Zebrafish: a model for the study of addiction genetics

Drug abuse and dependence are multifaceted disorders with complex genetic underpinnings. Identifying specific genetic correlates is challenging and may be more readily accomplished by defining endophenotypes specific for addictive disorders. Symptoms and syndromes, including acute drug response, consumption, preference, and withdrawal, are potential endophenotypes characterizing addiction that have been investigated using model organisms. We present a review of major genes involved in serotonergic, dopaminergic, GABAergic, and adrenoreceptor signaling that are considered to be directly involved in nicotine, opioid, cannabinoid, and ethanol use and dependence. The zebrafish genome encodes likely homologs of the vast majority of these loci. We also review the known expression patterns of these genes in zebrafish. The information presented in this review provides support for the use of zebrafish as a viable model for studying genetic factors related to drug addiction. Expansion of investigations into drug response using model organisms holds the potential to advance our understanding of drug response and addiction in humans.     

Rate and irregularity of electrical activation during atrial fibrillation affect myocardial NGF expression via different signalling routes

An irregular ventricular response during atrial fibrillation (AF) has been shown to mediate an increase in sympathetic nerve activity in human subjects. The molecular mechanisms remain unclear. This study aimed to investigate the impact of rate and irregularity on nerve growth factor (NGF) expression in cardiomyocytes, since NGF is known to be the main contributor to cardiac sympathetic innervation density. Cell cultures of neonatal rat ventricular myocytes were electrically stimulated for 48 h with increasing rates (0, 5 and 50 Hz) and irregularity (standard deviation (SD) = 5%, 25% and 50% of mean cycle length). Furthermore, we analyzed the calcineurin-NFAT and the endothelin-1 signalling pathways as possible contributors to NGF regulation during arrhythmic stimulation. We found that the increase of NGF expression reached its maximum at the irregularity of 25% SD by 5 Hz (NGF: 5 Hz 0% SD = 1 vs. 5 Hz 25% SD = 1.57, P < 0.05). Specific blockade of the ET-A receptor by BQ123 could abolish this NGF increase (NGF: 5 Hz 25% SD + BQ123 = 0.66, P < 0.05). High frequency electrical field stimulation (HFES) with 50 Hz decreased the NGF expression in a significant manner (NGF: 50 Hz = 0.55, P < 0.05). Inhibition of calcineurin-NFAT signalling with cyclosporine-A or 11R-VIVIT abolished the HFES induced NGF down-regulation (NGF: 50 Hz + CsA = 1.14, P < 0.05). In summary, this study reveals different signalling routes of NGF expression in cardiomyocytes exposed to increasing rates and irregularity. Whether this translates into different degrees of NGF expression and possibly neural sympathetic growth in various forms of ventricular rate control during AF remains to be elucidated in further studies.     

Small molecule structure correctors abolish detrimental effects of apolipoprotein E4 in cultured neurons

Apolipoprotein E4 (apoE4), the major genetic risk factor for late onset Alzheimer disease, assumes a pathological conformation, intramolecular domain interaction. ApoE4 domain interaction mediates the detrimental effects of apoE4, including decreased mitochondrial cytochrome c oxidase subunit 1 levels, reduced mitochondrial motility, and reduced neurite outgrowth in vitro. Mutant apoE4 (apoE4-R61T) lacks domain interaction, behaves like apoE3, and does not cause detrimental effects. To identify small molecules that inhibit domain interaction (i.e. structure correctors) and reverse the apoE4 detrimental effects, we established a high throughput cell-based FRET primary assay that determines apoE4 domain interaction and secondary cell- and function-based assays. Screening a ChemBridge library with the FRET assay identified CB9032258 (a phthalazinone derivative), which inhibits domain interaction in neuronal cells. In secondary functional assays, CB9032258 restored mitochondrial cytochrome c oxidase subunit 1 levels and rescued impairments of mitochondrial motility and neurite outgrowth in apoE4-expressing neuronal cells. These benefits were apoE4-specific and dose-dependent. Modifying CB9032258 yielded well defined structure-activity relationships and more active compounds with enhanced potencies in the FRET assay (IC(50) of 23 and 116 nm, respectively). These compounds efficiently restored functional activities of apoE4-expressing cells in secondary assays. An EPR binding assay showed that the apoE4 structure correction resulted from direct interaction of a phthalazinone. With these data, a six-feature pharmacophore model was constructed for future drug design. Our results serve as a proof of concept that pharmacological intervention with apoE4 structure correctors negates apoE4 detrimental effects in neuronal cells and could be further developed as an Alzheimer disease therapeutic.     

