Neurl1 is Localized in Postsynaptic Sites in the Adult Hippocampus
is expressed throughout the adult forebrain including the cerebral cortex, amygdala, striatum and hippocampus (). We also found Neurl1 protein in the CA1 area of the hippocampus ( & S1
). Within the CA1 region Neurl1 was detected in the cell bodies of the pyramidal neurons, and was distributed along their apical dendrites (). Consistent with its presence at postsynaptic sites, Neurl1 had a complementary immunostaining pattern with the presynaptic marker Synaptophysin (). The postsynaptic localization of Neurl1 was further documented by means of its presence in the Post-Synaptic Density fraction isolated from the hippocampus of adult mice and by colocalization with the postsynaptic protein PSD95 in cultured mouse hippocampal neurons ().
Neurl1 is expressed in the adult mouse forebrain and is localized in dendrites and at post-synaptic sites in hippocampal neurons
Synaptic Activity Increases Neurl1 Levels
In the fly Neuralized overexpression facilitates memory formation. This finding led us ask whether Neurl1 protein level is upregulated by synaptic activity. We found that 30 minutes after the induction of LTP Neurl1 protein level was significantly increased ().
Regulated Inhibition of Neurl1 in the Adult Forebrain Impairs Hippocampal-Dependent Memory
Activity-dependent upregulation of Neurl1 in the hippocampus suggested its possible involvement in mechanisms underlying synaptic plasticity and memory storage. Consistent with this view, Neuralized was found to be limiting for memory formation in Drosophila
(Pavlopoulos et al., 2008
). We therefore asked: is the function of Neurl1 also critical for memory and synaptic plasticity in the mammalian brain? To address this question, we inhibited Neurl1 in the adult mouse forebrain by generating double transgenic (DT) mice using the tetO/tTA system (Mayford et al., 1996
) that allowed us to regulate, reversibly, the expression of the transgene in the adult forebrain of DT mice (Figure S2A
). We inhibited Neurl1 by expressing a dominant negative form (Neurl1DN) that lacks its RING Zn finger, the domain that possesses the ubiquitin ligase activity (Koutelou et al., 2008
). A Flag epitope tag was fused at the C-terminus of Neurl1DN. Neurl1DN was expressed in the same CA1 neurons with Neurl1 and within those neurons in the same neuronal compartments (Figure S2C
). To avoid developmental effects, Neurl1DN
transcription was inhibited until day P40 (Figure S2B
We first assessed memory in the Morris water maze (Morris et al., 1982
). In the hidden version, which tests hippocampal-dependent spatial learning and memory, DT mice learned the task as well as their control littermates, but they had impaired memory of the platform position as evidenced by a probe trial one day after the end of training ( & S2D
). The mice were also impaired in forming a good memory of a new platform location (memory flexibility) and in novel object recognition, another hippocampal-dependent task ( & S2D
Inhibition of Neurl1 in the adult hippocampus impairs memory and synaptic plasticity
Non-cognitive parameters and anxiety levels were similar between all groups (Table S1
), suggesting that the impaired performance of DT mice results from impaired hippocampal-dependent memory. Moreover, when we blocked the expression of Neurl1DN in the adult forebrain by administering doxycycline (Figure S2B
), DT mice displayed cognitive performance similar to controls ( & S2E
). Thus, the expression of Neurl1DN in adult hippocampal neurons impairs hippocampal-dependent memory formation, indicating the requirement of Neurl1-mediated ubiquitination in this process.
Expression of Neurl1DN in The Adult Hippocampus Impairs Synaptic Plasticity at Schaffer Collateral Synapses
Is the hippocampal-dependent memory impairment observed in Neurl1DN expressing mice accompanied by alterations of hippocampal synaptic plasticity? We obtained extracellular recordings from hippocampal slices and induced LTP at the Schaffer collateral pathway using a single theta-burst stimulus (1TBS). We found that in Neurl1DN expressing mice the maintenance of L-LTP was impaired (). These effects were not related to a defect in basal synaptic properties, as both basal transmission and paired-pulse facilitation were unaltered (). LTP in these mice was restored to control levels when Neurl1DN expression was blocked by doxycycline (Figure S2F
In addition to their impaired memory, Neurl1DN mice exhibited an impairment of memory flexibility, an impairment thought to require LTD (Nicholls et al., 2008
; Malleret et al., 2010
). Consistent with this idea, we also found LTD to be impaired in these mice (). Taken together, our data suggest that Neurl1 is required for hippocampal synaptic plasticity, memory and memory flexibility, consistent with the observed activity-dependent upregulation of Neurl1 in wild type mice.
