Diana Le Duc

Altered microglial phagocytosis in GPR34-deficient mice

GPR34 is a Gi/o protein‐coupled receptor (GPCR) of the nucleotide receptor P2Y12‐like group. This receptor is highly expressed in microglia, however, the functional relevance of GPR34 in these glial cells is unknown. Previous results suggested an impaired immune response in GPR34‐deficient mice infected with Cryptococcus neoformans. Here we show that GPR34 deficiency results in morphological changes in retinal and cortical microglia. RNA sequencing analysis of microglia revealed a number of differentially expressed transcripts involved in cell motility and phagocytosis. We found no differences in microglial motility after entorhinal cortex lesion and in response to laser lesion. However, GPR34‐deficient microglia showed reduced phagocytosis activity in both retina and acutely isolated cortical slices. Our study identifies GPR34 as an important signaling component controlling microglial function, morphology and phagocytosis. GLIA 2015;63:206–215

Kiwi genome provides insights into evolution of a nocturnal lifestyle

BackgroundKiwi, comprising five species from the genus Apteryx, are endangered, ground-dwelling bird species endemic to New Zealand. They are the smallest and only nocturnal representatives of the ratites. The timing of kiwi adaptation to a nocturnal niche and the genomic innovations, which shaped sensory systems and morphology to allow this adaptation, are not yet fully understood.ResultsWe sequenced and assembled the brown kiwi genome to 150-fold coverage and annotated the genome using kiwi transcript data and non-redundant protein information from multiple bird species. We identified evolutionary sequence changes that underlie adaptation to nocturnality and estimated the onset time of these adaptations. Several opsin genes involved in color vision are inactivated in the kiwi. We date this inactivation to the Oligocene epoch, likely after the arrival of the ancestor of modern kiwi in New Zealand. Genome comparisons between kiwi and representatives of ratites, Galloanserae, and Neoaves, including nocturnal and song birds, show diversification of kiwi’s odorant receptors repertoire, which may reflect an increased reliance on olfaction rather than sight during foraging. Further, there is an enrichment of genes influencing mitochondrial function and energy expenditure among genes that are rapidly evolving specifically on the kiwi branch, which may also be linked to its nocturnal lifestyle.ConclusionsThe genomic changes in kiwi vision and olfaction are consistent with changes that are hypothesized to occur during adaptation to nocturnal lifestyle in mammals. The kiwi genome provides a valuable genomic resource for future genome-wide comparative analyses to other extinct and extant diurnal ratites.

The G Protein-coupled Receptor P2Y14 Influences Insulin Release and Smooth Muscle Function in Mice

Background: The relevance of the widely expressed GPCR P2Y14 is only partially understood. Results: Analysis of P2Y14-KO mice revealed decreased gastrointestinal emptying, reduced glucose tolerance, and insulin release. Conclusion: P2Y14 function is required for proper intestine emptying and adequate glucose response. Significance: P2Y14 plays a role in smooth muscle function and maintaining energy homeostasis by influencing insulin release. UDP sugars were identified as extracellular signaling molecules, assigning a new function to these compounds in addition to their well defined role in intracellular substrate metabolism and storage. Previously regarded as an orphan receptor, the G protein-coupled receptor P2Y14 (GPR105) was found to bind extracellular UDP and UDP sugars. Little is known about the physiological functions of this G protein-coupled receptor. To study its physiological role, we used a gene-deficient mouse strain expressing the bacterial LacZ reporter gene to monitor the physiological expression pattern of P2Y14. We found that P2Y14 is mainly expressed in pancreas and salivary glands and in subpopulations of smooth muscle cells of the gastrointestinal tract, blood vessels, lung, and uterus. Among other phenotypical differences, knock-out mice showed a significantly impaired glucose tolerance following oral and intraperitoneal glucose application. An unchanged insulin tolerance suggested altered pancreatic islet function. Transcriptome analysis of pancreatic islets showed that P2Y14 deficiency significantly changed expression of components involved in insulin secretion. Insulin secretion tests revealed a reduced insulin release from P2Y14-deficient islets, highlighting P2Y14 as a new modulator of proper insulin secretion.

Examining the Dynamic Evolution of G Protein-Coupled Receptors

The valuable source of large-scale genomic information initiated attempts to identify the origin(s) of G protein-coupled receptors (GPCR), count and categorize those genes, and follow their evolutionary history. Being present in fungi, plants, and unicellular eukaryotes, GPCR must have evolved before the plant-fungi-animal split about 1.5 billion years ago. Phylogenetic analyses revealed several kinds of evolutionary patterns that occurred during GPCR evolution including one-to-one orthologous relationships, species-specific gene expansion, and episodic duplication of the entire GPCR repertoire in certain species lineages. These data document the highly dynamic process of birth and death of GPCR genes since hundreds of millions of years. Genetic drift and selective forces have shaped the individual structure of a given receptor gene but also of the species-specific receptor repertoire - a process that is still ongoing. These processes have left footprints in the genomic sequence that can be detected by bioinformatic methods and may help to interpret receptor function in the light of a given species in its environment. Reasonable intraspecies sequence variability in GPCR is either physiologically tolerated or promotes individual phenotypes and adaptation, but also susceptibilities for diseases. Therefore, the impact of GPCR variants in epistatic networks will be an important task of future GPCR research. The chapter summarizes evolutionary processes working on GPCR genes and sheds light on their consequences at the levels of receptor structure and function.

