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

Aging Cell. 2025;24:e14355.   | 1 of 15 https://doi.org/10.1111/acel.14355 wileyonlinelibrary.com/journal/acel Received:10May2024 | Revised:30August2024 | Accepted:9September2024 DOI: 10.1111/acel.14355 RESEARCH ARTICLE Altered tubulin detyrosination due to SVBP malfunction induces cytokinesis failure and senescence, underlying a complex hereditary spastic paraplegia Nathalie Launay1,2 | Maria EspinosaAlcantud3 | Edgard Verdura1 | Gorka FernándezEulate4,5 | Jon Ondaro6,7 | Pablo Iruzubieta5,6,7,8 | Maria Marsal9 | Agatha Schlüter1 | Montserrat Ruiz1,2 | Stephane Fourcade1,2 | Agustí RodríguezPalmero1,2,10 | Miren Zulaica6 | Andone Sistiaga7,11 | Garazi Labayru7,11 | Pablo LozaAlvarez9 | Alejandro Vaquero3 | Adolfo Lopez de Munain5,6,7,8 | Aurora Pujol1,2,12 1NeurometabolicDiseasesLaboratory,Institutd'InvestigacióBiomèdicadeBellvitge(IDIBELL),HospitalDuraniReynals,Barcelona,Spain 2CenterforBiomedicalResearchonRareDiseases,(CIBERERU759)MinistryofScienceInnovationandUniversity,Madrid,Spain 3ChromatinBiologyLaboratory,JosepCarrerasLeukaemiaResearchInstitute,Badalona,Spain 4Nord-Est/Ile-de-FranceNeuromuscularReferenceCenter,InstituteofMyology,Pitié-SalpêtrièreHospital,Paris,France 5DepartmentofNeurology,HospitalUniversitarioDonostia,OSAKIDETZA-DepartmentofNeurosciences,UniversityoftheBasqueCpuntry,SanSebastian,Spain 6DepartmentofNeurosciences,InstitutoBiodonostia,SanSebastián,Spain 7CenterofBiomedicalResearchinNeurodegenerativeDiseases(CIBERNED),CIBER,MinistryofScience,InnovationandUniversity,Madrid,Spain 8DepartmentofMedicine,SchoolofMedicine,UniversityofDeusto,Bilbao,Spain 9ICFO-InstitutdeCienciesFotoniques,TheBarcelonaInstituteofScienceandTechnology,Castelldefels,Spain 10PediatricNeurologyUnit,DepartmentofPediatrics,UniversityHospitalGermansTriasiPujol,AutonomousUniversityofBarcelona,Badalona,Spain 11DepartmentofPersonality,AssessmentandPsychologicalTreatmentFacultyofPsychology,UniversityoftheBasqueCountry(UPV/EHU),SanSebastian,Spain 12CatalanInstitutionofResearchandAdvancedStudies(ICREA),Barcelona,Spain ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. ©2024TheAuthor(s).Aging CellpublishedbyAnatomicalSocietyandJohnWiley&SonsLtd. Abbreviations:AD,Alzheimer'sdisease;ALS,amyotrophiclateralsclerosis;CNS,thecentralnervoussystem;CSVS,TheCollaborativeSpanishVariantServer;CTL,control;ER,the endoplasmicreticulum;FBS,foetalbovineserum;FRDA,Friedreichataxia;FTD,Frontotemporaldementia;HSP,HereditarySpasticParaplegias;LOD,combinedlogarithmofodds;MS, multiplesclerosis;MTs,microtubules;PACT-RFP,theRFP-taggedPACTdomain;PBMCs,peripheralbloodmononuclearcells;PD,Parkinson'sdisease;Peric,Pericentrin;PN, Parthenolide;siRNAs,smallinterferingRNAs;SNPs,singlenucleotidepolymorphisms;SVBP,smallvasohibin-bindingprotein;TLL,tubulintyrosineligase;Veh,vehicle;WES,whole exomesequencing;βgal, βgalactosidase. Correspondence AuroraPujol,NeurometabolicDiseases Laboratory, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), HospitalDuraniReynals,GranVia,199, L'Hospitalet de Llobregat, Barcelona 08908,Spain. Email: [email protected] Funding information theSpanishMinistryofEconomyand CompetitivenessMINECO,Grant/Award Number:BES-2015-071251,PID2020- 117284RB-100andSEV-2015-0522; Abstract Senescence,markedbypermanentcellcyclearrestmaycontributetothedeclinein regenerative potential and neuronal function, thereby promoting neurodegenerative disorders.Inthisstudy,weemployedwholeexomesequencingtoidentifyapreviouslyunreportedbiallelicmissensevariantinSVBP(p.Leu49Pro)insixpatientsfrom threeunrelatedfamilies.Theseaffectedindividualspresentwithacomplexhereditaryspasticparaplegia(HSP),peripheralneuropathy,verbalapraxia,andintellectual disability,exhibitingamilderphenotypecomparedtopatientswithnonsenseSVBP mutationsdescribedpreviously.ConsistentwithSVBP'sprimaryroleasachaperone 2 of 15 | LAUNAY et al. 1 | INTRODUCTION Hereditaryspasticparaplegias(HSP)areagroupofinheritedneurodegenerativedisorderscharacterizedbythedegenerationofthe