alzheimer's disease – synergistic effects of glucose deficit, oxidative stress and advanced...

Alzheimer’s disease – synergistic effects 439J Neural Transm (1998) 105: 439–461

Alzheimer’s disease – synergistic effects of glucose deficit,oxidative stress and advanced glycation endproducts

G. Münch1, R. Schinzel1, C. Loske1, A. Wong1, N. Durany2, J. J. Li3,H. Vlassara3, M. A. Smith4, G. Perry4, and P. Riederer2

1 Physiological Chemistry, Biocenter, and 2 Clinical Neurochemistry, Department ofPsychiatry, University of Würzburg, Federal Republic of Germany

3 The Picower Institute for Medical Research, Manhasset, and 4 Department ofPathology, Case Western Reserve University, Cleveland, OH, USA

Accepted March 1, 1998; received November 15, 1997

Summary. Many approaches have been undertaken to understandAlzheimer’s disease (AD) but the heterogeneity of the etiologic factors makesit difficult to define the clincally most important factor determining the onsetand progression of the disease. However, there is increasing evidence that thepreviously so-called “secondary factors” such as a disturbed glucose metabo-lism, oxidative stress and formation of “advanced glycation endproducts”(AGEs) and their interaction in a vicious cycle are also important for theonset and progression of AD. AGEs are protein modifications that contributeto the formation of the histopathological and biochemical hallmarks of AD:amyloid plaques, neurofibrillary tangles and activated microglia. Oxidativemodifications are formed by a complex cascade of dehydration, oxidationand cyclisation reactions, subsequent to a non-enzymatic reaction of sugarswith amino groups of proteins. Accumulation of AGE-crosslinked proteinsthroughout life is a general phenomenon of ageing. However, AGEs are morethan just markers of ageing since they can also exert adverse biologic effectson tissues and cells, including the activation of intracellular signal transduc-tion pathways, leading to the upregulation of cytokine and free radical pro-duction (oxidative stress). Oxidative stress is involved in various divergentevents leading to cell damage, including an increase in membrane rigidity,DNA strand breaks and an impairment in glucose uptake. In addition, otherage-related metabolic changes such as depletion of antioxidants or decreasedenergy production by a disturbed glucose metabolism diminish the ability ofthe cell to cope with the effects of radical-induced membrane, protein andDNA damage. With our improving understanding of the molecular basisfor the clinical symptoms of dementia, it is hoped that the elucidation ofthe etiologic causes, particularly the positive feedback loops involvingradical damage and a reduced glucose metabolism, will help to develop novel

440 G. Münch et al.

“neuroprotective” treatment strategies able to interrupt this vicious cycle ofoxidative stress and energy shortage in AD.

Keywords: Oxidative stress, diabetes, aging, advanced glycation endproducts,lipid peroxidation.

Glucose metabolism in Alzheimer’s disease

One of the striking features of Alzheimer’s disease (AD) is the drastic reduc-tion of glucose metabolism in affected brain areas. In vivo imaging of patientsusing positron emission tomography (PET) with 2-[F-18]-fluoro-2-deoxy-D-glucose as label demonstrates progressive reductions in brain glucose metabo-lism and blood flow in relation to the severity of dementia. Interestingly, notonly patients, but also younger family members with familial AD (FAD;APP717 Val to Ile mutation) show regional brain glucose hypometabolism in apreclinical phase before the onset of the disease, suggesting that changes inthe processing of the amyloid precursor protein (APP) or the ratio of â-amyloid peptide (Aâ) derivatives are linked to the disturbed glucose metabo-lism in AD (Perani et al., 1997). Glucose metabolism in the brain limits thesynthesis of acetylcholine, glutamate, aspartate, gamma-aminobutyric acid,glycine, and ATP production. Whereas the cerebral energy pool is onlyslightly diminished during the normal ageing process, glucose metabolism andcellular energy production are severely reduced in sporadic AD (Hoyer, 1993,1996). This reduced brain glucose utilisation in the early stages of AD reflectspotentially reversible down-regulation of gene expression for oxidative phos-phorylation within neuronal mitochondria (Rapoport et al., 1996). This issupported by the fact that the CSF of AD patients shows higher levels ofpyruvate, which correlates significantly with the severity of dementia (Parnettiet al., 1995).

