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(The FASEB Journal. 2002;16:1526-1536.)
© 2002 FASEB

Plasma membrane cholesterol controls the cytotoxicity of Alzheimer’s disease AßP (1–40) and (1–42) peptides

NELSON ARISPE1 and MICHAEL DOH

Department of Anatomy, Physiology and Genetics, and Institute for Molecular Medicine, Uniformed Services University School of Medicine, USUHS, Bethesda, Maryland, USA

1Correspondence: Department of Anatomy, Physiology and Genetics, School of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd., Bethesda MD, 20814, USA. E-mail: narispe{at}usuhs.mil


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Cell degeneration in Alzheimer’s disease is mediated by a toxic mechanism that involves interaction of the AßP peptide with the plasma membrane of the target cell. We report here that PC12 cells become resistant to the cytotoxic action of AßP when incubated in a medium that enriches cholesterol levels of the surface membrane. On the other hand, making cholesterol-deficient membranes by either cholesterol extraction with cyclodextrin or by inhibiting de novo synthesis of cholesterol makes PC12 cells more vulnerable to the action of AßP. Increasing cholesterol content of PS liposomes also suppresses AßP-dependent liposome aggregation. We suggest that by modifying the fluidity of the neuronal membranes, cholesterol modulates the incorporation and pore formation of AßP into cell membranes. This idea is supported by our finding that the enhanced cytotoxicity generated by lowering the membrane cholesterol content can be reversed by AßP calcium channel blockers Zn2+ and tromethamine.—Arispe, N., Doh, M. Plasma membrane cholesterol controls the cytotoxicity of Alzheimer’s disease AßP (1–40) and (1–42) peptides.


Key Words: amyloid-ß-peptide • AßP ion channels • calcium • phosphatidylserine • membrane interaction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Alzheimer’s disease is characterized by extracellular plaques in specific parts of the brain, which are composed of amyloid ß-proteins (AßP) derived from proteolytic processing of amyloid precursor protein (APP) (1 , 2) . AßP(1–40) or AßP(1–42) interacts with the target cell membrane, which results in calcium influx and loss of intracellular calcium homeostasis (3) . This imbalance has been proposed to mediate AßP neurotoxicity (4) . The mechanism by which AßP interaction with the membrane results in the generation of a subsequent calcium influx remains elusive, and a variety of mechanisms have been proposed. Some investigators have proposed that the first step involves interaction of specific proteinaceous membrane receptors with AßP (4) . Others have postulated that the ion flux occurs after AßP interacts directly with components in the lipid bilayer matrix of the plasma membrane (3 , 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19) . We have proposed that AßP may act on nerve cells by directly interacting with the plasma membrane to form a toxic calcium channel (10 , 11 , 29) . Many subsequent reports have further demonstrated the ability of AßP to independently insert (20 , 21) and form ion channels in membranes (10 11 12 13 14 15 16 17 , 22 23 24 25 26 27 28 29 30) .

Changes in the lipid composition of the neuronal membranes may therefore have regulatory consequences for interactions of AßP with cells in the brain. For example, the presence of cholesterol in neuronal membranes is known to induce large changes in membrane fluidity (31 , 32) . The presence of cholesterol in artificial lipid bilayers inhibits the channel-forming activity of human amylin (62) and reduces the insertion and formation of ion channels by Aß25–35 (30) . Recently we reported that soluble cholesterol is an effective inhibitor of AßP-induced free calcium elevation in GT1–7 cells (33 , 34) and that phosphatidyl serine (PS) liposome aggregation by AßP (35) is modulated by cholesterol (36 37 38) . We have therefore hypothesized that AßP neurotoxicity may be modulated by the cholesterol content of the membrane. To test this hypothesis, we have investigated the ability of membrane cholesterol to affect AßP toxicity in differentiated and undifferentiated PC12 cells.

We report here that enriching PC12 cells with exogenous cholesterol makes the cell resistant to the cytotoxic action of AßP. On the other hand, reducing membrane cholesterol makes the cell more vulnerable to the action of AßP. This enhanced AßP cytotoxicity is suppressed by the addition to the cell culture medium of AßP calcium channels blockers such as Zn2+ and tromethamine. We suggest that by modifying the fluidity of the neuronal membranes, cholesterol controls the incorporation of AßP into cell membranes. This incorporation consequently results in formation of AßP calcium channels, and thus cell death.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Liposome preparations
Large unilamellar liposomes were prepared using the extrusion method. To form PS liposomes with cholesterol incorporated into the membrane, palmitoyl-oleoyl-phosphatidyl serine and cholesterol (Avanti Polar Lipids) dissolved in CHCl3 (10 mg/mL) were mixed at various ratios and air dried in N2 gas. The dried lipids were hydrated in a solution containing 250 mM NaCl and 25 mM TRIS-HCl to a final concentration of 10 mg of phospholipid/mL. For hydration, the phospholipids suspensions were kept at room temperature for 1 h (or at 4°C for 24 h) before they were passed through an Avanti Mini-Extruder (Avanti Polar Lipids, Alabaster, AL) apparatus. The suspension was passed 12 to 15 times through a 0.05 mm polycarbonate membrane to obtain a homogeneous size distribution of liposomes ~ 50 nm.

