Subcellular Fractionation for the Isolation of Synaptic Components from the Murine Brain

Synaptic terminals are the primary sites of neuronal communication. Synaptic dysfunction is a hallmark of many neuropsychiatric and neurological ...

Yale University

Synucleins are a vertebrate-specific family of abundant neuronal proteins. They comprise three closely related members, α-, β-, and γ-synuclein. α-Synuclein has been the focus of intense attention since mutations in it were identified as a cause for familial Parkinson's disease. Despite their disease relevance, the normal physiological function of synucleins has remained elusive. To address this, we generated and characterized αβγ-synuclein knockout mice, which lack all members of this protein family. Deletion of synucleins causes alterations in synaptic structure and transmission, age-dependent neuronal dysfunction, as well as diminished survival. Abrogation of synuclein expression decreased excitatory synapse size by ∼30% both in vivo and in vitro, revealing that synucleins are important determinants of presynaptic terminal size. Young synuclein null mice show improved basic transmission, whereas older mice show a pronounced decrement. The late onset phenotypes in synuclein null mice were not due to a loss of synapses or neurons but rather reflect specific changes in synaptic protein composition and axonal structure. Our results demonstrate that synucleins contribute importantly to the long-term operation of the nervous system and that alterations in their physiological function could contribute to the development of Parkinson's disease.

αβγ-Synuclein triple knockout mice reveal age-dependent neuronal dysfunction.

Synucleins &#xFF08;&alpha;&#x3001; &beta;&#x3001; &#x548C; &gamma;&#xFF09; &#x9AD8;&#x5EA6;&#x540C;&#x6E90;&#x86CB;&#x767D;&#x8D28;&#x60F3;&#x53D1;&#x6325;&#x8C03;&#x8282;&#x4F5C;&#x7528;&#x611F;&#x53D7;&#x5668;&#xFF0C;&#x5728;&#x7A81;&#x89E6;&#x524D;&#x7801;&#x5934;&#x4E2D;&#x6E05;&#x695A;&#x5730;&#x627E;&#x5230;&#x3002;&#x514B;&#x670D; synuclein &#x86CB;&#x767D;&#x529F;&#x80FD;&#x91CD;&#x53E0;&#xFF0C;&#x4E86;&#x89E3;&#x5176;&#x5728;&#x4ECE; mesostriatal &#x591A;&#x5DF4;&#x80FA;&#x80FD;&#x795E;&#x7ECF;&#x5143;&#x7A81;&#x89E6;&#x524D;&#x4FE1;&#x53F7;&#x4E2D;&#x7684;&#x4F5C;&#x7528;&#xFF0C;&#x6211;&#x4EEC;&#x5236;&#x4F5C;&#x4E86;&#x4E09;&#x4E2A;&#x5BB6;&#x5EAD;&#x6240;&#x6709;&#x6210;&#x5458;&#x7684; synuclein &#x90FD;&#x7F3A;&#x4E4F;&#x7684;&#x5C0F;&#x9F20;&#x3002;&#x5BF9; mesostriatal &#x7CFB;&#x7EDF;&#x7684;&#x5F71;&#x54CD;&#x8BC4;&#x4F30;&#x4E2D;&#x6210;&#x4EBA; (4-14 &#x4E2A;&#x6708;-&#x8001;) &#x52A8;&#x7269;&#x884C;&#x4E3A;&#x5B66;&#x3001; &#x751F;&#x7269;&#x5316;&#x5B66;&#xFF0C;&#x7EC4;&#x7EC7;&#x5B66;&#x3001; &#x548C;&#x7535;&#x5316;&#x5B66;&#x6280;&#x672F;&#x7ED3;&#x5408;&#x4F7F;&#x7528;&#x3002;&#x6210;&#x4EBA;&#x4E09;-synuclein-null &#xFF08;&#x5C06;&#x519B;&#x6FB3;&#xFF09; &#x5C0F;&#x9F20;&#x7684;&#x4E2D;&#x8111;&#x591A;&#x5DF4;&#x80FA;&#x80FD;&#x795E;&#x7ECF;&#x5143;&#x6570;&#x76EE;&#x663E;&#x793A;&#x6CA1;&#x6709;&#x516C;&#x5F00;&#x8868;&#x578B;&#x548C;&#x6CA1;&#x6709;&#x53D8;&#x5316;&#x3002;&#x5C06;&#x519B;&#x6FB3;&#x5C0F;&#x9F20;&#x52A8;&#x5728;&#x65B0;&#x73AF;&#x5883;&#x4E2D;&#xFF0C;&#x8868;&#x73B0;&#x51FA;&#x4E0E;&#x5FEB;&#x901F;&#x626B;&#x63CF;&#x5FAA;&#x73AF;&#x4F0F;&#x5B89;&#x6CD5;&#x68C0;&#x6D4B;&#x7EB9;&#x72B6;&#x4F53;&#x4E2D;&#x591A;&#x5DF4;&#x80FA;&#x542B;&#x91CF;&#x5347;&#x9AD8;&#x8BF1;&#x53D1;&#x7684;&#x91CA;&#x653E;&#x3002;&#x63D0;&#x5347;&#x591A;&#x5DF4;&#x80FA;&#x91CA;&#x653E;&#x662F;&#x7279;&#x5B9A;&#x4E8E;&#x80CC;&#x4E0D;&#x8179;&#x4FA7;&#x7EB9;&#x72B6;&#x4F53;&#x548C;&#x966A;&#x540C;&#x7684;&#x591A;&#x5DF4;&#x80FA;&#x7EC4;&#x7EC7;&#x542B;&#x91CF;&#x51CF;&#x5C11;&#x3002;&#x6211;&#x4EEC;&#x786E;&#x8BA4;&#x6B63;&#x5E38;&#x7684;&#x7A81;&#x89E6;&#x8D85;&#x5FAE;&#x7ED3;&#x6784;&#x548C;&#x80CC;&#x4FA7;&#x7EB9;&#x72B6;&#x4F53;&#x7684;&#x9677;&#x9631; (&#x53EF;&#x6EB6;&#x6027; N ethylmaleimide &#x654F;&#x611F;&#x56E0;&#x5B50;&#x9644;&#x4EF6;&#x86CB;&#x767D;&#x53D7;&#x4F53;) &#x86CB;&#x767D;&#x590D;&#x5408;&#x7269;&#x7684;&#x6B63;&#x5E38;&#x4E30;&#x5EA6;&#x3002;&#x5C06;&#x519B;&#x6FB3;&#x52A8;&#x7269;&#x5F71;&#x54CD;&#x591A;&#x5DF4;&#x80FA;&#x4EE3;&#x8C22;&#x7684;&#x836F;&#x7269;&#x6CBB;&#x7597;&#x663E;&#x793A;&#x5408;&#x6210;&#x6B63;&#x5E38;&#x901F;&#x5EA6;&#x3001; &#x589E;&#x5F3A;&#x7684;&#x8425;&#x4E1A;&#x989D;&#xFF0C;&#x800C;&#x4E14;&#x51CF;&#x5C11;&#x7A81;&#x89E6;&#x524D;&#x7EB9;&#x72B6;&#x4F53;&#x591A;&#x5DF4;&#x80FA;&#x5546;&#x5E97;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x6570;&#x636E;&#x552F;&#x4E00;&#x5730;&#x63ED;&#x793A; synuclein &#x86CB;&#x767D;&#x7684;&#x8C03;&#x63A7;&#x4ECE;&#x7279;&#x5B9A;&#x7FA4;&#x4F53;&#x4E2D;&#x8111;&#x591A;&#x5DF4;&#x80FA;&#x795E;&#x7ECF;&#x5143;&#x7684;&#x795E;&#x7ECF;&#x9012;&#x8D28;&#x91CA;&#x653E;&#x901A;&#x8FC7;&#x4E0D;&#x540C;&#x4E8E;&#x90A3;&#x4E9B;&#x5728;&#x5176;&#x4ED6;&#x795E;&#x7ECF;&#x5143;&#x4E2D;&#x62A5;&#x544A;&#x7684;&#x673A;&#x5236;&#x7684;&#x91CD;&#x8981;&#x6027;&#x3002;&#x8FD9;&#x4E00;&#x53D1;&#x73B0;&#x7684; synucleins &#x7684;&#x5B8C;&#x6574;&#x635F;&#x5931;&#x4F1A;&#x5BFC;&#x81F4;&#x591A;&#x5DF4;&#x80FA;&#x5904;&#x7406;&#xFF0C;&#x7279;&#x522B;&#x662F;&#x5728;&#x8FD9;&#x4E9B;&#x5730;&#x533A;&#x4F18;&#x5148;&#x8106;&#x5F31;&#x5728;&#x5E15;&#x91D1;&#x68EE;&#x75C5;&#x4E2D;&#x7684;&#x7A81;&#x89E6;&#x524D;&#x7801;&#x5934;&#x7684;&#x53D8;&#x5316;&#x53EF;&#x80FD;&#x6700;&#x7EC8;&#x544A;&#x77E5;&#x9009;&#x62E9;&#x6027;&#x7814;&#x7A76;&#x75BE;&#x75C5;&#x7684;&#x8FC7;&#x7A0B;&#x3002;

