Neuronal migration and disorders – an update

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Fiona Francis, Silvia Cappello

To cite this version:

Fiona Francis, Silvia Cappello. Neuronal migration and disorders – an update. Current Opinion in

Neurobiology, Elsevier, 2021, 66, pp.57 - 68. �10.1016/j.conb.2020.10.002�. �hal-03025850�

Neuronal

migration

and

disorders

–

an

update

Fiona

Francis

and

Silvia

Cappello

Thisreviewhighlightsgenes,proteinsandsubcellular mechanisms,recentlyshowntoinfluencecorticalneuronal migration.Acurrentviewonmechanismswhichbecome disruptedinadiversearrayofmigrationdisordersispresented. Themicrotubule(MT)cytoskeletonisamajorplayerin migratingneurons.Recently,variableimpactsonMTshave beenrevealedindifferentcellcompartments.Thustherearea multiplicityofeffectsinvolvingcentrosomal, microtubule-associated,aswellasmotorproteins.However,other causativefactorsalsoemerge,illuminatingcorticalneuronal migrationresearch.Theseincludedisruptionsoftheactin cytoskeleton,theextracellularmatrix,differentadhesion moleculesandsignalingpathways,especiallyrevealedin disorderssuchasperiventricularheterotopia.Theserecent advancesofteninvolvetheuseofhumaninvitromodelsaswell asmodelorganisms.Focusingoncell-typespecificknockouts andknockins,aswellasgeneratingomicsandfunctionaldata, allseemcriticalforanintegratedviewonneuronalmigration dysfunction.

Addresses

1_{INSERMU1270,Paris,France} 2

SorbonneUniversity,UMR-S1270,F-75005Paris,France

3

InstitutduFera` Moulin,Paris,France

4_{MaxPlanckInstituteofPsychiatry,Munich,Germany}

Correspondingauthor:Francis,Fiona(fiona.francis@inserm.fr)

CurrentOpinioninNeurobiology2021,66:xx–yy ThisreviewcomesfromathemedissueonDevelopmental neuroscience

EditedbyAlainChedotalandDenisJabaudon

https://doi.org/10.1016/j.conb.2020.10.002

0959-4388/_ã2020TheAuthors.PublishedbyElsevierLtd.Thisisan openaccessarticleundertheCCBY-NC-NDlicense( http://creative-commons.org/licenses/by-nc-nd/4.0/).

Introduction

Duringembryogenesis,corticalprojectionneuron

devel-opmentrequiresastrictcoordinationbetweenamplifying progenitorcells,neurogenesisandneuronalmigrationto superficialregionsofthebrain[1].Neocorticalexpansion

duringevolutionisconcomitantwithincreasednumbers

of progenitors and neurons, and a particular neuronal

organization which leads to the formation of gyri and

sulci[2,3].Afterbeinggeneratedinaventricular(VZ),or

subventricular zone(SVZ), neurons migrate through an

intermediate zone (IZ)to reach thedeveloping cortical

plate(CP).Apicalprogenitorsconsistofradialglia(RGs), exhibitingshortapicalandlongbasalprocessestomake

attachments with other cells and with the extracellular

matrix(ECM,[2]).Basalprocessesserveasasubstratefor radiallymigratingneurons(Figure1a).Basalprogenitors

found in the SVZ consist of intermediate progenitors

(IPs), or basal radial glia (bRGs, [3]), highly amplified

in number in gyrencephalic brains (exhibiting cortical

folds)[3,4].Ascorticogenesisproceeds,neuronsare gen-eratedeitherbydirect(fromRGs)orindirect(frombasal progenitors)neurogenesis[1].Overtime,successive gen-erationsofprogenitorswillproduceneuronsdestinedfor thedifferentlayersofthecortex,withdeeplayersformed beforeupperlayers[5].

Cortical malformations can show disrupted brain size

(microcephaly or macrocephaly), and/or morphology

[6–8].Abnormalcorticallayeringandfoldingareassociated

with epilepsy(oftenpharmaco-resistant),developmental

delay and intellectual disability [8]. The lissencephaly spectrum(corticalagyria,pachygyriaandsubcorticalband heterotopiaorSBH)ischaracterizedbylargenumbersof mis-positionedneurons,eitherpresentinthewhitematter, orinabnormalcorticallayers.Inperiventricularheterotopia (PH),populationsofmis-positionedneurons(nodules)are foundliningtheventricles[8].Polymicrogyriaisassociated withexcessivesmallfoldsonthesurfaceofthebrain.The patho-mechanismsgivingrisetothesephenotypesarestill beingelucidated.

PreviousgeneticstudieshaverevealedmanyMT cytoskel-etongenemutations(tubulinopathies[9,10])affecting cor-ticallaminationandfolding.Mutationsindifferenttubulins (TUBA1A, TUBB2B, TUBB3, TUBG1),

microtubule-asso-ciated proteins and motorproteins (LIS1,DCX, KIF2A,

KIF5C, DYNC1H1, EML1) have illuminated neuronal

migration disorderresearch[11,12]. InPH, themost fre-quentgeneshowingmutationsisFLNA,anactin cytoskel-etoncross-linkerregulatingadhesionmoleculessuchasthe integrins[13,14].MutationsinFLNAwerefoundin100% of familieswithX-linkedbilateralPHandin26%ofsporadic

PH patients [5]. Other PH genes are FAT4, DCHS1,

ARFGEF2, ERMARD, AKT3, INTS8, MCPH1,

NEDD4L and MAP1B [16] with diverse functions, for

example,inadhesion,trafficking,generegulationand sig-naling.Manymorepatientsexistforwhichthemutantgene isnotyetknown.Polymicrogyriageneshavediverse

func-tions and expression patterns. These include GPR56,

Figure1

(a)

(c)

(b)

Current Opinion in Neurobiology

Multipolar(Kif2A)andbipolar(Bicd2,Tubg1,Cep85L)neuronphenotypesrevealedbythestudyofMTcytoskeletalgenes.(a)Schemashowing keycelltypesinvolvedinrodentcorticaldevelopmentduringembryogenesis.InapicalregionsofRGs,MTs(orangelines)andaprimarycilium (bluetriangle)areindicated.Dync1h1,Bicd2,Kif2aandTubg1playrolesinRGs,althoughwefocusinthisreviewontheirroleduringmigration.It iscurrentlyunclearifCep85lplaysaroleinprogenitors.(b)AbipolarmigratingneuronisshownwithseveralMTfeaturesindicated.(c)Kif2a mutantcells,relatedtoalossofanMT-depolymerizingactivity,showlonger,nocodazole-resistantMTs,shownhereforsimplicityatthe

Reviewedherearenewgenes/mechanismsassociatedwith thesedisordersrevealingcorticaldevelopmentalprocesses.

MT

dynamics

and

function

in

migrating

neurons

Migrating corticalneurons undertake multiple dynamic

changes, first transitioning from multipolar to bipolar

morphologies[11], involvingprocess growthand

retrac-tion, and the polymerization and depolymerization of

MTs. Duringmigration,MTsaidforwardmovementof

the nucleus(nucleokinesis) following themovement of

the centrosome (the MT organizing centerinvolved in

nucleation)(Figure1b,[17]).Withinstableneuritic pro-cesses,MTtracksarerequiredfor traffickingofcargoes andorganelles.Anumberofrecentstudieshave

interro-gatedmutantMTandassociatedproteinsduring

migra-tion.Surprisingly,variableeffects areobserved showing thecomplexityofthecytoskeletonactingindifferentcell compartments(Table1).

Gilet et al. [18]studied KIF2A, akinesin MT

depoly-merizer. Mutations give rise to pachygyria and

micro-cephaly [19].Using anelegantconditionalknockin(KI)

strategy,miceweregeneratedexpressing apatient

mis-sensemutationinparticularcelltypes.Crerecombinase linesusedwere:ubiquitousRosa-Cre,progenitor

Nestin-Cre orimmatureneuronNex-Cre.Rosa-KIand

Nestin-KI miceare smaller in size and show microcephaly. At

E18.5inthesemice,corticesweremoreseverelyaffected thanNex-KImice,showingaroleforKif2ainprogenitors.

TheRGscaffoldrevealednomajoranomaliesin

Nestin-KImice,howevercelldeathwasgreatlyincreasedmainly

in IPsandneurons.Nex-KImicealsoshowedincreased

neurondeath. Time-lapseimagingandinutero

electro-porations(IUE)revealednormalneuronmigration

veloc-ityandpausing,butaprobleminthetransitionof

migrat-ing neurons from multipolar to bipolar morphologies

(Figure 1c). MTsshowedaresistance to

depolymeriza-tion, most probably via deficient ATP turnover and

persistent MT association of mutant KIF2A. Plus end

MT polymerization was also increased in mutant cells.

ThusarobustderegulationofMTdynamicsisobserved

duetoKIF2Amutations,affectingprogenitors,aswellas multipolarto bipolarneurontransitions.

MT motor proteins also play a key role in neuronal

migration.Dyneinandkinesinstransportcargoesin oppo-sitedirectionsalongMTs,transportMTsthemselves,and

are also implicatedin nuclearmovement [11].Denovo

heterozygotemutationsindynein(DYNC1H1)cangive

rise to a spectrum of brain phenotypes, and/ or spinal

muscularatrophy[19–22]andreferencestherein].Recent

studies focused on BICD2, a dynein adaptor protein

associatedwithspinalmuscularatrophy,butalso perisyl-vianpolymicrogyria[23].Willetal. [24]performed

cell-type specific knockouts (cKOs) of Bicd2 using Emx1

(earlyforebrain-specificprogenitors)andNex-Cremouse lines.Migrationdefectswereidentifiedinbipolarradially

migratingneuronsforbothcKOs.Althoughabnormalities

werealsoobservedinprogenitorsintheEmx1-CrecKO

(Bicd2 plays arole in interkinetic nuclear migration in

RGs), migration deficits were similar between the two

lines,revealingacell-intrinsicroleforBicd2inmigrating

neurons.Mutantmigratingneuronshadlongerandmore

branched leadingprocesses, as well as aberrantly

orga-nizedGolgiapparatuses(Figure1c).Thepolymicrogyria patientmutationinBICD2occursinabindingdomainfor

thesmallGTPaseRab6(involvedinGolgifunction)and

the nucleoporin RanBP2 (aiding dynein recruitment to

thenuclearenvelopeindividingcells).Thecontribution ofthedifferentorganellestothemigrationandprogenitor

defectsremainto beestablished.

Denovovariantsing-tubulin(TUBG1)werealso

iden-tified in patients with pachygyria and corpus callosum

abnormalities[19,25,26].g-tubulinisknownfor itsrole

nucleatingMTs,formingpartofthecentrosome.Tubg1

+/ mice show nocortical defects[27], howevereither

KDorKI(ubiquitousorneuron-specific)offour

hetero-zygote patient variants disrupted neuronal migration,

with one mutation also perturbing proliferation. Four

days afterIUE, unlikeWTneurons whichhad reached

theupperlayersoftheCP,mutantcellshadonlyreached

the IZand were arrestedin their migration:time-lapse

studies showed many non-migrating bipolar neurons.

Interestingly, mutant proteins were correctly localized

at the centrosome, and MT nucleation properties

appeared normal however,MT dynamics were altered,

with adecrease inpolymerization rate,resultingin less

organized and shorter MTs. Thus, studying different

models,mainlymigrationdefectsareobserved,associated

withdampenedMTpolymerization(duepotentiallytoa

non-centrosomal function of g-tubulin) in neurons of

normalbipolar morphology(Figure 1c).

Patients wereidentified withrareheterozygousvariants

in CEP85L, coding for acentrosome-associated protein

locatedtothemothercentriole[28,29].Mutationsgive

risetoanagyria,pachygyria-SBHspectrum,mostsevere

overoccipital,temporalandparietallobes.CEP85Lwas

(Figure1LegendContinued)centrosome.Thisperturbsthemultipolartobipolartransition.Bicd2bipolarneuronsareslowedintheirmigration andshowincreasedbranchingoftheirleadingprocesseswithaberrantlyorganizedGolgiapparatuses,relatedtodynein-dependentfunctions.In Tubg1mutantneurons,althoughMTnucleationseemstooccurproperly,thereisadecreaseinMTpolymerizationrate,MTsarehenceshorter,as wellasbeinglessstraight,eveninnon-centrosomalregions.Cep85LmutantcellsremainblockedintheIZ(multipolarversusbipolarneuronswere notassessed).Inthiscase,thereisacentrosomalaccumulationofvariousMTproteinsleadingtooverabundantMTs,sinceCep85Ltogetherwith Cdk5normallyrestrictsthebindingoftheseMTproteins.Corticalbipolarmigratingneuronshavethecentrosomeinfrontofthenucleus,oftenina visiblebulge.Nuclear-centrosomalcoordinationisimportantforcellmovement.

Table1

Humanmalformationandmousephenotypes

Gene Reference Mouse Phenotype Humanmodel Comments Pathology

BICD2 [23,24] cKO:Emx1-Cre; Nex-Cre

Intheneocortex, migrationdefectsin bipolarneurons-longer andmorebranched leadingprocesses.cKOs showedseveremigration defectsinupperlayer neurons,withaswella mixingofneuronsin layers5and6.Maturing neuronsincreased apoptosis.

– – Heterozygote.Bilateral

perisylvianPMG, enlargedlateral ventricles,thincorpus callosum,hypoplastic cerebellarvermis, retrocerebellarcyst

CEP85L [28,29] KD:shCep85l E13.5-E17.5

LessneuronsinCPmore inIZ,SVZ.Persistanceat P7

Expressionfrom fetalcortex lysates

– Heterozygous(canhave

autosomaldominant inheritance). Posterior-predominantpachygyria. Mildcerebellaratrophy. CEP83 [30,31] cKO:Emx1-Cre OverproliferationofRGin

medio-dorsalregions, macrocephaly – – Recessive.Infantile nephronophthisisand intellectualdisability ECE2 [49] KD:miRNAs directedagainst ECE-2and overexpression (E13-E16). DetachedRG,ectopic neurons,rosettes/ nodules,disruptedVS KD/Crispr-Cas9 KO/PHOSinh.in cerebral organoidsand 2Dcultures. Overexpression. DetachedRG,ectopic progenitorsandneurons, slowedmigration. Progenitorsincreased, neuronsdecreased(KD), oppositewith overexpression. Recessive.DiffusePH DCHS1/ FAT4 [39,41] KOsandKD: shRNAsdirected againstDchs1 andFat4(E13 -E16/E18).

