Inhibition of adenylyl cyclase by GTPase-deficient Gαi is mechanistically different from that mediated by receptor-activated Gαi.

Signal transduction through G protein-coupled receptors (GPCRs) has been a major focus in cell biology for decades. Numerous disorders are associated with GPCRs that utilize Gi proteins to inhibit adenylyl cyclase (AC) as well as regulate other effectors. Several early studies have successfully defined the AC-interacting domains of several members of Gαi by measuring the loss of activity upon homologous replacements of putative regions of constitutive active Gαi mutants. However, whether such findings can indeed be translated into the context of a receptor-activated Gαi have not been rigorously verified. To address this issue, an array of known and new chimeric mutations was introduced into GTPase-deficient Q204L (QL) and R178C (RC) mutants of Gαi1, followed by examinations on their ability to inhibit AC. Surprisingly, most chimeras failed to abolish the constitutive activity brought on by the QL mutation, while some were able to eliminate the inhibitory activity of RC mutants. Receptor-mediated inhibition of AC was similarly observed in the same chimeric constructs harbouring the pertussis toxin (PTX)-resistant C351I mutation. Moreover, RC-bearing loss-of-function chimeras appeared to be hyper-deactivated by endogenous RGS protein. Molecular docking revealed a potential interaction between AC and the α3/ß5 loop of Gαi1. Subsequent cAMP assays support a cooperative action of the α3/ß5 loop, the α4 helix, and the α4/ß6 loop in mediating AC inhibition by Gαi1-i3. Our results unveiled a notable functional divergence between constitutively active mutants and receptor-activated Gαi1 to inhibit AC, and identified a previously unknown AC-interacting domain of Gαi subunits. These results collectively provide valuable insights on the mechanism of AC inhibition in the cellular environment.

All-optical presynaptic plasticity induction by photoactivated adenylyl cyclase targeted to axon terminals.

Intracellular signaling plays essential roles in various cell types. In the central nervous system, signaling cascades are strictly regulated in a spatiotemporally specific manner to govern brain function; for example, presynaptic cyclic adenosine monophosphate (cAMP) can enhance the probability of neurotransmitter release. In the last decade, channelrhodopsin-2 has been engineered for subcellular targeting using localization tags, but optogenetic tools for intracellular signaling are not well developed. Therefore, we engineered a selective presynaptic fusion tag for photoactivated adenylyl cyclase (bPAC-Syn1a) and found its high localization at presynaptic terminals. Furthermore, an all-optical electrophysiological method revealed rapid and robust short-term potentiation by bPAC-Syn1a at brain stem-amygdala synapses in acute brain slices. Additionally, bPAC-Syn1a modulated mouse immobility behavior. These results indicate that bPAC-Syn1a can manipulate presynaptic cAMP signaling in vitro and in vivo. The all-optical manipulation technique developed in this study can help further elucidate the dynamic regulation of various cellular functions.

Mapping the Cellular Distribution of an Optogenetic Protein Using a Light-Stimulation Grid Mapping the Cellular Distribution of an Optogenetic Protein Using a Light-Stimulation Grid.

Our goal was to accurately track the cellular distribution of an optogenetic protein and evaluate its functionality within a specific cytoplasmic location. To achieve this, we co-transfected cells with nuclear-targeted cAMP sensors and our laboratory-developed optogenetic protein, bacterial photoactivatable adenylyl cyclase-nanoluciferase (bPAC-nLuc). bPAC-nLuc, when stimulated with 445 nm light or luciferase substrates, generates adenosine 3',5'-cyclic monophosphate (cAMP). We employed a solid-state laser illuminator connected to a point scanning system that allowed us to create a grid/matrix pattern of small illuminated spots (~1 µm2) throughout the cytoplasm of HC-1 cells. By doing so, we were able to effectively track the distribution of nuclear-targeted bPAC-nLuc and generate a comprehensive cAMP response map. This map accurately represented the cellular distribution of bPAC-nLuc, and its response to light stimulation varied according to the amount of protein in the illuminated spot. This innovative approach contributes to the expanding toolkit of techniques available for investigating cellular optogenetic proteins. The ability to map its distribution and response with high precision has far-reaching potential and could advance various fields of research.