&#x5728;&#x7F3A;&#x4E4F;&#x6240;&#x6709;&#x4E09;&#x4E2A;&#x5BB6;&#x5EAD;&#x6210;&#x5458;&#x7684; synuclein &#x5C0F;&#x9F20;&#x9ED1;&#x8D28;&#x7EB9;&#x72B6;&#x4F53;&#x7CFB;&#x7EDF;&#x4E2D;&#x7684;&#x529F;&#x80FD;&#x6539;&#x53D8;&#x3002;

The synucleins (Î±, Î², and Î³) are highly homologous proteins thought to play a role in regulating neurotransmission and are found abundantly in presynaptic terminals. To overcome functional overlap between synuclein proteins and to understand their role in presynaptic signaling from mesostriatal dopaminergic neurons, we produced mice lacking all three members of the synuclein family. The effect on the mesostriatal system was assessed in adult (4- to 14-month-old) animals using a combination of behavioral, biochemical, histological, and electrochemical techniques. Adult triple-synuclein-null (TKO) mice displayed no overt phenotype and no change in the number of midbrain dopaminergic neurons. TKO mice were hyperactive in novel environments and exhibited elevated evoked release of dopamine in the striatum detected with fast-scan cyclic voltammetry. Elevated dopamine release was specific to the dorsal not ventral striatum and was accompanied by a decrease of dopamine tissue content. We confirmed a normal synaptic ultrastructure and a normal abundance of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein complexes in the dorsal striatum. Treatment of TKO animals with drugs affecting dopamine metabolism revealed normal rate of synthesis, enhanced turnover, and reduced presynaptic striatal dopamine stores. Our data uniquely reveal the importance of the synuclein proteins in regulating neurotransmitter release from specific populations of midbrain dopamine neurons through mechanisms that differ from those reported in other neurons. The finding that the complete loss of synucleins leads to changes in dopamine handling by presynaptic terminals specifically in those regions preferentially vulnerable in Parkinson's disease may ultimately inform on the selectivity of the disease process.

Functional alterations to the nigrostriatal system in mice lacking all three members of the synuclein family.

Les synucleins (&#x3B1;, &#x3B2;, &#x3B3; et) sont hautement homologues des prot&#xE9;ines pens&#xE9; &#xE0; jouer un r&#xF4;le dans la r&#xE9;gulation de la neurotransmission et se trouvent en abondance dans les terminaisons pr&#xE9;synaptiques. Pour surmonter les chevauchements fonctionnels entre prot&#xE9;ines synucl&#xE9;ine et de comprendre leur r&#xF4;le dans la signalisation pr&#xE9;synaptiques de mesostriatal neurones dopaminergiques, nous avons produit des souris d&#xE9;pourvues de toutes trois membres de la famille synucl&#xE9;ine. L&#x26;#39;effet sur le syst&#xE8;me mesostriatal a &#xE9;t&#xE9; &#xE9;valu&#xE9;e dans des adultes (4 - &#xE0; 14-month-old) des animaux en utilisant une combinaison de techniques comportementales, biochimiques, histologiques et &#xE9;lectrochimique. Adultes triple-synucl&#xE9;ine-null (TKO) souris affich&#xE9; aucun ph&#xE9;notype manifeste, et aucun changement dans le nombre de neurones du m&#xE9;senc&#xE9;phale dopaminergiques. TKO souris &#xE9;taient hyperactifs dans des environnements nouveaux et expos&#xE9; &#xE9;lev&#xE9;e lib&#xE9;ration &#xE9;voqu&#xE9;e de la dopamine dans le striatum d&#xE9;tect&#xE9; par voltamm&#xE9;trie cyclique balayage rapide. La lib&#xE9;ration de dopamine &#xE9;lev&#xE9;e &#xE9;tait sp&#xE9;cifique &#xE0; l&#x26;#39;striatum dorsal pas ventrale et a &#xE9;t&#xE9; accompagn&#xE9;e par une diminution de la teneur des tissus dopamine. Nous avons confirm&#xE9; une ultrastructure normale synaptique et une abondance normale des complexes SNARE prot&#xE9;ines solubles (N-&#xE9;thylmal&#xE9;imide sensible r&#xE9;cepteur de prot&#xE9;ine facteur d&#x26;#39;attachement) dans le striatum dorsal. Traitement des animaux avec des m&#xE9;dicaments qui affectent TKO m&#xE9;tabolisme de la dopamine a r&#xE9;v&#xE9;l&#xE9; le taux normal de la synth&#xE8;se, le chiffre d&#x26;#39;affaires am&#xE9;lior&#xE9;, et la r&#xE9;duction des pr&#xE9;synaptiques magasins dopaminergiques du striatum. Nos donn&#xE9;es r&#xE9;v&#xE8;lent l&#x26;#39;importance unique des prot&#xE9;ines synucl&#xE9;ine dans la r&#xE9;gulation de la lib&#xE9;ration de neurotransmetteurs &#xE0; partir de populations sp&#xE9;cifiques de neurones dopaminergiques du m&#xE9;senc&#xE9;phale &#xE0; travers des m&#xE9;canismes qui diff&#xE8;rent de ceux rapport&#xE9;s dans d&#x26;#39;autres neurones. La constatation que la perte totale de synucleins conduit &#xE0; des changements dans la manipulation de dopamine par les terminaux pr&#xE9;synaptiques sp&#xE9;cifiquement dans ces r&#xE9;gions vuln&#xE9;rables de pr&#xE9;f&#xE9;rence dans la maladie de Parkinson pourrait finalement informer sur la s&#xE9;lectivit&#xE9; du processus de la maladie.

