NikiYazdani

BIOL4097
NIHGrantProposal2014
MitochondrialDNATransfersandAgingPrevention
Abstract
Themitochondriaofthecellismaternallyinheritedanditdrivestheproductionof
power for the cells biochemical reactions such as oxidative phosphorylation that
generatesadenosinetriphosphate,ironandcalciumhomeostasis,biosynthesisofsteroids,
pyrimidines, and heme, and apoptosis (Houten et al). Healthy mitochondrial DNA
(mtDNA)isimperativefornormalmitochondrialfunctionandwhenthereisamutation
inthisDNAthatproducesdisease,thediseasedindividualisfacedwithdeficitsinenergy
productionandashorterlifespan.Considerableevidencehasbeenfoundlinkingdeclines
inhumanmitochondrialoxidativephosphorylationcapacityduetoaging.Thiscorrelation
hasbeenhypothesizedtooccurbecauseoftheoccurrenceoflargedeletioninmtDNA
fromvarioustissuesbecauseoftheproductionofreactiveoxygenspecies.Theoxidative
damagethatoccurscanleadtopossiblecelldeath,whichwouldbeareasonforthe
declineofcellsobservedwithaging(Chomynetal).Thisprojectaimstoconcludeif
oxidativedamageistheprimarysourceofgenerationofmtDNA.
Withinthepastdecade,technologyhasallowedscientiststotransfermtDNAfrom
healthyembryostoembryoswithknowninfectedmtDNAresultingwithanembryothat
containsDNAfromthreedifferentindividuals.IfthismtDNAisaviablesolutionfor
individuals afflicted with possibility of giving rise to children with debilitating

mitochondrialdisease,researchdoneonmtDNAtransferscouldproduceabreakthrough
in the area of prevention of aging by manipulating the production of mitochondrial
oxidativephosphorylationcapacitythatslowsasanindividualages.Thisprojectalso
aims to determine the effects of mtDNA transfers on the aging process of mice by
comparingthedevelopmentofmiceembryoswithmutatedmtDNAandmiceembryos
with mutated mtDNA that underwent mtDNA transfer. It is expected that the mice
embryosthatunderwentmtDNAwillhaveacomparablylongerlifespanthanthemice
embryosthatlivedwithmutatedmtDNAduetoamorestablemitochondrialoxidative
phosphorylationcapacity.Thefindingsofthisprojectwilladvanceourunderstandingof
themitochondrialmechanismsandtheireffectonourcellularagingandapoptosis,which
canleadtoproceduresthatcanincreasethelifespansoffuturegenerations.

Specificaims
MitochondrialDNAarevitalcomponentstoourcellsandfurtherunderstanding
thereplacementofmutatedmtDNAcanenhanceourunderstandingtheeffectsofthis
repaironagingandembryosthathavethepotentialtoacquiremitochondrialdiseases.
Duringthetimeoftheproposedresearch,theintendedgoalsarethreefoldduringthe
periodofpredictedfundingbecausetodaywefacethechallengeofunderstandingthe
reasonswhythereisanincreaseinapoptosiswhenmtDNAismutated.
Theprimarygoalofthisresearchstudyistofindadirectlinkbetweenmutated
mtDNAandincreasedapoptosisleadingtocellularaging.Withthatinmind,istoresolve
if the accumulation of reactive oxygen species is the primary reason for the rise of

mutationstohealthymtDNA.ThiswillbedonebyexposinghealthymtDNAtovarying
levelsofreactiveoxygenspecies(ROS)viaionizingradiationandobservingmutations
thatemerge.ThefinalgoaloftheprojectseekstodetermineifhealthymtDNAtransfers
forknownmutatedmtDNAisfeasiblesolutiontoincreaselifespanofmicethatwould
normallybeafflictedwithmitochondrialdiseasesduetomaternalinheritance.Feasibility
willbegaugedbytheefficacyoftheprocedureandwhethertheextensionoflifeis
significant. ThecombinationoffindingthecorrelationbetweenmtDNAdamageand
apoptosis,determiningifROSresultinmtDNAmutations,andobservingtheeffectof
mtDNA transfers on lifespan will help the science community discern if continued
researchinmtDNAwillleadtoextensionstohumanslifespansandhealth.

