Journal of NeurochemistryLippincott.—Raven Publishers, Philadelphia© 1997 International Society for Neurochemistry

The Lipid Peroxidation Product, 4-Hydroxy-2-trans-Nonenal,Alters the Conformation of Cortical Synaptosomal

Membrane Proteins

*~RamSubramaniam, *ff~redRoediger, *Brad Jordan, t~MarkP. Mattson,

§Jeffrey N. Keller, IlGeorg Waeg, and *~D.Allan Butterfield

Departments of *Chemistry and tAnatomy and Neurobiology, tCenter of Membrane Science, and §Center on Aging,University of Kentucky, Lexington, Kentucky, U.S.A.; and ~Institutefor Biochemistry, University of Graz, Graz, Austria

Abstract: Alzheimer’s disease (AD) is widely held to bea disorder associated with oxidative stress due, in part,to the membrane action of amyloid ~3-peptide(A/3). A~-associated free radicals cause lipid peroxidation, a majorproduct of which is 4-hydroxy-2-trans-nonenal (HNE).We determined whether HNE would alter the conforma-tion of synaptosomal membrane proteins, which mightbe related to the known neurotoxicity of A~3and HNE.Electron paramagnetic resonance spectroscopy, using aprotein-specific spin label, MAL-6 (2,2 ,6,6-tetramethyl-4-maleimidopiperidin-1-oxyl), was used to probe confor-mational changes in gerbil cortical synaptosomal mem-brane proteins, and a lipid-specific stearic acid label, 5-nitroxide stearate, was used to probe for HNE-inducedalterations in the fluidity of the bilayer domain of thesemembranes. Synaptosomal membranes, incubated withlow concentrations of HNE, exhibited changes in proteinconformation and bilayer order and motion (fluidity). Thechanges in protein conformation were found to be con-centration- and time-dependent. Significant protein con-formational changes were observed at physiologically rel-evant concentrations of 1—10 p~MHNE, reminiscent ofsimilar changes in synaptosomal membrane proteinsfrom senile plaque- and A/3-rich AD hippocampal andinferior parietal brain regions. HNE-induced modificationsin the physical state of gerbil synaptosomal membraneproteins were prevented completely by using excess glu-tathione ethyl ester, known to protect neurons from HNE-caused neurotoxicity. Membrane fluidity was found to in-crease at higher concentrations of HNE (50 /2M). Theresults obtained are discussed with relevance to the hy-pothesis of A/3-induced free radical-mediated lipid perox-idation, leading to subsequent HNE-induced alterationsin the structure and function of key membrane proteinswith consequent neurotoxicity in AD brain. Key Words:Amyloid—Lipid peroxidation—Hydroxynonenal —Syn-aptosomal membranes—Protein alterations— Electronparamagnetic resonance—Alzheimer’s disease.J. Neurochem. 69, 1161—1169 (1997).

Several lines of evidence exist to suggest the impor-tance of amyloid /3-peptide (Aj3) in the neurodegener-

ation of Alzheimer’s disease (AD) (Selkoe, 1996).For example, familial AD is associated with geneticmutations in the amyloid precursor protein that leadto an overproduction of A/@ (Selkoe, 1996). Mutationsin the presenilin genes, in chromosomes 1 and 14, areassociated with AD and may result in excess amyloidproduction (Selkoe, 1996). Transgenic mice overex-pressing amyloid precursor protein have some brainpathology similar to that in AD (Games et al., 1995).A/3 is neurotoxic to cultured neurons (Yankner et al.,1990; Mattson et al., 1993; Harris et al., 1995), andA/3 neurotoxicity can be prevented by free radicalscavengers (for reviews, see Butterfield, 1996,1997a,b; Butterfield et al., 1996a, 1997a; Hensley etal., 1996).

We and others havereported, using the spin trappingagentN-tert-butyl-a-phenylnitrone (PBN) in conjunc-tion with electron paramagnetic resonance (EPR) spintrapping studies, that A/3 is associated with toxic freeradicals (Butterfield et al., 1994; Hensley et al., 1994a;Subramaniam et al., 1995; Tomiyama et al., 1996).The toxic properties of A/~-derivedfree radicals havebeen well documented (Butterfield et al., 1994; Harriset al., 1995, 1996; Mark et al., 1995). Among theseare the multiple membrane dysfunctions induced byA/3-derived free radicals, such as loss of key enzymaticactivities, lipid peroxidation, modification of structureof proteins, inhibition of Na ±-dependent glutamate up-

Received November 20, 1996; revised manuscript received March3, 1997; accepted April 16, 1997.

