Decreased glucocerebrosidase activity and substrate accumulation of glycosphingolipids in a novel GBA1 D409V knock-in mouse model.

Multiple mutations have been described in the human GBA1 gene, which encodes the lysosomal enzyme beta-glucocerebrosidase (GCase) that degrades glucosylceramide and is pivotal in glycosphingolipid substrate metabolism. Depletion of GCase, typically by homozygous mutations in GBA1, is linked to the lysosomal storage disorder Gaucher's disease (GD) and distinct or heterozygous mutations in GBA1 are associated with increased Parkinson's disease (PD) risk. While numerous genes have been linked to heritable PD, GBA1 mutations in aggregate are the single greatest risk factor for development of idiopathic PD. The importance of GCase in PD necessitates preclinical models in which to study GCase-related mechanisms and novel therapeutic approaches, as well as to elucidate the molecular mechanisms leading to enhanced PD risk in GBA1 mutation carriers. The aim of this study was to develop and characterize a novel GBA1 mouse model and to facilitate wide accessibility of the model with phenotypic data. Herein we describe the results of molecular, biochemical, histological, and behavioral phenotyping analyses in a GBA1 D409V knock-in (KI) mouse. This mouse model exhibited significantly decreased GCase activity in liver and brain, with substantial increases in glycosphingolipid substrates in the liver. While no changes in the number of dopamine neurons in the substantia nigra were noted, subtle changes in striatal neurotransmitters were observed in GBA1 D409V KI mice. Alpha-synuclein pathology and inflammation were not observed in the nigrostriatal system of this model. In summary, the GBA1 D409V KI mouse model provides an ideal model for studies aimed at pharmacodynamic assessments of potential therapies aiming to restore GCase.

The small molecule ferristatin II induces hepatic hepcidin expression in vivo and in vitro.

Previous studies have shown that administration of ferristatin II to rats is associated with decreased serum iron, reduced transferrin saturation, and increased hepatic hepcidin expression. BMP and IL-6 signaling act via Smad and Stat3 transcription factors, respectively, to increase expression of hepcidin, the master regulator of iron metabolism. In this study, we aimed to explore the underlying mechanism of ferristatin II action on hepcidin production. We found that ferristatin II greatly increased hepcidin expression both in vivo and in vitro. In the rat liver, ferristatin II treatment decreased expression of Smad downstream targets Smad7 and Id1 and increased expression of Stat3 downstream targets α-2-macroglobulin, α-1-acid glycoprotein, and C-reactive peptide. Ferristatin II also increased Stat3 phosphorylation in the rat liver without affecting serum or hepatic IL-6 levels. It is unclear whether the Stat3 activation observed in vivo is a cause or a consequence to hepcidin induction. Reporter gene expression studies demonstrated that ferristatin II synergized with BMP6 and IL-6 to enhance hepcidin expression in vitro. However, this synergy was not due to activation of either Smad or Stat3 signaling, raising the possibility that ferristatin II may activate a novel pathway for hepcidin regulation.

Ferristatin II promotes degradation of transferrin receptor-1 in vitro and in vivo.

Previous studies have shown that the small molecule iron transport inhibitor ferristatin (NSC30611) acts by down-regulating transferrin receptor-1 (TfR1) via receptor degradation. In this investigation, we show that another small molecule, ferristatin II (NSC8679), acts in a similar manner to degrade the receptor through a nystatin-sensitive lipid raft pathway. Structural domains of the receptor necessary for interactions with the clathrin pathway do not appear to be necessary for ferristatin II induced degradation of TfR1. While TfR1 constitutively traffics through clathrin-mediated endocytosis, with or without ligand, the presence of Tf blocked ferristatin II induced degradation of TfR1. This effect of Tf was lost in a ligand binding receptor mutant G647A TfR1, suggesting that Tf binding to its receptor interferes with the drug's activity. Rats treated with ferristatin II have lower TfR1 in liver. These effects are associated with reduced intestinal (59)Fe uptake, lower serum iron and transferrin saturation, but no change in liver non-heme iron stores. The observed hypoferremia promoted by degradation of TfR1 by ferristatin II appears to be due to induced hepcidin gene expression.

