No abstract

The mechanisms for neuronal apoptosis after axotomy and target deprivation in the adult central nervous system are poorly understood. We used a unilateral occipital cortex ablation model in the adult rat to test the hypothesis that apoptotic retrograde neurodegeneration in the dorsal lateral geniculate nucleus occurs in association with oxidative stress and mitochondrial abnormalities. Immunodetection of 8-hydroxy-2'-deoxyguanosine, a marker for oxidative injury to DNA, demonstrated that these apoptotic neurons undergo oxidative stress. Dual immunolabeling for the retrograde tracer Fluorogold to identify projection neurons and for 8-hydroxy-2'-deoxyguanosine demonstrated that apoptotic, oxidatively damaged neurons are geniculocortical projection neurons. By electron microscopy, degeneration of dorsal lateral geniculate nucleus neurons evolved in association with a transient increase in mitochondria within the perikaryon of dying neurons during the transition between chromatolysis and early apoptosis. The morphological integrity of mitochondria was preserved until late in the progression of apoptosis. The dorsal lateral geniculate nucleus ipsilateral to the cortical lesion had a transient increase in cytochrome c oxidase activity, and geniculocortical neurons at the transitional, early apoptotic stage accumulated cytochrome c oxidase activity. We conclude that axotomy-induced, retrograde neuronal apoptosis in the adult central nervous system occurs in association with the accumulation of functionally active mitochondria within the perikaryon and oxidative damage to nuclear DNA.

The mechanisms of injury-and disease-associated apoptosis of neurons within the CNS are not understood. We used a model of cortical injury in rat and mouse to induce retrograde neuronal apoptosis in thalamus. In this animal model, unilateral ablation of the occipital cortex induces apoptosis of corticopetal projection neurons in the dorsal lateral geniculate nucleus (LGN), by 7 days post-lesion, that is p53 modulated and Bax dependent. We tested the hypothesis that this degenerative process is initiated by oxidative stress and early formation of DNA damage and is accompanied by changes in the levels of pro-apoptotic mediators of cell death. Immunoblotting revealed that the protein profiles of Bax, Bak and Bad were different during the progression of neuronal apoptosis in the LGN. Bax underwent a subcellular redistribution by 1 day post-lesion, while Bak increased later. Bad showed an early sustained increase. Cleaved caspase-3 was elevated maximally at 5 and 6 days. Active caspase-3 underwent a subcellular translocation to the nucleus. A dramatic phosphorylation of p53 was detected at 4 days post-lesion. DNA damage was assessed immunocytochemically as hydroxyl radical adducts (8-hydroxy-2-deoxyguanosine) and singlestranded DNA. Both forms of DNA damage accumulated early in target-deprived LGN neurons. Transgenic overexpression of superoxide dismutase-1 provided significant protection against the apoptosis but antioxidant pharmacotreatments with trolox and ascorbate were ineffective. We conclude that overlapping and sequential signaling pathways are involved in the apoptosis of adult brain neurons and that DNA damage generated by superoxide derivatives is an upstream mechanism for p53-regulated, Bax-dependent apoptosis of targetdeprived neurons.