Lipocalin-2 inhibition alleviates neural injury by microglia ferroptosis suppression after experimental intracerebral hemorrhage in mice via enhancing ferritin light chain expression.

INTRODUCTION: Microglia play pivotal roles in post-intracerebral hemorrhage (ICH) neural injury. Iron metabolism, which is dysregulated after ICH, participates in microglial dysfunction. Previous studies have shown that iron metabolism-related lipocalin-2 (LCN2) is involved in regulating microglial function following ICH. In this study, we investigated the role of LCN2 in microglial function following ICH. METHODS: The BV2 (microglia) cell line, transfected with LCN2 for overexpression/interference, received a blood infusion from C57BL/6 mice in vitro. For the in vivo study of LCN2 function, an LCN2 knockout was conducted in mice. Liproxstatin-1 and RSL3 were used to manipulate ferroptosis and to study the effects of LCN2 on microglia after ICH. A BV2 (microglia) cell line, transfected with ferritin light chain (FTL) for overexpression/interference, was co-cultured with primary cultured neurons for a study on the mechanism of LCN2. Behavioral tests were conducted pre-ICH and on days 3, 7, and 28 post-ICH, and the brains and cultured cells were collected for protein, histological, and morphological studies. RESULTS: Brain LCN2 expression was upregulated in microglia, astrocytes, and neurons and played hazardous roles after ICH. In microglia, LCN2 promoted ferroptosis, which facilitated neural injury after ICH. LCN2-mediated FTL deficiency was shown to be responsible for microglial ferroptosis-induced neural injury. CONCLUSION: Our study suggests that LCN2-enhanced microglial ferroptosis plays a detrimental role by inducing FTL deficiency after ICH. The current study reveals a novel molecular mechanism involved in the pathophysiological progression of ICH.

Myeloid-specific ferritin light chain deletion does not exacerbate sepsis-associated AKI.

Sepsis-associated acute kidney injury (SA-AKI) is a key contributor to the life-threatening sequelae attributed to sepsis. Mechanistically, SA-AKI is a consequence of unabated myeloid cell activation and oxidative stress that induces tubular injury. Iron mediates inflammatory pathways directly and through regulating the expression of myeloid-derived ferritin, an iron storage protein comprising ferritin light (FtL) and ferritin heavy chain (FtH) subunits. Previous work revealed that myeloid FtH deletion leads to a compensatory increase in intracellular and circulating FtL and is associated with amelioration of SA-AKI. We designed this study to test the hypothesis that loss of myeloid FtL and subsequently, circulating FtL will exacerbate the sepsis-induced inflammatory response and worsen SA-AKI. We generated a novel myeloid-specific FtL knockout mouse (FtLLysM-/-) and induced sepsis via cecal ligation and puncture or lipopolysaccharide endotoxemia. As expected, serum ferritin levels were significantly lower in the knockout mice, suggesting that myeloid cells dominantly contribute to circulating ferritin. Interestingly, although sepsis induction led to a marked production of pro- and anti-inflammatory cytokines, there was no statistical difference between the genotypes. There was a similar loss of kidney function, as evidenced by a rise in serum creatinine and cystatin C and renal injury identified by expression of kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin. Finally, RNA sequencing revealed upregulation of pathways for cell cycle arrest and autophagy postsepsis, but no significant differences were observed between genotypes, including in key genes associated with ferroptosis, an iron-mediated form of cell death. The loss of FtL did not impact sepsis-mediated activation of NF-κB or HIF-1a signaling, key inflammatory pathways associated with dysregulated host response. Taken together, while FtL overexpression was shown to be protective against sepsis, the loss of FtL did not influence sepsis pathogenesis.NEW & NOTEWORTHY Hyperferritinemia in sepsis is often associated with a proinflammatory phenotype and poor prognosis. We previously showed the myeloid deletion of FtH results in a compensatory increase in FtL and is associated with reduced circulating cytokines and decreased rates of SA-AKI in animal sepsis models. Here, we show that myeloid deletion of FtL does not impact the severity of SA-AKI following CLP or LPS, suggesting that FtH plays the predominant role in propagating myeloid-induced proinflammatory pathways.

