Chaperone-Mediated Autophagy nyob rau hauv Neurodegenerative Diseases Thiab Mob Ntsws Neurological Insults nyob rau hauv Central Nervous System Part 2
Aug 05, 2024
4. Kev Tshawb Fawb Cov Cuab Yeej rau Assay CMA Kev Ua Haujlwm
Hauv seem no, peb piav qhia txog cov cuab yeej tshawb fawb txog kev sim muaj rau kev soj ntsuam CMA kev ua hauv vitro thiab hauv vivo. Cov txheej txheem feem ntau siv kom tau txais cov ntaub ntawv cuam tshuam nrog CMA kev ua haujlwm nrog rau CMA kev soj ntsuam ua haujlwm.
CMA (Certified Management Accountant) yog daim ntawv pov thawj kev tswj hwm tus accountant kev tshaj lij, uas yog lub hom phiaj los ua pov thawj cov neeg uas muaj kev txawj ua haujlwm txaus thiab kev paub txog kev paub txog kev tswj xyuas nyiaj txiag. Memory, tib neeg lub hlwb muaj peev xwm, hais txog kev muaj peev xwm nco qab cov ntaub ntawv, kev paub, lossis kev paub.
Muaj qee qhov kev sib raug zoo ntawm CMA thiab nco. Ua ntej tshaj plaws, qhov kev xeem CMA xav kom cov neeg sib tw yuav tsum nco ntsoov ntau cov ntsiab lus kev paub thiab cov ntsiab lus, thiab tib lub sijhawm xav kom cov neeg sib tw yuav tsum paub cov txuj ci los siv cov ntsiab lus kev paub no. Yog li ntawd, kev nco zoo heev yuav ua lub luag haujlwm tseem ceeb hauv kev ua tiav cov txiaj ntsig zoo hauv CMA kev xeem.
Qhov thib ob, kev nco tuaj yeem txhim kho los ntawm kev kawm thiab kev xyaum, uas tseem tuaj yeem pab kawm rau CMA kev xeem. Piv txwv li, los ntawm kev rov tshuaj xyuas cov ntsiab lus kev paub, kev kawm thiab kev nco txog cov ntaub ntawv sib txawv, thiab siv cov tswv yim nco, koj tuaj yeem txhim kho kev nco thiab pab cov neeg sib tw kom paub cov ntsiab lus thiab kev txawj ntse uas xav tau rau kev xeem CMA.
Thaum kawg, kev kawm CMA tsis yog ib qho kev xeem xwb, tab sis kuj yog kev txhim kho hauv kev ua haujlwm zoo. Hauv kev txhim kho kev ua haujlwm, yog tias koj xav ua kom koj lub peev xwm ua haujlwm tau zoo, koj ib txwm xav tau lub peev xwm nco tau los pab koj tus kheej kom ua tiav koj txoj haujlwm ua tiav.
Hauv ntej, muaj kev sib raug zoo ntawm CMA thiab nco. Sim txhim kho koj lub cim xeeb tsis yog tsuas yog pab koj kom tau qhab nia zoo xwb tab sis kuj tseem pab koj ua tiav koj cov haujlwm ua haujlwm zoo dua thiab paub txog koj tus kheej tus nqi thiab kev ua haujlwm tau zoo thaum koj txoj haujlwm txhim kho. Nws tuaj yeem pom tau tias peb yuav tsum txhim kho peb lub cim xeeb, thiab Cistanche deserticola tuaj yeem txhim kho kev nco zoo vim tias nws muaj cov tshuaj tiv thaiv antioxidant, tiv thaiv kev laus, thiab tiv thaiv kev laus, uas tuaj yeem pab txo qis oxidation thiab mob hauv lub hlwb, yog li tiv thaiv kev noj qab haus huv. lub paj hlwb. Tsis tas li ntawd, Cistanche deserticola kuj tseem tuaj yeem txhawb kev loj hlob thiab kho cov paj hlwb, yog li txhim kho kev sib txuas thiab kev ua haujlwm ntawm cov neural network. Cov teebmeem no tuaj yeem pab txhim kho kev nco, kev kawm muaj peev xwm, thiab kev xav nrawm, thiab tseem tuaj yeem tiv thaiv qhov tshwm sim ntawm kev paub tsis meej thiab cov kab mob neurodegenerative.
