Jountal of Neurochenaisln ,Raven Press, Ltd., New York

1995 International Society for Neurochemistry

Carbonyl-Related Posttranslational Modificationof Neurofilament Protein in the Neurofibrillary

Pathology of Alzheimer's Disease

Mark A. Smith, *Maria Rudnicka-Nawrot, Peggy L . Richey, Darja Praprotnik,Paul Mulvihill, *Carol A. Miller, tLawrence M . Sayre, and George Perry

Departments of Pathology and tCheinistry, Case Western Reserve School of Medicine, Cleveland, Ohio ;and *Department of Pathology, Universitv gf Southern California, Los Angeles, California, U.S.A .

Abstract: We present the first evidence for carbonyl-re-lated posttranslational modifications of neurofilaments inthe neurofibrillary pathology of Alzheimer's disease (AD) .Two distinct monoclonal antibodies that consistently la-beled neurofibrillary tangles (NFTs), neuropil threads,and granulovacuolar degeneration in sections of AD tis-sue also labeled the neurofilaments within axons of thewhite matter following modification by reducing sugars,glutaraldehyde, formaldehyde, or malondialdehyde. Theepitope recognized by these two antibodies shows astrict dependency for carbonyl modification of the neuro-filament heavy subunit. The in vivo occurrence of thisneurofilament modification in the neurofibrillary pathologyof AD suggests that carbonyl modification is associatedwith a generalized cytoskeletal abnormality that may becritical in the pathogenesis of neurofibrillary pathology.Furthermore, the data presented here support the ideathat extensive posttranslational modifications, includingoxidative stress-type mechanisms, through the formationof cross-links, might account for the biochemical proper-ties of NFTs and their resistance to degradation in vivo .Key Words: Neurofilaments-Alzheimer's disease-Neurofibrillary tangles-Carbonyl-related posttransla-tional modification-Cross-linking-Glycation .J. Neurochem. 64, 2660-2666 (1995) .

Alzheimer's disease (AD) is characterized by de-posits of insoluble proteins that form two main distinc-tive lesions-senile plaques and neurofibrillary tan-gles (NFTs) (reviewed by Perry and Smith, 1993) .The mechanism(s) by which normally soluble proteinsare accummulated as insoluble aggregates of proteinsin senile plaques and NFTs is unknown ; however, it isknown that there is a change to a 0-pleated secondaryprotein conformation that parallels insolubility (Glen-ner, 1980 ; lgbal et al ., 1984) . Recent data suggest thatß-amyloid deposition within senile plaques involvesspecific posttranslational modifications (Smith et al .,1994x ; Vitek et al ., 1994) that are mediated by oxida-tive stress and free radicals (Dyrks et al ., 1992 ; Smith

2660

et al ., 1994a-c), and similar mechanisms might alsobe responsible for NFT formation (Ledesma et al .,1994 ; Smith et al ., 1994a-c ; Yan et al ., 1994) .NFTs are insoluble in most protein solvents such as

sodium dodecyl sulfate (SDS), guanidine, and urea .This property has hindered the full characterization ofNFTs at the molecular level (Selkoe et al ., 1982) .Limited biochemical and immunological analyses ofNFT demonstrate a multitude of component and asso-ciated protein elements, including -r (Grundke-Igbal etal ., 1986), ubiquitin (Mori et al ., 1987 ; Perry et al .,1987), neurofilament heavy subunit (NFH) (Perry etal ., 1985), f-amyloid (Masters et al ., 1985 ; Tabatonet al ., 1991 ; Perry et al ., 1992), tropomyosin (Gallo-way et al ., 1990), serum amyloid P-component (Kala-ria et al ., 1991) and heparin and chondroitin sulfateproteoglycans (Snow et al ., 1990 ; Perry et al ., 1991 ;DeWitt et al ., 1993) . Oxidation-related posttransla-tional modifications are strongly implicated in the for-mation of NFTs, including the association of thesecomponents (Ledesma et al ., 1994 ; Smith et al .,1994a-c ; Yan et al ., 1994) .

In this study, we demonstrate carbonyl-related modi-fications in the neurofilament protein component ofNFT-related pathologies and discuss the implicationsof oxidative stress in the specific and general pathogen-esis of NFTs and AD, respectively .