Absorption of manganese and iron in a mouse model of hemochromatosis.

Hereditary hemochromatosis, an iron overload disease associated with excessive intestinal iron absorption, is commonly caused by loss of HFE gene function. Both iron and manganese absorption are regulated by iron status, but the relationships between the transport pathways of these metals and how they are affected by HFE-associated hemochromatosis remain poorly understood. Loss of HFE function is known to alter the intestinal expression of DMT1 (divalent metal transporter-1) and Fpn (ferroportin), transporters that have been implicated in absorption of both iron and manganese. Although the influence of HFE deficiency on dietary iron absorption has been characterized, potential effects on manganese metabolism have yet to be explored. To investigate the role of HFE in manganese absorption, we characterized the uptake and distribution of the metal in Hfe (-/-) knockout mice after intravenous, intragastric, and intranasal administration of (54)Mn. These values were compared to intravenous and intragastric administration of (59)Fe. Intestinal absorption of (59)Fe was increased and clearance of injected (59)Fe was also increased in Hfe(-/-) mice compared to controls. Hfe (-/-) mice displayed greater intestinal absorption of (54)Mn compared to wild-type Hfe(+/+) control mice. After intravenous injection, the distribution of (59)Fe to heart and liver was greater in Hfe (-/-) mice but no remarkable differences were observed for (54)Mn. Although olfactory absorption of (54)Mn into blood was unchanged in Hfe (-/-) mice, higher levels of intranasally-instilled (54)Mn were associated with Hfe(-/-) brain compared to controls. These results show that manganese transport and metabolism can be modified by HFE deficiency.

Iron loading impairs lipoprotein lipase activity and promotes hypertriglyceridemia.

Iron loading is associated with altered lipid metabolism, but underlying mechanisms remain unknown. We compared serum iron and triglycerides (TGs) in Belgrade rats, a genetic model of iron-loading anemia. Homozygous b/b rats had greater serum iron (68 vs. 28 µM; P=0.0004) and TG levels (180 vs. 84 mg/dl; P=0.014) compared to +/b controls. To confirm the association between iron loading and high TGs, Fischer rats were fed chow containing 1% carbonyl iron. Compared to controls pair-fed normal chow, carbonyl iron-fed rats had elevated serum iron (42 vs. 21 µM; P=0.007) and TGs (190 vs. 115 mg/dl; P=0.009). Despite normal hepatic production and secretion, TG clearance was lower in b/b than +/b rats due to reduced serum lipoprotein lipase (LPL) activity (3.1 vs. 5.0 mM/min; P=0.026). Likewise, LPL was lower in carbonyl iron-fed rats compared to controls (2.4 vs. 3.7 mM/min; P=0.017). Direct addition of iron to serum ex vivo or recombinant LPL in vitro decreased enzymatic activity in a dose-dependent manner. Lowering serum iron in Belgrade rats reduced TG levels (274 to 67 mg/dl, P=0.001). This study explains the relationship between iron status and lipid metabolism and provides mechanistic support for interventions that reduce serum iron levels in individuals at risk for hypertriglyceridemia.

Iron-responsive olfactory uptake of manganese improves motor function deficits associated with iron deficiency.

