Identification and inhibitory properties of a novel Ca(2+)/calmodulin antagonist.

We developed a high-throughput yeast-based assay to screen for chemical inhibitors of Ca(2+)/calmodulin-dependent kinase pathways. After screening two small libraries, we identified the novel antagonist 125-C9, a substituted ethyleneamine. In vitro kinase assays confirmed that 125-C9 inhibited several calmodulin-dependent kinases (CaMKs) competitively with Ca(2+)/calmodulin (Ca(2+)/CaM). This suggested that 125-C9 acted as an antagonist for Ca(2+)/CaM rather than for CaMKs. We confirmed this hypothesis by showing that 125-C9 binds directly to Ca(2+)/CaM using isothermal titration calorimetry. We further characterized binding of 125-C9 to Ca(2+)/CaM and compared its properties with those of two well-studied CaM antagonists: trifluoperazine (TFP) and W-13. Isothermal titration calorimetry revealed that binding of 125-C9 to CaM is absolutely Ca(2+)-dependent, likely occurs with a stoichiometry of five 125-C9 molecules to one CaM molecule, and involves an exchange of two protons at pH 7.0. Binding of 125-C9 is driven overall by entropy and appears to be competitive with TFP and W-13, which is consistent with occupation of similar binding sites. To test the effects of 125-C9 in living cells, we evaluated mitogen-stimulated re-entry of quiescent cells into proliferation and found similar, although slightly better, levels of inhibition by 125-C9 than by TFP and W-13. Our results not only define a novel Ca(2+)/CaM inhibitor but also reveal that chemically unique CaM antagonists can bind CaM by distinct mechanisms but similarly inhibit cellular actions of CaM.

Increasing arousal enhances inhibitory control in calm but not excitable dogs

© 2015, Springer-Verlag Berlin Heidelberg.The emotional-reactivity hypothesis proposes that problem-solving abilities can be constrained by temperament, within and across species. One way to test this hypothesis is with the predictions of the Yerkes–Dodson law. The law posits that arousal level, a component of temperament, affects problem solving in an inverted U-shaped relationship: Optimal performance is reached at intermediate levels of arousal and impeded by high and low levels. Thus, a powerful test of the emotional-reactivity hypothesis is to compare cognitive performance in dog populations that have been bred and trained based in part on their arousal levels. We therefore compared a group of pet dogs to a group of assistance dogs bred and trained for low arousal (N = 106) on a task of inhibitory control involving a detour response. Consistent with the Yerkes–Dodson law, assistance dogs, which began the test with lower levels of baseline arousal, showed improvements when arousal was artificially increased. In contrast, pet dogs, which began the test with higher levels of baseline arousal, were negatively affected when their arousal was increased. Furthermore, the dogs’ baseline levels of arousal, as measured in their rate of tail wagging, differed by population in the expected directions. Low-arousal assistance dogs showed the most inhibition in a detour task when humans eagerly encouraged them, while more highly aroused pet dogs performed worst on the same task with strong encouragement. Our findings support the hypothesis that selection on temperament can have important implications for cognitive performance.

Regulation of DLK-1 kinase activity by calcium-mediated dissociation from an inhibitory isoform.

MAPKKK dual leucine zipper-bearing kinases (DLKs) are regulators of synaptic development and axon regeneration. The mechanisms underlying their activation are not fully understood. Here, we show that C. elegans DLK-1 is activated by a Ca(2+)-dependent switch from inactive heteromeric to active homomeric protein complexes. We identify a DLK-1 isoform, DLK-1S, that shares identical kinase and leucine zipper domains with the previously described long isoform DLK-1L but acts to inhibit DLK-1 function by binding to DLK-1L. The switch between homo- or heteromeric DLK-1 complexes is influenced by Ca(2+) concentration. A conserved hexapeptide in the DLK-1L C terminus is essential for DLK-1 activity and is required for Ca(2+) regulation. The mammalian DLK-1 homolog MAP3K13 contains an identical C-terminal hexapeptide and can functionally complement dlk-1 mutants, suggesting that the DLK activation mechanism is conserved. The DLK activation mechanism is ideally suited for rapid and spatially controlled signal transduction in response to axonal injury and synaptic activity.

