PHD2 enzyme is an intracellular manganese sensor that initiates the homeostatic response against elevated manganese.

Intracellular sensors detect changes in levels of essential metals to initiate homeostatic responses. But, a mammalian manganese (Mn) sensor is unknown, representing a major gap in understanding of Mn homeostasis. Using human-relevant models, we recently reported that: 1) the primary homeostatic response to elevated Mn is upregulation of hypoxia-inducible factors (HIFs), which increases expression of the Mn efflux transporter SLC30A10; and 2) elevated Mn blocks the prolyl hydroxylation of HIFs by prolyl hydroxylase domain (PHD) enzymes, which otherwise targets HIFs for degradation. Thus, the mammalian mechanism for sensing elevated Mn likely relates to PHD inhibition. Moreover, 1) Mn substitutes for a catalytic iron (Fe) in PHD structures; and 2) exchangeable cellular levels of Fe and Mn are comparable. Therefore, we hypothesized that elevated Mn directly inhibits PHD by replacing its catalytic Fe. In vitro assays using catalytically active PHD2, the primary PHD isoform, revealed that Mn inhibited, and Fe supplementation rescued, PHD2 activity. However, a mutation in PHD2 (D315E) that selectively reduced Mn binding without substantially impacting Fe binding or enzymatic activity resulted in complete insensitivity of PHD2 to Mn in vitro. Additionally, hepatic cells expressing full-length PHD2D315E were less sensitive to Mn-induced HIF activation and SLC30A10 upregulation than PHD2wild-type. These results: 1) define a fundamental Mn sensing mechanism for controlling Mn homeostasis-elevated Mn inhibits PHD2, which functions as a Mn sensor, by outcompeting its catalytic Fe, and PHD2 inhibition activates HIF signaling to up-regulate SLC30A10; and 2) identify a unique mode of metal sensing that may have wide applicability.

A genetically encoded fluorescent sensor for manganese(II), engineered from lanmodulin.

The design of selective metal-binding sites is a challenge in both small-molecule and macromolecular chemistry. Selective recognition of manganese (II)-the first-row transition metal ion that tends to bind with the lowest affinity to ligands, as described by the Irving-Williams series-is particularly difficult. As a result, there is a dearth of chemical biology tools with which to study manganese physiology in live cells, which would advance understanding of photosynthesis, host-pathogen interactions, and neurobiology. Here we report the rational re-engineering of the lanthanide-binding protein, lanmodulin, into genetically encoded fluorescent sensors for MnII, MnLaMP1 and MnLaMP2. These sensors with effective Kd(MnII) of 29 and 7 µM, respectively, defy the Irving-Williams series to selectively detect MnII in vitro and in vivo. We apply both sensors to visualize kinetics of bacterial labile manganese pools. Biophysical studies indicate the importance of coordinated solvent and hydrophobic interactions in the sensors' selectivity. Our results establish lanmodulin as a versatile scaffold for design of selective protein-based biosensors and chelators for metals beyond the f-block.

Up-regulation of the manganese transporter SLC30A10 by hypoxia-inducible factors defines a homeostatic response to manganese toxicity.