Iron-responsive manganese uptake is increased in iron-deficient rats, suggesting that toxicity related to manganese exposure could be modified by iron status. To explore possible interactions, the distribution of intranasally-instilled manganese in control and iron-deficient rat brain was characterized by quantitative image analysis using T1-weighted magnetic resonance imaging (MRI). Manganese accumulation in the brain of iron-deficient rats was doubled after intranasal administration of MnCl(2) for 1- or 3-week. Enhanced manganese level was observed in specific brain regions of iron-deficient rats, including the striatum, hippocampus, and prefrontal cortex. Iron-deficient rats spent reduced time on a standard accelerating rotarod bar before falling and with lower peak speed compared to controls; unexpectedly, these measures of motor function significantly improved in iron-deficient rats intranasally-instilled with MnCl(2). Although tissue dopamine concentrations were similar in the striatum, dopamine transporter (DAT) and dopamine receptor D(1) (D1R) levels were reduced and dopamine receptor D(2) (D2R) levels were increased in manganese-instilled rats, suggesting that manganese-induced changes in post-synaptic dopaminergic signaling contribute to the compensatory effect. Enhanced olfactory manganese uptake during iron deficiency appears to be a programmed "rescue response" with beneficial influence on motor impairment due to low iron status.

Severe postnatal iron deficiency alters emotional behavior and dopamine levels in the prefrontal cortex of young male rats.

Iron deficiency in early human life is associated with abnormal neurological development. The objective of this study was to evaluate the effect of postnatal iron deficiency on emotional behavior and dopaminergic metabolism in the prefrontal cortex in a young male rodent model. Weanling, male, Sprague-Dawley rats were fed standard nonpurified diet (220 mg/kg iron) or an iron-deficient diet (2-6 mg/kg iron). After 1 mo, hematocrits were 0.42 ± 0.0043 and 0.16 ± 0.0068 (mean ± SEM; P < 0.05; n = 8), liver nonheme iron concentrations were 2.3 ± 0.24 and 0.21 ± 0.010 µmol/g liver (P < 0.05; n = 8), and serum iron concentrations were 47 ± 5.4 and 23 ± 7.1 µmol/L (P < 0.05; n = 8), respectively. An elevated plus maze was used to study emotional behavior. Iron-deficient rats displayed anxious behavior with fewer entries and less time spent in open arms compared to control rats (0.25 ± 0.25 vs. 1.8 ± 0.62 entries; 0.88 ± 0.88 vs. 13 ± 4.6 s; P < 0.05; n = 8). Iron-deficient rats also traveled with a lower velocity in the elevated plus maze (1.2 ± 0.15 vs. 1.7 ± 0.12 cm/s; P < 0.05; n = 8), behavior that reflected reduced motor function as measured on a standard accelerating rotarod device. Both the time on the rotarod bar before falling and the peak speed attained on rotarod by iron-deficient rats were lower than control rats (156 ± 12 vs. 194 ± 12 s; 23 ± 1.5 vs. 28 ± 1.6 rpm; P < 0.05; n = 7-8). Microdialysis experiments showed that these behavioral effects were associated with reduced concentrations of extracellular dopamine in the prefrontal cortex of the iron-deficient rats (79 ± 7.0 vs. 110 ± 14 ng/L; P < 0.05; n = 4). Altered dopaminergic signaling in the prefrontal cortex most likely contributes to the anxious behavior observed in young male rats with severe iron deficiency.

Influence of DMT1 and iron status on inflammatory responses in the lung.

Divalent metal transporter 1 (DMT1) is the major iron transporter responsible for duodenal dietary iron absorption and is required for erythropoiesis. Recent studies suggest that loss of DMT1 activity could be involved in metal-related lung injury, but little is known about the effects of iron status and DMT1 function on pulmonary inflammation. To better define the role of DMT1 and iron status in pulmonary inflammatory responses, we performed bronchoalveolar lavage (BAL) following intratracheal instillation of lipopolysaccharide (LPS) to the Belgrade rat, an animal model deficient in DMT1 function. In the basal state, the BAL fluid of Belgrade rats had more macrophages and higher lactate dehydrogenase, myeloperoxidase, albumin, and hemoglobin levels compared with heterozygote control rats. Following LPS instillation, the macrophage fraction relative to total BAL cell content and levels of albumin and IgM were increased in Belgrade rats compared with controls. In contrast, heterozygote Belgrade rats made anemic by diet-induced iron deficiency exhibited attenuated inflammatory responses to LPS. These combined results show that pulmonary inflammation can be modified by both DMT1 and iron status. Loss of DMT1 alters pulmonary responses necessary for lung homeostasis in the basal state and enhances LPS-induced inflammation and therefore would contribute to progression of lung injury.

