Elucidating ferroptosis mechanisms in heart failure through transcriptomics, single-cell sequencing, and experimental validation.

BACKGROUND: The mechanisms underlying ferroptosis in heart failure (HF) remain incompletely understood. METHODS: This study analyzed the heart failure dataset from the Gene Expression Omnibus to identify differentially expressed ferroptosis-related genes (DFRGs). Key DFRGs were selected using LASSO regression and SVM-RFE machine learning techniques. Their diagnostic accuracy was evaluated via ROC curve analysis. Single-cell sequencing data, heart failure cell, and mouse models were utilized to validate these key DFRGs. Additionally, potential non-coding RNAs targeting these genes were predicted, and analyses for gene set enrichment, immune cell infiltration, and drug targeting were conducted. RESULTS: A total of 127 DFRGs were identified, with 83 downregulated and 44 upregulated compared to controls. Seven key DFRGs (PTGS2, BECN1, SLC39A14, QSOX1, MLST8, TMSB4X, KDM4A) were identified, showing high diagnostic accuracy (AUC 0.988) in the GSE5406 dataset. GO and KEGG analyses linked these genes to ferroptosis, FoxO signaling, and autophagy pathways. A ceRNA network identified 217 miRNAs and 243 lncRNAs potentially targeting these genes, and 64 drugs were predicted as potential targets. Single-cell sequencing and in vitro experiments revealed differential expression of SLC39A14 and QSOX1, which was further confirmed in vivo. CONCLUSION: This study provides novel insights into the role of ferroptosis in heart failure by identifying and validating DFRGs that exhibit differential expression across various cell types. The differential expression patterns of these genes, particularly SLC39A14 and QSOX1, indicate their potential involvement in the pathophysiological mechanisms contributing to HF. These findings offer new insights for the development of targeted therapies for HF.

RELA-mediated upregulation of LINC03047 promotes ferroptosis in silica-induced pulmonary fibrosis via SLC39A14.

Long non-coding RNAs play a key role in silicosis, a fatal fibrotic lung disease, and there is an urgent need to develop new treatment targets. Long intergenic non-protein-coding RNA 3047 (LINC03047) is associated with cancer, but its role and mechanism in the progression of silicosis require further elucidation. This study investigated the function of LINC03047 in the epithelial-mesenchymal transition (EMT) during silicosis progression. LINC03047 expression was upregulated in SiO2-treated BEAS-2B and A549 cells, promoting SiO2-induced ferroptosis and subsequent EMT. Moreover, knockdown of LINC03047 significantly decreased the expression of solute carrier family 39 member 14 (SLC39A14), a ferrous iron transporter, and inhibition of SLC39A14 alleviated the ferroptosis and EMT caused by LINC03047 overexpression. We further investigated that NF-κB p65 (RELA) was critical for LINC03047 transcription in SiO2-treated BEAS-2B and A549 cells. In vivo experiments showed that SLC39A14 deficiency improved SiO2-induced lipid peroxidation and EMT. Collectively, our study reveals the function of the RELA/LINC03047/SLC39A14 axis in SiO2-induced ferroptosis and EMT, thereby contributing to the identification of novel drug targets for silicosis therapy.

Knockdown of SLC39A14 inhibits glioma progression by promoting erastin-induced ferroptosis SLC39A14 knockdown inhibits glioma progression.

