Exploiting Cell-Based Assays to Accelerate Drug Development for G Protein-Coupled Receptors.

G protein-coupled receptors (GPCRs) are relevant targets for health and disease as they regulate various aspects of metabolism, proliferation, differentiation, and immune pathways. They are implicated in several disease areas, including cancer, diabetes, cardiovascular diseases, and mental disorders. It is worth noting that about a third of all marketed drugs target GPCRs, making them prime pharmacological targets for drug discovery. Numerous functional assays have been developed to assess GPCR activity and GPCR signaling in living cells. Here, we review the current literature of genetically encoded cell-based assays to measure GPCR activation and downstream signaling at different hierarchical levels of signaling, from the receptor to transcription, via transducers, effectors, and second messengers. Singleplex assay formats provide one data point per experimental condition. Typical examples are bioluminescence resonance energy transfer (BRET) assays and protease cleavage assays (e.g., Tango or split TEV). By contrast, multiplex assay formats allow for the parallel measurement of multiple receptors and pathways and typically use molecular barcodes as transcriptional reporters in barcoded assays. This enables the efficient identification of desired on-target and on-pathway effects as well as detrimental off-target and off-pathway effects. Multiplex assays are anticipated to accelerate drug discovery for GPCRs as they provide a comprehensive and broad identification of compound effects.

Protocol for identifying properties of ERBB receptor antagonists using the barcoded ERBBprofiler assay.

The ERBBprofiler assay measures compound effects on ERBB family receptors and key downstream signaling pathways that are implicated in cancer or other complex diseases. Here, we present a protocol for identifying properties of ERBB receptor antagonists using the barcoded ERBBprofiler assay. We describe steps for in-solution transfection, cell treatment, combined processing of samples, amplification and indexing of PCRs, sequencing, and data analysis. This approach allows for the simultaneous assessment of drug effects and cell-type-dependent effects. For complete details on the use and execution of this protocol, please refer to Popovic et al.1.

Modulation of metastable ensemble dynamics explains optimal coding at moderate arousal in auditory cortex.

Performance during perceptual decision-making exhibits an inverted-U relationship with arousal, but the underlying network mechanisms remain unclear. Here, we recorded from auditory cortex (A1) of behaving mice during passive tone presentation, while tracking arousal via pupillometry. We found that tone discriminability in A1 ensembles was optimal at intermediate arousal, revealing a population-level neural correlate of the inverted-U relationship. We explained this arousal-dependent coding using a spiking network model with a clustered architecture. Specifically, we show that optimal stimulus discriminability is achieved near a transition between a multi-attractor phase with metastable cluster dynamics (low arousal) and a single-attractor phase (high arousal). Additional signatures of this transition include arousal-induced reductions of overall neural variability and the extent of stimulus-induced variability quenching, which we observed in the empirical data. Altogether, this study elucidates computational principles underlying interactions between pupil-linked arousal, sensory processing, and neural variability, and suggests a role for phase transitions in explaining nonlinear modulations of cortical computations.

Inhibiting Hippo pathway kinases releases WWC1 to promote AMPAR-dependent synaptic plasticity and long-term memory in mice.

The localization, number, and function of postsynaptic AMPA-type glutamate receptors (AMPARs) are crucial for synaptic plasticity, a cellular correlate for learning and memory. The Hippo pathway member WWC1 is an important component of AMPAR-containing protein complexes. However, the availability of WWC1 is constrained by its interaction with the Hippo pathway kinases LATS1 and LATS2 (LATS1/2). Here, we explored the biochemical regulation of this interaction and found that it is pharmacologically targetable in vivo. In primary hippocampal neurons, phosphorylation of LATS1/2 by the upstream kinases MST1 and MST2 (MST1/2) enhanced the interaction between WWC1 and LATS1/2, which sequestered WWC1. Pharmacologically inhibiting MST1/2 in male mice and in human brain-derived organoids promoted the dissociation of WWC1 from LATS1/2, leading to an increase in WWC1 in AMPAR-containing complexes. MST1/2 inhibition enhanced synaptic transmission in mouse hippocampal brain slices and improved cognition in healthy male mice and in male mouse models of Alzheimer's disease and aging. Thus, compounds that disrupt the interaction between WWC1 and LATS1/2 might be explored for development as cognitive enhancers.

