Iron regulates the quiescence of naive CD4 T cells by controlling mitochondria and cellular metabolism.

In response to an immune challenge, naive T cells undergo a transition from a quiescent to an activated state acquiring the effector function. Concurrently, these T cells reprogram cellular metabolism, which is regulated by iron. We and others have shown that iron homeostasis controls proliferation and mitochondrial function, but the underlying mechanisms are poorly understood. Given that iron derived from heme makes up a large portion of the cellular iron pool, we investigated iron homeostasis in T cells using mice with a T cell-specific deletion of the heme exporter, FLVCR1 [referred to as knockout (KO)]. Our finding revealed that maintaining heme and iron homeostasis is essential to keep naive T cells in a quiescent state. KO naive CD4 T cells exhibited an iron-overloaded phenotype, with increased spontaneous proliferation and hyperactive mitochondria. This was evidenced by reduced IL-7R and IL-15R levels but increased CD5 and Nur77 expression. Upon activation, however, KO CD4 T cells have defects in proliferation, IL-2 production, and mitochondrial functions. Iron-overloaded CD4 T cells failed to induce mitochondrial iron and exhibited more fragmented mitochondria after activation, making them susceptible to ferroptosis. Iron overload also led to inefficient glycolysis and glutaminolysis but heightened activity in the hexosamine biosynthetic pathway. Overall, these findings highlight the essential role of iron in controlling mitochondrial function and cellular metabolism in naive CD4 T cells, critical for maintaining their quiescent state.

Minimizing higher-order aggregation maximizes iron mobilization by small molecules.

The natural product hinokitiol mobilizes iron across lipid bilayers at low concentrations and restores hemoglobinization in iron transporter protein-deficient systems. But hinokitiol fails to similarly mobilize iron at higher concentrations, limiting its uses in chemical biology and medicine. Here we show that at higher concentrations, hinokitiol3:Fe(III) complexes form large, higher-order aggregates, leading to loss of transmembrane iron mobilization. Guided by this understanding and systematic structure-function studies enabled by modular synthesis, we identified FeM-1269, which minimally aggregates and dose-dependently mobilizes iron across lipid bilayers even at very high concentrations. In contrast to hinokitiol, FeM-1269 is also well-tolerated in animals at high doses for extended periods of time. In a mouse model of anemia of inflammation, FeM-1269 increases serum iron, transferrin saturation, hemoglobin and hematocrit. This rationally developed iron-mobilizing small molecule has enhanced potential as a molecular prosthetic for understanding and potentially treating iron transporter deficiencies.

A small molecule redistributes iron in ferroportin-deficient mice and patient-derived primary macrophages.

Deficiencies of the transmembrane iron-transporting protein ferroportin (FPN1) cause the iron misdistribution that underlies ferroportin disease, anemia of inflammation, and several other human diseases and conditions. A small molecule natural product, hinokitiol, was recently shown to serve as a surrogate transmembrane iron transporter that can restore hemoglobinization in zebrafish deficient in other iron transporting proteins and can increase gut iron absorption in FPN1-deficient flatiron mice. However, whether hinokitiol can restore normal iron physiology in FPN1-deficient animals or primary cells from patients and the mechanisms underlying such targeted activities remain unknown. Here, we show that hinokitiol redistributes iron from the liver to red blood cells in flatiron mice, thereby increasing hemoglobin and hematocrit. Mechanistic studies confirm that hinokitiol functions as a surrogate transmembrane iron transporter to release iron trapped within liver macrophages, that hinokitiol-Fe complexes transfer iron to transferrin, and that the resulting transferrin-Fe complexes drive red blood cell maturation in a transferrin-receptor-dependent manner. We also show in FPN1-deficient primary macrophages derived from patients with ferroportin disease that hinokitiol moves labile iron from inside to outside cells and decreases intracellular ferritin levels. The mobilization of nonlabile iron is accompanied by reductions in intracellular ferritin, consistent with the activation of regulated ferritin proteolysis. These findings collectively provide foundational support for the translation of small molecule iron transporters into therapies for human diseases caused by iron misdistribution.

Manganese and Sleep Outcomes in United States Adults: Results from the 2017-2020 National Health and Nutrition Examination Survey (NHANES).

