Altered tubulin detyrosination due to SVBP malfunction induces cytokinesis failure and senescence, underlying a complex hereditary spastic paraplegia

Aging Cell. 2025;24:e14355.  
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h ps://doi.o g/10.1111/acel.14355
wileyonlinelib a y.com/jou nal/acel
Recei ed:10May2024
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Re ised:30Augus 2024
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Accep ed:9Sep embe 2024
DOI: 10.1111/acel.14355
RESEARCH ARTICLE
Al e ed ubulin de y osina ion due o SVBP mal unc ion
induces cy okinesis ailu e and senescence, unde lying a
complex he edi a y spas ic pa aplegia
Na halie Launay1,2 | Ma ia Espinosa- Alcan ud3 | Edga d Ve du a1 |
Go ka Fe nández- Eula e4,5 | Jon Onda o6,7 | Pablo I uzubie a5,6,7,8 | Ma ia Ma sal9 |
Aga ha Schlü e 1 | Mon se a Ruiz1,2 | S ephane Fou cade1,2 |
Agus í Rod íguez- Palme o1,2,10 | Mi en Zulaica6 | Andone Sis iaga7,11 |
Ga azi Labay u7,11 | Pablo Loza- Al a ez9 | Alejand o Vaque o3 |
Adol o Lopez de Munain5,6,7,8 | Au o a Pujol1,2,12
1Neu ome abolicDiseasesLabo a o y,Ins i u d'In es igacióBiomèdicadeBell i ge(IDIBELL),Hospi alDu aniReynals,Ba celona,Spain
2Cen e  o BiomedicalResea chonRa eDiseases,(CIBERERU759)Minis yo ScienceInno a ionandUni e si y,Mad id,Spain
3Ch oma inBiologyLabo a o y,JosepCa e asLeukaemiaResea chIns i u e,Badalona,Spain
4No d-Es /Ile-de-F anceNeu omuscula Re e enceCen e ,Ins i u eo Myology,Pi ié-Salpê iè eHospi al,Pa is,F ance
5Depa men o Neu ology,Hospi alUni e si a ioDonos ia,OSAKIDETZA-Depa men o Neu osciences,Uni e si yo  heBasqueCpun y,SanSebas ian,Spain
6Depa men o Neu osciences,Ins i u oBiodonos ia,SanSebas ián,Spain
7Cen e o BiomedicalResea chinNeu odegene a i eDiseases(CIBERNED),CIBER,Minis yo Science,Inno a ionandUni e si y,Mad id,Spain
8Depa men o Medicine,Schoolo Medicine,Uni e si yo Deus o,Bilbao,Spain
9ICFO-Ins i u deCienciesFo oniques,TheBa celonaIns i u eo ScienceandTechnology,Cas ellde els,Spain
10Pedia icNeu ologyUni ,Depa men o Pedia ics,Uni e si yHospi alGe mansT iasiPujol,Au onomousUni e si yo Ba celona,Badalona,Spain
11Depa men o Pe sonali y,Assessmen andPsychologicalT ea men Facul yo Psychology,Uni e si yo  heBasqueCoun y(UPV/EHU),SanSebas ian,Spain
12Ca alanIns i u iono Resea chandAd ancedS udies(ICREA),Ba celona,Spain
Thisisanopenaccessa icleunde  he e mso  heC ea i eCommonsA ibu ion License, which pe mi s use, dis ibu ion and ep oduc ion in any medium,
p o ided he o iginal wo k is p ope ly ci ed.
©2024TheAu ho (s).Aging CellpublishedbyAna omicalSocie yandJohnWiley&SonsL d.
Abb e ia ions:AD,Alzheime 'sdisease;ALS,amyo ophicla e alscle osis;CNS, hecen alne oussys em;CSVS,TheCollabo a i eSpanishVa ian Se e ;CTL,con ol;ER, he
endoplasmic e iculum;FBS, oe albo inese um;FRDA,F ied eicha axia;FTD,F on o empo aldemen ia;HSP,He edi a ySpas icPa aplegias;LOD,combinedloga i hmo odds;MS,
mul iplescle osis;MTs,mic o ubules;PACT-RFP, heRFP- aggedPACTdomain;PBMCs,pe iphe albloodmononuclea cells;PD,Pa kinson'sdisease;Pe ic,Pe icen in;PN,
Pa henolide;siRNAs,smallin e e ingRNAs;SNPs,singlenucleo idepolymo phisms;SVBP,small asohibin-bindingp o ein;TLL, ubulin y osineligase;Veh, ehicle;WES,whole
exomesequencing;β- gal, β- galac osidase.
Co espondence
Au o aPujol,Neu ome abolicDiseases
Labo a o y, Ins i u d'In es igació
Biomèdica de Bell i ge (IDIBELL),
Hospi alDu aniReynals,G anVia,199,
L'Hospi ale de Llob ega , Ba celona
08908,Spain.
