aging as an event of proteostasis...

Aging as an Event of Proteostasis Collapse

Rebecca C. Taylor and Andrew Dillin

Howard Hughes Medical Institute, The Glenn Center for Aging Research, The Salk Institute for BiologicalStudies, La Jolla, California 92037

Correspondence: [email protected]

Aging cells accumulate damaged and misfolded proteins through a functional decline intheir protein homeostasis (proteostasis) machinery, leading to reduced cellular viabilityand the development of protein misfolding diseases such as Alzheimer’s and Huntington’s.Metabolic signaling pathways that regulate the aging process, mediated by insulin/IGF-1 sig-naling, dietary restriction, and reduced mitochondrial function, can modulate the proteosta-sis machinery in many ways to maintain ayouthful proteome for longer and prevent the onsetof age-associated diseases. These mechanisms therefore represent potential therapeutictargets in the prevention and treatment of such pathologies.

PATHWAYS THAT INFLUENCETHE AGING PROCESS

Aging was once regarded as a stochastic, pro-gressive decline. However, the discovery of

metabolic pathways able to modulate the agingprocess has challenged this view. Three mainsignaling pathways have been identified thatcan influence the rate of aging (Fig. 1) (Wolffand Dillin 2006). The first, dietary restriction(DR) has been shown to extend lifespan in mul-tiple species (Mair and Dillin 2008). The reduc-tion of dietary intake below unlimited or “adlibitum” levels causes an increase in lifespan,to an optimum point of consumption, typicallyaround 60% of ad libitum food intake. How-ever, although this extension of lifespan by DRhas been observed for several decades, its mech-anistic basis remains unclear. Evidence suggeststhat the amino-acid-sensing serine/threoninekinase mTOR, and the energy status-dependent

kinase AMPK, are involved in DR signaling(Kapahi and Zid 2004; Kahn et al. 2005). Inaddition, recent studies in Caenorhabditis ele-gans have suggested that two transcription fac-tors, PHA-4 and SKN-1, are essential for thisprocess, and act specifically in lifespan exten-sion by DR (Panowski et al. 2007; Bishop andGuarente 2007).

Another pathway mediating lifespan exten-sion, the insulin/IGF-1-like signaling pathway(IIS), has been remarkably well characterizedin recent years. Reduced IIS activity extendslifespan in both invertebrate and vertebratespecies (Bartke 2008). The role of the IIS systemin aging was first identified in C. elegans, andit has been best characterized in that species(Panowski and Dillin, 2008). Following ligandbinding, the C. elegans IIS receptor DAF-2recruits the insulin receptor substrate homologIST-1, and the PI3K AGE-1 (Wolkow and Ruv-kun 2002; Morris et al. 1996). Production of

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PI3 through AGE-1 activity, opposed by thephosphatase DAF-18, activates AKT kinases,which phosphorylate the transcription factorDAF-16 (Hertweck et al. 2004; Paradis and Ruv-kun 1998; Ogg and Ruvkun 1998; Hendersonand Johnson 2001; Lee et al. 2001; Lin et al.2001). Phosphorylation anchors DAF-16 inthe cytosol through interaction with 14-3-3proteins, whereas reduced IIS activity allows itto enter the nucleus, activating a diverse tran-scriptional profile that promotes extended life-span (Lin et al. 1997; Ogg et al. 1997; Cahillet al. 2001). DAF-16 is also regulated by mech-anisms other than localization, and requirescofactors such as SMK-1, HCF-1, and SIR-2for extension of longevity (Wolff et al. 2006;Tissenbaum and Guarente 2001). At least oneother transcription factor, the heat shock factorHSF-1, is also required for the IIS pathway to

extend lifespan, although how the pathway reg-ulates this transcription factor is unclear (Hsuet al. 2003). In addition, in turn, the IIS pathwayalso regulates localization of the SKN-1 tran-scription factor that is essential for the long life-span of DR animals (Tullet et al. 2008).

The third pathway enabling extension oflifespan acts through reduction in the activityof the mitochondrial electron transport chain(ETC). This was first shown, again, in C. elegans,in which reduced expression of several mito-chondrial genes including components of ETCcomplexes I, III, IV, and V by RNAi is sufficientto extend lifespan (Feng et al. 2001; Dillin et al.2002b; Lee et al. 2003). The role of mitochon-dria in lifespan has since been shown in Droso-phila and rodents (Liu et al. 2005; Copelandet al. 2009). However, the mechanisms and sig-naling pathways involved remain unknown.

P

PP

P

1ST-1PP

SKN-1

SKN-1

PHA-4Autophagy

Translation

Proteostasis& Longevity

4EBPS6K

TOR complex 1

RHEB

TSC1 TSC2

AMPK

Reducedmitochondrialfunction

High AMP:ATP

Dietary restrictionInsulin/IGF-like ligand

DAF-2

AKT

AGE-1

P13

DAF-18

P12

HSF-1

HSF-1

SMK-1 SIR-2

DAF-16

DAF-16

Figure 1. Pathways that regulate aging. The insulin-signaling pathway and dietary restriction pathway inducelongevity through mechanisms that are at least partially understood. Reduced mitochondrial function alsoincreases lifespan, but the signaling components are not clear.

