Theeffectofaketogenicdietinthetreatmentofsuccinicsemialdehydedehydrogenasedeficiencyinmice

KirkJonNylen

Thisthesisissubmittedinconformitywiththerequirementsforthedegreeof

DoctorateinPharmacology

GraduateDepartmentofPharmacology

UniversityofToronto

©CopyrightbyKirkNylen(2008)

ii

ABSTRACT

Theeffectofaketogenicdietinthetreatmentofsuccinicsemialdehyde

dehydrogenasedeficiencyinmice

KirkNylen

DoctorateofPhilosophy

DepartmentofPharmacology

FacultyofMedicine

UniversityofToronto

2008

Succinic semialdehyde dehydrogenase (ALDH5A1) deficiency (SSADH‐d) is

an autosomal recessive, inborn error of gamma‐aminobutyric acid (GABA)

metabolism that results inpsychomotor retardation, ataxiaandseizures.Amouse

modelofSSADH‐d(theAldh5a1‐/‐mouse)wascreatedtostudythepathophysiology

and treatment of SSADH‐d. Aldh5a1‐/‐ mice have psychomotor retardation and a

progressive seizure phenotype results in death around P25. The present

experimentstestedtheeffectsofaketogenicdietinthetreatmentofAldh5a1‐/‐mice.

TheKDwas found to prolong the lives ofAldh5a1‐/‐mice by >300%while

significantlydelayingtheonsettheataxiaandpreventingweightlossthatisseenin

untreatedAldh5a1‐/‐mice.Electrophysiologicalrecordingsrevealedacorresponding

decrease in seizures in KD fed mutants, as compared to control diet (CD) fed

mutants.Weassessed spontaneousminiaturepostsynaptic currents (mPSC) inCD

iii

and KD fed mutants. We found that CD fed mutants had significantly decreased

inhibitory mPSC (mIPSC) activity compared to CD fed wildtype controls. mIPSC

activity was restored in KD fed Aldh5a1‐/‐ mice. A similar effect was found in

[35S]TBPS binding experiments. TBPS bindingwas significantly reduced in CD fed

Aldh5a1‐/‐mice, but restored inKD fedmutants. Plasma analysis revealed that an

elevationofserumbeta‐hydroxybutyratemayplayaroleintheKD’seffects.TheKD

ledtoasignificantelevationinthenumberofhippocampalmitochondriainmutant

mice.Further,theKDwasabletonormalizethedeficienciesinthehippocampalATP

levelsseenintheAldh5a1‐/‐mice.

ThepresentdatasuggestthattheKDisabletosignificantlyimprovethe

Aldh5a1‐/‐phenotype.TheeffectoftheKDonmIPSCactivityisnovelandfurthers

ourunderstandingofhowtheKDmayexertitseffects.Themitochondrialstudies

confirmthefindingsofothers,thattheKDelevatesthenumberofmitochondria.The

KDalsorestoresATPdeficienciesinAldh5a1‐/‐mice,whichisanovelfinding.

Together,theseshowthattheKDmaybeaneffectivetreatmentforSSADH‐din

humans.ThesedataalsofurtherourunderstandingoftheKD’smechanismsof

action.

iv

ACKNOWLEDGEMENTS

TheoriginalgoalofmyPhDresearchwastodeterminetheinvitro

mechanismofacetoneasitpertainstotheanticonvulsantmechanismofthe

ketogenicdiet.Thiswastoinvolvewhole‐cellpatchclampingstudies.Myoriginal

PhDSupervisor,Dr.Burnham,didnothaveaninvitroelectrophysiologyrig—soDr.

Sneadallowedmetoworkonhislab’srigunderthehelpfulsupervisionofDr.Perez

Velazquez.Ittookmeafewmonthstodevelopmypatchingskillsand2daysto

figureoutthatyousimplycannotpatchcellsinthepresenceoftherapeutic

concentrationsofacetone.AllmyplannedexperimentswentoutthewindowandI

hadtocomeupwithanewproject.

Somelatenightwheelinganddealingwasdoneinabackalley.Lawyersdrew

upthepapers.ItwasdecidedthatIwouldjoinDr.Snead’slabtoworkon

experimentsfromtheirnewlyfundedgrantexploringtheroleofGABAAinmurine

succinicsemialdehydedehydrogenasedeficiency.Ididn’texactlydothese

experimentseither,thankstoDr.Snead’sgraceandabilitytoentrusthisstudents

withsufficientautonomytotesttheirownhair‐brainedideas.Asevidencedbythis

thesis,weallgotourways.Icontinuedmyresearchontheketogenicdietandwegot

alotofgooddatausingthemousemodelofsuccinicsemialdehydedehydrogenase

deficiency.

v

­Supervisors­

Iwouldlike,firstandforemost,tothankDr.Sneadfortakingmeonashis

student.Icanonlyhopetoachieveasimilarlevelofrespectandadmirationfrommy

past/present/futurecolleaguesandstudentsasyouhave.Yourmentorshipand

supporthavebeeninvaluable.

I’dalsoliketothankDr.JoseLuisPerezVelazquezforbeingmydefacto

advisorandprovidingmewithstrongguidanceandmentorship.Mostimportantly,

thankyouforintroducingmetothegreatwinesofRioja.Salut!

ThankyoutoDr.Burnhamforco‐supervisingmyPhDresearchandbeinga

mentorandguideduringmyentiregraduatestudentexperience.Heismystandard

forteachingexcellence.

­Committee­

Thankyoutomycommitteemembers:Dr.ElizabethDonner,Dr.Cindy

Woodland,Dr.PerezVelazquez,Dr.BurnhamandDr.Sneadfortakingthetimeto

meetwithmeandhelpdirectmyresearch.Dr.Woodland‐aspecialthanksforallof

yoursupportandencouragementoverthecourseofmygraduatestudies.

­LabMates‐

IwouldliketothankallofthefollowingmembersofDr.Snead’slaboratory

whohelpedmecompletemythesisinvariousways:Firstly,thankyoutoDr.Miguel

Cortezformanymotivatingdiscussionsandforprovidingmewithmuchsupport;

DickLiu(xiexiefororderingthingsandteachingmeTBPSbinding),Dr.YingWu(xie

vi

xieforthemanytipsandhelpfuldiscussions),YevgenLeshchenko(огpомное

спасибоforhelpwithelectrophysiologyandmanygreatchats…Мньощо),Lily

Shen(xiexieforallthegenotypingandalltheworkwiththemice),Larissa

Kokarovtseva(огpомноеспасибоLarissa,Неболтай!),LuisyRamon(gracias),Lee

(thanksforyourpatienceregardingthemanuscriptandforallowingmeto

collaboratewithyou),BarbaraZimnowodzkiandWendyRicketts‐thanksforyour

patienceandhelpwitheverything.

Tomy“other”labmatesinDr.Burnham’slab.SergeiandPeter(longlive

ketogroup!),Elan(thebestoffice‐mateever.Thankyouforallofthewonderful

conversationsandgut‐bustinglaughs.IlookforwardtomoreSANDandother

collaborations),Deborah(thanksforbeingawonderfulfriendandsupportoverthe

pastseveralyears.Canyoubelievethatwedidit?!),Brian(thanksforcountless

greattalksandforbeinganendlessfountainofknowledge),Kathryn(thanksforall

thehelpwithexperimentsandthegreattimeswithSACECandSAND),Christa

(thanksforshowingmethatyouaresimplyfaster,strongerandjustplainbetter),

Sofia(lestweforgetSAND06’)andJerome‐forkeepingthingstogether.

­Family‐

ToRenee,mysourceofgreathappinessandencouragement:Youleftfor

workintherainwhileIstayedathomeinmypajamastowrite‐andyouneveronce

complained.Iamaluckyguy.

vii

ToMomandDadforimmeasurablesupport.ToRob,Jill,Nick,Ethan,

Annaliese,andSeth.ToKristi,Dustin,Maddy,Emma,Olivia,SamandEva.ToKendra

andEvan.ToRick,myTorontofamily.ToDanandDoris.

InmemoryofJay‐thiswas“planB”tobecomingcustodianbrothers.

­Funding‐

IwouldliketothankallofthegroupsthathelpedfundmeduringmyPhD:

TheUniversityofToronto,DepartmentofPharmacology(numerousUofT

Fellowships,2004‐2007);TheHospitalforSickChildren(RestracompFellowship,

2006‐2007);TheSavoyFoundation(VanGelder‐SavoyAward2007);andCIHR

(DoctoralResearchAward,2008).

viii

TABLEOFCONTENTS

ABSTRACT iiACKNOWLEDGEMENTS iv

TABLEOFCONTENTS viii

LISTOFTABLES xiiLISTOFFIGURES xiii

LISTOFCHEMICALS xvLISTOFDRUGS xvi

CONTRIBUTIONSTOEXPERIMENTS xvii

CHAPTER1 1GENERALINTRODUCTION 11.1 SuccinicSemialdehydeDehydrogenaseDeficiency–TheClinicalSyndrome 11.2 SSADH­dEpidemiology 31.3 SSADH­dSymptoms 31.4 SSADH­dDiagnosis 41.5 EtiologyofSSADH­d 41.6 TreatmentofSSADH­d 51.7 SuccinicSemialdehydeDehydrogenaseDeficiency–TheAnimalModel 71.8 BiochemicalPerturbationsinAldh5a1­/­Mice 101.9 EffectsofSSADH­donFatOxidationinAldh5a1­/­Mice 161.10 EffectsofSSADH­donEnergyMetabolisminAldh5a1­/­Mice 171.11 AttemptedPharmacologicalTreatmentofSSADH­dinAldh5a1­/­Mice 171.12 RationaleforTestingtheKetogenicDietinAldh5a1­/­Mice 181.13 TheKetogenicDiet 191.14 HistoryoftheKD:FromFastingto4:1 191.15 ClinicalProfileoftheKD 211.16 TypesofKD 241.17 Side­EffectsoftheKD 251.18 TheKetogenicDiet’sMechanismofAction 261.18.1 TheBrainLipidsTheory 261.18.2 ThepHTheory/Keto‐acidosisTheory 271.18.3 TheGABAShuntTheory 271.18.4 TheEnergySubstrateTheory 301.18.5 TheKetonemiaTheory 311.18.6 TheAcetoneTheory 32

1.19 GeneralObjectives 351.20 SpecificObjectivesandPurposeofExperiments 35

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CHAPTER2 37

GENERALMETHODS 372.1 Subjects 372.2 Genotyping 372.3 Diets 382.3.1 4:1KetogenicDiet(KD) 382.3.2 ControlDiet(CD) 39

2.4 Statistics 39

CHAPTER3 42

EXPERIMENT1:THEEFFECTSOFAKETOGENICDIETONTHEALDH5A1­/­PHENOTYPE 423.1 Introduction&Rationale 423.2 Methods 443.2.1 Subjects 443.2.2 DeterminingLifespan 453.2.3 DeterminingWeights 453.2.4 DeterminingAtaxia 453.2.5 SurgeryandElectrocorticography(ECoG) 453.2.6 CharacterizationofSeizuresandConvulsions 46

3.3 Results 473.3.1 Lifespan 473.3.2 Weights 513.3.3 Ataxia 513.3.4 Electrocorticography 52

3.4 Discussion 593.4.1 Lifespan 593.4.2 Ataxia 613.4.3 Weights 623.4.4 Electrocorticography 633.4.5 Conclusions 65

CHAPTER4 66EXPERIMENT2:THEEFFECTSOFAKETOGENICDIETONMINIATUREPOST­SYNAPTICCURRENTSINALDH5A1­/­MICE 664.1 Introduction&Rationale 664.2 Methods 684.2.1 Subjects 684.2.2 Electrophysiology:BrainSlicesandSolutions 684.2.3 Electrophysiology:WholeCellRecordings 694.2.4 DataAnalysis 70

4.3 Results 704.3.1 MiniatureInhibitoryPost‐synapticCurrents(mIPSC) 704.3.2 MiniatureExcitatoryPost‐synapticCurrents(mEPSC) 75

4.4 Discussion 80

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4.4.1 mIPSC 804.4.2 mEPSC 834.4.3 Conclusions 83

CHAPTER5 85EXPERIMENT3:THEEFFECTSOFAKETOGENICDIETON[35S]TBPSBINDINGINALDH5A1­/­MICE 855.1 Introduction&Rationale 855.2 Methods 865.2.1 Subjects,SacrificeandPreparationofSlices 865.2.2 Ligands 875.2.3 T‐[35S]butylbicyclophosphorothionate([35S]TBPS)Autoradiography 875.2.4 [35S]TBPSQuantification 885.2.5 DataAnalysis 89

5.3 Results 895.4 Discussion 92

CHAPTER6 94

EXPERIMENT4:THEEFFECTSOFAKETOGENICDIETONSERUMANALYTESINALDH5A1­/­MICE 946.1 Introduction&Rationale 946.2 Methods 966.2.1 Subjects,SacrificeandCollectionofSerum 966.2.2 DeterminationofGlucoseinSerum 976.2.3 Determinationofβ‐HydroxybutyrateinSerum 976.2.4 DeterminationofNon‐EsterifiedFattyAcids(NEFA)inSerum 98

6.3 Results 1006.3.1 SerumGlucoseLevels 1006.3.2 SerumβOHBLevels 1006.3.3 SerumNEFALevels 101

6.4 Discussion 105

CHAPTER7 108

EXPERIMENT5:THEEFFECTSOFAKETOGENICDIETONMITOCHONDRIALNUMBERANDFUNCTIONINALDH5A1­/­MICE 1087.1 Introduction&Rationale 1087.2 Methods 1107.2.1 Subjects 1107.2.2 TissuePreparationforElectronMicroscopy 1107.2.3 ElectronMicroscopy 1127.2.4 AnalysisofMitochondrialCounts 1127.2.5 TissuePreparationforATPAssay 1127.2.6 ATPCalibrationCurves 1137.2.7 QuantificationofMitochondrialATPProduction 115

7.3 Results 1157.3.1 MitochondrialDensity 115

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7.3.2 MitochondrialArea 1167.3.3 ATPQuantificationinHippocampalTissue 120

7.4 Discussion 1227.4.1 MitochondrialNumberandSize 1227.4.2 MitochondrialATPLevelsinHippocampus 1247.4.3 Summary 125

CHAPTER8 126GENERALDISCUSSION 1268.1 GeneralHypothesis 1268.2 SummaryofExperiments 1268.2.1 Experiment1:Lifespan,Ataxia,WeightandECoG 1278.2.2 Experiment2:MiniaturePost‐SynapticCurrents 1328.2.3 Experiment3:[35S]TBPSBinding 1348.2.4 Experiment4:SerumAnalytes 1378.2.5 Experiment5:MitochondrialCountsandFunction 141

8.3 LimitationsofStudies 1438.4 InsightsintotheKD’sMechanismsofAction 1448.4.1GeneralComments 1448.4.2 TheMechanismsoftheKDinSSADH‐d 145

8.5 TheKetogenicDietintheClinicalTreatmentofSSADH­d 1478.6 FutureStudies 1488.7 Conclusions 152

REFERENCES 153LISTOFPUBLICATIONSANDABSTRACTS 172PublishedPapers(12). 172PublishedAbstracts(10). 173UnpublishedConferenceAbstracts(6). 174GraduateAwards 175

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LISTOFTABLES

Table1GHBandGABALevelsinWildtypeandAldh5a1‐/‐Mice p.11Table2ApproximateChangeinAminoAcidLevelsinAldh5a1‐/‐MiceComparedtoAldh5a1+/+Mice p.12Table3Compositionofthe4:1KetogenicDiet(KD) p.40Table4CompositionoftheControlDiet(CD) p.41Table5CalibrationTableforβOHBAssay p.98Table6CalibrationTableforNEFAAssay p.100

xiii

LISTOFFIGURES

Figure1TheRoleofSSADHDeficiencyintheElevationofGABAandGHB p.2

Figure2SizeDifferenceBetweenAldh5a1+/+andAldh5a1‐/‐Mice p.8

Figure3ElectrocorticographyRecordingsinWildtypeandAldh5a1‐/‐Mice p.9

Figure4MetabolicPathwayforSuccinicSemialdehydeDehydrogenase p.15

Figure5TheHistoricalEfficacyoftheKetogenicDiet p.23

Figure6BreakdownofaTypicalAmericanDietvs.KetogenicDiet p.24

Figure7TheGABAShunt p.29

Figure8.TheAnticonvulsantEffectsofAcetoneinAnimalModelsofEpilepsyp.34

Figure9PicturesofCDandKDfedAldh5a1‐/‐Mice p.48

Figure10AverageLifespansforCDandKDfedAldh5a1‐/‐Mice p.49

Figure11SurvivalCurvesforKDandCDFedAldh5a1‐/‐Mice p.50

Figure12ProgressionofAverageDailyWeightGaininAldh5a1‐/‐Mice p.53

Figure13AverageDailyWeightGaininAldh5a1‐/‐andAldh5a1+/+Mice(P12Onwards) p.54

Figure14AverageGroupAtaxiaScoresinAldh5a1‐/‐Mice p.55

Figure15ECoGRecordingsinAldh5a1‐/‐MiceFedEitheraCDorKD p.56

Figure16AverageNumberofConvulsionsDuring1hrECoGRecordingsinCDandKDfedAldh5a1‐/‐Mice p.58

Figure17TheEffectofa4:1KDonmIPSCCharacteristicsinAldh5a1‐/‐Mice p.72

Figure18TheEffectofa4:1KDonmEPSCCharacteristicsinAldh5a1‐/‐Mice p.77

Figure19RepresentativemIPSCandmEPSCTraces p.79

Figure20AverageGroup[35S]TBPSBinding p.91

Figure21MeanSerumGlucoseLevels p.102

xiv

Figure22MeanSerumβOHBLevels p.103

Figure23MeanSerumFreeFattyAcidLevels p.104

Figure24Figure25CalibrationCurveforATP‐GloAssay p.114

Figure25.ElectronMicrographofPyramidalNeuronandItsMitochondria p.117

Figure26AverageMitochondriaNumberPer10μm2ofSomaticArea p.118

Figure27PercentofSomaticAreaOccupiedbyMitochondria p.119

Figure28HippocampalATPLevels p.121

Figure29IllustrationofSeizureThreshold p.131

Figure30ProposedMechanismfortheKetogenicDiet’sEffects OnNeuralHyperexcitability p.136

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LISTOFCHEMICALS

Chemicals

Chemical CAS Company

CalciumChlorideDihydrate 10035‐04‐8 Fluka

CesiumChloride 7647‐17‐8 Riedel‐de‐Haën

CesiumHydroxide 21351‐79‐1 Aldrich

CesiumMethanesulfonate 2550‐61‐0 Sigma

EGTA 67‐42‐5 Sigma

Glucose 50‐99‐70 Sigma

Gluteraldehyde 111‐30‐8 Sigma

GTP 85737‐04‐8 Sigma

HEPES 7365‐45‐9 Sigma

Magnesium‐ATP 74804‐12‐9 Sigma

MagnesiumSulphate 7487‐88‐9 Fluka

OsmiumTetroxide 20816‐12‐0 Sigma

PotassiumChloride 7447‐40‐7 Riedel‐de‐Haën

PotassiumFerrocyanide 14459‐95‐1 Sigma

PotassiumPhosphateDibasic 7758‐11‐4 Sigma

PropyleneOxide 75‐56‐9 Fluka

SodiumBicarbonate 144‐55‐8 Fluka

SodiumCacodylateBuffer 97068 Sigma

SodiumChloride 7647‐14‐5 Sigma

SodiumPhosphateMonobasic 7558‐80‐7 Sigma

xvi

LISTOFDRUGS

Drug DIN Company

AP5 79055‐68‐8 TocrisD‐(‐)‐2‐Amino‐5‐phosphonopentanoicacid

BicucullineMethiodide 40709‐69‐1 Tocris[R‐(R*,S*)]‐5‐(6,8‐Dihydro‐8‐oxofuro[3,4‐e]‐1,3‐benzodioxol‐6‐yl)‐5,6,7,8‐tetrahydro‐6,6‐dimethyl‐1,3‐dioxolo[4,5‐g]isoquinoliniumiodideCNQX 115066‐14‐3 Tocris6‐Cyano‐7‐nitroquinoxaline‐2,3‐dionePicrotoxin 124‐87‐8 SigmaSodiumPentobarbital 00141690 MTCPharmaceuticals(Somnotol®)

[35S]TBPS NEG049000MC PerkinElmer[35S]tert‐butylbicyclophosphorothionate(65Ci/mmol;100μCiin0.05mlofethanol)

Tetrodotoxin 4368‐28‐9 Sigma

xvii

CONTRIBUTIONSTOEXPERIMENTS

GeneralContributions

AllgenotypingwasperformedbyLilyShen,Dr.Snead’stechnician.Imade

theketogenicdietandfedallanimalsonadailybasis.

Experiment1:TheEffectsofaKetogenicDietontheAldh5a1­/­Phenotype

IaidedDr.MiguelCortez,aclinicalneurophysiologistattheHospitalforSick

Children,withtheECoGelectrodeimplantationsurgeries.Togetherwecollectedthe

ECoG readings andevaluated/interpreted theECoG records. I performedall other

aspects of these experiments (weighing animals, ataxia assessment, lifespan

assessment).Thisstudyhasbeenpublishedinfull(Nylenetal.,2007).

Experiment2:TheEffectsofaKetogenicDietonMiniaturePost­SynapticPotentialsinAldh5a1­/­Mice

IwastaughtthepatchclampingmethodbyDr.JoseLuisPerezVelazquez.All

invitroelectrophysiologyexperimentswereprepared,performedandanalyzedby

me.Thisstudyhasbeenpublishedinfull(Nylenetal.,2007).

Experiment 3: The Effects of a Ketogenic Diet on [35S]TBPS Binding inAldh5a1­/­Mice

I prepared brain slices for the [35S]TBPS assay. I assisted Dick Liu (head

technicianforDr.Snead)inthe[35S]TBPSbindingassay.Mr.LiuandIanalyzedand

interpretedthe[35S]TBPSassaybindingtogether.Thisstudyhasbeenpublishedin

full(Nylenetal.,2007).

xviii

Experiment4:TheEffectsofaKetogenicDietonSerumAnalytesinAldh5a1­/­Mice

Icollectedandpreparedallbrainandserumsamplesforanalysis.Iassisted

Dr.SergeiLikhodii (clinical chemistat theHospital forSickChildren) inanalyzing

andinterpretingtheNEFA,βOHBandglucoselevels.Thisstudyhasbeenpublished

infull(Nylenetal.,2007).GHBandGABAlevelsinbraintissueandbloodarebeing

analyzedbyDr.EduardStruysinHolland.

Experiment 5: The Effects of a KetogenicDiet onMitochondriaNumber andFunctioninAldh5a1­/­Mice

I performedperfusions and collectedbrain samples forMr.RobertTemkin

(electronmicroscopytechnologistatMountSinaiHospital) toprepareforelectron

microscopy.Mr.Temkintaughtmehowtousetheelectronmicroscope.Iperformed

allmicroscopyanddataanalysis.IpreparedthetissueandperformedtheATP‐Glo

assay(includingstandardcurves).Iwasassistedbya4thyearprojectstudent,Venus

Sayed.Thisstudyiscurrentlybeingwrittenupforpublication.

Thesis

AllaspectsofthisthesiswerepreparedandwrittenbyKirkNylen.Thankyou

toDr.W.M.BurnhamandDr.OCSneadforhelpingeditthisthesis.

1

CHAPTER1

GENERALINTRODUCTION

1.1 SuccinicSemialdehydeDehydrogenaseDeficiency–TheClinical

Syndrome

Succinic semialdehyde dehydrogenase (SSADH; ALDH5A1) deficiency

(SSADH‐d;alsoknownas“γ‐hydroxybutyricaciduria”)isarare,autosomalrecessive

genetic disorder of γ‐aminobutyric acid (GABA) catabolism (Gibson et al., 1983;

Gibsonetal.,1997;GibsonandJakobs,2001;Gibsonetal.,2005).SSADH‐dispoorly

understood and requires significant research attention to understand the

pathophysiologyandtreatmentofGABAcatabolismdisorders.

SSADHisresponsibleformetabolizingsuccinicsemialdehydeintosuccinate.

AsshowninFigure1,deficienciesinthelevelofSSADHleadtoa“back‐log”inthe

pathwayandresultinasignificantincreaseinGABAandγ‐hydroxybutyrate(GHB)

levels (Gibson and Jakobs, 2001; Pearl et al., 2003). Previous studies have

demonstrated thatGABA is elevated three‐ to five‐fold abovenormal (Gibson and

Jakobs, 2001; Pearl et al., 2003). GHB is elevated 30‐ to 50‐fold above normal,

resulting in GHB aciduria (Gibson and Jakobs, 2001; Pearl et al., 2003). The

significantelevationoftheseneuroactivecompoundsisthoughttoplayaroleinthe

psychomotor retardation, ataxia and epilepsy—with seizure types ranging from

absence seizures to convulsive status epilepticus—seen in patients with this

disorder(Gibsonetal.,1998;Pearletal.,2003;Gordon,2004).Currently,thereisno

effectivetreatmentforhumanSSADH‐d(GibsonandJakobs,2001;Gropman,2003).

2

Figure1.TheRoleofSSADHDeficiencyintheElevationofGABAandGHB

TheGABAmetabolicpathwayissignificantlydisruptedinSSADH‐d.ThedeficiencyinSSADHlevelscausesa“back‐log”ofGABAandGHBlevelsinbothbrainandperipheraltissues.✠GABAtransaminaseisinhibitedbyvigabatrin(VGB)causingfurtherelevationsinGABA,butpreventingfurtherelevationsofGHB.

AdaptedfromGibsonetal.,1992andGuptaetal.,2004

Succinate

GABA-conjugates G

luta

mic

aci

d de

carb

oxyl

ase

Glutaminase Glutamine Glutamate

GABA *3-5 fold elevation*

Succinic Semialdehyde

*unchanged or only slightly elevated*

✠G

ABA

tran

sam

inas

e

Krebs Cycle

Succinic semialdehyde

dehydrogenase

Gamma-hydroxybutyrate

*30-50 fold elevation*

Succinic semialdehyde

reductase

3

1.2 SSADH­dEpidemiology

SSADH‐d is a rare disorder, with fewer than 500 cases ever reported

worldwide(Gropman,2003).TheactualprevalenceislikelymuchhigherasSSADH‐

d is probably under‐diagnosed, or mistaken for another disorder, because of its

broad,non‐specificneurologicalsymptoms.

1.3 SSADH­dSymptoms

ThemostcommonsymptomsofSSADH‐dincludepsychomotordelay,ataxia

and seizures (Gibsonet al., 1997).The “clinicalpicture”ofpatientswith SSADH‐d

varies greatly. Somepatientspresentwith severe symptomsand require full‐time

carewhileotherspresentwithmildersymptomsandcanliverelativelyindependent

lives.

BabieswithSSADH‐dmayhaveahigherriskofprematuredelivery,neonatal

lethargy,reducedfeeding,breathingproblemsandhypoglycemia(Gordon,2004).

In childhood, initial concerns are often generatedwhen the child begins to

miss certaindevelopmentalmilestones. Childrenwith SSADH‐dmayhavedelayed

motor function, speech and language (Jakobs et al., 1993). Other symptoms

commonlyseeninchildrenincludehallucinations,poormuscletone,dulledreflexes,

hyperkinesis, under‐developed cortex,myopathy, nystagmus, strabismus, retinitis,

“spiderveins”andseizures(Jakobsetal.,1993;Gordon,2004).

In adults, the disorder is characterized by gross psychomotor retardation,

hypotonia, ataxia, behavioral problems (e.g. heightened aggression) and seizures

(Gibson et al., 1997). The seizures may range from non‐convulsive (e.g., absence

4

seizures)toconvulsive(e.g.tonic‐clonicseizuresorstatusepilepticus)(Gibsonetal.,

1997).

1.4 SSADH­dDiagnosis

Clinically, SSADH‐d is very difficult to diagnose given its non‐specific,

heterogeneousneurologicalsymptoms.DetectionofexcessGHBintheurine isthe

most commonmethodused fordiagnosis (Gibsonet al., 1997). Levels ofGHBare

significantlyelevatedduetotheinabilityofsuccinicsemialdehydetobeconverted

to succinate (via SSADH) and the subsequent metabolic diversion of succinic

semialdehydetoGHBviasuccinicsemialdehydereductase(seeFigure1).

1.5 EtiologyofSSADH­d

SSADH‐d is a rare, autosomal recessive disorder. Consanguinity is very

common(~40%)amongstparentsofSSADH‐dpatients(Gibsonetal.,1997).

SSADH is a NAD+‐dependent mitochondrial enzyme responsible for the

biotransformation of succinic semialdehyde into succinate (Blasi et al., 2002).

Lacking this functional enzyme results in several different changes related to the

GABA catabolic pathway. Most notably, there is a three‐ to five‐fold increase in

blood,urineandcerebralspinal fluidGABAlevels(Gibsonetal.,2001;Pearletal.,

2003). There is also a 30‐ to 50‐fold increase in blood, urine and cerebral spinal

fluidGHBlevels(Gibsonetal.,2001;Pearletal.,2003).Interestingly,theredoesnot

appeartobeaconsistentincreaseinthelevelsofsuccinicsemialdehyde(Gibsonet

5

al.,1992),suggestingthatitistightlyregulatedandreadilyconvertedtoGABAand

GHB.

ThegenethatencodesSSADHinhumans(i.e.,ALDH5A1)hasbeenidentified

and is located on the short arm of chromosome 6p22 (Gordon, 2004). Blasi and

colleagues(2002)havecharacterizedthecompletecDNAandgenomicstructureof

theALDH5A1gene.Theyalsohaveidentifiedsplicemutations,missensemutations

and frame‐shift mutations of the ALDH5A1 gene as the genetic cause for the

heterogeneous clinical phenotype in SSADH‐d patients (Chambliss et al., 1998;

Akaboshietal.,2001;Blasietal.,2002).

Akaboshi et al. (2003) determined that less than 5% of SSADH enzyme

activity is required to confer the SSADH‐d phenotype. They identified several

SSADH‐d‐causing mutations, including seven splice mutations, seven nonsense

mutationsand12missensemutations.Nocommon,prevalentmutationwasfound

in the patients studied. The diversity in ALDH5A1 mutations could explain the

diversityinthegeneralfunctioningofpatientswithSSADH‐d.

TherearenoknownenvironmentalcausesofSSADH‐d.

1.6 TreatmentofSSADH­d

Most treatments for SSADH‐d are limited to treating the symptoms of the

disorder (Gibson et al., 2005). Anticonvulsant medications are used to treat the

seizures in patients with SSADH‐d. Antipsychotic drugs are used to treat the

aggressionoftenseen inadultswithSSADH‐d.Suchdrugshavebeensuccessful in

6

improvingSSADH‐dsymptoms,buttheydonothavedisease‐modifyingeffectsand

theirclinicalbenefitishighlyvaried(Gordon,2004;Gibsonetal.,2005).

The current “gold standard” treatment of SSADH‐d is vigabatrin (VGB).

ClinicaldatasuggestthatVGBhasmixedeffectsintreatingSSADH‐d.Somestudies

have reported abeneficial effect ofVGBon alleviating theneurological symptoms

and disrupted locomotion in patients with SSADH‐d (Jacobs et al. 1992). Other

studies have shown little or no benefit with VGB (Gibson et al., 1995; Gropman,

2003).

Mechanistically, VGB is thought to reduce GHB levels by irreversibly

inhibiting the GABA‐transaminase enzyme (see Figure 1). At the same time,

however, VGB also elevates GABA levels. This could, in part, explain its mixed

efficacy in SSADH‐d patients as VGB further elevates the GABA levels that are

thoughttocausesomeofthesymptomsinthesepatients.

A major concern with the use of VGB is the frequent visual field defects

caused by the drug. These include visual field loss, diplopia, nystagmus, reduced

color vision, reduced color discrimination, impaired visual evoked potentials,

reduced ocular blood flow and retinal atrophy (Gropman, 2003; Virrotti et al.,

2007).

Taken together, there is a need for a safer,more efficacious treatment for

SSADH‐d. Such a treatmentmight be evolved by studying animalmodels of this

disorder. To address this issue, Gibson and colleagues developed an SSADH

knockoutmouse(Aldh5a1‐/‐)thatmodelsclinicalSSADH‐d(Hogemaetal.,2001).

7

1.7 SuccinicSemialdehydeDehydrogenaseDeficiency–TheAnimalModel

SSADH‐dmicewerecreatedbyknockingoutthegenethatencodesSSADH—

theAldh5a1gene.TheresultantAldh5a1‐/‐mice(alsoreferredtointheliteratureas

SSADH‐/‐ mice, Oregon Mice; also referred to in this thesis as “mutants”) were

generated in order to explore the pathophysiology and possible treatment of

SSADH‐d(Hogemaetal.,2001).

Aldh5a1­/­ mice exhibit psychomotor retardation, stunted physical

development(seeFigure2 forsizecomparisonbetweenAldh5a1+/+andAldh5a1‐/‐

mice),ataxiaandaseizuredisorderthatprogressesfromabsenceseizures(~P15)

to lethal status epilepticus (~P25; Cortez et al., 2004). Figure 3 illustrates the

electrographicprogressionofseizuresinmutantmice.AlluntreatedAldh5a1‐/‐mice

diewithinthefirst25daysoflife.

8

Figure2.SizeDifferenceBetweenAldh5a1+/+andAldh5a1‐/‐Mice

Thisfiguredepictsthesizedifferencebetweenage‐matched(P33)Aldh5a1+/+(topright)andAldh5a1‐/‐(bottomleft)mice.TheAldh5a1‐/‐mousewaskeptaliveuntilP33withVGB.

FromGuptaetal.,2002.

9

Figure3.ElectrocorticographyRecordingsinWildtypeandAldh5a1‐/‐Mice.

This figure shows ECoG recordings from a P16 Aldh5a1+/+ mouse (A), a P16Aldh5a1‐/‐mouse(B)andaP20Aldh5a1‐/‐mouse(C).Thearrowsin(B)pointtothefrequent bursts of spike‐and‐wave discharge, which are often associated withhuman absence seizures, seen inAldh5a1‐/‐ mice. Note the transition from spike‐and‐wave activity in (B) to spikes often associated with tonic‐clonic seizures inhumansseenin(C).In(C),arrowsindicatethesustainedrhythmic,highamplitudespikesassociatedwithtonic‐clonicseizuresinAldh5a1‐/‐mice.LF‐RF=leftfrontaltorightfrontal;LP‐RP=leftparietaltorightparietal.AdaptedfromCortezetal.,2004

10

1.8 BiochemicalPerturbationsinAldh5a1­/­Mice

Severalstudieshaveinvestigatedthebiochemicalperturbationsthatoccurin

Aldh5a1‐/‐mice. Some of these perturbations are outlined in Table 1, Table 2 and

Figure3.

MostnotablearethesignificantelevationsinbrainandperipheralGHBand

GABA levels (Table1), similar to thoseseen inhumanswithSSADH‐d(Hogemaet

al., 2001; Gibson et al., 2002; Gibson et al., 2005). Previous studies have linked

elevations in GABA levels to decreased production of the neurosteroids

progesterone and allopregnanolone through inhibition of 3‐betahydroxysteroid

dehydrogenase(Do‐Regoetal.,2000).Guptaetal.(2003)haveshownasignificant

decrease in both progesterone and allopregnanolone from brain extracts of

Aldh5a1‐/‐mice.StudiesperformedbyLonsdaleandcolleagues(2005,2006,2007)

havehelpedrevealtheimportanceofneurosteroidsinsuppressingseizureactivity.

It is possible that perturbations in SSADH and the subsequent reductions in

neurosteroid levels contribute to the seizure disorder observed in the Aldh5a1‐/‐

mice.

Aldh5a1‐/‐ mice have been found to have significant disruptions in other

aminoacidsascompared towildtypecontrols.Table2outlines theseaminoacids

andtheirapproximatelevelchangewhencomparedtocontrols.

