Targeting mitochondria to oppose the progression of nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is a condition characterized by the excessive accumulation of triglycerides in hepatocytes. NAFLD is the most frequent chronic liver disease in developed countries, and is often associated with metabolic disorders such as obesity and type 2 diabetes. NAFLD definition encompasses a spectrum of chronic liver abnormalities, ranging from simple steatosis (NAFL), to steatohepatitis (NASH), significant liver fibrosis, cirrhosis, and hepatocellular carcinoma. NAFLD, therefore, represents a global public health issue. Mitochondrial dysfunction occurs in NAFLD, and contributes to the progression to the necro-inflammatory and fibrotic form (NASH). Disrupted mitochondrial function is associated with a decrease in the energy levels and impaired redox balance, and negatively affects cell survival by altering overall metabolism and subcellular trafficking. Such events reduce the tolerance of hepatocytes towards damaging hits, and favour the injurious effects of extra-cellular factors. Here, we discuss the role of mitochondria in NAFLD and focus on potential therapeutic approaches aimed at preserving mitochondrial function.

Selegiline induces a wake promoting effect in rats which is related to formation of its active metabolites.

The goal of the present work was to characterise the effects of selegiline on the rat sleep pattern. Furthermore, for comparative purposes, the pharmacokinetics of selegiline and its metabolites in brain and plasma were investigated, and microdialysis experiments were performed to examine the resulting effect on dopamine, noradrenaline and serotonin levels. Selegiline (1, 5, 10 and 30mg/kg) was found to dose-dependently increase the time spent awake following acute dosing. The pharmacokinetic assessment of selegiline showed that, following an oral dose of 5mg/kg, low circulating levels of the parent compound were found relative to those of biotransformed l-methamphetamine and l-amphetamine. The time course of selegiline-induced wakefulness was shown to follow the time course of l-methamphetamine and l-amphetamine in brain, suggesting that these metabolites are responsible for the modulation of sleep architecture. Furthermore, selegiline (5mg/kg) caused a significant increase of extracellular levels of DA (250%) and NA (200%), but not of 5-HT, in the rat prefrontal cortex. In summary, an integrated experimental approach was undertaken here to evaluate selegiline's effect on sleep architecture in rats in relation to its pharmacokinetics and changes in monoaminergic neurotransmitter levels in the brain. The effect of selegiline on sleep was likely mediated by an increase of dopamine and noradrenaline levels in the brain caused by the formed metabolites.

Oligomeric and phosphorylated alpha-synuclein as potential CSF biomarkers for Parkinson's disease.

BACKGROUND: Despite decades of intensive research, to date, there is no accepted diagnosis for Parkinson's disease (PD) based on biochemical analysis of blood or CSF. However, neurodegeneration in the brains of PD patients begins several years before the manifestation of the clinical symptoms, pointing to serious flaw/limitations in this approach. RESULTS: To explore the potential use of alpha-synuclein (α-syn) species as candidate biomarkers for PD, we generated specific antibodies directed against wide array of α-syn species, namely total-, oligomeric- and phosphorylated-Ser129-α-syn (t-, o- and p-S129-α-syn). Next we sought to employ our antibodies to develop highly specific ELISA assays to quantify α-syn species in biological samples. Finally we verified the usefulness of our assays in CSF samples from 46 PD patients and 48 age-matched healthy controls. We also assessed the discriminating power of combining multiple CSF α-syn species with classical Alzheimer's disease biomarkers. The combination of CSF o-/t-α-syn, p-S129-α-syn and p-tau provided the best fitting predictive model for discriminating PD patients from controls. Moreover, CSF o-α-syn levels correlated significantly with the severity of PD motor symptoms (r = -0.37). CONCLUSION: Our new ELISA assays can serve as research tools to address the unmet need for reliable CSF biomarkers for PD and related disorders.

In vitro and in vivo characterisation of Lu AF64280, a novel, brain penetrant phosphodiesterase (PDE) 2A inhibitor: potential relevance to cognitive deficits in schizophrenia.

