ABSTRACT
DNA repair plays a critical, but imprecisely defined role in excitotoxic injury and neuronal survival throughout adulthood. We utilized an excitotoxic injury model to compare the location and phenotype of degenerating neurons in mice (strain 129-C57BL) deficient in the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), an enzyme required for nonhomologous end joining (NHEJ). Brains from untreated adult heterozygous and DNA-PKcs null mice displayed comparable cytoarchitecture and undetectable levels of cell death. By day 1, and extending through 4 days following kainic acid-induced seizures, brains from DNA-PKcs null mice showed widespread neurodegeneration that encompassed the entire hippocampal CA1-CA3 pyramidal cell layer, entorhinal cortex, and lateral septum, with relative sparing of the dentate gyrus granule cell layer and hilus, as judged by toluidine blue, Fluoro-Jade B, and terminal dUTP nick end labeling staining. In contrast, seizure-related neurodegeneration in heterozygous littermates was limited to the CA3 region of the hippocampus. NeuN and calbindin staining revealed a selective decrease in the number and density of NeuN-positive neurons in the pyramidal layers of degenerating regions in both heterozygous and DNA-PKcs null mice. To elucidate the mechanisms leading to cell death, we examined an involvement of the p53 pathway, known to be induced by DNA damage. Addition of pifithrin-alpha, a p53 inhibitor, or expression of a dominant-negative p53 rescued neurons from kainate-induced excitotoxic cell death in primary cortical cultures derived from wildtype, DNA-PKcs heterozygous, or DNA-PKcs null neonatal mice. Moreover, pifithrin-alpha prevented kainate-induced loss of mitochondrial membrane potential, dendrite degeneration, and cell death. Results suggest that NHEJ plays a neuroprotective role in excitotoxicity, within the perforant, Schaffer collateral, hippocampal-septal, and temperoammonic pathways, in part by repairing DNA damage that would otherwise result in activation of a p53-dependent apoptotic cascade.
Subject(s)
DNA Damage/physiology , DNA Repair , DNA-Activated Protein Kinase/genetics , DNA-Activated Protein Kinase/physiology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Excitatory Amino Acid Agonists/toxicity , Hippocampus/pathology , Kainic Acid/toxicity , Neurons/physiology , Nuclear Proteins/genetics , Nuclear Proteins/physiology , Seizures/physiopathology , Animals , Benzothiazoles , Cell Death/drug effects , Cells, Cultured , DNA Damage/drug effects , DNA-Activated Protein Kinase/deficiency , DNA-Binding Proteins/deficiency , Entorhinal Cortex/drug effects , Entorhinal Cortex/pathology , Entorhinal Cortex/physiology , Heterozygote , Hippocampus/drug effects , Hippocampus/physiopathology , Immunohistochemistry , In Situ Nick-End Labeling , Kainic Acid/administration & dosage , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mice , Mice, Inbred C57BL , Mice, Neurologic Mutants , Mitochondria/drug effects , Mitochondria/physiology , Nerve Degeneration/physiopathology , Neurons/drug effects , Neurons/pathology , Nuclear Proteins/deficiency , Seizures/chemically induced , Seizures/pathology , Thiazoles/pharmacology , Toluene/analogs & derivatives , Toluene/pharmacology , Tumor Suppressor Protein p53/antagonists & inhibitors , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/physiologyABSTRACT
Poly(ADP-ribose)polymerase-1 (PARP-1) is a nuclear enzyme activated by DNA breaks and serves a role in DNA repair through the formation of polymers (poly(ADP)ribosylation) at sites of DNA damage. PARP-1 is activated by DNA damage in neurons of the hippocampus and cerebral cortex following excessive exposure to glutamate receptor agonists such as NMDA or kainic acid. In addition, recent studies suggest that degradation of PARP-1 occurs in cells that undergo apoptotic versus nonapoptotic forms of cell death. To investigate this process further, we examined the spatiotemporal aspects of excitotoxic injury in the rodent visual cortex by making focal intracerebral injections of kainic acid. These injections resulted in DNA damage, PARP-1 activation, and neuronal cell death over a 5-day period. Rapid neuronal cell injury assessed by Fluoro-Jade staining appeared within hours, but increased TUNEL staining occurred only after 24 h. A dramatic increase in caspase-3 activity, as well as an increase in the number of neurons containing active caspase-3, peaked 2 days after injury. Last, increased PARP-1 immunoreactivity and PARP-1 cleavage reached peak levels 2 to 3 days after delivering the excitotoxin. These findings suggest that increased caspase-3 activity may regulate the degradation of PARP-1 in subsets of cortical neurons during excitotoxic cell death.
