Original Contribution

DIMETHYLARGININASE, A NITRIC OXIDE REGULATORY PROTEIN,IN ALZHEIMER DISEASE

MARK A. SMITH,* M ILAN VASAK,† MARKUS KNIPP,† RUDY J. CASTELLANI,‡ and GEORGE PERRY**Institute of Pathology, Case Western Reserve University, Cleveland, OH, USA;†Institute of Biochemistry, University of Zu¨rich,Zurich, Switzerland; and‡Department of Pathology, Division of Anatomic Pathology, University of Maryland Medical System,

Baltimore, MD, USA

(Received20 March 1998;Revised24 April 1998;Accepted27 April 1998)

Abstract—In this study, we show that dimethylargininase, a zinc protein involved in the regulation of nitric oxidesynthase, is specifically elevated in neurons displaying cytoskeletal abnormalities and oxidative stress in Alzheimerdisease (AD) while none of this enzyme was found in neurons in age-matched control cases. Seen in the context ofearlier studies showing widespread nitric oxide related damage in AD and the role of dimethylargininase to activatenitric oxide synthetase, through catalytic removal of its endogenous inhibitors, these findings indicate major alterationsin nitric oxide regulation in AD. Further, that low levels of zinc specifically inhibit dimethylargininase may provide alink between the numerous studies showing specific abnormalities in zinc and oxidative stress. Finally, our resultsprovide additional evidence that oxidative stress- and nitric oxide-mediated events play important roles in thepathogenesis of AD. © 1998 Elsevier Science Inc.

Keywords—Alzheimer disease, nitric oxide, oxidative stress, transition metals, zinc

INTRODUCTION

In Alzheimer disease (AD), a great deal of oxidativedamage has been identified including glycation [1–4],nitration [5,6], lipid peroxidation [7,8] and carbonyl for-mation [9–11]. While damage is associated with neuro-fibrillary tangles (NFT) [1,3,4,12] and senile plaques[2,3], it is perhaps of greater significance that such dam-age is also found in all neuronal populations at risk ofdeath [10]. Neuronal oxidative stress is among the ear-liest cytopathological changes found in AD and may bea part of the spectrum of early changes that initiateneuronal damage and eventual death [6,10,11].

We found that redox-active iron is specifically asso-ciated with the lesions of AD [13,14] and subsequentlyshowed that this exchangeable iron can participate inredox reactions in vitro (Smith et al., unpublished obser-vation). The finding of iron alteration in AD raises thequestion of whether other metal abnormalities noted inAD are also related to oxidative stress [15–18]. The

recent finding that nitric oxide (NO)-mediated damagepresumably from peroxynitrite is prominent in AD pro-vided a vital clue to other metal involvement [5,13] sincethe zinc protein, dimethylargininase, which is primarilyexpressed in tissues containing the constitutive forms ofneuronal nitric oxide synthase (nNOS) like brain, kidneyand endothelium cells [19–21], regulates NO productionby hydrolyzing free methylated arginine derivatives, ef-fective endogenous inhibitors of NOS [22]. In this study,we show intraneuronal dimethylargininase is specificallyincreased in AD.

MATERIALS AND METHODS

Tissue

Hippocampal tissue from 11 cases of AD (ages 69–95yr) aged-matched controls (ages 68–80 yr) and 4 youngcontrols (ages 31–62 yr) with similar postmortem inter-vals between groups (2–24 h) were fixed in methacarn(methanol: acetic acid; 60:30:10) at 4°C overnight. Fol-lowing fixation, tissue was dehydrated through ascendingethanol, embedded in paraffin, and 6mm sections wereplaced on silane-coated slides.

Address correspondence to: Mark A. Smith, Institute of Pathology,Case Western Reserve University, 2085 Adelbert Road, Cleveland, OH44106, USA; Tel: (216) 368-3670; Fax: (216) 368-8964; E-Mail:mas21@po.cwru.edu.

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Antibodies, immunocytochemistry, and immunoblotting

Affinity purified rabbit antiserum raised to the di-methylargininase (L-Nv,Nv-dimethylarginine dimethylami-nohydrolase, EC 3.5.3.18) from bovine brain [23], wereused at a 1:100 dilution. Sections were immunostainedusing the peroxidase-antiperoxidase method with 3,39-dia-minobenzidine as cosubstrate [24]. Adjacent sections wereimmuno stained with antiserum to ubiquitin [25] or hemeoxygenase-1 (HO-1) [26] to confirm the identity and loca-tion of intraneuronal pathological structures.

Controls consisted of: I) absorption of the antibody(1:100) with 0.6 mg/ml dimethylargininase at 4°C over-night prior to application to the section; and ii) omittingthe primary antibody. The former procedure was per-formed in parallel with addition of 0.6 mg/ml dimethyl-argininase to the antisera to ubiquitin as a control againstnon-specific adsorption. Following immunostaining, insome cases, the sections were additionally stained withCongo red and viewed under cross-polarized light toshow NFT and amyloid-b deposits of senile plaques.

Immunoblots were prepared from gray matter of twocases of AD and two controls by SDS-PAGE [27] on10% gels followed by electrotransfer to Immobilont(Millipore) by standard procedures [28]. The specificityof immunoreaction was verified as above.

