Depressive disorder moderates the effect of the FTO gene on body mass index.

There is evidence that obesity-related disorders are increased among people with depression. Variation in the FTO (fat mass and obesity associated) gene has been shown to contribute to common forms of human obesity. This study aimed to investigate the genetic influence of polymorphisms in FTO in relation to body mass index (BMI) in two independent samples of major depressive disorder (MDD) cases and controls. We analysed 88 polymorphisms in the FTO gene in a clinically ascertained sample of 2442 MDD cases and 809 controls (Radiant Study). In all, 8 of the top 10 single-nucleotide polymorphisms (SNPs) showing the strongest associations with BMI were followed-up in a population-based cohort (PsyCoLaus Study) consisting of 1292 depression cases and 1690 controls. Linear regression analyses of the FTO variants and BMI yielded 10 SNPs significantly associated with increased BMI in the depressive group but not the control group in the Radiant sample. The same pattern was found in the PsyCoLaus sample. We found a significant interaction between genotype and affected status in relation to BMI for seven SNPs in Radiant (P<0.0057), with PsyCoLaus giving supportive evidence for five SNPs (P-values between 0.03 and 0.06), which increased in significance when the data were combined in a meta-analysis. This is the first study investigating FTO and BMI within the context of MDD, and the results indicate that having a history of depression moderates the effect of FTO on BMI. This finding suggests that FTO is involved in the mechanism underlying the association between mood disorders and obesity.

VEGF is a mediator of retinal pigment epithelium permeability leading to photoreceptor cell death in light-induced retinal degeneration : gene therapy strategy to prevent AMD-disorders

