Diesel Exhaust Exposure Hloov Cov Kev Qhia Txog Kev Sib Txuas Sib Txuas hauv Neurodegeneration hauv Zebrafish Brains Part 1

Abstract

Cov kab mob neurodegenerative yog ib qho laj thawj ntawm kev xiam oob khab hauv ntiaj teb, tab sis lawv cov etiologies feem ntau tseem tsis pom. Cov yam ntxwv ntawm caj ces tsuas tuaj yeem suav rau qee tus neeg tsawg ntawm kev pheej hmoo rau feem ntau ntawm cov kab mob no, qhia tias ib puag ncig yam tseem ceeb ua lub luag haujlwm tseem ceeb hauv kev txhim kho cov kab mob no.

Cov kab mob neurodegenerative hais txog ib hom kab mob uas ua rau degeneration ntawm hlwb hlwb vim yog vim li cas sib txawv. Feem ntau muaj xws li Alzheimer's disease, Parkinson's disease, Huntington's disease, thiab lwm yam. Cov kab mob no cuam tshuam rau cov neurons hauv lub hlwb, ua rau lub hlwb ua haujlwm tsis zoo thiab cov tsos mob xws li kev nco, kev xav, thiab kev hloov ntawm tus cwj pwm.

Txawm li cas los xij, peb tsis tas yuav poob peb tus cwj pwm zoo vim tias muaj cov mob no. Muaj ntau txoj hauv kev los pab cov neeg mob txhim kho kev nco thiab txo cov tsos mob. Cia peb saib qee yam ntawm cov txheej txheem no:

Ua ntej, tau txais kev tawm dag zog lub cev. Kev tshawb fawb qhia tau hais tias lub cev ua si txhim kho cov ntshav ncig hauv lub hlwb thiab nce kev tso tawm cov tshuaj xws li dopamine thiab endorphins, uas pab tiv thaiv thiab txhim kho cov haujlwm ntawm cov neurons. Tib lub sijhawm, kev tawm dag zog kuj tseem tuaj yeem pab txo cov teeb meem xws li kev ntxhov siab, kev nyuaj siab, thiab insomnia, ua rau cov neeg mob zoo siab rau lub neej.

Qhov thib ob, tswj kev sib raug zoo. Nws tseem yog ib qho tseem ceeb heev uas peb cov kev sib raug zoo sib raug zoo nrog rau cov cheeb tsam ntawm lub hlwb, thiab kev sib raug zoo tsis tuaj yeem tsuas yog txo cov kev ntxhov siab, tab sis kuj txhim kho lub hlwb kev noj qab haus huv los ntawm kev ua kom muaj kev zoo siab, sib ntaus sib tua, thiab lwm yam.

Thaum kawg, noj kom zoo thiab pw tsaug zog txaus. Ob qho no tuaj yeem muaj qhov cuam tshuam tseem ceeb rau lub hlwb kev noj qab haus huv. Kev noj zaub mov zoo tsis tsuas yog xav tau kom muaj cov as-ham txaus, tab sis nws kuj yuav tsum tsis txhob rov qab noj cov suab thaj ntau dhau, cov roj saturated, thiab cov roj cholesterol. Tsis tas li ntawd, kev pw tsaug zog txaus tuaj yeem pab tiv thaiv cov teeb meem nco ntawm ntau theem, tshwj xeeb tshaj yog cov kab mob xws li Alzheimer's disease.

Hauv luv luv, cov kab mob neurodegenerative tsis txhais hais tias muab kev cia siab rau lub neej. Thaum tuav tus cwj pwm zoo, nws kuj tseem yuav tsum tau txais cov kev tshawb fawb thiab kev ua tau zoo los txhim kho lub neej zoo. Kev luag thiab ntau lub hnub ci yuav pab koj noj qab haus huv. Nws tuaj yeem pom tias peb yuav tsum txhim kho kev nco, thiab Cistanche deserticola tuaj yeem txhim kho kev nco zoo vim Cistanche deserticola yog cov khoom siv tshuaj hauv Suav teb uas muaj ntau yam teebmeem, ib qho ntawm kev txhim kho kev nco. Kev ua tau zoo ntawm Cistanche deserticola los ntawm ntau yam khoom xyaw uas nws muaj, suav nrog tannic acid, polysaccharides, flavonoid glycosides, thiab lwm yam. Cov khoom xyaw no tuaj yeem txhawb lub hlwb kev noj qab haus huv los ntawm ntau txoj hauv kev.

