In many neurologic disorders, injury to neurons may be caused at least in part by overstimulation of receptors for excitatory amino acids, including glutamate and aspartate. These neurologic conditions range from acute insults such as stroke, hypoglycemia, trauma, and epilepsy (Table 1) to chronic neurodegenerative states such as Huntington's disease, the acquired immunodeficiency syndrome (AIDS) dementia complex, amyotrophic lateral sclerosis, and perhaps Alzheimer's disease (Table 2)¹³.Glutamate is the principal excitatory neurotransmitter in the brain, and its interactions with specific membrane receptors are responsible for many neurologic functions, including cognition, memory, movement, and sensation⁴. In addition, excitatory . . .

Ferrostatin-1 (Fer-1) inhibits ferroptosis, a form of regulated, oxidative, nonapoptotic cell death. We found that Fer-1 inhibited cell death in cellular models of Huntington’s disease (HD), periventricular leukomalacia (PVL), and kidney dysfunction; Fer-1 inhibited lipid peroxidation, but not mitochondrial reactive oxygen species formation or lysosomal membrane permeability. We developed a mechanistic model to explain the activity of Fer-1, which guided the development of ferrostatins with improved properties. These studies suggest numerous therapeutic uses for ferrostatins, and that lipid peroxidation mediates diverse disease phenotypes.

Innate immunity is an evolutionarily ancient system that provides organisms with immediately available defense mechanisms through recognition of pathogen-associated molecular patterns. We show that in the CNS, specific activation of innate immunity through a Toll-like receptor 4 (TLR4)-dependent pathway leads to neurodegeneration. We identify microglia as the major lipopolysaccharide (LPS)-responsive cell in the CNS. TLR4 activation leads to extensive neuronal deathin vitrothat depends on the presence of microglia. LPS leads to dramatic neuronal loss in cultures prepared from wild-type mice but does not induce neuronal injury in CNS cultures derived fromtlr4mutant mice. In anin vivomodel of neurodegeneration, stimulating the innate immune response with LPS converts a subthreshold hypoxic-ischemic insult from no discernable neuronal injury to severe axonal and neuronal loss. In contrast, animals bearing a loss-of-function mutation in thetlr4gene are resistant to neuronal injury in the same model. The present study demonstrates a mechanistic link among innate immunity, TLRs, and neurodegeneration.

The immediate or innate immune response is the first line of defense against diverse microbial pathogens and requires the expression of recently discovered toll-like receptors (TLRs). TLR4 serves as a specific receptor for lipopolysaccharide (LPS) and is localized on the surface of a subset of mammalian cells. Although innate immunity is a necessary host defense against microbial pathogens, the consequences of its activation in the CNS can be deleterious, as we show here in a developing neural model. We examined the major non-neuronal cell types in the CNS for expression of TLR4 and found that microglia expressed high levels, whereas astrocytes and oligodendrocytes expressed none. Consistent with TLR4 expression solely in microglia, we show that microglia are the only CNS glial cells that bind fluorescently tagged lipopolysaccharide. Lipopolysaccharide led to extensive oligodendrocyte death in culture only under conditions in which microglia were present. To determine whether TLR4 is necessary for lipopolysaccharide-induced oligodendrocyte death in mixed glial cultures, we studied cultures generated from mice bearing a loss-of-function mutation in thetlr4gene. Lipopolysaccharide failed to induce oligodendrocyte death in such cultures, in contrast to the death induced in cultures from wild-type mice. Finally, stereotactic intracerebral injection of lipopolysaccharide into the developing pericallosal white matter of immature rodents resulted in loss of oligodendrocytes and hypomyelination and periventricular cysts. Our data provide a general mechanistic link between (1) lipopolysaccharide and similar microbial molecular motifs and (2) injury to oligodendrocytes and myelin as occurs in periventricular leukomalacia and multiple sclerosis.

