Membrane topography and the overestimation of protein clustering in single molecule localisation microscopy - identification and correction.

According to single-molecule localisation microscopy almost all plasma membrane proteins are clustered. We demonstrate that clusters can arise from variations in membrane topography where the local density of a randomly distributed membrane molecule to a degree matches the variations in the local amount of membrane. Further, we demonstrate that this false clustering can be differentiated from genuine clustering by using a membrane marker to report on local variations in the amount of membrane. In dual colour live cell single molecule localisation microscopy using the membrane probe DiI alongside either the transferrin receptor or the GPI-anchored protein CD59, we found that pair correlation analysis reported both proteins and DiI as being clustered, as did its derivative pair correlation-photoactivation localisation microscopy and nearest neighbour analyses. After converting the localisations into images and using the DiI image to factor out topography variations, no CD59 clusters were visible, suggesting that the clustering reported by the other methods is an artefact. However, the TfR clusters persisted after topography variations were factored out. We demonstrate that membrane topography variations can make membrane molecules appear clustered and present a straightforward remedy suitable as the first step in the cluster analysis pipeline.

Targeted delivery of CEBPA-saRNA for the treatment of pancreatic ductal adenocarcinoma by transferrin receptor aptamer decorated tetrahedral framework nucleic acid.

Pancreatic cancer, predominantly pancreatic ductal adenocarcinoma (PDAC), remains a highly lethal malignancy with limited therapeutic options and a dismal prognosis. By targeting the underlying molecular abnormalities responsible for PDAC development and progression, gene therapy offers a promising strategy to overcome the challenges posed by conventional radiotherapy and chemotherapy. This study sought to explore the therapeutic potential of small activating RNAs (saRNAs) specifically targeting the CCAAT/enhancer-binding protein alpha (CEBPA) gene in PDAC. To overcome the challenges associated with saRNA delivery, tetrahedral framework nucleic acids (tFNAs) were rationally engineered as nanocarriers. These tFNAs were further functionalized with a truncated transferrin receptor aptamer (tTR14) to enhance targeting specificity for PDAC cells. The constructed tFNA-based saRNA formulation demonstrated exceptional stability, efficient saRNA release ability, substantial cellular uptake, biocompatibility, and nontoxicity. In vitro experiments revealed successful intracellular delivery of CEBPA-saRNA utilizing tTR14-decorated tFNA nanocarriers, resulting in significant activation of tumor suppressor genes, namely, CEBPA and its downstream effector P21, leading to notable inhibition of PDAC cell proliferation. Moreover, in a mouse model of PDAC, the tTR14-decorated tFNA-mediated delivery of CEBPA-saRNA effectively upregulated the expression of the CEBPA and P21 genes, consequently suppressing tumor growth. These compelling findings highlight the potential utility of saRNA delivered via a designed tFNA nanocarrier to induce the activation of tumor suppressor genes as an innovative therapeutic approach for PDAC.

Indium-111 radiolabelling of a brain-penetrant Aß antibody for SPECT imaging.

