ARTICLE OPEN ACCESS

HACE1 deficiency leads to structural andfunctional neurodevelopmental defectsVanja Nagy PhD Ronja Hollstein PhD Tsung-Pin Pai PhD Michel K Herde PhD Pisanu Buphamalai MSc

Paul Moeseneder MSc Ewelina Lenartowicz MSc Anoop Kavirayani DVM Dip ACVP

Georg Christoph Korenke MD Ivona Kozieradzki MSc Roberto Nitsch PhD Ana Cicvaric PhD

Francisco J Monje Quiroga PhD Matthew A Deardorff MD PhD Emma C Bedoukian MS Yun Li PhD

Gokhan Yigit PhD Jorg Menche PhD E Ferda Perccedilin MD PhD Bernd Wollnik MD

Christian Henneberger MD Frank J Kaiser PhD and Josef M Penninger MD

Neurol Genet 20195e330 doi101212NXG0000000000000330

Correspondence

Dr Penninger

josefpenningerimbaoeawacat

or Dr Nagy

vanjanagyrudlbgacat

AbstractObjectiveWe aim to characterize the causality and molecular and functional underpinnings of HACE1deficiency in a mouse model of a recessive neurodevelopmental syndrome called spasticparaplegia and psychomotor retardation with or without seizures (SPPRS)

MethodsBy exome sequencing we identified 2 novel homozygous truncating mutations in HACE1 in 3patients from 2 families pQ209 and pR332 Furthermore we performed detailed molecularand phenotypic analyses of Hace1 knock-out (KO) mice and SPPRS patient fibroblasts

ResultsWe show that Hace1 KO mice display many clinical features of SPPRS including enlargedventricles hypoplastic corpus callosum as well as locomotion and learning deficienciesMechanistically loss of HACE1 results in altered levels and activity of the small guanosinetriphosphate (GTP)ase RAC1 In addition HACE1 deficiency results in reduction in synapticpuncta number and long-term potentiation in the hippocampus Similarly in SPPRSpatientndashderived fibroblasts carrying a disruptive HACE1 mutation resembling loss of HACE1in KO mice we observed marked upregulation of the total and active GTP-bound form ofRAC1 along with an induction of RAC1-regulated downstream pathways

ConclusionsOur results provide a first animal model to dissect this complex human disease syndromeestablishing the first causal proof that a HACE1 deficiency results in decreased synapse numberand structural and behavioral neuropathologic features that resemble SPPRS patients

RELATED ARTICLE

EditorialHACE1 RAC1 and whatelse in the pathogenesis ofSPPRS

Page e326

From the IMBA (VN T-PP PM AK IK RN JMP) Institute of Molecular Biotechnology of the Austrian Academy of Sciences VBCmdashVienna BioCenter Campus AustriaDepartment of Medical Genetics (JMP) Life Science Institute University of British Columbia Vancouver Canada Ludwig Boltzmann Institute for Rare and Undiagnosed Diseases(VN EL) Vienna Austria Section for Functional Genetics at the Institute of HumanGenetics (RH FJK) University of Lubeck GermanCenter for Cardiovascular Research (DZHKeV)(FJK) Partner Site HamburgKielLubeck Lubeck Institute of Cellular Neurosciences (MKH CH) University of Bonn Medical School Germany Centre for Neuroendocrinology(MKH) Department of Physiology School of Biomedical Sciences University of Otago Dunedin New Zealand Department of Neurophysiology and Neuropharmacology (ACFJMQ) Center for Physiology and Pharmacology Medical University of Vienna Austria Drug Safety and Metabolism (RN) IMED Biotech Unit AstraZeneca Gothenburg SwedenDivision of Genetics and the Roberts Individualized Medical Genetics Center (MAD ECB) Childrenrsquos Hospital of Philadelphia PA Departments of Pediatrics (MAD) University ofPennsylvania Perelman School ofMedicine Philadelphia PA Institute of HumanGenetics (YL GY BW) UniversityMedical Center Gottingen Germany Institute of Neurology (CH)University College London UK German Center for Neurodegenerative Diseases (DZNE) (CH) Bonn Germany Zentrum fur Kinder- und Jugendmedizin (GCK) NeuropadiatrieKlinikum Oldenburg Germany Department of Medical Genetics (EFP) Faculty of Medicine Gazi University Ankara Turkey CeMM Research Center for Molecular Medicine of theAustrian Academy of Sciences (PB JM) Vienna Austria

Funding information and disclosures are provided at the end of the article Full disclosure form information provided by the authors is available with the full text of this article atNeurologyorgNG

The Article Processing Charge was funded by IMBA Austrian Academy of Sciences ERC

This is an open access article distributed under the terms of the Creative Commons Attribution License 40 (CC BY) which permits unrestricted use distribution and reproduction in anymedium provided the original work is properly cited

Copyright copy 2019 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology 1

Homologous to the E6-AP carboxyl terminus domain andankyrin repeat containing E3 ubiquitin-protein ligase 1 (HACE1)was identified to be downregulated in a number of tumors as wellas to play a role in inflammatory responses1ndash4 Analysis of Hace1knock-out (KO)mice showedHACE1 is ubiquitously expressedwith relatively high expression in the brain2

A neurodevelopmental disorder named spastic paraplegia andpsychomotor retardation with or without seizures (SPPRS)has recently been described in 15 patients of 6 unrelatedfamilies associated with mutations throughout the HACE1gene5ndash7 Mutations are predicted to be deleterious to HACE1protein function either disrupting folding or causing a frameshift and early termination of translation resulting in no de-tectable protein product in patient-derived fibroblasts67

Clinical manifestations are variable and include early-onsetdevelopmental delays severe intellectual disability epilepsyhypotonia spasticity ataxia and difficulty with verbal com-munication MRI findings include enlarged ventricles hypo-plastic corpus callosum and altered white and gray brainmatter ratios There are several known targets for HACE1ubiquitination including Ras-related C3 botulinum toxinsubstrate 1 (RAC1) small GTPase important for differentaspects of brain development and neuronal function48ndash11 Inaddition to cytoskeletal remodeling RAC1 can regulate re-active oxygen species (ROS) levels as part of the nicotinamideadenine dinucleotide phosphate oxidase (NADPH) com-plex12 Correspondingly Hace1 KO mice were reported tohave elevated ROS levels13 While the role for hace1 wasrecently described in embryonic development in Xenopuslaevis14 and murine HACE1 has been implicated in Hun-tington disease13 nothing is known about the role of HACE1during mammalian nervous system development or the mo-lecular underpinnings of SPPRS To elucidate the neuro-developmental pathophysiology of SPPRS we thereforeperformed detailed analysis ofHace1 KOmice and confirmedour findings in SPPRS patientndashderived fibroblasts

MethodsSee supplemental information (e-methods linkslwwcomNXGA151) for more details

Standard protocol approvals registrationsand patient consentsGenetic and clinical data of patients 6ndash8 were published6 Ournewly identified patients are labeled as patients 9ndash11 Geneticdata regarding patient 9 were produced as part of a clinicaldiagnostic service (GeneDx) and the research study including

patients 10 and 11 has been approved by the ethics committeeat The University of Gottingen Biological materials ofpatients and healthy donors (HDs) and written informedconsents were obtained in accordance with the Declaration ofHelsinki Fibroblasts from 2 female patients and 1 male pa-tient only 6 7 and 10 respectively are used in this study asno consent was available for the others

MiceAnimals were housed at the Institute of Molecular Bio-technology Vienna Austria maintained under a 12-hourlightdark cycle and provided with food and water ad libitumExperiments described in this study were approved by theBundesministerium fur Wissenschaft Forschung und Wirt-schaft (BMWFW-660150004-WFV3b2015) and per-formed according to EU-directive 201063EU

