An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

DUKE-NUS Graduate Medical School

We have previously demonstrated that cellular stimulation with GH results in the formation of a multiprotein signaling complex. One component of this multiprotein signaling complex is the adapter molecule c-Cbl. Here we have examined the role of c-Cbl in the mechanism of GH signal transduction. Forced expression of c-Cbl in NIH3T3 cells did not alter GH-stimulated Janus kinase 2 tyrosine phosphorylation nor GH-stimulated p44/42 MAPK activation and consequent Elk-1- mediated transcription. c-Cbl overexpression did, however, result in enhanced and prolonged GH-stimulated activation of phosphatidylinositol 3-kinase. Forced expression of c-Cbl did not affect GH-stimulated STAT5 tyrosine phosphorylation, nuclear translocation, nor binding to DNA but markedly abrogated GH-stimulated STAT5-mediated transactivation. c-Cbl overexpression resulted in increased ubiquitination and proteosomal degradation of STAT5 and increased degradation of GH-stimulated tyrosine phosphorylated STAT5. Cellular pretreatment with the proteosomal inhibitor MG132 reversed the effect of c-Cbl overexpression with prolonged duration of GH-stimulated STAT5 tyrosine phosphorylation and restoration of STAT5-mediated transcription. Thus, c-Cbl is a negative regulator of GH-stimulated STAT5-mediated transcription by direction of STAT5 for proteosomal degradation.

c-Cbl is a negative regulator of GH-stimulated STAT5-mediated transcription.

In the rat, a growth hormone-binding protein (GHBP) exists that is derived from the growth hormone (GH) receptor gene by an alternative mRNA splicing mechanism such that the transmembrane and intracellular domains of the GH receptor are replaced by a hydrophilic carboxyl terminus. In isolation, the GHBP is inactive, although it does compete with the receptor for ligand binding in the extracellular space and therefore inhibits the cellular response to GH. The GHBP is also located intracellularly and is translocated to the nucleus upon ligand stimulation. We show here that endogenously produced GHBP, in contrast to exogenous GHBP, was able to enhance the STAT5-mediated transcriptional response to GH. Interestingly, when the GHBP was targeted constitutively to the nucleus by the addition of the nuclear localization sequence of the SV40 large T antigen, greater enhancement of STAT5-mediated transcription was obtained. The transcriptional enhancement did not require GH per se and was not specific to the GH receptor, since similar enhancement of STAT5-mediated transcription by nuclear localized GHBP was obtained with specific ligand stimulation of both prolactin and erythropoietin receptors. Thus, the GHBP exerts divergent effects on STAT5-mediated transcription depending on its cellular location. The use of a soluble cytokine receptor as a location-dependent transcriptional enhancer and the ligand-independent involvement of the extracellular domain of a polypeptide ligand receptor in intracellular signal transduction provide additional novel mechanisms of transcriptional control.

The growth hormone-binding protein is a location-dependent cytokine receptor transcriptional enhancer.

We have exploited a discrepancy in the oncogenic potential of autocrine and exogenous human growth hormone (hGH) in an attempt to identify molecules that could potentially be involved in oncogenic transformation of the human mammary epithelial cell. Microarray analysis of 19,000 human genes identified a subset of 305 genes in a human mammary carcinoma cell line that were remarkably different in their response to autocrine and exogenous hGH. Autocrine and exogenous hGH also regulated 167 common genes. Semiquantitative reverse transcription-PCR confirmed differential regulation of genes by either autocrine or exogenous hGH. Functional analysis of one of the identified autocrine hGH-regulated genes, TFF3, determined that its expression is sufficient to support anchorage-independent growth of human mammary carcinoma cells. Small interfering RNA-mediated knockdown of TFF3 concordantly abrogated anchorage-independent growth of mammary carcinoma cells and abrogated the ability of autocrine hGH to stimulate oncogenic transformation of immortalized human mammary epithelial cells. Further functional characterization of the identified subset of specifically autocrine hGH regulated genes will delineate additional novel oncogenes for the human mammary epithelial cell.

Gene expression profiling to identify oncogenic determinants of autocrine human growth hormone in human mammary carcinoma.

