Ž .Brain Research 796 1998 307–310

Short communication

Advanced glycation endproducts are associated with Hirano bodies inAlzheimer’s disease

Gerald Munch a, Anne M. Cunningham b, Peter Riederer c, Eva Braak d,)¨a Physiological Chemistry I, Biocenter of the UniÕersity, Wurzburg, Germany¨

b Neurobiology Program, GarÕan Institute of Medical Research, Sydney, Australiac Clinical Neurochemistry, Dept. Psychiatry, UniÕersity Wurzburg, Wurzburg, Germany¨ ¨

d Department of Anatomy, J.W. Goethe UniÕersity, Theodor Stern-Kai 7, D-60590 Frankfurt, Germany

Accepted 24 March 1998

Abstract

One of the structural posttranslational modifications contributing to the formation of insoluble, and protease-resistant protein depositsŽ . Ž . Ž .in Alzheimer’s disease AD , such as neurofibrillary tangles NFT and b-amyloid plaques are ‘advanced glycation endproducts’ AGE .

Using a polyclonal antibody against AGE in frozen sections of fixed brain tissue from Alzheimer’s disease patients, AGE were identifiedin a further characteristic protein deposit in AD, namely in Hirano bodies. AGE are localized to ovoid, spherical, and rod-like Hiranobodies in the hippocampus, particularly numerous in the stratum lacunosum-moleculare of CA1. Since Hirano bodies are known tocontain mainly cytoskeletal and cytoplasmic components and are localized within the soma of neurons our study suggests that AGEformation and intracellular protein crosslinking represent early stages during neuronal degeneration. q 1998 Elsevier Science B.V. Allrights reserved.

Keywords: Advanced glycation endproduct; Alzheimer’s disease; Oxidative stress; Crosslinking; Hirano body

Ž .The histological hallmarks of Alzheimer’s disease ADinclude neurofibrillary changes, formation of amyloid de-posits, loss of synaptic plasticity, neuronal dysfunction andcell death. In the pathogenesis of AD, characteristiccrosslinked protein deposits, their constituents, and under-lying mechanisms for their formation from soluble precur-sors merit attention. One of the characteristic posttrans-lational modification of aged proteins is nonenzymatic

Ž .glycosylation glycation . Nucleophilic amino sidechains,such as arginine, lysine or histidine react with extracellularmonosaccharides including glucose and fructose or intra-cellular metabolites of glycolysis, such as hexose phos-phates, trioses and triose phosphates. Through subsequentoxidations and dehydrations, glycated proteins form het-erogeneous, fluorescent yellow-brown products known as

) Corresponding author. Fax: q49-69-6301-6425; E-mail:braak@em.uni-frankfurt.de

Ž . w x‘advanced glycation endproducts’ AGE 3,17 . AGE for-mation is irreversible and causes protease-resistant cross-linking of peptides and proteins, associated with variouspathological insoluble protein deposits in age and neurode-

w xgenerative diseases 14,15 . In particular, AGE formationŽ . w xhas been demonstrated in neurofibrillary tangles NFT 6 ,

w xin cortical amyloid plaques 6,11,21,22 , in Pick bodiesw x w x12 , and in Lewy bodies 4 . The protein constituents ofNFT are resistant to proteolytic removal, possibly as a

w xresult of extensive crosslinks 5 . A main component of theNFT is the microtubule-associated tau protein, which hasbeen shown to be subject to intracellular hyperglycation

w xand AGE formation 13 . In the electron microscope, theNFT appear as paired helical filaments and glycosylationappears to be responsible for the maintenance of these

w xstructures 23 .Not only NFT, but also Hirano bodies are found as

intracellular neuronal inclusions in neurodegenerative dis-eases, including AD. Hirano bodies have been shown toexpress epitopes for various cytoskeletal components like

0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.Ž .PII S0006-8993 98 00328-X

( )G. Munch et al.rBrain Research 796 1998 307–310¨308

actin, actin binding proteins, a-actinin, vinculin, low andmedium molecular weight neurofilament protein. In addi-tion, characteristic components of b-amyloid plaques andNFT, including a-1-anti-chymotrypsin, microtubule-asso-ciated tau-protein, and C-terminal sequences of b-amyloidprecursor protein, are likewise components of Hirano bod-

w xies 8,9,16,19,20 . Since these proteins are usually solubleit is likely that a posttranslational crosslinking processinduces formation of covalent bonds between the moleculesthereby leading to protein insolubility and deposition.

We demonstrate, in this study, that Hirano bodies,another intracellular protein deposit occurring in brains ofAlzheimer’s and other neurodegenerative diseases, also aremodified and crosslinked by AGE.

