Microcephaly: Methods and Protocols

173 Pages·2022·8.26 MB·English

by  Timothy Gershon

Methods in
Molecular Biology 2583
Timothy Gershon Editor
Microcephaly
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.
Microcephaly
Methods and Protocols
Edited by
Timothy Gershon
Department of Pediatrics, Children’s Center for Neuroscience Research, Emory University School
of Medicine, Atlanta, GA, USA
Editor
TimothyGershon
DepartmentofPediatrics,Children’s
CenterforNeuroscienceResearch
EmoryUniversitySchoolofMedicine
Atlanta,GA,USA
ISSN1064-3745 ISSN1940-6029 (electronic)
MethodsinMolecularBiology
ISBN978-1-0716-2751-8 ISBN978-1-0716-2752-5 (eBook)
https://doi.org/10.1007/978-1-0716-2752-5
©SpringerScience+BusinessMedia,LLC,partofSpringerNature2023
Thisworkissubjecttocopyright.AllrightsarereservedbythePublisher,whetherthewholeorpartofthematerialis
concerned,specificallytherightsoftranslation,reprinting,reuseofillustrations,recitation,broadcasting,reproduction
onmicrofilmsorinanyotherphysicalway,andtransmissionorinformationstorageandretrieval,electronicadaptation,
computersoftware,orbysimilarordissimilarmethodologynowknownorhereafterdeveloped.
Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnotimply,
evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsandregulations
andthereforefreeforgeneraluse.
Thepublisher,theauthors,andtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedto
betrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty,
expressedorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeen
made.Thepublisherremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations.
ThisHumanaimprintispublishedbytheregisteredcompanySpringerScience+BusinessMedia,LLC,partofSpringer
Nature.
Theregisteredcompanyaddressis:1NewYorkPlaza,NewYork,NY10004,U.S.A.
Preface
Microcephaly, which in children is defined by head circumference two to three standard
deviations smaller than the mean expected for age and gender, is a potentially disabling
developmental disorder [1]. Microcephaly may be congenital or acquired, and can result
from any process that impairs brain growth more than the growth of the rest of the body.
Causes of congenital microcephaly include genetic disorders, maternal infections, and
intrauterinetoxicexposures[2–5].Postnatalbraininjurymaycauseacquiredmicrocephaly,
as seen in hypoxic-ischemic encephalopathy [6–8]. The common link between these pro-
cesses is insufficient proliferation of neural stem or progenitor cells, resulting from a
combinationofinappropriatecelldeathandprematurelossofproliferativepotential[9].
Research on the pathogenesis of microcephaly addresses a significant human health
concern and at the same time provides information on fundamental processes of brain
growth [10]. Zika-virus associated microcephaly remains a threat to public health for
which new interventions are urgently needed [11, 12]. Genomic medicine has identified a
growing number of genetic microcephaly syndromes, and families with identified micro-
cephaly mutations need informed counseling and new experimental treatments. Studies of
Zika virus, genetic microcephaly, and other microcephaly-causing pathologies, moreover,
can reveal mechanisms and factors that are essential for normal brain growth [13, 14].
Insight from microcephaly studies may further inform brain tumor biology, providing
anotheropportunityforclinicalrelevance[14,15].Importantbasicandtranslationalinsight
canthusbelearnedfromstudiesofmicrocephalypathogenesis.
Here,weprovideasetofimagingandmoleculartechniquesforadvancingmicrocephaly
research by analyzing neural progenitor proliferation, survival, and gene expression. All of
these techniques envision using the mouse as a model system, and comparing mice with
microcephaly-inducingconditionstohealthy,age-matchedcontrols.Tocomplementcellu-
lar, metabolic, and transcriptomic analyses, we provide methods for analysis of three-
dimensional brain growth using MRI and developmentally adapted analysis of mouse
behavior in the neonatal period. These methods are intended as a starting point for
microcephaly investigations, through which diverse experimental conditions related to
microcephalycanbesubjectedtomechanisticanalyses.
Atlanta,GA,USA TimothyGershon
References
1. AshwalSetal(2009)Practiceparameter:Evaluationofthechildwithmicrocephaly(anevidence-based
review):reportoftheQualityStandardsSubcommitteeoftheAmericanAcademyofNeurologyand