11

Table1.GHBandGABALevelsinWildtypeandAldh5a1‐/‐Mice

Metabolite Fluid/Tissue Aldh5a1+/+ Aldh5a1+/­ Aldh5a1­/­

GHB Urinea 4.5,5.4(N=2) 3.3±0.7(N=7) 273±46(N=8)

GABA Urine 38±6.7(N=8) 43±6.0(N=8) 331±71(N=7)

GHB Brainb 0.13±0.02(N=8) 0.12±0.02(N=8) 5.6±1.8(N=8)

GABA Brain 53±3.2(N=9) 46±5.3(N=8) 148±16(N=8)

GHB Liverb 0.08±0.03(N=8) 0.06±0.01(N=8) 1.9±0.6(N=8)

GABA Liver 1.2±0.2(N=9) 1.0±0.2(N=8) 3.9±0.4(N=7)

ammol/molcreatinine.bµmol/gramprotein.GHB=gamma‐hydroxybutyrate,GABA=gamma‐aminobutyrate.

AdaptedfromHogemaetal.,2001

12

Table2.ApproximateChangeinAminoAcidLevelsinAldh5a1‐/‐MiceComparedtoAldh5a1+/+Mice

AminoAcid ~FoldChange

Glutamate +2.0*

Glutamine ‐7.0*

Aspartate +5.0

Homocarnosine +3.0

Arginine ‐4.0

AlllevelsdeterminedatP18.*Leveltakenfromwholebrain.Valuesotherwiserepresenthippocampallevels.

AdaptedfromGuptaetal.,2004

13

Beyond elevations in the above‐mentioned metabolites, several other

metabolites are altered in Aldh5a1‐/‐ mice. Succinic semialdehyde is elevated

approximately three‐fold in brain tissue fromAldh5a1‐/‐mice (Jansenet al., 2006;

Gibsonetal.,2006).Urineandcerebralspinalfluidlevelsofsuccinicsemialdehyde

are increased 10‐15‐fold and 100‐fold, respectively, in patients with SSADH‐d

(Struysetal.,2005).Resultsareinconsistent,however,asGibsonetal.(2002)found

nochangeinsuccinicsemialdehydelevelsinbrainextractsfromAldh5a1‐/‐mice.It

islikelythatsuccinicsemialdehydeistransientlyelevatedandrapidlyconvertedto

othermetabolites,suchasGABAandGHB,whicharethoughttoberesponsiblefor

thebehavioralabnormalitiesinAldh5a1‐/‐mice(Hogemaetal.,2001;Gibsonetal.,

2005).

4,5‐dihydroxyhexanoic acid (DHHA) is also significantly elevated in the

brainsofAldh5a1‐/‐mice(Brownetal.,1987;Gibsonetal.,2005).DHHAisaknown

inhibitor of mitochondrial electron transport chain activity (Okun et al., 2004;

Gibson et al., 2005). As such, it has a detrimental effect onmitochondrial energy

production.Saueretal.(2007)foundasignificantdecreaseinbrainglutathione,as

well as a significant reduction in complex I‐IV function in Aldh5a1‐/‐ mice. Their

results suggest that Aldh5a1‐/‐ mice have increased oxidative stress as well as

mitochondrialdysfunction,especially inthehippocampus.Significantelevations in

DHHA may contribute to the mitochondrial dysfunction found by Sauer and

colleagues.

D‐2‐hydroxyglutaric acid (D‐2‐HG) levels are also significantly elevated in

patientswithSSADH‐daswellasinAldh5a1‐/‐mice(Brownetal.,1987;Struysetal.,

14

2006).D‐2‐HGisformedduringthemetabolismofGHBandisaknownneurotoxin

(Struys et al., 2006). Significant elevations in brain D‐2‐HG and subsequent

neurotoxiceffectsmaycontributetotheAldh5a1‐/‐phenotype(Struysetal.,2006).

Disruption in theglutamate/glutaminecyclehasbeen found inbrain tissue

from Aldh5a1‐/‐ mice, as indicated by significantly reduced glutamine levels and

elevatedglutamate levels (seeTable2;Gibsonetal.,2002).Glutamine isnormally

takenupbyastroglia and shuttled toneurons. Significant reductions inglutamine

levelsmayfurtherimpactneurotransmitterbalanceinAldh5a1‐/‐mice(Pateletal.,

2001).

Taken together, there are multiple perturbations in the GABA related

catabolicpathwayandpathways involved inenergymetabolism(Tsacopoulosand

und Magistretti, 1996). These perturbations may create an imbalance in the

neuronal biochemical milieu and affect neural energy metabolism (Hassel et al.,

1998). The combination of these effects likely plays amajor role in the SSADH‐d

phenotype.

15

Figure4.MetabolicPathwayforSuccinicSemialdehydeDehydrogenase

This figuredepicts the relationship of SSADH toKrebs cycle and theGABA shunt.Upwardpointing arrowsdenote an elevation of succinic semialdehyde (SSA), 4,5‐dihydroxyhexanoic acid (DHHA), alanine (Ala), homocarnosine (HC), γ‐aminobutyrate (GABA), γ‐hydroxybutyrate (GHB). The downward pointing arrowdenotesadecreaseinglutamine(Gln).Abbreviations: α‐KG (alpha‐ketoglutarate), NADH (reduced nicotinamide adeninedinucleotide),ETC(electrontransportchain),ATP(adenosine50‐triphosphate),His(histidine), Glu (glutamate),NH4+ (ammonium), CO2 (carbondioxide). Thedashedarrowshowsareactionthathasnotbeenrigorouslyproven.

From:Saueretal.,2007.

16

1.9 EffectsofSSADH­donFatOxidationinAldh5a1­/­Mice

AsshowninFigure2,Aldh5a1‐/‐micearesignificantlysmallerthanwildtype

mice.DuringExperiment1(below)wenoticedthatAldh5a1‐/‐micerapidlylosttheir

bodyfatshortlyafterbeingweanedontoanormalmousechowdiet.Thislossoffat

contributestothelowbodyweightandstuntedgrowthseenintheAldh5a1‐/‐mice.

Untilrecently,itwasnotclearwhythemutantmicelosttheirbodyfat.

Undernormalphysiologicconditions,micepreferentiallyuseglucoseasan

energysubstrate.Whenthisprocessisimpaired,however,micecanoxidizefatasa

“substitute”energysubstrate.

ArecentstudybyChowdhuryandcolleagues(2007)hasshownthat

Aldh5a1‐/‐micehavesignificantlyimpairedglucosemetabolism.Assuch,themutant

miceappeartobeoxidizingtheirfatstoresforenergy.Thishypothesiswas

supportedbydatashowingasignificantelevationintheketonebodybeta‐

hydroxybutyrate(βOHB)—aproductoffatoxidation—inmutantsfedahigh

carbohydraterodentchowdiet(Chowdhuryetal.,2007;Nylenetal.,2007).

Itseemslikelythatanimpairedabilitytoefficientlyutilizeglucoseasan

energysubstratehasledthemutantmicetooxidizetheirfatstoresforenergy.

Dietaryfat,however,isverylimitedinthecontroldiet(CD).Assuch,CDfedmutants

mayeventuallyrunoutofoxidizableenergysubstrategiventheirinabilityto

efficientlyuseglucose,andthelowavailabilityofoxidizableenergysubstratein

theirdiet.Thisprocessmayplayanimportantroleintheprogressivephenotypic

declineseeninAldh5a1‐/‐mice.

17

1.10 EffectsofSSADH­donEnergyMetabolisminAldh5a1­/­Mice

Aldh5a1‐/‐micehavebeenshowntohavesignificantlyimpaired

mitochondriafunction(Saueretal.,2007).Thiswasevidencedbysignificant

impairmentsintheactivityofcomplexI‐IVinhippocampalmitochondria(other

brainregionsshowednormalactivity).

Takentogether,Aldh5a1‐/‐miceshowsignificantimpairmentsinenergy

metabolismandmitochondrialfunction.Thesubsequentimpactonenergy

availabilitylikelyplaysaroleintheprogressiveseizurephenotypeandpremature

deathseeninthemutantmice.

1.11 AttemptedPharmacologicalTreatmentofSSADH­dinAldh5a1­/­Mice

Several compounds have been used in attempts to rescue Aldh5a1­/­ mice

pharmacologically.Theseattempts,however,havehad limitedsuccess(Hogemaet

al., 2001; Cortez et al., 2004). Among the compounds tested were phenobarbital

(indirectGABAergicagonist),phenytoin(sodiumchannelmodulator),VGB(GABA‐

transaminase inhibitor), ethosuximide (T‐type calcium channel inhibitor) and

CGP35348(GABABantagonist).

Highdosesofphenobarbitalandphenytoinwereneithereffectiveatstopping

seizuresinAldh5a1‐/‐mice,norweretheyeffectiveatprolongingthelifespanofthe

mutant mice (Hogema et al., 2001). VGB significantly prolonged the lives of

Aldh5a1‐/‐ mice, but did not significantly improve weight gain, reduce ataxia or

prevent seizures in the mutant mice (Hogema et al., 2001). Ethosuximide and

CGP35348suppressedabsenceseizures(andbaclofen—aGABABreceptoragonist—

18

exacerbated absence seizures) inAldh5a1‐/‐mice but did not improve the overall

phenotype(Cortezetal.,2004).

Although VGBwasmildly effective in prolonging the lives ofmutantmice,

noneof thedrugswasable todecrease seizure frequency,decreaseataxiaand/or

improveweightgain(Hogemaetal.,2001;Cortezetal.,2004).

1.12 RationaleforTestingtheKetogenicDietinAldh5a1­/­Mice

Ithasbeenobservedthatpupsthatcontinuetosucklebeyondweaningage

(~P20)livelongerthantheirweanedcounterparts,suggestingthatthedamplaysa

role in delaying the progression of SSADH‐d (Hogema et al., 2001). It was first

hypothesizedthatthetaurine,anaminoacidfoundinthedam’smilk,wasactingto

prolong the mutants’ lives (Hogema et al., 2001). Taurine has purported

anticonvulsanteffects(HuxtableandLaird,1978;Kontroetal.,1983)andhasbeen

shown to have neuroprotective effects (Chen et al., 2001; Anderzhanova et al.,

2006).TaurinesignificantlyprolongedthelifespanofSSADHnullmice,however,it

did not improve weight gain—nor did it significantly reduce the frequency of

seizuresseeninAldh5a1‐/‐mice(Hogemaetal.,2001).Therefore,itappearsthatthe

beneficial effects of long‐term suckling in Aldh5a1‐/‐ may be due to some other

propertyofthedam’smilk.

Anotherpossibility is that thehigh fatcontentof thedam’smilkdelays the

developmentofSSADH‐dinAldh5a1‐/‐mice.Mousebreastmilkiscomprisedof69%

fat,23%proteinand8%carbohydrate(Dymszaetal.,1964;Silvermanetal.,1992).

Further,newbornmicearehighlyketoticandremaininketosisduringthesuckling

19

period(Nehlig,2004).Wehypothesized, therefore, that thehigh fatcontentof the

dam’smilkwasactinglikeaketogenicdiet(KD).

TheKDisahighfat,lowcarbohydrateandadequate‐proteindietthatisused

inthetreatmentofdrug‐resistantepilepsy(Vining,1999).Somestudieshaveshown

thattheKDhasanticonvulsanteffectsinanimalmodelsofepilepsy(Massieuetal.,

2003;Nohetal.,2003;Zeigleretal.,2003;Sullivanetal.,2004;Nohetal.,2005a;

Nohetal.,2005b;VanderAuweraetal.,2005;Nohetal.,2006;Zhaoetal.,2006;

Maaloufetal.,2007).TheKDhasalsobeenshowntohavedisease‐modifyingeffects

inanimalmodelsofneurodegenerativedisorders,suchasAlzheimer’sDiseaseand

Parkinson’sDisease(Gasioretal.,2006;Hartmanetal.,2007).

1.13 TheKetogenicDiet

Theketogenicdiet(KD)isahighfat,lowcarbohydrateandadequateprotein

diet. Although theKDhas traditionally beenused to treat drug‐resistant seizures,

new research demonstrates that theKDmay also be effective in the treatment of

other neurological disorders (Gasior et al., 2006; Gasior et al., 2007; Hartman &

Vining,2007).

1.14 HistoryoftheKD:FromFastingto4:1

The first report that fasting may suppress seizures comes from the Bible

(KingJamesVersion,Matthew17,14‐21).Intheseverses,afatherbringshissonto

Jesusandsaysthathissonissufferingfromseizures.Jesustellsthefatherthatitis

only through “prayer and fasting” that his son can be cured of his seizures. Of

20

course,mostreligiouspeoplewouldinterpretthistomeanthatthefathershouldbe

prayingand fasting forhis son‐but somescientistshave interpreted this tomean

thatthesonshouldbefastingandprayingtocurehisseizures.

Scientific study on “the effects of fasting on seizures” started in the 20th

Century.Intheearly1900s,twoFrenchphysicians,LaMarieandGuelpa,postulated

thatseizuresweretheresultof“intestinalintoxification”causedbyovereating,and

that fasting (and the subsequent cleansing of the intestines)would cure seizures

(Vining, 1999). Subsequently, Bernarr Macfadden (a faith healer) and Dr. Hugh

Conklin (an osteopathic doctor) worked together using prayer and fasting to

successfullytreatseizures(Vining,1999).

In 1921, Geyelin adopted a 3‐week fasting protocol—without prayer—that

was very successful in treating seizures in his patients. Some of his patients

remainedseizurefree,evenaftertheirnormaldietswereresumed(Vining,1999).It

wasclear,therefore,thatfastingpersehadabeneficialeffectonseizures.

In1921,Wilderdevelopedadiet thatmimicked thephysiologicaleffectsof

fasting.He termed it the “ketogenicdiet”.Thisdietelevatedsystemic levelsof the

ketonebodiesacetoacetate,beta‐hydroxybutyrate,andacetonebyforcingthebody

tousefatsratherthancarbohydrateasitsenergysource.Wilderintendedthisdiet

tohavea2:1,orideally,3:1ratiobetweenthe“ketogenicfoods”(i.e.fats)andnon‐

ketogenic foods (e.g. carbohydrates and proteins) (Vining, 1999; Nordli, 2002).

Today, the most commonly employed ketogenic diet—the so called “classic

ketogenicdiet”—usesa4:1ratiooffatstocombinedcarbohydrateandprotein(by

weight).

21

The KD served as a major treatment for seizure disorders until the

introduction of themodern anticonvulsant drugs in the 1930’s. After that its use

declined. Interest in theKD has recently revived, however, for several reasons. It

wasoriginallythoughtthattheanticonvulsantswouldstopallformsofepilepsy.The

new anticonvulsant medications, however, have turned out to be no more

efficaciousatstoppingseizuresthantheolderones(Lefevre&Aronson,2000).As

such, there remains a need to alternative treatment options, such as the KD.

Secondly, in1994, JimAbrahams(a famousHollywoodmovieproducer)produced

an NBC Dateline show on the anticonvulsant effects of the KD. He subsequently

produceda“made‐for‐tv”movieentitled“FirstDoNoHarm”,whichalsopromoted

the KD as an effective therapy for drug‐resistant seizures. These productions

sparkedconsiderable interest intheKD.Finally,adietcraze inthe1990’s ledtoa

widespreadbeliefthatdietisamorenaturalwaytocontrolseizuresthandrugs.

1.15 ClinicalProfileoftheKD

Clinically,theKDisusedasasecond‐orthird‐linetreatmentinpatientsthat

have failed to respond to anticonvulsant drugs (Nordli & De Vivo, 1997). Earlier

studies of the KD suggested that approximately 55% of these patients achieved

complete seizure control, while 26% achieved marked decreases in seizure

frequencyandseverity(Livingston,1977).Todayitisgenerallyacceptedthatabout

60%ofpatientsontheKDwillhaveagreaterthan50%reductionintheirseizures.

Of this group, 10‐15% will have a greater than 90% reduction in their seizures.

22

Forty percent of patients on the KDwill have a less than 50% reduction in their

seizures(seeFigure5;forreviews,see:Vining,1999;Thiele,2003).

The fact that the KD suppresses seizures in patients that have failed the

anticonvulsantdrugssuggeststhattheKDmayhaveanovelmechanismofaction.

The KD is most commonly used in children (Vining, 1999). Although the

strongestanticonvulsanteffectsareseeninchildrenbetweentheagesof2and10,

theKD has also been shown to have anticonvulsant actions in adults (Livingston,

1977; Swink et al., 1997; Vining, 1999; Sirven et al., 1999). Sirven et al. (1999)

reported that 3/11 adult patients had a >90% reduction in seizures on the KD.

Another3/11hada>50%reduction inseizuresontheKD,and1/11hada<50%

reduction in seizures on the diet. 4/10 adults in the Sirven study, however,

discontinuedtheKDduetothediet’sunpalatablenature.Pooradherence,duetothe

KD’sunpalatabilityandrigor,isbelievedtobethemainreasonwhyadultstendnot

todoaswellaschildrenontheKD(Vining,1999).

23

Figure5.TheHistoricalEfficacyoftheKetogenicDiet

Efficacyof theKDinclinicalstudiesbetween1925and2007,showingthevaryingdegreesofseizurecontrolobtainedinpatients.

24

1.16 TypesofKD

Several forms of theKD are used clinically.ModernKDs vary according to

their ratio of fat to combined carbohydrate and protein, ranging from 2:1 to 4:1

(fat:carbohydrate+protein,byweight).Theyalsovaryaccordingtothetypeoffat

used (e.g. long chain fatty acid, polyunsaturated fatty acid KD, etc.). One common

variant,the“MCT”KD,usesmediumchaintriglyceridesasthesourceoffat(Carroll

&Koenigsberger,1998).

Inmostclinics,patientsarestartedona“classic”4:1KD,withthefatbeing

providedby long chain fattyacids (e.g.meatanddairy fats) (Thiele,2003). In the

studiespresentedinthisthesis,a“classic”4:1ratioKDwasusedforallexperiments.

Abreakdownof“classicKD”vs.“typicalAmericandiet”isavailableinFigure6.

Figure6.BreakdownofaTypicalAmericanDietvs.KetogenicDiet

DistributionofthenutrientsinatypicalAmericandietanda“classic”4:1KD.From:Thiele,2003.Carbs=Carbohydrates.

25

1.17 Side­EffectsoftheKD

AnumberofsideeffectsoftheKDhavebeendocumented.Thesesideeffects,

however,tendtobeinfrequent,anddonotappeartooccurinadose‐relatedfashion

(Swinketal.,1997).Thisisincontrasttotheanticonvulsantdrugs,whichmaycause

bothseriousdose‐relatedanddose‐unrelatedsideeffects(Swinketal.,1997).

Potential short‐term risks of the KD often involve vomiting, hypoglycemia

anddehydrationduringinitialadministrationofthediet.Duetotheserisks,patients

arehospitalizedandmonitoredcloselywhile theKD is initiated. Inhospital, these

effectsmaybeminimalorabsent.

Apotentiallong‐termriskoftheKDishyperlipidemia(Dekaban,1966;

Chesneyetal.,1999),althoughSchwartzetal.(1989)andKatyaletal.(2000)failed

tofindsignificantincreasesinbloodlipidlevels.Kwiterovichetal.(2003)reported

thattheKDcausesasignificantincreaseinthebad,low‐densitylipoproteinsanda

significantdecreaseinthegood,high‐densitylipoproteins.Otherlong‐termrisks

associatedwiththeKDarekidneystones(Betseyetal.,1990;Herzbergetal.,1990;

Vining,1999;Furthetal.,2000;Nordli,2002;Kossoffetal.,2002),bone

demineralization(Nordli,2002;althoughHahnetal.(1979)showedthatthiscanbe

reversedwithco‐administrationofvitaminD),constipation(Swinketal.,1997),

stuntingofgrowth(Freemanetal.,1990;Liuetal.,2003)andketoacidosis(Swink

etal.,1997;Vining,1999).

Thompsonetal.(1998),however,determinedthatfewerthan6%ofpatients

discontinuetheKDduetosideeffects.Themostcommonreasonfordiscontinuing

theKDislackofefficacy(i.e.discontinuationishighinpatientsthatdonotachieve

26

seizure control on the KD). Eighty percent of patients experiencing a >90%

reductioninseizuresstayontheKDforatleastoneyear.Bycomparison,only20%

ofpatientsexperiencing<50%reductioninseizuresstayonthedietforatleastone

year(Vining,1999).

MostpatientsareweanedofftheKDafter2or3years,however,duetofears

over its long‐term effects on health—particularly those pertaining to potential

cardio‐vascularproblems(Vining,1999).

1.18 TheKetogenicDiet’sMechanismofAction

Themechanismsofactionof theKDarenot fullyunderstood.Anumberof

differenttheorieshavebeenproposed.

1.18.1 TheBrainLipidsTheory

Clinically,theKDhasbeenshowntoincreasebloodcholesteroland

triglyceridelevels(Dekaban,1966;Chesneyetal.,1999;Kwiterovichetal.,2003).It

washypothesizedthatthisincreaseinlipidlevelscontributestotheanticonvulsant

actionsoftheKD.Rabinovitzetal.(2004),forinstance,havesuggestedthatlipids

areincorporatedinthebrainandsubsequentlyalterthestructureandfunctionof

neuronalmembranes,causingchangestomembranefluidity,ionchannel

functioningandreceptor‐ligandaffinities.Theysuggestthesechangeshave

anticonvulsanteffects.

AmajorproblemwiththishypothesisisthatnotallKDselevatelipidlevelsin

asimilarfashion.Forexample,themediumchaintriglycerideKDdoesnotelevate

27

bloodtriglyceridelevels,butitstillhasgoodanticonvulsantactivity(Carroll&

Koenigsberger,1998).

1.18.2 ThepHTheory/Keto­acidosisTheory

Acidosiswasfirsthypothesizedastheanticonvulsantmechanismofaction

fortheKDin1931byBridgeandIob.WhenstartedontheKD,thepatient’s

metabolismswitchesfromtheusingcarbohydratetousingketonebodiesasan

energysubstrate.Theketonebodiesacetoacetateandbeta‐hydroxybutyrate,which

aremildacids,werehypothesizedtolowerbloodpHinpatientsontheKD.This

decreaseinpHwashypothesizedtoconferthediet’santiconvulsanteffects.For

example,lowpHhasbeenshowntoinhibitpH‐sensitiveNMDA‐typeglutamate

receptorsandpHsensitivegapjunctions,causingadecreaseinneuralexcitation

(Schwartzkroin,1999;PerezVelazquezandCarlen,2000).

Theacidosishypothesishaslargelybeenabandoned,however,asclinical

studieshavefailedtoshowlong‐term,KD‐inducedchangesinpH(Huttenlocher,

1976).Animalstudieshaveconfirmedthisfindingbydemonstratingthatthereisno

changeofbrainpHintheanimalsfedaKD(Al‐Mudallaletal.,1996).

1.18.3 TheGABAShuntTheory

TheGABAshunttheorysuggeststhattheKDleadstohigherlevelsofGABAin

thebrain.Nordli(2002)arguesthattheKDcausesincreasedlevelsofα‐

ketoglutarateinthebrain.Excessα‐ketoglutaratecanbeusedtoproduceGABAvia

28

theGABAshunt(seeFigure7).Nordli(2002)pointsoutthatelevatedGABAlevels

wouldthenelevateseizurethresholdinthebrain.

OneofthestrongestlinesofreasoningopposingtheGABAshunttheoryis

thattheKDisoftensuccessfulinpatientsthathavealreadyfailedtheanticonvulsant

medicationsthatelevateGABAlevelsinthebrain.Therefore,ifGABAagonistsdo

notcontrolthepatient’sseizuresandtheKDdoes,itwouldbeanonsequiturto

reasonthattheKDworksbyelevatingGABAlevels.

AnotherargumentagainsttheGABAshunttheoryisthatanimalstudieshave

shownthatGABAlevelsarenotincreasedinthebrainsofratsfedtheKD(Appleton

&DeVivo,1974;Al‐Mudallaletal.,1996).

29

Figure7.TheGABAShunt

TheGABAshuntinvolvesthebiotransformationofalpha‐ketoglutarate,anintermediateofKrebscycle,toglutamatevia

glutam

atedehydrogenase.GlutamateisthenbiotransformedintoGABAviaglutam

icaciddecarboxylase.GABAis

biotransformedtosuccinicsemialdehydeviaGABAtransaminase.Finally,succinicsem

ialdehydeisbiotransformedto

succinateviasuccinicsemialdehydedehydrogenase,completingtheGABAshunt’sre‐entrytoKrebscycle.CoA=coenzym

eA.

30

1.18.4 TheEnergySubstrateTheory

Anaerobicmetabolism—ormetabolismintheabsenceofoxygen—occurs

whenglucosemoleculesareoxidizedintotwopyruvatemoleculesoutsideofthe

mitochondria(Dioguardi,2004).Thisprocess,knownas“glycolysis”,yieldsasmall

butimmediatelyavailablesourceofenergyforthecell(~8molesofadenosine

triphosphatepermoleofglucose,ATP;Dioguardi,2004).

Aerobicmetabolism,however,requiresoxygenandoccursinmitochondria

viatheKrebscycleandtheelectrontransportchain.Undernormalconditions,most

ofthebrain’senergyisderivedfromtheaerobicoxidationofglucose,which

provideshigherlevelsofATP(~30moles;Greeneetal.,2003;Nehlig,2004;

Dioguardi,2004).Whendietarycarbohydratesarescarce—suchasinindividualson

aKD—thebrainbeginstouseketonebodiesforenergy.Ketonebodiescanbe

convertedtopyruvate,whichcansubsequentlybeusedintheKrebscycleandthe

electrontransportchaintomakeATP.Theconversionofketonebodiestopyruvate,

however,doesnotreleaseATPlikeglycolysisdoes.

Normally,glucoseservesasthepreferredenergysubstrateforthebrain.In

patientsfedaKD,however,ketonescansupplythebrainwithupto60%ofits

energyneeds(Veech,2004).Greeneetal.(2003)hypothesizedthatglucose

generatesboth“slow”energy(viaKrebscycle)and“fast”energy(viaglycolysis).

Ketones,however,yieldonly“slow”energy(viaKrebscycle).Theenergysubstrate

hypothesissuggests,therefore,thatalthoughketonesprovidesufficientenergyfor

regularbrainactivity,theydonotprovideenough“fast”energytosustainseizure

activity.

31

Theenergysubstratetheoryiscurrentlyoneofthemosthighlyaccepted

theoriesregardingtheKD’smechanismofaction.

1.18.5 TheKetonemiaTheory

Threeketonebodies,beta‐hydroxybutyrate(βOHB),acetoacetate(ACAC)

andacetonearesignificantlyelevatedinpatientsontheKD(Musa‐Velosoetal.,

2002;Kossoff,2004).AlthoughβOHBandACACareconsideredketonesbecauseof

theirinter‐conversionwithacetone,acetoneistheonly“true”ketone.βOHBand

ACACareorganicacidswithanextraalcoholgroupandanextraketonegroup,

respectively(Likhodii&Burnham,2004).

Theketonemiatheorypostulatesthatketonebodiesthemselvesare

anticonvulsant,andthattheKDiseffectivebecauseitelevatesketonebodiesinthe

bloodandbrain.Nospecificmechanismofaction,however,hasbeensuggested(i.e.

no“receptor”isknownthatketonesmightbind,toconfertheiranticonvulsant

activity.Forreview,see:Prasadetal.,1996).

Someclinicalstudiesandanimalstudieshavereportedsignificant

correlationsbetweenlevelsofβOHBorACACandseizureprotection(Huttenlocher,

1976;Boughetal.,1999a;Boughetal.,1999b;Whendonetal.,1999).Otherstudies,

however,havereportedalackofcorrelationbetweenβOHBorACACandseizure

protection(Likhodiietal.,2000;Boughetal.,2000;Eaglesetal.,2003).Atpresent,

therelationshipbetweenketosisandseizurecontrolremainsunclear.

OpponentstotheketonemiatheoryhavearguedthatβOHBorACACare

elevatedrapidlyinpatientsontheKD,butseizurecontrolcantakesomeweeksto

32

develop(Dekaban,1966;Schwartzetal.,1989;Sirvenetal.,1999;although,ithas

beensuggested,however,thatitmaytakethebrainafewweekstoadjusttothe

elevatedketonebodiesbeforeanticonvulsanteffectsareseen).Further,Rhoetal.

(2002)haveshownthatβOHBlacksantiseizureeffectsinvitro.

Historically,however,researchershaveneglectedthepossibleroleof

acetoneintheanticonvulsantmechanismoftheKD.

1.18.6 TheAcetoneTheory

AcetoneisaketoneelevatedinpatientsontheKD(Likhodiietal.,2002).The

ideathatacetonehasanticonvulsantpropertieswasfirstproposedbyHelmholtz

andKeithin1930.Theideawasthenignoredforsomeyears.Recently,however,

Seymouretal.(1999)havereportedthatacetonewaselevatedinthebrainsof

childrenontheKD.Also,Likhodiietal.(2003)foundthatacetonehadawide

spectrumofanticonvulsanteffectsinvariousanimalseizuremodels(seeFigure8).

Likhodiietal.(2003)proposedthe“acetonehypothesis”,whichstatesthatacetone

playsaroleintheanticonvulsantmechanismoftheKD.

Therearetwolinesofevidencefortheacetonehypothesis.Firstly,acetoneis

elevatedinfastedpatientsandpatientsfedtheKD(Seymouretal.,1999;Likhodiiet

al.,2002;Musa‐Velosoetal.,2002).ItisknownthatbothfastingandtheKDhave

anticonvulsantproperties(Vining,1999).

Secondly,acetonehasbeenshowntohaveabroadspectrumof

anticonvulsantaction,similartothatoftheKD.Intraperitonealinjectionsofacetone

havebeenshowntobeanticonvulsantintheamygdalakindlingpreparation(which

33

modelscomplex‐partialseizures),theMESpreparation(whichmodelstonic‐clonic

seizures),theAY9944preparation(whichmodelsatypicalabsenceseizures),and

thescPTZpreparation(whichmodelstypicalabsenceseizures)(Likhodiietal.,

2003).

Nylen et al. (2006) examined blood‐acetone levels in rats fed a KD. Unlike

humans on the KD, however, rats do not appear to develop high, anticonvulsant

concentrations(>2mM)ofacetoneinblood.

34

Figure8.TheAnticonvulsantEffectsofAcetoneinAnimalModelsofEpilepsy

Thisfigureillustratesthedose‐responsecurvesforacetoneintheamygdalakindlingmodel,MESmodel,AY‐9944modelandthePTZmodelofepilepsy.Theseresultsdemonstratethebroad‐spectrumanticonvulsantpropertiesofacetone,whicharesimilartothoseoftheKD.MES=maximalelectroshock,PTZ=pentylentetrazole.Toconvertmmol/kg(millimolesperkilogram)tomg/kg(milligramsperkilogram),multiplyby58.08.

From:Likhodiietal.,2003

35

1.19 GeneralObjectives

The followingexperimentsexamined theeffectsof aKD inAldh5a1‐/‐mice.

Aldh5a1‐/‐mice—as compared towildtypemice (Aldh5a1+/+)—havea significantly

shorter lifespan (Hogema et al., 2001), profound ataxia (Hogema et al., 2001),

stunted growth (Hogema et al., 2001), neural hyperexcitability (Wu et al., 2006),

elevatedserumketonebodies(Chowdhuryetal.,2007),impairedglucoseoxidation

(Chowdhury et al., 2007), reduced [35S]TBPS binding (Wu et al., 2006) and

decreasedhippocampalmitochondrialfunction(Saueretal.,2007).

Theobjectiveofthefollowingexperimentswastocharacterizetheeffectsof

the KD on these established deficits in Aldh5a1‐/‐ mice. If the KD can normalize

some, or all of these deficits, it may represent a novel therapy for the clinical

treatmentofSSADH‐d.

Any observed benefit of the KD on these deficits might also help further

elucidatethediet’smechanismsofactionorrevealnewmechanismsofaction.

1.20 SpecificObjectivesandPurposeofExperiments

Ourfirstobjectivewastodeterminetheeffectofa4:1KDontheAldh5a1‐/‐

mousephenotype.Experiment1 includedmultiple studies thatexamined lifespan,

weightgain, levelof ataxia, andelectrocorticographicactivity.Theseaspectswere

comparedbetweenAldh5a1‐/‐mice thatwere fedeitheracontroldiet (CD;mouse

chow)ora4:1KD.ThepurposeofExperiment1wastodeterminewhethertheKD

hasanyefficacyinthetreatmentofSSADH‐dinmice—withimplicationsforclinical

trialsinvolvingtheKDinhumansSSADH‐d.

36

The objective of Experiment 2 was to expand on the in vitro

electrophysiology findingsofWuetal. (2006),whichshowed thatAldh5a1‐/‐mice

havean imbalance inexcitatoryand inhibitoryneuronalactivity(Wuetal.,2006).

This experiments also sought to explore the effects of the KD on this imbalance.

Therefore,Experiment2measuredinhibitoryandexcitatoryminiaturepostsynaptic

currentsinCDfedAldh5a1+/+andAldh5a1‐/‐miceaswellasKDfedAldh5a1‐/‐mice.

TheobjectiveofExperiment3wastoassesstheeffectoftheKDon[35S]TBPS

bindinginAldh5a1‐/‐mice.[35S]TBPS—aligandfortheGABAAR‐associatedchloride

channel—issignificantlydecreasedinAldh5a1­/­mice(Wuetal.,2006).Experiment

3measured [35S]TBPSbinding inCD fedAldh5a1+/+andAldh5a1‐/‐miceaswellas

KDfedAldh5a1+/+andAldh5a1‐/‐mice.

TheobjectiveofExperiment4wastodeterminetheeffectofaKDonspecific

serumanalytesinAldh5a1‐/‐micethathavebeenimplicatedintheKD’smechanism

ofaction.Experiment4measuredlevelsofglucose,beta‐hydroxybutyrateandnon‐

esterifiedfreefattyacidsinCDfedAldh5a1+/+andAldh5a1‐/‐miceaswellasKDfed

Aldh5a1+/+andAldh5a1‐/‐mice.

The objective of Experiment 5 was to study the effect of SSADH‐d on the

number and function of mitochondria in Aldh5a1‐/‐ mice. Previous research has

shown that Aldh5a1‐/‐ mice have significant disruptions in their hippocampal

mitochondrial function (Sauer et al., 2007). Further, the KD has been shown to

improvemitochondrial function (Bough et al., 2006). Experiment 5 examined the

effectof theKDonthenumberand functionofmitochondria inCDfedAldh5a1+/+

andAldh5a1‐/‐miceaswellasKDfedAldh5a1+/+andAldh5a1‐/‐mice.

37

CHAPTER2

GENERALMETHODS

2.1 Subjects

Aldh5a1‐/‐micewithC57/129Svbackgroundwerefirstgeneratedinthe

OregonHealthandScienceUniversityinPortland(Hogemaetal.,2001).Five

heterozygous(Aldh5a1+/−)breedingpairsweretransportedtotheanimalcare

facilityattheHospitalforSickChildreninToronto.WemaintainedtheAldh5a1‐/‐

mouselinebyinbreedingheterozygouspairs.Allsubjectswerehousedina

pathogenfreeenvironmentwithcontrolledlighting(12hlight/12hdark,lightsonat

7am).Waterwasavailabletoallsubjectsadlibitum.

AllexperimentalprotocolswereapprovedbytheHospitalforSickChildren

LaboratoryAnimalServicesCommittee.Experimentswerecarriedoutin

accordancewiththeguidelinesoftheCanadianCouncilonAnimalCare.

2.2 Genotyping

PupshadtheirtailsclippedforDNAanalysisonpostnatalday(P)10.