Liposome aggregation assay
Aggregation of liposomes by the AßP was determined from the change in absorbance that follows the increase in turbidity of the liposome suspension. Absorbance was measured at 350 nm in a Hewlett Packard spectrophotometer and data were collected every 30 s. The aggregation reactions were performed at room temperature in 1 mm path-length optical glass cells. Standard assays were conducted in 300 mM sucrose-40 mM histidine-HCl, pH 6, in the presence of 0.5 mM MgCl2 and 1 mM CaCl2. AßP(1–40) and AßP(1–42), obtained from BACHEM (Torrance, CA) and from AnaSpec (San Jose, CA), were dissolved in water and added directly to the reaction cells to the desired final concentration. Finally, the aggregation reaction was initiated by addition of freshly prepared liposomes.

Cell culture
PC12 cells derived from a transplantable rat pheochromocytoma (ATCC # CRL 1721) were cultured in ATCC medium: Ham’s F12K medium (2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 82.5%; horse serum, 15%, fetal bovine serum, 2.5%) and subcultured in 24-well culture plates for viability tests. To subculture, cells were removed from dishes by pipetting with fresh medium and the suspension was passed several times through a 22 gauge needle and dispensed into the wells. Cell cultures were treated with or without nerve growth factor NGF (100 ng/mL) before addition of test agents. Differentiation states of cells had no consequence in terms of sensitivity to different test conditions. A subclone of PC12 cells, PC12s, was obtained from the National Institutes of Health, Bethesda, MD (courtesy of Dr. Stanley Rapaport) and was found to behave in similar fashion. PC12s cells have a greater intrinsic adhesive property than PC12 cells.

Changes in membrane cholesterol and cell viability
To enrich the cholesterol content of the membrane, PC12 cells were incubated in a cholesterol-enriched medium (33) . Water-soluble cholesterol (polyoxyethanyl-cholesteryl sebacate; Sigma, St. Louis, MO) was first dissolved in distilled water at a concentration of 10 mg/mL and added to the culture medium. Cells were incubated for 2 h in the media containing soluble cholesterol and washed with cholesterol-free media before the addition of AßP. To decrease the cholesterol content of the PC12 surface membranes, we used conventional methods with methyl-ß-cyclodextrin or mevastatin (39 40 41 42 , 58 , 59) . PC12 cells were cultured in the presence of either methyl-ß-cyclodextrin (Sigma), mevastatin (Sigma), or both at concentrations and times previously shown to yield maximal changes in cholesterol with minimal effects on cell viability. To prevent cell growth inhibition by mevastatin, mevalonate (500 µM) was simultaneously added to the culture medium (43) . Methyl-ß-cyclodextrin was routinely removed from the medium before the addition of AßP. Mevastatin and mevalonate were maintained in the medium for the duration of the experiments after the addition of AßP.

To quantify the cytotoxicity of AßP (1–40), a colorimetric XTT assay (Cell Proliferation Kit II from Roche Molecular Biochemicals, Nutley, NJ) was used. This metabolic activity assay is frequently used to measure factor-induced cytotoxicity. In our experiments this assay was applied after cells had been exposed to AßP and the floating dead cells removed. This approach provides an estimate of the cells that remain metabolically active and have not been fatally affected by AßP. Thus, there is a possibility of counting as viable cells those that are nearly dead. Consequently, the counts provided by the assay give the information required for the purpose of the experiments. PC12 cells subcultured in 24-well plates were exposed to different concentrations of the test drugs. To determine cell viability, the colorimetric assay was applied after the incubation period in the presence of the drugs and the results were expressed as a percentage of surviving control cells.

Determination of membrane cholesterol levels
To determine levels of cholesterol in the surface membrane, PC12 cells were grown in 96-well plates and treated as needed. Culture medium was removed and cells were incubated for 90 min in filipin (500 µg/mL) in PBS. Filipin was then removed and cells were washed twice with PBS. The fluorescence from each well, indicative of the level of cholesterol on the surface of the cells, was read in a Wallac plate reader (Victor2 multilevel counter) with a 355/460 filter.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSION
 REFERENCES
 
Cholesterol blocks AßP-induced PS liposome aggregation
The presence of cholesterol in membranes is known to induce changes in the structure and fluidity of the phospholipid bilayer. We have previously reported that AßP induces aggregation of PS liposomes (35) . We therefore examined the influence of cholesterol on AßP-induced liposome aggregation. As shown in Fig. 1 A, stepwise increments in cholesterol content result in proportional decreases in liposome aggregation induced by AßP(1–40). Figure 1B shows that similar results can be obtained using AßP(1–42). The initial rate of the aggregation reaction induced by both peptides as a function of the percentage of the cholesterol in the membrane is shown normalized in Fig. 1C . AßP(1–42) is relatively more potent than AßP(1–40). In either case, however, when the cholesterol concentration increases relative to the total lipid content, the liposome aggregation reaction tends to be progressively lower as a limit to the levels observed in controls.



