Experimental Gerontology 57 (2014) 122–131

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Experimental Gerontology

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Accumulation of modified proteins and aggregate formation in aging

Kerstin Nowotny, Tobias Jung, Tilman Grune, Annika Höhn ⁎Department of Nutritional Toxicology, Institute of Nutrition, Friedrich-Schiller-University Jena, 07743 Jena, Germany

Abbreviations: AD, Alzheimer's disease; AGE, advanceage-related macular degeneration; CML, Nε-(carboxymellitus; ECM, extracellular matrix; LDL, low density lipopRAGE, receptor for advanced glycation end products; RNSretinal pigment epithelium; ROS, reactive oxygen species;oxide dismutase.⁎ Corresponding author at: Department of Nutritional

University Jena, Dornburger Str. 24, 07743 Jena, GermanyE-mail address: annika.hoehn@uni-jena.de (A. Höhn).

http://dx.doi.org/10.1016/j.exger.2014.05.0160531-5565/© 2014 Elsevier Inc. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 1 April 2014Received in revised form 22 May 2014Accepted 26 May 2014Available online 28 May 2014

Section Editor: A. Simm

Keywords:AgingProtein oxidationProtein aggregatesLipofuscinAdvanced glycation end products

Increasing cellular damage during the aging process is considered to be one factor limiting the lifespan of organ-isms. Besides the DNA and lipids, proteins are frequent targets of non-enzymatic modifications by reactive sub-stances including oxidants and glycating agents. Non-enzymatic protein modifications may alter the proteinstructure often leading to impaired functionality. Although proteolytic systems ensure the removal of modifiedproteins, the activity of these proteases was shown to decline during the aging process. The additional age-related increase of reactive compounds as a result of impaired antioxidant systems leads to the accumulationof damaged proteins and the formation of protein aggregates. Both, non-enzymatic modified proteins and pro-tein aggregates impair cellular functions and tissue properties by a variety ofmechanisms. This is increasingly im-portant in aging and age-related diseases. In this review, we will give an overview on oxidation and glycation ofproteins and the function of modified proteins in aggregate formation. Furthermore, their effects as well as theirrole in aging and age-related diseases will be highlighted.

© 2014 Elsevier Inc. All rights reserved.

1. Accumulation of modified proteins and aggregate formation inaging: an introduction

Aging is a physiological and irreversible, progressive process involv-ing changes in the ability tomaintain cellular functionality, affecting tis-sues, organs and thewhole organism and thus finally causing death. It isaccepted that a series of complex reactions causes the aging process,however, the exact molecularmechanisms are not yet fully understood.During the last decades, over 300 different theories of aging have beendescribed (Medvedev, 1990). These theories can be grouped intothose emphasizing that aging is genetically programmed and into theso-called damage or error theories which consider the accumulationof cellular damage due to metabolism and/or environmental factors asa general cause of the aging process (Jin, 2010). Besides the DNA andlipids, it has been shown that proteins are primary targets of such dam-aging agents. Proteins becomemodified by different chemical reactionsand accumulate in aged individuals (Stadtman, 2006). Moreover,modified proteins were identified to influence physiological pathways

d glycation end product; AMD,methyl) lysine; DM, diabetesrotein; PD, Parkinson's disease;, reactive nitrogen species; RPE,RS, reactive species; SOD, super-

Toxicology, Friedrich-Schiller-.

and, therefore, to play an important role in aging and age-related dis-eases including cardiovascular (Simm, 2013; Stocker and Keaney,2004) and neurodegenerative diseases (Grimm et al., 2011; Li J. et al.,2012) and diabetes mellitus (DM) (Goh and Cooper, 2008). Accordingto the free radical theory of aging (Harman, 1956), reactive oxygen spe-cies (ROS) are proposed to be the major source of damage to proteins.Glycation is another important non-enzymatic modification of proteinsleading to the formation of altered proteins, known as advancedglycation end products (AGEs). Not only the protein function is im-paired due to these chemical modifications, proteolytic degradationcan also be affected leading to protein accumulation or aggregateformation during aging. To avoid accumulation of damaged, non-functional proteins, cells contain different proteolytic systems. Howev-er, there is persuasive evidence that the removal of damaged proteinsdeclines during the aging process, probably due to the decreased activ-ity of these proteolytic systems (Bulteau et al., 2002; Sitte et al., 2000a).Recently, López-Otín et al. categorized common aspects of aging and de-fined nine different hallmarks, among them the loss of proteostasis(Lopez-Otin et al., 2013). They reviewed that different stress factors, en-dogenous as well as exogenous, cause protein modifications associatedwith protein unfolding. Unfolded proteins are either refolded byproteins of the heat-shock family or degraded by the ubiquitin-proteasome or endosomal–lysosomal system. Failure in these processesfinally causes protein aggregation. In addition to the defective or de-clined degradation of modified, unfolded proteins, it was observedthat there is an age-related decrease in protein synthesis (Rattan,1996). Therefore, replacement of new functional proteins is reducedwhich in turn increases the ratio of damaged to functional proteins in

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cells. All mentioned factors cause or further accelerate aggregate forma-tion. This review focuses on the accumulation of modified proteins andtheir involvement in aggregate formation during the aging process.Protein modifications, especially reactions involving oxidation andglycation processes, are primary, causal events leading to increasingconcentrations of damaged proteins. Moreover, the potential functionof oxidized and glycated proteins and related protein aggregates, howthey influence physiological pathways and contribute to age-relateddiseases, will be discussed in the following.

