GRP78 Overexpression Triggers PINK1-IP3R-Mediated Neuroprotective Mitophagy Part 3

3.1.3. Mitophagy Induction los ntawm GRP78 Overexpression Mediates Neuroprotection

Peb xav tias GRP78-kev kho mob neuroprotection tuaj yeem yog los ntawm inducingselective autophagy ntawm mitochondria txhawm rau txhawm rau tshem tawm cov kab mob tsis zoo.

Nyob rau hauv xyoo tas los no, ntau thiab ntau cov kev tshawb fawb tau pom tias mitochondria ua lub luag haujlwm tseem ceeb hauv kev tsim thiab kho kev nco. Mitochondria yog ib qho tseem ceeb hauv cov hlwb uas muaj lub luag haujlwm tsim lub zog. Tsis tas li ntawd, mitochondria tseem koom nrog kev tswj hwm ntawm tag nrho cov cell metabolism thiab qhov sib npaug ntawm cov tshuaj tiv thaiv redox. Cov kev tshawb fawb tsis ntev los no kuj pom tau hais tias mitochondria tuaj yeem cuam tshuam rau kev ruaj ntseg ntawm kev nco mus ntev los ntawm kev tswj hwm kev hloov pauv hauv neuronal excitability.

Mitochondria yog lub luag haujlwm rau oxidative metabolism hauv hlwb, thiab lub zog ntawm cov hlwb feem ntau kis los ntawm adenosine triphosphate (ATP) hauv mitochondria. Neurons, raws li cov ntaub ntawv xa mus rau tib neeg lub cev, muaj lub zog tshwj xeeb, yog li kev ua haujlwm ntawm lawv cov mitochondria noj qab haus huv yog qhov tseem ceeb rau cov yam ntxwv ntawm lub cev ntawm cov neurons thiab kev ua haujlwm ntawm lub paj hlwb. Cov kws tshawb fawb tau pom tias nyob rau hauv lub xeev ntawm cov kab mob lossis kev laus, kev noj qab haus huv ntawm mitochondria yuav raug puas tsuaj thiab lawv txoj haujlwm yuav poob qis, ua rau muaj kev poob qis hauv kev ua haujlwm neuronal thiab cuam tshuam rau lub peev xwm nco.

Kev tsim thiab kev saib xyuas ntawm kev nco yuav tsum muaj kev sib koom tes ntawm cov neurons, synapses, thiab mitochondria tib lub sijhawm. Ntawm cov synapses ntawm qhov kawg ntawm cov neurons, mitochondria nyob nruab nrab ntawm cov neurons thiab glial hlwb uas txhawb nqa neurons. Cov synapses nthuav tawm cov qauv tshwj xeeb thiab yog cov yas zoo heev, uas tuaj yeem hloov kho raws li qhov xav tau ntawm kev nco thiab kev kawm. Mitochondria zoo ib yam li "qhov chaw tsim khoom" ntawm synapses, muab cov neurons thiab synapses nrog lub zog thiab cov khoom uas lawv xav tau, uas tuaj yeem tswj cov kev xav tau ntawm cov neurons sai thiab tswj xyuas cov yas ntev ntev thiab ruaj khov ntawm synapses.

Lwm qhov kev tshawb fawb qhia txog mitochondria thiab kev nco yog kho cov hlwb los ntawm kev hloov pauv cuam tshuam (ib hom protein). Qhov kev kho no tuaj yeem txhim kho mitochondrial muaj nuj nqi, nce metabolic tus nqi, thiab tiv thaiv kev noj qab haus huv ntawm neurons thiab synapses. Raws li cov kev tshawb fawb no, nws tau pom tias los ntawm kev ntxiv dag zog rau kev noj qab haus huv ntawm mitochondria, peb tuaj yeem txhim kho peb lub cim xeeb thiab kev txawj ntse thaum txo qis kev pheej hmoo ntawm Alzheimer's thiab lwm yam kab mob neurological.

Hauv cov ntsiab lus, mitochondria yog ib feem tseem ceeb ntawm kev tsim thiab kev saib xyuas. Kev noj qab haus huv ntawm mitochondria yog qhov tseem ceeb rau kev ua haujlwm ntawm cov neurons thiab synapses thiab tswj xyuas cov yas mus ntev thiab ruaj khov. Peb yuav tsum xyuam xim rau kev noj zaub mov, kev tawm dag zog, thiab kev ua neej nyob kom muaj kev noj qab haus huv ntawm mitochondria, txo kev pheej hmoo ntawm Alzheimer's thiab lwm yam kab mob paj hlwb, thiab txhim kho peb txoj kev txawj ntse thiab kev nco. Nws tuaj yeem pom tias peb yuav tsum txhim kho kev nco, thiab Cistanche tuaj yeem txhim kho kev nco zoo vim tias nws 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 deserticola 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 txoj hauv kev los txhim kho koj lub cim xeeb

Kev txhawb nqa ntawm qhov kev xav no, GRP78 tau raug pov thawj los txhawb macroautophagy [4] (tom qab no hu ua autophagy).

