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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Cell. Author manuscript; available in PMC 2012 April 15.
Published in final edited form as:
PMCID: PMC3085003
NIHMSID: NIHMS283311

The AAA+ ATPase, Thorase Regulates AMPA Receptor-Dependent Synaptic Plasticity and Behavior

SUMMARY

The synaptic insertion or removal of AMPA receptors (AMPAR) plays critical roles in the regulation of synaptic activity reflected in the expression of long-term potentiation (LTP) and long-term depression (LTD). The cellular events underlying this important process in learning and memory are still being revealed. Here we describe and characterize the AAA+ ATPase, Thorase, that regulates the expression of surface AMPAR. In an ATPase-dependent manner Thorase mediates the internalization of AMPAR by disassembling the AMPAR-GRIP1 complex. Following genetic deletion of Thorase, the internalization of AMPAR is substantially reduced, leading to increased amplitudes of miniature excitatory postsynaptic currents, enhancement of LTP and elimination of LTD. These molecular events are expressed as deficits in learning and memory in Thorase null mice. This study identifies an AAA+ ATPase that plays a critical role in regulating the surface expression of AMPAR and thereby regulates synaptic plasticity and learning and memory.

INTRODUCTION

The excitatory amino acid, glutamate, plays important roles in neuronal development, synaptic plasticity and neurodegeneration through activation of N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methylisoxazole-4-proprionate (AMPA) receptors (AMPAR) (Besancon et al., 2008; Kessels and Malinow, 2009). Synaptic strength is thought to be determined, in part, through the activity-dependent insertion and endocytosis of AMPARs (Kessels and Malinow, 2009), that regulates long-term potentiation (LTP) and long-term depression (LTD), and the initiation and formation of long-lasting memories (Kessels and Malinow, 2009). AMPARs are ionophores composed of a heteromeric complex of combinations of GluR1 through GluR4 subunits. A number of intracellular proteins regulate the trafficking of AMPARs thereby regulating neuronal excitability (Besancon et al., 2008; Kessels and Malinow, 2009).

The C-terminal PDZ binding domain of GluR2 receptors is important in AMPAR internalization by binding proteins such as glutamate receptor interacting protein (GRIP1) and protein interacting with C-kinase-1 (PICK1) (Newpher and Ehlers, 2009). Clathrin adaptor AP2, small GTPase Rab5, Homer, CPG2, dynamin 3, and Arc/Arg3.1 are also involved in controlling AMPAR endocytosis as is GluR1 AMPAR phosphorylation. These studies have provided insight into the protein machinery involved in AMPAR trafficking (Kessels and Malinow, 2009; Newpher and Ehlers, 2009). However, the specific mechanisms of AMPAR internalization are not well understood.

Here we describe and characterize neuroprotective gene 6 (NPG6) (EF688601), currently annotated as Atad1, which we named Thorase after Thor, the Norse God of Thunder and Lightening (Dai et al., 2010). Thorase controls AMPAR internalization in an ATPase-dependent manner by disassembling GluR2 and GRIP1 complexes. In the absence of Thorase, the internalization of AMPARs is decreased, leading to increased amplitude of miniature excitatory postsynaptic currents, enhanced LTP and impaired expression of LTD. These physiologic outcomes result in deficits in learning and memory. These results define an ATPase-dependent process that regulates the intracellular trafficking of AMPARs.

RESULTS

Thorase is an AAA+ ATPase

Thorase is a 361 amino acid protein (Figure S1A) containing an AAA+ ATPase domain composed of Walker A (ATP binding motif) and Walker B (ATP hydrolysis motif) motifs similar to other ATPases (Figure S1B). Consistent with general ATPase structures, Thorase contains an N-linker (NL) domain, which may transduce energy from ATP hydrolysis to the rest of the protein and a second region of homology (SRH) that differentiates classically defined AAA proteins from the broader AAA+ family (White and Lauring, 2007) (Figure 1A & S1A). Thorase possesses ATPase activity with a Km of 43.4 μM and a Vmax of 11.0 nM ATP/min/mg protein (Figure 1B & 1C). The ATPase activity of the Walker A (K193T) (mA-Thorase) mutant, or the Walker B (E139Q) (mB-Thorase) mutant are reduced by 60–70% and by greater than 92% in the mutant containing both mutations (mAB-Thorase) (Figure 1C).

