Shizuka Kobayashi

Kinase-Dead Knock-In Mouse Reveals an Essential Role of Kinase Activity of Ca2+/Calmodulin-Dependent Protein Kinase IIα in Dendritic Spine Enlargement, Long-Term Potentiation, and Learning

Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) is an essential mediator of activity-dependent synaptic plasticity that possesses multiple protein functions. So far, the autophosphorylation site-mutant mice targeted at T286 and at T305/306 have demonstrated the importance of the autonomous activity and Ca2+/calmodulin-binding capacity of CaMKIIα, respectively, in the induction of long-term potentiation (LTP) and hippocampus-dependent learning. However, kinase activity of CaMKIIα, the most essential enzymatic function, has not been genetically dissected yet. Here, we generated a novel CaMKIIα knock-in mouse that completely lacks its kinase activity by introducing K42R mutation and examined the effects on hippocampal synaptic plasticity and behavioral learning. In homozygous CaMKIIα (K42R) mice, kinase activity was reduced to the same level as in CaMKIIα-null mice, whereas CaMKII protein expression was well preserved. Tetanic stimulation failed to induce not only LTP but also sustained dendritic spine enlargement, a structural basis for LTP, at the Schaffer collateral–CA1 synapse, whereas activity-dependent postsynaptic translocation of CaMKIIα was preserved. In addition, CaMKIIα (K42R) mice showed a severe impairment in inhibitory avoidance learning, a form of memory that is dependent on the hippocampus. These results demonstrate that kinase activity of CaMKIIα is a common critical gate controlling structural, functional, and behavioral expression of synaptic memory.

Dysfunction of the RAR/RXR signaling pathway in the forebrain impairs hippocampal memory and synaptic plasticity

BackgroundRetinoid signaling pathways mediated by retinoic acid receptor (RAR)/retinoid × receptor (RXR)-mediated transcription play critical roles in hippocampal synaptic plasticity. Furthermore, recent studies have shown that treatment with retinoic acid alleviates age-related deficits in hippocampal long-term potentiation (LTP) and memory performance and, furthermore, memory deficits in a transgenic mouse model of Alzheimers disease. However, the roles of the RAR/RXR signaling pathway in learning and memory at the behavioral level have still not been well characterized in the adult brain. We here show essential roles for RAR/RXR in hippocampus-dependent learning and memory. In the current study, we generated transgenic mice in which the expression of dominant-negative RAR (dnRAR) could be induced in the mature brain using a tetracycline-dependent transcription factor and examined the effects of RAR/RXR loss.ResultsThe expression of dnRAR in the forebrain down-regulated the expression of RARβ, a target gene of RAR/RXR, indicating that dnRAR mice exhibit dysfunction of the RAR/RXR signaling pathway. Similar with previous findings, dnRAR mice displayed impaired LTP and AMPA-mediated synaptic transmission in the hippocampus. More importantly, these mutant mice displayed impaired hippocampus-dependent social recognition and spatial memory. However, these deficits of LTP and memory performance were rescued by stronger conditioning stimulation and spaced training, respectively. Finally, we found that pharmacological blockade of RARα in the hippocampus impairs social recognition memory.ConclusionsFrom these observations, we concluded that the RAR/RXR signaling pathway greatly contributes to learning and memory, and LTP in the hippocampus in the adult brain.

Neuronal Leucine-Rich Repeat Protein 4 Functions in Hippocampus-Dependent Long-Lasting Memory

ABSTRACT Neuronal leucine-rich repeat proteins (NLRRs) are type I transmembrane proteins and expressed in neuronal tissues, but their function remains unknown. Here, we describe the identification and characterization of a new member of the NLRR family, NLRR4, and its potential role in long-lasting memory. We generated NLRR4-deficient (NLRR4−/−) mice and found that they showed impaired memory retention. In hippocampus-dependent learning tasks, NLRR4−/− mice were able to learn and maintain the memories for one day but unable to retain the memories for four days after learning. In contrast, in a hippocampus-independent task, NLRR4−/− mice were able to retain the memory normally for at least seven days. These results suggest that NLRR4 plays a key role in hippocampus-dependent long-lasting memory.

CDKL5 controls postsynaptic localization of GluN2B-containing NMDA receptors in the hippocampus and regulates seizure susceptibility

