Mutations in the HECT Domain of NEDD4L Lead to AKT–Mtor

© 2016 Nature America, Inc. All rights reserved. addressed addressed to J.C. ( Hôpital Civil de Strasbourg, Hôpitaux Universitaires de Strasbourg, Strasbourg, France. Oxford, UK. Austria. de Génomique, CEA, Evry, France. Reims, Reims, France. 12 Department of Neurology, Boston Children’s Hospital, Boston, Massachusetts, USA. et d’Epileptologie Infantile, University Hospital of Montpellier, Montpellier, France. Centre for Human Genetics, University of Oxford, Oxford, UK. Central Manchester University Hospitals NHS Trust, Manchester Academic Health Science Centre, Manchester, UK. Strasbourg, Illkirch, France. 1 on based is classification evolving their and epilepsy, and disability microcephaly PNH (PMG), and includes polymicrogyria that (MCD) pachygyria, development lissencephaly, cortical of group malformations the of within classified are disorders these of Many disorders. developmental of range wide a in result can processes these of any populations neuronal distinct of differentiation as well as layering, and migration proliferation, include that developmental processes interdependent of regulation coordinated temporal and requires spatial cortex cerebral human the of Development Deciphering Developmental Disorders study Tristan Stemmelen Annapurna Poduri Jill Clayton-Smith BroixLoïc nodular periventricular heterotopia to AKT–mTOR pathway deregulation and cause Mutations in the HECT domain of Nature Ge Nature Received 1 April; accepted 24 August; published online 3 October 2016; Neurodevelopmental Izabela Sumara Laurent Nguyen Anne Boland insights while differential neuronal utero in delay. domain genetic Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France. Grappe Interdisciplinaire de Génoprotéomique Appliquée-Neurosciences, University of Liège, Liège, Belgium.

PNH-related 18 Cellular

causes

Laboratoire de Biochimie et Génétique Moléculaire, Hôpital Cochin, Paris, France. of into electroporation

3– 20 positioning n the Institut Imagine, INSERM U1163, Paris Descartes University, Hôpital Necker–Enfants Malades, Paris, France. 5

deregulation etics 1 . MCD are often associated with severe intellectual intellectual severe with associated often are MCD .

the – 5

[email protected]

encoded , and 15 remain 22

critical

, Robert , Olaso Robert , , Hélène Jagline 1 14 12 –

ADVANCE ONLINE PUBLICATION ONLINE ADVANCE

expression Paediatric Neurology Unit, A. Meyer Children’s Hospital, University of Florence, Florence, Italy. mutations 4 , Juliette Godin , , Maria-Victoria Hinckelmann 6

and 1 10 , , Alistair T Pagnamenta 5 disorders

Institut Cochin, INSERM U1016, CNRS U8104, Paris Descartes University, Paris, France. – in

4 , , Jenny C Taylor

role

, Gabrielle , RudolfGabrielle

approach of

many terminal

16 ). ubiquitin

pathways Institut Imagine, Bioinformatics Platform, Paris Descartes University, Paris, France.

are data

cases NEDD4L with

15 associated

showed translocation.

1 showed , Cecile Masson , Cecile

– 1 ligase

periventricular

unknown. 4 – involved. , 4 22 , Usha Kini

, , Ekaterina L Ivanova in 7

, , Peggy Tilly lead that

the sensitivity

1 with – 1

regulation Excess , PNH-related to Here Service de Génétique Médicale, University Hospital of Nantes, Nantes, France. , , Giuseppe Muraca

2 7

. Disruption of of Disruption . Further

, , Kay A Metcalfe

11 deregulation PNH

19 , Martin Moïse nodular

16 we , Patrick Nischké

of wild-type

1 , , Ratna Tripathy associated

–

show doi:10.1038/ng.367 PNH-associated investigations, 4 –

of & Jamel Chelly 4 , , Karine Poirier

heterotopia mutants mTOR CNRS U7104, Illkirch, France.

that

of 10 11

NEDD4L 1 Epilepsy Genetics Program, Division of Epilepsy and Clinical Neurophysiology,

A list of members and affiliations appears at a widely expressed 280-kDa actin-binding phosphoprotein, account account phosphoprotein, actin-binding 280-kDa expressed widely a features clinical common most the are difficulties learning and seizures although PNH are with in tions heterogeneous, patients presenta Clinical of ventricles. gray the nodules lateral matter lining ectopic by bilateral unpub characterized is PNH Anatomically, data). lished (R.G., MCD of 31% about represents PNH abnormalities, pathways biological and genes disrupted underlying the with the developmental process thought to be first affected in combination – mTORC

with 4 missense

So So far, mutations in the X-linked migration neuronal with associated MCD of group the Within pathways These authors contributed equally to this work. Correspondence should be , , Stéphane Schmucker 6 12

, Bertrand Isidor, Bertrand 5 and , , Benjamin Saintpierre , Nathalie Bednarek Weirauch 19

toe 16 including Department of Clinical Genetics, Oxford University Hospitals NHS Trust,

(PNH)

mutants 17 1 1 excess leads , Jean-François Deleuze

and , , David Keays syndactyly, 1

mutations – 5

NEDD4L and 4 , , Yoann Saillour ,

21 to AKT are

wild-type

their rapamycin-based to disruption

etiologically

activities. proteasome 13 3 INSERM U964, Illkirch, France. Hôpital Maison Blanche, University Hospital of

in cleft contributions 8 7 NIHR Biomedical Research Centre, Wellcome Trust , , Ulrike Walther Louvier

NEDD4L

NEDD4L 17 6 15 17

Manchester Centre for Genomic Medicine, palate

1 , Cherif , Beldjord Cherif Centre National de Génotypage, Institut of Institute of Molecular Pathology, Vienna, –

Altogether, 4 21

5 , , Nathalie Drouot Dab

heterogeneous, , , Adrienne Elmorjani Service de Diagnostic Génétique, FLNA degradation. , , Nicolas Lebrun lead

and http://dx.doi

mapping

experiments, 15 1 1 affect

in , Nadia Bahi-Buisson and gene (filamin A)

neurodevelopmental 13 cortical , Renzo Guerrini

mTORC these neurogenesis, 9

.org/10.1101/04905 to Unité de Neuropédiatrie

Moreover,

s e l c i t r A 4 the

Université de

18 data development. and

found , 1 1

HECT

1 5 pathways,

9 – their , provide , 6 4 , , encoding , 5

5 an 3

, .

20 .

, , - -

© 2016 Nature America, Inc. All rights reserved. four WW protein–protein interaction domains interaction WW four protein–protein heterozygous PNH-associated mutations. NEDD4L protein is characterized by an N-terminal C2 domain known to Ca known bind C2 domain by an N-terminal is characterized protein NEDD4L mutations. PNH-associated heterozygous ( photographs. patient to publish was obtained 12 months ( at the age of 8 ( performed were MRIs years arrows). PMG (white frontal shows the MRI section to PNH (arrowheads), ( thin show sections Sagittal (arrowheads). ventricles the lateral lining of heterotopia nodules confluent show section, or and one one coronal axial sections, two axial For patient, PNH. each and constant toe syndactyly frequent illustrating 1 Figure PNH. bilateral with patient affected single a had families All ing. and investigations genetic for selected 15 patient–parent trios for referred analysis by whole-exome sequenc MCD with patients from data (MRI) imaging resonance magnetic brain and Weclinical used and Patients RESULTS AKT pathways. signaling and mTOR the of dysregulation through likely processes, tal show that PNH-related NEDD4L mutants further We disrupt neurodevelopmen delay. neurodevelopmental and palate cleft syndactyly, in mutations missense of NEDD4L identified been has 6q27, gene, causal potential one only but studies, (aCGH) hybridization genomic comparative array through (GEF2) in mutations which encodes recessive the ADP–ribosylation factor with guanine exchange factor 2 associated PNH of forms familial rare of identification the following proposed were opment migration ronal the to adhesion its and neuroependymal membrane, neural progenitorscaffold proliferation and glia neu radial polarized the affecting that Ehler–Danlos females mostly associated affecting the conditions and PNH bilateral of 50% about for s e l c i t r A c b a

Here we provide evidence implicating the E3 ubiquitin ligase gene ligase E3 the ubiquitin implicating Here we evidence provide

mutations FLNA I-1 II-1

1 1 Mutations in Mutations

in the development of PNH. We report the identification identification the Wereport PNH. of development the in with . Finally, other genetic forms of PNH have been mapped mapped been have PNH of forms genetic other Finally, . II-2 c -related PNH results from the combination of defects defects of combination the from results PNH -related ), 7 months ( II-2

II-3 PNH I-2

8– in 1

0 NEDD4L . Other mechanisms contributing to PNH devel PNH to contributing mechanisms Other .

II-3 II-2 NEDD4L analyzed d ) and 8 months ( 1 2 NEDD4L . cause PNH and syndactyly.cause ( BRC217: c.2677G>A;p.Glu893Lys

P158: c.2690G>A;p.Arg897Gln P347: c.2677G>A;p.Glu893Lys by

whole-exome in patients with PNH, bilateral bilateral PNH, with patients in 7 C6orf70 . Compelling studies suggested suggested studies Compelling . e ). MRIs were not available for patient Pnh31124 with the c.2082G>T; p.Gln694His mutation. Written consent 1 6 f responsible for recognition of the substrate, and the C-terminal catalytic HECT domain HECT catalytic and the C-terminal of the substrate, for recognition responsible ) Linear representation of the NEDD4L polypeptide showing the positions in the HECT domain of in the the domain HECT the positions showing polypeptide of the NEDD4L ) representation Linear ( ERMARD

sequencing a – ) located at located ) e ARFGEF2 ) Photographs and representative sections of brain MRIs for affected individuals individuals for of MRIs affected brain sections and representative ) Photographs

