Thus, the signaling pathway for NMDAR-induced upregulation of Kv4.2 likely involves PP1 activation by NMDAR (Chung et al., 2009), leading to dephosphorylation of mTOR. This inhibition of the mTOR pathway then results in dephosphorylation of substrates of S6K1

downstream of mTOR, such as FMRP. To test whether regulation of FMRP phosphorylation at S499 might account for the regulation of Kv4.2-3′UTR-dependent translation, we compared the WT form of FMRP with mutant FMRP with S499 replaced by Alanine (S499A) or Aspartate (S499D) (Ceman et al., 2003), and performed a dual-luciferase reporter assay by cotransfecting HEK293 cells with Renilla Epacadostat luciferase-Kv4.2-3′UTR together with firefly luciferase, plus GFP-tagged FMRP (WT, S499A, S499D), or GFP alone as control. In contrast to the suppression of Kv4.2-3′UTR-dependent luciferase production by FMRP-WT and FMRP-S499D ( Figure 8C), FMRP-S499A showed much less suppression ( Figure 8C). Thus, FMRP phosphorylation at S499 appears to be crucial for FMRP suppression of

translation associated with Kv4.2-3′UTR. To test whether regulation of FMRP phosphorylation affects Kv4.2 channel density on neuronal dendrites, we transfected cultured hippocampal neurons from fmr1 KO mice with GFP-tagged FMRP (WT, S499A, S499D), or GFP alone as control, and used antibody against extracellular epitope of Kv4.2 to assess its surface expression level. In control BLZ945 experiments involving transfecting hippocampal neurons from WT or fmr1 KO mice with GFP, we found higher levels of surface expression of Kv4.2 on the dendrites of neurons from fmr1 KO mice ( Figure 8D). By introducing WT or mutant FMRP into hippocampal

neurons from fmr1 KO mice, we found that neurons expressing FMRP-WT or FMRP-S499D had similar levels of Kv4.2 surface expression whereas neurons expressing FMRP-S499A had significantly increased Kv4.2 protein levels on the surface of their dendrites ( Figure 8D), indicating that the S499D but not S499A mutant form of FMRP retains the ability to suppress Kv4.2. Taken together, our results suggest Kv4.2-3′UTR-dependent protein synthesis oxyclozanide as well as Kv4.2 channel density on neuronal dendrites depends on the status of FMRP phosphorylation. This study provides evidence for dendritic targeting of mRNA of the Kv4.2 dendritic voltage-gated potassium channel that is important for controlling dendritic excitability and synaptic plasticity. FMRP suppresses Kv4.2 expression in basal conditions, and is also involved in NMDAR-mediated Kv4.2 upregulation due to its dephosphorylation. Our study thus defines a signaling pathway linking FMRP with dendritic Kv4.2 regulation by synaptic activity, and provides a lead for consideration regarding the etiology of FXS. In addition to the interaction with FMRP for translation suppression, Kv4.2-3′UTR also mediates dendritic targeting (Jo et al., 2010) and increases steady state levels of mRNA.