Manganese (Mn) is an essential metal that induces incurable parkinsonism at elevated levels. However, unlike other essential metals, mechanisms that regulate mammalian Mn homeostasis are poorly understood, which has limited therapeutic development. Here, we discovered that the exposure of mice to a translationally relevant oral Mn regimen up-regulated expression of SLC30A10, a critical Mn efflux transporter, in the liver and intestines. Mechanistic studies in cell culture, including primary human hepatocytes, revealed that 1) elevated Mn transcriptionally up-regulated SLC30A10, 2) a hypoxia response element in the SLC30A10 promoter was necessary, 3) the transcriptional activities of hypoxia-inducible factor (HIF) 1 or HIF2 were required and sufficient for the SLC30A10 response, 4) elevated Mn activated HIF1/HIF2 by blocking the prolyl hydroxylation of HIF proteins necessary for their degradation, and 5) blocking the Mn-induced up-regulation of SLC30A10 increased intracellular Mn levels and enhanced Mn toxicity. Finally, prolyl hydroxylase inhibitors that stabilize HIF proteins and are in advanced clinical trials for other diseases reduced intracellular Mn levels and afforded cellular protection against Mn toxicity and also ameliorated the in vivo Mn-induced neuromotor deficits in mice. These findings define a fundamental homeostatic protective response to Mn toxicity-elevated Mn levels activate HIF1 and HIF2 to up-regulate SLC30A10, which in turn reduces cellular and organismal Mn levels, and further indicate that it may be possible to repurpose prolyl hydroxylase inhibitors for the management of Mn neurotoxicity.

Manganese efflux transporter SLC30A10 missense polymorphism T95I associated with liver injury retains manganese efflux activity.

The activity of the manganese (Mn) efflux transporter SLC30A10 in the liver and intestines is critical for Mn excretion and preventing Mn toxicity. Homozygous loss-of-function mutations in SLC30A10 are a well-established cause of hereditary Mn toxicity. But, the relationship between more common SLC30A10 polymorphisms, Mn homeostasis, and disease is only recently emerging. In 2021, the first coding SNP in SLC30A10 (T95I) was associated with liver disease raising the hypothesis that the T95I substitution may induce disease by inhibiting the Mn efflux function of SLC30A10. Here, we test this hypothesis using structural, viability, and metal quantification approaches. Analyses of a predicted structure of SLC30A10 revealed that the side chain of T95 pointed away from the putative Mn-binding cavity, raising doubts about the impact of the T95I substitution on SLC30A10 function. In HeLa or HepG2 cells, overexpression of SLC30A10-WT or T95I resulted in comparable reductions of intracellular Mn levels and protection against Mn-induced cell death. Furthermore, ΔSLC30A10 HepG2 cells, generated using CRISPR/Cas9, exhibited elevated Mn levels and heightened sensitivity to Mn-induced cell death, and these phenotypic changes were similarly rescued by expression of SLC30A10-WT or T95I. Finally, turnover rates of SLC30A10-WT or T95I were also comparable. In summary, our results indicate that the Mn transport activity of SLC30A10-T95I is essentially comparable to the WT protein. Our findings imply that SLC30A10-T95I either has a complex association with liver injury that extends beyond the simple reduction in SLC30A10 activity or alternatively the T95I mutation lacks a causal role in liver disease.NEW & NOTEWORTHY This study demonstrates that the T95I polymorphism in the manganese transporter SLC30A10, which has been associated with liver disease in human GWAS studies, does not impact transporter function in cell culture. These findings raise doubts about the causal relationship of the T95I polymorphism with human disease and highlight the importance of validating GWAS findings using mechanistic approaches.

Hepatic and intestinal manganese excretion are both required to regulate brain manganese during elevated manganese exposure.

Manganese (Mn) is essential but neurotoxic at elevated levels. Under physiological conditions, Mn is primarily excreted by the liver, with the intestines playing a secondary role. Recent analyses of tissue-specific Slc30a10 or Slc39a14 knockout mice (SLC30A10 and SLC39A14 are Mn transporters) revealed that, under physiological conditions: 1) excretion of Mn by the liver and intestines is a major pathway that regulates brain Mn; and surprisingly, 2) the intestines compensate for loss of hepatic Mn excretion in controlling brain Mn. The unexpected importance of the intestines in controlling physiological brain Mn led us to determine the role of hepatic and intestinal Mn excretion in regulating brain Mn during elevated Mn exposure. We used liver- or intestine-specific Slc30a10 knockout mice as models to inhibit hepatic or intestinal Mn excretion. Compared with littermates, both knockout strains exhibited similar increases in brain Mn after elevated Mn exposure in early or later life. Thus, unlike physiological conditions, both hepatic and intestinal Mn excretion are required to control brain Mn during elevated Mn exposure. However, brain Mn levels of littermates and both knockout strains exposed to elevated Mn only in early life were normalized in later life. Thus, hepatic and intestinal Mn excretion play compensatory roles in clearing brain Mn accumulated by early life Mn exposure. Finally, neuromotor assays provided evidence consistent with a role for hepatic and intestinal Mn excretion in functionally modulating Mn neurotoxicity during Mn exposure. Put together, these findings substantially enhance understanding of the regulation of brain Mn by excretion.NEW & NOTEWORTHY This article shows that, in contrast with expectations from prior studies and physiological conditions, excretion of manganese by the intestines and liver is equally important in controlling brain manganese during human-relevant manganese exposure. The results provide foundational insights about the interorgan mechanisms that control brain manganese homeostasis at the organism level and have important implications for the development of therapeutics to treat manganese-induced neurological disease.

Manganese and thyroid function in the national health and nutrition examination survey, 2011-2012.

CONTEXT: Manganese (Mn) exposure is prevalent, as it is found naturally as ionized trace elements and released into the environment as a byproduct of manufacturing and waste disposal. Animal and human studies have suggested variable effects on thyroid function, but the association of Mn exposure with thyroid function has not been evaluated in a national sample. OBJECTIVE: To investigate the associations between serum and urinary Mn levels and serum thyroid hormone concentrations in a nationally representative sample. DESIGN, SETTING, PARTICIPANTS, AND INTERVENTION: This was a cross-sectional analysis of data from the 2011-2012 National Health and Nutrition Examination Survey among 1360 participants. MAIN OUTCOME MEASURES: Serum thyroid stimulating hormone (TSH), total triiodothyronine (T3), total thyroxine (T4), free T3, and free T4. RESULTS: Serum Mn levels were positively associated with increasing total T4, free T3, and total T3 in the whole cohort (p < 0.01). Urinary Mn levels were not associated with thyroid hormone levels. When subgroup analyses were performed by gender, only males had total T4 associated with serum Mn [ß = 0.01, p < 0.01, confidence interval (CI): 0.004-0.018]. In individuals under 22 years old, serum Mn was significantly associated with total T4 (ß = 0.02, p = 0.002, CI: 0.008-0.029). Serum Mn was positively associated with Free T3 in both genders (ß = 0.07, p < 0.001). CONCLUSION: While our findings do not suggest clinical thyroid dysfunction, there is an association between serum Mn and subclinical changes in thyroid function that warrant further studies. Regulatory action should be considered as Mn-based organometallic compounds are being considered as replacements for lead in gasoline and may pose future risks to human health.

A three-pocket model for substrate coordination and selectivity by the nucleotide sugar transporters SLC35A1 and SLC35A2.

The CMP-sialic acid transporter SLC35A1 and UDP-galactose transporter SLC35A2 are two well-characterized nucleotide sugar transporters with distinctive substrate specificities. Mutations in either induce congenital disorders of glycosylation. Despite the biomedical relevance, mechanisms of substrate specificity are unclear. To address this critical issue, we utilized a structure-guided mutagenesis strategy and assayed a series of SLC35A2 and SLC35A1 mutants using a rescue approach. Our results suggest that three pockets in the central cavity of each transporter provide substrate specificity. The pockets comprise (1) nucleobase (residues E52, K55, and Y214 of SLC35A1; E75, K78, N235, and G239 of SLC35A2); (2) middle (residues Q101, N102, and T260 of SLC35A1; Q125, N126, Q129, Y130, and Q278 of SLC35A2); and (3) sugar (residues K124, T128, S188, and K272 of SLC35A1; K148, T152, S213, and K297 of SLC35A2) pockets. Within these pockets, two components appear to be especially critical for substrate specificity. Y214 (for SLC35A1) and G239 (for SLC35A2) in the nucleobase pocket appear to discriminate cytosine from uracil. Furthermore, Q129 and Q278 of SLC35A2 in the middle pocket appear to interact specifically with the ß-phosphate of UDP while the corresponding A105 and A253 residues in SLC35A1 do not interact with CMP, which lacks a ß-phosphate. Overall, our findings contribute to a molecular understanding of substrate specificity and coordination in SLC35A1 and SLC35A2 and have important implications for the understanding and treatment of diseases associated with mutations or dysregulations of these two transporters.