Simulated Microgravity and Hypergravity Affect the Expression Level of Soluble Guanylate Cyclase, Adenylate Cyclase, and Phosphodiesterase Genesin Rat Ventricular Cardiomyocytes.

Ion channels activity is regulated through soluble guanylate cyclase (sGC) and adenylate cyclase (AC) pathways, while phosphodiesterases (PDE) control the intracellular levels of cAMP and cGMP. Here we applied RNA transcriptome sequencing to study changes in the gene expression of the sGC, AC, and PDE isoforms in isolated rat ventricular cardiomyocytes under conditions of microgravity and hypergravity. Our results demonstrate that microgravity reduces the expression of sGC isoform genes, while hypergravity increases their expression. For a subset of AC isoforms, gene expression either increased or decreased under both microgravity and hypergravity conditions. The expression of genes encoding 10 PDE isoforms decreased under microgravity, but increased under hypergravity. However, under both microgravity and hypergravity, the gene expression increased for 7 PDE isoforms and decreased for 3 PDE isoforms. Overall, our findings indicate specific gravity-dependent changes in the expression of genes of isoforms associated with the studied enzymes.

ATP-dependent conformational dynamics in a photoactivated adenylate cyclase revealed by fluorescence spectroscopy and small-angle X-ray scattering.

Structural insights into the photoactivated adenylate cyclases can be used to develop new ways of controlling cellular cyclic adenosine monophosphate (cAMP) levels for optogenetic and other applications. In this work, we use an integrative approach that combines biophysical and structural biology methods to provide insight on the interaction of adenosine triphosphate (ATP) with the dark-adapted state of the photoactivated adenylate cyclase from the cyanobacterium Oscillatoria acuminata (OaPAC). A moderate affinity of the nucleotide for the enzyme was calculated and the thermodynamic parameters of the interaction have been obtained. Stopped-flow fluorescence spectroscopy and small-angle solution scattering have revealed significant conformational changes in the enzyme, presumably in the adenylate cyclase (AC) domain during the allosteric mechanism of ATP binding to OaPAC with small and large-scale movements observed to the best of our knowledge for the first time in the enzyme in solution upon ATP binding. These results are in line with previously reported drastic conformational changes taking place in several class III AC domains upon nucleotide binding.

Light-Mediated Enhancement of Glucose-Stimulated Insulin Release of Optogenetically Engineered Human Pancreatic Beta-Cells.

Enhancement of glucose-stimulated insulin secretion (GSIS) in exogenously delivered pancreatic ß-cells is desirable, for example, to overcome the insulin resistance manifested in type 2 diabetes or to reduce the number of ß-cells for supporting homeostasis of blood sugar in type 1 diabetes. Optogenetically engineered cells can potentiate their function with exposure to light. Given that cyclic adenosine monophosphate (cAMP) mediates GSIS, we surmised that optoamplification of GSIS is feasible in human ß-cells carrying a photoactivatable adenylyl cyclase (PAC). To this end, human EndoC-ßH3 cells were engineered to express a blue-light-activated PAC, and a workflow was established combining the scalable manufacturing of pseudoislets (PIs) with efficient adenoviral transduction, resulting in over 80% of cells carrying PAC. Changes in intracellular cAMP and GSIS were determined with the photoactivation of PAC in vitro as well as after encapsulation and implantation in mice with streptozotocin-induced diabetes. cAMP rapidly rose in ß-cells expressing PAC with illumination and quickly declined upon its termination. Light-induced amplification in cAMP was concomitant with a greater than 2-fold GSIS vs ß-cells without PAC in elevated glucose. The enhanced GSIS retained its biphasic pattern, and the rate of oxygen consumption remained unchanged. Diabetic mice receiving the engineered ß-cell PIs exhibited improved glucose tolerance upon illumination compared to those kept in the dark or not receiving cells. The findings support the use of optogenetics for molecular customization of the ß-cells toward better treatments for diabetes without the adverse effects of pharmacological approaches.

