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Applying Stereotactic Injection Technique to Study Genetic Effects on Animal Behaviors

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or ...

Telomerase is a specialized reverse transcriptase that extends telomeres of eukaryotic chromosomes. The catalytic core of human telomerase is composed of an RNA template known as hTER (human telomerase RNA) and a protein subunit named hTERT (human telomerase reverse transcriptase). We have been studying other functions of the telomerase besides its major role in telomere maintenance. In our previous work, we have demonstrated that the hTERT can functionally interact with a rabbit TER to regulate expression of other genes and also attenuate the induced apoptosis. Here we report that overexpression of hTERT in a human lens epithelial cell line accelerates growth of the transfected lens epithelial cells. Associated with the acceleration of cell growth, expression of p53, p21 and GCIP (Grap2 cyclin-D interacting protein) is downregulated in the hTERT-transfected cells. With the downregulation of p21 and GCIP, the retinoblastoma protein (RB) is completely hyperphosphorylated in the hTERT-transfected cells. As expected, in the presence of RB hyperphosphorylation, the E2F transactivity is upregulated. Inhibition of telomerase activity abolishes the observed growth acceleration and also the related molecular changes. Furthermore, expression of hTERT in telomerase-negative human lens epithelial cells derived from primary cultures also accelerates growth of the transfected cells. Taken together, our results suggest that hTERT, when overexpressed in human lens epithelial cells, accelerates cell growth rate through regulation of RB/E2F pathway and possibly other genes.

Human telomerase accelerates growth of lens epithelial cells through regulation of the genes mediating RB/E2F pathway.

Menschlichen Telomerase beschleunigt Wachstum der Linse Epithelzellen durch die Regulation der Gene vermittelnde Rb/E2F Weg.

Gab proteins amplify and integrate signals stimulated by many growth factors. In culture and animals, retinoic acid (RA) induces neuronal differentiation. We show that Gab2 expression is detected in neurons in three models of neuronal differentiation: embryonic carcinoma (EC) stem cells, embryonic stem cells, and primary neural stem cells (NSCs). RA treatment induces apoptosis, countered by basic FGF (bFGF). In EC cells, Gab2 silencing results in hypersensitivity to RA-induced apoptosis and abrogates the protection by bFGF. Gab2 suppression reduces bFGF-dependent activation of AKT but not ERK, and constitutively active AKT, but not constitutively active MEK1, reverses the hypersensitization. Thus, Gab2-mediated AKT activation is required for bFGF's protection. Moreover, Gab2 silencing impairs the differentiation of EC cells to neurons. Similarly, in NSCs, Gab2 suppression reduces bFGF-dependent proliferation as well as neuronal survival and production upon differentiation. Our findings provide the first evidence that Gab2 is an important player in neural differentiation, partly by acting downstream of bFGF to mediate survival through phosphoinositide 3 kinase-AKT.

A novel role for Gab2 in bFGF-mediated cell survival during retinoic acid-induced neuronal differentiation.

High-speed capillary electrophoresis (CE) was employed to detect binding and inhibition of SH2 domain proteins using fluorescently labeled phosphopeptides as affinity probes. Single SH2 protein-phosphopeptide complexes were detected and confirmed by competition and fluorescence anisotropy. The assay was then extended to a multiplexed system involving separation of three SH2 domain proteins: Src, SH2-Bbeta, and Fyn. The selectivity of the separation was improved by altering the charge of the peptide binding partners used, thus demonstrating a convenient way to control resolution for the multiplexed assay. The separation was completed within 6 s, allowing rapidly dissociating complexes to be detected. Two low molecular weight inhibitors were tested for inhibition selectivity and efficacy. One inhibitor interrupted binding interaction of all three proteins, while the other selectively inhibited Src only leaving SH2-Bbeta and Fyn complex barely affected. IC(50) of both selective and nonselective inhibitors were determined and compared for different proteins. The IC(50) of the nonselective inhibitor was 49 +/- 9, 323 +/- 42, and 228 +/- 19 microM (n = 3) for Src, SH2-Bbeta, and Fyn, respectively, indicating different efficacy of the nonselective inhibitor for different SH2 domain protein. It is concluded that high-speed CE has the potential for multiplexed screening of drugs that disrupt protein-protein interactions.

Multiplexed detection of protein-peptide interaction and inhibition using capillary electrophoresis.