Subjectswerealsoear‐clippedatthistimeforidentificationpurposes.Micewere

genotypedbytwo‐allele,three‐primerpolymerasechainreaction(PCR)using

genomicDNAfromthetailclips.WeemployedprimersspecificforAldh5a1common

towildtypeandtargetedalleles(sense,5´–GCTATGACTGTGAACTCTGTCAAGCCAC–

3�),theneocassette(antisense,5´–AGCGCATGCTCCAGACTGCCTTGG–3´)andapart

ofthePacIfragmentdeletedinthetargetingconstruct(antisense,5´–

38

CATGTTCGCAACTGCCTATAACTCCAGG–3´).Wecarriedout35cyclesofPCRwith

annealingat57°Cfor45sandsynthesisfor45s,generatingfragmentsof566bpfor

themutant,and653bpforthewildtypeallele(methodadaptedfromHogemaetal.,

2001).

2.3 Diets

A4:1ketogenicdiet(KD)servedastheKDinallexperiments.Standard

laboratorymousechowservedasthecontroldiet(CD)inallexperiments.Alldiets

wereavailabletothemiceadlibitum.DietcontentsaredetailedbelowinTable3

(KD)andTable4(CD).

2.3.1 4:1KetogenicDiet(KD)

Thecompositionofthe4:1KDispresentedinTable3.The4:1KDwas

composedoffourpartsfattoonepartofcombinedcarbohydrateandprotein(by

weight),toprovideaclassicKD.TheclassicKDisthemostoftenusedclinically

(Vining,1999).The4:1KDforrodentswasdevelopedbyDr.SergeiLikhodii

(Likhodii,2001)toaccuratelyreflectthefat,carbohydrateandproteinintakeof

patientsontheclassicKD.Powdercontainingproteinandmicronutrientssuchas

mineralandvitaminswasobtainedfromHarlanTeklad(Madison,WI;TD.03490)

andwasstoredat4˚Celsius.Fatsintheformofunsaltedbutter,lardandcanolaoil

wereaddedtothepowder(asdonebyLikhodii,2001;Nylenetal.,2005;Nylenet

al.,2006).TheKD,oncemade,wasstoredat4˚Celsius.

39

2.3.2 ControlDiet(CD)

ThecompositionoftheCDispresentedinTable4.Normallaboratoryrodent

chow(Purina,#5001)servedasthecontroldietforallexperiments.

2.4 Statistics

Lifespanandweightgaindatawereanalyzedusingtwo‐tailedt‐tests.Ataxia

datawereanalyzedusingarepeatedmeasures,two‐wayanalysisofvariance

(ANOVA).[35S]TBPSbindingandserumanalytedatawereanalyzedusingatwo‐way

ANOVAwithpost‐hoctests.Allotherdatawereanalyzedusingaone‐wayANOVA

withpost‐hoctests.Significancewasconsideredatp<0.05.GraphPadPrism

softwarewasusedtoconductallstatisticalanalyses(v.4.02,SanDiego,CA).

40

Table3.Compositionofthe4:1KetogenicDiet(KD)

Macronutrient Micronutrient 4:1KD Weightingrams

Casein 142.09ProteinL‐Cystine 4.887CornStarch ‐Sucrose ‐

Carbohydrate

Dextrin 30Fiber Cellulose 66.087

AIN‐93G 0.538VitaminsCholinebitartrate 4.073

Minerals AIN‐93G 44.472Fat Canolaoil 114.03 Lard 187.8 Butter 406Antioxidant tert‐

butylhydroquinine0.023

Thistableshowsthecompositionofthe4:1KD.TheAIN‐93Gvitaminmixcontainsnocarbohydrates.Canolaoilwasusedasacarrierforfat‐solublevitamins.Thetypeoffat(i.e.,butterorlard)canbevaried.Inthiscase,non‐saltedbutterservedasthemajorfatsource.

AdaptedfromNylenetal.,2005

41

Table4.CompositionoftheControlDiet(CD)

Macronutrient Micronutrient CD WeightingramsProtein ‐ 234Carbohydrate Dextrose 490Fiber ‐ 53MineralMix AIN‐93G 69VitaminMix AIN‐93G 54

Lard 15Butter 0

Fat

CornOil 85Thistableshowsthecompositionofstandardrodentchow,whichservedasthecontroldiet(CD)inourstudies.TheCDisLaboratoryRodentChow,Purina#5001.

AdaptedfromBoughetal.,1999

42

CHAPTER3

EXPERIMENT1:THEEFFECTSOFAKETOGENICDIETONTHE

ALDH5A1­/­PHENOTYPE

3.1 Introduction&Rationale

The Aldh5a1­/­ mouse provides an animal model in which to study the

pathophysiology and treatment of SSADH‐d. Aldh5a1­/­ mice exhibit pronounced

developmental delay, ataxia and a seizure disorder that progresses from absence

seizures—around post‐natal day (P) 15— to status epilepticus around day P25

(Cortez et al., 2004). All mice die in status epilepticus around P25 unless some

treatmentisadministered(Hogemaetal.,2001).Itishopedthatthetreatmentsthat

improvethephenotypeoftheAldh5a1‐/‐mousemayalsobeusefulinthetreatment

of clinical SSADH‐d. This is partially supported by data using vigabatrin (VGB;

Sabril®).

VGBhasbeenreportedtohavesomeefficacyinAldh5a1‐/‐mice(Hogemaet

al.,2001;Guptaetal.,2002)aswellasinthetreatmentofhumanSSADH‐d(Jaeken

etal.,1989;Gibsonetal.,1989,Howellsetal.,1992;Jacobsetal.,1992;Uzieletal.,

1993; Gibson et al., 1995). The data related to VGB in patients with SSADH‐d,

however,arelargelyanecdotal,andoutcomesarenotuniformlygood(Maternetal.,

1996;Gibsonetal.,1998;Guptaetal.,2002;Ergezingeretal.,2003;Gropman,2003;

Gordon,2004).

Hogema and colleagues (2001) also tested other potentially therapeutic

compoundstoseeiftheycouldimprovethephenotypeofAldh5a1‐/‐mice,butnone

43

of themwasparticularly successful.Among the compounds testedwereVGB,CGP

35348 (GABABR antagonist) and taurine (2‐aminoethanesulfonic acid).Homegaet

al. (2001) tested taurine following the observation that pups that continued to

suckle beyond weaning age (between P18‐P20) lived longer than their weaned

counterparts. Taurine is abundant in dam’s milk and it was hypothesized that

taurine might be acting to improve the Aldh5a1‐/‐ phenotype. Taurine has been

reported to have both anticonvulsant (Huxtable and Laird, 1978; Kontro et al.,

1983) and neuroprotective effects (Chen et al., 2001; Anderzhanova et al., 2006).

Taurine,however,hadlimitedeffectsintheAldh5a1‐/‐mice.

Despite taurine having limited effects in Aldh5a1‐/‐ mice, it remains an

intriguingfindingthatpupsthatcontinuedtosuckleshowedamarkedlyimproved

phenotypeascomparedtotheirweanedlittermates.Oneparticularfeatureofdam’s

milkthatisinterestingisthatitcontainshighconcentrationsoffat(69%)andlow

concentrationsofcarbohydrate(8%)(Dymszaetal.,1964;Silvermanetal.,1992).

In this way, it resembles a KD—a high fat diet used in the treatment of drug‐

resistantseizures.

Itwasthereforehypothesizedthatthehighfatdam’smilkisactinglikeaKD,

thus providing beneficial effects to the Aldh5a1‐/‐ mice. Other support for this

hypothesis is the observation that the onset of convulsions in Aldh5a1‐/‐ mice

coincideswiththetimethatmiceareweanedontonormalmousechow(i.e.,around

P20).

Herewe testedwhether administration of a 4:1KDmight be successful in

treatingSSADHdeficiency.

44

3.2 Methods

3.2.1 Subjects

Aldh5a1‐/‐ mice and Aldh5a1+/+ mice served as subjects for the present

experiments.Allmiceweregenotyped(seeGeneralMethods)byP10.Thesubjects

weresortedintothesethreegroups:1)CDfedAldh5a1+/+mice,2)CDfedAldh5a1‐/‐

miceand3)KDfedAldh5a1‐/‐mice.

MostlittersonlyyieldedoneortwoAldh5a1‐/‐mice.Thesemicewereoften

tooweak to competewith theirheterozygoteorhomozygotewildtype littermates

foraccess todam’smilk.Therefore,we transplantedAldh5a1‐/‐mice fromvarious

litters into a common cage tobe fed either aKDorCD.Each cagehada lactating

dam.

We did not use heterozygote (Aldh5a1+/‐)mice in our experiments as they

share the same phenotype as the wildtype mice. Heterozygote mice have been

showntohavenormallevelsofGHBandGABAinbrain,liverandurine(Hogemaet

al.,2001).NoKDfedwildtypemicewereusedfortheseexperimentsforreasonsof

practicality. Theywere later added to experimentswhere this control groupwas

deemedmoreimportant.

AllexperimentalsubjectsandtheirdamswerefedtheCDuntilP12.OnP12,

subjects intheKDfedgrouphadtheCDremovedandreplacedwiththeKD.From

thispointonwards,thepupsandthedamonlyhadaccesstotheKD.Thiswaswell

before the timeofweaningbut itensuredthatanynon‐suckling feedingwouldbe

ketogenicinnature.Micecontinuedtoreceivetheirrespectivedietsfortheduration

oftheirlives.KDdisheswerefilledeverymorning.

45

3.2.2 DeterminingLifespan

LifespanwasdeterminedinCDandKDfedAldh5a1‐/‐mice(Aldh5a1+/+mice

live a normal lifespan of approximately 2 years). Lifespan was determined by

counting the number of days that each subject lived.Miceweremonitored daily.

Whenmicewere founddead, their lifespanwas calculated as thenumberof days

frombirthuntilthedaythattheywerelastseenalive.

3.2.3 DeterminingWeights

Allanimalswereweighed,andtheirweightsrecorded,fivetimesperweek.

3.2.4 DeterminingAtaxia

Subjects were scored for ataxia during each weighing. Ataxia was scored

accordingtoLoscher’sscaleofsedation(Loscheretal.,1987).Thisscalehas6levels

of ataxia: 1—slight ataxia of hindlimbs, 2—dragging of the hindlimbs, 3—strong

ataxia and dragging of the hindlimbs, 4—marked ataxia and loss of balance, 5—

markedataxiawithnobalance,and6—lossofrightingreflexwithattemptstomove

forward.

3.2.5 SurgeryandElectrocorticography(ECoG)

OnP20‐P25,undersodiumpentobarbitalanesthesia(0.01mg/kg)micewere

stereotaxically implantedwith four epidural,monopolar electrodes. The electrode

tipswereimplantedbilaterallyandaimedatthefrontal(2electrodes)andparietal

(2electrodes)cortices.Theelectrodescoordinatesforthefrontalcortexwere1mm

46

deep, 2mm anterior to bregma and 2mm lateral from midline. The electrode

coordinatesforparietalcortexwere1mmdeep,2mmposteriortobregmaand2mm

lateralfrommidline(FranklinandPaxinos,1997).Thesurgerylastedapproximately

10min.

After surgery, animals were allowed to recover for 2h before ECoG

recordingswerebegun.Recordingsweremade1h,24hrsand48hrsafterrecovery

fromanesthesia.EachanimalwasplacedinanindividualPlexiglas™chamberfora

20minuteadaptationperiodpriortoECoGrecordings.Thiswasdonetominimize

movement artifact. ECoG activity was recorded both on paper, using a Grass

Polysomnographmachine(aspreviouslyreportedbyDepaulisetal.,1989;Cortezet

al., 2004), and digitally on a computer using the GrassLab Recorder (AstroMed,

West‐Warwick, RI, U.S.A.) (as previously reported by Cortez et al., 2001). All

baselineandtestrecordingswereperformedbetween10:00hto14:00htominimize

thecircadianvariationsreportedbyLoscherandFiedler(1996).

3.2.6 CharacterizationofSeizuresandConvulsions

Seizure activity was scored by a clinical neurophysiologist (Dr. Miguel

Cortez), using the same criteria as Cortez et al. (2004). Behavioural observations

were alsomade during the ECoG recordings. The presence or absence of seizure

activity was determined over the course of one hour. Absence seizures were

characterized by the presence of bilaterally synchronous 5–7 Hz spike‐and‐wave

discharges(SWD),associatedwithafrozenbehaviorandtwitchingofthevibrissae

lastingatleastonesecondinduration.

47

Convulsions—or behavioral seizures corresponding to ictal activity on the

ECoG—werealsorecorded.Themostcommonlyobservedconvulsionsweretonic‐

clonicinnature.CDfedAldh5a1‐/‐micealsohadrunningandjumpingfitsassociated

withhighfrequencyseizureactivityontheECoG.

3.3 Results

Figure9presentscomparativepicturesofKDfedAldh5a1‐/‐miceandCDfed

Aldh5a1‐/‐mice.

3.3.1 Lifespan

Figure10presentsdatarelatedtotheaveragelifespanforKDfedAldh5a1‐/‐

miceandCDfedAldh5a1‐/‐miceindays(±s.d.).Figure11providessurvivalcurves

forthesetwogroups.Lifespanwasdeterminedbysubtractingthedateofbirthfrom

thedateofdeathforeachsubject.AsindicatedbyFigure10,theCDfedAldh5a1‐/‐

mice lived an average of 23.78±8.12 (mean±s.d.) days (range: 19‐43) and KD fed

Aldh5a1‐/‐ mice lived an average of 85.56±31.12 days (range: 58‐146). KD fed

mutantsthuslived,onaverage,>300%longerthanCDfedcontrols(Figure10;n=9,

p<0.0001, two‐tailed t‐test). As indicated by Figure 11, by P50, all of the CD fed

mutantshaddiedwhilealloftheKDfedmutantsremainedalive.Thelongestliving

KDfedmutantlivedtoP146.

48

Figure9.PicturesofCDandKDfedAldh5a1‐/‐Mice

a

b

c

a)Aldh5a1+/‐ damwithAldh5a1‐/‐ pups consuming the KD in their home cage. b)Aldh5a1‐/‐ mouse fed a control diet (P38). Note the high degree of ataxia asevidenced by the subject’s posture. Themousewas unable towalk due to a highlevel of ataxia. c) Aldh5a1‐/‐ mouse fed KD (P42). Note the improved weight andposture.ThegreasyhairoccurredinKDfedmicebecausetheywalkedthroughtheirdietdishes.

49

Figure10.AverageLifespansforCDandKDfedAldh5a1‐/‐Mice

Thisfigurepresentsmean(±s.d.)lifespansindaysforCD(blackbar)andKD(graybar) fed Aldh5a1‐/‐ mice. KD fed Aldh5a1‐/‐ mice lived significantly longer thancontrol diet fedAldh5a1‐/‐mice. On average, CD fedAldh5a1‐/‐mice lived to P25,whileKDfedAldh5a1‐/‐micelivedtoanaverageofP85.d=days,***p<0.001.

50

Figure11.SurvivalCurvesforKDandCDFedAldh5a1‐/‐Mice

This figurepresentsthesurvivalcurves(indays) forCDfedAldh5a1‐/‐mice(solidline)andKDfedAldh5a1‐/‐mice(dashedline).AtP50,alloftheCDfedmutantshaddied,whereasall of theKD fedmutants remainedalive.The longest livingKD fedmutantlivedtoP146.

51

3.3.2 Weights

Figure 12 shows the average progression of daily weight gain for CD fed

Aldh5a1+/+ (wildtype)mice,CD fedAldh5a1‐/‐miceandKD fedAldh5a1‐/‐mice.As

indicatedbyFigure12,all subjectsgainedweightequallyuntilP10‐12.This is the

timewheremicebegintoconsumefoodotherthantheirdam’smilk,althoughthey

continuetosuckleuntilP18‐20.Arepeated‐measuresanalysisofvariance(ANOVA)

followed by Tukey’s post‐hoc tests revealed that group weights began to

significantlydivergearoundP20(p<0.05fordaysP20onwards).StartingatP12,CD

fedmutantsbegantoexhibitweight losswhileKDfedmutantscontinuedtoshow

weightgains.CDfedwildtypemiceshowedsignificantlymoreweightgainthanboth

groupsofmutantmice.

Figure 13 shows themean (±s.d.) weights (in grams) of CD fedAldh5a1‐/‐

mice,KDfedAldh5a1‐/‐miceandCDfedwildtypemice.As indicatedbyFigure13,

average daily weight loss in CD fed Aldh5a1‐/‐ mice was 0.003±0.013g/day

(mean±s.d.) while the average daily weight gain in KD fed mutants was

0.13±0.01g/day (Figure 13; p=0.0002, two‐tailed t‐test). Both the CD and KD fed

mutantgroupsshowedsignificantlylowerweightgainthanCDfedAldh5a1+/+mice.

3.3.3 Ataxia

Ataxia scores were assessed using a 6‐point rating scale developed by

Loscher and colleagues (1989). As indicated by Figure 14, CD fedAldh5a1­/­ mice

began to develop ataxia ~P18 (stage 2 and higher). The onset of ataxia was

significantly delayed in KD fed mutants as they only began to develop stage 2+

52

ataxia around P70 (p=0.0002, repeated‐measures two‐way ANOVA, Tukey’s post‐

hocanalysisrevealedsignificantdifferencesfordaysP17‐40).

3.3.4 Electrocorticography

Figure15presents representativeECoG records forCD fedAldh5a1‐/‐mice

andKD fedAldh5a1‐/‐mice.Electrocorticography (ECoG)wasperformedon freely

moving CD fedAldh5a1‐/‐ mice and KD fedAldh5a1‐/‐ mice—on days P20‐25—to

determinewhetherchanges in lifespan,weightgainandataxiacorrespondedwith

changesinbrainactivity.AsindicatedinFigure15,thebackgroundECoGbaselinein

CDfedAldh5a1‐/‐miceconsistedof35to60µVcorticalactivityatafrequencyof4to

7Hz with intermingled 3‐5Hz oscillations, often associated with fast frequency

oscillationsatthefrequencyof28Hzduringrestfulwakingconditions.Therewere

numerousinterruptionsofthebackgroundactivitybyintermittent,highamplitude

250‐300µV bursts of spontaneous, recurrent spike and wave discharges (SWD),

whoseonset/offsetwastime‐lockedwithabsence‐likeictalbehaviorthatconsisted

offrozenimmobility,facialmyoclonus,andtwitchingofthevibrissae.

Incontrast,thebaselineECoGactivityoftheKDfedAldh5a1‐/‐micewasfairly

normal,withwell‐regulated,lowamplitude35to50µVelectricalfieldsat4Hz.There

was an absence of both slower frequencies in the delta range or fast frequency

oscillationswithinthefrontalandparietalcortices.

Figure 16 indicates the number of convulsions seen in each group.

Convulsions were said to be present when they occurred simultaneously with

seizureactivityonECoG.TheKDfedmutantshadsignificantlyfewerconvulsionsas

comparedtotheCDfedmutants(p<0.05,t‐test).

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Figure12.ProgressionofAverageDailyWeightGaininAldh5a1‐/‐Mice

Figure12presentsmeanweights(±s.d.)ingramsfromP12toP150.CDfedmutantsgrewinweightfrombirthuntilP14‐18,atwhichpointtheirweightgainpeakedandthenregressed.KD fedAldh5a1‐/‐miceshowedsignificantly improvedweightgainas compared to CD fedmutants. Both the CD and KD fedAldh5a1‐/‐ mice groupsgrewsignificantlyslowerthanwildtypemice.Forpurposesofscale,weightdataforCD fed Aldh5a1+/+ mice were cut off at 20g. Mice from this group reached peakweightsofbetween30‐40g.N=9forallgroups.CD=controldiet,KD=4:1ketogenicdiet, d=days, g=grams.The solidblackbar indicates the ages atwhich thereweresignificantdifferencesbetweenCD fedAldh5a1‐/‐miceandKD fedAldh5a1‐/‐mice(p<0.05). The dashed line indicates the ages at which there were significantdifferencesbetweenCDfedwildtypemiceandbothAldh5a1‐/‐groups(p<0.05).

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Figure 13. Average Daily Weight Gain in Aldh5a1‐/‐ and Aldh5a1+/+ Mice (P12Onwards)

This figurepresentsmean(±s.d.)weightdata ingrams forCD fedAldh5a1‐/‐mice,KD fed Aldh5a1‐/‐ mice and CD fed Aldh5a1+/+ mice. From P12 onwards, CD fedmutants lost an average of 0.003±0.013g per day (n=9), while KD fed mutantsgained0.13±0.01gperday(n=9).CDfedwildtypemiceshowednormalweightgainforamouse,whichwassignificantlyhigherthaneithertheKDfedmutantsortheCDfedmutants.TheKD isgenerallyassociatedwithstuntedgrowth (both inanimalsandhumans).ThisheldtruehereasKDfedmutantsshowedsignificantlessweightgain than CD fed wildtype mice. g=grams, ***p<0.001 significant difference fromwildtypecontrols.#p<0.001significantdifferencefromKDfedmutants.

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Figure14.AverageGroupAtaxiaScoresinAldh5a1‐/‐Mice

Thisfigurepresentsmean(±s.d.)ataxiascoresforCDfedAldh5a1‐/‐miceandKDfedAldh5a1‐/‐mice.Ataxiawasassesseddaily.CDfedmutantsprogressedveryquicklytohigh levelsof ataxia (n=9).KD fedmutantseventually reached similar levelsofataxia,buttooksignificantlylongertogetthere(n=9).SignificantdifferencesweredetectedbetweengroupsfromP17‐40,atwhichtimealltheCDfedAldh5a1‐/‐grouphaddied.CD=controldiet,KD=4:1ketogenicdiet,d=days,g=grams.Thesolidblackbar indicates theagesatwhich therewere significantdifferencesbetweenCD fedAldh5a1‐/‐miceandKDfedAldh5a1‐/‐mice(p<0.05).

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Figure15.ECoGRecordingsinAldh5a1‐/‐MiceFedEitheraCDorKD

Figure15showsrepresentativeexamplesofECoGinCDfedAldh5a1‐/‐miceandKDfed Aldh5a1‐/‐ mice. The same ECoG trace is given at three different recordingspeeds: 30mm/sec (millimeters per second, top trace), 15mm/sec (middle trace)and 3mm/sec (bottom trace). In each case the top two trace lines are ECoGrecordings from CD fed Aldh5a1‐/‐ mice. The bottom two trace lines are ECoGrecordingsfromKDfedAldh5a1‐/‐mice.Differentrecordingspeedsarereportedtobettervisualizethemorphologyofthebrainwaves.Withineachgroup,thetoptrace

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is the ECoG activitymeasured between the left frontal lobe and left parietal lobe(LF‐P). The second trace is the ECoG activitymeasured between the right frontallobe and right parietal lobe (RF‐P). Notice the increase in spike frequency andamplitudeintheCDfedmutantsascomparedtotheKDfedmutants.Thegraybarinthe30mm/sectracerepresentstheportionofthattracemagnifiedinthe15mm/sectrace. The gray bar in the 15mm/sec trace represents the portion of that tracemagnified in the3mm/sectrace.Sensitivity=30µV/mm;LFF=1Hz;HFF=100Hz;NotchedFilter(60Hz)on;LFF=lowfrequencyfilter;HFF=highfrequencyfilter.

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Figure16.AverageNumberofConvulsionsDuring1hrECoGRecordingsinCDandKDfedAldh5a1‐/‐Mice

Figure16showsthemeannumberofconvulsions(±s.e.m.)witnessedinCDandKDfedmutantsmice during the one hour ECoG recordings. Convulsions consisted oftonic‐clonic posturing and was often followed by running fits and jumping fits.Thesebehaviorscorresponded tohigh frequency ictalactivityon theECoG. In thecase of CD fed mutants, electrographic seizures were often accompanied byconvulsions.InKDfedmutants,electrographicseizuresweremuchbrieferandwereseldom accompanied by convulsions. No convulsions were witnessed in wildtypecontrols.*p<0.05.

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UponcompletingourECoGstudiesinKDfedAldh5a1‐/‐miceweswitched

onemousebackontotheCD.ThiswasdonetodeterminewhethertheKD’sECoG‐

normalizingeffectswouldpersistevenafterbeingswitchedontoaCD.Withinaday

ofreceivingaCD,however,theECoGworsenedshowingburstsofspontaneous,

recurrentspikeandwavedischargesandhighfrequencyictalactivity.Themouse

diedafter4daysontheCD.

3.4 Discussion

3.4.1 Lifespan

ThisresultsofthepresentstudyshowedthattheKDcanprolongthelifespan

of Aldh5a1‐/‐ mice by over 300%. Previous research has shown that certain

pharmacological agents may also prolong lifespan of these mutants—albeit

modestly. Hogema et al. (2001) showed that about 50% of Aldh5a1‐/‐ mutants

treatedwithVGBortaurinelivedtoP50.VGBandtaurinehadnoeffectontheataxia

and weight gain of mutant mice. The present data are unique in that they

demonstrated that the KD greatly improved the general phenotype of Aldh5a1‐/‐

mice.Thiswasevidencedbyamuchgreaterincreaseinlifespan,asignificantdelay

intheonsetofataxia,improvementinweightgainandnormalizationoftheECoG.

AlthoughtheexactcauseofdeathinCDfedAldh5a1‐/‐mutantsisnotclear,it

is assumed that they die perhaps in cardiac or respiratory failure during status

epilepticus. After 3‐4 days of regular tonic‐clonic seizures (around P18‐23),

untreatedAldh5a1‐/‐ mice enter a period of sustained convulsive behavior. It has

beenshownthatprolongedstatusepilepticuscancauseneuronaldamageanddeath

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(Heinemann et al., 2002; Jiao&Nadler, 2007; Tsuchidaet al., 2007).Most deaths

wereunwitnessed.KDfedmutantsdidnotexperiencestatusepilepticusatP18‐23.

Theydid,however,becomeincreasinglysusceptibletoauditory‐andstress‐evoked

convulsionsovertime.Suchconvulsionswereonlyseen inanimalsP70andolder,

and they involved a tonic‐clonic component with jumping and running fits. The

exactcauseofdeathinKDfedmutantsisalsounknownanditispossibletheyalso

died inunwitnessedstatusepilepticus. It isalsopossiblethat theydiedduetoKD‐

relatedcomplicationssuchascardiacdisease.

RelativetopossibleKDinducingtoxicity,thepresentexperimentsinvolving

KDfedAldh5a1‐/‐micerepresentthelongesttimeanymousehasbeenmaintained

on the KD (the longest‐living KD fed mutant was maintained on the KD for 134

days—lifespanof146days,starteddietonP12).Thelong‐termeffectsofahigh‐fat

diet in mice have not been investigated. During the course of the present

experiment, the KD fed mutants became increasingly greasy in appearance and

somemicelosttheirfurduetoexcessivegrooming.

We investigated whether the greasy fur was caused by long‐term

consumptionofahigh fatdiet,orwhether itwas fromdirectcontactwiththeKD.

Micewereoftenseenwalkingthroughtheirfooddish.Totestthis,weremovedthe

KDovernight.Anylong‐termeffectsofdietshouldpersistintheabsenceofdiet.The

subjects’fur,however,wascompletelynormalafteronly1daywithouttheKD.This

suggestedthatdirectcontactwiththedietwascausingthemicetodevelopgreasy

fur.Tominimize this,webegan changing the cages every twodays topreventoil

buildupinthecage,anditstransfertotheanimals’fur.

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InmutantsmaintainedonaKDitbecomesdifficulttouncouplethecontinued

development of SSADH‐d with the toxic effects of long‐term high fat intake. In

humans, theKDhasbeenassociatedwith increasedriskofcoronaryheartdisease

(Bestetal.,2000;Bergqvistetal.,2003;Dashtietal.,2003;Kwiterovichetal.,2003).

It ispossible that suchanadverseeffectof thedietmayhaveplayeda role in the

deathof theKD fedmutants.Humanson theKDareusuallyweanedoff after 2‐3

years for fearof thedeleterious long‐termeffectsofhigh‐fatdietconsumption.As

proposed below (Future Studies), perhaps a more healthy form of the diet (e.g.,

polyunsaturatedfattyacidbaseddiet)wouldallowthemicetolivelongerwithout

thedeleteriouseffectsofadiethighinsaturatedfat.

3.4.2 Ataxia

The KD greatly delayed the onset of ataxia in Aldh5a1‐/‐ mice. CD fed

Aldh5a1‐/‐ mice developed marked ataxia by P15‐17. This was evidenced by

significantdraggingofthehindlimbs(stage2ataxia).Theataxiaquicklyworsened

toinvolvecompleteimmobilityforprolongedperiodswithunsuccessfulattemptsto

walk. Untreated mutants, by P25, had little control over their locomotion. When

ataxia was being determined, subjects were placed on a table and monitored

visually. CD fedmutants had to bewatched closely or theywouldwalk off of the

table.KDfedmutants,however,tooksignificantlylongertodevelopataxia.Stage2

ataxia did not occur until ~P70. Even at high levels of ataxia, KD fed mutants

maintainedenoughexteroceptiontoavoidtheedgeofthetableandnottofalloff.

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It remains unclear why Aldh5a1‐/‐ mice, and SSADH‐d patients, develop

ataxia. Gupta and colleagues (2003) have pointed out that neurotoxic molecules

accumulate in Aldh5a1‐/‐ mice similar to the molecules that have been shown to

accumulateinParkinson’sdisease.Interestingly,theKDhasrecentlybeenshownto

haveefficacy invariousmodelsofneurodegeneration, suchasParkinson’sdisease

(Massieu et al., 2003; Noh et al., 2003; Sullivan et al., 2004; Gasior et al., 2006;

Maaloufetal.,2007).

Alternatively,systemicadministrationofGHB,whichiselevatedinAldh5a1‐/‐

mice,hasalsobeenshowntodisruptcatecholaminemetabolism(Guptaetal.,2003;

Gibsonet al., 2003;Wonget al., 2004;Knerret al., 2007). It is possible that such

disruptionsofcatecholaminedegradationmightcausetheataxiaseeninAldh5a1‐/‐

mice,althoughthisispurelyspeculative.

3.4.3 Weights

Weightswere assessed inKD andCD fedAldh5a1‐/‐mice aswell as CD fed

wildtypemice.Thereareseveralstudies,bothclinicalandexperimental,thatreport

that KD fed subjects are lighter than normal diet fed subjects (Rho et al., 1999;

Bough & Eagles, 2001; Vining et al., 2002; Zhao et al., 2004; Nylen et al., 2005).

Despite having a significantly stunted growth pattern, rodents fed a KD do not

experienceanywherenearthestuntingseeninSSADHmutantsfedeitheraKDora

CD.

KDfedmutantsshowedsignificantweightgaincomparedtoCDfedmutants.

Clinically, the KD is associated with significantly attenuated growth compared to

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humansconsumingnon‐KD food(McDonald,1997;Couchetal.,1999;Williamset

al.,2002;Viningetal.,2002;Liuetal.,2003;Papandreouetal.,2006).Thishasalso

beenshowninratsfedaKD(Zhaoetal.,2004;Nylenetal.,2005;Nylenetal.,2006).

This perhaps held true in the present experiment as the KD fedmutants showed

significant stunting of growth as compared to CD fed wildtype mice. When

comparedtoCDfedAldh5a1‐/‐mice,however,KDfedmicegainedsignificantlymore

weight.

ItispossiblethatthecauseofthisweightgainissimplybecausetheKDfed

mutants consumed more food. Previous research from our group, however, has

measured food intake in rats on the KD and found that KD fed animals tend to

regulatetheircaloricintake(Likhodiietal.,2000).BecausetheKDisnearlytwiceas

calorie‐denseasthecontroldiet,thismeansthatKDfedanimalstendtoeatabout

halfasmuchfood.

Webelieve thatsignificantweight loss inuntreatedAldh5a1‐/‐micemaybe

causedbyimpairedglucosemetabolism(Chowdhuryetal.,inpress)andsubsequent

metabolism of fat and protein stores. Consumption of a high fat KD provides an

alternativeenergysourceforAldh5a1‐/‐mice,allowingsomegrowthtotakeplace.

3.4.4 Electrocorticography

KDfedmiceshowedaremarkablenormalizationintermsoftheirECoG.This

isconsistentwithotherstudies thathave foundEEG improvements inKDfedrats

(Raffoetal.,2007)andKDfedpatients(Cantelloetal.,2007;Hallbööketal.,2007).

ItisnotunderstoodhowtheKDconfersthesechanges.

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Arecentstudysuggeststhat,athighconcentrations,βOHBactsasanagonist

onKATPchannelsinthebrain,slowingthefiringratesofneurons(Maetal.,2007).

Ourdatashowareconsistentwiththispossibility,aswedemonstratedthatKDfed

mutantshadhighconcentrationsofβOHB(Chapter6,Experiment4).Suchaneffect

mayexplainwhytheECoGofAldh5a1‐/‐miceshowlessseizureactivity,andwhythe

micehavefewerconvulsions.

In general, we propose that the KD normalized ECoG by forestalling the

evolutionofalethalstatusepilepticusinthesemutantanimals.Evidenceinfavorof

thishypothesismaybefoundinthemarkeddiminutioninfrequencyandseverityof

epileptiformdischargesontheEEGofKDtreatedAldh5a1‐/‐miceandaconcomitant

decreaseintheoccurrenceofconvulsionsinKDtreatedmutantanimals.

UponswitchingKDfedAldh5a1‐/‐micebackontoaCD,wesawanimmediate

(within1day)worseningoftheECoGandreturnofconvulsions.Clinically, theKD

can cause some patients to become seizure free, and this seizure freedom may

persist after the KD is weaned. This raises the question of whether the KD has

“antiepileptogenic” (i.e., it prevents the genesis of seizures) effects or

“anticonvulsant”effects (i.e., it suppressesanexistingpropensity toseizures).Our

data demonstrate that the KD is anticonvulsant, but not antiepileptogenic (in the

Aldh5a1‐/‐mice)asbothelectrographicandbehavioralseizuresreappearafter the

KDisstopped.SimilarresultswerereportedbyHuttenlocher(1976)whosawthe

returnofseizuresafterusinganintravenousglucoseinfusiontoterminateketosisin

aboyontheketogenicdiet.

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3.4.5 Conclusions

Takentogether,theresultsfromthisstudylendconsiderablepromisetothe

hypothesisthattheKDmightbesuccessfulintheclinicaltreatmentofSSADH‐d.

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CHAPTER4

EXPERIMENT2:THEEFFECTSOFAKETOGENICDIETON

MINIATUREPOST­SYNAPTICCURRENTSINALDH5A1­/­MICE

4.1 Introduction&Rationale

Experiment1foundthattheKDwasabletosignificantlyprolongthelifespan

ofAldh5a1‐/‐mice.ItalsoshowedthattheKDsignificantlyimprovedweightgain,

delayedtheonsetofataxiaandnormalizedtheECoGinAldh5a1‐/‐miceascompared

toCDfedmutants.Thepresentstudywasdesignedtodeterminewhetherthese

significantchangesinphenotypewereaccompaniedbychangesininvitro

neuroelectrophysiology.

Wuetal.(2006)studiedhippocampalslicesfromAldh5a1+/+andAldh5a1‐/‐

mice.TheirworkshowedthatAldh5a1‐/‐micehavehyper‐excitableneurons(Wuet

al.,2006).ThiswasshowninslicestudieswhereAldh5a1‐/‐micehadsignificantly

enlargedCA1(cornuammonus)fieldpotentialsinAldh5a1‐/‐miceascomparedto

wildtypemice.WhentheSchaffercollateralswerestimulated,CA1pyramidal

neuronsfromAldh5a1‐/‐micenotonlyshowedenhancedfieldpotentials,butalso

seizure‐likeresponsesthatoutlastedthestimulation—whereascellsfromwildtype

miceonlygeneratedasingleevoked‐response(Wuetal.,2006).

Usingthewholecellcurrentclampmethod,Wuandcolleagues(2006)also

showedthatAldh5a1‐/‐micehavesignificantlyreducedevokedinhibitory

responses—i.e.,themutantsshowedlessGABAAreceptor‐linkedchloridechannel

ionflux,ascomparedtowildtypemice,whentheShaffercollateralswere

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stimulated.Takentogether,thesedatasuggestthatAldh5a1‐/‐micehavehyper‐

excitablehippocampalneuronsascomparedtowildtypecontrolmice.