Reprogramming of cardiac phosphoproteome, proteome, and transcriptome confers resilience to chronic adenylyl cyclase-driven stress.

Our prior study (Tarasov et al., 2022) discovered that numerous adaptive mechanisms emerge in response to cardiac-specific overexpression of adenylyl cyclase type 8 (TGAC8) which included overexpression of a large number of proteins. Here, we conducted an unbiased phosphoproteomics analysis in order to determine the role of altered protein phosphorylation in the adaptive heart performance and protection profile of adult TGAC8 left ventricle (LV) at 3-4 months of age, and integrated the phosphoproteome with transcriptome and proteome. Based on differentially regulated phosphoproteins by genotype, numerous stress-response pathways within reprogrammed TGAC8 LV, including PKA, PI3K, and AMPK signaling pathways, predicted upstream regulators (e.g. PDPK1, PAK1, and PTK2B), and downstream functions (e.g. cell viability, protein quality control), and metabolism were enriched. In addition to PKA, numerous other kinases and phosphatases were hyper-phosphorylated in TGAC8 vs. WT. Hyper-phosphorylated transcriptional factors in TGAC8 were associated with increased mRNA transcription, immune responses, and metabolic pathways. Combination of the phosphoproteome with its proteome and with the previously published TGAC8 transcriptome enabled the elucidation of cardiac performance and adaptive protection profiles coordinately regulated at post-translational modification (PTM) (phosphorylation), translational, and transcriptional levels. Many stress-response signaling pathways, i.e., PI3K/AKT, ERK/MAPK, and ubiquitin labeling, were consistently enriched and activated in the TGAC8 LV at transcriptional, translational, and PTM levels. Thus, reprogramming of the cardiac phosphoproteome, proteome, and transcriptome confers resilience to chronic adenylyl cyclase-driven stress. We identified numerous pathways/function predictions via gene sets, phosphopeptides, and phosphoproteins, which may point to potential novel therapeutic targets to enhance heart adaptivity, maintaining heart performance while avoiding cardiac dysfunction.

Two novel heterozygous ADCY10 variants identified in Chinese pediatric patients with absorptive hypercalciuria: case report and literature review.

Absorptive hypercalciuria (AH) is a prevalent cause of kidney stones, and the adenylate cyclase 10 (ADCY10) gene is a rare causative gene of AH. This study aims to investigate the genotypic and phenotypic characteristics of patients with AH caused by ADCY10 gene mutations. Whole-exome sequencing and Sanger sequencing were performed on the probands and their family members, respectively. Clinical and genetic data of patients with AH caused by ADCY10 gene mutations were collected and analysed retrospectively from the present study and published literature. Two female patients (6 years old and 1 year old) with multiple bilateral kidney stones were found to have a heterozygous c.3304T>C mutation and a heterozygous c.1726C>T mutation in the ADCY10 gene. Urinary metabolite analysis revealed that urine calcium / creatinine ratios were 0.95 mmol/mmol and 1.61 mmol/mmol, respectively. Both patients underwent thiazide intake postoperatively, and upon reexamination, urine calcium decreased to within the normal range. A total of 61 patients with AH were reported from previous and present studies. The sex ratio was 7:5 for males to females, and the mean age of onset was 23.61±20.08 years. A total of 16 ADCY10 gene mutations were identified, including seven missense (43.75%), five splicing (31.25%), two frameshift (12.50%) and two nonsense mutations (12.50%). Only two cases were identified as homozygous mutations (c.1205_1206del), and the others were heterozygous mutations. In summary, we identified two novel ADCY10 gene candidate pathogenic variants in Chinese pediatric patients, which expands the mutational spectrum of the ADCY10 gene and provides a potential diagnostic and therapeutic target.

Inhibition of adenylyl cyclase 8 prevents the upregulation of Orai1 channel, which improves cardiac function after myocardial infarction.