Upregulation of Neurl1 in the Adult Forebrain Enhances Hippocampal-Dependent Memory
The requirement of Neurl1 function in synaptic plasticity and memory, as revealed in the Neurl1DN mice, and the activity-dependent upregulation of Neurl1 level suggest that Neurl1 is likely limiting for these processes. We therefore asked: will overexpression of Neurl1 in the adult mouse forebrain similarly enhance memory and synaptic plasticity? We now generated a second line of DT mice: tetO-Neurl1-Flag/CaMKIIa-tTA
was expressed throughout the forebrain with a pattern similar to that of the endogenous Neurl1
). Both the wild type and the transgenic proteins were expressed in the same CA1 hippocampal neurons and in the same neuronal compartments (Figure S3A
). Neurl1 protein level in DT mice was 1.88 + 0.14 times more than controls (Figure S3A
). Similar to Neurl1DN, we activated the expression of Neurl1-Flag at day P40.
In marked contrast to Neurl1DN mice, Neurl1 overexpressing animals displayed an enhanced performance in learning and formed a good memory of the platform location in the hidden version of the Morris water maze. These mice also had significantly enhanced learning and memory flexibility ( & S3B
). Control mice needed almost twice as much training to reach a comparable level of performance. Moreover, even though by the end of training in the transfer phase both groups performed equally well, Neurl1 overexpressing mice again performed better in a probe trial eight days later ( & S3B
). Thus, Neurl1 overexpression also enhances the maintenance of long-term memory.
Overexpression of Neurl1 in the adult hippocampus results in enhanced learning and memory and increased synaptic plasticity
Neurl1 overexpressing mice also had enhanced memory for novel object recognition ().
Non-cognitive parameters and anxiety levels were similar between all groups (Table S2
). Inhibition of Neurl1-Flag expression and return of Neurl1 level to the wild type state in the adult forebrain of DT mice reversed their enhanced performance in the cognitive tasks ( & S3D
; Table S2
). Thus, consistent with the role of its Drosophila orthologue in flies, Neurl1 overexpression in adult hippocampal neurons facilitates hippocampal-dependent memory formation.
Neurl1 Upregulation in the Adult Hippocampus Enhances Synaptic Plasticity at Schaffer Collateral Synapses
Consistent with the results in Neurl1DN mice, L-LTP induced by 1TBS at the Schaeffer collateral pathway was increased in Neurl1 overexpressing animals (; Table S2
). In addition to an increase in the late phase, the early phase of LTP also was increased, and was present even when we used a weaker LTP induction protocol (1 train at 100Hz; ). This increase in the amplitude of E-LTP was likely due to a stronger depolarization produced by the tetanus. To test this idea more directly, we carried out recordings under whole-cell configuration using a pairing protocol consisting of a low-frequency presynaptic stimulation combined with sustained postsynaptic depolarization to 0 mV and found that indeed the magnitude of E-LTP was not different between Neurl1 overexpressing and control mice (). In further agreement with this idea, the basal synaptic transmission was slightly increased in Neurl1 overexpressing mice, which likely reflects a postsynaptic change as paired-pulse ratio was also slightly enhanced, consistent with a decrease in the probability of glutamate release ().
When we used 4 trains at 100Hz spaced by 5 minutes, which lead to the induction of L-LTP, we found in addition to the facilitation of the early phase a strong increase of the amplitude of L-LTP in Neurl1 overexpressing mice compared to controls (). The changes did not result from either impairment of inhibitory transmission or different basic membrane properties in DT mice (Figure S3C
), and they were reversed when Neurl1 overexpression was blocked in the adult hippocampus (Figure S3E
Consistent with the data obtained from Neurl1DN mice, the LTD also was significantly increased in Neurl1 overexpressing animals ().
