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

Abstract

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.

Non-invasive High Frequency Repetitive Transcranial Magnetic Stimulation (hfrTMS) Robustly Activates Molecular Pathways Implicated in Neuronal Growth and Synaptic Plasticity in Select Populations of Neurons

“…The stimulation paradigm was based on the patterns used to induce perforant path LTP, specifically delivery of trains of pulses (8 pulses at 400 Hz, 20 ms duration) at 10 s intervals for 60 or 180 hfrTMS trains. We chose this stimulation paradigm so as to link to our previous studies documenting activation of S6 phosphorylation in the dentate gyrus, which dissected signaling pathways and explored molecular mechanisms (Pirbhoy et al, 2016(Pirbhoy et al, , 2017. It should be noted that different patterns of stimulation are used to induce LTP in hippocampal slices including theta burst stimulation and stimulation with 100 Hz trains.…”

“…It should be noted that different patterns of stimulation are used to induce LTP in hippocampal slices including theta burst stimulation and stimulation with 100 Hz trains. However, consequences of these patterns of stimulation on S6 phosphorylation have not been extensively explored, and so we went with the 400 Hz paradigm where data on neurotransmitters and signaling mechanisms have been partially elucidated (Pirbhoy et al, 2017). The most extensive stimulation (180 trains) involved delivery of hfrTMS trains over a 30-min period, which is sufficient to robustly induce S6 phosphorylation in the dentate gyrus (Pirbhoy et al, 2016).…”

“…Because S6 phosphorylation is activated throughout the cell body and part of the dendritic tree, immunostaining provides information about the neuron types involved. In general, S6 phosphorylation can be activated by the AKT/mTOR pathway (Pende et al, 2004;Chowdhury and Köhler, 2015), but S6 phosphorylation in neurons can also be activated via MAP kinase/ERK 1-2 through NMDA receptordependent mechanisms (Roux et al, 2007;Pirbhoy et al, 2017). Of note, blockade of NMDA receptors with MK801 completely blocks synaptically-driven S6 phosphorylation in the dentate gyrus despite the fact that pharmacological evidence indicates that activation of phosphorylation is via both mTOR-and MAP kinase pathways (Pirbhoy et al, 2017).…”

Selective targeting of mRNA and following protein synthesis of CaMKIIα at the long-term potentiation-induced site

“…HFS induces localized phosphorylation of ribosomal protein S6, a component of the 40S ribosome detected in polyribosomeenriched fractions from cultured cortical neurons (Krichevsky and Kosik, 2001) and associated with initiating the translation of certain mRNAs (Pirbhoy et al, 2016(Pirbhoy et al, , 2017. Electron microscopy has revealed that the S6 immunoreactivity in dendritic spines is transiently increased at sites were LTP is induced (Nihonmatsu et al, 2015), with a time course similar to that for Camk2a mRNA.…”

The Tuberous Sclerosis gene, Tsc1, represses parvalbumin+/fast-spiking properties in somatostatin-lineage cortical interneurons

“…when PV expression first becomes evident during adolescence. This may be achieved as a consequence of activating certain growth factor receptors or potentially via synaptic activity 7,60 in prospective PV-lineages. However, given that Tsc1/2 and other components of the MTOR pathway are expressed in most cell types, there would need to be unique factors coupling MTOR signaling in PV-lineages that are different in SST-lineages.…”