Alt&#xE9;rations fonctionnelles au syst&#xE8;me nigro-stri&#xE9; chez les souris d&#xE9;pourvues Les trois membres de la famille Synucl&#xE9;ine

Die Synucleins (&alpha;, &beta; und &gamma;) sind hochgradig homologe Proteine spielen eine Rolle bei der Regulierung der Neurotransmission und findet man reichlich in pr&auml;synaptischen Klemmen. Funktionale &Uuml;berschneidungen zwischen Synuclein Proteine zu &uuml;berwinden und ihre Rolle in der pr&auml;synaptischen Signalisierung von Mesostriatal dopaminergen Neuronen verstehen, produzierten wir M&auml;usen fehlt alle drei Mitglieder der Familie Synuclein. Die Auswirkungen auf das Mesostriatal-System wurde in Erwachsene (4 bis 14-Monate alten) Tiere, die mit einer Kombination aus Verhaltens-, biochemische, histologische und elektrochemische Techniken bewertet. Erwachsene dreifach-Synuclein-Null (TKO) M&auml;use angezeigt keine offenkundige Ph&auml;notyp und keine &Auml;nderung der Zahl der Mittelhirn dopaminergen Neuronen. TKO M&auml;use wurden in neue Umgebungen hyperaktiv und stellte erh&ouml;hten evozierte Freisetzung von Dopamin im Striatum mit schnell-Scan Cyclovoltammetrie erkannt. Erh&ouml;hte Dopamin-Version wurde speziell f&uuml;r den dorsalen nicht ventralen Striatum und ging einher mit einer Abnahme der Dopamin-Gewebe-Inhalt. Wir best&auml;tigten eine normale synaptische Ultrastruktur und eine normale F&uuml;lle von SNARE (l&ouml;sliche N-Ethylmaleimid-Sensitive-Faktor-Anlage Protein-Rezeptor)-Proteinkomplexen im dorsalen Striatum. Behandlung der TKO-Tiere mit Medikamenten, die die Dopamin-Stoffwechsel offenbart Normaltarif der Synthese, verbessert Umsatz und reduziert pr&auml;synaptischen Striatale Dopamin L&auml;den. Unsere Daten zeigen eindeutig die Bedeutung der Synuclein-Proteine bei der Regulierung der Neurotransmitter-Freisetzung aus bestimmten Populationen von Mittelhirn Dopamin-Neuronen durch Mechanismen, die unterscheiden sich von denen in anderen Neuronen berichtet. Die Feststellung, die der v&ouml;lligen Verlust von Synucleins f&uuml;hrt zu &Auml;nderungen in der Handhabung von pr&auml;synaptischen Terminals speziell in diesen Regionen bevorzugt anf&auml;llig bei Parkinson-Krankheit Dopamin kann letztlich auf die Selektivit&auml;t der Krankheitsprozess informieren.

Funktionale &Auml;nderungen zum Nigrostriatal System in M&auml;usen fehlt alle drei Mitglieder der Familie Synuclein.

Synucleins (&alpha;, &beta; e &gamma;) sono proteine altamente omologhe pensate di giocare un ruolo nella regolazione della neurotrasmissione e si trovano abbondantemente nei terminali presinaptiche. Per superare le sovrapposizioni funzionali tra proteine sinucleina e capire il loro ruolo nella segnalazione presinaptica da Neuroni dopaminergici mesostriatal, abbiamo prodotto topi privi di tutti i tre membri della famiglia sinucleina. In animali adulti (4 - a 14 mesi) utilizzando una combinazione di tecniche comportamentali, biochimiche, istologiche ed elettrochimiche &egrave; stato valutato l&#39;effetto sul sistema mesostriatal. Topi adulti triple-synuclein-null (TKO) visualizzato nessun fenotipo palese e nessun cambiamento del numero di Neuroni dopaminergici mesencefalo. TKO topi erano iperattivi in nuovi ambienti ed esposta elevata evocato rilascio di dopamina nello striato rilevato con fast-scansione ciclovoltammetria. Rilascio di dopamina elevata era specifico per lo striato dorsale non ventrale e fu accompagnato da una diminuzione del contenuto di dopamina tessuto. Abbiamo confermato un normale ultrastruttura sinaptica e una normale abbondanza dei complessi della proteina SNARE (solubile N-ethylmaleimide-sensibile fattore accessorio proteina recettore) lo striato dorsale. Trattamento degli animali TKO con farmaci che influenzano il metabolismo della dopamina ha rivelato un tasso normale di sintesi, maggiore fatturato e ridotto striatal presinaptico della dopamina negozi. I nostri dati rivelano in modo univoco l&#39;importanza delle proteine sinucleina nella regolazione rilascio di neurotrasmettitore da popolazioni specifiche dei neuroni della dopamina mesencefalo attraverso meccanismi che differiscono da quelli riportati in altri neuroni. La constatazione che la perdita completa di synucleins porta a cambiamenti nella gestione terminali presinaptica in particolare in quelle regioni preferenzialmente vulnerabili nel morbo di Parkinson la dopamina in definitiva pu&ograve; informare sulla selettivit&agrave; del processo di malattia.

Alterazioni funzionali al sistema Sonografia in topi privi di tutti i tre membri della famiglia sinucleina.

