Part Ⅰ The Iron Chelator, PBT434, Modulates Transcellular Iron Trafficking in Brain Microvascular Endothelial Cells
Apr 28, 2023
Abstract
Iron and other transition metals, such as copper and manganese, are essential for supporting brain function, yet over-accumulation is cytotoxic. This over-accumulation of metals, particularly iron, is common to several neurological disorders; these include Alzheimer’s disease, Parkinson’s disease, Friedrich’s ataxia, and other disorders presenting with neurodegeneration and associated brain iron accumulation. The management of iron flux by the blood-brain barrier provides the first line of defense against the over-accumulation of iron in normal physiology and these pathological conditions. In this study, we determined that the iron chelator PBT434, which is currently being developed for the treatment of Parkinson’s disease and multiple system atrophy, modulates the uptake of iron by human brain microvascular endothelial cells (hBMVEC) by chelation of extracellular Fe2+. Treatment of hBMVEC with PBT434 results in an increase in the abundance of the transcripts for transferrin receptor (TfR) and ceruloplasmin (Cp). Western blot and ELISA analyses reveal a corresponding increase in the proteins as well. Within the cell, PBT434 increases the detectable level of charitable, labile Fe2+; data indicate that this Fe2+ is released from ferritin. In addition, PBT434 potentiates iron efflux likely due to the increase in cytosolic ferrous iron, the substrate for the iron exporter, ferroportin. PBT434 equilibrates rapidly and bi-directionally across an hBMVEC blood-brain barrier. These results indicate that the PBT434-iron complex is not a substrate for hBMVEC uptake and thus support a model in which PBT434 would chelate interstitial iron and inhibit re-uptake of iron by endothelial cells of the blood-brain barrier, as well as inhibit its uptake by the other cells of the neurovascular unit. Overall, this presents a novel and promising mechanism for therapeutic iron chelation.
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Introduction
Metal chelation therapy (MCT) has long been used as a treatment for transition metal poisoning and for genetic disorders in the metabolism of an essential metal ion that leads to the metal’s over-accumulation [1–3]. Two examples of the latter are the hyper-accumulation of copper in Wilson’s disease [4] and of iron in hereditary hemochromatosis [5]. Both copper and iron are catalysts of oxidative stress and thus are cytotoxic at concentrations that exceed the ability of the cell and organism to ‘chaperone’ these redox-active transition metals [6, 7]. Iron accumulation, in particular, is broadly idiopathic; indeed, an increase in iron is a hallmark of an aging brain [8–10]. Pathologically, this brain iron accumulation is a feature of mutations in genes unrelated to iron metabolism [11–15] as well as a variety of other neurodegenerative diseases, some of which lack a specific genetic link such as aging [16], Alzheimer’s Disease [17], Friedreich’s Ataxia [18] and Parkinson’s Disease [19]. As a group, such disorders can be thought of as neurodegeneration with brain iron accumulation (NBIA) although this acronym commonly has been restricted to those for which a genetic link has been identified [11, 13, 14].
In the case of iron overload, the objective is to ‘cleanse’ the body of excess iron due to a defect in cell iron uptake or efflux. Here the objective is to out-compete physiologic iron chelators with the drug; a compound that has good pharmacokinetics and a high affinity for ferrous iron is the target drug. Since the body is over-replete with the essential metal, there is little concern about inducing a deficiency in the course of treatment. Treating cerebral disease with iron chelation therapy requires a different strategy. This is not a problem of systemic iron overload, but of iron accumulation in areas of pathology with damaging downstream sequelae. Age-associated iron accumulation in Parkinson’s disease (PD), for instance, potentially contributes to oxidative stress-related cellular damage [20]. Excessive labile iron promotes the misfolding of α-synuclein in substantia nigral neurons. The use of a high-affinity chelator may lead to some reduction in the brain iron load but will most certainly induce an iron deficiency that in the aged population, at least, is contraindicated given the systemic iron deficiency common to that age group [21]. A chelator with an optimal affinity has the potential to reduce iron accumulation as well as the attendant oxidative stress due to excess labile iron and underlying disease processes.
