Why Quantification Matters: Characterization of Phenotypes at the <em>Drosophila</em> Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

University of Edinburgh

Signaling through tyrosine kinase receptors (TKRs) is thought to be modulated by receptor-mediated endocytosis and degradation of the receptor in the lysosome. However, factors that regulate endosomal sorting of TKRs are largely unknown. Here, we demonstrate that Hrs (Hepatocyte growth factor-regulated tyrosine kinase substrate) is one such factor. Electron microscopy studies of hrs mutant larvae reveal an impairment in endosome membrane invagination and formation of multivesicular bodies (MVBs). hrs mutant animals fail to degrade active epidermal growth factor (EGF) and Torso TKRs, leading to enhanced signaling and altered embryonic patterning. These data suggest that Hrs and MVB formation function to downregulate TKR signaling.

Hrs regulates endosome membrane invagination and tyrosine kinase receptor signaling in Drosophila.

Aplysia VAP-33 (VAMP-associated protein) has been previously proposed to be involved in the control of neurotransmitter release. Here, we show that a Drosophila homolog of VAP-33, DVAP-33A, is localized to neuromuscular junctions. Loss of DVAP-33A causes a severe decrease in the number of boutons and a corresponding increase in bouton size. Conversely, presynaptic overexpression of DVAP-33A induces an increase in the number of boutons and a decrease in their size. Gain-of-function experiments show that the presynaptic dose of DVAP-33A tightly modulates the number of synaptic boutons. Our data also indicate that the presynaptic microtubule architecture is severely compromised in DVAP-33A mutants. We propose that a DVAP-33A-mediated interaction between microtubules and presynaptic membrane plays a pivotal role during bouton budding.

Drosophila VAP-33A directs bouton formation at neuromuscular junctions in a dosage-dependent manner.

&#x8FD0;&#x52A8;&#x795E;&#x7ECF;&#x5143;&#x75BE;&#x75C5; (&#x65E5;&#x671F;) &#x662F;&#x9009;&#x62E9;&#x6027;&#x6B7B;&#x4EA1;&#x5BFC;&#x81F4;&#x75C9;&#x631B;&#x3001; &#x808C;&#x8089;&#x548C;&#x9EBB;&#x75F9;&#x7684;&#x8FD0;&#x52A8;&#x795E;&#x7ECF;&#x5143;&#x7684;&#x7279;&#x70B9;&#x9010;&#x6B65;&#x795E;&#x7ECF;&#x9000;&#x884C;&#x6027;&#x75BE;&#x75C5;&#x3002;&#x4EBA;&#x7C7B;&#x978B;&#x9762;&#x76F8;&#x5173;&#x86CB;&#x767D; B &#xFF08;hVAPB&#xFF09; &#x662F;&#x4E34;&#x5E8A;&#x4E0A;&#x5F62;&#x5F62;&#x8272;&#x8272;&#x7684;&#x65E5;&#x671F;&#x5305;&#x62EC;&#x808C;&#x840E;&#x7F29;&#x4FA7;&#x7D22;&#x786C;&#x5316; (ALS)&#x3001; &#x975E;&#x5178;&#x578B;&#x7684; ALS &#x548C;&#x8FDF;&#x53D1;&#x810A;&#x9AD3;&#x6027;&#x808C;&#x840E;&#x7F29;&#x75C7;&#x7684;&#x81F4;&#x75C5;&#x57FA;&#x56E0;&#x3002;&#x81F4;&#x75C5;&#x6027;&#x7A81;&#x53D8;&#x662F;&#x5360;&#x4E3B;&#x5BFC;&#x5730;&#x4F4D;&#x7684;&#x65B9;&#x5F0F;&#x7EE7;&#x627F;&#x7684;&#x3002;33 KDa &#x7684;&#x679C;&#x8747;&#x978B;&#x9762;&#x76F8;&#x5173;&#x86CB;&#x767D; (DVAP-33A) &#x662F;&#x7ED3;&#x6784;&#x540C;&#x7CFB;&#x7269;&#x7684; hVAPB &#x548C;&#x8C03;&#x8282;&#x7A81;&#x89E6;&#x91CD;&#x5851;&#x7684;&#x5F71;&#x54CD;&#x7684;&#x5927;&#x5C0F;&#x548C;&#x6570;&#x91CF;&#x7684; boutons &#x5728;&#x795E;&#x7ECF;&#x808C;&#x8089;&#x63A5;&#x5934;&#x3002;&#x8FD9;&#x4E9B;&#x7ED3;&#x6784;&#x6539;&#x52A8;&#x4E0E;&#x5173;&#x8054;&#x662F;&#x751F;&#x7406;&#x548C;&#x8D85;&#x5FAE;&#x7ED3;&#x6784;&#x7684;&#x7A81;&#x89E6;&#xFF0C;&#x5B83;&#x4FDD;&#x6301;&#x5728;&#x6B63;&#x5E38;&#x8303;&#x56F4;&#x5185;&#x7684;&#x8BF1;&#x53D1;&#x7684;&#x53CD;&#x5E94;&#x7684;&#x8865;&#x507F;&#x6027;&#x53D8;&#x5316;&#x3002;DVAP 33A &#x548C; hVAPB &#x529F;&#x80FD;&#x53EF;&#x4EE5;&#x4E92;&#x6362;&#xFF0C;&#x8F6C;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x7684; DVAP 33A &#x7A81;&#x53D8;&#x4F53;&#x795E;&#x7ECF;&#x5143;&#x7EAA;&#x8981;&#x7684;&#x4EBA;&#x7C7B;&#x75BE;&#x75C5;&#x5305;&#x62EC;&#x8FD0;&#x52A8;&#x7F3A;&#x9677;&#x3001; &#x795E;&#x7ECF;&#x5143;&#x6B7B;&#x4EA1;&#x548C;&#x805A;&#x5408;&#x5F62;&#x6210;&#x7684;&#x4E3B;&#x8981;&#x6807;&#x5FD7;&#x3002;&#x805A;&#x5408;&#x79EF;&#x7D2F;&#x966A;&#x540C;&#x5176;&#x6B63;&#x5E38;&#x7684;&#x672C;&#x5730;&#x5316;&#x7684;&#x5185;&#x6E90;&#x6027;&#x86CB;&#x767D;&#x7684;&#x6D88;&#x8017;&#x3002;&#x8FD9;&#x4E9B;&#x8C03;&#x67E5;&#x7ED3;&#x679C;&#x7CBE;&#x786E;&#x5B9A;&#x4F4D;&#x5230;&#x7684; hVAPB &#x5728;&#x7A81;&#x89E6;&#x7A33;&#x6001;&#x4E2D;&#x53EF;&#x80FD;&#x53D1;&#x6325;&#x7684;&#x4F5C;&#x7528;&#xFF0C;&#x5E76;&#x5F3A;&#x8C03;&#x6211;&#x4EEC;&#x98DE;&#x5728;&#x9610;&#x660E;&#x4EBA;&#x7C7B;&#x7684;&#x75C5;&#x7406;&#x751F;&#x7406;&#x5B66;&#x57FA;&#x7840;&#x8FD0;&#x52A8;&#x795E;&#x7ECF;&#x5143;&#x9000;&#x5316;&#x6A21;&#x578B;&#x7684;&#x76F8;&#x5173;&#x6027;&#x3002;