Here, we present the pharmacological characterisation of Lu AF64280, a novel, selective, brain penetrant phosphodiesterase (PDE) 2A inhibitor, in in vitro/in vivo assays indicative of PDE2A inhibition, and in vivo models/assays relevant to cognitive processing or antipsychotic-like activity. The in vitro selectivity of Lu AF64280 was determined against a panel of PDE enzymes and 3',5'-cyclic guanosine monophosphate (cGMP) levels in the hippocampus were determined using in vivo microdialysis. Lu AF64280 potently inhibited hPDE2A (Ki = 20 nM), 50-fold above moderate inhibition of both hPDE9A (Ki = 1,000 nM) and hPDE10A (Ki = 1,800 nM), and displayed a >250-fold selectivity over all other full-length human recombinant PDE family members (Ki above 5,000 nM). Lu AF64280 (20 mg/kg) significantly increased cGMP levels in the hippocampus (p < 0.01 versus vehicle-treated mice), attenuated sub-chronic phencyclidine-induced deficits in novel object exploration in rats (10 mg/kg, p < 0.001 versus vehicle-treated), blocked early postnatal phencyclidine-induced deficits in the intradimensional/extradimensional shift task in rats (1 and 10 mg/kg, p < 0.001 versus vehicle-treated) and attenuated spontaneous P20-N40 auditory gating deficits in DBA/2 mice (20 mg/kg, p < 0.05 versus vehicle-treated). In contrast, Lu AF64280 failed to attenuate phencyclidine-induced hyperactivity in mice, and was devoid of antipsychotic-like activity in the conditioned avoidance response paradigm in rats, at any dose tested. Lu AF64280 represents a novel tool compound for selective PDE2A inhibition that substantiates a critical role of this enzyme in cognitive processes under normal and pathological conditions.

23Na multiple quantum filtered NMR characterisation of Na+ binding and dynamics in animal cells: a comparative study and effect of Na+/Li + competition.

Double quantum and triple quantum filtered (23)Na nuclear magnetic resonance techniques were used to characterise in detail the isotropic and anisotropic binding and dynamics of intra- and extracellular Na(+) in different cellular systems, in the absence and presence of Li(+). The kinetics of Li(+) influx by different cell types was evaluated. At steady state, astrocytes accumulated more Li(+) than red blood cells (RBCs), while a higher intracellular Li(+) concentration was found in chromaffin than in SH-SY5Y cells. Anisotropic and isotropic motions were detected for extracellular Na(+) in all cellular systems studied. Isotropic intracellular Na(+) motions were observed in all types of cells, while anisotropic Na(+) motions in the intracellular compartment were only detected in RBCs. (23)Na triple quantum signal efficiency for intracellular Na(+) was SH-SY5Y > chromaffin > RBCs, while the reverse order was observed for the extracellular ions. (23)Na double quantum signal efficiency for intracellular Na(+) was non-zero only in RBCs, and for extracellular Na(+) the order RBCs > chromaffin > SH-SY5Y cells was observed. Li(+) loading generally decreased intracellular Na(+) isotropic movements in the cells, except for astrocytes incubated with a low Li(+) concentration and increased anisotropic intracellular Na(+) movements in RBCs. Li(+) effects on the extracellular signals were more complex, reflecting Li(+)/Na(+) competition for isotropic and anisotropic binding sites at the extracellular surface of cell membranes and also at the surface of the gel used for cell immobilisation. These results are relevant and contribute to the interpretation of the in vivo pharmacokinetics and sites of Li(+) action.

Vortioxetine (Lu AA21004), a novel multimodal antidepressant, enhances memory in rats.

The serotonergic system plays an important role in cognitive functions via various 5-HT receptors. Vortioxetine (Lu AA21004) in development as a novel multimodal antidepressant is a 5-HT3, 5-HT7 and 5-HT1D receptor antagonist, a 5-HT1B receptor partial agonist, a 5-HT1A receptor agonist and a 5-HT transporter (5-HTT) inhibitor in vitro. Preclinical studies suggest that 5-HT3 and 5-HT7 receptor antagonism as well as 5-HT1A receptor agonism may have a positive impact on cognitive functions including memory. Thus vortioxetine may potentially enhance memory. We investigated preclinical effects of vortioxetine (1-10mg/kg administered subcutaneously [s.c.]) on memory in behavioral tests, and on cortical neurotransmitter levels considered important in rat memory function. Contextual fear conditioning and novel object recognition tests were applied to assess memory in rats. Microdialysis studies were conducted to measure extracellular neurotransmitter levels in the rat medial prefrontal cortex. Vortioxetine administered 1h before or immediately after acquisition of contextual fear conditioning led to an increase in freezing time during the retention test. This mnemonic effect was not related to changes in pain sensitivity as measured in the hotplate test. Rats treated with vortioxetine 1h before training spent more time exploring the novel object in the novel object recognition test. In microdialysis studies of the rat medial prefrontal cortex, vortioxetine increased extracellular levels of acetylcholine and histamine. In conclusion, vortioxetine enhanced contextual and episodic memory in rat behavioral models. Further demonstration of its potential effect on memory functions in clinical settings is warranted.