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&#x3001;&#x884C;&#x52D5;&#x306E;&#x751F;&#x5316;&#x5B66;&#x7684;&#x3001;&#x7D44;&#x7E54;&#x5B66;&#x7684;&#x3001;&#x304A;&#x3088;&#x3073;&#x96FB;&#x6C17;&#x5316;&#x5B66;&#x7684;&#x624B;&#x6CD5;&#x306E;&#x7D44;&#x307F;&#x5408;&#x308F;&#x305B;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3001;&#x52D5;&#x7269;&#x3092; -  mesostriatal&#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x3078;&#x306E;&#x5F71;&#x97FF;&#x306F;&#xFF08;14&#x30AB;&#x6708;&#x9F62;&#x301C;4&#xFF09;&#x6210;&#x4EBA;&#x3067;&#x8A55;&#x4FA1;&#x3057;&#x305F;&#x3002;&#x5927;&#x4EBA;&#x306E;&#x30C8;&#x30EA;&#x30D7;&#x30EB;-&#x30B7;&#x30CC;&#x30AF;&#x30EC;&#x30A4;&#x30F3;&#x304C;null&#xFF08;TKO&#xFF09;&#x30DE;&#x30A6;&#x30B9;&#x306F;&#x660E;&#x767D;&#x306A;&#x8868;&#x73FE;&#x578B;&#x3068;&#x4E2D;&#x8133;&#x30C9;&#x30FC;&#x30D1;&#x30DF;&#x30F3;&#x4F5C;&#x52D5;&#x6027;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306E;&#x6570;&#x306E;&#x5909;&#x66F4;&#x306A;&#x3057;&#x306B;&#x8868;&#x793A;&#x3055;&#x308C;&#x307E;&#x305B;&#x3093;&#x3002; TKO&#x30DE;&#x30A6;&#x30B9;&#x306F;&#x3001;&#x65B0;&#x898F;&#x306E;&#x74B0;&#x5883;&#x3067;&#x306F;&#x975E;&#x5E38;&#x306B;&#x6D3B;&#x767A;&#x3067;&#x3042;&#x3063;&#x305F;&#x3068;&#x9AD8;&#x901F;&#x30B9;&#x30AD;&#x30E3;&#x30F3;&#x30B5;&#x30A4;&#x30AF;&#x30EA;&#x30C3;&#x30AF;&#x30DC;&#x30EB;&#x30BF;&#x30F3;&#x30E1;&#x30C8;&#x30EA;&#x30FC;&#x3067;&#x691C;&#x51FA;&#x7DDA;&#x6761;&#x4F53;&#x306B;&#x304A;&#x3051;&#x308B;&#x30C9;&#x30FC;&#x30D1;&#x30DF;&#x30F3;&#x306E;&#x4E0A;&#x6607;&#x8A98;&#x767A;&#x306E;&#x30EA;&#x30EA;&#x30FC;&#x30B9;&#x3092;&#x793A;&#x3057;&#x305F;&#x3002;&#x4E0A;&#x6607;&#x3057;&#x305F;&#x30C9;&#x30FC;&#x30D1;&#x30DF;&#x30F3;&#x306E;&#x30EA;&#x30EA;&#x30FC;&#x30B9;&#x3067;&#x306F;&#x3001;&#x80CC;&#x5074;&#x8179;&#x5074;&#x7DDA;&#x6761;&#x4F53;&#x306F;&#x306A;&#x3044;&#x306B;&#x7279;&#x7570;&#x7684;&#x3067;&#x3042;&#x3063;&#x305F;&#x3068;&#x30C9;&#x30FC;&#x30D1;&#x30DF;&#x30F3;&#x7D44;&#x7E54;&#x91CF;&#x306E;&#x6E1B;&#x5C11;&#x3092;&#x4F34;&#x3063;&#x3066;&#x3044;&#x305F;&#x3002;&#x6211;&#x3005;&#x306F;&#x3001;&#x901A;&#x5E38;&#x306E;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x306E;&#x5FAE;&#x7D30;&#x69CB;&#x9020;&#x3068;&#x80CC;&#x5074;&#x7DDA;&#x6761;&#x4F53;&#x306B;&#x304A;&#x3051;&#x308B;SNARE&#xFF08;&#x53EF;&#x6EB6;&#x6027;N-&#x30A8;&#x30C1;&#x30EB;&#x30DE;&#x30EC;&#x30A4;&#x30DF;&#x30C9;&#x611F;&#x53D7;&#x6027;&#x56E0;&#x5B50;&#x4ED8;&#x7740;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x53D7;&#x5BB9;&#x4F53;&#xFF09;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x8907;&#x5408;&#x4F53;&#x306E;&#x6B63;&#x5E38;&#x306A;&#x8C4A;&#x304B;&#x3055;&#x3092;&#x78BA;&#x8A8D;&#x3057;&#x305F;&#x3002;&#x30C9;&#x30FC;&#x30D1;&#x30DF;&#x30F3;&#x4EE3;&#x8B1D;&#x306B;&#x5F71;&#x97FF;&#x3092;&#x53CA;&#x307C;&#x3059;&#x85AC;&#x5264;&#x3068;TKO&#x52D5;&#x7269;&#x306E;&#x6CBB;&#x7642;&#x306F;&#x3001;&#x5408;&#x6210;&#x306E;&#x901A;&#x5E38;&#x306E;&#x30EC;&#x30FC;&#x30C8;&#x3001;&#x5F37;&#x5316;&#x3055;&#x308C;&#x305F;&#x58F2;&#x4E0A;&#x9AD8;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x3001;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x524D;&#x7DDA;&#x6761;&#x4F53;&#x30C9;&#x30FC;&#x30D1;&#x30DF;&#x30F3;&#x30B9;&#x30C8;&#x30A2;&#x3092;&#x6E1B;&#x5C11;&#x3055;&#x305B;&#x305F;&#x3002;&#x6211;&#x3005;&#x306E;&#x30C7;&#x30FC;&#x30BF;&#x3092;&#x4E00;&#x610F;&#x306B;&#x4ED6;&#x306E;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x3067;&#x5831;&#x544A;&#x3055;&#x308C;&#x305F;&#x3082;&#x306E;&#x3068;&#x306F;&#x7570;&#x306A;&#x308B;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x3092;&#x4ECB;&#x3057;&#x3066;&#x4E2D;&#x8133;&#x30C9;&#x30FC;&#x30D1;&#x30DF;&#x30F3;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306E;&#x7279;&#x5B9A;&#x306E;&#x96C6;&#x56E3;&#x304B;&#x3089;&#x795E;&#x7D4C;&#x4F1D;&#x9054;&#x7269;&#x8CEA;&#x306E;&#x653E;&#x51FA;&#x3092;&#x8ABF;&#x7BC0;&#x3059;&#x308B;&#x30B7;&#x30CC;&#x30AF;&#x30EC;&#x30A4;&#x30F3;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x91CD;&#x8981;&#x6027;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x305F;&#x3002;&#x30B7;&#x30CC;&#x30AF;&#x30EC;&#x30A4;&#x30F3;&#x306E;&#x5B8C;&#x5168;&#x306A;&#x640D;&#x5931;&#x306F;&#x3001;&#x7279;&#x306B;&#x30D1;&#x30FC;&#x30AD;&#x30F3;&#x30BD;&#x30F3;&#x75C5;&#x3067;&#x512A;&#x5148;&#x7684;&#x306B;&#x8106;&#x5F31;&#x306A;&#x5730;&#x57DF;&#x306B;&#x304A;&#x3051;&#x308B;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x524D;&#x7D42;&#x672B;&#x304B;&#x3089;&#x30C9;&#x30FC;&#x30D1;&#x30DF;&#x30F3;&#x306E;&#x53D6;&#x308A;&#x6271;&#x3044;&#x306E;&#x5909;&#x5316;&#x306B;&#x3064;&#x306A;&#x304C;&#x308B;&#x3068;&#x3044;&#x3046;&#x77E5;&#x898B;&#x306F;&#x3001;&#x6700;&#x7D42;&#x7684;&#x306B;&#x75C5;&#x6C17;&#x306E;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x306E;&#x9078;&#x629E;&#x306B;&#x77E5;&#x3089;&#x305B;&#x308B;&#x304B;&#x3082;&#x3057;&#x308C;&#x307E;&#x305B;&#x3093;&#x3002;

