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For more than 150 years, it is known that occupational overexposure of manganese (Mn) causes movement disorders resembling Parkinson's disease (PD) and PD-like syndromes. However, the mechanisms of Mn toxicity are still poorly understood. Here, we demonstrate that Mn dose- and time-dependently blocks the protein translation of amyloid precursor protein (APP) and heavy-chain Ferritin (H-Ferritin), both iron homeostatic proteins with neuroprotective features. APP and H-Ferritin are post-transcriptionally regulated by iron responsive proteins, which bind to homologous iron responsive elements (IREs) located in the 5'-untranslated regions (5'-UTRs) within their mRNA transcripts. Using reporter assays, we demonstrate that Mn exposure repressed the 5'-UTR-activity of APP and H-Ferritin, presumably via increased iron responsive proteins-iron responsive elements binding, ultimately blocking their protein translation. Using two specific Fe 2+ -specific probes (RhoNox-1 and IP-1) and ion chromatography inductively coupled plasma mass spectrometry (IC-ICP-MS), we show that loss of the protective axis of APP and H-Ferritin resulted in unchecked accumulation of redox-active ferrous iron (Fe 2+ ) fueling neurotoxic oxidative stress. Enforced APP expression partially attenuated Mn-induced generation of cellular and lipid reactive oxygen species and neurotoxicity. Lastly, we could validate the Mn-mediated suppression of APP and H-Ferritin in two rodent in vivo models (C57BL6/N mice and RjHan:SD rats) mimicking acute and chronic Mn exposure. Together, these results suggest that Mn-induced neurotoxicity is partly attributable to the translational inhibition of APP and H-Ferritin resulting in impaired iron metabolism and exacerbated neurotoxic oxidative stress. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/., (© 2018 International Society for Neurochemistry.)

Elevation of both neuronal iron and nitric oxide (NO) in the substantia nigra are associated with Parkinson's disease (PD) pathogenesis. We reported previously that the Alzheimer-associated β-amyloid precursor protein (APP) facilitates neuronal iron export. Here we report markedly decreased APP expression in dopaminergic neurons of human PD nigra and that APP(-/-) mice develop iron-dependent nigral cell loss. Conversely, APP-overexpressing mice are protected in the MPTP PD model. NO suppresses APP translation in mouse MPTP models, explaining how elevated NO causes iron-dependent neurodegeneration in PD., (Copyright © 2015 the authors 0270-6474/15/353591-07$15.00/0.)

The neurotoxicity of amyloid beta (Aβ), a major cleavage product of the amyloid precursor protein (APP), is enhanced by iron, as found in the amyloid plaques of Alzheimer's disease (AD) patients. By contrast, the long-known neuroprotective activity of APP is evident after α-secretase cleavage of the precursor to release sAPPα, and depends on the iron export actions of APP itself. The latter underlie its neurotrophic and protective effects in facilitating the homeostatic actions of ferroportin mediated-iron export. Thus APP-dependent iron export may alleviate oxidative stress by minimizing labile iron thus protecting neurons from iron overload during stroke and hemorrhage. Consistent with this, altered phosphorylation of iron-regulatory protein-1 (IRP1) and its signaling processes play a critical role in modulating APP translation via the 5' untranslated region (5'UTR) of its transcript. The APP 5'UTR region encodes a functional iron-responsive element (IRE) RNA stem loop that represents a potential target for modulating APP production. Targeted regulation of APP gene expression via the modulation of 5'UTR sequence function represents a novel approach for the potential treatment of AD since altering APP translation can be used to improve both the protective brain iron balance and provide anti-amyloid efficacy. Approved drugs including paroxetine and desferrioxamine and several novel compounds have been identified that suppress abnormal metal-promoted Aβ accumulation with a subset of these acting via APP 5'UTR-dependent mechanisms to modulate APP translation and cleavage to generate the non-toxic sAPPα., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: The homeostasis and physiological role of iron in Alzheimer's disease (AD) has been debated for decades. Overall, it has been difficult to reach a consensus to prove marked disease-associated changes in the iron content of the AD brain, blood, or cerebrospinal fluid (CSF)., Objectives: We sought to contribute to resolve this issue by quantifying the iron content in serum, CSF, and sub-regions of the AD brain., Methods: We conducted a comprehensive systematic meta-analysis and review of multiple observational studies till October 2013 that investigated the iron content in AD serum, CSF, or brain tissue., Results: 2,556 publications were screened. Forty-three eligible studies with 1,813 AD patients and 2,401 healthy controls were identified. Twenty-one studies investigated the serum iron in AD while seven and nineteen studies investigated the CSF iron and various brain regions iron respectively. Our meta-analysis showed that serum iron was significant lower in AD than healthy controls. CSF iron appeared not to be affected by AD although more studies are required due to the relative small number of CSF studies reported to date. We critically analyzed iron content in twelve selective brain regions by separated meta-analyses using cross-referenced statistical methods. We found that eight specific brain regions had higher iron concentrations that correlated with the clinical diagnosis of AD in a statistically validated manner., Conclusions: These data provided rigorous statistical support for the model that iron homeostasis was changed in AD patients, including the finding of lower iron in their serum and evidence for iron overload in several specific brain regions.

