Novel higher-order epigenetic regulation of the Bdnf gene upon seizures

Abstract

Studies in cultured cells have demonstrated the existence of higher-order epigenetic mechanisms, determining the relationship between expression of the gene and its position within the cell nucleus. It is unknown, whether such mechanisms operate in postmitotic, highly differentiated cell types, such as neurons in vivo. Accordingly, we examined whether the intranuclear positions of Bdnf and Trkb genes, encoding the major neurotrophin and its receptor respectively, change as a result of neuronal activity, and what functional consequences such movements may have. In a rat model of massive neuronal activation upon kainate-induced seizures we found that elevated neuronal expression of Bdnf is associated with its detachment from the nuclear lamina, and translocation toward the nucleus center. In contrast, the position of stably expressed Trkb remains unchanged after seizures. Our study demonstrates that activation-dependent architectural remodeling of the neuronal cell nucleus in vivo contributes to activity-dependent changes in gene expression in the brain.

Figure 1.

Spatial distributions of Bdnf and Trkb in the nuclei of rat hippocampal dentate gyrus neurons; effects of seizures. A, FISH labeling (maximum projection of five optical sections) of the control rat dentate gyrus for Bdnf (green) and Trkb (red); DNA was counterstained using TOPRO 3 (blue). Inset, A neuronal nucleus from control tissue with typical arrangement of Bdnf and Trkb alleles; it represents one of the 4571 nuclei used for and statistical analysis, obtained from 63 animals. Scale bar, 10 μm. B, C, Quantitative analysis of the intranuclear positions of Bdnf (B) and Trkb (C) alleles in the nuclei of the dentate gyrus granule neurons from control animals (blue lines), and animals killed 2 h after the onset of the status epilepticus (orange lines). The minimal distance between the respective alleles and the nucleus surface is presented in the normalized histogram. Red arrow in B indicates the distance of 350 nm from the nuclear envelope; the alleles that are positioned within this distance are considered to be present at the nuclear envelope. D, E, Quantitative analysis of the intranuclear positions of Bdnf (D) and Trkb (E) in the nuclei of the dentate gyrus neurons from control animals (blue bars), and animals killed 2 h, 1 d, 7 d, and 28 d after the onset of the status epilepticus (orange bars). The mean percentages of the nuclei with the minimum distance between respective alleles and the nucleus surface <350 nm are shown; Kruskal–Wallis test: p < 0.001, U test: *p < 0.05, ***p < 0.001. F, Detachment of Bdnf from nuclear lamina verified by chromatin immunoprecipitation assay. The results of ChIP analysis with anti-lamin antibodies demonstrate significant enrichment of Bdnf exons 2 and 9 in the immunoprecipitates from hippocampi of control rats compared with kainate-treated rats. Neither in control nor after seizures the enrichment of the Trkb sequence was observed. Kruskal–Wallis test: p < 0.001, U test: ***p < 0.001.

Figure 2.

The relationship between the intranuclear position of the Bdnf alleles and their activity, investigated by colocalization with immunoreactivity of activated RNA Polymerase II, and by RT-PCR. A, RT-PCR quantification of relative induction of Bdnf mRNA level relative to GAPDH mRNA in control hippocampi and upon seizures. B–E, Immuno-FISH for Bdnf (green) and activated RNA Polymerase II (red), p-Ser2 (B, C) or p-Ser5 (D, E). B, D, Control animals; C, E, animals killed 2 h after the onset of the status epilepticus. Two-channel- and three-channel overlays, and colocalization maps are shown. The approximate position of the border between the nuclear periphery and interior is indicated by dotted line. Scale bar, 5 μm. F, G, Quantitative analysis of the intensity of RNA Polymerase II p-Ser 2 (F) and p-Ser5 (G) signals colocalizing with the Bdnf alleles, in relation to the allele position. Clear bars, The alleles positioned <350 nm from the nucleus surface; dashed bars, >350 nm; blue bars, the control; orange bars, 2 h, 28 d, and 28 d + 2 h (the animals reexposed to kainate) time points. **p < 0.01, ***p < 0.001, t test. For clarity, the depicted differences are restricted to internal alleles.