Abstract
Fragile X syndrome, the most common form of inherited mental retardation, is caused by the absence of the RNA-binding protein fragile X mental retardation protein (FMRP). FMRP regulates local protein synthesis in dendritic spines. Dopamine (DA) is involved in the modulation of synaptic plasticity. Activation of DA receptors can regulate higher brain functions in a protein synthesis-dependent manner. Our recent study has shown that FMRP acts as a key messenger for DA modulation in forebrain neurons. Here, we demonstrate that FMRP is critical for DA D1 receptor-mediated synthesis of synapse-associated protein 90/PSD-95-associated protein 3 (SAPAP3) in the prefrontal cortex (PFC). DA D1 receptor stimulation induced dynamic changes of FMRP phosphorylation. The changes in FMRP phosphorylation temporally correspond with the expression of SAPAP3 after D1 receptor stimulation. Protein phosphatase 2A, ribosomal protein S6 kinase, and mammalian target of rapamycin are the key signaling molecules for FMRP linking DA D1 receptors to SAPAP3. Knockdown of SAPAP3 did not affect surface expression of alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-4-propionate (AMPA) GluR1 receptors induced by D1 receptor activation but impaired their subsequent internalization in cultured PFC neurons; the subsequent internalization of GluR1 was also impaired in Fmr1 knock-out PFC neurons, suggesting that FMRP may be involved in subsequent internalization of GluR1 through regulating the abundance of SAPAP3 after DA D1 receptor stimulation. Our study thus provides further insights into FMRP involvement in DA modulation and may help to reveal the molecular mechanisms underlying impaired learning and memory in fragile X syndrome.
Highlights
The phosphorylation status of fragile X mental retardation protein (FMRP) has been identified as a regulator for FMRP function [16, 17]
We demonstrate that DA D1 receptor stimulation could induce expression of synapse-associated protein 90/PSD95-associated protein 3 (SAPAP3) and dynamic protein 3; PFC, prefrontal cortex; PP2A, protein phosphatase 2A; mTOR, mammalian target of rapamycin; S6K1, ribosomal protein S6 kinase; KO, knock-out; GPCR, G protein-coupled receptor; mGluR, metabotropic glutamate receptor; AMPA, ␣-amino-3-hydroxyl-5-methyl-4-isoxazole-4-propionate; WT, wild type; siRNA, small interference RNA; RT, reverse transcription; PBS, phosphate-buffered saline; ACSF, artificial cerebrospinal fluid
To further elucidate the molecular mechanisms for FMRP involvement in DA modulation, we investigated whether DA receptor stimulation could induce synapse-associated protein expression in adult PFC slices
Summary
Animals—Adult male C57Bl/6 mice were used in most of the experiments. Fmr wild type (WT) and KO mice of the FVB.129P2-Fmr1tm1Cgr strain were generously provided by Dr W. The membranes were incubated in the appropriate horseradish peroxidase-coupled secondary antibody for 2 h at room temperature followed by ECL detection of the proteins with Western Lightning Plus ECL substrate (PerkinElmer Life Sciences) according to the manufacturer’s instructions. To immunoprecipitate PP1 or PP2A, rabbit anti-PP1 or mouse anti-PP2A antibodies were added to tissue lysate and gently mixed at 4 °C overnight, followed by 50% protein A-agarose bead slurry and incubation for 2 h at 4 °C. Surface Biotinylation Assay—Surface AMPA receptor subunits were detected by a biotinylation assay, followed by Western blot analysis [40]. Ice-cold PBS (with calcium and magnesium, pH 7.4; Invitrogen) were added to the cultures or slices to prevent receptor internalization. Biotinylated proteins were separated on 4 –12% SDS-PAGE gel and detected by Western blot using specific antibodies for AMPA receptor subunits.
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