Nce kev ua haujlwm ntawm kev sib xyaw ntsig-xyoob xwm Xinesmas 5 ua rau muaj kev sib tw ntawm cov yeeb yaj kiab uas muaj kabmob sib npaug (2005)

Proc Natl Acad Sci US A. 2005 Lub Ob Hlis 1; 102(5): 1737-1742.

Luam tawm online 2005 Lub Ib Hlis 21. doi:  10.1073 / pnas.0409456102
PMCID: PMC547862
neuroscience
Qhov no tsab xov xwm tau ua lawm sau los ntawm lwm cov xwm hauv PMC.

Abstract

Cocaine, ib qho yeeb tshuaj ntawm kev tsim txom, nce qib synaptic dopamine nyob rau hauv lub striatum los ntawm thaiv dopamine reuptake ntawm axon terminals. Cyclin-dependant kinase 5 (Cdk5) thiab nws txoj haujlwm p35, cov nqaijrog koom nrog phosphorylation ntawm cov kab mob hauv cov hauv qab postmitotic neurons, tau pom tias muaj kev tiv thaiv zoo tom qab cov neeg muaj yeeb dawb. Txhawm rau soj ntsuam ntxiv qhov cuam tshuam ntawm Cdk5 thiab p35 induction ntawm striatal dopamine signaling, peb tau tsim ob txoj kev ywj pheej transgenic nas cov kab uas Cdk5 lossis p35 tau mob siab rau tshwj xeeb hauv neurons. Peb tshaj tawm ntawm no tau nce Cdk5 kev ua haujlwm, ua rau p35 tab sis tsis yog Cdk5 kev ua haujlwm ntau, ua rau muaj kev ua yeeb yam ntawm cocaine-mediated dopamine cim. Nce Cdk5-mediated phosphorylation ntawm dopamine thiab cAMP-tswj hwm phosphoprotein, cov tshuaj loj me, 32 kDa (DARPP-32) ntawm Thr-75, tau nrog phosphorylation tsawg ntawm DARPP-32 ntawm Thr-34. Nce Cdk5-mediated phosphorylation ntawm kev tshaj tawm cov cim-kinase kinase 1 ntawm Thr-286 tau nrog los txo qis kev ua haujlwm ntawm cov teeb meem tsis qhia npe-tswj kinase 1 / 2. Cov kev cuam tshuam no ua rau muaj txiaj ntsig ntawm cocaine-hob phosphorylation ntawm cAMP cov lus teb-sib khi cov protein ntxiv nrog rau kev txo qis ntawm c-fos hauv striatum. Cov txiaj ntsig no txhawb nqa lub tswv yim tias Cdk5 kev ua haujlwm tau koom nrog kev hloov pauv ntawm cov noob keeb kwm tom qab raug ntev mus rau yeeb thiab vim li no cuam tshuam cov kev hloov mus ntev hauv cov kev ua kom cov neeg quav yeeb nyob rau hauv yeeb.

keywords: neeg quav yeeb quav cawv, phosphorylation, striatum

Cocaine nce synaptic dopamine qib nyob rau hauv striatum thiab hloov cov noob qhia nyob rau hauv lub dopaminoceptive neurons los ntawm kev ua kom muaj txoj hauv kev ua kom qhov kev tawm suab pib thaum pib los ntawm dopamine D1 receptor rau lub nucleus (1)). Kev tiv thaiv ntev ntev ntawm cocaine nce-tswj ntau ntau qhov hloov pauv ntawm cov ntawv, uas ua rau muaj kev hloov pauv ntev hauv cov noob qhia uas xav tias yuav ua raws li neuronal kev hloov kho hauv kev quav yeeb dawb (2)). OsFosB, txheeb tias yog qhov hloov tau (3), tau pom tias tau txhim kho tus cwj pwm kev coj ntawm cov tsiaj mus rau cocaine (4, 5)). Yog li, kev txheeb xyuas cov hom phiaj ntawm cov caj ces uas tswj los ntawm ΔFosB induction yog qhov xav kom ua rau muaj kev nkag siab ntau dua ntawm cov molecular mechanism nyob rau kev quav yeeb. Tsis ntev los no, kev kho mob ntev ntev ntawm cov tsiaj nrog cocaine tau pom tias nce siab-tswj cov lus qhia ntawm cyclin-dependant kinase 5 (Cdk5) thiab nws cov haujlwm p35 hauv striatum los ntawm kev tawm tsam ntawm ΔFosB (6, 7).