longdescendingaxonsofthecorticospinaluppermotorneurons,resulting in spasticity and weakness in the lower limbs (Fink, 2014).The clinicalandgeneticheterogeneityofHSPreflectstheinvolvementof diverse cellular pathways, encompassing membrane and cargo trafficking, mitochondrial function, organelle shaping, lipid metabolism, and autophagy (Blackstone, 2018; Lo Giudice et al., 2014).Among thesecellularpathways,microtubules(MTs)emergeasacrucialelement due to several factors: (i) their regulation directly influences intracellular transport, (ii) they interact with organelles such as the endoplasmic reticulum (ER) and mitochondria, and (iii) mutations affecting proteins that directly interact with MTs (such as SPAST, KIF5A,KIF1A,REEP1,andREEP2)arepresentinapproximatelyhalf of patients with genetically confirmed HSP (Blackstone, 2018; Lo Giudice et al., 2014). Microtubules are dynamic polymers composed of α/β tubulin dimers that serve as “railways” for motordriven intracellular transport. Theyplayavitalroleinintracellularorganizationandchromosome segregation.MTsexhibithighdynamics,withtheirgrowthandshrinkage regulated by (1) the addition and loss of αand βtubulin subunits, and (2) various types of posttranslational modifications (Janke, 2014). These modifications, such as detyrosination/tyrosination, acetylation, glycosylation, and (poly) glutamylation, collectively form a “tubulincode” that regulates interactions with molecular motors and otherMT-bindingproteins(Janke,2014;McKennaetal.,2023). Patients with loss-of-function mutations in SVBP (small vasohibin-bindingprotein)werereportedtoexhibitsymptomssuch asataxia,intellectualdisability,microcephaly,andmuscularhypotonia (Iqbal et al.,2019; Pagnamenta et al., 2019).SVBPinteracts withVASHproteinsinachaperone-likemanner,servingasacritical factor for the solubility/secretion and detyrosination activity of VASH1andVASH2(Aillaudetal.,2017; Nieuwenhuis et al., 2017). Detyrosinated tubulin is abundantly present in neuronal MTs and otherlong-livedMTpopulations,whereitplaysacrucialroleinaxonaltransportbyfacilitatingthetraffickingofkinesin-1(Konishi& Setou,2009). Defective detyrosination caused proliferative defects during neurogenesis, leading to microcephaly and abnormal behavior (Landskron et al., 2022). Moreover, spindle detyrosination is crucial for the accurate chromosome congression and symmetry breakage duringfemalemeiosis,underscoringtheessentialroleofMTdetyrosination in maintaining genomic stability (Barisic et al., 2015). In this study, we have identified a novel biallelic missense variant inSVBPamongsixindividualsexhibitingspasticparaparesisaccompaniedbysensorimotoraxonalneuropathy,verbalapraxia,epilepsy, andintellectualdisability,therebyexpandingthepreviouslyreported phenotype. Our findings, based on analyses of patientderived fibroblastsandSVBPknockdowncells,revealapivotalroleofSVBPin centrosome cohesion and chromosome segregation. Moreover, our results shed light for the first time on the potential involvement of cellular senescence as a driver of these neurological disorders. 2 | RESULTS 2.1 | Variant identification and clinical features Throughwholeexomesequencing(WES),wehaveidentifiedaconsistent biallelic SVBP variant in six individuals belonging to three unrelated families originating from the same geographic region in Northern Spain. The novel missense variant in the SVBP gene (Chr1:43273140A > G,NM_199342:exon3:c.146 T > C,p.Leu49Pro) is not present in control databases such as ExAC and 1000 genomes,withonlytwoheterozygouscarrierindividualsrecordedin the GnomAD database. Furthermore, this variant is absent from the Collaborative Spanish Variant Server (CSVS), which includes sequence datafrom 1644 unrelated individuals.Allsingle nucleotidepolymorphisms(SNPs)withinthehomozygousgenomicregion sharedbythepatientssequencedbyWES(spanningatleast2.32 Mb) exhibitidenticalgenotypesinthefiveanalyzedpatients(familiesA AssociationASL-HSPand‘LaMaratóde