Figure 1
The AAA+ ATPase, Thorase is a Cytosolic and Postsynaptic Protein that Regulates AMPAR Surface Expression

Expression pattern and synaptic enrichment of Thorase in mouse brain

Northern blot analysis shows that Thorase mRNA is expressed in many tissues with the highest expression in the brain and testes (Figure S1C). Polyclonal and monoclonal antibodies to Thorase recognize a single band on immunoblot analysis that is not present in Thorase gene deleted tissues (Figure S1D, see Figure S2D). Immunoblot analysis reveals a heterogeneous expression of Thorase in mouse brain (Figure S1E, F). Immunohistochemistry shows heterogeneous expression of Thorase with relatively high expression in hippocampal CA1 pyramidal cells (Figure S1G–J).

Subcellular fractionation from whole mouse brain shows Thorase segregates into the P2 fraction (crude synaptosome), but not in the P1 (nucleus) or the S2 (cytosol) fractions. Within the P2 fraction, Thorase segregates to the LP1 fraction (synaptosomal membranes) with minor segregation in the P3 (light membranes) and LP2 (synaptic vesicle-enriched fraction), and none in the S3 (cytosol) and LS2 (synaptic cytosol) (Figure 1D). The subcellular distribution of Thorase was compared to the synaptic proteins: GRIP1, GluR1, GluR2, postsynaptic density protein 95 (PSD95), vesicle-associated membrane protein 2 (VAMP2), α-synuclein, and synaptophysin 1. Histone was used as a nuclear marker and phosphoglycerate kinase 1 (PGK1) was used as a cytosolic marker (Figure 1D). The enrichment of these proteins in their respective fractions indicates that the fractionation was effective. Thorase co-segregates with the postsynaptic proteins PSD95, GRIP1, GluR1, and GluR2 in the LP1 fraction (Figure 1D). To investigate the localization of Thorase with synaptic proteins, primary mouse hippocampal neurons were co-stained with Thorase monoclonal antibodies and synaptic marker antibodies. By confocal microscopy, Thorase is juxtaposed to the presynaptic marker synapsin 1 but colocalizes with the postsynaptic marker PSD95 (Figure 1E and 1F) GluR1 and GluR2 (Figure 1G and 1H). Co-immunostaining of Thorase and the axon specific marker SMI312 shows that Thorase is not expressed in axons (Figure S1K). Co-expression of Thorase with postsynaptic proteins such as PSD-95, PICK1, GRIP1 and GRIP2 in HEK293 cells followed by co-immunoprecipitation indicates that Thorase specifically interacts with GRIP1, but fails to co-immunoprecipitate with GRIP2, Arc, Homer 1, Homer 2, PICK1 and PSD95 (Figure 1I). Domain mapping via co-immunoprecipitation experiments in HEK293 cells with Thorase and different PDZ domains of GRIP1 indicates that Thorase interacts with the PDZ5 domain of GRIP1 (Figure 1J). Co-immunoprecipitation experiments indicate that Thorase also binds to GluR2 (Figure 1K). GST-pull down of Thorase with different C-terminal truncations of GluR2 indicates that Thorase interacts with amino acids 832–839 of the C-terminus of GluR2 (Figure 1L). Thorase also interacts with GluR2 and GRIP in vivo (see Figure 5). These results taken together indicate Thorase is a cytosolic and postsynaptic protein that co-localizes with AMPARs and interacts with both GRIP1 and GluR2.

Figure 5
Thorase provides a driving force to disassemble the AMPAR complex

Thorase overexpression decreases surface AMPARs and AMPA currents

To explore the possibility that Thorase could regulate AMPAR surface expression in an ATPase-dependent manner, wild type (WT) Thorase or the Thorase ATPase deficient Walker AB mutant (mAB-Thorase) with or without a GFP-tag (Figure S1L & S1M) were overexpressed in high-density mouse cortical and hippocampal neural cultures via lentiviral transduction. Expression of Thorase with or without the GFP-tag decreases the level of surface GluR2 AMPARs as revealed by a surface biotinylation assay, whereas mAB-Thorase has no effect (Figure 1M & 1N). Expression of Thorase-GFP leads to a 28.1% reduction in surface GluR2 receptor intensity in low-density mouse hippocampal neurons by live cell immunohistochemistry with an anti-GluR2 N-terminal antibody compared to GFP control cultures (Figure 1O & 1P). Overexpression of mAB-Thorase-GFP has no effect on surface GluR2 expression compared to GFP control (Figure 1O & 1P). Adult mice were stereotaxically injected with lenti-GFP, lenti-Thorase-GFP or lenti-mAB-Thorase-GFP virus into the hippocampus. One week later hippocampal slices were prepared and NMDA and AMPA currents were recorded at 40 mV and −70 mV in GFP positive neurons (see Figure 6). NMDA currents are not affected by Thorase-GFP overexpression but AMPA currents are reduced by 20% compared to GFP controls (Figure 1Q & 1R). mAB-Thorase-GFP expression shows no effect on either NMDA and AMPA currents (Figure 1Q & 1R). Taken together these results suggest that Thorase specifically regulates AMPAR surface expression in an ATPase-dependent manner both in vitro and in vivo.

