Higher CSF Ferritin Heavy-Chain (Fth1) and Transferrin Predict Better Neurocognitive Performance in People with HIV.

HIV-associated neurocognitive disorder (HAND) remains prevalent despite antiretroviral therapy and involves white matter damage in the brain. Although iron is essential for myelination and myelin maintenance/repair, its role in HAND is largely unexplored. We tested the hypotheses that cerebrospinal fluid (CSF) heavy-chain ferritin (Fth1) and transferrin, proteins integral to iron delivery and myelination, are associated with neurocognitive performance in people with HIV (PWH). Fth1, transferrin, and the pro-inflammatory cytokines TNF-α and IL-6 were quantified in CSF at baseline (entry) in 403 PWH from a prospective observational study who underwent serial, comprehensive neurocognitive assessments. Associations of Fth1 and transferrin with Global Deficit Score (GDS)-defined neurocognitive performance at baseline and 30-42 months of follow-up were evaluated by multivariable regression. While not associated with neurocognitive performance at baseline, higher baseline CSF Fth1 predicted significantly better neurocognitive performance over 30 months in all PWH (p < 0.05), in PWH aged < 50 at 30, 36, and 42 months (all p < 0.05), and in virally suppressed PWH at all three visit time-points (all p < 0.01). Higher CSF transferrin was associated with superior neurocognitive performance at all visits, primarily in viremic individuals (all p < 0.05). All associations persisted after adjustment for neuro-inflammation. In summary, higher CSF Fth1 is neuroprotective over prolonged follow-up in all and virally suppressed PWH, while higher CSF transferrin may be most neuroprotective during viremia. We speculate that higher CSF levels of these critical iron-delivery proteins support improved myelination and consequently, neurocognitive performance in PWH, providing a rationale for investigating their role in interventions to prevent and/or treat HAND.

A potential role for zinc in restless legs syndrome.

STUDY OBJECTIVES: Evaluate serum and brain noniron metals in the pathology and genetics of restless legs syndrome (RLS). METHODS: In two independent studies (cohorts 1 and 2), in which subjects either remained on medications or tapered off medications, we analyzed serum levels of iron, calcium, magnesium, manganese, copper, and zinc both in RLS patients and controls, and assessed the prevalence of the MEIS1 and BTBD9 risk alleles previously established through genome-wide association studies. Human brain sections and a nematode genetic model were also quantified for metal levels using mass spectrometry. RESULTS: We found a significant enrichment for the BTBD9 risk genotype in the RLS affected group compared to control (p = 0.0252), consistent with previous literature. Serum (p = 0.0458 and p = 0.0139 for study cohorts 1 and 2, respectively) and brain (p = 0.0413) zinc levels were significantly elevated in the RLS patients versus control subjects. CONCLUSION: We show for the first time that serum and brain levels of zinc are elevated in RLS. Further, we confirm the BTBD9 genetic risk factor in a new population, although the zinc changes were not significantly associated with risk genotypes. Zinc and iron homeostasis are interrelated, and zinc biology impacts neurotransmitter systems previously linked to RLS. Given the modest albeit statistically significant increase in serum zinc of ~20%, and the lack of association with two known genetic risk factors, zinc may not represent a primary etiology for the syndrome. Further investigation into the pathogenetic role that zinc may play in restless legs syndrome is needed.

Evidence for communication of peripheral iron status to cerebrospinal fluid: clinical implications for therapeutic strategy.

BACKGROUND: Iron is crucial for proper functioning of all organs including the brain. Deficiencies and excess of iron are common and contribute to substantial morbidity and mortality. Whereas iron's involvement in erythropoiesis drives clinical practice, the guidelines informing interventional strategies for iron repletion in neurological disorders are poorly defined. The objective of this study was to determine if peripheral iron status is communicated to the brain. METHODS: We used a bi-chamber cell culture model of the blood-brain-barrier to determine transcytosis of iron delivered by transferrin as a metric of iron transport. In the apical chamber (representative of the blood) we placed transferrin complexed with iron59 and in the basal chamber (representative of the brain) we placed human cerebrospinal fluid. Cerebrospinal fluid (CSF) samples (N = 24) were collected via lumbar puncture. The integrity of the tight junctions were monitored throughout the experiments using RITC-Dextran. RESULTS: We demonstrate that iron transport correlates positively with plasma hemoglobin concentrations but not serum ferritin levels. CONCLUSIONS: The clinical ramifications of these findings are several- fold. They suggest that erythropoietic demands for iron take precedence over brain requirements, and that the metric traditionally considered to be the most specific test reflecting total body iron stores and relied upon to inform treatment decisions-i.e., serum ferritin-may not be the preferred peripheral indicator when attempting to promote brain iron uptake. The future direction of this line of investigation is to identify the factor(s) in the CSF that influence iron transport at the level of the BBB.

