Evaluation of Post‐Mortem Effects on Global Brain DNA Methylation and Hydroxymethylation

Abstract

The number of epigenetic studies on brain functions and diseases are dramatically increasing, but little is known about the impact of post-mortem intervals and post-sampling effects on DNA modifications such as 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Here, we examined post-mortem-induced changes in global brain 5mC and 5hmC levels at post-mortem intervals up to 540 min., and studied effects of tissue heat stabilization, using LUMA and ELISA. The global 5mC and 5hmC levels were generally higher in the cerebellum of adult rats than neonates. When measured by ELISA, the global 5mC content in adults, but not neonates, decreased with the post-mortem interval reaching a significantly lower level in cerebellum tissue at the post-mortem interval 540 min. (2.9 ± 0.7%; mean ± S.E.M.) compared to control (3.7 ± 0.6%). The global 5hmC levels increased with post-mortem interval reaching a significantly higher level at 540 min. (0.29 ± 0.06%) compared to control (0.19 ± 0.03%). This suggests that the post-mortem interval may confound 5mC and 5hmC analysis in human brain tissues as the post-mortem handling could vary substantially. The reactive oxygen species (ROS) level in cerebellum also increased over time, in particular in adults, and may be part of the mechanism that causes the observed post-mortem changes in 5mC and 5hmC. The global 5mC and 5hmC states were unaffected by heat stabilization, allowing analysis of tissues that are stabilized to preserve more labile analytes. Further studies in human samples are needed to confirm post-mortem effects on DNA methylation/hydroxymethylation and elucidate details of the underlying mechanisms.

DNA methylation and histone post-translational modification stability in post-mortem brain tissue

“…In a study of pig brains that remained in the skull at room temperature until the desired PMD time points were reached, up to 48% of DNA was degraded by 120 h post-mortem, and there was increased variance in DNA methylation [19]. In a study of neonatal and adult rat cerebellum left at room temperature for 0, 5, and 9 h post-mortem, there was a significant decrease in global 5mC at 9 h in the adult brains (2.9 ± 0.7% vs. 3.7 ± 0.6% in control), but no change in the neonates [25]. Not surprisingly, storage at cool temperature after death is an important factor, possibly because enzyme kinetics associated with autolysis are slowed.…”

“…Sirtuins are NAD+-dependent nuclear proteins; therefore, when energy production by mitochondria ceases after death, sirtuins are rendered inactive [26]. In mouse brain, HDAC 1 and 2 are the most prevalent, HDAC 3, 4, and 5 moderately so, and HDAC 6 is of low abundance [25]. In human frontal cortex, HDACs 1, 2, 5, and 6 are present, with HDAC2 the most highly expressed and HDAC 5 the least [27].…”

Genome-wide DNA methylation comparison between live human brain and peripheral tissues within individuals

“…Using human postmortem brain tissues, epigenetic studies among psychiatric populations have found differential DNA methylation (DNAm) changes in both candidate gene studies and genome-wide analyses, including in schizophrenia 1 , bipolar disorder 2 , and major depressive disorder (MDD) 3 , but access to brain tissue for psychiatric disorders is limited by a small number of postmortem brain samples. Additionally, considerations remain regarding the stability and the biological implications of measurements done on postmortem tissues, as the levels of global DNAm have been shown to change in relation to postmortem interval 4,5 . Consequently, psychiatric epigenetic studies with peripheral tissues, such as blood or saliva samples, have become common 6–12 .…”

Global DNA methylation and cognitive and behavioral outcomes at 4 years of age: A cross‐sectional study

“…Fisher et al (2015) recently demonstrated that the CpG site cg23933044 was hypomethylated in buccal cells in twins ( n = 24) with reported psychotic symptoms at 12 years of age and in postmortem brain tissue of adults with schizophrenia ( n = 38; Fisher et al, 2015). However, recent evidence suggests that postmortem intervals and postsampling effects may induce changes in global DNA methylation levels in the brain; therefore, these findings should be interpreted with caution (Pidsley & Mill, 2011; Sjoholm, Ransome, Ekstrom, & Karlsson, 2018). To address the limitations of using postmortem brain tissue, Braun et al (2019) compared genome‐wide DNA methylation in live brain tissue samples ( n = 27) from patients aged 5–61 years with medically intractable epilepsy undergoing brain resection, with peripheral tissues (blood, saliva, and buccal).…”