Patrick Walter

Pelizaeus-Merzbacher disease (PMD) is an X-linked leukodystrophy caused by mutations in Proteolipid Protein 1 (PLP1), encoding a major myelin protein, resulting in profound developmental delay and early lethality. Previous work showed involvement of unfolded protein response (UPR) and endoplasmic reticulum (ER) stress pathways, but poor PLP1 genotype-phenotype associations suggest additional pathogenetic mechanisms. Using induced pluripotent stem cell (iPSC) and gene-correction, we show that patient-derived oligodendrocytes can develop to the pre-myelinating stage, but subsequently undergo cell death. Mutant oligodendrocytes demonstrated key hallmarks of ferroptosis including lipid peroxidation, abnormal iron metabolism, and hypersensitivity to free iron. Iron chelation rescued mutant oligodendrocyte apoptosis, survival, and differentiation in vitro, and post-transplantation in vivo. Finally, systemic treatment of Plp1 mutant Jimpy mice with deferiprone, a small molecule iron chelator, reduced oligodendrocyte apoptosis and enabled myelin formation. Thus, oligodendrocyte iron-induced cell death and myelination is rescued by iron chelation in PMD pre-clinical models.

Phospholipase A2G6-associated neurodegeneration (PLAN) is a rare early-onset monogenic neurodegenerative movement disorder which targets the basal ganglia and other regions in the central and peripheral nervous system; presenting as a series of heterogenous subtypes in patients. We describe here a B6.C3-pla2g6m1J/CxRwb mouse model of PLAN, which presents with early-onset neurodegeneration at 90 days which is analogous of the disease progression that is observed in PLAN patients. Homozygous mice had a progressively worsening motor deficit, which presented as tremors starting at 65 days and progressed to severe motor dysfunction and increased falls on the wire hang test at 90 days. This motor deficit positively correlated with a reduction in tyrosine hydroxylase (TH) protein expression in dopaminergic neurons of the substantia nigra (SN) without any neuronal loss. Fluorescence imaging of Thy1-YFP revealed spheroid formation in the SN. The spheroids in homozygous mice strongly mirrors those observed in patients and were demonstrated to correlate strongly with the motor deficits as measured by the wire hang test. The appearance of spheroids preceded TH loss and increased spheroid numbers negatively correlated with TH expression. Perls/DAB staining revealed the presence of iron accumulation with in the SN of mice. This mouse model captures many of the major hallmarks of PLAN including severe-early onset neurodegeneration, a motor deficit that correlates directly to TH levels, spheroid formation and iron accumulation within the basal ganglia. Thus, this mouse line is a useful tool for further research efforts to improve understanding of how these disease mechanisms give rise to the disease presentations seen in PLAN patients as well as to test novel therapies.

Iron overload has been shown to induce DNA damage and may be implicated in leukocyte apoptosis. Previously we have shown that indirect markers of apoptosis such as caspase activity and Bax levels were higher in leukocytes of thalassemia patients. We have now assessed whether nucleosomal DNA fragmentation, a hallmark of apoptosis, is higher in leukocytes of iron-overloaded thalassemia patients compared with control subjects. Methods: Thalassemia Clinical Research Network patients participating in the Novartis CICL670A0107 trial (a randomized comparison of deferasirox, an oral iron chelator, vs. DFO) were eligible and 44 (25 male, mean age: 21.8 ± 11.1 yrs) were enrolled in the study. Fasting blood samples were obtained after a 5-day washout of DFO prior to commencing treatment with study drug, and 24 hours post-chelator at 1, 6, and 12 months on study. Thirty healthy controls matched for age, sex, race and antioxidant usage (15 male, mean age 24.5 ± 9 yrs) also supplied a blood sampl...