2. Protein oxidation

Damage to proteins, particularly oxidative damage, is proposed toplay an essential role in aging. Especially organisms living in an aerobicenvironment are continuously exposed to ROS. ROS are either formed asa by-product during metabolic processes of molecular oxygen or aregenerated due to exogenous sources including radiation, air pollutants,cigarette smoke or drugs. The intracellular production of ROS occurs en-zymatically and non-enzymatically in different cell organelles, but fourenzyme systems, the NADPH oxidase (Bedard and Krause, 2007), xan-thine oxidase (Harrison, 2002), uncoupled endothelial nitric oxide syn-thase (Landmesser et al., 2003), and the mitochondrial electrontransport chain (Liu et al., 2002) predominate (Fig. 1). In themitochon-dria, ROS are generated by components of the respiratory chain,especially complex I (Brandt, 2006) and III (Hunte et al., 2008), orother mitochondrial enzymes such as NADPH oxidase 4 (Bedard andKrause, 2007) and monoamino oxidase (Edmondson et al., 2009;Grivennikova and Vinogradov, 2013). ROS include among othersoxygen-containing free radicals, e.g. superoxide anion radical (O2

•−)or hydroxyl radical (•OH), and non-radical molecules such ashydrogen peroxide (H2O2), singlet oxygen (1O2) or hypochlorous acid(HOCl). O2

•−, the product of the reduction of oxygen, is one of the mostimportant free radicals and a precursor of many ROS. O2

•− is reducedto H2O2 in a spontaneous or by superoxide dismutase (SOD) catalyzedreaction. Since O2

•− is able to reduce iron, it also accelerates the produc-tion of highly reactive •OH via the Fenton reaction. Moreover, O2

•− reactswith nitric oxide (•NO) which leads to the formation of peroxynitrite(ONOO−), a very strong oxidant which belongs to the reactive nitrogen

Fig. 1. Enzymatic generation of ROS, modified according to Dikalov (2011). Among mformation of ROS: NADPH oxidase, xanthine oxidase, uncoupled endothelial nitric oxide synlead via ROS-mediated activation, stimulation or uncoupling of the others to increased ROS for

species (RNS). RNS are closely related to ROS and include, besides •NOand higher oxide radicals of nitrogen, non-radical species such asnitroxyl anion (NO−), nitrosonium cation (NO+), nitrous acid (HNO2)or dinitrogen tetroxide (N2O4). To summarizeROS andRNS, the term re-active species (RS) is used in the following. RS are known to play a dualrole: low levels of RS are indispensable for physiological cellular pro-cesses whereas high levels cause cellular damage. Beneficial effects ofRS rely on their function e.g. in cell signaling (Hancock et al., 2001)and regulation or pathogen defense (Torres et al., 2006). It has beenshown that low RS levels are inducedwhen cells are stimulated by cyto-kines or growth factors (Sauer et al., 2001). Thus, someRS act as second-ary messengers regulating cellular functions, including proliferation onone side and apoptosis on the other. However, RS are predominantly re-lated to cellular damage so that cells evolved various antioxidant sys-tems to delay, prevent or eliminate oxidative damage to the targetmolecules. On the one hand, there are proteins such as transferrin andalbumin that decrease RS formation by minimizing the availability ofmetal ions. On the other hand, there are pathways, e.g. glutathioneme-tabolism, SOD or catalase, which catalyze the removal of RS. Beside theelimination of RS, cells are able to repair or remove damaged moleculespreventing their accumulation and aggregation. One of themost impor-tant proteolytic systems for the degradation of modified proteins is theproteasomal system. It was reported that about 70–90% (Lee andGoldberg, 1998; Rock et al., 1994) of misfolded, damaged proteins aredegraded by the proteasome. The proteasomal system, essential forthe physiological cell function, includes the 20S core unit and severalregulator proteins which can bind to the 20S proteasome resulting indifferent proteasomal complexes such as the immunoproteasome or26S proteasome (Jung et al., 2009). Protein modifications may changethe conformation of the protein structure and cause the rearrangementof hydrophobic amino acids from the protein interior to the surface, andthus, modified proteins are recognized by the 20S proteasome (Gruneet al., 2003). When the antioxidative defense capacity is lower thanthe RS flux rate, the term oxidative or respectively nitrosative stress isused. The term oxidative stress was introduced by Helmut Sies in the1980s (Sies, 1985) and extended 2006 by Jones (2006) by includingthe disruption of redox signaling. Short-term oxidative stress wasshown to prolong the lifespan of organisms (Ristow and Zarse, 2010).

any others, there are four major intracellular enzyme systems which cause thethase and the mitochondrial electron transport chain. Activation of one ROS source canmation.

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However, under long-term or pathophysiological conditions oxidativestress leads to cellular damage.

Since proteins represent the largest group of macromolecules, theyare the most frequent targets of RS and as a major consequence, oxida-tively modified proteins are formed (Berlett and Stadtman, 1997). Pro-tein oxidation can either occur on the protein backbone or on specificamino acids side chains. Furthermore, protein oxidation can lead tofragmentation of polypeptide chains or to protein cross-linking(Berlett and Stadtman, 1997; Höhn et al., 2014). Protein oxidation canbe simplified into three different stages reflecting the susceptibility ofthe protein to proteolytic degradation as shown in Fig. 2 (Jung et al.,2009). Stage I of oxidation describes the state when proteins becomeslightly oxidized. In this stage, although the protein structure is still in-tact, the function/activity of the protein is already diminished. In stage II,increasing oxidative modifications cause protein unfolding which is ac-companied by the complete loss of protein activity. Protein unfoldingalso induces the rearrangement of hydrophobic structures from proteincore to the surface. As hydrophobic structures are well recognized bythe proteasome (Grune et al., 2001; Pacifici et al., 1993), oxidized,unfolded proteins are ideal substrates and become degraded. In conclu-sion, increasing concentrations of oxidants are correlated to an elevatedsusceptibility of proteins to proteolysis preventing the accumulation ofmodified, damaged proteins to maintain cell homeostasis. However, ifproteins are further oxidized, protein cross-linking occurs and the pro-teolytic susceptibility declines so that severe oxidized proteins are nolonger degraded by the proteasomal system. As a result, insoluble pro-tein aggregates accumulate in cells (stage III). Aggregates containinghigh amounts of oxidized proteins and accumulating during the agingprocess are lipofuscin.