ABSTRACTIn normal tissues, a balance between pro- and anti-angiogenic factors tightly controls angiogenesis. Alterations of this balance may have pathological consequences. For instance, concerning the retina, the vascular endothelial growth factor (VEGF) is a potent pro-angiogenic factor, and has been identified has a key player during ocular neovascularization implicated in a variety of retinal diseases. In the exudative form (wet-form) of age-related macular degeneration (AMD), neovascularizations occurring from the choroidal vessels are responsible for a quick and dramatic loss of visual acuity. In diabetic retinopathy and retinopathy of prematurity, sprouting from the retinal vessels leads to vision loss. Furthermore, the aging of the population, the increased- prevalence of diabetes and the better survival rate of premature infants will lead to an increasing rate of these conditions. In this way, anti-VEGF strategy represents an important therapeutic target to treat ocular neovascular disorders.In addition, the administration of Pigmented Epithelial growth factor, a neurotrophic and an anti- angiogenic factor, prevents photoreceptor cell death in a model of retinal degeneration induced by light. Previous results analyzing end point morphology reveal that the light damage (LD) model is used to mimic retinal degenerations arising from environmental insult, as well as aging and genetic disease such as advanced atrophic AMD. Moreover, light has been identified as a co-factor in a number of retinal diseases, speeding up the degeneration process. This protecting effect of PEDF in the LD retina raises the possibility of involvement of the balance between pro- and anti-angiogenic factors not only for angiogenesis, but also in cell survival and maintenance.The aim of the work presented here was to evaluate the importance of this balance in neurodegenerative processes. To this aim, a model of light-induced retinal degeneration was used and characterized, mainly focusing on factors simultaneously controlling neuron survival and angiogenesis, such as PEDF and VEGF.In most species, prolonged intense light exposure can lead to photoreceptor cell damage that can progress to cell death and vision loss. A protocol previously described to induce retinal degeneration in Balb/c mice was used. Retinas were characterized at different time points after light injury through several methods at the functional and molecular levels. Data obtained confirmed that toxic level of light induce PR cell death. Variations were observed in VEGF pathway players in both the neural retina and the eye-cup containing the retinal pigment epithelium (RPE), suggesting a flux of VEGF from the RPE towards the neuroretina. Concomitantly, the integrity of the outer blood-retinal-barrier (BRB) was altered, leading to extravascular albumin leakage from the choroid throughout the photoreceptor layer.To evaluate the importance of VEGF during light-induced retinal degeneration process, a lentiviral vector encoding the cDNA of a single chain antibody directed against all VEGF-A isoforms was developed (LV-V65). The bioactivity of this vector to block VEGF was validated in a mouse model of laser-induced choroidal neovascularization mediated by VEGF upregulation. The vector was then used in the LD model. The administration of the LV-V65 contributed to the maintenance of functional photoreceptors, which was assessed by ERG recording, visual acuity measurement and histological analyses. At the RPE level, the BRB integrity was preserved as shown by the absence of albumin leakage and the maintenance of RPE cell cohesion.These results taken together indicate that the VEGF is a mediator of light induced PR degeneration process and confirm the crucial role of the balance between pro- and anti-angiogenic factors in the PR cell survival. This work also highlights the prime importance of BRB integrity and functional coupling between RPE and PR cells to maintain the PR survival. VEGF dysregulation was already shown to be involved in wet AMD forms and our study suggests that VEGF dysregulation may also occur at early stages of AMD and could thus be a potential therapeutic target for several RPE related diseases.RESUMEDans les différents tissues de l'organisme, l'angiogenèse est strictement contrôlée par une balance entre les facteurs pro- et anti-angiogéniques. Des modifications survenant dans cette balance peuvent engendrer des conséquences pathologiques. Par exemple, concernant la rétine, le facteur de croissance de l'endothélium vasculaire (VEGF) est un facteur pro-angiogénique important. Ce facteur a été identifié comme un acteur majeur dans les néovascularisations oculaires et les processus pathologiques angiogéniques survenant dans l'oeil et responsables d'une grande variété de maladies rétiniennes. Dans la forme humide de la dégénérescence maculaire liée à l'âge (DMLA), la néovascularisation choroïdienne est responsable de la perte rapide et brutale de l'acuité visuelle chez les patients affectés. Dans la rétinopathie