Altered brain-derived neurotrophic factor blood levels and gene variability are associated with anorexia and bulimia

Murine models and association studies in eating disorder (ED) patients have shown a role for the brain-derived neurotrophic factor (BDNF) in eating behavior. Some studies have shown association of BDNF -270C/T single-nucleotide polymorphism (SNP) with bulimia nervosa (BN), while BDNF Val66Met variant has been shown to be associated with both BN and anorexia nervosa (AN). To further test the role of this neurotrophin in humans, we screened 36 SNPs in the BDNF gene and tested for their association with ED and plasma BDNF levels as a quantitative trait. We performed a family-based association study in 106 ED nuclear families and analyzed BDNF blood levels in 110 ED patients and in 50 sib pairs discordant for ED. The rs7124442T/rs11030102C/rs11030119G haplotype was found associated with high BDNF levels (mean BDNF TCG haplotype carriers = 43.6 ng/ml vs. mean others 23.0 ng/ml, P = 0.016) and BN (Z = 2.64; P recessive = 0.008), and the rs7934165A/270T haplotype was associated with AN (Z =-2.64; P additive = 0.008). The comparison of BDNF levels in 50 ED discordant sib pairs showed elevated plasma BDNF levels for the ED group (mean controls = 41.0 vs. mean ED = 52.7; P = 0.004). Our data strongly suggest that altered BDNF levels modulated by BDNF gene variability are associated with the susceptibility to ED, providing physiological evidence that BDNF plays a role in the development of AN and BN, and strongly arguing for its involvement in eating behavior and body weight regulation.

Impaired fast-spiking, suppressed cortical inhibition and increased susceptibility to seizures in mice lacking Kv3.2 K+ channel proteins

Voltage-gated K+ channels of the Kv3 subfamily have unusual electrophysiological properties, including activation at very depolarized voltages (positive to −10 mV) and very fast deactivation rates, suggesting special roles in neuronal excitability. In the brain, Kv3 channels are prominently expressed in select neuronal populations, which include fast-spiking (FS) GABAergic interneurons of the neocortex, hippocampus, and caudate, as well as other high-frequency firing neurons. Although evidence points to a key role in high-frequency firing, a definitive understanding of the function of these channels has been hampered by a lack of selective pharmacological tools. We therefore generated mouse lines in which one of the Kv3 genes, Kv3.2, was disrupted by gene-targeting methods. Whole-cell electrophysiological recording showed that the ability to fire spikes at high frequencies was impaired in immunocytochemically identified FS interneurons of deep cortical layers (5-6) in which Kv3.2 proteins are normally prominent. No such impairment was found for FS neurons of superficial layers (2-4) in which Kv3.2 proteins are normally only weakly expressed. These data directly support the hypothesis that Kv3 channels are necessary for high-frequency firing. Moreover, we found that Kv3.2 −/− mice showed specific alterations in their cortical EEG patterns and an increased susceptibility to epileptic seizures consistent with an impairment of cortical inhibitory mechanisms. This implies that, rather than producing hyperexcitability of the inhibitory interneurons, Kv3.2 channel elimination suppresses their activity. These data suggest that normal cortical operations depend on the ability of inhibitory interneurons to generate high-frequency firing.

Comparative gene finding in chicken indicates that we are closing in on the set of multi-exonic widely-expressed human genes

The recent availability of the chicken genome sequence poses the question of whether there are human protein-coding genes conserved in chicken that are currently not included in the human gene catalog. Here, we show, using comparative gene finding followed by experimental verification of exon pairs by RT–PCR, that the addition to the multi-exonic subset of this catalog could be as little as 0.2%, suggesting that we may be closing in on the human gene set. Our protocol, however, has two shortcomings: (i) the bioinformatic screening of the predicted genes, applied to filter out false positives, cannot handle intronless genes; and (ii) the experimental verification could fail to identify expression at a specific developmental time. This highlights the importance of developing methods that could provide a reliable estimate of the number of these two types of genes.