The present experiment was designed to extend these findings by

determining whether spontaneous miniature postsynaptic currents (mPSC) were

affected inAldh5a1‐/‐mice.Also,we sought todeterminewhether theKDhasany

effect on these currents. We measured spontaneous, inhibitory miniature

postsynapticcurrents(mIPSC)andspontaneous,excitatoryminiaturepostsynaptic

currents (mEPSC) in CD fed wildtype mice, CD fed Aldh5a1‐/‐ mice and KD fed

Aldh5a1‐/‐mice.

Miniaturepostsynaptic currents,or “minis”, arecausedby thespontaneous

release of neurotransmitter substance from the presynaptic neuron (Hirschet al.,

1999).Theseneurotransmittersare“leaked”intothesynapseintheabsenceofan

actionpotential.Thesecurrentsplayarole in“priming” thesynapsebyregulating

the expression and location of postsynaptic receptors (Verstreken and Bellen,

2002).

PaststudieshaveshownthatmPSCsplayacriticalrole in thedevelopment

andmaintenanceof synapses (Swanwicketal.,2006;Hartmanetal.,2006).Minis

are also thought to play a role in regulating synaptic strength (Verstreken and

Bellen,2002).mIPSCshelpregulate the inhibitory toneandmEPSCshelpregulate

theexcitatorytoneinthebrain.Therefore,itisnotsurprisingthatmPSCshavebeen

implicatedinthemechanismsofepileptogenesisandseizures.

Rats that develop spontaneous convulsions after kainic acid or pilocarpine

treatmentshowsignificantreductionsinmIPSCfrequency(Hirschetal.,1999;Shao

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andDudek,2005).KindledratsalsoshowsignificantreductionsinmIPSCfrequency

(WierengaandWadman,1999). Interestingly, thesekindled ratsdevelopedanew

typeofmIPSCcurrent,whichwasinfrequentbutverylargeinamplitude.

Wehypothesized that the severe seizures seen inAldh5a1‐/‐micemightbe

the result of either decreased mIPSC activity or increased mEPSC activity. We

further hypothesized—given the remarkable effects of the KD on the Aldh5a1‐/‐

phenotype—thattheKDmightnormalizeanyperturbationinthesecurrents.

4.2 Methods

4.2.1 Subjects

Aldh5a1‐/‐andAldh5a1+/+mice(P18‐22)servedassubjectsforthepresent

experiments.TheywereobtainedandhousedasdescribedintheGeneralMethods.

Subjectsweredividedintothreegroups:CDfedAldh5a1+/+,CDfedAldh5a1‐/‐and

KDfedAldh5a1‐/‐.

4.2.2 Electrophysiology:BrainSlicesandSolutions

Micewereanesthetizedwithhalothaneanddecapitatedtoobtain

hippocampalslices.Transversebrainslices(450µm)wereobtainedusinga

vibratome(Series1000;St.Louis,MO)andmaintainedinartificialcerebrospinal

fluid(aCSF)containing(mM)125NaCl,2.5KCl,1.25NaH2PO4,2MgSO4,2CaCl2,25

NaHCO3,and10glucose.aCSFwasbubbledwithcarbogen(95%O2,5%CO2)and

gravityfedtotherecordingchamberatarateof3‐4ml/min.

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Theinternalsolution—whichmimicsintracellularfluid—forrecording

mPSCsconsistedof(mM)20cesiummethanesulfonate,2Mg‐ATP,10HEPES,0.3

GTP,0.1EGTA,130CsCl.Osmolaritywas300±5mOsmandpHwasadjustedto7.2

usingcesiumhydroxide.FormIPSCrecordings,D‐2‐amino‐5‐phosphopentanoicacid

(D‐AP5;20µM,madeof50mMstocksolutionindistilledwater),6‐cyano‐7‐

nitroquinoxaline‐2,3‐dione(CNQX;100µM,madeof50mMstockin

dimethylsulfoxide)andtetrodotoxin(TTX;1µM,madeof1mMstockindH2O)were

addedtotheaCSFsuperfusate.GABAB‐mediatedpotassiumfluxeswereblockedby

cesiumchlorideintheinternalsolution.FormEPSCrecordings,bicuculline

methiodide(BMI;10µMmadeof10mMstockindH2O)wasaddedtoaCSF

superfusatealongwithTTX.ChemicalswereobtainedfromSigma.Aliquotswere

diluteddaily.

4.2.3 Electrophysiology:WholeCellRecordings

Neuronalrecordingswereobtainedwiththeuseofthewholecell

configurationofthepatchclamptechniquefromtheCA1hippocampalpyramidal

neurons.Electrodeshadtipresistancesbetween5and8MΩ.Neuronalresponses

wererecordedwiththeuseofaMulticlamp700B(MolecularDevices,Sunnyvale,

CA).Forextracellularstimulation,abipolarstimulationelectrode(catalog#:

CBARC100,FHCInc.,Bowdoin,ME)wasplacedintheSchaffercollaterals.

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4.2.4 DataAnalysis

mPSCtraceswereanalyzedusingMiniAnalysissoftware(Synaptosoft,

Decatur,GA).

4.3 Results

4.3.1 MiniatureInhibitoryPost­synapticCurrents(mIPSC)

mIPSCs were recorded from CA1 pyramidal cells of hippocampal slices.

Figure17 showsmean (±s.e.m.) groupdata formIPSCamplitude, frequency, area,

rise‐timeanddecay‐time.RepresentativemIPSCtracesfromCDfedwildtypemice,

CDfedmutantmiceandKDfedmutantmicecanbefoundinFigure19.

Figure 17a shows mean (±s.e.m.) group mIPSC amplitudes (measured in

picoamperes, pA) in CD fed Aldh5a1+/+ mice, CD fed Aldh5a1‐/‐ mice and KD fed

Aldh5a1‐/‐ mice. mIPSC amplitude is thought to reflect the number of opened

postsynaptic GABAAR associated chloride channels (Otis et al., 1994). mIPSC

amplitudewasanalyzedusingaone‐wayANOVA,whichrevealedanoveralleffect

(p=0.04). Tukey’s post‐hoc tests showed that CD fed Aldh5a1‐/‐ mice had

significantly larger mIPSC amplitudes than wildtype mice (p<0.05), but not as

compared to KD fed Aldh5a1‐/‐ mice. KD fed mutants and wildtype mice did not

differintermsofmIPSCamplitude(p>0.05).

Figure 17b shows mean (±s.e.m.) group mIPSC frequencies (measured in

Hertz,Hz)inCDfedAldh5a1+/+mice,CDfedAldh5a1‐/‐miceandKDfedAldh5a1‐/‐

mice. mIPSC frequency is thought to reflect the number of spontaneously active

synapses(WierengaandWadman,1999).Specifically,thiscanmeanachangeinthe

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number of axon terminals synapsing on the postsynaptic cell, changes in the

frequency of presynaptic neurotransmitter release—in this case GABA—or it can

reflect changes in postsynaptic receptor density or function—in this case, the

GABAAR and its associated chloride channel. Onemust examinemIPSC frequency

together with other mIPSC parameters (below) to get a better idea of which

parametermaybealtered.WeanalyzedmIPSCfrequencyusingaone‐wayanalysis

ofvariance(ANOVA),whichrevealedstatisticallysignificantdifferenceswithinthe

groups (p=0.04). Tukey’s post‐hoc tests revealed that mIPSC frequency in slices

fromCDfedAldh5a1‐/‐micewassignificantlydiminished(bygreaterthan2.5fold),

as compared to CD fedwildtypemice (p<0.05) or KD fedAldh5a1‐/‐mice.mIPSC

activity in KD fedAldh5a1‐/‐ mice, however, was completely restored towildtype

levelsanddidnotdifferfromCDfedwildtypemice(p>0.05).

Figure 17c shows mean (±s.e.m.) group mIPSC areas under the curve

(measuredinpicoamperespermillisecond,pAms)inCDfedAldh5a1+/+mice,CDfed

Aldh5a1‐/‐mice andKD fedAldh5a1‐/‐mice. The area under the curve formIPSCs

represents either the size of vesicular transmitter content or an enhanced

synchronization of presynaptic neurotransmitter release (Otis et al., 1994). An

ANOVArevealednosignificantdifferencesamongthegroups(p=0.19).

Figure 17d shows mean (±s.e.m.) group mIPSC rise times (measured in

milliseconds, ms) in CD fed Aldh5a1+/+ mice, CD fed Aldh5a1‐/‐ mice and KD fed

Aldh5a1‐/‐ mice. mIPSC rise‐time is thought to represent opening kinetics of the

GABAAR associated chloride channel (Otis et al., 1994). An ANOVA revealed no

significantdifferencesamongthegroups(p=0.85).

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Figure17eshowsmean(±s.e.m.)groupmIPSCdecaytimes(measuredinms)

inCDfedAldh5a1+/+mice,CDfedAldh5a1‐/‐miceandKDfedAldh5a1‐/‐mice.mIPSC

decay time is thought to represent the closing kinetics of the GABAAR associated

chloride channel (Otis et al., 1994). An ANOVA revealed a significant main effect

p=0.03).Tukey’spost‐hoctestsrevealedthatdecaytimesinCDfedAldh5a1‐/‐mice

weresignificantlylessthanthedecaytimesinKDfedAldh5a1‐/‐mice(p<0.05),but

notas compared toCD fedwildtypemice.Decay timesbetweenKD fedAldh5a1‐/‐

miceandCDfedwildtypemicedidnotdiffersignificantly.

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Figure 17. The Effect of a 4:1 KD on mIPSC Characteristics in Aldh5a1‐/‐ andAldh5a1+/+Mice

Figure 17 shows mIPSC properties for CD fed Aldh5a1+/+ mice (N=8), CD fedAldh5a1‐/‐mice(N=6)andKDfedAldh5a1‐/‐mice(N=6).N'sindicatethenumberofanimals.Recordingswerecollectedfromatleasttwocellsfromeachanimal.mIPSCswere recorded in the presence of the voltage gated sodium channel blocker TTX(1µM) and the glutamatergic blockers CNQX (100µM) and APV (20µM). ThepresenceofcesiuminthepatchingelectrodeblockedGABABRassociatedpotassiumchannel conductance. a) mIPSC amplitudes are significantly increased in CD fedmutantsascomparedtoCDfedwildtypemice.b)mIPSCfrequencyisdramaticallyreducedinCDfedAldh5a1‐/‐miceascomparedtoCDfedwildtypecontrolsandKDfed mutants. KD fed mutants had completely restored mIPSC activity. c) NodifferencewasdetectedbetweengroupsintermsofmIPSCarea.

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d)No differencewas detected between groups in terms ofmIPSC area. e)mIPSCdecay time was significantly longer in KD fed mutants as compared to CD fedmutants and did not differ significantly from CD fed wildtype mice. *p<0.05,**p<0.01.

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4.3.2 MiniatureExcitatoryPost­synapticCurrents(mEPSC)

Miniature excitatory post‐synaptic currents (mEPSC) were recorded from

CA1pyramidalcellsinhippocampalslices.Figure18showstheaveragegroupdata

for mEPSC frequency, amplitude, area, rise‐time and decay‐time. Representative

mEPSC traces fromCD fedwildtypemice,CD fedmutantmiceandKD fedmutant

micecanbefoundinFigure19.

Figure 18a showsmean (±s.e.m.) groupmEPSC amplitudes (pA) in CD fed

Aldh5a1+/+ mice, CD fed Aldh5a1‐/‐ mice and KD fed Aldh5a1‐/‐ mice. mEPSC

amplitudes are thought to reflect the number of opened postsynaptic glutamate

receptorlinkedionchannels(BekkersandStevens,1995).mEPSCamplitudeswere

analyzedusingaone‐wayANOVA.AlthoughbothAldh5a1‐/‐groups(CDandKDfed)

had lower mEPSC amplitudes, as compared to CD fed wildtype controls, no

statisticallysignificantdifferenceswerefound(p=0.11).

Figure 18b showsmean (±s.e.m.) groupmEPSC frequencies (Hz) in CD fed

Aldh5a1+/+ mice, CD fed Aldh5a1‐/‐ mice and KD fed Aldh5a1‐/‐ mice. mEPSC

frequency is thought to reflect the rate of presynaptic glutamate release and

subsequentpostsynapticglutamatereceptorbindingandchannelopening,allowing

theinfluxofcationsintothepostsynapticcell(BekkersandStevens,1995).mEPSCs

wereanalyzedusingaone‐wayANOVA.AlthoughcellsfromCDfedAldh5a1‐/‐mice

showeda somewhatdeceasedmEPSC frequency, as compared toCD fedwildtype

miceandKDfedAldh5a1‐/‐mice.Nosignificantdifferencesweredetectedamongthe

groups(p=0.46).

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Figure 18c shows mean (±s.e.m.) group mEPSC areas (pAms) in CD fed

Aldh5a1+/+mice,CDfedAldh5a1‐/‐miceandKDfedAldh5a1‐/‐mice.Theareaunder

thecurverepresentseitherthesizeofvesiculartransmittercontentoranenhanced

synchronization of presynaptic neurotransmitter release (Otis et al., 1994). An

ANOVArevealedsignificantdifferencesbetweengroups(p=0.02).Tukey’spost‐hoc

testsrevealedthattheCDfedAldh5a1‐/‐micehadsignificantlysmallermEPSCareas

thanCDfedwildtypemiceandKDfedAldh5a1‐/‐mice(p<0.05).mEPSCareadidnot

differ significantly between andKD fedAldh5a1‐/‐mice andCD fedwildtypemice

(p>0.05).

Figure 18d shows mean (±s.e.m.) group mEPSC rise times (ms) in CD fed

Aldh5a1+/+ mice, CD fed Aldh5a1‐/‐ mice and KD fed Aldh5a1‐/‐ mice. mEPSC rise

times were analyzed using a one‐way ANOVA. No significant differences were

detectedamongthegroups(p=0.90).

Figure18eshowsmean (±s.e.m.)groupmEPSCdecay times (ms) inCD fed

Aldh5a1+/+mice,CD fedAldh5a1‐/‐miceandKD fedAldh5a1‐/‐mice.mEPSCdecay

times were analyzed using a one‐way ANOVA. No significant differences were

detectedamongthegroups(p=0.24).

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Figure18.TheEffectofa4:1KDonmEPSCCharacteristicsinAldh5a1‐/‐andAldh5a1+/+Mice

Figure 18 shows mEPSC properties for CD fed Aldh5a1+/+ mice (N=8), CD fedAldh5a1‐/‐mice(N=8)andKDfedAldh5a1‐/‐mice(N=9).Recordingswerecollectedfromatleasttwocellsfromeachanimal.mEPSCswererecordedinthepresenceofthe voltage‐gated sodium channel blocker TTX (1µM) and the GABAergic blockerBMI (10µM). a) No significant differences were found in mEPSC amplitude. b)mEPSC activity is reduced non‐significantly in CD fed mutant mice. mEPSCfrequency is similar in KD fedmutantmice and CD fedwildtypemice. c)mEPSCareas were significantly lower in CD fed Aldh5a1‐/‐ mice as compared to KD fed

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Aldh5a1‐/‐miceandCD fedwildtypemice.d)Nodifferencewas found in termsofmEPSC rise times. e)No differencewas found in terms ofmEPSC decay times. **p<0.01.

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Figure19.RepresentativemIPSCandmEPSCTraces

Figure 19 shows (a) representative mIPSC and (b) mEPSC traces from CD fedAldh5a1+/+mice,CDfedAldh5a1‐/‐miceandKDfedAldh5a1‐/‐mice.(a)mIPSCtracesfromhippocampalpyramidalneurons.mIPSC frequencywas significantly reducedinCDfedmutantsascomparedtowildtypemicefedaCD.KDfedmutants,however,showrestoredmIPSCactivity.NotethelargeamplitudemIPSCcurrent(markedbythearrow)intheCDfedAldh5a1‐/‐group.(b)Nodifferencesweredetectedintermsof mEPSC frequency. CD=control diet, KD=ketogenic diet, pA=picoamperes,sec=second.

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4.4 Discussion

4.4.1 mIPSC

HerewereportforthefirsttimeasignificantdecreaseinmIPSCfrequencyin

Aldh5a1‐/‐ mice. This reflects a decrease in the number of spontaneously active

inhibitorysynapses(WierengaandWadman,1999).

Wu et al. (2006) used in vitro slice studies to show that the brains of

Aldh5a1‐/‐ mice are hyperexcitable as compared to the brains of wildtype mice.

Seizure frequencyandECoGdata fromExperiment1 furthersupportthe ideathat

brains of untreated mutants are hyperexcitable as compared to wildtype mice.

Experiment 1 showed that theKD is able to significantly decrease the number of

convulsions as well as normalize the ECoG in Aldh5a1‐/‐ mice. The results of

Experiment2areconsistentwiththeseobservations.

Experiment 2 showed that spontaneous inhibitory tone (i.e., mIPSC

frequency) is significantly reduced in Aldh5a1‐/‐ mice as compared to CD fed

wildtype mice. Previous research has suggested that such attenuation in mIPSCs

may be a mechanism for epileptogenesis, off‐setting the excitatory/inhibitory

balance in favor of excitation and leading to seizures (Shao et al., 2005). Other

experiments in animal models of epilepsy also support this theory, showing that

decreases in mIPSC frequency may play a role in epileptogenesis (Wierenga and

Wadman,1999; ShaoandDudek,2005). Inhibitory tonewas restored towildtype

control levels in the KD fedmutantmice. This restoration ofmIPSC activitymay

contributetothereductioninconvulsions,andsubsequentprolongationoflifespan,

inKDfedAldh5a1‐/‐mice.

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The mechanism behind the decrease in mIPSC frequency and KD induced

normalization of mIPSC frequency is not understood. Experiment 3 (below), an

examinationof[35S]TBPSbinding(aGABAAassociatedchloridechannelligand),may

providesome insight. [35S]TBPSbinds to thepostsynapticGABAAR‐linkedchloride

channel.Wuetal.(2006)haveshownthatthisbindingissignificantlyreducedinCD

fedAldh5a1‐/‐mice, suggesting thatmutantmicehave fewerpostsynapticchloride

channels. OurmIPSC data are consistentwith this, as CD fedmutantmice have a

significant lower frequency of mIPSC activity. If there are fewer postsynaptic

chloridechannels, thentherecouldbeacorrespondingdecrease inmIPSCs,which

aremediatedviathepostsynapticchloridechannel.

Thepresentdata show thatmIPSCactivity is restored inKD fedAldh5a1‐/‐

mice.OurdatainExperiment3(below)demonstratethattheKDrestores[35S]TBPS

binding in a region‐specific manner in Aldh5a1‐/‐ mice. If the KD leads to a

normalization of postsynaptic GABAAR‐linked chloride channels, then this could

explainthenormalizationofmIPSCfrequencyinKDfedmutants.

Another possibility is that mIPSC frequencies are reduced as a result of

significantelevations inGHB.AsmentionedintheGeneral Introduction,Aldh5a1‐/‐

mice have 50‐70‐fold elevations of GHB throughout their bodies (Hogema et al.,

2001). GHB is an agonist at the presynaptic GABAB receptor (Snead and Gibson,

2005). When activated, the presynaptic GABAB receptor inhibits the presynaptic

voltage‐gatedcalciumchannel(SneadandGibson,2005).Thepresynapticvoltage‐

gatedcalciumchannel,inturn,mediatescalciuminfluxandthesubsequentdocking

andreleaseoftransmittercontainingvesicles(SneadandGibson,2005).Thiscould

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inhibit the presynaptic release of GABA and explain the significant reduction in

mIPSCfrequency.Inthecontextofthismechanism,itisunclearhowtheKDmight

restoremIPSC frequencies. One possibility is that the KD lowers GHB levels. The

study to examineGHB levels inKD fedmutants is proposedbelow in the “Future

Studies”section.

The synaptic balance between excitation and inhibition also affects the

information that transfers through theneural networks (Somerset al., 1995) and

affects experience‐dependent plasticity (Hensch et al., 1998). Human SSADH‐d

patients experience psychomotor retardation as well as seizures. It is therefore

possiblethatthedecreasesinmIPSCfrequencymayalsoplayaroleinthecognitive

delay seen in humans with SSADH‐d. Recently, the KD was shown to improve

cognitionandmoodinpatientswithdrugresistantepilepsy(Farasatetal.,2006).

CorrespondingtothesignificantattenuationofmIPSCfrequencyin

Aldh5a1‐/‐micewasasignificantincreaseinmIPSCamplitude.Thisissimilartothe

resultsofWierengaandWadman(1999)whofoundasignificantdecreaseinmIPSC

frequency.Theyalso,however,sawtheformationofanew,largemIPSCcurrentin

kindledrats.AnexampleofalargeamplitudemIPSCcanbefoundinFigure19a(CD

fedAldh5a1‐/‐).ThisincreaseinmIPSCamplitudecouldreflectanincreaseinthe

postsynapticexpressionofGABAAreceptors(Edwards,1995).Thisseemsunlikely,

however,giventhatAldh5a1‐/‐micehavebeenshowntohavenochangeinGABAA

receptorexpression(Wuetal.,2006).Analternativeexplanationisthatthe

synchronousreleaseofGABA‐containingvesiclesfromseveralactivezonesis

responsiblefortheincreasedmIPSCamplitudesinCDfedAldh5a1‐/‐mice.Previous

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studieshaveshownthistobethecaseinkindledrats(Geinismanetal.,1990;

Nusseretal.,1997,Nusseretal.,1998).

4.4.2 mEPSC

OurstudyofmEPSCsshowedthatmEPSCareaissignificantlysmallerinCD

fedAldh5a1‐/‐miceascomparedtoKDfedAldh5a1‐/‐miceandCDfedwildtypemice.

Thissuggeststhattheremaybeeitherlesstransmittersubstancebeingreleasedor

that there is a significantly reduced synchronization of presynaptic transmitter

release.ItremainsunclearwhymEPSCareawouldbesmallerasnoneoftheother

mEPSCpropertiesweresignificantlychanged.

NoothersignificantchangestomEPSCpropertieswereseeninmutantmice

fedeitherdiet.ThissuggeststhatmEPSCdonotappeartoplayamajorroleinthe

hyperexcitabilitythathasbeenreportedinAldh5a1‐/‐mice.

4.4.3 Conclusions

This is the first study to show thatmIPSCactivity is impaired inAldh5a1‐/‐

mice.ItisalsothefirststudytoshowthattheKDcanrestoretowardsnormalmIPSC

activity inAldh5a1‐/‐ mice. Although restoration ofmIPSC frequency inAldh5a1‐/‐

micelikelyplaysaroleintheKD’smechanismofactioninAldh5a1‐/‐mice,itisnot

thediet’ssolemechanismofaction.TheKDappears tohavemanymechanismsof

action, some that are detailed in the General Introduction, and others that are

shownforthefirsttimeinthisthesis.

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Further studies will be required to determine why mIPSC frequency is

significantly reduced in Aldh5a1‐/‐ mice, and how the KD works to restore these

currents.

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CHAPTER5

EXPERIMENT3:THEEFFECTSOFAKETOGENICDIETON[35S]TBPS

BINDINGINALDH5A1­/­MICE

5.1 Introduction&Rationale

Workinginslices,Wuetal.(2006)showedthatthebindingof[35S]TBPS,a

markerfortheGABAAR‐associatedchloridechannel,issignificantlydecreasedin

Aldh5a1­/­mice.ThissuggeststhatAldh5a1­/­micemayhavefewerchloride

channelsintheirGABAergicsynapses.Fewerchloridechannelsshouldleadtoa

decreaseinpostsynapticchlorideinfluxfollowingstimulationoftheGABAAR,

resultinginlesshyperpolarizingcurrent.Thiscouldleadtoneuronal

hyperexcitability,whichcouldcontributetoseizuressuchasthoseseeninAldh5a1‐/‐

mice.

Experiment1showedthatCDfedAldh5a1‐/‐mice(P20‐25)have

spontaneous,recurrentabsence‐likeseizures,generalizedconvulsiveseizuresand

statusepilepticus(whilewildtypemicedonot).Experiment2showedthatAldh5a1‐/‐

micehavesignificantlyreducedmIPSCfrequenciesascomparedtowildtypemice.

TheseexperimentsalsodemonstratedthatseizuresandmIPSCfrequenciesare

normalizedinAldh5a1‐/‐micefedaKD.Apossiblemechanismforthisnormalization

couldbethattheKDrestoresthenumberofpost‐synapticGABAAR‐associated

chloridechannels.Thiswouldleadtotherestorationofhyperpolarizing(i.e.,

inhibitory)currents,makingthecellslessexcitable.

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ThepurposeofExperiment3wastodeterminewhetherAldh5a1‐/‐micefeda

KDhavehigher[35S]TBPSbindingascomparedtomutantsfedaCD.Giventhe

significantbeneficialeffectsoftheKDontheAldh5a1‐/‐phenotypewehypothesized

thattheKDwouldraise[35S]TBPSbindingtowardsnormallevelsinAldh5a1‐/‐

mutants,thusrestoringtheabilityofneuronstorespondtopresynapticGABAergic

signals.TissuefromCDandKDfedAldh5a1+/+miceprovidedanindexofnormal

bindinglevels.

5.2 Methods

5.2.1 Subjects,SacrificeandPreparationofSlices

SubjectswerehousedanddietswereformulatedasoutlinedintheGeneral

Methodssection.AllmiceweregenotypedbetweenP10‐12.Aldh5a1+/+miceand

Aldh5a1‐/‐micewereplacedoneitheraCDorKDbyP12andcontinuedontheir

respectivedietsuntilthetimeofsacrifice.

Subjects(P22‐25)wereanaesthetizedwithhalothaneanddecapitated.

Brainswereremovedimmediatelyandimmersedinisopentane,whichwaspre‐

cooledindry‐icefor20minutes.Brainswerethenstoredat‐80°Cuntilthetimeof

sectioning.

SectioningwasdoneusingaLeica(Wetzlar,Germany;model:CM1900)

cryostatat‐20°C.Coronalsectionswerecutatathicknessof30μmandthenthaw‐

mountedontogelatin‐coatedslides.Slideswereair‐driedandsubsequentlystored

at‐80°C.

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5.2.2 Ligands

Radioactivetert‐butylbicyclophosphorothionate([35S]TBPS)(65Ci/mmol;

100μCiin0.05mlofethanol)waspurchasedfromPerkinElmerInc.(Boston,MA).

K2HPO4,NaH2PO4,NaCl,EDTAandpicrotoxinwereallobtainedfromSigma

(Oakville,ON).

5.2.3 T­[35S]butylbicyclophosphorothionate([35S]TBPS)Autoradiography

The[35S]TBPSbindingprotocolwasperformedusingthemethodofBanerjee

etal.(1998).Slideswereremovedfromthefreezerandallowedtothawandair‐dry

inafume‐hoodfor1hour.Theywerethenpre‐incubatedfor10minutesina50mM

K2HPO4/NaH2PO4(adjustedtopH7.4)bufferthatcontained200mMNaCland1mM

EDTA.Followingthis,slideswereincubatedfor3hoursinthesamebuffer

(excludingEDTA)containing2nM[35S]TBPS.Thisdosewaschosenbasedondose‐

responsedata(2nMwastheBmaxdose)obtainedbyBanerjeeetal.(1998).

Incubationwasterminatedwithtwo15‐minutewashesintheabove‐mentioned

buffer.Slideswerethenrinsedindistilledwater.Theextentofnon‐specificbinding

wasdeterminedinadjacentslidesbyadding100µMpicrotoxinduringthe[35S]TBPS

incubationperiod.Allincubationprocedurestookplaceatroomtemperature.

Slideswereair‐driedovernightinafume‐hood.Thefollowingmorningthe

slideswereopposedtox‐rayfilm(KodakBiomaxMRfilm,Rochester,N.Y.),along

withstandardradioactivescales(AmershamLifeScience,IL),for3‐5daysatroom

temperature.Thefilmwasthendevelopedinadarkroomusinga3‐traymethod.

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Thefilmwasfirstplacedcarefully–toavoidagitation–inatray(a10”x16”Pyrex

cookingtray)containingKodakGBXDeveloper(approximately3cmdeep)untilthe

imageofthebrainsectionsbecameclearlyvisibleonthefilm(between30‐80

seconds).Thefilmwasthenmovedtoasecondtraycontaininganequalvolumeof

tapwater.Duringthisrinsingstep,thefilmwasagitatedbyhandfor30‐60seconds.

Finally,thefilmwassubmersedinKodakGBXFixersolutionandagitatedbyhand

forapproximately5minutesbeforebeingremoved.Filmswerethenplacedunder

runningwaterfor5minutesbeforebeingattachedtofilmclipsandair‐driedfor15

minutes.

5.2.4 [35S]TBPSQuantification

Forquantification,filmswereplacedonalighttable(model8‐95;MCID;

ImagingResearch;Ontario,Canada)anddigitizedusingaCCDcameraintandem

withacomputer.ImageAnalysis®software(Version2.2;ImageAnalysisInc.)was

calibratedfor[35S]TBPSbindingusing9standardcurves,generatingvaluesin

ƒmol/mg.

Theregionsanalyzedwere:1)thehippocampus,2)frontoparietalcortex,3)

thethalamus,and4)theamygdala.[35S]TBPSbindingvalues(ƒmol/mg)fromthe

non‐specificbindingslicesweresubtractedfromallvaluesobtainedfor2nM

[35S]TBPSslices.

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5.2.5 DataAnalysis

DataforCDfedAldh5a1+/+weresetas100%bindingandallothergroups

wereadjustedtoreflect“percentofcontrolvalues”.Atwo‐wayanalysisofvariance

(ANOVA)wasusedtodeterminedifferencesbetweenthegenotype/dietgroupsand

brainregionsexamined.ThiswasfollowedbyTukey’sposthoct‐tests.

5.3 Results

Figure20showsmean(±s.d.)[35S]TBPSbindinginthehippocampus,cortex,

amygdalaandthalamusofCDfedAldh5a1+/+miceandAldh5a1‐/‐miceaswellwas

KDfedAldh5a1+/+miceandAldh5a1‐/‐mice.Asshown inFigure20,weconfirmed

thefindingofWuetal.(2006)thatCDfedAldh5a1‐/‐micehavesignificantlyreduced

[35S]TBPSbindingascomparedtowildtypecontrolmice.Thisreductionwasseenin

allofthebrainareasstudied,withthelargestdecrease(about36%)beingseenin

thethalamus.[35S]TBPSbindingwascompletelyrestoredintheHPCandcortexof

KDfedmutantsandonlypartiallyrestoredintheAMYandTHALregionsofKDfed

mutants.

The2‐wayANOVA revealed significantdifferencesbetween the groups (F=

11.37,p<0.0099).Tukey’sposthoctestsshowedthatAldh5a1‐/‐micefedaCDhave

significantly lowered [35S]TBPS binding as compared to wildtype mice fed a CD

(p<0.001). The significant difference in [35S]TBPS binding between these groups

wasseeninallbrainregionsexamined.Aldh5a1‐/‐micefedaKDshownormalized

[35S]TBPS binding in the hippocampus and the cortex (p>0.05, as compared to

wildtype mice fed a CD). [35S]TBPS binding in the amygdala and thalamus of

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Aldh5a1‐/‐ mice fed a KD is partially elevated towards normal levels, however, it

remainedsignificantlydifferentfrom[35S]TBPSbindinglevelsintheamygdalaand

thalamusofwildtypemicefedaCD.[35S]TBPSbindinginKDfedwildtypemicedid

notdifferfrom[35S]TBPSbindinginCDfedwildtypemice.

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Figure20.AverageGroup[35S]TBPSBindinginCDandKDfedAldh5a1+/+MiceandCDandKDfedAldh5a1‐/‐Mice.

Figure20presents themean (±s.d.) [35S]TBPSbinding in thehippocampus (HPC),cortex,amygdala(AMY)andthalamus(THAL)ofCDfedAldh5a1+/+mice(N=10)andAldh5a1‐/‐mice(N=6),aswellasKDfedAldh5a1+/+mice(N=8)andAldh5a1‐/‐mice(N=6).InkeepingwithWuetal.(2006),TBPSbindingwassignificantlyreducedinCDfedAldh5a1‐/‐miceas compared to CD fed Aldh5a1+/+ mice in all regions.[35S]TBPS binding was completely restored in the HPC and cortex of KD fedAldh5a1‐/‐mice.[35S]TBPSbindingwasonlypartiallyrestoredintheAMYandTHALofKDfedmutantmice.*p<0.05,***p<0.001.KD=ketogenicdiet,CD=controldiet.

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5.4 Discussion

ThepurposeofthepresentexperimentwastodeterminetheeffectofaKD

on[35S]TBPSbindinginAldh5a1‐/‐andwildtypemice.

ThefindingsofWuetal.(2006)—showingthatCDfedAldh5a1‐/‐miceexhibit

significantlyreduced[35S]TBPSbindingascomparedtoCDfedwildtypecontrols—

were replicated. Significantly reduced [35S]TBPS binding was seen in all brain

regionsstudied inCDfedAldh5a1‐/‐mice,withthe largestdecrease(36%)seenin

thethalamus.[35S]TBPSbinding,however,wascompletelyrestoredintheHPCand

cortexofKDfedmutants,andpartiallyrestoredintheamygdalaandthalamusofKD

fedmutants.Experiment3isthefirsttoshowthataKDcanaffect[35S]TBPSbinding.

[35S]TBPS is a specific ligand for the GABAAR‐associated chloride channel

(Banerjee et al., 1998). Hence, these data suggest a reduction—and KD induced

normalization—of GABAAR‐associated chloride channel activity in mutant mice.

Interestingly, the KD had no effect on [35S]TBPS binding in wildtypemice. These

datasuggest, therefore, that theKDmayonlyelevate[35S]TBPSbindingwhen it is

significantlydecreased.

Howdothesechangesin[35S]TBPSbindingrelatetothemIPSCactivitydata

fromExperiment2?Althoughthepresentdatadonotdirectlyaddressthisissue,it

hasbeenreportedpreviouslythatdecreasesinmIPSCfrequencyareassociatedwith

decreasesinGABAARnumber(Kilmanetal.,2002).Intermsofpostsynapticcurrent

measurement,itisthechloridechannel’sactivity,andnottheactivityoftheGABAAR

perse,thatisresponsibleforpostsynapticcurrentchangesduetochlorideflux.Our

dataraisethequestionofwhetherpostsynapticchloridechannelexpressionplaysa

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roleinthesignificantlyreducedmIPSCfrequencyasseeninAldh5a1‐/‐mutantsfeda

CDascomparedtowildtypemicefedaCD.Thishypothesis isdiscussedfurtherin

theGeneralDiscussion.

A previous study has shown that the KD significantly increases protein

phosphorylationinthebrain(Ziegleretal.,2002).Althoughtheproteinsinvolvedin

chloride channel trafficking are not fully understood, protein kinase A has been

hypothesizedtoplayarole(Chappeetal.,2005).Wehypothesize,therefore,thatthe

KDmaybeincreasingphosphorylationofproteins(e.g.,proteinkinaseA)involved

inthetraffickingofchloridechannels,causingasubsequentincreaseincellsurface

chloride channel expression reflected by restored [35S]TBPS binding in KD fed

Aldh5a1‐/‐mice.

This is the first study to show the effects of a KD on [35S]TBPS binding.

Although [35S]TBPS binding may play a role in the KD’s mechanism of action in

Aldh5a1‐/‐ mice, it is increasingly apparent that the KD works by multiple

mechanisms.TheseincludethemechanismsoutlinedintheGeneralIntroduction,as

wellasthoserevealedforthefirsttimeinthisthesis.

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CHAPTER6

EXPERIMENT4:THEEFFECTSOFAKETOGENICDIETONSERUM

ANALYTESINALDH5A1­/­MICE

6.1 Introduction&Rationale

The mechanisms of the KD’s actions are not fully understood. There are,

however, a number of different diet‐induced serum changes thatmay play a role

(Kossoff,2004).ThetwochangesmostoftenassociatedwiththeKDaredecreased

blood glucose levels and increased beta‐hydroxybutyrate (βOHB) levels. More

recently,adiet‐inducedelevationoffattyacidshasalsobeenproposedtoplayarole

in theKD’smechanism (Cunnaneet al., 2002). In thepresent studywemeasured

serum levels of glucose, βOHB and free fatty acids in CD fed Aldh5a1+/+ and

Aldh5a1‐/‐miceaswellasKDfedAldh5a1+/+andAldh5a1‐/‐mice.Thiswasdoneto

determine whether any of these serum analytes (i.e., analyzed metabolites) are

alteredinAldh5a1‐/‐miceascomparedtowildtypemice,andtodeterminetheKD’s

effectontheseserumanalytesinAldh5a1‐/‐mice.

