Mosebetsi o eketsehileng oa kincine-dependent kinase 5 e lebisa tlhokomelong ea pontšo ea dopamine e sebelisitsoeng ke cocaine (2005)

Proc Natl Acad Sci US 2005 February 1; 102(5): 1737-1742.

E phatlalalitsoe marang-rang 2005 Januari 21. doi:  10.1073 / pnas.0409456102
PMCID: PMC547862
Khopolo-taba
Sehlooho sena se bile teng e boletsoeng ke lihlooho tse ling ho PMC.

inahaneloang

Cocaine, e leng lithethefatsi tsa tlhekefetso, e eketsa maemo a dopamine a dopamine ka striatum ka ho thibela ho pheta-pheta ha dopamine litsing tsa axon. Cyclin-based kinase 5 (Cdk5) le activator p35, liprotheine tse amehang phosphorylation ea substrates ho li-neuron tsa postmitotic, li fumanoe li le maemong a holimo ka mor'a ho pepesehela koae ka nako e telele. Ho tsoela pele ho hlahloba litlamorao tsa Cdk5 le p35 induction on striatal dopamine signing, re hlahisitse mela e 'meli e ikemetseng ea transgenic eo ho eona Cdk5 kapa p35 e neng e tebile ka ho khetheha ho li-neuron. Re tlaleha mona hore keketseho ea ts'ebetso ea Cdk5, ka lebaka la p35 empa eseng ea Cdk5 overexpression, e lebisa ho amoheloeng ha cocaine-mediated dopamine signaling. Phdk5-mediated phosphorylation ea dopamine le phamphory e laoloang ka cAMP, limolek'hule ea 32 kDa (DARPP-32) ho Thr-75, e ne e tsamaisana le phosphorylation ea DARPP-32 e neng e fokotsehile ho Thr-34. Keketseho e matla ea Cdk5-mediated phosphorylation ea kinase kinase 1 ho Thr-286 e ne e tsamaisana le ts'ebetso e fokotsehileng ea ts'ebetso ea extracellular sign-regated kinase 1 / 2. Litholoana tsena li tlatselitse ho kopeng phosphorylation ea cocaine e kenyang karolo ea "cAMP" e kopanyang protheine hape e kenyelletsa tlhahiso e fokolang ea c-fos ho striatum. Liphetho tsena li ts'ehetsa mohopolo oa hore ts'ebetso ea Cdk5 e kenella ho polelo ea tlhaho e fetotsoeng kamora ho pepesetsoa koae ka lebaka lena le amang liphetoho tse tšoarellang molemong oa ts'ebetso ea neuronal tlasa ts'ebeliso ea lithethefatsi tsa cocaine.

Keywords: lekhoba la koae, phosphorylation, striatum

Cocaine e eketsa maemo a dopamine a dopamine ka har'a striatum le alter gene ho "dopaminocept neurons" ka ho kenya tšebetsong marangrang a methapo ea methapo e phatlalatsang lets'oao la pele ho tsoa ho dopamine D1 receptor ho ea bokong (1). Ho pepesehela koae ka nako e sa lekanyetsoang ho laola lintlha tse 'maloa tse hlahisoang, ho etsa hore ho be le liphetoho tsa nako e telele puong ea gene eo ho nahanoang hore e fetoha liphetoho tsa methapo ea tahi ea cocaine (2). ΔFosB, e tšoailoe e le ntho ea bohlokoa ea mongolo (3), e bontšitsoe ho ntlafatsa boitšoaro ba liphoofolo ho arabela ho koae (cocaine)4, 5). Ka hona, ho tsebahatsoa ha mefuta ea sepheo e laoloang ke ΔFosB ho lebelletsoe ho kenya letsoho kutloisisong e kholo ea molek'hule o ka tlasa tahi ea koae. Haufinyane, kalafo e sa foleng ea liphoofolo tse nang le koae e bontšitsoe ho laola taolo ea cyclin-based kinase 5 (Cdk5) le activator ea eona ea P35 ho striatum ka ts'ebetso ea ΔFosB (6, 7).

Cdk5 ke setho sa lelapa la Cdk la serine / threonine kinases. Ho fapana le li-Cdks tse ling tseo e leng batsamaisi ba bohlokoa ba ho potoloha ha lisele, Cdk5 e ameha haholo phosphorylation ea substrates ho li-neuron tsa postmitotic (8). Ho hlaka ha neuronal ea ts'ebetso ea Cdk5 ho fihlelleha ka ho hokahana le li-activator tsa eona, ekaba p35 kapa p39, e hlahisoang haholo ke li-postmitotic neurons (8). Ntle le karolo ea bohlokoa ea Cdk5 kholisong ea boko (9, 10), iT hape e bile le tšusumetso phetisong ea dopaminergic bokong ba pelehi11, 12). Thibelo ea ts'ebetso ea Cdk5 e baka ho lokolloa ha dopamine ka har'a striatum, ho bonts'a ts'ebetso ea presynaptic ea Cdk5 e le molaoli ea seng molaong oa tokollo ea dopamine (11). Ntle le moo, Cdk5 e sebetsana le ho sebetsa ha li-postynaptic dopamine tse hlahisoang ke phosphorylating dopamine- le cAMP-e laoloang phosphoprotein, molek'hule ea 32 kDa (DARPP-32) ho Thr-75, e fetolang DARPP-32 ho inhibhibitara ea (PKAME ya CAMKA ya CPKMP ea CPkX. (12).