Potent and broad neutralizing activity of a single chain antibody fragment against cell-free and cell-associated HIV-1.

Several human monoclonal antibodies (hmAbs) exhibit relatively potent and broad neutralizing activity against HIV-1, but there has not been much success in using them as potential therapeutics. We have previously hypothesized and demonstrated that small engineered antibodies can target highly conserved epitopes that are not accessible by full-size antibodies. However, their potency has not been comparatively evaluated with known HIV-1-neutralizing hmAbs against large panels of primary isolates. We report here the inhibitory activity of an engineered single chain antibody fragment (scFv), m9, against several panels of primary HIV-1 isolates from group M (clades A-G) using cell-free and cell-associated virus in cell line-based assays. M9 was much more potent than scFv 17b, and more potent than or comparable to the best-characterized broadly neutralizing hmAbs IgG(1) b12, 2G12, 2F5 and 4E10. It also inhibited cell-to-cell transmission of HIV-1 with higher potency than enfuvirtide (T-20, Fuzeon). M9 competed with a sulfated CCR5 N-terminal peptide for binding to gp120-CD4 complex, suggesting an overlapping epitope with the coreceptor binding site. M9 did not react with phosphatidylserine (PS) and cardiolipin (CL), nor did it react with a panel of autoantigens in an antinuclear autoantibody (ANA) assay. We further found that escape mutants resistant to m9 did not emerge in an immune selection assay. These results suggest that m9 is a novel anti-HIV-1 candidate with potential therapeutic or prophylactic properties, and its epitope is a new target for drug or vaccine development.

The cytolytic molecules Fas ligand and TRAIL are required for murine thymic graft-versus-host disease.

Thymic graft-versus-host disease (tGVHD) can contribute to profound T cell deficiency and repertoire restriction after allogeneic BM transplantation (allo-BMT). However, the cellular mechanisms of tGVHD and interactions between donor alloreactive T cells and thymic tissues remain poorly defined. Using clinically relevant murine allo-BMT models, we show here that even minimal numbers of donor alloreactive T cells, which caused mild nonlethal systemic graft-versus-host disease, were sufficient to damage the thymus, delay T lineage reconstitution, and compromise donor peripheral T cell function. Furthermore, to mediate tGVHD, donor alloreactive T cells required trafficking molecules, including CCR9, L selectin, P selectin glycoprotein ligand-1, the integrin subunits alphaE and beta7, CCR2, and CXCR3, and costimulatory/inhibitory molecules, including Ox40 and carcinoembryonic antigen-associated cell adhesion molecule 1. We found that radiation in BMT conditioning regimens upregulated expression of the death receptors Fas and death receptor 5 (DR5) on thymic stromal cells (especially epithelium), while decreasing expression of the antiapoptotic regulator cellular caspase-8-like inhibitory protein. Donor alloreactive T cells used the cognate proteins FasL and TNF-related apoptosis-inducing ligand (TRAIL) (but not TNF or perforin) to mediate tGVHD, thereby damaging thymic stromal cells, cytoarchitecture, and function. Strategies that interfere with Fas/FasL and TRAIL/DR5 interactions may therefore represent a means to attenuate tGVHD and improve T cell reconstitution in allo-BMT recipients.

A novel, non-apoptotic role for Scythe/BAT3: a functional switch between the pro- and anti-proliferative roles of p21 during the cell cycle.

