Author Correction: A novel homozygous mutation in GAD1 gene described in a schizophrenic patient impairs activity and dimerization of GAD67 enzyme.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

Depression is a debilitating mental disease, characterized by persistent low mood and anhedonia. Stress represents a major environmental risk factor for depression; the complex interaction of stress with genetic factors results in different individual vulnerability or resilience to the disorder. Dysfunctions of the glutamate system have a primary role in depression. Clinical neuroimaging studies have consistently reported alterations in volume and connectivity of cortico-limbic areas, where glutamate neurons and synapses predominate. This is confirmed by preclinical studies in rodents, showing that repeated stress induces morphological and functional maladaptive changes in the same brain regions altered in humans. Confirming the key role of glutamatergic transmission in depression, compelling evidence has shown that the non-competitive NMDA receptor antagonist, ketamine, induces, at sub-anesthetic dose, rapid and sustained antidepressant response in both humans and rodents. We show here that the Chronic Mild Stress model of depression induces, only in stress-vulnerable rats, depressed-like anhedonic behavior, together with impairment of glutamate/GABA presynaptic release, BDNF mRNA trafficking in dendrites and dendritic morphology in hippocampus. Moreover, we show that a single administration of ketamine restores, in 24 h, normal behavior and most of the cellular/molecular maladaptive changes in vulnerable rats. Interestingly, ketamine treatment did not restore BDNF mRNA levels reduced by chronic stress but rescued dendritic trafficking of BDNF mRNA. The present results are consistent with a mechanism of ketamine involving rapid restoration of synaptic homeostasis, through re-equilibration of glutamate/GABA release and dendritic BDNF for synaptic translation and reversal of synaptic and circuitry impairment.

A novel homozygous mutation in GAD1 gene described in a schizophrenic patient impairs activity and dimerization of GAD67 enzyme.

Recently, by whole exome sequencing of schizophrenia (SCZ) patients, we identified a subject that was homozygous for a novel missense substitution (c.391 A > G) in the glutamate acid decarboxylase 1 (GAD1) gene. GAD1 encodes for GAD67 enzyme, catalyzing the production of gamma-aminobutyric acid (GABA) from L-glutamic acid. Here, we studied the impact of this mutation on GAD67 activity, dimerization and subcellular localization. Biochemical assay revealed that c.391 A > G reduces GAD67 enzymatic activity by ~30%, probably due to the impaired homodimerization of homozygous mutants as highlighted by proximity ligation assays. The mutational screening of 120 genes of the "GABAergic system" in a cohort of 4,225 SCZ cases and 5,834 controls (dbGaP: phs000473.v1.p2), did not identify other cases that were homozygous for ultra-rare variants in GAD1, but highlighted an increased frequency of cases that were homozygous for rare variants in genes of the GABA system (SCZ: 0.14% vs. Controls: 0.00%; p-value = 0.0055). In conclusion, this study demonstrates the functional impact of c.391 A > G variant and its biological effect makes it a good candidate as risk variant for SCZ. This study also supports an involvement of ultra-rare variants in GABAergic genes in the etiopathogenesis of SCZ.

Differential Enzymatic Activity of Rat ADAR2 Splicing Variants Is Due to Altered Capability to Interact with RNA in the Deaminase Domain.

In mammals, adenosine (A) to inosine (I) RNA editing is performed by adenosine deaminases acting on RNA (ADAR), ADAR1 and ADAR2 enzymes, encoded by mRNAs that might undergo splicing process. In rat, two splicing events produce several isoforms of ADAR2, called ADAR2a, ADAR2b, ADAR2e, and ADAR2f, but only ADAR2a and ADAR2b are translated into an active protein. In particular, they differ for ten amino acids located in the catalytic domain of ADAR2b. Here, we focused on these two isoforms, analyzing the splicing pattern and their different function during rat neuronal maturation. We found an increase of editing levels in cortical neurons overexpressing ADAR2a compared to those overexpressing ADAR2b. These results indicate ADAR2a isoform as the most active one, as reported for the homologous human short variant. Furthermore, we showed that the differential editing activity is not due to a different dimerization of the two isoforms; it seems to be linked to the ten amino acids loop of ADAR2b that might interfere with RNA binding, occupying the space volume in which the RNA should be present in case of binding. These data might shed light on the complexity of ADAR2 regulations.

Absence of the Fragile X Mental Retardation Protein results in defects of RNA editing of neuronal mRNAs in mouse.