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  		Figure 1. Influence of cholesterol in AßP-induced phosphatidylserine liposomes aggregation. A) Time course of the optical density change of a suspension of phosphatidylserine liposomes initiated by the addition of 10 µg/mL AßP(1–40). The numbers in the curves indicate the percentage of cholesterol in the membrane of the liposome (% mol/mol). B) Time course of the optical density change of a suspension of phosphatidylserine liposomes initiated by addition of 10 µg/mL AßP(1–42). The numbers in the curves indicate the percentage of cholesterol in the membrane of the liposome (% mol/mol). C) Normalized initial rate of the optical density change as a function of the percentage of cholesterol in the liposome membrane (% mol/mol).

Cholesterol elevation decreases the sensitivity of PC12 cells to AßP cytotoxicity
The results on liposome aggregation induced by AßP show that cholesterol reduces and can prevent PS-liposome aggregation reaction. To test whether cholesterol might similarly affect the association of AßP with cellular membranes, we examined the effect of cholesterol on the toxicity of AßP(1–40) and AßP(1–42) for PC12 cells. We used soluble cholesterol, which has been shown to readily incorporate into the surface membrane of the cells (33 , 34) . Viability tests performed after 24 h in cholesterol-enriched medium showed that additional incorporation of cholesterol into the surface membrane does not compromise the viability of the cells vs. the level of viability of cells in control condition. We therefore exposed PC12 cells for 24 h to different concentrations of AßP(1–40) or AßP(1–42) in the presence and the absence of soluble cholesterol (500 µM) in the culture medium. As shown in Fig. 2 A, AßP(1–40) reduces cell viability in a dose-dependent manner to values well below the control level. However, when soluble cholesterol (0.5 mM) was added simultaneously with AßP(1–40), the reduction of cell viability was highly attenuated. Determination of the amount of membrane cholesterol indicated that this procedure significantly increases the membrane cholesterol level by 27% ± 6.4 (P<0.0008) compared to control cells. The sole presence of this amount of cholesterol in the cell membrane did not affect the viability of PC12 cells compared to control cells.



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  		Figure 2. Effect of cholesterol-enriched medium on the sensitivity of PC12 cells to AßP toxicity. A) PC12 cells were incubated for 24 h with AßP(1–40) in a medium with (gray bars) or without (black bars) 0.5 mM soluble cholesterol. The percentage of viable cells after 24 h of incubation with 0.1 µM AßP(1–40) alone is 46% ± 6.3. When 0.5 mM cholesterol is in the medium, the percentage of cells is 94% ± 8.0 (*P<6x10-12 compared to AßP alone). The percentage of viable cells after 24 h of incubation with 1 µM AßP(1–40) alone is 39% ± 8.2. When 0.5 mM cholesterol is in the medium the percentage of cells is 85% ± 9.0 (*P<5x10-8 vs. AßP alone). Data are mean ± SE (n=4). B) PC12 cells were incubated for 24 h in a media containing several concentrations of AßP under three different experimental conditions. AßP alone (black bars), incubated for 24 h in media containing soluble cholesterol (0.5 mM) (dark gray bars) and exposed for 2 h to a medium containing soluble cholesterol (0.5 mM) before exposure to cholesterol-free AßP-containing medium (light gray bars). In both conditions in which cells were exposed to cholesterol, the percentage of viable cell is considerably improved and significantly different from the condition where cholesterol was not added (*P<0.0005, **P<0.003). Protection by cholesterol under the two different conditions gives significant results when cells are exposed to 1 µM AßP(1–42) (right end of the figure) (*P<0.0005 and **P<0.0004 compared to AßP alone).

It is possible that since soluble cholesterol was present in the medium for the entire time course, suppression of AßP toxicity could have been due to inactivating interactions between cholesterol and AßP in the medium. To exclude this possibility, we performed the experiment under three different experimental conditions. In the first condition, the cells were incubated for 24 h in media containing several concentrations of AßP. In the second condition, the cells were incubated for 24 h in media containing soluble cholesterol (0.5 mM) and several concentrations of AßP. In the third condition, cells were exposed for 2 h to a medium containing soluble cholesterol (0.5 mM) before exposure to cholesterol-free medium containing AßP. As shown in Fig. 2B , pretreatment with cholesterol for 2 h before addition of AßP(1–40 or AßP(1–42) resulted in vastly enhanced cellular survival. Determination of the amount of membrane cholesterol indicated that this procedure significantly increases the membrane cholesterol to the same levels observed after the 24 h of cholesterol exposure.

Finally, we were concerned that the effect of cholesterol addition might be due to transfer of cholesterol from the surface membrane into the cell interior. Progesterone has been used to control transfer of plasma membrane cholesterol to intracellular pools (44 , 45) . We therefore exposed PC12 cells to cholesterol-containing media for 2 h, then incubated the cells for 24 h with AßP in media with and without progesterone (4 µg/mL). The presence of progesterone in the media had no effect on the level of sensitivity of the cells toward the toxic effect of AßP (data not shown). The data suggests that cholesterol enrichment of intracellular membranes is not the basis of cholesterol-dependent suppression of AßP toxicity.