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One chelator approved for use in treating transfusion-induced iron overload in thalassemia patients is deferiprone (DFP, brand name Ferriprox) [5, 22]. DFP also has been used in treating Friedreich’s ataxia [23] and Parkinson’s disease [24, 25]. In a meta-analysis, DFP has been shown to provide significant reductions in myocardial iron content as well as greater cardiac protection in thalassemia patients than deferoxamine, the classical iron chelating agent [5]. On the other hand, DFP is rapidly metabolized by the liver [26] and more recent work has shown it chelates Fe2+ at the active site of iron-dependent histone lysine demethylases, an activity that correlates with a previously unrecognized cytotoxicity [27]. This finding underscores a key limitation in the use of iron chelation therapy, namely competition by the drug for physiologically-essential iron, whether in an iron store or a protein that harbors a prosthetic iron species. Nonetheless, DFP, for example, has shown efficacy in a Phase 2 trial treatment of Parkinson’s disease as indicated by both analytic (reduced brain iron load by T2- weighted MRI) and behavioral indices (cognitive and motor neuron function) [24, 25].
The affinity of DFP for Fe3+ remains a concern, however. The stable DFP-iron species is the tris-complex, [Fe(DFP)3] 0 [28]. While the neutrality of this complex is ideal for mobilizing iron out of the cell, the stability constant for it, ~1037, makes DFP a true iron scavenger; in this context, its inhibition of an iron enzyme like lysine demethylase is predictable [27]. This concern reflects the need to develop iron chelators that have the membrane permeability of DFP but a significantly weaker affinity for both Fe2+ and Fe3+. This latter feature limits drug scavenging of prosthetic metal and the thermodynamic potential of the chelating agent to catalyze the ferrous iron auto-oxidation that results in the production of reactive oxygen species. In essence, strong ferric iron chelators catalyze the pro-oxidant property of Fe2+ [29]. In this study, we report how such an iron chelator with moderate ferric and ferrous iron affinities modulates the flux of iron in the brain microvascular endothelial cells that form the blood-brain barrier (BBB).
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This drug, PBT434 [5,7-dichloro-2-((methylamino)methyl)-8-hydroxy-3-methylquinazolin-4 (3H)-one, Fig 1A], forms a bis-iron complex with log stability constants of ~11 and ~15 for Fe2+ and Fe3+, respectively [30]. PBT434 prevented loss of substantia nigra pars compacta (SNpc) neurons, lowered nigral α-synuclein accumulation, reduced PD disease model-related iron content in the midbrain, and rescued motor performance in two mouse models of Parkinson’s disease without any apparent depletion of systemic iron stores [30]. PBT434 is also efficacious in murine models of Multiple System Atrophy (MSA) [30, 31], a motor disorder similar in presentation to Parkinson’s but which is characterized by α-synuclein misfolding and subsequent accumulation causing the formation of glial cytoplasmic inclusions that are the hallmark pathology of the disease [32]. Significantly, PBT434 reduced markers of oxidative stress in mouse PD models [30] indicating that 1) PBT434 targeted iron stores that otherwise were primed to function as pro-oxidants and 2) PBT434 did not potentiate this nascent oxidation-based cytotoxicity. PBT434 has completed a Phase 1 study satisfactorily [33].
The work presented here was designed to interrogate the impact PBT434 has on iron trafficking in the brain’s barrier cells, the microvascular endothelial cells that together with underlying glia form the blood-brain barrier. These studies have used a well-validated immortalized endothelial cell line in both monolayer and transwell culture formats [34–37]. The primary objective of these studies was to determine the kinetics of iron uptake and efflux from these cells and their modulation by PBT434. The transwell BBB model was used also to demonstrate the bi-directional PBT434 transcellular flux across the endothelial cell barrier. The model demonstrated in molecular terms that PBT434 inhibits iron uptake by chelation while stimulating iron efflux. Cell imaging studies indicate that PBT434 accesses the same labile iron pool probed by a classic Fe2+ chelating agent, 2,2’-bipyridine or bipyridyl, and a fluorescent probe for ferrous iron. The results suggest a possible mechanism of action for PBT434 that includes inhibition of uptake of systemic iron at the BBB, and subsequent sequestration of brain iron in the interstitial space.
Results
1. PBT434 has no cytotoxic effects on brain microvascular endothelial cells
To determine an appropriate range of working concentrations for PBT434 in our in vitro cell culture, we utilized the MTT assay to monitor hBMVEC mitochondrial function in response to PBT434. Based on previous reports [30], have were treated with a range of PBT434 concentrations up to 100 μM for 24h. We observed no significant changes in hBMVEC viability with any concentration tested (Fig 2).
