title authors - biorxiv · 2017/12/16 · (abou jamra et al., 2011; tesson et al., 2015). ap-4...

1

Title

AP-4mediatedATG9Asortingunderliesaxonalandautophagosomebiogenesisdefectsina

mousemodelofAP-4deficiencysyndrome

Authors

DavorIvankovic1,GuillermoLópez-Doménech1,JamesDrew1,SharonA.Tooze2,andJosefT.Kittler1,3.1Neuroscience,PhysiologyandPharmacology,UCL,London,WC1E6BT,UK2TheFrancisCrickInstitute,London,NW11AT,UK3Correspondingauthor:[email protected]

RunningTitle

NeuronalAP-4mediatedATG9Asorting

Keywords

AP4E1/HSP/mAtg9/SPG51/TGN

Abstract

Adaptorprotein(AP)complexeshavecriticalrolesintransmembraneproteinsorting.AP-4

remainspoorlyunderstood inthebraindespite its lossof function leadingtoahereditary

spasticparaplegia termedAP-4deficiencysyndrome.Herewedemonstrate thatknockout

(KO) of AP-4 in amousemodel leads to thinning of the corpus callosum and ventricular

enlargement,anatomicaldefectspreviouslydescribed inpatients.At thecellular level,we

findthatAP-4KOleadstodefectsinaxonalextensionandbranching,inadditiontoaberrant

distal swellings. Interestingly, we show that ATG9A, a key protein in autophagosome

maturation, is critically dependent on AP-4 for its sorting from the trans-golgi network.

FailureofAP-4mediatedATG9Asortingresults in itsdramatic retention in the trans-golgi

networkinvitroandinvivoleadingtoaspecificreductionoftheaxonalpoolofATG9A.Asa

result, autophagosome biogenesis is aberrant in the axon of AP-4 deficient neurons. The

specific alteration to axonal integrity and axonal autophagosome maturation in AP-4

knockoutneuronsmayunderpinthepathologyofAP-4deficiency.

was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted December 16, 2017. ; https://doi.org/10.1101/235101doi: bioRxiv preprint

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Introduction

Adaptor protein (AP) complexes have roles in the selection of transmembrane proteins

(cargo) for inclusion into vesicles. AP complexes interact with sorting motifs within the

cytoplasmic facing tails of cargoes, leading to their specific enrichment at sites on donor

membranes. Upon motif recognition and binding to cargoes, AP complexes recruit coat

proteinswhichassembletogeneratefreevesicles(Bonifacino,2014).Ofthefivemembers

of theAP complex family, AP-1 andAP-2 are the best understood thusfar, functioning in

clathrin-dependent sorting fromthe trans-golginetwork (TGN)andendocyctosis fromthe

plasmamembranerespectively.Assemblingashetero-tetramers,APcomplexesrequirethe

presenceof all subunits for their function (Dell’Angelicaet al., 1998;Hardieset al., 2015;

Mitsunari et al., 2005). Mutations in genes encoding all subunits of AP-4 (ε; AP4E1, β4;

AP4B1,μ4;AP4M1andσ4;AP4S1)havebeenidentifiedasleadingtoacomplexhereditary

spastic paraplegia (HSP) termed AP-4 deficiency syndrome (henceforth: AP-4 deficiency)

(AbouJamraetal.,2011;Tessonetal.,2015).AP-4deficiencypatientspresentwithearly-

onsetsevereintellectualdisability,absenceofspeechandprogressivespasticityleadingto

para-ortetraplegia(Abdollahpouretal.,2015).Anatomically,characteristicthinningofthe

corpus callosum and ventriculomegaly is evident in patients with AP-4 deficiency

(Abdollahpouretal.,2015;Moreno-De-Lucaetal.,2011;Verkerketal.,2009).Despitethis

severe pathology little is known of AP-4 other than its localisation to the TGN and its

clathrin-independence(Dell’Angelicaetal.,1999;Hirstetal.,1999).Thecargoessortedby

AP-4 inneuronsandthefunctionalconsequenceoftheiralteredhandlingandsubsequent

traffickingasa resultofdisruptionof theAP-4 complex remainpoorlyunderstood.Given

the known roles ofAP complexes in transmembraneprotein sorting, identifyingneuronal

AP-4 cargoes will lead to a better understanding of the mechanisms underlying the

pathologyinAP-4deficiency.

Macro-autophagy (henceforth: autophagy), the process by which organelles and

macromoleculesarerecycledforthemaintenanceofcellularhomeostasiscanbesimplified

into three fundamental steps; induction, autophagosome biogenesis and lysosomal

degradation. Progression through the autophagy pathway is in part mediated by the

concerted recruitment of autophagy related (Atg) proteins (Mizushima et al., 2011), the

sequenceandnecessityofwhichareconservedintheneuron(MadayandHolzbaur,2014).


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After the induction of autophagy, membrane elongation of sites on the endoplasmic

reticulum(ER)formsaphagophorewhichincorporatescytosoliccomponents(Ktistakisand

Tooze, 2016). Enclosure of the expanding edges of the phagophore produces a double-

membranedautophagosome,whichthenmayfusewithlateendosomesandlysosomesto

formdegradativeautolysosomes(Galluzzietal.,2017).Intactandefficientautophagyisof

critical importance to post-mitotic neurons which cannot overcome proteotoxic burden

throughcellulardivision(VijayanandVerstreken,2017).Theaxoninparticularrepresentsa

uniquelogisticalchallengeforautophagyduetoitsextremelengthandarchitecture(Ariosa

andKlionsky,2015).Indeed,neuronshavecompartmentalisedspecialisationofautophagy;

axonally derived autophagosomes exhibiting distinct maturation states from those

generated somatodendritically (Maday and Holzbaur, 2016). Autophagosomes are

constitutively generated in the distal axon (Maday et al., 2012), and are subsequently

retrogradelytraffickedtowardthesomafortheirclearancebyresidentlysosomesinorder

to prevent distal accumulation (Xie et al., 2015). Thus, machineries necessary for

autophagosome generation must be delivered to the distal axon to maintain effective

biogenesiswithinthiscompartment.Giventhis,ATG9A isofparticular interestasthesole

mammaliantransmembraneAtgidentifiedtodate,sinceitreliesuponvesicularsortingand

trafficking mechanisms for its distribution. Given the roles of ATG9A in phagophore

extension and autophagosomematuration (Webber and Tooze, 2010a; Karanasios et al.,

2016),itsefficientsortingandsubsequentdeliverytotheaxonmaybeofcriticalimportance

for the maintenance of constitutive generation of autophagosomes in the distal axon.

Intriguingly,inAP-4βnullmicemissortedAMPAreceptorsaccumulatedinautophagosomes

inaxonalswellingspositiveforLC3(Matsudaetal.,2008).Whetherafailure inautophagy

underliesthepathologyinAP-4deficiencyremainstobeascertained.

Here we identify neuroanatomical defects in an AP-4ε knockout mousemodel mirroring

those of AP-4 deficiency patients. Hippocampal neurons cultured fromAP-4ε KO animals

exhibit defects in axonal extension and branching, alongwith sites of distal swelling.We

also show that functional AP-4 is critical for TGN exit of ATG9A, loss of which results in

ATG9A retention within the TGN and reduction in axonal ATG9A, leading to aberrant

autophagosomematuration in thedistal axon. The impairmentof axonal autophagosome

biogenesismayunderpintheseverepathologyevidentinAP-4deficiencypatients.


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Results

AP-4ε(-/-)micerecapitulatecharacteristicanatomicaldefectsofAP-4deficiency

GiventhestarkanatomicalfeaturesofAP-4deficiency,wesoughttocharacteriseanAP-4ε(-

/-)mousemodeltoelucidatetheAP-4dependentmechanismsunderpinningthepathology

in this condition. Heterozygousmice carrying one copy of the targeting cassette (Fig 1A)

were crossed, giving litterswith AP-4ε(+/+), AP-4ε(-/-), and AP-4ε(-/+) (hereafterWT, KO and

HETrespectively).KOwasconfirmedbyPCR(Fig1B)andAP-4εproteinshowntobeabsent

inKOembryosatE16(Fig1B).Brainregionsofadultmicewerealsoinvestigated,andAP-4ε

showntobeabsentat theprotein level (Fig1C).Toexaminewhether lossofAP-4εalters

brainanatomy, sectionswereprepared fromWTandKOanimalsatP30andstainedwith

NeuN and GFAP revealing brain morphology (Fig 1D). KO brains exhibited striking

enlargementofthelateralventriclesatthistimepoint(relativearea:WT1±0.14,KO10.12

± 2.5, p = 0.0064; t-test). Staining axonal neurofilament-200 (NF200) (Fig 1F) revealed

thinning of both the corpus callosumanddorsal fornix, axonal tracts projecting from the

cortex and hippocampus respectively (Fig 1G; thickness corpus callosum:WT 181.1 ± 5.7

μm,KO123±6.9μm,p=0.0002.Fig1H;thicknessdorsalfornix:WT119.2±6.1μm,KO

88.8±8.9μm,p=0.0223;t-test).Theidentificationofenlargementofthelateralventricles

andconcurrentthinningofthecorpuscallosumarehighlyreminiscentofthecharacteristic

featuresofAP-4deficiencypatients(Abdollahpouretal.,2015),supportingAP-4ε(-/-)miceas

amodelofAP-4deficiency.