Role of excretion in manganese homeostasis and neurotoxicity: a historical perspective.

The essential metal manganese (Mn) induces incurable neurotoxicity at elevated levels that manifests as parkinsonism in adults and fine motor and executive function deficits in children. Studies on Mn neurotoxicity have largely focused on the role and mechanisms of disease induced by elevated Mn exposure from occupational or environmental sources. In contrast, the critical role of excretion in regulating Mn homeostasis and neurotoxicity has received less attention although 1) studies on Mn excretion date back to the 1920s; 2) elegant radiotracer Mn excretion assays in the 1940s to 1960s established the routes of Mn excretion; and 3) studies on patients with liver cirrhosis in the 1990s to 2000s identified an association between decreased Mn excretion and the risk of developing Mn-induced parkinsonism in the absence of elevated Mn exposure. Notably, the last few years have seen renewed interest in Mn excretion largely driven by the discovery that hereditary Mn neurotoxicity due to mutations in SLC30A10 or SLC39A14 is caused, at least in part, by deficits in Mn excretion. Quite remarkably, some of the recent results on SLC30A10 and SLC39A14 provide explanations for observations made ∼40-50 years ago. The goal of the current review is to integrate the historic studies on Mn excretion with more contemporary recent work and provide a comprehensive state-of-the-art overview of Mn excretion and its role in regulating Mn homeostasis and neurotoxicity. A related goal is to discuss the significance of some of the foundational studies on Mn excretion so that these highly consequential earlier studies remain influential in the field.

Brain manganese and the balance between essential roles and neurotoxicity.

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.

SLC30A10 transporter in the digestive system regulates brain manganese under basal conditions while brain SLC30A10 protects against neurotoxicity.

The essential metal manganese becomes neurotoxic at elevated levels. Yet, the mechanisms by which brain manganese homeostasis is regulated are unclear. Loss-of-function mutations in SLC30A10, a cell surface-localized manganese efflux transporter in the brain and liver, induce familial manganese neurotoxicity. To elucidate the role of SLC30A10 in regulating brain manganese, we compared the phenotypes of whole-body and tissue-specific Slc30a10 knockout mice. Surprisingly, unlike whole-body knockouts, brain manganese levels were unaltered in pan-neuronal/glial Slc30a10 knockouts under basal physiological conditions. Further, although transport into bile is a major route of manganese excretion, manganese levels in the brain, blood, and liver of liver-specific Slc30a10 knockouts were only minimally elevated, suggesting that another organ compensated for loss-of-function in the liver. Additional assays revealed that SLC30A10 was also expressed in the gastrointestinal tract. In differentiated enterocytes, SLC30A10 localized to the apical/luminal domain and transported intracellular manganese to the lumen. Importantly, endoderm-specific knockouts, lacking SLC30A10 in the liver and gastrointestinal tract, had markedly elevated manganese levels in the brain, blood, and liver. Thus, under basal physiological conditions, brain manganese is regulated by activity of SLC30A10 in the liver and gastrointestinal tract, and not the brain or just the liver. Notably, however, brain manganese levels of endoderm-specific knockouts were lower than whole-body knockouts, and only whole-body knockouts exhibited manganese-induced neurobehavioral defects. Moreover, after elevated exposure, pan-neuronal/glial knockouts had higher manganese levels in the basal ganglia and thalamus than controls. Therefore, when manganese levels increase, activity of SLC30A10 in the brain protects against neurotoxicity.

