Alpha-Synuclein: Methods and Protocols

292 Pages·2019·8.25 MB·English

by  Tim Bartels

Methods in
Molecular Biology 1948
Tim Bartels Editor
Alpha-
Synuclein
Methods and Protocols
M M B
ETHODS IN OLECULAR IO LO GY
SeriesEditor
JohnM.Walker
School of Lifeand MedicalSciences
University ofHertfordshire
Hatfield, Hertfordshire,AL109AB,UK
Forfurther volumes:
http://www.springer.com/series/7651
Alpha-Synuclein
Methods and Protocols
Edited by
Tim Bartels
Dementia Research Institute, University College London, London, UK
Editor
TimBartels
DementiaResearchInstitute
UniversityCollegeLondon
London,UK
ISSN1064-3745 ISSN1940-6029 (electronic)
MethodsinMolecularBiology
ISBN978-1-4939-9123-5 ISBN978-1-4939-9124-2 (eBook)
https://doi.org/10.1007/978-1-4939-9124-2
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Preface
This book is a lab manual targeted at researchers working on topics related to alpha-
synuclein.Thissmallproteinistheobjectofintenseinvestigationinaplethoraoffieldsfor
thelasttwodecades.Thereasonliesinitsstillpuzzlingbiochemicalpropertiesandhardto
definephysiologicalfunction.
In biophysics, its properties as an “intrinsically disordered protein” have led to many
interesting results in the field of protein folding, while its amyloidogenic aggregation has
shedlightonproteinoligomerization.Inbiochemistry,itsspecificlipid-bindingcapabilities
and multiple proposed interactors have been broadly studied. In molecular biology, its
transient interaction with biological membranes and impact on vesicle trafficking have led
toimportantinsightsincellularfunction,whileitsmisfoldinghasshedlightonanumberof
mechanisms important for cell survival. Last but not least, major efforts to understand its
physiological role have stemmed from biomedicine. Alpha-synuclein seems to be a central
point of pathogenesis in Parkinson’s disease, Lewy body dementia, and multiple system
atrophy,diseasesthereforecommonlygroupedas“synucleinopathies.”
Hence,thisbookismeanttosupplythereaderwithdetailedprotocolsthatuseavariety
ofinvitroandinvivotechniquesandassaysthatareapplicableinreductionistsystemsaswell
asinhumansamples.Themethodstrytoencompassabroadspectrumoffunctionalaspects
of alpha-synuclein biology, from lipid interaction and misfolding to functional deficits in
cells and mice. While obviously not being able to cover all techniques used in this ever-
expandingfield,theexpertsthathavewrittenthechaptersandIhopetoenablethereaderto
get started in the most common methods if interested in a scientific question related to
alpha-synucleinbiology.
London,UK TimBartels
v
Contents
Preface ..................................................................... v
Contributors................................................................. ix
1 AssaysforNeuronalDefectsCausedbyEarlyFormationofα-Synuclein
InclusionsinPrimaryCulturedNeurons................................... 1
LauraA.Volpicelli-Daley
2 ImmunohistochemicalDetectionofAlpha-SynucleininUnfixed
HumanBrainTissue .................................................... 15
JohnB.Sanderson
3 KineticMeasurementsofEndocytosisandExocytosisinCultured
Neurons ............................................................... 23
KarinaJ.Vargas
4 DetectionofMisfoldedα-SynucleinAggregatesinCerebrospinal
FluidbytheProteinMisfoldingCyclicAmplificationPlatform................ 35
LuisConcha-Marambio,MohammadShahnawaz,andClaudioSoto
5 StereotaxicTargetingofAlpha-SynucleinPathologyinMouseBrain
UsingPreformedFibrils ................................................. 45
BinZhang,VictoriaKehm,RonGathagan,SusanN.Leight,
JohnQ.Trojanowski,VirginiaM.-Y.Lee,andKelvinC.Luk
