We propose a model for the bidirectional control of local translation that could underlie synaptic plasticity. NR2A mRNA, which contains CPEs in its 3′ UTR, has a short poly(A) tail and is translated inefficiently. The RNA is bound by CPEB, which in turn is associated with PARN, Gld2, symplekin, and Ngd; because Ngd is also bound to eIF4E, the cap binding factor, translation is blocked at initiation. TBS-evoked NMDAR activation promotes phosphorylation of CPEB and expulsion of PARN from the RNP complex. Gld2 then catalyzes poly(A) addition to NR2A mRNA, which we surmise leads to the displacement of Ngd from eIF4E, the binding of eIF4G to eIF4E, and translational enhancement of NR2A mRNA. Although we show that CPEB phosphorylation and Gld2-dependent polyadenylation occurs in dendrites, it is also possible that somatic mRNA regulation by the CPEB complex contributes to the observed affects. Nonetheless, the fact that dendritic translation mediates synaptic plasticity (Huber et al., 2000
; Kang and Schuman, 1996
) coupled with our data showing that CPEB, Gld2, and Ngd regulate dendritic mRNA polyadenylation and translation as well as synaptic efficacy suggests that local polyadenylation-induced translation could directly influence synaptic plasticity.
Gld2 is an important regulator of neuronal function as it enhances dendritic spine maturation, LTP, dendritic polyadenylation, and dendritic NR2A levels. In support of this assertion, a dominant negative Gld2 mutant inhibits long-term memory in Drosophila
(Kwak et al., 2008
). We have identified 102 mRNAs whose poly(A) tail size is reduced following depletion of Gld2, including several encoding plasticity-related proteins. For example, HuD is involved in dendritic morphogenesis and memory formation (Bolognani et al., 2007
), Sos1 links glutamate receptors to the Erk signaling pathway (Tian et al., 2004
), and Neto2 affects kainate receptor function (Zhang et al., 2009
). Gld2 also affects the stability of miR122 by regulating its monoadenylation in liver and primary fibroblasts (Katoh et al., 2009
; Burns et al., 2011
). Although there is very little miR122 in the brain, it is possible that Gld2 regulates the stability of other neuronal miRNAs, which could influence synaptic function.
We focused on NR2A as a target of Gld2 because NMDARs are implicated in synaptogenesis, synaptic plasticity and cognitive functions such as learning and memory. NMDARs are tetramers consisting of two NR1 subunits and two NR2 subunits, and NMDARs in the hippocampus are primarily NR1/NR2A, NR1/NR2B, or NR1/NR2A/NR2B hetero-trimers (Gray et al., 2011
). NR2A and NR2B differentially affect NMDAR channel properties, protein interactions, and subcellular localization, thus NR2A and NR2B subunit expression critically regulate synaptic function (Bellone and Nicoll, 2007
; Lau and Zukin, 2007
). LTP at hippocampal mossy fiber-CA3 synapses is expressed by insertion of NMDA receptors, thus NR2A-containing NMDARs can play a role in LTP expression as well as induction (Kwon and Castillo, 2008
; Rebola et al., 2008
). It is unknown whether local protein synthesis is involved in this process, but it is conceivable that a local pool of newly synthesized NR2A could contribute to activity-dependent NMDAR insertion. Indeed, LTP stimulation leads to NR2A production in the DG (Williams et al. 1998
; Wang et al. 2002
). It is also possible that NR2A synthesis might be required to enhance future synaptic responses. In any case, we envision that Gld2 helps maintain NR2A homeostasis in synapto-dendrites, and thus may act as a rheostat to provide the proper level and/or stoichiometry of NMDAR subunits. Even so, other Gld2 target mRNAs may also contribute to synaptic efficacy.
Other neuronal mRNAs polyadenylated in the cytoplasm such as those encoding α CaMKII (Wu et al., 1998
), AMPA receptor binding protein (Du and Richter, 2005
), and tissue plasminogen activator (Shin et al., 2004
) were not detected as having diminished poly(A) tail length following Gld2 knockdown. These results might indicate that a second poly(A) polymerase also functions in the cytoplasm of neurons; possible candidates include canonical poly(A) polymerase (Huang et al., 2002
) or Gld4 (Burns et al., 2011
). The extent to which CPEB mediates Gld2 function is also unclear. Gld2 has no RNA binding domain and must be tethered to RNA via an RNA binding protein. CPEB is one protein that anchors Gld2 to RNA, but Gld2 also interacts with the RNA binding proteins that may increase the repertoire of mRNAs that are regulated by polyadenylation (Kim et al., 2009
). In the mammalian brain, one could envision how Gld2 might interact with different RNA binding proteins, and thus, activate different mRNAs in a synaptic stimulation-dependent manner.
NMDA treatment of neurons causes CPEB phosphorylation, PARN expulsion from the CPEB-containing RNP complex, and mRNA polyadenylation. These observations suggest that PARN would play a critical role in dendritic translation and possibly synapse function. However, PARN knockdown had only a modest affect on dendritic spine morphology in cultured neurons and no significant effect on TBS-evoked LTP in the DG. It is possible that the PARN knockdown of ~50% was not sufficient to alter synaptic plasticity, or the loss of PARN may not have been sufficient to overcome other negative regulators of translation in the same RNP complexes, such as Ngd. Alternatively, PARN might control the deadenylation of mRNAs when LTP is induced by different stimulation protocols, or in response to stimuli that evoke LTD.
We have shown that Gld2 and Ngd mediate protein synthesis-dependent LTP in the DG. In our paradigm, LTP was protein synthesis-dependent shortly after TBS. In cultured neurons, NMDA stimulation elicited rapid CPEB phosphorylation, PARN exclusion, and dendritic polyadenylation, perhaps signifying that CPEB-mediated polyadenylation has an important early role during synapse potentiation in the DG. Moreover, Gld2 knockdown affected basal polyadenylation in addition to activity-induced poly(A) levels, which could indicate that steady-state Gld2 activity primes some dendritic mRNAs for impending stimulation-induced translation. It is also possible that CPEB complex proteins control the threshold for eliciting L-LTP by regulating basal translation as eIF2 phosphorylation does (Costa-Mattioli et al., 2007
In this report, we have defined a new molecular mechanism and identified critical factors controlling translation and synaptic plasticity in mammalian neurons. Translational control of any particular mRNA is often a complex process involving factors that influence different steps in translation. Indeed, NR2A translation is also regulated in part by an FMRP-microRNA pathway (Edbauer et al., 2010
). FMRP, like CPEB, regulates the translation of particular mRNAs in a stimulus-specific manner; it responds to mGluR-mediated signaling to regulate translation in dendrites (Bassell and Warren, 2008
). If one presumes that unique as well as shared mRNAs are translated in response to, for example, NMDAR- and mGluR-mediated signaling, then it becomes evident how combinations of newly synthesized proteins could impart characteristics to synapses that are exclusive to each signaling event.