Cdk5 yog ib tus tswv cuab ntawm Cdk tsev neeg ntawm serine / threonine kinases. Tsis zoo li lwm tus Cdks uas yog tus tsim txoj cai loj ntawm lub voj voog ncig ntawm tes, Cdk5 yog feem ntau koom nrog phosphorylation ntawm cov khoom me me hauv postmitotic neurons. (8)). Lub neuronal tshwj xeeb ntawm Cdk5 kev ua ub no tau ua tiav los ntawm kev koom nrog nws cov neeg ua haujlwm, tsis hais p35 lossis p39, uas feem ntau hais tawm nyob rau hauv postmitotic neurons (8)). Ntxiv rau txoj haujlwm tseem ceeb ntawm Cdk5 hauv kev tsim kho hlwb (9, 10), it kuj tau hais txog nyob rau hauv dopaminergic kis hauv postnatal paj hlwb (11, 12)). Kev txwv tsis pub los ntawm Cdk5 kev ua ub no ua rau muaj kev dopamine tso tawm ntau ntxiv hauv striatum, qhia txog qhov ua haujlwm tsis ua haujlwm ntawm Cdk5 ua tus tswj tsis zoo ntawm dopamine tso (11)). Txuas ntxiv, Cdk5 hloov qhov kev ua tau zoo ntawm postsynaptic dopamine teeb liab los ntawm phosphorylating dopamine- thiab cAMP-tswj hwm phosphoprotein, cov tshuaj molecular loj 32 kDa (DARPP-32) ntawm Thr-75, uas hloov DARPP-32 rau hauv cov tshuaj tiv thaiv cAMP-dependant kinase (12).

Cov kev soj ntsuam no pom zoo tias Cdk5 thiab p35 yog cov neeg tswj hwm qhov tseem ceeb ntawm kev ua kom ntev li ntawm dopamine cov paib tom qab mob ntev mus rau yeeb thiab yog li ntawm cov neeg quav yeeb. Txhawm rau hais ntxiv txog lub luag haujlwm ntawm Cdk5 ntawm striatal dopamine kev taw qhia, peb tau tsim ob txoj kab transgenic nas nyob rau hauv uas yog Cdk5 lossis p35 tau txhawb siab tshwj xeeb hauv neurons raws li kev tswj hwm ntawm p35 tshaj tawm. Peb qhov kev tshawb pom tau qhia tias Cdk5 kev ua haujlwm tau raug kho nrog kev nce qib p35 protein tab sis tsis yog Cdk5 protein, qhia tias qib p35 protein yog qhov txwv-npaum li cas rau Cdk5 kev ua ub no. Peb muab ntawm no nyob rau hauv vivo cov ntaub ntawv pov thawj uas tau nce Cdk5 kev ua haujlwm, vim yog p35 overexpression, ua rau kom muaj yeeb yam ntawm cocaine-mediated dopamine xa mus rau lub nucleus los ntawm kev txwv tsis pub PKA thiab cov paib ntxiv uas tswj cov kinase (ERK) cascades.

Ntaub ntawv thiab kev

Tshuaj tua kabmob. Cov tshuaj tiv thaiv polyclonal rau Cdk5 (C-8) thiab p35 (C-19) tau yuav los ntawm Santa Cruz Biotechnology. Cov tshuaj phosphorylation-dependant thiab -ntawm cov tshuaj tiv thaiv mus rau ERK kinase (MEK) 1 / 2, ERK1 / 2, thiab cAMP-teb cov ntsiab lus tiv thaiv protein (CREB) tau los ntawm Cell Signaling Technology (Beverly, MA). Cov tshuaj tiv thaiv rau phospho-Thr-34 DARPP 32 (13), phospho-Thr-75 DARPP-32 (12), tag nrho DARPP-32 (12), thiab c-fos (14) tau siv raws li tau piav qhia. Ib qho tshuaj tiv thaiv rau actin tau yuav los ntawm Sigma.

Sim Tsiaj. Peb ua ntej lawm cloned nas p35 noob Cdk5r1, uas encodes p35 protein, thiab nws cov xeeb ceem genomic (15)). Txhawm rau tsim cov pa transgenic nrog neuronal overexpression ntawm p35 (Tgp35), 6-kb echoRI-echoRI ib qho zuj zus uas muaj 1.2-kb cov chaw tshaj tawm hauv cheeb tsam tau raug xa mus rau hauv pGEM9Z (-) plasmid, thiab 45-bp tag muab tau los ntawm SV40 tau muab tso rau hauv KpnKuv xa nqes ntawm lub poly (A+) teeb liab (Daim duab. 1A)). Daim pib muaj a SpeKuv xaib rau genotyping ntawm cov tsiaj. 6-kb fragment tau dhau los ntawm plasmid thiab kev ua kom huv, tom qab los ntawm kev txhaj tshuaj ntawm transgene los tsim cov qog transgenic. Txhawm rau tshuaj xyuas qhov qhia tawm cov ntaub ntawv ntawm transgene nyob rau hauv kev tswj hwm txoj cai tswj hwm ntawm 1.2-kb p35 tshaj tawm nyob rau hauv vivo, ob lub transgenic nas (Tgp35; p35 - / -) tau tsim los ntxiv los ntawm kev siv ob-kauj ruam kev yug me nyuam uas tus Tgp35 nas tau tsim dua tshiab rau qhov keeb kwm yav dhau los p35-null. Lwm tus nas ua qauv siv hauv qhov kev tshawb fawb no suav nrog p35 +/–, p35 - / -, Cdk5 +/–, thiab nas transgenic nrog neuronal overexpression ntawm Cdk5 (TgCdk5) (9, 16, 17)). Genotypes ntawm cov nas tau txiav txim siab los ntawm kev ua haujlwm yav dhau los yav dhau los blot tsom xam los yog PCR ntawm genomic DNA cais tawm ntawm tus Tsov tus tw. Cov nas tau nyob hauv qab 12-h teeb / 12-h tsaus nti. Txhua qhov kev saib xyuas tau muab ua raws li National Institutes of Health txoj cai qhia txog kev saib xyuas thiab siv ntsuas hauv lub chaw kuaj thiab tsiaj.

