A Novel, Ataxia Mouse Model Of Ataxia Telangiectasia Ua Los Ntawm Kev Kho Mob Tsis Txaus Siab Hloov Kho (Part 2)

Xav paub ntau ntxiv plz hu raudavid.wan@wecistanche.com

3.0 Kev sib tham

Los ntawm kev ua kom muaj kev ntxhov siab genotoxic los ntawm kev sib ntxiv ntawm kev sib tw thib ob rau DDR txoj hauv kev, peb tau tsim cov qauv nas tshiab uas qhia txog cov txheej txheem AT cov tsos mob ntawm txhua tus qauv rau hnub tim. Qhov no suav nrog kev mob hnyav thiab hnyav ataxia cuam tshuam nrog cerebellar atrophy thiab perturbations ntawm PN cov khoom nrog rau kev mob qog noj ntshav siab thiab qhov tsis xws luag hauv lub cev tiv thaiv kab mob. Ua ke, cov comorbidities suav nrog peb qhov ua rau tuag ntxov ntxov hauvAT-txhua qhov kev txhawb nqatxog kwv yees li ib feem peb ntawm cov neeg tuag. Ntawm cov no, cov nyhuv incapacitating ntawm ataxia yog qhov nkag siab tshaj plaws thiab tau tshaj tawm los ntawm cov neeg mob thiab cov neeg saib xyuas tias muaj kev cuam tshuam loj tshaj plaws rau lawv lub neej zoo. Vim li no, lub xub ntiag ntawm ataxia thiab cerebellar atrophy hauv tus qauv nas tshiab no yog qhov tseem ceeb, vim nws muab thawj zaug uas muaj peev txheej tsis yog tsuas yog qhia cov txheej txheem ntawm kev ua haujlwm tsis zoo hauv lub paj hlwb, tab sis kuj tseem ceeb heev hauv vivo qauv rau kuaj mob hnyav xav tau AT kho mob, xws li cov ntawv nyeem-los ntawm kev sib txuas peb piav qhia ntawm no. Peb pom ntau qhov zoo sib xws ntawm qhov kev loj hlob tag nrho ntawm ataxia hauv Atm3535; Aptx nas thiabAT cov neeg mob. Hauv kev kho mob AT, lub cev muaj zog muaj peev xwm pom tau los ntawm kwv yees li 2 xyoos, thaum cov niam txiv thiab kws kho mob pom tias muaj peev xwm txo qis ntawm kev hloov pauv ntawm menyuam yaus mus rau ib qho chaw zoo, kev sib koom ua ke ntawm lub rooj vag - hmoov tsis zoo, me ntsis paub txog lub cev muaj zog tsis xws luag ntawm cov theem ua ntej vim cov kab mob. tsawg dua thiab tam sim no tsis muaj kev kuaj mob ntxov (Rothblum-Oviatt li al. 2016). Cov neeg mob feem ntau kawm taug kev yam tsis muaj kev pab thiab cov tsos mob ntawm lub paj hlwb yuav nyob ruaj khov los ntawm thawj 4 txog 5 xyoo ntawm lub neej (Rothblum-Oviatt et al. 2016). Peb pom qhov zoo sib xws thaum ntxov ntawm lub cev muaj zog tsis txaus hauv Atm735 × R3x; Aptx ^ 'cov nas, kuaj pom qhov tsis zoo ntawm lub cev muaj zog thaum ntxov ntawm P8 (txoj cai reflex tsis txaus) ua raws li lub sijhawm ntawm kev ruaj ntseg ntawm tus txheeb ze, ua ntej qhov pib ntawm kev loj hlob thiab loj ataxia tsim tom qab p210 uas suav nrog kev hloov pauv hauv gait, startle reflex, tremor, thiab locomotor kev ua si. Ntau cov lus nug tseem ceeb tshwm sim los ntawm qhov kev tshawb pom no, suav nrog seb ATM thiab / lossis APTX puas muaj lub luag haujlwm hauv kev tsim kho neurodevelopmental hauv

cerebellum. Cov kev tshawb fawb yav tom ntej tsom mus rau theem pib ntawm qhov teeb meem yuav yog qhov tseem ceeb hauv kev nkag siab yog tias lub cerebellum txhim kho ib txwm ua ntej kev ua haujlwm tsis zoo lossis seb qhov kev loj hlob tsis zoo yog thawj qhov ua rau. Peb kuj pom, zoo ib yam li cov neeg mob AT, tias qhov mob hnyav ntawm qhov kev txhim kho ataxia lig yog qhov sib txawv, nrog qee cov nas mbulating nrog ib tug clumsy, high-stepping rear rooj vag (Video 3) thiab lwm tus yuav luag tag nrho ntawm contortion ntawm lub rear lub cev ( Fig.1E thiab Video 4)(Rothblum-Oviatt et al.2016; Levy thiab Lang 2018; Boder thiab Sedgwick 1958). Zuag qhia tag nrho, peb pom tias Atm?35×xR35×; Aptx 'cov nas tau tsim qhov pom qhov pom tau zoo thiab ntsuas qhov kev poob qis hauv kev sib koom ua ke ntawm lub cev muaj zog zoo ib yam li tau pom hauv AT cov neeg mob, uas tau txais kev cawmdim los ntawm kev qhia tsawg kawg ib daim qauv ntawm Atm lossis Aptx tau txais nws. Kev poob ntawm kev sib koom ua ke ntawm lub cev muaj zog hauv AT tau raug ntaus nqi rau cerebellar degeneration vim nws cov kev xaiv neuropathology thoob plaws hauv lub hlwb thiab nws lub luag haujlwm ua rau muaj ntau yam sib txawv ntawm ataxia (Hoche et al.2012). Raws li nrogAT tus neeg mobneuroimaging kev tshawb fawb (Wallis et al.2007; Sahama et al.2015; Sahama li al. 2014; Dineen li al. 2020; Tavani li al. 2003; Quarantelli li al.2013), peb pom tias cerebellar loj 3 × 7. ; Aptx 'cov nas yog thawj zaug ib txwm, tab sis nce atrophies concurrently nrog kev hloov hauv kev ua haujlwm ntawm lub paj hlwb. Txawm hais tias qhov poob ntawm cerebellar cov ntaub so ntswg tau suav hais tias yog ib qho tseem ceeb ntawm ataxia hauv tib neeg, nws tsis paub meej los ntawm cov ntaub ntawv kho mob yog tias qhov hnyav ntawm ataxia yog ib qho kev kwv yees zoo ntawm qhov tshwm sim ntawm cerebellar degeneration pom postmortem (Aguilar et al. 1968b: Crawford et al, 2006. : Dineen et al.2020). Nyob rau hauv lub Atm? 35 × R35 ×; Aptx^ nas, peb pom qhov tseeb atrophy cuam tshuam nrog thinning ntawm Purkinje neuron dendrite txheej uas ua ntej lig, kev coj cwj pwm tsis zoo. Peb cov kev soj ntsuam histological hauv Atm?35xR35×; Aptx'mice qhia tias kev hloov pauv hauv cerebellar ua haujlwm nws tus kheej, tsis yog qhov poob ntawm cerebellar hlwb, txaus los ua rau ataxic phenotype, raws li kev soj ntsuam ntawm tus cwj pwm tsis zoo ua ntej PN poob ntau hauv SCAs (Shakkottai li al. 2011: Lorenzetti li al. 2000; Clark et al. 1997; Jayabal et al.2016). Qhov laj thawj yog vim li cas ATM thiab APTX tsis txaus yuav tsum tsim kom muaj ataxia hauv cov nas thaum poob ntawm ib qho txaus ua rau ataxia hauv tib neeg tseem tsis paub meej. Ib qho ua tau yog tias nas lub hlwb yuav hloov tau yooj yim siv txoj kev them nyiaj los yog cov protein ntau dua thaum teb rau 10-20k DNA qhov txhab uas cuam tshuam cov hlwb txhua hnub (Lindahl thiab Barnes 2000). Muaj ntau hom kev kho DNA uas muaj peev xwm ua tau raws li qhov kev sib tw no, suav nrog kev kho lub hauv paus excision (BER), nucleotide excision kho (NER),