Litlhokomeliso tsena li bonts'a hore Cdk5 le p35 ke batsamaisi ba tlase ba ts'ebetso ea ts'ebetso ea nako e telele ea dopamine e sa sebetseng kamora ho pepesetsoa nako e telele ea koae 'me ka hona ho lemalla koae. Ho nts'etsapele karolo ea Cdk5 ho lets'oao la dopamine, re hlahisitse mela e 'meli ea transgenic eo ho eona Cdk5 kapa p35 e ileng ea hlakisoa ka ho khetheha ka li-neurons tlasa taolo ea morekisi oa P35. Liphumano tsa rona li bonts'itse hore ts'ebetso ea Cdk5 e ne e tsamaisoa ka maemo a eketsehang a liprotheine tsa p35 empa eseng protheine ea Cdk5, e fana ka tlhahiso ea hore boemo ba protheine ea P35 e lekanyetsa mosebetsi oa Cdk5. Re fana ka mona Ka vivo bopaki bo eketsang ts'ebetso ea Cdk5, ka lebaka la p35 overexpression, e lebisa ho fihlelletsong ha dopamine ea cocaine-mediated e supang ho nucleus ka thibelo ea likhakanyo tsa PKA le li-extracellular sign-regated kinase (ERK).

Lisebelisoa le mekhoa

Li-antibodies. Li-antibodies tsa polyclonal ho Cdk5 (C-8) le p35 (C-19) li ile tsa rekoa ho Santa Cruz Biotechnology. Li-antibodies tse itšetlehileng ka le phosphorylation le -independent ho ERK kinase (MEK) 1 / 2, ERK1 / 2, le protein ea cAMP-reaction element-binding (CREB) li fumanoe ho Cell Signaling Technology (Beverly, MA). Li-antibodies ho phospho-Thr-34 DARPP 32 (13), phospho-Thr-75 DARPP-32 (12), kakaretso ea DARPP-32 (12), le c-fos (14) li sebelisitsoe joalo ka ha ho hlalositsoe. Ho ile ha rekoa antibody ea Actin ho Sigma

Liphoofolo tsa Tlhahlobo. Pejana re ile ra kopanya mofuta oa mouse p35 Cdk5r1, e emisang protheine ea P35, e bonts'a sebopeho sa eona sa genomic (15). Ho hlahisa mouse ea transgenic ka neuronal overexpression ea p35 (Tgp35), 6-kb EchoRI-EchoSekhechana sa RI se tšetseng sebaka sa papatso sa 1.2-kb se ile sa kenngoa ka har'a pGEM9Z (-) plasmid, 'me 45-bp tag e tsoang SV40 e kentsoe ka har'a KpnKe bula sebaka se tlase molemong oa mofuta ona (A+) lets'oao (Feie. 1A). Thepa e ne e na le a PuoKe bula sebaka sa liphoofolo. Sekhechana sa 6-kb se ne se tsoa ho plasmid mme se hloekisitsoe, se lateloe ka ente ea mantsoe a phetisetso ea transgene ho hlahisa litoeba tsa transgenic. Ho hlahloba boemo ba polelo ea transgene tlasa taolo ea taolo ea papatso ea 1.2-kb p35 Ka vivo, toeba e habeli ea transgenic (Tgp35; p35 - / -) e ile ea hlahisoa hape ka ho sebelisa leano la mehato e 'meli eo ka eona kokoana ea Tgp35 e ileng ea nchafatsoa ka morao molemong oa p35-null. Mefuta e meng ea mouse e sebelisitsoeng thutong ena e kenyelletsa p35 + / -, p35 - / -, Cdk5 +/-, le panya ea transgenic e nang le neuronal overexpression ea Cdk5 (TgCdk5) (9, 16, 17). Li-genotypes tsa litoeba tsena li ile tsa khethoa ka ho etsa tlhahlobo-leseling ea boroa kapa PCR ho DNA ea genomic e ikhethileng ho tsoa molekeng oa biopsies. Lice li ne li bolokiloe tlasa potoloho ea lefifi la 12-h / 12-h. Tlhokomelo eohle e ile ea fanoa ho latela litaelo tsa Naha tsa Naha tsa Bophelo mabapi le tlhokomelo le ts'ebeliso ea liphoofolo tsa mahlale le liteko.

Setšoantšo sa 1.  

