Graff et al (2009) sought to know the role of PP1 in chromatin remodeling as well as its possible contribution to epigenetic regulation of memory and learning. This investigation was to be accomplished by coming up with transgenic mice which would facilitate selective inhibition of PP1 in the nucleus of forebrain neurons. The authors of this study worked on the hypothesis that histone posttranslational modification (PTMs) in neurons is altered by PP1 inhibition in the nucleus of the forebrain thus impacting gene transcription and altering long-term memory.
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The authors prepared transgenic mice (NIPPI*-EGFP double transgenic mice). Control mice did not have the fragment contained in the transgenic mice. The researchers then conducted several procedures including reverse-transcription polymerase chain reaction (RT-PCR), immunohistochemistry, protein phosphate assay, coimmunoprecipitation, in vitro phosphorylation assay, HDAC activity assay, western blotting, quantitative RT-PCR, preparing lentivirus and injecting the same in hippocampal slices, and chromatin immunoprecipitation.
The behavior of the mice was assessed by testing for novel object recognition for five minutes in five sessions followed by memory testing where the mice were introduced to a novel object in addition to two already familiar objects. The discrimination ratio was then calculated as well as molecular analysis of the killed mice. Several statistical analyses were conducted including analysis of variance (ANOVA) with appropriate post hoc tests such as Tukey’s test, and 2-tailed paired t-test. Statistical significance was determined at.05,.01, and.001 levels.
It was identified that NPP1* was dominantly expressed mainly in the cortex and hippocampal formation but it was marginally present in the amygdala. Non-forebrain structures lacked the nuclear inhibitor NPP1*. In addition, NPP1* was only present in the nucleus of neurons thus only inhibition of PP1 was limited to the nucleus. Graff et al (2009) identified that nuclear PP1 forms an important component of the brain’s epigenetic machinery since inhibition of PP1 interfered with the association of PP1 and histone-modifying enzymes. Western blot analyses identified that multiple histone posttranslational modifications are altered by nuclear PP1 inhibition.
The authors of this study further noted (by the aid of ChIP assays) that inhibiting nuclear PP1 leads to altered gene expression (transcription to be specific) and this leads to altered memory formation since the genes responsible for memory formation are affected. In addition, the study revealed that all long-term memories depending on hippocampal structures as well as cortical structures are enhanced by nuclear PP1 inhibition. Lastly, Graff et al (2009) revealed that long-term memory induces specific histone PTMs including H4K5 acetylation and H3K36 trimethylation, all of which enhance CREB expression. Overall, the study shows that PP1 that is pooled in the nucleus of neurons in adults is crucial in epigenetic regulation of memory formation.
Koshibu, K., Graff, J., Beullens, M., Heitz, F. D. and Berchtold, D. et al. (2009). Protein Phosphatase 1 regulates the histone code for long-term memory. The Journal of Neuroscience, 29(41):13079 –13089.