1. Olfactory-Experience- and Developmental-Stage-Dependent Control of CPEB4 Regulates c-Fos mRNA Translation for Granule Cell Survival 
Ching-San Tseng, Hsu-Wen Chao, Hsien-Sung Huang, Yi-Shuian Huang 
Cell Reports 
Volume 21, Issue 8, Pages (November 2017)
DOI: /j.celrep
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2. Cell Reports 2017 21, 2264-2276DOI: (10.1016/j.celrep.2017.10.100)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1 Reduced OB Size and GCL Volume in Adult CPEB4-KO Mice
(A) Dorsal view of 3-month-old CPEB4-WT and CPEB4-KO brains. The decreased OB size in the KO brain is indicated by an arrowhead. Graphs show mean ± SEM body, brain, and OB weight of 3-month-old WT (n = 8) and KO (n = 9) male mice. ∗p < 0.05 (Student’s t test).
(B) Nissl-stained coronal sections of WT and KO OBs. Bulbar layer organization is denoted as follows: RMS, rostra migratory steam; GCL, granule cell layer; IPL, inner plexiform layer; MCL, mitral cell layer; EPL, external plexiform layer; GL, glomerular layer; and ONL, olfactory nerve fiber layer. Scale bars, 400 μm.
(C) A series of coronal OB sections (10 μm thick) selected from caudal to rostral (1 in 10 sections) was labeled with DAPI and measured for GCL area (n = 8 mice per group). GCL volume was estimated by multiplying the total GCL area calculated from selected sections by 10. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < (Student’s t test).
(D) Similar to (A), selected OB sections were immunolabeled with NeuN antibody. NeuN+ neurons were counted (n = 4 mice per group). Total GC number was estimated by multiplying the total NeuN+ cells by 10. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (Student’s t test).
Data are mean ± SEM.
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4. Figure 2 Normal Adult Neurogenesis in CPEB4-KO Mice
(A) Schematic distribution of neural stem cells (NSCs), neuroblasts, and mature neurons along the subventricular zone (SVZ)-OB neurogenic pathway. Immunostaining of glial fibrillary acidic protein (GFAP), doublecortin (Dcx), and neuronal nuclear antigen (NeuN) was used to distinguish the three cell types.
(B) Immunostaining images of CPEB4 in the SVZ and OB of adult mice. Magnified regions are shown in (B′) and (B″). LV, lateral ventricle; cc, corpus callosum; CPu, caudate putamen; TH, thalamus. Scale bars, 200 μm.
(C) Confocal images of CPEB4 with the NSC marker GFAP, neuroblast marker Dcx, or neuron marker NeuN in the SVZ or OB. Open and closed arrowheads indicate the absence and presence of the CPEB4 signal, respectively, in marker-positive cells. Scale bars, 20 μm.
(D) Immunostaining of Ki67 in the SVZ of adult mice. Ki67+ cells were counted to score proliferation (n = 4 mice per group). Scale bar, 100 μm.
(E) Schematic procedure of thymidine analog injection. The number of IdU- and CldU-labeled cells was measured in postnatal day 88 (P88) GCL. ∗p < 0.05 and ∗∗p < 0.01 (Student’s t test).
Data are mean ± SEM. See also Figures S1–S3.
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5. Figure 3
Normal Proliferation, but Enhanced Apoptosis, in the Early Postnatal CPEB4-KO GCL
(A) OBs from WT and KO mice of different ages were used for Ki67 immunostaining and TUNEL assay. Representative images from P3 OBs are shown. Scale bars, 100 μm.
(B) Quantification of Ki67-labeled cells in different laminar structures of OBs from WT (n = 4) and KO (n = 4) mice of different ages. SEL, subependymal layer; GCL, granule cell layer; MCL, mitral cell layer; PLs, internal and external plexiform layers; GL, glomerular layer.
(C) Quantification of TUNEL+ cells in different laminar structures of OBs from WT (n = 4) and KO (n = 4) mice. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < (two-way ANOVA).
Data are mean ± SEM. See also Figure S4.
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6. Figure 4
Decreased c-FOS Protein Levels and BDNF-TrkB Signaling in CPEB4-KO OBs
(A) A photo of a P14 mouse after unilateral naris occlusion (UNO) at P1. The occluded nostril is denoted by an arrowhead.
(B) Dorsal view of brains with occluded sides of OBs indicated by arrowheads. The weights of occluded (Occl) and control (Ctrl) sides of OBs are expressed as a relative ratio (Occl/Ctrl) (n = 6 animals per group).
(C) Quantification of apoptotic cells detected by TUNEL assay in the GCL of WT (n = 6) and KO (n = 7) mice expressed as a relative ratio (Occl/Ctrl).
(D) Western blot analysis of protein levels in OBs isolated from control and occluded sides of UNO-treated WT and KO mice (n = 4 animals per group). TH, tyrosine hydroxylase.
(E) qRT-PCR of mRNA levels of Cpeb4, c-Fos, and Gapdh in OBs from UNO-treated mice (n = 5 animals per group).
(F) Coronal OB sections collected for TUNEL assay in (C) used for c-FOS immunostaining. The density of c-FOS+ cells in the GCL was quantified. Scale bar, 50 μm.
(G) qRT-PCR of Bdnf mRNA levels in OBs isolated from P14 WT and KO mice (n = 4 animals per group).
(H) Immunoblotting of TrkB activation (i.e., Y816p-TrkB) in P14 WT and KO OBs (n = 4 mice per group). Data are expressed as the ratio of p-TrkB to TrkB levels.
(I) P0 mice were microinjected with PBS or BDNF (n = 8 pups per group) and then collected on P2 for a TUNEL assay in the GCL.
