Calcium and IL-6 regulate the anterograde trafficking and plasma membrane residence of the iron exporter ferroportin to modulate iron efflux.

Iron is an essential element for proper cell functioning, but unbalanced levels can cause cell death. Iron metabolism is controlled at the blood-tissue barriers provided by microvascular endothelial cells. Dysregulated iron metabolism at these barriers is a factor in both neurodegenerative and cardiovascular diseases. Mammalian iron efflux is mediated by the iron efflux transporter ferroportin (Fpn). Inflammation is a factor in many diseases and correlates with increased tissue iron accumulation. Evidence suggests treatment with interleukin 6 (IL-6) increases intracellular calcium levels and calcium is known to play an important role in protein trafficking. We have shown that calcium increases plasma membrane localization of the iron uptake proteins ZIP8 and ZIP14, but if and how calcium modulates Fpn trafficking is unknown. In this article, we examined the effects of IL-6 and calcium on Fpn localization to the plasma membrane. In HEK cells expressing a doxycycline-inducible GFP-tagged Fpn, calcium increased Fpn-GFP membrane presence by 2 h, while IL-6 increased membrane-localized Fpn-GFP by 3 h. Calcium pretreatment increased Fpn-GFP mediated 55Fe efflux from cells. Endoplasmic reticulum calcium stores were shown to be important for Fpn-GFP localization and iron efflux. Use of calmodulin pathway inhibitors showed that calcium signaling is important for IL-6-induced Fpn relocalization. Studies in brain microvascular endothelial cells in transwell culture demonstrated an initial increase in 55Fe flux with IL-6 that is reduced by 6 h coinciding with upregulation of hepcidin. Overall, this research details one pathway by which inflammatory signaling mediated by calcium can regulate iron metabolism, likely contributing to inflammatory disease mechanisms.

Calcium and the Ca-ATPase SPCA1 modulate plasma membrane abundance of ZIP8 and ZIP14 to regulate Mn(II) uptake in brain microvascular endothelial cells.

Manganese (II) accumulation in human brain microvascular endothelial cells is mediated by the metal-ion transporters ZRT IRT-like protein 8 (ZIP8) and ZRT IRT-like protein 14 (ZIP14). The plasma membrane occupancy of ZIP14, in particular, is increased in cells treated with Mn2+, lipopolysaccharide, or IL-6, but the mechanism of this regulation has not been elucidated. The calcium-transporting type 2C member 1 ATPase, SPCA1, is a Golgi-localized Ca2+-uptake transporter thought to support Golgi uptake of Mn2+ also. Here, we show using surface protein biotinylation, indirect immunofluorescence, and GFP-tagged proteins that cytoplasmic Ca2+ regulates ZIP8- and ZIP14-mediated manganese accumulation in human brain microvascular endothelial cells by increasing the plasma membrane localization of these transporters. We demonstrate that RNAi knockdown of SPCA1 expression results in an increase in cytoplasmic Ca2+ levels. In turn, we found increased cytoplasmic Ca2+ enhances membrane-localized ZIP8 and ZIP14 and a subsequent increase in 54Mn2+ uptake. Furthermore, overexpression of WT SPCA1 or a gain-of-function mutant resulted in a decrease in cytoplasmic Ca2+ and 54Mn2+ accumulation. While addition of Ca2+ positively regulated ZIP-mediated 54Mn2+ uptake, we show chelation of Ca2+ diminished manganese transport. In conclusion, the modulation of ZIP8 and ZIP14 membrane cycling by cytoplasmic calcium is a novel finding and provides new insight into the regulation of the uptake of Mn2+ and other divalent metal ions-mediated ZIP metal transporters.

Tuning the Type 1 Reduction Potential of Multicopper Oxidases: Uncoupling the Effects of Electrostatics and H-Bonding to Histidine Ligands.