AxonspecificdefectsinAP-4ε(-/-)neurons

Wenextsoughttoestablishwhetherdefectsatthecellular levelwereresponsibleforthe

thinningofthecommissuralaxonaltractsinAP-4KOanimals.WeexaminedGFP-transfected

neurons atDIV-4 to evaluate the integrity ofAP-4KOaxons (Fig 2A). KO axons exhibited

reducedextension(Fig2B;length:WT963.5±76.2μm,KO679.2±51.9μm,p=0.0055;t-

test)andbranching(Fig2C;number:WT5.6±3.2,KO3.7±1.9,p=0.023;Mann-WhitneyU

testU test).Together these reductions likelyunderpin the thinningofaxonal tracts inKO

animals. In quantifying axonal length and branching parameters, we also noticed distal

axonalswellings inKOneurons (Fig2D-E;number:WT1.5±0.61,KO7.2±1,p<0.0001;

Mann-WhitneyUtest).Despitethesealterationstotheaxonatthisageinculture,nascent

dendriticprocessesexhibitednoalteration intotal length(Fig2F; length:WT411.6±37.6


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μm,KO384.5±19.1μm,p=0.502;t-test),norbranching(Fig2G;number:WT10.41±1.1,

KO 10.04 ± 0.77, p = 0.772; t-test). Whether AP-4 KO had an impact upon developed

dendrites was investigated by transfecting hippocampal neurons with GFP, revealing

dendriticmorphology atDIV-14 (Fig 2H). Investigation of the dendritic arbour using Sholl

analysisdidnotrevealanyalterationtothecomplexityofKOneurons(Fig2I;NSbetweenall

concentric 10 μm regions; Two-way ANOVA), nor were any alterations in total dendritic

length (Fig 2J; length:WT 2807 ± 326.1 μm, KO 2730 ± 324.2 μm, p = 0.87; t-test), nor

branchesperneuron(Fig2K;number:WT55.6±4.9,KO51.3±5.1,p=0.56;t-test)found.

Notably we did not observe any dendritic swellings, despite axonal swellings still being

evidentatthistimepointinKOneurons(FigS1;per100μm:WT0.23±0.04,KO1.1±0.2,p

=0.005;t-test).Togethertheseparametershighlightthespecificalterationintheintegrity

ofaxonsinAP-4KOneurons,whereastherewasnoalterationtodendriticcomplexitynor

integrity.

ATG9AaccumulatesinAP-4εKOneurons

Interestingly,axonalswellingsreminiscentofthosewedescribehere inAP-4εKOneurons

have been identified in autophagy deficient models (Hara et al., 2006; Nishiyama et al.,

2007). Moreover, the transmembrane protein ATG9A was identified as a putative AP-4

interactorbymassspectroscopy(Matteraetal.,2015),suggestingthatATG9Atraffickingor

sortingmaybe altered inAP-4εKOneurons.We confirmedbiochemically the interaction

betweenATG9AandtheAP-4complexbyco-IPofATG9AandAP-4fromadultmousebrain

(Fig3A),suggestingthatATG9Ais indeedanAP-4cargointhebrain. Infurthersupportof

thiswefoundthatATG9AwasincreasedatproteinlevelinKOhippocampusatP30(Fig3B-

C;relativeprotein:WT1±0.23,KO2.33±0.05,p=0.0046;t-test),suggestingthatATG9A

levelsareaffectedbythelossofAP-4function.Inaccordancewiththis,insectionsprepared

at P30 we found that ATG9A accumulated in AP-4 KO mice within distinct structures in

neuronal cell layers (Fig 3D, S2), highlighting alteration to ATG9A localisation in vivo. To

better understand and confirm this accumulation within KO neurons, ATG9A levels and

localisation were examined in cultured hippocampal neurons. We found near 3-fold

accumulationofATG9AinKOneurons,andstarkretentionwithinareticularstructureinthe

soma(Fig3E-F;relativesignal:WT1±0.06,KO2.8±0.2,p<0.0001;t-test).Thesefindings


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provideevidencetowardsacriticalroleforAP-4inATG9Ahandlinginneuronsbothinvivo

andinculture.

FunctionalAP-4iscriticalforATG9AexitfromtheTGNinneurons

The known functions of AP complexes in transmembrane protein sorting and AP-4

localisationtotheTGNincelllinesleadustoinvestigatewhetherthesomaticaccumulation

ofATG9AwasduetoitsretentionwithintheTGNasaresultofthelossoffunctionofAP-4.

We firstly confirmedneuronalAP-4 localisation to theTGNandvesicles arising from it as

evidencedbyoverlapofAP-4εwiththeTGNmarkerGolgin-97(Golg97)(FigS3),supporting

aroleforAP-4intransmembraneproteinsortingfromtheTGNinneurons.Wenextsought

toidentifythecompartmentwithinwhichATG9AwasretainedincultureandinvivoinAP-

4εKO.Super-resolutionstructured-illumination imaging (SIM)of culturedneurons stained

against cis-golgi (GM-130) and TGN (Golg97) markers revealed ATG9A to be highly

associatedwiththeTGNinKOneurons(Fig4A-B),whereasinWTneuronsATG9Aexhibited

avesicularlocalisation.ThereticularATG9AstructuresevidentinKOneuronsinvivoandin

culturethusrepresentapoolofATG9AthatisretainedwithintheTGN,indicatingacritical

roleforAP-4inATG9AexitfromtheTGN.

WenextsoughttoestablishwhetherTGNATG9AretentionisafeatureinAP-4deficiency,

througharescueexperimentusingapathologicalmutationidentifiedinanAP-4deficiency

cohort (Fig S4) (Najmabadi et al., 2011). This homozygous 2-bp insertion resulting in

frameshift and premature stop was identified in a consanguineous family with 3 of 4

childrenpresentingwithsevereintellectualdisability,microcephalyandspasticparaplegia.

ThismutationresultsinterminationofAP-4εwithinthetrunkdomainatV454(FigS4)which

islikelynecessaryforcorrectAPcomplexassembly,stabilityandthusfunction(Pedenetal.,

2002). Given this, we hypothesised that the V454X-ε pathological mutant would fail to

rescueATG9Alocalisation inAP-4KOneurons.Tothisend,Myc-taggedfull-lengthε(FL-ε)

and pathology associatedV454X-εwere transfected in KOneurons, andATG9A retention

within the soma examined (Fig 4C, Fig S4). Restoration of ATG9A levels and vesicular

localisationwas evidentwith expression of FL-ε,whereas ATG9A remained TGN retained

when V454X-εwas expressed, to a similar level as untransfected cells (Fig 4C-D; relative

signal:FL1±0.06,V454X2.48±0.22,UT2.81±0.19,p<0.0001V454X/UTtoFL,NSbetween


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V454XandUT;Kruskall-Wallis).ThesefindingshighlightthenecessityoffunctionalAP-4for

ATG9Asorting,failureofwhichresultsinTGNATG9AretentioninAP-4KOneuronsandAP-4

deficiency.

ATG9ATGNconstraintresultsindefectiveaxonalautophagosomematuration

GiventhekeyrolesofATG9Ainautophagosomebiogenesis(Karanasiosetal.,2016;Orsiet

al.,2012;WebberandTooze,2010b)andovertaxonalswellingsreminiscentofautophagy

deficient models in AP-4 KO axons, we hypothesised that autophagosome biogenesis is

defectiveinKOneuronsasaresultoftheimpairedTGNexitofATG9A.Indeed,wefindthat

axonaldeliveryofATG9AisreducedinKOneurons(Fig5A,C;vesiclesper10μm2:WT5.56±

0.41, KO 3.49 ± 0.39, p = 0.0018; t-test), whereas dendritic ATG9A vesicle number is

unaffected(Fig5A,B;vesiclesper10μm2:WT4.47±0.25,KO4.18±0.26,p=0.43;t-test).

ThefailureofAP-4mediatedATG9AsortingfromtheTGNthusresultsinspecificreduction

ofATG9Atraffickingtotheaxon,whichmayaffectthecapacityofaxonalautophagosome

biogenesis. To further investigate this we imaged the dynamics of LC3 (Fig 5D), which

associateswithautophagosomes fromearly through to latematurationstatesprovidinga

robustmarkerfortrackingautophagosomesthroughouttheirlifespan(MadayandHolzbaur,

2014).NeuronsweretransfectedwithRFP-LC3andmoviescapturedwithinthedistalmost

100 μm of axon (Movies S1 and S2). Upon completion of biogenesis, autophagosomes

generatedinthedistalaxonmustbetraffickedretrogradelytowardslysosomesresidentin

thesoma for theirdegradation (Chengetal.,2015). InKOaxons,motileautophagosomes

were found to exhibit reduced absolute retrograde displacement (Fig 5E; retrograde

displacement:WT10.43±1.99μm,KO1.139±1.42μm,p<0.0001;Mann-WhitneyUtest).