Background: The development of bispecific antibodies that can traverse the blood-brain barrier has paved the way for brain-directed immunotherapy and when radiolabelled, immunoPET imaging. The objective of this study was to investigate how indium-111 (111In) radiolabelling with compatible chelators affects the brain delivery and peripheral biodistribution of the bispecific antibody RmAb158-scFv8D3, which binds to amyloid-beta (Aß) and the transferrin receptor (TfR), in Aß pathology-expressing tg-ArcSwe mice and aged-matched wild-type control mice. Methods: Bispecific RmAb158-scFv8D3 (biAb) was radiolabelled with 111In using CHX-A"-DTPA, DOTA, or DOTA-tetrazine (DOTA-Tz). Affinity toward TfR and Aß, as well as stability, was investigated in vitro. Mice were then intravenously administered with the three different radiolabelled biAb variants, and blood samples were collected for monitoring pharmacokinetics. Brain concentration was quantified after 2 and 72 h, and organ-specific retention was measured at 72 h by gamma counting. A subset of mice also underwent whole-body Single-photon emission computed tomography (SPECT) scanning at 72 h after injection. Following post-mortem isolation, the brains of tg-ArcSwe and WT mice were sectioned, and the spatial distribution of biAb was further investigated with autoradiography. Results: All three [111In]biAb variants displayed similar blood pharmacokinetics and brain uptake at 2 h after administration. Radiolabelling did not compromise affinity, and all variants showed good stability, especially the DOTA-Tz variant. Whole-body SPECT scanning indicated high liver, spleen, and bone accumulation of all [111In]biAb variants. Subsequent ex vivo measurement of organ retention confirmed SPECT data, with retention in the spleen, liver, and bone - with very high bone marrow retention. Ex vivo gamma measurement of brain tissue, isolated at 72 h post-injection, and ex vivo autoradiography showed that WT mice, despite the absence of Aß, exhibited comparable brain concentrations of [111In]biAb as those found in the tg-ArcSwe brain. Conclusions: The successful 111In-labelling of biAb with retained binding to TfR and Aß, and retained ability to enter the brain, demonstrated that 111In can be used to generate radioligands for brain imaging. A high degree of [111In]biAb in bone marrow and intracellular accumulation in brain tissue indicated some off-target interactions or potential interaction with intrabrain TfR resulting in a relatively high non-specific background signal.

Ferrostatin-1 inhibits fibroblast fibrosis in keloid by inhibiting ferroptosis.

Background: Keloid is a chronic proliferative fibrotic disease caused by abnormal fibroblasts proliferation and excessive extracellular matrix (ECM) production. Numerous fibrotic disorders are significantly influenced by ferroptosis, and targeting ferroptosis can effectively mitigate fibrosis development. This study aimed to investigate the role and mechanism of ferroptosis in keloid development. Methods: Keloid tissues from keloid patients and normal skin tissues from healthy controls were collected. Iron content, lipid peroxidation (LPO) level, and the mRNA and protein expression of ferroptosis-related genes including solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), transferrin receptor (TFRC), and nuclear factor erythroid 2-related factor 2 (Nrf2) were determined. Mitochondrial morphology was observed using transmission electron microscopy (TEM). Keloid fibroblasts (KFs) were isolated from keloid tissues, and treated with ferroptosis inhibitor ferrostatin-1 (fer-1) or ferroptosis activator erastin. Iron content, ferroptosis-related marker levels, LPO level, mitochondrial membrane potential, ATP content, and mitochondrial morphology in KFs were detected. Furthermore, the protein levels of α-smooth muscle actin (α-SMA), collagen I, and collagen III were measured to investigate whether ferroptosis affect fibrosis in KFs. Results: We found that iron content and LPO level were substantially elevated in keloid tissues and KFs. SLC7A11, GPX4, and Nrf2 were downregulated and TFRC was upregulated in keloid tissues and KFs. Mitochondria in keloid tissues and KFs exhibited ferroptosis-related pathology. Fer-1 treatment reduced iron content, restrained ferroptosis and mitochondrial dysfunction in KFs, Moreover, ferrostatin-1 restrained the protein expression of α-SMA, collagen I, and collagen III in KFs. Whereas erastin treatment showed the opposite results. Conclusion: Ferroptosis exists in keloid. Ferrostatin-1 restrained ECM deposition and fibrosis in keloid through inhibiting ferroptosis, and erastin induced ECM deposition and fibrosis through intensifying ferroptosis.

FTO deficiency in older livers exacerbates ferroptosis during ischaemia/reperfusion injury by upregulating ACSL4 and TFRC.