6 QuantificationofAlpha-SynucleininBiologicalFluids
byElectrochemiluminescence-BasedDetection............................. 59
NielsKruseandBritMollenhauer
7 Alpha-SynucleinProximityLigationAssay(AS-PLA)inBrainSections
toProbeforAlpha-SynucleinOligomers................................... 69
RosalindF.Roberts,NoraBengoa-Vergniory,
andJavierAlegre-Abarrategui
8 Studyingα-SynucleinConformationbyIntact-CellCross-Linking ............ 77
ThibautImberdis,SarannaFanning,AndrewNewman,
NagendranRamalingam,andUlfDettmer
9 C.elegansasaModelforSynucleinopathiesandOther
NeurodegenerativeDiseases:ToolsandTechniques......................... 93
MandyKoopman,Rene´eI.Seinstra,andEllenA.A.Nollen
10 RecombinantExpressionandPurificationofN-Acetylated
Alpha-Synuclein ........................................................ 113
TaraA.EastwoodandDanielP.Mulvihill
11 CircularDichroismandIsothermalTitrationCalorimetry
toStudytheInteractionofα-SynucleinwithMembranes .................... 123
MatteoRovere
12 Yeast-BasedScreenstoTargetAlpha-SynucleinToxicity ..................... 145
IneˆsCaldeiraBra´s,BlagovestaPopova,GerhardH.Braus,
andTiagoF.Outeiro
vii
viii Contents
13 ProbingStructuralChangesinAlpha-SynucleinbyNuclear
MagneticResonanceSpectroscopy........................................ 157
TapojyotiDasandDavidEliezer
14 MeasuringMitochondrialDysfunctionCausedbySoluble
α-SynucleinOligomers .................................................. 183
EricS.LuthandIrinaG.Stavrovskaya
15 Monitoringα-SynucleinProteotoxicityinDrosophilaModels................. 199
AronSzaboandGeorgeK.Tofaris
16 Detectionofα-SynucleininBiologicalSamplesUsingMass
Spectrometry........................................................... 209
AnnBrinkmalm,AnnikaO¨hrfelt,PayelBhattacharjee,
andHenrikZetterberg
17 Single-MoleculeFRETDetectionofEarly-StageConformations
inα-SynucleinAggregation .............................................. 221
MahdiMuhammadMoosa,JosephineC.Ferreon,
andAllanChrisM.Ferreon
18 InSituPeroxidaseLabelingandMassSpectrometryofAlpha-Synuclein
inRatCorticalNeurons ................................................. 235
RicardoSanz,PatrickOvando-Roche,NamrataD.Udeshi,
StevenA.Carr,CheeYeunChung,andVikramKhurana
19 ProbingAlpha-SynucleinConformationsbyElectronParamagnetic
Resonance(EPR)Spectroscopy........................................... 247
JuliaCattani,TheresaBraun,andMalteDrescher
20 ExpressionandPurificationofUntaggedα-Synuclein ....................... 261
AlexE.PowersandDushyantS.Patel
21 GenerationandAnalysisofViralVector-MediatedRodentModels
forParkinson’sDisease .................................................. 271
AkuaA.Karikari,JamesB.Koprich,andChiWangIp
Index ...................................................................... 287
Contributors
JAVIERALEGRE-ABARRATEGUI (cid:1) DivisionofBrainSciences,FacultyofMedicine,Imperial
CollegeLondon,London,UK
NORABENGOA-VERGNIORY (cid:1) DepartmentofPhysiology,AnatomyandGenetics,Oxford
Parkinson’sDiseaseCentre,UniversityofOxford,Oxford,UK
PAYELBHATTACHARJEE (cid:1) DepartmentofPsychiatryandNeurochemistry,Instituteof
NeuroscienceandPhysiology,TheSahlgrenskaAcademyattheUniversityofGothenburg,
Mo¨lndal,Sweden
INEˆSCALDEIRABRA´S (cid:1) DepartmentofExperimentalNeurodegeneration,Center for
BiostructuralImagingofNeurodegeneration,UniversityMedicalCenterGoettingen,
Goettingen,Germany
THERESABRAUN (cid:1) DepartmentofChemistryandKonstanzResearchSchoolChemicalBiology,
UniversityofKonstanz,Konstanz,Germany
GERHARDH.BRAUS (cid:1) DepartmentofMolecularMicrobiologyandGenetics,Instituteof
MicrobiologyandGenetics,Georg-August-Universita€tGo¨ttingen,Go¨ttingen,Germany;