2. PBT434 is rapidly taken up and trafficked across the hBMVEC barrier
PBT434 is an orally bioavailable drug that can readily penetrate the BBB, as seen in studies carried out in mice and humans [30, 38, 39]. We monitored the accumulation of PBT434 in hBMVEC grown in monolayers using 14C-labeled PBT434 as a radiotracer. The data indicated that in the first phase, 14C-PBT434 rapidly equilibrated between the uptake medium and the cell. This initial uptake was followed by an additional slow accumulation over 3 h which exhibited a rate of 30.1 ± 9.8 pmol/mg/h (Fig 3A). In the uptake protocol, uptake is quenched and cells are washed at 4˚C before processing for 14C-PBT434 accumulation (Methods). In a separate experiment, we examined the efflux of 14C-PBT434 from hBMVEC after a 30 min loading period. In the efflux protocol, cells are washed at 25˚C. The data in Fig 3B indicate that in the 25˚C wash, approximately 92% of the cell-accumulated 14C-PBT434 was lost (cf 550 pmol 14C-PBT434/mg protein in 3A at 30 min to 43 pmol 14C-PBT434/mg protein at t = 0 in 3B). There was a further slow loss of the remaining 14C-PBT434 (Fig 3B). The data suggest two aspects of the accumulation and efflux of PBT434 by hBMVEC. Flux across the plasma membrane is rapidly reaching what appears to be an equilibrium whether during uptake or efflux. However, in both processes, there appears another slower process. This suggests that within the cell, some fraction of cell PBT434 is in a locale/state that is in a kinetic steady-state relationship with the fraction in equilibrium with the extra-cellular milieu. The kinetic analysis noted in Fig 3B estimated this pool of PBT434 was represented by 27±4 pmol/mg protein in the cell lysate when cells were treated with 20 μM reagent.
To examine the transcellular flux of PBT434, we employed a well-validated in vitro BBB model using have grown on the apical side of a transwell membrane [35, 36, 40, 41]. The barrier properties of these transwell cultures were verified by quantification of their transendothelial electrical resistance (TEER) and impermeability to FITC-labelled dextran (S1 Fig). We compared 14C-PBT434 uptake at the luminal (or apical, blood side) (Fig 4A) to uptake at the abluminal (or basolateral, brain side) (Fig 4C) membrane. In the same experiment, the corresponding efflux (transcellular flux) was quantified by the appearance of 14C-PBT434 in the efflux chamber (Fig 4 panels B and D). The rates of these processes are provided in Table 1. The mass data illustrated in Fig 4 (panels B and D) show that the net flux of PBT434 across this model blood-brain barrier was the same in the two directions. There were 976±185 pmol 14C-PBT434 accumulated in the basal chamber (Fig 4B) and 1033±210 pmol quantified in the basal chamber (Fig 4D). This near equivalence was reflected also in the closely similar rates of PBT434 efflux at the two barrier membranes (Table 1). However, there was a significantly greater uptake of PBT434 at the basolateral membrane in this barrier model as illustrated by the ~50% greater loss of compound from the basal chamber (Fig 4C) that corresponded to a ~40% greater rate of apparent cell uptake (Table 1). A more robust uptake would be predicted to result in greater accumulation. Analysis of the cells at 3h showed that they retained ~6 μM PBT434 irrespective of the flux direction. The values were 8.1± 1.3 μM (apical to basal) and 4.7±1.2 μM (basal to apical). As noted above, this analysis follows the washing of the cells before lysis and quantification of total cell protein and 14C-PBT434. In addition, the medium in the apical chamber contained RPMI plus 10% FBS and 10% NuSerum whereas the basal, ‘brain’ chamber contained only RPMI (Methods). A reasonable inference was that the greater ‘uptake’ at the basal membrane reflected a cell surface adsorption of PBT434 that was limited in the apical chamber by the presence of protein components in the serum. Upon washing the cells for accumulation of PBT434, this adsorbed material (that registered as ‘uptake’) was removed. Repeating this flux experiment but with serum in the basal chamber demonstrated that, indeed, serum suppressed this likely cell surface PBT434 adsorption (S2 Fig).