Iron promotes ovarian cancer malignancy and advances platinum resistance by enhancing DNA repair via FTH1/FTL/POLQ/RAD51 axis.

Iron is crucial for cell DNA synthesis and repair, but an excess of free iron can lead to oxidative stress and subsequent cell death. Although several studies suggest that cancer cells display characteristics of 'Iron addiction', an ongoing debate surrounds the question of whether iron can influence the malignant properties of ovarian cancer. In the current study, we initially found iron levels increase during spheroid formation. Furthermore, iron supplementation can promote cancer cell survival, cancer spheroid growth, and migration; vice versa, iron chelators inhibit this process. Notably, iron reduces the sensitivity of ovarian cancer cells to platinum as well. Mechanistically, iron downregulates DNA homologous recombination (HR) inhibitor polymerase theta (POLQ) and relieves its antagonism against the HR repair enzyme RAD51, thereby promoting DNA damage repair to resist chemotherapy-induced damage. Additionally, iron tightly regulated by ferritin (FTH1/FTL) which is indispensable for iron-triggered DNA repair. Finally, we discovered that iron chelators combined with platinum exhibit a synergistic inhibitory effect on ovarian cancer in vitro and in vivo. Our findings affirm the pro-cancer role of iron in ovarian cancer and reveal that iron advances platinum resistance by promoting DNA damage repair through FTH1/FTL/POLQ/RAD51 pathway. Our findings highlight the significance of iron depletion therapy, revealing a promising avenue for advancing ovarian cancer treatment.

Preparation and brain targeting effects study of recombinant human ferritin nanoparticles.

Human heavy-chain ferritin is a naturally occurring protein with high stability and multifunctionality in biological systems. This study aims to utilize a prokaryotic expression system to produce recombinant human heavy-chain ferritin nanoparticles and investigate their targeting ability in brain tissue. The human heavy-chain ferritin gene was cloned into the prokaryotic expression vector pET28a and transformed into Escherichia coli BL21 (DE3) competent cells to explore optimal expression conditions. The recombinant protein was then purified to evaluate its immunoreactivity and characteristics. Additionally, the distribution of the administered protein in normal mice and its permeability in an in vitro blood-brain barrier (BBB) model were measured. The results demonstrate that the purified protein can self-assemble extracellularly into nano-cage structures of approximately 10 nm and is recognized by corresponding antibodies. The protein effectively penetrates the blood-brain barrier and exhibits slow clearance in mouse brain tissue, showing excellent permeability in the in vitro BBB model. This study highlights the stable expression of recombinant human heavy-chain ferritin using the Escherichia coli prokaryotic expression system, characterized by favorable nano-cage structures and biological activity. Its exceptional brain tissue targeting and slow metabolism lay an experimental foundation for its application in neuropharmaceutical delivery and vaccine development fields.

7T MRI detects widespread brain iron deposition in neuroferritinopathy.

Neuroferritinopathy is a disorder of neurodegeneration with brain iron accumulation that has no proven disease-modifying treatments. Clinical trials require biomarkers of iron deposition. We examined brain iron accumulation in one presymptomatic FTL mutation carrier, two individuals with neuroferritinopathy and one healthy control using ultra-high-field 7T MRI. There was increased magnetic susceptibility, suggestive of iron deposition, in superficial and deep gray matter in both presymptomatic and symptomatic neuroferritinopathy. Cavitation of the putamen and globus pallidus increased with disease stage and at follow up. The widespread brain iron deposition in presymptomatic and early disease provides an opportunity for monitoring disease-modifying intervention.

Ferritin heavy chain supports stability and function of the regulatory T cell lineage.