Center forNanoscaleMicroscopyandMolecularPhysiologyoftheBrain,Goettingen,
Germany
ANNBRINKMALM (cid:1) DepartmentofPsychiatryandNeurochemistry,InstituteofNeuroscience
andPhysiology,TheSahlgrenskaAcademyattheUniversityofGothenburg,Mo¨lndal,
Sweden
STEVENA.CARR (cid:1) BroadInstituteofMITandHarvard,Cambridge,MA,USA
JULIACATTANI (cid:1) DepartmentofChemistryandKonstanzResearchSchoolChemicalBiology,
UniversityofKonstanz,Konstanz,Germany
CHEEYEUNCHUNG (cid:1) YumanityTherapeutics,Cambridge,MA,USA
LUIS CONCHA-MARAMBIO (cid:1) AmprionInc.,SanDiego,CA,USA
TAPOJYOTIDAS (cid:1) WeillCornellMedicine,NewYork,NY,USA
ULFDETTMER (cid:1) AnnRomneyCenter forNeurologicDiseases,BrighamandWomen’s
HospitalandHarvardMedicalSchool,Boston,MA,USA
MALTEDRESCHER (cid:1) DepartmentofChemistryandKonstanzResearchSchoolChemical
Biology,UniversityofKonstanz,Konstanz,Germany
TARA A.EASTWOOD (cid:1) MolecularBiology,SchoolofBiosciences,UniversityofKent,
Canterbury,Kent,UK
DAVIDELIEZER (cid:1) WeillCornellMedicine,NewYork,NY,USA
SARANNAFANNING (cid:1) AnnRomneyCenter forNeurologicDiseases,BrighamandWomen’s
HospitalandHarvardMedicalSchool,Boston,MA,USA
ALLANCHRISM.FERREON (cid:1) DepartmentofPharmacologyandChemicalBiology,Baylor
CollegeofMedicine,Houston,TX,USA
JOSEPHINEC.FERREON (cid:1) DepartmentofPharmacologyandChemicalBiology,BaylorCollege
ofMedicine,Houston,TX,USA
RONGATHAGAN (cid:1) Center forNeurodegenerativeDiseaseResearch,InstituteonAging,
DepartmentofPathologyandLaboratoryMedicine,UniversityofPennsylvaniaPerelman
SchoolofMedicine,Philadelphia,PA,USA
THIBAUT IMBERDIS (cid:1) AnnRomneyCenter forNeurologicDiseases,BrighamandWomen’s
HospitalandHarvardMedicalSchool,Boston,MA,USA
ix
x Contributors
CHI WANGIP (cid:1) DepartmentofNeurology,UniversityHospitalofWu¨rzburg,Wu¨rzburg,
Germany
AKUAA.KARIKARI (cid:1) DepartmentofNeurology,UniversityHospitalofWu¨rzburg,Wu¨rzburg,
Germany
VICTORIA KEHM (cid:1) Center forNeurodegenerativeDiseaseResearch,InstituteonAging,
DepartmentofPathologyandLaboratoryMedicine,UniversityofPennsylvaniaPerelman
SchoolofMedicine,Philadelphia,PA,USA
VIKRAMKHURANA (cid:1) AnnRomneyCenter forNeurologicDisease,DepartmentofNeurology,
BrighamandWomen’sHospitalandHarvardMedicalSchool,Boston,MA,USA;Broad
InstituteofMITandHarvard,Cambridge,MA,USA;HarvardStemCellInstitute,
Cambridge,MA,USA
MANDYKOOPMAN (cid:1) UniversityofGroningen,UniversityMedicalCenterGroningen,
EuropeanResearchInstitutefor theBiologyofAging,Groningen,TheNetherlands
JAMESB.KOPRICH (cid:1) UniversityHealthNetwork,KrembilResearchInstitute,TorontoWestern
Hospital,Toronto,ON,Canada
NIELSKRUSE (cid:1) InstituteofNeuropathology,UniversityMedicalCenterGo¨ttingen,Go¨ttingen,
Germany
VIRGINIAM.-Y.LEE (cid:1) Center forNeurodegenerativeDiseaseResearch,InstituteonAging,
DepartmentofPathologyandLaboratoryMedicine,UniversityofPennsylvaniaPerelman
SchoolofMedicine,Philadelphia,PA,USA
SUSAN N.LEIGHT (cid:1) Center forNeurodegenerativeDiseaseResearch,InstituteonAging,
DepartmentofPathologyandLaboratoryMedicine,UniversityofPennsylvaniaPerelman
SchoolofMedicine,Philadelphia,PA,USA
KELVINC.LUK (cid:1) Center forNeurodegenerativeDiseaseResearch,InstituteonAging,
DepartmentofPathologyandLaboratoryMedicine,UniversityofPennsylvaniaPerelman
SchoolofMedicine,Philadelphia,PA,USA
ERICS.LUTH (cid:1) DepartmentofBiology,SimmonsUniversity,Boston,MA,USA
BRITMOLLENHAUER (cid:1) Paracelsus-Elena-Klinik,Kassel,Germany;DepartmentofNeurology,
UniversityMedicalCenterGo¨ttingen,Go¨ttingen,Germany
MAHDIMUHAMMADMOOSA (cid:1) DepartmentofPharmacologyandChemicalBiology,Baylor
CollegeofMedicine,Houston,TX,USA