3. Lipofuscin

The term lipofuscin, also known as ceroid (Levine et al., 1968) or agepigment (Samorajski et al., 1965), is derived from the Greek word ‘lipo’meaning fat and the Latin word ‘fuscus’ which means dark color and isnamed due to its yellow–brown appearance and high content of lipids.It was introduced in 1922 by Borst (1922), but already 80 years earlierthe first descriptionwas given by Hannover who observed this pigmentin the perikaryon of aged neurons (Hannover, 1842). In recent years,lipofuscin was found to accumulate intracellularly inmany different tis-sues in a time dependent manner but especially in non-dividing cells

Fig. 2. Influence of protein oxidation on the degradation of modified proteins, modified accorderation of oxidized proteins is often the result of a series of molecular events, in general three didized, their activity is reduced or lost, but the protein structure is not dramatically affected. Inaccompanied by protein unfolding which accelerates the susceptibility to proteolytic degradaoccur and inhibit protein digestion due to protein cross-linking finally leading to the formation

including neurons, skeletal muscle cells or cardiac myocytes (Beregiet al., 1988; Brunk and Ericsson, 1972; Del et al., 1991). In contrast tonon-dividing/postmitotic cells, all proliferating cells protect themselvesfrom lipofuscin accumulation due to cell division. Because of its cytotox-ic effects in postmitotic cells, lipofuscin has come into the focus of agingresearch as one major factor limiting the lifespan of cells. Today,lipofuscin is accepted as an important biomarker of aged individuals.Due to its content of fluorescent compounds, lipofuscin formation canbe detected by the measurement of its autofluorescence using fluores-cence microscopy or confocal laser scanning microscopy. In addition,histochemical staining for lipids or lipophilic substances using SudanBlack, Eosin, Berlin Blue or Fontana-Masson is an appropriate tool forlipofuscin detection (Jung et al., 2010).

Lipofuscin is a complex mixture containing oxidized, cross-linkedproteins (Schutt et al., 2003), lipids (Bourre et al., 1979) and lesseramounts of carbohydrates (Benavides et al., 2002). In addition, an incor-poration of transition metals such as iron, copper, manganese, calciumor zinc up to about two mass percent has been detected (Jolly et al.,1995). The composition of the components varies between differentcell types. However, all have in common the resistance to proteolyticdegradation. Moreover, cells do not exocytose lipofuscin leading to itsaccumulation (Terman and Brunk, 1998a,b). In the mitochondrial–lysosomal axis theory of aging, which was established by Brunk andTerman (2002), a model for the formation of lipofuscin is presented.They postulated that lipofuscin is formed in lysosomes, cellular compo-nents, inwhichmacromolecules and cell organelles are degraded by hy-drolytic enzymes. Brunk and Terman pointed out that the proteolysis ofiron-containing macromolecules such as ferritin or cytochrome c in thelysosomes cause the release of ironwhichmay be reduced, resulting in aredox active form, able to generate •OH (Brunk and Terman, 2002). •OHin turn react with surrounding macromolecules, e.g. unsaturated fattyacids, initiating a chain reactionwhich leads to the formation of reactivealdehydes, such as malondialdehyde or 4-hydroxynonenal, which areknown to cross-link proteins.

The involvement of mitochondria in lipofuscin formation is support-ed by the fact, that about 50% of some kinds of lipofuscin in vivo consistsof mitochondrial ATP synthase subunit c (Elleder et al., 1997). Mito-chondria are not only one of the main sources of free radicals in cells,they are also a main target. So, oxidatively damaged mitochondria aretaken up in the lysosomal system and, due to their high concentrationsof iron, it was assumed that formation of free radicals induces lipid

ing to Jung et al. (2009). Although protein oxidation is a continuous process and the gen-ifferent stages of protein oxidation can be distinguished. In stage I, proteins are mildly ox-creasing oxidative modifications of proteins cause in general the complete loss of activitytion (stage II). If proteins are not removed at this stage, further oxidative modificationsof protein aggregates (stage III).

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peroxidation and production of aldehydes as described above (Brunkand Terman, 2002). But the hypothesis about continuing redox reac-tions after the uptake of mitochondria into the lysosomal system re-quires the availability of an intact redox chain, reducing equivalentsand oxygen, which has not yet been confirmed (Jung et al., 2007). It is,however, unquestionable that lysosomes are the central structures oflipofuscin accumulation even if the formation mechanism is not fullyunderstood. It is still unclear whether the majority of cross-linkingsteps are occurring in the cytosol or solely after uptake of precursorsinto the lysosomal system. It was recently shown that lipofuscin canbe formed independently of the lysosomal system. In a study, blockingof lysosomal activity aswell as the knock-down of an enzyme necessaryfor the lysosomal uptake of macromolecules induced cytosolic accumu-lation of lipofuscin accompanied by an increased cellular toxicity (Höhnet al., 2012).

One mechanism which accelerates the formation of lipofuscin andcauses cellular damage is based on the ability of lipofuscin to inhibitproteolytic systems, including the proteasome and lysosomal enzymes(Ivy et al., 1984; Sitte et al., 2000b). It was shown that inhibition ofthe proteasome is probably due to the fact that the proteasome recog-nizes lipofuscin as a substrate by binding to hydrophobic structures ofmodified proteins on the lipofuscin surface (Höhn et al., 2011). Becausedegradation of these cross-linked structures is not possible, the protea-some becomes inhibited. As a result of proteasome inhibition, the deg-radation of oxidatively modified proteins is decreased which elevatesthe concentration of damaged proteins or rather increases the possibil-ity of further oxidative processes. In addition, it was demonstrated thatthe cytotoxic effect of lipofuscin is due to its ability to produce oxidants(Höhn et al., 2010). By incubating senescent fibroblasts with artificiallipofuscin it was observed that this material is able to incorporate ironions. Compared to iron-free lipofuscin, the redox-active form led to in-creased concentrations of oxidants due to the elevated Fenton reaction.Thus, all described abilities of lipofuscin create a vicious cycle, accelerat-ing not only lipofuscin formation, but also cellular damagewhich in turnmay limit cellular lifespan (Fig. 3).