TV3’Foundation,Grant/AwardNumber: 202006-30;InstitutodeSaludCarlosIII, Grant/AwardNumber:CD19/00221and MiguelServetprogramCP11/00080; FundaciónHesperiaandSecretariat for Universities and Research of the MinistryofBusinessandKnowledgeof the Government of Catalonia, Grant/ AwardNumber:2017SGR1206;URDcat programme,Grant/AwardNumber:PERIS SLT002/16/00174;CentreforBiomedical Research on Rare Diseases (CIBERER), Grant/AwardNumber:ACCI19-759; CatalangovernmentagencyAGAUR, Grant/AwardNumber:AGAUR2021-SGR- 01378 necessaryforVASH-mediatedtubulindetyrosination,bothpatientfibroblastswiththe p.Leu49Promutation,andHeLacellsharboringanSVBPknockdownexhibitmicrotubule dynamic instability and alterations in pericentriolar material (PCM) component trafficking and centrosome cohesion. In patient fibroblasts, structural abnormalities in the centrosome trigger mitotic errors and cellular senescence. Notably, premature senescencecharacterizedbyelevatedlevelsofp16INK4,wasalsoobservedinpatient peripheralbloodmononuclearcells(PBMCs).Takentogether,ourfindingsunderscore thecriticalroleofSVBPinthedevelopmentandmaintenanceofthecentralnervous system, providing novel insights associating cytokinesis failure with cortical motor neuron disease and intellectual disability. KEYWORDS centrosome,cytokinesisfailure,HSP,microtubuledetyrosination,senescence,SVBP 14749726, 2025, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/acel.14355 by Universidad Del Pais Vasco, Wiley Online Library on [26/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 3 of 15 LAUNAY et al. andB).Thisobservationsuggeststhatthevariantislocatedwithin thesamehaplotype,indicatingitasafoundervariant.Segregation analysis in all unaffected members of both families supports the recessivemodeofinheritancepreviouslyreportedforSVBP.Thecombined logarithm of odds (LOD) score, considering cosegregation data from all genotyped individuals, reached 2.73. Patientsfromfamilies A,B, and Cpresented at birthorchildhoodwithneurodevelopmentaldelaysandlaterexhibitedintellectual disabilities. Subsequently, they developed a complex spastic paraplegiasyndromeassociatedwithverbalapraxiaandaxonalneuropathy (Figure 1a). The clinical characteristics of all six patients aresummarizedinTable 1. Brain MRIs of patients P2, P4, and P6 wereanalyzed,revealingseveralsharedfeatures.Allthreeexhibited corpus callosum thinning, cerebellar atrophy, and ventriculomegaly (Figure 1b–d).Additionally,twopatientsdisplayedfrontalperiventricularhyperintensitiesonT2imaging,resemblingthe“earofthe lynx”sign(Figure 1c,d). Notably, half of the patients have a history of cancer, all of which are of epithelial origin. 2.2 | SVBP variant alters SVBP expression and impairs MT detyrosination Ourinsilicostructuralanalysisrevealedthatthep.Leu49Prosubstitution may disrupt intramolecular hydrophobic interactions and/ ordestabilizetheconservedα-helicalconformationofSVBP.This conformational change is crucial for its interaction with VASH1 and the detyrosination of the αtubulin peptide (Liao et al., 2019) (Figure 2a).Tovalidatethisprediction,wecotransfectedHeLacells withequimolaramountsofC-terminalFlag-taggedhumanVASH1 andeitherwild-typeorp.Leu49PromutantC-terminalFlag-tagged human SVBP. Subsequently, we analyzed the expression levels of VASH1 and SVBP by Western blot. In contrast to wild-type SVBP,themutantSVBPproteinwasundetectableinthecelllysate (Figure 2b),suggestingpossibledegradation oftheSVBPvariant. Moreover, a significant reduction in three posttranslational forms ofVASH1(42 kDa,36 kDa,and32 kDa)wasobservedincellstransfectedwiththemutantSVBP(Figure 2b), strongly indicating that FIGURE 1 ClinicalfeaturesoffamilieswithanovelSVBPmutation.(a)PedigreeforfamiliesA,B,andC.(b)AxialandsagittalT1MRI sequencesofpatientP2showingdiffusecerebellaratrophy,enlargedventricles,thincorpuscallosum,anddiffusecerebellaratrophy (c)SagittalT1andaxialFLAIRMRIofpatientP4showingcerebellarvermisatrophy,earsofthelynxsign(whitearrow),anasymmetrical ventricleenlargement.