Nyem qhov no kom paub ntau ntxiv txog Cistanche

as well as homologous thiab non-homologous kawg koom nrog (HEJ and NHEJ, ntsig txog), tag nrho cov ATM thiab APTX tau cuam tshuam rau hauv (Chou et al.2015; Caglayan et al.2017; Wakasugi et al. 2014; Tumbale et al. 2018; Chatterjee thiab Walker 2017) Xwb, nws yuav yog. rooj plaub uas tsis muaj peev xwm hauv ATM lossis APTX ib leeg tsis txaus cuam tshuam rau kev noj qab haus huv ntawm tes thaum lub sijhawm nas lub neej luv luv, thiab yog li tshem tawm ob qho proteins yog qhov tsim nyog kom ua tiav qhov txaus ntawm DNA puas tsuaj rau lub sijhawm no. Qhov muaj peev xwm no tau ntxiv dag zog los ntawm qhov tseeb tias ATM thiab APTX muaj cov khoom siv biochemical sib txawv thiab lub luag haujlwm ua haujlwm hauv DNA kev puas tsuaj teb, thiab yog li qhov tsis txaus ntawm ob qho tib si yuav raug kwv yees ua rau muaj kev cuam tshuam dav rau genome stability (xws li, nce genotoxic stress). Peb qhov kev tshawb pom tias ob lub genome ruaj khov txoj hauv kev muaj protein ntau yuav tsum tau ua rau muaj qhov tsis xws luag hauv cov nas pom tau hais tias nws yog qhov poob ntawm ATM's Oriole hauv DNA kho, es tsis muaj peev xwm ua haujlwm hauv oxidative stress signaling, mitophagy, los yog mitochondrial muaj nuj nqi uas ua rau cerebellar defects ( Xyoo 2020). Lwm txoj hauv kev, txawm li cas los xij, tsis tuaj yeem txiav txim siab tag nrho, raws li APTX, zoo li ATM, tau pom nyob rau hauv mitochondria ntawm lub hlwb hlwb, qhov uas nws xav tias yuav txhawb nqa cov txheej txheem ntawm mitochondrial DNA (Meagher thiab Lightowlers 2014; Sykora li al. 2011). Tus qauv nas tshiab no muab cov cuab yeej tshiab los tshawb txog qhov muaj peev xwm no thiab kev siv tshuab txhais tau li cas qhov poob ntawm ATM thiab APTX thaum kawg ua rau cerebellar tsis ua haujlwm. Lub biophysical perturbations pom hauv PNs kaw los ntawm AtmR35XR35X; Aptx nas zoo sib xws nyob rau hauv ob peb lwm nas qauv ntawm ataxia. Qhov no suav nrog cov kev hloov pauv uas peb pom hauv PN input resistance, membrane capacitance, thiab AP qhov pib thiab dav, uas kuj tau piav qhia hauv nas qauv ntawm SCA xws li 1, 3, thiab 7 (Stoyas li al.2020; Shakkottai li al.2011; Dell 'Orco et al. 2015). Tsis tas li ntawd, kev nce qib ntawm kev txo qis hauv PN kev ua haujlwm muaj peev xwm tua ntau zaus peb tau tshaj tawm zoo cuam tshuam nrog kev txhim kho ntawm ataxia hauv AtrnR35XR35X; Aptx'mice tau tshaj tawm nyob rau hauv ntau tus qauv ataxic nas, suav nrog SCAs 1, 2, 3,5, 6, thiab 13 nrog rau ob peb daim ntawv (saib tshuaj xyuas (Cook, Fields, thiab Watt 2021)). Muab qhov tseem ceeb sib tshooj hauv PN perturbations pom nyob thoob plaws ntau qhov sib txawv ataxias tshwm sim los ntawm cov xov tooj ntawm tes tsis sib xws, rov qab kho PN AP firing zaus tau raug suav tias yog txoj hauv kev kho dav dav. Txawm li cas los xij, nws tseem tsis tau paub meej tias qhov txo qis PN firing yog qhov laj thawj ntawm ataxia. Ntxiv mus, cov pov thawj kev sim qhia tau hais tias kev hloov pauv hauv PN kev ua si hauv qhov tseeb yuav yog cov lus teb dav dav rau kev tswj hwm homeostasis thaum muaj kab mob cuam tshuam txog kev puas tsuaj ntawm PN physiology (Dell'Orco et al.2015). Yog li, kev siv zog txuas ntxiv mus thoob plaws txhua lub cerebellar ataxia yog xav tau los txuas cov noob caj noob ces, molecular, thiab cellular cuam tshuam los ntawm tus kab mob mus rau cov kev hloov pauv hauv cerebellar neural signaling uas thaum kawg tsim cov ataxia. Qhov tseem ceeb ntawm qhov kev siv zog no yuav txiav txim siab seb puas muaj kab mob ua rau cerebellar tsis xws luag feem ntau lossis sib txawv ua rau ataxia los ntawm kev poob ntawm cerebellar muaj nuj nqi (xws li, poob ntawm kev sib koom tes thaum lub sij hawm txav), los yog los ntawm qhov tseem ceeb-tsis zoo (xws li, cuam tshuam downstream neural. circuits nrog txawv txav neural output qauv). Thaum kawg, thaum ib qho kev kho mob zoo tshaj plaws los daws cov kab mob cerebellar ataxias yuav muaj qhov cuam tshuam loj tshaj plaws, txoj hauv kev qhia uas hais txog qhov txawv ntawm caj ces thiab molecular ua rau cellular tsis ua hauj lwm thaum kawg yuav tsim nyog los txhim kho kev kho mob zoo. ruaj khov proteins zoo li ATM thiab APTX thiab PN kev ua haujlwm tsis zoo yog deb ntawm qhov tseeb. Peb cov txiaj ntsig tau qhia tias cov txiaj ntsig ntawm ATM thiab APTX poob ntawm PNs yog qhov tseem ceeb, vim peb tsis pom cov kev hloov pauv hauv cov khoom presynaptic ntawm granule cell lossis pov thawj ntawm lawv cov cellular poob (tsis muaj kev hloov pauv hauv GCL thickness). Tsis tas li ntawd, thaum peb pom qhov sib txawv ntawm lub sij hawm luv luv ntawm cov khoom siv tsis zoo ntawm cov txiv ntseej hauv ATM- thiab APTX-tsis muaj PNs thiab cov tsiaj qus, cov txiaj ntsig no yuav taw qhia txog kev cuam tshuam hauv Ca2* homeostasis uas muaj feem cuam tshuam los ntawm kev txo qis hauv Inositol 1,4,5- triphosphate receptor 1(ltpr1) kev qhia, zoo ib yam li cov tau pom hauv SCAs 1,2, thiab 3 nas qauv nrog rau ATM-tsis muaj nas (Kim li al.2020; Chen et al.2008; Demirci et al.2009; Shakkottai et al. 2011). Thaum qhov no muab txoj hauv kev zoo rau kev kuaj xyuas yav tom ntej thiab kev sib piv, nws tseem tsis tau paub meej, txawm tias SCAs, txawm tias kev hloov pauv hauv Ca² * homeostasis, yog qhov ua rau lossis tsuas yog lwm yam tsos mob lossis txawm tias cov lus teb ntawm cov kab mob lossis cuam tshuam PNs (Dell 'Orco et al. 2015). Hauv lub cev tiv thaiv kab mob, ATM cuam tshuam rau kev kho DNA tawg uas ib txwm tshwm sim thaum lub sijhawm hloov kho cov noob ntawm cov tshuaj antigen receptor genes hauv B-thiab T-cell precursors, qhov tshwm sim tseem ceeb rau antigen receptor (LG thiab TCR) ntau haiv neeg ntawm cov hlwb no. nrhiav qhov ntawdT-cellQhov feem pua ​​​​ntawm cov ntshav tau txo qis yog raws li kev tshawb fawb ua ntej hauv tib neeg thiab AT knockout nas (Schubert, Reichenbach, thiab Zielen 2002; Hathcock li al.2013; Chao, Yang, thiab Xu 2000; Barlow li al.1996). Qhov kev txo qis ntawm T-cells nyob rau hauv lub periphery yuav correlates nrog ib tug tsis xws luag ntawm cellular thiab humoral tiv thaiv. Qhov tseem ceeb, peb pom tias qhov kev nthuav qhia ntawm ib daim qauv ntawm ATM gene yog txaus los kho CD4 ntxiv rau cov ntshav tsis txaus uas qhia tau tias cov kev kho mob tuaj yeem kho cov kev qhia ATM ib nrab yuav muaj kev kho mob zoo. Txawm hais tias peb tsis tau soj ntsuam B-cell txoj kev loj hlob hauv daim ntawv no, nws yuav zoo li cov lus xaus zoo sib xws yuav siv rau cov txheej txheem uas tau muab lawv cov tshuab zoo sib xws (Marshall et al. 2018). Raws li qhov xav tau, qhov txo qis ntawm T-hlwb hauv cov ntshav peripheral cuam tshuam nrog kev txhim kho thymocyte tsis zoo. Hauv thymus, peb pom ob qhov tsis xws luag. Ib qho, induced feem ntau los ntawm APTX deficiency, manifests raws li ib tug tsis xws luag nyob rau hauv lub DN3 mus rau DN4 kev hloov pauv coinciding nrog thaum ntxov rearrangement ntawm TCR locus. Lwm qhov tsis xws luag, feem ntau tshwm sim los ntawm ATM tsis muaj peev xwm, cuam tshuam nrog kev txo qis ntawm ob qhov zoo CD4 * CD8 rau ib leeg-zoo hlwb, feem ntau CD4 'thymocytes. Thaum qhov kev tshawb pom APTX yog qhov xav tsis thoob, vim nws qhov tsis txaus (AOA 1) tsis cuam tshuam nrog kev tiv thaiv kab mob, APTX paub tias cuam tshuam nrog TCR gene rearrangement proteins, suav nrog XRCC4 (Clements li al. 2004). Cov kev tshawb fawb yav tom ntej txhawm rau txheeb xyuas APTX lub luag haujlwm hauv kev sib koom ua ke kawg thaum lub sijhawm TCR gene rearrangement yuav yog