Regulatory T (TREG) cells develop via a program orchestrated by the transcription factor forkhead box protein P3 (FOXP3). Maintenance of the TREG cell lineage relies on sustained FOXP3 transcription via a mechanism involving demethylation of cytosine-phosphate-guanine (CpG)-rich elements at conserved non-coding sequences (CNS) in the FOXP3 locus. This cytosine demethylation is catalyzed by the ten-eleven translocation (TET) family of dioxygenases, and it involves a redox reaction that uses iron (Fe) as an essential cofactor. Here, we establish that human and mouse TREG cells express Fe-regulatory genes, including that encoding ferritin heavy chain (FTH), at relatively high levels compared to conventional T helper cells. We show that FTH expression in TREG cells is essential for immune homeostasis. Mechanistically, FTH supports TET-catalyzed demethylation of CpG-rich sequences CNS1 and 2 in the FOXP3 locus, thereby promoting FOXP3 transcription and TREG cell stability. This process, which is essential for TREG lineage stability and function, limits the severity of autoimmune neuroinflammation and infectious diseases, and favors tumor progression. These findings suggest that the regulation of intracellular iron by FTH is a stable property of TREG cells that supports immune homeostasis and limits the pathological outcomes of immune-mediated inflammation.

Effects of ferritin heavy chain on oxidative stress, cell proliferation and apoptosis in geese follicular granulosa cells.

1. The ferritin heavy chain (FHC) has a vital impact on follicular development in geese, due to its ability to regulate apoptosis of granulosa cells (GCs) and follicular atresia. However, its specific regulatory mechanisms remain unclear. The present study characterised how FHC regulates oxidative stress, cell proliferation and apoptosis in goose GCs by interfering with and overexpressing the FHC gene.2. After 72 h of interference with FHC expression, the activity of GCs decreased remarkably (p < 0.05), reactive oxygen species (ROS) levels and the expression levels of antioxidant enzyme genes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) increased significantly (p < 0.05). The overexpression of FHC for 72 h was found to significantly reduce the expression of CAT and SOD genes (p < 0.05).3. Interfering with FHC expression revealed that the expression levels of the cell proliferation gene Aurora kinase A (AURORA-A) were significantly decreased (p < 0.05), while the expression levels of the apoptosis genes B-cell lymphoma-2 (BCL-2) and cysteine aspartate-specific protease 8 (CASPASE 8) increased (p < 0.05). Further research has shown that, when interfering with FHC expression for 72 h, apoptosis rate increased by 1.19-fold (p < 0.05), but the current data showed a lower apoptosis rate after FHC overexpression by 59.41%, 63.39%, and 52.31% at three different treatment times (p < 0.05).4. In conclusion, FHC improved the antioxidant capacity of GCs, promotes GCs proliferation, and inhibits GCs apoptosis of ovarian follicles in Sichuan white geese.

Quantitative MRI Evaluation of Ferritin Overexpression in Non-Small-Cell Lung Cancer.

Cancer cells frequently present elevated intracellular iron levels, which are thought to facilitate an enhanced proliferative capacity. Targeting iron metabolism within cancer cells presents an avenue to enhance therapeutic responses, necessitating the use of non-invasive models to modulate iron manipulation to predict responses. Moreover, the ubiquitous nature of iron necessitates the development of unique, non-invasive markers of metabolic disruptions to develop more personalized approaches and enhance the clinical utility of these approaches. Ferritin, an iron storage enzyme that is often upregulated as a response to iron accumulation, plays a central role in iron metabolism and has been frequently associated with unfavorable clinical outcomes in cancer. Herein, we demonstrate the successful utility, validation, and functionality of a doxycycline-inducible ferritin heavy chain (FtH) overexpression model in H1299T non-small-cell lung cancer (NSCLC) cells. Treatment with doxycycline increased the protein expression of FtH with a corresponding decrease in labile iron in vitro and in vivo, as determined by calcein-AM staining and EPR, respectively. Moreover, a subsequent increase in TfR expression was observed. Furthermore, T2* MR mapping effectively detected FtH expression in our in vivo model. These results demonstrate that T2* relaxation times can be used to monitor changes in FtH expression in tumors with bidirectional correlations depending on the model system. Overall, this study describes the development of an FtH overexpression NSCLC model and its correlation with T2* mapping for potential use in patients to interrogate iron metabolic alterations and predict clinical outcomes.

Adipose knockout of H-ferritin improves energy metabolism in mice.