4.1. Muaj txiaj ntsig Kev Ntsuam Xyuas rau Kev Saib Xyuas CMA Kev Ua Haujlwm
Kev ntsuas kev hloov pauv ntawm cov nyiaj ntawm cov khoom tseem ceeb ntawm CMA tuaj yeem siv los ua ib txoj hauv kev tsis ncaj ncees ntawm kev ntsuas CMA kev ua [46]. Tsis tas li ntawd, tus naj npawb thiab kev faib tawm ntawm CMA-active lysosomes tuaj yeem txheeb xyuas kom pom cov kev hloov pauv hauv CMA kev ua [46].
Txawm li cas los xij, cov kev txheeb xyuas no tsuas yog muab cov ntaub ntawv cuam tshuam nrog CMA xwm txheej. Yog li, cov kev txheeb xyuas no yuav tsum tau ua kom tiav nrog cov kev ntsuam xyuas ua haujlwm [47] .Immunoblotting thiab kev ntsuam xyuas rau kev ntsuam xyuas cov kev hloov pauv hauv CMA cov khoom tseem ceeb yog cov feem ntau siv txoj hauv kev los ntsuas CMA kev ua.
Txij li thaum lysosomallevels ntawm LAMP2A raug txwv rau CMA [48], kev hloov pauv hauv kev nplua nuj ntawm LAMP2Aprotein hauv lysosomes feem ntau cuam tshuam nrog kev ua haujlwm ntawm CMA. Yog li ntawd, nyob rau hauv kev ntsuam xyuas, lub xub ntiag ntawm LAMP2A ntawm lub lysosomal membrane yuav tsum tau soj ntsuam [47].
Immunoblotting rau LAMP2A siv lysosome-enriched fractions los yog tsawg kawg yog ib feem membranouscell yog cov ntaub ntawv ntau tshaj qhov uas siv tag nrho cov cell lysates [46].Levels ntawm lysosomal-hsc70 kuj muaj feem xyuam rau CMA kev ua [49].
Txawm li cas los xij, hsc70 yog ib qho ntawm cov xov tooj ntawm tes ntau tshaj plaws, thiab feem ntau nyob hauv lysosomes yog ib qho me me. Yog li, immunoblotting rau hsc70 nyob rau hauv tag nrho cov cellular lysates tsis qhia rau CMA [46]. Colocalization ntawm hsc70 nrog cov cim lysosomal (xws li, LAMP1) tuaj yeem siv los txheeb xyuas CMA-active lysosomes [49].
Tus naj npawb ntawm cov lysosomes colocalized withhsc70 hauv kev faib ua feem rau tag nrho lub pas dej lysosomal nce thaum CMA tau qhib [12]. Tsis tas li ntawd, ib qho kev tsom xam electron microscopic siv immunogold staining rau hsc70 tuaj yeem muab cov ntaub ntawv hais txog lub pas dej ntawm CMA-active lysosomes [49].
Hauv kev tshuaj xyuas siv cov lysosomes cais tawm ntawm cov hlwb lossis cov ntaub so ntswg, nce qib ntawm CMA substrates uas paub zoo (xws li, GAPDH) tuaj yeem qhia txog qhov txo qis ntawm CMA [46].
Ingeneral, CMA substrates yog sai degraded tom qab translocation [6]. Yog li, kev sib piv ntawm cov qib lysosomal ntawm CMA substrates hauv cov hlwb lossis cov tsiaj ntawm cov qauv kho lossis tsis kho nrog inhibitors ntawm lysosomal proteases (ie, leupeptin) tso cai rau kev ntsuas qhov flux hauv CMA qhov tseem ceeb [24].
4.2. Functional Assay
Ntau qhov kev ntsuam xyuas ua haujlwm pab kom taug qab ntawm CMA kev ua haujlwm dhau sijhawm hauv cov hlwb, cov ntaub so ntswg, thiab cov kab mob sib cais.
4.2.1. Intracellular Protein Degradation Assessment
Kwv yees li 30% ntawm tag nrho cov cytosolic proteins tuaj yeem degraded los ntawm CMA [9]. Txawm li cas los xij, qhov tseeb feem ntawm cytosolic protein degraded los ntawm CMA nws txawv nyob ntawm seb hom cell thiab cellular mob [6].