Cyclodextrin-mediated cholesterol extraction increases the sensitivity of PC12 cells to AßP toxicity
Based in the preceding data, we hypothesized that reducing the cholesterol content of the cells’ surface membrane below normal levels might have the opposite effect of increasing the sensitivity of the cells to AßP toxicity. To test this hypothesis, we extracted cholesterol from the surface membrane of cultured PC12 cells by treating the cells with methyl-ß-cyclodextrin (CD). This condition has the capacity to cause a reduction of cholesterol levels during the first 0.5 to 1 h of incubation while not affecting cell viability (39 , 40 , 46) . We performed preliminary experiments to determine the limits for safe use of CD on our PC12 cells. We observed that beyond certain limits of time and concentration, CD alone could affect the viability of PC12 cells. We found that 10 mM CD becomes toxic to cells when the exposure is > 60 min. Therefore, to reduce cholesterol from the surface membrane and maintain cell viability within the same levels of untreated cells, treatment of PC12 cells with CD never exceeded the limits of 60 min duration and 10 mM concentration. We then added AßP(1–40). As shown in Fig. 3 A, after 24 h, AßP(1–40) (1 µM) alone reduces the proportion of viable cells to 74%. However, pretreatment with 10 mM CD for up to 60 min increases the sensitivity of the cells to AßP in a time-dependent manner without significantly affecting the baseline viability of the cells. In this experiment, AßP reduces the percentage of viable cells pretreated with CD to 28% compared to 74% in the untreated cells (P<1x10-5).



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  		Figure 3. Cyclodextrin-mediated cholesterol extraction increases the sensitivity of PC12 cells to AßP toxicity. A) PC12 cells were treated for cholesterol extraction for different periods in a medium containing 10 mM methyl-ß-cyclodextrin, then incubated for 24 h in the presence of AßP(1–40) (black bars). The percentage of viable untreated cells after 24 h of incubation with 1 µM AßP(1–40) is 74% ± 5.5. Same concentration of AßP(1–40) reduces to 28% ± 6.0 the percentage of viable cells treated for 60 min with CD (*P<1x10-5 vs. untreated cells). Pretreatment with 10 mM CD for up to 60 min (gray bars) has no significant effect on percentage of viable cells after 24 h compared to control. B) PC12 cells were maintained for 60 min for cholesterol extraction in a medium containing different concentrations of methyl-ß-cyclodextrin, then incubated for 24 h in the presence of AßP(1–40) (black bars). The percentage of cholesterol that remains in the cell membrane (gray bars) after CD treatment is plotted for each corresponding CD concentration used. The percentage of viable cells after 24 h in the presence of 1 µM AßP(1–40) is 52% ± 3.45. When cells are pretreated for 60 min with 10 mM CD, the membrane cholesterol level is reduced to 38% of the untreated value (*P<7x10-7); 1 µM AßP(1–40) reduces the number of viable cells to 8% ± 7.1 (*P<1x10-5 vs. AßP on untreated cells). The insert shows the correlation between the percentage of cholesterol in the membrane (compared to control) and the percentage of viable cells after each CD treatment (empty circles) and after 2 h in cholesterol-enriched culture medium (filled circle). The straight line is the least-squares fit to the data (r2=0.995). For all other cell viability and membrane cholesterol values, *P << 1 x 10-3 compared to CD-untreated values. C) The viability of the PC12 cells treated and untreated for cholesterol extraction with 10 mM CD for 60 min was followed during the course of 24 h. At 2 h the reduction of the percentage of viable cells (81%±12.25) in the treated group (empty circles) was already significantly different (*P<0.002). At 24 h of exposure to AßP, the viability of untreated cells (filled circles) is significantly reduced to 76% ± 4.4 (*P<0.002 vs. time zero value). During the same period, the percentage of viable cells in the group of CD-treated cells is reduced to 52% ± 5.75 (*P<2x10-6 compared to zero time value).

We find that the effect of CD on AßP toxicity is dependent on CD concentration. As shown in Fig. 3B , increasing the concentration of CD in the pretreatment phase makes cells more sensitive to the toxic effect of AßP. This enhanced sensitivity becomes higher as the concentration of CD used in the pretreatment is raised. The figure also shows the corresponding reduced percentages of surface membrane cholesterol compared to control that remains after each progressively increased concentration of CD. As shown in the insert, there is a very good inverse correlation between the number of cells that remain viable after 24 h in the presence of AßP and the amount of cholesterol in the cell membrane. CD pretreatment accelerates development of the enhanced sensitivity of the cells. As shown in Fig. 3C , the time course for onset of the CD effect indicates that potentiation of AßP toxicity is evident as early as 2 h.

Pretreatment of PC12 cells with CD increases the sensitivity of the cells to AßP(1–40) and AßP(1–42). Figure 4 A, B shows the results from cells pretreated with 10 mM CD for 60 min before the media was replaced with CD-free media containing various concentrations of AßP(1–40) or AßP(1–42), respectively. The proportion of viable cells in the pretreated group measured after 24 h of exposure to AßP is always significantly smaller for any concentration of either peptide. The sensitivity of the cells to AßP(1–42) is relatively higher than AßP(1–40). However, CD pretreatment similarly affects sensitivity of the cells to any one of the peptides.



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  		Figure 4. Cyclodextrin-mediated cholesterol extraction increases the sensitivity of PC12 cells to both AßP(1–40) and AßP(1–42). A) The viability of the PC12 cells, treated (gray bars) and untreated (black bars) for cholesterol extraction with 10 mM CD for 60 min, was measured for different concentrations AßP(1–40). The percentage of viable cells in the treated group measured after 24 h of exposure to AßP(1–40) is always significantly smaller for any concentration (*P<0.006 compared to untreated cells). B) The viability of the PC12 cells treated (gray bars) and untreated (black bars) for cholesterol extraction with 10 mM CD for 60 min was measured after 24 h for different concentrations AßP(1–42). The percentage of viable cells in the treated group, measured after 24 h of exposure to AßP(1–40) is always significantly smaller for any concentration (*P<0.03 compared to untreated cells).

