Metal Ions, pH, and Cholesterol Regulate the Interactions of Alzheimer's Disease Amyloid-beta Peptide with Membrane Lipid*

Cyril C. CurtainDagger §, Fedá E. Ali, Danielle G. SmithDagger , Ashley I. BushDagger ||, Colin L. MastersDagger , and Kevin J. BarnhamDagger **

From the Dagger  Department of Pathology, University of Melbourne, and the Mental Health Research Institute of Victoria, Parkville, Victoria 3052, the § School of Physics and Materials Engineering, Monash University, Clayton, Victoria 3800, the  School of Chemistry, University of Melbourne, Parkville 3010, Victoria, Australia, and || Laboratory for Oxidation Biology, Genetics, and Aging Research Unit and Department of Psychiatry, Harvard Medical School, Massachusetts General Hospital East, Charlestown, Massachusetts 02129

Received for publication, June 3, 2002, and in revised form, September 27, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

The interaction of Abeta peptides with the lipid matrix of neuronal cell membranes plays an important role in the pathogenesis of Alzheimer's disease. By using EPR and CD spectroscopy, we found that in the presence of Cu2+ or Zn2+, pH, cholesterol, and the length of the peptide chain influenced the interaction of these peptides with lipid bilayers. In the presence of Zn2+, Abeta 40 and Abeta 42 both inserted into the bilayer over the pH range 5.5-7.5, as did Abeta 42 in the presence of Cu2+. However, Abeta 40 only penetrated the lipid bilayer in the presence of Cu2+ at pH 5.5-6.5; at higher pH there was a change in the Cu2+ coordination sphere that inhibited membrane insertion. In the absence of the metals, insertion of both peptides only occurred at pH < 5.5. Raising cholesterol to 0.2 mol fraction of the total lipid inhibited insertion of both peptides under all conditions investigated. Membrane insertion was accompanied by the formation of alpha -helical structures. The nature of these structures was the same irrespective of the conditions used, indicating a single low energy structure for Abeta in membranes. Peptides that did not insert into the membrane formed beta -sheet structures on the surface of the lipid.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Alzheimer's disease (AD)1 is a neurodegenerative disorder affecting the memory and cognitive functions of the brain. A characteristic central nervous system histologic marker in patients with AD is accumulation of the 39-43-residue amyloid beta -peptide (Abeta ) in morphologically heterogeneous neuritic plaques and cerebrovascular deposits (1). Abeta is derived from a proteolytic cleavage of the beta -amyloid precursor protein (APP). This is a highly conserved and widely expressed integral membrane protein with a single membrane-spanning domain. Possible mechanisms of Abeta toxicity include formation of plasma membrane channels (2) and generation of H2O2 through Cu2+ reduction by the peptide, which make cells more responsive to oxidative stress (3). The mechanisms may be related because reactive oxygen species may cause lipid peroxidation that leads to alterations in the order and fluidity of the bilayer lipids (4) and may also lead to an increase of membrane permeability. The common change observed in cell membrane permeability is an increased intracellular calcium level (5, 6) that could occur either indirectly through Abeta modulating an existing Ca2+ channel or directly through cation-selective channels formed by Abeta .

The supramolecular structure of membrane-associated Abeta , either as an ion channel or a fusogen, is unknown, although Durell et al. (7) have developed theoretical models for the structure of ion channels formed by the membrane-bound Abeta 40. Recently, Bhatia et al. (8) and Lin et al. (9) used atomic force microscopy, laser confocal microscopy, and fluorescent calcium imaging to examine in real time the acute effects of fresh and globular Abeta 40, Abeta 42, and Abeta 25-35 on cultured endothelial cells. Abeta peptides caused morphological changes within minutes after treatment and led to eventual cellular degeneration. Cellular morphological changes were most sensitive to Abeta 42, being seen at nanomolar concentrations and accompanied by an elevated intracellular calcium level. Atomic force microscopy of Abeta 42 reconstituted in a planar lipid bilayer showed multimeric channel-like structures. Biochemical analysis showed that the predominantly monomeric Abeta peptides in solution formed stable tetramers and hexamers after incorporation into lipid membranes.

