Neuralized1 activates CPEB3: a function for nonproteolytic ubiquitin in synaptic plasticity and memory storage

Abstract

The cytoplasmic polyadenylation element-binding protein 3 (CPEB3), a regulator of local protein synthesis, is the mouse homolog of ApCPEB, a functional prion protein in Aplysia. Here, we provide evidence that CPEB3 is activated by Neuralized1, an E3 ubiquitin ligase. In hippocampal cultures, CPEB3 activated by Neuralized1-mediated ubiquitination leads both to the growth of new dendritic spines and to an increase of the GluA1 and GluA2 subunits of AMPA receptors, two CPEB3 targets essential for synaptic plasticity. Conditional overexpression of Neuralized1 similarly increases GluA1 and GluA2 and the number of spines and functional synapses in the hippocampus and is reflected in enhanced hippocampal-dependent memory and synaptic plasticity. By contrast, inhibition of Neuralized1 reduces GluA1 and GluA2 levels and impairs hippocampal-dependent memory and synaptic plasticity. These results suggest a model whereby Neuralized1-dependent ubiquitination facilitates hippocampal plasticity and hippocampal-dependent memory storage by modulating the activity of CPEB3 and CPEB3-dependent protein synthesis and synapse formation.

Copyright © 2011 Elsevier Inc. All rights reserved.

Figures

Figure 1. Neurl1 is expressed in the adult mouse forebrain and is localized in dendrites and at post-synaptic sites in hippocampal neurons

(A) In situ hybridization analysis of Neurl1 mRNA expression on coronal (1&2) and sagittal (3) brain slices from adult (3 ½ months) wild type mice. cx: cerebral cortex, hp: hippocampus, st: striatum, am: amygdala. (B & C) Confocal sections of adult CA1 pyramidal neurons. Coimmunostaining for Neurl1 and the dendritic marker MAP2 or the presynaptic marker Synaptophysin (Syn). Lower panels: high magnification of apical dendrites. (D) Immunoblots of hippocampal homogenates (H) and PSD extracts. Neurl1 and PSD95 are enriched in the PSD fraction. Synaptophysin, the glial protein GFAP and histone 3 are controls. (E) Confocal sections of cultured hippocampal neurons (16 DIV) showing colocalization of endogenous Neurl1 and PSD95. (ii &iii) high magnification of dendrites. (F) Neurl1 protein level in the CA1 area is increased 30min after LTP induction at the Schaffer collateral pathway using 4 TBS. ctrl: unstimulated controls. Top: examples of western blots from 3 independent experiments. Bottom: Mean + SEM from 5 independent experiments (4 mice; *p

Figure 2. Inhibition of Neurl1 in the adult hippocampus impairs memory and synaptic plasticity

(A) Averaged data (+SEM) from the Morris water maze of Neurl1DN expressing mice (DT off dox) and control siblings. The performance of DT was significantly lower in the probe trial (quadrant*genotype effect: p0.4). (ii) Mice tested on dox. No differences were observed (p>0.6). (D) Expression of Neurl1DN in the hippocampus impairs the late phase of LTP and LTD at the Schaeffer collateral pathway. (i) LTP induction was not affected in DT (t-test 0-60min: p=0.17). L-LTP was impaired (t-test 60-120min & 120-180min; p0.63). Insets in (iii) & (iv): sample traces (PPR: traces for 10, 50, 100, 200 & 300 ms delays between pulses). See also Table S1 & Figure S2.

Figure 3. Overexpression of Neurl1 in the adult hippocampus results in enhanced learning and memory and increased synaptic plasticity

(A) Data from the Morris water maze of Neurl1 overexpressing mice (DT off dox) and control siblings (mean +SEM). Acquisition and transfer: DT reached minimal path lengths faster than controls (*p0.6). (C) (i) LTP induced by 1 TBS was enhanced in DT (mean % of baseline; ANOVA, p=0.01). Induction phase (0-60min) and maintenance (120-180min) were significantly enhanced in DT (mean % of baseline; unpaired t-test; induction: pA blockers. LTP was evoked with a pairing protocol consisting of a presynaptic stimulation (150 pulses at 2Hz) combined with postsynaptic depolarization at 0 mV. LTP magnitude was similar between DT and interleaved control experiments (p=0.9). Inset: sample traces. (v) Basal synaptic transmission was slightly increased in DT (5V: p=0.01; 10V: p=0.04; 15V: p<0.05; 20 and 30V: p=0.1). PPR (vi) was increased (ANOVA, p=0.04). Insets in (v) & (vi): sample traces (PPR: traces for 10, 50, 100, 200 & 300 ms delays between pulses). (vii) LTD induced by one 15min train at 1 Hz was facilitated in DT (mean % of baseline 40-90 min: unpaired t-test p<0.001). See also Figure S3 & Table S2.