BACKGROUND: Manganese (Mn) is an essential micronutrient, but inadequate or excess Mn intake can have a detrimental impact on human health. Despite the essentiality, little is known about the relationship between Mn and sleep. OBJECTIVE: This study aimed to examine the relationship between blood Mn concentrations and sleep outcomes in US adults. METHODS: This cross-sectional study used data on blood Mn and sleep from the 2017-2020 National Health and Nutrition Examination Survey (NHANES) (n = 8356, age ≥18 y). Multivariable logistic regression was used to examine associations between quintiles of blood Mn concentrations and subjective sleep outcomes (short sleep duration, late sleep midpoint, trouble sleeping, and obstructive sleep apnea [OSA] symptoms), adjusting for age, gender, body mass index, race/ethnicity, income, smoking, inflammation-adjusted serum ferritin concentration (iron status), caffeine, and alcohol intake. Gender-stratified models were used due to interactions with gender. RESULTS: The mean (SE) blood Mn concentration was 9.7 (0.1) µg/L in US adults. In males, a nonlinear association was noted in the relationship between blood Mn levels and short sleep duration on weekdays and weekends. The third Mn quintile (Q3) group had lower odds of short sleep duration (<7 h) on weekdays (odds ratio [OR]=0.6, 95% confidence interval [CI]: 0.4, 0.9) than the lowest Mn quintile (Q1, reference) after adjusting for covariates in males. The second Mn quintile (Q2) group had lower odds of late sleep midpoint on weekdays than Q1 (OR=0.6, 95% CI: 0.4, 0.8). In females, Q2 group had lower odds of OSA symptoms than Q1 (OR: 0.6, 95% CI: 0.4, 0.9). No relationship was noted between Mn and trouble sleeping. CONCLUSIONS: Gender differences exist in the association between Mn and sleep in adults. Q1 group had the poorest sleep outcomes, including higher odds of short sleep duration (in males), late sleep midpoint (in males), and OSA symptoms (in females).

A neurodegeneration gene, WDR45, links impaired ferritinophagy to iron accumulation.

Neurodegeneration with brain iron accumulation (NBIA) is a clinically and genetically heterogeneous group of neurodegenerative diseases characterized by the abnormal accumulation of brain iron and the progressive degeneration of the nervous system. One of the recently identified subtypes of NBIA is ß-propeller protein-associated neurodegeneration (BPAN). BPAN is caused by de novo mutations in the WDR45/WIPI4 (WD repeat domain 45) gene. WDR45 is one of the four mammalian homologs of yeast Atg18, a regulator of autophagy. WDR45 deficiency in BPAN patients and animal models may result in defects in autophagic flux. However, how WDR45 deficiency leads to brain iron overload remains unclear. To elucidate the role of WDR45, we generated a WDR45-knockout (KO) SH-SY5Y neuroblastoma cell line using CRISPR-Cas9-mediated genome editing. Using these cells, we demonstrated that the non-TF (transferrin)-bound iron pathway dominantly mediated the accumulation of iron. Moreover, the loss of WDR45 led to defects in ferritinophagy, a form of autophagy that degrades the iron storage protein ferritin. We showed that impaired ferritinophagy contributes to iron accumulation in WDR45-KO cells. Iron accumulation was also detected in the mitochondria, which was accompanied by impaired mitochondrial respiration, elevated reactive oxygen species, and increased cell death. Thus, our study links WDR45 to specific iron acquisition pathways and ferritinophagy. Cover Image for this issue: https://doi.org/10.1111/jnc.15388.

Cutting Edge: Activation-Induced Iron Flux Controls CD4 T Cell Proliferation by Promoting Proper IL-2R Signaling and Mitochondrial Function.

Iron has long been established as a critical mediator of T cell development and proliferation. However, the mechanisms by which iron controls CD4 T cell activation and expansion remain poorly understood. In this study, we show that stimulation of CD4 T cells from C57BL/6 mice not only decreases total and labile iron levels but also leads to changes in the expression of iron homeostatic machinery. Additionally, restraining iron availability in vitro severely inhibited CD4 T cell proliferation and cell cycle progression. Although modulating cellular iron levels increased IL-2 production by activated T lymphocytes, CD25 expression and pSTAT5 levels were decreased, indicating that iron is necessary for IL-2R-mediated signaling. We also found that iron deprivation during T cell stimulation negatively impacts mitochondrial function, which can be reversed by iron supplementation. In all, we show that iron contributes to activation-induced T cell expansion by positively regulating IL-2R signaling and mitochondrial function.

Impact of dietary manganese on experimental colitis in mice.

