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Methods in Molecular Biology 2561 Jerold Chun Editor Alzheimer’s Disease Methods and Protocols M M B ETHODS IN OLECULAR IO LO GY SeriesEditor JohnM.Walker School of Lifeand MedicalSciences University ofHertfordshire Hatfield, Hertfordshire, UK Forfurther volumes: http://www.springer.com/series/7651 For over 35 years, biological scientists have come to rely on the research protocols and methodologiesinthecriticallyacclaimedMethodsinMolecularBiologyseries.Theserieswas thefirsttointroducethestep-by-stepprotocolsapproachthathasbecomethestandardinall biomedicalprotocolpublishing.Eachprotocolisprovidedinreadily-reproduciblestep-by- step fashion, opening with an introductory overview, a list of the materials and reagents neededtocompletetheexperiment,andfollowedbyadetailedprocedurethatissupported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitutethekeyingredientineachandeveryvolumeoftheMethodsinMolecularBiology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexedinPubMed. Alzheimer’s Disease Methods and Protocols Edited by Jerold Chun Translational Neuroscience Initiative, Neuroscience Drug Discovery, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA Editor JeroldChun TranslationalNeuroscienceInitiative NeuroscienceDrugDiscovery SanfordBurnhamPrebysMedicalDiscoveryInstitute LaJolla,CA,USA ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-0716-2654-2 ISBN978-1-0716-2655-9 (eBook) https://doi.org/10.1007/978-1-0716-2655-9 ©TheEditor(s)(ifapplicable)andTheAuthor(s),underexclusivelicensetoSpringerScience+BusinessMedia,LLC,part ofSpringerNature2023 Thisworkissubjecttocopyright.AllrightsaresolelyandexclusivelylicensedbythePublisher,whetherthewholeorpart of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting,reproductionon microfilmsorinanyotherphysicalway,andtransmissionorinformation storageand retrieval,electronicadaptation, computersoftware,orbysimilar ordissimilar methodologynow knownorhereafter developed. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnotimply, evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsandregulations andthereforefreeforgeneraluse. Thepublisher,theauthors,andtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedto betrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty, expressedorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeen made.Thepublisherremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations. CoverCaption:VolumeimagingofneurovascularalterationsintheAlzheimer’sdiseasebrain.Fibrinogendeposits(red) aroundareasofvasculartortuosity(green)andAβplaques(blue).ImageCredit:MarioMerliniandKaterinaAkassoglou. ThisHumanaimprintispublishedbytheregisteredcompanySpringerScience+BusinessMedia,LLC,partofSpringer Nature. Theregisteredcompanyaddressis:1NewYorkPlaza,NewYork,NY10004,U.S.A. Preface Alzheimer’sdisease(AD)isthemostcommoncauseofdementiaworldwidewithenormous medicalandsocietalcoststhatimpactbothpatientsandtheirfamilies.Atthetimeofwriting, AD and related dementias remain without effective disease-modifying therapies, despite intensive research spanning well over a century, since the initial description of AD by Alzheimer and Fischer. The neuropathological identification of what is now recognized as senile plaques, neurofibrillary tangles, and neuronal loss came from careful examination of the human diseased brain itself, as did the identification of amyloid-beta (Aβ) by Glenner and Wong some eight decades later. Indeed, over the last decade, studies on the human brainhaveidentifiedcommoncomorbidbraindiseasesdistinctfromAD,conservedpatterns of lesions, neuroanatomical disease progression, and other key features, underscoring the essential importance of analyzing the actual diseased brain, with thoughtful use of experi- mentalsystemslikehumancellular models. ThefoundationalimportanceofhumanbrainstudiesforunderstandingADandrelated neurodegenerative disorders stimulated this collection of modern methodologies. Experts from around the globe have come together to share their expertise through 16 chapters, dividedinto5parts,forstudyingthediseasedhumanbrain:(I)BrainPreparations(through specialized imaging in, and single-cell isolation from, post-mortem brains); (II) Neural Cellular Models (using primary and human induced pluripotent stem