Txoj Kev Kom Txhim Kho Cov Kev Kawm Ua Ntej Ntawm Cov Hnub Nyoog Txog Cov Kab Mob Neurodegenerative: Ib Qhov Kev Pom Zoo Ntawm Cov Tsiaj Thiab HiPSC-Drived Models Part 3

Cov kab mob sib kis zoo sib xws tau raug soj ntsuam nyob rau hauv ntau yam qauv ntawm PD, nrog rau qhov ntxov tshaj plaws uas siv AAV2/2 lossis LVs.123–126Cov kab mob no tau txhaj rau hauv cov neeg laus nas hlwb tau siv los xa WT, A30P, lossis A53T mutants ntawm tib neeg a-syn rau tus kab mob. induction.

Kev sib raug zoo ntawm cov kab mob thiab kev nco yuav zoo li tsis zoo rau ntau tus neeg, vim cov kab mob feem ntau ua rau lub cev tsis xis nyob thiab mob. Txawm li cas los xij, cov kab mob kuj tuaj yeem muaj txiaj ntsig zoo rau kev txhim kho kev nco, feem ntau vim tias cov kab mob tuaj yeem txhawb kev ua haujlwm ntawm lub cev tiv thaiv kab mob.

Cov kev tshawb fawb tau pom tias kev tawm tsam kab mob tuaj yeem ua rau lub cev tiv thaiv kab mob, yog li txhim kho lub cev tiv thaiv kab mob. Qhov kev tiv thaiv kab mob no suav nrog kev nce cov qe ntshav dawb thiab lwm lub cev tiv thaiv kab mob tsim tawm, nrog rau kev tsim cov tshuaj tiv thaiv. Cov lus teb no tsis tsuas yog pab lub cev tiv thaiv kab mob cuam tshuam tab sis kuj pab txhawb kev ua haujlwm ntawm lub cev tiv thaiv kab mob thiab txhim kho kev noj qab haus huv tag nrho ntawm lub cev.

Viral tawm tsam kuj ua rau lub cev tiv thaiv kab mob hauv lub hlwb. Cov kab mob tiv thaiv kab mob no tuaj yeem tshem tawm cov khib nyiab thiab ua tsis zoo ntawm lub hlwb, txhim kho kev ua haujlwm ntawm lub paj hlwb, thiab txhawb nqa cov ntaub ntawv xa mus thiab kev ua haujlwm nco. Cov kev tshawb fawb kuj tau pom tias cov neeg muaj qee yam kab mob kis tau zoo dua thaum ua haujlwm nrog kev nco.

Tsis tas li ntawd, qee qhov kev tshawb fawb tau pom tias qee cov kab mob tuaj yeem txhawb lub cev los tsim cov paj hlwb-derived neurotrophic factor (BDNF), ib yam khoom uas txhawb kev loj hlob thiab kev loj hlob ntawm neurons. Cov tshuaj no muaj txiaj ntsig zoo rau lub hlwb ua haujlwm thiab tuaj yeem txhawb kev kho lub cev thiab kev nco txhim kho.

Tau kawg, hauv kev sib ntaus sib tua tiv thaiv tus kab mob, peb yuav tsum tau txais txoj hauv kev zoo thiab lub tswv yim, tswj tus cwj pwm kev nyiam huv, thiab tau txais kev pw tsaug zog txaus thiab tawm dag zog, kom peb lub cev tiv thaiv kab mob zoo tshaj plaws. Hauv luv luv, txawm hais tias tus kab mob no tuaj yeem ua rau peb lub cev tsis xis nyob, muaj cov pov thawj ntxiv tias nws tuaj yeem txhawb kev ua haujlwm ntawm lub cev tiv thaiv kab mob thiab muaj txiaj ntsig zoo rau peb kev noj qab haus huv thiab kev nco. Nws tuaj yeem pom tau tias peb yuav tsum txhim kho peb lub cim xeeb, thiab Cistanche tuaj yeem txhim kho peb lub cim xeeb zoo vim tias Cistanche tuaj yeem tswj hwm qhov sib npaug ntawm cov neurotransmitters, xws li nce qib ntawm acetylcholine thiab kev loj hlob, uas tseem ceeb heev rau kev nco thiab kev kawm. Tsis tas li ntawd, Cistanche tseem tuaj yeem txhim kho cov ntshav khiav thiab txhawb nqa cov pa oxygen, uas tuaj yeem ua kom lub hlwb tau txais cov khoom noj txaus thiab lub zog, yog li txhim kho lub hlwb tseem ceeb thiab kev ua siab ntev.