Address correspondence and reprint requests to Prof. D. A. But-terfield at Department of Chemistry and Center of Membrane Sci-ence, University of Kentucky, Lexington, KY 40506, U.S.A.

Abbreviations used: A/3, amyloid /3-peptide; AD, Alzheimer’sdisease; EPR, electron paramagnetic resonance; GEE, glutathioneethyl ester; HNE, 4-hydroxy-2-trans-nonenal; HWHH, half widthat half height; IRI, ischemia/reperfusion injury; MAL-6, 2,2,6,6-tetramethyl-4-maleimidopiperidin- I -oxyl; 5-NS, 5-nitroxide stea-rate; PBN, N-tert-butyl-cs-phenylnitrone; W/S, weakly/strongly im-mobilized MAL-6 spin label ratio.

1161

1162 R. SUBRAMANIAM ET AL.

take, disruption of multiple signaling pathways, proteinoxidation, generation of reactive oxygen species(ROS), inactivation of ion-motive ATPases, and ele-vated intracellular calcium levels (for reviews, seeButterfield, 1996, 1997a,b; Butterfield et al., 1996a,1997a; Hensley et al., 1996; Mattson et al., 1996).These free radical-mediated insults subsequently leadto cell death. We formulated an A/3-associated freeradical model of neuronal death in AD brain (But-terfield et al., 1994; Hensley et al., 1994a). Basically,A/3-associated free radicals are proposed to inducelipid and protein oxidation, leading to loss of ion ho-meostasis and cell death. A large number of studies onthe effects of Afi, and their prevention by free radicalantioxidants, in various cell types are consistent withthis model (for reviews, see Butterfield, 1996,l997a,b; Butterfield et al., 1996a, 1997a; Hensley etal., 1996).

Free radical-induced lipid peroxidation leads to theformation of several aldehydic products and hydroxy-alkenals (Esterbauer et al., 1991). Among these, 4-hydroxy-2-trans-nonenal (HNE) and malondialdehydehave been reviewed extensively in the literature (Es-terbauer et al., 1991). Reactive free radicals that trig-ger lipid peroxidation can eventually lead to the con-version of polyunsaturated fatty acids to lipid hydro-peroxides, which undergo the /3-cleavage reaction toform products, such as HNE and malondialdehyde(Frankel, 1982; Grosch, 1987;Esterbauer et al., 1990).HNE has been reported to be the most cytotoxic prod-uct of lipid peroxidation (Esterbauer et al., 1991). Thereactive sites on HNE, such as a — /3 unsaturation, alde-hydic functionality, and hydroxy group, allow for reac-tions with cysteines, histidines, and lysines (Friguet etal., 1994; Uchida et al., 1995; Butterfield and Stadt-man, 1997). In a protein molecule, this reaction canlead to structural modification and/or cross-linking ofproteins, which in turn can cause functional impair-ment (Butterfield and Stadtman, 1997). The activitiesof Na ±,K + -ATPase, Ca2~-ATPase, and the glutamatetransporter are inhibited by lipid peroxidation (Rohnet al., 1993; Volterra et al., 1994; Harris et al., 1996;Mark et al., 1997). HNE has been shown to lead tothe formation of cross-linked proteins, when incubatedwith glucose-6-phosphate dehydrogenase (Friguet etal., 1994), low-density lipoproteins (Nadkarni andSayre, 1995), and other proteins (Esterbauer et al.,1991). The activity of the HNE-conjugated proteinswere found to be diminished (Friguet et al., 1994;Nadkarni and Sayre, 1995). Isolated liver mitochon-dna, exposed to oxidative stress, were shown to formintra- and intermolecular protein—HNE cross-links(Cohn et al., 1996). It was proposed that HNE maytarget proteins in the lipid bilayer and alter their con-formation (Esterbauer et al., 1991). However, no studyhas been conducted to demonstrate that HNE affectsthe physical state of membrane proteins.

