1. CPEB2 Activates GRASP1 mRNA Translation and Promotes AMPA Receptor Surface Expression, Long-Term Potentiation, and Memory 
Wen-Hsin Lu, Nai-Hsing Yeh, Yi-Shuian Huang 
Cell Reports 
Volume 21, Issue 7, Pages (November 2017)
DOI: /j.celrep
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2. Cell Reports 2017 21, 1783-1794DOI: (10.1016/j.celrep.2017.10.073)
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3. Figure 1 Characterization of Forebrain-Specific CPEB2 cKO Mice
(A) The Cpeb2 gene contains 13 exons; exons 2–6 are shown. Mice carrying the Cpeb2 allele with two loxP sites flanking exons 3–5 (fCPEB2) were crossed with Camkii-Cre mice to generate CPEB2 cWT (Cpeb2f/f) and cKO (Cpeb2f/f, Camkii-Cre/+) mice.
(B) The designated areas isolated from 9- to 10-week-old cWT and cKO mice were used for immunoblotting. The protein level of CPEB2 was normalized to that of LRP130. Data are mean ± SEM (3 mice per group). ∗p < 0.05 and ∗∗∗p < 0.001, Student’s t test.
(C) CPEB2 immunohistochemistry of coronal brain slices from 6-week-old mice. Scale bars, 0.2 mm, unless specified. CA, Cornu Ammonis; DG, dentate gyrus.
See also Figures S1 and S2.
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4. Figure 2 Impaired Memory Consolidation in CPEB2 cKO Mice
(A) Open-field test. Representative moving traces in the open arena during the first 10 min. The entry times, staying duration, moving distance, and velocity in the center (gray region) are mean ± SEM (13 mice per group).
(B) Representative moving traces for 5 min in the elevated plus maze (EPM). The percentage of entry times, staying duration, and moving distance into the open arms versus total arms are mean ± SEM (13 mice per group).
(C) Contextual fear conditioning. The freezing response during habituation (hab) and acquisition was analyzed for 2 min per trial. A foot shock was given at the end of habituation and the first 3 conditioning trials. Mice were placed in the chamber for 6 min without reinforcing the shock on the next day. Data are mean ± SEM (6 mice per group).
(D) Shock reactivity. The current intensities required to evoke various responses are mean ± SEM (cWT/cKO, 6/12 mice).
(E–G) Both cWT and cKO mice learned to locate the hidden platform by using the surrounding cues after 4-day trainings (E). In the probe trial, the percentage of time that the mouse spent navigating each quadrant was analyzed (F). Swimming velocity and the latency to get on the visible platform were measured (G). Data are mean ± SEM (cWT/cKO, 15/18 mice). MWM, Morris water maze.
∗p < 0.05 and ∗∗p < 0.01, Student’s t test or Fisher’s least significant difference (LSD) post hoc test between cWT and cKO groups in all behavior assays.
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5. Figure 3 Impaired LTP Responses in CPEB2 cKO Hippocampal CA1 Neurons
(A–F) Hippocampal slices prepared from adult (3- to 4-month-old) mice were used for LTP recording in the SC-CA1 pathway.
(A and B) Basal synaptic transmission: input-output (I-O) curve (A) and paired-pulse facilitation (B) in the cWT and cKO hippocampi. Numbers in parentheses (n/N) represent the number of recorded slices (n) and mice (N). Sample traces are listed. Scale bars, 0.5 mV (vertical) and 20 ms (horizontal). Data are mean ± SEM. ∗∗∗p < 0.001, Fisher’s LSD post-hoc test.
(C and D) LTP evoked by one train of high-frequency stimulation (HFS) (C) or theta burst stimulation (TBS) (D) in CA1 neurons (n/N, slices/mice).
(E and F) Long-lasting LTP induced by 4 trains (4X) of HFS (E) or TBS (F) in CA1 neurons (n/N, slices/mice). The bar graphs present the percentages of LTP responses during the indicated periods. Data are mean ± SEM. ∗p < 0.05, Student’s t test.
Sample traces in (C)–(F) represent the average fEPSP of 5 min before (gray) and the very last 5 min (black) after stimulation. Scale bars, 0.5 mV (vertical) and 10 ms (horizontal).
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6. Figure 4
Elongated Spine Morphology and Decreased mEPSC Amplitude in CPEB2 KO Neurons
(A) The body weight of E18.5 embryos isolated from heterozygous CPEB2 matings. Numbers in parentheses represent the number of embryos.
(B) Immunoblotting of CPEB2 and LRP130 in protein lysates prepared from WT and KO neurons cultured for the indicated days in vitro (DIV).
(C) CPEB2 WT and KO neurons were transfected with the EGFP plasmid on DIV13 and fixed on DIV20 for EGFP immunostaining. Representative images of the whole neurons and dendritic spine area are shown. Scale bars, 20 μm. Dendritic spine morphology and density were quantified from ∼1,200–1,300 spines (n/N, the number of spines/neurons) from 4 independent cultures. Data are mean ± SEM. ∗∗∗p < 0.001, Student’s t test.
(D) Whole-cell patch recording. DIV14–22 neurons were patched to record mEPSCs at −70 mV. Representative recording traces from WT and KO neurons are shown. The amplitude and frequency of mEPSCs in 11 WT and 11 KO neurons from 7 independent cultures were quantified. Data are mean ± SEM. ∗p < 0.05, Student’s t test.
See also Figure S3.
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7. Figure 5 Decreased Surface Expression of AMPARs in CPEB2 KO Neurons
(A) Western blot analysis of GluA1 and GluA2 in DIV21 neurons. The protein levels were normalized to that of LRP130. Data are mean ± SEM from 8–9 independent cultures.
(B) Representative images of surface GluA1 (sGluA1) and GluA2 (sGluA2) immunolabeling. sGluA1 (n = 10 per group) and sGluA2 (n = 14 per group) signals in WT and KO neurons from 3 independent experiments were quantified. Scale bars, 5 μm. ∗p < 0.05 and ∗∗p < 0.01, Student’s t test. See also Figure S4.
(C) RNA immunoprecipitation (RNA-IP). DIV22–25 neuronal lysates were used for immunoprecipitation (IP) with control or CPEB2 IgG. qRT-PCR analysis of precipitated substances for level of denoted mRNA with the non-target control GAPDH mRNA as the reference. Data are mean ± SEM from 4 independent experiments. ∗p < 0.05 and ∗∗p < 0.01, Student’s t test. See also Figure S5.
(D) Western blot analysis of protein levels in DIV21 neurons. Data are mean ± SEM from 7-9 independent cultures. ∗∗∗p < 0.001, Student’s t test.
(E) Similar to (B), except DIV16 neurons were infected with lentiviruses expressing EGFP or 3xflag-GRASP1 and fixed on DIV21 for immunostaining of surface GluA1 and GluA2 (n = 15–25 infected neurons from 3–4 independent cultures). Scale bars, 5 μm. ∗p < 0.05 and ∗∗p < 0.01, Fisher’s LSD post hoc test.
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8. Figure 6
CPEB2-Activated GRASP1 mRNA Translation Contributes to AMPAR Recycling
(A) qRT-PCR analysis of GRASP1 and GAPDH mRNA level in DIV 21 neurons from 3 independent cultures.
(B) Dual luciferase reporter assay. The firefly luciferase reporter appended with mouse or rat GRASP1 3′ UTR and Renilla luciferase plasmids was co-transfected with the plasmid expressing EGFP, myc-tagged full-length (CP2), or C terminus (CP2C) of CPEB2 into HEK293T cells. Data are mean ± SEM from 5 independent experiments. ∗p < 0.05 and ∗∗∗p < 0.001, Student-Newman-Keuls post hoc test.
(C) Similar to Figure 5B, except neurons were treated ± 100 μM AMPA and 50 μM APV for 2 min and harvested for immunostaining of surface GluA1 (n = 9 per group) and GluA2 (n = 13 per group) 60 min later. Scale bars, 10 μm. ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001, Fisher’s LSD post hoc test.
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9. Figure 7
Reduced GRASP1 Expression Contributes to Synaptic Plasticity and Memory Impairment in CPEB2 cKO Mice
(A) CPEB2 cWT and cKO mice were transduced with adeno-associated virus (AAV) in the CA1 regions as illustrated. Expression of EGFP, myc-CPEB2, or 3xflag-GRASP1 was monitored by immunostaining at 3 weeks after AAV transduction. Scale bars, 20 μm.
(B) LTP induced by 4 trains (4X) of TBS in CA1 neurons (n/N, slices/mice). Sample traces represent the average fEPSP of 5 min before (gray) and the very last 5 min (black) after stimulation. Scale bars, 0.5 mV (vertical) and 10 ms (horizontal). The bar graphs present the percentages of LTP responses during the indicated periods. Data are mean ± SEM. ∗p < 0.05, Student’s t test.
(C–E) The AAV-transduced cWT and cKO mice were tested for spatial memory in the MWM task like in Figures 2E–2G. The escape latency (C), the percentage of time in navigating each quadrant (D), and swimming velocity and latency to the visible platform (E) were analyzed. The number of mice in each group is in parentheses. Data are mean ± SEM. ∗p < 0.05, Fisher’s LSD post hoc test.
Cell Reports  , DOI: ( /j.celrep )
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