Emily S. Gibson

cAMP-Dependent Protein Kinase Postsynaptic Localization Regulated by NMDA Receptor Activation through Translocation of an A-Kinase Anchoring Protein Scaffold Protein

NMDA receptor-dependent long-term potentiation and long-term depression (LTD) involve changes in AMPA receptor activity and postsynaptic localization that are in part controlled by glutamate receptor 1 (GluR1) subunit phosphorylation. The scaffolding molecule A-kinase anchoring protein (AKAP)79/150 targets both the cAMP-dependent protein kinase (PKA) and protein phosphatase 2B/calcineurin (PP2B/CaN) to AMPA receptors to regulate GluR1 phosphorylation. Here, we report that brief NMDA receptor activation leads to persistent redistribution of AKAP79/150 and PKA-RII, but not PP2B/CaN, from postsynaptic membranes to the cytoplasm in hippocampal slices. Similar to LTD, AKAP79/150 redistribution requires PP2B/CaN activation and is accompanied by GluR1 dephosphorylation and internalization. Using fluorescence resonance energy transfer microscopy in hippocampal neurons, we demonstrate that PKA anchoring to AKAP79/150 is required for NMDA receptor regulation of PKA-RII localization and that movement of AKAP–PKA complexes underlies PKA redistribution. These findings suggest that LTD involves removal of AKAP79/150 and PKA from synapses in addition to activation of PP2B/CaN. Movement of AKAP79/150–PKA complexes from the synapse could further favor the actions of phosphatases in maintaining dephosphorylation of postsynaptic substrates, such as GluR1, that are important for LTD induction and expression. In addition, our observations demonstrate that AKAPs serve not solely as stationary anchors in cells but also as dynamic signaling components.

AKAP150-Anchored Calcineurin Regulates Synaptic Plasticity by Limiting Synaptic Incorporation of Ca2+-Permeable AMPA Receptors

AMPA receptors (AMPARs) are tetrameric ion channels assembled from GluA1–GluA4 subunits that mediate the majority of fast excitatory synaptic transmission in the brain. In the hippocampus, most synaptic AMPARs are composed of GluA1/2 or GluA2/3 with the GluA2 subunit preventing Ca2+ influx. However, a small number of Ca2+-permeable GluA1 homomeric receptors reside in extrasynaptic locations where they can be rapidly recruited to synapses during synaptic plasticity. Phosphorylation of GluA1 S845 by the cAMP-dependent protein kinase (PKA) primes extrasynaptic receptors for synaptic insertion in response to NMDA receptor Ca2+ signaling during long-term potentiation (LTP), while phosphatases dephosphorylate S845 and remove synaptic and extrasynaptic GluA1 during long-term depression (LTD). PKA and the Ca2+-activated phosphatase calcineurin (CaN) are targeted to GluA1 through binding to A-kinase anchoring protein 150 (AKAP150) in a complex with PSD-95, but we do not understand how the opposing activities of these enzymes are balanced to control plasticity. Here, we generated AKAP150ΔPIX knock-in mice to selectively disrupt CaN anchoring in vivo. We found that AKAP150ΔPIX mice lack LTD but express enhanced LTP at CA1 synapses. Accordingly, basal GluA1 S845 phosphorylation is elevated in AKAP150ΔPIX hippocampus, and LTD-induced dephosphorylation and removal of GluA1, AKAP150, and PSD-95 from synapses are impaired. In addition, basal synaptic activity of GluA2-lacking AMPARs is increased in AKAP150ΔPIX mice and pharmacologic antagonism of these receptors restores normal LTD and inhibits the enhanced LTP. Thus, AKAP150-anchored CaN opposes PKA phosphorylation of GluA1 to restrict synaptic incorporation of Ca2+-permeable AMPARs both basally and during LTP and LTD.

A light-triggered protein secretion system

Secreted proteins fused to the plant photoreceptor protein UVR8 are conditionally sequestered in the ER until a pulse of light triggers trafficking through the secretory pathway, allowing precise control of forward secretory trafficking.

Palmitoylation of A-Kinase Anchoring Protein 79/150 Regulates Dendritic Endosomal Targeting and Synaptic Plasticity Mechanisms

NMDA receptor-dependent long-term potentiation (LTP) and depression (LTD) are forms of synaptic plasticity underlying learning and memory that are expressed through increases and decreases, respectively, in dendritic spine size and AMPA receptor (AMPAR) phosphorylation and postsynaptic localization. The A-kinase anchoring protein 79/150 (AKAP79/150) signaling scaffold regulates AMPAR phosphorylation, channel activity, and endosomal trafficking associated with LTP and LTD. AKAP79/150 is targeted to dendritic spine plasma membranes by an N-terminal polybasic domain that binds phosphoinositide lipids, F-actin, and cadherin cell adhesion molecules. However, we do not understand how regulation of AKAP targeting controls AMPAR endosomal trafficking. Here, we report that palmitoylation of the AKAP N-terminal polybasic domain targets it to postsynaptic lipid rafts and dendritic recycling endosomes. AKAP palmitoylation was regulated by seizure activity in vivo and LTP/LTD plasticity-inducing stimuli in cultured rat hippocampal neurons. With chemical LTP induction, we observed AKAP79 dendritic spine recruitment that required palmityolation and Rab11-regulated endosome recycling coincident with spine enlargement and AMPAR surface delivery. Importantly, a palmitoylation-deficient AKAP79 mutant impaired regulation of spine size, endosome recycling, AMPAR trafficking, and synaptic potentiation. These findings emphasize the emerging importance of palmitoylation in controlling synaptic function and reveal novel roles for the AKAP79/150 signaling complex in dendritic endosomes.

Regulation of Postsynaptic Structure and Function by an A-Kinase Anchoring Protein–Membrane-Associated Guanylate Kinase Scaffolding Complex

A-kinase anchoring protein (AKAP) 79/150 is a scaffold protein found in dendritic spines that recruits the cAMP-dependent protein kinase (PKA) and protein phosphatase 2B–calcineurin (CaN) to membrane-associated guanylate kinase (MAGUK)-linked AMPA receptors (AMPARs) to control receptor phosphorylation and synaptic plasticity. However, AKAP79/150 may also coordinate regulation of AMPAR activity with spine structure directly through MAGUK binding and membrane-cytoskeletal interactions of its N-terminal targeting domain. In cultured hippocampal neurons, we observed that rat AKAP150 expression was low early in development but then increased coincident with spine formation and maturation. Overexpression of human AKAP79 in immature or mature neurons increased the number of dendritic filopodia and spines and enlarged spine area. However, RNA interference knockdown of AKAP150 decreased dendritic spine area only in mature neurons. Importantly, AKAP79 overexpression in immature neurons increased AMPAR postsynaptic localization and activity. Neither the AKAP79 PKA nor CaN anchoring domain was required for increasing dendritic protrusion numbers, spine area, or AMPAR synaptic localization; however, an internal region identified as the MAGUK binding domain was found to be essential as shown by expression of a MAGUK binding mutant that formed mainly filopodia and decreased AMPAR synaptic localization and activity. Expression of the AKAP79 N-terminal targeting domain alone also increased filopodia numbers but not spine area. Overall, these results demonstrate a novel structural role for AKAP79/150 in which the N-terminal targeting domain induces dendritic filopodia and binding to MAGUKs promotes spine enlargement and AMPAR recruitment.

Phosphorylation Regulates Removal of Synaptic N-Methyl-d-Aspartate Receptors after Withdrawal from Chronic Ethanol Exposure

Alterations in N-methyl-d-aspartate receptor (NMDAR) protein levels or subcellular localization in brain after chronic ethanol exposure may contribute to withdrawal-associated seizures and neurotoxicity. We have investigated synaptic localization of NMDARs in cultured hippocampal pyramidal neurons after prolonged (7 days) exposure to, and acute withdrawal from, 80 mM ethanol using fluorescence immunocytochemistry techniques. After chronic ethanol exposure, there was a significant increase in the clustering of NR1 and NR2B subunits and their colocalization with the synaptic proteins synaptophysin and postsynaptic density protein 95, respectively. There was also increased expression of NR1 variants containing the C2′ cassette after chronic ethanol exposure. The ethanol-induced synaptic clustering and colocalization were rapidly reversed within 4 h after ethanol withdrawal. Surface labeling of NR2B subunits suggested that this rapid reversal involved lateral receptor movement to extrasynaptic sites rather than internalization of receptors. Receptor removal from the synapse during ethanol withdrawal was associated with changes in the phosphorylation state of NR2B Ser1480, controlled by the protein kinase CK2. The redistribution of NMDAR to synapses produced by long-term ethanol exposure, as well as the rapid removal during withdrawal, may not only affect neuronal withdrawal hyperexcitability but also may sensitize the system to subsequent synaptic plasticity.

Optogenetic Control of Synaptic Composition and Function

The molecular composition of the postsynaptic membrane is sculpted by synaptic activity. During synaptic plasticity at excitatory synapses, numerous structural, signaling, and receptor molecules concentrate at the postsynaptic density (PSD) to regulate synaptic strength. We developed an approach that uses light to tune the abundance of specific molecules in the PSD. We used this approach to investigate the relationship between the number of AMPA-type glutamate receptors in the PSD and synaptic strength. Surprisingly, adding more AMPA receptors to excitatory contacts had little effect on synaptic strength. Instead, we observed increased excitatory input through the apparent addition of new functional sites. Our data support a model where adding AMPA receptors is sufficient to activate synapses that had few receptors to begin with, but that additional remodeling events are required to strengthen established synapses. More broadly, this approach introduces the precise spatiotemporal control of optogenetics to the molecular control of synaptic function.