Figure 4. Neurl1 overexpression in the hippocampus increases the number of spines and functional synapses and the number of AMPAR subunits GluA1 and GluA2

(A) (i) Computer-assisted reconstructions of representative neurons in adult Neurl1 overexpressing (DT off dox) and control mice. (ii) Golgi stained apical dendrites from CA1 pyramidal neurons. 1-3: DT off dox. 4: DT on dox. 5: control on dox. 6: control off dox. (iii) Mean spine density (+ SEM). It was significantly increased in DT off dox. Inset: mean dendritic length and number of spines that were counted + SEM. *pNeurl1-Flag single transgenic mice. DT: double transgenic mice. (ii) mean fold difference + SEM (comparison with control off dox). Significant increase was observed only in DT off dox (overexpressed Neurl1; *p<0.0001). (D) (i) Western blot of hippocampal lysates (different mouse/lane) from DT and controls (single transgenic; st1-st3). st1 & st3: tetO-Neurl1-Flag, st2: CaMKIIα-tTA. (ii) Mean fold difference + SEM of GluA1 and GluA2 protein levels (comparison with control off dox). DT off dox showed significant increase (*p<0.0001). (E) Mean fold difference + SEM of mRNA levels in the hippocampus of Neurl1 overexpressing (DT) and control mice (real time qPCRs). No differences were found (p>0.3). (F) Time course for 35S-GluA1 and 35S-GluA2 levels in control (mock; infection with control lentivirus) and Neurl1 overexpressing hippocampal cultures (16DIV) after a 1hr pulse of 35S-Met/35S-Cys. (i) Immunoprecipitated 35S-GluA1 and 35S-GluA2 were analyzed by SDS-PAGE and phosphoimager. (ii) Mean half-life + SEM from 3 independent experiments. No differences were found (p>0.8). (G) Comparison of 35S-GluA1 and 35S-GluA2 at 0min. They were significantly increased in Neurl1 overexpressing neurons (*p<0.001). Real time qPCRs did not reveal differences at the mRNA level (inset; p>0.5). See also Figure S4 & Table S4.

Figure 5. Neurl1 interacts with and ubiquitinates CPEB3

(A) (i) Silver stain of hippocampal lysates from adult wild type mice after IP with anti-Neurl1 antibody. CPEB3 was detected as Neurl1-interacting protein. (ii) Co-IP from hippocampal lysates of adult wild type mice. (B) Confocal images of CA1 pyramidal neurons from adult Neurl1 overexpressing mice. CPEB3 and Neurl1 proteins colocalize next to presynaptic sites (synaptophysin puncta). Because CPEB3 and Neurl1 antibodies were produced in rabbit, we used anti-Flag antibody to detect Neurl1-Flag, which exhibited similar subcellular distribution with endogenous Neurl1 and interacted with endogenous CPEB3 (Figure S3A & S5B). (a-c; a’-c’): high magnification of apical dendrites. (a’-c’): cross sections. (C) Neurl1 promotes ubiquitination of CPEB3 in vivo. HEK293T cells were transiently expressing 6-his-tagged ubiquitin and the indicated proteins (i). Neurl1-F: Neurl1-Flag. Neurl1Rm-F: Neurl1-Flag without ubiquitin ligase activity. His-tagged ubiquitinated proteins were affinity-purified on nickel-agarose (Ni-NTA) under denaturing conditions and analyzed by immunoblotting with anti-CPEB3 antibody (ii). Anti-Ub antibody was used to test the recovery of ubiquitinated proteins. (iii) Immunoblot analysis of ubiquitinated species of CPEB3 next to lysate of cells transfected only with CPEB3. The most abundant ubiquitinated species of CPEB3 migrates in molecular size 8kD higher than the non-ubiquitinated protein, likely representing monoubiquitinated CPEB3 (arrow). (D) In vitro ubiquitination assays of CPEB3. Monoubiquitinated CPEB3 (arrowhead) is detected only when Neurl1-F and the rest of the reaction components are present. Anti-Ub antibody was used to test Neurl1-F activity, as it is known to form poly-Ub chains. (E) Isolation of ubiquitinated species of CPEB3 from adult hippocampal lysates using sequential IP with anti-CPEB3 and anti-Ub antibodies. (i) Normalized input of CPEB3 used in IPs. (ii) Detection of ubiquitinated CPEB3. CPEB3 (in vitro ub): Sample from in vitro ubiquitination of CPEB3 by Neurl1. CPEB3: non-ubiquitinated CPEB3 (in vitro assay without Neurl1). St1 & St2: single tetO-Neurl1-Flag and tetO-Neurl1DN-Flag mice, respectively. (iii) Western blot of monoubiquitinated CPEB3 isolated from hippocampal lysate (St1) next to the respective input. (iv) Mean + SEM from 3 independent experiments (1 mouse/genotype, treatment & experiment; controls: single tetO & tTA). Monoubiquitinated CPEB3 was increased in Neurl1 DT (*p<0.0016), while it was reduced in Neurl1DN DT (comparisons with respective control siblings and DT on dox; *p<0.0003). See also Figure S5 & Table S4-6.

