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. Author manuscript; available in PMC: 2016 Jan 7.
Published in final edited form as: Neuron. 2015 Jan 7;85(1):173–189. doi: 10.1016/j.neuron.2014.12.023

Rapid Dispersion of SynGAP from Synaptic Spines Triggers AMPA Receptor Insertion and Spine Enlargement during LTP

Yoichi Araki 1, Menglong Zeng 2, Mingjie Zhang 2, Richard L Huganir 1,*
PMCID: PMC4428669  NIHMSID: NIHMS649309  PMID: 25569349

SUMMARY

SynGAP is a Ras-GTPase activating protein highly enriched at excitatory synapses in the brain. Previous studies have shown that CaMKII and the RAS-ERK pathway are critical for several forms of synaptic plasticity including LTP. NMDA receptor-dependent calcium influx has been shown to regulate the RAS-ERK pathway and downstream events that result in AMPA receptor synaptic accumulation, spine enlargement, and synaptic strengthening during LTP. However, the cellular mechanisms whereby calcium influx and CaMKII control Ras activity remain elusive. Using live-imaging techniques, we have found that SynGAP is rapidly dispersed from spines upon LTP induction in hippocampal neurons, and this dispersion depends on phosphorylation of Syn-GAP by CaMKII. Moreover, the degree of acute dispersion predicts the maintenance of spine enlargement. Thus, the synaptic dispersion of Syn-GAP by CaMKII phosphorylation during LTP represents a key signaling component that transduces CaMKII activity to small G protein-mediated spine enlargement, AMPA receptor synaptic incorporation, and synaptic potentiation.

INTRODUCTION

Long-term changes in the strength of synaptic transmission in the brain, and the subsequent formation of neuronal circuits, are thought to be critical for learning and memory, activity-dependent development, and other higher brain processes (Huganir and Nicoll, 2013; Kessels and Malinow, 2009; Shepherd and Huganir, 2007). AMPA receptors (AMPARs) are the major excitatory neurotransmitter receptors in the central nervous system. The regulation of AMPAR number at synapses is thought to be a major determinant of synaptic strength and to mediate several forms of synaptic plasticity including long-term potentiation (LTP) and long-term depression (LTD) (Anggono and Huganir, 2012; Kessels and Malinow, 2009; Shepherd and Huganir, 2007). The most well-studied form of synaptic plasticity in the brain is NMDA receptor-dependent LTP. This form of plasticity requires the activation of the NMDA-type glutamate receptors (NMDARs), calcium influx and activation of CaM kinase II (CaM-KII), and the subsequent recruitment of AMPARs to the synapse (Anggono and Huganir, 2012; Kessels and Malinow, 2009; Shepherd and Huganir, 2007). Small G proteins such as Ras, Rac1, Cdc42, and RhoA are also essential modulators of synaptic strength and structure during NMDAR-dependent LTP (Qin et al., 2005; Tashiro et al., 2000; Wiens et al., 2005; Xie et al., 2007; Zhu et al., 2002). Ras-ERK signaling is thought to be critical for AMPAR recruitment to spines following LTP induction (Kim et al., 2005a; Patterson et al., 2010; Thomas and Huganir, 2004; Zhu et al., 2002), and several lines of evidence demonstrate that inhibition of Ras or ERK blocks LTP induction (Patterson et al., 2010; Zhu et al., 2002). On the other hand, activation of Rac1/Cdc42, during LTP, is critical for regulating the enlargement of dendritic spines, small membranous protrusions from neuronal dendrites that house the excitatory postsynapse (Murakoshi et al., 2011). Spine size and synaptic strength are significantly correlated (Colgan and Yasuda, 2013; Matsuzaki et al., 2001; Matsuzaki et al., 2004), and coordinated regulation of small G protein signal transduction is crucial for changes in spine size and synaptic strength during synaptic plasticity. Multiple imaging studies have demonstrated that shortly after LTP induction, CaMKII becomes activated (several seconds to 10 s after stimuli) and is followed by small G protein activation (approximately 1 min after stimuli) (Harvey et al., 2008; Lee et al., 2009; Murakoshi et al., 2011). However, the cellular mechanisms that coordinate CaMKII and small G protein activation as well as the critical CaMKII substrates required for LTP remain unclear.

SynGAP is a synaptic Ras-GTPase activating protein (GAP) that facilitates GTP hydrolysis to GDP and thereby negatively regulates Ras activity (Chen et al., 1998; Kim et al., 1998). Conversely, guanine nucleotide exchange factors (GEFs) are proteins that exchange GDP to GTP and thereby activate small G proteins. The activities of GAP and GEF proteins assure precise activation levels in neurons (Bos et al., 2007) and have profound effects on synaptic strength and plasticity. SYNGAP1 knockout mice show deficits in NMDAR-dependent LTP in a Ras-ERK-dependent manner (Kim et al., 2003; Komiyama et al., 2002) and have deficits in learning and memory (Komiyama et al., 2002). SynGAP regulates the baseline levels of Ras and Rac activity as well as the phosphorylation of Cofilin, a downstream target that regulates actin polymerization (Carlisle et al., 2008). SynGAP also regulates synaptic strength and Erk activity levels (Rumbaugh et al., 2006). Heterozygote SYNGAP1 knockout mice have premature dendritic spine formation in vitro (Vazquez et al., 2004) as well as accelerated functional maturation in the neocortex and altered duration of critical periods for cortical plasticity (Clement et al., 2013). Moreover, de novo loss-of-function mutations in SYNGAP1 have been identified in patients with intellectual disability (ID) and autism spectrum disorders (ASDs) (Berryer et al., 2012; Hamdan et al., 2011; Hamdan et al., 2009). In addition, conditional SYNGAP1 knockout mice recapitulate several characteristic cognitive deficits found in these patients (Clement et al., 2012).

Several lines of evidence have suggested that SynGAP transmits NMDA receptor and CaMKII activity to downstream small G proteins including the Ras-ERK, Ras-PI3K, and Rac1-PAK pathways (Carlisle et al., 2008; Chen et al., 1998; Kim et al., 1998; Krapivinsky et al., 2004; Oh et al., 2004; Qin et al., 2005; Rumbaugh et al., 2006; Zhu et al., 2005), but the precise molecular and cellular mechanisms of this signaling pathway is unknown.

To examine the role of SynGAP in LTP, we investigated the dynamics of the subcellular localization of SynGAP in response to LTP induction. We demonstrated that (i) SynGAP is rapidly dispersed from spines during and after chemical LTP; (ii) this dispersion predicts the long-lasting changes in spine size, suggesting SynGAP inhibits stable LTP; (iii) phosphorylation of Syn-GAP at Ser1108/1138 by CaMKII plays a crucial role in this dispersion; and (iv) this dispersion triggers Ras activation that regulates downstream cellular processes, including AMPAR insertion, spine enlargement, and increased synaptic strength, demonstrating that SynGAP is a critical CaMKII substrate for the expression of LTP.

