Pervasiveness and diagnostic potential of ferroptosis in human neuropathology

人类退行性疾病的病因学伴随着显著的细胞死亡。然而，除了凋亡细胞的TUNEL/切割的半胱天冬酶-3染色，由于技术上依赖于培养细胞中的瞬时标记物或抑制剂功效，排除了其他“程序性”死亡模式的体内分配。重要的是，通过排除，使用TUNEL的回顾性外推法揭示了中风、阿尔茨海默病、帕金森病和亨廷顿病中非凋亡性细胞死亡的实质性贡献。由于毒性脂质过氧化物和其他活性氧自由基的不受限制的铁依赖性催化导致的细胞死亡称为铁凋亡。由于其可能参与多种疾病，近年来对其机制的研究广泛增加。然而，尽管在体外进行了广泛的表征，但在人类的病理条件下尚未最终证实铁凋亡。这主要是由于缺乏可区分的铁凋亡细胞死亡标志物。明确确定这些疾病中的铁凋亡贡献将是直接进行临床病理学评估的合乎逻辑的第一步。为了鉴定这样的标记或多个标记，我们遵循双管齐下的策略：（a）在我们的初步工作中，我们已经基于细胞表面蛋白的生物素化、经由抗生物素蛋白珠的亲和纯化和质谱分析鉴定了对早期铁凋亡特异的蛋白。针对这些推定的标志物蛋白的抗体将在已建立的细胞培养物和3D类器官模型中针对铁凋亡的特异性进行深入剖析。在鉴定出可靠的标志物后，将在一组广泛的人类神经病理组织上检测抗体，以回顾性地对细胞死亡进行分类。(b)作为第二种方法，我们将表征细胞外囊泡（EV）中包含的标记蛋白或脂质，30-1000 nm的小膜实体。EV反映了其释放细胞的生理和代谢状态，并且可以容易地从体液中分离，因此它们是确定铁凋亡是否与任何上述病理相关的理想候选者。因此，我们将从遗传性铁凋亡小鼠模型的细胞培养上清液和血浆中鉴定EV标志物，并通过蛋白质和脂质质谱法对这些标志物进行分析。该分析将扩展到从患有各种神经/神经退行性疾病的人类患者的血浆中提取的EV。最终目标将是一个组织学或基于EV的工具包，用于大量患者来源的临床材料，以明确表征铁下垂对神经系统疾病的贡献。对于急性和回顾性分析，可接受的结局也可能是铁凋亡对人类神经病理学疾病无实质性贡献的明确证据，因此必须重新考虑其作用。

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.