The functions of sleep.

Here we report on The Satellite Symposium on Sleep Function that was held in Lausanne during 6(th) FENS forum and brought together neuroscientists from basic and clinical sleep research. We illustrate the principal questions that arose during this interdisciplinary gathering and introduce the contents of nine review articles on aspects of sleep that are contained in this Special Issue of the European Journal of Neuroscience.

Functions of peroxisome proliferator-activated receptors (PPAR) in skin homeostasis.

The peroxisome proliferator-activated receptors (PPAR) are ligand-activated transcription factors that belong to the nuclear hormone receptor family. Three isotypes (PPAR alpha, PPAR beta or delta, and PPAR gamma) with distinct tissue distributions and cellular functions have been found in vertebrates. All three PPAR isotypes are expressed in rodent and human skin. They were initially investigated for a possible function in the establishment of the permeability barrier in skin because of their known function in lipid metabolism in other cell types. In vitro studies using specific PPAR agonists and in vivo gene disruption approaches in mice indeed suggest an important contribution of PPAR alpha in the formation of the epidermal barrier and in sebocyte differentiation. The PPAR gamma isotype plays a role in stimulating sebocyte development and lipogenesis, but does not appear to contribute to epidermal tissue differentiation. The third isotype, PPAR beta, regulates the late stages of sebaceous cell differentiation, and is the most effective isotype in stimulating lipid production in these cells, both in rodents and in humans. In addition, PPAR beta activation has pro-differentiating effects in keratinocytes under normal and inflammatory conditions. Finally, preliminary studies also point to a potential role of PPAR in hair follicle growth and in melanocyte differentiation. By their diverse biological effects on cell proliferation and differentiation in the skin, PPAR agonists or antagonists may offer interesting opportunities for the treatment of various skin disorders characterized by inflammation, cell hyperproliferation, and aberrant differentiation.

Downregulation of cannabinoid receptor 1 from neuropeptide Y interneurons in the basal ganglia of patients with Huntington's disease and mouse models.

Cannabinoid receptor 1 (CB(1) receptor) controls several neuronal functions, including neurotransmitter release, synaptic plasticity, gene expression and neuronal viability. Downregulation of CB(1) expression in the basal ganglia of patients with Huntington's disease (HD) and animal models represents one of the earliest molecular events induced by mutant huntingtin (mHtt). This early disruption of neuronal CB(1) signaling is thought to contribute to HD symptoms and neurodegeneration. Here we determined whether CB(1) downregulation measured in patients with HD and mouse models was ubiquitous or restricted to specific striatal neuronal subpopulations. Using unbiased semi-quantitative immunohistochemistry, we confirmed previous studies showing that CB(1) expression is downregulated in medium spiny neurons of the indirect pathway, and found that CB(1) is also downregulated in neuropeptide Y (NPY)/neuronal nitric oxide synthase (nNOS)-expressing interneurons while remaining unchanged in parvalbumin- and calretinin-expressing interneurons. CB(1) downregulation in striatal NPY/nNOS-expressing interneurons occurs in R6/2 mice, Hdh(Q150/Q150) mice and the caudate nucleus of patients with HD. In R6/2 mice, CB(1) downregulation in NPY/nNOS-expressing interneurons correlates with diffuse expression of mHtt in the soma. This downregulation also occludes the ability of cannabinoid agonists to activate the pro-survival signaling molecule cAMP response element-binding protein in NPY/nNOS-expressing interneurons. Loss of CB(1) signaling in NPY/nNOS-expressing interneurons could contribute to the impairment of basal ganglia functions linked to HD.

Unique and conserved functions of B cell-activating factor of the TNF family (BAFF) in the chicken.

