AAV-mediated neuronal expression of an scFv antibody selective for Aß oligomers protects synapses and rescues memory in Alzheimer models.

The accumulation of soluble oligomers of the amyloid-ß peptide (AßOs) in the brain has been implicated in synapse failure and memory impairment in Alzheimer's disease. Here, we initially show that treatment with NUsc1, a single-chain variable-fragment antibody (scFv) that selectively targets a subpopulation of AßOs and shows minimal reactivity to Aß monomers and fibrils, prevents the inhibition of long-term potentiation in hippocampal slices and memory impairment induced by AßOs in mice. As a therapeutic approach for intracerebral antibody delivery, we developed an adeno-associated virus vector to drive neuronal expression of NUsc1 (AAV-NUsc1) within the brain. Transduction by AAV-NUsc1 induced NUsc1 expression and secretion in adult human brain slices and inhibited AßO binding to neurons and AßO-induced loss of dendritic spines in primary rat hippocampal cultures. Treatment of mice with AAV-NUsc1 prevented memory impairment induced by AßOs and, remarkably, reversed memory deficits in aged APPswe/PS1ΔE9 Alzheimer's disease model mice. These results support the feasibility of immunotherapy using viral vector-mediated gene delivery of NUsc1 or other AßO-specific single-chain antibodies as a potential therapeutic approach in Alzheimer's disease.

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.

GluN1 and GluN2A NMDA Receptor Subunits Increase in the Hippocampus during Memory Consolidation in the Rat.

It is widely accepted that NMDA receptors (NMDAR) are required for learning and memory formation, and for synaptic plasticity induction. We have previously shown that hippocampal GluN1 and GluN2A NMDAR subunits significantly increased following habituation of rats to an open field (OF), while GluN2B remained unchanged. Similar results were obtained after CA1-long-term potentiation (LTP) induction in rat hippocampal slices. Other studies have also shown NMDAR up regulation at earlier and later time points after LTP induction or learning acquisition. In this work, we have studied NMDAR subunits levels in the hippocampus and prefrontal cortex (PFC) after OF habituation and after object recognition (OR), to find out whether rising of NMDAR subunits is a general and structure-specific feature during memory formation. In 1, 2 and 3 month old rats there was an increase in hippocampal GluN1 and GluN2A, but not in GluN2B levels 70 min after OF habituation. This rise overlaps with early phase of memory consolidation, suggesting a putative relationship between them. The increases fell down to control levels 90 min after training. Similar results were obtained in the hippocampus of adult rats 70 min after OR training, without changes in PFC. Following OF test or OR discrimination phase, NMDAR subunits remained unchanged. Hence, rising of hippocampal GluN1 and GluN2A appears to be a general feature after novel "spatial/discrimination" memory acquisition. To start investigating the dynamics and possible mechanisms of these changes, we have studied hippocampal neuron cultures stimulated by KCl to induce plasticity. GluN1 and GluN2A increased both in dendrites and neuronal bodies, reaching a maximum 75 min later and returning to control levels at 90 min. Translation and/or transcription and mobilization differentially contribute to this rise in subunits in bodies and dendrites. Our results showed that the NMDAR subunits increase follows a similar time course both in vitro and in vivo. These changes happen in the hippocampus where a spatial representation of the environment is being formed making possible short term and long term memories (STM and LTM); appear to be structure-specific; are preserved along life; and could be related to synaptic tagging and/or to memory consolidation of new spatial/discrimination information.

Amnesia of inhibitory avoidance by scopolamine is overcome by previous open-field exposure.

The muscarinic cholinergic receptor (MAChR) blockade with scopolamine either extended or restricted to the hippocampus, before or after training in inhibitory avoidance (IA) caused anterograde or retrograde amnesia, respectively, in the rat, because there was no long-term memory (LTM) expression. Adult Wistar rats previously exposed to one or two open-field (OF) sessions of 3 min each (habituated), behaved as control animals after a weak though over-threshold training in IA. However, after OF exposure, IA LTM was formed and expressed in spite of an extensive or restricted to the hippocampus MAChR blockade. It was reported that during and after OF exposure and reexposure there was an increase in both hippocampal and cortical ACh release that would contribute to "prime the substrate," e.g., by lowering the synaptic threshold for plasticity, leading to LTM consolidation. In the frame of the "synaptic tagging and capture" hypothesis, plasticity-related proteins synthesized during/after the previous OF could facilitate synaptic plasticity for IA in the same structure. However, IA anterograde amnesia by hippocampal protein synthesis inhibition with anisomycin was also prevented by two OF exposures, strongly suggesting that there would be alternative interpretations for the role of protein synthesis in memory formation and that another structure could also be involved in this "OF effect."

Hippocampal NMDA receptors and the previous experience effect on memory.

N-methyl-D-aspartate receptors (NMDAR) are thought to be responsible for switching synaptic activity specific patterns into long-term changes in synaptic function and structure, which would support learning and memory. Hippocampal NMDAR blockade impairs memory consolidation in rodents, while NMDAR stimulation improves it. Adult rats that explored twice an open field (OF) before a weak though overthreshold training in inhibitory avoidance (IA), expressed IA long-term memory in spite of the hippocampal administration of MK-801, which currently leads to amnesia. Those processes would involve different NMDARs. The selective blockade of hippocampal GluN2B-containing NMDAR with ifenprodil after training promoted memory in an IA task when the training was weak, suggesting that this receptor negatively modulates consolidation. In vivo, after 1h of an OF exposure-with habituation to the environment-, there was an increase in GluN1 and GluN2A subunits in the rat hippocampus, without significant changes in GluN2B. Coincidentally, in vitro, in both rat hippocampal slices and neuron cultures there was an increase in GluN2A-NMDARs surface expression at 30min; an increase in GluN1 and GluN2A levels at about 1h after LTP induction was also shown. We hypothesize that those changes in NMDAR composition could be involved in the "anti-amnesic effect" of the previous OF. Along certain time interval, an increase in GluN1 and GluN2A would lead to an increase in synaptic NMDARs, facilitating synaptic plasticity and memory; while then, an increase in GluN2A/GluN2B ratio could protect the synapse and the already established plasticity, perhaps saving the specific trace.