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

Recently it was demonstrated that for the absence epilepsy characteristic spike-wave discharges initially emerge from the somatosensory cortex and quickly involve the rest of the cortex and cortico-thalamic network. This has led to the development of the focal theory of absence epilepsy. In this experiment, this theory was further investigated by studying the neuronal organization of the cortical focal zone, a non-focal zone in genetic epileptic WAG/Rij rats and functional related areas in non-epileptic age matched control rats. A classical Golgi staining technique was used to visualize whole cortical neurons with dendritic and axon arborisation. Apical dendrites of pyramidal cells in epileptic rats were often split, declined and were running in non-perpendicular directions. Quantitative differences between the strains were found for the length of neurons, between focal and control areas mainly for dendritic arborization. A significant "strain-zone" interaction was found for the maximal distance between two points of dendritic arborization, the mean length of a dendritic segment and the number of free terminations of apical dendrites. All this demonstrates that properties of dendrites in the cortical focal area of WAG/Rij rats were at variance with dendritic characteristics outside the focal area and with functional similar areas in non-epileptic controls. These features might reflect the hyperexcitability of somatosensory neurons, which underlie the initiation and spreading of spike-wave discharges in WAG/Rij rats. Finally, these results are in line with the cortical focus theory of absence epilepsy.

Morphometric Golgi study of cortical locations in WAG/Rij rats: the cortical focus theory.

Expression of synaptic plasticity involves the translation of mRNA into protein and, probably, active protein degradation via the proteasome pathway. Here, we report on the rapid activation of synthesis and degradation of a probe protein with the induction of long-term potentiation (LTP) in the hippocampal Schaffer collateral CA1 pathway. The proteasome inhibitor MG132 significantly reduced the field EPSP slope potentiation and LTP maintenance without acutely affecting basal synaptic transmission. To visualize protein dynamics, CA1 pyramidal cells of hippocampal slices were transfected with Semliki Forest virus particles expressing a recombinant RNA. This RNA contained the coding sequence for a degradable green fluorescence protein with a nuclear localization signal (NLS-d1EGFP) followed by a 3'- untranslated region dendritic targeting sequence. NLS-d1EGFP fluorescence remained stable in the low-frequency test stimulation but increased with LTP induction in the cell body and in most dendritic compartments of CA1 neurons. Applying anisomycin, a protein synthesis inhibitor, caused NLS-d1EGFP levels to decline; a proteasome inhibitor MG132 reversed this effect. In the presence of anisomycin, LTP induction accelerated the degradation of NLS-d1EGFP. When both inhibitors were present, NLS-d1EGFP levels remained unaffected by LTP induction. Moreover, LTP-induced acceleration of NLS-d1EGFP synthesis was blocked by rapamycin, which is consistent with the involvement of dendritic mammalian target of rapamycin in LTP-triggered translational activity. Our results clearly demonstrate that LTP induction not only leads to a rapid increase in the rate of protein synthesis but also accelerates protein degradation via the proteasome system.

Involvement of protein synthesis and degradation in long-term potentiation of Schaffer collateral CA1 synapses.

NMDA (N-methyl-D-aspartate) receptors and calcium can exert multiple and very divergent effects within neuronal cells, thereby impacting opposing occurrences such as synaptic plasticity and neuronal degeneration. The neuronal Ca2+ sensor Caldendrin is a postsynaptic density component with high similarity to calmodulin. Jacob, a recently identified Caldendrin binding partner, is a novel protein abundantly expressed in limbic brain and cerebral cortex. Strictly depending upon activation of NMDA-type glutamate receptors, Jacob is recruited to neuronal nuclei, resulting in a rapid stripping of synaptic contacts and in a drastically altered morphology of the dendritic tree. Jacob's nuclear trafficking from distal dendrites crucially requires the classical Importin pathway. Caldendrin binds to Jacob's nuclear localization signal in a Ca2+-dependent manner, thereby controlling Jacob's extranuclear localization by competing with the binding of Importin-alpha to Jacob's nuclear localization signal. This competition requires sustained synapto-dendritic Ca2+ levels, which presumably cannot be achieved by activation of extrasynaptic NMDA receptors, but are confined to Ca2+ microdomains such as postsynaptic spines. Extrasynaptic NMDA receptors, as opposed to their synaptic counterparts, trigger the cAMP response element-binding protein (CREB) shut-off pathway, and cell death. We found that nuclear knockdown of Jacob prevents CREB shut-off after extrasynaptic NMDA receptor activation, whereas its nuclear overexpression induces CREB shut-off without NMDA receptor stimulation. Importantly, nuclear knockdown of Jacob attenuates NMDA-induced loss of synaptic contacts, and neuronal degeneration. This defines a novel mechanism of synapse-to-nucleus communication via a synaptic Ca2+-sensor protein, which links the activity of NMDA receptors to nuclear signalling events involved in modelling synapto-dendritic input and NMDA receptor-induced cellular degeneration.