MATERIALS AND METHODSTissue preparation

Tissues were obtained from the CNS postmortem fromAD and age-matched control cases . The diagnosis of all

Received October 12, 1994 ; revised manuscript received January10, 1995 ; accepted January 14, 1995 .Address correspondence and reprint requests to Dr. M. A. Smith at

Department ofNeuropathology, Institute of Pathology, Case WesternReserve University, 2085 Adelbert Road, Cleveland, OH 44106,U.S .A .Abbreviations used: AD, Alzheimer's disease ; AGE, advanced

glycation end product; NFH, neurofilament heavy subunit; NFT,neurofibrillary tangle ; SDS, sodium dodecyl sulfate .

NEUROFILAMENT MODIFICATION IN ALZHEIMER'S DISEASE

cases was confirmed by neuropathological examination us-ing established diagnostic criteria (Khachaturian, 1985 ) . Forcryostat-cut sections, tissue was flash-frozen in liquid nitro-gen-chilled isopentane, and 20-N,m-thick sections were pre-pared. For paraffin sections, tissue was fixed in either 4%formaldehyde buffer (pH 7.4) or methacarn la nonaldehydefixative (methanol/chloroform/acetic acid, 6:3 :1 )], dehy-drated through a graded series of ethanol, xylene-treated,and embedded in paraffin, and 6-p,m-thick microtome sec-tions were prepared . For electron microscopy, 60-N,m-thickfree-floating sections were cut from formalin-fixed tissuewith a Vibratome (Oxford) .

AntibodiesThe monoclonal antibody NFT200 (Innogenetics, Zwijn-

drecht, Belgium) was generated using purified NFTs as im-munogen. Preliminary immunocytochemical analysis waspreviously described (Gheuens et al ., 1991) .

The monoclonal antibody 3A4 was generated as pre-viously described (Fujita et al ., 1982), and initial immuno-cytochemical analyses were documented (Miller et al ., 1987 ;Hinton et al ., 1988) .The monoclonal antibody Alz-50, an antibody against A68

protein, was used to localize intracellular NFTs (Wolozin etal ., 1986 ; Siedlak et al ., 1991 ; Tabaton et al ., 1991 ) .The monoclonal antibody SMI-34 (Sternberger Mono-

clonals, Baltimore, MD, U.S .A .), an antibody to phosphory-lated neurofilaments, was used to localize neuronal cell bod-ies (Sternberger and Sternberger, 1983) .

ImmunocytochemistryFollowing deparaffinization in xylene and rehydration

through graded ethanol for fixed tissue or a brief rinse in ice-cold acetone for cryostat-cut sections, tissue sections weresequentially incubated with 10% normal goat serum, primarymonoclonal antibody in 1 % normal goat serum, goat anti-mouse antibody in 1 % normal goat serum, mouse peroxi-dase-antiperoxidase complex in 1 % normal goat serum, andfinally diaminobenzidine/H Z0' (Sternberger, 1986) . Insome experiments, adjacent tissue sections were preincu-bated with 0.25 Mglutaraldehyde for 20 min at room temper-ature, 0.2 and 1 .0 M malondialdehyde for 20 min or 1 h atroom temperature, or 0.25M ribose for 3 days at 37°C beforeimmunocytochemical detection.

Neurofibrillary pathology was based on morphological cri-teria, congophilia, or immunoreactivity of adjacent tissuesections with Alz-50 or SMI-34 . Axons were identified bysimilar immunocytochemical labeling with SMI-34 .

Immunogold stainingVibratome-cut sections were treated at room temperature

for 10 min with 0.5% Triton X-100 in 0.15 M NaCl and50 mM Tris-HCI, pH 7.6 . They first were rinsed and thenincubated in the primary antibody for 2 days at 4°C followedby rinsing and subsequent incubation with the appropriatecolloidal gold antibody complex. Colloidal gold was pro-duced by citrate reduction and complexed to affinity-purifiedgoat anti-mouse IgG (Southern Laboratories) (DeMay,1983) . Sections were subsequently fixed in 2.5% glutaralde-hyde in 0.1 M phosphate buffer (pH 7.2) for 2 h followedby postfixation in 2% OS04 and flat-embedded in Spurr'smedium . Sections were viewed at 60 kV in a JEOL model100CX electron microscope.Immunodecoration of SDS-treated NFT fractionsGray matter from the hippocampus of an AD patient that

