The expression of genes in top obesity-associated loci is enriched in insula and substantia nigra brain regions involved in addiction and reward.

BACKGROUND: Genome-wide association studies (GWAS) have identified more than 250 loci associated with body mass index (BMI) and obesity. However, post-GWAS functional genomic investigations have been inadequate for understanding how these genetic loci physiologically impact disease development. METHODS: We performed a PCR-free expression assay targeting genes located nearby the GWAS-identified SNPs associated with BMI/obesity in a large panel of human tissues. Furthermore, we analyzed several genetic risk scores (GRS) summing GWAS-identified alleles associated with increased BMI in 4236 individuals. RESULTS: We found that the expression of BMI/obesity susceptibility genes was strongly enriched in the brain, especially in the insula (p = 4.7 × 10-9) and substantia nigra (p = 6.8 × 10-7), which are two brain regions involved in addiction and reward. Inversely, we found that top obesity/BMI-associated loci, including FTO, showed the strongest gene expression enrichment in the two brain regions. CONCLUSIONS: Our data suggest for the first time that the susceptibility genes for common obesity may have an effect on eating addiction and reward behaviors through their high expression in substantia nigra and insula, i.e., a different pattern from monogenic obesity genes that act in the hypothalamus and cause hyperphagia. Further epidemiological studies with relevant food behavior phenotypes are necessary to confirm these findings.

A Binary Genetic Approach to Characterize TRPM5 Cells in Mice.

Transient receptor potential channel subfamily M member 5 (TRPM5) is an important downstream signaling component in a subset of taste receptor cells making it a potential target for taste modulation. Interestingly, TRPM5 has been detected in extra-oral tissues; however, the function of extra-gustatory TRPM5-expressing cells is less well understood. To facilitate visualization and manipulation of TRPM5-expressing cells in mice, we generated a Cre knock-in TRPM5 allele by homologous recombination. We then used the novel TRPM5-IRES-Cre mouse strain to report TRPM5 expression by activating a τGFP transgene. To confirm faithful coexpression of τGFP and TRPM5 we generated and validated a new anti-TRPM5 antiserum enabling us to analyze acute TRPM5 protein expression. τGFP cells were found in taste bud cells of the vallate, foliate, and fungiform papillae as well as in the palate. We also detected TRPM5 expression in several other tissues such as in the septal organ of Masera. Interestingly, in the olfactory epithelium of adult mice acute TRPM5 expression was detected in only one (short microvillar cells) of two cell populations previously reported to express TRPM5. The TRPM5-IC mouse strain described here represents a novel genetic tool and will facilitate the study and tissue-specific manipulation of TRPM5-expressing cells in vivo.

Expression and functional assessment of candidate type 2 diabetes susceptibility genes identify four new genes contributing to human insulin secretion.

OBJECTIVES: Genome-wide association studies (GWAS) have identified >100 loci independently contributing to type 2 diabetes (T2D) risk. However, translational implications for precision medicine and for the development of novel treatments have been disappointing, due to poor knowledge of how these loci impact T2D pathophysiology. Here, we aimed to measure the expression of genes located nearby T2D associated signals and to assess their effect on insulin secretion from pancreatic beta cells. METHODS: The expression of 104 candidate T2D susceptibility genes was measured in a human multi-tissue panel, through PCR-free expression assay. The effects of the knockdown of beta-cell enriched genes were next investigated on insulin secretion from the human EndoC-ßH1 beta-cell line. Finally, we performed RNA-sequencing (RNA-seq) so as to assess the pathways affected by the knockdown of the new genes impacting insulin secretion from EndoC-ßH1, and we analyzed the expression of the new genes in mouse models with altered pancreatic beta-cell function. RESULTS: We found that the candidate T2D susceptibility genes' expression is significantly enriched in pancreatic beta cells obtained by laser capture microdissection or sorted by flow cytometry and in EndoC-ßH1 cells, but not in insulin sensitive tissues. Furthermore, the knockdown of seven T2D-susceptibility genes (CDKN2A, GCK, HNF4A, KCNK16, SLC30A8, TBC1D4, and TCF19) with already known expression and/or function in beta cells changed insulin secretion, supporting our functional approach. We showed first evidence for a role in insulin secretion of four candidate T2D-susceptibility genes (PRC1, SRR, ZFAND3, and ZFAND6) with no previous knowledge of presence and function in beta cells. RNA-seq in EndoC-ßH1 cells with decreased expression of PRC1, SRR, ZFAND6, or ZFAND3 identified specific gene networks related to T2D pathophysiology. Finally, a positive correlation between the expression of Ins2 and the expression of Prc1, Srr, Zfand6, and Zfand3 was found in mouse pancreatic islets with altered beta-cell function. CONCLUSIONS: This study showed the ability of post-GWAS functional studies to identify new genes and pathways involved in human pancreatic beta-cell function and in T2D pathophysiology.

Developmental pattern of NADPH-diaphorase positive neurons in chick optic tectum is sensitive to changes in visual stimulation.