Peb nrhiav kev txiav txim siab seb puas yog autophagy tsim nyog rau GRP78-induced neuroprotection siv ob peb yam zoo autophagy modulators: rapamycin, uas activates autophagy los ntawm inhibiting mTORC1, thiab ob inhibitors ntawm autophagy flux: 3-methyladenine ({{4 }}MA), tus inhibitor ntawm chav kawm III phosphatidylinositolkinase PI3K, thiab LY294002, tus inhibitor ntawm chav kawm I PI3K.

Tag nrho peb qhov autophagy modulators txo qis txoj sia nyob hauv GFP thiab GRP78 pawg hauv Tun-kho hlwb piv rau lub tsheb (Daim duab 4A). Cov txiaj ntsig no tau qhia tias qhov pib qhov tseeb ntawm autophagy, dhau ntawm PI3K-Beclin1 txoj hauv kev, thiab cov kua dej lig yog qhov tsim nyog, txawm tias tsis txaus, rau cov teebmeem kev tiv thaiv ntawm GRP78 overexpression nyob rau hauv ER-kev nyuaj siab hlwb.

Daim duab 3. GRP78 overexpression cawm mitochondrial dysfunction nyob rau hauv stressed NSC34 hlwb. (A) Immunoblot thiab bar graphs qhia cov qib ntawm GRP78 hauv NSC34 hlwb nucleofected nrog plasmid vector kom overexpress GRP78 lossis tsis muaj feem cuam tshuam nrog cov proteinas tswj (GFP). (B) Feem pua ​​​​ntawm kev muaj sia nyob ntawm GFP- lossis GRP78- qhia NSC34 hlwb (txhais tau tias ± SEM) tom qab 24 teev hauv qhov nruab nrab uas muaj 1 µg/mL tunicamycin (Tun) (sab laug) lossis 10 µM nocodazole (txoj cai) txiav txim siab siv MTT kev xeem (n=4; * p < 0.005,** p < 0.001 vs. GFP, Tub Kawm Ntawv t-test ). (C) Kev soj ntsuam ntau ntawm MitoSOX fluorescence nyob rau hauv hlwb overexpressing GFP los yog GRP78 thiab kho nrog Tun los yog lub tsheb (sab laug) los yog efavirenz (EFV) raws li kev tswj los yog lub tsheb (txoj cai). (D) Kev soj ntsuam ntau ntawm TMRM fluorescence hauv hlwb overexpressing GFP lossis GRP78 thiab kho nrog Tun lossis tsheb (sab laug) lossis EFV lossis tsheb (txoj cai) (GFP-Veh yog pawg tswj hwm). (E) Sab laug: Cov neeg sawv cev tsom xam ntawm O2 concentration (siv Clark-hom O2 electrode) asa muaj nuj nqi ntawm lub sij hawm hauv cov hlwb overexpressing GFP lossis GRP78 thiab kho nrog Tun. Txoj Cai: O2 noj tom qab 5 h ntawm Tuntreatment hauv hlwb overexpressing GFP lossis GRP78 (GFP-Veh yog pawg tswj hwm) (n=4; * p < 0.05 vs. Veh-GFP, # p < 0.05 vs.GRP78, ib-txoj kev ANOVA).

Daim duab 4. Mitochondria tagging rau mitophagy yog tsim nyog los ntawm GRP78 overexpression. (A) Bar graph ntawm feem pua ​​​​ntawm NSC34 hlwb (txhais tau tias ± SEM) nucleofected nrog GFP lossis GRP78 plasmids thiab kho nrog Tun lossis ua ke ntawmTun nrog autophagy modulators: rapamycin (RAPA), 3MA, lossis LY-902, hais txog tswj lub tsheb (Veh), txiav txim los ntawm MTT kev soj ntsuam 24 teev tom qab kev kho mob (GFP-Veh yog pawg tswj hwm) (n=4-8, * p < 0.05 vs.