Email: [email protected]
Funding in o ma ion
heSpanishMinis yo Economyand
Compe i i enessMINECO,G an /Awa d
Numbe :BES-2015-071251,PID2020-
117284RB-100andSEV-2015-0522;
Abs ac
Senescence,ma kedbype manen cellcyclea es maycon ibu e o hedeclinein
egene a i e po en ial and neu onal unc ion, he eby p omo ing neu odegene a i e
diso de s.In hiss udy,weemployedwholeexomesequencing oiden i yap e i-
ouslyun epo edbiallelicmissense a ian inSVBP(p.Leu49P o)insixpa ien s om
h eeun ela ed amilies.Thesea ec edindi idualsp esen wi hacomplexhe edi-
a yspas icpa aplegia(HSP),pe iphe alneu opa hy, e balap axia,andin ellec ual
disabili y,exhibi ingamilde pheno ypecompa ed opa ien swi hnonsenseSVBP
mu a ionsdesc ibedp e iously.Consis en wi hSVBP'sp ima y oleasachape one
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1 | INTRODUCTION
He edi a yspas icpa aplegias(HSP)a eag oupo inhe i edneu-
odegene a i ediso de scha ac e izedby hedegene a iono  he
longdescendingaxonso  heco icospinaluppe mo o neu ons, e-
sul ing in spas ici y and weakness in he lowe limbs (Fink, 2014).The
clinicalandgene iche e ogenei yo HSP e lec s hein ol emen o 
di e se cellula pa hways, encompassing memb ane and ca go a -
icking, mi ochond ial unc ion, o ganelle shaping, lipid me abolism,
and au ophagy (Blacks one, 2018; Lo Giudice e al., 2014).Among
hesecellula pa hways,mic o ubules(MTs)eme geasac ucialel-
emen due o se e al ac o s: (i) hei egula ion di ec ly in luences
in acellula anspo , (ii) hey in e ac wi h o ganelles such as he
endoplasmic e iculum (ER) and mi ochond ia, and (iii) mu a ions
a ec ing p o eins ha  di ec ly in e ac  wi h MTs (such as SPAST,
KIF5A,KIF1A,REEP1,andREEP2)a ep esen inapp oxima elyhal 
o  pa ien s wi h gene ically con i med HSP (Blacks one, 2018; Lo
Giudice e al., 2014).
Mic o ubules a e dynamic polyme s composed o α/β ubulin di-
me s ha se e as “ ailways” o mo o - d i en in acellula anspo .
Theyplaya i al oleinin acellula o ganiza ionandch omosome
seg ega ion.MTsexhibi highdynamics,wi h hei g ow handsh ink-
age egula ed by (1) he addi ion and loss o α- and β- ubulin subuni s,
and (2) a ious ypes o pos ansla ional modi ica ions (Janke, 2014).
These modi ica ions, such as de y osina ion/ y osina ion, ace yl-
a ion, glycosyla ion, and (poly) glu amyla ion, collec i ely o m a
“ ubulin- code” ha egula es in e ac ions wi h molecula mo o s and
o he MT-bindingp o eins(Janke,2014;McKennae al.,2023).
Pa ien s wi h loss-o - unc ion mu a ions in SVBP (small
asohibin-bindingp o ein)we e epo ed oexhibi symp omssuch
asa axia,in ellec ualdisabili y,mic ocephaly,andmuscula hypo-
onia (Iqbal e  al.,2019; Pagnamen a e al., 2019).SVBPin e ac s
wi hVASHp o einsinachape one-likemanne ,se ingasac i i-
cal ac o o he solubili y/sec e ion and de y osina ion ac i i y o
VASH1andVASH2(Aillaude al.,2017; Nieuwenhuis e al., 2017).
De y osina ed ubulin is abundan ly p esen  in neu onal MTs and
o he long-li edMTpopula ions,whe ei playsac ucial oleinax-
onal anspo by acili a ing he a ickingo kinesin-1(Konishi&
Se ou,2009). De ec i e de y osina ion caused p oli e a i e de ec s
du ing neu ogenesis, leading o mic ocephaly and abno mal beha io
(Landsk on e al., 2022). Mo eo e , spindle de y osina ion is c ucial
o he accu a e ch omosome cong ession and symme y b eakage
du ing emalemeiosis,unde sco ing heessen ial oleo MTde y-
osina ion in main aining genomic s abili y (Ba isic e al., 2015).
In his s udy, we ha e iden i ied a no el biallelic missense a ian
inSVBPamongsixindi idualsexhibi ingspas icpa apa esisaccom-
paniedbysenso imo o axonalneu opa hy, e balap axia,epilepsy,
andin ellec ualdisabili y, he ebyexpanding hep e iously epo ed
pheno ype. Ou indings, based on analyses o pa ien - de i ed ib o-
blas sandSVBPknockdowncells, e ealapi o al oleo SVBPin
cen osome cohesion and ch omosome seg ega ion. Mo eo e , ou
esul s shed ligh o he i s ime on he po en ial in ol emen o
cellula senescence as a d i e o hese neu ological diso de s.