Yog li ntawd, kev ntsuas ntawm lub pas dej ntawm cov cellular proteins uas undergo degradation los ntawm CMA yog ib tug ntau txoj kev los txiav txim lub zuag qhia tag nrhoactivity ntawm CMA txoj kev [46].
Pulse thiab chase thwmsim siv radiolabeled aminoacid thiab inhibitors ntawm lysosomal proteases lossis lwm txoj hauv kev autophagic tuaj yeem siv los cais cov proteins uas raug CMA degradation los ntawm cov tswj los ntawm lwm txoj kev [50].
4.2.2. Photoconvertible CMA Reporters
Tsis tas li ntawd, ib txoj hauv kev los saib xyuas lub koom haum lysosomal ntawm cov neeg sau xov xwm fluorescentCMA kuj tseem yuav pab tau rau kev taug qab cov substrate xa thiab degradation dhau CMA [51].
Siv photoconvertible fluorescent reporters [51], nws muaj peev xwm mus taug qab lub koom haum ntawm photoconverted protein nrog lysosomes nyob rau hauv ib tug txawv fluorescencechannel. Qhov nce ntawm cov fluorescent puncta ib lub xov tooj yuav yog qhov qhia tau zoo ntawm CMA ua kom [46].
4.2.3. Hauv Vitro Kev Ntsuam Xyuas ntawm CMA Siv Isolated Lysosomes
Kev sib tham ntawm txoj kev sib txawv ntawm autophagic ua rau nws nyuaj rau kev txheeb xyuas CMA cov haujlwm hauv cov hlwb tsis zoo [41]. Yog li ntawd, peb yuav tsum cais txhua qhov kev ua haujlwm uas koom nrog hauv cov txheej txheem degradation ntawm CMA txoj hauv kev [46,49].
Txoj kev ntseeg siab tshaj plaws rau kev tshuaj xyuas CMA kev ua haujlwm yog tau los ntawm kev hloov kho hauv vitro ntawm CMA nrog cais cov lysosomes [52] .Kev cais ntawm cov feem tshwj xeeb ntawm lysosomes nquag hauv CMA tso cai rau kev tshuaj xyuas cov ntsiab lus ntawm endogenous CMA substrates hauv CMA compartments [47].
Isolatedlysosomes kuj tso cai rau kev hloov kho ntawm CMA hauv vitro ua raws cov kauj ruam hauv CMA txheej txheem-substrate khi, lysosomal uptake, thiab lysosomal degradation [50].
Kev kho mob nrog lysosomal protease inhibitors ua raws li incubation nrog CMA substratew yuav tso cai rau kev ntsuas ntawm substrate-bound thiab translocated rau hauv lysosomes (kev khi thiab uptake) [39].
Los ntawm txo tus nqi ntawm cov substrate khi rau lysosomesin uas proteolysis tsis tau raug tiv thaiv, nws yuav ua tau rau xam cov uptake [39,53].
Cov lysosomal fractions cais kuj tso cai rau kev sib piv ncaj qha ntawm cov kev hloov pauv hauv cov ntsiab lus, kev hloov pauv tom qab kev hloov pauv, thiab lub koom haum ntawm CMA Cheebtsam ntawm thelysosomal membrane. Kev txo qis hauv lysosomal LAMP2A lossis lys-hsc70 qib hauv kev cais tawm yog qhov qhia tau tias txo qis CMA kev ua haujlwm [54,55], qhov nce hauv LAMP2Alevels qhia txog kev tswj hwm ntawm CMA kev ua haujlwm [56].
Tsis tas li ntawd, qhov piv ntawm lysosomal LAMP2A sib sau ua ke rau hauv ib qho multimeric complex ntawm ib lub sijhawm tuaj yeem txiav txim siab siv bluenative electrophoresis ntawm cais lysosomes thiab immunoblot rau LAMP-2A [57].
5. Cov Kab Mob Neurodegenerative thiab CMA
Neurons yog cov hlwb tom qab mitotic thiab xav tau cov tshuab ua kom muaj protein ntau kom tswj tau cov cellular homeostasis hauv kev ntxhov siab [58,59]. Kev puas tsuaj ntawm cov txheej txheem protein degradation hauv CNS ua rau kev sib sau ntawm cov proteins tsis zoo lossis puas tsuaj, uas yog qhov txawv ntawm ntau cov kab mob neurodegenerative.
