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

NMDAR subunits immunofluorescence in mature hipocampal neuron cultures stimulated by KCl.

<p><b><i>A.</i></b> Quantification of NMDAR subunit puncta at dendrites (n = 100 neurites/culture). A significant increase in GluN1 and GluN2A puncta was observed at 30 and 70 minutes after KCl stimulation (** p<0.05, *** p<0.001, Kruskal-Wallis test followed by Dunn’s Multiple Comparison Post-Test). <i>Insert on the top of each bar</i>: representative dendrite for each condition (bar: 2 µm). <b><i>B.</i></b> Total fluorescence quantification 30 and 70 minutes after KCl stimulation. There were significant increases in GluN1 and GluN2A 70 minutes after stimulation. There were no significant changes in GluN2B total immunofluorescence (* p<0.05, *** p<0.001, ONE WAY ANOVA, Dunnet Post-Test). <i>Right:</i> representative neurons for each condition.</p

NMDAR subunits modification in hippocampal slices after transcription or translation inhibition during LTP induction. <i>A.</i>

<p><i> Left:</i> Normalized slopes of evoked fEPSPs recorded as those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055244#pone-0055244-g003" target="_blank">Figure 3</a>, corresponding to the first pulse of the paired stimulation applied before and after TBS (arrow). Plots represent the average of five independent experiments over 90 minutes of recording (n = 5 for each group). <i>Black line</i>: drug perfusion. <i>Insert on top</i>: average traces of 10 individual recordings from a control slice and slices treated with ActD or CHX (black: 5 minutes before TBS; grey: 5 last minutes of recording). <i>Right:</i> Bars represent averages of normalized first pulse slopes of the 5 last minutes of recording for each group. LTP induction was blocked by 40 µg/ml CHX treatment (* p<0.05, one sample t test) compared to basal transmission (line referred to 1 in the graph). <b><i>B.</i></b> NMDAR subunits were evaluated by WB in same slices that in <b>A.</b> CHX treatment blocked GluN1 and GluN2A increase, while 50 µg/ml ActD only blocked GluN1 increase (* p<0,05 ONE WAY ANOVA, Dunnet Post-Test; n = 5 for each group). <i>Insert on top:</i> (from left to right): Representative GluN1 and GluN2A WB bands of +TBS+LTP slices (control), CHX and ActD +TBS+LTP slices treated slices. <b><i>C.</i></b> Table indicates mean ± SEM for GluN1/GAPDH (first row) or GluN2A/GAPDH (second row) in +TBS-LTP slices (n = 6) and +TBS+LTP slices without any drug treatment (Control in <b>B,</b> n = 9), or treated with ActD (n = 5) or CHX (n = 5) (*** p<0.0001; ONE WAY ANOVA - Newman Keuls Test).</p

NMDAR subunits changes after OF habituation. <i>A.</i>

<p>Habituation to the OF of rats exposed to a 5 minutes OF session (n = 16). Graphs show number of crossings (left panel) and rearings (right panel) <i>per</i> minute (bars indicates median with interquartile ranges). Crossings decreased significantly after 3 minutes, while rearings were only significantly decreased in the fifth minute. *** p<0.0001, ** p<0.01 by Friedman test followed by Dunn’s Multiple Comparison Test. <b><i>B.</i></b> Total crossings from rats exposed to the OF for 1 or 5 minutes (Training) and tested for STM 40 minutes later (n = 12) or LTM 24 h later (n = 16). There were significant differences in total number of crossings in the second session compared to the first, only in rats which spent 5 minutes in the OF in the training session, for STM (* p<0.05) as well as for LTM (*** p<0.0001) (Mann Whitney test). <b><i>C.</i></b> NMDAR subunits in the hippocampus of rats after OF exposure. Four groups of rats were analyzed: rats as in <b><i>A,</i></b> which were sacrificed at 0, 30 and 70 minutes after the task (5′-0′, 5′–30′ and 5′–70′ groups); and rats exposed for 1 minute to the OF, sacrificed 70 minutes later (1′–70′ group). WB analysis showed about a one fold increase in GluN1 and GluN2A level for <i>5′–70′</i> group, in 3 independent experiments (* p<0.05, ONE WAY ANOVA, Newman-Keuls Multiple Comparison Post-Test). <i>Insert on top:</i> representative WB bands for GluN1, GluN2A and GluN2B NMDAR subunits and GAPDH (internal control). <b><i>D</i></b>. NMDAR subunits analysis in the hippocampus of rats after two OF sessions. 4 groups of rats were analyzed: rats exposed to the OF 5 minutes and sacrificed immediately (5′-0′), 70 minutes (5′–70′), 24 h later (5′–24 h), or tested in the OF and sacrificed 70 minutes later (70′ postest-TE). * p<0.05 ONE WAY ANOVA, Dunnett’s Post-Test. <i>Insert on top:</i> representative WB bands for GluN1 and GluN2A NMDAR subunits and GAPDH (internal control).</p

