novel-whole-tissue-quantitative-assay-nitric-oxide-levels-drosophila

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in <em>Drosophila</em> Neuroinflammatory Response

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an ...

University of Alabama

The cofactor tetrahydrobiopterin plays critical roles in the modulation of the signaling molecules dopamine, serotonin, and nitric oxide. Deficits in cofactor synthesis have been associated with several human hereditary diseases. Responsibility for the regulation of cofactor pools resides with the first enzyme in its biosynthetic pathway, GTP cyclohydrolase I. Because organisms must be able to rapidly respond to environmental and developmental cues to adjust output of these signaling molecules, complex regulatory mechanisms are vital for signal modulation. Mammalian GTP cyclohydrolase is subject to end-product inhibition via an associated regulatory protein and to positive regulation via phosphorylation, although target residues are unknown. GTP cyclohydrolase is composed of a highly conserved homodecameric catalytic core and non-conserved N-terminal domains proposed to be regulatory sites. We demonstrate for the first time in any organism that the N-terminal arms of the protein serve regulatory functions. We identify two different modes of regulation of the enzyme mediated through the N-terminal domains. The first is end-product feedback inhibition, catalytically similar to that of the mammalian enzyme, except that feedback inhibition by the cofactor requires sequences in the N-terminal arms rather than a separate regulatory protein. The second is a novel inhibitory interaction between the N-terminal arms and the active sites, which can be alleviated through the phosphorylation of serine residues within the N termini. Both mechanisms allow for acute and highly responsive regulation of cofactor production as required by downstream signaling pathways.

A typical N-terminal extensions confer novel regulatory properties on GTP cyclohydrolase isoforms in Drosophila melanogaster.

Catastrophic loss of dopaminergic neurons is a hallmark of Parkinson's disease. Despite the recent identification of genes associated with familial parkinsonism, the etiology of most Parkinson's disease cases is not understood. Environmental toxins, such as the herbicide paraquat, appear to be risk factors, and it has been proposed that susceptibility is influenced by genetic background. The genetic model organism Drosophila is an advantageous system for the identification of genetic susceptibility factors. Genes that affect dopamine homeostasis are candidate susceptibility factors, because dopamine itself has been implicated in neuron damage. We find that paraquat can replicate a broad spectrum of parkinsonian behavioral symptoms in Drosophila that are associated with loss of specific subsets of dopaminergic neurons. In parallel with epidemiological studies that show an increased incidence of Parkinson's disease in males, male Drosophila exhibit paraquat symptoms earlier than females. We then tested the hypothesis that variation in dopamine-regulating genes, including those that regulate tetrahydrobiopterin, a requisite cofactor in dopamine synthesis, can alter susceptibility to paraquat-induced oxidative damage. Drosophila mutant strains that have increased or decreased dopamine and tetrahydrobiopterin production exhibit variation in susceptibility to paraquat. Surprisingly, protection against the neurotoxicity of paraquat is conferred by mutations that elevate dopamine pathway function, whereas mutations that diminish dopamine pools increase susceptibility. We also find that loss-of-function mutations in a negative regulator of dopamine production, Catecholamines-up, delay the onset of neurological symptoms, dopaminergic neuron death, and morbidity during paraquat exposure but confer sensitivity to hydrogen peroxide.

Interaction of genetic and environmental factors in a Drosophila parkinsonism model.

While studying the developmental functions of the Drosophila dopamine synthesis pathway genes, we noted interesting and unexpected mutant phenotypes in the developing trachea, a tubule network that has been studied as a model for branching morphogenesis. Specifically, Punch (Pu) and pale (ple) mutants with reduced dopamine synthesis show ectopic/aberrant migration, while Catecholamines up (Catsup) mutants that over-express dopamine show a characteristic loss of migration phenotype. We also demonstrate expression of Punch, Ple, Catsup and dopamine in tracheal cells. The dopamine pathway mutant phenotypes can be reproduced by pharmacological treatments of dopamine and a pathway inhibitor 3-iodotyrosine (3-IT), implicating dopamine as a direct mediator of the regulatory function. Furthermore, we show that these mutants genetically interact with components of the endocytic pathway, namely shibire/dynamin and awd/nm23, that promote endocytosis of the chemotactic signaling receptor Btl/FGFR. Consistent with the genetic results, the surface and total cellular levels of a Btl-GFP fusion protein in the tracheal cells and in cultured S2 cells are reduced upon dopamine treatment, and increased in the presence of 3-IT. Moreover, the transducer of Btl signaling, MAP kinase, is hyper-activated throughout the tracheal tube in the Pu mutant. Finally we show that dopamine regulates endocytosis via controlling the dynamin protein level.

Drosophila dopamine synthesis pathway genes regulate tracheal morphogenesis.

