Project description:Expression of VP16-CREB, a constitutively active form of CREB, in hippocampal neurons of the CA1 region lowers the threshold for eliciting the late, persistent phase of long-term potentiation (L-LTP) in the Schaffer collateral pathway. This VP16-CREB-mediated L-LTP differs from the conventional late phase of LTP in not being dependent on new transcription. This finding suggests that in the transgenic mice the mRNA transcript(s) encoding the protein(s) necessary for this form of L-LTP might already be present in CA1 neurons in the basal condition. We used high-density oligonucleotide arrays to identify the mRNAs differentially expressed in the hippocampus of transgenic and wild-type mice. Experiment Overall Design: To identify the specific gene whose induction in hippocampal neurons correlated with the facilitated L-LTP phenotype, we harvested hippocampal mRNA at time points assessed in our physiological study and carried out a gene expression analysis using oligonucleotide microarrays. To obtain a sufficient quantity of poly(A)-RNA and reduce the effect of the biological variability of the sample, hippocampi from 6 to 10 mice matched for genotype and time of induction were pooled together in each sample. The final data set included genechips for animals on dox (gene Off) and for animals that were one, two, and five weeks after removal of dox (gene On). We also included samples from wild-type mice maintained under identical conditions and from transgenic mice that expressed VP16-CREB for three weeks before turning the transgene off again for two weeks with dox (Rev, gene Off). Since the isolation of mRNA from the whole hippocampus favors genes that are more broadly over-expressed in the hippocampus of transgenic mice and, therefore, may lead to an underestimation of the total complement of genes showing an altered expression in specific hippocampal subregions, we extended our comparison between transgenic and wild-type expression profiles using microdissected CA1 regions. We obtained two samples corresponding to VP16-CREB mice three weeks after induction and one sample corresponding to wild-type mice kept in the same conditions. Experiment Overall Design: These twelve mRNA samples were analyzed using Affymetrix genechips MG-U74v2 setA.

Project description:We compare here the neurodegenerative processes observed in the hippocampus of bitransgenic mice with chronically altered levels of cAMP-response element-binding protein (CREB) function. The combination of genome-wide transcriptional profiling of degenerating hippocampal tissue with microscopy analyses reveals that the sustained inhibition of CREB function in A-CREB mice is associated with dark neuron degeneration, whereas its strong chronic activation in VP16-CREB mice primarily causes excitotoxic cell death and inflammation. Furthermore, the meta-analysis with gene expression profiles available in public databases identifies relevant common markers to other neurodegenerative processes and highlights the importance of the immune response in neurodegeneration. Overall, these analyses define the ultrastructural and transcriptional signatures associated with these two forms of hippocampal neurodegeneration, confirm the importance of fine-tuned regulation of CREBdependent gene expression for CA1 neuron survival and function, and provide novel insight into the function of CREB in the etiology of neurodegenerative processes. For each mouse genome 430 2.0 gene expression array (Affymetrix, Santa Clara, CA, USA), total RNA was extracted from the hippocampi of three to four mice with the same age, sex, and genotype to produce one pooled sample. We analyzed three late A-CREB pooled samples (6-week-old mice) and three late VP16-CREB pooled samples (3-week-off dox) with their corresponding control littermate samples (three pooled samples for each strain). To compare with early changes, we included two pooled A-CREB early samples (3-week-old mice) and two pooled VP16-CREB early samples (1-week-off dox) with their corresponding control littermate samples. In the case of A-CREB mice, we used the dataset GSE14320. We prepared new samples from bitransgenic mice and control littermates 1 week after transgene induction and hybridized them to mouse genome 430 2.0 genechips. The arrays were hybridized, washed, and screened for quality according to the manufacturerâs protocol.

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Mus musculus

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