Sickle cell disease (SCD) is a hemoglobinopathy characterized by micro-vascular hypoxia-reperfusion, inflammation and leukocytosis. Studies in SCD have shown that leukocytosis is a strong predictor of stroke and disease severity. It is known that leukocytosis and inflammation contribute to increased leukocyte-endothelial adhesion and vasoocclusive events. Leukocytosis or increased leukocyte number is determined by the balance between cell death programs (apoptosis) and proliferation. In this study, we examine markers of apoptosis and proliferation in SCD as compared to thalassemia (a hemoglobinopathy that is vasculitis negative) and a control group. Methods: Markers of leukocyte turnover, inflammation and free iron (NTBI, non-transferrin bound iron) were compared in 11 patients with SCD (7M, 13 ± 4 yrs), 18 with thalassemia (7M, 24 ± 9 yrs) and 10 disease-free controls (5M, 27 ± 12 yrs). All SCD and thalassemia patients were healthy and event free in the previous 4 months. Blood was...

Objective: We examined whether oxidant-stress and inflammation in β-thalassemia can be controlled by deferasirox as effectively as by deferoxamine (DFO) through analysis of body iron burden and biomarkers of lipid peroxidation and inflammation. Methods: Thalassemia Clinical Research Network patients participating in the Novartis CICL670A0107 trial (a randomized comparison of deferasirox, an oral iron chelator, vs. DFO) were eligible and 44 (25 male, 21.8±11.1 yrs) were enrolled in the study. Blood samples were obtained fasting after a 5-day washout of DFO prior to commencing treatment with study drug, and 24 hours post-chelator and antioxidant supplementation at 1, 6, and 12 months on study. Thirty healthy untreated controls matched for age, sex, and race (15 male, 24.5±9 yrs) were also studied. Plasma levels of malondialdehyde (MDA), a marker of lipid peroxidation, were determined by GC-MS; vitamin C by spectrophotometry; α-tocopherol by HPLC; and high-sensitivity C-reactive protei...

Objective: Chronic red blood cell transfusion therapy is life-saving for patients with β-thalassemia (THL) and sickle cell disease (SCD), but often results in severe iron overload. Clinical observations suggest that organ dysfunction (heart, liver, endocrine dysfunction, bone disease) resulting from iron overload is seen more often in patients with THL than SCD. This study examines the possible correlation between increased organ injury in these patients and oxidative stress, iron metabolism, inflammation and plasma vitamin E. Methods: Markers of oxidant stress were compared in 18 subjects with THL (7M, 24 ± 9 yrs) and 11 with SCD (7M, 13 ± 4 yrs) with 10 disease-free controls (5M, 27 ± 12 yrs). All THL and SCD patients admitted to the study were healthy and had not had a recent medical event including vascular crises for SCD within the last 4 months. Blood was drawn from fasted subjects, prior to blood transfusion, and plasma, serum and cells were separated by centrifugation. Plasm...

Introduction Complications of transfusional iron overload vary with the underlying hematologic condition and may be affected by inherent differences in non-transferrin bound iron (NTBI) generation between these conditions. We have previously reported high NTBI levels in Diamond Blackfan anemia (DBA) relative to sickle cell (SCD), despite lower serum ferritin and LIC associated with very low sTfR levels in DBA. This suggested that low transferrin iron utilization in DBA relative to SCD and thalassemia major (TM) accounted for high NTBI levels. It is known that pro-inflammatory cytokine IL6 will increase hepcidin synthesis thereby decreasing ferroportin expression on macrophages but it is not known whether high levels of inflammatory cytokines can overcome or limit the effects of iron overload in raising NTBI and transferrin saturation. Here we have examined additional factors that may affect NTBI levels in different forms of inherited anemias. The relationship of inflammatory markers...