&#x591A;&#x5DF4;&#x80FA;&#x7684;&#x4FE1;&#x53F7;&#x8F6C;&#x5BFC;&#x529F;&#x80FD;&#x5FEB;&#x901F;&#x611F;&#x5E94;&#x548C;&#x5236;&#x6B62;&#x7684;&#x8F93;&#x51FA;&#x9700;&#x8981;&#x7CBE;&#x5FC3;&#x7684;&#x76D1;&#x7BA1;&#x7F51;&#x7EDC;&#x3002;&#x8C03;&#x63A7;&#x7684;&#x4E3B;&#x8981;&#x76EE;&#x6807;&#x662F;&#x9176;&#x916A;&#x6C28;&#x9178;&#x7F9F;&#x5316;&#x9176;&#xFF0C;&#x901F;&#x7387;&#x9650;&#x5236;&#x9176;&#x4E2D;&#x591A;&#x5DF4;&#x80FA;&#x7684;&#x5408;&#x6210;&#xFF0C;&#x5B83;&#x662F;&#x7531;&#x78F7;&#x9178;&#x5316;&#x6FC0;&#x6D3B;&#x548C;&#x8C03;&#x5236;&#x800C;&#x6210;&#x5176;&#x8F85;&#x9176;&#xFF0C;&#x5464;&#x7684;&#x53EF;&#x7528;&#x6027;&#x3002;&#x7B2C;&#x4E00;&#x6B21;&#x8F85;&#x9176;&#x5408;&#x6210;&#x9014;&#x5F84;&#xFF0C;&#x7B2C;&#x4E00;&#x3001; &#x662F;&#x901A;&#x8FC7;&#x78F7;&#x9178;&#x5316;&#x6FC0;&#x6D3B;&#x548C;&#x6291;&#x5236;&#x5464; GTP &#x5408;&#x6210;&#x9176;&#x9176;&#x3002;&#x5148;&#x524D;&#x6211;&#x4EEC;&#x62A5;&#x9053;&#x4E2D; GTP &#x73AF;&#x6C34;&#x89E3;&#x9176;&#x6D3B;&#x6027;&#x7684;&#x679C;&#x8747;&#x7684;&#x8FD9;&#x79CD;&#x9176;&#x7684;&#x57FA;&#x56E0;&#x7684;&#x7A81;&#x53D8;&#x4F53;&#x7B49;&#x4F4D;&#x57FA;&#x56E0;&#x7684;&#x6742;&#x5408;&#x6027;&#x7684;&#x8D64;&#x5B57;&#x5BFC;&#x81F4;&#x4F53;&#x5185;&#x916A;&#x6C28;&#x9178;&#x7F9F;&#x5316;&#x9176;&#x6D3B;&#x6027;&#xFF0C;&#x4E0D;&#x80FD;&#x7531;&#x5916;&#x6E90;&#x56E0;&#x5B50;&#x6551;&#x51FA;&#x7D27;&#x76F8;&#x5E94;&#x51CF;&#x5C11;&#x3002;&#x6211;&#x4EEC;&#x8FD8;&#x53D1;&#x73B0;&#x4E24;&#x79CD;&#x9176;&#x53EF;&#x4EE5;&#x4ECE;&#x7EC4;&#x7EC7;&#x63D0;&#x53D6;&#x6DB2;&#x548C;&#x62DF;&#x8BAE;&#x529F;&#x80FD;&#x4E92;&#x52A8;&#x5173;&#x7CFB;&#x8D85;&#x8D8A;&#x8F85;&#x9176;&#x63D0;&#x4F9B;&#x4E00;&#x4E2A;&#x901A;&#x8DEF;&#x7684;&#x53E6;&#x4E00;&#x79CD;&#x7684;&#x9176; coimmunoprecipitated&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x786E;&#x8BA4;&#x8FD9;&#x4E9B;&#x9176;&#x7269;&#x7406;&#x534F;&#x4F1A;&#x786E;&#x5B9A;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x533A;&#x57DF;&#x90FD;&#xFF0C;&#x5728;&#x548C;&#x6211;&#x4EEC;&#x8BC1;&#x660E;&#x5176;&#x534F;&#x4F1A;&#x53EF;&#x4EE5;&#x53D7;&#x78F7;&#x9178;&#x5316;&#x3002;&#x4E92;&#x52A8;&#x529F;&#x80FD;&#x7684;&#x540E;&#x679C;&#x5305;&#x62EC;&#x589E;&#x52A0; GTP &#x73AF;&#x6C34;&#x89E3;&#x9176;&#x6D3B;&#x52A8;&#xFF0C;&#x4E0E;&#x76F8;&#x5E94;&#x7684;&#x4FDD;&#x62A4;&#xFF0C;&#x4ECE;&#x6700;&#x7EC8;&#x4EA7;&#x54C1;&#x7684;&#x53CD;&#x9988;&#x6291;&#x5236;&#x4F5C;&#x7528;&#x3002;&#x4F53;&#x5185;&#xFF0C;&#x8FD9;&#x79CD;&#x6548;&#x679C;&#x4F1A;&#x53CD;&#x8FC7;&#x6765;&#x63D0;&#x4F9B;&#x8DB3;&#x591F;&#x7684;&#x8F85;&#x9176;&#x65F6;&#x513F;&#x8336;&#x915A;&#x80FA;&#x5408;&#x6210;&#x9700;&#x6C42;&#x6700;&#x5927;&#x3002;&#x916A;&#x6C28;&#x9178;&#x7F9F;&#x5316;&#x9176;&#x7684;&#x6D3B;&#x6027;&#x4E5F;&#x5C31;&#x63D0;&#x9AD8;&#x4E86;&#x8FD9;&#x79CD;&#x4E92;&#x52A8;&#xFF0C;&#x8D85;&#x8FC7;&#x5355;&#x78F7;&#x9178;&#x5316;&#x6240;&#x5F15;&#x8D77;&#x7684;&#x523A;&#x6FC0;&#x3002;Vmax &#x4E3A;&#x9AD8;&#x67B6;&#x6865;&#x6881;&#xFF0C;&#x8FD9;&#x4E9B;&#x7ED3;&#x679C;&#x8868;&#x660E;&#x8FD9;&#x4E9B;&#x9176;&#x53C2;&#x4E0E;&#x76F8;&#x4E92;&#x79EF;&#x6781;&#x8C03;&#x63A7;&#x516C;&#x91CC;&#x6CA1;&#x6709;&#x53D8;&#x5316;&#x3002;

&#x76F4;&#x63A5;&#x7ED1;&#x5B9A;&#x7684; GTP &#x73AF;&#x6C34;&#x89E3;&#x9176;&#x3001; &#x916A;&#x6C28;&#x9178;&#x7F9F;&#x5316;&#x9176;&#xFF1A; &#x89C4;&#x7BA1;&#x591A;&#x5DF4;&#x80FA;&#x5408;&#x6210;&#x4E2D;&#x7684;&#x5173;&#x952E;&#x9176;&#x4E4B;&#x95F4;&#x7684;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x3002;