R.C. Taylor and A. Dillin

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Several lines of evidence suggest that theselifespan extension pathways are separate. Forone, genetic epistasis experiments show thatcombining different lifespan extension path-ways leads to additive increases in lifespan (Dil-lin et al. 2002b; Lakowski and Hekimi 1998).Additionally, temporal requirements for thepathways are different, reduction of IIS or DRhas important lifespan extension effects inadulthood, whereas the mitochondrial pathwayacts during development (Dillin et al. 2002a;Dillin et al. 2002b; Mair et al. 2003), and thecore components act separately. DAF-16 is dis-pensable for DR-induced lifespan extension,whereas PHA-4 plays a role specifically in DRand not the IIS and ETC pathways (Lakowskiand Hekimi 1998; Panowski et al. 2007).

THE IMPORTANCE OF PROTEOSTASISIN AGING

Although these lifespan extension pathways actindependently, it is logical to hypothesize thattheir underlying downstream mechanismsmight be the same. The phenotypes of long-lived animals overlap substantially, includingincreased stress resistance, altered metabolism,and delayed reproduction and development(Hekimi and Guarente 2003). One possibilityis that a major downstream function of all theseaging pathways is to change the way that the cel-lular proteome is maintained. Cellular proteinsare challenged throughout life by a multitude offactors that cause protein misfolding and aggre-gation, including translational error, the pres-ence of polymorphisms, and stresses that leadto covalent modifications such as oxidation.Misfolded proteins can have a range of negativeconsequences for the cell. Mutated and destabi-lized proteins with hydrophobic regions inap-propriately exposed tend to aggregate with thehydrophobic regions of other proteins, andthese protein aggregates can be directly cyto-toxic, through disruption of membranes andinteraction with cellular components (Chitiet al. 2003; Stefani and Dobson 2003). Theymay also create increased demands on the cell’sprotein homeostasis (proteostasis) machinery,titrating away components of this network and

consequently leading to further misfolding ofother proteins. The consequences of even alow level of misfolded protein in the cell canbe catastrophic—the expression of a metastableor misfolded protein in organismal models ofproteotoxic stress has been shown to destabilizeother, unrelated proteins in the proteome(Gidalevitz et al. 2006; Gidalevitz et al. 2009).

The importance of preventing protein mis-folding has been shown in evolutionary termsin a model of molecular evolution, studyingsynonymous codon substitutions (Drummondand Wilke 2008). Translational inaccuracy is amajor source of mutation, missense errors intranslation are believed to occur once every103–104 codons, suggesting that around 18%of translated proteins will contain an error,and some codons are translated more accuratelythan others. Mutation in turn leads to higherlevels of protein misfolding and aggregation.The model found that the avoidance of transla-tional error through the selection of the mostaccurately translated codons, driven by the neg-ative consequences of misfolded mutant pro-teins, was sufficient to account for observedrates of codon evolution. Consistent with thishypothesis, the most conserved proteins arenot those most essential for viability, but theones expressed at the highest rate, and thereforethe most damaging if mistranslated. Proteinsexpressed in long-lived postmitotic cells, inwhich the formation of misfolded proteinshas the most potential to do harm, were alsoextremely slow evolving. The fact that the avoid-ance of protein misfolding seems to have beensuch a driving force in evolution indicates inturn the danger that protein misfolding pre-sents to the cell.

Cells possess a complex network of mech-anisms designed to prevent and eliminate pro-tein misfolding, termed the proteostasis net-work, encompassing regulators of translation,protein folding, trafficking and secretion, andprotein degradation (Fig. 2) (Balch et al. 2008).However, despite the presence of these mecha-nisms, unwanted protein products accumulatewith age. Older cells contain more proteinsbearing oxidative modifications such as carbon-ylation, oxidized methionine, and glycation, as

Proteostasis Decline and Aging

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well as accumulating cross-linked and aggre-gated proteins, and less catalytically active en-zyme populations (Berlett and Stadtman 1997;Soskic et al. 2008). This accumulation of mis-folded proteins seen in elderly organisms seemshighly likely to contribute to tissue decline andloss of viability.

The implication is that a loss of function ofthe proteostasis machinery occurs with age, andrecent studies have supported this hypothesis.The causes of age-associated decline in proteo-stasis capacity are still largely unclear, but thereis mounting evidence that aging pathways candirectly modulate elements of the proteostasismachinery to extend healthy lifespan (Fig. 2).Given this connection between proteome main-tenance and the aging process, understandinghow pathways that can regulate aging affect the

proteostasis machinery is necessary in gainingan understanding of how to increase healthylifespan. It is this intersection between pathwaysthat modulate aging, and protein homeostasis,that will be discussed in the rest of this review.

RATES OF TRANSLATION

Modulating rates of protein synthesis is onemajor way in which aging pathways can alterproteostasis. Control of translation rate is gen-erally achieved at two stages of translation ini-tiation—the recruitment of the 40S ribosomalsubunit, and the loading of this subunit withthe initiator tRNA (Gebauer and Hentze 2004).Recruitment of the 40S subunit is controlledby the eIF4E translation initiation factor, whichis inhibited by the 4EBP binding proteins

Proteinmisfolding

Protein synthesis

Proteinunfolding

ProteinfoldingP

P

S6K

Proteintrafficking

Proteintrafficking

Proteindegradation

Proteinaggregation

Proteindisaggregation

Proteasomes

Chaperones

Chaperones

Initiationcomplex

Autophagy

Proteintrafficking

DAF-16?

DAF-16?

DAF-16HSF-1

mTOR

HSF-1

mTOR

Proteindamage

Figure 2. The life cycle of a protein. After synthesis, proteins require correct folding and trafficking to the correctcellular location. Components of the cell’s proteostasis machinery mediate these functions, as well as detoxifyingdamaged and misfolded proteins. Pathways that regulate aging can modulate many aspects of proteostasis,through transcription factors such as DAF-16 and HSF-1, and effector molecules such as the kinase mTOR.