Backgroundwithnumberedreferences
Mitochondriaaremostcommonlyknownasthepowerhouseofthecellbecause
of the major cellular processes that they drive including the generation of adenosine
triphosphateviaoxidativephosphorylation,biosynthesisofessentialcellmaterials,and
homeostasis of calcium and iron. Mitochondria are sometimes referred to as the
executioners in apoptosis because of their ability to release proteins that activate
programmedcelldeath1.ClaytonandFriedberginthe1970sdeterminedthatpyrimidine
dimers created by UVinduced lack repair function mtDNA and through continued
research it has become increasingly evident that mtDNA lacks nucleotide excision
repairingmechanisms,whicharetheprominentmechanismsforUVdamagetoDNA 2.
However, mtDNA is able toconduct basedamage repair, but is more susceptible to

damagecausedbyROSthanDNAbecausetheironcontentinmitochondriaishigh.High
ironcontentinthemitochondriaisaforceintheformationoffreeradicals.Ifoxidation
ofpyrimidineandpurinelesionsiscausedinmtDNA,thisresultsinthepreventionof
mtDNAtranscriptionbecauseRNApolymeraseisprohibitedtocontinueitsaction.ROS
propagationcascaderesultsbecauseofthelossofmtDNAencodedelectrontransport
proteins, which in turn create mtDNA oxidation. The mitochondrial catastrophe
hypothesis states that ultimately the inaccurate gene expression caused by oxidative
mtDNAdamageleadstokeyelectrontransportenzymesbeingdeficientandcelldeath 1.
The Free Radical Theory of Aging connects the previous hypothesis with aging by
suggesting that the mitochondrias decreased ability to deal with ROS leads to an
increaseinproductionofROS.ThedetrimentalcycleofROSproliferationcanthusbe
considered as a feature of aging because of the mitochondrias decline in repair of
mtDNA3.
Houten,Woshner,andSantoshypothesizethatifmtDNAisresponsibleforcell
death,then enhancement ofmtDNA repairshouldimprovecellsurvival. With this
conceptinmind,WilsonandLedouxconductedanexperimentinwhichtheytransfected
8oxodGglycosylase(OGG1)intomitochondria,whosemechanismisinvolvedinthe8
oxoguaninelesionexcision.Thisstudysawincreasedcellsurvivalduetorapidrepairof
mtDNA4. A review of this study by Houten, Woshner, and Santos concluded that
enhancedmtDNArepairprotectscellsfromcelldeath 1.Conversely,anincreaseincell
deathhasbeenevidentinstudiesinwhichmammaryadenocarcinomacellsmitochondria
weretargetedatexonucleaseIII,abacterialprotein,causedanincreaseincelldeathby

uncouplingoxidativeDNAdamagerepair.Theauthorsofthisstudybelievedthatabasic
sites that could notbe accessed for repair by mitochondrial mechanisms causing the
increaseincelldeath5.TofurthertheideathatmtDNAdamageispartlyresponsiblein
aginginvolvestheconsiderationofastudyconductedbyLarssonetal.wheremicethat
were deficient in 3exonuclease DNA polymerase experienced premature aging in
comparisontoitscounterpartswhowerenotdeficient.DNApolymeraseisofcoursea
necessityinDNArepairofmtDNAbecauseofitsabilitytobothreplicateandreplace
gaps6.
The aforementioned studies clarify that cell survival is dependent on mtDNA
repairmechanisms,andthataugmentedmtDNAisawaytoevadetheconsequencesof
mutated mitochondria due to damage. Some researchers have delved into bypassing
implementationofrepairmechanismsbytransplantinghealthymtDNAtoembryosthat
havediseasedandmutatedmtDNA.Thisresearchispartlyduetomitochondrialdisease
inhumansnotaffectingjustafewcells,buteitheramixtureofmutatedandwildtype
mtDNAarepresent(heteroplasy)orcopiesofmtDNAareallmutated(homoplasy).This
leads to people with mitochondrial disease being compromised and severely energy
deficientwithsymptomsincludingmuscleweakness,pain,motorcontrolloss,growth
deficiency, and gastrointestinal disorders to name a few complications depending on
whichcellsarespecificallyaffected.InthearticletitledPronucleartransferinhuman
embryos topreventtransmissionofmitochondrialDNA disease,researchersshowed
that transfer of pronuclei between abnormally fertilized human zygotes results in
minimalcarryoverofdonorzygotemtDNAandiscompatiblewithonwarddevelopment

totheblastocyststage invitroandtheyhypothesizethatpronucleartransferbetween
zygotes has the potential to prevent the transmission of mtDNA in humans with
increasedstudyandimprovementofprocedure7.
CurrentlyresearchersatNewcastleUniversityinnorthernEnglandareattempting
toperformmtDNAtransplantsfromembryoswithhealthymtDNAtodefectivemtDNA
and transferring these embryos into a womens womb for development, but face
tremendouscontroversyfromthecommunitywithsomebelievingthatthemanipulation
ofanembryosDNAisunethicalduetodamagingeffectsonthatpersonshealth
couldbepasseddownthegenerationstothatpersonsdescendants 8.Althoughthereis
controversyaboutthetrials,theEnglishgovernmentissaidtoallowthecontinuanceof
thestudy.Highimpactresearchthathasbeendonebyalloftheseresearchersshouldbe
consistently questioned by members of the scientific community for how ethical the
experimentsarebecauseoftheimplicationstheresultscouldhaveonhumanity.This
priorresearchthathasbeendonehasmotivatedtheaimsoftheproposedresearchthat
wouldliketobedonewiththehelpoftheappraisalofthisgrant.

