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  • Further our data demonstrate a role

    2021-10-13

    Further, our data demonstrate a role for mitochondrial AIF in oxidative cell death induced by RSL3. We found that AIF knockdown using siRNA completely protected the mlkl sale against RSL3 induced oxidative stress. Similar to other pathways of caspase-independent programmed cell death, mitochondrial damage and the consecutive AIF release is regarded a hallmark of the final steps leading to lethal signaling cascades. The present finding on protective effects of AIF siRNA is, thus, well in line with our earlier studies demonstrating cell death execution by AIF after oxidative damage induced by Xc- inhibition or genetic GPX4 deletion [16], [22], [26]. Our work supports the conclusion that mitochondrial damage represents the “point of no return” in RSL3-mediated ferroptosis. Using the CRISPR/Cas9 technology for genetic deletion of the pro-apoptotic protein BID, we found attenuated toxicity of RSL3 in HT22 Bid KO cells compared to WT cell lines. This was well in line with previous findings in models of glutamate or erastin toxicity where BID transactivation to mitochondria was revealed as a key mechanism of oxidative death signaling [22], [23], [24], [25]. Further, we tested the established BID inhibitor BI-6c9 [29], [31], and found significant protection against RSL3 toxicity, which confirmed BID as a key regulator of mitochondrial demise in oxidative cell death. Notably, targeting BID by BI-6c9 even rescued the cells when applied 6 h after onset of RSL3 exposure, rendering BID inhibition a promising therapeutic strategy in future approaches against ferroptosis. Indeed, BI-6c9 turned out to be as effective as commonly applied ferroptosis inhibitors against lipid peroxidation, mitochondrial ROS formation, and loss of mitochondrial membrane potential. Assuming that protecting mitochondria might prevent ferroptosis, we found significant protection by application of the radical scavenger MitoQ which accumulates in the mitochondria [30], [32]. Despite a considerable amount of studies investigating MitoQ [30], [32], no evidence was available for the detailed protective mechanism in regulated oxidative cell death. Our work shows that the cytoprotective effect of MitoQ was indeed mediated by selective attenuation of mitochondrial ROS formation during oxidative cell death through reduced mitochondrial respiration and concomitantly enhanced glycolytic function. We further demonstrated that MitoQ significantly protected from RSL3 induced lipid peroxidation, mitochondrial ROS formation, loss of mitochondrial membrane potential, and cell death. Notably, at low cytoprotective concentrations MitoQ rescued only mitochondrial parameters of oxidative cell death but did not affect RSL3-mediated increases in lipid peroxidation detected after BODIPY staining confirming selective ROS scavenging within the mitochondria. This finding thereby implies a mechanism of action through increased tolerance to oxidative stress at the level of mitochondria without affecting ROS formation in the cytosol. Such mitochondrial protection by MitoQ, however, was sufficient for cell survival despite GPX4 inhibition and pronounced lipid peroxidation. In addition, we identified a restricted concentration window for the protective effect of the already commercially available dietary supplement MitoQ in our model system of cultured neuronal cells. Although at low protective concentrations the ROS scavenger MitoQ rescued mitochondrial morphology, respiratory function and cell viability, MitoQ revealed opposite effects at higher concentrations, i.e. enhanced ROS production and accelerated loss of mitochondrial membrane potential [33]. Our findings on reduced mitochondrial respiration and the according increases in glycolysis activity after MitoQ exposure are in line with earlier findings showing MitoQ mediated mtDNA damage in MDA-MB-231 and H23 cancer cells, thereby decreasing expression of mitochondrial-encoded respiratory chain subunits which was also compensated by increased glycolytic activity [33].