was stored unfixed at -70°C was homogenized and treated

RESULTS

266 1

with SDS as previously described (Perry et al ., 1985) . Theinsoluble material was applied to carbon-coated nickel gridsand incubated for 1 h with the primary antibodies or controlsoutlined above, followed by a 1-h incubation with secondaryantibodies conjugated to colloidal gold . All the incubationswere at room temperature and were followed by three rinseswith 1% bovine serum albumin in 0.15 M NaCl and 50mM Tris-HCI, pH 7.6 . Immunostained preparations werenegatively stained with 2% phosphotungstic acid. Grids wereviewed at 80 kV in a JEOL model 1000X electron micro-scope.

Neurofilament preparationNeurofilaments were prepared as a cytoskeletal prepara-

tion from postmortem cauda equina tissue dissected fromneurologically normal individuals . In brief, excess nervesheath was removed, and the tissue was homogenized in 20volumes of solution A (50 mM Tris, 0.5% Triton X-100,and 2 mM EDTA, pH 6.8) . Following centrifugation at13,000 rpm (Beckman SW 28 rotor) for 15 min at 4°C, thepellet was rehomogenized with the same volume of solutionA made to 30% sucrose. Centrifugation as above removedexcess myelin and other contaminants . The resultant neuro-filament-enriched pellet was dissolved in Tris-HCI buffer,and the individual neurofilament subunits were separated byHPLC as previously described (Hui et al ., 1986) . Purifiedprotein was electrophoresed using a 10% acrylamide SDS-polyacrylamide gel electrophoresis mini-gel system (Laem-mli, 1970) and electroblotted (Towbin et al ., 1979) to lm-mobilon-P (Millipore, Bedford, MA, U .S.A .) .

ImmunoblottingReplica blots, after prewetting with methanol, were incu-

bated at room temperature with Tris-buffered saline con-taining 0.25Mglutaraldehyde for 20 min or 0.25 M glutaral-dehyde for 3 days at 37°C . Immunostaining was as pre-viously described (Miller et al ., 1987) except we used goatanti-mouse antibody conjugated to horseradish peroxidasefollowed by diaminobenzidine with H-O- .

ELISACrude neurofilament preparation or HPLC-purified neLu'o-

filament subunits were dissolved in phosphate-buffered sa-line, and samples were bound onto Nunc Maxisorb platesby overnight incubation at 4°C. The plates were washedwith phosphate-buffered saline and treated for 20 min withphosphate-buffered saline or 0.25 M glutaraldehyde . Non-specific binding sites were blocked by a 2-h incubation inphosphate-buffered saline containing 5% nonfat dried milkpowder, and the plates were sequentially incubated for 2 hwith primary monoclonal antibody (3A4, NFT200, or SMI-34) and then goat anti-mouse antibody conjugated to horse-radish peroxidase . Bound antibody was detected using 2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid) as chromogen[100 N,g/ml of citric acid/sodium phosphate buffer (pH 4.2)containing 0.003% H,O,], and the absorbante was measuredat 405 nm .

Formalin-fixed tissue sectionsImmunocytochemical analysis of formalin-fixed tis-

sue from AD cases with 3A4 and NFT200 monoclonalantibodies showed intense labeling of NFTs within theneuronal perikarya, neuropil threads, senile plaque

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M. A . SMITH ET AL.

FIG. 1. Light microscopy of 3A4 immunoreactivity, visualized by the peroxidase-antiperoxidase method . A: Adjacent serial sectionsof methacarn-fixed hippocamus from Alzheimer's brain show numerous densely stained pathological structures within the pyramidallayer, including NFTs, granulovacuolar degeneration, senile plaque neurites, and neuropil threads . Alv, alveus . B: Immunoreactivity ofaxons present in the white matter of the alveus is only apparent following preincubation of the tissue section with glutaraldehyde . Bar= 1 mm .

neurites, and granulovacuolar degeneration . In controlpatients, sporadic lesion-related immunoreactivity wasseen using these antibodies . Both antibodies showedintense axonal immunoreactivity in human cerebralcortex in control and AD tissue .