ImmunoblottingThe following antibodies were used for standard Westernblotting protocols anti-HACE1 at 11000 dilution (AbCam)anti-Cyclin D1 at 11000 (AbCam) anti-Glyceraldehyde-3-Phosphate Dehydrogenase at 11000 dilution (Cell signal-ling) anti-RAC1 at 11000 dilution (Millipore) anti-szlig-actinat 15000 dilution (Sigma clone AC-74) and appropriatesecondary horseradish peroxidasendashlinked whole antibodies(GE Healthcare)

HistologySelected brains were isolated processed embedded sec-tioned and stained by the Histopathology Facility at theVienna Biocenter Core Facilities (VBCF) member of theVienna Biocenter (VBC) Austria Briefly 2-μm-thick coronalor sagittal paraffin-embedded sections were prepared byroutine microtomy and stained with hematoxylin and eosin(HampE Shandon Harris Hematoxylin Acidified ThermoScientific Shandon Eosin Y Fisher Scientific) Luxol FastBluendashCresyl Violet (LFBCV Sigma) or with antindashMyelin BasicProtein antibody (Abcam 1100) Slides were imaged usinga Zeiss Axioskop 2 MOT microscope (Carl Zeiss Microscopy)and subsequently digitized with the Pannoramic FLASH 250 IIautomated slide scanner (3D Histech) Images were acquiredwith the Pannoramic Viewer software (3D Histech) and withSPOT Insight camera (Diagnostic Instruments Inc)

MRIAnesthetized male C57Bl6J mice 8ndash10 weeks of age andtheir Hace1 KO littermates were imaged in the PreclinicalImaging Facility at VBCF (pcPHENO VBCF) member ofthe VBC Austria using a 152 T MRI (Bruker BioSpec

GlossaryANOVA = analysis of variance HACE1 = HECT Domain And Ankyrin Repeat Containing E3 Ubiquitin Protein Ligase 1HD = healthy donorKO = knock out LTP = long-term potentiationNADPH = nicotinamide adenine dinucleotide phosphateoxidaseOFT = open-field test PPI = pre-pulse inhibitionRAC1 = Ras-related C3 botulinum toxin substrate 1ROS = reactiveoxygen species SPPRS = spastic paraplegia and psychomotor retardation with or without seizures WT = wild type

2 Neurology Genetics | Volume 5 Number 3 | June 2019 NeurologyorgNG

Ettlingen Germany) Values were normalized to brain sizeaveraged and presented as percentage of total brain volumeand unpaired Studentrsquos t test was used to determine statisticalsignificance

Behavioral testingOpen-field test (OFT) and elevated plus maze were performedas described previously using an automated activity system(TSE-Systems Germany)15Unpaired Studentrsquos t test was usedto determine significance Accelerating rotarod was performedon a 5ndash40 rpm accelerating apparatus (Ugo Basile) as describedpreviously16 Significance was determined by 2-way analysis ofvariance (ANOVA) with Sidakrsquos multiple comparisons In theladder rungwalking task (pcPHENOVBCF)micewere trainedto walk across a ladder (length 80 cm spacing of rungs 1 cm)toward an escape ladder in the TSE MotoRater system (tse-systemscom) Mice were recorded and manually scored usingSimiMotion software 85032717 Unpaired Studentrsquos t test wasused to determine significance T-maze was performed as de-scribed previously18 Significance of deviation of the number ofsuccesses inmutant vs wild-type (WT)mice was evaluated usinga binomial test For acoustic fear conditioning (pcPHENOVBCF) mice were trained to associate the conditioned soundstimulus (CS = 85 dB 10 kHz) with the unconditioned footshock stimulus delivered by the floor grid (US = 15 mA) asdescribed previously using Coulbourn Habitest operant cages(Coulbourn Instruments MA and FreezeFrame ActimetricsIL)19 Unpaired Studentrsquos t test was used to determine signifi-cance Mice were trained in the Morris water maze (pcPHENOVBCF) as described previously20 Unpaired Studentrsquos t test wasused to analyze the short- and long-termmemory data and 2-wayANOVAwith Sidakrsquos multiple comparison test for the latency toreach the platform Mice were tested for startle responses andprepulse inhibition (PPI) as described previously21 using the SR-LAB Startle Response System (San Diego Instruments) cham-ber Two-way ANOVA with Sidakrsquos multiple comparison testwas used to determine significance

Hippocampal slices preparation andelectrophysiologic recordingsElectrophysiologic field recordings in the stratum radiatum ofacute hippocampal slices were performed as described pre-viously22 Two-population Studentrsquos t test were used to de-termine significance

Synaptic number analysisHace1KO Thy1-green fluorescent protein (GFP) lineM andhace1 WT and Thy1-GFP line M16 were analyzed using theLSM700 Axio Imager Synapse number counts and mea-surement of neurite lengths were performed using ImageJ23

Statistical analysis was performed using Studentrsquos t test

Active RAC1 pulldownCellular levels of active RAC1 in fibroblasts were analyzedusing the Active Rac1 Pull-Down and Detection Kit(Thermo Fisher Scientific) according to the manufacturerrsquosinstructions

Migration assayPrimary dermal fibroblasts from patients 6 7 and 10 andHDswere seeded at high density in 24-well plates for 24 hours andscratched using a pipette tip The area of the scratch at 0 and24 hours following the scratch was measured digitally inpixels and the relative gap closure was calculated and nor-malized to wild-type cells Unpaired Studentrsquos t test was usedto determine significance

ROS detection in the mouse brain and humanprimary dermal fibroblastsTwenty-micrometer coronal mouse brain sections were washed2X in phosphate buffered saline and incubated in 10-mM dihy-droethidium for 15 minutes at 37degC Sections were washed twicein phosphate buffered saline and imaged with a Zeiss Axioplan2microscope Levels of ROS in fibroblasts were investigated usingCellROXDeep Red staining (Thermo Fischer) followed by flowcytometric analysis according to the manufacturerrsquos instructionsStatistical analysis was determined using unpaired Studentrsquos t test

ResultsIdentification of novel HACE1 mutations inSPPRS patientsFamily 1 originating from Saudi Arabia had 1 affected femaleindividual patient 9 born to healthy consanguineous parents(figure e-1A linkslwwcomNXGA148) The patient pre-sented with similar clinical symptoms as previously reportedcases of SPPRS including severe intellectual disability de-velopmental delay and inability to sit or speak by the age of 5years6 Cranial MRI revealed microcephaly and brachyceph-aly as well as hypoplastic corpus callosum and likely brain-stem abnormality small sella with ectopic neurohypophysisand mild ventriculomegaly (figure e-1B) Additional clinicalfindings include mild facial dysmorphia skeletal abnormalitiesand ulnar deviation of the wrists and small feet (figure e-1C) Inan unrelated consanguineous family 2 originating from Turkeyborn to a healthy father and mother with Hashimoto disease areone female (patient 10) and one male (patient 11) 4 and 6months of age at initial admission (Figure e-1D) During preg-nancy the mother was euthyroid and both children were carriedto term Patients had variable clinical symptoms in comparisonwith patient 9 and those previously reported including hypo-tonia small feet enlarged head circumference inverted and widespaced nipples facial dysmorphic features and strabismus(figures e-1 E and F) Considering the pronounced hypotoniain all 3 patients ataxia was difficult to determine For detailedclinical symptoms of all patients please refer to table

In these 3 patients mutations in HACE1 were identified byexome sequencing and verified by Sanger sequencing patient9 carries a homozygous c625CgtT pQ209 HACE1 muta-tion in exon 8 NM_0207713c625CgtT pQ209 (chr6104797 018 on the hg38 build) patients 10 and 11 carry a ho-mozygous c994CgtT pR332 HACE1 mutation in exon 11NM_0207713c994CgtT pR332 (chr6104 791 584)

NeurologyorgNG Neurology Genetics | Volume 5 Number 3 | June 2019 3

Table Comparison of clinical characteristics and genetic findings in reported patients