NMDAR subunits change after LTP induction and expression.

<p><b><i>A.</i></b> Evoked fEPSPs normalized slopes from fresh hippocampal slices corresponding to the first pulse of paired stimulation, before and after TBS (arrow). Plots represent the average of three independent experiments over 90 minutes of recording (n = 6 for each group). <i>Insert on top:</i> average traces of 10 individual recordings from a +TBS+LTP and a +TBS-LTP slices (black: 5 minutes before TBS; grey: 5 last minutes of recording). <b><i>B.</i></b> WB band densities quantification of samples from same slices that in <b>A.</b> A significant increase was only observed for +TBS+LTP slices (** p<0.01; *** p<0.001 ONE WAY ANOVA, Dunnet Post-Test; n = 6 for each group). <i>Insert on top:</i> (from left to right): representative GluN1 and GAPDH WB bands from: a −TBS slice, a +TBS-LTP slice and a +TBS+LTP slice. <b><i>C.</i></b> Evoked fEPSPs slopes corresponding to the first pulse of the paired stimulation before and after TBS (arrow). Plots represent the average of fEPSPs slopes over 50 and 90 minutes of recording, respectively (n = 6 for each group). <i>Right</i>: average traces of 10 individual recordings from a LTP-slice after 30 and 70 minutes TBS (black: 5 minutes before TBS; grey: 5 last minutes of recording). <b><i>D.</i></b> NMDAR subunits quantification by WB. Samples analyzed: slices used in <b>C</b>. (processed 30 or 70 minutes after TBS) and in −TBS slices (Control). Analysis of WB bands showed a significant increase in GluN1 and GluN2A level for the 70 minutes group in three independent experiments (* p<0,05; *** p<0,001 ONE WAY ANOVA-Dunnet Test). <i>Insert on top:</i> Representative WB bands for GluN1, GluN2A and GluN2B NMDAR subunits and GAPDH (internal control).</p

Recuperación de la memoria mediante terapia génica con un fragmento de anticuerpo selectivo para oligómeros de Aβ en un modelo de Alzheimer en ratas