&#x6210;&#x4EBA;&#x7684;&#x5927;&#x8111;&#xFF0C;&#x51FA;&#x751F;&#x548C;&#x96C6;&#x6210;&#x65B0;&#x7684;&#x795E;&#x7ECF;&#x5143;&#xFF0C;&#x4ECE;&#x6210;&#x4EBA;&#x795E;&#x7ECF;&#x5E72;&#x7EC6;&#x80DE;&#xFF0C;&#x5F15;&#x4EBA;&#x6CE8;&#x76EE;&#x7684;&#x7A97;&#x4F53;&#x7ED3;&#x6784;&#x7684;&#x53EF;&#x5851;&#x6027;&#xFF0C;&#x5E76;&#x7A81;&#x51FA;&#x663E;&#x793A;&#x5728;&#x6210;&#x5E74;&#x54FA;&#x4E73;&#x52A8;&#x7269;&#x7684;&#x5927;&#x8111;&#x5177;&#x6709;&#x518D;&#x751F;&#x80FD;&#x529B;&#x3002;&#x8D8A;&#x6765;&#x8D8A;&#x591A;&#x7684;&#x8BC1;&#x636E;&#x8868;&#x660E;&#x795E;&#x7ECF;&#x5143;&#x6D3B;&#x52A8;&#x89C4;&#x5B9A;&#x4E86;&#x6210;&#x4EBA;&#x5927;&#x8111;&#x548C;&#x65B0;&#x7684;&#x795E;&#x7ECF;&#x5143;&#x6709;&#x52A9;&#x4E8E;&#x7279;&#x5B9A;&#x8111;&#x529F;&#x80FD;&#x3002;&#x89C4;&#x7BA1;&#x65B0;&#x751F;&#x6210;&#x795E;&#x7ECF;&#x5143;&#x878D;&#x5165;&#x6210;&#x4EBA;&#x5927;&#x8111;&#x4E2D;&#x7684;&#x9884;&#x5148;&#x5B58;&#x5728;&#x7684;&#x529F;&#x80FD;&#x7535;&#x8DEF;&#x7684;&#x673A;&#x5236;&#x662F;&#x672A;&#x77E5;&#x7684;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#x6211;&#x4EEC;&#x663E;&#x793A;&#x6309;&#x987A;&#x5E8F;&#x88AB;&#x652F;&#x914D;&#x7684; GABA &#x548C;&#x8C37;&#x6C28;&#x9178;&#x4ECB;&#x5BFC;&#x7684;&#x7A81;&#x89E6;&#x6295;&#x5165;&#x4E4B;&#x524D; tonically &#x6FC0;&#x6D3B;&#x7684;&#x73AF;&#x5883; GABA &#xFF08;&#x6C28;&#x57FA;&#x4E01;&#x9178;&#xFF09; &#x65B0;&#x751F;&#x9897;&#x7C92;&#x7684;&#x6210;&#x5E74;&#x5927;&#x9F20;&#x6D77;&#x9A6C;&#x9F7F;&#x72B6;&#x56DE;&#x4E2D;&#x7684;&#x5355;&#x5143;&#x683C;&#x3002;&#x5728;&#x6210;&#x4EBA;&#x7684;&#x5927;&#x8111;&#x4E2D;&#xFF0C;&#x4E3B;&#x8981;&#x7684;&#x6291;&#x5236;&#x795E;&#x7ECF;&#x9012;&#x8D28; GABA&#xFF0C;&#x6700;&#x521D;&#x65BD;&#x52A0;&#x4E0A;&#x7531;&#x4E8E;&#x5176;&#x9AD8;&#x7684;&#x7EC6;&#x80DE;&#x8D28;&#x6C2F;&#x79BB;&#x5B50;&#x542B;&#x91CF;&#x7684;&#x65B0;&#x751F;&#x795E;&#x7ECF;&#x5143;&#x5174;&#x594B;&#x6027;&#x7684;&#x52A8;&#x4F5C;&#x3002;&#x8F6C;&#x6362;&#x6210;&#x6781;&#x5316; (&#x6291;&#x5236;) &#x4E2D;&#x7684;&#x65B0;&#x751F;&#x795E;&#x7ECF;&#x5143; GABA &#x8BF1;&#x5BFC;&#x6781;&#x5316; &#xFF08;&#x52B1;&#x78C1;&#xFF09; &#x5BFC;&#x81F4;&#x5176;&#x7A81;&#x89E6;&#x5F62;&#x6210;&#x548C;&#x4F53;&#x5185;&#x7684;&#x6811;&#x7A81;&#x53D1;&#x80B2;&#x7684;&#x660E;&#x663E;&#x7F3A;&#x9677;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x7814;&#x7A76;&#x65B0;&#x751F;&#x6210;&#x4E86;&#x6210;&#x4EBA;&#x5927;&#x8111;&#x4E2D;&#x795E;&#x7ECF;&#x5143;&#x7A81;&#x89E6;&#x6574;&#x5408;&#x4E3A; GABA &#x6807;&#x8BC6;&#x5FC5;&#x4E0D;&#x53EF;&#x5C11;&#x7684;&#x4F5C;&#x7528;&#xFF0C;&#x5E76;&#x5EFA;&#x8BAE;&#x610F;&#x5916;&#x6210;&#x4EBA;&#x795E;&#x7ECF;&#x518D;&#x751F;&#xFF0C;&#x65B0;&#x751F;&#x795E;&#x7ECF;&#x5143;&#x53EF;&#x80FD;&#x610F;&#x4E49;&#x4E0A;&#x901A;&#x8FC7;&#x8865;&#x54C1;&#x548C;&#x9636;&#x6BB5;&#x6027; GABA &#x6FC0;&#x6D3B;&#x795E;&#x7ECF;&#x5143;&#x7F51;&#x7EDC;&#x6D3B;&#x52A8;&#x4F9D;&#x8D56;&#x4E8E;&#x6D3B;&#x52A8;&#x7684;&#x76D1;&#x7BA1;&#x673A;&#x5236;&#x3002;

GABA &#x89C4;&#x7BA1;&#x6210;&#x4EBA;&#x5927;&#x8111;&#x4E2D;&#x65B0;&#x751F;&#x6210;&#x795E;&#x7ECF;&#x5143;&#x7A81;&#x89E6;&#x7684;&#x4E00;&#x4F53;&#x5316;&#x3002;

Adult neurogenesis, the birth and integration of new neurons from adult neural stem cells, is a striking form of structural plasticity and highlights the regenerative capacity of the adult mammalian brain. Accumulating evidence suggests that neuronal activity regulates adult neurogenesis and that new neurons contribute to specific brain functions. The mechanism that regulates the integration of newly generated neurons into the pre-existing functional circuitry in the adult brain is unknown. Here we show that newborn granule cells in the dentate gyrus of the adult hippocampus are tonically activated by ambient GABA (gamma-aminobutyric acid) before being sequentially innervated by GABA- and glutamate-mediated synaptic inputs. GABA, the major inhibitory neurotransmitter in the adult brain, initially exerts an excitatory action on newborn neurons owing to their high cytoplasmic chloride ion content. Conversion of GABA-induced depolarization (excitation) into hyperpolarization (inhibition) in newborn neurons leads to marked defects in their synapse formation and dendritic development in vivo. Our study identifies an essential role for GABA in the synaptic integration of newly generated neurons in the adult brain, and suggests an unexpected mechanism for activity-dependent regulation of adult neurogenesis, in which newborn neurons may sense neuronal network activity through tonic and phasic GABA activation.

GABA regulates synaptic integration of newly generated neurons in the adult brain.