Although introducing the term “diabetes mellitus of the brain” forAD seems speculative, but nevertheless interesting, there is more andmore evidence for parallel biochemical abnormalities between the glucosehypometabolism in AD and in non-insulin dependent diabetes (NIDDM)(Smith et al., 1996b). High blood and tissue glucose in NIDDM is caused byimpaired insulin signalling (“insulin resistance”) decreasing the ability of theglucose transport system to effectively transport glucose into the cells of theliver and muscle, which leads to a high concentration of extracellular glucoseand AGEs, causing increased oxidative stress (Table 1). Decreased intracellu-lar availability of glucose is particularly detrimental under conditions where itis vital for the cell to increase its cellular antioxidative capacity throughactivation of the pentose phosphate pathway to combat the damaging effectsof oxidative stress. In diabetes, accelerated AGE formation is caused primar-ily by a higher level of plasma glucose. However, accumulation of extracellu-lar AGEs in AD is more likely caused by accelerated oxidation of glycatedproteins (“glycoxidation”), e.g. by redox-active iron bound to proteins inamyloid plaques (Smith et al., 1997). Long-lived protein deposits such asâ-amyloid plaques with bound transition metals create an ideal chemicalenvironment for the formation of AGEs (Wells-Knecht, 1995b). Increased

Alzheimer’s disease – synergistic effects 441

Table 1. Comparable pathological events in AD and non-insulin dependent diabetesmellitus (NIDDM)

Glycation related factors in NIDDM in AD

Extracellular AGE modified plasma proteins proteins in amyloidproteins (e.g. albumin, plaques (AB, Apo E)

LDL, etc.)

Intracellular AGE modified peripheral neurons: myelin central neurons:proteins basic protein cytoskeletal proteins.

e.g. neurofilamentproteins, microtubuliassociated proteins

Major factor responsible for extracellular: high glucose extracellular: redox-increased AGE formation intracellular: reactive sugars active transition

(fructose-phosphates) metals, e.g. ironintracellular: reactivesugars (fructose-phosphates, trioses)?

Metabolic consequences Oxidative stress, glucose Oxidative stress,hypometabolism and glucoseimpaired cell function hypometabolism and

impaired cell function

accumulation of intracellular AGEs might be caused by an increase in con-centration of more active sugars or sugar fragmentation products (such asfructose and triose phosphates) e.g. by changes in metabolic pathways or ablockade of glycolytic enzymes.

The first type of evidence for the existence of a mechanistic link betweenoxidative stress caused by Aâ and impaired glucose transport was shown incultured neurons by a mechanism involving peroxidation of membrane lipidsleading to a decreased membrane fluidity and loss of protein function. Aâimpairs glucose transport followed by a decrease in cellular ATP levels. Thisimpairment of glucose transport, ATP depletion, and cell death can beprevented by antioxidants. Exposure of cultures to Aâ induces conjugationof 4-hydroxynonenal (HNE), bifunctional carbonyl product of lipid peroxi-dation, to membrane proteins including the GLUT 3 transporter. HNEinduces a concentration-dependent impairment of glucose transport andsubsequent ATP depletion. Lipid peroxidation caused by other sources ofoxidative stress e.g. activated microglia or free extracellular iron may thuscontribute to decreased glucose uptake and neuronal degeneration in AD(Mark et al., 1997). This is consistent with histopathological findings in AD,where a decreased membrane fluidity in mitochondria and an increase of theoxidised nucleoside 8-hydroxy-29-deoxyguanosine in mitochondrial DNA canbe observed, suggestive of a link between oxidative stress and energy produc-tion (Mecocci et al., 1997).

Additional support for parallel biochemical susceptibilities between ADand NIDDM comes from epidemiological data. In two large population based


studies in Rochester and Rotterdam it was convincingly shown, that adult-onset diabetes did not only increase the risk of vascular dementia but also ofAD (Ott et al., 1996; Leibson et al., 1997). Since diminished energy produc-tion by a disturbed glucose metabolism as an age- and disease-associatedchange is very likely to limit cellular capacity to cope with oxidative insults,this might thus be as important from a therapeutic view as the so-called“primary causes” (plaque and tangle formation), which may not be amenableto intervention (Finch and Cohen, 1997).

Advanced glycation endproducts: chemistry

There is more and more evidence that glucose is involved in the pathogenesisof AD in a different way. This involves a non-enzymatic reaction of glucose toform advanced glycation endproducts (AGEs) on long-lived protein deposits,which induce oxidative stress, subsequently disturbing glucose metabolism.AGEs are sugar-derived protein modifications able to irreversibly crosslinklong – lived proteins including the characteristic hallmarks of AD, i.e.â-amyloid plaques and NFT. AGE formation starts with the reaction ofthe amino groups of proteins, particularly the side chains of lysine, arginineand histidine, with reducing sugars, including glucose, fructose, hexose-phosphates, trioses and triose-phosphates. This modification, termed “non-enzymatic glycosylation”, “glycation” or “Maillard reaction”, leads viareversible Schiff-base adducts to protein-bound Amadori products. Throughsubsequent rearrangements, dehydrations and oxidations, a heterogeneousgroup of fluorescent and brown products is formed, the so-called “advancedglycation endproducts” (AGEs) (Brownlee, 1995; Buccala and Cerami, 1992).AGEs can be formed by non-oxidative and oxidative reaction pathways, thelatter being significantly accelerated by transition metals, such as copper andiron. In this “glycoxidation” reaction, protein-bound AGEs as well as soluble,highly reactive dicarbonyl products and oxygen free radicals are formed(Wells-Knecht et al., 1995a). Transition metals can also oxidise the monosac-charides directly in solution to form dicarbonyl products, which subsequentlycrosslink proteins through a process called “autooxidative glycosylation”. Theoxidation steps in both pathways can be inhibited by metal chelators,emphasising the significance of transition metals for AGE-formation (Wells-Knecht et al., 1995b). Among the physiologically relevant sugars, glucose isthe least reactive, presumably the reason for its selection by evolution as themain biological energy carrier. The rank order of reactivity for the othermonosaccharides increases from hexoses to trioses by several orders of mag-nitude (Namiki, 1988). AGE formation is irreversible and causes protease-resistant cross-linking of peptides and proteins, leading to protein depositionand amyloidosis (Smith et al., 1994b, 1995; Vitek et al., 1994) (Fig. 1).