2 | RESULTS
2.1  |  Va ian iden i ica ion and clinical ea u es
Th oughwholeexomesequencing(WES),weha eiden i iedacon-
sis en  biallelic SVBP a ian  in six indi iduals belonging o h ee
un ela ed amilies o igina ing om he same geog aphic egion
in No he n Spain. The no el missense a ian  in he SVBP gene
(Ch 1:43273140A > G,NM_199342:exon3:c.146 T > C,p.Leu49P o)
is no  p esen  in con ol da abases such as ExAC and 1000 ge-
nomes,wi honly wohe e ozygousca ie indi iduals eco dedin
he GnomAD da abase. Fu he mo e, his a ian  is absen  om
he Collabo a i e Spanish Va ian  Se e  (CSVS), which includes
sequence da a om 1644 un ela ed indi iduals.Allsingle nucleo-
idepolymo phisms(SNPs)wi hin hehomozygousgenomic egion
sha edby hepa ien ssequencedbyWES(spanninga leas 2.32 Mb)
exhibi iden icalgeno ypesin he i eanalyzedpa ien s( amiliesA
Associa ionASL-HSPand‘LaMa a óde
TV3’Founda ion,G an /Awa dNumbe :
202006-30;Ins i u odeSaludCa losIII,
G an /Awa dNumbe :CD19/00221and
MiguelSe e p og amCP11/00080;
FundaciónHespe iaandSec e a ia 
o Uni e si ies and Resea ch o he
Minis yo BusinessandKnowledgeo 
he Go e nmen o Ca alonia, G an /
Awa dNumbe :2017SGR1206;URDca 
p og amme,G an /Awa dNumbe :PERIS
SLT002/16/00174;Cen e o Biomedical
Resea ch on Ra e Diseases (CIBERER),
G an /Awa dNumbe :ACCI19-759;
Ca alango e nmen agencyAGAUR,
G an /Awa dNumbe :AGAUR2021-SGR-
01378
necessa y o VASH-media ed ubulinde y osina ion,bo hpa ien  ib oblas swi h he
p.Leu49P omu a ion,andHeLacellsha bo inganSVBPknockdownexhibi mic o u-
bule dynamic ins abili y and al e a ions in pe icen iola ma e ial (PCM) componen
a icking and cen osome cohesion. In pa ien ib oblas s, s uc u al abno mali ies
in he cen osome igge mi o ic e o s and cellula senescence. No ably, p ema u e
senescencecha ac e izedbyele a edle elso p16INK4,wasalsoobse edinpa ien 
pe iphe albloodmononuclea cells(PBMCs).Taken oge he ,ou  indingsunde sco e
hec i ical oleo SVBPin hede elopmen andmain enanceo  hecen alne ous
sys em, p o iding no el insigh s associa ing cy okinesis ailu e wi h co ical mo o
neu on disease and in ellec ual disabili y.
KEYWORDS
cen osome,cy okinesis ailu e,HSP,mic o ubulede y osina ion,senescence,SVBP
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LAUNAY e al.
andB).Thisobse a ionsugges s ha  he a ian isloca edwi hin
hesamehaplo ype,indica ingi asa ounde  a ian .Seg ega ion
analysis in all una ec ed membe s o bo h amilies suppo s he e-
cessi emodeo inhe i ancep e iously epo ed o SVBP.Thecom-
bined loga i hm o odds (LOD) sco e, conside ing coseg ega ion da a
om all geno yped indi iduals, eached 2.73.
Pa ien s om amilies A,B, and Cp esen ed a  bi ho child-
hoodwi hneu ode elopmen aldelaysandla e exhibi edin ellec-
ual disabili ies. Subsequen ly, hey de eloped a complex spas ic
pa aplegiasynd omeassocia edwi h e balap axiaandaxonalneu-
opa hy (Figu e 1a). The clinical cha ac e is ics o  all six pa ien s
a esumma izedinTable 1. B ain MRIs o pa ien s P2, P4, and P6
we eanalyzed, e ealingse e alsha ed ea u es.All h eeexhibi ed
co pus callosum hinning, ce ebella a ophy, and en iculomegaly
(Figu e 1b–d).Addi ionally, wopa ien sdisplayed on alpe i en-
icula hype in ensi iesonT2imaging, esembling he“ea o  he
lynx”sign(Figu e 1c,d). No ably, hal o he pa ien s ha e a his o y o
cance , all o which a e o epi helial o igin.
2.2  | SVBP a ian al e s SVBP exp ession and
impai s MT de y osina ion
Ou insilicos uc u alanalysis e ealed ha  hep.Leu49P osub-
s i u ion may dis up in amolecula hyd ophobic in e ac ions and/
o des abilize heconse edα-helicalcon o ma iono SVBP.This
con o ma ional change is c ucial o  i s in e ac ion wi h VASH1
and he de y osina ion o he α- ubulin pep ide (Liao e al., 2019)
(Figu e 2a).To alida e hisp edic ion,weco ans ec edHeLacells
wi hequimola amoun so C- e minalFlag- aggedhumanVASH1
andei he wild- ypeo p.Leu49P omu an C- e minalFlag- agged
human SVBP. Subsequen ly, we analyzed he exp ession le els
o  VASH1 and SVBP by Wes e n blo . In con as  o wild- ype
SVBP, hemu an SVBPp o einwasunde ec ablein hecelllysa e
(Figu e 2b),sugges ingpossibledeg ada ion o  heSVBP a ian .