Figure 6
Thorase Deficiency Enhances AMPAR Currents and LTP and Impairs LTD Without Affecting Basal Synaptic Transmission at CA1 Hippocampal Synapses

Generation of Conditional Thorase Knockout mice

Conditional Thorase knockout (KO) mice were generated by flanking exon 5 and exon 6 containing the AAA Walker A and Walker B domains with lox-P sites (Figure S2A). Genomic deletion of Thorase was achieved by breeding to male germ-line Protamine-Cre deletor mice. Southern blot analysis confirms the successful targeting with a 9.3 kb WT band and an 11.8 kb targeted band (Figure S2B). PCR confirms the KO of Thorase (Figure S2C). Immunoblot analysis and immunohistochemistry with a Thorase specific C-terminal antibody (see Figure S1D) reveals loss of protein expression in the KO animal (Figure S2D & S2E). Homozygote Thorase KO mice are viable, but significantly smaller than their WT littermates (Figure S2F). Approximately 80% of the Thorase KO mice die of a seizure like syndrome between postnatal day 19–25. The remaining 20% survive up to 8 weeks of age (Figure S2G). No gross developmental or behavioral defects are observed between heterozygotes and WT littermates. There are no obvious abnormalities in other tissues in Thorase KO mice at postnatal day 19 as accessed by a comprehensive pathologic necropsy of heart, lung, spleen, kidney, thymus, liver, intestine, testis, eyes and muscle. Golgi staining of the CA1 region of the hippocampus does not reveal any substantial difference in the dendritic complexity of the Thorase KO mice versus WT mice or in the number or size of dendritic spines (Figure S2H–S2K). Thorase KO mice also show normal density of synapses identified by colocalization of puncta containing both the presynaptic protein synapsin 1 and the postsynaptic protein PSD95 (Figure S2L and S2M). Hematoxylin and eosin (HE) or Nissl staining also show normal brain morphology and intact hippocampi and cortices from Thorase KO mice at P20 (Figure S2N and S2O).

Loss of Thorase results in an increase in surface AMPARs

Levels of AMPAR subunits and associated proteins were assessed in 19–23 day old Thorase KO mice and WT littermates via immunoblot analysis of whole brain lysate and the P2 fraction (Figure 2A). AMPAR subunits, GluR1 and GluR2, are increased in both whole brain lysate and the P2 fraction from Thorase KO mice compared to WT littermates (Figure 2A). There is a modest increase in the levels of GRIP1 in whole brain lysate and the P2 fraction in Thorase KO, but there are no obvious differences in PICK1, NMDA receptor 1 (NR1), NR2B, NR2A, PSD95 or synapsin-1 (Figure 2A). In Thorase KO low-density mouse hippocampal neurons live cell immuohistochemistry with anti-N-terminal GluR1 or GluR2 antibodies reveals a 23.8% increase in surface GluR1 (Figure 2B & 2C), and a 28.9% increase in surface GluR2 (Figure 2D & 2E). Specificity of these AMPAR increases in Thorase KO neurons is shown by similar expression of surface NR1 between Thorase KO and WT neurons (Figure 2F and 2G). In vivo AMPAR surface expression was assessed by a Bis(sulphosuccinimidyl)suberate (BS3) cross-linking assay that enables the quantification of surface and intracellular receptor pools. The ratio of surface GluR2/intracellular GluR2 is dramatically increased in the cortex, hippocampus and cerebellum from Thorase KO mice compared to WT littermates (Figure 2H & 2I). These data suggest that Thorase regulates the distribution of AMPARs and loss of Thorase results in an increase in the steady-state levels of these receptors at the cell surface.

Figure 2
Knockout of Thorase Increases AMPAR Surface Expression and AMPAR Currents

To evaluate the functional outcome of increased AMPAR surface receptor expression, spontaneous mEPSCs were assessed in CA1 hippocampal pyramidal neurons from Thorase KO versus WT littermates (Figure 2J–M). The amplitudes of mEPSCs are increased in Thorase KO compared to WT controls by 70% (Figure 2K) and the frequency of mEPSCs are similar in neurons from Thorase KO and WT mice (Figure 2L). There is a rightward shift of the cumulative probability distributions of mEPSCs in Thorase KO versus WT neurons indicating that the distribution of the increase in amplitude is distributed across the range of recorded events (Figure 2M). The increase in mEPSC amplitude is not due to altered presynaptic activity because paired-pulse facilitation (PPF) was not affected in Thorase KO neurons (see Figure 6B) and no significant difference in the input/output (I/O) curves or amplitude of the field EPSPs was found in slices from Thorase KO mice compared to WT mice (Figure 6A).

Thorase regulates synaptic scaling

Homeostatic scaling of AMPARs was assessed in WT and Thorase KO low-density hippocampal neurons treated for 48 h with TTX (1 μM), which blocks all evoked neuronal activity, or bicuculline (20 μM), which increases neuronal firing and blocks inhibitory neurotransmission mediated by GABAA receptors (Shepherd et al., 2006). WT neurons exhibit a robust increase in surface GluR1 (Figure 3A and 3B) and surface GluR2 (Figure 3C and 3D) with TTX treatment. Bicuculline treatment significantly lowers surface levels of GluR1 (Figure 3A and 3B) and GluR2 (figure 3C and 3D). In Thorase KO neurons TTX further increases the surface expression of GluR1 and GluR2 (Figure 3A–D) but bicuculline has no effect (Figure 3A–D). These results suggest that in response to activity, AMPARs can be inserted into surface membranes, but removal or endocytosis of AMPARs is blocked in Thorase KO neurons.

Figure 3
Deficits in Homeostatic Synaptic Scaling and Endocytosis of AMPARs in Thorase KO neurons

Loss of Thorase results in reduced endocytosis of AMPARs

An “antibody feeding” internalization assay for endocytosis of surface GluR2 receptors was performed in live neurons incubated with an anti-GluR2 N-terminal antibody followed by treatment with NMDA (20 μM) and glycine (10 μM) to trigger AMPAR endocytosis (Cottrell et al., 2004). In WT neurons there is a robust internalization of surface GluR2 but significantly less in Thorase KO neurons (Figure 3E) resulting in a 50% reduction in the internalization index in Thorase KO neurons compared to WT neurons (Figure 3F). These results suggest that Thorase regulates AMPAR scaling by controlling AMPAR endocytosis. No significant difference in internalization of transferrin receptors after stimulation with Alexa-568 conjugated-transferrin (Hanley et al., 2002) was detected between Thorase KO and WT neurons (Figure 3G and 3H), indicating Thorase actions on endocytosis are specific to AMPARs.

AMPAR Trafficking is ATPase Dependent

pH-sensitive GFP (pHluorin) fused at the N-terminal extracellular domain of GluR1 or GluR2 serves as spatiotemporal indicators of AMPAR distribution by fluorescing at pH 7.4 at the extracellular surface and becoming non-fluorescent in endosomes where the pH is less than 6.0 (Lin and Huganir, 2007). At baseline, Thorase KO neurons show significantly higher levels of pH-GluR1 and pH-GluR2 signal intensity than WT neurons (Figure S3A). In WT hippocampal cultures NMDA stimulated internalization of AMPARs leads to loss of the fluorescent signal for pH-GluR1 or pH-GluR2 (Figure 4A–E and S3B–F) and washout of NMDA leads to recovery of fluorescence consistent with AMPAR endocytosis and recycling as previously described (Lin and Huganir, 2007). In Thorase KO hippocampal cultures, NMDA causes a significantly smaller reduction in fluorescent intensity of both pH-GluR1 and pH-GluR2 and fluorescent recovery is significantly faster than in WT cultures (Figure 4A–E and S3B–F). These results indicate that Thorase is required for AMPAR internalization and that Thorase may inhibit GluR1 and GluR2 recycling back to the plasma membrane.

Figure 4
NMDA-induced AMPAR Internalization is Reduced in an ATPase-Dependent Manner in Thorase KO Neurons

Rescue experiments were performed in Thorase KO hippocampal cultures with a TdTomato WT Thorase fusion protein with similar ATPase activity to WT Thorase (Figure S3G) and a TdTomato mAB-Thorase fusion protein with an 80% reduction of ATPase activity (Figure S3G) in the presence of either the pH-GluR1 or pH-GluR2 in Thorase KO cultures (Figure 4F–I and S3G & S3H). NMDA stimulated AMPAR trafficking in Thorase KO cultures was restored to WT levels by WT Thorase whereas the mAB-Thorase fails to alter AMPAR trafficking deficits in Thorase KO cultures (Figure 4F–I and S3H). These results taken together indicate that NMDA stimulated AMPAR internalization is mediated by Thorase in an ATPase dependent manner and that inhibition of GluR1 and GluR2 recycling back to the plasma membrane by Thorase is ATPase dependent.

Thorase provides a driving force to disassemble the AMPAR complex

AAA+ ATPases often assist in the assembly, or disassembly of protein complexes (Ogura and Wilkinson, 2001; White and Lauring, 2007). To explore whether Thorase may function as an energy motor that dissociates the AMPAR complex to facilitate endocytosis, AMPAR complexes were prepared from brains of WT and Thorase KO mice in the presence or absence of ATP or non-hydrolyzable ATPγS (Figure 5A). Immunobloting with anti-GluR2 antibody shows that AMPAR complexes were well prepared and migrate at approximately 700 KDa. In the presence of ATP, a substantial portion of the AMPAR complex from WT mouse brains shifts to approximately 300 KDa (Figure 5A). However, in the presence of ATPγS, the position of the AMPAR complex remains stable. In contrast, AMPAR complexes from Thorase KO mouse brains show no difference in the presence or absence of ATP or ATPγS (Figure 5A). Co-immunoprecipitation from mouse hippocampal extracts with an anti-GluR2 antibody shows that GluR2, GRIP1 and Thorase coexist as a complex in vivo (Figure 5B). The binding of GRIP1 and Thorase to the GluR2 complex is sensitive to ATP hydrolysis as there is more binding in the presence of ATPγS and less binding in the presence of ATP (Figure 5B & 5C). These results taken together support the notion that the ATPase activity of Thorase is required for the disassembly of native AMPAR complexes in vivo.

To confirm if Thorase directly interacts with GluR2 and GRIP1, GST-GluR2 C terminus (R2C) and GST-GRIP1 were expressed and immobilized on the glutathione agarose beads, respectively. Thorase binds GluR2C in the presence of nonhydrolyzable Mg2+/ATPγS or EDTA/ATP (Figure 5D). In the presence of hydrolyzable ATP, Thorase is not retained by GST-GluR2C (Figure 5D). Thorase weakly binds GRIP1 in the presence of ATPγS (Figure 5E). However, when GluR2C was preincubated with GRIP1 to form GluR2C-GRIP1 complexes, Thorase shows robust binding to GluR2C-GRIP1 complexes in the presence of ATPγS (Figure 5F), indicating that Thorase prefers to bind to AMPAR complexes containing GRIP1. However, in the presence of ATP Thorase binding to GluR2C-GRIP1 complexes are disrupted, suggesting that this binding is sensitive to ATP hydrolysis (Figure 5F).

To test if in the presence of ATP Thorase might disrupt GluR2-GRIP1 interactions, His6-GRIP1 protein was immobilized on Dynabeads, incubated with GST-GluR2C to form His6-GRIP1/GST-GluR2C complexes and then incubated with GST-Thorase in the presence of ATP or ATPγS (Figure 5G). In the presence of ATPγS, the complex of His6-GRIP1/GST-GluR2C is stable (Figure 5Gb) but in the presence of ATP there is a striking dissociation of GST-GluR2C from His6-GRIP1 (Figure 5Gb). Additionally, in the presence of ATPγS, the His6-GRIP1/GST-GluR2C complexes are stable even at high concentration of GST-Thorase. In contrast, in the presence of ATP, 200 nM of GST-Thorase causes 75% disassembly of His6-GRIP1/GST-GluR2C complexes, with nearly complete disassembly in presence of 400 nM of GST-Thorase (Figure 5H). Taken together these results indicate that dissociation of GRIP1 and AMPAR complexes requires Thorase ATPase activity.

Loss of Thorase blocks LTD at Schaffer Collateral synapses

To determine whether Thorase plays a role in regulating synaptic plasticity, postsynaptic responses of CA1 neurons were recorded during and after stimulation of presynaptic Shaffer collateral axons in hippocampal slices from WT and Thorase KO mice. There are no significant differences in the input/output (I/O) curves or amplitudes of the field EPSPs or paired-pulse facilitation (PPF) in slices from Thorase KO mice compared to WT mice (Figure 6A–B). Collectively, these findings suggest that Thorase does not play a major role in basal transmission at CA1 synapses.

LTD and LTP were evaluated in Thorase KO and WT mice. LTD was induced in slices from WT littermates with persistent depression of evoked response (74.9 ± 12.1% of the baseline) while LTD was absent in slices from Thorase KO mice (105.7 ± 14.3% of the baseline) (Figure 6C). LTP was induced in slices from WT and Thorase KO mice with the magnitude of LTP greater in slices from Thorase KO mice (204.2 ± 28.6%) compared to WT mice (169.9 ± 27.7%) (Figure 6D). These changes were not due to alterations in gross anatomy (Figure S2N and S2O) or normal basal synaptic transmission.

