Elevated plasma levels of the sulfur-containing amino acid homocysteine increase the risk for atherosclerosis, stroke, and possibly Alzheimer9s disease, but the underlying mechanisms are unknown. We now report that homocysteine induces apoptosis in rat hippocampal neurons. DNA strand breaks and associated activation of poly-ADP-ribose polymerase (PARP) and NAD depletion occur rapidly after exposure to homocysteine and precede mitochondrial dysfunction, oxidative stress, and caspase activation. The PARP inhibitor 3-aminobenzamide (3AB) protects neurons against homocysteine-induced NAD depletion, loss of mitochondrial transmembrane potential, and cell death, demonstrating a requirement for PARP activation and/or NAD depletion in homocysteine-induced apoptosis. Caspase inhibition accelerates the loss of mitochondrial potential and shifts the mode of cell death to necrosis; inhibition of PARP with 3AB attenuates this effect of caspase inhibition. Homocysteine markedly increases the vulnerability of hippocampal neurons to excitotoxic and oxidative injury in cell culture and in vivo, suggesting a mechanism by which homocysteine may contribute to the pathogenesis of neurodegenerative disorders.

The transcription factor nuclear factor κB (NF‐κB) is moving to the forefront of the fields of apoptosis and neuronal plasticity because of recent findings showing that activation of NF‐κB prevents neuronal apoptosis in various cell culture and in vivo models and because NF‐κB is activated in association with synaptic plasticity. Activation of NF‐κB was first shown to mediate antiapoptotic actions of tumor necrosis factor in cultured neurons and was subsequently shown to prevent death of various nonneuronal cells. NF‐κB is activated by several cytokines and neurotrophic factors and in response to various cell stressors. Oxidative stress and elevation of intracellular calcium levels are particularly important inducers of NF‐κB activation. Activation of NF‐κB can interrupt apoptotic biochemical cascades at relatively early steps, before mitochondrial dysfunction and oxyradical production. Gene targets for NF‐κB that may mediate its anti‐apoptotic actions include the antioxidant enzyme manganese superoxide dismutase, members of the inhibitor of apoptosis family of proteins, and the calcium‐binding protein calbindin D28k. NF‐κB is activated by synaptic activity and may play important roles in the process of learning and memory. The available data identify NF‐κB as an important regulator of evolutionarily conserved biochemical and molecular cascades designed to prevent cell death and promote neuronal plasticity. Because NF‐κB may play roles in a range of neurological disorders that involve neuronal degeneration and/or perturbed synaptic function, pharmacological and genetic manipulations of NF‐κB signaling are being developed that may prove valuable in treating disorders ranging from Alzheimer’s disease to schizophrenia.

Ferroptosis has been defined as an oxidative and iron-dependent pathway of regulated cell death that is distinct from caspase-dependent apoptosis and established pathways of death receptor-mediated regulated necrosis. While emerging evidence linked features of ferroptosis induced e.g. by erastin-mediated inhibition of the Xc- system or inhibition of glutathione peroxidase 4 (Gpx4) to an increasing number of oxidative cell death paradigms in cancer cells, neurons or kidney cells, the biochemical pathways of oxidative cell death remained largely unclear. In particular, the role of mitochondrial damage in paradigms of ferroptosis needs further investigation. In the present study, we find that erastin-induced ferroptosis in neuronal cells was accompanied by BID transactivation to mitochondria, loss of mitochondrial membrane potential, enhanced mitochondrial fragmentation and reduced ATP levels. These hallmarks of mitochondrial demise are also established features of oxytosis, a paradigm of cell death induced by Xc- inhibition by millimolar concentrations of glutamate. Bid knockout using CRISPR/Cas9 approaches preserved mitochondrial integrity and function, and mediated neuroprotective effects against both, ferroptosis and oxytosis. Furthermore, the BID-inhibitor BI-6c9 inhibited erastin-induced ferroptosis, and, in turn, the ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 prevented mitochondrial dysfunction and cell death in the paradigm of oxytosis. These findings show that mitochondrial transactivation of BID links ferroptosis to mitochondrial damage as the final execution step in this paradigm of oxidative cell death.