GM2, GM3, disialoganglioside GD1a, trisialoganglioside GT1b, ceramide, sulfatide, and sphingomyelin were dissolved at a concentration of 5 mg/ml in either chloroform or chloroform:methanol (2:1). Bovine brain PC was purchased from Avanti Polar Lipids; all other lipids were purchased from Sigma. Mixed ganglioside preparations are composed of predominantly GM1, and the remainder consists of the GM, GD, and GT series of gangliosides as well as other minor gangliosides. An aliquot of each lipid was dried under a stream of nitrogen, lyophilized overnight, and suspended in PBS, pH 7.0, in the presence or absence of a saturated solution of 5-(and -6)-carboxy-2',7'-dichlorofluorescein (Molecular Probes Inc., Eugene, OR). Lipid suspensions were carried through 10 cycles of freeze-thaw in a acetone:dry ice bath. Unilamellar vesicles were then prepared by sonication for 30 min or until the solution cleared in a bath sonicator (Branson Ultrasonic Corp., Danbury, CT). Column chromatography on Sepharose 4B (Sigma) (1.5 × 12 cm) was used to separate free dye from dye-loaded vesicles.

Sucrose Density Gradient Centrifugation-- Discontinuous sucrose gradients were prepared by layering 10 and 40% sucrose solutions. To the top of each tube, 100 µl of sample was layered, and the gradients were centrifuged at 40,000 rpm for 18 h at 4 °C in an SW 55Ti rotor in a Beckman LA70 Ultracentrifuge. A total of 20 fractions were collected using an ISCO Density Gradient Fractionator model 640. The lipid vesicles were detected using the Bartlett assay for phosphorus (34), and the peptide was detected by Tyr absorbance at 275 nm. Fractions exhibiting Tyr absorbance were confirmed to contain Abeta peptide by dot-blot analysis using a polyclonal anti-Abeta antibody (Boehringer Mannheim) and ECL detection.

Membrane Disruption Assay-- Dye-loaded vesicles were diluted in PBS, pH 6.0 or pH 7.0, to a final lipid concentration of 20 µM (35). After 50 s incubation (with continual stirring), Abeta peptide samples, bee venom mellitin (Sigma), or solvent controls were added at a 1:20 peptide:lipid ratio. The final peptide concentration was 1.0 µM. To monitor dye leakage, we used a Photon Technology International Fluorimeter (London, Ontario, Canada). Excitation was at 506 nm, and emission was monitored at 524 nm with a 4-nm bandwidth. Data was collected at a rate of one data point every 5 s. At the end of the experiment, 10% Triton X-100 was added to a final concentration of 0.3% to obtain complete dye release and measure total fluorescence. Vesicles were not used unless the total fluorescence was greater than 200% of the initial fluorescence and the spontaneous diffusion of dye was less than 10% of the total fluorescence. The percentage of total dye release was defined as indicated in Equation 1.
<UP>% dye release</UP>=
   <UP>100% × </UP><FR><NU>(<UP>observed fluorescence</UP>−<UP>initial fluorescence</UP>)</NU><DE>(<UP>total fluorescence</UP>−<UP>initial fluorescence</UP>)</DE></FR> (Eq. 1)

Circular Dichroism-- CD spectra were recorded on an Aviv Circular Dichroism Spectrometer model 62DS (Lakewood, NJ) at 25 °C. Spectra were obtained from 200 to 260 nm, with a 0.5-nm step, 1-nm bandwidth, and 20-s collection time per step. Peptide:lipid ratios were maintained at 1:20 with a final peptide concentration of 10 µM. The effect of various lipids on peptide conformation was determined by adding an aliquot of stock peptide solutions to lipid vesicles suspended in PBS with continuous stirring. The contribution of lipid vesicles to the CD signal was removed by subtracting the CD spectra of pure lipid vesicles from the CD spectra of peptide:lipid suspensions. Peptide conformations in 40% TFE:H2O were determined under the same conditions. When ellipticity at either 218 or 222 nm was measured, data were collected at a rate of 1 s/point for 300 data points.

Electron Microscopy-- For negative staining, carbon-coated pioloform grids were floated on aqueous solutions of peptides (30 µg/ml) in the presence and absence of lipids (1:20, peptide:lipid ratio). After grids were blotted and air-dried, the samples were stained with 1% (w/v) phosphotungstic acid. The peptide assemblies were observed in a Hitachi 7000 operated at 75 kV.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Abeta -ganglioside Binding Studies-- We have used synthetic lipid vesicles as a model system to investigate the interactions of Abeta with ganglioside containing cellular membranes. Gangliosides alone are unable to form stable vesicles (36); consequently, the various gangliosides were mixed with PC in a 1:1 ratio (by weight) to produce stable vesicles. We have previously demonstrated that Abeta 40 and Abeta 42 are unable to cause membrane disruption and aggregation of PC vesicles (19, 28). The ability of Abeta 40 and Abeta 42 to bind PC, mixed gangliosides, and GM1 was investigated using a binding assay that utilized sucrose density gradient centrifugation.

