Summary The molecular mechanism how RISC and microRNAs selectively and reversibly regulate mRNA translation in response to receptor signaling is unknown but could provide a means for temporal and spatial control of translation. Here we show that miR-125a targeting PSD-95 mRNA allows reversible inhibition of translation and regulation by mGluR signaling. Inhibition of miR-125a increased PSD-95 levels in dendrites and altered dendritic spine morphology. Bidirectional control of PSD-95 expression depends on miR-125a and FMRP phosphorylation status. miR-125a levels at synapses and its association with AGO2 is reduced in Fmr1 KO. FMRP phosphorylation promotes the formation of an AGO2-miR-125a inhibitory complex on PSD-95 mRNA, whereas mGluR signaling of translation requires FMRP dephosphorylation and release of AGO2 from the mRNA. These findings reveal a novel mechanism whereby FMRP phosphorylation provides a reversible switch for AGO2 and microRNA to selectively regulate mRNA translation at synapses in response to receptor activation.

Fragile X syndrome is a common form of inherited mental retardation and is caused by loss of fragile X mental retardation protein (FMRP), a selective RNA-binding protein that influences the translation of target messages. Here, we identify protein phosphatase 2A (PP2A) as an FMRP phosphatase and report rapid FMRP dephosphorylation after immediate group I metabotropic glutamate receptor (mGluR) stimulation (Ͻ1 min) in neurons caused by enhanced PP2A enzymatic activity. In contrast, extended mGluR activation (1-5 min) resulted in mammalian target of rapamycin (mTOR)-mediated PP2A suppression and FMRP rephosphorylation. These activitydependent changes in FMRP phosphorylation were also observed in dendrites and showed a temporal correlation with the translational profile of select FMRP target transcripts. Collectively, these data reveal an immediate-early signaling pathway linking group I mGluR activity to rapid FMRP phosphorylation dynamics mediated by mTOR and PP2A.

Fragile X syndrome is a common form of cognitive deficit caused by the functional absence of fragile X mental retardation protein (FMRP), a dendritic RNA-binding protein that represses translation of specific messages. Although FMRP is phosphorylated in a group I metabotropic glutamate receptor (mGluR) activity-dependent manner following brief protein phosphatase 2A (PP2A)-mediated dephosphorylation, the kinase regulating FMRP function in neuronal protein synthesis is unclear. Here we identify ribosomal protein S6 kinase (S6K1) as a major FMRP kinase in the mouse hippocampus, finding that activity-dependent phosphorylation of FMRP by S6K1 requires signaling inputs from mammalian target of rapamycin (mTOR), ERK1/2, and PP2A. Further, the loss of hippocampal S6K1 and the subsequent absence of phospho-FMRP mimic FMRP loss in the increased expression of SAPAP3, a synapse-associated FMRP target mRNA. Together these data reveal a S6K1-PP2A signaling module regulating FMRP function and place FMRP phosphorylation in the mGluR-triggered signaling cascade required for proteinsynthesis-dependent synaptic plasticity.Fragile X syndrome is the most common form of inherited mental retardation and is caused by a functional absence of the RNA-binding protein, fragile X mental retardation protein, FMRP.3 The protein, FMRP, is known to associate with ϳ3% of the mammalian brain mRNAs, repressing their translation; many of these target mRNAs indeed appear overtranslated in the absence of FMRP (1). Electrophysiology of the fragile X mouse model has revealed a synaptic deficit in the hippocampus with elevated group I metabotropic glutamate receptor (mGluR)-mediated long term depression. Accordingly, ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor internalization is enhanced in the absence of FMRP, all thought due to constitutive overtranslation of an long-term depression (LTD)-required FMRP target mRNA(s) (2-4). Group I mGluR activity is also known to influence FMRP transport and synthesis (5, 6); however, little is known about regulation of FMRP itself.Post-translational modifications are known regulators of activity-dependent protein synthesis (7), and FMRP is known to be phosphorylated at a highly conserved serine at position 499. The effects of FMRP phosphorylation on translation by ribosomal run-off assays suggested that non-phosphorylated FMRP associates with actively translating ribosomes, whereas phosphorylated FMRP is found in the context of potentially stalled ribosomes (8). We recently determined that FMRP is dephosphorylated by protein phosphatase 2A (PP2A) and, immediately following mGluR stimulation, PP2A dephosphorylates FMRP, corresponding with the translation of SAPAP3, an FMRP target mRNA (9). SAPAP3 is a post-synaptic scaffolding protein associated with PSD95, whose message was previously identified as an FMRP target mRNA in the mouse brain following FMRP immunoprecipitation and microchip analyses (10,35,36). Less than 2 min following mGluR activation, FMRP is rephosphorylated in a PP2A-and mamm...

Fragile X syndrome (FXS), a common inherited form of mental retardation, is caused by the functional absence of the fragile X mental retardation protein (FMRP), an RNA-binding protein that regulates the translation of specific mRNAs at synapses. Altered synaptic plasticity has been described in a mouse FXS model. However, the mechanism by which the loss of FMRP alters synaptic function, and subsequently causes the mental impairment, is unknown. Here, in cultured hippocampal neurons, we used siRNAs against Fmr1 to demonstrate that a reduction of FMRP in dendrites leads to an increase in internalization of the ␣-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) subunit, GluR1, in dendrites. This abnormal AMPAR trafficking was caused by spontaneous action potential-driven network activity without synaptic stimulation by an exogenous agonist and was rescued by 2-methyl-6-phenylethynyl-pyridine (MPEP), an mGluR5-specific inverse agonist. Because AMPAR internalization depends on local protein synthesis after mGluR5 stimulation, FMRP, a negative regulator of translation, may be viewed as a counterbalancing signal, wherein the absence of FMRP leads to an apparent excess of mGluR5 signaling in dendrites. Because AMPAR trafficking is a driving process for synaptic plasticity underlying learning and memory, our data suggest that hypersensitive AMPAR internalization in response to excess mGluR signaling may represent a principal cellular defect in FXS, which may be corrected by using mGluR antagonists.fragile X syndrome ͉ autism

Summary Translational control of mRNAs in dendrites is essential for certain forms of synaptic plasticity and learning and memory. CPEB is an RNA-binding protein that regulates local translation in dendrites. Here, we identify poly(A) polymerase Gld2, deadenylase PARN, and translation inhibitory factor neuroguidin (Ngd) as components of a dendritic CPEB-associated polyadenylation apparatus. Synaptic stimulation induces phosphorylation of CPEB, PARN expulsion from the ribonucleoprotein complex, and polyadenylation in dendrites. A screen for mRNAs whose polyadenylation is altered by Gld2 depletion identified >100 transcripts including one encoding NR2A, an NMDA receptor subunit. shRNA depletion studies demonstrate that Gld2 promotes and Ngd inhibits dendritic NR2A expression. Finally, shRNA-mediated depletion of Gld2 in vivo attenuates protein synthesis-dependent long-term potentiation (LTP) at hippocampal dentate gyrus synapses; conversely Ngd depletion enhances LTP. These results identify a pivotal role for polyadenylation and the opposing effects of Gld2 and Ngd in hippocampal synaptic plasticity.