- - - - ,

f e d Supplementary Note Supplementary traces consistently showed an imbalance in the height of the peaks peaks the of height the in imbalance an showed consistently traces father. and For mother, however, the sister sequencing healthy of the brother but state a from in absent the DNAthe affected heterozygous iant in all members of the family. We found the variant to be present in parents ( healthy and girl healthy a brother), her and (P347 children affected Note Supplementary ( delay neurodevelopmental and toes, third and second the of syndactyly bilateral PNH, bilateral by characterized also was 1c Fig. Supplementary (P347; patient detected additional (already one in mutation BRC217) in c.2677G>A the identified and MCD with patients 96 of cohort a in sequences) flanking their and exons sequencing ( neu BRC217; and and (P158 toes, delay third and rodevelopmental second the of syndactyly bilateral PNH, in changes sense Weidentified patients. unrelated in gene same the in of recurrence for searched and data exome tered findings significant validate and variants sequence prioritize and detect to workflow experimental described previously a applied we approach, sequencing whole-exome the For DNA by digital droplet PCR and found that the frequency of the the of frequency the that found and PCR droplet digital mother’s by the DNA analyzed we mosaicism, somatic of level the evaluate somatic and a of mosaicism germline of suggestive were results these Altogether, corresponding to normal and variant alleles ( 7 C2 c ) or dysmorphic ( ) or dysmorphic 109 Supplementary Supplementary Fig. 1c Supplementary Fig. 1a 193 NEDD4L

a WW1 226 DVANCE ONLINE PUBLICATION ONLINE DVANCE NEDD4L

a 385 ). ). As the family of patient P347 consisted of two

WW2 Sanger by mutations the of validation After ). and

418 BRC-DDDP110533: c.2036A>G;p.Tyr679Cys ). Interestingly, the phenotype of this patient patient this of phenotype the Interestingly, ). variant in the mother. To confirm this and and this To mother. the confirm in variant in two patients with bilateral contiguous contiguous bilateral with patients two in c , II-3) corpus callosum. In callosum. , corpus II-3) 497 PNC: c.2677G>A;p.Glu893Lys WW3 530 ), ), we tested the segregation of the var 548 a WW4 , ), 12 months ), ( 12 months

581 b p.Tyr679Cys ), we screened 2+ and phospholipids 13 p.Gln694His , 1 Supplementary Supplementary Fig. 1c

640 4 Fig. 1a Fig. . We then analyzed fil analyzed We . then N lobe Fig. 1 Fig.

b Nature Ge Nature de novo de ), ), 9 and 1 8 NEDD4L HECT p.Glu893Lys . d , b , in addition , in addition c , , , , (3×) de novo de Table Table 859 Table C lobe mutations mutations

1 p.Arg897Gln 7 , , two to (coding n 1 1 etics 1 mis and and and and 975 and

). ). - - - -

© 2016 Nature America, Inc. All rights reserved. present; −,absent.Transcript PNH, periventricularnodularheterotopia;PMG,polymycrogyria;CC,corpuscallosum;GW, gestationalweek;HC,headcircumference;IS,infantilespasm;NA,notavailable;+, CC anomalies PNH Brain MRIfindings Seizures Developmental delay Syndactyly distributed in the cortical plate, ventricular zone and ganglionicemiplate,andventricularzonecortical the distributedin zation at embryonic day (E) 15), of development. expression As illustrated the in analyzed first We Functional ( domain HECT of the modeling informatics predictions (from the MutationTester tool) and structural of conservation of the affected residues ( degree by high the was suggested effect pathogenic of their relevance tions associated with PNH mapped to the HECT domain ( processes and pathways cellular mental the turnover of and function a number of proteins involved in funda to known regulate ligases of E3 ubiquitin HECT-type family NEDD4 Table delay ( and neurodevelopmental with syndactyly associated PNH of presence the showed also all results MRI For brain patients, studies. three Consortium Epi4K and Disorders Deciphering Developmental the of part as analyzed and sequenced exome trios of and ( we which included in genes of panel MCD-related an of screening targeted 1 Fig. PNH ( with patients unrelated three of normal. be to found were evaluations behavioral and cognitive neurological, and reexamined was mother the data, molecular these ( 16% around was allele mutated Other Cerebellum anomalies Cortex anomalies Weight (kg) Height (cm) HC (cm) Age atlastexamination Cleft palate Hypotonia Birth weight Birth (GW) Sex Inheritance Patients withPNH T Nature Ge Nature nences.Moreover, analysis of Fig. Fig. 1 able able 1 NEDD4L implication the reinforced we findings, initial these after Shortly NEDD4L Supplementary Fig. 1f Fig. Supplementary d 1 e , , and and s , , Table Table NEDD4L ummary ummary of clinical and neuroimaging features of patients harboring n

1 by the identification of additional additional of identification the by effects Supplementary Note Supplementary 5 etics (also known as as known (also 1 1 and and

. The second and third patients—DDDP110533 patients—DDDP110533 third and second The .

of Supplementary Fig. 1d Fig. Supplementary ADVANCE ONLINE PUBLICATION ONLINE ADVANCE Supplementary Fig. 1e Supplementary P158 c.2690G>A

16.5 (−1.5s.d.) 1.08 (−1.5s.d.) wild-type 54 (+1.5s.d.) NM_001144967. p. Arg897Gln + (bilateral) + (atbirth) At 8years De novo 6 years Myopia 3,750 )—were identified through data sharing sharing data through identified )—were 40 Supplementary Figure 3a M − − − − + − + Nedd4l NEDD4-2 Nedd4l Supplementary Table 1 Supplementary Supplementary Fig. 2b Fig. Supplementary

NEDD4L ). Table expression byreal-time qPCRin

2 transcripts were homogenously Cerebral atrophy 125 (−3.5s.d.) (ENST00000400345)andprotein p.Glu893Lys P347-II.2P347-II.3 Nedd4l 25 (−2.5s.d.) 50 (−2.4s.d.) Supplementary Supplementary Fig. 2a Optic atrophy + (lateonset) 16– At 9months + (bilateral) impairment ) encodes a member of the the of member a encodes ) mosaicism + (severe) Maternal 12 years Hearing 1 1 3,160 9 ). The first patient (PNC: (PNC: patient ). The first

NA 41 ) ) and ( Pnh31124 and ) was identified through through ) identified was . Interestingly, all muta all Interestingly, . − − + + F P347 c.2677G>A uig os brain mouse during de novo de

NEDD4L

( 10.9 (+0.7s.d.) 49.8 (+0.7s.d.) in situ cryptorchidism mutations in in mutations 88 (+0.8s.d.) At 2months + (bilateral) Strabismus Convergent Fig. 1 – ). In view of ). In view Bifid uvula mosaicism + (severe) Maternal j 2 years ).

Fig. Fig. 1d mutants NA NA 41 hybridi M − − − + + Table

f ), ), bio ), and

e Q96PU 1 - - - - - ,

++ (at2months) 5.29 (−1.1s.d.) Frontal cortical 39 (−1.2s.d.) At 12months and its localization was in line with what was previously reported ( expressed highly was NEDD4L and levels expression localization by compared immunofluorescence ( and mutants different three and NEDD4L wild-type for cDNA constructs with cells N2A transfected ized by both proliferation and migration. sion at E16.5 ( developing mouse cortex from E12.5 to E18.5 showed a peak of expres we analyzed the expression of ( NEDD4L wild-type of those to similar were constructs NEDD4L of versions control different the for levels Cellular expression and domain). localization HECT the in and (located p.Ser179Thr p.Gly872Ser c.2614G>A, c.535T>A, p.Ser233Leu; c.698C>T, are variants to Referring databases. (ExAC) Consortium Aggregation Exome ClinVarand/or the in reported variants control represents a disease-relevant phenotype, we tested the expression of three ( mutants NEDD4L PNH-related of expression of levels high experiments immunoblot and rescence with the proteasome inhibitor MG132. We by observed immunofluo and examined NEDD4L protein expression in transfected cells treated unstable are mutants PNH-associated that hypothesized we results, using protein N2A from cells ( transfected extracts immunoblotting by expression NEDD4L mutant of lack the mouse cultured neurons ( ( vector empty control with transfected cells of signal background the to comparable a faint signal, we only detected 48 h transfection, after contrast, PNH-related mutant proteins were hardly detectable. Indeed, p.Glu893Lys impairment, + (bilateral) c.2677G>A Dysmorphic Supplementary Fig. 5a Fig. Fig. 2 5 4 months dysplasia BRC217 wereusedforannotationofnucleotideandproteinchanges,respectively. De novo Hearing To assess the cellular consequences of PNH-related mutations, we mutations, of PNH-related consequences To cellular the assess To strengthen the implication of a post-transcriptional mechanism, To further ascertain that the instability of NEDD4L mutant proteins 61 (M) 3,120 41 − + − + + F a ). Similar results were observed upon transfection of primary upon transfection were results ). observed Similar NEDD4L

Supplementary Fig. 3b + (axialhypotonia PNC c.2677G>A 14 (−0.8s.d.) 48 (−1.2s.d.) at 34months) 100 (+2s.d.) p.Glu893Lys At 7months + (bilateral) Dysmorphic Dysmorphic mutations features De novo 4 years 3,360 PMG 38 − − + + − F , b Supplementary Fig. 4 Supplementary ). NEDD4L

+ (ISat5months) evoked potentials, optic nervepallor Abnormal visual 68 (−0.5s.d.) Fig. 2 Fig. p.Gln694His 7.6 (−2s.d.) At 8months 43 (−1s.d.) Fig. 2a Fig. + (bilateral) c.2082G>T Pnh31124 8 months Full term De novo ), a developmental stage character Fig. NA + − − − + + + F transcripts by qRT–PCR using a and and , b 2 ). ). ). We found that wild-type Fig. Fig. 2 Supplementary Fig. 4 Fig. Supplementary NM_00114496 ). ). We confirmed also + (unabletositorwalk cryptorchidism, ptosis, + (withmicrognathia) complex strabismus s e l c i t r A b 49.7(−2.32 s.d.) ). ). In view of these 20 (−0.51s.d.) DDDP110533 Arthrogyposis, At 8months + (bilateral) c.2036A>G at 6years) Tyr679Cys + (severe) 6.2 years De novo 3,000 NA 40 M + − − − + 7 , these these ,

0 . . In ), ), - - -

© 2016 Nature America, Inc. All rights reserved. mutant and compared by two different approaches the ubiquitination Arg897Gln the on focused we end, Tothis self-ubiquitination. and activity enzymatic for variants disease-causing of consequences the and to E3 lead NEDD4L ligase protein instability, we to sought assess of mutantcomplete the absence almost the not proteins. explain mutant ( levels NEDD4L a as normalizer. GFP We reporter cotransfected found wild-type that MG132. with cells N2A of treatment upon detectable become mutants NEDD4L PNH-associated that Note protein. NEDD4L for expression of level a high to led NEDD4L wild-type for transfection while mutants, PNH-associated of expression of lack show Blots MG132. or DMSO either of presence the in cultured and NEDD4L mutant and wild-type encoding constructs with transfected bar, 50 Scale detectable. not are mutants Arg897Gln and Glu893Lys Gln694His, the whereas NEDD4L, wild-type for cells N2A of periphery the in enrichment with distribution a cytoplasmic shows immunostaining NEDD4L MG132. or DMSO either with treated were cells cultured construct, each For NEDD4L. mutant or (WT) wild-type encoding constructs cDNA or vector empty with 2 Figure s e l c i t r A mutant. the and NEDD4L wild-type of ability Because all Because the mutations map to the catalytic HECT domain of the

