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.

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.

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.

Increased 5-lipoxygenase immunoreactivity in the hippocampus of patients with Alzheimer's disease.

The proinflammatory enzyme 5-lipoxygenase (5-LOX) is upregulated in Alzheimer's disease (AD), but its localization and association with the hallmark lesions of the disease, beta-amyloid (Abeta) plaques and neurofibrillary tangles (NFTs), is unknown. This study examined the distribution and cellular localization of 5-LOX in the medial temporal lobe from AD and control subjects. The spatial relationship between 5-LOX immunoreactive structures and AD lesions was also examined. We report that, in AD subjects, 5-LOX immunoreactivity is elevated relative to controls, and its localization is dependent on the antibody-targeted portion of the 5-LOX amino acid sequence. Carboxy terminus-directed antibodies detected 5-LOX in glial cells and neurons, but less frequently in neurons with dystrophic (NFT) morphology. In contrast, immunoreactivity observed using 5-LOX amino terminus-directed antibodies was virtually absent in neurons and abundant in NFTs, neuritic plaques, and glia. Double-labeling studies showed a close association of 5-LOX-immunoreactive processes and glial cells with Abeta immunoreactive plaques and vasculature and also detected 5-LOX in tau immunoreactive and amyloid containing NFTs. Different immunolabeling patterns with antibodies against carboxy vs amino terminus of 5-LOX may be caused by post-translational modifications of 5-LOX protein in Abeta plaques and NFTs. The relationship between elevated intracellular 5-LOX and hallmark AD pathological lesions provides further evidence that neuroinflammatory pathways contribute to the pathogenesis of AD.

Melatonin signaling in mouse cerebellar granule cells with variable native MT1 and MT2 melatonin receptors.

Although G protein-coupled MT1 and MT2 melatonin receptors are expressed in neurons of the mammalian brain including in humans, relatively little is known about the influence of native MT1 and MT2 melatonin receptors on neuronal melatonin signaling. Whereas human cerebellar granule cells (CGC) express only MT1 receptors, mouse CGC express both MT1 and MT2. To study the effects of altered neuronal MT1/MT2 receptors, we used CGC cultures prepared from immature cerebella of wild-type mice (MT1/MT2 CGC) and MT1- and MT2-knockout mice (MT2 and MT1 CGC, respectively). Here we report that in MT1/MT2 cultures, physiological (low nanomolar) concentrations of melatonin decrease the activity (phosphorylation) of extracellular-signal-regulated kinase (ERK) whereas a micromolar concentration was ineffective. Both MT1 and MT2 deficiencies transformed the melatonin inhibition of ERK into melatonin-induced ERK activation. In MT1/MT2 CGC, 1 nM melatonin inhibited serine/threonine kinase Akt, whereas in MT1 and MT2 CGC, this concentration was ineffective. Under these conditions, both MT1 and MT2 deficiencies prevented melatonin from inhibiting forskolin-stimulated cAMP levels and cFos immunoreactivity. We demonstrated that selective removal of native neuronal MT1 and MT2 receptors has a profound effect on the intracellular actions of low/physiological concentrations of melatonin. Since the expression of MT1 and MT2 receptors is cell-type-specific and species-dependent, we postulate that the pattern of expression of neuronal melatonin receptor types in different brain areas and cells could determine the capabilities of endogenous melatonin in regulating neuronal functioning.

Stimulatory effects of a melatonin receptor agonist, ramelteon, on BDNF in mouse cerebellar granule cells.

Melatonin receptor activation has been linked to the regulation of neurotrophic factors, including the brain-derived neurotrophic factor (BDNF). To further characterize the effects of melatonin receptor stimulation on neuronal BDNF, we used a clinically available novel agonist for MT1 and MT2 melatonin receptors, ramelteon. Primary cultures of cerebellar granule cells (CGC) have been established as an in vitro model for studying neuronal BDNF. We took advantage of the availability of MT1- and MT2-deficient (knockout; KO) mice to employ primary CGC prepared from wild type (WT), MT1 KO, and MT2 KO mice. We investigated the effects of ramelteon on BDNF protein and mRNA content. Administered in a low nanomolar range, ramelteon increased BDNF protein content in all three types of mouse CGC. This ramelteon-triggered BDNF protein elevation was not preceded by a BDNF mRNA increase. However, it was prevented by treatment of cultures with a protein synthesis inhibitor cycloheximide. These results demonstrated that the MT1/MT2 melatonin receptor agonist ramelteon is capable of increasing BDNF protein in neurons expressing either of the two melatonin receptor types and that this action of ramelteon involves translational mechanisms. Further research is needed to explore the putative influence of ramelteon on BDNF-associated neuroplasticity.