Patient 6 Patient 7 Patient 9 Patient 10 Patient 11

Background HACE1 mutation Nonsense c2242C gt T pR748and Frameshiftc2019_2020insTTTAGGTATTTTTAGGTATT pP674Ffs5 (Hollsteinet al6)

Nonsense c2242C gt TpR748 and Frameshiftc2019_2020insTTTAGGTATTTTTAGGTATT pP674Ffs5(Hollstein et al6)

Nonsense c625C gt T pQ209X

Saudi Arabia

Family 1

Nonsense c994C gt T pR332X

Turkey

Family 2

Nonsense c994C gt T pR332X

Turkey

Family 2

Sex F M F F M

Birth weight (kg) 337 34 30 4140 (97th percentile) 3480

Birth OFC (cm) 33 34 UK 37 cm (97th percentile) UK

Gestational age 39 40 40 40 40

Development Recent growth parameters

Age 10 y 11 mo 7 y 8 mo 5 y 4 mo 4 mo 6 mo

Height (cm) BMI 248 BMI 2117 865 Z = minus567 (scoliosis) 67 (gt97th percentile) 66 (75th percentile)

Weight (kg) 155 Z = minus16 95 (gt97th percentile) 97 (gt97th percentile)

OFC (cm) 54 (+05 SD) 53 (+02 SD) 4y 44 Z = minus372 44 (gt97th percentile) 435 (75th percentile)

Neurology Sitting and walking 4 y 3ndash4 y Not at 5 y Not at 4 mo Not at 6 mo

Language Inarticulate speech Inarticulate speech None at 5 y Too young to be evaluated Too young to be evaluated

General Developmental delay Developmental delay Developmental delay Developmental delay Developmental Delay

Epilepsy None Myoclonic seizures andfocal epilepsy

None None None

Stereotypies None None Bruxism None None

Mobility Hypotonia and unstable ataxicgait

Hypotonia and unstableataxic gait

Hypotonia Hypotonia Hypotonia

Ophthalmic findings None None Retinal dystrophy Strabismus of the right eye Bilateral strabismus

Hearing No abnormality No abnormality Bilateral sensorineural loss notedat 4 y

No abnormality No abnormality

Intellectual disability Yes Yes Yes Too young to be evaluated Too young to be evaluated

Neuroradiology MRI Hypoplastic corpus callosum Hypoplastic corpuscallosum

Microcephaly and brachycephalyhypoplastic corpus callosum andlikely brainstem abnormalitysmall sella with ectopicneurohypophysis mildventriculomegaly

Normal Normal

Continued

4NeurologyG

enetics

|Vo

lume5N

umber

3|

June2019

NeurologyorgN

G

(figure 1A) Both are nonsense mutations predicted to resultin a truncated HACE1 protein product with no catalytic ac-tivity Parents in both families were determined to be het-erozygous carriers Figure 1A also indicates all previouslyreported HACE1 mutations in SPPRS patients56 Thus wehave identified three new cases with biallelic HACE1 muta-tions associated with marked and diverse neurologic as well asnon-neurologic abnormalities diagnosed to be SPPRS

Hace1-mutantmice exhibit brainmorphologiessimilar to SPPRS patientsTo determine whether Hace1 KO mice develop any clinicalsymptoms of SPPRS patients we first analyzed structural andcellular features of mutant mouse brains using 152 T MRIandor histologic staining at different developmental stagesHistologic examination of mouse brains at embryonic day (E)185 postnatal day (P) 05 and P145 revealed no evidentdefects cortical lamination cerebellar foliation hippocampalarchitecture or cellular aberration and density in any de-velopmental period examined (figure 1 BndashD) Howeverenlarged ventricles and hypoplastic corpus callosum (figure 1B and C) were noted in Hace1 KO animals as compared withWT littermates as early as E185 and P05 respectively

Western blot analyses of 8-week-old mouse brain lysatesrevealed HACE1 to be expressed throughout the adult mousebrain which was absent in the brains of Hace1 KO mice(figure 2A) Total volume and morphology of the Hace1 KOadult mouse brain as compared with WT littermates mea-sured by MRI andor histologic examination revealed nowhole-brain volumetric or cellularity differences as comparedwith WT littermates (figure 2 B and C) Review of histologicsections also revealed no abnormality in cortical laminar or-ganization and cellular density in the adult brain (figure 2D)Detailed analysis of different brain regions via MRI alsoshowed no significant differences in the volumes of the cortexcolliculi thalamus and putamen comparing adult WT andHace1 KO brains with a small decrease in the volume of theolfactory bulb (data not shown) The adult cerebellar volumeand foliation as well as cortical laminar organization werecomparable between Hace1 KOs and their control WT lit-termates (figure 2 E and F) Hippocampal volume was sig-nificantly decreased as measured by MRI (figure 2G)however there were no evident aberrations in cellularity andarchitecture on microscopic examination of hippocampi inadult animals (figure 2H)

Of interest volumetric MRI reconstructions revealed a sig-nificant reduction in whole-brain white matter (figure e-2AndashD linkslwwcomNXGA149) inHace1 KO adult mice ascompared with their WT littermates substantiated by myelinbasic protein staining and volumetric MRI analysis (figure e-2BndashD) similar to findings in reported SPPRS patients5ndash7 andpatient 9 described here (figure 1C and table) Also notedwas a reduction of the anterior commissure inHace1KOmiceas compared with WT littermates (figure e-2C) In additionin line with findings in SPPRS patients as well as adult andTa

ble

Comparisonofclinical

charac

teristicsan

dge

neticfin

dings

inreported

patients

(con

tinue

d)

Gastro

inte

stinal

Neu

roge

nicbladder

and

esophag

ealreflux

Norm

alIntestinal

pse

udo-obstru

ction

neu

roge

nicbladder

andbladder

stroma

Norm

alAcces

sory

spleen

bila

teral

ureteralreflux

ingu

inal

hernia

Skeleta

lLu

mbar

lord

osis

Pes

planus

Bila

teralh

ipdisloca

tion

thoraco

lumbar

kyphoscolio

sis

ulnar

dev

iationofthewristsan

dsm

allfee

t

Norm

alDelay

edclosu

reofthe

anteriorfontanelle

Endocrine

Norm

alNorm

alHyp

othyroidisma

drenal

insu

fficiency

Norm

alNorm

al

Facialfeatu

res

Norm

alNorm

alDownturn

edmouth

Frontalb

ossing

prominen

tglab

ellad

epressed

andsh

ort

nas

albridge

anteve

rted

nares

eve

rted

lower

liph

igh

palate

anddysplasticea

rs

Frontalb

ossing

prominen

tglab

ellad

epressed

nas

albridge

anteve

rted

nares

and

evertedlower

lip

Abbreviations

BMI=

bodymas

sindex

OFC

=occipital

frontalcircu

mference

Su

mmaryofclinical

featuresofthepreviouslypublished

Patient6

713that

hav

edonated

fibroblastsforthestudy

aswella

snew

lyiden

tified

threeaffected

individualsfrom

families

1an

d2

Featureswereleftblankifthe

patients

weretooyo

ungfordiagn

osisat

timeofad

missionorlabeled

aslsquounkn

ownrsquoifthefeatureswerenotnotedat

timeofdoctorrsquos

visit

NeurologyorgNG Neurology Genetics | Volume 5 Number 3 | June 2019 5

Figure 1 Histologic analysis of brain regions in neonatal and young Hace1 knock-out (KO) mice

