J Korean Med Sci.  2012 Oct;27(10):1241-1247. 10.3346/jkms.2012.27.10.1241.

Dendritic eIF4E-binding Protein 1 (eIF4E-BP1) mRNA Is Upregulated by Neuronal Activation

• Moon IS
• Lee HJ
• Park IS

• KMID: 1778833
• DOI: http://doi.org/10.3346/jkms.2012.27.10.1241

Abstract

Long-term synaptic plasticity requires addition of new proteins at the synaptic site. The local protein synthesis at subsynaptic sites confers advantageous mechanisms that would regulate the protein composition in local domains on a moment-by-moment basis. However, our information on the identities of 'dendritic' mRNAs is very limited. In this study we investigated the expression of the protein and mRNA for eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4EBP1) in cultured rat hippocampal neurons. Immunocytochemistry (ICC) showed that 4EBP1 protein is highly localized to the nucleus. In dendrites most 4EBP1 punctae were not colocalized with those of eIF4E. In situ hybridization (ISH) and Fluorescence ISH (FISH) revealed that 4EBP1 mRNA was present in dendrites. The FISH signals formed clusters along dendrites that colocalized with ICC signals for Staufen, a marker for RNA granules. The neuronal activation by KCl (60 mM, 10 min) significantly increased the density of 4EBP1 FISH signals in the nucleus after 2 hr, and both in the nucleus and dendrites after 6 hr. Our results indicate that 4EBP1 and its mRNA are present in dendrites, and the mRNA is upregulated and transported to dendritic domains in RNA granules upon neuronal activation.

Keyword

Dendritic mRNA; eIF4E; 4EBP1; FISH; Hippocampal neuron; Immunocytochemistry; ISH

MeSH Terms

Animals
Carrier Proteins/genetics/*metabolism
Cell Nucleus/metabolism
Cells, Cultured
Dendrites/*metabolism
Hippocampus/cytology/drug effects/*metabolism
Immunohistochemistry
In Situ Hybridization, Fluorescence
Phosphoproteins/genetics/*metabolism
Potassium Chloride/pharmacology
RNA, Messenger/*metabolism
RNA-Binding Proteins/metabolism
Rats
Rats, Sprague-Dawley
Up-Regulation/drug effects
Carrier Proteins
Eif4ebp1 protein, rat
Phosphoproteins
RNA, Messenger
RNA-Binding Proteins
Potassium Chloride

Figure

• 

  Fig. 1 Expression of 4EBP1 and eIF4E. Confocal micrographs of dissociate rat hippocampal neurons (DIV16) double-labeled with 4EBP1 (A) and eIF4E (B). A merge image (C) is shown and the insets show the boxed area enlarged in single channels. The boxed area in the inset was further enlarged on the right. The punctae with overlapping signals are marked with arrowheads. * in A, nucleus. * in B, perikaryon. Scale bar, 75 µm.

• 

  Fig. 2 ISH and FISH showing dendritic localization of 4EBP1 mRNA. (A) ISH. DIV16 dissociated rat hippocampal neurons were hybridized with DIG-labeled antisense (a) or sense (b) cRNA probes prepared by in vitro transcription. The boxed area was shown enlarged in the inset. Sister hippocampal neurons were hybridized with DIG-labeled antisense (c) or sense (d) eIF4E cRNA probes. The soma and dendrites are marked by an arrow and an arrowhead, respectively. (B) Colocalization of FISH signals with staufen immunopuncta. Sister hippocampal neurons were hybridized with DIG-labeled anti-sense 4EBP1 cRNA probes (a) followed by ICC with antibodies against staufen (b). The 4EBP1 mRNA clusters colocalizing with staufen immunopuncta were marked by arrowheads in single (a, b) and merge images (c). Scale bars; 75 µm.

• 

  Fig. 3 Subcellular localization of 4EBP1 mRNA. DIV16 dissociated rat hippocampal neurons were hybridized with the DIG-labeled antisense 4EBP1 cRNA probe followed by ICC with antibodies against PSD-95. Confocal micrographs of FISH (A) and ICC (B) images are shown along with a merge (C). The soma and dendrite are marked with an asterisk and an arrowhead, respectively, in A. The inset in C shows the boxed area enlarged in single channels or merge. A FISH signal colocalizing with the ICC signal is marked with an arrowhead. A surface plot of the boxed area in A is shown in D. The positions of nucleus (Nuc) and dendrite (Den) are marked. Scale bars; 75 µm.

• 

  Fig. 4 Upregulation of 4EBP1 mRNA clusters in dendrites of rat hippocampal neurons by KCl treatment. (A) 2 hr post-KCl exposure. Dissociated rat hippocampal cultures (DIV16) were treated with KCl (60 mM, 10 min). After 2 hr, cells were hybridized with DIG-labeled anti-sense 4EBP1 (AS-4EBP1) or sense (S-4EBP1) cRNA probes (FISH) followed by ICC with antibodies against PSD-95 (α-PSD95). Confocal images of single channels or merge are shown. The neuronal and non-neuronal cells are marked with an arrow and an arrowhead, respectively. (B) 6 hr post-KCl exposure. Presentation and markings are similar to A. Neuronal soma and nucleus are marked with an arrow and asterisk, respectively. The large clusters in the neuronal and non-neuronal nuclei (arrow and arrowhead, respectively) are marked in the S-4EBP1 panel. (C) Surface plots. The surface plots of the boxed areas in A and B are shown. The positions of neuronal (Neu-S) and glial (Gli-S) soma are indicated, along with dendrites (Den). (D) Statistics. Statistical significance was assessed by Mann-Whitney U-test. *P < 0.01. Scale bars; 75 µm.

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