Daim duab 1.  

Lub cim ntawm transgenic nas nrog neuronal overexpression ntawm p35 qhia los ntawm p35 kev nthuav dav (Tgp35). (A) Qhov kev sib hloov transgene tau qhia nrog cov qauv schematic ntawm cov tsiaj qus-hom thiab tsom p35 alleles. Cov kab liab qhia txog kev sojntsuam uas siv rau kev txiav txim genotyping. ...

Kev Tshawb Xyuas Yav Qab Teb Blot. Genomic DNA muab rho tawm los ntawm tus Tsov tus tw me me tau zom nrog echoRI thiab SpeKuv, electrophoresed ntawm 0.9% agarose gel, thiab hloov mus rau lub nylon daim nyias nyias. Lub membrane tau hybridized nrog ib tug random-primed 32P-labeled sojntsuam ntawm 42 ° C thaum hmo ntuj. 485-bp kev sojntsuam rau kev ua txhaum ntawm p35 knockout (p35 - / -) thiab Tgp35 cov nas tau tsim los ntawm PCR los ntawm kev siv cov khoom siv hauv qab no: 5′-ACATCCTGCTGCCACGGTGAC-3 ′ thiab 5′-CCACTGTAAAAGCAACAAGA-XNUM ′-3′-CCACTGTAAAAGCAACAAGA. Cov qog ua ke tau ntxuav ob zaug hauv 2 × SSC / 0.1% SDS ntawm 42 ° C rau 10 min, thiab ob zaug hauv 0.1 × SSC / 0.1% SDS ntawm 65 ° C rau 20 min, thiab nthuav tawm x-ray zaj duab xis.

Tshuaj Kho Mob. Cocaine (Sigma) tau yaj hauv cov ntsev ua kom tsis muaj menyuam. Cov tsiaj tau ip-txhaj nrog cocaine (15 mg / kg) lossis sib npaug ntawm cov kua ntsev ntawm lub hnub nyoog ntawm 3 lub hlis thiab raug tua los ntawm decapitation ntawm cov sij hawm sib txawv (15, 30, 60, thiab 120 min) tom qab kev txhaj tshuaj. Hlwb raug tshem tawm sai thiab ntuag hauv PBS-txias. Lub striata tau ces dissected tawm thiab raug rau qaum teb lossis Western blot tsom. Txog kev txhaj tshuaj immunohistochemical, cov ntu sib nrug tau los ntawm nas 2 h tom qab txhaj.

Kev Tshawb Fawb Qaum Qaum Teb. Tag nrho RNA tau muab rho tawm los ntawm striata nrog TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA) thiab raug rau Northern blot tsom xam raws li tau piav qhia (18)). Txhawm rau nrhiav c-fos mRNA, ib qho 189-bp tawg ntawm nas c-fos cDNA tau siv los ua kev sojntsuam raws li tau piav (19)). Cov theem ntawm c-fos mRNA tau suav los ntawm kev ntsuas qhov ntsuas ntom ntom ntom ntom ntom ntawm cov kab tshwj xeeb tshwj xeeb los ntawm kev siv cov tsom xam cov duab nrog nih duab software, Version 1.62.

Western Blot Kev Ntsuam Xyuas. Striatal cov ntaub so ntswg tau sonicated hauv 1% SDS thiab rhaub rau 10 min. Cov protein ntau hauv txhua qhov qauv tau txiav txim los ntawm BCA protein soj ntsuam (Pierce). Sib npaug zos rau cov protein tau sib cais los ntawm SDS / PAGE ua ntej hloov mus rau hauv nitrocellulose membrane. Cov puas tau raug thaiv hauv 1 × PBS muaj 5% skim mis thiab 0.05% Tween 20 thiab muab cov khoom muaj cov tshuaj tiv thaiv thaum hmo ntuj tas ntawm 4 ° C. Kev tsim tawm nrog peroxidase-sib txuas sib tua tiv thaiv nas los yog luav IgG (Sigma) tau ua nyob rau hauv chav tsev kub rau 60 min. Ib lub teeb liab raug tshawb pom los ntawm kev txhim kho chemiluminescence (Pierce), thiab cov qhov ntom ntom ntom ntaws ntawm cov kab txuas tau raug pom muaj nuj nqis raws li tau piav saum toj no.