&#x30B7;&#x30CC;&#x30AF;&#x30EC;&#x30A4;&#x30F3;&#x30D5;&#x30A1;&#x30DF;&#x30EA;&#x30FC;&#x306E;3&#x3064;&#x306E;&#x30E1;&#x30F3;&#x30D0;&#x30FC;&#x3092;&#x6B20;&#x3044;&#x3066;&#x3044;&#x308B;&#x30DE;&#x30A6;&#x30B9;&#x306E;&#x9ED2;&#x8CEA;&#x7DDA;&#x6761;&#x4F53;&#x7CFB;&#x306E;&#x6A5F;&#x80FD;&#x5909;&#x5316;

Synucleins (&alpha;, &beta;, &gamma;) &#xB192;&#xC740; &#xB3D9;&#xC885; &#xB2E8;&#xBC31;&#xC9C8; neurotransmission &#xC870;&#xC808;&#xC5D0; &#xC5ED;&#xD560;&#xC744; &#xD560; &#xAC83;&#xC73C;&#xB85C; &#xC0DD;&#xAC01; &#xB418;&#xBA70; presynaptic &#xD130;&#xBBF8;&#xB110;&#xC5D0;&#xC11C; &#xD48D;&#xBD80; &#xD558; &#xAC8C; &#xBC1C;&#xACAC; &#xB41C;&#xB2E4;. Synuclein &#xB2E8;&#xBC31;&#xC9C8; &#xC0AC;&#xC774;&#xC758; &#xAE30;&#xB2A5; &#xC911;&#xBCF5;&#xC744; &#xADF9;&#xBCF5; &#xD558; &#xACE0; mesostriatal dopaminergic &#xC2E0;&#xACBD; &#xC138;&#xD3EC;&#xC5D0;&#xC11C; presynaptic &#xC2E0;&#xD638;&#xC5D0;&#xC11C; &#xADF8;&#xB4E4;&#xC758; &#xC5ED;&#xD560;&#xC744; &#xC774;&#xD574; &#xD558; &#xC6B0;&#xB9AC; &#xC950; synuclein &#xAC00;&#xC871;&#xC758; &#xBAA8;&#xB4E0; &#xC138; &#xAC00;&#xC9C0; &#xBA64;&#xBC84;&#xB97C; &#xBD80;&#xC871; &#xD55C; &#xC0DD;&#xC0B0;. &#xC131;&#xC778; (4-&#xC5D0; 14 &#xAC1C;&#xC6D4;) &#xB3D9;&#xBB3C; &#xD589;&#xB3D9;, &#xC0DD;&#xD654;&#xD559;, &#xC870;&#xC9C1;&#xD559;, &#xBC0F; &#xC804;&#xAE30; &#xD654;&#xD559; &#xAE30;&#xC220; &#xD568;&#xAED8; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; mesostriatal &#xC2DC;&#xC2A4;&#xD15C;&#xC5D0; &#xBBF8;&#xCE58;&#xB294; &#xC601;&#xD5A5; &#xD3C9;&#xAC00; &#xB418;&#xC5C8;&#xB2E4;. &#xC131;&#xC778; &#xD2B8;&#xB9AC;&#xD50C;-synuclein-null (&#xB9DD;&#xAC00;) &#xB9C8;&#xC6B0;&#xC2A4; &#xD45C;&#xC2DC; &#xC5C6;&#xC74C; &#xBA85;&#xBC31;&#xD55C; &#xD45C;&#xD604; &#xD615; &#xBCC0;&#xD654; midbrain dopaminergic &#xC2E0;&#xACBD; &#xC138;&#xD3EC;&#xC758; &#xC218;&#xC5D0;. &#xB9DD;&#xAC00; &#xC950; &#xC18C;&#xC124; &#xD658;&#xACBD;&#xC5D0;&#xC11C; &#xD65C;&#xB3D9;&#xC801;&#xC778; &#xB418;&#xC5C8;&#xACE0; &#xB192;&#xC740; evoked &#xCD9C;&#xC2DC;&#xAC00; &#xBE60;&#xB978; &#xC2A4;&#xCE94; &#xC8FC;&#xAE30;&#xC801; voltammetry &#xBC1C;&#xACAC;&#xC5D0;&#xC11C; &#xB3C4;&#xD30C;&#xBBFC;&#xC758; &#xC804;&#xC2DC;. &#xC0C1;&#xC2B9; &#xB41C; &#xB3C4;&#xD30C;&#xBBFC; &#xBC29;&#xCD9C; &#xAD00;&#xB828; &#xC9C0; &#xD558;&#xC9C0; &#xBCF5; &#xBD80;&#xAC00; &#xB418;&#xC5C8;&#xACE0; &#xB3C4;&#xD30C;&#xBBFC; &#xC870;&#xC9C1;&#xC758; &#xCF58;&#xD150;&#xCE20;&#xC758; &#xAC10;&#xC18C;&#xC640; &#xD568;&#xAED8; &#xD588;&#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; &#xC815;&#xC0C1;&#xC801;&#xC778; &#xC2DC; &#xB0C5; &#xC2A4; &#xB300;&#xAD8C; &#xACE0; &#xC9C0;&#xAC00;&#xC5D0; &#xC62C; &#xBB34; (&#xC218;&#xC6A9; &#xC131; N ethylmaleimide &#xAD6C;&#xBD84; &#xC778;&#xC790; &#xCCA8;&#xBD80; &#xC218;&#xC6A9; &#xB2E8;&#xBC31;&#xC9C8; &#xCCB4;) &#xB2E8;&#xBC31;&#xC9C8; &#xBCF5;&#xD569;&#xBB3C;&#xC758; &#xC815;&#xC0C1;&#xC801;&#xC778; &#xD48D;&#xBD80;&#xD55C; &#xD655;&#xC778;. &#xB3C4;&#xD30C;&#xBBFC; &#xB300;&#xC0AC;&#xC5D0; &#xC601;&#xD5A5;&#xC744; &#xBBF8;&#xCE58;&#xB294; &#xC57D;&#xBB3C;&#xC73C;&#xB85C; &#xB9DD;&#xAC00; &#xB3D9;&#xBB3C;&#xC758; &#xCE58;&#xB8CC; &#xD569;&#xC131;&#xC758; &#xC815;&#xC0C1;&#xC801;&#xC778; &#xC18D;&#xB3C4; &#xACF5;&#xAC1C; &#xD558; &#xACE0; &#xB9E4;&#xCD9C;, &#xD5A5;&#xC0C1; &#xB41C; presynaptic striatal &#xB3C4;&#xD30C;&#xBBFC; &#xB9E4;&#xC7A5; &#xAC10;&#xC18C;. &#xC6B0;&#xB9AC;&#xC758; &#xB370;&#xC774;&#xD130; &#xACE0;&#xC720; &#xD558; &#xAC8C; &#xB2E4;&#xB978; &#xB274;&#xB7F0;&#xC5D0;&#xC11C; &#xBCF4;&#xACE0; &#xADF8;&#xC640; &#xB2E4;&#xB978; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC744; &#xD1B5;&#xD574; midbrain &#xB3C4;&#xD30C;&#xBBFC; &#xC2E0;&#xACBD; &#xC138;&#xD3EC;&#xC758; &#xD2B9;&#xC815; &#xC778;&#xAD6C;&#xC5D0;&#xC11C; &#xC2E0;&#xACBD; &#xC804;&#xB2EC; &#xBB3C;&#xC9C8; &#xBC29;&#xCD9C; &#xC870;&#xC808;&#xC5D0; synuclein &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xC911;&#xC694;&#xC131;&#xC744; &#xACC4;&#xC2DC; &#xD55C;&#xB2E4;. Synucleins&#xC758; &#xC644;&#xC804; &#xD55C; &#xC190;&#xC2E4; presynaptic &#xB2E8;&#xB9D0;&#xAE30; &#xD2B9;&#xBCC4;&#xD788; &#xADF8; &#xC9C0;&#xC5ED; &#xC6B0;&#xC120;&#xC801;&#xC73C;&#xB85C; &#xD30C; &#xD0A8; &#xC2A8; &#xBCD1;&#xC5D0; &#xCDE8;&#xC57D; &#xD558; &#xC5EC; &#xCC98;&#xB9AC; &#xD558;&#xB294; &#xB3C4;&#xD30C;&#xBBFC;&#xC758; &#xBCC0;&#xD654;&#xB97C; &#xB9AC;&#xB4DC; &#xCC3E;&#xB294; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xAD81;&#xADF9;&#xC801;&#xC73C;&#xB85C; &#xC9C8;&#xBCD1; &#xACFC;&#xC815;&#xC758; &#xC120;&#xD0DD;&#xB3C4;&#xC5D0; &#xD1B5;&#xBCF4; &#xB429;&#xB2C8;&#xB2E4;.

