2-3′UTR and examined the distribution of endogenous Kv4.2 mRNA in dendrites.
We found similar dendritic localization and punctate pattern of Kv4.2 mRNA in WT and fmr1 KO neurons ( Figure 4C), and similar Kv4.2 mRNA levels along dendrites of neurons with or without FMRP ( Figure 4C). Taken together, these results suggest that FMRP is not essential for Kv4.2 mRNA dendritic targeting or stability in basal conditions. To test for FMRP regulation of Kv4.2 protein expression we compared click here Kv4.2 levels in the hippocampus from adult WT and fmr1 mutant mice, using Kv4.2 KO mice as control for Kv4.2 antibody specificity ( Figure 5A and 5B). We found ∼1.5–2-fold increase of Kv4.2 immunoreactivity in the CA1 dendritic field of the hippocampus from 3-week-old and 2-month-old fmr1 KO mice ( Figure 5A), and a similar increase of Kv4.2 protein levels in the hippocampus from adult fmr1 KO mice ( Figure 5B). Next, we performed surface biotinylation ABT-263 order on cultured hippocampal neurons and used actin both as loading control and to confirm that our biotinylation protocol results in biotinylation of surface but not cytosolic proteins. We found higher surface as well as total Kv4.2 levels in DIV14 hippocampal neurons without FMRP (Figure 5C). Using antibody against an extracellular
epitope of Kv4.2 for immunostaining of unpermeabilized DIV14 hippocampal neurons, we found higher levels of Kv4.2 on the dendritic surface of neurons without FMRP (Figure 5D). Because both total Kv4.2 protein levels and the surface expression of Kv4.2 on dendrites are elevated in the absence of FMRP whereas the relative proportions of surface and total Kv4.2 protein levels were not significantly altered, these findings indicate that FMRP suppresses Kv4.2 production. To test whether FMRP binding to Kv4.2-3′UTR could suppress protein production, we performed an in vitro translation assay using Renilla luciferase transcript fused to Kv4.2-3′UTR together with firefly luciferase transcript
for normalization, and included either purified GST as control or purified GST-mouse full-length FMRP, and nuclease-treated rabbit reticulocyte lysates. We found that FMRP suppressed Kv4.2-3′UTR-dependent translation by 60% (p < 0.001, n = 4) ( Figure 5F). Moreover, expression of MS2BS(6X)-Kv4.2-S.3′UTR but not MS2BS(6X) alone led to an increase of surface Kv4.2 expression ( Figure 5E), indicating that FMRP during suppression of Kv4.2 is relieved by disruption of FMRP interaction with the 3′UTR of Kv4.2 mRNA via MS2BS(6X)-Kv4.2-S.3′UTR. Taken together, these findings support the notion that FMRP suppression of Kv4.2 protein expression in neuronal dendrites is due to translational repression via its association with Kv4.2-3′UTR. To look for evidence of Kv4.2 local translation in the dendrites of cultured hippocampal neurons, we expressed Dendra-Kv4.2 in these neurons, severed a dendrite via UV illumination from the 2-photon microscope, photo-converted the existing Dendra-Kv4.