Mutations in the Cyclin-dependent kinase-like 5 (CDKL5) gene cause severe neurodevelopmental disorders accompanied by intractable epilepsies, i.e. West syndrome or atypical Rett syndrome. Here we report generation of the Cdkl5 knockout mouse and show that CDKL5 controls postsynaptic localization of GluN2B-containing N-methyl-d-aspartate (NMDA) receptors in the hippocampus and regulates seizure susceptibility. Cdkl5 -/Y mice showed normal sensitivity to kainic acid; however, they displayed significant hyperexcitability to NMDA. In concordance with this result, electrophysiological analysis in the hippocampal CA1 region disclosed an increased ratio of NMDA/α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated excitatory postsynaptic currents (EPSCs) and a significantly larger decay time constant of NMDA receptor-mediated EPSCs (NMDA-EPSCs) as well as a stronger inhibition of the NMDA-EPSCs by the GluN2B-selective antagonist ifenprodil in Cdkl5 -/Y mice. Subcellular fractionation of the hippocampus from Cdkl5 -/Y mice revealed a significant increase of GluN2B and SAP102 in the PSD (postsynaptic density)-1T fraction, without changes in the S1 (post-nuclear) fraction or mRNA transcripts, indicating an intracellular distribution shift of these proteins to the PSD. Immunoelectron microscopic analysis of the hippocampal CA1 region further confirmed postsynaptic overaccumulation of GluN2B and SAP102 in Cdkl5 -/Y mice. Furthermore, ifenprodil abrogated the NMDA-induced hyperexcitability in Cdkl5 -/Y mice, suggesting that upregulation of GluN2B accounts for the enhanced seizure susceptibility. These data indicate that CDKL5 plays an important role in controlling postsynaptic localization of the GluN2B-SAP102 complex in the hippocampus and thereby regulates seizure susceptibility, and that aberrant NMDA receptor-mediated synaptic transmission underlies the pathological mechanisms of the CDKL5 loss-of-function.

LMTK3 Deficiency Causes Pronounced Locomotor Hyperactivity and Impairs Endocytic Trafficking

LMTK3 belongs to the LMTK family of protein kinases that are predominantly expressed in the brain. Physiological functions of LMTK3 and other members of the LMTK family in the CNS remain unknown. In this study, we performed a battery of behavioral analyses using Lmtk3−/− mice and showed that these mice exhibit abnormal behaviors, including pronounced locomotor hyperactivity, reduced anxiety behavior, and decreased depression-like behavior. Concurrently, the dopamine metabolite levels and dopamine turnover rate are increased in the striata of Lmtk3−/− mice compared with wild-type controls. In addition, using cultured primary neurons from Lmtk3−/− mice, we found that LMTK3 is involved in the endocytic trafficking of N-methyl-d-aspartate receptors, a type of ionotropic glutamate receptor. Altered membrane traffic of the receptor in Lmtk3−/− neurons may underlie behavioral abnormalities in the mutant animals. Together, our data suggest that LMTK3 plays an important role in regulating locomotor behavior in mice.

The active zone protein CAST regulates synaptic vesicle recycling and quantal size in the mouse hippocampus

Synaptic efficacy is determined by various factors, including the quantal size, which is dependent on the amount of neurotransmitters in synaptic vesicles at the presynaptic terminal. It is essential for stable synaptic transmission that the quantal size is kept within a constant range and that synaptic efficacy during and after repetitive synaptic activation is maintained by replenishing release sites with synaptic vesicles. However, the mechanisms for these fundamental properties have still been undetermined. We found that the active zone protein CAST (cytomatrix at the active zone structural protein) played pivotal roles in both presynaptic regulation of quantal size and recycling of endocytosed synaptic vesicles. In the CA1 region of hippocampal slices of the CAST knockout mice, miniature excitatory synaptic responses were increased in size, and synaptic depression after prolonged synaptic activation was larger, which was attributable to selective impairment of synaptic vesicle trafficking via the endosome in the presynaptic terminal likely mediated by Rab6. Therefore, CAST serves as a key molecule that regulates dynamics and neurotransmitter contents of synaptic vesicles in the excitatory presynaptic terminal in the central nervous system.

SNAP-25 phosphorylation at Ser187 regulates synaptic facilitation and short-term plasticity in an age-dependent manner

Neurotransmitter release is mediated by the SNARE complex, but the role of its phosphorylation has scarcely been elucidated. Although PKC activators are known to facilitate synaptic transmission, there has been a heated debate on whether PKC mediates facilitation of neurotransmitter release through phosphorylation. One of the SNARE proteins, SNAP-25, is phosphorylated at the residue serine-187 by PKC, but its physiological significance has been unclear. To examine these issues, we analyzed mutant mice lacking the phosphorylation of SNAP-25 serine-187 and found that they exhibited reduced release probability and enhanced presynaptic short-term plasticity, suggesting that not only the release process, but also the dynamics of synaptic vesicles was regulated by the phosphorylation. Furthermore, it has been known that the release probability changes with development, but the precise mechanism has been unclear, and we found that developmental changes in release probability of neurotransmitters were regulated by the phosphorylation. These results indicate that SNAP-25 phosphorylation developmentally facilitates neurotransmitter release but strongly inhibits presynaptic short-term plasticity via modification of the dynamics of synaptic vesicles in presynaptic terminals.

Kinase activity of CaMKIIα is essential for structural, functional and behavioral expression of synaptic memory

has various effects including antagonism of adenosine receptors, it remains to be determined whether the caffeine-induced synaptic enhancement was mediated by activation of ryanodine receptors. To assess relative contribution of these effects, we applied the adenosine A1 receptor antagonist 8-cyclopenthyl-1, 3-dipropylxanthine (DPCPX) to block A1 receptors before application of caffeine. Application of 1 M DPCPX increased EPSPs to 156 ± 23% of control, whereas subsequent application of 10 mM caffeine in the presence of DPCPX further enhanced EPSPs to 313 ± 24% (n = 4). This result suggests that caffeine enhances the mossy fiber transmission not only by blocking A1 receptors, but also probably by activating ryanodine receptors.