Overexpression of Neurl1 Increases the Number of Synapses in the Adult Hippocampus
The increase in basal transmission accompanied by a decrease in glutamate release probability and the bidirectional facilitation of plasticity in Neurl1 overexpressing mice could reflect an increase of the number of functional synapses. To explore this possibility, we investigated the density of spines of the apical dendrites of CA1 pyramidal neurons in Neurl1 overexpressing mice and found it to be significantly increased (54.3%), and to return to control levels in DT mice on dox ( & S4A
Neurl1 overexpression in the hippocampus increases the number of spines and functional synapses and the number of AMPAR subunits GluA1 and GluA2
To determine whether the increased spine number resulted in an increase in the number of functional synapses, we performed whole-cell recordings and monitored miniature EPSCs (mEPSCs) independent of action potentials. We found that the amplitude of mEPSCs was similar in controls and Neurl1 overexpressing mice, suggesting that the number of synaptic glutamate AMPA receptors (AMPAR) was likely not affected by Neurl1 overexpression (). However, the frequency of mEPSCs was significantly increased in the Neurl1 overexpressing animals (). Because release probability is likely not increased in these mice, these results suggest that Neurl1 overexpression in the adult hippocampus, leads to an increase in functional synapses, but not in the number of AMPAR per synapse.
In addition, the levels of three markers of synaptic density, the two post-synaptic markers PSD95 and Shank, and the pre-synaptic marker synaptophysin (Sala et al., 2001
; Shimada et al., 2003
), were significantly increased, and returned back to control levels when the overexpression of Neurl1 was blocked ().
The involvement of Neurl1 in the regulation of synapse formation is consistent with the role of Neurl1 in the maintenance of LTP and LTD (see discussion), and may also explain the facilitation of E-LTP in the Neurl1 overexpressing mice, as more synaptic contacts tend to increase the slope of EPSPs and facilitate the induction of LTP.
Neurl1 Overexpression Increases the Number of AMPA-Type Receptor Subunits GluA1 and GluA2
The increase of the number of functional synapses and the accompanying unaltered amplitude of mEPSCs compared to controls suggest that the overall synaptic population of AMPAR -critical components for synaptic plasticity- is likely increased in Neurl1 overexpressing hippocampal neurons. What is responsible for this increase? We found that the upregulation of Neurl1 in the adult hippocampus significantly increased both the GluA1 and the GluA2 subunits of AMPAR (). We confirmed these observations using cultured hippocampal neurons. We also found that Neurl1 overexpression does not affect the trafficking and endocytosis of AMPAR (Table S3
; Figure S4B&C
). Thus, Neurl1 is involved in regulating the overall protein levels of AMPAR. Perhaps most important, AMPAR levels and spine formation follow similar patterns of change, suggesting that Neurl1 may act on common or parallel molecular mechanisms to regulate these processes.
Neurl1 Facilitates Protein Synthesis of GluA1 and GluA2
In addition to not affecting the mRNA levels of GluA1 and GluA2 in the hippocampus (), Neurl1 overexpression did not affect the rate of degradation of GluA1 and GluA2 proteins. This was evident by: 1) the similar net decrease of GluA1 and GluA2 protein levels in the hippocampus of Neurl1 overexpressing and control mice one and a half hour after the administration of the protein synthesis inhibitor anisomycin, and 2) the similar rates of degradation and half-life of metabolically labelled (35
S) GluA1 and GluA2 proteins in Neurl1 overexpressing and control cultured hippocampal neurons ( & S4D
). Importantly, the levels of newly synthesized 35
S-GluA1 and 35
S-GluA2 in neurons overexpressing Neurl1 were significantly higher than controls (). This increase was not due to differences of the mRNA levels of GluA1 and GluA2 (), suggesting that Neurl1 overexpression augments the protein levels of GluA1 and GluA2 by modulating their translation.
Consistent with the involvement of Neurl1 in the regulation of AMPAR protein synthesis, inhibiting Neurl1 reduced the synthesis of GluA1 and GluA2 proteins in cultured hippocampal neurons without affecting their rate of degradation. Similarly, the protein, but not the mRNA levels, of GluA1 and GluA2 were significantly reduced in the hippocampus of adult Neurl1DN expressing mice (Figure S4E&F
). The fact that reduced basal levels of AMPAR in Neurl1DN mice do not alter basal transmission and the induction of LTP is not surprising as the synaptic population of AMPAR could remain unchanged due to insertion of extrasynaptic AMPAR in the synapse.
