Essential role of systemic iron mobilization and redistribution for adaptive thermogenesis through HIF2-α/hepcidin axis.

Iron is an essential biometal, but is toxic if it exists in excess. Therefore, iron content is tightly regulated at cellular and systemic levels to meet metabolic demands but to avoid toxicity. We have recently reported that adaptive thermogenesis, a critical metabolic pathway to maintain whole-body energy homeostasis, is an iron-demanding process for rapid biogenesis of mitochondria. However, little information is available on iron mobilization from storage sites to thermogenic fat. This study aimed to determine the iron-regulatory network that underlies beige adipogenesis. We hypothesized that thermogenic stimulus initiates the signaling interplay between adipocyte iron demands and systemic iron liberation, resulting in iron redistribution into beige fat. To test this hypothesis, we induced reversible activation of beige adipogenesis in C57BL/6 mice by administering a ß3-adrenoreceptor agonist CL 316,243 (CL). Our results revealed that CL stimulation induced the iron-regulatory protein-mediated iron import into adipocytes, suppressed hepcidin transcription, and mobilized iron from the spleen. Mechanistically, CL stimulation induced an acute activation of hypoxia-inducible factor 2-α (HIF2-α), erythropoietin production, and splenic erythroid maturation, leading to hepcidin suppression. Disruption of systemic iron homeostasis by pharmacological HIF2-α inhibitor PT2385 or exogenous administration of hepcidin-25 significantly impaired beige fat development. Our findings suggest that securing iron availability via coordinated interplay between renal hypoxia and hepcidin down-regulation is a fundamental mechanism to activate adaptive thermogenesis. It also provides an insight into the effects of adaptive thermogenesis on systemic iron mobilization and redistribution.

The thermogenic characteristics of adipocytes are dependent on the regulation of iron homeostasis.

The development of thermogenic adipocytes concurs with mitochondrial biogenesis, an iron-dependent pathway. Iron regulatory proteins (IRP) 1 and 2 are RNA-binding proteins that regulate intracellular iron homeostasis. IRPs bind to the iron-response element (IRE) of their target mRNAs, balancing iron uptake and deposition at the posttranscriptional levels. However, IRP/IRE-dependent iron regulation in adipocytes is largely unknown. We hypothesized that iron demands are higher in brown/beige adipocytes than white adipocytes to maintain the thermogenic mitochondrial capacity. To test this hypothesis, we investigated the IRP/IRE regulatory system in different depots of adipose tissue. Our results revealed that 1) IRP/IRE interaction was increased in proportional to the thermogenic function of the adipose depot, 2) adipose iron content was increased in adipose tissue browning upon ß3-adrenoceptor stimulation, while decreased in thermoneutral conditions, and 3) modulation of iron content was linked with mitochondrial biogenesis. Moreover, the iron requirement was higher in HIB1B brown adipocytes than 3T3-L1 white adipocytes during differentiation. The reduction of the labile iron pool (LIP) suppressed the differentiation of brown/beige adipocytes and mitochondrial biogenesis. Using the 59Fe-Tf, we also demonstrated that thermogenic stimuli triggered cell-autonomous iron uptake and mitochondrial compartmentalization as well as enhanced mitochondrial respiration. Collectively, our work demonstrated that IRP/IRE signaling and subsequent adaptation in iron metabolism are a critical determinant for the thermogenic function of adipocytes.

Optimal priority assignment for real-time systems: a coevolution-based approach.

In real-time systems, priorities assigned to real-time tasks determine the order of task executions, by relying on an underlying task scheduling policy. Assigning optimal priority values to tasks is critical to allow the tasks to complete their executions while maximizing safety margins from their specified deadlines. This enables real-time systems to tolerate unexpected overheads in task executions and still meet their deadlines. In practice, priority assignments result from an interactive process between the development and testing teams. In this article, we propose an automated method that aims to identify the best possible priority assignments in real-time systems, accounting for multiple objectives regarding safety margins and engineering constraints. Our approach is based on a multi-objective, competitive coevolutionary algorithm mimicking the interactive priority assignment process between the development and testing teams. We evaluate our approach by applying it to six industrial systems from different domains and several synthetic systems. The results indicate that our approach significantly outperforms both our baselines, i.e., random search and sequential search, and solutions defined by practitioners. Our approach scales to complex industrial systems as an offline analysis method that attempts to find near-optimal solutions within acceptable time, i.e., less than 16 hours.