Synuclein &#xC81C;&#xD488;&#xAD70;&#xC758; &#xBAA8;&#xB4E0; &#xC138; &#xBA85;&#xC758; &#xBA64;&#xBC84;&#xAC00; &#xBD80;&#xC871; &#xD55C; &#xC950;&#xC5D0; nigrostriatal &#xC2DC;&#xC2A4;&#xD15C; &#xAE30;&#xB2A5; &#xBCC0;&#xACBD;.

Los synucleins (&#x3B1;, &#x3B2;, &#x3B3;, y) son altamente hom&#xF3;logos prote&#xED;nas se cree que desempe&#xF1;an un papel en la regulaci&#xF3;n de la neurotransmisi&#xF3;n y se encuentran abundantemente en las terminales presin&#xE1;pticas. Para superar la superposici&#xF3;n de funciones entre las prote&#xED;nas sinucle&#xED;na y comprender su papel en la se&#xF1;alizaci&#xF3;n presin&#xE1;ptica de mesoestriatales las neuronas dopamin&#xE9;rgicas, que producen ratones que carec&#xED;an de los tres miembros de la familia sinucle&#xED;na. El efecto sobre el sistema mesoestriatales se evalu&#xF3; en adultos (4 - a 14 meses de edad) los animales mediante una combinaci&#xF3;n de t&#xE9;cnicas conductuales, bioqu&#xED;micos, histol&#xF3;gicos y electroqu&#xED;mica. Adultos triple-sinucle&#xED;na-null (TKO) los ratones no mostraron fenotipo abierta y ning&#xFA;n cambio en el n&#xFA;mero de neuronas del cerebro medio dopamin&#xE9;rgicos. Los ratones eran hiperactivos TKO en entornos nuevos y exhibi&#xF3; liberaci&#xF3;n elevada evocado de la dopamina en el estriado detectado con voltametr&#xED;a c&#xED;clica r&#xE1;pida exploraci&#xF3;n. La liberaci&#xF3;n de dopamina elevada era espec&#xED;fico para el estriado dorsal ventral y no fue acompa&#xF1;ado por una disminuci&#xF3;n del contenido de tejido dopamina. Se confirm&#xF3; un ultraestructura sin&#xE1;ptica normal, y una abundancia normal de las prote&#xED;nas SNARE complejos (soluble N-etilmaleimida sensible a la uni&#xF3;n del receptor del factor de prote&#xED;na) en el estriado dorsal. El tratamiento de animales con TKO f&#xE1;rmacos que afectan el metabolismo de la dopamina revel&#xF3; ritmo normal de la s&#xED;ntesis, la rotaci&#xF3;n mejorada y la reducci&#xF3;n de las tiendas de presin&#xE1;ptica de dopamina estriatal. Nuestros datos revelan la importancia &#xFA;nica de las prote&#xED;nas sinucle&#xED;na en la regulaci&#xF3;n de la liberaci&#xF3;n de neurotransmisores en poblaciones espec&#xED;ficas de neuronas dopamin&#xE9;rgicas del cerebro medio a trav&#xE9;s de mecanismos que difieren de los reportados en otras neuronas. El hallazgo de que la p&#xE9;rdida completa de synucleins conduce a cambios en el manejo de la dopamina presin&#xE1;ptica terminales espec&#xED;ficamente en las regiones vulnerables preferentemente en la enfermedad de Parkinson en &#xFA;ltima instancia, podr&#xE1; informar sobre la selectividad del proceso de la enfermedad.

Las alteraciones funcionales al sistema nigroestriatal en ratones que carecen los tres miembros de la Familia sinucle&#xED;na

Cysteine string protein α (CSPα), a presynaptic cochaperone for Hsc70, is required for synapse maintenance. Deletion of CSPα leads to neuronal dysfunction, synapse loss, and neurodegeneration. We utilized unbiased, systematic proteomics to identify putative CSPα protein clients. We found 22 such proteins whose levels are selectively decreased in CSPα knockout synapses. Of these putative CSPα protein clients, two directly bind to the CSPα chaperone complex and are bona fide clients. They are the t-SNARE SNAP-25 and the GTPase dynamin 1, which are necessary for synaptic vesicle fusion and fission, respectively. Using hippocampal cultures, we show that CSPα regulates the stability of client proteins and synaptic vesicle number. Our analysis of CSPα-dynamin 1 interactions reveals unexpectedly that CSPα regulates the polymerization of dynamin 1. CSPα, therefore, participates in synaptic vesicle endocytosis and may facilitate exo- and endocytic coupling. These findings advance the understanding of how synapses are functionally and structurally maintained.

Identification of CSPα clients reveals a role in dynamin 1 regulation.