The effects of domestication on the scaling of below- vs. aboveground biomass in four selected wheat (Triticum; Poaceae) genotypes

? Premise of the study: Theory and empirical studies have shown that, on average, belowground biomass (M(B)) scales one-to-one (isometrically) with aboveground biomass (M(A)) within and across plant species both at the individual and population level, i.e., M(B) ∝ M(A)(α=1), where α is the scaling exponent. However, little is known about how domestication affects this relationship. ? Methods: To examine the effects of domestication, we investigated the root vs. shoot biomass relationship during the first 30 d of growth of four wheat genotypes: two older genotypes, MO4 (T. monococcum, a diploid) and DM31 (T. dicoccum, a tetraploid) and two more recent genotypes, DX24 and L8275 (T. aestivum, both hexaploids). ? Results: Biomass allocation to roots scaled more or less isometrically with respect to shoot biomass allocation during the first 30 d of growth for both of the older genotypes, whereas shoot biomass allocation exceeded root allocation for the two more recent genotypes. This difference was attributable to the first 15 d of growth. Although root biomass allocation exceeded shoot biomass allocation during the first 15 d of growth for the two older genotypes, shoot biomass exceeded root biomass allocation during this critical phase of development for the two more recent genotypes. ? Conclusions: Based on a very limited sample of wheat genotypes, these results indicate that domestication has resulted in an increased biomass allocation to shoots compared to root biomass allocation. This shift possibly reflects artificial selection under agricultural conditions (for which water and nutrients are not limiting) favoring higher crop yields.     

Systematics and evolution of arctic-alpine Arabis alpina (Brassicaceae) and its closest relatives in the eastern Mediterranean

? Premise of the study: The high mountains in southern Anatolia and the eastern Mediterranean are assumed to play a major role as a primary center of genetic diversity and species richness in Eurasia. We tested this hypothesis by focusing on the widespread perennial arctic-alpine Arabis alpina and its sympatrically distributed closest relatives in the eastern Mediterranean. ? Methods: Plastid (trnL intron, trnL-F intergenic spacer) and nuclear (ITS) DNA sequence analysis was used for phylogenetic reconstruction. Broad-scale plastid haplotype analyses were conducted to infer ancestral biogeographic patterns. ? Key results: Five Arabis species, identified from the eastern Mediterranean (Turkey mainland and Cyprus), evolved directly and independently from A. alpina, leaving Arabis alpina as a paraphyletic taxon. These species are not affected by hybridization or introgression, and species divergence took place at the diploid level during the Pleistocene. ? Conclusions: Pleistocene climate fluctuations produced local altitudinal range-shifts among mountain glacial survival areas, resulting not only in the accumulation of intraspecific genotype diversity but also in the formation of five local species. We also show that the closest sister group of Arabis alpina consists exclusively of annuals/winter annuals and diverged prior to Pleistocene climatic fluctuations during the colonization of the lowland Mediterranean landscape. These findings highlight that Anatolia is not only a center of species richness but also a center for life-history diversification.     

The Escherichia coli cell division protein ZipA forms homodimers prior to association with FtsZ

ZipA is an essential component of the cell division machinery in E. coli and other closely related bacteria. It is an integral membrane protein that binds to FtsZ, tethering it to the inner membrane. ZipA also induces bundling of FtsZ protofilaments and may play a role in regulating FtsA activity; however, the molecular details behind these observations are not clear. In this study we have analyzed the oligomeric state of ZipA in vivo, by chemical cross-linking, and in vitro, by native gel electrophoresis (BN-PAGE). Our data indicate that ZipA can self-associate as a homodimer and that this self-interaction is not dependent on the FtsZ-binding domain. This observation rules out the possibility that FtsZ polymers mediate the ZipA self-interaction. Given this observation, it is possible that a certain population of ZipA is recruited to the division septum in a homodimeric form.     

Influence of squalene on lipid particle/droplet and membrane organization in the yeast Saccharomyces cerevisiae

In a previous study (Spanova et al., 2010, J. Biol. Chem., 285, 6127-6133) we demonstrated that squalene, an intermediate of sterol biosynthesis, accumulates in yeast strains bearing a deletion of the HEM1 gene. In such strains, the vast majority of squalene is stored in lipid particles/droplets together with triacylglycerols and steryl esters. In mutants lacking the ability to form lipid particles, however, substantial amounts of squalene accumulate in organelle membranes. In the present study, we investigated the effect of squalene on biophysical properties of lipid particles and biological membranes and compared these results to artificial membranes. Our experiments showed that squalene together with triacylglycerols forms the fluid core of lipid particles surrounded by only a few steryl ester shells which transform into a fluid phase below growth temperature. In the hem1? deletion mutant a slight disordering effect on steryl esters was observed indicated by loss of the high temperature transition. Also in biological membranes from the hem1? mutant strain the effect of squalene per se is difficult to pinpoint because multiple effects such as levels of sterols and unsaturated fatty acids contribute to physical membrane properties. Fluorescence spectroscopic studies using endoplasmic reticulum, plasma membrane and artificial membranes revealed that it is not the absolute squalene level in membranes but rather the squalene to sterol ratio which mainly affects membrane fluidity/rigidity. In a fluid membrane environment squalene induces rigidity of the membrane, whereas in rigid membranes there is almost no additive effect of squalene. In summary, our results demonstrate that squalene (i) can be well accommodated in yeast lipid particles and organelle membranes without causing deleterious effects; and (ii) although not being a typical membrane lipid may be regarded as a mild modulator of biophysical membrane properties.     