(A) Schematic representation of HACE1 proteinand locations of mutations of previously pub-lished (in gray) and novel mutations reportedhere (red arrows black letters) (B) HampE-stainedsections of wild-type (WT) and Hace1 KO mice atE185 reveal no major structural abnormalityacross the regions studied with slight ven-triculomegaly in theHace1 KO brain as comparedwith controls left panel (yellow arrows) (C) HampE-stained coronal sections of the newborn brain ofWT and Hace1 KO mice at P05 reveal ventriculardilation in theHace1 KObrain (yellow arrows) andattenuation of the corpus callosum (blackarrows) (D) HampE-stained sections of WT andHace1 KO mice at P145 Cortical laminationhippocampal formation and the cerebellar ver-mis are comparable betweenWTandHace1KO inall 3 age groups studied All brain regions are la-beled in the figure and magnifications are as in-dicated A = ankyrin domain HampE = hematoxylinand eosin HECT = homologous to the E6-APcarboxyl terminus MID = middle region

6 Neurology Genetics | Volume 5 Number 3 | June 2019 NeurologyorgNG

developing mouse brains we observed enlarged ventricles inHace1 KO mice as compared with control littermates (figure2C) These data show that Hace1 KO mice phenocopy keystructural features associated with SPPRS patients

HACE1-deficient mice exhibit locomotion andassociative learning disabilitiesTo assess what if any behavioral defects can be detected inHace1 KO mice we subjected cohorts to an extensive battery

of behavioral assays The most notable clinical features ofSPPRS are intellectual disability and ataxia5ndash7 we thereforefocused on hippocampal- and cerebellar-dependent tasksprincipal brain regions regulating those behaviors in miceHace1 KO mice generated in our group previously are gen-erally healthy viable and fertile2 First we found no signifi-cant differences in anxiety levels in Hace1 WT and KOlittermates analyzed by the OFT and the elevated plus maze(figure e-3 A and B linkslwwcomNXGA150) however

Figure 2 Morphology of the Hace1 KO mouse brain

(A) Western blot analysis depicting HACE1abundance in the indicated 8ndash12-week-oldmalemouse brain regions of wild-type (WT)and Hace1 knock-out (KO) mice (B) Pseu-docolored 3D reconstruction of lateral MRIimages of representative whole mousebrains and quantification (mean values plusmnSEM) showing no difference in total brainvolumes in 8ndash12-week-old male WT andHace1 KO littermates Orangemdashcerebellum yellowmdashhippocampus redmdashcorpus callosum light bluemdashthalamuspurplemdashcolliculi turquoisemdasholfactorybulbs and dark bluemdashputamen WT n = 12and KO n = 9 p value as indicated in the bargraph unpaired Studentrsquos t test (C) LFB-CVndashstained coronal sections of whole WTand Hace1 KO adult mouse brains Ven-triculomegaly (red arrows) is evident in theKO brain as compared with the WT litter-mate (D) LFB-CVndashstained coronal sectionsof the adult cortex show no defects in cel-lularity or lamination (laminae I to VI) inHace1 KO mice (E) Pseudocolored (orange)3D reconstructed MRI images of represen-tative lateral views of WT and Hace1 KO lit-termate cerebelli showing no significantvolumetric differences in Hace1 KO vs WTcontrol littermates Right quantification ofvolumetric differencesmean values plusmn SEMWT n = 12 KO n = 9 p value as indicated inthe figure unpaired Studentrsquos t test (F) Se-lected LFB-CVndashstained sagittal sections ofthe cerebellum showing no apparent dif-ferences betweenWT and Hace1 KO brains(G) Pseudocolored (yellow) 3D re-construction of MRI images of representa-tive lateral view of hippocampi of WT andHace1 KO mice shows a significant re-duction in hippocampal volume in Hace1KO brains quantification is shown in theright panel mean values plusmn SEM WT n = 12and KOn = 9 p le 005 unpaired Studentrsquos ttest (H) LFB-CVndashstained coronal sections ofthe hippocampus show no notable mor-phological difference in cellularity betweenWT and Hace1 KO brains Scale bars as la-beled in the images CA = cornu ammonisCC = corpus callosum cereb = cerebellumCtx = cortex DG = dentate gyrus hipp =hippocampus hypo = hypothalamus LFB-CV = luxol fast blue - cresyl violet LV = lat-eral ventricle stria = striatum WM = whitematter

NeurologyorgNG Neurology Genetics | Volume 5 Number 3 | June 2019 7

we noted a reduction in the distance traveled during thetesting (figure 3A) These data indicated that while our miceexhibited normal anxiety levels there may be a locomotiondefect Therefore we tested Hace1 KO mice for any loco-motion and gait defects WT and Hace1 KOmice were testedin the accelerating rotarod task a standard assay to determinelocomotion and gait defects16 While Hace1 KO mice andcontrol littermates had similar latencies to fall off the rotating

rod in the first trialHace1KOmice exhibited shorter latenciesto fall on all subsequent trials (figure 3B) We further analyzedthe mice on a ladder rung walking task which enables theobserver to analyze coordinated front and hind paw place-ment on each individual rung of the ladder17 While Hace1KO animals were capable of performing the task withoutfalling off the apparatus and showed similar percentages ofcorrect placement over all limbs analyzed percent of major

Figure 3 Behavioral analysis of Hace1 KO mice reveals deficits in specific locomotion learning and memory tasks

(A) Open-field maze (OFM) and elevated plus maze (EPM) showed slight and significant reductions in the distances Hace1 KOmice had traveled as comparedwith their WT littermates respectively data are shown as mean values plusmn SEM WT n = 10 and KO n = 10 p le 005 or as indicated in the bar graphs unpairedStudentrsquos t test (B) Hace1 KO mice show significantly shorter latencies to fall off the accelerating rotarod mean values plusmn SEM WT n = 19 and KO n = 21 p le005 2-way ANOVAwith Sidakrsquosmultiple comparison test (C) Ladder rungwalking task illustrates a significant increase in the percentage of totalmisses deepslips andor slight slips (0ndash2 score) in Hace1 KOmice compared with WT littermate controls mean values plusmn SEM WT n = 12 and KO n = 9 p le 005 unpairedStudentrsquos t test No significant differences were noted in the 3ndash4 score (replacement and correction) 5 score (partial placement) or 6 score (correctplacement) Bar graphs depictmean values of all paws plusmn SEM p values are indicated in figuresWT n = 12 and KOn = 9 unpaired Studentrsquos t test (D) Significantdeficiency in long-term contextual memory as measured by auditory fear conditioning was detected in Hace1 KOmice as compared with WT littermates (leftpanel)mean values plusmn SEMWTn=16 andKOn= 13 ple 005 unpaired Studentrsquos t test Cued fear conditioningwasnot significantly different inHace1KOmiceas comparedwithWT littermatesmean values plusmn SEMWTn=16 KOn= 13p value = 021 unpaired Studentrsquos t test (E)Hace1KOs show significant reduction inacoustic startle inhibition at 110 and 120 dB as comparedwithWT littermatesMean values plusmn SEMWT n = 14 KO n = 11 p le 005 p le 0001 2-way ANOVAwith Sidakrsquos multiple comparison test ANOVA = analysis of variance KO = knock out WT = wild type

8 Neurology Genetics | Volume 5 Number 3 | June 2019 NeurologyorgNG

missteps made were significantly higher as compared withWT littermates Both groups of animals had similar placementand correction scores while WT animals had a higher per-centage of partial placement on rungs (figure 3C) ThusHace1 KO mice exhibit locomotion and gait deficits

The second notable clinical feature of SPPRS patients is in-tellectual disability (table)5ndash7 To determine whether learningand memory processes are altered in Hace1 KO animals weanalyzed mutant mice in various hippocampal-dependentbehavioral tasks In theMorris water maze KOmice were ableto swim normally and there were no apparent differences inlatencies between WT and Hace1 KO littermates to find thevisible platform (not shown) in escape latencies during any ofthe training days (figure e-3C linkslwwcomNXGA150)nor in short- or long-termmemory (figure e-3C right panels)Likewise there were no differences in T-maze between WTand Hace1 KO animals (figure e-3D) However subjectingthe animals to cued and contextual fear conditioning revealedsignificantly reduced freezing responses in the contextual as-pect of the task in Hace1 KO mice compared with WT lit-termates (figure 3D)

