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SUMOylation Is an Inhibitory Constraint that Regulates the Prion-like Aggregation and Activity of CPEB3

Drisaldi, Bettina, Colnaghi, Luca, Fioriti, Luana, Rao, Nishta, Myers, Cory, Snyder, Anna M., Metzger, Daniel J., Tarasoff, Jenna, Konstantinov, Edward, Fraser, Paul E., Manley, James L., Kandel, Eric R.·2015·Cell Reports
DOI 10.1016/j.celrep.2015.04.061PMC PMC5477225
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Abstract

Protein synthesis is crucial for the maintenance of long-term-memory-related synaptic plasticity. The prion-like cytoplasmic polyadenylation element-binding protein 3 (CPEB3) regulates the translation of several mRNAs important for long-term synaptic plasticity in the hippocampus. Here, we provide evidence that the prion-like aggregation and activity of CPEB3 is controlled by SUMOylation. In the basal state, CPEB3 is a repressor and is soluble. Under these circumstances, CPEB3 is SUMOylated in hippocampal neurons both in vitro and in vivo. Following neuronal stimulation, CPEB3 is converted into an active form that promotes the translation of target mRNAs, and this is associated with a decrease of SUMOylation and an increase of aggregation. A chimeric CPEB3 protein fused to SUMO cannot form aggregates and cannot activate the translation of target mRNAs. These findings suggest a model whereby SUMO regulates translation of mRNAs and structural synaptic plasticity by modulating the aggregation of the prion-like protein CPEB3.

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Crossref · 10.1016/j.celrep.2015.04.061