Profiling of ERBB receptors and downstream pathways reveals selectivity and hidden properties of ERBB4 antagonists.

ERBB receptor tyrosine kinases are involved in development and diseases like cancer, cardiovascular, neurodevelopmental, and mental disorders. Although existing drugs target ERBB receptors, the next generation of drugs requires enhanced selectivity and understanding of physiological pathway responses to improve efficiency and reduce side effects. To address this, we developed a multilevel barcoded reporter profiling assay, termed 'ERBBprofiler', in living cells to monitor the activity of all ERBB targets and key physiological pathways simultaneously. This assay helps differentiate on-target therapeutic effects from off-target and off-pathway side effects of ERBB antagonists. To challenge the assay, eight established ERBB antagonists were profiled. Known effects were confirmed, and previously uncharacterized properties were discovered, such as pyrotinib's preference for ERBB4 over EGFR. Additionally, two lead compounds selectively targeting ERBB4 were profiled, showing promise for clinical trials. Taken together, this multiparametric profiling approach can guide early-stage drug development and lead to improved future therapeutic interventions.

Polygenic risk for schizophrenia converges on alternative polyadenylation as molecular mechanism underlying synaptic impairment.

Schizophrenia (SCZ) is a genetically heterogenous psychiatric disorder of highly polygenic nature. Correlative evidence from genetic studies indicate that the aggregated effects of distinct genetic risk factor combinations found in each patient converge onto common molecular mechanisms. To prove this on a functional level, we employed a reductionistic cellular model system for polygenic risk by differentiating induced pluripotent stem cells (iPSCs) from 104 individuals with high polygenic risk load and controls into cortical glutamatergic neurons (iNs). Multi-omics profiling identified widespread differences in alternative polyadenylation (APA) in the 3' untranslated region of many synaptic transcripts between iNs from SCZ patients and healthy donors. On the cellular level, 3'APA was associated with a reduction in synaptic density of iNs. Importantly, differential APA was largely conserved between postmortem human prefrontal cortex from SCZ patients and healthy donors, and strongly enriched for transcripts related to synapse biology. 3'APA was highly correlated with SCZ polygenic risk and affected genes were significantly enriched for SCZ associated common genetic variation. Integrative functional genomic analysis identified the RNA binding protein and SCZ GWAS risk gene PTBP2 as a critical trans-acting factor mediating 3'APA of synaptic genes in SCZ subjects. Functional characterization of PTBP2 in iNs confirmed its key role in 3'APA of synaptic transcripts and regulation of synapse density. Jointly, our findings show that the aggregated effects of polygenic risk converge on 3'APA as one common molecular mechanism that underlies synaptic impairments in SCZ.

Comprehensive split TEV based protein-protein interaction screening reveals TAOK2 as a key modulator of Hippo signalling to limit growth.

The conserved Hippo signalling pathway plays a crucial role in tumour formation by limiting tissue growth and proliferation. At the core of this pathway are tumour suppressor kinases STK3/4 and LATS1/2, which limit the activity of the oncogene YAP1, the primary downstream effector. Here, we employed a split TEV-based protein-protein interaction screen to assess the physical interactions among 28 key Hippo pathway components and potential upstream modulators. This screen led us to the discovery of TAOK2 as pivotal modulator of Hippo signalling, as it binds to the pathway's core kinases, STK3/4 and LATS1/2, and leads to their phosphorylation. Specifically, our findings revealed that TAOK2 binds to and phosphorylates LATS1, resulting in the reduction of YAP1 phosphorylation and subsequent transcription of oncogenes. Consequently, this decrease led to a decrease in cell proliferation and migration. Interestingly, a correlation was observed between reduced TAOK2 expression and decreased patient survival time in certain types of human cancers, including lung and kidney cancer as well as glioma. Moreover, in cellular models corresponding to these cancer types the downregulation of TAOK2 by CRISPR inhibition led to reduced phosphorylation of LATS1 and increased proliferation rates, supporting TAOK2's role as tumour suppressor gene. By contrast, overexpression of TAOK2 in these cellular models lead to increased phospho-LATS1 but reduced cell proliferation. As TAOK2 is a druggable kinase, targeting TAOK2 could serve as an attractive pharmacological approach to modulate cell growth and potentially offer strategies for combating cancer.

