Zhaoqing Zheng

AMPA receptor trafficking and learning.

In the last few years it has become clear that AMPA‐type glutamate neurotransmitter receptors are rapidly transported into and out of synapses to strengthen or weaken their function. The remarkable dynamics of AMPA receptor (AMPAR) synaptic localization provides a compelling mechanism for understanding the cellular basis of learning and memory, as well as disease states involving cognitive dysfunction. Here, we summarize the evidence for AMPAR trafficking as a mechanism underlying a variety of learned responses derived from both behavioral and cellular studies. Evidence is also reviewed supporting synaptic dysfunction related to impaired AMPAR trafficking as a mechanism underlying learning and memory deficits in Alzheimer’s disease. We conclude that emerging data support the concept of multistage AMPAR trafficking during learning and that a broad approach to include examination of all of the AMPAR subunits will provide a more complete view of the mechanisms underlying multiple forms of learning.

PKA Has a Critical Role in Synaptic Delivery of GluR1- and GluR4-Containing AMPARs During Initial Stages of Acquisition of In Vitro Classical Conditioning

The cyclic AMP-dependent protein kinase (PKA) signaling pathway has been shown to be important in mechanisms of synaptic plasticity, although its direct and downstream signaling effects are not well understood. Using an in vitro model of eyeblink classical conditioning, we report that PKA has a critical role in initiating a signaling cascade that results in synaptic delivery of glutamate receptor 1 (GluR1)- and GluR4-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) in abducens motor neurons during conditioning. PKA and the Ca(2+)-calmodulin-dependent protein kinases (CaMKs) II and IV are activated early in conditioning and are required for acquisition and expression of conditioned responses (CRs). cAMP-response-element-binding protein (CREB) is also activated early in conditioning but is blocked by coapplication of inhibitors to PKA and the CaMKs, suggesting that CREB is downstream of those signaling cascades. Moreover, evidence suggests that PKA activates extracellular signal-regulated kinase, which is also required for conditioning. Imaging studies after conditioning further indicate that colocalization of GluR1 AMPAR subunits with the synaptic marker synaptophysin requires PKA, but is insensitive to the N-methyl-d-aspartate receptor (NMDAR) inhibitor d,l-AP5. PKA activation also leads to synaptic localization of GluR4 subunits that, unlike GluR1, is dependent on NMDARs and is mediated by CaMKII. Together with previous studies, our findings support a two-stage model of AMPAR synaptic delivery during acquisition of classical conditioning. The first stage involves synaptic incorporation of GluR1-containing AMPARs that serves to activate silent synapses. This allows a second stage of NMDAR- and protein kinase C-dependent delivery of GluR4 AMPAR subunits that supports the acquisition of CRs.

Cleavage of proBDNF to BDNF by a Tolloid-Like Metalloproteinase Is Required for Acquisition of In Vitro Eyeblink Classical Conditioning

The tolloid/bone morphogenetic protein-1 family of metalloproteinases have an important role in the regulation of embryonic pattern formation and tissue morphogenesis. Studies suggest that they participate in mechanisms of synaptic plasticity in adults, but very little is known about their function. Recently, we isolated a reptilian ortholog of the tolloid gene family designated turtle tolloid-like gene (tTll). Here, we examined the role of tTLL in an in vitro model of eyeblink classical conditioning using an isolated brainstem preparation to assess its role in synaptic plasticity during conditioning. Analysis by real-time reverse transcription-PCR shows that an extracellularly secreted form of tTLL, tTLLs, is transiently expressed in the early stages of conditioning during conditioned response acquisition, whereas a cytosolic form, tTLLc, is not. Short interfering RNA (siRNA)-directed gene knockdown and rescue of tTLL expression demonstrate that it is required for conditioning. Significantly, we show that tTLLs cleaves the precursor proBDNF into mature BDNF in cleavage assay studies, and application of recombinant tTLLs protein alone to preparations results in induction of mature BDNF expression. The mature form of BDNF is minimally expressed in preparations treated with anti-tTLL siRNA, and the synaptic incorporation of both GluR1- and GluR4-containing AMPA receptors is significantly reduced, resulting in suppression of conditioning. This is the first study to demonstrate that expression of an extracellularly secreted tolloid-like metalloproteinase is regulated in the early stages of classical conditioning and functions in the conversion of proBDNF to mature BDNF. The mature form of BDNF is required for synaptic delivery of AMPA receptors and acquisition of conditioned responses.