Dendritic Cells Regulate GPR34 through Mitogenic Signals and Undergo Apoptosis in Its Absence

Dendritic cells (DCs) are specifically equipped with the G protein–coupled receptor 34 (GPR34). Tight regulation of GPR34 gene expression seems highly important for proper immunological functions, because the absence of this receptor leads to an alteration of the immune response, whereas overexpression was reported to be involved in neuroinflammation. However, the regulatory mechanism of GPR34 expression has not yet been investigated. Whole-transcriptome RNA sequencing analysis from spleens and DCs of GPR34 knockout and wild-type mice, combined with protein–protein interaction data, revealed functional modules affected by the absence of this receptor. Among these, NF-κB, MAPK, and apoptosis-signaling pathways showed high significance. Using murine DCs we experimentally show that NF-κB and MAPK pathways are involved in the downregulation of GPR34. DCs lacking GPR34 have a higher caspase-3/7 activity and increased apoptosis levels. Our study reveals a novel role of GPR34 in the fate of DCs and identifies a regulatory mechanism that could be relevant for treatment of GPR34-overexpressing pathologies, such as neuroinflammatory or cancer conditions.

Behavioral and molecular effects of prenatal continuous light exposure in the adult rat

Disruption of the maternal environment during pregnancy leads to behavioral changes and diseases in the adult offspring. To explore the influence of prenatal continuous light exposure (PCLE) on the adult offspring, we exposed pregnant Wistar rats to constant light during late gestation. Adult PCLE offspring showed an anxiety-like behavior and impairment of short-term memory in different tests. Measurements in the whole brain homogenates from newborn and adult offspring indicated decreased melatonin and serotonin levels and increased reactive oxygen species level in PCLE offspring. Further, we determined melatonin-, serotonin-, oxidative stress-, apoptosis-, and circadian system-related genes expression in different brain areas of adult offspring. The serotonin reuptaker Slc6a4 displayed a decreased expression in the prefrontal cortex of PCLE group. The circadian rhythm-related gene Rora was upregulated in the amygdala of PCLE offspring. Our results point to adverse behavioral effects of PCLE on adult offspring, involving serotonin and melatonin signaling dysregulation, increased chronic oxidative stress, and altered gene expression.

Luciferase activity under direct ligand-dependent control of a muscarinic acetylcholine receptor

BackgroundControlling enzyme activity by ligand binding to a regulatory domain of choice may have many applications e.g. as biosensors and as tools in regulating cellular functions. However, until now only a small number of ligand-binding domains have been successfully linked to enzyme activity. G protein-coupled receptors (GPCR) are capable of recognizing an extraordinary structural variety of extracellular signals including inorganic and organic molecules. Ligand binding to GPCR results in conformational changes involving the transmembrane helices. Here, we assessed whether ligand-induced conformational changes within the GPCR helix bundle can be utilized to control the activity of an integrated enzyme.ResultsAs a proof of principle, we inserted the luciferase amino acid sequence into the third intracellular loop of the M3 muscarinic acetylcholine receptor. This fusion protein retained both receptor and enzyme function. Receptor blockers slightly but significantly reduced enzyme activity. By successive deletion mutagenesis the enzyme activity was optimally coupled to ligand-induced conformational helix movements.ConclusionOur results demonstrate that in engineered GPCR-enzyme chimeras, intracellular enzyme activity can be directly controlled by a GPCR serving as the extracellular ligand-binding domain.

Adaptation to nocturnality – learning from avian genomes

The recent availability of multiple avian genomes has laid the foundation for a huge variety of comparative genomics analyses including scans for changes and signatures of selection that arose from adaptions to new ecological niches. Nocturnal adaptation in birds, unlike in mammals, is comparatively recent, a fact that makes birds good candidates for identifying early genetic changes that support adaptation to dim‐light environments. In this review, we give examples of comparative genomics analyses that could shed light on mechanisms of adaptation to nocturnality. We present advantages and disadvantages of both “data‐driven” and “hypothesis‐driven” approaches that lead to the discovery of candidate genes and genetic changes promoting nocturnality. We anticipate that the accessibility of multiple genomes from the Genome 10K Project will allow a better understanding of evolutionary mechanisms and adaptation in general.

Fetal alcohol spectrum disorder: molecular insights into neural damage reduction

Fetal alcohol spectrum disorders (FASD) is a group of entirely preventable, lifelong conditions, which occur upon maternal alcohol use during pregnancy. This can result in severe consequences for the newborn and ultimately the family. It is usually characterized by delays in development and motor function, craniofacial abnormalities, and difficulties with learning, memory, speech, and academic achievement. According to the German guidelines for fetal alcohol syndrome (FAS) diagnosis, the prevalence of FASD ranges between 0.02–0.8% of all annual births and often the disorder is not recognized (Landgraf et al., 2013). The U.S. National Institutes of Health regard FAS as the most common nonhereditary cause of mental retardation. Thus, preventing programs, like the one undertaken by the Australian Government, which appointed a National FASD Technical Network (Elliott, 2015), may seem a very reasonable strategy. However, preventing programs for FASD focus mainly either on primary prevention, by increasing pregnant womens awareness of ethanol consumption risks, or on tertiary prevention which supposes early recognition of the condition and social support in the form of an improved developmental framework of the affected individual. Secondary prevention of the disorder, which includes early detection by screening pregnant women for ethanol consumption and control of the progression from a pre- clinical pathological condition to a severe form of the disease, is considered to be most challenging (Elliott, 2015). Although the most important form of prevention is abstinence from alcohol during pregnancy, offsprings from women with known gestational alcohol misuse could still benefit from a secondary form of FASD prevention, which might hinder the progression to FAS. However, therapy approaches are precluded by our limited knowledge of molecular mechanisms responsible for neuronal damage.