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Figures

Figure 1 CPEB3 Is SUMOylated and Aggregates in Cultured Neurons (A) Western blotting (WB) showing detergentinsolubility assay of neuronal cells stimulated for0 and 30min with glycine. Soluble (S) and insoluble(I) fractions were probed with endogenous CPEB3antibody. (B) Graph showing the percent of CPEB3 in theinsoluble fraction as a mean of five independenttrials ± SE (t- Stud p < 0.0001). (C) WB showing immunoprecipitation of endogenous CPEB3 from cultured neurons stimulatedwith glycine for 0 and 15 min (CPEB3, immunoprecipitation using an antibody against CPEB3;IgG, IgG negative control; In, Input). The top panelshows WB using an antibody raised againstSUMO-2/3. The bottom panel shows WB using anantibody raised against CPEB3.
Figure 1 CPEB3 Is SUMOylated and Aggregates in Cultured Neurons (A) Western blotting (WB) showing detergentinsolubility assay of neuronal cells stimulated for0 and 30min with glycine. Soluble (S) and insoluble(I) fractions were probed with endogenous CPEB3antibody. (B) Graph showing the percent of CPEB3 in theinsoluble fraction as a mean of five independenttrials ± SE (t- Stud p < 0.0001). (C) WB showing immunoprecipitation of endogenous CPEB3 from cultured neurons stimulatedwith glycine for 0 and 15 min (CPEB3, immunoprecipitation using an antibody against CPEB3;IgG, IgG negative control; In, Input). The top panelshows WB using an antibody raised againstSUMO-2/3. The bottom panel shows WB using anantibody raised against CPEB3.CC BY-NC-ND · PubMed Central
Figure 2 Fear Conditioning Induces CPEB3 Aggregation and a Decrease of SUMOylation in the Hippocampus (A) WB showing immunoprecipitation of endogenous CPEB3 from hippocampal extracts 30min after chamber exposure and foot shock (FS).The experiment wasdone in duplicate. An antibody raised against SUMO-2/3 was used in the top panel. The bottom panel shows WB using an antibody raised against CPEB3. (B) WB showing immunoprecipitation of endogenous CPEB3 from hippocampal extracts 30 min after chamber exposure and FS. An antibody raised againstubiquitin was used in the top panel. CPEB3 was used for the bottom panel. (C) WB showing detergent insolubility assay of hippocampal extracts 30 min after chamber exposure or foot shock. S and I fractions were probed with antibodyagainst endogenous CPEB3. (D) Graph showing the percentage of CPEB3 in the insoluble fraction as a mean of three independent trials ± SE (t-Stud p = 0.023). (E) WB showing the fractionation of cortical and hippocampal tissue in P1 (nuclear fraction), S1 (cytoplasmic fraction), and P2 (synaptosome fraction andmitochondria). (F) Immunoprecipitation of CPEB3 from synaptosome fraction (P2). (G) WB showing detergent insolubility of CPEB3 and PSD95 in synaptosomes (P2).
Figure 2 Fear Conditioning Induces CPEB3 Aggregation and a Decrease of SUMOylation in the Hippocampus (A) WB showing immunoprecipitation of endogenous CPEB3 from hippocampal extracts 30min after chamber exposure and foot shock (FS).The experiment wasdone in duplicate. An antibody raised against SUMO-2/3 was used in the top panel. The bottom panel shows WB using an antibody raised against CPEB3. (B) WB showing immunoprecipitation of endogenous CPEB3 from hippocampal extracts 30 min after chamber exposure and FS. An antibody raised againstubiquitin was used in the top panel. CPEB3 was used for the bottom panel. (C) WB showing detergent insolubility assay of hippocampal extracts 30 min after chamber exposure or foot shock. S and I fractions were probed with antibodyagainst endogenous CPEB3. (D) Graph showing the percentage of CPEB3 in the insoluble fraction as a mean of three independent trials ± SE (t-Stud p = 0.023). (E) WB showing the fractionation of cortical and hippocampal tissue in P1 (nuclear fraction), S1 (cytoplasmic fraction), and P2 (synaptosome fraction andmitochondria). (F) Immunoprecipitation of CPEB3 from synaptosome fraction (P2). (G) WB showing detergent insolubility of CPEB3 and PSD95 in synaptosomes (P2).CC BY-NC-ND · PubMed Central
Figure 3 SUMOylation of CPEB3 Decreases Its Aggregation (A) WB showing detergent insolubility assay of in-vitro-translated CPEB3-HA, SUMO-1-CPEB3-HA,and SUMO-2-CPEB3-HA fusion proteins in thepresence of two different concentrations of SDS(0.1% and 0.2%). (B) WB showing detergent insolubility assay ofHEK293 cells extracts overexpressing CPEB3-HAand SUMO-2-CPEB3-HA fusion proteins. An HAantibody was used to detect the constructs. (C) Left panel shows a WB of a 2D agarose gelshowing aggregates formation for CPEB3-HA andSUMO-2-CPEB3-HA in HEK293 cells. Right panelshows a WB of the input material. (D) WB showing detergent insolubility assay ofHeLa cells extracts overexpressing CPEB3 alone,CPEB3-HA and SUMO-2, and CPEB3-HA andSENP1.
Figure 3 SUMOylation of CPEB3 Decreases Its Aggregation (A) WB showing detergent insolubility assay of in-vitro-translated CPEB3-HA, SUMO-1-CPEB3-HA,and SUMO-2-CPEB3-HA fusion proteins in thepresence of two different concentrations of SDS(0.1% and 0.2%). (B) WB showing detergent insolubility assay ofHEK293 cells extracts overexpressing CPEB3-HAand SUMO-2-CPEB3-HA fusion proteins. An HAantibody was used to detect the constructs. (C) Left panel shows a WB of a 2D agarose gelshowing aggregates formation for CPEB3-HA andSUMO-2-CPEB3-HA in HEK293 cells. Right panelshows a WB of the input material. (D) WB showing detergent insolubility assay ofHeLa cells extracts overexpressing CPEB3 alone,CPEB3-HA and SUMO-2, and CPEB3-HA andSENP1.CC BY-NC-ND · PubMed Central
Figure 4 SUMOylation Prevents Translation of CPEB3 mRNA Targets (A) Luciferase assay of actin UTR in 293 with overexpression of CPEB3 or SUMO-2-CPEB3. n = 3 experiments; n = 8 replicates. ANOVA shows a significant interaction between the four conditions (p = 0.0003). (B and C) Filopodia counts. Primary hippocampal neurons express CPEB3 or SUMO-2-CPEB3. ANOVA shows a significant interaction between the four conditions (p = 0.0335). (D and E) Luciferase assay of SUMO-2 UTR or SUMO-2 CPE mutant UTR in 293 with over-expression ofCPEB3.n=3 replicates (p<0.0001). (F and G) Luciferase assay of SUMO-2 UTR in primary hippocampal neurons with over-expression of CPEB3 and stimulation with glycine. n = 3 replicates (p < 0.0001).
Figure 4 SUMOylation Prevents Translation of CPEB3 mRNA Targets (A) Luciferase assay of actin UTR in 293 with overexpression of CPEB3 or SUMO-2-CPEB3. n = 3 experiments; n = 8 replicates. ANOVA shows a significant interaction between the four conditions (p = 0.0003). (B and C) Filopodia counts. Primary hippocampal neurons express CPEB3 or SUMO-2-CPEB3. ANOVA shows a significant interaction between the four conditions (p = 0.0335). (D and E) Luciferase assay of SUMO-2 UTR or SUMO-2 CPE mutant UTR in 293 with over-expression ofCPEB3.n=3 replicates (p<0.0001). (F and G) Luciferase assay of SUMO-2 UTR in primary hippocampal neurons with over-expression of CPEB3 and stimulation with glycine. n = 3 replicates (p < 0.0001).CC BY-NC-ND · PubMed Central
Figure 5 CPEB3 and SUMO-2 Regulate Each Other SUMO-2 prevents aggregation of CPEB3 molecules before treatment. DeSUMOylation of the protein facilitates aggregation and mRNA translation. The SUMO-2 mRNA is a target of CPEB3, and it is induced upon stimulation of neurons and deSUMOylation of CPEB3.
Figure 5 CPEB3 and SUMO-2 Regulate Each Other SUMO-2 prevents aggregation of CPEB3 molecules before treatment. DeSUMOylation of the protein facilitates aggregation and mRNA translation. The SUMO-2 mRNA is a target of CPEB3, and it is induced upon stimulation of neurons and deSUMOylation of CPEB3.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Drisaldi, B., Colnaghi, L., Fioriti, L., Rao, N., Myers, C., Snyder, A. M., Metzger, D. J., Tarasoff, J., Konstantinov, E., Fraser, P. E., Manley, J. L., & Kandel, E. R.
Venue
Cell Reports
Publisher
Elsevier BV
Published
2015-06
Volume
11
Issue
11
Pages
1694-1702
ISSN
2211-1247
DOI
10.1016/j.celrep.2015.04.061
PMC
PMC5477225
Cited by
120
References
34
Language
en
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