Reduced expression of GluN2A induces a delay in neuron maturation.

NMDA receptors (NMDARs) play an important role in synaptic plasticity both in physiological and pathological conditions. GluN2A and GluN2B are the most expressed NMDAR regulatory subunits, in the hippocampus and other cognitive-related brain structures. GluN2B is characteristic of immature structures and GluN2A of mature ones. Changes in GluN2A expression were associated with complex phenotypes that led to complex neurodevelopmental disorders, including the occurrence of seizures. However, little is known about the role of GluN2A in these phenotypes. In this work, we reduced GluN2A expression in mature neuronal cultures and observed an altered GluN2A/GluN2B ratio. Furthermore, those neurons exhibit an increase in immature dendritic spines and dendritic branching, as well as an increased response to glutamate stimulus. This phenotype (considering GluN2A/GluN2B ratio, index branching and glutamate response) resembles those observed at immature neuronal stages in vitro. We propose that this immature phenotype led to a higher response to glutamate stimulus which, in vivo, would be the basis of reduced threshold for seizure onset in GluN2A-pathological conditions.

Impaired fasting glucose, oxidative distress, and cognitive impairment. Is this the starting point on DBT cognitive decline?

Different studies performed in human patients, animal models, and in vitro cell cultures, show a correlation between type 2 diabetes (DBT2) and certain neurodegenerative pathologies. Also, it was proposed that increased inflammation and- or oxidative distress are a possible cause of DBT2-accelerated cognitive decline. The onset of DBT2 is characterized by an increase in blood glucose levels due to (an inability of the body's cells to use insulin properly) called impaired fasting glucose (IFG). Genetic and/or molecular causes of IFG have not yet been established, but metabolic syndrome, obesity, unbalanced diets, and sedentary lifestyle would be responsible, at least in part, for the multiplication in the number of this disease. It has been proposed that hyperglycemia itself causes an imbalance in the redox state and could compromise blood-brain barrier (BBB) causing neurodegeneration. For this reason, we propose, in this review, to evaluate the available data about redox state and neurocognitive studies during the IFG period.

Reduced Expression of Hippocampal GluN2A-NMDAR Increases Seizure Susceptibility and Causes Deficits in Contextual Memory.

N-methyl-D-aspartate receptors are heterotetramers composed of two GluN1 obligatory subunits and two regulatory subunits. In cognitive-related brain structures, GluN2A and GluN2B are the most abundant regulatory subunits, and their expression is subjected to tight regulation. During development, GluN2B expression is characteristic of immature synapses, whereas GluN2A is present in mature ones. This change in expression induces a shift in GluN2A/GluN2B ratio known as developmental switch. Moreover, modifications in this relationship have been associated with learning and memory, as well as different pathologies. In this work, we used a specific shRNA to induce a reduction in GluN2A expression after the developmental switch, both in vitro in primary cultured hippocampal neurons and in vivo in adult male Wistar rats. After in vitro characterization, we performed a cognitive profile and evaluated seizure susceptibility in vivo. Our in vitro results showed that the decrease in the expression of GluN2A changes GluN2A/GluN2B ratio without altering the expression of other regulatory subunits. Moreover, rats expressing the anti-GluN2A shRNA in vivo displayed an impaired contextual fear-conditioning memory. In addition, these animals showed increased seizure susceptibility, in terms of both time and intensity, which led us to conclude that deregulation in GluN2A expression at the hippocampus is associated with seizure susceptibility and learning-memory mechanisms.

Early Long-Term Memory Impairment and Changes in the Expression of Synaptic Plasticity-Associated Genes, in the McGill-R-Thy1-APP Rat Model of Alzheimer's-Like Brain Amyloidosis.

Accruing evidence supports the hypothesis that memory deficits in early Alzheimer Disease (AD) might be due to synaptic failure caused by accumulation of intracellular amyloid beta (Aß) oligomers, then secreted to the extracellular media. Transgenic mouse AD models provide valuable information on AD pathology. However, the failure to translate these findings to humans calls for models that better recapitulate the human pathology. McGill-R-Thy1-APP transgenic (Tg) rat expresses the human amyloid precursor protein (APP751) with the Swedish and Indiana mutations (of familial AD), leading to an AD-like slow-progressing brain amyloid pathology. Therefore, it offers a unique opportunity to investigate learning and memory abilities at early stages of AD, when Aß accumulation is restricted to the intracellular compartment, prior to plaque deposition. Our goal was to further investigate early deficits in memory, particularly long-term memory in McGill-R-Thy1-APP heterozygous (Tg+/-) rats. Short-term- and long-term habituation to an open field were preserved in 3-, 4-, and 6-month-old (Tg+/-). However, long-term memory of inhibitory avoidance to a foot-shock, novel object-recognition and social approaching behavior were seriously impaired in 4-month-old (Tg+/-) male rats, suggesting that they are unable to either consolidate and/or evoke such associative and discriminative memories with aversive, emotional and spatial components. The long-term memory deficits were accompanied by increased transcript levels of genes relevant to synaptic plasticity, learning and memory processing in the hippocampus, such as Grin2b, Dlg4, Camk2b, and Syn1. Our findings indicate that in addition to the previously well-documented deficits in learning and memory, McGill-R-Thy1-APP rats display particular long-term-memory deficits and deep social behavior alterations at pre-plaque early stages of the pathology. This highlights the importance of Aß oligomers and emphasizes the validity of the model to study AD-like early processes, with potentially predictive value.

