Alternative splicing in the variable domain of CaMKIIß affects the level of F-actin association in developing neurons.

The Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) ß has an essential function in dendritic spines via binding to and reorganization of the actin cytoskeleton during plasticity events not shared by CaMKIIα isoform. CaMKIIß and CaMKIIα isoforms have remarkable structural differences within the variable region. Three exons (E1, E3, and E4) are present in CaMKIIß but not in CaMKIIα gene. Four splice variants of CaMKIIß isoforms (CaMKIIß, ß', ße and ß'e) were discovered in embryonic and adult brains. Exons E1 (lacked in ße and ß'e) and E4 (lacked in ß' and ß'e) are subject to differential alternative splicing. We hypothesized that the sequences encoded by exons E1, E3, and/or E4 are involved in CaMKIIß-specific bundling to the F-actin cytoskeleton. We tested the colocalization and association of these CaMKIIß variants within an F-actin-rich structure (microspike) in CaMKIIα free embryonic day 18 (E-18) rat cortical neurons. Our results showed that CaMKIIß and CaMKIIß' containing exon E1 displayed an association with F-actin, while CaMKIIße and CaMKIIß'e lacking E1 did not. Moreover, CaMKIIß' lacking exon E4 but having E1 showed decreased actin bindingcapacity compared to WT CaMKIIß. This suggested E1 is required for the association between CaMKIIß and F-actin, while E4 assists CaMKIIß to associate with F-actin better. Thus, alternative splicing of CaMKIIß variants in developing neurons may serve as a developmental switch for actin cytoskeleton-associated isoforms and therefore correlated with dendritic arborization and synapse formation during LTP.

Neuronal CaMKII acts as a structural kinase.

CaMKII, calcium/calmodulin dependent protein kinase, is an active kinase in the cell that phosphorylates a number of substrates including several cytoskeletal and signaling proteins. In addition to kinase activity, the beta isoform of CaMKII also contains an F-actin binding region. We recently identified a new F-actin rich structure in developing cortical neurons that endogenous CaMKIIbeta bound. In nonneuronal cells and dendrite spines of hippocampal neurons where an interaction between CaMKIIbeta and F-actin has been identified, CaMKIIbeta was involved in regulating the differentiation of dendrite spines and formation of synapses. In this study, we took advantage of the temporal and spatial regulation of CaMKII isoforms to reveal a specific role for CaMKIIbeta in binding and stability of a novel F-actin rich structure. We used FRAP and colocalization assays in this CaMKIIbeta rich system to demonstrate a structural, rather than enzymatic, role of CaMKIIbeta. In this addendum, we further discuss the significance of this study and the possible implication to the field.

CaMKIIbeta binding to stable F-actin in vivo regulates F-actin filament stability.

Ca2(+)/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine kinase that is best known for its role in synaptic plasticity and memory. Multiple roles of CaMKII have been identified in the hippocampus, yet its role in developing neurons is less well understood. We show here that endogenous CaMKIIbeta, but not CaMKIIalpha, localized to prominent F-actin-rich structures at the soma in embryonic cortical neurons. Fluorescence recovery after photobleaching analyses of GFP-CaMKIIbeta binding interactions with F-actin in this CaMKIIalpha-free system indicated CaMKIIbeta binding depended upon a putative F-actin binding domain in the variable region of CaMKIIbeta. Furthermore, CaMKIIalpha decreased CaMKIIbeta binding to F-actin. We examined the interaction of CaMKIIbeta with stable and dynamic actin and show that CaMKIIbeta binding to F-actin was dramatically prolonged when F-actin was stabilized. CaMKIIbeta binding to stable F-actin was disrupted when it was bound by Ca2(+)/calmodulin or when it was highly phosphorylated, but not by kinase inactivity. Whereas CaMKIIbeta over-expression increased the prevalence of the F-actin-rich structures, disruption of CaMKIIbeta binding to F-actin reduced them. Taken together, these data suggest that CaMKIIbeta binding to stable F-actin is important for the in vivo maintenance of polymerized F-actin.

ERK mediates activity dependent neuronal complexity via sustained activity and CREB-mediated signaling.

