Longitudinal <em>In Vivo</em> Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Institute of Functional Genomics

UMR-5203

Université de Montpellier

The etiology of major depression remains unknown, but dysfunction of serotonergic signaling has long been implicated in the pathophysiology of this disorder. p11 is an S100 family member recently identified as a serotonin 1B [5-hydroxytryptamine 1B (5-HT(1B))] and serotonin 4 (5-HT(4)) receptor-binding protein. Mutant mice in which p11 is deleted show depression-like behaviors, suggesting that p11 may be a mediator of affective disorder pathophysiology. Using somatic gene transfer, we have now identified the nucleus accumbens as a key site of p11 action. Reduction of p11 with adeno-associated virus (AAV)-mediated RNA interference in the nucleus accumbens, but not in the anterior cingulate, of normal adult mice resulted in depression-like behaviors nearly identical to those seen in p11 knockout mice. Restoration of p11 expression specifically in the nucleus accumbens of p11 knockout mice normalized depression-like behaviors. Human nucleus accumbens tissue shows a significant reduction of p11 protein in depressed patients when compared to matched healthy controls. These results suggest that p11 loss in rodent and human nucleus accumbens may contribute to the pathophysiology of depression. Normalization of p11 expression within this brain region with AAV-mediated gene therapy may be of therapeutic value.

Reversal of depressed behaviors in mice by p11 gene therapy in the nucleus accumbens.

&#x636E;&#x5047;&#x5B9A;&#x8111;&#x80BF;&#x7624;&#x5E72;&#x7EC6;&#x80DE; (TSCs) &#x53EF;&#x80FD;&#x662F;&#x4EBA;&#x53E3;&#x7684;&#x7EC6;&#x80DE;&#x8D1F;&#x8D23;&#x7EF4;&#x62A4;&#x4E0E;&#x590D;&#x53D1;&#x7684;&#x591A;&#x5F62;&#x6027;&#x80F6;&#x8D28;&#x6BCD;&#x7EC6;&#x80DE;&#x7624; (GBM)&#x3002;&#x8FD9;&#x9879;&#x7814;&#x7A76;&#x7684;&#x76EE;&#x7684;&#x662F;&#x4F18;&#x5316;&#x5176;&#x968F;&#x540E;&#x8F6C;&#x67D3;&#x6216;&#x8F6C;&#x5BFC;&#x751F;&#x6210; TSCs &#x91CD;&#x73B0;&#x534F;&#x8BAE;&#x3002;&#x75C5;&#x4EBA; GBMs &#x88AB;&#x9176;&#x548C;&#x673A;&#x68B0;&#x5206;&#x79BB;&#x548C; resuspended &#x5728;&#x9002;&#x5F53;&#x7684;&#x5A92;&#x4F53;&#x548C;&#x5206;&#x6790;&#xFF0C;&#x4EE5;&#x786E;&#x4FDD;&#x5B83;&#x4EEC;&#x7B26;&#x5408;&#x6807;&#x51C6;&#x5E72;&#x7EC6;&#x80DE;&#x80BF;&#x7624;&#x9886;&#x57DF;&#x4E86;&#x3002;&#x8FD9;&#x4E9B;&#x7EC6;&#x80DE;&#x7136;&#x540E;&#x88AB;&#x8D28;&#x7C92;&#x8F6C;&#x67D3;&#x6216;&#x67D3;&#x75C5;&#x6BD2;&#x8F7D;&#x4F53;&#x4ECB;&#x7ECD;&#x4EE5;&#x524D;&#x7F3A;&#x5E2D;&#x57FA;&#x56E0;&#xFF0C;&#x7136;&#x540E;&#x5141;&#x8BB8;&#x5411;&#x7A97;&#x4F53;&#x80BF;&#x7624;&#x9886;&#x57DF;&#x3002;&#x80BF;&#x7624;&#x9886;&#x57DF;&#x662F;&#x4ECE;&#x65E0;&#x6C61;&#x67D3;&#x7684;&#x75C5;&#x4EBA; GBMs &#x751F;&#x6210;&#x7684;&#x3002;&#x8FD9;&#x4E9B;&#x5355;&#x5143;&#x683C;&#x4F5C;&#x4E3A;&#x5E72;&#x7EC6;&#x80DE;&#xFF0C;&#x5305;&#x62EC; multipotentiality &#x548C;&#x81EA;&#x6211;&#x66F4;&#x65B0;&#x6EE1;&#x8DB3;&#x4E25;&#x683C;&#x7684;&#x6807;&#x51C6;&#x3002;&#x9AD8;&#x6548;&#x7387;&#x8F6C;&#x67D3;&#x3001; &#x8F6C;&#x5BFC;&#x80BF;&#x7624;&#x9886;&#x57DF;&#x662F;&#x53EF;&#x80FD;&#x7684;&#x5373;&#x4F7F;&#x662F;&#x5728;&#x7403;&#x7684;&#x6838;&#x5FC3;&#x3002;&#x8FD9;&#x5141;&#x8BB8;&#x4E3A;&#x65B0;&#x57FA;&#x56E0;&#x7B80;&#x4ECB; Tsc&#xFF0C;&#x8367;&#x5149;&#x663E;&#x5FAE;&#x955C;&#x548C;&#x5370;&#x8FF9;&#x5206;&#x6790;&#x53EF;&#x89C1;&#x4E00;&#x6591;&#x3002;&#x8FD9;&#x9879;&#x7814;&#x7A76;&#x662F;&#x75C5;&#x4EBA;&#x7684;&#x6307;&#x5357;&#xFF0C;&#x4EE5;&#x4F18;&#x5316;&#x751F;&#x6210;&#x6D3E;&#x751F;&#x5927;&#x7D2B;&#x8346;&#x52CB;&#x8D24;&#x80BF;&#x7624;&#x9886;&#x57DF;&#x6CA1;&#x6709;&#x7EA2;&#x7EC6;&#x80DE;&#x548C;&#x6B7B;&#x7EC6;&#x80DE;&#x6C61;&#x67D3;&#x3002;&#x5927;&#x7D2B;&#x8346;&#x52CB;&#x8D24; TSCs &#x80BF;&#x7624;&#x9886;&#x57DF;&#x5185;&#x53EF;&#x4EE5;&#x8F7B;&#x677E;&#x8D28;&#x7C92;&#x8F6C;&#x67D3;&#x6216;&#x67D3;&#x75C5;&#x6BD2;&#x3002;&#x8FD9;&#x662F;&#x91CD;&#x8981;&#x4ECE;&#x6CBB;&#x7597;&#x7684;&#x89D2;&#x5EA6;&#x6765;&#x770B;&#xFF0C;&#x5982;&#x679C;&#x57FA;&#x56E0;&#x7F6E;&#x6362;&#x662F;&#x8981;&#x6210;&#x529F;&#x5730;&#x53D6;&#x4EE3;&#x5927;&#x7D2B;&#x8346;&#x52CB;&#x8D24;&#x7EA7;&#x6570;&#x4E2D;&#x4E22;&#x5931;&#x7684;&#x57FA;&#x56E0;&#x6216;&#x51FB;&#x5012;&#x6216;&#x6C89;&#x9ED8;&#x662F; over-expressed &#x5728;&#x6076;&#x6027;&#x8111;&#x80BF;&#x7624;&#x7684;&#x57FA;&#x56E0;&#x3002;

