isolation-ca1-nuclear-enriched-fractions-from-hippocampal-slices-to

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular ...

Leibniz Institute for Neurobiology

&#x8FD1;&#x51E0;&#x5E74;&#x7684;&#x7279;&#x70B9;&#x662F;&#xFF0C;&#x5927;&#x91CF;&#x7684;&#x6F5C;&#x5728;&#x7684;&#x7A81;&#x89E6;&#x6838;&#x86CB;&#x767D;&#x4F7F;&#x8005;&#xFF0C;&#x88AB;&#x8BA4;&#x4E3A;&#x662F;&#x53C2;&#x4E0E;&#x53EF;&#x5851;&#x6027;&#x76F8;&#x5173;&#x57FA;&#x56E0;&#x7684;&#x8868;&#x8FBE;&#xFF0C;&#x800C;&#x4E14;&#x5177;&#x6709;&#x80FD;&#x529B;&#x7684;&#x79C9;&#x6301;-&#x4ECB;&#x5BFC;&#x548C;&#x6D3B;&#x6027;&#x4F9D;&#x8D56;&#x6838;&#x5BFC;&#x5165;&#x3002;&#x4E0D;&#x8FC7;&#xFF0C;&#x5F88;&#x4EE4;&#x4EBA;&#x60CA;&#x8BB6;&#x7684;&#x5B66;&#x4E60;&#x548C;&#x8BB0;&#x5FC6;&#x7684;&#x7EC6;&#x80DE;&#x6A21;&#x578B;&#x4E2D;&#x663E;&#x793A;&#x8FD9;&#x79CD;&#x86CB;&#x767D;&#x7684;&#x6838;&#x5BFC;&#x5165;&#x6570;&#x636E;&#x7F3A;&#x4E4F;&#x3002;&#x53EA;&#x662F;&#x6700;&#x8FD1;&#x624D;&#x53D1;&#x73B0;&#x5411;&#x6838;&#x8FC7;&#x5883;&#x7684;&#x8F6C;&#x5F55;&#x56E0;&#x5B50;&#x73AF;&#x78F7;&#x9178;&#x817A;&#x82F7;&#x53CD;&#x5E94;&#x5143;&#x4EF6;&#x7ED3;&#x5408;&#x86CB;&#x767D; 2 (CREB2) &#x671F;&#x95F4;&#x957F;&#x671F;&#x8427;&#x6761; (&#x6709;&#x9650;&#x516C;&#x53F8;)&#xFF0C;&#x4F46;&#x4E0D;&#x662F;&#x5728;&#x957F;&#x671F;&#x957F;&#x65F6;&#x7A0B;&#x589E;&#x5F3A; (LTP) &#x6D77;&#x9A6C;&#x795E;&#x7ECF;&#x5143;&#x7A81;&#x89E6;&#x4F20;&#x9012;&#x7684;&#x8FC7;&#x7A0B;&#x3002;&#x96C5;&#x5404;&#x5E03;&#x662F;&#x60C5;&#x4FA3; NMDA &#x53D7;&#x4F53;&#x6D3B;&#x6027;&#x7684;&#x6838;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x7684;&#x53E6;&#x4E00;&#x4E2A;&#x4F7F;&#x8005;&#x3002;&#x56E0;&#x6B64;&#xFF0C;&#x6211;&#x4EEC;&#x65E8;&#x5728;&#x7814;&#x7A76;&#x662F;&#x5426;&#x96C5;&#x5404;&#x7D2F;&#x79EF;&#x6D3B;&#x52A8;&#x4F9D;&#x8D56;&#x7684;&#x7A81;&#x89E6;&#x53EF;&#x5851;&#x6027;&#x76F8;&#x5173;&#x7684;&#x751F;&#x7406;&#x6A21;&#x578B;&#x5728;&#x7EC6;&#x80DE;&#x6838;&#x4E2D;&#x3002;