Strong evidence supports the hypothesis that synapse damage and memory impairment in early Alzheimer disease (AD) might be due to synaptic failure caused by amyloid beta oligomers (AβOs). We demonstrated the preclinical efficacy of a single-chain variable-fragment (scFv) antibody NUsc1 that selectively targets a subpopulation of AβOs; NUsc1 prevented AβO-induced inhibition of long-term potentiation in hippocampal slices and short-term memory impairment in mice. Since specific targeting of AβOs by NUsc1 may be a substantial improvement in target engagement and efficacy for AD therapy, we developed an adeno-associated virus (AAV) vector to drive neuronal expression of NUsc1 within the brain. AAV-NUsc1 rescued short-term memory (STM) for objects and congeners interaction in mice AD models. Purpose: In heterozygous McGill-R-Thy1-APP transgenic (Tg+/–) rat model of AD, progressive amyloid pathology is accompanied by cognitive impairment involving long-term memory (LTM) decline. LTM in a novel-object-recognition (NOR) task was impaired in 4-month-old (Tg+/–) male rats, suggesting that they are unable to form/evoke such discriminative memories. Hence, we investigated if AAV-NUsc1 treatment could rescued this memory. Methods: 10-12 weeks-old either Tg or wild type male rats were i.c.v. infused with AAV-NUsc1. Two months later, short-term exploratory behavior, habituation to an open field (OF), object discrimination and LTM for objects were assessed. Results: AAV-NUsc1 treated Tg rats were able to successfully perform the task 24 h after training, denoting recovery of LT discrimination capacity and LTM formation. Wild type rats successfully performed the task either treated or not with AAV-NUsc1. Also, exploration and short-term habituation to an open field was preserved in Tg+/– rats either treated or not. Conclusions: Our present and previous results suggest that AAV-NUsc1 represents a significant advance in gene therapy, supporting the feasibility of immunotherapy using viral vector-mediated NUsc1 gene delivery as a potential therapeutic approach in AD.Evidencias conspicuas respaldan la hipótesis de que la presencia de oligómeros beta-amiloides (AβO) ocasiona un deterioro sináptico y de la memoria en etapas tempranas de la Enfermedad de Alzheimer (AD). Se ha demostrado que el anticuerpo de cadena única y Fragmento variable (scFv) NUsc1, que une selectivamente una subpoblación de AβO, evitó el deterioro de la memoria a corto plazo, inducido por AβO en ratones. Dado que la selectividad de NUsc1 mejora sustancialmente la detección del AβO, y consecuentemente su eficacia terapéutica para AD, se ha desarrollado un vector derivado de Virus Adenoasociado para expresar NUsc1 (AAV-NUsc1) en el cerebro. AAV-NUsc1 rescató la Memoria de Corto Plazo (STM) para el reconocimiento de objetos e interacción con congéneres en ratones modelo de AD. La rata McGill-R-Thy1-APP transgénica heterocigota (Tg+/–) modelo de AD, sufre una patología amiloide progresiva acompañada de deterioro cognitivo, que afecta la Memoria de Largo Plazo (LTM) de Reconocimiento de Objetos (NOR) evitando su formación/evocación. Evaluando si el tratamiento con AAV-NUsc1 podría rescatar la LTM de reconocimiento en ratas macho (Tg+/–) de 4 meses. Ratas macho Tg y de genotipo salvaje (Wt) de 10-12 semanas fueron infundidas i.c.v con AAV-NUsc1. Dos meses más tarde, se evaluaron: el comportamiento exploratorio a corto plazo, la habituación a un Campo Abierto (OF), la discriminación y LTM para objetos. 24 h después del entrenamiento se observó que las ratas Tg tratadas con AAV-NUsc1 recuperaron la capacidad de expresar una LTM y de reconocer objetos novedosos. De manera similar, las ratas Wt tratadas con AAV-NUsc1 y las del grupo control, realizaron la tarea con éxito. La exploración y habituación al OF fueron similares para ratas Tg+/– y Wt, tratadas y control. Nuestros resultados sugieren que AAV-NUsc1 representa un avance significativo en terapia génica, respaldando la viabilidad de la inmunoterapia mediada por vectores virales aportando el gen de NUsc1 como posible enfoque terapéutico para AD

Recuperación de la memoria mediante terapia génica con un fragmento de anticuerpo monocatenario selectivo para oligómeros de Aβ en un modelo de Alzheimer en ratas