&#x591A;&#x5F62;&#x6027;&#x80F6;&#x8D28;&#x6BCD;&#x7EC6;&#x80DE;&#x7624;&#x5E72;&#x7EC6;&#x80DE;&#x5206;&#x79BB;&#x3001; &#x8F6C;&#x67D3;&#x3001; &#x8F6C;&#x5BFC;&#x7684;&#x4F18;&#x5316;&#x3002;

It has been postulated that brain tumor stem cells (TSCs) may be the population of cells responsible for the maintenance and recurrence of glioblastoma multiforme (GBM). The purpose of this study was to optimize a reproducible protocol for generating TSCs for their subsequent transfection or transduction. Patient GBMs were enzymatically and mechanically dissociated and tumor spheres were resuspended in appropriate media and analyzed to ensure they met stem cell criteria. These cells were then transfected with a plasmid or transduced with a viral vector to introduce a previously absent gene and then allowed to form tumor spheres. Tumor spheres were generated from patient GBMs without contamination. These cells met stringent criteria as stem cells, including multipotentiality and self-renewal. High efficiency transfection and transduction of tumor spheres was possible, even at the core of the sphere. This allowed for the introduction of new genes to the TSCs, as evidenced by fluorescent microscopy and Western blot analysis. This study is a guide to optimize the generation of patient derived GBM tumor spheres without RBC and dead cell contamination. GBM TSCs within tumor spheres can easily be transfected with plasmids or transduced with a virus. This is important from a therapeutic perspective if gene replacement is to be successful in replacing genes lost in GBM progression or to knock down or silence genes that are over-expressed in malignant brain tumors.

Optimization of glioblastoma multiforme stem cell isolation, transfection, and transduction.

Il a &eacute;t&eacute; postul&eacute; que les cellules souches du cerveau tumeur (TSCs) peut &ecirc;tre la population des cellules responsables de la maintenance et la r&eacute;currence du glioblastome multiforme (GBM). Cette &eacute;tude visait &agrave; optimiser un protocole reproductible pour g&eacute;n&eacute;rer des TSCs pour leur transfection ult&eacute;rieure ou la transduction. GBM patients ont &eacute;t&eacute; dissoci&eacute;es enzymatiquement et m&eacute;caniquement et les sph&egrave;res de la tumeur ont &eacute;t&eacute; remis en suspension dans un milieu appropri&eacute; et analys&eacute;es afin de garantir qu&#39;ils r&eacute;pondent &agrave; des crit&egrave;res de cellules souches. Ces cellules ont &eacute;t&eacute; ensuite transfected avec un plasmide ou transduites avec un vecteur viral d&#39;introduire un pr&eacute;c&eacute;demment absente de g&egrave;ne et le droit de sph&egrave;res de tumeur de forme. Sph&egrave;res de la tumeur ont &eacute;t&eacute; g&eacute;n&eacute;r&eacute;s &agrave; partir de patient GBM sans contamination. Ces cellules correspond aux crit&egrave;res rigoureuses comme des cellules souches, y compris multipotentiality et auto-renouvellement. Transfection de rendement &eacute;lev&eacute; et la transduction des sph&egrave;res de la tumeur n&#39;a pas &eacute;t&eacute; possible, m&ecirc;me au c&oelig;ur de la sph&egrave;re. Ceci a permis l&#39;introduction de nouveaux g&egrave;nes aux TSCs, comme en t&eacute;moignent la microscopie fluorescente et analyse par Western blot. Cette &eacute;tude est un guide pour optimiser la g&eacute;n&eacute;ration de patient provenant de sph&egrave;res tumeur GBM sans RBC et contamination de la cellule morte. GBM TSCs dans les sph&egrave;res de la tumeur peut facilement &ecirc;tre transfect&eacute;es avec les plasmides ou transduites avec un virus. C&#39;est important du point de vue th&eacute;rapeutique si le remplacement de g&egrave;ne est de r&eacute;ussir dans le remplacement de g&egrave;nes, perdus dans la progression de GBM ou &agrave; abattre ou le silence des g&egrave;nes qui sont surexprim&eacute;es dans les tumeurs malignes du cerveau.

