Role of p90 ribosomal S6 kinase in long-term synaptic facilitation and enhanced neuronal excitability

Abstract

Multiple kinases converge on the transcription factor cAMP response element-binding protein (CREB) to enhance the expression of proteins essential for long-term synaptic plasticity and memory. The p90 ribosomal S6 kinase (RSK) is one of these kinases, although its role is poorly understood. The present study exploited the technical advantages of the Aplysia sensorimotor culture system to examine the role of RSK in long-term synaptic facilitation (LTF) and long-term enhancement of neuronal excitability (LTEE), two correlates of long-term memory (LTM). Inhibition of RSK expression or RSK activity both significantly reduced CREB1 phosphorylation, LTF, and LTEE, suggesting RSK is required for learning-related synaptic plasticity and enhancement in neuronal excitability. In addition, knock down of RSK by RNAi in Aplysia sensory neurons impairs LTF, suggesting that this may be a useful single-cell system to study aspects of defective synaptic plasticity in Coffin-Lowry Syndrome (CLS), a cognitive disorder that is caused by mutations in rsk2 and associated with deficits in learning and memory. We found that the impairments in LTF and LTEE can be rescued by a computationally designed spaced training protocol, which was previously demonstrated to augment normal LTF and LTM.

Figure 1

5-HT induced phosphorylation of CREB1 was attenuated by MEK inhibition. (A) Representative confocal images of pCREB1 immunofluorescence in SNs 2 h after 5 pulses of Veh or 5-HT. Immunoreactivity was located in the nucleus. Scale bar, 20 μm. (B) Summary data. 5-HT induced increase in pCREB1 was blocked by the MEK inhibitor U0126 (U0) (n = 5 independent experiments and in each experiment 5–10 SNs were analyzed in each group). In this and subsequent illustrations, significant differences are indicated by * for P < 0.05, and N.S. indicates that the difference between two groups is not significant.

Figure 2

5-HT-induced increase in pRSK was blocked by a MEK inhibitor. (A) 5-HT increased pRSK immunoreactivity without affecting tRSK. (A1) Representative confocal images of pRSK immunofluorescence in SNs 1 h after 5 pulses of Veh or 5-HT. Immunoreactivity was located in both the cytoplasm and nucleus. (A2) Immunoreactivity for total RSK. (A3) Summary data. Significant differences between 5-HT and Veh are indicated by * for P < 0.05 (Paired t-test, n = 5 independent experiments). (B) Immunoreactivity for pRSK after 5-HT was blocked by U0126 (U0). (B1) Protocol for U0126 application with 50 μM 5-HT or Veh. (B2) Representative confocal images of pRSK in SNs 1 h after treatment. (B3) Summary data. 5-HT-induced increase in pRSK was blocked by preincubation with U0 (n = 4 independent experiments). Scale bar, 20 μm.

Figure 3

Biphasic regulation of pRSK by the Standard protocol (five 5-min pulses of 5-HT, 50 μM). (A) Representative confocal images of pRSK immunofluorescence in SNs at different times after the end of 5-HT. Scale bar, 20 μm. (B) Summary data. The percent change was calculated as the change of pRSK level after 5-HT compared to the control level. pRSK was elevated at 1 h after treatment, followed by a decrease at 2 h. A second wave of increase was evident at 5 h, and pRSK levels returned to basal levels at 24 h. Statistical analysis (Wilcoxon signed rank test with Bonferroni correction) revealed significant differences between Veh and 5-HT treatment groups at 1 h and 5 h.

Figure 4

Inhibition of RSK reduced the 5-HT-induced phosphorylation of CREB1 and reduced LTF. (A) BID, a RSK inhibitor, blocked 5-HT-induced increases in pCREB1. (A1) Protocol for BID application with 5-HT or Veh. (A2) Representative confocal images of pCREB1 in SNs 2 h after treatment. (A3) Summary data. 5-HT-induced increase in pCREB1 was blocked by pre-incubation with BID (n = 6 independent experiments). Scale bar, 20 μm. (B) BID attenuated LTF. (B1) Protocol for BID application with 5-HT or Veh and EPSP recording. (B2) Representative EPSPs recorded immediately before (pre) and 24 h after treatment with 5-HT or Veh. Dashed lines represent the amplitude of the pre-test EPSPs. (B3) Summary data. Application of BID significantly reduced LTF produced by the S protocol (Veh, n = 7; 5-HT, n = 8; BID, n = 7; BID + 5-HT, n = 7).

Figure 5

Knock down of the expression of RSK by siRNA injection reduced LTF. (A) Representative EPSPs recorded immediately before (pre-test) and 24 h after (post-test) treatment with 5-HT. Dashed lines represent the amplitude of the pre-test EPSP. (B) Summary data. RSK-siRNA significantly reduced 5-HT-induced LTF [(Con-siRNA, 5-HT), n= 6; (RSK-siRNA, Veh), n= 6; (RSK-siRNA, 5-HT), n = 6].

Figure 6

RSK-siRNA impaired LTF is restored by an Enhanced protocol. (A) Two 5-HT protocols. The patterns of 5-HT pulses are illustrated in each panel. The Standard protocol (S) had uniform ISIs of 20 min, whereas the Enhanced protocol (E) had non-uniform ISIs of 10, 10, 5 and 30 min. (B) The deficit in LTF induced by RSK-siRNA injection can be rescued by the Enhanced protocol. (B1) Representative EPSPs recorded immediately before (pre-test) and 24 h after (post-test) treatment with 5-HT or Veh. Dashed lines represent the amplitude of the pre-test EPSPs. (B2) Summary data. In each SN-MN pair, the EPSPs that were measured 24 h post 5-HT treatment were normalized to the pre-test EPSP. A value of 0% represents no change in EPSP amplitude. RSK-siRNA injection reduced S protocol-induced LTF, whereas this attenuation in EPSP amplitude was significantly rescued by the E protocol (Con-siRNA + S, n = 7; RSK-siRNA + S, n = 7; RSK-siRNA + E, n = 7). (C) The decreased pCREB1 in RSK siRNA-injected, S treated SNs was restored by the E protocol (Con-siRNA + S, n = 5; RSK-siRNA + S, n = 4; RSK-siRNA + E, n = 4).

Figure 7

Enhanced training protocol rescued BID-induced impairment of LTEE. (A) BID blocked the S protocol-induced LTEE in isolated SNs. (A1) Action potentials were recorded from cultured SNs before (pre), and 24 h after treatment with S or vehicle (post). (A2) Summary data (24 h post-test). A value of 100% represented no change in cell excitability. A significant increase in cell excitability was found in the S group, but this LTEE was blocked by BID (Veh, n = 5; BID, n = 5; S, n = 6; BID + S, n = 5 independent experiments). (B) BID-blocked LTEE was restored by the E protocol. (B1) Electrophysiological recording of action potentials (APs). (B2) Summary data. The E protocol rescued BID-impaired LTEE (S, n = 8; BID + S, n = 8; E, n = 6; BID+E, n = 6 independent experiments).

Figure 8

Schematic model describing activation of Aplysia CREB1, deactivation of Aplysia CREB2, and consequent induction of genes regulated by a cAMP response element (CRE) that is required for the induction of LTF. CREB1 can be phosphorylated by both PKA and RSK. Phosphorylation of CREB1 is assumed necessary for induction of gene expression. CREB2 represses gene expression. Phosphorylation of CREB2 by ERK removes this repression. Arrows and circles indicate positive and negative regulation of transcription, respectively.