Methacarn/cryostat-cut tissue sectionMethacarn-fixed or cryostat-cut sections from AD

and control cases show intense immunostaining ofpathological structures such as NFTs, neuropil threads,senile plaque neurites, and granulovacuolar degenera-tion with the two antibodies, 3A4 and NFT200 (Fig .]A) . However, somewhat curiously, in marked con-trast to the formalin-fixed tissue, both white and graymatter axons were unstained using methacarn-fixed orcryostat-cut sections .

Effects of carbonyl-related epitope modificationThe discrepancy in axonal staining between alde-

hyde-fixed tissue and methacarn-fixed or acetone-fixedcryostat-cut tissue led us to speculate that the epi-tope(s) recognized by 3A4 and NFT200 was fixationdependent. To test this proposal, we pretreated metha-carn-fixed and cryostat-cut tissue sections with car-bonyl compounds before immunocytochemical analy-sis . Our data clearly showed that the epitopes for 3A4and NFT200 could be created by pretreatment with aselection of carbonyl compounds, including glutaralde-hyde, malondialdehyde, glyceraldehyde, and ribose(Fig . 113) . These carbonyl pretreatments showed aconcentration and time dependence (data not shown) .Indeed, methacarn-fixed section postfixed with thesecompounds were virtually indishinguishable from for-malin-fixed tissue, whereas closely related controlcompounds such as the malondialdehyde bis-diethylac-etal precursor were unable to create the 3A4 orNFT200 epitope in axons . The presence of the 3A4and NFT200 epitope in non-carbonyl-treated tissue,i .e ., methacarn-fixed or cryostat-cut sections, suggeststhat this particular modification exists in vivo.

.1. Neurochem., Vol. 64, No. 6, 1995

Electron microscopyThe gold immunodecoration of NFT by monoclonal

antibodies 3A4 and NFT200 was visible in the lightmicroscope as a red stain restricted to neurofibrillarypathology and axons (data not shown) . At the ultra-structural level, both antibodies decorated paired heli-cal filaments (Fig . 2A) and the straight filamentswithin Alzheimer's NFTs . In addition to the neurofi-brillary pathology-specific immunodecoration, therewas also strong labeling of the neurofilaments presentwithin axons (Fig . 2B) . No staining was observedwhen either nonimmune mouse serum or an irreleventmonoclonal antibody was substituted for the primaryantibody in control preparations .

Preparations of SDS-extracted Alzheimer's NFTfilaments were strongly immunodecorated by 3A4 andNFT200, indicating that the epitopes recognized area closely associated component of the paired helicalfilaments (Fig . 2C) . The 3A4 and NFT200 epitope(s)were not removed by detergent extraction with SDSor sarcosyl (Lee et al ., 1991) . The cerebral amyloidpresent in these preparations was not labeled .Immunoblot analysisThe differential carbonyl-dependent axonal immu-

nostaining patterns of monoclonal antibodies 3A4 andNFT200 were further evaluated by immunoblot analy-sis . Confirming the role of carbonyl dependency of the3A4 and NFT200 epitope, we found that immunoblotsof purified axonal proteins as well as isolated NFHlabel a band migrating with an apparent molecularmass of 200 kDa only after carbonyl pretreatment withglutaraldehyde, glyceraldehyde, malondialdehyde, orribose (Fig . 3, lanes A-D) . The band recognized by3A4 and NFT200 co-migrates with a band recognizedby SMl-34, an antibody that recognizes the 200-kDaheavy subunit of the neurofilament triplet, NFH (Fig .3, lane E) (Sternberger and Sternberger, 1983) .ELISAWith crude neurotilament preparations, in inival ex-

periments, or HPLC-purified NFH, in later experi-

FIG. 3. Immunoblotting with NFT200 (lanesA and B) or 3A4 (lanes C and D) antibodiesof a neurofilament-en rich ed, cytoskeletalpreparation demonstrates that without pre-treatment (lanes B and D) of the blot withglutaraldehyde (lanes A and C) or othercarbonyl-containing compounds, there isno staining of the 200-kDa band that comi-grates with the SMI-34-positive neurofila-ment heavy subunit (lane E) .