Recently, it has been shown that A/3 induces a largeincrease in levels of free and protein-bound HNE

(Mark et al., 1997). Separate studies demonstrated thatboth A,@ and HNE can disrupt ion homeostasis andcause neuronal cell death (Mark et al., 1995, 1997).In P19 mixed neuronal cultures, HNE was shown tocross-link cytoskeletal proteins, including ‘r, a majorprecursor of the neurofibrillary tangles found in ADbrain, into high-molecular-weight species (Montine etal., 1996). Recently, Keller et al. (1997) showed thatHNE impairs glutamate transport in rat cortical synap-tosomes by directly conjugating to the glutamate trans-port protein GLT-1.

Combining all these data, we hypothesized that lipidperoxidation products can be a “second toxic trigger”for oxidative stress (Butterfield, l997b). Specifically,in AD, our model predicts that A~3-associatedfree radi-cals (first toxic trigger) can lead to the multiple mem-brane lipid peroxidation, protein oxidation, and othermembrane dysfunctions listed above. Lipid peroxida-tion, one of the major responses to A/3-induced oxida-tive stress, will eventually lead to formation of cyto-toxic products, especially HNE, which in turn causethe structure—function alterations in the membraneproteins. The current article reports research designedto test this idea and indicates that HNE, at physiologi-cally relevant concentrations, is capable of significantmodification of the structure of cortical synaptosomalmembrane proteins.

MATERIALS AND METHODS

ChemicalsHNE was obtained from Cayman Chemical Co. (Ann

Arbor, MI, U.S.A.). Five milligrams of HNE was suppliedin 500 ~ilof ethanol (10 mg/ml) and stored at —80°C.Su-crose used in synaptosome isolation was obtained fromSigma Chemical Co. (St. Louis, MO, U.S.A.). Protease in-hibitors and chelating agents were obtained from ICN Bio-chemicals (Costa Mesa, CA, U.S.A.). All other chemicalswere of thehighest grade purity from either Sigma or AldrichChemical Co. (Milwaukee, WI, U.S.A.).

AnimalsAll procedures involving animals were approved by the

University of Kentucky Animal Care and Use Committee.Male Mongolian gerbils, 3—4 months of age, were obtainedfrom Tumblebrook Farms (West Brookfield, MA, U.S.A.),kept under 12-h light/dark conditions in the University ofKentucky Central Animal Facility, and fed standard PurinaRodent Laboratory Chow. During the light phase, the ani-mals were decapitated and the brains quickly dissected onice according to the method developed in our laboratory(Hensley et al., l994b). The neocortex was isolated, imme-diately suspended in 20 ml of ice-cold isolation buffer (0.32M sucrose containing 4 ~zg/mlleupeptin, 4 ~sgfmlpepstatin,5 ~ig/mlaprotinin, 20 ~zg/mltrypsin inhibitor, 0.2 mM phe-nylmethylsulfonyl fluoride, 2 mM EDTA, 2 mM EGTA, and20 mM HEPES), and homogenized by 12 passes with amotor-driven Teflon pestle. Samples from different gerbilswere kept separately.

Synaptosome preparationSynaptosomes were purified from homogenized neocorti-

ces by ultracentrifugation across discontinuous sucrose gra-

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HNE-INDUCED ALTERATIONS OF BRAIN MEMBRANE PROTEINS 1163

dients, as described previously (Hensley et al., 1994b).Crude homogenate was centrifuged at 3,500 rpm for 10 mmat 4°Cin a Du Pont Sorvall RC5C refrigerated centrifuge.The pellet was discarded, and the supernatant was spun at13,500 rpm for 10 mm at 4°Cin the same machine. Theresulting pellet was resuspended in isolation buffer and lay-ered onto discontinuous sucrose density gradients. Sampleswere then spun for 2 h in an SW28 rotor in a Beckman L7-55 refrigerated ultracentrifuge. Removal of the 1.18 M/ 1.10Minterface yielded thepurified synaptosomes used in furtherstudies.

HNE dose—response studyPurified synaptosomes were washed with oxidation buffer

(10 mM HEPES, 137 mM NaC1, 4.6 mM KC1, 1.1 mMKH2PO4, 0.6 mMMgCl2, and 1.1 mMEDTA, pH 7.4), firstwithout protease inhibitors, followed by another wash inthe same buffer containing protease inhibitors. Total proteinconcentration in the isolated synaptosomes was measured bymethods described previously (Lowry et al., 1951). Sampleswere divided into two aliquots, and the protein concentrationin all the samples was adjusted to 8 mg/ml. One part of thesample was treated with the vehicle for HNE (ethanol), andthe other part was treated with HNE. In this study, the sam-ples were incubated with varying concentrations of HNE (1,5, 30, 40, and 50 ~zM) for 60 mm at room temperaturewith constant stirring. At the end of this time, samples werecentrifuged at 4°C in an Eppendorf centrifuge and thenwashed three times with lysing buffer.