Cov ntaub ntawv pov thawj tseem ceeb tau sau tseg tias kev ua haujlwm tsis zoo ntawm CMA yog txuam nrog cov kab mob sib txawv hauv ntau yam kab mob neurodegenerative cuam tshuam rau CNS [1,14].
Hauv cov kab mob no, ntau yam kab mob pathogenic tau raug txheeb xyuas raws li cov substrates ntawm CMA, xws li -synuclein hauv PD [60], Tauprotein hauv AD [61], Huningtin (Htt) hauv HD [62,63], thiab TDP{{5} } hauv ALS thiab FTLD [64,65].
5.1. Tus kab mob Parkinson
PD yog ib qho ntawm cov kab mob neurodegenerative tshaj plaws. Lub ntsiab pathological yam ntxwv ntawm PD yog maj mam poob ntawm dopaminergic neurons nyob rau hauv lub substantia nigra thiab aggregation ntawm cov protein -synuclein nyob rau hauv Lewy lub cev.
Ntau cov kev tshawb fawb tau pom tias qhov tsis zoo ntawm CMA yog cuam tshuam nrog lub ntsiab pathogenesis ntawm PD [4,66]. Hauv cov neeg mob nrog PD, qib ntawm LAMP2A cov protein tau txo qis hauv lub hlwb, qhia tias CMA kev ua haujlwm tau txo qis [67,68].
Ntau cov kev tshawb fawb yav dhau los tau qhia tias inhibition ntawm CMAdegradation txoj hauv kev ua rau cov tsub zuj zuj ntawm -synuclein, uas cuam tshuam nrog kev poob qis ntawm dopaminergic neurons [8].
Qhov tseem ceeb, cov ntawv hloov pauv A53T thiab A30P ntawm -synuclein txheeb xyuas hauv tsev neeg PD tsis tuaj yeem degraded los ntawm CMA. Tsis tas li ntawd, cov ntaub ntawv mutant no nruj nreem khi rau LAMP2A ntawm lub lysosomal daim nyias nyias thiab thiaj li inhibit qhov kev degradation ntawm lwm yam CMA substrates hauv vitro [60,69].G2019S kev hloov pauv hauv leucine-nplua nuj rov kinase 2 protein (LRRK2) tuaj yeem ua rau pathologicalcause ntawm [70].
G2019S mutant inhibits lub dynamic sib dhos ntawm CMAtranslocation complex ntawm lub lysosomal membrane, ua rau lub dysfunction ntawm CMA nyob rau hauv amouse qauv ntawm PD thiab lub hlwb ntawm mutant LRRK2 PD cov neeg mob [25]. Tsis tas li ntawd, cov kab mob mutant ntawm LRRK2 khi rau cytosolic Hsc70 thiab cuam tshuam txawv txav nrog CMA Cheebtsam, thaiv kev degradation ntawm lwm yam CMA substrates thiab neuronal proteinhomeostasis hauv vitro [25,71].
Ubiquitin C-terminal hydrolase L1 (UCH-L1) lub cev cuam tshuam nrog LAMP-2A, Hsc70, thiab Hsp90 thiab koom nrog hauv kev tswj hwm txoj cai ntawm CMA txoj hauv kev [72]. Hauv kev kawm dhau los, daim ntawv I93M mutant ntawm UCH-L1 tau txheeb xyuas hauv ib tsev neeg PD [73].
Nws kuj tau tshaj tawm tias I93M kev hloov pauv hauv UCH-L1 abnormallyenhanced kev sib cuam tshuam nrog thaj tsam cytosolic ntawm LAMP2A, inhibiting CMA txoj hauv kev hauv vitro [74]. Tsis tas li ntawd, qhov kev qhia ntawm I93M mutant daim ntawv ntawm UCH-L1 inmammalian hlwb induced CMA inhibition-koom nrog nce ntawm cov synuclein [74].