In multicopper oxidases (MCOs), the type 1 (T1) Cu accepts electrons from the substrate and transfers these to the trinuclear Cu cluster (TNC) where O2 is reduced to H2O. The T1 potential in MCOs varies from 340 to 780 mV, a range not explained by the existing literature. This study focused on the ∼350 mV difference in potential of the T1 center in Fet3p and Trametes versicolor laccase (TvL) that have the same 2His1Cys ligand set. A range of spectroscopies performed on the oxidized and reduced T1 sites in these MCOs shows that they have equivalent geometric and electronic structures. However, the two His ligands of the T1 Cu in Fet3p are H-bonded to carboxylate residues, while in TvL they are H-bonded to noncharged groups. Electron spin echo envelope modulation spectroscopy shows that there are significant differences in the second-sphere H-bonding interactions in the two T1 centers. Redox titrations on type 2-depleted derivatives of Fet3p and its D409A and E185A variants reveal that the two carboxylates (D409 and E185) lower the T1 potential by 110 and 255-285 mV, respectively. Density functional theory calculations uncouple the effects of the charge of the carboxylates and their difference in H-bonding interactions with the His ligands on the T1 potential, indicating 90-150 mV for anionic charge and ∼100 mV for a strong H-bond. Finally, this study provides an explanation for the generally low potentials of metallooxidases relative to the wide range of potentials of the organic oxidases in terms of different oxidized states of their TNCs involved in catalytic turnover.

Energy metabolism, oxygen flux, and iron in bacteria: The Mössbauer report.

Iron is the most common transition metal cofactor across biological systems. As the earth transitioned from an anaerobic to aerobic environment, cellular mechanisms evolved to protect against iron-mediated oxidative damage, but the molecular details of these protective strategies remain unclear. In this report, the Lindahl group has combined spectroscopic, biochemical, and genetic approaches to inventory iron in Escherichia coli as a function of bacterial oxygen metabolism. Their results suggest that ferrous iron functions as an oxygen sink that is modulated by a "respiratory shield" of electron flux in the bacterial plasma membrane.

The solute carriers ZIP8 and ZIP14 regulate manganese accumulation in brain microvascular endothelial cells and control brain manganese levels.

Manganese supports numerous neuronal functions but in excess is neurotoxic. Consequently, regulation of manganese flux at the blood-brain barrier (BBB) is critical to brain homeostasis. However, the molecular pathways supporting the transcellular trafficking of divalent manganese ions within the microvascular capillary endothelial cells (BMVECs) that constitute the BBB have not been examined. In this study, we have determined that ZIP8 and ZIP14 (Zrt- and Irt-like proteins 8 and 14) support Mn2+ uptake by BMVECs and that neither DMT1 nor an endocytosis-dependent pathway play any significant role in Mn2+ uptake. Specifically, siRNA-mediated knockdown of ZIP8 and ZIP14 coincided with a decrease in manganese uptake, and kinetic analyses revealed that manganese uptake depends on pH and bicarbonate and is up-regulated by lipopolysaccharide, all biochemical markers of ZIP8 or ZIP14 activity. Mn2+ uptake also was associated with cell-surface membrane presentation of ZIP8 and ZIP14, as indicated by membrane protein biotinylation. Importantly, surface ZIP8 and ZIP14 biotinylation and Mn2+-uptake experiments together revealed that these transporters support manganese uptake at both the apical, blood and basal, brain sides of BMVECs. This indicated that in the BMVECs of the BBB, these two transporters support a bidirectional Mn2+ flux. We conclude that BMVECs play a critical role in controlling manganese homeostasis in the brain.

Fluorescence resonance energy transfer links membrane ferroportin, hephaestin but not ferroportin, amyloid precursor protein complex with iron efflux.

Iron efflux from mammalian cells is supported by the synergistic actions of the ferrous iron efflux transporter, ferroportin (Fpn) and a multicopper ferroxidase, that is, hephaestin (Heph), ceruloplasmin (Cp) or both. The two proteins stabilize Fpn in the plasma membrane and catalyze extracellular Fe3+ release. The membrane stabilization of Fpn is also stimulated by its interaction with a 22-amino acid synthetic peptide based on a short sequence in the extracellular E2 domain of the amyloid precursor protein (APP). However, whether APP family members interact with Fpn in vivo is unclear. Here, using cyan fluorescent protein (CFP)-tagged Fpn in conjunction with yellow fluorescent protein (YFP) fusions of Heph and APP family members APP, APLP1, and APLP2 in HEK293T cells we used fluorescence and surface biotinylation to quantify Fpn membrane occupancy and also measured 59Fe efflux. We demonstrate that Fpn and Heph co-localize, and FRET analysis indicated that the two proteins form an iron-efflux complex. In contrast, none of the full-length, cellular APP proteins exhibited Fpn co-localization or FRET. Moreover, iron supplementation increased surface expression of the iron-efflux complex, and copper depletion knocked down Heph activity and decreased Fpn membrane localization. Whereas cellular APP species had no effects on Fpn and Heph localization, addition of soluble E2 elements derived from APP and APLP2, but not APLP1, increased Fpn membrane occupancy. We conclude that a ferroportin-targeting sequence, (K/R)EWEE, present in APP and APLP2, but not APLP1, helps modulate Fpn-dependent iron efflux in the presence of an active multicopper ferroxidase.