Strong evidence supports the hypothesis that synapse damage and memory impairment in early Alzheimer disease (AD) might be due to synaptic failure caused by amyloid beta oligomers (AβOs). We demonstrated the preclinical efficacy of a single-chain variable-fragment (scFv) antibody NUsc1 that selectively targets a subpopulation of AβOs; NUsc1 prevented AβO-induced inhibition of long-term potentiation in hippocampal slices and short-term memory impairment in mice. Since specific targeting of AβOs by NUsc1 may be a substantial improvement in target engagement and efficacy for AD therapy, we developed an adeno-associated virus (AAV) vector to drive neuronal expression of NUsc1 within the brain. AAV-NUsc1 rescued short-term memory (STM) for objects and congeners interaction in mice AD models. Purpose: In heterozygous McGill-R-Thy1-APP transgenic (Tg+/–) rat model of AD, progressive amyloid pathology is accompanied by cognitive impairment involving long-term memory (LTM) decline. LTM in a novel-object-recognition (NOR) task was impaired in 4-month-old (Tg+/–) male rats, suggesting that they are unable to form/evoke such discriminative memories. Hence, we investigated if AAV-NUsc1 treatment could rescued this memory. Methods: 10-12 weeks-old either Tg or wild type male rats were i.c.v. infused with AAV-NUsc1. Two months later, short-term exploratory behavior, habituation to an open field (OF), object discrimination and LTM for objects were assessed. Results: AAV-NUsc1 treated Tg rats were able to successfully perform the task 24 h after training, denoting recovery of LT discrimination capacity and LTM formation. Wild type rats successfully performed the task either treated or not with AAV-NUsc1. Also, exploration and short-term habituation to an open field was preserved in Tg+/– rats either treated or not. Conclusions: Our present and previous results suggest that AAV-NUsc1 represents a significant advance in gene therapy, supporting the feasibility of immunotherapy using viral vector-mediated NUsc1 gene delivery as a potential therapeutic approach in AD.Conspicua evidencia apoya la hipótesis de que el danio sináptico y el deterioro de la memoria en etapas tempranas de la enfermedad de Alzheimer (AD) podrían deberse a falla sináptica causada por oligómeros beta-amiloides (AβO). Demostramos la eficacia preclínica del anticuerpo de cadena única y fragmento variable (scFv) NUsc1, que une selectivamente una subpoblación de AβO; NUsc1 evitó el deterioro de la memoria a corto plazo, inducido por AβO en ratones. Como la selectividad por AβO por parte de NUsc1 puede mejorar sustancialmente la detección del blanco y la eficacia terapéutica para AD, desarrollamos un vector derivado de virus adenoasociado (AAV), para expresión neuronal de NUsc1 en cerebro. AAV-NUsc1 rescató la memoria de corto plazo (STM) para objetos, e interacción con congéneres en ratones modelo de AD. Propósito: La rata McGill-R-Thy1-APP transgénica heterocigota (Tg+/–) modelo de AD, sufre patología amiloide progresiva acompañada de deterioro cognitivo, incluyendo memoria de largo plazo (LTM) de reconocimiento de objetos (NOR). Esta LTM se vio afectada en ratas macho (Tg+/–) de 4 meses, lo que sugiere que no pueden formar/evocar dicha memoria. Investigamos si el tratamiento con AAV-NUsc1 podría rescatar esa memoria. Métodos: Ratas macho Tg y de genotipo salvaje (Wt) de 10-12 semanas fueron infundidas i.c.v con AAV-NUsc1. Dos meses más tarde, se evaluaron: comportamiento exploratorio a corto plazo, habituación a un campo abierto (OF), discriminación y LTM para objetos. Resultados: Las ratas Tg tratadas con AAV-NUsc1 fueron exitosas 24 h después del entrenamiento, mostrando recuperación de la capacidad de discriminación y formación de LTM. Las ratas Wt tratadas o no con AAV-NUsc1, realizaron con éxito la tarea. La exploración y habituación al OF fueron similares para ratas Tg+/– y Wt, tratadas o no. Conclusiones: Nuestros resultados sugieren que AAV-NUsc1 representa un avance significativo en terapia génica, respaldando la viabilidad de la inmunoterapia mediada por vectores virales aportando el gen de NUsc1 como posible enfoque terapéutico para AD

Recuperación de la memoria mediante terapia génica con un fragmento de anticuerpo selectivo para oligómeros de Aβ en un modelo de Alzheimer en ratas