Cdk5 Kinase Assay. Striatal lysates tau npaj nrog lysis tsis xws luag nrog 50 mM Tris · HCl, pH 7.4 / 50 mM NaCl / 5 mM EDTA / 1% Triton X-100 / 1mMDTT / 1 mM phenylmethylsulfonyl fluoride / XNUMin μ apμμ ml leupeptin / phosphatase inhibitors (phosphatase inhibitor sib xyaw I thiab II, Sigma). Cov lysates tau txhaj tshuaj tiv thaiv nrog los tiv thaiv Cdk1 (C-1) lossis tshuaj tiv thaiv p5 (C-8). Cdk35 immunoprecipitates tau npaj los ntawm kev tsim kom loj hlob ntawm 19 μl ntawm lysate (sib haum rau 5 μg ntawm cov protein) nrog cov tshuaj tiv thaiv Cdk300 (300 μg) thaum hmo ntuj ntawm 5 ° C ua raws li kev tawm tsam ntxiv nrog 3 μl ntawm Protein A-agarose hlaws (X % slurry hauv lysis tsis; Santa Santa Cruz Biotechnology) rau 4 h ntawm 25 ° C. Txog kev npaj p50 immunoprecipitates, 3 μl ntawm lysate (sib haum rau 4 mg ntawm cov protein) tau tawm nrog cov tshuaj tiv thaiv p35 (500 μg) raws li tau piav saum toj no. Lub immunoprecipitates tau raug ntxuav ob zaug nrog lysis tsis thiab ob zaug nrog kinase tsis muaj ntawm 1 mM Tris · HCl, pH 35 / 3 mM MgCl2/ 1 mM EDTA / 1 mM EGTA / 1 mM DTT, rov kho dua hauv 60 μl ntawm kinase tsis. Kev ua haujlwm Kinase tau ntsuas los ntawm kev siv histone H1 ua duab qis (18).

Immunohistochemistry. Mice tau tso tshuaj pleev ib ce los ntawm ip kev txhaj tshuaj ntawm avertin (250 mg / kg, Fluka) thiab cov tshuaj txau hloov pauv nrog 0.1 M sodium phosphate tsis, pH 7.4, tom qab ntawd Streck Tissue Fixative (Streck Laboratories, La Vista, NE), uas tsis muaj qhov txuas hla. Cov hlwb tawg tau raug kho ntxiv rau hauv tib txoj kev txhim kho thaum hmo ntuj ntawm 37 ° C. Tom qab ntawd, cov paj hlwb tau muab tso rau hauv cov roj av paraffin, txiav rau hauv 5-μm-tuab coronal ntu thiab raug rau immunohistochemistry los ntawm kev siv avidin-biotin-peroxidase complex txheej txheem (Vector Laboratories) nrog diaminobenzidine ua cov hauv qab. Cov seem tau muab pov tseg nrog kev sib raug zoo-purified polyclonal antibody tiv thaiv c-fos thaum hmo ntuj ntawm 4 ° C. Lub staining tshwj xeeb tau raug txheeb xyuas los ntawm kev tsis lees paub ntawm thawj cov tshuaj tiv thaiv.

tau

Lub Cim Ntawm Transgenic Mice nrog Neuronal Overexpression ntawm p35. Lub transgene tau siv los ua kom tiav cov kev qhia txog neuronal ntawm p35 muaj 6-kb tawg ntawm cov cloned nas p35 gene uas muaj 1.2-kb tus tshaj tawm thiab tag nrho cov lej lej ntawm p35 (Daim duab. 1 A)). Cov genotypes ntawm nas tau txiav txim siab los ntawm Southern blot tsom xam los ntawm kev siv cov kev sojntsuam uas tau tsim los kom paub qhov txawv p35 - / - thiab Tgp35 cov nas los ntawm cov tsiaj qus-hom nas (Daim duab. 1 A thiab B)Cov. Txhawm rau tshuaj xyuas cov transgene qhia nyob rau hauv kev tswj hwm ntawm 1.2-kb p35 tus tshaj tawm, peb tau tsim ob npaug ntawm cov nas transgenic (Tgp35; p35 - / -) nyob rau hauv uas p35 qhia tau tsav tsuas yog los ntawm transgene. Lub p35 qhia nyob rau hauv Tgp35; p35 - / - nas tau tshawb pom tsuas yog hauv lub hlwb (Daim duab. 1C), qhov chaw hais txog qhov sib txawv tau sib thooj ntawm cov nas-tsiaj qus hom (Daim duab. 1D)). Tsis muaj p35 tsis tau pom tias ua rau cov txheej txheem txawv txav hauv lub cerebral cortex thiab hippocampus ntawm cov nas (10)). Txawm li cas los xij, Tgp35; p35 - / - nas tau pom qhov ua tiav kev dim ntawm p35 - / - lub hlwb phenotype (Daim duab. 1E)). Cov ntaub ntawv no qhia tias 1.2-kb p35 cov neeg txhawb nqa tau tswj cov lus qhia ntawm transgene uas muaj cov ntaub ntawv zoo sib xws ntawm cov p35 los ntawm cov noob p35 noob qes.