(Richter and Sonenberg 2005). tRNA loading isregulated by the eIF2a subunit, which controlsthe recycling of eIF2 (Gebauer and Hentze2004).

The IIS and DR pathways both affect trans-lation rates. Protein synthesis is reduced underDR, possibly through reduced activation ofthe mTOR kinase (Zid et al. 2009; Wullschlegeret al. 2006). mTOR regulates translation byphosphorylation of its targets, 4EBP-1, and theribosomal kinase S6K. Phosphorylation of4EBP-1 dissociates it from the translation ini-tiation complex, whereas phosphorylation ofS6K activates this kinase, allowing it to phos-phorylate translational targets that includeribosomal protein S6 and the elongation factor2 kinase. Both events promote translation, andconversely, reduction of mTOR activity by DRdecreases translation initiation. In Drosophila,d4EBP-1 is required for lifespan extensionunder DR, and is under the transcriptional con-trol of the FOXO transcription factor (Zid et al.2009; Puig et al. 2003). Insulin can regulate TORactivity through modifying the activity of theupstream TSC1/TSC2 regulatory complex andphosphorylation of S6K, and reduced Insulin/IGF-1 signaling in long-lived Snell and Amesdwarf mice leads to reduced translation (Man-ning 2004; Hsieh and Papaconstantinou 2004;Sharp and Bartke 2005).

Indeed, reducing translation rates alone issufficient to extend lifespan. Reducing TORfunction directly increases longevity in multiplespecies, as does reduced S6K activity (Vellaiet al. 2003; Kapahi et al. 2004; Kaeberlein et al.2005; Jia et al. 2004; Hansen et al. 2007; Selmanet al. 2009). In yeast, deletion or inhibition ofribosomal protein levels increases replicativelifespan (Chiocchetti et al. 2007). In addition,in C. elegans, an RNAi screen for longevity-enhancing genes identified many translationregulators (Curran and Ruvkun, 2007). Addi-tionally, reduction of the worm homologs oftranslation initiation factors eIF4E (ife-2),eIF-4G (ifg-1), eIF-2B (iftb-1), and a range ofribosomal proteins, increases lifespan, whereasifg-1 is reduced in long-lived dauers (Hansenet al. 2007; Pan et al. 2007; Syntichaki et al.2007). Initiation factor knockdown may be

dependent on DAF-16 to increase lifespan,whereas ribosomal proteins and S6K are inde-pendent of DAF-16, suggesting a complicatedrelationship between IIS and the control oftranslation. In addition, lifespan extension bytranslational inhibition and DR or mTOR re-duction are additive, suggesting that these sig-naling pathways also invoke other mechanismsto extend lifespan (Hansen et al. 2007).

It seems that reduction in translation is animportant mechanism in the extension of life-span. What therefore underlies this mechanisticimportance? One explanation might be that re-duced protein synthesis reduces the load on theremainder of the proteostasis machinery, allow-ing more efficient protein folding and degrada-tion, for example, and consequently reducingthe load of misfolded and damaged proteins.This is substantiated by the observation thatreducing translation rates renders cells tolerantto elevated temperature, a stress that causes pro-tein misfolding (Hansen et al. 2007).

Reducing translation also causes preferen-tial translation of certain mRNAs. DR in fliesallows preferential translation of mitochondrialgenes, including components of complexes I andIV, dependent on 4EBP1 and therefore reducedtranslation. This altered translational profileenhances mitochondrial activity and facilitateslifespan extension (Zid et al. 2009). In yeast,deletion of 60S ribosomal subunits increaseslifespan through the preferential translationof the GCN4 transcription factor, inducing atranscriptional profile necessary for full lifespanextension by DR (Steffen et al. 2008). And fi-nally, recent work suggests that reduced IIS mayincrease intrinsic thermotolerance and lifespanthrough preferential translation of specific tran-scripts (McColl et al. 2010).

PROTEIN FOLDING

Once proteins have been synthesized, the ma-jority do not fold spontaneously into the appro-priate conformation. They require assistance,provided in the form of molecular chaperones,and this assisted protein folding is crucial tomaintaining protein homeostasis (Morimoto2008). The binding of chaperones to nascent





polypeptides both prevents folding into inap-propriate forms, and facilitates the assembly ofthe correct structure. This assembly involvesthe stabilization of folding-competent inter-mediate states, and can also involve active ATP-driven folding into the native form, accom-plished by the HSP70 family of chaperones(Buckau et al. 2006). In the case of proteinsthat have formed unwanted and potentiallytoxic aggregates, some chaperones, such asHSP104, are able to disassemble these aggre-gates into intermediate forms that can then beassisted in refolding to the native state. On theother hand, at high concentrations of aggrega-tive proteins, HSP104, TRiC, and potentiallyother chaperones, can actively aggregate thesetoxic species into less toxic types of aggregates(Behrends et al. 2006; Shorter and Lindquist2004).

Chaperones represent many of the tran-scriptional targets of the C. elegans DAF-16 IIStranscription factor. Heat shock proteins, par-ticularly of the HSP-16 and HSP-12 families,are prominently up-regulated by reduction ofdaf-2 signaling as determined by microarray,SAGE, CHIP, and mass spectrometry analysis(Murphy et al. 2003; McElwhee et al. 2003;Halaschek-Wiener et al. 2005; Oh et al. 2006;Dong et al. 2007). Heat shock proteins are infact required for full lifespan extension indaf-2 mutants, and over-expression of HSP-16is sufficient to extend lifespan in both wildtype and long-lived mutant worms (Murphyet al. 2003; Morley and Morimoto 2004; Walkerand Lithgow 2003). Expression of small heatshock proteins is also sufficient to extend life-span in Drosophila (Morrow et al. 2004). Thissuggests that the ability to correctly fold pro-teins is important in maintaining youthful cells,and is a target of lifespan extension pathways.