&#x6838;&#x96C5;&#x5404;&#x7684;&#x6D77;&#x9A6C;&#x795E;&#x7ECF;&#x5143;&#x540E;&#x79FB;&#x4F4D;&#x523A;&#x6FC0;&#x8BF1;&#x5BFC;&#x957F;&#x65F6;&#x7A0B;&#x589E;&#x5F3A;&#xFF0C;&#x4F46;&#x4E0D;&#x662F;&#x957F;&#x671F;&#x8427;&#x6761;&#x3002;

In recent years a number of potential synapto-nuclear protein messengers have been characterized that are thought to be involved in plasticity-related gene expression, and that have the capacity of importin- mediated and activity-dependent nuclear import. However, there is a surprising paucity of data showing the nuclear import of such proteins in cellular models of learning and memory. Only recently it was found that the transcription factor cyclic AMP response element binding protein 2 (CREB2) transits to the nucleus during long-term depression (LTD), but not during long-term potentiation (LTP) of synaptic transmission in hippocampal primary neurons. Jacob is another messenger that couples NMDA-receptor-activity to nuclear gene expression. We therefore aimed to study whether Jacob accumulates in the nucleus in physiological relevant models of activity-dependent synaptic plasticity.

Nuclear translocation of jacob in hippocampal neurons after stimuli inducing long-term potentiation but not long-term depression.

Ces derni&egrave;res ann&eacute;es qu&#39;un certain nombre de messagers de prot&eacute;ines nucl&eacute;aires synapto potentiels ont &eacute;t&eacute; caract&eacute;ris&eacute; qui sont cens&eacute;es &ecirc;tre impliqu&eacute;s dans l&#39;expression des g&egrave;nes de plasticit&eacute;-connexes et qui ont la capacit&eacute; de l&#39;important - m&eacute;diation et d&eacute;pendante de l&#39;activit&eacute; nucl&eacute;aire importer. Cependant, il y a un manque surprenant de donn&eacute;es montrant l&#39;importation nucl&eacute;aire de ces prot&eacute;ines dans des mod&egrave;les cellulaires de l&#39;apprentissage et la m&eacute;moire. R&eacute;cemment, il a &eacute;t&eacute; constat&eacute; que la transcription factor AMP cyclique response element binding protein 2 (CREB2) transite vers le noyau au cours de la d&eacute;pression &agrave; long terme (DLT), mais pas au cours de la potentialisation &agrave; long terme (LTP), de la transmission synaptique dans des neurones hippocampal primaires. Jacob est un autre messager qui couple NMDA-r&eacute;cepteur-activity &agrave; l&#39;expression des g&egrave;nes nucl&eacute;aires. L&#39;&eacute;tude visait donc &agrave; &eacute;tudier si Jacob s&#39;accumule dans le noyau dans des mod&egrave;les physiologiques pertinents de la plasticit&eacute; synaptique d&eacute;pendante de l&#39;activit&eacute;.

Translocation nucl&eacute;aire de jacob dans les neurones de l&#39;hippocampe apr&egrave;s stimulus induisant la potentialisation &agrave; long terme mais pas &agrave; long terme d&eacute;pression.

In den letzten Jahren, die eine Reihe von potenziellen Synapto-nukleare Protein boten gepr&auml;gt haben, die man Plastizit&auml;t-bezogene Genexpression, und beteiligt, die die Kapazit&auml;t von importin - vermittelte und leistungsabh&auml;ngige nukleare importieren. Allerdings gibt es ein &uuml;berraschender Mangel an Daten zeigen den nuklearen Import solcher Proteine in zellul&auml;ren Modellen von lernen und Ged&auml;chtnis. Erst vor kurzem wurde festgestellt, dass das Transkription Faktor zyklischem AMP Antwort Element verbindliches Protein 2 (CREB2) zum Kern, w&auml;hrend langfristige Depression (LTD), aber nicht w&auml;hrend langfristige Erm&ouml;glichung (LTP) der synaptischen &Uuml;bertragung in prim&auml;ren Neuronen l&auml;uft. Jacob ist eine andere Messenger, der NMDA-Rezeptor-Aktivit&auml;t zu nuklearen Genexpression Paare. Wir wollten daher, ob Jacob im Zellkern in physiologischen relevanten Modelle der leistungsabh&auml;ngige synaptische Plastizit&auml;t sammelt zu studieren.

