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

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

Synaptically driven phosphorylation of ribosomal protein S6 is differentially regulated at active synapses versus dendrites and cell bodies by MAPK and PI3K/mTOR signaling pathways

High-frequency stimulation of the medial perforant path triggers robust phosphorylation of ribosomal protein S6 (rpS6) in activated dendritic domains and granule cell bodies. Here we dissect the signaling pathways responsible for synaptically driven rpS6 phosphorylation in the dentate gyrus using pharmacological agents to inhibit PI3-kinase/mTOR and MAPK/ERK-dependent kinases. Using phospho-specific antibodies for rpS6 at different sites (ser235/236 versus ser240/244), we show that delivery of the PI3-kinase inhibitor, wortmannin, decreased rpS6 phosphorylation throughout the somatodendritic compartment (granule cell layer, inner molecular layer, outer molecular layer), especially in granule cell bodies while sparing phosphorylation at activated synapses (middle molecular layer). In contrast, delivery of U0126, an MEK inhibitor, attenuated rpS6 phosphorylation specifically in the dendritic laminae leaving phosphorylation in the granule cell bodies intact. Delivery of the mTOR inhibitor, rapamycin, abolished activation of rpS6 phosphorylation in granule cell bodies and dendrites, whereas delivery of a selective S6K1 inhibitor, PF4708671, or RSK inhibitor, SL0101-1, attenuated rpS6 phosphorylation throughout the postsynaptic cell. These results reveal that MAPK/ERK-dependent signaling is predominately responsible for the selective induction of rpS6 phosphorylation at active synapses. In contrast, PI3-kinase/mTOR-dependent signaling induces rpS6 phosphorylation throughout the somatodendritic compartment but plays a minimal role at active synapses. Collectively, these results suggest a potential mechanism by which PI3-kinase/mTOR and MAPK/ERK pathways regulate translation at specific subcellular compartments in response to synaptic activity

Synaptic activation of ribosomal protein S6 phosphorylation occurs locally in activated dendritic domains

Previous studies have shown that induction of long-term potentiation (LTP) induces phosphorylation of ribosomal protein S6 (rpS6) in postsynaptic neurons, but the functional significance of rpS6 phosphorylation is poorly understood. Here, we show that synaptic stimulation that induces perforant path LTP triggers phosphorylation of rpS6 (p-rpS6) locally near active synapses. Using antibodies specific for phosphorylation at different sites (ser235/236 versus ser240/244), we show that strong synaptic activation led to dramatic increases in immunostaining throughout postsynaptic neurons with selectively higher staining for p-ser235/236 in the activated dendritic lamina. Following LTP induction, phosphorylation at ser235/236 was detectable by 5 min, peaked at 30 min, and was maintained for hours. Phosphorylation at both sites was completely blocked by local infusion of the NMDA receptor antagonist, APV. Despite robust induction of p-rpS6 following high frequency stimulation, assessment of protein synthesis by autoradiography revealed no detectable increases. Exploration of a novel environment led to increases in the number of p-rpS6-positive neurons throughout the forebrain in a pattern reminiscent of immediate early gene induction and many individual neurons that were p-rpS6-positive coexpressed Arc protein. Our results constrain hypotheses about the possible role of rpS6 phosphorylation in regulating postsynaptic protein synthesis during induction of synaptic plasticity