OBJECTIVE: Ferritin, the principal iron storage protein, is essential to iron homeostasis. How iron homeostasis affects the adipose tissue is not well understood. We investigated the role of ferritin heavy chain in adipocytes in energy metabolism. METHODS: We generated adipocyte-specific ferritin heavy chain (Fth, also known as Fth1) knockout mice, herein referred to as FthAKO. These mice were analyzed for iron homeostasis, oxidative stress, mitochondrial biogenesis and activity, adaptive thermogenesis, insulin sensitivity, and metabolic measurements. Mouse embryonic fibroblasts and primary mouse adipocytes were used for in vitro experiments. RESULTS: In FthAKO mice, the adipose iron homeostasis was disrupted, accompanied by elevated expression of adipokines, dramatically induced heme oxygenase 1(Hmox1) expression, and a notable decrease in the mitochondrial ROS level. Cytosolic ROS elevation in the adipose tissue of FthAKO mice was very mild, and we only observed this in the brown adipose tissue (BAT) but not in the white adipose tissue (WAT). FthAKO mice presented an altered metabolic profile and showed increased insulin sensitivity, glucose tolerance, and improved adaptive thermogenesis. Interestingly, loss of ferritin resulted in enhanced mitochondrial respiration capacity and a preference for lipid metabolism. CONCLUSIONS: These findings indicate that ferritin in adipocytes is indispensable to intracellular iron homeostasis and regulates systemic lipid and glucose metabolism.

A newly identified ferritin L-subunit variant results in increased proteasomal subunit degradation, impaired complex assembly, and severe hypoferritinemia.

Ferritin is a hetero-oligomeric nanocage, composed of 24 subunits of two types, FTH1 and FTL. It protects the cell from excess reactive iron, by storing iron in its cavity. FTH1 is essential for the recruitment of iron into the ferritin nanocage and for cellular ferritin trafficking, whereas FTL contributes to nanocage stability and iron nucleation inside the cavity. Here we describe a female patient with a medical history of severe hypoferritinemia without anemia. Following inadequate heavy IV iron supplementation, the patient developed severe iron overload and musculoskeletal manifestations. However, her serum ferritin levels rose only to normal range. Genetic analyses revealed an undescribed homozygous variant of FTL (c.92A > G), which resulted in a Tyr31Cys substitution (FTLY31C ). Analysis of the FTL structure predicted that the Y31C mutation will reduce the variant's stability. Expression of the FTLY31C variant resulted in significantly lower cellular ferritin levels compared with the expression of wild-type FTL (FTLWT ). Proteasomal inhibition significantly increased the initial levels of FTLY31C , but could not protect FTLY31C subunits from successive degradation. Further, variant subunits successfully incorporated into hetero-polymeric nanocages in the presence of sufficient levels of FTH1. However, FTLY31C subunits poorly assembled into nanocages when FTH1 subunit levels were low. These results indicate an increased susceptibility of unassembled monomeric FTLY31C subunits to proteasomal degradation. The decreased cellular assembly of FTLY31C -rich nanocages may explain the low serum ferritin levels in this patient and emphasize the importance of a broader diagnostic approach of hypoferritinemia without anemia, before IV iron supplementation.

Sexually dimorphic effect of H-ferritin genetic manipulation on survival and tumor microenvironment in a mouse model of glioblastoma.

PURPOSE: Iron plays a crucial role in various biological mechanisms and has been found to promote tumor growth. Recent research has shown that the H-ferritin (FTH1) protein, traditionally recognized as an essential iron storage protein, can transport iron to GBM cancer stem cells, reducing their invasion activity. Moreover, the binding of extracellular FTH1 to human GBM tissues, and brain iron delivery in general, has been found to have a sex bias. These observations raise questions, addressed in this study, about whether H-ferritin levels extrinsic to the tumor can affect tumor cell pathways and if this impact is sex-specific. METHODS: To interrogate the role of systemic H-ferritin in GBM we introduce a mouse model in which H-ferritin levels are genetically manipulated. Mice that were genetically manipulated to be heterozygous for H-ferritin (Fth1+/-) gene expression were orthotopically implanted with a mouse GBM cell line (GL261). Littermate Fth1 +/+ mice were used as controls. The animals were evaluated for survival and the tumors were subjected to RNA sequencing protocols. We analyzed the resulting data utilizing the murine Microenvironment Cell Population (mMCP) method for in silico immune deconvolution. mMCP analysis estimates the abundance of tissue infiltrating immune and stromal populations based on cell-specific gene expression signatures. RESULTS: There was a clear sex bias in survival. Female Fth1+/- mice had significantly poorer survival than control females (Fth1+/+). The Fth1 genetic status did not affect survival in males. The mMCP analysis revealed a significant reduction in T cells and CD8 + T cell infiltration in the tumors of females with Fth1+/- background as compared to the Fth1+/+. Mast and fibroblast cell infiltration was increased in females and males with Fth1+/- background, respectively, compared to Fth1+/+ mice. CONCLUSION: Genetic manipulation of Fth1 which leads to reduced systemic levels of FTH1 protein had a sexually dimorphic impact on survival. Fth1 heterozygosity significantly worsened survival in females but did not affect survival in male GBMs. Furthermore, the genetic manipulation of Fth1 significantly affected tumor infiltration of T-cells, CD8 + T cells, fibroblasts, and mast cells in a sexually dimorphic manner. These results demonstrate a role for FTH1 and presumably iron status in establishing the tumor cellular landscape that ultimately impacts survival and further reveals a sex bias that may inform the population studies showing a sex effect on the prevalence of brain tumors.