Neurogen&#xE8;se adulte, la naissance et l&#x26;#39;int&#xE9;gration des nouveaux neurones &#xE0; partir de cellules souches neurales adultes, est une forme frappante de la plasticit&#xE9; structurelle et met en &#xE9;vidence la capacit&#xE9; de r&#xE9;g&#xE9;n&#xE9;ration du cerveau adulte des mammif&#xE8;res. Les preuves qui s&#x26;#39;accumulent sugg&#xE8;rent que l&#x26;#39;activit&#xE9; neuronale r&#xE9;glemente la neurogen&#xE8;se adulte et que de nouveaux neurones contribuent &#xE0; des fonctions sp&#xE9;cifiques du cerveau. Le m&#xE9;canisme qui r&#xE9;gule l&#x26;#39;int&#xE9;gration des neurones nouvellement g&#xE9;n&#xE9;r&#xE9;es dans le circuit de pr&#xE9;-existant fonctionnelle dans le cerveau de l&#x26;#39;adulte est inconnue. Ici, nous montrons que les cellules granulaires du nouveau-n&#xE9; dans le gyrus dent&#xE9; de l&#x26;#39;hippocampe adulte sont toniquement activ&#xE9; par ambiante GABA (acide gamma-aminobutyrique), avant d&#x26;#39;&#xEA;tre successivement innerv&#xE9; par GABA-et induite par le glutamate entr&#xE9;es synaptiques. GABA, principal neurotransmetteur inhibiteur dans le cerveau adulte, exerce d&#x26;#39;abord une action excitatrice sur les neurones nouveau-n&#xE9;s en raison de leur forte teneur en chlorure de cytoplasmique d&#x26;#39;ions. Conversion de GABA induite par d&#xE9;polarisation (excitation) dans une hyperpolarisation (inhibition) dans les neurones nouveau-n&#xE9;s conduit &#xE0; des d&#xE9;fauts marqu&#xE9;s dans leur formation de la synapse et le d&#xE9;veloppement dendritique in vivo. Notre &#xE9;tude identifie un r&#xF4;le essentiel pour le GABA dans l&#x26;#39;int&#xE9;gration synaptique des neurones nouvellement g&#xE9;n&#xE9;r&#xE9;s dans le cerveau adulte, et sugg&#xE8;re un m&#xE9;canisme inattendu de l&#x26;#39;activit&#xE9; r&#xE9;gulation d&#xE9;pendante de la neurogen&#xE8;se adulte, dans lequel les nouveaux neurones peuvent d&#xE9;tecter l&#x26;#39;activit&#xE9; du r&#xE9;seau neuronal par le biais tonique et phasique activation GABA .

GABA R&#xE9;gule int&#xE9;gration synaptique des neurones nouvellement g&#xE9;n&#xE9;r&#xE9;s dans le cerveau adulte

Adulten Neurogenese, die Geburt und die Integration neuer Nervenzellen von adulten neuralen Stammzellen ist eine auffallende strukturelle Plastizit&auml;t und highlights der Regenerationsf&auml;higkeit von den erwachsenen Gehirn von S&auml;ugetieren. Sammeln Beweise darauf hin, dass neuronaler Aktivit&auml;t adulten Neurogenese regelt und neue Nervenzellen bestimmter Hirnfunktionen beitragen. Der Mechanismus, der die Integration der neu generierten Neuronen in die bereits vorhandenen funktionellen Schaltungen im erwachsenen Gehirn regelt, ist unbekannt. Hier zeigen wir, dass Neugeborene Untermodul Zellen in den Gyrus dentatus des Erwachsenen Hippocampus tonisch durch Umgebungstemperatur GABA (Gamma - Aminobutters&auml;ure) aktiviert werden, bevor Sie wird nacheinander von GABA und Glutamat-vermittelte synaptische Eing&auml;nge innerviert. GABA, wichtigsten inhibitorischen Neurotransmitters im erwachsenen Gehirn, &uuml;bt zun&auml;chst eine erregenden Aktion auf der neugeborenen Neuronen aufgrund ihrer hohen zytoplasmatischen Chloridgehalt Ion. Umwandlung von GABA-induzierte Depolarisierung (Erregung) in einem Gatter (Hemmung) in neugeborenen Neuronen f&uuml;hrt zu deutlichen M&auml;ngel in ihre Synapse Bildung und in-vivo dendritische Entwicklung. Unsere Studie identifiziert eine wesentliche Rolle f&uuml;r GABA in die synaptische Integration der neu generierten Nervenzellen im erwachsenen Gehirn und schl&auml;gt einen unerwarteten Mechanismus f&uuml;r leistungsabh&auml;ngige Regelung der adulten Neurogenese, in dem Neugeborene Neuronen neuronale Netzwerk-Aktivit&auml;t durch tonische und phasische Aktivierung der GABA sp&uuml;ren k&ouml;nnen.

GABA regelt synaptische Integration der neu generierten Nervenzellen im erwachsenen Gehirn.

Neurogenesi adulta, la nascita e l&#39;integrazione di nuovi neuroni da cellule staminali neurali adulte, &egrave; una straordinaria forma di plasticit&agrave; strutturale e mette in evidenza la capacit&agrave; rigenerativa del cervello dei mammiferi adulti. Accumulando prove suggerisce che l&#39;attivit&agrave; neuronale regola neurogenesi adulta e che nuovi neuroni contribuiscono alle funzioni specifiche del cervello. Il meccanismo che regola l&#39;integrazione dei neuroni appena generati nel circuito funzionale preesistente nel cervello adulto &egrave; sconosciuto. Qui vi mostriamo che cellule del giro dentato dell&#39;ippocampo adulto neonato granulo tonicamente sono attivate da ambiente GABA (acido gamma - aminobutirrico) prima di essere sequenzialmente innervate da ingressi sinaptica mediata da GABA e glutammato. GABA, il principale neurotrasmettitore inibitorio nel cervello adulto, inizialmente esercita un&#39;azione eccitatoria sui neuroni neonati a causa del loro contenuto di ioni cloruro citoplasmatica alta. Conversione della depolarizzazione indotta dal GABA (eccitazione) in hyperpolarization (inibizione) in neuroni neonati porta a difetti contrassegnati nella loro formazione sinapsi dendritico sviluppo in vivo. Il nostro studio identifica un ruolo essenziale per GABA nell&#39;integrazione sinaptica dei neuroni appena generati nel cervello adulto e suggerisce un meccanismo inaspettato per la regolazione di attivit&agrave;-dipendente della neurogenesi adulta, in cui neuroni neonati possono percepire attivit&agrave; di rete neuronale attraverso toniche e fasiche attivazione GABA.