hVAPB&#xFF0C;&#x81F4;&#x75C5;&#x57FA;&#x56E0;&#x7684;&#x5F02;&#x6784;&#x7EC4;&#x8FD0;&#x52A8;&#x795E;&#x7ECF;&#x5143;&#x75BE;&#x75C5;&#x5728;&#x4EBA;&#x7C7B;&#x4E2D;&#xFF0C;&#x662F;&#x529F;&#x80FD;&#x4E0A;&#x53EF;&#x4EE5;&#x4E92;&#x6362;&#x7684;&#x795E;&#x7ECF;&#x808C;&#x8089;&#x63A5;&#x5934;&#x5904;&#x53CA;&#x5176;&#x679C;&#x8747;&#x540C;&#x7CFB;&#x7269; DVAP-33a &#x6761;&#x3002;

Motor neuron diseases (MNDs) are progressive neurodegenerative disorders characterized by selective death of motor neurons leading to spasticity, muscle wasting and paralysis. Human VAMP-associated protein B (hVAPB) is the causative gene of a clinically diverse group of MNDs including amyotrophic lateral sclerosis (ALS), atypical ALS and late-onset spinal muscular atrophy. The pathogenic mutation is inherited in a dominant manner. Drosophila VAMP-associated protein of 33 kDa A (DVAP-33A) is the structural homologue of hVAPB and regulates synaptic remodeling by affecting the size and number of boutons at neuromuscular junctions. Associated with these structural alterations are compensatory changes in the physiology and ultrastructure of synapses, which maintain evoked responses within normal boundaries. DVAP-33A and hVAPB are functionally interchangeable and transgenic expression of mutant DVAP-33A in neurons recapitulates major hallmarks of the human diseases including locomotion defects, neuronal death and aggregate formation. Aggregate accumulation is accompanied by a depletion of the endogenous protein from its normal localization. These findings pinpoint to a possible role of hVAPB in synaptic homeostasis and emphasize the relevance of our fly model in elucidating the patho-physiology underlying motor neuron degeneration in humans.

hVAPB, the causative gene of a heterogeneous group of motor neuron diseases in humans, is functionally interchangeable with its Drosophila homologue DVAP-33A at the neuromuscular junction.