Optimisation de l&#39;isolement de cellules souches glioblastome multiforme, transfection et transduction.

Es hat postuliert worden, dass Gehirn Tumor-Stammzellen (TSCs) m&ouml;glicherweise die Bev&ouml;lkerung der Zellen verantwortlich f&uuml;r die Instandhaltung und Wiederauftreten des Glioblastoma Multiforme (GBM). Der Zweck dieser Studie war es, eine reproduzierbare Protokoll zum Generieren von TSCs f&uuml;r ihre sp&auml;tere Transfektion oder Transduktion zu optimieren. Patienten GBMs wurden enzymatisch und mechanisch getrennt und Tumor-Kugeln waren in entsprechende Medium resuspendiert und analysiert, um sicherzustellen, dass sie Stammzell-Kriterien erf&uuml;llen. Diese Zellen wurden dann die mit einem transfected oder septischen mit einem viralen Vektor zur Einf&uuml;hrung einer zuvor abwesend gen und dann erlaubt, Form Tumor Sph&auml;ren. Tumor-Kugeln wurden vom Patienten GBMs ohne Verunreinigung erzeugt. Diese Zellen erf&uuml;llt die strenge Kriterien wie Stammzellen, einschlie&szlig;lich Multipotentiality und Selbsterneuerung. Hocheffiziente Transfection und Transduktion von Tumor-Kugeln waren m&ouml;glich, sogar im Mittelpunkt der Kugel. Dies erm&ouml;glichte die Einf&uuml;hrung der neuen Gene in die TSCs wie Fluoreszenz-Mikroskopie und Western-Blot-Analyse belegt. Diese Studie ist ein Leitfaden f&uuml;r die Generation der Patienten zu optimieren GBM Tumor Kugeln ohne RBC und tote Zelle Kontamination. GBM TSCs im Tumor-Kugeln k&ouml;nnen problemlos die mit Plasmiden transfected oder transduzierten mit einem Virus. Dies ist wichtig f&uuml;r therapeutische, wenn Gen-Ersatz ist erfolgreich in Gene verloren in GBM Fortschreiten zu ersetzen oder das umwerfen oder Gene, die over-expressed in b&ouml;sartige Hirntumoren sind, zum Schweigen zu bringen.

Optimierung von Glioblastoma Multiforme Stammzellen isoliert, Transfektion und Transduktion.

&Egrave; stato ipotizzato che il cervello tumore cellule staminali (TSCs) pu&ograve; essere la popolazione di cellule responsabili per la manutenzione e la recidiva di glioblastoma multiforme (GBM). Lo scopo di questo studio era quello di ottimizzare un protocollo riproducibile per la generazione di TSCs per la loro successiva transfezione o trasduzione. Paziente GBMs erano enzimaticamente e meccanicamente dissociato e tumore sfere sono state risospese nel supporto appropriato e analizzate per assicurare che hanno incontrato i criteri delle cellule staminali. Queste cellule sono state poi transfected con un plasmide o transduced con un vettore virale per introdurre un precedentemente assente gene e quindi autorizzati a sfere di forma tumorale. Sfere di tumore sono state generate dal paziente GBMs senza contaminazione. Queste cellule si incontrarono criteri rigorosi come le cellule staminali, tra cui multipotentiality e auto-rinnovamento. Transfezione di alta efficienza e trasduzione delle sfere di tumore era possibile, anche al centro della sfera. Questo ha permesso l&#39;introduzione di nuovi geni per il TSCs, come evidenziato da microscopia di fluorescenza e analisi Western blot. Questo studio &egrave; una guida per ottimizzare la generazione di paziente derivato sfere tumore GBM senza RBC e contaminazione di cellule morte. GBM TSCs entro sfere di tumore pu&ograve; facilmente essere transfected con plasmidi o transduced con un virus. Questo &egrave; importante dal punto di vista terapeutico se sostituzione genica &egrave; per avere successo in sostituzione di geni perduti in progressione GBM o per abbattere o silenziare i geni che sono sovraespresso in tumori maligni cerebrali.

Ottimizzazione dell&#39;isolamento delle cellule staminali di glioblastoma multiforme, transfezione e trasduzione.