In our initial study using the EPR technique (10), we found that, in the presence of Cu2+ and Zn2+, Abeta 42 formed allosterically ordered alpha -helical structures that penetrated negatively charged membranes. This paper extends that study to include the influence of pH, metal concentration, peptide length, and cholesterol on the conformation and incorporation of the peptide into the lipid bilayer. We show that penetration of the bilayer is closely related to conditions favorable to oligomerization of the peptide. The significance of this finding is that metals play an important part in the formation of amyloid plaques, the end point of oligomerization. Cherny et al. (11) have shown that administration of a lipophilic Cu2+/Zn2+ chelator (5-chloro-7-iodo-8-hydroxyquinoline) that crosses the blood-brain barrier attenuates plaque formation in APP2576 transgenic mice expressing human APP. Recently, the importance of Zn2+ in plaque formation has been emphasized by the finding that the age and female sex-related plaque formation characteristic of these mice was greatly reduced if they lacked zinc transporter 3, which is required for zinc transport into synaptic vesicles (12).

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Materials and Methods-- Peptides were obtained from AusPep (Melbourne, Australia) and from the W. M. Keck Laboratory, Yale University. The acidic phospholipid spin label 16NPS was synthesized according to Hubbell and McConnell (13). The water-soluble spin label TCC was obtained from Molecular Probes. All spin probes were checked for purity and to ensure that their number of spins/mol were >90% of theory (14). Cholesterol and synthetic POPC was purchased from Sigma, and POPS was purchased from Avanti Polar Lipids (Pelham, AL). LUV were prepared by the method described by Hope et al. (15). Peptides as a freeze-dried powder were added to the desired concentration to a suspension of LUV in buffer, and the mixture was vortexed under N2 for 10 min at 305 K in polypropylene tubes. One hundred-millimolar stock solutions of analytical reagent grade CuCl2 or ZnCl2 were prepared, and the desired amount was added to the LUV after addition of the peptide. 0.05 M of the chelator EGTA was added to all control solutions to counter the possible effects of any trace metals present. For copper EPR measurements, 99.99% pure 65Cu (Cambridge Isotopes) was used. Metal concentrations were measured by ICP-MS (model 700, Varian), and peptide concentrations were determined by quantitative amino acid analysis. Uptake of peptide by the LUV was estimated by density gradient centrifugation in MetrizamideTM (Nyegaard, Oslo, Norway)-Tris buffer (pH 7.0, 0.05 M) as described by Cornell et al. (16) and then measuring the extinction at 210 nm of the sub-phase. The protein concentration was estimated by using an E<UP><SUB>1 cm</SUB><SUP>1%</SUP></UP> of 204 (17). It was found that 10-15% of the added peptide was not taken up by the LUV.

EPR Spectroscopy-- Continuous wave X-band EPR spectra were obtained using a Bruker ECS106 spectrometer equipped with a temperature controller and flow-through liquid nitrogen cryostat. Cu2+ spectra were collected at 110 K from samples contained in 4-mm inner diameter "Suprasil" quartz EPR tubes (Wilmad). In order to eliminate the possibility that any line broadening observed in the spectra might be due to freezing-induced localized concentrations of sample, 10% glycerol was added to aqueous peptide buffer solutions. Labeled lipid samples (25 µl) were contained in 0.8-mm inner diameter quartz capillaries (Wilmad) and handled as described by Gordon et al. (18) to ensure reproducibility. Other procedures, including adding to the spin-labeled SUV a small amount of the water-soluble, non-membrane penetrant spin probe TCC to ensure x axis reproducibility, have been described previously (10, 19).

The analysis of EPR spectra of peptide/lipid mixtures was carried out using the spectral subtraction and addition methods described by Marsh (20). As a check on the validity of these procedures as applied to our system where an MRLC was observed, the lipid spin label spectra were simulated using the modified Bloch equations as described by Davoust and Devaux (21). Their model I was used in the simulations where the unique director orientation in the fluid component, corresponding to the nitroxide axes being aligned along the membrane normal in the MRLC, was assumed to be preserved on exchange.

CD Spectroscopy-- CD spectra of peptides in the LUV were obtained using a CD spectropolarimeter model 62DS (AVIV, Lakewood, NJ) as described previously (10).

