an-engulfment-assay-protocol-to-assess-interactions-between-cns

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and ...

Boston Children's Hospital, Harvard Medical School

The presynaptic regions of axons accumulate synaptic vesicles, active zone proteins and periactive zone proteins. However, the rules for orderly recruitment of presynaptic components are not well understood. We systematically examined molecular mechanisms of presynaptic development in egg-laying synapses of Caenorhabditis elegans, demonstrating that two scaffolding molecules, SYD-1 and SYD-2, have key roles in presynaptic assembly. SYD-2 (liprin-alpha) was previously shown to regulate the size and the shape of active zones. We now show that in syd-1 and syd-2 mutants, synaptic vesicles and numerous other presynaptic proteins fail to accumulate at presynaptic sites. SYD-1 and SYD-2 function cell-autonomously at presynaptic terminals, downstream of synaptic specificity molecule SYG-1. SYD-1 is likely to act upstream of SYD-2 to positively regulate its synaptic assembly activity. These data imply a hierarchical organization of presynaptic assembly, in which transmembrane specificity molecules initiate synaptogenesis by recruiting a few key scaffolding proteins, which in turn assemble other presynaptic components.

Hierarchical assembly of presynaptic components in defined C. elegans synapses.

&#x6781;&#x5316;&#x8D29;&#x5356;&#x5230;&#x795E;&#x7ECF;&#x8F74;&#x7A81;&#x548C;&#x6811;&#x7A81;&#x7A81;&#x89E6;&#x86CB;&#x767D;&#x662F;&#x795E;&#x7ECF;&#x5143;&#x529F;&#x80FD;&#x7684;&#x5173;&#x952E;&#x3002;&#x901A;&#x8FC7;&#x8F6C;&#x53D1;&#x7EBF;&#x866B;&#x4E2D;&#x7684;&#x9057;&#x4F20;&#x5206;&#x6790;&#xFF0C;&#x6211;&#x4EEC;&#x53D1;&#x73B0;&#x7EC6;&#x80DE;&#x5468;&#x671F;&#x86CB;&#x767D; (CYY-1&#xFF09; &#x548C;&#x7EC6;&#x80DE;&#x5468;&#x671F;&#x86CB;&#x767D;&#x4F9D;&#x8D56;&#x6027; Pctaire &#x6FC0;&#x9176; (PCT-1&#xFF09; &#x6709;&#x5FC5;&#x8981;&#x9488;&#x5BF9;&#x8F74;&#x7A81;&#x7684;&#x7A81;&#x89E6;&#x524D;&#x7EC4;&#x4EF6;&#x3002;&#x53E6;&#x4E00;&#x79CD;&#x7EC6;&#x80DE;&#x5468;&#x671F;&#x86CB;&#x767D;&#x4F9D;&#x8D56;&#x6027;&#x6FC0;&#x9176;&#x3001; CDK-5 &#x548C;&#x5176;&#x6D3B;&#x5316;&#x5242; p35 &#x884C;&#x4E8B;&#x5E76;&#x884C;&#x548C;&#x90E8;&#x5206;&#x5197;&#x4F59;&#x4E0E; CYY-1/PCT-1 &#x901A;&#x8DEF;&#x3002;&#x7A81;&#x89E6;&#x5C0F;&#x6CE1;&#x548C;&#x6D3B;&#x52A8;&#x533A;&#x57DF;&#x86CB;&#x767D;&#x5927;&#x591A;&#x662F; mislocalize &#x6811;&#x7A81; PCT 1 &#x548C; CDK 5 &#x7684;&#x8DEF;&#x5F84;&#x4E3A;&#x6709;&#x7F3A;&#x9677;&#x7684;&#x52A8;&#x7269;&#x3002;&#x4E0E;&#x4E0D;&#x540C;&#x7684;&#x9A71;&#x52A8;&#x86CB;&#x767D;-3 &#x7535;&#x673A;&#x3001; unc-104/Kif1a &#x7684;&#x7A81;&#x53D8;&#x79CD;&#xFF0C;cyy 1 cdk 5 &#x53CC;&#x7A81;&#x53D8;&#x4F53;&#x4E2D; anterogradely &#x79FB;&#x52A8;&#x7A81;&#x89E6;&#x56CA;&#x6CE1;&#x524D;&#x4F53; &#xFF08;SVPs&#xFF09; &#x52A8;&#x6001;&#x5F71;&#x50CF;&#x6240;&#x8BF4;&#x7684;&#x90A3;&#x6837;&#x6709;&#x6CA1;&#x6709;&#x51CF;&#x5C11;&#x3002;&#x76F8;&#x53CD;&#xFF0C;&#x9006;&#x884C;&#x79FB;&#x52A8; SVPs &#x7684;&#x4EBA;&#x6570;&#x662F;&#x5927;&#x5927;&#x589E;&#x52A0;&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x6B64;&#x5B9A;&#x4F4D;&#x7F3A;&#x9677;&#x88AB;&#x6291;&#x5236;&#x5E72;&#x6270;&#x7684;&#x9006;&#x884C;&#x7684;&#x9A6C;&#x8FBE;&#xFF0C;&#x590D;&#x6742;&#x7684;&#x7EC6;&#x80DE;&#x8D28;&#x808C;&#x3002;&#x56E0;&#x6B64;&#xFF0C;PCT 1 CDK 5 &#x9014;&#x5F84;&#x76F4;&#x63A5;&#x548C;&#x6781;&#x5316;&#x7684;&#x7A81;&#x89E6;&#x524D;&#x6210;&#x5206;&#x6291;&#x5236;&#x808C;&#x4ECB;&#x5BFC;&#x9006;&#x884C;&#x8FD0;&#x8F93;&#x548C;&#x8D29;&#x5356;&#x8BBE;&#x7F6E;&#x987A;&#x884C;&#x548C;&#x9006;&#x884C;&#x7535;&#x673A;&#x4E4B;&#x95F4;&#x7684;&#x5E73;&#x8861;&#x3002;