&#x8133;&#x816B;&#x760D;&#x5E79;&#x7D30;&#x80DE; (Tsc) &#x7D30;&#x80DE;&#x3092;&#x7DAD;&#x6301;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E;&#x8CAC;&#x4EFB;&#x306E;&#x4EBA;&#x53E3;&#x3068;&#x81A0; &#xFF08;GBM&#xFF09; &#x306E;&#x518D;&#x767A;&#x3092;&#x304B;&#x3082;&#x3057;&#x308C;&#x306A;&#x3044;&#x3053;&#x3068;&#x304C;&#x4EEE;&#x5B9A;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x672C;&#x7814;&#x7A76;&#x306E;&#x76EE;&#x7684;&#x306F;&#x3001;&#x5F8C;&#x7D9A;&#x306E;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30D5;&#x30A7;&#x30AF;&#x30B7;&#x30E7;&#x30F3;&#x3084;&#x4F1D;&#x9054;&#x7528; Tsc &#x3092;&#x751F;&#x6210;&#x3059;&#x308B;&#x518D;&#x73FE;&#x6027;&#x306E;&#x3042;&#x308B;&#x30D7;&#x30ED;&#x30C8;&#x30B3;&#x30EB;&#x3092;&#x6700;&#x9069;&#x5316;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x3060;&#x3063;&#x305F;&#x3002;&#x60A3;&#x8005; GBMs &#x9175;&#x7D20;&#x7684;&#x304A;&#x3088;&#x3073;&#x6A5F;&#x68B0;&#x7684;&#x306B;&#x89E3;&#x96E2;&#x3055;&#x308C;&#x305F;&#x3001;&#x816B;&#x760D;&#x7403;&#x3067;&#x9069;&#x5207;&#x306A;&#x30E1;&#x30C7;&#x30A3;&#x30A2;&#x3092;&#x518D;&#x505C;&#x6B62;&#x3057;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x57FA;&#x6E96;&#x3092;&#x6E80;&#x305F;&#x3057;&#x3066;&#x78BA;&#x4FDD;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x5206;&#x6790;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x7D30;&#x80DE;&#x306E;&#x3068;&#x30D7;&#x30E9;&#x30B9;&#x30DF;&#x30C9;&#x3092;&#x5C0E;&#x5165;&#x307E;&#x305F;&#x306F;&#x5C0E;&#x5165;&#x3059;&#x308B;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x6027;&#x30D9;&#x30AF;&#x30BF;&#x30FC;&#x304C;&#x5C0E;&#x5165;&#x3055;&#x308C;&#x305F; [&#x3001;&#x907A;&#x4F1D;&#x5B50;&#x4EE5;&#x524D;&#x4E0D;&#x5728;&#x3057;&#x3001;&#x30D5;&#x30A9;&#x30FC;&#x30E0;&#x816B;&#x760D;&#x7403;&#x3059;&#x308B;&#x3092;&#x8A31;&#x53EF;&#x3057;&#x307E;&#x3059;&#x3002;&#x7403;&#x816B;&#x760D;&#x60A3;&#x8005; GBMs &#x6C5A;&#x67D3;&#x306A;&#x3057;&#x304B;&#x3089;&#x751F;&#x6210;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x7D30;&#x80DE;&#x306F;&#x53B3;&#x3057;&#x3044;&#x57FA;&#x6E96;&#x30A4;&#x30E2;&#x30EA;&#x3068;&#x81EA;&#x5DF1;&#x8907;&#x88FD;&#x306A;&#x3069;&#x3092;&#x542B;&#x3080;&#x3001;&#x5E79;&#x7D30;&#x80DE;&#x3068;&#x3057;&#x3066;&#x4F1A;&#x3063;&#x305F;&#x3002;&#x9AD8;&#x52B9;&#x7387;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30D5;&#x30A7;&#x30AF;&#x30B7;&#x30E7;&#x30F3;&#x3068;&#x4F1D;&#x9054;&#x816B;&#x760D;&#x7403;&#x306E;&#x7403;&#x306E;&#x30B3;&#x30A2;&#x3067;&#x3055;&#x3048;&#x53EF;&#x80FD;&#x3060;&#x3063;&#x305F;&#x3002;&#x3053;&#x308C;&#x306F;&#x3001;&#x86CD;&#x5149;&#x9855;&#x5FAE;&#x93E1;&#x304A;&#x3088;&#x3073;&#x897F;&#x90E8;&#x306E;&#x3057;&#x307F;&#x306E;&#x5206;&#x6790;&#x306B;&#x3088;&#x3063;&#x3066;&#x8A3C;&#x660E;&#x3055;&#x308C;&#x308B;&#x3088;&#x3046;&#x65B0;&#x3057;&#x3044;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x5C0E;&#x5165;&#x3001;&#x5206;&#x6975;&#x3059;&#x308B;&#x305F;&#x3081;&#x8A31;&#x53EF;&#x3002;&#x3053;&#x306E;&#x7814;&#x7A76;&#x306F;&#x3001;&#x60A3;&#x8005;&#x306E;&#x751F;&#x6210;&#x3092;&#x6700;&#x9069;&#x5316;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E;&#x30AC;&#x30A4;&#x30C9; GBM &#x816B;&#x760D;&#x7403; RBC &#x3068;&#x6B7B;&#x3093;&#x3060;&#x7D30;&#x80DE;&#x6C5A;&#x67D3;&#x306A;&#x3057;&#x6D3E;&#x751F;&#x3067;&#x3059;&#x3002;GBM Tsc &#x5185;&#x816B;&#x760D;&#x7403;&#x3067;&#x7C21;&#x5358;&#x306B;&#x30D7;&#x30E9;&#x30B9;&#x30DF;&#x30C9;&#x3092;&#x5C0E;&#x5165;&#x3057;&#x305F;&#x308A;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x3092;&#x5C0E;&#x5165;&#x3057;&#x307E;&#x3059;&#x3002;&#x907A;&#x4F1D;&#x5B50;&#x7F6E;&#x63DB; GBM &#x9032;&#x884C;&#x3067;&#x5931;&#x308F;&#x308C;&#x305F;&#x907A;&#x4F1D;&#x5B50;&#x3092;&#x4EA4;&#x63DB;&#x3059;&#x308B;&#x3053;&#x3068;&#x3067;&#x6210;&#x529F;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x30C0;&#x30A6;&#x30F3; &#x30CE;&#x30C3;&#x30AF;&#x307E;&#x305F;&#x306F;&#x60AA;&#x6027;&#x8133;&#x816B;&#x760D;&#x306B;&#x5BFE;&#x3059;&#x308B; over-expressed &#x306E;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x6C88;&#x9ED9;&#x306B;&#x3042;&#x308B;&#x5834;&#x5408;&#x3001;&#x3053;&#x308C;&#x306F;&#x6CBB;&#x7642;&#x306E;&#x89B3;&#x70B9;&#x304B;&#x3089;&#x91CD;&#x8981;&#x3067;&#x3059;&#x3002;