Interaction of AGEs with cells

It is becoming increasingly clear how AGEs are involved in the formation ofthe abnormal filamentous lesions, NFT and senile plaques, in and outsideneurons in the brains of afflicted individuals. However, the effects of AGEs


Fig. 1. Chemical reactions leading to the formation of advanced glycation endproducts

on cells, particularly their interaction with cell surface receptors and intracel-lular signal transduction, is a relatively new direction of research which mighthelp to solve certain aspects of the etiopathogenesis of AD. While it has beenargued a few years ago that the AGEs on these deposits are static by-productsof the disease rather than dynamic participants in neuronal death, it is becom-ing more and more obvious that AGEs in senile plaques and NFT exertmultiple detrimental effects on cells. For example:

Glycated proteins produce radicals through oxidation reactions

Formation of oxygen free radicals is associated with the Maillard reaction.Glycated proteins produce nearly 50 fold more radicals than non-glycatedproteins (Mullarkey et al., 1990). This process commences with the produc-tion of superoxide radicals by the transition metal-catalysed autoxidation ofprotein-bound Amadori products, followed by the dismutation of superoxideto hydrogen peroxide, and the generation of hydroxyl radicals by the Fentonreaction. This results in a site-specific attack on proteins, with consequentprotein damage and lipid peroxidation, as well as damage to other cell com-ponents, such as DNA (Smith et al., 1995).

AGEs activate microglia and induce cytokine production andrelease of oxygen free radicals

Interaction of AGEs with cells increases oxidative stress. It is not yet clearwhether this occurs simply by the binding of AGEs to the cell surface andsubsequent diffusion of chemically produced free radicals across the mem-


brane, or by a receptor-mediated oxidative signalling pathway. In addition tothe recently described AGE-receptor, RAGE (Schmidt et al., 1993, 1994),various other receptors with AGE binding properties have been described(Vlassara et al., 1995). Interestingly, the macrophage scavenger receptorwhich is associated with senile plaques in AD (Christie et al., 1996), alsomediates the endocytic uptake and degradation of AGE proteins (Araki et al.,1995). These AGE-mediated oxidative signalling effects can be blocked bythe addition of antioxidants and antibodies to the AGE-receptor (Wautier etal., 1994). In cell culture experiments, PHF-tau isolated from post-mortemtissue and AGE- modified recombinant MAP-tau each generate oxygen freeradicals, thereby not only activating transcription via NFκB, but also inducingthe release of the characteristic 4kDa Aâ peptides (Schmidt et al., 1994;Yan et al., 1995).

What may be the most important contribution of AGEs to theetiopathogenesis of AD is free radical production via the innate immunesystem. It has been shown by several groups that AGE-modified proteins canelicit an acute phase response in microglial cells, generate a “respiratoryburst” and cause “bystander-lysis” of neighbouring neurons (McMillian et al.,1995). These findings corroborate with earlier studies showing that AGEsinduce chemotaxis (Schmidt et al., 1993) and inflammatory responses in mac-rophages including secretion of interleukin 1 and 6, and TNF-α (Vlassara etal., 1988; Guzdek and Stalinska, 1992) (Fig. 2).

The Aâ deposition in Alzheimer’s disease bears similarity to otheramyloidoses such as the hemodialysis-associated amyloidosis (HAA), a com-plication of long-term hemodialysis whereby â2-microglobulin (â2M) is depos-ited in â-sheeted amyloid fibrils. â2M in the amyloid deposits of HAA is alsomodified by AGEs. AGE-â2M enhances directed migration (chemotaxis) andrandom cell migration (chemokinesis) of human monocytes. In addition,AGE-â2M increases the secretion of TNF-α and IL-1â from macrophages.These findings suggest that AGE-â2M, a major component of amyloiddeposits, participates in the pathogenesis of HAA as foci where monocytes/macrophages accumulation initiates an inflammatory response that leads tobone/joint destruction (Miyata et al., 1996).