GABA regola integrazione sinaptica dei neuroni appena generati nel cervello adulto.

&#x5927;&#x4EBA;&#x306E;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x65B0;&#x751F;&#x306F;&#x3001;&#x6210;&#x4EBA;&#x306E;&#x795E;&#x7D4C;&#x5E79;&#x7D30;&#x80DE;&#x304B;&#x3089;&#x65B0;&#x3057;&#x3044;&#x795E;&#x7D4C;&#x7D30;&#x80DE;&#x306E;&#x8A95;&#x751F;&#x3068;&#x306E;&#x7D71;&#x5408;&#x306F;&#x3001;&#x69CB;&#x9020;&#x53EF;&#x5851;&#x6027;&#x306E;&#x5370;&#x8C61;&#x7684;&#x306A;&#x5F62;&#x5F0F;&#x3067;&#x3042;&#x308A;&#x3001;&#x54FA;&#x4E73;&#x985E;&#x6210;&#x4F53;&#x8133;&#x306E;&#x518D;&#x751F;&#x80FD;&#x529B;&#x3092;&#x5F37;&#x8ABF;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x8A3C;&#x62E0;&#x3092;&#x84C4;&#x7A4D;&#x3059;&#x308B;&#x3068;&#x3001;&#x305D;&#x306E;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306E;&#x6D3B;&#x52D5;&#x306F;&#x3001;&#x6210;&#x4F53;&#x306E;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x65B0;&#x751F;&#x3092;&#x8ABF;&#x7BC0;&#x3057;&#x3001;&#x65B0;&#x3057;&#x3044;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306F;&#x7279;&#x5B9A;&#x306E;&#x8133;&#x6A5F;&#x80FD;&#x306B;&#x5BC4;&#x4E0E;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x5506;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002;&#x6210;&#x4F53;&#x8133;&#x306B;&#x304A;&#x3051;&#x308B;&#x65E2;&#x5B58;&#x306E;&#x6A5F;&#x80FD;&#x56DE;&#x8DEF;&#x306B;&#x65B0;&#x305F;&#x306B;&#x751F;&#x6210;&#x3055;&#x308C;&#x305F;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306E;&#x7D71;&#x5408;&#x3092;&#x8ABF;&#x7BC0;&#x3059;&#x308B;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x306F;&#x4E0D;&#x660E;&#x3067;&#x3042;&#x308B;&#x3002;&#x3053;&#x3053;&#x3067;&#x6211;&#x3005;&#x306F;&#x3001;&#x6210;&#x4F53;&#x6D77;&#x99AC;&#x306E;&#x6B6F;&#x72B6;&#x56DE;&#x306B;&#x304A;&#x3051;&#x308B;&#x65B0;&#x751F;&#x5150;&#x306E;&#x9846;&#x7C92;&#x7D30;&#x80DE;&#x306F;tonically&#x9806;&#x6B21;GABA&#x3068;&#x30B0;&#x30EB;&#x30BF;&#x30DF;&#x30F3;&#x9178;&#x3092;&#x4ECB;&#x3057;&#x305F;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x5165;&#x529B;&#x306B;&#x3088;&#x3063;&#x3066;&#x652F;&#x914D;&#x3055;&#x308C;&#x308B;&#x524D;&#x306B;&#x5468;&#x56F2;&#x306E;GABA&#xFF08;&#x3B3;-&#x30A2;&#x30DF;&#x30CE;&#x916A;&#x9178;&#xFF09;&#x306B;&#x3088;&#x3063;&#x3066;&#x6D3B;&#x6027;&#x5316;&#x3055;&#x308C;&#x8868;&#x793A;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002; GABA&#x3001;&#x6210;&#x4F53;&#x8133;&#x306B;&#x304A;&#x3051;&#x308B;&#x4E3B;&#x8981;&#x306A;&#x6291;&#x5236;&#x6027;&#x795E;&#x7D4C;&#x4F1D;&#x9054;&#x7269;&#x8CEA;&#x306F;&#x3001;&#x5F53;&#x521D;&#x306F;&#x3001;&#x305D;&#x306E;&#x9AD8;&#x3044;&#x8CEA;&#x306E;&#x5869;&#x5316;&#x7269;&#x30A4;&#x30AA;&#x30F3;&#x542B;&#x6709;&#x91CF;&#x306E;&#x304A;&#x304B;&#x3052;&#x3067;&#x65B0;&#x751F;&#x5150;&#x306E;&#x795E;&#x7D4C;&#x7D30;&#x80DE;&#x306B;&#x8208;&#x596E;&#x6027;&#x4F5C;&#x7528;&#x3092;&#x767A;&#x63EE;&#x3059;&#x308B;&#x3002;&#x65B0;&#x751F;&#x5150;&#x306E;&#x795E;&#x7D4C;&#x7D30;&#x80DE;&#x306E;&#x904E;&#x5206;&#x6975;&#xFF08;&#x6291;&#x5236;&#xFF09;&#x3078;&#x306E;GABA&#x8A98;&#x767A;&#x6027;&#x8131;&#x5206;&#x6975;&#xFF08;&#x52B1;&#x8D77;&#xFF09;&#x306E;&#x5909;&#x63DB;&#x306F;&#x3001;in vivo&#x3067;&#x306E;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x5F62;&#x6210;&#x3068;&#x6A39;&#x72B6;&#x7A81;&#x8D77;&#x767A;&#x9054;&#x306E;&#x9855;&#x8457;&#x306A;&#x6B20;&#x9665;&#x306B;&#x3064;&#x306A;&#x304C;&#x308B;&#x3002;&#x6211;&#x3005;&#x306E;&#x7814;&#x7A76;&#x306F;&#x3001;&#x6210;&#x4EBA;&#x306E;&#x8133;&#x3067;&#x3001;&#x65B0;&#x3057;&#x304F;&#x751F;&#x6210;&#x3055;&#x308C;&#x305F;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306E;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x7D71;&#x5408;&#x306B;&#x304A;&#x3051;&#x308B;GABA&#x306E;&#x305F;&#x3081;&#x306B;&#x91CD;&#x8981;&#x306A;&#x5F79;&#x5272;&#x3092;&#x8B58;&#x5225;&#x3057;&#x3001;&#x6210;&#x4EBA;&#x306E;&#x795E;&#x7D4C;&#x306E;&#x6D3B;&#x52D5;&#x4F9D;&#x5B58;&#x7684;&#x8ABF;&#x7BC0;&#x306E;&#x305F;&#x3081;&#x306E;&#x4E88;&#x671F;&#x3057;&#x306A;&#x3044;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x3092;&#x793A;&#x5506;&#x3057;&#x3001;&#x65B0;&#x751F;&#x5150;&#x306E;&#x795E;&#x7D4C;&#x7D30;&#x80DE;&#x306F;&#x5F37;&#x58EE;&#x5264;&#x3068;&#x76F8;&#x6027;GABA&#x6D3B;&#x6027;&#x5316;&#x3092;&#x4ECB;&#x3057;&#x3066;&#x795E;&#x7D4C;&#x7D30;&#x80DE;&#x306E;&#x30CD;&#x30C3;&#x30C8;&#x30EF;&#x30FC;&#x30AF;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D3;&#x30C6;&#x30A3;&#x3092;&#x691C;&#x51FA;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3042;&#x3063;&#x305F;&#x3002;

