Prevalence and influence of cys407* Grm2 mutation in Hannover-derived Wistar rats: mGlu2 receptor loss links to alcohol intake, risk taking and emotional behaviour.

Modulation of metabotropic glutamate 2 (mGlu2) receptor function has huge potential for treating psychiatric and neurological diseases. Development of drugs acting on mGlu2 receptors depends on the development and use of translatable animal models of disease. We report here a stop codon mutation at cysteine 407 in Grm2 (cys407*) that is common in some Wistar rats. Therefore, researchers in this field need to be aware of strains with this mutation. Our genotypic survey found widespread prevalence of the mutation in commercial Wistar strains, particularly those known as Han Wistar. Such Han Wistar rats are ideal for research into the separate roles of mGlu2 and mGlu3 receptors in CNS function. Previous investigations, unknowingly using such mGlu2 receptor-lacking rats, provide insights into the role of mGlu2 receptors in behaviour. The Grm2 mutant rats, which dominate some selectively bred lines, display characteristics of altered emotionality, impulsivity and risk-related behaviours and increased voluntary alcohol intake compared with their mGlu2 receptor-competent counterparts. In addition, the data further emphasize the potential therapeutic role of mGlu2 receptors in psychiatric and neurological disease, and indicate novel methods of studying the role of mGlu2 and mGlu3 receptors. This article is part of the Special Issue entitled 'Metabotropic Glutamate Receptors, 5 years on'.

ApCPEB4, a non-prion domain containing homolog of ApCPEB, is involved in the initiation of long-term facilitation.

Two pharmacologically distinct types of local protein synthesis are required for synapse- specific long-term synaptic facilitation (LTF) in Aplysia: one for initiation and the other for maintenance. ApCPEB, a rapamycin sensitive prion-like molecule regulates a form of local protein synthesis that is specifically required for the maintenance of the LTF. However, the molecular component of the local protein synthesis that is required for the initiation of LTF and that is sensitive to emetine is not known. Here, we identify a homolog of ApCPEB responsible for the initiation of LTF. ApCPEB4 which we have named after its mammalian CPEB4-like homolog lacks a prion-like domain, is responsive to 5-hydroxytryptamine, and is translated (but not transcribed) in an emetine-sensitive, rapamycin-insensitive, and PKA-dependent manner. The ApCPEB4 binds to different target RNAs than does ApCPEB. Knock-down of ApCPEB4 blocked the induction of LTF, whereas overexpression of ApCPEB4 reduces the threshold of the formation of LTF. Thus, our findings suggest that the two different forms of CPEBs play distinct roles in LTF; ApCPEB is required for maintenance of LTF, whereas the ApCPEB4, which lacks a prion-like domain, is required for the initiation of LTF.

The role of nuclear PKMζ in memory maintenance.

Recently, protein kinase M ζ (PKMζ) has emerged as an important player for maintaining memory. It has been reported that PKMζ regulates the trafficking of GluA2 in postsynaptic membranes to maintain memory. However, there has been no study on PKMζ outside the synaptic region regarding memory maintenance. Here, we found that PKMζ is transported to the nucleus in a neural activity-dependent manner. Moreover, we found that PKMζ phosphorylates CREB-binding protein (CBP) at serine residues and that PKMζ inhibition reduces the acetylation of histone H2B and H3. Finally, we showed that the amnesic effect of PKMζ inhibition can be rescued by enhancing histone acetylation level. These results suggest the possibility that nuclear PKMζ has a crucial role in memory maintenance.

Down-regulation of p21-activated serine/threonine kinase 1 is involved in loss of mesencephalic dopamine neurons.

BACKGROUND: Although the roles of p21-activated serine/threonine kinase 1 (PAK1) have been reported in some neurodegenerative diseases, details regarding neurodegeneration are still limited. Hence, we tried to determine the role of PAK1 and molecular mechanisms of neuronal death involved in neurodegeneration. RESULTS: Expression of a dominant-negative form of PAK1 (PAK1(H83,86L, K229R), PAK1-DN) decreased the cell viability and increased cell death induced by oxidative stress. Indeed, oxidative stress decreased the phosphorylation of PAK1 in neuroblastoma cells, cultured dopamine (DA) neurons, or rat midbrains. PAK1-DN reduced the level of Bcl-2 protein, through an ubiquitin/proteasome-dependent mechanism. The level of Bcl-2 may be regulated by PAK1-ERK signaling and/or PAK1, directly. Conversely, expression of an active form of PAK1 (PAK1(T423E), PAK1-CA) could recover both loss of DA neurons in the substantia nigra (SN) and behavioral defects in a 6-OHDA-induced hemiparkinsonian rat model. CONCLUSIONS: Our data suggest that the oxidative stress-induced down-regulation of PAK1 activity could be involved in the loss of mesencephalic DA neurons through modulation of neuronal death, suggesting a novel role of PAK1 as a molecular determinant and mechanisms in the pathogenesis of Parkinson's disease.