Mixed ganglioside:PC, GM1:PC, and PC vesicles were incubated with either Abeta 40 or Abeta 42 (Fig. 1). The Abeta -bound vesicles were separated from unbound vesicles by sucrose density gradient centrifugation in a 10-40% discontinuous sucrose gradient. The presence of peptide in the various fractions was detected by Tyr absorbance and dot-blot analyses with an anti-Abeta polyclonal antibody; lipid was detected by phosphorus assay. When samples containing Abeta 40 and PC vesicles were examined, all the peptide was recovered near the top of the gradient, although the vesicles were found near the bottom of the gradient (Fig. 1A). Similar results were seen for Abeta 42 incubated with PC vesicles (data not shown).


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 1.   Sucrose density gradient of Abeta 40 in the presence of lipid vesicles consisting of either PC (A) or mixed ganglioside:PC (B). Abeta 40 was detected by Tyr absorbance at 275 nm (dashed line). Lipid vesicles were detected by the Bartlett assay for phosphorus content (solid line). Dot-blot analysis of fractions containing Tyr absorbance confirmed the presence of Abeta 40.

However, when vesicles were made with mixed gangliosides and PC, the Abeta 40 peptide was recovered further down the sucrose gradient coincident with fractions containing vesicles (Fig. 1B). Similar results were seen for Abeta 42-mixed ganglioside:PC vesicles and when both peptides were incubated with GM1:PC vesicles (data not shown). These results demonstrate that although Abeta 40 and Abeta 42 do not bind to PC vesicles, they bind ganglioside:PC vesicles, suggesting that any effects observed with ganglioside:PC vesicles will be solely the result of Abeta -ganglioside interactions.

Abeta -induced Dye Release from Lipid Vesicles-- To investigate further the Abeta -ganglioside interaction, we employed a dye release assay. 5-(and 6-)Carboxy-2',7'-dichlorocarboxyfluorescein is incorporated into various lipid vesicles at a concentration that induces self-quenching. The addition of a membrane disrupting molecule results in the release of dye and an increase in fluorescence. The integrity of the vesicles was determined by monitoring the spontaneous diffusion of dye over time. If the spontaneous dye release exceeded 10% of the total release or if bee venom mellitin did not induce 100% dye release, then the vesicles were not used in this study. The interaction of peptides with lipid vesicles may induce dye leakage as a result of peptide surface binding that disrupts vesicle integrity, penetration of the lipid membrane, or micellar fusion (37). We investigated the ability of Abeta 40 and Abeta 42 to induce dye leakage from lipid vesicles at pH 7.0 (Table I).

                              
View this table:
[in this window]
[in a new window]
 
Table I
Percentage dye release
Abeta peptides 1-40 and 1-42 (dissolved in 40% TFE) were added to lipid vesicles in a 1:20 peptide to lipid ratio with a final peptide concentration of 1 µM. Dye release was measured at pH 7.0. Mellitin was used as a positive control, and 40% TFE was used as a negative solvent control for dye release. Data points were collected continuously for 10 min, and the data are reported as the maximal percent dye release. S.D. were calculated from at least five experiments for each lipid system. Mixed, mixed ganglioside preparation. Sph, sphingomyelin.

We have previously demonstrated that Abeta 40 is able to induce dye release from mixed ganglioside:PC and GM1:PC vesicles at pH 7.0, whereas Abeta 42 could not (19). To determine if this is a general phenomenon associated with all gangliosides or if there is a dependence on the carbohydrate and sialic acid content, we investigated the ability of Abeta 40 and Abeta 42 to induce dye leakage from vesicles containing GM2, GM3, GD1a, and GT1b (Table I). Abeta 40 was able to induce dye leakage from GM2 containing vesicles but not GM3, GD1a, and GT1b containing vesicles. These results indicate that the position and amount of carbohydrate affect the Abeta 40-ganglioside interaction. The inability of Abeta 40 to induce leakage from ceramide containing vesicles confirms that the ganglioside head group, which is missing in ceramide, is critical for Abeta -ganglioside interaction. Abeta 42 was unable to induce dye leakage from all ganglioside containing vesicles. On the glycosphingolipid synthetic pathway, ceramide is a precursor of gangliosides, cerebrosides, or sphingomyelin. We found that Abeta 40 and Abeta 42 were able to cause dye leakage from sphingomyelin vesicles (Table I). These results are surprising since the head group of sphingomyelin is either phosphorylethanolamine or phosphorylcholine, and their physical properties are very similar to phosphatidylethanolamine (PE) and phosphatidylcholine. Previously, we have shown that Abeta 40 and Abeta 42 do not disrupt PE or PC vesicles (28). The difference between sphingomyelin and PC:PE is the fatty acyl composition, the former containing one fatty acyl chain and one hydrocarbon chain of sphingosine, whereas the latter contains 2 fatty acyl chains. Variation in the dye release assay may result from the inability of the sphingosine group to pack efficiently in vesicle systems, thereby allowing access to the hydrophobic core of the lipid.

