Biology subjects

Hebert, T.

Publications and source records attributed to Hebert, T..

9 recordsLinked to original sources

bioRxiv2024

The Orphan G Protein-Coupled Receptor GPR52 is a Novel Regulator of Breast Cancer Multicellular Organization

Statement of SignificanceWe showed that loss of the orphan G protein-coupled receptor GPR52 in human breast cell lines leads to increased cell clustering, hybrid/partial EMT, and increased tumor burden in zebrafish. BackgroundG protein-coupled receptors (GPCRs) are the largest class of membrane-bound receptors that transmit critical signals from extracellular to intracellular spaces. Transcriptomic data of resected breast tumors show that low mRNA expression of orphan GPCR GPR52 correlates with reduced overall survival in patients with breast cancer, leading to the hypothesis that loss of GPR52 supports breast cancer progression. MethodsCRISPR-Cas9 was used to knockout GPR52 in the human triple-negative breast cancer (TNBC) cell lines MDA-MB-468 and MDA-MB-231, and in the non-cancerous breast epithelial cell line MCF10A. 2D and 3D in vitro studies, electron microscopy, Matrigel culture, and a zebrafish xenograft model were used to assess the morphology and behavior of GPR52 KO cells. RNA-sequencing and proteomic analyses were also conducted on these cell lines, and transcriptomic data from The Cancer Genome Atlas (TCGA) database were used to compare GPR52-null and wild-type (WT) signatures in breast cancer. ResultsLoss of GPR52 was found to be associated with increased cell-cell interaction in 2D cultures, altered 3D spheroid morphology, and increased propensity to organize and invade collectively in Matrigel. Furthermore, GPR52 loss was associated with features of EMT in MDA-MB-468 cells, and zebrafish injected with GPR52 KO cells developed a greater total cancer area than those injected with control cells. RNA sequencing and proteomic analyses of GPR52-null breast cancer cells revealed an increased cAMP signaling signature. Consistently, we found that treatment of wild-type (WT) cells with forskolin, which stimulates the production of cAMP, induces phenotypic changes associated with GPR52 loss, and inhibition of cAMP production rescued some GPR52 KO phenotypes. ConclusionGPR52 is an orphan GPCR and its role in cancer progression has not been previously characterized. We found that GPR52 loss in breast cancer cells can lead to increased cell clustering, collective invasion, and EMT in vitro. These are features of increased cancer aggression. Our results reveal that GPR52 loss is a potential mechanism by which breast cancer progression may occur and support the investigation of GPR52 agonism as a therapeutic option for breast cancer. O_FIG O_LINKSMALLFIG WIDTH=169 HEIGHT=200 SRC="FIGDIR/small/604482v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@8b0f55org.highwire.dtl.DTLVardef@19bd139org.highwire.dtl.DTLVardef@c52117org.highwire.dtl.DTLVardef@1ffa2bd_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

cancer biology↗

bioRxiv2022

Comparing the signaling and transcriptome profiling landscapes of human iPSC-derived and primary rat neonatal cardiomyocytes

The inaccessibility of human cardiomyocytes significantly hindered years of cardiovascular research efforts. Post-mortem tissue or biopsies from diseased patients, which remain scarcely available, rendered it possible to study end-stage heart disease yet the inclusion of healthy human cardiac materials for basic science research was beyond reach. To overcome these limitations, non-human cell sources were used as proxies to study heart function and associated diseases. Rodent models became increasingly acceptable surrogates to model the human heart either in vivo or through in vitro cultures. More recently, due to concerns regarding animal to human translation, including cross-species differences, the use of human inducible stem cell derived cardiomyocytes presented a renewed opportunity. We think it necessary to conduct a comparative study, assessing cellular signalling through cardiac G protein-coupled receptors and bulk transcriptomics of traditional rat neonatal cardiomyocytes and human iPSC-CMs. Genetically-encoded biosensors were used to interrogate nuclear protein kinase A (PKA) and extracellular signal-regulated kinase 1/ 2 (ERK1/2) in rat and human-derived cardiomyocyte populations. To increase data granularity, a single-cell analytical approach was conducted for an in-depth examination of existing differences between both in vitro cardiomyocyte models. Using automated high content microscopy, our analyses of nuclear PKA and ERK1/2 signaling revealed distinct response clusters in rat and human CMs. In line with this, bulk RNA-seq demonstrated key differences regarding the expression patterns of GPCRs, G proteins and effectors. Overall, our study demonstrates that human stem cell derived models of the cardiomyocyte do provide significant advantages and should be taken advantage of.

biochemistry↗

bioRxiv2022

Effective use of genetically-encoded optical biosensors for profiling signalling signatures in iPSC-CMs derived from idiopathic dilated cardiomyopathy patients