Dietary Iron Deficiency Modulates Adipocyte Iron Homeostasis, Adaptive Thermogenesis, and Obesity in C57BL/6 Mice.

BACKGROUND: Adaptive thermogenesis is an iron-demanding pathway, significantly contributing to whole-body energy expenditure. However, the effects of iron-deficient diets on adaptive thermogenesis and obesity remain unknown. OBJECTIVES: We aimed to determine the impact of dietary iron deficiency on iron homeostasis in adipocytes, adaptive thermogenic capacity, and metabolic consequences in obesity. METHODS: C57BL/6 male mice were assigned to either the iron-adequate (IA, 35 ppm) or the iron-deficient group (ID, 3 ppm) at weaning. Upon 8 wk of age, both IA and ID groups received an isocaloric high-fat diet (45% kcal from fat) for 10 wk, maintaining the same iron content. Mice (n = 8) were used to determine the iron status at the systemic and tissue levels and lipid metabolism and inflammatory signaling in adipose tissue. The same mice were used to evaluate cold tolerance (4°C) for 3 h. For assessing adaptive thermogenesis, mice (n = 5) received an intraperitoneal injection of ß3-adrenoceptor agonist CL316243 (CL) for 5 d. RESULTS: Compared with the IA group, the ID group had nonanemic iron deficiency, lower serum ferritin (42.8%, P < 0.01), and greater weight gain (8.67%, P < 0.05) and insulin resistance (159%, P < 0.01), partly due to reduced AMP-activated protein kinase activation (61.0%, P < 0.05). Upon cold exposure, the ID group maintained a core body temperature 2°C lower than the IA group. The ID group had lower iron content (47.0%, P < 0.01) in the inguinal adipose tissue (iWAT) than the IA group, which was associated with impaired adaptive thermogenesis. In response to CL, ID mice showed decreased heat production (P < 0.01) and defective upregulation of beige adipocyte-specific markers, including uncoupling protein 1 (41.1%, P < 0.001), transferrin receptor 1 (47.5%, P < 0.001), and mitochondrial respiratory chain complexes (P < 0.05) compared with IA mice. CONCLUSIONS: Dietary iron deficiency deregulates iron balance in the iWAT and impairs adaptive thermogenesis, thereby escalating the diet-induced weight gain in C57BL/6 mice.

Sestrin2 Phosphorylation by ULK1 Induces Autophagic Degradation of Mitochondria Damaged by Copper-Induced Oxidative Stress.

Selective autolysosomal degradation of damaged mitochondria, also called mitophagy, is an indispensable process for maintaining integrity and homeostasis of mitochondria. One well-established mechanism mediating selective removal of mitochondria under relatively mild mitochondria-depolarizing stress is PINK1-Parkin-mediated or ubiquitin-dependent mitophagy. However, additional mechanisms such as LC3-mediated or ubiquitin-independent mitophagy induction by heavy environmental stress exist and remain poorly understood. The present study unravels a novel role of stress-inducible protein Sestrin2 in degradation of mitochondria damaged by transition metal stress. By utilizing proteomic methods and studies in cell culture and rodent models, we identify autophagy kinase ULK1-mediated phosphorylation sites of Sestrin2 and demonstrate Sestrin2 association with mitochondria adaptor proteins in HEK293 cells. We show that Ser-73 and Ser-254 residues of Sestrin2 are phosphorylated by ULK1, and a pool of Sestrin2 is strongly associated with mitochondrial ATP5A in response to Cu-induced oxidative stress. Subsequently, this interaction promotes association with LC3-coated autolysosomes to induce degradation of mitochondria damaged by Cu-induced ROS. Treatment of cells with antioxidants or a Cu chelator significantly reduces Sestrin2 association with mitochondria. These results highlight the ULK1-Sestrin2 pathway as a novel stress-sensing mechanism that can rapidly induce autophagic degradation of mitochondria under severe heavy metal stress.