The Endolysosomal Transporter DMT1 is Required for Morphine Regulation of Neuronal Ferritin Heavy Chain.

NeuroHIV and other neurologic disorders present with altered iron metabolism in central nervous system neurons. Many people with HIV also use opioids, which can worsen neuroHIV symptoms by further dysregulating neuronal iron metabolism. Our previous work demonstrated that the µ-opioid agonist morphine causes neuronal endolysosomes to release their iron stores, and neurons respond by upregulating ferritin heavy chain (FHC), an iron storage protein associated with cognitive impairment in neuroHIV. Here, we investigated if this process required divalent metal transporter 1 (DMT1), a well-known iron transporter expressed on endolysosomes. We first optimized conditions to detect DMT1 isoforms (DMT1 1B ± iron responsive element) using fluorescently labeled rat DMT1 constructs expressed in HEK-293 cells. We also expressed these constructs in primary rat cortical neurons to compare their expression and subcellular distribution with endogenous DMT1 isoforms. We found endogenous DMT1 isoforms in the cytoplasm that colocalized with lysosomal-associated protein 1 (LAMP1), a marker of endolysosomes. Next, we blocked endogenous DMT1 isoforms using ebselen, a potent pharmacological inhibitor of DMT1 iron transport. Ebselen pre-treatment blocked morphine's ability to upregulate FHC protein, suggesting this pathway requires DMT1 iron transport from endolysosomes. This was further validated using viral-mediated genetic silencing of DMT1±IRE in cortical neurons, which also blocked FHC upregulation in the presence of morphine. Overall, our work demonstrates that the µ-opioid agonist morphine utilizes the endolysosomal iron transporter DMT1 to modulate neuronal cellular iron metabolism, upregulate FHC protein, and contribute to cognitive decline in neuroHIV. Morphine requires DMT1 to upregulate neuronal FHC. Cortical neurons treated with morphine release their endolysosomal iron stores to the cytoplasm and upregulate FHC, an iron storage protein associated with dendritic spine deficits and cognitive impairment in neuroHIV. This pathway requires the endolysosomal iron transporter DMT1, as pharmacological and genetic inhibitors of the transporter completely block morphine's ability to upregulate FHC. Created with BioRender.com .

Overexpression of ferritin light chain as a poor prognostic factor for breast cancer.

