Reduction of Cellular Lipid Content by a Knockdown of Drosophila PDP1 γ and Mammalian Hepatic Leukemia Factor.

In exploring the utility of double-stranded RNA (dsRNA) injections for silencing the PAR-domain protein 1 (Pdp1) gene in adult Drosophila, we noticed a dramatic loss of fat tissue lipids. To verify that our RNAi approach produced the expected Pdp1 knockdown, the abdominal fat tissues sections were stained with PDP1 antibodies. PDP1 protein immunostaining was absent in flies injected with dsRNA targeting a sequence common to all known Pdp1 isoforms. Subsequent experiments revealed that lipid staining is reduced in flies injected with dsRNA against Pdp1 γ (fat body specific) and not against Pdp1 ε (predominantly involved in circadian mechanisms). Drosophila PDP1 γ protein shows a high homology to mammalian thyrotroph embryonic factor (TEF), albumin D site-binding protein (DBP), and hepatic leukemia factor (HLF) transcription factors. In an in vitro model of drug- (olanzapine-) induced adiposity in mouse 3T3-L1 cells, the mRNA content of HLF but not TEF and DBP was increased by the drug treatment. A knockdown of the HLF mRNA by transfecting the cultures with HLF dsRNA significantly reduced their lipid content. Furthermore, the HLF RNAi prevented olanzapine from increasing the cell lipid content. These results suggest that the PDP1/HLF system may play a role in physiological and drug-influenced lipid regulation.

5-lipoxygenase-activating protein as a modulator of olanzapine-induced lipid accumulation in adipocyte.

Experiments were performed in 3T3-L1 preadipocytes differentiated in vitro into adipocytes. Cells were treated with olanzapine and a 5-lipoxygenase (5-LOX) activating protein (FLAP) inhibitor MK-886. Lipid content was measured using an Oil Red O assay; 5-LOX and FLAP mRNA content was measured using quantitative real-time PCR; the corresponding protein contents were measured using quantitative Western blot assay. Olanzapine did not affect the cell content of 5-LOX mRNA and protein; it decreased FLAP mRNA and protein content at day five but not 24 hours after olanzapine addition. In the absence of MK-886, low concentrations of olanzapine increased lipid content only slightly, whereas a 56% increase was induced by 50 µ M olanzapine. A 5-day cotreatment with 10 µ M MK-886 potentiated the lipid increasing action of low concentrations of olanzapine. In contrast, in the presence of 50 µ M olanzapine nanomolar and low micromolar concentrations of MK-886 reduced lipid content. These data suggest that FLAP system in adipocytes is affected by olanzapine and that it may modify how these cells respond to the second-generation antipsychotic drugs (SGADs). Clinical studies could evaluate whether the FLAP/5-LOX system could play a role in setting a variable individual susceptibility to the metabolic side effects of SGADs.

Progress in mitochondrial epigenetics.

Mitochondria, intracellular organelles with their own genome, have been shown capable of interacting with epigenetic mechanisms in at least four different ways. First, epigenetic mechanisms that regulate the expression of nuclear genome influence mitochondria by modulating the expression of nuclear-encoded mitochondrial genes. Second, a cell-specific mitochondrial DNA content (copy number) and mitochondrial activity determine the methylation pattern of nuclear genes. Third, mitochondrial DNA variants influence the nuclear gene expression patterns and the nuclear DNA (ncDNA) methylation levels. Fourth and most recent line of evidence indicates that mitochondrial DNA similar to ncDNA also is subject to epigenetic modifications, particularly by the 5-methylcytosine and 5-hydroxymethylcytosine marks. The latter interaction of mitochondria with epigenetics has been termed 'mitochondrial epigenetics'. Here we summarize recent developments in this particular area of epigenetic research. Furthermore, we propose the term 'mitoepigenetics' to include all four above-noted types of interactions between mitochondria and epigenetics, and we suggest a more restricted usage of the term 'mitochondrial epigenetics' for molecular events dealing solely with the intra-mitochondrial epigenetics and the modifications of mitochondrial genome.

Effect of valproic acid on mitochondrial epigenetics.