&#x81A0;&#x82BD;&#x816B;&#x306E;&#x591A;&#x5F62;&#x306E;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x9694;&#x96E2;&#x3001;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30D5;&#x30A7;&#x30AF;&#x30B7;&#x30E7;&#x30F3;&#x3001;&#x304A;&#x3088;&#x3073;&#x4F1D;&#x9054;&#x306E;&#x6700;&#x9069;&#x5316;&#x3002;

&#xADF8;&#xAC83;&#xC740; &#xB1CC; &#xC885;&#xC591; &#xC904;&#xAE30; &#xC138;&#xD3EC; (Tsc) &#xC140; &#xC720;&#xC9C0; &#xBCF4;&#xC218;&#xC5D0; &#xB300; &#xD55C; &#xCC45;&#xC784;&#xC758; &#xC778;&#xAD6C;&#xC640; glioblastoma &#xB2D8; (GBM)&#xC758; &#xC7AC;&#xBC1C; &#xB420; &#xC218; postulated &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC774; &#xC5F0;&#xAD6C;&#xC758; &#xBAA9;&#xC801;&#xC740; &#xADF8;&#xB4E4;&#xC758; &#xD6C4;&#xC18D; transfection &#xB610;&#xB294; &#xBCC0;&#xD658;&#xC5D0; &#xB300; &#xD55C; Tsc&#xB97C; &#xC0DD;&#xC131; &#xD558;&#xAE30; &#xC704;&#xD55C; &#xC7AC;&#xD604; &#xAC00;&#xB2A5;&#xD55C; &#xD504;&#xB85C;&#xD1A0;&#xCF5C;&#xC744; &#xCD5C;&#xC801;&#xD654; &#xD588;&#xB2E4;. &#xD658;&#xC790; GBMs &#xD588;&#xB2E4; enzymatically &#xADF8;&#xB9AC;&#xACE0; &#xAE30;&#xACC4;&#xC801;&#xC73C;&#xB85C; &#xC5F0;&#xACB0; &#xD558; &#xACE0; &#xC885;&#xC591; &#xBD84;&#xC57C;&#xC5D0;&#xC11C; &#xC801;&#xC808; &#xD55C; &#xBBF8;&#xB514;&#xC5B4; resuspended &#xB418;&#xC5C8;&#xACE0; &#xADF8;&#xB4E4;&#xC740; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xC870;&#xAC74; &#xCDA9;&#xC871; &#xBD84;&#xC11D;. &#xC774; &#xC138;&#xD3EC;&#xB294; &#xD50C;&#xB77C;&#xC2A4; &#xBBF8;&#xB4DC;&#xC640; &#xD398; &#xB610;&#xB294; &#xC18C;&#xAC1C; &#xD558;&#xB294; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xC131; &#xBCA1;&#xD130;&#xC640; &#xBD88;&#xB9AC;&#xACE0; &#xB2E4;&#xC74C; &#xD55C; &#xC9C4; &#xC774;&#xC804;&#xC5D0; &#xACB0; &#xC11D; &#xD558; &#xACE0; &#xB2E4;&#xC74C; &#xC591;&#xC2DD; &#xC885;&#xC591; &#xBD84;&#xC57C; &#xD5C8;&#xC6A9;. &#xC885;&#xC591; &#xBD84;&#xC57C;&#xB294; &#xC624;&#xC5FC; &#xC5C6;&#xC774; &#xD658;&#xC790; Gbms&#xC5D0;&#xC11C;&#xC5D0;&#xC11C; &#xC0DD;&#xC131; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC774;&#xB7EC;&#xD55C; &#xC140; &#xC904;&#xAE30; &#xC138;&#xD3EC;, multipotentiality &#xB4F1; &#xC790;&#xCCB4; &#xAC31;&#xC2E0;&#xC73C;&#xB85C; &#xC5C4;&#xACA9;&#xD55C; &#xC870;&#xAC74;. &#xACE0;&#xD6A8;&#xC728; transfection &#xBC0F; &#xC885;&#xC591; &#xBD84;&#xC57C;&#xC758; &#xBCC0;&#xD658; &#xBD84;&#xC57C;&#xC758; &#xD575;&#xC2EC;&#xC5D0;&#xB3C4; &#xAC00;&#xB2A5; &#xD588;&#xB2E4;. &#xD615;&#xAD11; &#xD604;&#xBBF8;&#xACBD; &#xAC80;&#xC0AC; &#xBC95; &#xBC0F; &#xC11C;&#xCABD; &#xC624; &#xC810; &#xBD84;&#xC11D;&#xC5D0; &#xC758;&#xD574; &#xC785;&#xC99D;&#xC774; Tsc, &#xC0C8;&#xB85C;&#xC6B4; &#xC720;&#xC804;&#xC790;&#xC758; &#xB3C4;&#xC785;&#xC5D0; &#xB300; &#xD55C; &#xD5C8;&#xC6A9;. &#xC774; &#xC5F0;&#xAD6C;&#xB294; &#xD658;&#xC790;&#xC758; &#xC0DD;&#xC131;&#xC744; &#xCD5C;&#xC801;&#xD654; &#xD558;&#xAE30; &#xAC00;&#xC774;&#xB4DC; &#xD30C;&#xC0DD; &#xB41C; RBC&#xC640; &#xC8FD;&#xC740; &#xC138;&#xD3EC; &#xC624;&#xC5FC; &#xC5C6;&#xC774; GBM &#xC885;&#xC591; &#xBD84;&#xC57C;. GBM Tsc &#xC885;&#xC591; &#xBD84;&#xC57C; &#xB0B4;&#xC5D0;&#xC11C; &#xD50C;&#xB77C;&#xC2A4; &#xBBF8;&#xB4DC;&#xC640; &#xD398; &#xB610;&#xB294; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xBD88;&#xB9AC;&#xACE0; &#xC27D;&#xAC8C; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC720;&#xC804;&#xC790; &#xAD50;&#xCCB4; &#xC131;&#xACF5; &#xC720;&#xC804;&#xC790; GBM &#xC9C4;&#xD589;&#xC5D0;&#xC11C; &#xAE38;&#xC744; &#xC783; &#xC5C8; &#xAD50;&#xCCB4;&#xC5D0; &#xD5C8;&#xBB3C;&#xACE0; &#xB610;&#xB294; &#xC545;&#xC131; &#xB1CC;&#xC885;&#xC591;&#xC5D0; over-expressed &#xC720;&#xC804;&#xC790; &#xCE68;&#xBB35; &#xD558;&#xB294; &#xACBD;&#xC6B0; &#xCE58;&#xB8CC; &#xC801; &#xAD00;&#xC810;&#xC5D0;&#xC11C; &#xC911;&#xC694; &#xD558;&#xB2E4;.

