Adenylyl cyclase 2 expression and function in neurological diseases.

Adenylyl cyclases (Adcys) catalyze the formation of cAMP, a secondary messenger essential for cell survival and neurotransmission pathways in the CNS. Adcy2, one of ten Adcy isoforms, is highly expressed in the CNS. Abnormal Adcy2 expression and mutations have been reported in various neurological disorders in both rodents and humans. However, due to the lack of genetic tools, loss-of-function studies of Adcy2 are scarce. In this review, we summarize recent findings on Adcy2 expression and function in neurological diseases. Specifically, we first introduce the biochemistry, structure, and function of Adcy2 briefly. Next, the expression and association of Adcy2 in human patients and rodent models of neurodegenerative diseases (Alzheimer's disease and Parkinson's disease), psychiatric disorders (Tourette syndrome, schizophrenia, and bipolar disorder), and other neurological conditions (stress-associated disorders, stroke, epilepsy, and Lesch-Nyhan Syndrome) are elaborated. Furthermore, we discuss the pros and cons of current studies as well as key questions that need to be answered in the future. We hope to provide a focused review on Adcy2 that promotes future research in the field.

KCTD proteins regulate morphine dependence via heterologous sensitization of adenylyl cyclase 1 in mice.

Heterologous sensitization of adenylyl cyclase (AC) results in elevated cAMP signaling transduction that contributes to drug dependence. Inhibiting cullin3-RING ligases by blocking the neddylation of cullin3 abolishes heterologous sensitization, however, the modulating mechanism remains uncharted. Here, we report an essential role of the potassium channel tetramerization domain (KCTD) protein 2, 5, and 17, especially the dominant isoform KCTD5 in regulating heterologous sensitization of AC1 and morphine dependence via working with cullin3 and the cullin-associated and neddylation-dissociated 1 (CAND1) protein. In cellular models, we observed enhanced association of KCTD5 with Gß and cullin3, along with elevated dissociation of Gß from AC1 as well as of CAND1 from cullin3 in heterologous sensitization of AC1. Given binding of CAND1 inhibits the neddylation of cullin3, we further elucidated that the enhanced interaction of KCTD5 with both Gß and cullin3 promoted the dissociation of CAND1 from cullin3, attenuated the inhibitory effect of CAND1 on cullin3 neddylation, ultimately resulted in heterologous sensitization of AC1. The paraventricular thalamic nucleus (PVT) plays an important role in mediating morphine dependence. Through pharmacological and biochemical approaches, we then demonstrated that KCTD5/cullin3 regulates morphine dependence via modulating heterologous sensitization of AC, likely AC1 in PVT in mice. In summary, the present study revealed the underlying mechanism of heterologous sensitization of AC1 mediated by cullin3 and discovered the role of KCTD proteins in regulating morphine dependence in mice.

Inhibition of calcium-stimulated adenylyl cyclase subtype 1 (AC1) for the treatment of pain and anxiety symptoms in Parkinson's disease mice model.

Pain and anxiety are two common and undertreated non-motor symptoms in Parkinson's disease (PD), which affect the life quality of PD patients, and the underlying mechanisms remain unclear. As an important subtype of adenylyl cyclases (ACs), adenylyl cyclase subtype 1 (AC1) is critical for the induction of cortical long-term potentiation (LTP) and injury induced synaptic potentiation in the cortical areas including anterior cingulate cortex (ACC) and insular cortex (IC). Genetic deletion of AC1 or pharmacological inhibition of AC1 improved chronic pain and anxiety in different animal models. In this study, we proved the motor deficit, pain and anxiety symptoms of PD in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice model. As a lead candidate AC1 inhibitor, oral administration (1 dose and seven doses) of NB001 (20 and 40 mg/kg) showed significant analgesic effect in MPTP-treated mice, and the anxiety behavior was also reduced (40 mg/kg). By using genetic knockout mice, we found that AC1 knockout mice showed reduced pain and anxiety symptoms after MPTP administration, but not AC8 knockout mice. In summary, genetic deletion of AC1 or pharmacological inhibition of AC1 improved pain and anxiety symptoms in PD model mice, but didn't affect motor function. These results suggest that NB001 is a potential drug for the treatment of pain and anxiety symptoms in PD patients by inhibiting AC1 target.

A CatSper-Uninvolved Mechanism to Induce Forward Sperm Motility in the Internal Fertilization.

