Yon Novel, Ataksik Modèl sourit Ataksya Telangiectasia ki te koze pa yon mitasyon san sans ki enpòtan nan klinik (Pati 1)

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Résumé

Ataksya Telangiectasia (AT) ak Ataksya ak Ocular Apraxia Kalite 1 (AOA1) se maladi newolojik devastatè ki te koze pa mitasyon nil nan jèn estabilite genomic, AT mutated(ATM) ak Aprataxin (APTX), respektivman. Konpreyansyon mekanistik nou ak repètwa terapetik pou trete maladi sa yo manke anpil, an gran pati akòz echèk modèl bèt anvan yo ak mitasyon nil menm jan an pou refè pèt karakteristik kowòdinasyon motè (sa vle di, ataksya) ak domaj serebeleux ki asosye yo. Pa ogmantejenotoksikestrès atravè ensèsyon nan mitasyon nil nan tou de jèn yo Atm (sen sans) ak Aptx (knockout) nan menm bèt la, nou te pwodwi yon modèl sourit roman ki pou premye fwa devlope yon fenotip ataksik progresivman grav ki asosye ak atrofi molekilè serebeleux. kouch. Nou jwenn pwopriyete byofizik nan newòn Purkinje serebeleux yo siyifikativman perturbe (egzanp, kapasite manbràn redwi, pi ba papòt potansyèl aksyon, elatriye), pandan y ap pwopriyete nan entrain sinaptik rete lajman chanje. Sa yoperturbasyonsiyifikativman chanje Purkinie newòn aktivite neral, ki gen ladan yon rediksyon pwogresif nan aksyon espontane potansyèl tire

frekans lan ki korelasyon ak tou de atrofi serebeleux ak ataksya sou premye ane nan lavi bèt la. Doub sourit mutan tou montre yon gwo predispozisyon pou devlope kansè (timom) ak anomali iminitè (enfimite devlopman thymocyte bonè ak spirasyon selil T), sentòm karakteristik AT. Anfen, lè nou mete yon mitasyon nil ki gen rapò ak san sans nan Atm, nou demontre ke ti molekil Read-Through (SMRT) konpoze ka retabli pwodiksyon ATM, ki endike potansyèl yo kòm yon fiti AT terapetik.

Klike la a pou aprann plis sou Cistanche

1.0 Entwodiksyon

Ataxia Telangiectasia (AT) se yon bagay ki ra (1 nan ~ 100,000)(Swift et al. 1986), yon maladi jenetik otozomal resesif ki karakterize pa predispozisyon kansè, defisyans iminitè, ak yon ataksya grav pwogresif ak grav ki lye. nan atrofi serebeleux (Rothblum-Oviatt et al. 2016; Boder and Sedgwick 1958; Levy and Lang 2018). Pasyan AT yo anjeneral mouri nan dezyèm oswa twazyèm deseni yo nan lavi yo (Crawford et al.2006) nan kansè lenfatik, enfeksyon respiratwa, oswa konplikasyon nan ataksya - malerezman, sivivabilite pa te chanje dramatikman depi ane 1950 yo (Micol et al.2011; Rothblum-). Oviatt et al.2016). Pandan ke pwogresyon maladi ak kòz lanmò yo varye anpil atravè pasyan yo, n bès pwogresif trè pénétrant nan kowòdinasyon motè rapòte kòm gen pi gwo enpak negatif sou kalite lavi yon pasyan (Jackson et al.2016). Swen an jeneral se palyatif, ki dirije pou diminye, limite, oswa elimine kansè oswa enfeksyon. Pa gen okenn terapi alontèm ki disponib pou trete ataksya a ak malfonksyònman serebeleux ki asosye ak atrofi. AT se koze pa defisi oswa malfonksyònman nan ATM la (AT mitasyon) pwoteyin (Savitsky et al. 1995). Kodon tèminasyon prematire (PTC) ki lakòz mitasyon istwa san sans reprezante jiska mwatye nan ka li te ye, ak mitasyon missense ak sipresyon tou kontribye (Concannon and Gatti 1997; Sandoval et al. 1999). ATM se yon serin/treonin kinaz fanmi PIKK ki jwe yon wòl kle nan repons domaj ADN (DDR), pwoteje selil yo kont dè dizèn de milye blesi ADN ki fèt chak jou (Lindahl and Barnes 2000; Kastan and Bartek 2004; Shiloh and Ziv). 2013).Nan fòm monomerik aktif la, ATM fosforil plizyè pwoteyin kle ki sispann pwodiksyon nouvo ADN (arestasyon sik selil) (Ando et al.2012), ak Lè sa a, depann sou gravite domaj la, inisye reparasyon ADN oswa selil pwograme. lanmò (sa vle di, apoptoz) (Ando et al.2012; Rashi-Elkeles et al.2006). Yo te idantifye plizyè sib chemen DDR en ATM, tankou p53, CHK2, BRCA1, SMC1, ak NBS1(Matsuoka et al.2007).Wòl ATM nan reparasyon ADN enplike tou nan devlopman nòmal sistèm iminitè, kote li pwopoze pou kontribye. nan rekonbinasyon nan splicing ADN natirèl ki fèt pandan reyajman jèn nan spirasyon lenfosit T ak B (Chao, Yang, and Xu 2000; Matei, Guidos, and Danska 2006; Vacchio et al.2007; Schubert, Reichenbach, and Zielen 2002). ). Malgre ke wòl li yo toujou ap parèt, ATM te enplike tou nan omeyostaz estrès oksidatif (Guo et al. 2010) ak mitofaji (Valentin-Vega and Kastan 2012; Pizzamiglio, Focchi, and Antonucci 2020). Li pa klè poukisa ATM deficiency lakòz ataksya, men li se lwen sèlman pwoteyin DDR ki lye ak ataksya, kòm Aprataxin (APTX) (Aicardi et al.1988), Meyotic recombination 11 homolog 1 (MRE11) (Sedghi et al.2018), Nibrin (NBS1) (van der Burgt et al.1996), Senataxin (SETX) (Moreira et al.2004) , ak Tyrosyl-DNA Phosphodiesterase 1 (TDP1) (Takashima et al. 2002) lè absan oswa disfonksyonèl ka lakòz ataksya serebeleux ki gen rapò. Sa a sijere ke karakteristik newolojik sendwòm enstabilite genòm gen yon kòz komen ki kache, byenke sa a poko demontre mekanikman (McKinnon 2009; Rass, Ahel, and West 2007). Yon gwo faktè ki limite kapasite nou pou defini poukisa pèt pwoteyin DDR, tankou ATM, afekte serebelo a oaza epi lakòz ataksya pwogresif se mank de yon modèl bèt ki refè sentòm newolojik sa yo (Lavin 2013). Plizyè modèl rat AT yo te kreye pandan plizyè ane ki sot pase yo lè yo mete mitasyon jèn ki lakòz malfonksyònman pwoteyin (mank aktivite kinaz) oswa defisi konplè (Herzog et al.1998; Xu and Baltimore 1996; Elson et al.1996; Spring et al. 2001; Campbell et al.2015; Quek et al.2016; Tal et al. 2018; Lavin 2013); yon minipig te tou dènyèman rapòte (Beraldi et al. 2017). Sepandan, pa gen okenn devlope ataksya pwogresif aklè ak malfonksyònman serebeleux ak atrofi ki rezime maladi imen an, menmsi lòt aspè nan maladi a tankou kansè tiwoyid, lakòz, ak anomali iminitè yo devlope. Li rete klè poukisa modèl bèt sa yo anvan yo pa montre fenotip ataksik pwogresif la (Lavin 2013). Li posib ke konpansasyon molekilè espesifik espès nan sourit bay èkse oswa chemen altènatif minimize efè deficiency ATM nan sèvo a (El-Brolosy and Stainier 2017). Li posib tou ke vi ki pi kout nan modèl anvan yo (Barlow et al. 1996) se twò kout pou mekanis stochastic yo kondwi malfonksyònman serebeleux ak atrofi akimile ak enpak sou konpòtman motè. Lòt defi gen ladan manipilasyon jenetik potansyèlman koule ki lakòz nivo ki ba nan pwoteyin ATM oswa fragman aktif ak aktivite kinaz rezidyèl, kidonk limite neropatoloji (Li et al. 2011). Enpak la nan manke tankou yon modèl bèt enpòtan genyen