KOs:cortexnormal structureatE18.Lethality. Overproliferation.Many cellsremainin proliferativezones.KDs: changesinRG morphology, overproliferation (Yap-mediated),neuronal migrationdefects. Patient mutations/ Crispr-Cas9KO/ KD,incerebral organoidsand 2Dcultures. Neuronalnodulesat ventricularpositions. Poorlyorganized germinalzone.Disrupted RGmorphology(e.g. processestwisted, FAT4).Perturbedcilia. Slowedmigration.

Recessive.Van Maldergemsyndrome. PHtypicallyaround posteriorhornsbutcan extendanteriorly. Simplifiedgyriinaffected regions. EML1 [32,33,34] Spontaneous mutation(HeCo) Neocorticalheterotopia. DampenedMT polymerization(EB3). Perturbedspindles,cilia andGolgiapparatus.

Fibroblastsand humandorsal cortical progenitors

Defectiveciliogenesis andGolgiapparatus

Recessive.

Megalencephalywitha characteristicribbon-like subcorticalandPH combinedwithpartialor completecallosal agenesisandanoverlying PMG-likecortical malformation. Hydrocephalus. FLNA [12,13,14,15] KO:Flna,cKO:

FlnaEmx1Cre; cKO/KO:Flna/ Flnbmice

KO:Embryoniclethal, abnormalvessels,cardiac defects;thinCP;cKO: Mildphenotypein neuroependyma.Flnb KO/FlnacKOPH, mislocalizedandexcess IPs,lossofepithelial-like featuresofmutantRG cells

– – X-linked.PH

predominantlyliningthe anteriorhornsand ventricularbodiesofthe lateralventricles. Hypoplasiaofthe cerebellarvermisand posteriorfossacystsare common

Table1 (Continued)

Gene Reference Mouse Phenotype Humanmodel Comments Pathology

KIF2A [18] cKI:Nestin,Nexor Rosa-Cre Microcephalyand disorganizedlayers (Rosa,Nestin-Cre). Hippocampal heterotopia.Problemin multipolar-bipolar transition.Celldeath increased.MTsresistant todepolymerization. Fibroblasts Defective depolymerizationofMTs (nocodazoleassay). Increasedpolymerization (EB3). Heterozygote.Posterior pachygyriaand microcephaly MAP1B [16,44] Map1B-deficient mice Defectsinneuron migration,neurite extensionandsynapse development Co-expression analysesofPH genesbasedon brain-specific human transcriptomic resources

FLNAandINTS8similar pathologyand expression

Heterozygous.Bilateral frontal-predominantPH, canbeassociatedwith perisylvian/insularPMG

MOB2 [43] KD:miRNAs directedagainst Mob2IUE(E13 -E16).Dchs1KD also. IncreasedcellsinVZ, decreaseinouterCP. Nuclear-ciliadistance increasedinmigrating neurons(andsometimes multi-cilia).Positionand/ ornumberofciliain migratingneuronswas aberrantuponDchs1KD.

Humancerebral organoid cultures

Defectsincilianumber uponMOB2knockdown

Recessive.BilateralPH

NEDD4L [50] KO:Nedd4l;KD: shRNA. Overexpressionof mutantvariants. IUEE14-E16or E18. KOperinatallethal;KDno migrationdefect; overexpressionmutants: migrationarrestinVZ/ SVZ/IZ.Proliferation defectsatE16. Rapamycin-rescued neuronalposition – – Heterozygous.Bilateral frontal-predominantPH, cleftpalateandmildtoe syndactyly PCDH19 [54] KD:Inutero injectionof retrovirusshRNA (E11)and overexpression KD:Fewerneuronsin clones.InSVZ/IZneurons hadmoreneurites.More laterallydispersed. Impactonsisterneuron connectivity.

Overexpression:more neuronsinclonesand laterallyclustered. Increasedconnectivity.

Femalesaffected. Relatedtomosaicism andabnormalcellsorting. PCDH19isaTBR2target.

X-linked.Early-onset epilepsyandintellectual disabilityinfemales,FCD, cortexthickeningand abnormalfolding, occasionalmicrocephaly PLEKHG6 [48] KD:miRNA (isoform1),and overexpressionof PLEKHG6_4in mouse(E13-E16) Overexpressiondisrupts VZintegrity.Plekhg6_1 KDdisrupts neuroprogenitor differentiationand neuronalmigration Overexpression inhuman cerebral organoid cultures Impairedventricular surfaceintegrityandPH formation

Recessive.Bilateral, posterior-predominant, PH.

TUBG1 [26] KD:shRNA,KI: variantsbyIUE: andconstitutiveKI mousemodel (Tubg1-Y92C). Dcxpromoterfor KI. Mainlypost-mitotic defects.Migration defectsinbipolar neurons.AtE18.5,layer 5wasmorespread-out andlayer6was decreased,thelatter defectsalsoobservedin theadultbrain.The cerebellum,anterior commissure,fimbriaand hippocampuswerealso affected.DecreasedMT polymerization.