Generation ea transgenic mouse e nang le neuronal overexpression ea p35 e tataisoang ke morekisi oa p35 (Tgp35). (A) Moetso oa transgene o bonts'oa le meralo ea meralo ea mofuta oa hlaha le mofuta oa li-p35 tse boletsoeng. Mekoallo e khubelu e bonts'a probe e sebelisitsoeng bakeng sa genotyping. ...

Tlhahlobo ea Blot ea Boroa. DNA ea genomic e ntšitsoeng mohloling oa biopsies e ile ea chekoa ka eona EchoRI le Puo'Na, ke kentse motlakase ka sekhoneng sa agarose sa 0.9%,' me ka fetisetsoa membrane ea nylon. Letsoalo le ne le kopantsoe ka mokhoa o ikhethileng 32Bopaki ba projeke ea 42 ° C bosiu bo le bong. Probe ea 485-bp bakeng sa genotyping ea p35 Knoutout (p35 - / -) le litoeba tsa Tgp35 li hlahisitsoe ke PCR ka ho sebelisa li-primers tse latelang: 5'-ACATCCTGCTGCCACGGTGAC-3 ′ le 5'-CCNGXA-GCAUMX'A-CECUMX'A-GNUMX'-XCAUM''CCA. Membrane e kopanetsoeng e ile ea hlatsuoa makhetlo a mabeli ho 3 × SSC / 2% SDS ho 0.1 ° C bakeng sa 42 min, le makhetlo a mabeli ho 10 × SSC / 0.1% SDS ho 0.1 ° C bakeng sa 65 min, mme ea pepesetsoa filimi ea x-ray.

Phekolo ea Lithethefatsi. Cocaine (Sigma) e ile ea qhibiliha ka letsoai le letsoai. Liphoofolo li ile tsa kenngoa ka har'a k'hok'heine ka koae (15 mg / kg) kapa palo e lekanang ea letsoai a le lilemo li likhoeli tsa 3 mme a bolaoa ka ho lihla nako ka linako tse fapaneng (15, 30, 60, le 120 min) kamora ho entoa. Mabili a ile a tlosoa kapele mme a chesoa ke PBS e batang haholo. Li-striata li ile tsa qhaloa 'me tsa hlahlojoa ka Leboea kapa Bophirima. Bakeng sa tlhahlobo ea immunohistochemical, likarolo tsa striatal li ile tsa fumanoa ho litoeba 2 h ka mor'a ente.

Tlhahlobo ea Blot ea Leboea. Kakaretso ea RNA e nkuoe striata e nang le TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA) mme ea etsoa tlhatlhobo ea North blot joalo ka ha ho hlalositsoe (18). Bakeng sa ho fumana c-fos mRNA, sekhechana sa 189-bp sa c-fos cDNA se sebelisitsoe joalo ka probe joalokaha ho hlalositsoe (19). Maemo a c-fos mRNA a ile a hlakoloa ka ho lekanya boteng ba sehlopha se khethehileng ka ho sebelisa sistimi ea tlhaiso ea litšoantšo ka software ea nih, Mofuta oa 1.62.

Tlhahlobo ea Blot ea Bophirimela. Li-tishu tsa striatal li ile tsa kenngoa ho 1% SDS mme li phehiloe bakeng sa 10 min. Tekanyo ea protheine ka sampole e 'ngoe le e' ngoe e ile ea khethoa ke mofuta oa protheine ea BCA (Pierce). Liprotheine tse lekanang li ne li arotsoe ke SDS / PAGE pele li fetisoa ka har'a membrane ea nitrocellulose. Litho tsa kahare li ne li koetsoe ka 1 × PBS e nang le lebese la 5% skim le 0.05% Pakeng tsa 20 mme li kenelletse ka li-antibodies tsa mantlha bosiu bo le bong ho 4 ° C. Ho incubation ka peroxidase-conjugated anti-mouse kapa mmutla IgG (Sigma) e ne e etsoa ka mocheso oa kamore bakeng sa 60 min. Letšoao le fumanoe ke chemiluminescence (Pierce) e ntlafalitsoeng, 'me methapo ea methapo ea methapo e ile ea hlakoloa joalo ka ha ho hlalositsoe kaholimo.

