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
57
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.
74
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.
84
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
90
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.
95
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
100
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
106
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.
113
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).
122
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).
129
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.
131
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.
132
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.
134
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
135
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.
139
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
140
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
143
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.
144
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
145
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
146
(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
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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