Effects of plus-maze experience and chlordiazepoxide on anxiety-like behavior and serotonin neural activity in the dorsal raphe nucleus in rats.

The extent to which rats express anxiety-like behavior on the elevated plus-maze (EPM) depends on their previous maze experience. Open-arm avoidance develops in maze-experienced rats, and is often accompanied by a diminished anxiolytic response to benzodiazepines. Regions of the dorsal raphe nucleus (DRN) were examined in male Sprague-Dawley rats using c-Fos and serotonin immunohistochemistry following a single exposure, a second exposure or no exposure to the EPM. We then examined the effect of the benzodiazepine anxiolytic chlordiazepoxide (CDP, 5 mg/kg) on EPM behavior and DRN neural activity. Enhanced open-arm avoidance was evident on the second EPM trial in both experiments. The observed pattern of c-Fos expression suggests that the first exposure to the maze activates serotonin cells in the rostral and dorsal regions of the DRN and that only the dorsal subregion is activated by a second exposure. CDP increased open-arm exploration during the first trial, which corresponded to decreased 5-hydroxytryptamine (5-HT) activity in the rostral and ventral subregions of the DRN. However, 5-HT activity in the DRN was reduced in rats on the second maze trial compared with the first trial, when CDP had no effect on open-arm exploration. These results suggest that open-arm avoidance in maze-experienced rats can be characterized as a coping response that is mediated by specific populations of 5-HT neurons in the DRN.

Comorbidities, treatment, and pathophysiology in restless legs syndrome.

Restless legs syndrome, also known as Willis-Ekbom disease, is a common neurological condition whose manifestation is affected by complex environmental and genetic interactions. Restless legs syndrome can occur on its own, mostly at a young age, or with comorbidities such as cardiovascular disease, diabetes, and arterial hypertension, making it a difficult condition to properly diagnose. However, the concept of restless legs syndrome as being two entities, primary or secondary to another condition, has been challenged with genetic data providing further insight into the pathophysiology of the condition. Although dopaminergic treatment was formerly the first-line therapy, prolonged use can result in a serious worsening of symptoms known as augmentation. Clinical studies on pregabalin, gabapentin enacarbil, oxycodone-naloxone, and iron preparations have provided new treatment options, but most patients still report inadequate long-term management of symptoms. Studies of the hypoxic pathway activation and iron deficiency have provided valuable information about the pathophysiology of restless legs syndrome that should now be translated into new, more effective treatments for restless legs syndrome.

Quantifying impacts of white-tailed deer (Odocoileus virginianus Zimmerman) browse using forest inventory and socio-environmental datasets.

Elevated population levels of white-tailed deer (Odocoileus virginianus Zimmerman) can drastically alter forest ecosystems and negatively impact society through human interactions such as deer vehicle collisions. It is currently difficult to estimate deer populations at multiple scales ranging from stand, county, state, and regional levels. This presents a challenge as natural resource managers develop silvicultural prescriptions and forest management practices aimed at successfully regenerating tree species in the face of deer browsing. This study utilized measurements of deer browse impact from the new tree regeneration indicator developed by the United States Department of Agriculture Forest Service Forest Inventory and Analysis (FIA) program. Seedling and sapling abundance and other plot-level characteristics were analyzed across three states (Michigan, Minnesota, and Wisconsin) in the Great Lakes Region of the United States. Socio-environmental datasets (Lyme disease cases, deer vehicle collisions, and deer density estimates) were used in conjunction with FIA data to determine their predictive power in estimating deer browse impacts by county. Predictions from random forests models indicate that using Lyme disease case reports, the number of deer-vehicle collisions, deer density estimates, and forest inventory information correctly predicted deer browse impact 70-90% of the time. Deer-vehicle collisions per county ranked highly important in the random forests for predicting deer browse impacts in all three states. Lyme disease cases ranked high in importance for the Lake States combined and for Minnesota and Wisconsin, separately. Results show the effectiveness of predicting deer browse impacts using a suite of freely available forest inventory and other socio-environmental information.