In addition Hace1 KO mice exhibited significantly enhancedstartle amplitude responses or reduced startle inhibition inthe acoustic startle task at presentation of 110- and 120-dBstartle sounds as compared with their WT littermates (figure3E) These results also suggestHace1KO animals do not haveany hearing deficiencies PPI was reduced for all prepulseintensities presented (80 85 90 and 95 dB) however thisreduction was not significant when comparing Hace1 KOanimals withWT controls (figure e-3E linkslwwcomNXGA150) These data indicate that besides locomotion defectsassociative learning is altered in Hace1 KO mice parallelingbehavioral findings in human SPPRS patients

Hace1 deficiency results in alteredhippocampal synaptic transmission andreduced synapse numbersWe and others have previously shown that altered synaptictransmission and plasticity at CA3-CA1 Schaffer collateralscan underlie deficits of contextual fear memory2224We testedwhether synaptic transmission and plasticity of this pathwayare affected in Hace1 KO mice Recordings of field excitatorypost synaptic potentials in response to CA3-CA1 synapsestimulation (schematic of the set-up in figure 4A) revealed anupregulation of excitatory synaptic transmission in hippo-campal slices from Hace1 KO mice (figure 4 A and B) Fur-ther analysis showed that the increase in synaptic transmissionat the CA3-CA1 synaptic pathway was most likely due toa postsynaptic effect of theHace1 deletion because neither theaxonal fiber volley (figure 4C) nor the paired-pulse ratio anindirect measure of the presynaptic release probability25 wasaffected (interstimulus interval 50 ms paired-pulse ratio WT170 plusmn 0066 n = 19Hace1 KO 166 plusmn 0036 n = 20 p = 0642-population t test not shown) We then probed long-term po-tentiation (LTP) a cellular correlate of memory formation26 of

this synaptic pathway and found that LTP is significantly reducedin acute hippocampal slices fromHace1KOmice compared withWT littermates (figure 4 D and E) Of note nerve conductionvelocity was not affected in Hace1 KO recordings as comparedwith controls (data not shown) in line with the report that nerveconduction velocity is unaffected in SPPRS patients6

Because we observed hippocampal-dependent contextual fearlearning deficiency and dysfunctions in electrophysiologicproperties of pyramidal cells within the CA1 region of thehippocampus we analyzed morphological properties of py-ramidal neurons in this brain region To this end we crossedHace1 KO mice with Thy1-GFP-M mice to drive neuronalGFP expression which allowed us to visualize neuronalmorphology including spines along dendrites of CA1 pyra-midal cells27 Quantification of selected dendritic segmentsindeed revealed a significant reduction in spine numbers inHace1 KO neurons as compared with hippocampal neuronsfrom WT littermates (figure 4F) Whether such reduction inthe synapse number is also present throughout the brain andmight explain defective locomotion in Hace1 KO mice needsto be determined These data indicate that as a correlate tohippocampus-dependent impaired associate learningHACE1 deficiency leads to a deficit of synaptic plasticity anda marked reduction of synapses in the hippocampus

Elevated levels of active Rac1 in Hace1 KOmouse brains and SPPRSpatientndashderived fibroblastsTo determine molecular alterations downstream of HACE1deficiency we focused on Ras-related C3 botulinum toxinsubstrate 1 RAC1 a well-characterized substrate for HACE1-mediated proteasomal degradation89 Indeed Western blotanalysis showed that RAC1 protein abundance was increasedin Hace1 KO brains when compared with WT littermates(figure 5A) In addition Cyclin D1 abundance and ROSlevels as downstream read-outs for RAC1 activity12 werelikewise increased in Hace1 KO brain regions as comparedwith controls (figure 5 A and B)

The role of RAC1 has been extensively studied in the contextof mouse brain development1011 Of interest perturbation ofRAC1 levels either overexpression or downregulation resultsin decrease in dendritic spines in the mouse brain similar toour Hace1 KOmice1028 To determine whether RAC1 is alsochanged in HACE1-mutant SPPRS patients we performedWestern blotting analysis of SPPRS patientndashderived fibro-blasts isolated from patients 6 7 and 10 (table) Of note inthese patients the mutations result in undetectable HACE1protein expression (figure 5C) resembling our Hace1 KOmice In all patientsrsquo fibroblasts total abundance of RAC1 andCyclin D1 was increased as compared with fibroblasts fromHD controls (figure 5C) It is important that increasedabundance of active GTP-bound RAC1 was detected in all 3patient fibroblasts (figure 5D) As a functional consequence ofincreased RAC1 activity ROS upregulation was also evidentin SPPRS patientndashderived fibroblasts (figure 5E) In addition

NeurologyorgNG Neurology Genetics | Volume 5 Number 3 | June 2019 9

Figure 4 Altered long-term potentiation and reduced hippocampal synapse numbers in Hace1 KOmice as compared withWT controls

(A) Upper left panel schematic fEPSP recording configuration fromacute hippocampal slices stimulation (redbolt) of CA3-CA1 Schaffer collaterals (green) thefEPSP extracellular recording electrode in the CA1 region (white cone ep = extracellular pipette) Synaptic transmission at CA3-CA1 hippocampal synapses isincreased in Hace1 KO mice compared with WT littermates fEPSP slopes were normalized to axonal fiber volleys to account for variations in axonalstimulation efficacy between slice preparations and animals Analysis was performed over a range of stimulus intensities Average fEPSP slopes weresignificantly higher inHace1 KO slices mean values plusmn SEMWTn = 19 and KOn = 20 p le 005 2-population Studentrsquos t test (B) Representative traces of fEPSPsin response to CA3-CA1 synapse stimulation obtained from WT (black trace) and Hace1 KO (red trace) mice Field EPSP (black arrow) recordings werenormalized to fiber volleys (gray arrow) to account for varying stimulation efficiencies across experiments and preparations Time scale of recording isindicated (C) No significant difference was found between fiber volley amplitudes (mean values plusmn SEM) between the indicated genotypesWT n = 19 and KO n= 20 (D) Long-term potentiation (LTP) of synaptic transmission was induced by HFS after a 10-minute baseline recording fEPSP slopes were normalized totheir baseline levels and plotted over time LTP was reduced in Hace1 KO animals mean values plusmn SEM WT n = 19 and KO n = 18 p le 001 2-populationStudentrsquos t test (E) Representative fEPSP traces illustrating LTP experiments displayed in panel (D) Solid lines represent baseline fEPSPs and dashed linesrepresent fEPSPs 30minutes after LTP induction (Hace1WT black upper panelHace1 KO red lower panel) (F) Representative 3D composite of z-stacked times63magnified (zoom factor of 2) confocal images of hippocampal CA1 pyramidal cell distal dendrites ofHace1++Thy1-GFP andHace1minusminusThy1-GFPmice Uncoloreddendritic segments are on the left synaptic spines are pseudocolored in magenta on the right panels scale bar is 25 μm Right bar graphs show quantifi-cations of spine numbersmean values plusmn SEMWTn= 3 and KOn= 3 p le 001 unpaired Studentrsquos t test fEPSPs = field excitatory post synaptic potentials KO= knock out WT = wild type

10 Neurology Genetics | Volume 5 Number 3 | June 2019 NeurologyorgNG

in a scratch assay patient fibroblasts migrated significantlyfaster as compared with control fibroblasts (figure 5 F andG) confirming enhanced RAC1 activity29ndash32 These data

show that loss of HACE1 expression results in increased levelsof active RAC1 in bothHace1 KOmouse brains andHACE1-mutant SPPRS patientndashderived fibroblasts