A transducible nuclear/nucleolar protein, mLLP, regulates neuronal morphogenesis and synaptic transmission.

Cell-permeable proteins are emerging as unconventional regulators of signal transduction and providing a potential for therapeutic applications. However, only a few of them are identified and studied in detail. We identify a novel cell-permeable protein, mouse LLP homolog (mLLP), and uncover its roles in regulating neural development. We found that mLLP is strongly expressed in developing nervous system and that mLLP knockdown or overexpression during maturation of cultured neurons affected the neuronal growth and synaptic transmission. Interestingly, extracellular addition of mLLP protein enhanced dendritic arborization, demonstrating the non-cell-autonomous effect of mLLP. Moreover, mLLP interacts with CCCTC-binding factor (CTCF) as well as transcriptional machineries and modulates gene expression involved in neuronal growth. Together, these results illustrate the characteristics and roles of previously unknown cell-permeable protein mLLP in modulating neural development.

Calcium-Permeable AMPA Receptors Mediate the Induction of the Protein Kinase A-Dependent Component of Long-Term Potentiation in the Hippocampus.

Two forms of NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) at hippocampal CA1 synapses can be distinguished based on their sensitivity to inhibitors of protein kinase A (PKA). The PKA-dependent form requires multiple episodes of high-frequency stimulation (HFS) or theta burst stimuli (TBS) with a spacing between episodes in the order of minutes. To investigate the mechanism by which spaced episodes induce the PKA-dependent form of LTP, we have compared, in interleaved experiments, spaced (s) and compressed (c) TBS protocols in the rat CA1 synapses. We find that LTP induced by sTBS, but not that induced by cTBS, involves the insertion of calcium-permeable (CP) AMPARs, as assessed using pharmacological and electrophysiological criteria. Furthermore, a single TBS when paired with rolipram [4-(3-(cyclopentyloxy)-4-methoxyphenyl)pyrrolidin-2-one], to activate PKA, generates an LTP that also involves the insertion of CP-AMPARs. These data demonstrate that the involvement of CP-AMPARs in LTP is critically determined by the timing of the induction trigger and is associated specifically with the PKA-dependent form of LTP. SIGNIFICANCE STATEMENT: Long-term potentiation is a family of synaptic mechanisms that are believed to be important for learning and memory. Two of the most extensively studied forms are triggered by the synaptic activation of NMDA receptors and expressed by changes in AMPA receptor function. They can be distinguished on the basis of their requirement for activation of a protein kinase, PKA. We show that the PKA-dependent form also involves the transient insertion of calcium-permeable AMPA receptors. These results have implications for relating synaptic plasticity to learning and memory and suggest a specific linkage between PKA activation and the rapid synaptic insertion of calcium-permeable AMPA receptors during long-term potentiation.

Elevated RalA activity in the hippocampus of PI3Kγ knock-out mice lacking NMDAR-dependent long-term depression.

Phosphoinositide 3-kinases (PI3Ks) play key roles in synaptic plasticity and cognitive functions in the brain. We recently found that genetic deletion of PI3Kγ, the only known member of class IB PI3Ks, results in impaired N-methyl-D-aspartate receptor-dependent long-term depression (NMDAR-LTD) in the hippocampus. The activity of RalA, a small GTP-binding protein, increases following NMDAR-LTD inducing stimuli, and this increase in RalA activity is essential for inducing NMDAR-LTD. We found that RalA activity increased significantly in PI3Kγ knockout mice. Furthermore, NMDAR-LTD-inducing stimuli did not increase RalA activity in PI3Kγ knockout mice. These results suggest that constitutively increased RalA activity occludes further increases in RalA activity during induction of LTD, causing impaired NMDAR-LTD. We propose that PI3Kγ regulates the activity of RalA, which is one of the molecular mechanisms inducing NMDAR dependent LTD.