Maladies des neurones moteurs (DMN) sont des troubles neurod&eacute;g&eacute;n&eacute;rative progressive caract&eacute;ris&eacute;es par la mort s&eacute;lective des motoneurones, conduisant &agrave; une spasticit&eacute;, une perte musculaire et paralysie. La prot&eacute;ine humaine associ&eacute;e &agrave; VAMP B (hVAPB) est le g&egrave;ne responsable d&#39;un groupe diversifi&eacute; sur le plan clinique des multinationales, y compris la scl&eacute;rose lat&eacute;rale amyotrophique (SLA), ALS atypique et l&#39;atrophie musculaire spinale tardives. La mutation pathog&egrave;ne est h&eacute;rit&eacute;e de fa&ccedil;on dominante. Prot&eacute;ine associ&eacute;e &agrave; la VAMP de drosophile de 33 kDa un (message-33 a) est l&#39;homologue structural de hVAPB et r&eacute;glemente remodelage synaptique en agissant sur la taille et le nombre de boutons au niveau des jonctions neuromusculaires. Associ&eacute; &agrave; ces alt&eacute;rations structurales sont des changements compensatoires dans la physiologie et l&#39;ultrastructure des synapses, qui maintiennent des r&eacute;ponses &eacute;voqu&eacute;es dans les limites normales. VIOLENCE-33 a et hVAPB sont fonctionnellement interchangeables et expression transg&eacute;nique de mutant message-33 a de neurones r&eacute;capitule les grands poin&ccedil;ons des maladies humaines, y compris les d&eacute;fauts de locomotion, la mort neuronale et la formation globale. Accumulation globale s&#39;accompagne d&#39;une d&eacute;pl&eacute;tion de la prot&eacute;ine endog&egrave;ne de sa localisation normale. Ces r&eacute;sultats identifier un possible r&ocirc;le de hVAPB dans l&#39;hom&eacute;ostasie synaptique et mettent l&#39;accent sur la pertinence de notre mod&egrave;le de mouche dans l&#39;&eacute;lucidation de la d&eacute;g&eacute;n&eacute;rescence de neurones moteurs sous-jacents physiopathologie chez l&#39;homme.

hVAPB, le g&egrave;ne responsable d&#39;un groupe h&eacute;t&eacute;rog&egrave;ne de maladies des neurones moteurs chez l&#39;homme, est fonctionnellement interchangeable avec son homologue de la drosophile message-33 a &agrave; la jonction neuromusculaire.

Motoneuron-Erkrankungen (MNDs) sind progressive Neurodegenerative Erkrankungen mit selektiven Tod von Motoneuronen und Spastizit&auml;t, Muskelschwund und L&auml;hmungen f&uuml;hren. Menschliche VAMP-assoziierte Protein B (hVAPB) ist das urs&auml;chliche Gen eine klinisch heterogene Gruppe von MNDs einschlie&szlig;lich der amyotrophen Lateralsklerose (ALS), atypische ALS und sp&auml;t beginnende Muskelatrophie. Die Pathogene Mutation ist eine dominant vererbt. Taufliege VAMP-assoziierte Protein von 33 kDa (DVAP-33A) ist die strukturelle Homolog von hVAPB und reguliert synaptische Umbau durch Einwirkung auf die Gr&ouml;&szlig;e und Anzahl der Bottoni an. Mit dieser strukturellen &Auml;nderungen verbunden sind kompensatorische Ver&auml;nderungen der Physiologie und Ultrastruktur der Synapsen, die evozierte Antworten in normalen Grenzen zu halten. DVAP-33A und hVAPB sind funktional austauschbar und transgene Ausdruck der Mutant DVAP-33A in Neuronen rekapituliert die wichtige Kennzeichen der menschlichen Krankheiten einschlie&szlig;lich Fortbewegung M&auml;ngel, neuronale Tod und aggregierte Bildung. Aggregat Anh&auml;ufung ist eine Entleerung des endogenen Proteins von seiner normalen Lokalisierung einher. Diese Befunde auf eine m&ouml;gliche Rolle des hVAPB im synaptischen Hom&ouml;ostase zu ermitteln und betonen die Relevanz unseres fliegen-Modells in die Patho-Physiologie zugrunde liegenden Motorneuron Degeneration beim Menschen elucidating.

hVAPB, das urs&auml;chliche Gen einer heterogenen Gruppe von Motoneuron-Erkrankungen beim Menschen, ist funktional identisch mit seinem Drosophila-Homolog DVAP-33A an der neuromuskul&auml;ren Synapse.