Dilated cardiomyopathy (DCM) is a cardiovascular condition that develops when the left ventricle of the heart enlarges, compromising its function and diminishing its capacity to pump oxygenated blood throughout the body. After patients are diagnosed with DCM, disease progression can lead to heart failure and the need for a heart transplantation. DCM is a complex disease where underlying causes can be idiopathic, genetic, or environmental. An incomplete molecular understanding of disease progression poses challenges for drug discovery efforts as effective therapeutics strategies remain elusive. Decades of research using primary cells or animal models have increased our understanding of DCM but has been hampered due to the inaccessibility of human cardiomyocytes, to model cardiac disease, in vitro, in a dish. Here, our goal is to leverage patient-derived hiPSC-CMs and to combine them with biosensors to understand how cellular signalling is altered in DCM. With high sensitivity and versatility, optical biosensors represent the ideal tools to dissect the molecular determinants of cardiovascular disease, in an unbiased manner and in real-time at the level of single cells. By characterizing the pathobiology of dilated cardiomyopathy in a patient-specific manner using high content biosensor-based assays, we aim to uncover personalized mechanisms for the occurrence and development of DCM and as a pathway to development of personalized therapeutics.

biochemistry↗

bioRxiv2021

Addition of a carboxy terminal tail to the normally tailless gonadotropin-releasing hormone receptor impairs fertility in female mice

Gonadotropin-releasing hormone (GnRH) is the primary neuropeptide controlling reproduction in vertebrates. GnRH stimulates follicle-stimulating hormone (FSH) and luteinizing hormone (LH) synthesis via a G protein-coupled receptor, GnRHR, in the pituitary gland. In mammals, GnRHR lacks a C-terminal cytosolic tail (Ctail) and does not exhibit homologous desensitization. This might be an evolutionary adaptation that enables LH surge generation and ovulation. To test this idea, we fused the chicken GnRHR Ctail to the endogenous murine GnRHR in a transgenic model. The LH surge was blunted, but not blocked in these mice. In contrast, they showed reductions in FSH production, ovarian follicle development, and fertility. Addition of the Ctail altered the nature of agonist-induced calcium signaling required for normal FSH production. The loss of the GnRHR Ctail during mammalian evolution is unlikely to have conferred a selective advantage by enabling the LH surge. The adaptive significance of this specialization remains to be determined.

physiology↗

bioRxiv2021

Dopamine D1 receptor activation and cAMP/PKA signalling mediate Brd4 recruitment to chromatin to regulate gene expression in rat striatal neurons

The activity of striatal medium-spiny projection neurons is regulated by dopamine acting principally at D1 and D2 dopamine receptors. The dopamine D1 receptor (D1R) is a Gs/olf-coupled GPCR which activates a cAMP/PKA/DARPP-32 signalling cascade that increases excitability and facilitates plasticity, partly through the regulation of transcription. Transcriptional regulation downstream of the D1R involves the activation of PKA, which can translocate to the nucleus to phosphorylate various targets. The chromatin reader Brd4 regulates transcription induced by neurotrophic factors in cortical neurons and has also been implicated in dopamine-dependent striatal functions. Brd4 is activated by phosphorylation; this facilitates its binding to acetylated histones at promoters and enhancers. In non-neuronal cells, Brd4 is recruited to chromatin in response to PKA signalling. However, it is unknown whether Brd4 is involved in transcriptional activation by the D1R in striatal neurons. Here, we demonstrate that cAMP/PKA signalling increases Brd4 recruitment to dopamine-induced genes in striatal neurons, and that knockdown or inhibition of Brd4 modulated D1R-induced gene expression. Specifically, inhibition of Brd4 with the bromodomain inhibitor JQ1 suppressed the expression of [~]25% of D1R-upregulated genes, while increasing the expression of a subset of immediate-early genes, including Fos and Jun. This pro-transcriptional effect of JQ1 was P-TEFb-dependent, and mediated through inhibition of the BD1 bromodomain of Brd4. Finally, we report that JQ1 treatment downregulated expression of many GPCRs and also impaired ERK1/2 signalling in striatal neurons. Our findings identify Brd4 as a novel regulator of D1R-dependent transcription and delineate complex bi-directional effects of bromodomain inhibitors on neuronal transcription.

neuroscience↗

bioRxiv2021

Single cell analysis of population-wide nuclear and cytosolic drug responses using high-content FRET imaging: measuring protein kinase activation in rat primary striatal neurons