The signaling functions of dopamine require a finely tuned regulatory network for rapid induction and suppression of output. A key target of regulation is the enzyme tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, which is activated by phosphorylation and modulated by the availability of its cofactor, tetrahydrobiopterin. The first enzyme in the cofactor synthesis pathway, GTP cyclohydrolase I, is activated by phosphorylation and inhibited by tetrahydrobiopterin. We previously reported that deficits in GTP cyclohydrolase activity in Drosophila heterozygous for mutant alleles of the gene encoding this enzyme led to tightly corresponding diminution of in vivo tyrosine hydroxylase activity that could not be rescued by exogenous cofactor. We also found that the two enzymes could be coimmunoprecipitated from tissue extracts and proposed functional interactions between the enzymes that extended beyond provision of cofactor by one pathway for another. Here, we confirm the physical association of these enzymes, identifying interacting regions in both, and we demonstrate that their association can be regulated by phosphorylation. The functional consequences of the interaction include an increase in GTP cyclohydrolase activity, with concomitant protection from end-product feedback inhibition. In vivo, this effect would in turn provide sufficient cofactor when demand for catecholamine synthesis is greatest. The activity of tyrosine hydroxylase is also increased by this interaction, in excess of the stimulation resulting from phosphorylation alone. Vmax is elevated, with no change in Km. These results demonstrate that these enzymes engage in mutual positive regulation.

Direct binding of GTP cyclohydrolase and tyrosine hydroxylase: regulatory interactions between key enzymes in dopamine biosynthesis.

Les fonctions de signalisation de la dopamine requi&egrave;rent un r&eacute;seau finement r&eacute;gl&eacute; de r&eacute;gulation pour induction rapide et l&#39;&eacute;limination de la production. Un objectif cl&eacute; du r&egrave;glement est l&#39;enzyme tyrosine hydroxylase, l&#39;enzyme limitante dans la synth&egrave;se de dopamine, qui est activ&eacute;e par phosphorylation et modul&eacute;e par la disponibilit&eacute; de son cofacteur, la t&eacute;trahydrobiopt&eacute;rine. La premi&egrave;re enzyme dans la voie de synth&egrave;se du cofacteur, GTP cyclohydrolase I, est activ&eacute;e par phosphorylation et inhib&eacute;e par la t&eacute;trahydrobiopt&eacute;rine. Nous avons d&eacute;j&agrave; indiqu&eacute; que les d&eacute;ficits dans l&#39;activit&eacute; de la GTP cyclohydrolase chez la drosophile h&eacute;t&eacute;rozygote pour les all&egrave;les mutants du g&egrave;ne codant pour cette enzyme conduit &agrave; &eacute;troitement correspondante de diminution de l&#39;activit&eacute; in vivo de tyrosine hydroxylase qui ne puisse pas &ecirc;tre sauv&eacute;e par cofacteur exog&egrave;ne. Nous avons &eacute;galement constat&eacute; que les deux enzymes pourraient &ecirc;tre coimmunoprecipitated d&#39;extraits de tissus et propos&eacute;es les interactions fonctionnelles entre les enzymes qui s&#39;&eacute;tendait au-del&agrave; de la prestation du cofacteur par une voie d&#39;autre part. Ici, nous confirmons l&#39;association physique de ces enzymes, identifier les r&eacute;gions qui interagissent dans les deux, et nous d&eacute;montrons que leur association peut &ecirc;tre r&eacute;gl&eacute;e par la phosphorylation. Les cons&eacute;quences fonctionnelles de l&#39;interaction comprennent une augmentation de l&#39;activit&eacute; GTP cyclohydrolase, avec protection concomitante de r&eacute;troinhibition de produit fini. In vivo, cet effet &agrave; son tour fournirait suffisamment cofacteur lorsque la demande pour la synth&egrave;se des cat&eacute;cholamines est plus grande. L&#39;activit&eacute; de la tyrosine hydroxylase est &eacute;galement augment&eacute;e par cette interaction, d&eacute;passant la stimulation r&eacute;sultant de phosphorylation seule. Vmax est &eacute;lev&eacute;e, sans changement de km, ces r&eacute;sultats d&eacute;montrent que ces enzymes s&#39;engagent dans une r&eacute;gulation positive mutuelle.

Liaison du GTP cyclohydrolase et la tyrosine hydroxylase directe : interactions r&eacute;glementaires entre les enzymes cl&eacute;s dans la biosynth&egrave;se de la dopamine.

Die Signalisierung Funktionen des Dopamin erfordern eine fein abgestimmte regulatorischen Netzwerk f&uuml;r schnelle Induktion und Unterdr&uuml;ckung der Ausgabe. Ein Ziel der Verordnung ist das Enzym Tyrosin-Hydroxylase, das geschwindigkeitsbestimmende Enzym Dopamin-Synthese, die durch Phosphorylierung aktiviert und moduliert durch die Verf&uuml;gbarkeit seiner Adjunkte Tetrahydrobiopterin. Das erste Enzym in der Cofaktor Syntheseweg, GTP-Cyclohydrolase I, wird durch Phosphorylierung aktiviert und durch Tetrahydrobiopterin gehemmt. Wir bereits berichtet, dass Defizite in der GTP-Cyclohydrolase-Aktivit&auml;t in Drosophila heterozygote f&uuml;r mutierte Allele des Gens Kodierung dieses Enzym zu eng entsprechende Verringerung der in-vivo-Tyrosin-Hydroxylase-Aktivit&auml;t, die nicht durch exogene Cofaktor gerettet werden konnte. Wir fanden auch, dass die zwei Enzyme, die Coimmunoprecipitated von Gewebe-Extrakte und vorgeschlagenen funktionalen Wechselwirkungen zwischen der Enzyme, die &uuml;ber Bereitstellung von Cofaktor sich eine Option zum anderen hinaus sein k&ouml;nnte. Hier best&auml;tigen wir die k&ouml;rperliche Vereinigung dieser Enzyme identifizieren von interagierende Regionen in beiden, und wir zeigen, dass deren Assoziation durch Phosphorylierung reguliert werden kann. Die funktionellen Konsequenzen der Interaktion umfassen eine Erh&ouml;hung an GTP Cyclohydrolase-Aktivit&auml;t, mit gleichzeitigen Schutz vor-End-Produkt-Feedback-Hemmung. In-vivo, w&uuml;rde dieser Effekt wiederum ausreichend Cofaktor bereitstellen, wenn Nachfrage nach Katecholamin-Synthese am gr&ouml;&szlig;ten ist. Diese Interaktion &uuml;ber die Stimulation durch Phosphorylierung allein erh&ouml;ht auch die Aktivit&auml;t der Tyrosin-Hydroxylase. Vmax ist mit unver&auml;nderter km erh&ouml;ht, die diese Ergebnisse zeigen, dass diese Enzyme in gegenseitigen positiven Verordnung zu engagieren.