NEUROFILAMENT MODIFICATION IN ALZHEIMER'S DISEASE

2663

FIG. 2. A: Immunogold decoration of Alzheimer's NFTs with NFT200 antibody clearly shows that the NFT200 epitope is present directlyon the paired helical filaments and straight filaments rather than peripherally . B: Similarly, the NFT200 epitope maps to the neurofilamentspresent within normal neuronal axons. C: Note the close association of this epitope to SIDS-extracted filaments of Alzheimer's pairedhelical filaments. Bars = 1 Nm .

rnents, immunoreactivity with 3A4 and NFT200 wasapparent only after preincubation of the neurofilamentprotein with glutaraldehyde . Neither 3A4 nor NFT200recognized unmodified protein, whereas both antibod-ies strongly recognized the glutaraldehyde-modifiedform (Fig . 4) . There was no significant difference be-tween native and glutaraldehyde-fixed protein usingSMI-34 (Fig. 4) .

DISCUSSION

In this report we demonstrate that two distinct mono-clonal antibodies, designated 3A4 and NFT200, recog-nize a carbonyl-modified form of NFH. The epitope(s)recognized can be created in vitro by the reaction be-tween reactive aldehydes and NFH . However, this car-bonyl-related posttranslational modification or an epi-topically indistinguishable modification exists in vivowithin the neurofilament component of the neurofibril-lary pathology found in AD. Electron microscopic lo-calization of 3A4 and NFT200 labeling shows that themodified neurofilament epitope forms a tightly associ-

ated component of the paired helical filaments withinthe NFT and the neurofilaments present within axonsin carbonyl-treated samples .

Several posttranslational protein modifications havebeen considered to contribute to the decreased proteinsolubility and increased protease resistance character-istic of NFT . However, increased phosphorylation ofT protein (Grundke-Igbal et al ., 1986) does not, as wasinitially thought (Goedert et al ., 1991 ), effect proteininsolubility (Gustke et al ., 1992 ; Goedert et al ., 1993),and neither racemization (Fisher et al ., 1992) nor isom-erization (Payan et al ., 1992) have any effect on thesolubility properties of NFT proteins . However, thepresence of advanced glycation end product (AGE)posttranslational modifications (Smith et al ., 1994x ;Vitek et al ., 1994 ; Yan et al ., 1994) is associated withcross-link formation, decreased protein solubility, andincreased protease resistance, i .e ., the same biochemi-cal features displayed by the pathological lesions inAD. The chemical reaction responsible for initiatingAGE modifications is termed the Maillard reaction andinvolves the nonenzymatic condensation of a reducingsugar (ribose and glyceraldehyde were used here) witha protein lysine c-amino group to form a Schiff base,which then undergoes an Amadori rearrangement toregenerate carbonyl reactivity . This results in a cascadeof subsequent chemical reactions, including inter- andintramolecular cross-linking . These may include homo-and heteropolymers of each neurofilament protein . Inaddition to reducing sugars, antibody recognition wasinduced by formaldehyde and glutaraldehyde, whoseability to fix tissue is a consequence of characteristiccarbonyl condensation-dependent inter- and intramo-lecular cross-linking, e.g ., the lysine-derived pyridin-ium cross-links produced by glutaraldehyde . Further-more, antibody recognition was also effected by ma-londialdehyde, the most well-known lysine-dependentcross-linking agent generated from lipid peroxidation .

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These data suggest that the common feature of epitoperecognition by 3A4 and NFT200 is carbonyl reactivitywith lysyl c-amino groups to form stable cross-links .Because the cross-linking moieties are structurally di-verse, it is probably the altered secondary structure inthe lysine-rich region of the neuronal proteins that isrecognized . Although the epitope for 3A4 and NFT200appears to be specific for the 200-kDa NFH, glycationof the medium (160-kDa) and light (68-kDa) neuro-filament subunits cannot be excluded .