Veh-GFP; # p < 0.05 vs. Tun-GFP).(B) Cov neeg sawv cev ntawm cov duab ntawm cov cell nucleofected nrog PARK2-HA plasmid ib leeg (sab saum toj, nruab nrab panels, tswj) orwith GRP78 plasmid (hauv qab vaj huam sib luag) kho nrog lub tsheb (Veh) lossis Tun (5 h) thiab tiv thaiv kab mob rau HA (ntsuab) thiab HSP60 (liab); scale bar=10 µm. (C) Nruab Nrab ± SEM ntawm Pearson tus correlation coefficient ntawm Parkin thiab Hsp60 co-localizationfrom Veh, Tun, thiab Tun + GRP78 pawg (n=4; * p < 0.05 vs. Parkin-Veh, # p < 0.05 vs.

Parkin-Tun, ib-txoj kev ANOVA). (D) Sab laug, Western blots rau qhia cov proteins nyob rau hauv lub mitochondrial (mito) thiab cytosolic (cyto) pooled feem ntawm GFP- los yog GRP78- overexpressing hlwb kho nrog tsheb los yog Tun rau 5 h. Cov proteins uas txheeb xyuas yog OPA-1, CV-, PDI, GRP78, Ubiquitinated residues, LC3, thiab actin. Txoj cai, bar graphs ntawm qhov nruab nrab fold hloov ntawm GRP78 nyob rau hauv ob qho tib si pooled feem thiab LC{{1{{20}}}}}II nyob rau hauv mitochondrial feem txheeb ze rau actin (cytosol) los yog beta subunit ntawm complex V (CV-B) (mitochondria) hauv pawg GFP. (E) Bar graph ntawm feem pua ​​​​ntawm cov hlwb siv tau (txhais tau tias ± SEM) overexpressing GFP los yog GRP78 proteins thiab kho nrog Tun ib leeg los yog ua ke nrog CCCP, CSA, los yog BAPTA-AM, txiav txim los ntawm MTT assay 24 teev tom qab kev kho mob txog kev tswj tsheb-kho. hlwb. (F) Ib yam li daim duab ntawm cell viability rau cov hlwb kho nrog thapsigargin (THA) nrog lossis tsis muaj CCCP (GFP-Veh yog pawg tswj hwm) (n=4-8, hauv 3 qhov kev sim sib txawv, * p < 0.05 vs . tswj-Veh;# p < 0.05 vs. GRP78-Tun).

Parkin tsis tas yuav koom nrog hauv mitophagy vim tias nws tuaj yeem tshwm sim los ntawm lwm txoj hauv kev [46]. Txhawm rau kom paub tseeb tias Parkin puas koom nrog GRP78- cov nyhuv kho tau, peb tau txiav txim siab qhov chaw subcellular ntawm HA-tagged Parkin qhia hauv NSC34 hlwb nrog thiab tsis muaj ER kev ntxhov siab.

Los ntawm confocal microscopy, peb pom tias Parkin raug faib thoob plaws hauv NSC34 cell tom qab 5 teev ntawm lub tsheb lossis Tun kho (Daim duab 4B).Tshwj xeeb, hauv cov hlwb overexpressing GRP78, muaj kev nce hauv co-localization ntawm Parkin nrog HSP60, mitochondrial protein. , tawm tswv yim rau lub hom phiaj ntawm lub organelle formitophagy (Daim duab 4B, C).

Tsis tas li ntawd, peb cais cov mitochondrial thiab cytosolic feem ntawm cov hlwb uas tshaj tawm GFP lossis GRP78 thiab tau kho nrog Tun lossis lub tsheb raws li kev tswj ntawm 5 h tom qab kev thuam. Lub purity ntawm pooled mitochondrial fractions tau lees paub los ntawm kev txheeb xyuas qhov muaj OPA1 thiab CV, thiab qhov tsis muaj ER-neeg nyob hauv cov protein, cov proteindisulfide isomerase (PDI) (Daim duab 4D).

Interestingly, peb kuj pom ib tug nyiam nce GRP78 abundance nyob rau hauv cov pooled mitochondrial feem ntawm ER-ntses hlwb piv rau kev tswj. Nco ntsoov tias txawm hais tias Tun stimulus kuj tuaj yeem ua rau muaj kev nce ntxiv hauv GRP78 qib hauv cytosol, qhov no tsis tau pom ntawm mitochondrial feem.

Kev quab yuam overexpression ntawm GRP78 txhawb nqa ib qho kev nyiam kom nce ob leeg PINK1 thiab Parkin tsub zuj zuj hauv cov mitochondrial feem (Daim duab 4D thiab daim duab S4). Nws tau tshaj tawm tias qhov muaj Parkinamplifies qhov tsub zuj zuj ntawm Ub thiab txhim kho mitophagy piv rau qhov muaj ntawm PINK ib leeg [42,47]. Raws li, Ubiquitin (Ub) immunoblotting qhia qhov sib txawv ntawm nws qhov profile hauv mitochondrial feem ntawm GRP78- qhia cov hlwb piv rau GFP pawg (Daim duab 4D).