Diet plays a significant role in the pathogenesis of inflammatory bowel disease (IBD). A recent epidemiological study has shown an inverse relationship between nutritional manganese (Mn) status and IBD patients. Mn is an essential micronutrient required for normal cell function and physiological processes. To date, the roles of Mn in intestinal homeostasis remain unknown and the contribution of Mn to IBD has yet to be explored. Here, we provide evidence that Mn is critical for the maintenance of the intestinal barrier and that Mn deficiency exacerbates dextran sulfate sodium (DSS)-induced colitis in mice. Specifically, when treated with DSS, Mn-deficient mice showed increased morbidity, weight loss, and colon injury, with a concomitant increase in inflammatory cytokine levels and oxidative and DNA damage. Even without DSS treatment, dietary Mn deficiency alone increased intestinal permeability by impairing intestinal tight junctions. In contrast, mice fed a Mn-supplemented diet showed slightly increased tolerance to DSS-induced experimental colitis, as judged by the colon length. Despite the well-appreciated roles of intestinal microbiota in driving inflammation in IBD, the gut microbiome composition was not altered by changes in dietary Mn. We conclude that Mn is necessary for proper maintenance of the intestinal barrier and provides protection against DSS-induced colon injury.

Serum selenium and non-alcoholic fatty liver disease (NAFLD) in U.S. adults: National Health and Nutrition Examination Survey (NHANES) 2011-2016.

BACKGROUND: Selenium is an essential trace element that shows beneficial or adverse health effects depending on the dose. Laboratory studies suggest that high selenium may contribute to the development of non-alcoholic fatty liver disease (NAFLD). However, human evidence is limited. We evaluated the associations of serum selenium level with serum alanine aminotransferase (ALT) activity and suspected NAFLD prevalence in U.S. adults. METHODS: We conducted the cross-sectional analysis in 3827 adults aged 20 years and older without viral hepatitis, hemochromatosis, or alcoholic liver disease who participated in the National Health and Nutrition Examination Survey (NHANES) 2011-2012, 2013-2014, and 2015-2016. Serum selenium was measured using inductively coupled plasma dynamic reaction cell mass spectrometry. Suspected NAFLD cases were defined in the presence of serum ALT >30 international units (IU)/L in men and >19 I.U./L in women in the absence of other identifiable causes of liver disease. RESULTS: The median (interquartile range) of serum selenium level was 127.9 (117.9, 139.4) µg/L. Non-linear associations of serum selenium with NAFLD prevalence and serum ALT activity were observed in the generalized additive models with penalized splines. After adjustment for sociodemographic variables, lifestyle factors, body mass index, and NHANES survey cycles, positive associations were found at > ~130 µg/L serum selenium with both NAFLD and ALT, whereas the associations were flattened at < ~130 µg/L. CONCLUSIONS: Our findings provide evidence of non-linear associations of serum selenium with ALT activity and NAFLD prevalence. In particular, positive associations were found above serum selenium level of 130 µg/L, whereas no association was observed below this value. This finding requires confirmation in future prospective cohort studies.

Genome-Wide Association Meta-Analysis of Individuals of European Ancestry Identifies Suggestive Loci for Sodium Intake, Potassium Intake, and Their Ratio Measured from 24-Hour or Half-Day Urine Samples.

BACKGROUND: Excess sodium intake and insufficient potassium intake are risk factors for hypertension, but there is limited knowledge regarding genetic factors that influence intake. Twenty-hour or half-day urine samples provide robust estimates of sodium and potassium intake, outperforming other measures such as spot urine samples and dietary self-reporting. OBJECTIVE: The aim of this study was to investigate genomic regions associated with sodium intake, potassium intake, and sodium-to-potassium ratio measured from 24-h or half-day urine samples. METHODS: Using samples of European ancestry (mean age: 54.2 y; 52.3% women), we conducted a meta-analysis of genome-wide association studies in 4 cohorts with 24-h or half-day urine samples (n = 6,519), followed by gene-based analysis. Suggestive loci (P < 10-6) were examined in additional European (n = 844), African (n = 1,246), and Asian (n = 2,475) ancestry samples. RESULTS: We found suggestive loci (P < 10-6) for all 3 traits, including 7 for 24-h sodium excretion, 4 for 24-h potassium excretion, and 4 for sodium-to-potassium ratio. The most significant locus was rs77958157 near cocaine- and amphetamine-regulated transcript prepropeptide (CARTPT) , a gene involved in eating behavior and appetite regulation (P = 2.3 × 10-8 with sodium-to-potassium ratio). Two suggestive loci were replicated in additional samples: for sodium excretion, rs12094702 near zinc finger SWIM-type containing 5 (ZSWIM5) was replicated in the Asian ancestry sample reaching Bonferroni-corrected significance (P = 0.007), and for potassium excretion rs34473523 near sodium leak channel (NALCN) was associated at a nominal P value with potassium excretion both in European (P = 0.043) and African (P = 0.043) ancestry cohorts. Gene-based tests identified 1 significant gene for sodium excretion, CDC42 small effector 1 (CDC42SE1), which is associated with blood pressure regulation. CONCLUSIONS: We identified multiple suggestive loci for sodium and potassium intake near genes associated with eating behavior, nervous system development and function, and blood pressure regulation in individuals of European ancestry. Further research is needed to replicate these findings and to provide insight into the underlying genetic mechanisms by which these genomic regions influence sodium and potassium intake.

