Global epigenetic analysis of BDNF Val66Met mice hippocampus reveals changes in dendrite and spine remodeling genes.

Brain-derived neurotrophic factor (BDNF), a neurotrophin highly expressed in the hippocampus, plays crucial roles in cognition, neuroplasticity, synaptic function, and dendritic remodeling. The common human Val66Met polymorphism of BDNF has been implicated in the pathophysiology of neuropsychiatric and neurodegenerative disorders, and in the outcome of pro-adaptive and therapeutic treatments. Altered gene-expression profile has been previously shown in BDNF Val66Met knock-in mice, which recapitulate the phenotypic hallmarks of individuals carrying the BDNF Met allele. The aim of this study was to investigate the impact of the BDNF Val66Met polymorphism in the knock-in mouse model on two hippocampal epigenetic marks for transcriptional repression and activation, respectively: trimethylation of lysine 27 on histone H3 (H3K27me3) and acetylation of histone H3 (AcH3), using a genome-wide approach. Chromatin immunoprecipitation followed by deep sequencing of immunoprecipitated DNA (ChIP-Seq) was carried out with specific antibodies for H3K27me3 and AcH3. Our results revealed broad alteration of H3K27me3 and AcH3 marks association profiles in BDNFMet/Met , compared to BDNFVal/Val mice. Bioinformatics analysis showed changes in several biological functions and related pathways, affected by the presence of the polymorphism. In particular, a number of networks of functional interaction contained BDNF as central node. Quantitative PCR analysis confirmed epigenetically related significant changes in the expression of five genes: Dvl1, Nos3, Reln, Lypd6, and Sh3gl2. The first three are involved in dendrite and spine remodeling, morphological features altered in BDNFMet/Met mice. This work in homozygous knock-in mice shows that the human BDNF Val66Met polymorphism induces an array of histone H3 epigenetic modifications, in turn altering the expression of select genes crucial for structural and functional neuronal features.

Time-dependent activation of MAPK/Erk1/2 and Akt/GSK3 cascades: modulation by agomelatine.

BACKGROUND: The novel antidepressant agomelatine, a melatonergic MT1/MT2 agonist combined with 5-HT2c serotonin antagonist properties, showed antidepressant action in preclinical and clinical studies. There is a general agreement that the therapeutic action of antidepressants needs the activation of slow-onset adaptations in downstream signalling pathways finally regulating neuroplasticity. In the last several years, particular attention was given to cAMP-responsive element binding protein (CREB)-related pathways, since it was shown that chronic antidepressants increase CREB phosphorylation and transcriptional activity, through the activation of calcium/calmodulin-dependent (CaM) and mitogen activated protein kinase cascades (MAPK/Erk1/2). Aim of this work was to analyse possible effects of chronic agomelatine on time-dependent changes of different intracellular signalling pathways in hippocampus and prefrontal/frontal cortex of male rats. To this end, measurements were performed 1 h or 16 h after the last agomelatine or vehicle injection. RESULTS: We have found that in naïve rats chronic agomelatine, contrary to traditional antidepressants, did not increase CREB phosphorylation, but modulates the time-dependent regulation of MAPK/Erk1/2 and Akt/glycogen synthase kinase-3 (GSK-3) pathways. CONCLUSION: Our results suggest that the intracellular molecular mechanisms modulated by chronic agomelatine may be partly different from those of traditional antidepressants and involve the time-dependent regulation of MAPK/Erk1/2 and Akt/GSK-3 signalling pathways. This could exert a role in the antidepressant efficacy of the drug.

Chronic treatment with agomelatine or venlafaxine reduces depolarization-evoked glutamate release from hippocampal synaptosomes.

BACKGROUND: Growing compelling evidence from clinical and preclinical studies has demonstrated the primary role of alterations of glutamatergic transmission in cortical and limbic areas in the pathophysiology of mood disorders. Chronic antidepressants have been shown to dampen endogenous glutamate release from rat hippocampal synaptic terminals and to prevent the marked increase of glutamate overflow induced by acute behavioral stress in frontal/prefrontal cortex. Agomelatine, a new antidepressant endowed with MT1/MT2 agonist and 5-HT2C serotonergic antagonist properties, has shown efficacy at both preclinical and clinical levels. RESULTS: Chronic treatment with agomelatine, or with the reference drug venlafaxine, induced a marked decrease of depolarization-evoked endogenous glutamate release from purified hippocampal synaptic terminals in superfusion. No changes were observed in GABA release. This effect was accompanied by reduced accumulation of SNARE protein complexes, the key molecular effector of vesicle docking, priming and fusion at presynaptic membranes. CONCLUSIONS: Our data suggest that the novel antidepressant agomelatine share with other classes of antidepressants the ability to modulate glutamatergic transmission in hippocampus. Its action seems to be mediated by molecular mechanisms located on the presynaptic membrane and related with the size of the vesicle pool ready for release.

Abnormal exocytotic release of glutamate in a mouse model of amyotrophic lateral sclerosis.