Inhibition of cholesterol synthesis increases sensitivity of PC12 cells to AßP
Limiting the cholesterol supply by inhibiting the de novo synthesis with HMG-CoA reductase inhibitors has been successful in reducing the content of cholesterol in the plasma membrane. By combining pretreatment with CD, followed by inhibition of HMG-CoA reductase with statins, a 70% reduction in cellular cholesterol levels has been obtained in a variety of cells. This procedure has been used without significantly affecting cell viability and integrity (39 , 40 , 47 , 48) . Based on these results, we followed similar procedures to test whether limiting cholesterol synthesis could also increase sensitivity of PC12 cells to AßP. We treated the cells with mevastatin alone and in combination with CD. Mevastatin is an effective inhibitor of HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis (43) . Mevastatin also inhibits cell growth, which can be prevented by the addition of mevalonate (43) . The use of mevastatin in our experiments was always accompanied by the simultaneous addition of mevalonate (500 µM) to the culture medium. We preincubated PC12 cells for 24 h in a serum-free medium containing 5 µM mevastatin, then the medium was replaced with a medium containing mevastatin (5 µM) and serum. Under these conditions, PC12 cells maintain the same viability levels as cells growing in the absence of mevastatin. The cells were then exposed to different concentration of AßP(1–40) and AßP(1–42). Figures 5 A, B show the percentages of viable cells remaining after 24 h in the presence of AßP(1–40) and AßP(1–42), respectively. For all AßP concentrations examined, the cells treated with mevastatin showed a higher sensitivity to the toxic effect of both AßP(1–40) and AßP(1–42). The counts of viable cells after 24 h in the presence of AßP were significantly lower (P<0.008 and 0.003 for AßP(1–40) and AßP(1–42), respectively) when the cells had been cultured in the presence of the cholesterol synthesis inhibitor. The data plotted have been normalized because a small reduction (<10%) in the viability of the cells was observed after 24 h pretreatment with serum-free and mevastatin-containing media. Our experiments therefore show that either CD or mevastatin are effective in making cells more sensitive to the toxic effect of AßP.



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  		Figure 5. Inhibition of cholesterol synthesis with mevastatin increases the sensitivity of PC12 cells to both AßP(1–40) and AßP(1–42). A) Viability of the PC12 cells incubated in the presence and absence of 5 µM mevastatin for inhibition of the synthesis of cholesterol was measured for different concentrations AßP(1–40). Percentages of viable cells measured after 24 h of incubation in media containing mevastatin and different concentrations of AßP(1–40) (gray bars) are always significantly smaller (*P<0.006) than those of the groups incubated in the absence of mevastatin (black bars). B) The viability of the PC12 cells incubated in the presence and in the absence of 5 µM mevastatin for inhibition of the synthesis of cholesterol was measured for different concentrations AßP(1–42). The percentages of viable cells measured after 24 h of incubation in media containing mevastatin and different concentrations of AßP(1–42) (gray bars) are always significantly smaller (*P<0.003) than those of the groups incubated in the absence of mevastatin (black bars).

To test whether a combination of CD and mevastatin might potentiate sensitivity of PC12 cells to AßP toxicity more profoundly than either one alone, we incubated PC12 cells in a medium containing CD (10 mM) for 1 h, then replaced the media with serum-free, mevastatin (5 µM) -containing media. After 24 h the media were replaced again with mevastatin, serum, and AßP-containing media. Figure 6 shows the results of this experiment for AßP(1–40) and AßP(1–42) (0.1 µM). In all experimental conditions, treated PC12 cells were significantly more sensitive to AßP(1–40) and AßP(1–42) than were untreated cells. These results are compared with the results from simultaneous experiments in which the cells were treated with CD alone or mevastatin alone. To make the conditions equivalent for all the cells, cells treated with CD were maintained afterward for 24 h in a serum-free medium before the addition of serum and AßP containing media. Results are expressed in percentage of viable cells remaining after 24 h under the toxic action of the peptides. The result is that the combined treatment doubles the sensitivity of the cells to either of the AßPs compared to untreated cells.



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  		Figure 6. Cyclodextrin-mediated cholesterol extraction combined with inhibition of cholesterol synthesis with mevastatin increases the sensitivity of PC12 cells to AßP(1–40) and to AßP(1–42). A) PC12 cells were incubated in a medium containing CD (10 mM) for 1 h, then the media was replaced with a serum-free mevastatin (5 µM) -containing media. After 24 h, the media was replaced again with mevastatin, serum, and AßP-containing media. Compared to the percentage of viable cells exposed to AßP alone (black bars), the percentage of viable cells in the group submitted to combined cholesterol extraction and inhibition of cholesterol synthesis (horizontal striped bars) is significantly smaller (***P<1x10-4 and 2x10-6 for AßP(1–40) and AßP(1–42), respectively) than the percentage of viable cells in the group submitted to cholesterol extraction with CD alone (diagonal striped bars) (*P<0.06 and 4x10-4 for AßP(1–40) and AßP(1–42, respectively) and the percentage of viable cells in the group submitted to inhibition of cholesterol synthesis with mevastatin alone (gray bars) (**P<0.02 and 8x10-4 for AßP(1–40) and AßP(1–42), respectively).