Effects of MK-886, a 5-lipoxygenase activating protein (FLAP) inhibitor, and 5-lipoxygenase deficiency on the forced swimming behavior of mice.

A common biological pathway may contribute to the comorbidity of atherosclerosis and depression. Increased activity of the enzymatic 5-lipoxygenase (5-LOX, 5LO) pathway is a contributing factor in atherosclerosis and a 5-LOX inhibitor, MK-886, is beneficial in animal models of atherosclerosis. In the brain, MK-886 increases phosphorylation of the glutamate receptor subunit GluR1, and the increased phosphorylation of this receptor has been associated with antidepressant treatment. In this work, we evaluated the behavioral effects of MK-886 in an automated assay of mouse forced swimming, which identifies antidepressant activity as increased climbing behavior and/or decreased rest time. Whereas a single injection of MK-886 (3 and 10 mg/kg) did not affect forced swimming behaviors assayed 30 min later, six daily injections of 3 mg/kg MK-886 slightly increased climbing and significantly reduced rest time in wild-type mice but not in 5-LOX-deficient mice. A diet delivery of MK-886, 4 micro/(100 mg(body-weight)day), required 3 weeks to affect forced swimming; it increased climbing behavior. Climbing behavior was also increased in naive 5-LOX-deficient mice compared to naive wild-type controls. These results suggest that 5-LOX inhibition and deficiency may be associated with antidepressant activity. Increased climbing in a forced swimming assay is a typical outcome of antidepressants that increase noradrenergic and dopaminergic activity. Interestingly, 5-LOX deficiency and MK-886 treatment have been shown to be capable of increasing the behavioral effects of a noradrenaline/dopamine-potentiating drug, cocaine. Future research is needed to evaluate the clinical relevance of our findings.

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.

Clock genes: influencing and being influenced by psychoactive drugs.

Although clock genes are the hallmark of circadian rhythms, they are also currently considered as transcription factors that have a prominent role in the pharmacology of the CNS. The expression of these transcription factors in the mammalian brain is not only intrinsically rhythmic but is also modulated by external inputs and hormones. An altered expression of clock genes, as evidenced in transgenic mice, has a profound influence on the behavioral effects of psychoactive drugs. Focusing on clock genes expressed in the brain might lead to the discovery of novel drug-target pathways.

5-Lipoxygenase inhibitor MK-886 increases GluR1 phosphorylation in neuronal cultures in vitro and in the mouse cortex in vivo.

Modifications of AMPA glutamate receptor GluR1 phosphorylation are critical for neuroplastic mechanisms. Previous in vitro studies in brain slices employed MK-886, a functional inhibitor of the enzyme 5-lipoxygenase (5-LOX), and found increased GluR1 phosphorylation. Since slice preparations have accompanying postmortem phosphorylation changes, e.g., decreased GluR1 phosphorylation, it remains to be clarified whether MK-886 can affect GluR1 phosphorylation in intact neurons and in the brain in vivo. We used primary neuronal cultures prepared from embryonic mouse brain and in vivo drug administration to investigate the effects of MK-886 on GluR1 phosphorylation using quantitative Western immunoblotting assays. In vitro, MK-886 increased GluR1 phosphorylation at both serine 831 and serine 845. In vivo, repeated but not a single MK-886 injection increased GluR1 phosphorylation in the prefrontal cortex. These findings indicate that MK-886 has an intrinsic effect on neuronal phosphorylation both in vitro and in vivo and support the use of MK-886 as a pharmacological tool in studies of not only the 5-LOX pathway but also neuronal GluR1 functioning.

Possible role for interactions between 5-lipoxygenase (5-LOX) and AMPA GluR1 receptors in depression and in antidepressant therapy.

Emerging evidence suggests that 5-lipoxygenase (5-LOX) plays a role in central nervous system functioning. It has been shown that 5-LOX metabolic products can decrease the phosphorylation of the glutamate receptor subunit GluR1, and that this effect can be antagonized by 5-LOX inhibitors. Recent concepts about the pathobiological mechanisms of depression and the molecular mechanisms of antidepressant activity postulate a significant role for glutamatergic neurotransmission and the GluR1 receptor. Regulation of GluR1 phosphorylation, i.e., enhancement of this phosphorylation, may be a part of antidepressant activity. On the other hand, reduced GluR1 phosphorylation may be a pathobiological mechanism contributing to depression. Since 5-LOX inhibitors, along with antidepressants share the capacity to increase GluR1 phosphorylation, we hypothesize that they may also have antidepressant properties. Furthermore, we postulate that increased brain 5-LOX expression may lead to decreased GluR1 phosphorylation and favor the development of depression. For example, brain 5-LOX expression is stimulated by stress hormone glucocorticoids, and stress is a known contributing factor in depression.