Fluorescence anisotropy (FA), non-equilibrium CE of equilibrium mixtures (NECEEM) and high-speed CE were evaluated for measuring dissociation kinetics of peptide-protein binding systems. Fyn-SH3-SH2, a protein construct consisting of the src homology 2 (SH2) and 3 (SH3) domain of the protein Fyn, and a fluorescein-labeled phosphopeptide were used as a model system. All three methods gave comparable half-life of approximately 53 s for Fyn-SH3-SH2:peptide complex. Achieving satisfactory results by NECEEM required columns over 30 cm long. When using Fyn-SH2-SH3 tagged with glutathione S-transferase (GST) as the binding protein, both FA and NECEEM assays gave evidence of two complexes forming with the peptide, yet neither method allowed accurate measurement of dissociation rates for both complexes because of a lack of resolution. High-speed CE, with a 7 s separation time, enabled separation of both complexes and allowed determination of dissociation rate of both complexes independently. The two complexes had half-lives of 22.0+/-2.7 and 58.8+/-6.1 s, respectively. Concentration studies revealed that the GST-Fyn-SH3-SH2 protein formed a dimer so that complexes had binding ratios of 2:1 (protein-to-peptide ratio) and 2:2. Our results demonstrate that although all methods are suitable for 1:1 binding systems, high-speed CE is unique in allowing multiple complexes to be resolved simultaneously. This property allows determination of binding kinetics of complicated systems and makes the technique useful for discovering novel affinity interactions.

Measurement of dissociation rate of biomolecular complexes using CE.

The Disrupted in Schizophrenia 1 (DISC1) gene is disrupted by a balanced chromosomal translocation (1; 11) (q42; q14.3) in a Scottish family with a high incidence of major depression, schizophrenia, and bipolar disorder. Subsequent studies provided indications that DISC1 plays a role in brain development. Here, we demonstrate that suppression of DISC1 expression reduces neural progenitor proliferation, leading to premature cell cycle exit and differentiation. Several lines of evidence suggest that DISC1 mediates this function by regulating GSK3beta. First, DISC1 inhibits GSK3beta activity through direct physical interaction, which reduces beta-catenin phosphorylation and stabilizes beta-catenin. Importantly, expression of stabilized beta-catenin overrides the impairment of progenitor proliferation caused by DISC1 loss of function. Furthermore, GSK3 inhibitors normalize progenitor proliferation and behavioral defects caused by DISC1 loss of function. Together, these results implicate DISC1 in GSK3beta/beta-catenin signaling pathways and provide a framework for understanding how alterations in this pathway may contribute to the etiology of psychiatric disorders.

Disrupted in schizophrenia 1 regulates neuronal progenitor proliferation via modulation of GSK3beta/beta-catenin signaling.

The psychiatric illness risk gene Disrupted in Schizophrenia-1 (DISC1) plays an important role in brain development; however, it is unclear how DISC1 is regulated during cortical development. Here, we report that DISC1 is regulated during embryonic neural progenitor proliferation and neuronal migration through an interaction with DIX domain containing-1 (Dixdc1), the third mammalian gene discovered to contain a Disheveled-Axin (DIX) domain. We determined that Dixdc1 functionally interacts with DISC1 to regulate neural progenitor proliferation by co-modulating Wnt-GSK3beta/beta-catenin signaling. However, DISC1 and Dixdc1 do not regulate migration via this pathway. During neuronal migration, we discovered that phosphorylation of Dixdc1 by cyclin-dependent kinase 5 (Cdk5) facilitates its interaction with the DISC1-binding partner Ndel1. Furthermore, Dixdc1 phosphorylation and its interaction with DISC1/Ndel1 in vivo is required for neuronal migration. Together, these data reveal that Dixdc1 integrates DISC1 into Wnt-GSK3beta/beta-catenin-dependent and -independent signaling pathways during cortical development and further delineate how DISC1 contributes to neuropsychiatric disorders.

Dixdc1 is a critical regulator of DISC1 and embryonic cortical development.

Disc1 is a schizophrenia risk gene that engages multiple signaling pathways during neurogenesis and brain development. Using the zebrafish as a tool, we analyze the function of zebrafish Disc1 (zDisc1) at the earliest stages of brain and body development. We define a "tool" as a biological system that gives insight into mechanisms underlying a human disorder, although the system does not phenocopy the disorder. A zDisc1 peptide binds to GSK3Î², and zDisc1 directs early brain development and neurogenesis, by promoting Î²-catenin-mediated Wnt signaling and inhibiting GSK3Î² activity. zDisc1 loss-of-function embryos additionally display a convergence and extension phenotype, demonstrated by abnormal movement of dorsolateral cells during gastrulation, through changes in gene expression, and later through formation of abnormal, U-shaped muscle segments, and a truncated tail. These phenotypes are caused by alterations in the noncanonical Wnt pathway, via Daam and Rho signaling. The convergence and extension phenotype can be rescued by a dominant negative GSK3Î² construct, suggesting that zDisc1 inhibits GSK3Î² activity during noncanonical Wnt signaling. This is the first demonstration that Disc1 modulates the noncanonical Wnt pathway and suggests a previously unconsidered mechanism by which Disc1 may contribute to the etiology of neuropsychiatric disorders.