Ferroportin disease mutations influence manganese accumulation and cytotoxicity.

Hemochromatosis is a frequent genetic disorder, characterized by the accumulation of excess iron across tissues. Mutations in the FPN1 gene, encoding a cell surface iron exporter [ferroportin (Fpn)], are responsible for hemochromatosis type 4, also known as ferroportin disease. Recently, Fpn has been implicated in the regulation of manganese (Mn), another essential nutrient required for numerous cellular enzymes. However, the roles of Fpn in Mn regulation remain ill-defined, and the impact of disease mutations on cellular Mn levels is unknown. Here, we provide evidence that Fpn can export Mn from cells into extracellular space. Fpn seems to play protective roles in Mn-induced cellular toxicity and oxidative stress. Finally, disease mutations interfere with the role of Fpn in controlling Mn levels as well as the stability of Fpn. These results define the function of Fpn as an exporter of both iron and Mn and highlight the potential involvement of Mn dysregulation in ferroportin disease.-Choi, E.-K., Nguyen, T.-T., Iwase, S., Seo, Y. A. Ferroportin disease mutations influence manganese accumulation and cytotoxicity.

Regulation of divalent metal transporter-1 by serine phosphorylation.

Divalent metal transporter-1 (DMT1) mediates dietary iron uptake across the intestinal mucosa and facilitates peripheral delivery of iron released by transferrin in the endosome. Here, we report that classical cannabinoids (Δ9-tetrahydrocannabinol, Δ9-THC), nonclassical cannabinoids (CP 55,940), aminoalkylindoles (WIN 55,212-2) and endocannabinoids (anandamide) reduce 55Fe and 54Mn uptake by HEK293T(DMT1) cells stably expressing the transporter. siRNA knockdown of cannabinoid receptor type 2 (CB2) abrogated inhibition. CB2 is a G-protein (GTP-binding protein)-coupled receptor that negatively regulates signal transduction cascades involving serine/threonine kinases. Immunoprecipitation experiments showed that DMT1 is serine-phosphorylated under basal conditions, but that treatment with Δ9-THC reduced phosphorylation. Site-directed mutation of predicted DMT1 phosphosites further showed that substitution of serine with alanine at N-terminal position 43 (S43A) abolished basal phosphorylation. Concordantly, both the rate and extent of 55Fe uptake in cells expressing DMT1(S43A) was reduced compared with those expressing wild-type DMT1. Among kinase inhibitors that affected DMT1-mediated iron uptake, staurosporine also reduced DMT1 phosphorylation confirming a role for serine phosphorylation in iron transport regulation. These combined data indicate that phosphorylation at serine 43 of DMT1 promotes transport activity, whereas dephosphorylation is associated with loss of iron uptake. Since anti-inflammatory actions mediated through CB2 would be associated with reduced DMT1 phosphorylation, we postulate that this pathway provides a means to reduce oxidative stress by limiting iron uptake.

Dietary supplementation with ipriflavone decreases hepatic iron stores in wild type mice.