CHCA-1 is a copper-regulated CTR1 homolog required for normal development, copper accumulation, and copper-sensing behavior in Caenorhabditis elegans.

Copper plays key roles in catalytic and regulatory biochemical reactions essential for normal growth, development, and health. Dietary copper deficiencies or mutations in copper homeostasis genes can lead to abnormal musculoskeletal development, cognitive disorders, and poor growth. In yeast and mammals, copper is acquired through the activities of the CTR1 family of high-affinity copper transporters. However, the mechanisms of systemic responses to dietary or tissue-specific copper deficiency remain unclear. Here, taking advantage of the animal model Caenorhabditis elegans for studying whole-body copper homeostasis, we investigated the role of a C. elegans CTR1 homolog, CHCA-1, in copper acquisition and in worm growth, development, and behavior. Using sequence homology searches, we identified 10 potential orthologs to mammalian CTR1 Among these genes, we found that chca-1, which is transcriptionally up-regulated in the intestine and hypodermis of C. elegans during copper deficiency, is required for normal growth, reproduction, and maintenance of systemic copper balance under copper deprivation. The intestinal copper transporter CUA-1 normally traffics to endosomes to sequester excess copper, and we found here that loss of chca-1 caused CUA-1 to mislocalize to the basolateral membrane under copper overload conditions. Moreover, animals lacking chca-1 exhibited significantly reduced copper avoidance behavior in response to toxic copper conditions compared with WT worms. These results establish that CHCA-1-mediated copper acquisition in C. elegans is crucial for normal growth, development, and copper-sensing behavior.

Gene duplication and neo-functionalization in the evolutionary and functional divergence of the metazoan copper transporters Ctr1 and Ctr2.

Copper is an essential element for proper organismal development and is involved in a range of processes, including oxidative phosphorylation, neuropeptide biogenesis, and connective tissue maturation. The copper transporter (Ctr) family of integral membrane proteins is ubiquitously found in eukaryotes and mediates the high-affinity transport of Cu+ across both the plasma membrane and endomembranes. Although mammalian Ctr1 functions as a Cu+ transporter for Cu acquisition and is essential for embryonic development, a homologous protein, Ctr2, has been proposed to function as a low-affinity Cu transporter, a lysosomal Cu exporter, or a regulator of Ctr1 activity, but its functional and evolutionary relationship to Ctr1 is unclear. Here we report a biochemical, genetic, and phylogenetic comparison of metazoan Ctr1 and Ctr2, suggesting that Ctr2 arose over 550 million years ago as a result of a gene duplication event followed by loss of Cu+ transport activity. Using a random mutagenesis and growth selection approach, we identified amino acid substitutions in human and mouse Ctr2 proteins that support copper-dependent growth in yeast and enhance copper accumulation in Ctr1-/- mouse embryonic fibroblasts. These mutations revert Ctr2 to a more ancestral Ctr1-like state while maintaining endogenous functions, such as stimulating Ctr1 cleavage. We suggest key structural aspects of metazoan Ctr1 and Ctr2 that discriminate between their biological roles, providing mechanistic insights into the evolutionary, biochemical, and functional relationships between these two related proteins.

The Intestinal Copper Exporter CUA-1 Is Required for Systemic Copper Homeostasis in Caenorhabditis elegans.

Copper plays key catalytic and regulatory roles in biochemical processes essential for normal growth, development, and health. Defects in copper metabolism cause Menkes and Wilson's disease, myeloneuropathy, and cardiovascular disease and are associated with other pathophysiological states. Consequently, it is critical to understand the mechanisms by which organisms control the acquisition, distribution, and utilization of copper. The intestinal enterocyte is a key regulatory point for copper absorption into the body; however, the mechanisms by which intestinal cells transport copper to maintain organismal copper homeostasis are poorly understood. Here, we identify a mechanism by which organismal copper homeostasis is maintained by intestinal copper exporter trafficking that is coordinated with extraintestinal copper levels in Caenorhabditis elegans Specifically, we show that CUA-1, the C. elegans homolog of ATP7A/B, localizes to lysosome-like organelles (gut granules) in the intestine under copper overload conditions for copper detoxification, whereas copper deficiency results in a redistribution of CUA-1 to basolateral membranes for copper efflux to peripheral tissues. Worms defective in gut granule biogenesis exhibit defects in copper sequestration and increased susceptibility to toxic copper levels. Interestingly, however, a splice isoform CUA-1.2 that lacks a portion of the N-terminal domain is targeted constitutively to the basolateral membrane irrespective of dietary copper concentration. Our studies establish that CUA-1 is a key intestinal copper exporter and that its trafficking is regulated to maintain systemic copper homeostasis. C. elegans could therefore be exploited as a whole-animal model system to study regulation of intra- and intercellular copper trafficking pathways.