HNE time-course studySamples, prepared as described above, were treated with

5 ~iMHNE and allowed to react for different times (0, 1, 3,and 6 h). The treated samples were then centrifuged andlysed as described above.

Glutathione ethyl ester (GEE) protection studyAfter the protein concentration in the sample was adjusted

as described above, the samples were divided into four ali-quots. Each aliquot was incubated at room temperature for3 h. Aliquots I and 2 were incubated in buffer, whereasaliquots 3 and4 were incubated with 2 mM GEE. Followingthe 3-h incubation period, all samples were incubated for anadditional 1 h. During the fourth hour, samples 1 and 3 wereincubated with the vehicle for HNE, and samples 2 and 4were treated with 50 ~zMHNE.

Spin labelingThe lysed synaptosomes were labeled with the paramag-

netic spin labels 2,2,6,6-tetramethyl-4-maleimidopiperidin-1-oxyl (MAL-6; Fig. la), which specifically labels proteinthiols, or 5-nitroxide stearate (5-NS; Fig. lb), which interca-lates in the lipid bilayer. The procedures used for spin label-ing synaptosomal membranes with MAL-6 or 5-NS weredescribed before (Hensley et al., 1994b; Howardet al., 1996;Butterfield et al., 1997b).

Protein carbonyl measurementsThe 2,4-dinitrophenylhydrazine adducts ofprotein carbon-

yls were determined spectroscopically as previously de-scribed (Howard et al., 1996; Butterfield and Stadtman,1997; Butterfield et al., l997b).

Western blot analysisThesemethods were similar to those described previously

(Mark et al., 1996). In brief, total synaptosomal proteins

FIG. 1. a: Structure of MAL-6. b: Structure of 5-NS: 5-nitroxidestearate. C: EPR spectrum of MAL-6-labeled synaptosomalmembranes. Boxed region of spectrum is expanded in the bot-tom panel showing measurements necessary to calculate theW/S ratio. Samples were spin-labeled as described previously(Hall et al., 1995a; Howard et al., 1996; Butterfield et al.,1997a,b). d: EPR spectrum of 5-NS-labeled synaptosomes.Boxed region of spectrum is expanded in the right-hand sideshowing the HWHH. Samples were spin-labeled as describedpreviously (Hall et al., 1995a).

were separated by electrophoresis in a 7.5% polyacrylamidegel, transferred to a nitrocellulose sheet, and immunoreactedwith an antibody against HNE-conjugated proteins (Waeg etal., 1996). The blot was processed further using horseradishperoxidase-conjugated secondary antibody and achemilumi-nescence detection kit (Amersham, Arlington Heights, IL,U.S.A.).

Data analysisData were analyzed by ANOVA followed by post-hoc

analysis. The Student’s t test was used to assess individualconcentrations of HNE. A value ofp < 0.05 was consideredto indicate statistical difference between data sets.

RESULTS

To determine whether HNE conjugates to proteinsin synaptosomes, and therefore has the potential tomodify their structure and function, synaptosomeswere exposed to HNE and western blots were per-formed using an antibody that recognizes HNE—pro-tein conjugates (Waeg et al., 1996). HNE conjugatedto many different proteins across a range of molecularweights, but HNE-conjugated proteins of higher mo-

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1164 R. SUBRAMANIAM ET AL.

FIG. 2. HNE conjugates to multiple proteinsin cortical synaptosomes. Synaptosomeswere exposed for 2 h to vehicle (control) or10 jiM HNE. Synaptosomes were then solu-bilized, and proteins were separated by so-dium dodecyl sulfate—polyacrylamide gelelectrophoresis, transferred to nitrocellulose,and immunoblotted with an anti-HNE anti-body (see Materials and Methods). Note thatmany different synaptosomal proteins exhib-ited HNE immunoreactivity.

lecular weight were particularly abundant (Fig. 2).HNE contains a carbonyl functional group. Cytosolicproteins also reacted with HNE, as evidenced by theincreased carbonyl content of HNE-treated samples(Fig. 3): synaptosomes treated with 50 ~aM HNEhad a significantly larger carbonyl content (p < 0.01)than untreated controls. To determine if, in bindingto synaptosomal membrane proteins, conformationalchanges occur, which conceivably could be related toHNE-induced neurotoxicity in neurons undergoing ox-idative stress, EPR protein-specific spin labeling wasused.