Glutamate-mediated excitotoxicity plays a major role in the degeneration of motor neurons in amyotrophic lateral sclerosis and reduced astrocytary glutamate transport, which in turn increases the synaptic availability of the amino acid neurotransmitter, was suggested as a cause. Alternatively, here we report our studies on the exocytotic release of glutamate as a possible source of excessive glutamate transmission. The basal glutamate efflux from spinal cord nerve terminals of mice-expressing human soluble superoxide dismutase (SOD1) with the G93A mutation [SOD1/G93A(+)], a transgenic model of amyotrophic lateral sclerosis, was elevated when compared with transgenic mice expressing the wild-type human SOD1 or to non-transgenic controls. Exposure to 15 mM KCl or 0.3 µM ionomycin provoked Ca(2+)-dependent glutamate release that was dramatically increased in late symptomatic and in pre-symptomatic SOD1/G93A(+) mice. Increased Ca(2+) levels were detected in SOD1/G93A(+) mouse spinal cord nerve terminals, accompanied by increased activation of Ca(2+)/calmodulin-dependent kinase II and increased phosphorylation of synapsin I. In line with these findings, release experiments suggested that the glutamate release augmentation involves the readily releasable pool of vesicles and a greater capability of these vesicles to fuse upon stimulation in SOD1/G93A(+) mice.

Antidepressant treatments change 5-HT2C receptor mRNA expression in rat prefrontal/frontal cortex and hippocampus.

BACKGROUND/AIMS: Compelling evidence would suggest the involvement of the serotonin 2C receptor in the pathophysiology of affective disorders and in the action of antidepressants. We analyzed the time course of 5-HT2C receptor (5-HTR2C) mRNA expression during antidepressant treatment in the prefrontal/frontal cortex (P/FC) and in the hippocampus (HC) of rats chronically treated with fluoxetine (a selective serotonin reuptake inhibitor) and reboxetine (a selective noradrenaline reuptake inhibitor). We also analyzed the 5-HTR2C RNA-editing levels at the sites called A, B, C, C' and D, which are known to modulate 5-HTR2C receptor function. RESULTS: The expression profile of 5-HTR2C mRNA was modified during treatment with both antidepressants. In particular, we found a general down-regulation of 5-HTR2C mRNA expression in P/FC, which became significant after 3 weeks of treatment with both antidepressants and persisted after a fourth week of drug withdrawal (-46% with fluoxetine, -41% with reboxetine, p < 0.05). In HC, however, reboxetine induced significant down-regulation (-56%, p < 0.05) of 5-HTR2C mRNA after 3 weeks, while fluoxetine induced threefold up-regulation (p < 0.01) by the 2nd and 3rd week, returning to the base level after drug withdrawal of both antidepressants. Moreover, the frequency of 5-HTR2C-edited isoforms showed no significant alterations, although analysis of the RNA-editing level at the single editing sites showed small decreases in the C' and D sites induced by reboxetine in P/FC. CONCLUSION: Our results suggest that chronic administration of antidepressants in rats slightly modifies the editing levels of 5-HT2C receptor but has considerable influence on its mRNA expression patterns in a way that is area- and time-specific.

miR-9-5p is involved in the rescue of stress-dependent dendritic shortening of hippocampal pyramidal neurons induced by acute antidepressant treatment with ketamine.

Converging clinical and preclinical evidence demonstrates that depressive phenotypes are associated with synaptic dysfunction and dendritic simplification in cortico-limbic glutamatergic areas. On the other hand, the rapid antidepressant effect of acute ketamine is consistently reported to occur together with the rescue of dendritic atrophy and reduction of spine number induced by chronic stress in the hippocampus and prefrontal cortex of animal models of depression. Nevertheless, the molecular mechanisms underlying these morphological alterations remain largely unknown. Here, we found that miR-9-5p levels were selectively reduced in the hippocampus of rats vulnerable to Chronic Mild Stress (CMS), while acute subanesthetic ketamine restored its levels to basal condition in just 24h; miR-9-5p expression inversely correlated with the anhedonic phenotype. A decrease of miR-9-5p was reproduced in an in vitro model of stress, based on primary hippocampal neurons incubated with the stress hormone corticosterone. In both CMS animals and primary neurons, decreased miR-9-5p levels were associated with dendritic simplification, while treatment with ketamine completely rescued the changes. In vitro modulation of miR-9-5p expression showed a direct role of miR-9-5p in regulating dendritic length and spine density in mature primary hippocampal neurons. Among the putative target genes tested, Rest and Sirt1 were validated as biological targets in primary neuronal cultures. Moreover, in line with miR-9-5p changes, REST protein expression levels were remarkably increased in both CMS vulnerable animals and corticosterone-treated neurons, while ketamine completely abolished this alteration. Finally, the shortening of dendritic length in corticosterone-treated neurons was shown to be partly rescued by miR-9-5p overexpression and dependent on REST protein expression. Overall, our data unveiled the functional role of miR-9-5p in the remodeling of dendritic arbor induced by stress/corticosterone in vulnerable animals and its rescue by acute antidepressant treatment with ketamine.