Fibroblasts Lessstraightandshorter MTs Heterozygote. Posterior-predominantpachygyria, corpuscallosum abnormalitiesand microcephaly

Abbreviations:CPcorticalplate;FCDfocalcorticaldysplasia;IPintermediateprogenitor;IUEinutero;electroporation;IZintermediatezone;KD knockdown;KOknockout;MTmicrotubule;PHperiventricularheterotopia;PMGpolymicrogyria;RGradialglialcell;SVZsubventricularzone;VZ ventricularzone.

Figure2 (a) (c) (b)

Delaminating

cell

Delaminating

cell

Radial glial cell

Apical endfeet

Adhesive contacts

Current Opinion in Neurobiology

(a)SchemafocusingonapicalendfeetofRGs,whichcontainacentrosomeorganizinganMTnetwork(goldlinesinenlargedendfoot).Molecular alterationsinapicalendfeetcanfavorizecelldelamination.(b)Cep83mutantendfeetarelarger,duetoaslightlymorebasalpositionofthe centrosomewhichweakenstheMTs,andhasanimpactontheactin-richcellcortex.Changedmechanicaltensionsincreasethesizeofthe endfeet,activatingtheHipposignalingpathway.(c)ECE2mutantcellshaveachangedsecretome(ECMrepresentedinorangeandgrey surroundingendfeet),aswellashavinganalteredactincytoskeleton(pink,shownjustsurroundingadherensjunctions,representedinblue).ECE2 mutationalterstheECMandcytoskeleton(actinandMTs),perturbingadhesionandleadingtoRGdelamination.Migratingneuronsarealso abnormalmostlikelyduetocytoskeletonandadhesionchanges(representedintheleadingprocesses).

foundtobemorestronglyexpressedinhumanvisualthan in frontal cortex [29] which might help explain the

milderor unaffected frontallobes.WithKD byIUEin

themouse,Cep85l-depletedcellswerefoundblockedin

theIZ[28,29].Inscratch-woundmigrationassays,cells

were found to correctly orient their centrosomes,

how-ever,impairedMTdynamicswereidentifiedsurrounding

thecentrosomewithanoverabundanceof

newly-synthe-sized MTs[29]. CEP85L was shown to interact with

LIS1,NDE1,KIF2A,DYNC1H1andCDK5[29],with

CDK5activitynecessarytorestrictthecentrosomal

accu-mulation of these lissencephaly proteins. They hence

accumulateatthecentrosomeinCEP85L-depletedcells

andoverabundantMTsresult.Interestingly,

phosphory-latedCDK5 alsoshowedastrongerexpression invisual

than in frontal cortex, which might help explain the

posterior-predominant phenotype in CEP85L patients.

This study hence links together functions of several

lissencephaly-relatedgenesandnotablytheirroles

influ-encing MTdynamicsatthecentrosome(Figure 1c).

Together, study of these genes reveals individual

phe-notypes at the subcellular level. Abnormal MTs were

foundinvariouscellcompartments,aswellasindifferent celltypesorstages(progenitorsandmultipolarorbipolar

neurons).ClearlyvariableMTimpactscanhavethesame

end-resultof abnormalmigration.

Proteins

influencing

the

centrosome

in

progenitors

Centrosomes (made up of centrioles) are positioned in

apical endfeet of RGs during interphase (Figure 2a).

They are important for ciliogenesis and contribute to

RGpolarity,influencingalsoRGcell-contactsand

attach-ment in theVZ[1]. DisruptedRGcentrosomal

mecha-nisms can also impact neuron production (including in

microcephaly,notfurthermentionedhere),organization

andmigration,oftennon-cellautonomously. Several-spe-cificcases arementionedhere.

CEP83isassociatedwithintellectualdisability[30],and

was recently shown to be involved in mother centriole

function[31].Aforebrain-specificcKOledto

overpro-liferation and enlarged mouse brains (macrocephaly),

especially affecting medio-dorsal regions which even

exhibited folding. There were increased RGs, as well

as IPsfromearly-midcorticogenesis.Centrosomeswere

found to be marginally displaced in apical endfeet of

mutantRGs(Figure2b).MTssupportingthecellcortex wereaffected,influencingtherigidityoftheapical mem-braneandactivatingsignalingpathways(suchasHippo), in turninfluencing proliferation.Thisstudyemphasizes

the intricate relationship between the interdependent

actinand MTcytoskeletons.

Mutations in an MT-binding proteinEML1 leadingto

human subcortical heterotopia, polymicrogyria and

macrocephaly [32]also affect RG apical endfeet in the mouse [33,34]. In this case, centrosome position and primary ciliaare severely disrupted and aproportion of

RGs detach (Figure 2a). Apical end feet size are also

enlarged which may be a consequence of cell

detach-ment. Ultimately non cellautonomous neuronal

migra-tion defects causing heterotopia are observed in the

mouse [32], appearing to closely mimic the human

phenotype.

