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.

Effective delivery of anti-PD-L1 siRNA with human heavy chain ferritin (HFn) in acute myeloid leukemia cell lines.

Because of the high biocompatibility, self-assembly capability, and CD71-mediated endocytosis, using human heavy chain ferritin (HFn) as a nanocarrier would greatly increase therapeutic effectiveness and reduce possible adverse events. Anti-PD-L1 siRNA can downregulate the level of PD-L1 on tumor cells, resulting in the activation of effector T cells against leukemia. Therefore, this study aimed to produce the tumor-targeting siPD-L1/HFn nanocarrier. Briefly, the HFn coding sequence was cloned into a pET-28a, and the constructed expression plasmid was subsequently transformed into E. coli BL21. After induction of Isopropyl ß-D-1-thiogalactopyranoside (IPTG), HFn was purified with Ni-affinity chromatography and dialyzed against PBS. The protein characteristics were analyzed using SDS-PAGE, Western Blot, and Dynamic light scattering (DLS). The final concentration was assessed using the Bicinchoninic acid (BCA) assay. The encapsulation was performed using the standard pH system. The treatment effects of siPD-L1/HFn were carried out on HL-60 and K-562 cancer cell lines. The RT-PCR was used to determine the mRNA expression of PD-L1. The biocompatibility and excretion of siPD-L1/HFn have also been evaluated. The expression and purity of HFn were well verified through SDS-PAGE, WB, and DLS. RT-PCR analyses also showed significant siRNA-mediated PD-L1 silencing in both HL-60 and K-562 cells. Our study suggested a promising approach for siRNA delivery. This efficient delivery system can pave the way for the co-delivery of siRNAs and multiple chemotherapies to address the emerging needs of cancer combination therapy.

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.

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.

HMOX1 regulates ferroptosis via mic14 and its impact on chemotherapy resistance in small-cell lung cancer.

This study aimed to investigate the role and molecular mechanism of heme oxygenase-1 (HMOX1) in chemotherapy resistance in small-cell lung cancer (SCLC). Employed bioinformatics, qPCR, and Western Blot to assess HMOX1 levels in SCLC versus normal tissues and its prognostic relevance. CCK-8, flow cytometry, and thiobarbituric acid assays determined HMOX1's impact on SCLC chemosensitivity, ferroptosis markers, lipid peroxidation, and mic14's role in chemoresistance. In the GSE40275 and GSE60052 cohorts, HMOX1 expression was downregulated in SCLC tissues compared to normal tissues. Higher HMOX1 expression was associated with improved prognosis in the Sun Yat-sen University Cancer Hospital cohort and GSE60052 cohort. The RNA and protein levels of HMOX1 were reduced in drug-resistant SCLC cell lines compared to chemosensitive cell lines. Upregulation of HMOX1 increased chemosensitivity and reduced drug resistance in SCLC, while downregulation of HMOX1 decreased chemosensitivity and increased drug resistance. Upregulation of HMOX1 elevated the expression of ferroptosis-related proteins ACSL4, CD71, Transferrin, Ferritin Heavy Chain, and Ferritin Light Chain, while decreasing the expression of GPX4 and xCT. Conversely, downregulation of HMOX1 decreased the expression of ACSL4, CD71, Transferrin, Ferritin Heavy Chain, and Ferritin Light Chain, while increasing the expression of GPX4 and xCT. Upregulation of HMOX1 promoted cellular lipid peroxidation, whereas downregulation of HMOX1 inhibited cellular lipid peroxidation. Upregulation of HMOX1 reduced the RNA level of mic14, while downregulation of HMOX1 increased the RNA level of mic14. mic14 exhibited inhibitory effects on cellular lipid peroxidation in SCLC cells and contributed to reduced chemosensitivity and increased drug resistance in chemoresistant SCLC cell lines. HMOX1 plays a role in ferroptosis by regulating mic14 expression, thereby reversing chemoresistance in SCLC.

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.

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.

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.