Figure 5 Hace1 KO mouse brains and SPPRS patientndashderived fibroblasts have elevated Rac1 and ROS levels

(A) Western blot analysis of the cortex (ctx) hippocampus (hipp) and cerebellum (cereb) fromWT andHace1 KOmice revealed a strong upregulation of totalRAC1 abundance (lower band arrow) and Cyclin D1 protein in the absence of HACE1 in all indicated brain regions tested Actin was used as a loading control(B) ROS (detected by DHE staining) is elevated in the hippocampus of Hace1 KO mice as compared with WT littermates times20 magnification (C) Fibroblastsharvested from SPPRS patients (P6 P7 and P10) have no detectable HACE1 protein product as compared with normal donor fibroblasts (ND) as revealed byWestern blotting using an antibody recognizing the HACE1 C-terminus however patient fibroblasts express elevated total abundance of RAC1 and Cyclin D1protein product GAPDH was used as a loading control (D) SPPRS patientndashderived fibroblasts exhibit elevated abundance of active RAC1 in comparison withnormal donor fibroblasts as measured by the Active RAC1 Pull-Down and Detection Kit (E) Left panel FACS analysis of SPPRS patientndashderived fibroblastsincubated with CellROX deep red reagent revealed significantly more ROS in patient cells as compared with normal donor (ND) fibroblasts Right panelquantification of ROS production in the indicated patient and control fibroblasts mean values plusmn SD n = 7 for ND P6 and P7 fibroblasts and n = 3 biologicalreplicates for patient 10 fibroblasts p le 005 unpaired Studentrsquos t test (F and G) Confluent SPPRS patient P6 P7 P10 and ND-derived fibroblasts weresubjected to scratch assays and migration was monitored Panel (F) shows a quantification of fibroblast migration rates mean values plusmn SD n = 3 p le 001and p le 0001 comparing patient fibroblastswith theND controls unpaired Studentrsquos t test Representative images are shown in (G) KO = knock out ROS =reactive oxygen species SPPRS = spastic paraplegia and psychomotor retardation with or without seizures WT = wild type

NeurologyorgNG Neurology Genetics | Volume 5 Number 3 | June 2019 11

DiscussionHere we report 2 novel mutations in HACE1 pQ209 andpR332 discovered in 3 patients from 2 unrelated consan-guineous families with a complex neurodevelopmental dis-order These new patients have variable clinical symptomsoverlapping with previous descriptions of SPPRS5ndash7 Thegenotypendashphenotype correlations of the different mutationsneed to be addressed in future studies as more SPPRSpatients are diagnosed We find that HACE1 is expressedthroughout the adult mouse brain Hace1 KO mice havedefects in basic sensorimotor processing deficiencies in spe-cific learning andmemory tasks reduced LTP of hippocampalCA3-CA1 synapses and fewer synaptic spines at CA1 pyra-midal neurons In addition they exhibit locomotion defectsas compared with littermate controls Furthermore weshow a marked upregulation of RAC1 levels throughoutthe mutant mouse brain and elevated ROS levels Verifyingthat our findings are relevant in humans active RAC1abundance downstream signaling components ROSproduction and cellular migration in SPPRS patientndashderived fibroblasts are likewise dysregulated all indicativeof a hyperactive RAC1 pathway Thus HACE1 deficiencyleads to neuroradiologic and behavioral manifestations inmice reminiscent of the clinical features seen in SPPRSpatients

A well-characterized target for HACE1 ubiquitination andsubsequent proteasomal degradation is RAC1 a small mem-ber of the Rho family of GTPases critical for neurogenesismigration axonal elongation synaptogenesis and activity-driven plasticity11 HACE1 is therefore poised to regulatemanyfunctions of RAC1 by modulating its active GTP-bound levelsincluding NADPH-dependent ROS production1213

Mutations in several genes encoding guanine exchange factorsand GTPase-activating proteins that regulate the onoff stateof RAC1 have been identified to cause rare neuronaldisorders3334 Indeed analysis of a proteinndashprotein interactionsubnetwork extracted from the human-integrated proteinndashprotein interaction reference database35 reveals RAC1 to have 25first-order interactors reported to be causative of intellectualdisabilities Neuronally expressed RAC1 first-order interactorsare enriched in GTPase signal transduction by Gene Ontologyanalysis Furthermore RAC1 was also identified as a candidategene for intellectual disability in a meta-analysis of severalthousand trios36 and RAC1 missense mutations were recentlyreported to cause a neurodevelopmental disorder with variablesymptoms in 7 individuals some of which are overlapping withSPPRS patients37 Of interest mutations in RAC1 caused re-markably different neurologic phenotypes that ranged frommicrocephaly to normal head circumference to macrocephaly aswell as variable intellectual disability (47) hypotonia (47)epilepsy (37) behavioral problems (37) and stereotypicmovements (27)37 The individuals who were determined tocarry gain-of-function RAC1 mutations (37) had normal headcircumference (13) or megalocephaly (23) in contrast to the

3 newly reported SPPRS patients here who either had micro-cephaly (13) or normal head circumference (23) Hypoplasticcorpus callosum hypotonia intellectual disability and hearingimpairment were noted in some but not all patients carryinggain-of-function RAC1mutations requiring further examinationof the specific or common molecular pathology of RAC1-associated neurodevelopmental disorder and SPPRS

Importantly it has been previously established that dysregu-lation of RAC1 abundance in mouse models results in a de-crease in dendritic spine number as well as reduction inhippocampal plasticity as we have uncovered in our Hace1KO mice102838 We therefore speculate that RAC1 is a keyfactor underlining neuronal pathology inHace1 KO mice andHACE1-deficient patients Whether RAC1-dependent cyto-skeletal modulation andor ROS homeostasis is the key de-terminant feature of HACE1-dependent neurodevelopmentaldeficiencies remains to be elucidated The present study addsHACE1 to the large RAC1 regulatory complex which whenperturbed has deleterious consequence to brain developmentand neuronal function

AcknowledgmentThe authors thank patients and their families for theirparticipation members of the IMP Biooptics facility for theirtechnical advice and assistance Dr David Keays IMPVienna for the use of behavioral equipment and members ofhis team for helpful discussions In addition the authors thankDr Daniel Lin from Sunjin Lab for the technical support with3D Images of dendritic spines The work was supported bythe Human Frontiers Science Program (HFSP to CH) theGerman Research Foundation (DFG SPP1757 SFB1089 toCH) FWF Lise Meitner Postdoctoral Fellowship to VNand EMBO Long-Term Fellowship to T-PP FJK issupported by the German Research Foundation (DFG)Research Unit 2488 ProtectMoversquo JMP is supported byThe Austrian Academy of Sciences the European Commun-ityrsquos Seventh Framework ProgrammeAdvanced ERC grantand Era of HopeDOD Excellence grant

Study fundingNo targeted funding reported

DisclosureV Nagy has received government research funding from theAustrian Science Fund and the Lise Meitner PostdoctoralFellowship and has received foundationsociety researchsupport from the Ludwig Boltzmann Society R Hollstein T-P Pai MK Herde P Buphamalai and P Moeseneder reportno disclosures E Lenartowicz has received foundationsociety research support from the Ludwig Boltzmann SocietyAnoop Kavirayani is employed with VBCFs which receivesgovernment funding from the Austrian Federal Ministry ofEducation Science and Research and the City of Vienna GChristoph Korenke I Kozieradzki R Nitsch A Cicvaric FJMonje Quiroga MA Deardorff EC Bedoukian Y Li and GYigit report no disclosures Jorg Menche has received

12 Neurology Genetics | Volume 5 Number 3 | June 2019 NeurologyorgNG

government research support from the Vienna Science andTechnology Fund E Ferda Perccedilin and B Wollnik report nodisclosures Christian Henneberger has served on the editorialboard for Frontiers in Molecular Neuroscience and Brain ResearchBulletin has received government research support from NRW-Ruckkehrerprogramm and has received foundationsociety re-search support from the Human Frontiers Science Program FJKaiser has received government research support from theGerman Research Foundation JM Penninger reports no dis-closures Go to NeurologyorgNG for full disclosures