Cerebrospinal fluid (CSF) biomarkers of iron status are associated with CSF viral load, antiretroviral therapy, and demographic factors in HIV-infected adults.

BACKGROUND: HIV-associated neurocognitive disorder (HAND) remains common, despite antiretroviral therapy (ART). HIV dysregulates iron metabolism, but cerebrospinal fluid (CSF) levels of iron and iron-transport proteins in HIV-infected (HIV+) persons are largely unknown. The objectives of this study were to characterize CSF iron-related biomarkers in HIV+ adults and explore their relationships to known predictors of HAND. METHODS: We quantified total iron, transferrin and heavy-chain (H)-ferritin by immunoassay in CSF sampled by lumbar puncture in 403 HIV+ participants in a multi-center, observational study and evaluated biomarker associations with demographic and HIV-related correlates of HAND [e.g., age, sex, self-reported race/ethnicity, ART, and detectable plasma virus and CSF viral load (VL)] by multivariable regression. In a subset (N = 110) with existing CSF: serum albumin (QAlb) measurements, QAlb and comorbidity severity were also included as covariates to account for variability in the blood-CSF-barrier. RESULTS: Among 403 individuals (median age 43 years, 19% women, 56% non-Whites, median nadir CD4+ T cell count 180 cells/µL, 46% with undetectable plasma virus), men had 25% higher CSF transferrin (median 18.1 vs. 14.5 µg/mL), and 71% higher H-ferritin (median 2.9 vs. 1.7 ng/mL) than women (both p-values ≤0.01). CSF iron was 41% higher in self-reported Hispanics and 27% higher in (non-Hispanic) Whites than in (non-Hispanic) Blacks (median 5.2 and 4.7 µg/dL in Hispanics and Whites, respectively, vs. 3.7 µg/dL in Blacks, both p ≤ 0.01); these findings persisted after adjustment for age, sex, and HIV-specific factors. Median H-ferritin was 25% higher (p < 0.05), and transferrin 14% higher (p = 0.06), in Whites than Blacks. Transferrin and H-ferritin were 33 and 50% higher, respectively, in older (age > 50 years) than in younger persons (age ≤ 35 years; both p < 0.01), but these findings lost statistical significance in subset analyses that adjusted for QAlb and comorbidity. After these additional adjustments, associations were observed for CSF iron and transferrin with race/ethnicity as well as CSF VL, for transferrin with sex and ART, and for H-ferritin with plasma virus detectability and significant comorbidity (all p < 0.05). CONCLUSIONS: CSF iron biomarkers are associated with demographic factors, ART, and CSF VL in HIV+ adults. Future studies should investigate a role for CNS iron dysregulation, to which an altered blood-CSF barrier may contribute, in HAND.

Cerebrospinal fluid cell-free mitochondrial DNA is associated with HIV replication, iron transport, and mild HIV-associated neurocognitive impairment.

BACKGROUND: Mitochondria are abundant organelles critical for energy metabolism and brain function. Mitochondrial DNA (mtDNA), released during cellular injury and as part of the innate immune response to viral pathogens, contains CpG motifs that act as TLR-9 ligands. We investigated relationships between cerebrospinal fluid (CSF) cell-free mtDNA levels and HIV viral load (VL), biomarkers of inflammation and iron transport, and neurocognitive (NC) function in the CNS HIV Antiretroviral Therapy Effects Research (CHARTER) cohort. METHODS: We quantified cell-free mtDNA in CSF by droplet digital PCR in 332 CHARTER participants who underwent comprehensive neuropsychiatric evaluation. NC performance was assessed using the global deficit score (GDS) as either a continuous or a binary measure (GDS ≥ 0.5, impaired vs. GDS < 0.5, unimpaired). CSF, clinical, and biomarker data from the earliest available time point were analyzed. Cell-free mtDNA associations with CSF inflammation and iron-related biomarkers [CXCL10, IL-6, IL-8, TNF-a, transferrin (TF), ceruloplasmin (CP), and vascular endothelial growth factor (VEGF)], VL, and GDS were evaluated by multivariable regression. RESULTS: CSF cell-free mtDNA levels were significantly lower in participants with undetectable (vs. detectable) VL in either plasma (p < 0.001) or CSF (p < 0.001) and in those on antiretroviral therapy (ART; p < 0.001). Participants on ART with undetectable VL in both CSF and plasma had lower mtDNA levels than those with detectable VL in both compartments (p = 0.001). Higher mtDNA levels were observed in participants in the highest vs. lowest tertile (T3 vs. T1) of CSF CXCL10 (T3 vs. T1, p < 0.001) and TNF-a (T3 vs. T1, p < 0.05) in unadjusted analyses. MtDNA levels also correlated with CSF leukocyte count. After adjusting for CSF leukocyte count and VL, mtDNA levels were also associated with other inflammation- and iron-related biomarkers in CSF, including TF (T3 vs. T1, p < 0.05) and CP (T3 vs. T1, p < 0.05). With additional correction for ART use, mtDNA was also negatively associated with CSF VEGF (p < 0.05) and IL-6 (p = 0.05). We observed no associations of CSF mtDNA levels with age or GDS-defined NC impairment. CONCLUSIONS: CSF cell-free mtDNA levels were associated with HIV RNA and ART status, as well as with biomarkers of iron transport and VEGF, a growth factor with known effects on mitochondrial integrity and autophagy. CSF mtDNA may be a biomarker of iron dysregulation and/or neuroinflammation during HIV infection.