Spin labeling cortical synaptosomal membraneproteins with MAL-6

One of the most widely used protein-specific spinlabels to study membrane protein conformationalchanges is MAL-6 (Butterfield, 1982) (Fig. Ia). Thespin label selectively binds covalently to the thiolgroups on protein sulfhydryls located on two differentkinds of environments. Sulfhydryls located on the sur-face of the protein molecules are capable of onlyweakly restricting the motion of the spin label,allowing free rotations of MAL-6, which are mani-fested as relatively narrow lines in the EPR spectrumof a labeled protein. The steric hindrance to motionprovided by sulfhydryls buried in a deep pocket in theprotein molecule is capable of strongly immobilizingthe rotational motion of the spin label, which is mani-fested as broadened lines in the EPR spectrum of alabeled protein. Due to these two different binding siteson protein molecules, an EPR spectrum reflecting bothenvironments results (Fig. lc). The ratio of the EPRspectral amplitudes of the weakly immobilized line(W) to that of the strongly immobilized line (5), inthe low-field region of the EPR spectrum of MAL-6-labeled synaptosomal membranes, is the W/S ratio.This parameter is known to be highly sensitive to pro-tein conformational changes and protein—protein inter-actions (Butterfield, 1982; Trad and Butterfield, 1994;Bellary et al., 1995; Hall et al., 1995a—c, 1997; How-ard et al., 1996; Butterfield et al., 1997b). Increasedprotein—protein interactions, decreased segmental mo-tion, and/or conformational changes in the spin-la-

beled proteins lead to decreased molecular motion re-sulting in a lowering of the W/S ratio (Butterfield,1982; Wyse and Butterfield, 1988; Trad and But-terfield, 1994; Bellary et al., 1995; Hall et al., 1995a—c, 1997; Howard et al., 1996; Butterfield et al., l997b).

Concentration dependence of HNE on thephysical state of membrane proteins

HNE was able to induce significant changes in themeasured parameter for protein conformation (W/Sratio), at all concentrations from I to 50 ,uM (Fig.4a), with the percent decrease in W/S ratio beinglarger at higher concentrations of HNE. Statisticalanalyses showed no significant difference in the HNE-induced effects observed for HNE concentrations of>30 itM. At all concentrations, the maximum amountof the vehicle (ethanol) added to the synaptosomeswas 5 p~l,and this did not cause any significant changein the W/S ratio of the MAL-6 spectrum (data notshown). The results are consistent with HNE inducingslower motion of spin-labeled proteins, concordantwith the hypothesis that HNE can bind to and/or cross-link proteins (Esterbauer et al., 1991; Butterfield andStadtman, 1997).

To be certain that the observed decrease in the W/Sratio of MAL-6-labeled synaptosomal membrane pro-teins following treatment with HNE was not due to thebinding of HNE to the cysteine residues that wouldnormally be bound by the protein-thiol-specific spin la-bel MAL-6, we performed double-integration of theEPR spectra of the control and HNE-treated samples.The double-integrated area is a measure of the concen-tration of the protein-bound spin label. Both control andHNE-treated samples showed nearly identical double-integrated intensity, i.e., spin-label incorporation (data

FIG. 3. Protein carbonyl content of control and 50 jiM HNE-treated synaptosomes. Carbonyl levels were determined as the2,4-dinitrophenylhydrazine adduct (Butterfield and Stadtman,1997). The carbonyl content is expressed as nanomoles of car-bonyls per milligram of protein in the sample. HNE-treated sam-ples show a significantly larger level of protein carbonyls (°p<0.01, n = 4).

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HNE-INDUCED ALTERATIONS OF BRAIN MEMBRANE PROTEINS 1165

FIG. 4. a: HNE decreases the W/S ratio of MAL-6 covalentlybound to synaptosomal membrane proteins. The W/S valuesare expressed as a percentage of the respective controls foreach treatment. Spin-labeled synaptosomal membranes weretreated with varying concentrations of HNE, and theW/S ratio,a measure of protein conformation, showed a significant de-crease at all concentrations (°p< 0.01, “p < 0.0003, n = 6 or7). b: Effect of time on the W/S ratio of MAL-6-labeled corticalsynaptosomal membranes treated with 5 jiM HNE. The maximaldecrease in the W/S ratio was observed at 6 h (see text).

not shown), suggesting that the lowering of the W/Sratio observed in the HNE-treated samples is not dueto a lower spin-label incorporation in the HNE-treatedsamples, but rather due to an altered conformation and/or protein—protein interaction caused by the reaction ofmembrane proteins with HNE.