Local and Use-Dependent Effects of β-Amyloid Oligomers on NMDA Receptor Function Revealed by Optical Quantal Analysis

Beta amyloid (Aβ) triggers the elimination of excitatory synaptic connections in the CNS, an early manifestation of Alzheimers disease. Oligomeric assemblies of Aβ peptide associate with excitatory synapses resulting in synapse elimination through a process that requires NMDA-type glutamate receptor activation. Whether Aβ affects synaptic NMDA receptor (NMDAR) function directly and acts locally at synapses to which it has bound and whether synaptic activity influences Aβ synaptic binding and synaptotoxicity have remained fundamental questions. Here, we used subcellular Ca2+ imaging in rat hippocampal neurons to visualize NMDAR function at individual synapses before and after Aβ application. Aβ triggered a robust impairment of NMDAR Ca2+ entry at most, but not all, synapses. NMDAR function was more severely impaired at highly active synapses and synapses with bound Aβ, but activity was not required for Aβ synapse binding. Blocking NMDARs during Aβ exposure prevented Aβ-mediated impairment. Finally, Aβ impaired NMDAR Ca2+ entry at doses much lower than those required for NMDAR internalization, revealing a novel, potent mode of NMDAR regulation by Aβ. SIGNIFICANCE STATEMENT Amyloid β (Aβ) is strongly implicated in Alzheimers disease. Aβ triggers the elimination of excitatory synapses through a mechanism that requires NMDA receptors (NMDARs). However, little is known about how or whether Aβ influences synaptic NMDAR function. We used an imaging-based assay to investigate the relationship among Aβ binding, activity, and NMDAR function at individual synapses. Aβ triggered a robust impairment of NMDAR Ca2+ entry at most, but not all, synapses. NMDAR function was more severely impaired at highly active synapses and synapses with bound Aβ. Blocking NMDARs during Aβ exposure prevented Aβ-mediated impairment. Together, our experiments reveal a novel use-dependent, potent, and local mode of Aβ-mediated NMDAR impairment.

L-Type Voltage-Gated Ca2+ Channels Regulate Synaptic Activity-Triggered Recycling Endosome Fusion in Neuronal Dendrites

The repertoire and abundance of proteins displayed on the surface of neuronal dendrites are tuned by regulated fusion of recycling endosomes (REs) with the dendritic plasma membrane. While this process is critical for neuronal function and plasticity, how synaptic activity drives RE fusion remains unexplored. We demonstrate a multistep fusion mechanism that requires Ca2+ from distinct sources. NMDA receptor Ca2+ initiates RE fusion with the plasma membrane, while L-type voltage-gated Ca2+ channels (L-VGCCs) regulate whether fused REs collapse into the membrane or reform without transferring their cargo to the cell surface. Accordingly, NMDA receptor activation triggered AMPA-type glutamate receptor trafficking to the dendritic surface in an L-VGCC-dependent manner. Conversely, potentiating L-VGCCs enhanced AMPA receptor surface expression only when NMDA receptors were also active. Thus L-VGCCs play a role in tuning activity-triggered surface expression of key synaptic proteins by gating the mode of RE fusion.

Inhibition of the Motor Protein Eg5/Kinesin-5 in Amyloid β-Mediated Impairment of Hippocampal Long-Term Potentiation and Dendritic Spine Loss

Alzheimer’s disease (AD) is characterized by neurofibrillary tangles, amyloid plaques, and neurodegeneration. However, this pathology is preceded by increased soluble amyloid beta (Aβ) 1−42 oligomers that interfere with the glutamatergic synaptic plasticity required for learning and memory, including N-methyl-d-aspartate receptor (NMDAR)-dependent long-term potentiation (LTP). In particular, soluble Aβ(1–42) acutely inhibits LTP and chronically causes synapse loss. Many mechanisms have been proposed for Aβ-induced synaptic dysfunction, but we recently found that Aβ(1–42) inhibits the microtubule motor protein Eg5/kinesin-5. Here we compared the impacts of Aβ(1–42) and monastrol, a small-molecule Eg5 inhibitor, on LTP in hippocampal slices and synapse loss in neuronal cultures. Acute (20-minute) treatment with monastrol, like Aβ, completely inhibited LTP at doses >100 nM. In addition, 1 nM Aβ(1–42) or 50 nM monastrol inhibited LTP #x223c;50%, and when applied together caused complete LTP inhibition. At concentrations that impaired LTP, neither Aβ(1–42) nor monastrol inhibited NMDAR synaptic responses until #x223c;60 minutes, when only #x223c;25% inhibition was seen for monastrol, indicating that NMDAR inhibition was not responsible for LTP inhibition by either agent when applied for only 20 minutes. Finally, 48 hours of treatment with either 0.5–1.0 μM Aβ(1–42) or 1–5 μM monastrol reduced the dendritic spine/synapse density in hippocampal cultures up to a maximum of #x223c;40%, and when applied together at maximal concentrations, no additional spine loss resulted. Thus, monastrol can mimic and in some cases occlude the impact of Aβ on LTP and synapse loss, suggesting that Aβ induces acute and chronic synaptic dysfunction in part through inhibiting Eg5.