Evidencias conspicuas respaldan la hipótesis de que la presencia de oligómeros beta-amiloides (AβO) ocasiona un deterioro sináptico y de la memoria en etapas tempranas de la Enfermedad de Alzheimer (AD). Se ha demostrado que el anticuerpo de cadena única y Fragmento variable (scFv) NUsc1, que une selectivamente una subpoblación de AβO, evitó el deterioro de la memoria a corto plazo, inducido por AβO en ratones. Dado que la selectividad de NUsc1 mejora sustancialmente la detección del AβO, y consecuentemente su eficacia terapéutica para AD, se ha desarrollado un vector derivado de Virus Adenoasociado para expresar NUsc1 (AAV-NUsc1) en el cerebro. AAV-NUsc1 rescató la Memoria de Corto Plazo (STM) para el reconocimiento de objetos e interacción con congéneres en ratones modelo de AD. La rata McGill-R-Thy1-APP transgénica heterocigota (Tg+/–) modelo de AD, sufre una patología amiloide progresiva acompañada de deterioro cognitivo, que afecta la Memoria de Largo Plazo (LTM) de Reconocimiento de Objetos (NOR) evitando su formación/evocación. Evaluando si el tratamiento con AAV-NUsc1 podría rescatar la LTM de reconocimiento en ratas macho (Tg+/–) de 4 meses. Ratas macho Tg y de genotipo salvaje (Wt) de 10-12 semanas fueron infundidas i.c.v con AAV-NUsc1. Dos meses más tarde, se evaluaron: el comportamiento exploratorio a corto plazo, la habituación a un Campo Abierto (OF), la discriminación y LTM para objetos. 24 h después del entrenamiento se observó que las ratas Tg tratadas con AAV-NUsc1 recuperaron la capacidad de expresar una LTM y de reconocer objetos novedosos. De manera similar, las ratas Wt tratadas con AAV-NUsc1 y las del grupo control, realizaron la tarea con éxito. La exploración y habituación al OF fueron similares para ratas Tg+/– y Wt, tratadas y control. Nuestros resultados sugieren que AAV-NUsc1 representa un avance significativo en terapia génica, respaldando la viabilidad de la inmunoterapia mediada por vectores virales aportando el gen de NUsc1 como posible enfoque terapéutico para AD.Strong evidence supports the hypothesis that synapse damage and memory impairment in early Alzheimer disease (AD) might be due to synaptic failure caused by amyloid beta oligomers (AβOs). We demonstrated the preclinical efficacy of a single-chain variable-fragment (scFv) antibody NUsc1 that selectively targets a subpopulation of AβOs; NUsc1 prevented AβO-induced inhibition of long-term potentiation in hippocampal slices and short-term memory impairment in mice. Since specific targeting of AβOs by NUsc1 may be a substantial improvement in target engagement and efficacy for AD therapy, we developed an adeno-associated virus (AAV) vector to drive neuronal expression of NUsc1 within the brain. AAV-NUsc1 rescued short-term memory (STM) for objects and congeners interaction in mice AD models. Purpose: In heterozygous McGill-R-Thy1-APP transgenic (Tg+/–) rat model of AD, progressive amyloid pathology is accompanied by cognitive impairment involving long-term memory (LTM) decline. LTM in a novel-object-recognition (NOR) task was impaired in 4-month-old (Tg+/–) male rats, suggesting that they are unable to form/evoke such discriminative memories. Hence, we investigated if AAV-NUsc1 treatment could rescued this memory. Methods: 10-12 weeks-old either Tg or wild type male rats were i.c.v. infused with AAV-NUsc1. Two months later, short-term exploratory behavior, habituation to an open field (OF), object discrimination and LTM for objects were assessed. Results: AAV-NUsc1 treated Tg rats were able to successfully perform the task 24 h after training, denoting recovery of LT discrimination capacity and LTM formation. Wild type rats successfully performed the task either treated or not with AAV-NUsc1. Also, exploration and short-term habituation to an open field was preserved in Tg+/– rats either treated or not. Conclusions: Our present and previous results suggest that AAV-NUsc1 represents a significant advance in gene therapy, supporting the feasibility of immunotherapy using viral vector-mediated NUsc1 gene delivery as a potential therapeutic approach in AD.Fil: Colettis, Natalia Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Oberholzer, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Cercato, Magalí Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Habif, Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Selles, Maria Clara. Universidade Federal do Rio de Janeiro; BrasilFil: Salas, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Sebollela, Adriano. Universidade de Sao Paulo; BrasilFil: Klein, William L.. Northwestern University; Estados UnidosFil: Epstein, Alberto Luis. Université Claude Bernard Lyon 1; FranciaFil: Salvetti, Anna. Université Claude Bernard Lyon 1; FranciaFil: Ferreira, Sergio Teixeira. Universidade Federal do Rio de Janeiro; BrasilFil: Jerusalinsky, Diana Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentin