Loss of AKAP150 perturbs distinct neuronal processes in mice.

A-Kinase Anchoring Proteins (AKAPs) ensure the fidelity of second messenger signaling events by directing protein kinases and phosphatases toward their preferred substrates. AKAP150 brings protein kinase A (PKA), the calcium/calmodulin dependent phosphatase PP2B and protein kinase C (PKC) to postsynaptic membranes where they facilitate the phosphorylation dependent modulation of certain ion channels. Immunofluorescence and electrophysiological recordings were combined with behavioral analyses to assess whether removal of AKAP150 by gene targeting in mice changes the signaling environment to affect excitatory and inhibitory neuronal processes. Mislocalization of PKA in AKAP150 null hippocampal neurons alters the bidirectional modulation of postsynaptic AMPA receptors with concomitant changes in synaptic transmission and memory retention. AKAP150 null mice also exhibit deficits in motor coordination and strength that are consistent with a role for the anchoring protein in the cerebellum. Loss of AKAP150 in sympathetic cervical ganglion (SCG) neurons reduces muscarinic suppression of inhibitory M currents and provides these animals with a measure of resistance to seizures induced by the non-selective muscarinic agonist pilocarpine. These studies argue that distinct AKAP150-enzyme complexes regulate context-dependent neuronal signaling events in vivo.

Recruitment of calcium-permeable AMPA receptors during synaptic potentiation is regulated by CaM-kinase I.

Ca(2+)-permeable AMPA receptors (CP-AMPARs) at central glutamatergic synapses are of special interest because of their unique biophysical and signaling properties that contribute to synaptic plasticity and their roles in multiple neuropathologies. However, intracellular signaling pathways that recruit synaptic CP-AMPARs are unknown, and involvement of CP-AMPARs in hippocampal region CA1 synaptic plasticity is controversial. Here, we report that intracellular infusion of active CaM-kinase I (CaMKI) into cultured hippocampal neurons enhances miniature EPSC amplitude because of recruitment of CP-AMPARs, likely from an extrasynaptic pool. The ability of CaMKI, which regulates the actin cytoskeleton, to recruit synaptic CP-AMPARs was blocked by inhibiting actin polymerization with latrunculin A. CaMK regulation of CP-AMPARs was also confirmed in hippocampal slices. CA1 long-term potentiation (LTP) after theta bursts, but not high-frequency tetani, produced a rapid, transient expression of synaptic CP-AMPARs that facilitated LTP. This component of TBS LTP was blocked by inhibition of CaM-kinase kinase (CaMKK), the upstream activator of CaMKI. Our calculations show that adding CP-AMPARs numbering <5% of existing synaptic AMPARs is sufficient to account for the potentiation observed in LTP. Thus, synaptic expression of CP-AMPARs is a very efficient mechanism for rapid enhancement of synaptic strength that depends on CaMKK/CaMKI signaling, actin dynamics, and the pattern of synaptic activity used to induce CA1 LTP.

Regulatory mechanisms of AMPA receptors in synaptic plasticity.

Activity-dependent changes in the strength of excitatory synapses are a cellular mechanism for the plasticity of neuronal networks that is widely recognized to underlie cognitive functions such as learning and memory. AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-type glutamate receptors (AMPARs) are the main transducers of rapid excitatory transmission in the mammalian CNS, and recent discoveries indicate that the mechanisms which regulate AMPARs are more complex than previously thought. This review focuses on recent evidence that alterations to AMPAR functional properties are coupled to their trafficking, cytoskeletal dynamics and local protein synthesis. These relationships offer new insights into the regulation of AMPARs and synaptic strength by cellular signalling.

A WAVE-1 and WRP signaling complex regulates spine density, synaptic plasticity, and memory.

The scaffolding protein WAVE-1 (Wiskott-Aldrich syndrome protein family member 1) directs signals from the GTPase Rac through the Arp2/3 complex to facilitate neuronal actin remodeling. The WAVE-associated GTPase activating protein called WRP is implicated in human mental retardation, and WAVE-1 knock-out mice have altered behavior. Neuronal time-lapse imaging, behavioral analyses, and electrophysiological recordings from genetically modified mice were used to show that WAVE-1 signaling complexes control aspects of neuronal morphogenesis and synaptic plasticity. Gene targeting experiments in mice demonstrate that WRP anchoring to WAVE-1 is a homeostatic mechanism that contributes to neuronal development and the fidelity of synaptic connectivity. This implies that signaling through WAVE-1 complexes is essential for neural plasticity and cognitive behavior.

Extrasynaptic membrane trafficking regulated by GluR1 serine 845 phosphorylation primes AMPA receptors for long-term potentiation.