Hepcidin, a peptide produced in the liver, decreases intestinal iron absorption and macrophage iron release by causing degradation of the iron exporter, ferroportin. Because its levels are inappropriately low in patients with iron overload syndromes, hepcidin is a potential drug target. We previously conducted a chemical screen that revealed ipriflavone, an orally available small molecule, as a potent inducer of hepcidin expression. To evaluate ipriflavone's effect on iron homeostasis, we placed groups of 5-week old wild type or thalassemia intermedia (Hbb(Th3+/-)) mice on a soy-free, iron-sufficient diet, AIN-93G containing 220mg iron and 0-750mgipriflavone/kg of food for 50days. Ipriflavone 500mg/kg significantly reduced liver iron stores and intestinal ferroportin expression in WT mice, while increasing the ratio of hepcidin transcript levels to liver iron stores. Ipriflavone supplementation in Hbb(Th3+/-) mice failed to alleviate iron overload and was associated with a milder reduction in intestinal ferroportin and a failure to alter the ratio of hepcidin transcript levels to liver iron stores or splenic expression of the hepcidin-regulatory hormone, erythroferrone. These data suggest that dietary supplementation with ipriflavone alone would not be sufficient to treat iron overload in thalassemia intermedia.

Ferroportin deficiency impairs manganese metabolism in flatiron mice.

We examined the physiologic role of ferroportin (Fpn) in manganese (Mn) export using flatiron (ffe/+) mice, a genetic model of Fpn deficiency. Blood (0.0123 vs. 0.0107 mg/kg; P = 0.0003), hepatic (1.06 vs. 0.96 mg/kg; P = 0.0125), and bile Mn levels (79 vs. 38 mg/kg; P = 0.0204) were reduced in ffe/+ mice compared to +/+ controls. Erythrocyte Mn-superoxide dismutase was also reduced at 6 (0.154 vs. 0.096, P = 0.0101), 9 (0.131 vs. 0.089, P = 0.0162), and 16 weeks of age (0.170 vs. 0.090 units/mg protein/min; P < 0.0001). (54)Mn uptake after intragastric gavage was markedly reduced in ffe/+ mice (0.0187 vs. 0.0066% dose; P = 0.0243), while clearance of injected isotope was similar in ffe/+ and +/+ mice. These values were compared to intestinal absorption of (59)Fe, which was significantly reduced in ffe/+ mice (8.751 vs. 3.978% dose; P = 0.0458). The influence of the ffe mutation was examined in dopaminergic SH-SY5Y cells and human embryonic HEK293T cells. While expression of wild-type Fpn reversed Mn-induced cytotoxicity, ffe mutant H32R failed to confer protection. These combined results demonstrate that Fpn plays a central role in Mn transport and that flatiron mice provide an excellent genetic model to explore the role of this exporter in Mn homeostasis. -

Distribution of manganese and other biometals in flatiron mice.

Flatiron (ffe) mice display features of "ferroportin disease" or Type IV hereditary hemochromatosis. While it is known that both Fe and Mn metabolism are impaired in flatiron mice, the effects of ferroportin (Fpn) deficiency on physiological distribution of these and other biometals is unknown. We hypothesized that Fe, Mn, Zn and/or Cu distribution would be altered in ffe/+ compared to wild-type (+/+) mice. ICP-MS analysis showed that Mn, Zn and Cu levels were significantly reduced in femurs from ffe/+ mice. Bone deposits reflect metal accumulation, therefore these data indicate that Mn, Zn and Cu metabolism are affected by Fpn deficiency. The observations that muscle Cu, lung Mn, and kidney Cu and Zn levels were reduced in ffe/+ mice support the idea that metal metabolism is impaired. While all four biometals appeared to accumulate in brains of flatiron mice, significant gender effects were observed for Mn and Zn levels in male ffe/+ mice. Metals were higher in olfactory bulbs of ffe/+ mice regardless of gender. To further study brain metal distribution, (54)MnCl2 was administered by intravenous injection and total brain (54)Mn was measured over time. At 72 h, (54)Mn was significantly greater in brains of ffe/+ mice compared to +/+ mice while blood (54)Mn was cleared to the same levels by 24 h. Taken together, these results indicate that Fpn deficiency decreases Mn trafficking out of the brain, alters body Fe, Mn, Zn and Cu levels, and promotes metal accumulation in olfactory bulbs.

Prolactin (PRL)-stimulated ubiquitination of ZnT2 mediates a transient increase in zinc secretion followed by ZnT2 degradation in mammary epithelial cells.