RESULTS

CaMKII Dependent Dispersion of SynGAP from Synapses during LTP

To investigate the mechanism underlying SynGAP activity during LTP, we studied the dynamics of the subcellular localization of SynGAP before and after LTP induction using live imaging techniques. We expressed GFP-tagged SynGAP and mCherry (as a morphology marker) in cultured hippocampal neurons and induced LTP chemically using a standard protocol that selectively activates synaptic NMDARs (chemLTP) (Liao et al., 2001; Lu et al., 2001). In this method, the magnesium in the media was withdrawn in conjunction with glycine perfusion. With spontaneous glutamate release from axonal terminals, glycine strongly and specifically stimulates synaptic NMDA receptors (Liao et al., 2001; Lu et al., 2001). This results in recruitment of AMPARs to synapses, spine enlargement, and synaptic potentiation. In our experiments, we observed chemLTP induced a rapid and sustained increase in dendritic spine size that lasted for at least 40 min (Figures 1A and 1B). Under resting conditions, GFP-SynGAP showed a clear punctate localization in dendritic spine heads consistent with previous studies on SynGAP’s enrichment in dendritic spines (Chen et al., 1998; Kim et al., 1998). Surprisingly, upon chemLTP induction, SynGAP was rapidly dispersed from spines (Figure 1A; Movie S1 available online). This dispersion occurred within minutes after chemLTP induction and lasted for at least 1 hr. We quantified the total Syn-GAP content in spines (as a measure of total signal from green channel) as well as spine volume (as a measure of total signal from red channel) during chemLTP (Figures 1B and S1A). Interestingly, SynGAP concentration in spines was rapidly decreased upon chemLTP while SynGAP concentration in dendritic shafts was increased but did not surpass the initial concentration in spines, suggesting that this dispersion is a passive diffusion of SynGAP upon chemLTP (Figure S1B). Biochemical isolation of postsynaptic density (PSD) fractions from these cultures also revealed the rapid dispersion of SynGAP from the PSD (Figure 1C). In Figure 1D, we plotted the correlation between SynGAP dispersion and spine enlargement 1 hr after LTP induction. The degree of dispersion of SynGAP was significantly correlated with the degree of spine enlargement (R2 = 0.73, correlation coefficient; p < 0.001), suggesting that SynGAP dispersion may be important for LTP-induced changes in spine size. Rapid SynGAP dispersion from spines was also observed in glutamate-uncaging-induced single spine LTP (ssLTP) (Figure S1C). In this protocol, caged glutamate was photolysed repeatedly (0.5 Hz × 30 times) on the spine head to induce LTP in specific spines. The kinetics of spine dispersion and recovery of SynGAP was quite similar to those during chemLTP (Figures 1B and S1D). Notably, this rapid dispersion did not occur during chemLTD (Figure S1E). Using electron microscopy, it was reported that chemLTD stimulation reduced SynGAP levels in the PSD core in a CaMKII-dependent manner (Yang et al., 2013; Yang et al., 2011); however, this microrelocalization may not be observed at the resolution of light microscopy. We confirmed endogenous SynGAP was also dispersed from spines upon chemLTP stimulation (Figures S2A and S2B).

Figure 1. Dynamic Dispersion of SynGAP from Spines during LTP.

Figure 1

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(A) Dispersion of SynGAP from synapses upon LTP stimulation. GFP-tagged SynGAP was dynamically dispersed upon LTP. mCherry was used as a morphology marker to show spine enlargement during LTP. Enlarged spines (e.g., spines 1–4) dispersed SynGAP. Some “no-response spines” (e.g., spines a–b) failed to disperse SynGAP. Correlations are shown in (D). Scale bar, 5 μm.

(B) Time course of averages of all spine size changes and SynGAP dispersion during LTP (n = 3 independent experiments/neurons that contain 35 spines).

(C) PSD fractionation during LTP also showed dynamic SynGAP dispersion from PSDs during LTP. Error bars indicate ± SEM.

(D) The relationship of “Dispersion of SynGAP from spine” and “Spine enlargement” in sustained phase (60 min) showed a strong and significant positive correlation between SynGAP dispersion and spine enlargement (n = 91 spines from seven independent experiments/neurons, R2 = 0.7288, p < 0.01). Spines 1–4 and a–b in (A) are also displayed. Note that the y intercept of trend line is nearly zero, showing that there was no spine enlargement if the spine failed to disperse SynGAP.

(E) Effects of pharmacological inhibition of “NMDAR-CaMKII,” “small G proteins,” and “actin polymerization” on Spine volume and SynGAP dispersion in the Sustained phase (60 min) (Spine volume: Drug F(9, 40) = 23.97, p < 0.001; SynGAP dispersion: Drug F(9, 40) = 28.00, p < 0.001). These results showed NMDAR-CaMKII pathway involved both in spine enlargement and SynGAP dispersion. Inhibition of “small G proteins, downstream kinase” and “actin polymerization” inhibited spine enlargement but not SynGAP dispersion, suggesting SynGAP dispersion is upstream cellular process of small G protein activation and actin polymerization. Error bars indicate ± SEM.

(F) Effects of pharmacological inhibition of “NMDAR-CaMKII,” “small G proteins,” and “actin polymerization” on Spine volume and SynGAP dispersion in the Acute phase (10 min) (Spine volume: Drug F(9, 40) = 42.13, p < 0.001; SynGAP dispersion: Drug F(9, 40) = 17.44, p < 0.001). Note that spine size enlargement was insensitive to CaMKII inhibition, Rac1 inhibition, and low dose of Latrunculin A (20 nM) treatment only in Acute phase, whereas SynGAP dispersion was still inhibited by CaMKII inhibition also in acute phase, suggesting CaMKII activity is essential for SynGAP dispersion (n = 5 independent experiments/neurons in all conditions, which contain 61 [Ctrl], 60 [APV], 52 [W7], 48 [KN62-4 μM], 50 [KN62-20 μM], 59 [RasDN], 62 [RacDN], 60 [G1152], 51 [LatA-20nM], and 60 [LatA-100nM] spines in total, respectively). Error bars indicate ± SEM.

To begin to dissect the molecular mechanisms underlying SynGAP dispersion, we examined the pharmacology of this process (Figures 1E and 1F). The NMDA receptor antagonist (APV), CaM inhibitor (W7), and CaMKII inhibitor (KN62) each completely blocked chemLTP-induced SynGAP dispersion indicating that NMDAR, CaM, and CaMKII function are critical for processes upstream of SynGAP spine dispersion. As reported previously, the CaMKII inhibitor does not block spine enlargement in the acute phase (10 min) but blocked sustained spine enlargement (60 min) (Matsuzaki et al., 2001; Murakoshi et al., 2011). In contrast, we found that inhibition of small G proteins such as Ras and Rac by expressing dominant-negative forms of Ras and Rac, or inhibition of the Rac downstream target protein ROCK, blocked spine enlargement in the sustained phase without affecting SynGAP dispersion (Figures 1E and 1F). These data suggest that SynGAP dispersion is upstream of small G protein activation. Inhibition of actin polymerization by Latrunculin A (LatA) also blocked both the acute and sustained phases of spine enlargement at a high concentration (100 nM) or only the sustained phase at a lower concentration (20 nM). However, both of these treatments failed to block SynGAP spine dispersion (Figures 1E and 1F). Together, these results indicate that chemLTP-induced dispersion of SynGAP from dendritic spines is downstream of NMDAR and CaMKII activation and upstream of small G protein activation and actin polymerization.