BACKGROUND: Ferroptosis is a newly classified form of regulated cell death with implications in various tumor progression pathways. However, the roles and mechanisms of ferroptosis-related genes in glioma remain unclear. METHODS: Bioinformatics analysis was employed to identify differentially expressed ferroptosis-related genes in glioma. The expression levels of hub genes were assessed using real-time reverse transcriptase-polymerase chain reaction (RT-qPCR). To explore the role of SLC39A14 in glioma, a series of in vitro assays were conducted, including cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, wound healing, and Transwell assays. Enzyme-linked immunosorbent assay (ELISA) was utilized to measure the levels of indicators associated with ferroptosis. Hematoxylin-eosin (HE) and immunohistochemistry (IHC) staining were performed to illustrate the clinicopathological features of the mouse transplantation tumor model. Additionally, Western blot analysis was used to assess the expression of the cGMP-PKG pathway-related proteins. RESULTS: Seven ferroptosis-related hub genes, namely SLC39A14, WWTR1, STEAP3, NOTCH2, IREB2, HIF1A, and FANCD2, were identified, all of which were highly expressed in glioma. Knockdown of SLC39A14 inhibited glioma cell proliferation, migration, and invasion, while promoting apoptosis. Moreover, SLC39A14 knockdown also facilitated erastin-induced ferroptosis, leading to the suppression of mouse transplantation tumor growth. Mechanistically, SLC39A14 knockdown inhibited the cGMP-PKG signaling pathway activation. CONCLUSION: Silencing SLC39A14 inhibits ferroptosis and tumor progression, potentially involving the regulation of the cGMP-PKG signaling pathway.

A simple and highly reproducible method for the detection of erythrocyte membrane ZIP metal transporters by immunoblotting.

Manganese is one of the essential trace elements found in erythrocytes. Metal transporters situated on the plasma membrane generally facilitate the movement of manganese into and out of cells. This study aims at determining whether two recently discovered manganese importers, ZIP8 and ZIP14, are located in the erythrocyte membrane. We outline a simple, effective and repeatable method for the isolation of erythrocyte membrane from a minimum of 50 µL mouse blood, followed by the identification of ZIP metal transporters using immunoblotting. Our results revealed that ZIP8 is expressed within the erythrocyte membrane, in contrast to ZIP14 which is not identified using immunoblotting approach. A direct measurement of the ZIP8 protein expression in erythrocyte membranes could provide valuable information for further analyzing its biological function.

Sucrose-induced hyperglycemia dysregulates intestinal zinc metabolism and integrity: risk factors for chronic diseases.

Objective: Zinc is an essential micronutrient that is critical for many physiological processes, including glucose metabolism, regulation of inflammation, and intestinal barrier function. Further, zinc dysregulation is associated with an increased risk of chronic inflammatory diseases such as type II diabetes, obesity, and inflammatory bowel disease. However, whether altered zinc status is a symptom or cause of disease onset remains unclear. Common symptoms of these three chronic diseases include the onset of increased intestinal permeability and zinc dyshomeostasis. The specific focus of this work is to investigate how dietary sources of intestinal permeability, such as high sucrose consumption, impact transporter-mediated zinc homeostasis and subsequent zinc-dependent physiology contributing to disease development. Method: We used in vivo subchronic sucrose treatment, ex vivo intestinal organoid culture, and in vitro cell systems. We analyze the alterations in zinc metabolism and intestinal permeability and metabolic outcomes. Results: We found that subchronic sucrose treatment resulted in systemic changes in steady-state zinc distribution and increased 65Zn transport (blood-to-intestine) along with greater ZIP14 expression at the basolateral membrane of the intestine. Further, sucrose treatment enhanced cell survival of intestinal epithelial cells, activation of the EGFR-AKT-STAT3 pathway, and intestinal permeability. Conclusion: Our work suggests that subchronic high sucrose consumption alters systemic and intestinal zinc homeostasis linking diet-induced changes in zinc homeostasis to the intestinal permeability and onset of precursors for chronic disease.

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.

Metal-ion transporter SLC39A8 is required for brain manganese uptake and accumulation.

Manganese (Mn) is an essential nutrient, but is toxic in excess. Whole-body Mn levels are regulated in part by the metal-ion influx transporter SLC39A8, which plays an essential role in the liver by reclaiming Mn from bile. Physiological roles of SLC39A8 in Mn homeostasis in other tissues, however, remain largely unknown. To screen for extrahepatic requirements for SLC39A8 in tissue Mn homeostasis, we crossed Slc39a8-inducible global-KO (Slc39a8 iKO) mice with Slc39a14 KO mice, which display markedly elevated blood and tissue Mn levels. Tissues were then analyzed by inductively coupled plasma-mass spectrometry to determine levels of Mn. Although Slc39a14 KO; Slc39a8 iKO mice exhibited systemic hypermanganesemia and increased Mn loading in the bone and kidney due to Slc39a14 deficiency, we show Mn loading was markedly decreased in the brains of these animals, suggesting a role for SLC39A8 in brain Mn accumulation. Levels of other divalent metals in the brain were unaffected, indicating a specific effect of SLC39A8 on Mn. In vivo radiotracer studies using 54Mn in Slc39a8 iKO mice revealed that SLC39A8 is required for Mn uptake by the brain, but not most other tissues. Furthermore, decreased 54Mn uptake in the brains of Slc39a8 iKO mice was associated with efficient inactivation of Slc39a8 in isolated brain microvessels but not in isolated choroid plexus, suggesting SLC39A8 mediates brain Mn uptake via the blood-brain barrier. These findings establish SLC39A8 as a candidate therapeutic target for mitigating Mn uptake and accumulation in the brain, the primary organ of Mn toxicity.