The fragile X syndrome (FXS), the most common form of inherited intellectual disability, is due to the absence of FMRP, a protein regulating RNA metabolism. Recently, an unexpected function of FMRP in modulating the activity of Adenosine Deaminase Acting on RNA (ADAR) enzymes has been reported both in Drosophila and Zebrafish. ADARs are RNA-binding proteins that increase transcriptional complexity through a post-transcriptional mechanism called RNA editing. To evaluate the ADAR2-FMRP interaction in mammals we analyzed several RNA editing re-coding sites in the fmr1 knockout (KO) mice. Ex vivo and in vitro analysis revealed that absence of FMRP leads to an increase in the editing levels of brain specific mRNAs, indicating that FMRP might act as an inhibitor of editing activity. Proximity Ligation Assay (PLA) in mouse primary cortical neurons and in non-neuronal cells revealed that ADAR2 and FMRP co-localize in the nucleus. The ADAR2-FMRP co-localization was further observed by double-immunogold Electron Microscopy (EM) in the hippocampus. Moreover, ADAR2-FMRP interaction appeared to be RNA independent. Because changes in the editing pattern are associated with neuropsychiatric and neurodevelopmental disorders, we propose that the increased editing observed in the fmr1-KO mice might contribute to the FXS molecular phenotypes.

The Good and the Bad of Glutamate Receptor RNA Editing.

Glutamate receptors play a key role in excitatory synaptic transmission and plasticity in the central nervous system (CNS). Their channel properties are largely dictated by the subunit composition of tetrameric receptors. Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate channels are assembled from GluA1-4 AMPA or GluK1-5 kainate receptor subunits. However, their functional properties are highly modulated by a post-transcriptional mechanism called RNA editing. This process involves the enzymatic deamination of specific adenosines (A) into inosines (I) in pre-messenger RNA. This post-transcriptional modification leads to critical amino acid substitutions in the receptor subunits, which induce profound alterations of the channel properties. Three of the four AMPA and two of the five kainate receptor subunits are subjected to RNA editing. This study reviews the advances in understanding the importance of glutamate receptor RNA editing in finely tuning glutamatergic neurotransmission under physiological conditions and discusses the way in which the dis-regulation of RNA editing may be involved in neurological pathology.

Acute Footshock Stress Induces Time-Dependent Modifications of AMPA/NMDA Protein Expression and AMPA Phosphorylation.

Clinical studies on patients with stress-related neuropsychiatric disorders reported functional and morphological changes in brain areas where glutamatergic transmission is predominant, including frontal and prefrontal areas. In line with this evidence, several preclinical works suggest that glutamate receptors are targets of both rapid and long-lasting effects of stress. Here we found that acute footshock- (FS-) stress, although inducing no transcriptional and RNA editing alterations of ionotropic AMPA and NMDA glutamate receptor subunits, rapidly and transiently modulates their protein expression, phosphorylation, and localization at postsynaptic spines in prefrontal and frontal cortex. In total extract, FS-stress increased the phosphorylation levels of GluA1 AMPA subunit at Ser(845) immediately after stress and of GluA2 Ser(880) 2 h after start of stress. At postsynaptic spines, stress induced a rapid decrease of GluA2 expression, together with an increase of its phosphorylation at Ser(880), suggesting internalization of GluA2 AMPA containing receptors. GluN1 and GluN2A NMDA receptor subunits were found markedly upregulated in postsynaptic spines, 2 h after start of stress. These results suggest selected time-dependent changes in glutamatergic receptor subunits induced by acute stress, which may suggest early and transient enhancement of AMPA-mediated currents, followed by a transient activation of NMDA receptors.

Molecular characterization of LASP-1 expression reveals vimentin as its new partner in human hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related mortality worldwide. We have previously reported that LASP-1 is a downstream protein of the urokinase type plasminogen activator (uPA). Here we investigated the role of LASP-1 in HCC by a molecular and biological characterization of LASP-1 expression in human HCC specimens and in cultured HCC cells. We determined the LASP-1 mRNA expression levels in 55 HCC cases with different hepatic background disease. We identified 3 groups of patients with high, equal or low LASP-1 mRNA levels in HCC tissues compared to the peritumoral (PT) tissues. In particular we found that i) the HCCs displayed a higher LASP-1 mRNA level in HCC compared to PT tissues; ii) the expression levels of LASP-1 mRNA in female HCCs were significantly higher compared to male HCCs; iii) the cirrhotic HCCs displayed a higher LASP-1 mRNA. Further, the biological characterization of the ectopic LASP-1 overexpression in HCC cells, using MALDI-TOF mass spectrometer on the LASP-1 co-immunoprecipitated fractions, displayed vimentin as a novel putative partner of LASP-1. Our results suggest that LASP-1 mRNA overexpression may be mainly implicated in female HCCs and cirrhotic HCCs; and that LASP1 may play its role with vimentin in HCC cells.