Abeta 40-induced dye release from mixed ganglioside and GM1 containing vesicles plateaus before 100% dye release; this may be the result of multiple vesicle types present in our system, one of which is accessible to disruption and another which is inaccessible. Alternatively, Abeta 40 may have a strong interaction with a limited number of lipid molecules where it becomes sequestered, thereby leaving a number of vesicles unaffected. To differentiate between these two possibilities, the dependence of dye release on peptide concentration was examined. Incremental increases in concentrations of Abeta 40 resulted in a stepwise increase in dye release from both mixed ganglioside:PC and GM1:PC vesicles (Fig. 2A). The step-shaped Abeta 40-ganglioside dye release curves suggested that binding of Abeta 40 to mixed ganglioside or GM1 containing vesicles is effectively irreversible. Increasing concentrations of Abeta 42 failed to induce dye leakage from either mixed ganglioside:PC or GM1:PC vesicles (Fig. 2B). The data from the dye release and the sucrose gradient assays suggest that although Abeta 42 is able to bind gangliosides, its interaction with the ganglioside head group does not induce disruption of the bilayer.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2.   The concentration dependence of Abeta 40 (A) and Abeta 42 (B) membrane interaction was investigated by means of the dye release assay. The initial lipid concentration was held constant at 20 µM. The initial peptide concentration was 1 µM. Incremental amounts of peptide were added to mixed ganglioside:PC (dashed line) or GM1:PC (solid line) vesicles when the dye release had stabilized. Equal volumes of 40% trifluoroethanol (dotted line) were included to monitor vesicle stability. These results are representative of at least three experiments.

Ganglioside-induced Abeta Structural Transitions-- The conformation of Abeta 40 and Abeta 42 in the presence of GM2, GM3, GD1a, GT1b containing vesicles was investigated by CD spectroscopy. As reported previously, Abeta 40 (Fig. 3A) and Abeta 42 (Fig. 4A) in 40% trifluoroethanol are partly alpha -helical (19, 38). Upon dilution into PBS, at pH 6.0 or pH 7.0, both Abeta 40 and Abeta 42 adopt a random structure at early time points (Figs. 3A and 4A). We have previously demonstrated that the interaction of Abeta 40 and Abeta 42 with mixed ganglioside:PC (Figs. 3B and 4B) and GM1:PC vesicles results in a alpha /beta conformation at pH 7.0 but a beta -structured conformation at pH 6.0 (19). Although Abeta 40 interacts with GM2:PC vesicles, as demonstrated by the dye release assay, this is not accompanied by a significant change in the CD spectrum at either pH 6.0 or pH 7.0 (Fig. 3, C and D). GM3:PC vesicles were also unable to induce CD changes. Similarly, Abeta 42 did not undergo significant changes in CD in the presence of GM2:PC or GM3:PC vesicles at both pH 6.0 and pH 7.0 (Fig. 4, C and D).


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 3.   Circular dichroism spectra of Abeta 40 in the presence or absence of various lipids. Lipids were present at a 1:20 peptide to lipid ratio with a final peptide concentration of 10 µM. CD spectra of Abeta 40 in PBS, pH 6.0 (solid line), PBS, pH 7.0 (dotted line), and 40% TFE (dashed line) are shown in A. CD spectra of Abeta 40 in the presence of mixed ganglioside:PC (B), GM2:PC (C), GM3:PC (D), GD1a:PC (E), and GT1b:PC (F) at both pH 6.0 (solid line) and pH 7.0 (dotted line).


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 4.   Circular dichroism spectra of Abeta 42 in the presence or absence of various lipids. Lipids were present at a 1:20 peptide to lipid ratio with a final peptide concentration of 10 µM. CD spectra of Abeta 42 in PBS, pH 6.0 (solid line), PBS, pH 7.0 (dotted line), and 40% TFE (dashed line) are shown in A. CD spectra of Abeta 42 in the presence of mixed ganglioside:PC (B), GM2:PC (C), GM3:PC (D), GD1a:PC (E), and GT1b:PC (F) at both pH 6.0 (solid line) and pH 7.0 (dotted line).

The interaction of Abeta 40 with GD1a containing vesicles does not induce a structural transition at either pH (Fig. 3E). Although Abeta 40 did not induce dye release from GT1b:PC vesicles, the interaction with GT1b containing vesicles induces beta -structure at pH 6.0 (Fig. 3F). Abeta 42 was unable to induce membrane disruption of GD1a:PC and GT1b:PC vesicles, but these interactions induce beta -structure formation at both pH 6.0 and pH 7.0 (Fig. 4, E and F). It is interesting that the extent of beta -structure is identical at pH 6.0 and pH 7.0; this is similar to the structural transition of Abeta 42 in the presence of phosphatidylinositol vesicles (28). These results imply that Abeta 42 is interacting with the ganglioside head group and that induction of beta -structure does not affect membrane integrity.

We have previously demonstrated that asialo-GM1:PC vesicles were unable to induce the structural transitions of both Abeta 40 and Abeta 42, implying a critical role for the sialic acid moiety (19). Neither ceramide nor sialic acid alone could induce the partly helical structure seen in Abeta 40- and Abeta 42-mixed ganglioside-PC interactions at pH 7.0 (Fig. 5). Similarly, these components could not induce beta -structure in Abeta 40 at pH 6.0. These results imply that although the sialic acid is critical for structure formation, its association with the carbohydrate backbone is also necessary. Both ceramide and sialic acid induced a small change in the structure of Abeta 42 at pH 6.0. The ability of ceramide alone to induce a structural change may be the result of the presence of both a hydrogen acceptor (amide carbonyl) and a hydrogen donor (hydroxyl group) on ceramide, which we have previously shown to be necessary for beta -structural transitions (28).


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 5.   Circular dichroism spectra of Abeta 40 (A, B, and C) and Abeta 42 (D, E, and F) in the presence of ceramide (A and D), sialic acid (B and E) and sphingomyelin (C and F). Lipids and sialic acid were present at a 1:20 peptide:reagent ratio with a final peptide concentration of 10 µM. CD spectra were obtained in PBS at pH 6.0 (solid line) and pH 7.0 (dashed line).