Expression and cellular localization of wild-type and mutant NEDD4L. ( NEDD4L. mutant and wild-type of localization cellular and Expression transcript levels were approximately 1.5- to 2-fold higher than b a NEDD4L Tomato/DAPI Supplementary Fig. 6 Supplementary DMSO NEDD4L Gln694His Actin

NEDD4L Tomato/DAPI DMSO Empty vector ), ), a range of that does difference MG132 DMSO MG132 Empty vector DMSO WT MG132 MG132 DMSO Glu893Lys DMSO p.Gln694His parable to that for the wild-type protein ( protein wild-type the for that to parable the intensity of the by smear detected to antibody ubiquitin was com amount, low its and protein mutant V5-tagged the of instability the self- representing despite Interestingly, NEDD4L. mainly mutant and wild-type ubiquitinated smear high-molecular-mass a showed immunoblots the for times exposure Long detect ubiquitin. of to conjugation ubiquitin to antibody an with immunoblotted and V5 to NEDD4L, immunoprecipitated NEDD4L from lysates using antibody V5-tagged N-terminally encoding constructs cDNA with cells N2A First, to test specifically for NEDD4L ubiquitination, we transfected a MG132 ) Tomato/DAPI and NEDD4L detection in N2A cells transfected transfected cells N2A in detection NEDD4L and ) Tomato/DAPI MG132 DMSO µ m. ( m. DMSO b p.Glu893Lys ) Immunoblots using protein extracts from N2A cells cells N2A from extracts protein using ) Immunoblots a WT DVANCE ONLINE PUBLICATION ONLINE DVANCE MG132 MG132 DMSO DMSO p.Arg897Gln Arg897Gln MG132 Supplementary Fig. 7a Fig. Supplementary MG132

Nature Ge Nature n etics ). -

© 2016 Nature America, Inc. All rights reserved. neurons neurons P in highlighted bar, 100 Scale zone. zone/subventricular ventricular VZ/SVZ, zone; intermediate IZ, plate; CP, construct. cortical a Tomato with reporter combination in constructs NEDD4L mutant or NEDD4L wild-type vector, empty with IUEP 4 d after 4 Figure NEDD4L mutant and wild-type both ubiquitin, of presence the In or with without recombinant ubiquitin. enzymes and E1 (UbcH7) E2 and type NEDD4L (wild mutant)V5-tagged in an than for NEDD4L. mutant using four NEDD4L for times mixture performed less wild-type reaction as signals, shown in immunoblot of and amounts and NEDD4L mutant wild-type corresponding of of in the and NEDD4L mutant instability imbalance the resulting to with V5 antibodies and Note NEDD4L. immunoblotting that, because (+) by were or analyzed mixtures (Ub). (−) Reaction without ubiquitin cells with and ATP,incubated E1 and enzyme E2 with enzyme (UbcH7) from the N2A of lysates transfected immunopurified NEDD4L mutant in activity an ubiquitination NEDD4L and V5 to detect ubiquitinated NEDD4L (FT,(IB) by usinganalyzed immunoblot precipitates antibodiesflow through). to ubiquitin ( assay (IP; to using antibody with V5 NEDD4L), tagged to precipitate Figure 3 Nature Ge Nature * Glu893Lys, vs. EV Tbr2: in as zone Glu893Lys, Fig. 9d in as zone ventricular the in index) (mitotic cells electroporated all against marker PH3 the for positive neurons electroporated of bar, 5 Scale constructs. NEDD4L with E14.5 at electroporated brains E18.5 of zone intermediate the in neurons Tomato-positive 130 kDa 130 kDa a P < 0.0001; CP: EV vs. WT, vs. EV CP: < 0.0001;

Second, Second, we compared the ubiquitination ability of immunopurified ≤ 0.05, ** 0.05, ): EV vs. WT,

± Ubiquitination activity of mutant NEDD4L. ( Effect of wild-type and mutant NEDD4L on neuronal position and progenitor proliferation. ( proliferation. progenitor and position neuronal on NEDD4L mutant and wild-type of Effect s.e.m. of independent brains (EV, brains independent of s.e.m. Input a Empty vector c a n ( n

= 4; Arg897Gln, Tomato/DAPI

S WT

P WT Empty vector etics Tomato a upplementary Fig. 9e Fig. upplementary VZ/SVZ IZ CP Arg897Gln : VZ/SVZ: empty vector (EV) vs. Glu893Lys, Glu893Lys, vs. (EV) vector empty : VZ/SVZ: ≤ E14

0.01, *** 0.01, Empty vector Empty vector IP

P WT

S < 0.0001, EV vs. Glu893Lys, NEDD4L

E18 Arg897Gln upplementary Figure 7 Figure upplementary ADVANCE ONLINE PUBLICATION ONLINE ADVANCE Empty vector FT P WT = 0.0147, EV vs. Arg897Gln, Arg897Gln, vs. EV = 0.0147,

P Arg897Gln P

Merge ≤ exposure) (long IB: V5 IP: V5 exposure) (short IB: V5 IP: V5 < 0.0001, EV vs. Glu893Lys, Glu893Lys, vs. EV < 0.0001, n 0.001, **** 0.001, = 3). Error bars, s.e.m. ( ) 2 d after electroporation at E14.5: Pax6: EV vs. Glu893Lys, Glu893Lys, vs. EV Pax6: E14.5: at electroporation ) 2 d after

Arg897Gln Glu893Lys in vitro Tomato WT 130 kDa 130 kDa b P assay using wild-type and assay using wild-type , immunoblot analysis was , analysis immunoblot n NEDD4L

≤ = 4; WT,= 4; 0.0001. Ub P = 0.0021, EV vs. Arg897Gln, + – a Merge ) Immunoprecipitation Empty vector in vitro Glu893Lys e b n ) Quantification of Pax6 + – ) Analysis ) of Analysis = 3; Glu893Lys, Glu893Lys, = 3;

= 0.0014 (EV, = 0.0014 WT P P d = 0.0078; IZ: EV vs. WT, vs. EV IZ: = 0.0078; + – < 0.0001, EV vs. Arg897Gln, Arg897Gln, vs. EV < 0.0001, Mitotic index (%) assay using 15 10 Arg897Gln 0 5 NEDD4L V5 E14 E16 Arg897Gln n n

** = 4; WT,= 4; = 4; Arg897Gln, Arg897Gln, = 4; * tion disorders and PNH are not among the features associated with with associated features the among not are PNH and disorders tion plate cortical to the properly migrate mice) both Nedd4-1 for deficient hypothesis neurons this postmitotic made instance, For data questionable. our and findings reported previously unstable led us to consider a haploinsufficiency mechanism. However, the despite protein. the of instability activity ubiquitination of level substantial the account into take we if enhanced possibly and preserved is NEDD4L mutant NEDD4L. and V5 to antibodies mutant immunoprecipitation NEDD4L induced a high-molecular-mass smear detected both with difficult, ( is activities ubiquitination NEDD4L mutant and wild-type of comparison accurate Although ( NEDD4L mutant with one the to than NEDD4L wild-type to with assay the products protein less four times we in loaded which of series another and performed immunoprecipitation we mutant, NEDD4L the of activity smear detected by antibody to NEDD4L ( exhibited ubiquitination activity as illustrated by the high-molecular-mass have been excluded. Finally, we used an an used we Finally, excluded. been have disease pediatric severe by affected individuals which from set data one in ExAC Moreover, the were reported mutations frameshift and three stop-gain databases. DECIPHER in reported (CNVs) ations vari number copy heterozygous from resulting haploinsufficiency Fig. Fig. 3 + P Tomato At first sight, the fact that PNH-related NEDD4L mutants are are mutants NEDD4L PNH-related that fact the sight, first At of activity ubiquitination the that suggest results These ubiquitination the visualize better and confirm further To = 0.0001. Numbers of analyzed brains are as follows: EV, ** a n ) ) and Arg897Gln Glu893Lys WT Empty vector = 3; Glu893Lys, Glu893Lys, = 3; + **** P and and b and Tbr2

< 0.0001, EV vs. Glu893Lys, Glu893Lys, vs. EV < 0.0001, Percent of electroporated cells 100 µ 20 40 60 80 m. ( m. 0 in vitro P n n = Nedd4l < 0.0001. Bars represent the means of fluorescent fluorescent of means the represent Bars < 0.0001. b E14 3). ( 3). VZ/SVZ ) Fluorescent neurons were quantified in the regions regions the in quantified were neurons ) Fluorescent + P Tomato = 0.0003, EV vs. Arg897Gln, Arg897Gln, vs. EV = 0.0003, e ubiquitination ( ** a Percent of electroporated E18 ) Coronal sections of mouse brains at E18.5, E18.5, at brains mouse of sections ) Coronal c (analyzed in conditional double-knockout double-knockout conditional in (analyzed

) Immunofluorescence staining of NEDD4L in NEDD4L of staining ) Immunofluorescence cells in VZ/SVZ n 20 40 60 80 + = 4; Arg897Gln, Arg897Gln, = 4; 0 cells in the ventricular zone/subventricular in vitro in E14 **** Pax6 IZ **** E16 ** assays and immunoblot analysis analysis immunoblot and assays **** + * Fig. Fig. 3 ** P < 0.0001, EV vs. Arg897Gln, Arg897Gln, vs. EV < 0.0001, * Supplementary Fig. 7b in utero in n b = 3). Error bars, s.e.m. s.e.m. bars, Error = 3). 2 ) ) approaches showed that 1 ** ** . Also, neuronal migra neuronal . Also, CP **** a µ P Tbr2 ( m. ( m. **** Arg897Gln Glu893Lys WT Empty vector Arg897Gln Glu893Lys WT Empty vector = 0.0001; = 0.0001; S RNA interference interference RNA s e l c i t r A upplementary upplementary * + d n ) Percentage ) Percentage ** = 4; WT,

Fig. Fig.