Nyem paub ntxiv los txhim kho kev nco

Cov kws sau ntawv pom tau hais tias muaj txiaj ntsig zoo ntawm a-syn hauv nigral DA neurons, nrog rau cov kab mob molecular thiab cellular thiab nigralDA degeneration uas tau hloov zuj zus zuj zus mus.

Txawm li cas los xij, thawj tiam ntawm rAAV2/2 vectors siv nyob rau hauv cov kev tshawb fawb no pom tau tias muaj kev puas tsuaj neuronal, txawm hais tias cov neurodegeneration ntawm tyrosine-hydroxylase-positive neurons, nrog rau lub sij hawm kawm, nws kuj sib txawv (25% -80% thiab 6 lub lis piam mus rau 1 xyoo. ib.), 123, 124

Nyob rau hauv sib piv, LVs encoding WT-, A30P-, los yog A53T-mutated a-synwere muaj peev xwm ntawm inducing neuronal cell poob nyob rau hauv nas nyob rau hauv ib tug ntau ncua sij hawm thiab zoo ib yam (25% -40% thiab 5 lub hlis, raws li).126 Ob qho kev tshawb fawb pom. ib tug zuj zus poob nyob rau hauv neurite lengthand o perikarya nyob rau hauv seem DA neurons.

Tsis tas li ntawd, loj cytoplasmic accumulations ntawm a-syn tau pom nyob rau hauv ob qho tib si cellbodies thiab neurites. Cov txiaj ntsig no tau rov ua dua tsis ntev los no inmultiple kev tshawb fawb uas siv ob lub viral platforms.127–131

LVs kuj tau siv ntau hom nas PD. Forexample, Lauwers thiab cov npoj yaig125 tau pom tias kev txhaj tshuaj ntawm LV nqa WT lossis A30P mutant ntawm a-syn hauv striatum, amygdala, lossis SNof nas muaj peev xwm ua rau muaj kev hloov pauv neurodegenerative cuam tshuam nrog PD nyob rau lub sijhawm thiab suav nrog kev ywj pheej ntawm neurtic o. thiab cytoplasmic inclusions.

Tsis tas li ntawd, txoj kev tshawb no pom tau hais tias nigral overexpression ntawm A30P-a-syn ua rau kwv yees li 25% ntawm tes poob ntawm 10-12 lub hlis.125 Kev xa ntawm A30P, E57K, thiab E35K mus rau SN ntawm tus nas siv LV tau pom qhov zoo sib xws hauv DA neuron. poob (kwv yees li 50%) piv nrog WT (30%), whereas lub ceev fibril-forming mutant A53T siv nyob rau hauv tib txoj kev tshawb no tsis qhia ib tug tseem ceeb txo nyob rau hauv DA cell xov tooj.

Interestingly, tag nrho cov yam ntxwv neuropathological pom nyob rau hauv nas (piv txwv li, onsetand mob hnyav) tshwm sim tsis tshua muaj hnyav piv rau cov nas, txawm hais tias qhov sib txawv no tej zaum yuav yog ib qho khoom cuav ntawm tus kab mob purity los yog ntau lawm titers ntawm tus kab mob txhaj thiab / los yog lwm yam cuam tshuam kev tsis sib xws.