Rapid Decay of the Native Intermediate in the Metallooxidase Fet3p Enables Controlled FeII Oxidation for Efficient Metabolism.

The multicopper oxidases (MCOs) couple four 1e- oxidations of substrate to the 4e- reduction of O2 to H2O. These divide into two groups: those that oxidize organic substrates with high turnover frequencies (TOFs) up to 560 s-1 and those that oxidize metal ions with low TOFs, ∼1 s-1 or less. The catalytic mechanism of the organic oxidases has been elucidated, and the high TOF is achieved through rapid intramolecular electron transfer (IET) to the native intermediate (NI), which only slowly decays to the resting form. Here, we uncover the factors that govern the low TOF in Fet3p, a prototypical metallooxidase, in the context of the MCO mechanism. We determine that the NI decays rapidly under optimal turnover conditions, and the mechanism thereby becomes rate-limited by slow IET to the resting enzyme. Development of a catalytic model leads to the important conclusions that proton delivery to the NI controls the mechanism and enables the slow turnover in Fet3p that is functionally significant in Fe metabolism enabling efficient ferroxidase activity while avoiding ROS generation.

For Cryptococcus neoformans, responding to the copper status in a colonization niche is not just about copper.

Most fungi express two transcription factors that regulate the expression of genes associated with copper uptake for nutritional needs, and with copper resistance when copper approaches a cytotoxic level. These factors are characterized by cysteine-rich motifs which are associated with copper-sensing, DNA-binding and release, and/or cytoplasmic retention. Cryptococcus neoformans differs from most in that it expresses a single such copper-sensing trans-factor, Cuf1, a protein that up-regulates copper uptake when copper is scarce, and up-regulates copper sequestration when cells become super-replete. For C. neoformans this is an essential task in as much as copper is relatively bioavailable in lung airways while the brain interstitium can be copper-limiting for growth. While fungal dependence on and sensitivity to copper have long been considered targets for anti-fungal chemistry, fungi have proven adept at finding 'work arounds' by using a chelated form of copper as nutrient or adapting to a copper-surfaced hospital bed by increased resistance. However, the cohort of Cuf1 targets identified in this report represent far more than just the uptake and sequestration machinery, but include additional loci that, perhaps, are less easily 'defended' by the fungus. Garcia-Santamarina et al. provide that list and thus lay the ground-work for developing novel anti-fungal reagents.

Is brain iron trafficking part of the physiology of the amyloid precursor protein?

The amyloid precursor protein is so named, because a proteolytic fragment of it was found associated with a neuropathic disorder now known as Alzheimer's disease. This fragment, Aß, along with tau makes up the plaques and tangles that are the hallmark of AD. Iron (and other first-row transition metals) is found associated with these proteinaceous deposits. Much research has focused on the relationship of the plaques and iron to the etiology of the disease. This commentary asks another question, one only more recently addressed namely, what is the physiologic function of the amyloid precursor protein (APP) and of its secretase-generated soluble species? Overall, the data make clear that APP and its products have neurotrophic functions and some data indicate one of these may be to modulate the trafficking of iron in the brain.

The Ferroxidase Hephaestin But Not Amyloid Precursor Protein is Required for Ferroportin-Supported Iron Efflux in Primary Hippocampal Neurons.

Iron efflux in mammalian cells is mediated by the ferrous iron exporter ferroportin (Fpn); Fpn plasma membrane localization and function are supported by a multicopper ferroxidase and/or the soluble amyloid precursor protein (sAPP). Fpn and APP are ubiquitously expressed in all cell types in the central nervous system including neurons. In contrast, neuronal ferroxidase(s) expression has not been well characterized. Using primary cultures of hippocampal neurons, we examined the molecular mechanism of neuronal Fe efflux in detail. Developmental increases of Fpn, APP, and the ferroxidase hephaestin (Hp) were observed in hippocampal neurons. Iron efflux in these neurons depended on the level of Fpn localized at the cell surface; as noted, Fpn stability is supported by ferroxidase activity, an enzymatic activity that is required for Fe efflux. Iron accumulation increases and iron efflux decreases in Hp knockout neurons. In contrast, suppression of endogenous APP by RNAi knockdown does not affect surface Fpn stability or Fe efflux. These data support the model that the neuronal ferroxidase Hp plays a unique role in support of Fpn-mediated Fe efflux in primary hippocampal neurons. Our data also demonstrate that Hp ferroxidase activity relies on copper bioavailability, which suggests neuronal iron homeostasis will be modulated by cellular copper status.