Iron and restless legs syndrome: treatment, genetics and pathophysiology.

In this article, we review the original findings from MRI and autopsy studies that demonstrated brain iron status is insufficient in individuals with restless legs syndrome (RLS). The concept of deficient brain iron status is supported by proteomic studies from cerebrospinal fluid (CSF) and from the clinical findings where intervention with iron, either dietary or intravenous, can improve RLS symptoms. Therefore, we include a section on peripheral iron status and how peripheral status may influence both the RLS symptoms and treatment strategy. Given the impact of iron in RLS, we have evaluated genetic data to determine if genes are directly involved in iron regulatory pathways. The result was negative. In fact, even the HFE mutation C282Y could not be shown to have a protective effect. Lastly, a consistent finding in conditions of low iron is increased expression of proteins in the hypoxia pathway. Although there is lack of clinical data that RLS patients are hypoxic, there are intriguing observations that environmental hypoxic conditions worsen RLS symptoms; in this chapter we review very compelling data for activation of hypoxic pathways in the brain in RLS patients. In general, the data in RLS point to a pathophysiology that involves decreased acquisition of iron by cells in the brain. Whether the decreased ability is genetically driven, activation of pathways (eg, hypoxia) that are designed to limit cellular uptake is unknown at this time; however, the data strongly support a functional rather than structural defect in RLS, suggesting that an effective treatment is possible.

Proteomic analysis of the cerebrospinal fluid of patients with restless legs syndrome/Willis-Ekbom disease.

BACKGROUND: Restless Legs Syndrome/Willis-Ekbom Disease (RLS/WED) is a sensorimotor disorder that causes patients to experience overwhelming and distressing sensations in the legs compelling the patient to move their legs to provide relief. The purpose of this study was to determine if biomarkers in the cerebrospinal fluid can distinguish RLS/WED patients from neurological controls. METHODS: We obtained CSF samples by lumbar puncture from 5 early-onset RLS/WED patients and 5 controls. We performed 2-dimensional difference in-gel electrophoresis (2D-DIGE). Proteins that were significantly altered were identified by Student's t-test. Protein spots that were differentially expressed (p ≤ 0.05, Av. Ratio ≥ 2.0) between RLS/WED and control CSF samples were identified using MALDI-TOF-MS. Statistical analyses of the validation immunoblot assays were performed using Student's t-test. RESULTS: In this discovery study we identified 6 candidate CSF protein markers for early-onset RLS/WED. Four proteins (Cystatin C, Lipocalin-type Prostaglandin D2 Synthase, Vitamin D binding Protein, and ß-Hemoglobin) were increased and 2 proteins (Apolipoprotein A1 and α-1-acid Glycoprotein) were decreased in RLS/WED patients. CONCLUSIONS: Our results reveal a protein profile in the RLS/WED CSF that is consistent with clinical findings of disruptive sleep, cardiovascular dysfunction and painful symptoms. Moreover, protein profiles are consistent with neuropathological findings of activation of hypoxia inducible factor (HIF) pathways and alterations in dopaminergic systems. These data indicate the CSF of RLS/WED patients may provide information relevant to biological basis for RLS/WED, treatment strategies and potential new treatment targets.