Chronic glutamate treatment selectively modulates AMPA RNA editing and ADAR expression and activity in primary cortical neurons.

Adenosine-to-inosine RNA editing is a post-transcriptional process, catalyzed by ADAR enzymes, with an important role in diversifying the number of proteins derived from a single gene. In neurons, editing of ionotropic AMPA glutamate receptors has been shown to be altered under several experimental conditions, including severe pathologies, thus highlighting the potential significance of its modulation. In this study, we treated rat primary cortical cell cultures with a sub-lethal dose of glutamate (10 µM), focusing on RNA editing and ADAR activity. We found that chronic glutamate treatment down-regulates RNA editing levels at the R/G site of GluA2-4 subunits of AMPA receptors and at the K/E site of CYFIP2. These changes are site-specific since they were not observed either for the GluA2 Q/R site or for other non-glutamatergic sites. Glutamate treatment also down-regulates the protein expression levels of both ADAR1 and ADAR2 enzymes, through a pathway that is Ca(2+)- and calpain-dependent. Given that AMPA receptors containing unedited subunits show a slower recovery rate from desensitization compared to those containing edited forms, the reduced editing at the R/G site may, at least in part, compensate for glutamate over-stimulation, perhaps through the reduced activation of postsynaptic receptors. In summary, our data provide direct evidence of the involvement of ADAR1 and ADAR2 activity as a possible compensatory mechanism for neuronal protection following glutamate over-stimulation.

Kainate receptor RNA editing is markedly altered by acute spinal cord injury.

We have previously observed changes in the RNA editing of AMPA receptors after acute spinal cord injury (SCI); this implies that post-transcriptional modifications are capable of affecting the physiological properties of glutamate receptor channels and related signal transduction in this neurodegenerative condition. Here, we report that the editing of the ionotropic KAR is markedly decreased at both GluK1 and GluK2 Q/R sites in the epicenter of the lesion and with distinct magnitude and kinetics also in the caudal and rostral portions of the injured cord. These effects are persistent, being observed as late as 30 days after lesioning. In addition, also the I/V and Y/C sites of GluK2 were severely affected after SCI. These findings add novel information to the relevance of editing of glutamate receptors following acute SCI, thus expanding the recently emerged role of post-transcriptional mechanisms under these experimental conditions.

AMPA receptor properties are modulated in the early stages following pilocarpine-induced status epilepticus.

Glutamate over-activation and the consequent neuronal excitotoxicity have been identified as crucial players in brain dysfunctions such as status epilepticus (SE). Owing to the central function of 2-amino-3-(hydroxyl-5-methylisoxazole-4-yl) propionic acid receptors (AMPARs) in fast excitatory neurotransmission, these receptors have been recognized to play a prominent role in the development and generation of epileptic seizure. This study was undertaken to investigate both the early changes that affect glutamatergic neurons in the rat cerebral cortex and hippocampus and the level and channel properties of AMPARs in response to SE. The results obtained after 3 h of pilocarpine (PILO)-induced SE showed a disorganization of glutamatergic neurons in the CA3 and a thinner neuronal cell layer in the dentate gyrus (DG) region as compared with controls. A significant increase in AMPAR GluA2 protein expression, a decrease in GluA1, GluA3, and GluA4 expression, and a reduction in the phosphorylation of Ser831-GluA1 and Ser880-GluA2 were also observed. In addition, we report a downregulation of R/G editing levels and of Flip splicing isoforms, with a prominent effect on the hippocampus of PILO-treated rats. Our results suggest the presence of an attenuation of AMPARs' post-synaptic excitatory response to glutamate after PILO treatment, thus conferring neuronal protection from the excitotoxic conditions observed in the SE. This study suggests a role for AMPARs in alterations of the glutamatergic pathway during the onset and early progression of epilepsy, thus indicating additional targets for potential therapeutic interventions.

Modulation of dendritic AMPA receptor mRNA trafficking by RNA splicing and editing.

RNA trafficking to dendrites and local translation are crucial processes for superior neuronal functions. To date, several α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR) mRNAs have been detected in dendrites and are subject to local protein synthesis. Here, we report the presence of all AMPAR GluA1-4 mRNAs in hippocampal and cortical rat synaptic spines by synaptoneurosomes analysis. In particular, we showed that dendritic AMPAR mRNAs are present in the Flip versions in the cortex and hippocampus. To further confirm these data, we demonstrate, using in situ hybridization, the dendritic localization of the GluA2 Flip isoform in vitro and in vivo, whereas the Flop variant is restricted mainly to the soma. In addition, we report that dendritic AMPA mRNAs are edited at low levels at their R/G sites; this result was also supported with transfection experiments using chimeric GluA2 DNA vectors, showing that transcripts carrying an unedited nucleotide at the R/G site, in combination with the Flip exon, are more efficiently targeted to dendrites when compared with the edited-Flip versions. Our data show that post-transcriptional regulations such as RNA splicing, editing and trafficking might be mutually coordinated and that the localization of different AMPAR isoforms in dendrites might play a functional role in the regulation of neuronal transmission.