Effect of the Spray Distance on the Properties of High Velocity Oxygen-Fuel (HVOF) Sprayed WC-12Co Coatings.

In this study influence of spray distance on the properties of WC-12Co coatings deposited by HVOF was investigated. WC-12Co coating was sprayed at spray distance of 300, 385 and 450 mm. From microstructure observation, it is confirmed that the porosity of coatings increases with increasing the spray distance. The X-ray diffraction patterns indicate that the coatings consist of pure WC, W, and Co as well as W2C and Co6W6C phases. The increase of the spray distance accelerated the decarburization of coatings. From micro hardness tests, it was found that the hardness and the fracture toughness decreased with increasing spray distance. These mechanical properties would be related with not only porosity but also the degree of decarburization.

Preparation and Properties of WC-Based Alloy Coatings with Controlled Co and Cr via High Velocity Oxy-Fuel Spray Technique.

WC based alloy coatings included different mass percent of Co and Cr have been synthesized on high carbon steel by using a facile high velocity oxy-fuel spray method. The mechanical nature of the coating films has been investigated by micro vickers hardness and fracture toughness. X-ray diffraction (XRD) and EDX analyses indicate that the three different samples (WC-10Co-4Cr, WC-17Co, and WC-12Co) consist of pure WC, W, Cr, and Co constituents as well as W2C and Co6W6C phases. The SEM and image analysis results show that WC-10Co-4Cr condition has higher porosity than those of WC-17Co, and WC-12Co coatings. WC-17Co coating showed the highest value in the hardness and fracture toughness test among three different samples. The obtained results revealed that the mechanical properties of WC based alloy coatings synthesized by a facile high velocity oxy-fuel spray method is very sensitive to Co content.

Cadmium and Secondary Structure-dependent Function of a Degron in the Pca1p Cadmium Exporter.

Protein turnover is a critical cellular process regulating biochemical pathways and destroying terminally misfolded or damaged proteins. Pca1p, a cadmium exporter in the yeast Saccharomyces cerevisiae, is rapidly degraded by the endoplasmic reticulum-associated degradation (ERAD) system via a cis-acting degron that exists at the 250-350 amino acid region of Pca1p and is transferable to other proteins to serve as a degradation signal. Cadmium stabilizes Pca1p in a manner dependent on the degron. This suggested that cadmium-mediated masking of the degron impedes its interaction with the molecular factors involved in the ERAD. The characteristics and mechanisms of action of the degron in Pca1p and most of those in other proteins however remain to be determined. The results presented here indicate that specific cysteine residues in a degron of Pca1p sense cadmium. An unbiased approach selecting non-functional degrons indicated a critical role of hydrophobic amino acids in the degron for its function. A secondary structure modeling predicted the formation of an amphipathic helix. Site-directed mutagenesis confirmed the functional significance of the hydrophobic patch. Last, hydrophobic amino acids in the degron- and cadmium-binding region affected the interaction of Pca1p with the Ssa1p molecular chaperone, which is involved in ERAD. These results reveal the mechanism of action of the degron, which might be useful for the identification and characterization of other degrons.

Endoplasmic Reticulum-associated Degradation of Pca1p, a Polytopic Protein, via Interaction with the Proteasome at the Membrane.