There is more and more evidence that additionally to cross-linkingaldehydes (derived from lipid peroxidation) AGEs may play a role inlipofuscin formation and the subsequent cytotoxic effects of lipofuscin(Fig. 3). Researchers were able to identify AGE deposits associated

Fig. 3. Cytotoxic effects of lipofuscin, modified according to Höhn and Grune (2013). In vitro stuability (Höhn et al., 2010, 2011). Cytotoxicity of lipofuscin is mediated by several mechanismslipofuscin to inhibit proteolytic systems such as the proteasome. Furthermore, glycoxidation andin lipofuscin and may also contribute to the toxic potential of lipofuscin.

with lipofuscin, e.g. in the human retinal pigment epithelium (RPE)(Schutt et al., 2003), heart (Nozynski et al., 2013) or brain (Horieet al., 1997). Glycoxidation products, such as pentosidine (Sell andMonnier, 1989) and Nε-(carboxymethyl) lysine (CML) (Ahmed et al.,1986), were used as markers for AGE detection (Horie et al., 1997;Nozynski et al., 2013). These AGEs are formed due to both, glycationand oxidation processes, so that oxidative stress triggers their formation(Baynes, 1991). As mentioned above, enhanced oxidative stress duringaging is causal for lipofuscin formation, thus, an involvement of AGEsin lipofuscin formation is comprehensible. Some AGEs, includingpentosidine, are known to cross-link proteins and may contribute tothe resistance of lipofuscin against proteolytic degradation. Further-more, AGE deposits in lipofuscin may at least partly explain the fluores-cent properties of lipofuscin.

Whether and in whichmanner lipofuscin contributes to age-relateddiseases such as age-related macular degeneration (AMD) is still con-troversial. Lipofuscin is known to accumulate in cells of the RPE andA2E was identified as a fluorophore in lipofuscin of RPE (Eldred, 1993;Eldred and Lasky, 1993), which, when excited by blue light has photo-toxic properties perhaps contributing to the development of AMD(Sparrow and Cai, 2001; Sparrow et al., 2000). In contrast to these ob-servations, Ablonczy et al. recently published that there is no correlationbetween the localization of lipofuscin and A2E in the human retina(Ablonczy et al., 2013). However, as already mentioned, lipofuscincontains probably other structures which may play a role in the patho-genesis. Further investigations are necessary to analyze lipofuscin com-pounds helping to understand the role and function of lipofuscin inaging and age-related diseases.

4. Advanced glycation end products and their role in aging

Already in the early twentieth century, Louis-Camille Maillard madethe important discovery that heated amino acids form brown-coloredproducts in the presence of sugars (Maillard, 1912). Although thefirst glycated protein, namely hemoglobin, was identified in 1955 byKunkel and Wallenius (1955), it took almost another 30 years to ob-serve that the browning reaction, which is associated with foods andtheir processing and storage, is linked to the non-enzymatic glycationin the human body (Monnier and Cerami, 1981). Monnier and Cerami

dies revealed that cell exposure to artificial lipofuscin increases apoptosis and impairs vi-including ROS formation due to the incorporation of transition metals and the ability oflipid peroxidation products which are able to cross-link cellularmoleculeswere identified

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postulated that these glycated proteins play an important role in theaging process and age-related pathologies, mainly due to the formationof irreversible, partly cross-linked protein adducts, meanwhile knownas AGEs (Monnier and Cerami, 1981). In the following years, studiesconfirmed that AGEs accumulate during aging and contribute to compli-cations in age-related diseases such as DM (Goh and Cooper, 2008)or neurogenerative diseases including Alzheimer's disease (AD) andParkinson's disease (PD) (Li J. et al., 2012).

4.1. Formation and structural diversity

In general, AGEs are a very heterogenous class of compounds formedin different pathways either exogenously e.g. in food or tobacco smoke(Cerami et al., 1997; Goldberg et al., 2004) or via endogenous mecha-nisms. Themost studied pathway leading to AGE formation over a seriesof reactions is the Maillard reaction. The underlying chemical reactionswere first described by Hodge and summarized in the so-called Hodgescheme (Hodge, 1953). In the initial step of the Maillard reaction, a nu-cleophilic addition between the carbon atom of the carbonyl group of areducing sugar and the nitrogen atom of the amino group of nucleicacids or proteins generates an imine derivate, also called Schiff base.This reaction takes place in only a few hours, however, the so-produced imine is unstable, so that it rearranges to more stable prod-ucts. If the reducing sugar is an aldose, for example D-glucose, theimine spontaneously undergoes Amadori rearrangement to generate1-amino-2-desoxy-2-ketose, which can cyclize to Amadori products.Although these products are more stable, they generate further inter-mediates via oxidative cleavage or retro-aldol fragmentation (Davideket al., 2006; Thornalley et al., 1999). These intermediates, for exampleglyoxal or methylglyoxal, are very reactive and, in addition to theMaillard reaction, the polyol pathway, the autoxidation of glucose andthe lipid peroxidation cause the formation of these reactive dicarbonylcompounds, which finally generate AGEs (Fu et al., 1996; Thornalleyet al., 1999). Thus, oxidative stress is also involved in the formation ofAGEs. Even if oxidation is not essential for AGE formation, many ofthe generated AGEs are a result of the combination of oxidative andglycation reactions, named glycoxidation products (Baynes, 1991).Two common markers for glycoxidation processes are CML andpentosidine. AGE formation can occur under non-oxidative pathways,but if they are formed from ascorbate or glucose oxidative conditionsare required (Baynes and Thorpe, 1999; Dunn et al., 1990).