Oligomeric Amyloid-β Inhibits the Proteolytic Conversion of Brain-derived Neurotrophic Factor (BDNF), AMPA Receptor Trafficking, and Classical Conditioning

Amyloid-β (Aβ) peptide is thought to have a significant role in the progressive memory loss observed in patients with Alzheimer disease and inhibits synaptic plasticity in animal models of learning. We previously demonstrated that brain-derived neurotrophic factor (BDNF) is critical for synaptic AMPA receptor delivery in an in vitro model of eyeblink classical conditioning. Here, we report that acquisition of conditioned responses was significantly attenuated by bath application of oligomeric (200 nm), but not fibrillar, Aβ peptide. Western blotting revealed that BDNF protein expression during conditioning is significantly reduced by treatment with oligomeric Aβ, as were phosphorylation levels of cAMP-response element-binding protein (CREB), Ca2+/calmodulin-dependent protein kinase II (CaMKII), Ca2+/calmodulin-dependent protein kinase IV (CaMKIV), and ERK. However, levels of PKA and PKCζ/λ were unaffected, as was PDK-1. Protein localization studies using confocal imaging indicate that oligomeric Aβ, but not fibrillar or scrambled forms, suppresses colocalization of GluR1 and GluR4 AMPA receptor subunits with synaptophysin, indicating that trafficking of these subunits to synapses during the conditioning procedure is blocked. In contrast, coapplication of BDNF with oligomeric Aβ significantly reversed these findings. Interestingly, a tolloid-like metalloproteinase in turtle, tTLLs (turtle tolloid-like protein), which normally processes the precursor proBDNF into mature BDNF, was found to degrade oligomeric Aβ into small fragments. These data suggest that an Aβ-induced reduction in BDNF, perhaps due to interference in the proteolytic conversion of proBDNF to BDNF, results in inhibition of synaptic AMPA receptor delivery and suppression of the acquisition of conditioning.

Protein kinase C-dependent and independent signaling pathways regulate synaptic GluR1 and GluR4 AMPAR subunits during in vitro classical conditioning.

Protein kinase C (PKC) signal transduction pathways have been implicated in mechanisms of synaptic plasticity and learning, however, the roles of the different PKC family isoforms remain to be clarified. Previous studies showed that NMDAR-mediated trafficking of GluR4-containing AMPARs supports conditioning and that the mitogen-activated protein kinases (MAPKs) have a central role in the synaptic delivery of GluR4 subunits. Here, an in vitro model of classical conditioning in pond turtles, Pseudemys scripta elegans, was used to assess the role of PKC isoforms in mechanisms underlying this form of learning. We show that the PKC antagonists chelerythrine and bisindolylmaleimide I attenuated conditioned response (CR) acquisition and expression, as did the PKCzeta pseudosubstrate peptide inhibitor ZIP. Analysis of protein expression revealed that PKCzeta is activated in early stages of conditioning followed shortly afterward by increased levels of PKCalpha/beta and activation of ERK MAPK. Data also suggest that PKCzeta is upstream from and activates ERK. Finally, protein localization studies using confocal imaging indicate that inhibitors of ERK, but not PKC, suppress colocalization of GluR1 with synaptophysin while inhibitors of PKC and ERK attenuate colocalization of GluR4 with synaptophysin. Together, these data suggest that acquisition of conditioning proceeds by two stages of AMPAR trafficking. The first is PKC-independent and ERK-dependent synaptic delivery of GluR1 subunits to activate silent synapses. This is followed by PKC-dependent and ERK-dependent synthesis and delivery of GluR4 subunits that supports the acquisition of CRs. Therefore, there is a selective role for PKC and MAPK signaling pathways in multistep AMPAR trafficking that mediates acquisition of classical conditioning.