The effects of acute treatment with escitalopram on the different stages of contextual fear conditioning are reversed by atomoxetine.

RATIONALE: Although the antidepressant and anxiolytic effects of selective serotonin reuptake inhibitors and serotonin-noradrenaline reuptake inhibitors are well-documented, less is known about their cognitive effects. OBJECTIVE: Escitalopram, a selective serotonin reuptake inhibitor, and atomoxetine, a selective noradrenaline reuptake inhibitor, were used to evaluate the interaction between noradrenergic and serotonergic neurotransmission in the modulation of contextual fear conditioning in rats. METHODS: Contextual fear-conditioning test was used to investigate the acute effects of escitalopram, alone or in combination with atomoxetine, in different stages of learning and memory in rats. Furthermore, microdialysis in freely moving animals was used to investigate the effect of escitalopram on serotonin, dopamine, and noradrenaline levels in the rat hippocampus. RESULTS: Escitalopram significantly increased conditioned responses when applied before the acquisition, but decreased responses, when applied before the recall test. When administered during memory consolidation, escitalopram dose-dependently enhanced conditioned responding. These effects were blocked by atomoxetine. Escitalopram (at a dose that affects memory consolidation) increased hippocampal serotonin levels fourfold without changing dopamine or noradrenaline. Atomoxetine, at dose levels that blocked the effects of escitalopram on contextual fear conditioning, increased the extracellular levels of noradrenaline eightfold but did not change dopamine or serotonin. A combined treatment of escitalopram and atomoxetine caused a significant attenuation of escitalopram-induced increase in serotonin levels, while noradrenaline levels were not affected. CONCLUSIONS: These findings indicate that escitalopram affects fear memory in rats, likely modulated by increases in serotonin levels in the brain. This effect is impaired by atomoxetine, probably due to a noradrenaline-mediated decrease in serotonin levels. Further studies are warranted to study the effects of potential differences among antidepressant therapies on long-term cognitive outcomes.

Phosphodiesterase 10A inhibition modulates the sensitivity of the mesolimbic dopaminergic system to D-amphetamine: involvement of the D1-regulated feedback control of midbrain dopamine neurons.

Phosphodiesterase (PDE) 10A is highly expressed in medium spiny neurons of the striatum, at the confluence of the corticostriatal glutamatergic and the midbrain dopaminergic pathways, both believed to be involved in the physiopathology of schizophrenia. There is a growing body of evidence suggesting that targeting PDE10A may be beneficial for the treatment of positive symptoms in schizophrenia. The aim of the present study was to investigate how PDE10A inhibition modulates mesolimbic dopaminergic neurotransmission. We found that the selective PDE10A inhibitor, MP-10, blocked D-amphetamine-induced hyperactivity as well as D-amphetamine-induced dopamine efflux in the nucleus accumbens in a dose-dependent manner. We further investigated the mechanism by which PDE10A inhibition affects dopaminergic neurotransmission. We report that MP-10 potentiated the effect of a high but not a low dose of D-amphetamine on the mean firing rate of dopaminergic neurons recorded from the ventral tegmental area. Similarly, the effect of a high, but not a low dose of D-amphetamine, was completely reversed by the selective D(1) antagonist, SCH23390. These data suggest that the D(1)-regulated feedback control of midbrain dopamine neurons is a critical pathway involved in the modulation of the response of mesolimbic dopamine neurons to D-amphetamine by PDE10A inhibition.

HIF prolyl hydroxylase inhibition augments dopamine release in the rat brain in vivo.