Gene finding in the chicken genome

Background: Despite the continuous production of genome sequence for a number of organisms,reliable, comprehensive, and cost effective gene prediction remains problematic. This is particularlytrue for genomes for which there is not a large collection of known gene sequences, such as therecently published chicken genome. We used the chicken sequence to test comparative andhomology-based gene-finding methods followed by experimental validation as an effective genomeannotation method.Results: We performed experimental evaluation by RT-PCR of three different computational genefinders, Ensembl, SGP2 and TWINSCAN, applied to the chicken genome. A Venn diagram wascomputed and each component of it was evaluated. The results showed that de novo comparativemethods can identify up to about 700 chicken genes with no previous evidence of expression, andcan correctly extend about 40% of homology-based predictions at the 5' end.Conclusions: De novo comparative gene prediction followed by experimental verification iseffective at enhancing the annotation of the newly sequenced genomes provided by standardhomology-based methods.

Use of a short hPDE6b promoter for rod gene transfer in a model of severe retinal cystrophy, the Rd10 mouse

Purpose: Gene therapy of severe retinal dystrophies directly affecting photoreceptor is still a challenge in terms of clinical application. One of the main hurdles is to generate high transgene expression specifically in rods or cones. In the present study, we are investigating the possibility to drive hPDE6b expression in the Rd10 mouse retina using a specific sequence of the human PDE6b promoter. Methods: Two 5' flanking fragments of the human PDE6b gene: (-93 to +53 (146 bp) and -297 to +53 (350 bp, see Di Polo and Farber, 1995) were cloned in different plasmids in order to check their expression in vitro and in vivo. These elements drove the activity of either luciferase (pGL3 plasmids) or EGFP (AAV2/8 backbone). Then, an AAV2/8 vector carrying the PDE6b cDNA was tested with subretinal injections at P9 in the Rd10 eyes. Eye fundus, OCT, ERG recordings and histological investigations were performed to assess the efficacy of the gene transfer. Results: The short PDE6b promoter containing 146bp (-93 to +53) showed the highest activity in the Y-79 cells, as described previously (Di Polo and Farber, 1995). Subretinal administrations of AAV2/8-PDE6bpromoter-EGFP allowed a rapid expression specifically in rods and not in cones. The expression is faster than a vector containing the CMV promoter. The AAV2/8-PDE6bpromoter-PDE6b and the control vector were injected at P9 in the Rd10 mouse retina and investigated 5 weeks post-injection. Out of 14 eyes, 6 presented an increased rod sensitivity of about 300 fold, and increased a- and b-wave responses in ERG recordings. Flicker stimulations revealed that cones are also functional. OCT images and histological analyses revealed an increased ONL size in the injected area. The retina treated with the therapeutic vector presented 4-6 rows of photoreceptors with outersegments containing PDE6b. In the control eyes, only 2-4 rows of photoreceptors with almost no OS were observed . Conclusions: The 146 bp promoter sequence (-93 to + 53) is the shortest regulatory element described to date which allows to obtain efficient rod-specific expression in the context of somatic gene transfer. This first result is of great interest for AAV vector design in general allowing more space for the accommodation of transgenes of interest and good expression in rods. Moreover we showed the proof of principle of the efficacy of AAV2/8-PDE6bp-PDE6b vector in the Rd10 mouse model of severe photoreceptor degeneration without using neither AAV mutated capsids, nor self-complementary vectors.

Sleep deprivation effects on circadian clock gene expression in the cerebral cortex parallel electroencephalographic differences among mouse strains.

Sleep deprivation (SD) results in increased electroencephalographic (EEG) delta power during subsequent non-rapid eye movement sleep (NREMS) and is associated with changes in the expression of circadian clock-related genes in the cerebral cortex. The increase of NREMS delta power as a function of previous wake duration varies among inbred mouse strains. We sought to determine whether SD-dependent changes in circadian clock gene expression parallel this strain difference described previously at the EEG level. The effects of enforced wakefulness of incremental durations of up to 6 h on the expression of circadian clock genes (bmal1, clock, cry1, cry2, csnk1epsilon, npas2, per1, and per2) were assessed in AKR/J, C57BL/6J, and DBA/2J mice, three strains that exhibit distinct EEG responses to SD. Cortical expression of clock genes subsequent to SD was proportional to the increase in delta power that occurs in inbred strains: the strain that exhibits the most robust EEG response to SD (AKR/J) exhibited dramatic increases in expression of bmal1, clock, cry2, csnkIepsilon, and npas2, whereas the strain with the least robust response to SD (DBA/2) exhibited either no change or a decrease in expression of these genes and cry1. The effect of SD on circadian clock gene expression was maintained in mice in which both of the cryptochrome genes were genetically inactivated. cry1 and cry2 appear to be redundant in sleep regulation as elimination of either of these genes did not result in a significant deficit in sleep homeostasis. These data demonstrate transcriptional regulatory correlates to previously described strain differences at the EEG level and raise the possibility that genetic differences underlying circadian clock gene expression may drive the EEG differences among these strains.