NMDA and AMPA currents were recorded simultaneously by whole-cell patch-clamp during a series of voltage steps. The NMDA receptor antagonist AP-5 blocks the current induced at a positive potential (Figure 6E). The AMPAR antagonist, CNQX, abolishes currents at all stimulation voltages; this effect is reversed during a 20–30 minute washout period (Figure 6E). The maximum amplitude of the AMPA current is significantly greater in slices from the Thorase KO mice compared to the WT mice at all stimulus voltages tested (Figure 6F). No significant change was found in the amplitude of NMDA currents. The ratio of AMPA current/NMDA current was increased about 30% (Figure 6F). There is a significant rightward shift in the I-V curve at negative stimulation potentials in slices from Thorase KO mice compared to WT mice (Figure 6G). There is still a significant rightward shift in the I-V curve at negative stimulation potentials in the presence of the NMDA inhibitor AP-5 in Thorase KO slices, confirming that the increased AMPA current is caused by increased surface AMPARs (Figure 6H).

The AMPAR rectification index was measured by dividing the EPSC amplitude at +40 mV by that at −70 mV (Isaac et al., 2007). There is no significant difference in the rectification index between WT and Thorase KO mice (Figure 6I), suggesting that there is no alteration in the synaptic AMPAR composition in Thorase KO mice. Philanthotoxin 433 (PhTx), a specific antagonist of the GluR2-lacking AMPARs did not alter the I-V curves in neurons from WT and KO mice (Figure S4A–C), suggesting that GluR2-containing AMPARs predominated in both WT and KO neurons. Although the surface levels of GluR1 and GluR2 in KO neurons are higher than those of WT neurons evaluated by immunohistochemistry, there is no significant change in the ratio of surface GluR2 to GluR1 between WT and KO neurons (Figure S4D and S4E). These results taken together indicate that knockout of Thorase does not affect AMPAR subunit composition.

Thorase was reintroduced into Thorase KO hippocampus by stereotaxic injection of lenti-GFP, lenti-Thorase-GFP and lenti-mAB-Thorase-GFP virus. One week later hippocampal slices were generated for physiology. There is no effect on AMPAR currents from lenti-GFP (Figure 6J and 6K), however Thorase-GFP restores AMPAR currents back to WT levels, whereas, mAB-Thorase-GFP has no rescue effect (Figure 6J and 6K). In hippocampal culture, the increased surface expression of GluR2 in Thorase KO neurons could also be rescued by lentiviral expression of Thorase-GFP but not by mAB-Thorase-GFP or GFP (Figure S4F and S4G). Taken together these results indicate that the increase in AMPA current in Thorase KO mice results from lack of Thorase and not a non-specific process due to the absence of Thorase and the functional effects are ATPase-dependent.

Thorase is essential for learning and memory

To avoid the lethality of Thorase KO mice, Thorase flox/+ mice were crossed with CaMKIIa-iCre transgenic mice to restrict the temporal and spatial deletion of Thorase to the adult forebrain. Immunoblot analysis and immunohistochemistry verifies that Thorase is largely eliminated from forebrain structures including the hippocampus and cortex but not in other regions such as the cerebellum (Figure S5). There are no gross anatomical changes in the hippocampus or other forebrain structures in the conditional Thorase KO (cKO) mice compared to WT control (Figure S5).

The general activity of Thorase cKO mice was assessed in open field testing (Pletnikov et al., 2008) in which Thorase cKO mice spend more time at the margins of the open field, less time in the center and show less rearing activity (Figure 7A–C). This is not due to altered anxiety as there was no significant difference in performance on the elevated plus maze (Walf and Frye, 2007) in the total distance, percent open time, percent entrance to open arms, or the number of rearing behaviors (Figure 7D–G). These results suggest that the deficits in the open field could be due to the inability to form immediate memories in a novel environment. Assessment of spatial memory using the Morris Water Maze test was not possible due to seizures in the conditional Thorase KO mice. Accordingly, spatial memory was determined in spontaneous alteration in a Y-maze using a continuous trials procedure. The Thorase cKO mice have a lower percentage of correct alternating arm entries compared to the littermate WT mice (Figure 7H), indicating that the Thorase cKO mice are amnesic when reintroduced into a previously novel environment. Seven days after exposure to the Y-maze, mice were placed for 5 minutes into a Y-maze in which the third arm was closed to acquire contextual memory (Etkin et al., 2006). While the WT mice spent more time in the novel arm that was previously blocked, the Thorase cKO mice spent significantly less time in the novel arm (Figure 7I) suggesting deficits in short term memory. To exclude the possibility for a Y-maze side preference, a T-maze non-matching to place task was performed to test hippocampal dependent spatial working memory (Deacon and Rawlins, 2006; Reisel et al., 2002). WT mice show a strong non-place matching performance for a sweet milk reward attaining a 90% correct choice by the end of training (Figure 7J). In contrast, the Thorase cKO mice are impaired and remain at chance levels at the end of training (Figure 7J) (58%, F1, 44 = 15.1, p < 0.002). These behavioral data indicate that the knockout of Thorase has profound effects on learning and memory, consistent with the effects of Thorase on AMPAR function.