Glioblastoma &#xB2D8; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xBD84;&#xB9AC;, transfection, &#xC804;&#xB2EC;&#xC758; &#xCD5C;&#xC801;&#xD654;.

Se ha postulado que c&eacute;lulas de tumor de cerebro (TSC) puede ser la poblaci&oacute;n de c&eacute;lulas responsables del mantenimiento y la recurrencia de glioblastoma multiforme (GBM). El prop&oacute;sito de este estudio fue optimizar un protocolo reproducible para generar TSC para su posterior transfecci&oacute;n o transducci&oacute;n. GBMs pacientes fueron enzim&aacute;tico y mec&aacute;nicamente disociados y esferas del tumor se resuspendi&oacute; en medios apropiados y se analizaron para cumplir los criterios de la c&eacute;lula de v&aacute;stago. Estas c&eacute;lulas entonces fueron transfectadas con un pl&aacute;smido o transduced con un vector viral para introducir un ausente previamente gene y luego permiti&oacute; a esferas de tumor de forma. Se obtuvieron esferas del tumor del paciente GBMs sin contaminaci&oacute;n. Estas c&eacute;lulas cumplen estrictos criterios como c&eacute;lulas, incluyendo multipotentiality y auto renovaci&oacute;n. Transfecci&oacute;n de alta eficiencia y transducci&oacute;n de esferas de tumor era posible, incluso en el centro de la esfera. Esto permiti&oacute; la introducci&oacute;n de nuevos genes en el TSC, evidenciada por microscop&iacute;a fluorescente y an&aacute;lisis de Western blot. Este estudio es una gu&iacute;a para optimizar la generaci&oacute;n de paciente derivado esferas de tumor GBM sin RBC y contaminaci&oacute;n de c&eacute;lulas muertas. GBM TSC en &aacute;mbitos del tumor puede ser f&aacute;cilmente transfectada con pl&aacute;smidos o transduced con un virus. Esto es importante desde una perspectiva terap&eacute;utica si el reemplazo de genes es para tener &eacute;xito en el reemplazo de genes perdidos en progresi&oacute;n de GBM o derribar o silenciar genes que est&aacute;n sobreexpresados en tumores cerebrales malignos.

Optimizaci&oacute;n de aislamiento de c&eacute;lulas de glioblastoma multiforme, transfecci&oacute;n y transducci&oacute;n de se&ntilde;ales.

A large number of studies have demonstrated that the nucleus accumbens (NAC) is a critical site in the neuronal circuits controlling reward responses, motivation, and mood, but the neuronal cell type(s) underlying these processes are not yet known. Identification of the neuronal cell types that regulate depression-like states will guide us in understanding the biological basis of mood and its regulation by diseases like major depressive disorder. Taking advantage of recent findings demonstrating that the serotonin receptor chaperone, p11, is an important molecular regulator of depression-like states, here we identify cholinergic interneurons (CINs) as a primary site of action for p11 in the NAC. Depression-like behavior is observed in mice after decrease of p11 levels in NAC CINs. This phenotype is recapitulated by silencing neuronal transmission in these cells, demonstrating that accumbal cholinergic neuronal activity regulates depression-like behaviors and suggesting that accumbal CIN activity is crucial for the regulation of mood and motivation.

Cholinergic interneurons in the nucleus accumbens regulate depression-like behavior.

The high rate of comorbidity between depression and cocaine addiction suggests shared molecular mechanisms and anatomical pathways. Limbic structures, such as the nucleus accumbens (NAc), play a crucial role in both disorders, yet how different cell types within these structures contribute to the pathogenesis remains elusive. Downregulation of p11 (S100A10), specifically in the NAc, elicits depressive-like behaviors in mice, but its role in drug addiction is unknown.

Cell-type specific expression of p11 controls cocaine reward.