Older livers are more prone to hepatic ischaemia/reperfusion injury (HIRI), which severely limits their utilization in liver transplantation. The potential mechanism remains unclear. Here, we demonstrate older livers exhibit increased ferroptosis during HIRI. Inhibiting ferroptosis significantly attenuates older HIRI phenotypes. Mass spectrometry reveals that fat mass and obesity-associated gene (FTO) expression is downregulated in older livers, especially during HIRI. Overexpressing FTO improves older HIRI phenotypes by inhibiting ferroptosis. Mechanistically, acyl-CoA synthetase long chain family 4 (ACSL4) and transferrin receptor protein 1 (TFRC), two key positive contributors to ferroptosis, are FTO targets. For ameliorative effect, FTO requires the inhibition of Acsl4 and Tfrc mRNA stability in a m6A-dependent manner. Furthermore, we demonstrate nicotinamide mononucleotide can upregulate FTO demethylase activity, suppressing ferroptosis and decreasing older HIRI. Collectively, these findings reveal an FTO-ACSL4/TFRC regulatory pathway that contributes to the pathogenesis of older HIRI, providing insight into the clinical translation of strategies related to the demethylase activity of FTO to improve graft function after older donor liver transplantation.

Peripheral expression of brain-penetrant progranulin rescues pathologies in mouse models of frontotemporal lobar degeneration.

Progranulin (PGRN) haploinsufficiency is a major risk factor for frontotemporal lobar degeneration with TAR DNA-binding protein 43 (TDP-43) pathology (FTLD-GRN). Multiple therapeutic strategies are in clinical development to restore PGRN in the CNS, including gene therapy. However, a limitation of current gene therapy approaches aimed to alleviate FTLD-associated pathologies may be their inefficient brain exposure and biodistribution. We therefore developed an adeno-associated virus (AAV) targeting the liver (L) to achieve sustained peripheral expression of a transferrin receptor (TfR) binding, brain-penetrant (b) PGRN variant [AAV(L):bPGRN] in two mouse models of FTLD-GRN, namely, Grn knockout and GrnxTmem106b double knockout mice. This therapeutic strategy avoids potential safety and biodistribution issues of CNS-administered AAVs and maintains sustained concentrations of PGRN in the brain after a single dose. AAV(L):bPGRN treatment reduced several FTLD-GRN-associated pathologies including severe motor function deficits, aberrant TDP-43 phosphorylation, dysfunctional protein degradation, lipid metabolism, gliosis, and neurodegeneration in the brain. The potential translatability of our findings was tested in an in vitro model using cocultured human induced pluripotent stem cell (hiPSC)-derived microglia lacking PGRN and TMEM106B and wild-type hiPSC-derived neurons. As in mice, aberrant TDP-43, lysosomal dysfunction, and neuronal loss were ameliorated after treatment with exogenous TfR-binding protein transport vehicle fused to PGRN (PTV:PGRN). Together, our studies suggest that peripherally administered brain-penetrant PGRN replacement strategies ameliorate FTLD-GRN relevant phenotypes including TDP-43 pathology, neurodegeneration, and behavioral deficits. Our data provide preclinical proof of concept for the use of this AAV platform for treatment of FTLD-GRN and potentially other CNS disorders.

Generation of a bloodstream form Trypanosoma brucei double glycosyltransferase null mutant competent in receptor-mediated endocytosis of transferrin.