Endoplasmic reticulum-associated degradation (ERAD) plays a critical role in the destruction of terminally misfolded proteins at the secretory pathway. The system also regulates expression levels of several proteins such as Pca1p, a cadmium exporter in yeast. To gain better insight into the mechanisms underlying ERAD of Pca1p and other polytopic proteins by the proteasome in the cytosol, our study determined the roles for the molecular factors of ERAD in dislodging Pca1p from the endoplasmic reticulum (ER). Inactivation of the 20S proteasome leads to accumulation of ubiquitinated Pca1p in the ER membrane, suggesting a role for the proteasome in extraction of Pca1p from the ER. Pca1p formed a complex with the proteasome at the membrane in a Doa10p E3 ligase-dependent manner. Cdc48p is required for recruiting the proteasome to Pca1p. Although the Ufd2p E4 ubiquitin chain extension enzyme is involved in efficient degradation of Pca1p, Ufd2p-deficient cells did not affect the formation of a complex between Pca1p and the proteasome. Two other polytopic membrane proteins undergoing ERAD, Ste6*p and Hmg2p, also displayed the same outcomes observed for Pca1p. However, poly-ubiquitinated Cpy1*p, a luminal ERAD substrate, was detected in the cytosol independent of proteolytic activities of the proteasome. These results indicate that extraction and degradation of polytopic membrane proteins at the ER is a coupled event. This mechanism would relieve the cost of exposed hydrophobic domains in the cytosol during ERAD.

Potassium and the K+/H+ Exchanger Kha1p Promote Binding of Copper to ApoFet3p Multi-copper Ferroxidase.

Acquisition and distribution of metal ions support a number of biological processes. Here we show that respiratory growth of and iron acquisition by the yeast Saccharomyces cerevisiae relies on potassium (K(+)) compartmentalization to the trans-Golgi network via Kha1p, a K(+)/H(+) exchanger. K(+) in the trans-Golgi network facilitates binding of copper to the Fet3p multi-copper ferroxidase. The effect of K(+) is not dependent on stable binding with Fet3p or alteration of the characteristics of the secretory pathway. The data suggest that K(+) acts as a chemical factor in Fet3p maturation, a role similar to that of cations in folding of nucleic acids. Up-regulation of KHA1 gene in response to iron limitation via iron-specific transcription factors indicates that K(+) compartmentalization is linked to cellular iron homeostasis. Our study reveals a novel functional role of K(+) in the binding of copper to apoFet3p and identifies a K(+)/H(+) exchanger at the secretory pathway as a new molecular factor associated with iron uptake in yeast.

Iron chaperones PCBP1 and PCBP2 mediate the metallation of the dinuclear iron enzyme deoxyhypusine hydroxylase.

Although cells express hundreds of metalloenzymes, the mechanisms by which apoenzymes receive their metal cofactors are largely unknown. Poly(rC)-binding proteins PCBP1 and PCBP2 are multifunctional adaptor proteins that bind iron and deliver it to ferritin for storage or to prolyl and asparagyl hydroxylases to metallate the mononuclear iron center. Here, we show that PCBP1 and PCBP2 also deliver iron to deoxyhypusine hydroxylase (DOHH), the dinuclear iron enzyme required for hypusine modification of the translation factor eukaryotic initiation factor 5A. Cells depleted of PCBP1 or PCBP2 exhibited loss of DOHH activity and loss of the holo form of the enzyme in cells, particularly when cells were made mildly iron-deficient. Lysates containing PCBP1 and PCBP2 converted apo-DOHH to holo-DOHH in vitro with greater efficiency than lysates lacking PCBP1 or PCBP2. PCBP1 bound to DOHH in iron-treated cells but not in control or iron-deficient cells. Depletion of PCBP1 or PCBP2 had no effect on the cytosolic Fe-S cluster enzyme xanthine oxidase but led to loss of cytosolic aconitase activity. Loss of aconitase activity was not accompanied by gain of RNA-binding activity, a pattern suggesting the incomplete disassembly of the [4Fe-4S] cluster. PCBP depletions had minimal effects on total cellular iron, mitochondrial iron levels, and heme synthesis. Thus, PCBP1 and PCBP2 may serve as iron chaperones to multiple classes of cytosolic nonheme iron enzymes and may have a particular role in restoring metal cofactors that are spontaneously lost in iron deficient cells.

Imaging of the Finger Vein and Blood Flow for Anti-Spoofing Authentication Using a Laser and a MEMS Scanner.