&#x4E24;&#x4E2A;&#x7EC6;&#x80DE;&#x5468;&#x671F;&#x86CB;&#x767D;&#x4F9D;&#x8D56;&#x6027;&#x6FC0;&#x9176;&#x901A;&#x8DEF;&#x5BF9;&#x7A81;&#x89E6;&#x524D;&#x6210;&#x5206;&#x7684;&#x6781;&#x5316;&#x8D29;&#x8FD0;&#x81F3;&#x5173;&#x91CD;&#x8981;&#x3002;

Polarized trafficking of synaptic proteins to axons and dendrites is crucial to neuronal function. Through forward genetic analysis in C. elegans, we identified a cyclin (CYY-1) and a cyclin-dependent Pctaire kinase (PCT-1) necessary for targeting presynaptic components to the axon. Another cyclin-dependent kinase, CDK-5, and its activator p35, act in parallel to and partially redundantly with the CYY-1/PCT-1 pathway. Synaptic vesicles and active zone proteins mostly mislocalize to dendrites in animals defective for both PCT-1 and CDK-5 pathways. Unlike the kinesin-3 motor, unc-104/Kif1a mutant, cyy-1 cdk-5 double mutants have no reduction in anterogradely moving synaptic vesicle precursors (SVPs) as observed by dynamic imaging. Instead, the number of retrogradely moving SVPs is dramatically increased. Furthermore, this mislocalization defect is suppressed by disrupting the retrograde motor, the cytoplasmic dynein complex. Thus, PCT-1 and CDK-5 pathways direct polarized trafficking of presynaptic components by inhibiting dynein-mediated retrograde transport and setting the balance between anterograde and retrograde motors.

Two cyclin-dependent kinase pathways are essential for polarized trafficking of presynaptic components.