Quantitative proteomic analyses of cerebrospinal fluid using iTRAQ in a primate model of iron deficiency anemia.

Iron deficiency affects nearly 2 billion people worldwide, with pregnant women and young children being most severely impacted. Sustained anemia during the first year of life can cause cognitive, attention and motor deficits, which may persist despite iron supplementation. We conducted iTRAQ analyses on cerebrospinal fluid (CSF) from infant monkeys (Macaca mulatta) to identify differential protein expression associated with early iron deficiency. CSF was collected from 5 iron-sufficient and 8 iron-deficient anemic monkeys at weaning age (6-7 months) and again at 12-14 months. Despite consumption of iron-fortified food after weaning, which restored hematological indices into the normal range, expression of 5 proteins in the CSF remained altered. Most of the proteins identified are involved in neurite outgrowth, migration or synapse formation. The results reveal novel ways in which iron deficiency undermines brain growth and results in aberrant neuronal migration and connections. Taken together with gene expression data from rodent models of iron deficiency, we conclude that significant alterations in neuroconnectivity occur in the iron-deficient brain, which may persist even after resolution of the hematological anemia. The compromised brain infrastructure could account for observations of behavioral deficits in children during and after the period of anemia.

Profile of altered brain iron acquisition in restless legs syndrome.

Restless legs syndrome is a neurological disorder characterized by an urgency to move the legs during periods of rest. Data from a variety of sources provide a compelling argument that the amount of iron in the brain is lower in individuals with restless legs syndrome compared with neurologically normal individuals. Moreover, a significant percentage of patients with restless legs syndrome are responsive to intravenous iron therapy. The mechanism underlying the decreased iron concentrations in restless legs syndrome brains is unknown. We hypothesize that the source of the brain iron deficit is at the blood-brain interface. Thus we analysed the expression of iron management proteins in the epithelial cells of the choroid plexus and the brain microvasculature in post-mortem tissues. The choroid plexus, obtained at autopsy, from 18 neurologically normal controls and 14 individuals who had primary restless legs syndrome was subjected to histochemical staining for iron and immunostaining for iron management proteins. Iron and heavy chain ferritin staining was reduced in the epithelial cells of choroid plexus in restless legs syndrome. Divalent metal transporter, ferroportin, transferrin and its receptor were upregulated in the choroid plexus in restless legs syndrome. Microvessels were isolated from the motor cortex of 11 restless legs syndrome and 14 control brains obtained at autopsy and quantitative immunoblot analyses was performed. Expression of heavy chain ferritin, transferrin and its receptor in the microvessels from restless legs syndrome was significantly decreased compared with the controls but divalent metal protein 1, ferroportin, prohepcidin, mitochondrial ferritin and light-chain ferritin remained unchanged. The presence of an iron regulatory protein was demonstrated in the brain microvasculature and the activity of this protein is decreased in restless legs syndrome; a finding similar to our earlier report in neuromelanin cells from the substantia nigra of restless legs syndrome brains. This study reveals that there are alterations in the iron management protein profile in restless legs syndrome compared with controls at the site of blood-brain interface suggesting fundamental differences in brain iron acquisition in individuals with restless legs syndrome. Furthermore, the decrease in transferrin receptor expression in the microvasculature in the presence of relative brain iron deficiency reported in restless legs syndrome brains may underlie the problems associated with brain iron acquisition in restless legs syndrome. The consistent finding of loss of iron regulatory protein activity in restless legs syndrome brain tissue further implicates this protein as a factor in the underlying cause of the iron deficiency in the restless legs syndrome brain. The data herein provide evidence for regulation of iron uptake and storage within brain microvessels that challenge the existing paradigm that the blood-brain barrier is merely a transport system.

Mitochondrial ferritin in the substantia nigra in restless legs syndrome.