Kev txiav txim siab tseem ceeb rau AD thiab PD Viral Vector Models

Txoj kev loj hlob ntawm cov serotypes tshiab, suav nrog AAV2/1, AAV2/5, AAV2/6, AAV2/8, AAVrh10, DJ, thiab DJ8, tau nthuav dav cellulartargets thiab txhim kho AAV transduction efficiency.

Cov AAVs serotyped nrog cov capsids tshiab no tau raug sim hauv nas thiab cov neeg tsis yog tib neeg los ntawm overexpress a-syn.132-137 Overexpression ntawm S129Aform xa los ntawm AAV2/5 thiab AAV2/6 tsis tu ncua qhia toxicity hauv cov txheej txheem neurodegeneration.134,138

Cov kev sim no kuj pom tau hais tias kev ceev faj ntawm kev noj cov tshuaj overexpression transgene yog qhov tseem ceeb, txawm tias tswj cov vectors nqa tsuas yog cov neeg sau xov xwm fluorescent xa ntawm cov titers siab tuaj yeem ua rau muaj kev cuam tshuam tsis zoo.135

Qhov kev ceeb toom no yuav tsum tau ua tib zoo xav thaum tsim kev sim cawm siav raws li AAV. Piv txwv li, nws tau pom tias qhov kev kho mob AAV harboring me interferingRNA (siRNA) lub hom phiaj SNCA ua rau muaj kev toxicity siab thiab ua rau muaj kev poob loj ntawm nigrostriatal DA neurons. Qhov kev tshawb fawb ntxiv no tau qhia txog cov kev txwv ntawm AAV daim ntawv thov hauv tsiaj txhu.

Txawm li cas los xij, nws tsis tuaj yeem raug cais tawm tias cov neurotoxicity tau tshwm sim los ntawm kev txo qis ntawm SNCA qib hauv cov qauv nas, asa-syn plays lub luag haujlwm tseem ceeb hauv DA neurons, thiab nws cov robustreduction tuaj yeem txo cov cell viability.139 Ua ke, qhov no qhia txog qhov tseem ceeb ntawm tsim ib lub cuab yeej rau kev kho kom zoo rau kev qhia txog cov kab mob uas cuam tshuam nrog cov noob rau kev tsim cov tshuaj kho noob.

Hauv qhov no, qhov tsis ntev los no CRISPR-dCas9-raws li qhov system tuaj yeem nthuav qhia qhov txiaj ntsig zoo dua rau kev hloov pauv cov noob qhia hauv qhov tseeb thiab zoo-tuned zam.

Raws li cov kab no, peb nyuam qhuav tsim LV-CRISPR-Cas9 system kom ua tiav qhov tseeb thiab zoo-tuned tswj ntawm SNCA noob qhia. Txoj kev tshawb no tau muab pov thawj ntawm kev xav tias kev tswj hwm ntawm cov noob qhia (xws li, rov qab los ntawm kev tshaj tawm) los ntawm kev kho cov kab mob epigenome yog ib qho tseem ceeb kho mob rau cov kab mob neurological, xws li PD, 140 tshwm sim los ntawm cov noob dysregulation.

Raws li xws li, tus kab mob vector PD qauv siv LV thiab AAV rau kev tshaj tawm ntawm a-syn protein zaub PD pathology txuam nrog DA neurodegeneration, thiab kev siv cov AAV tshiab serotypes txhim kho a-syn qhia hauv DA neurons piv rau AAV2/2.

Zuag qhia tag nrho, ob qho tib si AAVs thiab LVs muaj peev xwm inducing transgene qhia thiab qhia tau hais tias ib theem siab ntawm tropism rau DA neurons, ua rau cov qib zoo sib xws ntawm neurodegeneration. Kev txhim kho ntxiv ntawm cov kab mob kab mob no yuav tsum tau ua kom tsis txhob ua kom qeeb ntawm cov cellular PD phenotypes thiab cov theem qis ntawm kev coj cwj pwm tsis zoo.