A variant in the gene FUT9 is associated with susceptibility to placental malaria infection

Malaria in pregnancy forms a substantial part of the worldwide burden of malaria, with an estimated annual death toll of up to 200,000 infants, as well as increased maternal morbidity and mortality. Studies of genetic susceptibility to malaria have so far focused on infant malaria, with only a few studies investigating the genetic basis of placental malaria, focusing only on a limited number of candidate genes. The aim of this study therefore was to identify novel host genetic factors involved in placental malaria infection. To this end we carried out a nested case-control study on 180 Mozambican pregnant women with placental malaria infection, and 180 controls within an intervention trial of malaria prevention. We genotyped 880 SNPs in a set of 64 functionally related genes involved in glycosylation and innate immunity. A SNP located in the gene FUT9, rs3811070, was significantly associated with placental malaria infection (OR = 2.31, permutation p-value = 0.028). Haplotypic analysis revealed a similarly strong association of a common haplotype of four SNPs including rs3811070. FUT9 codes for a fucosyl-transferase that is catalyzing the last step in the biosynthesis of the Lewis-x antigen, which forms part of the Lewis blood group-related antigens. These results therefore suggest an involvement of this antigen in the pathogenesis of placental malaria infection.

Characterization of human gene expression changes after olive oil ingestion: an exploratory approach

Olive oil consumption is protective against risk factors for cardiovascular and cancer diseases. A nutrigenomic approach was performed to assess whether changes in gene expression could occur in human peripheral blood mononuclear cells after oli ve oil ingestion at postprandial state. Six healthy male volunteers ingested, at fasting state, 50 ml of olive oil. Prior to intervention a 1-week washout period with a controlled diet and sunflower oil as the only source of fat was followed. During the 3 days before and on the intervention day, a very low-phenolic compound diet was followed. At baseline (0 h) and at post-ingestion (6 h), total RNA was isolated and gene expression (29,082 genes) was evaluated by microarray. From microarray data, nutrient-gene interactions were observed in genes related to metabolism, cellular processes, cancer, and atherosclerosis (e.g. USP48 by 2.16; OGT by 1.68-fold change) and associated processes such as inflammation (e.g. AKAP13 by 2.30; IL-10 by 1.66-fold change) and DNA damage (e.g. DCLRE1C by 1.47; POLK by 1.44- fold change). When results obtained by microarray were verified by qRT-PCR in nine genes, full concordance was achieved only in the case of up-regulated genes. Changes were observed at a real-life dose of olive oil, as it is daily consumed in some Mediterranean areas. Our results support the hypothesis that postprandial protective changes related to olive oil consumption could be mediated through gene expression changes.

OSIRISv1.2: a named entity recognition system for sequence variants of genes in biomedical literature

Background: Single Nucleotide Polymorphisms, among other type of sequence variants, constitute key elements in genetic epidemiology and pharmacogenomics. While sequence data about genetic variation is found at databases such as dbSNP, clues about the functional and phenotypic consequences of the variations are generally found in biomedical literature. The identification of the relevant documents and the extraction of the information from them are hampered by the large size of literature databases and the lack of widely accepted standard notation for biomedical entities. Thus, automatic systems for the identification of citations of allelic variants of genes in biomedical texts are required. Results: Our group has previously reported the development of OSIRIS, a system aimed at the retrieval of literature about allelic variants of genes http://ibi.imim.es/osirisform.html. Here we describe the development of a new version of OSIRIS (OSIRISv1.2, http://ibi.imim.es/OSIRISv1.2.html webcite) which incorporates a new entity recognition module and is built on top of a local mirror of the MEDLINE collection and HgenetInfoDB: a database that collects data on human gene sequence variations. The new entity recognition module is based on a pattern-based search algorithm for the identification of variation terms in the texts and their mapping to dbSNP identifiers. The performance of OSIRISv1.2 was evaluated on a manually annotated corpus, resulting in 99% precision, 82% recall, and an F-score of 0.89. As an example, the application of the system for collecting literature citations for the allelic variants of genes related to the diseases intracranial aneurysm and breast cancer is presented. Conclusion: OSIRISv1.2 can be used to link literature references to dbSNP database entries with high accuracy, and therefore is suitable for collecting current knowledge on gene sequence variations and supporting the functional annotation of variation databases. The application of OSIRISv1.2 in combination with controlled vocabularies like MeSH provides a way to identify associations of biomedical interest, such as those that relate SNPs with diseases.