Synucleins are a family of presynaptic membrane binding proteins. α-Synuclein, the principal member of this family, is mutated in familial Parkinson disease. To gain insight into the molecular functions of synucleins, we performed an unbiased proteomic screen and identified synaptic protein changes in αβγ-synuclein knock-out brains. We observed increases in the levels of select membrane curvature sensing/generating proteins. One of the most prominent changes was for the N-BAR protein endophilin A1. Here we demonstrate that the levels of synucleins and endophilin A1 are reciprocally regulated and that they are functionally related. We show that all synucleins can robustly generate membrane curvature similar to endophilins. However, only monomeric but not tetrameric α-synuclein can bend membranes. Further, A30P α-synuclein, a Parkinson disease mutant that disrupts protein folding, is also deficient in this activity. This suggests that synucleins generate membrane curvature through the asymmetric insertion of their N-terminal amphipathic helix. Based on our findings, we propose to include synucleins in the class of amphipathic helix-containing proteins that sense and generate membrane curvature. These results advance our understanding of the physiological function of synucleins.

Monomeric synucleins generate membrane curvature.

Genetic and pathological studies link α-synuclein to the etiology of Parkinson's disease (PD), but the normal function of this presynaptic protein remains unknown. α-Synuclein, an acidic lipid binding protein, shares high sequence identity with β- and γ-synuclein. Previous studies have implicated synucleins in synaptic vesicle (SV) trafficking, although the precise site of synuclein action continues to be unclear. Here we show, using optical imaging, electron microscopy, and slice electrophysiology, that synucleins are required for the fast kinetics of SV endocytosis. Slowed endocytosis observed in synuclein null cultures can be rescued by individually expressing mouse α-, β-, or γ-synuclein, indicating they are functionally redundant. Through comparisons to dynamin knock-out synapses and biochemical experiments, we suggest that synucleins act at early steps of SV endocytosis. Our results categorize α-synuclein with other familial PD genes known to regulate SV endocytosis, implicating this pathway in PD. 

Synucleins regulate the kinetics of synaptic vesicle endocytosis.

Cysteine String Protein alpha (CSPα) is a palmitoylated, synaptic vesicle co-chaperone that is essential for neuroprotection. Two mutations in CSPα - L115R and L116Δ - cause adult neuronal ceroid lipofuscinosis (ANCL), a dominantly-inherited neurodegenerative disease. To elucidate the pathogenesis of ANCL, the intrinsic biochemical properties of human wildtype (WT) and disease mutant CSPα were examined. Mutant proteins purified from Escherichia coli exhibited high potency to oligomerize in a concentration, temperature, and time dependent manner, with L115R possessing the greatest potency. When freshly purified, ANCL mutant proteins displayed normal co-chaperone activity and substrate recognition similar to WT. However, co-chaperone activity was impaired for both CSPα mutants upon oligomerization. When WT and mutant CSPα were mixed together they co-oligomerized leading to an overall decrease of co-chaperone activity. The oligomerization properties of ANCL mutants were faithfully replicated in HEK 293T cells. Interestingly, the oligomers were covalently tagged by ubiquitination instead of palmitoylation. Taken together, ANCL mutations result in both a gain and partial loss-of-function. 

Oligomerization of Cysteine String Protein alpha mutants causing adult neuronal ceroid lipofuscinosis.

Neuronal ceroid lipofuscinoses (NCL) are a group of inherited neurodegenerative disorders with lysosomal pathology (CLN1-14). Recently, mutations in the DNAJC5/CLN4 gene, which encodes the presynaptic co-chaperone CSPα were shown to cause autosomal-dominant NCL. Although 14 NCL genes have been identified, it is unknown if they act in common disease pathways. Here we show that two disease-associated proteins, CSPα and the depalmitoylating enzyme palmitoyl-protein thioesterase 1 (PPT1/CLN1) are biochemically linked. We find that in DNAJC5/CLN4 patient brains, PPT1 is massively increased and mis-localized. Surprisingly, the specific enzymatic activity of PPT1 is dramatically reduced. Notably, we demonstrate that CSPα is depalmitoylated by PPT1 and hence its substrate. To determine the consequences of PPT1 accumulation, we compared the palmitomes from control and DNAJC5/CLN4 patient brains by quantitative proteomics. We discovered global changes in protein palmitoylation, mainly involving lysosomal and synaptic proteins. Our findings establish a functional link between two forms of NCL and serve as a springboard for investigations of NCL disease pathways. 

Neuronal ceroid lipofuscinosis with DNAJC5/CSPα mutation has PPT1 pathology and exhibit aberrant protein palmitoylation.

Synucleins (α, β, γ-synuclein) are a family of abundant presynaptic proteins. α-Synuclein is causally linked to the pathogenesis of Parkinson's disease (PD). In an effort to define their physiological and pathological function or functions, we investigated the effects of deleting synucleins and overexpressing α-synuclein PD mutations, in mice, on synapse architecture using electron microscopy (EM) and cryoelectron tomography (cryo-ET). We show that synucleins are regulators of presynapse size and synaptic vesicle (SV) pool organization. Using cryo-ET, we observed that deletion of synucleins increases SV tethering to the active zone but decreases the inter-linking of SVs by short connectors. These ultrastructural changes were correlated with discrete protein phosphorylation changes in αβγ-synuclein-/- neurons. We also determined that α-synuclein PD mutants (PARK1/hA30P and PARK4/hα-syn) primarily affected presynaptic cytomatrix proximal to the active zone, congruent with previous findings that these PD mutations decrease neurotransmission. Collectively, our results suggest that synucleins are important orchestrators of presynaptic terminal topography.

Synucleins Have Multiple Effects on Presynaptic Architecture.

Glucocerebrosidase 1 () mutations responsible for Gaucher disease (GD) are the most common genetic risk factor for Parkinson's disease (PD). Although the genetic link between GD and PD is well established, the underlying molecular mechanism(s) are not well understood. We propose that glucosylsphingosine, a sphingolipid accumulating in GD, mediates PD pathology in -associated PD. We show that, whereas GD-related sphingolipids (glucosylceramide, glucosylsphingosine, sphingosine, sphingosine-1-phosphate) promote α-synuclein aggregation , glucosylsphingosine triggers the formation of oligomeric α-synuclein species capable of templating in human cells and neurons. Using newly generated GD/PD mouse lines of either sex [ mutant (N370S, L444P, KO) crossed to α-synuclein transgenics], we show that  mutations predispose to PD through a loss-of-function mechanism. We further demonstrate that glucosylsphingosine specifically accumulates in young GD/PD mouse brain. With age, brains exhibit glucosylceramide accumulations colocalized with α-synuclein pathology. These findings indicate that glucosylsphingosine promotes pathological aggregation of α-synuclein, increasing PD risk in GD patients and carriers. Parkinson's disease (PD) is a prevalent neurodegenerative disorder in the aging population. Glucocerebrosidase 1 mutations, which cause Gaucher disease, are the most common genetic risk factor for PD, underscoring the importance of delineating the mechanisms underlying mutant -associated PD. We show that lipids accumulating in Gaucher disease, especially glucosylsphingosine, play a key role in PD pathology in the brain. These data indicate that ASAH1 (acid ceramidase 1) and GBA2 (glucocerebrosidase 2) enzymes that mediate glucosylsphingosine production and metabolism are attractive therapeutic targets for treating mutant -associated PD.