BACKGROUND: Scythe/BAT3 is a member of the BAG protein family whose role in apoptosis has been extensively studied. However, since the developmental defects observed in Bat3-null mouse embryos cannot be explained solely by defects in apoptosis, we investigated whether BAT3 is also involved in cell-cycle progression. METHODS/PRINCIPAL FINDINGS: Using a stable-inducible Bat3-knockdown cellular system, we demonstrated that reduced BAT3 protein level causes a delay in both G1/S transition and G2/M progression. Concurrent with these changes in cell-cycle progression, we observed a reduction in the turnover and phosphorylation of the CDK inhibitor p21, which is best known as an inhibitor of DNA replication; however, phosphorylated p21 has also been shown to promote G2/M progression. Our findings indicate that in Bat3-knockdown cells, p21 continues to be synthesized during cell-cycle phases that do not normally require p21, resulting in p21 protein accumulation and a subsequent delay in cell-cycle progression. Finally, we showed that BAT3 co-localizes with p21 during the cell cycle and is required for the translocation of p21 from the cytoplasm to the nucleus during the G1/S transition and G2/M progression. CONCLUSION: Our study reveals a novel, non-apoptotic role for BAT3 in cell-cycle regulation. By maintaining a low p21 protein level during the G1/S transition, BAT3 counteracts the inhibitory effect of p21 on DNA replication and thus enables the cells to progress from G1 to S phase. Conversely, during G2/M progression, BAT3 facilitates p21 phosphorylation by cyclin A/Cdk2, an event required for G2/M progression. BAT3 modulates these pro- and anti-proliferative roles of p21 at least in part by regulating cyclin A abundance, as well as p21 translocation between the cytoplasm and the nucleus to ensure that it functions in the appropriate intracellular compartment during each phase of the cell cycle.

Coupling of beta2-adrenoceptor to Gi proteins and its physiological relevance in murine cardiac myocytes.

-Transgenic mouse models have been developed to manipulate beta-adrenergic receptor (betaAR) signal transduction. Although several of these models have altered betaAR subtypes, the specific functional sequelae of betaAR stimulation in murine heart, particularly those of beta2-adrenergic receptor (beta2AR) stimulation, have not been characterized. In the present study, we investigated effects of beta2AR stimulation on contraction, [Ca2+]i transient, and L-type Ca2+ currents (ICa) in single ventricular myocytes isolated from transgenic mice overexpressing human beta2AR (TG4 mice) and wild-type (WT) littermates. Baseline contractility of TG4 heart cells was increased by 3-fold relative to WT controls as a result of the presence of spontaneous beta2AR activation. In contrast, beta2AR stimulation by zinterol or isoproterenol plus a selective beta1-adrenergic receptor (beta1AR) antagonist CGP 20712A failed to enhance the contractility in TG4 myocytes, and more surprisingly, beta2AR stimulation was also ineffective in increasing contractility in WT myocytes. Pertussis toxin (PTX) treatment fully rescued the ICa, [Ca2+]i, and contractile responses to beta2AR agonists in both WT and TG4 cells. The PTX-rescued murine cardiac beta2AR response is mediated by cAMP-dependent mechanisms, because it was totally blocked by the inhibitory cAMP analog Rp-cAMPS. These results suggest that PTX-sensitive G proteins are responsible for the unresponsiveness of mouse heart to agonist-induced beta2AR stimulation. This was further corroborated by an increased incorporation of the photoreactive GTP analog [gamma-32P]GTP azidoanilide into alpha subunits of Gi2 and Gi3 after beta2AR stimulation by zinterol or isoproterenol plus the beta1AR blocker CGP 20712A. This effect to activate Gi proteins was abolished by a selective beta2AR blocker ICI 118,551 or by PTX treatment. Thus, we conclude that (1) beta2ARs in murine cardiac myocytes couple to concurrent Gs and Gi signaling, resulting in null inotropic response, unless the Gi signaling is inhibited; (2) as a special case, the lack of cardiac contractile response to beta2AR agonists in TG4 mice is not due to a saturation of cell contractility or of the cAMP signaling cascade but rather to an activation of beta2AR-coupled Gi proteins; and (3) spontaneous beta2AR activation may differ from agonist-stimulated beta2AR signaling.

beta-Arrestin-mediated PDE4 cAMP phosphodiesterase recruitment regulates beta-adrenoceptor switching from Gs to Gi.