995 Background: β-thalassemia major (TM) is the paradigm for chronic transfusional iron overload, in which the extra-hepatic organ failure is best described. In Sickle Cell Disease (SCD), these consequences appear later and at a lower frequency. In chronically transfused Diamond Blackfan Anemia (DBA), extra-hepatic iron overload, although less well documented, appears to occur early and at high frequency. A Multicenter Study of Iron Overload (MCSIO) aims to explore how key candidate factors affect iron distribution; including inflammation, ineffective erythropoiesis, level of iron overload, and hepcidin synthesis. Plasma non-transferrin bound iron (NTBI) could be a key mechanism by which iron is delivered to tissues and may determine the propensity for extra-hepatic iron distribution. Here we focus on how markers of ineffective erythropoiesis (IE) and erythron expansion impact iron distribution, with particular reference to NTBI and iron distribution determined by MRI. Methods: Iron...

Introduction Complications of transfusional iron overload vary with the underlying hematologic condition and may be affected by inherent differences in non-transferrin bound iron (NTBI) generation between these conditions. We have previously reported high NTBI levels in Diamond Blackfan anemia (DBA) relative to sickle cell (SCD), despite lower serum ferritin and LIC associated with very low sTfR levels in DBA. This suggested that low transferrin iron utilization in DBA relative to SCD and thalassemia major (TM) accounted for high NTBI levels. It is known that pro-inflammatory cytokine IL6 will increase hepcidin synthesis thereby decreasing ferroportin expression on macrophages but it is not known whether high levels of inflammatory cytokines can overcome or limit the effects of iron overload in raising NTBI and transferrin saturation. Here we have examined additional factors that may affect NTBI levels in different forms of inherited anemias. The relationship of inflammatory markers, the hepcidin/ferritin ratio and monocyte ferroportin in these conditions to NTBI levels have been examined. The possible effect of vitamin C or vitamin D deficiency on NTBI has also been examined. Methods 15 iron-overloaded patients (5 from each group of TM, SCD, and DBA) with ferritin…

Abstract 995 Background: β-thalassemia major (TM) is the paradigm for chronic transfusional iron overload, in which the extra-hepatic organ failure is best described. In Sickle Cell Disease (SCD), these consequences appear later and at a lower frequency. In chronically transfused Diamond Blackfan Anemia (DBA), extra-hepatic iron overload, although less well documented, appears to occur early and at high frequency. A Multicenter Study of Iron Overload (MCSIO) aims to explore how key candidate factors affect iron distribution; including inflammation, ineffective erythropoiesis, level of iron overload, and hepcidin synthesis. Plasma non-transferrin bound iron (NTBI) could be a key mechanism by which iron is delivered to tissues and may determine the propensity for extra-hepatic iron distribution. Here we focus on how markers of ineffective erythropoiesis (IE) and erythron expansion impact iron distribution, with particular reference to NTBI and iron distribution determined by MRI. Methods: Iron-overloaded patients (5 TM, 5 SCD, and 5 DBA) with ferritin > 1500 g/dl or LIC > 7 mg/g dry wt, age ≥16, age 0 to 9 at initiation of transfusion and 10 to 20 years of transfusion exposure were enrolled from 3 sites in the US and Europe. 5 non-transfused healthy controls were also enrolled. A detailed medical, transfusion and chelation history were obtained with standardized MRI evaluations for hepatic, cardiac, and pituitary iron deposition. Fasting, early morning blood samples were obtained one day prior to transfusion. Chelation was held for 72 hours prior to each sample. Results: Results are shown in the table as median values. DBA patients had the highest NTBI prior to transfusion despite having the lowest ferritin and LIC levels. GDF15 levels were highest in TM, with similar levels in SCD and DBA. EPO levels were nearly two orders of magnitude higher in DBA than TM or SCD. DBA patients also had the highest median cardiac R2*; two patients showing values above the control range. Whereas the median pituitary R2 in DBA was not above control, two of the patients had the highest R2 values, suggesting heavy iron deposition. EPO values in DBA are nearly two orders of magnitude higher that in SCD or TM despite similar pre-transfusion Hb values. GDF15 values are approximately three times controls, while soluble transferrin receptors (sTfR) values are almost undetectable. With SCD, no patients had increased cardiac iron loading, despite median SF and LIC being the highest in this group. Surprisingly all SCD patients had pituitary R2 values above the upper limit of normal. 1 TM patient had increased cardiac R2* whereas three had increased pituitary iron. In TM, NTBI was strongly correlated with GDF15 (Pearson's Rho=0.93) but in DBA, GDF15 was inversely correlated with NTBI (-.95). Conclusions: High GDF15 levels have been reported in conditions associated with IE, such as TM, but not in DBA. GDF15 reputedly suppresses hepcidin synthesis, thereby increasing iron absorption and potentially NTBI levels. The increased GDF15 in DBA, while sTfr remain less than controls, suggests that erythropoietic precursors do not reach the stage where sTfr are expressed and that this occurs at a later differentiation stage than GDF15. Increasing NTBI in TM with increasing GDF15 is consistent with IE contributing to NTBI formation, but the lack of this relationship in DBA suggest another mechanism for high NTBI. As the erythron is destroyed at a pre-hemoglobinised stage in DBA, IE would not contribute directly to NTBI formation. However, the extremely high EPO levels in DBA may inhibit hepcidin synthesis, as in other conditions, thereby increasing NTBI. This in turn may account for the extra-hepatic iron distribution demonstrated by MRI in DBA. The increased pituitary iron without cardiac loading in the heavily loaded SCD patients suggests that with prolonged exposure to heavy iron overload, the pituitary iron loading may be the first indicator of extra-hepatic deposition. Disclosures: Porter: Novartis: Consultancy, Research Funding. Walter:Novartis: Research Funding. Harmatz:Novartis: Research Funding; Ferrokin: Research Funding. Wood:Ferrokin Biosciences: Consultancy; Shire:…