Potassium and the K+/H+ Exchanger Kha1p Promote Binding of Copper to ApoFet3p Multi-copper Ferroxidase.

Acquisition and distribution of metal ions support a number of biological processes. Here we show that respiratory growth of and iron acquisition by the yeast Saccharomyces cerevisiae relies on potassium (K(+)) compartmentalization to the trans-Golgi network via Kha1p, a K(+)/H(+) exchanger. K(+) in the trans-Golgi network facilitates binding of copper to the Fet3p multi-copper ferroxidase. The effect of K(+) is not dependent on stable binding with Fet3p or alteration of the characteristics of the secretory pathway. The data suggest that K(+) acts as a chemical factor in Fet3p maturation, a role similar to that of cations in folding of nucleic acids. Up-regulation of KHA1 gene in response to iron limitation via iron-specific transcription factors indicates that K(+) compartmentalization is linked to cellular iron homeostasis. Our study reveals a novel functional role of K(+) in the binding of copper to apoFet3p and identifies a K(+)/H(+) exchanger at the secretory pathway as a new molecular factor associated with iron uptake in yeast.

sAPP modulates iron efflux from brain microvascular endothelial cells by stabilizing the ferrous iron exporter ferroportin.

A sequence within the E2 domain of soluble amyloid precursor protein (sAPP) stimulates iron efflux. This activity has been attributed to a ferroxidase activity suggested for this motif. We demonstrate that the stimulation of efflux supported by this peptide and by sAPPα is due to their stabilization of the ferrous iron exporter, ferroportin (Fpn), in the plasma membrane of human brain microvascular endothelial cells (hBMVEC). The peptide does not bind ferric iron explaining why it does not and thermodynamically cannot promote ferrous iron autoxidation. This peptide specifically pulls Fpn down from the plasma membrane of hBMVEC; based on these results, FTP, for ferroportin-targeting peptide, correctly identifies the function of this peptide. The data suggest that in stabilizing Fpn via the targeting due to the FTP sequence, sAPP will increase the flux of iron into the cerebral interstitium. This inference correlates with the observation of significant iron deposition in the amyloid plaques characteristic of Alzheimer's disease.

Iron transport across the blood-brain barrier: development, neurovascular regulation and cerebral amyloid angiopathy.

There are two barriers for iron entry into the brain: (1) the brain-cerebrospinal fluid (CSF) barrier and (2) the blood-brain barrier (BBB). Here, we review the literature on developmental iron accumulation by the brain, focusing on the transport of iron through the brain microvascular endothelial cells (BMVEC) of the BBB. We review the iron trafficking proteins which may be involved in the iron flux across BMVEC and discuss the plausible mechanisms of BMVEC iron uptake and efflux. We suggest a model for how BMVEC iron uptake and efflux are regulated and a mechanism by which the majority of iron is trafficked across the developing BBB under the direct guidance of neighboring astrocytes. Thus, we place brain iron uptake in the context of the neurovascular unit of the adult brain. Last, we propose that BMVEC iron is involved in the aggregation of amyloid-ß peptides leading to the progression of cerebral amyloid angiopathy which often occurs prior to dementia and the onset of Alzheimer's disease.

Molecular mechanisms of non-transferrin-bound and transferring-bound iron uptake in primary hippocampal neurons.