Small molecule inhibitors of divalent metal transporter-1.

Divalent metal transporter-1 (DMT1) is a divalent cation transporter that plays a key role in iron metabolism by mediating ferrous iron uptake across the small intestine. We have previously identified several small molecule inhibitors of iron uptake (4). Using a cell line that stably overexpresses DMT1, we screened the ability of these inhibitors to specifically block this transporter's activity. One compound, NSC306711, inhibited DMT1-mediated iron uptake in a reversible and competitive manner. This inhibitor is a polysulfonated dye containing two copper centers. Although one of these two sites could be chelated by Triethylenetetramine copper chelation did not perturb NSC306711 inhibition of DMT1 activity. Several other polysulfonated dyes with structural features similar to NSC306711 were identified as potential DMT1 transport inhibitors. This study characterizes important pharmacological tools that can be used to probe DMT1's mechanism of iron transport and its role in iron metabolism.

Small-molecule screening identifies the selanazal drug ebselen as a potent inhibitor of DMT1-mediated iron uptake.

HEK293T cells overexpressing divalent metal transporter-1 (DMT1) were established to screen for small-molecule inhibitors of iron uptake. Using a fluorescence-based assay, we tested 2000 known bioactive compounds to find 3 small molecules that potently block ferrous iron uptake. One of the inhibitors, ebselen, is a seleno compound used in clinical trials as a protective agent against ischemic stroke. Ebselen inhibited Fe(II) uptake (IC(50) of approximately 0.22 microM), but did not influence Fe(III) transport or DMT1-mediated manganese uptake. An unrelated antioxidant, pyrrolidine dithiobarbamate (PDTC), also inhibited DMT1 activity (IC(50) of approximately 1.54 microM). Both ebselen and PDTC increased cellular levels of reduced glutathione. These observations indicate that Fe(II) transport by DMT1 can be modulated by cellular redox status and suggest that ebselen may act therapeutically to limit iron-catalyzed damage due to transport inhibition.

Single-cell FRET imaging of transferrin receptor trafficking dynamics by Sfp-catalyzed, site-specific protein labeling.

Fluorescence imaging of living cells depends on an efficient and specific method for labeling the target cellular protein with fluorophores. Here we show that Sfp phosphopantetheinyl transferase-catalyzed protein labeling is suitable for fluorescence imaging of membrane proteins that spend at least part of their membrane trafficking cycle at the cell surface. In this study, transferrin receptor 1 (TfR1) was fused to peptide carrier protein (PCP), and the TfR1-PCP fusion protein was specifically labeled with fluorophore Alexa 488 by Sfp. The trafficking of transferrin-TfR1-PCP complex during the process of transferrin-mediated iron uptake was imaged by fluorescence resonance energy transfer between the fluorescently labeled transferrin ligand and TfR1 receptor. We thus demonstrated that Sfp-catalyzed small molecule labeling of the PCP tag represents a practical and efficient tool for molecular imaging studies in living cells.

Chemical genetic screening identifies sulfonamides that raise organellar pH and interfere with membrane traffic.