Neurl1 Interacts with and Ubiquitinates a Translational Regulator of GluA1 and GluA2: the Cytoplasmic Polyadenylation Element-Binding Protein 3
To investigate further how Neurl1 regulates GluA1 and GluA2 proteins, we applied Neurl1-specific immunoprecipitation and mass spectrometry to examine the proteins that interact with Neurl1 in the hippocampus of adult wild type mice (Extended Experimental Procedures). This analysis identified the Cytoplasmic Polyadenylation Element Binding Protein 3 (CPEB3; Theis et al., 2003
) as one of the proteins interacting with Neurl1 (; Table S5
). CPEB3 is an RNA binding protein that is a member of a protein family that controls polyadenylation of mRNAs in the cytoplasm, a widely used mechanism for the activation of the translation of dormant mRNAs critical for certain forms of synaptic plasticity in the mouse, in the fly, and in Aplysia
). Of the seven Neurl1-interacting proteins that we identified, only CPEB3 was functionally related to the effect of Neurl1 overexpression in synapse formation and in causing an increase in GluA1 and GluA2 translation. Indeed, CPEB3 is a known translational regulator of GluA2 (Huang et al., 2006
) while GluA1 has been recently identified in our laboratory as an additional target (Figure S5A
). Moreover, CPEB3 is a potential functional homologue of CPEB in Aplysia
(Theis et al., 2003
), where it serves as a regulator of translation important for the maintenance of synaptic growth (Miniaci et al, 2008
; Si et al., 2003
). We, therefore, explored the possibility that CPEB3 may be the functional link between the increase of GluA1 and GluA2 translation and the formation of new synapses in Neurl1 overexpressing mice.
Neurl1 interacts with and ubiquitinates CPEB3
We first confirmed the interaction of Neurl1 with CPEB3. We immunoprecipitated CPEB3 from adult hippocampal lysates of wild type mice and detected Neurl1 in the same macromolecular complex (). We also found that CPEB3 and Neurl1 colocalize along the apical dendrites of adult CA1 neurons, in opposition to Synaptophysin ().
Using in vivo ubiquitination assays in HEK293T cells, we found that Neurl1 promotes ubiquitination of CPEB3 and that this depends on its ubiquitin ligase activity (). We also observed that the most abundant form of ubiquitinated CPEB3 migrated at a molecular size that was approximately 8kD higher than the non-ubiquitinated protein, which suggests that it may represent monoubiquitinated CPEB3.
Using in vitro
ubiquitination assays, we found that Neurl1 targets CPEB3 directly for monoubiquitination (). Importantly, we detected monoubiquitinated CPEB3 in the adult hippocampus and found that it was significantly increased in Neurl1 overexpressing mice while it was significantly reduced in Neurl1DN animals (Neurl1: 3.01% + 0.31 of total CPEB3 vs 1.39% + 0.13 in controls; Neurl1DN: 0.48% +0.09 vs 1.37+0.15 in controls; p<0.0004; & S5C
). When the expression of the transgenes in the adult hippocampus was blocked by doxycycline, monoubiquitinated CPEB3 returned back to wild type levels ( & S5C
). Consistent with our data, synaptic stimulation with glutamate in cultured hippocampal neurons led to upregulation of the protein levels of endogenous Neurl1 and increased monoubiquitination of CPEB3. Importantly, this activity-dependent increase of monoubiquitinated CPEB3 was blocked by lentiviral expression of Neurl1DN (Figure S5D&E
Neurl1-dependent Ubiquitination Modulates the Activity of CPEB3 on its Translational Targets GluA1 and GluA2 and Increases their Protein Levels
A well-established function of ubiquitination is the degradation of proteins (DiAntonio and Hicke, 2004
). In the basal state CPEB3 negatively regulates the translation of GluA1 and GluA2 (Huang et al., 2006
; E.R.K. & L. F., unpublished data). Indeed, Neurl1-dependent degradation of CPEB3 might explain how overexpression of Neurl1 increases the levels of GluA1 and GluA2. However, we found this not to be case. In mice overexpressing Neurl1 the protein levels of CPEB3 in the hippocampus were not reduced; rather they were increased and this increase was not due to differences at the mRNA level (Figure S5F
). Yet, in the hippocampus of Neurl1DN mice, we consistently found a reduction of CPEB3, without any alteration at the mRNA level (Figure S4E
). Similarly, the activity-dependent upregulation of Neurl1 in cultured hippocampal neurons was accompanied by Neurl1-mediated increase of the overall protein levels of CPEB3 (Figure S5D
). This paradox could be explained by a CPEB3-independent regulation of GluA1 and GluA2. Alternatively, Neurl1-dependent ubiquitination might affect CPEB3 translational activity by leading to the activation of CPEB3.