The molecular mechanisms of iron trafficking in neurons have not been elucidated. In this study, we characterized the expression and localization of ferrous iron transporters Zip8, Zip14 and divalent metal transporter 1 (DMT1), and ferrireductases Steap2 and stromal cell-derived receptor 2 in primary rat hippocampal neurons. Steap2 and Zip8 partially co-localize, indicating these two proteins may function in Fe(3+) reduction prior to Fe(2+) permeation. Zip8, DMT1, and Steap2 co-localize with the transferrin receptor/transferrin complex, suggesting they may be involved in transferrin receptor/transferrin-mediated iron assimilation. In brain interstitial fluid, transferring-bound iron (TBI) and non-transferrin-bound iron (NTBI) exist as potential iron sources. Primary hippocampal neurons exhibit significant iron uptake from TBI (Transferrin-(59) Fe(3+)) and NTBI, whether presented as (59) Fe(2+) -citrate or (59) Fe(3+) -citrate; reductase-independent (59) Fe(2+) uptake was the most efficient uptake pathway of the three. Kinetic analysis of Zn(2+) inhibition of Fe(2+) uptake indicated that DMT1 plays only a minor role in the uptake of NTBI. In contrast, localization and knockdown data indicate that Zip8 makes a major contribution. Data suggest also that cell accumulation of (59) Fe from TBI relies at least in part on an endocytosis-independent pathway. These data suggest that Zip8 and Steap2 play a major role in iron accumulation from NTBI and TBI by hippocampal neurons. Analysis of the expression and localization of known iron uptake transporters demonstrated that Zip8 makes a major contribution to iron accumulation in primary cultures of rat embryonic hippocampal neurons. These cells exhibit uptake pathways for ferrous and ferric iron (non-transferrin-bound iron, NTBI in figure) and for transferrin-bound iron; the ferrireductases Steap2 and SDR2 support the uptake of ferric iron substrates. Zip8 and Steap2 are strongly expressed in the plasma membrane of both soma and processes, implying a crucial role in iron accumulation from NTBI and transferrin-bound iron (TBI) by hippocampal neurons.

Ferroportin and exocytoplasmic ferroxidase activity are required for brain microvascular endothelial cell iron efflux.

The mechanism(s) of iron flux across the brain microvasculature endothelial cells (BMVEC) of the blood-brain barrier remains unknown. Although both hephaestin (Hp) and the ferrous iron permease ferroportin (Fpn) have been identified in BMVEC, their roles in iron efflux have not been examined. Using a human BMVEC line (hBMVEC), we have demonstrated that these proteins are required for iron efflux from these cells. Expression of both Hp and Fpn protein was confirmed in hBMVEC by immunoblot and indirect immunofluorescence; we show that hBMVEC express soluble ceruloplasmin (Cp) transcript as well. Depletion of endogenous Hp and Cp via copper chelation leads to the reduction of hBMVEC Fpn protein levels as well as a complete inhibition of (59)Fe efflux. Both hBMVEC Fpn protein and (59)Fe efflux activity are restored upon incubation with 6.6 nm soluble plasma Cp. These results are independent of the source of cell iron, whether delivered as transferrin- or non-transferrin-bound (59)Fe. Our results demonstrate that iron efflux from hBMVEC Fpn requires the action of an exocytoplasmic ferroxidase, which can be either endogenous Hp or extracellular Cp.

Activation of C6 glioblastoma cell ceruloplasmin expression by neighboring human brain endothelia-derived interleukins in an in vitro blood-brain barrier model system.

BACKGROUND: Iron transport across the blood-brain barrier (BBB) involves the cooperation of brain microvascular endothelial cells (BMVEC) and their neighboring astrocytes. Astrocytes secrete a soluble form of ceruloplasmin (sCp) which, in turn, acts to export iron from ferroportin (Fpn) on the basolateral surface of BMVEC. Although regulation of astrocyte sCp gene expression has been demonstrated to be influenced by interleukin-1 beta (IL-1ß) and interleukin-6 (IL-6), the role of neighboring BMVEC in this regulation has yet to be determined and is the basis for this work. RESULTS: We provide evidence that human BMVEC (hBMVEC) IL-1ß and IL-6 positively influence the expression of sCp transcript by neighboring C6 glioma cells (astrocytes). The effect of hBMVEC on C6 glioma sCp expression at the level of transcript and protein was repressed via the addition of IL-1ß and IL-6 pathway inhibitors (IL-1 receptor antagonist protein and SC144, respectively). Stimulation of hBMVEC interleukin gene expression by apical exposure to bacterial endotoxin lipopolysaccharide significantly enhanced hBMVEC-mediated C6 glioma sCp gene expression. CONCLUSION: hBMVEC influence the gene expression of neighboring C6 glioma sCp. This change in gene expression is mediated by the secretion of IL-1ß and IL-6 from hBMVEC. Furthermore, the hBMVEC-induced increase in neighboring C6 glioma sCp gene expression leads to an increased rate of hBMVEC iron efflux. Taken together, our results indicate that hBMVEC-secreted cytokine activity increases the gene expression of neighboring C6 glioma sCp, which reciprocally acts on basolateral hBMVEC Fpn to enhance brain iron import.

Modified reactivity toward O2 in first shell variants of Fet3p: geometric and electronic structure requirements for a functioning trinuclear copper cluster.