AßP ion channels blockers attenuate AßP toxicity on cells
Numerous reports demonstrate that in bilayers as well as cells, AßP forms ion channels in membranes. It was therefore possible that the level of cholesterol in the plasma membrane could modulate the insertion of AßP oligomers to form toxic ion channels. AßP channels are blocked by zinc and tromethamine. Therefore, we predicted that blocking these channels with either Zn2+ or tromethamine would attenuate the enhanced cytotoxicity due to reduced cholesterol. To test this hypothesis, the toxicity of AßP(1–40) was examined in PC12 cells that had been pretreated with CD to remove the plasma cholesterol then incubated in media containing either Zn2+ ions or tromethamine and AßP(1–40). Figure 7 A shows that in the absence of zinc, exposure to AßP(1–40) for 24 and 48 h reduces the percentage of viable cells to 65 and 45% of the control level, respectively. When 5 µM ZnCl2 is present in the incubation media, the reduction in the percentage of cells after exposure to AßP(1–40) for 24 and 48 h is attenuated to only 6 and 19% of the control, respectively. Thus Zn2+ blocks CD-potentiated AßP cytotoxicity.



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  		Figure 7. Zinc attenuates the effect of AßP on the viability of PC12 cells. A) PC12 cells were grown in a medium containing 1 µM of AßP(1–40) and different concentrations of ZnCl2; viability of the cells was measured after 24 (dark gray bars) and 48 (light gray bars) h of incubation. In the absence of zinc, exposure to AßP(1–40) for 24 and 48 h reduces the percentage of viable cells to 65% ± 3.55 and 45% ± 7.0, respectively. The addition of zinc to the media significantly attenuates the effect of AßP on the viability of PC12 cells; for instance, the percentages of viable cells in a media containing 5 µM ZnCl2 are 95% ± 3.2 and 81% ± 4.1 after 24 and 48 h, respectively. These percentages are significantly much higher compared to the percentages in the absence of zinc (+ P<5x10-5 and 2x10-5 for 24 and 48 h, respectively). For all other concentrations of zinc used, the attenuation of the effect of AßP is also significant (*<0.02, **P<0.002, ***P<5x10-4). B) PC12 cells were treated for 60 min with different concentrations of CD for cholesterol extraction, then incubated for 24 h in a medium containing 1 µM of AßP(1–40) to study the effect of the presence of zinc in the media. In the absence of zinc in the medium (black bars), 1 µM AßP(1–40) reduces the percentage of cells not treated with CD to 37% ± 8.5. After addition of 0.5 µM of ZnCl2 to the incubation medium (gray bars), reduction in the percentage of viable cells is attenuated to 57% ± 8.0. Attenuation of the effect AßP(1–40) by the presence of zinc in the media is observed despite the intensity of the treatment with CD. For instance, 1 µM AßP(1–40) reduces the percentage of viable cells treated with 10 mM CD to 1.2%. Addition of 0.5 µM of ZnCl2 significantly attenuates this reduction to 33% ± 5.8 (***P<2x10-5, **P<6x10-4, *P<0.02 compared to the effect of AßP in the absence of zinc).

Figure 7B shows the result of an experiment where cells were pretreated with different concentrations of CD for 60 min before incubation for 24 h in the presence and the absence of 5 µM of ZnCl2 and 1 µM AßP(1–40). In the absence of zinc, exposure of the cells to AßP(1–40) reduced the percentage of viable cells in the CD-untreated group to 37% of the control. When Zn2+ was present in the media, the reduction by AßP(1–40) was attenuated to 57%. As anticipated, CD-pretreated cells show an increased sensitivity to AßP(1–40). The sensitivity of the cells to AßP increases as the concentration of CD in the pretreatment is made higher. However, despite the increasing effect of the concentration of CD in the pretreatment, this enhanced sensitivity is attenuated when zinc is also present in the incubation medium.

Turning to the effect of blocker tromethamine, Fig. 8 A shows that addition of tromethamine to the media attenuates AßP toxicity. The magnitude of the tromethamine attenuation is highly significant for all the AßP concentrations tested (*P<0.007). The effect of tromethamine on the toxicity of AßP(1–40) was also examined in PC12 cells that had been pretreated with CD to remove cholesterol from the plasma membrane. Figure 8B shows the results from an experiment in which PC12 cells were treated for 60 min with 10 mM CD for cholesterol extraction and incubated for 24 h in a medium containing 5 µM of AßP(1–40). When 30 mM of tromethamine is present in the incubation medium, the reduction in the percentage of viable cells by AßP in CD-treated and untreated groups of cells is significantly attenuated. For instance, after 24 h of exposure to AßP, the percentage of viable cells is 91% vs. 77% for non-CD-treated and -treated cells, respectively. It is clear from the results of these experiments that zinc and tromethamine, AßP ion channel blockers, attenuate the effect of increasing the sensitivity of PC12 cells to AßP due to removal of cholesterol from the cells’ surface membrane.