If a pathophysiological cascade similar to HAA is also occurring in AD, itmight explain why such an autodestructive process, involving reactive astro-cytes and microglial cells, reflected by an increase in pro-inflammatorycytokines in the CSF (Blum-Degen et al., 1995), exists in the AD brain(McGeer et al., 1994).

AGE receptors – physiological role and pathophysiological effects

AGEs are markers of protein ageing. In view of the close association of AGEswith cells in the body and their known adverse biologic effects on cells, manystudies have focused on searching for AGE binding proteins. The rationale isthat AGE binding proteins might play important roles in the removal ofAGEs from the tissue, thus indirectly determining the ageing process per se inthe body (Monnier and Cerami, 1981; Monnier et al., 1991).


Fig. 2. Direct and indirect toxic effects of AGEs through crosslinking of Aâ peptide

Binding and internalisation of AGE-modified proteins is mediatedby several AGE- specific cell surface receptors, including AGE-R1 (OST-48/p60), AGE-R2 (80K-H/p90) and AGE-R3 (galectin-3) (Yang et al., 1991;Vlassara et al., 1995; Li et al., 1996), scavenger-receptor (Smedsrod et al.,1997), and a receptor for AGEs (RAGE) (Schmidt et al., 1996; Miyata et al.,1996; Yan et al., 1996). These receptors are widely distributed amongdifferent cell types including hemopoetic, endothelial, mesangial, neuronaland glial cells (Imani et al., 1993; Li et al., 1996; Yan et al., 1996). There isincreasing evidence that AGE receptors play important roles in the catabo-lism of AGEs in the body (Yang et al., 1991). Smedsrod et al. show that ratliver scavenger receptors are capable of eliminating AGE-modified bovineserum albumin from the blood (Smedsrod et al., 1997). We also have evidencesuggesting that AGE receptors on astrocytes participate in the degradation ofAGE-modified proteins in human brain (Li et al., 1998). It has also beenshown that AGE-R1-3 expression is a dynamic process and can be up-


regulated by cytokines (Vlassara et al., 1989). AGE-R3 expression can beenhanced by GM-CSF in human mononuclear phagocytes, but not in astro-cytes (Li et al., 1998). Our results corroborate well with the fact that GM-CSFcan up – regulate AGE-R3 (MAC-2) expression in macrophages and Schwanncells (Saada et al., 1996). The most interesting AGE receptor due to its abilityto take part in signal transduction across the membrane is RAGE (Yan et al.,1996; Bierhaus et al., 1997). In addition to binding to AGE-modified proteins,RAGE can also interact with amphoterin to regulate neurite outgrowthduring nerve development (Hori et al., 1995), and with â-amyloid peptide toinduce intracellular oxidative stress and to activate NFκB regulated geneexpression (Yan et al., 1996).

Cellular signalling of AGEs has recently emerged as an important linkin the pathophysiological events of AD, showing activation of cellular tran-scription mechanisms in the response to an elevated AGE microenviroment.AGEs have been shown to activate two major signal transduction pathways:a redox-sensitive pathway involving the transcription factor NFκB (Schmidtet al., 1996) and a mitogenic pathway involving protein kinases includingERK-2 and the transcription factor AP-1 (Lander et al., 1997; Simm et al.,1997) (Fig. 3). Both signal transduction pathways could be involved in thepathophysiological events in AD.

NADPH-oxidase/superoxide/NFκB-pathway

The redox-sensitive NFκB pathway is likely to be important in neurons,astroglia and microglia where AGEs or Aâ might cause the release ofcytokines and free radicals. Activation of the p65 NFκB subunit was observedin the neurons and astroglia of brain sections from patients with AD, wherebyactivated NFκB was shown to be restricted to cells in the close vicinity of earlysenile plaques (Kaltschmidt et al., 1997). The NADPH-oxidase/superoxide/NFκB-pathway can be initiated by both AGEs and Aâ and uses reactiveoxygen intermediates as messengers (Yan et al., 1996). Maximal activation ofNFκB requires 0.1µM Aâ (1–40) which is about 10–20 times less than the EC50

for direct neurotoxicity (Kaltschmidt et al., 1997). A similar ratio betweenactivation and toxicity thresholds can be observed with model-AGEs whereupregulation of the inducible nitric oxide synthase, a NFκB regulated gene, isabout 50 times lower than the AGE concentration necessary for the inductionof significant cell death (Münch et al., 1997b).