Acute Dispersion of SynGAP Predicts the Long-Term Maintenance of Spine Enlargement

Interestingly, we noticed three classes of spines regarding the degree of spine enlargement and dispersion of SynGAP following LTP induction (Figures 2A and 2B). Most spines showed stable responses (57.6% ± 2.9% of total population) and increased in size and dispersed SynGAP in the acute and sustained phase of the response (60 min). Some spines showed no response (27.8% ± 3.4% of total population), while the remaining population (14.6% ± 2.0% of total population) had a transient response in which spines enlarged after LTP induction in the acute phase, but this growth was not sustained. Interestingly, all of the “transient” spines failed to disperse SynGAP in response to LTP induction. This suggests that dispersion of Syn-GAP in the acute phase predicts the long-term stability of the increased spine size.

Figure 2. Degree of SynGAP Dispersion Foretells the Long-Term Spine Enlargement and Maintenance Showing SynGAP as “Negative Synaptic Marker” for Sustained Phase.

Figure 2

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(A) Three typical spine responses during LTP.

(A1) “Stable” synapses (57.6% ± 2.9%) dispersed SynGAP after LTP and spines size enlargement was well retained for sustained phase.

(A2) “Transient” synapses (14.4% ± 2.0%) failed to disperse SynGAP, but spine enlargement in acute phase was normal. All transient spines which failed to disperse SynGAP shrank back to the basal level in the sustained phase.

(A3) Some portions of spines (28.0% ± 3.6%) were “No response” (No Res.) type, where both spine size change and SynGAP dispersion did not occur. (Stable: Spines with volume increased over 15% both at 10 and 60 min; Transient: Spines with volume increased over 15% at 10 min, but back to less than 15% changes at 60 min; No Res.: Spines with volume increased less than <15% at 10 and 60 min). Scale bar, 5 μm.

(B) Relationships between SynGAP dispersion in acute phase and spine volume changes. There is strong and significant positive correlation between SynGAP dispersion in “acute” phase and spine enlargement in “sustained” phase (R2 = 0.73, p < 0.001). There was less positive correlations between SynGAP dispersion and spine enlargement in “acute” phase (R2 = 0.35, p < 0.001), suggesting that SynGAP dispersion in “acute” phase predicts spine enlargement and maintenance for long term rather than in acute phase. Ninety percent prediction bands of trend lines are also displayed. Examples of track changes (arrows) of unique “Stable” spines (S 1–3) and “Transient” spines (T 1–3) are presented (n = 103 spines from nine independent experiments/neurons).

CaMKII Inhibitor KN62 Inhibits SynGAP Dispersion and Converts “Stable” Spines to “Transient” Spines

When we treated neurons with a CaMKII inhibitor, SynGAP dispersion was almost completely blocked during both acute and sustained phases (Figures 3A and 3B). However, as previously reported, CaMKII inhibition did not block acute increases in spine size but only blocked long lasting spine enlargement during the sustained phase (Figures 3A and 3B) (Lee et al., 2009; Matsuzaki et al., 2004). Thus, CaMKII inhibition converted “stable” spines to “transient” spines (Figure 3C). These results suggest that SynGAP dispersion is required for spine enlargement during the sustained phase and that the presence of Syn-GAP in spines inhibits long lasting changes in spine structure.

Figure 3. CaMKII Inhibitor KN62 Maintained SynGAP in Spines and Changed “Stable” to “Transient” Synapses.

Figure 3

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(A) CaMKII inhibitor blocked SynGAP dispersion from spines. Spines were still enlarged in the acute phase but returned to the basal level in the sustained phase. Scale bar, 5 μm.

(B) Time course of spine enlargement and SynGAP dispersion with or without CaMKII inhibitor. Note that the CaMKII inhibitor blocks SynGAP dispersion both in acute and sustained phase, and spine size returned to the basal level in the sustained phase (Ctrl: n = 3 independent experiments/neurons that contain 35 spines, KN62: n = 3 independent experiments/neurons that contain 39 spines). Error bars indicate ± SEM.

(C) Population of “Stable,” “Transient,” and “No response” synapses with or without CaMKII inhibitor. KN-62 dramatically reduced “Stable” synapses and changed them into “Transient” synapses (Ctrl: n = 103 spines from nine independent experiments/neurons, KN62: n = 50 spines from five independent experiments/neurons). Error bars indicate ± SEM.

Phosphorylation of SynGAP at Ser1108 and Ser1138 by CaMKII Regulates Synaptic Localization of SynGAP during LTP through Binding to PSD-95

Two CaMKII phosphorylation sites (Ser780 and Ser1138; numbering according to NP_851606) have been discovered on SynGAP (Carlisle et al., 2008; Oh et al., 2004). We identified an additional well-conserved CaMKII consensus site (S1108) (Figure S3A) that is phosphorylated in vitro and in vivo. Among these three sites, phospho-mimetic mutants of S1108 and S1138 but not S780 affected SynGAP localization and were less enriched in spines (Figure S3B). Thus, we chose the S1108 and S1138 sites for further analysis. We generated double phospho-deficient (2SA; S1108/1138A) and phospho-mimetic (2SD; S1108/1138D) mutants of SynGAP and investigated the synaptic localization of these constructs in the basal or potentiated state (Figures 4A and 4B). The double phospho-mimetic and the single phospho-mimetic mutants of SynGAP were less enriched at synapses, suggesting both phosphorylation events can regulate synaptic dispersion of SynGAP. Moreover, there was no additional reduction in synaptic content of the mutant SynGAP 2SD upon chemLTP (Figures 4A and 4B). In contrast, the phospho-deficient mutant of SynGAP was enriched in spines to the same degree as wild-type (WT) under basal conditions. Upon LTP induction there was no dispersion of SynGAP 2SA (Figures 4A and 4B), suggesting that dynamic changes in SynGAP phosphorylation are required for dispersion of SynGAP. Collectively, these findings indicate that dynamic modulation of these two phosphorylation sites plays a major role in SynGAP synaptic localization.

Figure 4. Phosphorylation of SynGAP Regulates Its Synaptic Localization.

Figure 4

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(A) Localization of SynGAP WT, phospho-deficient (2SA; S1108/1138A), and phospho-mimetic (2SD; S1108/1138D) before and after LTP stimulus. Note that 2SA failed to be dispersed upon LTP and cells expressing S2A showed a failure of spine enlargement. 2SD was not concentrated even in basal state, and spines were already enlarged.

