Thyroid Hormone Protects Primary Cortical Neurons Exposed to Hypoxia by Reducing DNA Methylation and Apoptosis

Traumatic brain injury (TBI) is associated with disruption of cerebral blood flow leading to localized brain hypoxia. Thyroid hormone (TH) treatment, administered shortly after injury, has been shown to promote neural protection in rodent TBI models. The mechanism of TH protection, however, is not established. We used mouse primary cortical neurons to investigate the effectiveness and possible pathways of T3-promoted cell survival after exposure to hypoxic injury. Cultured primary cortical neurons were exposed to hypoxia (0.2% oxygen) for 7 hours with or without T3 (5 nM). T3 treatment enhanced DNA 5-hydroxymethylcytosine levels and attenuated the hypoxia-induced increase in DNA 5-methylcytosine (5-mc). In the presence of T3, mRNA expression of Tet family genes was increased and DNA methyltransferase (Dnmt) 3a and Dnmt3b were downregulated, compared with conditions in the absence of T3. These T3-induced changes decreased hypoxia-induced DNA de novo methylation, which reduced hypoxia-induced neuronal damage and apoptosis. We used RNA sequencing to characterize T3-regulated genes in cortical neurons under hypoxic conditions and identified 22 genes that were upregulated and 15 genes that were downregulated. Krüppel-like factor 9 (KLF9), a multifunctional transcription factor that plays a key role in central nervous system development, was highly upregulated by T3 treatment in hypoxic conditions. Knockdown of the KLF9 gene resulted in early apoptosis and abolished the beneficial role of T3 in neuronal survival. KLF9 mediates, in part, the neuronal protective role of T3. T3 treatment reduces hypoxic damage, although pathways that reduce DNA methylation and apoptosis remain to be elucidated.