Recent progress toward understanding the role of ZIP14 in regulating systemic manganese homeostasis.

ZIP14 is a metal transporter essential for the regulation of body manganese homeostasis. The physiological functions of ZIP14 have been uncovered mainly through two lines of in vivo studies that examined the phenotypes of ZIP14 loss, including studies of humans with ZIP14 mutations and animals with ZIP14 deficiency. This mini review aims at presenting an updated view of the important advances made towards understanding the genetic and pathological mechanisms of brain manganese overload caused by ZIP14 deficiency.

Hereditary Disorders of Manganese Metabolism: Pathophysiology of Childhood-Onset Dystonia-Parkinsonism in SLC39A14 Mutation Carriers and Genetic Animal Models.

Over the last decade, several clinical reports have outlined cases of childhood-onset manganese (Mn)-induced dystonia-parkinsonism, resulting from loss-of-function mutations in the Mn influx transporter gene SLC39A14. These clinical cases have provided a wealth of knowledge on Mn toxicity and homeostasis. However, our current understanding of the underlying neuropathophysiology is severely lacking. The recent availability of Slc39a14 knockout (KO) murine and zebrafish animal models provide a powerful platform to investigate the neurological effects of elevated blood and brain Mn concentrations in vivo. As such, the objective of this review was to organize and summarize the current clinical literature and studies utilizing Slc39a14-KO animal models and assess the validity of the animal models based on the clinical presentation of the disease in human mutation carriers.

Hypermanganesemia with Dystonia Type 2: A Potentially Treatable Neurodegenerative Disorder: A Case Series in a Tertiary University Hospital.

IMPORTANCE: Hypermanganesemia with dystonia type 2 is a rare autosomal recessive neurodegenerative disorder characterized by the loss of previously acquired milestones, dystonia, parkinsonian features, a high serum manganese level, and characteristic neuroimaging findings such as bilateral and symmetrically increased T1 and decreased T2/fluid-attenuated inversion recovery signal intensity in the basal ganglia. This condition is secondary to a mutation in the SLC39A14 gene. OBJECTIVE: To present a series of three cases of hypermanganesemia with dystonia type 2, which was genetically confirmed secondary to a mutation in the SLC39A14 gene, and to describe the treatment and clinical course in these cases. DESIGN: A retrospective case series. SETTING: University, Tertiary hospital. PARTICIPANTS: Three unrelated pediatric patients with hypermanganesemia with dystonia type 2, genetically confirmed to be secondary to a mutation in the SLC39A14 gene. EXPOSURES: Chelation therapy using calcium disodium edetate. MAIN OUTCOME(S) AND MEASURE(S): The response to chelation therapy based on clinical improvements in motor and cognition developments. RESULTS: All three patients were started on chelation therapy using calcium disodium edetate, and two of them showed an improvement in their clinical course. The chelation therapy could alter the course of the disease and prevent deterioration in the clinical setting. CONCLUSIONS AND RELEVANCE: Early diagnosis and intervention with chelating agents, such as calcium disodium edetate, will help change the outcome in patients with hypermanganesemia with dystonia type 2. This finding highlights the importance of early diagnosis and treatment in improving the outcomes of patients with treatable neurodegenerative disorders.

Pathophysiological studies of aging Slc39a14 knockout mice to assess the progression of manganese-induced dystonia-parkinsonism.