Physiologically, aldehyde residues are associatedwith oxidative stress, free radical production, metal-cat-alyzed oxidation, and lipid peroxidation (Esterbauer etal ., 1982 ; Levine, 1984 ; Oliver et al ., 1987 ; Starke-Reedand Oliver, 1989 ; Olivero et al ., 1992 ; Stadtman, 1992) .Therefore, our data also further the notion that oxidativestress is an important pathogenic event in AD (Smithet al ., 1994b,c) . The changes in AD are superimposedon the increased oxidative stress in certain brain nucleias a result of aging, with a several hundredfold increasein the mitochondrial 5-kb "common" deletion (Corral-Debrinski et al ., 1992 ; Soong et al ., 1992) . The evidencethat free radicals and oxidative stress are important inAD includes an increased number of activated microgliathat produce large quantities of free radicals (Coltonand Gilbert, 1987), the presence of elevated levels ofiron, a free radical catalyst, in NFT-bearing neurons(Landsberg et al ., 1990 ; Good et al ., 1992), and thepresence of the antioxidant enzyme heme oxygenase-1in all neurons that contain neurofibrillary abnormalities(Smith et al ., 1994c) . It is interesting that heme oxy-genase-1, a microsomal enzyme, oxidatively cleavesheme to produce a potent antioxidant and also generatescarbon monoxide, a putative neurotransmitter (Vertnaet al ., 1993) . In a previous study, we suggested thatthe increase in heme oxygenase-1 activity in AD couldgenerate excitotoxic levels of carbon monoxide and con-tribute toward the progression of neuronal degeneration(Smith et al ., 1994b,c) .

In another study, we showed that paired helical fil-aments, the fibrillar component of NFTs, are insolublein ionic detergents, chaotrophes, chelators, and dena-

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M. A. SMITH ET AL.

FIG. 4. Effect of glutaraldehyde modifica-tion of HPLC-purified NFH on the 3A4 andSMI-34 epitope(s) . Unmodified protein isnot recognized by 3A4, whereas SMI-34recognizes both modified and unmodifiedforms of the protein .

turants but are readily soluble at high pH (P . Mulvihillet al ., unpublished data) . It is interesting that in thatstudy, AGE modifications were also found to be labileunder conditions of high pH . Therefore, we suggestthat PHF insolubility is mediated, at least in part, bycarbonyl protein modifications, including those derivedfrom lysine oxidation or reaction of sugar or lipid per-oxidation products with amino residues .The presence of carbonyl-related posttranslational

modification in the neurofilament component of NFTis not totally unexpected because we previouslyshowed that T, the main proteinacious component ofNFTs, is modified by AGE (Yan et al ., 1994) . Com-pared with other cellular proteins, neurofilaments andcytoskeletal proteins such as T are highly susceptibleto cumulative oxidative damage and carbonyl modifi-cation because they have relatively long half-lives andcontain an abundance of lysines (Lasek and Black,1988 ; Shaw, 1991 ) . Although the multiple lysine resi-dues of neurofilaments play an integral role in supra-molecular cytoskeletal organization, they are the tar-gets for modification by neurotoxic chemicals knownto disrupt neurofilament transport (Sayre et al ., 1985) .

In conclusion, the findings presented in this report,together with previous findings, suggest that the lesionsof AD are insoluble owing to carbonyl modification .The modification of protein by carbonyl-related mech-anisms and the formation of associated cross-linkswould explain the resistance to intracellular removal,even though the NFTs are extensively ubiquitinated( Mori et al ., 1987 ; Perry et al ., 1987 ), and to extracel-lular removal following neuronal death, where they areassociated with reactive microglia (Cras et al ., 1995) .Furthermore, the carbonyl modification of neurofila-ment protein, along with other susceptible proteins,could play an important role in cytoskeletal disruptionand the aggregation and deposition of insoluble pro-tein, i .e ., the genesis of Alzheimer's NFTs .

Acknowledgment : The authors wish to thank Dr . Sey-mour Benzer at Caltech for his generous gift of the mono-clonal antibody 3A4 . This work was supported by grants

NEUROFILAMENT MODIFICATION IN ALZHEIMER'S DISEASE

AG07552, AG09287, AGO8992 (to M.A.S ., P.L.R ., andG.P .), NS22688 (to L.M.S .), and AG05142 and 5-R37-MH39145 (to C.A.M.) from the National Institutes of Healthand a grant from the Sankyo Corporation (to C.A.M .) .

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