Transsynaptic EphB/Ephrin-B Signaling Regulates Growth of Presynaptic Boutons Required for Classical Conditioning

Learning-related presynaptic remodeling has been documented in only a few systems, and its molecular mechanisms are largely unknown. Here we describe a role for the bidirectional EphB/ephrin-B signaling system in structural plasticity of presynaptic nerve terminals using an in vitro model of classical conditioning. Conditioning or BDNF application induced significant growth of auditory nerve presynaptic boutons that convey the conditioned stimulus to abducens motor neurons. Interestingly, bouton enlargement occurred only for those synapses apposed to motor neuron dendrites rather than to somata. Phosphorylation of ephrin-B1, but not EphB2, was induced by both conditioning and BDNF application and was inhibited by postsynaptic injections of ephrin-B antibody. Finally, suppression of postsynaptic ephrin-B function inhibited presynaptic bouton enlargement that was rescued by activation of EphB2 by ephrin-B1-Fc. These data provide evidence for ephrin-B-induced EphB2 forward signaling in presynaptic structural plasticity during classical conditioning. They also reveal a functional interaction between BDNF/TrkB and the Eph/ephrin signaling systems in the coordination of presynaptic and postsynaptic modifications during conditioning.

Sequential Delivery of Synaptic GluA1- and GluA4-containing AMPA Receptors (AMPARs) by SAP97 Anchored Protein Complexes in Classical Conditioning

Background: Subunit-specific synaptic delivery of AMPARs during associative learning is not well characterized. Results: SAP97-AKAP/PKA-GluA1 followed by SAP97-KSR1/PKC-GluA4 complexes function for surface delivery of AMPARs. Conclusion: SAP97 interacts with AKAP and KSR1 proteins to coordinate the sequential synaptic delivery of AMPAR subunits during in vitro classical conditioning. Significance: A cooperative interaction of multiple scaffolding proteins selectively delivers AMPARs to synapses during conditioning. Multiple signaling pathways are involved in AMPAR trafficking to synapses during synaptic plasticity and learning. The mechanisms for how these pathways are coordinated in parallel but maintain their functional specificity involves subcellular compartmentalization of kinase function by scaffolding proteins, but how this is accomplished is not well understood. Here, we focused on characterizing the molecular machinery that functions in the sequential synaptic delivery of GluA1- and GluA4-containing AMPARs using an in vitro model of eyeblink classical conditioning. We show that conditioning induces the interaction of selective protein complexes with the key structural protein SAP97, which tightly regulates the synaptic delivery of GluA1 and GluA4 AMPAR subunits. The results demonstrate that in the early stages of conditioning the initial activation of PKA stimulates the formation of a SAP97-AKAP/PKA-GluA1 protein complex leading to synaptic delivery of GluA1-containing AMPARs through a SAP97-PSD95 interaction. This is followed shortly thereafter by generation of a SAP97-KSR1/PKC-GluA4 complex for GluA4 AMPAR subunit delivery again through a SAP97-PSD95 interaction. These data suggest that SAP97 forms the molecular backbone of a protein scaffold critical for delivery of AMPARs to the PSD during conditioning. Together, the findings reveal a cooperative interaction of multiple scaffolding proteins for appropriately timed delivery of subunit-specific AMPARs to synapses and support a sequential two-stage model of AMPAR synaptic delivery during classical conditioning.

Two-stage AMPA receptor trafficking in classical conditioning and selective role for glutamate receptor subunit 4 (tGluA4) flop splice variant

