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.
Subject(s)
Animals , Rats , Carrier Proteins/genetics , Cell Nucleus/metabolism , Cells, Cultured , Dendrites/metabolism , Hippocampus/cytology , Immunohistochemistry , In Situ Hybridization, Fluorescence , Phosphoproteins/genetics , Potassium Chloride/pharmacology , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Rats, Sprague-Dawley , Up-Regulation/drug effectsABSTRACT
Objective To study the localizations and changes of mRNA for nNOS-interacting DHHC domain-containing protein with dendritic mRNA(NIDD) in normal and injured peripheral nerves. Methods Real-time fluorescence quantitative PCR(FQ-PCR) and a combination of in situ hybridization and indirect immunofluorescence were used to detect the localizations and changes of mRNA for nNOS and NIDD. Results It was found that nNOS and NIDD were mainly localized in Schwann cells(SCs) of normal sciatic nerve,and the expression peaked in crushed sections of sciatic nerve in the 2nd week after injury and in proximate or distal transected stumps in the 1st week after injury respectively.Expressions of mRNA for nNOS and NIDD were detected in the perinuclear plasm of primary SCs as well.Conclusion The increase in mRNA for NIDD in SCs may play an important role in axonal regeneration after nerve injury.