diabétique et celle lié à la prématurité, l'émergence de néovaisseaux rétiniens est la cause de la perte de la vision. Les néovascularisations oculaires représentent la principale cause de cécité dans les pays développés. De plus, l'âge croissant de la population, la progression de la prévalence du diabète et la meilleure survie des enfants prématurés mèneront sans doute à l'augmentation de ces pathologies dans les années futures. Dans ces conditions, les thérapies anti- angiogéniques visant à inhiber le VEGF représentent une importante cible thérapeutique pour le traitement de ces pathologies.Plusieurs facteurs anti-angiogéniques ont été identifiés. Parmi eux, le facteur de l'épithélium pigmentaire (PEDF) est à la fois un facteur neuro-trophique et anti-angiogénique, et l'administration de ce facteur au niveau de la rétine dans un modèle de dégénérescence rétinienne induite par la lumière protège les photorécepteurs de la mort cellulaire. Des études antérieures basées sur l'analyse morphologique ont révélé que les modifications survenant lors de la dégénération induite suite à l'exposition à des doses toxiques de lumière représente un remarquable modèle pour l'étude des dégénérations rétiniennes suite à des lésions environnementales, à l'âge ou encore aux maladies génétiques telle que la forme atrophique avancée de la DMLA. De plus, la lumière a été identifiée comme un co-facteur impliqué dans un grand nombre de maladies rétiniennes, accélérant le processus de dégénération. L'effet protecteur du PEDF dans les rétines lésées suite à l'exposition de des doses toxiques de lumière suscite la possibilité que la balance entre les facteurs pro- et anti-angiogéniques soit impliquée non seulement dans les processus angiogéniques, mais également dans le maintient et la survie des cellules.Le but de ce projet consiste donc à évaluer l'implication de cette balance lors des processus neurodégénératifs. Pour cela, un modèle de dégénération induite par la lumière à été utilisé et caractérisé, avec un intérêt particulier pour les facteurs comme le PEDF et le VEGF contrôlant simultanément la survie des neurones et l'angiogenèse.Dans la plupart des espèces, l'exposition prolongée à une lumière intense peut provoquer des dommages au niveau des cellules photoréceptrices de l'oeil, qui peut mener à leur mort, et par conséquent à la perte de la vision. Un protocole préalablement décrit a été utilisé pour induire la dégénération rétinienne dans les souris albinos Balb/c. Les rétines ont été analysées à différents moments après la lésion par différentes techniques, aussi bien au niveau moléculaire que fonctionnel. Les résultats obtenus ont confirmé que des doses toxiques de lumière induisent la mort des photorécepteurs, mais altèrent également la voie de signalisation du VEGF, aussi bien dans la neuro-rétine que dans le reste de l'oeil, contenant l'épithélium pigmentaire (EP), et suggérant un flux de VEGF provenant de ΙΈΡ en direction de la neuro-rétine. Simultanément, il se produit une altération de l'intégrité de la barrière hémato-rétinienne externe, menant à la fuite de protéine telle que l'albumine, provenant de la choroïde et retrouvée dans les compartiments extravasculaires de la rétine, telle que dans la couche des photorécepteurs.Pour déterminer l'importance et le rôle du VEGF, un vecteur lentiviral codant pour un anticorps neutralisant dirigée contre tous les isoformes du VEGF a été développé (LV-V65). La bio-activité de ce vecteur a été testé et validée dans un modèle de laser, connu pour induire des néovascularisations choroïdiennes chez la souris suite à l'augmentation du VEGF. Ce vecteur a ensuite été utilisé dans le modèle de dégénération induite par la lumière. Les résultats des électrorétinogrammes, les mesures de l'acuité visuelle et les analyses histologiques ont montré que l'injection du LV-V65 contribue à la maintenance de photorécepteurs fonctionnels. Au niveau de l'EP, l'absence d'albumine et la maintenance des jonctions cellulaires des cellules de l'EP ont démontré que l'intégrité de la barrière hémato-rétinienne externe est préservée suite au traitement.Par conséquent, tous les résultats obtenus indiquent que le VEGF est un médiateur important impliquée dans le processus de dégénération induit par la lumière et confirme le rôle cruciale de la balance entre les facteurs pro- et anti-angiogéniques dans la survie des photorécepteurs. Cette étude révèle également l'importance de l'intégrité de la barrière hémato-rétinienne et l'importance du lien fonctionnel et structurel entre l'EP et les photorécepteurs, essentiel pour la survie de ces derniers. Par ailleurs, Cette étude suggère que des dérèglements au niveau de l'équilibre du VEGF ne sont pas seulement impliqués dans la forme humide de la DMLA, comme déjà démontré dans des études antérieures, mais pourraient également contribuer et survenir dans des formes précoces de la DMLA, et par conséquent le VEGF représente une cible thérapeutique potentielle pour les maladies associées à des anomalies au niveau de l'EP.