Malattie del motoneurone (MNDs) sono malattie neurodegenerative progressive, caratterizzate da morte selettiva dei motoneuroni portando a spasticit&agrave;, atrofia muscolare e paralisi. Human VAMP-associated protein B (hVAPB) &egrave; il gene responsabile di un gruppo clinicamente eterogeneo di MNDs tra cui la sclerosi laterale amiotrofica (SLA), ALS atipica e atrofia muscolare spinale ad esordio tardivo. La mutazione patogena &egrave; ereditata in maniera dominante. Drosofila VAMP-associated protein di 33 kDa (DVAP-33A) &egrave; l&#39;omologo strutturale di hVAPB e regola il rimodellamento sinaptico interessando la dimensione e il numero di bottoni nelle giunzioni neuromuscolari. Associato a queste alterazioni strutturali sono compensativi cambiamenti nella fisiologia e ultrastruttura della sinapsi, che mantenere evocate risposte entro i limiti normali. DVAP-33A e hVAPB sono funzionalmente intercambiabili ed espressione transgenica del mutante DVAP-33A in neuroni riassume caratteristiche principali delle malattie umane tra cui difetti di locomozione, morte neuronale e formazione di aggregati. Accumulazione aggregata &egrave; accompagnato da un impoverimento della proteina endogena dalla sua localizzazione normale. Questi risultati individuare un possibile ruolo di hVAPB nell&#39;omeostasi sinaptica e sottolineano l&#39;importanza del nostro modello di vola nel chiarire la degenerazione del motoneurone sottostante patho-fisiologia negli esseri umani.

hVAPB, il gene responsabile di un gruppo eterogeneo di malattie del motoneurone in esseri umani, &egrave; funzionalmente intercambiabili con il suo omologo della drosofila DVAP-33A presso la giunzione neuromuscolare.

&#x904B;&#x52D5;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x75BE;&#x60A3; &#xFF08;MNDs&#xFF09; &#x306F;&#x3001;&#x75D9;&#x76F4;&#x3001;&#x7B4B;&#x840E;&#x7E2E;&#x3084;&#x9EBB;&#x75FA;&#x306B;&#x3064;&#x306A;&#x304C;&#x308B;&#x904B;&#x52D5;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306E;&#x9078;&#x629E;&#x7684;&#x306A;&#x6B7B;&#x306B;&#x3088;&#x3063;&#x3066;&#x7279;&#x5FB4;&#x3065;&#x3051;&#x3089;&#x308C;&#x308B;&#x9032;&#x884C;&#x6027;&#x795E;&#x7D4C;&#x5909;&#x6027;&#x75BE;&#x60A3;&#x3067;&#x3059;&#x3002;&#x4EBA;&#x9593;&#x30F4;&#x30A1;&#x30F3;&#x30D7;&#x95A2;&#x9023;&#x86CB;&#x767D; B (hVAPB) MNDs &#x840E;&#x7E2E;&#x6027;&#x5074;&#x7D22;&#x786C;&#x5316;&#x75C7; (ALS)&#x3001;&#x975E;&#x5B9A;&#x578B;&#x306E; ALS&#x3001;&#x9045;&#x767A;&#x6027;&#x810A;&#x9AC4;&#x6027;&#x7B4B;&#x840E;&#x7E2E;&#x306A;&#x3069;&#x306E;&#x81E8;&#x5E8A;&#x7684;&#x306B;&#x591A;&#x69D8;&#x306A;&#x30B0;&#x30EB;&#x30FC;&#x30D7;&#x306E;&#x539F;&#x56E0;&#x907A;&#x4F1D;&#x5B50;&#x3067;&#x3059;&#x3002;&#x75C5;&#x539F;&#x6027;&#x306E;&#x5909;&#x7570;&#x306F;&#x3001;&#x652F;&#x914D;&#x7684;&#x306A;&#x65B9;&#x6CD5;&#x3067;&#x7D99;&#x627F;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x30B7;&#x30E7;&#x30A6;&#x30B8;&#x30E7;&#x30A6;&#x30D0;&#x30A8; &#x30F4;&#x30A1;&#x30F3;&#x30D7;&#x95A2;&#x9023;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA; 