As previously noted, ceramide is a biosynthetic precursor to gangliosides, sphingomyelin and cerebrosides. To determine if the ability of Abeta 40 and Abeta 42 to induce dye release from sphingomyelin vesicles at pH 7.0 has a structural correlate, we investigated the ability of sphingomyelin to induce structure in Abeta 40 and Abeta 42 (Fig. 5, C and F). beta -Structure was evident in Abeta 42 in the presence of sphingomyelin at pH 6.0 but not pH 7.0. The ability of sulfated cerebrosides (sulfatides) to induce beta -structure in Abeta 40 and Abeta 42 was investigated in light of the importance of sulfated glycosaminoglycans to enhance Abeta -fibrillogenesis (10, 11, 39). We were unable to detect a structural change in either Abeta 40 or Abeta 42 in the presence of sulfatide containing vesicles (data not shown). These results suggest that sulfated cerebrosides do not function in a similar manner to sulfated glycosaminoglycans during fibrillogenesis.

Effect of Relative Ganglioside Concentration on Abeta -Ganglioside Interactions-- The relative concentration of ganglioside required to induce the structural transitions in Abeta 40 was determined at both pH 7.0 and pH 6.0 (Fig. 6). The ganglioside content was decreased by decreasing the ganglioside:PC ratio. The ability of mixed gangliosides or GM1 to induce alpha -helical structure was monitored at 222 nm. Decreasing the GM1:PC ratio of the lipid vesicles, from 0.5 to 0.33, eliminated the Abeta 40 structural transition to alpha -helix at pH 7.0 (Fig. 6A). On the other hand, decreasing the mixed ganglioside:PC ratio from 0.5 to 0.1 reduced helical content by only 36% (Fig. 6B). These results may suggest that there is an unidentified ganglioside that induces more stable helix formation than GM1, or alternatively, mixed gangliosides can segregate within PC vesicles to a higher extent than GM1 enabling the necessary environment for the structural transition (36). Similar to the GM1 results, when the mixed ganglioside:PC ratio is decreased from 0.5 to 0.3, the ability to induce beta -structure in Abeta 40 at pH 6.0 is eliminated (Fig. 6C). The monotonic relationship between beta -structure and ganglioside content suggests that the structural transition does not involve the formation of a discrete peptide-ganglioside complex but is an interaction between the peptide and negatively charged regions of the membrane. These results are consistent with the beta -structural transitions induced by acidic phospholipids (28).


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 6.   The dependence of circular dichroism at 222 and 218 nm of Abeta 40:lipid mixtures on ganglioside:PC ratios. The total lipid concentration was at a 1:20 peptide:lipid ratio with a final peptide concentration of 10 µM. The ganglioside:PC ratio was varied by increasing concentrations of PC. The alpha -helical content of Abeta 40 in the presence of GM1 (A) and mixed gangliosides (B) was monitored at pH 7.0, whereas the amount of beta -structure in the presence of mixed gangliosides (C) was monitored at pH 6.0. Data points are reported as the mean of 300 data points.

Ionic Strength Dependence of Abeta -Ganglioside Interactions-- The interaction of Abeta 40 with mixed ganglioside containing vesicles may be the result of electrostatic and/or hydrophobic interactions. The ability of increasing concentrations of NaCl to shield charges on both Abeta and lipid vesicles can give an indication of the contribution of electrostatic interactions to the stability of the Abeta -ganglioside complex. The alpha -helical content of Abeta 40 in the presence of increasing concentrations of NaCl was monitored at 222 nm, and beta -structure was monitored at 218 nm. At pH 7.0, the helical structure was retained even at 1.0 M NaCl (Fig. 7A). These results imply that the alpha -helical structure is not stabilized by charge interactions. At pH 6.0, the amount of beta -structure present in Abeta 40 decreased by one-third at 120 mM NaCl and did not change significantly up to 1 M NaCl (Fig. 7B). These results indicate that the beta -structure seen in Abeta 40-ganglioside interactions is stabilized by both electrostatic and hydrophobic interactions. When Abeta 42-ganglioside interactions were monitored in NaCl concentrations up to 1 M and at pH 6.0, the amount of beta -structure decreased. These results suggest that electrostatic interactions are involved in stabilizing the beta -conformation of Abeta 42.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 7.   The dependence of circular dichroism at 222 and 218 nm of Abeta 40:lipid mixtures on ionic strength. The ability of mixed ganglioside:PC vesicles to induce alpha -helix (A) at pH 7.0 and beta -structure (B) at pH 6.0 was monitored at 222 and 218 nm, respectively. Data points are reported as the mean of 300 data points.