). n

= 3;

3 ). ). - -

© 2016 Nature America, Inc. All rights reserved. these these cell populations only in brains electroporated with PNH-related of number increased an found and zone subventricular and zone lar ventricu the in cells Tbr2-positive and Pax6-positive electroporated with wild-type NEDD4L and PNH-related mutants. We also quantified gested an mitotic increased index of apical progenitors electroporated empty vector ( control the to Tomato-positive compared expressing when zone cells ventricular the in mutants HECT-domain or protein wild-type ing centage of Tomato-positive and PH3-positive co-labeled cells express against PH3, Pax6, Tbr2 and Ki67. We found a significantly higher per lected embryos 48 h later for immunohistochemistry using antibodies col and E14.5 at IUEP performed we proliferation, progenitor basal disrupted. was translocation terminal ( region Cux1 lower the in enrichment a deviating with cells of neuronal distribution abnormal We found that expression of and wild-type mutant NEDD4L led to an layer ( of parts Cux1-positive the remaining and to in the upper and two equal lower Cux1 corresponding regions (PCZ) zone cortical primitive as the defined previously region the (ECM) matrix in to the extracellular adhering with a process leading the pups. We then the assessed distribution of electroporated neurons from (P2) later d 6 brains collected and at E14.5 constructs different expressed endogenously to correspond ( NEDD4L could that signal faint a detected only we constructs, mutant with electroporated neurons in Fig. 9c ited strong cytoplasmic immunolabeling ( be that cells electroporated could with construct for NEDD4L wild-type exhib effect same the whether observed assessed also we unstable, are plate the cortical ( of VI and V layers in and matter white the in distributed abnormally with constructs for wild-type NEDD4L and PNH-related mutants were layers II–IV of the cortical plate, we found that neurons electroporated superficial in located mainly were vector empty with electroporated neurons Whereas at E18.5. observed those thatparalleled differences the effect on neuron positioning at postnatal day (P) 2 and highlighted responding depletion in the cortical plate ( tricular zone and subventricular zone and intermediate zone with cor PNH-related mutants induced significant arrest of cells within the ven the cortical plate ( we observed that neurons electroporated with the empty vector reached and brainsembryo analyzed 4 d later In (E18.5). E18.5 brain sections, genitor cells located in the ventricular zone of E14.5 mouse neocortices pro into Tomatoconstruct with combination reporter in constructs on projection effects neuron we positioning, electroporated Toassess translocation. terminal and proliferation progenitor ronal neu positioning, neuronal for Arg897Gln) and (Glu893Lys mutants and NEDD4L of comparedexpression consequences of the wild-type We an processes. used of PNH-related mutationsconsequences the for neurodevelopmental ( targeting hairpin short (shRNA) with RNA transfected brains embryo significant no between found and difference mice in migration neuronal cortical on of consequences the analyze to approach s e l c i t r A ( mutant constructs Supplementary Fig. 8a Fig. Supplementary

To test the consequences of the the of consequences To the test translocation terminal study To As our that data mutantsNEDD4L PNH-related cellular suggested study to sought then we arguments, convergent these of view In ), ), consistent with a high level of NEDD4L expression. However, in vivo Fig. 4 Fig. Fig. 4 in electroporated migrating neuronal cells. We found c Fig. 4a and and Supplementary Fig. 9a Supplementary Fig. 4 Fig. d and in uteroin Supplementary Fig. 9c Fig. Supplementary , b , e b ). However, IUEP of wild-type NEDD4L and Supplementary Supplementary Fig. 9d and ). Supplementary Fig. 10 Fig. Supplementary Nedd4l electroporation (IUEP) approachand (IUEP) electroporation Supplementary Fig. 9e Fig. Supplementary NEDD4L and scrambled shRNA control control shRNA scrambled and 22– 2 5 , we conducted IUEP of the the of IUEP conducted we , , Fig. Fig. 4 Fig. 4a b ). Supplementary Fig. 10 Supplementary mutations on apical and and apical on mutations Nedd4l ). c and , b ). ). These results sug ). ). We also assessed ), suggesting that that suggesting ), downregulation downregulation Supplementary Supplementary ). NEDD4L 2 4 ). ). ------,

on mTOR-dependent pathways for the following main reasons. reasons. main following the for pathways mTOR-dependent on NEDD4L mutant and wild-type of consequences the investigate to opmental processes has recently emerged evidence demonstrating crucial regulatory roles of activity NEDD4L in devel ENaC elevated by caused hypertension tary a shown to (ENaC), syndrome, channel heredi be involved in Liddle sodium epithelial the is of NEDD4L target known best the Although causing Pathways constructs shown). not different (data the with electroporation following no difference found and rate death cell evaluate to caspase-3 cleaved to body are ( layers upper for cortex fated and the differentiated of layers deep in neurons misplaced that indicating Cux1, expressed mutants PNH-related or NEDD4L wild-type with electroporated neurons arrested that observed and E18.5 at marker ( and mutant NEDD4L wild-type encoding constructs cDNA and vector empty control with poration in both regions, we did any electro following not observe differences both Tomato and Ki67 in the ventricular zone and subventricular zone; *** and Methods (Online means represent Data phosphorylation. Akt and S6 illustrating measurements densitometric ( Thr308/Ser473) (p-Akt; Akt phosphorylated ( activity) mTORC1 reflecting (p-S6; S6 phosphorylated of levels the on NEDD4L mutant and wild-type of effect the showing NEDD4L, mutant and wild-type for constructs or vector empty with transfected cells N2A from extracts protein using immunoblots ( activities. AKT and mTORC1 of deregulation 5 Figure b a p-Akt (Ser473) p-Akt (Thr308) ial, e aee ban lcs E65 n E85 wt anti with E18.5) and (E16.5 slices brain labeled we Finally, Cux1 the against immunohistochemistry performed also We To assess the pool of proliferating cells, we evaluated cells positive for P

NEDD4L NEDD4L ≤ 0.001, **** 0.001,

Actin Actin p-S6

Wild-type NEDD4L and PNH-related mutants induce induce mutants PNH-related and NEDD4L Wild-type PNH Akt S6

disrupted

Empty vector Empty vector a DVANCE ONLINE PUBLICATION ONLINE DVANCE

P WT WT

S ≤ ± upplementary upplementary 0.0001.

s.e.m. from three independent experiments experiments independent three from s.e.m. Gln694His by Gln694His

wild-type Glu893Lys Glu893Lys

Arg897Gln Arg897Gln Supplementary Fig. 9f Fig. Supplementary T able 2 able

and c

19 ). * ). mutant Supplementary Fig. 9h Fig. Supplementary b Akt-pSer473/Akt Akt-pThr308/Akt p-S6/S6 a , 2 ). ( ). , 7 b (% relative to empty vector) (% from empty vector) (% from empty vector) P

. . In this study, we sought Empty vector Empty vector

) Representative ) Representative Empty vector 100 150 200 250 200 400 600 200 400 600

c ≤ 50 ) Histograms of ) Histograms 0 0 0 0.05, ** 0.05,

NEDD4L Nature Ge Nature a

) and ) and Gln694His Gln694His WT W Gln694HisWT **** 2 T 6 P , compelling compelling , Glu893Lys Glu893Lys **** , *** **

g Glu893Lys ≤ ). 0.01, 0.01,

Arg897Gln * ****

Arg897Gln

**** * Arg897Gln n etics ****

** **** ).

- - - -

© 2016 Nature America, Inc. All rights reserved. expression to the above highlighted findings, we tested the effect of of effect the tested we findings, highlighted above the to expression at ( Ser473 and Thr308 phosphorylated forms Akt of levels increased significantly with trast, for Akt, only PNH-associated NEDD4L mutants were associated ( activity mTORC1 signaling elevated reflects that (Ser240/Ser244) S6 phosphorylated of level increased an observed we mutants, PNH-associated and NEDD4L wild-type for constructs and Akt mTORC1, on NEDD4L Smad2/3 signaling Followingactivities. transfection of mutant N2A cells with and wild-type of effect pathways signaling TGF- and PI3K–mTORC2 between crosstalk the of regulation in player critical a as identified recently was NEDD4L Third, pathways. signaling Dab1 mTORand Dab1 and expression Cul5 expression ligase ubiquitin E3 signaling, in mTOR deficit migration neuronal with tuberous complex a sclerosis involving pathology (TSC) cascade linking the insights migration, reported neuronal recently modulate that pathways signaling tified pathways (PI3K)–AKT–mTOR 3-kinase phosphatidylinositol of the interdependent by dysfunctions caused disorders body and brain of developmental range wide the in condition particular a represent abnormalities developmental these whether wondered we study, this in described patients in observed First, in view of the association of MCD and distal-limb abnormalities *** 100 bars, Scale plate). cortical the of neurons extent, a lesser to and, zone intermediate the of neurons arrested in (mainly cytoplasm the of periphery the in enrichment its and distribution Dab1 of pattern specific the note mice, non-treated from NEDD4L wild-type for construct with electroporated neurons arrested In Dab1. of distribution the showing (2) zone intermediate and (1) plate cortical the in boxes white in enclosed regions the of views higher-magnification are section coronal each of right the to Panels ( brains. independent rapamycin, Arg897Gln vs. DMSO Arg897Gln rapamycin, Glu893Lys in indicated regions the in bar, 100 Scale mg/kg/d). (0.5 rapamycin or (DMSO) vehicle either with treated were mice pregnant IUEP, After vector. a Tomato with reporter together NEDD4L mutant and wild-type for constructs or vector empty with E14.5 at electroporated brains 6 Figure Nature Ge Nature found and phosphorylation Akt and on S6 variants control three the c a

To reinforce the relevance and specificity of NEDD4L mutant mutant NEDD4L of specificity and relevance the reinforce To the assays immunoblot and cellular by tested first we Therefore, Dab1/Tomato Tomato/DAPI P Empty vector VZ/SVZ IZ CP E14

DMSO E14 ≤ 0.001, **** 0.001,

Effect of rapamycin treatment on neuronal positioning and Dab1 localization. ( localization. Dab1 and positioning neuronal on treatment rapamycin of Effect Empty vector E18 E18 2 n 8 also led us to consider a potential effect of NEDD4L on of NEDD4L effect a potential consider to us led also etics Rapamycin Dab1 Dab1 1 2 P

c ≤ ADVANCE ONLINE PUBLICATION ONLINE ADVANCE Fig. 5b, Fig. ) Immunolabeling of Dab1 on cortical slices at E18.5 from the brains of embryos subjected to DMSO or rapamycin treatment. treatment. rapamycin or DMSO to subjected embryos of brains the from E18.5 at slices cortical on Dab1 of ) Immunolabeling , 0.0001. P 3 0 = 0.0377, Arg897Gln DMSO vs. Arg897Gln rapamycin, rapamycin, Arg897Gln vs. DMSO Arg897Gln = 0.0377, . a : VZ/SVZ: WT DMSO vs. WT rapamycin, rapamycin, WT vs. DMSO WT : VZ/SVZ: WT DMSO c β ). –activin–Smad2–Smad3 (Smad2/3) (Smad2/3) –activin–Smad2–Smad3 5 , 1 Dab1 Dab1 4 WT . Second, among the many iden many the among Second, . 2 1 Rapamycin P Glu893Lys = 0.0013. Bars represent the means of electroporated neurons in each region region each in neurons electroporated of means the represent Bars = 0.0013. DMSO Fig. 5a Fig.