ABSTRACT Mucopolysaccharidosis IVA (MPSIVA) is a recessive lysosomal storage disease characterized by a deficiency in N-acetylgalactosamine-6-sulfate sulfatase (GALNS), the enzyme partially responsible for the catabolism of keratan sulfate (KS) and chondroitin-6-sulfate (C6S). Point mutations in GALNS cause misfolding, targeting the enzyme for degradation by the proteosome before its physiological role can be fulfilled. Pharmacological chaperones and antioxidants have been shown to enhance enzyme activity and prevent protein misfolding both in vitro and in vivo in some patients with other lysosomal storage disorders, such as Gauchers disease. Methods: Using an in vitro model analyzing enzyme activity in MPSIVA fibroblasts and the monocytic cell line THP-1, we are examining enzyme enhancement therapy using the chaperones 1-deoxygalactonojirimycin (DGJ) and galactose-6-sulfate (G6S). These chaperones are competitive inhibitors that bind directly to the active site of possibly misfolded GALNS, thereby increasing the stability and allowing it to evade proteosome destruction. We also investigated the use of the antioxidant glutathione (GSH) and N-acetylcysteine (NAC, GSH precursor) to enhance the activity of GALNS by maintaining disulfide bridges and stabilizing quaternary structure. GALNS activity was measured after culture in either of DGJ, G6S, GSH, NAC and/or buthionine sulfoximine (BSO, induces GSH depletion). Additional work examined the effects of the variation of KS and CS on GALNS activity. Analysis of fibroblasts from MPSIVA and control subjects is ongoing. Results: In THP-1 cells, 10 μM DGJ induced a 124% increase of GALNS activity (p<0.05) compared to controls without DGJ. Similarly, 5 mM GSH supplementation increased GALNS activity 91% (p<0.5). Low doses of NAC also stimulated activity while 5mM BSO decreased activity 2.7 fold (p<0.05). A positive correlation was observed when cells were exposed to increasing doses of KS and C6S levels and enzyme activity. This work supports the possible usage of chaperones and/or glutathione enhancement in restoring enzyme activity possibly through maintenance of cellular redox balance or maintenance of enzyme structure.