Immunostaining was performed with a polyclonal anti-body against AGE. The polyclonal antibody was raised in

Žrabbits against AGE-keyhole limpet hemocyanin AGE-. ŽKLH , which has been prepared by incubating KLH 10.mgrml with 1 M glucose for 3 months at 508C. The

specificity of the antibody was verified by ELISA andWestern blotting, whereas model proteins, such as bovine

Ž .serum albumin-AGE BSA-AGE , chicken egg albumin-AGE and lysozyme-AGE can be detected, but no immuno-reactivity is observed towards unmodified proteins. Thisantibody also was used to detect elevated AGE levels inserum of hemodialysis patients by means of a competitive

w xELISA 18 .The investigation was performed on postmortem brain

tissue of six individuals: four brains had AD-related neu-w xrofibrillary changes corresponding to stage IV to V 1 and

two brains were devoid of neurofibrillary changes.At the time of autopsy, the brains were cut in the frontal

plane into 0.5-cm thick slices. The slices were fixed forŽ .two days in a mixture of 4% paraformaldehyde 7 ml ,

Ž .saturated aqueous picric acid 3 ml and 0.1 M sodiumŽ .phosphate buffer 10 ml, pH 7.2 ; cryoprotected with

saccharose, and frozen. Afterwards, 40–80-mm thick sec-tions were cut, containing the entorhinal region, the amyg-dala, and the hippocampus. The free-floating sections were

w xtreated according to a published protocol 2 . Three typesof pretreatments were performed prior to incubation with

Žthe primary antibody directed against AGE 1:500; charges. Ž .K 22.1; K 14.11 : sections were pretreated with a targetŽ . Žunmasking fluid TUF Kreatech Diagnostics, Amster-

. Ž .dam according to the supplier’s instructions, b 10%Ž .NaOH for 10 min, or c 80% formic acid for 10 min.

Immunoreaction was visualized with the ABC-complexŽ . YVector and the chromogen 3,3 -diaminobenzidine-tetra-chloride. The detection of AGE immunoreactivity was notpossible in paraffin sections or in sections not pretreatedwith TUF. A similar observation was made by Dickson et

w xal. 6 , showing that protease pretreatment is necessary todocument AGE epitopes in extracellular and intracellularNFT.

In brain tissue from AD patients, AGE immunoreactiv-ity was localized in dystrophic neurites arranged in plaque-

like formations, whereas only a diffuse immunoreactivityŽ .was seen in control tissue data not shown , consistent with

w xprevious observations by other groups 6,21 .In addition, AGE immunoreactivity was detected in

Hirano bodies in the hippocampal sector CA1 of the ADbrain tissue. Distinct AGE staining was observed in spheri-cal to ovoid inclusions adjacent or within the soma of

Ž .pyramid-shaped neurons Figs. 2–4 . Furthermore, rod-shaped inclusions of various sizes, oriented with their longaxis in various directions, were mainly present within thestratum lacunosum, probably within neuronal processes in

Ž .the neuropil arrows, Figs. 1, 2 and 4 . The localizationand shape of the inclusions indicate that they are Hiranobodies.

Most of the neurons containing Hirano bodies appear tohave an intact cell membrane. This suggests that formationof Hirano bodies, together with their modification andcrosslinking by AGE, takes place within a living cell in anearly stage of neurodegeneration. One interesting questionin that respect is whether AGE are actively promoting thecrosslinking process of the proteins deposited in Hiranobodies or whether they are formed on already existingdeposits. One might conclude from various reports that theabnormal protein deposits found in AD, including NFTand amyloid plaques, have a very low protein turnover and

Figs. 1–4. AGE immunoreactivity in Hirano bodies in sector CA1 of theAmmon’s horn, which are ovoid within the soma and rod-shaped within

Ž . Žprocesses arrows . Nomarski phase contrast; stage IV-AD-case; sections.pretreated with TUF, 80 mm thick .

( )G. Munch et al.rBrain Research 796 1998 307–310¨ 309

thus provide an opportunity for extensive crosslinking overtime. Whereas AGE formation mainly has been observedon extracellular proteins, there is more evidence that intra-cellular AGE-mediated crosslinking contributes to the for-mation of deposits, such as NFT, Lewy bodies and Hiranobodies in age-related neurodegeneration. It is unclear,however, as to whether AGE accumulate owing to theslow turnover, cause the slow turnover by decreasingproteolysis, or actively induce protein crosslinking.

ŽThe reasons for increased AGE accumulation intra-.and extracellular in AD remain to be elucidated. In dia-

betes, increased sorbitol pathway metabolites have beenimplicated by many investigators in the extracellular hy-perglycation underlying the pathogenesis of vascular andneural dysfunction. Since no increase in glucose is seen inAD, oxidative stress may be the most important factorcontributing to AGE mediated protein crosslinking in AD.This may occur through acceleration of the glycoxidation

w xreaction through an increase in free transition metals 24or else through a depletion of the antiglycation substances,e.g., the histidine dipeptides including carnosine and anser-

w xine or thiol antioxidants, such as reduced glutathione 7 .In cell culture experiments, injection into cells of NFT-tauisolated from postmortem tissue and recombinant AGE-taugenerate oxygen free radicals, thereby not only activatingtranscription via nuclear factor NF-k B, but also inducingthe release of the characteristic 4 kDa amyloid-b peptidesw x25 . Accordingly, a positive feedback loop involving ox-idative stress, possibly with underlying age-related de-

w xcreases in cellular energy 10 , may be an importantpathogenic factor in diseases characterized by intracellulardeposit formation, which act upon cytoskeletal proteins,and by the formation of intracellular inclusions with im-pairment of axonal transport leading to subsequent cellulardysfunction and death.

Acknowledgements

We thank J. Shine, J. Michaelis, U. Schindler, andparticularly R. Holliday for helpful and stimulating discus-sions, and A. Biczysko for her skillful technical assistance.This work was supported by the Hirnliga e.V., the ‘Claus-

Žsen-Stiftung,’ the ‘Universitatsbund Wurzburg’ G.M.,¨ ¨.P.R. , the Garnett Passe and Rodney Williams Memorial

Ž .Foundation A.M.C. , and the Deutsche Forschungsge-Ž .meinschaft E.B. .

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