    RESULTS
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INTRODUCTION
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Effect of pH and Peptide Length on Interaction of Abeta Peptides with LUV-- Fig. 1 shows the EPR spectra recorded at 305 K of 16NPS in POPS/POPC (1/1) LUV and the same LUV containing Abeta 42 before and after the addition of Cu2+ (0.3/1 M peptide) at pH 7.5, 7.0, 6.5, 6.0, and 5.5. The sharp lines due to the TCC marker and the curved base line due to the broad Cu2+ line have been subtracted as described previously (10). It can be seen that adding Cu2+ to the vesicles at all pH values caused the appearance of the MRLC (marked with an arrow) that is characteristic of annular phospholipid surrounding a rigid peptide inserted into the bilayer (20). At pH 5.5 the spectrum of the non-Cu2+-treated Abeta -containing LUV clearly shows the MRLC, at a slightly lower intensity than in the Cu2+-treated sample at the same pH. The control spectra (set A) were subtracted from those of the Cu2+-treated LUV and from the pH 5.5 peptide-containing LUV to give the difference spectra (set D). These experiments were repeated over the same range of pH with Zn2+ (4/1 M peptide) and Abeta 42 and with Cu2+ and Zn2+ at the respective 0.3 and 4.0 molar ratios with Abeta 40. No MRLC was found with rat Abeta 40 with both metals over the whole pH range (data not shown). By performing the subtraction with spectra of control LUV made at 286 K, as done previously with both Abeta 42 (10) and a viral peptide (19), it was shown that the peptides did not perturb the bulk lipid, as suggested by McIntyre et al. (22). All of the spectra could be simulated over the range of peptide/lipid mixtures used, as described by Davoust and Devaux (21), further discounting long range perturbation effects on the bulk lipid by the peptide at high peptide/lipid. The on- and off-rate constants were estimated from the simulation; the former were in the range 6-5.5 × 106 (s-1), and the latter were 5.5-5 × 106 (s-1). These values are similar to those published for other lipid/protein systems (23).


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Fig. 1.   EPR spectra of 16NPS in POPS/POPC LUV over the pH range 5.5-7.5 (A), after the addition of Abeta 42 (1 M peptide/30 M lipid) (B), after the addition of 0.3 M Cu2+/1 M peptide to the samples in B (C), and difference spectra obtained by subtracting the spectra in A from the corresponding spectra in C (D). Spectra were recorded at 305 K. Instrument conditions are as follows: microwave frequency 9.237 MHz, modulation frequency 100 KHz, modulation amplitude 3.2 G, microwave power 6 milliwatts, sweep time 400 s, and time constant 0.5 s. Scan width was 100 G and mid-field 3,300 G.

The proportion of MRLC to total lipid was calculated by double-integrating the five sets of difference spectra thus obtained and expressing the number of spins as a percentage of the number of spins in each experimental spectrum. The values are given in Table I. It can be seen that, with Abeta 42, decreasing pH with Cu2+ and Zn2+ leads to an increase in the MRLC. It is notable that there is a significant MRLC at pH 5.5 in the absence of either metal. With Abeta 40, however, there is a marked difference between the behavior of Cu2+ with pH in that there is negligible MRLC at pH 7.0 and 7.5 with the Cu2+, although with the Zn2+ the proportion of MRLC at all pH values is similar to that found with Zn2+ and Abeta 42. Adding Zn2+ to the pH 7.0 and 7.5 Abeta 40/Cu2+ samples (2 M Zn2+/0.3 M Cu2+) resulted in the appearance of the MRLC. At the lipid/peptide ratio employed (30/1), the proportion of MRLC corresponded approximately to four lipid molecules/peptide. This value was previously observed to remain constant for Abeta 42 (10) over a range of lipid/peptide ratios, and it can be satisfied on structural grounds by postulating that the basic membrane penetrant unit is a alpha -helical hexamer surrounded by 24 lipids (10).

                              
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Table I
Effect of pH on percentages of MRLC in EPR spectra and beta -strand and alpha -helix content of Abeta 40 and Abeta 42 in LUV
Figures at left margin of each column show % MRLC, figures in parentheses show % beta -strand (italics) and alpha -helix (boldface). Difference between 100% and sum of alpha  and beta  was due to conformational flexibility.