Rescue of Comorbid Behavioral and Metabolic Phenotypes of Arrhythmic Mice by Restoring Circadian Cry1/2 Expression in the Suprachiasmatic Nucleus.

Background: Psychiatric and metabolic disorders occur disproportionately often comorbidly, which poses particular hurdles for patients and therapists. However, the mechanisms that promote such comorbidities are largely unknown and therefore cannot yet be therapeutically targeted for the simultaneous treatment of both conditions. Because circadian clocks regulate most physiological processes and their disruption is a risk factor for both psychiatric and metabolic disorders, they may be considered as a potential mechanism for the development of comorbidities and a therapeutic target. In the current study, we investigated the latter assumption in Cry1/2-/- mice, which exhibit substantially disrupted endogenous circadian rhythms and marked metabolic and behavioral deficits. Methods: By targeted virus-induced restoration of circadian rhythms in their suprachiasmatic nucleus, we can restore behavioral as well as several metabolic processes of these animals to near-normal circadian rhythmicity. Results: Importantly, by rescuing suprachiasmatic nucleus rhythms, several of their anxiety-like behavioral as well as diabetes- and energy homeostasis-related deficits were significantly improved. Interestingly, however, this did not affect all deficits typical of Cry1/2-/- mice; for example, restlessness and body weight remained unaffected. Conclusions: Taken together, the results of this study demonstrate, on the one hand, that restoration of disturbed circadian rhythms can be used to simultaneously treat psychiatric and metabolic deficits. On the other hand, the results also allow us to distinguish processes that depend more on local canonical clocks from those that depend more on suprachiasmatic nucleus rhythms.

Hippo-released WWC1 facilitates AMPA receptor regulatory complexes for hippocampal learning.

Learning and memory rely on changes in postsynaptic glutamergic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type receptor (AMPAR) number, spatial organization, and function. The Hippo pathway component WW and C2 domain-containing protein 1 (WWC1) regulates AMPAR surface expression and impacts on memory performance. However, synaptic binding partners of WWC1 and its hierarchical position in AMPAR complexes are largely unclear. Using cell-surface proteomics in hippocampal tissue of Wwc1-deficient mice and by generating a hippocampus-specific interactome, we show that WWC1 is a major regulatory platform in AMPAR signaling networks. Under basal conditions, the Hippo pathway members WWC1 and large tumor-suppressor kinase (LATS) are associated, which might prevent WWC1 effects on synaptic proteins. Reduction of WWC1/LATS binding through a point mutation at WWC1 elevates the abundance of WWC1 in AMPAR complexes and improves hippocampal-dependent learning and memory. Thus, uncoupling of WWC1 from the Hippo pathway to AMPAR-regulatory complexes provides an innovative strategy to enhance synaptic transmission.

A genetically identified population of layer 4 neurons in auditory cortex that contributes to pre-pulse inhibition of the acoustic startle response.

A fundamental task faced by the auditory system is the detection of events that are signaled by fluctuations in sound. Spiking in auditory cortical neurons is critical for sound detection, but the causal roles of specific cell types and circuits are still mostly unknown. Here we tested the role of a genetically identified population of layer 4 auditory cortical neurons in sound detection. We measured sound detection using a common variant of pre-pulse inhibition of the acoustic startle response, in which a silent gap in background noise acts as a cue that attenuates startle. We used a Gpr26-Cre driver line, which we found expressed predominantly in layer 4 of auditory cortex. Photostimulation of these cells, which were responsive to gaps in noise, was sufficient to attenuate the startle reflex. Photosuppression of these cells reduced neural responses to gaps throughout cortex, and impaired behavioral gap detection. These data demonstrate that cortical Gpr26 neurons are both necessary and sufficient for top-down modulation of the acoustic startle reflex, and are thus likely to be involved in sound detection.