The chicken represents the best-characterized animal model for B cell development in the so-called gut-associated lymphoid tissue (GALT) and the molecular processes leading to B cell receptor diversification in this species are well investigated. However, the mechanisms regulating B cell development and homeostasis in GALT species are largely unknown. Here we investigate the role played by the avian homologue of B cell-activating factor of the tumor necrosis factor family (BAFF). Flow cytometric analysis showed that the receptor for chicken B cell-activating factor of the tumor necrosis factor family (chBAFF) is expressed by mature and immature B cells. Unlike murine and human BAFF, chBAFF is primarily produced by B cells both in peripheral lymphoid organs and in the bursa of Fabricius, the chicken's unique primary lymphoid organ. In vitro and in vivo studies revealed that chBAFF is required for mature B cell survival. In addition, in vivo neutralization with a decoy receptor led to a reduction of the size and number of B cell follicles in the bursa, demonstrating that, in contrast to humans and mice, in chickens BAFF is also required for the development of immature B cells. Collectively, we show that chBAFF has phylogenetically conserved functions in mature B cell homeostasis but displays unique and thus far unknown properties in the regulation of B cell development in birds.

Non-invasive diagnostic biomarkers for estimating the onset time of permanent cerebral ischemia.

The treatments for ischemic stroke can only be administered in a narrow time-window. However, the ischemia onset time is unknown in ~30% of stroke patients (wake-up strokes). The objective of this study was to determine whether MR spectra of ischemic brains might allow the precise estimation of cerebral ischemia onset time. We modeled ischemic stroke in male ICR-CD1 mice using a permanent middle cerebral artery filament occlusion model with laser Doppler control of the regional cerebral blood flow. Mice were then subjected to repeated MRS measurements of ipsilateral striatum at 14.1 T. A striking initial increase in γ-aminobutyric acid (GABA) and no increase in glutamine were observed. A steady decline was observed for taurine (Tau), N-acetyl-aspartate (NAA) and similarly for the sum of NAA+Tau+glutamate that mimicked an exponential function. The estimation of the time of onset of permanent ischemia within 6 hours in a blinded experiment with mice showed an accuracy of 33±10 minutes. A plot of GABA, Tau, and neuronal marker concentrations against the ratio of acetate/NAA allowed precise separation of mice whose ischemia onset lay within arbitrarily chosen time-windows. We conclude that (1)H-MRS has the potential to detect the clinically relevant time of onset of ischemic stroke.

MACROPHAGES FROM CROHN'S DISEASE PATIENTS EXHIBIT DEFICIENT REPAIR FUNCTIONS

AbstractBackground: Mucosal healing is becoming a major goal in the treatment of Crohn's disease. It has been previously reported that myeloid cells induce mucosal healing in a mouse model of acute colitis. The aim in this study is to investigate the pro-repair function of myeloid cells in healthy donors (HD) and Crohn's disease patients (CD).Methods: Peripheral blood mononuclear cells (PBMC) from HD and CD patients were isolated from blood samples and tested either directly or after differentiation ex-vivo into macrophages (Μφ). Intestinal macrophages (IMACs) were isolated from the bowel mucosa of patients undergoing intestinal surgical resections. Through an in vitro wound healing assay the repairing ability of these various human myeloid cells and the mechanisms responsible of wound healing were evaluated.Results: PBMC and myeloid CD14+ cells from HD and CD were not able to repair at any tested cell concentration. Μφ from HD and ulcerative colitis (UC) patients were able to induce wound healing and this capacity was partially mediated by Hepatocyte Growth Factor (HGF). Remarkably, CD Μφ were unable to promote wound healing and produced lower levels of HGF as compared to Μφ from HD or UC patients. In particular, Μφ from CD in active phase (ACD) exhibited the weakest repair function, but this defect was rescued if rh- GM-CSF was added during the differentiation of PBMCs. Interestingly, IMACs from HD promoted wound healing and produced HGF.Conclusion: We demonstrated that CD Μφ, unlike HD or UC Μφ, were defective in promoting wound healing, in particular if coming from an ACD. This deficient pro-repair function was related to a lower production of HGF. IMACs from HD colonic mucosa induced wound healing, confirming the results obtained with Μφ. Our results are in keeping with the current theory of CD as an innate immunodeficiency. In this context, Μφ may be responsible for the mucosal repair defects observed in CD patients and for the subsequent chronic activation of the adaptive immune response.