Additionally, themean retrograde run length per autophagosomewas reduced, whereas

anterograderunlengthswereunaltered(Fig5F;anterogradelength:WT15.89±1.28μm,

KO11.96±1.16μm,p=0.686.retrograde length:WT25.75±1.59μm,KO12.58±1.055

μm,p<0.0001;Mann-WhitneyUtest).Theseanalysesidentifiedaspecificreductioninthe

propensityofautophagosomes tomove toward thesoma inKOaxons, suggestingaltered

maturation state. We also found that autophagosomes in KO axons were less motile,

exhibitingreductionintotaldistancetravelled(Fig5G;length:WT41.64±2.11,KO24.54±

1.72, p < 0.0001; Mann-Whitney U test). Indeed, KO autophagosomes spent more time

stationary(Fig5H%time:WT58.75±1.6%,KO65.82±2.077%,p=0.0055;Mann-Whitney


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Utest)andexclusivelylesstimemovingretrogradely(Fig5H;%timeretrograde:WT23.45±

1.29%,KO15.52±1.13%,p=0.0003.%timeanterograde:WT17.80±1.17%,KO18.66±

1.55%,p=0.45;Mann-WhitneyUtest).Takentogether,weidentifyautophagosomestobe

lessmotile, and a specific reduction in the propensity tomove retrogradely in distal KO

axons.Thesefindingssuggestalteredkineticsofautophagosomesduringbiogenesis inthe

distalaxon,inaccordancewiththespecificreductioninaxonalATG9Aandaccumulationin

thesoma.


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Discussion

Despite the mounting evidence of impaired autophagy in neurodevelopmental and

neurodegenerativedisease (Alvarez-Ervitietal.,2010;Leeetal.,2011;Nixonetal.,2005;

Shibata et al., 2006; Winslow et al., 2010; Xie et al., 2015), mechanistically autophagy

remains poorly understood in the neuron.Neurons have a limited capacity to upregulate

autophagy (Maday and Holzbaur, 2016), and as such may be particularly vulnerable to

impairedautophagicflux.Giventhespatialrestrictionofautophagosomebiogenesisandthe

extremelengthoftheaxon,itiscriticalthatthedeliveryofnewlysynthesizedcargoesfrom

thesoma is tightlybalancedwithefficientclearancetopreventaccumulationat thedistal

axon. In the present study, we identify a critical role of AP-4 in axonal autophagosome

biogenesisthroughthesortingofATG9AfromtheTGN.DefectiveaxonaldeliveryofATG9A

whereAP-4functionislostresultsinaberrantautophagosomematurationinthedistalaxon

which may underpin the defects in axonal integrity in AP-4 deficiency. These findings

strengthentheemerginglinksbetweenHSPandautophagy(Changetal.,2014;Khundadze

etal.,2013;Oz-Levietal.,2012;Vantaggiatoetal.,2013;Vargaetal.,2015),highlighting

theimportanceofautophagyinthedevelopmentandmaintenanceofaxonalintegrity.

Autophagosomegenerationisconstitutivewithintheaxon,predominantlyoccurringwithin

thedistal-mostregionsandatpresynapticsites(MadayandHolzbaur,2014;Okerlundetal.,

2017). Maturing autophagosomes initially exhibit bidirectional movement (Maday et al.,

2012)priortoswitchingtorobustdyneindrivenretrogrademovementmediatedbyJIP1(Fu

etal.,2014).Interestingly,wefoundthatafunctionalconsequenceofthereductioninthe

axonaldeliveryofATG9AinAP-4εKOneuronswerealterationstoautophagosomekinetics

inthedistalaxon.Autophagosomes inKOaxonswerenotonly lessmotile,butalsospent

proportionally less time moving retrogradely and exhibited reduced net retrograde

displacement. This specific reduction in the propensity of autophagosomes to move

retrogradely in KO axons supports impaired autophagosome maturation in this

compartment,inaccordancewiththespecificreductionintheprovisionofATG9A.Indeed,

in C.eleganswhere AP-4 is not evolutionarily conserved (Boehm and Bonifacino, 2002),

axonal delivery of ATG9A is also critical for axonal autophagosome biogenesis and axon

outgrowth (Stavoe et al., 2016). It is also of note that in AP-4β deficient neurons AMPA

receptorsaremissortedtoaxons,wheretheyco-localisewithLC3positiveautophagosomal


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accumulations through altered TARP (transmembrane AMPA receptor regulatory protein)

dependent sorting (Matsuda et al., 2008). Given our identification of defective axonal

autophagosomebiogenesisinAP-4εKOneurons,theselikelyaccumulateasaresultofthe

impairedautophagosomeclearanceduetoATG9ATGNretention.Whilstwecannotentirely

rule out whether another unidentified AP-4 cargo contributes to alterations in

autophagosomegenerationevidentintheaxon,theknownrolesofATG9Amakeitaprime

candidateinourAP-4deficiencymodel.Indeed,inagreementwiththisATG9Alossleadsto

axon swellings and thin corpus callosum in a CNS-specific knockout mouse model

(Yamaguchietal.2017).

ThedistalaxonalswellingevidentinAP-4εKOneuronshavealsobeenidentifiedinstudies

wherecomponents critical for theearly stagesofautophagosomebiogenesisareablated,

namely ATG5 and ATG7 (Nishiyama et al. 2007; Komatsu et al. 2006). Given that

autophagosomesmature from ER sites (Ktistakis and Tooze, 2016;Maday and Holzbaur,

2014), it is intriguing that accumulation of expanded ER and ‘autophagosome-like’

structuresareobservedintheswellingsofATG5nullaxons(Nishiyamaetal.,2007)andthat

ER expansion is evident where ATG5 or Beclin-1 are silenced (Khaminets et al., 2015).

NotablyAtlastin-1,REEP1andspastin,accountingforover50%ofHSP,allhaverolesinER

shapingandremodeling(Botzolakisetal.,2011;Montenegroetal.,2012;Parketal.,2010;

RenvoiséandBlackstone,2010), andaxonal swellingshavealsobeen identified in several

HSP models (Fassier et al., 2013; Tarrade et al., 2006;Watanabe et al., 2013). Whether

axonal swellings comprise expanded ER as a result of impaired axonal autophagosome

biogenesis remains tobeelucidated. In thepresent study,our results are consistentwith

axonal swellingsarisingasa resultof slowedorstalledautophagosomematuration in the

axonsofAP-4εKOneurons.

70%ofcomplexHSPpatientspresentingwithprogressivespasticity,intellectualimpairment

and thin corpus callosumare accounted for bymutations in SPG11 and SPG15, encoding

spatacsin and spastizin respectively (Tesson et al., 2015). Notably, both spatacsin and

spastizin have roles in autophagosome maturation and endolysosomal function

(Vantaggiato et al., 2013). More recently, thin corpus callosum has been identified in

autophagy-relatedCNS specific KOmousemodels includingULK1/ULK2dKO (Wanget al.,


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2017;Yamaguchietal.,2017).Importantly,axonalextensionisreducedinculturedprimary

neuronspreparedfromalloftheselines(Khundadzeetal.,2013;Pérez-Brangulíetal.,2014;

Yamaguchi et al., 2017; Zhou et al., 2007). Given this similarity to AP-4ε KOmice in thin

corpuscallosumandconcomitantreductioninaxonalextension,weproposethatthinning

ofthecorpuscallosuminAP-4deficiency isduetotheaxonalextensiondefectelicitedby

failureofAP-4mediatedATG9Asorting.Notably,constitutiveknockoutofbothATG9Aand

ULK1 leads to peri-natal lethality (Cheong et al., 2014; Kojima et al., 2015; Saitoh et al.,

2009). Thus in AP-4 deficiency, the remaining pools of ATG9A that are not TGN retained

likelyaccountforthesurvivalofAP-4deficiencypatientsandAP-4εKOmiceintoadulthood.

Wespeculate that thedramatic increase inATG9Awithin theTGNmembrane leads to its

stochastic incorporationintovesiclesemanatingfromtheTGNmediatedbyothercarriers.

As a result, sufficient vesicular ATG9A is delivered somatodendritically in KO neurons to

maintaineffectiveautophagywithinthiscompartment.Inaccordancewiththisthereisno

defect indendritic integrity inAP-4εKOneuronsdespitetheovertaxonaldefectsevident.

Alternatively,dendriticdeliveryofATG9Amaybemediatedbyanothercarrier,whichwould

alsoaccountforthemaintenanceofthedendriticATG9Apool.Specificaxonalexclusionof

somatodendritically destined ATG9A vesicles at the peri-axonal exclusion zone (PAEZ)

(Farías et al., 2015) or the trafficking challenge posed by the axon may account for the

reducedprovisionofATG9Atothedistalaxoninthisscenario.

During the preparation of thismanuscript,we became aware of a study identifying AP-4

mediatedTGNsortingofATG9A inmousefibroblastandHEKcell linesresulting inslowed

autophagosome biogenesis (Mattera et al., 2017). The additional logistical challenge

imposed by the great length of the axon would make autophagosome generation more

critically dependent onAP-4mediated TGNexit of ATG9A for neurons. Thiswork further

supportsourfindingsinvitroandinvivo,lendingweighttotheimplicationofthefailureof

AP-4mediatedATG9AsortinginourmousemodelofAP-4deficiency.

Insummary,thepresentstudyrevealsacriticalroleofAP-4insortingATG9AfromtheTGN

in neurons. Impairment of this function as evident in our AP-4 deficiencymodel leads to

accumulationofATG9AwithintheTGNinvivoandinvitro,leadingtoaspecificreductionto

theaxonaldeliveryofATG9A.Resultingdefectiveaxonalautophagosomebiogenesis likely


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underlies the axonal defects evident in AP-4ε KO mice, providing evidence towards a

mechanismofpathologyinAP-4deficiency.


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MaterialsandMethods

Animals

AP-4ε knockout (AP4E1-/- C57BL/6J [Ap4e1tm1b(KOMP)Wtsi], KO) were generated using the

knockout-first tm1a allele system (Skarnes et al., 2011) by the International Mouse

PhenotypingConsortium (IMPC)MRCHarwell.Animalsweremaintainedunder controlled

12:12hour light-darkcyclesatatemperatureof20±2oCwithfoodandwaterad libitum.