Mo eo e , a signi ican educ ion in h ee pos ansla ional o ms
o VASH1(42 kDa,36 kDa,and32 kDa)wasobse edincells ans-
ec edwi h hemu an SVBP(Figu e 2b), s ongly indica ing ha
FIGURE 1 Clinical ea u eso  amilieswi hano elSVBPmu a ion.(a)Pedig ee o  amiliesA,B,andC.(b)Axialandsagi alT1MRI
sequenceso pa ien P2showingdi usece ebella a ophy,enla ged en icles, hinco puscallosum,anddi usece ebella a ophy
(c)Sagi alT1andaxialFLAIRMRIo pa ien P4showingce ebella  e misa ophy,ea so  helynxsign(whi ea ow),anasymme ical
en icleenla gemen .(d)AxialT2andsagi alT1MRIsequences ompa ien P6showingea so  helynxsign(whi ea ow),enla ged
en icles, and e mis ce ebella a ophy.
14749726, 2025, 1, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1111/acel.14355 by Uni e sidad Del Pais Vasco, Wiley Online Lib a y on [26/02/2025]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
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TABLE 1 Mainclinical ea u eso  amiliesA,B,andC.
Family A B C
Pa ien P1 P2 P3 P4 P5 P6
Mu a ion c.146 T > CHom
p.Leu49P o
c.146 T > CHom
p.Leu49P o
c.146 T > CHom
p.Leu49P o
c.146 T > CHom
p.Leu49P o
c.146 T > CHom
p.Leu49P o
c.146 T > CHom
p.Leu49P o
Gende M M F F F F
E hnici y Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian
Pa en al
Consanguini y
No No No No No No
Fi s symp oms and
age o onse
Neu ode elopmen al delay
since bi h
Neu ode elopmen al delay
since bi h
Neu ode elopmen al delay
since bi h
Mild clumsy gai and oo
de o mi y since childhood
Neu ode elopmen al
delay since bi h
Neu ode elopmen al delay
since bi h
Psychomo o
de elopmen
Delayed Delayed Delayed N/A Delayed Delayed
In ellec ual disabili y + + + + + +
Mo o examina ion Spas icpa apa esis
A e lexia
Wheelchai -bound(adul )
Spas icpa apa esis
B adykinesia, dis al lowe
limb weakness
Wheelchai -bound(adul )
Spas icpa apa esis Spas icpa apa esis
Dis al lowe limb amyo ophy
andweakness,anklea e lexia
Spas icpa apa esis
Dis al lowe limb
weakness,a e lexia
A axic-spas icgai .B isk
e lexesexcep  o Achilles
e lex(absen )
A axia ++N/A +−+
Ve balap axia − +−+ + N/A
Epilepsy −+ + N/A +−
Agg essi ebeha io −+ + − − +
Oph halmologic
mani es a ions
−S abismus,Nys agmus N/A −Nys agmus Nys agmus
O he clinical
mani es a ions
Hamme oes
Adenoca cinomawi hhepa ic
me as asis.
Deceaseda age59 yea s
Scoliosis,lumba 
hype lo dosis, pes planus,
Hamme oes
Colonic ubula adenoma
Deceaseda 52 yea s
Deceaseda age59 yea s Pes ca us, Hamme oes
Fee hypoalgesia and
hypopalles hesia
Cold, e y hema ous ee
Pes ca us. Hamme oes.
Hypopalles hesia. B eas
cance (a 51 yea s)
MRI N/A Ce ebella a ophy,
en iculomegaly, middle
ce ebella peduncles a ophy.
Ca um sep um pellucidum
pe sis ence
N/A B ain a ophy,
en iculomegaly.
Ea o  helynxsign
N/A Ce ebella a ophy,
en iculomegaly, Ea o
helynxsign
Ne e Conduc ion
S udies(NCS)
N/A Lowe limbaxonal
senso imo o neu opa hy
N/A Axonalsenso imo o 
neu opa hy
Axonalsenso imo o 
neu opa hy
Axonalsenso imo o 
neu opa hy
Abb e ia ions:F, emale;M,male;N/A,no a ailable;Y,yea s.
14749726, 2025, 1, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1111/acel.14355 by Uni e sidad Del Pais Vasco, Wiley Online Lib a y on [26/02/2025]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
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he SVBP mu a ion impai s VASH1 sec e ion and/o  solubili y.
Immuno luo escenceandWes e nblo analysesdemons a edde-
c easedSVBPle elsinpa ien  ib oblas s, u he con i ming ha 
hep.Leu49P omu a iona ec sp o eins abili yandleads oi s
deg ada ion (Figu e 2c,d).
Toin es iga e hepo en ialimpac o  heSVBPmu an onMT
de y osina ion,weemployedpacli axel oele a ede y osina ed u-
bulinle elsin ib oblas s.Pacli axel unc ionsbys abilizingMTsand
emo ing he ee α/β- ubulin dime s, which se e as subs a es o
ubulin y osineligase(TTL)(P o ae al.,2013). Immuno luo escence
and Wes e n blo  expe imen s e ealed a signi ican  educ ion in
de y osina ed α- ubulin le els in pa ien ib oblas s compa ed o
con ols (Figu e 2e, ), p o iding e idence ha  he SVBP mu a ion
dis up sMTde y osina ion.Collec i ely, hese indingsunde sco e
hec i ical oleo  esidueLeu49inSVBP o  hein e ac ionwi h
VASH1,sugges ing ha i ssubs i u ionwi hP ohampe sMTde-
y osina ion by educing he abundance o  ac i e SVBP-VASH1
he e odime s.