Sperm-specific cation channel (CatSper), sperm-specific Na + /H + exchanger (sNHE), and soluble adenylyl cyclase (sAC) are necessary in the signaling pathways to control sperm motility in many animals, whereas some animals have lost some or all of them. In the present study, we examined CatSper-uninvolved signaling for vigorous undulation of the undulating membrane that is attached to the sperm tail and gives thrust for forward motility in the internally fertilizing newt Cynops pyrrhogaster. Reverse-transcription PCR failed to detect sNHE in the newt sperm. However, the pH of sperm cytoplasm was raised under a high extracellular pH equivalent to that of egg jelly, where sperm motility is initiated by sperm motility-initiating substance (SMIS). Carbonic anhydrase XII/ XVI and SLC4A4/8 were suggested to be present in the sperm, and transported bicarbonates raised the intracellular pH. In egg jelly extract that contained SMIS, the anion transporter inhibitor DIDS weakened the undulation of the undulating membrane, while bicarbonates enhanced it. The cyclic AMP concentration was found to increase in sperm cytoplasm in the egg-jelly extract. An inhibitor of sAC (KH7) weakened the undulation of the undulating membrane, and dibutyryl cyclic AMP blocked the inhibitory effect. Inhibitor of transmembrane AC (DDA) limitedly affected the undulation. The undulation was weakened by an inhibitor of protein kinase A (H89), and by an inhibitor of transient receptor potential (TRP) channels (RN1747). Our results support the conclusions that the high pH of the egg jelly triggers a signaling pathway through sAC, PKA, and TRP channels, and coacts with SMIS to induce forward sperm motility.

Glucose and HODEs regulate Aspergillus ochraceus quorum sensing through the GprC-AcyA pathway.

Aspergillus ochraceus is the traditional ochratoxin A (OTA)-producing fungus with density-dependent behaviors, which is known as quorum sensing (QS) that is mediated by signaling molecules. Individual cells trend to adapt environmental changes in a "whole" flora through communications, allowing fungus to occupy an important ecological niche. Signals perception, transmission, and feedback are all rely on a signal network that constituted by membrane receptors and intracellular effectors. However, the interference of density information in signal transduction, which regulates most life activities of Aspergillus, have yet to be elucidated. Here we show that the G protein-coupled receptor (GPCR) to cAMP pathway is responsible for transmitting density information, and regulates the key point in life cycle of A. ochraceus. Firstly, the quorum sensing phenomenon of A. ochraceus is confirmed, and identified the density threshold is 103 spores/mL, which represents the low density that produces the most OTA in a series quorum density. Moreover, the GprC that classified as sugar sensor, and intracellular adenylate cyclase (AcyA)-cAMP-PKA pathway that in response to ligands glucose and HODEs are verified. Furthermore, GprC and AcyA regulate the primary metabolism as well as secondary metabolism, and further affects the growth of A. ochraceus during the entire life cycle. These studies highlight a crucial G protein signaling pathway for cell communication that is mediated by carbohydrate and oxylipins, and clarified a comprehensive effect of fungal development, which include the direct gene regulation and indirect substrate or energy supply. Our work revealed more signal molecules that mediated density information and connected effects on important adaptive behaviors of Aspergillus ochraceus, hoping to achieve comprehensive prevention and control of mycotoxin pollution from interrupting cell communication.

Systems Genetics Analyses Reveals Key Genes Related to Behavioral Traits in the Striatum of CFW Mice.

The striatum plays a central role in directing many complex behaviors ranging from motor control to action choice and reward learning. In our study, we used 55 male CFW mice with rapid decay linkage disequilibrium to systematically mine the striatum-related behavioral functional genes by analyzing their striatal transcriptomes and 79 measured behavioral phenotypic data. By constructing a gene coexpression network, we clustered the genes into 13 modules, with most of them being positively correlated with motor traits. Based on functional annotations as well as Fisher's exact and hypergeometric distribution tests, brown and magenta modules were identified as core modules. They were significantly enriched for striatal-related functional genes. Subsequent Mendelian randomization analysis verified the causal relationship between the core modules and dyskinesia. Through the intramodular gene connectivity analysis, Adcy5 and Kcnma1 were identified as brown and magenta module hub genes, respectively. Knock outs of both Adcy5 and Kcnma1 lead to motor dysfunction in mice, and KCNMA1 acts as a risk gene for schizophrenia and smoking addiction in humans. We also evaluated the cellular composition of each module and identified oligodendrocytes in the striatum to have a positive role in motor regulation.

GDF15 controls primary cilia morphology and function thereby affecting progenitor proliferation.