Differentiation of trophoblast giant cells and their metabolic functions are dependent on peroxisome proliferator-activated receptor beta/delta.

Mutation of the nuclear receptor peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) severely affects placenta development, leading to embryonic death at embryonic day 9.5 (E9.5) to E10.5 of most, but not all, PPARbeta/delta-null mutant embryos. While very little is known at present about the pathway governed by PPARbeta/delta in the developing placenta, this paper demonstrates that the main alteration of the placenta of PPARbeta/delta-null embryos is found in the giant cell layer. PPARbeta/delta activity is in fact essential for the differentiation of the Rcho-1 cells in giant cells, as shown by the severe inhibition of differentiation once PPARbeta/delta is silenced. Conversely, exposure of Rcho-1 cells to a PPARbeta/delta agonist triggers a massive differentiation via increased expression of 3-phosphoinositide-dependent kinase 1 and integrin-linked kinase and subsequent phosphorylation of Akt. The links between PPARbeta/delta activity in giant cells and its role on Akt activity are further strengthened by the remarkable pattern of phospho-Akt expression in vivo at E9.5, specifically in the nucleus of the giant cells. In addition to this phosphatidylinositol 3-kinase/Akt main pathway, PPARbeta/delta also induced giant cell differentiation via increased expression of I-mfa, an inhibitor of Mash-2 activity. Finally, giant cell differentiation at E9.5 is accompanied by a PPARbeta/delta-dependent accumulation of lipid droplets and an increased expression of the adipose differentiation-related protein (also called adipophilin), which may participate to lipid metabolism and/or steroidogenesis. Altogether, this important role of PPARbeta/delta in placenta development and giant cell differentiation should be considered when contemplating the potency of PPARbeta/delta agonist as therapeutic agents of broad application.

Predicting stroke through genetic risk functions: the CHARGE Risk Score Project.

BACKGROUND AND PURPOSE: Beyond the Framingham Stroke Risk Score, prediction of future stroke may improve with a genetic risk score (GRS) based on single-nucleotide polymorphisms associated with stroke and its risk factors. METHODS: The study includes 4 population-based cohorts with 2047 first incident strokes from 22,720 initially stroke-free European origin participants aged ≥55 years, who were followed for up to 20 years. GRSs were constructed with 324 single-nucleotide polymorphisms implicated in stroke and 9 risk factors. The association of the GRS to first incident stroke was tested using Cox regression; the GRS predictive properties were assessed with area under the curve statistics comparing the GRS with age and sex, Framingham Stroke Risk Score models, and reclassification statistics. These analyses were performed per cohort and in a meta-analysis of pooled data. Replication was sought in a case-control study of ischemic stroke. RESULTS: In the meta-analysis, adding the GRS to the Framingham Stroke Risk Score, age and sex model resulted in a significant improvement in discrimination (all stroke: Δjoint area under the curve=0.016, P=2.3×10(-6); ischemic stroke: Δjoint area under the curve=0.021, P=3.7×10(-7)), although the overall area under the curve remained low. In all the studies, there was a highly significantly improved net reclassification index (P<10(-4)). CONCLUSIONS: The single-nucleotide polymorphisms associated with stroke and its risk factors result only in a small improvement in prediction of future stroke compared with the classical epidemiological risk factors for stroke.

Estimating the net contribution of interleukin-28B variation to spontaneous hepatitis C virus clearance.