The transcription factor hypoxia-inducible factor (HIF) is essential for the activation of several genes that promote the survival of cells exposed to oxidative stress. Expression of tyrosine hydroxylase (TH), which is the rate-limiting enzyme in the dopamine (DA) synthesis, is one of the genes that are positively regulated by HIF. Accordingly, HIF induction results in elevated DA release in various cell lines in vitro. HIF prolyl hydroxylase (HPH) is critically involved in the negative regulation of HIF levels. We investigated the in vivo effects of the HPH inhibitor FG0041 on brain DA function in rats by microdialysis in freely moving rats, locomotor activity, and Western blot analysis. Administration of FG0041 (10 mg/kg i.p.), as an acute (single injection), or as subchronic (once daily for 6 days) treatment and cobalt chloride (CoCl2) (60 mg/kg s.c.) potentiated potassium (K+) induced increases in extracellular levels of DA levels in the rat striatum. The increase in extracellular DA of freely moving rats was sought in relationship to locomotor activity in rats. A significant increase in locomotor activity was observed in FG0041-treated rats compared with vehicle on a cocaine challenge. In support of these findings, protein levels of TH in the rat brain stem were increased after treatment with FG0041. These data indicate that FG0041 augments DA function in the rat brain. Inhibition of HPH enhances DA function by increasing DA release, which has implications for the use of HIF induction in the treatment of neurodegenerative diseases.

Antipsychotic-like effect of retigabine [N-(2-Amino-4-(fluorobenzylamino)-phenyl)carbamic acid ester], a KCNQ potassium channel opener, via modulation of mesolimbic dopaminergic neurotransmission.

Dopaminergic (DAergic) neurons in the ventral tegmental area express both KCNQ2 and KCNQ4 channels, which opening is expected to decrease neuronal excitability via neuronal hyper-polarization. Because psychotic symptoms are believed to be associated with an increased excitability of dopamine (DA) cells in the mesencephalon, KCNQ channels might represent a new potential target for the treatment of psychosis. The aim of our study was to investigate the antipsychotic-like potential of KCNQ channel opening via modulation of neuronal activity within the mesolimbic DAergic system. We report that retigabine [N-(2-amino-4-(fluorobenzylamino)-phenyl)carbamic acid ester], a KCNQ opener, dose-dependently reduced basal DA firing rate and more potently suppressed burst firing activity in the ventral tegmental area, whereas XE-991 [10,10-bis(pyridinylmethyl)-9(10H)-anthracenone], a selective KCNQ blocker, induced opposite effects. In addition, retigabine prevented d-amphetamine-induced DA efflux in the nucleus accumbens and d-amphetamine-induced locomotor hyperactivity. In contrast, XE-991 potentiated both the locomotor hyperactivity and DA efflux evoked by d-amphetamine. These data strongly suggest that the activation of KCNQ channels attenuates DAergic neurotransmission in the mesolimbic system, particularly in conditions of excessive DAergic activity. In a model predictive of antipsychotic activity, the conditioned avoidance response paradigm, retigabine was found to inhibit avoidance responses, an effect blocked by coadministration of XE-991. Furthermore, retigabine was found to significantly inhibit the hyperlocomotor response to a phencyclidine (PCP) challenge in PCP-sensitized animals, considered as a disease model for schizophrenia. Taken together, our studies provide evidence that KCNQ channel openers represent a potential new class of antipsychotics.

Effects of mood stabilizers on the inhibition of adenylate cyclase via dopamine D(2)-like receptors.

OBJECTIVE: The mood stabilizing drugs lithium, carbamazepine and valproate modulate brain adenosine monophosphate (cAMP) levels, which are assumed to be elevated in bipolar disorder patients. The aim of this work was to investigate how these three mood stabilizing agents affect the regulation of cAMP levels by dopamine D(2)-like receptors in vitro in rat cortical neurons in culture and in vivo in the rat prefrontal cortex. METHODS: The production of cAMP was measured in the cultured cortical neurons or in microdialysis samples collected from the prefrontal cortex of freely moving rats using the [8-(3)H] and [(125)I] radioimmunoassay kits. RESULTS: In vitro and in vivo data showed that the treatment with the mood stabilizing drugs had no effect on basal cAMP levels in vitro, but had differential effects in vivo. Direct stimulation of adenylate cyclase (AC) with forskolin increased cAMP levels both in vitro and in vivo, and this effect was significantly inhibited by all three mood stabilizers. Activation of dopamine D(2)-like receptors with quinpirole partially inhibited forskolin-induced increase in cAMP in untreated cultures, but no effect was observed in cortical neuron cultures treated with the mood stabilizing drugs. Similar results were obtained by chronic treatment with lithium and valproate in the prefrontal cortex in vivo. However, surprisingly, in carbamazepine-treated rats the activation of dopamine D(2)-like receptors enhanced the responsiveness of AC to subsequent activation by forskolin, possibly as a consequence of chronic inhibition of the activity of the enzyme. CONCLUSIONS: It was shown that each of these drugs affects basal- and forskolin-evoked cAMP levels in a distinct way, resulting in differential responses to dopamine D(2)-like receptors activation.