p21(ras)/MAP-kinase (ERK) pathway

Studies in rat pulmonary artery smooth muscle cells have shown that AGEsare able to activate p21(ras) as well as the MAP-kinases Erk 1 and ERK2 viaa RAGE-dependent pathway (Lander et al., 1997). This result is supported bystudies in the renal tubulus cell line LLC-PK1, where AGEs were shown toactivate p42(MAP) kinase (ERK 2) and its downstream target, the AP-1complex (Simm et al., 1997). This might explain one of the paradox findings inAD, namely the activation of mitogenic signal transduction pathways in ADneurons. For example, the expression of the small G-protein p21(ras), a


critical regulator of cell proliferation and differentiation is increased withinneuritic plaques as well as in neurons and glial cells closely associated withplaques. Neurons containing tangle-bearing material also show a high level ofp21(ras) expression (Gärtner et al., 1995). The downstream members of thep21(ras) pathway are the mitogen-activated protein kinase kinase (MAPK-kinase) and its substrate, the mitogen-activated protein kinase (MAP-kinaseor ERK), which is able to phosphorylate MAP-tau, thereby generating abnor-mally hyperphosphorylated tau species that are similar to PHF-tau found inAD. Immunoreactive neurons are localized in the direct vicinity of neuriticsenile plaques (Arendt et al., 1995). In addition, the cyclin-dependent kinaseinhibitor p16, a regulator of the orderly progression through the cell cycle, isaccumulated in both neurofibrillary tangles and neuritic components of senileplaques (Arendt et al., 1996; McShea et al., 1997). A second important regu-lator of the cell cycle, cyclin-dependent kinase-4 (cdk-4) was shown to beincreased in the brains of cases of AD patients compared with age-matchedcontrols. Both proteins are increased in the pyramidal neurons of the hippo-

Fig. 3. Schematic illustration of putative AGE-induced signal transduction pathways


campus, including those neurons containing NFT and granulovacuolar degen-eration (McShea et al., 1997). As p16 is not normally found in terminallydifferentiated neurons, it seems paradoxical that it is increased in AD. Induc-tion of p16, a protein that signals re-entry and progression through the cellcycle, may itself be the consequence of a response to a growth stimulus. Re-entry into the cell cycle is likely deleterious in terminally differentiated neu-rons and may contribute to the biochemical abnormalities, particularly axonalsprouting and hyperphosphorylation of tau as well as the neuronal degenera-tion characteristic of the pathology of AD (McShea et al., 1998). Thus it isvery likely that the activation of the MAP kinase cascade by AGEs is criticallyinvolved in the processes of neurodegeneration and aberrant repair.

Advanced glycation endproducts in AD

AGEs accumulate in pyramidal (glutaminergic) neurons in aged animals andhumans, but their intracellular distribution pattern is quite different betweenanimal and man. In the human pyramidal neuron, AGEs exhibit a granular,perikaryonal distribution, whereas in animal brains, AGEs show a nuclearstaining pattern (Li et al., 1994). Pyramidal neurons selectively accumulateAGE-containing vesicles in an age-dependent manner presumably in endo-somes or lysosomes (Kimura et al., 1996; Li et al., 1995). In addition to intra-cellular cytoplasmic accumulation of AGEs during ageing, proteins presentin senile plaques and NFT have been shown to contain significant amountsof AGEs:

Senile plaques

The major proteinaceous component of the amyloid deposits which accumu-late extracellularly in the AD brain, is Aâ. This 39–42 amino acid peptideconsists of 28 extramembrananal amino acids plus 11 to 14 residues of thehydrophobic transmembrane domain of its precursor, amyloid precursor pro-tein, APP (Price and Sisodia, 1994). Genetic studies have linked some familialcases of early onset Alzheimer’s disease to the APP-locus on chromosome 21(,1% of all AD cases), where mutations in the APP-gene influence theprocessing of APP with subsequent changes in Aâ concentration or the ratioof the 1–42 to 1–40 Aâ form (Hardy, 1997; Citron et al., 1997). In analogy,mutations in the presenilin genes change the ratio of the 1–42 to 1–40 Aâform, most likely by influencing APP processing in the endoplasmatic reticu-lum (Hartmann et al., 1997).

Two major biochemical mechanisms have been proposed by which Aâmay be involved in neuronal cell death: direct neurotoxicity and/or indirect,immune system-mediated neurotoxicity as the result of microglial cell activa-tion. Firstly, several studies have demonstrated that Aâ peptide is both toxicto neurons in primary culture and to neuronal cell lines (Lorenzo andYankner, 1996; Zhang et al., 1996). The neurotoxic effect appears to correlatewith the aggregation state and degree of â-sheet conformation (Pike et al.,1995). In addition, oxidative stress (Behl et al., 1994), apoptosis (Loo et al.,1993) and ion channel formation (Pollard et al., 1995) can combine to mediate


Aâ neurotoxicity. However, it appears that overexpression of APP intransgenic mice with resulting Aâ deposition does not cause neuronal celldeath and behavioural changes related to “dementia”, although the â-amyloiddeposits are associated with gliosis and neuritic dystrophy, suggesting thatadditional factors are necessary to promote the progression of the disease(Irizarry et al., 1997). One of these additional factors may be the activation ofresting microglia cells by the Aâ deposits; as a result, many inflammatorymediators, such as oxygen free radicals, NO, cytokines and complementproteins are released. These inflammatory mediators can subsequently dam-age surrounding neurons through “bystander lysis” (McGeer et al., 1994,1997).