te siyifikatif, sevè limite etid eksperimantal nan idantifye mekanis selilè ak molekilè ak anpeche devlopman pre-klinik ak tès nan terapetik ki nesesè anpil. Nou teste isit la si ogmante estrès jenotoksik, lè nou mete mitasyon nil nan pa sèlman jèn Atm, men tou, jèn Aptx ki gen rapò, mennen nan yon modèl sourit ki pi reprezantan ki montre malfonksyònman serebeleux, atrofi, ak devlopman ataksya pwogresif. Nou te chwazi anplis frape Aptx paske deficiency li lakòz yon twoub tankou AT nan imen yo rele ataksya ak apraksya okilè tip 1 (AOA1), which does not feature A-T's other system defects that could increase the potential for prenatal lethality or early death (e.g., immunodeficiency and cancer predisposition)(Coutinho P 2002).Moreover, APTX is a phosphodiesterase involved in DNA reassembly after double-and single-stranded repair, and has a function downstream of—but not directly regulated by or related to—ATM (Gueven et al.2004; Schellenberg, Tumbale, and Williams 2015; Ahel et al.2006).We hypothesized that the functional expression of both proteins would have an additive effect and induce neurological dysfunction. Our results indeed demonstrate that mice deficient in ATM and APTX develop cerebellar dysfunction, atrophy, and progressive and profound ataxia, while mice deficient in either protein alone do not Additionally, double mutants displayed other characteristic symptoms of A-T, including defects in immune maturation and a high incidence of cancer (thymomas), making it the most representative model, from a phenotypic standpoint to date. Finally, we designed this new mouse model to test our recently developed Small Molecule Read-Through Compounds(SMRT)that enable translation through premature termination codons(Du et al.2013). Thus, we inserted a premature termination-causing nonsense mutation (103C>T) nan jèn nan Atm komen nan yon gwo fanmi nan North Afriken pasyan AT (Gilad, Bar-Shira, et al.1996). Mitasyon sa a rezilta nan yon kodon tèminasyon twò bonè (PTC) nan sa ki ta nòmalman asid amine 35 ak pèt tradiksyon ATM. Isit la, nou rapòte eksperyans prèv prensip ki montre ke mitasyon jenetik ki enpòtan nan klinik ki enkòpore nan modèl AT sourit yo kapab li nan konpoze e konsa apwopriye pou tès preklinik nan konpoze SMRT.

2.0 Rezilta yo

2.1 Creation of a new A-T mutant mouse model expressing a clinically relevant nonsense mutation To create a more clinically relevant mouse model of A-T we used a gateway recombination cloning and site-directed mutagenesis method to recapitulate a c.103C>T(p.R35X) mutation in the ATM gene found in a large population of North African A-T patients(Fig.1A and Methods)(Gilad, Khosravi, et al. 1996). The insertion of thymine in place of cytosine at this site in exon 3 results in a premature termination codon(PTC)-causing nonsense mutation in the ATM gene. Since the c.103C>T mitasyon rezilta nan diferan PTC nan jèn imen an konpare ak sourit Atm lajèn-TGA vs. TAG, respectively—we created two different mice by exchanging the mouse Atm exon 3 with either a human or mouse exon 3 variant with the c.103C>T mutation(Fig.1B). In the human variant, a 103C>T mutation of the mouse codon, where the arginine(R) encoding codon(CGA) becomes a TGA stop codon, results in a mouse we denote as AtmR35×(officially Atm'm103cAc)rGAMag). In the mouse variant, the c.103C>Mitasyon T transfòme glutamin (Q)-kodaj kodon CAG nan yon kodon sispann TAG epi li deziye Atm235×(ofisyèlman Atmm1.103c) tan). Prezans PTC a lakòz yon pèt ekspresyon ATM, swa redwi apeprè mwatye nan eterozigòt ki eksprime yon kopi nòmal sourit la.

oswa Atm Q35v35×) (Fig. oswa Atm 835×), oswa konplètman nan omozigòt la (Atm?35×R35×Atm jèn (Atm35× plis 1C).