Cdk5 Kinase Assay. Li-striatal lysates li ne li hlophisitsoe ka mokhoa oa ho kuta oa lysis o kenyelletseng 50 mM Tris · HCl, pH 7.4 / 50 mM NaCl / 5 mM EDTA / 1% Triton X-100 / 1mMDTT / 1 mM phenylmeth / 1 / XLUMX / XLUMX / XLUMX / XLUMX / 1 ml leupeptin / phosphatase inhibitors (motsoako oa phosphatase inhibitor I le II, Sigma). Li-lysates li ne li entsoe ka mokhoa oa anti-Cdk5 (C-8) kapa anti-p35 (C-19) li-antibodies. Li-immunoprecipitates tsa Cdk5 li ne li lokisitsoe ke ho kenella ha 300 μl ea lysate (e tsamaellanang le 300 μg ea protheine) e nang le anti-Cdk5 antibody (3 μg) bosiu bo le bong ho 4 ° C e lateloe ke ho kenella ka ho eketsehileng ka 25 μl ea Prote % slurry in the lysis buffer; Santa Cruz Biotechnology) ea 50 h ho 3 ° C. Bakeng sa ho hlophisoa ha li-immunoprecipitates tsa P4, 35 μl ea lysate (e tsamaellanang le 500 mg ea protheine) e ne e entsoe ka anti-p1 antibody (35 μg) joalokaha ho hlalositsoe ka holimo. Li-immunoprecipitates li ile tsa hlatsuoa habeli ka sesebelisoa sa lysis mme habeli ka buase ea kinase e entsoeng ka 3 mM Tris · HCl, pH 50 / 7.4 mM MgCl2/ 1 mM EDTA / 1 mM EGTA / 1 mM DTT, e tsosolositsoe ho 60 μl ea buase ea kinase. Ts'ebetso ea Kinase e lekantsoe ka ho sebelisa histone H1 joalo ka substrate (18).

Immunohistochemistry. Litsuonyana li ne li sa anngoe ke liente tsa ipod ea avertin (250 mg / kg, Fluka) mme li entsoe ka mokhoa o phethahetseng ka 0.1 M sodium phosphate buffer, pH 7.4, e lateloe ke Streck Tissue Fixative (Streck Laboratories, La Vista, NE), fixative e sa sebetseng. Masapo a lahliloeng a ile a hlophisoa ka mokhoa o tšoanang bosiu bo le bong ho 37 ° C. Ebe, masapo a kelello a kenngoa ka parafini, a khaoloa likarolo tsa coronal tsa 5-μm mme a kenngoa ka har'a immunohistochemistry ka ho sebelisa mokhoa o rarahaneng oa avidin-biotin-peroxidase (Vector Laboratories) e nang le diaminobenzidine e le karoloana. Likarolo tsena li ne li entsoe ka antibody e hloekisitsoeng ea polyclonal khahlanong le c-fos bosiu bo le bong ho 4 ° C. Boemo ba lits'oants'o bo ile ba hlahlojoa ka ho siea ha motho ea thibeloang letsoho ea mantlha.

Results

Moloko oa Transgenic litoeba ka Neuronal Overexpression ea p35. Transgene e sebelisitsoeng ho fihlela polelo e eketsehang ea neuronal ea p35 e ne e na le sekhechana sa 6-kb sa mofuta o hlophisitsoeng oa p35 mofuta o kenyelletsang 1.2-kb khihtori le tatelano eohle ea podNUMX (Feie. 1 A). Litoeba tsa genotypes li ile tsa khethoa ke tlhahlobo ea boroa ka ho sebelisa mofuta oa probe o neng o etselitsoe ho khetholla lithunya tsa p35 - / - le Tkup35 ho litoeba tsa mofuta oa hlaha (Feie. 1 A 'me B). Ho hlahloba polelo ea transgene tlasa taolo ea morekisi oa 1.2-kb p35, re hlahisitse litoeba tse tsoang habeli tsa transgenic (Tgp35; p35 - / -) moo polelo ea p35 e neng e tsamaisoa feela ho tsoa ho transgene. Polelo ea p35 e Tgp35; p35 - / - litoeba e ile ea bonoa bokong feela (Feie. 1C), moo sebopeho sa polelo ea sebaka se neng se tšoana le sa litoeba tsa mofuta oa hlaha (Feie. 1D). Ho haelloa ke p35 ho bontšitsoe ho fella ka sebopeho se sa tloaelehang mokokotlong oa mokokotlo le hippocampus ea litoeba (10). Leha ho le joalo, lithunya tsa Tgp35; p35 - / - - li bontšitse poloko e felletseng ea p35 - / - - - - - phenotype ea boko (Feie. 1E). Lintlha tsena li bonts'a hore morekisi oa 1.2-kb p35 o ne a laola polelo ea transgene ka polelo e ts'oanang ea polelo ho ea p35 ho tsoa mofuta oa mofuta oa p35.

Tekanyetso ea liprotheine tsa p35 e na le litekanyetso tsa ho phahama bakeng sa Ts'ebetso ea Cdk5. Re ile ra hlahloba litla-morao tsa gene-dosage tsa mofuta oa mofuta oa p35 le Cdk5 ho polelo ea protheine lits'ebetsong tse tsoang ho p35 - / -, p35 +/-, mofuta oa hlaha, Tgp35, Cdk5 +/--, le TgCdk5 litso. Mefuta ea liprotheine tsa p3 le Cdk35 li hokahane hantle le litekanyetso tsa gene, ka ho latellana (Feie. 2 A 'me B). Litoeba tsa Tkup35 li bontšitse keketseho ea ≈1.6-fold ea p35 boemo ba protheine ha e bapisoa le litoeba tsa mofuta o hlaha, athe liprotheine tsa Cdk5 li ne li sa angoe ke maemo a fapaneng a protheine ea P35 Litoeba tsa TgCdk5 li bontšitse keketseho ea ≈1.9-protein ea Cdk5 ea protheine ha e bapisoa le litoeba tsa mofuta o hlaha, athe liprotheine tsa p35 li ne li sa angoe ke maemo a fapaneng a protheine ea Cdk5. Ho hlahloba litlamorao tsa lipalo tse fapaneng tsa proteni ea p35 ts'ebetsong ea Cdk5, Cdk5 e ile ea kenngoa lits'ebetsong tsa striatal ka anti-Cdk5 antibody and shughuli ea kinase e lekantsoe. Ka mokhoa o ts'oanang, ho hlahloba litlamorao tsa lipalo tse fapaneng tsa protheine ea Cdk5 ho ts'ebetso ea kinase, p35 e ile ea thibeloa ho tsoa litsing tsa striatal ka anti-p35 antibody, mme ketsahalo ea kinase e lekantsoe. Ts'ebetso ea Cdk5 e hokahane hantle le boemo ba protheine ea p35 empa eseng joalo ka protheine ea Cdk5 (Feie. 2 C 'me D). Liphetho tsena li bonts'a hore palo ea proteni ea p35 ke ntho e fokotsang sekhahla bakeng sa ts'ebetso ea Cdk5. Ka hona re sebelisitse litoeba tsa Tkup35 ho etsa lipatlisiso ka litlamorao tsa ho eketseha ha Cdk5 ts'ebetsong ho saena matšoao a driamine.

Setšoantšo sa 2.  

Tekanyetso ea mesebetsi ea Cdk5 e lekanyelitsoe ke boemo ba liprotheine tsa P35. (A) Bophirimela ba Bophirima bo bonts'a maemo a liprotheine tsa p35 le Cdk5 li-genetic dosages tsa mofuta oa p35 le Cdk5, ka ho latellana. (BMaemo a amanang le P35 kapa protheine ea Cdk5 ...

Phosphorylation ea Cocaine-E kenyellelitsoeng ho DARPP-32 ho Thr-34 E Akoetsoe ho Tkup35 litoeba. Ts'ebetso ea DARPP-32 e ipapisitse le boemo ba eona ba phosphorylation libakeng tse ngata (20). PhKA phosphorylates DARPP-32 ho Thr-34, athe Cdk5 phosphorylates DARPP-32 ho Thr-75. Kahoo, re ile ra hlahloba boemo ba phosphorylation ba DARPP-32 ka lintlha tse qotsitsoeng ho litoeba tse hlaha le litoeba tsa Tgp35. Boemo ba phospho-Thr-75 DARPP-32 e ne e phahame ho litoeba tsa Thup35 (Feie. 3A; 1.6 ± 0.2-fold holimo ka holim'a boleng ba litoeba tsa mofuta o hlaha). Ka mor'a moo re ile ra lekola litlamorao tsa ho eketseha ha Cdk5 ts'ebetsong ho saena matšoao a driamine. Re ile ra hlahloba ts'ebetso ea PKA e hlahisang koae ka koae ea Tkup35 ka ho sekaseka boemo ba phosphorylation ba DARPP-32 ho Thr-34. Boemo ba phospho-Thr-34 DARPP-32 bo ile ba eketseha ka mofuta oa litoeba tsa mofuta oa hlaha oa 15 min kamora ho ts'oaroa ka ente ea cocaine (Feie. 3B; 1.8 ± 0.2-fold in over the basal level). Leha ho le joalo, phello ea koae ho Thr-34 phosphorylation ea DARPP-32 e ile ea fumaneha ka litoeba tsa Tkup35 (1.2 ± 0.3-fold over the basal level). Liphetho tsena li bonts'itse hore keketseho ea ts'ebetso ea Cdk5 e kentse ts'ebetso ea PKA e khothalletsoang ke koae mohlomong ka phosphorylation ea DARPP-32 ho Thr-75 (6, 12). Hape ho ka etsahala hore keketseho ea mosebetsi oa presynaptic Cdk5 e lebise ho fokotseheng ha dopamine, le hore sena se kenya letsoho phello e fokotsang ea koae. Haholo, ente e le 'ngoe ea koae ha ea ka ea ama maemo a protheine ea p35 le Cdk5 hammoho le ts'ebetso ea kinase (Feie. 3 C 'me D). Sena se fapane le phuputso e fetileng eo ho eona ho pepesitsoe checocaine e sa ts'oaneng ho tsamaisang polelo ea p35 le Cdk5 (6).

Setšoantšo sa 3.  

Up-regulation ea mesebetsi ea Cdk5 e eketsa boemo ba phospho-Thr-75 DARPP-32 mme e bonts'a ts'ebetso ea PKA ea koae. (A) Immunoblot e bonts'ang phosphorylation e eketsehileng ea DARPP-32 ho Thr-75 (P-D32 Thr-75) lits'ebetsong tse tsoang ho litoeba tsa Tkup35. Ho ...

