Sayaka Takemoto-Kimura

Regulation of osteoclast differentiation and function by the CaMK-CREB pathway

Calcium (Ca2+) signaling is essential for a variety of cellular responses and higher biological functions. Ca2+/calmodulin-dependent kinases (CaMKs) and the phosphatase calcineurin activate distinct downstream pathways that are mediated by the transcription factors cAMP response element (CRE)-binding protein (CREB) and nuclear factor of activated T cells (NFAT), respectively. The importance of the calcineurin-NFAT pathway in bone metabolism has been demonstrated in osteoclasts, osteoblasts and chondrocytes. However, the contribution of the CaMK-CREB pathway is poorly understood, partly because of the difficulty of dissecting the functions of homologous family members. Here we show that the CaMKIV-CREB pathway is crucial for osteoclast differentiation and function. Pharmacological inhibition of CaMKs as well as the genetic ablation of Camk4 reduced CREB phosphorylation and downregulated the expression of c-Fos, which is required for the induction of NFATc1 (the master transcription factor for osteoclastogenesis) that is activated by receptor activator of NF-κB ligand (RANKL). Furthermore, CREB together with NFATc1 induced the expression of specific genes expressed by differentiated osteoclasts. Thus, the CaMK-CREB pathway biphasically functions to regulate the transcriptional program of osteoclastic bone resorption, by not only enhancing induction of NFATc1 but also facilitating NFATc1-dependent gene regulation once its expression is induced. This provides a molecular basis for a new therapeutic strategy for bone diseases.

Control of axon elongation via an SDF-1α/Rho/mDia pathway in cultured cerebellar granule neurons

Rho–GTPase has been implicated in axon outgrowth. However, not all of the critical steps controlled by Rho have been well characterized. Using cultured cerebellar granule neurons, we show here that stromal cell–derived factor (SDF)-1α, a neural chemokine, is a physiological ligand that can turn on two distinct Rho-dependent pathways with opposite consequences. A low concentration of the ligand stimulated a Rho-dependent pathway that mediated facilitation of axon elongation. In contrast, Rho/ROCK activation achieved by a higher concentration of SDF-1α caused repression of axon formation and induced no more increase in axon length. However, even at this higher concentration a Rho-dependent axon elongating activity could be recovered upon removal of ROCK activity using Y-27632. SDF-1α–induced axon elongating activity under ROCK inhibition was replicated by the dominant-active form of the mammalian homologue of the Drosophila gene Diaphanous (mDia)1 and counteracted by its dominant-negative form. Furthermore, RNAi knockdown of mDia1 abolished SDF-1α–induced axon elongation. Together, our results support a critical role for an SDF-1α/Rho/mDia1 pathway in mediating axon elongation.

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.

Ca2+/CREB/CBP-dependent gene regulation: a shared mechanism critical in long-term synaptic plasticity and neuronal survival

CREB is a transcription factor critical for long-term synaptic plasticity. Intriguingly, recent work has elucidated a role for CREB, as well as upstream CREB kinases, in the control of activity-dependent neuronal survival. Additionally, analysis of the molecular pathology of polyglutamine-repeat diseases suggest that alteration of pCREB-CBP function may underlie, at least in part, the neurodegenerative process. Taken together, these new findings support the idea that Ca(2+)/CREB/CBP-dependent gene regulation might be a shared mechanism critical in both long-term synaptic plasticity and neuronal survival.

Rational design of a high-affinity, fast, red calcium indicator R-CaMP2

Fluorescent Ca2+ reporters are widely used as readouts of neuronal activities. Here we designed R-CaMP2, a high-affinity red genetically encoded calcium indicator (GECI) with a Hill coefficient near 1. Use of the calmodulin-binding sequence of CaMKK-α and CaMKK-β in lieu of an M13 sequence resulted in threefold faster rise and decay times of Ca2+ transients than R-CaMP1.07. These features allowed resolving single action potentials (APs) and recording fast AP trains up to 20–40 Hz in cortical slices. Somatic and synaptic activities of a cortical neuronal ensemble in vivo were imaged with similar efficacy as with previously reported sensitive green GECIs. Combining green and red GECIs, we successfully achieved dual-color monitoring of neuronal activities of distinct cell types, both in the mouse cortex and in freely moving Caenorhabditis elegans. Dual imaging using R-CaMP2 and green GECIs provides a powerful means to interrogate orthogonal and hierarchical neuronal ensembles in vivo.

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.