Death of oligodendrocyte (OL) precursors can be triggered in vitro by cystine deprivation, a form of oxidative stress that involves depletion of intracellular glutathione. We report here that OLs demonstrate maturation-dependent differences in survival when subjected to free radical-mediated injury induced by glutathione depletion. Using immunopanning to isolate rat preoligodendrocytes (preOLs), we generated highly enriched populations of preOLs and mature OLs under chemically defined conditions. Cystine deprivation caused a similar decrease in glutathione levels in OLs at both stages. However, preOLs were completely killed by cystine deprivation, whereas mature OLs remained viable. Although the glutathione-depleting agents buthionine sulfoximine and diethylmaleate were more potent in depleting glutathione in mature OLs, both agents were significantly more toxic to preOLs. Glutathione depletion markedly increased intracellular free radical generation in preOLs, but not in mature OLs, as indicated by oxidation of the redox-sensitive probe dihydrorhodamine 123. The antioxidants α-tocopherol, idebenone, and glutathione monoethylester prevented the oxidation of dihydrorhodamine in cystine-depleted preOLs and markedly protected against cell death. When the intracellular glutathione level was not manipulated, preOLs were also more vulnerable than mature OLs to exogenous free radical toxicity generated by a xanthine–xanthine oxidase system. Ultrastructural features of free radical-mediated injury in glutathione-depleted preOLs included nuclear condensation, margination of chromatin, and mitochondrial swelling. These observations indicate that preOLs are significantly more sensitive to the toxic effects of glutathione depletion and that oligodendroglial maturation is associated with decreased susceptibility to oxidative stress.

Periventricular white matter injury, the principal variety of brain injury of the human premature infant, involves differentiating oligodendroglia. Nothing is known of the biochemical mechanism of oligodendroglial death in this disorder. Because an early event in periventricular white matter injury is ischemia-induced axonal disruption and because such axonal destruction could lead to a marked increase in local concentrations of glutamate, we evaluated the vulnerability of differentiating oligodendroglia to glutamate in a culture model. Oligodendroglia were isolated from mixed-glial primary cultures by a selective detachment technique and grown in a primary culture under conditions that lead to differentiation. These oligodendroglia were found to be highly vulnerable to glutamate-induced cell death. The EC50 for glutamate for a 24 hr exposure was approximately 200 microM, comparable to the value reported for neurons in conventional cerebral cortical cultures. Astrocytes, in contrast, were shown to be resistant to as much as 5 mM glutamate. Study of glutamate receptor antagonists and glutamate transport substrates showed that the glutamate-induced oligodendroglial death was not related to a receptor mechanism, as operates in neurons, but rather was secondary to glutamate uptake by the oligodendroglia. Glutamate transport by high-affinity, sodium-dependent and by sodium-independent systems was shown. The central importance of glutamate uptake for the toxic effect of glutamate was shown by total prevention of the oligodendroglial toxicity by the simultaneous inhibition of glutamate uptake by the specific inhibitor D,L-threo-beta-hydroxyaspartate. Subsequent observations showed that the toxicity of glutamate was mediated by free radical attack, the consequence of glutathione depletion, apparently caused by the action of a glutamate-cystine exchange mechanism that results in cystine and thereby glutathione depletion. Thus, addition of cystine or cysteine totally prevented the glutamate toxicity to oligodendroglia. Second, glutamate exposure led to cystine efflux. Third, glutathione levels decreased markedly in cells exposed to glutamate, and this marked decrease preceded the loss of cell viability. Fourth, glutamate toxicity could be prevented totally by exposure to different free radical scavengers, vitamin E and idebenone. The data thus show that glutamate is highly toxic to oligodendroglia. Moreover, the findings raise the possibilities that such glutamate toxicity is operative in the oligodendroglial cell death associated with ischemic processes that disrupt axons, such as periventricular white matter injury of the premature infant, and that novel therapies directed against glutamate transport, glutathione depletion, and free radical attack might be beneficial in prevention of that injury.