Cytoplasmic Labile Iron Accumulates in Aging Stem Cells Perturbing a Key Rheostat for Identity Control

Bone marrow resident and rarely dividing hematopoietic stem cells (HSC) harbor an extensive self-renewal capacity to sustain life-long blood formation, albeit their function declines during ageing. Various molecular mechanisms confer stem cell identity, ensure long-term maintenance and are known to be deregulated in aged stem cells. How these programs are coordinated, particularly during cell division, and what triggers their ageing-associated dysfunction has been unknown. We have previously uncovered that iron chelator exposure increases the number of functional HSC ex vivo and in vivo (Kao et al., Science Transl Med 2018). While ensuring a sufficient amount of redox active, readily available iron which is required in numerous electron transfer reactions governing fundamental cellular processes, cells tightly regulate the size of the intracellular labile iron pool (LIP) to limit adverse ROS generation. Perturbations in the ability to limit intracellular iron is detrimental for cells and known to compromise HSC maintenance and function via altered redox signaling and increased macromolecule oxidation and damage. The HSC stimulatory effects of iron chelator (IC) treatment and the well characterized central roles of redox active intracellular iron in sustaining basic cell function prompted us to examine a potential regulatory role of the LIP in controlling somatic stem cell function. In this study, we quantified LIP in young and aged HSC and monitored iron homoeostasis pathway activation, hallmarked by the stabilization of transferrin receptor (Tfrc) mRNA, in stem cells for which we developed a single molecule RNA fluorescence in situ hybridization (smRNA FISH) assay enabling the quantification of Tfrc dynamics with unparalleled resolution and sensitivity. We have further used experimental LIP modulation in primary hematopoietic stem cell models to characterize the consequences of iron homeostasis pathway activation in young and aged stem cells; and employed integrated comparative quantitative transcriptomics (single cell RNA-seq) and proteomics along with genetic and pharmacological rescue models to identify the consequences and mechanisms of LIP size alterations. Our findings demonstrate that HSC, containing the lowest amount of cytoplasmic chelatable iron hematopoietic cells, activate a limited iron response during mitosis. Engagement of this iron homeostasis pathway elicits mobilization and β-oxidation of arachidonic acid and enhances stem cell-defining transcriptional programs governed by histone acetyl transferase Tip60/KAT5. We further find an age-associated expansion of the labile iron pool, along with loss of Tip60/KAT5-dependent gene regulation to contribute to the functional decline of ageing HSC, which can be mitigated by iron chelation. Together, our work reveals cytoplasmic redox active iron as a novel rheostat in adult stem cells; it demonstrates a role for the intracellular labile iron pool in coordinating a cascade of molecular events which reinforces HSC identity during cell division and to drive stem cell ageing when perturbed. As loss of iron homeostasis is commonly observed in the elderly, we anticipate these findings to trigger further studies into understanding and therapeutic mitigation of labile iron pool-dependent hematopoietic stem cell dysfunction in a wide range of degenerative and malignant hematologic pathologies. Disclosures D'Alessandro: Omix Thecnologies: Other: Co-founder; Rubius Therapeutics: Consultancy; Forma Therapeutics: Membership on an entity's Board of Directors or advisory committees.

The Labile Iron Pool Reacts Rapidly and Catalytically with Peroxynitrite

While investigating peroxynitrite-dependent oxidation in murine RAW 264.7 macrophage cells, we observed that removal of the Labile Iron Pool (LIP) by chelation increases the intracellular oxidation of the fluorescent indicator H2DCF, so we concluded that the LIP reacts with peroxynitrite and decreases the yield of peroxynitrite-derived oxidants. This was a paradigm-shifting finding in LIP biochemistry and raised many questions. In this follow-up study, we address fundamental properties of the interaction between the LIP and peroxynitrite by using the same cellular model and fluorescence methodology. We have identified that the reaction between the LIP and peroxynitrite has catalytic characteristics, and we have estimated that the rate constant of the reaction is in the range of 106 to 107 M−1s−1. Together, these observations suggest that the LIP represents a constitutive peroxynitrite reductase system in RAW 264.7 cells.