BACKGROUND: Ferritin light chain (FTL) is involved in tumor progression, but the specific molecular processes by which FTL affects the development of breast cancer (BRCA) have remained unknown. In this research, the clinicopathological significance of FTL overexpression in BRCA was investigated. METHODS: To investigate the role of FTL in BRCA, we utilized multiple online databases to analyse FTL expression levels in BRCA. Next, we reviewed the expression and localization of the FTL protein in BRCA by immunohistochemistry (IHC), Western blot (WB) and immunofluorescence (IF) staining. To assess the impact of FTL on patient prognosis, we conducted Kaplan‒Meier, univariate and multivariate survival analyses. The relationship between FTL and immune infiltration in BRCA was also analysed in the TISCH and SangerBox databases. MTT, malondialdehyde (MDA) and reactive oxygen species (ROS) assays were carried out to investigate the molecular mechanisms of FTL action in BRCA cells. RESULTS: FTL was significantly upregulated in BRCA compared to normal tissues. Its expression significantly linked to histological grade (P = 0.038), PR expression (P = 0.021), Her2 expression (P = 0.012) and Ki-67 expression (P = 0.040) in patients with BRCA. Furthermore, the expression of the FTL protein was higher in the BRCA cell lines than in the normal breast cells and mainly localized in the cytoplasm. Compared to patients with a low level of FTL expression, patients with a high level of FTL expression showed lower overall survival (OS). More convincingly, univariate and multivariate statistical analyses revealed that FTL expression (P = 0.000), ER expression (P = 0.036) and Her2 expression (P = 0.028) were meaningful independent prognostic factors in patients with BRCA. FTL was associated with immune infiltration in BRCA. Functional experiments further revealed that FTL knockdown inhibited the capacity of proliferation and increased the level of oxidative stress in BRCA cells. CONCLUSIONS: Overexpression of FTL was associated with the progression of BRCA. FTL overexpression may become a biomarker for the evaluation of poor prognosis in patients with BRCA.

Microglial CD68 and L-ferritin upregulation in response to phosphorylated-TDP-43 pathology in the amyotrophic lateral sclerosis brain.

Microglia, the innate immune cells of the brain, are activated by damage or disease. In mouse models of amyotrophic lateral sclerosis (ALS), microglia shift from neurotrophic to neurotoxic states with disease progression. It remains unclear how human microglia change relative to the TAR DNA-binding protein 43 (TDP-43) aggregation that occurs in 97% of ALS cases. Here we examine spatial relationships between microglial activation and TDP-43 pathology in brain tissue from people with ALS and from a TDP-43-driven ALS mouse model. Post-mortem human brain tissue from the Neurological Foundation Human Brain Bank was obtained from 10 control and 10 ALS cases in parallel with brain tissue from a bigenic NEFH-tTA/tetO-hTDP-43∆NLS (rNLS) mouse model of ALS at disease onset, early disease, and late disease stages. The spatiotemporal relationship between microglial activation and ALS pathology was determined by investigating microglial functional marker expression in brain regions with low and high TDP-43 burden at end-stage human disease: hippocampus and motor cortex, respectively. Sections were immunohistochemically labelled with a two-round multiplexed antibody panel against; microglial functional markers (L-ferritin, HLA-DR, CD74, CD68, and Iba1), a neuronal marker, an astrocyte marker, and pathological phosphorylated TDP-43 (pTDP-43). Single-cell levels of microglial functional markers were quantified using custom analysis pipelines and mapped to anatomical regions and ALS pathology. We identified a significant increase in microglial Iba1 and CD68 expression in the human ALS motor cortex, with microglial CD68 being significantly correlated with pTDP-43 pathology load. We also identified two subpopulations of microglia enriched in the ALS motor cortex that were defined by high L-ferritin expression. A similar pattern of microglial changes was observed in the rNLS mouse, with an increase first in CD68 and then in L-ferritin expression, with both occurring only after pTDP-43 inclusions were detectable. Our data strongly suggest that microglia are phagocytic at early-stage ALS but transition to a dysfunctional state at end-stage disease, and that these functional states are driven by pTDP-43 aggregation. Overall, these findings enhance our understanding of microglial phenotypes and function in ALS.

Ferritin heavy chain participated in ameliorating 3-nitropropionic acid-induced oxidative stress and apoptosis of goose follicular granulosa cells.

Oxidative stress is the major culprits responsible for ovarian dysfunction by damaging granulosa cells (GCs). Ferritin heavy chain (FHC) may participate in the regulation of ovarian function by mediating GCs apoptosis. However, the specific regulatory function of FHC in follicular GCs remains unclear. Here, 3-nitropropionic acid (3-NPA) was utilized to establish an oxidative stress model of follicular GCs of Sichuan white geese. To explore the regulatory effects of FHC on oxidative stress and apoptosis of primary GCs in geese by interfering or overexpressing FHC gene. After transfection of siRNA-FHC to GCs for 60 h, the expressions of FHC gene and protein decreased significantly (P < 0.05). After FHC overexpression for 72 h, the expressions of FHC mRNA and protein upregulated considerably (P < 0.05). The activity of GCs was impaired after interfering with FHC and 3-NPA coincubated (P < 0.05). When overexpression of FHC combined with 3-NPA treatment, the activity of GCs was remarkably enhanced (P < 0.05). After interference FHC and 3-NPA treatment, NF-κB and NRF2 gene expression decreased (P < 0.05), the intracellular reactive oxygen species (ROS) level increased greatly (P < 0.05), BCL-2 expression reduced, BAX/BCL-2 ratio intensified (P < 0.05), the mitochondrial membrane potential decreased notably (P < 0.05), and the apoptosis rate of GCs aggravated (P < 0.05). While overexpression of FHC combined with 3-NPA treatment could promote BCL-2 protein expression and reduce BAX/BCL-2 ratio, indicating that FHC regulated the mitochondrial membrane potential and apoptosis of GCs by mediating the expression of BCL-2. Taken together, our research manifested that FHC alleviated the inhibitory effect of 3-NPA on the activity of GCs. FHC knockdown could suppress the expression of NRF2 and NF-κB genes, reduce BCL-2 expression and augment BAX/BCL-2 ratio, contributing to the accumulation of ROS and jeopardizing mitochondrial membrane potential, as well as exacerbating GCs apoptosis.

WDR45 mutation dysregulates iron homeostasis by promoting the chaperone-mediated autophagic degradation of ferritin heavy chain in an ER stress/p38 dependent mechanism.

Ferritin is the main iron storage protein that plays a pivotal role in the regulation of iron homeostasis. Mutations in the autophagy protein WD repeat domain 45 (WDR45) that lead to iron overload is associated with the human ß-propeller protein-associated neurodegeneration (BPAN). Previous studies have demonstrated that ferritin was decreased in WDR45 deficient cells, but the mechanism remains unclear. In this study, we have demonstrated that the ferritin heavy chain (FTH) could be degraded via chaperone-mediated autophagy (CMA) in ER stress/p38-dependent pathway. In HeLa cells, inducing the ER stress activated CMA, therefore facilitated the degradation of FTH, and increased the content of Fe2+. However, the increased CMA activity and Fe2+ as well as the decreased FTH by ER stress inducer were restored by pre-treatment with p38 inhibitor. Overexpression of a mutant WDR45 activated CMA thus promoted the degradation of FTH. Furthermore, inhibition of ER stress/p38 pathway resulted in reduced activity of CMA, which consequently elevated the protein level of FTH but reduced the Fe2+ level. Our results revealed that WDR45 mutation dysregulates iron homeostasis by activating CMA, and promotes FTH degradation through ER stress/p38 signaling pathway.

Iron-siRNA Nanohybrids for Enhanced Chemodynamic Therapy via Ferritin Heavy Chain Downregulation.

Ferrous iron (Fe2+ ) has more potent hydroxyl radical (⋅OH)-generating ability than other Fenton-type metal ions, making Fe-based nanomaterials attractive for chemodynamic therapy (CDT). However, because Fe2+ can be converted by ferritin heavy chain (FHC) to nontoxic ferric form and then sequestered in ferritin, therapeutic outcomes of Fe-mediated CDT agents are still far from satisfactory. Here we report the synthesis of siRNA-embedded Fe0 nanoparticles (Fe0 -siRNA NPs) for self-reinforcing CDT via FHC downregulation. Upon internalization by cancer cells, pH-responsive Fe0 -siRNA NPs are degraded to release Fe2+ and FHC siRNA in acidic endo/lysosomes with the aid of oxygen (O2 ). The accompanied O2 depletion causes an intracellular pH decrease, which further promotes the degradation of Fe0 -siRNA NPs. In addition to initiating chemodynamic process, Fe2+ -catalyzed ⋅OH generation facilitates endo/lysosomal escape of siRNA by disrupting the membranes, enabling FHC downregulation-enhanced CDT.