Time Dependence of Abeta -Ganglioside Interactions-- The stability of the Abeta 40 and Abeta 42-mixed ganglioside-PC interaction over a period of 96 h was also investigated (Fig. 8). It is well documented that Abeta alone or in the presence of kinetic enhancers undergoes a structural transition from random coil to beta -structure that is necessary for fibril formation. In the absence of lipid vesicles and at pH 7.0, Abeta 40 was randomly structured for up to 72 h. At 96 h, beta -structure could be detected in Abeta 40 (Fig. 8C). On the other hand, Abeta 40 remained alpha -helical in the presence of mixed ganglioside:PC vesicles for the entire 96-h time course (Fig. 8). The magnitude of theta 222 nm, which is indicative of helical content, did not vary over time indicating that Abeta 40 was not lost due to aggregation or precipitation. These results suggest that when monomeric Abeta 40 interacts with gangliosides it forms a stable alpha -helical structure that does not progress to the beta -sheet structure of amyloid fibrils.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 8.   The stability of Abeta 40-mixed ganglioside interactions over a 96-h period, monitored by circular dichroism spectroscopy. Abeta 40 alone (dashed line) and in the presence of mixed ganglioside:PC vesicles (solid line) were incubated at 10 µM peptide concentration in PBS, pH 7.0. Abeta 40 was unstructured at t = 0 h (A) and remained unstructured for up to 48 h (B), whereas at 96 h (C) beta -structure was apparent. In contrast, Abeta 40 in the presence of mixed ganglioside:PC vesicles remained alpha -helical over the entire 96 h.

In the absence of lipid vesicles and at pH 7.0, Abeta 42 was randomly structured when diluted into PBS. At 48 h, beta -structure could be detected in Abeta 42 (data not shown). The amount of CD signal at 218 nm decreased significantly over the 96-h period indicating that aggregation was occurring. The alpha -helical content of Abeta 42 in the presence of mixed ganglioside:PC vesicles decreased steadily over the 96 h. CD signal could be detected at 218 nm indicating a shift to beta -structure (data not shown). These results suggest that although Abeta 42-ganglioside interactions initially form an alpha -helical structure, this may proceed to the beta -structure over time.

Abeta -Fibril Formation-- The structural characteristics of Abeta 40 and Abeta 42 fibrils in the presence and absence of gangliosides were examined by means of electron microscopy. Negative stain preparations examined by electron microscopy indicated that Abeta 40 and Abeta 42 exhibited distinct abilities to assemble into fibrous structures in the presence of gangliosides. Initial studies involved the incubation of unseeded Abeta peptides in the presence of PC and mixed ganglioside:PC vesicles. Negative stain electron microscopy demonstrated that Abeta 40 and Abeta 42 formed short, indistinct fibers that were highly aggregated when incubated alone or in the presence of PC and mixed ganglioside:PC vesicles (data not shown). All fibers were detected in areas devoid of vesicles.

The structural characteristics of fibrils formed from seeded Abeta in the presence and absence of gangliosides was also determined using negative stain electron microscopy. Abeta 42 incubated alone produces short aggregated fibers (data not shown). When seeded Abeta 42 was incubated in the presence of PC vesicles, no fibers were seen in association with lipid vesicles. Some fibers could be detected in areas that were devoid of lipid and were indistinguishable from those of Abeta 42 incubated alone. The mixed ganglioside:PC vesicles present with Abeta 42 were starting to collapse but were similar to the structure of mixed ganglioside:PC vesicles incubated alone (Fig. 9A). At initial times, no fibrils could be detected when Abeta 42 was added to mixed ganglioside:PC vesicles (Fig. 9B). When Abeta 42 was incubated for longer periods in the presence of mixed ganglioside:PC vesicles, many long fibers could be seen arranged along the surface of the lipid vesicles (Fig. 9C). The Abeta 42 fibrils had some lateral aggregation present. The mixed ganglioside:PC vesicles are no longer small sonicated vesicles but seem to have coalesced into larger vesicles with increased surface area. These results suggest that the glycolipids are creating a support for the fibrils to extend across.


View larger version (69K):
[in this window]
[in a new window]
 
Fig. 9.   Negative stain electron microscopy of Abeta 42 and in the presence of mixed ganglioside:PC vesicles. Mixed ganglioside:PC preparations contained both unilamellar (arrows) and multilamellar vesicles (arrowheads). Abeta 42 incubated in buffer alone demonstrates small thin fibers. When Abeta 42 was incubated in the presence of PC vesicles (B) no fibers could be detected although many long aggregated fibers were arranged along the surface of mixed gangliosides:PC vesicles (C). Magnification, 87,000 (A and B) and 116,000 × (C).

When Abeta 40 was incubated alone, it also produced short aggregated fibers (Fig. 10A). In the presence of PC vesicles, many fibers could be seen arranged along the surface of the vesicles (Fig. 10C). Fibrils appeared to have a lot of lateral aggregation present. These results suggest that once Abeta 40 is seeded then it will form fibrils across the lipid membrane surface as a result of hydrophobic interactions. When Abeta 40 was incubated in the presence of mixed ganglioside:PC vesicles, a few long fibers could be detected (Fig. 10B). The fibrils were present as singular fibers that transversed the lipids rather than staying at the edge of the membrane as seen with PC vesicles alone. As detected with Abeta 42, the mixed ganglioside:PC vesicles appeared to have coalesced into large structures with an increased surface area. These results are consistent with gangliosides acting as an inhibitor of nucleation, since a lower number of fibrils were present and they were of greater length (9).