Qib p35 Qib Txo Qib Yog Tus Nqi-Txwv Rau Txoj Cai Tshaj Tawm Ntawm Cdk5 Kev Ua Haujlwm. Peb tau tshuaj xyuas qhov kev txiav txim siab ntawm gene-dosage cov kev sib raug zoo ntawm cov noob encoding p35 thiab Cdk5 ntawm cov protein qhia hauv striatal extracts los ntawm p35 - / -, p35 +/–, hom tsiaj qus, Tgp35, Cdk5 +/–, thiab TgCdk5 nas thaum muaj hnub nyoog ntawm 3 lub hlis. Cov qib ntawm p35 thiab Cdk5 protein correlated zoo nrog cov gene dosages, feem (Daim duab. 2 A thiab B)). Tgp35 cov nas tsuag tau pom tias ≈1.6-nce p35 qib protein ntau npaum li piv nrog cov nas-tsiaj qus, hos Cdk5 qib protein tsis raug cuam tshuam los ntawm ntau qib p35 protein. TgCdk5 cov nas tsuag tau pom tias ≈1.9-nce qib hauv Cdk5 qib protein piv nrog cov nas-tsiaj qus, qhov sib txawv p35 protein tsis raug cuam tshuam los ntawm ntau qib Cdk5 protein. Txheeb xyuas qhov tshwm sim ntawm cov nqi sib txawv ntawm p35 protein ntawm Cdk5 kev ua si, Cdk5 tau txhaj tshuaj los ntawm striatal extract nrog anti-Cdk5 antibody thiab kinase kev ua haujlwm tau raug ntsuas. Zoo li no, txhawm rau tshuaj xyuas qhov tshwm sim ntawm cov nqi sib txawv ntawm Cdk5 protein ntawm kinase kev ua si, p35 tau txhaj tshuaj tiv thaiv los ntawm striatal extract nrog anti-p35 antibody, thiab kinase kev ua haujlwm tau raug ntsuas. Kev ua haujlwm Cdk5 cuam tshuam zoo nrog qib uas p35 protein tab sis tsis nrog qib Cdk5 protein (Daim duab. 2 C thiab D)). Cov txiaj ntsig no tau qhia tias tus nqi ntawm p35 protein yog qhov ntsuas tus lej rau Cdk5 kev ua haujlwm. Yog li peb thiaj li siv Tgp35 cov nas los tshawb xyuas qhov tshwm sim ntawm kev ua si Cdk5 ntau ntxiv ntawm striatal dopamine signaling.

Daim duab 2.  

Kev cai nce qib ntawm Cdk5 kev ua haujlwm yog tus nqi-txwv los ntawm p35 qib protein. (A) Cov lus qhia Western pom tias cov qib protein ntawm p35 thiab Cdk5 cuam tshuam nrog cov noob tshuaj ntawm p35 thiab Cdk5 noob, feem. (B) Cov theem sib txawv ntawm p35 lossis Cdk5 protein ...

Cocaine-Induced Phosphorylation ntawm DARPP-32 ntawm Thr-34 Yog Attenuated hauv Tgp35 Mice. Kev ua haujlwm ntawm DARPP-32 nyob ntawm nws lub phosphorylation xeev ntawm ntau qhov chaw (20)). PKA phosphorylates DARPP-32 ntawm Thr-34, hos Cdk5 phosphorylates DARPP-32 ntawm Thr-75. Yog li, peb tau soj ntsuam phosphorylation xeev ntawm DARPP-32 hauv striatal extracts los ntawm hom qus thiab Tgp35 nas. Qib ntawm phospho-Thr-75 DARPP-32 tau siab dua hauv Tgp35 cov nas (Daim duab. 3A; 1.6 ± 0.2-quav saum tus nqi ntawm cov tsiaj qus hom nas). Peb tom ntej no los soj ntsuam qhov tshwm sim ntawm kev ua si Cdk5 ntau ntxiv ntawm striatal dopamine signaling. Peb tau kuaj xyuas qhov kev xoom xaim ntawm PKA kev ua haujlwm hauv Tgp35 cov nas los ntawm kev txheeb xyuas phosphorylation lub xeev ntawm DARPP-32 ntawm Thr-34. Qib ntawm phospho-Thr-34 DARPP-32 tau nce rau hauv cov tsiaj qus hom nas 15 min tom qab muab cov yeeb dawb (Daim duab. 3B; 1.8 ± 0.2-quav saum cov qib hauv qab). Txawm li cas los xij, qhov cuam tshuam ntawm cocaine ntawm Thr-34 phosphorylation ntawm DARPP-32 tau ntxim nyiam hauv Tgp35 cov nas (1.2 ± 0.3-quav saum theem qib hauv qab). Cov txiaj ntsig no tau taw qhia tias qhov kev nce ntawm Cdk5 kev ua haujlwm tau nqis peev rau qhov ua kom neeg nkag rau PKA kev ua haujlwm tej zaum dhau los ntawm DARPP-32 phosphorylation ntawm Thr-75 (6, 12)). Nws tseem tuaj yeem hais tias qhov nce ntxiv ntawm presynaptic Cdk5 kev ua haujlwm ua rau txo qis dopamine tso, thiab tias qhov no ua rau muaj kev txo qis ntawm cov yeeb dawb. Qhov zoo tshaj plaws, kev txhaj tshuaj ib zaug ntawm cov yeeb dawb tsis cuam tshuam rau theem ntawm p35 thiab Cdk5 protein nrog rau kev ua haujlwm kinase (Daim duab. 3 C thiab D)). Qhov no yog qhov sib piv rau cov kev tshawb fawb dhau los nyob rau hauv uas ntev mus rau neeg quav yeeb tau qhia txog kev tswj hwm kev qhia ntawm p35 thiab Cdk5 (6).

Daim duab 3.  

Kev cai nce qib ntawm Cdk5 kev ua si nce qib ntawm phospho-Thr-75 DARPP-32 thiab attenuates cocaine-hob kev ua kom PKA. (A) Immunoblot qhia kev nce phosphorylation ntawm DARPP-32 ntawm Thr-75 (P-D32 Thr-75) hauv striatal extracts los ntawm Tgp35 cov nas. Hauv ...