Atm35x7R35×; Aptx ^ (doub mutan) sourit yo te kreye pa premye kwaze yon sèl mutan AtmF3xR3x (konjenik sou background nan C57BL / 6J) ak Aptx' (melanje C57BL / 6J ak 129 background) sourit sourit Aptx *. F1-5 littermate Atm"; Lè sa a, sourit Aptx* yo te kwaze pou jenere eterozigòt Atm nan fatra yo pou kreye ase kantite jenotip eksperimantal yo vle ak kontwole pou detèmine kijan pèt diferan kantite ATM ak APTX afekte fenotip bèt la ( Fig.1D).

Menm jan ak modèl AT sourit anvan ATM-defisi, deficiency ATM oswa APTX pou kont li pa te lakòz sourit ak ataksya (Videyo 1 ak 2). Sepandan, defisi nan tou de pwoteyin (Atm35k35x; Aptx ') rezilta nan devlopman nan yon fenotip grav ak pwogresif ataksik (Fig.1E, Videyo 3 ak 4).

2.2 ATM-defisi sourit yo te bese sivivabilite ak yon ensidans segondè nan timom

Nou evalye sante jeneral ak devlopman kontwòl ak sourit eksperimantal eksprime diferan nivo ATM ak APTX (Fig.2). Nou te jwenn ke Atm735xR35×; Sourit aptx yo te grandi ~ 55 pousan pi dousman epi yo te rive jwenn pwa plato estime ki te ~ 35 pousan mwens pase jenotip kontwòl (log-rank, n=21 a 40,p<0.0001; fig.="" 2a).="" these="" differences="" in="" weight="" were="" a="" postnatal="" phenomenon,="" as="" no="" significant="" weight="" differences="" were="" detected="" just="" after="" birth="" (p8)="" across="" all="" genotypes(1-way="" anova,n="5" to="" 23,="" p~0.23).="" adolescent="" double="" mutant="" mice="" at="" postnatal="" day="" 45(p45)weighed="" on="" average="" 30%="" less="" than="" male="" mice="" [double="" mutant:14.4±1.0g="" (n="13)vs." wildtype:20.2±0.5="" g(n="16),"><0.0001] and="" 25%less="" in="" females="" [double="" mutant:12.7±0.6g="" (n="17)" vs.wildtype:17.0±0.2g(n="15)," test,=""><0.0001; fig.="" 1a].differences="" across="" the="" control="" genotypes="" were="" observed,="" but="" they="" were="" small="" and="" not="" consistent="" across="" time="" points="" or="" sex="" and="" therefore="" judged="" to="" not="" be="" physiologically="" relevant="" (fig.="" 2a).="" survivability="" of="" the="" atm735xr35×;="" aptx*="" mice="" was="" significantly="" reduced="" compared="" to="" atm*;="" aptx*="" mice,="" with="" 53%="" of="" mice="" still="" alive="" at="" 400="" days="" of="" age,="" compared="" to="" 97%="" of="" atm*;="" aptx*="" mice="" at="" the="" same="" time="" point="" (fig.2b).atm="" deficiency="" alone="" was="" sufficient="" to="" reduce="" survivability;="" as="" compared="" to="" atm*;="" aptx*="" mice,="" both="" atm35xr35×;="" aptx*="" and="" atm735x/r35x;="" aptx*="" mice="" had="" significantly="" reduced="" survivability="" [42%,log-rank,xu5="13.49,p=0.0002" and="" 52%,log-rank,xu,53)=""><0.0001,respectively]. no="" significant="" difference="" in="" survivability="" between="" atm-deficient="" mice="" with="" partial="" or="" complete="" aptx="" deficiency="" was="" detected="" [log-rank,="" x(2.85)="1.01,p=0.6]." conversely,="" mice="" harboring="">

least one functional copy of the Atm gene had normal survivability, regardless of whether they expressed APTX or not [log-rank, X(3 131)=3.08, p=0.4]. No significant difference between male and female mice was observed, and thus data were pooled [log-rank, p>0.4 pou tout konparezon par; Fig.2-fig.S1B]. Anjeneral, yon tyè nan sourit ki gen deficiency ATM te mouri nan konplikasyon ki gen rapò ak gwo kansè timik (timom) yo te jwenn nan kavite thoracic la (Fig.2C). Prezans oswa absans APTX pa te gen enpak sou prévalence kansè, ak sourit ki gen omwen yon transkripsyon Atm yo te san kansè jiska omwen P400. An jeneral, defisi ATM men pa APTX te gen efè grav sou sante ak sivivabilite sourit yo.

