Brain Phenotype of Transgenic Mice Overexpressing Cystathionine β-Synthase

The cystathionine β-synthase (CBS) gene, located on human chromosome 21q22.3, is a good candidate for playing a role in the Down Syndrome (DS) cognitive profile: it is overexpressed in the brain of individuals with DS, and it encodes a key enzyme of sulfur-containing amino acid (SAA) metabolism, a pathway important for several brain physiological processes.Here, we have studied the neural consequences of CBS overexpression in a transgenic mouse line (60.4P102D1) expressing the human CBS gene under the control of its endogenous regulatory regions. These mice displayed a ∼2-fold increase in total CBS proteins in different brain areas and a ∼1.3-fold increase in CBS activity in the cerebellum and the hippocampus. No major disturbance of SAA metabolism was observed, and the transgenic mice showed normal behavior in the rotarod and passive avoidance tests. However, we found that hippocampal synaptic plasticity is facilitated in the 60.4P102D1 line.We demonstrate that CBS overexpression has functional consequences on hippocampal neuronal networks. These results shed new light on the function of the CBS gene, and raise the interesting possibility that CBS overexpression might have an advantageous effect on some cognitive functions in DS

Genetic diversity, linkage disequilibrium and power of a large grapevine (Vitis vinifera L) diversity panel newly designed for association studies

UMR-AGAP Equipe DAVV (Diversité, adaptation et amélioration de la vigne) ; équipe ID (Intégration de Données)International audienceAbstractBackgroundAs for many crops, new high-quality grapevine varieties requiring less pesticide and adapted to climate change are needed. In perennial species, breeding is a long process which can be speeded up by gaining knowledge about quantitative trait loci linked to agronomic traits variation. However, due to the long juvenile period of these species, establishing numerous highly recombinant populations for high resolution mapping is both costly and time-consuming. Genome wide association studies in germplasm panels is an alternative method of choice, since it allows identifying the main quantitative trait loci with high resolution by exploiting past recombination events between cultivars. Such studies require adequate panel design to represent most of the available genetic and phenotypic diversity. Assessing linkage disequilibrium extent and panel power is also needed to determine the marker density required for association studies.ResultsStarting from the largest grapevine collection worldwide maintained in Vassal (France), we designed a diversity panel of 279 cultivars with limited relatedness, reflecting the low structuration in three genetic pools resulting from different uses (table vs wine) and geographical origin (East vs West), and including the major founders of modern cultivars. With 20 simple sequence repeat markers and five quantitative traits, we showed that our panel adequately captured most of the genetic and phenotypic diversity existing within the entire Vassal collection. To assess linkage disequilibrium extent and panel power, we genotyped single nucleotide polymorphisms: 372 over four genomic regions and 129 distributed over the whole genome. Linkage disequilibrium, measured by correlation corrected for kinship, reached 0.2 for a physical distance between 9 and 458 Kb depending on genetic pool and genomic region, with varying size of linkage disequilibrium blocks. This panel achieved reasonable power to detect associations between traits with high broad-sense heritability (> 0.7) and causal loci with intermediate allelic frequency and strong effect (explaining > 10 % of total variance).ConclusionsOur association panel constitutes a new, highly valuable resource for genetic association studies in grapevine, and deserves dissemination to diverse field and greenhouse trials to gain more insight into the genetic control of many agronomic traits and their interaction with the environment

NLRP6 negatively regulates type 2 immune responses in mice

International audienceBackground: Inflammasomes are large protein complexes that assemble in the cytosol in response to danger such as tissue damage or infection. Following activation, inflammasomes trigger cell death and the release of biologically active forms of pro-inflammatory cytokines interleukin (IL)-1β and IL-18. NOD-like receptor family pyrin domain containing 6 (NLRP6) inflammasome is required for IL-18 secretion by intestinal epithelial cells, macrophages, and T cells, contributing to homeostasis and self-defense against pathogenic microbes. However, the involvement of NLRP6 in type 2 lung inflammation remains elusive.Methods: Wild-type (WT) and Nlrp6−/− mice were used. Birch pollen extract (BPE)-induced allergic lung inflammation, eosinophil recruitment, Th2-related cytokine and chemokine production, airway hyperresponsiveness, and lung histopathology, Th2 cell differentiation, GATA3, and Th2 cytokines expression, were determined. Nippostrongylus brasiliensis (Nb) infection, worm count in intestine, type 2 innate lymphoid cell (ILC2), and Th2 cells in lungs were evaluated.Results: We demonstrate in Nlrp6−/− mice that a mixed Th2/Th17 immune responses prevailed following birch pollen challenge with increased eosinophils, ILC2, Th2, and Th17 cell induction and reduced IL-18 production. Nippostrongylus brasiliensis infected Nlrp6−/− mice featured enhanced early expulsion of the parasite due to enhanced type 2 immune responses compared to WT hosts. In vitro, NLRP6 repressed Th2 polarization, as shown by increased Th2 cytokines and higher expression of the transcription factor GATA3 in the absence of NLRP6. Exogenous IL-18 administration partially reduced the enhanced airways inflammation in Nlrp6−/− mice.Conclusions: In summary, our data identify NLRP6 as a negative regulator of type 2 immune responses

Protein kinase C θ controls type 2 innate lymphoid cell and TH2 responses to house dust mite allergen.