AMPA receptor regulation at the mRNA and protein level in rat primary cortical cultures.

Ionotropic glutamate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors are the major mediators of fast synaptic neurotransmission. In this work, we used primary cortical cultures from rats as a model system to study AMPA receptor regulation during in vitro cell maturation and after synaptic activity modifications. The levels of AMPA receptor mRNA and protein, along with the alternative splicing and RNA editing of the AMPA receptor subunit (GluR1-4) mRNAs, were analyzed in immature (DIV5) and mature (DIV26) rat neuronal cultures. We observed an increase in the expression of all four AMPA receptor subunits during in vitro neuronal maturation. This finding might be due to the formation of new synapses between neurons during the development of a complex neuronal network. We also analyzed the effects of stimulation (KCl and glutamate) and inhibition (APV/TTX) on rat mature neuronal cultures (DIV26): stimulation with KCl led to an overall down-regulation of GluR1 and GluR3 AMPA receptor subunits and an up-regulation of the GluR2 subunit. Similarly, glutamate treatment induced a significant down-regulation of GluR1 together with an up-regulation of GluR2. In contrast, the chronic blockade of neuronal activity that resulted from APV/TTX treatment up-regulated GluR1 and GluR3 with a parallel down-regulation of GluR2 and GluR4. RNA editing at the R/G site increased during neuronal cell maturation for all AMPA receptors (from 8-39% at DIV5 to 28-67% at DIV26). Unexpectedly, all the treatments tested induced a marked reduction (ranging from -9% to -52%) of R/G editing levels in mature neurons, primarily for the mRNA flip variant. In summary, we showed that cultured rat cortical neurons are able to vary the stoichiometric ratios of the AMPA receptor subunits and to control post-transcriptional processes to adapt fast synaptic transmission under different environmental conditions.

Chronic antidepressant treatments induce a time-dependent up-regulation of AMPA receptor subunit protein levels.

Growing evidence suggests a pivotal role for glutamatergic neurotransmission in the pathophysiology of major depressive disorder and in the action of antidepressants. The main aim of this study was to elucidate the temporal profile of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors expression and their functional regulation in prefrontal/frontal cortex (P/FC) and hippocampus (HC) of rats chronically treated with two different antidepressants: fluoxetine (FLX) and reboxetine (RBX). Rat groups were treated for 1, 2 or 3 weeks with the two drugs and, in additional groups, the treatments were followed by 1 week of drug washout (3+1). We found that both drugs induced strong increases in AMPAR subunit protein expression that were time dependent and subunit specific. Especially in P/FC, FLX had the main effect on GluA2 and GluA4 subunits, reaching a 5-fold increase after the drug washout; RBX mostly affected GluA1 and GluA3, reaching a 4-fold increase at the end of the treatment. Furthermore, in HC, the two drugs induced a time specific increase in subunit protein levels, with GluA3 and GluA4 presenting the main changes, albeit with different kinetics. In addition, our data indicate that antidepressants might alter, though by small changes, the R/G editing levels for GluA2, mostly in P/FC, and in turn may induce fine-tuning of glutamate neurotransmission. Overall, we showed that antidepressant treatments induced marked changes in AMPA receptor subunits expression, with time-dependent effects that are consistent with the onset of therapeutic effect of these drugs. These data confirm the involvement of glutamate neurotransmission in the effects of these drugs and further suggest the targeting of AMPA receptors as a therapeutic approach for the treatment of depression.

Mesenchymal cells from human amniotic fluid survive and migrate after transplantation into adult rat brain.

Amniotic fluid has been recently suggested as an alternative source of mesenchymal stem cells. However, the fate of amniotic fluid-derived mesenchymal stem cells (AF-MSCs) after in vivo transplantation has yet to be determined. In the present study we explored whether human AF-MSCs could survive and migrate following transplantation into the striatum of normal and ischemic rat. We found that the grafted cells could survive and migrate towards multiple brain regions in the normal animals, while they moved towards the injured region in the ischemic rat. Double-immunostaining analyses showed that the implanted human AF-MSCs express markers for immature neurons (Doublecortin) at 10 days, and for astrocytes (GFAP) at 10, 30 and 90 after transplantation. This study provides the first evidence that human amniotic fluid contains cells having the potential to survive and integrate into adult rat brain tissue and, therefore, to function as effective stem cells for therapeutic strategies.