The regulated activity of transcription factors plays crucial roles in the development and maintenance of connections in the nervous system. Neuronal activity, in the form of membrane depolarization, is known to influence transcription factor activity (Greer and Greenberg 2008). Membrane depolarization activates voltage-gated calcium channels such as the NMDA receptor to initiate signaling cascades, including the activation of downstream kinases and phosphatases, to alter transcription factor function (Morishita et al. 2001; Genoux et al. 2002; Cohen and Greenberg 2008; Hardingham 2009; Paoletti et al. 2013). Thus, many of the profound effects of the NMDA receptor on neuronal development, viability, and plasticity are mediated, in part, through the regulated post-translational modification of transcription factors.
Sp4 is a zinc finger transcription factor that is highly expressed in neurons (Mao et al. 2007). Alterations at the Sp4 gene locus have been linked to psychiatric disorders, including bipolar disorder, major depressive disorder, and schizophrenia (Zhou et al. 2009; Tam et al. 2010; Shi et al. 2011; Shyn et al. 2011). Reduced levels of the Sp4 protein have been directly observed in the cerebellum and pre-frontal cortex of bipolar disorder subjects, and Sp4 levels in the cerebellum are inversely correlated with severe negative symptoms in schizophrenia (Pinacho et al. 2011, 2013). Mice with reduced Sp4 expression displayed deficits in learning and memory and impaired pre-pulse inhibition, a suggested endophenotype for schizophrenia and other psychiatric disorders (Zhou et al. 2005). Consistent with observed memory deficits, Sp4 hypomorphs exhibited decreased long-term potentiation in hippocampal slice recordings (Zhou et al. 2010).
Sp4 activity is likely to be highly dependent on the cellular and developmental contexts of its expression. In dentate granule neurons of the hippocampus, Sp4 promotes dendrite outgrowth and branching (Zhou et al. 2007). We have previously shown that in developing cerebellar granule (CG) neurons, Sp4 is required for dendritic morphogenesis by limiting dendrite branching and promoting the elimination of excess primary dendrites (Ramos et al. 2007, 2009). The maturation of CG neuron dendrites is concomitant with the arrival of excitatory mossy fibers, and this process is regulated in vitro by membrane depolarization. These observations suggested that depolarization regulates Sp4 activity, and, indeed, depolarization enhances the stability of the Sp4 protein (Pinacho et al. 2011). The specific pathways that regulate the stability and activity of the Sp4 protein in response to extracellular signals, however, are unknown.
Here, we identify a site of phosphorylation on Sp4 at S770 that is reduced in response to membrane depolarization. We provide evidence that the NMDA receptor-dependent activation of a protein phosphatase 1/2A (PP1/PP2A) signaling pathway reduces Sp4 phosphorylation at S770. Inhibition of the NMDA receptor increased Sp4 S770 phosphorylation while having no effect on the levels of the protein, indicating that S770 phosphorylation and degradation are separable processes. A non-phosphorylatable mutant of Sp4 promoted CG neuron maturation, whereas a phosphomimetic Sp4 mutant impaired this function, suggesting that the phosphorylation state of Sp4 S770 influences the dendritic maturation of CG neurons. These data describe Sp4 as a transcription factor regulated downstream of NMDA receptor activation, revealing new mechanisms by which neuronal activity informs the gene expression programs of the nervous system.
- Top of page
- Materials and methods
- Acknowledgments and conflict of interest disclosure
- Supporting Information
We present three major findings in this work: First, we identify a novel site of phosphorylation on transcription factor Sp4 at S770. Second, using a phosphospecific antibody we find that Sp4 phosphorylation at S770 is reduced by the activation of the NMDA receptor through a PP1-dependent signaling pathway. Third, we provide evidence that phosphorylation impairs the function of Sp4 in promoting the developmental maturation of CG neuron dendrites.
The role of NMDA receptor signaling in neuronal development is largely context dependent. Activation of the receptor has been shown to promote dendrite outgrowth in some circumstances (Sin et al. 2002; Lei et al. 2006; Sepulveda et al. 2010), and to enhance dendrite elimination in others (Datwani et al. 2002; Monnerie et al. 2003; Lee et al. 2005; Espinosa et al. 2009). The NMDA receptor regulates multiple transcription factors to alter cellular gene expression programs, and it is likely that the specific regulation of these transcription factors contributes to the varied physiological outcomes of receptor activation (Lyons and West 2011). Although degradation of Sp4 has been reported in response to excitotoxic glutamate receptor activation (Mao et al. 2007), we show here for the first time that Sp4 is a transcription factor regulated specifically by NMDA receptor signaling in non-excitotoxic conditions. As Sp4 activity is also context dependent, our data support the view that the specific downstream signaling from the NMDA receptor may influence the outcome of Sp4 activity.