Maintaining Translational Relevance in Animal Models of Manganese Neurotoxicity.

Manganese is an essential metal, but elevated brain Mn concentrations produce a parkinsonian-like movement disorder in adults and fine motor, attentional, cognitive, and intellectual deficits in children. Human Mn neurotoxicity occurs owing to elevated exposure from occupational or environmental sources, defective excretion (e.g., due to cirrhosis), or loss-of-function mutations in the Mn transporters solute carrier family 30 member 10 or solute carrier family 39 member 14. Animal models are essential to study Mn neurotoxicity, but in order to be translationally relevant, such models should utilize environmentally relevant Mn exposure regimens that reproduce changes in brain Mn concentrations and neurological function evident in human patients. Here, we provide guidelines for Mn exposure in mice, rats, nematodes, and zebrafish so that brain Mn concentrations and neurobehavioral sequelae remain directly relatable to the human phenotype.

Functional analyses of the UDP-galactose transporter SLC35A2 using the binding of bacterial Shiga toxins as a novel activity assay.

SLC35A2 transports UDP-galactose from the cytosol to the lumen of the Golgi apparatus and endoplasmic reticulum for glycosylation. Mutations in SLC35A2 induce a congenital disorder of glycosylation. Despite the biomedical relevance, mechanisms of transport via SLC35A2 and the impact of disease-associated mutations on activity are unclear. To address these issues, we generated a predicted structure of SLC35A2 and assayed for the effects of a set of structural and disease-associated mutations. Activity assays were performed using a rescue approach in ΔSLC35A2 cells and took advantage of the fact that SLC35A2 is required for expression of the glycosphingolipid globotriaosylceramide (Gb3), the cell surface receptor for Shiga toxin 1 (STx1) and 2 (STx2). The N- and C-terminal cytoplasmic loops of SLC35A2 were dispensable for activity, but two critical glycine (Gly-202 and Gly-214) and lysine (Lys-78 and Lys-297) residues in transmembrane segments were required. Residues corresponding to Gly-202 and Gly-214 in the related transporter SLC35A1 form a substrate-translocating channel, suggesting that a similar mechanism may be involved in SLC35A2. Among the eight disease-associated mutations tested, SLC35A2 function was completely inhibited by two (S213F and G282R) and partially inhibited by three (R55L, G266V, and S304P), providing a straight-forward mechanism of disease. Interestingly, the remaining three (V331I, V258M, and Y267C) did not impact SLC35A2 function, suggesting that complexities beyond loss of transporter activity may underlie disease due to these mutations. Overall, our results provide new insights into the mechanisms of transport of SLC35A2 and improve understanding of the relationship between SLC35A2 mutations and disease.

Hypothyroidism induced by loss of the manganese efflux transporter SLC30A10 may be explained by reduced thyroxine production.

SLC30A10 and SLC39A14 are manganese efflux and influx transporters, respectively. Loss-of-function mutations in genes encoding either transporter induce hereditary manganese toxicity. Patients have elevated manganese in the blood and brain and develop neurotoxicity. Liver manganese is increased in patients lacking SLC30A10 but not SLC39A14. These organ-specific changes in manganese were recently recapitulated in knockout mice. Surprisingly, Slc30a10 knockouts also had elevated thyroid manganese and developed hypothyroidism. To determine the mechanisms of manganese-induced hypothyroidism and understand how SLC30A10 and SLC39A14 cooperatively mediate manganese detoxification, here we produced Slc39a14 single and Slc30a10/Slc39a14 double knockout mice and compared their phenotypes with that of Slc30a10 single knockouts. Compared with wild-type controls, Slc39a14 single and Slc30a10/Slc39a14 double knockouts had higher manganese levels in the blood and brain but not in the liver. In contrast, Slc30a10 single knockouts had elevated manganese levels in the liver as well as in the blood and brain. Furthermore, SLC30A10 and SLC39A14 localized to the canalicular and basolateral domains of polarized hepatic cells, respectively. Thus, transport activities of both SLC39A14 and SLC30A10 are required for hepatic manganese excretion. Compared with Slc30a10 single knockouts, Slc39a14 single and Slc30a10/Slc39a14 double knockouts had lower thyroid manganese levels and normal thyroid function. Moreover, intrathyroid thyroxine levels of Slc30a10 single knockouts were lower than those of controls. Thus, the hypothyroidism phenotype of Slc30a10 single knockouts is induced by elevated thyroid manganese, which blocks thyroxine production. These findings provide new insights into the mechanisms of manganese detoxification and manganese-induced thyroid dysfunction.