Nyob rau hauv cov kev ntxhov siab, mitochondrial protein ubiquitylation ua rau muaj kev tsim khoom ntawm autophagosome machinery Cheebtsam uas pib nrog cov lipidated isoform ntawm LC3, thiab LC3II [42,48].

Ntau cov protein ntau LC3II tau pom nyob rau hauv mitochondrial feem nrog ib qho kev nyiam ua kom muaj ntau dua nyob rau hauv cov hlwb uas muaj kev ntxhov siab uas overexpress GRP78 txog cov neeg uas muaj GFP ntawm cov xwm txheej zoo sib xws (Daim duab 4D).Koom ua ke, cov ntaub ntawv no tau qhia tias qhov yuam kev ntawm GRP78 tuaj yeem pab txhawb kev hloov mus rau mitochondrial thiab tagging rau mitophagy.

Txhawm rau txiav txim siab qhov cuam tshuam ntawm mitophagy induction rau cov neuroprotectiveeffect mediated los ntawm GRP78 overexpression, peb tau soj ntsuam cov kev hloov pauv los ntawm cov activators thiab inhibitors ntawm mitophagy. Peb siv carbonyl cyanide m-chlorophenyl hydrazine (CCCP) rau chemically uncouple oxidative phosphorylation thiab induce mitophagy los ntawm collapsing mitochondrial membrane muaj peev xwm [49,50], nrog rau ob mitophagy inhibitors: BAPTA-AM, cell-permeant chelular Ca 2+ thiab cyclosporin A (CSA), themitochondrial permeability transition pore (mPTP) inhibitor uas thaiv Ca2+ efflux los ntawm mitochondria [51].

Txawm hais tias CCCP kev kho mob hauv NSC34 tsis txaus los txo qhov muaj txiaj ntsig zoo, raws li tau tshaj tawm dav dav, peb pom tias tib yam kev kho mob ntawm ER-kev ntxhov siab tau nce nws txoj sia nyob, tsis hais seb GRP78 tau dhau los (Daim duab 4E).

Qhov tseem ceeb, cov nyhuv neuroprotective txhawb nqa los ntawm GRP78 overexpression onER-kev ntxhov siab hlwb raug tshem tawm nyob rau hauv lub xub ntiag ntawm CSA los yog BAPTA-AM (Daim duab 4E).Cov kev tshawb pom no qhia tias mitophagy induction tso cai rau cov hlwb los tiv thaiv ER kev nyuaj siab thiab Ca2+-flux. yog ib qho tseem ceeb rau cov nyhuv neuroprotective txhawb los ntawm GRP78.

Ib qho ua tau rau GRP78 los ntxias mitophagy tuaj yeem ua kom yooj yim rau qhov tsim nyogCa2+-kev ua haujlwm kho mob, tej zaum los ntawm qhov kev txiav txim ntawm ER-neeg nyob inositol 1,4,5 triphosphate receptor (IP3R) uas nws qhib tso cai rau Ca2+ ntws. los ntawm ER mus rau mitochondria.Peb xav tshawb nrhiav qhov ua tau los ntawm kev cuam tshuam kev ua kom IP3R siv thapsigargin(THA), Ca2+ ATPase inhibitor [52].

Thapsigargin tseem paub tias tsim ER kev ntxhov siab hauv cov hlwb txij li qhov kev kho mob no depletes Ca2+ khw muag khoom los ntawm ER, thiab yog li ntawd nce GRP78expression ua ib feem ntawm cov lus teb canonical unfolded protein [53]. Daim duab 4F qhia tau hais tias thapsigargin cuam tshuam rau cell viability zoo ib yam li tunicamycin kev kho mob hauv GFP-tswj cov cell thiab tias CCCP kev kho sib xyaw ua ke tiv thaiv kev tuag ntawm tes raws li pom nrog Tun.

Txawm li cas los xij, kev quab yuam overexpressing ntawm GRP78 tsis muaj peev xwm los thaiv qhov cuam tshuam thapsigargin zoo li nws tau ua nrog kev thuam ntawm tunicamycin. Qhov kev soj ntsuam no lees paub qhov tsim nyog kev koom tes ntawm Ca2+ flux hauv cov nyhuv neuroprotective ntawm GRP78, tej zaum los ntawm IP3R.