thePracticeCommitteeoftheChildNeurologySociety.Neurology73:887–897
2. Schuler-Faccini L etal (2022)Neurodevelopment in children exposed toZika in utero: clinical and
molecularaspects.FrontGenet13:758715
3. Kempinska W, Korta K, Marchaj M, Paprocka J (2022) Microcephaly in neurometabolic diseases.
Children(Basel)9(1):97.https://doi.org/10.3390/children9010097
v
vi Preface
4. GulloGetal(2022)CongenitalZikasyndrome:geneticavenuesfordiagnosisandtherapy,possible
management and long-term outcomes. J Clin Med 11(5):1351. https://doi.org/10.3390/
jcm11051351
5. ZaqoutS,KaindlAM(2021)Autosomalrecessiveprimarymicrocephaly:notjustasmallbrain.Front
CellDevBiol9:784700
6. Preeti S et al (2019) Anthropometric measures as biomarkers of neurodevelopmental outcomes of
newborns with moderate to severe hypoxic ischemic encephalopathy. J Neonatal Perinatal Med 12:
127–134
7. Rousset CI, Baburamani AA, Thornton C, Hagberg H (2012) Mitochondria and perinatal brain
injury.JMaternFetalNeonatalMed25(Suppl1):35–38
8. MercuriEetal(2000)Headgrowthininfantswithhypoxic-ischemicencephalopathy:correlationwith
neonatalmagneticresonanceimaging.Pediatrics106:235–243
9. JamuarSS, WalshCA (2015)Genomicvariants andvariations in malformationsofcorticaldevelop-
ment.PediatrClinNorthAm62:571–585
10. Becerra-Solano LE, Mateos-Sanchez L, Lopez-Munoz E (2021) Microcephaly, an etiopathogenic
vision.PediatrNeonatol62:354–360
11. Wilder-SmithAetal(2021)ThelegacyofZikaPLAN:atransnationalresearchconsortiumaddressing
Zika. Glob Health Action 2021; 14(Suppl): 2008139. https://doi.org/10.1080/16549716.2021.
2008139
12. SantiagoHC,Pereira-NetoTA,Goncalves-PereiraMH,TerzianACB,DurbinAP(2022)Peculiarities
ofZikaimmunityandvaccinedevelopment:lessonsfromdengueandthecontributionfromcontrolled
humaninfectionmodel.Pathogens11
13. Komarasamy TV, Adnan NAA, James W, Balasubramaniam V (2022) Zika virus neuropathogenesis:
thedifferentbraincells,hostfactorsandmechanismsinvolved.FrontImmunol13:773191
14. LangPY,GershonTR(2018)Anewwaytotreatbraintumors:targetingproteinscodedbymicro-
cephalygenes?:braintumorsandmicrocephalyarisefromopposingderangementsregulatingprogeni-
torgrowth.Driversofmicrocephalycouldbeattractivebraintumortargets.Bioessays40:e1700243
15. IegianiG,DiCuntoF,PallaviciniG(2021)Inhibitingmicrocephalygenesasalternativetomicrotu-
buletargetingagentstotreatbraintumors.CellDeathDis12:956
Contents
Preface ..................................................................... v
Contributors................................................................. ix
PART I CELLULAR ANALYSES OF NEURAL PROGENITORS
1 DissociationofCerebellarGranuleNeuronProgenitorsforCulture,
FACS,Transcriptomics,andMolecularBiology...... ........ ....... ........ 3
JenniferKarinOcasio
2 MaintainingCerebellarGranuleNeuronProgenitorsinCellCulture.......... 9
JenniferKarinOcasio
3 ProliferationAnalysisofCerebellarGranuleNeuronProgenitors
forMicrocephalyResearch,UsingImmunofluorescentStaining
andFlowCytometry ....... ........ ....... ....... ........ ....... ........ 13
JenniferKarinOcasio
4 ConventionalandSpectralKaryotypingofMurineCerebellar
GranuleNeuronProgenitors........ ....... ..... .. ...... .. ...... ......... 25
GouriNanjangud
PART II ANALYSES OF NEURAL PROGENITORS IN SITU
5 ProcessingNeonatalMouseBrainsforImmunohistochemicalAnalysis:
AsRequiredforIncludingSpindleOrientationAnalysisandAnalysis
ofDNADamageandApoptosis ..... ....... ....... ........ ....... ........ 49
GabrielaDelaCruz,NanaNikolaishvili-Feinberg,
ScottE.Williams,andTimothyR.Gershon
6 AutomatedImmunofluorescenceStainingforAnalysisofDNADamage
andApoptosisinBrainSections...... ....... ....... ........ ....... ........ 55
GabrielaDelaCruz,NanaNikolaishvili-Feinberg,
andTimothyR.Gershon
7 AutomatedImmunofluorescenceStainingforAnalysisofMitoticStages
andDivisionOrientationinBrainSections.......... ... ..... ....... ........ 63
GabrielaDelaCruz,NanaNikolaishviliFeinberg,
andScottE.Williams
PART III RNA-SEQ ANALYSIS WITH SINGLE-CELL RESOLUTION (SCRNA-SEQ)