Genotyping was carried out using the following primers; AP4E1-5arm-WTF:

GCCTCTGTTTAGTTTGCGATG, AP4E1-Crit-WTR: CGTGCACAGACAGGTTTGAT and 5mut-R1:

GAACTTCGGAATAGGAACTTCG. Littermate matched controls were used for primary

neuronal cultures and immunocytochemistry experiments. All experimental procedures

were in accordance with UCL institutional animal welfare guidelines, and under the UK

HomeOfficelicenceinaccordancewiththeAnimals(ScientificProcedures)Act1986.

AntibodiesandDNAConstructs

Antibodies: For immunocytochemistry (ICC), Immunohistochemistry (IHC) and western

blotting (WB)antibodieswereusedwith the followingdilutions;Actin (SigmaA2066;WB:

1/1,000), AP-4ε (BDBiosciences 612018;WB: 1/300, ICC: 1/250), ATG9A (Rabbit STO-219

WB:1/2000,IF:1/2000(Youngetal.,2006)),ATG9A(Hamster14F28B1;IF:1/500(Younget

al., 2006)),GFP (Nescalai Tesque04404-84; ICC: 1/1000),GFAP (Dako Z0334; ICC: 1/300),

GM130 (BD Biosciences 610822; ICC: 1/1000), Golgin-97 (CST 13193; ICC: 1/250), MAP2

(Synaptic Systems 188-004, ICC: 1/500), NeuN (Chemicon MAB377; IHC: 1/300), NF200

(Abcamab4680; IF:1/500IHC:1/500)andMYC(NeuroMab9E10;WB:1/100, ICC:1/100).

HRP-Conjugatedanti-mouse/rabbitantibodieswereused forwesternblottingat1/10,000

(JacksonLaboratories).FluorescentAlexaFluorconjugatedsecondaryantibodies(Invitrogen

andAbcam) for ICC, IHC and Super-resolution imagingwereused as follows; anti-chicken

405and647, anti-GuineaPig 405and647, anti-Mouse488and647, anti-Rabbit 555and

647. Anti-Armenian Hamster conjugated to Cy3 was used for super-resolution imaging

(Jackson).

DNA Constructs: CAG-GFP (Addgene plasmid #16664), pmRFP-LC3 (Atkin et al.,

2012)(Addgeneplasmid#21075). Full-lengthN-terminallyMyc taggedεwasgeneratedby

cloning the coding sequence of AP4E1 (Cusabio; CSB-CL890772HU, cDNA clone


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MGC:163338) into pRK6-Myc. V454X-ε was then generated by reverse mutagenesis

methods replicating the reported 2 nucleotide insertion leading to frameshift induced

prematurestopatV454(Najmabadietal.2011).

BrainlysatepreparationforCo-Immunoprecipitation

Lysate Preparation: Brains to be used for co-immunoprecipitation were removed from

animalsandhomogenizedinicecoldHEPESbuffer(50mMHEPES,0.5%tritonx-100,150mM

NaCl, 1mM EDTA, 1mM PMSF, 50μl Antipain/Pepstatin/Leupeptin in ddH2O). Lysate was

solubilisedby rotation for2hoursat4oCprior toultracentrifugationat38,000rpm for40

minutes.ProteincontentdeterminedusingaBCAassaykit(Promega).

Co-Immunoprecipitation:5mgofbrainlysatewasincubatedwith1μgofantibodyinHEPES

buffer for 12hat 4oCwith rotation, and1μg IgG control (rabbit)was incubatedwithWT

brain lysate in tandem. Input sampleswere incubated in the samemanneratall stepsas

immunoprecipitationsamples.ProteinAagarosebeads(Generon)wereaddedfor4hours

to IP samples, beads washed in HEPES buffer and suspended in protein sample buffer

(150mMtrispH8,6%SDS,300mMDTT,30%glycerol,0.3%bromophenolblue)andheated

to95oCfor7minutespriortoSDS-Pageandwesternblotting.

BrainLysatepreparation,SDS-PageandWesternBlotting

LysatePreparation:Brainstobeusedforwesternblottingwereremovedfromanimalsand

snap frozen at -80oC. For preparation of lysates, brains were defrosted, relevant regions

dissectedandkeptonicethroughout.Tissuewashomogenizedbysonicationinlysisbuffer

(50mM HEPES, 0.5% Triton-X100, 150mM NaCl, 1mM EDTA, 1mM PMSF and Antipain,

pepstatin and leupeptin), and debris pelleted at 38,000g for 10 minutes at 4oC. Lysate

proteincontentwasdeterminedusingacommercialBCAassaykit(Promeaga)andsamples

denaturedfor7minutesat95oCinproteinsamplebuffer.Sampleswerestoredat-20oCor-

80oC.

SDS-PageandWesternBlotting:20-40μgofproteinofproteinlysatewasseparatedbySDS-

PAGE using XCell Minicell II systems (Novex) and transferred onto nitrocellulose (GE

healthcare)or0.45μmporePVDF(forLC3,GEHealthcare).Membraneswereblockedinmilk

(4%non-fatmilkpowder,0.05%Tween-20 inPBS) for1hourand incubatedwithprimary


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15

antibodies at empirically determined dilutions as above overnight with agitation at 4oC.

Membranes were then washed, secondary HRP-conjugated antibodies applied in milk at

1/10,000 and after a finalwashing steps bands visualised by application of ECL substrate

(LuminataCrescendo,Millipore)andimagingusingaCCDbasedsystem(QuantLAS4000,GE

Healthcare).DensitometricanalysiswasperformedusingFIJIsoftware(NIH).

Hippocampalneuronalcultureandtransienttransfection

Hippocampalneuronalcultures:HippocampalculturesfromcrossesofheterozygousAP-4ε

animals were prepared from embryos at E16 as described previously (Davenport et al.,

2017; López-Doménech et al., 2016; Vaccaro et al., 2017). Briefly, hippocampi were

dissectedinice-coldHBSS(Gibco)supplementedwith10mMHEPESandincubatedin0.25%

trypsin for 15minutes prior to trituration.Dissociated neuronswere seededonto Poly-L-

Lysine (0.5mg/ml in 0.1M borate buffer, pH 8) coated coverslips at a density of 30-

50,000/cm2 in attachment medium (10% horse serum, 10mM sodium pyruvate, 0.6%

glucose inMEM (Gibco).Attachmentmediawas replaced thenextdaywithMaintenance

medium(2%B27,2mMglutamax,100μg/mlPenicillin/StreptomycininNeurobasal(Gibco).

50%of themaintenancemedium replacedevery4days after the firstweek in culture to

maintaincellhealth.

Transienttransfection:Neuronsweretransfectedusinglipofectamine2000(ThermoFisher)

according tomanufacturersprotocols,atanempiricallydeterminedratioof lipofectamine

to DNA per construct used (GFP 0.25μg, RFP-LC3 0.25μg and ε constructs 1μg per 2

coverslips,1μllipofectaminepercoverslip).Neuronswerelefttoexpressconstructsfor2-3

dayspriortofurtherexperimentation.

Immunocytochemistryandimmunohistochemistry

Immunocytochemistry(ICC):Hippocampalculturesoncoverslipswerefixedpriortostaining

with 4% PFA with 4% sucrose in PBS for 7 minutes at RT. Post-fixation coverslips were

washedinPBSandpermeabilisedfor10minutesinblockingsolution(1%BSA,10%horse

serum, 0.1% Triton-X100 in PBS). Primary antibodieswere diluted in blocking solution at

empiricallydetermineddilutionsandappliedfor1houratRTinadarkhumidifiedchamber.

Coverslipswerewashed inPBSand fluorescent-conjugated secondary antibodies as listed

abovewereusedataconcentrationof1/1000andappliedfor1houratRTinahumidified


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chamber.CoverslipsweremountedinProLongGoldmountingmedium(Invitrogen,P36930)

andallowedtodryovernightatRTpriortoimaging.

Immunohistochemistry(IHC):Brainswereremovedfromanimalsandfixedbyimmersionin

4%PFAfor24hat4oC,cryoprotectedin30%Sucrose-PBSfor24handfrozenandstoredat-

80oC. Frozen brains were embedded into OCT compound and serially cryosectioned into

30μmsectionsinaBrightOTF-ASCryostat(BrightInstruments)andstoredat-20oCpriorto

staining in cryoprotective solution (30% Glycerol, 30% PEG in PBS). IHC staining was

performedwithfree-floatingsectionsatRTwithgentleagitation.Sectionswerewashedand

permeabilised inPBS-Tx (0.5%Triton-X100 inPBS) for30minutesprior toblocking in IHC

blockingsolution(3%BSA,10%FetalBovineSerum,0.2MGlycineinPBS-Tx)for3hours.A

secondblockwasappliedfor3hoursaspriorbutwiththeadditionofgoatanti-mouseFab-

fragment(JacksonImmunoresearch)at50μg/mltoreducedendogenousbackgroundwhen

using antibodies raised in mouse. Sections were washed for 30 minutes and primary

antibodies applied at concentrations as listed above in IHC blocking solution for 4 hours.

Sectionswerewashedfor30minutesandfluorescentantibodiesappliedfor4hourspriorto

mountingontoglassslideswithMowiol(Calbiochem)medium.Slideswereallowedtodryat

RTovernightpriortoimaging.