Peroxisome proliferator-activated receptor beta regulates acyl-CoA synthetase 2 in reaggregated rat brain cell cultures.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that regulate the expression of many genes involved in lipid metabolism. The biological roles of PPARalpha and PPARgamma are relatively well understood, but little is known about the function of PPARbeta. To address this question, and because PPARbeta is expressed to a high level in the developing brain, we used reaggregated brain cell cultures prepared from dissociated fetal rat telencephalon as experimental model. In these primary cultures, the fetal cells initially form random aggregates, which progressively acquire a tissue-specific pattern resembling that of the brain. PPARs are differentially expressed in these aggregates, with PPARbeta being the prevalent isotype. PPARalpha is present at a very low level, and PPARgamma is absent. Cell type-specific expression analyses revealed that PPARbeta is ubiquitous and most abundant in some neurons, whereas PPARalpha is predominantly astrocytic. We chose acyl-CoA synthetases (ACSs) 1, 2, and 3 as potential target genes of PPARbeta and first analyzed their temporal and cell type-specific pattern. This analysis indicated that ACS2 and PPARbeta mRNAs have overlapping expression patterns, thus designating the ACS2 gene as a putative target of PPARbeta. Using a selective PPARbeta activator, we found that the ACS2 gene is transcriptionally regulated by PPARbeta, demonstrating a role for PPARbeta in brain lipid metabolism.

Neuroprotective gene therapy for Huntington's disease, using polymer-encapsulated cells engineered to secrete human ciliary neurotrophic factor: results of a phase I study.

Huntington's disease (HD) is a monogenic neurodegenerative disease that affects the efferent neurons of the striatum. The protracted evolution of the pathology over 15 to 20 years, after clinical onset in adulthood, underscores the potential of therapeutic tools that would aim at protecting striatal neurons. Proteins with neuroprotective effects in the adult brain have been identified, among them ciliary neurotrophic factor (CNTF), which protected striatal neurons in animal models of HD. Accordingly, we have carried out a phase I study evaluating the safety of intracerebral administration of this protein in subjects with HD, using a device formed by a semipermeable membrane encapsulating a BHK cell line engineered to synthesize CNTF. Six subjects with stage 1 or 2 HD had one capsule implanted into the right lateral ventricle; the capsule was retrieved and exchanged for a new one every 6 months, over a total period of 2 years. No sign of CNTF-induced toxicity was observed; however, depression occurred in three subjects after removal of the last capsule, which may have correlated with the lack of any future therapeutic option. All retrieved capsules were intact but contained variable numbers of surviving cells, and CNTF release was low in 13 of 24 cases. Improvements in electrophysiological results were observed, and were correlated with capsules releasing the largest amount of CNTF. This phase I study shows the safety, feasibility, and tolerability of this gene therapy procedure. Heterogeneous cell survival, however, stresses the need for improving the technique.

Restricted gene flow and subpopulation differentiation in Silene dioica

The size of breeding units and the hierarchical population structure of the dioecious perennial herb Silene dioica were investigated on four closely situated island populations in the Skeppsvik Archipelago in northern Sweden. F-statistics analyses of nine polymorphic allozyme loci revealed that plants on single islands are divided into many small breeding units, between 0.2 m2 and 6 m2. Hierarchical analyses showed that levels of differentiation among subpopulations within islands (FPL=0.080) were about twice as high as among islands (FLT=0.048). These results are discussed in the light of what is known about pollen and seed movement in the archipelago.

FIST/HIPK3: a Fas/FADD-interacting serine/threonine kinase that induces FADD phosphorylation and inhibits fas-mediated Jun NH(2)-terminal kinase activation.