GABA&#x306F;&#x3001;&#x6210;&#x4F53;&#x8133;&#x3067;&#x65B0;&#x3057;&#x304F;&#x4F5C;&#x6210;&#x3055;&#x308C;&#x305F;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306E;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x7D71;&#x5408;&#x3092;&#x8ABF;&#x7BC0;&#x3059;&#x308B;

&#xC131;&#xCCB4; &#xD0C4;&#xC0DD;&#xACFC; &#xD1B5;&#xD569; &#xC131;&#xC778; &#xC2E0;&#xACBD; &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xC5D0;&#xC11C; &#xC0C8;&#xB85C;&#xC6B4; &#xC2E0;&#xACBD; &#xC138;&#xD3EC;&#xC758; &#xAD6C;&#xC870;&#xC801; &#xC18C;&#xC131;&#xC758; &#xB208;&#xC5D0; &#xB744;&#xB294; &#xD615;&#xD0DC; &#xC774;&#xBA70; &#xC131;&#xC778; &#xD3EC;&#xC720;&#xB958; &#xB450;&#xB1CC;&#xC758; &#xC7AC;&#xC0DD; &#xB2A5;&#xB825;&#xC744; &#xAC15;&#xC870;. &#xC99D;&#xAC70;&#xB97C; &#xCD95;&#xC801; &#xAC74;&#xC758; &#xC2E0;&#xACBD; &#xD65C;&#xB3D9; &#xADDC;&#xC81C; &#xC131;&#xCCB4; &#xC0C8;&#xB85C;&#xC6B4; &#xC2E0;&#xACBD; &#xD2B9;&#xC815; &#xB1CC; &#xAE30;&#xB2A5;&#xC5D0; &#xAE30;&#xC5EC; &#xD55C;&#xB2E4;. &#xC131;&#xC778; &#xB450;&#xB1CC;&#xC5D0;&#xC11C; &#xAE30;&#xC874;&#xC758; &#xAE30;&#xB2A5; &#xD68C;&#xB85C;&#xC5D0; &#xC0C8;&#xB85C; &#xC0DD;&#xC131; &#xB41C; &#xC2E0;&#xACBD; &#xC138;&#xD3EC;&#xC758; &#xD1B5;&#xD569; &#xC870;&#xC808; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC744; &#xC54C; &#xC218; &#xC788;&#xB2E4;. &#xC5EC;&#xAE30; &#xC6B0;&#xB9AC;&#xAC00; &#xC131;&#xC778; &#xD574; &#xB9C8;&#xAC00; &#xB1CC;&#xC5D0;&#xC11C; &#xC2E0;&#xC0DD;&#xC544;&#xACFC; &#xB9BD; &#xC138;&#xD3EC;&#xB294; GABA&#xC640; &#xC870;&#xBBF8;&#xB8CC; &#xC911;&#xC7AC; &#xC2DC; &#xB0C5; &#xC2A4; &#xC785;&#xB825;&#xC5D0; &#xC758;&#xD574; innervated &#xC21C;&#xCC28;&#xC801;&#xC73C;&#xB85C; &#xB418; &#xACE0; &#xC804;&#xC5D0; tonically &#xC8FC;&#xBCC0; GABA (&#xAC10;&#xB9C8;-aminobutyric &#xC0B0;)&#xC5D0; &#xC758;&#xD574; &#xD65C;&#xC131;&#xD654; &#xB429;&#xB2C8;&#xB2E4; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;. &#xC131;&#xC778; &#xB1CC;&#xC758; &#xC8FC;&#xC694; &#xC5B5;&#xC81C; &#xC2E0;&#xACBD; &#xC804;&#xB2EC; &#xBB3C;&#xC9C8; GABA &#xCC98;&#xC74C; &#xB54C;&#xBB38;&#xC5D0; &#xADF8;&#xB4E4;&#xC758; &#xB192;&#xC740; &#xC138;&#xD3EC;&#xC9C8; &#xC5FC;&#xD654; &#xC774;&#xC628; &#xCF58;&#xD150;&#xCE20; &#xC2E0;&#xC0DD; &#xD558;&#xB294; &#xC2E0;&#xACBD;&#xC5D0; &#xD765;&#xBD84; &#xC131;&#xC758; &#xD589;&#xB3D9;&#xC744; &#xBBF8;&#xCE58;&#xB294;. GABA &#xC720;&#xB3C4; (&#xC5EC;&#xAE30;) depolarization hyperpolarization (&#xC5B5;&#xC81C;)&#xC5D0;&#xC11C; &#xC2E0;&#xC0DD; &#xD558;&#xB294; &#xC2E0;&#xACBD;&#xC73C;&#xB85C;&#xC758; &#xBCC0;&#xD658; &#xC2DC; &#xB0C5; &#xC2A4; &#xD615;&#xC131; &#xBC0F; &#xC218;&#xC9C0;&#xC0C1; &#xAC1C;&#xBC1C; vivo&#xC5D0;&#xC11C; &#xD45C;&#xC2DC; &#xB41C; &#xACB0;&#xD568;&#xC73C;&#xB85C; &#xC774;&#xC5B4;&#xC9D1;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xC758; &#xC5F0;&#xAD6C; &#xC131;&#xC778; &#xB450;&#xB1CC;&#xC5D0;&#xC11C; &#xC0C8;&#xB85C; &#xC0DD;&#xC131; &#xB41C; &#xC2E0;&#xACBD; &#xC138;&#xD3EC;&#xC758; &#xC2DC; &#xB0C5; &#xC2A4; &#xD1B5;&#xD569;&#xC5D0; GABA&#xC5D0; &#xB300; &#xD55C; &#xC911;&#xC694; &#xD55C; &#xC5ED;&#xD560;&#xC744; &#xC2DD;&#xBCC4; &#xD558; &#xACE0; &#xC2E0;&#xC0DD; &#xD558;&#xB294; &#xC2E0;&#xACBD; &#xD1A0; &#xB2C9;&#xACFC; phasic GABA &#xD65C;&#xC131;&#xD654;&#xB97C; &#xD1B5;&#xD574; &#xC2E0;&#xACBD; &#xB124;&#xD2B8;&#xC6CC;&#xD06C; &#xD65C;&#xB3D9;&#xC744; &#xB290;&#xB084; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xC131;&#xCCB4;&#xC758; &#xD65C;&#xB3D9;&#xC5D0; &#xC885;&#xC18D;&#xC801;&#xC778; &#xADDC;&#xC81C;&#xC5D0; &#xB300; &#xD55C; &#xC608;&#xAE30;&#xCE58; &#xC54A;&#xC740; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC744; &#xC81C;&#xC548; &#xD569;&#xB2C8;&#xB2E4;.