Vitellogenin B2 gene in Xenopus laevis: isolation, in vitro transcription and relation to other vitellogenin genes.

The isolation of the four Xenopus laevis vitellogenin genes has been completed by the purification from a DNA library of the B2 gene together with its flanking sequences. The overlapping DNA fragments analyzed cover 34 kilobases. The B2 gene which has a length of 17.5 kilobases was characterized by heteroduplex and R-loop mapping in the electron microscope and by in vitro transcription in a HeLa whole-cell extract. Its structural organization is compared with that of the closely related B1 gene. The mRNA-coding sequence of about 6 kilobases is interrupted 34 times in the B1 gene and 33 times in the B2 gene. Sequence homology between the two genes was not only found in exons. In addition, 54% of the intron sequences as well as 63% and 48.5% respectively of the 5' and 3' flanking sequences, show enough homology to form stable duplexes. These findings are compared with earlier results obtained with the two other closely related members of the vitellogenin gene family, the A1 and the A2 genes.

Simultaneous coexpression of memory-related and effector-related genes by individual human CD8 T cells depends on antigen specificity and differentiation.

Phenotypic and functional cell properties are usually analyzed at the level of defined cell populations but not single cells. Yet, large differences between individual cells may have important functional consequences. It is likely that T-cell-mediated immunity depends on the polyfunctionality of individual T cells, rather than the sum of functions of responding T-cell subpopulations. We performed highly sensitive single-cell gene expression profiling, allowing the direct ex vivo characterization of individual virus-specific and tumor-specific T cells from healthy donors and melanoma patients. We have previously shown that vaccination with the natural tumor peptide Melan-A-induced T cells with superior effector functions as compared with vaccination with the analog peptide optimized for enhanced HLA-A*0201 binding. Here we found that natural peptide vaccination induced tumor-reactive CD8 T cells with frequent coexpression of both memory/homing-associated genes (CD27, IL7R, EOMES, CXCR3, and CCR5) and effector-related genes (IFNG, KLRD1, PRF1, and GZMB), comparable with protective Epstein-Barr virus-specific and cytomegalovirus-specific T cells. In contrast, memory/homing-associated and effector-associated genes were less frequently coexpressed after vaccination with the analog peptide. Remarkably, these findings reveal a previously unknown level of gene expression diversity among vaccine-specific and virus-specific T cells with the simultaneous coexpression of multiple memory/homing-related and effector-related genes by the same cell. Such broad functional gene expression signatures within antigen-specific T cells may be critical for mounting efficient responses to pathogens or tumors. In summary, direct ex vivo high-resolution molecular characterization of individual T cells provides key insights into the processes shaping the functional properties of tumor-specific and virus-specific T cells.

Vitellogenesis and the vitellogenin gene family.

Vitellogenin is synthesized under estrogen control in the liver, extensively modified, transported to the ovary, and there processed to the yolk proteins lipovitellin and phosvitin. In the frog Xenopus laevis there are at least four distinct but related vitellogenin genes. The two genes A1 and A2 have a 95 percent sequence homology in their messenger RNA coding regions, and contain 33 introns that interrupt the coding region (exons) at homologous positions. Sequences and lengths of analogous introns differ, and many introns contain repetitive DNA elements. The introns in these two genes that have apparently arisen by duplication have diverged extensively by events that include deletions, insertions, and probably duplications. Rapid evolutionary change involving rearrangements and the presence of repeated DNA suggests that the bulk of the sequences within introns may not have any specific function.

The distribution of the dinucleotide CpG and cytosine methylation in the vitellogenin gene family.

Sequence data from regions of five vertebrate vitellogenin genes were used to examine the frequency, distribution, and mutability of the dinucleotide CpG, the preferred modification site for eukaryotic DNA methyltransferases. The observed level of the CpG dinucleotide in all five genes was markedly lower than that expected from the known mononucleotide frequencies. CpG suppression was greater in introns than in exons. CpG-containing codons were found to be avoided in the vitellogenin genes, but not completely despite the redundancy of the genetic code. Frequency and distribution patterns of this dinucleotide varied dramatically among these otherwise closely related genes. Dense clusters of CpG dinucleotides tended to appear in regions of either functional or structural interest (e.g., in the transposon-like Vi-element of Xenopus) and these clusters contained 5-methylcytosine (5 mC). 5 mC is known to undergo deamination to form thymidine, but the extent to which this transition occurs in the heavily methylated genomes of vertebrates and its contribution to CpG suppression are still unclear. Sequence comparison of the methylated vitellogenin gene regions identified C----T and G----A substitutions that were found to occur at relatively high frequencies. The predicted products of CpG deamination, TpG and CpA, were elevated. These findings are consistent with the view that CpG distribution and methylation are interdependent and that deamination of 5 mC plays an important role in promoting evolutionary change at the nucleotide sequence level.

Lack of hypotriglyceridemic effect of gemfibrozil as a consequence of age-related changes in rat liver PPARalpha.

We have investigated if changes in hepatic lipid metabolism produced by old age are related to changes in liver peroxisome proliferator-activated receptor alpha (PPARalpha). Our results indicate that 18-month-old rats showed a marked decrease in the expression and activity of liver PPARalpha, as shown by significant reductions in PPARalpha mRNA, protein and binding activity, resulting in a reduction in the relative mRNA levels of PPARalpha target genes, such as liver-carnitine-palmitoyl transferase-I (CPT-I) and mitochondrial medium-chain acyl-CoA dehydrogenase (MCAD). Further, in accordance with a liver PPARalpha deficiency in old rats, treatment of old animals with a therapeutic dose of gemfibrozil (GFB) (3mg/kg per day, 21 days) was ineffective in reducing plasma triglyceride concentrations (TG), despite attaining a 50% reduction in TG when GFB was administered to young animals at the same dose and length of treatment. We hypothesize that the decrease in hepatic PPARalpha can be related to a state of leptin resistance present in old animals.

