It is well known that when animals are treated with protein synthesis inhibitors, such as anisomycin, which stop the production of proteins in the brain, these animals lose their long-term memory. This observation has led us to predict that the formation of long-term memory requires new protein synthesis. Furthermore, certain types of memories are dependent on the hippocampus 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. This accumulating evidence has suggested that continued protein synthesis is essential for the normal function of the brain. The caveat when interpreting the research of this field, however, is that most of the studies use chemicals such as anisomycin, emetine, and cycloheximide to inhibit protein synthesis. Recent studies have demonstrated that these protein synthesis chemical inhibitors can induce mRNA expression, a process called super-induction. 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 the 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 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 eIF2alpha, 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 hr 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 alphaCaM Kinase II promoter, into mouse eggs to create transgenic mice. She has since obtained several lines which shows high expression of LacZ in the mouse forebrain. One of transgenic strains showed relatively restricted expression of LacZ in hippocampal CA1 pyramidal cells, and dentate granule cells. This floxed-PKR mice was crossed with forebrain restricted Cre transgenic mice, T29-1, to restrict PKR expression to forebrain, in particular to CA1 in the hippocampus. In order to address whether de novo protein synthesis inhibition efficiently occurs in the mouse brain following intra-peritoneal administration of AP20187, we took two approaches after mutant (Cre+/floxed-PKR+) and control (Cre-/floxed-PKR+) mice were intra-cerebroventricularly injected with drug inducer, AP20187. First, hippocampal protein homogenates on the SDS-PAGE were immunostained with anti-phospho-eIF2alpha 20 min after induction, and in vivo induction of eIF2alpha phosphorylation was confirmed. Second, after AP20187 administration, S35-Methionine was infused into lateral ventricles to label the de novo protein synthesis. We found that de novo protein synthesis was inhibited by approximately 30% overall in the mutant hippocampal homogenates 4 hr after AP20187 infusion. De novo synthesis of some of proteins was inhibited over 80% on the SDS-PAGE gels. For a more accurate measure, we are currently evaluating regional protein synthesis inhibition using the C14-Leucine method in collaboration with Dr. Carolyn Beebe Smith at NIMH. Also, preliminary results in collaboration with Bai Lu at NICHD suggest that late phase LTP in area CA1 is absent in the mutants after AP20187 application to hippocampal slices. In a separate project (MH002824-04), Zhihong Jiang has attempted to create hippocampal CA1-restricted Cre transgenic lines using BAC transgenic technology. Since the number of transgenic pups we obtained from the Transgenic Core facility was limited, we need to continue screening the Cre transgenic lines using our BAC11-Cre construct under control of the region-specific keratocan promoter.
| 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 |
