5 Enhancing the Expression of the Dna Repair/Redox Enzyme, Ape1/Ref-1, Reduces Neurotoxicity Induced by Ionizing Radiation: Implications for Decreasing Pediatric Neurocognitive Dysfunction.

Abstract

Neurotoxicity which includes both neurocognitive dysfunction commonly called “chemobrain” and peripheral neuropathy, occurs frequently in pediatric patients who receive ionizing radiation (IR) for the treatment of cancer. Despite the prevalence of these side effects, little work has been done to elucidate the mechanisms of or interventions to prevent neurotoxicity. Apurinic/apyrimidinic endonuclease/redox effector factor (Ape1) repairs AP sites in DNA secondary to oxidative damage and regulates the redox state of transcription factors. Because IR produces reactive oxygen species and DNA damage, we examined whether overexpressing Ape1 in primary neuronal cultures reduces toxicity after IR. To decrease the expression of Ape1, rat hippocampal or sensory neuronal cultures were exposed to small interfering RNA to Ape1 (Ape1siRNA) or to scramble siRNA (SCsiRNA) as a control. To overexpress Ape1, cells treated with siRNA were subsequently exposed to adenoviral constructs containing Ape1 or to a vector control. Cultured cells in the absence or presence of altered expression of Ape1 were then exposed to IR and three endpoints examined, cell viability, DNA double strand breaks, and neurotransmitter release. Exposing neurons to increasing amounts of IR resulted in a dose–dependent increase in cell death 24 hours after the treatment. Reducing Ape1 expression with Ape1siRNA by ∼ 85-95% significantly augmented IR-induced cell death compared to cultures treated with SCsiRNA. In a similar manner, reductions in Ape1 in neuronal cultures doubled the IR-induced phosphorylation of histone 2A.X, indicating an increase in DNA double-strand breaks. Overexpressing Ape1 in neuronal cultures treated with SCsiRNA or Ape1siRNA resulted in a significant increase in cell viability and a 4-6 fold reduction in H2A.X phosphorylation after exposure to IR. IR significantly reduced the stimulated release if calcitonin gene-related peptide (CGRP) from sensory neurons from 10.2 ± 0.5 5 of the total content of CGRP to 7.5 0.7%. This reduction in release was augmented by reducing Ape1 expression by 85% and revered by overexpressing Ape1. These data demonstrate that Ape1 plays an important role in minimizing neurotoxicity secondary to oxidative stress and modulation of Ape1's functions could lead to protection of neuronal cell damage and killing during IR treatments.