Publication historyReceived by Neurology Genetics July 19 2018 Accepted in final formFebruary 5 2019

Appendix Authors

Name Location Role Contribution

Vanja NagyPhD

IMBA Institute ofMolecularBiotechnology of theAustrian Academy ofSciences VBCmdashViennaBioCenter CampusAustria and LBI-RUDVienna Austria

First and co-correspondingauthor

Conceptualizedand coordinatedthe projectPerformed allmouse behavioralexperimentsanalyzed the dataand wrote themanuscriptAssisted withmanuscript editing

RonjaHollstein PhD

University of LuebeckGermany

Author Performed allexperimentspertaining tohuman fibroblastsShe assisted incoordinatingclinical input andassisted with dataanalysis Assistedwith manuscriptediting

Tsung-Pin PaiPhD

IMBA Institute ofMolecularBiotechnology of theAustrian Academy ofSciences VBCmdashViennaBioCenter CampusAustria

Author Performed imageacquisition andanalysis of dendriticspines Assistedwith manuscriptediting

Michel KHerde PhD

University of BonnMedical SchoolGermany and CentreforNeuroendocrinologyDepartment ofPhysiology School ofBiomedical SciencesUniversity of OtagoDunedin NewZealand

Author Designedperformed andanalyzedelectrophysiologicexperimentsAssisted withmanuscript editing

PisanuBuphamalaiMSc

CeMM ViennaAustria

Author Performed andanalyzed the RAC1network analysisAssisted withmanuscriptediting

PaulMoesenederMSc

IMBA Institute ofMolecularBiotechnology of theAustrian Academy ofSciencesVBCmdashViennaBioCenter CampusAustria

Author Assisted withbehavioralexperimentsAssisted withmanuscriptediting

Appendix (continued)

Name Location Role Contribution

EwelinaLenartowiczMSc

LBI-RUD ViennaAustria

Author Assisted withbehavioralexperimentsAssisted withmanuscriptediting

AnoopKavirayaniDVM DipACVP

IMBA Institute ofMolecularBiotechnology of theAustrian Academy ofSciences VBCmdashViennaBioCenter CampusAustria

Author Coordinated andanalyzed mousehistologic findingsAssisted withmanuscript editing

Georg CKorenke MD

Klinikum OldenburgOldenburg Germany

Author Diagnosed andtreated thepatients obtainedconsents andorbiomaterials andanalyzed patientclinical dataAssisted withmanuscript editing

IvonaKozieradzkiMSc

IMBA Institute ofMolecularBiotechnology of theAustrian Academy ofSciences VBCmdashViennaBioCenter CampusAustria

Author Assisted withWestern blottingAssisted withmanuscriptediting

RobertoNitsch PhD

IMBA Institute ofMolecularBiotechnology of theAustrian Academy ofSciencesVBCmdashViennaBioCenter CampusAustria andAstraZenecaGothenburg Sweden

Author Assisted with earlyconceptualizationof the project andprovided molecularbiology toolsAssisted withmanuscript editing

Ana CicvaricPhD

Medical University ofVienna Austria

Author Designedperformed andanalyzedelectrophysiologicexperimentsAssisted withmanuscript editing

Francisco JMonjeQuiroga PhD

Medical University ofVienna Austria

Author Designedperformed andanalyzedelectrophysiologicexperimentsAssisted withmanuscriptediting

Matthew ADeardorff MD

Univ of PennPerelman School ofMed and ChildrenrsquosHospital ofPhiladelphia

Author Diagnosed andtreated thepatientsobtainedconsents andorbiomaterials andanalyzed patientclinical dataAssisted withmanuscriptediting

Emma CBedoukianMS

Childrenrsquos Hospital ofPhiladelphia PA

Author Diagnosed andtreated thepatients obtainedconsents andorbiomaterials andanalyzed patientclinical dataAssisted withmanuscriptediting

Continued

NeurologyorgNG Neurology Genetics | Volume 5 Number 3 | June 2019 13

References1 Anglesio MS Evdokimova V Melnyk N et al Differential expression of a novel

ankyrin containing E3 ubiquitin-protein ligase Hace1 in sporadic Wilmsrsquo tumorversus normal kidney Hum Mol Genet 2004132061ndash2074

2 Zhang L Anglesio MS OrsquoSullivan M et al The E3 ligase HACE1 is a critical chro-mosome 6q21 tumor suppressor involved in multiple cancers Nat Med 2007131060ndash1069

3 Diskin SJ Capasso M Schnepp RW et al Common variation at 6q16 within HACE1and LIN28B influences susceptibility to neuroblastoma Nat Genet 2012441126ndash1130

4 Tortola L Nitsch R Bertrand MJ et al The tumor suppressor Hace1 is a criticalregulator of TNFR1-mediated cell fate Cell Rep 2016151481ndash1492

5 Akawi N McRae J Ansari M et al Discovery of four recessive developmental dis-orders using probabilistic genotype and phenotype matching among 4125 familiesNat Genet 2015471363ndash1369

6 Hollstein R Parry DA Nalbach L et al HACE1 deficiency causes an autosomalrecessive neurodevelopmental syndrome J Med Genet 201552797ndash803

7 Hariharan N Ravi S Pradeep BE et al A novel loss-of-functionmutation in HACE1 islinked to a genetic disorder in a patient from India Hum Genome 2018517061

8 Torrino S Visvikis O Doye A et al The E3 ubiquitin-ligase HACE1 catalyzes theubiquitylation of active Rac1 Dev Cell 201121959ndash965

9 Castillo-Lluva S Tan CT Daugaard M Sorensen PH Malliri A The tumour sup-pressor HACE1 controls cell migration by regulating Rac1 degradation Oncogene2013321735ndash1742

10 Luo L Hensch TK Ackerman L Barbel S Jan LY Jan YN Differential effects of the RacGTPase on Purkinje cell axons and dendritic trunks and spines Nature 1996379837ndash840

11 Stankiewicz TR Linseman DA Rho family GTPases key players in neuronal de-velopment neuronal survival and neurodegeneration Front Cel Neurosci 20148314

12 Daugaard M Nitsch R Razaghi B et al Hace1 controls ROS generation of vertebrateRac1-dependent NADPH oxidase complexes Nat Commun 201342180

13 Rotblat B Southwell AL Ehrnhoefer DE et al HACE1 reduces oxidative stress andmutant Huntington toxicity by promoting the NRF2 response Proc Natl Acad Sci U SA 20141113032ndash3037

14 Iimura A Yamazaki F Suzuki T Endo T Nishida E Kusakabe M The E3 ubiquitinligase Hace1 is required for early embryonic development in Xenopus laevis BMCDev Biol 20161631

15 Carola V DrsquoOlimpio F Brunamonti E Mangia F Renzi P Evaluation of the elevatedplus-maze and open-field tests for the assessment of anxiety-related behaviour ininbred mice Behav Brain Res 200213449ndash57

16 Deacon RM Measuring motor coordination in mice J Vis Exp 2013e260917 Metz GA Whishaw IQ The ladder rung walking task a scoring system and its

practical application J Vis Exp 2009120418 Deacon RM Rawlins JN T-maze alternation in the rodent Nat Protoc 200617ndash1219 Wehner JM Radcliffe RA Cued and contextual fear conditioning in mice Curr Protoc

Neurosci 2004 chapter 8Unit 85C20 Vorhees CV Williams MT Assessing spatial learning and memory in rodents ILAR J

201455310ndash33221 Breuss MW Hansen AH Landler L Keays DA Brain-specific knockin of the path-

ogenic Tubb5 E401K allele causes defects in motor coordination and prepulse in-hibition Behav Brain Res 201732347ndash55