The bloodstream form of Trypanosoma brucei expresses large poly-N-acetyllactosamine (pNAL) chains on complex N-glycans of a subset of glycoproteins. It has been hypothesised that pNAL may be required for receptor-mediated endocytosis. African trypanosomes contain a unique family of glycosyltransferases, the GT67 family. Two of these, TbGT10 and TbGT8, have been shown to be involved in pNAL biosynthesis in bloodstream form Trypanosoma brucei, raising the possibility that deleting both enzymes simultaneously might abolish pNAL biosynthesis and provide clues to pNAL function and/or essentiality. In this paper, we describe the creation of a TbGT10 null mutant containing a single TbGT8 allele that can be excised upon the addition of rapamycin and, from that, a TbGT10 and TbGT8 double null mutant. These mutants were analysed by lectin blotting, glycopeptide methylation linkage analysis and flow cytometry. The data show that the mutants are defective, but not abrogated, in pNAL synthesis, suggesting that other GT67 family members can compensate to some degree for loss of TbGT10 and TbGT8. Despite there being residual pNAL synthesis in these mutants, certain glycoproteins appear to be particularly affected. These include the lysosomal CBP1B serine carboxypeptidase, cell surface ESAG2 and the ESAG6 subunit of the essential parasite transferrin receptor (TfR). The pNAL deficient TfR in the mutants continued to function normally with respect to protein stability, transferrin binding, receptor mediated endocytosis of transferrin and subcellular localisation. Further the pNAL deficient mutants were as viable as wild type parasites in vitro and in in vivo mouse infection experiments. Although we were able to reproduce the inhibition of transferrin uptake with high concentrations of pNAL structural analogues (N-acetylchito-oligosaccharides), this effect disappeared at lower concentrations that still inhibited tomato lectin uptake, i.e., at concentrations able to outcompete lectin-pNAL binding. Based on these findings, we recommend revision of the pNAL-dependent receptor mediated endocytosis hypothesis.

Confinement energy landscape classification reveals membrane receptor nano-organization mechanisms.

The cell membrane organization has an essential functional role through the control of membrane receptor confinement in micro- or nanodomains. Several mechanisms have been proposed to account for these properties, although some features have remained controversial, notably the nature, size, and stability of cholesterol- and sphingolipid-rich domains or lipid rafts. Here, we probed the effective energy landscape acting on single-nanoparticle-labeled membrane receptors confined in raft nanodomains- epidermal growth factor receptor (EGFR), Clostridium perfringens ε-toxin receptor (CPεTR), and Clostridium septicum α-toxin receptor (CSαTR)-and compared it with hop-diffusing transferrin receptors. By establishing a new analysis pipeline combining Bayesian inference, decision trees, and clustering approaches, we systematically classified single-protein trajectories according to the type of effective confining energy landscape. This revealed the existence of only two distinct organization modalities: confinement in a quadratic energy landscape for EGFR, CPεTR, and CSαTR (A), and free diffusion in confinement domains resulting from the steric hindrance due to F-actin barriers for transferrin receptor (B). The further characterization of effective confinement energy landscapes by Bayesian inference revealed the role of interactions with the domain environment in cholesterol- and sphingolipid-rich domains with (EGFR) or without (CPεTR and CSαTR) interactions with F-actin to regulate the confinement energy depth. These two distinct mechanisms result in the same organization type (A). We revealed that the apparent domain sizes for these receptor trajectories resulted from Brownian exploration of the energy landscape in a steady-state-like regime at a common effective temperature, independently of the underlying molecular mechanisms. These results highlight that confinement domains may be adequately described as interaction hotspots rather than rafts with abrupt domain boundaries. Altogether, these results support a new model for functional receptor confinement in membrane nanodomains and pave the way to the constitution of an atlas of membrane protein organization.

Longitudinal changes in iron homeostasis in human experimental and clinical malaria.