Direkte Bindung von GTP-Cyclohydrolase und Tyrosin-Hydroxylase: regulatorische Interaktionen zwischen wichtige Enzyme in Dopamin Biosynthese.

Le funzioni di segnalazione della dopamina richiedono una rete regolamentare finemente sintonizzata per rapida induzione e soppressione dell&#39;output. Un obiettivo chiave del regolamento &egrave; l&#39;enzima tirosina idrossilasi, l&#39;enzima di limitazione della velocit&agrave; nella sintesi della dopamina, che &egrave; attivata da fosforilazione e modulata dalla disponibilit&agrave; del suo cofattore, tetraidrobiopterina. Il primo enzima nella via di sintesi del cofattore, deficit di GTP cicloidrolasi I, &egrave; attivata da fosforilazione e inibito da tetraidrobiopterina. Abbiamo precedentemente riportato che deficit nell&#39;attivit&agrave; di GTP cicloidrolasi in drosofila eterozigote per gli alleli mutanti del gene che codifica per questo enzima ha condotto a strettamente corrispondente diminuzione di attivit&agrave; idrossilasi tirosina in vivo che non poteva essere salvato da cofattore esogeno. Abbiamo anche trovato che i due enzimi potrebbero essere coimmunoprecipitated da estratti di tessuto e proposte interazioni funzionali tra gli enzimi che si estendeva oltre la fornitura di cofattore di una via per un altro. Qui, confermiamo l&#39;associazione fisica di questi enzimi, identificando regioni interagenti in entrambi, e dimostriamo che la loro associazione pu&ograve; essere regolato dalla fosforilazione. Le conseguenze funzionali dell&#39;interazione includono un aumento nell&#39;attivit&agrave; di GTP cicloidrolasi, con concomitante protezione da inibizione feedback prodotto finale. In vivo, questo effetto sarebbe a sua volta fornire sufficiente cofattore quando la richiesta per la sintesi delle catecolamine &egrave; pi&ugrave; grande. L&#39;attivit&agrave; della tirosina idrossilasi &egrave; aumentata da questa interazione, eccesso di stimolazione derivanti dalla fosforilazione da solo. Vmax &egrave; elevata, con nessun cambiamento nel km. questi risultati dimostrano che questi enzimi impegnano in reciproca regolazione positiva.

Diretto legame del GTP cicloidrolasi e tirosina idrossilasi: normative interazioni tra enzimi chiave nella biosintesi della dopamina.