Beside the endogenous AGE formation, AGEs are formed during theprocessing of foods. Cooking as well as baking can influence the foods

Non-fluorescent

Fluorescent -/ nonfluorescent cross-lin

N -ε ε(carboxymethyl) lysine (CML) N -(carboxyethyl) l

GlucosepaPentosidine

COOH C

CHH C3

H C3

HN HN

R RLYS R LYS

2

LYSHN

N

NH ARG

HO

HO

N

NN

LYS

N+

Fig. 4. Structures and properties of relevant AGEs, mod

color, flavor, taste or texture changing the sensory and nutritional qual-ity of the food. Thereby, the height of the temperature, the incubationtime, and particularly the composition of foods is crucial for the contentof AGEs (Goldberg et al., 2004). High fat foods such as butter, oils or nutscontain the highest concentrations of AGEs. Medium AGE concentra-tions are found in foods with high amounts of protein (cheese, meat,fish, and eggs), while carbohydrate-rich foods are associated with lowAGE levels (Goldberg et al., 2004). Studies, which investigated the influ-ence of dietary AGEs on the plasma AGE level in human subjects, dem-onstrated that there is an increased level after oral administration ofAGE-rich foods (Koschinsky et al., 1997; Vlassara et al., 2002). Animalstudies were able to confirm these findings (Lin et al., 2003; Peppaet al., 2003; Zheng et al., 2002). Even if the exact mechanism of absorp-tion in the intestinal tract is not fully understood yet, it can be assumedthat both, dietary and endogenous formed AGEs, contribute to the AGEpool of the human body.

AGEs include many heterogenous compounds, which show im-mense structural differences and, depending on the structure, possessvarious properties (Fig. 4). It must be considered that due to the com-plex and diverse possibilities of formation, AGEs are probably only par-tially identified and chemically characterized (Schmitt et al., 2005). Thefirst isolated and characterized AGE was CML in 1986 (Ahmed et al.,1986) and, still today, it is one of the best known AGEs. CML neither in-duces protein cross-links nor is fluorescent, but due to the existence of areliable antibody, it is one of the main biomarkers for AGE formation inbiological systems. Another well characterized AGE is pentosidine,which generates protein cross-links and is fluorescent (Sell andMonnier, 1989). Moreover, there are AGEs, which are non-fluorescentcross-linkers (e.g. glucosepane (Biemel et al., 2002)), or showfluorescent properties, but do not induce cross-links between proteins(e.g. argpyrimidine (Shipanova et al., 1997)).

4.2. Effects of AGEs on tissue and cellular level

In the last three decades, research focused on AGEs because the im-portance of these modified compounds in the aging process and age-related diseases became clear. AGE-modified proteins are characterizedby a loss of functional and structural properties. Since the formation ofAGEs is a prolonged process, which takes weeks to months, long-livedproteins, such as extracellular matrix (ECM) proteins (e.g. collagen(Dyer et al., 1992), elastin (Konova et al., 2004)) or lens crystallins(Araki et al., 1992), are frequent targets for modifications. AGE forma-tion on collagen induces cross-links impairing enzymatic digestion

Fluorescentkers

ysine (CEL) Argpyrimidine

ne

OOH

H N COOH

CH

CH

2

H C2

H C3

2

CH

NH

HO

N N

2

CH3

R

NH ARG

ified according to Ahmed and Thornalley (2007).

Fig. 5. Factors associated with AGE accumulation, modified according to Tessier (2010).

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(Nowotny and Grune, 2013) that may contribute to vascular and tissuestiffening (Sell and Monnier, 2012; Verzijl et al., 2002). Moreover, AGEformation on ECM proteins leads to impaired matrix–matrix interac-tions. For example, AGE-modified laminin or vitronectin decreasesbinding to collagen or heparan sulfate proteoglycan (Charonis et al.,1990; Hammes et al., 1996). Matrix–cell interactions may also be dis-turbed by glycation, which influence cell adhesion and proliferation(McCarthy et al., 2001; Pozzi et al., 2009). It was recently shown thatglycated ECM proteins inhibit not only cell adhesion, but alsomigrationof Jurkat T-cells accompanied by decreased actin polymerization(Haucke et al., 2014).

In addition to the direct damage to protein function and structure,the toxic potential of AGEs is based on numerous cellular responses in-duced byAGE binding to cell surface receptors. Themajor and best stud-ied receptor for AGEs is RAGE (receptor for advanced glycation endproducts) (Schmidt et al., 1996), a multi-ligand receptor of the immu-noglobulin superfamily, which is also able to bind agonists like S100proteins (Hofmann et al., 1999), amyloid-β peptide (Yan et al., 1996)or amphoterin (Hori et al., 1995). The receptor is expressed on the sur-face of different cell types, such as smoothmuscle cells, endothelial cells,neurons, microglia and leukocytes including T-cells, monocytes/macro-phages, granulocytes and dendritic cells (Kierdorf and Fritz, 2013). De-pending on the cell type ligand binding to RAGE upregulates differentsignaling pathways, which induce the activation of various transcrip-tional factors. AGE-dependent RAGE signaling involves, for example, ac-tivation of extracellular-signal-regulated kinase 1/2, p38, rho-GTPasesand janus kinase (Grimm et al., 2012a; Hirose et al., 2010; Li et al.,2004; Tanikawa et al., 2009). It has been shown that downstream acti-vation of nuclear factor kappa B (NFκB) is a main target of RAGE(Bierhaus et al., 2001) inducing secretion of proinflammatory andprothromboticmolecules, growth factors and the expression of RAGE it-self (Berbaum et al., 2008; Goldin et al., 2006; Li and Schmidt, 1997).This in turn triggers an inflammatory response and converts a short-term proinflammatory situation into a chronic state of inflammation.

AGEs and some of their precursors are proposed to be one source ofoxidative stress (Yan et al., 1994). AGE binding to RAGE induces theNADPH oxidase system (Guimaraes et al., 2010). This causes anenhanced generation of ROS, so that AGE formation, which is increasedunder oxidative stress, is induced creating another positive feed-back cycle. Beside RAGE, further receptors of AGEs have been identified(Ott et al., 2014). These include oligosaccharyl transferase-48 (AGE-R1),80 K-H (AGE-R2), and galectin-3 (AGE-R3), which presumably form theAGE receptor complex (Li et al., 1996; Radoff et al., 1990; Vlassara et al.,1995; Yang et al., 1991). AGE-R1 and AGE-R3 are believed to function asclearance receptors, whereas AGE-R2 is assigned to the inflammatorytype of receptors, such as RAGE (Vlassara and Palace, 2002). Further-more, different scavenger receptors, among them CD36 (Kuniyasuet al., 2003; Ohgami et al., 2001a), SR-A (Araki et al., 1995) or SR-BI(Ohgami et al., 2001b), are described tomediate AGE uptake by endocy-tosis after receptor binding. Endocytotic uptake of AGEs is a protectivecellular mechanism necessary for the degradation of AGEs. However, ithas been recently shown that scavenger receptors are not only involvedin AGE uptake, but may also play a role in cell signaling. Zhu et al. dem-onstrated the involvement of CD36 in signaling and hyperreactivity ofplatelets by inducing JNK2 phosphorylation (Zhu et al., 2012).