View larger version (60K):
[in this window]
[in a new window]
 
Fig. 10.   Negative stain electron microscopy of Abeta 40 in the presence of PC and mixed ganglioside:PC vesicles. Abeta 40 incubated in buffer alone (A) demonstrates short thin fibers. When seeded Abeta 40 was incubated in the presence of PC vesicles (C) long fibers were present along the surface of the vesicles, probably as a result of hydrophobic interactions. Alternatively, only a few thin fibers could be detected in the presence of mixed ganglioside:PC vesicles (B). Magnification, 116,000 (A) and 87,000 × (B and C).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Abeta peptides have been reported to exert many effects on cell function. It has been proposed that Abeta exerts these effects by initially disrupting the membrane lipid environment that in turn would affect the activity of various proteins involved in signal transduction and ion fluxes (40). Abeta peptides may alter the neuronal membrane lipid environment by inducing lipid peroxidation and/or changing lipid fluidity. It has been shown previously that Abeta 40 increased both annular and bulk fluidity of synaptic plasma membranes (29). In contrast, Abeta 40 also was shown to decrease the bulk fluidity of lymphocyte membranes and of membranes isolated from cortex, hippocampus, striatum, and cerebellum (41). The disparity between these systems may originate from differences in the lipid composition of the membranes. The synaptic plasma membranes were isolated directly, whereas membranes isolated from various regions of the brain are homogenates containing plasma membranes, intracellular membranes, and various organelles. With particular relevance to the results reported here, synaptic plasma membranes have a relatively high concentration of gangliosides with respect to other cellular membranes.

We have shown that Abeta 40 is able to induce dye leakage from ganglioside containing vesicles and requires the presence of a single sialic acid molecule and a carbohydrate backbone. If the vesicles contain more complex gangliosides (i.e. GM3, GD1b, and GT1a) then Abeta 40 is unable to induce membrane disruption. The Abeta 40-ganglioside interaction forms a stable, specific complex of Abeta 40 with a limited number of gangliosides. We have shown that mixed gangliosides and GM1 are able to form this complex. In contrast, we found that Abeta 42 was unable to induce dye release from all ganglioside containing vesicles. These results suggested that Abeta 42 bound to the surface of gangliosides and does not affect bilayer integrity; however, the possibility that it affects membrane fluidity cannot be ruled out. The interaction of Abeta with gangliosides in the neuronal membrane may represent one mechanism by which Abeta disrupts the membrane fluidity and/or integrity thus explaining the increased permeability to ions and dysregulation of signal transduction systems seen in the presence of Abeta (40).

The high binding potential of gangliosides has been attributed to the general surfactant character of the gangliosides and the direct involvement of the ceramide or oligosaccharide portions of the molecules in binding (42). Whereas certain toxins, interferons, and hormones interact primarily with the oligosaccharides of gangliosides, certain antibiotics and membrane proteins interact with the ceramide group. The carbohydrate moiety of gangliosides is a low affinity receptor for the hormone, thyrotropin, and it induces the conformational change necessary for thyrotropin's insertion into the lipid bilayer (43). The antibiotic, valinomycin, interacts with the fatty acyl chains of gangliosides and fits into a cavity created by the ganglioside packing in the hydrophobic region of the lipid (44). This interaction does not disrupt the lipid bilayer organization.

Our results demonstrate that both Abeta 40 and Abeta 42 bind gangliosides. The interactions of Abeta 40 and Abeta 42 with mixed gangliosides may explain some of the different effects of these peptides in vitro and in vivo. The binding of Abeta 40 to mixed gangliosides or GM1 containing vesicles induces an alpha -helical structure at pH 7.0 and beta -structure at pH 6.0. The helical structure was stable over time and resistant to large changes in ionic strengths but required proper positioning of sialic acid on the carbohydrate moiety. These results suggest that stable alpha -helical structure is stabilized by both polar and hydrophobic interactions. Gangliosides could not induce the alpha -helical structure if the Abeta was already aggregated in a beta -structured form. Conversely, the helical Abeta 40-ganglioside complex does not proceed to beta -structure, at least over a period of 4 days. These results were confirmed using electron microscopy in that unseeded Abeta 40 did not progress to form fibrils, whereas some fibrils formed if seeded Abeta 40 was present with mixed ganglioside:PC vesicles. The decreased number but increased length of Abeta 40 fibrils in the presence of mixed gangliosides:PC vesicles in comparison to Abeta 40 alone suggests that mixed gangliosides:PC vesicles are acting as an inhibitor of the fibril nucleation step.

The interaction of Abeta 42 with gangliosides also induces an increased amount of alpha -helical structure at pH 7.0 and beta -structure at pH 6.0. Yanagisawa and co-workers (45) reported that Abeta 42 was isolated in association with GM1 from human brain that had a high proportion of diffuse plaques. They speculated that this may be a specific form of Abeta 42 that led to formation of diffuse plaques. The presence of Abeta 42-ganglioside complexes with an increased alpha -helical content may help explain the lack of cell death associated with diffuse plaques. Previous studies have demonstrated that Abeta neurotoxicity is associated with the formation of beta -structure, Abeta aggregation, and fibril formation (12, 32, 33). The alpha -helical structure of Abeta bound to gangliosides may therefore be a non-toxic conformation.

It is well documented that Abeta 42 has a greater propensity to form beta -structure than Abeta 40 (46). The ability of gangliosides to induce beta -structure in Abeta 42 at both pH 6.0 and pH 7.0 is proportional to the number of negative charges. Increasing the number of sialic acid residues on the carbohydrate backbone (i.e. with GD1a and GT1b) resulted in formation of beta -structure at pH 7.0. The presence of a high net negative surface charge on the lipid vesicles will favor a beta -structured transition and prevent the formation of the alpha -helical structure. The requirement for hydrophobic interactions to stabilize the helical conformation and the inherent properties of Abeta 42 alone suggest that formation of the helical Abeta 42-ganglioside complex will be disfavored when there is an elevation of Abeta 42 concentrations, as reported in early onset Alzheimer's cases (47).