BACKGROUND: The interaction between iron status and malaria is incompletely understood. We evaluated longitudinal changes in iron homeostasis in volunteers enrolled in malaria volunteer infection studies (VIS) and in Malaysian patients with falciparum and vivax malaria. METHODS: We retrieved data and samples from 55 participants (19 female) enrolled in malaria VIS, and 171 patients (45 female) with malaria and 30 healthy controls (13 female) enrolled in clinical studies in Malaysia. Ferritin, hepcidin, erythropoietin, and soluble transferrin receptor (sTfR) were measured by ELISA. FINDINGS: In the VIS, participants' parasitaemia was correlated with baseline mean corpuscular volume (MCV), but not iron status (ferritin, hepcidin or sTfR). Ferritin, hepcidin and sTfR all increased during the VIS. Ferritin and hepcidin normalised by day 28, while sTfR remained elevated. In VIS participants, baseline ferritin was associated with post-treatment increases in liver transaminase levels. In Malaysian patients with malaria, hepcidin and ferritin were elevated on admission compared to healthy controls, while sTfR increased following admission. By day 28, hepcidin had normalised; however, ferritin and sTfR both remained elevated. INTERPRETATION: Our findings demonstrate that parasitaemia is associated with an individual's MCV rather than iron status. The persistent elevation in sTfR 4 weeks post-infection in both malaria VIS and clinical malaria may reflect a causal link between malaria and iron deficiency. FUNDING: National Health and Medical Research Council (Program Grant 1037304, Project Grants 1045156 and 1156809; Investigator Grants 2016792 to BEB, 2016396 to JCM, 2017436 to MJG); US National Institute of Health (R01-AI116472-03); Malaysian Ministry of Health (BP00500420).

Fucoxanthin Induces Ferroptosis in Cancer Cells via Downregulation of the Nrf2/HO-1/GPX4 Pathway.

This study investigated the mechanism by which fucoxanthin acts as a novel ferroptosis inducer to inhibit tongue cancer. The MTT assay was used to detect the inhibitory effects of fucoxanthin on SCC-25 human tongue squamous carcinoma cells. The levels of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), and total iron were measured. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting were used to assess glutathione peroxidase 4 (GPX4), nuclear factor erythroid 2-related factor 2 (Nrf2), Keap1, solute carrier family 7 member 11 (SLC7A11), transferrin receptor protein 1 (TFR1), p53, and heme oxygenase 1 (HO-1) expression. Molecular docking was performed to validate interactions. Compared with the control group, the activity of fucoxanthin-treated SCC-25 cells significantly decreased in a dose- and time-dependent manner. The levels of MMP, GSH, and SOD significantly decreased in fucoxanthin-treated SCC-25 cells; the levels of ROS, MDA, and total iron significantly increased. mRNA and protein expression levels of Keap1, GPX4, Nrf2, and HO-1 in fucoxanthin-treated cells were significantly decreased, whereas levels of TFR1 and p53 were significantly increased, in a concentration-dependent manner. Molecular docking analysis revealed that binding free energies of fucoxanthin with p53, SLC7A11, GPX4, Nrf2, Keap1, HO-1, and TFR1 were below -5 kcal/mol, primarily based on active site hydrogen bonding. Our findings suggest that fucoxanthin can induce ferroptosis in SCC-25 cells, highlighting its potential as a treatment for tongue cancer.

Gene expression of iron transporters, markers of vascularization and structural integrity in placenta of mothers with and without anemia.

OBJECTIVE: To compare the mRNA expression of placental iron transporters (TfR-1 and FPN), markers of placental vascularization (VEGF and sFLT1) and marker of structural integrity (LMN-A) in term women with and without iron deficiency anemia. MATERIALS AND METHODS: A total of 30 pregnant women were enrolled; 15 cases of iron deficiency anemia (Hb 7-10.9 gm/dL) and 15 gestational age matched healthy controls (Hb ≥ 11 gm/dL). Peripheral venous blood was collected for assessment of hemoglobin levels and serum iron profile. Placental tissue was used for assessing the mRNA expression of TfR-1, FPN, VEGF, sFLT-1 and LMN-A via real time PCR. RESULTS: Placental expression of TfR-1, VEGF and LMN-A was increased in pregnant women with anemia compared to healthy pregnant controls. Placental expression of sFLT-1 was decreased in pregnant women with anemia compared to healthy pregnant controls. There was no change in the placental expression of FPN. CONCLUSION: The increased expression of TfR-1, VEGF and LMN-A in cases of iron deficiency anemia are most likely to be compensatory in nature to help maintain adequate fetal iron delivery. WHAT DOES THIS STUDY ADDS TO THE CLINICAL WORK: Compensatory changes in the placenta aimed at buffering transport of iron to the fetus are seen in pregnant women with anemia compared to healthy pregnant controls.