The EPR results of synaptosomes treated with 5 ~ttMHNE for different periods of time are shown in Fig.4b. There was a steady decrease in the W/S ratio,compared with control, over time with a maximal de-crease observed at 6 h of incubation.

Effect of preincubation of synaptosomes withGEE

It has been proposed that glutathione protects neu-rons against HNE toxicity (Esterbauer et al., 1991),

and this was observed in cultured hippocampal neurons(Mark et al., 1997). We reasoned that if HNE-inducedprotein conformational alterations were important inneurotoxicity, glutathione should modulate the effecton protein conformation caused by HNE. Synapto-somes were treated with 2 mM GEE for 3 h and thentreated with 50 ,uM HNE. GEE protected against theHNE-induced decrease in the W/S ratio of MAL-6-labeled proteins, i.e., prevented the conformational al-terations by HNE (Fig. 5). The protection from GEEwas observed even for the highest concentration ofHNE studied (50 pM).

Effect of HNE treatment on motion and order ofphospholipids

To obtain information about the synaptosomal mem-brane lipid dynamics and order, nitroxide stearate mol-ecules are used as probes (Butterfield, 1982). WhereasMAL-6 binds to membrane proteins via a covalentbond, the nitroxide stearates intercalate in the bilayerwith the charged head group near the head group ofthe phospholipids and the acyl chain of the probealigned with the lipid acyl chains. The probe used inthis study was the amphipathic molecule 5-NS, which,upon intercalation in the bilayer, has its paramagneticcenter close to the lipid/water interface. The EPR spec-trum of 5-NS-labeled synaptosomal membranes isshown in Fig. id. The measured parameter in the spec-trum is the half width at half height (HWHH) of thelow-field resonance line. The membrane dynamics andthe order are related to the motion of the spin label.A decrease in the order of the lipids will lead to anincrease in the lipid motion, i.e., a decrease in the

FIG. 5. Effect ofpreincubation of synaptosomes with 2 mM GEEprior to treatment with 50 jiM HNE on the W/S ratio of MAL-6spin-labeled synaptosomal membrane proteins. Group A: Sam-ples treated with 50 jiM HNE (black column) show a significantdecrease in the W/S ratio (°p< 0.02, n = 4) compared withcontrols (gray column). Group B: Both controls (gray column)and 50 jiM HNE-treated samples (black column) were pretreatedwith 2 mM GEE for 3 h followed by a 1-h incubation with HNE(see Materials and Methods). There is no difference in the WIS ratio of control and 50 jiM HNE-treated samples that werepretreated with GEE.

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1166 R. SUBRAMANIAM ET AL.

FIG. 6. Significant increase in the HWHH (G) is observed forsynaptosomes treated with 50 jiM HNE for 1 h, suggesting in-creased membrane fluidity relative to control (°p< 0.005, n = 4).

lifetime of a particular orientation of the principal axisof the spin probe with respect to the static externalmagnetic field (Butterfield, 1982). In the EPR spec-trum, this effect manifests as a line broadening, conse-quently leading to an increase in the HWHH (But-terfield, 1982; Hall et al., 1995a,b).

Because HNE is reactive toward proteins, we rea-soned that when HNE binds to transmembrane proteinsin the lipid bilayer domain, lipid acyl chain packingof bulk lipids might be disturbed and lead to increasedfluidity. Synaptosomes were spin-labeled with lipidspecific 5-NS and treated with 50 ,aM HNE. TheHWHH for 50

1uM HNE-treated samples increased bynearly 16% compared with that for untreated controls(Fig. 6; n = 4, p < 0.005). This result is consistentwith our hypothesis and suggests an HNE-induced in-crease in membrane fluidity near the membrane lipid/water interface, the region probed by 5-NS.