Amyloid Pathology in the Central Auditory Pathway of 5XFAD Mice Appears First in Auditory Cortex.

BACKGROUND: Effective treatment of Alzheimer's disease (AD) will hinge on early detection. This has led to the search for early biomarkers that use non-invasive testing. One possible early biomarker is auditory temporal processing deficits, which reflect central auditory pathway dysfunction and precede cognitive and memory declines in AD. Gap detection is a measure of auditory temporal processing, is impaired in human AD, and is also impaired in the 5XFAD mouse model of AD. Gap detection deficits appear as early as postnatal day 60 in 5XFAD mice, months before cognitive deficits or cell death, supporting gap detection as an early biomarker. However, it remains unclear how gap detection deficits relate to the progression of amyloid pathology in the auditory system. OBJECTIVE: To determine the progression of amyloid pathology throughout the central auditory system and across age in 5XFAD mice. METHODS: We quantified intracellular and extracellular antibody labelling of Aß42 in 6 regions of the central auditory system from p14 to p150. RESULTS: Pathology appeared first in primary auditory cortex (A1) as intracellular accumulation of Aß42 in layer 5 pyramidal neurons by age p21. Extracellular plaques appeared later, by age p90, in A1, medial geniculate body, and inferior colliculus. Auditory brainstem structures showed minimal amyloid pathology. We also observed pathology in the caudal pontine reticular nucleus, a brainstem structure that is outside of the central auditory pathway but which is involved in the acoustic startle reflex. CONCLUSION: These results suggest that Aß42 accumulation, but not plaques, may impair gap detection.

Expression of Lineage Transcription Factors Identifies Differences in Transition States of Induced Human Oligodendrocyte Differentiation.

Oligodendrocytes (OLs) are critical for myelination and are implicated in several brain disorders. Directed differentiation of human-induced OLs (iOLs) from pluripotent stem cells can be achieved by forced expression of different combinations of the transcription factors SOX10 (S), OLIG2 (O), and NKX6.2 (N). Here, we applied quantitative image analysis and single-cell transcriptomics to compare different transcription factor (TF) combinations for their efficacy towards robust OL lineage conversion. Compared with S alone, the combination of SON increases the number of iOLs and generates iOLs with a more complex morphology and higher expression levels of myelin-marker genes. RNA velocity analysis of individual cells reveals that S generates a population of oligodendrocyte-precursor cells (OPCs) that appear to be more immature than those generated by SON and to display distinct molecular properties. Our work highlights that TFs for generating iOPCs or iOLs should be chosen depending on the intended application or research question, and that SON might be beneficial to study more mature iOLs while S might be better suited to investigate iOPC biology.

Dissecting intercellular and intracellular signaling networks with barcoded genetic tools.

The power of next-generation sequencing has stimulated the development of many analysis techniques for transcriptomics and genomics. More recently, the concept of 'molecular barcoding' has broadened the spectrum of sequencing-based applications to dissect different aspects of intracellular and intercellular signaling. In these assay formats, barcode reporters replace standard reporter genes. The virtually infinitive number of expressed barcode sequences allows high levels of multiplexing, hence accelerating experimental progress. Furthermore, reporter barcodes are used to quantitatively monitor a variety of biological events in living cells which has already provided much insight into complex cellular signaling and will further increase our knowledge in the future.

Improved Split TEV GPCR ß-arrestin-2 Recruitment Assays via Systematic Analysis of Signal Peptide and ß-arrestin Binding Motif Variants.

