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 death in vitro that 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 from tlr4 mutant mice. In an in vivo model 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 the tlr4 gene are resistant to neuronal injury in the same model. The present study demonstrates a mechanistic link among innate immunity, TLRs, and neurodegeneration. Systemic infection is associated with sustained worsening in many diseases of the CNS, yet the molecular and cellular relationship between infection outside the CNS and potential neuronal loss within the CNS is elusive. Activation of microglia, bone marrow-derived macrophage-like cells that function as the resident immune defense system of the brain (1), is a characteristic feature of most neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, multiple sclerosis, AIDS dementia complex, and amyotrophic lateral sclerosis as well as ischemia and posttraumatic brain injury (2-4). Neurotoxicity induced by ␤-amyloid or HIV proteins in mixed CNS cultures depends on the presence and activation of microglia (5, 6). Liberatore et al. (7) demonstrated in vivo that microglial inducible nitric oxide synthase plays a crucial role in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurodegeneration in the MPTP mouse model of Parkinson's disease.The evolutionarily ancient innate immune system provides the first line of host defense against a large variety of pathogens and also controls many aspects of the adaptive immune response (8). Cells of the innate immune system recognize invariant molecular structures of pathogens termed pathogen-associated molecular patterns through a series of genetically conserved and stable cell-surface receptors related to the Drosophila gene toll that thus are referred to as Toll-like receptors (TLRs) (9).TLR4 functions as the signal-transducing receptor for the endotoxin lipopolysaccharide (LPS) (10), which is a major component of the outer membrane of Gram-negative bacteria. LPS binds to the serum protein LPS-binding protein and the soluble or glycosylphosphatidylinositol-anchored CD14. This complex in turn binds to TLR4 (11) and initiates an intracellular signaling pathway that regulates gene expression through derepression of the transcriptional a...

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.

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 the tlr4 gene. 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 redoxsensitive probe dihydrorhodamine 123. The antioxidants ␣-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 leukomalacia (PVL), the major substrate of cerebral palsy in survivors of prematurity, is defined as focal periventricular necrosis and diffuse gliosis in immature cerebral white matter. We propose that nitrosative and/or oxidative stress to premyelinating oligodendrocytes complicating cerebral ischemia in the sick premature infant is a key mechanism of injury interfering with maturation of these cells to myelin-producing oligodendrocytes and subsequent myelination. Using immunocytochemical markers in autopsy brain tissue from 17 PVL cases and 28 non-PVL controls, we found in the PVL cases: 1) selective regionalization of white matter injury, including preferential involvement of the deep compared to intragyral white matter; 2) prominent activation of microglia diffusely throughout the white matter; 3) protein nitration and lipid peroxidation in premyelinating oligodendrocytes in the diffuse component; 4) preferential death of premyelinating oligodendrocytes diffusely; and 5) virtual sparing of the overlying cerebral cortex, as demonstrated by markers of activated astrocytes and microglia. These data establish that PVL is primarily a white matter disease that involves injury to premyelinating oligodendrocytes, potentially through activation of microglia and release of reactive oxygen and nitrogen species. Agents that prevent nitrosative and oxidative stress may play a key role in ameliorating PVL in premature infants in the intensive care nursery.

The excitatory neurotransmitter glutamate is released from axons and glia under hypoxic/ischemic conditions. In vitro, oligodendrocytes (OLs) express non-NMDA glutamate receptors (GluRs) and are susceptible to GluR-mediated excitotoxicity. We evaluated the role of GluR-mediated OL excitotoxicity in hypoxic/ ischemic white matter injury in the developing brain. Hypoxic/ ischemic white matter injury is thought to mediate periventricular leukomalacia, an age-dependent white matter lesion seen in preterm infants and a common antecedent to cerebral palsy. Hypoxia/ischemia in rat pups at postnatal day 7 (P7) produced selective white matter lesions and OL death. Furthermore, OLs in pericallosal white matter express non-NMDA GluRs at P7. Unilateral carotid ligation in combination with hypoxia (6% O 2 for 1 hr) resulted in selective, subcortical white matter injury with a marked ipsilateral decrease in immature and myelin basic protein-expressing OLs that was also significantly attenuated by 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2,3-dione (NBQX). Intracerebral AMPA demonstrated greater susceptibility to OL injury at P7 than in younger or older pups, and this was attenuated by systemic pretreatment with the AMPA antagonist NBQX. These results indicate a parallel, maturation-dependent susceptibility of immature OLs to AMPA and hypoxia/ischemia. The protective efficacy of NBQX suggests a role for glutamate receptormediated excitotoxic OL injury in immature white matter in vivo.Key words: white matter; glutamate receptors; AMPA; NBQX; oligodendrocytes; excitotoxicity Differential regional vulnerability to hypoxic/ischemic brain injury may be dependent on the maturational stage of the neuronal and non-neuronal cells in a given region. A common example of such age-dependent regional susceptibility is the exclusively white matter injury seen in infants as a complication of premature birth, referred to as periventricular leukomalacia (PVL). PVL is the principal neuropathological correlate of cerebral palsy. The lesion is defined by focal necrosis of the deep periventricular white matter involving all cellular components, combined with a more diffuse white matter injury that appears selective for developing oligodendrocytes (OLs) (Gilles and Averill, 1977;Dambska et al., 1989;Rorke, 1998). Reduced cerebral myelin is the most prominent subsequent cerebral abnormality observed in premature infants with evidence of PVL in the neonatal period (Paneth et al., 1990;Rorke, 1992;Iida et al., 1995;Olsen et al., 1997;Skranes et al., 1997;Inder et al., 1999).A propensity to cerebral ischemia caused by impaired cerebrovascular autoregulation, combined with a selective vulnerability of immature OLs to ischemic injury (Volpe, 1997), may contribute to the prevalence of this lesion in the preterm infant. Developing OLs in vitro have been demonstrated to be more vulnerable than are mature, myelin basic protein (MBP)-expressing OLs to oxidative stress and to glutamate receptor (GluR)-mediated ischemic death (Fern and Moller, 2000). OLs appear ...

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.

Brain injury in the premature infant, a problem of enormous importance, is associated with a high risk of neurodevelopmental disability. The major type of injury involves cerebral white matter and the principal cellular target is the developing oligodendrocyte. The specific phase of the oligodendroglial lineage affected has been defined from study of both human brain and experimental models. This premyelinating cell (pre-OL) is vulnerable because of a series of maturation-dependent events. The pathogenesis of pre-OL injury relates to operation of two upstream mechanisms, hypoxia-ischemia and systemic infection/inflammation, both of which are common occurrences in premature infants. The focus of this review and of our research over the past 15-20 years has been the cellular and molecular bases for the maturation-dependent vulnerability of the pre-OL to the action of the two upstream mechanisms. Three downstream mechanisms have been identified, i.e., microglial activation, excitotoxicity and free radical attack. The work in both experimental models and human brain has identified a remarkable confluence of maturation-dependent factors that render the pre-OL so exquisitely vulnerable to these downstream mechanisms. Most importantly, elucidation of these factors has led to delineation of a series of potential therapeutic interventions, which in experimental models show marked protective properties. The critical next step, i.e., clinical trials in the living infant, is now on the horizon.