Increased brain α-synuclein (SNCA) protein expression resulting from gene duplication and triplication can cause a familial form of Parkinson's disease (PD). Dopaminergic neurons exhibit elevated iron levels that can accelerate toxic SNCA fibril formation. Examinations of human post mortem brain have shown that while mRNA levels for SNCA in PD have been shown to be either unchanged or decreased with respect to healthy controls, higher levels of insoluble protein occurs during PD progression. We show evidence that SNCA can be regulated via the 5'untranslated region (5'UTR) of its transcript, which we modeled to fold into a unique RNA stem loop with a CAGUGN apical loop similar to that encoded in the canonical iron-responsive element (IRE) of L- and H-ferritin mRNAs. The SNCA IRE-like stem loop spans the two exons that encode its 5'UTR, whereas, by contrast, the H-ferritin 5'UTR is encoded by a single first exon. We screened a library of 720 natural products (NPs) for their capacity to inhibit SNCA 5'UTR driven luciferase expression. This screen identified several classes of NPs, including the plant cardiac glycosides, mycophenolic acid (an immunosuppressant and Fe chelator), and, additionally, posiphen was identified to repress SNCA 5'UTR conferred translation. Western blotting confirmed that Posiphen and the cardiac glycoside, strophanthidine, selectively blocked SNCA expression (~1 μM IC(50)) in neural cells. For Posiphen this inhibition was accelerated in the presence of iron, thus providing a known APP-directed lead with potential for use as a SNCA blocker for PD therapy. These are candidate drugs with the potential to limit toxic SNCA expression in the brains of PD patients and animal models in vivo.

Recent studies that alleles in the hemochromatosis gene may accelerate the onset of Alzheimer's disease by five years have validated interest in the model in which metals (particularly iron) accelerate disease course. Biochemical and biophysical measurements demonstrated the presence of elevated levels of neurotoxic copper zinc and iron in the brains of AD patients. Intracellular levels of APP holoprotein were shown to be modulated by iron by a mechanism that is similar to the translation control of the ferritin L- and H mRNAs by iron-responsive element (IRE) RNA stem loops in their 5' untranslated regions (5'UTRs). More recently a putative IRE-like sequence was hypothesized present in the Parkinsons's alpha synuclein (ASYN) transcript (see [A.L. Friedlich, R.E. Tanzi, J.T. Rogers, The 5'-untranslated region of Parkinson's disease alpha-synuclein messenger RNA contains a predicted iron responsive element, Mol. Psychiatry 12 (2007) 222-223. [6]]). Together with the demonstration of metal dependent translation of APP mRNA, the involvement of metals in the plaque of AD patients and of increased iron in striatal neurons in the substantia nigra (SN) of Parkinson's disease patients have stimulated the development of metal attenuating agents and iron chelators as a major new therapeutic strategy for the treatment of these neurodegenerative diseases. In the case of AD, metal based therapeutics may ultimately prove more cost effective than the use of an amyloid vaccine as the preferred anti-amyloid therapeutic strategy to ameliorate the cognitive decline of AD patients.