The upregulation of Orai1 and subsequent store-operated Ca2+ entry (SOCE) has been associated with adverse cardiac remodeling and heart failure (HF). However, the mechanism underlying Orai1 upregulation and its role in myocardial infarction remains unclear. Our study investigated the role of Orai1 in activating adenylyl cyclase 8 (AC8) and cyclic AMP (cAMP) response element-binding protein (CREB), as well as its contribution to cardiac dysfunction induced by ischemia and reperfusion (I/R). We found that I/R evoked an increase in the expression of Orai1 and AC8 in rats' hearts, resulting in a substantial rise in diastolic Ca2+ concentration ([Ca2+]i), and reduced ventricular contractions. The expression of Orai1 and AC8 was also increased in ventricular biopsies of post-ischemic HF patients. Mechanistically, we demonstrate that I/R activation of Orai1 stimulated AC8, which produced cAMP and phosphorylated CREB. Subsequently, p-CREB activated the ORAI1 promoter, resulting in Orai1 upregulation and SOCE exacerbation. Intramyocardial administration of AAV9 carrying AC8 short hairpin RNA decreased the expression of AC8, Orai1 and CREB, which restored diastolic [Ca2+]i and improved cardiac contraction. Therefore, our data suggests that the axis composed by Orai1/AC8/CREB plays a critical role in I/R-induced cardiac dysfunction, representing a potential new therapeutic target to limit the progression of the disease toward HF.

Loss-of-function of GNAL dystonia gene impairs striatal dopamine receptors-mediated adenylyl cyclase/ cyclic AMP signaling pathway.

Loss-of-function mutations in the GNAL gene are responsible for DYT-GNAL dystonia. However, how GNAL mutations contribute to synaptic dysfunction is still unclear. The GNAL gene encodes the Gαolf protein, an isoform of stimulatory Gαs enriched in the striatum, with a key role in the regulation of cAMP signaling. Here, we used a combined biochemical and electrophysiological approach to study GPCR-mediated AC-cAMP cascade in the striatum of the heterozygous GNAL (GNAL+/-) rat model. We first analyzed adenosine type 2 (A2AR), and dopamine type 1 (D1R) receptors, which are directly coupled to Gαolf, and observed that the total levels of A2AR were increased, whereas D1R level was unaltered in GNAL+/- rats. In addition, the striatal isoform of adenylyl cyclase (AC5) was reduced, despite unaltered basal cAMP levels. Notably, the protein expression level of dopamine type 2 receptor (D2R), that inhibits the AC5-cAMP signaling pathway, was also reduced, similar to what observed in different DYT-TOR1A dystonia models. Accordingly, in the GNAL+/- rat striatum we found altered levels of the D2R regulatory proteins, RGS9-2, spinophilin, Gß5 and ß-arrestin2, suggesting a downregulation of D2R signaling cascade. Additionally, by analyzing the responses of striatal cholinergic interneurons to D2R activation, we found that the receptor-mediated inhibitory effect is significantly attenuated in GNAL+/- interneurons. Altogether, our findings demonstrate a profound alteration in the A2AR/D2R-AC-cAMP cascade in the striatum of the rat DYT-GNAL dystonia model, and provide a plausible explanation for our previous findings on the loss of dopamine D2R-dependent corticostriatal long-term depression.

Adenylate cyclase 3: a potential genetic link between obesity and major depressive disorder.

Obesity and major depressive disorder (MDD) are both significant health issues that have been increasing in prevalence and are associated with multiple comorbidities. Obesity and MDD have been shown to be bidirectionally associated, and they are both influenced by genetics and environmental factors. However, the molecular mechanisms that link these two diseases are not yet fully understood. It is possible that these diseases are connected through the actions of the cAMP/protein kinase A (PKA) pathway. Within this pathway, adenylate cyclase 3 (Adcy3) has emerged as a key player in both obesity and MDD. Numerous genetic variants in Adcy3 have been identified in humans in association with obesity. Rodent knockout studies have also validated the importance of this gene for energy homeostasis. Furthermore, Adcy3 has been identified as a top candidate gene and even a potential blood biomarker for MDD. Adcy3 and the cAMP/PKA pathway may therefore serve as an important genetic and functional link between these two diseases. In this mini-review, we discuss the role of both Adcy3 and the cAMP/PKA pathway, including specific genetic mutations, in both diseases. Understanding the role that Adcy3 mutations play in obesity and MDD could open the door for precision medicine approaches and treatments for both diseases that target this gene.