Hauv Vitro Cell-Culture ModelsTam sim no Hauv Vitro AD thiab PD Cov Qauv: Cov Kev Txwv thiab Cov Sijhawm

Hauv plaub lub xyoos dhau los, ntau yam AD thiab PD hauv vitro qauv tau tsim los tshuaj xyuas kev sib cuam tshuam ntawm cov ntsiab lus tseem ceeb ntawm lawv cov kab mob pathophysiology thiab tom qab siv rau kev tshuaj ntsuam xyuas preclinical.

Tam sim no AD thiab PD hauv vitro qauv feem ntau tsom rau kev rov ua cov kab mob molecular thiab cellular, xws li mitochondrial tsis ua haujlwm, oxidative stress, cell survival, thiab muaj cov kab mob hauv nruab nrog cev thiab / lossis cov protein ntau ntxiv. Forexample, qhov tseem ceeb tshaj plaws thiab dav siv cellular qauv ntawm ADare raws li induction ntawm Ab40 42 aggregates los yog tau hyperphosphorylation thiab aggregation141,142 (Table 1).

Ib yam li ntawd, cov qauv hauv vitro ntawm PD yog ua raws li cov lus hais los ntawm cov tshuaj lom neeg ua rau mitochondrialdysfunction (piv txwv li, MPTP, rotenone, paraquat, 6-OHDA) thiab cuam tshuam cov proteostasis (xws li, thapsigargin, ionomycin, tunicamycin), a-synexpression overexpression. ntawm kev hloov pauv PD tsev neeg cov noob (piv txwv li, a-syn, Parkin, PINK1)92 (Table 2).

Txawm li cas los xij cov qauv ntawm tes-kev coj noj coj ua no tsis muaj nuj nqis hauv kev txhim kho peb txoj kev nkag siab ntawm pertinentneurobiology, lawv tseem tsis tau ua rau muaj txiaj ntsig DMTs tsom rau kev tiv thaiv, ncua kev pib, lossis qeeb kev kis kab mob, hu rau cov lus nug txog lawv qhov cuam tshuam thiab kev txhais lus siv tau.

Ntau qhov kev txwv meej hauv cov qauv no yog qhov tseem ceeb kom lees paub los txhim kho cov kev coj ua tam sim no.

Piv txwv li, cov qauv raws li overexpression thiab knockout/down of pertinent kab mob-koom nrog noob yog notoriously variable nyob rau hauv lub ntau yam ntawm cov kev hloov pauv rau cov qib protein thiab cov txheej txheem siv los induces hloov mus rau gene qhia (ie, viral-mediated, yug me nyuam-raws li, constitutive, los yog transient qhia) uas yuav ua rau contradictorydata thiab inferences.92

Qhov tsis muaj qhov tsis sib xws hauv kev tshem tawm cov kab mob cuam tshuam nrog cov phenotypes qhia txog qhov tsis txaus ntseeg rau cov kev tshawb fawb ua ntej siv cov qauv no los ntsuas qhov muaj peev xwm DMTs. Ntxiv mus, qhov sib txawv ntawm cov cellularphenotypes thiab cov txheej txheem tau pom nyob rau hauv preclinical NDD qauv (piv txwv li, hloov pauv ntawm cov protein ntau dhau los yog knockdown) qhia tau hais tias muaj ntau dua genetic heterogeneity uas tsis tuaj yeem tshawb pom hauv cov qauv ntawm tes tam sim no.

Qhov no yog qhov nyuaj ntxiv los ntawm kev nthuav dav ntawm cov qauv ntawm tes raws li cov noob caj noob ces pom nyob rau hauv tsev neeg pawg, txawm hais tias tsev neeg sib txawv sawv cev rau cov haiv neeg tsawg ntawm tag nrho cov xwm txheej NDD.109,143

Vim yog qhov tsis paub txog caj ces, nws tseem nyuaj rau qhov tseeb sawv cev rau cov pawg NDD lossis cov kab mob subtypes nrog cov caj ces tsis yog Mendelian hauv cov qauv ua ntej, tsim kom muaj kev cuam tshuam tseem ceeb hauv kev nthuav dav cov kab mob sib txawv, txheeb xyuas cov hom phiaj kho mob, thiab kev tsim tshuaj.