A major question in the process of dendrite development and complexity is not whether neuronal activity plays a role, but how it contributes to specific components of the mature dendrite pattern. Neurons interpret activity into the influx of calcium ions leading to activation of signaling pathways. The dynamics of calcium-activated signaling pathways after neuronal activity and the contribution to formation of dendrite complexity remain unclear. Here, we show that one calcium activated signaling pathway, extracellular signal-regulated kinase (ERK), showed differential activity in cortical neurons. In response to depolarizing stimuli, ERK was active for less than an hour in most neurons, whereas in others ERK remained active for several hours. Further, neurons in which ERK activity was sustained, displayed greater dendrite complexity than neurons that did not display sustained ERK activity. Interestingly, this difference in dendrite complexity was detected in some, but not all, morphological parameters. Pharmacological inhibition of sustained ERK activity inhibited calcium-activated dendrite complexity. Increasing the duration and degree of ERK phosphorylation, and thus activity, with dominant negative MAP kinase phosphatase-1 accentuated dendrite complexity. Neurons in which ERK activity was sustained activated downstream nuclear targets including RSK, MSK, cAMP response element binding protein (CREB), CRE-mediated gene transcription, and stabilized c-Fos. Further, the increase in dendrite complexity mediated by sustained ERK activity was inhibited by expression of a dominant negative CREB. These data indicate that ERK-mediated activity induced dendrite complexity via sustained signaling and CREB-mediated signaling.

Translating neuronal activity into dendrite elaboration: signaling to the nucleus.

Growth and elaboration of neuronal processes is key to establishing neuronal connectivity critical for an optimally functioning nervous system. Neuronal activity clearly influences neuronal connectivity and does so via intracellular calcium signaling. A number of CaMKs and MAPKs convey the calcium signal initiated by neuronal activity. Several of these kinases interact with substrates in close proximity to the plasma membrane and alter dendrite structure locally via these local interactions. However, many calcium-activated kinases, such as Ras-MAPK and CaMKIV, target proteins in the nucleus, either by activating a downstream substrate that is a component of a signaling cascade or by directly acting within the nucleus. It is the activation of nuclear signaling and gene transcription that is thought to mediate global changes in dendrite complexity. The identification of calcium-sensitive transcription factors and transcriptional coactivators provides substantial evidence that gene transcription is a prevalent mechanism by which neuronal activity is translated into changes in dendrite complexity. The present review presents an overview of the role of neuronal activity in the development of neuronal dendrites, the signaling mechanisms that translate neuronal activity into gene transcription, and the transcribed effectors that regulate dendrite complexity.

Regulation of dendritic development by calcium signaling.

Neuronal activity can have profound effects on dendrite morphology in the developing brain. The effects of neuronal activity on dendritic morphology are mediated by calcium signaling. While many effects of calcium on dendrite structure occur locally at the site of calcium entry into the cytoplasmic milieu, elevation of cytoplasmic calcium is also translated into changes in gene transcription. Decoding the calcium signal into specific changes in gene transcription involve coordinating the action of a number of kinases, phosphatases, transcription factors and transcriptional coactivators. This review focuses on the contribution of calcium-dependent transcription on the control of dendritic morphology.

Calcium regulation of dendritic growth via CaM kinase IV and CREB-mediated transcription.

We report that CaM kinase IV and CREB play a critical role in mediating calcium-induced dendritic growth in cortical neurons. Calcium-dependent dendritic growth is suppressed by CaM kinase inhibitors, a constitutively active form of CaM kinase IV induces dendritic growth in the absence of extracellular stimulation, and a kinase-dead form of CaM kinase IV suppresses dendritic growth induced by calcium influx. CaM kinase IV activates the transcription factor CREB, and expression of a dominant negative form of CREB blocks calcium- and CaM kinase IV-induced dendritic growth. In cortical slice cultures, dendritic growth is attenuated by inhibitors of voltage-sensitive calcium channels and by dominant negative CREB. These experiments indicate that calcium-induced dendritic growth is regulated by activation of a transcriptional program that involves CaM kinase IV and CREB-mediated signaling to the nucleus.