33 kDa &#x306F; &#xFF08;&#x30B1;&#x30A2;&#x30BB;&#x30F3;&#x30BF;&#x30FC; 33 a) hVAPB &#x306E;&#x69CB;&#x9020;&#x306E;&#x76F8;&#x540C;&#x3042;&#x308A;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x795E;&#x7D4C;&#x7B4B;&#x63A5;&#x5408;&#x90E8;&#x3067;&#x306E;&#x30DC;&#x30BF;&#x30F3;&#x306E;&#x6570;&#x3001;&#x30B5;&#x30A4;&#x30BA;&#x306B;&#x5F71;&#x97FF;&#x3059;&#x308B;&#x6539;&#x9020;&#x3092;&#x8ABF;&#x7BC0;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x69CB;&#x9020;&#x5909;&#x5316;&#x3068;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x3066;&#x3044;&#x308B;&#x751F;&#x7406;&#x3068;&#x901A;&#x5E38;&#x5883;&#x754C;&#x5185;&#x8A98;&#x767A;&#x5FDC;&#x7B54;&#x3092;&#x7DAD;&#x6301;&#x3059;&#x308B;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x306E;&#x5FAE;&#x7D30;&#x69CB;&#x9020;&#x306E;&#x4EE3;&#x511F;&#x7684;&#x306A;&#x5909;&#x5316;&#x3067;&#x3059;&#x3002;&#x30B1;&#x30A2;&#x30BB;&#x30F3;&#x30BF;&#x30FC; 33 a &#x3068; hVAPB &#x306F;&#x6A5F;&#x80FD;&#x7684;&#x306B;&#x4EA4;&#x63DB;&#x53EF;&#x80FD;&#x3068;&#x907A;&#x4F1D;&#x5B50;&#x7D44;&#x63DB;&#x3048;&#x767A;&#x73FE;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306E;&#x30B1;&#x30A2;&#x30BB;&#x30F3;&#x30BF;&#x30FC; 33 a &#x5909;&#x7570;&#x4F53;&#x306E;&#x6B69;&#x884C;&#x306E;&#x6B20;&#x9665;&#x3001;&#x795E;&#x7D4C;&#x7D30;&#x80DE;&#x6B7B;&#x3068;&#x7C92;&#x306E;&#x5F62;&#x6210;&#x3092;&#x542B;&#x3080;&#x4EBA;&#x9593;&#x306E;&#x75C5;&#x6C17;&#x306E;&#x4E3B;&#x8981;&#x306A;&#x7279;&#x5FB4;&#x304B;&#x3082;&#x3057;&#x308C;&#x307E;&#x305B;&#x3093;&#x3002;&#x96C6;&#x8A08;&#x84C4;&#x7A4D;&#x5185;&#x56E0;&#x6027;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x67AF;&#x6E07;&#x306B;&#x3088;&#x308A;&#x3001;&#x901A;&#x5E38;&#x306E;&#x30ED;&#x30FC;&#x30AB;&#x30E9;&#x30A4;&#x30BA;&#x304B;&#x3089;&#x4ED8;&#x5C5E;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x8ABF;&#x67FB;&#x7D50;&#x679C;&#x306F;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x306E;&#x6052;&#x5E38;&#x6027;&#x306E; hVAPB &#x306E;&#x5F79;&#x5272;&#x3092;&#x7279;&#x5B9A;&#x3057;&#x3001;&#x75C5;&#x614B;&#x751F;&#x7406;&#x306E;&#x57FA;&#x306B;&#x306A;&#x308B;&#x904B;&#x52D5;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x5909;&#x6027;&#x30D2;&#x30C8;&#x3092;&#x89E3;&#x660E;&#x306B;&#x304A;&#x3051;&#x308B;&#x6211;&#x3005; &#x30D5;&#x30E9;&#x30A4;&#x306E;&#x30E2;&#x30C7;&#x30EB;&#x3068;&#x306E;&#x95A2;&#x9023;&#x6027;&#x3092;&#x5F37;&#x8ABF;&#x3057;&#x307E;&#x3059;&#x3002;