Engineered human H-chain ferritin with reversed charge of the internal cavity exhibits RNA-mediated spongelike effect for loading RNA/DNA-binding molecules.

Ferritins are globular proteins with an internal cavity that enables the encapsulation of a plethora of low-mass compounds. Unfortunately, the overall negative surface charge of ferritin's internal cavity hampers efficient loading of negatively charged molecules. Therefore, we produced a genetically engineered human H-chain ferritin containing a cationic RKRK domain, reversing the natural net charge of the cavity to positive, thus allowing for efficient encapsulation of negatively charged siRNA. Due to the reversed, positive charge mediated by RKRK domains, the recombinant ferritin produced in E. coli inherently carries a load of bacterial RNA inside its cavity, turning the protein into an effective sponge possessing high affinity for DNA/RNA-binding substances that can be loaded with markedly higher efficiency compared to the wildtype protein. Using doxorubicin as payload, we show that due to its loading through the RNA sponge, doxorubicin is released in a sustained manner, with a cytotoxicity profile similar to the free drug. In summary, this is the first report demonstrating a ferritin/nucleic acid hybrid delivery vehicle with a broad spectrum of properties exploitable in various fields of biomedical applications.

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.

Evaluation of the expression of genes associated with iron metabolism in peripheral blood mononuclear cells from Type 2 diabetes mellitus patients.

AIMS: Type 2 Diabetes (T2DM) has been linked to ferroptosis. This study aimed to assess expression levels of genes linked with iron metabolism in peripheral blood mononuclear cells (PBMCs) from T2DM patients and to investigate the association of these expression levels with anthropometric and clinical parameters. METHODS: Gene expression of iron metabolism genes (Ferritin Light Chain, FTL; Ferritin Heavy Chain, FTH1; Transferrin Receptor, TFRC; Divalent Metal Transporter 1, SLC11A2; Ferroportin, SLC40A1) in archival PBMCs was assessed using quantitative real-time PCR assays. Correlations of gene expression with anthropometric/biochemical patient data were evaluated. RESULTS: The study included 36 (18 male/18 female) T2DM patients and 45 (28 male/17 female) normoglycemic (NGT) subjects with a mean age of 38.1 ± 6.8 years and 47.6 ± 8.6 years respectively. Relative expression of FTL was significantly lower in T2DM females compared to that in NGT females (P = 0.027). Relative expression of SLC40A1 was significantly lower in the T2DM group (P = 0.043) and in the T2DM females (P = 0.021). Relative expression of SLC11A2 was negatively correlated with systolic blood pressure in T2DM male patients. Relative expression of SLC40A1 was negatively associated with serum phosphorous and positively associated with serum thyroid stimulating hormone in male T2DM patients. CONCLUSIONS: Our findings indicate a reduction in the expression of FTL in perimenopausal T2DM females. Also, in male T2DM patients and NGT subjects, biochemical markers are significantly correlated with the expression of FTL, FTH1, SLC11A2, and SLC40A1 in PBMCs.

Genetic insights into associations of multisite chronic pain with common diseases and biomarkers using data from the UK Biobank.

BACKGROUND: Chronic pain is a common, complex, and challenging condition, for which specialised healthcare is required. We investigated the relationship between multisite chronic pain (MCP) and different disease traits identify safe biomarker interventions that can prevent MCP. METHODS: Univariable and multivariable Mendelian randomisation (MR) analysis were conducted to investigate associations between MCP and 36 common diseases in the UK Biobank. Subsequently, we estimated the potential effect of expression of 4774 proteins on MCP utilising existing plasma protein quantitative trait locus data. For the significant biomarkers, we performed phenome-wide MR (Phe-MR) with 1658 outcomes to predict potential safety profiles linked to biomarker intervention. RESULTS: Multisite chronic pain had a substantial impact on psychiatric and neurodevelopmental traits (major depression and attention deficit hyperactivity disorder), cardiovascular diseases (myocardial infarction, coronary artery disease, and heart failure), respiratory outcomes (asthma, chronic obstructive pulmonary disease, and sleep apnoea), arthropathies, type 2 diabetes mellitus, and cholelithiasis. Higher genetically predicted levels of S100A6, DOCK9, ferritin, and ferritin light chain were associated with a risk of MCP, whereas PTN9 and NEUG were linked to decreased MCP risk. Phe-MR results suggested that genetic inhibition of DOCK9 increased the risk of 21 types of disease, whereas the other biomarker interventions were relatively safe. CONCLUSIONS: We established that MCP has an effect on health conditions covering various physiological systems and identified six novel biomarkers for intervention. In particular, S100A6, PTN9, NEUG, and ferritin light chain represent promising targets for MCP prevention, as no significant side-effects were predicted in our study.