ABSTRACT MRM proteomics was used to investigate the systemic state of iron trafficking, OS and inflammation in an NBIA patient with PLA2G6 Associated Neurodegeneration (PLAN). Patients with PLAN show progressive Parkinsonism and dystonia and are characterized by abnormal iron accumulation and increased OS and inflammation in the Substantia Nigra (SN).Plasma samples were obtained from a patient with a heterozygous mutation in the PLA2G6 gene and a matched control. MRM is a mass spectrometry technique that can be applied to accurately measure a large number of target proteins in a sample such as plasma. For this pilot trial we used MRM to measure proteins of inflammation (Lipocalin-2), OS (glutathione peroxidase (GP)) and iron trafficking (hemopexin, ceruloplasmin, transferrin(Tf) and soluble transferrin receptor(STf)). Additional measurements of the OS markers malondialdehyde (MDA) and 4-Hydroxynonenal (4-HNE) and the inflammatory markers IL-6 and c-reactive protein (CRP) were also performed. Proteomic analysis revealed that the PLAN patient had a 129% increase in the expression of the heme trafficking proteins haptoglobin and 39% increase in the ferroxidase ceruloplasmin relative to a healthy control. Tf expression was decreased by 26%, but STf expression increased 47% relative to the control. A marker of inflammation, Lipocalin-2 was increased 62% and the antioxidant protein GP was increased by 14% relative to the control. These proteomic changes in OS and inflammation were confirmed by a 78% increase in MDA and a 4-fold increase in 4-HNE and IL-6 compared to that of the control as well as a CRP of 4.0. MRM proteomic analysis showed changes in the expression of iron trafficking proteins and increased systemic OS and inflammation in the patient. This preliminary pilot trial showed that MRM has the potential to identify disease biomarkers in NBIA patients. Expanded biomarker analysis of NBIA patients using MRM is ongoing.

Plasma and urinary levels of malondialdehyde-like products (MDA) and isoprostanes were identified as markers of in vivo lipid peroxidation in an animal model of CCl4 poisoning. We sought to determine the extent to which the formation of these oxidation products is influenced by inhibition of the cyclooxygenase enzymes which catalytically generate proinflammatory lipid peroxidation products known as prostaglandins and thromboxane. In the present studies, after induction of oxidant stress in rats with CCl4, lipid peroxidation products measured in plasma and urine demonstrate that isoprostanes and MDA can be partially inhibited by cyclooxygenase inhibitors, albeit to different extents. The lowering of isoprostane and MDA formation, however, may not to due primarily to the diminution of catalytic generation of isoprostanes or MDA by the cyclooxygenases but, rather, may be the result of the suppression of nonenzymatic lipid peroxidation. This is suggested since 8,12-iso-iPF2alpha-VI is also reduced by indomethacin, yet, unlike other isoprostanes and MDA, it is not generated catalytically by the cyclooxygenase. Thus, although the two cyclooxygenase inhibitors we tested have statistically significant effects on the measurements of both isoprostanes and MDA in this study, the results provide evidence that these lipid-degradation products primarily constitute markers of oxidative stress.

The aim of this study was to determine whether women with low iron stores (plasma ferritin ≤ 20 μg/L) receiving a daily iron supplement for 8 wks at a level commonly used to treat poor iron status develop increased lipid peroxidation as measured by ethane exhalation rates and plasma malondialdehyde. The women served as their own control as pre- and post-supplementation periods were compared. Twelve women participated in the study for a 70-day period and consumed daily iron supplements (98 mg of iron as ferrous sulfate) from day 14 to day 70. Baseline blood and expired air samples were obtained on days 1 and 14; measurements during supplementation were performed on days 56 and 70, that is at 6 and 8 weeks of supplementation. Iron status improved during the iron supplementation period; biochemical indicators of lipid peroxidation also increased. After 6 wks of iron supplementation, serum ferritin almost doubled and body iron more than doubled. Hemoglobin levels increased slightly and ...