ImageAnalysis

Allimagingandimageanalysistechniqueswereperformedblinded.AllWTandKOembryos

generated per genotype were used, and cell numbers kept consistent between embryos

ratherthangenotypes(asaresultofblindingatacquisitionstage).Between3and6images

weretakenperconditionandsamplessizeskeptconsistentacrossexperimentaltechniques.

AllmicroscopicimagingunlessstatedotherwisewasusinganuprightZeissLSM700upright

confocalmicroscope.ImagesweredigitallycapturedusingZen2010Software(Zeiss),using

oilimmersionobjectives:63x;1.4NA,40x1.3NAandairobjectives;10x0.3NA,5x0.16NA.

Brain measurements: Quantification of the thickness of the corpus callosum and dorsal

fornixwasperformedmanuallyusingFiji.Atleast2brainsectionsperanimalwereanalysed

andthemeanmeasurementusedastherepresentativevalue.

AxonalLength,Branchingandswellings:GFP-filledneuronsatDIV-4werefixedandimaged

usinga40xobjective,andimagesstitchedusing‘MosaicJ’or‘Pairwisestitching’(Preibischet

al., 2009) plugins in FIJI as required. Entire lengths of axons including all branches was


https://doi.org/10.1101/235101

17

measuredmanuallyusingFIJI,andbranchesquantifiedexcludinganyprocessbelow20μm.

Axonal swellings were defined as a compartment >2x the width of the axon shaft, and

numbers counted manually. For DIV 14 swellings quantification, fields of view were

captured, total axonal length and numbers of swellings presentwithin the field captured

werecountedtodetermineswellingsper100μmofaxon.

Dendriticmorphologyandcomplexity:DIV14GFP-filledneuronswerefixedandimaged,and

images stitched as previously described where necessary. Dendritic morphology was

reconstructedusingNeuronstudio (CNIC) and inbuilt analysis toolsused to ascertain total

dendriticlengthandbranchesasdescribedpreviously(Norkettetal.,2016;Pathaniaetal.,

2014).Shollanalysisofintersectionswasperformedusingthe‘SimpleNeuriteTracer’plugin

inFIJI,withashollradiusof10μm.Imageswerestitchedwherenecessary.

Nascentdendriticlengthandbranching:DIV-4GFPfilledneuronswerereconstructedusing

Neuronstudio and total length and branches of nascent dendritic processes per neuron

quantifiedusinginbuilttools.

ICC quantification of total fluorescent signal: For quantification of dendritic and axonal

vesiclenumbers,regionspositiveforcompartmentmarkers(MAP2andNF200respectively)

were outlined manually per image, and values normalised to area. Total fluorescence

(ATG9Ainsoma)wasquantifiedbyoutliningthecellsomaandmeasuringtotalfluorescence

usinginbuiltFIJItools.

LiveImagingandautophagosomemotilityanalysis

Live Imagingof autophagosomematuration: For autophagosomematuration andmotility

experiments, cultured hippocampal neurons were transfected at DIV-4 with RFP-LC3 as

described, to be imaged at DIV 6-7. Imaging was carried out under perfusion with ACSF

(124mMNaCl2, 2.5mMCaCl2, 2.5mMKCl, 1mMMgCl2, 10mMD-Glucose,25mMNaHCO3,

1mM NaHPO4) at 37oC with a flow-rate of 1-2ml/min and aerated (5% CO2, 95% O2)

throughout.Growth-coneswereidentifiedandtheRFP-LC3signalinatleastthedistalmost

100μmofaxoncapturedusingaEM-CCDcamerasystem(iXon,Andortechnology)mounted

toanOlympusmicroscope(BX60M)witha60xobjective,asdescribedpreviously(Norkett

etal.,2016).Amercuryarc lampwithfilteringprovidedexcitationoftheRFPfluorophore

(Cairn Research). Images were acquired using MicroManager (Opensource, Micro-

manager.org)(Edelsteinetal.,2014)for6minutesat1frameevery1.5s.


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18

Autophagosome motility analysis: Movies generated from distal axons used to generate

kymographs using the ‘Multiple Kymograph’ plugin. Resulting kymographs represent

autophagosomemotionastimeontheyaxis(1.5s/px)anddistanceonthex(0.1333μm/px).

Trajectoriesweremanuallytrackedandanalysedusinganin-houseMATLABscript.Briefly,

themotionof an autophagosome is possible to defineprecisely by thepositional change

fromco-ordinates x1/y1 to x2/y2.Calculatingall of the individual trajectory changes foran

individual autophagosome’s trackwewere able to ascertain; velocity, proportion of time

spent moving, directionality etc. Per track, portions of time spent moving at less than

0.05μm/swereclassedasstationary.

StructuredIlluminationImaging(SIM)

SIMwasperformedonacommerciallydevelopedZeissElyraPS.1invertedmicroscopeusing

aZeiss63xoilobjectivelens(NA:1.4)andpco.edgeCMOScameraandZENBlacksoftware

(Zeiss)asdescribedpreviously (Davenportetal.,2017;Norkettetal.,2016). Imageswere

captured using SIMparadigms (34-μm grating, three rotations and five lateral shifts) and

processedusing the SIM reconstructionmodulewithin ZENBlackwith default theoretical

PSFandothersettings.Shiftsbetweenacquiredchannelswerecorrectedforusing100nm

Tetraspecfluorescentmicrospheres(MolecularProbes).

StatisticalAnalysis

ResultswereanalysedusingGraphpadPrism6(GraphpadSoftwareInc).Dataispresented

asmean±SEM.Wherenormalized,valuesarepresentedrelativetotheaverageofcontrol

valuesunlessstatedotherwise.Datawastestedfornormalitypriortostatisticaltesting,and

appropriatestatisticaltestsused.Fordifferencesbetweentwogroupsstatisticalsignificance

wasdeterminedusingunpaired two-tailedStudent’s t-testswhenparametric.Twogroups

were tested using two-tailedMann-Whitney U tests where at least one group was non-

parametric. For threeormoregroups, statistical significancewasdeterminedby two-way

ANOVAs with Bonferroni post-hoc testing where data was parametric. Kruskall-Wallis H

testswereusedforcomparisonofthreeormoregroupswhereatleastonegroupwasnon-

parametric.Significanceisrepresentedas;p*<0.05,p**<0.01andp***<0.001.


https://doi.org/10.1101/235101

19

Acknowledgements

TheauthorswouldliketothankallmembersoftheKittlerLabforinvaluablediscussionsand

suggestions.WeextendthankstoLorenaArancibia-Carcamoforsupportwithscriptdesign

andanalysismethodologies.ThisworkwassupportedbygrantsfromtheMedicalResearch

Council(MR/N025644/1)andERC(FuellingSynapses)toJ.T.K..D.I.andJ.D.wereontheUCL

ClinicalNeuroscienceProgramfundedbyaBrainResearchTrustPhDScholarshipandMRC

PhD studentship, respectively.We thank theUCL Super-resolution Facility (fundedby the

MRCNextGenerationOpticalMicroscopy Initiative) and theMRC LMCB LightMicroscopy

stafffortheircontributions.

AuthorContributions

ThisstudywasconceivedbyD.I.andJ.T.K.ExperimentsweredesignedD.I.,G.L.D.andJ.T.K.

andperformedandanalysedbyD.I.andG.L.D.AnalysisscriptsweredevelopedbyJ.D.and

D.I..S.T.providedessentialadvice,toolsandreagents.D.I.andJ.T.K.wrotethepaper.

ConflictofInterest

Theauthorsdeclarenoconflictsofinterest


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References:

Abdollahpour,H.,Alawi,M.,Kortüm,F.,Beckstette,M.,Seemanova,E.,Komárek,V.,Rosenberger,G.,andKutsche,K.(2015).AnAP4B1frameshiftmutationinsiblingswithintellectualdisabilityandspastictetraplegiafurtherdelineatestheAP-4deficiencysyndrome.EurJHumGenet23,256–259.AbouJamra,R.,Philippe,O.,Raas-Rothschild,A.,Eck,S.H.,Graf,E.,Buchert,R.,Borck,G.,Ekici,A.,Brockschmidt,F.F.,Nöthen,M.M.,etal.(2011).Adaptorproteincomplex4deficiencycausessevereautosomal-recessiveintellectualdisability,progressivespasticparaplegia,shycharacter,andshortstature.AmJHumGenet88,788–795.Alvarez-Erviti,L.,Rodriguez-Oroz,M.C.,Cooper,J.M.,Caballero,C.,Ferrer,I.,Obeso,J.A.,andSchapira,A.H.V.(2010).Chaperone-mediatedautophagymarkersinParkinsondiseasebrains.ArchNeurol67,1464–1472.Ariosa,A.R.,andKlionsky,D.J.(2015).Long-distanceautophagy.Autophagy11,193–194.Atkin,T.A.,Brandon,N.J.,andKittler,J.T.(2012).DisruptedinSchizophrenia1formspathologicalaggresomesthatdisruptitsfunctioninintracellulartransport.HumMolGenet21,2017–2028.Boehm,M.,andBonifacino,J.S.(2002).Geneticanalysesofadaptinfunctionfromyeasttomammals.Gene286,175–186.Bonifacino,J.S.(2014).Adaptorproteinsinvolvedinpolarizedsorting.JCellBiol204,7–17.Botzolakis,E.J.,Zhao,J.,Gurba,K.N.,Macdonald,R.L.,andHedera,P.(2011).TheeffectofHSP-causingmutationsinSPG3AandNIPA1ontheassembly,trafficking,andinteractionbetweenatlastin-1andNIPA1.MolCellNeurosci46,122–135.Chang,J.,Lee,S.,andBlackstone,C.(2014).Spasticparaplegiaproteinsspastizinandspatacsinmediateautophagiclysosomereformation.JClinInvest124,5249–5262.Cheng,X.-T.,Zhou,B.,Lin,M.-Y.,Cai,Q.,andSheng,Z.-H.(2015).Axonalautophagosomesusetheride-onserviceforretrogradetransporttowardthesoma.Autophagy11,1434–1436.Cheong,H.,Wu,J.,Gonzales,L.K.,Guttentag,S.H.,Thompson,C.B.,andLindsten,T.(2014).Analysisofalungdefectinautophagy-deficientmousestrains.Autophagy10,45–56.Davenport,E.C.,Pendolino,V.,Kontou,G.,McGee,T.P.,Sheehan,D.F.,López-Doménech,G.,Farrant,M.,andKittler,J.T.(2017).AnEssentialRolefortheTetraspaninLHFPL4intheCell-Type-SpecificTargetingandClusteringofSynapticGABAAReceptors.CellRep21,70–83.Dell’Angelica,E.C.,Klumperman,J.,Stoorvogel,W.,andBonifacino,J.S.(1998).AssociationoftheAP-3adaptorcomplexwithclathrin.Science280,431–434.