The essential metals iron, zinc and copper deposit near the Abeta (amyloid beta-peptide) plaques in the brain cortex of AD (Alzheimer's disease) patients. Plaque-associated iron and zinc are in neurotoxic excess at 1 mM concentrations. APP (amyloid precursor protein) is a single transmembrane metalloprotein cleaved to generate the 40-42-amino-acid Abetas, which exhibit metal-catalysed neurotoxicity. In health, ubiquitous APP is cleaved in a non-amyloidogenic pathway within its Abeta domain to release the neuroprotective APP ectodomain, APP(s). To adapt and counteract metal-catalysed oxidative stress, as during reperfusion from stroke, iron and cytokines induce the translation of both APP and ferritin (an iron storage protein) by similar mechanisms. We reported that APP was regulated at the translational level by active IL (interleukin)-1 (IL-1-responsive acute box) and IRE (iron-responsive element) RNA stem-loops in the 5' untranslated region of APP mRNA. The APP IRE is homologous with the canonical IRE RNA stem-loop that binds the iron regulatory proteins (IRP1 and IRP2) to control intracellular iron homoeostasis by modulating ferritin mRNA translation and transferrin receptor mRNA stability. The APP IRE interacts with IRP1 (cytoplasmic cis-aconitase), whereas the canonical H-ferritin IRE RNA stem-loop binds to IRP2 in neural cell lines, and in human brain cortex tissue and in human blood lysates. The same constellation of RNA-binding proteins [IRP1/IRP2/poly(C) binding protein] control ferritin and APP translation with implications for the biology of metals in AD.

Iron-responsive elements (IREs) are the RNA stem loops that control cellular iron homeostasis by regulating ferritin translation and transferrin receptor mRNA stability. We mapped a novel iron-responsive element (IRE-Type II) within the 5'-untranslated region (5'-UTR) of the Alzheimer's amyloid precursor protein (APP) transcript (+51 to +94 from the 5'-cap site). The APP mRNA IRE is located immediately upstream of an interleukin-1 responsive acute box domain (+101 to +146). APP 5'-UTR conferred translation was selectively down-regulated in response to intracellular iron chelation using three separate reporter assays (chloramphenicol acetyltransferase, luciferase, and red fluorescent protein reflecting an inhibition of APP holoprotein translation in response to iron chelation. Iron influx reversed this inhibition. As an internal control to ensure specificity, a viral internal ribosome entry sequence was unresponsive to intracellular iron chelation with desferrioxamine. Using RNA mobility shift assays, the APP 5'-UTRs, encompassing the IRE, bind specifically to recombinant iron-regulatory proteins (IRP) and to IRP from neuroblastoma cell lysates. IRP binding to the APP 5'-UTR is reduced after treatment of cells with desferrioxamine and increased after interleukin-1 stimulation. IRP binding is abrogated when APP cRNA probe is mutated in the core IRE domain (Delta4 bases:Delta83AGAG86). Iron regulation of APP mRNA through the APP 5'-UTR points to a role for iron in the metabolism of APP and confirms that this RNA structure can be a target for the selection of small molecule drugs, such as desferrioxamine (Fe chelator) and clioquinol (Fe, Cu, and Zn chelator), which reduce Abeta peptide burden during Alzheimer's disease.