Nyem paub 10 txoj hauv kev los txhim kho kev nco

Tsis ntev los no tau pom tias muaj kev cuam tshuam rau huab cua tsis zoo los ua kom muaj kev pheej hmoo ntawm Alzheimer's thiab Parkinson cov kab mob, tab sis cov txheej txheem molecular uas nws ua tsis tau zoo.

Zebrafishembryos raug rau cov khoom siv hluav taws xob tso pa tawm (DEPe) ua rau tsis ua haujlwm autophagy thiab neuronal poob. Ntawm no, peb nthuav tawm zebrafish embryos mus rau DEPe thiab ua tau zoo dhau los ntawm cov khoom siv proteomic thiab transcriptomic qhia tshuaj ntsuam xyuas los ntawm lawv lub hlwb txhawm rau txheeb xyuas cov kab mob pathogenic tshwm sim los ntawm huab cua phem. Kev kho DEPe tau hloov pauv ntau yam txheej txheem lom neeg thiab kev qhia txog txoj hauv kev cuam tshuam rau cov txheej txheem neurodegenerative, suav nrog xenobiotic metabolism, phagosome maturation, thiab amyloid ua. Qhov loj tshaj induction ntawm noob qhia hauv hlwb yog nyob rau hauv Cyp1A (tshaj 30-fold).

Qhov cuam tshuam ntawm qhov kev hloov pauv no tau lees paub los ntawm kev thaiv induction siv CRISPR / Cas9, uas ua rau muaj kev cuam tshuam loj heev rau DEPe toxicity, lees paub tias Cyp1A induction yog ib qho kev tiv thaiv kev tiv thaiv.

Cov kev tshawb fawb no tau txheeb xyuas cov kev cuam tshuam ntawm cov molecular txoj hauv kev uas tuaj yeem ua rau muaj kev cuam tshuam ntawm cov kab mob neurodegenerative disorders. Thaum kawg, kev txiav txim siab lub hauv paus molecular ntawm howair pollution ua rau muaj kev pheej hmoo ntawm neurodegeneration yuav pab txhim kho cov kab mob-modifyingtherapies.

Ntsiab lus

Cov pa phem · Dementia · Parkinson's Disease · Alzheimer's disease · Transcriptomics · Proteomics.

Taw qhia

Cov pa phem yog qhov tseem ceeb rau kev tuag thiab muaj feem xyuam nrog cov kab mob ua pa, mob plawv, mob stroke, mob ntsws cancer, thiab ntshav qab zib. Cov ntaub ntawv pov thawj tshwm sim los txhawb kev sib txuas ntawm huab cua paug thiab kev txhim kho cov kab mob neurodegenerative, suav nrog Alzheimer's thiab Parkinson's kab mob (AD thiab PD) (Fu li al. 2019). Muaj qhov tsis txaus ntseeg ntawm kev tshawb fawb tshawb xyuas cov txheej txheem uas cov pa phem tuaj yeem ua rau muaj kev pheej hmoo ntawm AD thiab PD, tab sis qee qhov uas muaj nyob ntawd txhawb nqa kev sib raug zoo (Fu li al. 2019).

Kev sib sau ntawm cov protein suav nrog hauv lub hlwb yog ib qho kev qhia thoob ntiaj teb ntawm neurodegenerativedisorders thiab yog ib txoj hauv kev uas ua rau kev ua haujlwm tsis zoo ntawm lub hlwb thiab kev tuag. Piv txwv li, theformation ntawm amyloid-beta (A-beta) thiab tau cov qauv yog cov qauv pathological ntawm AD thiab -synuclein (-syn) aggregates lossis Lewy Bodies inPD (Ross and Poirier 2004; Woulfe 2008).