Learning-related synaptic growth mediated by internalization of Aplysia cell adhesion molecule is controlled by membrane phosphatidylinositol 4,5-bisphosphate synthetic pathway.

Long-term facilitation in Aplysia is accompanied by the growth of new synaptic connections between the sensory and motor neurons of the gill-withdrawal reflex. One of the initial steps leading to the growth of these synapses is the internalization, induced by 5-HT, of the transmembrane isoform of Aplysia cell-adhesion molecule (TM-apCAM) from the plasma membrane of sensory neurons (Bailey et al., 1992). However, the mechanisms that govern the internalization of TM-apCAM and how this internalization is coupled to the molecular events that initiate the structural changes are not fully understood. Here, we report that the synthesis of membrane phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], which is known to be mediated by a signaling cascade through Aplysia Sec7 protein (ApSec7) and phosphatidylinositol-4-phosphate 5-kinase type I α (PIP5KIα) is required for both the internalization of TM-apCAM and the initiation of synaptic growth during 5-HT-induced long-term facilitation. Pharmacological blockade of PI(4,5)P(2) synthesis by the application of the inhibitor phenylarsine oxide blocked the internalization of apCAM. Furthermore, perturbation of the endogenous activation of ApSec7 and its downstream target PIP5KIα also blocked 5-HT-mediated internalization of TM-apCAM and synaptic growth. Finally, long-term facilitation was specifically impaired by blocking the ApSec7 signaling pathway at sensory-to-motor neuron synapses. These data indicate that the ApSec7/PIP5KIα signaling pathway is actively recruited during learning-related 5-HT signaling and acts as a key regulator of apCAM internalization associated with the formation of new synaptic connections during long-term facilitation.

AU-rich element-binding protein negatively regulates CCAAT enhancer-binding protein mRNA stability during long-term synaptic plasticity in Aplysia.

The consolidation of long-term memory for sensitization and synaptic facilitation in Aplysia requires synthesis of new mRNA including the immediate early gene Aplysia CCAAT enhancer-binding protein (ApC/EBP). After the rapid induction of ApC/EBP expression in response to repeated treatments of 5-hydroxytryptamine (5-HT), ApC/EBP mRNA is temporarily expressed in sensory neurons of sensory-to-motor synapses. However, the molecular mechanism underlying the rapid degradation of ApC/EBP transcript is not known. Here, we cloned an AU-rich element (ARE)-binding protein, ApAUF1, which functions as a destabilizing factor for ApC/EBP mRNA. ApAUF1 was found to bind to the 3' UTR of ApC/EBP mRNA that contains AREs and subsequently reduces the expression of ApC/EBP 3' UTR-containing reporter genes. Moreover, overexpression of ApAUF1 inhibited the induction of ApC/EBP mRNA in sensory neurons and also impaired long-term facilitation of sensory-to-motor synapses by repetitive 5-HT treatments. These results provide evidence for a critical role of the posttranscriptional modification of ApC/EBP mRNA during the consolidation of synaptic plasticity.

A cellular model of memory reconsolidation involves reactivation-induced destabilization and restabilization at the sensorimotor synapse in Aplysia.

The memory reconsolidation hypothesis suggests that a memory trace becomes labile after retrieval and needs to be reconsolidated before it can be stabilized. However, it is unclear from earlier studies whether the same synapses involved in encoding the memory trace are those that are destabilized and restabilized after the synaptic reactivation that accompanies memory retrieval, or whether new and different synapses are recruited. To address this issue, we studied a simple nonassociative form of memory, long-term sensitization of the gill- and siphon-withdrawal reflex in Aplysia, and its cellular analog, long-term facilitation at the sensory-to-motor neuron synapse. We found that after memory retrieval, behavioral long-term sensitization in Aplysia becomes labile via ubiquitin/proteasome-dependent protein degradation and is reconsolidated by means of de novo protein synthesis. In parallel, we found that on the cellular level, long-term facilitation at the sensory-to-motor neuron synapse that mediates long-term sensitization is also destabilized by protein degradation and is restabilized by protein synthesis after synaptic reactivation, a procedure that parallels memory retrieval or retraining evident on the behavioral level. These results provide direct evidence that the same synapses that store the long-term memory trace encoded by changes in the strength of synaptic connections critical for sensitization are disrupted and reconstructed after signal retrieval.