A new authentication method employing a laser and a scanner is proposed to improve image contrast of the finger vein and to extract blood flow pattern for liveness detection. A micromirror reflects a laser beam and performs a uniform raster scan. Transmissive vein images were obtained, and compared with those of an LED. Blood flow patterns were also obtained based on speckle images in perfusion and occlusion. Curvature ratios of the finger vein and blood flow intensities were found to be nearly constant, regardless of the vein size, which validated the high repeatability of this scheme for identity authentication with anti-spoofing.

X-linked spinal muscular atrophy in mice caused by autonomous loss of ATP7A in the motor neuron.

ATP7A is a copper-transporting P-type ATPase that is essential for cellular copper homeostasis. Loss-of-function mutations in the ATP7A gene result in Menkes disease, a fatal neurodegenerative disorder resulting in seizures, hypotonia and failure to thrive, due to systemic copper deficiency. Most recently, rare missense mutations in ATP7A that do not impact systemic copper homeostasis have been shown to cause X-linked spinal muscular atrophy type 3 (SMAX3), a distal hereditary motor neuropathy. An understanding of the mechanistic and pathophysiological basis of SMAX3 is currently lacking, in part because the disease-causing mutations have been shown to confer both loss- and gain-of-function properties to ATP7A, and because there is currently no animal model of the disease. In this study, the Atp7a gene was specifically deleted in the motor neurons of mice, resulting in a degenerative phenotype consistent with the clinical features in affected patients with SMAX3, including the progressive deterioration of gait, age-dependent muscle atrophy, denervation of neuromuscular junctions and a loss of motor neuron cell bodies. Taken together, these data reveal autonomous requirements for ATP7A that reveal essential roles for copper in the maintenance and function of the motor neuron, and suggest that SMAX3 is caused by a loss of ATP7A function that specifically impacts the spinal motor neuron.

Autonomous requirements of the Menkes disease protein in the nervous system.

Menkes disease is a fatal neurodegenerative disorder arising from a systemic copper deficiency caused by loss-of-function mutations in a ubiquitously expressed copper transporter, ATP7A. Although this disorder reveals an essential role for copper in the developing human nervous system, the role of ATP7A in the pathogenesis of signs and symptoms in affected patients, including severe mental retardation, ataxia, and excitotoxic seizures, remains unknown. To directly examine the role of ATP7A within the central nervous system, we generated Atp7a(Nes) mice, in which the Atp7a gene was specifically deleted within neural and glial cell precursors without impairing systemic copper homeostasis, and compared these mice with the mottled brindle (mo-br) mutant, a murine model of Menkes disease in which Atp7a is defective in all cells. Whereas mo-br mice displayed neurodegeneration, demyelination, and 100% mortality prior to weaning, the Atp7a(Nes) mice showed none of these phenotypes, exhibiting only mild sensorimotor deficits, increased anxiety, and susceptibility to NMDA-induced seizure. Our results indicate that the pathophysiology of severe neurological signs and symptoms in Menkes disease is the result of copper deficiency within the central nervous system secondary to impaired systemic copper homeostasis and does not arise from an intrinsic lack of ATP7A within the developing brain. Furthermore, the sensorimotor deficits, hypophagia, anxiety, and sensitivity to NMDA-induced seizure in the Atp7a(Nes) mice reveal unique autonomous requirements for ATP7A in the nervous system. Taken together, these data reveal essential roles for copper acquisition in the central nervous system in early development and suggest novel therapeutic approaches in affected patients.

Expression of iron homeostasis proteins in the spinal cord in experimental autoimmune encephalomyelitis and their implications for iron accumulation.