Notch1 is asymmetrically distributed from the beginning of embryogenesis and controls the ventral center.

Based on functional evidence, we have previously demonstrated that early ventral Notch1 activity restricts dorsoanterior development in Xenopus We found that Notch1 has ventralizing properties and abolishes the dorsalizing activity of ß-catenin by reducing its steady state levels, in a process that does not require ß-catenin phosphorylation by glycogen synthase kinase 3ß. In the present work, we demonstrate that Notch1 mRNA and protein are enriched in the ventral region from the beginning of embryogenesis in Xenopus This is the earliest sign of ventral development, preceding the localized expression of wnt8a, bmp4 and Ventx genes in the ventral center and the dorsal accumulation of nuclear ß-catenin. Knockdown experiments indicate that Notch1 is necessary for the normal expression of genes essential for ventral-posterior development. These results indicate that during early embryogenesis ventrally located Notch1 promotes the development of the ventral center. Together with our previous evidence, these results suggest that ventral enrichment of Notch1 underlies the process by which Notch1 participates in restricting nuclear accumulation of ß-catenin to the dorsal side.

NMDA Receptor Subunits Change after Synaptic Plasticity Induction and Learning and Memory Acquisition.

NMDA ionotropic glutamate receptors (NMDARs) are crucial in activity-dependent synaptic changes and in learning and memory. NMDARs are composed of two GluN1 essential subunits and two regulatory subunits which define their pharmacological and physiological profile. In CNS structures involved in cognitive functions as the hippocampus and prefrontal cortex, GluN2A and GluN2B are major regulatory subunits; their expression is dynamic and tightly regulated, but little is known about specific changes after plasticity induction or memory acquisition. Data strongly suggest that following appropriate stimulation, there is a rapid increase in surface GluN2A-NMDAR at the postsynapses, attributed to lateral receptor mobilization from adjacent locations. Whenever synaptic plasticity is induced or memory is consolidated, more GluN2A-NMDARs are assembled likely using GluN2A from a local translation and GluN1 from local ER. Later on, NMDARs are mobilized from other pools, and there are de novo syntheses at the neuron soma. Changes in GluN1 or NMDAR levels induced by synaptic plasticity and by spatial memory formation seem to occur in different waves of NMDAR transport/expression/degradation, with a net increase at the postsynaptic side and a rise in expression at both the spine and neuronal soma. This review aims to put together that information and the proposed hypotheses.

NMDA receptor subunits in the adult rat hippocampus undergo similar changes after 5 minutes in an open field and after LTP induction.

NMDA receptor subunits change during development and their synaptic expression is modified rapidly after synaptic plasticity induction in hippocampal slices. However, there is scarce information on subunits expression after synaptic plasticity induction or memory acquisition, particularly in adults. GluN1, GluN2A and GluN2B NMDA receptor subunits were assessed by western blot in 1) adult rats that had explored an open field (OF) for 5 minutes, a time sufficient to induce habituation, 2) mature rat hippocampal neuron cultures depolarized by KCl and 3) hippocampal slices from adult rats where long term potentiation (LTP) was induced by theta-burst stimulation (TBS). GluN1 and GluN2A, though not GluN2B, were significantly higher 70 minutes--but not 30 minutes--after a 5 minutes session in an OF. GluN1 and GluN2A total immunofluorescence and puncta in neurites increased in cultures, as evaluated 70 minutes after KCl stimulation. Similar changes were found in hippocampal slices 70 minutes after LTP induction. To start to explore underlying mechanisms, hippocampal slices were treated either with cycloheximide (a translation inhibitor) or actinomycin D (a transcription inhibitor) during electrophysiological assays. It was corroborated that translation was necessary for LTP induction and expression. The rise in GluN1 depends on transcription and translation, while the increase in GluN2A appears to mainly depend on translation, though a contribution of some remaining transcriptional activity during actinomycin D treatment could not be rouled out. LTP effective induction was required for the subunits to increase. Although in the three models same subunits suffered modifications in the same direction, within an apparently similar temporal course, further investigation is required to reveal if they are related processes and to find out whether they are causally related with synaptic plasticity, learning and memory.