The aim of the present work was to modulate in vivo the level of hepatic mitochondrial glutathione (GSH). Rats were given phorone (diisopropylidene acetone), which in vivo becomes enzymatically conjugated to GSH, and were subsequently treated with N-acetylcysteine (NAC) to rescue GSH. In liver homogenate, a rapid and biphasic (T1/2 less than or equal to 15 min and 1.5 hr) drop of GSH was observed upon phorone administration. NAC treatment led to a restoration (T1/2 about 1 hr) of GSH in the homogenate above control values within 3 hr. The mitochondrial GSH level decreased with T1/2 of about 1.5 hr upon phorone treatment, and was 75% restored by NAC treatment within 3 hr. Hydroperoxide-induced mitochondrial pyridine nucleotide oxidation and Ca2+ release were impeded in GSH-depleted organelles, and NAC treatment restored these processes. The GSH status had no influence on mitochondrial pyridine nucleotide oxidation and Ca2+ released induced by alloxan, which reacts directly and non-enzymatically with pyridine nucleotides. It is concluded that NAC is able to rescue mitochondrial GSH in vivo and restore important mitochondrial functions. The data suggest that NAC may be a useful antidote in oxidative stress-related diseases.

Oxidative DNA damage is important in aging and the degenerative diseases of aging such as cancer. Estimates commonly rely on measurements of 8-oxo-2′-deoxyguanosine (oxo 8 dG), an adduct that occurs in DNA and is also excreted in urine after DNA repair. Here we ...

Objective: We examined whether oxidant-stress and inflammation in β-thalassemia can be controlled by deferasirox as effectively as by deferoxamine (DFO) through analysis of body iron burden and biomarkers of lipid peroxidation and inflammation. Methods: Thalassemia Clinical ...

In transfusional iron overload, extra-hepatic iron distribution differs, depending on the underlying condition. Relative mechanisms of plasma non-transferrin bound iron (NTBI) generation may account for these differences. Markers of iron metabolism (plasma NTBI, labile iron, hepcidin, transferrin, monocyte SLC40A1 [ferroportin]), erythropoiesis (growth differentiation factor 15, soluble transferrin receptor) and tissue hypoxia (erythropoietin) were compared in patients with Thalassaemia Major (TM), Sickle Cell Disease and Diamond-Blackfan Anaemia (DBA), with matched transfusion histories. The most striking differences between these conditions were relationships of NTBI to erythropoietic markers, leading us to propose three mechanisms of NTBI generation: iron overload (all), ineffective erythropoiesis (predominantly TM) and low transferrin-iron utilization (DBA).

Blood transfusion therapy is life-saving for patients with β-thalassaemia and sickle cell disease (SCD), but often results in severe iron overload. This pilot study examined whether the biomarkers of tissue injury or inflammation differ in these two diseases. Plasma malondialdehyde (MDA) was significantly increased 1·8-fold in thalassaemia relative to control patients. In contrast, MDA in SCD was not significantly different from controls. In multivariate analysis, the strongest predictors of elevated MDA were liver iron concentration (P < 0·001) and specific diagnosis (P = 0·019). A significant 2-fold elevation of non-transferrin bound iron (NTBI) was observed in thalassaemia relative to SCD. NTBI was not a significant predictor of high MDA in multivariate analysis. SCD patients showed a significant 2·2-fold elevation of the inflammatory marker interleukin (IL)-6 relative to controls, and a 3·6- and 1·7-fold increase in IL-5 and IL-10 relative to thalassaemia. Although α-tocopherol was significantly decreased by at least 32% in both thalassaemia and SCD, indicating ongoing oxidant stress and antioxidant consumption, γ-tocopherol, a nitric oxide-selective antioxidant, was increased 36% in SCD relative to thalassaemia. These results demonstrate that thalassaemia patients have increased MDA and circulating NTBI relative to SCD patients and lower levels of some cytokines and γ-tocopherol. This supports the hypothesis that the biology of SCD may show increased inflammation and increased levels of protective antioxidants compared with thalassaemia.