3.1.4. Neuroprotection Mediated los ntawm GRP78 Nyob ntawm PINK1 thiab IP3R

Txhawm rau pib qhov kev txheeb xyuas tus neeg nruab nrab ntawm GRP78 cov txiaj ntsig neuroprotective, weused shRNA thev naus laus zis. Ua ntej, peb tau txheeb xyuas tias shRNA tau xaiv txo qis kev qhia ntawm GRP78, PINK1, lossis IP3R, raws li (Daim duab S4A–C) thiab tias lawv tsis cuam tshuam txog kev tiv thaiv ntawm lub hlwb (Daim duab S4D). Peb thawj zaug tau lees paub tias qhov kev qhia ntawm GRP78 nws tus kheej tsim nyog rau ob qho tib si GRP78- thiab CCCP-mediated neuroprotection kom ntsib Tun-inducedER kev nyuaj siab (Daim duab 5A).

Tom ntej no, kev ntsiag to PINK1 lossis IP3R thaiv cov neuroprotectiveeffect txhawb nqa los ntawm GRP78 overexpression lossis CCCP kev kho mob (Daim duab 5A). Tsis tas li ntawd, peb pom GRP78 hloov pauv, ib txwm muab faib rau hauv qhov sib luag ntawm qhov chaw nyob ib puag ncig ntawm lub nucleus, yuav tsum tau sib koom ua ke rau IP3R hauv speckle foci thaum nws overexpressed (Daim duab 5B).

Ua ke cov txiaj ntsig no tau qhia tias GRP78 hloov kho kev tiv thaiv mitophagy hauv peb cov qauv hauv vitro, thiab nws ua rau qhov kev tiv thaiv neuroprotection hauv PINK1- thiab IP3R-nyob ntawm hom.

4. Kev sib tham

Kev txhawb nqa cov txheej txheem endogenous ntawm neuroprotection tuaj yeem ua rau cov cuab yeej kho mob tau zoo los tiv thaiv cov txheej txheem neurodegenerative tom qab raug mob los yog kab mob [3,54,55].Tus ER-neeg nyob hauv chaperone GRP78 yog nyob rau ntawm txoj kev sib tshuam ntawm ntau yam ntawm cov txheej txheem no txhawb kev tiv thaiv neuroprotection thaum overexpressed nyob rau hauv ntau yam kab mob. qauv [4].

Txhawm rau txiav txim siab cov txheej txheem hauv qab, peb siv tus qauv ntawm tus txha caj qaum avulsion [55] uas cuam tshuam kev sib txuas ntawm motoneuron ua rau cov txheej txheem retrograde neurodegenerative [20].

Motoneuron kev tuag nyob rau hauv cov qauv no yog tus cwj pwm los ntawm ER kev nyuaj siab, ib qho thaiv autophagy flux, thiab yog nonnapoptotic vim tsis muaj cov ntaub ntawv nquag ntawm caspase 3 lossis 12 tau pom, qhia tias apoptosisis tsis yog qhov kawg executor ntawm neuronal demise [20]. Raws li, peb yav dhau los tau txheeb xyuas nrog proteomics txoj hauv kev uas cov yam ntxwv tiv thaiv apoptotic tshwm sim nyob rau tib lub sijhawm rau apoptotic ones afterRA, thaiv kev ua kom zoo apoptosis [25].

Nyob rau hauv cov ntsiab lus neurodegenerative, theoverexpression ntawm GRP78 exerted motor neuroprotection [20,26,27]. Ntawm no, thawj zaug, peb siv cov kev sib piv thiab kev txheeb xyuas ntau ntawm cov proteome los nthuav tawm lub hauv paus tseem ceeb rau kev tiv thaiv neuroprotection.

Kev npaj txhij txog, mitochondria yog lub hom phiaj tseem ceeb, nrog rau cov ntsiab lus ntawm cov organelle protein ntau nrog los ntawm kev kho kom zoo nkauj ntawm mitochondriainto vesicles hauv cov motoneurons puas uas overexpressed GRP78.

Cov kev soj ntsuam no tau pom tias muaj mitophagy uas yog lub hauv paus tseem ceeb rau kev tiv thaiv kab mob. Deeperin vitro trials validated no hypothesis txij li thaum GRP78 mediated neuroprotection los ntawm (i) restoring mitochondria respiration thiab ROS theem, (ii) stimulating PINK1/PARKINmitochondria translocation, tagging lub organelle rau mitophagy, thiab (iii) nyob ntawm IP3R muaj nuj nqi raws li ib tug tseem ceeb mediator.

