Pervasiveness and diagnostic potential of ferroptosis in human neuropathology

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The etiology of human degenerative diseases is accompanied by prominent cell death. However, with exception of TUNEL/cleaved caspase-3 staining for apoptotic cells, in vivo assignment of other “programmed” death modalities are precluded due to technical reliance on transient markers or inhibitor efficacy in cultured cells. Importantly, retrospective extrapolation using TUNEL has revealed, by exclusion, a substantial contribution of non-apoptotic cell death in stroke, Alzheimer’s, Parkinson’s, and Huntington’s disease. Cell death due to unrestrained iron-dependent catalysis of toxic lipid peroxides and other reactive oxygen radicals is termed ferroptosis. Due to its possible involvement in multiple diseases, recent years have seen an extensive increase in studies on its mechanisms. However, although extensively characterized in vitro, ferroptosis has not been conclusively demonstrated under pathological conditions in humans. This is primarily due to a lack of discriminating ferroptosis cell death markers. An unambiguous determination of ferroptosis contribution in these diseases would be a logical first step to directly enable clinicopathological assessment. For the identification of such a marker or multiple markers we follow a two-pronged strategy: (a) In our preliminary work, we have already identified proteins specific to early stage ferroptosis based on biotinylation of cell surface proteins, affinity purification via avidin beads, and mass spectrometry analysis. Antibodies against these putative marker proteins will be deeply profiled for specificity to ferroptosis in established cell culture and 3D organoid models. Following the identification of faithful markers, antibodies will be then tested on an extensive panel of human neuropathological tissues to retrospectively categorize cell death. (b) As a second approach, we will characterize marker proteins or lipids contained in extracellular vesicles (EVs), small membrane entities of 30-1000 nm. EVs reflect the physiological and metabolic state of their releasing cells and can be easily isolated from body fluids, hence they are ideal candidates to establish whether ferroptosis is associated with any of the aforementioned pathologies. Thus, we will identify EV markers from cell culture supernatants and plasma of genetic ferroptosis mouse models and additionally analyze these by protein- and lipid-mass spectrometry. This analysis will be extended to EVs extracted from the plasma of human patients with various neurological/neurodegenerative disease. The final goal will be a histological or EV-based toolkit to be used on a large collection of patient-derived clinical material to definitively characterize the contribution of ferroptosis to neurological disease. For both acute and retrospective analyses, an acceptable outcome could also be definitive proof that ferroptosis does not contribute substantially to human neuropathological diseases and therefore its role would have to be reconsidered.