(B) Quantification of (A) (n = 5 independent experiments/neurons in each condition that contains 48 [WT], 59 [2SA], and 50 [2SD] spines in total, respectively) showing relative SynGAP enrichment and spine size change upon LTP for each SynGAP construct transfected. Two-way ANOVA followed by Tukey’s post hoc test was performed (for SynGAP enrichment [left panel], Phospho-mutation F(2, 24) = 20.82, p < 0.001; chemLTP F(1, 24) = 7.98, p < 0.001; Interaction F(2, 24) = 4.49, p < 0.001; for Spine size area [right panel], Phospho-mutation F(2, 24) = 45.23, p < 0.001; chemLTP F(1, 24) = 14.07, p < 0.001; Interaction F(2, 24) = 17.18, p < 0.001). Error bars indicate ± SEM.

(C) Rapid phosphorylation at Ser1108 and 1138 upon LTP.

(D) PSD fractionations from neurons with basal state or after LTP. Note that SynGAP was dispersed from PSD fraction and moved to triton-soluble synaptosomes (Syn/Tx). Phosphorylated SynGAP after LTP was mainly located in cytosolic fraction (S2) and Syn/Tx. (PNS, postnuclear supernatant; P2, membrane fraction; S2, cytosolic fraction; Syn, total synaptosomal fraction; Syn/Tx, triton soluble synaptosomal fraction; PSD, postsynaptic density fraction.)

(E) HEK cells cotransfected with myc-tagged SynGAP and constitutive active CaMKII (T286D) were lysed and blotted with indicated antibodies. Only WT SynGAP was phosphorylated by active CaMKII.

(F) Coimmunoprecipitation of PSD-95 and SynGAP from transfected HEK293 cells with or without active (T286D) or inactive (K42M) CaMKII constructs. Myc-PSD95 coprecipitates SynGAP (Lane4). Interaction was disrupted by active CaMKII T286D (Lane6) but not inactive CaMKII (Lane5).

(G) Coimmunoprecipitation of PSD-95 and various SynGAP constructs expressed in HEK293 cells. Interaction was diminished in phospho-mimetic SynGAP 2SD (Lane 6) compared to WT (Lane 4) or phospho-deficient (Lane 5) constructs.

(H) Rapid dissociation of SynGAP from PSD-95 upon LTP stimulus in neurons. During LTP, levels of phosphorylation at S1108 and 1138 were increased, and SynGAP was concurrently released from PSD-95 (Lanes 2–5). Inhibition of CaMKII by KN62 blocked this dissociation (Lane 6).

We raised phospho-specific antibodies against S1108 and S1138 in SynGAP in order to examine the changes in phosphorylation upon LTP induction. CaMKII inhibition or lambda-phosphatase treatment abolished antibody recognition demonstrating the specificity of our phospho-antibodies (Figure S3C). Indeed, phosphorylation of each of these sites was increased upon LTP, and these effects were blocked by the CaMKII inhibitor KN62 (Figures 4C and S4A). Biochemical PSD fractionation showed that the population of phosphorylated SynGAP was efficiently dispersed from the PSD upon LTP (Figure 4D and S4B). CaMKII-dependent phosphorylation of SynGAP was further confirmed by coexpressing constitutively active CaMKII (T286D) with either WT or phospho-deficient SynGAP in HEK cells. CaMKII T286D dramatically increased phosphorylation of S1108 and S1138 specifically upon coexpression with WT SynGAP, but not with the 2SA mutant SynGAP (Figure 4E). These results indicate S1108 and S1138 are sites of CaMKII-dependent phosphorylation and further support the specificity of our antibodies.

Next, we investigated how SynGAP phosphorylation may regulate its synaptic dispersion. SynGAP binds to PSD95 and SAP102 two of the major synaptic scaffolding proteins at excitatory synapses (Kim et al., 1998). To examine if phosphorylation regulates the interaction between SynGAP and PSD95, we co-transfected myc-PSD95 and GFP-SynGAP with either inactive (K42M) or constitutively active (T286D) CaMKII mutants in HEK293 cells and examined their interaction using coimmuno-precipitation assays (Figure 4F). Expression of constitutively active CaMKII disrupted the interaction between PSD95 and SynGAP (lanes 4–6). However, active CaMKII failed to disrupt the interaction between PSD95 and SynGAP 2SA phospho-deficient mutant (2SA; lanes 7–9). We also directly confirmed that phospho-mimetic mutant SynGAP had a weaker affinity for PSD95 (Figure 4G). In addition, isothermal titration calorimetric in vitro using purified proteins showed that the phospho-mimetic SynGAP had a reduced affinity with PSD95, compared with WT or phospho-deficient SynGAP (Figure S3D). Taken together, these results suggest that phosphorylation of SynGAP at S1108/1138 by CaMKII triggers its dissociation from PSD95, thereby promoting the dispersion of phosphorylated SynGAP from spines. Finally, we investigated the time course of the changes in SynGAP phosphorylation and PSD95 interaction after chemLTP induction (Figures 4H and S4C). CaMKII rapidly phosphorylated S1108 and S1138 upon LTP induction and Syn-GAP was simultaneously and rapidly released from the PSD95 complex. Moreover, this dissociation was CaMKII dependent, as KN62 completely blocked this process (Figure 4H). To confirm if this dispersion occurs in vivo, we treated mice with electroconvulsive therapy (ECT) stimulation and examined SynGAP levels in PSD fraction (Figure S5). Indeed, ECT stimulation reduced SynGAP levels in PSD, suggesting that neuronal activity in vivo facilitates this dispersion process.

Phosphorylation of SynGAP Induced SynGAP Dispersion from Spines and Increases Synaptic Ras Activity during LTP