The first step in intestinal iron absorption is mediated by the H(+)-coupled Fe(2+) transporter called divalent cation transporter 1/divalent metal ion transporter 1 (DCT1/DMT1) (also known as natural resistance-associated macrophage protein 2). DCT1/DMT1 mRNA levels in the duodenum strongly increase in response to iron depletion. To study the mechanism of iron-dependent DCT1/DMT1 mRNA regulation, we investigated the endogenous expression of DCT1/DMT1 mRNA in various cell types. We found that only the iron responsive element (IRE)-containing form, which corresponds to one of two splice forms of DCT1/DMT1, is responsive to iron treatment and this responsiveness was cell type specific. We also examined the interaction of the putative 3'-UTR IRE with iron responsive binding proteins (IRP1 and IRP2), and found that IRP1 binds to the DCT1/DMT1-IRE with higher affinity compared to IRP2. This differential binding of IRP1 and IRP2 was also reported for the IREs of transferrin receptors, erythroid 5-aminolevulinate synthase and mitochondrial aconitase. We propose that regulation of DCT1/DMT1 mRNA by iron involves post-transcriptional regulation through the binding of IRP1 to the transporter's IRE, as well as other as yet unknown factors.

Ferritin and transferrin receptor expression is post-transcriptionally regulated by a conserved mRNA sequence termed the iron-responsive element (IRE), to which a transacting protein called the iron-regulatory protein (IRP) is bound. Our data demonstrate that hypoxia powerfully enhances IRE/IRP-1 binding in human cell lines. Using the human hepatoma cell line Hep3B as a model, we found that 16 h in a 1% oxygen atmosphere markedly increases IRE/IRP-1 binding as assessed by electromobility shift assay. Hypoxia also decreased cytosolic aconitase activity. The hypoxia-enhanced IRE/IRP-1 binding stabilized the transferrin receptor message, increased the cellular mRNA content by over 10-fold, and doubled surface receptor expression. Simultaneously, hypoxia suppressed ferritin message translation. Hypoxia's effect was most strikingly depicted by the absence of ferritin synthesis in cells challenged with inorganic iron. Our results contrast with previously reported data (Hanson, E. S., and Leibold, E. A. (1998) J. Biol. Chem. 273, 7588-7593) in which a 3% oxygen atmosphere reduced IRE/IRP-1 binding in rat hepatoma cells. We discuss some possible reasons for the differences. In aggregate with other investigations involving responses to hypoxia, iron, or nitric oxide, our data indicate that cellular iron metabolic responses are complex and that IRE/IRP-1 interactions vary between cell lines and perhaps between species.

Ascorbate is an important cofactor in many cellular metabolic reactions and is intimately linked to iron homeostasis. Continuously cultured cells are ascorbate deficient due to the lability of the vitamin in solution and to the fact that daily supplementation of media with ascorbate is unusual. We found that ascorbate repletion alone did not alter ferritin synthesis. However, ascorbate-replete human hepatoma cells, Hep3B and HepG2, as well as K562 human leukemia cells achieved a substantially higher cellular ferritin content in response to a challenge with iron than did their ascorbate-deficient counterparts grown under standard culture conditions. Most of the elevation in ferritin content was due to an increase in de novo ferritin synthesis of greater than 50-fold, as shown by in vivo labeling with [35S]methionine and immunoprecipitation. RNA-blot analysis showed only minor changes in steady state levels of ferritin mRNA, suggesting that ascorbate enhances iron-induced ferritin synthesis primarily by post-transcriptional events. Transient gene expression experiments using chloramphenicol acetyltransferase reporter gene constructs showed that the ascorbate effect on ferritin translation is not mediated through the stem-loop near the translational start site that transduces ferritin synthesis in response to cytokines. The data suggest that ascorbate possibly modifies the action of the iron-responsive element on ferritin translation, although more precise structure-function studies are needed to clarify this issue. These data demonstrate a novel role of ascorbate as a signaling molecule in post-transcriptional gene regulation. The mechanism by which ascorbate modulates cellular iron metabolism is complex and requires additional detailed investigation.