&#x30C9;&#x30FC;&#x30D1;&#x30DF;&#x30F3;&#x306E;&#x30B7;&#x30B0;&#x30CA;&#x30EA;&#x30F3;&#x30B0;&#x6A5F;&#x80FD;&#x306F;&#x6025;&#x901F;&#x306A;&#x8A98;&#x5C0E;&#x3068;&#x6291;&#x5236;&#x306E;&#x51FA;&#x529B;&#x3092;&#x7D30;&#x304B;&#x304F;&#x8ABF;&#x6574;&#x3055;&#x308C;&#x305F;&#x8ABF;&#x7BC0;&#x30CD;&#x30C3;&#x30C8;&#x30EF;&#x30FC;&#x30AF;&#x304C;&#x5FC5;&#x8981;&#x3067;&#x3059;&#x3002;&#x898F;&#x5236;&#x306E;&#x30AD;&#x30FC;&#x306E;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x9175;&#x7D20;&#x30C1;&#x30ED;&#x30B7;&#x30F3;&#x6C34;&#x9178;&#x5316;&#x9175;&#x7D20;&#x3001;&#x30EC;&#x30FC;&#x30C8;&#x5236;&#x9650;&#x9175;&#x7D20;&#x306B;&#x3088;&#x308B;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x6D3B;&#x6027;&#x5316;&#x3068;&#x305D;&#x306E;&#x88DC;&#x9175;&#x7D20;&#x306E;&#x30C6;&#x30C8;&#x30E9;&#x30D2;&#x30C9;&#x30ED;&#x30D3;&#x30AA;&#x30D7;&#x30C6;&#x30EA;&#x30F3;&#x306E;&#x53EF;&#x7528;&#x6027;&#x306B;&#x3088;&#x3063;&#x3066;&#x5909;&#x8ABF;&#x3055;&#x308C;&#x305F;&#x30C9;&#x30FC;&#x30D1;&#x30DF;&#x30F3;&#x306E;&#x5408;&#x6210;&#x3067;&#x3059;&#x3002;&#x88DC;&#x56E0;&#x5B50;&#x5408;&#x6210;&#x7D4C;&#x8DEF;&#x3001;&#x79C1;&#x306F;&#x3001;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x306B;&#x3088;&#x3063;&#x3066;&#x6D3B;&#x6027;&#x5316;&#x3055;&#x308C;&#x30C6;&#x30C8;&#x30E9;&#x30D2;&#x30C9;&#x30ED;&#x30D3;&#x30AA;&#x30D7;&#x30C6;&#x30EA;&#x30F3;&#x306B;&#x3088;&#x308B;&#x6291;&#x5236;&#x30B7;&#x30AF;&#x30ED;&#x30D2;&#x30C9;&#x30ED;&#x30E9;&#x30FC;&#x30BC;&#x306E;&#x6700;&#x521D;&#x306E;&#x9175;&#x7D20;&#x3002;&#x30B7;&#x30E7;&#x30A6;&#x30B8;&#x30E7;&#x30A6;&#x30D0;&#x30A8;&#x3053;&#x306E;&#x9175;&#x7D20;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x7A81;&#x7136;&#x5909;&#x7570;&#x4F53;&#x306E;&#x5BFE;&#x7ACB;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x30D8;&#x30C6;&#x30ED;&#x63A5;&#x5408;&#x4F53;&#x306B;&#x304A;&#x3051;&#x308B; GTP &#x30B7;&#x30AF;&#x30ED;&#x30D2;&#x30C9;&#x30ED;&#x30E9;&#x30FC;&#x30BC;&#x6D3B;&#x6027;&#x306E;&#x8D64;&#x5B57;&#x306B;&#x3064;&#x306A;&#x304C;&#x3063;&#x305F;&#x3053;&#x3068;&#x3092;&#x65E2;&#x5831;&#x5916;&#x56E0;&#x6027;&#x56E0;&#x5B50;&#x306B;&#x3088;&#x3063;&#x3066;&#x6551;&#x51FA;&#x3055;&#x308C;&#x308B;&#x53EF;&#x80FD;&#x6027;&#x306F;&#x3042;&#x308A;&#x307E;&#x305B;&#x3093; in vivo &#x30C1;&#x30ED;&#x30B7;&#x30F3;&#x6C34;&#x9178;&#x5316;&#x9175;&#x7D20;&#x6D3B;&#x6027;&#x306E;&#x3057;&#x3063;&#x304B;&#x308A;&#x3068;&#x5BFE;&#x5FDC;&#x3059;&#x308B;&#x6E1B;&#x5C11;&#x3002;&#x6211;&#x3005; &#x3082;&#x3001;2 &#x3064;&#x306E;&#x9175;&#x7D20;&#x306E;&#x30C6;&#x30A3;&#x30C3;&#x30B7;&#x30E5; &#x30A8;&#x30AD;&#x30B9;&#x3068;&#x63D0;&#x6848;&#x306E;&#x6A5F;&#x80FD;&#x7684;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x56E0;&#x5B50;&#x306E;&#x898F;&#x5B9A;&#x3092;&#x8D85;&#x3048;&#x3066; 1 &#x3064;&#x306E;&#x7D4C;&#x8DEF;&#x306B;&#x3088;&#x3063;&#x3066;&#x5225;&#x306E;&#x5EF6;&#x9577;&#x9175;&#x7D20;&#x304B;&#x3089; coimmunoprecipitated &#x3053;&#x3068;&#x304C;&#x308F;&#x304B;&#x3063;&#x305F;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x6211;&#x3005; &#x7269;&#x7406;&#x7684;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3053;&#x308C;&#x3089;&#x306E;&#x9175;&#x7D20;&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x9818;&#x57DF;&#x306E;&#x4E21;&#x65B9;&#x3067;&#x3001;&#x8B58;&#x5225;&#x3092;&#x78BA;&#x8A8D;&#x3057;&#x3066;&#x79C1;&#x305F;&#x3061;&#x306E;&#x5354;&#x4F1A;&#x306B;&#x3088;&#x308B;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x8ABF;&#x7BC0;&#x3067;&#x304D;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x6A5F;&#x80FD;&#x7684;&#x306A;&#x7D50;&#x679C;&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x306E;&#x5897;&#x52A0;&#x4F75;&#x7528;&#x4FDD;&#x8B77;&#x7D42;&#x308F;&#x308A;&#x88FD;&#x54C1;&#x30D5;&#x30A3;&#x30FC;&#x30C9;&#x30D0;&#x30C3;&#x30AF;&#x963B;&#x5BB3;&#x304B;&#x3089;&#x306E; GTP &#x30B7;&#x30AF;&#x30ED;&#x30D2;&#x30C9;&#x30ED;&#x30E9;&#x30FC;&#x30BC; &#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D3;&#x30C6;&#x30A3;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x30AB;&#x30C6;&#x30B3;&#x30FC;&#x30EB;&#x30A2;&#x30DF;&#x30F3;&#x5408;&#x6210;&#x306E;&#x305F;&#x3081;&#x306E;&#x9700;&#x8981;&#x304C;&#x6700;&#x5927;&#x306E;&#x3068;&#x304D;&#x306F;&#x3001;in vivo &#x3067;&#x3053;&#x306E;&#x52B9;&#x679C;&#x9806;&#x756A;&#x306B;&#x5341;&#x5206;&#x306A;&#x88DC;&#x56E0;&#x5B50;&#x63D0;&#x4F9B;&#x3067;&#x3057;&#x3087;&#x3046;&#x3002;&#x30C1;&#x30ED;&#x30B7;&#x30F3;&#x6C34;&#x9178;&#x5316;&#x9175;&#x7D20;&#x306E;&#x6D3B;&#x6027;&#x306F;&#x307E;&#x305F;&#x5358;&#x72EC;&#x3067;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x304B;&#x3089;&#x751F;&#x3058;&#x308B;&#x523A;&#x6FC0;&#x3092;&#x8D85;&#x3048;&#x308B;&#x3053;&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x306B;&#x3088;&#x3063;&#x3066;&#x5897;&#x52A0;&#x3057;&#x307E;&#x3059;&#x3002;Vmax &#x306F;&#x5909;&#x66F4;&#x305B;&#x305A;&#x306B;&#x3053;&#x308C;&#x3089;&#x306E;&#x7D50;&#x679C;&#x304B;&#x3089;&#x3053;&#x308C;&#x3089;&#x306E;&#x9175;&#x7D20;&#x304C;&#x76F8;&#x4E92;&#x306E;&#x80AF;&#x5B9A;&#x7684;&#x306A;&#x898F;&#x5236;&#x3067;&#x5F93;&#x4E8B;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3059; Km. &#x9AD8;&#x67B6;&#x3067;&#x3059;&#x3002;