Cytoplasmic labile iron accumulates in aging stem cells perturbing a key rheostat for identity control

Bone marrow resident and rarely dividing haematopoietic stem cells (HSC) harbour an extensive self-renewal capacity to sustain life-long blood formation; albeit their function declines during ageing. Various molecular mechanisms confer stem cell identity, ensure long-term maintenance and are known to be deregulated in aged stem cells. How these programs are coordinated, particularly during cell division, and what triggers their ageing-associated dysfunction has been unknown. Here, we demonstrate that HSC, containing the lowest amount of cytoplasmic chelatable iron (labile iron pool) among hematopoietic cells, activate a limited iron response during mitosis. Engagement of this iron homeostasis pathway elicits mobilization and β-oxidation of arachidonic acid and enhances stem cell-defining transcriptional programs governed by histone acetyl transferase Tip60/KAT5. We further find an age-associated expansion of the labile iron pool, along with loss of Tip60/KAT5-dependent gene regulation to contribute to the functional decline of ageing HSC, which can be mitigated by iron chelation. Together, our work reveals cytoplasmic redox active iron as a novel rheostat in adult stem cells; it demonstrates a role for the intracellular labile iron pool in coordinating a cascade of molecular events which reinforces HSC identity during cell division and to drive stem cell ageing when perturbed. As loss of iron homeostasis is commonly observed in the elderly, we anticipate these findings to trigger further studies into understanding and therapeutic mitigation of labile iron pool-dependent stem cell dysfunction in a wide range of degenerative and malignant pathologies.

TRIB2 desensitizes ferroptosis via βTrCP-mediated TFRC ubiquitiantion in liver cancer cells

Tribbles homolog 2 (TRIB2) is known to boost liver tumorigenesis via regulating Ubiquitin (Ub) proteasome system (UPS). At least two ways are involved, i.e., acts as an adaptor protein to modulate ubiquitination functions of certain ubiquitin E3 ligases (E3s) and reduces global Ub levels via increasing the proteolysis activity of proteasome. Recently, we have identified the role of TRIB2 to relieve oxidative damage via reducing the availability of Ub that is essential for the ubiquitination and subsequent degradation of Glutathione peroxidase 4 (GPX4). Although GPX4 is a critical antioxidant factor to protect against ferroptosis, the exact evidence showing that TRIB2 desensitizes ferroptosis is lacking. Also, whether such function is via E3 remains unclear. Here, we demonstrated that deletion of TRIB2 sensitized ferroptosis via lifting labile iron in liver cancer cells. By contrast, overexpression of TRIB2 led to the opposite outcome. We further demonstrated that transferrin receptor (TFRC) was required for TRIB2 to desensitize the cells to ferroptosis. Without TFRC, the labile iron pool could not be reduced by overexpressing TRIB2. We also found that beta-transducin repeat containing E3 ubiqutin protein ligase (βTrCP) was a genuine E3 for the ubiquitination of TFRC, and TRIB2 was unable to decline labile iron level once upon βTrCP was knocked out. In addition, we confirmed that the opposite effects on ferroptosis and ferroptosis-associated lipid reactive oxygen species (ROS) generation resulted from knockout and overexpression of TRIB2 were all indispensible of TFRC and βTrCP. Finally, we demonstrated that TRIB2 exclusively manipulated RSL3- and erastin-induced-ferroptosis independent of GPX4 and glutathione (GSH). In conclusion, we elucidated a novel role of TRIB2 to desensitize ferroptosis via E3 βTrCP, by which facilitates TFRC ubiquitiation and finally decreases labile iron in liver cancer cells.