Up-Kev Cai ntawm Cdk5 Kev Ua Haujlwm Attenuates Cocaine-Ua Rau Raug Kev Tshawb Fawb ntawm ERK1 / 2. Cov ntawv pov thawj tsis ntev los no qhia tias dopamine receptor ua kom muaj zog hauv striatum kuj ua kom lwm yam ua paib, nrog rau ERK txoj hauv kev (21, 22), uas muaj lub luag haujlwm tseem ceeb hauv kev coj tus cwj pwm teb rau cocaine (23)). Yog li peb tau kuaj xyuas seb Cdk5 kev ua haujlwm puas yuav cuam tshuam rau qhov muaj cov yeeb dawb (cocaine) kev ua kom rov qab los ntawm ERK txoj kev taug. Kev ua kom zoo ntawm ERK txoj hauv kev tau pom tom qab kev muab tshuaj yeeb hauv kev txhaj tshuaj hauv striatal extracts los ntawm cov tsiaj qus-nas, raws li pom tseeb los ntawm kev nce phosphorylation ntawm MEK1 / 2 ntawm Ser-217 thiab Ser-221 (1.5 ± 0.2-fold saum theem qib) thiab ntawm ERK1 / 2 ntawm Thr-202 thiab Tyr-204 (ERK2 phosphorylation: 1.5 ± 0.2-quav saum cov qib hauv qab) ()Daim duab. 4 A thiab B)). Txawm li cas los xij, kev tawm dag zog ntawm cocaine ntawm MEK1 / 2 (1.2 ± 0.2-quav saum theem qib hauv qab) thiab ntawm ERK1 / 2 (ERK2 phosphorylation: 1.2 ± 0.2-fold saum toj ntawm qib hauv qab) tau muab tso rau hauv Tgp35 cov nas (Daim duab. 4 A thiab B)). Ntxiv mus, cov qib theem pib ntawm phospho-ERK1 / 2 tau qis dua hauv Tgp35 cov nas (0.8 ± 0.2-quav qis dua tus nqi ntawm cov tsiaj qus hom nas), qhov tseeb no tsis tseem ceeb. Qhov kawg tshwm sim no tuaj yeem raug cuam tshuam los ntawm Cdk5-dependant phosphorylation ntawm MEK1 ntawm Thr-286, uas ua rau muaj kev txo qis ntawm cov kev ua haujlwm catalytic (24)). Txhawm rau ntsuas qhov ua tau, peb tau tshuaj xyuas phosphorylation lub xeev ntawm MEK1 ntawm Thr-286 thiab pom tias cov qib siab ntawm phospho-Thr-286 MEK1 tau tshwm sim hauv striatal extracts los ntawm Tgp35 cov nas (Daim duab. 4C; 1.3 ± 0.1-quav saum tus nqi ntawm cov tsiaj qus hom nas). Tsis tas li ntawd, phosphorylation xeev ntawm MEK1 ntawm Thr-286 tsis tau hloov kho los ntawm ib qho kev txhaj tshuaj yeeb dawb, raws li qhov tshawb pom tias Cdk5 kev ua haujlwm tsis cuam tshuam los ntawm kev kho mob (Daim duab. 3D).

Daim duab 4.  

Cdk5-mediated inhibition of MEK1 / 2 ua rau attenuation ntawm kev ua kom muaj cocaine ntawm ERK1 / 2. Striatal extracts tau npaj los ntawm cov tsiaj qus-hom (WT) thiab Tgp35 nas 15 min tom qab txhaj ntawm cocaine lossis saline thiab raug rau immunoblotting ...

Kev Tshaj Tawm ntawm Dopamine Kev Coj Mus Rau Nucleus Yog Attenuated los ntawm Kev Nce Cdk5 Kev Ua Haujlwm. Kev pib ua haujlwm hauv cocaine ntawm ntau cov paib uas muaj PKA thiab ERK ua rau kom muaj kev cuam tshuam txog kev hloov pauv ntawm CREB hauv lub keeb los ntawm nws phosphorylation ntawm Ser-133 (22, 25)). Txheeb xyuas seb puas yog Cdk5-mediated inhibitory cov teebmeem ntawm PKA thiab ERK kev ua haujlwm cascades tuaj yeem hloov ntawm CREB phosphorylation hauv nucleus, peb tau soj ntsuam phosphorylation lub xeev ntawm CREB ntawm Ser-133 hauv striatal extracts los ntawm cov tsiaj qus hom thiab Tgp35 cov nas. Qib theem ntawm phospho-CREB tau qis dua hauv Tgp35 cov nas (0.7 ± 0.1-quav ntawm cov nqi ntawm cov tsiaj qus-hom nas) ()Daim duab. 5)). Teb rau kev txhaj tshuaj yeeb dawb, qib ntawm phospho-CREB tau nce nyob rau hauv striatum ntawm cov tsiaj qus-nas (1.5 ± 0.1-quav saum cov qib hauv qab), tab sis cov lus teb no rau cocaine tau raug mob nyob rau hauv Tgp35 cov nas (1.2 ± 0.1- quav saum toj saud) (Daim duab. 5).

Daim duab 5.  