Consumption of a KD causes slight—albeit statistically significant—

reductions in blood glucose levels (Vining, 1999; Nylen et al., 2005). The diet’s

ability to lower glucose levels has been hypothesized to confer anticonvulsant

effects by altering brain energymetabolism—specifically, by bypassing glycolysis

(PfeiferandThiele,2005;Garriga‐Canutetal.,2006;Freemanetal.,2007;Lianetal.,

2007). Therefore, we measured glucose levels in serum to determine whether a

dropinglucosemayplayaroleinthebeneficialeffectsoftheKDinAldh5a1‐/‐mice.

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Correspondingtodecreasesinbloodglucoseareelevationsinbloodketone

levels(Huttenlocher,1976;Boughetal.,1999;Nylenetal.,2005).Aketonethathas

been implicated in theKD’smechanismof action is βOHB. There are at least two

hypothesesabouthowβOHBmayrelatetotheKD’santiconvulsanteffects.Greene

etal. (2003)haveproposed thatβOHB’sactionsare indirect.βOHB inter‐converts

with acetoacetate (in an NAD+/NADH dependent manner), which is readily

metabolizedtoacetylCoA—asubstrateintheKrebscycle.Greeneetal.(2003)have

argued that bypassing glycolysis and forcing the body to utilize fats as an energy

substrateresults inarelatively“slower”poolofenergy,rendering thesystemless

prone toseizures.Maetal. (2007), in contrast,havedemonstratedadirectacting

agonistic effect of βOHB on KATP channels, which serves to hyperpolarize cells

making them lessprone to therapid firingrequired for seizureactivity.Clinically,

blood βOHB levels are monitored closely and are thought to relate to the diet’s

anticonvulsantactivity(Huttenlocher,1976;Gilbertetal.,2000).

Previous reportshaveshown thatCD fedAldh5a1‐/‐micehavesignificantly

elevatedβOHBlevels(Chowdhuryetal.,2007).Chowdhuryandcolleagues(2007)

showedthatthisincreaseinketosisistheresultoftheinabilityofAldh5a1‐/‐miceto

efficientlymetabolizeglucoseforenergy.Thisresultsinanenergydeficiencyanda

compensatoryincreaseinfatoxidationtoprovideanalternativeenergysourcefor

thebrain.Forthesereasons,wemeasuredβOHBlevelsinthepresentexperiment.

Elevations in free fatty acid levelsmay also contribute to the KD’s actions

(Cunnaneetal.,2002).Clinically,Fraseretal.(2003)demonstratedthatbloodlevels

of arachidonic acid correlate significantlywith seizure suppression in childrenon

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theKD.Somefattyacidsaredirectlyanticonvulsant.Valproicacid,forexample,isa

fatty acid in structure and is one of the most widely used anticonvulsant

medications(Ben‐Menachemetal.,2006).Giventhepotentialroleof fattyacids in

themechanismof theKD,wemeasured free fattyacid levels inCD fedAldh5a1+/+

andAldh5a1‐/‐miceaswellasKDfedAldh5a1+/+andAldh5a1‐/‐mice.

ThepurposeofExperiment4,therefore,wastodeterminewhetherchanges

in glucose, βOHB or free fatty acids might contribute to the effects of the KD in

Aldh5a1‐/‐mice.Wehypothesized that, inkeepingwithprevious study results,KD

fed Aldh5a1‐/‐ mice would have decreased blood glucose levels, increased βOHB

levelsandincreasedfreefattyacidslevelsascomparedtoCDfedcontrols.

6.2 Methods

6.2.1 Subjects,SacrificeandCollectionofSerum

Fourgroupsofmice(P20‐25)wereusedforthesestudies:CDAldh5a1+/+

mice(n=4),KDfedAldh5a1+/+mice(n=5),CDfedAldh5a1‐/‐mice(n=5)andKDfed

Aldh5a1‐/‐mice(n=4).Micewereobtained,fedandhousedasdescribedinthe

GeneralMethodssection.

Onthedayofsacrifice,micewereanesthetizedusinghalothaneandrapidly

decapitated.Trunkbloodwascollectedin1.5mLvialsandallowedtocoagulate.

Vialswerethenspunat9200gfor10minutes.Serumwaspipettedinto150µlvials

andstoredinat‐80˚Cuntilthetimeofanalysis.

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6.2.2 DeterminationofGlucoseinSerum

GlucoselevelsweredeterminedinanassayestablishedbyDr.Sergei

Likhodii.Theassayusestheconversionofβ‐D‐glucosegluconatetoH2O2viaglucose

oxidase.Theresultinghydrogenperoxidethenoxidizesthechemical4‐

aminoantipyrine,1,7dihydroxynaphthalenewhichresultsinacoloreddye.The

densityoftheresultingdyecomplexisrelatedtotheconcentrationofglucoseinthe

specimenandismeasuredbyreflectancespectrophotometryat540nm(User

DefinedAssayReferenceGuideforVitros5,1FSChemistrySystem.Ortho‐Clinical

Diagnostics).

6.2.3 Determinationofβ­HydroxybutyrateinSerum

Theassayusedtodetectβ‐hydroxybutyrate(βOHB)inserumwasdeveloped

byDr.SergeiLikhodii,andisbasedonthefollowingreaction:

ThisreactioniscatalyzedbytheenzymeβOHB‐dehydrogenaseandit

involvestheoxidationofβOHBtoacetoacetate.Thiscanbedetectedusinga

spectrophotometerbymeasuringthechangesinabsorbanceat340nmcausedby

thereductionofNAD+toNADH.Thisconversioniscorrelateddirectlywiththe

originalconcentrationofβOHB.ThisassayisperformedusingaVitrosChemistry

System5.1FusionSeries(Ortho‐ClinicalDiagnostics,N.J.).Beforeanysampleswere

analyzed,theVitrossystemwascalibratedusinga3‐pointcalibrationwithtwo

β­Hydroxybutyrate+NAD+ Acetoacetate+H++NADH

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replicatespercalibrationlevel.Calibrationsolutionswerepreparedaccordingtothe

tablebelow.Calibrationwasperformedeverytimeanewbottleofreagentwas

used.

Table5.CalibrationTableforβOHBAssay

CalibrationLevel Volumeofkitcalibrator(µl)

Volumeofwater(µl)

Concentration(mM)

1 0 100 0.002 100 100 0.503 100 0 1.00

Aftercalibrationwascomplete,theassaywasperformed.TheVitrossystem

wasloadedwithaRandoxRanbut™reagentkit(CatalogNo.RB1007)andareagent

boatfortheβOHBassay.Samplecupswerefilledwith70μlofserumandloadedinto

theVitros.TheVitrossystemperformedspectrophotometricreadingsaftertwo

incubationsof52.25and66.50secondsinduration(UserDefinedAssayReference

GuideforVitros5.1FSChemistrySystem.Ortho‐ClinicalDiagnostics).Thesystem

generatedresultsforβOHBinmmol/L(mM).

6.2.4 DeterminationofNon­EsterifiedFattyAcids(NEFA)inSerum

Thespecificmethodfordeterminingnon‐esterifiedfattyacids(NEFA,orfree

fattyacids)inserumwasdevelopedbyDr.SergeiLikhodii.Thisassayinvolvesthe

acylationofcoenzymeAbyfattyacidsinthepresenceofacyl‐CoAsynthetase.The

acyl‐CoAproducedinthereactionisfurtheroxidizedbyacyl‐CoAoxidase,which

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generatedH2O2(hydrogenperoxide)asaby‐product.Hydrogenperoxide,inthe

presenceofperoxidasepermitstheoxidativecondensationofN‐ethyl‐N‐(2hydroxy‐

3‐sulphopropyl)‐m‐toluidinewith4‐aminoantipyrine(4‐AAP)toformapurple

colouredadduct.Thiscolourcanbemeasuredspectrophotometricallyat550nm.

Belowarethechemicalreactionsinvolvedinthisassay:

TheVitroswascalibratedasfollowspriortoanalyzingsamples.Thesystem

wasloadedwithaready‐to‐usereagentkitaswellas1.0mMoffreefattyacids.A

two‐pointcalibration—with3replicatespercalibrationlevel—wasperformed.

DeionizedwaterwasusedasacalibratorfortheLevel1,asshowninTable6.

CalibrationwasperformedeachtimeanewbottleofreagentR1wasadded.

Table6.CalibrationTableforNEFAAssay

CalibrationLevel Volumeofkitcalibrator(µl)

Volumeofwater(µl)

Concentration(mM)

1 0 100 0.002 100 0 1.00

NEFA+ATP+CoA AcylCoA+AMP+PPi

AcylCoA+O2+CoA 2,3,­trans­Enoyl­CoA+H2O2

2H2O2+TOOS+4­AAP purpleadduct+4H2O

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Sera(70μl)werepipettedintosamplecupsandloadedintheVitros.The

Vitrosemployeda2‐point‐rateassaymodelwithtwospectrophotometricreadings

at550nmandtwoincubationsof608.0and598.5secondsinduration.

6.3 Results

6.3.1 SerumGlucoseLevels

Figure21showsmean(±s.e.m.)serumglucoselevelsinmillimolar(mM)as

measuredinCDfedAldh5a1+/+andAldh5a1‐/‐miceaswellasKDfedAldh5a1+/+and

Aldh5a1‐/‐ mice. As indicated in the figure, the CD fed mutants had lower serum

glucose levels than the other three groups, which had glucose levels that were

similar toeachother.Aone‐wayANOVArevealedasignificantdifferenceamongst

the groups (p=0.006). Tukey’s post‐hoc analyses revealed that CD fed Aldh5a1‐/‐

mice had significantly lower serum glucose levels than both KD fed groups (i.e.,

Aldh5a1‐/‐ andAldh5a1+/+; p<0.05). None of the groups differed significantly from

theCDfedwildtypecontrolmice(p>0.05).

6.3.2 SerumβOHBLevels

Figure 22 showsmean (±s.e.m.) serumβOHB levels inmillimolar (mM) as

measuredinCDfedAldh5a1+/+andAldh5a1‐/‐miceaswellasKDfedAldh5a1+/+and

Aldh5a1‐/‐ mice. As shown in the figure, βOHB levels were lowest in the CD fed

wildtypemice,intermediateintheCDfedmutantsandtheKDfedwildtypemiceand

the highest in the KD fed mutants. A one‐way ANOVA revealed a significant

differenceamongstthegroups(p=0.0004).Tukey’spost‐hocanalysesrevealedthat

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CD fed Aldh5a1‐/‐ mice had significantly elevated serum βOHB levels when

compared to CD fed wildtype mice (p<0.05). KD fed wildtype mice also had

significantly elevated βOHB levels when compared to CD fed Aldh5a1+/+ mice

(p<0.05).Aldh5a1‐/‐mice fedaKDhad thehighestβOHB levelsofall, significantly

higherthanCDfedwildtypemice(p<0.01).

6.3.3 SerumNEFALevels

Figure23showsmean(±s.e.m.)serumNEFAlevels(mM)asmeasuredinCD

fedAldh5a1+/+andAldh5a1‐/‐miceaswellasKDfedAldh5a1+/+andAldh5a1‐/‐mice.

As indicated in the figure, NEFA levelswere lowest in the CD fedAldh5a1+/+ and

Aldh5a1‐/‐miceandslightlyhigher in theKDfedAldh5a1+/+andAldh5a1‐/‐mice.A

one‐wayANOVArevealedthatnosignificantdifferencesexistedamongstthegroups

(p=0.229).

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Figure21.MeanSerumGlucoseLevels

This figure shows mean (±s.e.m.) serum glucose levels in millimolar (mM) asmeasuredinCDfedAldh5a1+/+andAldh5a1‐/‐miceaswellasKDfedAldh5a1+/+andAldh5a1‐/‐ mice. Serum glucose levels in CD fedmutants were significantly lowerthan those in the KD fed groups (p<0.05). There was no significant differencebetweenCDfedwildtypemiceandKDfedgroupsintermsofbloodglucoselevels.mM=millimolar,CD=controldiet,KD=ketogenicdiet.**p<0.01.

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Figure22.MeanSerumβOHBLevels

This figure shows mean (±s.e.m.) serum βOHB levels in millimolar (mM) asmeasuredinCDfedAldh5a1+/+andAldh5a1‐/‐miceaswellasKDfedAldh5a1+/+andAldh5a1‐/‐mice.SerumβOHBwassignificantlyelevatedinCDandKDfedAldh5a1‐/‐mice and KD fed wildtype mice as compared to CD fed wildtype mice. CD fedAldh5a1‐/‐miceare thought tobeketoticdue toaglucosemetabolizingdeficiencyandthesubsequentcompensatoryincreaseinfattyacidoxidation.KDfedmutantsshowtheadditiveeffectofincreasedfattyacidoxidationplusaKD.mM=millimolar,CD=controldiet,KD=ketogenicdiet.*p<0.05,***p<0.001.

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Figure23.MeanSerumFreeFattyAcidLevels

This figure shows mean (±s.e.m.) serum NEFA levels in millimolar (mM) asmeasuredinCDfedAldh5a1+/+andAldh5a1‐/‐miceaswellasKDfedAldh5a1+/+andAldh5a1‐/‐ mice. Serum NEFAs did not differ significantly amongst the groups.Interestingly,NEFAlevelswereonlyslightlyincreasedinanimalsconsumingahighfat,KD.

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6.4 Discussion

Thepresentstudywasdesignedtoexamineserumlevelsofglucose,βOHB

andNEFAsinCDfedAldh5a1+/+andAldh5a1‐/‐miceaswellasKDfedAldh5a1+/+

andAldh5a1‐/‐mice.Itwashypothesizedthatglucoselevelswouldbesignificantly

decreasedinKDfedgroups,βOHBlevelswouldbesignificantlyincreasedinKDfed

groupsandNEFAswouldbesignificantlyincreasedinKDfedgroups.

NoneofthegroupsdifferedsignificantlyfromCDfedwildtypemiceinterms

ofserumglucoselevels.ThiswascontrarytoourhypothesisthatKDfedmicewould

havesignificantlyreducedbloodglucoselevels.Previousreportshaveconsistently

shownthatbloodglucoselevelsaresignificantlylowerinKDfedrodentsas

comparedtoCDfedcontrols(AppletonandDeVivo,1974;Todorova,2000;Likhodii

etal.,2000;Nylenetal.,2005).OneexplanationforwhytheKDfedgroupsdidnot

havesignificantlylowerbloodglucoselevelshastodowiththemethodof

phlebotomy.Thestudiesmentionedaboveusedvenousbloodtoassayserum

glucoselevels.Duetothesmallsizeofthemutantmice,however,wewererequired

tousetrunkblood—whichincludesvenousandarterialblood—toobtainalarge

enoughsampletoassay.Trunkbloodisthoughttoyieldhigherlevelsofblood

glucosethanvenousblood(Dr.SergeiLikhodii,personalcommunication).

SerumglucoselevelsweresignificantlylowerinCDfedAldh5a1‐/‐groupas

comparedtobothKDfedgroups.CDfedAldh5a1‐/‐micehavebeenshowntohave

animpairedabilitytometabolizeglucose(Chowdhuryetal.,2007).Theeffectthat

thedeficiencyinglucosemetabolismwillhaveonbloodglucoselevelslargely

dependsonwhere(inthemetabolicpathway)thedeficiencyexists(unknown).If

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thedeficiencyexistsintheglycolysispathway,thenglucosewouldbeshunted

towardsglycogenstorageinsteadofbeingmetabolizedforenergyproduction.As

such,thedeficiencyinglucosemetabolismmayplayaroleintheslightloweringof

bloodglucoselevelsinCDfedAldh5a1‐/‐group.Itisimportanttonote,however,that

noneofthegroupsdifferedsignificantlyfromtheCDfedwildtypegroupintermsof

bloodglucoselevels.

βOHBlevelsweresignificantlyelevatedinCDfedAldh5a1‐/‐miceas

comparedtoCDfedwildtypemice.Thiswasexpectedanditsupportsaprevious

findingshowingthatimpairedglucoseoxidationinAldh5a1‐/‐leadstoasignificant

elevationofβOHBlevels(Chowdhuryetal.,2007).Presumably,ifAldh5a1‐/‐mice

areunabletooxidizedietarycarbohydrateeffectivelythentheybegintooxidize

theirfatstoresforenergy,resultinginketosis.TheKDwasabletoelevateβOHB

levelsinKDfedwildtypemiceduetotheketogenicnatureofthediet.βOHBlevelsin

KDfedmutantswereveryhigh.Thiswaspossiblycausedbyacompoundedeffectof

elevatedketosis—duetoimpairedglucoseoxidationandthesubsequent

upregulationoffatoxidizingenzymes—coupledwiththeketogenicnatureofthe

diet.

ThesechangesinβOHBlevelshaveimportantimplicationsforthe

mechanismoftheKDinAldh5a1‐/‐mice.Itispossiblethatgivingthemutantmicea

highfatdietsimplygivesthemanimportant,newenergysourcethattheycan’tget

fromcarbohydraterichmousechow.Thispossibilityisdiscussedfurtherinthe

GeneralDiscussionsection.

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An unexpected findingwas an absence of increased bloodNEFA in KD fed

mice.ItwasexpectedthatKDfedmicewouldhavesignificantlyhigherbloodNEFA

levels. It was, however, found that KD fed mice only had slight, non statistically

significant elevations in blood NEFA levels. Similar findings, however, have been

reported by Klepper and colleagues (2004) using the KD to treat human glucose

transporterdeficiency.WhereasβOHBrosesignificantlyintheirpatients,therewas

nocorrespondingincreaseinserumNEFAs.Inourstudy,weassumenodifferences

between genotypes for lipoprotein lipase activity or its ability to activate in

response to diet. One explanation for the absence of increasedplasmaNEFAmay

resideinthecapacityofAldh5a1‐/‐micetomoreeffectivelyconvertNEFAtoβOHB.

TheratiosofmeanNEFAtoβOHBwere:Aldh5a1+/+(CD),1.20;Aldh5a1‐/‐(CD),0.73;

Aldh5a1+/+(KD),1.04;Aldh5a1‐/‐(KD),0.46.WhiletheratioforAldh5a1+/+micewas

comparable despite diet, the same value inAldh5a1‐/‐micewas lower on CD and

decreased and additional 40% with KD intervention, suggesting that the KD fed

Aldh5a1‐/‐micemayhaveanenhancedabilitytooxidizefattyacidstoketonebodies.

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CHAPTER7

EXPERIMENT5:THEEFFECTSOFAKETOGENICDIETON

MITOCHONDRIALNUMBERANDFUNCTIONINALDH5A1­/­MICE

7.1 Introduction&Rationale

Mitochondriaprovidethemajorityofenergyforcellularfunction.Theenergy

comes fromadenosine triphosphate (ATP),which is producedbyKrebs cycle and

the electron transport chain in mitochondria, through the oxidation of fats,

carbohydratesandproteins(RicquierandBouillaud,2000).

Aldh5a1‐/‐micehavebeenshown tohavea significantly impairedability to

oxidizeglucoseascomparedtowildtypemice(Chowdhuryetal.,2007).Whenthese

mutants are fed a CD—which is high carbohydrate and low in fat—they show

significantly reduced levels of the important substrates used by Krebs cycle. This

mayleadtopoormitochondrialfunction.

Sauer and colleagues (2007) found a significant, hippocampal‐specific

impairment ofmitochondrial function inAldh5a1‐/‐mice as compared towildtype

mice.Otherdeficitshavebeenfoundthat implicatemitochondrial function.Gibson

and colleagues (2005) reported a significant decrease in glutathione levels and

increased apoptotic cell death in the hippocampus of Aldh5a1‐/‐ mice, which is

consistentwithdiminishedmitochondrialfunction(Gibsonetal.,2005).Hogemaet

al. (2001) likewise showed significant levels of gliosis in the hippocampi of

Aldh5a1‐/‐mice.Bothapoptosisandgliosiscanbecausedbyreactiveoxygenspecies,

whichareproducedbyunhealthymitochondria(Moroetal.,2005).

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The KD has also been shown to cause mitochondrial biogenesis (i.e., it

increasesthenumberandfunctionofmitochondria).Boughandcolleagues(2006)

haveshownthattheKDcauseda50%increaseinthetotalnumberofmitochondria.

Theauthorsalsofoundacorrespondingsignificantup‐regulationofmitochondrial‐

associatedmRNA(Boughetal.,2006).Masinoetal.(2007)havealsofoundthatthe

KD causes a significant increase in brain ATP levels. They concluded that these

changesinbrainenergymetabolismmayunderlietheKD’smechanismofaction.

Perhaps because of its effects onmitochondria, theKD has been shown to

increase theantioxidantcapabilitiesofblood inhumans(Nazarewiczetal.,2007).

TheKDhas alsobeen shown to significantly increase the levelsof glutathione, an

endogenousanti‐oxidant,inthehippocampiofKDfedrats(Schutzmanetal.,2007).

Further,acetoacetateandbeta‐hydroxybutyrate—ketonessignificantlyelevatedby

theKD—havebeenshowntohavesignificantantioxidanteffectsinanimalmodelsof

epilepsy(MaaloufandRho,inpress).

Given the disruptions in hippocampal mitochondrial function reported in

Aldh5a1‐/‐miceandthereportedbeneficialeffectoftheKDonmitochondria,itwas

hypothesizedthattheKDwouldamelioratethediminishedmitochondrial function

inAldh5a1‐/‐mice.Thepresentexperiment,therefore,usedelectronmicroscopyto

quantifythenumberofmitochondriainhippocampalCA1pyramidalneuronsofKD

fed Aldh5a1‐/‐ mice, as well as CD fed Aldh5a1‐/‐ mice and Aldh5a1+/+ mice.

Quantificationinvolvedtakinghighmagnificationimagesofthecellbodiesofthese

neuronsandusingcomputersoftware todeterminethedensityand%‐areaof the

mitochondria.

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Mitochondrialfunctionwasmeasuredinhippocampaltissuefromtheabove‐

mentionedgroupssinceanetincreaseinthenumberofmitochondriamaynot

equatetoanetincreaseintheproductionofATP.

WethereforehypothesizedthatAldh5a1‐/‐micewouldhavesignificantly

reducednumberandfunctionofmitochondriainthehippocampus.Also,inkeeping

withpreviousreports(Boughetal.,2006)wehypothesizedthatKDfedmutants

wouldshowarestorednumberofmitochondria,andasignificantelevationofATP

levels.

7.2 Methods

7.2.1 Subjects

Aldh5a1‐/‐andAldh5a1+/+miceservedassubjectsforthepresent

experiments.TheywereobtainedandhousedasdescribedintheGeneralMethods.

Fortheelectronmicroscopyexperiment,subjectsweredividedintothreegroups:

CDfedAldh5a1+/+(N=3),CDfedAldh5a1‐/‐(N=3)andKDfedAldh5a1‐/‐(N=3).For

theATPexperiment,subjectsweredividedintofourgroups:CDfedAldh5a1+/+

(N=13),CDfedAldh5a1‐/‐(N=11),KDfedAldh5a1+/+(N=15)andKDfedAldh5a1‐/‐

(N=12).

7.2.2 TissuePreparationforElectronMicroscopy

Between22‐25daysofage,micewereanesthetizedwith0.1mg/kgsodium

pentobarbital(diluted10xwithwater,injectedi.p.).Uponreachingasurgicalplane

ofanesthesia,micewereperfusedtranscardiallywith0.1Mphosphatebufferfor5

111

minutesatarateof5ml/minusingavaristaticinfusionpump(Model72‐315‐000

Manostat™,BarnantCompany,Barrington,IL,USA).Thiswasfollowedbya10

minuteperfusionwith2%glutaraldehydein0.1Mphosphatebuffer(pH7.4)atthe

samerate.Uponcompletionoftheperfusion,brainswereextractedandpost‐fixed

bysubmersionintheglutaraldehydefixativesolutionforatleasttwodays.

Transversecoronalsectionsweresubsequentlycutat50μmusinga

Vibrotome(Series1000;TechnicalProductsInternational;Ellisville,MD).

Hippocampiwerethendissectedoutofthesesectionsandwashed3timeswith

0.1Msodiumcacodylatebuffer,pH7.3.Threesectionsweretakenfromeachbrain.

Thetissuewasthentreatedwith1%osmiumtetroxideand1.25%potassium

ferrocyanideincacodylatebufferfor1.5hoursatroomtemperature.Itwasthen

washed3timeswithcacodylatebuffer.Thehippocampalsectionswerethen

dehydratedthroughagradedseriesofethanol(EtOH)asfollows:70%EtOH(2x10

minutes),90%EtOH(2x10minutes)and100%EtOH(3x10minutes).Samples

weretheninfiltratedwithEPON™resin(HexionInc,HoustonTX,USA)asfollows:

100%propyleneoxide(3x10minutes),1:1EPON™topropyleneoxidefor2hours,

3:1EPON™topropyleneoxidefor2hours,100%EPON™overnightandfinally,

100%EPON™for4hours.SampleswerethenplacedinfreshEPON™whichwas

polymerizedovernightina70°Coven.

Sectionswerefurthercutat80nmusinganUltraCutLeicaEMFCSsystem

(LeicaMircosystems;Wetzlar,Germany)andcollectedoncoppergridsandstained

withuranylacetateandleadcitrate.

112

7.2.3 ElectronMicroscopy

10CA1pyramidalcellsomaswereimagedfromeachsubject,withthree

subjectspergroup.SectionswereexaminedusingaFEITecnaiG2F20transmission

electronMicroscope(FEICompany,Hillsboro,Oregon).TheCA1pyramidalcell

regionofthehippocampuswaslocated.Picturesofsomaticmitochondriawere

obtainedatamagnificationof6900xto8500x.

7.2.4 AnalysisofMitochondrialCounts

Thenumberofmitochondriapersomawasblindlycountedbytwo

independentresearchers.Themeanofthetworesearchers’countswasusedfor

subsequentanalyses.ImageJ(v.1.38X,NationalInstitutesofHealth,USA)image

analysissoftwarewasusedtodeterminethedensityofmitochondriainthesoma,as

wellasthetotalareaofthesoma—excludingthenucleus,whichdoesnotcontain

mitochondria—thatwasoccupiedbymitochondria.Thiscalculationgavethe%‐

areathatthemitochondriaoccupiedinthecellbody.

7.2.5 TissuePreparationforATPAssay

BetweenP22‐25,separategroupsofmicewereinjectedwith0.1mg/kg

sodiumpentobarbital(diluted10xwithwater).Uponasurgicalplaneofanesthesia,

subjectsweredecapitatedandtheirbrainswereextracted.Between5‐20µgof

tissuewasextractedfromtheleftandrighthippocampi.Uponremoval,thetissue

wasimmediatelyflashfrozenbysubmersioninliquidnitrogen.Sampleswere

storedat‐80°untiltheassaywasperformed.

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7.2.6 ATPCalibrationCurves

Immediatelybeforerunningtheassay,sampleswereremovedfromthe

freezerandthawedoncrushedice.ATPlevelsweredeterminedusinga

commerciallyavailableATP‐GloTMBioluminometricCellViabilityAssaykit

(#30020‐1,BiotiumInc.,Hayward,CA,USA).

ATPcalibrationcurvesweregeneratedaccordingtokit.Theassayusesfirefly

luciferaseinthepresenceofATP,whichoxidizesD‐luciferinresultinginthe

emissionoflight.Lightemissionlevelsweremeasuredusingaluminometer(Turner

Designs,Inc.,Sunnyvale,CA).

Aseriesoften‐foldtitrationsfrom100ρmoles(picomoles)to0.01ρmolesof

ATPwerepreparedin100µLofdistilledwater(DH2O)foreachsampleina1.5mL

microfugetube.100µLofATP‐GloTMdetectioncocktailwasthenaddedtoeach

microfugetubecontainingtheindicatedamountofATP.Eachmicrofugetubewas

flickedthreetimesusingtheexperimenter’sfinger.Thiswasdonetoensure

thoroughmixingbeforethetubewasplacedintheluminometer.Lightemissionwas

integratedover10secondswithnopre‐readdelay.Asensitivitysettingof31%was

used.Figure24showsthestandardcurvefortheATP‐GloTMassay.

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Figure24.CalibrationCurveforATP‐GloAssay

ThisfigureshowsthecalibrationcurvefortheATP‐GloTMassay.Alinearregressionwasperformed,yieldingaregressionlinewithaslopeof65.64±0.9528withaY‐interceptwhenX=0.0of‐3.227±42.82andanX‐interceptwhenY=0.0of0.04916.1/slopewas0.01523.95%ConfidenceIntervalswereassessed(dottedlines)withaslopebetween63.58to67.70,aY‐interceptwhenX=0.0at‐95.73to89.27andanX‐interceptwhenY=0.0at‐2.206to2.261.TheGoodnessofFitgeneratedanr²valueof0.9973.Fivestandardswereusedtoassessthisregression(0.01,0.1,1,10,100picomoles)witheachstandardbeingperformedintriplicate(i.e.,3Yreplicates).

115

7.2.7 QuantificationofMitochondrialATPProduction

Afterthecalibrationcurveswererun,ATPlevelsfromthetissuesamples

werequantified.Fireflyluciferasewasaddedtotheluciferin–containingATP‐GloTM

assaysolutioninaratioof1uLto100uL(25µLluciferasefor2.5mLoftheATP‐

GloTMassaysolution).TheATP‐GloTMDetectionCocktailwaspreparedimmediately

beforeeachuseaccordingtothemanufacturer’sdirections.

Theluminometerwasalwaysadjustedtothesettingsobtainedwhenrunning

thestandardsamples.Assuch,theluminometerwassetwithadelaytimeof0

secondsandanintegrationtimeof10seconds.Thesensitivitysettingwas31%.

Sampleswererun,one‐at‐a‐time,inthesameorderthattheywereprepared.

OnehundredμLofATP‐GloTMDetectionCocktailwasaddedtoeachsample.Each

tubewasmixedbymanuallyagitatingthetube.Thetubewasthenplacedinthe

luminometerandmeasurementwasinitiated.Therelativeluminescenceactivity

wasrecordedandthenextsamplewasthenprepared.Relativeluminescencewas

translatedintoATPconcentrationusingthecalibrationcurvesconstructedearlier.

7.3 Results

7.3.1 MitochondrialDensity

Figure25showsarepresentativeelectronmicrograph(6900xmagnification)

ofaCA1pyramidalcell.Usingelectronmicroscopywecountedthenumberof

mitochondriapresentinthesomataofCA1pyramidalcellsfromthebrainsofCDfed

Aldh5a1+/+mice,CDfedAldh5a1‐/‐miceandKDfedAldh5a1‐/‐mice.

116

Figure26showsthemean(±s.d.)numberofmitochondriaper10µm2in

electronmicrographstakenfromCDfedAldh5a1+/+mice,CDfedAldh5a1‐/‐miceand

KDfedAldh5a1‐/‐mice.Thisgivesameasureofmitochondrialdensity.Asindicated,

themeannumberofmitochondriawaslowestinCDfedwildtypemice,intermediate

intheCDfedmutantsandthehighestintheKDfedmutants.Aone‐wayANOVAwas

usedtocomparegroupmeans.Asignificantdifferencewasdetectedamongthe

groups(F=4.569,p=0.019).Tukey’spost‐hocanalysesrevealedthatKDfed

Aldh5a1‐/‐mice(mean±s.d;2.58±0.52)hadasignificantlyhigherdensityof

mitochondriathanAldh5a1+/+mice(2.06±0.13;p<0.05).TheCDfedAldh5a1‐/‐mice

didnotdiffersignificantlyfromeitheroftheothergroups(p>0.05).

7.3.2 MitochondrialArea

Figure27showsthemean(±s.d.)somaticareaoccupiedbymitochondria

(calculatedasapercentageoftotalsomaticarea)inCDfedAldh5a1+/+mice,CDfed

Aldh5a1‐/‐miceandKDfedAldh5a1‐/‐mice.ThemeansomaticareawaslowestinCD

fedwildtypemice,similarlylowintheCDfedmutantsandsignificantlyelevatedin

theKDfedmutants.Aone‐wayANOVAdetectedasignificantdifferenceamongthe

groups(F=6.626,p=0.0046).Tukey’spost‐hocanalysesshowedthatmitochondria

occupyasignificantlylargerareaofthesomainKDfedmutantmice(mean±s.d.;

8.00±2.25)thanCDfedwildtypemice(6.03±0.55;p<0.05).Thedifferencebetween

KDfedmutantmiceandCDfedmutantmiceapproachedsignificance(p=0.06),but

wasnotstatisticallysignificant.TherewasnostatisticaldifferencebetweenCDfed

mutantmiceandCDfedwildtypemice.

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Figure25.ElectronMicrographofPyramidalNeuronandItsMitochondria

ThisfigureshowsaselectronmicrographofaCA1hippocampalpyramidalneuronfrom a CD fed Aldh5a1‐/‐ mouse. c=cytosol m= mitochondria n=nucleusno=nucleolus.Scalebar=2μm.Phototakenat6900Xmagnification.

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Figure26.AverageMitochondriaNumberPer10μm2ofSomaticArea

Figure26showsthemean(±s.d.)numberofhippocampalmitochondriaper10µm2inelectronmicrographstakenfromCDfedAldh5a1+/+mice,CDfedAldh5a1‐/‐miceandKDfedAldh5a1‐/‐mice.KDfedmutantshadsignificantlymoremitochondriathanCDfedwildtypemice.CDfedmutantsdidnotdifferfromeitheroftheothergroups.Tencellswereexaminedfromeachsubject(N=3pergroup)withfourtofiveimagesanalyzedpercell.#=number,μm2=squaremicrometers,CD=controldiet,KD=ketogenicdiet,**p<0.01

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Figure27.PercentofSomaticAreaOccupiedbyMitochondria

Figure27showsthemean(±s.d.)hippocampalCA1somaticareaoccupiedbymitochondria(calculatedasapercentageoftotalsomaticarea)inCDfedAldh5a1+/+mice,CDfedAldh5a1‐/‐miceandKDfedAldh5a1‐/‐mice.KDfedmutantshadasignificantlyhigherdensityofmitochondriainthecellbodythandidthewildtypecontrolgroupfedacontroldiet.ThedifferencebetweenCDfedmutantsandKDfedmutantsapproachedsignificance(p=0.06).Tencellswereexaminedfromeachsubject(N=3pergroup)withfourtofiveimagesanalyzedpercell.%=percent,CD=controldiet,KD=ketogenicdiet,*p<0.05

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7.3.3 ATPQuantificationinHippocampalTissue

Figure28showsthemean(±s.e.m.)hippocampalATPlevels(expressedas

picomolesATPperdecigramoftissue)inCDfedAldh5a1+/+mice,CDfedAldh5a1‐/‐

mice,KDfedAldh5a1+/+miceandKDfedAldh5a1‐/‐mice.Asindicated,hippocampal

ATPlevelsarehighinCDfedwildtypemiceandKDfedmutantmice,intermediatein

KDfedwildtypemiceandlowinCDfedmutantmice.Aone‐wayANOVAwasusedto

comparegroupmeans.Inhippocampaltissue,nosignificantdifferencewasdetected

amongstthegroups(F=2.046,p=0.12,n.s.).Althoughtherewasnostatistically

significanteffect,therewasanapparenttrend.Therefore,weranindividualt‐tests

betweenthegroups.Thesetests,revealedthatCDfedmutantshadsignificantly

lowerATPlevelsascomparedtoCDfedwildtypemice(p=0.039).t‐testsalso

suggestedthataCDfedmutantshadsignificantlylowerATPlevelsthanKDfed

mutants(p=0.015).Regardlessofthestatisticused,thereisanapparentdecreasein

hippocampalATPlevelsinCDfedAldh5a1‐/‐mice.TheKDappearstorestorethese

levelstowardnormalinmutantmice.