4.3. AGE accumulation in aging

During aging, proteins are continuously exposed to reactivecompounds including glycating agents such as reducing sugars or theeven more reactive carbonyl compounds glyoxal, methylglyoxal and3-desoxyosone causing AGE formation. An age-related accumulationhas been shown in almost every tissue in the body. In particular,Albon et al. demonstrated in the aging human lamina cribrosa thatthere is an accumulation of pentosidine (Albon et al., 1995). Further-more, an accumulation of AGEs e.g. in rodent and human skin (Dyer

et al., 1992; Nowotny and Grune, 2013), in lens proteins (Dunn et al.,1989), in renal arteries (Schleicher et al., 1997) as well as in the inter-vertebral disk (Schleicher et al., 1997) was observed. The accumulationof AGE-modified proteins is dependent on many different factors(Fig. 5) (Tessier, 2010). The turnover of proteins plays an importantrole. There are various studies, which pointed out, that the concentra-tion of AGEs in tissue correlates with the half-life of proteins. For exam-ple, cartridge collagen which is associated with a very long lifetime(half-life of 117 years) was found to have much higher levels of AGEscompared to collagen of the skin with a half-life of 15 years (Verzijlet al., 2000). The other way around, intracellular proteins are primarilycharacterized by a fast protein turnover which indirectly protects theseproteins from molecular damage. However, there is evidence that theprotein turnover in general decreases with age. Both, protein synthesisas well as protein degradation, decline during the aging process, sothat proteins serve as targets for posttranslational modification longer(Rattan, 1996). This once more leads to the accumulation of damagedproteins including AGEs. A second major factor which influences thebody's AGE pool is the type or rather the reactivity of the glycatingagent. It was found that the reactivity of monosaccharides depends onthe extent to which they are present in the open chain form (Bunnand Higgins, 1981). Glucose, the most common metabolic sugar inhumans, is the least reactive one due to the fact that under physiologicalcircumstances it exists to over 90% in a ring structure. This makes glu-cose highly stable and limits non-enzymatic glycation of proteins. Com-pared to glucose, D-fructose and D-ribose are more reactive sugarswhich cause an enhanced formation of AGEs (Bunn and Higgins,1981). In addition to monosaccharides, glycolytic intermediates andα-dicarbonyl compounds are able to generate AGEs and are substantial-ly more reactive compared to glucose (Rabbani and Thornalley, 2008).The grade of AGE accumulation also depends on the extent of exposureof proteins to the glycating agent. Serum proteins and proteins of theECM are directly affected by glycating compounds in the blood, whileglycation of intracellular proteins is dependent on their cellular uptake.Inmuscle cell, for example, glucose uptake is regulated by glucose trans-porters. Although muscle proteins have a shorter protein turnover andintracellular enzymes may degrade modified proteins, the lower intra-cellular glucose concentration may be an important point why lessAGE formation in muscle proteins compared to skin collagen was ob-served by Alt et al. (2004). Another factor associated with AGE accumu-lation is oxidative stress. As mentioned above, oxidative stress isimplicated in AGE formation and AGE formation itself induces oxidativestress.

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Beside an increased formation of AGEs, the accumulation of glycatedproteins is dependent on their degradation and renal clearance as wellas on the elimination of the glycating agents. Due to cross-links whichblock enzyme binding sites, AGE-proteins are more resistant to enzy-matic degradation (Nowotny and Grune, 2013). In this way, proteinturnover is prolonged anddamagedproteins can become further targetsfor protein modifications causing finally protein aggregates. It has beenshown that AGEs can be degraded at least partially by the endosomal–lysosomal system after cellular uptake. Here, cathepsin D and L wereidentified as important detoxification enzymes of AGE-modified BSAin RAW 264.7 cells (Grimm et al., 2012b). Even though an age-relatedincrease of cathepsin D activity was observed, for example in the brainof rats (Nakanishi et al., 1994), studies demonstrated that lysosomalproteases are inhibited by glycating agents and AGE-modified proteins,thus, affecting AGE degradation and inducing their accumulation(Stolzing et al., 2006; Zeng et al., 2006). It is assumed that after digestionthe degradation products of AGEs are released in the extracellular spaceand circulate in the bloodstream until they are excreted by the kidney.Thus, the kidney is continuously exposed to AGEs and one preferentialtarget of AGE-mediateddamage in aging. AGEs induce thickening of glo-merular basement membranes and mesangial matrix expansion(Vlassara et al., 1994) and in turn affect renal function and AGE levels.That there is a correlation between renal clearance and the body's AGEpool was confirmed in studies showing that patients with limitedrenal clearance have increased serum AGE levels (Miyata et al., 1996;Odetti et al., 1992). In conclusion, there aremany factors which contrib-ute to AGE accumulation whereas the protein turnover as well as theconcentration and reactivity of glycating agents are probably the mostimportant ones. However, it must be considered that all factors de-scribed to cause AGE accumulation should not be regarded separatelysince they can influence the accumulation in a synergistic or antagonis-tic way.