8 scRNA-seqforMicrocephalyResearch[I]:Single-CellDroplet
Encapsulation,mRNACapture,andcDNASynthesis ........ ....... ........ 83
BenjaminBabcockandSethWeir
9 scRNA-seqforMicrocephalyResearch[II]:PreparationofSingle-Cell
cDNALibraries..... ....... ........ ....... ....... ........ ....... ........ 99
BenjaminBabcockandSethWeir
vii
viii Contents
10 scRNA-seqforMicrocephalyResearch[III]:ComputationalAnalysis
ofscRNA-seqData......... ........ ....... ....... ........ ....... ........ 105
BenjaminBabcockandDanielMalawsky
11 scRNA-seqforMicrocephalyResearch[IV]:DirichletRegression
forSingle-CellPopulationDifferences....... ....... ........ ....... ........ 123
DanielMalawskyandTimothyR.Gershon
PART IV MRI ANALYSES OF THE MOUSE BRAIN
12 MouseBrainMRI:IncludingInVivo,ExVivo,andfcMRI
for theStudyofMicrocephaly....... ....... ....... ..... ... ...... ......... 129
MartinJ.MacKinnon,Tzu-WenW.Wang,andYen-YuI.Shih
13 Whole-BodyMouseFluxomicAnalysistoDetectMetabolic
DisruptionsAssociatedwithMicrocephaly:Using13CIsotopes ...... ........ 149
AndreyP.Tikunov
PART V BEHAVIORAL PHENOTYPING IN NEONATAL MICE
14 NeonatalBehavioralScreenforMouseModelsofNeurodevelopmental
Disorders .......... ....... ........ ....... ....... ........ ....... ........ 159
KathrynM.Harper,ViktoriyaD.Nikolova,
MonikaE.Conrad,andSherylS.Moy
Index ...................................................................... 175
Contributors
BENJAMINBABCOCK • DepartmentofMedicine,DivisionofImmunology,LowanceCenter
forHumanImmunology,EmoryUniversitySchoolofMedicine,Atlanta,GA,USA;
DepartmentofNeurology,UniversityofNorthCarolinaMedicalSchool,ChapelHill,NC,
USA
MONIKAE.CONRAD • DepartmentofPsychiatry,TheUniversityofNorthCarolinaatChapel
Hill,ChapelHill,NC,USA;CarolinaInstituteforDevelopmentalDisabilities,The
UniversityofNorthCarolinaatChapelHill,ChapelHill,NC,USA
GABRIELADELACRUZ • DepartmentofPathology&LaboratoryMedicine,TheUniversityof
NorthCarolinaatChapelHill,ChapelHill,NC,USA;PathologyServicesCore,The
UniversityofNorthCarolinaatChapelHill,ChapelHill,NC,USA;Lineberger
ComprehensiveCancerCenter,TheUniversityofNorthCarolinaatChapelHill,Chapel
Hill,NC,USA;TranslationalPathologyLaboratory,TheUniversityofNorthCarolinaat
ChapelHill,ChapelHill,NC,USA
TIMOTHYR.GERSHON • LinebergerComprehensiveCancerCenter,TheUniversityofNorth
CarolinaatChapelHill,ChapelHill,NC,USA;DepartmentofNeurology,The
UniversityofNorthCarolinaatChapelHill,ChapelHill,NC,USA;UNCNeuroscience
Center,TheUniversityofNorthCarolinaatChapelHill,ChapelHill,NC,USA
KATHRYNM.HARPER • DepartmentofPsychiatry,TheUniversityofNorthCarolinaat
ChapelHill,ChapelHill,NC,USA;CarolinaInstituteforDevelopmentalDisabilities,
TheUniversityofNorthCarolinaatChapelHill,ChapelHill,NC,USA
MARTINJ.MACKINNON • Center forAnimalMRI,UniversityofNorthCarolinaatChapel
Hill,ChapelHill,NC,USA;BiomedicalResearchImagingCenter,UniversityofNorth
CarolinaatChapelHill,ChapelHill,NC,USA;JointDepartmentofBiomedical
Engineering,UniversityofNorthCarolinaatChapelHill,ChapelHill,NC,USA;
DepartmentofNeurology,UniversityofNorthCarolinaatChapelHill,ChapelHill,NC,
USA
DANIELMALAWSKY • DepartmentofNeurology,UniversityofNorthCarolinaMedicalSchool,
ChapelHill,NC,USA;WellcomeSangerInstitute,WellcomeGenomeCampus,Hinxton,
UK
SHERYLS.MOY • DepartmentofPsychiatry,TheUniversityofNorthCarolinaatChapel
Hill,ChapelHill,NC,USA;CarolinaInstituteforDevelopmentalDisabilities,The
UniversityofNorthCarolinaatChapelHill,ChapelHill,NC,USA
GOURINANJANGUD • MemorialSloan-KetteringCancerCenter,NewYork,NY,USA
NANANIKOLAISHVILI-FEINBERG • DepartmentofPathology&LaboratoryMedicine,The
UniversityofNorthCarolinaatChapelHill,ChapelHill,NC,USA;Translational
PathologyLaboratory,TheUniversityofNorthCarolinaatChapelHill,ChapelHill,NC,
USA;PathologyServicesCore,TheUniversityofNorthCarolinaatChapelHill,Chapel
Hill,NC,USA
VIKTORIYAD.NIKOLOVA • DepartmentofPsychiatry,TheUniversityofNorthCarolinaat
ChapelHill,ChapelHill,NC,USA;CarolinaInstituteforDevelopmentalDisabilities,
TheUniversityofNorthCarolinaatChapelHill,ChapelHill,NC,USA
ix