Sp4 has been implicated in the regulation of dendrite patterning, the induction of long-term potentiation, contributions to behavior including learning and memory, as well as psychiatric disorders such as bipolar disorder and schizophrenia (Zhou et al. 2005, 2007, 2009, 2010; Ramos et al. 2007; Pinacho et al. 2011). The NMDA receptor has also been linked to many of these processes (Paoletti et al. 2013). In fact, altered NMDA receptor signaling was suggested to contribute to some phenotypes in Sp4 mutant mice, as reduced levels of the NMDA receptor subunit 1 subunit were observed in these animals (Zhou et al. 2010). On the basis of our data, we hypothesize that the modification state of Sp4, informed by NMDA receptor signaling, influences Sp4 transcriptional activity and ultimately contributes to NMDA receptor-dependent dendrite elimination.
Our results also implicate the NMDA receptor-dependent activation of the PP1/PP2A phosphatase in the regulated dephosphorylation of Sp4. Because of the pharmacological and molecular-genetic limitations of studying phosphatases, we cannot rule out a contribution of PP2A to the dephosphorylation of Sp4; however, our data strongly implicate the activity of PP1. PP1 has been shown to specifically contribute to certain forms of NMDA receptor-dependent long-term depression (Mulkey et al. 1994; Morishita et al. 2001; Genoux et al. 2002). NMDA receptor activation of PP1 is often observed in response to stimulation paradigms that activate long-term depression and require calcineurin activation. Our data suggest a calcineurin-independent pathway regulating PP1 activity in CG neurons. Calcineurin-independent activation of PP1 downstream of the NMDA receptor has also been observed, although the specific mechanisms mediating this activation are currently unknown (Sala et al. 2000). An important unanswered question is the identity of the kinase regulating Sp4 S770 phosphorylation. Experiments using pharmacological inhibitors targeting predicted candidate kinases have not yet identified the relevant kinase, and this remains an important ongoing effort.
We show here that both membrane depolarization and NMDA receptor signaling reduce Sp4 S770 phosphorylation. Only in the absence of membrane depolarization, however, is Sp4 protein degradation observed, as inhibition of the NMDA receptor did not reduce Sp4 protein levels (Fig. 4b). These observations suggest that Sp4 phosphorylation at S770 alone is not sufficient to mediate Sp4 protein degradation. In support of this, we did not observe changes in steady-state expression of full-length FLAG-Sp4 when S770 was mutated to alanine or aspartic acid (Figure S1a). The regulation of Sp4 levels and activity by multiple cell signaling pathways likely allows for more precise control of this transcription factor. Given our finding of an Sp4 phosphorylation site at S136, it is highly likely that additional post-translational modifications contribute to regulating the activity and stability of Sp4.
We investigated the function of Sp4 phosphorylation at S770 using point mutants. We provide evidence that while the non-phosphorylatable S770A mutant was functional, the phosphomimetic S770D mutant failed to promote CG neuron dendritic maturation. Phosphomimetics are often useful to assay the function of phosphorylation, however, it is a concern that a mutation may destroy the function of the protein or that the mutant will not recapitulate the true activity of a phosphoprotein (Tarrant et al. 2012). We observed that both the S770A and S770D mutants were expressed and had a similar ability as wild type to activate a reporter gene, indicating that many Sp4 functions were preserved in the phosphomimetic mutant (Figure S1b). Thus, while the exact mechanism by which Sp4 phosphorylation at S770 impairs CG neuron maturation remains unclear, we propose that it involves the failure to activate a specific subset of Sp4 target genes. As there are currently very few described target genes of Sp4, further studies are needed to identify the specific genes regulating dendrite pruning.
In conclusion, this study identifies a signal-dependent phosphorylation of the transcription factor Sp4 at S770 that impairs the Sp4-dependent maturation of CG neurons and is regulated by an NMDA-receptor/PP1 signaling pathway. Our results describe a new transcriptional component downstream of NMDA receptor signaling, and also suggest that the phosphorylation state of Sp4 is a mechanism regulating Sp4 activity. These results expand our understanding of the signal-dependent mechanisms regulating neuronal gene expression, which have broad implications for neuronal development and disease.