Researchstrategy
Thesignificanceofthefollowingresearchstrategycomparedtootherresearchis
thatitseekstoconnecttheaffectsofmtDNAdamageonmorethanonelevelbecausethe
firstaimoftheexperimentisneededinorderforthefinalaimoftheproject.Thiswill
clearlybeexplainedinthiscomponentoftheproposal.Inordertoclarifythecorrelation
betweenapoptosisandmtDNAdamage,cytoplasmichybridswithmutatedmtDNAwill

betransplantedinto200micewithhealthymtDNAandwillbecomparedtothelifespan
ofmicewithhealthymtDNA.Theleveloffunctionandmobilityofeachmousewillbe
measuredandrecordedtodeterminethesignificancethatdamagedmtDNAhasona
diseasedmouse.Themethodsofthisaimoftheexperimentwillbesimilartothemethods
completedbyInoueetal.,butwillhavetheaddedaspectofbehavioralobservation.
GenerationofmicewithmutatedmtDNAwillbeginbyintroducingmutatedpathogenic
mtDNAintomicezygotesbyelectrofusionofpronucleusstageembryoswithenucleated
cytoplastsofthepathogenicmtDNA.Theseembryosarethentransferredtotheoviduct
of psuedopregnant females9. Pathophysiological consequences will be noted in these
cytoplasmichybrids.Thenumberofprogenythatsurvivetoadulthoodwillbemeasured
andtheprogenythatdosurvivewillbeobservedtoseetheapoptoticactivityintheir
cells.Detectionofmitochondrialactivitywillbedoneusinghomogenouscolorimetric
assay. Mitochondrial assays utilize cationic dye that fluoresces one color to indicate
healthycellsandadifferentcolorthatsindicativeofapoptoticcells.Thismethodutilizes
thedifferentmembranepotentialsseenwhenthemitochondriaishealthyasopposedtoa
mitochondria undergoing apoptosis10. MitoCaptureTM Mitochondrial Apoptosis
DetectionFluorometricKitmaybeused.Acomparisonofthephysicalactivityofthe
controlmiceandthemutantmicewillallowtheresearchteamtomakeconclusionsofthe
detrimentaleffectsofthemutantmtDNA.Ifcontinuedresearchisallowed,someofthe
progenyofthemutatedmicewillundergotransplantsofhealthymtDNAtothefemales
embryosinordertohavegenerationsofdataoftheimpactoftransplantingmtDNA.

Ourproposedresearchalsoaimstoaccumulatedatathatpointstowhetherornot
ROSaretheprimarysourceofmutatedmtDNA.Thefocusofthisproposedresearchis
on varied amounts of ionizing radiation exposure to healthy mtDNA of 200 mice.
Through the various amounts of IR exposure, researchers can see what amount of
radiationhasthegreatestagingconsequenceandifthereisanypointwhereIReffects
plateauorbecomeinsignificant.Theresults fromthis partoftheexperimentwillbe
comparedtowildtypemtDNAundergoingnormalreplicationandtherateofmutationin
multipleconsecutivegenerations willbenotedandanalyzed.Thenaturallyoccurring
mutationswillbeanalyzedtoseewhatkindofmutationsareoccurringincludingpoint
mutations,rearrangement,anddeletions.
Thefinalaspectofthisprojectaimsatdeterminingtheefficacyoftransplantation
ofhealthymtDNAintoembryoswithknowndetrimentalmtDNAintheirmitochondrial
genomes.Asstatedbefore,someofthemiceprogenythatsurvivedfromthefirstpartof
the experiment will have healthy mtDNA transplanted and will replace the damaged
mtDNA. This will also be done to embryos that have naturally damaged mtDNA
maternallyinherited,themousemodelofmitochondrialdiseasesuchasJAXMicestrain
B6.129S7Sod2tm1Leb/Jbeingapotentialstrain.ThepercentageofdonormtDNAcarrying
over to the fertilized embryo will be determined through analysis. This part of this
experimentwillallowresearcherstotracethedevelopmentofmtDNAmutationsthrough
severalgenerationsandthelengthoftheextensionoflifespansofthemicewilldetermine
thesignificanceandefficacyofmtDNAtransplants.Ifallgoeswellinthisseriesof
experiments,humanmtDNAtransplantationtrialscouldbethetopicofanothergrant

proposalfromourresearchteam,butonlyiftheextensionoflifespanissignificantinour
studyandthesafetyoftheembryosisnotcompromised.