Also, important in progenitors, thecentrosomal protein

Akna associateswith themothercentriole[35].There

arehigherlevelsofAknawhenRGstransitiontobecome

basalprogenitors(Figure2a).AknarecruitsMTandactin

cytoskeleton components from RG apical endfeet cell

junctions to the centrosome. Its action also then helps

retaincellsintheSVZ.Thus,withitsKDthereisafaster multipolar to bipolar transition. This is hence a highly dynamicprotein,regulatingmultiplestepsof corticogen-esis.Plekha7alsoactsatcelljunctionsinRGs,makinga linkwiththeMTcytoskeleton[36,37].Itsmutationby

Crispr/Cas9 also causes RG delamination from the VZ

mostlikelybydismantlingadhesivejunctionscombined

withcytoskeletalrearrangements[36].

Thus, mechanisms are emerging by thestudy of these

genes, which influence adhesive, cytoskeletal and

bio-physical aspects of RGs, especially in apical endfeet,

henceregulating celldetachment.

Cell

interactions,

adhesion

and

the

cytoskeleton

Cytoskeletalmechanismsinfluencingadhesion,linked toPH

Celladhesionisanessentialprocess duringbrain devel-opmentasitprovidesnotonlystability,particularlyatthe apicalsidewhereRGsdonothaveaphysicalstructureto

attach to, but also mechanical signals which result in

intracellularresponses[1].PHgeneshavepossibleroles

in actin remodeling and vesicle trafficking, likely to

control cell adhesion. For example, the first PH gene

identified,FLNAencodesalargephosphoproteinwhich

cross-linksactinfilamentsintonetworksandreorganizes

thembyinteractingwithplasmamembraneproteinssuch

as integrins[11,12,38].ARFGEF2, thesecondPHgene

identified, codes for brefeldin-A-inhibited guanine

exchange factor-2 and seems likely to play a role in

endocytosis[13].Aquestionhasbeeniftheimportance

of these processes is in migrating neurons and/or

peri-ventricularRGs,asbothcouldberelevantforthis disor-der[13].Cell-typespecificdefectsarebeingrevealedfor

certain genes in mouse and human models (Table 1),

aidedbysinglecelltranscriptomics.Recentstudiesalso identifyraregeneticvariantsgivinghintsofwider

Aheterophiliccadherinreceptor-ligandpair,DCHS1and

FAT4, exhibit mutations in a PH-associated syndrome

(VanMaldergem,[39]).Amousemodelshowedarolefor thesemoleculesinRGs[39]andtheywerealsoshownto

be involved in apically-located adhesive complexes in

other cell types [40]. A more recent studyemphasized

their importance in migrating neurons using human in

vitromodels[41].InDCHS1(homozygous)andFAT4

(compoundheterozygous)patientmutation,or

CRISPR-Cas9 generated KO organoids, RG processes and the

apicalsurfacewereshowntobedisrupted,and

periven-tricularnoduleswererevealed.SinglecellRNA

sequenc-ing of mutant organoids revealed transcriptional

signa-turesofdelaminatedmutantprogenitorsandanexcessof

differentiated neurons. In mutant migrating neurons,

time-lapsemicroscopyshowedmorepausesand

tortuos-ity,aswellasdecreasedvelocity.Interestingly, hierarchi-calclusteringanalysis revealedan alteredpopulation of

neurons unexpectedly expressing ROBO3 with other

dysregulated adhesion and guidance molecules, which

could suggest incorrect regulation of specification and

guidance. Similarto Cep83, in mouse, Dchs1and Fat4

canalsoinfluencetheHipposignalingpathwayandlead

to hyperproliferation [39,42]. This information helped

identify rare biallelic variants in aHippo factor MOB2

in one PH patient [43]. Mouse Mob2 KD showed a

proportionofcellsblockedintheVZandIZ,inparticular,

migrating neurons with reduced levels of Mob2 show

centrosome/cilia defects, and similar defects were

observed in cerebral organoids. Mob2 was shown to be

linkedtoNdr1/2kinases,abranchoftheHipposignaling

pathway controlling actin-cytoskeletal arrangements.

FlnA phosphorylation was also increased with Mob2

KD, expectedto impairFlnA turnover, elevateprotein

levels, thus increasingactin cytoskeletalassembly,

sug-gestingconvergingmechanismsleadingtotheformation

of PH in the case of FLNA, DCHS1, FAT4 and MOB2

mutations[38,41,43].

RarePHvariants were identified in theMT-associated

proteingene,MAP1B[16],whichisexpressedinneural progenitorsanddeveloping/regeneratingneurons[44,45].