The zinc transporter ZnT2 imports zinc into secretory vesicles and regulates zinc export from the mammary epithelial cell. Mutations in ZnT2 substantially impair zinc secretion into milk. The lactogenic hormone prolactin (PRL) transcriptionally increases ZnT2 expression through the Jak2/STAT5 signaling pathway, increasing zinc accumulation in secretory vesicles and zinc secretion. Herein, we report that PRL post-translationally stimulated ZnT2 ubiquitination, which altered ZnT2 trafficking and augmented vesicular zinc accumulation and secretion from mammary epithelial cells in a transient manner. Ubiquitination then down-regulated zinc secretion by stimulating degradation of ZnT2. Mutagenesis of two N-terminal lysine residues (K4R and K6R) inhibited ZnT2 ubiquitination, vesicular zinc accumulation and secretion, and protein degradation. These findings establish that PRL post-translationally regulates ZnT2-mediated zinc secretion in a multifactorial manner, first by enhancing zinc accumulation in vesicles to transiently enhance zinc secretion and then by activating ubiquitin-dependent ZnT2 degradation. This provides insight into novel mechanisms through which ZnT2 and zinc transport is tightly regulated in mammary epithelial cells.

The manganese transporter SLC39A8 links alkaline ceramidase 1 to inflammatory bowel disease.

The metal ion transporter SLC39A8 is associated with physiological traits and diseases, including blood manganese (Mn) levels and inflammatory bowel diseases (IBD). The mechanisms by which SLC39A8 controls Mn homeostasis and epithelial integrity remain elusive. Here, we generate Slc39a8 intestinal epithelial cell-specific-knockout (Slc39a8-IEC KO) mice, which display markedly decreased Mn levels in blood and most organs. Radiotracer studies reveal impaired intestinal absorption of dietary Mn in Slc39a8-IEC KO mice. SLC39A8 is localized to the apical membrane and mediates 54Mn uptake in intestinal organoid monolayer cultures. Unbiased transcriptomic analysis identifies alkaline ceramidase 1 (ACER1), a key enzyme in sphingolipid metabolism, as a potential therapeutic target for SLC39A8-associated IBDs. Importantly, treatment with an ACER1 inhibitor attenuates colitis in Slc39a8-IEC KO mice by remedying barrier dysfunction. Our results highlight the essential roles of SLC39A8 in intestinal Mn absorption and epithelial integrity and offer a therapeutic target for IBD associated with impaired Mn homeostasis.

A dominant negative heterozygous G87R mutation in the zinc transporter, ZnT-2 (SLC30A2), results in transient neonatal zinc deficiency.

Zinc is an essential mineral, and infants are particularly vulnerable to zinc deficiency as they require large amounts of zinc for their normal growth and development. We have recently described the first loss-of-function mutation (H54R) in the zinc transporter ZnT-2 (SLC30A2) in mothers with infants harboring transient neonatal zinc deficiency (TNZD). Here we identified and characterized a novel heterozygous G87R ZnT-2 mutation in two unrelated Ashkenazi Jewish mothers with infants displaying TNZD. Transient transfection of G87R ZnT-2 resulted in endoplasmic reticulum-Golgi retention, whereas the WT transporter properly localized to intracellular secretory vesicles in HC11 and MCF-7 cells. Consequently, G87R ZnT-2 showed decreased stability compared with WT ZnT-2 as revealed by Western blot analysis. Three-dimensional homology modeling based on the crystal structure of YiiP, a close zinc transporter homologue from Escherichia coli, revealed that the basic arginine residue of the mutant G87R points toward the membrane lipid core, suggesting misfolding and possible loss-of-function. Indeed, functional assays including vesicular zinc accumulation, zinc secretion, and cytoplasmic zinc pool assessment revealed markedly impaired zinc transport in G87R ZnT-2 transfectants. Moreover, co-transfection experiments with both mutant and WT transporters revealed a dominant negative effect of G87R ZnT-2 over the WT ZnT-2; this was associated with mislocalization, decreased stability, and loss of zinc transport activity of the WT ZnT-2 due to homodimerization observed upon immunoprecipitation experiments. These findings establish that inactivating ZnT-2 mutations are an underlying basis of TNZD and provide the first evidence for the dominant inheritance of heterozygous ZnT-2 mutations via negative dominance due to homodimer formation.

In vitro studies of manganese transport and homeostasis.