hVAPB &#x7570;&#x7A2E;&#x30B0;&#x30EB;&#x30FC;&#x30D7;&#x306E;&#x30D2;&#x30C8;&#x306E;&#x904B;&#x52D5;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x75BE;&#x60A3;&#x306E;&#x539F;&#x56E0;&#x907A;&#x4F1D;&#x5B50;&#x3068;&#x305D;&#x306E;&#x795E;&#x7D4C;&#x7B4B;&#x63A5;&#x5408;&#x90E8;&#x3067;&#x306E;&#x30B7;&#x30E7;&#x30A6;&#x30B8;&#x30E7;&#x30A6;&#x30D0;&#x30A8; &#x30DB;&#x30E2;&#x30ED;&#x30B0; &#x30B1;&#x30A2;&#x30BB;&#x30F3;&#x30BF;&#x30FC; 33 a &#x6A5F;&#x80FD;&#x7684;&#x306B;&#x4EA4;&#x63DB;&#x53EF;&#x80FD;&#x3067;&#x3059;&#x3002;

&#xBAA8;&#xD130; &#xC2E0;&#xACBD; &#xC9C8;&#xBCD1; (MNDs)&#xC740; spasticity, &#xADFC;&#xC721;&#xC744; &#xB0AD;&#xBE44; &#xD558; &#xACE0; &#xB9C8;&#xBE44;&#xB97C; &#xC120;&#xB3C4; &#xD558;&#xB294; &#xC6B4;&#xB3D9; &#xC2E0;&#xACBD;&#xC758; &#xC120;&#xD0DD;&#xC801; &#xC8FD;&#xC74C;&#xC73C;&#xB85C; &#xD2B9;&#xC9D5; &#xC9C4;&#xBCF4;&#xC801;&#xC778; &#xC2E0;&#xACBD; &#xD1F4;&#xD589; &#xC131; &#xC9C8;&#xD658;. &#xC778;&#xAC04; &#xBC40;&#xD30C;&#xC774;&#xC5B4; &#xAD00;&#xB828; &#xB2E8;&#xBC31;&#xC9C8; B (hVAPB)&#xB294; MNDs &#xB8E8; &#xAC8C; &#xB9AD; &#xBCD1; &#xCE21;&#xBA74; &#xACBD;&#xD654; &#xC99D; (ALS), &#xBE44;&#xC815;&#xD615; ALS &#xB2A6;&#xC740; &#xBC1C;&#xBCD1; &#xCC99;&#xC218; &#xADFC;&#xC721; &#xC704;&#xCD95; &#xC99D; &#xB4F1; &#xC784;&#xC0C1;&#xC801;&#xC73C;&#xB85C; &#xB2E4;&#xC591; &#xD55C; &#xADF8;&#xB8F9;&#xC758; &#xC6D0;&#xC778;&#xC774; &#xB418;&#xB294; &#xC720;&#xC804;&#xC790;. &#xBCD1;&#xC6D0; &#xC131; &#xBCC0;&#xC774; &#xC9C0;&#xBC30;&#xC801;&#xC778; &#xBC29;&#xC2DD;&#xC73C;&#xB85C; &#xC0C1;&#xC18D; &#xB429;&#xB2C8;&#xB2E4;. 33 Kda&#xC758; &#xCD08;&#xD30C;&#xB9AC; &#xBC40;&#xD30C;&#xC774;&#xC5B4; &#xAD00;&#xB828; &#xB2E8;&#xBC31;&#xC9C8; (DVAP-33A) Hvapb&#xC758; &#xAD6C;&#xC870;&#xC801; homologue &#xC774;&#xBA70;, &#xD06C;&#xAE30;&#xC640; &#xC218;&#xC5D0; &#xC2E0;&#xACBD; &#xADFC;&#xC721; &#xD559; &#xC811;&#xD569; boutons&#xC758; &#xC601;&#xD5A5;&#xC5D0; &#xC758;&#xD574; &#xC2DC; &#xB0C5; &#xC2A4; &#xB9AC; &#xBAA8;&#xB378;&#xB9C1; &#xADDC;&#xC81C;. &#xC774;&#xB7EC;&#xD55C; &#xAD6C;&#xC870; &#xBCC0;&#xACBD; &#xC5F0;&#xAD00; &#xC0DD;&#xB9AC;&#xD559; &#xBC0F; &#xC815;&#xC0C1;&#xC801;&#xC778; &#xACBD;&#xACC4; &#xB0B4;&#xC5D0;&#xC11C; &#xAC16;&#xB294; &#xC751;&#xB2F5; &#xC720;&#xC9C0; &#xB0C5;&#xC758; &#xB300;&#xAD8C;&#xC758; &#xBCF4;&#xC0C1; &#xBCC0;&#xD654;&#xB294;. DVAP-33a&#xC640; Hvapb&#xB294; &#xAE30;&#xB2A5;&#xC801;&#xC73C;&#xB85C; &#xC0C1;&#xD638; &#xAD50;&#xD658; &#xD558; &#xACE0; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; DVAP-33A &#xC2E0;&#xACBD; &#xC138;&#xD3EC;&#xC5D0;&#xC11C;&#xC758; &#xC720;&#xC804;&#xC790; &#xBCC0;&#xD615; &#xC2DD; &#xBC1C;&#xC0DD; &#xC6B4;&#xB3D9; &#xACB0;&#xD568;, &#xC9D1;&#xACC4; &#xD615;&#xC131;, &#xC2E0;&#xACBD; &#xC8FD;&#xC74C; &#xB4F1; &#xC778;&#xAC04;&#xC758; &#xC9C8;&#xBCD1;&#xC758; &#xC8FC;&#xC694; &#xD2B9;&#xC9D5;. &#xC9D1;&#xACC4; &#xCD95;&#xC801; &#xC0DD; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xACE0;&#xAC08;&#xC758; &#xC815;&#xC0C1;&#xC801;&#xC778; &#xC9C0;&#xC5ED;&#xD654;&#xC5D0;&#xC11C; &#xD568;&#xAED8; &#xD569;&#xB2C8;&#xB2E4;. &#xC774;&#xB7EC;&#xD55C; &#xC5F0;&#xAD6C; &#xACB0;&#xACFC; &#xC2DC; &#xB0C5; &#xC2A4; &#xD56D;&#xC0C1;&#xC131;&#xC5D0;&#xC11C; Hvapb&#xC758; &#xAC00;&#xB2A5;&#xD55C; &#xC5ED;&#xD560;&#xC744; &#xC815;&#xD655;&#xD788; &#xC9C0;&#xC801; &#xD558; &#xACE0; &#xC6B0;&#xB9AC;&#xC758; &#xBE44;&#xD589; &#xBAA8;&#xB378; elucidating &#xC778;&#xAC04;&#xC5D0;&#xC11C; patho &#xC0DD;&#xB9AC;&#xD559; &#xAE30;&#xBCF8; &#xBAA8;&#xD130; &#xC2E0;&#xACBD; &#xBCC0;&#xC131;&#xC5D0;&#xC758; &#xAD00;&#xB828;&#xC131;&#xC744; &#xAC15;&#xC870;.

hVAPB, &#xC778;&#xAC04;, &#xBAA8;&#xD130; &#xC2E0;&#xACBD; &#xC9C8;&#xBCD1;&#xC758; &#xC720;&#xD615;&#xC774; &#xB2E4;&#xB978; &#xADF8;&#xB8F9;&#xC758; &#xC6D0;&#xC778;&#xC774; &#xC720;&#xC804;&#xC790;&#xB294; &#xC2E0;&#xACBD; &#xADFC;&#xC721; &#xD559; &#xAD50;&#xCC28;&#xB85C;&#xC5D0; &#xCD08;&#xD30C;&#xB9AC; homologue DVAP-33a&#xC640; &#xAE30;&#xB2A5;&#xC801;&#xC73C;&#xB85C; &#xC0C1;&#xD638; &#xAD50;&#xD658;.