2.3 Tou de deficiency ATM ak APTX yo nesesè pou pwodwi pwogresif malfonksyònman motè

Devlopman pwogresif ataksya grav se yon karakteristik karakteristik AT ki rezime nan Atm la? 35/R35x; Sourit Aptx 'men pa youn nan lòt jenotip kontwòl nou teste yo. An jeneral, nou jwenn defisi kowòdinasyon motè parèt ant 210 ak 400 jou apre nesans nan Atm735Xk3x; Aptxmice epi pa jwenn okenn prèv ataksya nan sourit ak omwen yon kopi jèn Atm oswa Aptx (Fig.3A, B). Pou tès poto vètikal la, Atm735xR3x×; Sourit Aptx yo te pran de fwa plis tan pou yo desann nan P400 konpare ak Atm*;Aptx*,Atm*; Aptx',Atm735xR35×; Aptx*, oswa Atm35×*; Sourit Aptx ' [Gason: 29.1±0.9s(n=3) vs.7.5±0.4s(n=12),12.5±2.5s(n{=9),9.2±0.9s (n=10),8.6±0.9s(n{=11),1-fason ANOVA,F4.40=19.9,p<0.001;female:19.0±4.0s(n=4)was.7.5±0.4s(n=12),7.8±0.4s(n=10),10.5±1.2s(n=6), 8.2±0.5="" s(n="8),1-way" anova,="" f4,="" 35="13.9,"><0.0001]. an="" examination="" of="" gait="" indicated="" thatatm?35×r35×;="" aptx^="" mice="" at="" p400,="" but="" not="" p210="" need="" additional="" stabilization="" during="" ambulation,="" as="" they="" spend="" twice="" as="" much="" time="" with="" 3="" paws,="" rather="" than="" the="" normal="" 2,="" in="" contact="" with="" the="" ground="" as="" they="" walk="" across="" the="" gait="" analysis="" platform="" [male:="" 56.2="" vs.="" 26.4="" to="" 32.2="" %,1-way="" anova,="" f4.54)="14.3,"><0.0001; female:="" 58.4="" vs.18.9="" to="" 28.8="" %,1-way="" anova,="" f3.178)="95.5,"><0.0001; fig.3b].="" atm35xr35×;="" aptx'="" also="" display="" a="" slower="" cadence="" and="" average="" speed="" across="" the="" platform="" compared="" to="" all="" other="" genotypes="" at="" p400="" [cadence,="" male:9.5="" vs.="" 13.3="" to="" 15.9="" steps/s,="" 1-way="" anova,="" f3.204)=""><0.0001;female: 9.1="" vs.14.2="" to="" 15.9="" steps/s,1-way="" anova,f,204)="39.7,"><0.0001;speed, male:8.8="" vs.22="" to="" 26="" cm/s,1-way="" anova,="" f4.50)=""><0.0001;female: 58.4vs.18.9="" to="" 28.8="" cm/s,1-way="" anova,="" f3.178)="39.7,"><0.0001;fig.3b; fig.3-fig.="" s1].="" this="" difference="" in="" speed="" and="" cadence="" is="" unlikely="" to="" be="" caused="" by="" the="" animal="" size,="" as="" there="" are="" no="" significant="" differences="" in="" these="" parameters="" at="" earlier="" time="" points="" when="" the="" difference="" in="" size="" is="" significant(fig.2a).="" these="" observations="" across="" the="" two="" behavioral="" tests="" were="" found="" in="" both="" male="" and="" female="" mice="" at="" each="" of="" their="" respective="" time="" points,="" consistent="" with="" the="" lack="" of="" sex="" differences="" observed="" in="" a-t="" patients.="" we="" further="" examined="" behavioral="" differences="" between="" the="" atm735xr35x;="" aptx'="" and="" atm*;="" aptx*="" mice="" using="" a="" standardized="" set="" of="" experimental="" procedures="" used="" to="" phenotype="" genetically="" modified="" mice="" (i.e.,="" sherpa;="" fig.3c;="" fig.3-fig.s1)(rogers="" et="" al.1997).="" we="" first="" detected="" differences="" in="" motor="" function="" at="" p8,="" where="" atm35xr35×;aptx'="" mice="" took="" 3-4="" times="" longer="" on="" average="" to="" right="" themselves="" compared="" to="" atm*;aptx*mice="" [male:="" 6.4±1.1="" s(n="24)vs.1.5±0.1" s(n=""><0.0002;female: 11.1±1.9s(n="21)was.2.4±0.3s" (n=""><0.0002;fig.3c bottom].at="" 30="" days="" of="" age,="" we="" detected="" x;="" aptx*="" and="" atm*+;="" aptx**="" mice="" in="" behavioral="" tests="" that="" significant="" differences="" between="" atm735×r35×;="" qualitatively="" measured="" body="" position="" and="" spontaneous="" activity(fig.3c).="" striking="" differences="" in="" atm35×r35×;="" aptx^'="" compared="" to="" atm*;="" aptx*"="" mice="" were="" observed="" at="" p400,="" especially="" for="" behaviors="" related="" to="" movement,="" including="" locomotor="" activity,="" body="" position,="" and="" gait(fig.3c).="" the="" results="" from="" this="" battery="" of="" tests="" demonstrate="" that="" atm35×r35x;="" aptx*mice="" develop="" a="" severe="" change="" in="" behavior="" by="" p400,="" consistent="" with="" purely="" visual="" observations="" of="" significant="" motor="" coordination="" deficits="" in="" the="" mice="" up="" to="" this="" time="" point.="" importantly,="" we="" do="" not="" find="" any="" significant="" differences="" between="" the="" other="" control="" genotypes,="" including="" atm35×;="" aptx*="" mice="" that="" express="" at="" least="" some="" atm="" but="" no="" aptx="" protein="" (fig.="">

2.4 Pwopriyete manbràn ak sinaptik yo twouble nan newòn Purkinje ATM- ak APTX-defisi

Newòn Purkinje (PN) se yon sous-tip kle newòn ki sitiye nan cortical serebeleux la. Yo montre konsiderab eksitasyon intrinsèque, tire potansyèl aksyon espontaneman nan pousantaj siyifikativman pi wo pase pifò lòt newòn nan sèvo a (50 a 100 Hz plis nan anpil ka). Aktivite yo fòme pwodiksyon serebeleuz atravè anpèchman tonik nan newòn nan nwayo serebeleux yo, ki pwojte nan sant kowòdinasyon motè nan sèvo avan, tij sèvo, ak mwal epinyè. Serebeleux PN malfonksyònman asosye ak plizyè fòm ataksya epi li enplike nan AT(Hoxha et al.2018: Cook. Fields. and Watt 2021: Shiloh Aptx*-X/R35X.2020). Se poutèt sa, nou te egzamine si pwopriyete elektwofizyolojik PN nan serebeleu Atm yo te nòmal. Depi aktivite debaz PN ak repons nan opinyon yo medyatè pa yon seri debaz pwopriyete manbràn pasif ak aktif (Fig.4), nou dirèkteman anrejistre ak konpare pwopriyete manbràn PNs nan seksyon serebeleux egi rekòlte nan Atm35XR35×; Aptx* ak Atm*; Aptx* sourit (P350 a 400). PN anrejistre nan Atm35xR35×; Sourit Aptx te gen siyifikativman "pi sere" manbràn, montre pi wo manbràn D 'resistances(Rm) pase sa yo ki soti nan Atm; Aptx * sourit [47.7±5.6 (n=15) vs.