AGE accumulation has been demonstrated in senile plaques in differentcortical areas (Smith et al., 1994b,c; Vitek et al., 1994) in primitive plaques,coronas of classic plaques and some glial cells of AD brain (Kimura et al.,1995). The higher AGE level in AD tissue is reflected by increased AGE-Aâlevels in the CSF of AD patients (Li J. et al., unpublished data). We and othershave demonstrated in vitro that nucleation-dependent polymerisation of Aâ,the major component of senile plaques is significantly accelerated by AGE-mediated crosslinking (Vitek et al., 1994; Münch et al., 1997a). Aâ aggrega-tion follows a classic nucleation-dependent polymerisation process, consistingof two distinctive steps, an initial slow nucleus formation step, followed by arapid elongation phase (Jarett and Lansbury, 1993). The slow nucleus forma-tion stage is a reversible process depending on the concentration of themonomer. Therefore, at a sub-threshold peptide concentration, irreversiblecovalent crosslinking by AGEs play a crucial role in stabilising monomernucleation as well as growth of the larger aggregates, the amyloidplaques. Since the aggregation of Aâ therefore is critical for its toxiceffects via the stimulation of either direct or indirect neurotoxicity, the rate ofsenile plaque formation is likely to be important for the progression ofthe disease. This suggests that AGEs may indeed represent a driving force inthe acceleration of â-amyloid deposition and senile plaque formation (Smithet al., 1995).

It is now well accepted that AD susceptibility is correlated with the doseof the ε4 allele of apolipoprotein E (Apo E) (Czech et al., 1995). Substitutionof cysteines by arginines creates more glycation sites and hence, facilitates theAGE-mediated crosslinking of ApoE to the insoluble amyloid fibrils andaccelerates senile plaque formation (Harrington and Colaco, 1994; Li andDickson, 1997).

Neurofibrillary tangles (NFTs)

NFTs and neuropil threads are also histopathological hallmarks of AD. Theinterrelationships between the rate of NFT formation and the progression ofAD remain unknown (Cras et al., 1995). Nonetheless, early signs of tangleformation in certain brain regions such as the entorhinal cortex precede theclinical diagnosis of AD by decades (Braak and Braak, 1995). The majorcomponent of NFT is hyperphosphorylated microtubule-associated protein


tau (MAP-tau). This abnormal MAP-tau is resistant to proteolytic enzymesand it is suggested that glycation, disulphide bond formation, phosphorylation(Smith et al., 1994c, 1995; Mandelkow et al., 1996; Schweers et al., 1995) and/or formation of core fragments of MAP-tau (Novak et al., 1993) can allcontribute to extensive cross-linking between MAP-tau subunits.

NFT isolated from brain tissue of AD patients are detergent-insoluble andprotease-resistant and have characteristic fluorescent spectra of brown pig-ments similar to synthetic AGEs (Colaco et al., 1996). The major componentof the NFT is MAP-tau, which has been shown to be subject to intracellularglycation and AGE formation (Ledesma et al., 1994). The relevance ofglycation as an additional pathological modification of MAP-tau in vitro issupported by the immunohistochemical co-localization of AGEs with NFT(Smith et al., 1994c; Dickson et al., 1996). MAP-tau can be glycated in vitro,inhibiting its ability to bind to microtubules; in addition, paired helical fila-ment (PHT)-tau isolated from the brains of AD patients is glycated in thetubulin-binding region (Ledesma et al., 1995). Interestingly, only the glycationof MAP-tau gives rise to the formation of fibrils which resemble NFT in theAD brain (Colaco et al., 1996). Finally, the introduction of glycated MAP-tau into cells can generate oxygen-free radicals capable of disturbingneuronal function, including up-regulation of APP and release of Aâ (Yanet al., 1995).