The pattern of melatonin receptor expression in the brain may influence antidepressant treatment.

The pineal hormone melatonin produces most of its biological effects via G protein-coupled receptors MT1 and MT2. In mammals, these receptors are expressed in various tissues and organs including in the brain. Recent research points to a putative role of MT1/MT2 dimerization as a mechanism that could determine the receptor-mediated biological effects of melatonin. Brain content and the ratios between MT1 and MT2 receptors are affected by illness, e.g., Alzheimer's disease, and by prolonged drug treatment, e.g., antidepressants. New drugs with antidepressant properties that bind and activate melatonin receptors have been discovered. We hypothesize that endogenous, i.e., low, levels of melatonin could contribute to antidepressant effects depending on the expression pattern of melatonin receptors in the brain. Hence, we propose that a prolonged treatment with classical antidepressant drugs alters the brain ratio of MT1/MT2 receptors to enable the endogenous melatonin, which is secreted during the night, to further improve the antidepressant effects. A corollary of this hypothesis is that antidepressants would be less effective in conditions of pathologically altered brain melatonin receptors, e.g., in Alzheimer's patients or due to genetic polymorphisms. If our hypothesis is confirmed, supplementing classical antidepressant treatment with an appropriate dose of a melatonin receptor agonist might be used to improve antidepressant effects in subjects with a susceptible pattern of brain melatonin receptor expression.

Impaired hippocampal long-term potentiation in melatonin MT2 receptor-deficient mice.

The pineal product melatonin that acts on specific melatonin receptors has been implicated in pathobiological mechanisms of neuropsychiatric disorders including Alzheimer's disease. We used mice lacking melatonin MT(2) receptors (MT(2) knockouts) to investigate the role of these receptors in synaptic plasticity and learning-dependent behavior. In field CA1 of hippocampal slices from wild-type mice, theta burst stimulation induced robust and stable long-term potentiation that was smaller and decremental in slices from MT(2) knockouts. Tested in an elevated plus-maze on two consecutive days, wild-type mice showed shorter transfer latencies to enter a closed arm on the second day; this experience-dependent behavior did not occur in MT(2) knockouts. These results suggest that MT(2) receptors participate in hippocampal synaptic plasticity and in memory processes.

5-Lipoxygenase (ALOX5) and FLAP (ALOX5AP) gene polymorphisms as factors in vascular pathology and Alzheimer's disease.

We first hypothesized in 2000 that a polymorphism of the human gene encoding the enzyme 5-lipoxygenase (5-LOX) might be associated with Alzheimer's disease. Only a little progress has been made in directly testing our proposal. However, additional important new data lead us to hypothesize that genetic variability not only in the 5-LOX gene, i.e., ALOX5, but also in polymorphism of the five-lipoxygenase activating protein (FLAP) gene, i.e., ALOX5AP, may be associated with Alzheimer's pathology. Studies in mice followed by several extensive clinical studies have identified ALOX5 and ALOX5AP polymorphisms as strong risk factors for atherosclerosis and cerebrovascular pathologies. New data point to a significant aggregation of vascular risk factors and risk of Alzheimer's disease. Preliminary findings in postmortem brain of Alzheimer's patients identified elevated 5-LOX immunostaining in this disease. We suggest that our hypothesis of a link between the ALOX5 and ALOX5AP gene polymorphisms and Alzheimer's disease could be tested in a clinical setting and in animal models, i.e., transgenic mice could be produced by crossing the available 5-LOX-deficient mice with the available transgenic mice models of Alzheimer's disease.

Techniques: fruit flies as models for neuropharmacological research.

An unlikely animal model is gaining popularity in neuropharmacological research: the 2-mm fruit fly (Drosophila melanogaster). Drugs have been administered to adult flies in their food and, more recently, via gasses and injections. Pharmacological tools have introduced behavioral alterations in Drosophila reminiscent of human behavior, rescued flies from gene-alteration-triggered neuropathologies, and triggered gene silencing. Combined, these methods hold promise for significant neuropharmacological advancement.

The regional and cellular expression profile of the melatonin receptor MT1 in the central dopaminergic system.