Multiple spatiotemporal modes of actin reorganization by NMDA receptors and voltage-gated Ca2+ channels

Cytoskeleton is believed to contribute to activity-dependent processes underlying neuronal plasticity, such as regulations of cellular morphology and localization of signaling proteins. However, how neuronal activity controls actin cytoskeleton remains obscure. Taking advantage of confocal imaging of enhanced GFP-actin in the primary culture of hippocampal neurons, we show that synaptic activity induces multiple types of actin reorganization, both at the spines and at the somatic periphery. Activation of N-methyl-d-aspartate receptors, accompanied with a local rise in [Ca2+]i, was sufficient to trigger a slow and sustained recruitment of actin into dendritic spines. In contrast, opening of voltage-gated Ca2+ channels rapidly and reversibly enhanced cortical actin at the somatic periphery but not in the spines, in keeping with a high transient rise in somatic [Ca2+]i. These data suggest that spatiotemporal dynamics of [Ca2+]i, triggered by activation of N-methyl-d-aspartate receptors and voltage-gated Ca2+ channels, provides the molecular basis for activity-dependent actin remodeling.

Functional labeling of neurons and their projections using the synthetic activity–dependent promoter E-SARE

Identifying the neuronal ensembles that respond to specific stimuli and mapping their projection patterns in living animals are fundamental challenges in neuroscience. To this end, we engineered a synthetic promoter, the enhanced synaptic activity–responsive element (E-SARE), that drives neuronal activity–dependent gene expression more potently than other existing immediate-early gene promoters. Expression of a drug-inducible Cre recombinase downstream of E-SARE enabled imaging of neuronal populations that respond to monocular visual stimulation and tracking of their long-distance thalamocortical projections in living mice. Targeted cell-attached recordings and calcium imaging of neurons in sensory cortices revealed that E-SARE reporter expression correlates with sensory-evoked neuronal activity at the single-cell level and is highly specific to the type of stimuli presented to the animals. This activity-dependent promoter can expand the repertoire of genetic approaches for high-resolution anatomical and functional analysis of neural circuits.

Control of Cortical Axon Elongation by a GABA-Driven Ca2+/Calmodulin-Dependent Protein Kinase Cascade

Ca2+ signaling plays important roles during both axonal and dendritic growth. Yet whether and how Ca2+ rises may trigger and contribute to the development of long-range cortical connections remains mostly unknown. Here, we demonstrate that two separate limbs of the Ca2+/calmodulin-dependent protein kinase kinase (CaMKK)–CaMKI cascades, CaMKK–CaMKIα and CaMKK–CaMKIγ, critically coordinate axonal and dendritic morphogenesis of cortical neurons, respectively. The axon-specific morphological phenotype required a diffuse cytoplasmic localization and a strikingly α-isoform-specific kinase activity of CaMKI. Unexpectedly, treatment with muscimol, a GABAA receptor agonist, selectively stimulated elongation of axons but not of dendrites, and the CaMKK–CaMKIα cascade critically mediated this axonogenic effect. Consistent with these findings, during early brain development, in vivo knockdown of CaMKIα significantly impaired the terminal axonal extension and thereby perturbed the refinement of the interhemispheric callosal projections into the contralateral cortices. Our findings thus indicate a novel role for the GABA-driven CaMKK–CaMKIα cascade as a mechanism critical for accurate cortical axon pathfinding, an essential process that may contribute to fine-tuning the formation of interhemispheric connectivity during the perinatal development of the CNS.

Molecular Cloning and Characterization of CLICK-III/CaMKIγ, a Novel Membrane-anchored Neuronal Ca2+/Calmodulin-dependent Protein Kinase (CaMK)

During a screen for novel putative Ca2+/calmodulin-dependent protein kinase (CaMK)-like CREB kinases (CLICKs), we have cloned a full-length cDNA for CLICK-III/CaMKIγ, an isoform of the CaMKI family with an extended C-terminal domain ending with CAAX motif (whereAA is aliphatic acid). As expected from the similarity of its kinase domain with the other CaMKI isoforms, full activation of CLICK-III/CaMKIγ required both Ca2+/CaM and phosphorylation by CaMKK. We also found that Ca2+/cAMP-response element-binding protein (CREB) was a good substrate for CLICK-III/CaMKIγ, at least in vitro. Interestingly enough, CLICK-III/CaMKIγ transcripts were most abundant in neurons, with the highest levels in limited nuclei such as the central nucleus of the amygdala (CeA) and the ventromedial hypothalamus. Consistent with the presence of the CAAXmotif, CLICK-III/CaMKIγ was found to be anchored to various membrane compartments, especially to Golgi and plasma membranes. Both point mutation in the CAAX motif and treatment with compactin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, disrupted such membrane localization, suggesting that membrane localization of CLICK-III/CaMKIγ occurred in a prenylation-dependent way. These findings provide a novel mechanism by which neuronal CaMK activity could be targeted to specific membrane compartments.