Glu893Lys Dab1 Dab1 2 1 , c Rapamycin ). In con In ). Arg897Gln P = 0.012, IZ: WT DMSO vs. WT rapamycin, rapamycin, WT vs. DMSO WT IZ: = 0.012, - -

µ m and 10 10 m and DMSO electroporated at E14.5 with wild-type NEDD4L and subjected to to subjected and NEDD4L wild-type with E14.5 at electroporated embryos from sections brain E18.5 in Interestingly, positioning. ron inhibitor) on pregnant mice and analyzed the effect on projectionin neu approach IUEP an combination with treatment used with rapamycin (a we well-known mTORC1 above, described defects opmental in signaling Smad2/3 and activin–TGF- Akt between crosstalk regulated the disruption of to leads mutant NEDD4L the of expression that gested ( Smad2/3 phosphorylated of levels stable and Akt phosphorylated of levels in the decrease substantial a with associated was mutant of NEDD4L expression the whereas Smad2/3, and Akt the phosphorylated in of increase levels an with associated was NEDD4L wild-type of sion TGF- the of A activin by tion ( Smad2/3 phosphorylated of levels the in increase substantial a mutant with of associated was expression NEDD4L whereas Smad2/3, of level activation the effect on no had NEDD4L wild-type of overexpression that found we and Ser425 Akt at phosphorylated UnderSer473. the condition, basal at Ser423/ phosphorylated Smad3 at Ser465/Ser467, phosphorylated of the TGF- activation upon and condition basal the under NEDD4L Arg897Gln ( NEDD4L wild-type one with to the similar was that pattern a phosphorylation that overexpression of the three NEDD4L variants was associated with Dab1 Dab1 To assess the To the assess and wild-type of effect the tested we activity, Smad2/3 For 2 1 P Arg897Gln = 0.0004; CP: WT DMSO vs. WT rapamycin, rapamycin, WT vs. DMSO WT CP: = 0.0004; µ Rapamycin a m (higher-magnification images). * images). m (higher-magnification Dab1/Tomato ) Confocal microscopy images of coronal sections from E18.5 E18.5 from sections coronal of images microscopy ) Confocal Supplementary Fig. 11 Fig. Supplementary Supplementary Fig. 5b Fig. Supplementary β Empty vector E14 pathway by activin A on the expression levels of A pathway levels Smad2 by on the expression activin β pathways. in vivo in E18+ rapamycin

Empty vector + DMSO b

µ Empty vector + rapamycin

m. ( m. Percent of contributions of these pathways to the devel the to pathways these of contributions electroporated cells 100 b 20 40 60 80 ) Electroporated neurons were quantified quantified were neurons ) Electroporated 0 Supplementary Fig. 11 Fig. Supplementary WT

β WT + DMSO WT + rapamycin pathway, we found that overexpres that found we pathway, P

< 0.0001, Glu893Lys DMSO vs. vs. DMSO Glu893Lys < 0.0001, Glu893Lys + DMSO Glu893Lys + rapamycin ). ). Collectively, these results sug results these Collectively, ). Dab1 Dab1

2 1 **** * ****

Arg897Gln + DMSO Arg897Gln + rapamycin P

≤ ± Glu893Lys

0.05, ** 0.05, s.e.m. from three three from s.e.m. * P < 0.0001, < 0.0001,

s e l c i t r A

** . pn activa Upon ). *** P

≤ 0.01, 0.01, 0 20 40 60 80 100 Arg897Gln

VZ/SVZ IZ CP

- - - - -

© 2016 Nature America, Inc. All rights reserved. Our findings demonstrate the critical role of NEDD4L in the regu the in NEDD4L of role critical the demonstrate findings Our DISCUSSION ( plate cortical the reached that neurons rescued the in or neurons in either misplaced was observed, localization cellular Dab1 with constructs for mutants,PNH-associated no significant change in ( localization Dab1 pattern of diffuse the of restoration found we level NEDD4L, high a wild-type of expressed still that and plate cortical the reached that of neurons pool predominant the in treatment, rapamycin following rated ( empty with or vector mutant constructs found in electropo neurons pattern from the diffuse distinguishable of of Dab1 in a that the the cytoplasm, periphery distribution is easily localization exclusive nearly and enrichment suggesting labeling ing electroporated with construct for NEDD4L, wild-type we found strik and in neurons. distribution In electroporated arrested neuronal cells neuronal position rapamycin-rescued correlates with relevant changeswhether in Dab1 expression assessed and Dab1 against antibodies ( NEDD4L of level high a expressed still NEDD4L wild-type for construct with porated mutants (data PNH-associated not shown), neurons whereas electro for constructs with electroporated neurons in expression NEDD4L of an absence and found treatment rapamycin after plate cortical the also analyzed the expression of NEDD4L in neuronal cells that reached ( increased significantly was plate cortical the reached that was neurons of number the while zone, migration intermediate the in reduced delayed with neurons Tomato-positive of number with IUEP ( from mutants PNH-related resulting for constructs defect position neuronal the of rescue ( similar was treatment rapamycin without or with cells neuronal of ( vector empty control with electroporated in embryos to what similar was observed across the cells cortex of with a ing electroporated defect distribution rapamycin treatment, we found clear rescue of the neuronal position s e l c i t r A implicate and development cortex in involved processes of lation between components of signaling pathways. signaling of components between relationships understood poorly and indirect depict arrows dotted and proteins, deregulated indicate contours Red (right). mutants NEDD4L related ( degradation. and autoubiquitination trigger and state active open, the to state inactive the from transition favor that changes conformational to lead to predicted ( 7 Figure a a Ub Fig. 6a Fig.

) Wild-type NEDD4L (left) is shown in its closed and inactive conformation, and mutant NEDD4L (right) is shown with an alteration in the domain HECT an is with alteration shown (right) NEDD4L and mutant conformation, and is in inactive shown its closed (left) ) NEDD4L Wild-type We then analyzed brain sections by immunohistochemistry using using by immunohistochemistry sections brain We analyzed then C2

, Models depicting the consequences of PNH-related mutations for NEDD4L stability and PI3K–Akt–mTOR and TGF- and PI3K–Akt–mTOR and stability NEDD4L for mutations PNH-related of consequences the depicting Models b ). However, rapamycin treatment induced only a partial partial a only induced treatment rapamycin However, ). Inactive conformatio WT NEDD4L WW b ) Overview of observed deregulations under basal conditions resulting from an excess of wild-type NEDD4L (left) and expression of PNH- of expression and (left) NEDD4L wild-type of excess an from resulting conditions basal under deregulations observed of ) Overview Ub Fig. 6a Fig. WW HEC Supplementary Fig. 12 Fig. Supplementary T WW PY , Fig. 6 Fig. b WW ). For this latter condition, the distribution distribution the condition, latter this For ). n c ). However, in neurons electroporated electroporated neurons in However, ). C2 C2 Fig. 6a Fig. in theHECTdomain Missense mutations Ub proteasomal degradation Autoubiquitination and ). WW WW Ub , b WW Fig. Fig. 6 WW

). We found that the the that Wefound ). HECT

WW HECT WW c

). ). Interestingly, PY

Fig. 6a Fig. PY WW Fig. 6 Fig. , b b Smad2/3 ). We). c ). activin TGF - - - - -

- WT NEDD4Loverexpression β A , Thr220/179 Smad2/3 mic Dab1 in the cytoplasm of postmitotic neurons and its inhibitory inhibitory its and neurons postmitotic of cytoplasm the in Dab1 mic studies that demonstrated association between excess nucleocytoplas worth mentioning that is finding in this line with previously reported is It distribution. Dab1 of pattern distinct a observed we NEDD4L, wild-type overexpressing cells neuronal misplaced the In cells. nal neuro postmitotic of defect positioning a to corresponds NEDD4L mutants. NEDD4L unstable the and NEDD4L wild-type stable resulting from defects IUEP of neurodevelopmental similar observed the regarding discrepancy apparent the partially, least at reconcile, the overexpression of wild-type NEDD4L. This model could therefore from resulting activity increased the mimic could mutants NEDD4L tion and the degradation, state predicted of constitutive activation for autoubiquitina despite Moreover, activity. ubiquitination in deficit a to lead to expected is mechanism loss-of-function conventional a whereas substrates, NEDD4L other of but ubiquitination aberrant autoubiquitination also only not expect could one domain, HECT conformation NEDD4L on dependent is ligases, E3 HECT of studies suggesting that catalytic activity, including autoubiquitination and their degradation. This hypothesis variants is based mutant on previously NEDD4L reported of autoubiquitination trigger in could which turn function, catalytic of activation constitutive and changes amino acid changes in the HECT domain could lead to conformation in model the by illustrated hypothesis to the propose mutants we sensitivity, this To corresponding explain degradation. the proteasome of sensitivity in increase likely the ( families unrelated three in found was that tion one of these mutations, c.2677G>A, p.Glu893Lys, is a recurrent muta and the domain HECT of NEDD4L, encoding the E3 ligase ubiquitin of region the in located are phenotype this with associated mutations All patients). seven of six (in syndactyly and palate cleft disability, by PNH, intellectual characterized phenotype tinguishable patients (two siblings and five unrelated patients) with a common dis and of five one transmitted identification of domain HECT the in mutations mTORC2 P nte fnig ht a osre wt wl-ye n mutant and wild-type with observed was that finding Another the in mutations to due activation constitutive of model the With was mutations PNH-related the of effects intriguing the of One S6K S6 P hormones, cytokines... P P Ser X Ser Growth factors, Ser473 mTORC1 P TSC2 Rheb PDK1 Pl3K Ak P TSC1 t Thr308 a Complete DVANCE ONLINE PUBLICATION ONLINE DVANCE rescu e Dab1 Reelin Rapamycin VLDLR Dab1 APOER2 Smad2/3 activin TGF- NEDD4L β A , NEDD4L HECTmutants Figure 7 Figure Smad2/3 Thr220/179 mTORC2 P de de novo S6K β S6 hormones, cytokines... P P P –Smad pathways. pathways. –Smad in PNH. We report the the We PNH. report in Ser X Ser Growth factors, Ser473 a