Chemical genetics seeks to identify small molecules that afford functional dissection of cell biological pathways. Previous screens for small molecule inhibitors of exocytic membrane traffic yielded the identification and characterization of several compounds that block traffic from the Golgi to the cell surface as well as transport from the endoplasmic reticulum to the Golgi network [Feng et al. Proc Natl Acad Sci USA 2003;100:6469-6474; Yarrow et al. Comb Chem High Throughput Screen 2003;6:279-286; Feng et al. EMBO Reports 2004: in press]. Here, we screened these inhibitors for potential effects on endocytic membrane traffic. Two structurally related sulfonamides were found to be potent and reversible inhibitors of transferrin-mediated iron uptake. These inhibitors do not block endoplasmic reticulum-to-Golgi transport, but do disrupt Golgi-to-cell surface traffic. The compounds are members of a novel class of sulfonamides that elevate endosomal and lysosomal pH, down-regulate cell surface receptors, and impair recycling of internalized transferrin receptors to the plasma membrane. In vitro experiments revealed that the sulfonamides directly inhibit adenosine triphosphate (ATP) hydrolysis by the V-ATPase and that they also possess a potent proton ionophore activity. While maintenance of organellar pH is known to be a critical factor in both endocytosis and exocytosis, the precise role of acidification, beyond the uncoupling of ligands from their receptors, remains largely unknown. Identification of this novel class of sulfonamide inhibitors provides new chemical tools to better understand the function of organelle pH in membrane traffic and the activity of V-ATPases in particular.

Identification of small molecule inhibitors that distinguish between non-transferrin bound iron uptake and transferrin-mediated iron transport.

Chemical genetics is an emerging field that takes advantage of combinatorial chemical and small molecule libraries to dissect complex biological processes. Here we establish a fluorescence-based assay to screen for inhibitors of iron uptake by mammalian cells. Using this approach, we screened the National Cancer Institute's Diversity Set library for inhibitors of non-transferrin bound iron uptake. This screen identified 10 novel small molecule inhibitors of iron transport with IC(50) values that ranged from 5 to 30 microM. Of these ten compounds, only two blocked uptake of iron mediated by transferrin. Thus, this study characterizes the first small molecule inhibitors that distinguish between different pathways of iron transport.

In vivo gene transfer of Kv1.5 normalizes action potential duration and shortens QT interval in mice with long QT phenotype.

Mutations in cardiac voltage-gated K+ channels cause long QT syndrome (LQTS) and sudden death. We created a transgenic mouse with a long QT phenotype (Kv1DN) by overexpression of a truncated K+ channel in the heart and investigated whether the dominant negative effect of the transgene would be overcome by the direct injection of adenoviral vectors expressing wild-type Kv1.5 (AV-Kv1.5) into the myocardium. End points at 3-10 days included electrophysiology in isolated cardiomyocytes, surface ECG, programmed stimulation of the right ventricle, and in vivo optical mapping of action potentials and repolarization gradients in Langendorff-perfused hearts. Overexpression of Kv1.5 reconstituted a 4-aminopyridine-sensitive outward K+ current, shortened the action potential duration, eliminated early afterdepolarizations, shortened the QT interval, decreased dispersion of repolarization, and increased the heart rate. Each of these changes is consistent with a physiologically significant primary effect of adenoviral expression of Kv1.5 on ventricular repolarization of Kv1DN mice.

Regional upregulation of Kv2.1-encoded current, IK,slow2, in Kv1DN mice is abolished by crossbreeding with Kv2DN mice.

Overexpression of a truncated Kv1.1 channel transgene in the heart (Kv1DN) resulted in mice with a prolonged action potential duration due to marked attenuation of a rapidly activating, slowly inactivating potassium current (I(K,slow1)) in ventricular myocytes. Optical mapping and programmed electrical stimulation revealed inducible ventricular tachycardia due to spatial dispersion of repolarization and refractoriness. Here we show that a delayed rectifier with slower inactivation kinetics (I(K,slow2)) was selectively upregulated in Kv1DN cardiocytes. This electrical remodeling was spatially restricted to myocytes derived from the apex of the left ventricle. Biophysical and pharmacological studies of I(K,slow2) indicate that it resembles Kv2-encoded currents. Northern blot analyses and real-time PCR revealed upregulation of Kv2.1 transcript in Kv1DN mice. Crossbreeding of Kv1DN mice with mice expressing a truncated Kv2.1 polypeptide (Kv2DN) eliminated I(K,slow2). In summary, our data indicate that the spatially restrictive upregulation of Kv2.1-encoded currents underlies the increased dispersion of the repolarization observed in Kv1DN mice.