Trafic polaris&eacute;e de prot&eacute;ines synaptiques d&#39;axones et les dendrites est crucial pour la fonction neuronale. Par analyse g&eacute;n&eacute;tique avant chez c. elegans, nous avons identifi&eacute; une cycline (CYY-1) et une kinase de Pctaire cycline-d&eacute;pendantes (PCT-1) n&eacute;cessaire pour cibler des composants &agrave; l&#39;axone pr&eacute;synaptiques. Une autre kinase cycline-d&eacute;pendante, CDK-5 et son activateur p35, agissent en parall&egrave;le et partiellement redondante avec la voie CYY 1/PCT-1. Les v&eacute;sicules synaptiques et les prot&eacute;ines de la zone active principalement mislocalize &agrave; dendrites chez les animaux d&eacute;fectueux pour les PCT-1 et CDK-5 voies. &Agrave; la diff&eacute;rence du moteur de kinesin-3, unc-104/Kif1a mutant, cyy-1 cdk-5 doubles mutants n&#39;ont aucune r&eacute;duction dans axonales se d&eacute;pla&ccedil;ant pr&eacute;curseurs (pression) de la v&eacute;sicule synaptique, tel qu&#39;observ&eacute; par imagerie dynamique. Au lieu de cela, le nombre des cas marqu&eacute;s en mouvement est consid&eacute;rablement augment&eacute;. En outre, ce d&eacute;faut localis&eacute;e est supprim&eacute; en perturbant le moteur r&eacute;trograde, la dyn&eacute;ine cytoplasmique complexe. Ainsi, PCT- 1et/CDK-5 voies directement polaris&eacute;e traite des composants pr&eacute;synaptiques en inhibant le transport r&eacute;trograde m&eacute;di&eacute;e par dyn&eacute;ine et en affectant l&#39;&eacute;quilibre entre ant&eacute;rograde et r&eacute;trogrades moteurs.

Deux voies de kinase cycline-d&eacute;pendante sont essentiels pour trafic polaris&eacute;e de composants pr&eacute;synaptiques.

Polarisierte Handel mit synaptischen Proteinen in Axonen und Dendriten ist neuronale Funktion entscheidend. Durch forward genetische Analyse in C. Elegans identifizierten wir ein cyclin (CYY-1) und ein cyclin-abh&auml;ngige Pctaire Kinase (PCT-1) notwendig f&uuml;r targeting pr&auml;synaptische Komponenten mit dem Axon. Ein weiterer cyclin-abh&auml;ngige Kinase CDK-5 und seine p35 Aktivator agieren parallel und teilweise redundant mit der CYY-1/PCT-1-Weg. Synaptische Vesikel und aktive Zone Proteine mislocalize meist an Dendriten bei Tieren f&uuml;r PCT-1 und CDK-5 Bahnen defekt. Im Gegensatz zu Kinesin-3 Motor Unc-104/Kif1a Mutant, Cyy-1 Cdk-5 doppelte-Mutanten haben keine Reduzierung in Anterogradely synaptische Vesikel Vorstufen (SVP) zu verschieben, wie durch dynamische Bildgebung beobachtet. Stattdessen wird die Anzahl der retrogradely Bewegung SVP drastisch erh&ouml;ht. Dar&uuml;ber hinaus ist dieser Mislocalization Fehler st&ouml;ren den retrograden Motor, die komplexe cytoplasmatische Dynein unterdr&uuml;ckt. Somit direkte PCT-1 und CDK-5 Wege, polarisierte Menschenhandel pr&auml;synaptischen Komponenten indem retrograde Verkehr Dynein-vermittelte Hemmung und das Gleichgewicht zwischen Anterograde und retrograde Motoren.

Zwei cyclin-abh&auml;ngige Kinase-Wege sind f&uuml;r polarisierte Menschenhandel pr&auml;synaptischen Komponenten.

Traffico polarizzata di proteine sinaptiche di assoni e dendriti &egrave; cruciale alla funzione neuronale. Attraverso l&#39;analisi genetica avanti in c. elegans, abbiamo identificato un cyclin (CYY-1) e una chinasi Pctaire chinasi ciclina-dipendenti (PCT-1) necessaria per il targeting presinaptico componenti per l&#39;assone. Un&#39;altra chinasi ciclina-dipendente CDK-5 e relativo attivatore p35, agiscono in parallelo e parzialmente ridondante con il pathway CYY-1/PCT-1. Vescicole sinaptiche e proteine zona attiva principalmente mislocalize di dendriti negli animali difettosi per vie PCT-1 e di CDK-5. A differenza del motore 3 chinesina, unc-104/Kif1a mutante, cyy-1 cdk-5 doppi mutanti non hanno nessuna riduzione in anterogradely lo spostamento precursori della vescicola sinaptica (SVP), come osservato da imaging dinamico. Invece, il numero di retrogradely movimento SVP &egrave; aumentato drammaticamente. Inoltre, questo difetto mislocalization &egrave; soppressa interrompendo il motore retrogrado, la dineina citoplasmatica complessa. Cos&igrave;, PCT-1 e CDK-5 vie diretta traffico polarizzata di componenti presinaptico inibendo il trasporto retrogrado dineina-mediata e impostando il bilanciamento tra anterograda e retrogradi motori.