Significant differences (analyzed by two-way ANOVA in D–F and I and Student’s t test in B, C, G and H) are denoted by number signs between control and treatment (UNO or BDNF) groups or asterisks between WT and KO groups (# or ∗, p < 0.05; ## or ∗∗, p < 0.01; and ### or ∗∗∗, p < 0.001). Data are mean ± SEM. See also Figures S5–S7.
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7. Figure 5
CPEB4 Binds to and Activates Polyadenylation-Induced Translation of c-Fos RNA
(A) Two conserved CPE sequences in the 3′ UTR of mouse, rat, and human c-Fos RNA.
(B) RNA immunoprecipitation. P7 OB lysates were precipitated with control (Ctrl) or CPEB4 (CP4) immunoglobulin G (IgG). qRT-PCR of c-Fos mRNA levels in immunoprecipitates expressed as relative ratio to the non-target control, Gapdh. IgG H.C., IgG heavy chain. Data are from 3 independent experiments. ∗p < 0.05 (Student’s t test).
(C) RNA stability assay. Reporter plasmids, firefly luciferase (FLuc) appended with the control SV40 or mouse c-Fos 3′ UTR and Renilla luciferase (RLuc), were co-transfected with the myc or myc-CPEB4 plasmid into HEK293T cells. qRT-PCR of FLuc and RLuc mRNA levels in cells. Data are from 4 independent experiments.
(D) Dual-luciferase reporter assay. The reporter plasmids FLuc-c-Fos 3′ UTR and RLuc were co-transfected into HEK293T cells with a plasmid expressing myc-tag, or myc-tagged full-length CPEB4 (myc-CP4), N-terminal CPEB4 (myc-CP4N), or C-terminal CPEB4 (myc-CP4C). Activity (protein levels) of FLuc and RLuc was assessed in cells. Data are from 4 independent experiments. ∗∗p < 0.01 (Student’s t test).
(E) Various FLuc reporters carrying the c-Fos 3′-UTR sequence containing wild-type (CPE, UUUUAAU) or mutant CPE (mtCPE, UCCCAU) or the mutant poly(A) signal (mtHEX, AACGGA) were used for the dual-luciferase reporter assay and qRT-PCR. Data are from 4 independent experiments. ∗p < 0.05 and ∗∗p < 0.01 (Student’s t test).
Data are mean ± SEM.
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8. Figure 6
GABAergic-Neuron-Specific Depletion of CPEB4 Sufficiently Recapitulates Developing Defects in the OB
(A) Primary cultured OB neurons at 10 days in vitro (DIV10) were stimulated with 10 μM NMDA for 30 min before measuring the poly(A) tail length of c-Fos mRNA.
(B) Similar to (A), except the NMDA treatment lasted for 60 min before western blot analysis. Data are mean ± SEM from 3 independent cultures. Two-way ANOVA, ∗p < 0.05 and ##p < 0.01.
(C) CPEB4 immunostaining and DAPI labeling in coronal OB sections prepared from P14 conditional WT (cWT, Cpeb4f/f, +/+) and KO (cKO, Cpeb4f/f, Gad65-cre/+) mice. Scale bar, 100 μm. Magnified images of selected areas are shown in (C′) and (C″).
(D) Dorsal view of the rostral brains and graphs showing mean ± SEM OB weight in P14 cWT and cKO mice (n = 8 per group). ∗p < 0.05 (Student’s t test).
(E) Western blot analysis of protein levels of CPEB4, c-Fos and GAPDH in OBs from P14 cWT and cKO mice (n = 8 per group). ∗p < 0.05 and ∗∗∗p < (Student’s t test).
(F) TUNEL+ and Ki67+ cell density in the GCL of P14 cWT and cKO OBs (n = 6 mice per group). ∗p < 0.05 (Student’s t test).
Data are mean ± SEM. See also Figure S8.
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9. Figure 7
CREB- and CPEB4-Dependent c-FOS Expression in P3, but Not Adult, OBs
(A) Adult CPEB4-WT and CPEB4-KO OBs were used for immunoblotting. The c-FOS protein level is expressed as relative ratio after normalization to GAPDH level (n = 5 mice per group).
(B) OBs isolated from P3, P14, and adult WT mice (n = 4 per group) were used for immunoblotting. Protein levels are expressed as relative ratio after normalization to GAPDH level. ∗p < 0.05 and ∗∗∗p < (Student’s t test).
(C) Co-immunoprecipitation (co-IP). OB lysates were precipitated with control (Ctrl) or CPEB4 (CP4) IgG and immunoblotted with the denoted proteins.
(D) Co-IP to detect the CPEB4-CREB interaction in DIV10 primary OB neurons stimulated with NMDA for 30 or 60 min.
(E) RIP. OB lysates were precipitated with Ctrl or CP4 IgG, and the pull-down substances were used for immunoblotting and qRT-PCR of denoted targets. Data are from 3 independent experiments. ∗p < 0.05 (Student’s t test).
(F) Chromatin-IP. Crosslinked OB lysates were precipitated with Ctrl, CREB, or CP4 IgG. The precipitates were used for immunoblotting and qPCR. Data are from 3 independent experiments. TRE/CRE, TPA/cAMP response element. ∗∗∗p < (Student’s t test).
(G) Olfactory experience activates NMDA receptors of GCs, presumably via the release of glutamate from mitral cells. NMDA-receptor signaling somehow enhances the CREB and CPEB4 interaction and facilitates CPEB4-mediated polyadenylation-induced translation of c-Fos mRNA. CREB and CPEB4-dependent c-FOS expression occurs in the early postnatal stage to maintain neurotrophic signaling for GC survival.
Data are mean ± SEM. See also Figure S9.
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