30.2±1.47(n=23)MQ, t-tès,p-0.008; Fig.4B] epi li montre yon tan konstan manbràn pi rapid () [3.6±0.4(n=15)vs.5.1±0.3(n=23)ms, t-test, p=0 .009;Fig.4B]. Rezilta sa yo endike ke kapasite total manbràn (Cm=t/Rm) nan Atm35x/35x; Aptx^PNs yo redwi anpil [98.25±19.23(n=15)vs.175.6± 12.67(n). =23)pF,t-test,p=0.0025;Fig.4B].Nan nivo selilè a, sa sijere ke PN ki defisi ATM ak APTX yo gen mwens (sètadi, diminye zòn) oswa manbràn mens

pase sa yo ki nan PNs sovaj, yon rezilta sijere yon defisi devlopman oswa pwosesis neurodegenerative; Aptx/-and (DellOrco et al.2015). Apre sa, nou te evalye eksitabilite intrinsèque PN nan AtmR35xR3x;

Atm*;Aptx* sourit lè yo egzamine jenerasyon ak dinamik potansyèl aksyon PN (AP). Defisi siyifikatif nan kapasite PN yo tire kontinyèlman an repons a piki aktyèl yo te obsève nan Atm?35 × 3x;Aptx ^ sourit (Fig.4C). Defisi sa yo te asosye ak perturbasyon enpòtan nan anplitid, papòt, ak zòn nan potansyèl aksyon evoke [anplitid: 66.2± 0.7 (n=14) vs. 72.1±1.4 (n=13)AmV,t-tès,p=0.003;papòt:-55.2±1.5vs.-48. 61±1.9 mV,t-tès,p=0.0196; zòn: 17.96±0.6vs.

20.63± 1.0 mV*ms, t-tès, p=0.048; Fig.4C]. Ansanm, eksperyans sa yo demontre perturbasyon enpòtan nan pwopriyete fizyolojik PN ki gen anpil chans deranje kapasite yo pou yo fonksyone nòmalman nan serebeleu At/mR35×R35×; Aptx' sourit.

Apre sa, nou te teste si pwopriyete PN ekstèn ak/oswa sinaptik yo te afekte tou nan AtmR35xR3sx. Aptx* sourit. Nou premye egzamine espontane eksitasyon pòs sinaptik kouran (iPSC) ki te pwodwi pa granules selil-a-PN sinaps (sa vle di, paralèl fib entrées). Pa gen okenn diferans nan gwosè UPSC te detekte, ki endike fonksyon an nan tèminal axon selil granules (sa vle di fib paralèl) te relativman nòmal nan AtmR35KR35X; Aptx'cerebellum [18.92±1.3(n{{10}}) te. 23.4±3.3(n=11)pA,t-test,p=0.477;Fig.4D](Yamasaki, Hashimoto, and Kano 20{{5{{58} }}}6). sEPSCfrequency, sepandan, yo te jwenn yo te ogmante siyifikativman, yon fenomèn ki ta ka atribiye a swa yon ogmantasyon nan kantite total sinaps, yon ogmantasyon nan gwosè a nan pisin nan vesik sinaptik ki fasil pou libere, oswa yon ogmantasyon nan pwobabilite pou nerotransmeteur. lage nan PN nan fAtmR35XR35X; Aptx'sourit [18.75±2.8Hz(n=11) kont 11.4±1.0Hz(n{=11), t-tès,p=0.{{ 69}}47; Fig.4D]. Apre sa, nou te eksplore liberasyon sinaptik evoke ak plastisit a kout tèm nan anrejistreman an menm tan nan PN yo ak elektrik stimile swa selil granules (sa vle di, fib paralèl) oswa enferyè olivary (sa vle di, k ap grenpe fib) axons ak yon pete batman pè (2- pulsasyon, 50 ms apa). Pwopriyete sinaptik fib paralèl yo te jwenn yo nòmal, pa montre okenn diferans enpòtan nan fasilitasyon kout tèm espere (Atluri and Regehr 1996) oswa mwatye lajè ak konstan tan pouri nan EPSC evoke [PPR: 1.3±0. 03 (n{=10) kont 1.4±{{9{0}}.{{1{{1{{106}}5}}1 }}5(n=13),t-tès,p=0.162;mwatye:3.9±0.6 vs.4.9±0.4 ms,t -test,p=0.175;tan konstan: 3.5±0.5 vs.4.7±0.4 ms,t-test,p=0.054;Fig. 4E]. An konparezon, nou te jwenn k ap grenpe fib-a-PN repons sinaptik, ki nòmalman montre depresyon pè-puls (Hansel and Linden 2000), t depresyon nan grandè siyifikativman pi gwo nan Atm/3338/R35X; Aptx'sourit [PPR:0.6±0.03 (n=6)vs.0.7±0.02(n=9),t-test,p=0.03;Fig.4F].Lajè an jeneral ak konstan tan pouri nan evoke a kouran yo te tou pi piti [mwatye lajè: 2.3±0.6(n =6).3.0±0.2(n=9)ms,t-test,p=0.004;tan konstan (vit) :1.1±0.14 vs.2.9±0.4ms,t-tès, p=0.001]. Pandan ke rezilta sa yo ta ka koze pa yon defisi presinaptik, tankou depo vesik redui nan tèminal axon fib k ap grenpe, grandè inisyal EPSC la pa afekte [2.4±0.4(n=6)vs. 1.9 ± 0.2 nA (n=9), t-tès, p=0.3] montre yon defisi ki pi intrinsèques, tankou yon aflu Ca2* ki redui nan retikul andoplasmik la, ki ta ka gen yon enpak siyifikativ long. -tèm plastisit sinaptik kritik fonksyon serebeleux (Hoxha et al. 2018; Kano and Watanabe 2017). An jeneral, perturbasyon yo nan pwopriyete yo pasif ak aktif PN ke nou obsève isit la gen anpil chans bay monte nan malfonksyònman serebeleux enpòtan nan AtmR35X / R35X; Aptx'sourit.