Phosphorylation of the beta(2) adrenoreceptor (beta(2)AR) by cAMP-activated protein kinase A (PKA) switches its predominant coupling from stimulatory guanine nucleotide regulatory protein (G(s)) to inhibitory guanine nucleotide regulatory protein (G(i)). beta-Arrestins recruit the cAMP-degrading PDE4 phosphodiesterases to the beta(2)AR, thus controlling PKA activity at the membrane. Here we investigate a role for PDE4 recruitment in regulating G protein switching by the beta(2)AR. In human embryonic kidney 293 cells overexpressing a recombinant beta(2)AR, stimulation with isoprenaline recruits beta-arrestins 1 and 2 as well as both PDE4D3 and PDE4D5 to the receptor and stimulates receptor phosphorylation by PKA. The PKA phosphorylation status of the beta(2)AR is enhanced markedly when cells are treated with the selective PDE4-inhibitor rolipram or when they are transfected with a catalytically inactive PDE4D mutant (PDE4D5-D556A) that competitively inhibits isoprenaline-stimulated recruitment of native PDE4 to the beta(2)AR. Rolipram and PDE4D5-D556A also enhance beta(2)AR-mediated activation of extracellular signal-regulated kinases ERK12. This is consistent with a switch in coupling of the receptor from G(s) to G(i), because the ERK12 activation is sensitive to both inhibitors of PKA (H89) and G(i) (pertussis toxin). In cardiac myocytes, the beta(2)AR also switches from G(s) to G(i) coupling. Treating primary cardiac myocytes with isoprenaline induces recruitment of PDE4D3 and PDE4D5 to membranes and activates ERK12. Rolipram robustly enhances this activation in a manner sensitive to both pertussis toxin and H89. Adenovirus-mediated expression of PDE4D5-D556A also potentiates ERK12 activation. Thus, receptor-stimulated beta-arrestin-mediated recruitment of PDE4 plays a central role in the regulation of G protein switching by the beta(2)AR in a physiological system, the cardiac myocyte.

Activation and targeting of extracellular signal-regulated kinases by beta-arrestin scaffolds.

Using both confocal immunofluorescence microscopy and biochemical approaches, we have examined the role of beta-arrestins in the activation and targeting of extracellular signal-regulated kinase 2 (ERK2) following stimulation of angiotensin II type 1a receptors (AT1aR). In HEK-293 cells expressing hemagglutinin-tagged AT1aR, angiotensin stimulation triggered beta-arrestin-2 binding to the receptor and internalization of AT1aR-beta-arrestin complexes. Using red fluorescent protein-tagged ERK2 to track the subcellular distribution of ERK2, we found that angiotensin treatment caused the redistribution of activated ERK2 into endosomal vesicles that also contained AT1aR-beta-arrestin complexes. This targeting of ERK2 reflects the formation of multiprotein complexes containing AT1aR, beta-arrestin-2, and the component kinases of the ERK cascade, cRaf-1, MEK1, and ERK2. Myc-tagged cRaf-1, MEK1, and green fluorescent protein-tagged ERK2 coprecipitated with Flag-tagged beta-arrestin-2 from transfected COS-7 cells. Coprecipitation of cRaf-1 with beta-arrestin-2 was independent of MEK1 and ERK2, whereas the coprecipitation of MEK1 and ERK2 with beta-arrestin-2 was significantly enhanced in the presence of overexpressed cRaf-1, suggesting that binding of cRaf-1 to beta-arrestin facilitates the assembly of a cRaf-1, MEK1, ERK2 complex. The phosphorylation of ERK2 in beta-arrestin complexes was markedly enhanced by coexpression of cRaf-1, and this effect is blocked by expression of a catalytically inactive dominant inhibitory mutant of MEK1. Stimulation with angiotensin increased the binding of both cRaf-1 and ERK2 to beta-arrestin-2, and the association of beta-arrestin-2, cRaf-1, and ERK2 with AT1aR. These data suggest that beta-arrestins function both as scaffolds to enhance cRaf-1 and MEK-dependent activation of ERK2, and as targeting proteins that direct activated ERK to specific subcellular locations.

