OL precursors are highly sensitive to oxidative stress-induced damage, in part due to a weaker antioxidant defense system as compared to mature OLs [41
]. This developmental stage specific vulnerability of preOLs to oxidative stress and to AMPA/kainate excitotoxicity predisposes them to various types of injury that lead to mye-lination abnormalities [5
]. In this study, we investigated key signaling events involved in arachidonic acid-induced oxidative death of preOLs in culture. We demonstrate that both arachidonic acid and GSH depletion trigger a programmed necrotic death pathway that is regulated by the kinase activity of RIP1. Specific inhibition of RIP1 kinase activity with Nec-1 markedly prevents cell death via blocking ROS production and JNK activation.
RIP1 belongs to the RIP family of serine/ threonine kinases that are involved in innate and adaptive immunity. RIP1 contains an N-terminal kinase domain, an intermediate domain, a RIP homotypic motif that enables its interaction with RIP3, and a C-terminal death domain (DD), through which it interacts with death receptor TNFR1 and also with DD-containing adaptor proteins such as TRADD and FADD [25
]. Recent studies identified RIP1 and RIP3 as key regulators of programmed necrosis induced by TNF, Fas, Toll-like receptor 3 (TLR3) and 4. RIP1 has now emerged as a tightly regulated signaling molecule with bifurcated functions, the kinase-independent proinflammatory function and the kinase-dependent pro-necrosis function, under the control of caspases and ubiquitination. The identification of Nec-1 as a small-molecule that allosterically inhibits the kinase activity of RIP1 without affecting RI P1-mediated activation of NFkB allows one to dissect the role of RIP1 in necrotic cell death [23
]. It should be noted that Nec-1 is not an anti-oxidant [23
]. Our finding that arachidonic acid-induced oxidative death of preOLs is prevented by Nec-1 reveals that programmed necrosis may be a common pathway for certain oxidative cell death and suggests that receptor-independent activation of RIP1 kinase may occur under GSH depletion paradigms. At present, it is unclear how arachidonic acid or GSH depletion activates RIP1 kinase. The possibility that arachidonic acid induces RIP1 kinase activation indirectly through TNF production is highly unlikely in our experimental conditions since direct addition of TNF to preOLs fails to cause significant loss of preOL viability [43
]. Furthermore, Nec-1 does not prevent lipopolysaccha-ride-induced, TNF-dependent killing of preOLs in mixed glial cultures (Li, unpublished data). Thus, a receptor-independent RIP1 activation seems operative in arachidonic acid-challenged or GSH -depleted preOLs. Since RIP1 is regulated by ubiquitination, and deubiquitination enables its dissociation from the TNF receptor complex and subsequent interaction with RIP3 to form necro-some [25
], one potential mechanism is that the redox status of preOLs regulates RIP1 kinase activation indirectly through affecting the ubiquitination pathway. The ubiquitination machinery has in fact been previously shown to be under redox control [44
]. Intracellular GSH depletion dose-dependently reduces the levels of endogenous ubiquitinated protein conjugates. A20, a RIP1 deubiquitination enzyme, is upregu-lated by oxidative stress and promotes necrotic cell death [45
]. Recently, in a genome-wide siRNA screen for regulators of programmed necrosis induced by the caspase inhibitor zVAD, Hitomi et al
] identified the involvement of the GSH metabolic pathway in RIP1 kinase-dependent necrosis. Reducing glutathione per-oxidase and glutathione S-transferases via siRNA, and presumably the subsequent increase of free GSH level, protected L929 cells against zVAD-induced necrosis [46
]. This observation is consistent with our finding that arachidonic acid and GSH depletion trigger a receptor-independent, RIP1 kinase-dependent necrosis, and is in line with our speculation that RIP1 kinase activation may be under redox control. A similar protective effect of Nec-1 has also been found in glutamate-induced GSH depletion and oxidative death of HT-22 cells [42
]. Moreover, nitric oxide-induced endothelial cell death has recently been shown to be mediated by RIP 1/3 and blocked by over-expression of mitochondrial superoxide dismutase [47
]. Interestingly, nitric oxide toxicity has been linked to GSH depletion and 12-LOX activation in primary midbrain cultures [19
]. Apparently, further work is needed to unravel the mechanism of receptor-independent activation of RIP1 kinase and its yet-to-be identified downstream substrate.
Our results revealed a causal role for RIP1 kinase in arachidonic acid-, cystine deprivation-, and BSO-induced oxidative damage to preOLs. In all three cell death paradigms, 12-LOX activation is essential. Blocking either 12-LOX or RIP1 kinase confers significant protection. For oxidative cell death that is not dependent on 12-LOX, such as that induced by H2O2, Nec-1 is ineffective. Furthermore, our preliminary data suggest that Nec-1 has no effect on kainate-triggered excitotoxicity to preOLs, which is 12-LOX independent (Li unpublished data). The functional relationship between 12-LOX and RIP1 kinase activation is currently unknown and requires further investigation. Our identification of an essential role for RIP1 kinase in arachidonic acid-induced oxidative damage to primary preOLs has important implications for conditions like hypoxic-ischemic injury where increased arachidonic acid and oxidative stress are key pathogenic events. Necro-statins have been shown to reduce tissue damage in transient ischemic brain injury in mice [23
], traumatic brain injury in mice [48
], myo-cardial ischemia-reperfusion injury [49
], and retinal ischemia/reperfusion injury [50
]. It is of note that blockade of 12/15-LOX or 12/15-LOX deficiency also protects mice against transient focal ischemia in a mouse model of stroke [21
]. Given our current findings and the known intrinsic vulnerability of preOLs to oxidative stress, blocking RIP1 kinase activity with necrostatins may offer beneficial effects in white matter injury. Therefore, it would be interesting to test whether RIP1-dependent programmed necrosis pathway contributes to hypoxic/ischemic white matter injury and other pathological conditions such as multiple sclerosis and spinal cord injury.