Dulka Karolina, Lajkó Noémi, Nacsa Kálmán, Gulya Karoly
Department of Cell Biology and Molecular Medicine, University of Szeged, 6720 Szeged, Hungary.
Epigenomes. 2024 Mar 18;8(1):11. doi: 10.3390/epigenomes8010011.
Temporal and spatial epigenetic modifications in the brain occur during ontogenetic development, pathophysiological disorders, and aging. When epigenetic marks, such as histone methylations, in brain autopsies or biopsy samples are studied, it is critical to understand their postmortem/surgical stability. For this study, the frontal cortex and hippocampus of adult rats were removed immediately (controls) or after a postmortem delay of 15, 30, 60, 90, 120, or 150 min. The patterns of unmodified H3 and its trimethylated form H3K9me3 were analyzed in frozen samples for Western blot analysis and in formalin-fixed tissues embedded in paraffin for confocal microscopy. We found that both the unmodified H3 and H3K9me3 showed time-dependent but opposite changes and were altered differently in the frontal cortex and hippocampus with respect to postmortem delay. In the frontal cortex, the H3K9me3 marks increased approximately 450% with a slow parallel 20% decrease in the unmodified H3 histones after 150 min. In the hippocampus, the change was opposite, since H3K9me3 marks decreased steadily by approximately 65% after 150 min with a concomitant rapid increase of 20-25% in H3 histones at the same time. Confocal microscopy located H3K9me3 marks in the heterochromatic regions of the nuclei of all major cell types in the control brains: oligodendrocytes, astrocytes, neurons, and microglia. Therefore, epigenetic marks could be affected differently by postmortem delay in different parts of the brain.
大脑中的时空表观遗传修饰发生在个体发育、病理生理紊乱和衰老过程中。在研究脑尸检或活检样本中的表观遗传标记（如组蛋白甲基化）时，了解它们的死后/手术稳定性至关重要。在本研究中，成年大鼠的额叶皮质和海马体在死后立即取出（对照组），或在死后延迟15、30、60、90、120或150分钟后取出。对冷冻样本进行蛋白质免疫印迹分析，以分析未修饰的H3及其三甲基化形式H3K9me3的模式，并对石蜡包埋的福尔马林固定组织进行共聚焦显微镜检查。我们发现，未修饰的H3和H3K9me3均呈现出时间依赖性的相反变化，并且在额叶皮质和海马体中，相对于死后延迟，它们的变化有所不同。在额叶皮质中，150分钟后，H3K9me3标记增加了约450%，而未修饰的H3组蛋白缓慢平行下降了20%。在海马体中，变化则相反，因为150分钟后，H3K9me3标记稳定下降了约65%，同时H3组蛋白迅速增加了20%-25%。共聚焦显微镜检查发现，在对照大脑的所有主要细胞类型（少突胶质细胞、星形胶质细胞、神经元和小胶质细胞）的细胞核异染色质区域中都有H3K9me3标记。因此，死后延迟可能会对大脑不同部位的表观遗传标记产生不同的影响。