STRESS RESPONSES

The level of chaperones in a cell is believed tobe finely tuned to that cells individual proteinfolding requirements. Introduction of a new,folding-sensitive protein to cells can result in asubsequent destabilization and decreased fold-ing of the rest of the proteome, suggesting that

normal chaperone capacity can be easily ex-ceeded by increases in demand (Gidalevitzet al. 2006; Gidalevitz et al. 2009). Many stressesperturb protein folding, necessitating a dra-matic up-regulation of chaperones and otherproteostasis components if cells are to survivethis challenge to their proteome. The responsesto different cellular stresses are coordinated byupstream transcription factors that respond todistinct stimuli, and several of these stress re-sponses have been implicated in aging and theextension of longevity (Morimoto 2008).

THE HEAT SHOCK RESPONSE

The heat shock response coordinates the rapidinduction of HSPs in response to thermal stress,and is controlled by the HSF transcriptionfactor family. Mammals possess HSFs 1, 2, and4, with HSF1 acting in a ubiquitous stress-inducible manner, whereas Drosophila, C. ele-gans, and yeast possess only HSF1 orthologs(Pirkkala et al. 2001; Anckar and Sistonen2007; McMillan et al. 1998).

In C. elegans the HSF-1 transcription factoris required for lifespan extension by both the IISand DR pathways, whereas its reduction byRNAi in wild type worms causes a prematureaging phenotype (Hsu et al. 2003; Morley andMorimoto 2004; Steinkraus et al. 2008). DAF-16 and HSF-1 function in a very connectedway—as well as a requirement for HSF-1 inmediating DAF-16-dependent lifespan exten-sion, DAF-16 activation confers heat shockresistance, and both appear to function in con-cert with the sirtuin SIRT1 (Hsu et al. 2003;Morley and Morimoto 2004; Lithgow et al.1995; Tissenbaum et al. 2001; Westerheide et al.2009). In turn, over-expression of HSF-1 en-hances the folding of aggregative proteins, andcan extend lifespan, as can administration of asublethal dose of heat shock in both C. elegansand Drosophila (Hsu et al. 2003; Morley andMorimoto 2004; Lithgow et al. 1995; Hercuset al. 2003). The importance of the heat shockresponse in lifespan is reflected in a screen forthermal stress resistant worms, a high percent-age of which also showed a lifespan extensionof more than 15% (Munoz and Riddle 2003).





The heat shock response therefore seems toform an important component of lifespanextension pathways, and without the HSR, ani-mals age at an accelerated rate. The ability toinduce the HSR declines with age and thisappears to be a relatively early event in the agingprocess, occurring shortly after the reproductiveperiod in C. elegans (Ben-Zvi et al. 2009). Themechanisms underlying the loss of the responseare unclear, but it seems likely that losing theability to induce the HSR and therefore copewith increases in protein misfolding mightunderlie deterioration associated with aging.

THE HYPOXIA RESPONSE

The stress response to low oxygen conditions,termed hypoxia, is regulated by the HIF1 tran-scription factor (Lendahl et al. 2009). Underconditions of normoxia, prolyl hydroxylaseshydroxylate HIF1 at specific proline residues.This hydroxylation targets HIF1 for proteo-somal degradation through recognition by theVHL E3 ubiquitin ligase. In hypoxia HIF1 isnot targeted for degradation and is able to acti-vate a transcriptional program required for sur-vival in low oxygen conditions. This programincludes the up-regulation of HSPs, which isin turn partly dependent on up-regulation ofHSF1 transcription by HIF1 (Baird et al. 2006).

Recent data suggests that HIF-1 plays a rolein regulating longevity in C. elegans. The pic-ture, however, is confusing. Although one re-port has suggested that increasing active HIF-1by knockdown of its negative regulator VHL-1increases lifespan, independent of other life-span extension pathways, another report sug-gests the opposite—that null mutations inHIF-1 increase lifespan, in a manner not addi-tive to the lifespan extension observed in S6Kmutants, suggesting that it lies downstream ofthe TOR pathway (Mehta et al. 2009; Chenet al. 2009). In this study, knockdown of theHIF-1-specific PH family member EGL-9,increasing levels of active HIF-1, has no effecton wild type lifespan, but decreases lifespanextension by DR. The discrepancies might beexplained by technical differences between thestudies, including the different temperatures at

which the lifespan assays were performed. Inaddition, a third study found that both HIF-1over-expression, and hif-1 loss of function mu-tations, extend lifespan, through different path-ways (Zhang et al. 2009). Therefore, althoughthe HIF-1 hypoxia pathway seems to influencelifespan, the nature of this influence and itis interaction with aging pathways is not un-derstood, and may lie in a complex interac-tion between hypoxia, nutrient availability,and temperature.

THE UPRER

Although many proteins require chaperonesfor folding, this is particularly true of proteinstrafficking through the secretory pathway. Theendoplasmic reticulum (ER) maintains a set ofchaperones to guide the folding of secreted andmembrane proteins. In addition, the ER has itsown stress response, enabling the organelle tocope with increased flux, for example duringdevelopment or in cell types with heavy secre-tory loads, or during stresses such as heat shockthat increase protein misfolding (Schroder andKaufman 2005). This ER stress response (theunfolded protein response, or UPR) is regulatedby three upstream sensors, IRE1, PERK, andATF-6, and has several outcomes, including re-duced translation rates; mRNA degradation;transcriptional up-regulation of a range ofgenes, including many chaperones; and afterexcessive or prolonged ER stress, apoptosis.