Blockade of stress-induced increase of glutamate release in the rat prefrontal/frontal cortex by agomelatine involves synergy between melatonergic and 5-HT2C receptor-dependent pathways.

BACKGROUND: Agomelatine is a melatonergic receptor agonist and a 5HT2C receptor antagonist that has shown antidepressant efficacy. In order to analyze separately the effect of the two receptorial components, rats were chronically treated with agomelatine, melatonin (endogenous melatonergic agonist), or S32006 (5-HT2C antagonist), and then subjected to acute footshock-stress. RESULTS: Only chronic agomelatine, but not melatonin or S32006, completely prevented the stress-induced increase of glutamate release in the rat prefrontal/frontal cortex. CONCLUSIONS: These results suggest a potential synergy between melatonergic and serotonergic pathways in the action of agomelatine.

Blues in the Brain and Beyond: Molecular Bases of Major Depressive Disorder and Relative Pharmacological and Non-Pharmacological Treatments.

Despite the extensive research conducted in recent decades, the molecular mechanisms underlying major depressive disorder (MDD) and relative evidence-based treatments remain unclear. Various hypotheses have been successively proposed, involving different biological systems. This narrative review aims to critically illustrate the main pathogenic hypotheses of MDD, ranging from the historical ones based on the monoaminergic and neurotrophic theories, through the subsequent neurodevelopmental, glutamatergic, GABAergic, inflammatory/immune and endocrine explanations, until the most recent evidence postulating a role for fatty acids and the gut microbiota. Moreover, the molecular effects of established both pharmacological and non-pharmacological approaches for MDD are also reviewed. Overall, the existing literature indicates that the molecular mechanisms described in the context of these different hypotheses, rather than representing alternative ones to each other, are likely to contribute together, often with reciprocal interactions, to the development of MDD and to the effectiveness of treatments, and points at the need for further research efforts in this field.

Early raise of BDNF in hippocampus suggests induction of posttranscriptional mechanisms by antidepressants.

BACKGROUND: The neurotrophin BDNF has been implicated in the regulation of neuroplasticity, gene expression, and synaptic function in the adult brain, as well as in the pathophysiology of neuropsychiatric disorders and the mechanism of action of antidepressants. Antidepressant treatments have been shown to increase the expression of BDNF mRNA, although the changes measured were found to be different depending on various factors. A few studies only have measured levels of BDNF protein after antidepressant treatments, and poor correlation was found between mRNA and protein changes. We studied the time course of expression of BDNF mRNA and protein during drug treatments, in order to elucidate the temporal profile of regulation of this effector and whether mRNA and protein levels correlate. Rat groups were treated for 1, 2 or 3 weeks with fluoxetine or reboxetine; in additional groups drug treatment was followed by a washout week (3+1). Total BDNF mRNA was measured by Real Time PCR, pro- and mature BDNF proteins were measured by Western blot. RESULTS: We found that mature BDNF protein is induced more rapidly than mRNA, by both drugs in hippocampus (weeks 1-2) and by reboxetine in prefrontal/frontal cortex (week 1). The temporal profile of BDNF protein expression was largely inconsistent with that of mRNA, which followed the protein induction and reached a peak at week 3. CONCLUSION: These results suggest that BDNF protein is rapidly elevated by antidepressant treatments by posttranscriptional mechanisms, and that induction of BDNF mRNA is a slower process.

Early induction of CREB activation and CREB-regulating signalling by antidepressants.

Converging evidence points to adaptive changes in neuroplasticity and gene expression as mediators of therapeutic action of antidepressants. Activation of cAMP response-element binding protein (CREB) and CREB-regulating signalling are considered main effectors in these mechanisms. We analysed the temporal profile of intracellular changes induced by antidepressants, by measuring activation of major CREB-regulating signalling cascades and activation (Ser133 phosphorylation) of CREB. The main aims of the study were to investigate how these different variables are modulated with time, whether stronger activation of signalling cascades corresponds to stronger activation of CREB, and whether these changes are different in distinct brain areas. Rat groups were treated for 1, 2 or 3 wk with the antidepressants fluoxetine or reboxetine; in additional groups drug treatment was followed by a washout week (3+1). Activation of CREB and major effectors in signalling cascades were analysed by Western blot analysis with phospho-antibodies, in nuclear and cytosolic fractions from hippocampus and prefrontal/frontal cortex (P/FC). Surprisingly, CREB activation was already maximal after 1-wk treatment. In hippocampus early and stronger CREB activation was consistent with early and stronger activation of signalling. For both drugs, the profile of activation in P/FC was different from that observed in hippocampus. The results also showed that, contrary to the activatory role of MAP-ERKs and CaM kinase IV, nuclear alphaCaM kinase II was inactivated in parallel with activation of CREB.