Oxidative stress in Alzheimer’s disease

The first hints for oxidative stress in AD came from studies of fibroblasts fromAD patients where several key enzyme abnormalities were found. Two oxida-tive abnormalities were consistently noted; deficiencies in thiamine-requiringenzymes (Gibson et al., 1988) and changes in the mitochondrial respiratorychain (Sheu et al., 1994). Uncoupling of electron transport from oxidativephosphorylation leads to incomplete reduction of oxygen, producing straysuperoxide radicals. The addition of chemical uncoupling agents to neuroblas-toma cell lines leads to the appearance of abnormal filaments similar to thoseforming NFT (Blass et al., 1990). These findings have been applied to rodentsby Gibson and co-workers who fed rats or mice a thiamine-deficient diet andfound clusters of dystrophic neurites similar to those in senile plaques in AD(Calingasan et al., 1995, 1996). That oxidatively-compromised animals de-velop AD-type neuritic dystrophy suggests that disturbed energy metabolismand subsequent oxidative stress may be a common denominator of neuriticdystrophy. Recent studies suggest that oxidative stress may also play a role inDown’s syndrome which is trisomic for superoxide dismutase. Moreover,neurons in culture from Down’s syndrome cases undergo a prematureapoptotic death that is delayed by antioxidants (Busciglio and Yankner,1995).

There is overwhelming evidence that tissue in AD is exposed to oxidativestress during the course of the disease (Fig. 4). NFT bear the footprints ofoxidative membrane damage since they contain adducts of malondialdehyde,the most well known lipid peroxidation product (Yan et al., 1994), and


Fig. 4. Sources and effects of oxidative stress on a molecular and cellular level

hydroxynonenal, the most highly reactive lipid peroxidation product(Montine et al., 1996a; Sayre et al., 1997). Furthermore, dystrophic neurites ofsenile plaques that contain the PHF filaments also show greater membranedamage than those that lack abnormal filaments (Praprotnik et al., 1996).

AD tissue shows an imbalance in radical detoxifying enzymes. The ratio ofsuperoxide dismutase to catalase decreases, leading to a buildup of hydrogenperoxide, which is particularly dangerous in the presence of free iron due tosubsequent production of hydroxyl radicals (Götz et al., 1994; Gerlach et al.,1994; Gsell et al., 1995).

Increased levels of DNA strand breaks have been found in AD using theTUNEL labelling technique.While DNA strand cleavage by endonucleases isa part of apoptosis, strand breaks in AD are found in nearly all brain cells,which is surprising for a chronic condition (Anderson et al., 1996). Therefore,it is likely that oxidative damage to DNA, rather than apoptosis, is responsiblefor DNA breakage in AD and this is consistent with the increased freecarbonyls in the nuclei of neurons and glia in AD (Smith et al., 1996a).

The induction of heme oxygenase-1, an antioxidant enzyme involved inthe conversion of heme to bilirubin, is a robust indicator of the oxidative stressresponse in cells (Maines, 1988). In previous studies, we demonstratedthat the heme oxygenase-1 protein (Smith et al., 1994a, 1995) and mRNA(Premkumar et al., 1995) were increased in AD brains and this increase wastightly correlated with regions of NFT pathology. Furthermore, glycated taufound in neurons in AD (Smith et al., 1994b; Yan et al., 1994; Ledesma et al.,1994) causes an oxidative response in neuroblastoma cells including inductionof heme oxygenase-1, translocation of NFκB and peroxidation of membranelipids (Yan et al., 1994, 1995).

Evidence continues to mount that bifunctional 4-hydroxy-2-alkenals, suchas 4-hydroxy-2-nonenal (HNE), as opposed to other reactive lipid-derivedaldehydes such as malondialdehyde (MDA) or 2-alkenals, are the majorcytotoxic products of lipid peroxidation (Esterbauer et al., 1991) (Fig. 5).Following lipid peroxidation, a 2-pentylpyrrole modification of lysine is theonly presently known “advanced” (stable end-product) adduct that formsfrom the modification of proteins by HNE (Sayre et al., 1993). Rabbit


Fig. 5. Reactions scheme for the formation of HNE modified proteins

polyclonal antibodies specific to this HNE-pyrrole modification label not onlyneurofibrillary pathology but also pyramidal neurons in cases of AD, but notcontrol patients (Sayre et al., 1997). These findings, together with the recentdemonstration that HNE is cytotoxic to neurons (Montine et al., 1996a,b) andthat it impairs the function of membrane proteins including the neuronalglucose transporter 3 (GLUT 3) (Mark et al., 1997), indicate that HNEis not only a characteristic marker but also a major toxin leading toneurodegenerative events in AD.

Since oxidative stress is characterized by the imbalance of radical produc-tion and antioxidative defense, the latter is considered to be also a majorplayer in the process of age-related neurodegeneration. Reducing equivalentsare responsible for the endogenous redox potential in the living cell which iskept under tight regulation. Among their various roles is the ability to donateelectrons to reactive oxygen species and by doing so, to scavenge them. Mostof the reducing equivalents in the cell are molecules with low molecularweights, such as glutathione and NADH which are synthesized by the cell(Kohen et al., 1997).

It has been shown that the antioxidant cell defense system increaseswith age but the rate of reactive oxygen species generation exceeds theinduced antioxidant ability, generating a situation that favours oxidativestress and peroxidation. The decrease in the concentration of reduced glu-tathione is dramatic; it reaches up to 50% (human erythrocytes) or 70% (rathepathocytes) (Richards et al., 1997; Sanz et al., 1997).

