Molecular insights from the crab Neohelice memory model.

Memory acquisition, formation and maintenance depend on synaptic post-translational machinery and regulation of gene expression triggered by several transduction pathways. In turns, these processes lead to stabilization of synaptic modifications in neurons in the activated circuits. In order to study the molecular mechanisms involved in acquisition and memory, we have taken advantage of the context-signal associative learning and, more recently, the place preference task, of the crab Neohelice granulata. In this model organism, we studied several molecular processes, including activation of extracellular signal-regulated kinase (ERK) and the nuclear factor kappa light chain enhancer of activated B cells (NF-κB) transcription factor, involvement of synaptic proteins such as NMDA receptors and neuroepigenetic regulation of gene expression. All these studies allowed description of key plasticity mechanisms involved in memory, including consolidation, reconsolidation and extinction. This article is aimed at review the most salient findings obtained over decades of research in this memory model.

Contributions of extracellular-signal regulated kinase 1/2 activity to the memory trace.

The ability to learn from experience and consequently adapt our behavior is one of the most fundamental capacities enabled by complex and plastic nervous systems. Next to cellular and systems-level changes, learning and memory formation crucially depends on molecular signaling mechanisms. In particular, the extracellular-signal regulated kinase 1/2 (ERK), historically studied in the context of tumor growth and proliferation, has been shown to affect synaptic transmission, regulation of neuronal gene expression and protein synthesis leading to structural synaptic changes. However, to what extent the effects of ERK are specifically related to memory formation and stabilization, or merely the result of general neuronal activation, remains unknown. Here, we review the signals leading to ERK activation in the nervous system, the subcellular ERK targets associated with learning-related plasticity, and how neurons with activated ERK signaling may contribute to the formation of the memory trace.

Two spaced training trials induce associative ERK-dependent long term memory in Neohelice granulata.

Memory formation depends upon several parametric training conditions. Among them, trial number and inter-trial interval (ITI) are key factors to induce long-term retention. However, it is still unclear how individual training trials contribute to mechanisms underlying memory formation and stabilization. Contextual conditioning in Neohelice granulata has traditionally elicited associative long-term memory (LTM) after 15 spaced (ITI = 3 min) trials. Here, we show that LTM in crabs can be induced after only two training trials by increasing the ITI to 45 min (2t-LTM) and maintaining the same training duration as in traditional protocols. This newly observed LTM was preserved for at least 96 h, exhibiting protein synthesis dependence during consolidation and reconsolidation as well as context-specificity. Moreover, we demonstrate that 2t-LTM depends on inter-trial and post-training ERK activation showing a faster phosphorylation after the second trial compared to the first one. In summary, we present a new training protocol in crabs through a reduced number of trials showing associative features similar to traditional spaced training. This novel protocol allows for intra-training manipulation and the assessment of individual trial contribution to LTM formation.

Relevance of ERK1/2 Post-retrieval Participation on Memory Processes: Insights in Their Particular Role on Reconsolidation and Persistence of Memories.

Back in 1968, Misanin and his group posited that reactivation of consolidated memories could support changes in that trace, similar to what might happen during the consolidation process. Not until 2000, when Nader et al. (2000) studied the behavioral effect of a protein synthesis inhibitor on retrieved memories, could this previous statement be taken under consideration once again; suggesting that consolidated memories can become labile after reactivation. The process of strengthening after memory labilization was named memory reconsolidation. In recent years, many studies pointed towards a critical participation of the extracellular signal-regulated kinase (ERK)/mitogen activated protein kinases (MAPKs) pathway in different memory processes (e.g., consolidation, extinction, reconsolidation, among others). In this review article, we will focus on how this system might be modulating the processes triggered after retrieval of well-consolidated memories in mice.