Deficiency in the manganese efflux transporter SLC30A10 induces severe hypothyroidism in mice.

Manganese is an essential metal that becomes toxic at elevated levels. Loss-of-function mutations in SLC30A10, a cell-surface-localized manganese efflux transporter, cause a heritable manganese metabolism disorder resulting in elevated manganese levels and parkinsonian-like movement deficits. The underlying disease mechanisms are unclear; therefore, treatment is challenging. To understand the consequences of loss of SLC30A10 function at the organism level, we generated Slc30a10 knock-out mice. During early development, knock-outs were indistinguishable from controls. Surprisingly, however, after weaning and compared with controls, knock-out mice failed to gain weight, were smaller, and died prematurely (by ∼6-8 weeks of age). At 6 weeks, manganese levels in the brain, blood, and liver of the knock-outs were ∼20-60-fold higher than controls. Unexpectedly, histological analyses revealed that the brain and liver of the knock-outs were largely unaffected, but their thyroid exhibited extensive alterations. Because hypothyroidism leads to growth defects and premature death in mice, we assayed for changes in thyroid and pituitary hormones. At 6 weeks and compared with controls, the knock-outs had markedly reduced thyroxine levels (∼50-80%) and profoundly increased thyroid-stimulating hormone levels (∼800-1000-fold), indicating that Slc30a10 knock-out mice develop hypothyroidism. Importantly, a low-manganese diet produced lower tissue manganese levels in the knock-outs and rescued the phenotype, suggesting that manganese toxicity was the underlying cause. Our unanticipated discovery highlights the importance of determining the role of thyroid dysfunction in the onset and progression of manganese-induced disease and identifies Slc30a10 knock-out mice as a new model for studying thyroid biology.

A Conserved Structural Motif Mediates Retrograde Trafficking of Shiga Toxin Types 1 and 2.

Shiga toxin-producing Escherichia coli (STEC) produce two types of Shiga toxin (STx): STx1 and STx2. The toxin A-subunits block protein synthesis, while the B-subunits mediate retrograde trafficking. STEC infections do not have definitive treatments, and there is growing interest in generating toxin transport inhibitors for therapy. However, a comprehensive understanding of the mechanisms of toxin trafficking is essential for drug development. While STx2 is more toxic in vivo, prior studies focused on STx1 B-subunit (STx1B) trafficking. Here, we show that, compared with STx1B, trafficking of the B-subunit of STx2 (STx2B) to the Golgi occurs with slower kinetics. Despite this difference, similar to STx1B, endosome-to-Golgi transport of STx2B does not involve transit through degradative late endosomes and is dependent on dynamin II, epsinR, retromer and syntaxin5. Importantly, additional experiments show that a surface-exposed loop in STx2B (ß4-ß5 loop) is required for its endosome-to-Golgi trafficking. We previously demonstrated that residues in the corresponding ß4-ß5 loop of STx1B are required for interaction with GPP130, the STx1B-specific endosomal receptor, and for endosome-to-Golgi transport. Overall, STx1B and STx2B share a common pathway and use a similar structural motif to traffic to the Golgi, suggesting that the underlying mechanisms of endosomal sorting may be evolutionarily conserved.