4.4. AGEs in age-related diseases and diabetes complications

AGEs do not only accumulate during the aging process, they alsohave a significant role in the development of age-related pathologies.For example, AGEs are involved in the genesis of neurodegenerativediseases including AD and PD (Li J. et al., 2012; Srikanth et al., 2011).AD and PD are the most common neurodegenerative diseases in the el-derly inwhich protein aggregation is observed. In patientswith AD, AGEdeposits and associated protein cross-linking have been shown inextracellular plaques as well as intracellular neurofibrillary tangles(NFTs) (Li et al., 1994; Smith et al., 1994). Senile plaques are primarilycomposed of β-amyloid and its polymerizationwas found to be acceler-ated by AGE-mediated cross-linking (Munch et al., 1997). Moreover,AGEs lead to an increased level of β-amyloid per se due to the upregu-lation of the amyloid precursor protein. Ko et al. were able to demon-strate that especially the induction of ROS is an important mechanismsince upregulation of amyloid precursor protein by AGEs was blockedwhen using the antioxidant N-acetyl-L-cysteine (Ko et al., 2010). In ad-dition to the amyloid plaque formation, AGEs are proposed to partici-pate in the development of NFTs. In particular, it was shown that thereaction of tau with AGE precursors such as methylglyoxal contributesto NFT formation (Kuhla et al., 2007). Tau protein is a microtubules-associated protein and is responsible for the stability of the cytoskele-ton. In its hyperphosphorylated form it is the main componentof NFTs, which are composed of paired helical filaments (PHF).Hyperphosphorylation of tau inhibits the binding step to microtubulesand may induce self-assembly of tau protein into filaments. Accordingto Li et al., methylglyoxal induces hyperphosphorylation of tau in neuro-blastoma cells due to the formation of AGEs (Li X.H. et al., 2012a). AGEtreatment of primary neurons, neuroblastoma cells and rats confirmedthe finding that AGEs mediate tau hyperphosphorylation (Li X.H. et al.,2012b). Furthermore, they showed that this is a result of the activationof glycogen synthase kinase-3β, a protein kinase which is able to

phosphorylate tau, via RAGE (Li X.H. et al., 2012b). In addition to tauhyperphosphorylation, tau was found to be the direct target of AGE for-mation (Ledesma et al., 1995). Glycation of PHF-tau probably stabilizesthese filaments and, therefore, contributes to NFT formation in AD.Moreover, glycated tau induces oxidative stress which may contributeto the toxic potential of NFTs (Ledesma et al., 1995). Protein aggregateswhich are associated with PD are the so-called Lewy bodies. Lewy bod-ies contain different neurofilament proteins among them α-synuclein.In vitro studies showed that AGEs induce α-synuclein aggregation(Lee et al., 2009; Padmaraju et al., 2011). Moreover, ribose-modifiedα-synuclein caused the generation of aggregates whichwere highly cy-totoxic and promote cellular oxidative stress (Chen et al., 2010). It is notyet established, but more and more studies indicate evidence that pa-tients with DM have an elevated risk of developing neurodegenerativediseases (Arvanitakis et al., 2004; Valente et al., 2010). Resulting fromelevated serum glucose and oxidative stress, patients with DM have in-creased plasma AGE levels which accelerate the describedmechanisms.In addition, DM is associated with many other complications and AGEsare supposed to have a causal function in their pathogenesis. Since hy-perglycemia correlates with diabetic complications, it is evident thatalso AGEs are one of the causal factors leading to complications in DM.It was recently shown that AGEs may even promote the developmentof DM. Zhao et al. observed that AGEs impair the function of murinepancreatic β-cells by inhibiting ATP production and the activity of cyto-chrome c (Zhao et al., 2009). This resulted in higher glucose levels due todecreased insulin secretionmediated by elevated •NO formation and ex-pression of inducible nitric oxide synthase. These findings are in agree-ment with a study of Coughlan et al., who demonstrated that thedeficient insulin secretion in β-cells and isolated mice islets exposedto AGEs is due to impaired mitochondrial functions (Coughlan et al.,2011). The same effects were shown, when rats were fed with AGE-enriched food (Coughlan et al., 2011).

In recent years, the involvement of AGEs in diabetes complicationswas investigated to a large extent, in vitro and in vivo. Diabetic compli-cations primarily affect the vascular system and can be divided into mi-croangiopathies and macroangiopathies. Microangiopathies includediseases which are due to damage on small blood vessels in eyes(retinopathies), kidneys (nephropathies) or the nervous system(neuropathies). Damage to the arteries cause macroangiopathies suchas cardiovascular or cerebrovascular diseaseswhich can lead tomyocar-dial infarction or stroke due to vascular occlusion. Vascular stiffening aswell as endothelial dysfunction are important pathophysiological pro-cesses associated with diabetic long-term complications and vasculardamage in general. Based on experimental studies, it is accepted thatAGEs contribute at least partially to structural and functional changesinvolved in these mechanisms (Fig. 6). AGE deposits in arterial wallsare accelerated in DM and as described above, AGE cross-linking ofECM proteins in the blood vessel wall plays an important role in vascu-lar stiffening (Sell and Monnier, 2012). That there is a correlation be-tween the stiffness of the aorta and cross-linking compounds derivedfrom glycation processes was demonstrated in a study where increasedAGE concentrations in the aorta from diabetic compared to the controlsubjects were found (Sims et al., 1996). Furthermore, the causal roleof AGEs in arterial stiffness was indicated in studies showing that treat-ment of diabetic rats with substances which either inhibit AGE forma-tion (aminoguanidine) or break AGE crosslinks (ALT-711) reverseddiabetes-induced arterial stiffness (Brownlee et al., 1986; Huijbertset al., 1993; Wolffenbuttel et al., 1998). AGE-induced ROS formationmay also contribute to arterial stiffening due to the formation of lipidperoxide which may cross-link proteins in the vessel wall.