FIGURE 2 Pa hogenici yo SVBP a ian on heVASH1sec e ionandMTde y osina ionac i i y.(a)Close-up iewso  heVASH1–SVBP
in e ace,wi hin e ac ing esiduesshownass icks.VASH1 esiduesa ecolo edblueandlabeledwi hbluele e s,whileSVBP esiduesa e
colo edo angeandlabeledwi ho angele e s.(b)HeLacells ans ec edwi h ec o sdi ec ing heexp essiono FLAG- aggedSVBPWT,
SVBPL49PandVASH1,o combina ions he eo ,we esubjec ed oimmunoblo analysiswi han i-Flagan ibody.To alamoun so β- ac in
we eusedasaloadingcon ol.(c)Con ol(CTL)andpa ien (P2andP4) ib oblas swe es ainedwi han i-SVBP( ed)andan i-α- ubulin (α-
ub;g een)an ibodiesandDAPI(blue).Scaleba s:10 μm.(d)Con ol(CTL)andpa ien (P2andP4) ib oblas swe esubjec ed oimmunoblo 
analysisusing hean i-SVBPan ibody.To alamoun so α- ubulinwe eusedasaloadingcon ol.(e)Con ol(CTL)andpa ien (P2and
P4) ib oblas swe e ea edwi h ehicleo pacli axelands ainedwi han i-α- ubulin (α- ub; ed) and an i- de y osina ed ubulin (de y -
ub;g een)an ibodiesandDAPI(blue).Scaleba s:50 μm.( )Con ol(CTL)andpa ien (P2andP4) ib oblas swe e ea edwi h ehicleo 
pacli axelandsubjec ed oimmunoblo analysisusingan ibodiesdi ec edagains de y osina ed(de y - ubulin)andα- ubulin.To alamoun s
o β-ac inwe eusedasaloadingcon ol.The ela i e a ioso de y osina ed e sus o al⍺- ubulin le els a e indica ed (n = 3).
14749726, 2025, 1, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1111/acel.14355 by Uni e sidad Del Pais Vasco, Wiley Online Lib a y on [26/02/2025]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License

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2.3  | SVBP mu an induces MT dynamic
ins abili y and cen osome cohesion de ici
Gi en hees ablishedassocia ionbe weenhighle elso MTde y os-
ina ion and cen ioles (Janke, 2014;Song&B ady,2015), sugges ing
api o al oleo MTde y osina ionincen osomes abiliza ionand
in eg i y,weop ed oin es iga e heimpac o  heSVBPmu a ion
on cen osome cohesion. In iguingly, immuno luo escence analysis
e ealed p ema u e cen osome sepa a ion in pa ien ib oblas s
du ing in e phase. The p opo ion o  cells exhibi ing cen osomes
sepa a ed by >2 μm inc eased om 4% in con ols o >20% in pa ien
cells (Figu e 3a). Consis en wi h hese indings, pa henolide, an in-
hibi o o MTde y osina ion(F eunde al.,2020), induced abno mal
cen iole sepa a ion in nea ly 20% o con ol ib oblas s (Figu e 3b),
mi o ing he obse ed a io in pa ien cells.
Subsequen ly,wequan i iedpe icen inle els oe alua ecen o-
some in eg i y. Ou analysis e ealed a signi ican educ ion in cen o-
somal pe icen in s aining in pa ien ib oblas s compa ed o con ols
(Figu e 3a), indica i e o dis up ed pe icen iola ma e ial (PCM) as-
sembly due o he SVBP mu a ion. Consis en ly, high- esolu ion
luo escence mic oscopy un eiled an inc ease in pe iphe al and cy-
oplasmic pe icen in oci along he MT cy oskele on in in e phase
pa ien  ib oblas s,sugges ingade ec inMT-dependen  a icking
whe e PCM componen s a e inadequa ely anspo ed o cen o-
somes (Figu e S1).Fu he mo e,weobse edMTdepolyme iza ion
and deg ada ion in pa ien ib oblas s, e iden om he punc ua ed
pa e no MTs aining(Figu e S1).Consequen ly,weconclude ha  he
SVBP a ian inducesMTdynamicins abili y,leading oal e a ionsin
PCM componen a icking and a de ici in cen osome cohesion.
2.4  | SVBP mu an leads o spindle mo phology
al e a ion, cy okinesis ailu e, and ch omosome
ins abili y
ThePCMse esasac ucialhub o MTnuclea ionand egula es
he numbe  and composi ion o  MTs h oughou  he cell cycle
(Zimme mane al.,2004). Consis en ly, s udies employing small in-
e e ingRNAs(siRNAs) a ge ingbo hpe icen iniso o ms(Aand
B)ha edemons a eda educ ioninas alMTsandmi o icspindle
leng hinSAOScells(Zimme mane al.,2004). Co espondingly, ou
analysis e ealed ha pa ien  ib oblas sexhibi edsho e mi o ic
spindlesandimpai ed o ma iono MTas e s(Figu e 3c), sugges ing
ha  heSVBPmu a ional e sMTnuclea ion.Theseobse a ions
align wi h a ecen s udy demons a ing ha deple ion o asohibin
inU2OScells esul edinsho e mi o icspindles,accompaniedbya
signi ican  educ ioninas alMTs(Liaoe al.,2019).