Kev hloov pauv hauv proteostasis tshwm sim tsawg kawg yog ib feem ntawm kev tsim cov aggregates, tab sis cov txheej txheem meej tseem tsis tau paub thiab yuav txawv ntawm cov tib neeg nyob ntawm caj ces thiab ib puag ncig kev pheej hmoo. Peb paub tias kev nthuav tawm ntau ntxiv lossis txo qis ntawm A-betaor -syn tuaj yeem ua rau AD thiab PD (Bostancıklıoğlu2019; Johnson li al. 2019).

Lwm cov nta hauv cov kab mob neurodegenerative yog o thiab oxidative kev nyuaj siab. Activated microglia, cov kab mob inflammatory hauv CNS, tuaj yeem txaus siab rau hauv autopsies ntawm AD thiab PD lub hlwb thiab zoo li yuav pab txhawb rau cov kab mob ntawm neurodegeneration (Kannarkat li al. 2013). Qhov no inflammatory thiab tej zaum mitochondrial dysfunction yog xav tias yuav ua rau oxidative kev nyuaj siab uas kuj txaus siab rau autopsied hlwb (Picca li al. 2020).

Feem ntau ntawm cov kev tshawb fawb mechanistic ntawm huab cua paug thiab neurodegeneration rau hnub tim tau tsom mus rau qhov mob (Jayaraj li al. 2017). Cov pa phem zoo li ua rau CNS inflammatory thiab oxidative stress nyob rau hauv tsiaj thiab tib neeg lub hlwb (Calderón-Garcidueñas li al. 2004, 2007, 2008a, b thiab c; Levesque et al. 2011a, b, 2013; Moultonand Yang M. 201. 2017; Yokota et al.2013a, b).

Ntawm qhov kev txaus siab tshwj xeeb yog qhov kev tshawb pom tias huab cua muaj kuab paug nce qhov kev qhia ntawm qee cov noob caj noob ces nyob rau hauv olfactory noob (OB) ofmice, ib cheeb tsam ntawm lub paj hlwb uas PD pathology yog pom thaum ntxov ntawm tus kab mob (Levesque et al. 2011a, b; Yokota et al. 2013a) . Kev mob tshwm sim kuj tau pom cov indogs nyob hauv nroog piv rau cov neeg nyob hauv cov nroog nyob deb nroog, thiab cov kws sau ntawv xav tias cov kev hloov pauv no yog vim muaj huab cua ntau (Calderón-Garcidueñas li al. 2003).

Cov tib neeg sau ntawv kuj tau hais tias -syn sau rau hauv lub hlwb ntawm cov neeg nyob hauv cov nroog vim muaj pa phem (Calderón-Garcidueñas li al. 2004, 2008a, b). Hauv kev tshawb fawb tsis ntev los no hauv tus qauv zebrafish, exposureto DEPe tau tshaj tawm tias yuav ua rau muaj kev puas tsuaj rau neurotoxicity thiab txo qis hauv cov xov tooj neuron los ntawm kev cuam tshuam autophagy (Barnhill li al. 2020).

DEPe muaj ntau yam tshuaj lom ntawm cov pa phem thiab feem ntau yog siv los ua qauv nthuav tawm hauv cov kab lis kev cai ntawm tes (Costa li al. 2014; Hesterberg li al. 2010; Levesque et al. 2011b). Autophagy yog ib qho tseem ceeb hauv cov txheej txheem rau kev tshem tawm thiab tshem tawm cov misfolded proteins thiab puas organelles.

Tseeb tiag, qhov tseeb autophagic kev ua si yog tsim nyog rau cellular homeostasis, thiab dysfunctionalautophagy nyob rau hauv neurons ua rau hloov pauv ciaj sia taus thiab neurodegeneration (Kesidou li al. 2013).Muaj tseeb cov kev txwv nyob rau hauv ntau yam ntawm cov kev tshawb fawb no, tab sis lawv tag nrho qhia hais tias huab cua muaj kuab paug tshwm sim ntawm AD. thiab PD. Peb txoj kev nkag siab ntawm cov txheej txheem uas ua rau muaj kuab paug ua rau qhov kev pheej hmoo no txwv, tab sis cov khoom siv ultrafine thiab ntau yam ntawm cov pa phem tuaj yeem nkag mus rau hauv lub hlwb ncaj qha los ntawm olfactory bulbor los ntawm lub ntsws ntawm cov hlab ntsha (Kilian andKitazawa 2018).