The comorbidity of depression and cocaine addiction suggests shared mechanisms and anatomical pathways. Specifically, the limbic structures, such as the nucleus accumbens (NAc), play a crucial role in both disorders. P11 (S100A10) is a promising target for manipulating depression and addiction in mice. We summarized the recent genetic and viral strategies used to determine how the titration of p11 levels within the NAc affects hedonic behavior and cocaine reward learning in mice. In particular, p11 in the ChAT+ cells or DRD1+ MSN of the NAc, controls depressive-like behavior or cocaine reward, respectively. Treatments to counter maladaptation of p11 levels in the NAc could provide novel therapeutic opportunities for depression and cocaine addiction in humans.

[Depression and addiction comorbidity: towards a common molecular target?].

Well-defined as signaling hormones for the programming of cell type-specific and context-dependent gene expression signatures, glucocorticoids control experience-driven allostasis. One unifying model is that glucocorticoids help maintaining the integrity and plasticity of cellular networks in changing environments through the mobilization of cellular energy stores, profiling of gene expression, and changes in the electrical and morphological properties of cells. The nucleus is their primary site of action, yet recent discoveries point to additional gene transcription-independent functions at the plasma membrane of neuronal synapses. Glucocorticoids are secreted factors that reflect intrinsically the changes coming from the external world, temporally and regionally, during development and adulthood. In this review, we will enumerate the properties and signaling attributes of glucocorticoids and their receptors that characterize them as allostatic modulators. The molecular mechanisms used to support their role at the synapse will be highlighted.

Molecular Biology of Glucocorticoid Signaling.

Neurotrophins and glucocorticoids are robust synaptic modifiers, and deregulation of their activities is a risk factor for developing stress-related disorders. Low levels of brain-derived neurotrophic factor (BDNF) increase the desensitization of glucocorticoid receptors (GR) and vulnerability to stress, whereas higher levels of BDNF facilitate GR-mediated signaling and the response to antidepressants. However, the molecular mechanism underlying neurotrophic-priming of GR function is poorly understood. Here we provide evidence that activation of a TrkB-MAPK pathway, when paired with the deactivation of a GR-protein phosphatase 5 pathway, resulted in sustained GR phosphorylation at BDNF-sensitive sites that is essential for the transcription of neuronal plasticity genes. Genetic strategies that disrupted GR phosphorylation or TrkB signaling in vivo impaired the neuroplasticity to chronic stress and the effects of the antidepressant fluoxetine. Our findings reveal that the coordinated actions of BDNF and glucocorticoids promote neuronal plasticity and that disruption in either pathway could set the stage for the development of stress-induced psychiatric diseases.

Neurotrophic-priming of glucocorticoid receptor signaling is essential for neuronal plasticity to stress and antidepressant treatment.

Clinical and experimental evidence point to a possible role of cerebrovascular dysfunction in Alzheimer's disease (AD). The 5xFAD mouse model of AD expresses human amyloid precursor protein and presenilin genes with mutations found in AD patients. It remains unknown whether amyloid deposition driven by these mutations is associated with cerebrovascular changes. 5xFAD and wild type mice (2 to 12months old; M2 to M12) were used. Thinned skull in vivo 2-photon microscopy was used to determine Aβ accumulation on leptomeningeal or superficial cortical vessels over time. Parenchymal microvascular damage was assessed using FITC-microangiography. Collagen-IV and CD31 were used to stain basal lamina and endothelial cells. Methoxy-XO4, Thioflavin-S or 6E10 were used to visualize Aβ accumulation in living mice or in fixed brain tissues. Positioning of reactive IBA1 microglia and GFAP astrocytes at the vasculature was rendered using confocal microscopy. Platelet-derived growth factor receptor beta (PDGFRβ) staining was used to visualize perivascular pericytes. In vivo 2-photon microscopy revealed Methoxy-XO4(+) amyloid perivascular deposits on leptomeningeal and penetrating cortical vessels in 5xFAD mice, typical of cerebral amyloid angiopathy (CAA). Amyloid deposits were visible in vivo at M3 and aggravated over time. Progressive microvascular damage was concomitant to parenchymal Aβ plaque accumulation in 5xFAD mice. Microvascular inflammation in 5xFAD mice presented with sporadic FITC-albumin leakages at M4 becoming more prevalent at M9 and M12. 3D colocalization showed inflammatory IBA1(+) microglia proximal to microvascular FITC-albumin leaks. The number of perivascular PDGFRβ(+) pericytes was significantly decreased at M4 in the fronto-parietal cortices, with a trend decrease observed in the other structures. At M9-M12, PDGFRβ(+) pericytes displayed hypertrophic perivascular ramifications contiguous to reactive microglia. Cerebral amyloid angiopathy and microvascular inflammation occur in 5xFAD mice concomitantly to parenchymal plaque deposition. The prospect of cerebrovascular pharmacology in AD is discussed.

Cerebrovascular pathology during the progression of experimental Alzheimer's disease.

Palmitoylation is involved in several neuropsychiatric and movement disorders for which a dysfunctional signaling of the dopamine D3 receptor (Drd3) is hypothesized. Computational modeling of Drd3's homologue, Drd2, has shed some light on the putative role of palmitoylation as a reversible switch for dopaminergic receptor signaling. Drd3 is presumed to be palmitoylated, based on sequence homology with Drd2, but the functional attributes afforded by Drd3 palmitoylation have not been studied. Since these receptors are major targets of antipsychotic and anti-Parkinsonian drugs, a better characterization of Drd3 signaling and posttranslational modifications, like palmitoylation, may improve the prospects for drug development. Using molecular dynamics simulations, we evaluated in silico how Drd3 palmitoylation could elicit significant remodeling of the C-terminal cytoplasmic domain to expose docking sites for signaling proteins. We tested this model in cellulo by using the interaction of Drd3 with the G-alpha interacting protein (GAIP) C terminus 1 (GIPC1) as a template. From a series of biochemical studies, live imaging, and analyses of mutant proteins, we propose that Drd3 palmitoylation acts as a molecular switch for Drd3-biased signaling via a GIPC1-dependent route, which is likely to affect the mode of action of antipsychotic drugs.

