Phosphorylation of ribosomal protein S6 as a mechanism to regulate translation at activated synapses
Long-term potentiation (LTP) is a key candidate for cellular and molecular mechanisms underlying memory formation. LTP induction results in functional and structural changes at synapses, but mechanisms connecting signal transduction pathways to processes responsible for these synaptic modifications remain obscure. The present studies investigate the induction of a post-translational modification, phosphorylation of ribosomal protein S6 (rpS6), in response to synaptic activity and its role in the initiation of translation. Using acute neurophysiological techniques, LTP was induced in the dentate gyrus (DG) of intact, anesthetized rats by delivering high-frequency stimulation (HFS) to the medial perforant path. Immunohistochemical analysis using phospho-specific antibodies that detect phosphorylation at subsets of serine residues, ser235/236, and ser240/244, were used to assess levels of rpS6 phosphorylation. Chapter 2 presents experiments detailing activation parameters of rpS6 phosphorylation in response to synaptic activity. Results show that HFS induces robust phosphorylation of rpS6 (p-rpS6) in granule cell bodies and a selective accumulation in the portion of the dendrites contacted by active synapses. Surprisingly, the selective accumulation at activated synapses was primarily observed with ser235/236. Also, synaptically-driven rpS6 phosphorylation is dependent on NMDA receptor activation and persists for hours after LTP induction. Interestingly, despite the robust induction of p-rpS6 following HFS, assessment of protein synthesis by autoradiography revealed no increases in protein synthesis. Furthermore, a learning experience triggered p-rpS6 in individual neurons in a pattern similar to that of immediate early gene (IEG) induction. Chapter 3 presents studies that investigate the induction of p-rpS6 by PI3K/mTOR and MAPK/ERK-dependent signaling pathways. Local infusions of selective pharmacological agents were delivered into the DG to inhibit fundamental kinases. Results show that PI3K/mTOR and MAPK/ERK signaling pathways regulate p-rpS6 differentially in granule cell bodies and dendrites. Surprisingly, local delivery of the PI3-kinase inhibitor, wortmannin, abolished p-rpS6 specifically in the cell bodies, while sparing p-rpS6 in the dendrites. Results from the local infusion of the MEK inhibitor, U0126, provided further support by revealing a selective reduction of p-rpS6 in the dendrites. Together, these results point to phosphorylation of rpS6 as a mechanism by which PI3K/mTOR and MAPK/ERK signaling regulates translational control in response to neuronal activity.