Valproic acid (valproate), an anticonvulsant and a mood stabilizer, is a potent histone deacetylase inhibitor and a widely utilized pharmacological tool for neuroepigenetic research including DNA methylation. However, only nuclear but not mitochondrial DNA (mtDNA) has been investigated for the effects of valproate on the formation of 5-methylcytosine (5 mC) and 5-hydroxymethylcytosine (5 hmC). Using mouse 3T3-L1 cells, we investigated the effects of short (1 day) and prolonged (3 days) valproate treatment on global mtDNA 5 mC content, global and mtDNA sequence-specific 5 hmC content, mRNA levels for ten-eleven-translocation (TET) enzymes involved in 5 hmC formation, and the mitochondrial content of TET proteins. Only 5 hmC but not 5 mC content in mtDNA was affected (decreased) by valproate, and only after the prolonged treatment. This action of valproate was mimicked by MS-275, a class I histone deacetylase inhibitor. The prolonged but not the short valproate treatment decreased the expression of Tet1 mRNA and reduced the mitochondrial content of the TET1 protein. Hence, a likely scenario for a valproate-induced 5 hmC decrease in mtDNA may involve nuclear histone deacetylase inhibition (mitochondria do not contain histones) causing the initial increase of Tet1 transcription, which is followed by a delayed compensatory decrease of Tet1 expression and a reduced presence of TET1 protein in mitochondria. Further research is needed to elucidate the functional implications of epigenetic modifications of mtDNA. The observed effects of valproate on mitochondrial epigenetics may have implications for a better understanding of both therapeutic and unwanted effects of this drug and possibly other histone deacetylase inhibitors.

Mitochondrial DNA: A Blind Spot in Neuroepigenetics.

Neuroepigenetics, which includes nuclear DNA modifications such as 5-methylcytosine and 5-hydoxymethylcytosine and modifications of nuclear proteins such as histones, is emerging as the leading field in molecular neuroscience. Historically, a functional role for epigenetic mechanisms, including in neuroepigenetics, has been sought in the area of the regulation of nuclear transcription. However, one important compartment of mammalian cell DNA, different from nuclear but equally important for physiological and pathological processes (including in the brain), mitochondrial DNA has for the most part not had a systematic epigenetic characterization. The importance of mitochondria and mitochondrial DNA (particularly its mutations) in central nervous system physiology and pathology has long been recognized. Only recently have mechanisms of mitochondrial DNA methylation and hydroxymethylation, including the discovery of mitochondrial DNA-methyltransferases and the presence and the functionality of 5-methylcytosine and 5-hydroxymethylcytosine in mitochondrial DNA (e.g., in modifying the transcription of mitochondrial genome), been unequivocally recognized as a part of mammalian mitochondrial physiology. Here we summarize for the first time evidence supporting the existence of these mechanisms and we propose the term "mitochondrial epigenetics" to be used when referring to them. Currently, neuroepigenetics does not include mitochondrial epigenetics - a gap that we expect to close in the near future.

Effect of aging on 5-hydroxymethylcytosine in brain mitochondria.

Nuclear epigenetics of the mammalian brain is modified during aging. Little is known about epigenetic modifications of mitochondrial DNA (mtDNA). We analyzed brain samples of 4- and 24-month-old mice and found that aging decreased mtDNA 5-hydroxymethylcytosine (5hmC) but not 5-methylcytosine (5mC) levels in the frontal cortex but not the cerebellum. Transcript levels of selected mtDNA-encoded genes increased during aging in the frontal cortex only. Aging affected the expression of enzymes involved in 5-methylcytosine and 5-hydroxymethylcytosine synthesis (mitochondrial DNA methyltransferase 1 [mtDNMT1] and ten-eleven-translocation [TET]1-TET3, respectively). In the frontal cortex, aging decreased mtDNMT1 messenger RNA (mRNA) levels without affecting TET1-TET3 mRNAs. In the cerebellum, TET2 and TET3 mRNA content was increased but mtDNMT1 mRNA was unaffected. Using Western immunoblotting of samples from primary neuronal cultures, we found TET immunoreactivity in the mitochondrial fraction. At the single cell level, TET immunoreactivity was detected in the nucleus and in the perinuclear/intraneurite areas where it frequently colocalized with a mitochondrial marker. Our results demonstrated the presence and susceptibility to aging of mitochondrial epigenetic mechanisms in the mammalian brain.