Cov khoom no yog tsim los ntawm acarbon core thiab adsorbed tebchaw xws li polycyclic aromatic hydrocarbons, hlau, nitrate, sulfate, thiab lwm yam ntsiab lus thiab ua ib feem tseem ceeb ntawm cov pa phem, tshwj xeeb tshaj yog nyob rau hauv nroog.Ntau yam ntawm cov tebchaw no tau implicated inoxidative kev nyuaj siab thiab cellular. toxicity ntxiv plausibility rau ib tug causal koom haum ntawm huab cua pa phem thiab neurodegeneration.

Kev tshawb nrhiav ntau yam kev qhuab qhia, suav nrog cov qauv ntawm tes thiab tsiaj txhu, tuaj yeem muab kev nkag siab txog qhov ua rau muaj kab mob thiab ncaj qha rau kev kho mob yav tom ntej (Cannon and Greenamyre 2011). Ntawm cov qauv kab mob uas siv rau hauv kev tshawb fawb vivo, Danio rerio (zebrafish) yog tus qauv muaj zog rau kev kawm toxicology, molecular genetics, toxicogenomics, thiab drugdiscovery.

Feem ntau txoj hauv kev lom neeg tau txais txiaj ntsig zoo hauv vertebrates, thiab feem ntau ntawm cov noob caj noob ces muaj homologs hauv zebrafish (Vaz li al.2018). Tsis tas li ntawd, zebrafish embryos loj hlob sai thiab ntawm nws tus kheej, tso cai rau kev tshuaj xyuas ntawm txhua qhov kev loj hlob theem. Cov yam ntxwv no, ua ke nrog kev loj hlob sai thiab tus nqi qis ntawm kev ua liaj ua teb, ua rau zebrafish ua qauv zoo rau kev kawm txog cov teebmeem ntawm ib puag ncig.

Txhawm rau kom nkag siab zoo dua cov txheej txheem molecular tom qab lub pathogenesis ntawm tus kab mob, kev nthuav dav ntawm cov noob qoob loo nthuav qhia profileingapproaches tau tsim. Mass spectrometry-based proteomics thiab high-throughput RNAsequencing tuav cov lus cog tseg tshwj xeeb hauv kev txheeb xyuas txoj hauv kev lom neeg. Cov tswv yim no tau siv dav los nkag siab txog cov lus teb ntawm cellular thiab tsiaj qauv rau ib puag ncig stimuli (Duan li al. 2017; García-Estrada li al. 2013; Jamiet al. 2014a, b, 2015; Kosalková et al. 2012).

Hauv qhov kev tshawb fawb no, peb tau ua raws li kev tshuaj ntsuam xyuas cov txiaj ntsig ntawm kev kho DEPe ntawm ob qho tib si proteome thiab cov ntaub ntawv transscriptome nyob rau hauv lub taub hau ntawm zebrafishembryos. Kev cais tawm ntawm cov qauv ntawm lub taub hau uas feem ntau yog tsim los ntawm cov ntaub so ntswg ntawm lub paj hlwb tso cai rau kom tau txais cov ntaub so ntswg tshwj xeeb thiab cov teebmeem me me uas tshwm sim los ntawm lwm cov ntaub so ntswg. Peb piav qhia ntawm no hloov pauv cov noob qhia thiab cov protein ntau hauv ntau txoj hauv kev cuam tshuam hauv neurodegenerationin lub taub hau ntawm DEPe-exposed zebrafish. Cov txiaj ntsig no muab kev nkag siab tshiab rau hauv cov txheej txheem ua tau los ntawm kev ua pa phem ua rau muaj kev pheej hmoo ntawm AD thiab PD.

Khoom siv thiab cov txheej txheem

Ntses kev kho mob thiab CRISPR/Cas9-kev kho mob geneknockdown

Txhua qhov kev tshawb fawb tau pom zoo los ntawm UCLA AnimalRights Committee. Zebrafish (AB) tau bred bylight stimulation rau 1 h, thiab tag nrho cov naj npawb ntawm 200 qe tau incubated rau 24 teev ntawm 28 degree. Qhov tshwm sim embryos tau dechorionated hauv pronase (2 mg / ml) thiab kho nrog DEPe (Standard Reference Materials, NIST, Gaithersburg, MD) ntawm qhov kawg concentration ntawm 20 ug / ml lossis tsheb mus txog 5 hnub tom qab yug me nyuam (DPF).