We examined the ability of Abeta 42 to form fibrils similar to amyloid. When Abeta 42 was incubated in the presence of mixed ganglioside:PC vesicles, many fibrils could be detected that lie along the surface of the lipid vesicles. These data are consistent with the ability of gangliosides to affect both the induction of a structural transition in and the lack of membrane disruption by Abeta 42. The ability of Abeta to form fibrils with a high degree of lateral aggregation on the surface of the vesicles may help explain association of plaques to cellular plasma membrane. Finally, although we have only discussed the interaction of Abeta with plasma membranes, the Abeta -ganglioside interaction may also be important in the Golgi apparatus or the transport vesicles. Recently, it has been shown that Abeta can be generated in the trans Golgi network in the absence of transport vesicles (48, 49); this is also the site of ganglioside biosynthesis (42).

    FOOTNOTES

* This work was supported in part by grants from the Alzheimer Association, U. S. A. (to A. C.), the Ontario Mental Health Foundation (to P. E. F.), and the National Science and Engineering Council of Canada (to P. E. F.).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.

§ Recipient of a Postdoctoral Fellowship from the Alzheimer's Society of Canada.

par Supported by the Alzheimer Society of Ontario.

** To whom correspondence should be addressed: Ontario Cancer Institute, 610 University Ave., Toronto, Ontario M5G 2M9, Canada. Tel.: 416-946-2000 (ext. 4910); Fax: 416-946-6529; E-mail: chakrab{at}oci.utoronto.ca.

1 The abbreviations used are: Abeta , beta -amyloid; TFE, trifluoroethanol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PBS, phosphate-buffered saline.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

