Target identification for a Hedgehog pathway inhibitor reveals the receptor GPR39.

Hedgehog (Hh) signaling determines cell fate during development and can drive tumorigenesis. We performed a screen for new compounds that can impinge on Hh signaling downstream of Smoothened (Smo). A series of cyclohexyl-methyl aminopyrimidine chemotype compounds ('CMAPs') were identified that could block pathway signaling in a Smo-independent manner. In addition to inhibiting Hh signaling, the compounds generated inositol phosphates through an unknown GPCR. Correlation of GPCR mRNA expression levels with compound activity across cell lines suggested the target to be the orphan receptor GPR39. RNA interference or cDNA overexpression of GPR39 demonstrated that the receptor is necessary for compound activity. We propose a model in which CMAPs activate GPR39, which signals to the Gli transcription factors and blocks signaling. In addition to the discovery of GPR39 as a new target that impinges on Hh signaling, we report on small-molecule modulators of the receptor that will enable in vitro interrogation of GPR39 signaling in different cellular contexts.

Identification of the C3a receptor (C3AR1) as the target of the VGF-derived peptide TLQP-21 in rodent cells.

TLQP-21, a peptide derived from VGF (non-acronymic) by proteolytic processing, has been shown to modulate energy metabolism, differentiation, and cellular response to stress. Although extensively investigated, the receptor for this endogenous peptide has not previously been described. This study describes the use of a series of studies that show G protein-coupled receptor-mediated biological activity of TLQP-21 signaling in CHO-K1 cells. Unbiased genome-wide sequencing of the transcriptome from responsive CHO-K1 cells identified a prioritized list of possible G protein-coupled receptors bringing about this activity. Further experiments using a series of defined receptor antagonists and siRNAs led to the identification of complement C3a receptor-1 (C3AR1) as a target for TLQP-21 in rodents. We have not been able to demonstrate so far that this finding is translatable to the human receptor. Our results are in line with a large number of physiological observations in rodent models of food intake and metabolic control, where TLQP-21 shows activity. In addition, the sensitivity of TLQP-21 signaling to pertussis toxin is consistent with the known signaling pathway of C3AR1. The binding of TLQP-21 to C3AR1 not only has effects on signaling but also modulates cellular functions, as TLQP-21 was shown to have a role in directing migration of mouse RAW264.7 cells.

Simultaneous control of attention by multiple working memory representations.

Working memory representations play a key role in controlling attention by making it possible to shift attention to task-relevant objects. Visual working memory has a capacity of three to four objects, but recent studies suggest that only one representation can guide attention at a given moment. We directly tested this proposal by monitoring eye movements while observers performed a visual search task in which they attempted to limit attention to objects drawn in two colors. When the observers were motivated to attend to one color at a time, they searched many consecutive items of one color (long run lengths) and exhibited a delay prior to switching gaze from one color to the other (switch cost). In contrast, when they were motivated to attend to both colors simultaneously, observers' gaze switched back and forth between the two colors frequently (short run lengths), with no switch cost. Thus, multiple working memory representations can concurrently guide attention.

Oculomotor capture reveals trial-by-trial neural correlates of attentional guidance by contents of visual working memory.

Evidence from attentional and oculomotor capture, contingent capture, and other paradigms suggests that mechanisms supporting human visual working memory (VWM) and visual attention are intertwined. Features held in VWM bias guidance toward matching items even when those features are task irrelevant. However, the neural basis of this interaction is underspecified. Prior examinations using fMRI have primarily relied on coarse comparisons across experimental conditions that produce varying amounts of capture. To examine the neural dynamics of attentional capture on a trial-by-trial basis, we applied an oculomotor paradigm that produced discrete measures of capture. On each trial, subjects were shown a memory item, followed by a blank retention interval, then a saccade target that appeared to the left or right. On some trials, an irrelevant distractor appeared above or below fixation. Once the saccade target was fixated, subjects completed a forced-choice memory test. Critically, either the target or distractor could match the feature held in VWM. Although task irrelevant, this manipulation produced differences in behavior: participants were more likely to saccade first to an irrelevant VWM-matching distractor compared with a non-matching distractor - providing a discrete measure of capture. We replicated this finding while recording eye movements and scanning participants' brains using fMRI. To examine the neural basis of oculomotor capture, we separately modeled the retention interval for capture and non-capture trials within the distractor-match condition. We found that frontal activity, including anterior cingulate cortex and superior frontal gyrus regions, differentially predicted subsequent oculomotor capture by a memory-matching distractor. Other regions previously implicated as involved in attentional capture by VWM-matching items showed no differential activity across capture and non-capture trials, even at a liberal threshold. Our findings demonstrate the power of trial-by-trial analyses of oculomotor capture as a means to examine the underlying relationship between VWM and attentional guidance systems.