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Figure28.HippocampalATPLevels

Figure28showsthemean(±s.e.m.)hippocampalATPlevels(expressedaspicomolesATPperdecigramoftissue)inCDfedAldh5a1+/+mice(N=13),CDfedAldh5a1‐/‐mice(N=11),KDfedAldh5a1+/+mice(N=15)andAldh5a1‐/‐mice(N=12).AlthoughatrendtowardslowerATPlevelsinAldh5a1‐/‐miceexists,itdidnotreachstatisticalsignificanceusinganANOVA.t‐tests,however,suggestedthatATPlevelsmaybesignificantlylowerinCDfedmutantsascomparedtoCDfedwildtypemice.Further,ATPlevelsarecompletelyrestoredinKDfedmutants.CD=controldiet,KD=ketogenicdiet,ρ=pico,dg=decigram.*p<0.05(t‐tests).

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7.4 Discussion

ThepresentexperimentsweredesignedtoexplorewhetherKD‐induced

changestomitochondriainAldh5a1‐/‐miceplayaroleinthediet’smechanismof

actioninthesemutantmice.

7.4.1 MitochondrialNumberandSize

Experimentsonmitochondrialprofilesweredesignedtodeterminewhether

Aldh5a1‐/‐micehadnormalmitochondrialnumbersandsizeascomparedto

wildtypecontrols.Thisexperimentwasalsodesignedtoreplicatethefindingsof

Boughetal.(2006),whoshowedthattheKDcausesasignificantincreasein

mitochondrialnumber.WehypothesizedthatAldh5a1‐/‐micewouldhave

significantlyfewerhippocampalmitochondriaandthattheKDwouldelevate

mitochondrialnumbersinAldh5a1‐/‐mice.

Mitochondrial Number

OurelectronmicroscopystudiesrevealedthatCDfedwildtypemicehad,on

average,approximately2mitochondriaper10µm2ofCA1somaticcellarea.CDfed

mutantsshowedanon‐significantincreaseinmitochondrialnumber,withameanof

approximately2.3mitochondriaper10µm2ofsomaticcellarea.KDfedmutantshad

significantlymoremitochondriathanwildtypecontrolsandslightlymorethanCD

fedmutants,withameanofapproximately2.6mitochondriaper10µm2ofsomatic

cellarea.

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Interestingly,mitochondrialnumberisnotlowerinCDfedmutants,as

expected.Saueretal.(2006)showedthatCDfedmutantshaveimpaired

hippocampalmitochondrialfunction.Ourdatasuggestthatthisimpairmentisnot

causedbyadecreaseinthenumberofmitochondria.

ThepresentstudyconfirmsthefindingofBoughetal.(2006)byshowing

thattheKDincreasesthenumberofhippocampalmitochondria.Boughand

colleaguesreporteda45‐50%increaseinthetotalnumberofmitochondriawhereas

thepresentstudyonlyfounda25‐30%increase.ThesubjectsofBough’sstudies

wereadultrats.Thespeciesdifferencebetweenthestudiesmayexplainthe

differencesinthemagnitudebetweentheresultsofthetwostudies.

Mitochondrial Size

AlthoughtheabovestudyshowedthatKDfedmutantshadsignificantlymore

mitochondria,itwasnotclearwhetherthesemitochondriawerenormalinsize.

Therefore,thenextstudywastoanalyzethesizeofmitochondria.Todothis,we

determinedthepercentofthesomaticareathatisoccupiedbymitochondria.We

foundthatapproximately6%ofthesomawasoccupiedbymitochondriainCDfed

wildtypemiceandCDfedmutantmice.Thisjumped,however,toapproximately8%

inKDfedmutantmice.

ThesedatashowthattheKD‐inducedincreaseinmitochondrialnumber

correspondstoanincreaseinthepercent‐areaofthesomaoccupiedby

mitochondria.ThissuggeststhatmitochondriageneratedbytheKDarenormalin

size.

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7.4.2 MitochondrialATPLevelsinHippocampus

ThisexperimentwasperformedtotestthehypothesesthatCDfedmutant

micewouldhavelowerATPlevelsthanwildtypemice,andthattheKDwould

restoreATPlevelsinthemutantmice.WefoundthathippocampalATPlevelsare

highinCDfedwildtypemiceandKDfedmutantmice,intermediateinKDfed

wildtypemiceandlowinCDfedmutantmice.AnANOVAwasunabletodetect

significantdifferencesamongthegroups.Therewas,however,acleartrendtowards

areductioninhippocampalATPlevelsinCDfedmutantmice.Whenindividualt‐

testswererunbetweenthegroups,itwasfoundthatCDfedmutantmicemayhave

significantlylowerATPlevelsthanCDfedwildtypemutants(p=0.039).t‐testsalso

showedthatCDfedmutantshadsignificantlylowerATPlevelsascomparedtoKD

fedmutants(p=0.015),whohavesimilarATPlevelstoCDfedwildtypemice.

Saueretal.(2006)showedthathippocampalneuronsfromCDfedAldh5a1‐/‐

micehavesignificantlyimpairedmitochondrialfunction.Specifically,theyidentified

deficienciesincomplexI‐IVoftheelectrontransportchain,whichisessentialforthe

aerobicproductionofATP.OurdataextendthefindingsofSaueretal.(2006)to

showthatCDfedmutantshadsignificantlylowerhippocampalATPlevels.

SignificantlyreducedhippocampalATPmayplayaroleinthephenotypeof

Aldh5a1‐/‐mice.Further,theKD‐inducednormalizationofhippocampalATPlevels

mayexplainwhytheKDshifts‐toward‐normaltheSSADH‐dphenotype.

PreviousreportshavesuggestedthattheKDelevatesATPlevelsinthebrain

(AppletonandDeVivo,1974;Masinoetal.,2007),whileothergroupshavefailedto

showsuchanelevation(Boughetal.,personalcommunication).AlthoughBough

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andcolleaguesdidnotfindelevatedATPlevels,theydidfindasignificantelevation

inthephosphocreatine:creatineratio(Boughetal.,2006).Phosphocreatineisa

moleculethatactsasanenergystore.ItcanbeusedtoanaerobicallycreateATP

throughthefollowingreversiblereaction:

InagreementwithBoughetal.,thepresentstudysuggeststhattheKDdoes

notelevatehippocampalATPlevelsinwildtypemice.Inmutantmice,however,the

KDelevatesATPtothelevelsseeninwildtypecontrols,butnothigher.Thepresent

studydidnotexaminephosphocreatinelevels.SimilartoBoughetal.(2006),

however,itispossiblethatATPlevelsinKDfedwildtypemicewerenotelevatedby

theKD,butphosphocreatinelevelswere.Thishasbeenproposedbelowinthe

FutureStudysection.

7.4.3 Summary

ThepresentstudyfoundthattheKDdoesacttoincreasethenumberof

mitochondriaintheCA1pyramidalcellsofAldh5a1‐/‐mice.TheKDalsonormalizes

thedeficitsinhippocampalATPlevelsthatareseeninCDfedAldh5a1‐/‐mice.Taken

together,theKD’sbeneficialeffectsinAldh5a1‐/‐micemaybemediated,inpart,

throughthediet’sactionsonmitochondria.

Phosphocreatine+ADPcreatinephosphokinaseCreatine+ATP

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CHAPTER8

GENERALDISCUSSION

ThepresentexperimentsweredesignedtoexaminetheeffectsofaKDin

Aldh5a1‐/‐mice.ItwasfoundthattheKDprolongedthelifespanandreverseda

numberofabnormalitiesinthemutantmice.Anumberofpossibleexplanations

exist,andwillbeconsideredbelow.Webelieve,however,thatthemost

parsimoniousexplanationisthattheKDactsbybypassingtheflawedglucose

metabolismfoundinthemutant’sbrains.Thisisourgeneralhypothesis.

8.1 GeneralHypothesis

OurgeneralhypothesisisthattheKDworksinAldh5a1‐/‐micebysupplying

themutantswithanabundant,alternativeenergysubstrate(i.e.,ketonebodies).We

further hypothesize that the results of Experiments 1‐5 can be explained in the

contextofthishypothesis.

This hypothesis is discussed below in greater detail below. Also discussed

belowarethechangesfoundinExperiments1‐5andhowtheyrelatetoourgeneral

hypothesis, aswell as how theymight relate to other hypotheses that have been

proposedintheliterature.

8.2 SummaryofExperiments

Thepresent researchhasshown thata4:1KD isaneffective treatment for

murineSSADH‐d.ThedataprovidesupportforthepotentialusefulnessoftheKDin

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theclinicaltreatmentofSSADH‐dandtheyprovidenovelinsightsintothetreatment

ofthisdisorderaswellasthemechanismsoftheKDinSSADH‐d.

8.2.1 Experiment1:Lifespan,Ataxia,WeightandECoG

The purpose of Experiment 1was to determine the effects of a KD on the

SSADH‐dphenotypeinmice.TherationalefortestingtheKDinthesemutantswas

related to the observation that Aldh5a1‐/‐ mice that are left with their dams live

significantly longer than their weaned littermates. We hypothesized that dam’s

milk—which is high in fat—was acting like a high fat KD. If this hypothesiswere

true, then a KD should also significantly improve the general phenotype of

Aldh5a1‐/‐mice.

Lifespan

Inagreementwiththehypothesis,thelifespanofKDfedAldh5a1‐/‐micewas

significantlyincreased,ascomparedtothelifespanofCDfedAldh5a1‐/‐mice.

The effect of theKDwas considerably greater than the effects of the other

strategies that have been used in attempts to rescue these mutants. Other

treatments(e.g.,VGBandtaurine)havealsobeenshowntoprolongthelifespanof

Aldh5a1‐/‐mice,buttheyhavehadlittleeffectonotheraspectsofthisdisordersuch

as ataxia,weight loss andEEG (Hogemaet al., 2001). In contrast, theKD led to a

significantimprovementinweightgain,adelayinthedevelopmentofataxiaanda

remarkablenormalizationofEEGactivity.

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Ataxia

Asmentionedabove,theKDcausedasignificantdelayintheonsetofataxia

inAldh5a1‐/‐mice.TheonsetofataxiawasseenatP15‐17intheCDfedmutantsand

aroundP70intheKDfedmutants.AlthoughKDfedmutantsultimatelydeveloped

levelsofataxiaashighasthoseseenintheCDfedAldh5a1‐/‐mice,theonsetofataxia

wasdelayedbymorethan50days.

An interestingobservation is thatevenafter theonsetofataxia, theKD fed

mutants continued to have better sensory awareness than the CD fed mutant

controls.Whenplacedonatabletop,theCDfedmutantshadtobecloselymonitored

because they would walk off the table’s edge and fall to the floor. The KD fed

mutants,however,neverwalkedofftheedge.

Weights

Experiment1alsoshowedthatKDfedAldh5a1‐/‐micehadimprovedweight

gain,ascomparedtoCD fedAldh5a1‐/‐mice.KD fedmutantsgainedanaverageof

0.13gramsperday,whileCDfedmutants lostanaverageof0.003gramsperday.

This improvement inweight gainwas seen despite the fact that the KD normally

decreasesweightgain.

Normally, the KD is associated with stunted growth, both in humans

(McDonald,1997;Couchetal.,1999;Williamsetal.,2002;Viningetal.,2002;Liuet

al., 2003; Papandreouet al., 2006) and in rodents (Zhaoet al., 2004;Nylenet al.,

2005;Nylenetal.,2006).

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ECoG

Experiment1also replicated the findingsofCortezetal. (2004),whohave

demonstrated frequent electrographic seizure activity in the ECoG of CD fed

Aldh5a1‐/‐mice.TheECoGinthesemiceshowedspike‐and‐wavedischarges,aswell

as intermittent, high frequency ictal activity that corresponded to behavioral

convulsions.AscomparedtoCDfedmutants,KDfedmutantshadalmostnoseizure

activityintheECoGandalmostnobehavioralconvulsions.

TheKDnormalized theECoG inAldh5a1‐/‐mice.Thisraises thequestionof

whethertheKDisactingdirectlyasananticonvulsant.Therearemixedreportsasto

whethertheKDelevates“seizurethreshold”inrodents.AsdepictedinFigure29,the

seizurethresholdisdefinedastheminimumstimulusrequiredtoelicitaseizure.In

rodents, approximately half of the KD studies, using experimentally induced

seizures,haveshownsignificantanticonvulsanteffectsofaKD(e.g.,Horietal.,1997;

Bough et al., 1999; Thavendiranathan et al., 2000), while half have failed to find

significant anticonvulsant effects (e.g., Thavendiranathan et al., 2000;Nylen et al.,

2005;Nylenetal.,2006).IthasbeenconcludedthatiftheKDcausesanelevationof

seizurethresholdinrodents,itmustbeasmallelevation(Thavendiranathanetal.,

2000).

A small elevationwouldprobablynotproduce thedramatic suppressionof

seizuresseeninthepresentstudy.Norwouldanticonvulsanteffectsbeexpectedto

producethechangesin[35S]TBPS,mitochondria,ATP,asseeninthepresentstudies.

130

As an alternative to proposing a direct anticonvulsant effect of theKD, the

presentdatamaybeexplainedbythegeneralhypothesis.Insufficientenergyinthe

brain can lead to neuronal dysfunction and seizures (Ziegler et al., 2002). By

providinganabundant,alternative formofenergythebrain’snormal functioncan

be restored and seizure activity can be decreased. Reversing themutants’ energy

deficitmightalsoreverseanumberoftheotherdeficitsseeninthemutantmice.

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Figure29.IllustrationofSeizureThreshold

Thisfiguredepictstheconceptof“seizurethreshold”.Seizurethresholdisdefinedastheminimalstimulusrequiredtoelicitaseizure.Thesestimulicantakemanyforms,e.g., noises, lights, touch, stress. In a seizure‐prone animal, these stimuli may besufficienttotriggeraseizure,whereas itwouldnottriggeraseizure ina“normal”individual.Abnormallystrongstimulicanbeusedtotriggeraseizureinanyone,asseeninelectroconvulsiveshocktherapy.

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8.2.2 Experiment2:MiniaturePost­SynapticCurrents

mIPSCs

Experiment 2 was designed to explore the effects of the KD on miniature

postsynaptic currents in Aldh5a1‐/‐ mice. We hypothesized at the time that the

severeseizuresseeninAldh5a1‐/‐micemightbearesultofeitherdecreasedmIPSC

activityorincreasedmEPSCactivity.Wefurtherhypothesized,giventheremarkable

effects of the KD on the Aldh5a1‐/‐ phenotype, that the KD would normalize any

perturbation inthesecurrents.This isdiscussedfurtherbelowincontextwiththe

[35S]TBPSbindingdata.

mIPSCs frequency was significantly reduced in CD fed mutant mice, as

compared to CD fed wildtypemice. The KD restoredmIPSC frequency inmutant

mice to the levels seen in CD fed wildtype mice. Experiment 2 also showed that

mIPSCamplitudesweresignificantlylargerinCDfedmutantmice,ascomparedto

CD fedmutants.mIPSC amplitudes inKD fedmutantswere restored to the levels

seeninCDfedwildtypemice.

Experiment2isthefirststudytoshowthatmIPSCfrequencyandamplitude

aresignificantlyperturbedinAldh5a1‐/‐mice.ItisalsothefirsttoshowthattheKD

cannormalizemIPSCfrequencyandamplitudeinAldh5a1‐/‐mice.

mIPSCs play an key role in themaintenance and development of synapses

(Swanwick et al., 2006;Hartman et al., 2006).mIPSCs also play a critical role in

regulating synaptic strength in the brain (Verstreken andBellen, 2002), and they

havebeenshowntoplayan importantrole inepileptogenesis(Hirschetal.,1999;

Wierenga and Wadman, 1999; Shao and Dudek, 2005; Rajasekaran et al., 2007).

133

Further,neurosteroidssuchasallopregnanaloneandtetrahydrodeoxcorticosterone

—which are known anticonvulsants—have been shown to work by enhancing

mIPSCsaspartoftheiranticonvulsantmechanismofaction(Schwabeetal.,2005).

Another anticonvulsant drug, stiripentol, has also been shown to enhancemIPSC

activity(Quilichinietal.,2006).

The impairment of mIPSCs in the mutant mice might explain the seizures

seen in these subjects, and the ability of the KD to reverse these deficits might

explain the decrease in seizure activity in KD fed mutants. These results can be

related to the general hypothesis when viewed together with the results of

Experiment3.Therefore, thesedatawill bediscussed furtherbelow.Nonetheless,

the ability of the KD to restore the significant reductions in mIPSC frequency in

Aldh5a1‐/‐miceisanovelfindingthatprovidesinsightintothemechanismoftheKD

inthismodel.

mEPSCs

LessdramaticeffectsofSSADH‐dwereseeninourstudyofmEPSCs.mEPSC

area was significantly smaller in CD fed Aldh5a1‐/‐ mice, as compared to KD fed

Aldh5a1‐/‐mice andCD fedwildtypemice. This suggests that theremaybe either

less transmitter substance being released or that there is a significantly reduced

synchronizationofpresynaptictransmitterrelease. ItremainsunclearwhymEPSC

area would be smaller as none of the other mEPSC properties were significantly

changed. No other significant changes to mEPSC properties were seen in mutant

micefedeitherdiet.

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AreductioninmEPSCswouldbeexpectedtohaveanticonvulsanteffects.The

changeinmEPSCs,however,waslessdramaticthanthechangeinmIPSCs,andwas

notreversedbytheKD.ThissuggeststhatmEPSCdonotappeartoplayamajorrole

in SSADH‐dmice. Further, it suggests that the impact of SSADH‐d—and theKD—

maybemainlylimitedtoGABAergicsynapses.

8.2.3 Experiment3:[35S]TBPSBinding

Wuetal.(2006)showedthat[35S]TBPSbindingwassignificantlydecreased

inAldh5a1‐/‐mice,ascomparedtowildtypecontrols,suggestingthatthemutant

micehavefewerligand‐gatedchloridechannels.Wuetal.(2006)hypothesizedthat

thisplayedacriticalroleintheneuralhyperexcitationandseizuresseenin

Aldh5a1‐/‐mice.

Experiments1and2demonstratedthattheKDhadsignificant,normalizing

effectsonbothinvivoelectrophysiology(ECoG)andinvitroelectrophysiology

(mIPSCs)inAldh5a1‐/‐mice.Wewondered,therefore,whethertheKDmight

normalize[35S]TBPSbindinginAldh5a1‐/‐mutants.Incombinationwiththeeffects

ofmIPSCs,thiscouldexplainwhyKDfedmutantsshowednormalizedECoGactivity

andhadfewerconvulsions,ascomparedtoCDfedmutants.

SimilartoWuetal.(2006),wefound[35S]TBPSbindingtobesignificantly

decreasedinAldh5a1‐/‐mice.Consistentwithourhypothesis,wealsofoundthatthe

KDrestored[35S]TBPSbindinginAldh5a1‐/‐mice.Therestoration,however,

occurredinaregion‐specificmanner.Bindingwascompletelyrestoredinthe

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hippocampusandcortexofKDfedAldh5a1‐/‐mice,butitwasonlypartialrestored

intheamygdalaandthalamus.

It is unclear why the KD would fully restore [35S]TBPS binding in the

hippocampus and cortex, but only partially restore binding in the amygdala and

thalamus. Future studies investigating the mechanism behind the KD’s ability to

restore [35S]TBPS binding may reveal why it occurs more readily in the

hippocampusandcortexthantheamygdalaandthalamus.

How do defects in [35S]TBPS binding relate to the decreases we found in

mIPSC frequency? How does the KD reverse both of these abnormalities? One

possibility relates to the KD’s ability to significantly increase protein

phosphorylation, which is an energy dependent process (Ziegler et al., 2002).

Phosphorylationplaysaroleinthetraffickingofchannelstothemembrane(Jacob

etal.,2008).TheKDmaycauseanincreaseinchloridechanneltraffickingtothecell

surfacethroughaphosphorylationmechanism,causingasubsequentincreaseincell

surfacechloridechannelexpressionreflectedbyrestored [35S]TBPSbinding inKD

fedAldh5a1‐/‐mice.This,inturn,maycausearestorationofmIPSCcurrents,asthe

number of post‐synaptic chloride channels—which mediate the mIPSCs—is

restored.

Thishypothesisisonlyspeculative,however.Theexactproteinsinvolvedin

thetransportofchloridechannelsarenotfullyknown.Ziegleretal.(2005)showed

that theKDcauses a≥40% increase in thephosphorylationof the sevendifferent

proteins theyexamined,so itappears that theKDmaybeable tophosphorylatea

largenumberofproteins.

13

6

Figure30.ProposedMechanismfortheKetogenicDiet’sEffectsonNeuralHyperexcitability

137

AnotherpossibleexplanationthatlinksthemPSCdataand[35S]TBPSdata

relatestothehighlevelsofGHBandGABAinAldh5a1‐/‐mice.BothGABAandGHB

(perhapsthroughitsconversionintoGABA)bindthepresynapticGABAB

autoreceptoratGABAergicsynapses,andpresynapticGABABheteroreceptorat

glutamatergicsynapses(BettlerandTiao,2006;UlrichandBettler,2007).

StimulationofthesepresynapticGABABreceptorsreducesthepresynapticrelease

ofneurotransmitterviainhibitionofthepresynapticcalciumchannel(Bettlerand

Tiao,2006;UlrichandBettler,2007).Thismayberesponsibleforthedecreaseseen

inbothmIPSCandmEPSCactivity.ThemIPSCactivity,however,mayhavebeen

furtherloweredbythesignificantdown‐regulationofpostsynapticligand‐gated

chloridechannels,asdiscussedabove.

8.2.4 Experiment4:SerumAnalytes

Inkeepingwiththepreviouspublishedreports,wehypothesizedthatKDfed

animals would have significantly decreased blood glucose levels (Appleton and

DeVivo, 1974; Nylen et al., 2005; Nylen et al., 2006) and increased βOHB levels

(Bough et al., 2000; Nylen et al., 2005; Nylen et al., 2006).We examined both of

these in Experiment 4.We also examined free fatty acid levels, given that recent

studieshaveproposed that theymayplay a role in theKD’smechanismof action

(Cunnaneetal.,2002;Tahaetal.,2005).

Experiment4showedthatbloodglucose levels inCDfedmutantsandboth

KD fed groupswere not different fromCD fedwildtypemice. Blood glucosewas,

however, significantly lower in the CD fed mutants as compared to the KD fed

138

groups.BloodβOHBlevelsweresignificantlyelevated inCDfedmutantsandboth

KD fed groups as compared to CD fed wildtypemice. Blood NEFA levels did not

differsignificantlyamongstthegroups.

GlucoseLevels

NoneofthegroupsdifferedsignificantlyfromCDfedwildtypemiceinterms

ofserumglucoselevels.CDfedwildtypemicehadserumglucoseofabout9mM,CD

fedmutantshadlowerserumglucoselevelsofabout7mM,butthisdifferencewas

notsignificant.TheKD,however,elevatedglucoselevelsinboththemutantand

wildtypesubjectstoabout10mM.ThedifferencesbetweenCDfedmutantsandboth

KDfedgroupsweresignificant.

ThefindingofhigherserumglucoselevelsinKDfedsubjectswas

unexpected,aspreviousreportshaveshownthatbloodglucoselevelstendtobe

lowerinKDfedwildtyperodentsascomparedtoCDfedwildtypecontrols

(AppletonandDeVivo,1974;Todorova,2000;Likhodiietal.,2000;Nylenetal.,

2005).

GlucosedataintheCDfedmutants,however,arenotinconsistentwiththe

generalhypothesis.AlthoughAldh5a1‐/‐micehavenormalbloodglucoselevels,they

alsohaveasignificantlyimpairedabilitytoconvertglucoseintoenergy(Chowdhury

etal.,2006).Therefore,normalbloodglucoselevelsmaynotmeannormalglucose

utilizationforenergy.

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βOHBLevels

Asexpected,βOHBlevelswererelativelylowinCDfedwildtypemice.They

weresignificantlyelevatedinCDfedmutantsandKDfedwildtypemiceandfurther

elevatedinKDfedmutants.Itwasinterestingtonotethatmutantshadsignificantly

elevated βOHB levels even on the CD. This has been reported previously

(Chowdhuryetal.,2006),andithasbeensuggestedthatitiscausedbyadeficiency

inglucosemetabolisminAldh5a1‐/‐mice.ElevationsinβOHBintheKDfedgroups

occurredasexpected.

HowmightthesechangesinβOHBberelatedtoimprovementoftheSSADH‐

d phenotype? There are a number of possibilities. The first, which relates to the

generalhypothesis,isthattheincreaseinβOHBisasignthatthebodyisoxidizing

fats into ketone bodies. This is part of the metabolic shift away from the use of

glucoseandtowardstheuseofketonebodiesasanenergysubstrate.βOHB,along

withtheotherketonebodies,canbeconvertedintoacetylCoAandutilizedbythe

Krebs cycle in the production of energy (Nehlig, 2004). This would supply the

glucose‐starvedmutantbrainswithanalternatesourceofenergyandmightexplain

the improvement seen in a number of parameters measured in the present

experiments.

ThereareotherpossibilitiesworthconsideringastohowβOHBmayworkin

SSADH‐d. One relates to βOHB’s actions on KATP channels. Ma et al. (2007) have

shownthatβOHB—atconcentrationssimilartothoseseenintheKDfedAldh5a1‐/‐

mice—acts as an agonist at KATP channels. Activation of these channels causes

neuronstofirelessfrequently.βOHB’sactionsatthesechannelsmightplayarolein

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theKD’seffectsinAldh5a1‐/‐mice.Thiscouldexplainwhytheyshowfewerseizures,

however,thismechanismwouldnotexplainotherchangesobservedinthepresent

experiments(e.g.ataxia,weightgain,[35S]TBPSbinding,ATPlevels,etc.).

Another possibility to be considered relates to the antioxidant effects of

ketonebodies.RodentsfedaKDhavesignificantlyelevatedlevelsoftheantioxidant

glutathione (Schutzmani et al., 2007; Jarrett et al., 2008). This effect is primarily

mediatedthroughtheelevationoftheketonebodiesβOHBandacetoacetate,bothof

which have been shown to elevate glutathione levels inmitochondria, which has

anti‐oxidant effects (Haces et al., 2008; Jarrett et al., 2008; Maalouf and Rho, in

press).Althoughthemechanismofthiseffectremainsunknown,Jarrettetal(2008)

showed that only mitochondrial glutathione levels (not cytosolic) are increased,

suggesting that the KD might be increasing the transport of glutathione into

mitochondria.

Aldh5a1‐/‐micehavebeenshowntohavesignificantlydecreasedglutathione

levelsaswellasincreasedapoptoticcelldeathfromoxidativestress(Gibsonetal.,

2005). Both increased oxidative stress and cell death are implicated in

epileptogenesis(KimandRho,2008).TheKD’sbeneficialeffectsinAldh5a1‐/‐mice

may be related to the diet’s ability to elevate ketone bodies, which increases

mitochondrial glutathione levels resulting in decreased oxidative stress, therefore

preventingepileptogenesis.

Although the KD’s affects on glutathione levels may help explain the

reduction in seizures inAldh5a1‐/‐ mice, they do not explain the numerous other

beneficialeffectsoftheKDinthismodel.

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NEFALevels

Aninterestingfindingwasthatserumfreefattyacidswerenotsignificantly

elevatedinmicefedahighfatKD.ThisisconsistentwiththefindingsofDelletal.

(2001),whofailedtoseeanincreaseinfreefattyacidsinKDfedrats.Itshouldbe

noted, however, that there was a trend towards increase in both KD fed groups,

whichmighthavebecomesignificantwithlargergroupnumbers.

The finding of no dramatic increase in KD fed subjects suggests that

increasedfreefattyacidsdonotplayadirectroleinthebeneficialeffectsoftheKD

inAldh5a1‐/‐mice. It ispossible,however, thatNEFAscontribute in indirectways.

Forexample,Aldh5a1‐/‐micemayberapidlyconvertingNEFAstoβOHB.Theratios

of mean NEFA to βOHB were: Aldh5a1+/+ (CD), 1.20; Aldh5a1‐/‐ (CD), 0.73;

Aldh5a1+/+ (KD), 1.04;Aldh5a1‐/‐ (KD), 0.46. The ratios forAldh5a1+/+micewere

comparableonbothdiets.TheratiosinAldh5a1‐/‐micewerelower,andtheywere

particularlylowinmutantmiceontheKD,suggestingenhancedβ‐oxidationoffatty

acidsintheAldh5a1‐/‐mice.

8.2.5 Experiment5:MitochondrialCountsandFunction

MitochondrialCounts

Experiment5involvedadeterminationofthenumberofmitochondriain

hippocampalCA1pyramidalcellsfromCDfedAldh5a1+/+mice,CDfedAldh5a1‐/‐

miceandKDfedAldh5a1‐/‐mice.GiventheworkofSaueretal.(2007)thatshowed

decreasedmitochondrialfunctioninhippocampaltissuefromAldh5a1‐/‐mice,we

hypothesizedthatCDfedAldh5a1‐/‐micewouldhavefewerhippocampal

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mitochondria,ascomparedtoCDfedwildtypemice.Inkeepingwithaprevious

report(Boughetal.,2006),wefurtherhypothesizedthatKDfedsubjectswould

showanincreasednumberofmitochondria.

Contrarytoourhypothesis,CDfedAldh5a1‐/‐micedidnothavesignificantly

fewermitochondriathanCDfedwildtypemice.Rather,CDfedmutantshadslightly

moremitochondriathanwildtypemice,althoughthiseffectwasnotstatistically

significant.

TheKDfedmutantshadsignificantlymoremitochondriathanCDfed

wildtypemice.ThisisinagreementwithapreviousreportthattheKDincreasesthe

numberofmitochondria.Boughandcolleagues(2006)whoreporteda50%

increaseinthetotalnumberofhippocampalmitochondriainKDfedmutants.

Experiment5foundasmallerincrease(~25‐30%)inthetotalnumberof

hippocampalmitochondria.Thereasonasmallerincreasewasfoundmayrelateto

ouruseofmutantmice,whereasBoughworkedinhealthyrats.

ItisnotknownhowtheKDmightelevatethenumberofmitochondria.

Boughandcolleagues(2006)havesuggestedthatchronicketosismayplayarole.

DatafromExperiment5areinagreementwiththishypothesisastheCDfedmutant

mice,whoareketotic,hadslightlyelevatedmitochondrialnumbers.

MitochondrialATPLevels

Experiment5alsoexaminedhippocampalATPlevelsinCDfedAldh5a1+/+

miceandCDfedAldh5a1‐/‐miceaswellasKDfedAldh5a1+/+miceandKDfed

Aldh5a1‐/‐mice.Duetothereporteddeficiencyinmutantmice,wehypothesized

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thatCDfedAldh5a1‐/‐micewouldhavesignificantlylowerATPlevelsascompared

toCDfedwildtypemice.SincetheKDprovidesanalternativeenergysource,wealso

hypothesizedthattheKDwouldresultinarestorationofATPlevelsinAldh5a1‐/‐

mice.

OurresultsshowedthatCDfedAldh5a1‐/‐micedidhavesignificantlylower

levelsofhippocampalATPascomparedtoCDfedwildtypemice.Thesedata

complimentandextendthoseofSaueretal.(2006),whoshowedthathippocampal

mitochondriainAldh5a1‐/‐micehavesignificantlyimpairedelectrontransportchain

function(complexesI‐IV).

Thesedataareinagreementwiththegeneralhypothesisframedabove.CD

fedmutantmicehadsignificantlyreducedATPlevels,ascomparedtowildtype

controls.AlthoughitisunknownwhatlevelsofATParerequiredfor“normal”brain

function,thereductionsinhippocampalATP,seeninCDfedAldh5a1‐/‐mice,might

playaroleintheSSADH‐dphenotype.TheKDsignificantlyelevatedhippocampal

ATPlevelsinAldh5a1‐/‐mice,whichlikelyplayacriticalroleinthenormalizationof

SSADH‐dphenotype.

8.3 LimitationsofStudies

Thepresentstudiesarenotwithoutlimitations.Previousworkhasshown

thatVGBiseffectiveatprolongingthelifespanofAldh5a1‐/‐mice.Ourworkshows

thattheKDalsoprolongsthelifespanofmutantmice.Itwouldhavebeengoodto

comparedirectlytheeffectsofVGBandtheKDinAldh5a1‐/‐mice.

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AllstudiesinvolvedstudyingAldh5a1‐/‐betweentheagesofP18‐25.Thisis

whentheuntreatedmutantmicebegintoexperiencetonic‐clonicseizuresand

statusepilepticus.AlthoughtheuntreatedmiceweresickerthantheKDtreated

mice,wedidnotusemiceinstatusepilepticusforanyofourexperiments.

Althoughourstudieshaveclinicalrelevance,theAldh5a1‐/‐micehaveavery

severeSSADH‐dphenotypethatmaynotbeparalleledinhumans,whovaryinterms

oftheseverityofthedisorder.Itisnotknownwhetherhumanswithmoresevere

formsofSSADH‐ddieatanearlyage.OurhypothesisthattheKDmaybeeffectivein

patientswithSSADH‐dremainstobetested(seeFutureStudiesbelow).

8.4 InsightsintotheKD’sMechanismsofActioninAldh5a1­/­Mice

8.4.1GeneralComments

StudiesontheKDoftenseektheonemechanismthroughwhichtheKD

exertsitseffects.Inreviewingtheliterature,itbecomesreadilyapparentthattheKD

appearstoworkviaoneverygeneralmechanism,i.e.,shiftingenergymetabolism

awayfromglucoseandtowardsfats.This,however,causesalargecascadeofevents

thatmayplayaroleinthediet’smechanism(tonameafew:elevatedketosis,

anticonvulsantactionsofacetone,antioxidantactionsofβOHBandacetoacetate,

glycolysisinhibition,increasedproteinphosphorylation,mitochondrialbiogenesis,

etc.).

Withregardstothepresentstudies,ourdatahaveidentifiedanovel

observationinvolvedinthecascadediscussedabove.ThisistheKD’sabilityto

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restoreGABAergicfunction—asevidencedbynormalized[35S]TBPSbindingand

normalizedmIPSCactivity.

OneinterestingobservationinthepresentexperimentsisthattheKDtends

to“normalize”themutants,butnotpushthembeyondnormal.TheKDnormalized

[35S]TBPSbinding,mIPSCfrequencies,ECoGactivityandATPlevels.Itdidnot,

however,causesignificantlyenhanced[35S]TBPSbinding,mIPSCfrequencies,ECoG

activityorATPlevelsascomparedtowildtypecontrols.Themainexceptiontothis

findingisthesignificantelevationsinbloodβOHBlevels.OtherwisetheKDappears

topushsystemstowardshomeostasis—butnotbeyondhomeostaticlevels—in

Aldh5a1‐/‐mice.

AsimilarargumentcanbeappliedtowardtheKDinthetreatmentofother

neurologicaldisorders,suchasepilepsy.TheKDmightsuppressseizures,butunlike

theanticonvulsantdrugs,itdoesnotsedateorcausereducedcognitivefunctionin

childrenontheKD(Farasatetal.,2006).

8.4.2 TheMechanismsoftheKDinSSADH­d

PreviousresearchhasshownthatAldh5a1‐/‐micehaveasignificantly

impairedabilitytooxidizeglucoseasenergy(Chowdhuryetal.,2007).We

hypothesizethattherapidprogressionofdiseaseinthesemutantsiscausedbytheir

inabilitytoefficientlyutilizeglucose,coupledwiththefactthattheyaregenerally

fedahighcarbohydratediet(i.e.,theCD).Inanattempttoobtainenergy,themice

oxidizetheirfatstores.Thistheoryissupportedbytherapidworseningofthe

SSADH‐dphenotype(e.g.,onsetofgeneralizedconvulsions)atthetimeofweaning

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(P18‐20).Itisfurthersupportedbyweightdatafromthepresentstudy,whichshow

thatAldh5a1‐/‐mutantsbegintoloseweightaroundthetimeofweaning.Autopsies

ofCDfedAldh5a1‐/‐mutantsshowanearcompleteabsencetobodyfat.Finally,

Aldh5a1‐/‐micehavesignificantlyelevatedβOHBlevels,evenwhilebeingfedahigh

carbohydrateCD,clearlysuggestingthattheyarenotabletoefficientlyutilize

dietarycarbohydratesforenergy.