From membrane to nucleus: A three-wave hypothesis of cAMP signaling.

For many decades, our understanding of G protein-coupled receptor (GPCR) activity and cyclic AMP (cAMP) signaling was limited exclusively to the plasma membrane. However, a growing body of evidence has challenged this view by introducing the concept of endocytosis-dependent GPCR signaling. This emerging paradigm emphasizes not only the sustained production of cAMP but also its precise subcellular localization, thus transforming our understanding of the spatiotemporal organization of this process. Starting from this alternative point of view, our recent work sheds light on the role of an endocytosis-dependent calcium release from the endoplasmic reticulum in the control of nuclear cAMP levels. This is achieved through the activation of local soluble adenylyl cyclase, which in turn regulates the activation of local protein kinase A (PKA) and downstream transcriptional events. In this review, we explore the dynamic evolution of research on cyclic AMP signaling, including the findings that led us to formulate the novel three-wave hypothesis. We delve into how we abandoned the paradigm of cAMP generation limited to the plasma membrane and the changing perspectives on the rate-limiting step in nuclear PKA activation.

Encystation stimuli sensing is mediated by adenylate cyclase AC2-dependent cAMP signaling in Giardia.

Protozoan parasites use cAMP signaling to precisely regulate the place and time of developmental differentiation, yet it is unclear how this signaling is initiated. Encystation of the intestinal parasite Giardia lamblia can be activated by multiple stimuli, which we hypothesize result in a common physiological change. We demonstrate that bile alters plasma membrane fluidity by reducing cholesterol-rich lipid microdomains, while alkaline pH enhances bile function. Through depletion of the cAMP producing enzyme Adenylate Cyclase 2 (AC2) and the use of a newly developed Giardia-specific cAMP sensor, we show that AC2 is necessary for encystation stimuli-induced cAMP upregulation and activation of downstream signaling. Conversely, over expression of AC2 or exogenous cAMP were sufficient to initiate encystation. Our findings indicate that encystation stimuli induce membrane reorganization, trigger AC2-dependent cAMP upregulation, and initiate encystation-specific gene expression, thereby advancing our understanding of a critical stage in the life cycle of a globally important parasite.

Cholesterol is required for activity-dependent synaptic growth.

Changes in cholesterol content of neuronal membranes occur during development and brain aging. Little is known about whether synaptic activity regulates cholesterol levels in neuronal membranes and whether these changes affect neuronal development and function. We generated transgenic flies that express the cholesterol-binding D4H domain of perfringolysin O toxin and found increased levels of cholesterol in presynaptic terminals of Drosophila larval neuromuscular junctions following increased synaptic activity. Reduced cholesterol impaired synaptic growth and largely prevented activity-dependent synaptic growth. Presynaptic knockdown of adenylyl cyclase phenocopied the impaired synaptic growth caused by reducing cholesterol. Furthermore, the effects of knocking down adenylyl cyclase and reducing cholesterol were not additive, suggesting that they function in the same pathway. Increasing cAMP levels using a dunce mutant with reduced phosphodiesterase activity failed to rescue this impaired synaptic growth, suggesting that cholesterol functions downstream of cAMP. We used a protein kinase A (PKA) sensor to show that reducing cholesterol levels reduced presynaptic PKA activity. Collectively, our results demonstrate that enhanced synaptic activity increased cholesterol levels in presynaptic terminals and that these changes likely activate the cAMP-PKA pathway during activity-dependent growth.

Genes positively regulated by Mef2c in cortical neurons are enriched for common genetic variation associated with IQ and educational attainment.