Qhov kev xav tau no tau raug xaiv vim nws ua rau cov neuron poob raws li tau hais dhau los tab sis tsis muaj kev tuag tseem ceeb (Barnhillet al. 2020).Cyp1A knockdown ntses tau npaj raws li hauv qab no.Siv cov cuab yeej muaj nyob ntawm http://crispr.mit.edu, theCRISPR RNA (Cyp1A-crRNA ) tau tsim los raws li qhov chaw ntawm qhov chaw tsim nyog PAM hauv thawj exonof Cyp1A noob.

Qhov zoo tshaj plaws tsim tau yuav los ntawm IDT DNA (www.idtdna.com) ua ke nrog tus qauv CRISPR-Cas9 tracrRNA. Lub crRNA rau Cyp1Agene thiab tracrRNA tau cais tawm rov qab nyob rau hauv 10 mM Tris-EDTA kom tau txais 100 μM qhov kawg concentration. Tom qab ntawd, tracrRNA tau ua ke nrog crRNAin 1 × duplex buffer (100 mM potassium acetate; 30 mM HEPES, pH 7.5; IDT DNA) kom ua tiav qhov kawg ntawm 10 μM. Cov sib tov tau rhaub rau 95 degree rau 5 min nyob rau hauv ib lub boiling dej da dej thiab maj mam txias mus rau chav tsev kub kom tso cai rau lub annealing ntawm complementary nucleotide sequences incrRNA thiab tracrRNA thiab tsim functionalguide RNA (gRNA).

Ib qhov ntim ntawm 3 μL ntawm annealedgRNA tau ua ke nrog tib lub ntim ntawm tus neeg pub dawb ib leeg-strand DNA (ssDNA; 200 μM) raws li tus qauv ua ntu zus rau mutant Cyp1A. Ib qho nyiaj ntawm 3 ug ntawm Cas9 Nuclease (IDT) hauv 1 × txhaj tshuaj tsis haum (5 mM KCl; 0.1 M sodium phosphate, pH 6.8) tau ntxiv rau qhov sib tov los ua qhov kawg CRISPR / Cas9ribonucleoprotein (Cyp1A-RNP) complexes.

Rau kev npaj ntawm Scramble RNP (SC-RNP), qhov tshwj xeeb Cyp1A-crRNA tau hloov los ntawm kev lag luam Scramble-crRNA uas tsis muaj lub hom phiaj ntawm genome. Cov txiaj ntsig ntawm tag nrho cov oligonucleotides siv rau hauv txoj haujlwm no yog teev nyob rau hauv Cov Lus Qhia Ntxiv 1.Eggs raug txhaj rau hauv stereomicroscope nrog ib ncig ntawm 3 nL ntawm RNP txhaj tshuaj sib tov.

Fivebiological replicate txhaj (txhua qhov kev hloov pauv suav nrog 400-600 qe txhaj nrog Cyp1A-RNPand 100-200 txhaj nrog SC-RNP) tau ua, thiab tag nrho cov microinjections tau ua tiav hauv 60 min. Cov qe tau muab tso rau hauv 100 hli yas Petri tais diav thiab incubated ntawm 28 degree nyob rau hauv lub qe dej.Qhov ciaj sia taus ntawm embryos tau saib xyuas txog li 7 hnub.

Cov duab ntawm cov ntses tseem muaj sia nyob tau raug tshuaj xyuas rau cov qhab nia malformation. Txhua tus menyuam yaus tau qhab nia qhov muag tsis pom kev rau qhov muaj 4 qhov kev loj hlob malformations xws li lub taub hau malformation, tail malformation, cardiac edema, thiab yolk edema.Larvae nrog tag nrho 4 malformations yuav tau qhab nia 4, whereas ib txwm loj hlob larvae yuav tau qhab nia 0. Qhov ntsuas T-test tau siv los ntsuas qhov sib txawv tseem ceeb.

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