Abstract

Long-term facilitation (LTF) in Aplysia is a leading cellular model for elucidating the biochemical mechanisms of synaptic plasticity underlying learning. In Aplysia, LTF requires translational control downstream of the target of rapamycin (TOR) complex 1 (TORC1). The major known downstream targets of TORC1 are 4E binding protein (4E-BP) and S6 kinase (S6K). By removing the site within these regulators required for their interaction with TORC1, we have generated dominant negative proteins that disrupt specific pathways downstream of TORC1. Expression of dominant negative S6K, but not dominant negative 4E-BP, in Aplysia sensory neurons (SNs) blocked 24-h LTF. TORC1 is directly activated by the small GTP-binding protein, Ras homologue enriched in brain (Rheb). To determine the effects of TORC1 activation on translation in Aplysia neurons, we have examined the effects of expressing a constitutively active form of the Aplysia orthologue of Rheb, ApRheb (ApRheb(Q63L)). Expression of ApRheb(Q63L) increased 4E-BP phosphorylation and the level of general, cap-dependent translation within the SN cell soma in a rapamycin-sensitive manner. This increase in cap-dependent translation was blocked neither by dominant negative 4E-BP nor dominant negative S6K. Thus, we demonstrate that S6K is an important downstream target of TORC1 in Aplysia and that it is necessary for 24-h LTF, but not for TORC1-mediated increases in somatic cap-dependent translation.

Highlights

  • We have demonstrated that S6K, but not 4E-BP, plays a critical role during 24-h LTF and have shown that neither S6K nor 4E-BP are involved in TORC1mediated increases in general, somatic translation

  • We will gain a clearer understanding of the significance of this key switch that helps our brains grow and generate imprints of our experiences

  • Acknowledgment—We acknowledge Jake Ormond for the initial degenerate PCR cloning of a fragment of ApRheb

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Summary

EXPERIMENTAL PROCEDURES

Generation of Constructs and Cloning of Aplysia Rheb—The dominant negative 4E-BP construct (4E-BP(⌬TOS)) has previously been described (11). In experiments examining phosphorylation of eEF2, 2 days after microinjection, a single distal neurite was isolated from each SN cell soma, through transection with a micropipette, and allowed to recover for 2 h After this recovery period, the SNs, together with their intact and isolated neurites, were treated with either a single, 10-min pulse of 5-HT (10 ␮M) or a similar mock treatment with artificial sea water (ASW). The mean pixel intensity within the entire cell soma or, in the case of phospho-4E-BP staining and S6K(⌬TOS)-mRFP fluorescence, just within the cytoplasm was measured, and the mean pixel intensity of nonexpressing cells within the same experiment and the mean pixel intensity of the surrounding background were subtracted to account for endogenous fluorescence within the cell and fluorescence from the surrounding media The mean of this net fluorescence in control (vector alone/mRFP construct)-injected cells was calculated, and the values of all individual cells from all groups, including the control group, were normalized to this mean. One-tailed t tests were used only when testing a hypothesis that the means would differ in a given direction, otherwise a two-tailed t test was employed

RESULTS
SN property
DISCUSSION
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