Caldendrin-Jacob: a protein liaison that couples NMDA receptor signalling to the nucleus.

Jacob is a recently identified plasticity-related protein that couples N-methyl-d-aspartate receptor activity to nuclear gene expression. An expression analysis by Northern blot and in situ hybridization shows that Jacob is almost exclusively present in brain, in particular in the cortex and the limbic system. Alternative splicing gives rise to multiple mRNA variants, all of which exhibit a prominent dendritic localization in the hippocampus. Functional analysis in primary hippocampal neurons revealed that a predominant cis-acting dendritic targeting element in the 3'-untranslated region of Jacob mRNAs is responsible for dendritic mRNA localization. In the mouse brain, Jacob transcripts are associated with both the fragile X mental retardation protein, a well described trans-acting factor regulating dendritic mRNA targeting and translation, and the kinesin family member 5C motor complex, which is known to mediate dendritic mRNA transport. Jacob is susceptible to rapid protein degradation in a Ca(2+)- and Calpain-dependent manner, and Calpain-mediated clipping of the myristoylated N terminus of Jacob is required for its nuclear translocation after N-methyl-d-aspartate receptor activation. Our data suggest that local synthesis in dendrites may be necessary to replenish dendritic Jacob pools after truncation of the N-terminal membrane anchor and concomitant translocation of Jacob to the nucleus.

Dendritic mRNA targeting of Jacob and N-methyl-d-aspartate-induced nuclear translocation after calpain-mediated proteolysis.

&#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.

The communication between synapses and the cell nucleus has attracted considerable interest for many years. This interest is largely fueled by the idea that synapse-to-nucleus signaling might specifically induce the expression of genes that make long-term memory "stick." However, despite many years of research, it is still essentially unclear how synaptic signals are conveyed to the nucleus, and it remains to a large degree enigmatic how activity-induced gene expression feeds back to synaptic function. In this chapter, we will focus on the activity-dependent synapto-nuclear trafficking of protein messengers and discuss the underlying mechanisms of their retrograde transport and their supposed functional role in neuronal plasticity.

Long-distance signaling from synapse to nucleus via protein messengers.

Calcium (Ca(2+)) signaling in neurons is mediated by plethora of calcium binding proteins with many of them belonging to the Calmodulin family of calcium sensors. Many studies have shown that the subcellular localization of neuronal EF-hand Ca(2+)-sensors is crucial for their cellular function. To overcome the resolution limit of classical fluorescence and confocal microscopy various imaging techniques have been developed recently that improve the resolution by an order of magnitude in all dimensions. This new microscope techniques make co-localization studies of Ca(2+)-binding proteins more reliable and help to get insights into the macromolecular organization of intracellular structures and signaling pathways beyond the diffraction limit of visible light.

Super-resolution microscopy of the neuronal calcium-binding proteins Calneuron-1 and Caldendrin.

The activation of N-methyl-D-aspartate-receptors (NMDARs) in synapses provides plasticity and cell survival signals, whereas NMDARs residing in the neuronal membrane outside synapses trigger neurodegeneration. At present, it is unclear how these opposing signals are transduced to and discriminated by the nucleus. In this study, we demonstrate that Jacob is a protein messenger that encodes the origin of synaptic versus extrasynaptic NMDAR signals and delivers them to the nucleus. Exclusively synaptic, but not extrasynaptic, NMDAR activation induces phosphorylation of Jacob at serine-180 by ERK1/2. Long-distance trafficking of Jacob from synaptic, but not extrasynaptic, sites depends on ERK activity, and association with fragments of the intermediate filament α-internexin hinders dephosphorylation of the Jacob/ERK complex during nuclear transit. In the nucleus, the phosphorylation state of Jacob determines whether it induces cell death or promotes cell survival and enhances synaptic plasticity.