A Simple and Efficient CRISPR Technique for Protein Tagging.

Genetic knock-in using homology-directed repair is an inefficient process, requiring the selection of few modified cells and hindering its application to primary cells. Here, we describe Homology independent gene Tagging (HiTag), a method to tag a protein of interest by CRISPR in up to 66% of transfected cells with one single electroporation. The technique has proven effective in various cell types and can be used to knock in a fluorescent protein for live cell imaging, to modify the cellular location of a target protein and to monitor the levels of a protein of interest by a luciferase assay in primary cells.

Multiple states in visual working memory: Evidence from oculomotor capture by memory-matching distractors.

Visual working memory (VWM) representations interact with attentional guidance, but there is controversy over whether multiple VWM items simultaneously influence attentional guidance. Extant studies relied on continuous variables like response times, which can obscure capture - especially if VWM representations cycle through interactive and non-interactive states. Previous conflicting findings regarding guidance when under high working memory (WM) load may be due to the use of noisier response time measures that mix capture and non-capture trials. Thus, we employed an oculomotor paradigm to characterize discrete attentional capture events under both high and low VWM load. Participants held one or two colors in memory, then executed a saccade to a target disk. On some trials, a distractor (sometimes VWM-matching) appeared simultaneously with the target. Eye movements were more frequently directed to a VWM-matching than a non-matching distractor for both load conditions. However, oculomotor capture by a VWM-matching distractor occurred less frequently under high compared with low load. These results suggest that attention is automatically guided toward items matching only one of two colors held in memory at a time, suggesting that items in VWM may cycle through attention-guiding and not-guiding states when more than one item is held in VWM and the task does not require that multiple items be maintained in an active, attention-guiding state.

UTS2B Defines a Novel Enteroendocrine Cell Population and Regulates GLP-1 Secretion Through SSTR5 in Male Mice.

The gut-pancreas axis plays a key role in the regulation of glucose homeostasis and may be therapeutically exploited to treat not only type 2 diabetes but also hypoglycemia and hyperinsulinemia. We identify a novel enteroendocrine cell type expressing the peptide hormone urotensin 2B (UTS2B). UTS2B inhibits glucagon-like peptide-1 (GLP-1) secretion in mouse intestinal crypts and organoids, not by signaling through its cognate receptor UTS2R but through the activation of the somatostatin receptor (SSTR) 5. Circulating UTS2B concentrations in mice are physiologically regulated during starvation, further linking this peptide hormone to metabolism. Furthermore, administration of UTS2B to starved mice demonstrates that it is capable of regulating blood glucose and plasma concentrations of GLP-1 and insulin in vivo. Altogether, our results identify a novel cellular source of UTS2B in the gut, which acts in a paracrine manner to regulate GLP-1 secretion through SSTR5. These findings uncover a fine-tuning mechanism mediated by a ligand-receptor pair in the regulation of gut hormone secretion, which can potentially be exploited to correct metabolic unbalance caused by overactivation of the gut-pancreas axis.

The architecture of interaction between visual working memory and visual attention.

In five experiments, we examined whether a task-irrelevant item in visual working memory (VWM) interacts with perceptual selection when VWM must also be used to maintain a template representation of a search target. This question is critical to distinguishing between competing theories specifying the architecture of interaction between VWM and attention. The single-item template hypothesis (SIT) posits that only a single item in VWM can be maintained in a state that interacts with attention. Thus, the secondary item should be inert with respect to attentional guidance. The multiple-item template hypothesis (MIT) posits that multiple items can be maintained in a state that interacts with attention; thus, both the target representation and the secondary item should be capable of guiding selection. This question has been addressed previously in attention capture studies, but the results have been ambiguous. Here, we modified these earlier paradigms to optimize sensitivity to capture. Capture by a distractor matching the secondary item in VWM was observed consistently across multiple types of search task (abstract arrays and natural scenes), multiple dependent measures (search reaction time (RT) and oculomotor capture), multiple memory dimensions (color and shape), and multiple search stimulus dimensions (color, shape, common objects), providing strong support for the MIT. (PsycINFO Database Record

Whatever you do, don't look at the...: Evaluating guidance by an exclusionary attentional template.