Nukleare Translokation von Jacob in Neuronen nach Reize induzierende langfristige Erm&ouml;glichung aber nicht langfristige Depression.

Negli ultimi anni un numero di potenziali messaggeri synapto-nucleare della proteina &egrave; stato caratterizzato che si pensiero siano coinvolti nell&#39;espressione genica plasticit&agrave;-correlati e che hanno la capacit&agrave; di importin - mediata e importare attivit&agrave;-dipendente nucleare. Tuttavia, c&#39;&egrave; una sorprendente scarsit&agrave; di dati visualizzando l&#39;importazione di tali proteine nucleare in modelli cellulari dell&#39;apprendimento e della memoria. Solo recentemente si &egrave; riscontrato che la trascrizione fattore AMP ciclico response element binding protein 2 (CREB2) transita al nucleo durante la depressione a lungo termine (LTD), ma non durante il potenziamento a lungo termine (LTP) della trasmissione sinaptica nei neuroni hippocampal primari. Jacob &egrave; un altro Messaggero che le coppie NMDA-recettori-attivit&agrave; di espressione del gene nucleare. Abbiamo quindi rivolto a studiare se Jacob si accumula nel nucleo in modelli fisiologici rilevanti della plasticit&agrave; sinaptica attivit&agrave;-dipendente.

Traslocazione nucleare di jacob nei neuroni hippocampal dopo stimoli che inducono fenomeni di potenziamento a lungo termine, ma non a lungo termine depressione.

