A rat brain bicistronic gene with an internal ribosome entry site codes for a phencyclidine-binding protein with cytotoxic activity.

The cloning and characterization of the gene for the fourth subunit of a glutamate-binding protein complex in rat brain synaptic membranes are described. The cloned rat brain cDNA contained two open reading frames (ORFs) encoding 8.9- (PRO1) and 9.5-kDa (PRO2) proteins. The cDNA sequence matched contiguous genomic DNA sequences in rat chromosome 17. Both ORFs were expressed within the structure of a single brain mRNA and antibodies against unique sequences in PRO1- and PRO2-labeled brain neurons in situ, indicative of bicistronic gene expression. Dicistronic vectors in which ORF1 and ORF2 were substituted by either two different fluorescent proteins or two luciferases indicated concurrent, yet independent translation of the two ORFs. Transfection with noncapped mRNA led to cap-independent translation of only ORF2 through an internal ribosome entry sequence preceding ORF2. In vitro or cell expression of the cloned cDNA led to the formation of multimeric protein complexes containing both PRO1 and PRO2. These complexes had low affinity (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801)-sensitive phencyclidine-binding sites. Overexpression of PRO1 and PRO2 in CHO cells, but not neuroblastoma cells, caused cell death within 24-48 h. The cytotoxicity was blocked by concurrent treatment with MK-801 or by two tetrahydroisoquinolines that bind to phencyclidine sites in neuronal membranes. Co-expression of two of the other subunits of the protein complex together with PRO1/PRO2 abrogated the cytotoxic effect without altering PRO1/PRO2 protein levels. Thus, this rare mammalian bicistronic gene coded for two tightly interacting brain proteins forming a low affinity phencyclidine-binding entity in a synaptic membrane complex.

Genome-wide transcriptome profiling of region-specific vulnerability to oxidative stress in the hippocampus.

Neurons in the hippocampal CA1 region are particularly sensitive to oxidative stress (OS), whereas those in CA3 are resistant. To uncover mechanisms for selective CA1 vulnerability to OS, we treated organotypic hippocampal slices with duroquinone and compared transcriptional profiles of CA1 vs CA3 cells at various intervals. Gene Ontology and Biological Pathway analyses of differentially expressed genes showed that at all time points, CA1 had higher transcriptional activity for stress/inflammatory response, transition metal transport, ferroxidase, and presynaptic signaling activity, while CA3 had higher GABA-signaling, postsynaptic, and calcium and potassium channel activity. Real-time PCR and immunoblots confirmed the transcriptome data and the induction of OS by duroquinone in both hippocampal regions. Our functional genomics approach has identified in CA1 cells molecular pathways as well as unique genes, such as guanosine deaminase, lipocalin 2, synaptotagmin 4, and latrophilin 2, whose time-dependent induction following the initiation of OS may represent attempts at neurite outgrowth, synaptic recovery, and resistance against OS.

High intrinsic oxidative stress may underlie selective vulnerability of the hippocampal CA1 region.

Oxidative stress (OS) causes extensive cell death in the CA1 but not the CA3 region of the hippocampus. We found that the CA1 region of hippocampus explants, cultured under normal conditions, had significantly higher superoxide levels and expressed both anti-oxidant genes and genes related to the generation of reactive oxygen species at significantly higher levels than the CA3. These observations were indicative of high intrinsic OS in CA1.

Selective dendrite-targeting of mRNAs of NR1 splice variants without exon 5: identification of a cis-acting sequence and isolation of sequence-binding proteins.

Both protein and mRNA for the NR1 subunit of N-methyl-D-aspartate receptors are present in neuronal dendrites and undergo changes in distribution following synaptic excitation. However, the expression of all exonic splice variants of NR1 in dendrites has not been determined. In the present study, antibodies against the exon 5 (ex5) peptide sequence labeled proteins mostly in the soma of hippocampus neurons, whereas antibodies against ex21 or ex22 labeled cell bodies and dendrites. Antisense cRNAs for ex5 hybridized with mRNAs in cell bodies, whereas cRNAs for ex21 with mRNAs in both cell bodies and dendrites. Antisense DNA to a 24-base sequence identified as being present only in the 5'-UTR of cDNAs lacking ex5 (ex5(-)), hybridized with mRNAs in soma and dendrites and this labeling was coincident, mostly, with RNA granules. Insertion of the 24-base DNA ahead of that for enhanced green fluorescent protein (EGFP) increased the transport of EGFP mRNA and the expression of EGFP in neurites of neurons in culture. Fluorescent sense mRNA that contained the 24-base sequence bound to proteins in dendrites and to two proteins, 60 and 70 kDa, in brain microsomes. Proteins of similar size were also labeled by [32P]CTP-mRNA for NR1-(1a), which contains the 24-base 5'-UTR sequence, but not for NR1-(2b), which does not. Biotinylated 24-base sense mRNA was used to purify from brain microsomes two RNA-binding proteins (60 and 70 kDa). We concluded that the 24-base sequence in 5'-UTR of ex5(-) mRNA functioned as a cis-acting, dendrite-targeting element recognized selectively by two microsome proteins.