DISCUSSION

This study shows that HNE significantly alters theconformation of cortical synaptosomal membrane pro-teins as assessed by the W/S ratio of MAL-6-labeledmembrane proteins using EPR spectroscopy. This ef-fect of HNE occurred with concentrations previouslyshown to be produced in neurons by A~3(Mark et al.,1997) and formed in other cell types (Esterbauer etal., 1991) exposed to oxidative insults. Decreases inthe W/S ratio of MAL-6 observed in dose—responsestudies of HNE are due to structural modification orprotein cross-linking due to HNE. The reactive siteson HNE (aldehyde functionality, double bond, and hy-droxy functionality) can cause protein side chains tobecome conjugated to HNE by Michael addition andcause further cross-linking through the c-amino groupon the side chain of lysine residues on other proteinmolecules via formation of a Schiff base (Esterbaueret al., 1991; Butterfield and Stadtman, 1997). These

reactions have been well characterized, and it is wellknown that HNE can react with cysteine, histidine,and lysine residues on protein side chains by Michaeladdition (Esterbauer et al., 1991; Butterfield and Stadt-man, 1997). Modified and/or cross-linked proteinslead to proteins with altered conformation and/orcause increased protein—protein interactions and arethe likely explanation for the observed decrease in theW/S ratio of MAL-6-labeled synaptosomal mem-branes treated with HNE. Previous EPR results fromour laboratory have shown that when membrane pro-tein—protein interactions are increased by cross-link-ing, e.g., by the polyamine spermine (Wyse and But-terfield, 1988), the W/S ratio of MAL-6 is decreased.Conversely, when protein—protein interactions are de-creased, e.g., by inositol hexaphosphate, the W/S ratio,as predicted, is increased (Wyse et al., 1987). In thecurrent study, it is clear from Figs. 2 and 3 that HNEis bound to synaptosomal membrane proteins. Thus,we suggest that the ability of HNE to reduce the W/S ratio of MAL-6-labeled membrane proteins is due toincreased protein—protein interactions caused by HNE.

The production of HNE during lipid peroxidationoccurs within the lipid bilayer where the concentrationof HNE could potentially rise to 30—50 bIM. Hence,it is reasonable that protein conformational changesreflected by a decrease in the W/S ratio would beexpected in a membrane undergoing lipid peroxidation.The time-dependent decrease in the W/S ratio of 5 ,tiMHNE-treated synaptosomes suggests a time-dependenteffect of HNE on membrane proteins. It is unlikelythat a concentration of 5 ~tiM would be maintainedwithin the membrane for longer than 6 h, because even-tually HNE diffuses out of the bilayer and is dilutedin the cytosol or extracellular fluid (Esterbauer et al.,1991). Consistent with this observation, cytosolic pro-tein carbonyl levels were increased by HNE (Fig. 3).

HNE is thought to react with glutathione by forminga thio-ether linkage at the double bond, followed byring closure to a five-membered hemiacetal (Sayre etal., 1993; Nadkarni and Sayre, 1995). The thio-etherlinkage is highly stable compared with Schiff ‘s baselinkage. Therefore, the protection against HNE-in-duced protein conformational changes afforded byGEE (Fig. 5) is likely due to the inhibition of HNEreactivity upon formation of this cyclic adduct. ThatHNE can induce alterations in the order and dynamicsof the lipid bilayer is evident from the increase in theHWHH for 50 1iM HNE-treated samples. Buko et al.(1996) also used the 5-NS spin probe to investigatethe effect of HNE on the fluidity of liver plasma mem-branes. These researchers found that HNE caused anincrease in membrane fluidity near the lipid/water in-terface, consistent with our findings that HNE causedan increase in the fluidity of synaptosomal membranesprobed by 5-NS.

The amyloid-associated free radical hypothesis ofAD (Butterfield et al., 1994, 1996a, l997a; Hensleyet al., 1994a; Butterfield, 1996, l997a,b) suggests that

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HNE-INDUCED ALTERATIONS OF BRAIN MEMBRANE PROTEINS 1167

FIG. 7. Model showing free radical(R~)generation from Aj3, which leadsto lipid peroxidation, HNE formation,and also direct protein oxidation. HNEfrom lipid peroxidation is capable ofindependently altering proteins in themembrane, a process that is pre-vented by GEE (see text). HNE-in-duced alteration of the physical stateof synaptosomal membrane proteinscould explain the HNE-associatedloss of activity of ion-motive AlPases(Market al., 1995, 1997) and inhibitionof the glutamate transporter (Harris etal., 1996; Kelleretal., 1997).APP,am-yloid precursor protein; MDA, malon-dialdehyde; ROS, reactive oxygenspecies.