We have shown previously (10) that the appearance of the MRLC in the Abeta 42 negatively charged LUV system was associated with an increase in alpha -helicity of the peptide. The % alpha -helix (bold type) and beta -structure (italics) are shown alongside the MRLC data in Table I. It can be seen that wherever significant MRLC is present, the peptide showed increased alpha -helicity. Wherever the peptide does not show a MRLC, there is a preponderance of beta -structure.

Effect of pH on the Coordination Sphere of Cu2+ in Both Peptides in the LUV-- EPR spectra taken at 110 K of 65Cu2+-Abeta 28 in buffer over the pH range 5.5-7.5 (Cu2+/peptide 0.3/1 M) are shown in Fig. 2A. Identical spectra were obtained at the same pH values for Abeta 40 and Abeta 42 in the LUV and for Abeta 16 in buffer. All of the spectra are characteristic of type 2 square planar copper. However, the notable feature of the series is a decrease in g∥ (marked with arrow in Fig. 2A) between pH 5.5 and 6.0 and a further decrease between pH 7.0 and 7.5 indicating a change in the 65Cu2+ coordination sphere over these two pH ranges. The spectral parameters are given in Table II. As observed previously (10) for Abeta 42, spectra for 65Cu2+-Abeta 40 in 16NPS-LUV showed no signs of line broadening due to dipolar-induced spin lattice relaxation by the nitroxide-free radical (24), indicating that the Cu2+-binding site of the peptide did not penetrate the bilayer and come into the proximity of the nitroxide spin label. However, Cu2+ molar ratio-dependent line broadening due to Heisenberg exchange, the result of Cu2+ atoms being close in space, had been observed previously in Abeta complexes (10). Those spectra had been recorded of samples in pH 6.9 buffer. Fig. 2B shows the effect of a range of pH on this line broadening. It can be seen that, at the 0.7 Cu2+ molar ratio used, line broadening was present from pH 6 to 7, much reduced at pH 7.5, and eliminated at pH 8. The broadened spectrum at pH 5, which is markedly different to that seen at pH 6-7, was due to the presence of unbound Cu2+, indicating a weakening of the strong binding to the metal observed at the higher pH values, and corresponding to the protonation of the imidazole side chain of the histidine residues. There were no peaks at g >4 (not shown in Fig. 2B), indicating the absence of axial Cu2+ dimers, nor was there any marked reduction of signal intensity, diagnostic of the formation of EPR "silent" dicopper (25).


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Fig. 2.   A, EPR spectra taken at 110 K of Cu2+-Abeta 28 in aqueous buffer over the pH range 5.5-7.5 (Cu2+/peptide 0.3/1 M). B, EPR spectra taken at 110 K of Cu2+-Abeta 28 in aqueous buffer over the pH range 5.0-8.0 (Cu2+/peptide 0.7/1 M). Instrument settings are as follows: frequency A, 9.7660 GHz; frequency B, 9.7655, modulation frequency 100 KHz, modulation amplitude 1.011 G, microwave power 2 milliwatts, sweep time 83.886 s, time constant 10.240 ms. Spectra averaged over 36 sweeps.

                              
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Table II
EPR parameters of the type-2 Cu2+ Abeta complexes at different pH values
The hyperfine data are given for 65Cu.

From Fig. 3 it can be seen that the increase in the amount of MRLC at pH 6.0 with both peptides reaches a maximum at a Cu2+ concentration of 1 mol/mol, after which it remains constant. The effect of Zn2+ follows a similar pattern, albeit at higher concentrations, requiring almost 3 eq in keeping with the lower affinity of Abeta for Zn2+ (26).


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Fig. 3.   Effect of increasing metal concentration on proportion of MRLC in spectra of 16NPS in POPS/POPC LUV at pH 6.5. Closed circles, Abeta 42 + Cu2+; closed squares, Abeta 40 + Cu2+; open circles, Abeta 42 + Zn2+; open squares, Abeta 40 + Zn2+. Peptide/lipid, 1/30.