Up-Regulation of Cdk5 Activity Attenuates Cocaine-Induction activation of ERK1 / 2. Bopaki ba morao-rao bo bonts'a hore ts'ebetso ea dopamine receptor ka har'a striatum e boetse e kenya li-cascade tse ling tse saenneng, ho kenyelletsa le tsela ea ERK (21, 22), e nang le karolo ea bohlokoa karabong ea boits'oaro ho koae (cocaine)23). Ka hona re ile ra hlahloba hore na ts'ebetso ea Cdk5 e kanna ea ama ts'ebetso e khothalletsoang ke koae tseleng ea ERK. Ts'ebetso ea tsela ea ERK e hlokometsoe kamora ho kenngoa ka motsoako oa koae ho litoeba tsa mofuta oa hlaha, joalo ka ha ho bonahala ka phosphorylation ea MEK1 / 2 ho Ser-217 le Ser-221 (1.5 ± 0.2-fold over the basal level) and of ERK1 / 2 ho Thr-202 le Tyr-204 (phosphorylation ea ERK2: 1.5 ± 0.2-fold in over the basal level) (Feie. 4 A 'me B). Leha ho le joalo, ts'ebetso ea ts'ebeliso ea koae ea MEK1 / 2 (1.2 ± 0.2-fold over level basal) le ea ERK1 / 2 (phosphorylation ea ERK2: ka 1.2 bas 0.2-fold over level basal)Feie. 4 A 'me B). Ntle le moo, litheko tsa basele tsa phospho-ERK1 / 2 li ne li le tlase ho litoeba tsa Thup35 (0.8 ± 0.2-fold tlase ho boleng ba litoeba tsa mofuta o hlaha), athe mokhoa ona o ne o se bohlokoa lipalo. Sephetho sena sa ho qetela se ka bakoa ke phosphorylation e itšetlehileng ka Cdk5 ea MEK1 ho Thr-286, e bakang ho fokotseha hoa ts'ebetso ea matla (24). Ho lekola monyetla ona, re ile ra hlahloba boemo ba phosphorylation ba MEK1 ho Thr-286 mme ra fumana hore maemo a phahameng a phospho-Thr-286 MEK1 a ne a le teng litšoantšong tse tsoang ho litoeba tsa Tkup35 (Feie. 4C; 1.3 ± 0.1-fold holimo ka holim'a boleng ba litoeba tsa mofuta o hlaha). Ho feta moo, boemo ba phosphorylation ba MEK1 ho Thr-286 ha boa ka ba fetoloa ka ente e le 'ngoe ea koae, e tsamaellanang le ho fumana hore ts'ebetso ea Cdk5 ha e anngoe ke kalafo (Feie. 3D).

Setšoantšo sa 4.  

Cdk5-Mediated inhibition ea MEK1 / 2 e lebisa pakong ea ts'ebetso e khothalletsoang ea koae ea ERK1 / 2. Li-extracts tsa striatal li ile tsa lokisoa ho tloha ho mofuta oa hlaha (WT) le Tgp35 litoeba tsa 15 kamora ho kenngoa ka motsoako oa cocaine kapa letsoai le ho kenngoa immunoblotting ...

Keketseho ea Pontšo ea Dopamine ho Nyutlelie e Hlahisoa ke Ts'ebetso e Eketsehileng ea Cdk5. Ts'usumetso e khothalletsoang ke cocaine ea li-cascade tse ngata tse saenneng tse amang PKA le ERK li lebisa ts'ebetsong e latelang ea transcript factor CREB bokong ka phosphorylation ea eona ho Ser-133 (22, 25). Ho etsa lipatlisiso tsa hore na litlamorao tsa Cdk5-Mediated inhibitory in PKA le li-cascade tsa activation tsa ERK li ka fetohela phosphorylation ea CREB ka har'a nucleus, re ile ra hlahloba boemo ba phosphorylation ba CREB ho Ser-133 lithutong tse tsoang mats'oafong ho tsoa ho mofuta oa hlaha le litoeba tsa Tgp35. Boleng ba basotho ba phospho-CREB bo ne bo le tlase ho litloholo tsa Thup35 (0.7 ± 0.1-boleng ba litoeba tsa mofuta o hlaha) (Feie. 5). Ha a ts'oaroa ka ente ea koae, boemo ba phospho-CREB bo ile ba eketseha maemong a litoeba tsa mofuta oa hlaha (1.5 ± 0.1-fold over-level of basal), empa karabelo ena ea koae e ile ea fumaneha ho litoeba tsa Tgp35 (1.2 ± 0.1- mena ka holimo ho basal)Feie. 5).

Setšoantšo sa 5.  

