Michiko Okamura

Synaptic activity-responsive element in the Arc/Arg3.1 promoter essential for synapse-to-nucleus signaling in activated neurons

The neuronal immediate early gene Arc/Arg-3.1 is widely used as one of the most reliable molecular markers for intense synaptic activity in vivo. However, the cis-acting elements responsible for such stringent activity dependence have not been firmly identified. Here we combined luciferase reporter assays in cultured cortical neurons and comparative genome mapping to identify the critical synaptic activity-responsive elements (SARE) of the Arc/Arg-3.1 gene. A major SARE was found as a unique ≈100-bp element located at >5 kb upstream of the Arc/Arg-3.1 transcription initiation site in the mouse genome. This single element, when positioned immediately upstream of a minimal promoter, was necessary and sufficient to replicate crucial properties of endogenous Arc/Arg-3.1s transcriptional regulation, including rapid onset of transcription triggered by synaptic activity and low basal expression during synaptic inactivity. We identified the major determinants of SARE as a unique cluster of neuronal activity-dependent cis-regulatory elements consisting of closely localized binding sites for CREB, MEF2, and SRF. Consistently, a SARE reporter could readily trace and mark an ensemble of cells that have experienced intense activity in the recent past in vivo. Taken together, our work uncovers a novel transcriptional mechanism by which a critical 100-bp element, SARE, mediates a predominant component of the synapse-to-nucleus signaling in ensembles of Arc/Arg-3.1-positive activated neurons.

Regulation of Dendritogenesis via a Lipid-Raft-Associated Ca2+/Calmodulin-Dependent Protein Kinase CLICK-III/CaMKIγ

Ca(2+) signaling plays a central role in activity-dependent regulation of dendritic arborization, but key molecular mechanisms downstream of calcium elevation remain poorly understood. Here we show that the C-terminal region of the Ca(2+)/calmodulin-dependent protein kinase CLICK-III (CL3)/CaMKIgamma, a membrane-anchored CaMK, was uniquely modified by two sequential lipidification steps: prenylation followed by a kinase-activity-regulated palmitoylation. These modifications were essential for CL3 membrane anchoring and targeting into detergent-resistant lipid microdomains (or rafts) in the dendrites. We found that CL3 critically contributed to BDNF-stimulated dendritic growth. Raft insertion of CL3 specifically promoted dendritogenesis of cortical neurons by acting upstream of RacGEF STEF and Rac, both present in lipid rafts. Thus, CL3 may represent a key element in the Ca(2+)-dependent and lipid-raft-delineated switch that turns on extrinsic activity-regulated dendrite formation in developing cortical neurons.

Region-specific activation of CRTC1-CREB signaling mediates long-term fear memory.

CREB is a pivotal mediator of activity-regulated gene transcription that underlies memory formation and allocation. The contribution of a key CREB cofactor, CREB-regulated transcription coactivator 1 (CRTC1), has, however, remained elusive. Here we show that several constitutive kinase pathways and an activity-regulated phosphatase, calcineurin, converge to determine the nucleocytoplasmic shuttling of CRTC1. This, in turn, triggered an activity-dependent association of CRTC1 with CREB-dependent regulatory elements found on IEG promoters. Forced expression of nuclear CRTC1 in hippocampal neurons activated CREB-dependent transcription, and was sufficient to enhance contextual fear memory. Surprisingly, during contextual fear conditioning, we found evidence of nuclear recruitment of endogenous CRTC1 only in the basolateral amygdala, and not in the hippocampus. Consistently, CRTC1 knockdown in the amygdala, but not in the hippocampus, significantly attenuated fear memory. Thus, CRTC1 has a wide impact on CREB-dependent memory processes, but fine-tunes CREB output in a region-specific manner.

Molecular identification and characterization of a family of kinases with homology to Ca2+/calmodulin-dependent protein kinases I/IV.

Despite the critical importance of Ca2+/calmodulin (CaM)-dependent protein kinase (CaMK) II signaling in neuroplasticity, only a limited amount of work has so far been available regarding the presence and significance of another predominant CaMK subfamily, the CaMKI/CaMKIV family, in the central nervous system. We here searched for kinases with a core catalytic structure similar to CaMKI and CaMKIV. We isolated full-length cDNAs encoding three mouse CaMKI/CaMKIV-related kinases, CLICK-I (CL1)/doublecortin and CaM kinase-Like (DCAMKL)1, CLICK-II (CL2)/DCAMKL2, and CLICK-I,II-related (CLr)/DCAMKL3, the kinase domains of which had an intermediate homology not only to CaMKI/CaMKIV but also to CaMKII. Furthermore, CL1, CL2, and CLr were highly expressed in the central nervous system, in a neuron-specific fashion. CL1α and CL1β were shorter isoforms of DCAMKL1, which lacked the doublecortin-like domain (Dx). In contrast, CL2α and CL2β contained a full N-terminal Dx, whereas CLr only possessed a partial and dysfunctional Dx. Interestingly, despite a large similarity in the kinase domain, CL1/CL2/CLr had an impact on CRE-dependent gene expression distinct from that of the related CaMKI/CaMKIV and CaMKII. Although these were previously shown to activate Ca2+/cAMP-response element-binding protein (CREB)-dependent transcription, we here show that CL1 and CL2 were unable to significantly phosphorylate CREB Ser-133 and rather inhibited CRE-dependent gene expression by a dominant mechanism that bypassed CREB and was mediated by phosphorylated TORC2.