It was recently shown that in C. elegans, theability to mount a response to ER stress declineswith age, at a similar rate to the loss of activity inthe HSR (Ben-Zvi et al. 2009). It seems likely,therefore, that the UPR might also play animportant role in aging and lifespan extension.Indeed, recent findings suggest that in C. elegansloss of IRE-1 in the worm abolishes lifespanextension by DR and by loss of hif-1 at elevatedtemperatures, and that IRE-1 and XBP-1 areboth required for full lifespan extension byreduced IIS (Chen et al. 2009; Henis-Korenblitet al. 2010). It will be interesting to explore fur-ther the roles that this stress response pathwayplays in longevity.





PROTEIN TRAFFICKING

Another important aspect of protein homeosta-sis is the delivery of proteins to their correctcompartments. Although it is currently unclearwhether the protein trafficking machinerychanges with age, there is evidence that compo-nents of this machinery are crucial to the life-span extension induced by reductions in IISactivity. In a study of genes required for daf-2mediated lifespan extension, a large number ofthe genes identified were involved in endolyso-somal trafficking and vesicle membrane fusion,including subunits of the HOPS and ESCRTcomplexes (Samuelson et al. 2007). Knockdownof most of these genes alter vesicular trafficking.Genes acting in the fusion of vesicles with lyso-somes appeared to function within the IIS path-way, whereas other genes functioned in parallel,but converging with the IIS system. Interest-ingly, inactivation of some of the genes affectingvesicle-lysosome fusion also prevented the up-regulation of a known DAF-16 target, SOD-3,suggesting that these genes might influence IISsignaling itself. Trafficking to the lysosometherefore seems important in maintaining ayouthful proteome. This may reflect the im-portance of being able to degrade misfoldedsecreted or membrane proteins through endo-somal uptake, maturation and fusion with thelysosome. These results are also interesting inlight of the importance of the autophagy path-way in lifespan extension, discussed below.

PROTEIN DEGRADATION

Despite the mechanisms in place to prevent pro-tein misfolding, some proteins slip through thissafety net, or become irrevocably damaged overtime. To remove and recycle these terminallyaltered proteins, the cell has pathways thatmediate protein degradation. The best charac-terized are the ubiquitin-proteasome system,in which proteins are specifically tagged withchains of ubiquitin and directed to the pro-teasome for degradation, and autophagy, aprocess of cellular “eating” in which cellularconstituents are engulfed in membrane-boundautophagosomes that are then targeted to

lysosomes for proteolytic breakdown of theirconstituents (Ciechanover 2005). Both path-ways have been implicated in the aging process.

AUTOPHAGY

The role of autophagy in aging has been closelyexamined in recent years (Cuervo 2008). Therehave been three main types of autophagycharacterized thus far. In chaperone-mediatedautophagy, specific proteins are recognized bychaperones and delivered to the lysosome,where they bind to the lysosomal LAMP2Areceptor and are translocated into the lysosomefor degradation. Microautophagy is a poorlycharacterized process, involving the engulfmentof regions of the cytosol by the lysosomal mem-brane itself. Macroautophagy, the most studiedform of autophagy, involves the formation of anovel membrane that encloses cytosol, dam-aged proteins, and sometimes organelles, toform an autophagic vesicle that then fuses withthe lysosome. Formation of the autophagosomeis regulated by the beclin-VPS34 and the mTORkinase complexes (Yorimitsu and Klionsky2005).

The best characterized of these processes inthe context of aging is macroautophagy. It hasbeen known for some time that in older cells,lysosome function and autophagy decrease(Vittorini et al. 1999; Terman 1995). It is notentirely clear what underlies this decrease. Stud-ies in fly neurons have shown a decline in levelsof several autophagy proteins with age (Simon-sen et al. 2008). In addition, mammalian stud-ies have indicated a decreased ability to clearautophagosomes, and to respond to hormonalautophagy cues, in older cells (Terman 1995;Brunk and Terman 2002; Donati et al. 2001;Donati et al. 2008).

The interactions between macroautophagyand aging pathways have been largely elucidatedin C. elegans. Macroautophagy is necessary forboth lifespan extension and formation of thelong-lived dauer life cycle stage in daf-2 IIS mu-tants, and large numbers of autophagosomesare observed in the cells of these animals(Melendez et al. 2003). This autophagy is inde-pendent of the DAF-16 transcription factor,





however, and given that DAF-16 is essential forlifespan extension in this model, this suggeststhat increased autophagy is not sufficient forlifespan extension (Hansen et al. 2008).

Macroautophagy is also required for DR-induced longevity (Jia and Levine 2007;Hansen et al. 2008). Worms subjected to DRhave higher rates of protein turnover, reducedlevels of oxidatively damaged proteins, andlarger numbers of autophagosomes. This ele-vated autophagy is dependent on the RAB-10small GTPase and the PHA-4 transcriptionfactor, but the specific downstream targets ofPHA-4 involved in the regulation of autophagyare not clear (Hansen et al. 2008). The inhibi-tion of mTOR under low nutrient conditionsalso results in enhanced autophagy, and thisautophagy is required for the longevity of ani-mals with reduced mTOR activity (Hansenet al. 2008).

