Biology subjects

Connor, J. R.

Publications and source records attributed to Connor, J. R..

7 recordsLinked to original sources

bioRxiv2025

Tumor-associated macrophages protect glioblastoma cells from ferroptosis by inducing the release of ferritin-bound iron via exosomes.

Tumor-associated macrophages (TAMs) are the most abundant non-tumor cell type in glioblastoma (GBM) and act as the pivotal cell type in regulating iron metabolism in the GBM tumor microenvironment. High TAM infiltration into the TME is also associated with increased resistance to ferroptosis, an iron-dependent cell death. However, the exact mechanism by which TAMs make the cancer cells resistant to ferroptosis remains relatively unexplored. Here, we have investigated how TAMs modify iron metabolism in GBM cells to make them more resistant to ferroptotic stress. We utilized GL261 cells, a GBM cell line derived from C57BL6 mice, and syngeneic primary murine bone marrow-derived macrophages (BMDM) to study GBM-TAM interactions in vitro. We found that male macrophages exhibited higher iron uptake, greater iron storage, and a larger labile iron pool compared to female macrophages, indicating intrinsic sex biases in macrophage iron metabolism. Subsequently, we used co-culture experiments to study how macrophages regulate the iron and ferroptotic status of GL261 cells. We discovered that GL261 cells cocultured with BMDMs showed higher resistance to RSL3-induced ferroptotic stress. Mechanistically, BMDMs caused a decrease in total cellular iron in GL261 cells by inducing increased H-ferritin-bound iron release via CD63-positive exosomes; thus, limiting the amount of iron that is available for lipid peroxidation during ferroptosis. This process was moderately sex biased in favor of male macrophages. Finally, we show that this mechanism of BMDM-induced resistance to ferroptosis is independent of Hepcidin regulation and can act as a possible pathway by which GBM cells escape ferroptotic stress during proinflammatory conditions.

cancer biology↗

bioRxiv2023

Amyloid-β exposed astrocytes induce iron transport from endothelial cells at the blood-brain barrier by altering the ratio of apo- and holo-transferrin

Excessive brain iron accumulation is observed in early in the onset of Alzheimers disease, notably prior to widespread proteinopathy. These findings suggest that increases in brain iron levels are due to a dysregulation of the iron transport mechanism at the blood-brain barrier. Astrocytes release signals (apo- and holo-transferrin) that communicate brain iron needs to endothelial cells in order to modulate iron transport. Here we use iPSC-derived astrocytes and endothelial cells to investigate how early-disease levels of amyloid-{beta} disrupt iron transport signals secreted by astrocytes to stimulate iron transport from endothelial cells. We demonstrate that conditioned media from astrocytes treated with amyloid-{beta} stimulates iron transport from endothelial cells and induces changes in iron transport pathway protein levels. The mechanism underlying this response begins with increased iron uptake and mitochondrial activity by the astrocytes which in turn increases levels of apo-transferrin in the amyloid-{beta} conditioned astrocyte media leading to increased iron transport from endothelial cells. These novel findings offer a potential explanation for the initiation of excessive iron accumulation in early stages of Alzheimers disease. Whats more, these data provide the first example of how the mechanism of iron transport regulation by apo- and holo-transferrin becomes misappropriated in disease to detrimental ends. The clinical benefit from understanding early dysregulation in brain iron transport in AD cannot be understated. If therapeutics can target this early process, they could possibly prevent the detrimental cascade that occurs with excessive iron accumulation. Significance StatementExcessive brain iron accumulation is hallmark pathology of Alzheimers disease that occurs early in the disease staging and before widespread proteinopathy deposition. This overabundance of brain iron has been implicated to contribute to disease progression, thus understandingthe mechanism of early iron accumulation has significant therapeutic potential to slow to halt disease progression. Here, we show that in response to low levels of amyloid-{beta} exposure, astrocytes increase their mitochondrial activity and iron uptake, resulting in iron deficient conditions. Elevated levels of apo (iron free)-transferrin stimulate iron release from endothelial cells. These data are the first to propose a mechanism for the initiation of iron accumulation and the misappropriation of iron transport signaling leading to dysfunctional brain iron homeostasis and resultant disease pathology.