Rau peb txoj kev paub, qhov no yog thawj txoj kev tshawb fawb uas qhia tias GRP78 overexpression txhawb kev tiv thaiv mitophagy.Ntxiv rau GRP78 lub luag haujlwm hauv kev ua haujlwm tsis zoo ntawm cov proteins thiab lwm yam kev ua haujlwm ntawm lub hli uas yog calcium-binding protein [4,56], peb ntxiv nws cov pro-active acting. txhawb mitophagy. Ntxiv mus, peb txoj kev tshawb fawb koom nrog cov kev tshawb fawb tsis ntev los no txuas ER kev nyuaj siab rau mitochondrial dysfunction [57–59].

Peb pom cov cim tseem ceeb rau mitophagy hauv vivo thaum GRP78 tau tshaj tawm thiab txhawb nqa neuroprotection. Tib yam tau pom siv hauv vitro qauv uas ua raws li qee yam ntawm cov txheej txheem neurodegenerative uas tshwm sim tom qab RA xws li ER kev nyuaj siab [20,26].

Kev tswj xyuas zoo ntawm mitochondria los ntawm mitophagy yog qhov tseem ceeb los saib xyuas cov ntsiab lus mitochondrial thiab cov metabolism hauv homeostasis. Nyob rau hauv cov ntaub ntawv, nws tseem yog controverted seb mitophagy induction txhawb kev ciaj sia los yog cell tuag inseveral kab mob vim nws nws kim heev crosstalk nrog apoptosis signaling [60], txawm hais tias nws tau piav qhia tias kev tshem tawm ntawm puas mitochondria muaj lub luag haujlwm tseem ceeb hauv cov kab mob neurodegenerative xws li Alzheimer's kab mob. , Parkinson's disease, lossis inaging [61].

Yav dhau los, nws tau raug tshaj tawm tias GRP78 inhibits apoptosis tshwm sim los ntawm ER kev ntxhov siab los ntawm kev tiv thaiv CHOP induction [62,63], ua kom cov caspase 7 [5], lossis byactivating PI3K-AKT-mTOR signaling axis [4]. Nws kuj tau ua pov thawj los tswj cov qib oxidative kev nyuaj siab thiab DNA puas [4,64]. Tshwj xeeb, hauv cov qog nqaij hlav cancer, nws tau pom tias GRP78 attenuates ROS los ntawm kev ua kom muaj protein ntau kinase RNA-likeendoplasmic reticulum kinase (PERK)-NRF2 cov cim [65,66], ua rau kev txhim kho ntawm cov tshuaj tiv thaiv kab mob cuam tshuam nrog rau kev txhim kho cov protein ntau. glycolytic enzymes [67]

Hloov chaw, peb tau pom tias GRP78 overexpression downregulated glycolytic enzymes (piv txwv li, ENO1 thiab ENO2) (cov lus ntxiv). Cov txiaj ntsig no yuav qhia tau tias nyob rau hauv peb cov qauv, qhov pom kev txo qis ntawm ROS tej zaum yuav muaj feem cuam tshuam rau kev nce mitophagy, txawm hais tias cov ntsiab lus ntawm cov txheej txheem koom nrog yuav tsum tau tshawb xyuas ntxiv.Mitochondrial dysfunction yog ib qho mob hauv cov kab mob neurodegenerative [68], thiab mitophagy tau koom nrog. hauv Parkinson's [49,69], Huntington's [70,71], thiab Alzheimer's kab mob thiab Tauopathies [72].

Ntau tus kws sau ntawv tau taw qhia tias los ntawm kev tswj hwm txoj hauv kev mitophagy, lub cev tuaj yeem zam cov kab mob oxidative, thiab tswj cov redox tshuav nyiaj li cas thiab homeostasis [73]. Yog li, GRP78 overexpression los ntawm qhov kev tswj mitophagy induction tej zaum yuav raug suav hais tias yog ib qho kev tiv thaiv neuroprotective hauv cov kab mob.Neuroprotection mediated los ntawm GRP78 overexpression zoo li nyob ntawm ona Ca2+ flux txij li thaum nws tau thaiv los ntawm BAPTA-AM. BAPTA-AM chelating ntawm Ca2+ tej zaum yuav cuam tshuam rau ntawm ob peb kauj ruam sib txawv raws txoj kev autophagic flux, thaiv tsis yog qhov tshwm sim ntawm autophagy tab sis kuj inhibiting cov kauj ruam hauv kev tsim thiab ua cov autophagosomes [74].

Hauv qhov sib piv, kev siv lwm tus neeg sawv cev cuam tshuam rau Ca2+ flux, xws li asthapsigargin, tau txais txiaj ntsig zoo heev. Thapsigargin blocks sarco-endoplasmic reticulumCa2+ ATPases (SERCAs), txhawb nqa cytosolic Ca2+ nce, tab sis nws kuj ua rau ER kev ntxhov siab dhau los ntawm kev tsis txaus ntawm cov khoom siv hauv lub cev Ca2+ thiab cov khoom ntim ntawm cov protein ntau.