GABA&#xB294; &#xC131;&#xC778; &#xB450;&#xB1CC;&#xC5D0;&#xC11C; &#xC0C8;&#xB85C; &#xC0DD;&#xC131; &#xB41C; &#xC2E0;&#xACBD; &#xC138;&#xD3EC;&#xC758; &#xC2DC; &#xB0C5; &#xC2A4; &#xD1B5;&#xD569;&#xC744; &#xC870;&#xC808;&#xD569;&#xB2C8;&#xB2E4;.

Neurog&#xE9;nesis adulta, el nacimiento y la integraci&#xF3;n de nuevas neuronas de c&#xE9;lulas madre neurales adultas, es una forma llamativa de la plasticidad estructural y pone de manifiesto la capacidad regenerativa del cerebro adulto de mam&#xED;feros. La acumulaci&#xF3;n de pruebas sugiere que la actividad neuronal regula la neurog&#xE9;nesis adulta y que las nuevas neuronas contribuyen a las funciones espec&#xED;ficas del cerebro. El mecanismo que regula la integraci&#xF3;n de las neuronas reci&#xE9;n generadas en el circuito funcional pre-existente en el cerebro adulto es desconocido. Aqu&#xED; nos muestran que las c&#xE9;lulas granulares del reci&#xE9;n nacido en el giro dentado del hipocampo adulto est&#xE1;n t&#xF3;nica del ambiente activado por el GABA (&#xE1;cido gamma-aminobut&#xED;rico), antes de ser secuencial inervado por GABA-glutamato y mediada por las entradas sin&#xE1;pticas. GABA, el principal neurotransmisor inhibidor en el cerebro adulto, en un principio su acci&#xF3;n excitadora en las neuronas reci&#xE9;n nacidos debido a su alto contenido en iones cloruro citoplasm&#xE1;tica. La conversi&#xF3;n de GABA inducida por la despolarizaci&#xF3;n (excitaci&#xF3;n) en hiperpolarizaci&#xF3;n (inhibici&#xF3;n) en las neuronas reci&#xE9;n nacidos conduce a defectos marcados en su formaci&#xF3;n de sinapsis y el desarrollo dendr&#xED;ticas in vivo. Nuestro estudio identifica un papel esencial para el GABA en la integraci&#xF3;n sin&#xE1;ptica de las neuronas reci&#xE9;n generadas en el cerebro adulto, y sugiere un mecanismo inesperado de la actividad dependen de la regulaci&#xF3;n de la neurog&#xE9;nesis adulta, en la que las neuronas reci&#xE9;n nacidas puede detectar la actividad de la red neuronal a trav&#xE9;s de la activaci&#xF3;n t&#xF3;nica y f&#xE1;sica GABA .

