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Brain manganese and the balance between essential roles and neurotoxicity

Open AccessPublished:March 18, 2020DOI:https://doi.org/10.1074/jbc.REV119.009453
      Manganese (Mn) is an essential micronutrient required for the normal development of many organs, including the brain. Although its roles as a cofactor in several enzymes and in maintaining optimal physiology are well-known, the overall biological functions of Mn are rather poorly understood. Alterations in body Mn status are associated with altered neuronal physiology and cognition in humans, and either overexposure or (more rarely) insufficiency can cause neurological dysfunction. The resultant balancing act can be viewed as a hormetic U-shaped relationship for biological Mn status and optimal brain health, with changes in the brain leading to physiological effects throughout the body and vice versa. This review discusses Mn homeostasis, biomarkers, molecular mechanisms of cellular transport, and neuropathological changes associated with disruptions of Mn homeostasis, especially in its excess, and identifies gaps in our understanding of the molecular and biochemical mechanisms underlying Mn homeostasis and neurotoxicity.

      Introduction

      Manganese is essential for numerous vital process including nerve and brain development and cognitive functioning. For most people, dietary consumption generally fulfills the requisite Mn intake (
      • Aschner J.L.
      • Aschner M.
      Nutritional aspects of manganese homeostasis.
      ). Even though Mn is crucial for maintaining optimal physiology, several aspects of the biology of the homeostatic control and toxicity of Mn remain unclear (
      • Underwood M.J.
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      • Thompson M.M.
      • Gershlick A.H.
      Reduction of vein graft intimal hyperplasia by ex vivo treatment with desferrioxamine manganese.
      ,
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      Manganese action in brain function.
      ). Unanswered questions include the subcellular and organelle distribution of Mn and the nature of cellular events that occur when deviations from Mn homeostasis occur (e.g. with exposure to chronic low levels of Mn). Mn has been implicated in the metabolism of proteins, lipids, and carbohydrates and acts as a cofactor for numerous kinases and other enzymes (
      • Wedler F.C.
      Biological significance of manganese in mammalian systems.
      ,
      • Keen C.L.
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      • Clegg M.S.
      Manganese metabolism in animals and humans including the toxicity of manganese.
      ). Because magnesium (Mg) and Mn share a resemblance in their physicochemical properties, a vast majority of enzymes can use Mg in lieu of Mn for their activation (
      • Takeda A.
      Manganese action in brain function.
      ).
      Mn also plays unique roles that cannot be replaced by other metals, such as in Mn-dependent enzymes, including arginase, agmatinase, glutamine synthetase, and Mn superoxide dismutase (MnSOD).
      The abbreviations used are: SOD
      superoxide dismutase
      DAergic
      dopaminergic
      PD
      Parkinson's disease
      MRI
      magnetic resonance imaging
      GABA
      γ-aminobutyric acid
      TH
      tyrosine hydroxylase
      DA
      dopamine
      D1R and D2R
      DA subtype 1 and 2 receptor, respectively
      DAT
      dopamine transporter
      IGF
      insulin-like growth factor
      AD
      Alzheimer's disease.
      As a result, its presence at optimal levels to support these functions is required, and the lack of this essential micronutrient can give rise to cognitive deficits (
      • Vollet K.
      • Haynes E.N.
      • Dietrich K.N.
      Manganese exposure and cognition across the lifespan: contemporary review and argument for biphasic dose-response health effects.
      ). Excess cellular levels of Mn are also detrimental, and this aspect of Mn-induced disease has gained attention in the field of toxicology. Mn accumulates in specific regions of the brain to selectively alter neurophysiology. Elevated brain Mn levels usually occur only following overexposure, which can result from numerous sources, including environmental sources, occupational exposures, or dietary exposures to Mn such as from contaminated drinking water.
      The consequences of Mn overexposure occur throughout the nervous system and can affect both motor function and higher-order cognitive functions. Motor control is disrupted via disruption of dopaminergic (DAergic) function (
      • Guilarte T.R.
      Manganese neurotoxicity: new perspectives from behavioral, neuroimaging, and neuropathological studies in humans and non-human primates.
      ). This includes clinical expression of parkinsonism in occupationally exposed workers (
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      Prevalence of parkinsonism and relationship to exposure in a large sample of Alabama welders.
      ). Occupational exposure to Mn has been linked to other unfavorable outcomes, including learning deficits and neurodegeneration (
      • Caito S.
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      Neurotoxicity of metals.
      ). The consequences of Mn overexposure occur throughout the nervous system and affect motor functions. This review is focused on neurotoxicity and presents evidence that Mn plays a key role in the maintenance of brain physiological homeostasis and is a primary target of this metal (
      • Roh E.
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      • Kim M.S.
      Emerging role of the brain in the homeostatic regulation of energy and glucose metabolism.
      ). Recent identification of genetic disorders of Mn metabolism combined with studies providing insights into the mechanisms of Mn neurotoxicity make a comprehensive review on this topic timely. This review illuminates the unfavorable outcomes Mn exposure causes. There is urgency to explore and address the potential changes that are occurring at a molecular level, as these unfavorable outcomes are leading to increased risk of diseases. This attempt requires an interdisciplinary approach where scientists with expertise in neuroscience, biology, and chemistry to come together to think about the problem at hand.

      Routes of manganese exposure and accumulation in the brain

      Beyond occupational exposures, excessive dietary or drinking water uptake of Mn is another source of overexposure. The World Health Organization recommends that the daily Mn consumption for an adult be between 0.7 and 10.9 mg (
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      Manganese in drinking-water: background document for development of WHO.
      ). Although use of Mn dietary supplementation containing greater than 20 mg of Mn has been reported in cases of osteoarthritis and osteoporosis (
      • Lehmann T.
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      IFNα treatment in systemic mastocytosis.
      ,
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      • Hammad T.A.
      Efficacy of a combination of FCHG49 glucosamine hydrochloride, TRH122 low molecular weight sodium chondroitin sulfate and manganese ascorbate in the management of knee osteoarthritis.
      ), to our knowledge, naturally occurring Mn deficiency has never been reported in humans. Ingested Mn has an absorption rate of 3–5% through the gastrointestinal tract and is subject to tight homeostatic control in vivo (
      • Finley J.W.
      • Johnson P.E.
      • Johnson L.K.
      Sex affects manganese absorption and retention by humans from a diet adequate in manganese.
      ,
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      • Zech L.
      • Greger J.L.
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      ). Systemic homeostatic control of Mn is a balance between transport across the enterocytes lining the intestinal wall and removal by the liver (
      • Papavasiliou P.S.
      • Miller S.T.
      • Cotzias G.C.
      Role of liver in regulating distribution and excretion of manganese.
      ). Several factors can influence the oral uptake of Mn, including iron (Fe) status, dietary matrix, bioavailability, and existing body burden of Mn (
      • Horning K.J.
      • Caito S.W.
      • Tipps K.G.
      • Bowman A.B.
      • Aschner M.
      Manganese is essential for neuronal health.
      ). Following oral intake, Mn is distributed widely to tissues, and its metabolism may also involve cycling between Mn2+ and Mn3+, although only a small fraction is found in the 3+ oxidation state (
      • Gibbons R.A.
      • Dixon S.N.
      • Hallis K.
      • Russell A.M.
      • Sansom B.F.
      • Symonds H.W.
      Manganese metabolism in cows and goats.
      ). The oxidation state of Mn exposure is a critical determinant of Mn toxicokinetics, tissue toxicodynamics, and toxicity (
      • Reaney S.H.
      • Bench G.
      • Smith D.R.
      Brain accumulation and toxicity of Mn(II) and Mn(III) exposures.
      ). Contaminated drinking water is another source of Mn exposure (
      • Wasserman G.A.
      • Liu X.
      • Parvez F.
      • Ahsan H.
      • Levy D.
      • Factor-Litvak P.
      • Kline J.
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      • Slavkovich V.
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      • Cheng Z.
      • Zheng Y.
      • Graziano J.H.
      Water manganese exposure and children's intellectual function in Araihazar, Bangladesh.
      ,
      • Khan K.
      • Wasserman G.A.
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      • Ahmed E.
      • Parvez F.
      • Slavkovich V.
      • Levy D.
      • Mey J.
      • van Geen A.
      • Graziano J.H.
      • Factor-Litvak P.
      Manganese exposure from drinking water and children's academic achievement.
      ). Metal concentrations in water vary by location, with Mn ranging from 0.0001 to 0.1 mg/liter (
      • Horning K.J.
      • Caito S.W.
      • Tipps K.G.
      • Bowman A.B.
      • Aschner M.
      Manganese is essential for neuronal health.
      ). Although the United States Environmental Protection Agency does not consider Mn to be a primary drinking water contaminant, the standard concentration of allowable Mn is 0.05 mg/liter (
      • National Center for Environmental Assessment
      Manganese (CASRN 7439-96-5).
      ,
      • Environmental Protection Agency
      ). There are arguments that the current Mn reference concentration guidelines need to be re-evaluated to consider Mn as a potential contaminant (
      • Ljung K.
      • Vahter M.
      Time to re-evaluate the guideline value for manganese in drinking water?.
      ), specifically when using water for infant formulas that already contain high levels of Mn (
      • Collipp P.J.
      • Chen S.Y.
      • Maitinsky S.
      Manganese in infant formulas and learning disability.
      ). Inhalation is the predominant route of exposure associated with the toxic effects of Mn in adults. Current data suggest that Mn exposure via drinking water in children/adolescents is as important as inhalation exposure under environmental and occupational settings (
      • Leonhard M.J.
      • Chang E.T.
      • Loccisano A.E.
      • Garry M.R.
      A systematic literature review of epidemiologic studies of developmental manganese exposure and neurodevelopmental outcomes.
      ). Excess environmental exposure may arise from air pollution (
      • Haynes E.N.
      • Sucharew H.
      • Kuhnell P.
      • Alden J.
      • Barnas M.
      • Wright R.O.
      • Parsons P.J.
      • Aldous K.M.
      • Praamsma M.L.
      • Beidler C.
      • Dietrich K.N.
      Manganese exposure and neurocognitive outcomes in rural school-age children: the communities actively researching exposure study (Ohio, U.S.A.).
      ,
      • Torres-Agustín R.
      • Rodríguez-Agudelo Y.
      • Schilmann A.
      • Solís-Vivanco R.
      • Montes S.
      • Riojas-Rodríguez H.
      • Cortez-Lugo M.
      • Ríos C.
      Effect of environmental manganese exposure on verbal learning and memory in Mexican children.
      ,
      • Carvalho C.F.
      • Menezes-Filho J.A.
      • de Matos V.P.
      • Bessa J.R.
      • Coelho-Santos J.
      • Viana G.F.
      • Argollo N.
      • Abreu N.
      Elevated airborne manganese and low executive function in school-aged children in Brazil.
      ,
      • Solís-Vivanco R.
      • Rodríguez-Agudelo Y.
      • Riojas-Rodríguez H.
      • Ríos C.
      • Rosas I.
      • Montes S.
      Cognitive impairment in an adult Mexican population non-occupationally exposed to manganese.
      ), gasoline enhanced with methylcyclopentadienyl manganese tricarbonyl (MMT) (
      • Davis J.M.
      Methylcyclopentadienyl manganese tricarbonyl: health risk uncertainties and research directions.
      ,
      • Gulson B.
      • Mizon K.
      • Taylor A.
      • Korsch M.
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      • Davis J.M.
      • Louie H.
      • Wu M.
      • Swan H.
      Changes in manganese and lead in the environment and young children associated with the introduction of methylcyclopentadienyl manganese tricarbonyl in gasoline–preliminary results.
      ), and agricultural fungicides (
      • Mora A.M.
      • Arora M.
      • Harley K.G.
      • Kogut K.
      • Parra K.
      • Hernández-Bonilla D.
      • Gunier R.B.
      • Bradman A.
      • Smith D.R.
      • Eskenazi B.
      Prenatal and postnatal manganese teeth levels and neurodevelopment at 7, 9, and 10.5 years in the CHAMACOS cohort.
      ,
      • Gunier R.B.
      • Arora M.
      • Jerrett M.
      • Bradman A.
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      • Kogut K.
      • Hubbard A.
      • Austin C.
      • Holland N.
      • Eskenazi B.
      Manganese in teeth and neurodevelopment in young Mexican-American children.
      ). Many of the initial epidemiologic studies of Mn and health outcomes examined occupational inhalation exposures within industries such as ferromanganese welding, mining, and refining (
      • Rodier J.
      Manganese poisoning in Moroccan miners.
      ,
      • Zoni S.
      • Albini E.
      • Lucchini R.
      Neuropsychological testing for the assessment of manganese neurotoxicity: a review and a proposal.
      ,
      • Iregren A.
      Psychological test performance in foundry workers exposed to low levels of manganese.
      ,
      • Roels H.
      • Lauwerys R.
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      • Genet P.
      • Sarhan M.J.
      • Hanotiau I.
      • de Fays M.
      • Bernard A.
      • Stanescu D.
      Epidemiological survey among workers exposed to manganese: effects on lung, central nervous system, and some biological indices.
      ,
      • Racette B.A.
      • Searles Nielsen S.
      • Criswell S.R.
      • Sheppard L.
      • Seixas N.
      • Warden M.N.
      • Checkoway H.
      Dose-dependent progression of parkinsonism in manganese-exposed welders.
      ). Inhaled Mn enters the circulatory system through the nasal mucosa, bypasses the biliary excretion mechanism, and can cross the blood-brain barrier via several pathways, including facilitated diffusion and active transport from the olfactory bulb to the cerebral cortex (
      • Aschner M.
      The transport of manganese across the blood-brain barrier.
      ,
      • Davis J.M.
      Inhalation health risks of manganese: an EPA perspective.
      ,
      • Elder A.
      • Gelein R.
      • Silva V.
      • Feikert T.
      • Opanashuk L.
      • Carter J.
      • Potter R.
      • Maynard A.
      • Ito Y.
      • Finkelstein J.
      • Oberdörster G.
      Translocation of inhaled ultrafine manganese oxide particles to the central nervous system.
      ,
      • Dorman D.C.
      • Struve M.F.
      • Wong B.A.
      Brain manganese concentrations in rats following manganese tetroxide inhalation are unaffected by dietary manganese intake.
      ). Mn accumulates in Fe-rich brain regions of the basal ganglia: caudate, putamen, globus pallidus, substantia nigra, and subthalamic nuclei of the brain (
      • Elder A.
      • Gelein R.
      • Silva V.
      • Feikert T.
      • Opanashuk L.
      • Carter J.
      • Potter R.
      • Maynard A.
      • Ito Y.
      • Finkelstein J.
      • Oberdörster G.
      Translocation of inhaled ultrafine manganese oxide particles to the central nervous system.
      ,
      • Bock N.A.
      • Paiva F.F.
      • Nascimento G.C.
      • Newman J.D.
      • Silva A.C.
      Cerebrospinal fluid to brain transport of manganese in a non-human primate revealed by MRI.
      ,
      • Eriksson H.
      • Gillberg P.G.
      • Aquilonius S.M.
      • Hedström K.G.
      • Heilbronn E.
      Receptor alterations in manganese intoxicated monkeys.
      ). Under normal conditions, estimated concentrations of Mn in the human brain range from 5.32 to 14.03 ng of Mn/mg of protein, with 15.96–42.09 ng of Mn/mg of protein being the estimated pathophysiological threshold (
      • Williams M.
      • Todd G.D.
      • Roney N.
      • Crawford J.
      • Coles C.
      • McClure P.R.
      • Garey J.D.
      • Zaccaria K.
      • Citra M.
      ). Based on occupational studies of Mn exposure, the Occupational Safety and Health Administration exposure limit for general industry, construction industry, and shipyard employment is 5 mg/m3 (RRID:SCR_018203). However, even with Mn airborne levels near the Environmental Protection Agency's reference concentration of 0.05 μg/m3 (
      • National Center for Environmental Assessment
      Manganese (CASRN 7439-96-5).
      ), exposure can result in deficits in postural balance and neuropsychological and motor functions (
      • Hernández-Bonilla D.
      • Schilmann A.
      • Montes S.
      • Rodríguez-Agudelo Y.
      • Rodríguez-Dozal S.
      • Solís-Vivanco R.
      • Ríos C.
      • Riojas-Rodríguez H.
      Environmental exposure to manganese and motor function of children in Mexico.
      ,
      • Standridge J.S.
      • Bhattacharya A.
      • Succop P.
      • Cox C.
      • Haynes E.
      Effect of chronic low level manganese exposure on postural balance: a pilot study of residents in southern Ohio.
      ,
      • Lucchini R.G.
      • Guazzetti S.
      • Zoni S.
      • Donna F.
      • Peter S.
      • Zacco A.
      • Salmistraro M.
      • Bontempi E.
      • Zimmerman N.J.
      • Smith D.R.
      Tremor, olfactory and motor changes in Italian adolescents exposed to historical ferro-manganese emission.
      ) and increased risk for physician diagnosis of Parkinson's disease (PD) (
      • Finkelstein M.M.
      • Jerrett M.
      A study of the relationships between Parkinson's disease and markers of traffic-derived and environmental manganese air pollution in two Canadian cities.
      ). Deficits in neuromotor function are similar among adult Mn-exposed workers (
      • Huang C.C.
      Parkinsonism induced by chronic manganese intoxication—an experience in Taiwan.
      ,
      • Huang C.C.
      • Chu N.S.
      • Lu C.S.
      • Chen R.S.
      • Schulzer M.
      • Calne D.B.
      The natural history of neurological manganism over 18 years.
      ) and older PD patients (
      • Revilla F.J.
      • Larsh T.R.
      • Mani A.
      • Duker A.P.
      • Cox C.
      • Succop P.
      • Gartner M.
      • Jarrin Tejada C.
      • Bhattacharya A.
      Effect of dopaminergic medication on postural sway in advanced Parkinson's disease.
      ). Longitudinal and cross-sectional environmental exposure studies link low-level Mn exposure to deficits in intellectual development (
      • Vollet K.
      • Haynes E.N.
      • Dietrich K.N.
      Manganese exposure and cognition across the lifespan: contemporary review and argument for biphasic dose-response health effects.
      ), neurobehavior (
      • Oulhote Y.
      • Mergler D.
      • Barbeau B.
      • Bellinger D.C.
      • Bouffard T.
      • Brodeur M.È.
      • Saint-Amour D.
      • Legrand M.
      • Sauvé S.
      • Bouchard M.F.
      Neurobehavioral function in school-age children exposed to manganese in drinking water.
      ), neuromotor function (
      • Rugless F.
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      • Succop P.
      • Dietrich K.N.
      • Cox C.
      • Alden J.
      • Kuhnell P.
      • Barnas M.
      • Wright R.
      • Parsons P.J.
      • Praamsma M.L.
      • Palmer C.D.
      • Beidler C.
      • Wittberg R.
      • Haynes E.N.
      Childhood exposure to manganese and postural instability in children living near a ferromanganese refinery in Southeastern Ohio.
      ), and neuropsychiatric changes with respect to attention and mood (
      • Laohaudomchok W.
      • Lin X.
      • Herrick R.F.
      • Fang S.C.
      • Cavallari J.M.
      • Shrairman R.
      • Landau A.
      • Christiani D.C.
      • Weisskopf M.G.
      Neuropsychological effects of low-level manganese exposure in welders.
      ).
      Using magnetic resonance imaging (MRI), several studies have shown that significant brain accumulation of Mn in both humans and other animal models is associated with increased risk of neurotoxicity. These subjects often show a characteristic accumulation of manganese in the basal ganglia, particularly in the globus pallidus (
      • Ma R.E.
      • Ward E.J.
      • Yeh C.L.
      • Snyder S.
      • Long Z.
      • Gokalp Yavuz F.
      • Zauber S.E.
      • Dydak U.
      Thalamic GABA levels and occupational manganese neurotoxicity: association with exposure levels and brain MRI.
      ,
      • Olanow C.W.
      Manganese-induced parkinsonism and Parkinson's disease.
      ,
      • Criswell S.R.
      • Perlmutter J.S.
      • Huang J.L.
      • Golchin N.
      • Flores H.P.
      • Hobson A.
      • Aschner M.
      • Erikson K.M.
      • Checkoway H.
      • Racette B.A.
      Basal ganglia intensity indices and diffusion weighted imaging in manganese-exposed welders.
      ). Mn accumulation was also observed in the frontal cortex (Fig. 1) (
      • Ma R.E.
      • Ward E.J.
      • Yeh C.L.
      • Snyder S.
      • Long Z.
      • Gokalp Yavuz F.
      • Zauber S.E.
      • Dydak U.
      Thalamic GABA levels and occupational manganese neurotoxicity: association with exposure levels and brain MRI.
      ). Although recovery upon cessation is possible (
      • Han J.H.
      • Chung Y.H.
      • Park J.D.
      • Kim C.Y.
      • Yang S.O.
      • Khang H.S.
      • Cheong H.K.
      • Lee J.S.
      • Ha C.S.
      • Song C.W.
      • Kwon I.H.
      • Sung J.H.
      • Heo J.D.
      • Kim N.Y.
      • Huang M.
      • et al.
      Recovery from welding-fume-exposure-induced MRI T1 signal intensities after cessation of welding-fume exposure in brains of cynomolgus monkeys.
      ), this rarely happens in cases of occupational exposures, which are prolonged and cumulative. In rodent studies, areas of notable accumulation include the olfactory bulb, cerebellum, hippocampus and dentate gyrus, and pituitary gland in addition to the basal ganglia (
      • Finkelstein Y.
      • Zhang N.
      • Fitsanakis V.A.
      • Avison M.J.
      • Gore J.C.
      • Aschner M.
      Differential deposition of manganese in the rat brain following subchronic exposure to manganese: a T1-weighted magnetic resonance imaging study.
      ,
      • Fitsanakis V.A.
      • Zhang N.
      • Anderson J.G.
      • Erikson K.M.
      • Avison M.J.
      • Gore J.C.
      • Aschner M.
      Measuring brain manganese and iron accumulation in rats following 14 weeks of low-dose manganese treatment using atomic absorption spectroscopy and magnetic resonance imaging.
      ). Other studies in nonhuman primates have shown that inhaled ultrafine Mn particles translocate and accumulate in the olfactory bulb in addition to the striatum, frontal cortex, and cerebellum (
      • Elder A.
      • Gelein R.
      • Silva V.
      • Feikert T.
      • Opanashuk L.
      • Carter J.
      • Potter R.
      • Maynard A.
      • Ito Y.
      • Finkelstein J.
      • Oberdörster G.
      Translocation of inhaled ultrafine manganese oxide particles to the central nervous system.
      ). In addition to using MRI to analyze brain Mn levels, other noninvasive methods of measuring total body Mn burden based on bone levels are being used based on neutron activation analysis (
      • Liu Y.
      • Rolle-McFarland D.
      • Mostafaei F.
      • Zhou Y.
      • Li Y.
      • Zheng W.
      • Wells E.
      • Nie L.H.
      In vivo neutron activation analysis of bone manganese in workers.
      ). The next section details the biomarkers that are currently being used to assess Mn biological status.
      Figure thumbnail gr1
      Figure 1Schematic showing the sagittal section of human brain showing the brain regions where Mn predominantly accumulates (
      • Blomlie V.
      • Sivanandan R.
      • Jynge P.
      Manganese uptake and accumulation in the human brain.
      ,
      • Long Z.
      • Jiang Y.M.
      • Li X.R.
      • Fadel W.
      • Xu J.
      • Yeh C.L.
      • Long L.L.
      • Luo H.L.
      • Harezlak J.
      • Murdoch J.B.
      • Zheng W.
      • Dydak U.
      Vulnerability of welders to manganese exposure—a neuroimaging study.
      ). Dopamine is a key neurotransmitter that is produced in the substantia nigra, and the dopaminergic neurotransmitters project to the basal ganglia region.

      Biomarkers of brain and body Mn status

      Blood Mn levels are the most used indicator of exposure and can characterize the difference between exposed and unexposed subjects (
      • Baker M.G.
      • Simpson C.D.
      • Sheppard L.
      • Stover B.
      • Morton J.
      • Cocker J.
      • Seixas N.
      Variance components of short-term biomarkers of manganese exposure in an inception cohort of welding trainees.
      ). Blood Mn is reflective of recent exposure rather than total body burden due to the short half-life of Mn in blood, which is less than 2 h owing to rapid hepatic clearance (
      • Baker M.G.
      • Simpson C.D.
      • Stover B.
      • Sheppard L.
      • Checkoway H.
      • Racette B.A.
      • Seixas N.S.
      Blood manganese as an exposure biomarker: state of the evidence.
      ,
      • O'Neal S.L.
      • Zheng W.
      Manganese toxicity upon overexposure: a decade in review.
      ,
      • Smith D.
      • Gwiazda R.
      • Bowler R.
      • Roels H.
      • Park R.
      • Taicher C.
      • Lucchini R.
      Biomarkers of Mn exposure in humans.
      ). Hair Mn levels have been widely used to quantify chronic low levels of exposure, such as those commonly associated with deficits in cognition in children (
      • Haynes E.N.
      • Sucharew H.
      • Kuhnell P.
      • Alden J.
      • Barnas M.
      • Wright R.O.
      • Parsons P.J.
      • Aldous K.M.
      • Praamsma M.L.
      • Beidler C.
      • Dietrich K.N.
      Manganese exposure and neurocognitive outcomes in rural school-age children: the communities actively researching exposure study (Ohio, U.S.A.).
      ,
      • Torres-Agustín R.
      • Rodríguez-Agudelo Y.
      • Schilmann A.
      • Solís-Vivanco R.
      • Montes S.
      • Riojas-Rodríguez H.
      • Cortez-Lugo M.
      • Ríos C.
      Effect of environmental manganese exposure on verbal learning and memory in Mexican children.
      ,
      • Carvalho C.F.
      • Menezes-Filho J.A.
      • de Matos V.P.
      • Bessa J.R.
      • Coelho-Santos J.
      • Viana G.F.
      • Argollo N.
      • Abreu N.
      Elevated airborne manganese and low executive function in school-aged children in Brazil.
      ,
      • Betancourt Ó.
      • Tapia M.
      • Méndez I.
      Decline of general intelligence in children exposed to manganese from mining contamination in Puyango River Basin, Southern Ecuador.
      ,
      • Rink S.M.
      • Ardoino G.
      • Queirolo E.I.
      • Cicariello D.
      • Mañay N.
      • Kordas K.
      Associations between hair manganese levels and cognitive, language, and motor development in preschool children from Montevideo, Uruguay.
      ), but evidence that the hair Mn levels reflect internal exposure dose versus external exposure dose is lacking. Mn levels in dentin of deciduous teeth have also been used to quantify both prenatal and postnatal accumulation (
      • Mora A.M.
      • Arora M.
      • Harley K.G.
      • Kogut K.
      • Parra K.
      • Hernández-Bonilla D.
      • Gunier R.B.
      • Bradman A.
      • Smith D.R.
      • Eskenazi B.
      Prenatal and postnatal manganese teeth levels and neurodevelopment at 7, 9, and 10.5 years in the CHAMACOS cohort.
      ,
      • Gunier R.B.
      • Arora M.
      • Jerrett M.
      • Bradman A.
      • Harley K.G.
      • Mora A.M.
      • Kogut K.
      • Hubbard A.
      • Austin C.
      • Holland N.
      • Eskenazi B.
      Manganese in teeth and neurodevelopment in young Mexican-American children.
      ). Fingernails and toenails are noninvasive indicators of internal Mn exposure and hold the potential to quantify long-term exposure for up to a year (
      • Hassani H.
      • Golbabaei F.
      • Shirkhanloo H.
      • Tehrani-Doust M.
      Relations of biomarkers of manganese exposure and neuropsychological effects among welders and ferroalloy smelters.
      ,
      • Laohaudomchok W.
      • Lin X.
      • Herrick R.F.
      • Fang S.C.
      • Cavallari J.M.
      • Christiani D.C.
      • Weisskopf M.G.
      Toenail, blood and urine as biomarkers of manganese exposure.
      ). Due to the high rate of Mn elimination through bile to feces (>95%) and short half-life, urine Mn concentration is not recommended as an optimal medium for internal Mn exposure assessment (
      • Smith D.
      • Gwiazda R.
      • Bowler R.
      • Roels H.
      • Park R.
      • Taicher C.
      • Lucchini R.
      Biomarkers of Mn exposure in humans.
      ,
      • Laohaudomchok W.
      • Lin X.
      • Herrick R.F.
      • Fang S.C.
      • Cavallari J.M.
      • Christiani D.C.
      • Weisskopf M.G.
      Toenail, blood and urine as biomarkers of manganese exposure.
      ,
      • Zheng W.
      • Fu S.X.
      • Dydak U.
      • Cowan D.M.
      Biomarkers of manganese intoxication.
      ). Only limited research is available on saliva and the hormone prolactin as alternate biomarkers for Mn exposure (
      • O'Neal S.L.
      • Zheng W.
      Manganese toxicity upon overexposure: a decade in review.
      ,
      • Zheng W.
      • Fu S.X.
      • Dydak U.
      • Cowan D.M.
      Biomarkers of manganese intoxication.
      ,
      • Marreilha Dos Santos A.P.
      • Lopes Santos M.
      • Batoréu M.C.
      • Aschner M.
      Prolactin is a peripheral marker of manganese neurotoxicity.
      ).

      Biphasic relationship

      Mn exhibits negative health effects at both deficient and excess exposures (
      • Vollet K.
      • Haynes E.N.
      • Dietrich K.N.
      Manganese exposure and cognition across the lifespan: contemporary review and argument for biphasic dose-response health effects.
      ,
      • Kang J.O.
      Chronic iron overload and toxicity: clinical chemistry perspective.
      ,
      • Ghosh K.
      Non haematological effects of iron deficiency—a perspective.
      ). In 1999, Mergler et al. (
      • Mergler D.
      • Baldwin M.
      • Bélanger S.
      • Larribe F.
      • Beuter A.
      • Bowler R.
      • Panisset M.
      • Edwards R.
      • de Geoffroy A.
      • Sassine M.P.
      • Hudnell K.
      Manganese neurotoxicity, a continuum of dysfunction: results from a community based study.
      ) coined the term “a continuum of dysfunction” to describe the manifestations of Mn neurotoxicity along three toxicological outcomes: manganism, a neurological disorder in response to high exposures of Mn; neuropsychological abnormalities; and idiopathic Parkinson's disease. Vollet et al. (
      • Vollet K.
      • Haynes E.N.
      • Dietrich K.N.
      Manganese exposure and cognition across the lifespan: contemporary review and argument for biphasic dose-response health effects.
      ) describe this dose-response relationship of Mn focusing on neurocognitive outcomes (Fig. 2). In recent years, an inverse U-shaped association has been reported between Mn exposure and adverse neurodevelopmental effects in infants and children, suggesting adverse effects of both low and high Mn exposures (Fig. 2). This pattern is consistent with Mn acting as both an essential nutrient and a toxicant (
      • Vollet K.
      • Haynes E.N.
      • Dietrich K.N.
      Manganese exposure and cognition across the lifespan: contemporary review and argument for biphasic dose-response health effects.
      ,
      • Haynes E.N.
      • Sucharew H.
      • Kuhnell P.
      • Alden J.
      • Barnas M.
      • Wright R.O.
      • Parsons P.J.
      • Aldous K.M.
      • Praamsma M.L.
      • Beidler C.
      • Dietrich K.N.
      Manganese exposure and neurocognitive outcomes in rural school-age children: the communities actively researching exposure study (Ohio, U.S.A.).
      ,
      • Claus Henn B.
      • Ettinger A.S.
      • Schwartz J.
      • Téllez-Rojo M.M.
      • Lamadrid-Figueroa H.
      • Hernández-Avila M.
      • Schnaas L.
      • Amarasiriwardena C.
      • Bellinger D.C.
      • Hu H.
      • Wright R.O.
      Early postnatal blood manganese levels and children's neurodevelopment.
      ). This biphasic dose response between Mn exposure and neurotoxic effects is observed in both human and animal studies (
      • Haynes E.N.
      • Sucharew H.
      • Kuhnell P.
      • Alden J.
      • Barnas M.
      • Wright R.O.
      • Parsons P.J.
      • Aldous K.M.
      • Praamsma M.L.
      • Beidler C.
      • Dietrich K.N.
      Manganese exposure and neurocognitive outcomes in rural school-age children: the communities actively researching exposure study (Ohio, U.S.A.).
      ,
      • Beaudin S.A.
      • Strupp B.J.
      • Strawderman M.
      • Smith D.R.
      Early postnatal manganese exposure causes lasting impairment of selective and focused attention and arousal regulation in adult rats.
      ,
      • Bhang S.Y.
      • Cho S.C.
      • Kim J.W.
      • Hong Y.C.
      • Shin M.S.
      • Yoo H.J.
      • Cho I.H.
      • Kim Y.
      • Kim B.N.
      Relationship between blood manganese levels and children's attention, cognition, behavior, and academic performance—a nationwide cross-sectional study.
      ). Additional studies are needed to determine the full extent of Mn exposure effects on health outcomes. Future prospective field-based exposure studies can provide substantial insights regarding chronic low-level impacts and eventual progression in terms of disability and disease (
      • Mergler D.
      • Baldwin M.
      • Bélanger S.
      • Larribe F.
      • Beuter A.
      • Bowler R.
      • Panisset M.
      • Edwards R.
      • de Geoffroy A.
      • Sassine M.P.
      • Hudnell K.
      Manganese neurotoxicity, a continuum of dysfunction: results from a community based study.
      ). Future biochemical research should include examination of mechanisms of nonlinear relationships and cell/molecular consequences of exposures across the lifespan, particularly during critical developmental windows.
      Figure thumbnail gr2
      Figure 2Mn exhibits hormetic dose response, which means an inverted “U-shaped” curve. Deficits in neurocognition are seen at both lower and higher doses, with maximum function being at the top of the inverted U-shaped curve. Adapted from Vollet et al. (
      • Vollet K.
      • Haynes E.N.
      • Dietrich K.N.
      Manganese exposure and cognition across the lifespan: contemporary review and argument for biphasic dose-response health effects.
      ). This research was originally published in Current Environmental Health Reports. Vollet, K., Haynes, E. N., and Dietrich, K. N. Manganese exposure and cognition across the lifespan: contemporary review and argument for biphasic dose-response health effects. Curr. Environ. Health Rep. 2016; 3:392–404. © Springer.

      Effects of Mn on neurotransmitters

      Accumulation of Mn in the brain could potentially alter multiple neurotransmitter systems and their activity in the brain. Mn predominantly accumulates in the basal ganglia region of the brain, including the substantia nigra, striatum, and pallidum, when in excess (
      • Madison J.L.
      • Wegrzynowicz M.
      • Aschner M.
      • Bowman A.B.
      Disease-toxicant interactions in manganese exposed Huntington disease mice: early changes in striatal neuron morphology and dopamine metabolism.
      ,
      • Madison J.L.
      • Wegrzynowicz M.
      • Aschner M.
      • Bowman A.B.
      Gender and manganese exposure interactions on mouse striatal neuron morphology.
      ,
      • Robison G.
      • Sullivan B.
      • Cannon J.R.
      • Pushkar Y.
      Identification of dopaminergic neurons of the substantia nigra pars compacta as a target of manganese accumulation.
      ,
      • Park J.D.
      • Chung Y.H.
      • Kim C.Y.
      • Ha C.S.
      • Yang S.O.
      • Khang H.S.
      • Yu I.K.
      • Cheong H.K.
      • Lee J.S.
      • Song C.W.
      • Kwon I.H.
      • Han J.H.
      • Sung J.H.
      • Heo J.D.
      • Choi B.S.
      • et al.
      Comparison of high MRI T1 signals with manganese concentration in brains of cynomolgus monkeys after 8 months of stainless steel welding-fume exposure.
      ). The basal ganglia have an intricate network of neurotransmitters that could potentially be altered and cause deviations in optimal physiology and behavior.
      γ-Aminobutyric acid (GABA) is an inhibitory neurotransmitter seen in abundance in globus pallidus and substantia nigra pars reticulata that receives inputs from the striatum (caudate nucleus and putamen) (
      • Stanwood G.D.
      • Leitch D.B.
      • Savchenko V.
      • Wu J.
      • Fitsanakis V.A.
      • Anderson D.J.
      • Stankowski J.N.
      • Aschner M.
      • McLaughlin B.
      Manganese exposure is cytotoxic and alters dopaminergic and GABAergic neurons within the basal ganglia.
      ,
      • Fitsanakis V.A.
      • Au C.
      • Erikson K.M.
      • Aschner M.
      The effects of manganese on glutamate, dopamine and γ-aminobutyric acid regulation.
      ). Mn exposures, even at levels that are not cytotoxic to neurons (e.g. 100 μm), cause early and profound changes in neurite length and integrity, thus subsequently altering GABA levels (
      • Stanwood G.D.
      • Leitch D.B.
      • Savchenko V.
      • Wu J.
      • Fitsanakis V.A.
      • Anderson D.J.
      • Stankowski J.N.
      • Aschner M.
      • McLaughlin B.
      Manganese exposure is cytotoxic and alters dopaminergic and GABAergic neurons within the basal ganglia.
      ). These deviations in GABAergic neurotransmission cause disinhibition of excitatory neurotransmitters. Stanwood et al. (
      • Stanwood G.D.
      • Leitch D.B.
      • Savchenko V.
      • Wu J.
      • Fitsanakis V.A.
      • Anderson D.J.
      • Stankowski J.N.
      • Aschner M.
      • McLaughlin B.
      Manganese exposure is cytotoxic and alters dopaminergic and GABAergic neurons within the basal ganglia.
      ) showed that acute Mn treatment was neurotoxic in vitro, causing death in tyrosine hydroxylase (TH)-positive, presumptive dopamine (DA) neurons, along with loss of glutamic acid decarboxylase–positive neurons in the basal ganglia. Dopaminergic neurons present in the basal ganglia express TH, which is the rate-limiting enzyme for the synthesis of dopamine. Their study indicates that Mn toxicity acts on the neurocircuitry of this brain region to alter homeostasis and mediate neurodegeneration in the brain (
      • Stanwood G.D.
      • Leitch D.B.
      • Savchenko V.
      • Wu J.
      • Fitsanakis V.A.
      • Anderson D.J.
      • Stankowski J.N.
      • Aschner M.
      • McLaughlin B.
      Manganese exposure is cytotoxic and alters dopaminergic and GABAergic neurons within the basal ganglia.
      ). Mn also inhibited GABA transport in the rat forebrain, confirming that Mn neurotoxicity perturbs the GABAergic neurotransmission (
      • Wong P.C.L.
      • Lai J.C.K.
      • Lim L.
      • Davison A.N.
      Selective inhibition of l-glutamate and gammaaminobutyrate transport in nerve ending particles by aluminium, manganese, and cadmium chloride.
      ).
      Glutamate is the primary excitatory neurotransmitter and a critical signaling molecule. Mn can trigger neurotoxicity by release of excessive amounts of glutamate in the extracellular space, potentially leading to activation of glutamate receptors and subsequent downstream processes (
      • Danbolt N.C.
      Glutamate uptake.
      ). Mn exposure leads to altered synaptic neurotransmission via increased sensitivity of postsynaptic glutamate receptors and activation of neurons in the globus pallidus (
      • Spadoni F.
      • Stefani A.
      • Morello M.
      • Lavaroni F.
      • Giacomini P.
      • Sancesario G.
      Selective vulnerability of pallidal neurons in the early phases of manganese intoxication.
      ,
      • Takeda A.
      • Sotogaku N.
      • Oku N.
      Manganese influences the levels of neurotransmitters in synapses in rat brain.
      ,
      • Takeda A.
      • Sotogaku N.
      • Oku N.
      Influence of manganese on the release of neurotransmitters in rat striatum.
      ). Mn is also a cofactor for glutamine synthetase, and Mn disruption of astrocyte metabolism also leads to unavailability of the necessary components for neurotransmitter and GSH metabolism (
      • Zwingmann C.
      • Leibfritz D.
      • Hazell A.S.
      Energy metabolism in astrocytes and neurons treated with manganese: relation among cell-specific energy failure, glucose metabolism, and intercellular trafficking using multinuclear NMR-spectroscopic analysis.
      ).
      The effects of Mn on brain cholinergic systems could add to the understanding of Mn neurotoxicity. Mn binds to the choline transporter and reduces choline uptake. Additionally, Mn influences regional choline uptake in the hippocampus, the frontal and parietal cortices, the caudate, and the putamen. These results suggest that choline uptake across the blood-brain barrier is likely inhibited by Mn (
      • Eriksson H.
      • Morath C.
      • Heilbronn E.
      Effects of manganese on the nervous system.
      ). The deficit of choline, a key component needed for the synthesis of the neurotransmitter acetylcholine, could potentially contribute to deficits in both behavior and physiology. Mn has a greater neurotoxic effect on cholinergic neurons in the developing brain, as there is decrease in the enzyme choline acetyltransferase specifically in the midbrain and the cerebellar region (
      • Lai J.C.K.
      • Leung T.K.C.
      • Lim L.
      Differences in the neurotoxic effects of manganese during development and aging—some observations on brain regional neurotransmitter and non-neurotransmitter metabolism in a developmental rat model of chronic manganese encephalopathy.
      ). In adult rats, Mn may further contribute to regional specific (e.g. striatum and cerebellum) increases in the acetylcholine-degrading enzyme acetylcholinesterase (
      • Lai J.C.K.
      • Leung T.K.C.
      • Lim L.
      Differences in the neurotoxic effects of manganese during development and aging—some observations on brain regional neurotransmitter and non-neurotransmitter metabolism in a developmental rat model of chronic manganese encephalopathy.
      ,
      • Yousefi Babadi V.
      • Sadeghi L.
      • Shirani K.
      • Malekirad A.A.
      • Rezaei M.
      The toxic effect of manganese on the acetylcholinesterase activity in rat brains.
      ,
      • Lai J.C.K.
      • Chan A.W.K.
      • Leung T.K.C.
      • Minski M.J.
      • Lim L.
      Neurochemical changes in rats chronically treated with a high-concentration of manganese chloride.
      ). Further study is needed to corroborate the neurotoxic effects of Mn on cholinergic neurons and the interactions it has with the other neurotransmitter systems in the brain.
      The current body of literature has predominantly focused on the effects of Mn neurotoxicity on DA. As discussed earlier in this work, Mn accumulates in the basal ganglia, a DA-rich region. This accumulation has been shown to correlate inversely with DA levels in both neonates (
      • Tran T.T.
      • Chowanadisai W.
      • Crinella F.M.
      • Chicz-DeMet A.
      • Lönnerdal B.
      Effect of high dietary manganese intake of neonatal rats on tissue mineral accumulation, striatal dopamine levels, and neurodevelopmental status.
      ) and adult rats (
      • Seth P.K.
      • Chandra S.V.
      Neurotransmitters and neurotransmitter receptors in developing and adult rats during manganese poisoning.
      ). Research has predominantly focused on DA subtype 2 receptor (D2R) and the DA transporter (DAT) responsible for Mn transport into DA neurons (
      • Ingersoll R.T.
      • Montgomery Jr., E.B.
      • Aposhian H.V.
      Central nervous system toxicity of manganese. II: Cocaine or reserpine inhibit manganese concentration in the rat brain.
      ). Nelson et al. showed that D2R are involved in Mn toxicity, at the receptor level or at a point downstream in the signaling cascade that does not involve adenylyl cyclase (
      • Nelson M.
      • Adams T.
      • Ojo C.
      • Carroll M.A.
      • Catapane E.J.
      Manganese toxicity is targeting an early step in the dopamine signal transduction pathway that controls lateral cilia activity in the bivalve mollusc Crassostrea virginica.
      ). MnCl2 exposure can lead to DA depletion and blunt the efficacy of DAergic neurotransmission, leading to up-regulation of the post-synaptic dopamine receptors and causing behavioral alterations including hypoactivity, cognitive impairments, and altered sensorimotor function (
      • Vezér T.
      • Kurunczi A.
      • Náray M.
      • Papp A.
      • Nagymajtényi L.
      Behavioral effects of subchronic inorganic manganese exposure in rats.
      ). DA release kinetics in rat striatum are also impacted by sub-acute exposure to Mn due to accumulation within the striatum, with decreased basal levels and lower stimulated release observed up to 3 weeks post-treatment (
      • Khalid M.
      • Aoun R.A.
      • Mathews T.A.
      Altered striatal dopamine release following a sub-acute exposure to manganese.
      ).
      DAT density is highest in the caudate, putamen, and nucleus accumbens, and this density increases with age under normal physiological conditions. Examination of DAT in Mn-intoxicated patients revealed a slight decrease in DAT density (
      • Huang C.C.
      • Weng Y.H.
      • Lu C.S.
      • Chu N.S.
      • Yen T.C.
      Dopamine transporter binding in chronic manganese intoxication.
      ). The authors also noted significantly greater DAT in striatum of patients with Parkinson's disease compared with those with Mn intoxication. Nonhuman primate studies have shown that DAT in striatum is a target for Mn and could potentially indicate an early event in the damage of DAergic neurons by increased uptake of DA and/or Mn (
      • Chen M.K.
      • Lee J.S.
      • McGlothan J.L.
      • Furukawa E.
      • Adams R.J.
      • Alexander M.
      • Wong D.F.
      • Guilarte T.R.
      Acute manganese administration alters dopamine transporter levels in the non-human primate striatum.
      ). Low doses of Mn do not kill DAergic neurons in vitro but can impair TH activity through activation of protein kinase Cδ and protein phosphatase 2A even in the absence of frank toxicity (
      • Zhang D.
      • Kanthasamy A.
      • Anantharam V.
      • Kanthasamy A.
      Effects of manganese on tyrosine hydroxylase (TH) activity and TH-phosphorylation in a dopaminergic neural cell line.
      ). Mn exposure in vivo increased DA and the DA metabolite 3,4-dihydroxyphenylacetic acid in adult rats (
      • Amos-Kroohs R.M.
      • Davenport L.L.
      • Gutierrez A.
      • Hufgard J.R.
      • Vorhees C.V.
      • Williams M.T.
      Developmental manganese exposure in combination with developmental stress and iron deficiency: effects on behavior and monoamines.
      ). Tran et al. (
      • Tran T.T.
      • Chowanadisai W.
      • Lönnerdal B.
      • Le L.
      • Parker M.
      • Chicz-Demet A.
      • Crinella F.M.
      Effects of neonatal dietary manganese exposure on brain dopamine levels and neurocognitive functions.
      ) showed that dietary Mn exposure caused disruption to the DA system that subsequently altered executive function. Developmental exposure of Mn caused cognitive deficits that implicate DA and brain-derived neurotrophic factor (
      • Bailey R.A.
      • Gutierrez A.
      • Kyser T.L.
      • Hemmerle A.M.
      • Hufgard J.R.
      • Seroogy K.B.
      • Vorhees C.V.
      • Williams M.T.
      Effects of preweaning manganese in combination with adult striatal dopamine lesions on monoamines, BDNF, TrkB, and cognitive function in Sprague-Dawley rats.
      ). Mn accumulation in the DAergic cells of the substantia nigra pars compacta in a rodent model points to a biological basis for deficits in motor skills seen in association with manganism (
      • Robison G.
      • Sullivan B.
      • Cannon J.R.
      • Pushkar Y.
      Identification of dopaminergic neurons of the substantia nigra pars compacta as a target of manganese accumulation.
      ). Reported effects of Mn neurotoxicity on DA include decreased DA levels (
      • Autissier N.
      • Rochette L.
      • Dumas P.
      • Beley A.
      • Loireau A.
      • Bralet J.
      Dopamine and norepinephrine turnover in various regions of the rat brain after chronic manganese chloride administration.
      ,
      • Eriksson H.
      • Mägiste K.
      • Plantin L.-O.
      • Fonnum F.
      • Hedström K.-G.
      • Theodorsson-Norheim E.
      • Kristensson K.
      • Stålberg E.
      • Heilbronn E.
      Effects of manganese oxide on monkeys as revealed by a combined neurochemical, histological and neurophysiological evaluation.
      ,
      • Sistrunk S.C.
      • Ross M.K.
      • Filipov N.M.
      Direct effects of manganese compounds on dopamine and its metabolite Dopac: an in vitro study.
      ), increased DA levels (
      • Tomás-Camardiel M.
      • Herrera A.J.
      • Venero J.L.
      • Cruz Sánchez-Hidalgo M.
      • Cano J.
      • Machado A.
      Differential regulation of glutamic acid decarboxylase mRNA and tyrosine hydroxylase mRNA expression in the aged manganese-treated rats.
      ), or no modification (
      • Gwiazda R.H.
      • Lee D.
      • Sheridan J.
      • Smith D.R.
      Low cumulative manganese exposure affects striatal GABA but not dopamine.
      ) in the substantia nigra or striatum. These differences may be due to the specific exposure paradigm, including route of exposure, concentration, duration, Mn compound, animal model, species age, and sex of the subjects (
      • Avila Costa M.R.
      • Gutierrez-Valdez A.
      • Anaya V.
      • Ordoñez Librado J.
      • Sanchez Betancourt J.
      • Montiel-Flores E.
      • Aley-Medina P.
      • Reynoso-Erazo L.
      • Espinosa-Villanueva J.
      • Tron-Alvarez R.
      • Rodriguez Lara V.
      Manganese inhalation induces dopaminergic cell loss: relevance to Parkinson's disease.
      ). An alternate mode of cytotoxicity is by promoting oxidation of DA and other catecholamines and altering the cellular protective mechanisms that cause the formation of ROS due to higher Mn dosages (
      • Stredrick D.L.
      • Stokes A.H.
      • Worst T.J.
      • Freeman W.M.
      • Johnson E.A.
      • Lash L.H.
      • Aschner M.
      • Vrana K.E.
      Manganese-induced cytotoxicity in dopamine-producing cells.
      ). Mn may directly contribute to dopaminergic cell death by disturbing mitochondrial respiration and antioxidant systems following accumulation within mitochondria (
      • Smith M.R.
      • Fernandes J.
      • Go Y.M.
      • Jones D.P.
      Redox dynamics of manganese as a mitochondrial life-death switch.
      ). Cellular DA also potentiates the cytotoxicity caused by Mn through the oxidative stress–dependent NF-κB signaling cascade (
      • Prabhakaran K.
      • Ghosh D.
      • Chapman G.D.
      • Gunasekar P.G.
      Molecular mechanism of manganese exposure-induced dopaminergic toxicity.
      ). Related to this, antioxidants like taurine may provide neuroprotective effects against Mn neurotoxicity (
      • Ommati M.M.
      • Heidari R.
      • Ghanbarinejad V.
      • Abdoli N.
      • Niknahad H.
      Taurine treatment provides neuroprotection in a mouse model of manganism.
      ).

      Mn transporters and deviations of Mn homeostasis

      Nongenetic influences on Mn toxicity

      Mn and Fe share common absorption and transport pathways (
      • Fitsanakis V.A.
      • Zhang N.
      • Garcia S.
      • Aschner M.
      Manganese (Mn) and iron (Fe): interdependency of transport and regulation.
      ). The absorption of Mn is closely linked with absorption of Fe (
      • Sandstrom B.
      • Davidsson L.
      • Cederblad A.
      • Eriksson R.
      • Lonnerdal B.
      Manganese absorption and metabolism in man.
      ,
      • Finley J.W.
      Manganese absorption and retention by young women is associated with serum ferritin concentration.
      ,
      • Thomson A.B.
      • Olatunbosun D.
      • Valverg L.S.
      Interrelation of intestinal transport system for manganese and iron.
      ). Fe-deficient diets lead to increased absorption of Mn (
      • Rehnberg G.L.
      • Hein J.F.
      • Carter S.D.
      • Linko R.S.
      • Laskey J.W.
      Chronic ingestion of Mn3O4 by rats: tissue accumulation and distribution of manganese in two generations.
      ), and conversely, large amounts of dietary Fe have been shown to inhibit Mn absorption (
      • Schroeder H.A.
      • Balassa J.J.
      • Tipton I.H.
      Essential trace metals in man: manganese. A study in homeostasis.
      ,
      • Lutz T.A.
      • Schroff A.
      • Scharrer E.
      Effects of calcium and sugars on intestinal manganese absorption.
      ,
      • McDermott S.D.
      • Kies C.
      Manganese usage in humans as affected by use of calcium supplements.
      ). This observation was confirmed by a study that showed that as Fe content increased, absorption of Mn decreased (
      • Keen C.L.
      • Zidenberg-Cherr S.
      Manganese.
      ). Fe supplementation of 60 mg/day over 4 months was associated with decreased blood Mn levels and an overall reduction in Mn nutritional status as shown by decreased Mn superoxide dismutase activity in white blood cells (
      • Davis C.D.
      • Greger J.L.
      Longitudinal changes of manganese-dependent superoxide dismutase and other indexes of manganese and iron status in women.
      ). In addition, individual Fe body concentrations affect Mn bioavailability. Fe deficiency increases intestinal absorption of Mn, and increased Fe storage measured by ferritin levels is associated with a decrease in Mn absorption (
      • Finley J.W.
      Manganese absorption and retention by young women is associated with serum ferritin concentration.
      ). Men generally have higher Fe stores than women, in part due to the loss of Fe during menstruation. This may be why men generally absorb less Mn than women (
      • Finley J.W.
      • Johnson P.E.
      • Johnson L.K.
      Sex affects manganese absorption and retention by humans from a diet adequate in manganese.
      ). Further, Fe deficiency has been linked to increased risk of Mn accumulation in the brain (
      • Aschner M.
      • Dorman D.C.
      Manganese: pharmacokinetics and molecular mechanisms of brain uptake.
      ).

      Genetic modifiers of Mn toxicity

      Whereas some mammalian Mn transporters were characterized over the last 2 decades, the recent identification of three hereditary disorders of Mn metabolism is beginning to provide a clearer understanding of the mechanisms that regulate Mn homeostasis in mammalian cells and organisms. Loss-of-function mutations in the Mn efflux transporter SLC30A10 or the Mn influx transporter SLC39A14 increase Mn levels in the body to induce neurotoxicity. In contrast, loss-of-function mutations in another Mn influx transporter, SLC39A8, induce Mn deficiency. The function of these transporters and the underlying disease mechanisms are described below (Fig. 3).
      Figure thumbnail gr3
      Figure 3Function of SLC30A10, SLC39A14, and SLC39A8. SLC30A10 and SLC39A14 synergistically mediate Mn excretion. SLC39A14 transports Mn from blood into hepatocytes and enterocytes for subsequent excretion by SLC30A10 into bile and feces. SLC30A10 also mediates Mn efflux from neuronal cells. In contrast, SLC39A8 reclaims Mn lost in bile. Elevated brain Mn levels and neurotoxicity evident on loss-of-function of SLC30A10 or SLC39A14 is a consequence of an inhibition of Mn excretion and, for SLC30A10, a block in Mn efflux from neurons. Loss-of-function of SLC39A8, in contrast, produces Mn deficiency.

      SLC30A10

      Results from human patients

      A detailed clinical description of a patient who harbored homozygous loss-of-function mutations in SLC30A10 was first reported in 2008 (
      • Tuschl K.
      • Mills P.B.
      • Parsons H.
      • Malone M.
      • Fowler D.
      • Bitner-Glindzicz M.
      • Clayton P.T.
      Hepatic cirrhosis, dystonia, polycythaemia and hypermanganesaemia—a new metabolic disorder.
      ). The patient was born to consanguineous parents, which was suggestive of an autosomal recessive disorder. On clinical examination, notable findings were that the patient exhibited motor abnormalities that influenced gait and fine movements of hands and dystonia that affected all four limbs. Blood Mn levels were ∼10-fold higher than normal. MRI was indicative of basal ganglia Mn deposition. Additionally, there was evidence of cirrhosis and increased liver Mn levels on biopsy. The patient was not environmentally exposed to high Mn, and plasma copper and zinc (Zn) levels were normal (
      • Tuschl K.
      • Mills P.B.
      • Parsons H.
      • Malone M.
      • Fowler D.
      • Bitner-Glindzicz M.
      • Clayton P.T.
      Hepatic cirrhosis, dystonia, polycythaemia and hypermanganesaemia—a new metabolic disorder.
      ).
      Subsequent studies reported on additional individuals with the above-described clinical features (
      • Quadri M.
      • Federico A.
      • Zhao T.
      • Breedveld G.J.
      • Battisti C.
      • Delnooz C.
      • Severijnen L.A.
      • Di Toro Mammarella L.
      • Mignarri A.
      • Monti L.
      • Sanna A.
      • Lu P.
      • Punzo F.
      • Cossu G.
      • Willemsen R.
      • et al.
      Mutations in SLC30A10 cause parkinsonism and dystonia with hypermanganesemia, polycythemia, and chronic liver disease.
      ,
      • Tuschl K.
      • Clayton P.T.
      • Gospe Jr., S.M.
      • Gulab S.
      • Ibrahim S.
      • Singhi P.
      • Aulakh R.
      • Ribeiro R.T.
      • Barsottini O.G.
      • Zaki M.S.
      • Del Rosario M.L.
      • Dyack S.
      • Price V.
      • Rideout A.
      • Gordon K.
      • et al.
      Syndrome of hepatic cirrhosis, dystonia, polycythemia, and hypermanganesemia caused by mutations in SLC30A10, a manganese transporter in man.
      ). MRI provided evidence for the accumulation of manganese in numerous brain regions, including the caudate and lentiform nuclei, thalamus, cortico-spinal tract, and substantia nigra (
      • Quadri M.
      • Federico A.
      • Zhao T.
      • Breedveld G.J.
      • Battisti C.
      • Delnooz C.
      • Severijnen L.A.
      • Di Toro Mammarella L.
      • Mignarri A.
      • Monti L.
      • Sanna A.
      • Lu P.
      • Punzo F.
      • Cossu G.
      • Willemsen R.
      • et al.
      Mutations in SLC30A10 cause parkinsonism and dystonia with hypermanganesemia, polycythemia, and chronic liver disease.
      ). Accumulation in areas beyond the basal ganglia may reflect the substantial elevations in body manganese levels in the genetic disease. Through whole-genome homozygosity mapping and exome sequencing, homozygous mutations in SLC30A10 were identified in affected patients. As expected, the disease exhibited an autosomal recessive form of inheritance (
      • Quadri M.
      • Federico A.
      • Zhao T.
      • Breedveld G.J.
      • Battisti C.
      • Delnooz C.
      • Severijnen L.A.
      • Di Toro Mammarella L.
      • Mignarri A.
      • Monti L.
      • Sanna A.
      • Lu P.
      • Punzo F.
      • Cossu G.
      • Willemsen R.
      • et al.
      Mutations in SLC30A10 cause parkinsonism and dystonia with hypermanganesemia, polycythemia, and chronic liver disease.
      ,
      • Tuschl K.
      • Clayton P.T.
      • Gospe Jr., S.M.
      • Gulab S.
      • Ibrahim S.
      • Singhi P.
      • Aulakh R.
      • Ribeiro R.T.
      • Barsottini O.G.
      • Zaki M.S.
      • Del Rosario M.L.
      • Dyack S.
      • Price V.
      • Rideout A.
      • Gordon K.
      • et al.
      Syndrome of hepatic cirrhosis, dystonia, polycythemia, and hypermanganesemia caused by mutations in SLC30A10, a manganese transporter in man.
      ). Additional cases of patients harboring homozygous mutations in SLC30A10 and suffering from Mn toxicity were later identified (
      • Gulab S.
      • Kayyali H.R.
      • Al-Said Y.
      Atypical neurologic phenotype and novel SLC30A10 mutation in two brothers with hereditary hypermanganesemia.
      ,
      • Quadri M.
      • Kamate M.
      • Sharma S.
      • Olgiati S.
      • Graafland J.
      • Breedveld G.J.
      • Kori I.
      • Hattiholi V.
      • Jain P.
      • Aneja S.
      • Kumar A.
      • Gulati P.
      • Goel M.
      • Talukdar B.
      • Bonifati V.
      Manganese transport disorder: novel SLC30A10 mutations and early phenotypes.
      ,
      • Zaki M.S.
      • Issa M.Y.
      • Elbendary H.M.
      • El-Karaksy H.
      • Hosny H.
      • Ghobrial C.
      • El Safty A.
      • El-Hennawy A.
      • Oraby A.
      • Selim L.
      • Abdel-Hamid M.S.
      Hypermanganesemia with dystonia, polycythemia and cirrhosis in 10 patients: six novel SLC30A10 mutations and further phenotype delineation.
      ). Autopsy findings from a patient with homozygous SLC30A10 mutations revealed marked elevations in Mn levels in the brain and liver with normal brain Zn and Fe levels (
      • Gospe Jr., S.M.
      • Caruso R.D.
      • Clegg M.S.
      • Keen C.L.
      • Pimstone N.R.
      • Ducore J.M.
      • Gettner S.S.
      • Kreutzer R.A.
      Paraparesis, hypermanganesaemia, and polycythaemia: a novel presentation of cirrhosis.
      ). There was loss of neurons in the globus pallidus, which is also a characteristic feature of Mn toxicity secondary to occupational overexposure (
      • Olanow C.W.
      Manganese-induced parkinsonism and Parkinson's disease.
      ,
      • Perl D.P.
      • Olanow C.W.
      The neuropathology of manganese-induced parkinsonism.
      ); depigmentation without neuronal loss in the substantia nigra; and hepatomegaly and cirrhosis (
      • Lechpammer M.
      • Clegg M.S.
      • Muzar Z.
      • Huebner P.A.
      • Jin L.W.
      • Gospe Jr., S.M.
      Pathology of inherited manganese transporter deficiency.
      ). Overall, the major implication of the human studies is that loss-of-function mutations in SLC30A10 alter Mn homeostasis in a manner that leads to the retention of Mn in the body. Accumulation of Mn in the brain, particularly in the basal ganglia, and liver likely cause neurotoxicity and hepatic damage, respectively. Notably, whereas the above discussion relates to rare homozygous mutations in SLC30A10, more recently, widely prevalent SNPs in SLC30A10 associated with altered blood manganese and neurological function have been identified (
      • Wahlberg K.E.
      • Guazzetti S.
      • Pineda D.
      • Larsson S.C.
      • Fedrighi C.
      • Cagna G.
      • Zoni S.
      • Placidi D.
      • Wright R.O.
      • Smith D.R.
      • Lucchini R.G.
      • Broberg K.
      Polymorphisms in manganese transporters SLC30A10 and SLC39A8 are associated with children's neurodevelopment by influencing manganese homeostasis.
      ,
      • Wahlberg K.
      • Kippler M.
      • Alhamdow A.
      • Rahman S.M.
      • Smith D.R.
      • Vahter M.
      • Lucchini R.G.
      • Broberg K.
      Common polymorphisms in the solute carrier SLC30A10 are associated with blood manganese and neurological function.
      ), suggesting that changes in SLC30A10 function likely influence Mn neurotoxicity in the general population as well.

      Characterization of SLC30A10 as a Mn efflux transporter from cell culture assays

      Determination of the molecular mechanism of action of SLC30A10 came from studies in cell culture. SLC30A10 is a member of the SLC30 family of metal transporters that usually transport Zn (
      • Huang L.
      • Tepaamorndech S.
      The SLC30 family of zinc transporters: a review of current understanding of their biological and pathophysiological roles.
      ). Initial studies also characterized SLC30A10 as a Zn efflux transporter (
      • Bosomworth H.J.
      • Thornton J.K.
      • Coneyworth L.J.
      • Ford D.
      • Valentine R.A.
      Efflux function, tissue-specific expression and intracellular trafficking of the Zn transporter ZnT10 indicate roles in adult Zn homeostasis.
      ). However, doubts were raised by the fact that human patients had elevated Mn, but not Zn, levels. Evidence generated over the last few years indicates that SLC30A10 is a specific Mn efflux transporter. In multiple different cell types, WT SLC30A10 localized to the cell surface, decreased cellular Mn levels, and protected against Mn-induced toxicity (
      • Leyva-Illades D.
      • Chen P.
      • Zogzas C.E.
      • Hutchens S.
      • Mercado J.M.
      • Swaim C.D.
      • Morrisett R.A.
      • Bowman A.B.
      • Aschner M.
      • Mukhopadhyay S.
      SLC30A10 is a cell surface-localized manganese efflux transporter, and parkinsonism-causing mutations block its intracellular trafficking and efflux activity.
      ). Disease-causing SLC30A10 mutants were retained in the endoplasmic reticulum and did not mediate Mn efflux (
      • Leyva-Illades D.
      • Chen P.
      • Zogzas C.E.
      • Hutchens S.
      • Mercado J.M.
      • Swaim C.D.
      • Morrisett R.A.
      • Bowman A.B.
      • Aschner M.
      • Mukhopadhyay S.
      SLC30A10 is a cell surface-localized manganese efflux transporter, and parkinsonism-causing mutations block its intracellular trafficking and efflux activity.
      ). In GABAergic primary mouse midbrain neurons, expression of SLC30A10WT, but not a disease-causing mutant, protected against Mn-induced neurotoxicity (
      • Leyva-Illades D.
      • Chen P.
      • Zogzas C.E.
      • Hutchens S.
      • Mercado J.M.
      • Swaim C.D.
      • Morrisett R.A.
      • Bowman A.B.
      • Aschner M.
      • Mukhopadhyay S.
      SLC30A10 is a cell surface-localized manganese efflux transporter, and parkinsonism-causing mutations block its intracellular trafficking and efflux activity.
      ). In neuronal AF5 cells, knockdown of SLC30A10 elevated cellular Mn levels and increased sensitivity to Mn toxicity (
      • Leyva-Illades D.
      • Chen P.
      • Zogzas C.E.
      • Hutchens S.
      • Mercado J.M.
      • Swaim C.D.
      • Morrisett R.A.
      • Bowman A.B.
      • Aschner M.
      • Mukhopadhyay S.
      SLC30A10 is a cell surface-localized manganese efflux transporter, and parkinsonism-causing mutations block its intracellular trafficking and efflux activity.
      ). Importantly, overexpression of SLC30A10WT did not impact intracellular Zn levels or protect against Zn-induced cell death (
      • Leyva-Illades D.
      • Chen P.
      • Zogzas C.E.
      • Hutchens S.
      • Mercado J.M.
      • Swaim C.D.
      • Morrisett R.A.
      • Bowman A.B.
      • Aschner M.
      • Mukhopadhyay S.
      SLC30A10 is a cell surface-localized manganese efflux transporter, and parkinsonism-causing mutations block its intracellular trafficking and efflux activity.
      ,
      • Zogzas C.E.
      • Aschner M.
      • Mukhopadhyay S.
      Structural elements in the transmembrane and cytoplasmic domains of the metal transporter SLC30A10 are required for its manganese efflux activity.
      ). Mechanisms that confer Mn transport specificity to SLC30A10 are unclear, but analyses of the predicted structure of SLC30A10 and mutational assays suggest that the metal-binding site within its transmembrane domain is substantially different from that of related Zn transporters (
      • Zogzas C.E.
      • Aschner M.
      • Mukhopadhyay S.
      Structural elements in the transmembrane and cytoplasmic domains of the metal transporter SLC30A10 are required for its manganese efflux activity.
      ,
      • Nishito Y.
      • Tsuji N.
      • Fujishiro H.
      • Takeda T.A.
      • Yamazaki T.
      • Teranishi F.
      • Okazaki F.
      • Matsunaga A.
      • Tuschl K.
      • Rao R.
      • Kono S.
      • Miyajima H.
      • Narita H.
      • Himeno S.
      • Kambe T.
      Direct comparison of manganese detoxification/efflux proteins and molecular characterization of ZnT10 protein as a manganese transporter.
      ). Orientation of amino acids within the transmembrane domain of SLC30A10 may favor Mn binding while simultaneously disfavoring association of other metals, such as Zn. Notably, mutations in residues required for, or adjacent to those required for, the Mn transport activity of SLC30A10 have been identified to induce disease in humans (
      • Zaki M.S.
      • Issa M.Y.
      • Elbendary H.M.
      • El-Karaksy H.
      • Hosny H.
      • Ghobrial C.
      • El Safty A.
      • El-Hennawy A.
      • Oraby A.
      • Selim L.
      • Abdel-Hamid M.S.
      Hypermanganesemia with dystonia, polycythemia and cirrhosis in 10 patients: six novel SLC30A10 mutations and further phenotype delineation.
      ,
      • Zogzas C.E.
      • Aschner M.
      • Mukhopadhyay S.
      Structural elements in the transmembrane and cytoplasmic domains of the metal transporter SLC30A10 are required for its manganese efflux activity.
      ). Liposome-based transport assays and crystallization may provide the information necessary to better-understand the mechanisms that confer specific Mn transport capability to SLC30A10.

      Understanding the function of SLC30A10 at the organism level using mouse, nematode, and zebrafish models

      Full-body Slc30a10 knockout mice were generated and characterized in 2017 (
      • Hutchens S.
      • Liu C.
      • Jursa T.
      • Shawlot W.
      • Chaffee B.K.
      • Yin W.
      • Gore A.C.
      • Aschner M.
      • Smith D.R.
      • Mukhopadhyay S.
      Deficiency in the manganese efflux transporter SLC30A10 induces severe hypothyroidism in mice.
      ). Knockout animals had ∼20–60-fold increases in brain, liver, and blood Mn levels (
      • Hutchens S.
      • Liu C.
      • Jursa T.
      • Shawlot W.
      • Chaffee B.K.
      • Yin W.
      • Gore A.C.
      • Aschner M.
      • Smith D.R.
      • Mukhopadhyay S.
      Deficiency in the manganese efflux transporter SLC30A10 induces severe hypothyroidism in mice.
      ). Other essential metals (Zn, Cu, and Fe) were largely unaffected (
      • Hutchens S.
      • Liu C.
      • Jursa T.
      • Shawlot W.
      • Chaffee B.K.
      • Yin W.
      • Gore A.C.
      • Aschner M.
      • Smith D.R.
      • Mukhopadhyay S.
      Deficiency in the manganese efflux transporter SLC30A10 induces severe hypothyroidism in mice.
      ). The full-body knockouts exhibited a failure-to-thrive phenotype and died between 7 and 8 weeks of age (
      • Hutchens S.
      • Liu C.
      • Jursa T.
      • Shawlot W.
      • Chaffee B.K.
      • Yin W.
      • Gore A.C.
      • Aschner M.
      • Smith D.R.
      • Mukhopadhyay S.
      Deficiency in the manganese efflux transporter SLC30A10 induces severe hypothyroidism in mice.
      ). Histological analyses provided signs of thyroid dysfunction, and hormone assays demonstrated that the animals suffered from severe hypothyroidism (
      • Hutchens S.
      • Liu C.
      • Jursa T.
      • Shawlot W.
      • Chaffee B.K.
      • Yin W.
      • Gore A.C.
      • Aschner M.
      • Smith D.R.
      • Mukhopadhyay S.
      Deficiency in the manganese efflux transporter SLC30A10 induces severe hypothyroidism in mice.
      ). Further analyses revealed that the knockouts accumulated high levels of Mn in the thyroid, which blocked thyroxine biosynthesis (
      • Hutchens S.
      • Liu C.
      • Jursa T.
      • Shawlot W.
      • Chaffee B.K.
      • Yin W.
      • Gore A.C.
      • Aschner M.
      • Smith D.R.
      • Mukhopadhyay S.
      Deficiency in the manganese efflux transporter SLC30A10 induces severe hypothyroidism in mice.
      ,
      • Liu C.
      • Hutchens S.
      • Jursa T.
      • Shawlot W.
      • Polishchuk E.V.
      • Polishchuk R.S.
      • Dray B.K.
      • Gore A.C.
      • Aschner M.
      • Smith D.R.
      • Mukhopadhyay S.
      Hypothyroidism induced by loss of the manganese efflux transporter SLC30A10 may be explained by reduced thyroxine production.
      ). The phenotype was rescued when animals were fed a low-Mn diet, which decreased tissue Mn levels (
      • Hutchens S.
      • Liu C.
      • Jursa T.
      • Shawlot W.
      • Chaffee B.K.
      • Yin W.
      • Gore A.C.
      • Aschner M.
      • Smith D.R.
      • Mukhopadhyay S.
      Deficiency in the manganese efflux transporter SLC30A10 induces severe hypothyroidism in mice.
      ). Additionally, knockout of the Mn importer SLC39A14 in the Slc30a10 knockouts (i.e. Slc30a10/Slc39a14 double knockouts; see below for a more detailed description of these double knockouts) reduced thyroid Mn levels and also rescued the hypothyroidism phenotype (
      • Liu C.
      • Hutchens S.
      • Jursa T.
      • Shawlot W.
      • Polishchuk E.V.
      • Polishchuk R.S.
      • Dray B.K.
      • Gore A.C.
      • Aschner M.
      • Smith D.R.
      • Mukhopadhyay S.
      Hypothyroidism induced by loss of the manganese efflux transporter SLC30A10 may be explained by reduced thyroxine production.
      ). The novel phenotype of Slc30a10 knockout mice raises the hypothesis that thyroid dysfunction may be an understudied aspect of Mn-induced disease that exacerbates the direct neurotoxic effects of Mn. At least one human patient with homozygous SLC30A10 mutations has now been reported to also have hypothyroidism (
      • Anagianni S.
      • Tuschl K.
      Genetic disorders of manganese metabolism.
      ).
      As detailed below, further understanding of the mechanisms leading to Mn retention upon loss-of-function of SLC30A10 are derived from analyses of tissue-specific Slc30a10 knockout mice. Mn is excreted by the liver and intestines into bile and feces, with biliary excretion being the predominant route of Mn elimination (
      • Papavasiliou P.S.
      • Miller S.T.
      • Cotzias G.C.
      Role of liver in regulating distribution and excretion of manganese.
      ,
      • Greenberg D.M.
      • Copp D.H.
      • Cuthbertson E.M.
      Studies in mineral metabolism with the aid of artificial radioactive isotopes. 7. The distribution and execration, particularly by way of the bile, of iron, cobalt, and manganese.
      ,
      • Ballatori N.
      • Miles E.
      • Clarkson T.W.
      Homeostatic control of manganese excretion in the neonatal rat.
      ,
      • Klaassen C.
      Biliary excretion of manganese in rats, rabbits, and dogs.
      ,
      • Bertinchamps A.J.
      • Miller S.T.
      • Cotzias G.C.
      Interdependence of routes excreting manganese.
      ). In mice and humans, strong expression of SLC30A10 was detectable in the liver and intestines (
      • Quadri M.
      • Federico A.
      • Zhao T.
      • Breedveld G.J.
      • Battisti C.
      • Delnooz C.
      • Severijnen L.A.
      • Di Toro Mammarella L.
      • Mignarri A.
      • Monti L.
      • Sanna A.
      • Lu P.
      • Punzo F.
      • Cossu G.
      • Willemsen R.
      • et al.
      Mutations in SLC30A10 cause parkinsonism and dystonia with hypermanganesemia, polycythemia, and chronic liver disease.
      ,
      • Lechpammer M.
      • Clegg M.S.
      • Muzar Z.
      • Huebner P.A.
      • Jin L.W.
      • Gospe Jr., S.M.
      Pathology of inherited manganese transporter deficiency.
      ,
      • Hutchens S.
      • Liu C.
      • Jursa T.
      • Shawlot W.
      • Chaffee B.K.
      • Yin W.
      • Gore A.C.
      • Aschner M.
      • Smith D.R.
      • Mukhopadhyay S.
      Deficiency in the manganese efflux transporter SLC30A10 induces severe hypothyroidism in mice.
      ,
      • Liu C.
      • Hutchens S.
      • Jursa T.
      • Shawlot W.
      • Polishchuk E.V.
      • Polishchuk R.S.
      • Dray B.K.
      • Gore A.C.
      • Aschner M.
      • Smith D.R.
      • Mukhopadhyay S.
      Hypothyroidism induced by loss of the manganese efflux transporter SLC30A10 may be explained by reduced thyroxine production.
      ,
      • Taylor C.A.
      • Hutchens S.
      • Liu C.
      • Jursa T.
      • Shawlot W.
      • Aschner M.
      • Smith D.R.
      • Mukhopadhyay S.
      SLC30A10 transporter in the digestive system regulates brain manganese under basal conditions while brain SLC30A10 protects against neurotoxicity.
      ,
      • Uhlén M.
      • Fagerberg L.
      • Hallström B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • Sivertsson A.
      • Kampf C.
      • Sjöstedt E.
      • Asplund A.
      • Olsson I.
      • Edlund K.
      • Lundberg E.
      • Navani S.
      • Szigyarto C.A.
      • et al.
      Proteomics: tissue-based map of the human proteome.
      ). Moreover, SLC30A10 localized to the apical/canalicular aspect of polarized HepG2 cells that model hepatocytes (
      • Liu C.
      • Hutchens S.
      • Jursa T.
      • Shawlot W.
      • Polishchuk E.V.
      • Polishchuk R.S.
      • Dray B.K.
      • Gore A.C.
      • Aschner M.
      • Smith D.R.
      • Mukhopadhyay S.
      Hypothyroidism induced by loss of the manganese efflux transporter SLC30A10 may be explained by reduced thyroxine production.
      ) and CaCo2 cells that model enterocytes (
      • Taylor C.A.
      • Hutchens S.
      • Liu C.
      • Jursa T.
      • Shawlot W.
      • Aschner M.
      • Smith D.R.
      • Mukhopadhyay S.
      SLC30A10 transporter in the digestive system regulates brain manganese under basal conditions while brain SLC30A10 protects against neurotoxicity.
      ), raising the hypothesis that the transport activity of SLC30A10 mediates hepatic and intestinal Mn excretion. Consistent with this, tissue-specific Slc30a10 knockout mice lacking SLC30A10 in both the liver and intestines (generated using an endoderm-specific Cre) exhibited marked increases in blood and brain Mn levels and had reduced fecal Mn levels (
      • Taylor C.A.
      • Hutchens S.
      • Liu C.
      • Jursa T.
      • Shawlot W.
      • Aschner M.
      • Smith D.R.
      • Mukhopadhyay S.
      SLC30A10 transporter in the digestive system regulates brain manganese under basal conditions while brain SLC30A10 protects against neurotoxicity.
      ). The above data suggest that loss-of-function of SLC30A10 blocks Mn excretion, which leads to a build-up of Mn within the body, and the retained Mn accumulates in the brain to induce neurotoxicity (Fig. 3). The excretory function of SLC30A10 was validated in 2019 using radioactive Mn excretion and surgical approaches (
      • Mercadante C.J.
      • Prajapati M.
      • Conboy H.L.
      • Dash M.E.
      • Herrera C.
      • Pettiglio M.A.
      • Cintron-Rivera L.
      • Salesky M.A.
      • Rao D.B.
      • Bartnikas T.B.
      Manganese transporter Slc30a10 controls physiological manganese excretion and toxicity.
      ).
      In addition to its role in Mn excretion, SLC30A10 has an additional neuroprotective function in the brain (Fig. 3) (
      • Taylor C.A.
      • Hutchens S.
      • Liu C.
      • Jursa T.
      • Shawlot W.
      • Aschner M.
      • Smith D.R.
      • Mukhopadhyay S.
      SLC30A10 transporter in the digestive system regulates brain manganese under basal conditions while brain SLC30A10 protects against neurotoxicity.
      ). Robust expression of SLC30A10 was detected in the human and mouse brain, including neurons of the basal ganglia (
      • Quadri M.
      • Federico A.
      • Zhao T.
      • Breedveld G.J.
      • Battisti C.
      • Delnooz C.
      • Severijnen L.A.
      • Di Toro Mammarella L.
      • Mignarri A.
      • Monti L.
      • Sanna A.
      • Lu P.
      • Punzo F.
      • Cossu G.
      • Willemsen R.
      • et al.
      Mutations in SLC30A10 cause parkinsonism and dystonia with hypermanganesemia, polycythemia, and chronic liver disease.
      ,
      • Lechpammer M.
      • Clegg M.S.
      • Muzar Z.
      • Huebner P.A.
      • Jin L.W.
      • Gospe Jr., S.M.
      Pathology of inherited manganese transporter deficiency.
      ,
      • Hutchens S.
      • Liu C.
      • Jursa T.
      • Shawlot W.
      • Chaffee B.K.
      • Yin W.
      • Gore A.C.
      • Aschner M.
      • Smith D.R.
      • Mukhopadhyay S.
      Deficiency in the manganese efflux transporter SLC30A10 induces severe hypothyroidism in mice.
      ,
      • Liu C.
      • Hutchens S.
      • Jursa T.
      • Shawlot W.
      • Polishchuk E.V.
      • Polishchuk R.S.
      • Dray B.K.
      • Gore A.C.
      • Aschner M.
      • Smith D.R.
      • Mukhopadhyay S.
      Hypothyroidism induced by loss of the manganese efflux transporter SLC30A10 may be explained by reduced thyroxine production.
      ,
      • Taylor C.A.
      • Hutchens S.
      • Liu C.
      • Jursa T.
      • Shawlot W.
      • Aschner M.
      • Smith D.R.
      • Mukhopadhyay S.
      SLC30A10 transporter in the digestive system regulates brain manganese under basal conditions while brain SLC30A10 protects against neurotoxicity.
      ,
      • Uhlén M.
      • Fagerberg L.
      • Hallström B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • Sivertsson A.
      • Kampf C.
      • Sjöstedt E.
      • Asplund A.
      • Olsson I.
      • Edlund K.
      • Lundberg E.
      • Navani S.
      • Szigyarto C.A.
      • et al.
      Proteomics: tissue-based map of the human proteome.
      ). Mn levels in the basal ganglia of pan-neuronal/glial Slc30a10 knockout mice, lacking SLC30A10 in all neurons and glia, were comparable with littermate controls (
      • Taylor C.A.
      • Hutchens S.
      • Liu C.
      • Jursa T.
      • Shawlot W.
      • Aschner M.
      • Smith D.R.
      • Mukhopadhyay S.
      SLC30A10 transporter in the digestive system regulates brain manganese under basal conditions while brain SLC30A10 protects against neurotoxicity.
      ). However, exposure to a sub-chronic Mn regimen produced larger increases in basal ganglia Mn levels of the pan/neuronal-glial knockouts than littermates. These findings suggest that activity of SLC30A10 in the brain is likely important to reduce Mn levels and protect against neurotoxicity when body Mn levels become elevated (Fig. 3). In totality, the data available to date suggest that neurotoxicity on loss-of-function of SLC30A10 is a consequence of an inhibition of hepatic and intestinal Mn excretion combined with a block in the efflux of Mn from vulnerable basal ganglia neurons (Fig. 3).
      Results in other organisms support findings obtained in mice. As examples, in Caenorhabditis elegans, SLC30A10WT protected DAergic neurons against Mn toxicity, rescued a Mn-induced behavioral defect, and increased viability on exposure to elevated levels of Mn, whereas a disease-causing mutant failed to exert these protective effects (
      • Leyva-Illades D.
      • Chen P.
      • Zogzas C.E.
      • Hutchens S.
      • Mercado J.M.
      • Swaim C.D.
      • Morrisett R.A.
      • Bowman A.B.
      • Aschner M.
      • Mukhopadhyay S.
      SLC30A10 is a cell surface-localized manganese efflux transporter, and parkinsonism-causing mutations block its intracellular trafficking and efflux activity.
      ). There was no effect of SLC30A10WT expression on Zn toxicity in the nematodes (
      • Chen P.
      • Bowman A.B.
      • Mukhopadhyay S.
      • Aschner M.
      SLC30A10: a novel manganese transporter.
      ). Additionally, depletion of SLC30A10 in zebrafish also induced Mn toxicity (
      • Xia Z.
      • Wei J.
      • Li Y.
      • Wang J.
      • Li W.
      • Wang K.
      • Hong X.
      • Zhao L.
      • Chen C.
      • Min J.
      • Wang F.
      Zebrafish slc30a10 deficiency revealed a novel compensatory mechanism of Atp2c1 in maintaining manganese homeostasis.
      ).

      SLC39A14

      Results from human patients

      In 2016, homozygous loss-of-function mutations in SLC39A14 were reported to induce another inherited form of Mn neurotoxicity (
      • Tuschl K.
      • Meyer E.
      • Valdivia L.E.
      • Zhao N.
      • Dadswell C.
      • Abdul-Sada A.
      • Hung C.Y.
      • Simpson M.A.
      • Chong W.K.
      • Jacques T.S.
      • Woltjer R.L.
      • Eaton S.
      • Gregory A.
      • Sanford L.
      • Kara E.
      • et al.
      Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia.
      ). SLC39A14 is a member of the SLC39 family of metal transporters (
      • Jeong J.
      • Eide D.J.
      The SLC39 family of zinc transporters.
      ). Unlike SLC30 proteins, members of the SLC39 family mediate metal influx; most members mediate Zn influx, but SLC39A14 can also mediate influx of Mn, Fe, and cadmium (Cd). Within the first decade of life, affected patients exhibited severe neurological dysfunction, including developmental deficits, dystonia, bulbar defects, spasticity, scoliosis, and loss of independent ambulatory activity (
      • Tuschl K.
      • Meyer E.
      • Valdivia L.E.
      • Zhao N.
      • Dadswell C.
      • Abdul-Sada A.
      • Hung C.Y.
      • Simpson M.A.
      • Chong W.K.
      • Jacques T.S.
      • Woltjer R.L.
      • Eaton S.
      • Gregory A.
      • Sanford L.
      • Kara E.
      • et al.
      Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia.
      ). Parkinsonian features were evident in some (
      • Tuschl K.
      • Meyer E.
      • Valdivia L.E.
      • Zhao N.
      • Dadswell C.
      • Abdul-Sada A.
      • Hung C.Y.
      • Simpson M.A.
      • Chong W.K.
      • Jacques T.S.
      • Woltjer R.L.
      • Eaton S.
      • Gregory A.
      • Sanford L.
      • Kara E.
      • et al.
      Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia.
      ). Blood Mn levels of patients were elevated, but importantly, Fe, Zn, and Cd in blood were within normal limits when tested (
      • Tuschl K.
      • Meyer E.
      • Valdivia L.E.
      • Zhao N.
      • Dadswell C.
      • Abdul-Sada A.
      • Hung C.Y.
      • Simpson M.A.
      • Chong W.K.
      • Jacques T.S.
      • Woltjer R.L.
      • Eaton S.
      • Gregory A.
      • Sanford L.
      • Kara E.
      • et al.
      Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia.
      ). MRI indicated that there was accumulation of Mn in the brain, including in the globus pallidus and striatum (
      • Tuschl K.
      • Meyer E.
      • Valdivia L.E.
      • Zhao N.
      • Dadswell C.
      • Abdul-Sada A.
      • Hung C.Y.
      • Simpson M.A.
      • Chong W.K.
      • Jacques T.S.
      • Woltjer R.L.
      • Eaton S.
      • Gregory A.
      • Sanford L.
      • Kara E.
      • et al.
      Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia.
      ). An important distinction from disease induced by SLC30A10 mutations was the lack of deposition of Mn in the liver and, consequently, lack of liver dysfunction (
      • Tuschl K.
      • Meyer E.
      • Valdivia L.E.
      • Zhao N.
      • Dadswell C.
      • Abdul-Sada A.
      • Hung C.Y.
      • Simpson M.A.
      • Chong W.K.
      • Jacques T.S.
      • Woltjer R.L.
      • Eaton S.
      • Gregory A.
      • Sanford L.
      • Kara E.
      • et al.
      Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia.
      ). This clinical finding provided the first clue that activity of SLC39A14 may be necessary to transport Mn from blood into hepatocytes (Fig. 3). Post-mortem analyses of one patient showed evidence of neuronal degeneration in the globus pallidus (
      • Tuschl K.
      • Meyer E.
      • Valdivia L.E.
      • Zhao N.
      • Dadswell C.
      • Abdul-Sada A.
      • Hung C.Y.
      • Simpson M.A.
      • Chong W.K.
      • Jacques T.S.
      • Woltjer R.L.
      • Eaton S.
      • Gregory A.
      • Sanford L.
      • Kara E.
      • et al.
      Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia.
      ). Additional descriptions of patients suffering from Mn toxicity due to mutations in SLC39A14 were reported in 2018 (
      • Juneja M.
      • Shamim U.
      • Joshi A.
      • Mathur A.
      • Uppili B.
      • Sairam S.
      • Ambawat S.
      • Dixit R.
      • Faruq M.
      A novel mutation in SLC39A14 causing hypermanganesemia associated with infantile onset dystonia.
      ,
      • Marti-Sanchez L.
      • Ortigoza-Escobar J.D.
      • Darling A.
      • Villaronga M.
      • Baide H.
      • Molero-Luis M.
      • Batllori M.
      • Vanegas M.I.
      • Muchart J.
      • Aquino L.
      • Artuch R.
      • Macaya A.
      • Kurian M.A.
      • Dueñas P.
      Hypermanganesemia due to mutations in SLC39A14: further insights into Mn deposition in the central nervous system.
      ).

      Mechanistic in vivo and in vitro assays

      Studies performed prior to discovery of the genetic disease had already demonstrated that SLC39A14 could mediate influx of Zn, Mn, Fe, and Cd (
      • Jeong J.
      • Eide D.J.
      The SLC39 family of zinc transporters.
      ,
      • Girijashanker K.
      • He L.
      • Soleimani M.
      • Reed J.M.
      • Li H.
      • Liu Z.
      • Wang B.
      • Dalton T.P.
      • Nebert D.W.
      Slc39a14 gene encodes ZIP14, a metal/bicarbonate symporter: similarities to the ZIP8 transporter.
      ,
      • Jenkitkasemwong S.
      • Wang C.Y.
      • Mackenzie B.
      • Knutson M.D.
      Physiologic implications of metal-ion transport by ZIP14 and ZIP8.
      ,
      • Liuzzi J.P.
      • Aydemir F.
      • Nam H.
      • Knutson M.D.
      • Cousins R.J.
      Zip14 (Slc39a14) mediates non-transferrin-bound iron uptake into cells.
      ,
      • Liuzzi J.P.
      • Lichten L.A.
      • Rivera S.
      • Blanchard R.K.
      • Aydemir T.B.
      • Knutson M.D.
      • Ganz T.
      • Cousins R.J.
      Interleukin-6 regulates the zinc transporter Zip14 in liver and contributes to the hypozincemia of the acute-phase response.
      ,
      • Pinilla-Tenas J.J.
      • Sparkman B.K.
      • Shawki A.
      • Illing A.C.
      • Mitchell C.J.
      • Zhao N.
      • Liuzzi J.P.
      • Cousins R.J.
      • Knutson M.D.
      • Mackenzie B.
      Zip14 is a complex broad-scope metal-ion transporter whose functional properties support roles in the cellular uptake of zinc and nontransferrin-bound iron.
      ,
      • Taylor K.M.
      • Morgan H.E.
      • Johnson A.
      • Nicholson R.I.
      Structure-function analysis of a novel member of the LIV-1 subfamily of zinc transporters, ZIP14.
      ). SLC39A14WT and disease-causing mutants localized to the cell surface, but the Mn transport capacity of the mutants was lower than for the WT protein (
      • Tuschl K.
      • Meyer E.
      • Valdivia L.E.
      • Zhao N.
      • Dadswell C.
      • Abdul-Sada A.
      • Hung C.Y.
      • Simpson M.A.
      • Chong W.K.
      • Jacques T.S.
      • Woltjer R.L.
      • Eaton S.
      • Gregory A.
      • Sanford L.
      • Kara E.
      • et al.
      Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia.
      ). Combined with the fact that levels of Fe, Cd, and Zn were unaltered in patients, these results suggested that a defect in Mn influx underlies the observed disease phenotype.
      Findings from Slc39a14 knockout mice were first reported in 2010 and 2011 (
      • Hojyo S.
      • Fukada T.
      • Shimoda S.
      • Ohashi W.
      • Bin B.H.
      • Koseki H.
      • Hirano T.
      The zinc transporter SLC39A14/ZIP14 controls G-protein coupled receptor-mediated signaling required for systemic growth.
      ,
      • Tang T.
      • Li L.
      • Tang J.
      • Li Y.
      • Lin W.Y.
      • Martin F.
      • Grant D.
      • Solloway M.
      • Parker L.
      • Ye W.
      • Forrest W.
      • Ghilardi N.
      • Oravecz T.
      • Platt K.A.
      • Rice D.S.
      • et al.
      A mouse knockout library for secreted and transmembrane proteins.
      ), but changes in Mn homeostasis were identified in 2017 by three independent groups (
      • Aydemir T.B.
      • Kim M.H.
      • Kim J.
      • Colon-Perez L.M.
      • Banan G.
      • Mareci T.H.
      • Febo M.
      • Cousins R.J.
      Metal transporter Zip14 (Slc39a14) deletion in mice increases manganese deposition and produces neurotoxic signatures and diminished motor activity.
      ,
      • Jenkitkasemwong S.
      • Akinyode A.
      • Paulus E.
      • Weiskirchen R.
      • Hojyo S.
      • Fukada T.
      • Giraldo G.
      • Schrier J.
      • Garcia A.
      • Janus C.
      • Giasson B.
      • Knutson M.D.
      SLC39A14 deficiency alters manganese homeostasis and excretion resulting in brain manganese accumulation and motor deficits in mice.
      ,
      • Xin Y.
      • Gao H.
      • Wang J.
      • Qiang Y.
      • Imam M.U.
      • Li Y.
      • Wang J.
      • Zhang R.
      • Zhang H.
      • Yu Y.
      • Wang H.
      • Luo H.
      • Shi C.
      • Xu Y.
      • Hojyo S.
      • et al.
      Manganese transporter Slc39a14 deficiency revealed its key role in maintaining manganese homeostasis in mice.
      ). Consistent with observations in human patients, knockout mice exhibited increased Mn levels in blood and brain, but this increase was not evident in the liver (
      • Aydemir T.B.
      • Kim M.H.
      • Kim J.
      • Colon-Perez L.M.
      • Banan G.
      • Mareci T.H.
      • Febo M.
      • Cousins R.J.
      Metal transporter Zip14 (Slc39a14) deletion in mice increases manganese deposition and produces neurotoxic signatures and diminished motor activity.
      ,
      • Jenkitkasemwong S.
      • Akinyode A.
      • Paulus E.
      • Weiskirchen R.
      • Hojyo S.
      • Fukada T.
      • Giraldo G.
      • Schrier J.
      • Garcia A.
      • Janus C.
      • Giasson B.
      • Knutson M.D.
      SLC39A14 deficiency alters manganese homeostasis and excretion resulting in brain manganese accumulation and motor deficits in mice.
      ,
      • Xin Y.
      • Gao H.
      • Wang J.
      • Qiang Y.
      • Imam M.U.
      • Li Y.
      • Wang J.
      • Zhang R.
      • Zhang H.
      • Yu Y.
      • Wang H.
      • Luo H.
      • Shi C.
      • Xu Y.
      • Hojyo S.
      • et al.
      Manganese transporter Slc39a14 deficiency revealed its key role in maintaining manganese homeostasis in mice.
      ). These findings were also consistent with results in zebrafish depleted in SLC39A14 in which Mn levels in the brain, but not abdominal viscera, were elevated (
      • Tuschl K.
      • Meyer E.
      • Valdivia L.E.
      • Zhao N.
      • Dadswell C.
      • Abdul-Sada A.
      • Hung C.Y.
      • Simpson M.A.
      • Chong W.K.
      • Jacques T.S.
      • Woltjer R.L.
      • Eaton S.
      • Gregory A.
      • Sanford L.
      • Kara E.
      • et al.
      Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia.
      ).
      A straightforward hypothesis emerged from the above-described findings in human patients and model systems lacking SLC30A10 or SLC39A14: SLC39A14 and SLC30A10 likely function cooperatively to mediate Mn excretion, with SLC39A14 transporting Mn from blood into hepatocytes and enterocytes and SLC30A10 transporting Mn into bile and feces (Fig. 3). This hypothesis supported two important predictions. First, SLC39A14 and SLC30A10 should localize to the basolateral and apical domains of polarized hepatocytes and enterocytes, respectively (Fig. 3). Second, liver and intestine Mn levels of Slc30a10/Slc39a14 double knockouts should not be elevated (
      • Liu C.
      • Hutchens S.
      • Jursa T.
      • Shawlot W.
      • Polishchuk E.V.
      • Polishchuk R.S.
      • Dray B.K.
      • Gore A.C.
      • Aschner M.
      • Smith D.R.
      • Mukhopadhyay S.
      Hypothyroidism induced by loss of the manganese efflux transporter SLC30A10 may be explained by reduced thyroxine production.
      ). As mentioned above, in polarized HepG2 cells, SLC30A10 was detected in the apical/luminal domain (
      • Liu C.
      • Hutchens S.
      • Jursa T.
      • Shawlot W.
      • Polishchuk E.V.
      • Polishchuk R.S.
      • Dray B.K.
      • Gore A.C.
      • Aschner M.
      • Smith D.R.
      • Mukhopadhyay S.
      Hypothyroidism induced by loss of the manganese efflux transporter SLC30A10 may be explained by reduced thyroxine production.
      ). In the same system, SLC39A14 localized to the basolateral aspect (
      • Liu C.
      • Hutchens S.
      • Jursa T.
      • Shawlot W.
      • Polishchuk E.V.
      • Polishchuk R.S.
      • Dray B.K.
      • Gore A.C.
      • Aschner M.
      • Smith D.R.
      • Mukhopadhyay S.
      Hypothyroidism induced by loss of the manganese efflux transporter SLC30A10 may be explained by reduced thyroxine production.
      ). Basolateral localization of SLC39A14 in polarized HepG2 cells was consistent with earlier observations in rat liver sections (
      • Nam H.
      • Wang C.Y.
      • Zhang L.
      • Zhang W.
      • Hojyo S.
      • Fukada T.
      • Knutson M.D.
      ZIP14 and DMT1 in the liver, pancreas, and heart are differentially regulated by iron deficiency and overload: implications for tissue iron uptake in iron-related disorders.
      ). A similar localization of SLC30A10 and SLC39A14 was also reported in Caco-2 enterocytes (
      • Taylor C.A.
      • Hutchens S.
      • Liu C.
      • Jursa T.
      • Shawlot W.
      • Aschner M.
      • Smith D.R.
      • Mukhopadhyay S.
      SLC30A10 transporter in the digestive system regulates brain manganese under basal conditions while brain SLC30A10 protects against neurotoxicity.
      ,
      • Guthrie G.J.
      • Aydemir T.B.
      • Troche C.
      • Martin A.B.
      • Chang S.M.
      • Cousins R.J.
      Influence of ZIP14 (slc39A14) on intestinal zinc processing and barrier function.
      ). Analyses of Slc30a10/Slc39a14 double-knockout mice provided direct support for the above hypothesis. In Slc30a10/Slc39a14 double knockouts, liver Mn levels were not elevated (
      • Liu C.
      • Hutchens S.
      • Jursa T.
      • Shawlot W.
      • Polishchuk E.V.
      • Polishchuk R.S.
      • Dray B.K.
      • Gore A.C.
      • Aschner M.
      • Smith D.R.
      • Mukhopadhyay S.
      Hypothyroidism induced by loss of the manganese efflux transporter SLC30A10 may be explained by reduced thyroxine production.
      ). This effect was specific because, in the same experiment, liver Mn levels of Slc30a10 single knockouts were substantially higher than WT, but no change was evident in Slc39a14 single knockouts (
      • Liu C.
      • Hutchens S.
      • Jursa T.
      • Shawlot W.
      • Polishchuk E.V.
      • Polishchuk R.S.
      • Dray B.K.
      • Gore A.C.
      • Aschner M.
      • Smith D.R.
      • Mukhopadhyay S.
      Hypothyroidism induced by loss of the manganese efflux transporter SLC30A10 may be explained by reduced thyroxine production.
      ). Furthermore, blood and brain Mn levels of the double knockouts were higher than WT controls and both single knockouts (
      • Liu C.
      • Hutchens S.
      • Jursa T.
      • Shawlot W.
      • Polishchuk E.V.
      • Polishchuk R.S.
      • Dray B.K.
      • Gore A.C.
      • Aschner M.
      • Smith D.R.
      • Mukhopadhyay S.
      Hypothyroidism induced by loss of the manganese efflux transporter SLC30A10 may be explained by reduced thyroxine production.
      ). Results from the double knockouts imply that activity of SLC39A14 is necessary to transport Mn into hepatocytes for subsequent excretion by SLC30A10 (Fig. 3). Whereas intestinal Mn levels were not analyzed in the double knockouts, the differential localization of SLC30A10 and SLC39A14 in enterocytes described above, data from tissue-specific Slc30a10 knockout mice (
      • Taylor C.A.
      • Hutchens S.
      • Liu C.
      • Jursa T.
      • Shawlot W.
      • Aschner M.
      • Smith D.R.
      • Mukhopadhyay S.
      SLC30A10 transporter in the digestive system regulates brain manganese under basal conditions while brain SLC30A10 protects against neurotoxicity.
      ,
      • Mercadante C.J.
      • Prajapati M.
      • Conboy H.L.
      • Dash M.E.
      • Herrera C.
      • Pettiglio M.A.
      • Cintron-Rivera L.
      • Salesky M.A.
      • Rao D.B.
      • Bartnikas T.B.
      Manganese transporter Slc30a10 controls physiological manganese excretion and toxicity.
      ), and recent findings showing that SLC39A14 is required for the transport of Mn from blood into enterocytes (
      • Scheiber I.F.
      • Wu Y.
      • Morgan S.E.
      • Zhao N.
      The intestinal metal transporter ZIP14 maintains systemic manganese homeostasis.
      ), put together, suggest that SLC30A10 and SLC39A14 likely also act cooperatively to mediate intestinal Mn excretion (Fig. 3). We note an additional interesting feature of the Slc30a10/Slc39a14 double knockouts. SLC39A14, but not SLC30A10, was detected in the thyroid (
      • Liu C.
      • Hutchens S.
      • Jursa T.
      • Shawlot W.
      • Polishchuk E.V.
      • Polishchuk R.S.
      • Dray B.K.
      • Gore A.C.
      • Aschner M.
      • Smith D.R.
      • Mukhopadhyay S.
      Hypothyroidism induced by loss of the manganese efflux transporter SLC30A10 may be explained by reduced thyroxine production.
      ). Consequently, thyroid Mn levels of Slc39a14 single and Slc30a10/Slc39a14 double knockouts were lower than Slc30a10 single knockouts, and both Slc39a14 single and Slc30a10/Slc39a14 double knockouts had functioning thyroid hormone (
      • Liu C.
      • Hutchens S.
      • Jursa T.
      • Shawlot W.
      • Polishchuk E.V.
      • Polishchuk R.S.
      • Dray B.K.
      • Gore A.C.
      • Aschner M.
      • Smith D.R.
      • Mukhopadhyay S.
      Hypothyroidism induced by loss of the manganese efflux transporter SLC30A10 may be explained by reduced thyroxine production.
      ). In sum, available data support the model presented in Fig. 3 and suggest that the neurotoxicity evident in patients with SLC39A14 mutations is an effect of a defect in Mn excretion.

      SLC39A8

      Results from human patients

      In 2015, two companion papers reported that mutations in another member of the SLC39 family, SLC39A8, induce an inherited disorder of Mn deficiency (
      • Boycott K.M.
      • Beaulieu C.L.
      • Kernohan K.D.
      • Gebril O.H.
      • Mhanni A.
      • Chudley A.E.
      • Redl D.
      • Qin W.
      • Hampson S.
      • Küry S.
      • Tetreault M.
      • Puffenberger E.G.
      • Scott J.N.
      • Bezieau S.
      • Reis A.
      • et al.
      Autosomal-recessive intellectual disability with cerebellar atrophy syndrome caused by mutation of the manganese and zinc transporter gene SLC39A8.
      ,
      • Park J.H.
      • Hogrebe M.
      • Grüneberg M.
      • DuChesne I.
      • von der Heiden A.L.
      • Reunert J.
      • Schlingmann K.P.
      • Boycott K.M.
      • Beaulieu C.L.
      • Mhanni A.A.
      • Innes A.M.
      • Hörtnagel K.
      • Biskup S.
      • Gleixner E.M.
      • Kurlemann G.
      • et al.
      SLC39A8 deficiency: a disorder of manganese transport and glycosylation.
      ). SLC39A14 mediates influx of several metals: Zn, Mn, Fe, and Cd as well as cobalt (
      • Jeong J.
      • Eide D.J.
      The SLC39 family of zinc transporters.
      ,
      • Jenkitkasemwong S.
      • Wang C.Y.
      • Mackenzie B.
      • Knutson M.D.
      Physiologic implications of metal-ion transport by ZIP14 and ZIP8.
      ,
      • Wang C.Y.
      • Jenkitkasemwong S.
      • Duarte S.
      • Sparkman B.K.
      • Shawki A.
      • Mackenzie B.
      • Knutson M.D.
      ZIP8 is an iron and zinc transporter whose cell-surface expression is up-regulated by cellular iron loading.
      ). One of the papers reported detailed findings from an infant who had cranial asymmetry, severe infantile spasms with hypsarrhythmia, and disproportionate dwarfism (
      • Park J.H.
      • Hogrebe M.
      • Grüneberg M.
      • DuChesne I.
      • von der Heiden A.L.
      • Reunert J.
      • Schlingmann K.P.
      • Boycott K.M.
      • Beaulieu C.L.
      • Mhanni A.A.
      • Innes A.M.
      • Hörtnagel K.
      • Biskup S.
      • Gleixner E.M.
      • Kurlemann G.
      • et al.
      SLC39A8 deficiency: a disorder of manganese transport and glycosylation.
      ). Atrophic changes were seen in the brain on computerized tomography and MRI (
      • Park J.H.
      • Hogrebe M.
      • Grüneberg M.
      • DuChesne I.
      • von der Heiden A.L.
      • Reunert J.
      • Schlingmann K.P.
      • Boycott K.M.
      • Beaulieu C.L.
      • Mhanni A.A.
      • Innes A.M.
      • Hörtnagel K.
      • Biskup S.
      • Gleixner E.M.
      • Kurlemann G.
      • et al.
      SLC39A8 deficiency: a disorder of manganese transport and glycosylation.
      ). Plasma and urine Mn levels were below detection, but serum Zn levels and Fe metabolism parameters were unaffected (
      • Park J.H.
      • Hogrebe M.
      • Grüneberg M.
      • DuChesne I.
      • von der Heiden A.L.
      • Reunert J.
      • Schlingmann K.P.
      • Boycott K.M.
      • Beaulieu C.L.
      • Mhanni A.A.
      • Innes A.M.
      • Hörtnagel K.
      • Biskup S.
      • Gleixner E.M.
      • Kurlemann G.
      • et al.
      SLC39A8 deficiency: a disorder of manganese transport and glycosylation.
      ). As part of the clinical analyses, glycosylation of serum transferrin (a common biomarker used to screen for congenital disorders of glycosylation) was found to be defective (
      • Park J.H.
      • Hogrebe M.
      • Grüneberg M.
      • DuChesne I.
      • von der Heiden A.L.
      • Reunert J.
      • Schlingmann K.P.
      • Boycott K.M.
      • Beaulieu C.L.
      • Mhanni A.A.
      • Innes A.M.
      • Hörtnagel K.
      • Biskup S.
      • Gleixner E.M.
      • Kurlemann G.
      • et al.
      SLC39A8 deficiency: a disorder of manganese transport and glycosylation.
      ). Sequencing revealed that the patient carried homozygous mutations in SLC39A8. Subsequent examination of additional patients with unexplained defects in transferrin glycosylation led to the identification of a second infant with homozygous mutations in SLC39A8, severe neurological deficits, and undetectable Mn levels in whole blood and urine (
      • Park J.H.
      • Hogrebe M.
      • Grüneberg M.
      • DuChesne I.
      • von der Heiden A.L.
      • Reunert J.
      • Schlingmann K.P.
      • Boycott K.M.
      • Beaulieu C.L.
      • Mhanni A.A.
      • Innes A.M.
      • Hörtnagel K.
      • Biskup S.
      • Gleixner E.M.
      • Kurlemann G.
      • et al.
      SLC39A8 deficiency: a disorder of manganese transport and glycosylation.
      ).
      The second paper described findings from six children who belonged to the genetically isolated Hutterite ethnoreligious group and two other siblings born to consanguineous parents (
      • Boycott K.M.
      • Beaulieu C.L.
      • Kernohan K.D.
      • Gebril O.H.
      • Mhanni A.
      • Chudley A.E.
      • Redl D.
      • Qin W.
      • Hampson S.
      • Küry S.
      • Tetreault M.
      • Puffenberger E.G.
      • Scott J.N.
      • Bezieau S.
      • Reis A.
      • et al.
      Autosomal-recessive intellectual disability with cerebellar atrophy syndrome caused by mutation of the manganese and zinc transporter gene SLC39A8.
      ). Clinical features included severe intellectual and developmental disabilities, atrophy of the cerebellum, cross-eyed features, and hypotonia with signs being evident as early as birth (
      • Boycott K.M.
      • Beaulieu C.L.
      • Kernohan K.D.
      • Gebril O.H.
      • Mhanni A.
      • Chudley A.E.
      • Redl D.
      • Qin W.
      • Hampson S.
      • Küry S.
      • Tetreault M.
      • Puffenberger E.G.
      • Scott J.N.
      • Bezieau S.
      • Reis A.
      • et al.
      Autosomal-recessive intellectual disability with cerebellar atrophy syndrome caused by mutation of the manganese and zinc transporter gene SLC39A8.
      ). Sequencing identified homozygous mutations in SLC39A8 (
      • Boycott K.M.
      • Beaulieu C.L.
      • Kernohan K.D.
      • Gebril O.H.
      • Mhanni A.
      • Chudley A.E.
      • Redl D.
      • Qin W.
      • Hampson S.
      • Küry S.
      • Tetreault M.
      • Puffenberger E.G.
      • Scott J.N.
      • Bezieau S.
      • Reis A.
      • et al.
      Autosomal-recessive intellectual disability with cerebellar atrophy syndrome caused by mutation of the manganese and zinc transporter gene SLC39A8.
      ). Of the eight patients, Mn levels in blood or erythrocytes were decreased in four, at the lower end of the normal range in three, and not determined in one (
      • Boycott K.M.
      • Beaulieu C.L.
      • Kernohan K.D.
      • Gebril O.H.
      • Mhanni A.
      • Chudley A.E.
      • Redl D.
      • Qin W.
      • Hampson S.
      • Küry S.
      • Tetreault M.
      • Puffenberger E.G.
      • Scott J.N.
      • Bezieau S.
      • Reis A.
      • et al.
      Autosomal-recessive intellectual disability with cerebellar atrophy syndrome caused by mutation of the manganese and zinc transporter gene SLC39A8.
      ). Authors of the first paper performed glycosylation assays on some of the patients described in the second study and discovered that glycosylation of transferrin was defective (
      • Park J.H.
      • Hogrebe M.
      • Grüneberg M.
      • DuChesne I.
      • von der Heiden A.L.
      • Reunert J.
      • Schlingmann K.P.
      • Boycott K.M.
      • Beaulieu C.L.
      • Mhanni A.A.
      • Innes A.M.
      • Hörtnagel K.
      • Biskup S.
      • Gleixner E.M.
      • Kurlemann G.
      • et al.
      SLC39A8 deficiency: a disorder of manganese transport and glycosylation.
      ). Overall, the clinical studies suggest that mutations in SLC39A8 induce Mn deficiency, which leads to deficits in glycosylation as several Golgi-localized enzymes involved in the glycosylation pathway require Mn for activity (
      • Wagner R.R.
      • Cynkin M.A.
      Glycoprotein metabolism: a UDP-galactose-glycoprotein galactosyltransferase of rat serum.
      ,
      • Schachter H.
      • McGuire E.J.
      • Roseman S.
      Sialic acids. 13. A uridine diphosphate d-galactose: mucin galactosyltransferase from porcine submaxillary gland.
      ,
      • Bendiak B.
      • Schachter H.
      Control of glycoprotein synthesis. Kinetic mechanism, substrate specificity, and inhibition characteristics of UDP-N-acetylglucosamine:α-d-mannoside β1–2 N-acetylglucosaminyltransferase II from rat liver.
      ). Defective glycosylation, in turn, induces neurological disease.

      Mechanistic in vivo and in vitro assays

      In HeLa cells, SLC39A8WT localized to the cell surface (
      • Choi E.K.
      • Nguyen T.T.
      • Gupta N.
      • Iwase S.
      • Seo Y.A.
      Functional analysis of SLC39A8 mutations and their implications for manganese deficiency and mitochondrial disorders.
      ). In contrast, disease-causing mutations trapped the transporter in the endoplasmic reticulum and blocked its capability to mediate Mn influx (
      • Choi E.K.
      • Nguyen T.T.
      • Gupta N.
      • Iwase S.
      • Seo Y.A.
      Functional analysis of SLC39A8 mutations and their implications for manganese deficiency and mitochondrial disorders.
      ). Further mechanistic insights came from analyses of Slc39a8 knockout mice. Full-body depletion of Slc39a8, using a tamoxifen-inducible system, decreased tissue Mn levels and induced glycosylation defects, but did not impact Zn or Fe levels (
      • Lin W.
      • Vann D.R.
      • Doulias P.T.
      • Wang T.
      • Landesberg G.
      • Li X.
      • Ricciotti E.
      • Scalia R.
      • He M.
      • Hand N.J.
      • Rader D.J.
      Hepatic metal ion transporter ZIP8 regulates manganese homeostasis and manganese-dependent enzyme activity.
      ). Constitutive liver-specific Slc39a8 knockouts exhibited reductions in Mn levels not only in the liver but also in other tissues, such as the brain and intestines, and overexpression of Slc39a8 in the liver increased Mn levels in the liver as well as in extrahepatic organs (
      • Lin W.
      • Vann D.R.
      • Doulias P.T.
      • Wang T.
      • Landesberg G.
      • Li X.
      • Ricciotti E.
      • Scalia R.
      • He M.
      • Hand N.J.
      • Rader D.J.
      Hepatic metal ion transporter ZIP8 regulates manganese homeostasis and manganese-dependent enzyme activity.