The interaction between dopamine D2-like and beta-adrenergic receptors in the prefrontal cortex is altered by mood-stabilizing agents.

Several studies have suggested the involvement of biogenic monoaminergic neurotransmission in bipolar disorder and in the therapy for this disease. In this study, the effects of the mood-stabilizing drugs lithium, carbamazepine or valproate on the dopaminergic and adrenergic systems, particularly on D2-like and beta-adrenergic receptors, were studied both in cultured rat cortical neurones and in rat prefrontal cortex. In vitro and in vivo data showed that stimulation of beta-adrenergic receptors with isoproterenol increased cyclic adenosine monophosphate (cAMP) levels and this effect was significantly inhibited by lithium, carbamazepine or valproate. The activation of dopamine D2-like receptors with quinpirole decreased the isoproterenol-induced rise in cAMP in control conditions. This inhibition was observed in vivo after chronic treatment of the rats with carbamazepine or valproate, but not after treatment with lithium or in cultured rat cortical neurones after 48 h exposure to the three mood stabilizers. Dopamine D2 and beta1-adrenergic receptors were found to be co-localized in prefrontal cortical cells, as determined by immunohistochemistry, but western blot experiments revealed that receptor levels were differentially affected by treatment with the three mood stabilizers. These data show that mood stabilizers affect D2 receptor-mediated regulation of beta-adrenergic signalling and that each drug acts by a unique mechanism.

Tricarboxylic acid cycle inhibition by Li+ in the human neuroblastoma SH-SY5Y cell line: a 13C NMR isotopomer analysis.

Li+ effects on glucose metabolism and on the competitive metabolism of glucose and lactate were investigated in the human neuroblastoma SH-SY5Y cell line using 13C NMR spectroscopy. The metabolic model proposed for glucose and lactate metabolism in these cells, based on tcaCALC best fitting solutions, for both control and Li+ conditions, was consistent with: (i) a single pyruvate pool; (ii) anaplerotic flux from endogenous unlabelled substrates; (iii) no cycling between pyruvate and oxaloacetate. Li+ was shown to induce a 38 and 53% decrease, for 1 and 15 mM Li+, respectively, in the rate of glucose conversion into pyruvate, when [U-13C]glucose was present, while no effects on lactate production were observed. Pyruvate oxidation by the tricarboxylic acid cycle and citrate synthase flux were shown to be significantly reduced by 64 and 84% in the presence of 1 and 15 mM Li+, respectively, suggesting a direct inhibitory effect of Li+ on tricarboxylic acid cycle flux. This work also showed that when both glucose and lactate are present as energetic substrates, SH-SY5Y cells preferentially consumed exogenous lactate over glucose, as 62% of the acetyl-CoA was derived from [3-13C]lactate while only 26% was derived from [U-13C]glucose. Li+ did not significantly affect the relative utilisation of these two substrates by the cells or the residual contribution of unlabelled endogenous sources for the acetyl-CoA pool.

Quantification and localization of intracellular free mg in bovine chromaffin cells.

Magnesium is an essential element for all living systems. The quantification of free intracellular Mg(2+) concentration ([Mg(2+)](i)) is of utmost importance since changes in its basal value may be an indication of different pathologies due to abnormalities of Mg(2+) metabolism. In this work we used (31)P NMR and fluorescence spectroscopy to determine the resting [Mg(2+)](i) in bovine chromaffin cells, a neuron-like cellular model, as well as confocal laser scanning microscopy to study the free Mg(2+) spatial distribution in these cells. (31)P NMR spectroscopy did not prove to be effective for the determination of [Mg(2+)](i) in this particular case due to some special morphological and physiological properties of this cell type. A basal [Mg(2+)](i) value of 0.551 +/- 0.008 mM was found for these cells using fluorescence spectroscopy and the Mg(2+)-sensitive probe furaptra; this value falls in the concentration range reported in the literature for neurons from different sources. This technique proved to be an accurate and sensitive tool to determine the [Mg(2+)](i).lntraceilular free Mg(2+) seems to be essentially localized in the nucleus and around it, as shown by confocal microscopy with the Mg(2+)-sensitive probe Magnesium Green. It was not possible to derive any conclusion about free Mg(2+) localization inside the chromaffin granules and/or in the cytoplasm due to the lack of sufficient spatial resolution and to probe compartmentalization.