Kev cai nce qib ntawm Cdk5 kev ua haujlwm ua rau muaj phosphorylation tsawg ntawm CREB ntawm Ser-133 hauv cov nas nrog kev txhaj ntawm cov ntsev los yog cocaine. Striatal extracts tau npaj los ntawm cov tsiaj qus-hom (WT) thiab Tgp35 nas 30 min tom qab txhaj tshuaj thiab raug txhaj tshuaj tiv thaiv kab mob ...

Phosphorylation ntawm CREB ntawm Ser-133 ntxiv rau nws cov kev hloov pauv ntawm cAMP cov lus teb hauv cov cheeb tsam nce qib ntawm qee cov noob, suav nrog c-fos noob (26)). Yog li peb tau kuaj xyuas qhov induction ntawm c-fos hauv striatum ntawm cov tsiaj qus hom thiab Tgp35 cov nas tom qab muab cov yeeb dawb. Hauv cov nas-tsiaj qus hom, qib ntawm c-fos mRNA tau nce mus rau tus nqi siab tshaj plaws (1.8 ± 0.2-quav saum toj theem hauv qab) 30 min tom qab txhaj cov yeeb dawb, thiab tom qab ntawd rov mus rau qib hauv qab los ntawm 120 min tom qab txhaj (Daim duab. 6 A thiab B)). Txawm li cas los xij, cov theem ntawm c-fos mRNA tau ≈30% qis dua hauv Tgp35 cov nas ntau dua li cov nas-tsiaj qus kom txog thaum 30 min tom qab txhaj (Daim duab. 6 A thiab B)). Kev ua haujlwm ntawm c-fos tsawg dua hauv Tgp35 cov nas tau muab kho ntxiv los ntawm tshuaj tiv thaiv kabmob (Daim duab. 6 C-F)). Cov neeg tswj hwm cocaine tau nce c-fos immunoreactivity, muaj zog nyob rau hauv qhov chaw dorsomedial – dorsocentral ntawm striatum thiab tsis muaj zog ntawm cov tog tom ntej, hauv ob hom tsiaj qus thiab Tgp35 cov nas. Txawm li cas los xij, qhov pib ua kom pom kev zais rau ntau ntawm c-fos-immunopositive hlwb tsis pom zoo nyob rau hauv qhov striatum ntawm Tgp35 cov nas (Daim duab. 6G)). Ua ke, cov txiaj ntsig no qhia tau hais tias cocaine-kev sib kho txhim kho kom zoo dua ntawm striatal dopamine xa tuaj rau nucleus tau raug muab tso rau hauv Tgp35 cov nas, qhov yuav tshwm sim los ntawm Cdk5 kev ua ub no ntxiv.

Daim duab 6.  

Kev cai nce qib ntawm Cdk5 kev ua haujlwm tau ua rau txo qis ntawm striatal c-fos qhia thiab nws txoj kev paub qis dua tom qab kev tswj hwm cocaine. (A) Yav qaum teb blot uas qhia lub sijhawm kawm ntawm c-fos induction hauv qus-hom (WT) thiab Tgp35 (Tg) cov nas tom qab muab yeeb dawb. ...

kev sib tham

Cdk5 thiab nws cov neeg ua haujlwm p35 tau txheeb pom tias yog cov hom phiaj hauv nruab nrog uas tau hloov kho los ntawm kev ua kom mob ntev ntev los ntawm yeeb dawb (6). Peb tshaj tawm cov ntaub ntawv pov thawj no uas tau nce Cdk5 kev ua haujlwm, qhov txiaj ntsig ntawm p35 tshaj-txoj cai tsis yog Cdk5 txog txoj cai, ua rau attenuation ntawm cocaine-mediated dopamine signaling hauv striatal neurons. Txhawm rau tshuaj xyuas qhov tshwm sim ntawm kev tshaj tawm cov lus hais ntawm Cdk5 lossis p35 ntawm striatal dopamine xaib, ob kab kev sib hloov nas, TgCdk5 thiab Tgp35 nas, tau txheeb xyuas. Peb pom tias Cdk5 kev ua haujlwm tau raug tswj hwm raws li qhov feem ntau ntawm p35 protein tab sis tsis raug cuam tshuam los ntawm kev nce qib ntawm Cdk5 protein. Peb daim ntawv tshaj tawm dhau los tseem tau qhia tias Cdk5 kev ua haujlwm hauv TgCdk5 nas lub hlwb qis dua hauv nas hom nas hlwb thaum cov haujlwm tau ntsuas los ntawm kev siv Cdk5 immunoprecipitates (17), hais tias Cdk5 overexpression tsub kom kev nce qib ntawm monomeric Cdk5 yog p35 qib tsis tau nce ntxiv. Cov txiaj ntsig no tau qhia tias qib p35 protein yog qhov ntsuas tus lej rau Cdk5 kev ua haujlwm.

Tgp35 cov nas tau nthuav tawm qhov ua tsis tau zoo dua ntawm ob txoj kev CREB phosphorylation thiab c-fos hauv striatum tom qab kev txhaj tshuaj ntawm cocaine, hais qhia tias cov lus teb rov rau cocaine tau raug txwv los ntawm kev ua si Cdk5 ntau. Qhov xeeb ceem ntawm cocaine-mediated dopamine xaib nyob rau hauv Tgp35 cov nas twb yuav tshwm sim los ntawm Cdk5-mediated inhibition ntawm ntau cov paib uas muaj DARPP-32, PKA, thiab ERK. Cov neeg tswj hwm hauv cocaine tau nce PKA phosphorylation ntawm DARPP-32 ntawm Thr-34 hauv cov tsiaj qus, cov lus teb no tau muab tso rau hauv Tgp35 cov nas. PKA phosphorylation ntawm DARPP-32 ntawm Thr-34 tau pom tias inhibit qhov kev ua haujlwm ntawm protein phosphatase 1 (PP1), lub enzyme lub luag haujlwm rau lub dephosphorylation ntawm Ser-133 ntawm CREB (27)). Yog li, kev ua haujlwm PP1 yuav tsis raug thaiv ntawm DARPP-32 / PP1 txoj hauv kev hauv Tgp35 cov nas.

Cov neeg mob xoom xoom ntawm ERK1 / 2 kuj tau ua rau hauv Tgp35 cov nas. Muaj ob peb lub tswv yim sib txawv uas Cdk5 tuaj yeem cuam tshuam qhov kev ua haujlwm ntawm cocaine-vim raug yuam kev ntawm ERK1 / 2. Ua ntej, Cdk5-dependant phosphorylation ntawm DARPP-32 ntawm Thr-75 tuaj yeem cuam tshuam PKA, ua rau muaj kev cuam tshuam tom ntej ntawm ib qho PKA-mediated MEK1 / 2 kev ua haujlwm uas xav tau rau ERK1 / 2 kev ua haujlwm. Kev tshawb nrhiav tsis ntev los no kuj tau pom tias phosphorylation ntawm DARPP-32 ntawm Thr-34 yog yuav tsum rau cocaine-mediated kev ua haujlwm ntawm ERK1 / 2 los ntawm ntau txoj hauv kev uas muaj feem cuam tshuam ntawm kev tswj hwm ntawm MEK kev ua haujlwm nrog rau kev tswj hwm ntawm striatal-enriched phosphatase, ib qho tyrosine phosphatase uas ua ncaj qha rau ERK1 / 2 (28)). Kev txhawb nqa rau qhov ua tau no yog qhia los ntawm kev tshawb pom tias cocaine-hob phosphorylation ntawm MEK1 / 2 ntawm Ser-217 thiab Ser-221 tau muab tshem tawm hauv Tgp35 cov nas. Lwm txoj kev yuav tshwm sim yog los ntawm Cdk5-dependant phosphorylation ntawm MEK1 ntawm Thr-286, uas yuav ua rau nws txo qis ntawm nws cov kev ua haujlwm catalytic thiab ua rau muaj kev cuam tshuam ntawm ERK1 / 2 kev ua si (24).

Kev txwv tsis pub cov kev ua si Cdk5 hauv striatum tau pom tias muaj kev coj ua lub luag haujlwm ntawm kev muaj yeeb muaj tshuaj ntau hauv cov tsiaj (6)). Raws li lub tswv yim xav tias kev ua raws cai ntawm Cdk5 kev ua ub no yuav ua rau kev hloov kho neuronal rau kev cuam tshuam cov kev cuam tshuam ntawm kev tswj yeeb cocaine ntxiv (6), peb pom tias Cdk5-mediated phosphorylation ntawm DARPP-32 thiab MEK1 ua rau kom muaj kev txhawb nqa ntawm cocaine-vim kev ua kom muaj zog ntawm ERK1 / 2, uas ua rau muaj kev cuam tshuam qis dua ntawm CREB phosphorylation thiab c-fos hauv striatum. Peb qhov kev tshawb pom tau txhawb nqa lub tswv yim uas nce Cdk5 kev ua haujlwm, vim yog p35 txog kev cai, tej zaum yuav hloov cov noob keeb kwm nyob rau hauv striatum tom qab mob raug cocaine. Qhov no tuaj yeem tshwm sim los ntawm kev hloov pauv hauv cov kev ua ntawm cov kev hloov pauv tseem ceeb xws li CREB thiab c-fos. Yog li, Cdk5 tus neeg ua haujlwm p35, los ntawm kev tsim txiaj ntawm nws cov txiaj ntsig tus cwj pwm ntawm Cdk5 kev ua haujlwm, yuav ua rau muaj kev hloov pauv ntev ntev ntawm cov kev ua rau neuronal nyob rau kev quav yeeb..

ACKNOWLEDGMENTS

Peb ua tsaug rau Drs. Mary Jo Danton, Philip Grant, thiab Sashi Kesavapany rau kev nyeem cov ntawv sau tseem ceeb. Qhov haujlwm no tau txhawb nqa los ntawm National Institutes of Health Grant Z01DE00664-05 (rau ABK), Asmeskas Kev Pabcuam Kev Noj Qab Haus Huv Pab Nyiaj DA10044, thiab nyiaj pab los ntawm Simons Foundation, Peter J. Sharp Foundation, thiab Picower Foundation (rau PG).

Sau ntawv

Kev hais luv luv: Cdk5, cyclin-depend kinase 5; ERK, cov cim hluav taws xob ntxiv-kinase; DARPP-32, dopamine thiab cAMP-tswj hwm phosphoprotein, cov tshuaj molecular loj 32 kDa; PKA, cAMP-depend kinase; MEK, ERK kinase; CREB, cAMP-teb cov lus-tiv thaiv protein.

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