Enfermedades de la neurona motora (compararon) son trastornos neurodegenerativa progresiva caracterizados por la muerte selectiva de las neuronas motoras que conducen a la espasticidad, atrofia muscular y par&aacute;lisis. Prote&iacute;na humana VAMP B (hVAPB) es el gen causante de un grupo cl&iacute;nico diverso de compararon como esclerosis lateral amiotr&oacute;fica (ELA), ALS anormal y atrofia muscular de aparici&oacute;n tard&iacute;a. La mutaci&oacute;n patog&eacute;nica se hereda de forma dominante. Prote&iacute;na VAMP Drosophila de 33 kDa (DVAP-33A) es el hom&oacute;logo estructural de hVAPB y regula la remodelaci&oacute;n sin&aacute;ptica afectando el tama&ntilde;o y el n&uacute;mero de botones en las ensambladuras neuromusculares. Asociados con estas alteraciones estructurales son cambios compensatorios en la fisiolog&iacute;a y la ultraestructura de las sinapsis, que mantienen las respuestas evocadas dentro de l&iacute;mites normales. DVAP-33A y hVAPB son funcionalmente intercambiables y recapitula la expresi&oacute;n transg&eacute;nica del mutante DVAP-33A en neuronas principales caracter&iacute;sticas distintivas de las enfermedades humanas, incluyendo defectos de locomoci&oacute;n, la muerte neuronal y formaci&oacute;n agregada. Acumulaci&oacute;n total se acompa&ntilde;a de una disminuci&oacute;n de la prote&iacute;na end&oacute;gena de su localizaci&oacute;n normal. Estos resultados identificar a un posible papel de hVAPB en la homeostasis sin&aacute;ptica y enfatizan la importancia de nuestro modelo de mosca en la aclaraci&oacute;n de la degeneraci&oacute;n patho-fisiolog&iacute;a de la neurona motora subyacente en los seres humanos.

hVAPB, el gen causante de un grupo heterog&eacute;neo de enfermedades de la neurona motora en los seres humanos, es funcionalmente intercambiable con su hom&oacute;logo de Drosophila DVAP-33A en la Uni&oacute;n neuromuscular.

Following the mutation screening of genes known to cause amyotrophic lateral sclerosis (ALS) in index cases from 107 familial ALS (FALS) kindred, a point mutation was identified in vesicle-associated membrane protein-associated protein B (VAPB), or VAMP-associated protein B, causing an amino acid change from threonine to isoleucine at codon 46 (T46I) in one FALS case but not in 257 controls. This is an important finding because it is only the second mutation identified in this gene that causes ALS. In order to investigate the pathogenic effects of this mutation, we have used a motor neuron cell line and tissue-specific expression of the mutant protein in Drosophila. We provide substantial evidence for the pathogenic effects of this mutation in abolishing the effect of wild type VAPB in the unfolded protein response, promoting ubiquitin aggregate formation, and activating neuronal cell death. We also report that expression of the mutant protein in the Drosophila motor system induces aggregate deposition, endoplasmic reticulum disorganization, and chaperone up-regulation both in neurons and in muscles. Our integrated analysis of the pathogenic effect of the T46I mutation and the previously identified P56S mutation indicate extensive commonalities in the disease mechanism for these two mutations. In summary, we show that this newly identified mutation in human FALS has a pathogenic effect, supporting and reinforcing the role of VAPB as a causative gene of ALS.

Characterization of the properties of a novel mutation in VAPB in familial amyotrophic lateral sclerosis.

The Vesicle-associated membrane protein (VAMP)-Associated Protein B (VAPB) is the causative gene of amyotrophic lateral sclerosis 8 (ALS8) in humans. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective death of motor neurons leading to spasticity, muscle atrophy and paralysis. VAP proteins have been implicated in various cellular processes, including intercellular signalling, synaptic remodelling, lipid transport and membrane trafficking and yet, the molecular mechanisms underlying ALS8 pathogenesis remain poorly understood. We identified the conserved phosphoinositide phosphatase Sac1 as a Drosophila VAP (DVAP)-binding partner and showed that DVAP is required to maintain normal levels of phosphoinositides. Downregulating either Sac1 or DVAP disrupts axonal transport, synaptic growth, synaptic microtubule integrity and the localization of several postsynaptic components. Expression of the disease-causing allele (DVAP-P58S) in a fly model for ALS8 induces neurodegeneration, elicits synaptic defects similar to those of DVAP or Sac1 downregulation and increases phosphoinositide levels. Consistent with a role for Sac1-mediated increase of phosphoinositide levels in ALS8 pathogenesis, we found that Sac1 downregulation induces neurodegeneration in a dosage-dependent manner. In addition, we report that Sac1 is sequestered into the DVAP-P58S-induced aggregates and that reducing phosphoinositide levels rescues the neurodegeneration and suppresses the synaptic phenotypes associated with DVAP-P58S transgenic expression. These data underscore the importance of DVAP-Sac1 interaction in controlling phosphoinositide metabolism and provide mechanistic evidence for a crucial role of phosphoinositide levels in VAP-induced ALS.