A GIPC1-Palmitate Switch Modulates Dopamine Drd3 Receptor Trafficking and Signaling.

Complications of dopamine replacement for Parkinson's disease (PD) can limit therapeutic options, leading to interest in identifying novel pathways that can be exploited to improve treatment. p11 (S100A10) is a cellular scaffold protein that binds to and potentiates the activity of various ion channels and neurotransmitter receptors. We have previously reported that p11 can influence ventral striatal function in models of depression and drug addiction, and thus we hypothesized that dorsal striatal p11 might mediate motor function and drug responses in parkinsonian mice. To focally inhibit p11 expression in the dorsal striatum, we injected an adeno-associated virus (AAV) vector producing a short hairpin RNA (AAV.sh.p11). This intervention reduced the impairment in motor function on forced tasks, such as rotarod and treadmill tests, caused by substantia nigra lesioning in mice. Measures of spontaneous movement and gait in an open-field test declined as expected in control lesioned mice, whereas AAV.sh.p11 mice remained at or near normal baseline. Mice with unilateral lesions were then challenged with l-dopa (levodopa) and various dopamine receptor agonists, and resulting rotational behaviors were significantly reduced after ipsilateral inhibition of dorsal striatal p11 expression. Finally, p11 knockdown in the dorsal striatum dramatically reduced l-dopa-induced abnormal involuntary movements compared with control mice. These data indicate that focal inhibition of p11 action in the dorsal striatum could be a promising PD therapeutic target to improve motor function while reducing l-dopa-induced dyskinesias.

Gene therapy blockade of dorsal striatal p11 improves motor function and dyskinesia in parkinsonian mice.

The brain evolved cellular mechanisms for adapting synaptic function to energy supply. This is particularly evident when homeostasis is challenged by stress. Signaling loops between the mitochondria and synapses scale neuronal connectivity with bioenergetics capacity. A biphasic "inverted U shape" response to the stress hormone glucocorticoids is demonstrated in mitochondria and at synapses, modulating neural plasticity and physiological responses. Low dose enhances neurotransmission, synaptic growth, mitochondrial functions, learning, and memory whereas chronic, higher doses produce inhibition of these functions. The range of physiological effects by stress and glucocorticoid depends on the dose, duration, and context at exposure. These criteria are met by neuronal activity and the circadian, stress-sensitive and ultradian, stress-insensitive modes of glucocorticoid secretion. A major hallmark of stress-related neuropsychiatric disorders is the disrupted glucocorticoid rhythms and tissue resistance to signaling with the glucocorticoid receptor (GR). GR resistance could result from the loss of context-dependent glucocorticoid signaling mediated by the downregulation of the activity-dependent neurotrophin BDNF. The coincidence of BDNF and GR signaling changes glucocorticoid signaling output with consequences on mitochondrial respiration efficiency, synaptic plasticity, and adaptive trajectories.

Linking Mitochondria to Synapses: New Insights for Stress-Related Neuropsychiatric Disorders.

Glucocorticoid actions are tailored to the organs and cells responding thanks to complex integration with ongoing signaling mediated by cytokines, hormones, neurotransmitters, and growth factors. Disruption of: (1) the amount of signaling molecules available locally; (2) the timing with other signaling pathways; (3) the post-translational modifications on glucocorticoid receptors; and (4) the receptors-interacting proteins within cellular organelles and functional compartments, can modify the sensitivity and efficacy of glucocorticoid responses with implications in physiology, diseases and treatments. Tissue sensitivity to glucocorticoids is sustained by multiple systems that do not operate in isolation. We take the example of the interplay between the glucocorticoid and brain-derived neurotrophic factor signaling pathways to deconstruct context-dependent glucocorticoid responses that play key roles in physiology, diseases and therapies.

Timing and crosstalk of glucocorticoid signaling with cytokines, neurotransmitters and growth factors.

Glucocorticoid resistance is a risk factor for Alzheimer's disease (AD). Molecular and cellular mechanisms of glucocorticoid resistance in the brain have remained unknown and are potential therapeutic targets. Phosphorylation of glucocorticoid receptors (GR) by brain-derived neurotrophic factor (BDNF) signaling integrates both pathways for remodeling synaptic structure and plasticity. The goal of this study is to test the role of the BDNF-dependent pathway on glucocorticoid signaling in a mouse model of glucocorticoid resistance. We report that deletion of GR phosphorylation at BDNF-responding sites and downstream signaling via the MAPK-phosphatase DUSP1 triggers tau phosphorylation and dendritic spine atrophy in mouse cortex. In human cortex, DUSP1 protein expression correlates with tau phosphorylation, synaptic defects and cognitive decline in subjects diagnosed with AD. These findings provide evidence for a causal role of BDNF-dependent GR signaling in tau neuropathology and indicate that DUSP1 is a potential target for therapeutic interventions.

Margarita Arango-Lievano

Inserm,
U1191,
Inserm, U1191

Institute of Functional Genomics

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Margarita Arango-Lievano

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Inserm,U1191,Inserm, U1191

Institute of Functional Genomics

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Inserm,
U1191,
Inserm, U1191