50 Harvard Science Review • fall 2006

understanding how and why we age

WHO WANTS TO LIVE

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By Angelo S. Mao

‘Tell me, how joinedst thou the Assembly of the gods.In thy quest of life?’Utnapishtim said to him, to Gilgamesh:’I will reveal to thee, Gilgamesh, a hidden matterAnd a secret of the gods will I tell thee…’

Overthecourseof humanexistence,manyhavetriedtodiscoverthesecrettoimmortality.AncientChinesealchemistsmadecocktailsof mercury andpowderedgold,whileKingArthur’sknightsscatteredfarandwideinsearchof theHolyGrail.Butwhatisthekeytoeverlastinglife?Newperspectivesandunderstandingsonhowandwhyourmostfundamentalbuilding blocks age—why our cells senesce—may bring uscloser to finding an answer to this eternal question.

Hayflick’s Limit and Harman’s PuzzleOne of the first pieces to the puzzle was uncovered in 1961 when Leonard Hayflick and Paul Moorhead at the Wistar Institutepublishedapaperthatoverturnedthedogmaof thetime. Cells isolated from multicellular organisms—such ashumans—were thought tobe innately “immortal” with theability to divide indefinitely. As it turned out, however, cells could only divide a finite number of times before stopping at the so-called “Hayflick limit” (1).

Ataboutthesametime,DenhamHarman,thenaresearchassociateattheUniversityof CaliforniaBerkeley,noticedthata variety of diseases, ranging from cancer to Alzheimer’s, were connectedbyacommonfactor:agroupof moleculesknownasfreeradicals.Becausefreeradicals lackacompleteouter-mostshellof electrons,theyarehighlyreactiveandcanattackthe cellularmacromolecules essential for life.Harman triedtoincreasethemaximumlifespanof micebyfeedingthemelementsthatpreventfreeradicaldamage,calledantioxidants;his results revealed thateven though themice’s average lifespanincreased,theirmaximumlifespandidnot.Somehow,freeradicalsweregeneratedsomewhereinthebodythatanti-oxidantscouldnotreach.

FOREVER?

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Figure 1. Mitochondria create reactive oxygen species, which may severely damage macromol-ecules or organelles including the mitochondria themselves.

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Suffering from Cellular ExhaustThe answer lay in mitochondria, thepowerhouses of the cell. These spe-cialized intracellular structures release energyfromorganiccompoundsandeffectively use oxygen molecules aselectron dumps. During the electrontransport chain at the heart of themitochondria’s function, eachoxygenmoleculereceivesfourelectrons,even-tuallyformingwater.However,astheelectronspassthroughthemitochon-drialmembrane,someof theminevi-tablycombinewithoxygentoproducesuperoxide,whichgivesrisetomanyof the other reactive oxygen species (ROS) inthecell.Inadditiontotheactivityof the mitochondria’s inner membrane,the outer membrane oxidizes amines to form another ROS, hydrogen peroxide (2). Together, these two processes ac-count for an estimated 90% of the free radicals found in the body (3).

If unchecked, ROS would severely damage all organic macromolecules,including mitochondrial DNA, pro-teins,carbohydrates,andlipids.BecausemitochondrialDNAislocatedclosetothe site of ROS formation and lacks the stringent error-checking mechanismsfoundinnuclearDNA,itisespeciallyat

risk (4). To counter this production of ROS, however, the body has developed numerousantioxidantdefenses,includ-ing the enzymes superoxide dismutases (SODs), catalase, and the molecules vitamins E and C. SODs, which require metalliccofactors suchasmanganeseor zinc to function, convert superoxide intothelessreactivehydrogenperoxide.Catalase, a common enzyme in cells, then decomposes hydrogen peroxideintooxygenandwater.VitaminCandvitaminEworkcooperativelytoprotectlipidsandreduceradicalsfromsourcesthroughout the cell (5).