It has been suggested that the increased AGE formation in diabeticpatients is one factor causing endothelial dysfunction. The vascular en-dothelium is exposed to circulating AGEs in the blood stream as wellas AGEs localized in the matrix of subendothelial layers. Endothelialcells express receptors for AGEs mediating AGE uptake, transcytosisand cellular signal transduction. Especially AGE binding to RAGE

Fig. 6. AGE-mediated mechanisms inducing vascular pathologies, modified according to Goldin et al. (2006). Vascular AGE accumulation has been found in almost every organ systemincluding the eye, brain, kidney and cardiovascular system. AGEs may contribute to vascular damage and angiopathies due to two major events: vascular stiffening and endothelial dys-function. AGE formation occurs on components of the ECM of the vessel wall such as collagen and elastin. In addition to protein cross-linking by AGEs, AGE-mediate ROS formation causesthe formation of cross-linking lipid peroxidation products, increasing the stiffness of blood vessels. AGEs primary contribute to endothelial dysfunction due to interactions with AGE re-ceptors. By activating NFκB via RAGE and increased ROS formation, the transcription of adhesion molecules, coagulant factors, growth factors, vasoconstrictors etc. is induced. Thus,thrombogenesis, cell proliferation, adhesion as well as migration are stimulated by AGEs. •NO quenching and RAGE-mediated reduced expression/activity of eNOS leads to decreased•NO concentrations by AGEs which in turn cause vasoconstriction and cell proliferation.

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induces several cellular reactions. AGE–RAGE interaction increases ROSformation, activates NFκB and, therefore, elevates the transcription ofNFκB-related genes including adhesionsmolecules, which induce adhe-sion of circulating immune cells (Schmidt et al., 1995). AGEs induceprocoagulant tissue factor and lead to the reduction of thrombomodulinpromoting thrombogenicity (Bierhaus et al., 1997; Esposito et al., 1989).It has been shown that AGE-RAGE interaction disrupts the endothelialbarrier function by increasing the permeability of the endothelium(Wautier et al., 1996). It has also been observed that AGEs notonly quench •NO (Bucala et al., 1991), but also decrease •NO synthesisby reducing the expression/activity of endothelial •NO synthase(Chakravarthy et al., 1998; Xuet al., 2003). Decreased •NOconcentrationcauses reduced vasodilation and induces proliferation of vascularsmooth muscle (David et al., 2008; Satoh et al., 1997) which may resultin vascular thickening associated with hypertension, reduced elasticityand endothelial dysfunction. In parallel, vasoconstriction is amplifiedby AGE-mediated RAGE signaling inducing the expression of the vaso-constrictor endothelin-1 (Quehenberger et al., 2000). Since endothelialdysfunction is involved in early stages of atherosclerosis, it is compre-hensible that atherosclerosis is accelerated in DM and, therefore, be-longs to the most frequent diabetic complications (Basta et al., 2004).AGE deposits have been localized in athereosclerotic lesions (Kumeet al., 1995) and beside the involvement of AGEs in mechanism leadingto endothelial dysfunction, AGEs are also important in further processesof the pathogenesis of atherosclerosis. For example, AGEs accelerate thedevelopment of atherosclerotic plaques by glycating low density lipo-proteins (LDL). After the discovery that an AGE-modified form of LDLcirculates in the plasma of diabetics Bucala et al. (1993), Bucala et al.were able to show that AGE modification occurs on apoprotein B andleads to structural changes which impair LDL clearance by blockingthe receptor-mediated uptake (Bucala et al., 1995). However, uptakeof AGE-LDL in macrophages is elevated compared to unmodified LDL(Klein et al., 1995). AGE incubation of monocyte-derived macrophagescontributes to increased gene expression of CD36, a receptor which is

also involved in the uptake of oxidized LDL (Iwashima et al., 2000). Asa result, transformation into foam cells is accelerated. In addition, solu-ble AGEs such as CML have been described to activate macrophages viaRAGE, whereas AGEs associated with the basement membrane inhibittheir migration, allowing them to adhere and to become activated bycirculating AGEs (Goldin et al., 2006).

Besides atherosclerosis, AGE-associated vascular dysfunction is in-volved in diabetic retinopathy, neuropathy and nephropathy. To gain adeeper insight into the role of AGEs in theses complications see reviews(Forbes et al., 2003; Singh et al., 2001; Wada and Yagihashi, 2005).

Taken together, pathogenic effects of AGEs are related to a widerange of age-related diseases and diabetic complications. Most in vivostudies focused on the pathological mechanisms of AGEs in diabetesdue to the accelerated AGE formation which cause tissue damage in ashorter period. However, AGEs also accumulate in normal aged individ-uals and, therefore, would be expected to induce damage by the samemechanisms as observed in diabetes. Thereby, AGEs directly lead tochanges at cellular and tissue level by affecting the protein structureand function or by a series of receptor-mediated cellular reactions.

5. Conclusion

Understanding the aging process and its causal molecular mecha-nism is the goal of research all over the world. Numerous of differentaging theories have been described in the past, clearly demonstratingthe complexity of this biological phenomenon. Most of the theoriesagree that accumulation of cellular and tissue damage is responsiblefor the decline of biological functions in aging, however, their underly-ing mechanisms are controversially discussed. Protein modificationsare primary events leading to cellular or tissue damage. In particular, ox-idative and glycative modifications have been independently proposedas the primary cause of the aging process. Protein oxidation proceedsunder conditions of oxidative stress when the formation of RS over-comes mechanisms of antioxidative defense systems leading to the

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generation and aggregation of oxidativelymodified proteins. Proteins thatbecome glycated by reducing sugars cause the formation of AGEs, whichtend to accumulate during aging and mediate various cellular reactions.However, oxidation and glycation were shown to interact synergistically.Oxidative pathways are involved in AGE formation. Moreover, AGEsthemselves induce the formation of RS accelerating their own formationbut also other oxidative modifications. In addition, there is evidence thatoxidation and glycationmay be involved in lipofuscinogenesis. Therefore,Kristal et al. proposed already 20 years ago that the Free Radical andGlycation Theory of Aging should be regarded as a combined ‘Free Radi-cal–Glycation’ Theory of Aging (Kristal and Yu, 1992). Recent studies in-vestigating the role of chemical modifications in aging support thehypothesis that age-relatedpathologies are due todamageby free radicalsas well as glycative modifications.

Conflict of interest

There are no conflicts of interests for all authors.

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