DANIELP.MULVIHILL (cid:1) MolecularBiology,SchoolofBiosciences,UniversityofKent,
Canterbury,Kent,UK
ANDREWNEWMAN (cid:1) AnnRomneyCenter forNeurologicDiseases,BrighamandWomen’s
HospitalandHarvardMedicalSchool,Boston,MA,USA
ELLENA.A.NOLLEN (cid:1) UniversityofGroningen,UniversityMedicalCenterGroningen,
EuropeanResearchInstitutefor theBiologyofAging,Groningen,TheNetherlands
ANNIKAO¨HRFELT (cid:1) DepartmentofPsychiatryandNeurochemistry,InstituteofNeuroscience
andPhysiology,TheSahlgrenskaAcademyattheUniversityofGothenburg,Mo¨lndal,
Sweden
TIAGOF.OUTEIRO (cid:1) DepartmentofExperimentalNeurodegeneration,Center for
BiostructuralImagingofNeurodegeneration,UniversityMedicalCenterGoettingen,
Goettingen,Germany;Center forNanoscaleMicroscopyandMolecularPhysiologyofthe
Brain,Goettingen,Germany;MaxPlanckInstituteforExperimentalMedicine,
Goettingen,Germany
PATRICKOVANDO-ROCHE (cid:1) AnnRomneyCenter forNeurologicDisease,Departmentof
Neurology,BrighamandWomen’sHospitalandHarvardMedicalSchool,Boston,MA,
USA
Contributors xi
DUSHYANTS.PATEL (cid:1) AnnRomneyCenter forNeurologicDiseases,BrighamandWomen’s
Hospital,HarvardMedicalSchool,Boston,MA,USA
BLAGOVESTAPOPOVA (cid:1) DepartmentofMolecularMicrobiologyandGenetics,Instituteof
MicrobiologyandGenetics,Georg-August-Universita€tGo¨ttingen,Go¨ttingen,Germany;
Center forNanoscaleMicroscopyandMolecularPhysiologyoftheBrain,Goettingen,
Germany
ALEXE.POWERS (cid:1) AnnRomneyCenter forNeurologicDiseases,BrighamandWomen’s
Hospital,HarvardMedicalSchool,Boston,MA,USA
NAGENDRANRAMALINGAM (cid:1) AnnRomneyCenter forNeurologicDiseases,Brighamand
Women’sHospitalandHarvardMedicalSchool,Boston,MA,USA
ROSALINDF.ROBERTS (cid:1) MontrealNeurologicalInstitute,McGillUniversity,Montre´al,QC,
Canada
MATTEOROVERE (cid:1) AnnRomneyCenter forNeurologicDiseases,BrighamandWomen’s
HospitalandHarvardMedicalSchool,Boston,MA,USA
JOHNB.SANDERSON (cid:1) TheWarrenAlpertMedicalSchoolofBrownUniversity,Providence,
RI,USA
RICARDO SANZ (cid:1) AnnRomneyCenter forNeurologicDisease,DepartmentofNeurology,
BrighamandWomen’sHospitalandHarvardMedicalSchool,Boston,MA,USA
RENE´EI.SEINSTRA (cid:1) UniversityofGroningen,UniversityMedicalCenterGroningen,
EuropeanResearchInstitutefor theBiologyofAging,Groningen,TheNetherlands
MOHAMMADSHAHNAWAZ (cid:1) MitchellCenter forAlzheimer’sDiseaseandRelatedBrain
Disorders,DepartmentofNeurology,UniversityofTexasHoustonMedicalSchool,Houston,
TX,USA
CLAUDIOSOTO (cid:1) AmprionInc.,SanDiego,CA,USA;MitchellCenter forAlzheimer’s
DiseaseandRelatedBrainDisorders,DepartmentofNeurology,UniversityofTexas
HoustonMedicalSchool,Houston,TX,USA
IRINA G.STAVROVSKAYA (cid:1) DepartmentofNeurology,ColumbiaUniversityMedicalCenter,
NewYork,NY,USA
ARONSZABO (cid:1) InstituteofGenetics,BiologicalResearchCentreoftheHungarianAcademyof
Sciences,Szeged,Hungary
GEORGEK.TOFARIS (cid:1) NuffieldDepartmentofClinicalNeurosciences,UniversityofOxford,
JohnRadcliffeHospital,Oxford,UK
JOHNQ.TROJANOWSKI (cid:1) CenterforNeurodegenerativeDiseaseResearch,InstituteonAging,
DepartmentofPathologyandLaboratoryMedicine,UniversityofPennsylvaniaPerelman
SchoolofMedicine,Philadelphia,PA,USA
NAMRATAD.UDESHI (cid:1) BroadInstituteofMITandHarvard,Cambridge,MA,USA
KARINAJ.VARGAS (cid:1) DepartmentofNeurology,YaleUniversity,NewHaven,CT,USA;
PrograminCellularNeuroscience,NeurodegenerationandRepair,YaleUniversity,New
Haven,CT,USA;EugeneBellCenter forRegenerativeBiologyandTissueEngineering,
MarineBiologyLaboratory,WoodsHole,MA,USA
LAURAA.VOLPICELLI-DALEY (cid:1) DepartmentofNeurology,CenterforNeurodegenerationand
ExperimentalTherapeutics,UniversityofAlabamaatBirmingham,Birmingham,AL,
USA