  1. Selkoe, D. J. (1991) Neuron 6, 487-498[Medline] [Order article via Infotrieve]
  2. Terry, R. D. (1994) Prog. Brain Res. 101, 383-390[Medline] [Order article via Infotrieve]
  3. Glenner, G. G., and Wong, C. W. (1984) Biochem. Biophys. Res. Commun. 120, 883-890
  4. Esch, F., Keim, P. S., Beattie, E. C., Blacher, R. W., Culwell, A. R. (1990) Science 248, 1122-1128[Medline] [Order article via Infotrieve]
  5. 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., Selkoe, D. J. (1992) Nature 359, 322-325[CrossRef][Medline] [Order article via Infotrieve]
  6. Roher, A. E., Palmer, K. C., Yurewicz, E. C., Ball, M. J., Greenberg, B. D. (1993) J. Neurochem. 61, 1916-1926[Medline] [Order article via Infotrieve]
  7. Kirschner, D. A., Abraham, C., and Selkoe, D. J. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 503-507[Abstract]
  8. Jarrett, J. T., and Lansbury, P. T. (1993) Cell 73, 1055-1058[Medline] [Order article via Infotrieve]
  9. Lomatin, A., Chung, D. S., Benedek, G. B., Kirschner, D. A., Teplow, D. B. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1125-1129[Abstract/Free Full Text]
  10. Fraser, P. E., Nguyen, J. T., Chin, D. T., Kirschner, D. A. (1992) J. Neurochem. 59, 1531-1540[Medline] [Order article via Infotrieve]
  11. Brunden, K. R., Richter-Cook, N. J., Chaturvedi, N., Frederickson, R. C. A. (1993) J. Neurochem. 61, 2147-2154[Medline] [Order article via Infotrieve]
  12. 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. (1994) Mol. Pharmacol. 45, 373-379[Abstract]
  13. Evans, K. C., Berger, E. P., Cho, C.-G., Weisgraber, K. H., Lansbury, P. T., Jr. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 763-767[Abstract]
  14. Soto, C., and Frangione, B. (1995) Neurosci Lett. 186, 115-118[CrossRef][Medline] [Order article via Infotrieve]
  15. Fraser, P. E., McLachlan, D. R., Surewicz, W. K., Mizzen, C. A., Snow, A. D., Nguyen, J. T., Kirschner, D. A. (1994) Mol. Biol. (Moscow) 244, 64-73
  16. Levy, E., Carman, M., Fernandez-Madrid, I., Power, M., Lieberburg, I., vanDuinen, S., Gerard, T., Bots, A., Luyendijk, W., and Frangione, B. (1990) Science 248, 1124-1128[Medline] [Order article via Infotrieve]
  17. Wisniewski, T., Ghiso, J., and Frangione, B. (1991) Biochem. Biophys. Res. Commun. 179, 1247-1254[Medline] [Order article via Infotrieve]
  18. Soto, C., Castano, E. M., Frangione, B., and Inestrosa, N. C. (1995) J. Biol. Chem. 270, 3063-3067[Abstract/Free Full Text]
  19. McLaurin, J., and Chakrabartty, A. (1996) J. Biol. Chem. 271, 26482-26489[Abstract/Free Full Text]
  20. Yamaguchi, H., Nakazato, Y., Hirai, S., Shoji, M., and Harigaya, Y. (1989) Am. J. Pathol. 135, 593-597[Abstract]
  21. Weigel, J., and Wisniewski, H. M. (1990) Acta Neuropathol. 81, 116-124[Medline] [Order article via Infotrieve]
  22. Maestre, G. E., Tate, B. A., Majocha, R. E., Marotta, C. A. (1992) Brain Res. 599, 64-72[CrossRef][Medline] [Order article via Infotrieve]
  23. Maestre, G. E., Tate, B. A., Majocha, R. E., Marotta, C. A. (1993) Brain Res. 621, 145-149[CrossRef][Medline] [Order article via Infotrieve]
  24. Tate, B., Majocha, R. E., Maestre, G. E., Marotta, C. A. (1992) Bull. Clin. Neurosci. 56, 131-139
  25. Arispe, N., Pollard, H. B., and Rojas, E. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10573-10577[Abstract]
  26. Terzi, E., Holzemann, G., and Seelig, J. (1995) J. Mol. Biol. 252, 633-642[CrossRef][Medline] [Order article via Infotrieve]
  27. Pillot, T., Goethals, M., Vanloo, B., Talussot, C., Brasseur, R., Vandekerckhove, J., Rosseneu, M., and Lin, L. (1996) J. Biol. Chem. 271, 28757-28765[Abstract/Free Full Text]
  28. McLaurin, J., and Chakrabartty, A. (1997) Eur. J. Biochem. 245, 355-363[Abstract]
  29. Avdulov, N. A., Chochina, S. V., Igbavboa, U., O'Hare, E. O., Schroeder, F., Cleary, J. P., Wood, W. G. (1997) J. Neurochem. 68, 2086-2091[Medline] [Order article via Infotrieve]
  30. Blanc, E. M., Toborek, M., Mark, R. J., Hennig, B., Mattson, M. P. (1997) J. Neurochem. 68, 1870-1881[Medline] [Order article via Infotrieve]
  31. Choo-Smith, L.-P., and Surewicz, W. K. (1997) FEBS Lett. 402, 95-98[CrossRef][Medline] [Order article via Infotrieve]
  32. Pike, C. J., Burdick, D., Walencewicz, A. J., Glabe, C. G., Cotman, C. W. (1993) J. Neurosci. 13, 1676-1687[Abstract]
  33. Lorenzo, A., and Yankner, B. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 12243-12247[Abstract/Free Full Text]
  34. Barlett, G. R. (1959) J. Biol. Chem. 234, 466-468[Free Full Text]
  35. McLean, L. R., Baron, B. M., Buck, S. H., Krstenansky, J. L. (1990) Biochim Biophys. Acta 1024, 1-4[Medline] [Order article via Infotrieve]
  36. Sarti, P., Antonini, G., Malatesta, F., Vallone, B., Brunori, M., Masserini, M., Palestini, P., and Tettamanti, G. (1990) Biochem. J. 267, 413-416[Medline] [Order article via Infotrieve]
  37. Dempsey, C. E. (1990) Biochim. Biophys. Acta 1031, 143-161[Medline] [Order article via Infotrieve]
  38. Barrow, C. J., and Zagorski, M. G. (1991) Science 253, 179-182[Medline] [Order article via Infotrieve]
  39. Snow, A. D., Mar, H., Nochlin, D., Kimata, K., Kato, M., Suzuki, S., Hassell, J., and Wight, T. N. (1988) Am. J. Pathol. 133, 456-463[Abstract]
  40. Roth, G. S., Joseph, J. A., and Mason, R. P. (1995) Trends Neurosci. 18, 203-206[CrossRef][Medline] [Order article via Infotrieve]
  41. Muller, W. E., Koch, S., Eckert, A., Hartmann, H., and Scheuer, K. (1995) Brain Res. 674, 133-136[CrossRef][Medline] [Order article via Infotrieve]
  42. Tettamanti, G., and Riboni, L. (1993) Adv. Lipid Res. 25, 235-267[Medline] [Order article via Infotrieve]
  43. Kohn, L. D., Consiflio, E., DeWolf, M. J. S., Grollman, E. F., Ledley, F. D., Lee, G., Morris, N. P. (1980) in Structure and Function of Gangliosides (Svennerholm, L., Mandel, P., Dreyfus, H., and Urban, P.-F., eds), pp. 333-339, Plenum Publishing Corp., New York
  44. Rahmann, H., Schifferer, F., and Beitinger, H. (1992) Neurochem. Int. 20, 323-338[Medline] [Order article via Infotrieve]
  45. Yanagisawa, K., Odaka, A., Suzuki, N., and Ihara, Y. (1995) Nat. Med. 1, 1062-1066[Medline] [Order article via Infotrieve]
  46. Hilbich, C., Kisters-Woike, B., Reed, J., Masters, C. L., Beyreuther, K. (1991) J. Mol. Biol. 218, 149-163[Medline] [Order article via Infotrieve]
  47. Selkoe, D. J. (1997) Science 275, 630-631[Free Full Text]
  48. Tienari, P. J., Ida, N., Ikonen, E., Simons, M., Weidemann, A., Multhaup, G., Masters, C. L., Dotti, C. G., Beyreuther, K. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4125-4130[Abstract/Free Full Text]
  49. Xu, H., Sweeney, D., Wang, R., Thinakaran, G., Lo, A. C. Y., Sisodia, S. S., Greengard, P., Gandy, S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3748-3752[Abstract/Free Full Text]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.