&#x76F4;&#x63A5;&#x30D0;&#x30A4;&#x30F3;&#x30C9; GTP &#x30B7;&#x30AF;&#x30ED;&#x30D2;&#x30C9;&#x30ED;&#x30E9;&#x30FC;&#x30BC; &#x30C1;&#x30ED;&#x30B7;&#x30F3;&#x6C34;&#x9178;&#x5316;&#x9175;&#x7D20;&#x306E;&#xFF1A; &#x898F;&#x5236;&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x306B;&#x304A;&#x3051;&#x308B;&#x30C9;&#x30D1;&#x30DF;&#x30F3;&#x751F;&#x5408;&#x6210;&#x9375;&#x9175;&#x7D20;&#x3002;

&#xB3C4;&#xD30C;&#xBBFC;&#xC758; &#xC2E0;&#xD638; &#xAE30;&#xB2A5; &#xC2E0;&#xC18D;&#xD55C; &#xC720;&#xB3C4; &#xBC0F; &#xC5B5;&#xC81C; &#xCD9C;&#xB825;&#xC758; &#xAC00;&#xB298;&#xAC8C; &#xC870;&#xC815;&#xB41C; &#xADDC;&#xC81C; &#xB124;&#xD2B8;&#xC6CC;&#xD06C;&#xAC00; &#xD544;&#xC694;&#xD569;&#xB2C8;&#xB2E4;. &#xADDC;&#xC81C;&#xC758; &#xC8FC;&#xC694; &#xB300;&#xC0C1; &#xD6A8;&#xC18C; &#xD2F0;&#xB85C;&#xC2E0; hydroxylase &#xC778; &#xC0B0;&#xD654;&#xC5D0; &#xC758;&#xD574; &#xD65C;&#xC131;&#xD654; &#xBC0F; &#xADF8;&#xAC83;&#xC758; &#xACF5;&#xB3D9; &#xC778;&#xC790;, tetrahydrobiopterin&#xC758; &#xAC00;&#xC6A9;&#xC131;&#xC5D0; &#xC758;&#xD574; &#xBCC0;&#xC870; &#xD558;&#xB294; &#xB3C4;&#xD30C;&#xBBFC; &#xD569;&#xC131;&#xC758; &#xC18D;&#xB3C4; &#xC81C;&#xD55C; &#xD6A8;&#xC18C; &#xC774;&#xB2E4;. GTP cyclohydrolase, &#xC778; &#xC0B0;&#xD654;&#xC5D0; &#xC758;&#xD574; &#xD65C;&#xC131;&#xD654; &#xBC0F; tetrahydrobiopterin&#xC5D0; &#xC758;&#xD574; &#xC800;&#xD574; cofactor &#xD569;&#xC131; &#xACBD;&#xB85C;&#xC5D0;&#xC11C; &#xCCAB; &#xBC88;&#xC9F8; &#xD6A8;&#xC18C;. &#xC6B0;&#xB9AC;&#xB294; &#xC774;&#xC804;&#xC5D0; &#xC778;&#xCF54;&#xB529;&#xC774; &#xD6A8;&#xC18C; &#xC720;&#xC804;&#xC790;&#xC758; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xCCB4; &#xB300;&#xB9BD; heterozygous &#xCD08;&#xD30C;&#xB9AC;&#xC5D0;&#xC11C; GTP cyclohydrolase &#xD65C;&#xB3D9;&#xC5D0;&#xC11C; &#xC801;&#xC790;&#xB97C; &#xC8FC;&#xB3C4; &#xBCF4;&#xACE0; exogenous &#xACF5;&#xB3D9; &#xC778;&#xC790;&#xC5D0; &#xC758;&#xD574; &#xAD6C;&#xCD9C; &#xD558;&#xC9C0; &#xC218; vivo&#xC5D0;&#xC11C; &#xD2F0;&#xB85C;&#xC2E0; hydroxylase &#xD65C;&#xB3D9;&#xC758; &#xAC10;&#xC18C;&#xB294; &#xBC00;&#xC811; &#xD558; &#xAC8C; &#xD574;&#xB2F9;. &#xC6B0;&#xB9AC;&#xB294; &#xB610;&#xD55C; &#xB450; &#xD6A8;&#xC18C; &#xC870;&#xC9C1; &#xCD94;&#xCD9C; &#xBB3C;&#xACFC; &#xD6A8;&#xC18C;&#xC758; cofactor&#xC758; &#xD55C; &#xD1B5;&#xB85C; &#xC758;&#xD574; &#xB610; &#xB2E4;&#xB978; &#xD655;&#xC7A5; &#xC0AC;&#xC774; &#xC81C;&#xC548; &#xB41C; &#xAE30;&#xB2A5; &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC5D0;&#xC11C; coimmunoprecipitated &#xC218; &#xBC1C;&#xACAC;. &#xC5EC;&#xAE30;, &#xC6B0;&#xB9AC; &#xBAA8;&#xB450;&#xC5D0;&#xC11C; &#xC0C1;&#xD638; &#xC791;&#xC6A9; &#xC601;&#xC5ED;&#xC744; &#xC2DD;&#xBCC4; &#xC774;&#xB7EC;&#xD55C; &#xD6A8;&#xC18C;&#xC758; &#xC2E4;&#xC81C; &#xC5F0;&#xACB0;&#xC744; &#xD655;&#xC778; &#xD558; &#xACE0; &#xADF8;&#xB4E4;&#xC758; &#xD611;&#xD68C; &#xC778; &#xC0B0;&#xD654;&#xC5D0; &#xC758;&#xD574; &#xD1B5;&#xC81C; &#xB420; &#xC218; &#xBCF4;&#xC5EC; &#xC90D;&#xB2C8;&#xB2E4;. &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC758; &#xAE30;&#xB2A5;&#xC801; &#xACB0;&#xACFC; &#xCD5C;&#xC885; &#xC81C;&#xD488; &#xD53C;&#xB4DC;&#xBC31; &#xC5B5;&#xC81C;&#xC5D0;&#xC11C; &#xBD80;&#xC218;&#xC801;&#xC778; &#xBCF4;&#xD638;&#xC640; GTP cyclohydrolase &#xD65C;&#xB3D9;&#xC5D0; &#xC99D;&#xAC00; &#xD3EC;&#xD568;&#xD569;&#xB2C8;&#xB2E4;. Vivo&#xC5D0;&#xC11C;&#xC774; &#xD6A8;&#xACFC; &#xC81C;&#xACF5; &#xD558;&#xB294; &#xAC83; &#xCDA9;&#xBD84; &#xD55C; cofactor &#xCE74; &#xD14C; &#xCF5C; &#xC544;&#xBBFC; &#xD569;&#xC131;&#xC5D0; &#xB300; &#xD55C; &#xC218;&#xC694;&#xAC00; &#xAC00;&#xC7A5; &#xD070; &#xACBD;&#xC6B0;. &#xD2F0;&#xB85C;&#xC2E0; hydroxylase &#xD65C;&#xB3D9; &#xB610;&#xD55C; &#xD63C;&#xC790; &#xC778; &#xC0B0;&#xD654;&#xC5D0;&#xC11C; &#xBC1C;&#xC0DD; &#xD558;&#xB294; &#xC790;&#xADF9; &#xCD08;&#xACFC;&#xC774; &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC5D0; &#xC758;&#xD574; &#xC99D;&#xAC00; &#xD588;&#xB2E4;. Vmax&#xB294; &#xC774;&#xB7EC;&#xD55C; &#xACB0;&#xACFC; &#xC785;&#xC99D;&#xC774; &#xD6A8;&#xC18C; &#xC0C1;&#xD638; &#xAE0D;&#xC815;&#xC801;&#xC778; &#xADDC;&#xC81C;&#xC5D0; &#xC885;&#xC0AC; &#xD0AC;&#xB85C;&#xBBF8;&#xD130;&#xC5D0; &#xBCC0;&#xD654; &#xC5C6;&#xC774; &#xC0C1;&#xC2B9; &#xB41C;.