tseem ceeb, thiab muaj peev xwm uas lwm txoj kev sib koom ua ke, xws li kev siv cov microhomologies, suav txog qhov tsis muaj kev tiv thaiv kab mob hauv nws qhov tsis tuaj yeem xav tau kev tshawb fawb ntxiv (Bogue et al. 1997). Kev muaj sia nyob ntawm Atm? 35 × / R35 ×; Aptx'mice yog qhov ntev dua li cov qauv AT nas ua ntej. Hauv kev sib piv, thawjUA KOnas qauv qhia los ntawm Barlow et al. tuag los ntawm thymomas feem ntau tsis pub dhau 2-4 hli tom qab yug me nyuam (Barlow li al. 1996). Kev txo qis ntawm kev muaj sia nyob hauv qhov no thiab ntau lwm qhov kev sib tw AT nas qauv yog qhov caj ces, raws li keeb kwm yav dhau los harboring qhov kev hloov pauv tau pom tias muaj kev cuam tshuam loj rau kev mob qog noj ntshav thiab muaj sia nyob, nrog A / J thiab C57BL / 6.

Cov keeb kwm yav dhau los muaj kev muaj sia nyob ntau dua li BALBC thiab 129S hom kab mob (Genik li al. 2014). Qhov tseeb tias peb cov ATM-tsis muaj nas tau tsim los ntawm C57BL / 6 keeb kwm yav dhau los yuav muaj tseeb; tias Aptx * t nas tsis tsim ataxia, nws yog lawv qhov kev sib piv ntev ntev. Muab hais tias Atm35xR35 ×; Tsis zoo li qhov kev tuag ntxov hauv AT KO nas tiv thaiv kev soj ntsuam ntawm ataic phenotype uas yuav txwv tsis pub tsim hauv cov nas no. Txawm li cas los xij, nws tsis paub meej tias C57BL / 6 keeb kwm yav dhau los cuam tshuam rau kev txhim kho ataxia, zoo li nws ua rau mob qog noj ntshav. Kev txhais cov caj ces los yog tej zaum yuav muaj cov kab mob epigenetic uas cuam tshuam rau qhov mob hnyav tuaj yeem muab txoj hauv kev rau kev kho mob yav tom ntej. Muab qhov xwm txheej thoob ntiaj teb ntawm ATM thiab APTX tsis muaj kev hloov pauv hauv peb tus qauv nas, peb tsis tuaj yeem txiav txim siab tag nrho tias qhov tsis xws luag ntawm cerebellar kuj tseem tuaj yeem ua rau tus mob ataxic phenotype, thiab yog li yav tom ntej kev ntsuam xyuas sab nraum lub cerebellum nyob rau hauv lub forebrain, brainstem, qaum qaum. , thiab txawm tias cov leeg yuav tsum tau ua. Nyob rau hauv lub cerebellum, thaum peb pom ib co anatomical sib txawv nyob rau hauv lub PN firing zog nyob rau hauv sib txawv cheeb tsam ntawm lub cerebellum, peb tsis tau pom lub regional sib txawv nyob rau hauv ML dav los yog PN ceev. Txawm li cas los xij, muaj kev sib tw hauv kev siv lub cev lub cev hauv cheeb tsam los ua ib pawg hauv cerebellum, vim tias lub cev ntawm cov ntaub so ntswg tsis tas yuav cuam tshuam nrog thaj tsam ntawm kev ua haujlwm, molecular qhia, lossis cov khoom siv lub cev uas tau piav qhia (Apps and Hawkes 2009 ; Tsutsumi et al.2015; Gao, van Beugen, and De Zeeuw 2012; Zhou et al.2014). Cov kev sim tsom mus rau kev tshuaj xyuas qhov cuam tshuam ntawm cerebellar nyob rau hauv cov chaw no yuav yog ib qho tseem ceeb hauv kev tshawb fawb yav tom ntej thiab muab piv rau cov ntaub ntawv qhia txog qhov sib txawv ntawm anatomical hauv cov neeg mob AT (Verhagen li al.2012; De Leon, Grover, and Huff 1976; Aromin, Boder, thiab Teplitz 1979; Monaco li al. 1988; Terplan thiab Krauss 1969; Strich 1966; Solitare 1968; Solitare thiab Lopez 1967; Aguilar et al. 1968a; Paula-Barbosa et al. 19). Thaum peb tshawb pom ob lub sijhawm muaj peev xwm ntawm kev nce qib ntawm ataxia hauv Atm735wR35x; Aptx nas, tom qab theem ntawm ataxia loj hlob tuaj rau cov neeg laus hauv cov nas, piv rau cov menyuam yaus pib tshwm sim hauv tib neeg. Qhov no tuaj yeem txwv nws siv hauv qee qhov kev tshawb fawb neurodevelopmental. Tsis tas li ntawd, kev txhais cov kev sim yav tom ntej yuav tsum ua tib zoo saib xyuas qhov tseeb tias tus qauv tshiab no qhia txog kev hloov pauv tsis zoo hauv ob lub genome ruaj khov noob nyob rau tib lub sijhawm, qhov xwm txheej uas tsis tau kuaj pom hauv tib neeg cov neeg mob nrog AT lossis AOA1. Thaum kawg, taw qhia qhov twg, thaum twg, thiab yuav ua li cas ATM tsis muaj peev xwm ua rau kab mob cerebellar thiab ataxia yog qhov nyuaj, vim tias ua ntej ATM-tsis muaj nas feem ntau tsis muaj cov yam ntxwv uas xav tau los ua kom muaj kev sib txuas ntawm cellular thiab molecular tsis txaus rau ataxia phenotype. Ntau qhov kev cog lus ntawm kev tshawb nrhiav tau raug txiav txim siab, suav nrog cov tsom mus rau qib neuronal, qhov twg ATM cuam tshuam rau oxidative stress signaling (Chen et al.2003) thiab synaptic function (Li et al.2009; Vail et al.2016), as zoo li glial muaj nuj nqi, qhov uas tsis ntev los no pov thawj qhia glial pathology tej zaum yuav yog ib qho tseem ceeb ntawm cerebellar pathology (Kaminsky li al. 2016; Campbell li al.2016; Petersen, Rimkus, thiab Wassarman 2012; Weyemi li al.2015). Tus qauv tsiaj tshiab no muab cov cuab yeej tshiab los ntsuas cov kev xav ntawm lub tshuab ATM uas tsis muaj peev xwm ua rau cerebellar pathology thiab ataxia. Tsis tas li ntawd, tus qauv no tuaj yeem ua haujlwm tseem ceeb tshaj plaws raws li cov cuab yeej tseem ceeb hauv kev sim tshuaj ntsuam xyuas yav dhau los cov neeg sib tw kho mob (Browne li al.2004; Chen et al.2003) thiab peb tus kheej SMRT compounds (Du et al. 2013). Cov kev txwv loj ntawm tsis muaj tus qauv tsim nyog rau kev kuaj mob, tshwj xeeb tshaj yog rau cov kab mob tsis tshua muaj xws li AT thiab AOA1, tsis tuaj yeem hais ntau dhau.