Functionally active targeting domain of the beta-adrenergic receptor kinase: an inhibitor of G beta gamma-mediated stimulation of type II adenylyl cyclase.

The beta-adrenergic receptor kinase (beta ARK) phosphorylates its membrane-associated receptor substrates, such as the beta-adrenergic receptor, triggering events leading to receptor desensitization. beta ARK activity is markedly stimulated by the isoprenylated beta gamma subunit complex of heterotrimeric guanine nucleotide-binding proteins (G beta gamma), which translocates the kinase to the plasma membrane and thereby targets it to its receptor substrate. The amino-terminal two-thirds of beta ARK1 composes the receptor recognition and catalytic domains, while the carboxyl third contains the G beta gamma binding sequences, the targeting domain. We prepared this domain as a recombinant His6 fusion protein from Escherichia coli and found that it had both independent secondary structure and functional activity. We demonstrated the inhibitory properties of this domain against G beta gamma activation of type II adenylyl cyclase both in a reconstituted system utilizing Sf9 insect cell membranes and in a permeabilized 293 human embryonic kidney cell system. Gi alpha-mediated inhibition of adenylyl cyclase was not affected. These data suggest that this His6 fusion protein derived from the carboxyl terminus of beta ARK1 provides a specific probe for defining G beta gamma-mediated processes and for studying the structural features of a G beta gamma-binding domain.

Frontal-striatal circuits in cognitive aging: Evidence for caudate involvement

Changes in cognition with aging have been claimed to be due in large part to a decline in frontal lobe function. However, at our present state of knowledge, the emphasis on the frontal lobes to the exclusion of the rest of the frontal-striatal circuits of which they are a part is unwarranted. To argue this point, I consider another anatomical candidate within these circuits, the caudate. Evidence is presented that the caudate decreases in size with age as much as the frontal lobes and that damage to either the frontal lobes or the caudate is accompanied by declines in inhibitory processes, executive control, and cognitive speed similar to those seen in normal aging. Separating the unique contributions of the frontal lobes and the caudate to these circuits is difficult but should be the focus of future studies of the biological basis of cognitive aging.

Advances in understanding mechanisms of thalamic relays in cognition and behavior.

The main impetus for a mini-symposium on corticothalamic interrelationships was the recent number of studies highlighting the role of the thalamus in aspects of cognition beyond sensory processing. The thalamus contributes to a range of basic cognitive behaviors that include learning and memory, inhibitory control, decision-making, and the control of visual orienting responses. Its functions are deeply intertwined with those of the better studied cortex, although the principles governing its coordination with the cortex remain opaque, particularly in higher-level aspects of cognition. How should the thalamus be viewed in the context of the rest of the brain? Although its role extends well beyond relaying of sensory information from the periphery, the main function of many of its subdivisions does appear to be that of a relay station, transmitting neural signals primarily to the cerebral cortex from a number of brain areas. In cognition, its main contribution may thus be to coordinate signals between diverse regions of the telencephalon, including the neocortex, hippocampus, amygdala, and striatum. This central coordination is further subject to considerable extrinsic control, for example, inhibition from the basal ganglia, zona incerta, and pretectal regions, and chemical modulation from ascending neurotransmitter systems. What follows is a brief review on the role of the thalamus in aspects of cognition and behavior, focusing on a summary of the topics covered in a mini-symposium held at the Society for Neuroscience meeting, 2014.

Division of labor in frontal eye field neurons during presaccadic remapping of visual receptive fields.

