Neuronal activation increases the density of eukaryotic translation initiation factor 4E mRNA clusters in dendrites of cultured hippocampal neurons

Activity-dependent dendritic translation in CNS neurons is important for the synapse-specific provision of proteins that may be necessary for strengthening of synaptic connections. A major rate-limiting factor during protein synthesis is the availability of eukaryotic translation initiation factor 4E (eIF4E), an mRNA 5'-cap-binding protein. In this study we show by fluorescence in situ hybridization (FISH) that the mRNA for eIF4E is present in the dendrites of cultured rat hippocampal neurons. Under basal culture conditions, 58.7 +/- 11.6% of the eIF4E mRNA clusters localize with or immediately adjacent to PSD-95 clusters. Neuronal activation with KCl (60 mM, 10 min) very significantly increases the number of eIF4E mRNA clusters in dendrites by 50.1 and 74.5% at 2 and 6 h after treatment, respectively. In addition, the proportion of eIF4E mRNA clusters that localize with PSD-95 increases to 74.4 +/- 7.7% and 77.8 +/- 7.6% of the eIF4E clusters at 2 and 6 h after KCl treatment, respectively. Our results demonstrate the presence of eIF4E mRNA in dendrites and an activity-dependent increase of these clusters at synaptic sites. This provides a potential mechanism by which protein translation at synapses may be enhanced in response to synaptic stimulation.

Nonspecific association of 2',3'-cyclic nucleotide 3'-phosphodiesterase with the rat forebrain postsynaptic density fraction

The 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), a protein of unknown function in vivo, is abundantly expressed in myelinating glia in two isoforms, CNP1 and CNP2. In this study, immunoblot analysis showed that CNP1 is the major isoform in adult forebrain, and that both isoforms are included in the postsynaptic density (PSD) fraction and tyrosine-phosphorylated at the basal level. However, subcellular distribution and detergent extraction data showed that CNP is nonspecifically associated with the PSD fraction. Immunocytochemistry revealed that CNP is detected, in a weak but punctate pattern, in dissociated rat hippocampal neurons of 3 days to 2 weeks in vitro. The CNP-positive punctae were distributed throughout soma and dendrites, and distinct from PSD95-positive ones. Immunoblot analysis indicated that CNP is also expressed in neuronal stem cell lines, HiB5 and F11. Interestingly, in addition to the known two isoforms, a new CNP isoform of MW 45 kDa was expressed in these cell lines and was the major type of isoform in F11 cells. Taken together, our data suggest that CNP is expressed in the early stage of in vitro development and nonspecifically included in the adult rat PSD fraction.