Raws li cov kev cov nyom no, cov kws tshawb fawb tau raug yuam kom ntsuas cov qauv no txhawm rau txheeb xyuas thaj chaw ntawm kev txhim kho thiab ntsuas cov cib fim uas nthuav tawm nrog cov txheej txheem tshiab ntawm tes-kev coj noj coj ua.

Xyoo tsis ntev los no, peb tau tsim los ntawm cov qauv no, suav nrog cov thev naus laus zis hloov kho tshiab thiab ntsuas cov koom haum tseem ceeb ntawm cov kab mob caj ces tau txais hauv GWASs hauv kev txhim kho cov ntaub so ntswg-culturemodels.144,145

Kev sib koom ua ke ntawm hiPSC thev naus laus zis sawv cev rau kev nce qib tseem ceeb hauv kev ua haujlwm ntawm NDD hauv vitro qauv thiab muab cov sijhawm yav dhau los tsis muaj peev xwm los siv cov kab mob ntsig txog cov xov tooj ntawm tes los ntawm cov neeg mob los tshawb txog kab mob pathogenesis hauv cov ntsiab lus ntawm tib neeg genome.

Tag nrho cov spectrum ntawm qhov zoo thiab cov kev txwv ntawm hiPSC-los ntawm NDD qauv, tshwj xeeb tshaj yog cov hais txog AD thiab PD, tau tshawb nrhiav tag nrho hauv seem tom ntej.

HiPSC-Drived Models ntawm NDDs: Ntau yam thiab Genetically "Faithful"

Kev nce qib niaj hnub hauv kev siv thev naus laus zis ntawm kev coj noj coj ua sawv cev yog ib qho kev ua tiav zoo tshaj plaws hauv kev tshawb fawb ntawm NDDs, muab lub sijhawm rau kev txhim kho tus qauv kab mob, tshuaj xyuas tshuaj, thiab tshuaj kho tus kheej.

Txog thaum tsis ntev los no, postmortem hlwb kuaj tau txiav txim siab kub-tus qauv tib neeg-raws li cov khoom siv lom neeg rau kev nkag siab txog cov txheej txheem pathological ntawm tib neeg cov kab mob neurodegenerative.

Txawm li cas los xij, cov thev naus laus zis niaj hnub no, suav nrog kev tshuaj xyuas ntawm cov neeg mob tshwj xeeb neurons thiab glial hlwb, tau qhib txoj hauv kev tshiab rau kev tshawb xyuas cov txheej txheem pathological ntawm NDDs thiab tau nce ntxiv hauv kev txiav tawm molecular ntawm NDDs nrog kev txhim kho caj ces nyuaj, xws li AD thiab PD.

Los ntawm ib qho yooj yim, noninvasive biopsy ntawm tus neeg mob daim tawv nqaij, ntxiv rau ntau yam ntawm lwm yam tshuaj lom neeg cov qauv, tiam ntawm hiPSCs ua rau cov kws tshawb fawb los qhia txog molecularpathways thiab kab mob mechanisms uas nyob rau hauv NDDs nyob rau hauv ib txoj kev uas yog tsis yooj yim sua nrog yav dhau los cell-culture txoj kev.

Tam sim no, cov hlwb pub dawb tshaj plaws yog fibroblasts, uas tau siv ntau dua 80% ntawm tag nrho cov kev sim reprogramming luam tawm; 146 txawm li cas los xij, hiPSCs tuaj yeem tau txais los ntawm ntau qhov chaw, suav nrog embryonic qaum stem cells, umbilical cord blood, corneal epithelial cells, thiab cov qe ntshav, xws li peripheral ntshav mononuclear cells147–149 (Daim duab 1).