Effect of aging on 5-hydroxymethylcytosine in the mouse hippocampus.

PURPOSE: Aging is believed to affect epigenetic marking of brain DNA with 5-methylcytosine (5mC) and possibly via the 5mC to 5-hydroxymethylcytosine (5hmC) conversion by TET (ten-eleven translocation) enzymes. We investigated the impact of aging on hippocampal DNA 5-hydroxymethylation including in the sequence of aging-susceptible 5-lipoxygenase (5-LOX) gene. METHODS: Hippocampal samples were obtained from C57BL6 mice. Cellular 5hmC localization was determined by immunofluorescence. The global 5mC and 5hmC contents were measured with the corresponding ELISA. The 5-LOX 5hmC content was measured using a glucosyltransferase/enzymatic restriction digest assay. TET mRNA was measured using qRT-PCR. RESULTS: Global hippocampal 5hmC content increased during aging as did the 5hmC content in the 5-LOX gene. This occurred without alterations of TET1-3 mRNAs and without changes in the content of 8-hydroxy-2-deoxy-guanosine, a marker of non-enzymatic DNA oxidation. CONCLUSIONS: The aging-associated increase of hippocampal 5hmC content (global and 5-LOX) appears to be unrelated to oxidative stress. It may be driven by an altered activity but not by the increased expression of the three TET enzymes. Global 5hmC content was increased during aging in the absence of 5mC decrease, suggesting that 5hmC could act as an epigenetic marker and not only as an intermediary in DNA demethylation. Further research is needed to elucidate the functional implications of the impact of aging on hippocampal cytosine hydroxymethylation.

Effects of minocycline on cocaine sensitization and phosphorylation of GluR1 receptors in 5-lipoxygenase deficient mice.

In wild-type (WT) mice, the antibiotic minocycline inhibits development of cocaine-induced locomotor sensitization. Some of the actions of minocycline may involve the 5-lipoxygenase (5-LOX) pathway. We used the model of 5-LOX-deficient mice to investigate whether 5-LOX participates in minocycline's influence on the effects of cocaine. Locomotor sensitization was induced by 4 daily cocaine injections and the phosphorylation status of GluR1 glutamate receptors was assayed in brain samples. Minocycline failed to affect cocaine sensitization in 5-LOX-deficient mice. In these mice, neither cocaine nor minocycline 4-day treatment altered GluR1 phosphorylation. In WT mice in which minocycline inhibited development of cocaine sensitization, a 4-day cocaine treatment increased GluR1 phosphorylation at both Ser831 and Ser845 sites in the frontal cortex but not the striatum; further, this effect was prevented by minocycline. Under basal conditions and in response to a single cocaine injection the levels of GluR1, GluR2, and GluR3 AMPA receptor subunits did not differ between WT and 5-LOX-deficient mice, but the response of GluR1 phosphorylation to a single cocaine injection was greater under the 5-LOX deficiency. Hence, in WT mice GluR1 phosphorylation increased only in the frontal cortex and only at the Ser831 site. In 5-LOX-deficient mice, acute cocaine injection increased both Ser831 and Ser845 phosphorylation both in the frontal cortex and in the striatum. We suggest that in studying minocycline's action on cocaine's effects and/or addiction in humans, it would be important to consider the characterization of the subjects' 5-LOX system. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

Cyclooxygenases and 5-lipoxygenase in Alzheimer's disease.