The identification of associations between interleukin-28B (IL-28B) variants and the spontaneous clearance of hepatitis C virus (HCV) raises the issues of causality and the net contribution of host genetics to the trait. To estimate more precisely the net effect of IL-28B genetic variation on HCV clearance, we optimized genotyping and compared the host contributions in multiple- and single-source cohorts to control for viral and demographic effects. The analysis included individuals with chronic or spontaneously cleared HCV infections from a multiple-source cohort (n = 389) and a single-source cohort (n = 71). We performed detailed genotyping in the coding region of IL-28B and searched for copy number variations to identify the genetic variant or haplotype carrying the strongest association with viral clearance. This analysis was used to compare the effects of IL-28B variation in the two cohorts. Haplotypes characterized by carriage of the major alleles at IL-28B single-nucleotide polymorphisms (SNPs) were highly overrepresented in individuals with spontaneous clearance versus those with chronic HCV infections (66.1% versus 38.6%, P = 6 × 10(-9) ). The odds ratios for clearance were 2.1 [95% confidence interval (CI) = 1.6-3.0] and 3.9 (95% CI = 1.5-10.2) in the multiple- and single-source cohorts, respectively. Protective haplotypes were in perfect linkage (r(2) = 1.0) with a nonsynonymous coding variant (rs8103142). Copy number variants were not detected. We identified IL-28B haplotypes highly predictive of spontaneous HCV clearance. The high linkage disequilibrium between IL-28B SNPs indicates that association studies need to be complemented by functional experiments to identify single causal variants. The point estimate for the genetic effect was higher in the single-source cohort, which was used to effectively control for viral diversity, sex, and coinfections and, therefore, offered a precise estimate of the net host genetic contribution.

Ly49D engagement on T lymphocytes induces TCR-independent activation and CD8 effector functions that control tumor growth.

Recent data showing expression of activating NK receptors (NKR) by conventional T lymphocytes raise the question of their role in the triggering of TCR-independent responses that could be damaging for the host. Transgenic mice expressing the activating receptor Ly49D/DAP12 offer the opportunity to better understand the relevance of ITAM signaling in the biology of T cells. In vitro experiments showed that Ly49D engagement on T lymphocytes by a cognate MHC class I ligand expressed by Chinese hamster ovary (CHO) cells or by specific Ab triggered cellular activation of both CD4 and CD8 populations with modulation of activation markers and cytokine production. The forced expression of the ITAM signaling chain DAP12 is mandatory for Ly49D-transgenic T cell activation. In addition, Ly49D stimulation induced T lymphocyte proliferation, which was much stronger for CD8 T cells. Phenotypic analysis of anti-Ly49D-stimulated CD8 T cells and their ability to produce high levels of IFN-gamma and to kill target cells indicate that Ly49D ligation generates effector cytotoxic CD8 T cells. Ly49D engagement by itself also triggered cytotoxic activity of activated CD8 T cells. Adoptive transfer experiments confirmed that Ly49D-transgenic CD8 T cells are able to control growth of CHO tumor cells or RMA cells transfected with Hm1-C4, the Ly49D ligand normally expressed by CHO. In conclusion, Ly49D engagement on T cells leads to T cell activation and to a full range of TCR-independent effector functions of CD8 T cells.

A cnidarian homologue of an insect gustatory receptor functions in developmental body patterning.

Insect gustatory and odorant receptors (GRs and ORs) form a superfamily of novel transmembrane proteins, which are expressed in chemosensory neurons that detect environmental stimuli. Here we identify homologues of GRs (Gustatory receptor-like (Grl) genes) in genomes across Protostomia, Deuterostomia and non-Bilateria. Surprisingly, two Grls in the cnidarian Nematostella vectensis, NvecGrl1 and NvecGrl2, are expressed early in development, in the blastula and gastrula, but not at later stages when a putative chemosensory organ forms. NvecGrl1 transcripts are detected around the aboral pole, considered the equivalent to the head-forming region of Bilateria. Morpholino-mediated knockdown of NvecGrl1 causes developmental patterning defects of this region, leading to animals lacking the apical sensory organ. A deuterostome Grl from the sea urchin Strongylocentrotus purpuratus displays similar patterns of developmental expression. These results reveal an early evolutionary origin of the insect chemosensory receptor family and raise the possibility that their ancestral role was in embryonic development.

