Altered Mechanisms Underlying the Abnormal Glutamate Release in Amyotrophic Lateral Sclerosis at a Pre-Symptomatic Stage of the Disease.

Abnormal Glu release occurs in the spinal cord of SOD1(G93A) mice, a transgenic animal model for human ALS. Here we studied the mechanisms underlying Glu release in spinal cord nerve terminals of SOD1(G93A) mice at a pre-symptomatic disease stage (30days) and found that the basal release of Glu was more elevated in SOD1(G93A) with respect to SOD1 mice, and that the surplus of release relies on synaptic vesicle exocytosis. Exposure to high KCl or ionomycin provoked Ca(2+)-dependent Glu release that was likewise augmented in SOD1(G93A) mice. Equally, the Ca(2+)-independent hypertonic sucrose-induced Glu release was abnormally elevated in SOD1(G93A) mice. Also in this case, the surplus of Glu release was exocytotic in nature. We could determine elevated cytosolic Ca(2+) levels, increased phosphorylation of Synapsin-I, which was causally related to the abnormal Glu release measured in spinal cord synaptosomes of pre-symptomatic SOD1(G93A) mice, and increased phosphorylation of glycogen synthase kinase-3 at the inhibitory sites, an event that favours SNARE protein assembly. Western blot experiments revealed an increased number of SNARE protein complexes at the nerve terminal membrane, with no changes of the three SNARE proteins and increased expression of synaptotagmin-1 and \u3b2-Actin, but not of an array of other release-related presynaptic proteins. These results indicate that the abnormal exocytotic Glu release in spinal cord of pre-symptomatic SOD1(G93A) mice is mainly based on the increased size of the readily releasable pool of vesicles and release facilitation, supported by plastic changes of specific presynaptic mechanism

Analysis of miRNome expression profile in hippocampus of rats treated with antidepressants

MicroRNAs (miRNAs) are small, non-coding RNAs with a key role in regulating gene expression at post-transcriptional level. Recent investigations suggested that dysregulation in miRNA expression may be critical for mental disorders pathophysiology, including mood disorders, and in psychotropic drugs action, such as mood stabilizers and antidepressants (ADs). Aim of the studies presented was to analyze whether modulation of hippocampal miRNome could be part of the mode of action of different ADs, fluoxetine, desipramine and agomelatine. Moreover, in order to assess the time course of ADs effects, treatments were performed for various time lengths (3,7 or 21 days). Rat hippocampal miRNome expression was investigated by means of TaqMan Rodent MicroRNA Arrays. We found that all ADs induced a significant modulation of hippocampal miRNome, although with different time-dependent effects. Indeed, all ADs significant modulated miRNA expression already after 3 days of treatment with the most pronounced effect by agomelatine (37 miRNAs, mainly down-regulated); whereas both fluoxetine and desipramine modified the expression of 8 different miRNAs. After 7 days, the greater number of miRNAs was modulated by fluoxetine (35 miRNAs, mainly up-regulated), while desipramine and agomelatine modulated the expression of 13 and 22 miRNAs, respectively. Finally, 21 days of agomelatine treatment modulated the expression of 6 miRNAs. Interestingly, some of the miRNAs were found to be similarly affected by at least two of the different ADs. A bio-informatic analysis allowed the identification of putative miRNA targets and signalling pathways modulated by the identified miRNAs. The predicted target genes of miRNAs are involved in several pathways related to neuronal functions and ADs mechanism of action; moreover, many of the putative target genes, in particular those of miRs modulated by agomelatine, are enriched in pathways related to DNA/RNA regulation and epigenetic mechanisms. A number of putative miRNA target genes have been selected for validation studies by means of mRNA/protein expression analyses. The results of these work, showing that AD treatments induce early and time-dependent modifications in the expression of hippocampal miRNAs, could be of help to better clarify the ADs mechanism of action and to identify new putative targets for the development of novel drugs with greater efficacy and a more rapid onset of action

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

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

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.Peer reviewe

Chronic mild stress induces anhedonic behavior and changes in glutamate release, BDNF trafficking and dendrite morphology only in stress vulnerable rats. The rapid restorative action of ketamine

17siopenopenTornese, Paolo; Sala, Nathalie; Bonini, Daniela; Bonifacino, Tiziana; La Via, Luca; Milanese, Marco; Treccani, Giulia; Seguini, Mara; Ieraci, Alessandro; Mingardi, Jessica; Nyengaard, Jens R.; Calza, Stefano; Bonanno, Giambattista; Wegener, Gregers; Barbon, Alessandro; Popoli, Maurizio; Musazzi, LauraTornese, Paolo; Sala, Nathalie; Bonini, Daniela; Bonifacino, Tiziana; La Via, Luca; Milanese, Marco; Treccani, Giulia; Seguini, Mara; Ieraci, Alessandro; Mingardi, Jessica; Nyengaard, Jens R.; Calza, Stefano; Bonanno, Giambattista; Wegener, Gregers; Barbon, Alessandro; Popoli, Maurizio; Musazzi, Laur