Structural Elements in the Transmembrane and Cytoplasmic Domains of the Metal Transporter SLC30A10 Are Required for Its Manganese Efflux Activity.

Homozygous mutations in SLC30A10 lead to the development of familial manganese-induced parkinsonism. We previously demonstrated that SLC30A10 is a cell surface-localized manganese efflux transporter, and parkinsonism-causing mutations block its trafficking and efflux activity. Interestingly, other transporters in the SLC30 family mediate zinc efflux. Determining the mechanisms that allow SLC30A10 to transport manganese, which are unclear, is essential to understand its role in parkinsonism. Here, we generated a predicted structure of SLC30A10, based on the structure of the bacterial zinc transporter YiiP, and performed functional studies. In YiiP, side chains of residues Asp-45 and Asp-49 in the second and His-153 and Asp-157 in the fifth transmembrane segments coordinate zinc and are required for transport. In SLC30A10, the corresponding residues are Asn-43 and Asp-47 in the second and His-244 and Asp-248 in the fifth transmembrane segments. Surprisingly, although alanine substitution of Asp-248 abolished manganese efflux, that of Asn-43 and Asp-47 did not. Instead, side chains of charged or polar residues adjacent to Asp-248 in the first (Glu-25) or fourth (Asn-127) transmembrane segments were required. Further analyses revealed that residues His-333 and His-350 in the cytoplasmic C-terminal domain were required for full activity. However, the C-terminal domain failed to transfer manganese transport capability to a related zinc transporter. Overall, our results indicate that residues in the transmembrane and C-terminal domains together confer optimal manganese transport capability to SLC30A10 and suggest that the mechanism of ion coordination in the transmembrane domain of SLC30A10 may be substantially different from that in YiiP/other SLC30 proteins.

SLC30A10 is a cell surface-localized manganese efflux transporter, and parkinsonism-causing mutations block its intracellular trafficking and efflux activity.

Manganese (Mn) is an essential metal, but elevated cellular levels are toxic and may lead to the development of an irreversible parkinsonian-like syndrome that has no treatment. Mn-induced parkinsonism generally occurs as a result of exposure to elevated Mn levels in occupational or environmental settings. Additionally, patients with compromised liver function attributable to diseases, such as cirrhosis, fail to excrete Mn and may develop Mn-induced parkinsonism in the absence of exposure to elevated Mn. Recently, a new form of familial parkinsonism was reported to occur as a result of mutations in SLC30A10. The cellular function of SLC30A10 and the mechanisms by which mutations in this protein cause parkinsonism are unclear. Here, using a combination of mechanistic and functional studies in cell culture, Caenorhabditis elegans, and primary midbrain neurons, we show that SLC30A10 is a cell surface-localized Mn efflux transporter that reduces cellular Mn levels and protects against Mn-induced toxicity. Importantly, mutations in SLC30A10 that cause familial parkinsonism blocked the ability of the transporter to traffic to the cell surface and to mediate Mn efflux. Although expression of disease-causing SLC30A10 mutations were not deleterious by themselves, neurons and worms expressing these mutants exhibited enhanced sensitivity to Mn toxicity. Our results provide novel insights into the mechanisms involved in the onset of a familial form of parkinsonism and highlight the possibility of using enhanced Mn efflux as a therapeutic strategy for the potential management of Mn-induced parkinsonism, including that occurring as a result of mutations in SLC30A10.

Manganese homeostasis in the nervous system.