Pharmacological interference with AGE formation or signalling as anovel treatment strategy for AD

The focus of drug development for AD remains at a very unsatisfying state,primarily to an emphasis on pallative treatments with cholinesterase-inhibitors or cholinergic agonists. However, pharmacological approachestargeting oxidative stress, protein glycation, glucose metabolism and the acute


phase response offer new opportunities for the treatment of AD (Heidrichet al., 1997).

AGE-Inhibitors

Inhibitors of the Maillard reaction such as aminoguanidine are currently beingassessed in clinical trials for the treatment of diabetic complications. In con-trast, their potential for the treatment of AD is only just being recognised. TheAGE-inhibitor Tenilsetam has been shown to improve cognitive abilities andmemory constantly over a time span of three months in two phase II trials(Dierks et al., 1989; Ihl et al., 1989; Münch et al., 1994; Shoda et al., 1997).These recent studies indicate possible neuroprotective approaches to thedisease, based on inhibition of the action of AGEs by the use of AGE-bindingcompounds and the modulation of degradation of AGE-cross-linked proteins(Colaco and Harrington, 1996; Münch et al., 1997b).

Anti-inflammatory drugs

Similar beneficial results for AD patients are seen with the non-steroidal anti-inflammatory drugs (NSAIDs) indomethacin, ibuprofen, tenidap and acetyl-salicylic acid (Lieb et al., 1997). Although a direct inhibitory role of theNSAIDs on glycation is discussed (Colaco and Harrington, 1996; Colaco et al.,1996), their primary targets are signal transduction pathway involvingprostaglandins, e.g. between AGE-receptors and downstream effectors in-volved in cytokine and radical production (Gonzales et al., 1994; McGeer andMcGeer, 1997).

Antioxidants

There is evidence that vitamins that protect against oxidative damage orinhibit radical producing enzymes may reduce the neuronal damage and slowthe progression of AD (Frölich and Riederer, 1995). In a recent controversialstudy, patients with moderate AD receiving the monoamine oxidase B(MAO-B) inhibitor selegiline, α-tocopherol (vitamin E, 2000IU per day)were shown to deteriorate slower than the respective placebo group (Sano etal., 1997). In addition, two novel antioxidants possess not merely passiveradical scavenging capabilities, but can also block the redox-sensitive NFκBmediated signal transduction pathways of AGEs. One of them, thioctic (α-lipoic) acid, combines antioxidant with energy enhancing effects, includingupregulation of glucose metabolism, direct radical scavenging, redox modula-tion of cell metabolism, and the potential to inhibit oxidatively-induced injury.Reduction of lipoate to dihydrolipoate occurs by specific NADH andNADPH dependent enzymes and is an important requirement (Haramaki etal., 1997; Roy et al., 1997), since only the reduced form of thioctic acid inhibitsAGE-induced activation of NFκB (Bierhaus et al., 1997). A second class ofnovel and promising antioxidants are estrogen derivatives such as 17â-estradiol, which are excellent candidates for intracellular radical scavenging(Behl et al., 1997). This might also explain the reduced risk for AD in post-


menopausal women undergoing estrogen replacement therapy (Ohkura et al.,1994).

Although it remains a very speculative hypothesis, interference withAGE-induced signalling might explain the success of some of the treatmentstrategies mentioned above. AGE inhibitors might be able to stop formationof AGE-Aâ deposits or modify their structure causing loss of binding to AGEreceptors, thus interrupting the AGE-induced signal transduction pathway atthe earliest possible step. Antioxidants scavenge intracellular and extracellu-lar radicals before they damage cell constituents or, especially in microglia,induce cytokine and radical production (Yan et al., 1996). A similar interfer-ence with proinflammatory signal transduction might be the predominant wayof action for the NSIADs. However, future studies need to prove that thesesubstances are truely neuroprotective, demonstrated not only by improve-ment of cognitive function in AD patients, but also by a decelerated rate ofneural cell death, evident by low levels of MAP-tau (released from dyingcells) in the CSF or by a delayed progression of lesion formation monitored byimaging techniques.

Acknowledgements

We thank A. Hipkiss, J. Michaelis, R. Holliday, U. Schindler, A. M. Cunningham, G.Grigg, F. Pemper and D. Palm for valuable and critical discussions and the “Claussen-Stiftung” and the “Hirnliga e.V.” for funding. This work was supported by a grant fromBIOMED I Nr. BMH 1-CT 94-1563.

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Authors’ address: Dr. G. Münch, Physiological Chemistry I, Biocenter Am Hubland,D-97074 Würzburg, Federal Republic of Germany.

alzheimer's disease – synergistic effects of glucose deficit, oxidative stress and advanced...

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