https://doi.org/10.1101/235101

21

Dell’Angelica,E.C.,Mullins,C.,andBonifacino,J.S.(1999).AP-4,anovelproteincomplexrelatedtoclathrinadaptors.JBiolChem274,7278–7285.Edelstein,A.D.,Tsuchida,M.A.,Amodaj,N.,Pinkard,H.,Vale,R.D.,andStuurman,N.(2014).AdvancedmethodsofmicroscopecontrolusingμManagersoftware.JBiolMethods1.Farías,G.G.,Guardia,C.M.,Britt,D.J.,Guo,X.,andBonifacino,J.S.(2015).SortingofDendriticandAxonalVesiclesatthePre-axonalExclusionZone.CellRep13,1221–1232.Fassier,C.,Tarrade,A.,Peris,L.,Courageot,S.,Mailly,P.,Dalard,C.,Delga,S.,Roblot,N.,Lefèvre,J.,Job,D.,etal.(2013).Microtubule-targetingdrugsrescueaxonalswellingsincorticalneuronsfromspastinknockoutmice.DisModelMech6,72–83.Fu,M.-M.,Nirschl,J.J.,andHolzbaur,E.L.F.(2014).LC3bindingtothescaffoldingproteinJIP1regulatesprocessivedynein-driventransportofautophagosomes.DevCell29,577–590.Galluzzi,L.,Baehrecke,E.H.,Ballabio,A.,Boya,P.,Bravo-SanPedro,J.M.,Cecconi,F.,Choi,A.M.,Chu,C.T.,Codogno,P.,Colombo,M.I.,etal.(2017).Moleculardefinitionsofautophagyandrelatedprocesses.EMBOJ36,1811–1836.Hara,T.,Nakamura,K.,Matsui,M.,Yamamoto,A.,Nakahara,Y.,Suzuki-Migishima,R.,Yokoyama,M.,Mishima,K.,Saito,I.,Okano,H.,etal.(2006).Suppressionofbasalautophagyinneuralcellscausesneurodegenerativediseaseinmice.Nature441,885–889.Hardies,K.,May,P.,Djémié,T.,Tarta-Arsene,O.,Deconinck,T.,Craiu,D.,ARworkinggroupoftheEuroEPINOMICSRESConsortium,Helbig,I.,Suls,A.,Balling,R.,etal.(2015).Recessiveloss-of-functionmutationsinAP4S1causemildfever-sensitiveseizures,developmentaldelayandspasticparaplegiathroughlossofAP-4complexassembly.HumMolGenet24,2218–2227.Hirst,J.,Bright,N.A.,Rous,B.,andRobinson,M.S.(1999).Characterizationofafourthadaptor-relatedproteincomplex.MolBiolCell10,2787–2802.Karanasios,E.,Walker,S.A.,Okkenhaug,H.,Manifava,M.,Hummel,E.,Zimmermann,H.,Ahmed,Q.,Domart,M.-C.,Collinson,L.,andKtistakis,N.T.(2016).AutophagyinitiationbyULKcomplexassemblyonERtubulovesicularregionsmarkedbyATG9vesicles.NatCommun7,12420.Khaminets,A.,Heinrich,T.,Mari,M.,Grumati,P.,Huebner,A.K.,Akutsu,M.,Liebmann,L.,Stolz,A.,Nietzsche,S.,Koch,N.,etal.(2015).Regulationofendoplasmicreticulumturnoverbyselectiveautophagy.Nature522,354–358.Khundadze,M.,Kollmann,K.,Koch,N.,Biskup,C.,Nietzsche,S.,Zimmer,G.,Hennings,J.C.,Huebner,A.K.,Symmank,J.,Jahic,A.,etal.(2013).AhereditaryspasticparaplegiamousemodelsupportsaroleofZFYVE26/SPASTIZINfortheendolysosomalsystem.PLoSGenet9,e1003988.


https://doi.org/10.1101/235101

22

Kojima,T.,Yamada,T.,Akaishi,R.,Furuta,I.,Saitoh,T.,Nakabayashi,K.,Nakayama,K.I.,Nakayama,K.,Akira,S.,andMinakami,H.(2015).RoleoftheAtg9ageneinintrauterinegrowthandsurvivaloffetalmice.ReprodBiol15,131–138.Komatsu,M.,Waguri,S.,Chiba,T.,Murata,S.,Iwata,J.,Tanida,I.,Ueno,T.,Koike,M.,Uchiyama,Y.,Kominami,E.,etal.(2006).Lossofautophagyinthecentralnervoussystemcausesneurodegenerationinmice.Nature441,880–884.Ktistakis,N.T.,andTooze,S.A.(2016).Digestingtheexpandingmechanismsofautophagy.TrendsCellBiol26,624–635.Lee,S.,Sato,Y.,andNixon,R.A.(2011).LysosomalproteolysisinhibitionselectivelydisruptsaxonaltransportofdegradativeorganellesandcausesanAlzheimer’s-likeaxonaldystrophy.JNeurosci31,7817–7830.López-Doménech,G.,Higgs,N.F.,Vaccaro,V.,Roš,H.,Arancibia-Cárcamo,I.L.,MacAskill,A.F.,andKittler,J.T.(2016).LossofDendriticComplexityPrecedesNeurodegenerationinaMouseModelwithDisruptedMitochondrialDistributioninMatureDendrites.CellRep17,317–327.Maday,S.,andHolzbaur,E.L.F.(2014).Autophagosomebiogenesisinprimaryneuronsfollowsanorderedandspatiallyregulatedpathway.DevCell30,71–85.Maday,S.,andHolzbaur,E.L.F.(2016).Compartment-SpecificRegulationofAutophagyinPrimaryNeurons.JNeurosci36,5933–5945.Maday,S.,Wallace,K.E.,andHolzbaur,E.L.F.(2012).Autophagosomesinitiatedistallyandmatureduringtransporttowardthecellsomainprimaryneurons.JCellBiol196,407–417.Matsuda,S.,Miura,E.,Matsuda,K.,Kakegawa,W.,Kohda,K.,Watanabe,M.,andYuzaki,M.(2008).AccumulationofAMPAreceptorsinautophagosomesinneuronalaxonslackingadaptorproteinAP-4.Neuron57,730–745.Mattera,R.,Guardia,C.M.,Sidhu,S.S.,andBonifacino,J.S.(2015).BivalentMotif-EarInteractionsMediatetheAssociationoftheAccessoryProteinTepsinwiththeAP-4AdaptorComplex.JBiolChem290,30736–30749.Mattera,R.,Park,S.Y.,DePace,R.,Guardia,C.M.,andBonifacino,J.S.(2017).AP-4mediatesexportofATG9Afromthetrans-Golginetworktopromoteautophagosomeformation.ProcNatlAcadSciUSA114,E10697–E10706.Mitsunari,T.,Nakatsu,F.,Shioda,N.,Love,P.E.,Grinberg,A.,Bonifacino,J.S.,andOhno,H.(2005).ClathrinadaptorAP-2isessentialforearlyembryonaldevelopment.MolCellBiol25,9318–9323.Mizushima,N.,Yoshimori,T.,andOhsumi,Y.(2011).TheroleofAtgproteinsin