&#xC9C1;&#xC811; GTP cyclohydrolase &#xBC0F; &#xD2F0;&#xB85C;&#xC2E0; hydroxylase &#xBC14;&#xC778;&#xB529;: &#xB3C4;&#xD30C;&#xBBFC; &#xC0DD; &#xD569;&#xC131;&#xC758; &#xC8FC;&#xC694; &#xD6A8;&#xC18C; &#xAC04;&#xC758; &#xADDC;&#xC81C; &#xC0C1;&#xD638; &#xC791;&#xC6A9;.

Las funciones de se&ntilde;alizaci&oacute;n de la dopamina requieren una red regulador finamente sintonizada para inducci&oacute;n r&aacute;pida y supresi&oacute;n de la producci&oacute;n. Un objetivo clave del Reglamento es la enzima tirosina hidroxilasa, la enzima tarifa-limitadora en s&iacute;ntesis de la dopamina, que es activado por fosforilaci&oacute;n y modulada por la disponibilidad de su cofactor, tetrahidrobiopterina. La primera enzima en la v&iacute;a de s&iacute;ntesis de cofactor, GTP ciclohidrolasa I, se activa por fosforilaci&oacute;n e inhibida por tetrahidrobiopterina. Divulgamos previamente que en la actividad de GTP ciclohidrolasa en Drosophila heterocigoto para alelos mutantes del gen que codifica esta enzima propici&oacute; que firmemente correspondiente disminuci&oacute;n de la actividad tirosina in vivo de la hidroxilasa que no pudo ser rescatada por cofactor ex&oacute;gena. Tambi&eacute;n encontramos que las dos enzimas podr&iacute;an ser coinmunoprecipit&oacute; de extractos de tejido y propuestas interacciones funcionales entre las enzimas que se extend&iacute;a m&aacute;s all&aacute; de la disposici&oacute;n del cofactor por una v&iacute;a para el otro. Aqu&iacute;, nos confirman la Asociaci&oacute;n f&iacute;sica de estas enzimas, identificar las regiones de interacci&oacute;n en ambos, y demostramos que su asociaci&oacute;n puede ser regulada por fosforilaci&oacute;n. Las consecuencias funcionales de la interacci&oacute;n incluyen un aumento en la actividad de GTP ciclohidrolasa, con protecci&oacute;n concomitante de inhibici&oacute;n de la regeneraci&oacute;n del producto final. In vivo, este efecto a su vez dar&iacute;a cofactor suficiente cuando es mayor la demanda de s&iacute;ntesis de catecolaminas. Esta interacci&oacute;n, por encima de la estimulaci&oacute;n resultante de fosforilaci&oacute;n sola tambi&eacute;n se incrementa la actividad de la tirosina hidroxilasa. Vmax es elevado, sin cambio en km., estos resultados demuestran que estas enzimas practiquen la mutua regulaci&oacute;n positiva.

Uni&oacute;n de GTP ciclohidrolasa y tirosina hidroxilasa directa: interacciones reguladoras entre enzimas clave en la bios&iacute;ntesis de la dopamina.

The underlying defects in Angelman syndrome (AS) and autism spectrum disorder (ASD) may be in part due to basic defects in synaptic plasticity and function. In some individuals serotonin reuptake inhibitors, which decrease pre-synaptic re-uptake of serotonin, can ameliorate symptoms, as can resperidone, which blocks both dopamine and serotonin receptors. Loss of maternal UBE3A expression causes AS, while maternal duplications of chromosome 15q11.2-q13 that include the UBE3A gene cause ASD, implicating the maternally expressed UBE3A gene in the ASD phenotype. In a Drosophila screen for proteins regulated by UBE3A, we identified a key regulator of monoamine synthesis, the gene Punch, or GCH1, encoding the enzyme GTP cyclohydrolase I. Here we show that Dube3a, the fly UBE3A orthologue, regulates Punch/GCH1 in the fly brain. Over-expression of Dube3a elevates tetrahydrobiopterin (THB), the rate-limiting cofactor in monoamine synthesis while loss of Dube3a has the opposite effect. The fluctuations in dopamine levels were associated with hyper- and hypoactivity, respectively, in flies. We show that changes in Punch/GCH1 and dopamine levels do not depend on the ubiquitin ligase catalytic domain of Dube3a. In addition, both wild type Dube3a and a ubiquitination-defective Dube3a-C/A form were found at high levels in nuclear fractions and appear to be poly-ubiquitinated in vivo by endogenous Dube3a. We propose that the transcriptional co-activation function of Dube3a may regulate GCH1 activity in the brain. These results provide a connection between monoamine synthesis (dopamine/serotonin) and Dube3a expression that may explain why some individuals with ASD or AS respond better to selective serotonin reuptake inhibitors than others.