cells); (III) Nucleic AcidAnalyses(transcriptomicandsomaticgenomicchanges);(IV)LipidAnalyses(viamass spectrometry);and(V)ProteinAnalyses(particularlyexaminingAβandTau,includingtheir prionforms). Detailedstep-by-step protocolsdesigned for clarityhavebeen supplemented withexplanatoryfootnotesandgeneroususeofcomplementaryfigures. Sincerethanksareduetomanywhoenabledthisbooktomaterialize.Foremostarethe brain donors and their families who selflessly and generously donated brains for scientific study. The availability of these precious samples has been facilitated by brain banks and disease-related organizations, both public and private, and we thank them here as well as specifically within the book’s chapters. Thanks go to all of the authors who contributed to the effort during the Covid-19 pandemic: hailing from 12 countries beyond the United States,thismeantnavigatinganunpredictableandconstantlychanginglandscapeofhealth andregulatorychallengesduringthecreationofthisvolumeofMethodsinMolecularBiology. With sadness, we respectfully offer our condolences to the family of author Professor SvetlanaG.Vorsanova(Chapter10)whowastragicallylosttoCovid-19.Dr.LauraWolszon wastheformalassociateeditorofthisbook,beinginstrumentalinitsassemblyandcomple- tion.FurtherthanksgototheexpertandpatienteditorialstaffatSpringer,whoenabledthe book’s completion: John Walker, Patrick Marton, and Anna Rakovsky. We gratefully acknowledge the many funding agencies around the world, particularly the National Insti- tutes of Health and the National Institutes on Aging, for supporting the germane science. Last and by no means least, we thank past and current members of the Chun laboratory, particularlyDr.GwendolynKaeserwhohelpedtogetthisprojectoffthegroundalongwith Dr.LauraWolszon,whotookonassociateeditor responsibilities. v vi Preface Wehopethatallreadersmaybenefitfromthisbook,towardsexpandingknowledgeand helping patients and their families through new understanding, new treatments, and, one day,curingAlzheimer’sdiseaseandrelateddementias. LaJolla,CA,USA JeroldChun Contents Preface ..................................................................... v Contributors................................................................. ix PART I HUMAN BRAIN PREPARATIONS 1 Protocolfor theSystematicFixation,Circuit-BasedSampling, andQualitativeandQuantitativeNeuropathologicalAnalysis ofHumanBrainTissue..... ........ ....... ....... ........ ....... ........ 3 CaitlinS.Latimer,EricaJ.Melief,JeanelleAriza-Torres,KimHoward, AmandaR.Keen,LisaM.Keene,AimeeM.Schantz,TrevorM.Sytsma, AngelaM.Wilson,ThomasJ.Grabowski,MartinDarvas, KristenDamsO’Connor,AmberL.Nolan,BrianL.Edlow, ChristineL.MacDonald,andC.DirkKeene 2 ExtractionandPurificationofSingleNucleifromFrozen HumanBrainTissue ....... ........ ....... ....... ........ ....... ........ 31 CarterR.PalmerandJeroldChun 3 IsolationofHumanMicrogliafromNeuropathologicallyDiagnosed CasesintheSingle-CellEra ......... .... ... ...... .... ..... ....... ........ 43 Lih-FenLue,DouglasG.Walker,SuetThengBeh,andThomasG.Beach 4 MicrogliainHumanPostmortemBrainSamples:Quantitative UltrastructuralAnalysisofScanningElectronMicroscopyImages..... ........ 63 Marie-KimSt-Pierre,EvaSˇimoncˇicˇova´, MicaëlCarrier,andMarie-E`veTremblay 5 Three-DimensionalImagingofFibrinogenandNeurovascular AlterationsinAlzheimer’sDisease.... ....... ....... ........ ....... ........ 87 MarioMerlini,ElifG.Sozmen,KeshavS.Subramanian, AlissaL.Nana,WilliamW.Seeley,andKaterinaAkassoglou PART II HUMAN NEURAL CELLULAR MODELS 6 Human-InducedPluripotentStemCell(hiPSC)-DerivedNeurons andGliafor theElucidationofPathogenicMechanisms inAlzheimer’sDisease...... ........ ....... ...... ..... .... ....... ........ 105 JessicaE.YoungandLawrenceS.B.Goldstein 7 ModelingAlzheimer’sDiseaseUsingHumanBrainOrganoids....... ........ 135 KarinaKarmirian,MarianaHolubiec,LiviaGoto-Silva, IvanFernandezBessone,GabrielaVit(cid:1)oria,BeatrizMello, MatiasAlloatti,BartVanderborght,Toma´sL.Falzone,andStevensRehen vii viii Contents 8 Three-DimensionalBiohybridStarPEG–HeparinHydrogelCultures forModelingHumanNeuronalDevelopmentandAlzheimer’s DiseasePathology.......... ........ ....... ....... ........ ....... ........ 