Fromprevious studies it is known that,as well as

per-turbedMTbinding,RacandCdc42activitiesarereduced

in Map1b-deficient neurons, while RhoA activity is

increased [46]. Indeed, dosage of RhoA is critical for migration[47].However,asevereRG-relatedheterotopia

phenotypeisobservedinaRhoA(Emx1-Cre)cKOmouse,

with neurons arrested in the VZ [47]. One of the first

observed changes was perturbed cell-cell adhesion

between RGs at the apical side, supporting a primary

role in the maintenance of adhesion. Furthermore,

O’Neill et al. [48] also showed a rare homozygous PH

mutation in theRhoA activator, PLEKHG6,leading to

thelossof aprimate-specific isoformof this protein.As

wellasaroleforPLEKHG6inmigration,cerebral

orga-noidand overexpression/KDmouse modelsshow a

dis-ruptedbalancebetweenapicalandbasalprogenitorsand

neurons,alsomediatedbylossofcell-celladhesionatthe

apical side. RhoA and PLEKHG6 are thus pleiotropic

proteinsplayingarolein bothprogenitorsand neurons,

underlining the complexity of cortical development

phenotypes.

Furthermore,anewPHgene,ECE2,hasrecently been

associatedwithrolesinactinandMTcytoskeletonaswell

as adhesion mechanisms, influencing neurogenesis and

migration [49]. KO, overexpression and/or KD in

Figure3

Anterior

Posterior

Current Opinion in Neurobiology

Manycorticalmalformationsshowanterior-posteriorgradientsofseverity.Severalgenesarenoted(notexhaustive)toillustratethispoint.Of these,fewgeneshavebeenassociatedwithparticularexpressionpatternsorinteractingpartners,toexplainthegradient.Itishoweverthecase forGPR56(bilateralfrontoparietalpolymicrogyriaorperisylvianpolymicrogyria)whichisregulatedbytheRFXtranscriptionfactorswhichshow regionalexpressionpatterns[63].ItisalsothecaseforCEP85LwhichinteractswithCDK5,bothofwhicharemorehighlyexpressedinvisual thanfrontalcortex[28].

cerebral organoids and the mouse cortex showed

detachedRGsandectopicneurons,formingproliferative

rosettes and neuronal nodules in the mouse.

Interest-ingly, many ectopic cells had notdirectly received the

mutantconstructrevealinghenceanon-cellautonomous

effect. Furthermore, proteomics analyses showed that

ECE2 is involved in the generation and secretion of

extracellular matrix(ECM)componentsandthesewere

downregulated inthemutant situation,potentially

con-tributingtothephenotypesobserved(Figure2c).Thus,it

seemsimportanttotakeintoaccountextracellular(ECM

as well as cell-cell contacts) effects when deciphering

thesedisorders.

Adhesivecomplexesandsignalingrelatedtofocal corticaldysplasia(FCD)

Perturbed mTOR signaling is also associated with PH

[50].Inaddition,somaticmutationsinthemTOR path-waycanleadtofocalcorticaldysplasia(FCD),associated

withabnormal neuronalmigrationandlocallyperturbed

cortical lamination. It was shown that hyperactivated

mTORsignaling disruptsciliogenesis and the

multi-to-bipolar transition (via altered Wnt signaling)disrupting hencecorticallamination[51].Perturbedneuronal

cilio-genesiswascausedbydisruptedautophagymakingalink

betweenthisprocess andcorrectcorticallamination. MutationsinPCDH19(acelladhesionmolecule,Table1) werealsoidentifiedinfemaleswithFCDassociatedwith

a thickened cortex and abnormal folding, as well as

epilepsy and intellectual disability phenotypes [52,53].

ArecentpapershowsthatPCDH19isregulatedbyTBR2

[54],atranscriptionfactorexpressedinIPs.TBR2 muta-tions cangiverise to perisylvian polymicrogyria, micro-cephalyandcorpuscallosumagenesis[55].Interestingly,

in Tbr2mousemutants,neuronsshow reducedlevelsof

Pcdh19 andbecome morelaterallydispersed, impairing

clonally-related synapse development [54]. Neurons in

theSVZ/IZshowincreasedprocessesandthisislikelyto

contribute to the dispersed tangential distribution.

Branching of leading processes and lateral dispersion

wasalsoanalyzedbyMartinez-Martinezetal.[56],finding

more intheferretthanmouse cortex.

Conclusions

Newstudiespresentedhereexplorethesubcellular

mech-anisms critical to either progenitors and/ or migrating

neurons.Complexityisrevealedwithpleiotropicproteins playingaroleinmultiplecelltypes.Conditionalmutants andsinglecellanalysesarecriticaltoidentifycell autono-mousphenotypes,withextracellularrolescontributingas welltonon-cellautonomousdefects.Futurestudiesneed to comprehensively addressthese differentpossibilities. Additionalasyetunidentifiedcentralplayersmayalsobe

hiddenduetohighheterogeneity.Newglobalapproaches

canhelptoidentifytheseactors,forexample,Luetal.[57]

whotookadvantageoftransposon-mediatedmutagenesis

in mouse,to exploregenes withpreviously unidentified

rolesincorticaldevelopment.Furtherunbiasedanalyses willhelpinterpretgeneticsdatafrompatientstoidentify rarevariants,helpingtopiecetogetherpathwaysimportant forcorticaldevelopment.