A/3, the major component of senile plaques found inAD brain, is associated with free radicals, which areneurotoxic and cytotoxic. The model proposes that A~3-associated free radicals lead to both lipid peroxidationand protein oxidation, which was found in isolatedsynaptosomes and culturedhippocampal neurons (But-terfield et al., 1994, 1996b; Harris et al., 1995), cul-tured astrocytes (Harris et al., 1996), and AD brain(Smith et al., 1991; Hensley et al., 1995). The A/3-associated free radical model for AD neurotoxicity alsopredicts that the multiple membrane abnormalities inAD enzymes, transport functions, lipids, etc., can berationalized as discrete manifestations of free radicalsor free radical-induced lipid peroxidation products,e.g., HNE. A~3,in ways completely inhibitable by freeradical antioxidants, was shown to inhibit the activitiesof ion-motive ATPases, lead to increased intracellularCa2~,and cause neuronal death (Mark et al., 1995).In more recent studies, it is reported that A~3caninducethe formation of HNE in hippocampal neuronal cellcultures and synaptosomal membranes (Mark et al.,1997) and directly impair glutamate transport function(Keller et al., 1997), previously shown to be alteredin cultured astrocytes by A/3 (Harris et al., 1996).HNE by itself is capable of inducing several of theoxidative-stress responses previously shown for A/3,such as disruption of ion homeostasis and neuronaldeath (Mark et al., 1997).

Based on this evidence and the current research, wenow propose that even though the generation of freeradicals by A/3 will still serve as the first toxic triggerof AD pathology, impairment of protein functions bythe lipid peroxidation product HNE, the second toxictrigger, in the cascade may be of critical significance(Fig. 7). HNE, generated due to the initial free radicalinsult from A/3, can subsequently conjugate with van-

ous membrane proteins. Ifgenerated at high concentra-tions, HNE can also lead to alteration in the physicalstate of lipids, which can affect the function of othertransmembrane proteins. The proteins conjugated withHNE are modified structurally and functionally. Al-though further work is necessary, our results are con-sistent with the concept that HNE could be of greatimportance in understanding the multiple membranedysfunctions, including Ca2~accumulation, observedin AD brain membranes. Investigations designed toresearch this possibility are in progress.

In addition to AD, oxidative stress is thought to beimportant in several degenerative neurological disor-ders, one of which is ischemia/reperfusion injury (IRI)as a model for stroke (Hall et al., l995a—c, 1997).Synaptosomal membrane proteins and lipids have al-tered conformation in IRI (Hall et al., 1995a), whichcan be prevented by pretreatment with the free radicalscavenger PBN (Hall et al., 1995b) and exacerbatedby diminution of intracellular glutathione (Hall et al.,1997). Consequently, based on the results of the cur-rent study, in IRI in vivo interaction of the major freeradical lipid peroxidation product, HNE, with synapto-somal membrane proteins conceivably could accountfor both the alterations in the physical state of mem-brane proteins and the increased synaptosomal mem-brane fluidity reported (Hall et al., 1995a). We predictthat similar considerations may apply to oxidativestress in amyotrophic lateral sclerosis, and studies totest this prediction are in progress.

Protein-specific spin labeling studies using MAL-6consistently showed decreased W/S ratios in mem-branes in conditions of oxidative stress induced eitherin vitro (Hensley et al., l994b; Trad and Butterfield,1994; Bellary et al., 1995) or in vivo (Hall et al.,1995a—c, 1997; Howard et al., 1996; Butterfield et al.,

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1168 R. SUBRAMANIAM ET AL.

1997b). The decreased WIS ratios of MAL-6 in rodentcortical synaptosomal membranes returned towardcontrol values when animals exposed to hyperoxia orIRI or senescent-accelerated animals were previouslyinjected withbrain-accessible PBN (Hall et al., 1995b;Howard et al., 1996; Butterfield et al., 1997b). Theseresults raise the possibility that free radical-associatedoxidative stress, leading to increased HNE productionwith subsequent reaction with membrane proteins, maybe a common mechanism to explain altered conforma-tion and function of brain membrane proteins in oxida-tive stress conditions.

Acknowledgment: This work was supported in part byNIH grants AG-05ll9 and AG-10836.

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