Typically, cyclooxygenases (COXs) and 5-lipoxygenase (5-LOX), enzymes that generate biologically active lipid molecules termed eicosanoids, are considered inflammatory. Hence, their putative role in Alzheimer's disease (AD) has been explored in the framework of possible inflammatory mechanisms of AD pathobiology. More recent data indicate that these enzymes and the biologically active lipid molecules they generate could influence the functioning of the central nervous system and the pathobiology of neurodegenerative disorders such as AD via mechanisms different from classical inflammation. These mechanisms include the cell-specific localization of COXs and 5-LOX in the brain, the type of lipid molecules generated by the activity of these enzymes, the type and the localization of receptors selective for a type of lipid molecule, and the putative interactions of the COXs and 5-LOX pathways with intracellular components relevant for AD such as the gamma-secretase complex. Considering the importance of these multiple and not necessarily inflammatory mechanisms may help us delineate the exact nature of the involvement of the brain COXs and 5-LOX in AD and would reinvigorate the search for novel targets for AD therapy.

GABA-B receptors in Drosophila.

Drosophila melanogaster, the "fruit fly," is being increasingly used as an experimental model in neurosciences, including neuropharmacology. The advantages of Drosophila over typical mammalian models in neuropharmacology include better access to genetic manipulation and the availability of almost unlimited numbers of experimental subjects at relatively low cost and with minimal regulatory restrictions. Nevertheless, one should remain cognizant of the substantial differences between insects and mammals. Insects, including Drosophila, utilize gamma-aminobutyric acid (GABA) as a neurotransmitter and express both ionotropic GABA receptors and metabotropic GABA-B receptors. Before cloning of the Drosophila GABA-B receptors (subunits 1-3), it had been assumed that flies did not express these receptors since baclofen, a typical agonist for mammalian GABA-B receptors, does not produce any effects in insects. Subsequently, it was confirmed that cloned Drosophila GABA-B receptors exhibit a unique pharmacology. Using Drosophila as a model, it has been shown that GABA-B receptors are involved in the behavioral actions of alcohol and gamma-hydroxybutyric acid, and possibly in pain. Furthermore, recent research suggests that in flies these receptors may play an important developmental role and that they participate in olfaction and in regulation of circadian rhythms.

Benefits of neuropsychiatric phenomics: example of the 5-lipoxygenase-leptin-Alzheimer connection.

Phenomics is a systematic study of phenotypes on a genomewide scale that is expected to unravel, as of yet, unsuspected functional roles of the genome. It remains to be determined how to optimally approach and analyze the available phenomics databases to spearhead innovation in neuropsychiatry. By serendipitously connecting two unrelated phenotypes of increased blood levels of the adipokine leptin, a molecule that regulates appetite, in 5-lipoxygenase- (5-LOX) deficient mice and patients with a lower risk for Alzheimer's disease (AD), we postulated a leptin-mediated basis for beneficial effects of ALOX5 (a gene encoding 5-LOX) gene-deficiency in AD. We suggest that it might be possible to avoid relying on serendipity and develop data-mining tools capable of extracting from phenomics databases indications for such novel hypotheses. Hence, we provide an example of using a free-access Arrowsmith two-node search interface to identify ALOX5 as unsuspected putative mechanisms for the previously described clinical association between increased plasma levels of leptin and a lower risk of incident dementia and AD.

Interactions with GluR1 AMPA receptors could influence the therapeutic usefulness of minocycline in ALS.

Despite the beneficial effects of minocycline seen in animal models of ALS, initial clinical trials of minocycline in ALS patients failed to support its expected therapeutic usefulness. Here we discuss new results from recent preclinical studies of the molecular neuronal effects of minocycline pertinent for better understanding of the therapeutic potential of this antibiotic.

Minocycline affects cocaine sensitization in mice.

Growing evidence has pointed to an interaction between the tetracycline antibiotic minocycline and drugs with abuse liability such as opioids and amphetamines. In this work, we tested the hypothesis that similar to its effects on methamphetamine-induced locomotor sensitization, minocycline may influence the behavioral effects of cocaine. Experiments were performed in male C57BL/6J mice using an automated system to measure locomotor activity. We found that 80 mg/kg minocycline significantly reduced locomotor activity when administered either alone or injected 30 min prior to cocaine, which increased locomotor activity. To investigate whether minocycline selectively affects the development of locomotor sensitization induced by four daily injections of 10mg/kg cocaine, we sought a schedule of minocycline administration that does not per se affect locomotor activity. Thus, we selected 40 mg/kg minocycline administered 3h prior to cocaine; minocycline did not affect cocaine-stimulated locomotor activity on the first day of administration but prevented the development of cocaine sensitization. We also tested whether minocycline would affect an already established cocaine sensitization. After establishing the sensitization effect by four daily injections, cocaine treatment was discontinued and mice were treated with minocycline daily (days 5-11) or on day 11 only. There was no effect of minocycline treatment on the response of cocaine-sensitized mice to the challenge dose of cocaine on day 11. The mechanisms by which minocycline interferes with the development of cocaine sensitization need to be characterized.