2.5 Deficiency ATM ak APTX lakòz yon perturbation pwogresif nan aktivite neral PN ki asosye ak dendritik.retresi ak an jeneral atrofi serebeleux Decreased rates of spontaneous PN action potential firing, which can be indicative of PN dysfunction, have been observed in several mouse models of ataxia, including spinocerebellar ataxias (SCA)2,3,5,6,13,27, several models of episodic ataxia (e.g., leaner, ducky, and tottering), and autosomal- recessive spastic ataxia of the Charlevoix-Saguenay (Hourez et al.2011; Hansen et al. 2013; Dell'Orco, Pulst, and Shakkottai 2017; Kasumu and Bezprozvanny 2012; Liu et al.2009; Perkins et al.2010; Shakkottai et al. 2011; Jayabal et al.2016; Stoyas et al.2020; Hurlock, McMahon, and Joho 2008; Shakkottai et al.2009; Bosch et al.2015; Walter et al.2006; Alvina and Khodakhah 2010; Ady et al.2018; Lariviere et al.2019; Cook, Fields, and Watt 2021). We, therefore, used this biomarker to characterize the progression of PN perturbation in Atm35XR35; Aptx'mice and assess whether deficits were restricted to ATM- and APTX-deficient mice, consistent with the behavioral results(Fig.2,3). We additionally examined whether decreased PN activity differed across the cerebellum, as anecdotal clinical pathology reports suggest degeneration may occur asymmetrically across the cerebellum, with the anterior and posterior vermis and middle cerebellar hemispheres affected the most, although no systematic analysis has been performed, and the consistency of results across patients is highly variable (Verhagen et al.2012; De Leon, Grover, and Huff 1976; Amromin, Boder, and Teplitz 1979; Monaco et al.1988; Terplan and Krauss 1969; Strich 1966; Solitare 1968; Solitare and Lopez 1967; Aguilar et al. 1968a; Paula-Barbosa et al. 1983). Using extracellular recording methods in the acute slice, we recorded spontaneous action potentials Aptx^ and 3 others from 3,300 PNs(Fig. 4G) across 188 animals, encompassing Atm35×R35×; genotypes at four different time points (P45, 120,210, and 400). We visually selected "healthy cells (see Methods) located deeper in the slice, that consistently fired during the extent of the 60-second recording period. Qualitatively, tissue and cell quality did not visually differ across genotypes under DIC microscopy. Cells were sampled in a distributed fashion across the lateral, intermediate, and medial (vermis)cerebellum of each mouse to assess whether changes in PN firing activity were ubiquitous or anatomically restricted. Regions were segregated based on gross anatomical domains in the mouse defined by natural anatomical boundaries (e.g., foliation) and their general connectivity with different regions of the nervous system (e.g., forebrain, brainstem, etc.)(Voogd and Glickstein 1998). We found that a complete deficiency of both ATM and APTX, consistent with the behavioral results, was necessary to produce a significantly reduced spontaneous PN firing frequency (Fig. 4G, H). Although the trend of slower PN firing rates were observed across most regions of the cerebellum, some subregions appeared to be less or minimally impacted, including several areas of the lateral cerebellum, including the paraflocculus, paramedian, and crus I and Ⅱ (Fig. 4-fig. S2). Significant age-dependent changes in firing frequency were also commonly observed in AtmR35XR35X; Aptx'mice(Fig. 4H), with the most significant decline occurring betweenP120 and 210 [medial: 50.3±2.4Hz(n=61)vs. 36.9±2.2Hz(n=31),t-test,p=0.0006].No significant difference in PN firing frequency was detected between male and female mice within each genotype, thus the data were pooled (2-way ANOVA, p>0.3 atravè tout konparezon par; Figi 4-fig. S3). Etid anvan yo atravè plizyè modèl sourit nan ataksya eritye, ki gen ladan ataksya epizod ak plizyè varyant nan ataksya spinocerebellar, jwenn ke dezòd fizyolojik nan tire PN pa sèlman chanje frekans li yo, men tou, regilarite li (Kasumu and Bezprozvanny 2012; Jayabal et al. 2016; Stoyas). et al 2020; Cook, Fields, and Watt 2021). Nou konpare tou de koyefisyan varyasyon (CV) ak varyasyon nan entèval adjasan (CV2) ant AtmR35X/R35X; Aptx/ ak kontwòl sourit (fig. 4-figs.S4, S5). Pa gen okenn diferans nan paramèt sa yo atravè sèks, laj, oswa jenotip yo te detekte. Konfòm ak rezilta konpòtman yo, yo te jwenn malfonksyònman serebeleux sèlman nan AtmR35XR35X la; Aptx'sourit ki devlope ataksya epi yo pa nan sourit ak ekspresyon an pasyèl oswa konplè nan ATM oswa APTX.