Iron accumulation occurs in the CNS in multiple sclerosis (MS) and in experimental autoimmune encephalomyelitis (EAE). However, the mechanisms underlying such iron accumulation are not fully understood. We studied the expression and cellular localization of molecules involved in cellular iron influx, storage, and efflux. This was assessed in two mouse models of EAE: relapsing-remitting (RR-EAE) and chronic (CH-EAE). The expression of molecules involved in iron homeostasis was assessed at the onset, peak, remission/progressive and late stages of the disease. We provide several lines of evidence for iron accumulation in the EAE spinal cord which increases with disease progression and duration, is worse in CH-EAE, and is localized in macrophages and microglia. We also provide evidence that there is a disruption of the iron efflux mechanism in macrophages/microglia that underlie the iron accumulation seen in these cells. Macrophages/microglia also lack expression of the ferroxidases (ceruloplasmin and hephaestin) which have antioxidant effects. In contrast, astrocytes which do not accumulate iron, show robust expression of several iron influx and efflux proteins and the ferroxidase ceruloplasmin which detoxifies ferrous iron. Astrocytes therefore are capable of efficiently recycling iron from sites of EAE lesions likely into the circulation. We also provide evidence of marked dysregulation of mitochondrial function and energy metabolism genes, as well as of NADPH oxidase genes in the EAE spinal cord. This data provides the basis for the selective iron accumulation in macrophage/microglia and further evidence of severe mitochondrial dysfunction in EAE. It may provide insights into processes underling iron accumulation in MS and other neurodegenerative diseases in which iron accumulation occurs.

Overexpression of alpha-synuclein at non-toxic levels increases dopaminergic cell death induced by copper exposure via modulation of protein degradation pathways.

Gene multiplications or point mutations in alpha (α)-synuclein are associated with familial and sporadic Parkinson's disease (PD). An increase in copper (Cu) levels has been reported in the cerebrospinal fluid and blood of PD patients, while occupational exposure to Cu has been suggested to augment the risk to develop PD. We aimed to elucidate the mechanisms by which α-synuclein and Cu regulate dopaminergic cell death. Short-term overexpression of wild type (WT) or mutant A53T α-synuclein had no toxic effect in human dopaminergic cells and primary midbrain cultures, but it exerted a synergistic effect on Cu-induced cell death. Cell death induced by Cu was potentiated by overexpression of the Cu transporter protein 1 (Ctr1) and depletion of intracellular glutathione (GSH) indicating that the toxic effects of Cu are linked to alterations in its intracellular homeostasis. Using the redox sensor roGFP, we demonstrated that Cu-induced oxidative stress was primarily localized in the cytosol and not in the mitochondria. However, α-synuclein overexpression had no effect on Cu-induced oxidative stress. WT or A53T α-synuclein overexpression exacerbated Cu toxicity in dopaminergic and yeast cells in the absence of α-synuclein aggregation. Cu increased autophagic flux and protein ubiquitination. Impairment of autophagy by overexpression of a dominant negative Atg5 form or inhibition of the ubiquitin/proteasome system (UPS) with MG132 enhanced Cu-induced cell death. However, only inhibition of the UPS stimulated the synergistic toxic effects of Cu and α-synuclein overexpression. Our results demonstrate that α-synuclein stimulates Cu toxicity in dopaminergic cells independent from its aggregation via modulation of protein degradation pathways.

Cellular Zinc Deficiency Impairs Heme Biosynthesis in Developing Erythroid Progenitors.

Anemia is the most prevalent nutrition-related disorder worldwide. Zinc is an essential trace element for various biological processes in the body, and zinc deficiency has been associated with anemia in humans. However, the molecular mechanisms by which zinc availability alters red blood cell development remain uncertain. The present study identifies the essentiality of zinc during erythroid development, particularly for normal heme biosynthesis. G1E-ER4 mouse cells were used as an in vitro model of terminal erythroid differentiation, which featured elevated cellular zinc content by development. Restriction of zinc import compromised the rate of heme and α-globin production and, thus, the hemoglobinization of the erythroid progenitors. Heme is synthesized by the incorporation of iron into protoporphyrin. The lower heme production under zinc restriction was not due to changes in iron but was attributable to less porphyrin synthesis. The requirement of adequate zinc for erythroid heme metabolism was confirmed in another erythropoietic cell model, MEL-DS19. Additionally, we found that a conventional marker of iron deficiency anemia, the ZnPP-to-heme ratio, responded to zinc restriction differently from iron deficiency. Collectively, our findings define zinc as an essential nutrient integral to erythroid heme biosynthesis and, thus, a potential therapeutic target for treating anemia and other erythrocyte-related disorders.