RESULTS

In all cases of AD examined, the antibody to dim-ethylargininase recognized the cytoplasm of neurons inhippocampal sections in cases of AD (Fig. 1A) while,

conversely, in control cases no specific cells or structureswere recognized (Fig. 1B). Comparison of adjacent sec-tions stained with ubiquitin or HO-1 showed essentiallythe same pattern of neuronal cell bodies stained as withdimethylargininase. In sections taken from AD cases thatwere counterstained with Congo red, the most intenselydimethylargininase positive neurons generally containedNFT. Yet in addition neurons lacking Congo red posi-tivity but displaying ubiquitin immunoreactivity whenviewed on the adjacent sections, pre-NFT, also containdimethylargininase immunoreactivity. No reaction wasnoted to the amyloid deposits or dystrophic neurites ofsenile plaques. This distribution is essentially the same asthe distribution of HO-1 in AD, which was also localizedto neuronal cell bodies of neurons displaying AD pathol-ogy thereby suggesting a link to oxidative stress [26,29].

Dimethylargininase immunoreactivity is specificsince (i) preabsorption of the antibodies with dimethyl-argininase blocked recognition (Fig. 2) and (ii) no im-munostaining was noted with omission of the primaryantibody.

Immunoblotting with the antibody showed a singleband at 35 kDa that was present with equal intensity inthe control and AD cases (data not shown). Dimethyl-argininase immunoreactivity of the band was greatlyreduced by preabsorbtion of the antibody with dimethy-largininase.

DISCUSSION

This study demonstrates that the level of dimethyl-argininase is increased in the cytoplasm of neurons with

Fig. 1. Neurons containing neurofibrillary tangles are strongly recognized by the antibody to dimethylargininase (A), while in the brainsof control individuals (B), no specific staining was noted. Scale bar5 50 mm.

899Dimethylargininase in AD

cytoskeletal pathology, including NFT and pre-NFT. Instark contrast, dimethylargininase immunoreactivity isundetectable in the neurons of cerebral cortex of controlslacking AD pathology. While this contrast is marked,immunoblotting shows the bulk of dimethylargininase incerebral cortex is unchanged in AD, suggesting eitherthat we are noting a change in distribution of the enzymerather than induction or that whatever increase there is inneurons is not detectable when combined with the gliaand neuropil that dominate bulk analysis of tissue. Thesefindings are specific to dimethylargininase since similarimmunostaining was not seen with omission of the anti-body or its preabsorption with dimethylargininase. Fur-ther, in immunoblots, the antibody recognized a single35 kDa band, absorbable with the antigen, correspondingto the known molecular weight of dimethylargininase.

Dimethylargininase is a monomeric enzyme contain-ing one non-catalytic zinc [21]. The enzyme is involvedin the hydrolyses of MMA (L-Nv-monomethylarginine)and ADMA (L-Nv,Nv-dimethylarginine, asymmetricdimethylatedL-arginine), both potent endogenous inhib-itors of NOS that occur at high enough concentrations inbrain to be effective in regulating NOS activity [30].Thus, using a specific inhibitor of dimethylargininaseincreased intracellular levels of ADMA and MMA led toa substantially reduced production of NO [22]. More-over, a similar effect has been observed upon extracel-lular administration of ADMA and MMA [31]. Conse-quently, the observed local accumulation ofdimethylargininase in NFT would result in the removalof both arginine analogues and hence in elevated NOsynthesis. Therefore, abnormalities in dimethylargini-

nase may not be surprising in AD where nitration, pre-sumably resulting from peroxynitrite-related damage, theproduct of the reaction of NO with superoxide, is in-creased in all neurons at risk of death in AD [6]. Whilewe do not know the source of NO causing this damage,the low diffusion of ROS suggests that the ROS source,like the distribution of nitro tyrosine, is confined to theneuronal soma.

Interestingly, dimethylargininase in this respect hasthe same distribution since it, as nitrotyrosine, is absentfrom senile plaques. Yet it differs from peroxynitritewhich involves all neurons at risk of death in AD whiledimethylargininase is only increased in those neuronsdisplaying cytoskeletal abnormalities. In that regard, thefindings are similar to those with the oxidative stressresponse protein, HO-1. Intriguingly, it could be thataltered dimethylargininase activity is an important re-sponse to oxidative stress. Therefore, the specific sparingof NOS-expressing neurons during AD could reflect theirNO-mediated protection from ROS [32,33]. While theformation of NO can exacerbate oxidative damage, it canalso play a role in removing reactive oxygen species [32]consistent with our observation of increased dimethyl-argininase in neurons undergoing oxidative stress in AD.In this connection it should be noted that, in AD, iNOSis found in NFT-bearing neurons but not in controlpatients [34]. Further studies that compare the distribu-tion of NOS and dimethylargininase are clearly war-ranted.

Besides its tightly bound non-catalytic zinc, dimethyl-argininase is competitively inhibited by low concentra-tions of free zinc (Ki 5 2.0 mM) [21,35]. Abnormalities

Fig. 2. The specificity of the antibody to recognize neurons (A) in AD was demonstrated by omission of the antibody, not shown, orby blockage of immunoreaction by preabsorption with dimethylargininase (B). Adjacent serial section with two vessels (*) indicatedas landmarks. Scale bar5 50 mm.

900 M. A. SMITH et al.

in zinc metabolism in AD [36–40] are well documented,and free zinc levels increase during oxidative stress [16,18]. It is of particular interest that the symptoms of ADpatients treated with zinc worsen (C. Masters, personalcommunication) while metal chelation with deferox-amine leads to improvement [41]. Could it be that zinc isactivated to inhibit dimethylargininase with consequentdown regulation of NOS? That intraneuronal redox-ac-tive iron, HO-1 and iron response protein-2 [42], dem-onstrate similar distributions to dimethylargininase isalso significant. These findings implicate abnormalitiesin the handling of transition metals integrally involved inthe oxidative stress of AD.

Acknowledgements—This work was supported through grants from theNational Institutes of Health (MAS, GP), the American Health Assis-tance Foundation (MAS, GP) and by financial support from the SwissNational Science Foundation Grant 31-49460.096 (MV).

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