(d)AxialT2andsagittalT1MRIsequencesfrompatientP6showingearsofthelynxsign(whitearrow),enlarged ventricles, and vermis cerebellar atrophy. 14749726, 2025, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/acel.14355 by Universidad Del Pais Vasco, Wiley Online Library on [26/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 4 of 15 | LAUNAY et al. TABLE 1 MainclinicalfeaturesoffamiliesA,B,andC. Family A B C Patient P1 P2 P3 P4 P5 P6 Mutation c.146 T > CHom p.Leu49Pro c.146 T > CHom p.Leu49Pro c.146 T > CHom p.Leu49Pro c.146 T > CHom p.Leu49Pro c.146 T > CHom p.Leu49Pro c.146 T > CHom p.Leu49Pro Gender M M F F F F Ethnicity Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Parental Consanguinity No No No No No No First symptoms and age of onset Neurodevelopmental delay since birth Neurodevelopmental delay since birth Neurodevelopmental delay since birth Mild clumsy gait and foot deformity since childhood Neurodevelopmental delay since birth Neurodevelopmental delay since birth Psychomotor development Delayed Delayed Delayed N/A Delayed Delayed Intellectual disability + + + + + + Motorexamination Spasticparaparesis Areflexia Wheelchair-bound(adult) Spasticparaparesis Bradykinesia, distal lower limb weakness Wheelchair-bound(adult) Spasticparaparesis Spasticparaparesis Distal lower limb amyotrophy andweakness,ankleareflexia Spasticparaparesis Distal lower limb weakness,areflexia Ataxic-spasticgait.Brisk reflexesexceptforAchilles reflex(absent) Ataxia ++N/A +−+ Verbalapraxia − +−+ + N/A Epilepsy −+ + N/A +− Aggressivebehavior −+ + − − + Ophthalmologic manifestations −Strabismus,Nystagmus N/A −Nystagmus Nystagmus Other clinical manifestations Hammer toes Adenocarcinomawithhepatic metastasis. Deceasedatage59 years Scoliosis,lumbar hyperlordosis, pes planus, Hammer toes Colonic tubular adenoma Deceasedat52 years Deceasedatage59 years Pes cavus, Hammer toes Feet hypoalgesia and hypopallesthesia Cold, erythematous feet Pes cavus. Hammer toes. Hypopallesthesia. Breast cancer(at51 years) MRI N/A Cerebellar atrophy, ventriculomegaly, middle cerebellar peduncles atrophy. Cavum septum pellucidum persistence N/A Brain atrophy, ventriculomegaly. Earofthelynxsign N/A Cerebellar atrophy, ventriculomegaly, Ear of thelynxsign Nerve Conduction Studies(NCS) N/A Lowerlimbaxonal sensorimotor neuropathy N/A Axonalsensorimotor neuropathy Axonalsensorimotor neuropathy Axonalsensorimotor neuropathy Abbreviations:F,female;M,male;N/A,notavailable;Y,years. 14749726, 2025, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/acel.14355 by Universidad Del Pais Vasco, Wiley Online Library on [26/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 5 of 15 LAUNAY et al. the SVBP mutation impairs VASH1 secretion and/or solubility. ImmunofluorescenceandWesternblotanalysesdemonstrateddecreasedSVBPlevelsinpatientfibroblasts,furtherconfirmingthat thep.Leu49Promutationaffectsproteinstabilityandleadstoits degradation (Figure 2c,d). ToinvestigatethepotentialimpactoftheSVBPmutantonMT detyrosination,weemployedpaclitaxeltoelevatedetyrosinatedtubulinlevelsinfibroblasts.PaclitaxelfunctionsbystabilizingMTsand removing the free α/βtubulin dimers, which serve as substrates for tubulintyrosineligase(TTL)(Protaetal.,2013). Immunofluorescence and Western blot experiments revealed a significant reduction in detyrosinated αtubulin levels in patient fibroblasts compared to controls (Figure 2e,f), providing evidence that the SVBP mutation disruptsMTdetyrosination.Collectively,thesefindingsunderscore thecriticalroleofresidueLeu49inSVBPfortheinteractionwith VASH1,suggestingthatitssubstitutionwithProhampersMTdetyrosination by reducing the abundance of active SVBP-VASH1 heterodimers. FIGURE 2 PathogenicityofSVBPvariantontheVASH1secretionandMTdetyrosinationactivity.(a)Close-upviewsoftheVASH1–SVBP interface,withinteractingresiduesshownassticks.VASH1residuesarecoloredblueandlabeledwithblueletters,whileSVBPresiduesare coloredorangeandlabeledwithorangeletters.