Herpes simplex virus type 1-based amplicon vectors for fundamental research in neurosciences and gene therapy of neurological diseases.

Somatic manipulation of the nervous system without the involvement of the germinal line appears as a powerful counterpart of the transgenic strategy. The use of viral vectors to produce specific, transient and localized knockout, knockdown, ectopic expression or overexpression of a gene, leads to the possibility of analyzing both in vitro and in vivo molecular basis of neural function. In this approach, viral particles engineered to carry transgenic sequences are delivered into discrete brain regions, to transduce cells that will express the transgenic products. Amplicons are replication-incompetent helper-dependent vectors derived from herpes simplex virus type 1 (HSV-1), with several advantages that potentiate their use in neurosciences: (1) minimal toxicity: amplicons do not encode any virus proteins, are neither toxic for the infected cells nor pathogenic for the inoculated animals and elicit low levels of adaptive immune responses; (2) extensive transgene capacity to carry up to 150-kb of foreign DNA; i.e., entire genes with regulatory sequences could be delivered; (3) widespread cellular tropism: amplicons can experimentally infect several cell types including glial cells, though naturally the virus infects mainly neurons and epithelial cells; (4) since the viral genome does not integrate into cellular chromosomes there is low probability to induce insertional mutagenesis. Recent investigations on gene transfer into the brain using these vectors, have focused on gene therapy of inherited genetic diseases affecting the nervous system, such as ataxias, or on neurodegenerative disorders using experimental models of Parkinson's or Alzheimer's disease. Another group of studies used amplicons to investigate complex neural functions such as neuroplasticity, anxiety, learning and memory. In this short review, we summarize recent data supporting the potential of HSV-1 based amplicon vector model for gene delivery and modulation of gene expression in primary cultures of neuronal cells and into the brain of living animals.

Staufen: from embryo polarity to cellular stress and neurodegeneration.

Staufen is a double-stranded RNA-binding protein that forms RNA granules by RNA-dependent and -independent interactions. Staufen was initially described in Drosophila as a key molecule for targeting maternal mRNAs. In vertebrates, two highly similar paralogs with several splicing variants mediate mRNA transport, thus affecting neuron plasticity, learning and memory. Staufen also regulates translation and mRNA decay. In recent years, Staufen was shown to be an important regulatory component of stress granules (SGs), which are large aggregates of silenced mRNPs specifically induced upon acute cellular stress. SGs contribute to cell survival by reprogramming translation and inhibiting pro-apoptotic pathways, and Staufen appears to negatively modulate SG formation by several mechanisms. More recently, mammalian Staufen was found in RNA granules and pathological cytoplasmic aggregates related to SGs containing huntingtin, TDP43, FUS/TLS or FMRP. In addition, Staufen binds CUG repeats present in mutant RNAs causative of degenerative conditions, thus ameliorating disease. Finally, Staufen affects HIV and influenza infection at several levels. Collectively, these observations unveil important roles for Staufen-mediated post-transcriptional regulation in a growing number of human diseases.

Smaug1 mRNA-silencing foci respond to NMDA and modulate synapse formation.

Mammalian Smaug1/Samd4A is a translational repressor. Here we show that Smaug1 forms mRNA-silencing foci located at postsynapses of hippocampal neurons. These structures, which we have named S-foci, are distinct from P-bodies, stress granules, or other neuronal RNA granules hitherto described, and are the first described mRNA-silencing foci specific to neurons. RNA binding was not required for aggregation, which indicates that S-foci formation is not a consequence of mRNA silencing. N-methyl-D-aspartic acid (NMDA) receptor stimulation provoked a rapid and reversible disassembly of S-foci, transiently releasing transcripts (the CaMKIIα mRNA among others) to allow their translation. Simultaneously, NMDA triggered global translational silencing, which suggests the specific activation of Smaug1-repressed transcripts. Smaug1 is expressed during synaptogenesis, and Smaug1 knockdown affected the number and size of synapses, and also provoked an impaired response to repetitive depolarizing stimuli, as indicated by a reduced induction of Arc/Arg3.1. Our results suggest that S-foci control local translation, specifically responding to NMDA receptor stimulation and affecting synaptic plasticity.