Cov kev tshawb pom no qhia tias kev sib cuam tshuam tsis zoo ntawm I93M mutant daim ntawv ntawm UCH-L1 nrog CMA machinery tej zaum yuav ua rau lub pathogenesis ntawm PD cuam tshuam nrog kev sib sau ntawm -synuclein.Parkinson tus kab mob protein 7 (PARK7), tseem hu ua DJ-1, yog multifunctional protein koom nrog ntau yam kev ua haujlwm ntawm tes, suav nrog oxidation tsis kam [75].
PARK7/DJ-1 muaj lub luag haujlwm tseem ceeb hauv kev tswj hwm mitochondrial homeostasis [75]. Nws tau raug tshaj tawm tias kev hloov pauv hauv DJ-1 gene mediates autosomal recessive thiab ntxov cov ntaub ntawv ntawm PD [76].DJ-1 deficiency ua rau kom lub degradation ntawm LAMP2A hauv lysosomes, ua rau cov aggregation ntawm -synuclein [77] ].
Hauv qhov sib piv, DJ-1 muaj peev xwm inhibit qhov tsub zuj zuj ntawm -synuclein los ntawm regulating CMA [78].Txhua yam, ntau yam molecular mechanisms ua rau dysfunction ntawm CMA yog suav tias yog underlie lub pathogenesis ntawm PD.
Txawm li cas los xij, cov txheej txheem pathological cuam tshuam nrog CMA tseem tsis meej. Kev tshawb fawb ntxiv yuav xav tau los qhia meej txog kev sib raug zoo ntawm CMA txheej txheem thiab cov kab mob tiag tiag ntawm PD.
5.2. Alzheimer's Disease
AD yog cov kab mob neurodegenerative tshaj plaws hauv cov neeg laus. Lub ntsiab pathogenesis ntawm AD yog amyloid-plaque tsim thiab Tau aggregation tshwm sim los ntawm kev puas tsuaj ntawm protein homeostasis. Ntau cov proteins uas cuam tshuam nrog AD tau raug txheeb xyuas tias yog CMA substrates.Qhov CMA degradation ntawm cov protein substrates tau pom tias muaj kev puas tsuaj rau cov neeg mob nrog AD [45,61,79].
Kev nce zuj zus ntawm amyloid-oligomers yog qhov tshwm sim hauv nruab nrab hauv AD [80,81]. Ib txoj kev tshawb fawb tsis ntev los no tau pom tias tagging amyloid-oligomers nrog ntau tus KFERQ motifs txhawb lawv txoj kev nkag mus rau hauv endosomes thiab lysosomes, tiv thaiv tib neeg cov kab mob cortical neurons los ntawm neurotoxicity [82].Amyloid precursor protein (APP) yog ib qho tseem ceeb pathogenic molecule hauv AD vim tias nws tuaj yeem ua tiav los tsim cov kab mob. amyloid - [83].
APP muaj KFERQ zoo li motif ntawm nws Cterminus. Qhov motif no yog qhov tseem ceeb rau kev ua haujlwm ib txwm ua thiab degradation ntawm APP los tiv thaiv kev sib sau ntawm APP-C-terminal fragments [84]. Kev tshawb fawb tsis ntev los no tau qhia tias APP yog CMA substrate uas khi rau Hsc70 [85].
Qhov inhibition ntawm CMA degradation ntawm APP txhim kho nws cytotoxicity. Tsis tas li ntawd, kev ua kom CMA los ntawm Hsc70 overexpressionor Metformin txo qis lub hlwb amyloid-plaque ntau ntau thiab thim rov qab thev naus laus zis thiab kev coj tus cwj pwm AD phenotypes hauv tus qauv nas ntawm AD [85].Tau yog ib qho cytosolic protein uas feem ntau stabilizes microtubules hauv cov hlwb neuronal.
Tauprotein muaj CMA-targeting motifs thiab tuaj yeem degraded los ntawm CMA txoj kev [79]. Aggregation ntawm mutant Tau proteins uas ua rau Tau hyperphosphorylation thiab tsim ntawm neurofibrillary tangles yog ib qho cim ntawm AD thiab lwm yam tauopathies [86,87].
Tsis tas li ntawd, themutant Tau proteins tuaj yeem cuam tshuam txawv txav nrog LAMP2A thiab inhibit translocation rau hauv lysosome lumen, impairing CMA kev ua [79]. Tus tswj hwm ntawm calcineurin 1 (RCAN1) yog lub substrate ntawm CMA [45] thiab tau nce siab hauv cov neeg mob AD [88].