The physiological effects of pineal melatonin are primarily mediated by melatonin receptors located in the brain and periphery. Even though there are a number of studies demonstrating the regulatory role of melatonin in the development of dopaminergic behaviors, such as psychostimulant-induced diurnal locomotor sensitization or drug seeking, little is known about the contribution of melatonin receptors (i.e., MT1) to this role. Therefore, as a first step in understanding the functional role of melatonin receptors in dopaminergic behaviors, we focused on determining the expression pattern of MT1 receptors in the dopaminergic system of the human and rodent brain. Regional (e.g., nucleus accumbens shell) and cellular (e.g., tyrosine hydroxylase immunopositive cells) expression of MT1 mRNA was characterized by applying the immuno-laser capture microdissection (immuno-LCM) technique coupled with nested RT-PCR. Moreover, employing quantitative Western immunoblotting and RT-PCR, we found that the mouse MT1 receptor expression presents diurnal variations (i.e., low mRNA and high protein levels at night, ZT21). The dopaminergic system-based presence of MT1 receptor proteins was not limited to rodents; we found these receptors in postmortem human brain as well. Further research is needed to understand the regional/cellular functional role of melatonin receptors in the regulation of dopaminergic behaviors, using models such as melatonin receptor knockout mice.

Reliability assessment of an automated forced swim test device using two mouse strains.

The Porsolt forced swim test (FST) is one of the most widely used behavioral tests in the evaluation of the antidepressant effects of drugs. It is based on the fact that these drugs reduce the depression-related behaviors of learned helplessness. The model has been modified for use in mice. In contrast to rats, mice are exposed to forced swimming only once and their immobility behavior is measured and considered a "depression-like" phenotype. Like many other behavioral tests, FST can be affected by observer-related artifacts. In recent years, automated testing systems have been developed to decrease artifacts that may greatly influence the interpretation of results. In this work, we used two strains of mice, i.e., C3H/HeJ and C57BL/6J, which differ in their FST immobility times. We employed a new commercially available automated FST device and a blinded observer-based FST, and we examined their ability to measure behavioral differences between these two mouse strains. Our results suggest that the tested automated FST system generates reliable data comparable to results obtained by trained observers.

Drosophila GABA(B) receptors are involved in behavioral effects of gamma-hydroxybutyric acid (GHB).

Gamma-hydroxybutyric acid (GHB) can be synthesized in the brain but is also a known drug of abuse. Although putative GHB receptors have been cloned, it has been proposed that, similar to the behavior-impairing effects of ethanol, the in vivo effects of pharmacological GHB may involve metabotropic gamma-aminobutyric acid (GABA) GABA(B) receptors. We developed a fruitfly (Drosophila melanogater) model to investigate the role of these receptors in the behavioral effects of exogenous GHB. Injecting GHB into male flies produced a dose-dependent motor impairment (measured with a computer-assisted automated system), which was greater in ethanol-sensitive cheapdate mutants than in wild-type flies. These effects of pharmacological concentrations of GHB require the presence and activation of GABA(B) receptors. The evidence for this was obtained by pharmacological antagonism of GABA(B) receptors with CGP54626 and by RNA interference (RNAi)-induced knockdown of the GABA(B(1)) receptor subtype. Both procedures inhibited the behavioral effects of GHB. GHB pretreatment diminished the behavioral response to subsequent GHB injections; i.e., it triggered GHB tolerance, but did not produce ethanol tolerance. On the other hand, ethanol pretreatment produced both ethanol and GHB tolerance. It appears that in spite of many similarities between ethanol and GHB, the primary sites of their action may differ and that recently cloned putative GHB receptors may participate in actions of GHB that are not mediated by GABA(B) receptors. These receptors do not have a Drosophila orthologue. Whether Drosophila express a different GHB receptor should be explored.

Developmental role of GABAB(1) receptors in Drosophila.

Previously, an RNA interference (RNAi) knockdown of GABAB(1) subunit in adult Drosophila was used for behavioral studies. Here we report on developmental deficits caused by embryonic Drosophila GABAB(1) RNAi and drug antagonism. Injecting embryos with CGP54626 (a GABAB receptor antagonist) reduced hatching and caused lethality. Similar effects were produced by injecting embryos with GABAB(1) double-stranded RNA (RNAi). The surviving GABAB(1) RNAi larvae were significantly smaller than controls and showed a peculiar phenotype; their tracheae were folded. Our results suggest that GABAB receptors are required for normal development and that the Drosophila model could be used to investigate the participating molecular mechanisms.

Fruit flies for anti-pain drug discovery.

Recent work has indicated that fruit flies (Drosophila melanogaster) can be used in nociception research. Genetic screening identified a gene, painless, that is required for thermal and mechanical nociception in Drosophila larvae. On the other hand, pharmacological techniques and noxious heat were used to assay antinocieceptive behavior in intact adult Drosophila. In general, animal models for pain research are bound by ethical concerns. Since no serious ethical controversies have been raised regarding experiments in insects, Drosophila may be, for the time being an ethically acceptable animal model for combined genetic and pharmacological analgesia research.