Disc1 regulates both Î²-catenin-mediated and noncanonical Wnt signaling during vertebrate embryogenesis.

Disc1 ist ein Schizophrenie-Risiko-gen, das mehrere Signalwege w&auml;hrend der Neurogenese und Gehirn-Entwicklung besch&auml;ftigt. Mit dem Zebrafisch als ein Werkzeug, analysieren wir die Funktion der Zebrafisch Disc1 (zDisc1) an den fr&uuml;hesten Stadien der Entwicklung von Gehirn und K&ouml;rper. Wir definieren ein &quot;Werkzeug&quot; als ein biologisches System, das Einblick in die Mechanismen einer menschlichen St&ouml;rung gibt, obwohl das System nicht Ph&auml;nokopie die Erkrankung f&uuml;hrt. Ein zDisc1 Peptid bindet an GSK3&beta; und zDisc1 leitet die fr&uuml;he Entwicklung des Gehirns und Neurogenese, durch die F&ouml;rderung von &beta;-Catenin-vermittelter Wnt signalisieren und hemmende GSK3&beta;-Aktivit&auml;t. zDisc1 Loss of Function Embryonen zeigen zus&auml;tzlich einen Konvergenz und Erweiterung Ph&auml;notyp, nachgewiesen durch abnorme Bewegung der dorsolateralen Zellen w&auml;hrend der Gastrulation, durch &Auml;nderungen in der Genexpression, und sp&auml;ter durch Bildung von abnormen, u-f&ouml;rmige Muskel-Segmente und eine abgeschnittene Schwanz. Diese Ph&auml;notypen entstehen durch Ver&auml;nderungen in der Mitgliedattribut Wnt-Signalweg, &uuml;ber Daam und Rho signalisieren. Der Konvergenz und Erweiterung Ph&auml;notyp kann gerettet werden durch ein dominant negative GSK3&beta;-Konstrukt, was darauf hindeutet, dass zDisc1 hemmt GSK3&beta; Aktivit&auml;t w&auml;hrend Mitgliedattribut Wnt signalisieren. Dies ist die erste Demonstration, dass Disc1 die Mitgliedattribut Wnt-Signalweg moduliert und schl&auml;gt einen zuvor unber&uuml;cksichtigt Mechanismus, durch den Disc1, die &Auml;tiologie der neuropsychiatrischen Erkrankungen beitragen k&ouml;nnen.

Disc1 regelt &beta;-Catenin-vermittelter und Mitgliedattribut Wnt signalisieren w&auml;hrend der Embryogenese der Wirbeltiere.

Colony-stimulating factor 1 (CSF-1) receptor (CSF-1R, or macrophage CSF receptor [M-CSFR]) is the primary regulator of the proliferation, survival, and differentiation of mononuclear phagocytes (MNPs), but the critical CSF-1 signals for these functions are unclear. The scaffold protein Gab2 is a major tyrosyl phosphoprotein in the CSF-1R signaling network. Here we demonstrate that Gab2 deficiency results in profoundly defective expansion of CSF-1R-dependent MNP progenitors in the bone marrow, through decreased proliferation and survival. Reconstitution and phospho-flow studies show that downstream of CSF-1R, Gab2 uses phosphatidylinositol 3-kinase (PI3K)-Akt and extracellular signal-regulated kinase (Erk) to regulate MNP progenitor expansion. Unexpectedly, Gab2 ablation enhances Jun N-terminal protein kinase 1 (JNK1) phosphorylation in differentiated MNPs but reduces their proliferation; inhibition of JNK signaling or reduction of JNK1 levels restores proliferation. MNP recruitment to inflammatory sites and the corresponding bone marrow response is strongly impaired in Gab2-deficient mice. Our data provide genetic and biochemical evidence that CSF-1R, through Gab2, utilizes different effectors at different stages of MNP development to promote their expansion.

Gab2 promotes colony-stimulating factor 1-regulated macrophage expansion via alternate effectors at different stages of development.

Disrupted in Schizophrenia-1 (DISC1) is a candidate gene for psychiatric disorders and has many roles during brain development. Common DISC1 polymorphisms (variants) are associated with neuropsychiatric phenotypes including altered cognition, brain structure, and function; however, it is unknown how this occurs. Here, we demonstrate using mouse, zebrafish, and human model systems that DISC1 variants are loss of function in Wnt/GSK3Î² signaling and disrupt brain development. The DISC1 variants A83V, R264Q, and L607F, but not S704C, do not activate Wnt signaling compared with wild-type DISC1 resulting in decreased neural progenitor proliferation. In zebrafish, R264Q and L607F could not rescue DISC1 knockdown-mediated aberrant brain development. Furthermore, human lymphoblast cell lines endogenously expressing R264Q displayed impaired Wnt signaling. Interestingly, S704C inhibited the migration of neurons in the developing neocortex. Our data demonstrate DISC1 variants impair Wnt signaling and brain development and elucidateÂ a possible mechanism for their role in neuropsychiatric phenotypes.