Hauv qhov xwm txheej no, peb pom tias GRP78 overexpression tsis tuaj yeem cawm cov dyingcells, thiab peb xav tias nws yuav yog vim thapsigargin tseem cuam tshuam IP3R kev ua haujlwm [52].Basal IP3R kev ua haujlwm thiab tsis tu ncua qis qis Ca2+ flux los ntawm ER mus rau mitochondria. Nws yog ib qho tseem ceeb los txhawb mitochondrial ua pa thiab cell bioenergetics [75].

GRP78 interactsat MAMs nrog lub complex tsim los ntawm sigma -1 receptor (SIGR1) thiab IP3R [76,77]. Thaum ERstress, SIGR1-GRP78 kev sib cuam tshuam txo qis los ntawm Ca2+ depletion tom qab boosting Ca2+ flux los ntawm ER mus rau mitochondria los ntawm IP3Rs [76]. Tsis tas li ntawd, nws kuj tau qhia tias IP3R yog qhov tsim nyog rau Parkin-induced mitophagy tshwj xeeb rau kev tso cai rau mitochondrial pawg hauv qab dej Parkin recruitment [78].

Peb cov txiaj ntsig tau pom zoo txij li qhov txo qis ntawm IP3R los ntawm shRNA thev naus laus zis tshem tawm cov kev tiv thaiv neuroprotection los ntawm GRP78 overexpression nyob rau hauv ER-kev ntxhov siab hlwb.Qhov kev soj ntsuam tias GRP78 tuaj yeem nce ntawm mitochondria feem nws tus kheej yog qhov tseem ceeb thiab. GRP78 tau raug kuaj pom nyob rau hauv daim nyias nyias nruab nrab nruab nrab ntawm qhov chaw thiab matrix ntawm mitochondria, txawm hais tias nws txoj haujlwm tseem yuav tau piav qhia [79].

Txawm li cas los xij, thiab hais txog nws ntau txoj haujlwm, peb xav tias nws tuaj yeem ua rau bufferingCa2+. Yog tias qhov no yog qhov teeb meem, nws muaj ntau ntxiv nyob rau hauv mitochondria hauv ER-kev nyuaj siab hlwb thaum overexpressed yuav ua rau Ca2+ influx loj heev kom tsis txhob tsav tsheb apoptosis. Kev sim ua ntxiv rau qhov ntawd yuav muaj txiaj ntsig heev.

Kawm Kev Txwv thiab Kev Tshawb Fawb Yav Tom Ntej

Txoj kev tshawb no nthuav txoj hauv kev rau yav tom ntej kev txheeb xyuas ntawm lub luag haujlwm neuroprotective ntawm GRP78-nyob ntawm mitophagy hauv neuronal tuag tom qab neurotrauma. Thawj qhov kev txwv ntawm qhov kev tshawb fawb no yog tias mitophagy tau txheeb xyuas nyob rau hauv lub sijhawm tshwj xeeb qhov rais tom qab RA, thiab txij li mitophagy yog cov txheej txheem tsis muaj zog, nws yuav txaus siab rau kev txiav txim siab nws cov dej ntws thiab cov molecules koom nrog.

Yog li, kev sim ntxiv kev hloov kho mitophagy (los ntawm cov tshuaj pharmacological lossis caj ces) yog xav tau los txiav txim seb qhov teebmeem tshwm sim vivo puas yog nyob ntawm mitophagy.

Qhov no yuav tso cai rau peb kom paub meej tias kev nthuav tawm ntau dhau ntawm GRP78 txhawb nqa MN ciaj sia los ntawm mitophagy. Txij li thaum muaj ib qho kev piav qhia zoo ntawm mitochondria thiab apoptosis, ntxiv rau hauv vitro thiab hauv vivo manipulations yog qhov tseem ceeb rau kev txiav txim siab uas txoj kev tuag ua rau MN tuag hauv peb tus qauv. Qhov no yuav pab kom paub meej seb GRP78 ncaj qha hloov kho lub xov tooj ntawm tes kev tuag los ntawm kev thaiv nws lossis seb nws lub xub ntiag tso cai rau MNs tiv thaiv nws thiab muaj sia nyob.