Yog li ntawd, cov qauv hiPSC-derived sawv cev rau ntau yam kev hloov pauv rau cov qauv kab mob hauv cellular uas twb muaj lawm thiab tau txais los ntawm cov neeg mob somatic cov ntaub somatic uas raug quab yuam rau hauv pluripotency los ntawm kev siv ntau yam khoom siv reprogramming, xws li plasmids, vectors (piv txwv li, episomal), thiab kis kab mob (xws li. , adenovirus, Sendai tus kab mob, lentivirus;

Cov hlwb no yog tus kheej-renewing thiab muab lwm txoj rau embryonic stem cells (ESCs), uas yog nrog rau kev coj ncaj ncees thiab kev txhawj xeeb txog kev sib raug zoo. Tam sim no muaj ntau lub chaw khaws ntaub ntawv hiPSC uas sau thiab faib cov kab hiPSC los ntawm cov neeg mob uas muaj NDDs thiab kev noj qab haus huv muaj hnub nyoog sib luag los tswj cov qauv hauv nruab nrab thiab ua kom yooj yim rau cov kws tshawb fawb (Table S2).

Ib qho ntawm cov kev siv tseem ceeb ntawm hiPSCs yog qhov sib txawv ntawm cov xov tooj ntawm tes, uas yog li ntawd tau siv nyob rau hauv ntau yam kev lom neeg cov ntsiab lus, suav nrog neurodevelopmental152 thiab neurodegeneration kev tshawb fawb, 144 ex vivo transplantation, 153,154 kab mob qauv, lub hom phiaj validation, thiab tshuaj nrhiav pom.155

Txog niaj hnub no, ntau cov txheej txheem sib txawv ntawm tes tau raug tsim los uas tsis yog tsuas yog cov neurons nkaus xwb tab sis kuj tseem muaj cov neeg mob hlwb tshwj xeeb, suav nrog excitatory, cholinergic, DAs, thiab inhibitoryGABAergic neurons.156-158 Ntau yam tshwj xeeb glial subpopulationcells kuj tau muab los ntawm hiPSCs, suav nrog astrocytes, 159–161oligodendrocytes, 162,163 thiab microglia.164–166

Ntau yam kev hloov kho tshiab tshiab, suav nrog CRISPR-Cas9, zinc-ntiv tes nucleases (ZFN), thiab transcription activator-zoo li effector nucleases (TALENS), tau qhia txog qhov ntev ntxiv rau hiPSC-derivedsystem-kev tsim cov qauv isogenic uas muaj tib yam geneticbackground. thiab tsuas yog txawv ntawm qhov kev hloov pauv.167

Kev sib txawv ntawm cov txheej txheem sib txawv ntawm tes tau pab txhawb kev tshawb fawb txog kev siv tshuab, thiab muaj cov kab isogenic tau txhawb nqa kev tshuaj ntsuam genetic analyses of gene function and its pathogenic role in NDDs. hiPSC-derivedmodels yog tsim nyog rau cov kev tshawb fawb no, raws li lawv pom cov yam ntxwv tseem ceeb ntsig txog kab mob.

Piv txwv li, fAD- thiab sAD-derived hiPSCneuronal qauv nqa PS1 thiab PS2 kev hloov pauv (A246E thiab N14II, raws li) 168 thiab APP duplication mutation169 qhov tseem ceeb AD biochemical nta, suav nrog Ab secretion, nce Ab42: Ab40 ratio, thiab horylevated hyperpho.

Nws yog ib qho tseem ceeb uas yuav tsum nco ntsoov tias kev siv cov qauv hiPSC uas tsim cov phenotypes uas muaj feem cuam tshuam yog txhais tau tias yog kev tshawb nrhiav lub luag haujlwm ntawm Ab qhia hauv AD neurodegeneration thaum piv rau cov qauv ectopic qhia lossis cov qauv uas xav tau ntxiv thiab / lossis induction ntawm Ab aggregates, xws li cov no txais yuav qhov "natural" tshwm sim cov txheej txheem pathological ntsig txog Ab deposits.

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