Effect of Cholesterol on the MRLC-- In the light of numerous reports on the significance of cholesterol on Abeta -membrane interactions in both biological membranes and model systems (27-29), the effect of adding cholesterol to the LUV on MRLC formation was investigated. The results are summarized in Table III where it can be seen that at each pH value and metal where the component was observed in the absence of cholesterol it was reduced to zero when cholesterol represented 0.2 mol fraction of the total lipid, i.e. cholesterol reduced the stability of the membrane-penetrant form of the peptide.

                              
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Table III
Effect of adding cholesterol to LUV on percentages of MRLC in EPR spectra of Abeta 40 and Abeta 42

The Effect of Adding Cu2+- to Zn2+-Abeta 40 in the Spin-labeled LUV-- Because it had been observed that the appearance of MRLC in the spin-labeled LUV after the addition of Zn2+-Abeta 40 was independent of pH, and it was pH-dependent after the addition of Cu2+-Abeta 40 (Table I), the effect of adding Cu2+ to Zn2+-Abeta 40 LUV was explored. It can be seen (Fig. 4) that at pH 7.0 the addition of Cu2+ markedly reduced the percent MRLC until none was detectable at M Cu2+/M of peptide. This result indicates that Cu2+ competes with Zn2+ for at least one of the His residues of Abeta 40, but with a coordination sphere such that the stability of the complex is to too low to promote membrane insertion.


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Fig. 4.   The effect of adding Cu2+ to Zn2+-Abeta 40 in spin-labeled LUV at pH 7.0 on the percentage of MRLC. Lipid/peptide, 1/30 (M/M), metal/peptide, 4/1 (mol/mol), temperature 294 K.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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pH, Metal-induced Membrane Penetration-- The addition of metal ion and pH are two conditions that govern the formation of the MRLC indicating peptide penetration of the hydrocarbon core of the membrane by Abeta peptides in LUV (Fig. 1), and these conditions closely parallel those governing Abeta oligomerization (30). In the absence of metals and in the presence of the chelating agent EGTA, both Abeta 40 and Abeta 42 penetrate the membrane at pH 5.5 but not at higher pH values. Both peptides aggregate in the absence of either metal at this pH (30). In the presence of Zn2+ both peptides penetrated the membrane over the entire range from pH 5.5 to 7.5. Zn2+ aggregates Abeta across this pH range (31). Abeta 40 in the presence of Cu2+ only penetrated the membrane at pH 6.5 and below, pH values at which it has been reported to oligomerize (32). On the other hand, Abeta 42 in the presence of Cu2+ penetrates and is oligomerized in solution at all pH values. It should be noted that rat Abeta 40, which has a 2N2O coordination (10) and much-reduced metal-induced oligomerization (33), did not produce an MRLC with either metal at any pH. The significance of this result is that the selectivity of human over rat Abeta 40 is one of the key metal-differentiating effects that argues strongly for metals being germane to the pathophysiology of AD. The induction of MRLC by copper-bound Abeta 42 was saturated at 1 eq (Fig. 3); this parallels our previous observation that the cooperative binding of Cu2+ to Abeta was also saturated at 1 eq (10).

Our results showed that membrane penetration was associated with an increase in alpha -helicity as determined by CD spectroscopy, whereas the CD spectra of peptide associated with liposomes showing no evidence of MRLC indicated predominantly beta -structure (Table I). These results indicated that peptide association with the hydrocarbon region of the lipid bilayer facilitated alpha -helix formation. Previously published CD studies of the interaction between the Abeta peptides and lipid vesicles of varying composition have suggested that the peptides associated with the lipids are in the beta -strand conformation (34-36). Most of these studies were limited to Abeta 25-35, a peptide that is too short to be able to span the lipid bilayer in an alpha -helical conformation. On the other hand, NMR structural studies in SDS micelle systems show that residues 15-36 are alpha -helical with a kink in the 25-27 region (37, 38).