(b)HeLacellstransfectedwithvectorsdirectingtheexpressionofFLAG-taggedSVBPWT, SVBPL49PandVASH1,orcombinationsthereof,weresubjectedtoimmunoblotanalysiswithanti-Flagantibody.Totalamountsofβactin wereusedasaloadingcontrol.(c)Control(CTL)andpatient(P2andP4)fibroblastswerestainedwithanti-SVBP(red)andanti-αtubulin (αtub;green)antibodiesandDAPI(blue).Scalebars:10 μm.(d)Control(CTL)andpatient(P2andP4)fibroblastsweresubjectedtoimmunoblot analysisusingtheanti-SVBPantibody.Totalamountsofα-tubulinwereusedasaloadingcontrol.(e)Control(CTL)andpatient(P2and P4)fibroblastsweretreatedwithvehicleorpaclitaxelandstainedwithanti-αtubulin (αtub; red) and antidetyrosinated tubulin (detyrtub;green)antibodiesandDAPI(blue).Scalebars:50 μm.(f)Control(CTL)andpatient(P2andP4)fibroblastsweretreatedwithvehicleor paclitaxelandsubjectedtoimmunoblotanalysisusingantibodiesdirectedagainstdetyrosinated(detyr-tubulin)andα-tubulin.Totalamounts of β-actinwereusedasaloadingcontrol.Therelativeratiosofdetyrosinatedversustotal⍺- tubulin levels are indicated (n = 3). 14749726, 2025, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/acel.14355 by Universidad Del Pais Vasco, Wiley Online Library on [26/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 6 of 15 | LAUNAY et al. 2.3 | SVBP mutant induces MT dynamic instability and centrosome cohesion deficit GiventheestablishedassociationbetweenhighlevelsofMTdetyrosination and centrioles (Janke, 2014;Song&Brady,2015), suggesting apivotalroleofMTdetyrosinationincentrosomestabilizationand integrity,weoptedtoinvestigatetheimpactoftheSVBPmutation on centrosome cohesion. Intriguingly, immunofluorescence analysis revealed premature centrosome separation in patient fibroblasts during interphase. The proportion of cells exhibiting centrosomes separated by >2 μm increased from 4% in controls to >20% in patient cells (Figure 3a). Consistent with these findings, parthenolide, an inhibitorofMTdetyrosination(Freundetal.,2020), induced abnormal centriole separation in nearly 20% of control fibroblasts (Figure 3b), mirroring the observed ratio in patient cells. Subsequently,wequantifiedpericentrinlevelstoevaluatecentrosome integrity. Our analysis revealed a significant reduction in centrosomal pericentrin staining in patient fibroblasts compared to controls (Figure 3a), indicative of disrupted pericentriolar material (PCM) assembly due to the SVBP mutation. Consistently, high-resolution fluorescence microscopy unveiled an increase in peripheral and cytoplasmic pericentrin foci along the MT cytoskeleton in interphase patientfibroblasts,suggestingadefectinMT-dependenttrafficking where PCM components are inadequately transported to centrosomes (Figure S1).Furthermore,weobservedMTdepolymerization and degradation in patient fibroblasts, evident from the punctuated patternofMTstaining(Figure S1).Consequently,weconcludethatthe SVBPvariantinducesMTdynamicinstability,leadingtoalterationsin PCM component trafficking and a deficit in centrosome cohesion. 2.4 | SVBP mutant leads to spindle morphology alteration, cytokinesis failure, and chromosome instability ThePCMservesasacrucialhubforMTnucleationandregulates the number and composition of MTs throughout the cell cycle (Zimmermanetal.,2004). Consistently, studies employing small interferingRNAs(siRNAs)targetingbothpericentrinisoforms(Aand B)havedemonstratedareductioninastralMTsandmitoticspindle lengthinSAOScells(Zimmermanetal.,2004). Correspondingly, our analysisrevealedthatpatientfibroblastsexhibitedshortermitotic spindlesandimpairedformationofMTasters(Figure 3c), suggesting thattheSVBPmutationaltersMTnucleation.Theseobservations align with a recent study demonstrating that depletion of vasohibin inU2OScellsresultedinshortermitoticspindles,accompaniedbya significantreductioninastralMTs(Liaoetal.,2019). Remarkably, our immunofluorescence experiments uncovered that 80% of patient fibroblasts experienced cytokinesis failure, evidenced by intercellular cytoplasmic bridges and the presence of binucleated cells (Figure 3c,d). Furthermore, the elevated rate of micronucleus formation (~10%) observed in patient fibroblasts compared to control cells indicated compromised chromosome stability (Figure 3d). Collectively, these findings suggest a crucial role ofSVBPinensuringaccuratemitosisandestablishalinkbetween SVBPdeficiencyandalterationsinspindlemorphology,cytokinesis failure, and chromosome instability. 