https://doi.org/10.1101/235101

23

autophagosomeformation.AnnuRevCellDevBiol27,107–132.Montenegro,G.,Rebelo,A.P.,Connell,J.,Allison,R.,Babalini,C.,D’Aloia,M.,Montieri,P.,Schüle,R.,Ishiura,H.,Price,J.,etal.(2012).MutationsintheER-shapingproteinreticulon2causetheaxon-degenerativedisorderhereditaryspasticparaplegiatype12.JClinInvest122,538–544.Moreno-De-Luca,A.,Helmers,S.L.,Mao,H.,Burns,T.G.,Melton,A.M.A.,Schmidt,K.R.,Fernhoff,P.M.,Ledbetter,D.H.,andMartin,C.L.(2011).Adaptorproteincomplex-4(AP-4)deficiencycausesanovelautosomalrecessivecerebralpalsysyndromewithmicrocephalyandintellectualdisability.JMedGenet48,141–144.Najmabadi,H.,Hu,H.,Garshasbi,M.,Zemojtel,T.,Abedini,S.S.,Chen,W.,Hosseini,M.,Behjati,F.,Haas,S.,Jamali,P.,etal.(2011).Deepsequencingreveals50novelgenesforrecessivecognitivedisorders.Nature478,57–63.Nishiyama,J.,Miura,E.,Mizushima,N.,Watanabe,M.,andYuzaki,M.(2007).AberrantMembranesandDouble-MembraneStructuresAccumulateintheAxonsofAtg5-NullPurkinjeCellsbeforeNeuronalDeath.Autophagy3,591–596.Nixon,R.A.,Wegiel,J.,Kumar,A.,Yu,W.H.,Peterhoff,C.,Cataldo,A.,andCuervo,A.M.(2005).ExtensiveinvolvementofautophagyinAlzheimerdisease:animmuno-electronmicroscopystudy.JNeuropatholExpNeurol64,113–122.Norkett,R.,Modi,S.,Birsa,N.,Atkin,T.A.,Ivankovic,D.,Pathania,M.,Trossbach,S.V.,Korth,C.,Hirst,W.D.,andKittler,J.T.(2016).DISC1-dependentRegulationofMitochondrialDynamicsControlstheMorphogenesisofComplexNeuronalDendrites.JBiolChem291,613–629.Okerlund,N.D.,Schneider,K.,Leal-Ortiz,S.,Montenegro-Venegas,C.,Kim,S.A.,Garner,L.C.,Gundelfinger,E.D.,Reimer,R.J.,andGarner,C.C.(2017).BassoonControlsPresynapticAutophagythroughAtg5.Neuron93,897–913.e7.Orsi,A.,Razi,M.,Dooley,H.C.,Robinson,D.,Weston,A.E.,Collinson,L.M.,andTooze,S.A.(2012).DynamicandtransientinteractionsofAtg9withautophagosomes,butnotmembraneintegration,arerequiredforautophagy.MolBiolCell23,1860–1873.Oz-Levi,D.,Ben-Zeev,B.,Ruzzo,E.K.,Hitomi,Y.,Gelman,A.,Pelak,K.,Anikster,Y.,Reznik-Wolf,H.,Bar-Joseph,I.,Olender,T.,etal.(2012).MutationinTECPR2revealsaroleforautophagyinhereditaryspasticparaparesis.AmJHumGenet91,1065–1072.Park,S.H.,Zhu,P.-P.,Parker,R.L.,andBlackstone,C.(2010).HereditaryspasticparaplegiaproteinsREEP1,spastin,andatlastin-1coordinatemicrotubuleinteractionswiththetubularERnetwork.JClinInvest120,1097–1110.Pathania,M.,Davenport,E.C.,Muir,J.,Sheehan,D.F.,López-Doménech,G.,andKittler,J.T.(2014).TheautismandschizophreniaassociatedgeneCYFIP1iscriticalforthemaintenance


https://doi.org/10.1101/235101

24

ofdendriticcomplexityandthestabilizationofmaturespines.TranslPsychiatry4,e374.Peden,A.A.,Rudge,R.E.,Lui,W.W.Y.,andRobinson,M.S.(2002).AssemblyandfunctionofAP-3complexesincellsexpressingmutantsubunits.JCellBiol156,327–336.Pérez-Brangulí,F.,Mishra,H.K.,Prots,I.,Havlicek,S.,Kohl,Z.,Saul,D.,Rummel,C.,Dorca-Arevalo,J.,Regensburger,M.,Graef,D.,etal.(2014).DysfunctionofspatacsinleadstoaxonalpathologyinSPG11-linkedhereditaryspasticparaplegia.HumMolGenet23,4859–4874.Preibisch,S.,Saalfeld,S.,andTomancak,P.(2009).Globallyoptimalstitchingoftiled3Dmicroscopicimageacquisitions.Bioinformatics25,1463–1465.Renvoisé,B.,andBlackstone,C.(2010).EmergingthemesofERorganizationinthedevelopmentandmaintenanceofaxons.CurrOpinNeurobiol20,531–537.Saitoh,T.,Fujita,N.,Hayashi,T.,Takahara,K.,Satoh,T.,Lee,H.,Matsunaga,K.,Kageyama,S.,Omori,H.,Noda,T.,etal.(2009).Atg9acontrolsdsDNA-drivendynamictranslocationofSTINGandtheinnateimmuneresponse.ProcNatlAcadSciUSA106,20842–20846.Shibata,M.,Lu,T.,Furuya,T.,Degterev,A.,Mizushima,N.,Yoshimori,T.,MacDonald,M.,Yankner,B.,andYuan,J.(2006).RegulationofintracellularaccumulationofmutantHuntingtinbyBeclin1.JBiolChem281,14474–14485.Skarnes,W.C.,Rosen,B.,West,A.P.,Koutsourakis,M.,Bushell,W.,Iyer,V.,Mujica,A.O.,Thomas,M.,Harrow,J.,Cox,T.,etal.(2011).Aconditionalknockoutresourceforthegenome-widestudyofmousegenefunction.Nature474,337–342.Stavoe,A.K.H.,Hill,S.E.,Hall,D.H.,andColón-Ramos,D.A.(2016).KIF1A/UNC-104TransportsATG-9toRegulateNeurodevelopmentandAutophagyatSynapses.DevCell38,171–185.Tarrade,A.,Fassier,C.,Courageot,S.,Charvin,D.,Vitte,J.,Peris,L.,Thorel,A.,Mouisel,E.,Fonknechten,N.,Roblot,N.,etal.(2006).Amutationofspastinisresponsibleforswellingsandimpairmentoftransportinaregionofaxoncharacterizedbychangesinmicrotubulecomposition.HumMolGenet15,3544–3558.Tesson,C.,Koht,J.,andStevanin,G.(2015).Delvingintothecomplexityofhereditaryspasticparaplegias:howunexpectedphenotypesandinheritancemodesarerevolutionizingtheirnosology.HumGenet134,511–538.Vaccaro,V.,Devine,M.J.,Higgs,N.F.,andKittler,J.T.(2017).Miro1-dependentmitochondrialpositioningdrivestherescalingofpresynapticCa2+signalsduringhomeostaticplasticity.EMBORep18,231–240.Vantaggiato,C.,Crimella,C.,Airoldi,G.,Polishchuk,R.,Bonato,S.,Brighina,E.,Scarlato,M.,Musumeci,O.,Toscano,A.,Martinuzzi,A.,etal.(2013).Defectiveautophagyinspastizin


https://doi.org/10.1101/235101

25

mutatedpatientswithhereditaryspasticparaparesistype15.Brain136,3119–3139.Varga,R.-E.,Khundadze,M.,Damme,M.,Nietzsche,S.,Hoffmann,B.,Stauber,T.,Koch,N.,Hennings,J.C.,Franzka,P.,Huebner,A.K.,etal.(2015).InvivoevidenceforlysosomedepletionandimpairedautophagicclearanceinhereditaryspasticparaplegiatypeSPG11.PLoSGenet11,e1005454.Verkerk,A.J.M.H.,Schot,R.,Dumee,B.,Schellekens,K.,Swagemakers,S.,Bertoli-Avella,A.M.,Lequin,M.H.,Dudink,J.,Govaert,P.,vanZwol,A.L.,etal.(2009).MutationintheAP4M1geneprovidesamodelforneuroaxonalinjuryincerebralpalsy.AmJHumGenet85,40–52.Vijayan,V.,andVerstreken,P.(2017).Autophagyinthepresynapticcompartmentinhealthanddisease.JCellBiol216,1895–1906.Wang,B.,Iyengar,R.,Li-Harms,X.,Joo,J.H.,Wright,C.,Lavado,A.,Horner,L.,Yang,M.,Guan,J.-L.,Frase,S.,etal.(2017).TheAutophagy-InducingKinases,ULK1andULK2,RegulateAxonGuidanceintheDevelopingMouseForebrainviaaNoncanonicalPathway.AutophagypreprintWatanabe,F.,Arnold,W.D.,Hammer,R.E.,Ghodsizadeh,O.,Moti,H.,Schumer,M.,Hashmi,A.,Hernandez,A.,Sneh,A.,Sahenk,Z.,etal.(2013).Pathogenesisofautosomaldominanthereditaryspasticparaplegia(SPG6)revealedbyaratmodel.JNeuropatholExpNeurol72,1016–1028.Webber,J.L.,andTooze,S.A.(2010a).NewinsightsintothefunctionofAtg9.FEBSLett584,1319–1326.Webber,J.L.,andTooze,S.A.(2010b).Coordinatedregulationofautophagybyp38alphaMAPKthroughmAtg9andp38IP.EMBOJ29,27–40.Winslow,A.R.,Chen,C.-W.,Corrochano,S.,Acevedo-Arozena,A.,Gordon,D.E.,Peden,A.A.,Lichtenberg,M.,Menzies,F.M.,Ravikumar,B.,Imarisio,S.,etal.(2010).α-Synucleinimpairsmacroautophagy:implicationsforParkinson’sdisease.JCellBiol190,1023–1037.Xie,Y.,Zhou,B.,Lin,M.-Y.,Wang,S.,Foust,K.D.,andSheng,Z.-H.(2015).EndolysosomalDeficitsAugmentMitochondriaPathologyinSpinalMotorNeuronsofAsymptomaticfALSMice.Neuron87,355–370.Yamaguchi,J.,Suzuki,C.,Nanao,T.,Kakuta,S.,Ozawa,K.,Tanida,I.,Saitoh,T.,Sunabori,T.,Komatsu,M.,Tanaka,K.,etal.(2017).Atg9adeficiencycausesaxon-specificlesionsincludingneuronalcircuitdysgenesis.AutophagypreprintYoung,A.R.J.,Chan,E.Y.W.,Hu,X.W.,Köchl,R.,Crawshaw,S.G.,High,S.,Hailey,D.W.,Lippincott-Schwartz,J.,andTooze,S.A.(2006).StarvationandULK1-dependentcyclingofmammalianAtg9betweentheTGNandendosomes.JCellSci119,3888–3900.


https://doi.org/10.1101/235101

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Zhou,X.,Babu,J.R.,daSilva,S.,Shu,Q.,Graef,I.A.,Oliver,T.,Tomoda,T.,Tani,T.,Wooten,M.W.,andWang,F.(2007).Unc-51-likekinase1/2-mediatedendocyticprocessesregulatefilopodiaextensionandbranchingofsensoryaxons.ProcNatlAcadSciUSA104,5842–5847.