GABA regula la integraci&#xF3;n sin&#xE1;ptica de las neuronas reci&#xE9;n generadas en el cerebro adulto

Several myelin-associated factors that inhibit axon growth of mature neurons, including Nogo66, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp), can associate with a common GPI-linked protein Nogo-66 receptor (NgR). Accumulating evidence suggests that myelin inhibitors also signal through unknown NgR-independent mechanisms. Here we show that MAG, a RGD tri-peptide containing protein, forms a complex with β1-integrin to mediate axonal growth cone turning responses of several neuronal types. Mutations that alter the RGD motif in MAG or inhibition of β1-integrin function, but not removal of NgRs, abolish these MAG-dependent events. In contrast, OMgp-induced repulsion is not affected by inhibition of b1-integrin function. We further show that MAG stimulates tyrosine phosphorylation of focal adhesion kinase (FAK), which in turn is required for MAG-induced growth cone turning. These studies identify β1-integrin as a specific mediator for MAG in growth cone turning responses, acting through FAK activation.

beta1-integrin mediates myelin-associated glycoprotein signaling in neuronal growth cones.

During development and tissue repair, progenitor cells are guided by both biochemical and biophysical cues of their microenvironment, including topographical signals. The topographical cues have been shown to play an important role in controlling the fate of cells. Systematic investigation of topographical structures with different geometries and sizes under the identical experimental conditions on the same chip will enhance the understanding of the role of shape and size in cell-topography interactions. A simple customizable multi-architecture chip (MARC) array is therefore developed to incorporate, on a single chip, distinct topographies of various architectural complexities, including both isotropic and anisotropic features, in nano- to micrometer dimensions, with different aspect ratios and hierarchical structures. Polydimethylsiloxane (PDMS) replicas of MARC are used to investigate the influence of different geometries and sizes in neural differentiation of primary murine neural progenitor cells (mNPCs). Anisotropic gratings (2 μm gratings, 250 nm gratings) and isotropic 1 μm pillars significantly promote differentiation of mNPCs into neurons, as indicated by expression of β-III-tubulin (59%, 58%, and 58%, respectively, compared to 30% on the control). In contrast, glial differentiation is enhanced on isotropic 2 μm holes and 1 μm pillars. These results illustrate that anisotropic topographies enhance neuronal differentiation while isotropic topographies enhance glial differentiation on the same chip under the same conditions. MARC enables simultaneous cost-effective investigation of multiple topographies, allowing efficient optimization of topographical and biochemical cues to modulate cell differentiation.

Microarray with micro- and nano-topographies enables identification of the optimal topography for directing the differentiation of primary murine neural progenitor cells.

Taurine is a sulfur-containing amino acid present in high concentrations in mammalian tissues. It has been implicated in several processes involving brain development and neurotransmission. However, the role of taurine in hippocampal neurogenesis during brain development is still unknown. Here we show that taurine regulates neural progenitor cell (NPC) proliferation in the dentate gyrus of the developing brain as well as in cultured early postnatal (P5) hippocampal progenitor cells and hippocampal slices derived from P5 mice brains. Taurine increased cell proliferation without having a significant effect on neural differentiation both in cultured P5 NPCs as well as cultured hippocampal slices and in vivo. Expression level analysis of synaptic proteins revealed that taurine increases the expression of Synapsin 1 and PSD 95. We also found that taurine stimulates the phosphorylation of ERK1/2 indicating a possible role of the ERK pathway in mediating the changes that we observed, especially in proliferation. Taken together, our results demonstrate a role for taurine in neural stem/progenitor cell proliferation in developing brain and suggest the involvement of the ERK1/2 pathways in mediating these actions. Our study also shows that taurine influences the levels of proteins associated with synapse development. This is the first evidence showing the effect of taurine on early postnatal neuronal development using a combination of in vitro, ex-vivo and in vivo systems.

Taurine induces proliferation of neural stem cells and synapse development in the developing mouse brain.

Mitochondrial energy production is a tightly regulated process involving the coordinated transcription of several genes, catalysis of a plethora of posttranslational modifications, and the formation of very large molecular supercomplexes. The regulation of mitochondrial activity is particularly important for the brain, which is a high-energy-consuming organ that depends on oxidative phosphorylation to generate ATP. Here we show that brain mitochondrial ATP production is controlled by the cytoplasmic polyadenylation-induced translation of an mRNA encoding NDUFV2, a key mitochondrial protein. Knockout mice lacking the Cytoplasmic Polyadenylation Element Binding protein 1 (CPEB1) have brain-specific dysfunctional mitochondria and reduced ATP levels, which is due to defective polyadenylation-induced translation of electron transport chain complex I protein NDUFV2 mRNA. This reduced ATP results in defective dendrite morphogenesis of hippocampal neurons both in vitro and in vivo. These and other results demonstrate that CPEB1 control of mitochondrial activity is essential for normal brain development.

Translational control of mitochondrial energy production mediates neuron morphogenesis.

Class 3 semaphorins are well-known axonal guidance cues during the embryonic development of mammalian nervous system. However, their activity on postnatally differentiated neurons in neurogenic regions of adult brains has not been characterized. We found that silencing of semaphorin receptors neuropilins (NRP) 1 or 2 in neural progenitors at the adult mouse dentate gyrus resulted in newly differentiated neurons with shorter dendrites and simpler branching in vivo. Tyrosine phosphorylation (Tyr 397) and serine phosphorylation (Ser 732) of FAK were essential for these effects. Semaphorin 3A and 3F mediate serine phosphorylation of FAK through the activation of Cdk5. Silencing of either Cdk5 or FAK in newborn neurons phenocopied the defects in dendritic development seen upon silencing of NRP1 or NRP2. Furthermore, in vivo overexpression of Cdk5 or FAK rescued the dendritic phenotypes seen in NRP1 and NRP2 deficient neurons. These results point to a novel role for class 3 semaphorins in promoting dendritic growth and branching during adult hippocampal neurogenesis through the activation of Cdk5-FAK signaling pathway.