The myocyte enhancer factor 2 C (MEF2C) gene encodes a transcription factor important for neurogenesis and synapse development and contains common variants associated with intelligence (IQ) and educational attainment (EA). Here, we took gene expression data from the mouse cortex of a Mef2c mouse model with a heterozygous DNA binding-deficient mutation of Mef2c (Mef2c-het) and combined these data with MEF2C ChIP-seq data from cortical neurons and single-cell data from the mouse brain. This enabled us to create a set of genes that were differentially regulated in Mef2c-het mice, represented direct target genes of MEF2C and had elevated in expression in cortical neurons. We found this gene-set to be enriched for genes containing common genetic variation associated with IQ and EA. Genes within this gene-set that were down-regulated, i.e. have reduced expression in Mef2c-het mice versus controls, were specifically significantly enriched for both EA and IQ associated genes. These down-regulated genes were enriched for functionality in the adenylyl cyclase signalling system, which is known to positively regulate synaptic transmission and has been linked to learning and memory. Within the adenylyl cyclase signalling system, three genes regulated by MEF2C, CRHR1, RGS6, and GABRG3, are associated at genome-wide significant levels with IQ and/or EA. Our results indicate that genetic variation in MEF2C and its direct target genes within cortical neurons contribute to variance in cognition within the general population, and the molecular mechanisms involved include the adenylyl cyclase signalling system's role in synaptic function.

Plant adenylate cyclases have come full circle.

In bacteria, fungi and animals, 3'-5'-cyclic adenosine monophosphate (cAMP) and adenylate cyclases (ACs), enzymes that catalyse the formation of 3',5'-cAMP from ATP, are recognized as key signalling components. In contrast, the presence of cAMP and its biological roles in higher plants have long been a matter of controversy due to the generally lower amounts in plant tissues compared with that in animal and bacterial cells, and a lack of clarity on the molecular nature of the generating and degrading enzymes, as well as downstream effectors. While treatment with 3',5'-cAMP elicited many plant responses, ACs were, however, somewhat elusive. This changed when systematic searches with amino acid motifs deduced from the conserved catalytic centres of annotated ACs from animals and bacteria identified candidate proteins in higher plants that were subsequently shown to have AC activities in vitro and in vivo. The identification of active ACs moonlighting within complex multifunctional proteins is consistent with their roles as molecular tuners and regulators of cellular and physiological functions. Furthermore, the increasing number of ACs identified as part of proteins with different domain architectures suggests that there are many more hidden ACs in plant proteomes and they may affect a multitude of mechanisms and processes at the molecular and systems levels.

Time-resolved study on signaling pathway of photoactivated adenylate cyclase and its nonlinear optical response.

Photoactivated adenylate cyclases (PACs) are multidomain BLUF proteins that regulate the cellular levels of cAMP in a light-dependent manner. The signaling route and dynamics of PAC from Oscillatoria acuminata (OaPAC), which consists of a light sensor BLUF domain, an adenylate cyclase domain, and a connector helix (α3-helix), were studied by detecting conformational changes in the protein moiety. Although circular dichroism and small-angle X-ray scattering measurements did not show significant changes upon light illumination, the transient grating method successfully detected light-induced changes in the diffusion coefficient (diffusion-sensitive conformational change (DSCC)) of full-length OaPAC and the BLUF domain with the α3-helix. DSCC of full-length OaPAC was observed only when both protomers in a dimer were photoconverted. This light intensity dependence suggests that OaPAC is a cyclase with a nonlinear light intensity response. The enzymatic activity indeed nonlinearly depends on light intensity, that is, OaPAC is activated under strong light conditions. It was also found that both DSCC and enzymatic activity were suppressed by a mutation in the W90 residue, indicating the importance of the highly conserved Trp in many BLUF domains for the function. Based on these findings, a reaction scheme was proposed together with the reaction dynamics.

A signaling complex of adenylate cyclase CyaC of Sinorhizobium meliloti with cAMP and the transcriptional regulators Clr and CycR.