Due percorsi di chinasi ciclina-dipendenti sono essenziali per traffico polarizzata di componenti presinaptiche.

&#x504F;&#x5149;&#x8EF8;&#x7D22;&#x3068;&#x6A39;&#x72B6;&#x7A81;&#x8D77;&#x306E;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x4EBA;&#x8EAB;&#x58F2;&#x8CB7;&#x306F;&#x795E;&#x7D4C;&#x7D30;&#x80DE;&#x306E;&#x6A5F;&#x80FD;&#x306B;&#x91CD;&#x5927;&#x3067;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x7DDA;&#x866B;&#x306E;&#x524D;&#x65B9;&#x306E;&#x907A;&#x4F1D;&#x306E;&#x5206;&#x6790;&#x3092;&#x901A;&#x3057;&#x3066;&#x3001;&#x6211;&#x3005; &#x306F;&#x30B5;&#x30A4;&#x30AF;&#x30EA;&#x30F3; (CYY 1&#xFF09; &#x3068;&#x306F;&#x30B5;&#x30A4;&#x30AF;&#x30EA;&#x30F3;&#x4F9D;&#x5B58;&#x6027; Pctaire &#x30AD;&#x30CA;&#x30FC;&#x30BC; (PCT 1) &#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x524D;&#x30B3;&#x30F3;&#x30DD;&#x30FC;&#x30CD;&#x30F3;&#x30C8;&#x8EF8;&#x7D22;&#x3078;&#x306E;&#x5BFE;&#x8C61;&#x5316;&#x306B;&#x5FC5;&#x8981;&#x306A;&#x8B58;&#x5225;&#x3002;&#x5225;&#x306E;&#x30B5;&#x30A4;&#x30AF;&#x30EA;&#x30F3;&#x4F9D;&#x5B58;&#x6027;&#x30AD;&#x30CA;&#x30FC;&#x30BC; CDK 5&#x3001;&#x305D;&#x306E;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D9;&#x30FC;&#x30BF;&#x30FC; p35 &#x3092;&#x4E26;&#x884C;&#x3057;&#x3066;&#x90E8;&#x5206;&#x7684;&#x306B;&#x91CD;&#x8907;&#x3057;&#x3066; CYY 1/PCT 1 &#x7D4C;&#x8DEF;&#x3068;&#x884C;&#x52D5;&#x3057;&#x307E;&#x3059;&#x3002;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x5C0F;&#x80DE;&#x304A;&#x3088;&#x3073;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;&#x306A;&#x30BE;&#x30FC;&#x30F3;&#x86CB;&#x767D;&#x8CEA;&#x4E3B;&#x52D5;&#x7269;&#x306E;&#x305F;&#x3081;&#x306E; PCT 1 &#x304A;&#x3088;&#x3073; CDK 5 &#x7D4C;&#x8DEF;&#x6B20;&#x9665;&#x306E;&#x6A39;&#x72B6;&#x7A81;&#x8D77;&#x306B; mislocalize &#x3057;&#x307E;&#x3059;&#x3002;&#x30AD;&#x30CD;&#x30B7;&#x30F3; 3 &#x30E2;&#x30FC;&#x30BF;&#x3068;&#x306F;&#x7570;&#x306A;&#x308A;&#x304C; unc-104/Kif1a &#x5909;&#x7570; cyy 1 cdk 5 &#x4E8C;&#x91CD;&#x7A81;&#x7136;&#x5909;&#x7570;&#x4F53;&#x3042;&#x308B;&#x524A;&#x6E1B;&#x30C0;&#x30A4;&#x30CA;&#x30DF;&#x30C3;&#x30AF; &#x30A4;&#x30E1;&#x30FC;&#x30B8;&#x30F3;&#x30B0;&#x306B;&#x3088;&#x308B;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x5C0F;&#x80DE;&#x306E;&#x524D;&#x99C6;&#x4F53; (SVPs) &#x3092;&#x79FB;&#x52D5; anterogradely &#x3067;&#x3002;&#x4EE3;&#x308F;&#x308A;&#x306B;&#x3001;&#x9006;&#x884C;&#x6027; SVPs &#x306E;&#x79FB;&#x52D5;&#x306E;&#x6570;&#x304C;&#x5927;&#x5E45;&#x306B;&#x5411;&#x4E0A;&#x3057;&#x307E;&#x3059;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#x3053;&#x306E;&#x30B5;&#x30C3;&#x30AB;&#x30FC;&#x30C9;&#x6B20;&#x9665;&#x9006;&#x884C;&#x30E2;&#x30FC;&#x30BF;&#x30FC;&#x3001;&#x7D30;&#x80DE;&#x8CEA;&#x30C0;&#x30A4;&#x30CB;&#x30F3;&#x8907;&#x5408;&#x3092;&#x6DF7;&#x4E71;&#x3055;&#x305B;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x6291;&#x5236;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;PCT 1 &#x304A;&#x3088;&#x3073; CDK 5 &#x7D4C;&#x8DEF;&#x306E;&#x76F4;&#x63A5;&#x504F;&#x5149;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x524D;&#x306E;&#x30B3;&#x30F3;&#x30DD;&#x30FC;&#x30CD;&#x30F3;&#x30C8;&#x306E;&#x30C0;&#x30A4;&#x30CB;&#x30F3;&#x3092;&#x4ECB;&#x3057;&#x305F;&#x9006;&#x884C;&#x6027;&#x8F38;&#x9001;&#x3092;&#x963B;&#x5BB3;&#x3057;&#x524D;&#x5411;&#x6027;&#x3068;&#x9006;&#x884C;&#x6027;&#x30E2;&#x30FC;&#x30BF;&#x30FC;&#x306E;&#x30D0;&#x30E9;&#x30F3;&#x30B9;&#x3092;&#x8A2D;&#x5B9A;&#x3059;&#x308B;&#x3068;&#x4EBA;&#x8EAB;&#x58F2;&#x8CB7;&#x3057;&#x307E;&#x3059;&#x3002;