Figure 7
Thorase is required for spatial working memory

DISCUSSION

The major finding of this paper is that AAA+ ATPase, Thorase is a significant and important regulator of AMPAR trafficking, synaptic plasticity and behavior. A major mechanism regulating synaptic strength is the net balance between the insertion and internalization of AMPARs in the postsynaptic membrane (Gong and De Camilli, 2008). Our data shows that surface levels of AMPARs in Thorase KO neurons are increased with chronic TTX treatment, whereas bicuculline treatment failed to lower the surface levels of AMPARs in Thorase KO neurons, indicating that the process of AMPAR insertion is intact and that internalization is impaired. This is also strongly supported by the impaired NMDA-stimulated internalization of GluR2 receptors as detected by the “antibody feeding” assay. Moreover, the spatiotemporal indicators of AMPAR distribution, pH-GluR1 or pH-GluR2 indicate that Thorase is required for AMPAR internalization and that Thorase may inhibit GluR1 and GluR2 recycling back to the plasma membrane in an ATPase-dependent manner. In addition, the impaired LTD in Thorase KO neurons occurs immediately during the 1 Hz stimulation protocol, indicating that the absence of LTD is due to a deficit in the mechanism of LTD induction. Since LTD in hippocampal CA1 neurons is mainly initiated postsynaptically by the removal of surface AMPARs through endocytosis (Massey and Bashir, 2007), these findings taken together suggest that Thorase is involved in the process of AMPAR endocytosis and the lack of Thorase causes impaired internalization of AMPARs.

Thorase belongs to an AAA+ ATPase family which often performs chaperone-like functions, including membrane dynamics, protein transport and assembly or disassembly of protein complexes without unfolding or destroying their target proteins (Ogura and Wilkinson, 2001; White and Lauring, 2007). Numerous ATPases are expressed in neurons that regulate many key cellular functions. In this study, two-dimensional gel separations shows that the ATPase activity of Thorase is required for the disassembly of AMPAR complexes in vivo, suggesting that Thorase plays a crucial role in disassembly of the AMPAR complex. Endogenous coimmunoprecipitation assays show that GluR2, GRIP1 and Thorase coexist as a complex in vivo and the binding of Thorase to GluR2 is sensitive to ATP hydrolysis. In vitro binding experiments further confirmed that Thorase can directly bind to GluR2 and GRIP1 and that this binding is highly sensitive to ATP hydrolysis. Intriguingly, Thorase prefers to bind the GluR2-GRIP1 complex in the presence of nonhydrolyzable ATPγS and Thorase robustly disassembles GluR2-GRIP1 complexes in the presence of ATP, indicating that Thorase may mainly function on surface AMPAR complexes. GRIP1 acts as an AMPAR anchor stabilizing AMPARs at the plasma membrane and limits the endocytosis rate of AMPARs by its association with GluR2 (Osten et al., 2000). Therefore, we propose a mechanism in which GluR2, GRIP1 and Thorase form a complex in the presence of ATP and Thorase provides the ATP-driving force to disrupt GluR2-GRIP1 interactions resulting in dissociation of the AMPAR complex. Thorase-mediated disassembly of AMPAR complexes appears to play a crucial role in limiting endocytosis of AMPARs to adjust the level of AMPARs at the synaptic membrane. The regulation of disassembly of AMPARs by Thorase is likely to account for its essential roles in LTD and regulation of synaptic plasticity and learning memory since LTD is at least partly expressed by removal of AMPARs from the synapse by endocytosis (Wang and Linden, 2000).

The related AAA+ ATPase, NSF is another postsynaptic protein that is required for disassembly of SNARE complex (Ungermann et al., 1998) and for AMPAR trafficking (Nishimune et al., 1998). NSF binds to the AMPAR subunit GluR2 and disassembles the GluR2-PICK1 protein complex and regulates excitatory synaptic plasticity by stabilizing or recycling AMPARs into postsynaptic membranes (Huang et al., 2005). In contrast, Thorase acts at GluR2-GRIP1 complexes to control the endocytosis and removal of AMPARs from the postsynaptic membrane. The identification of Thorase as a major regulator of AMPAR trafficking offers new opportunities to enhance the understanding and to identify additional regulators of AMPAR turnover.