Increased levels of phosphoinositides cause neurodegeneration in a Drosophila model of amyotrophic lateral sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a motor neuron degenerative disease characterized by a progressive, and ultimately fatal, muscle paralysis. The human VAMP-Associated Protein B (hVAPB) is the causative gene of ALS type 8. Previous studies have shown that a loss-of-function mechanism is responsible for VAPB-induced ALS. Recently, a novel mutation in hVAPB (V234I) has been identified but its pathogenic potential has not been assessed. We found that neuronal expression of the V234I mutant allele in Drosophila (DVAP-V260I) induces defects in synaptic structure and microtubule architecture that are opposite to those associated with DVAP mutants and transgenic expression of other ALS-linked alleles. Expression of DVAP-V260I also induces aggregate formation, reduced viability, wing postural defects, abnormal locomotion behavior, nuclear abnormalities, neurodegeneration and upregulation of the heat-shock-mediated stress response. Similar, albeit milder, phenotypes are associated with the overexpression of the wild-type protein. These data show that overexpressing the wild-type DVAP is sufficient to induce the disease and that DVAP-V260I is a pathogenic allele with increased wild-type activity. We propose that a combination of gain- and loss-of-function mechanisms is responsible for VAPB-induced ALS.

Gain-of-function mutations in the ALS8 causative gene VAPB have detrimental effects on neurons and muscles.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective loss of motor neurons, muscle atrophy and paralysis. Mutations in the human VAMP-associated protein B (hVAPB) cause a heterogeneous group of motor neuron diseases including ALS8. Despite extensive research, the molecular mechanisms underlying ALS pathogenesis remain largely unknown. Genetic screens for key interactors of hVAPB activity in the intact nervous system, however, represent a fundamental approach towards understanding the in vivo function of hVAPB and its role in ALS pathogenesis. Targeted expression of the disease-causing allele leads to neurodegeneration and progressive decline in motor performance when expressed in the adult Drosophila, eye or in its entire nervous system, respectively. By using these two phenotypic readouts, we carried out a systematic survey of the Drosophila genome to identify modifiers of hVAPB-induced neurotoxicity. Modifiers cluster in a diverse array of biological functions including processes and genes that have been previously linked to hVAPB function, such as proteolysis and vesicular trafficking. In addition to established mechanisms, the screen identified endocytic trafficking and genes controlling proliferation and apoptosis as potent modifiers of ALS8-mediated defects. Surprisingly, the list of modifiers was mostly enriched for proteins linked to lipid droplet biogenesis and dynamics. Computational analysis reveals that most modifiers can be linked into a complex network of interacting genes, and that the human genes homologous to the Drosophila modifiers can be assembled into an interacting network largely overlapping with that in flies. Identity markers of the endocytic process were also found to abnormally accumulate in ALS patients, further supporting the relevance of the fly data for human biology. Collectively, these results not only lead to a better understanding of hVAPB function but also point to potentially relevant targets for therapeutic intervention.

Network analyses reveal novel aspects of ALS pathogenesis.

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease causing the death of motor neurons with consequent muscle atrophy and paralysis. Several neurodegenerative diseases have been modeled in Drosophila and genetic studies on this model organism led to the elucidation of crucial aspects of disease mechanisms. ALS, however, has lagged somewhat behind possibly because of the lack of a suitable genetic model. We were the first to develop a fly model for ALS and over the last few years, we have implemented and used this model for a large scale, unbiased modifier screen. We also report an extensive bioinformatic analysis of the genetic modifiers and we show that most of them are associated in a network of interacting genes controlling known as well as novel cellular processes involved in ALS pathogenesis. A similar analysis for the human homologues of the Drosophila modifiers and the validation of a subset of them in human tissues confirm and expand the significance of the data for the human disease. Finally, we analyze a possible application of the model in the process of therapeutic discovery in ALS and we discuss the importance of novel "non-obvious" models for the disease.

Giuseppa Pennetta

Euan MacDonald Centre for Motor Neurone Disease Research

University of Edinburgh

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Giuseppa Pennetta

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Euan MacDonald Centre for Motor Neurone Disease Research

University of Edinburgh

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Euan MacDonald Centre for Motor Neurone Disease Research