MYCN mediates TFRC-dependent ferroptosis and reveals vulnerabilities in neuroblastoma

MYCN amplification is tightly associated with the poor prognosis of pediatric neuroblastoma (NB). The regulation of NB cell death by MYCN represents an important aspect, as it directly contributes to tumor progression and therapeutic resistance. However, the relationship between MYCN and cell death remains elusive. Ferroptosis is a newly identified cell death mode featured by lipid peroxide accumulation that can be attenuated by GPX4, yet whether and how MYCN regulates ferroptosis are not fully understood. Here, we report that MYCN-amplified NB cells are sensitive to GPX4-targeting ferroptosis inducers. Mechanically, MYCN expression reprograms the cellular iron metabolism by upregulating the expression of TFRC, which encodes transferrin receptor 1 as a key iron transporter on the cell membrane. Further, the increased iron uptake promotes the accumulation of labile iron pool, leading to enhanced lipid peroxide production. Consistently, TFRC overexpression in NB cells also induces selective sensitivity to GPX4 inhibition and ferroptosis. Moreover, we found that MYCN fails to alter the general lipid metabolism and the amount of cystine imported by System Xc(−) for glutathione synthesis, both of which contribute to ferroptosis in alternative contexts. In conclusion, NB cells harboring MYCN amplification are prone to undergo ferroptosis conferred by TFRC upregulation, suggesting that GPX4-targeting ferroptosis inducers or TFRC agonists can be potential strategies in treating MYCN-amplified NB.

Caffeic Acid Phenyl Ester (CAPE) Protects against Iron-Mediated Cellular DNA Damage through Its Strong Iron-Binding Ability and High Lipophilicity

Caffeic acid phenethyl ester (CAPE) and its structurally-related caffeic acid (CA), ferulic acid (FA) and ethyl ferulate (EF) are constituents of honeybee propolis that have important pharmacological activities. This study found that CAPE—but not CA, FA, and EF—could effectively prevent cellular DNA damage induced by overloaded iron through decreasing the labile iron pool (LIP) levels in HeLa cells. Interestingly, CAPE was found to be more effective than CA in protecting against plasmid DNA damage induced by Fe(II)–H2O2 or Fe(III)–citrate–ascorbate-H2O2 via the inhibition of hydroxyl radical (•OH) production. We further provided more direct and unequivocal experimental evidences for the formation of inactive CAPE/CA–iron complexes. CAPE was found to have a stronger iron-binding ability and a much higher lipophilicity than CA. Taken together, we propose that the esterification of the carboxylic moiety with phenethyl significantly enhanced the iron-binding ability and lipophilicity of CAPE, which is also responsible for its potent protection against iron-mediated cellular DNA damage. A study on the iron coordination mechanism of such natural polyphenol antioxidants will help to design more effective antioxidants for the treatment and prevention of diseases caused by metal-induced oxidative stress, as well as help to understand the structure–activity relationships of these compounds.

Protection of Iron-Induced Oxidative Damage in Neuroblastoma (SH-SY5Y) Cells by Combination of 1-(N-Acetyl-6-aminohexyl)-3-hydroxy-2-methylpyridin-4-one and Green Tea Extract

Iron is a crucial trace element and essential for many cellular processes; however, excessive iron accumulation can induce oxidative stress and cell damage. Neurodegenerative disorders, such as Alzheimer’s disease and Parkinson’s disease, have been associated with altered iron homoeostasis causing altered iron distribution and accumulation in brain tissue. This study aims to investigate the protective effect of 1-(N-acetyl-6-aminohexyl)-3-hydroxy-2-methylpyridin-4-one (CM1) in combination with green tea extract (GTE) on iron-induced oxidative stress in neuroblastoma (SH-SY5Y) cells. Cells were cultured in medium with or without ferric chloride loading. Their viability and mitochondrial activity were assessed using MTT and JC-1 staining methods. Levels of the cellular labile iron pool (LIP), reactive oxygen species (ROS), and lipid-peroxidation products were determined using calcein acetoxymethyl ester, 2′,7′-dichlorohydrofluorescein diacetate, and TBARS-based assays, respectively. The viability of iron-loaded cells was found to be significantly increased after treatment with CM1 (10 µM) for 24 h. CM1 co-treatment with GTE resulted in a greater protective effect than their monotherapy. Combination of CM1 and GTE also reduced mitochondrial disruption and LIP content and ROS and TBARS production. In conclusion, the combination of CM1 and GTE exhibits protection against iron-induced oxidative stress in neuroblastoma cells.

