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. 2009 Oct;12(10):1275-84.
doi: 10.1038/nn.2386. Epub 2009 Sep 6.

Epac2 induces synapse remodeling and depression and its disease-associated forms alter spines

Kevin M Woolfrey  1 , Deepak P Srivastava, Huzefa Photowala, Megumi Yamashita, Maria V Barbolina, Michael E Cahill, Zhong Xie, Kelly A Jones, Lawrence A Quilliam, Murali Prakriya, Peter Penzes
Affiliations

Epac2 induces synapse remodeling and depression and its disease-associated forms alter spines

Kevin M Woolfrey et al. Nat Neurosci. 2009 Oct.

Abstract

Dynamic remodeling of spiny synapses is crucial for cortical circuit development, refinement and plasticity, whereas abnormal morphogenesis is associated with neuropsychiatric disorders. We found that activation of Epac2, a PKA-independent cAMP target and Rap guanine-nucleotide exchange factor (GEF), in cultured rat cortical neurons induced spine shrinkage, increased spine motility, removed synaptic GluR2/3-containing AMPA receptors and depressed excitatory transmission, whereas its inhibition promoted spine enlargement and stabilization. Epac2 was required for dopamine D1-like receptor-dependent spine shrinkage and GluR2 removal from spines. Epac2 interaction with neuroligin promoted its membrane recruitment and enhanced its GEF activity. Rare missense mutations in the EPAC2 (also known as RAPGEF4) gene, previously found in individuals with autism, affected basal and neuroligin-stimulated GEF activity, dendritic Rap signaling, synaptic protein distribution and spine morphology. Thus, we identify a previously unknown mechanism that promotes dynamic remodeling and depression of spiny synapses, disruption of which may contribute to some aspects of disease.

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Figures

Figure 1
Figure 1
Epac2 is present in synapses in cultured cortical pyramidal neurons. (a) Domain structure of Epac2. (b) Quantitative PCR analysis of Epac1 and Epac2 mRNA in cortical neurons (div 28) demonstrates the relative enrichment of Epac2. (c) Western blot detection of Epac2 in rat forebrain homogenate. (d) Localization of Epac2 in cultured cortical pyramidal neurons (div 28); colocalization with GluR2/3. White arrowheads, colocalization; green arrowheads, non-colocalized Epac2 puncta. (e) Double immunofluorescence with antibodies for synaptic proteins bassoon, NR1 and PSD-95. (f) Epac2 activation by 8-CPT (50 µM, 1 hr) in cortical neurons; endogenous Rap activation was measured. Fold Rap activation compared to control: 1.57±0.11 fold increase, *P<0.001, n = 4. (g) Specificity of 8-CPT for Epac2 in neurons: effect of 8-CPT or BDNF on CREB phosphorylation, n = 3 (h) Effect of incubation with 8-CPT (50 µM, 1 hr) on the phosphorylation of the Rap target B-Raf in situ in pyramidal neuronal dendrites. (i) Effect of incubation with 8-CPT (50 µM, 1 hr) on B-Raf phosphorylation in dendrites of neurons expressing Epac2 RNAi. (j) Quantification of B-Raf fluorescence intensities in h-i (*P<0.001), n = 9–12 cells per condition, 3 experiments. Error bars: s.e.m. Scale bars: d, 15 µm; d-zoom, e, h, i, 5 µm.
Figure 2
Figure 2
Epac2 activation induces dendritic spine shrinkage, reduces presynaptic contact and enhances spine motility and turnover. (a) Effect of incubation with 8-CPT (50 µM, 1 hr), in absence or presence of Epac2 RNAi or rescue RNAi, on spine morphology. (b) Quantification of average spine areas in a; area (µm2): control, 0.92±0.04; 8-CPT, 0.68±0.04; Epac2 RNAi, 1.10±0.05; Epac2 RNAi+8-CPT, 1.04±0.07; Epac2 RNAi+rescue, 0.87±0.03, *P<0.001. n = 102–252 spines, 5–10 cells per condition, 3 experiments (see also Supplementary Fig 7a,c). (c) Epac2 lacking the GEF domain (Epac2-ΔGEF) prevents 8-CPT-induced spine shrinkage. (d) Quantification of c; area (µm2): Epac2, 0.88±0.05; Epac2+8-CPT, 0.72±0.03; Epac2ΔGEF, 1.24±0.06; Epac2 ΔGEF+8-CPT, 1.17±0.05, *P<0.001, n = 169–274 spines, 5–9 cells per condition, 3 experiments. All neurons were analyzed at div 28. (e) Time-lapse imaging of spine dynamics in GFP-expressing cortical pyramidal neurons (div 25) pretreated with or without 8-CPT (50 µM). Visualization of spine dynamics from the beginning, middle and end of 80-minute imaging sessions; red: retracting, green: transient, blue: newly extended. (f) Quantification of total spine motility, expressed as fraction of spines undergoing extension, retraction or head morphing, and fraction of spines undergoing extensions or retractions; normalized total motility: control, 0.21±0.02; 8-CPT, 0.34±0.01, *P<0.001, n = 1218 spines, 5 cells per condition. (g) Example of time-lapse imaging of an individual spine before and after 8-CPT (50 µM, 1 hr) incubation; spine shrinks following 8-CPT treatment. (h) Quantification of g; -60 min, 100%; 0 min 97.9±3.0%; 60 min, 87.5±2.4%, *P<0.01, n = 84 spines, 3 experiments. Error bars: s.e.m. Scale bars: a, c, e 5µm; g, 2.5µm.
Figure 3
Figure 3
Epac2 interacts with GluR2/3-containing AMPAR and removes them from spines. (a) Coimmunoprecipitation of Epac2 with GluR2/3 but not GluR1 from cortical neurons (div 28); myc, control antibody. (b) Effects of 8-CPT (50 µM, 1 hr) and Epac2 RNAi knockdown on GluR2/3 content in spine heads. GluR2/3 clusters were visualized in spines outlined by GFP (arrowhead, clusters in spines; open arrowhead, shafts) (c) Quantification of the effects in b on GluR2/3 signal intensity in spines (top) and shaft (bottom); GluR2/3 immunofluorescence (a.u.): control, 2.94±0.27; 8-CPT, 1.78±0.13; Epac2 RNAi, 2.81±.37; Epac2 RNAi+8-CPT, 2.61±0.24,*P<0.01, n = 10–14 cells, 3 experiments. (d) GluR1 and NR1 cluster intensity was not affected: GluR1 immunofluorescence (a.u.): control, 1.29±0.12; 8-CPT, 1.08±0.11; NR1 immunofluorescence (a.u.): control, 0.95±0.04; 8-CPT, 1.05±0.08. n = 8–14 cells per condition. (e) Effect of 8-CPT on GluR2/3 colocalization with bassoon (arrowhead, clusters on spines; open arrowhead, shafts). Percent GluR2/3 puncta overlapping bassoon: control, 0.89± 0.02; 8-CPT, 0.80±0.03 (*P<0.05), n = 17 cells per condition. Error bars: s.e.m. Scale bars: 5µm. a.u.: arbitrary units.
Figure 4
Figure 4
Epac2 activation depresses AMPAR-mediated synaptic transmission. (a) Effect of 8-CPT on AMPAR-mediated mEPSC amplitudes and frequencies in pyramidal neurons (div 28). Synaptic currents were recorded in single cells pretreated with vehicle or 8-CPT (50 µM, 1 hr). Traces show representative recordings. Bar graphs: quantification of mean amplitudes (control (pA), 13.85±2.12; 8-CPT, 9.19±0.53, *P<0.05) and frequencies (control (events/s), 13.40±2.00; 8-CPT, 5.55±1.06, *P<0.01) of AMPAR-mediated mEPSCs. n = 9–11 cells per condition. Epac2 RNAi blocks 8-CPT-induced decrease in AMPAR-mediated mEPSC amplitude (RNAi+vehicle (pA), 12.54±0.98; RNAi+8-CPT, 12.74±1.80), but not mEPSC frequency (RNAi+vehicle (events/s), 12.98±1.09; RNAi+8-CPT, 6.70±0.67,*P<0.01) n = 5 cells per condition. Cumulative probability plots show a shift of mEPSC amplitudes toward smaller values in response to 8-CPT treatment (left), while in Epac2 RNAi expressing cells, no difference in mEPSC amplitude distribution is detected (right). (b) Synaptic currents were recorded in single cells before (gray) and 10–15 min after (black) perfusion with 8-CPT (50 µM). This resulted in a rapid reduction of mean amplitude of AMPAR-mediated mEPSCs (post 8-CPT treatment, −16.51±3.65% relative to control mean pA, *P<0.05), but not frequency (post 8-CPT, +13.84±12.99% relative to control events/s). Insets: mEPSC rise and decay time. n = 3. Error bars: s.e.m.
Figure 5
Figure 5
Dopamine D1/D5-like receptors modulate Rap activity, spine morphology, and GluR2 surface expression. (a) Rap activation by SKF-38393 (20 µM, 30 min) in cortical neurons. Fold Rap activation compared to control: 1.62±08 fold increase, *P<0.05, n = 4 experiments. (b) Effect of SKF-38393 (20 µM, 30 min) on B-Raf phosphorylation in situ in dendrites. B-Raf immunofluorescence (a.u.): control, 68.52±4.6; SKF-38393, 141.55±15.2, *P<0.001, n = 6–9 cells per condition, 2–3 experiments. (c) Effect of SKF-38393 (20 µM, 30 min) on spine morphology and surface GluR2 in spines in cortical neurons (div 28). Epac2 knockdown prevents SKF-38393-dependent spine remodeling and AMPAR removal. GluR2 was detected using an antibody to its extracellular N-terminus (GluR2-n) in non-permeabilized cells. (arrowhead, clusters in spines; open arrowhead, shafts) (d) Quantification of spine areas in e; area (µm2): control, 0.74±0.02; SKF-38393, 0.59±0.01, Epac2 RNAi, 0.86±0.03; Epac RNAi+SKF-38393, 0.90±0.04, *P<0.001, n = 308–426 spines from 9–13 cells per condition, 4 experiments. (e) Quantification of surface GluR2 (GluR2-n) clusters in e; GluR2-n immunofluorescence (a.u.): control, 60.6±3.53; SKF-38393, 36.9±1.65; Epac2 RNAi, 67.7±4.48; Epac2 RNAi+SKF-38393, 59.6±3.94, *P < 0.001, n = 9–13 cells per condition, 4 experiments. Error bars: s.e.m. Scale bars: 5µm.
Figure 6
Figure 6