People can use a target template consisting of one or more features to guide attention and gaze to matching objects in a search array. But can we also use feature information to guide attention away from known irrelevant items? Some studies found a benefit from foreknowledge of a distractor feature, whereas others found a cost. Importantly, previous work has largely relied on end-of-trial manual responses; it is unclear how feature-guided avoidance might unfold as candidate objects are inspected. In the current experiments, participants were cued with a distractor feature to avoid, then performed a visual search task while eye movements were recorded. Participants initially fixated a to-be-avoided object more frequently than predicted by chance, but they also demonstrated avoidance of cue-matching objects later in the trial. When provided more time between cue stimulus and search array, participants continued to be initially captured by a cued-color item. Furthermore, avoidance of cue-matching objects later in the trial was not contingent on initial capture by a cue-matching object. These results suggest that the conflicting findings in previous negative-cue experiments may be explained by a mixture of two independent processes: initial attentional capture by memory-matching items and later avoidance of known irrelevant items. (PsycINFO Database Record

Competition in saccade target selection reveals attentional guidance by simultaneously active working memory representations.

The content of visual working memory (VWM) guides attention, but whether this interaction is limited to a single VWM representation or functional for multiple VWM representations is under debate. To test this issue, we developed a gaze-contingent search paradigm to directly manipulate selection history and examine the competition between multiple cue-matching saccade target objects. Participants first saw a dual-color cue followed by two pairs of colored objects presented sequentially. For each pair, participants selectively fixated an object that matched one of the cued colors. Critically, for the second pair, the cued color from the first pair was presented either with a new distractor color or with the second cued color. In the latter case, if two cued colors in VWM interact with selection simultaneously, we expected the second cued color object to generate substantial competition for selection, even though the first cued color was used to guide attention in the immediately previous pair. Indeed, in the second pair, selection probability of the first cued color was substantially reduced in the presence of the second cued color. This competition between cue-matching objects provides strong evidence that both VWM representations interacted simultaneously with selection. (PsycINFO Database Record

A natural ligand for the orphan receptor GPR15 modulates lymphocyte recruitment to epithelia.

GPR15 is an orphan G protein-coupled receptor (GPCR) that is found in lymphocytes. It functions as a co-receptor of simian immunodeficiency virus and HIV-2 and plays a role in the trafficking of T cells to the lamina propria in the colon and to the skin. We describe the purification from porcine colonic tissue extracts of an agonistic ligand for GPR15 and its functional characterization. In humans, this ligand, which we named GPR15L, is encoded by the gene C10ORF99 and has some features similar to the CC family of chemokines. GPR15L was found in some human and mouse epithelia exposed to the environment, such as the colon and skin. In humans, GPR15L was also abundant in the cervix. In skin, GPR15L was readily detected after immunologic challenge and in human disease, for example, in psoriatic lesions. Allotransplantation of skin from Gpr15l-deficient mice onto wild-type mice resulted in substantial graft protection, suggesting nonredundant roles for GPR15 and GPR15L in the generation of effector T cell responses. Together, these data identify a receptor-ligand pair that is required for immune homeostasis at epithelia and whose modulation may represent an alternative approach to treating conditions affecting the skin such as psoriasis.

Memory-based attention capture when multiple items are maintained in visual working memory.

Efficient visual search requires that attention is guided strategically to relevant objects, and most theories of visual search implement this function by means of a target template maintained in visual working memory (VWM). However, there is currently debate over the architecture of VWM-based attentional guidance. We contrasted a single-item-template hypothesis with a multiple-item-template hypothesis, which differ in their claims about structural limits on the interaction between VWM representations and perceptual selection. Recent evidence from van Moorselaar, Theeuwes, and Olivers (2014) indicated that memory-based capture during search, an index of VWM guidance, is not observed when memory set size is increased beyond a single item, suggesting that multiple items in VWM do not guide attention. In the present study, we maximized the overlap between multiple colors held in VWM and the colors of distractors in a search array. Reliable capture was observed when 2 colors were held in VWM and both colors were present as distractors, using both the original van Moorselaar et al. singleton-shape search task and a search task that required focal attention to array elements (gap location in outline square stimuli). In the latter task, memory-based capture was consistent with the simultaneous guidance of attention by multiple VWM representations. (PsycINFO Database Record

Evidence for negative feature guidance in visual search is explained by spatial recoding.

Theories of attention and visual search explain how attention is guided toward objects with known target features. But can attention be directed away from objects with a feature known to be associated only with distractors? Most studies have found that the demand to maintain the to-be-avoided feature in visual working memory biases attention toward matching objects rather than away from them. In contrast, Arita, Carlisle, and Woodman (2012) claimed that attention can be configured to selectively avoid objects that match a cued distractor color, and they reported evidence that this type of negative cue generates search benefits. However, the colors of the search array items in Arita et al. (2012) were segregated by hemifield (e.g., blue items on the left, red on the right), which allowed for a strategy of translating the feature-cue information into a simple spatial template (e.g., avoid right, or attend left). In the present study, we replicated the negative cue benefit using the Arita et al. (2012), method (albeit within a subset of participants who reliably used the color cues to guide attention). Then, we eliminated the benefit by using search arrays that could not be grouped by hemifield. Our results suggest that feature-guided avoidance is implemented only indirectly, in this case by translating feature-cue information into a spatial template.

Hyperfocusing in schizophrenia: Evidence from interactions between working memory and eye movements.

Recent research suggests that processing resources are focused more narrowly but more intensely in people with schizophrenia (PSZ) than in healthy control subjects (HCS), possibly reflecting local cortical circuit abnormalities. This hyperfocusing hypothesis leads to the counterintuitive prediction that, although PSZ cannot store as much information in working memory as HCS, the working memory representations that are present in PSZ may be more intense than those in HCS. To test this hypothesis, we used a task in which participants make a saccadic eye movement to a peripheral target and avoid a parafoveal nontarget while they are holding a color in working memory. Previous research with this task has shown that the parafoveal nontarget is more distracting when it matches the color being held in working memory. This effect should be enhanced in PSZ if their working memory representations are more intense. Consistent with this prediction, we found that the effect of a match between the distractor color and the memory color was larger in PSZ than in HCS. We also observed evidence that PSZ hyperfocused spatially on the region surrounding the fixation point. These results provide further evidence that some aspects of cognitive dysfunction in schizophrenia may be a result of a narrower and more intense focusing of processing resources.

Control of working memory content in schizophrenia.

People with schizophrenia (PSZ) exhibit signs of reduced working memory (WM) capacity. However, this may reflect an impairment in managing its content, e.g. preventing irrelevant information from taking up available storage space, rather than a true capacity reduction. We tested the ability to eliminate and update WM content in 38 PSZ and 30 healthy control subjects (HCS). Images of real-world objects were presented consecutively, and a tone cued the item most likely to be tested for memory. On half the trials, randomly intermixed, a second tone occurred. Participants were informed that the item cued by the second tone was now the most likely to be tested, and the item cued by the first tone now the least likely, providing incentive to eliminate the first cued item from WM. Both HCS and PSZ displayed a robust performance advantage for cued items. Unexpectedly, PSZ more efficiently removed the no-longer-essential item from WM than HCS. The magnitude of the WM clearance of this first cued item correlated with memory performance for the newly prioritized second cued item in PSZ, indicating that it was adaptive. However, WM clearance was not associated with WM capacity, ruling out the need to budget limited resources as an explanation for greater clearance in PSZ. A robust correlation between WM clearance and poverty of speech in PSZ instead suggests that the propensity to rapidly clear non-essential information and minimize the number of items in WM may be the reflection of a negative symptom trait. This finding may reflect a more general tendency of PSZ to focus processing more narrowly than HCS.

Response activation impairments in schizophrenia: evidence from the lateralized readiness potential.

Previous research has demonstrated deficits in preresponse motor activity in schizophrenia, as evidenced by a reduced lateralized readiness potential (LRP). The LRP deficit could be due to increased activation of the incorrect response (e.g., failure to suppress competition) or to reduced activation of the correct response (e.g., a low-level impairment in response preparation). To distinguish these possibilities, we asked whether the LRP impairment is increased under conditions of strong response competition. We manipulated the compatibility of stimulus-response mappings (Experiment 1) and the compatibility of the target with flankers (Experiment 2). In both experiments, the patient LRP was reduced as much under conditions of low response competition as under high competition. These results are incompatible with a failure of patients to suppress competition and are instead consistent with a deficit in activating the correct response.

R-Spondin potentiates Wnt/ß-catenin signaling through orphan receptors LGR4 and LGR5.