2.5 | CRISPR/Cas9mediated knockout of SVBP in HeLa cells replicates centrosome cohesion deficit and mitosis abnormalities ToconfirmthattheobserveddefectsinpatientfibroblastswereindeedattributedtoSVBPdeficiency,weemployedtheCRISPR/Cas9 techniquetogenerateanSVBPknockoutinHeLacells(Figure S2). Consistent with our earlier findings, we noted a significant increase in centrosome splitting and a reduction in centrosomal pericentrin staining during interphase in SVBP-KO HeLa cells compared to wildtype cells (Figure 4a).Similaroutcomeswereobservedwhen SVBP-KO HeLa cells were transfected with an expression vector encoding the RFP-tagged PACT domain (PACT-RFP) (Figure 4b), a conservedmotifknowntotargetcentrosomesbytaggingAKAP450 andpericentrin(Gillingham&Munro,2000).Additionally,SVBP-KO FIGURE 3 SVBP mutant induces centrosome cohesion deficit, mitotic spindle abnormalities, cytokinesis failure, and chromosome instability.(a)Interphasecontrol(CTL)andpatient(P2andP4)fibroblastswerestainedwithalpha-tubulin(αtub; green) and antipericentrin (Peric.;red)antibodiesandDAPI(blue)atcellpassages<10 (inset shows enlargement of the PCM, asterisk (*) mark the position of centrosome).Scalebars,10 μm.Percentageofcontrol(CTL)andpatientfibroblasts(P2andP4)withsplitcentrosomesandcentrosomal pericentrinintensitywerequantified.n ≥ 50cells/100centrosomes;mean ± SD;*p < 0.05;**p < 0.01byone-wayANOVAwithTukeyposthoc tests.(b)Representativepictureofcontrolfibroblasts(CTL)treatedwithvehicle(Veh.)orparthenolide(PN,5 μM,24 h)andstainedwithanti- pericentrin(Peric.;green)antibodiesandDAPI(blue)(whitearrowsindicatethepositionofthecentrosome).Scalebars,20 μm. Percentage of cellstreatedornotwithparthenolide(PN)wasquantified.n ≥ 20cells/40centrosomes;mean ± SD;**p < 0.01bytwo-tailedt test. (c) Control (CTL)andpatient(P2andP4)fibroblastswerestainedatdifferentmitoticphases(prophase,metaphase,andcytokinesis)withalpha-tubulin (α-tub;green)andanti-pericentrin(Peric.;red)antibodiesandDAPI(blue)atcellpassages<10 (inset shows enlargement of the PCM, asterisk (*)markthepositionofcentrosome).Scalebars,5 μm.Mitoticdefects(spindleabnormalitiesandcytokinesisdefects)werequantifiedin control(CTL)andpatient(P2andP4fibroblasts).n ≥ 10cellsforeachmitoticphase;Mean ± SD;*p < 0.05;*** < p < 0.001byone-wayANOVA withTukeyposthoctests.(d)Representativepicturesofcontrol(CTL)andpatient(P2andP4)fibroblastsatcellpassages>10 stained with antiαtubulin (α-tub;green)andDAPI(blue),showingthepresenceofmicronucleiandbinucleatedcells(whitearrows)(insertshows enlargementoftheindicatedarea).Scalebars,50or10 μm.Thepercentageofcellswithmicronucleiandbinucleatedcellswerequantified. n ≥ 50;Mean ± SD;*p < 0.05;**p < 0.01byone-wayANOVAwithTukeyposthoctests. 14749726, 2025, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/acel.14355 by Universidad Del Pais Vasco, Wiley Online Library on [26/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 7 of 15 LAUNAY et al. 14749726, 2025, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/acel.14355 by Universidad Del Pais Vasco, Wiley Online Library on [26/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 8 of 15 | LAUNAY et al. FIGURE 4 CRISPR/Cas9knockoutofSVBPinHeLacellsinducescentrosomeabnormalitiesandaberrantmitosis.(a)InterphaseWild-type (WT)andSVBP-KO(SVBPko) HeLa cells were stained with alphatubulin (α-tub;green)andanti-pericentrin(Peric.;red)antibodiesandDAPI (blue)(insetshowsenlargementofthePCM;theasterisk(*)marksthepositionofthecentrosome).Scalebars,10 μm. Percentage of wildtype(WT)andSVBP-KO(SVBPko)HeLacellswithsplitcentrosomesandcentrosomalpericentrinintensitywerequantified.n ≥ 30cells/60 centrosomespercondition;Mean ± SD;**p < 0.01bytwo-tailedttest.