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FigureLegends

Figure1:AP-4ε(-/-)micerecapitulatecharacteristicanatomicaldefectsofAP-4deficiency

A.SchematicoftheKOtm1ballele,showingremovalofcriticalexon3.

B. Representative genotyping PCR of litter used for E16 hippocampal neuronal culture

showingAP-4εWT (+/+), KO (-/-) andHet (+/-) embryos.Bottompanel,westernblot showing

lossofεproteininKOembryos.

C.εproteinlevelinbrainregionsofadultmice(n=3repeats).

D,E.SectionspreparedfromanimalsatP30stainedagainstNeuNandGFAPshowinglateral

ventricular enlargement in KO. Scale bar = 200 μm. (E) Quantification of relative area of

lateralventricle(n=5animalsWT/KO).

F-H.Commissuralcrossingaxons,stainedagainstNeurofilament-200(NF200).Scalebar=

100μm.(G)QuantificationofthicknessofCCand(H)DF(n=5animalsWT/KO).

Quantifieddataisexpressedasmean±SEM.(E)presentedrelativetocontrolvalue(n=5

animals per genotype). Statistical analysis: Two-tailed unpaired Student’s t-test, *p<0.05,**P<0.01and***P<0.001.CC–corpuscallosum,DF–dorsalfornix.

Figure2:AxonspecificdefectsinAP-4ε(-/-)neurons

A-C.CulturedGFP-filledDIV-4hippocampalneuronsstainedagainstGFPshowingneuronal

morphology.Scalebar=50μm.(B)Quantificationoftotalaxonallengthand(C)branches(n

=22/18neuronsWT/KO).

D,E. Insetmagnifiedpanel from (A)ofdistalaxonal regions, redarrows indicatingaxonal

swelling.Scalebar=20μm.(E)Quantificationofnumberofswellingsperneuron(n=20/17

neuronsWT/KO).

F,G.Quantificationoftotalnascentdendriticlength(F)andbranchesperneuron(G)atDIV-

4.(n=22/27neuronsWT/KO).

H - K. Cultured GFP-filled DIV-14 hippocampal neurons stained against GFP showing

neuronalmorphology.Scalebar=100μm.(I)Analysisofdendriticcomplexityusing10μm

concentric sholl intersections. Quantification of (J) total dendritic length and (K) total

branchesperneuron(n=12/17neuronsWT/KO).

Quantifieddataisexpressedasmean±SEM,fromthreeindependentexperimentalrepeats.

Statistical analysis: (I) Two-wayANOVAwithBonferroni post-hoc test. (B, F,G, J, K) Two-


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tailedunpairedStudent’s t-test. (C,E)Two-tailedMann-WhitneyUtest test, *p<0.05, **P<

0.01and***P<0.001.

Figure3:ATG9AaccumulatesinAP-4ε(-/-)neurons

A. Endogenous co-immunoprecipitation of AP-4εwith ATG9A frommouse brain, showing

interactionbetweenAP-4andATG9A(n=3repeats).

B, C. ATG9A protein accumulation in hippocampal lysates from KO animals and (C)

densitometricquantification(n=3animals).

D.SectionsstainedagainstATG9AandNF200revealingbrainmorphologyandaccumulation

ofATG9Awithincell layersofthehippocampus.Highmagnificationpanelsshowincreased

ATG9AimmunoreactivitywithinthepyramidalCA1celllayer.Scalebars=200μm,Highmag

20μm(n=3animalsWT/KO).

E, F. DIV-8 cultured hippocampal neurons stained against ATG9A and MAP2 revealing

dendriticmorphology. Inset panels show crops of cell body and accumulation of ATG9A.

Scalebars=20μm,crop5μm(F)quantificationoftotalATG9Asignalinneuronalsoma(n=

40/20neuronsWT/KO).


Statisticalanalysis:Two-tailedunpairedStudent’st-test,**P<0.01and***P<0.001.

Figure4:FunctionalAP-4iscriticalforATG9AexitfromtheTGNinneurons

A,B. SIMofDIV-8 culturedhippocampalneurons stainedagainstATG9A,cis-golgimarker

GM130and trans-golgimarkerGolg97.Dashedboxes indicate region inmagnifiedpanels,

showingvesicularATG9AinWTneurons,andreticularATG9AoverlappingGolg97inKO.(B)

IntensitylinescandemonstratesATG9AretentionwithintheTGNinKOneurons.Scalebars=

5μm,0.5μmcrop(n=3repeats).

C,D.ATG9Aconstraintrescuebyexpressionofmyc-taggedFLandpathologicalV454xAP-4ε

constructs.Numbereddashedboxesindicateregioninmagnifiedpanels.Scalebars=5μm,

crop2μm.(D)QuantificationofrelativeATG9AinsomaofKOneuronsinrescueconditions

(n=26/26/23neuronsFL/V454X/UT).

(D) Quantified data is expressed as mean ± SEM, relative to FL rescue value from three

independentexperimentalrepeats.Statisticalanalysis:Kruskall-Wallistest,***P<0.001.


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Figure5:ATG9Aconstraintresultsindefectiveaxonalautophagosomematuration

A – C. ATG9A vesicles in axons and dendrites. Quantification of vesicular density in (B)

dendrites and (C) axons. Scale bar = 5 μm. (Dendrite; 28/24 WT/KO, Axon; n = 19/12

WT/KO).

D-H.Liveimagingofautophagosomemotilityatthegrowthconeanddistalmost100μmof

axon. Movies generated over 6 minutes from cultured hippocampal neurons at DIV-6/7

transfected with RFP-LC3. First frames and resulting kymographs shown with pseudo-

colouring of RFP-LC3 signal. X-axis scale bar = 10 μm, Y-axis represents time (1px/1.5s).

Quantificationof;(E)absoluteretrogradedisplacement(F)anterogradeandretrograderun

lengthpermotileautophagosome,(G)totaldistancetravelledpermotileautophagosome,

(H)proportionoftimespentstationary,ormovinganterogradelyorretrogradelypermotile

autophagosome.(n=227/117motileautophagosomesfrom46/36neuronsWT/KO).


Statisticalanalysis:(B,C)Two-tailedunpairedStudent’st-test.(E,F,G,H)Two-tailedMann-

WhitneyUtest,**P<0.01and***P<0.001.


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SupplementaryFigureLegends

FigureS1–AxonalswellingsareevidentinDIV-14hippocampalcultures

A, B. Axonal field of culturedGFP-filledDIV-14 hippocampal neurons stained againstGFP

showingaxonalmorphology. Scalebar=20μm (B)Quantificationof axonal swellingsper

100μm.(n=3/5embryosWT/KO).

Quantifieddataexpressedasmean±SEM,fromthree independentexperimentalrepeats.

Statisticalanalysis:Two-tailedunpairedStudent’st-test,**P<0.01.

FigureS2–ATG9Aaccumulatesinpyramidalcelllayersofthehippocampus

A.SectionsstainedagainstATG9AandNF200revealingbrainmorphologyandaccumulation

ofATG9AwithincelllayersofthehippocampusatP30inAP-4KO.Highmagnificationpanels

showincreasedATG9Aimmunoreactivitywithinthepyramidaldentategyruscelllayer(n=3

animalsWT/KO).

FigureS3–AP-4islocalisedtotheTGNinneurons

A.WTDIV-8culturedhippocampalneuronstainedagainstεandGolgin-97(Golg97).Dashed

boxshowsmagnifiedregionandlocalisationofεattheTGN.Scalebars=10μm,2μmcrop

(n=3repeats).

FigureS4–ReconstitutionofAP-4complexrescuesATG9ATGNconstraint

A. Schematic of n-terminally myc tagged constructs generated for this study, showing

structuralpositionofpathologicalprematurestopmutation.

B. Related to figure 4C.Wide-field panels of exogenously expressedMyc constructs and

endogenousATG9A,showingrescueofTGNconstraintonlyintransfectedcells.

Movies S1 and S2 – Defective distal axonal autophagosome maturation in AP-4 KO

neurons

MoviesshowingDIV6-7culturedhippocampalneuronstransfectedwithRFP-LC3,pseudo-

coloured for clarity. Rightward motion is retrograde towards the soma, leftward is

anterogradetowardsthegrowthcone.1frame/1.5sfor240frames,playbackat20frames/

second.S1isrepresentativemoviefromWTandS2isrepresentativemoviefromKO.


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https://doi.org/10.1101/235101

title authors - biorxiv · 2017/12/16 · (abou jamra et al., 2011; tesson et al., 2015). ap-4...

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