Drosophila Ube3a regulates monoamine synthesis by increasing GTP cyclohydrolase I activity via a non-ubiquitin ligase mechanism.

The highly reactive nature of dopamine renders dopaminergic neurons vulnerable to oxidative damage. We recently demonstrated that loss-of-function mutations in the Drosophila gene Catecholamines up (Catsup) elevate dopamine pools but, paradoxically, also confer resistance to paraquat, an herbicide that induces oxidative stress-mediated toxicity in dopaminergic neurons. We now report a novel association of the membrane protein, Catsup, with GTP cyclohydrolase rate-limiting enzyme for tetrahydrobiopterin (BH(4)) biosynthesis and tyrosine hydroxylase, rate-limiting enzyme for dopamine biosynthesis, which requires BH(4) as a cofactor. Loss-of-function Catsup mutations cause dominant hyperactivation of both enzymes. Elevated dopamine levels in Catsup mutants coincide with several distinct characteristics, including hypermobility, minimal basal levels of 3,4-dihydroxy-phenylacetic acid, an oxidative metabolite of dopamine, and resistance to the vesicular monoamine transporter inhibitor, reserpine, suggesting that excess dopamine is synaptically active and that Catsup functions in the regulation of synaptic vesicle loading and release of dopamine. We conclude that Catsup regulates and links the dopamine synthesis and transport networks.

Catecholamines up integrates dopamine synthesis and synaptic trafficking.

Dystonia represents the third most common movement disorder in humans. At least 15 genetic loci (DYT1-15) have been identified and some of these genes have been cloned. TOR1A (formally DYT1), the gene responsible for the most common primary hereditary dystonia, encodes torsinA, an AAA ATPase family protein. However, the function of torsinA has yet to be fully understood. Here, we have generated and characterized a complete loss-of-function mutant for dtorsin, the only Drosophila ortholog of TOR1A. Null mutation of the X-linked dtorsin was semi-lethal with most male flies dying by the pre-pupal stage and the few surviving adults being sterile and slow moving, with reduced cuticle pigmentation and thin, short bristles. Third instar male larvae exhibited locomotion defects that were rescued by feeding dopamine. Moreover, biochemical analysis revealed that the brains of third instar larvae and adults heterozygous for the loss-of-function dtorsin mutation had significantly reduced dopamine levels. The dtorsin mutant showed a very strong genetic interaction with Pu (Punch: GTP cyclohydrolase), the ortholog of the human gene underlying DYT14 dystonia. Biochemical analyses revealed a severe reduction of GTP cyclohydrolase protein and activity, suggesting that dtorsin plays a novel role in dopamine metabolism as a positive-regulator of GTP cyclohydrolase protein. This dtorsin mutant line will be valuable for understanding this relationship and potentially other novel torsin functions that could play a role in human dystonia.

Dtorsin, the Drosophila ortholog of the early-onset dystonia TOR1A (DYT1), plays a novel role in dopamine metabolism.

The neurological movement disorder dystonia is an umbrella term for a heterogeneous group of related conditions where at least 20 monogenic forms have been identified. Despite the substantial advances resulting from the identification of these loci, the function of many DYT gene products remains unclear. Comparative genomics using simple animal models to examine the evolutionarily conserved functional relationships with monogenic dystonias represents a rapid route toward a comprehensive understanding of these movement disorders. Current studies using the invertebrate animal models Caenorhabditis elegans and Drosophila melanogaster are uncovering cellular functions and mechanisms associated with mutant forms of the well-conserved gene products corresponding to DYT1, DYT5a, DYT5b, and DYT12 dystonias. Here we review recent findings from the invertebrate literature pertaining to molecular mechanisms of these gene products, torsinA, GTP cyclohydrolase I, tyrosine hydroxylase, and the alpha subunit of Na+/K ATPase, respectively. In each study, the application of powerful genetic tools developed over decades of intensive work with both of these invertebrate systems has led to mechanistic insights into these human disorders. These models are particularly amenable to large-scale genetic screens for modifiers or additional alleles, which are bolstering our understanding of the molecular functions associated with these gene products. Moreover, the use of invertebrate models for the evaluation of DYT genetic loci and their genetic interaction networks has predictive value and can provide a path forward for therapeutic intervention.

Article	Year
A typical N-terminal extensions confer novel regulatory properties on GTP cyclohydrolase isoforms in Drosophila melanogaster. The Journal of biological chemistry\| PubMed ID: 16966327	2006
Interaction of genetic and environmental factors in a Drosophila parkinsonism model. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 17344383	2007
Drosophila dopamine synthesis pathway genes regulate tracheal morphogenesis. Developmental biology\| PubMed ID: 17585895	2007
Direct binding of GTP cyclohydrolase and tyrosine hydroxylase: regulatory interactions between key enzymes in dopamine biosynthesis. The Journal of biological chemistry\| PubMed ID: 18801743	2008
Drosophila Ube3a regulates monoamine synthesis by increasing GTP cyclohydrolase I activity via a non-ubiquitin ligase mechanism. Neurobiology of disease\| PubMed ID: 21147225	2011
Catecholamines up integrates dopamine synthesis and synaptic trafficking. Journal of neurochemistry\| PubMed ID: 21985068	2011
Dtorsin, the Drosophila ortholog of the early-onset dystonia TOR1A (DYT1), plays a novel role in dopamine metabolism. PloS one\| PubMed ID: 22022556	2011
Invertebrate models of dystonia. Current neuropharmacology\| PubMed ID: 23814534	2013

Janis M. O'Donnell

Biography

JoVE Journal Publications

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