OurgeneralhypothesisisthattheyKD’sbeneficialeffectsinAldh5a1‐/‐mice

arearesultofthediet’sabilitytoofferanalternateoxidizablesubstrate,i.e.,fat.This

yieldsasignificantincreaseinketonebodies,whichcanbeoxidizedforenergy

productioninplaceofglucose.Thisrestorestheamountofenergyavailabletothe

mice,allowingprocessesthatwereperturbed,duetoinadequateenergy,tobegin

functioningmorenormally.Thiswouldexplainthenormalizationofchloride

channelexpressionandmIPSCactivity.Thisoveralleffectcausesthemutantstobe

healthier,whichprolongsthelifespanofKDfedmutants.

Thishypothesisisnotwithoutitsproblems,however,asitdoesnotexplain

whyAldh5a1‐/‐micestillprogressintheirdiseasestate—albeitsignificantlymore

slowly—whensuppliedwithanalternativeenergysourcethroughadministrationof

theKD.Onepossibleexplanationrelatestothefactthatketonescanonlyaccountfor

30‐60%oftotalbrainenergy(Nehlig,2004).Thisisduetotherate‐limitingstepin

ketonebodyutilizationinthebrain,whichisthetransportofacetoacetateandbeta‐

hydroxybutyrateintothebrainviathemonocarboxyllictransporter(Nehlig,2004).

Mutantmicehavebeenshowntohaveasignificantlyimpairedabilitytoutilize

glucoseasanenergysubstrate(Chowdhuryetal.,2007).Ifmutantmicearenot

147

usingglucoseefficiently,andketonescanonlyaccountfor30‐60%oftotalbrain

energy,thenperhapsbrainfunctionbeginstobreakdownafterprolongedexposure

toinadequatelevelsofenergysubstrate.Thismayexplainwhymutantmicefeda

KDultimatelysuccumbtothesamefate,albeitsignificantlylaterinlife,thanCDfed

Aldh5a1‐/‐mice.

AnotherpossibilityworthconsideringisthattheKDitselfmighthave

detrimentaleffectsonthemiceafterlong‐termexposure.Ourlifespanstudy

involvedkeepingmiceontheKDfromP12untiltheendoftheirlife.Inonecase,this

meantthemousewasmaintainedontheKDforover130days.Inhumans,the

effectsofchronicconsumptionofahighfatdietareassociatedwithstuntedgrowth,

increasedriskofcardiovasculardiseaseandvitamindeficiencies(Vining,1999).In

rodents,Zhaoetal.(2004)reportedthatadministrationofaKDtoratsfor1month

ledtosignificantlysmallerbrainsandsignificantimpairmentsinvisual‐spatial

memoryascomparedtoCDfedcontrols.Anotherstudyshowedsignificantliver

diseaseinmicefedahighfatdietfor5months(Nevesetal.,2006).Itisworth

consideringthattheKDbenefitstheAldh5a1‐/‐mousebyrescuingtheSSADH‐d

phenotype—butitmayhavesomeharmfuleffectsinthelongterm.Amorehealthy

formofaKD,suchasapoly‐unsaturatedfattyacid‐basedKD,mighthelpanswerthis

question.Suchexperimentsareproposedbelow.

8.5 TheKetogenicDietintheClinicalTreatmentofSSADH­d

There is a need for better treatments for SSADH‐d inhumans. The current

treatmentofchoiceisVGB(Gordon,2004).VGB,however,isnotveryeffectiveand

148

carriestheriskofirreversiblevisualfielddamage.Basedonanimalstudies,VGBhas

beenshowntoprolongthelivesofAldh5a1‐/‐miceby~20%(Hogemaetal.,2001).

ThepresentworkhasshownthattheKDhasamuchgreaterabilitytodelay

theprogressionofSSADH‐dinmice.Lifespanofthemutantmicewereextendedby

over300%.AclinicaltrialoftheeffectsofaKDinSSADH‐disclearlyindicated.As

such,wearecurrentlycollaboratingonapilotstudytoexaminetheeffectsoftheKD

inpatientswithSSADH‐d.

8.6 FutureStudies

Experiment 1. The efficacy of the KD in clinical SSADH‐d. A future study that is

alreadyintheplanningstageistodeterminetheefficacyofaKDinthetreatmentof

clinicalSSADH‐d.ConsideringtheefficacyoftheKDinmutantmice,theKDmaybea

feasibletreatmentoptionforhumans.

Experiment 2. The efficacy of the Atkins diet and the polyunsaturated fatty acid

based KD inAldh5a1‐/‐ mice. A second future experiment would be to determine

whether other forms of anticonvulsant diet are able to rescue the Aldh5a1‐/‐

phenotype.ThereisoftenseriousreservationinplacingapatientontheKDdueto

thediet’shighfatcontentandthedifficultyinvolvedinadministeringtheclassicKD.

Certaindietshaverecentlybeenshowntohaveanticonvulsantefficacyagainstdrug‐

resistant seizures, for example, theAtkinsdiet (Kossoffetal., 2003;Kossoffetal.,

2006;Kossoffetal.,2007;Kossoffetal.,2008)andthepolyunsaturatedfattyacid‐

basedKD(Tahaetal.,2005).TheAtkinsdietismuchlessrestrictivethantheclassic

149

KD and it would be less rigorous to administer. The polyunsaturated fatty acid‐

basedKDwouldnotraisecholesterollevels.

Like the KD, all of these diets would elevate ketone bodies and thus

compensateforthelackofglucosemetabolism(forenergy)inAldh5a1‐/‐mice.

Experiment3.TheeffectofaKDonbloodandbrainGABAandGHBlevels.Athird

futurestudy,thatisalreadyunderway,woulddeterminetheeffectofaKDonGHB

andGABA levels.BothhumanswithSSADH‐dandAldh5a1‐/‐micehavesignificant

elevations inGABA(3‐5x increase)andGHB(30‐50x increase) inbloodandbrain

(Gibsonand Jakobs,2001;Hogemaetal., 2001;Pearletal., 2003).The significant

elevation of these neuroactive compounds may play a significant role in the

pathophysiologyofSSADH‐d.Onepossibleexplanation for thebeneficialeffectsof

the KD in Aldh5a1‐/‐ mice is that the KD acts to lower GHB and GABA levels.

Therefore,wearecurrentlyexaminingthelevelsofGHBandGABAinthebloodand

brain tissueofCDandKD fedAldh5a1‐/‐mice, aswell asCDandKD fedwildtype

mice. Brain and blood samples have been sent to Holland where they are being

analyzedbyDr.Struys’group.

Experiment4.TheeffectofaKDonenergymetabolismintermediatesinAldh5a1‐/‐

mice.AfourthfutureexperimentwouldbetoexaminetheeffectofaKDonallthe

intermediatesinvolvesintheKrebscycleaswellastheGABAshunt.Thepurposeof

this future experiment would be to perform a more in‐depth analysis of energy

metabolismincludingthemetabolicintermediatesastheyfluctuateoverthecourse

150

of KD administration. Chowdhury et al. (2007) showed thatAldh5a1‐/‐mice have

impaired glucose oxidation, which may explain why Aldh5a1‐/‐ mice become

progressivelymoreimpairedaroundthetimeofweaningontoahighcarbohydrate

diet. As the present thesis shows, weaning the mutants onto a high fat diet

significantlydelaystheprogressionofSSADH‐d.Wehypothesizethatthisisdueto

an increase in non‐carbohydrate energy substrate availability, but this is not yet

proven.Toaddress thisquestion,wepropose to infuse [13C]beta‐hydroxybutyrate

or[13C]glucoseinalive,anaesthetizedCDfedorKDfedmutantmice.Brianextracts

wouldbestudiedusing1H‐[13C]NMRspectroscopy.Wepredictthat[13C]frombeta‐

hydroxybutyratewould be found in the intermediates of the Krebs cycle and the

GABAshunt inKDfedmutants,whereas [13C] fromglucosewouldnotbe found in

these intermediates (as previously shown by Chowdhury and colleagues, 2006).

Thesestudiesarecurrentlybeingplanned.

Experiment 5. Elucidating the role of mitochondria in the ketogenic diet’s

mechanismofactioninthetreatmentofAldh5a1‐/‐mice.Afifthfuturestudywould

examine the precise role of mitochondria in rescuing the phenotype of KD fed

Aldh5a1‐/‐mice.CDandKDfedwildtypemice,aswellasCDandKDfedmutantmice,

wouldbeused.ApositivecontrolgroupofAldh5a1+/+micetreatedwithresveratrol,

a chemical shown to improvemitochondrial function would be used, as would a

negative control group of Aldh5a1+/+ mice treated with 3‐nitropropionic acid, a

chemicalknowntodecreasemitochondrialfunction(Lagougeetal.,2006).

151

Inthesegroupsofmice,wewouldtestseveralparametersofmitochondrial

function.First,wewouldassess theeffectofeach treatmenton themitochondrial

membranepotential.Themitochondrialmembranepotential(ψm)isanindicatorof

mitochondrial health. A decrease in ψm indicates mitochondrial dysfunction and

subsequentapoptosis/necrosis.MitoTrackerRedCM‐H2XRosdyewouldbeusedto

show changes in mitochondrial membrane potential in mice from each group

(Santraetal.,2004).

We would also determine the effect of each treatment on mitochondrial

cytochromec release.Mitochondrialcytochromec functionsasanelectroncarrier

intherespiratorychain.Ittranslocatestothecytosolincellsundergoingapoptosis.

SelectFXAlexaFluor488canbeusedtotagcytochromecandvisualizedifferences

betweentheabove‐mentionedgroups.

Wewouldalso like toassess theeffectof each treatmentonmitochondrial

respiration. Respiration (i.e., oxygen consumption) will be measured in a sealed,

continuously stirred chamber using a Clark‐type electrode. Oxygraphs will be

constructedandcomparedbetweengroups.

Experiment 6. Determining the role of mitochondria in rescuing the SSADH‐d

phenotype. A sixth future experiment would examine whether improving

mitochondrialfunctionaloneissufficienttonormalizetheAldh5a1‐/‐phenotype.On

P12,CDandKDfedAldh5a1‐/‐micewouldbeimplantedwithosmoticmini‐pumps

filled with resveratrol. Resveratrol has been shown to significantly improve

mitochondrial function and causemitochondria biogenesis (Lagouge et al, 2006).

152

These pumpswould allow constant and continuous administration of resveratrol.

We will then assess and compare the lifespan of all groups. If improved

mitochondrial function is the main mechanism behind the KD’s effects, then

resveratrolshouldhavesimilarlybeneficialeffectsinAldh5a1‐/‐miceastheKD.

8.7 Conclusions

Thepresentstudieshaveshown,forthefirsttime,thataKDissuccessfulin

prolonging the livesofAldh5a1‐/‐mice.Thedata representan important first step

towardsthebetterunderstandingandtreatmentofhumanSSADH‐d.

153

REFERENCES

AnderzhanovaE,SaransaariP,OjaSS.2006.Neuroprotectivemechanismsoftaurineinvivo.Adv.Exp.Med.Biol.583,377‐87.AkaboshiS,HogemaBM,NovellettoA,MalaspinaP,SalomonsGS,MaropoulosGD,JakobsC,GrompeM,GibsonKM.2003.Mutationalspectrumofthesuccinatesemialdehydedehydrogenase(ALDH5A1)geneandfunctionalanalysisof27noveldisease‐causingmutationsinpatientswithSSADHdeficiency.HumMutat.22,442‐450.AkaboshiS,HogemaBM,SalomonsGS,JakobsC,MalaspinaP,NovellettoA,GrompeM,GibsonKM.2002.Identificationandinvitroexpressionofmutantsuccinicsemialdehydedehydrogenase(SSADH)alleles.J.Inher.Metab.Dis.24,63.Al‐MudallalAS,LaMannaJC,LustWD,HarikSI.1996.Diet‐inducedketosisdoesnotcausecerebralacidosis.Epilepsia.37,258‐261.AppletonDB,DeVivoDC.1974.Ananimalmodeloftheketogenicdiet.Epilepsia.15,211‐227.Banerjee PK, Snead OC. 1995. Presynaptic γ‐hydroxybutyric acid (GHB) and γ‐aminobutyric acid B (GABAB) receptor‐mediated release of GABA and glutamate(GLU) in rat thalamic ventrobasal nucleus (VB): a possible mechanism for thegeneration of absence‐like seizures induced by GHB. J. Pharmacol. Exp. Ther. 273,1534‐1543.BanerjeePK,OlsenRW,TillakaratneNJ,BrailowskyS,TobinAJ,SneadOC3rd.1998.Absenceseizuresdecreasesteroidmodulationoft‐[35S]butylbicyclophosphorothionatebindinginthalamicrelaynuclei.J.Pharmacol.Exp.Ther.287,766–772.BekkersJM,Stevens,CF.1995.QuantalanalysisofEPSCsrecordedfromsmallnumbersofsynapsesinhippocampalcultures.J.Neurophys.73,1145‐1156.Ben‐MenachemE,SchmitzB,TomsonT,VajdaF.2006.Roleofvalproateacrosstheages.Treatmentofepilepsyinadults.ActaNeurol.Scand.Suppl.184,14‐27.BergqvistAG,CheeCM,LutchkaL,RychikJ,StallingsVA.2003.Seleniumdeficiencyassociatedwithcardiomyopathy:acomplicationoftheketogenicdiet.Epilepsia.44,618‐20.BergqvistAG,SchallJI,GallagherPR,CnaanA,StallingsVA.2005.Fastingversusgradualinitiationoftheketogenicdiet:aprospective,randomizedclinicaltrialofefficacy.Epilepsia.46,1810‐9.

154

BestTH,FranzDN,GilbertDL,NelsonDP,EpsteinMR.2000.Cardiaccomplicationsinpediatricpatientsontheketogenicdiet.Neurology.54,2328‐30.BettlerB,TiaoJY‐H.2006.Moleculardiversity,traffickingandsubcellularlocalizationofGABABreceptors.Pharmacol.Therapeutics.110,533–543.BetseyD,DeMenezesM,StapletonB.1998.Nephrolithiasisassociatedwithketogenicdiettreatment.Epilepsia.39,168.BhattS,RameshwarP,SiegelA.2004.Amygdaloidkindledseizuresinduceweightgainthatreflectslefthemispheredominanceinrats.Physiol.Behav.82,581‐587.BoughKJ,ChenRS,EaglesDA.1999a.Pathanalysisshowsthatincreasingketogenicratio,butnotbeta‐hydroxybutyrate,elevatesseizurethresholdintherat.Dev.Neurosci.21,400‐406.BoughKJ,EaglesDA.2001.Comparisonoftheanticonvulsantefficaciesandneurotoxiceffectsofvalproicacid,phenytoin,andtheketogenicdiet.Epilepsia.42,1345‐1353.BoughKJ,EaglesDA.1999.Aketogenicdietincreasesresistancetopentylenetetrazole‐inducedseizuresintherat.Epilepsia.40,138‐143.BoughKJ,MatthewsPJ,EaglesDA.2000a.Aketogenicdiethasdifferenteffectsuponseizuresinducedbymaximalelectroshockandbypentylenetetrazoleinfusion.EpilepsyRes.38,105‐114.BoughKJ,ValiyilR,HanFT,EaglesD.1999b.Seizureresistanceisdependentuponageandcalorierestrictioninratsfedaketogenicdiet.EpilepsyRes.35,21‐28.BoughKJ,WetheringtonJ,HasselB,PareJF,GawrylukJW,GreeneJG,ShawR,SmithY,GeigerJD,DingledineRJ.2006.Mitochondrialbiogenesisintheanticonvulsantmechanismoftheketogenicdiet.Ann.Neurol.60,223‐35.BoughKJ,YaoSG,EaglesDA.2000b.Higherketogenicratiosconferprotectionfromseizureswithoutneurotoxicity.EpilepsyRes.38,15‐25.BrownGK,CrombyCH,ManningNJ,PollittRJ.1987.Urinaryorganicacidsinsuccinicsemialdehydedehydrogenasedeficiency:evidenceofalpha‐oxidationof4‐hydroxybutyricacid,interactionofsuccinicsemialdehydewithpyruvatedehydrogenaseandpossiblesecondaryinhibitionofmitochondrialbeta‐oxidation.J.Inherit.Metab.Dis.10,367–375.Buzzi A, Wu Y, Frantseva MV, Perez‐Velazquez J‐L, Cortez MA, Liu CC, Shen LQ,Gibson KM, Snead OC. 2006. Succinic semialdehyde dehydrogenase deficiency:GABABreceptormediatedfunction.BrainRes.1090,15‐22.

155

CantelloR,VarrasiC,TarlettiR,CecchinM,D'AndreaF,VeggiottiP,BellomoG,MonacoF.2007.Ketogenicdiet:electrophysiologicaleffectsonthenormalhumancortex.Epilepsia.48,1756‐1763.CarrollJ,KoenigsbergerD.1998.Theketogenicdiet:apracticalguideforcaregivers.J.Am.DietAssoc.98,316‐321.ChamblissKL,HinsonDD,TrettelF,MalaspinaP,NovellettoA,JakobsC,GibsonKM.1998.Twoexon‐skippingmutationsasthemolecularbasisofsuccinicsemialdehydedehydrogenasedeficiency(4‐hydroxybutyricaciduria).Am.J.Hum.Genet.63,399‐408.ChappeV,IrvineT,LiaoJ,EvagelidisA,HanrahanJW.2005.PhosphorylationofCFTRbyPKApromotesbindingoftheregulatorydomain.EMBOJ.24,2730‐2740.ChenL,LeeM,HongJY,HuangW,WangE,YangCS.1994.RelationshipbetweencytochromeP4502E1andacetonecatabolisminratsasstudiedwithdiallylsulfideasaninhibitor.Biochem.Pharmacol.48,2199‐2205.ChenWQ,JinH,NguyenM,CarrJ,LeeYJ,HsuCC,FaimanMD,SchlossJV,WuJY.2001.Roleoftaurineinregulationofintracellularcalciumlevelandneuroprotectivefunctioninculturedneurons.J.Neurosci.Res.66,612‐619.ChesneyD,BrouhardBH,WyllieE,PowaskiK.1999.Biochemicalabnormalitiesoftheketogenicdietinchildren.ClinPediatr(Phila)38,107‐109.ChowdhuryGMI,GuptaM,GibsonKM,PatelAB,BeharKL(inpress).Alteredglucoseandacetatemetabolisminsuccinicsemialdehydedehydrogenase(SSADH)deficientmice: evidence for glial dysfunction and reduced glutamate/glutamine cycling. J.Neurochem.CoppolaG,VeggiottiP,CusmaiR,BertoliS,CardinaliS,Dionisi‐ViciC,EliaM,LispiML,SarnelliC,TagliabueA,ToraldoC,PascottoA.2002.Theketogenicdietinchildren,adolescentsandyoungadultswithrefractoryepilepsy:anItalianmulticentricexperience.EpilepsyRes.48,221‐7.CortezMA,McKerlieC,SneadIIIOC.2001.Amodelofatypicalabsenceseizures:EEG,pharmacologyanddevelopmentalcharacterization.Neurology.56,341–349.CortezMA,WuY,GibsonKM,Snead,OC.2004.Absenceseizuresinsuccinicsemialdehydedehydrogenase‐deficientmice:Amodelofjuvenileabsenceepilepsy.Pharmacol.Biochem.Behav.79,547‐553.CouchSC,SchwarzmanF,CarrollJ,KoenigsbergerD,NordliDR,DeckelbaumRJ,DeFeliceAR. 1999.Growthandnutritionaloutcomesofchildrentreatedwiththeketogenicdiet.J.Am.DietAssoc.99,1573‐5.

156

CunnaneSC,LikhodiiSS.2004.Claimstoidentifydetrimentaleffectsoftheketogenicdiet(KD)oncognitivefunctioninrats.Pediatr.Res.56,663‐664.CunnaneSC,MusaK,RyanMA,WhitingS,FraserDD.2002.Potentialroleofpolyunsaturatesinseizureprotectionachievedwiththeketogenicdiet.ProstaglandinsLeukot.Essent.FattyAcids.67,131‐5.DashtiHM,Bo‐AbbasYY,AsfarSK,MathewTC,HusseinT,BehbahaniA,KhoursheedMA,Al‐SayerHM,Al‐ZaidNS.2003.Ketogenicdietmodifiestheriskfactorsofheartdiseaseinobesepatients.Nutrition.19,901‐2.DekabanAS.1966.Plasmalipidsinepilepticchildrentreatedwiththehighdiet.Arch.Neurol.15,177‐184.DepaulisA,SneadIIIOC,MarescauxC,VergnesM.1989.Suppressiveeffectsofintranigralinjectionofmuscimolinthreemodelsofgeneralizednonconvulsiveepilepsyinducedbychemicalagents.BrainRes.498,64–72.Do‐RegoJL,Mensah‐NyaganGA,BeaujeanD,VaudryD,SieghartW,Luu‐TheV,PelletierG,VaudryH.2000.γ‐Aminobutyricacid,actingthroughgamma‐aminobutyricacidtypeAreceptors,inhibitsthebiosynthesisofneurosteroidsinthefroghypothalamus.Proc.Natl.Acad.Sci.97,13925‐13930.DioguardiFS.2004.Wastingandthesubstrate‐to‐energycontrolledpathway:Aroleforinsulinresistanceandaminoacids.Am.J.Cardiol.93,6A‐12A.Doherty JD, Hattox SE, Snead OC, Roth RH. 1978. Identification of endogenousgamma‐hydroxybutyrateinhumanandbovinebrainanditsregionaldistributioninhuman,guineapigandrhesusmonkeybrain.J.Pharmacol.Exp.Ther.207,130‐139.DymszaHA,CzajkaDM,MillerSA.1964.InfluenceofArtificialDietonWeightGainandBodyCompositionoftheNeonatalRat.J.Nutrition.84,100‐106.EaglesDA,BoydSJ,KotakA,AllanF.2003.Calorierestrictionofahigh‐carbohydratedietelevatesthethresholdofPTZ‐inducedseizurestovaluesequaltothoseseenwithaketogenicdiet.EpilepsyRes.54,41‐52.EdwardsFA.1995.Anatomyandelectrophysiologyoffastcentralsynapsesleadtoastructuralmodelforlong‐termpotentiation.Physiol.Rev.75,759‐787.ErgezingerK,JeschkeR,Frauendienst‐EggerG,KorallH,GibsonKM,SchusterVH..2003.Monitoringof4‐hydroxybutyricacidlevelsinbodyfluidsduringvigabatrintreatmentinsuccinicsemialdehydedehydrogenasedeficiency.Ann.Neurol.54,686‐689.

157

FarasatS,KossoffEH,PillasDJ,RubensteinJE,ViningEP,FreemanJM.2006.Theimportanceofparentalexpectationsofcognitiveimprovementfortheirchildrenwithepilepsypriortostartingtheketogenicdiet.EpilepsyBehav.8,406‐410.FranklinK,PaxinosG.Themousebraininstereotaxiccoordinates.SanDiego,AcademicPress;1997.FraserDD,WhitingS,AndrewRD,MacdonaldEA,Musa‐VelosoK,CunnaneSC.2003.Elevatedpolyunsaturatedfattyacidsinbloodserumobtainedfromchildrenontheketogenicdiet.Neurology.60,1026‐9.FreemanJM,KossoffEH,HartmanAL.2007.Theketogenicdiet:onedecadelater.Pediatrics.119,535‐543.FurthSL,CaseyJC,PyzikPL,NeuAM,DocimoSG,ViningEP,FreemanJM,FivushBA.2000.Riskfactorsforurolithiasisinchildrenontheketogenicdiet.Pediatr.Nephrol.15,125‐128.Garriga‐CanutM,SchoenikeB,QaziR,BergendahlK,DaleyTJ,PfenderRM,MorrisonJF,OckulyJ,StafstromC,SutulaT,RoopraA.2006.2‐Deoxy‐D‐glucosereducesepilepsyprogressionbyNRSF‐CtBP‐dependentmetabolicregulationofchromatinstructure.Nat.Neurosci.9,1382‐7.GasiorM,FrenchA,JoyMT,TangRS,HartmanAL,RogawskiMA.2007.Theanticonvulsantactivityofacetone,themajorketonebodyintheketogenicdiet,isnotdependentonitsmetabolitesacetol,1,2‐propanediol,methylglyoxal,orpyruvicacid.Epilepsia.48,793‐800.GasiorM,RogawskiMA,HartmanAL.2006.Neuroprotectiveanddisease‐modifyingeffectsoftheketogenicdiet.Behav.Pharmacol.17,431‐9.GeinismanY,MorellF,DeToledo‐MorellL.1990.Increaseintherelativeproportionofperforatedaxospinoussynapsesfollowinghippocampalkindlingisspecificforthesynapticfieldofstimulatedaxons.BrainRes.507,325‐331.GibsonKM,ChristensenE,JakobsC,FowlerB,ClarkeMA,HammersenG,RaabK,KoboriJ,MoosaA,VollmerB,RossierE,IafollaKA,MaternD,BrouwerOF,FinkelsteinJ,AksuF,WeberH‐P,BakkerenJAJM,FonsJ,GabreelsM,BluestoneD,BarronTF,BeauvaisP,RabierD,SantosC,UmanskyR,LehnertW.1997.(4‐HydroxybutyricAciduria):CaseReportsof23NewPatients.Pediatrics.99,667‐574.GibsonKM,DeVivoDC,JakobsC.1989.Vigabatrintherapyinapatientwithsuccinicsemialdehydedehydrogenasedeficiency.Lancet.2,1105–1106.

158

GibsonKM,GuptaM,SenephansiriH,JansenEEW,MontineTJ,HylandK,SwitzerRC,SneadOC,JakobsC.2006.Oxidantstressandneurodegenerationinmurinesuccinicsemialdehydedehydrogenase(SSADH)deficiency.In:Hoffmann,G.F.(Ed.),DiseasesofNeurotransmission—FromBenchtoBed.SPSVerlagsgesellschaft,Heilbronn,pp.199–212.GibsonKM,GuptaM,PearlPL,TuchmanM,VezinaLG,SneadOC3rd,SmitLM,JakobsC.2003.Significantbehavioraldisturbancesinsuccinicsemialdehydedehydrogenase(SSADH)deficiency(gamma‐hydroxybutyricaciduria).Biol.Psychiatry.54,763‐8.GibsonKM,GuptaM,SenephansiriH,JansenEEW,MontineTJ,HylandK,SwitzerRC,SneadOC,JakobsC.2004.Oxidantstressandneurodegenerationinmurinesuccinatesemialdehydedehydrogenase(SSADH)deficiency.ProceedingsoftheInternationalSymposium‘‘DiseasesofNeurotransmissionfrombenchtobed’’,Fulda,Germany,17–19November,2005.GibsonKM,JakobsC,OgierH,HagenfeldtL,Eeg‐OlofssonKE,Eeg‐OlofssonO,AksuF,WeberHP,RossierE,VollmerB,etal.1995.Vigabatrintherapyinsixpatientswithsuccinicsemialdehydedehydrogenasedeficiency.J.Inherit.Metab.Dis.18,143–146.GibsonKM,JakobsC.Disordersofbeta‐andgammaaminoacidsinfreeandpeptide‐linkedforms.inThemetabolicandmolecularbasesofinheriteddisease.8thedn(edsScriver,C.R.,Beaudet,A.L.,Sly,W.S.&Valle,D.)2079–2105(McGraw‐Hill,NewYork,2001).GibsonKM,JakobsC,PearlPL,SneadOC.2005.MurineSuccinateSemialdehydeDehydrogenase(SSADH)Deficiency,aHeritableDisorderofGABAMetabolismwithEpilepticPhenotype.IUBMBLife57,639‐644.GibsonKM,SchorDSM,GuptaM,GuerandWS,SenephansiriH,BurlingameTG,BartelsH,HogemaBM,BottiglieriT,FroestlW,SneadOC,GrompeM,andJakobsC.2002.Focalneurotransmitteralterationsinmicedeficientforsuccinatesemialdehydedehydrogenase.J.Neurochem.81,71‐79.GilbertDL,PyzikPL,FreemanJM.2000.Theketogenicdiet:seizurecontrolcorrelatesbetterwithserumbeta‐hydroxybutyratethanwithurineketones.J.ChildNeurol.15,787‐90.GordonN.2004.Succinicsemialdehydedehydrogenasedeficiency(SSADH)(4‐hydroxybutyricaciduria,gamma‐hydroxybutyricaciduria).Eur.J.Paediatr.Neurol.8,261‐5.

159

GreeneAE,TodorovaMT,SeyfriedTN.2003.Perspectivesonthemetabolicmanagementofepilepsythroughdietaryreductionofglucoseandelevationofketonebodies.J.Neurochem.86,529‐37.GropmanA.2003.Vigabatrinandnewerinterventionsinsuccinicsemialdehydedehydrogenasedeficiency.Ann.Neurol.54,S66‐S72.GuptaM,GrevenR,JansenEE,JakobsC,HogemaBM,FroestlW,SneadOC,BartelsH,GrompeM,GibsonKM.2002.Therapeuticinterventioninmicedeficientforsuccinatesemialdehydedehydrogenase(gamma‐hydroxybutyricaciduria).J.Pharmacol.Exp.Ther.302,180‐187.GuptaM,HogemaBM,GrompeM,BottiglieriTG,ConcasA,BiggioG,SoglianoC,RigamontiAE,PearlPL,SneadOC3rd,JakobsC,GibsonKM.2003.Murinesuccinatesemialdehydedehydrogenasedeficiency.Ann.Neurol.54(Suppl6),S81‐90.GuptaM,PolinskyM,SenephansiriH,SneadOC,JansenEE,JakobsC,GibsonKM.2004.Seizureevolutionandaminoacidimbalancesinmurinesuccinatesemialdehydedehydrogenase(SSADH)deficiency.Neurobiol.Dis.16,556‐62.HacesML,Hernández‐FonsecaK,Medina‐CamposON,MontielT,Pedraza‐ChaverriJ,MassieuL.Antioxidantcapacitycontributestoprotectionofketonebodiesagainstoxidativedamageinducedduringhypoglycemicconditions.2008.Exp.Neurol.(epubaheadofprint).HaggardHW,GreenbergLA,TurnerJM.1944.Thephysiologicalprinciplesgoverningtheactionofacetonetogetherwithdeterminationoftoxicity.J.Ind.Hyg.Toxicol.26,133‐151.HallböökT,KöhlerS,RosénI,LundgrenJ.2007.Effectsofketogenicdietonepileptiformactivityinchildrenwiththerapyresistantepilepsy.EpilepsyRes.77,134‐140.HaranoY,OhtsukiM,IdaM,KojimaH,HaradaM,OkanishiT,KashiwagiA,OchiY,UnoS,ShigetaY.1985.Directautomatedassaymethodforserumorurinelevelsofketonebodies.Clin.Chim.Acta.151,177–183.HarneyJP,MadaraJ,MadaraJ,I'AnsonH.2002.Effectsofacuteinhibitionoffattyacidoxidationonlatencytoseizureandconcentrationsofbetahydroxybutyrateinplasmaofratsmaintainedoncalorierestrictionand/ortheketogenicdiet.EpilepsyRes.49,239‐246.HartmanKN,PalSK,BurroneJ,MurthyVN.2006.Activity‐dependentregulationofinhibitorysynaptictransmissioninhippocampalneurons.Nat.Neurosci.9,642‐9.

160

HartmanAL,GasiorM,ViningEP,RogawskiMA.2007.Theneuropharmacologyoftheketogenicdiet.Pediatr.Neurol.36,281‐92.HartmanAL,ViningEP.2007.Clinicalaspectsoftheketogenicdiet.Epilepsia.48,31‐42.HasselB,JohannessenCU,SonnewaldU,FonnumF.1998.QuantificationoftheGABAshuntandtheimportanceoftheGABAshuntversusthe2‐oxoglutaratedehydrogenasepathwayinGABAergicneurons.J.Neurochem.71,1511–1518.HeinemannU,BuchheimK,GabrielS,KannO,KovacsR,SchuchmannS.2002.Celldeathandmetabolicactivityduringepileptiformdischargesandstatusepilepticusinthehippocampus.Prog.BrainRes.135,197‐210.HelmholtzHF,KeithHM.1930.Eightyears’experiencewiththeketogenicdietinthetreatmentofepilepsy.JAMA.95,707‐709.HenschTK,FagioliniM,MatagaN,StrykerMP,BaekkeskovS,KashSF. 1998.LocalGABAcircuitcontrolofexperience‐dependentplasticityindevelopingvisualcortex.Science.282,1504‐8.HirschJC,AgassandianC,Merchan‐PerezA,Ben‐AriY,DeFelipeJ,EsclapezM,BernardC.1999.DeficitofquantalreleaseofGABAinexperimentalmodelsoftemporallobeepilepsy.Nat.Neurosci.2,499‐500.HogemaBM,GuptaM,SenephansiriH,BurlingameTG,TaylorM,JakobsC,SchutgensRB,FroestlW,SneadOC,Diaz‐ArrastiaR,BottiglieriT,GrompeM,GibsonKM.2001.Pharmacologicrescueoflethalseizuresinmicedeficientinsuccinatesemialdehydedehydrogenase.Nat.Genet.29,212–216.HoriA,TandonP,HolmesGL,StafstromCE.1997.Ketogenicdiet:effectsonexpressionofkindledseizuresandbehaviorinadultrats.Epilepsia.38,750‐758.HowellsDW,JakobsC,KokR.1992.Vigabatrintherapyinsuccinicsemialdehydedehydrogenasedeficiency.Mol.Neuropharmacol.2,181–184.HuttenlocherPR.1976.Ketonemiaandseizures:metabolicandanticonvulsanteffectsoftwoketogenicdietsinchildhoodepilepsy.Pediat.Res.10,536‐540.HuxtableR,LairdH.1978.Theprolongedanticonvulsantactionoftaurineongeneticallydeterminedseizure‐susceptibility.Can.J.Neurol.Sci.5,215‐21.JaekenJ,CasaerP,deCockP,FrancoisB.1989.VigabatrininGABAmetabolismdisorders.Lancet.1,1074.JacobTC,MossSJ,JurdR.2008.GABA(A)receptortraffickinganditsroleinthedynamicmodulationofneuronalinhibition.Nat.Rev.Neurosci.9,331‐343.

