Table_1_Quantitative omics analyses of NCOA4 deficiency reveal an integral role of ferritinophagy in iron homeostasis of hippocampal neuronal HT22 cells.XLSX

IntroductionNeurons require iron to support their metabolism, growth, and differentiation, but are also susceptible to iron-induced oxidative stress and cytotoxicity. Ferritin, a cytosolic iron storage unit, mediates cellular adaptation to fluctuations in iron delivery. NCOA4 has been characterized as a selective autophagic cargo receptor facilitating the mobilization of intracellular iron from ferritin. This process named ferritinophagy results in the degradation of ferritin and the consequent release of iron into the cytosol.
MethodsHere we demonstrate that NCOA4 is important for the adaptation of the HT22 mouse hippocampal neuronal cell line to cellular iron restriction. Additionally, we determined the pathophysiological implications of impaired ferritinophagy via functional analysis of the omics profile of HT22 cells deficient in NCOA4.
ResultsNCOA4 silencing impaired ferritin turnover and was cytotoxic when cells were restricted of iron. Quantitative proteomics identified IRP2 accumulation among the most prominent protein responses produced by NCOA4 depletion in HT22 cells, which is indicative of functional iron deficiency. Additionally, proteins of apoptotic signaling pathway were enriched by those responsive to NCOA4 deficiency. Transcriptome profiles of NCOA4 depletion revealed neuronal cell death, differentiation of neurons, and development of neurons as potential diseases and bio functions affected by impaired ferritinophagy, particularly, when iron was restricted.
DiscussionThese findings identify an integral role of NCOA4-mediated ferritinophagy in the maintenance of iron homeostasis by HT22 cells, and its potential implications in controlling genetic pathways of neurodevelopment and neurodegenerative diseases.

引言
神经元依赖铁以维持其代谢、生长与分化过程，但同时也易遭受铁诱导的氧化应激与细胞毒性损害。铁蛋白（Ferritin）作为胞质铁储存单元，可介导细胞应对铁供给的波动变化。核受体辅激活因子4（NCOA4）已被鉴定为一种选择性自噬货物受体，可促进细胞内铁从铁蛋白中动员释放。这一被称为铁自噬（ferritinophagy）的过程会导致铁蛋白降解，并随之将铁释放至胞质溶胶中。
方法
本研究证实，核受体辅激活因子4（NCOA4）对于HT22小鼠海马神经元细胞系适应细胞铁限制环境至关重要。此外，我们通过对NCOA4缺陷型HT22细胞的多组学谱（omics profile）进行功能分析，阐明了铁自噬受损的病理生理学意义。
结果
NCOA4基因沉默会损害铁蛋白周转过程，且在细胞处于铁限制状态时引发细胞毒性。定量蛋白质组学分析显示，HT22细胞中NCOA4缺失所引发的最显著蛋白应答中，铁调节蛋白2（IRP2）的蓄积最为突出，这一现象提示存在功能性铁缺乏。此外，响应NCOA4缺失的差异蛋白显著富集于凋亡信号通路相关蛋白中。对NCOA4缺失细胞的转录组谱分析显示，铁自噬受损所影响的潜在疾病与生物功能包括神经元死亡、神经元分化及神经元发育，且在铁限制条件下这一效应尤为显著。
讨论
本研究结果证实，NCOA4介导的铁自噬在HT22细胞维持铁稳态（iron homeostasis）过程中发挥不可或缺的作用，其潜在意义在于调控神经发育与神经退行性疾病相关的遗传通路。