Over the last decade, several clinical reports have outlined cases of early-onset manganese (Mn)-induced dystonia-parkinsonism, resulting from loss of function mutations of the Mn transporter gene SLC39A14. Previously, we have performed characterization of the behavioral, neurochemical, and neuropathological changes in 60-day old (PN60) Slc39a14-knockout (KO) murine model of the human disease. Here, we extend our studies to aging Slc39a14-KO mice to assess the progression of the disease. Our results indicate that 365-day old (PN365) Slc39a14-KO mice present with markedly elevated blood and brain Mn levels, similar to those found in the PN60 mice and representative of the human cases of the disease. Furthermore, aging Slc39a14-KO mice consistently manifest a hypoactive and dystonic behavioral deficits, similar to the PN60 animals, suggesting that the behavioral changes are established early in life without further age-associated deterioration. Neurochemical, neuropathological, and functional assessment of the dopaminergic system of the basal ganglia revealed absence of neurodegenerative changes of dopamine (DA) neurons in the substantia nigra pars compacta (SNc), with no changes in DA or metabolite concentrations in the striatum of Slc39a14-KO mice relative to wildtype (WT). Similar to the PN60 animals, aging Slc39a14-KO mice expressed a marked inhibition of potassium-stimulated DA release in the striatum. Together our findings indicate that the pathophysiological changes observed in the basal ganglia of aging Slc39a14-KO animals are similar to those at PN60 and aging does not have a significant effect on these parameters.

Calcium and the Ca-ATPase SPCA1 modulate plasma membrane abundance of ZIP8 and ZIP14 to regulate Mn(II) uptake in brain microvascular endothelial cells.

Manganese (II) accumulation in human brain microvascular endothelial cells is mediated by the metal-ion transporters ZRT IRT-like protein 8 (ZIP8) and ZRT IRT-like protein 14 (ZIP14). The plasma membrane occupancy of ZIP14, in particular, is increased in cells treated with Mn2+, lipopolysaccharide, or IL-6, but the mechanism of this regulation has not been elucidated. The calcium-transporting type 2C member 1 ATPase, SPCA1, is a Golgi-localized Ca2+-uptake transporter thought to support Golgi uptake of Mn2+ also. Here, we show using surface protein biotinylation, indirect immunofluorescence, and GFP-tagged proteins that cytoplasmic Ca2+ regulates ZIP8- and ZIP14-mediated manganese accumulation in human brain microvascular endothelial cells by increasing the plasma membrane localization of these transporters. We demonstrate that RNAi knockdown of SPCA1 expression results in an increase in cytoplasmic Ca2+ levels. In turn, we found increased cytoplasmic Ca2+ enhances membrane-localized ZIP8 and ZIP14 and a subsequent increase in 54Mn2+ uptake. Furthermore, overexpression of WT SPCA1 or a gain-of-function mutant resulted in a decrease in cytoplasmic Ca2+ and 54Mn2+ accumulation. While addition of Ca2+ positively regulated ZIP-mediated 54Mn2+ uptake, we show chelation of Ca2+ diminished manganese transport. In conclusion, the modulation of ZIP8 and ZIP14 membrane cycling by cytoplasmic calcium is a novel finding and provides new insight into the regulation of the uptake of Mn2+ and other divalent metal ions-mediated ZIP metal transporters.

The Combined Inactivation of Intestinal and Hepatic ZIP14 Exacerbates Manganese Overload in Mice.

ZIP14 is a newly identified manganese transporter with high levels of expression in the small intestine and the liver. Loss-of-function mutations in ZIP14 can lead to systemic manganese overload, which primarily affects the central nervous system, causing neurological disorders. To elucidate the roles of intestinal ZIP14 and hepatic ZIP14 in maintaining systemic manganese homeostasis, we generated mice with single-tissue or two-tissue Zip14 knockout, including intestine-specific (Zip14-In-KO), liver-specific (Zip14-L-KO), and double (intestine and liver) Zip14-knockout (Zip14-DKO) mice. Zip14flox/flox mice were used as the control. Tissue manganese contents in these mice were compared using inductively coupled plasma mass spectrometry (ICP-MS) analysis. We discovered that although the deletion of intestinal ZIP14 only moderately increased systemic manganese loading, the deletion of both intestinal and hepatic ZIP14 greatly exacerbated the body's manganese burden. Our results provide new knowledge to further the understanding of manganese metabolism, and offer important insights into the mechanisms underlying systemic manganese overload caused by the loss of ZIP14.