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.

Hydrophobicity-enhanced ferritin nanoparticles for efficient encapsulation and targeted delivery of hydrophobic drugs to tumor cells.

Ferritin, a naturally occurring iron storage protein, has gained significant attention as a drug delivery platform due to its inherent biocompatibility and capacity to encapsulate therapeutic agents. In this study, we successfully genetically engineered human H ferritin by incorporating 4 or 6 tryptophan residues per subunit, strategically oriented towards the inner cavity of the nanoparticle. This modification aimed to enhance the encapsulation of hydrophobic drugs into the ferritin cage. Comprehensive characterization of the mutants revealed that only the variant carrying four tryptophan substitutions per subunit retained the ability to disassemble and reassemble properly. As a proof of concept, we evaluated the loading capacity of this mutant with ellipticine, a natural hydrophobic indole alkaloid with multimodal anticancer activity. Our data demonstrated that this specific mutant exhibited significantly higher efficiency in loading ellipticine compared to human H ferritin. Furthermore, to evaluate the versatility of this hydrophobicity-enhanced ferritin nanoparticle as a drug carrier, we conducted a comparative study by also encapsulating doxorubicin, a commonly used anticancer drug. Subsequently, we tested both ellipticine and doxorubicin-loaded nanoparticles on a promyelocytic leukemia cell line, demonstrating efficient uptake by these cells and resulting in the expected cytotoxic effect.

Heterozygous nonsense variants in the ferritin heavy-chain gene FTH1 cause a neuroferritinopathy.

Ferritin, the iron-storage protein, is composed of light- and heavy-chain subunits, encoded by FTL and FTH1, respectively. Heterozygous variants in FTL cause hereditary neuroferritinopathy, a type of neurodegeneration with brain iron accumulation (NBIA). Variants in FTH1 have not been previously associated with neurologic disease. We describe the clinical, neuroimaging, and neuropathology findings of five unrelated pediatric patients with de novo heterozygous FTH1 variants. Children presented with developmental delay, epilepsy, and progressive neurologic decline. Nonsense FTH1 variants were identified using whole-exome sequencing, with a recurrent variant (p.Phe171∗) identified in four unrelated individuals. Neuroimaging revealed diffuse volume loss, features of pontocerebellar hypoplasia, and iron accumulation in the basal ganglia. Neuropathology demonstrated widespread ferritin inclusions in the brain. Patient-derived fibroblasts were assayed for ferritin expression, susceptibility to iron accumulation, and oxidative stress. Variant FTH1 mRNA transcripts escape nonsense-mediated decay (NMD), and fibroblasts show elevated ferritin protein levels, markers of oxidative stress, and increased susceptibility to iron accumulation. C-terminal variants in FTH1 truncate ferritin's E helix, altering the 4-fold symmetric pores of the heteropolymer, and likely diminish iron-storage capacity. FTH1 pathogenic variants appear to act by a dominant, toxic gain-of-function mechanism. The data support the conclusion that truncating variants in the last exon of FTH1 cause a disorder in the spectrum of NBIA. Targeted knockdown of mutant FTH1 transcript with antisense oligonucleotides rescues cellular phenotypes and suggests a potential therapeutic strategy for this pediatric neurodegenerative disorder.

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.