Effect of aging on 5-hydroxymethylcytosine in brain mitochondria.

Nuclear epigenetics of the mammalian brain is modified during aging. Little is known about epigenetic modifications of mitochondrial DNA (mtDNA). We analyzed brain samples of 4- and 24-month-old mice and found that aging decreased mtDNA 5-hydroxymethylcytosine (5hmC) but not 5-methylcytosine (5mC) levels in the frontal cortex but not the cerebellum. Transcript levels of selected mtDNA-encoded genes increased during aging in the frontal cortex only. Aging affected the expression of enzymes involved in 5-methylcytosine and 5-hydroxymethylcytosine synthesis (mitochondrial DNA methyltransferase 1 [mtDNMT1] and ten-eleven-translocation [TET]1-TET3, respectively). In the frontal cortex, aging decreased mtDNMT1 messenger RNA (mRNA) levels without affecting TET1-TET3 mRNAs. In the cerebellum, TET2 and TET3 mRNA content was increased but mtDNMT1 mRNA was unaffected. Using Western immunoblotting of samples from primary neuronal cultures, we found TET immunoreactivity in the mitochondrial fraction. At the single cell level, TET immunoreactivity was detected in the nucleus and in the perinuclear/intraneurite areas where it frequently colocalized with a mitochondrial marker. Our results demonstrated the presence and susceptibility to aging of mitochondrial epigenetic mechanisms in the mammalian brain.