2.5 ATM ak deficiency APTX pwovoke atrofi serebeleux

In A-T patients, ataxia is usually detected between1 two 2-years of age and is associated with little to no cerebellar atrophy (Tavani et al. 2003; Taylor et all. 2015). Significant structural changes and atrophy are usually first detected via neuroimaging between 5 and to 10-years of age (Demaerel, Kendall, and Kingsley 1992; Tavani et al. 2003). Postmortemclinical histopathology in A-T patients points to significant changes in PN morphology and density, however, these reports primarily detail patients at late stages of the disorder, and the relationship between the severity of PN pathology and ataxia is not clear (Verhagen et al. 2012; De Leon, Grover, and Huff 1976; Amromin, Boder, and Teplitz 1979; Monaco et al. 1988; Terplan and Krauss 1969; Strich 1966; Solitare 1968; Solitare and Lopez 1967:Aguilar et al. 1968a; Paula-Barbosa et al. 1983; Gatti and Vinters 1985). In the Atm735X7k3×; Aptx^ mice, we found the gross size of the cerebellum to be normal early in life, but significant atrophy developed as the severity of ataxia increased (Fig.5A). At early stages(P45-P210), the size of the cerebellum in Atm3xR35×; Aptx^ mice did not differ from mice with at least one copy of the Atm gene(2-way ANOVA, Fa.s52)=1.0, p-0.4). However, by P210, the overall size of the cerebellum in Atm735XR35×; Aptx* mice was significantly reduced(1-way ANOVA, F(3,37= 1.4,p=0.3), with the degenerative process continuing to at least the last time point investigated (P460). To rule out the possibility that reduced cerebellar size was related to the smaller stature of Atm735xR35×; Aptx" mice, we examined, animal weight and cerebellar size and found no correlation [Pearson's correlation, p>0.3 pou tout 4 jenotip nan P460, n=10 rive 20]. Anplis de sa, nou te jwenn ke gwosè serebeleu pa t diferan ant gason ak sourit fi yo an mwayèn 22 pousan ki pi piti atravè jenotip nan laj sa a [2-fason ANOVA, F(2. 153)=1.9, p. =0.2]. Se poutèt sa, neurodegeneration serebeleux nan Atm35XR35× la; Aptx * sourit, ki kòmanse apre P120, gen rapò ak ATM ak deficiency APTX. Nou te jwenn atrofi serebeleu ki asosye ak yon rediksyon selektif nan lajè kouch molekilè a kote dendrit PN abite (figi 5B). Konfòm ak chanjman tanporèl nan gwosè serebeleux brit, frekans tire PN, ak konpòtman, lajè ML nan Atm35 × R35X; Sourit Aptx* yo te nòmal nan sourit ki pi piti yo men yo te piti piti diminye nan lajè kòm gravite ataksya a te ogmante 【P400∶120.2±2.1 μm （n=5）vs.140.2±4.8 μm（n=5）, {31}}fason ANOVA,Fu42=45.04,p<0.0001;fig.5b, fig.5-figs.s1a】.in="" contrast,="" no="" difference="" in="" the="" width="" of="" the="" granular="" cell="" layer(gcl)across="" age="" was="" observed="" in="" the="" atm35xr3x;="" aptx*mice="" 【p400∶135.5±2.4="" μm（n="6）v.127.5±4.3" μm（n="5）,2-way" anova,fu="" 42="3.3,p=0.08;Fig.5B," fig.5-figs.s1a].="" cerebellar="" atrophy,="" however,="" was="" not="" due="" to="" pn="" cell="" death,="" as="" pn="" density="" did="" not="" significantly="" differ="" between="" atm735×r35x;="" aptx'="" and="" atm*;="" aptx*="" mice="" [p400:4.5="" ±0.3（n="9）vs.4.5±0.2（n=7）PNs/4000" μm²,welch's="" t-test,="" p="0.9;Fig.5C," d="" fig.5-figs.s1b】.moreover,="" we="" found="" no="" evidence="" from="" immunohistological="" experiments="" for="" increased="" levels="" of="" aptx"-="" mice="" (fig.5-figs.s2).="" at="" the="" programmed="" cell="" death="" or="" microglial="" activation="" in="" the="" atm?35x/r35x="" anatomical="" level,="" we="" found="" pathological="" changes="" in="" pn="" morphology,="" as="" the="" somatic="" size="" was="" reduced(15.3±0.3（n="5）vs.17.8±0.1（n=5）μm," welch's="" t-test,="" p="0.0004;" fig.5e）and="" the="" primary="" and="" secondary="" dendrites="" were="" abnormally="" large="" in="" caliber="" in="" the="" atm35xxr35x;="" aptx'mice="" (3.1±0.2(n="6)vs.2.8±0.1" （n="6）μm," welch's="" t-test,="" p-0.003;="" fig.5f,="" fig.5-figs.s2c）.="" overall,="" we="" found="" a="" good="" correlation="" between="" the="" abnormal="" structural="" and="" electrophysiological="" properties="" of="" the="" cerebellum="" and="" the="" progression="" of="" motor="" deficits.="" 2.6="" differential="" disruption="" of="" thymocyte="" development="" in="" atm-deficient="" vs.="" aptx-deficient="">

