| 1. |
- Olszewski, Pawel K., et al.
(författare)
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Neurobeachin, a Regulator of Synaptic Protein Targeting, Is Associated with Body Fat Mass and Feeding Behavior in Mice and Body-Mass Index in Humans
- 2012
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Ingår i: PLoS Genetics. - : Public Library of Science (PLoS). - 1553-7390 .- 1553-7404. ; 8:3, s. e1002568-
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Tidskriftsartikel (refereegranskat)abstract
- Neurobeachin (Nbea) regulates neuronal membrane protein trafficking and is required for the development and functioning of central and neuromuscular synapses. In homozygous knockout (KO) mice, Nbea deficiency causes perinatal death. Here, we report that heterozygous KO mice haploinsufficient for Nbea have higher body weight due to increased adipose tissue mass. In several feeding paradigms, heterozygous KO mice consumed more food than wild-type (WT) controls, and this consumption was primarily driven by calories rather than palatability. Expression analysis of feeding-related genes in the hypothalamus and brainstem with real-time PCR showed differential expression of a subset of neuropeptide or neuropeptide receptor mRNAs between WT and Nbea+/- mice in the sated state and in response to food deprivation, but not to feeding reward. In humans, we identified two intronic NBEA single-nucleotide polymorphisms (SNPs) that are significantly associated with body-mass index (BMI) in adult and juvenile cohorts. Overall, data obtained in mice and humans suggest that variation of Nbea abundance or activity critically affects body weight, presumably by influencing the activity of feeding-related neural circuits. Our study emphasizes the importance of neural mechanisms in body weight control and points out NBEA as a potential risk gene in human obesity.
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| 2. |
- Andreasson, Claes, et al.
(författare)
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Direct Cloning of Isogenic Murine DNA in Yeast and Relevance of Isogenicity for Targeting in Embryonic Stem Cells
- 2013
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:9, s. e74207-
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Tidskriftsartikel (refereegranskat)abstract
- Efficient gene targeting in embryonic stem cells requires that modifying DNA sequences are identical to those in the targeted chromosomal locus. Yet, there is a paucity of isogenic genomic clones for human cell lines and PCR amplification cannot be used in many mutation-sensitive applications. Here, we describe a novel method for the direct cloning of genomic DNA into a targeting vector, pRTVIR, using oligonucleotide-directed homologous recombination in yeast. We demonstrate the applicability of the method by constructing functional targeting vectors for mammalian genes Uhrf1 and Gfap. Whereas the isogenic targeting of the gene Uhrf1 showed a substantial increase in targeting efficiency compared to non-isogenic DNA in mouse E14 cells, E14-derived DNA performed better than the isogenic DNA in JM8 cells for both Uhrf1 and Gfap. Analysis of 70 C57BL/6-derived targeting vectors electroporated in JM8 and E14 cell lines in parallel showed a clear dependence on isogenicity for targeting, but for three genes isogenic DNA was found to be inhibitory. In summary, this study provides a straightforward methodological approach for the direct generation of isogenic gene targeting vectors.
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| 3. |
- Refojo, Damian, et al.
(författare)
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Glutamatergic and Dopaminergic Neurons Mediate Anxiogenic and Anxiolytic Effects of CRHR1
- 2011
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 333:6051, s. 1903-1907
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Tidskriftsartikel (refereegranskat)abstract
- The corticotropin-releasing hormone receptor 1 (CRHR1) critically controls behavioral adaptation to stress and is causally linked to emotional disorders. Using neurochemical and genetic tools, we determined that CRHR1 is expressed in forebrain glutamatergic and gamma-aminobutyric acid-containing (GABAergic) neurons as well as in midbrain dopaminergic neurons. Via specific CRHR1 deletions in glutamatergic, GABAergic, dopaminergic, and serotonergic cells, we found that the lack of CRHR1 in forebrain glutamatergic circuits reduces anxiety and impairs neurotransmission in the amygdala and hippocampus. Selective deletion of CRHR1 in midbrain dopaminergic neurons increases anxiety-like behavior and reduces dopamine release in the prefrontal cortex. These results define a bidirectional model for the role of CRHR1 in anxiety and suggest that an imbalance between CRHR1-controlled anxiogenic glutamatergic and anxiolytic dopaminergic systems might lead to emotional disorders.
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| 4. |
- Shimokawa, Noriaki, et al.
(författare)
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CIN85 regulates dopamine receptor endocytosis and governs behaviour in mice
- 2010
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Ingår i: EMBO Journal. - : European Molecular Biology Organization. - 0261-4189 .- 1460-2075. ; 29:14, s. 2421-2432
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Tidskriftsartikel (refereegranskat)abstract
- Despite extensive investigations of Cbl-interacting protein of 85 kDa (CIN85) in receptor trafficking and cytoskeletal dynamics, little is known about its functions in vivo. Here, we report the study of a mouse deficient of the two CIN85 isoforms expressed in the central nervous system, exposing a function of CIN85 in dopamine receptor endocytosis. Mice lacking CIN85 exon 2 (CIN85(Deltaex2)) show hyperactivity phenotypes, characterized by increased physical activity and exploratory behaviour. Interestingly, CIN85(Deltaex2) animals display abnormally high levels of dopamine and D2 dopamine receptors (D2DRs) in the striatum, an important centre for the coordination of animal behaviour. Importantly, CIN85 localizes to the post-synaptic compartment of striatal neurons in which it co-clusters with D2DRs. Moreover, it interacts with endocytic regulators such as dynamin and endophilins in the striatum. Absence of striatal CIN85 causes insufficient complex formation of endophilins with D2DRs in the striatum and ultimately decreased D2DR endocytosis in striatal neurons in response to dopamine stimulation. These findings indicate an important function of CIN85 in the regulation of dopamine receptor functions and provide a molecular explanation for the hyperactive behaviour of CIN85(Deltaex2) mice.