Enhancement of synaptic transmission, as occurs in long-term potentiation (LTP), can result from several mechanisms that are regulated by phosphorylation of the AMPA-type glutamate receptor (AMPAR). Using a quantitative assay of net serine 845 (Ser-845) phosphorylation in the GluR1 subunit of AMPARs, we investigated the relationship between phospho-Ser-845, GluR1 surface expression, and synaptic strength in hippocampal neurons. About 15% of surface AMPARs in cultured neurons were phosphorylated at Ser-845 basally, whereas chemical potentiation (forskolin/rolipram treatment) persistently increased this to 60% and chemical depression (N-methyl-D-aspartate treatment) decreased it to 10%. These changes in Ser-845 phosphorylation were paralleled by corresponding changes in the surface expression of AMPARs in both cultured neurons and hippocampal slices. For every 1% increase in net phospho-Ser-845, there was 0.75% increase in the surface fraction of GluR1. Phosphorylation of Ser-845 correlated with a selective delivery of AMPARs to extrasynaptic sites, and their synaptic localization required coincident synaptic activity. Furthermore, increasing the extrasynaptic pool of AMPA receptors resulted in stronger theta burst LTP. Our results support a two-step model for delivery of GluR1-containing AMPARs to synapses during activity-dependent LTP, where Ser-845 phosphorylation can traffic AMPARs to extrasynaptic sites for subsequent delivery to synapses during LTP.

Calmodulin-dependent kinase kinase/calmodulin kinase I activity gates extracellular-regulated kinase-dependent long-term potentiation.

Intracellular Ca2+ and protein phosphorylation play pivotal roles in long-term potentiation (LTP), a cellular model of learning and memory. Ca2+ regulates multiple intracellular pathways, including the calmodulin-dependent kinases (CaMKs) and the ERKs (extracellular signal-regulated kinases), both of which are required for LTP. However, the mechanism by which Ca2+ activates ERK during LTP remains unknown. Here, we describe a requirement for the CaMK-kinase (CaMKK) pathway upstream of ERK in LTP induction. Both the pharmacological inhibitor of CaMKK, STO-609, and dominant-negative CaMKI (dnCaMKI), a downstream target of CaMKK, blocked neuronal NMDA receptor-dependent ERK activation. In contrast, an inhibitor of CaMKII and nuclear-localized dnCaMKIV had no effect on ERK activation. NMDA receptor-dependent LTP induction robustly activated CaMKI, the Ca2+-stimulated Ras activator Ras-GRF1 (Ras-guanyl-nucleotide releasing factor), and ERK. STO-609 blocked the activation of all three enzymes during LTP without affecting basal synaptic transmission, activation of CaMKII, or cAMP-dependent activation of ERK. LTP induction itself was suppressed 50% by STO-609 in a manner identical to the ERK inhibitor U0126: either inhibitor occluded the effect of the other, suggesting they are part of the same signaling pathway in LTP induction. STO-609 also suppressed regulatory phosphorylation of two downstream ERK targets during LTP, the general translation factors eIF4E (eukaryotic initiation factor 4) and its binding protein 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1). These data indicate an essential role for CaMKK and CaMKI to link NMDA receptor-mediated Ca2+ elevation with ERK-dependent LTP.

Inhibition of calcium/calmodulin-dependent protein kinase kinase by protein 14-3-3.

Intracellular calcium concentrations regulate diverse cellular events including cytoskeletal dynamics, gene transcription, and synaptic plasticity. The calcium signal is transduced in part by the calcium/calmodulin-dependent protein kinase (CaMK) cascade that is comprised of CaMK kinase (CaMKK) and its primary downstream substrates, CaMKI and CaMKIV. The CaMK cascade also participates in cross-talk with other signaling pathways: CaMKK/CaMKI can activate the mitogen-activated protein kinase pathway and cAMP-dependent protein kinase (PKA) can directly phosphorylate two inhibitory sites (Thr108 and Ser458) in CaMKK. Here we report an additional PKA-dependent regulation of CaMKK through its interaction with protein 14-3-3. CaMKK and 14-3-3 co-immunoprecipitated from co-transfected heterologous cells as well as from rat brain homogenate, and site-directed mutagenesis studies identified phospho-Ser74 in CaMKK as the primary 14-3-3 binding site. In cultured rat hippocampal neurons and acute hippocampal slices this interaction was robustly stimulated by activation of PKA through forskolin treatment and was blocked by inhibition of PKA. Interaction of 14-3-3 with CaMKK had two regulatory consequences in vitro. It directly inhibited CaMKK activity, and it also blocked dephosphorylation of Thr108, an inhibitory PKA phosphorylation site. In human embryonic kidney 293 cells transfected with CaMKK and stimulated with forskolin, co-transfection with 14-3-3 prevented dephosphorylation of Thr108 to the same extent as did inhibition of protein phosphatases with okadaic acid. We conclude that binding of 14-3-3 to CaMKK stabilizes its inhibition by PKA-mediated phosphorylation, which may have important consequences in the regulation of CaMKI, CaMKIV, protein kinase B, and ERK signaling pathways.