&#x53EF;&#x5851;&#x6027;&#x95A2;&#x9023;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x767A;&#x73FE;&#x306B;&#x95A2;&#x4E0E;&#x3059;&#x308B;&#x3068;&#x8003;&#x3048;&#x3089;&#x308C;&#x308B;&#x6F5C;&#x5728;&#x7684;&#x306A; synapto &#x6838;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x4F7F;&#x5F92;&#x306E;&#x6570;&#x7279;&#x5FB4;&#x3065;&#x3051;&#x3089;&#x308C;&#x3066;&#x3044;&#x308B;&#x6700;&#x8FD1;&#x306E;&#x5E74;&#x3067;&#x30A4;&#x30F3;&#x30DD;&#x30FC;&#x30C1;&#x30F3;&#x306E;&#x5BB9;&#x91CF;&#x304C;&#x3042;&#x308B; - &#x3092;&#x4ECB;&#x3057;&#x305F;&#x3057;&#x3001;&#x6838;&#x6D3B;&#x52D5;&#x4F9D;&#x5B58;&#x3092;&#x30A4;&#x30F3;&#x30DD;&#x30FC;&#x30C8;&#x3057;&#x307E;&#x3059;&#x3002;&#x3057;&#x304B;&#x3057;&#x3001;&#x643A;&#x5E2F;&#x96FB;&#x8A71;&#x306E;&#x30E2;&#x30C7;&#x30EB;&#x5B66;&#x7FD2;&#x3068;&#x8A18;&#x61B6;&#x306E;&#x3088;&#x3046;&#x306A;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x6838;&#x306E;&#x8F38;&#x5165;&#x3092;&#x793A;&#x3059;&#x30C7;&#x30FC;&#x30BF;&#x306E;&#x9A5A;&#x304F;&#x3079;&#x304D;&#x4E0D;&#x8DB3;&#x3067;&#x3059;&#x3002;&#x3054;&#x304F;&#x6700;&#x8FD1;&#x3001;&#x8EE2;&#x5199;&#x56E0;&#x5B50;&#x30B5;&#x30A4;&#x30AF;&#x30EA;&#x30C3;&#x30AF; AMP &#x5FDC;&#x7B54;&#x8981;&#x7D20;&#x7D50;&#x5408;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA; 2 (CREB2) &#x306B;&#x6838;&#x9577;&#x671F;&#x6291;&#x5727; (LTD) &#x4E2D;&#x304C;&#x6D77;&#x99AC;&#x306E;&#x4E00;&#x6B21;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306B;&#x304A;&#x3051;&#x308B;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x4F1D;&#x9054;&#x306E;&#x9577;&#x671F;&#x5897;&#x5F37; (LTP) &#x4E2D;&#x3067;&#x306F;&#x306A;&#x304F;&#x9077;&#x79FB;&#x3053;&#x3068;&#x304C;&#x898B;&#x3064;&#x304B;&#x308A;&#x307E;&#x305B;&#x3093;&#x3067;&#x3057;&#x305F;&#x3002;&#x30B8;&#x30A7;&#x30A4;&#x30B3;&#x30D6;&#x6838;&#x907A;&#x4F1D;&#x5B50;&#x767A;&#x73FE;&#x3059;&#x308B; NMDA &#x53D7;&#x5BB9;&#x4F53;&#x306E;&#x6D3B;&#x6027;&#x3092;&#x30AB;&#x30C3;&#x30D7;&#x30EB;&#x5225;&#x306E;&#x30E1;&#x30C3;&#x30BB;&#x30F3;&#x30B8;&#x30E3;&#x30FC;&#x3067;&#x3059;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;&#x30E4;&#x30B3;&#x30D6;&#x6838;&#x6D3B;&#x6027;&#x4F9D;&#x5B58;&#x6027;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x53EF;&#x5851;&#x6027;&#x306E;&#x751F;&#x7406;&#x7684;&#x95A2;&#x9023;&#x6027;&#x306E;&#x9AD8;&#x3044;&#x30E2;&#x30C7;&#x30EB;&#x3067;&#x84C4;&#x7A4D;&#x3059;&#x308B;&#x304B;&#x3069;&#x3046;&#x304B;&#x3092;&#x691C;&#x8A0E;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x76EE;&#x7684;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;

&#x30B8;&#x30A7;&#x30A4;&#x30B3;&#x30D6;&#x6D77;&#x99AC;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306B;&#x304A;&#x3051;&#x308B;&#x9577;&#x671F;&#x5897;&#x5F37;&#x3067;&#x3059;&#x304C;&#x306A;&#x3044;&#x9577;&#x671F;&#x7684;&#x306A;&#x4E0D;&#x6CC1;&#x306E;&#x8A98;&#x767A;&#x523A;&#x6FC0;&#x5F8C;&#x6838;&#x8EE2;&#x5EA7;&#x306F;&#x3002;