Calculation of the annular lipid/peptide ratio allows an estimate of the size of the molecule/complex penetrating the membrane. For all the spectra exhibiting MLRC the same value of 4-4.5/1 was obtained, consistent with an alpha -helical hexameric peptide structure inserted into the bilayer in line with our previous results (10). The constancy of this ratio over a range of peptide/lipid suggested that formation of multimers occurs at relatively low value of the latter and that their size remains constant with increasing proportion of peptide in the bilayer. Factors influencing the interpretation of the stoichiometry of lipid-protein interactions determined by EPR have been discussed in detail by Marsh and Horváth (39).

It has been postulated that the ion channels that have been observed in various membrane systems could consist of either strands arranged in a beta -barrel conformation or as an alpha -helical assemblage, probably a tetramer or hexamer (see Refs. 7-10 and the data presented here). It has been shown by Horváth et al. (23) and Wolfs et al. (40), however, that the motion of the MRLC observed with membrane penetrant segments of known beta -barrel structure is much more restricted than with alpha -helical segments. This is because of a slower on-off rate for the lipids in the former case. The on-off rates calculated by simulation in our case are close to those observed for other alpha -helical systems (20).

Adding metal ions or lowering the pH are two different methods of aggregating Abeta . In the absence of lipid, metal ions induce amorphous aggregates of Abeta , and these oligomers are different from those formed in the pH-induced oligomerization of Abeta . These have a fibrillar beta -sheet structure as shown by Congo Red birefringence (41). In lipid systems, however, both methods of Abeta oligomerization give the same membrane penetrating alpha -helical structure, suggesting a well defined low energy structure for Abeta in membranes. The observation of an alpha -helical hexamer is consistent with the results of the atomic force microscopy study by Lin et al. (9) that showed that Abeta peptides were able to form hexameric channel-like structures. The greater stability of Abeta 42 in the lipid is also consistent with the observation by Bhatia et al. (8) showing a higher tendency by Abeta 42 to form channel-like structures and a greater ability to disrupt cellular Ca2+ regulation than Abeta 40. The two extra hydrophobic residues at the C terminus of Abeta 42 would result in this peptide having a higher preference for the hydrophobic core of the lipid bilayer. The effect of the extra two hydrophobic residues is amplified in the hexameric unit, as there are 12 extra hydrophobic residues per unit for Abeta 42 versus Abeta 40.

We have shown that Zn2+ will induce Abeta penetration of the lipid with the formation of structures that are similar to the channels proposed by Lin et al. (9). However, their results showed that Zn2+ and an antibody directed against the N terminus had an inhibitory effect on Abeta channel activity (9). Although our results show that metal ions can induce oligomerization and membrane penetration, the metal-binding site itself is outside the lipid bilayer (10). The antagonistic action of Zn2+ may be due to its ability to extensively cross-link the three histidine residues of Abeta to form a cap over the channel, physically blocking it.

pH-induced Differences between Abeta 40 and Abeta 42-- There is a significant reduction in Cu2+-facilitated membrane insertion of Abeta 40 at pH values of 7.0 and above, contrasting with Abeta 42 that showed consistent membrane insertion across the pH range investigated. Our present and earlier EPR data (10) indicated that Cu2+ formed type 2 square planar complexes with Abeta peptides. The spectra were independent of the peptide chain length and the presence or absence of lipid, but were influenced by pH. For type 2 complexes the g|| and A|| values correlated with the types of equatorially coordinated atoms. The decrease in g|| (Fig. 2A and Table II) between pH 6.0 and 5.5 and a further decrease between pH 7.0 and 7.5 indicated a change in the 65Cu2+ coordination sphere over these two pH ranges. This is further illustrated in Fig. 2B where the Cu2+ molar fraction-dependent line broadening attributed to Heisenberg exchange effects (10) diminishes with increasing pH, suggesting a change in the coordination sphere such that the Cu2+ atoms are no longer close in space. This would be consistent with reduction in the extent of histidine bridging between Cu2+ centers, although this was not eliminated until pH 8. Such a change in the coordination mode reflects a change in the Cu2+ from a coordination sphere that promotes peptide oligomerization such as with a histidine residue bridging copper atoms (a coordination sphere that resembles the active site of superoxide dismutase) to a mode that does not. Miura et al. (42) and Suzuki et al. (43) have shown by using Raman spectroscopy that Cu2+ binds to the Ntau of the three His residues of Abeta at pH 5.8, whereas at pH 6.6 and 7.4 it could bind to the Npi and an as yet unidentified backbone amide nitrogen. Zn2+, on the other hand, binds to the Ntau over the pH range 5.8-7.4. A change in the coordination mode of the Cu2+ bound to Abeta at higher pH that also reduces the oligomerization of the peptides would explain the observed reduction of copper-induced MLRC by Abeta 40 at these pH values. These observations are consistent with the reduced aggregation of Abeta by copper at higher pH (32). The extra hydrophobic stabilization of Abeta 42 versus Abeta 40 is able to shift the pH-induced change in equilibrium between the different coordination modes of Cu2+ back to the form that favors membrane penetration and peptide oligomerization; this is a form that resembles the active site of superoxide dismutase as we have shown previously (10). Zn, which does not undergo a pH-induced change in metal coordination, induces peptide insertion into the membrane with both Abeta 40 and Abeta 42 over the pH range investigated. However, from the data shown in Fig. 4 it appears that the 1 eq of copper at pH 7.0 will displace the zinc and inhibit Abeta 40 insertion into the membrane. Competition between Cu2+ and Zn2+ at this pH for binding to at least one of the histidine residues would explain these results. The different Cu2+ coordination modes and, hence, lipid interactions may explain the increased toxicity normally associated with Abeta 42 because this peptide is more stable in the lipid environment.