Previously, we proposed a two-stage model for an in vitro neural correlate of eyeblink classical conditioning involving the initial synaptic incorporation of glutamate receptor A1 (GluA1)-containing α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid type receptors (AMPARs) followed by delivery of GluA4-containing AMPARs that support acquisition of conditioned responses. To test specific elements of our model for conditioning, selective knockdown of GluA4 AMPAR subunits was used using small-interfering RNAs (siRNAs). Recently, we sequenced and characterized the GluA4 subunit and its splice variants from pond turtles, Trachemys scripta elegans (tGluA4). Analysis of the relative abundance of mRNA expression by real-time RT-PCR showed that the flip/flop variants of tGluA4, tGluA4c, and a novel truncated variant tGluA4trc1 are major isoforms in the turtle brain. Here, transfection of in vitro brain stem preparations with anti-tGluA4 siRNA suppressed conditioning, tGluA4 mRNA and protein expression, and synaptic delivery of tGluA4-containing AMPARs but not tGluA1 subunits. Significantly, transfection of abducens motor neurons by nerve injections of tGluA4 flop rescue plasmid prior to anti-tGluA4 siRNA application restored conditioning and synaptic incorporation of tGluA4-containing AMPARs. In contrast, treatment with rescue plasmids for tGluA4 flip or tGluA4trc1 failed to rescue conditioning. Finally, treatment with a siRNA directed against GluA1 subunits inhibited conditioning and synaptic delivery of tGluA1-containing AMPARs and importantly, those containing tGluA4. These data strongly support our two-stage model of conditioning and our hypothesis that synaptic incorporation of tGluA4-containing AMPARs underlies the acquisition of in vitro classical conditioning. Furthermore, they suggest that tGluA4 flop may have a critical role in conditioning mechanisms compared with the other tGluA4 splice variants.

SYNAPTIC LOCALIZATION OF GLUR4-CONTAINING AMPARS AND ARC DURING ACQUISITION, EXTINCTION AND REACQUISITION OF IN VITRO CLASSICAL CONDITIONING

Data suggest that modulation of synaptic strength by incorporation of GluR4-containing AMPARs occurs during conditioned response (CR) acquisition in an in vitro model of classical conditioning. Here we extend these findings by showing that synaptically localized GluR4 subunits parallels the expression of CRs during conditioning training in which there is differential expression of CRs, such as during acquisition, extinction, and reacquisition. Moreover, colocalization and coimmunoprecipitation data suggest that Arc associates with GluR4-containing AMPARs during these different training procedures. Once induced, Arc remains present in synapses during these phases of conditioning. The results are consistent with the interpretation that synaptic incorporation of GluR4-containing AMPARs supports the expression of CRs in this preparation, and that Arc may be involved in trafficking of GluR4 subunits during conditioning. Moreover, the maintained presence of synaptically localized Arc during all phases of conditioning examined indicates that synapses do not return to their naïve state after extinction and that, given the potential trafficking function of Arc, may facilitate relearning after extinction.

Regulation of BDNF chromatin status and promoter accessibility in a neural correlate of associative learning

Brain-derived neurotrophic factor (BDNF) gene expression critically controls learning and its aberrant regulation is implicated in Alzheimers disease and a host of neurodevelopmental disorders. The BDNF gene is target of known DNA regulatory mechanisms but details of its activity-dependent regulation are not fully characterized. We performed a comprehensive analysis of the epigenetic regulation of the turtle BDNF gene (tBDNF) during a neural correlate of associative learning using an in vitro model of eye blink classical conditioning. Shortly after conditioning onset, the results from ChIP-qPCR show conditioning-dependent increases in methyl-CpG-binding protein 2 (MeCP2) and repressor basic helix-loop-helix binding protein 2 (BHLHB2) binding to tBDNF promoter II that corresponds with transcriptional repression. In contrast, enhanced binding of ten-eleven translocation protein 1 (Tet1), extracellular signal-regulated kinase 1/2 (ERK1/2), and cAMP response element-binding protein (CREB) to promoter III corresponds with transcriptional activation. These actions are accompanied by rapid modifications in histone methylation and phosphorylation status of RNA polymerase II (RNAP II). Significantly, these remarkably coordinated changes in epigenetic factors for two alternatively regulated tBDNF promoters during conditioning are controlled by Tet1 and ERK1/2. Our findings indicate that Tet1 and ERK1/2 are critical partners that, through complementary functions, control learning-dependent tBDNF promoter accessibility required for rapid transcription and acquisition of classical conditioning.