Genesis and evolution of the Evx and Mox genes and the extended Hox and ParaHox gene clusters

Background: Hox and ParaHox gene clusters are thought to have resulted from the duplication of a ProtoHox gene cluster early in metazoan evolution. However, the origin and evolution of the other genes belonging to the extended Hox group of homeobox-containing genes, that is, Mox and Evx, remains obscure. We constructed phylogenetic trees with mouse, amphioxus and Drosophila extended Hox and other related Antennapedia-type homeobox gene sequences and analyzed the linkage data available for such genes.Results: We claim that neither Mox nor Evx is a Hox or ParaHox gene. We propose a scenariothat reconciles phylogeny with linkage data, in which an Evx/Mox ancestor gene linked to aProtoHox cluster was involved in a segmental tandem duplication event that generated an arrayof all Hox-like genes, referred to as the `coupled¿ cluster. A chromosomal breakage within thiscluster explains the current composition of the extended Hox cluster (with Evx, Hox and Moxgenes) and the ParaHox cluster.Conclusions: Most studies dealing with the origin and evolution of Hox and ParaHox clustershave not included the Hox-related genes Mox and Evx. Our phylogenetic analyses and theavailable linkage data in mammalian genomes support an evolutionary scenario in which anancestor of Evx and Mox was linked to the ProtoHox cluster, and that a tandem duplication of alarge genomic region early in metazoan evolution generated the Hox and ParaHox clusters, plusthe cluster-neighbors Evx and Mox. The large `coupled¿ Hox-like cluster EvxHox/MoxParaHox wassubsequently broken, thus grouping the Mox and Evx genes to the Hox clusters, and isolating theParaHox cluster.

Thermal evolution of gene expression profiles in Drosophila subobscura

Background: Despite its pervasiveness, the genetic basis of adaptation resulting in variation directly or indirectly related to temperature (climatic) gradients is poorly understood. By using 3-fold replicated laboratory thermal stocks covering much of the physiologically tolerable temperature range for the temperate (i.e., cold tolerant) species Drosophila subobscura we have assessed whole-genome transcriptional responses after three years of thermal adaptation, when the populations had already diverged for inversion frequencies, pre-adult life history components, and morphological traits. Total mRNA from each population was compared to a reference pool mRNA in a standard, highly replicated two-colour competitive hybridization experiment using cDNA microarrays.Results: A total of 306 (6.6%) cDNA clones were identified as 'differentially expressed' (following a false discovery rate correction) after contrasting the two furthest apart thermal selection regimes (i.e., 13°C vs . 22°C), also including four previously reported candidate genes for thermotolerance in Drosophila (Hsp26, Hsp68, Fst, and Treh). On the other hand, correlated patterns of gene expression were similar in cold- and warm-adapted populations. Analysis of functional categories defined by the Gene Ontology project point to an overrepresentation of genes involved in carbohydrate metabolism, nucleic acids metabolism and regulation of transcription among other categories. Although the location of differently expressed genes was approximately at random with respect to chromosomes, a physical mapping of 88 probes to the polytene chromosomes of D. subobscura has shown that a larger than expected number mapped inside inverted chromosomal segments.Conclusion: Our data suggest that a sizeable number of genes appear to be involved in thermal adaptation in Drosophila, with a substantial fraction implicated in metabolism. This apparently illustrates the formidable challenge to understanding the adaptive evolution of complex trait variation. Furthermore, some clustering of genes within inverted chromosomal sections was detected. Disentangling the effects of inversions will be obviously required in any future approach if we want to identify the relevant candidate genes.

Is the Gibraltar strait a barrier to gene flow for the bat Myotis myotis (Chiroptera: Vespertilionidae)?