Rema kably, ou  immuno luo escence expe imen s unco e ed
ha  80% o  pa ien  ib oblas s expe ienced cy okinesis ailu e,
e idenced by in e cellula cy oplasmic b idges and he p esence
o binuclea ed cells (Figu e 3c,d). Fu he mo e, he ele a ed a e
o mic onucleus o ma ion (~10%) obse ed in pa ien ib oblas s
compa ed o con ol cells indica ed comp omised ch omosome s a-
bili y (Figu e 3d). Collec i ely, hese indings sugges a c ucial ole
o SVBPinensu ingaccu a emi osisandes ablishalinkbe ween
SVBPde iciencyandal e a ionsinspindlemo phology,cy okinesis
ailu e, and ch omosome ins abili y.
2.5  |  CRISPR/Cas9- media ed knockou o SVBP in
HeLa cells eplica es cen osome cohesion de ici and
mi osis abno mali ies
Tocon i m ha  heobse edde ec sinpa ien  ib oblas swe ein-
deeda ibu ed oSVBPde iciency,weemployed heCRISPR/Cas9
echnique ogene a eanSVBPknockou inHeLacells(Figu e S2).
Consis en wi h ou ea lie indings, we no ed a signi ican inc ease
in cen osome spli ing and a educ ion in cen osomal pe icen in
s aining du ing in e phase in SVBP-KO HeLa cells compa ed o
wild- ype cells (Figu e 4a).Simila ou comeswe eobse edwhen
SVBP-KO HeLa cells we e ans ec ed wi h an exp ession ec o 
encoding he RFP- agged PACT domain (PACT-RFP) (Figu e 4b), a
conse edmo i known o a ge cen osomesby aggingAKAP450
andpe icen in(Gillingham&Mun o,2000).Addi ionally,SVBP-KO
FIGURE 3 SVBP mu an induces cen osome cohesion de ici , mi o ic spindle abno mali ies, cy okinesis ailu e, and ch omosome
ins abili y.(a)In e phasecon ol(CTL)andpa ien (P2andP4) ib oblas swe es ainedwi halpha- ubulin(α- ub; g een) and an i- pe icen in
(Pe ic.; ed)an ibodiesandDAPI(blue)a cellpassages<10 (inse shows enla gemen o he PCM, as e isk (*) ma k he posi ion o
cen osome).Scaleba s,10 μm.Pe cen ageo con ol(CTL)andpa ien  ib oblas s(P2andP4)wi hspli cen osomesandcen osomal
pe icen inin ensi ywe equan i ied.n ≥ 50cells/100cen osomes;mean ± SD;*p < 0.05;**p < 0.01byone-wayANOVAwi hTukeypos hoc
es s.(b)Rep esen a i epic u eo con ol ib oblas s(CTL) ea edwi h ehicle(Veh.)o pa henolide(PN,5 μM,24 h)ands ainedwi han i-
pe icen in(Pe ic.;g een)an ibodiesandDAPI(blue)(whi ea owsindica e heposi iono  hecen osome).Scaleba s,20 μm. Pe cen age o
cells ea edo no wi hpa henolide(PN)wasquan i ied.n ≥ 20cells/40cen osomes;mean ± SD;**p < 0.01by wo- ailed es . (c) Con ol
(CTL)andpa ien (P2andP4) ib oblas swe es aineda di e en mi o icphases(p ophase,me aphase,andcy okinesis)wi halpha- ubulin
(α- ub;g een)andan i-pe icen in(Pe ic.; ed)an ibodiesandDAPI(blue)a cellpassages<10 (inse shows enla gemen o he PCM, as e isk
(*)ma k heposi iono cen osome).Scaleba s,5 μm.Mi o icde ec s(spindleabno mali iesandcy okinesisde ec s)we equan i iedin
con ol(CTL)andpa ien (P2andP4 ib oblas s).n ≥ 10cells o eachmi o icphase;Mean ± SD;*p < 0.05;*** < p < 0.001byone-wayANOVA
wi hTukeypos hoc es s.(d)Rep esen a i epic u eso con ol(CTL)andpa ien (P2andP4) ib oblas sa cellpassages>10 s ained
wi h an i- α- ubulin (α- ub;g een)andDAPI(blue),showing hep esenceo mic onucleiandbinuclea edcells(whi ea ows)(inse shows
enla gemen o  heindica eda ea).Scaleba s,50o 10 μm.Thepe cen ageo cellswi hmic onucleiandbinuclea edcellswe equan i ied.
n ≥ 50;Mean ± SD;*p < 0.05;**p < 0.01byone-wayANOVAwi hTukeypos hoc es s.