Manganese (Mn) is an essential heavy metal that is naturally found in the environment. Daily intake through dietary sources provides the necessary amount required for several key physiological processes, including antioxidant defense, energy metabolism, immune function and others. However, overexposure from environmental sources can result in a condition known as manganism that features symptomatology similar to Parkinson's disease (PD). This disorder presents with debilitating motor and cognitive deficits that arise from a neurodegenerative process. In order to maintain a balance between its essentiality and neurotoxicity, several mechanisms exist to properly buffer cellular Mn levels. These include transporters involved in Mn uptake, and newly discovered Mn efflux mechanisms. This review will focus on current studies related to mechanisms underlying Mn import and export, primarily the Mn transporters, and their function and roles in Mn-induced neurotoxicity. Though and essential metal, overexposure to manganese may result in neurodegenerative disease analogous to Parkinson's disease. Manganese homeostasis is tightly regulated by transporters, including transmembrane importers (divalent metal transporter 1, transferrin and its receptor, zinc transporters ZIP8 and Zip14, dopamine transporter, calcium channels, choline transporters and citrate transporters) and exporters (ferroportin and SLC30A10), as well as the intracellular trafficking proteins (SPCA1 and ATP12A2). A manganese-specific sensor, GPP130, has been identified, which affords means for monitoring intracellular levels of this metal.

Identification of a gain-of-function mutation in a Golgi P-type ATPase that enhances Mn2+ efflux and protects against toxicity.

P-type ATPases transport a wide array of ions, regulate diverse cellular processes, and are implicated in a number of human diseases. However, mechanisms that increase ion transport by these ubiquitous proteins are not known. SPCA1 is a P-type pump that transports Mn(2+) from the cytosol into the Golgi. We developed an intra-Golgi Mn(2+) sensor and used it to screen for mutations introduced in SPCA1, on the basis of its predicted structure, which could increase its Mn(2+) pumping activity. Remarkably, a point mutation (Q747A) predicted to increase the size of its ion permeation cavity enhanced the sensor response and a compensatory mutation restoring the cavity to its original size abolished this effect. In vivo and in vitro Mn(2+) transport assays confirmed the hyperactivity of SPCA1-Q747A. Furthermore, increasing Golgi Mn(2+) transport by expression of SPCA1-Q747A increased cell viability upon Mn(2+) exposure, supporting the therapeutic potential of increased Mn(2+) uptake by the Golgi in the management of Mn(2+)-induced neurotoxicity.

Elevated thyroid manganese reduces thyroid iodine to induce hypothyroidism in mice, but not rats, lacking SLC30A10 transporter.

Elevated manganese (Mn) accumulates in the brain and induces neurotoxicity. SLC30A10 is an Mn efflux transporter that controls body Mn levels. We previously reported that full-body Slc30a10 knockout mice (1) recapitulate the body Mn retention phenotype of humans with loss-of-function SLC30A10 mutations and (2) unexpectedly develop hypothyroidism induced by Mn accumulation in the thyroid, which reduces intra-thyroid thyroxine. Subsequent analyses of National Health and Nutrition Examination Survey data identified an association between serum Mn and subclinical thyroid changes. The emergence of thyroid deficits as a feature of Mn toxicity suggests that changes in thyroid function may be an underappreciated, but critical, modulator of Mn-induced disease. To better understand the relationship between thyroid function and Mn toxicity, here we further defined the mechanism of Mn-induced hypothyroidism using mouse and rat models. Slc30a10 knockout mice exhibited a profound deficit in thyroid iodine levels that occurred contemporaneously with increases in thyroid Mn levels and preceded the onset of overt hypothyroidism. Wild-type Mn-exposed mice also exhibited increased thyroid Mn levels, an inverse correlation between thyroid Mn and iodine levels, and subclinical hypothyroidism. In contrast, thyroid iodine levels were unaltered in newly generated Slc30a10 knockout rats despite an increase in thyroid Mn levels, and the knockout rats were euthyroid. Thus, Mn-induced thyroid dysfunction in genetic or Mn exposure-induced mouse models occurs due to a reduction in thyroid iodine subsequent to an increase in thyroid Mn levels. Moreover, rat and mouse thyroids have differential sensitivities to Mn, which may impact the manifestations of Mn-induced disease in these routinely used animal models.