Because of their role in limiting gene flow, geographical barriers like mountains or seas often coincide with intraspecific genetic discontinuities. Although the Strait of Gibraltar represents such a potential barrier for both plants and animals, few studies have been conducted on its impact on gene flow. Here we test this effect on a bat species (Myotis myotis) which is apparently distributed on both sides of the strait. Six colonies of 20 Myotis myotis each were sampled in southern Spain and northern Morocco along a linear transect of 1350 km. Results based on six nuclear microsatellite loci reveal no significant population structure within regions, but a complete isolation between bats sampled on each side of the strait. Variability at 600 bp of a mitochondrial gene (cytochrome b) confirms the existence of two genetically distinct and perfectly segregating clades, which diverged several million years ago. Despite the narrowness of the Gibraltar Strait (14 km), these molecular data suggest that neither males, nor females from either region have ever reproduced on the opposite side of the strait. Comparisons of molecular divergence with bats from a closely related species (M. blythii) suggest that the North African clade is possibly a distinct taxon warranting full species rank. We provisionally refer to it as Myotis cf punicus Felten 1977, but a definitive systematic understanding of the whole Mouse-eared bat species complex awaits further genetic sampling, especially in the Eastern Mediterranean areas.

Genetic variants of the FADS gene cluster and ELOVL gene family, colostrums LC-PUFA levels, breastfeeding, and child cognition

Introduction: Breastfeeding effects on cognition are attributed to long-chain polyunsaturated fatty acids (LC-PUFAs), but controversy persists. Genetic variation in fatty acid desaturase (FADS) and elongase (ELOVL) enzymes has been overlooked when studying the effects of LC-PUFAs supply on cognition. We aimed to: 1) to determine whether maternal genetic variants in the FADS cluster and ELOVL genes contribute to differences in LC-PUFA levels in colostrum; 2) to analyze whether these maternal variants are related to child cognition; and 3) to assess whether children's variants modify breastfeeding effects on cognition. Methods: Data come from two population-based birth cohorts (n = 400 mother-child pairs from INMA-Sabadell; and n = 340 children from INMA-Menorca). LC-PUFAs were measured in 270 colostrum samples from INMA-Sabadell. Tag SNPs were genotyped both in mothers and children (13 in the FADS cluster, 6 in ELOVL2, and 7 in ELOVL5). Child cognition was assessed at 14 mo and 4 y using the Bayley Scales of Infant Development and the McCarthy Scales of Children"s Abilities, respectively. Results: Children of mothers carrying genetic variants associated with lower FADS1 activity (regulating AA and EPA synthesis), higher FADS2 activity (regulating DHA synthesis), and with higher EPA/AA and DHA/AA ratios in colostrum showed a significant advantage in cognition at 14 mo (3.5 to 5.3 points). Not being breastfed conferred an 8- to 9-point disadvantage in cognition among children GG homozygote for rs174468 (low FADS1 activity) but not among those with the A allele. Moreover, not being breastfed resulted in a disadvantage in cognition (5 to 8 points) among children CC homozygote for rs2397142 (low ELOVL5 activity), but not among those carrying the G allele. Conclusion: Genetically determined maternal supplies of LC-PUFAs during pregnancy and lactation appear to be crucial for child cognition. Breastfeeding effects on cognition are modified by child genetic variation in fatty acid desaturase and elongase enzymes.

Stem cell-related "self-renewal" signature and high epidermal growth factor receptor expression associated with resistance to concomitant chemoradiotherapy in glioblastoma.

PURPOSE: Glioblastomas are notorious for resistance to therapy, which has been attributed to DNA-repair proficiency, a multitude of deregulated molecular pathways, and, more recently, to the particular biologic behavior of tumor stem-like cells. Here, we aimed to identify molecular profiles specific for treatment resistance to the current standard of care of concomitant chemoradiotherapy with the alkylating agent temozolomide. PATIENTS AND METHODS: Gene expression profiles of 80 glioblastomas were interrogated for associations with resistance to therapy. Patients were treated within clinical trials testing the addition of concomitant and adjuvant temozolomide to radiotherapy. RESULTS: An expression signature dominated by HOX genes, which comprises Prominin-1 (CD133), emerged as a predictor for poor survival in patients treated with concomitant chemoradiotherapy (n = 42; hazard ratio = 2.69; 95% CI, 1.38 to 5.26; P = .004). This association could be validated in an independent data set. Provocatively, the HOX cluster was reminiscent of a "self-renewal" signature (P = .008; Gene Set Enrichment Analysis) recently characterized in a mouse leukemia model. The HOX signature and EGFR expression were independent prognostic factors in multivariate analysis, adjusted for the O-6-methylguanine-DNA methyltransferase (MGMT) methylation status, a known predictive factor for benefit from temozolomide, and age. Better outcome was associated with gene clusters characterizing features of tumor-host interaction including tumor vascularization and cell adhesion, and innate immune response. CONCLUSION: This study provides first clinical evidence for the implication of a "glioma stem cell" or "self-renewal" phenotype in treatment resistance of glioblastoma. Biologic mechanisms identified here to be relevant for resistance will guide future targeted therapies and respective marker development for individualized treatment and patient selection.

