The involvement of RARα in RA-mediated homeostatic synaptic plasticity was demonstrated previously using a shRNA-based knockdown method (Aoto et al., 2008
). Although rescue experiments with a shRNA-resistant version of full-length RARα was done in that study, the complexity of RNAi experiments does not allow us to completely exclude the possibility of an off-target effect. We intended to achieve two goals in the current study: to validate the shRNA results using a genetic approach, and to perform structure-function analysis of RARα. Indeed, results from the RARα KO neurons confirmed our previous findings, validating the conclusion that RARα is required for homeostatic up-regulation of synaptic strength. Importantly, co-expression of full-length RARα together with the Cre-recombinase successfully rescued synaptic scaling, making this an ideal system for subsequent structure-function analyses.
Our results provide strong evidence for a non-genomic role of RARα in regulating excitatory synaptic strength. Specifically, we show that knocking out RARα in mature neurons acutely blocks homeostatic up-regulation of synaptic strength, a process mediated by RA. The rescue experiments with various forms of mutant RARα revealed that individual RARα domains perform differential functions in RA-mediated homeostatic synaptic plasticity—the DNA-binding activity of RARα was dispensable, while the RNA-binding activity of the F-domain and the RA-binding activity of the LBD were both required. The LBD and F-domains of the receptor do not participate in DNA-binding, and, therefore, are not known to be directly mediating the transcriptional regulation by RARα. Instead, our previous work demonstrated that the F-domain has mRNA binding abilities and that binding occurs in a sequence specific manner. The consensus sequences for binding are potentially present in many dendritically localized mRNAs, in particular the ones encoding proteins known to be involved in synaptic scaling, such as the mRNA coding for the GluA1 subunit of AMPA receptors (Poon and Chen, 2008
). In vitro
studies suggested that binding of the LBD/F-domain to mRNA regulates translation in an RA-dependent manner so that addition of RA leads to translational de-repression and increased GluA1 protein levels (Poon and Chen, 2008
). Recent evidence from our lab and others indicate that RARα protein is not restricted in the nucleus in mature neurons, but can be found also in neuronal dendrites (Huang et al., 2008
; Maghsoodi et al., 2008
), further supporting a possible non-genomic function of RARα in mature brains. Taken together, we hypothesize that direct binding of RARα to GluA1 mRNA through the F-domain and the ability of RARα to localize to dendrites provides a repertoire of dendritic GluA1 mRNA that is translationally dormant under basal synaptic activity. Another important piece of the puzzle is the regulation of RA synthesis by changes in synaptic activity. We have shown that blocking excitatory synaptic transmission leads to rapid up-regulation of RA synthesis (Aoto et al., 2008
), a process that is tightly controlled by dendritic calcium levels (Wang et al., 2011
). The RA thus made binds to the LBD domain of RARα and reduces its mRNA-binding affinity, allowing rapid increase of dendritic GluA1 synthesis through translational de-repression (Poon and Chen, 2008
). Synaptic insertion of newly synthesized GluA1 homomeric AMPA receptors compensates the decrease in excitatory synaptic transmission. Calcium-permeable AMPA receptors have been implicated in many forms of synaptic plasticity and diseases (Isaac et al., 2007
; Liu and Zukin, 2007
). In this case, the calcium-permeable nature of these receptors sends a negative feedback signal to reduce and eventually halt RA synthesis, thus stabilizing synaptic strength. In this context, findings from this study on the critical role of the RARα LBD/F-domains in synaptic scaling provide an important functional correlate to the observations previously made in vitro
, and also suggest a basic mechanism for translational regulation that allows neurons to quickly respond to changes in activity with increased protein levels.
An unexpected finding of our study is the impact of RARα expression levels on synaptic scaling—synaptic scaling is fully rescued when full-length RARα or the LBD/F-domain of the RARα is expressed in RARα KO neurons, but is impaired by expression of full-length RARα or the LBD/F-domains in WT neurons containing endogenous RARα (Figures and ). A possible explanation for this observation is that there may be two limiting factors in dendrites for synaptic RA-signaling: the amount of RA produced during activity blockade-induced synthesis, and the amount of mRNAs in dendrites that serve as RARα substrates. When RARα is expressed at higher levels, these two factors can limit synaptic scaling through two non-mutually exclusive mechanisms. If the amount of GluA1 mRNA is limiting, there will be a fraction of RARα that is not mRNA-bound. Additionally, a large part of GluA1 mRNA may fail to localize to dendrites because of their association with somatically localized RARα due to high RARα expression levels. If the amount of RA is limiting, on the other hand, part of dendritic RARα that is GluA1 mRNA-bound may not be activated by RA produced during activity blockade. Additionally, the mRNA-free RARα will further exacerbate the situation by competing with GluA1-bound RARα for RA binding. One or both limiting factors could explain the lack of synaptic scaling upon TTX + APV treatment in the presence of excess RARα. The fact that synaptic scaling induced by direct application of exogenous RA is impaired in RARα full-length and LBD/F-domain expressing WT neurons argues that simply supplying more RA is not enough, and that dendritically localized GluA1 mRNA may be the other limiting factor. The developmentally regulated reduction of RARα expression as neurons become mature (Huang et al., 2008
) supports this notion, and suggests that protein expression levels can be tightly coupled with switches of function during development.
In summary, we have in the present study pursued two goals: first, to use rigorous genetic tools to test our hypothesis that RARα, previously only known to function as a nuclear RA-activated transcription factor, leads a double life as a dendritic repressor of protein translation whose repressive activity is reversed by RA (Aoto et al., 2008
); and second, to examine whether the same protein domains of RARα are required for this double life, or whether there is a dissociation in domain requirements for its two functions. Our data confirm RARα's double life, validating that this RA-regulated protein exhibits an amazing and unexpected versatility in which RA activates transcription of genes but inactivates repression of protein translation. Moreover, together with previous studies on the structure/function relationship of RARα as a transcription factor (Evans, 1988
; Green and Chambon, 1988
; Tora et al., 1988a
; Tasset et al., 1990
), our data reveal that the two functional lives of RARα depend on distinct protein domains, whereby RA-binding to the LBD serves as the central event that regulates either an N-terminal transcription activity (via the DBD that is not needed for the translational function of RARα ) or a C-terminal translation repression activity (via the F-domain). Thus, the architecture of RARα exhibits an exquisite symmetry in which the same central regulatory domain (the LBD) acts either on a transcriptional or translational control element, both of which affect gene expression as the final readout but operate by completely different mechanisms.
Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.