2 &#x3064;&#x306E;&#x30B5;&#x30A4;&#x30AF;&#x30EA;&#x30F3;&#x4F9D;&#x5B58;&#x6027;&#x30AD;&#x30CA;&#x30FC;&#x30BC;&#x7D4C;&#x8DEF;&#x306F;&#x504F;&#x5149;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x524D;&#x306E;&#x30B3;&#x30F3;&#x30DD;&#x30FC;&#x30CD;&#x30F3;&#x30C8;&#x306E;&#x4EBA;&#x8EAB;&#x58F2;&#x8CB7;&#x306E;&#x305F;&#x3081;&#x4E0D;&#x53EF;&#x6B20;&#x3067;&#x3059;&#x3002;

&#xCD95; &#xC0AD;&#xACFC; dendrites&#xC744; &#xC2DC; &#xB0C5; &#xC2A4; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xD3B8;&#xAD11; &#xBC00;&#xB9E4; &#xC2E0;&#xACBD; &#xAE30;&#xB2A5;&#xC5D0; &#xACB0;&#xC815;&#xC801; &#xC774;&#xB2E4;. C. elegans&#xC5D0; &#xC55E;&#xC73C;&#xB85C; &#xC720;&#xC804;&#xC790; &#xBD84;&#xC11D;&#xC744; &#xD1B5;&#xD574; cyclin (CYY-1)&#xC640; &#xF06C; &#xC885;&#xC18D; Pctaire &#xD0A4; (%1) &#xCD95; &#xC0AD;&#xC744; presynaptic &#xAD6C;&#xC131; &#xC694;&#xC18C;&#xB97C; &#xB300;&#xC0C1;&#xC73C;&#xB85C; &#xD558;&#xB294; &#xB370; &#xD544;&#xC694;&#xD55C; &#xC2DD;&#xBCC4;. &#xB2E4;&#xB978; &#xF06C; &#xC885;&#xC18D; &#xD0A4;, CDK 5&#xC640; &#xD65C;&#xC131; &#xC81C; p 35&#xB294; &#xBCD1;&#xB82C;&#xB85C; &#xD558; &#xACE0; &#xBD80;&#xBD84;&#xC801;&#xC73C;&#xB85C; &#xC911;&#xBCF5; CYY 1/PCT 1 &#xD1B5;&#xB85C; &#xC5ED;&#xD560;. &#xC2DC; &#xB0C5; &#xC2A4; &#xC18C;&#xD3EC; &#xBC0F; &#xD65C;&#xC131; &#xC601;&#xC5ED; &#xB2E8;&#xBC31;&#xC9C8; &#xC8FC;&#xB85C; &#xB3D9;&#xBB3C; PCT 1&#xACFC; CDK 5 &#xACBD;&#xB85C; &#xB300; &#xD55C; &#xACB0;&#xD568;&#xC774; dendrites&#xC744; mislocalize. Kinesin 3 &#xBAA8;&#xD130;&#xC640;&#xB294; &#xB2EC;&#xB9AC; unc-104/Kif1a &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; anterogradely &#xB3D9;&#xC801; &#xC601;&#xC0C1;&#xC73C;&#xB85C; &#xAD00;&#xCC30; &#xC2DC; &#xB0C5; &#xC2A4; &#xC18C;&#xD3EC; &#xC120;&#xAD6C;&#xC790; (SVPs) &#xC774;&#xB3D9;&#xC5D0; cyy 1 cdk 5 &#xC774;&#xC911; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xAC10;&#xC18C; &#xD569;&#xB2C8;&#xB2E4;. &#xB300;&#xC2E0;, retrogradely SVPs &#xC774;&#xB3D9; &#xC218;&#xAC00; &#xADF9;&#xC801;&#xC73C;&#xB85C; &#xC99D;&#xAC00; &#xD588;&#xB2E4;. &#xB610;&#xD55C;, mislocalization &#xACB0;&#xD568;&#xC774;&#xB294; &#xD1F4;&#xD589; &#xC131; &#xBAA8;&#xD130;, &#xBCF5;&#xC7A1; &#xD55C; &#xC138;&#xD3EC;&#xC9C8; &#xD68C;&#xB97C; &#xBC29;&#xD574; &#xD558; &#xC5EC; &#xD45C;&#xC2DC; &#xB418;&#xC9C0; &#xC54A;&#xC2B5;&#xB2C8;&#xB2E4;. &#xB530;&#xB77C;&#xC11C;, PCT 1 CDK 5 &#xACBD;&#xB85C; &#xC9C1;&#xC811; &#xC5B5;&#xC81C; &#xC5ED;&#xD589; &#xC804;&#xC1A1; &#xD68C; &#xC911;&#xC7AC; &#xCC38;&#xAC00;&#xC790;&#xC640; &#xC5ED;&#xD589; &#xBAA8;&#xD130; &#xC0AC;&#xC774;&#xC758; &#xADE0;&#xD615;&#xC744; &#xC124;&#xC815; &#xD558; &#xC5EC; presynaptic &#xCEF4;&#xD3EC;&#xB10C;&#xD2B8;&#xC758; &#xD3B8;&#xAD11; &#xC778;&#xC2E0; &#xB9E4;&#xB9E4;.

&#xB450; &#xF06C; &#xC885;&#xC18D; &#xD0A4; &#xACBD;&#xB85C; presynaptic &#xCEF4;&#xD3EC;&#xB10C;&#xD2B8;&#xC758; &#xD3B8;&#xAD11; &#xC778;&#xC2E0; &#xB9E4;&#xB9E4;&#xC5D0; &#xB300; &#xD55C; &#xD544;&#xC218;&#xC801;&#xC785;&#xB2C8;&#xB2E4;.

Tr&aacute;fico polarizado de prote&iacute;nas sin&aacute;pticas axones y dendritas es crucial para la funci&oacute;n neuronal. A trav&eacute;s de an&aacute;lisis gen&eacute;tico hacia adelante en C. elegans, identificamos una ciclina (CYY-1) y una necesaria para apuntar el ax&oacute;n presin&aacute;pticos componentes quinasa de Pctaire dependiente de ciclina (PCT-1). Otra quinasa dependiente de ciclina CDK-5 y su activador p35, act&uacute;an paralelamente y parcialmente redundante con la v&iacute;a CYY-1/PCT-1. Ves&iacute;culas sin&aacute;pticas y zona activa prote&iacute;nas mislocalize sobre todo a las dendritas en animales defectuosos para v&iacute;as PCT-1 tanto CDK-5. A diferencia del quinesina-3 motor, unc-104/Kif1a mutante, cyy-1 cdk-5 dobles mutantes no tienen ninguna reducci&oacute;n en anterogradely movimiento de precursores de la ves&iacute;cula sin&aacute;ptica (SVPs) seg&uacute;n lo observado por la proyecci&oacute;n de imagen din&aacute;mica. En cambio, el n&uacute;mero de m&oacute;vil retrogradely SVPs se incrementa dram&aacute;ticamente. Adem&aacute;s, este defecto de mislocalization se suprime interrumpiendo el motor retr&oacute;grado, la dine&iacute;na citoplasm&aacute;tica complejo. As&iacute;, PCT-1 y CDK-5 v&iacute;as dirigen tr&aacute;fico polarizado de componentes presin&aacute;pticos inhibiendo el transporte retr&oacute;grado mediada por dine&iacute;na y establecer el equilibrio entre anter&oacute;grada y retr&oacute;grados motores.