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| 5. |
- Vogel, Katharina U, et al.
(författare)
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Roquin paralogs 1 and 2 redundantly repress the Icos and Ox40 costimulator mRNAs and control follicular helper T cell differentiation
- 2013
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Ingår i: Immunity. - Cambridge, USA : Cell Press. - 1074-7613 .- 1097-4180. ; 38:4, s. 655-68
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Tidskriftsartikel (refereegranskat)abstract
- The Roquin-1 protein binds to messenger RNAs (mRNAs) and regulates gene expression posttranscriptionally. A single point mutation in Roquin-1, but not gene ablation, increases follicular helper T (Tfh) cell numbers and causes lupus-like autoimmune disease in mice. In T cells, we did not identify a unique role for the much lower expressed paralog Roquin-2. However, combined ablation of both genes induced accumulation of T cells with an effector and follicular helper phenotype. We showed that Roquin-1 and Roquin-2 proteins redundantly repressed the mRNA of inducible costimulator (Icos) and identified the Ox40 costimulatory receptor as another shared mRNA target. Combined acute deletion increased Ox40 signaling, as well as Irf4 expression, and imposed Tfh differentiation on CD4(+) T cells. These data imply that both proteins maintain tolerance by preventing inappropriate T cell activation and Tfh cell differentiation, and that Roquin-2 compensates in the absence of Roquin-1, but not in the presence of its mutated form.
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| 6. |
- Wurst, Friedrich M, et al.
(författare)
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Characterization of Sialic Acid Index of Plasma Apolipoprotein J and Phosphatidylethanol During Alcohol Detoxification-A Pilot Study.
- 2012
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Ingår i: Alcoholism: Clinical and Experimental Research. - : Wiley. - 0145-6008. ; 36:2, s. 251-257
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Tidskriftsartikel (refereegranskat)abstract
- Background: Apolipoprotein J (ApoJ) is a component of plasma high-density lipoproteins. Previous studies have shown progressive recovery of ApoJ sialic acid content with increased duration of alcohol abstinence. Therefore, the sialic acid index of plasma apolipoprotein J (SIJ) seems to be a promising alcohol biomarker. Phosphatidylethanol (PEth) is a direct ethanol metabolite and has recently attracted attention as a biomarker of prolonged intake of higher amounts of alcohol. The aim of the pilot study was to explore sensitivity, specificity, and normalization of SIJ and PEth in comparison with traditional and emerging biomarkers. Methods: Five male alcohol-dependent patients (International Classification of Diseases 10, F 10.25) were included (median: 40 years old; Alcohol Use Disorders Identification Test value, 30; alcohol consumption in the previous 7 days, 1,680 g). SIJ, PEth, urinary ethyl glucuronide (UEtG), urinary ethyl sulfate (UEtS), and gamma glutamyl-transpeptidase (GGT) were determined at days 1, 3, 7, 10, 14, 21, and 28. Results: At study entry, SIJ, PEth, UEtG, and UEtS were positive in all subjects, whereas GGT and mean corpuscular volume were positive in 3 of 5 (60%) of the subjects. Individual SIJ levels increased between day 1 and 28 between 13.7 and 44.3%, respectively. For SIJ and PEth, the ANOVA (p < 0.005) showed a significant trend with the average subject's SIJ and PEth changing 1.22 and 1.02, respectively, per week. Conclusions: Our preliminary data suggest that SIJ and PEth might hold potential as markers of heavy ethanol intake.
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| 7. |
- Wurst, Friedrich Martin, et al.
(författare)
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Phosphatidylethanol: normalization during detoxification, gender aspects and correlation with other biomarkers and self-reports
- 2010
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Ingår i: Addiction Biology. - : Wiley. - 1369-1600 .- 1355-6215. ; 15:1, s. 88-95
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Tidskriftsartikel (refereegranskat)abstract
- Phosphatidylethanol (PEth) is a direct ethanol metabolite, and has recently attracted attention as biomarker of ethanol intake. The aims of the current study are: (1) to characterize the normalization time of PEth in larger samples than previously conducted; (2) to elucidate potential gender differences; and (3) to report the correlation of PEth with other biomarkers and self-reported alcohol consumption. Fifty-seven alcohol-dependent patients (ICD 10 F 10.25; 9 females, 48 males) entering medical detoxification at three study sites were enrolled. The study sample was comprised of 48 males and 9 females, with mean age 43.5. Mean gamma glutamyl transpeptidase (GGT) was 209.61 U/l, average mean corpuscular volume (MCV) was 97.35 fl, mean carbohydrate deficient transferrin (%CDT) was 8.68, and mean total ethanol intake in the last 7 days was 1653 g. PEth was measured in heparinized whole blood with a high-pressure liquid chromatography method, while GGT, MCV and %CDT were measured using routine methods. PEth levels at day 1 of detoxification ranged between 0.63 and 26.95 mu mol/l (6.22 mean, 4.70 median, SD 4.97). There were no false negatives at day 1. Sensitivities for the other biomarkers were 40.4% for MCV, 73.1% for GGT and 69.2% for %CDT, respectively. No gender differences were found for PEth levels at any time point. Our data suggest that PEth is (1) a suitable intermediate term marker of ethanol intake in both sexes; and (2) sensitivity is extraordinary high in alcohol dependent patients. The results add further evidence to the data that suggest that PEth has potential as a candidate for a sensitive and specific biomarker, which reflects longer-lasting intake of higher amounts of alcohol and seemingly has the above mentioned certain advantages over traditional biomarkers.
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