Expectedoutcome
Sincethisprojectfocusesonthreeaims,thissectionwillclarifytheactionsthat
willbetakeninresponsetopossibleoutcomesfromtheexperiment.Ifthecytoplasmic
hybridsfailtothriveintheexperiment,meaningthattheyareunabletotakethenew
enucleated cytoplasm, then we will attempt to use a mouse model of mitochondrial
disease such as JAX Mice strain B6.129S7Sod2tm1Leb/J. These mitochondrial mouse
modelsexhibitclassicsignsofmitochondrialdiseasebyexhibitingseverewastingand
novelpathogenicphenotypes,includingsevereanemia,degenerationofneuronsinthe
basal ganglia and brainstem and progressive motor disturbances, and myocardial
injury11.Thelevelofmitochondrialmetabolismandapoptosiswillbemeasuredinthese
mutantmiceandtheresultswillbecomparedtowildtypemiceapoptosislevelsasthey
age.
Themousemodelsofmitochondrialdiseasecanalsobeusedforthefinalportion
oftheproject.TheywouldbeusedtotransplanthealthymtDNAintotheirmitochondrial
genomesfromhealthyembryos.Thedownsideofusingthesemodelsisthatwewould
have to continue the experiment for many more generations in order to receive an
adequateunderstandingoftheaffectsfromtheexperiment.Useofthesemodelsmaybe
implementedearlierintheexperiment,butthatisuptotheresearchteamsdiscretion.If
there is no significant change in metabolic activity during the ROS exposure to the

mtDNA,theresearchteamwillemploymorethoroughmethodstoevaluatetheimpact
ROShasonthehealthymtDNA.ProceduresuchasmitochondrialDNAisolationand
cytochromeoxidaseapoptosiskitsmaybeuseddependingontheoutcomeoftheresults.
The expected outcome is that the cytoplasmic hybrids will have significantly
higherlevelsofapoptosisandagingwillbefastercomparedtothewildtypemice,ROS
willbetheprimarysourceformutationinhealthymtDNA,andmtDNAtransplantswill
provetobemoreefficientbythefertilizedembryohavinggreatercarryoverofdonor
mtDNAthaninpreviousfindings.

RealisticBudget
The realistic budget is as follows: 50% of the Principal Investigators salary,
whichis40,000,onepostdoctoralat$50,000/year,onePhDstudentat$25,000,34%
Fringebenefitsonsalaries, $15,000peryearinresearchsupplies,$3,000fortravelto
scientific meetings. The allocation of other costs includes $5,000 for miscellaneous
items,and48%InstitutionalOverhead(theLSUrate)onthewholebudget. TheLSU
CollegeofScience,BiologicalSciencesDepartmenthasalltheequipmentnecessaryfor
theprojectwithoutpurchase.Thetotalrealisticbudgetfortheprojectedgrantthreeyear
periodincludingFacilitiesandAdministrativecostsis$ 753,204.

BudgetJustification

Theresearchteamconsistsofthreevitalmembers:thePrincipalInvestigator,one
postdoctoral and one PhD student. The Principal Investigator will primarily oversee
experimentalproceduresandadjusttheprojectonanasneededbasis,providelaboratory
research staff with protocols, ensure safety practices and techniques are employed,
complywithandensuretheintegrityoftheresearchresults,remainincommunication
withNIHinordertoupdateonresearchfindingsandreportsignificantproblems.The
Principal Investigator is available and should be utilized by the other research team
memberstobeasoundingboardforideasandresults.
ThepostdoctoralwillassistthePrincipalInvestigatorsfortheabovedutiesand
willperformtheproceduresandassessmentsnecessaryforeachofthecomponentsofthe
experiment.ThepostdoctoralwillbeanadviseranddelegatorforthePhDstudentand
willanalyzetheinformationallocatedfromtheresearchexperiment.ThePhDstudent
willperformdaytodayproceduresanddocumentfindingsonacontinuousbasisinorder
toanalyzethedata.ThePhDstudentwilladjusttheconditionsofeachcomponentofthe
experimentandwillassistthepostdoctoralinmtDNAtransplantationaswellasperform
themitochondrialmetabolismassay.Thethreemembersoftheresearchteamwillwork
togetherandutilizeeachothersstrengthsinordertoproducethemostpreciseresults
fromtheexperimentalprocedure.

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