Decrease of ERK/MAPK overactivation in prefrontal cortex reverses early memory deficit in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) can be considered as a disease of memory in its initial clinical stages. Amyloid-ß (Aß) peptide accumulation is central to the disease initiation leading later to intracellular neurofibrillary tangles (NFTs) of cytoskeletal tau protein formation. It is under discussion whether different Aß levels of aggregation, concentration, brain area, and/or time of exposure might be critical to the disease progression, as well as which intracellular pathways it activates. The aim of the present work was to study memory-related early molecular and behavioral alterations in a mouse model of AD, in which a subtle deregulation of the physiologic function of Aß can be inferred. For this purpose we used triple-transgenic (3xTg) mice, which develop Aß and tau pathology resembling the disease progression in humans. Memory impairment in novel object recognition task was evident by 5 months of age in 3xTg mice. Hippocampus and prefrontal cortex extra-nuclear protein extracts developed differential patterns of Aß aggregation. ERK1/MAPK showed higher levels of cytosolic activity at 3 months and higher levels of nuclear activity at 6 months in the prefrontal cortex. No significant differences were found in JNK and NF-κB activity and in calcineurin protein levels. Finally, intra-PFC administration of a MEK inhibitor in 6-month-old 3xTg mice was able to reverse memory impairment, suggesting that ERK pathway alterations might at least partially explain memory deficits observed in this model, likely as a consequence of memory trace disruption.

Effect on memory of acute administration of naturally secreted fibrils and synthetic amyloid-beta peptides in an invertebrate model.

Amyloid beta peptide (Abeta) is considered one of the main agents of Alzheimer's disease pathogenesis. Recently, it has been proposed that memory deficits are caused by different stages of Abeta aggregation, particularly by oligomers. In addition, although memory impairment was found after Abeta administration in rodents and chicks, the nature of the memory deficits induced in invertebrates by acute administration of mammalian Abeta peptides is not well understood. Previously, we reported the amnesic effect of acute pre-training administration of naturally formed fibrils (NF) in crab memory. Here we evaluate the effect of NF and synthetic Abeta peptides administration at different times before and after training in this well characterized invertebrate memory model, the context-signal memory of the crab Chasmagnathus. We found a clear amnesic effect at very low doses of naturally Abeta NF only when administered immediately pre- and post-training, but not 24 h and 18 h before or 6h after training. Activation of ERK/MAPK (a protein kinase required for memory formation in this model) 60 min after administration was found. In contrast, neither JNK/SAPK nor NF-kappaB transcription factor were activated. Furthermore, synthetic Abeta1-42 and Abetapy3-42 administration induced amnesia when used after a protocol for fibrillation but not after a protocol for oligomerization. On the contrary, no amnestic effect was found when fibrillated Abeta1-40 and Abetapy11-42 peptides were used. Thus, Abeta1-42 and Abetapy3-42 peptides impaired memory and the effects were only found when highly aggregated peptides, which may include fibrils, protofibrils and oligomers, were administered. These temporally- and signaling-specific effects suggest that Abeta impairs memory by inducing transient physiological, rather than permanent neuropathological, alterations of the brain and this effect is achieved through generalized ERK activation.

Lessons from a crab: molecular mechanisms in different memory phases of Chasmagnathus.

Consolidation of long-term memory requires the activation of several transduction pathways that lead to post-translational modifications of synaptic proteins and to regulation of gene expression, both of which promote stabilization of specific changes in the activated circuits. In search of the molecular mechanisms involved in such processes, we used the context-signal associative learning paradigm of the crab Chasmagnathus. In this model, we studied the role of some molecular mechanisms, namely cAMP-dependent protein kinase (PKA), extracellular-signal-regulated kinase (ERK), the nuclear factor kappa B (NF-kappaB) transcription factor, and the role of synaptic proteins such as amyloid beta precursor protein, with the object of describing key mechanisms involved in memory processing. In this article we review the most salient results obtained over a decade of research in this memory model.

Phosphorylation of extra-nuclear ERK/MAPK is required for long-term memory consolidation in the crab Chasmagnathus.

It was previously demonstrated that mitogen-activated protein kinase (MAPK) signaling plays a pivotal role in neural plasticity and memory processes both in rodents and mollusks. Although the MAPK pathways are highly conserved, no evidence was found for its participation in memory models in other animal groups. Here we found ERK-like and JNK-like cross-immunoreactivity in the crab brain with phospho-specific antibodies and we estimated ERK and JNK activity during long-term memory consolidation in the context-signal learning paradigm of the crab Chasmagnathus. At 0, 1, 3 and 6h after training ERK and JNK activity was measured. ERK-like activation was found 1h after spaced training in cytosolic but not in nuclear fractions of brain homogenates, while JNK activity remained unchanged in both fractions. Passive (context exposure) and active (continuous stimulation) controls showed cytosolic ERK and JNK activation immediately after training, which decayed 1h later. In coincidence with this time course of activity, an ERK1/2 pathway inhibitor, PD098059, induced amnesia only when administered 45 min after training but not when administered immediately pre- or post-training. These data support that: (1) cytoplasmic but not nuclear ERK substrates must be differentially phosphorylated during memory consolidation, and (2) ERK phosphorylation and consequent activation 1h after training is necessary for long-term memory consolidation in this arthropod model.