Tlhahiso-leseling ea tšebetso ea Cdk5 e etsa hore phosphorylation ea CREB e fokotsehe ho Ser-133 ka litoeba ka ente ea saline kapa cocaine. Li-extracts tsa striatal li lokiselitsoe ho tsoa ho mofuta oa hlaha (WT) le Tgp35 litoeba tsa 30 ka mor'a ho kenngoa le ho kenngoa ka entunoblotting ...

Phosphorylation ea CREB ho Ser-133 e ntlafatsa mosebetsi oa eona oa mongolo ka ntho ea karabelo ea cAMP tikolohong ea papatso ea liphatsa tsa lefutso tse itseng, ho kenyeletsoa mofuta oa c-fos (26). Ka hona, re ile ra hlahloba ho kenngoa ha li-c-fos bothateng ba mofuta oa hlaha le litoeba tsa Tgp35 kamora ho entetsoa ka koae. Ho litoeba tsa mofuta oa hlaha, boemo ba c-fos mRNA bo ile ba eketseha ho fihlela boleng ba tlhoro (1.8 ± 0.2-fold over-level of basal) 30 min kamora jekese ea cocaine, mme kamora moo ba khutlela boemong ba basal ka 120 min kamora ho ente (Feie. 6 A 'me B). Leha ho le joalo, maemo a c-fos mRNA a ne a le ≈30% tlase ho litoeba tsa Tgp35 ho feta ho litoeba tsa mofuta o hlaha ho fihlela 30 min kamora ho entoa (Feie. 6 A 'me B). Ts'ebetso e nyane haholo ea li-c-fos tsa Tonk35 litoeba li ile tsa ntlafatsoa hape ke immunohistochemistry (Feie. 6 C-F). Tsamaiso ea cocoaine e ile ea eketsa cun fis immunoreactivity, ka matla likarolong tsa dorsomedial-dorsocentral le ka mokhoa o fokolang likarolong tsa morao, ka mefuta ea hlaha le mofuta oa Tgp35. Leha ho le joalo, keketseho e khothalletsoang ea koae ka palo ea lisele tsa c-fos-immunopositive e ile ea kenella haholo lithutong tsa litoeba tsa Thup35 (Feie. 6G). Ka kopanelo, liphetho tsena li bonts'itse hore ntlafatso ea cocaine e kopantsoeng le "dopamine signaling" e ne e thibetsoe ho litoeba tsa Tkup35, e ka bang sephetho sa ts'ebetso ea Cdk5 e eketsehileng.

Setšoantšo sa 6.  

Keketseho ea ts'ebetso ea Cdk5 e baka ho fokotseha ha polelo ea c-fos ea striatal le ho kenella ha eona hanyane ka mor'a taolo ea koae. (A) Lebala le ka Leboea le bonts'a nako ea c-fos induction ka mofuta oa hlaha (WT) le litoeba tsa Tgp35 (Tg) kamora ho entetsoa ka koae. ...

Puisano

Cdk5 le activator ea eona p35 li khethollotsoe e le mefuta e lebisitsoeng e laoloang ke ho pepeseha ha koae (6). Re tlaleha mona bopaki bo eketsang ts'ebetso ea Cdk5, ka lebaka la p35 up-regulation ho fapana le Cdk5 up-regulation, e lebisang ho fihlelleng ha dopamine ea cocaine-mediated dopamine e bontšang li-neurons tsa striatal. Ho hlahloba litlamorao tsa polelo e ntlafalitsoeng ea Cdk5 kapa p35 ho matšoao a dopamine dopamine, mela e 'meli ea transgenic, TgCdk5 le Tgp35 litoeba. Re fumane hore ts'ebetso ea Cdk5 e ne e tsamaisoa ka mokhoa o lekanang le boemo bo eketsehang ba protheine ea p35 empa ha ea ka ea angoa ke boemo bo eketsehileng ba proteni ea Cdk5. Tlaleho ea rona e fetileng e boetse e bonts'itse hore ts'ebetso ea Cdk5 ho TgCdk5 boko ba panya e ne e le tlase ho feta bokong ba mofuta oa hlaha ha ts'ebetso e lekantsoe ka ho sebelisa Cdk5 immunoprecipitates (17), ho fana ka tlhahiso ea hore Cdk5 overexpression e hlahisa boemo bo eketsehang ba monomeric Cdk5 haeba boemo ba p35 bo sa eketsehe. Liphetho tsena li bonts'a hore boemo ba protheine ea p35 ke ntho e fokotsang sekhahla bakeng sa ts'ebetso ea Cdk5.