3. PBT434, unlike bipyridyl, does not limit the intracellular availability of labile iron
Since PBT434 has a more moderate affinity for iron compared to classical iron chelators such as deferiprone or bipyridyl, we examined how that difference was reflected in the PBT434 effect on the cellular labile iron pool (LIP) of hBMVEC. To do this, we took advantage of the permeable, Fe2+-specific fluorescent dye FerroOrange, which reacts with charitable cytoplasmic iron. We saw a significant ablation of fluorescence in cells when treated with bipyridyl, consistent with the chelation of the LIP by this high-affinity ferrous iron chelator and thus blocking the action of the fluorescent iron indicator (Fig 5A). In contrast, PBT434 did not compete with FerroOrange for Fe2+, a behavior consistent with its more moderate affinity [30]. The results showed that PBT434, but not the PBT434-met inactive derivative, induced a 34 ± 9% increase in FerroOrange-accessible Fe2+ suggesting this chelating agent mobilized iron within the cell without concurrent toxicity. The data presented below suggests this iron came from ferritin.
PBT434 was shown previously to restore depleted ferroportin protein expression in MPTP-treated mice to a level similar to that of unlesioned mice [30]. This result, along with the increase in intracellular ferrous iron staining in response to PBT434, suggested a potential effect on the cellular iron response system and function of downstream iron-related proteins. To assess this, we first conducted quantitative PCR (qPCR) analysis of the PBT434 effect on the abundance of the transcripts for several iron-handling proteins (Fig 6). While the transcripts for the iron efflux protein, ferroportin (Fpn), and the two cytoplasmic iron chaperones, PCBP1 and 2 were unaffected, the abundance of the mRNAs for the transferrin receptor (TfR), and the ferroxidase, ceruloplasmin (Cp), did change. TfR and Cp transcripts increased by 2.8 and 3.6-fold, respectively. Transferrin receptor (TfR) expression is linked to the iron-responsive element (IRE)/iron regulatory protein (IRP) system [42–44]. The increase in TfR mRNA suggests that PBT434 competes with the PCBP1-dependent delivery of iron for the assembly of the Fe, S cluster that converts the regulatory IREBP from an RNA-binding protein to cytosolic aconitase [45]. Thus, PBT434 shifts this regulatory modulation towards RNA-binding and the corresponding inhibition of TfR mRNA degradation. In cell iron deficiency, Cp expression is, in part, regulated by HIF-1α [46]. An increase in HIF-1α function follows from the knock-down of its hydroxylation by prolyl hydroxylase activity in an iron-dependent reaction [47]. As in the case of the IREBP, PBT434 appears to diminish the pool of iron that serves as a co-factor in HIF-1α hydroxylation and degradation. In this model, the increase in the steady-state level of this transcriptional activator increases Cp transcription.
Using a combination of ELISA analysis and western blotting, we probed the expression of iron-handling proteins in PBT434 or PBT434-met treated hBMVEC; examples of the WB analyses are given in Fig 7A. The data showed the abundance of TfR monomer and dimer were significantly increased by 24h as was Cp (Fig 7B and 7C). Both increases paralleled the PBT434-dependent increase in the respective transcripts (Fig 6), In contrast, the expression of the iron efflux protein, Fpn, was insensitive to PBT434 treatment (Fig 7D).
We used ELISA as an additional method to quantify the fold changes indicated by the western blot data. Thus, hBMVEC were treated with PBT434 for 24h and cell lysates were assayed by ELISA for TfR (Fig 8A). The fold increase in TfR in response to PBT434 treatment quantified by ELISA was equivalent to that provided by analysis of the western blots (Fig 7B). ELISA was used also to assess secreted and GPI-linked Cp protein abundance, using HepG2 cells as a positive control. Concerning Cp secreted into growth media, this approach was limited in that sCp abundance in both HepG2 and hBMVEC conditioned media were at or below the lower sensitivity limit of this assay (S3 Fig). However, it did enable the assessment of GPI-Cp abundance. In this method, cells were treated with phosphatidylinositol-specific phospholipase C (PI-PLC), which cleaves the GPI anchor; the media thus conditioned was concentrated and analyzed by the Cp-ELISA. While this approach demonstrated PBT434 increased the amount of GPI-Cp in HepG2 cells, it again failed to detect any Cp released by the PI-PLC (Fig 8B). ELISA also afforded a direct method for the quantification of ferritin. To do so, hBMVEC were loaded with 1 uM Fe-citrate for 24h, followed by treatment in the absence or presence of PBT434 for an additional 1 h. The resulting cell lysates were subjected to ELISA analysis for ferritin (Fig 8C). In contrast to the increase in TfR, treatment with PBT434 knocked down ferritin (Ft) protein by ~18%. Indeed, this loss of Ft protein was apparent after only 1h treatment with the reagent. The temporal nature of this result can be correlated with the increase in charitable Fe2+ noted above following a 30 min treatment with PBT434. As discussed later, the knockdown of ferritin has been demonstrated following treatment with other cell-permeant Fe2+ chelating agents [48].