neuroscience↗

bioRxiv2023

Apo- and holo- transferrin differentially interact with ferroportin and hephaestin to regulate iron release at the blood-brain barrier

AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSBackgroundC_ST_ABSApo- (iron free) and holo- (iron bound) transferrin (Tf) participate in precise regulation of brain iron uptake at endothelial cells of the blood-brain barrier. Apo-Tf indicates an iron deficient environment and stimulates iron release, while holo-Tf indicates an iron sufficient environment and suppresses additional iron release. Free iron is exported through ferroportin, with hephaestin as an aid to the process. Until now, the molecular mechanism of apo- and holo-Tfs influence on iron release was largely unknown. MethodsHere we use a variety of cell culture techniques, including co-immunoprecipitation and proximity ligation assay, in iPSC-derived endothelial cells and HEK 293 cells to investigate the mechanism of apo- and holo-Tfs influence over iron release. We placed our findings in physiological context by further deciphering how hepcidin played a role in this mechanism as well. ResultsWe demonstrate that holo-Tf induces the internalization of ferroportin through the established ferroportin degradation pathway. Furthermore, holo-Tf directly binds to ferroportin, whereas apo-Tf directly binds to hephaestin. Only pathological levels of hepcidin disrupt the interaction between holo-Tf and ferroportin, and no amount of hepcidin disrupts the interaction between apo-Tf and hephaestin. The disruption of the holo-Tf and ferroportin interaction by hepcidin is due to hepcidins ability to rapidly internalize ferroportin compared to holo-Tf. ConclusionsThese novel findings provide a molecular mechanism for apo- and holo-Tf regulation of iron release from endothelial cells. They further demonstrate how hepcidin impacts these protein-protein interactions, and offer a model for how holo-Tf and hepcidin corporate to suppress iron release. We have established a more thorough understanding of the mechanisms behind iron release regulation with great clinical impact for a variety of neurological conditions in which iron release is dysregulated.

cell biology↗

bioRxiv2022

Iron Inhibits Glioblastoma Cell Migration and Polarization

Glioblastoma is one of the deadliest malignancies facing modern oncology today. The ability of glioblastoma cells to diffusely spread into neighboring healthy brain makes complete surgical resection nearly impossible and contributes to the recurrent disease faced by most patients. Although research into the impact of iron on glioblastoma has addressed proliferation, there has been little investigation into how cellular iron impacts the ability of glioblastoma cells to migrate - a key question especially in the context of the diffuse spread observed in these tumors. Herein, we show that increasing cellular iron content results in decreased migratory capacity of human glioblastoma cells. The decrease in migratory capacity was accompanied by a decrease in cellular polarization in the direction of movement. Expression of CDC42, a Rho GTPase that is essential for both cellular migration and establishment of polarity in the direction of cell movement, was reduced upon iron treatment. Bioinformatic analysis of CDC42 mRNA revealed a potential iron-responsive element that may contribute to the regulation of CDC42 by iron. We then analyzed a single-cell RNA-seq dataset of human glioblastoma samples and found that cells at the tumor periphery had a gene signature that is consistent with having lower levels of cellular iron. Altogether, our results suggest that cellular iron content is impacting glioblastoma cell migratory capacity and that cells with higher iron levels exhibit reduced motility.