Reactive oxygen species-mediated phosphorylation of JNK is involved in the regulation of BmFerHCH on Bombyx mori nucleopolyhedrovirus proliferation.

c-Jun N-terminal kinase (JNK) phosphorylation is widely observed during virus infection, modulating various aspects of the virus-host interaction. In our previous research, we have proved that B. mori ferritin heavy-chain homolog (BmFerHCH), an inhibitor of reactive oxygen species (ROS), facilitates B. mori nucleopolyhedrovirus (BmNPV) proliferation. However, one question remains: Which downstream signaling pathways does BmFerHCH regulate by inhibiting ROS? Here, we first determined that silencing BmFerHCH inhibits BmNPV proliferation, and this inhibition depends on ROS. Then, we substantiated that BmNPV infection activates the JNK signaling pathway. Interestingly, the JNK phosphorylation during BmNPV infection is activated by ROS. Further, we found that the enhanced nuclear translocation of phospho-JNK induced by BmNPV infection was dramatically reduced by pretreatment with the antioxidant N-acetylcysteine (NAC), whereas there was more detectable phospho-JNK in the cytoplasm. Next, we investigated how changes in BmFerHCH expression affect JNK phosphorylation. BmFerHCH overexpression suppressed the phosphorylation of JNK and nuclear translocation of phospho-JNK during BmNPV infection, whereas BmFerHCH knockdown facilitated phosphorylation of JNK and nuclear translocation of phospho-JNK. By measuring the viral load, we found the inhibitory effect of BmFerHCH knockdown on BmNPV infection depends on phosphorylated JNK. In addition, the JNK signaling pathway was involved in BmNPV-triggered apoptosis. Hence, we hypothesize that ROS-mediated JNK phosphorylation is involved in the regulation of BmFerHCH on BmNPV proliferation. These results elucidate the molecular mechanisms and signaling pathways of BmFerHCH-mediated response to BmNPV infection.

Cyclosporine A-loaded apoferritin alleviates myocardial ischemia-reperfusion injury by simultaneously blocking ferroptosis and apoptosis of cardiomyocytes.

Myocardial ischemia-reperfusion injury (MI/RI) seriously restricts the therapeutic effect of reperfusion. It is demonstrated that ferroptosis and apoptosis of cardiomyocytes are widely involved in MI/RI. Therefore, simultaneous inhibition of ferroptosis and apoptosis of cardiomyocytes can be a promising strategy to treat MI/RI. Besides, transferrin receptor 1 (TfR1) is highly expressed in ischemic myocardium, and apoferritin (ApoFn) is a ligand of the transferrin receptor. In this study, CsA@ApoFn was prepared by wrapping cyclosporin A (CsA) with ApoFn and actively accumulated in ischemic cardiomyocytes through TfR1 mediated endoctosis in MI/RI mice. After entering cardiomyocytes, ApoFn in CsA@ApoFn inhibited ferroptosis of ischemic cardiomyocytes by increasing the protein expression of GPX4 and reducing the content of labile iron pool and lipid peroxides. At the same time, CsA in CsA@ApoFn attenuated the apoptosis of ischemic cardiomyocytes through recovering mitochondrial membrane potential and reducing the level of reactive oxygen species, which played a synergistic role with ApoFn in the treatment of MI/RI. In conclusion, CsA@ApoFn restored cardiac function of MI/RI mice by simultaneously blocking ferroptosis and apoptosis of cardiomyocytes. ApoFn itself not only served as a safe carrier to specifically deliver CsA to ischemic cardiomyocytes but also played a therapeutic role on MI/RI. CsA@ApoFn is proved as an effective drug delivery platform for the treatment of MI/RI. STATEMENT OF SIGNIFICANCE: Recent studies have shown that ferroptosis is an important mechanism of myocardial ischemia-reperfusion injury (MI/RI). Therefore, simultaneous inhibition of ferroptosis and apoptosis of cardiomyocytes can be a promising strategy to treat MI/RI. Apoferritin, as a delivery carrier, can actively target to ischemic myocardium through binding with highly expressed transferrin receptor on ischemic cardiomyocytes. At the same time, apoferritin plays a protective role on ischemic cardiomyocytes by inhibiting ferroptosis. This strategy of killing two birds with one stone significantly improves the therapeutic effect on MI/RI while does not need more pharmaceutical excipients, which has the prospect of clinical transformation.