Table . Mutations causing causing Mutations . mutations in seven in mutations seven Nature Ge Nature P mTORC1 TSC2 Rheb PDK1 Pl3K Ak P TSC1 t 1 ). rescu Partial Thr308 NEDD4L e Dab1 n Reelin Rapamycin 31– VLDLR etics Dab1 3

4 APOER2 . - - - - -

© 2016 Nature America, Inc. All rights reserved. providing humanV5-tagged Nedd4-2 constructs (pcDNA3.1-nV5 wild type, We thank D. Rotin and C. Jiang (The Hospital for Sick Children) for kindly We are grateful to the patients and their families for their participation. onl Note: Any Supplementary Information and Source Data files are available in the ( Accessioncodes. the pape the of in version available are references associated any and Methods M portal, http://www.ddduk http://www.epgp.o DECIPHER, broadinstitute.org URLs. from different haploinsufficiency. are constitutive that consequences functional with but protein, muta missense state and of loss which active to the mutant lead a might tions constitutively in mechanism, molecular novel disease-causing potential a identified study our Moreover, pathways. naling of consequences disruptive the highlighting evidence reported also we encompassing duplications with associated phenotypes of understanding better a for basis a provides therefore finding latter This functioning. and development brain for eterious del to be is likely NEDD4L excess that and PNH with are associated in mutations that shown time MCD of features phenotypic with associated in mutations vating acti by caused syndromes neurodevelopmental of spectrum large a in pathways signaling PI3K–AKT of implication established the and development cortical underlying processes neurodevelopmental regulation of the in NEDD4L of role the further understand to point TGF- upon and conditions mTORC1, the deregulation of Akt and under Smad2/3 activities basal NEDD4L could be mediated by mTORC1 pathway deregulation. wild-type of overexpression by induced defects positioning and tion migra the that rapamycinsuggest bytreatment Dab1 of distribution cellular the of restoration and defect position neuronal the of rescue ing signal PI3K–AKT regulates and PIK3CA ubiquitinationof catalyzes these hypotheses are pathways, supported these by aaffect recent variants study and overexpression showing NEDD4L that which NEDD4L investigationsfurther are mechanisms required exact bythe to define mTORC2 and TGF- PNH-related mutants might act through deregulation of mTORC1, Akt,tion of signaling pathways regulated by mTORC1 and Dab1. Insuggesting contrast, that excess wild-type NEDD4L is associated with the disrup overexpression of wild-type NEDD4L. In favor of this view is evidence could be mediated by a mechanism divergent from the one disrupted by bymutants NEDD4L caused neuronal defect thatmigrationgests the rated with NEDD4L mutants, the unchanged distribution of Dab1 sug migration neuronal on effect Nature Ge Nature A NM_0 cknowledgments ethods NEDD4L As a whole and to the best of our knowledge, we have for the first first the for have we knowledge, our of best the to and whole a As of activity increased the to addition in mutants, PNH-related For ine version of the pape 3 5 and by our rapamycin-based experiments. Indeed, the consistent Exome Aggregation Consortium (ExAC), (ExAC), Consortium Aggregation Exome http://www 0111438 n mutations on the AKT–mTOR and TGF- etics https:// 6 ). ClinVar accessions: Human / .org .sbg.bio.ic.ac.uk/ph

rg ; MutationTester, ; r β . r ADVANCE ONLINE PUBLICATION ONLINE ADVANCE / AKT3 –Smad2/3 pathway activities ( . ; Deciphering Developmental Disorders Study, Disorders Developmental Deciphering ; / decipher.sanger.ac.u ; SubRVIS, NEDD4L β , , PIK3R2 activation provides an interesting entry entry an interesting provides activation 3 5 . In arrested neuronal cells electropo cells neuronal arrested In . NEDD4L http://www.sub ( and and NM_00114496 http://www.mutation SCV00026710 yre2 altering the HECT domain domain HECT the altering PIK3C / . k / NEDD4L 5 ; Epi4K Consortium, Consortium, Epi4K ; , 14 , , some of which are are which of some , 36 rvis.org , Fig. 7 3 6 7 7 – ), mouse ),

. β SCV00026710 . In this study, this In . –Smad2/3 sig –Smad2/3 b / http://exac. ). Although ; Phyre web taster.org

Nedd4l online online

9 / ------; .

13. 12. B.I., U.W.L., A.P., N.B.W., R.G., D.K., C.B., the DDD study, U.K.performed expressionand N.B.-B. and genetic studies, and prepared reagents. J.C.-S., K.A.M., performed ubiquitination experiments. N.D. provided technical assistance, to cellular, IUEP and functional studies. S.S. provided technical assistance and the experiments, performed statistical analysis and analyzed the data related L.B., H.J., E.L.I. and M.-V.H. conceived and designed the experiments, performed UK Department of Health. in this publication are ofthose the authors and not ofnecessarily those the Health’s NIHR Biomedical Research Centre funding scheme. The expressed views Biomedical Research Centre Oxford with funding from the UK Department of NS077303 and This NS077276). study was also supported in part by the NIHR and the Epilepsy Phenome/Genome Project NS077364, (NS053998, NS077274, National Institute of Neurological Disorders and Stroke to the Epi4k Consortium 241995) and DESIRE (grant agreement 602531), and funding provided from the ANR-13-BSV-0009-01) and European Union FP7 project GENECODYS (grant (ANR Blanc 1103 01, project R11039KK; ANR projectE-Rare-012-01, E10107KP; Rares, the Fondation NRJ–Institut de France, Agence National de Recherche of the program Equipe FRM; J.C–DEQ20130326477), the Fondation Maladies Fondation pour la Recherche Médicale (FRM funding within the framework the frame program Investissements d’Avenir ANR-10-IDEX-0002-02, the a French State Fund managed by the Agence Nationale de la Recherche under funding from Strasbourg University and grant ANR-10-LABX-0030-INRT, NEDD4L Consortium and the Epilepsy Phenome/Genome Project for contributing for their thoughtful comments and help. We thank investigators from the Epi4K CS and Y971A constructs) and L. Lindner, M. Ruff, M. Macias and F. Francis 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. reprints/index.htm at online available is information permissions and Reprints The authors declare no competing interests.financial the study, designed experiments, analyzed data and wrote the manuscript. designed ubiquitination experiments. J.C. conceived, coordinated and supervised sequencing, bioinformatics tools and analysis of sequencing data. I.S. conceived and A.B., R.O., C.M., P.N. and J.-F.D. contributed to the generation of whole-exome material, as well as critical suggestions for functional studies. T.S., the DDD study, expression studies during brain development. L.N. and J.G. contributed reagents and in candidate genes and screened DNA from subjects. M.M. and J.G. performed G.R., B.S., A.E. and R.T. contributed to genetic studies and analysis of variants P.T. and G.M. provided assistance for IUEP studies. A.T.P., J.C.T., contributedK.P., clinical andY.S., imaging data andN.L., follow-up of patients and families. AUTHOR CONTRIBUTIONS COMPETING FINANCIAL INTERESTS FINANCIAL COMPETING

ore, K. Poirier, V. Conti, he, V.L. Sheen, G. Lian, A. Carabalona, R.J. Ferland, Parrini, E. Fox, J.W. Guerrini, R. & Dobyns, W.B. Malformations of cortical development: clinical features F.Francis, W.B. Dobyns, & G.D. Jackson, R.I., Kuzniecky, R., Guerrini, A.J., Barkovich, P.Rakic, mutants V.S.neurological Caviness, from & view Jr.development: Cortical Caviness, V.S. Jr.,Takahashi, T. neocortical and time R.S. Numbers, & Nowakowski, (2013). malformations of cortical development and microcephaly. the of cortex. 36 cerebral human the in migration and proliferation progenitor (2012). phosphorylation. Cdk1 Wee1-dependent through filamin-A. of expression impaired by (2009). subventricular zones in periventricular heterotopia. mutations. A filamin with heterotopia. periventricular human in causes. genetic and Neurosci. J. Eur. 2012. update A developmental and genetic classification for malformations of cortical development: later. decades two 18 model. evolutionary and developmental general a neuronogenesis: , 69–76 (2004). 69–76 , (1995). 379–383 , -related genotype and phenotype data. This work was supported by C6orf70 et al. et et al. t al. et et al. et al. et t al. et t al. et Filamin A regulates neural progenitor proliferation and cortical size cortical and proliferation progenitor neural regulates A Filamin Mutations in filamin 1 prevent migration of cerebral cortical neurons t al. et Brain eietiua htrtpa n q emnl eein ydoe role syndrome: deletion terminal 6q in heterotopia Periventricular l gene. Periventricular heterotopia: phenotypic heterogeneity and correlation . t al. et Human disorders of cortical development: from past to present. to past from development: cortical of disorders Human

23 uain in Mutations uain in Mutations Neuron

irpin f erl rgntr aog h vnrclr and ventricular the along progenitors neural of Disruption , 877–893 (2006). 877–893 , Lancet Neurol. Lancet 135 gil rgn o prvnrclr oua htrtpa caused heterotopia nodular periventricular for origin glial A Brain , 1348–1369 (2012). 1348–1369 ,

Brain 14

136 , 1101–1104 (1995). 1101–1104 ,

, 3378–3394 (2013). 3378–3394 , 129 ARFGEF2 TUBG1

13 , 1892–1906 (2006). 1892–1906 , Hum. Mol. Genet. Mol. Hum. Neuron , 710–726 (2014). 710–726 , , implicate vesicle trafficking in neural in trafficking vesicle implicate DYNC1H1

21 , 1315–1325 (1998). 1315–1325 , Hum. Mol. Genet. . Neurosci. J. ,

21 KIF5C Nat. Genet. http://www.nature. , 1004–1017 (2012). 1004–1017 , s e l c i t r A and

32 TrendsNeurosci. 7672–7684 ,

18 45 KIF2A a. Genet. Nat. , 497–516 , 639–647

cause

com/

Sekine, K.,Honda,T., Kawauchi,T., Kubo,K.&Nakajima,Theoutermostregion Olson, E.C., Kim, S. & Walsh, C.A. Impaired neuronal positioning and dendritogenesis U. Müller, & S.R. Harkins-Perry, C., Gil-Sanz, I., Martinez-Garay, S.J., Franco, H.E. Hsia, A. Rohrwasser, & J.M. Lalouel, R.B., Weiss, B.L., Blazer-Yost, N.F., Garrone, for ligase ubiquitin the (NEDD4L): NEDD4-2 S. Kumar, & J.A. P.,Manning, Goel, L. Huang, Ca universal a of function and structure C2-domains, T.C. Südhof, & J. Rizo, of Characterization P. Bork, & with A. Einbond, ofgenes C., Bougeret, H.I., Chen, aset M., Sudol, of Identification M. Noda, & Y. Tomooka, S., Kumar, G.M. Mirzaa, 31 mode and the Dab1-dependent “inside-out” lamination in the neocortex. migration radial the of switch the both for crucial is plate cortical developing the of suppression. Dab1 cell-autonomous after (2006). 1767–1775 neocortex the in migration neocortex. neuronal the in control lamination and to Dab1/Rap1 via function cadherin regulates Reelin USA growth. neurite promote to signaling PI3K/PTEN–mTORC1 two domain. C2 a to lack or leading contain that by isoforms targeting subcellular NEDD4L affects polymorphism human A proteins. membrane multiple cascade. enzyme E2–E3 the by domain. binding protein-bindingnovela module—thedomain.WW Commun. Res. brain. mouse the in expression down-regulated developmentally study. sequencing subunit, regulatory , 9426–9439 (2011). 9426–9439 ,