Enfeksyon sinopulmonè kwonik ki asosye ak iminodefisyans yo se youn nan kòz prensipal lanmò nan pasyan AT (Morrell, Cromartie, and Swift 1986; Bhatt and Bush 2014). Iminodefisyans lye ak defisi nan jenerasyon lenfosit B ak T ki te lye ak defo nan pwosesis reranje jèn reseptè antijèn yo pandan jenerasyon selil sa yo nan mwèl zo a ak tim, respektivman (Staples et al. 2008). Defo yo ki kapab lakòz nan nimewo lenfosit ki gen matirite yo enkli diminisyon nan selil T asistan CD4t ak selil CD8 * T asasen (Schubert, Reichenbach, and Zielen 2002). Se poutèt sa, nou te egzamine pousantaj selil T nan san periferik ak diferan subpopulations nan timis Atm35xR35×; Aptx*'sourit ki itilize reseptè antijèn selil T (TCR) ak ekspresyon ko-reseptè CD4/CD8. Nan san an periferik, nou te obsève yon rediksyon enpòtan nan fraksyon total CD3 * T-selil nan sourit ak ekspresyon ATM redwi oswa absan konpare ak sourit sovaj-kalite (Fig.6). Te rediksyon sa a plis agrave pa defisi a koncomitan nan APTX. Defisyans ATM ak APTX redwi selil T nan san periferik pa plis pase 65 pousan konpare ak kontwòl kalite sovaj. Efè a nan deficiency APTX te aditif ak sa yo ki nan deficiency ATM, sijere yon mekanis diferan nan aksyon pou chak nan de pwoteyin sa yo sou jenerasyon T-selil. Rediksyon an nan pousantaj selil T nan san periferik te sitou asosye ak yon rediksyon nan popilasyon an CD4 plis moun k ap ede T-selil (Fig.6B). Nan enterè, pwopòsyon an nan CD8 plis T-selil te ogmante sèlman nan AtmR35X / R35 ×; Aptx* sourit(Fig.6B). Yon fwa ankò, nou te obsève yon efè diferans nan defisyans ATM ak APTX jan yo wè pou efè mitasyon sa yo sou fraksyon total T-cel la. Etandone rediksyon nan popilasyon selil T nan san an, nou te evalye devlopman selil T nan timis la. Nan ògàn sa a, progenitors selil T ki sòti nan mwèl zo yo sibi reyajman jèn TCR ki te swiv pa seleksyon pozitif pou restriksyon MHC ak seleksyon negatif klon autoreactive. Faz devlopman thymocyte yo ka swiv pa kontwole ekspresyon CD4 ak CD8 ekspresyon nan thymocytes. Pwogresyon pwogram devlopman sa a soti nan timosit doub negatif (CD4CD8) pou rive nan timosit doub-pozitif (CD4*CD8) epi answit pou ale nan timosit sèl-pozitif (CD4*oswa CD8*). Anplis de sa, nan etap doub-negatif la, yo ka idantifye kat subpopulasyon diferan, ki baze sou ekspresyon CD25 ak CD44, ke yo rekonèt kòm DN1 (CD44 * CD25), DN2 (CD44 * CD25 *), DN3 (CD44 CD25 *) ak DN4 (CD44 degre CD25) (Germain 2002). Reyajman jèn pandan devlopman thymocyte rive de fwa—yon fwa nan etap doub thymocyte negatif nan etap CD25 * CD44 (Krangel 2009) epi answit ankò nan etap thymocytes doub-pozitif anvan pwogrese nan yon sèl popilasyon pozitif CD4 * ak CD8t (Livak et al. 1999). Defisi ATM yo te lye ak defo nan tou de bout nan rearanjman nan sourit (Vachio 2007, Hathcock 2013). Se poutèt sa, nou konpare pwopòsyon selil ki nan timis la ki eksprime makè sifas selil devlopman diferan sa yo nan sourit ATM nou yo ak kontwòl (Fig.7). AtmR35xR35×; Aptx ak Atm35×; Aptx^ men se pa AtmR35XR35; Sourit Aptx * te gen pwopòsyon siyifikativman elve nan CD44 * CD25, CD44 * CD25t, ak CD44CD25 * selil konpare ak sovaj (Fig.7A) Pwopòsyon ogmante sa yo parèt akòz yon anpèchman nan selil CD44CD25t matirite nan CD44CD25 selil doub negatif, kòm fraksyon nan selil CD44CD25 soti nan Atm735xR35×; Aptx'and Atm35× plis ; Aptx'mice se siyifikativman pi ba pase wildtype(Fig.7A).Nan enterè, APTX deficiency pou kont li te gen pi gwo efè sou pèt la nan selil DN4, sijere ke deficiency APTX, olye ke deficiency ATM, se responsab pou efè sa a. Dapre nou konnen, konklizyon sa a enplike pou premye fwa APTX nan reyajman jèn pandan pwosesis la nan rekonbinasyon TCR. Apre sa, nou te gade pwopòsyon CD4 * CD8t thymocytes konpare ak CD4tCD8 ak CD4CD8 plis sèl-pozitif thymocytes nan kat diferan tansyon sa yo. An akò ak rezilta nou yo nan san an ak etid anvan yo, nou te jwenn ke sourit ATM-ensufizant men pa sourit kontwòl parèt diminye ekspresyon nan CD4 * CD8 ak CD4CD8 * sèl thymocytes pozitif (Fig.7B). Rezilta sa yo sipòte wòl ATM nan TCR a/δgene reyaji pandan devlopman thymocyte （Bredemeyer et al.2006）, yon wòl ki endepandan de wòl APTX jwe nan bonè thymocyte spirasyon.

2.7 Molekil Read-through simonte PTC pou retabli ekspresyon ATM

Rezon prensipal nou an pou mete yon mitasyon san sans ki enpòtan klinikman nan jèn Atm la se te jenere yon sourit ki kapab fè tès pre-klinik kritik nan yon seri nouvo konpoze SMRT. Nou te deja demontre ke konpoze SMRT refè pwodiksyon an nan pwoteyin ATM nan liy selil lenfoblastoid AT ki sòti nan pasyan lè yo simonte kodon tèminasyon twò bonè (PTC) ki te koze pa mitasyon istwa san sans (Du et al. 2013). Pou demontre konvnab nouvo modèl bèt AT sa a pou tès konpoze SMRT nou te chwazi dirèkteman egzamine kapasite yo pou retabli ekspresyon ATM lè l sèvi avèk yon apwòch eksplant ki kontourne defi ki gen rapò ak livrezon in vivo (egzanp, byodisponibilite, wout livrezon, elatriye). Ekspresyon ATM te mezire nan echantiyon ki soti nan larat la, kote ATM nòmalman eksprime nan nivo segondè, ak serebeleu a, yon tisi sib kle pou maladi a. Èske nou te ekspoze tisi eksplant sa yo, rekòlte nan omozigòt Atm?35× ak Atm? 35 sourit ak swa yon konpoze SMRT kandida (GJ103) oswa yon aminoglikozid ki te deja konnen ki gen pwopriyete lekti (G418) ak Lè sa a, mezire ekspresyon ATM pa immunoblot pou evalye restorasyon. Nan tou de kalite ATM-defisi sourit, ekspresyon ATM te toujou retabli nan larat la ak serebeleu pa tou de G418 ak GJ103 (Fig. 8). Rezilta sa yo demontre ke konpoze SMRT nou yo ka pèmèt lekti nan omwen 2 nan 3 mitasyon san sans posib ki lakòz PTC yo epi yo bay rezon pou tès efikasite in vivo nan etid swiv-n.