MARK2 phosphorylates KIF13A at a 14-3-3 binding site to polarize vesicular transport of transferrin receptor within dendrites.

Neurons regulate the microtubule-based transport of certain vesicles selectively into axons or dendrites to ensure proper polarization of function. The mechanism of this polarized vesicle transport is still not fully elucidated, though it is known to involve kinesins, which drive anterograde transport on microtubules. Here, we explore how the kinesin-3 family member KIF13A is regulated such that vesicles containing transferrin receptor (TfR) travel only to dendrites. In experiments involving live-cell imaging, knockout of KIF13A, BioID assay, we found that the kinase MARK2 phosphorylates KIF13A at a 14-3-3 binding motif, strengthening interaction of KIF13A with 14-3-3 such that it dissociates from TfR-containing vesicles, which therefore cannot enter axons. Overexpression of KIF13A or knockout of MARK2 leads to axonal transport of TfR-containing vesicles. These results suggest a unique kinesin-based mechanism for polarized transport of vesicles to dendrites.

Iron controls the development of airway hyperreactivity by regulating ILC2 metabolism and effector function.

Group 2 innate lymphoid cells (ILC2s) rapidly induce a type 2 inflammation in the lungs in response to allergens. Here, we focused on the role of iron, a critical nutritional trace element, on ILC2 function and asthma pathogenesis. We found that transferrin receptor 1 (TfR1) is rapidly up-regulated and functional during ILC2 activation in the lungs, and blocking transferrin uptake reduces ILC2 expansion and activation. Iron deprivation reprogrammed ILC2 metabolism, inducing a HIF-1α-driven up-regulation of glycolysis and inhibition of oxidative mitochondrial activity. Consequently, we observed that in vivo iron chelation or induction of hypoferremia reduced the development of airway hyperreactivity in experimental models of ILC2-driven allergic asthma. Human circulating ILC2s rapidly induced TfR1 during activation, whereas inhibition of iron uptake or iron deprivation reduced effector functions. Last, we found a negative relationship between circulating ILC2 TfR1 expression and airway function in cohorts of patients with asthma. Collectively, our studies define cellular iron as a critical regulator of ILC2 function.

Internalization of transferrin-tagged Myxococcus xanthus encapsulins into mesenchymal stem cells.

Currently, various functionalized nanocarrier systems are extensively studied for targeted delivery of drugs, peptides, and nucleic acids. Joining the approaches of genetic and chemical engineering may produce novel carriers for precise targeting different cellular proteins, which is important for both therapy and diagnosis of various pathologies. Here we present the novel nanocontainers based on vectorized genetically encoded Myxococcus xanthus (Mx) encapsulin, confining a fluorescent photoactivatable mCherry (PAmCherry) protein. The shells of such encapsulins were modified using chemical conjugation of human transferrin (Tf) prelabeled with a fluorescein-6 (FAM) maleimide acting as a vector. We demonstrate that the vectorized encapsulin specifically binds to transferrin receptors (TfRs) on the membranes of mesenchymal stromal/stem cells (MSCs) followed by internalization into cells. Two spectrally separated fluorescent signals from Tf-FAM and PAmCherry are clearly distinguishable and co-localized. It is shown that Tf-tagged Mx encapsulins are internalized by MSCs much more efficiently than by fibroblasts. It has been also found that unlabeled Tf effectively competes with the conjugated Mx-Tf-FAM formulations. That indicates the conjugate internalization into cells by Tf-TfR endocytosis pathway. The developed nanoplatform can be used as an alternative to conventional nanocarriers for targeted delivery of, e.g., genetic material to MSCs.

Combined Ferritin Nanocarriers with ICG for Effective Phototherapy Against Breast Cancer.