Fas is a cell surface death receptor that signals apoptosis. Several proteins have been identified that bind to the cytoplasmic death domain of Fas. Fas-associated death domain (FADD), which couples Fas to procaspase-8, and Daxx, which couples Fas to the Jun NH(2)-terminal kinase pathway, bind independently to the Fas death domain. We have identified a 130-kD kinase designated Fas-interacting serine/threonine kinase/homeodomain-interacting protein kinase (FIST/HIPK3) as a novel Fas-interacting protein. Binding to Fas is mediated by a conserved sequence in the COOH terminus of the protein. FIST/HIPK3 is widely expressed in mammalian tissues and is localized both in the nucleus and in the cytoplasm. In transfected cell lines, FIST/HIPK3 causes FADD phosphorylation, thereby promoting FIST/HIPK3-FADD-Fas interaction. Although Fas ligand-induced activation of Jun NH(2)-terminal kinase is impaired by overexpressed active FIST/HIPK3, cell death is not affected. These results suggest that Fas-associated FIST/HIPK3 modulates one of the two major signaling pathways of Fas.

Dispensable role of myeloid differentiation primary response gene 88 (MyD88) and MyD88-dependent toll-like receptors (TLRs) in a murine model of osteoarthritis.

OBJECTIVES: The aim of our study was to evaluate the role of cell-membrane expressed TLRs and the signaling molecule MyD88 in a murine model of OA induced by knee menisectomy (surgical partial removal of the medial meniscus [MNX]). METHODS: OA was induced in 8-10weeks old C57Bl/6 wild-type (WT) female (n=7) mice and in knockout (KO) TLR-1 (n=7), -2 (n=8), -4 (n=9) -6 (n=5), MyD88 (n=8) mice by medial menisectomy, using the sham-operated contralateral knee as a control. Cartilage destruction and synovial inflammation were evaluated by knee joint histology using the OARSI scoring method. Apoptotic chondrocytes and cartilage metabolism (collagen II synthesis and MMP-mediated aggrecan degradation) were analyzed using immunohistochemistry. RESULTS: Operated knees exhibited OA features at 8weeks post-surgery compared to sham-operated ones. In menisectomized TLR-1, -2, -4, and -6 deficient mice, cartilage lesions, synovial inflammation and cartilage metabolism were similar to that in operated WT mice. Accordingly, using the same approach, we found no significant protection in MyD88-deficient mice in terms of OA progression as compared to WT littermates. CONCLUSIONS: Deficiency of TLRs or their signalling molecule MyD88 did not impact on the severity of experimental OA. Our results demonstrate that MyD88-dependent TLRs are not involved in this murine OA model. Moreover, the dispensable role of MyD88, which is also an adaptor for IL-1 receptor signaling, suggests that IL-1 is not a key mediator in the development of OA. This latter hypothesis is strengthened by the lack of efficiency of IL-1β antagonist in the treatment of OA.

Beta(1)-selective agonist (-)-1-(3,4-dimethoxyphenetylamino)-3-(3,4-dihydroxy)-2-propanol [(-)-RO363] differentially interacts with key amino acids responsible for beta(1)-selective binding in resting and active states.

(-)-1-(3,4-Dimethoxyphenetylamino)-3-(3,4-dihydroxy)-2-propanol [(-)-RO363] is a highly selective beta(1)-adrenergic receptor (beta(1)AR) agonist. To study the binding site of beta(1)-selective agonist, chimeric beta(1)/beta(2)ARs and Ala-substituted beta(1)ARs were constructed. Several key residues of beta(1)AR [Leu(110) and Thr(117) in transmembrane domain (TMD) 2], and Phe(359) in TMD 7] were found to be responsible for beta(1)-selective binding of (-)-RO363, as determined by competitive binding. Based on these results, we built a three-dimensional model of the binding domain for (-)-RO363. The model indicated that TMD 2 and TMD 7 of beta(1)AR form a binding pocket; the methoxyphenyl group of N-substituent of (-)-RO363 seems to locate within the cavity surrounded by Leu(110), Thr(117), and Phe(359). The amino acids Leu(110) and Phe(359) interact with the phenyl ring of (-)-RO363, whereas Thr(117) forms hydrogen bond with the methoxy group of (-)-RO363. To examine the interaction of these residues with beta(1)AR in an active state, each of the amino acids was changed to Ala in a constitutively active (CA)-beta(1)AR mutant. The degree of decrease in the affinity of CA-beta(1)AR for (-)-RO363 was essentially the same as that of wild-type beta(1)AR when mutated at Leu(110) and Thr(117). However, the affinity was decreased in Ala-substituted mutant of Phe(359) compared with that of wild-type beta(1)AR. These results indicated that Leu(110) and Thr(117) are necessary for the initial binding of (-)-RO363 with beta(1)-selectivity, and interaction of Phe(359) with the N-substituent of (-)-RO363 in an active state is stronger than in the resting state.