Figure 6. Neurl1-dependent ubiquitination and ubiquitin modulate the activity of CPEB3 and increase CPEB3-dependent polyadenylation and translation of GluA1 and GluA2 leading to an increase of their protein levels

(A) Western blots (i-iv) and quantitative (v) analysis of GluA1 and GluA2 proteins in lysates of cultured hippocampal neurons. The neurons were infected with control lentivirus (mock) or lentiviruses expressing the indicated proteins. Anti-Flag antibody was used for the detection of Neurl1-Flag (Neurl1-F) and Neurl1Rm-Flag (Neurl1Rm-F). CPEB3-UbKO: chimeric CPEB3 having fused at its C-terminus single ubiquitin that cannot form poly-Ub chains. CPEB3-EGFP & CPEB3-SUMO: controls. (v) Averaged data from 4 independent experiments. Black asterisks: comparisons with mock. *p<0.0007. (B) 35S-GluA1 and 35S-GluA2 immediately after a 1hr 35S-Met/35S-Cys pulse in cultured hippocampal neurons (16DIV) expressing the indicated proteins by viral gene transfer (i). (ii) Averaged data (+SEM) from 3 independent experiments. Compared to mock, 35S-GluA1 and 35S-GluA2 were significantly increased in neurons overexpressing Neurl1 (Neurl1-F expressing neurons), Neurl1 and CPEB3 together, and CPEB3-UbKO (p<0.008). 35S-GluA1 and 35S-GluA2 in the rest of neurons were similar and significantly lower compared to mock (*p<0.009). (C) Poly(A) assay. (i) Schematic representation of the assay. Universal primer: poly(A)-specific primer. (ii) Assays in cultured hippocampal neurons expressing the indicated proteins. Mock: neurons expressing control lentivirus. (iii) Poly(A) tails of hippocampal GluA1 and GluA2 mRNAs from adult Neurl1 and Neurl1DN double transgenic mice (DT) and respective controls (single tetO; st) kept either off dox (transgene ON in DT) or on dox (transgene OFF in DT). See also Figure S6 & Table S4.

Figure 7. Neurl1 and ubiquitin increase spine number by modulating the translational activity of CPEB3

(A) Dendrites of cultured hippocampal neurons (11DIV) expressing EGFP alone (mock) or EGFP and the indicated proteins. Branches of two neurons are shown. Neurl1-F: Neurons expressing Neurl1-F (Neurl1-Flag; Neurl1 overexpression). Neurl1Rm-F: mutant Neurl1-F. (B) Averaged density of dendritic protrusions + SEM [mock: 12 neurons, 2 branches/neuron (12×2×50μm); all the others: 8 neurons, 2 branches/neuron (8×2×50μm)]. The spine density of neurons overexpressing Neurl1, Neurl1+CPEB3, and CPEB3-UbKO were significantly increased compared to mock (CPEB3+Neurl1-F: p=0.041; all the others: p<0.0001). The difference between neurons overexpressing CPEB3 and neurons overexpressing CPEB3+Neurl1 was highly significant (p<0.0001). In the rest of the neurons, the density of protrusions was similar (p=0.6165), and significantly lower than mock (p<0.0003). See also Table S4 and Figure S7.