Our percept of visual stability across saccadic eye movements may be mediated by presaccadic remapping. Just before a saccade, neurons that remap become visually responsive at a future field (FF), which anticipates the saccade vector. Hence, the neurons use corollary discharge of saccades. Many of the neurons also decrease their response at the receptive field (RF). Presaccadic remapping occurs in several brain areas including the frontal eye field (FEF), which receives corollary discharge of saccades in its layer IV from a collicular-thalamic pathway. We studied, at two levels, the microcircuitry of remapping in the FEF. At the laminar level, we compared remapping between layers IV and V. At the cellular level, we compared remapping between different neuron types of layer IV. In the FEF in four monkeys (Macaca mulatta), we identified 27 layer IV neurons with orthodromic stimulation and 57 layer V neurons with antidromic stimulation from the superior colliculus. With the use of established criteria, we classified the layer IV neurons as putative excitatory (n = 11), putative inhibitory (n = 12), or ambiguous (n = 4). We found that just before a saccade, putative excitatory neurons increased their visual response at the RF, putative inhibitory neurons showed no change, and ambiguous neurons increased their visual response at the FF. None of the neurons showed presaccadic visual changes at both RF and FF. In contrast, neurons in layer V showed full remapping (at both the RF and FF). Our data suggest that elemental signals for remapping are distributed across neuron types in early cortical processing and combined in later stages of cortical microcircuitry.

Selected Gamma Aminobutyric Acid (GABA) Esters may Provide Analgesia for Some Central Pain Conditions.

Central pain is an enigmatic, intractable condition, related to destruction of thalamic areas, resulting in likely loss of inhibitory synaptic transmission mediated by GABA. It is proposed that treatment of central pain, a localized process, may be treated by GABA supplementation, like Parkinson's disease and depression. At physiologic pH, GABA exists as a zwitterion that is poorly permeable to the blood brain barrier (BBB). Because the pH of the cerebral spinal fluid (CSF) is acidic relative to the plasma, ion trapping may allow a GABA ester prodrug to accumulate and be hydrolyzed within the CSF. Previous investigations with ester local anesthetics may be applicable to some GABA esters since they are weak bases, hydrolyzed by esterases and cross the BBB. Potential non-toxic GABA esters are discussed. Many GABA esters were investigated in the 1980s and it is hoped that this paper may spark renewed interest in their development.

Inhibition-Induced Forgetting Results from Resource Competition between Response Inhibition and Memory Encoding Processes.

UNLABELLED: Response inhibition is a key component of executive control, but its relation to other cognitive processes is not well understood. We recently documented the "inhibition-induced forgetting effect": no-go cues are remembered more poorly than go cues. We attributed this effect to central-resource competition, whereby response inhibition saps attention away from memory encoding. However, this proposal is difficult to test with behavioral means alone. We therefore used fMRI in humans to test two neural predictions of the "common resource hypothesis": (1) brain regions associated with response inhibition should exhibit greater resource demands during encoding of subsequently forgotten than remembered no-go cues; and (2) this higher inhibitory resource demand should lead to memory encoding regions having less resources available during encoding of subsequently forgotten no-go cues. Participants categorized face stimuli by gender in a go/no-go task and, following a delay, performed a surprise recognition memory test for those faces. Replicating previous findings, memory was worse for no-go than for go stimuli. Crucially, forgetting of no-go cues was predicted by high inhibitory resource demand, as quantified by the trial-by-trial ratio of activity in neural "no-go" versus "go" networks. Moreover, this index of inhibitory demand exhibited an inverse trial-by-trial relationship with activity in brain regions responsible for the encoding of no-go cues into memory, notably the ventrolateral prefrontal cortex. This seesaw pattern between the neural resource demand of response inhibition and activity related to memory encoding directly supports the hypothesis that response inhibition temporarily saps attentional resources away from stimulus processing. SIGNIFICANCE STATEMENT: Recent behavioral experiments showed that inhibiting a motor response to a stimulus (a "no-go cue") impairs subsequent memory for that cue. Here, we used fMRI to test whether this "inhibition-induced forgetting effect" is caused by competition for neural resources between the processes of response inhibition and memory encoding. We found that trial-by-trial variations in neural inhibitory resource demand predicted subsequent forgetting of no-go cues and that higher inhibitory demand was furthermore associated with lower concurrent activation in brain regions responsible for successful memory encoding of no-go cues. Thus, motor inhibition and stimulus encoding appear to compete with each other: when more resources have to be devoted to inhibiting action, less are available for encoding sensory stimuli.