BACKGROUND: Adenylate cyclases (ACs) generate the second messenger cyclic AMP (cAMP), which is found in all domains of life and is involved in the regulation of various cell physiological and metabolic processes. In the plant symbiotic bacterium Sinorhizobium meliloti, synthesis of cAMP by the membrane-bound AC CyaC responds to the redox state of the respiratory chain and the respiratory quinones. However, nothing is known about the signaling cascade that is initiated by cAMP produced by CyaC. RESULTS: Here, the CRP-like transcriptional regulator Clr and the TetR-like regulator CycR (TR01819 protein) were identified to interact with CyaC using the bacterial two-hybrid system (BACTH), co-sedimentation assays, and surface plasmon resonance spectroscopy. Interaction of CycR with Clr, and of CyaC with Clr requires the presence of cAMP and of ATP, respectively, whereas that of CyaC with CycR was independent of the nucleotides. CONCLUSION: The data implicate a ternary CyaC×CycR×cAMP-Clr complex, functioning as a specific signaling cascade which is formed after activation of CyaC and synthesis of cAMP. cAMP-Clr is thought to work in complex with CycR to regulate a subset of genes of the cAMP-Clr regulon in S. meliloti.

Optogenetic manipulation of neuronal and cardiomyocyte functions in zebrafish using microbial rhodopsins and adenylyl cyclases.

Even though microbial photosensitive proteins have been used for optogenetics, their use should be optimized to precisely control cell and tissue functions in vivo. We exploited GtCCR4 and KnChR, cation channelrhodopsins from algae, BeGC1, a guanylyl cyclase rhodopsin from a fungus, and photoactivated adenylyl cyclases (PACs) from cyanobacteria (OaPAC) or bacteria (bPAC), to control cell functions in zebrafish. Optical activation of GtCCR4 and KnChR in the hindbrain reticulospinal V2a neurons, which are involved in locomotion, induced swimming behavior at relatively short latencies, whereas activation of BeGC1 or PACs achieved it at long latencies. Activation of GtCCR4 and KnChR in cardiomyocytes induced cardiac arrest, whereas activation of bPAC gradually induced bradycardia. KnChR activation led to an increase in intracellular Ca2+ in the heart, suggesting that depolarization caused cardiac arrest. These data suggest that these optogenetic tools can be used to reveal the function and regulation of zebrafish neurons and cardiomyocytes.

The psychosis risk factor RBM12 encodes a novel repressor of GPCR/cAMP signal transduction.

RBM12 is a high-penetrance risk factor for familial schizophrenia and psychosis, yet its precise cellular functions and the pathways to which it belongs are not known. We utilize two complementary models, HEK293 cells and human iPSC-derived neurons, and delineate RBM12 as a novel repressor of the G protein-coupled receptor/cAMP/PKA (GPCR/cAMP/PKA) signaling axis. We establish that loss of RBM12 leads to hyperactive cAMP production and increased PKA activity as well as altered neuronal transcriptional responses to GPCR stimulation. Notably, the cAMP and transcriptional signaling steps are subject to discrete RBM12-dependent regulation. We further demonstrate that the two RBM12 truncating variants linked to familial psychosis impact this interplay, as the mutants fail to rescue GPCR/cAMP signaling hyperactivity in cells depleted of RBM12. Lastly, we present a mechanism underlying the impaired signaling phenotypes. In agreement with its activity as an RNA-binding protein, loss of RBM12 leads to altered gene expression, including that of multiple effectors of established significance within the receptor pathway. Specifically, the abundance of adenylyl cyclases, phosphodiesterase isoforms, and PKA regulatory and catalytic subunits is impacted by RBM12 depletion. We note that these expression changes are fully consistent with the entire gamut of hyperactive signaling outputs. In summary, the current study identifies a previously unappreciated role for RBM12 in the context of the GPCR-cAMP pathway that could be explored further as a tentative molecular mechanism underlying the functions of this factor in neuronal physiology and pathophysiology.