&#xCD5C;&#xADFC; &#xBA87; &#xB144;&#xAC04;&#xC774; &#xC18C;&#xC131; &#xAD00;&#xB828; &#xC720;&#xC804;&#xC790; &#xBC1C;&#xD604;&#xC5D0; &#xAD00;&#xC5EC; &#xB420; &#xAC83;&#xC73C;&#xB85C; &#xC0DD;&#xAC01; &#xB418;&#xB294; &#xB2E4;&#xC591; &#xD55C; &#xC7A0;&#xC7AC;&#xC801;&#xC778; synapto &#xD575; &#xB2E8;&#xBC31;&#xC9C8; &#xBA54;&#xC2E0;&#xC800; &#xD2B9;&#xC9D5; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4; importin&#xC758; &#xB2A5;&#xB825;&#xC744;&#xAC00;&#xC9C0;&#xACE0;-&#xC911;&#xC7AC; &#xBC0F; &#xD65C;&#xB3D9; &#xC885;&#xC18D;&#xC801;&#xC778; &#xD575;&#xC744; &#xAC00;&#xC838;&#xC62C;. &#xADF8;&#xB7EC;&#xB098;, &#xC774;&#xB7EC;&#xD55C; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xD575; &#xAC00;&#xC838;&#xC624;&#xAE30; &#xD559;&#xC2B5; &#xBC0F; &#xBA54;&#xBAA8;&#xB9AC;&#xC758; &#xD734;&#xB300; &#xC804;&#xD654; &#xBAA8;&#xB378;&#xC744; &#xBCF4;&#xC5EC;&#xC8FC;&#xB294; &#xB370;&#xC774;&#xD130;&#xC758; &#xB180;&#xB77C;&#xC6B4; &#xC18C;&#xC218;&#xAC00; &#xC774;&#xB2E4;. &#xCD5C;&#xADFC; &#xC7A5;&#xAE30; &#xBD88;&#xD669; (&#xACF5;&#xC0AC;) &#xD558;&#xB294; &#xB3D9;&#xC548; &#xD558;&#xC9C0;&#xB9CC; &#xC7A5;&#xAE30; potentiation (LTP) hippocampal &#xAE30;&#xBCF8; &#xC2E0;&#xACBD; &#xC138;&#xD3EC;&#xC5D0; &#xC2DC; &#xB0C5; &#xC2A4; &#xC804;&#xC1A1; &#xC911;&#xC774; &#xC544;&#xB2C8;&#xB77C; &#xC804;&#xC0AC; &#xC778;&#xC790; &#xC21C;&#xD658; &#xC570;&#xD504; &#xC751;&#xB2F5; &#xC694;&#xC18C; &#xBC14;&#xC778;&#xB529; &#xB2E8;&#xBC31;&#xC9C8; 2 (CREB2) &#xD575;&#xC744; &#xC804;&#xC1A1; &#xBC1C;&#xACAC;. &#xC57C; &#xACF1;&#xC774; &#xB2E4;&#xB978; &#xBA54;&#xC2E0;&#xC800; &#xD575; &#xC720;&#xC804;&#xC790; &#xBC1C;&#xD604;&#xC5D0; NMDA &#xC218;&#xC6A9; &#xCCB4; &#xD65C;&#xB3D9;&#xC744; &#xCEE4;&#xD50C;. &#xC6B0;&#xB9AC;&#xB294; &#xB530;&#xB77C;&#xC11C; &#xC81C;&#xC774;&#xCF65; &#xC0DD;&#xB9AC; &#xAD00;&#xB828; &#xBAA8;&#xB378; &#xD65C;&#xB3D9;&#xC5D0; &#xC885;&#xC18D; &#xC2DC; &#xB0C5; &#xC2A4;&#xAC00; &#xC18C;&#xC131;&#xC758; &#xD575;&#xC5D0; &#xCD95;&#xC801; &#xC5EC;&#xBD80;&#xB97C; &#xC5F0;&#xAD6C; &#xBAA9;&#xC801;.

&#xD575; &#xC804; &#xC81C;&#xC774;&#xCF65;&#xC758; hippocampal &#xB274;&#xB7F0;&#xC5D0;&#xC11C; &#xC790;&#xADF9; &#xC7A5;&#xAE30; potentiation &#xD558;&#xC9C0;&#xB9CC; &#xD558;&#xC9C0; &#xC7A5;&#xAE30; &#xBD88;&#xD669;&#xC744; &#xC720;&#xB3C4; &#xD55C; &#xD6C4;.