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  		Figure 8. Tromethamine attenuates the effect of AßP on the viability of PC12 cells. A) The viability of the PC12 cells incubated in the presence (gray bars) and the absence (black bars) of 20 mM tromethamine to block AßP ion channels was measured after 24 h incubation in media containing different concentrations of AßP(1–40). Addition of tromethamine to the media attenuates the reduction in the percentage of viable cells produced by AßP. The attenuation by tromethamine is highly significant for all AßP concentrations (*P<0.007). B) PC12 cells were treated for 60 min with 10 mM CD for cholesterol extraction, then incubated for 24 h in a medium containing 5 µM of AßP(1–40) to study the effect of the presence of tromethamine in the media. In the absence of tromethamine, 5 µM AßP(1–40) reduces the percentage of cells not treated with CD (black bars) to 71% ± 10.1 and CD-treated cells (gray bars) to 30% ± 9.9. When 30 mM of tromethamine is in the incubation medium, the reduction is significantly attenuated to 91% ± 1.65 (*P<0.02) and to 77 ± 9.3 (*P<0.0002) for non-CD-treated and CD-treated cells, respectively.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSION
 REFERENCES
 
Modulation of the sensitivity of PC12 cells to AßP by membrane cholesterol
These data show that levels of cellular cholesterol control the sensitivity of both differentiated and undifferentiated PC12 cells to the cytotoxicity of AßP peptides. This conclusion is shown most profoundly in Fig. 3B , where the sensitivity of the cells to the cytotoxicity of AßP is progressively enhanced by lowering the levels of cholesterol by either cyclodextrin extraction and/or suppression of cholesterol biosynthesis with mevastatin. By contrast, increasing the levels of cholesterol leads to suppression of AßP toxicity. We interpret these data to indicate that the mechanism of the cholesterol effect is due to well-characterized, cholesterol-dependent changes in the properties of the affected membranes.

It has been long appreciated that one of the consequences of loading membranes with cholesterol is to increase membrane stiffness and modulate membrane protein functions (49) . We used the liposome aggregation assay to analyze the effect of different contents of cholesterol in a pure acidic phospholipid membrane and thereby model the interaction of AßP with a membrane. At the level of the well-defined PS liposome system, our data show that PS liposome aggregation and fusion by AßP in vitro are also suppressed by increasing the mole fraction of cholesterol in the liposomes. Despite the unphysiological conditions, the conclusion is that without intervention of any other mechanisms, the level of cholesterol in a membrane can dramatically affect AßP-lipid interaction. It is likely that the ability of AßP to aggregate and fuse liposome depends on the ability of the peptide to penetrate into the membrane partners. Therefore, The novelty and physiological relevance of these results is that cholesterol controls membrane penetration by AßP and therefore cell sensitivity to this peptide. It follows that the cytotoxic effect of AßP may be dependent on the ability of the peptide to insert into the target cell membrane (9) . The important thing is that the model membrane study and the cell study lead to the same general conclusion.

Cholesterol is the predominant sterol in the plasma membrane and is required for numerous cellular functions (49) . Changes in cholesterol content of the membrane can modulate lipid fluidity, affecting various cellular functions related to the plasma membrane, including endocytosis, enzyme activities, receptor functions, etc. To our knowledge, little is known about alterations of other membrane lipids as a consequence of natural changes in cholesterol content of the membrane. It has been reported that in Alzheimer’s disease brains, the cholesterol/phospholipid ratio is reduced by as much as 30% whereas the phospholipid/protein ratio remains unchanged (63) . We have found that after CD extraction of cholesterol from PC12 cells’ plasma membrane, there is a slight increase in the detectable level of phosphatidyl serine in the outer side of the membrane (unpublished results). This negatively charged phospholipid increases the net negative charge of the membrane surface, thus facilitating the interaction of AßP with the membrane. Such facilitation has been observed with the AßP(25–35) fragment (30) and with the full AßP(1–40) (35) . The increased presence of these toxic peptides on the membrane will facilitate the formation of the damaging AßP ion channels. We have previously proposed that cytotoxicity of AßP is due to AßP calcium channels formed in the membrane of target cells. Our data consistently indicate that AßP channel blockers such as Zn2+ and tromethamine (10 , 11) also block the enhanced cytotoxicity of AßP(1–40) and AßP(1–42) when added to cholesterol-depleted cells. We and others have found that zinc blocks ion currents through AßP channels at concentrations well below those required to precipitate soluble AßP from aqueous solution (12 , 16) . The very low concentrations of zinc (<5 µM) necessary to attenuate cytotoxicity of both AßP(1–40) and AßP(1–42) suggest that this effect of zinc may occur by means of interference with ion flow through AßP channels.