22 Minge D Senkov O Kaushik R et al Heparan sulfates support pyramidal cell ex-citability synaptic plasticity and context discrimination Cereb Cortex 201727903ndash918

23 Schneider CA Rasband WS Eliceiri KW NIH Image to ImageJ 25 years of imageanalysis Nat Methods 20129671ndash675

24 Kochlamazashvili G Henneberger C Bukalo O et al The extracellular matrix mol-ecule hyaluronic acid regulates hippocampal synaptic plasticity by modulating post-synaptic L-type Ca(2+) channels Neuron 201067116ndash128

25 Regehr WG Short-term presynaptic plasticity Cold Spring Harb Perspect Biol 20124a005702

26 Bliss TV Gardner-Medwin AR Long-lasting potentiation of synaptic transmission inthe dentate area of the unanaesthetized rabbit following stimulation of the perforantpath J Physiol (lond) 1973232357ndash374

27 Feng G Mellor RH Bernstein M et al Imaging neuronal subsets in transgenic miceexpressing multiple spectral variants of GFP Neuron 20002841ndash51

28 Bongmba OY Martinez LA Elhardt ME Butler K Tejada-Simon MV Modulation ofdendritic spines and synaptic function by Rac1 a possible link to Fragile X syndromepathology Brain Res 2011139979ndash95

29 Nobes CD Hall A Rho GTPases control polarity protrusion and adhesion duringcell movement J Cel Biol 19991441235ndash1244

30 Kraynov VS Chamberlain C Bokoch GM Schwartz MA Slabaugh S Hahn KM Lo-calized Rac activation dynamics visualized in living cells Science 2000290333ndash337

31 Liu S KapoorM Leask A Rac1 expression by fibroblasts is required for tissue repair invivo Am J Pathol 20091741847ndash1856

32 Kaverina I Krylyshkina O Small JV Regulation of substrate adhesion dynamicsduring cell motility Int J Biochem Cel Biol 200234746ndash761

33 van Bokhoven H Genetic and epigenetic networks in intellectual disabilities AnnuRev Genet 20114581ndash104

34 PengellyRJ Greville-Heygate S Schmidt S et alMutations specific to theRac-GEFdomainof TRIO cause intellectual disability and microcephaly J Med Genet 201653735ndash742

35 Alanis-Lobato G Andrade-Navarro MA Schaefer MH HIPPIE v20 enhancingmeaningfulness and reliability of protein-protein interaction networks Nucleic AcidsRes 201745D408ndashD414

36 Lelieveld SH Reijnders MR Pfundt R et al Meta-analysis of 2104 trios providessupport for 10 new genes for intellectual disability Nat Neurosci 2016191194ndash1196

37 Reijnders MRF Ansor NM Kousi M et al RAC1 missense mutations in de-velopmental disorders with diverse phenotypes Am JHumGenet 2017101466ndash477

38 Zamboni V Armentano M Berto G et al Hyperactivity of Rac1-GTPase pathwayimpairs neuritogenesis of cortical neurons by altering actin dynamics Sci Rep 201887254

Appendix (continued)

Name Location Role Contribution

Yun Li PhD University MedicalCenter GottingenGermany

Author Analyzed exomedata of family 2identified thecausative mutationand confirmedcosegregation in thefamily Assisted withmanuscript editing

GoekhanYigit PhD

University MedicalCenter GoettingenGoettingen Germany

Author Analyzed exomedata of family 2identified thecausative mutationand confirmedcosegregation in thefamily Assisted withmanuscript editing

Jorg MenchePhD

CeMM ViennaAustria

Author Performed andanalyzed the RAC1network analysisAssisted withmanuscriptediting

E FerdaPercin MDPhD

Gazi UniversityAnkara Turkey

Author Diagnosed andtreated thepatients obtainedconsents andorbiomaterials andanalyzed patientclinical dataAssisted withmanuscript editing

BerndWollnik MD

University MedicalCenter GoettingenGoettingen Germany

Author Analyzed exomedata of family 2identified thecausative mutationand confirmedcosegregation in thefamily Assisted withmanuscript editing

ChristianHennebergerMD

University of BonnMedical School BonnGermany andUniversity CollegeLondon London UKand German CenterforNeurodegenerativeDiseases (DZNE)Bonn Germany

Author Designedperformed andanalyzedelectrophysiologicexperimentsAssisted withmanuscript editing

Frank JKasier PhD

University of LuebeckLuebeck GermanyDZHK eV (GermanCenter forCardiovascularResearch) PartnerSite HamburgKielLubeck LubeckGermany

Author Assisted withprojectcoordinationclinicalcollaborations dataanalysis andwriting themanuscriptAssisted withmanuscript editing

Josef MPenningerMD

IMBA Vienna AustriaDepartment ofMedical Genetics LifeScience InstituteUniversity of BritishColumbia VancouverCanada

Co-correspondingauthor

Conceived thestudy and wrotethe manuscripttogether with VNAssisted withmanuscript editing

14 Neurology Genetics | Volume 5 Number 3 | June 2019 NeurologyorgNG

DOI 101212NXG000000000000033020195 Neurol Genet

Vanja Nagy Ronja Hollstein Tsung-Pin Pai et al HACE1 deficiency leads to structural and functional neurodevelopmental defects

This information is current as of April 30 2019

reserved Online ISSN 2376-7839Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology All rightsan open-access online-only continuous publication journal Copyright Copyright copy 2019 The Author(s)

is an official journal of the American Academy of Neurology Published since April 2015 it isNeurol Genet

ServicesUpdated Information amp

httpngneurologyorgcontent53e330fullhtmlincluding high resolution figures can be found at

References httpngneurologyorgcontent53e330fullhtmlref-list-1

This article cites 36 articles 6 of which you can access for free at

Citations httpngneurologyorgcontent53e330fullhtmlotherarticles

This article has been cited by 2 HighWire-hosted articles

Subspecialty Collections

httpngneurologyorgcgicollectionspastic_paraplegiaSpastic paraplegia

httpngneurologyorgcgicollectionmental_retardationMental retardation

httpngneurologyorgcgicollectionintelligenceIntelligence

httpngneurologyorgcgicollectiongait_disorders_ataxiaGait disordersataxia

httpngneurologyorgcgicollectionall_geneticsAll Geneticsfollowing collection(s) This article along with others on similar topics appears in the

Permissions amp Licensing

httpngneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in

Reprints

httpngneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online

reserved Online ISSN 2376-7839Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology All rightsan open-access online-only continuous publication journal Copyright Copyright copy 2019 The Author(s)

is an official journal of the American Academy of Neurology Published since April 2015 it isNeurol Genet

ServicesUpdated Information amp

httpngneurologyorgcontent53e330fullhtmlincluding high resolution figures can be found at

References httpngneurologyorgcontent53e330fullhtmlref-list-1

This article cites 36 articles 6 of which you can access for free at

Citations httpngneurologyorgcontent53e330fullhtmlotherarticles

This article has been cited by 2 HighWire-hosted articles

Subspecialty Collections

httpngneurologyorgcgicollectionspastic_paraplegiaSpastic paraplegia

httpngneurologyorgcgicollectionmental_retardationMental retardation

httpngneurologyorgcgicollectionintelligenceIntelligence

httpngneurologyorgcgicollectiongait_disorders_ataxiaGait disordersataxia

httpngneurologyorgcgicollectionall_geneticsAll Geneticsfollowing collection(s) This article along with others on similar topics appears in the

Permissions amp Licensing

httpngneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in

Reprints

httpngneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online

reserved Online ISSN 2376-7839Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology All rightsan open-access online-only continuous publication journal Copyright Copyright copy 2019 The Author(s)

is an official journal of the American Academy of Neurology Published since April 2015 it isNeurol Genet