The Effect of Cholesterol on Membrane Penetration-- Cholesterol is a major component of mammalian cell membranes and is known to affect the dynamics and thickness of the hydrocarbon region of the membrane. It has been widely reported to modulate Abeta -membrane interactions, and there is strong circumstantial evidence that it has a role to play in AD (27-29). Increased membrane cholesterol reduces the membrane disordering effects of Abeta , as shown by fluorescence polarization techniques, and inhibits its effects on Ca2+ signaling (35). Our results show that enhanced membrane cholesterol has the marked effect of decreasing MRLC formation by Abeta . Penetration of the membrane by the peptides would depend on a balance between the rigidifying effects of cholesterol and the possible better match of the hydrophobic region of the peptide by the increased bilayer thickness. Because the alpha -helical hydrophobic regions of Abeta peptides span 22-25 residues (37), close to the 20-23-residue width of the unexpanded bilayer, it is likely that the rigidifying effects will predominate, resulting in peptide exclusion. Such exclusion in the presence of cholesterol would explain the inhibitory effects of the latter on Ca2+ signaling, because Abeta -induced channels would be destabilized in lipid rich in cholesterol.

Overall, the data suggest the location and stability of Abeta peptides within the membrane is delicately balanced, and as such, small changes in pH and lipid composition would be able to affect the stability of the Abeta -lipid interactions. Because neuronal cells are in a dynamic environment, with respect to pH and lipid composition that varies with different sub-cellular membrane fractions, it is not surprising that other workers (44) have found wide variations in the effects of Abeta on membrane order and fluidity in such fractions. A challenge for the future will be to define the links between the biophysical observations made in these diverse systems with the pathophysiology of AD.

    FOOTNOTES

* This work was supported in part by Prana Biotechnology Ltd., NIA Grant 2RO1AG12686 from the National Institutes of Health (to A. I. B.), and the National Health and Medical Research Council, Canberra, Australia.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. Fax: 61 3 99039655; E-mail: kbarnham@unimelb.edu.au.

Published, JBC Papers in Press, November 14, 2002, DOI 10.1074/jbc.M205455200

    ABBREVIATIONS

The abbreviations used are: AD, Alzheimer's disease; Abeta , human amyloid beta -peptide; LUV, large unilamellar vesicles; MRLC, motionally restricted lipid component; POPC palmitoyloleoyl phosphatidylcholine, POPS; palmitoyloleoyl phosphatidylserine, 16NPS, 1 palmitoyl-2-(16-doxyl stearoyl) phosphatidylserine; TCC, TEMPO choline chloride, 4-(N,N-dimethyl-N-(2-hydroxyethyl)ammonium)-2,2,6,6-tetramethylpiperidine-1-oxyl; APP, beta -amyloid precursor protein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. 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]
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