Regional and cellular gene expression changes in human Huntington's disease brain.

Huntington's disease (HD) pathology is well understood at a histological level but a comprehensive molecular analysis of the effect of the disease in the human brain has not previously been available. To elucidate the molecular phenotype of HD on a genome-wide scale, we compared mRNA profiles from 44 human HD brains with those from 36 unaffected controls using microarray analysis. Four brain regions were analyzed: caudate nucleus, cerebellum, prefrontal association cortex [Brodmann's area 9 (BA9)] and motor cortex [Brodmann's area 4 (BA4)]. The greatest number and magnitude of differentially expressed mRNAs were detected in the caudate nucleus, followed by motor cortex, then cerebellum. Thus, the molecular phenotype of HD generally parallels established neuropathology. Surprisingly, no mRNA changes were detected in prefrontal association cortex, thereby revealing subtleties of pathology not previously disclosed by histological methods. To establish that the observed changes were not simply the result of cell loss, we examined mRNA levels in laser-capture microdissected neurons from Grade 1 HD caudate compared to control. These analyses confirmed changes in expression seen in tissue homogenates; we thus conclude that mRNA changes are not attributable to cell loss alone. These data from bona fide HD brains comprise an important reference for hypotheses related to HD and other neurodegenerative diseases.

Role of β-catenin and γ-catenin in haematopoiesis : Generation of floxed Delta like 4 gene targeted mice