We recently reported that growth/differentiation factor 15 (GDF15) and its receptor GDNF family receptor alpha-like (GFRAL) are expressed in the periventricular germinal epithelium thereby regulating apical progenitor proliferation. However, the mechanisms are unknown. We now found GFRAL in primary cilia and altered cilia morphology upon GDF15 ablation. Mutant progenitors also displayed increased histone deacetylase 6 (Hdac6) and ciliary adenylate cyclase 3 (Adcy3) transcript levels. Consistently, microtubule acetylation, endogenous sonic hedgehog (SHH) activation and ciliary ADCY3 were all affected in this group. Application of exogenous GDF15 or pharmacological antagonists of either HDAC6 or ADCY3 similarly normalized ciliary morphology, proliferation and SHH signalling. Notably, Gdf15 ablation affected Hdac6 expression and cilia length only in the mutant periventricular niche, in concomitance with ciliary localization of GFRAL. In contrast, in the hippocampus, where GFRAL was not expressed in the cilium, progenitors displayed altered Adcy3 expression and SHH signalling, but Hdac6 expression, cilia morphology and ciliary ADCY3 levels remained unchanged. Thus, ciliary signalling underlies the effect of GDF15 on primary cilia elongation and proliferation in apical progenitors.

Regulation of cardiac fibrosis in mice with TAC/DOCA-induced HFpEF by resistin-like molecule gamma and adenylate cyclase 1.

Heart failure with preserved ejection fraction (HFpEF) is one of the major subtypes of heart failure (HF) and no effective treatments for this common disease exist to date. Cardiac fibrosis is central to the pathology of HF and a potential avenue for the treatment of HFpEF. To explore key fibrosis-related genes and pathways in the pathophysiological process of HFpEF, a mouse model of HFpEF was constructed. The relevant gene expression profiles were downloaded from the Gene Expression Omnibus database, and single-sample Gene Set Enrichment Analysis (ssGSEA) was performed targeting fibrosis-related pathways to explore differentially expressed genes (DEGs) in healthy control and HFpEF heart tissues with cross-tabulation analysis of fibrosis-related genes. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on the identified fibrosis-related genes. The two most significant DEGs were selected, and further validation was conducted in HFpEF mice. The results indicated that myocardial fibrosis was significantly upregulated in HFpEF mice compared to healthy controls, while the ssGSEA results revealed significant differences in the enrichment of nine fibrosis-related pathways in HFpEF myocardial tissue, with 112 out of 798 DEGs being related to fibrosis. The in vivo results demonstrated that expression levels of resistin-like molecule gamma (Relmg) and adenylate cyclase 1 (Adcy1) in the heart tissues of HFpEF mice were significantly higher and lower, respectively, compared to healthy controls. Taken together, these results suggest that Relmg and Acdy1 as well as the fibrosis process may be potential targets for HFpEF treatment.

Mendelian Randomization Analysis Identifies Inverse Causal Relationship between External Eating and Metabolic Phenotypes.

Disordered eating contributes to weight gain, obesity, and type 2 diabetes (T2D), but the precise mechanisms underlying the development of different eating patterns and connecting them to specific metabolic phenotypes remain unclear. We aimed to identify genetic variants linked to eating behaviour and investigate its causal relationships with metabolic traits using Mendelian randomization (MR). We tested associations between 30 genetic variants and eating patterns in individuals with T2D from the Volga-Ural region and investigated causal relationships between variants associated with eating patterns and various metabolic and anthropometric traits using data from the Volga-Ural population and large international consortia. We detected associations between HTR1D and CDKAL1 and external eating; between HTR2A and emotional eating; between HTR2A, NPY2R, HTR1F, HTR3A, HTR2C, CXCR2, and T2D. Further analyses in a separate group revealed significant associations between metabolic syndrome (MetS) and the loci in CRP, ADCY3, GHRL, CDKAL1, BDNF, CHRM4, CHRM1, HTR3A, and AKT1 genes. MR results demonstrated an inverse causal relationship between external eating and glycated haemoglobin levels in the Volga-Ural sample. External eating influenced anthropometric traits such as body mass index, height, hip circumference, waist circumference, and weight in GWAS cohorts. Our findings suggest that eating patterns impact both anthropometric and metabolic traits.

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.

Optogenetic induction of chronic glucocorticoid exposure in early-life leads to blunted stress-response in larval zebrafish.

Early life stress (ELS) exposure alters stress susceptibility in later life and affects vulnerability to stress-related disorders, but how ELS changes the long-lasting responsiveness of the stress system is not well understood. Zebrafish provides an opportunity to study conserved mechanisms underlying the development and function of the stress response that is regulated largely by the neuroendocrine hypothalamus-pituitary-adrenal/interrenal (HPA/I) axis, with glucocorticoids (GC) as the final effector. In this study, we established a method to chronically elevate endogenous GC levels during early life in larval zebrafish. To this end, we employed an optogenetic actuator, beggiatoa photoactivated adenylyl cyclase, specifically expressed in the interrenal cells of zebrafish and demonstrate that its chronic activation leads to hypercortisolaemia and dampens the acute-stress evoked cortisol levels, across a variety of stressor modalities during early life. This blunting of stress-response was conserved in ontogeny at a later developmental stage. Furthermore, we observe a strong reduction of proopiomelanocortin (pomc)-expression in the pituitary as well as upregulation of fkbp5 gene expression. Going forward, we propose that this model can be leveraged to tease apart the mechanisms underlying developmental programming of the HPA/I axis by early-life GC exposure and its implications for vulnerability and resilience to stress in adulthood.