The Wnt/ß-catenin signaling pathbway controls many important biological processes. R-Spondin (RSPO) proteins are a family of secreted molecules that strongly potentiate Wnt/ß-catenin signaling, however, the molecular mechanism of RSPO action is not yet fully understood. We performed an unbiased siRNA screen to identify molecules specifically required for RSPO, but not Wnt, induced ß-catenin signaling. From this screen, we identified LGR4, then an orphan G protein-coupled receptor (GPCR), as the cognate receptor of RSPO. Depletion of LGR4 completely abolished RSPO-induced ß-catenin signaling. The loss of LGR4 could be compensated by overexpression of LGR5, suggesting that LGR4 and LGR5 are functional homologs. We further demonstrated that RSPO binds to the extracellular domain of LGR4 and LGR5, and that overexpression of LGR4 strongly sensitizes cells to RSPO-activated ß-catenin signaling. Supporting the physiological significance of RSPO-LGR4 interaction, Lgr4-/- crypt cultures failed to grow in RSPO-containing intestinal crypt culture medium. No coupling between LGR4 and heterotrimeric G proteins could be detected in RSPO-treated cells, suggesting that LGR4 mediates RSPO signaling through a novel mechanism. Identification of LGR4 and its relative LGR5, an adult stem cell marker, as the receptors of RSPO will facilitate the further characterization of these receptor/ligand pairs in regenerative medicine applications.

Reduced capacity but spared precision and maintenance of working memory representations in schizophrenia.

CONTEXT: Working memory deficits are considered a core feature of schizophrenia. Several recent integrative articles have offered mechanistic computational and neurobiological models of the origins of this cognitive deficit. OBJECTIVE: To test predictions of these models using a new experimental paradigm from the basic science literature that makes it possible to determine whether patients with schizophrenia show (1) deficits in working memory storage capacity, (2) deficits in the precision of working memory representations, and (3) an amplification of these deficits as the retention interval increases. DESIGN: Case-control design. All subjects performed a color working memory test in which they were asked to recall 3 or 4 items after a 1- or 4-second delay. All subjects also received a standard measure of intelligence and the Matrics Consensus Cognitive Battery. SETTING: A tertiary care research outpatient clinic. Patients A total of 31 clinically stable patients with a DSM-IV diagnosis of schizophrenia or schizoaffective disorder and 26 healthy volunteers participated. The 2 groups were similar in age, sex, and ethnicity distribution. MAIN OUTCOME MEASURES: (1) The number of items stored in working memory and (2) the precision of the working memory representations. RESULTS: Patients showed a clear reduction in the number of items stored in working memory. Patients did not differ from controls in the precision of their working memory representations. There was no evidence of delay-related amplification of impairment in either capacity or precision. CONCLUSIONS: Patients do not show the type of imprecision or delay-dependent amplification of impairment that is predicted on the basis of current models of the neurobiology of schizophrenia. The models need to be revised to account for a pure reduction in the number of items that patients are able to store in working memory.

Failure of schizophrenia patients to overcome salient distractors during working memory encoding.

BACKGROUND: Prior demonstrations of impaired attentional control in schizophrenia focused on conditions in which top-down control is needed to overcome prepotent response tendencies. Attentional control over stimulus processing has received little investigation. Here, we test whether attentional control is impaired during working memory encoding when salient distractors compete with less salient task-relevant stimuli. METHODS: Patients with schizophrenia (n = 28) and healthy control subjects (n = 25) performed a visuospatial working memory paradigm in which half of the to-be-encoded stimuli flickered to increase their salience. After a 2-second delay, stimuli reappeared and participants had to decide whether or not a probed item had shifted location. RESULTS: In the unbiased condition where flickering and nonflickering stimuli were equally likely to be probed, both groups displayed a trend toward better memory for the flickering items. In the flicker-bias condition in which the flickering stimuli were likely to be probed, both groups displayed a robust selection advantage for the flickering items. However, in the nonflicker-bias condition in which the nonflickering stimuli were likely to be probed, only healthy control subjects showed selection of the nonflickering items. Patients displayed a trend toward preferential memory for the flickering items, as in the unbiased condition. CONCLUSIONS: Both groups were able to select salient over nonsalient stimuli, but patients with schizophrenia were unable to select nonsalient over salient stimuli, consistent with impairment in the effortful control of attention. These findings demonstrate the generality of top-down control failure in schizophrenia in the face of bottom-up competition from salient stimuli as with prepotent response tendencies.