Manganese causes neurotoxic iron accumulation via translational repression of Amyloid Precursor Protein (APP) and H-Ferritin

Manganese causes neurotoxic iron ABSTRACT 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 (IRPs), 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 IRP-IRE 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 (ROS) 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 role and functions of APP and H-Ferritin in orchestrating iron homeostasis and responses, we here investigated if Mn interferes with the translational control of both proteins resulting in blocked iron efflux and depleted iron storage in neuronal-like SH-SY5Y cells and in two different rodent in vivo models. Cell viability data are presented as relative changes in % compared to untreated controls. In parallel, cells were grown in 12-well plates for cell number analysis. Repetitive aliquots were taken and counted using a hemocytometer with trypan blue exclusion assay. For each treatment condition, triplicate wells were counted, and values were averaged. Data were presented as relative values in % compared to untreated controls. Speciation and quantification of Fe 2+ , Fe 3+ and Mn 2+ was performed by ion chromatography inductively coupled plasma mass spectrometry (IC-ICP-MS) as described previously (Fernsebner et al. 2014, Solovyev et al. 2017) and in addition with capillary electrophoresis inductively coupled plasma mass spectrometry (CE-ICP-MS). For redox speciation of Fe 2+ vs. Fe 3+ the IC-ICP-MS method used a Thermo-Dionex guard column IonPac™ CG5A column and an IonPac™ CS5A RFIC 4*250 mm analytical column, directly hyphenated to high-resolution sectorfield ICP-sf-MS (Element II, Thermo Scientific). Experimental conditions were analogous to Redox species were separated isocratically using an eluent consisting of 50 mM ammonium citrate, acid pH 4.2 at a flow rate of 0.8 ml/min. Detection was performed on 56Fe isotope at the ICP-sf-MS in medium resolution mode. For cross-validation a coupling of capillary electrophoresis Prince Technologies) with ICP-qMS Perkin Elmer) The CE-ICP-MS method operated in positive electrophoretic mode at acidic pH (background electrolyte: 20 mM HCl, +25 kV). Monitored isotopes were 55 Mn, 56 Fe and 57 Fe, using DRC cell technology with as DRC gas in to for removing interferences at 585 nm (FL-2)). Data were analyzed by FlowJo software and presented as relative induced ROS compared to untreated controls. For Lipid-ROS the fluorescence ratio FL1/FL2 reflecting the oxidation of the probe were presented either in arbitrary units or as relative induced lipid ROS compared to untreated controls. model for Mn-induced neurotoxicity. (a) Under physiological conditions, free ferrous iron (Fe 2+ ) causes IRP1 release from iron responsive elements (IRE) that are located within the 5’-untranslated regions (5’-UTRs) of APP and H-Ferritin mRNA inducing enhanced ribosomal protein translation. To counteract toxic redox-active Fe 2+ levels, APP facilitates the stabilization of membrane-bound Fe 2+ -exporter ferroportin (FPN) while H-Ferritin safely sequesters Fe 2+ via conversion to redox-inactive Fe 3+ . (b) Manganese (Mn) decreases APP and H-Ferritin protein translation by increasing the binding of IRP1 to the IRE on the 5’-UTR of APP and H-Ferritin mRNA. Translational blockage of APP and H-Ferritin results in accumulation of toxic Fe 2+ that fuels the generation of reactive oxygen species (ROS), ultimately resulting in neurotoxicity.