Manganese (Mn) is an essential micronutrient required for fundamental cell functions and vital physiological processes. More than a dozen putative Mn transporters have been described over the last two decades, but few have been thoroughly evaluated. Recent genetic studies have revealed vital roles for solute carrier family 39, member 8 (SLC39A8) in Mn homeostasis. SLC39A8 can mediate the cellular uptake of the essential metals zinc, iron, and Mn, as well as the non-essential metal cadmium. However, loss-of-function mutations in SLC39A8 have been found in patients with severe Mn deficiency in the blood without affecting other metals. An in vitro study from our laboratory showed that SLC39A8 is a cell-surface transporter that strongly stimulates 54Mn incorporation into cells (Choi, Nguyen, Gupta, Iwase, & Seo, 2018). By contrast, the disease-associated mutations completely abrogated the cellular uptake of 54Mn (Choi et al., 2018), thereby providing a causal link between SLC39A8 deficiency and Mn deficiency. The importance of SLC39A8 is now increasingly recognized in multiple disease processes, and SLC39A8 has emerged as a critical regulator of Mn homeostasis. Thus, exploring the function of SLC39A8 in cellular Mn homeostasis is of significant research interest. This chapter describes the advanced methods used in our laboratory to examine Mn homeostasis and transport. Specifically, genetic and molecular approaches are described in HeLa cells overexpressing SLC39A8 and disease-associated SLC39A8 mutants. These methods are useful for characterizing the roles of Mn in diverse cellular events.

Marginal maternal zinc deficiency in lactating mice reduces secretory capacity and alters milk composition.

Dietary analysis predicts that marginal Zn deficiency is common in women of reproductive age. The lack of reliable biomarkers limits the capacity to assess Zn status and consequently understand effects of maternal Zn deficiency. We determined effects of marginal maternal Zn deficiency on mammary gland function, milk secretion, and milk composition in mice. Mice (n = 12/diet) were fed marginal (ZD; 15 mg Zn/kg diet) or adequate (ZA; 30 mg Zn/kg diet) Zn diets for 30 d prior to conception through mid-lactation. Mice fed the ZD had a higher plasma Zn concentration (~20%; P < 0.05) but lower milk Zn concentration (~15%; P < 0.05) compared with mice fed the ZA. ZnT2 abundance was higher (P < 0.05) in mice fed the ZD compared with mice fed the ZA; no effect on ZnT4 abundance was detected. The Zn concentration of mammary gland mitochondria tended to be ~40% greater in mice fed ZD (P = 0.07); this was associated with apoptosis and lower milk secretion (~80%; P < 0.01). Total milk protein was ~25% higher (P < 0.05), although the abundance of the major milk proteins (caseins and whey acidic protein) was lower (P < 0.05) in mice fed the ZD. Proteomic analysis of milk proteins revealed an increase (P < 0.05) in four proteins in mice fed the ZD. These findings illustrate that marginal maternal Zn deficiency compromises mammary gland function and milk secretion and alters milk composition. This suggests that lactating women who consume inadequate Zn may not produce and/or secrete an adequate amount of high quality milk to provide optimal nutrition to their developing infant.

A histidine-rich motif mediates mitochondrial localization of ZnT2 to modulate mitochondrial function.

Female reproductive tissues such as mammary glands, ovaries, uterus, and placenta are phenotypically dynamic, requiring tight integration of bioenergetic and apoptotic mechanisms. Mitochondrial zinc (Zn) pools have emerged as a central player in regulating bioenergetics and apoptosis. Zn must first be imported into mitochondria to modulate mitochondrion-specific functions; however, mitochondrial Zn import mechanisms have not been identified. Here we documented that the Zn transporter ZnT2 is associated with the inner mitochondrial membrane and acts as an auxiliary Zn importer into mitochondria in mammary cells. We found that attenuation of ZnT2 expression significantly reduced mitochondrial Zn uptake and total mitochondrial Zn pools. Moreover, expression of a ZnT2-hemagglutinin (HA) fusion protein was localized to mitochondria and significantly increased Zn uptake and mitochondrial Zn pools, directly implicating ZnT2 in Zn import into mitochondria. Confocal microscopy of truncated and point mutants of ZnT2-green fluorescent protein (GFP) fusion proteins revealed a histidine-rich motif ((51)HHXH(54)) in the NH(2) terminus that is important for mitochondrial targeting of ZnT2. More importantly, the expansion of mitochondrial Zn pools by ZnT2 overexpression significantly reduced ATP biogenesis and mitochondrial oxidation concurrent with increased apoptosis, suggesting a functional role for ZnT2-mediated Zn import into mitochondria. These results identify the first Zn transporter directly associated with mitochondria and suggest that unique secretory tissues such as the mammary gland require novel mechanisms to modulate mitochondrion-specific functions.