Restless legs syndrome (RLS) is a neurological disorder that is thought to involve decreased iron availability in the brain. Iron is required for oxidative metabolism and plays a critical role in redox reactions in mitochondria. The recent discovery of mitochondrial ferritin (FtMt) provided the opportunity to identify a potential correlation between iron and mitochondrial function in RLS. Human substantia nigra (SN) and putamen autopsy samples from 8 RLS cases and 8 controls were analyzed. Mitochondrial ferritin levels in RLS SN tissue homogenate samples assessed by immunoblots had more FtMt than control samples (p < 0.01), whereas there were no significant differences in FtMt in the putamen samples. By immunohistochemistry, neuromelanin-containing neurons in the SN were the predominant cell type expressing FtMt. Staining in neurons in RLS samples was consistently greater than that in controls. Cytochrome c oxidase staining, which reflects numbers of mitochondria, showed a similar staining pattern to that of FtMt, whereas there was less immunostaining in the RLS cases for cytosolic H-ferritin. These results suggest that increased numbers of mitochondria in neurons in RLS and increased FtMt might contribute to insufficient cytosolic iron levels in RLS SN neurons; they are consistent with the hypothesis that energy insufficiency in these neurons may be involved in the pathogenesis of RLS.

Altered iron metabolism in lymphocytes from subjects with restless legs syndrome.

OBJECTIVE: Studies using cerebrospinal fluid, magnetic resonance imaging, and autopsy tissue have implicated a primary role for brain iron insufficiency in restless legs syndrome (RLS). If the abnormalities of brain iron regulation reflect a basic disturbance of iron metabolism, then this might be expressed at least partially in some peripheral systems. Thus the study aim was to determine whether patients with RLS and control subjects show differences in lymphocyte iron regulator proteins. METHODS: Fasting morning blood samples were used to obtain common serum measures of iron status and to determine lymphocyte iron management proteins. Twenty-four women with early-onset RLS and 25 control women without RLS symptoms were studied. RESULTS: RLS and control subjects were matched for age, hemoglobin, and serum iron profile. However, transferrin receptor (TfR) and DMT1 (divalent metal transporter 1 protein) levels in lymphocytes were significantly higher for RLS patients than for controls. No significant differences in ferritin subtypes or transferrin levels were found. No significant correlations were found between lymphocyte and serum indices of iron status. INTERPRETATION: RLS lymphocytes showed an increase in ferroportin, implying increased cellular iron excretion, in the face of increased iron need (increased TfR and DMT1). In the absence of changes in H-ferritin, the findings indicate a balance between input and output with no net iron change but probable overall increase in iron turnover. The lack of any significant correlation between serum and lymphocyte iron indices indicates that iron management proteins from lymphocytes are at a minimum an alternative and independent marker of cellular iron metabolism.

Consequences of expressing mutants of the hemochromatosis gene (HFE) into a human neuronal cell line lacking endogenous HFE.

HFE mutations have traditionally been associated with the iron overload disorder known as hemochromatosis. Recently, it has become clear that the two most common mutations in the HFE gene, H63D and C282Y, may be genetic modifiers for risk of neurodegenerative disorders and cancer, respectively. We developed human neuroblastoma stable cell lines that express either wild-type (WT) or mutant HFE to determine the cellular consequences of the mutant forms of HFE. The presence of the C282Y mutation was associated with relatively higher labile iron pool and iron regulatory protein activity than WT or H63D HFE. Targeted gene arrays revealed that the signal transduction pathway was up-regulated in the C282Y cells. H63D cells had higher levels of lipid peroxidation, protein oxidation, and lower mitochondrial membrane potential, suggesting higher baseline stress. This cell line was also more vulnerable to exposure to oxidative stress agents and more responsive to iron chelation than the C282Y cells. These data demonstrate that the different mutations in the HFE gene have unique effects on the cells and provide insights into how the different mutations may have different clinical consequences. The results also raise multiple novel questions for future study about the function of the HFE protein.

Hypoxia-inducible factor prolyl 4-hydroxylase inhibition. A target for neuroprotection in the central nervous system.

Hypoxia-inducible factor (HIF) prolyl 4-hydroxylases are a family of iron- and 2-oxoglutarate-dependent dioxygenases that negatively regulate the stability of several proteins that have established roles in adaptation to hypoxic or oxidative stress. These proteins include the transcriptional activators HIF-1alpha and HIF-2alpha. The ability of the inhibitors of HIF prolyl 4-hydroxylases to stabilize proteins involved in adaptation in neurons and to prevent neuronal injury remains unclear. We reported that structurally diverse low molecular weight or peptide inhibitors of the HIF prolyl 4-hydroxylases stabilize HIF-1alpha and up-regulate HIF-dependent target genes (e.g. enolase, p21(waf1/cip1), vascular endothelial growth factor, or erythropoietin) in embryonic cortical neurons in vitro or in adult rat brains in vivo. We also showed that structurally diverse HIF prolyl 4-hydroxylase inhibitors prevent oxidative death in vitro and ischemic injury in vivo. Taken together these findings identified low molecular weight and peptide HIF prolyl 4-hydroxylase inhibitors as novel neurological therapeutics for stroke as well as other diseases associated with oxidative stress.