5. Cov lus xaus

Txoj kev tshawb no yog thawj zaug piav qhia txog lub luag haujlwm tshiab rau GRP78 hauv kev hloov kho mitophagy kom ua tiav kev tiv thaiv motoneuronal. Peb cov txiaj ntsig tau nthuav tawm tias GRP78 tuaj yeem tsav mitophagy los txhawb kev tiv thaiv neuroprotection ntawm degenerating lub cev muaj zog neurons raws li kev raug mob ntawm lub paj hlwb, rov ua kom puas mitochondrial muaj nuj nqi hauv neuronalcells. Ntxiv mus, qhov GRP no 78-kev kho mob neuroprotection yog nyob ntawm PINK1 thiab IP3R.Yog li ntawd, qhov kev ua kom zoo ntawm cov mitophagy, los ntawm kev kho noob nrog GPR78 lossis lwm tus neeg sib tw, yuav raug siv los ua kev kho tshiab rau kev raug mob ntawm lub paj hlwb. .

Cov Khoom Siv Ntxiv: Cov hauv qab no muaj nyob online ntawm https://www.mdpi.com/article/10.3390/biomedicines9081039/s1. Daim duab S1: Bioinformatic tsom xam. Daim duab S2: Endogenous GRP78levels tom qab Tun kho. Daim duab S3: Tun thiab EFV ua rau mitochondrial dysfunction hauv NSC34 hlwb.Figure S4: Tsis muaj npe. Daim duab S5: Kev soj ntsuam ntawm cov cell viability tom qab shRNA nucleofection Cov khoom siv ntxiv kuj suav nrog cov ntxhuav nrog tag nrho cov proteins pom hauv kev tsom xam proteomic.

Tus sau kev koom tes: TL-R. thiab DR-G. tsim thiab ua qhov kev sim, txheeb xyuas cov txiaj ntsig, thiab sau ib feem ntawm cov ntawv sau. MH-G. thiab JF tau pab nrog hauv vivo kev sim.PM-G. thiab JML tau ua mitochondrial purifications. MP, CB, thiab NA pab nrog mitochondria functional tsom xam. VP thiab AB tau npaj tag nrho GRP78 plasmids thiab viralvectors siv. CC tau tsim, tsim, saib xyuas, thiab tshuaj xyuas txhua qhov kev sim thiab sau cov ntawv sau. Txhua tus kws sau ntawv tau nyeem thiab pom zoo rau cov ntawv luam tawm ntawm cov ntawv sau.

Cov Nyiaj Txiag: Txoj haujlwm no feem ntau yog txhawb nqa los ntawm Ministerio de Economía y Competitividad ntawm Spain (#SAF 2014-59701) thiab los ntawm Marató de TV3 (#201607.10). Peb kuj ua tsaug rau kev txhawb nqa los ntawm CIBERNED thiab Generalitat de Catalunya nyiaj txiag. CB yog tus tau txais Miguel Servet daim ntawv cog lus (CP19/00077) los ntawm Carlos III Health Institute.

Institutional Review Board Statement: Tag nrho cov txheej txheem uas koom nrog cov tsiaj tau pom zoo los ntawm Universitat Autònoma de Barcelona thiab Generalitat de Catalunya thiab ua raws li EuropeanCommunity Council Directive 2010/63/EU.

Cov Lus Qhia Txog Kev Pom Zoo: Tsis siv tau.

Cov Lus Qhia Txog Cov Ntaub Ntawv: Txhua cov ntaub ntawv tsim los yog tshuaj xyuas thaum lub sijhawm kawm no suav nrog hauv tsab xov xwm no thiab nws cov ntaub ntawv ntxiv.

Kev lees paub: Peb ua tsaug Marta Monserrat, Ariadna Aransanz, Miguel Chillón, Julia Lorenzo, Sara Marmolejo, thiab Neuroplasticity thiab Regeneration Group ntawm UAB. Peb ua tsaug tshwj xeeb rau Carlos Guillem (University Complutense de Madrid) rau kev ua siab zoo muab PARK2-HAplasmid thiab rau Elena Galea thiab Esther Dalfo ntawm UAB rau cov tshuaj tiv thaiv.

Kev tsis sib haum xeeb ntawm kev txaus siab: Cov neeg sau ntawv tshaj tawm tsis muaj teeb meem ntawm kev txaus siab.

Cov Lus Qhia Tshwj Xeeb: Cov kws sau ntawv xav muab qhov tshwj xeeb hais hauv kev nco txog Caty Casas, tus kws tshawb fawb muaj txiaj ntsig, thiab tus neeg zoo thiab siab tawv, uas tau tuag ntxov ntxov thaum muaj hnub nyoog 54 xyoo. lub neej, nws txoj kev muaj peev xwm, thiab nws txoj kev coj ncaj ncees thiab tib neeg muaj nuj nqis.

Cov ntaub ntawv

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