Receptor-Gα fusion proteins as a tool for ligand screening

Abstract We have prepared fusion proteins of muscarinic M 1 - M 5 receptors with α subunits of G proteins G i1 , G i2 , G s , G 11 , G 16 and chimera of G protein α subunits using the bacurovirus-Sf9 expression system. In fusion proteins such as M 2 -G i1 α and M 4 -G i1 α, agonist caused the decrease in the apparent affinity for GDP of these fusion proteins and then the increase in [ 35 S]GTPγS binding in the presence of GDP. Thus we could use the membrane preparation expressing these fusion proteins as a tool to screen agonists and antagonists. On the other hand, the effect of agonists to decrease the apparent affinity for GDP was not clearly observed in fusion proteins of G q /G 11 -coupled receptors such as M 1 -G 11 α, M 3 -G 11 α, and M 5 -G 11 α. The effect of agonists could be observed for fusion proteins with G 16 α of muscarinic M 1 , M 2 and adrenergic β2 receptors, but the extent of the effect was much less than that for fusion proteins with G i1 α of G i /G o -coupled receptors. Fusion proteins of M 1 receptors with G i1 α or chimera of G 16 α and G i2 α were also not effective in detecting the action of agonists.

Control elements of muscarinic receptor gene expression.

Studies describing the structures of the M1, M2 and M4 muscarinic acetylcholine receptors (mAChR) genes and the genetic elements that control their expression are reviewed. In particular, we focus on the role of the neuron-restrictive silencer element/restriction element-1 (NRSE/RE-1) in the regulation of the M4 mAChR gene. The NRSE/RE-1 was first identified as a genetic control element that prevents the expression of the SCG-10 and type II sodium channel (NaII) genes in non-neuronal cells in culture. The NRSE/RE-1 inhibits gene expression by binding the repressor/silencer protein NRSF/REST, which is present in many non-neuronal cell lines and tissues. Our studies show that although the expression of the M4 mAChR gene is inhibited by NRSF/REST, this inhibition is not always complete. Rather, the efficiency of silencing by NRSF/REST is different in different cells. A plausible explanation for this differential silencing is that the NRSF/RE-1 interacts with distinct sets of promoter binding proteins in different types of cells. We hypothesize that modulation of NRSF/REST silencing activity by these proteins contributes to the cell-specific pattern of expression of the M4 mAChR in neuronal and non-neuronal cells. Recent studies that suggest a more complex role for the NRSE/RE-1 in regulating gene expression are also discussed.

Molecular dissection of the critical role of CRTC1-CREB in regulating neuronal immediate early gene expression

of LTP inhibition by melatonin, this study electrophysiologically examined whether melatonin inhibits hippocampal LTP by way of the NO signaling pathway, because melatonin has the ability to scavenge free radicals such as nitric oxide (NO) and because NO has been suggested to be an important contributor to LTP induction. Field EPSPs at Schaffer collateral CA1 pyramidal cell synapses were recorded, and LTP was induced by tetanic stimulation (100 Hz, 1 sec). Melatonin (100 nM) reduced the degree of LTP, and L-NAME (100 M), an inhibitor of NO synthase, also reduced LTP, but simultaneous application of melatonin and L-NAME did not evoke any additional reduction of LTP in comparison with only melatonin or only L-NAME were applied. Furthermore, the inhibition of LTP by the application of melatonin and LNAME was disrupted by the application of an NO donor, DEA/NO (3 M). The paired-pulse (ISI 50 msec) facilitation ratios before and after LTP induction by tetanic stimulation in the absence and presence of L-NAME were nearly identical, showing that under our experimental conditions the transmitter release probability was not changed by tetanic stimulation and that presynaptic sites were not affected by L-NAME application. These findings demonstrate that the inhibition of LTP in the presence of melatonin is due to the action of melatonin on the postsynaptic NO signaling pathway.