(b)Representativeimageofwild-type(WT)andSVBP-KO(SVBPko) expressingPACT-RFP(asterisk(*)markthepositionofcentrosome).Twenty-fourhoursaftertransfection,cellswerefixedandstainedfor DNAcontent(DAPI;blue).Scalebars,10 μm.Percentageofwild-type(WT)andSVBP-KO(SVBPko)HeLacellsexpressingPACT-RFPwith splitcentrosomeswasquantified.n ≥ 10cells/20centrosomespercondition;Mean ± SD;**p < 0.01bytwo-tailedttest.(c)Wild-type(WT) and SVBP-KO(SVBPko) HeLa cells were stained at different mitotic phases (prophase, metaphase, and cytokinesis) with alphatubulin (αtub; green)andanti-pericentrin(Peric.,red)antibodiesandDAPI(blue)(insetshowsenlargementofthePCM;theasterisk(*)markstheposition ofthecentrosome).Mitoticdefects(spindleabnormalitiesandcytokinesisdefects)inwild-type(WT)andSVBP-KO(SVBPko) HeLa cells werequantified.n ≥ 10cellsforeachmitoticphase;Mean ± SD;*p < 0.05;*** < p < 0.001bytwo-tailedt test. (d) Representative pictures of wild-type(WT)andSVBP-KO(SVBPko) HeLa cells stained with antialphatubulin (α-tub,green)andDAPI(blue)atpassages>10, showing the presenceofmultinucleatedcells(whitearrowsindicatemultinucleatedcells);insertshowsenlargementoftheindicatedarea.Scalebars,50 or10 μm.Thepercentageofmultinucleatedandmicronucleatedwild-type(WT)andSVBP-KO(SVBPko)HeLacellswerequantified.n ≥ 50; Mean ± SD;*p < 0.05;**p < 0.01bytwo-tailedt test. 14749726, 2025, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/acel.14355 by Universidad Del Pais Vasco, Wiley Online Library on [26/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 9 of 15 LAUNAY et al. HeLa cells displayed a shorter mitotic spindle size, reduced astral MTs, cytokinesis failure, and increased micronuclei formation (Figure 4c,d), collectively mimicking the phenotype observed in patient fibroblasts. 2.6 | SVBP mutant induces cell cycle arrest and senescence Centrosome alterations or mitotic errors typically activate the apoptosis machinery through p53 (Imreh et al., 2016).Therefore,we examined p53 expression in patient's fibroblasts by Western Blot analysis.Whilenochangewasobservedinearlycellpassages(<10), the decreased protein and mRNA expression levels of p53 in patient fibroblasts at cell passage >10 suggested the progressive inactivation of the p53dependent apoptosis response (Figure 5a,b; Figure S3a,b). Consistently, flow cytometry apoptosis assays showed a slight increase in apoptotic cell levels in patient fibroblasts at late passage compared to controls (Figure 5c), indicating that apoptosis isaminoroutcomeinSVBPmutantcells. WetheninvestigatedtheimpactoftheSVBPmutationoncell proliferation. EdU incorporation and histone H3-Serine 28 phosphorylation levels (H3Ser28P)wereusedtomonitor S phaseprogression and the lateG(2)/M status of cells, respectively. Flow cytometryanalysisrevealedequivalentEdUincorporationinearly passages of control and patient fibroblasts (Figure S3c). However, by cell passages >10, patient fibroblasts displayed decreased EdU incorporation compared to control fibroblasts (Figure 5d,e).Similarly, we foundthat the phosphorylationofH3Ser28 decreased significantly from cell passages >10 in patient fibroblasts (Figure 5f,g; Figure S3d).Therefore,ourresultsindicatedthatpatientfibroblasts progressively arrested in G1/G0 phase. Toassesssenescencelevels,wefirstperformedβgalactosidase (βgal) staining, which revealed increased senescenceassociated βgalactosidase activity in patient cells, correlated with an increasing number of cell passages (Figure 5h).mRNAlevelsoflmnb1 were decreased while p21 levels were raised, consistent with augmented βgalactosidase staining with increasing cell passages (Figure 5i). Remarkably, we observed a very high expression level of IL6, a senescence-associated inflammatory mediator molecule (SASP) in lowpassage patient fibroblasts (Figure 5i, Figure S3c). Moreover, we observed a correlation between increased