Epac2 interacts with neuroligins. (a) Coimmunoprecipitation of NL1-3 with Epac2 but not Epac1 from cortical neurons (div 28). (b) Reverse coimmunoprecipitation of NL1-3 with Epac2 from cortical neurons (div 28). (c) Coimmunoprecipitation of Epac2 with NL1–3 from rat forebrain; myc, control antibody. All coimmunoprecipitation experiments were performed 3 times, Western Blots show typical results. (d) Immunofluorescence colocalization of Epac2 and NL 1 and 3 on dendrites of cortical pyramidal neurons. (e) NL3 affects Epac2 localization. Epitope-tagged Epac2 and NL3 were expressed individually or together in COS7 cells, and visualized by immunostaining. (open arrowhead, cytosolic expression; arrowhead, membrane expression) (f) Treatment with 8-CPT (50 µM) increases Epac2/NL3 colocalization along dendrites of mature cortical neurons. Epac2/NL3 colocalized puncta: control, 7.69±0.21; 8-CPT, 11.53±0.58, *P<0.001, n = 7–8 cells per condition, 2–3 experiments. (g) Coexpression of Epac2 with NL3 in hEK293 cells enhances its Rap-GEF activity. (h) Quantification of Rap-GTP in g; fold increase in Rap activity relative to control: NL3, 1.08±0.23, Epac2, 2.61±0.19, NL3+Epac2, 6.45±0.08, *P<0.001, n = 3 experiments. Error bars: s.e.m. Scale bars: d, f, 5µm; e 15µm.
Figure 7
Figure 7
Disease-associated missense mutations affect Epac2 function. (a) Epac2 mutations affected Rap-GEF activity in hEK293 cells transfected with Epac2 or its mutants. (b) Quantification of Rap1-GTP in a: reduced Rap1 activation by Epac2-V646F; fold increase in Rap activity relative to control: Epac2-WT, 2.67±0.40, Epac2-M165T, 2.52±0.50, Epac2-V646F, 0.55±0.17, Epac2-G706R, 2.62±0.45, Epac2-T809S, 1.80±0.43, *P<0.01, n = 3 experiments. (c) Effect of Epac2’s missense mutations on NL3-dependent stimulation of its GEF activity. (d) Quantification of Rap1-GTP in c: NL3-enhanced Rap activation is reduced in Epac2-V646F and increased in Epac2-T809S; fold increase in Rap activity relative to control: Epac2-WT, 5.98±0.24, Epac2-M165T, 5.56±0.76, Epac2-V646F, 3.26±0.38, Epac2-G706R, 4.93±0.50, Epac2-T809S, 15.21±2.32, *P<0.001, n = 3 experiments. (e) Effect of Epac2 mutations on dendritic B-Raf phosphorylation (p-B-Raf) in cortical pyramidal neurons (div 28). (f) Quantification of dendritic B-Raf fluorescence intensities in e. Epac2-V646F reduced and Epac2-T809S increased dendritic phospho-B-Raf immunofluorescence; control, 232.2±11.4 Epac2-WT, 272.2±17.3, Epac2-M165T, 265.3±34.2, Epac2-V646F, 145.7±10.5, Epac2-G706R, 255.6±32.0, Epac2-T809S, 528.1±49.5, *P<0.001, n = 6–8 cells per condition from 3 experiments. Error bars: s.e.m. Scale bar: 5µm.
Figure 8
Figure 8
Epac2 missense mutants affect spine morphology. (a) Co-expression of HA-tagged Epac2 or its disease-associated mutants with GFP in cortical pyramidal neurons (div 28); Epac2 mutants Epac2-V646F and Epac2-T809S alter dendritic spine morphology. (b) Quantification of the effects on spine area and number in a; area (µm2): GFP, 0.67±0.02; Epac2-WT, 0.66±0.02; Epac2-M165T, 0.70±0.02; Epac2-V646F, 0.82±0.02; Epac2-G706R, 0.67±0.02; Epac2-T809S, 0.66±0.02, *P<0.001; spines/10µm: GFP, 5.98±0.37; Epac2-WT, 7.00±0.32; Epac2-M165T, 6.07±0.34; Epac2-V646F, 6.66±0.52; Epac2-G706R, 5.60±0.40; Epac2-T809S, 8.23±0.31, *P<0.001, n = 406-592 spines from 9 cells per condition, 4 experiments. (c) Effects of expression of HA-Epac2 mutations on the average intensity and number of endogenous PSD-95 immunofluorescent puncta in dendrites. (d) Quantification of average immunofluorescence intensity and linear density of PSD-95 puncta in c. Epac2-V646F increased PSD-95 average intensity in individual puncta (a.u.); Epac2-WT, 170.5±7.07; Epac2-M165T, 152.4±9.16; Epac2-V646F, 291.0±26.0; Epac2-G706R, 203.7±8.24; Epac2-T809S, 205.3±7.46, *P<0.001. Epac2-T809S increased the number of PSD-95 clusters: Epac2-WT, 8.26±0.76; Epac2-M165T, 7.80±0.29; Epac2-V646F, 10.05±0.93; Epac2-G706R, 8.34±0.98; Epac2-T809S, 14.5±1.01, *P<0.001, n = 4–8 cells, 2–3 experiments. Error bars: s.e.m. Error bars: s.e.m. Scale bars: 5µm.
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