Genetically-encoded biosensors are used to track biochemical activities in living cells by measuring changes in fluorescence emitted by one or more fluorescent proteins. In the present article, we describe the application of genetically-encoded FRET biosensors with high content microscopy to image the signaling responses of thousands of neurons in response to drug treatments. We applied this approach to reveal intercellular variation in signaling responses among cultured striatal neurons stimulated with multiple drugs. The striatum is largely composed of medium-spiny GABAergic neurons which are divided into two broad sub-types based in part on their expression of dopamine D1 vs. D2 receptors. Using high content FRET imaging and immunofluorescence, we identified neuronal sub-populations with unique responses to pharmacological manipulation. Focusing on dopamine- and glutamate-regulated PKA and ERK1/2 signaling in both the cytoplasm and nucleus, we identified pronounced intercellular differences, in both the magnitude and kinetics of signaling responses to drug application. Importantly, we found that a conventional "bulk" analysis that included all cells in culture yielded a different rank order of drug potency than that revealed by our single-cell analysis. The high degree of heterogeneity that we observed at the single cell level would not have been detectable using common population-level analyses, derived for example from western blotting or plate reader-based measurements. In conclusion, our single-cell analytical approach highlights the limitations of population-level analyses, and provides a novel way to study signaling biology.

neuroscience↗

bioRxiv2020

Differential activation of P-TEFb complexes in the development of cardiomyocyte hypertrophy following activation of distinct GPCRs

Pathological cardiac hypertrophy is driven by neurohormonal activation of specific G protein-coupled receptors (GPCRs) in cardiomyocytes and is accompanied by large-scale changes in cardiomyocyte gene expression. These transcriptional changes require activity of positive transcription elongation factor b (P-TEFb), which is recruited to target genes by the bromodomain protein Brd4 or the Super Elongation Complex (SEC). Here we describe GPCR-specific regulation of these P-TEFb complexes and a novel mechanism for activating Brd4 in primary neonatal rat cardiomyocytes. The SEC was required for the hypertrophic response downstream of either the 1-adrenergic receptor (1-AR) or the endothelin receptor (ETR). In contrast, Brd4 inhibition selectively impaired the 1-AR response. This was corroborated by the finding that activation of 1-AR, but not ETR, increased Brd4 occupancy at promoters and super enhancers of hypertrophic genes. Transcriptome analysis demonstrated that activation of both receptors initiated similar gene expression programs, but that Brd4 inhibition attenuated hypertrophic genes more robustly following 1-AR activation. Finally, we show that protein kinase A (PKA) is required for 1-AR stimulation of Brd4 chromatin occupancy. The differential role of the Brd4/P-TEFb complex in response to distinct GPCR pathways has potential clinical implications as therapies targeting this complex are currently being explored for heart failure.

molecular biology↗

bioRxiv2019

A comparison of eDNA to camera trapping for assessment of terrestrial mammal diversity

Environmental DNA (eDNA) is one of the most promising approaches to meet the demand for the fast and frequent monitoring of ecosystems needed to tackle the current decline in biodiversity. However, before eDNA can establish itself as a robust alternative for mammal monitoring, comparison with existing approaches is necessary, yet has not been done. Moreover, much is unknown regarding the nature, spread and persistence of DNA shed by animals into terrestrial environments, or the optimal experimental design for understanding these potential biases.\n\nTo address some of these challenges, we compared the detection of terrestrial mammals using eDNA analysis of soil samples against confirmed species observations from a long-term ([~]9-yr) camera trapping study. At the same time, we considered multiple experimental parameters, including two sampling designs, two DNA extraction kits and two metabarcodes of different sizes.\n\nAll mammals consistently recorded with cameras were detected in eDNA. In addition, eDNA reported many small mammals not recorded by camera traps, but whose presence in the study area is otherwise documented. A long metabarcode ({approx}220bp) offering a high taxonomic resolution, achieved a similar efficiency as a shorter one ({approx}70bp) and a phosphate buffer-based extraction gave similar results as a total DNA extraction method for a fraction of the price. Our results support that eDNA-based monitoring should become a valuable part of terrestrial mammal surveys. Yet, the lack of coverage of mammal mitochondrial genomes in public databases must be addressed before eDNA can be used to its full potential.

ecology↗

bioRxiv2019

Return of an apex predator to a suburban preserve triggers a rapid trophic cascade

Absence of apex predators simplifies food chains, leading to trophic degradation of ecosystems and diminution of the services they provide1. However, most predators do not coexist well with humans, which has resulted in a decline of carnivores and functional ecosystems worldwide2. In some instances, cryptic carnivores manage to survive amidst human settlements, finding refuge in small biological islands surrounded by urban landscapes. In such a system, we used two non-invasive data collection methods (camera trapping and fecal sampling) to investigate the multiannual relationship between predators and prey, and between competitors, through analysis of: (1) relative abundance and detection probability of species over time, (2) causal interactions via empirical dynamic modeling, (3) diet, and (4) diel activity patterns. All approaches show concordance in the results: the natural return of an apex predator, the puma (Puma concolor), triggered a trophic cascade, affecting the abundance and behavior of its main prey, subordinate predators and other prey in the studied system. Our study demonstrates that trophic recovery can occur rapidly following the return of a top predator, even in small protected areas in increasingly urbanized landscapes.

ecology↗