Oxidative DNA damage is concurrently repaired by base excision repair (BER) and apyrimidinic endonuclease 1 (APE1)-initiated nonhomologous end joining (NHEJ) in cortical neurons.

AIMS: Accumulating studies have suggested that base excision repair (BER) is the major repair pathway of oxidative DNA damage in neurons, and neurons are deficient in other DNA repair pathways, including nucleotide excision repair and homologous recombination repair. However, some studies have demonstrated that neurons could efficiently repair glutamate- and menadione-induced double-strand breaks (DSBs), suggesting that the DSB repair mechanisms might be implicated in neuronal health. In this study, we hypothesized that BER and nonhomologous end joining (NHEJ) work together to repair oxidative DNA damage in neurons. METHODS: Immunohistochemistry and confocal microscopy were employed to examine the colocalization of apyrimidinic endonuclease 1 (APE1), histone variant 2AX (γH2AX) and phosphorylated p53-binding protein (53BP1). APE1 inhibitor and shRNA were respectively applied to suppress APE1 activity and protein expression to determine the correlation of APE1 and DSB formation. The neutral comet assay was used to determine and quantitate the formation of DSB. RESULTS: Both γH2AX and 53BP1 were upregulated and colocalized with APE1 in the nuclei of rat cortical neurons subjected to menadione-induced oxidative insults. Phospho53BP1 foci were efficiently abolished, but γH2AX foci persisted following the suppression of APE1 activity. Comet assays demonstrated that the inhibition of APE1 decreased the DSB formation. CONCLUSIONS: Our results indicate that APE1 can engage the NHEJ mechanism in the repair of oxidative DNA damage in neurons. These findings provide insights into the mechanisms underlying the efficient repair of oxidative DNA damage in neurons despite the high oxidative burden.

Tachykinins and tachykinin-receptors in the rat pineal gland.

High-pressure liquid chromatography of extracts of rat pineal glands, followed by radio immunological analysis with antibodies against tachykinins, demonstrated the presence of substance P, neurokinin A and neurokinin B in the superficial rat pineal gland. Immunohistochemistry on perfusion-fixed rat brain sections showed substance P and neurokinin A to be present in nerve fibers located both in the perivascular spaces as well as intraparenchymally between the pinealocytes. After extracting total RNA, followed by reverse transcription and polymerase chain reaction amplification with primers specific for NK1-, NK2- and NK3-receptors, agarose gel analysis of the reaction products showed the presence of mRNA encoding all three neurokinin receptors. Immunohistochemical analysis showed NK1 receptor to be located in the interstitial cells of the gland. This location was confirmed by use of in situ hybridization using radioactively labeled antisense oligonucleotide probes. Double immunohistochemical stainings showed that the NK1-immunoreactive cells were not a part of the macrophages or antigen-presenting cells of the gland. Our study suggests that tachykinins, after release from intrapineal nerve fibers, are involved in an up to now unknown function, different from that of melatonin synthesis.