IF 13.081International audienceBACKGROUND:Protein kinase C (PKC) θ, a serine/threonine kinase, is involved in TH2 cell activation and proliferation. Type 2 innate lymphoid cells (ILC2s) resemble TH2 cells and produce the TH2 cytokines IL-5 and IL-13 but lack antigen-specific receptors. The mechanism by which PKC-θ drives innate immune cells to instruct TH2 responses in patients with allergic lung inflammation remains unknown.OBJECTIVES:We hypothesized that PKC-θ contributes to ILC2 activation and might be necessary for ILC2s to instruct the TH2 response.METHODS:PRKCQ gene expression was assessed in innate lymphoid cell subsets purified from human PBMCs and mouse lung ILC2s. ILC2 activation and eosinophil recruitment, TH2-related cytokine and chemokine production, lung histopathology, interferon regulatory factor 4 (IRF4) mRNA expression, and nuclear factor of activated T cells (NFAT1) protein expression were determined. Adoptive transfer of ILC2s from wild-type mice was performed in wild-type and PKC-θ-deficient (PKC-θ-/-) mice.RESULTS:Here we report that PKC-θ is expressed in both human and mouse ILC2s. Mice lacking PKC-θ had reduced ILC2 numbers, TH2 cell numbers and activation, airway hyperresponsiveness, and expression of the transcription factors IRF4 and NFAT1. Importantly, adoptive transfer of ILC2s restored eosinophil influx and IL-4, IL-5 and IL-13 production in lung tissue, as well as TH2 cell activation. The pharmacologic PKC-θ inhibitor (Compound 20) administered during allergen challenge reduced ILC2 numbers and activation, as well as airway inflammation and IRF4 and NFAT1 expression.CONCLUSIONS:Therefore our findings identify PKC-θ as a critical factor for ILC2 activation that contributes to TH2 cell differentiation, which is associated with IRF4 and NFAT1 expression in allergic lung inflammation

Theta-burst-induced LTP in Tg<i>hCBS</i>60.4 mice.

<p>Comparison of averaged LTP expressed as percent change in the slope of fEPSP <i>vs</i> time, induced by theta-burst stimulation (TBS, arrow) of glutamate afferents and recorded in slices from Tg<i>hCBS</i>60.4 (12 slices/9 animals) and control (10 slices/7 animals) mice. In the insert, representative traces of fEPSPs, recorded before and 60 min after TBS, are superimposed.</p

Involvement of CBS in sulfur-containing amino acid metabolism.

<p>From Stipanuk <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029056#pone.0029056-Stipanuk1" target="_blank">[54]</a>.</p

Basal synaptic transmission in Tg<i>hCBS</i>60.4 mice.

<p>(A) Superimposed sample traces of evoked AMPA-R-mediated fEPSPs induced in a control (left) and a Tg<i>hCBS</i>60.4 mouse (right) by increased intensities of electrical stimulation of glutamate afferents (arrow). Traces are averages of 3 consecutive responses. (B) Comparison of synaptic efficacy as determined by the fEPSP/PFV ratio calculated at a stimulus intensity from 500 to 900 µA in Tg<i>hCBS</i>60.4 (23 slices/5 animals) and control (22 slices/5 animals) mice.</p

Concentrations (nmoles/mg protein) of 30 amino acids and metabolite compounds in cerebellum extracts of Tg<i>hCBS</i>60.4 and control mice.

<p>Data correspond to mean ± S.E.M. BD: below detection. ND: not determined.</p

Behavioral assessments in Tg<i>hCBS</i>60.4 mice.

<p>The analysis was conducted on 8 Tg<i>hCBS</i>60.4 and 8 control mice. Data represent mean+S.E.M. for each group. (A) Rotarod test: latencies of first fall, second fall and third fall recorded in the 10 min accelerating periods of the two rotarod sessions (S1, S2) are presented. (B) Passive avoidance test: latency to enter into the dark compartment in acquisition session and in retention session was recorded.</p

CBS expression and activity in brain regions of Tg<i>hCBS</i>60.4 mice.

<p>(A, top) Immunoblots for CBS and β-actin in cerebellum (left panel), hippocampus (middle panel) and forebrain (right panel). Total CBS proteins (human CBS plus mouse CBS) were detected using the anti-hCBS polyclonal antibody that recognises both the human and mouse proteins. (A, bottom) Quantification of CBS proteins normalized to β-actin and relative to control mice in the three brain regions (<i>n</i> = 3 Tg<i>hCBS</i>60.4 and <i>n</i> = 3 control) (B) CBS activity in cerebellum (left panel), hippocampus (middle panel) and forebrain (right panel). Units are nanomoles of cystathionine formed per milligram of protein extract per hour. For cerebellum and forebrain, data represent mean ± S.E.M. of activity measurements in each group (<i>n</i> = 9 Tg<i>hCBS</i>60.4 and <i>n</i> = 7 control; <i>n</i> = 8 Tg<i>hCBS</i>60.4 and <i>n</i> = 8 control, respectively). For hippocampus, data represent mean ± S.E.M. of three experimental assays performed on same pooled hippocampi (<i>n</i> = 7 Tg<i>hCBS</i>60.4 and <i>n</i> = 8 control). *for <i>p</i><0.05. ***for <i>p</i><0.001.</p