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FIGURE 4 CRISPR/Cas9knockou o SVBPinHeLacellsinducescen osomeabno mali iesandabe an mi osis.(a)In e phaseWild- ype
(WT)andSVBP-KO(SVBPko) HeLa cells we e s ained wi h alpha- ubulin (α- ub;g een)andan i-pe icen in(Pe ic.; ed)an ibodiesandDAPI
(blue)(inse showsenla gemen o  hePCM; heas e isk(*)ma ks heposi iono  hecen osome).Scaleba s,10 μm. Pe cen age o wild-
ype(WT)andSVBP-KO(SVBPko)HeLacellswi hspli cen osomesandcen osomalpe icen inin ensi ywe equan i ied.n ≥ 30cells/60
cen osomespe condi ion;Mean ± SD;**p < 0.01by wo- ailed  es .(b)Rep esen a i eimageo wild- ype(WT)andSVBP-KO(SVBPko)
exp essingPACT-RFP(as e isk(*)ma k heposi iono cen osome).Twen y- ou hou sa e  ans ec ion,cellswe e ixedands ained o 
DNAcon en (DAPI;blue).Scaleba s,10 μm.Pe cen ageo wild- ype(WT)andSVBP-KO(SVBPko)HeLacellsexp essingPACT-RFPwi h
spli cen osomeswasquan i ied.n ≥ 10cells/20cen osomespe condi ion;Mean ± SD;**p < 0.01by wo- ailed  es .(c)Wild- ype(WT)
and SVBP-KO(SVBPko) HeLa cells we e s ained a di e en mi o ic phases (p ophase, me aphase, and cy okinesis) wi h alpha- ubulin (α- ub;
g een)andan i-pe icen in(Pe ic., ed)an ibodiesandDAPI(blue)(inse showsenla gemen o  hePCM; heas e isk(*)ma ks heposi ion
o  hecen osome).Mi o icde ec s(spindleabno mali iesandcy okinesisde ec s)inwild- ype(WT)andSVBP-KO(SVBPko) HeLa cells
we equan i ied.n ≥ 10cells o eachmi o icphase;Mean ± SD;*p < 0.05;*** < p < 0.001by wo- ailed es . (d) Rep esen a i e pic u es o
wild- ype(WT)andSVBP-KO(SVBPko) HeLa cells s ained wi h an i- alpha- ubulin (α- ub,g een)andDAPI(blue)a passages>10, showing he
p esenceo mul inuclea edcells(whi ea owsindica emul inuclea edcells);inse showsenla gemen o  heindica eda ea.Scaleba s,50
o 10 μm.Thepe cen ageo mul inuclea edandmic onuclea edwild- ype(WT)andSVBP-KO(SVBPko)HeLacellswe equan i ied.n ≥ 50;
Mean ± SD;*p < 0.05;**p < 0.01by wo- ailed es .
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HeLa cells displayed a sho e  mi o ic spindle size, educed as-
al MTs, cy okinesis ailu e, and inc eased mic onuclei o ma ion
(Figu e 4c,d), collec i ely mimicking he pheno ype obse ed in pa-
ien ib oblas s.
2.6  | SVBP mu an induces cell cycle a es and
senescence
Cen osome al e a ions o mi o ic e o s ypically ac i a e he ap-
op osis machine y h ough p53 (Im eh e al., 2016).The e o e,we
examined p53 exp ession in pa ien 's ib oblas s by Wes e n Blo 
analysis.Whilenochangewasobse edinea lycellpassages(<10),
he dec eased p o ein and mRNA exp ession le els o  p53 in pa-
ien ib oblas s a cell passage >10 sugges ed he p og essi e in-
ac i a ion o he p53- dependen apop osis esponse (Figu e 5a,b;
Figu e S3a,b). Consis en ly, low cy ome y apop osis assays showed
a sligh inc ease in apop o ic cell le els in pa ien ib oblas s a la e
passage compa ed o con ols (Figu e 5c), indica ing ha apop osis
isamino ou comeinSVBPmu an cells.
We henin es iga ed heimpac o  heSVBPmu a iononcell
p oli e a ion. EdU inco po a ion and his one H3-Se ine 28 phos-
pho yla ion le els (H3Se 28P)we eused omoni o  S phasep o-
g ession and he la e- G(2)/M s a us o cells, espec i ely. Flow
cy ome yanalysis e ealedequi alen EdUinco po a ioninea ly
passages o con ol and pa ien ib oblas s (Figu e S3c). Howe e , by
cell passages >10, pa ien ib oblas s displayed dec eased EdU in-
co po a ion compa ed o con ol ib oblas s (Figu e 5d,e).Simila ly,
we ound ha  he phospho yla iono H3Se 28 dec eased signi i-
can ly om cell passages >10 in pa ien ib oblas s (Figu e 5 ,g;
Figu e S3d).The e o e,ou  esul sindica ed ha pa ien  ib oblas s
p og essi ely a es ed in G1/G0 phase.
Toassesssenescencele els,we i s pe o medβ- galac osidase
(β- gal) s aining, which e ealed inc eased senescence- associa ed
β- galac osidase ac i i y in pa ien cells, co ela ed wi h an inc eas-
ing numbe o cell passages (Figu e 5h).mRNAle elso lmnb1 we e
dec eased while p21 le els we e aised, consis en wi h augmen ed
β- galac osidase s aining wi h inc easing cell passages (Figu e 5i).