Implication of Dietary Iron-Chelating Bioactive Compounds in Molecular Mechanisms of Oxidative Stress-Induced Cell Ageing

One of the prevailing perceptions regarding the ageing of cells and organisms is the intracellular gradual accumulation of oxidatively damaged macromolecules, leading to the decline of cell and organ function (free radical theory of ageing). This chemically undefined material known as “lipofuscin,” “ceroid,” or “age pigment” is mainly formed through unregulated and nonspecific oxidative modifications of cellular macromolecules that are induced by highly reactive free radicals. A necessary precondition for reactive free radical generation and lipofuscin formation is the intracellular availability of ferrous iron (Fe2+) (“labile iron”), catalyzing the conversion of weak oxidants such as peroxides, to extremely reactive ones like hydroxyl (HO•) or alcoxyl (RO•) radicals. If the oxidized materials remain unrepaired for extended periods of time, they can be further oxidized to generate ultimate over-oxidized products that are unable to be repaired, degraded, or exocytosed by the relevant cellular systems. Additionally, over-oxidized materials might inactivate cellular protection and repair mechanisms, thus allowing for futile cycles of increasingly rapid lipofuscin accumulation. In this review paper, we present evidence that the modulation of the labile iron pool distribution by nutritional or pharmacological means represents a hitherto unappreciated target for hampering lipofuscin accumulation and cellular ageing.

A facile colorimetric method for the quantification of labile iron pool and total iron in cells and tissue specimens

Quantification of iron is an important step to assess the iron burden in patients suffering from iron overload diseases, as well as tremendous value in understanding the underlying role of iron in the pathophysiology of these diseases. Current iron determination of total or labile iron, requires extensive sample handling and specialized instruments, whilst being time consuming and laborious. Moreover, there is minimal to no overlap between total iron and labile iron quantification methodologies—i.e. requiring entirely separate protocols, techniques and instruments. Herein, we report a unified-ferene (u-ferene) assay that enables a 2-in-1 quantification of both labile and total iron from the same preparation of a biological specimen. We demonstrate that labile iron concentrations determined from the u-ferene assay is in agreement with confocal laser scanning microscopy techniques employed within the literature. Further, this assay offers the same sensitivity as the current gold standard, inductively coupled plasma mass spectrometry (ICP-MS), for total iron measurements. The new u-ferene assay will have tremendous value for the wider scientific community as it offers an economic and readily accessible method for convenient 2-in-1 measurement of total and labile iron from biological samples, whilst maintaining the precision and sensitivity, as compared to ICP-MS.

Glutathione and the intracellular labile heme pool

One candidate for the cytosolic labile iron pool is iron(II)glutathione. There is also a widely held opinion that an equivalent cytosolic labile heme pool exists and that this pool is important for the intracellular transfer of heme. Here we describe a study designed to characterise conjugates that form between heme and glutathione. In contrast to hydrated iron(II), heme reacts with glutathione, under aerobic conditions, to form the stable hematin–glutathione complex, which contains iron(III). Thus, glutathione is clearly not the cytosolic ligand for heme, indeed we demonstrate that the rate of heme degradation is enhanced in the presence of glutathione. We suggest that the concentration of heme in the cytosol is extremely low and that intracellular heme transfer occurs via intracellular membrane structures. Should any heme inadvertently escape into the cytosol, it would be rapidly conjugated to glutathione thereby protecting the cell from the toxic effects of heme.