Model for participation of cholesterol in the mechanism of interaction of AßP peptides with membranes based on the AßP ion channel hypothesis
Indirect and unspecific actions of cholesterol and zinc and tromethamine can be alleged to explain the modulating effects of these agents on the PC12 cells sensitivity to AßP. However, numerous reports indicate that AßP directly interacts with components in the lipid matrix of the plasma membrane (3 , 5 , 19 , 20 , 21) and that AßP may directly form ion channels (10 11 12 13 14 15 16 17 , 22 23 24 25 26 27 28 29 30) . Many other reports show that micromolar concentrations of zinc (12 , 14 , 15 , 16) and tromethamine interfere with ionic flow through AßP channels (10 , 12 , 14 15 16 , 26 , 36) and that membrane cholesterol interferes with the insertion of peptides to increase membrane conductance (30 , 62) . We can therefore be led to consider that this information can be used to construct a testable model for the participation of cholesterol in AßP cytotoxicity. As shown in Fig. 9 , AßP polymerizes into oligomers (51) and arrives at the membrane surface, guided by acidic phospholipid receptors (35) (1). The polymerization process, which also leads to increase AßP toxicity, can be blocked by either millimolar concentration of Zn2+ (16) or Congo red (26) . The AßP oligomer then inserts into the membrane matrix (20 , 21) . When the level of cholesterol in the membrane is higher than normal, the insertion process is prevented by the enhanced stiffness of the membrane (2) . On the other hand, if the AßP oligomer encounters a membrane with reduced fluidity due to a lower than normal level of cholesterol, the insertion process occurs (3) and a AßP calcium channel is formed (10 11 12 13 14 15 16 17 , 22 23 24 25 26 27 28 29 30 , 37 , 38) . Opening of this newly formed ion channel permits gradient-driven inflow of calcium ion, which can reach uncontrollable toxic levels (4) (11) . The ionic currents through the transmembrane AßP Ca2+ channel can be blocked by either Zn2+ or tromethamine (10 11 12 , 14) . This mechanism underlies the ß-amyloid calcium channel hypothesis for cellular degeneration in Alzheimer’s disease (22) and other amyloid related diseases (52 , 53) .



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  		Figure 9. Model for participation of cholesterol in the mechanism of interaction of AßP peptides with membranes, based in the AßP ion channel hypothesis. 1) AßP oligomer approaches the target membrane. 2) The AßP oligomer insertion process is prevented by the stiffness of the cholesterol-enriched membrane. 3) The AßP oligomer insertion process is eased by the fluidity of a cholesterol-depleted membrane. AßP forms a closed calcium channel. 4) AßP(1–40) channel opens to allow Ca2+ ions to flow across the membrane. Channels are blocked by Zn2+and tromethamine.

Alternative mechanisms for interaction of AßP with cholesterol have been proposed. For example, binding of AßP and cholesterol is known to occur when the two agents are incubated together for more than 6 h (9) . However, the protective effect by cholesterol is observed even after it is removed from the medium before exposure of the system to AßP. So although such interactions may occur in other circumstances, they are very unlikely to explain the present set of experiments.

Role of cholesterol in Alzheimer’s disease
Recent publications report a modulating effect of membrane cholesterol on the processing of the APP (40 , 44 , 47 , 48 , 54) . The stiffening of the membrane produced by loading the membrane with cholesterol is reported to decrease the generation of soluble APP by the {alpha}-secretase cleavage of the transmembrane APP (44 , 54 , 61) . Depleting the membrane of cholesterol by inhibiting the enzyme of cholesterol biosynthesis, alone or in combination with cyclodextrin, reduces the level of AßP(1–40) and AßP(1–42) and stimulates the {alpha}-secretase pathway to generate soluble APP (40 , 47 , 48) .

On the basis of those results and the example we are presenting here, it appears that modifying cholesterol in the membrane may have a dual effect on the cells. On one hand, cholesterol modulates the enzymatic cleavage of the amyloid precursor protein, altering the production of the toxic AßP(1–40) and AßP(1–42). On the other hand, lowering cholesterol increases the sensitivity of the cells to the toxic effects of both AßP(1–40) and AßP(1–42). It has been published that statin use represents a valid pharmacological tool to significantly modulate brain cholesterol levels (31) and that peripheral and central cholesterol homeostasis are independent from each other (55) . Therefore, the possibility of considering the reduction of cholesterol for therapeutic purposes on the treatment of Alzheimer’s disease will depend on which one of the dual actions of cholesterol prevails.

The majority of sporadic Alzheimer’s disease patients have been reported to exhibit a loss of the HMG-CoA reductase activity in the frontal and temporal cortex. This loss of activity leads to alteration in lipid homeostasis, particularly reduction of cholesterol in areas of vulnerability (56) . Furthermore, it has been shown that disturbances in the cholesterol metabolism interact with lysosomal dysfunction to promote pathologies that characterize Alzheimer’s disease (57) . So far, however, it has not been shown that elevated membrane cholesterol elevates the amount of AßPs generated.


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
REFERENCES
 
In conclusion, the results from all the experiments discussed here demonstrate that the magnitude of the toxicity expressed by AßP on PC12 cells is modulated by the amount of cholesterol in the surface membrane. By affecting the physical properties of the membrane, cholesterol modulates the interaction and possibly the incorporation of AßP ion channel protein into the cell membrane. The reduction of cell viability generated by the interaction of AßP with the cells in culture can be attenuated with the use of substances that have been demonstrated to prevent the ionic flow through the AßP channels.


   ACKNOWLEDGMENTS
 
The authors thank Dr. H. B. Pollard for invaluable comments and discussion. This work was supported by a grant from The Alzheimer’s Association of America.

Received for publication April 11, 2002. Revision received May 23, 2002.
   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
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