To distinguish between these alternatives, we overexpressed Neurl1 in cultured hippocampal neurons using lentiviral gene transfer. Neurl1 overexpression resulted in increased levels of GluA1 and GluA2 proteins (). CPEB3 levels were increased similar to what we observed in the adult hippocampus. But, when we overexpressed CPEB3 alone we found that the levels of GluA1 and GluA2 were dramatically reduced (). This effect of CPEB3 was rescued when Neurl1 was coexpressed with CPEB3 (). To rule out the possibility that Neurl1 affects GluA1 and GluA2 levels by a mechanism not related to the modulation of CPEB3 translational activity, we overexpressed Neurl1 and a truncated form of CPEB3 that retains its RNA binding properties but lacks the first 222 N-terminal aminoacids, a putative prion domain (CPEB3ΔNter; Huang et al., 2006
). The deletion of this domain in Drosophila CPEB (orb2) resulted in impaired long-term memory formation, indicating a critical memory function of this protein region (Keleman et al., 2007
). Importantly, we found that this domain is critical for both the interaction of CPEB3 with Neurl1 and for its ubiquitination (Figure S5G
). If Neurl1 interacts and modulates the activity of CPEB3 and increases the translation of GluA1 and GluA2 mRNAs, this effect would be blocked if we abolished the interaction between CPEB3 and Neurl1. Indeed, CPEB3ΔNter was still capable of reducing the levels of GluA1 and GluA2 but this effect was not reversed by Neurl1 overexpression in contrast to full length CPEB3 ().
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
We also found that Neurl1 regulated CPEB3 activity via ubiquitination. Neurl1Rm-Flag, which does not ubiquitinate CPEB3 (), did not reverse the effect of CPEB3 overexpression on the levels of GluA1 and GluA2 (). Our results are consistent with the idea that Neurl1 ubiquitinates CPEB3 and that the ubiquitinated form of CPEB3 upregulates GluA1 and GluA2 protein synthesis.
Ubiquitin Modulates the Activity of CPEB3 on its Translational Targets GluA1 and GluA2
To test the idea that the addition of a single ubiquitin to CPEB3 is sufficient to modulate the activity of CPEB3 on GluA1 and GluA2 and mimic the effect of Neurl1 overexpression, we generated chimeric CPEB3 fused at its C-terminus to one moiety of ubiquitin with its seven lysines mutated to arginines and incapable to form polyubiquitin chains (CPEB3-UbKO
). This approach has been used to study protein modification by ubiquitin as Ub fusion proteins have been shown to mimic ubiquitination in some instances (Qian et al. 2002
; Li et al., 2003
; Carter and Vousden, 2008
). In contrast to wild type CPEB3, CPEB3-UbKO
increased the protein levels of GluA1 and GluA2 in dissociated hippocampal neurons (). The effect of ubiquitin on CPEB3 activity was specific, and not an artifact of the fusion or a general effect of ubiquitin-like molecules, as chimeric CPEB3-EGFP and CPEB3-SUMO proteins reduced the levels of GluA1 and GluA2, similar to CPEB3 overexpression (). Thus, single ubiquitin is sufficient to activate CPEB3 and CPEB3-dependent translation of GluA1 and GluA2 and to increase their protein levels.