The role of macroautophagy in lifespan reg-ulation is not specific to C. elegans promotingautophagy in neurons of Drosophila throughenhanced expression of autophagy proteinsthat decline with age, leads to increased longev-ity, and reduction of autophagy in this speciesmay cause premature aging (Simonsen et al.2008). Perhaps the most persuasive indicationof the importance of macroautophagy in aginghas been the increased longevity seen folowingapplication of autophagy-inducing drugs. Theapplication of rapamycin, a natural inhibitorof mTOR, to mice at a relatively late age (600days) significantly increased longevity (Harri-son et al. 2009). In a study of many differentpotentially lifespan-extending agents, usinglarge cohorts of mice, this was the only onewith such a profound effect on lifespan. In ad-dition, a separate study has shown that treat-ment with spermidine, a polyamine the cellularlevels of which decrease during aging, increaseslongevity of yeast, worms, flies, and humanimmune cells, and this enhanced longevity isdependent on the induction of autophagy(Eisenberg et al. 2009).

Finally, chaperone-mediated autophagy(CMA) has also proven to be important in theaging process. Rates of CMA decrease withage, because of decreased levels of the lysosomal

LAMP-2A receptor through impaired transit tothe lysosomal membrane, and reduced receptoractivation (Cuervo and Dice 2000; Kiffin et al.2007). Rescue of LAMP-2A levels in mouse liv-ers at 10 months old, the age at which LAMP-2Alevels begin to decline, maintains youthful liverfunction in these animals, with an increasedability to respond to stress-induced damage(Zhang and Cuervo 2008). An interesting aspectof this study was that the function of otheraspects of the quality control machinery,including macroautophagy and the ubiquitin-proteasome system, were also improved, sug-gesting that the repair of individual elementsof the proteostasis machinery could have indi-rect effects in preserving other components.

PROTEASOMAL DEGRADATION

The ubiquitin-proteasosome system (UPS)targets proteins for degradation by the pro-teasome, a large proteolytic complex, and theimportance of this degradation pathway in theremoval of misfolded proteins and in cellularsignaling have been well characterized. Theeffects of aging on proteasome activity, however,are only just becoming clear. Conflicting reportshave previously suggested that proteasomeactivity is increased, decreased, or constantwith increasing age (Carrard et al. 2002; Fer-rington et al. 2005; Keller et al. 2004). Thesestudies have been complicated by tissue-specificdifferences, and a comprehensive look at pro-teasome activity with age in different tissueshas been lacking. A new tool in C. elegans,however, holds promise in redressing this, andsuggests that there are indeed significanttissue-specific differences in the effect of ageon UPS activity. Using a photoconvertiblefluorescent moiety, Dendra 2, conjugated to amutant form of ubiquitin, Hamer et al. haveshown that neurons of aging worms show adecreased level of UPS-mediated proteolysis,whereas muscle cells remain unaffected in thisregard (Hamer et al. 2010). This line of researchseems likely to contribute substantially to under-standing the effects of age on UPS activity.

The ubiquitin-proteasome degradation path-way also interacts directly with aging signaling





pathways through the targeting of their com-ponents for degradation by specific E3 ligases.As discussed already, the VHL-1 E3 ligase affectslongevity through its effects on HIF-1 (Mehtaet al. 2009). Also, the DAF-16 transcription fac-tor is ubiquitinated and targeted for degrada-tion by an E3 ligase, RLE-1 (Li et al. 2007).Mutations in rle-1 result in lifespan extension,dependent on an increase in DAF-16 proteinlevels. In addition, another C. elegans E3 ligase,WWP-1, along with an E2 ubiquitin-conju-gating enzyme, UBC-18, have been identifiedas factors essential for the extension of life-span through DR (Carrano et al. 2009). Over-expression of WWP-1 is able to increase lifespanby about 20%, and this lifespan extension isdependent on PHA-4 and the catalytic E3 ligaseactivity of WWP-1. The effect of age-dependentchanges in UPS activity on signaling withinthese pathways might therefore represent aninteresting area for future research.

DISEASES OF PROTEIN MISFOLDING

An important consequence of declining proteo-stasis capacity is the emergence of a variety ofprotein misfolding maladies at advanced ages.These diseases are characterized by the aggrega-tion of a particular protein, or proteins, in spe-cific cell types. Despite the involvement of amultitude of disparate toxic proteins, the lateage of onset is remarkably similar in many ofthese conditions, including Alzheimer’s, Par-kinson’s, and Huntington’s diseases. It is alsobecoming increasingly clear that signalingpathways that regulate aging can alter suscepti-bility to these diseases, and that this may bethrough their regulation of the proteostasismachinery.

NEURODEGENERATIVE DISEASE

Neurons seem to be particularly vulnerable todiseases of protein misfolding (Drummondand Wilke 2008; Lee et al. 2006). Major break-throughs in understanding the influence ofaging on neurodegenerative protein misfoldingdiseases have come about through the study of

invertebrate models of these pathologies. Usingorganisms such as C. elegans or Drosophilaallows the study of basic cellular disease proc-esses in the context of postmitotic cells withina multicellular organism. Many studies haveshown that the expression of human diseaseproteins in C. elegans or Drosophila can be toxic,and results in scoreable physiological andbehavioral readouts (Morley et al. 2002; Cohenet al. 2006; Kerr et al. 2009). Aging and proteo-stasis pathways can be easily manipulated inthese systems, to study their effects on proteo-toxicity phenotypes.

The first such study used C. elegans express-ing polyQ repeats, seen in CAG expansion dis-orders such as Huntington’s disease, fused toYFP in body wall muscle cells (Morley et al.2002). This shows that polyQ becomes ag-gregated in a length-dependent manner, withaggregation accompanied by loss of the animal’smotility. Aggregation was also age-dependent.At a threshold length of 35-40 polyQ, aggregatesaccumulate progressively throughout life. Inthis study, reducing IIS pathway activity wasable to delay aggregation and toxicity, suggest-ing that this aging pathway can modify the pro-teostasis machinery sufficiently to enable cellsto deal with highly toxic proteins to a muchlater age. Subsequent studies have shown thatHSF-1 over-expression is also highly protectiveagainst polyQ aggregation, and that many othercomponents of the proteostasis network aremodulators of polyQ toxicity, including genesinvolved in RNA metabolism and proteinsynthesis, folding, trafficking, and degradation(Hsu et al. 2003; Nollen et al. 2004; Jia et al.2007). Interestingly, similar gene categorieshave also been identified as regulators ofa-synuclein toxicity in C. elegans models ofParkinson’s disease (Hamamichi et al. 2008;van Ham et al. 2008).

Further mechanistic insights have comefrom studies of a model of Alzheimer’s diseasein C. elegans. In this system, the toxic disease-associated Ab1-42 peptide is expressed in thebody wall muscle cells, producing a progressiveparalysis phenotype (Link 1995; Cohen et al.2006). Again, reduced function of the IIS path-way can protect against toxicity, dependent on





the DAF-16 and HSF-1 transcription factors(Cohen et al. 2006). Interestingly, althoughdaf-2 knockdown decreased toxicity, it in-creased the amount of high molecular weightAb aggregates, whereas daf-16 knockdownincreased toxicity and decreased the presenceof these high molecular weight species. Andhsf-1 knockdown both dramatically increasedtoxicity, and greatly increased the amount ofboth large and small aggregates. This has sug-gested a model in which HSF-1 and DAF-16both transcriptionally regulate detoxificationactivities, with HSF-1 mediating a disaggrega-tion and degradation activity, and DAF-16inducing a secondary active aggregation activitythat assembles small oligomers into larger, lesstoxic structures (Fig. 3). Studies showing thatHSP104 and TRiC can actively aggregate pro-teins suggest that similar chaperones might beemployed under the transcriptional control ofDAF-16 (Behrends et al. 2006; Shorter andLindquist 2004). Recent work has also separatedthe HSF-1-controlled activity into separate Ab

disaggregation and proteolytic degradationactivities, moving closer to an identification ofthe molecular components involved (Bieschkeet al. 2009).

Other studies on the Ab worm have shownthat DR is also protective against Ab toxicity,and that HSF-1 is again required for this protec-tion (Steinkraus et al. 2008). However, it isinteresting to note that DR was not protectiveagainst Ab neurotoxicity in a Drosophila model(Kerr et al. 2009). It has also been suggested thatautophagy is required for the protective effectof daf-2 knockdown in Ab-expressing worms(Florez-McClure et al. 2007). Autophagic ves-icles are seen in Ab-expressing cells, and knock-down of autophagy regulators enhances toxicity.Curiously, in this study, reduced daf-2 expres-sion actually reduced the number of autophagicvesicles in Ab-expressing cells, suggesting amodel in which IIS reduction increases theefficiency of autophagosome-vacuole fusion,which is consistent with a proposed role forincreased vesicle-lysosome fusion efficiency in

Secretion?Disaggregation?Degradation?

DisaggregationActiveaggregation

Toxicaggregates

Less toxicaggregates

HSF-1DAF-16

Aggregation

Aβ peptide production

Protease-mediateddegradation

Autophagy?

Figure 3. Detoxification of Ab aggregates by DAF-16 and HSF-1. The Ab is produced throughout life by pro-teolysis of the amyloid precursor protein. In youthful animals, Ab aggregates are believed to be detoxified byan active aggregation activity, regulated by the transcription factor DAF-16, and a disaggregation/proteolyticdegradation pathway downstream of the transcription factor HSF-1.





daf-2 lifespan extension (Samuelson et al.2007).

These results have been substantiated inrodents, in which a heterozygous mutation inthe IGF-1 receptor is substantially protectivein a mouse model of AD (Cohen et al. 2009).Although behavioral phenotypes of AD are sub-stantially delayed, there appears to be no de-crease in Ab production. Instead, Ab fibrilsare packed into dense areas, leaving surround-ing brain tissue intact. This is consistent withthe existence of a conserved Ab-aggregativeactivity downstream of reduced IIS. Theseresults are, however, inconsistent with otherdata suggesting that increased IGF signalingcan increase Ab and polyQ clearance (Carroet al. 2002; Yamamoto et al. 2006). The answerto this anomaly may lie in the different effectsof different levels of IIS in different organs,as discussed elsewhere (Cohen and Dillon2008). Regardless, the observation that interfer-ence with the aging process through reductionin IIS can modify Ab toxicity in mammals sug-gests that this may represent a promising avenuefor future therapeutics.

OTHER DISEASES

Although the links between neurodegenerativediseases and age-associated proteostasis declineare the best characterized, it is possible thatother late onset diseases are also diseases of pro-teostasis failure. Type II diabetes is one example.A component of the group of late-onset diseasesknown as “metabolic syndrome,” diabetes andinsulin resistance have been associated with adecline in UPR and autophagy capacity (Ozcanet al. 2004; Jung and Lee 2010). The decline ofthese proteostasis functions with age couldtherefore predispose toward diabetes. In turn,this suggests that enhancement of proteostasisin older individals could protect against a vari-ety of different but associated protein misfold-ing diseases.

The temptation to see increased proteostasiscapacity as a panacea for all diseases of old age,however, should be avoided. The restricted ex-pression of proteostasis regulators may actually

help to limit the development of cancers. Can-cer cells have been shown to depend on theactivity of the HSF1 and UPR pathways for via-bility, and susceptibility to Alzheimer’s diseaseand cancer show an inverse correlation (Biet al. 2005; Dai et al. 2007; Roe et al. 2010).This suggests that increasing the capacity ofthese pathways, while protecting against somediseases, could also potentiate tumorigenesis.

EVOLUTIONARY TRADEOFFS?

It is interesting to ask why, given the importantrole of proteostasis in longevity and protectionagainst disease, components of the proteostasismachinery are not constitutively expressed athigh levels. The potentiation of cancerousgrowth in mammals might be one explanation;however, restricted capacity of the proteostasismachinery has been shown throughout the evo-lutionary tree, for example in C. elegans (Gi-dalevitz et al. 2006; Gidalevitz et al. 2009).The answer might lie in the concept of a trade-off between somatic maintenance and repro-duction. On an evolutionary level, faced withfinite resources, organisms must balance thecostly demands of reproduction and somaticmaintenance, including maintenance of appro-priate protein homeostasis (Kirkwood 2005).This balance takes into account extrinsic mor-tality, the chance that an organism will diebecause of external factors such as predation,to create the best evolutionary strategy for max-imizing offspring. Evidence suggests that alter-ing the proteostasis machinery tomimic conditions that extend lifespan, forexample, overexpression of chaperones, activa-tion of stress responses, or reduction of transla-tion rates, reduces fertility and increasesdevelopment time. Indeed, reduced IIS, DR,or reduced mitochondrial function, have allbeen shown to reduce fecundity as well asextending lifespan, although it is important tonote that increased lifespan and reducedfecundity are not inseparable (Dillin et al.2002a; Partridge et al. 2005; Grandison et al.2009). This concept may explain the restrainedexpression of many components of the proteo-stasis machinery.





CONCLUSIONS

The many and intimate connections betweenpathways that regulate aging, and the cellularprotein homeostasis machinery, suggest thatmaintenance of a healthy proteostasis state isessential for longevity. It also seems that thesepathways may play an important role in the pre-vention of age-associated protein misfoldingdiseases. This makes pathways that regulateaging, as well as the proteostasis machineryitself, promising targets for future therapeutics.

In fact, several drugs targeting various com-ponents of the proteostasis machinery arecurrently in clinical trials (Balch et al. 2008).However, it is clear is that the agents and path-ways that influence proteostasis act as an inte-grated network. Affecting one component haseffects throughout the entire system. Thispresents both opportunities, and problems. Asthe expression of one misfolded protein has adestabilizing effect on proteostasis throughoutthe proteome, it could be that by aiding thefolding of just one protein, a range of relatedpathologies, and symptoms could be alleviated.In addition, an understanding of upstream pro-teostasis regulators can enable intervention inthe whole network simultaneously. In thesecontexts, invertebrate models should prove use-ful tools in screening drugs and interventionsthat can protect against proteotoxic diseases.

However, given the potential negative effectsthat manipulation of aging and proteostasispathways can have on, as mentioned, growthand fertility, caution is required in interferingwith these programs. In addition, althoughmany age-associated diseases seem likely tobenefit from an increase in proteostasis ca-pacity, this may also play into the hands of othermaladies, including cancer. With these caveatsin mind, however, modulation of the proteinhomeostasis network is a promising avenuefor the treatment of some of the biggest medicalproblems facing our aging society.

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published online February 16, 2011Cold Spring Harb Perspect Biol Rebecca C. Taylor and Andrew Dillin Aging as an Event of Proteostasis Collapse

Subject Collection Protein Homeostasis

ChaperonesModulation of Amyloid States by Molecular

Bernd BukauAnne Wentink, Carmen Nussbaum-Krammer and

Mitochondrial Proteolysis and Metabolic ControlSofia Ahola, Thomas Langer and Thomas MacVicar

StrategyProtein Phase Separation as a Stress Survival

Titus M. Franzmann and Simon AlbertiUnfolded Protein ResponseSignaling and Regulation of the Mitochondrial

Nandhitha Uma Naresh and Cole M. Haynes

Ameliorate Protein Conformational DiseasesProteostasis in the Secretory Pathway to Pharmacologic Approaches for Adapting

Jeffery W. Kelly

Functional AmyloidsDaniel Otzen and Roland Riek

ProcessesRole of Polyphosphate in Amyloidogenic

Justine Lempart and Ursula JakobEndoplasmic Reticulum HomeostasisRedox-Mediated Regulatory Mechanisms of

Ryo Ushioda and Kazuhiro Nagata

in Aging and DiseaseCell-Nonautonomous Regulation of Proteostasis

Richard I. MorimotoHSP90 and Beyond−−in Cancer

A Chemical Biology Approach to the Chaperome

Tony Taldone, Tai Wang, Anna Rodina, et al.

NeurodegenerationThe Autophagy Lysosomal Pathway and

Steven FinkbeinerBiology and MedicineThe Amyloid Phenomenon and Its Significance in

Michele VendruscoloChristopher M. Dobson, Tuomas P.J. Knowles and

Shock FactorsTailoring of Proteostasis Networks with Heat

Jenny Joutsen and Lea SistonenUnfolded Protein ResponseEarly Events in the Endoplasmic Reticulum

Steffen Preissler and David Ron

Proteostasis in Living CellsProteome-Scale Mapping of Perturbed

Isabel Lam, Erinc Hallacli and Vikram KhuranaChaperone Interplay−Complex Virus

Proteostasis in Viral Infection: Unfolding the

Ranen Aviner and Judith Frydman

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