Dos v&iacute;as de la quinasa dependiente de ciclina son esenciales para tr&aacute;fico polarizado de componentes presin&aacute;pticos.

Presynaptic assembly requires the packaging of requisite proteins into vesicular cargoes in the cell soma, their long-distance microtubule-dependent transport down the axon, and, finally, their reconstitution into functional complexes at prespecified sites. Despite the identification of several molecules that contribute to these events, the regulatory mechanisms defining such discrete states remain elusive. We report the characterization of an Arf-like small G protein, ARL-8, required during this process. arl-8 mutants prematurely accumulate presynaptic cargoes within the proximal axon of several neuronal classes, with a corresponding failure to assemble presynapses distally. This proximal accumulation requires the activity of several molecules known to catalyze presynaptic assembly. Dynamic imaging studies reveal that arl-8 mutant vesicles exhibit an increased tendency to form immotile aggregates during transport. Together, these results suggest that arl-8 promotes a trafficking identity for presynaptic cargoes, facilitating their efficient transport by repressing premature self-association.

An Arf-like small G protein, ARL-8, promotes the axonal transport of presynaptic cargoes by suppressing vesicle aggregation.

Microglia are the resident CNS immune cells and active surveyors of the extracellular environment. While past work has focused on the role of these cells during disease, recent imaging studies reveal dynamic interactions between microglia and synaptic elements in the healthy brain. Despite these intriguing observations, the precise function of microglia at remodeling synapses and the mechanisms that underlie microglia-synapse interactions remain elusive. In the current study, we demonstrate a role for microglia in activity-dependent synaptic pruning in the postnatal retinogeniculate system. We show that microglia engulf presynaptic inputs during peak retinogeniculate pruning and that engulfment is dependent upon neural activity and the microglia-specific phagocytic signaling pathway, complement receptor 3(CR3)/C3. Furthermore, disrupting microglia-specific CR3/C3 signaling resulted in sustained deficits in synaptic connectivity. These results define a role for microglia during postnatal development and identify underlying mechanisms by which microglia engulf and remodel developing synapses.

Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner.

Microglia are the resident immune cells and phagocytes of our central nervous system (CNS). While most work has focused on the rapid and robust responses of microglia during CNS disease and injury, emerging evidence suggests that these mysterious cells have important roles at CNS synapses in the healthy, intact CNS. Groundbreaking live imaging studies in the anesthetized, adult mouse demonstrated that microglia processes dynamically survey their environment and interact with other brain cells including neurons and astrocytes. More recent imaging studies have revealed that microglia dynamically interact with synapses where they appear to serve as "synaptic sensors," responding to changes in neural activity and neurotransmitter release. In the following review, we discuss the most recent work demonstrating that microglia play active roles at developing and mature synapses. We first discuss the important imaging studies that have led us to better understand the physical relationship between microglia and synapses in the healthy brain. Following this discussion, we review known molecular mechanisms and functional consequences of microglia-synapse interactions in the developing and mature CNS. Our current knowledge sheds new light on the critical functions of these mysterious cells in synapse development and function in the healthy CNS, but has also incited several new and interesting questions that remain to be explored. We discuss these open questions, and how the most recent findings in the healthy CNS may be related to pathologies associated with abnormal and/or loss of neural circuits.

Emily K. Lehrman

Department of Neurology,
F.M. Kirby Neurobiology Center,
Department of Neurology, F.M. Kirby Neurobiology Center

Boston Children's Hospital, Harvard Medical School

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Emily K. Lehrman

Department of Neurology,F.M. Kirby Neurobiology Center,Department of Neurology, F.M. Kirby Neurobiology Center

Boston Children's Hospital, Harvard Medical School

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Department of Neurology,
F.M. Kirby Neurobiology Center,
Department of Neurology, F.M. Kirby Neurobiology Center