cancer biology↗

bioRxiv2022

Tumor cell-intrinsic HFE drives glioblastoma growth

BackgroundGlioblastoma (GBM) tumor cells modulate expression of iron-associated genes to enhance iron uptake from the surrounding microenvironment, driving proliferation and tumor growth. The homeostatic iron regulator (HFE) gene, encoding the iron sensing HFE protein, is upregulated in GBM and correlates with poor survival outcomes. However, the molecular mechanisms underlying these observations remain unclear. Identification of pathways for targeting iron dependence in GBM tumors is therefore a critical area of investigation. MethodsWe interrogated the impact of cell-intrinsic Hfe expression on proliferation and tumor growth through genetic loss and gain of function approaches in syngeneic mouse glioma models. We determined the expression of iron-associated genes and their relationship with survival in GBM using public datasets and identified differentially expressed pathways in Hfe knockdown cells through Nanostring transcriptional profiling. ResultsLoss of Hfe induced apoptotic cell death in vitro and inhibited tumor growth in vivo while overexpression of Hfe accelerated both proliferation and tumor growth. Analysis of iron gene signatures in Hfe knockdown cells revealed alterations in the expression of several iron-associated genes, suggesting global disruption of intracellular iron homeostasis. Analyzing differentially expressed pathways further identified oxidative stress as the top pathway upregulated with Hfe loss. Enhanced 55Fe uptake and generation of reactive oxygen species (ROS) were found with Hfe knockdown, implicating toxic iron overload resulting in apoptotic cell death. ConclusionsCollectively, these findings identify a novel role for HFE in regulating iron homeostasis in GBM tumors and provide a potential avenue for future therapeutic development. Key PointsO_LIHFE is an iron sensor that is upregulated in GBM and negatively impacts survival. C_LIO_LIHFE overexpression drives proliferation and tumor growth in vivo. C_LIO_LILoss of HFE increases production of reactive oxygen species and induces apoptosis, extending survival in vivo. C_LI Importance of StudyDysregulation of iron metabolism is an important feature of GBM contributing to tumor growth and negatively impacting survival. The identification of key iron regulators controlling this process is therefore important for therapeutic targeting. We identify HFE as an important regulator of iron homeostasis in GBM and suggest a role for sexual dimorphism in HFE-mediated tumor iron regulation that ultimately results in differential survival outcomes. Our findings demonstrate that HFE drives tumor cell proliferation and survival in GBM and may be a viable target for modulating tumor iron flux and inducing apoptosis in tumor cells.

cancer biology↗

bioRxiv2019

JAM-A functions as a female microglial tumor suppressor in glioblastoma

Glioblastoma (GBM) remains refractory to treatment. In addition to its cellular and molecular heterogeneity, epidemiological studies indicate the presence of additional complexity associated with biological sex. GBM is more prevalent and aggressive in male compared to female patients, suggesting the existence of sex-specific growth, invasion, and therapeutic resistance mechanisms. While sex-specific molecular mechanisms have been reported at a tumor cell-intrinsic level, sex-specific differences in the tumor microenvironment have not been investigated. Using transgenic mouse models, we demonstrate that deficiency of junctional adhesion molecule-A (JAM-A) in female mice enhances microglia activation, GBM cell proliferation, and tumor growth. Mechanistically, JAM-A suppresses anti-inflammatory/pro-tumorigenic gene activation via interferon-activated gene 202b (Ifi202b) and found in inflammatory zone (Fizz1) in female microglia. Our findings suggest that cell adhesion mechanisms function to suppress pathogenic microglial activation in the female tumor microenvironment, which highlights an emerging role for sex differences in the GBM microenvironment and suggests that sex differences extend beyond previously reported tumor cell intrinsic differences.\n\nSummaryTuraga et al. demonstrate that female microglia drive a more aggressive glioblastoma phenotype in the context of JAM-A deficiency. These findings highlight a sex-specific role for JAM-A and represent the first evidence of sexual dimorphism in the glioblastoma microenvironment.

cancer biology↗

bioRxiv2017

Inhibition of the Platelet-activating factor receptor for the treatment of Amyotrophic Lateral Sclerosis?

Cerebrospinal Fluids (CSF) of Amyotrophic Lateral Sclerosis (ALS) patients have increased levels of the inflammatory cytokine IL-18. Because IL-18 is produced by dendritic cells stimulated by the Platelet-activating factor (PAF), a major neuroinflammatory mediator, it is expected that PAF is involved in ALS. Pilot experimental data on amplification of PAF receptor (PAFR) mRNA by RT-PCR show that PAFR is overexpressed, as compared to age matched controls, in the spinal cords of transgenic ALS mouse model SOD1-G93A, suggesting PAF mediation. Although anti-inflammatory drugs have been tested for ALS before, no clinical trial has been conducted using PAFR specific inhibitors. Therefore, we hypothesize that administration of PAFR inhibitors, such as Ginkgolide B, PCA 4248 and WEB 2086, have potential to function as a novel therapy for ALS, particularly in SOD1 familial ALS forms. Because currently there are only two approved drugs with modest effectiveness for ALS therapy, a search for novel drugs and targets is essential.

neuroscience↗