Neuronal Intrinsic Apoptosis Mechanisms and Their Modulation by Sp1 Inhibition

Abstract

Drug-induced DNA damage, reactive oxygen species (ROS), inflammatory mediators and central neurotoxicity have all been shown to play a role in cognitive impairments after chemotherapy, after CNS injury or in neurodegenerative diseases. Intrinsic apoptosis is a regulated cell death pathway implicated in many of these conditions. This pathway proceeds sequentially through DNA damage responses, including phosphorylation of ATM, H2AX and Tumor Protein 53 (p53), transcriptional activation of pro-apoptotic BH3-only proteins, and mitochondrial outer membrane permeabilization (MOMP), activating caspase-dependent and caspase-independent apoptosis. Previous work has indicated that Sp1 may regulate p53’s transcriptional profile and ability to promote apoptosis after DNA damage. To examine the role of Sp1 in DNA-damage-induced apoptosis, my work has utilized Mithramycin, a drug that binds G-C rich DNA to compete with Sp1 chromatin binding. Our hypothesis is that Mithramycin competes with Sp1 chromatin binding and thus protects neurons from DNA damage-induced, p53-dependent intrinsic apoptosis. However, it is vital to recognize that neurons increase their resistance to cytotoxic stimuli as they mature, leading to decreased levels of apoptosis. Thus, we additionally characterized the changes in the intrinsic apoptosis pathway upon neuronal maturation. Despite significant attenuation of the intrinsic apoptosis pathway in mature neurons, we found evidence that Mithramycin can still attenuate DNA-damage dependent intrinsic apoptosis in this paradigm to the extent that it is induced by neurons. Additional experiments showed that mature animal cortices or hippocampi do not demonstrate evidence of end-stage apoptosis (caspase activity) in response to DNA damage, unlike those in immature animals. However, in vivo administration of Mithramycin was able to attenuate cortical expression of p53-dependent genes. Thus, our studies provide evidence that: 1) Mithramycin attenuates activation of the DNA damage-dependent intrinsic apoptosis pathway via indirect inhibition of p53-dependent transcription and 2) Mature neurons restrict intrinsic apoptosis pathway activation in response to DNA damage. Therefore, Sp1 is likely to play a significant role in DNA damage-dependent neuronal intrinsic apoptosis, and Mithramycin treatment is protective when this pathway is activated. However, mature neurons significantly downregulate this pathway, thus limiting the potential therapeutic effects of Mithramycin in adult animals.