RCAN1 yog ib qho inhibitor ntawm calcineurin-dephosphorylation ntawm Tau proteins. Qhov tseem ceeb, CMA kev ua haujlwm tuaj yeem cuam tshuam los ntawm kev nce qib ntawm RCAN1, yog li cuam tshuam qhov degradation ntawm lwm cov substrates ntawm CMA [45].
5.3. Huntington's Disease
HD yog ib qho kab mob neurodegenerative lig tshwm sim los ntawm kev tswj tsis tau txav, dementia, thiab kev ntxhov siab. HD yog ib yam kab mob uas muaj feem cuam tshuam los ntawm kev sib sau thiab sib sau ua ke ntawm mutant Htt protein nyob rau hauv striatal thiab cortical neurons.
Htt muaj qhov txawv txav N-terminal polyglutamine (polyQ) tract [62,63,89] .Dysfunction ntawm Htt degradation yog pom zoo raws li lub ntsiab pathogenesis ntawm HD.
Cov kev tshawb fawb yav dhau los tau pom tias CMA tau koom nrog hauv kev degradation ntawm mutant Htt hauv cellular thiabmouse qauv ntawm HD [62]. Htt harbors a putative KFERQ motif and interacts with the keycomponents of CMA, Hsc70, and LAMP2A. Tsis tas li ntawd, mutant Htt nrog kev nthuav dav ntawm polyQ tract qhia txog kev cuam tshuam los ntawm CMA hauv vitro [89].
Tsis yog CMA nkaus xwb tab sis macroautophagy koom nrog hauv kev degradation ntawm Htt [90]. Htt tuaj yeem khi rau ob qho tib si LAMP2A thiab macroautophagy-txog protein Atg7 hauv qhov txheej txheem degradation [89,91,92].
CMA kev ua ub no tau tshaj tawm txog kev tswj hwm hauv cellular thiab tsiaj qauv ntawm HD hauv thawj theem ntawm tus kab mob. Txawm li cas los xij, kev ua haujlwm ntawm CMA txo qis thaum lub sijhawm kawg ntawm tus kab mob [91]. Cov kev tshawb pom no qhia tias qhov nce thaum ntxov hauv CMA kev ua haujlwm yuav yog ib qho kev cai them nyiaj hauv kev teb rau qhov tsis muaj peev xwm ntawm macroautophagy. Kev poob qis hauv qib ntawm lysosomal LAMP2A qhia tias muaj qhov poob ntawm CMA ua haujlwm nyob rau theem kawg ntawm HD [91].
5.4. Amyotrophic Lateral Sclerosis thiab Frontotemporal Lobar Degeneration
ALS thiab FTLD yog cov kab mob neurodegenerative nrog ntau yam kev kho mob thiab kab mob zoo sib xws [93]. Transactivation teb DNA-binding protein 43 kDa (TDP-43) isa ribonuclear protein regulating ntau yam ntawm RNA metabolism.
Kev sib sau ntawm TDP-43 C-terminal fragments nyob rau hauv neuronal hlwb feem ntau pom nyob rau hauv cov neeg mob ALSand FTLD [65]. Yog li, TDP-43 tsub zuj zuj yog dav suav hais tias yog lub cim ntawm cov kab mob no.
TDP-43 protein muaj KFERQ-zoo li motif khi rau Hsc70 thiab tuaj yeem degraded los ntawm cov txheej txheem CMA [94,95]. Hsc70 qhia tau hais tias txo qis hauv cov lymphomonocytes ntawm cov neeg mob ALS tsis sib xws thiab pab txhawb rau TDP-43 tsub zuj zuj [64].
Ib qho kev hloov pauv hauv KFERQ-zoo li motif hauv TDP-43 tuaj yeem cuam tshuam nws qhov degradation ntawm CMA, inducing tsub zuj zuj ntawm TDP-43 thiab inhibition ntawm CMA hauv kab lis kev cai hlwb [94]. CMA tuaj yeem pab tswj kev hloov pauv ntawm lub cev thiab cov kab mob pathological ntawm TDP-43 [94].
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