The chick retinotectal system is a suitable model to investigate the mechanisms involved in the establishment of synaptic connections in whose refinement nitric oxide was implicated. The purpose of this work was to describe the developmental pattern of the nitric oxide synthase (NOS)-positive neurons as well as to determine if it is sensitive to changes in visual stimulation. The NADPH-diaphorase histochemical method was used to describe and quantify NOS neurons in normally stimulated and subnormally stimulated chickens. Nine types of NOS neurons were identified; seven of them express NOS until adulthood, while two of them show only a transient expression. The developmental pattern of NOS neurons follows the process of laminar segregation. It can be divided into three phases. The first includes the onset of NOS expression in periventricular neurons and the formation of a deep network of NOS fibers during early development. These neurons do not show any significant change in subnormally stimulated animals. The second phase includes the appearance of two transient NOS populations of bipolar neurons that occupy the intermediate layers during the optic fibers ingrowth. One of them significantly changes in subnormally stimulated chicks. The third phase occurs when the transitory expression of bipolar neurons decreases. It includes NOS expression in six neuronal populations that innervate the superficial retinorecipient layers. Most of these cells suffer plastic changes in subnormally stimulated chicks. The diversity of neuronal types with regard to their morphology, location, and sensitivity to visual stimulation strongly suggests that they serve different functions.

EphA3 expressed in the chicken tectum stimulates nasal retinal ganglion cell axon growth and is required for retinotectal topographic map formation.

BACKGROUND: Retinotopic projection onto the tectum/colliculus constitutes the most studied model of topographic mapping and Eph receptors and their ligands, the ephrins, are the best characterized molecular system involved in this process. Ephrin-As, expressed in an increasing rostro-caudal gradient in the tectum/colliculus, repel temporal retinal ganglion cell (RGC) axons from the caudal tectum and inhibit their branching posterior to their termination zones. However, there are conflicting data regarding the nature of the second force that guides nasal axons to invade and branch only in the caudal tectum/colliculus. The predominant model postulates that this second force is produced by a decreasing rostro-caudal gradient of EphA7 which repels nasal optic fibers and prevents their branching in the rostral tectum/colliculus. However, as optic fibers invade the tectum/colliculus growing throughout this gradient, this model cannot explain how the axons grow throughout this repellent molecule. METHODOLOGY/PRINCIPAL FINDINGS: By using chicken retinal cultures we showed that EphA3 ectodomain stimulates nasal RGC axon growth in a concentration dependent way. Moreover, we showed that nasal axons choose growing on EphA3-expressing cells and that EphA3 diminishes the density of interstitial filopodia in nasal RGC axons. Accordingly, in vivo EphA3 ectodomain misexpression directs nasal optic fibers toward the caudal tectum preventing their branching in the rostral tectum. CONCLUSIONS: We demonstrated in vitro and in vivo that EphA3 ectodomain (which is expressed in a decreasing rostro-caudal gradient in the tectum) is necessary for topographic mapping by stimulating the nasal axon growth toward the caudal tectum and inhibiting their branching in the rostral tectum. Furthermore, the ability of EphA3 of stimulating axon growth allows understanding how optic fibers invade the tectum growing throughout this molecular gradient. Therefore, opposing tectal gradients of repellent ephrin-As and of axon growth stimulating EphA3 complement each other to map optic fibers along the rostro-caudal tectal axis.

The chick optic tectum developmental stages. A dynamic table based on temporal- and spatial-dependent histogenetic changes: A structural, morphometric and immunocytochemical analysis.

Development is often described as temporal sequences of developmental stages (DSs). When tables of DS are defined exclusively in the time domain they cannot discriminate histogenetic differences between different positions along a spatial reference axis. We introduce a table of DSs for the developing chick optic tectum (OT) based on time- and space-dependent changes in quantitative morphometric parameters, qualitative histogenetic features and immunocytochemical pattern of several developmentally active molecules (Notch1, Hes5, NeuroD1, ß-III-Tubulin, synaptotagmin-I and neurofilament-M). Seven DSs and four transitional stages were defined from ED2 to ED12, when the basic OT cortical organization is established, along a spatial developmental gradient axis extending between a zone of maximal and a zone of minimal development. The table of DSs reveals that DSs do not only progress as a function of time but also display a spatially organized propagation along the developmental gradient axis. The complex and dynamic character of the OT development is documented by the fact that several DSs are simultaneously present at any ED or any embryonic stage. The table of DSs allows interpreting how developmental cell behaviors are temporally and spatially organized and explains how different DSs appear as a function of both time and space. The table of DSs provides a reference system to characterize the OT corticogenesis and to reliably compare observations made in different specimens.

Key roles of Ephs and ephrins in retinotectal topographic map formation.

Cellular and molecular mechanisms involved in the development of topographic ordered connections in the central nervous system (CNS) constitute a key issue in neurobiology because neural connectivities are the base of the CNS normal function. We discuss the roles of the Eph/ephrin system in the establishment of retinotopic projections onto the tectum/colliculus, the most detailed studied model of topographic mapping. The expression patterns of Ephs and ephrins in opposing gradients both in the retina and the tectum/colliculus, label the local addresses on the target and give specific sensitivities to growth cones according to their topographic origin in the retina. We postulate that the highest levels of these gradients could signal both the entry as well as the limiting boundaries of the target. Since Ephs and ephrins are membrane-bound molecules, they may function as both receptors and ligands producing repulsive or attractant responses according to their microenvironment and play central roles in a variety of developmental events such as axon guidance, synapse formation and remodeling. Due to different experimental approaches and the inherent species-specific differences, some results appear contradictory and should be reanalyzed. Nevertheless, these studies about the roles of the Eph/ephrin system in retinotectal/collicular mapping support general principles in order to understand CNS development and could be useful to design regeneration therapies.