G protein-coupled receptors (GPCRs) are major disease-relevant drug targets; robust monitoring of their activities upon drug treatment is key to drug discovery. The split TEV cell-based assay technique monitors the interaction of an activated GPCR with ß-arrestin-2 through TEV protein fragment complementation using a luminescent signal as the readout. In this work, split TEV GPCR ß-arrestin-2 recruitment assays were optimized to monitor the endogenous ligand-induced activities of six GPCRs (DRD1, DRD2, HTR2A, GCGR, AVPR2, and GLP1R). Each GPCR was tested in four forms; i.e., its wildtype form, a variant with a signal peptide (SP) to facilitate receptor expression, a variant containing the C-terminal tail from the V2 vasopressin receptor (V2R tail) to promote ß-arrestin-2 recruitment, and a variant containing both the SP and V2R tail. These 24 GPCR variants were systematically tested for assay performance in four cell lines (HEK-293, PC12 Tet-Off, U-2 OS, and HeLa). We found that the assay performance differed significantly for each GPCR variant and was dependent on the cell line. We found that V2R improved the DRD2 split TEV assays and that HEK-293 cells were the preferred cell line across the GPCRs tested. When taking these considerations into account, the defined selection of assay modifications and conditions may improve the performance of drug development campaigns that apply the split TEV technique as a screening tool.

Effects of Locomotion in Auditory Cortex Are Not Mediated by the VIP Network.

Movement has a prominent impact on activity in sensory cortex, but has opposing effects on visual and auditory cortex. Both cortical areas feature a vasoactive intestinal peptide-expressing (VIP) disinhibitory circuit, which in visual cortex contributes to the effect of running. In auditory cortex, however, the role of VIP circuitry in running effects remains poorly understood. Running and optogenetic VIP activation are known to differentially modulate sound-evoked activity in auditory cortex, but it is unknown how these effects vary across cortical layers, and whether laminar differences in the roles of VIP circuitry could contribute to the substantial diversity that has been observed in the effects of both movement and VIP activation. Here we asked whether VIP neurons contribute to the effects of running, across the layers of auditory cortex. We found that both running and optogenetic activation of VIP neurons produced diverse changes in the firing rates of auditory cortical neurons, but with distinct effects on spontaneous and evoked activity and with different patterns across cortical layers. On average, running increased spontaneous firing rates but decreased evoked firing rates, resulting in a reduction of the neuronal encoding of sound. This reduction in sound encoding was observed in all cortical layers, but was most pronounced in layer 2/3. In contrast, VIP activation increased both spontaneous and evoked firing rates, and had no net population-wide effect on sound encoding, but strongly suppressed sound encoding in layer 4 narrow-spiking neurons. These results suggest that VIP activation and running act independently, which we then tested by comparing the arithmetic sum of the two effects measured separately to the actual combined effect of running and VIP activation, which were closely matched. We conclude that the effects of locomotion in auditory cortex are not mediated by the VIP network.

A Layer 3→5 Circuit in Auditory Cortex That Contributes to Pre-pulse Inhibition of the Acoustic Startle Response.

While connectivity within sensory cortical circuits has been studied extensively, how these connections contribute to perception and behavior is not well understood. Here we tested the role of a circuit between layers 3 and 5 of auditory cortex in sound detection. We measured sound detection using a common variant of pre-pulse inhibition of the acoustic startle response, in which a silent gap in background noise acts as a cue that attenuates startle. We used the Nr5a-Cre driver line, which we found drove expression in the auditory cortex restricted predominantly to layer 3. Photoactivation of these cells evoked short-latency, highly reliable spiking in downstream layer 5 neurons, and attenuated startle responses similarly to gaps in noise. Photosuppression of these cells did not affect behavioral gap detection. Our data provide the first demonstration that direct activation of auditory cortical neurons is sufficient to attenuate the acoustic startle response, similar to the detection of a sound.

5XFAD mice show early-onset gap encoding deficits in the auditory cortex.

Early detection will be crucial for effective treatment or prevention of Alzheimer's disease. The identification and validation of early, noninvasive biomarkers is therefore key to avoiding the most devastating aspects of Alzheimer's disease. Measures of central auditory processing such as gap detection have recently emerged as potential biomarkers in both human patients and the 5XFAD mouse model of Alzheimer's disease. Full validation of gap detection deficits as a biomarker will require detailed understanding of the underlying neuropathology, including which brain structures are involved and how the operation of neural circuits is affected. Here we show that 5XFAD mice exhibit gap detection deficits as early as 2 months of age, well before development of Alzheimer's disease-associated pathology. We then examined responses of neurons in the auditory cortex to gaps in white noise. Both gap responses and baseline firing rates were robustly and progressively degraded in 5XFAD mice compared to littermate controls. These impairments were first evident at 2-4 months of age in males, and 4-6 months in females. This demonstrates early-onset impairments to the central auditory system, which could be due to damage in the auditory cortex, upstream subcortical structures, or both.

