Pervasiveness and diagnostic potential of ferroptosis in human neuropathology

铁死亡在人类神经病理学中的普遍性和诊断潜力

• 批准号: 501860452
• 负责人: Dr. Thomas Arzberger
• 金额: --
• 依托单位: Institut für Molekulare Toxikologie und Pharmakologie (TOXI)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/501860452?language=en
• 关键词: Pervasiveness; diagnostic; potential; ferroptosis; human

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.

人类退行性疾病的病因是伴随着显著的细胞死亡。然而，除了凋亡细胞的TUNEL/cleaved caspase-3染色外，由于技术上依赖于培养细胞的瞬时标记或抑制剂功效，因此排除了其他“程序性”死亡模式的体内分配。重要的是，使用TUNEL进行回顾性外推，通过排除，揭示了非凋亡细胞死亡在中风、阿尔茨海默病、帕金森病和亨廷顿病中的重要作用。由于毒性脂质过氧化物和其他活性氧自由基的无限制铁依赖性催化而导致的细胞死亡被称为铁凋亡。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，尽管在体外广泛表征，铁下垂尚未在人类病理条件下得到最终证实。这主要是由于缺乏鉴别的铁下垂细胞死亡标记物。明确确定铁下垂在这些疾病中的作用将是直接进行临床病理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，我们已经根据细胞表面蛋白的生物素化，亲和纯化通过亲和蛋白珠和质谱分析鉴定了早期铁死亡的特异性蛋白质。针对这些假定的标记蛋白的抗体将在已建立的细胞培养和3D类器官模型中深入分析对铁下垂的特异性。在确定可靠的标记物之后，抗体将在广泛的人类神经病理组织上进行测试，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（EVs）中含有的标记蛋白或脂质，EVs是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，并且可以很容易地从体液中分离出来，因此它们是确定铁下垂是否与上述任何病理相关的理想候选物。因此，我们将从遗传性铁下垂小鼠模型的细胞培养上清和血浆中鉴定EV标记物，并通过蛋白质和脂质谱分析这些标记物。该分析将扩展到从患有各种神经系统/神经退行性疾病的人类患者血浆中提取的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，一个可接受的结果也可能是明确的证据，证明铁下垂不会对人类神经病理疾病产生重大影响，因此必须重新考虑其作用。