Several lines of evidence indicate that SynGAP is required for LTP expression and that SynGAP phosphorylation is likely key to this process. Thus, to investigate the physiological role of rapid SynGAP phosphorylation and dispersion upon LTP, we first examined how SynGAP phosphorylation regulates Ras activity during LTP. We performed molecular replacement experiments by knocking down endogenous SynGAP using shRNA and then rescued the neurons with WT or phosphor-mutant Syn-GAP constructs (Figure 5A). We screened nine candidate shRNAs and selected shRNA #5 for use in further experiments (Figure S2C). We confirmed this shRNA#5 effectively knocked down endogenous SynGAP in neurons (Figure S2D). To measure Ras activity, we employed Raichu-Ras, a FRET-based sensor for active Ras (Penzes and Jones, 2008; Penzes et al., 2009). The Raichui-Ras probe contains Ras and the Raf-Ras binding domain (RBD) aligned in tandem and flanked by the fluorescent proteins CFP and YFP. Upon binding GTP, activated Ras interacts with the Raf domain causing a structural change that brings CFP and YFP into close proximity and thereby increases FRET efficiency. In control neurons (Figure 5A1; pLKO ctrl), Ras was activated in response to LTP (from 0.17 ± 0.01 to 0.76 ± 0.04 at 10 min and 0.42 ± 0.03 at 60 min for FRET efficiency). However, in SynGAP knockdown cells (Figure 5A2; pLKO shRNA-SG#5), the basal Ras activity was already elevated compared to control conditions (0.73 ± 0.05 for FRET efficiency), and there was no further increase after LTP, showing that SynGAP knockdown results in occlusion of LTP-induced changes in Ras activity. Expression of shRNA-resistant SynGAP WT rescued these deficits by reducing basal Ras activity (Figure 5A3; pLKO shRNA-SG#5, SynGAP-WT). Interestingly, phospho-deficient SynGAP expression (Figure 5A4; pLKO shRNA-SG#5, SynGAP-2SA) also lowered basal Ras activity but failed to increase Ras activity in response to LTP, likely due to phospho-deficient SynGAP’s retention at synapses (Figure 4A, SynGAP-2SA). In contrast, phospho-mimetic SynGAP (Figure 5A5; pLKO shRNA-SG#5, SynGAP-2SD) failed to decrease basal Ras activity and also occluded LTP-induced Ras activation. This effect is likely due to the reduced synaptic targeting of SynGAP S2D (Figure 4A, SynGAP-2SD). We investigated if phosphorylation of SynGAP affects RasGAP activity in vitro by overexpressing SynGAP with H-Ras in HEK cells (Figure S6). SynGAP WT, 2SA, or 2SD reduced active Ras-GTP levels, suggesting that phosphorylation does not change enzymatic RasGAP activity itself; rather, localization of SynGAP might regulate active Ras levels in spines. Collectively, these results indicate that dynamic changes in SynGAP phosphorylation status are a prerequisite for Ras activation during LTP in hippocampal neurons.

Figure 5. Phosphorylation of SynGAP Regulates Ras Activity during LTP.

Figure 5

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(A) Imaging of Raichu-Ras, a FRET-based sensor for cellular Ras activity with or without SynGAP knockdown as well as rescued by WT or phospho-mutants. Error bars indicate ± SEM.

(A1) Control: Upon LTP stimulus, synaptic Ras activity was increased.

(A2) Knockdown of SynGAP: (shRNA-SG#5) increased the basal Ras activity, thus occluding the Ras activity change upon LTP.

(A3) Knockdown of SynGAP rescued with WT: shRNA-resistant SynGAP WT rescued this occlusion.

(A4) Knockdown of SynGAP rescued with phospho-deficient SynGAP 2SA: shRNA-resistant SynGAP 2SA failed to rescue Ras activation upon LTP, likely because this mutant could not be dispersed, since it cannot be phosphorylated.

(A5) Knockdown of SynGAP rescued with phospho-mimetic SynGAP 2SD: shRNA-resistant SynGAP 2SD failed to rescue, likely because 2SD could not localize to spines (N = 7 independent experiments/neurons respectively that contain 72 [A1], 81 [A2], 75 [A3], 76 [A4], and 95 [A5] spines in total, respectively). Two-way ANOVA followed by Tukey’s post hoc test was performed (shRNA+Rescue F(4, 90) = 76.64, p < 0.001; Time F(2, 90) = 55.79, p < 0.001; Interaction F(8, 90) = 10.07, p < 0.001). Scale bar, 2 μm.

(B) Amount of GTP bound (active) Ras was quantified by pull-down assay using Raf-RBD (Ras effector domain) beads with or without electroporation of SynGAP knockdown constructs and shRNA-resistant SynGAP rescues (N = 6 independent experiments). Two-way ANOVA followed by Tukey’s post hoc test was performed (shRNA+Rescue F(4, 75) = 63.53, p < 0.001; Time F(2, 75) = 46.56, p < 0.001; Interaction F(8, 75) = 7.526, p < 0.001). Error bars indicate ± SEM.

We also examined the activity changes of Ras during LTP using biochemical methods (Figure 5B). Beads covalently bound with Raf-RBD were used for precipitation of active GTP-Ras after lysis of hippocampal neurons. We electroporated the pLKO-shRNA knockdown construct in conjunction with SynGAP rescue constructs using an Amaxa electroporator. Neurons were lysed prior to stimulation and either 10 or 60 min after LTP induction. We confirmed knockdown of SynGAP in pLKO-shRNA#5-transfected neurons (lowered expression in 2 compared to Figure 5B1) as well as expression of SynGAP rescue constructs (Figure 5B3–5B5). Under control conditions (Figure 5B1), active Ras-GTP was augmented in response to LTP. SynGAP knockdown caused an increase in basal Ras-GTP forms, again suggesting that SynGAP knockdown occluded the LTP-induced increase in Ras activity (Figure 5B2). SynGAP WT expression rescued this effect (Figure 5B3), whereas expression of phospho-deficient S2A blocked the response to LTP (Figure 5B4) and phospho-mimetic S2D again resulted in occlusion of any effect of LTP induction (Figure 5B5). Thus, our biochemical experiments corroborate our results from Raichu-Ras experiments (Figure 5A). These results suggest that the removal of SynGAP from synapses either by knocking down or phospho-mimetic mutants is sufficient to activate Ras.

Dynamic Changes of SynGAP Phosphorylation Status Regulates AMPAR Trafficking and Spine Enlargement during LTP

It is widely believed that AMPAR recruitment to synapses is the molecular basis of increased synaptic strength in response to LTP induction. Spine enlargement occurs concurrently with AMPAR recruitment leading to correlated spine size and synaptic transmission (Matsuzaki et al., 2001). We next examined the role of SynGAP in these plastic changes using techniques similar to those described above. To visualize spine structure and AMPAR trafficking, we transfected mCherry (Figure 6A) and SEP-GluA1 (Figure 6B) with pLKO-shRNA and SynGAP rescue constructs. Under control conditions, spines were enlarged (Figure 6A1) and AMPARs were recruited to spines after LTP induction (Figure 6B1). SynGAP knockdown caused enlarged spines (Figure 6A2) with concentrated levels of AMPARs (Figure 6B2) in the basal state, which occluded further increases in spine size and receptor content upon LTP induction. SynGAP WT expression rescued this phenotype (Figures 6A3 and 6B3). Phospho-deficient SynGAP decreased spine size and receptor content but failed to respond to the chemLTP stimulus (Figure 6A4 and 6B4), likely due to the deficits in SynGAP dispersion. In contrast, the phospho-mimetic mutant did not lower basal spine size (Figure 6A5) or receptor content (Figure 6B5) compared to the knockdown-only condition. These results suggest that dynamic changes in SynGAP phosphorylation and spine dispersion regulate spine size and AMPAR content in response to LTP induction (Figure 6).

Figure 6. Phosphorylation of SynGAP Regulates Spine Enlargement and GluA1 Trafficking during LTP.

Figure 6

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(A and B) (A) Spine enlargement and (B) GluA1 trafficking upon LTP with or without SynGAP ([A1] and [B1]) Control: (shRNA-Ctrl), ([A2] and [B2]) Knockdown of SynGAP: (shRNA-SG#5), and ([A3] and [B3]) Knockdown of SynGAP rescued with WT: shRNA-resistant SynGAP WT rescue. ([A4] and [B4]) Knockdown of SynGAP rescued with phospho-deficient SynGAP 2SA; ([A5] and [B5]) Knockdown of SynGAP rescued with phospho-mimetic SynGAP 2SD. N = 7 independent experiments/neurons that contain 70, 66, 68, 72, and 78 spines in total, respectively. Two-way ANOVA followed by Tukey’s post hoc test was performed (shRNA+Rescue F(4, 90) = 68.76 [A]/73.30 [B], p < 0.001 [A, B]; Time F(2, 90) = 22.54 [A]/22.75 [B], p < 0.001 [A, B]; Interaction F(8, 90) = 8.88 [A]/10.90 [B], p < 0.001 [A, B]). Scale bar, 2 μm. Error bars indicate ± SEM.

Dynamic Changes of SynGAP Phosphorylation Controls Synaptic Strength in Single Spines after LTP Induction

We also examined the roles of SynGAP dispersion and phosphorylation on synaptic strength using glutamate uncaging techniques before and after LTP induction. Caged glutamate was photolysed to mimic presynaptic glutamate release and evoke EPSCs (uEPSC) by delivering a millisecond two-photon laser pulse to individual spines. We measured uEPSCs at each synapse before and after LTP to compare these changes at each synapse (Figure 7A). Representative spine images and uncaging locations are shown in the upper panel. Under control conditions, the uEPSC was increased by 68.1% ± 12.9% in response to chemLTP (Figure 7A1). SynGAP knockdown eliminated these LTP-induced changes, likely due to the occlusion of LTP-induced changes in Ras activity described above (Figure 7A2). Similar to the results on spine size and Ras activity, SynGAP WT rescued this phenotype (Figure 7A3), whereas phospho-deficient (Figure 7A4) and phospho-mimetic (Figure 7A5) failed to rescue LTP induced changes in synaptic strength, likely because of their deficiency in SynGAP spine dispersion or reduced synaptic localization, respectively. We investigated if SynGAP phosphorylation affects the structural plasticity in CA1 pyramidal neurons in organotypic hippocampal culture models during uncaging evoked ssLTP (Figure S7A). After invoking ssLTP, spines were enlarged for at least 45 min postinduction while SynGAP knockdown abolished this structural plasticity. Notably, WT Syn-GAP rescued this deficit whereas 2SA and 2SD did not (Figures S7B and S7C). Taken together, these results also suggest that SynGAP dispersion upon phosphorylation plays a key role in synaptic potentiation after LTP induction.

Figure 7. Phosphorylation of SynGAP Regulates Synaptic Strength during LTP.

Figure 7

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(A) Uncaging EPSC (uEPSC) changes upon LTP. Caged-glutamate was uncaged at each spine before or after chemLTP induction, and the changes were compared (n = 24, 20, 26, 20, and 20 spines from three independent experiments/neurons, respectively; shRNA+Rescue F(4, 105) = 14.49, p < 0.001). (A1–A5) Constructs were the same as Figures 5 and 6. Scale bar, 5 μm.

(B) Location of SynGAP nonsense mutation found in human intellectual disability patients that is used in this study (S738X, L813RfsX22 [frame shift at L813 position that leads to premature stop codon after 22 amino acids], and Q893RfsX184 [frame shift at Q893 position that leads to premature stop codon after 184 amino acids]). Note that all mutants lack the phosphorylation sites by CaMKII.

(C) Synaptic targeting of SynGAP mutants found in the patients of human intellectual disability patients. Scale bar, 2 μm.

(D) Synaptic dispersions of SynGAP and spine enlargements were abolished in the mutants of human intellectual ability.

(D1) Representative images of SynGAP localization and spine shapes during LTP.

(D2) Quantification of SynGAP dispersion and spine enlargement during LTP. Two-way ANOVA followed by Tukey’s post hoc test was performed (n = 4 independent experiments/neurons that contain 39, 41, 45, and 42 spines, respectively. Mutation F(3, 24) = 22.92 [enrichment]/5.671 [size], p < 0.001; chemLTP F(1, 24) = 15.54[enrichment]/7.05[size], p < 0.001; Interaction F (3, 24) = 11.06[enrichment]/5.50 [size], p < 0.001). Scale bar, 1 μm.

Human Truncation Mutations in SynGAP Associated with ID and ASD Eliminate SynGAP Synaptic Targeting and Synaptic Dispersion during LTP

Finally, recent genetic studies have shown that mutations in Syn-GAP are associated with ID and ASDs (Berryer et al., 2012; Hamdan et al., 2011; Hamdan et al., 2009). Several of these mutations found in ID and ASD patients are C-terminal deletions in SynGAP that truncate the region containing the CaMKII sites that regulate synaptic dispersion described in this manuscript. To test the effect of these mutations on SynGAP synaptic targeting, we expressed three different truncation mutants (Figure 7B; S738X, L813RfsX22, and Q893RfsX184) and examined their subcellular localization and synaptic dispersion during chemLTP. All three truncation mutants of SynGAP were less enriched at synapses, indicating that these mutations inhibit the proper synaptic targeting of SynGAP (Figure 7C). Moreover, there was no additional reduction in synaptic content of the truncated SynGAP mutants upon chemLTP (Figure 7D). Additionally, these mutant SynGAP disrupted spine enlargements upon LTP even though endogenous SynGAP was intact, which indicates that these mutants have dominant-negative effects on synaptic plasticity process. We confirmed these three constructs expressed almost equally in cells (Figure S7D). These results suggest that SynGAP synaptic targeting and CaMKII-dependent SynGAP dispersion is disrupted in these cases of ID and ASD and that the etiology of Syngap1-deficient human ID/ASD may not be due to an insufficiency of GAP activity but due to a lack of synaptic targeting and CaMKII-dependent SynGAP dispersion during LTP, affecting synaptic plasticity and cognition.

SynGAP Synaptic Dispersion Is Required for LTP

Finally, to more directly link phosphorylation, SynGAP dispersion, and receptor trafficking/spine enlargement, we made a mutant SynGAP that is artificially targeted to spines in both the basal and chemLTP states (Figures S8 and 8A). We added a glycine linker and the NR2B C-terminal PDZ ligand that maintained SynGAP targeting to spines even during chemLTP (Figure S8). This WT:GL-NR2B construct decreased spine size and receptor content in the basal state (Figure 8A) and blocked spine enlargement and receptor insertion during LTP with phosphorylation sites intact (Figures 8A and S8D). These results suggest that SynGAP relocation is required to trigger its downstream effects.

Figure 8. CaMKII Phosphorylation of SynGAP Transmit Signals to Small G Protein.

Figure 8

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(A) Top panel: Artificially highly concentrated SynGAP reduced spine size and SEP-GluA1 at the basal state and blocked spine enlargement and insertion of SEP-GluA1 into spines during chemLTP. Dynamics of AMRAR trafficking (SEP-GluA1 channel) and spine enlargement (mCherry channel) during chemLTP were observed when endogenous SynGAP was replaced with WTRes#5 GFP-SynGAP or with WTRes#5:GL-NR2BC GFP-SynGAP. Yellow arrows indicate spines with newly inserted SEP-GluA1 upon chemLTP, while green arrowheads indicate spines without newly inserted SEP-GluA1 upon chemLTP. Note that the population of the spines with green arrowheads was increased with WTRes#5:GL-NR2BC compared to WT Res#5 SynGAP replacement. Scale bar, 2 μm. Bottom panel: Quantification of the relative ratio of SEP-GluA1 per spine before/after chemLTP with WT Res#5 or WTRes#5:GL-NR2BC SynGAP replacement. Error bars indicate ± SEM.

(B) Schematic diagram of relationships between CaMKII activity and small G protein activation that leads to cellular changes upon LTP.

(C) Schematic model of the cellular events that link CaMKII activity, SynGAP dispersion, and small G protein activation.

DISCUSSION

CaMKII and Small G-Protein Dependence of LTP Induction

NMDAR-dependent calcium influx and activation of CaMKII are well known to be required for the induction of LTP (Lisman et al., 2012). However, the downstream CaMKII substrates and signaling pathways that actually invoke the wide variety of cellular events that occur during LTP, including AMPAR recruitment to synapses and reorganization of the cytoskeleton for spine enlargement, remain elusive. Recent studies have shown that several small G proteins such as Ras, Rac1, Cdc42, and RhoA are essential components to induce synaptic changes during LTP. The Ras-ERK or Ras-PI3K pathways trigger AMPAR recruitment (Man et al., 2003; Patterson et al., 2010; Zhu et al., 2002), while the Rac1-PAK or RhoA-ROCK pathways are essential for the induction and maintenance of spine enlargement (Corbetta et al., 2009; Penzes et al., 2001; Saneyoshi et al., 2008; Tashiro and Yuste, 2008; Xie et al., 2007). Inhibitors of CaMKII or small G proteins block the induction of LTP (Lisman et al., 2012; Zhu et al., 2002). Studies of the time course of LTP induction clearly show that CaMKII activation (~10 s) occurs ahead of the activation of small G proteins (~1 min) (Murakoshi et al., 2011), suggesting that CaMKII activation is upstream of small G protein activation. However, the actual molecular and mechanistic link between CaMKII and the activation of these small G proteins is unclear.

Synaptic Dispersion of SynGAP Is Required for LTP Induction

In the present study, we investigated whether SynGAP, a Ras-GAP protein enriched in synapses, is a key CaMKII substrate for LTP induction and provides a mechanistic “missing link” between CaMKII and small G protein activation. SynGAP was identified as a protein that is associated with the synaptic scaffolding protein SAP102 and other MAGUK family members and is highly enriched at excitatory synapses and in PSD fractions (Chen et al., 1998; Kim et al., 1998). SynGAP is known to be a CaMKII substrate (Carlisle et al., 2008; Oh et al., 2004), and the addition of active CaMKII to biochemically purified PSD fractions has been reported to suppress RasGAP activity in the PSD; however, it is not clear if this effect was mediated through SynGAP or another CaMKII target (Chen et al., 1998), and this result was also not reproducible (see erratum of Chen et al., 1998). A subsequent report showed that phosphorylation of SynGAP increased its RasGAP activity using an in vitro assay (Carlisle et al., 2008; Oh et al., 2004). These reports suggested phosphorylation of SynGAP at S1138 by CaMKII likely increases its RasGAP activity, thus inhibiting Ras signaling. This result suggests that CaMKII phosphorylation should inhibit Ras signaling and LTP induction and is inconsistent with CaMKII phosphorylation of SynGAP playing a positive role in the induction of LTP.

In contrast, our results demonstrate that CaMKII phosphorylation has dramatic effects on the synaptic localization of SynGAP. Our results show that the dispersion of SynGAP from synapses was dependent on NMDAR and CaMKII activation. Compared to previous studies, we could not confirm changes in RasGAP activity upon phosphorylation; rather, phosphorylation profoundly affects SynGAP targeting. However, we could not exclude the possibility that phosphorylation sites other than S1108 and S1138 regulate SynGAP enzymatic activity. SynGAP has many splice variants consisting of combinations of N-terminal variants A, B, C, and D isoforms as well as C-tail α1, α2, β1, β2, β3, β4, and γ isoforms (Li et al., 2001; McMahon et al., 2012). We used GFP-SynGAP Bα1 isoform in our live imaging; however, for endogenous staining and biochemical PSD fractionation assays, we used an antibody that detects all of SynGAP splice variants. We confirmed SynGAP dispersion also occurred using endogenous staining and PSD fractionation, suggesting that most SynGAP variants share this dispersion mechanism.

To examine the physiological effects of CaMKII phosphorylation of SynGAP, we performed molecular replacement experiments where we knocked down endogenous SynGAP and rescued it with various SynGAP mutations and investigated the role of phosphorylation on Ras activity, spine enlargement, and receptor trafficking. We could rescue SynGAP knockdown phenotype with shRNA-resistant WT SynGAP but not phospho-deficient or phosphomimetic SynGAP showing that dynamic changes of phosphorylation (and thus relocation) are essential for these LTP-related responses in neurons. To confirm the causal link where SynGAP relocation by phosphorylation triggers downstream events, we used an artificial SynGAP construct that remained in spines even during chemLTP by adding a glycine linker plus the NR2B C-tail (GL-NR2BC) at the end of SynGAP. Rescue experiments with GL-NR2BC blocked downstream events such as spine enlargement and receptor trafficking, suggesting that SynGAP relocation is required for triggering the downstream events. We cannot exclude the possibility that phosphorylation might regulate other cellular process than the SynGAP relocation and GAP activity to evoke synaptic plasticity. However, with the SynGAP-NR2B experiment, we think the relocation of SynGAP plays substantial role in triggering downstream events.

Collectively, these data suggest that SynGAP dispersion constitutes a key missing link between CaMKII and small G protein activation during LTP that is important for synaptic plasticity (see schematic model in Figures 8B and 8C).

Downstream Signaling from SynGAP and Its Functional Consequences

Although we have clearly demonstrated that SynGAP is a key CaMKII substrate required for LTP induction and maintenance, the key signaling components down stream of SynGAP that are required for LTP expression are still not clear. The major downstream target of SynGAP is Ras and the ERK pathway (Chen et al., 1998; Kim et al., 1998, 2003; Komiyama et al., 2002; Rumbaugh et al., 2006), and many studies have indicated that the ERK pathway is critical for LTP expression (Kim et al., 2005b; Patterson et al., 2010; Thomas and Huganir, 2004; Zhu et al., 2002). SynGAP has also been reported to have a promiscuous GTPase activity that directly or indirectly regulates the small G-proteins Rap, Rac1, and Rab5 (Carlisle et al., 2008; Krapivinsky et al., 2004; Pena et al., 2008; Tomoda et al., 2004), which may in turn regulate the actin cytoskeleton and membrane trafficking involved in LTP-induced increases in spine size and AMPAR recruitment. For example, SynGAP has been show to indirectly regulate Rac1 and PAK phosphorylation of Cofilin, which controls actin polymerization and synaptic spine size. The ability of SynGAP to regulate Rac1 may result from the direct interaction and activation of Tiam1, a RacGEF, by Ras (Lambert et al., 2002). Alternatively, Ras can activate PI3K, leading to PIP3 production that stimulates Tiam1 activity (Fleming et al., 2004). Whatever the pathway, there is a significant increase in both Ras and Rac1 activity in SynGAP heterozygote mice (Carlisle et al., 2008).