WediscussedheremechanismsaffectingMTsin

migrat-ingneurons. Theseand previousworksshow thatMTs

canbetoostraight(TUBA1A,[58]),notstraightenough

(TUBG1), too polymerized (KIF2A), too abundant

(CEP85L), not polymerized enough (TUBG1). There

hence canbemanyimpactsand theaffectedMT

com-partment in the cell (outside the centrosome) can be

variable. Independently of the way MTs are impaired,

commonglobalphenotypescanbeobserved(e.g.

neuro-nal migration). Of note, systematically analyzing each

stageof migration,forexample,themultipolar– bipolar transition,aswellasbipolarmigration,couldshedfurther lightoncontributingphenotypes.Post-translational

mod-ifications of MTs could also play a role and influence

corticalmalformations(e.g.acetylation,phosphorylation,

tubulinchaperones,whichallregulateMTs)andwecan

expectthatmutationsinsomeoftheseregulatingfactors willbeidentified(e.g.TBCD,[59]).Also,tangential(but

not radial) migration of interneurons has recentlybeen

shown to be fine-tuned by the CCP1 enzyme, which

promotesglutamylation,apost-translationalmodification of MTs [60]. Further data of this nature will almost

certainlyappear moreabundantly inthefuture.

Cortical malformations can be associated with intrinsic

mechanisms in migrating neurons and this seems less

complexthan,forexample,PHpathogenesis.Indeed,PH

proteinsmayinfluencetheactinandMTcytoskeletons,

adhesion,signalingaswellasECM,andcanhave

poten-tiallydifferentimpactsinprogenitorsandmigrating

neu-rons. Gene dosage and splicing, and primate-specific

isoforms, are further mechanisms to take into account

for understandingPHpathogenesis.ECMandadhesive

mechanismscouldrepresentahubforthisdisorderwith

downstreamsignalingimpactingthecytoskeleton.

Recent work from Long and colleagues [61] has

highlightedsomeoftheessentialspecies-specific

differ-encesinthecompositionofECM.Somehuman

compo-nentsarecriticalforlocalmodificationsinstiffness, sup-porting the generation of folds and fissures, typical of

gyrencephalic species. Extracellular signals have not

been deeply characterized in the context of neuronal

migration disorders but they could be an interesting

sourceof differencesbetweenmouse and humanand a

possible explanationofdiscrepanciesamongst the

mod-els. Modeling lissencephaly Karzbrun and colleagues

[62]showeda clearenrichmentin dysregulated ECM

genes in organoids derived from LIS1 patients.

progenitor-basedstructures,duetoalteredmechanicalforces,

possi-blymediatedbydifferencesinthematrixcomposition.

Brain folding, disrupted in many disorders, is a rather

complex process not present in rodents, and several

mechanismsunderlyingthisprocesshavebeenidentified:

apical RG detachment, essential for the formation of

bRGs [4]; local modifications of ECM components

[63];andadhesivemechanismsregulatingneuronal orga-nization,forexample, shownby theinactivationof Flrt

molecules in the mouse leading to abnormal neuron

clusteringandtheformationofgyri[64].Humanmodels

of PH, as well as progenitor abnormalities, can show

dysregulated neuronal adhesion/axon guidance

mole-cules, suggesting that processes mediating neuron-RG

celladhesioncouldcontributetothecorrectmigrationof

some neuron populations. Thus, processes associated

withbrainfoldingemergeaspotentiallyimportant

mech-anismsinneuronalmigrationdisordersandadapted

mod-els are required to study them [65]. Furthermore, each malformationcaneitherbediffusewith similarseverity acrossthecortex,or canshowan anterior–posterior gra-dient(Figure3).Moreinformationisrequiredonhuman

gene expression (genes and partners) to help explain

thesegradients[66,29].Theseperspectiveswillgreatly

contributeto decipheringpathomechanisms.

CRediT

author

statement

FFandSCwrotethisreview.

Conflict

of

interest

statement

Nothingdeclared.

Acknowledgements

FFwasassociatedwiththeBioPsyLabexprojectandtheEcoledes NeurosciencesdeParisIle-de-France(ENP)network.Salariesandthelab weresupportedbyInserm,Centrenationaldelarecherchescientifique (CNRS),SorbonneUniversity.FFandSCaresupportedbytheE-Rare-3 theERA-NetforResearchonRareDiseases(ERARE18-049,

HETEROMICS,ANRtoFF),aswellastheEuropeanCooperationon ScienceandTechnology(COSTActionCA16118NEURO-MIG).FFis supportedbytheJTC2015NeurodevelopmentalDisordersaffiliatedwith theANR(forNEURON8-Full-815-006STEM-MCD).

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