Résumé La voie de signalisation de Wnt est extrêmement conservée au cours de l'évolution. Les protéines Wnt sont des molécules sécrétées qui se lient à la famille de récepteurs Frizzled. Cette interaction mène à la stabilisation de la protéine β-caténine, qui va s'accumuler dans le cytoplasme puis migrer dans le noyau où elle peut s'hétérodimériser avec les facteurs de transcription de la famille TCF/LEF. Il a été démontré que cette voie de signalisation joue un rôle important durant la lymphopoïèse et de récents résultats suggèrent un rôle clé de cette voie dans le renouvellement des Cellules Souches Hématopoïétique (CSH). Des études se basant sur un système de surexpression de protéines montrent clairement que la voie Wnt peut influencer l'hématopoïèse. Cependant, le rôle de la protéine β-caténine dans le système hématopoïétique n'a jamais été testé directement. Ce projet de thèse se propose d'étudier la fonction de la protéine β-caténine par sa délétion inductible via le système Cre-loxP. De façon surprenante, nous avons pu démontrer que les progéniteurs de la moelle osseuse, déficients en β-caténine, ne montrent aucune altération dans leur capacité à s'auto-renouveler et/ou à reconstituer toutes les lignées hématopoïétiques (myéloïde, érythroïde et lymphoïde) dans les souris-chimères. De plus, le développement, la survie des thymocytes ainsi que la prolifération des cellules T périphériques induite par un antigène, sont indépendants de β-caténine. Ces résultats suggèrent soit que la protéine β-caténine ne joue pas un rôle primordial dans le système hématopoiétique, soit que son absence pourrait être compensée par une autre protéine. Un candidat privilégié susceptible de se substituer à β-caténine, serait plakoglobine, aussi connu sous le nom de γ-caténine. En effet, ces deux protéines partagent de multiples caractéristiques structurelles. Afin de démontrer que la protéine γ-caténine peut compenser l'absence de β-caténine, nous avons généré des souris dans lesquelles, le système hématopoïétique est déficient pour ces deux protéines. Cette déficience combinée de β- caténine et γ-caténine ne perturbe pas la capacité des Cellules Souche Hématopoïétique-Long Terme (CSH-LT) de se renouveler, par contre elle agit sur un progéniteur précoce déjà différencié de la moelle osseuse. Ces résultats mettent en évidence que la protéine γ-caténine est capable de compenser l'absence de protéine β-caténine dans le système hématopoïétique. Par conséquent, ce travail contribue à une meilleure connaissance de la cascade Wnt dans l'hématopoïèse. Summary The canonical Wnt signal transduction pathway is a developmentally highly conserved. Wnts are secreted molecules which bind to the family of Frizzled receptors in a complex with the low density lipoprotein receptor related protein (LRP-5/6). This initial activation step leads to the stabilization and accumulation of β-catenin, first in the cytoplasm and subsequently in the nucleus where it forms heterodimers with TCF/LEF transcription factor family members. Wnt signalling has been shown to be important during early lymphopoiesis and has more recently, been suggested to be a key player in self-renewal of haematopoietic stem cells (HSCs). Although mostly gain of function studies indicate that components of the Wnt signalling pathway can influence the haematopoietic system, the role of β-catenin has never been directly investigated. The aim of this thesis project is to investigate the putatively critical role of β-catenin in vivo using the Cre-loxP mediated conditional loss of function approach. Surprisingly, β-catenin deficient bone marrow (BM) progenitors arc not impaired in their ability to self-renew and/or to reconstitute all haematopoietic lineages (myeloid, erythroid and lymphoid) in both mixed and straight bone marrow chimeras. In addition, both thymocyte development and survival, and antigen-induced proliferation of peripheral T cells are β- catenin independent. Our results do not necessarily exclude the possibility of an important function for β-catenin mediated Wnt signalling in the haematopoietic system, it rather raises the question that β-catenin is compensated for by another protein. A prime candidate that may take over the function of β-catenin in its absence, is the close relative plakoglobin, also know as γ-catenin. This protein shares multiple structural features with β-catenin. In order to investigate whether γ-catenin can compensate for the loss of β-catenin we have generated mice in which the haematopoietic compartment is deficient for both proteins. Combined deficiency of β-catenin and γ-catenin does not perturb Long Term-Haematopoietic Stem Cells (LT-HSC) self renewal, but affects an already lineage committed progenitor population within the BM. Our results demonstrate that y-catenin can indeed compensate for the loss of β-catenin within the haematopoietie system.

Gene therapy regenerates protein expression in cone photoreceptors in Rpe65(R91W/R91W) mice.

Cone photoreceptors mediate visual acuity under daylight conditions, so loss of cone-mediated central vision of course dramatically affects the quality of life of patients suffering from retinal degeneration. Therefore, promoting cone survival has become the goal of many ocular therapies and defining the stage of degeneration that still allows cell rescue is of prime importance. Using the Rpe65(R91W/R91W) mouse, which carries a mutation in the Rpe65 gene leading to progressive photoreceptor degeneration in both patients and mice, we defined stages of retinal degeneration that still allow cone rescue. We evaluated the therapeutic window within which cones can be rescued, using a subretinal injection of a lentiviral vector driving expression of RPE65 in the Rpe65(R91W/R91W) mice. Surprisingly, when applied to adult mice (1 month) this treatment not only stalls or slows cone degeneration but, actually, induces cone-specific protein expression that was previously absent. Before the intervention only part of the cones (40% of the number found in wild-type animals) in the Rpe65(R91W/R91W) mice expressed cone transducin (GNAT2); this fraction increased to 64% after treatment. Correct S-opsin localization is also recovered in the transduced region. In consequence these results represent an extended therapeutic window compared to the Rpe65(-/-) mice, implying that patients suffering from missense mutations might also benefit from a prolonged therapeutic window. Moreover, cones are not only rescued during the course of the degeneration, but can actually recover their initial status, meaning that a proportion of altered cones in chromophore deficiency-related disease can be rehabilitated even though they are severely affected.