H2A histone (γ- H 2 A X )  phosphorylation,amarkerofDNAdamage(Biranetal.,2017), and the accumulation of senescent cells in patients (Figure 5j; Figure S3a). Tocorroborateourinvitrofindings,weassessedtheexpression of CDKN2A/p16INK4 in peripheral blood mononuclear cells (PBMCs) from both control individuals and patients. p16INK4 serves as a wellestablishedbiomarkerofsenescenceinvarioustissues,includingT cells,whereitrestrictstheirreplicativecapacity(Y.Liuetal.,2009). Remarkably, the relative expression of CDKN2A/p16INK4 was significantlyhigherinpatientPBMCs(3.4 ± 0.1fold)comparedtocontrols (Figure 5k), aligning with the senescence phenotype observed in patient fibroblasts. 3 | DISCUSSION Using whole-exome sequencing (WES), we uncovered a novel bi- allelicSVBPvariantinsixindividualsfromthreeunrelatedfamilies. Our functional investigations provide compelling evidence suggestingthatcentrosomeabnormalitiesandsubsequentsenescencemay serveasunderlyingdriversofHSP.Whenconsideringthefindingsof IqbalandPagnamenta(Iqbaletal.,2019; Pagnamenta et al., 2019), alongwithourcohortofsixpatients,acollectionofcommonclinical features emerges. All patients exhibited intellectual disability along with delayed gross motor and speech development. Notably, patients harboring the missense p.Leu49Pro variant manifested a motor-predominantphenotypecharacterizedbycomplexHSPand axonalneuropathy,significantlyimpactingtheirmobilitycompared to previously reported individuals who presented with hypotonia, dysmorphia,andseverecognitiveimpairment.Theearlieronsetand more severe neurodevelopmental features observed in the previously reported cases could be attributed to their lossoffunction variants. From a neuroimaging perspective, several common features were observed among our patients, including thin corpus callosum, ears of thelynxsign, and enlarged ventricles or cerebellar atrophy. These characteristics are also evident in the MRI findings of previouslydescribedSVBPpatients(Iqbaletal.,2019; Pagnamenta et al., 2019), indicating a distinct pattern that could aid in clinical diagnosisanddifferentiatefromotherformsofHSPwithcorpuscallosumthinningandearsofthelynxsign(Ebrahimi-Fakharietal.,2021; Pascual et al., 2019). The implication of dysfunctional SVBP in inducing structural centrosome defects and premature centrosome separation strongly suggestsacrucialroleofMTdetyrosinationinthecentrosomecycle. Dysfunctional centrosomes not only impair the longterm proliferativecapacitybutalsothepolarizationofneuralstemcells,leadingto conditions such as microcephaly and neurodegenerative disorders like Parkinson's disease (Goundiam & Basto, 2021; Madero-Pérez et al., 2018).Thisisfurthersupportedbythefactthatmanygenes associated with microcephaly encode centrosome proteins (CPAP, CEP152,CEP135,STIL,andCDK5RAP2)involvedincentriolebiogenesis and centrosome maturation (Naveed et al., 2018). Previous researchbyMadero-Perezetal.hasrevealedthatpathogenicLRRK2 in Parkinson's disease causes centrosomal polarity and cohesion deficits in both dividing and nondividing cells, resulting in impaired neurite outgrowth, cell polarization, and migration (Madero-Pérez et al., 2018).Intriguingly,downregulationofSVBPhasbeenshown to disrupt neuronal migration in the developing mouse neocortex (Pagnamenta et al., 2019). Furthermore, cultured neurons lacking SVBPdisplayedacleardelayinaxondifferentiationandseveremorphological defects, suggesting that the brain atrophy observed in SVBPknockoutmicelikelyoriginatesfromabnormaldifferentiation and maturation of deficient neurons (Aillaud et al., 2017). Hence, wepropose thatdysfunctionalcentrosomesandsubsequentinaccurate cytokinesis in mitosis, induced by SVBP malfunction, may disrupt progenitor proliferation and polarity of neural cells, thereby 14749726, 2025, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/acel.14355 by Universidad Del Pais Vasco, Wiley Online Library on [26/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License