Flawed Elixir: Antioxidant TherapyIn lightof themitochondria’s role inROS generation, Harman proposed that aging resultedwhen theproduc-tion of ROS overwhelmed the ability of antioxidants to convert them intosafer end products. This theory wassupported by strong correlations be-tweenoxidativeaccumulationandtheage of organisms (6). Another possible support for this theory was the find-ingthatlimitingdietaryintakeextendslifespan by as much as 40%, ostensibly by minimizing ROS produced during

the oxidation of food (6)With the recognition of the role

antioxidants play, why not use anti-oxidants to vanquish ROS and live forever? Unfortunately, the benefit of antioxidant therapy—and indeed thecause-and-effect relationshipbetweenagingandantioxidantlevels—hasbeenambiguous at best. While oxidative damageincreaseswithage,nomatchingcorrelationforadeclineinantioxidantshas been observed (7). Though a recent studyshowedthatthenematodeCae-norhabditiseleganstreatedwithSODand catalase lived about fifty percent longer than average (8), later studies donebothindifferentorganismsandC. elegans could not duplicate theresults (9).

Mitochondria-produced ROS are known to accumulate with age. Why, then,doantioxidanttherapiesfallshortof expectations? One explanation isthatantioxidantscannotreachthemi-tochondrianestled insidecells,whichhaspromptedscientiststoinvestigateSzeto-Schiller peptides, antioxidant peptidesthatcanpermeatecellmem-branesandtargetthemitochondria,asapotentialtherapy.Anotherexplanationis the so-called antioxidant paradox.Thisstatesthat,becausemanyantioxi-dantsmustreverttostatesthatpromoteoxidants as part of the reduction of freeradicals,largedosesof antioxidantsmay actually increase oxidation (10).

Antioxidant-basedtherapiesarenotwithout promise, however. Recently, BruceSpeigelmanof theDana-FarberCancer Institute found thataproteincalled PGC-1α, which regulates the expression of genes that control mi-tochondrial activity, also plays a rolein regulating ROS levels. Though high levels of PGC-1α increases electron transport activity and ostensibly theamount of ROS generated, raising levels of PGC-1α also induces cells to expressproteinsthat,likeSOD,protectcellular mechanisms from ROS. Since it maintains normal mitochondrialfunction, PGC-1α does not interfere with the body’s normal metabolism

52 Harvard Science Review • fall 2006

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Figure 2. With each reproduction, the telomere of somatic cells diminishes.

whenitreducesfreeradicals.BecausePGC-1α can clear ROS without reduc-ingmitochondria activity, theproteinshows great potential as a therapycountering ROS-associated diseases and aging (11).

In Our GenesThoughmitochondriahavebeenshowntocontributetosenescence,scientistshavefoundthatanotheragentof agingliesinthenucleus.Abouttwentyyearsafter Hayflick made his observation that cells reached senescence after a finite number of divisions, James Watson, oneof thediscoverersof DNAstruc-ture,raisedtheso-calledendreplicationproblem: DNA polymerase, the enzyme behind DNA replication, could onlysynthesize in one direction. To polym-erize in the opposite direction, RNA-capped Okazaki fragments form and must be joined later by transformingthese RNA caps (called primers) into DNA. However, this process requires that therebeanupstreamDNAseg-mentinorderforthesefragmentstobejoined.Howdoesthecelldealwiththeendof aDNAmolecule,wherethereisnoDNAstrandtofacilitatethistrans-formation?Halfwayacrosstheworld,aRussian biologist by the name of Alexey Olovnikov made a startling connec-tion: perhaps Watson’s end replication

problemexplainedwhycellsinculturescould not divide indefinitely (12).

Severalscientists,includingBarbaraMcClintock, had previously hypoth-esized the presence of special tips at theendsof chromosomes,butthelinkbetween the shorteningof these tipsandcellularagingwasonlycementedtwenty years after Olovnikov’s first ob-servations.Thesetips,calledtelomeres,areactually“ribonucleoproteins,”DNAstructures that also contain an RNA primerandproteincomponentstopro-tectthechromosomalterminalend.

In the past few years, scientistshavefoundthatstressfactorscorrelatestronglywithshortertelomeres.ABrit-ish study involving more than five hun-dredpairsof twinsrevealedthatthosewhosmokedandsufferedfromobesitylost about five more base pairs of DNA per year, an increase of 18%, than their healthiercounterparts,resultinginanetdifference of about 240 base pairs after age adjustment (13). Another study showedthatwomenwhoexperiencedhighlevelsof psychologicalstress,suchas having to care for a chronically illchild,had telomeres shortenedby anequivalent of one decade of aging in comparison to low-stress women (14). Scientists have also determined that,besides external influences, telomere lengthdependsonheredity,oxidative

stress,andgender,withwomengener-allyhavinglongertelomeresthanmen(15).

Thoughourtelomeresshrinkwitheveryroundof replication,ourbodieshavethecapabilitytoextendtelomeresusingspecialstructurescalledtelomer-ases.Thesecomplexesof proteinsandnucleic acids are composed of a TERC (TElomerase RNA Component) RNA templaterichincytosineandguanine,and a TERT (TElomerase Reverse Transcriptase) enzyme, which uses the TERC template to create more DNA and belongs to the same classof proteins as the HIV-utilized RNA transcriptase (16). Recently, researchers havealsofoundthatotherchemicals,including a form of vitamin B3, can reduce telomere shortening withoutactivating telomerases (17).

Though the body has the capac-itytocounteracttelomereshortening,studies show that not all lengths areequal. One reason is that only specific typesof cellsactivatetelomerases.Inmulticellularorganisms,thesearegermcells,whichdividerapidlytoreplenishthe body. Normal cells, on the otherhand,ceasedividingandmayevenenterprogrammedcelldeathaftertheirtelo-meresshortenandchromosomeendsunwind. Why would most cells in the bodywillinglyshortentheirreproduc-

fall 2006 • Harvard Science Review 53

pleiotropytheory,whichpostulatedthatgenes conferring benefits to organisms earlyintheirlivescouldleadtodelete-riouseffectslateron,afterindividualscouldnolongerservetoaddtothegenepool. As an example, a hypotheticalgenethatincreasedcalciumdepositioncould both build stronger bones andinduce the formation of hard plaques inbloodvessels, leadingtoheartdis-ease (19).

Though Medawar and Williams’ theoriesarethemostwidelyacceptedtoday,cracksstillremainintheirexpla-nation of why we age. For example,arecentstudyonguppiesinTrinidadshowedthatthoselivinginpredation-highwatersactuallyhadalongernaturallifespanthanthoselivinginwaterswithrelatively less predation. By Medawar’s theory,theguppieslivinginwaterwithhighpredationshouldhaveshorterlifespansbecauseevolutionneverhadthechanceto“eliminate”deleteriousmu-tationsthatwouldplaguetheguppieslaterintheirlives.Clearly,atleastinthisparticular instance, the evolutionarymechanismsatworkcannotbesosim-plyexplainedby theoriesof extrinsicmortality and pleiotropy (20).

Despiteadvancesinourknowledgeof cellular mechanisms that have al-lowedustobetterunderstandhowweage and why cells senesce, a detailedappreciation of why we, as organ-isms,agehoversbeyondourreach.Isour mortality encoded in our genesordictatedbyourenvironment?Is ita biological inevitability or a diseaseto be cured? Will there ultimately be a tradeoff between an evolutionarysafeguardagainstdangerssuchascan-cer proliferation and the fulfillment of anancientdream?Untilwecanpiecetogether more pieces of this puzzle, aging remains an unavoidable realitythat—for now at least—eludes ourdreamsof eternallife.