4. 55Fe2+ uptake is inhibited by complexation with PBT434
Given the rapid equilibration of PBT434 in hBMVEC within 30 min, in comparison to the slow, biphasic uptake and equilibration of Fe2+ over 24h [49], we hypothesized that PBT434 and Fe2+ did not share the same uptake mechanism. To test this, monolayers were incubated with radiolabeled 55Fe2+ in the absence or presence of PBT434 or PBT434-met, and 55Fe2+ uptake over 3 h was monitored (Fig 9A). PBT434 significantly decreased the rate of 55Fe2+ uptake, as well as decreased the overall accumulation of 55Fe2+ in cell lysates (Fig 9C). This effect was not seen with PBT434-met. Comparison of PBT434 to 55Fe-uptake rates indicates that PBT434 and Fe2+ are taken up by separate transport pathways. Furthermore, the inhibition of 55Fe-uptake in the presence of PBT434 but not PBT434-met suggests that an extracellular PBT434-iron complex is not a ligand for the ferrous iron transporters in hBMVEC, namely ZIP8, and ZIP14.
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To further examine the role of PBT434 in iron accumulation, we tested the effect its preexposure had on 55Fe2+ uptake. Cells pre-treated with PBT434 which were, after washing, exposed to 55Fe2+ displayed an increase in the rate of uptake and accumulation of 55Fe2+ after 3h (Fig 9, panels B and D). This increased accumulation was maintained for at least 24h. These data suggest that pre-exposure of cells to PBT434 transiently potentiates iron uptake. Unexpectedly, PBT434-met pre-treatment also showed an increase in both uptake and accumulation (Fig 9B), but this effect was not as significant or persistent as that shown by PBT434.
We have shown that the uptake of iron from 59Fe-transferrin is supported by ferri-reduction and ferro-permeation at the plasma membrane of have [50, 51]. One experimental outcome in support of this TBI iron uptake model was the knockdown of this uptake by inhibition of extra-cytoplasmic ferrireductase activity; another result was a 60% inhibition of TBI iron uptake by ferrozine, a strong ferrous iron chelating agent [50]. This latter strategy was utilized to demonstrate that PBT434, but not PBT434-met, also inhibited TBI iron uptake (Fig 10).
5. PBT434 stimulates Fpn-dependent 55Fe2+ efflux
PBT434 has approximately 20% of the ability of deferiprone to produce an apparent stimulation of Fe2+ efflux from neuronal cells [30]. We assessed the efflux of 55Fe2+ from hBMVEC in the absence or presence of PBT434 in control cells or cells treated with a mini-hepcidin, PR73. Hepcidin is a peptide hormone found both systemically and in the brain interstitium that binds to Fpn and targets the transporter for degradation. The effects of hepcidin on the iron export function of Fpn have been extensively studied [52–54]. We have previously shown efflux of Fe2+ from hBMVEC is Fpn-dependent [35, 49]. PR73 has an EC50 of ~4 nM for Fpn degradation in a GFP reporter assay [55]. hBMVEC in monolayers were loaded with 55Fe2+ for 24 h in the absence or presence of PR73. 55Fe-efflux was then quantified over a 5h period in the continued absence or presence of PR73 in combination with the absence and presence of PBT434 (Fig 11). While PR73 knocked down 55Fe efflux from both the control and PBT434-treated cultures, PBT434 partially suppressed the inhibition due to the mini-hepcidin. In the absence of PBT434, iron efflux from the PR73-treated cultures was knocked down by ~75% while knockdown in the PBT434-treated cultures was only ~50% (Fig 11 and Table 2). Two inferences can be drawn from these results. First, the knockdown of Fpn by PR73 down-regulates 55Fe-efflux in the presence as well as the absence of PBT434. Second, under either condition, PBT434 supports a significant albeit small stimulation of iron efflux.
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Danielle K. BaileyID, Whitney Clark, Daniel J. Kosman
Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY, United States of America