Multiparametric Assays for Accelerating Early Drug Discovery.

Drug discovery campaigns are hampered by substantial attrition rates largely due to a lack of efficacy and safety reasons associated with candidate drugs. This is true in particular for genetically complex diseases, where insufficient knowledge of the modulatory actions of candidate drugs on targets and entire target pathways further adds to the problem of attrition. To better profile compound actions on targets, potential off-targets, and disease-linked pathways, new innovative technologies need to be developed that can elucidate the complex cellular signaling networks in health and disease. Here, we discuss progress in genetically encoded multiparametric assays and mass spectrometry (MS)-based proteomics, which both represent promising toolkits to profile multifactorial actions of drug candidates in disease-relevant cellular systems to promote drug discovery and personalized medicine.

Add-on spironolactone as antagonist of the NRG1-ERBB4 signaling pathway for the treatment of schizophrenia: Study design and methodology of a multicenter randomized, placebo-controlled trial.

BACKGROUND: Preclinical studies recently showed that the mineralocorticoid antagonist spironolactone acts also as an antagonist of the NRG1-ERBB4 signaling pathway and improves schizophrenia-like behaviour in Nrg1 transgenic mouse model. As this signaling pathway is critically linked to the pathophysiology of schizophrenia, especially in the context of working-memory dysfunction, spironolactone may be a novel treatment option for patients with schizophrenia targeting cognitive impairments. AIMS: To evaluate whether spironolactone added to an ongoing antipsychotic treatment improves cognitive functioning in schizophrenia. METHODS: The add-on spironolactone for the treatment of schizophrenia trial (SPIRO-TREAT) is a multicenter randomized, placebo-controlled trial with three arms (spironolactone 100 mg, spironolactone 200 mg and placebo). Schizophrenia patients are treated for three weeks and then followed-up for additional nine weeks. As primary outcome, we investigate changes in working memory before and at the end of the intervention phase. We will randomize 90 patients. Eighty-one patients are intended to reach the primary endpoint measure at the end of the three-week intervention period. Secondary endpoints include other measures of cognition, psychopathology, safety measures and biological measures. CONCLUSIONS: SPIRO-TREAT is the first study evaluating the efficacy of the mineralocorticoid receptor antagonist spironolactone to improve cognitive impairments in schizophrenia patients targeting the NRG1-ERBB4 signaling pathway. With SPIRO-TREAT, we intend to investigate a novel treatment option for cognitive impairments in schizophrenia that goes beyond the established concepts of interfering with dopaminergic neurotransmission as key pathway in schizophrenia treatment. CLINICAL TRIAL REGISTRATION: International Clinical Trials Registry Platform: http://apps.who.int/trialsearch/Trial2.aspx?TrialID=EUCTR2014-001968-35-DE.

A Cortico-Collicular Amplification Mechanism for Gap Detection.

Auditory cortex (AC) is necessary for the detection of brief gaps in ongoing sounds, but not for the detection of longer gaps or other stimuli such as tones or noise. It remains unclear why this is so, and what is special about brief gaps in particular. Here, we used both optogenetic suppression and conventional lesions to show that the cortical dependence of brief gap detection hinges specifically on gap termination. We then identified a cortico-collicular gap detection circuit that amplifies cortical gap termination responses before projecting to inferior colliculus (IC) to impact behavior. We found that gaps evoked off-responses and on-responses in cortical neurons, which temporally overlapped for brief gaps, but not long gaps. This overlap specifically enhanced cortical responses to brief gaps, whereas IC neurons preferred longer gaps. Optogenetic suppression of AC reduced collicular responses specifically to brief gaps, indicating that under normal conditions, the enhanced cortical representation of brief gaps amplifies collicular gap responses. Together these mechanisms explain how and why AC contributes to the behavioral detection of brief gaps, which are critical cues for speech perception, perceptual grouping, and auditory scene analysis.