En los &uacute;ltimos a&ntilde;os se ha caracterizado una serie de posibles mensajeros de la prote&iacute;na nuclear synapto que se cree que son involucrados en la expresi&oacute;n de genes relacionados con la plasticidad y tienen la capacidad de importin - mediada y dependientes de actividad nuclear de importaci&oacute;n. Sin embargo, hay una sorprendente falta de datos que muestran la importaci&oacute;n nuclear de tales prote&iacute;nas en modelos celulares del aprendizaje y la memoria. S&oacute;lo recientemente se encontr&oacute; que la transcripci&oacute;n factor AMP c&iacute;clico respuesta elemento prote&iacute;na 2 (CREB2) transita al n&uacute;cleo durante la depresi&oacute;n a largo plazo (LTD), pero no durante la potenciaci&oacute;n a largo plazo (LTP) de la transmisi&oacute;n sin&aacute;ptica en neuronas hipocampales de primarias. Jacob es otro mensajero que parejas la actividad del receptor de NMDA a la expresi&oacute;n de genes nucleares. Por lo tanto decidimos estudiar si Jacob se acumula en el n&uacute;cleo en modelos fisiol&oacute;gicos relevantes de plasticidad sin&aacute;ptica dependiente de la actividad.

Translocaci&oacute;n nuclear de jacob en neuronas de hipocampales tras est&iacute;mulos induciendo potenciaci&oacute;n a largo plazo pero no a largo plazo depresi&oacute;n.

Activity-dependent synaptic plasticity is associated with synaptic protein turnover involving the ubiquitin proteasome system (UPS) for protein degradation. In primary hippocampal cell culture, it has been shown that increased or decreased activity of synaptic transmission can regulate the amount of postsynaptic density (PSD) proteins via UPS. However, the specific spatio-temporal dynamic of PSD protein degradation after LTP induction and its downstream signaling pathways remains to be clarify. We used confocal microscopy to monitor levels of eGFP-tagged SPAR (spine-associated Rap GTPase activating protein) expressed in acute hippocampal slices and found that LTP induction triggered a UPS-dependent decay of eGFP-SPAR fluorescence. SPAR degradation was reduced upon inhibition of cyclin-dependent kinase 5 (CDK5) as well as by a protein synthesis inhibitor. Comparison of eGFP-tagged SPAR levels with those obtained in control experiments with eGFP revealed a protein synthesis-independent component of LTP-associated SPAR degradation. This second component required UPS and NMDA receptor activation but not CDK5. We conclude that LTP triggers a down regulation of SPAR by two complementary mechanisms, one of which has previously been described to mediate homeostatic plasticity.

Hippocampal LTP triggers proteasome-mediated SPAR degradation in CA1 neurons.

In previous work, we found that the protein messenger Jacob is involved in N-methyl-D-aspartate receptor (NMDAR) signaling to the nucleus and cAMP response element-binding protein (CREB) mediated gene expression in hippocampal primary neurons. Particularly, extrasynaptic NMDAR activation drives Jacob efficiently into the nucleus where it then induces gene expression that promotes neurodegeneration. However, the protein also translocates to the nucleus in CA1 neurons after Schaffer collateral long-term potentiation (LTP) but not long-term depression (LTD), suggesting that Jacob might be involved in hippocampal and LTP-dependent learning and memory processes. Not much is known about the cellular and subcellular distribution of the protein in brain. In this paper, we provide an overview of the expression of Jacob in rat brain with special emphasis on the hippocampus. We show that Jacob is abundant in hippocampal pyramidal neurons and interneurons but absent from astrocytes and microglia. Interestingly, we found that Jacob is also present in mossy fiber axons. Double immunofluorescence confocal laser scans with presynaptic markers demonstrate that Jacob is indeed found at excitatory but not inhibitory presynaptic sites. Accordingly, we found no substantial co-localization of Jacob with a postsynaptic marker of inhibitory synapses, gephyrin. In contrast, almost all postsynaptic density protein 95 (PSD-95) positive excitatory postsynaptic sites also exhibited strong Jacob-immunofluorescence. Taken together, these data support a synaptic and nuclear role of Jacob that implicates long-distance NMDAR signaling to the nucleus in excitatory neurons.

Pingan Yuanxiang

RG Neuroplasticity

Leibniz Institute for Neurobiology

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins