Zebrafish behavioral profiling identifies multitarget antipsychotic-like compounds.

Many psychiatric drugs act on multiple targets and therefore require screening assays that encompass a wide target space. With sufficiently rich phenotyping and a large sampling of compounds, it should be possible to identify compounds with desired mechanisms of action on the basis of behavioral profiles alone. Although zebrafish (Danio rerio) behavior has been used to rapidly identify neuroactive compounds, it is not clear what types of behavioral assays would be necessary to identify multitarget compounds such as antipsychotics. Here we developed a battery of behavioral assays in larval zebrafish to determine whether behavioral profiles can provide sufficient phenotypic resolution to identify and classify psychiatric drugs. Using the antipsychotic drug haloperidol as a test case, we found that behavioral profiles of haloperidol-treated zebrafish could be used to identify previously uncharacterized compounds with desired antipsychotic-like activities and multitarget mechanisms of action.

Protein kinase C isoform ε negatively regulates ADP-induced calcium mobilization and thromboxane generation in platelets.

OBJECTIVE: Members of the protein kinase C (PKC) family are shown to positively and negatively regulate platelet activation. Although positive regulatory roles are extensively studied, negative regulatory roles of PKCs are poorly understood. We investigated the mechanism and specific isoforms involved in PKC-mediated negative regulation of ADP-induced functional responses. METHODS AND RESULTS: A pan-PKC inhibitor, GF109203X, potentiated ADP-induced cPLA(2) phosphorylation and thromboxane generation as well as ERK activation and intracellular calcium (Ca(2+)(i)) mobilization, 2 signaling molecules, upstream of cPLA(2) activation. Thus, PKCs inhibit cPLA(2) activation by inhibiting ERK and Ca(2+)(i) mobilization. Because the inhibitor of classic PKC isoforms, GO-6976, did not affect ADP-mediated thromboxane generation, we investigated the role of novel class of PKC isoforms. ADP-induced thromboxane generation, calcium mobilization, and ERK phosphorylation were potentiated in PKCε null murine platelets compared with platelets from wild-type littermates. Interestingly, when thromboxane release is blocked, ADP-induced aggregation in PKCε knockout and wild-type was similar, suggesting that PKCε does not affect ADP-induced aggregation directly. PKCε knockout mice exhibited shorter times to occlusion in an FeCl(3)-induced arterial injury model and shorter bleeding times in tail-bleeding experiments. CONCLUSIONS: We conclude that PKCε negatively regulates ADP-induced thromboxane generation in platelets and offers protection against thrombosis.

Discovering novel neuroactive drugs through high-throughput behavior-based chemical screening in the zebrafish.

Most neuroactive drugs were discovered through unexpected behavioral observations. Systematic behavioral screening is inefficient in most model organisms. But, automated technologies are enabling a new phase of discovery-based research in central nervous system (CNS) pharmacology. Researchers are using large-scale behavior-based chemical screens in zebrafish to discover compounds with new structures, targets, and functions. These compounds are powerful tools for understanding CNS signaling pathways. Substantial differences between human and zebrafish biology will make it difficult to translate these discoveries to clinical medicine. However, given the molecular genetic similarities between humans and zebrafish, it is likely that some of these compounds will have translational utility. We predict that the greatest new successes in CNS drug discovery will leverage many model systems, including in vitro assays, cells, rodents, and zebrafish.

Effects of preexisting autoimmunity on heart graft prolongation after donor-specific transfusion and anti-CD154.

BACKGROUND: Alloreactive memory T cells prevent costimulatory blockade-induced heart graft survival in mice, but whether and how preexisting autoreactive T cells affect solid-organ transplants under these conditions is unknown. METHODS: We tested the impact of preexisting cardiac myosin (CM)-specific immunity on murine heart transplant recipients treated with donor-specific transfusion (DST) plus anti-CD154 monoclonal antibody MR1. RESULTS: Preimmunization with CM but not control ovalbumin abrogated the graft prolonging effects of DST/MR1, whether administered 2 weeks or more than 6 weeks before transplantation. Adoptive transfer of spleen cells from CM-immunized mice into naïve recipients had similar effects. CM-specific immunity did not cross-react with donor antigens and preimmunization with CM had no impact on the survival or histology of DST/MR1-treated syngeneic heart grafts, the latter indicating that persistent autoimmunity is insufficient to cause rejection in the context of costimulatory blockade. We observed that the CM preimmunized mice produced higher frequencies of donor-reactive T cells with higher ratios of CD8/CD4Foxp3 cells, suggesting that the autoreactive memory T cells provide help for activation of alloreactive T cells despite the costimulatory blockade. CONCLUSIONS: These mechanistic insights linking autoimmunity and alloimmunity in a model of murine heart transplantation have clinical relevance to the known association between autoimmunity and an elevated risk of acute and chronic heart transplant injury in humans.

G(12/13) signaling pathways substitute for integrin αIIbß3-signaling for thromboxane generation in platelets.

BACKGROUND: We have previously shown that ADP-induced TXA(2) generation requires signaling from αIIbß3 integrin in platelets. Here we observed that, unlike ADP, protease-activated receptor (PAR)-mediated TXA(2) generation occurs independently of αIIbß3. PAR agonists, but not ADP, activate G(12/13) signaling pathways. Hence, we evaluated the role of these pathways in TXA(2) generation. PRINCIPAL FINDINGS: Inhibition of ADP-induced thromboxane generation by fibrinogen receptor antagonist SC57101 was rescued by co-stimulation of G(12/13) pathways with YFLLRNP. This observation suggested an existence of a common signaling effector downstream of integrins and G(12/13) pathways. Hence, we evaluated role of three potential tyrosine kinases; c-Src, Syk and FAK (Focal Adhesion Kinase) that are known to be activated by integrins. c-Src and Syk kinase did not play a role in ADP-induced functional responses in platelets. Selective activation of G(12/13) pathways resulted in the activation of FAK, in the absence of integrin signaling. Interestingly, αIIbß3-mediated FAK activation occurred in a Src family kinase (SFK)-independent manner whereas G(12/13) pathway caused FAK activation in a SFK and RhoA-dependent manner. A FAK selective inhibitor TAE-226, blocked TXA(2) generation. However, in comparison to WT mice, Pf4-Cre/Fak-Floxed mice did not show any difference in platelet TXA(2) generation. CONCLUSIONS: Therefore, we conclude that differential activation of FAK occurs downstream of Integrins and G(12/13) pathways. However, the common effector molecule, possibly a tyrosine kinase downstream of integrins and G(12/13) pathways contributing to TXA(2) generation in platelets remains elusive.

G-protein-gated inwardly rectifying potassium channels regulate ADP-induced cPLA2 activity in platelets through Src family kinases.

ADP-induced TXA2 generation requires the costimulation of P2Y1, P2Y12, and the GPIIb/IIIa receptors. Signaling events downstream of the P2Y receptors that contribute to ADP-induced TXA2 generation have not been clearly delineated. In this study, we have investigated the role of G-protein-gated inwardly rectifying potassium channels (GIRKs), a recently identified functional effector for the P2Y12 receptor, in the regulation of ADP-induced TXA2 generation. At 10-microM concentrations, the 2 structurally distinct GIRK channel blockers, SCH23390 and U50488H, caused complete inhibition of ADP-induced cPLA2 phosphorylation and TXA2 generation, without affecting the conversion of AA to TXA2 or ADP-induced primary platelet aggregation in aspirin-treated platelets. In addition, Src family kinase selective inhibitors abolished 2MeSADP-mediated cPLA2 phosphorylation and TXA2 generation. Furthermore, these GIRK channel blockers completely blocked Gi-mediated Src kinase activation, suggesting that GIRK channels are upstream of Src family tyrosine kinase activation. In weaver mouse platelets, which have dysfunctional GIRK2 subunits, ADP-induced TXA2 generation was impaired. However, we did not observe any defect in 2MeSADP-induced platelet functional responses in GIRK2-null mouse platelets, suggesting that functional channels composed of other GIRK subunits contribute to ADP-induced TXA2 generation, via the regulation of the Src and cPLA2 activity.