Introduction: Photodynamic Therapy (PDT) is a promising, minimally invasive treatment for cancer with high immunostimulatory potential, no reported drug resistance, and reduced side effects. Indocyanine Green (ICG) has been used as a photosensitizer (PS) for PDT, although its poor stability and low tumor-target specificity strongly limit its efficacy. To overcome these limitations, ICG can be formulated as a tumor-targeting nanoparticle (NP). Methods: We nanoformulated ICG into recombinant heavy-ferritin nanocages (HFn-ICG). HFn has a specific interaction with transferrin receptor 1 (TfR1), which is overexpressed in most tumors, thus increasing HFn tumor tropism. First, we tested the properties of HFn-ICG as a PS upon irradiation with a continuous-wave diode laser. Then, we evaluated PDT efficacy in two breast cancer (BC) cell lines with different TfR1 expression levels. Finally, we measured the levels of intracellular endogenous heavy ferritin (H-Fn) after PDT treatment. In fact, it is known that cells undergoing ROS-induced autophagy, as in PDT, tend to increase their ferritin levels as a defence mechanism. By measuring intracellular H-Fn, we verified whether this interplay between internalized HFn and endogenous H-Fn could be used to maximize HFn uptake and PDT efficacy. Results: We previously demonstrated that HFn-ICG stabilized ICG molecules and increased their delivery to the target site in vitro and in vivo for fluorescence guided surgery. Here, with the aim of using HFn-ICG for PDT, we showed that HFn-ICG improved treatment efficacy in BC cells, depending on their TfR1 expression. Our data revealed that endogenous H-Fn levels were increased after PDT treatment, suggesting that this defence reaction against oxidative stress could be used to enhance HFn-ICG uptake in cells, increasing treatment efficacy. Conclusion: The strong PDT efficacy and peculiar Trojan horse-like mechanism, that we revealed for the first time in literature, confirmed the promising application of HFn-ICG in PDT.

An AAV capsid reprogrammed to bind human transferrin receptor mediates brain-wide gene delivery.

Developing vehicles that efficiently deliver genes throughout the human central nervous system (CNS) will broaden the range of treatable genetic diseases. We engineered an adeno-associated virus (AAV) capsid, BI-hTFR1, that binds human transferrin receptor (TfR1), a protein expressed on the blood-brain barrier. BI-hTFR1 was actively transported across human brain endothelial cells and, relative to AAV9, provided 40 to 50 times greater reporter expression in the CNS of human TFRC knockin mice. The enhanced tropism was CNS-specific and absent in wild-type mice. When used to deliver GBA1, mutations of which cause Gaucher disease and are linked to Parkinson's disease, BI-hTFR1 substantially increased brain and cerebrospinal fluid glucocerebrosidase activity compared with AAV9. These findings establish BI-hTFR1 as a potential vector for human CNS gene therapy.

Glycosylation and Characterization of Human Transferrin in an End-Stage Kidney Disease.

Chronic kidney disease (CKD) is a global health concern affecting approximately one billion individuals worldwide. End-stage kidney disease (ESKD), the most severe form of CKD, is often accompanied by anemia. Peritoneal dialysis (PD), a common treatment for ESKD, utilizes the peritoneum for solute transfer but is associated with complications including protein loss, including transferrin (Tf) a key protein involved in iron transport. This study investigated Tf characteristics in ESKD patients compared to healthy individuals using lectin microarray, spectroscopic techniques and immunocytochemical analysis to assess Tf interaction with transferrin receptors (TfRs). ESKD patients exhibited altered Tf glycosylation patterns, evidenced by significant changes in lectin reactivity compared to healthy controls. However, structural analyses revealed no significant differences in the Tf secondary or tertiary structures between the two groups. A functional analysis demonstrated comparable Tf-TfR interaction in both PD and healthy samples. Despite significant alterations in Tf glycosylation, structural integrity and Tf-TfR interaction remained preserved in PD patients. These findings suggest that while glycosylation changes may influence iron metabolism, they do not impair Tf function. The study highlights the importance of a glucose-free dialysis solutions in managing anemia exacerbation in PD patients with poorly controlled anemia, potentially offering a targeted therapeutic approach to improve patient outcomes.

