Presence of translation elongation factor-1A (eEF1A) in the excitatory postsynaptic density of rat cerebral cortex.

The postsynaptic density (PSD) is a proteinaceous cellular structure that is specialized for postsynaptic signal transduction. Here, we show that eukaryotic translation factor-1A (eEF1A; formerly known as eEF-1alpha) is associated with the excitatory PSD in rat forebrain. Immunoblot analysis showed that eEF1A in the PSD fraction is enriched over homogenate. Salt (1.0M NaCl), but not non-ionic detergents such as Triton X-100 (1.0%) and n-octyl glucoside (1.0%), could dissociate eEF1A from the PSD core. In cultured cortical neurons, eEF1A was colocalized with postsynaptic markers (PSD95 and SynGAPalpha), but not with a presynaptic marker (synaptophysin). These results indicate that eEF1A is present in the PSD of the excitatory synapses.

Carbofuran induces apoptosis of rat cortical neurons and down-regulates surface alpha7 subunit of acetylcholine receptors.

Carbofuran (CF), an anticholinesterase carbamate, is one of the most widely used N-methylcarbamate esters in insect and nematode control. Despite its serious adverse health effects on wildlife and humans, cellular and molecular studies of the damage of CF to CNS neurons are very limited. We have examined the cytotoxic effects of CF on cultured rat cortical cells, and the expression of the alpha7 subunit of the nicotinic acetylcholine receptor (alpha7 nAChR) in hippocampal neurons. CF was cytotoxic with an IC50 approximately 730 and approximately 640 microm when assessed by the lactate dehydrogenase (LDH) assay and propidium iodide (PI) staining, respectively, 3 days after treatment. CF induced DNA fragmentation and exposure of phosphatidyl serine (PS) on the cell surface. Surface labeling of the alpha7 nAChR with Alexa Fluor 488-conjugated alpha-bungarotoxin (alphaBgt) revealed a significant decrease in the density of the subunits in treated (500 microm CF) hippocampal neurons. Our data indicate that CF induces neuronal death by apoptosis and down-regulates nAChRs.

Presence of translation elongation factor-1A in the rat cerebellar postsynaptic density.

This study examined a 55 kDa protein in the rat cerebellar postsynaptic density (PSD) fraction. The amino acid sequence of an HPLC-purified peptide derived from tryptic digestion of the protein was contained in eukaryotic translation elongation factor-1A (eEF1A, formerly known as eEF-1alpha). Immunoblot analysis showed that eEF1A is enriched in the PSD fraction and is tightly associated with the PSD 'core'. The association of eEF1A with the PSD was further evidenced by colocalization of the protein with PSD95, a PSD marker, in dissociated cerebellar cultures and immunoelectron microscopy of the adult rat cerebellum. Combined, our results indicate that eEF1A is associated with the PSD.

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.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the expression of synaptic proteins in dissociated rat cortical cells.

We investigated the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the expression of synaptic proteins in dissociated E18 rat cortical cells. TCDD (0-50 nM) was added to plating media, and cell viability and expression of synaptic proteins were assayed on 4 and 7 days in vitro (DIV), respectively. TCDD had no apparent effect on early neurite outgrowth 12 h after plating. However, on 4 DIV, cell viability was reduced significantly, and neurons often revealed vacuoles in 20 or 50 nM culture and had limited secondary or higher order dendritic processes in 20 or 50 nM culture. Immunoblot analyses of cell homogenates indicated upregulation of NMDA receptor subunits (NR1, NR2A, and NR2B), but downregulation of synaptic organizing proteins (PSD-95, densin-180, and septin6 homologue) and a synapse-enriched enzyme (alphaCaMKII). Changes in the expression of synaptic proteins may be a underlying mechanism for altered synaptic transmission and neuropathy by TCDD.