Similar to Neurl1, overexpression of CPEB3-Ub KO increased the surface population of AMPAR without affecting their trafficking and endocytosis (Table S3
; Figure S4B
Neurl1 and Ubiquitin Activate CPEB3-dependent Translation of GluA1 and GluA2 mRNAs
Our data suggest that Neurl1-dependent ubiquitination and ubiquitin modulate CPEB3 translational activity and activate CPEB3-dependent protein synthesis of GluA1 and GluA2. We tested this directly by using 35
S-Cysteine metabolic labelling and examining the levels of newly synthesized GluA1 and GluA2. We obtained complementary results (). The observed effects were due to modulation of GluA1 and GluA2 translation, as we observed no differences at the level of mRNA (Figure S6A
). The changes were specific. We tested the translation of actin and found it unaltered (Figure S6B
). Using reporter assays in HEK293T cells we also found that the translational regulation of GluA1 and GluA2 mRNAs by Neurl1 and ubiquitin was mediated specifically by CPEB3 and required their 3′-UTRs (Table S3
; Figure S6C
), consistent with the role of CPEB3 as an RNA binding protein that binds the 3′-UTR of GluA1 and GluA2 mRNAs (Huang et al., 2006
& figure S5A
). Taken together, these results directly demonstrate that Neurl1 and ubiquitin modulate CPEB3-dependent translation of GluA1 and GluA2.
Neurl1 and Ubiquitin Activate CPEB3-dependent Polyadenylation of GluA1 and GluA2 mRNAs
We next asked: Does ubiquitinated CPEB3 activity promote polyadenylation and increased translation of GluA1 and GluA2 mRNAs? Using polyadenylation assays in cultured hippocampal neurons, we found that consistent with its inhibitory role on GluA1 and GluA2 translation at the basal state overexpression of CPEB3 alone induced shortening of the poly(A) tails of GluA1 and GluA2 mRNAs ( & S6D
). This action of CPEB3 was reversed when Neurl1 was coexpressed. The effect of Neurl1 on CPEB3 activity was blocked when the N-terminal domain of CPEB3 was missing, and it was dependent on its ubiquitin ligase activity (). Expressing CPEB3 fused to single ubiquitin, but not SUMO and EGFP, also led to the elongation of the poly(A) tails of GluA1 and GluA2 mRNAs ().
GluA1 and GluA2 mRNA poly(A) tails were also elongated in the adult hippocampus of mice overexpressing Neurl1, while they were shortened in the hippocampus of mice expressing Neurl1DN (). The observed changes were specific, as the poly(A) length of actin mRNA was unaltered either in cultured neurons or the adult hippocampus (Figure S6E
). Thus, Neurl1-mediated ubiquitination modulates CPEB3-dependent polyadenylation of GluA1 and GluA2 mRNAs.
Neurl1 and Ubiquitin Facilitate Spine Growth by Modulating CPEB3 Translational Activity
In addition to the increased protein levels of GluA1 and GluA2, the number of functional synapses increases in the Neurl1 overexpressing mice. Does Neurl1 also regulate spine formation by modulating CPEB3 translational activity? This modulation of translation would be consistent with a Neurl1-dependent molecular mechanism which links AMPA receptor translation and synapse formation to the role of local polyadenylation and protein synthesis in synaptic growth and plasticity (Sutton and Schuman, 2006
). In agreement with and parallel to our observation in Neurl1 overexpressing mice, we found that Neurl1 overexpression in cultured hippocampal neurons resulted in significantly increased number of spines and filopodia-like protrusions (). This number was reduced when CPEB3 was overexpressed alone. This inhibition was reversed when Neurl1 was coexpressed with CPEB3, similar to what we observed for Neurl1 enhancement of CPEB3 activity on GluA1 and GluA2 (). Expression of CPEB3ΔNter in turn significantly reduced the number of spines and filopodia compared to control neurons and Neurl1 overexpression did not reverse this effect. Neurl1Rm
-Flag was similarly not sufficient to reverse the inhibitory effect of CPEB3. However, expressing CPEB3-UbKO
resulted in pronounced increase of number of spines and filopodia, whereas CPEB3 fused to either DsRed or SUMO resulted in a phenotype similar to wild type CPEB3 (). Taken together, our data suggest that the ability of Neurl1 to ubiquitinate CPEB3 is a limiting factor and facilitates hippocampal synaptic plasticity and hippocampal-dependent memory by modulating CPEB3 translational activity through ubiquitination with consequent induction of an increase of GluA1 and GluA2 protein levels and the formation of new spines.
Neurl1 and ubiquitin increase spine number by modulating the translational activity of CPEB3