Lithunya tsa Tgn35 li bonts'itse ts'ebetso e nyane ea COSB phosphorylation le c-fos ho striatum kamora ho kenngoa ka mokhoa o matla oa cocaine, ho fana ka maikutlo a hore karabelo ea boikoetliso ho koaeine e thibetsoe ke ts'ebetso e eketsehileng ea Cdk5. Khokahano ea matšoao a dopamine ea cocaine-mediated dopamine ho litloholo tsa Thup35 e kanna ea fihlelloa ka Cdk5-Mediated inhibition ea li-cascade tse ngata tsa ho saena tse amanang le DARPP-32, PKA, le ERK. Tsamaiso ea koae e ile ea eketsa phosphorylation ea PKA ea DARPP-32 ho Thr-34 ka litoeba tsa mofuta o hlaha, athe karabelo ena e ile ea fumaneha ho litoeba tsa Tgp35. PKA phosphorylation ea DARPP-32 ho Thr-34 e bontšitsoe ho thibela ts'ebetso ea protheine phosphatase 1 (PP1), enzyme e ikarabellang bakeng sa dephosphorylation ea Ser-133 ea CREB (27). Kahoo, ts'ebetso ea PP1 e ne e ke ke ea hanyetsoa ka tsela ea DARPP-32 / PP1 tseleng ea litoeba tsa Tgp35.

Ts'usumetso e khothalletsoang ke Cocaine e susumetsoang ea ERK1 / 2 le eona e ile ea bontšoa ka litoeba tsa Tgp35. Ho na le mekhoa e fapaneng e fapaneng eo Cdk5 e ka thibelang ts'ebetso ea ts'ebeliso ea koae ea ERK1 / 2. Taba ea pele, phdphXlNUMX e itšetlehileng ka Cdk5 ea DARPP-32 ho Thr-75 e ka thibela PKA, ea lebisa ho thibelo e latelang ea PKA-Mediated MEK1 / 2 activation e hlokahalang bakeng sa ts'ebetso ea ERK1 / 2. Phuputso ea morao-rao e boetse e fumane hore phosphorylation ea DARPP-32 ho Thr-34 e hlokahala bakeng sa ts'ebetso ea ts'ebeliso ea koae ea ERK1 / 2 ka mekhoa e mengata e kenyelletsang taolo e sa sebetseng ea ts'ebetso ea MEK le ho kenyelletsa taolo ea phosphatase e matlafalitsoeng, mofuta oa tyrosine. phosphatase e sebetsang ka kotloloho ho ERK1 / 2 (28). Ts'ehetso ea monyetla ona e khothaletsoa ke ho fumana hore phosphorylation ea koae ea MEK1 / 2 ho Ser-217 le Ser-221 e felisitsoe ho litoeba tsa Tkup35. Tsela e 'ngoe e ka etsahalang ke ka phosphorylation e itšetlehileng ka Cdk5 ea MEK1 ho Thr-286, e ka lebisang ho fokotseha ha ts'ebetso ea eona ea phello mme ea lebisa ho sitiseng mosebetsi oa ERK1 / 2 (24).

Thibelo ea ts'ebetso ea Cdk5 ho striatum e bontšitsoe hore e na le litlamorao tsa boits'oaro ba kalafo ea koae e sa foleng ho liphoofolo (6). E lumellana le ts'oaetso ea hore ts'ebetso ea kemiso ea Cdk5 e ka kenya letsoho ho fetoheng ha methapo ea kutlo bakeng sa ho loants'a litlamorao tsa tsamaiso ea koae e phetoang hangata. (6), re fumane hore Cdk5-Mediated phosphorylation ea DARPP-32 le MEK1 li kentse letsoho ho amoheloa ha ts'ebetso ea cocaine e kenyellelitsoeng ea ERK1 / 2, e bakileng ho kenella ho fokolang ha phofo ea CREB le c-fos ho striatum. Liphumano tsa rona li ts'ehetsa mohopolo o ileng oa eketsa ts'ebetso ea Cdk5, ka lebaka la p35 up-regulation, e ka fetola polelo ea mofuta ho striatum kamora ho pepesetsoa nako e telele ea koae. Sena se ka hlaha ka liphetoho mesebetsing ea lingoloa tse kang CREB le c-fos. Kahoo, Cdk5 activator p35, ka lebaka la litekanyetso tsa eona tse fokotsang ts'ebetso ea Cdk5, e kanna ea kenya letsoho phetohong e sa feleng ea ts'ebetso ea neuronal tlasa ts'ebetso ea lithethefatsi tsa cocaine.

lumela hore baa fokola

Re leboha Drs. Mary Jo Danton, Philip Grant le Sashi Kesavapany bakeng sa ho bala ka hloko buka e ngotsoeng ka letsoho. Mosebetsi ona o tšehelitsoe ke National Institutes of Health Grant Z01DE00664-05 (ho ABK), US Public Health Service Grant DA10044, le lithuso tse tsoang Simons Foundation, Peter J. Sharp Foundation, le Picower Foundation (ho PG).

Notes

Maelefatso: Cdk5, cyclin-based kinase 5; ERK, kinase e laoloang ke li-extracellular signature; DARPP-32, phophoprotein e laoloang ke cAMP, boima ba limolek'hule 32 kDa; PKA, kinase e itšetlehileng ka cAMP; MEK, ERK kinase; CREB, protheine e kopanyang likarolo tsa cAMP.

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