Rema kably, we obse ed a e y high exp ession le el o  IL- 6, a
senescence-associa ed in lamma o y media o  molecule (SASP) in
low- passage pa ien ib oblas s (Figu e 5i, Figu e S3c). Mo eo e , we
obse ed a co ela ion be ween inc eased H2A his one (γ- H 2 A X ) 
phospho yla ion,ama ke o DNAdamage(Bi ane al.,2017), and
he accumula ion o senescen cells in pa ien s (Figu e 5j; Figu e S3a).
Toco obo a eou in i o indings,weassessed heexp ession
o CDKN2A/p16INK4 in pe iphe al blood mononuclea cells (PBMCs)
om bo h con ol indi iduals and pa ien s. p16INK4 se es as a well-
es ablishedbioma ke o senescencein a ious issues,includingT
cells,whe ei  es ic s hei  eplica i ecapaci y(Y.Liue al.,2009).
Rema kably, he ela i e exp ession o  CDKN2A/p16INK4 was sig-
ni ican lyhighe inpa ien PBMCs(3.4 ± 0.1 old)compa ed ocon-
ols (Figu e 5k), aligning wi h he senescence pheno ype obse ed
in pa ien ib oblas s.
3 | DISCUSSION
Using whole-exome sequencing (WES), we unco e ed a no el bi-
allelicSVBP a ian insixindi iduals om h eeun ela ed amilies.
Ou unc ional in es iga ions p o ide compelling e idence sugges -
ing ha cen osomeabno mali iesandsubsequen senescencemay
se easunde lyingd i e so HSP.Whenconside ing he indingso 
IqbalandPagnamen a(Iqbale al.,2019; Pagnamen a e al., 2019),
alongwi hou coho o sixpa ien s,acollec iono commonclini-
cal ea u es eme ges. All pa ien s exhibi ed in ellec ual disabili y
along wi h delayed g oss mo o and speech de elopmen . No ably,
pa ien s ha bo ing he missense p.Leu49P o a ian  mani es ed a
mo o -p edominan pheno ypecha ac e izedbycomplexHSPand
axonalneu opa hy,signi ican lyimpac ing hei mobili ycompa ed
o p e iously epo ed indi iduals who p esen ed wi h hypo onia,
dysmo phia,andse e ecogni i eimpai men .Theea lie onse and
mo e se e e neu ode elopmen al ea u es obse ed in he p e i-
ously epo ed cases could be a ibu ed o hei loss- o - unc ion
a ian s. F om a neu oimaging pe spec i e, se e al common ea-
u es we e obse ed among ou pa ien s, including hin co pus cal-
losum, ea s o  helynxsign, and enla ged en icles o  ce ebella 
a ophy.
These cha ac e is ics a e also e iden  in he MRI indings o 
p e iouslydesc ibedSVBPpa ien s(Iqbale al.,2019; Pagnamen a
e al., 2019), indica ing a dis inc pa e n ha could aid in clinical di-
agnosisanddi e en ia e omo he  o mso HSPwi hco puscallo-
sum hinningandea so  helynxsign(Eb ahimi-Fakha ie al.,2021;
Pascual e al., 2019).
The implica ion o  dys unc ional SVBP in inducing s uc u al
cen osome de ec s and p ema u e cen osome sepa a ion s ongly
sugges sac ucial oleo MTde y osina ionin hecen osomecycle.
Dys unc ional cen osomes no only impai he long- e m p oli e a-
i ecapaci ybu also hepola iza iono neu als emcells,leading o
condi ions such as mic ocephaly and neu odegene a i e diso de s
like Pa kinson's disease (Goundiam & Bas o, 2021; Made o-Pé ez
e al., 2018).Thisis u he suppo edby he ac  ha manygenes
associa ed wi h mic ocephaly encode cen osome p o eins (CPAP,
CEP152,CEP135,STIL,andCDK5RAP2)in ol edincen iolebio-
genesis and cen osome ma u a ion (Na eed e al., 2018). P e ious
esea chbyMade o-Pe eze al.has e ealed ha pa hogenicLRRK2
in Pa kinson's disease causes cen osomal pola i y and cohesion
de ici s in bo h di iding and nondi iding cells, esul ing in impai ed
neu i e ou g ow h, cell pola iza ion, and mig a ion (Made o-Pé ez
e al., 2018).In iguingly,down egula iono SVBPhasbeenshown
o dis up  neu onal mig a ion in he de eloping mouse neoco ex
(Pagnamen a e al., 2019). Fu he mo e, cul u ed neu ons lacking
SVBPdisplayedaclea delayinaxondi e en ia ionandse e emo -
phological de ec s, sugges ing ha he b ain a ophy obse ed in
SVBPknockou micelikelyo igina es omabno maldi e en ia ion
and ma u a ion o  de icien  neu ons (Aillaud e  al., 2017). Hence,
wep opose ha dys unc ionalcen osomesandsubsequen inac-
cu a e cy okinesis in mi osis, induced by SVBP mal unc ion, may
dis up p ogeni o p oli e a ion and pola i y o neu al cells, he eby
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