EXPERIMENTAL PROCEDURES

Antibodies

Monoclonal and polyclonal Thorase antibodies were generated as described in supplemental information. GluR1-N (mAb) was a gift from Dr. Richard L. Huganir. Other antibodies were acquired commercially: GluR2-N (mAb, Chemicon) and GluR2 (pAb, Millipore), PSD95 (mAb, NeuroMab), synaptophysin 1 (mAb, Sigma), synapsin 1 (pAb, mAb, BD Tranduction Laboratories), PICK1 (mAB, NeuroMab), NR1 (pAb, Sigma), NR2A (mAb, Sigma), NR-2B (mAb, NeuroMab), β-tubulin (mAb, Sigma), GRIP1(pAb, Chemicon).

ATPase activity assay

The ATPase activities of Thorase and its mutants were determined by the measurement of the amount of [γ-32P]-Pi obtained from [γ-32P]-ATP hydrolysis using EasyRad Phosphate Assay Biochem Kit (Cytoskeleton Inc, Denver, CO) according to the instruction from the manufacturer (Supplemental information).

Primary Neuronal Cultures and Transduction of Cultured Neurons with Recombinant Lentivirus

Primary cortical neuron cultures were prepared from embryonic day 15 (E15) mouse pups as described (Shepherd et al., 2006). Low-density hippocampal neurons from E18 or postnatal day 0–1 mouse pups were prepared as reported previously (Shepherd et al., 2006). Neurons were infected with different lentivirus 10 days after plating.

Electrophysiology

Thorase KO and WT littermates with the age of postnatal day 19–22 were used for all electrophysiological experiments. Transverse hippocampal slices (350 μm) were prepared and maintained recordings were performed as described in our previous studies (Wang et al., 2004) (Supplemental information). Excitatory postsynaptic currents (EPSCs) were recorded from CA1 pyramidal neurons (Wang et al., 2009) (Supplemental information).

Behavioral experiments

All behavioral experiments were done in 3- to 4-month-old cKO (ThoraseFlox/Flox, cre+) mice (n = 20, 10 males and 10 females) and WT (Thorase+/+, cre+) littermates (n = 19, 9 males and 10 females) which were obtained by mating heterozygous ThoraseFlox/+, cre+ mice. Both the sex and the age were matched between two groups.

Open field

Open field test was performed to assess the general activity of Thorase KO mice using Photobeam Activity System (San Diego Instruments, La Jolla, CA, USA) which records both horizontal and vertical activities simultaneously (Supplemental information).

Elevated Plus Maze

Anxiety-like behavior was evaluated in Elevated Plus Maze (San Diego Instruments Inc., CA, USA) as described previously(Singer et al., 2009) (Supplemental information).

Y maze

Spontaneous alternation was assessed by Y-maze test using a protocol similar to those previously described (Holcomb et al., 1998; Hsiao et al., 1996) (Supplemental information).

T maze

To avoid side preferences of some animals in Y-maze test, spatial non-matching-to-place testing was performed on an elevated T-maze according to the protocol previously described (Deacon and Rawlins, 2006; Reisel et al., 2002) (Supplemental information).

Statistics

Quantitative data is presented as the mean ± S.E.M. Statistical significance was either assessed via an unpaired two-tailed Student’s t-test or an ANOVA test with Tukey-Kramer post-hoc analysis. All behavioral data were first subjected to a Kolmogorov-Smirnov normality and an equal variance test. For open field and T-maze test, repeated measures two-way ANOVAs were conducted and significant differences were determined at p < 0.05 for group main effects. The points with significant differences were identified by post hoc analysis using the Holm-Sidak method for multiple comparisons. For elevated plus maze and Y-maze tests, all data were analyzed using a Student’s t-test. Assessments were considered significant with a p < 0.05.

Supplementary Material

01

Inventory of Supplemental Information:

1. Figure Legends, Extended Experimental Procedures.SupplementalReferences

2. Figure S1

3. Figure S2

4. Figure S3

5. Figure S4

6. Figure S5

Acknowledgments

We thank Dr. Richard Huganir for the generous gift of monoclonal anti-GluR1 N-terminal antibody and Dr. Edward B. Ziff (New York University School of Medicine) for the GST-GluR2C plasmid. Research was supported by the Intramural Research Program of the National Institute on Aging, American Heart Association Postdoctoral Fellowship to JZ (0725470U) USPHS AG029368, and DA00266 and the Simon’s Foundation Autism Research Initiative. We thank the Comparative Medicine Section of NIA for technical support. T.M.D. is the Leonard and Madlyn Abramson Professor in Neurodegenerative Diseases.

Footnotes

SUPPLEMENTAL INFORMATION

Supplemental information includes extended Experimental Procedures, 5 Figures.

The authors declare that they have no competing financial interest.

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