5-Lipoxygenase DNA methylation and mRNA content in the brain and heart of young and old mice.

The expression of 5-lipoxygenase (5-LOX) is affected by aging and regulated by epigenetic mechanisms including DNA methylation. We used methylation-sensitive restriction endonucleases (AciI, BstUI, HpaII, and HinP1I) to assess 5-LOX DNA methylation in brain and heart tissue samples from young (2 months) and old (22 months) mice. We also measured mRNA content for 5-LOX and the DNA methyltransferases DNMT1 and DNMT3a. In young mice, the 5-LOX mRNA content was significantly greater in the heart compared to the brain; 5-LOX DNA methylation was lower, except in the AciI assay in which it was higher in the heart. Aging decreased 5-LOX mRNA content in the heart and increased it in the brain. Aging also increased 5-LOX DNA methylation and this effect was site- (i.e., enzyme) and tissue-specific. Generally, DNMT1 and DNMT3a mRNA content was lower in the brain regions compared to the heart; the only effect of aging was observed in the mRNA content of DNMT3a, which was decreased in the heart of old mice. These results indicate a complex tissue-specific and aging-dependent interplay between the DNA methylation system and 5-LOX mRNA content. Interpretation of this data must take into account that the tissue samples contained a mixture of various cell types.

The melatonin receptor MT1 is required for the differential regulatory actions of melatonin on neuronal 'clock' gene expression in striatal neurons in vitro.

Through inhibitory G protein-coupled melatonin receptors, melatonin regulates intracellular signaling systems and also the transcriptional activity of certain genes. Clock genes are proposed as regulatory factors in forming dopamine-related behaviors and mood and melatonin has the ability to regulate these processes. Melatonin-mediated changes in clock gene expression have been reported in brain regions, including the striatum, that are crucial for the development of dopaminergic behaviors and mood. However, it is not known whether melatonin receptors present in striatum mediate these effects. Therefore, we investigated the role of the melatonin/melatonin receptor system on clock gene expression using a model of primary neuronal cultures prepared from striatum. We found that melatonin at the receptor affinity range (i.e., nm) affects the expression of the clock genes mPer1, mClock, mBmal1 and mNPAS2 (neuronal PAS domain protein 2) differentially in a pertussis toxin-sensitive manner: a decrease in Per1 and Clock, an increase in NPAS2 and no change in Bmal1 expression. Furthermore, mutating MT1 melatonin receptor (i.e., MT1 knockouts, MT1(-/-)) reversed melatonin-induced changes, indicating the involvement of MT1 receptor in the regulatory action of melatonin on neuronal clock gene expression. Therefore, by controlling clock gene expression we propose melatonin receptors (i.e., MT1) as novel therapeutic targets for the pathobiologies of dopamine-related behaviors and mood.

Minocycline increases phosphorylation and membrane insertion of neuronal GluR1 receptors.

The tetracycline antibiotic minocycline beneficially affects neuronal functioning and also inhibits the enzyme 5-lipoxygenase (5-LOX). We hypothesized that similar to 5-LOX inhibitors, minocycline may increase phosphorylation and membrane insertion of the glutamate receptor GluR1. The experiments were performed in primary cultures of mouse striatal neurons and in the prefrontal cortex and striatum of minocycline-treated mice. In vitro, low micromolar minocycline concentrations increased GluR1 phosphorylation at Ser845 and Ser831 and increased the surface content of GluR1. Minocycline also increased GluR1 phosphorylation in vivo. Increased GluR1 phosphorylation and minocycline treatment have been associated with antidepressant and memory-enhancing activities. Direct consequences of minocycline-increased GluR1 phosphorylation are yet to be established.