161

JakobsC,JaekenJ,GibsonKM.1993.InheriteddisordersofGABAmetabolism.J.Inherit.Metab.Dis.16,704‐15.JakobsC,MichaelT,JaegerE,JaekenJ,GibsonKM.1992.FurtherevaluationofVigabatrintherapyin4‐hydroxybutyricaciduria.Eur.J.Pediatr.151,466–468.JansenEE,VerhoevenNM,JakobsC,SchulzeA,SenephansiriH,GuptaM,SneadOC,GibsonKM.2006.Increasedguanidinospeciesinmurineandhumansuccinatesemialdehydedehydrogenase(SSADH)deficiency.Biochim.Biophys.Acta.1762,494‐8.JarrettSG,MilderJB,LiangLP,PatelM.2008.Theketogenicdietincreasesmitochondrialglutathionelevels.J.Neurochem.(e‐pubaheadofprint).JiaoY,NadlerJV.2007.StereologicalanalysisofGluR2‐immunoreactivehilarneuronsinthepilocarpinemodeloftemporallobeepilepsy:correlationofcelllosswithmossyfibersprouting.Exp.Neurol.205,569‐82.KangHC,KimYJ,KimDW,KimHD.2005.Efficacyandsafetyoftheketogenicdietforintractablechildhoodepilepsy:Koreanmulticentricexperience.Epilepsia.46,272‐9.KatyalNG,KoehlerAN,McGheeB,FoleyCM,CrumrinePK.2000.Theketogenicdietinrefractoryepilepsy:theexperienceofChildren’sHospitalofPittsburgh.Clin.Pediatr.(Phila).39,153‐159.KilmanV,vanRossumMC,TurrigianoGG.2002.ActivitydeprivationreducesminiatureIPSCamplitudebydecreasingthenumberofpostsynapticGABA(A)receptorsclusteredatneocorticalsynapses.J.Neurosci.22,1328‐37.KimdoY,RhoJM.2008.Theketogenicdietandepilepsy.Curr.Opin.Clin.Nutr.Metab.Care.11,113‐120.KnerrI,PearlPL,BottiglieriT,SneadOC,JakobsC,GibsonKM. 2007.Therapeuticconceptsinsuccinatesemialdehydedehydrogenase(SSADH;ALDH5A1)deficiency(gamma‐hydroxybutyricaciduria).Hypothesesevolvedfrom25yearsofpatientevaluation,studiesinAldh5a1‐/‐miceandcharacterizationofgamma‐hydroxybutyricacidpharmacology.J.Inherit.Metab.Dis.30,279‐94.KontroP,LindenIB,GothoniG,OjaSS.1983.Novelanticonvulsanttaurinederivatives.Prog.Clin.Biol.Res.125,211‐20.KossoffEH.2004.Morefatandfewerseizures:dietarytherapiesforepilepsy.LancetNeurol.3,415‐420.KossoffEH,KraussGL,McGroganJR,FreemanJM.2003.EfficacyoftheAtkinsdietastherapyforintractableepilepsy.Neurology.61,1789‐1791.

162

KossoffEH,McGroganJR,BlumlRM,PillasDJ,RubensteinJE,ViningEP.2006.AmodifiedAtkinsdietiseffectiveforthetreatmentofintractablepediatricepilepsy.Epilepsia.47,421‐424.KossoffEH,PyzikPL,FurthSL,HladkyHD,FreemanJM,ViningEP.2002.Kidneystones,carbonicanhydraseinhibitors,andtheketogenicdiet.Epilepsia.15,125‐128.KossoffEH,RowleyH,SinhaSR,ViningEPG.2008.AprospectivestudyofthemodifiedAtkinsdietforintractableepilepsyinadults.Epilepsia.49,316‐319.KossoffEH,TurnerZ,BlumlRM,PyzikPL,ViningEP.2007.Arandomized,crossovercomparisonofdailycarbohydratelimitsusingthemodifiedAtkinsdiet.EpilepsyBehav.10,432‐436.KwiterovichPOJr,ViningEP,PyzikP,SkolaskyRJr,FreemanJM.2003.Effectofahigh‐fatketogenicdietonplasmalevelsoflipids,lipoproteins,andapolipoproteinsinchildren.JAMA.290,912‐920.LagougeM,ArgmannC,Gerhart‐HinesZ,MezianeH,LerinC,DaussinF,MessadeqN,MilneJ,LambertP,ElliottP,GenyB,LaaksoM,PuigserverP,AuwerxJ.2006.ResveratrolimprovesmitochondrialfunctionandprotectsagainstmetabolicdiseasebyactivatingSIRT1andPGC‐1alpha.Cell.127,1109‐1122.LefevreF,AronsonN.2000.Ketogenicdietforthetreatmentofrefractoryepilepsyinchildren:Asystematicreviewofefficacy.Pediatrics.105,E46.LianXY,KhanFA,StringerJL.2007.Fructose‐1,6‐bisphosphatehasanticonvulsantactivityinmodelsofacuteseizuresinadultrats.J.Neurosci.27,12007‐12011.LikhodiiSS,MusaK,MendoncaA,DellC,BurnhamWM,CunnaneSC.2000.Dietaryfat,ketosis,andseizureresistanceinratsontheketogenicdiet.Epilepsia.41,1400‐1410.LikhodiiSS.2001.Experimentsintheratpentylenetetrazoleinfusionthresholdmodeloftheketogenicdiet.EpilepsyRes.44,83‐86.LikhodiiSS,BurnhamWM.2004.Theeffectsofketonebodiesonneuronalexcitability.In:EpilepsyandtheKetogenicDiet(Eds.StafstromCE,RhoJM).HumanaPress.LikhodiiSS,BurnhamWM.2003.CalorierestrictionmaynotelevatethethresholdofPTZ‐inducedseizures.EpilepsyRes.57,77‐78.LikhodiiSS,BurnhamWM.2002a.Ketogenicdiet:Doesacetonestopseizures?Med.Sci.Monit.8,HY19‐24.

163

LikhodiiSS,BurnhamWM.2002b.Ontheanticonvulsanteffectofacetoneandtheketogenicdiet.Epilepsia.43,1596.LikhodiiSS,MusaK,CunnaneSC.2002.Breathacetoneasameasureofsystemicketosisassessedinaratmodeloftheketogenicdiet.Clin.Chem.48,115‐120.LikhodiiSS,MusaK,MendoncaA,DellC,BurnhamWM,CunnaneSC.2000.Dietaryfat,ketosis,andseizureresistanceinratsontheketogenicdiet.Epilepsia.41,1400‐1410.LikhodiiSS,SerbanescuI,CortezMA,MurphyP,SneadIIIOC,BurnhamWM.2003.Anticonvulsantpropertiesofacetone,abrainketoneelevatedbytheketogenicdiet.Ann.Neurol.54,219‐226.LingenhoehlK,BromR,HeidJ,BeckP,FroestlW,KaupmannK,BettlerB,MosbacherJ.1999.Gamma‐hydroxybutyrateisaweakagonistatrecombinantGABA(B)receptors.Neuropharmacology38,1667‐1673.LiuYM,WilliamsS,Basualdo‐HammondC,StephensD,CurtisR.2003.Aprospectivestudy:growthandnutritionalstatusofchildrentreatedwiththeketogenicdiet.J.AM.DietAssoc.103,707‐712.LiuL,RapoportSI,ChandrasekaranK.1999.RegulationofMitochondrialGeneExpressioninDifferentiatedPC12Cells.Ann.N.Y.Acad.Sci.893,341–344.LiuYM,WilliamsS,Basualdo‐HammondC,StephensD,CurtisR.2003.Aprospectivestudy:growthandnutritionalstatusofchildrentreatedwiththeketogenicdiet.J.Am.DietAssoc.103,707‐12.LivingstonS.1977.Ketogenicdietinthetreatmentofchildhoodepilepsy.Dev.Med.ChildNeurol.19,833‐834.LonsdaleD,BurnhamWM.2003.Theanticonvulsanteffectsofprogesteroneand5alpha‐dihydroprogesteroneonamygdala‐kindledseizuresinrats.Epilepsia.44,1494‐1499.LonsdaleD,NylenK,McIntyreBurnhamW.Theanticonvulsanteffectsofprogesteroneanditsmetabolitesonamygdala‐kindledseizuresinmalerats.BrainRes.1101,110‐116.LonsdaleD,BurnhamWM.2007.Theanticonvulsanteffectsofallopregnanoloneagainstamygdala‐kindledseizuresinfemalerats.Neurosci.Lett.411‐147‐151.LoscherW,BrandtC,EbertU.2003.Excessiveweightgaininratsoverextendedkindlingofthebasolateralamygdala.Neuroreport.14,1829‐1832.

164

LoscherW,FiedlerM.1996.Theroleoftechnical,biologicalandpharmacologicalfactorsinthelaboratoryevaluationofanticonvulsantdrugs:VISeasonalinfluencesonmaximalelectroshockandpentylenetetrazoleseizurethresholds.EpilepsyRes.25,3‐10.LoscherW,HonackD,HashemA.1987.Anticonvulsantefficacyofclonazepamandtheβ‐carbolineZK93423duringchronictreatmentinamygdala‐kindledrats.Eur.J.Pharmacol.143,403–414.MaaloufM,RhoJM.OxidativeImpairmentofHippocampalLong‐termPotentiationInvolvesActivationofProteinPhosphatase2AandIsPreventedbyKetoneBodies.JournalNeuroscienceResearch(inpress).MaaloufM,SullivanPG,DavisL,KimDY,RhoJM.2007.KetonesinhibitmitochondrialproductionofreactiveoxygenspeciesproductionfollowingglutamateexcitotoxicitybyincreasingNADHoxidation.Neuroscience.145,256‐64.MaW,BergJ,YellenG.2007.KetogenicdietmetabolitesreducefiringincentralneuronsbyopeningK(ATP)channels.JNeurosci.27,3618‐25.MakSC,ChiCS,WanCJ.1999.Clinicalexperienceofketogenicdietonchildrenwithrefractoryepilepsy.Acta.Paediatr.Taiwan.40,97‐100.MasinoSA,GockelSA,WasserCA,PomeroyLT,WagenerJF,GawrylukJW,GeigerJD.TherelationshipamongATP,adenosineandaketogenicdiet.ProgramNo.595.12/X25.2007NeuroscienceMeetingPlanner.SanDiego,CA:SocietyforNeuroscience,2007.Online.MassieuL,HacesML,MontielT,Hernandez‐FonsecaK.2003.Acetoacetateprotectshippocampalneuronsagainstglutamate‐mediatedneuronaldamageduringglycolysisinhibition.Neuroscience.120,365‐78.MaternD,LehnertW,GibsonKM,KorinthenbergR.1996.Seizuresinaboywithsuccinicsemialdehydedehydrogenasedeficiencytreatedwithvigabatrin(gamma‐vinyl‐GABA).JInheritMetabDis.19,313‐318.McDonaldME.1997.Useoftheketogenicdietintreatingchildrenwithseizures.Pediatr.Nurs.23,461‐4.MoroMA,AlmeidaA,BolanosJP,LizasoainI.2005.Mitochondrialrespiratorychainandfreeradicalgenerationinstroke.FreeRadicBiolMed.39,1291‐1304.MurphyP,LikhodiiS,NylenK,BurnhamWM.2004.Theantidepressantpropertiesoftheketogenicdiet.Biol.Psychiatry.56,981‐983.

165

Musa‐VelosoK,LikhodiiSS,CunnaneSC.2002.Breathacetoneisareliableindicatorofketosisinadultsconsumingketogenicmeals.Am.J.Clin.Nutr.76,65‐70.Musa‐VelosoK.2004.Non‐invasivedetectionofketosisanditsapplicationinrefractoryepilepsy.ProstaglandinsLeukot.Essent.FattyAcids.70,329‐335.NehligA.2004.Brainuptakeandmetabolismofketonebodiesinanimalmodels.ProstaglandinsLeukot.Essent.FattyAcids.70,265‐275.NazarewiczRR,ZiolkowskiW,VaccaroPS,GhafourifarP.2007.Effectofshort‐termketogenicdietonredoxstatusofhumanblood.RejuvenationRes.10,435‐440.NevesaRH,deBarrosAC,AlencaraM,Costa‐SilvaaM,ÁguilabMB,de‐LacerdabCAM,Machado‐SilvaaJR,GomescDC.2006.Long‐termfeedingahigh‐fatdietcauseshistologicalandparasitologicaleffectsonmurineschistosomiasismansonioutcome.Exp.Parasitol.115,324‐332.NordliDR.2002.Theketogenicdiet:usesandabuses.Neurology.58,S21‐S24.NohHS,KimYS,LeeHP,ChungKM,KimDW,KangSS,ChoGJ,ChoiWS.2003.Theprotectiveeffectofaketogenicdietonkainicacid‐inducedhippocampalcelldeathinthemaleICRmice.EpilepsyRes.53,119‐28.NohHS,KimDW,KangSS,ChoGJ,ChoiWS.2005a.KetogenicdietpreventsclusterinaccumulationinducedbykainicacidinthehippocampusofmaleICRmice.BrainRes.1042,114‐118.NohHS,KangSS,KimDW,KimYH,ParkCH,HanJY,ChoGJ,ChoiWS.2005b.Ketogenicdietincreasescalbindin‐D28kinthehippocampiofmaleICRmicewithkainicacidseizures.EpilepsyRes.65,153‐159. NohHS,KimDW,KangSS,KimYH,ChoGJ,ChoiWS.2006.KetogenicdietdecreasesthelevelofproenkephalinmRNAinducedbykainicacidinthemousehippocampus.Neurosci.Lett.395,87‐92.NordliDR,DeVivoD.1997.Theketogenicdietrevisited:backtothefuture.Epilepsia.38,743‐749.NusserZ,Cull‐CandyS,FarrantM.1997.DifferencesinsynapticGABAAreceptornumberunderlievariationinGABAminiamplitude.Neuron.19,697‐709.NusserZH,AjosN,SomogyiP,ModyI.1998.IncreasednumberofsynapticGABAAreceptorsunderliespotentiationathippocampalinhibitorysynapses.Nature.395,172‐177.

166

NylenK,LikhodiiSS,AbdelmalikPA,BurnhamWM.2005.Acomparisonoftheabilityofa4:1ketogenicdietanda6.3:1ketogenicdiettoelevateseizurethresholdsinadultandyoungrats.Epilepsia.46,1198‐1204.Nylen K, Likhodii SS, Hum KM, BurnhamWM. 2006. A ketogenic diet and diallylsulfidedonotelevateafterdischarge thresholds inadultkindledrats.EpilepsyRes.71,23‐31.NylenK,VelazquezJL,LikhodiiSS,CortezMA,ShenL,LeshchenkoY,AdeliK,GibsonKM,BurnhamWM,SneadOC3rd.2007.Aketogenicdietrescuesthemurinesuccinicsemialdehydedehydrogenasedeficientphenotype.Exp.Neurol.210,449‐457.Okun JG,SauerS, tenBrinkHJ, JakobsC,HoffmannGF,GibsonKM,andKoelkerS.2004. SSADH deficiency: 4,5‐Dihydroxyhexanoic acid inhibits ubiquinone‐dependentrespiratorychainsI–III.J.Inher.Metab.Dis.27(Suppl.1),87.PapandreouD,PavlouE,KalimeriE,MavromichalisI.2006.Theketogenicdietinchildrenwithepilepsy.Br.J.Nutr.95,5‐13.PatelAB,RothmanDL,ClineGW,BeharKL.2001.GlutamineisthemajorprecursorforGABAsynthesisinratneocortexinvivofollowingacuteGABA‐transaminaseinhibition.BrainRes.919,207‐220.PearlPL,GibsonKM,AcostaMT,VezinaLG,TheodoreWH,RogawskiMA,NovotnyEJ,GropmanA,ConryJA,BerryGT,TuchmanM.2003.Clinicalspectrumofsuccinicsemialdehydedehydrogenasedeficiency.Neurology.60,1413–7.PerezVelazquezJL,CarlenPL.2000.Gapjunctions,synchronyandseizures.TrendsNeurosci.23,68‐74.PfeiferHH,ThieleEA.2005.Low‐glycemic‐indextreatment:aliberalizedketogenicdietfortreatmentofintractableepilepsy.Neurology.65,1810‐1812.PinelJP,SkeltonR,MuchaRF.1976.Kindling‐relatedchangesinafterdischargethresholds.Epilepsia.17,197‐206.PrasadAN,StafstromCF,HolmesGL.1996.Alternativeepilepsytherapies:theketogenicdiet,immunoglobulins,andsteroids.Epilepsia.37(Suppl1),S81‐S95.PriceTD,RittenbergD.1950.Themetabolismofacetone:I.Grossaspectsofcatabolismandexcretion.J.Biol.Chem.185,449‐459.PulsiferMB,GordonJM,BrandtJ,ViningEP,FreemanJM.2001.EffectsofketogenicdietondevelopmentandbehaviorPreliminaryreportofaprospectivestudy.Dev.Med.ChildNeurol.43,301–306.

167

RabinovitzS,MostofskyDI,YehudaS.2004.Anticonvulsantefficiency,behavioralperformanceandcortisollevels:acomparisonofcarbamazepine(CBZ)andafattyacidcompound(SR‐3).Psychoneuroendocrinology.29,113‐124.RacineRJ.1972.Modificationofseizureactivitybyelectricalstimulation,II:motorseizure.Electroencephalogr.Clin.Neurophysiol.32,281‐294.RaffoE,FrançoisJ,FerrandonA,KoningE,NehligA.2007.Calorie‐restrictedketogenicdietincreasesthresholdstoallpatternsofpentylenetetrazol‐inducedseizures:Criticalimportanceofelectroclinicalassessment.Epilepsia.49,320‐328.RhoJM,KimDW,RobbinsCA,AndersonGD,SchwartzkroinPA.1999.Age‐dependentdifferencesinflurothylseizuresensitivityinmicetreatedwithaketogenicdiet.EpilepsyRes.37,233‐240.RhoJM,AndersonGD,DonevanSD,WhiteHS.2002.Acetoacetate,acetone,anddibenzylamine(acontaminantinl‐(+)‐beta‐hydroxybutyrate)exhibitdirectanticonvulsantactionsinvivo.Epilepsia.43,358‐61.RicquierD,BouillaudF.2000.Mitochondrialuncouplingproteins:frommitochondriatotheregulationofenergybalance.J.Physiol.15,3‐10.RumignyJF,CashC,MandelP,VincendonG,MaitrenM.1981.Evidencethataspecificsuccinicsemialdehydereductaseisresponsibleforgamma‐hydroxybutyratesynthesisinbraintissueslices.FEBSLett.134,96‐98.SaitoeM,SchwarzTL,UmbachJA,GundersonCB,KidokoroY.2001.TINS.25,385‐387.SantraS,GilkersonRW,DavidsonM,SchonEA.2004.KetogenictreatmentreducesdeletedmitochondrialDNAsinculturedhumancells.Ann.Neurol.56,662‐669.SauerSW,KölkerS,HoffmannGF,TenBrinkHJ,JakobsC,GibsonKM,OkunJG.2007.Enzymaticandmetabolicevidenceforaregionspecificmitochondrialdysfunctioninbrainsofmurinesuccinicsemialdehydedehydrogenasedeficiency(Aldh5a1‐/‐mice).NeurochemInt.50,653‐659.SchutzmaniJ,JarrettSG,LiangLP,PatelM.Upregulationofglutathionebiosynthesisanddecreasedmitochondrialoxidativestressinratsfedtheketogenicdiet.ProgramNo.595.14/X27.2007NeuroscienceMeetingPlanner.SanDiego,CA:SocietyforNeuroscience,2007.Online.SchwartzRM,BoyesS,Aynsley‐GreenA.1989.Metaboliceffectsofthreeketogenicdietsinthetreatmentofsevereepilepsy.Dev.Med.ChildNeurol.31,152‐160.

168

SchwartzkroinPA.1999.Mechanismsunderlyingtheanti‐epilepticefficacyoftheketogenicdiet.EpilepsyRes.37‐171‐180.ScriverCR,GibsonKM.Disordersofb‐andg‐aminoacidsinfreeandpeptide‐linkedforms.In:ScriverCR,BeaudetAL,SlyWS,ValleD,eds.TheMetabolicandMolecularBasisofInheritedDisease.7thed.NewYork,NY:McGraw‐Hill;1995:1349–1368.SeoJH,LeeYM,LeeJS,KangHC,KimHD.2007.Efficacyandtolerabilityoftheketogenicdietaccordingtolipid:nonlipidratios‐‐comparisonof3:1with4:1diet.Epilepsia.48,801‐5.SeymourKJ,BlumlS,SutheringJ,SutherlingW,RossBD.1999.Identificationofcerebralacetoneby1H‐MRSinpatientswithepilepsycontrolledbyketogenicdiet.MAGMA.8,33‐42.ShaoLR,DudekFE.2005.InputtoDentateGranuleCellsandPossibleCompensatoryResponses.J.Neurophysiol.94,952‐960.SilvermanJI,StoneDW,PowersJD.1992.Thelipidcompositionofmilkfrommicefedhighorlowfatdiets.Lab.Animals.26,127‐131.SirvenJ,WhedonB,CaplanD,LiporaceJ,GlosserD,O'DwyerJ,SperlingMR.1999.Theketogenicdietforintractableepilepsyinadults:preliminaryresults.Epilepsia.40,1721‐1726.Snead OC. 1996. Presynaptic GABAB‐ and g‐hydroxybutyric acid mediatedmechanismsingeneralizedabsenceseizures.Neuropharmacology.35,259‐367.Snead OC 3rd.1999. The gamma‐hydroxybutyrate model of absence seizures:correlation of regional brain levels of gamma‐hydroxybutyric acid and gamma‐butyrolactonewithspikewavedischarges.Neuropharmacology.30,161‐167.SneadOC3rd, GibsonKM. 2005. Gamma‐hydroxybutyric acid.N. Engl. J.Med. 352,2721‐2732.SomersDC,NelsonSB,SurM.Anemergentmodeloforientationselectivityincatvisualcorticalsimplecells.J.Neurosci.15,5448‐65.SpiegelmanBM,FlierJS.2001.Obesityandtheregulationofenergybalance.Cell.104,531–543.StruysEA,JansenEEW,GibsonKM,JakobsC.2005.DeterminationoftheGABAanaloguesuccinicsemialdehydeinurineandcerebrospinalfluidbydinitrophenylhydrazinederivatizationandliquidchromatography–tandemmassspectrometry:ApplicationtoSSADHdeficiency.J.Inherit.Metab.Dis.28,913–920.

169

SullivanPG,RippyNA,DorenbosK,ConcepcionRC,AgarwalAK,RhoJM.2004.Theketogenicdietincreasesmitochondrialuncouplingproteinlevelsandactivity.Ann.Neurol.55,576‐80.SwanwickCC,MurthyNR,MtchedlishviliZ,SieghartW,KapurJ.2006.Developmentofgamma‐aminobutyricacidergicsynapsesinculturedhippocampalneurons.J.Comp.Neurol.495,497‐510.SwinkTD,ViningEP,FreemanJM.1997.Theketogenicdiet:1997.Adv.Pediatr.44,297‐329.TahaAY,RyanMA,CunnaneSC.2005.Despitetransientketosis,theclassichigh‐fatketogenicdietinducesmarkedchangesinfattyacidmetabolisminrats.Metabolism.54,1127‐1132.ThavendiranathanP,MendoncaA,DellC,LikhodiiSS,MusaK,IracleousC,CunnaneSC,BurnhamWM.2000.TheMCTketogenicdiet:effectsonanimalseizuremodels.Exp.Neurol.161,696‐703.ThavendiranathanP,ChowC,CunnaneS,BurnhamWM.2003.Theeffectofthe‘classic’ketogenicdietonanimalseizuremodels.BrainRes.959,206‐213.ThieleE.2003.Assessingtheefficacyofantiepileptictreatments:Theketogenicdiet.Epilepsia.44,26‐69.Thompson,J.A.,VanOrman,C.B.,Peterson,P.,1998.Ketogenicdiet:long‐termefficacyandpatienttoleranceinthetreatmentofintractablepediatricepilepsy.Epilepsia.39,167.TsacopoulosM,undMagistrettiPJ.1996.Metaboliccouplingbetweengliaandneurons.J.Neurosci.16,877–885.TsuchidaTN,BarkovichAJ,BollenAW,HartAP,FerrieroDM.2007.Childhoodstatusepilepticusandexcitotoxicneuronalinjury.Pediatr.Neurol.36,253‐7.UhlemannER,NeimsAH.1972.Anticonvulsantpropertiesoftheketogenicdietinmice.J.Pharmacol.Exp.Ther.180,231‐238.UlrichD,BettlerB.2007.GABABreceptors:synapticfunctionsandmechanismsofdiversity.Curr.Opin.Neurobiol.17,298–303.UzielG,BardelliP,PantaleoniC,RimoldiM,SavoiardoM.1993.4‐Hydroxybutyricaciduria:clinicalfindingsandVigabatrintherapy.J.Inher.Metab.Dis.16,520–522.

170

VanderAuweraI,WeraS,VanLeuvenF,HendersonST.2005.Aketogenicdietreducesamyloidbeta40and42inamousemodelofAlzheimer'sdisease.Nutr.Metab.(Lond).17,2‐28.VeechRL.2004.Thetherapeuticimplicationsofketonebodies:theeffectsofketonebodiesinpathologicalconditions:ketosis,ketogenicdiet,redoxstates,insulinresistance,andmitochondrialmetabolism.ProstaglandinsLeukot.Essent.FattyAcids.70,309‐19.VeechRL,ChanceB,KashiwayaY,LardyHA,CahillGFJr.2001.Ketonebodies,potentialtherapeuticuses.IUBMBLife.51,241–247.VerrottiA,MancoR,MatricardiS,FranzoniE,ChiarelliF.2007.Antiepilepticdrugsandvisualfunction.PediatrNeurol.36,353‐360.VerstrekenP,BellenHJ.2002.Meaninglessminis?Mechanismsofneurotransmitter‐receptorclustering.TrendsNeurosci.25,383‐5.ViningE.1999.Clinicalefficacyoftheketogenicdiet.EpilepsyRes.37,181‐190.ViningEP,PyzikP,McGroganJ,HladkyH,AnandA,KrieglerS,FreemanJM.2002.Growthofchildrenontheketogenicdiet.Dev.Med.ChildNeurol.44,796‐802.WallaceDC.2005.Amitochondrialparadigmofmetabolicanddegenerativediseases,aging,andcancer:adawnforevolutionarymedicine.Annu.Rev.Genet.39,359‐407.WetheringtonJP,LambertNA.2002.GABA(B)receptoractivationdesensitizespostsynapticGABA(B)andA(1)adenosineresponsesinrathippocampalneurones.J.Physiol.544,459‐467.WierengaCJ,WadmanWJ.1999.MiniatureinhibitorypostsynapticcurrentsinCA1pyramidalneuronsafterkindlingepileptogenesis.J.Neurophysiol.82,1352‐62.WilderRM.1921.Theeffectofketonemiaonthecourseofepilepsy.MayoClinicBulletin.2,307.WilliamsS,Basualdo‐HammondC,CurtisR,SchullerR.2002.Growthretardationinchildrenwithepilepsyontheketogenicdiet:aretrospectivechartreview.J.Am.DietAssoc.102,405‐7.WongCG,ChanKF,GibsonKM,SneadOC.2004.Gamma‐hydroxybutyricacid:neurobiologyandtoxicologyofarecreationaldrug.Toxicol.Rev.23,3‐20.

171

WuY,AliS,AhmadianG,LiuCC,WangYT,GibsonKM,CalverAR,FrancisJ,PangalosMN, Snead OC 3rd. 2004. Gamma‐hydroxybutyric acid (GHB) and gamma‐aminobutyric acidB receptor (GABABR) binding sites are distinctive from oneanother:molecularevidence.Neuropharmacology.47,1146‐1156.WuY,BuzziA,FrantsevaM,VelazquezJP,CortezM,LiuC,ShenL,GibsonKM,SneadOC3rd.2006.Statusepilepticusinmicedeficientforsuccinatesemialdehydedehydrogenase:GABAAreceptor‐mediatedmechanisms.Ann.Neurol.59,42‐52.ZieglerDR,AraújoE,RottaLN,PerryML,GonçalvesCA.AKetogenicDietIncreasesProteinPhosphorylationinBrainSlicesofRats.2002.J.Nutrit.132,483‐487.ZieglerDR,RibeiroLC,HagennM,SiqueiraIR,AraujoE,TorresIL,GottfriedC,NettoCA,GoncalvesCA.2003.Ketogenicdietincreasesglutathioneperoxidaseactivityinrathippocampus.Neurochem.Res.28,1793‐1797.ZhaoZ,LangeDJ,VoustianioukA,MacGroganD,HoL,SuhJ,HumalaN,ThiyagarajanM,WangJ,PasinettiGM.2006.Aketogenicdietasapotentialnoveltherapeuticinterventioninamyotrophiclateralsclerosis.BMCNeurosci.3,7‐29.ZhaoQ,StafstromCE,FuDD,HuY,HolmesGL.2004.Detrimentaleffectsoftheketogenicdietoncognitivefunctioninrats.Pediatr.Res.55,498‐506.

172

LISTOFPUBLICATIONSANDABSTRACTS

PublishedPapers(12).

StewartLS,NylenK,CortezMA,GibsonKM,SneadOC3rd.Circadiandistributionofgeneralizedtonic‐clonicseizuresassociatedwithmurinesuccinicsemialdehydedehydrogenasedeficiency,adisorderofGABAmetabolism.EpilepsyandBehav.(inpress).NylenK,PerezVelazquezJL,LikhodiiSS,CortezMA,ShenL,LeshchenkoY,AdeliK,GibsonKM,BurnhamWM,SneadOC3rd.2007.Aketogenicdietrescuesthemurinesuccinicsemialdehydedehydrogenasedeficientphenotype.Exp.Neurol.210,449‐457.TahaAY,BaghiaB,LuiR,NylenK,MaD,BurnhamWM.2006.Lackofbenefitoflinoleicandalpha‐linolenicpolyunsaturatedfattyacidsonseizurelatency,duration,severityorincidenceinrats.EpilepsyRes.71,40‐46.LonsdaleD,NylenK,BurnhamWM.2006.Theanticonvulsanteffectsofprogesteroneanditsmetabolitesonamygdalakindledseizuresinmalerats.BrainRes.1101,110‐116.NylenK,LikhodiiSS,HumK,BurnhamWM.2006.Aketogenicdietanddiallylsulphidedonotelevateafterdischargethresholdsinadultkindledrats.EpilepsyRes.71,23‐31.NylenK,LikhodiiSS,BurnhamWM.2006.Theutilityoftestingpentylenetetrazolethreshold.Epilepsia.47,663‐664.NylenK,LikhodiiSS,AbdelmalikPA,ClarkeJ,BurnhamWM.2005.Acomparisonoftheabilityofa4:1ketogenicdietanda6.3:1ketogenicdiettoelevateseizurethresholdsinadultandyoungrats.Epilepsia.46,1198‐1204.MurphyP,LikhodiiSS,NylenK,BurnhamWM.2004.Moodstabilizingpropertiesoftheketogenicdiet.Biol.Psychiatry.56,981‐983.SheerinAH,NylenK,ZhangX,SaucierDM,CorcoranME.2004.Furtherevidenceforaroleoftheclaustruminepileptogenesis.Neuroscience.125,57‐62.EliasL,SaucierD,NylenK,CheesemanJ.2003.Anexaminationofthefemaleadvantageinspeededcolournaming:Isitduetoaspecialnamingfactororsuperiormotorsequencing?PerceptualandMotorSkills.96,955‐961.

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SaucierD,NylenK,EliasL.2002.Arecoloursspecial?Anexaminationofthefemaleadvantageforspeededcolournaming.PersonalityandIndividualDifferences.32,27‐35.Chlan‐FourneyJ,AsheP,NylenK,JuorioAV,LiX.2002.DifferentialregulationofhippocampalBDNFmRNAbyacuteandchronicantipsychoticadministration.BrainRes.954,11‐20.PublishedAbstracts(10).

LikhodiiSS,NylenK,BurnhamWM.2008.Theanticonvulsantactionsofactetone.Epilepsia(inpress).1stInternationalSymposiumontheDietaryTreatmentofEpilepsyandOtherNeurologicalDisorders(Phoenix,AZ).NylenK,LikhodiiSS,PerezVelasquezJL,BurnhamWM,GibsonKM,SneadOCIII.2007.Theketogenicdietrescuesthelethalphenotypeandrestoressynapticactivityinsuccinicsemialdehydedehydrogenasedeficientmice.AmericanEpilepsySocietyAnnualMeeting.(Philadelphia,PA).NylenK,LikhodiiSS,PerezVelasquezJL,BurnhamWM,GibsonKM,SneadOCIII.2007.Theketogenicdietrescuesthelethalphenotypeandrestoressynapticactivityinsuccinicsemialdehydedehydrogenasedeficientmice.SocietyforNeuroscience.(SocietyforNeuroscienceMeeting,SanDiego,CA)NylenK,LikhodiiSS,PerezVelasquezJL,BurnhamWM,GibsonKM,SneadOCIII.2007.Theketogenicdietrescuesthelethalphenotypeandrestoressynapticactivityinsuccinicsemialdehydedehydrogenasedeficientmice.Clin.Neurophysiol.118,e187.(EasternAssociationofElectroencephalographers,NewYork,NewYork,U.S.A.)NylenK,HumK,LikhodiiSS,BurnhamWM.2006.Aketogenicdietanddiallylsulphidedonotelevateafterdischargethresholds.Clin.Neurophysiol.117,e18.(EasternAssociationofElectroencephalographers,NewYork,NewYork,U.S.A.)LonsdaleD,NylenK,BurnhamWM.2006.Theanticonvulsanteffectsofprogesteroneanditsmetabolitesonamygdalakindledseizuresinmalerats.Clin.Neurophysiol.117,e17.(EasternAssociationofElectroencephalographers,NewYork,NewYork,U.S.A.)NylenK,HumK,LikhodiiSS,BurnhamWM.2005.Effectofelevatedblood‐acetonelevelsonseizurethresholdinkindledrats:implicationsfortheroleofacetoneintheanticonvulsantmechanismoftheketogenicdiet.Epilepsia.46,61.(InternationalConferenceonEpilepsy,Paris,France).

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NylenK,LikhodiiSS,BurnhamWM.2005.Acetoneandtheanticonvulsantmechanismoftheketogenicdiet.Clin.Neurophysiol.116,e15‐16.(EasternAssociationofElectroencephalographers,NewYork,NewYork,U.S.A.)NylenK,LikhodiiSS,AbdelmalikP,ClarkeJ,BurnhamWM.2003.Istheketogenicdietanticonvulsantinallanimalspecies?Clin.Neurophysiol.115,2427.(EasternAssociationofElectroencephalographers,NewYork,NewYork,U.S.A.)SheerinAH,NylenK,ZhangX,SaucierD,CorcoranME.2001.DynamicregulationofKCC2inresponsetokindling.SocietyforNeuroscience.(SocietyforNeuroscienceMeeting,SanDiego,CA)UnpublishedConferenceAbstracts(6).

NylenK,LikhodiiSS,PerezVelasquezJL,BurnhamWM,GibsonKM,SneadOCIII.2007.Theketogenicdietrescuesthelethalphenotypeandrestoressynapticactivityinsuccinicsemialdehydedehydrogenasedeficientmice.UniversityofTorontoEpilepsyResearchProgram(UTERP)AnnualScienceDay,UniversityofToronto,Toronto,Ontario,Canada.NylenK,LikhodiiSS,PerezVelasquezJL,BurnhamWM,GibsonKM,SneadOCIII.2007.Theketogenicdietrescuesthelethalphenotypeandrestoressynapticactivityinsuccinicsemialdehydedehydrogenasedeficientmice.VisionsinPharmacologyAnnualConference,UniversityofToronto,Toronto,Ontario,Canada.NylenK,LikhodiiSS,PerezVelasquezJL,BurnhamWM,GibsonKM,SneadOCIII.2007.Theketogenicdietrescuesthelethalphenotypeandrestoressynapticactivityinsuccinicsemialdehydedehydrogenasedeficientmice.CuringEpilepsy2007:NationalInstitutesofHealth(NIH),Bethesda,Maryland,U.S.A.NylenK.2006.UsingDiettoTreatSeizureDisorders:ImplicationsforAutonomy.PrinciplesofAutonomousNeurodynamicsConference,Eilat,Israel.NylenK,LikhodiiSS.2004. TheKetogenicDietandEpilepsy:ProbingtheMechanismsofAction.PrinciplesofAutonomousNeurodynamicsConference,UniversityofToronto,Toronto,Ontario,Canada.NylenK,LikhodiiSS,HumK,BurnhamWM.2004.Effectofelevatedblood‐acetonelevelsonseizurethresholdinkindledrats:implicationsfortheroleofacetoneintheanticonvulsantmechanismoftheketogenicdiet.VisionsinPharmacologyAnnualConference,UniversityofToronto,Toronto,Ontario,Canada.

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GraduateAwards

Award Amount Held

CIHRDoctoralResearchAward $21,000+$1000 2008–current

VanGelder‐SavoyAward $12,000+$1000 2007‐2008

SickKidsFoundationRestracomp

$19,000 2006(full)2007(partialwithSavoy)

MargaretandHowardGambleOSOTF

$20,000 2005

UofTOpenFellowships $3000‐$5000perannum

2003‐2007