Multicopper oxidases (MCOs) carry out the most energy efficient reduction of O2 to H2O known, i.e., with the lowest overpotential. This four-electron process requires an electron mediating type 1 (T1) Cu site and an oxygen reducing trinuclear Cu cluster (TNC), consisting of a binuclear type 3 (T3)- and a mononuclear type 2 (T2) Cu center. The rate-determining step in O2 reduction is the first two-electron transfer from one of the T3 Cu's (T3ß) and the T2 Cu, forming a bridged peroxide intermediate (PI). This reaction has been investigated in T3ß Cu variants of the Fet3p, where a first shell His ligand is mutated to Glu or Gln. This converts the fast two-electron reaction of the wild-type (WT) enzyme to a slow one-electron oxidation of the TNC. Both variants initially react to form a common T3ß Cu(II) intermediate that converts to the Glu or Gln bound resting state. From spectroscopic evaluation, the nonmutated His ligands coordinate linearly to the T3ß Cu in the reduced TNCs in the two variants, in contrast to the trigonal arrangement observed in the WT enzyme. This structural perturbation is found to significantly alter the electronic structure of the reduced TNC, which is no longer capable of rapidly transferring two electrons to the two perpendicular half occupied π*-orbitals of O2, in contrast to the WT enzyme. This study provides new insight into the geometric and electronic structure requirements of a fully functional TNC for the rate determining two-electron reduction of O2 in the MCOs.

Iron metabolism in aerobes: managing ferric iron hydrolysis and ferrous iron autoxidation.

Aerobes and anaerobes alike express a plethora of essential iron enzymes; in the resting state, the iron atom(s) in these proteins are in the ferrous state. For aerobes, ferric iron is the predominant environmental valence form which, given ferric iron's aqueous chemistry, occurs as 'rust', insoluble, bio-inert polymeric ferric oxide that results from the hydrolysis of [Fe(H(2)O)(6)](3+). Mobilizing this iron requires bio-ferrireduction which in turn requires managing the rapid autoxidation of the resulting Fe(II) which occurs at pH > 6. This review examines the aqueous redox chemistry of iron and the mechanisms evolved in aerobes to suppress the 'rusting out' of Fe(III) and the ROS-generating autoxidation of Fe(II) so as to make this metal ion available as the most ubiquitous prosthetic group in metallobiology.

Frataxin-deficient human brain microvascular endothelial cells lose polymerized actin and are paracellularly permeable -implications for blood-brain barrier integrity in Friedreich's Ataxia.

Background: Friedreich's Ataxia (FRDA) is the most prevalent inherited ataxia; the disease results from loss of Frataxin, an essential mitochondrial iron trafficking protein. FRDA presents as neurodegeneration of the dorsal root ganglion and cerebellar dentate nuclei, followed by brain iron accumulation in the latter. End stage disease includes cardiac fibrosis that contributes to hypertrophic cardiomyopathy. The microvasculature plays an essential barrier role in both the brain and heart, thus an investigation of this tissue system in FRDA is essential to the delineation of the cellular dysfunction in this genetic disorder. Here, we investigate brain microvascular endothelial cell integrity in FRDA in a model of the blood-brain barrier (BBB). Methods: We used lentiviral mediated shRNA delivery to generate a novel FRDA model in immortalized human brain microvascular endothelial cells (hBMVEC) that compose the microcapillaries of the BBB. We verified known cellular pathophysiologies of FXN knockdown including increased oxidative stress, loss of energy metabolism, and increased cell size. Furthermore, we investigated cytoskeletal architecture including the abundance and organization of filamentous actin, and barrier physiology via transendothelial electrical resistance and fluorescent tracer flux. Results: shFXN hBMVEC display the known FRDA cell morbidity including increased oxidative stress, decreased energy metabolism, and an increase in cell size. We demonstrate that shFXN hBMVEC have less overall filamentous actin, and that filamentous actin is lost at the cell membrane and cortical actin ring. Consistent with loss of cytoskeletal structure and anchorage, we found decreased barrier strength and increased paracellular tracer flux in the shFXN hBMVEC transwell model. Conclusion: We identified that insufficient FXN levels in the hBMVEC BBB model causes changes in cytoskeletal architecture and increased barrier permeability, cell pathologies that may be related to patient brain iron accumulation, neuroinflammation, neurodegeneration, and stroke. Our findings implicate other barrier cells, e.g., the cardiac microvasculature, likely contributory also to disease pathology in FRDA.