O-GlcNAcylation regulates the stability of transferrin receptor (TFRC) to control the ferroptosis in hepatocellular carcinoma cells.

Ferroptosis is an iron-dependent programmed cell death (PCD) enforced by lipid peroxidation accumulation. Transferrin receptor (TFRC), one of the signature proteins of ferroptosis, is abundantly expressed in hepatocellular carcinoma (HCC). However, post-translational modification (PTM) of TFRC and the underlying mechanisms for ferroptosis regulation remain less understood. In this study, we found that TFRC undergoes O-GlcNAcylation, influencing Erastin-induced ferroptosis sensitivity in hepatocytes. Further mechanistic studies found that Erastin can trigger de-O-GlcNAcylation of TFRC at serine 687 (Ser687), which diminishes the binding of ubiquitin E3 ligase membrane-associated RING-CH8 (MARCH8) and decreases polyubiquitination on lysine 665 (Lys665), thereby enhancing TFRC stability that favors labile iron accumulation. Therefore, our findings report O-GlcNAcylation on an important regulatory protein of ferroptosis and reveal an intriguing mechanism by which HCC ferroptosis is controlled by an iron metabolism pathway.

Inflammation Triggers Chondrocyte Ferroptosis in TMJOA via HIF-1α/TFRC.

Inflammation and loss of articular cartilage are considered the major cause of temporomandibular joint osteoarthritis (TMJOA), a painful condition of the temporomandibular joint (TMJ). To determine the cause of TMJ osteoarthritis in these patients, synovial fluid of TMJOA patients was compared prior to and after hyaluronic lavage, revealing substantially elevated levels of interleukin (IL) 1ß, reactive oxidative stress (ROS), and an overload of Fe3+ and Fe2+ prior to lavage, indicative of ferroptosis as a mode of chondrocyte cell death. To ask whether prolonged inflammatory conditions resulted in ferroptosis-like transformation in vitro, we subjected TMJ chondrocytes to IL-1ß treatment, resulting in a shift in messenger RNA sequencing gene ontologies related to iron homeostasis and oxidative stress-related cell death. Exposure to rat unilateral anterior crossbite conditions resulted in reduced COL2A1 expression, fewer chondrocytes, glutathione peroxidase 4 (GPX4) downregulation, and 4-hydroxynonenal (4-HNE) upregulation, an effect that was reversed after intra-articular injections of the ferroptosis inhibitor ferrostatin 1 (Fer-1). Our study demonstrated that ferroptosis conditions affected mitochondrial structure and function, while the inhibitor Fer-1 restored mitochondrial structure and the inhibition of hypoxia-inducible factor 1α (HIF-1α) or the transferrin receptor 1 (TFRC) rescued IL-1ß-induced loss of mitochondrial membrane potential. Inhibition of HIF-1α downregulated IL-1ß-induced TFRC expression, while inhibition of TFRC did not downregulate IL-1ß-induced HIF-1α expression in chondrocytes. Moreover, inhibition of HIF-1α or TFRC downregulated the IL-1ß-induced MMP13 expression in chondrocytes, while inhibition of HIF-1α or TFRC rescued IL-1ß-inhibited COL2A1 expression in chondrocytes. Furthermore, upregulation of TFRC promoted Fe2+ entry into chondrocytes, inducing the Fenton reaction and lipid peroxidation, which in turn caused ferroptosis, a disruption in chondrocyte functions, and an exacerbation of condylar cartilage degeneration. Together, these findings illustrate the far-reaching effects of chondrocyte ferroptosis in TMJOA as a mechanism causing chondrocyte death through iron overload, oxidative stress, and articular cartilage degeneration and a potential major cause of TMJOA.