Subcellular localization of iron regulatory proteins to Golgi and ER membranes.

Interaction between iron regulatory proteins and iron responsive elements on certain mRNAs is at the core of regulation of intracellular iron homeostasis. Previous results suggested that in cultured cells iron regulatory proteins (IRPs) exist in cytosolic and microsomal subcellular locations and that this distribution is affected by cellular iron status. In this study, we tested the hypothesis that the membrane-associated fractions of iron regulatory proteins are specifically in the endoplasmic reticulum and Golgi membranes. Confocal microscopy revealed that IRP1 could be co-localized to the endoplasmic reticulum and the Golgi apparatus. To examine the intracellular distribution of IRPs biochemically, we used rats fed normal or iron-deficient diets. As expected, the IRPs were found predominantly in the cytosolic fraction. However, subfractionation of crude microsomal preparations revealed IRP1 in the Golgi apparatus. In animals fed an iron-deficient diet, IRP1 was found in the Golgi apparatus and the endoplasmic reticulum. To identify the mechanisms and factors involved in the localization of iron regulatory proteins in the cytosol and membrane fractions, cells were treated with a phorbol ester, a protein kinase C inhibitor (chelerythrine), hydrogen peroxide, interleukin-1beta, and 1,2-bis-(o-aminophenoxy)-ethane-N,N,-N'N'-tetraacetic acid tetraacetoxy-methyl ester. The results indicate that iron-regulatory-protein-binding activity in the membrane fraction can be altered by cell stress or iron status and that phosphorylation plays a role in the translocation. As a result of this study we propose a novel model for intracellular distribution of IRPs and identify differences between the two iron regulatory proteins.

Development of a fluorescent reporter to assess iron regulatory protein activity in living cells.

Through the insertion of an iron responsive element (IRE) into a pd2ECFP vector, we demonstrate a noninvasive method for determining alterations in iron regulatory protein (IRP) activity that results in changes in protein translation in living cells. This construct takes advantage of the specifically iron-dependent interaction between IRPs that bind IREs on mRNAs to posttranscriptionally regulate protein expression in a manner similar to ferritin production. In this report, we demonstrate, using HEK-293 cells, that an IRE-driven fluorescent reporter can be used to observe changes in cellular iron status that are sufficient to alter protein synthesis. When iron availability was decreased, there was less cyan fluorescent protein (CFP) expression, suggesting that IRPs bind to the IRE and block protein translation. Conversely, exposing the cells to iron increased CFP fluorescence. This construct has advantages over traditionally used dyes and existing IRE driven constructs because it can be used to repeatedly study iron-influenced protein production over extended periods of time. The future applications of this construct include investigation of how mutations in cells may impact cellular iron metabolism and how various types of exogenously applied trophic, stress, and therapeutic agents may impact cellular iron metabolism.

Characterization of nuclear ferritin and mechanism of translocation.

Ferritin, normally considered a cytoplasmic iron-storage protein, is also found in cell nuclei. It is an established fact that H-ferritin is the major form of nuclear ferritin, but little is known about the roles of ferritin in nuclei or about the mechanisms that control its appearance within the nuclear volume. In the present study, we show that, for human SW1088 astrocytoma cells, the nuclear and cytoplasmic forms of H-ferritin are products of the same mRNA. Histochemical and biochemical evidence is presented showing that ferritin is distributed non-randomly within the nuclear volume and that it preferentially associates with heterochromatin. Both cytoplasmic and nuclear populations of H-ferritin contain mixtures of non- and O-glycosylated forms, but the nuclear population is enriched in O-glycosylated forms. Cells treated with alloxan, a potent inhibitor of O-glycosylation, contained significantly less nuclear ferritin compared with cells grown in control media. Alloxan inhibited the reappearance of H-ferritin in nuclei of cells released from conditions of iron depletion, but did not prevent its disappearance from nuclei of cells undergoing iron depletion. These results suggest that O-glycosylation accompanies the transfer of ferritin from the cytoplasm to the nucleus, but does not influence the reverse process. The picture that emerges is one in which ferritin translocation between the cytoplasm and the nucleus is post-translationally regulated and responds to environmental and nutritional cues.