Estudio de factores genético-moleculares del virus de la hepatitis C asociados a la actividad de telomerasa hepática en el hepatocarcinoma / Study of genetic-molecular factors of the hepatitis C virus associated to the hepatic telomerase activity in hepatocarcinoma

El virus de la hepatitis C (HCV) constituye en el mundo el agente etiológico asociado a la mayor incidencia de hepatitis crónica que puede conducir la cirrosis hepática y, eventualmente al hepatocarcinoma. Más de 300 millones de personas padecen infección crónica por este virus, representando una causa significativa de morbi-mortalidad, frente a la cual urgen medidas de control efectivas. Originalmente, la magnitud de la heterogeneidad genética del virus no fue claramente advertida. Sin embargo, esta característica irrumpió con sus inherentes implicancias en la patogenia, el diagnóstico, la terapéutica y la inmunoprofilaxis. En cada paciente infectado, el HCV circula como una población heterogénea de variantes virales genéticamente relacionadas que se denominan cuasiespecies. Las mismas se exhiben dinámicas en el curso de la infección cambiando en forma progresiva, alentando una estrategia capaz de evadir los mecanismos de inmunidad humoral y celular desarrollados por el hospedador, y consecuentemente, estableciendo una infección persistente. Existen múltiples evidencias que demuestran que la caracterización genómica de HCV es relevante desde el punto de vista clínico. Importantes diferencias han surgido respecto del curso natural y la severidad de la infección, la evolución post-transplante hepático, el diagnóstico molecular e inmunoserológico, la vía de infección y aún la eficacia de la terapéutica con interferón alfa. El desarrollo de herramientas terapéuticas y profilácticas capaces de limitar la infección por HCV, debe estar sustentado por minuciosos estudios que consideren la heterogeneidad genética exhibida por este agente. En diversas neoplasias -incluídos los hepatocarcinomas (HCC)- se ha documentado la actividad de la oncoenzima telomerasa. Esta ribonucleoproteína con actividad telómero. La hipótesis de la senescencia celular asume que el acortamiento progresivo del telómero de células somáticas de organismos multicelulares, genera una señal que las inhabilita para el ingreso a sucesivos ciclos celulares. De este modo, la pérdida del telómero está asociada in vivo con el envejecimiento de modo tal que con posterioridad a un cierto número de duplicaciones, la célula detendrá su división, entrando en senescencia. Diferencialmente, en las células inmortalizadas, hematopoyéticas y reproductivas, la longitud del telómero está estabilizada, por la actividad de la telomerasa...(TRUNCADO)

Estudio de factores genético-moleculares del virus de la hepatitis C asociados a la actividad de telomerasa hepática en el hepatocarcinoma / Study of genetic-molecular factors of the hepatitis C virus associated to the hepatic telomerase activity in hepatocarcinoma

El virus de la hepatitis C (HCV) constituye en el mundo el agente etiológico asociado a la mayor incidencia de hepatitis crónica que puede conducir la cirrosis hepática y, eventualmente al hepatocarcinoma. Más de 300 millones de personas padecen infección crónica por este virus, representando una causa significativa de morbi-mortalidad, frente a la cual urgen medidas de control efectivas. Originalmente, la magnitud de la heterogeneidad genética del virus no fue claramente advertida. Sin embargo, esta característica irrumpió con sus inherentes implicancias en la patogenia, el diagnóstico, la terapéutica y la inmunoprofilaxis. En cada paciente infectado, el HCV circula como una población heterogénea de variantes virales genéticamente relacionadas que se denominan cuasiespecies. Las mismas se exhiben dinámicas en el curso de la infección cambiando en forma progresiva, alentando una estrategia capaz de evadir los mecanismos de inmunidad humoral y celular desarrollados por el hospedador, y consecuentemente, estableciendo una infección persistente. Existen múltiples evidencias que demuestran que la caracterización genómica de HCV es relevante desde el punto de vista clínico. Importantes diferencias han surgido respecto del curso natural y la severidad de la infección, la evolución post-transplante hepático, el diagnóstico molecular e inmunoserológico, la vía de infección y aún la eficacia de la terapéutica con interferón alfa. El desarrollo de herramientas terapéuticas y profilácticas capaces de limitar la infección por HCV, debe estar sustentado por minuciosos estudios que consideren la heterogeneidad genética exhibida por este agente. En diversas neoplasias -incluídos los hepatocarcinomas (HCC)- se ha documentado la actividad de la oncoenzima telomerasa. Esta ribonucleoproteína con actividad telómero. La hipótesis de la senescencia celular asume que el acortamiento progresivo del telómero de células somáticas de organismos multicelulares, genera una señal que las inhabilita para el ingreso a sucesivos ciclos celulares. De este modo, la pérdida del telómero está asociada in vivo con el envejecimiento de modo tal que con posterioridad a un cierto número de duplicaciones, la célula detendrá su división, entrando en senescencia. Diferencialmente, en las células inmortalizadas, hematopoyéticas y reproductivas, la longitud del telómero está estabilizada, por la actividad de la telomerasa...(TRUNCADO)(AU)

Bases genómicas y filogenéticas para la propuesta de un nuevo método de caracterización molecular del virus de la hepatitis G / Genomic and phylogenetic bases for the proposal of a new method of molecular characterization of the hepatitis G virus

El agente GBV-C / virus de hepatitis G (HGV) ha sido identificado como infeccioso para el ser humano, aunque su potencial participación como virus patógeno es aún motivo de controversia. Basándose en estudios clínicos y epidemiológicos en pacientes politransfundidos, receptores de hemoderivados de este agente por vía parenteral. La clasificación taxonómica de GBV-C/HGV como miembro de la familia Flaviviridae, lo relaciona, aunque en forma distante, con el virus de hepatitis C (HCV), demostrándose similitudes en la organización genómica y en el mecanismo propuesto de replicación viral. En ésta se enfatiza la participación de una RNA polimerasa viral RNA dependiente carente de lectura de prueba (actividad de proofreading) lo cual constituye el principal sustento de la heterogeneidad nucleotídica observada entre los aislamietos. Hasta el presente, se ha demostrado la existencia de tres genotipos mediante secuenciación nucleotídica (a partir del DNA copia obtenido por retrotranscripción [RT] y amplificación por PCR del RNA viral) o a través del análisis del polimorfismo de la longitud de los fragmentos de restricción (RFLP) mediante uso de endonucleasas sobre amplímeros obtenidos por PCR. Existe una asociación entre el genotipo involucrado y el origen geográfico de los mismos, según se desprende de estudios de filogenia entre los aislamientos caracterizados del virus... (TRUNCADO)

Bases genómicas y filogenéticas para la propuesta de un nuevo método de caracterización molecular del virus de la hepatitis G / Genomic and phylogenetic bases for the proposal of a new method of molecular characterization of the hepatitis G virus

El agente GBV-C / virus de hepatitis G (HGV) ha sido identificado como infeccioso para el ser humano, aunque su potencial participación como virus patógeno es aún motivo de controversia. Basándose en estudios clínicos y epidemiológicos en pacientes politransfundidos, receptores de hemoderivados de este agente por vía parenteral. La clasificación taxonómica de GBV-C/HGV como miembro de la familia Flaviviridae, lo relaciona, aunque en forma distante, con el virus de hepatitis C (HCV), demostrándose similitudes en la organización genómica y en el mecanismo propuesto de replicación viral. En ésta se enfatiza la participación de una RNA polimerasa viral RNA dependiente carente de lectura de prueba (actividad de proofreading) lo cual constituye el principal sustento de la heterogeneidad nucleotídica observada entre los aislamietos. Hasta el presente, se ha demostrado la existencia de tres genotipos mediante secuenciación nucleotídica (a partir del DNA copia obtenido por retrotranscripción [RT] y amplificación por PCR del RNA viral) o a través del análisis del polimorfismo de la longitud de los fragmentos de restricción (RFLP) mediante uso de endonucleasas sobre amplímeros obtenidos por PCR. Existe una asociación entre el genotipo involucrado y el origen geográfico de los mismos, según se desprende de estudios de filogenia entre los aislamientos caracterizados del virus... (TRUNCADO)(AU)