The Jak/STAT pathway is involved in synaptic plasticity.

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway is involved in many cellular processes, including cell growth and differentiation, immune functions and cancer. It is activated by various cytokines, growth factors, and protein tyrosine kinases (PTKs) and regulates the transcription of many genes. Of the four JAK isoforms and seven STAT isoforms known, JAK2 and STAT3 are highly expressed in the brain where they are present in the postsynaptic density (PSD). Here, we demonstrate a new neuronal function for the JAK/STAT pathway. Using a variety of complementary approaches, we show that the JAK/STAT pathway plays an essential role in the induction of NMDA-receptor dependent long-term depression (NMDAR-LTD) in the hippocampus. Therefore, in addition to established roles in cytokine signaling, the JAK/STAT pathway is involved in synaptic plasticity in the brain.

Assessment of the effects of virus-mediated limited Oct4 overexpression on the structure of the hippocampus and behavior in mice.

Recently, pluripotency induction or cellular reprogramming by introducing critical transcription factors has been extensively studied, but has been demonstrated only in vitro. Based on reports that Oct4 is critically involved in transforming neural stem cells into pluripotent cells, we used the lentiviral vector to introduce the Oct4 gene into the hippocampal dentate gyrus (DG) of adult mice. We examined whether this manipulation led to cellular or behavioral changes, possibly through processes involving the transformation of NS cells into pluripotent cells. The Oct4 lentivirus-infused group and the green fluorescent protein lentivirus-infused group showed a similar thickness of the DG and a comparable level of synaptophysin expression in the DG. Furthermore, our behavioral analyses did not show any differences between the groups concerning exploratory activity, anxiety, or memory abilities. This first trial for pluripotency induction in vivo, despite negative results, provides implications and information for future studies on in vivo cellular reprogramming.

N termini of apPDE4 isoforms are responsible for targeting the isoforms to different cellular membranes.

Phosphodiesterases (PDEs) are known to play a key role in the compartmentalization of cAMP signaling; however, the molecular mechanisms underlying intracellular localization of different PDE isoforms are not understood. In this study, we have found that each of the supershort, short, and long forms of apPDE4 showed distinct localization in the cytoplasm, plasma membrane, and both plasma membrane and presynaptic terminals, respectively. The N-terminal 20 amino acids of the long form of apPDE4 were involved in presynaptic terminal targeting by binding to several lipids. In addition, the N terminus of the short form of apPDE4 bound to several lipids including phosphoinositols, thereby targeting the plasma membrane. Overexpression of the long and the short forms, but not the supershort form attenuated 5-HT-induced membrane hyperexcitability. Finally, the knockdown of apPDE4s in sensory neurons impaired both short-term and long-term facilitation. Thus, these results suggest that apPDE4s can participate in the regulation of cAMP signaling through specific subcellular localization by means of lipid binding activities.

Differential evolutionary rates of neuronal transcriptome in Aplysia kurodai and Aplysia californica as a tool for gene mining.

The marine mollusk Aplysia is a fascinating model organism for studying molecular mechanisms underlying learning and memory. However, evolutionary studies about Aplysia have been limited by the lack of its genomic information. Recently, large-scale expressed sequence tag (EST) databases have been acquired by sequencing cDNA libraries from A. californica and A. kurodai. The closeness between the two species allowed us to investigate rapidly evolving genes by comparing their orthologs. Using this method, we found that a subset of signal transduction genes in neurons showed rates of protein evolution higher than those of housekeeping genes. Moreover, we were also able to find several candidate genes that may be involved in learning and memory or synaptic plasticity among genes showing relatively higher K(a)/K(s) ratios. We also investigated the relationship between evolutionary rates and tissue distribution of Aplysia genes. They propose that the estimation of evolutionary rates cannot be a good marker to assess neuronal expression; however, it still can be an efficient way to narrow down the pool of candidate genes involved in neuronal functions for the further studies.

Transcriptome and protein domain analyses in Aplysia nervous system with evolutionary implications.

The sea hare Aplysia is a powerful model organism for studying the structure and function of the nervous system. Recently, the genomic characterization of Aplysia has been facilitated: A large scale EST sequences was acquired by sequencing cDNA libraries from A. californica and a parallel EST database of the closely related species A. kurodai was reported. These EST databases provide useful tools for both molecular biology and bioinformatics. In our previous report, we demonstrated the utility of the database by screening the candidate genes for the synaptic plasticity and the behavioral sensitization using the microarray containing A. kurodai ESTs. In this addendum, we have expanded our study to show that the protein domain repertoire and the abundance of regulatory genes displayed a linear relationship with the evolution of the complex brains in different lineages. This distinct set of protein domains may play critical roles in evolution of the nervous systems.

Identification of a serotonin receptor coupled to adenylyl cyclase involved in learning-related heterosynaptic facilitation in Aplysia.

Serotonin (5-HT) plays a critical role in modulating synaptic plasticity in the marine mollusc Aplysia and in the mammalian nervous system. In Aplysia sensory neurons, 5-HT can activate several signal cascades, including PKA and PKC, presumably via distinct types of G protein-coupled receptors. However, the molecular identities of these receptors have not yet been identified. We here report the cloning and functional characterization of a 5-HT receptor that is positively coupled to adenylyl cyclase in Aplysia neurons. The cloned receptor, 5-HT(apAC1), stimulates the production of cAMP in HEK293T cells and in Xenopus oocytes. Moreover, the knockdown of 5-HT(apAC1) expression by RNA interference blocked 5-HT-induced cAMP production in Aplysia sensory neurons and blocked synaptic facilitation in nondepressed or partially depressed sensory-to-motor neuron synapses. These data implicate 5-HT(apAC1) as a major modulator of learning related synaptic facilitation in the direct sensory to motor neuron pathway of the gill withdrawal reflex.

Transcriptome analysis and identification of regulators for long-term plasticity in Aplysia kurodai.

The marine mollusk Aplysia is a useful model organism for studying the cellular bases of behavior and plasticity. However, molecular studies of Aplysia have been limited by the lack of genomic information. Recently, a large scale characterization of neuronal transcripts was performed in A. californica. Here, we report the analysis of a parallel set of neuronal transcripts from a closely related species A. kurodai found in the northwestern Pacific. We collected 4,859 nonredundant sequences from the nervous system tissue of A. kurodai. By performing microarray and real-time PCR analyses, we found that ApC/EBP, matrilin, antistasin, and eIF3e clones were significantly up-regulated and a BAT1 homologous clone was significantly down-regulated by 5-HT treatment. Among these, we further demonstrated that the Ap-eIF3e plays a key role in 5-HT-induced long-term facilitation (LTF) as a positive regulator.

A novel conditional genetic system reveals that increasing neuronal cAMP enhances memory and retrieval.

Consistent evidence from pharmacological and genetic studies shows that cAMP is a critical modulator of synaptic plasticity and memory formation. However, the potential of the cAMP signaling pathway as a target for memory enhancement remains unclear because of contradictory findings from pharmacological and genetic approaches. To address these issues, we have developed a novel conditional genetic system in mice based on the heterologous expression of an Aplysia octopamine receptor, a G-protein-coupled receptor whose activation by its natural ligand octopamine leads to rapid and transient increases in cAMP. We find that activation of this receptor transgenically expressed in mouse forebrain neurons induces a rapid elevation of hippocampal cAMP levels, facilitates hippocampus synaptic plasticity, and enhances the consolidation and retrieval of fear memory. Our findings clearly demonstrate that acute increases in cAMP levels selectively in neurons facilitate synaptic plasticity and memory, and illustrate the potential of this heterologous system to study cAMP-mediated processes in mammalian systems.

Synaptic protein degradation underlies destabilization of retrieved fear memory.

Reactivated memory undergoes a rebuilding process that depends on de novo protein synthesis. This suggests that retrieval is dynamic and serves to incorporate new information into preexisting memories. However, little is known about whether or not protein degradation is involved in the reorganization of retrieved memory. We found that postsynaptic proteins were degraded in the hippocampus by polyubiquitination after retrieval of contextual fear memory. Moreover, the infusion of proteasome inhibitor into the CA1 region immediately after retrieval prevented anisomycin-induced memory impairment, as well as the extinction of fear memory. This suggests that ubiquitin- and proteasome-dependent protein degradation underlies destabilization processes after fear memory retrieval. It also provides strong evidence for the existence of reorganization processes whereby preexisting memory is disrupted by protein degradation, and updated memory is reconsolidated by protein synthesis.