References1. Hayflick, L., and Moorhead, P. S. (1961) “The serial cultivation of human diploid cell strains.” Exp Cell Res 25:585-6212. Hauptmann D., Grimsby J., Shih J., and Cadenas E. The Metabolism of Tyramine by Monoamine Oxidase A/B Causes Oxidative Damage to Mitochondrial DNA. Archives of Biochemistry and Biophysics. 335:2 (1996) Pages 295-304.3. Balaban, R., Nemoto, S., and Finkel, T. Mito-chondria, Oxidants, and Aging. Cell. 120:4 (2005) 483-495.4. Cadenas, E. Mitochondrial free radical generation, oxidative stress, and aging. Free Radical Biology and Medicine. 29:3-4 (2000) Pages 222-2305. Finkel T, Holbrook NJ. “Oxidants, oxidative stress and the biology of ageing”. Nature 408:6809 (2000) 239-47.6. Kregel, K. and Zhang, H. Oxidative Stress in Aging. Am J Physiol Regul Integr Comp Physiol (August 17, 2006) in press upon retrieval of source7. Mattson, Mark P. Energy intake, meal frequency, and health: a neurobiological perspective. Annual Rev. of Nutrr. (2005) 25: 237-2608. Melov S, Ravenscroft J, Malik S, Gill MS, Walker DW, Clayton PE, Wallace DC, Malfroy B, Doctrow SR, and Lithgow GJ. Extension of life-span with superoxide dismutase/catalase mimetics. Science 289 (2000) 1567-1569.9. Keaney M, Matthijssens F, Sharpe M, Vanfleteren J, and Gems D. Superoxide dismutase mimetics elevate superoxide dismutase activity in vivo but do not re-tard aging in the nematode Caenorhabditis elegans. Free Rad Biol Med 37 (2004) 239-250.10. Szeto, Hazel H. Cell-permeable, Mitochondrial-targeted, Peptide Antioxidants. AAPS J. (2006) 8(2):E277-83.11. De Bono, D.P. Olovnikov’s clock: telomeres and vascular biology. Heart (1998) 80:110-11112. João Pedro de Magalhães. www.senescence.info (2004, 2005)13. Campisi, Judith. Senescent Cells, Tumor Sup-pression, and Organismal Aging: Good Citizens, Bad Neighbors. Cell (2005) 120(4):513-2214. Gavrilov, Leonid A. and Gavrilova, Natalia S. Evolutionary Theories of Aging and Longevity. The Scientific World JOURNAL (2002) 2, 339-356.15. Reznick, D. N., Bryant, M. J., Roff, D., Ghalambor, C. K. & Ghalambor, D. E. Nature 431 (2004) 1095-1099, as referenced by Peter A. Abrams (Nature 2004 431:1048).

—Angelo S. Mao ’10 is a Chemical Physical Biology concentrator in Greenough.

tivelifespan?

Give Me Fidelity, or Give Me DeathIn the years following Hayflick’s ob-servations, scientistsnoticed that thesenescentresponsearosefromvariousphenomena,includingdamagedDNA,oxidative lesionsdue to free radicals,andoverexpressionof oncogenes,allof which are potential precursors tocancer. Moreover, two important cel-lularpathwaysthathavebeenfoundtoplay a large role in senescence, the p53 and p16 routes, are central to major tumor suppression and are dysfunc-tional in many cases of cancer (18). Sig-nificantly, many cancerous cells possess activated telomerases, whereas theirmortalsomaticneighborsdonot.

As early as the 1960s, Hayflick pro-posedthatcellularsenescenceevolvedas a response to cancer suppression.Recent studies have strengthened the evidencebehindtheideathatcellsceasetodividewhentheriskthattheymaypass down damaged DNA becomestoogreat.Viewedmorebroadly,evolu-tionhasprovidedcellswithaginganddeathasaperennialdefenseagainstthegreaterperilsof passingdownharmfulgeneticmaterialthatmighteventuallycauseacelltobecomecancerous.

From Cell to SpeciesIf weconsidercellularsenescenceasameans to increase the longevityof theorganism,whatcanwesayabouttheagingof anindividualorganismasit reflects upon the evolution of the species? In the 1950s, Peter Medawar, a Brazilian-born British scientist who would later win the Nobel Prize in medicine,pointedoutthatspeciesfac-inghighextrinsicmortalitypossessedshorterintrinsiclifespan.Forexample,the brown bat has far fewer preda-tors than the common field mouse; incidentally, it lives up to ten timeslonger than its rodent relative. Soonafterwards,Americanevolutionarybi-ologist George Wiliams complemented Medawar’s theory with the antagonistic