Other Possible Bridging Molecules between CaMKII and Small G Proteins

Additional key molecules that might transmit CaMKII signals to small G proteins during NMDAR-dependent plasticity are Kalirin-7 and Tiam1. It has been reported that phosphorylation of Kalirin-7 at Thr95 by CaMKII increases its Rac1 GEF activity to induce Rac1 activation (Xie et al., 2007). Another line of evidence showed that NMDAR-dependent phosphorylation of Tiam1 regulates Rac1 activity during the bath application of glutamate (Tolias et al., 2005). Although there is likely significant crosstalk between these pathways, SynGAP, Kalirin-7, and Tiam1 may be involved in different plasticity processes by differentially regulating Ras, Rac1, Rho, or Cdc42 signaling and thus have differential effects on spine maintenance, enlargement, synaptogenesis, and LTP or LTD. In addition, these different GEFs may play differential roles in distinct brain regions or developmental stages. Recent reports suggested SynGAP might play a more prominent role in the regulation of synaptic connectivity during neonatal brain development (Clement et al., 2012) and critical-period synaptic plasticity (Clement et al., 2013). This is reasonable since SynGAP expression is high in very early in life (Liu et al., 2012) and plays an important role in cortical synaptic development (Clement et al., 2013), whereas Kalirin-7 expression is increased in adulthood (Penzes et al., 2008).

SynGAP and Cognition

In summary, SynGAP plays a critical role in development, synaptic plasticity, learning, and memory, as well as in several neurological and psychiatric diseases. SYNGAP1 knockout mice have impaired LTP induction as well as learning and memory deficits (Kim et al., 2003; Komiyama et al., 2002). All SYNGAP1 mutations found in ASD/ID require only monoallelic loss of function (i.e., haploinsufficiency) to produce these disorders (Berryer et al., 2012; Hamdan et al., 2009, 2011; Ozkan et al., 2014). Our results indicate that CaMKII-dependent SynGAP dispersions were abolished in affected protein (S738X, L813RfsX22, and Q893RfsX183). These findings strongly suggest that the location and synaptic dispersion of SynGAP in neurons and its regulation by CaMKII play a central role in the determination of synaptic connectivity, plasticity, cognition, and social behavior in humans.

EXPERIMENTAL PROCEDURES

Reagents

All restriction enzymes were from New England Biolabs. Chemicals were obtained from SIGMA-Aldrich unless otherwise specified. APV, W7, KN62, Glycyl-H-1152, and Latrunculin A were from TOCRIS Bioscience. DNA sequencing was performed at the Johns Hopkins University School of Medicine Sequencing Facility.

Animal Care

All animals were treated in accordance with the Johns Hopkins University Animal Care and Use Committee Guidelines.

Neuronal Cultures, Induction of LTP, and Time-Lapse Imaging

Hippocampal neurons from embryonic day 18 (E18) rats were seeded on poly-L-lysine coated coverslips. The cells were plated in Neurobasal media (GIBCO) containing 50 U/ml penicillin, 50 mg/ml streptomycin, and 2 mM GlutaMax supplemented with 2% B27 (GIBCO) and 5% horse serum (Hyclone). At DIV6, cells were maintained in glia-conditioned NM1 (neurobasal media with 2 mM GlutaMax, 1% FBS, 2% B27, 1 × FDU, 5 mM uridine [SIGMA F0503], and 5 mM 5-Fluro-2′-deoxyuridine [SIGMA U3003]). Cells were transfected at DIV17-19 with LipofectAMINE2000 (Invitrogen) in accordance with manufacture’s manual. After 2 days, cells were perfused with basal ECS (143 mM NaCl, 5 mM KCl, 10 mM HEPES [pH 7.42], 10 mM Glucose, 2 mM CaCl2, 1 mM MgCl2, 0.5 μM TTX, 1 μM Strychnine, and 20 μM Bicuculline), and time-lapse images were captured with either LSM510 or Spinning disk confocal system (Zeiss). Following 5–10 min of basal recording, cells were perfused with 10 ml of glycine/0 Mg ECS (143 mM NaCl, 5 mM KCl, 10 mM HEPES [pH 7.42], 10 mM Glucose, 2 mM CaCl2, 0 mM MgCl2, 0.5 μM TTX, 1 μM Strychnine, 20 μM Bicuculline, and 200 μM Glycine) for 10 min. See Supplemental Information for detailed procedures.

Supplementary Material

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Download video file (46MB, avi)

Highlights.

  • SynGAP is dispersed from synapses by NMDAR and CaMKII activation

  • SynGAP dispersion activates synaptic Ras and induces LTP

  • Rapid dispersion of SynGAP predicts the maintenance of potentiated synapse

  • SynGAP mutations found in intellectual disability disrupt this mechanism

Acknowledgments

We thank all members of the Huganir lab for discussion and support throughout this work—especially Drs. Graham Diering, Ingie Hong, Lenora Volk, Olof Lagerlöf, and Natasha Hussain for critical reading of the manuscript. Dominant-negative forms of Ras (RasDN; S17N) and Rac1 (Rac1DN; T17N) were generous gift from Dr. Takanari Inoue (Johns Hopkins University). Raichu-Ras was generous gift from Dr. Michiyuki Matsuda (Kyoto University). This work was supported by grants from National Institute of Health (MH64856, NS036715 to R.L.H.), JSPS (Y.A.), the Research Grant Council of Hong Kong (AoE/M09/12 to M.Z.), and the Howard Hughes Medical Institute (R.L.H.).

Footnotes

SUPPLEMENTAL INFORMATION

Supplemental Information includes eight figures, one movie, and Supplemental Experimental Procedures and can be found with this article online at http://dx.doi.org/10.1016/j.neuron.2014.12.023.

AUTHOR CONTRIBUTIONS

Y.A. and R.H. designed research. Y.A. and M.L.Z. performed experiments. Y.A., M.L.Z., M.Z., and R.H. contributed new reagents/analytic tools. Y.A., M.L.Z., M.Z., and R.H. analyzed data. Y.A. and R.H. wrote the paper.

Under a licensing agreement between Millipore Corporation and The Johns Hopkins University, R.L.H. is entitled to a share of royalties received by the University on sales of products described in this article. R.L.H. is a paid consultant to Millipore Corporation. The terms of this arrangement are being managed by The Johns Hopkins University in accordance with its conflict-of-interest policies.

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