It is well known that when animals are treated with protein synthesis inhibitors, such as anisomycin, which stop the production of proteins in the animals brains, these animals have long-term memory impairments. Furthermore, certain types of memories are dependent on the hippocampal protein synthesis for a short period of time following training, after which they are no longer susceptible to hippocampal manipulations. This process has been called systems consolidation, a process by which memory eventually depends more on the cortex than the hippocampus. However, recent studies have suggested that, after having completed the initial systems consolidation process, a memory once again engages the hippocampus when recalled. The accumulating evidence has suggested that continued protein synthesis is essential for the normal function of the brain. The caveat when interpreting the research in this field, however, is that most of the studies use chemicals such as anisomycin, emetine, and cycloheximide, to inhibit protein synthesis which can affect intracellular signaling pathways. For instance, anisomycin has been shown to activate MAP kinase pathways in mammalian cells. In order to overcome this significant drawback, inducible genetic manipulation of protein synthesis knockdown in live animals is desired for the study of memory consolidation at both a cellular and systems level. Genetic manipulation would also allow us to inhibit protein synthesis in a cell-type specific manner, which is hard to achieve by conventional pharmacological intervention.? Since fall of 2003, we have been working on a project to develop inducible genetic suppression of protein synthesis in the mouse brain. It is known that double-strand RNA-dependent protein kinase R (PKR) inhibits synthesis of most proteins in a cell by phosphorylating eIF2α, a key factor to initiate peptide elongation during protein translation process. Taking the advantage of inducible dimerization of PKR domain upon drug administration, we used a chemically induced dimerization system, FKBP12, to control the activity of PKR. First, Zhihong Jiang prepared a cDNA construct of HA-FKBP-PKR under the control of cytomegalovirus (CMV) promoter, and then transfected this construct into a human neuroblastoma SH-SY5Y cell line. She confirmed de novo protein synthesis inhibition 16 hrs after AP20187 treatment. She then asked the Transgenic Core Facility (Dr. Jim Pickel) to inject the construct of loxP-LacZ-loxP-FKBP-PKR, under the control of αCaM Kinase II promoter, into mouse eggs to create transgenic mice. She has since obtained several lines which show high expression of LacZ in the mouse forebrain. One of transgenic strains, called PKR6, showed restricted expression of LacZ in hippocampal CA1 pyramidal cells, and dentate granule cells. Double immunostaining with antibodies against beta-galactosidase and GAD67 (inhibitory interneuron marker) or CaMKII2alpha (pyramidal neuron marker) confirmed that beta-gal positive cells are all excitatory pyramidal (in CA1) and granule cells (in DG). This floxed-PKR mouse line was crossed with forebrain restricted Cre transgenic mice, T29-1, to restrict PKR expression to CA1 and DG in the hippocampus. RT-PCR and western blotting using specific primers and human PKR specific antibody both indicated that PKR was expressed in the hippocampus of the double transgenic line (PKR6/cre+). When dimerizer AP20187 was injected into the lateral ventricle of mutant mouse brain, increased phosphorylation of eIF2alpha was observed in the hippocampus, suggesting that PKR is functionally activated in the mouse brain. To evaluate the effect of our genetic manipulation on the hippocampal dependent behavior, several behavior tests have been conducted. Hippocampal dependent spatial habituation was tested in the open field, and mutant mice after AP20187 infusion showed slower habituation than control mice. Accordingly, mutant behaved as if nave when exposed to the same open field 24 h later. In context discrimination in fear conditioning task, mutants with the drug infused showed a significant decrease in the freezing response during the context test while fear memory associated with a tone was not affected, suggesting an impairment in hippocampal dependent consolidation. Using these system, we are evaluating how long this memory is dependent on the hippocampus before being transferred to other brain areas.
| Nakazawa, Kazu; McHugh, Thomas J; Wilson, Matthew A et al. (2004) NMDA receptors, place cells and hippocampal spatial memory. Nat Rev Neurosci 5:361-72 |