111 , 13205–13210 (2014). 13205–13210 , et al. et t al. et et al. et

Structure of an E6AP–UbcH7 complex: insights into ubiquitination into insights complex: E6AP–UbcH7 an of Structure 185 J. Biol. Chem. Biol. J. bqii E lgs Nd41 cs s dwsra tre of target downstream a as acts Nedd4-1 ligase E3 Ubiquitin Lancet Neurol. Lancet Characterisation of mutations of the phosphoinositide-3-kinase the of mutations of Characterisation , 1155–1161 (1992). 1155–1161 , PIK3R2 i prslin oyirgra a next-generation a polymicrogyria: perisylvian in , Gene

273 Science

Neuron 14

557 , 15879–15882 (1998). 15879–15882 , , 1182–1195 (2015). 1182–1195 , , 1–10 (2015). 1–10 ,

286 69 BMC Cell Biol. Cell BMC , 482–497 (2011). 482–497 , , 1321–1326 (1999). 1321–1326 , FEBS Lett.FEBS

10 rc Nt. cd Sci. Acad. Natl. Proc.

369 , 26 (2009). 26 , Biochem. Biophys. Biochem. . Neurosci. J. , 67–71 (1995).67–71, J. Neurosci.

26 2+ - ,

37. 36. 35. 34. 33. 32. 31. 30. 29. 28. 27. 26. 25.

asn L.A. Jansen, J.B. Rivière, regulated is which disabled-1, cytosolic of level Proper K. Nakajima, & T. Honda, A. Escobedo, J. Wang, M.C. Bruce, Wiesner,S. J.S. Yu, S. Gao, U.Y.Moon, human ubiquitin– of of family a of member mechanisms founding the NEDD4: S. Kumar, Molecular & N.A. Boase, D.S. Geller, & A.G. Gharavi, R.P., Lifton, K. Sekine, afrain fo mglnehl t fcl otcl dysplasia. cortical focal to (2015). 1613–1628 megalencephaly from malformations 44 and Cortex Cereb. by dual nuclear translocation pathways, is important for cortical neuronal migration. Nedd4L. ligase ubiquitin E3 the of (2010). auto-inhibition. domain–mediated C2 the HECT-domain.its within located motif domain. C2 its phosphorylation. linker via activity Smad2/3 signaling. complex. sclerosis tuberous of deficits ligases. protein hypertension. integrin of activation inside-out via adhesion , 934–940 (2012). 934–940 , PIK3CA t al. et t al. et Mol. Cell Mol. t al. et et al. et et al. et t al. et cause a spectrum of related megalencephaly syndromes. megalencephaly related of spectrum a cause et al. et et al. et

bqii lgs Nd4 tres ciae Sa23 o ii TGF- limit to Smad2/3 activated targets Nedd4L ligase Ubiquitin et al. et Cell 26 t al. et IKmOC rglts TGF- regulates PI3K/mTORC2 Cell Gene a acu atvts ed E uiutn iae b releasing by ligases ubiquitin E3 Nedd4 activates Calcium Impaired reelin-Dab1 signaling contributes to neuronal migration neuronal to contributes signaling reelin-Dab1 Impaired , 3219–3236 (2016). 3219–3236 , Autoinhibition of the HECT-type ubiquitin ligase Smurf2 through Smurf2 ligase HECT-typeubiquitin the of Autoinhibition eln otos ernl oiinn b pooig cell-matrix promoting by positioning neuronal controls Reelin

DVANCE ONLINE PUBLICATION ONLINE DVANCE Regulation of Nedd4-2 self-ubiquitination and stability by a PY a by stability and self-ubiquitination Nedd4-2 of Regulation

104 Structural basis of the activation and degradation mechanisms degradation and activation the of basis Structural De novo De

IKAT aha mttos as a pcrm f brain of spectrum a cause mutations pathway PI3K/AKT 36 130

557 , 545–556 (2001). 545–556 , , 457–468 (2009). 457–468 , , 651–662 (2007). 651–662 , , 113–122 (2015). 113–122 , germline and postzygotic mutations in mutations postzygotic and germline Structure Cell Rep. Cell Biochem. J. Biochem. . il Chem. Biol. J. Nat. Commun. Nat. α

22 5 β β

atvn inlig y modulating by signalling /activin , 1446–1457 (2014). 1446–1457 , 1. 12

415 Neuron , 965–978 (2015). 965–978 , , 155–163 (2008). 155–163 ,

Nature Ge Nature

76 6 285 , 7212 (2015). 7212 , , 353–369 (2012). 353–369 , 12279–12288 , AKT3 Brain Nat. Genet. Nat. , n PIK3R2 etics

138 β ,

© 2016 Nature America, Inc. All rights reserved. Master Mix (Roche). For transfection-based experiments, we used GFP as a as GFP used we experiments, transfection-based For (Roche). Mix Master Green SYBR using (Roche) instrument PCR LightCycler a in qRT–PCR performed was (Invitrogen). Transcriptase Reverse syn II were SuperScript samples with cDNA thetized and Scientific), Fisher (Thermo reagent TRIzol with cells transfected and of from points cultured time development different qRT–PCR. described situ in pJET2.1- pJET2.1- from (Roche) polymerase transcript of 1,470 to 462 In situ analysis. immunoblot and cells N2A in constructs human wild-type with cotransfection using conducted were experiments efficiency NEDD4L, of expression of level low sequence was checked for specificity in sequence databases. Because of the very the under The promoter. vector U6 the of control psiSTRIKE into inserted were (TG505433), vector V-RS in by the p-GFP- and provided Origene designed commercially both cassette, mouse cells. electroporated visualize to vector pCAGGS-IRES-Tomato a with combination in electroporated were vectors psiSTRIKE- CAG promoter, For experiments, the IUEP pCDNA3-nV5. of control the under vector psiSTRIKE of site multiple-cloning the into Wild-type and mutagenesis mutated cDNAs human encoding NEDD4L were then inserted site-directed by Technologies). kit (Agilent Mutagenesis Site-Directed introduced QuikChange the using were Mutations Origene. from constructs. ( plasmid and Cloning Chimera. using Bank Data Protein code Bioinformatics (PDB) Structural for Collaboratory Research using modeling homology by built were Models portal. web Phyre the using protein for structure domain the catalytic of the human E3 NEDD4-like ligase modeling. Protein Research). Science Life (Bio-Rad v.1.4 software QuantaSoft with analyzed vidual) and primers to specific the variant and Data sequences. were wild-type indi unaffected and the parents the patients, two (the of family the members all of blood peripheral the from DNA extracted using Research) Science Life Bio-Rad System, PCR Digital Droplet (QX100 approach PCR digital droplet by of the variant of was the estimation performed the bearing percentage cells affected siblings and suspected maternal somatic mosaicism, confirmation and using sequencing DNA from patients and their parents. In the family with two frequency a with variants 1%. than exclude greater and variants exome filter to used database) were platform Bioinformatics Descartes Paris local a and Consortium Aggregation Exome Server, Variant Exome Project, Genomes (dbSNP,1000 Sanger the Sequencing and Centre as previously platform described Bioinformatics Descartes Paris the France), Evry, (CNG, for Genotyping Centre National at French performed the analyzed were and data were parents their and subjects affected comprising trios in ing generation, library processing, exome enrichment and whole-exome sequenc DNA excluded. been previously had (CNVs) variations number copy genic ( genes PNH-related known in and/or epilepsy, 3 of had PMG. For the also mutations 15 patients patients, all For the 15 selected patients with PNH in association with developmental delay photographs. patient to publish Written obtained was boards. consent review institutional site-specific with accordance in participants all from obtained samples from affected individuals and their parents and informed consent validation. were variant and sequencing whole-exome Subjects, ONLINE doi: For as qRT–PCRa reporter). cotransfected is, systematically (that normalizer NM_001144967. For RNA interference (RNAi) experiments, a 29-mer sequence targeting targeting sequence 29-mer a experiments, (RNAi) interference RNA For 10.1038/ng.3676 hybridization on frozen brain sections was performed as previously previously as performed was sections brain frozen on hybridization hybridization. Nedd4l Nedd4l 3

9 METHODS Total RNA was prepared from the brains of mouse embryos at embryos mouse of brains the from prepared was RNA Total . 2ON mRNA and a HuSH 29-mer non-effective shRNA scrambled scrambled shRNA non-effective 29-mer HuSH a and mRNA (nucleotides 1,470 to 462) plasmids. Non-radioactive RNA Non-radioactive plasmids. 462) to 1,470 (nucleotides 1 De novo De I ) cloned into pCMV6-Entry vector (SC326303) was purchased . The images in in images The . Amino acid substitutions were plotted onto the solved solved the onto plotted were substitutions acid Amino Mouse variants were analyzed by PCR and direct Sanger Sanger direct and PCR by analyzed were variants NM_00111438 Nedd4l FLNA Nedd4l Supplementary Figure 2 Figure Supplementary Nedd4l sense and antisense probes (nucleotides (nucleotides probes and antisense sense Human untagged untagged Human , , ARFGEF2 shRNA directed against the coding coding the against directed shRNA 6 13 ) were synthesized using T7 RNA T7 using synthesized were ) , (nucleotides 462 to 1,470) and and 1,470) to 462 (nucleotides 14 , 3 NEDD4L 8 . . Available genomic databases and and C6orf70 cDNA and shRNA shRNA and cDNA NEDD4L Blood or DNA or Blood were rendered rendered were ) and patho and ) NEDD4L cDNA cDNA - - - -