Glucosylsphingosine Promotes α-Synuclein Pathology in Mutant GBA-Associated Parkinson's Disease.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders, affecting 1-1.5% of the total population. While progress has been made in understanding the neurodegenerative mechanisms that lead to cell death in late stages of PD, mechanisms for early, causal pathogenic events are still elusive. Recent developments in PD genetics increasingly point at endolysosomal (E-L) system dysfunction as the early pathomechanism and key pathway affected in PD. Clathrin-mediated synaptic endocytosis, an integral part of the neuronal E-L system, is probably the main early target as evident in auxilin, RME-8, and synaptojanin-1 mutations that cause PD. Autophagy, another important pathway in the E-L system, is crucial in maintaining proteostasis and a healthy mitochondrial pool, especially in neurons considering their inability to divide and requirement to function an entire life-time. PINK1 and Parkin mutations severely perturb autophagy of dysfunctional mitochondria (mitophagy), both in the cell body and synaptic terminals of dopaminergic neurons, leading to PD. Endolysosomal sorting and trafficking is also crucial, which is complex in multi-compartmentalized neurons. VPS35 and VPS13C mutations noted in PD target these mechanisms. Mutations in GBA comprise the most common risk factor for PD and initiate pathology by compromising lysosomal function. This is also the case for ATP13A2 mutations. Interestingly, α-synuclein and LRRK2, key proteins involved in PD, function in different steps of the E-L pathway and target their components to induce disease pathogenesis. In this review, we discuss these E-L system genes that are linked to PD and how their dysfunction results in PD pathogenesis. This article is part of the Special Issue "Synuclein".

Role of the endolysosomal system in Parkinson's disease.

The autosomal dominant neuronal ceroid lipofuscinoses (NCL)  is caused by mutations in the synaptic vesicle (SV) protein CSPα. We developed animal models of  by expressing  mutant human CSPα (hCSPα) in  neurons. Similar to patients,  mutations induced excessive oligomerization of hCSPα and premature lethality in a dose-dependent manner. Instead of being localized to SVs, most  mutant hCSPα accumulated abnormally, and co-localized with ubiquitinated proteins and the prelysosomal markers HRS and LAMP1. Ultrastructural examination revealed frequent abnormal membrane structures in axons and neuronal somata. The lethality, oligomerization and prelysosomal accumulation induced by  mutations was attenuated by reducing endogenous wild type (WT) dCSP levels and enhanced by increasing WT levels. Furthermore, reducing the gene dosage of Hsc70 also attenuated  phenotypes. Taken together, we suggest that  alleles resemble dominant hypermorphic gain of function mutations that drive excessive oligomerization and impair membrane trafficking.

A  model of neuronal ceroid lipofuscinosis  reveals a hypermorphic gain of function mechanism.

Parkinson's disease is characterized by the aggregation of the presynaptic protein α-synuclein and its deposition into pathologic Lewy bodies. While extensive research has been carried out on mediators of α-synuclein aggregation, molecular facilitators of α-synuclein disaggregation are still generally unknown. We investigated the role of molecular chaperones in both preventing and disaggregating α-synuclein oligomers and fibrils, with a focus on the mammalian disaggregase complex. Here, we show that overexpression of the chaperone Hsp110 is sufficient to reduce α-synuclein aggregation in a mammalian cell culture model. Additionally, we demonstrate that Hsp110 effectively mitigates α-synuclein pathology in vivo through the characterization of transgenic Hsp110 and double-transgenic α-synuclein/Hsp110 mouse models. Unbiased analysis of the synaptic proteome of these mice revealed that overexpression of Hsp110 can override the protein changes driven by the α-synuclein transgene. Furthermore, overexpression of Hsp110 is sufficient to prevent endogenous α-synuclein templating and spread following injection of aggregated α-synuclein seeds into brain, supporting a role for Hsp110 in the prevention and/or disaggregation of α-synuclein pathology.

Hsp110 mitigates α-synuclein pathology in vivo.

Loss-of-function mutations in the depalmitoylating enzyme palmitoyl protein thioesterase 1 (PPT1) cause neuronal ceroid lipofuscinosis (NCL), a devastating neurodegenerative disease. The substrates of PPT1 are largely undescribed, posing a limitation on molecular dissection of disease mechanisms and therapeutic development. Here, we provide a resource identifying >100 novel PPT1 substrates. We utilized Acyl Resin-Assisted Capture (Acyl RAC) and mass spectrometry to identify proteins with increased in vivo palmitoylation in PPT1 knockout (KO) mouse brains. We then validated putative substrates through direct depalmitoylation with recombinant PPT1. This stringent screen elucidated diverse PPT1 substrates at the synapse, including channels and transporters, G-protein-associated molecules, endo/exocytic components, synaptic adhesion molecules, and mitochondrial proteins. Cysteine depalmitoylation sites in transmembrane PPT1 substrates frequently participate in disulfide bonds in the mature protein. We confirmed that depalmitoylation plays a role in disulfide bond formation in a tertiary screen analyzing posttranslational modifications (PTMs). Collectively, these data highlight the role of PPT1 in mediating synapse functions, implicate molecular pathways in the etiology of NCL and other neurodegenerative diseases, and advance our basic understanding of the purpose of depalmitoylation.

Identification of substrates of palmitoyl protein thioesterase 1 highlights roles of depalmitoylation in disulfide bond formation and synaptic function.

The development of single-cell RNA-sequencing (scRNA-seq) technologies has offered insights into complex biological systems at the single-cell resolution. In particular, these techniques facilitate the identifications of genes showing cell-type-specific differential expressions (DE). In this paper, we introduce MARBLES, a novel statistical model for cross-condition DE gene detection from scRNA-seq data. MARBLES employs a Markov Random Field model to borrow information across similar cell types and utilizes cell-type-specific pseudobulk count to account for sample-level variability. Our simulation results showed that MARBLES is more powerful than existing methods to detect DE genes with an appropriate control of false positive rate. Applications of MARBLES to real data identified novel disease-related DE genes and biological pathways from both a single-cell lipopolysaccharide mouse dataset with 24 381 cells and 11 076 genes and a Parkinson's disease human data set with 76 212 cells and 15 891 genes. Overall, MARBLES is a powerful tool to identify cell-type-specific DE genes across conditions from scRNA-seq data.

Sreeganga S. Chandra

Department of Neurology,
Department of Neuroscience

Yale University

Subcellular Fractionation for the Isolation of Synaptic Components from the Murine Brain

Sreeganga S. Chandra

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Neurology,Department of Neuroscience

Yale University

Subcellular Fractionation for the Isolation of Synaptic Components from the Murine Brain

Department of Neurology,
Department of Neuroscience