Class 3 semaphorin mediates dendrite growth in adult newborn neurons through Cdk5/FAK pathway.

Docosahexaenoic acid (DHA) is an omega-3 fatty acid that is essential for normal brain growth and cognitive function. Consistent with its importance in the brain, DHA is highly enriched in brain phospholipids. Despite being an abundant fatty acid in brain phospholipids, DHA cannot be de novo synthesized in brain and must be imported across the blood-brain barrier, but mechanisms for DHA uptake in brain have remained enigmatic. Here we identify a member of the major facilitator superfamily--Mfsd2a (previously an orphan transporter)--as the major transporter for DHA uptake into brain. Mfsd2a is found to be expressed exclusively in endothelium of the blood-brain barrier of micro-vessels. Lipidomic analysis indicates that Mfsd2a-deficient (Mfsd2a-knockout) mice show markedly reduced levels of DHA in brain accompanied by neuronal cell loss in hippocampus and cerebellum, as well as cognitive deficits and severe anxiety, and microcephaly. Unexpectedly, cell-based studies indicate that Mfsd2a transports DHA in the form of lysophosphatidylcholine (LPC), but not unesterified fatty acid, in a sodium-dependent manner. Notably, Mfsd2a transports common plasma LPCs carrying long-chain fatty acids such LPC oleate and LPC palmitate, but not LPCs with less than a 14-carbon acyl chain. Moreover, we determine that the phosphor-zwitterionic headgroup of LPC is critical for transport. Importantly, Mfsd2a-knockout mice have markedly reduced uptake of labelled LPC DHA, and other LPCs, from plasma into brain, demonstrating that Mfsd2a is required for brain uptake of DHA. Our findings reveal an unexpected essential physiological role of plasma-derived LPCs in brain growth and function.

Mfsd2a is a transporter for the essential omega-3 fatty acid docosahexaenoic acid.

The topography of the extracellular microenvironment influences cell morphology, provides conduct guidance and directs cell differentiation. Aspect ratio and dimension of topography have been shown to affect cell behaviours, but the ability and mechanism of depth-sensing is not clearly understood. We showed that murine neural progenitor cells (mNPCs) can sense the depth of the micro-gratings. Neurite elongation, alignment and neuronal differentiation were observed to increase with grating depth. We proposed a mechanism for depth-sensing by growing neurites: filopodial adhesion in the growth cones favour elongation but the bending rigidity of the neurite cytoskeleton resists it. Thus, perpendicular extension on deeper grooves is unfavourable as neurites need to bend over a larger angle. A quantitative model was developed and its prediction of neurite growth on gratings fit well with the experimental data. The results indicated that mNPC fate can be directed by appropriately designed patterned surfaces.

Extending neurites sense the depth of the underlying topography during neuronal differentiation and contact guidance.

Neural stem/progenitor cells (NSC) have the potential for treatment of a wide range of neurological diseases such as Parkinson Disease and multiple sclerosis. Currently, NSC have been isolated only from hippocampus and subventricular zone (SVZ) of the adult brain. It is not known whether NSC can be found in all parts of the developing mid-trimester central nervous system (CNS) when the brain undergoes massive transformation and growth. Multipotent NSC from the mid-trimester cerebra, thalamus, SVZ, hippocampus, thalamus, cerebellum, brain stem and spinal cord can be derived and propagated as clonal neurospheres with increasing frequencies with increasing gestations. These NSC can undergo multi-lineage differentiation both in vitro and in vivo, and engraft in a developmental murine model. Regionally-derived NSC are phenotypically distinct, with hippocampal NSC having a significantly higher neurogenic potential (53.6%) over other sources (range of 0%-27.5%, p<0.004). Whole genome expression analysis showed differential gene expression between these regionally-derived NSC, which involved the Notch, epidermal growth factor as well as interleukin pathways. We have shown the presence of phenotypically-distinct regionally-derived NSC from the mid-trimester CNS, which may reflect the ontological differences occurring within the CNS. Aside from informing on the role of such cells during fetal growth, they may be useful for different cellular therapy applications.

Regionally-specified second trimester fetal neural stem cells reveals differential neurogenic programming.

Parkinson's disease (PD) is a neurodegenerative disease attributed to the loss of midbrain dopaminergic (DA) neurons. The current lack of predictive models for this disease has been hampered by the acquirement of robust cells, posing a major barrier to drug development. Differentiation of stem cells into subtype specific cells may be guided by appropriate topographical cues but the role of topography has hitherto not been well understood. We used a Multi-Architecture (MARC) chip with various topographical structures and identified three topographies, which generate DA neurons from murine hippocampal neural progenitor cells with the highest percentage of neuronal (β-III-tubulin positive) and dopaminergic (tyrosine hydroxylase positive) populations. Analysis on single pattern structures showed that 2 μm gratings with 2 μm spacing and 2 μm height (2 μm gratings) and 2 μm gratings with hierarchical structure produced cells with the highest gene expression of TH and PITX3, with the longest neurite and highest percentage of alignment. Quantitative image analysis showed the 2 μm gratings produced cells with the highest expression of pituitary homeobox 3 (PITX3), LIM homeobox transcription factor 1 alpha (LMX1a), aldehyde dehydrogenase 1 family member A1 (ALDH1a1) and microtubule associated protein 2 (MAP2), as compared to nano-gratings and unpatterned controls. These patterns also enhance DA neuron differentiation on different substrate rigidities, as seen on both poly-dimethylsiloxane (PDMS) and tissue culture polystyrene (TCPS) substrates. These results show the use of topographical influence for neuronal subtype specification, which could be translated into a wide range of clinical applications for PD.

Eyleen L. K. Goh

Neuroscience & Behavioral Disorders

DUKE-NUS Graduate Medical School

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Eyleen L. K. Goh

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Neuroscience & Behavioral Disorders

DUKE-NUS Graduate Medical School

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Neuroscience & Behavioral Disorders