Structure of adenylyl cyclase 5 in complex with Gßγ offers insights into ADCY5-related dyskinesia.

The nine different membrane-anchored adenylyl cyclase isoforms (AC1-9) in mammals are stimulated by the heterotrimeric G protein, Gαs, but their response to Gßγ regulation is isoform specific. In the present study, we report cryo-electron microscope structures of ligand-free AC5 in complex with Gßγ and a dimeric form of AC5 that could be involved in its regulation. Gßγ binds to a coiled-coil domain that links the AC transmembrane region to its catalytic core as well as to a region (C1b) that is known to be a hub for isoform-specific regulation. We confirmed the Gßγ interaction with both purified proteins and cell-based assays. Gain-of-function mutations in AC5 associated with human familial dyskinesia are located at the interface of AC5 with Gßγ and show reduced conditional activation by Gßγ, emphasizing the importance of the observed interaction for motor function in humans. We propose a molecular mechanism wherein Gßγ either prevents dimerization of AC5 or allosterically modulates the coiled-coil domain, and hence the catalytic core. As our mechanistic understanding of how individual AC isoforms are uniquely regulated is limited, studies such as this may provide new avenues for isoform-specific drug development.

Cold-induced expression of a truncated adenylyl cyclase 3 acts as rheostat to brown fat function.

Promoting brown adipose tissue (BAT) activity innovatively targets obesity and metabolic disease. While thermogenic activation of BAT is well understood, the rheostatic regulation of BAT to avoid excessive energy dissipation remains ill-defined. Here, we demonstrate that adenylyl cyclase 3 (AC3) is key for BAT function. We identified a cold-inducible promoter that generates a 5' truncated AC3 mRNA isoform (Adcy3-at), whose expression is driven by a cold-induced, truncated isoform of PPARGC1A (PPARGC1A-AT). Male mice lacking Adcy3-at display increased energy expenditure and are resistant to obesity and ensuing metabolic imbalances. Mouse and human AC3-AT are retained in the endoplasmic reticulum, unable to translocate to the plasma membrane and lack enzymatic activity. AC3-AT interacts with AC3 and sequesters it in the endoplasmic reticulum, reducing the pool of adenylyl cyclases available for G-protein-mediated cAMP synthesis. Thus, AC3-AT acts as a cold-induced rheostat in BAT, limiting adverse consequences of cAMP activity during chronic BAT activation.

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.

RelA-mediated signaling connects adaptation to chronic cardiomyocyte stress with myocardial and systemic inflammation in the ADCY8 model of accelerated aging.

Mice with cardiac-specific overexpression of adenylyl cyclase (AC) type 8 (TGAC8) are under a constant state of severe myocardial stress. They have a remarkable ability to adapt to this stress, but they eventually develop accelerated cardiac aging and experience reduced longevity. We have previously demonstrated through bioinformatics that constitutive adenylyl cyclase activation in TGAC8 mice is associated with the activation of inflammation-related signaling pathways. However, the immune response associated with chronic myocardial stress in the TGAC8 mouse remains unexplored. Here we demonstrate that chronic activation of adenylyl cyclase in cardiomyocytes of TGAC8 mice results in activation of cell-autonomous RelA-mediated NF-κB signaling. This is associated with non-cell-autonomous activation of proinflammatory and age-associated signaling in myocardial endothelial cells and myocardial smooth muscle cells, expansion of myocardial immune cells, increase in serum levels of inflammatory cytokines, and changes in the size or composition of lymphoid organs. All these changes precede the appearance of cardiac fibrosis. We provide evidence indicating that RelA activation in cardiomyocytes with chronic activation of adenylyl cyclase is mediated by calcium-protein Kinase A (PKA) signaling. Using a model of chronic cardiomyocyte stress and accelerated aging, we highlight a novel, calcium/PKA/RelA-dependent connection between cardiomyocyte stress, myocardial inflammation, and systemic inflammation. These findings suggest that RelA-mediated signaling in cardiomyocytes might be an adaptive response to stress that, when chronically activated, ultimately contributes to both cardiac and systemic aging.

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.

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.

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.

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.