Inhibition of autophagy rescues HT22 hippocampal neurons from erastin-induced ferroptosis.

Ferroptosis is a regulated form of cell death which is considered an oxidative iron-dependent process. The lipid hydroperoxidase glutathione peroxidase 4 prevents the iron (Fe2+)-dependent formation of toxic lipid reactive oxygen species. While emerging evidence indicates that inhibition of glutathione peroxidase 4 as a hallmark of ferroptosis in many cancer cell lines, the involvement of this biochemical pathway in neuronal death remains largely unclear. Here, we investigate, first whether the ferroptosis key players are involved in the neuronal cell death induced by erastin. The second objective was to examine whether there is a cross talk between ferroptosis and autophagy. The third main was to address neuron response to erastin, with a special focus on ferritin and nuclear receptor coactivator 4-mediated ferritinophagy. To test this in neurons, erastin (0.5-8 µM) was applied to hippocampal HT22 neurons for 16 hours. In addition, cells were cultured with the autophagy inhibitor, 3-methyladenin (10 mM) and/or ferroptosis inhibitors, ferrostatin 1 (10-20 µM) or deferoxamine (10-200 µM) before exposure to erastin. In this study, we demonstrated by immunofluorescence and western blot analysis, that erastin downregulates dramatically the expression of glutathione peroxidase 4, the sodium-independent cystine-glutamate antiporter and nuclear receptor coactivator 4. The protein levels of ferritin and mitochondrial ferritin in HT22 hippocampal neurons did not remarkably change following erastin treatment. In addition, we demonstrated that not only the ferroptosis inhibitor, ferrostatin1/deferoxamine abrogated the ferroptotic cell death induced by erastin in hippocampal HT22 neurons, but also the potent autophagy inhibitor, 3-methyladenin. We conclude that (1) erastin-induced ferroptosis in hippocampal HT22 neurons, despite reduced nuclear receptor coactivator 4 levels, (2) that either nuclear receptor coactivator 4-mediated ferritinophagy does not occur or is of secondary importance in this model, (3) that ferroptosis seems to share some features of the autophagic cell death process.

Cerebral Ischemia Induces Iron Deposit, Ferritin Accumulation, Nuclear Receptor Coactivator 4-depletion, and Ferroptosis.

BACKGROUND: The neuronal death upon cerebral ischemia shares not only characteristics of necrosis, apoptosis, and autophagy but also exhibits biochemical and morphological characteristics of ferroptosis. Ferroptosis is a regulated form of cell death that is considered to be an oxidative iron-dependent process. It is now commonly accepted that iron and free radicals are considered to cause lipid peroxidation as well as the oxidation of proteins and nucleic acids, leading to increased membrane and enzymatic dysfunction and finally contributing to cell death. Although ferroptosis was first described in cancer cells, emerging evidence now links mechanisms of ferroptosis to many different diseases, including cerebral ischemia. METHODS: The objective of this study was to identify the key players and underlying biochemical pathways of ferroptosis, leading to cell death upon focal cerebral ischemia in mice by using immunofluorescence, Western blotting, histochemistry, and densitometry. RESULTS: In this study, we demonstrated that cerebral ischemia induced iron-deposition, downregulated dramatically the expression of the glutathione peroxidase 4 (GPX4), decreased the expression of the nuclear receptor coactivator 4 (NCOA4), and induced inappropriate accumulation of ferritin in the ischemic brain. This supports the hypothesis that an ischemic insult may induce ferroptosis through inhibition of GPX4. CONCLUSION: We conclude that iron excess following cerebral ischemia leads to cell death despite activating compensatory mechanisms for iron homeostasis, as illustrated by the accumulation of ferritins. These data emphasized the presence of a cellular mechanism that allows neuronal cells to buffer iron levels.

Evolution of resistance mutations during low-level viral replication in HIV-1-infected patients treated with zidovudine/lamivudine/abacavir as a first-line regimen.

OBJECTIVE: Long-term evaluation of viral evolution in patients who continued first-line therapy with zidovudine/lamivudine/abacavir (Trizivir [TZV]) in the presence of low-level viral replication and assessment of the impact of mutational patterns selected under TZV on viral load (VL), CD4+ T-cell count (CD4) and subsequent therapeutic options. DESIGN: Analysis of viral evolution based on genotypic resistance tests (GRT) from samples collected during non-suppressive first-line therapy with TZV. METHODS: Patients from the Frankfurt HIV cohort with at least 3 months uninterrupted first-line therapy with TZV in whom VL and CD4 measurements were performed at baseline and at follow up were identified. Criteria for virological failure (VF) were two consecutive VL >400 copies/ml. GRTs were required at baseline, VF and last visit (LV). RESULTS: Initially, 23/119 patients were classified as VF; 4/23 were lost to follow up. Median time to VF was 48 weeks. Because of the observed virological and immunological benefit, patients continued TZV for a median of 87 weeks despite detectable viraemia. Median CD4 increase and VL reduction at LV were 120 cells/mm3 and 317,100 copies/ml, respectively, compared to baseline. After 54 weeks of treatment with detectable VL, three mutational patterns were observed: Group A (n=4) characterized by M184V without further regimen-associated mutations, group B (n=9) by M184V accompanied by one to three thymidine analogue mutations (TAMs), and group C (n=6) by M184V and four to six TAMs. No virological or CD4 parameters correlated with these patterns. Group A remained unchanged, thus preserving activity of most nucleoside analogues (NA). However, in the majority of patients (groups B and C) accumulation of mutations at different rates was observed, leading to a sequential loss of NA options. CONCLUSIONS: Continuous treatment with TZV in the presence of viral replication is associated with a stepwise accumulation of resistance mutations. M184V was present in all cases, not followed by further selection of TAMs in a small, unpredictable subgroup of patients. However, in the majority of patients selection of M184V was associated with accumulation of TAMs at different rates leading to a substantial loss of active NAs, despite continuous virological and immunological benefit when compared with baseline.