Immunoblotting. Microsystems). (Leica microscope a using TCS SP5 confocal and photographed were antibody. observed Results secondary the as dilution) 1:800 Technologies; Life (A-21208, 647 IgG rabbit anti- (13690-1-AP, and donkey antibody primary as the dilution) Nedd4l 1:200 Proteintech; rabbit, to antibody using procedures standard to according was performed Immunocytochemistry parafor 96 h electroporation. maldehyde after 4% 1% in fixed B27, were 2% Cells with penicillin-streptomycin. 1% and supplemented glutamine medium Neurobasal in maintained and (Lonza) kit Nucleofector mouse Amaxa the using electroporated were (E17) immunoblotting. and by immunocytochemistry transfection after h 48 analyzed was genes transfected of Expression culture. at a MG132 10 with treated were dissolved in DMSO solution buffer. DMSO was used as a control vehicle. Cells using transfected 2000 Lipofectamine (Invitrogen). MG132 was (Calbiochem) and FCS 5% with supplemented (Gibco) DMEM in cultured were cells N2A culture, and Cell transfections immunofluorescence. to specific primers and transcriptase reverse without or with obtained products reaction cDNA the using performed was triplicate) (in qPCR real-time sample each for constructs, transfected from expressed transcripts amplified we that ensure to Also, DNA. plasmid from amplification avoid to Technology) Life Turbo (Ambion, with DNase treated of transcripts expressed from transfected cDNA constructs, RNA samples were ing analysis was achieved with ImageJ software (NIH) and proliferation proliferation and (NIH) Graphs Microsystems). (Leica software software AF LAS with performed was ImageJ analysis with achieved was analysis position ing and Microsystems), (Leica microscope confocal SP8 TCS a using acquired were images All (Interchim). with medium mounted mounting then and Fluoromount-G washed were Sections temperature. room at h 1.5 for PBS-T in 1:500 diluted Technologies) Life from all 647; IgG anti-mouse donkey A-31571, 488; IgG anti-rabbit donkey A-21206, 488; IgG anti-rat key don A-21208, 647; IgG anti-rabbit donkey (A-31573, antibodies secondary After washes in 1× were PBS, sections with incubated Alexa Fluor–conjugated Millipore; 1:500 dilution) and TBR2 (14-4875, rat, eBioscience; 1:200 dilution). rabbit, PH3 (06-570, Pax6 (PRB-278P,dilution), tion), 1:200 Covance; rabbit, dilu 1:100 Millipore; mouse, (MAB377, NeuN dilution), 1:300 Proteintech; rabbit, (13690-1-AP, NEDD4L dilution), 1:250 Laboratories; Bethyl rabbit, (sc-13024, rabbit, Santa Cruz Biotechnology; 1:100 dilution), Ki67 (IHC-00375, Cux1 dilution), 1:500 Millipore; rabbit, (AB5840, Dab1 were used: antibodies primary following The °C. 4 at overnight sections with incubated and NGS PBS-T- in diluted were antibodies Primary temperature. room at min 30 for (PBS-T-NGS) TritonX-100 0.3% and Dutscher) (Dominique serum donkey normal 2% with PBS 1× with blocked were sections immunodetection, For buffer. in PBS azide in 0.01% maintained were Sections Microsystems). Leica (80 coronal sections into cut and (Bio-Rad) agarose low-melting 4% of solution a in placed were night at 4 °C in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Brains Immunohistochemistry. replicates. independent three at of least consisted experiments blot immuno All dilution). 1:5,000 Invitrogen; mouse, and V5 dilution) (R96025, 1:250 dilution), 1:1,000 mouse, ubiquitin Santa (sc-8017, Cruz Technology; Biotechnology; Signaling Cell rabbit, (2217, S6 dilution), 1:1,000 Technology; Signaling Cell rabbit, (2211, S6-pSer236/236 dilution), 1:1,000 Technology; Cell rabbit, (2965, Signaling Technology; Akt-pThr308 1:1,000 dilution), Akt (pan) (4691, Cell rabbit, dilution), Cell Signaling rabbit, 1:1,000 (4060, Technology; Akt-pSer473 Signaling dilution), 1:1,000 IGBMC; (mouse, specified actin the dilution), at 1:1,000 Proteintech; antibodies rabbit, (13690-1-AP, Nedd4l primary concentrations: following the then and using Tween 0.1% with immunoblotted buffer TBS in milk nonfat 5% in blocked were Membranes membranes. nitrocellulose onto transferred and SDS–PAGE by reagent. assay Samples were protein denatured at 95 Bio-Rad °C for 10 using min in loading buffer and measured then resolved was concentration (Sigma-Aldrich). Protein inhibitors phosphatases and (Roche) inhibitors protease with 1 EDTAmM M NaCl, supplemented Triton0.15 1% and 7.7, pH X-100) For primary cultures of neuronal cells, embryonic mouse cortical neurons neurons cortical mouse embryonic cells, of neuronal cultures For primary Cells were lysed in RIPA buffer (50 mM Tris-HCl, Tris-HCl, mM (50 buffer RIPA in lysed were Cells µ m) using a vibrating-blade microtome (Leica VT1000S, VT1000S, microtome (Leica m) using a vibrating-blade Mouse embryo brains were fixed by incubation over µ M concentration for 15 h before the end of end the for 15 h before M concentration NEDD4L and and Mouse neuroblastoma Nature Ge Nature GFP . n etics ------

© 2016 Nature America, Inc. All rights reserved. in 4% paraformaldehyde in PBS overnight. PBS in paraformaldehyde 4% in to E18 E16, or continue until or and P2. Embryo pup fixed allowed brains were dissected was development and cavity abdominal the in back placed were using a embryos After electroporation, electroporator CUY21EDIT (Sonidel). the to adjacent progenitors ventricle by pulses atdelivering five electric 50 V for 50 neuronal ms at 950-ms intervals the into electroporated further were fluorescent Tomato protein was used to visualize electroporated cells. Plasmids expression vector pCAGGS-Tomato was systematically co-electroporated, and The Nagel). (Macherey kit purification plasmid EndoFree the with prepared 1 of concentration final a with combined Sigma) each (2 Green Fast of with capillaries ventricle pulled-glass using lateral injected was embryo a and exposed, surgery; were during horns uterine isoflurane The 2% Animal and Minerve). sleep during has isoflurane 4% study oxygen, of The min per l (2 isoflurane with facilities. anaesthetized were (E14.5) mice pregnant Briefly, timed (2014-059). animal approval Committee Ethics IGBMC Research Experimentation the at performed previously In utero NEDD4L. and V5 ubiquitin, to antibodies with immunoblotting by analyzed and Tris-HCl °C 30 at h 1 mM for incubated (25 0.1 buffer NaCl, mM reaction 50 in 7.5), ATP (pH mM 2 and (Sigma) ubiquitin 400 400 nM BostonBiochem), E2 (UbcH7; enzyme ubiquitin-conjugating reaction mixtures in containing 200 incubated nM was E1 ubiquitin-activating cells enzymeN2A (BostonBiochem), transfected from NEDD4L nopurified SDS–PAGE.to For subjected and °C 95 at buffer SDS Laemmli in eluted were proteins Immunoprecipitated buffer. for 2 in RIPAh Sigma) rotation at constant 4 °C (A7345, under beads agarose (25 lysed with RIPA buffer supplemented with protease inhibitors (Roche), MG132 Ubiquitination assays. Systems). (Adobe 13.0.1 Photoshop Adobe with were generated in GraphPad Prism 6 (GraphPad), and images were assembled Nature Ge Nature µ M) and PR-619 (20 mM) and protein extracts were incubated with anti-V5 electroporation. 13 , n 4 0 etics using Swiss mice (Janvier). Animal experimentations were were experimentations Animal (Janvier). mice Swiss using For immunoprecipitation assays, transfected cells were

In utero µ M DTT and 4 mM MgCl mM 4 and DTT M electroporation was performed as described in in vitro ubiquitination assays, immu assays, ubiquitination µ g/ µ l of DNA constructs constructs DNA of l 2 ). Reactions were were Reactions ). µ g/ml; g/ml; µ M -

patients to deposit the data in a repository. a in data the deposit to patients accession the from obtained was no consent approach, under sequencing by whole-exome the (EGA) Archive EGAD0000100184 Genome-phenome European the and accession study under (dbGaP) and of Phenotypes in the database Genotypes deposited deposition. data sequencing Exome in included are details Statistical analysis. and collection retained data for be applied not to was were Blinding assumed normal. was but tested not efficiencies was distribution and Data quantification. regions for electroporated comparable brains only with examination, histological After necessary. be would condition per embryos three least at that considered we laboratory, our in performed *** comparisons. dual Student’s two-tailed unpaired whereas respectively, tests, for performed multiple comparisons followed by Dunnett’s or Sidak’s are as presented the mean percentages at Statistics. collected were Embryos E14.5. at analysis. for E18.5 electroporated females into pregnant E17.5 to E15.5 from intra daily mg/kg injected 0.5 of then concentration a and at PBS peritoneally in diluted previously was Sigma) DMSO; in treatment. Rapamycin 40. 39. 38.

P ilr M. Kielar, F.G., Fode,C.&Guillemot, Cau, E.,Gradwohl, bHLH Mash1activatesacascadeof C.F.Wright, heterotopia in mouse and human. and mouse in heterotopia progenitors. neuron (1997). olfactory in regulators data. research genome-wide (2015). of analysis scalable a < 0.001, **** 0.001, < All statistics were calculated with GraphPad Prism 6. Final counts counts Final 6. Prism GraphPad with calculated were statistics All phs000653.v1.p t al. et et al. et 8 P uain in Mutations P Genetic diagnosis of developmental disorders in the DDD study: DDD the in disorders developmental of diagnosis Genetic for the DDDP110533 trios. For the other patients analyzed < 0.0001. On the basis of previous IUEP experiments experiments IUEP previous of basis the On 0.0001. < < 0.05 was considered significant: * significant: considered was 0.05 < Supplementary Table 2 Supplementary Rapamycin Ready-Made Solution (2.5 mg/ml mg/ml (2.5 Solution Ready-Made Rapamycin 1 for the Pnh31124 trio (proband EPGP012746) EPGP012746) (proband trio Pnh31124 the for Eml1 Nat. Neurosci. Nat. ed o coi poeios n neuronal and progenitors ectopic to lead ± Exome sequencing data have been been have data sequencing Exome s.e.m. One- or One- ANOVAs.e.m. two-way was Development .

17 , 923–933 (2014). 923–933 , Lancet P doi: t tests were used for used were tests

< 0.05, ** 0.05, <

124 385 10.1038/ng.3676 1611–1621 , 1305–1314 , P post < 0.01, 0.01, < - hoc -