Accurate calculations of intermolecular interaction energies using explicitly correlated coupled cluster wave functions and a dispersion-weighted MP2 method.

Explicitly correlated coupled-cluster calculations of intermolecular interaction energies for the S22 benchmark set of Jurecka, Sponer, Cerny, and Hobza (Chem. Phys. Phys. Chem. 2006, 8, 1985) are presented. Results obtained with the recently proposed CCSD(T)-F12a method and augmented double-zeta basis sets are found to be in very close agreement with basis set extrapolated conventional CCSD(T) results. Furthermore, we propose a dispersion-weighted MP2 (DW-MP2) approximation that combines the good accuracy of MP2 for complexes with predominately electrostatic bonding and SCS-MP2 for dispersion-dominated ones. The MP2-F12 and SCS-MP2-F12 correlation energies are weighted by a switching function that depends on the relative HF and correlation contributions to the interaction energy. For the S22 set, this yields a mean absolute deviation of 0.2 kcal/mol from the CCSD(T)-F12a results. The method, which allows obtaining accurate results at low cost, is also tested for a number of dimers that are not in the training set.

Estimating sex-specific dispersal rates with autosomal markers in hierarchically structured populations.

A recent study suggests that sex-specific dispersal rates can be quantitatively estimated on the basis of sex- and state-specific (pre- vs. postdispersal) F-statistics. In the present paper, we extend this approach to account for the hierarchical structure of natural populations, and we validate it through individual-based simulations. The model is applied to an empirical data set consisting of 536 individuals (males, females, and predispersal juveniles) of greater white-toothed shrews (Crocidura russula), sampled according to a hierarchical design and typed for seven autosomal microsatellite loci. From this dataset, dispersal is significantly female biased at the local scale (breeding-group level), but not at the larger scale (among local populations). We argue that selective pressures on dispersal are likely to depend on the spatial scale considered, and that short-distance dispersal should mainly respond to kin interactions (inbreeding or kin competition avoidance), which exert differential pressure on males and females.

Genetic differentiation for size at first reproduction through male versus female functions in the widespread Mediterranean tree Pinus pinaster.

BACKGROUND AND AIMS: The study of local adaptation in plant reproductive traits has received substantial attention in short-lived species, but studies conducted on forest trees are scarce. This lack of research on long-lived species represents an important gap in our knowledge, because inferences about selection on the reproduction and life history of short-lived species cannot necessarily be extrapolated to trees. This study considers whether the size for first reproduction is locally adapted across a broad geographical range of the Mediterranean conifer species Pinus pinaster. In particular, the study investigates whether this monoecious species varies genetically among populations in terms of whether individuals start to reproduce through their male function, their female function or both sexual functions simultaneously. Whether differences among populations could be attributed to local adaptation across a climatic gradient is then considered. METHODS: Male and female reproduction and growth were measured during early stages of sexual maturity of a P. pinaster common garden comprising 23 populations sampled across the species range. Generalized linear mixed models were used to assess genetic variability of early reproductive life-history traits. Environmental correlations with reproductive life-history traits were tested after controlling for neutral genetic structure provided by 12 nuclear simple sequence repeat markers. KEY RESULTS: Trees tended to reproduce first through their male function, at a size (height) that varied little among source populations. The transition to female reproduction was slower, showed higher levels of variability and was negatively correlated with vegetative growth traits. Several female reproductive traits were correlated with a gradient of growth conditions, even after accounting for neutral genetic structure, with populations from more unfavourable sites tending to commence female reproduction at a lower individual size. CONCLUSIONS: The study represents the first report of genetic variability among populations for differences in the threshold size for first reproduction between male and female sexual functions in a tree species. The relatively uniform size at which individuals begin reproducing through their male function probably represents the fact that pollen dispersal is also relatively invariant among sites. However, the genetic variability in the timing of female reproduction probably reflects environment-dependent costs of cone production. The results also suggest that early sex allocation in this species might evolve under constraints that do not apply to other conifers.