Common DISC1 polymorphisms disrupt Wnt/GSK3Î² signaling and brain development.

Schizophrenie-1 gest&ouml;rt (DISC1) ist ein Kandidat-gen f&uuml;r psychiatrische Erkrankungen und hat viele Rollen w&auml;hrend der Entwicklung des Gehirns. Gemeinsame DISC1-Polymorphismen (Varianten) stehen im Zusammenhang mit neuropsychiatrischen Ph&auml;notypen ver&auml;nderte Wahrnehmung, Gehirnstruktur und-Funktion; Es ist jedoch unbekannt, wie dies geschieht. Hier zeigen wir Maus, Zebrafisch und menschlichen Modellsysteme, dass DISC1-Varianten Funktionsverlust in Wnt/GSK3&beta; Signalisierung sind und Gehirnentwicklung st&ouml;ren. Die DISC1-Varianten A83V, R264Q, und L607F, aber nicht S704C, nicht aktivieren Wnt signalisieren, im Vergleich zu Wildtyp DISC1 resultierenden in neuronalen Vorl&auml;uferzellen Verbreitung abgenommen. Im Zebrafisch konnte R264Q und L607F DISC1 Niederschlag-vermittelte aberrante Hirnentwicklung nicht retten. Dar&uuml;ber hinaus beeintr&auml;chtigt die menschliche Lymphozyt-Zelllinien, die endogen Ausdr&uuml;cken R264Q angezeigt Wnt signalisieren. Interessanterweise gehemmt S704C die Migration der Neuronen in Entwicklungsl&auml;ndern Neocortex. Unsere Daten zeigen DISC1 Varianten Wnt signalisieren und Gehirn Entwicklung beeintr&auml;chtigen und einen m&ouml;glichen Mechanismus f&uuml;r ihre Rolle in der neuropsychiatrischen Ph&auml;notypen zu erhellen.

Gemeinsame DISC1-Polymorphismen st&ouml;ren Wnt/GSK3&beta;-Signalisierung und Gehirn-Entwicklung.

The formation of layered structure of the mammalian neocortex requires a fine organized migration of post-mitotic neurons during early development. However, whether the radial migration is regulated by NMDA receptor and specific subunits remains contradictory and unknown. Here, we reported that in the developing rat cortex, migration of presumptive layer II/III neurons to their deserved destination was regulated by NMDA receptors with GluN2B but not GluN2A subunit. Using in utero electroporation of small interference RNA (siRNA) of distinct NMDA receptor subunits, we found that knockdown GluN1 and GluN2B subunits dramatically delayed the neuronal migration to proper layer II/III, while improperly stayed at lower layers or even the germinal regions, without changing the cell fate. In contrast, knockdown of GluN2A subunit did not impair the neuronal migration. Additionally, the ecotopic neurons by GluN2B RNAi developed to well dendritic differentiation, while the ones by GluN1 RNAi still kept morphology of migrating neurons. Therefore, GluN2B subunit of NMDA receptor plays an essential role in regulating proper neuronal migration and cortical lamination.

Article	Year
Human telomerase accelerates growth of lens epithelial cells through regulation of the genes mediating RB/E2F pathway. Oncogene\| PubMed ID: 12032846	2002
A novel role for Gab2 in bFGF-mediated cell survival during retinoic acid-induced neuronal differentiation. The Journal of cell biology\| PubMed ID: 16009726	2005
Multiplexed detection of protein-peptide interaction and inhibition using capillary electrophoresis. Analytical chemistry\| PubMed ID: 17297974	2007
Measurement of dissociation rate of biomolecular complexes using CE. Electrophoresis\| PubMed ID: 19148904	2009
Disrupted in schizophrenia 1 regulates neuronal progenitor proliferation via modulation of GSK3beta/beta-catenin signaling. Cell\| PubMed ID: 19303846	2009
Dixdc1 is a critical regulator of DISC1 and embryonic cortical development. Neuron\| PubMed ID: 20624590	2010
Disc1 regulates both Î²-catenin-mediated and noncanonical Wnt signaling during vertebrate embryogenesis. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 21859895	2011
Gab2 promotes colony-stimulating factor 1-regulated macrophage expansion via alternate effectors at different stages of development. Molecular and cellular biology\| PubMed ID: 21930791	2011
Common DISC1 polymorphisms disrupt Wnt/GSK3Î² signaling and brain development. Neuron\| PubMed ID: 22099458	2011
The GluN2B subunit of N-methy-D-asparate receptor regulates the radial migration of cortical neurons in vivo. Brain research\| PubMed ID: 25838242	2015

Yingwei Mao

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