Vascular Endothelial Cell-specific MicroRNA-15a Inhibits Angiogenesis in Hindlimb Ischemia

The effects and potential mechanisms of the vascular endothelial cell (EC)-enriched microRNA-15a (miR-15a) on angiogenesis remain unclear. Here, we show a novel finding that EC-selective miR-15a transgenic overexpression leads to reduced blood vessel formation and local blood flow perfusion in mouse hindlimbs at 1-3 weeks after hindlimb ischemia. Mechanistically, gain- or loss-of-miR-15a function by lentiviral infection in ECs significantly reduces or increases tube formation, cell migration, and cell differentiation, respectively. By FGF2 and VEGF 3'-UTR luciferase reporter assays, Real-time PCR, and immunoassays, we further identified that the miR-15a directly targets FGF2 and VEGF to facilitate its anti-angiogenic effects. Our data suggest that the miR-15a in ECs can significantly suppress cell-autonomous angiogenesis through direct inhibition of endogenous endothelial FGF2 and VEGF activities. Pharmacological modulation of miR-15a function may provide a new therapeutic strategy to intervene against angiogenesis in a variety of pathological conditions.     

Both estrogen receptor subtypes, ERα and ERβ, prevent aldosterone-induced oxidative stress in VSMC via increased NADPH bioavailability

Activation of vascular mineralocorticoid (MR) or estrogen receptors (ER) exerts opposing effects on vascular remodeling. As we have previously shown, activation of either estrogen receptor subtype, ERα or ERβ, is fully sufficient to attenuate vascular remodeling in aldosterone salt-treated rats. To further elucidate the underlying mechanism(s) we tested the hypothesis that ER and MR activation might differentially modulate vascular reactive oxygen species (ROS) generation. In support of this concept, aldosterone increased ROS generation in vascular smooth muscle cells as determined by quantitative dihydroethidium fluorescence microscopy. Co-treatment with the selective ERα agonist 16α-LE2, the selective ERβ agonist 8β-VE2 or the non-selective ER agonist 17β-estradiol (E2) significantly reduced aldosterone-induced ROS generation. The pure ER antagonist ICI 182,780 completely blocked these salutary effects of E2, 16α-LE2 and 8β-VE2. Activation of ERα or ERβ fully blocked the reduction of intracellular nicotinamide adenine dinucleotide phosphate (NADPH) levels observed in aldosterone treated vascular smooth muscle cells. Intracellular NADPH levels were closely associated with expression and activity of the NADPH generating enzyme glucose-6-phosphate dehydrogenase. In conclusion, estrogens attenuate the detrimental vascular effects of excessive MR activation at least in part by preventing the depletion of intracellular NADPH levels.     

Involvement of cytochrome c CymA in the anaerobic metabolism of RDX by Shewanella oneidensis MR-1

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a cyclic nitramine explosive commonly used for military applications that is responsible for severe soil and groundwater contamination. In this study, Shewanella oneidensis MR-1 was shown to efficiently degrade RDX anaerobically (3.5?μmol·h(-1)·(g protein)(-1)) via two initial routes: (1) sequential N-NO(2) reductions to the corresponding nitroso (N-NO) derivatives (94% of initial RDX degradation) and (2) denitration followed by ring cleavage. To identify genes involved in the anaerobic metabolism of RDX, a library of ~2500 mutants of MR-1 was constructed by random transposon mutagenesis and screened for mutants with a reduced ability to degrade RDX compared with the wild type. An RDX-defective mutant (C9) was isolated that had the transposon inserted in the c-type cytochrome gene cymA. C9 transformed RDX at ~10% of the wild-type rate, with degradation occurring mostly via early ring cleavage caused by initial denitration leading to the formation of methylenedinitramine, 4-nitro-2,4-diazabutanal, formaldehyde, nitrous oxide, and ammonia. Genetic complementation of mutant C9 restored the wild-type phenotype, providing evidence that electron transport components have a role in the anaerobic reduction of RDX by MR-1.