Encoding and transducing the synaptic or extrasynaptic origin of NMDA receptor signals to the nucleus.

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.

Cellular distribution of the NMDA-receptor activated synapto-nuclear messenger Jacob in the rat brain.

Gephyrin is the key scaffolding molecule organizing the postsynaptic density at inhibitory synapses. Utilizing localization microscopy, Specht et al. (2013) report in this issue of Neuron on the quantitative assessment of gephyrin clusters and associated glycine receptors and GABAA receptors.

Counting gephyrins, one at a time: a nanoscale view on the inhibitory postsynapse.

In Alzheimer's disease (AD), the β-amyloid peptide (Aβ) has been causally linked to synaptic dysfunction and cognitive impairment. Several studies have shown that N-Methyl-D-Aspartate receptors (NMDAR) activation is involved in the detrimental actions of Aβ. Polyamines, like spermidine and spermine, are positive modulators of NMDAR function and it has been shown that their levels are regulated by Aβ. In this study we show here that interruption of NMDAR modulation by polyamines through blockade of its binding site at NMDAR by arcaine (0.02 nmol/site), or inhibition of polyamine synthesis by DFMO (2.7 nmol/site), reverses Aβ25-35-induced memory impairment in mice in a novel object recognition task. Incubation of hippocampal cell cultures with Aβ25-35 (10 µM) significantly increased the nuclear accumulation of Jacob, which is a hallmark of NMDAR activation. The Aβ-induced nuclear translocation of Jacob was blocked upon application of traxoprodil (4 nM), arcaine (4 µM) or DFMO (5 µM), suggesting that activation of the polyamine binding site at NMDAR located probably at extrasynaptic sites might underlie the cognitive deficits of Aβ25-35-treated mice. Extrasynaptic NMDAR activation in primary neurons results in a stripping of synaptic contacts and simplification of neuronal cytoarchitecture. Aβ25-35 application in hippocampal primary cell cultures reduced dendritic spine density and induced alterations on spine morphology. Application of traxoprodil (4 nM), arcaine (4 µM) or DFMO (5 µM) reversed these effects of Aβ25-35. Taken together these data provide evidence that polyamine modulation of extrasynaptic NMDAR signaling might be involved in Aβ pathology.

Article	Year
Morphometric Golgi study of cortical locations in WAG/Rij rats: the cortical focus theory. Neuroscience research\| PubMed ID: 15681029	2005
Involvement of protein synthesis and degradation in long-term potentiation of Schaffer collateral CA1 synapses. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 16672670	2006
Caldendrin-Jacob: a protein liaison that couples NMDA receptor signalling to the nucleus. PLoS biology\| PubMed ID: 18303947	2008
Dendritic mRNA targeting of Jacob and N-methyl-d-aspartate-induced nuclear translocation after calpain-mediated proteolysis. The Journal of biological chemistry\| PubMed ID: 19608740	2009
Nuclear translocation of jacob in hippocampal neurons after stimuli inducing long-term potentiation but not long-term depression. PloS one\| PubMed ID: 21364755	2011
Long-distance signaling from synapse to nucleus via protein messengers. Advances in experimental medicine and biology\| PubMed ID: 22351064	2012
Super-resolution microscopy of the neuronal calcium-binding proteins Calneuron-1 and Caldendrin. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 23296610	2013
Encoding and transducing the synaptic or extrasynaptic origin of NMDA receptor signals to the nucleus. Cell\| PubMed ID: 23452857	2013
Cellular distribution of the NMDA-receptor activated synapto-nuclear messenger Jacob in the rat brain. Brain structure & function\| PubMed ID: 23539133	2014
Counting gephyrins, one at a time: a nanoscale view on the inhibitory postsynapse. Neuron\| PubMed ID: 23889929	2013
Inhibition of the polyamine system counteracts β-amyloid peptide-induced memory impairment in mice: involvement of extrasynaptic NMDA receptors. PloS one\| PubMed ID: 24921942	2014

Anna Karpova

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