4.0 Cov ntaub ntawv thiab cov txheej txheem

4.1 Nqe lus hais txog Ethics

Txoj kev tshawb no tau ua tiav raws li cov lus pom zoo hauv Phau Ntawv Qhia Txog Kev Saib Xyuas thiab Kev Siv Cov Tsiaj Txhaum Cai ntawm National Institutes of Health. Tag nrho cov tsiaj tau raug tswj raws li Pawg Saib Xyuas Tsiaj Saib Xyuas Tsiaj thiab Siv Siv (IACUC) kev pom zoo ntawm Lub Tsev Haujlwm Lundquist (31374-03,31773-02) thiab UCLA(ARC-2007-082, ARC{{3} }). Cov txheej txheem tau pom zoo los ntawm Pawg Neeg Saib Xyuas Kev Ncaj Ncees ntawm Tsiaj Kev sim ntawm Lundquist Institute (Assurance Number: D16-00213). Txhua qhov kev siv zog tau ua kom txo qis qhov mob thiab kev txom nyem los ntawm kev txhawb nqa thaum tsim nyog thiab xaiv qhov kawg ntawm kev coj ncaj ncees.

4.2 mus

All mice were group-housed and kept under a 12-h day/night cycle with food and water available ad libitum. Animals were housed within the general mouse house population, and not in specialized pathogen-free rooms. Older animals were made available wetted food or food gel packs on the ground of the cages as ataxia developed. Atm35 and Atm935×mice were created and provided by Dr. Hicks and colleagues at the University of Manitoba. These mice were created to contain the c.103C>T kev hloov pauv pom nyob rau hauv cov neeg coob ntawm North AfricanATcov neeg mob using recombineering Gateway technology and site-directed mutagenesis. A C>T mutation at this position in the mouse Atm gene creates a TAG G stop codon. The same mutation in the human ATM gene produces a TGA G stop codon. In consideration of the use of these models for therapeutic interventions, we chose to create a mouse model for each of the two PTC codons(Fig.1A). A modified Gateway R3-R4-destination vector was used to pull out the desired region of the mouse Atm gene from a Bacterial Artificial Chromosome (BAC) and subsequently mutated to create either a TAG G stop codon at codon 35(M00001, position 103(C>T))or a TGA G stop codon (M00002, position 103 (CAG>TGA), replicating tib neeg AT PTC). Cov genomic alleles ces tau cloned rau hauv ib qho kev hloov kho ntawm NorCOMM mammalian targeting vector siv 3-way Gateway Reaction(Bradley li al. 2012). Cov txiaj ntsig tau pom cov vectors tau electroporated rau hauv C2 ES hlwb (C57BI/6N, muab tau los ntawm A. Nagy lab, Toronto, Canada) thiab ua tiav cov phiaj xwm clones raug txheeb xyuas los ntawm kev xaiv nrog G418 (Gertsenstein li al.2010). Kev sib koom ua ke ntawm kev hloov pauv lub hom phiaj rau hauv Atm gene locus tau lees paub los ntawm Southern blot, thiab los ntawm kev sib luag ntawm PCR cov khoom kom paub meej tias muaj kev hloov pauv ntawm Atm PTC, tsis muaj qhov yuam kev tsis muaj phiaj xwm rau hauv Atm locus, thiab tsis muaj qhov yuam kev tsis ua haujlwm ntawm cov khoom siv.

vector (cov ntaub ntawv tsis qhia). Qhov zoo ES clones tau siv rau kev txhaj tshuaj blastocyst kom tau txais cov kab hloov pauv. Lub transgenic allele muaj ib tug floxed human beta-actin promoter-deltaTK1-Neo cassette in the intron upstream of the region containing the mutated exon. This floxed cassette was subsequently excised by crossing with a Cre driver mouse(B6.C-Tg(CMV-are)1Cgn/J) to generate Atm3x+ and AtmM1(103CTMFcc, respectively) mouse lines Atm35×+ (MGI nomenclature: AtmTM1(103CAG>TGA)MFGCC;(Fig.1A). Genotyping ntawm ob kab Atm tau ua los ntawm kev siv cov primers hauv qab no ntawm Tm 62 degree degree: Atm noob rau pem hauv ntej (F) primer: 5'-CCTTTTGAGGCAATAAGTTGCAACTTG-3'; thiab Atm gene thim rov qab (R) primer: 5'-GTACAGTGTATCAGGTTAGGCATGC-3', tsim cov khoom lag luam tsiaj qus ntawm 151bp lossis cov khoom lag luam tsom ntawm 241bp (Figs. 1A, 1B).Atmn35 ~ thiab Atm035 × v tau rov qab hla nrog C57Bl / 6J nas rau 9 tiam (99.2 feem pua ​​​​ isogenic) ua ntej cryopreservation thiab rov qab rov qab siv C57BI / 6J surrogate niam. Atm735 × thiab Atm?35xand Atm 35 × / Q35 × yog cov neeg yug tsiaj tau los ntawm F1 kwv tij Atm35 thiab Atm835X ntxiv rau nas. AtmR35 × k35 ×: ob qho tib si tau pom tias muaj txiaj ntsig. Aptx knockout (Aptx) nas tau tsim thiab muab rau Dr. Mathews ua embryos los ntawm Dr. McKinnon (Ahel et al.2006), thiab tom qab ntawd tau muab rov qab los ntawm C57Bl/6J cov niam uas muaj menyuam. Aptx cov nas nyob ntawm C57Bl/6 thiab 129 sib xyaw keeb kwm yav dhau. yam 35k35;. Aptx nas ntawm ntau hom tsiaj qus, heterozygous, thiab homozygous ua ke tau tsim los ntawm Atm35X; Aptxt breeders tau tsim los ntawm hla Atm?35 × R35 thiab Aptx^ nas. Ib cohort ntawm ob chav mutant thiab cov nas tswj tau raug siv nyob rau hauv txoj kev tshawb fawb txog kev coj tus cwj pwm ntev rau kev soj ntsuam gait thiab SHERPA kuaj (Figs.2,3). Ntau pawg ntxiv ntawm cov hnub nyoog sib piv ob chav mutant thiab cov nas tswj tau siv rau kev sim electrophysiological, immunohistological, thiab Vertical Pole test thwmsim (Figs.4, 7). Kev sim tshuaj tiv thaiv kab mob thiab cov protein tau ua los ntawm kev siv cov nas bred los ntawm thawj AtmR35 × thiab Atm35 × cov nas rov qab (Figs.5, 6, thiab 8). Genotyping tau ua los ntawm cov qauv pob ntseg ntawm P8-11 nas. Cov txheej txheem PCR tiag tiag ua los ntawm Transnetyx Inc. tau siv los txiav txim siab txhua tus tsiaj lub genotype. Tsiaj txhu tau txheeb xyuas los ntawm cov ntiv taw tattoos muab rau tib lub sijhawm raws li pob ntseg biopsy. Cov cim tshwj xeeb rau Atm35 × thiab Atm35 × tau suav thiab siv los txheeb xyuas cov tsiaj qus, heterozygous thiab homozygous nas (cov npe saum toj no). Aptx'and Aptx*" primers tau siv los ntsuas lawv cov genotypes.

4.3 Tsiaj Noj Qab Haus Huv

Tsiaj txhu raug ntsuas los ntawm qhov ntsuas digital ntawm P8, 45, 120,210,400. Cov tsiaj tuag tau sau tseg raws li hnub pom tuag, lossis hnub ntawm euthanization thaum cov tsiaj mus txog qhov kawg ntawm tib neeg (tsiaj tsis tuaj yeem ua nws tus kheej hauv 60s, cov plaub hau tseem ceeb uas qhia tias tsis muaj kev tu tus kheej lossis kev ntxhov siab ntau dhau raws li tau sau tseg los ntawm kws kho tsiaj. cov neeg ua haujlwm). Tsiaj carcasses tau khov tam sim ntawd thaum tuag, thiab postmortem necropsies tau nqa tawm hauv cov khoom siv. Qhov tshwm sim ntawm kev tuag tau txiav txim siab rau qhov zoo tshaj plaws ntawm peb lub peev xwm hauv kev koom tes nrog cov neeg ua haujlwm kws kho tsiaj (Dr. Catalina Guerra) los ntawm kev tshuaj xyuas qhov muag ntawm lub cev. Qee cov nas tau raug tua los yog raug pov tseg los ntawm cov neeg ua haujlwm vivarium thiab tau

Yog li ntawd sau npe tias "tam sim no." Cov nas uas tsis pom qhov pom kev tuag tau sau tias "indeterminable." Cov nas uas tau pom nrog cov thoracic loj nyob ze qhov chaw uas cov thymus ib txwm nyob hauv cov nas me tau teev tias muaj "mob qog noj ntshav." Tag nrho lwm yam tau txheeb xyuas. Tej yam tshwm sim ntawm kev tuag (xws li, lub siab loj, urinary blockage) tau sau tias "lwm yam."

4.4 Kev coj cwj pwm

Ua ntej ua ib qho kev ntsuam xyuas tus cwj pwm, cov nas tau txais txiaj ntsig zoo rau qhov kev coj cwj pwm rau ~ 20 feeb. Cov nas tau sim nyob rau lub sijhawm sib txawv ntawm hnub, raws li lawv lub voj voog txhua hnub. Lub roj teeb ntawm kev ntsuas tus cwj pwm tau ua rau ntawm cov nas tsis paub ob npaug ntawm cov genotypes qhia nyob rau ntau lub sijhawm cov ntsiab lus nyob ntawm tus cwj pwm tab sis nyob rau tib pawg nas. Lub roj teeb ntawm cov kev xeem suav nrog Catwalk Gait kev ntsuam xyuas (P45, 120,210, 400) thiab ib pawg ntawm SmithKline-Beecham Harwell Imperial-College thiab Royal-London-Hospital Phenotype Assessment (SHERPA) tests (P30 thiab 400). Cov kev xeem no tau ua los ntawm UCLA Behavioral Core. Ob chav mutant thiab cov nas tswj tau raug tshuaj xyuas ntxiv ntawm Vertical Pole test. Tag nrho cov cuab yeej coj tus cwj pwm raug tshem tawm nrog ethanol (70 feem pua) ntawm txhua qhov kev xeem.

Gait Analysis

Peb tau siv Noldus Catwalk Gait tsom xam qhov system tsim los ntsuas ib nrab tsis siv neeg ntsuas thiab txheeb xyuas cov nas thaum lub sijhawm ua haujlwm ib txwm muaj. Luv luv, kev txav ntawm nas hla lub iav-hauv qab txoj hauv kev yog video kaw los ntawm qhov chaw nruab nrab. Paw prints yog highlighted nyob rau hauv cov yees duab vim lub teeb illumination hla lub iav taug kev platform. Txhua kauj ruam nas nyob rau hauv ib qho vis dis aus tom qab kuaj pom siv Catwalk XT (Noldus) hauv ib nrab-automated zam. Ib qho kev khiav rau txhua tus nas muaj 3 qhov kev sim ua kom zoo ib yam thoob plaws lub platform saib xyuas. Tsuas yog cov kev sim sib raug zoo tau lees txais, thiab cov nas tuaj yeem siv sijhawm txog 10 qhov kev sim ua kom tiav 3 qhov kev sim ua raws li ob qho kev taw qhia thoob plaws txoj kev hauv tsev. Cov kev sim ua raws cai tau txhais tias yog cov uas muaj kev txav mus los thoob plaws lub platform hauv qab 5 s-ntev thiab tsis muaj ntau tshaj 60 feem pua ​​​​ntawm kev hloov pauv ceev. Thaum muab tso rau hauv lub platform, cov nas feem ntau khiav rov qab thiab tawm mus yam tsis xav tau kev sim ua kom sai.

Ncej Ncej

Cov nas yog muab tso rau saum lub qhov siab ntawm 80 cm nrog lawv lub qhov ntswg tig mus thiab hind paws ze rau sab saum toj li sai tau. Cov nas raug tso tawm tam sim ntawd, thiab lub sijhawm pib tam sim ntawd thaum muab tso rau.

Lub sij hawm nres thaum thawj forepaw kov saum npoo hauv qab tus ncej. Ib tug nas lub ntuj predilection yog tam sim ntawd nce tus ncej, thiab lawv raug muab mus txog 60s mus hla tus ncej, txwv tsis pub, lawv raug pab tawm ntawm tus ncej. Ib qho kev sim tsis tiav yog tau txais ib lub sijhawm ntawm 30s, raws li 95 feem pua ​​​​ntawm cov nas uas tsis tau nqis los hauv 30s tseem nyob ntawm tus ncej ntawm 60s cim. SHIRPA Behavioral xeem tau ua los ntawm University of California, Los Angeles Behavioral Core ntawm P30

thiab P400.Txhua qhov ntsuas tau raug ntsuas los muab kev ntsuas ntau. uas ua rau muaj kev sib piv ntawm cov txiaj ntsig ob lub sijhawm thiab ntawm cov chaw sim sib txawv. Txhua tus nas tau raug sim ua ntu zus hla txhua tus cwj pwm hauv ~20-min lub sijhawm ua ntej txav mus rau tus nas tom ntej. Tus kws sim tau dig muag rau tsiaj genotype. Qhov kev tshuaj ntsuam tau ua raws li tau piav qhia yav dhau los (Rogers li al. 1997).

Kev soj ntsuam tus cwj pwm

Lub vijtsam tseem ceeb muab qhov kev soj ntsuam tus cwj pwm thiab kev ntsuas ntawm txhua tus tsiaj pib los ntawm kev soj ntsuam tus cwj pwm tsis muaj kev cuam tshuam hauv lub thawv saib (10 cm inch) rau 5 min. Ntxiv nrog rau cov qhab nia ntawm lub cev txoj hauj lwm, kev ua haujlwm ntawm tus kheej, kev ua pa, thiab kev tshee, tus neeg soj ntsuam teev cov xwm txheej ntawm tus cwj pwm txawv txawv los yog tsis meej pem thiab convulsions, compulsive licking, self-destructive tom, thiab retropulsion (taug kev rov qab) thiab indications ntawm spatial. disorientation.

Arena cwj pwm

Tom qab ntawd, tus nas raug xa mus rau qhov chaw ua yeeb yam (30 cm x 50 cm) rau kev ntsuas kev hloov pauv arousal thiab kev soj ntsuam ntawm tus cwj pwm zoo. Lub arena yog cim rau hauv daim phiaj ntawm 10 x 10 cm² squares los ntsuas qhov kev ua haujlwm ntawm locomotor hauv 30 s-lub sijhawm. Thaum tus nas ua haujlwm nyob rau hauv qhov chaw ua si, kev ntsuas ntawm cov lus teb pib, gait, pelvic elevation, thiab tus Tsov tus tw nce.

Supine Restraint

Tus tsiaj yog txwv nyob rau hauv ib tug supine txoj hauj lwm los sau autonomic cwj pwm. Thaum qhov kev ntsuam xyuas no, tuav lub zog, lub cev lub suab, pinna reflex, corneal reflex, ntiv taw pinch, hlau maneuver, thiab lub plawv dhia tau raug soj ntsuam.

Balance thiab Orientation

Thaum kawg, ntau qhov kev ntsuas ntawm vestibular system ua haujlwm tau ua. Txoj cai reflex, hu rau righting reflex, thiab tsis zoo geotaxis kuaj tau ua. Thoob plaws hauv txoj kev no, kev tso zis, thiab kev ntshai, kev chim siab, lossis kev ua phem raug kaw.

Cov cuab yeej siv

1.Clear Plexiglas arena (kwv yees qhov ntev 55x33 x 18 cm). Nyob rau hauv pem teb ntawm lub arena yog ib daim ntawv Plexiglas cim nrog 15 squares (11 cm). Ib txoj kab rov tav kab rov tav (3 hli txoj kab uas hla) tau ruaj ntseg hla lub nraub qaum sab xis xws li cov tsiaj tsis tuaj yeem kov ob sab thaum lub xaim maneuver. Ib daim phiaj (40x 20 cm) nrog 12 hli mesh (kwv yees) yog ruaj khov hla qhov dav ntawm lub thawv rau ntsuas tus Tsov tus tw thiab tuav lub zog tuav. 2. Lub tog raj kheej Plexiglas ntshiab (15 x ll cm) tau siv los ua lub thawv saib. 3. Ib daim phiaj hauv pem teb (40x 20 cm) nrog 12mm meshes ntawm qhov pom cov thawv sawv.4. Plaub cylindrical stainless-steel txhawb nqa (3 cm ntev x 2.5 cm txoj kab uas hla) los tsa daim phiaj tawm ntawm lub rooj ntev zaum. 5. Ib square (13 cm) stainless hlau phaj rau kev hloov cov tsiaj mus rau lub arena. 6. Txiav ntev ntawm 3/0 Mersilk tuav nyob rau hauv lub forceps rau corneal thiab pinna reflex kev ntsuam xyuas 7. Ib tug yas dowel pas nrig yog sharpened mus rau ib tug mem hluav taw tes mus kuaj salivation thiab tom.8. Ib khub ntawm cov cuab yeej txiav tawm, nkhaus nrog cov ntsiab lus zoo (125 mm forceps, Philip Harris Scientific, Cat. No. D46-174), rau ntiv taw pinch. 9.A stopwatch.10. Ib lub thawv IHR Nyem yog siv los ntsuas cov lus teb tsis txaus ntseeg. Nyem Box tsim ib lub suab luv luv 20 kHz ntawm 90dB SPL thaum tuav 30cm saum tus nas. Hu rau Prof. KP Steel, MRC Lub Tsev Kawm Ntawv ntawm Kev Tshawb Fawb Kev Hnov Lus, University Park, Nottingham NG7 2RD. 11.A ruler.12.A 30 cm ntshiab Plexiglas raj nrog ib sab hauv ntawm 2.5 cm rau kev sib cuag righting reflex.4.5 Electrophysiology Kev npaj ntawm mob cerebellar hlais ntxim hlub parasagittal slices ntawm 300 μm thickness tau npaj los ntawm cerebellum ntawm kev sim thiab tswj littermate nas. los ntawm kev ua raws li cov txheej txheem luam tawm (Hansen li al., 2013). Hauv luv luv, cerebella tau raug tshem tawm sai sai thiab muab tso rau hauv cov dej khov-txias extracellular tov nrog cov muaj pes tsawg leeg ntawm (mM): 119 NaCl, 26 NaHCOg, 11 qabzib, 2.5 KCl, 2.5 CaCla, 1.3 MgCla, thiab 1 NaHzPOa, pH7.4 thaum gassed nrog 5% CO-/95% O2. Cerebella tau muab faib ua parasagittally siv lub vibratome (Leica VT-100, Leica Biosystems, Nussloch, Lub teb chaws Yelemees) thiab pib incubated ntawm 35 degree rau -30 min, thiab tom qab ntawd sib npaug thiab khaws cia hauv chav sov kom txog thaum siv. Extracellular Electrophysiology Extracellular thiab intracellular recordings tau txais los ntawm Purkinje neurons (PNs) nyob rau hauv slices tas li perfused nrog ib tug carbogen-bubbled extracellular tov thiab tswj ntawm 37 degree C (extracellular) los yog 32 degree (intracellular) ± 1 degree (saib saum toj). Cells tau pom nrog DIC optics thiab dej-immersion 40 × lub hom phiaj (NA 0.75) siv Zeiss Examiner microscope. Cov iav pipettes ntawm ~ 3 MQ tsis kam (Model P-1000, Sutter Instruments, Novato, CA) tau ntim nrog cov tshuaj extracellular thiab muab tso rau ze ntawm PN axon hillocks txhawm rau ntsuas qhov muaj peev xwm cuam tshuam txog kev cuam tshuam tam sim no hauv voltage-clamp hom nrog lub peev xwm pipette tuav ntawm 0 mV. Rau tag nrho-cell patch-clamp kaw, pipettes tau ntim nrog cov tshuaj intracellular (mM): 140 lub hlis (CH3KO3S), 10 NaCl, 2 MgCl2,0.2 CaClz, 10 HEPES, 14 Phosphocreatine (tris ntsev), 1 EGTA, 4 Mg -ATP,0.4 Na-GTP.100 μM Picrotoxin (Sigma) tau ntxiv los thaiv inhibitory GABAegeric synaptic inputs. Cov ntaub ntawv tau txais los ntawm kev siv

MultiClamp 700B amplifier ntawm 20 lossis 100 kHz hauv voltage lossis tam sim no-clamp hom, Digidata 1440 nrog pClamp10 (Molecular Devices, Sunnyvale, CA) thiab lim ntawm 2 txog 4 kHz. Lub series kuj feem ntau yog nyob nruab nrab ntawm 10 thiab 15 MQ. Series kuj tau them nyiaj ntawm 80 feem pua ​​​​rau cov kev sim plasticity luv luv nkaus xwb. Rau cov ntaub ntawv kaw tseg, tag nrho ntawm 20 txog 45 PNs raug kaw los ntawm txhua tus tsiaj thoob plaws txhua hom genotypes, poj niam txiv neej, thiab hnub nyoog pawg. Cov ntaub ntawv kaw tseg tau muab faib thoob plaws ob qho tib si nruab nrab- sab nraud thiab rostrocaudal axis ntawm cerebellum. Tsuas yog cov hlwb uas muaj "kev noj qab haus huv" zoo nkaus li (tsis tshua muaj qhov sib txawv ntawm cov ciam teb ntawm tes) thiab tsis tu ncua, tsis muaj kev cuam tshuam kev tua hluav taws raug tshuaj xyuas. Thaum lub sij hawm soj ntsuam, ob peb lub hlwb tau pom tias muaj qhov sib txawv ntawm kev tua ntau dua 2 vib nas this, thiab cov hlwb no raug tshem tawm los ntawm kev tshuaj ntsuam, vim tias hom kev tua hluav taws no cuam tshuam nrog kev ua "tsis zoo." Ob chav mutant cov ntaub so ntswg tsis zoo sib txawv nyob rau hauv. Cov tsos mob nyob rau hauv DIC microscopy ua ntej cov ntaub ntawv kaw tseg, tsis yog tus naj npawb ntawm cov hlwb "tsis zoo" ntau dua li ntawm lwm cov genotypes (7 feem pua ​​​​vs 4 rau 11 feem pua ​​​​ntawm tag nrho cov hlwb hla kev tswj genotypes ntawm P400). Kev sib piv ntawm cov neural kev ua haujlwm tau txais los ntawm kev sau cia los ntawm cov ntu ntu hauv flocculus, lateral (2 "d lossis 3'), nruab nrab (6" lossis 7th), thiab medial (11" lossis 12t) daim. cov hnub nyoog qis dua (P45 thiab 110) kom sib npaug ntawm qhov sib txheeb ntawm cov ntaub ntawv kaw tseg thoob plaws pawg hnub nyoog. 0-3 cov ntaub ntawv kaw tau tsim los ntawm txhua lub lobule nyob rau hauv txhua daim nyob ntawm cov ntaub so ntswg zoo thiab kev noj qab haus huv. Txhua cov ntaub ntawv kaw tau ntev rau {{29} } feeb. 3 mus rau 5 nas tau siv rau txhua pab pawg hnub nyoog, thiab tus kws sim tau ua rau qhov muag tsis pom qhov genotype, hnub nyoog, thiab poj niam txiv neej.

Intracellular recordings tau txais los ntawm PNs nyob rau hauv ob qho tib si lobule IIl los yog VIl ntawm medial cerebellum (ie, vermis); Tsis muaj qhov sib txawv ntawm cov khoom raug pom ntawm lobules.

Kev tshuaj xyuas

Kev txiav txim siab ua rau muaj peev xwm cuam tshuam nrog lub sijhawm tau kuaj pom thiab tshuaj xyuas siv cov qauv thiab kev cai niaj hnub hauv ClampFit (Molecular Device), GoPro (Wavemetrics), thiab Excel (Microsoft). Tshwj xeeb, qhov ua tau zoo tau raug kuaj pom, thiab cov txheeb cais tawm (piv txwv li, zaus thiab lub sijhawm ntev) tau txiav txim siab sivadaptedlgorProroutines(TaroTools;https://sites.google.com/site/tarotoolsregister/). Qhov sib txawv ntawm qhov sib txawv ntawm qhov nruab nrab nruab nrab ntawm qhov nruab nrab (CV) thiab qhov nruab nrab nruab nrab ntawm qhov nruab nrab (CV2=2 ISIn ntxiv rau 1-ISInl/(ISIn ntxiv rau 1 ntxiv rau ISIn)) tau suav hauv Excel siv kev cai macro. Cov txheej txheem membrane cov khoom raug tshuaj xyuas siv lgorPro. RM tau txiav txim siab los ntawm qhov nruab nrab 3 qhov hluav taws xob taug qab cov lus teb rau -5 mV kauj ruam mem tes los ntawm -80 mV tuav lub peev xwm thiab ntsuas qhov tshwm sim tam sim no deflection ntawm 900 thiab 1000 ms tom qab pib. Lub sij hawm daim nyias nyias tau ntsuas los ntawm kev haum ib qho exponential rau thawj theem kev lwj ntawm 90 feem pua ​​​​rau 10 feem pua ​​​​ntawm cov ncov. CM tau suav los ntawm kev faib lub sijhawm membrane tas li los ntawm RM.sEPSC cov xwm txheej tau sau tseg dhau ntawm 1- feeb sijhawm thiab kuaj pom thiab ntsuas siv Neuroexpress (v21.1.13). Parallel thiab nce toj fiber ntau axons tau txhawb nqa siv theta-iav electrodes (WPI) thiab TTL-tswj stimulus cais tawm (ISO-Flex, AMPI). Evoked EPSC amplitudes thiab lwj lub sij hawm tsis tu ncua (1 exp. rau parallel thiab 2 exp. rau nce fibers) tau soj ntsuam siv cov kev cai niaj hnub hauv lgorPro. Cov peev xwm ua tau raug tshuaj xyuas raws li ib feem ntawm cov txheej txheem ntawm 1 s tam sim no txhaj tshuaj ntawm -500 thiab 2250 pA (250 pA cov kauj ruam) nrog lub tuav tam sim no kho kom muaj peev xwm ~ 70 mV. Kev txiav txim muaj peev xwm waveforms tau ntsuas los ntawm kev cai

cov txheej txheem hauv lgorPro. Kev ua haujlwm muaj peev xwm pib tau txhais tau tias yog thawj daim nyias nyias qhov hluav taws xob nyob rau hauv uas thawj derivative tshaj 30 mV / ms (Zhu li al.2006).

4.6 Kev kuaj mob Cerebellar Atrophy

Cerebellar loj Tam sim ntawd tom qab lub hlwb tshem tawm ntawm pob txha taub hau, ib tug dorsal, tag nrho-mount duab tau txais. Cov duab tau ua tiav siv Fiji (NIH). Lub forebrain thiab cerebellar qhov ntau thiab tsawg tau soj ntsuam los ntawm kev piav qhia lawv 2- qhov chaw seem thiab tom qab ntawd suav cov cheeb tsam. Peb normalized rau qhov sib txawv ntawm lub hlwb tag nrho los ntawm kev faib cov txiaj ntsig ntawm cerebellum los ntawm forebrain loj los tsim ib tus txheeb ze cerebellum-rau-forebrain ratio. Cov kws sim tau qhov muag tsis pom qhov genotype ntawm tus tsiaj. Immunohistochemistry Ntawm qhov kawg ntawm txoj kev kawm (P45, 120,210,4{{7{{1{108}}4}}}}}0), txiv neej thiab poj niam nas ntawm tag nrho cov genotypes sawv cev rau hauv txoj kev tshawb no tau tshuaj loog nrog isoflurane thiab underwent transcardial perfusion nrog phosphate-buffered saline ua raws li 4 feem pua ​​(w/v) buffered paraformaldehyde (PFA) thiab tom qab ntawd txiav mus rau hauv lub hlwb. Cov duab ntawm tag nrho lub hlwb raug coj tam sim ntawd tom qab tshem tawm lub hlwb ntawm lub pob txha taub hau thiab lub hlwb ces submerged hauv 4 feem pua ​​​​PFA rau 24 teev, thiab ces cryoprotected hauv Tris-buffered saline (TBS) nrog 0.05 feem pua ​​​​azide thiab 30 feem pua ​​sucrose rau 72. teev thiab khaws cia ntawm 4 degree kom txog thaum siv ntxiv. Lub cerebellum tau muab cais tawm ntawm lub forebrain thiab parasagittal sectioned siv lub zawv zawg microtome (Microm HM 430, Thermo Scientific) teem rau ntu ntawm 40 μm thickness. Cerebellum seem tau sau nyob rau hauv ib tug series ntawm rau thiab muab cia rau hauv TBS-AF (TBS nrog 30 feem pua ​​sucrose, 0.05 feem pua ​​sodium azide, thiab 30 feem pua ​​​​ethylene glycol) ntawm 4 degree los yog -20 degree mus txog rau thaum siv ntxiv. Rau immunofluorescent visualization ntawm Purkinje neurons, cerebellum seem ntawm ob Atm *; Aptx*t thiab Atm?35×R35×; Aptx^(n=5 ib genotype) raug ntxuav rau 5 min hauv TBS peb zaug thiab tom qab ntawd thaiv hauv 15 feem pua ​​​​tshuaj tshis ib txwm nyob hauv chav sov li 30 min tom qab los ntawm qhov dej dawb-floating incubation hauv luav lossis nas anti-calbindin D -28k (1:1000) rau 1 teev ntawm chav tsev kub ntawm lub orbital shaker. Tom qab ntawd cov seem raug ntxuav rau 5 feeb nrog TBS peb zaug, ua raws li cov dej ntws tawm hauv cov tshis los tiv thaiv luav lossis nas Alexa Fluor 488 (1: 1000) rau 1 h hauv qhov tsaus ntuj ntawm chav tsev kub ntawm lub orbital shaker. Tom qab cov tshuaj tiv thaiv kab mob thib ob, ntu tau ntxuav rau 5 feeb hauv TBS peb zaug, tom qab ntawd txuas thiab npog-slipped nrog Fluoromount-G nrog DAPI. Rau qee ntu, cov tshuaj tiv thaiv kab mob Caspase-3(1:200) thiab anti-CD68(1:400) cov tshuaj tiv thaiv ntxiv tau raug soj ntsuam ua ke nrog Calbindin siv Alexa Fluor 647 (1:500) cov tshuaj tiv thaiv thib ob. Slides raug tshuaj xyuas siv Stereo Investigator (MBF Bioscience, ver.2020) ntawm Zeiss microscope nruab nrog ApoTome 2 (Carl Zeiss Microscopy, Axio Imager.M2) siv 2.5, 10, 20, 40, lossis 63x lub hom phiaj, thiab cov duab ntes nrog Hamamatsu CMOS lub koob yees duab (Hamamatsu Photonics, ORCA Flash 4.0 LT plus ).Kom kom muaj nuj nqis ntawm calbindin-reactive hlwb hauv txhua lub lobule hauv cov duab tshwm sim, peb siv Stereo Investigator randomly kos 2 kab ntawm 300 txog 500 μm ntev hauv txhua lobule thiab manually suav tag nrho cov naj npawb ntawm PNs raws li qhov ntev li ntawm 40 um thickness ntawm cov ntaub so ntswg hlais nyob rau hauv 40x magnification.2D ceev (# ntawm PNs / (linear ntev * 40 um thickness)) ntawm ob qho tib si kuaj ib lobule ces nruab nrab rau kev sib piv ntxiv ntawm lobules thiab tsiaj txhu. Calbindin zoo PN dendrite qhov dav tau ntsuas ntawm qhov chaw nyob hauv qhov chaw nyob hauv lobule VI los ntawm txhua tus tsiaj hauv 25 lossis 40 um cov ntaub so ntswg tuab hauv qab 20x magnification. Dendritic widths ntawm thawj thiab theem nrab ceg tau ntsuas nyob rau hauv nruab nrab ntawm lub PN cell lub cev thiab ntug ntawm lub molecular txheej. Nruab nrab ntawm 7 thiab 13 dendrites tau ntsuas rau ib ntu, ib ntu rau ib tus tsiaj. Rau PN somatic kev ntsuas, Stereo Investigator tau siv los xaiv PNs random faib thoob plaws hauv seem nruab nrab hauv qab 20x magnification. Qhov nruab nrab PN dav rau ib tus tsiaj tau txiav txim siab los ntawm qhov nruab nrab ntawm qhov nruab nrab ntawm 3 ntu ntu (16 txog 37 PNs ib ntu). Molecular txheej thiab granule cell txheej (visualized nrog Calbindin thiab DAPI stains, ntsig txog) qhov dav tau soj ntsuam hauv Stereo Investigator los ntawm qhov nruab nrab ob qhov dav ntsuas ntawm qhov chaw ua ntej rau txhua lub lobule, kwv yees li ib nrab nrog rau qhov ntev ntawm txhua lub lobule hauv qab 2.5x magnification.CD68 Qhov zoo ntawm cov kab mob cerebellar tau ntsuas los ntawm kev ntsuas tag nrho cov feem pua ​​​​ntawm CD68 ntxiv rau qhov zoo staining hla tag nrho cov kab mob hauv nruab nrab. 10x stitched dluab tau pib rau kev tswj tsis zoo thiab suav nrog siv ImageJ, ib ntu rau ib tus tsiaj. Txhawm rau kom suav cov feem pua ​​​​ntawm Calbindin-zoo PNs uas zoo rau cleaved Caspase-3 peb suav PNs hla tag nrho cov cerebellum siv Stereo Investigator. Peb, 20x magnification stitched dluab ib tug tsiaj raug kuaj xyuas thiab cov txiaj ntsig tau nruab nrab. Qhov chaw pib rau Caspase-3 qhov zoo tau tsim los ntawm kev tswj cov seem uas tsuas yog cov tshuaj tiv thaiv kab mob thib ob. Rau kev tshuaj ntsuam xyuas tsis muaj fluorescent histological, 25-um-tuab, cov ntaub so ntswg dawb-floating mus rau qhov zoo nqi slides thiab cua-dried hmo ntuj. Cov ntaub so ntswg tau ntxuav hauv Phosphate-buffered saline (PBS) ob zaug rau 5 feeb, tom qab ntawd stained nrog 0.1 feem pua ​​​​Hematoxylin hauv 95 feem pua ​​​​ethanol rau ~ 25s thiab 0.5 feem pua ​​​​Eosin hauv 95 feem pua ​​​​ethanol rau ~ 3s thiab ntxuav hauv ob-distilled dej tom qab txhua stain. Cov ntaub so ntswg tom qab lub cev qhuav dej rau 1 min hauv 95 feem pua ​​​​ethanol, 100 feem pua ​​ethanol, thiab 100 feem pua ​​Xylene ntxuav, ces npog nrog Permount. Slides tau yees duab siv lub koob yees duab xim (Q Imaging, MBF Biosciences) ntawm tib Zeiss microscope thiab MBF nrhiav software. Cov kws sim tau ua qhov muag tsis pom rau tus nas genotype nyob rau hauv cov ntu uas tau kuaj xyuas, thiab qhov kev txiav txim ntawm kev ntsuam xyuas tau cuam tshuam rau txhua qhov kev ntsuas histological.

4.7 Flow Cytometry Kev Ntsuas

Flow cytometry tsom xam ntawm cov ntshav thiab thymus hlwb tau ua los ntawm staining nrog cov tshuaj tiv thaiv nas tshwj xeeb∶CD4, CD8, CD3, CD44, thiab CD25. Luv luv, tag nrho cov ntshav kuaj (50 μ) tau stained siv cov tshuaj tiv thaiv fluorescent-labeled, tom qab ntawd cov qe ntshav liab tau lysed siv cov tshuaj BD lysing thaum cov qe ntshav dawb nyob tau stained siv cov stain. Thymi tau mechanically dissociated. 1 txog 2 lab thymus hlwb tau zoo sib xws siv cov tshuaj tiv thaiv tshwj xeeb rau CD4, CD8, CD44, thiab CD25. Kev soj ntsuam ntawm cov tshuaj tiv thaiv kab mob ntshav dawb los yog cov qauv thymus tau ua tiav siv FACS ARIA l thiab cov ntaub ntawv raug tshuaj xyuas siv FlowJo software raws li tau tshaj tawm yav dhau los (Sanghez li al. 2017).

4.8 Western BIots

Protein extracts (cells/tissues) tau homogenized nyob rau hauv radioimmunoprecipitation assay (RIPA) lysis buffer (150 mM NaCl,1 feem pua ​​Nonidet P-40 [NP-40],0 .5 feem pua ​​deoxycholate, 0.1 feem pua ​​SDS, 50 mM Tris, pH 8.0) nrog protease inhibitors (10 ug/ml AEBSF, 10 ug/ml leupeptin, 5 ug/ml pepstatin, 5 ug/ml chymotrypsin, 10 ug/ml aprotinin). Cov protein rho tawm tau sonicated thiab ces pelleted los ntawm centrifugation ntawm 13, 000 rpm rau 15 min ntawm 4C. Kev ntsuam xyuas BCA protein tau siv los ntsuas cov protein ntau. Cov qauv uas muaj qhov sib npaug ntawm cov protein 50 mus rau 100 ug ib txoj kab tau sib cais siv 4 mus rau 12 feem pua ​​​​gradient TGX precast gels BioRad ces pauv los ntawm TransBlot Semi-Dry BioRad system siv Nitrocellulose hloov pob. Hloov cov blots tau stained los ntawm Ponceau S stain rau qhov sib npaug ntawm cov protein thauj khoom ces ntxuav thiab thaiv nrog 5 feem pua ​​​​cov mis tsis muaj roj qhuav hauv TBST rau 60 min ntawm chav tsev kub. Thawj cov tshuaj tiv thaiv kab mob tau tsim nrog co thaum hmo ntuj ntawm 4 degree. Blots raug soj ntsuam rau cov tshuaj tiv thaiv hauv qab no: ATM (D2E2) Luav mAb Cell Signaling, ntawm 1: 1000 dilution, -Actin (D6A8)Lab mAb Cell Signaling, GAPDH (D16H11) Luav mAb Cell Signaling ua raws li qhov tsim nyog horseradish peroxidase (- HRP) thib ob Anti-luav, Anti-nas rau 2 teev ntawm chav tsev kub. Tom qab ntau qhov kev ntxuav nrog TBST, Protein qhia tau pom los ntawm Radiance Plus chemiluminescence substrate siv Azure c400 thiab BioRad ChemiDoc imaging systems. Kev tsom xam Densitometric ntawm ATM tau ua tiav siv ImageJ. Cov kev sim tau ua nrog 2 cov txuj ci thiab 2-3 cov khoom siv roj ntsha raws li qhia.

4.9 Kev Ntsuas Kev Ntsuas

Tus naj npawb ntawm cov tsiaj uas tau xaiv rau txhua pab pawg tau ua raws li qhov kev ntsuam xyuas lub zog ua ntej siv GPower v3.1 raws li qhov loj ntawm 0.5, lub zog ntawm 0.8, thiab qhov loj me uas kwv yees los ntawm cov ntaub ntawv ua ntej los yog kev kawm ua ntej. Peb tau siv ob qho tib si parametric (1- thiab 2- txoj kev ANOVA) rau ib txwm faib thiab tsis yog parametric (Kruskal Wallace) cov txheej txheem rau cov ntaub ntawv ncua sij hawm los ntsuas qhov sib txawv ntawm cov pab pawg ua raws li kev sib piv ntau qhov kev sib piv raws li qhia hauv cov ntawv. Outliers rau cov ntaub ntawv tiv thaiv kab mob hauv Fig. 6 thiab 7 raug cais tawm ntawm txoj kev ROUT (Q=2 feem pua ​​). Cov kev txheeb xyuas tshwj xeeb siv rau txhua cov ntaub ntawv teev tseg hauv txhua daim duab lus dab neeg.Rau txhua daim duab: ns tsis tseem ceeb, *p Tsawg dua lossis sib npaug rau 0.05,** p<0.01,***><0.001,****><0.0001. data="" are="" reported="" as="" mean="" ±="" sem="" and="" box="" and="" whisker="" plots="" indicate="" the="" minimum,="" first="" quartile,="" median,="" third="" quartile,="" and="" maximum="" data="" values.="" all="" figures="" and="" statistical="" analyses="" were="" completed="" using="" excel="" (microsoft="" 360)="" or="" prism="" v8="" and="" 9="">