159 TohidSiddiqui,HilalCelikkaya,ZeynepTansuAtasavum, StanislavaPopova,UweFreudenberg,CarstenWerner,andCaghanKizil PART III NUCLEIC ACID ANALYSES OF HUMAN BRAIN 9 ComputationalApproachestoAssessAbnormalMetabolism inAlzheimer’sDiseaseUsingTranscriptomics....... ........ ....... ........ 173 HaticeBu¨s¸raLu¨leci,DilaraUzuner,TunahanÇakır, andMadhavThambisetty 10 FISHingforChromosomeInstabilityandAneuploidyintheAlzheimer’s DiseaseBrain....... ....... ........ ....... .. ..... ...... .. ..... .. ........ 191 YuriB.Yurov,SvetlanaG.Vorsanova,andIvanY.Iourov 11 SomaticCNVDetectionbySingle-CellWhole-GenomeSequencing inPostmortemHumanBrain ....... ....... ....... ........ ....... ........ 205 DiegoPerez-Rodriguez,MariaKalyva,CatherineSantucci, andChristosProukakis PART IV LIPIDS ANALYSES OF HUMAN BRAIN 12 MassSpectrometryAnalysisoftheHumanBrainSphingolipidome ........... 233 XinYingChua,RyanHuang,DeronHerr, MitchellK.P.Lai,MarkusR.Wenk,andFedericoTorta 13 MassSpectrometryfor theAdvancementofLipidAnalysis inAlzheimer’sResearch .... ........ ....... ....... ....... ....... .... ..... 245 JonatanMartıńez-Gardeazabal,MartaMoreno-Rodrı´guez, Estı´balizGonza´lezdeSanRomań,BeatrizAbad,IvańManuel, andRafaelRodrı´guez-Puertas PART V PROTEIN ANALYSES OF HUMAN BRAIN 14 UseofAffinityPurification–MassSpectrometrytoIdentify PhosphorylatedTauInteractorsinAlzheimer’sDisease....... ....... ........ 263 Geoffrey Pires,BeatrixUeberheide,ThomasWisniewski, andEleanorDrummond 15 InVitroAmplificationofPathogenicTauSeedsfromNeurodegenerative DiseasePatientBrains ...... ........ ....... ....... ........ ....... ........ 279 HongXuandVirginiaM.-Y.Lee 16 AβandTauPrionsCausingAlzheimer’sDisease..... ........ ....... ..... .. . 293 CarloCondello,GregoryE.Merz,AtsushiAoyagi, WilliamF.DeGrado,andStanleyB.Prusiner Index ...................................................................... 339 Contributors BEATRIZABAD • FacultyofScienceandTechnology,CentralAnalysisService,Universityofthe BasqueCountry(UPV/EHU),Leioa,Spain KATERINAAKASSOGLOU • GladstoneUCSFCenter forNeurovascularBrainImmunology, SanFrancisco,CA,USA;GladstoneInstituteofNeurologicalDisease,SanFrancisco,CA, USA;DepartmentofNeurology,WeillInstituteofNeurosciences,UniversityofCalifornia, SanFrancisco,CA,USA MATIASALLOATTI • InstitutodeBiologıáCelularyNeurocienciaIBCN(UBA-CONICET), FacultaddeMedicina,UniversidaddeBuenosAires,BuenosAires,Argentina ATSUSHIAOYAGI • InstituteforNeurodegenerativeDiseases,WeillInstituteforNeurosciences, UniversityofCaliforniaSanFrancisco,SanFrancisco,CA,USA;DaiichiSankyoCo., Ltd.,Tokyo,Japan JEANELLEARIZA-TORRES • UniversityofWashington,DepartmentofLaboratoryMedicine andPathology,Seattle,WA,USA ZEYNEPTANSUATASAVUM • GermanCenter forNeurodegenerativeDiseases(DZNE) Dresden,HelmholtzAssociation,Dresden,Germany;Leibniz-Institutfu¨r PolymerforschungDresdene.V.,MaxBergmannCenterofBiomaterialsDresden,Dresden, Germany THOMASG.BEACH • CivinNeuropathologyLaboratory,BannerSunHealthResearch Institute,SunCity,AZ,USA SUET THENGBEH • HumanCellCoreforTranslationalResearch,BannerSunHealth ResearchInstitute,SunCity,AZ,USA TUNAHANÇAKIR • DepartmentofBioengineering,GebzeTechnicalUniversity,Gebze, Kocaeli,Turkey MICAE¨LCARRIER • Axeneurosciences,CentrederechercheduCHUdeQue´bec-Universite´ Laval,Que´bec,QC,Canada;De´partementdeme´decinemolećulaire,Faculte´deme´decine, Universite´Laval,Que´bec,QC,Canada;DivisionofMedicalSciences,Universityof Victoria,Victoria,BC,Canada HILALCELIKKAYA • GermanCenter forNeurodegenerativeDiseases(DZNE)Dresden, HelmholtzAssociation,Dresden,Germany XINYINGCHUA • DepartmentofPharmacology,YongLooLinSchoolofMedicine,National UniversityofSingapore,Singapore,Singapore;MemoryAgingandCognitionCentre, DepartmentofPsychologicalMedicine,YongLooLinSchoolofMedicine,National UniversityofSingapore,Singapore,Singapore JEROLDCHUN • TranslationalNeuroscienceInitiative,NeuroscienceDrugDiscovery, SanfordBurnhamPrebysMedicalDiscoveryInstitute,LaJolla,CA,USA CARLOCONDELLO • InstituteforNeurodegenerativeDiseases,WeillInstitutefor Neurosciences,UniversityofCaliforniaSanFrancisco,SanFrancisco,CA,USA; DepartmentofNeurology,WeillInstituteforNeurosciences,UniversityofCaliforniaSan Francisco,SanFrancisco,CA,USA MARTINDARVAS • UniversityofWashington,DepartmentofLaboratoryMedicineand Pathology,Seattle,WA,USA ESTI´BALIZGONZA´LEZDESANROMAŃ • DepartmentofPharmacology,FacultyofMedicine andNursing,UniversityoftheBasqueCountry(UPV/EHU),Leioa,Spain ix