The MAPK/ERK cascade targets both Elk-1 and cAMP response element-binding protein to control long-term potentiation-dependent gene expression in the dentate gyrus in vivo

Abstract

The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling cascade contributes to synaptic plasticity and to long-term memory formation, yet whether MAPK/ERK controls activity-dependent gene expression critical for long-lasting changes at the synapse and what the events underlying transduction of the signal are remain uncertain. Here we show that induction of long-term potentiation (LTP) in the dentate gyrus in vivo leads to rapid phosphorylation and nuclear translocation of MAPK/ERK. Following a similar time course, the two downstream transcriptional targets of MAPK/ERK, cAMP response element-binding protein (CREB) and the ternary complex factor Elk-1, a key transcriptional-regulator of serum response element (SRE)-driven gene expression, were hyperphosphorylated and the immediate early gene zif268 was upregulated. The mRNA encoding MAP kinase phosphatase MKP-1 was upregulated at the time point when MAPK/ERK phosphorylation had returned to basal levels, suggesting a negative feedback loop to regulate deactivation of MAPK/ERK. We also show that inhibition of the MAPK/ERK cascade by the MAPK kinase MEK inhibitor SL327 prevented CREB and Elk-1 phosphorylation, and LTP-dependent gene induction, resulting in rapidly decaying LTP. In conclusion, we suggest that Elk-1 forms an important link in the MAP kinase pathway to transduce signals from the cell surface to the nucleus to activate the genetic machinery necessary for the maintenance of synaptic plasticity in the dentate gyrus. Thus, MAPK/ERK activation is required for LTP-dependent transcriptional regulation and we suggest this is regulated by two parallel signaling pathways, the MAPK/ERK-Elk-1 pathway targeting SRE and the MAPK/ERK-CREB pathway targeting CRE.

Fig. 1.

LTP in the dentate gyrus in vivoand corresponding upregulation of zif268 mRNA expression. a, The slope of the EPSP is plotted as a percentage change against the baseline before high-frequency tetanic stimulation (LTP) or a pseudotetanus (control). Each point represents an average of four consecutive evoked responses. The white bar indicates the 10 min period in which the tetanus or the pseudotetanus was delivered. Rats were killed either immediately after the last train of tetani (LTP 0), or 15 (LTP 15) or 60 (LTP 60) min after the tetanus. In rats that were killed immediately after the tetanus, an average of four responses were measured between each tetani. b, Autoradiograms shows upregulation ofzif268 mRNA at different time points after the induction of LTP compared with control rats (CT 0). There were three to four rats in each group at each of the different time points.

Fig. 2.

Increase in p-MAPK/ERK after the induction of LTP.a, Immunocytochemical images representing p-MAPK/ERK in the ipsilateral dentate gyrus show that it is increased at LTP 0 compared with the contralateral side. No change was observed in the controls or LTP 15. b, High magnification (630×) of images show p-MAPK/ERK labeling in both cell bodies and dendrites of the granule cells in the ipsilateral dentate gyrus. c, Densitometric quantification of immunolabeled sections shows that p-MAPK/ERK is significantly increased only at LTP 0 (p < 0.05). d, Western blots of p-ERK1 and p-ERK2 confirm immunohistochemical results showing that the increase in p-MAPK/ERK occurs at LTP 0 (top panel), and this increase was only observed with the activated forms (bottom panel).

Fig. 3.

Expression of MKP-1 mRNA after induction of LTP in the dentate gyrus. In situ hybridization shows that MKP-1 is upregulated at LTP 15 and LTP 60 and that the increase is restricted to the potentiated side of the dentate gyrus. No difference was observed in the control stimulated rats at either time point. There were three to four rats in each group.

Fig. 4.

Increase in p-Elk-1 after the induction of LTP.a, Immunocytochemical images in the ipsilateral dentate gyrus shows an increase in p-Elk-1 at LTP 0 and to a lesser extent at LTP 15 compared with the contralateral side. No change was observed in the control groups. b, High magnification (630×) of these images shows p-Elk-1 labeling in both cell bodies and dendrites in the ipsilateral dentate gyrus. c, Densitometric quantification of immunolabeling shows that p-Elk-1 is significantly increased at LTP 0 and LTP 15. d, Western blots of p-Elk-1 confirm immunohistochemical results showing increased p-Elk-1 at LTP 0 (top panel), and this increase was only observed with the activated form, with no change in total Elk-1 (bottom panel).

Fig. 5.

Inactivation of MEK with SL327 results in disruption of the downstream activation of p-MAPK/ERK, p-Elk-1, andzif268 mRNA expression induced by LTP. a, After the induction of LTP in the presence of SL327, the EPSP declined to basal levels within 60 min, and there was no effect on LTP after injections of DMSO. b, Immunocytochemical images of p-MAPK/ERK in the dentate gyrus at LTP 0 shows that DMSO had no effect on LTP-induced p-MAPK/ERK, whereas injections of SL327 resulted in inhibition of p-MAPK/ERK. c, Similarly, p-Elk-1 was also inhibited by SL327 at LTP 0, with no effect on LTP-induced activation of p-Elk-1 after DMSO. d, In situhybridization images of zif268 at LTP 0 and LTP 60 in rats injected with either SL327 or DMSO. Zif268 was upregulated in the potentiated dentate gyrus in the DMSO control group at both time points, whereas in the SL327 group, LTP-induced upregulation of zif268 was blocked at both time points.e, Quantification of densitometric measures of p-MAPK/ERK and p-Elk-1 at LTP 0 and optical density measures of the expression of zif268 mRNA at LTP 0 and LTP 60 confirm blockade of MAPK/ERK and Elk-1 phosphorylation, and ofzif268 induction in the presence of SL327 (asterisks indicate significant difference from DMSO controls).

Fig. 6.

High magnification of phosphorylated MAPK/ERK and Elk-1, and expression of zif268 at LTP 0 in the presence of DMSO or SL327. a, In the presence of DMSO, there is heavy labeling of p-MAPK/ERK in both dendrites and the cell bodies of granule cells. In addition, there is hyperphosphorylation of Elk-1 (b) and an upregulation of zif268mRNA (c). d, In some cases, there was some labeling of p-MAPK/ERK in the SL327-treated rats, but this was restricted to the dendrites. In these rats, p-Elk-1 was greatly reduced (e), and there was no upregulation ofzif268 mRNA (f), suggesting the need for translocation of MAPK/ERK to the nucleus to phosphorylate Elk-1 and upregulate zif268.

Fig. 7.

Phosphorylation of CREB after induction of LTP and in the presence of SL327. a, b, An increase in p-CREB labeling was observed on the potentiated side at LTP 0 compared with the contralateral dentate gyrus. c,d, Western blots confirm that p-CREB is increased only at this time point compared with control rats, and quantification showed that this was a significant increase. e,f, The level of p-CREB was greatly attenuated in SL327-treated rats compared with DMSO controls.