Loss of slc39a14 causes simultaneous manganese hypersensitivity and deficiency in zebrafish.

Manganese neurotoxicity is a hallmark of hypermanganesemia with dystonia 2, an inherited manganese transporter defect caused by mutations in SLC39A14. To identify novel potential targets of manganese neurotoxicity, we performed transcriptome analysis of slc39a14-/- mutant zebrafish that were exposed to MnCl2. Differentially expressed genes mapped to the central nervous system and eye, and pathway analysis suggested that Ca2+ dyshomeostasis and activation of the unfolded protein response are key features of manganese neurotoxicity. Consistent with this interpretation, MnCl2 exposure led to decreased whole-animal Ca2+ levels, locomotor defects and changes in neuronal activity within the telencephalon and optic tectum. In accordance with reduced tectal activity, slc39a14-/- zebrafish showed changes in visual phototransduction gene expression, absence of visual background adaptation and a diminished optokinetic reflex. Finally, numerous differentially expressed genes in mutant larvae normalised upon MnCl2 treatment indicating that, in addition to neurotoxicity, manganese deficiency is present either subcellularly or in specific cells or tissues. Overall, we assembled a comprehensive set of genes that mediate manganese-systemic responses and found a highly correlated and modulated network associated with Ca2+ dyshomeostasis and cellular stress. This article has an associated First Person interview with the first author of the paper.

Effect of Chelation Therapy on a Korean Patient With Brain Manganese Deposition Resulting From a Compound Heterozygous Mutation in the SLC39A14 Gene.

Mutations in the manganese transporter gene SLC39A14 lead to inherited disorders of manganese metabolism. Chelation therapy with edetate calcium disodium (CaNa2EDTA) is known to effectively reduce manganese deposition. We describe the first identified Korean case of SLC39A14-associated manganism and the treatment response to a 5-year chelation therapy. An 18-year-old female presented with childhood-onset dystonia. Magnetic resonance imaging showed T1 hyperintensity throughout the basal ganglia, brainstem, cerebellum, cerebral and cerebellar white matter, and pituitary gland. Blood manganese levels were elevated, and whole-exome sequencing revealed compound heterozygous mutations in SLC39A14. Treatment with intravenous CaNa2EDTA led to a significant reduction in serum manganese levels and T1 hyperintensities. However, her dystonia improved insignificantly. Hence, early diagnosis of this genetic disorder is essential because it is potentially treatable. Even though our treatment did not significantly reverse the establish deficits, chelation therapy could have been more effective if it was started at an earlier stage of the disease.

"Cock-walk" gait and "horseshoe moustache" sign on MRI in inherited hypermanganesemia.

Exogenous manganese (Mn) intoxication leads only to neurotoxicity, whereas inherited hypermanganesemia additionally can cause cirrhosis and polycythemia. We report two affected siblings in a family from South India with severe dysarthria, without dysphagia, generalized dystonia, and characteristic "cock-walk" gait which are clinical clues. Genetic study showed homozygous mutation in the first exon of solute carrier family 30 member 10 (SLC30A10) gene (c.134T>C) confirming the diagnosis of inherited hypermanganesaemia with dystonia 1 (HMNDYT1). Characteristic brain MRI finding is involvement of pontine tegmentum on T1 axial images (due to affliction of central tegmental tract [CTT]) with sparing of ventral pons giving rise to "horseshoe moustache" sign. Symmetric hyperintensities in dentate nucleus, globus pallidus, and putamen while relatively sparing caudate nucleus on T1 without signal intensity abnormalities on T2 images are highly suggestive of hypermanganesaemia. Axial diffusion tensor imaging confirmed the "horseshoe moustache" sign to be constituted by the affected CTT. Hypermanganesaemia-induced CTT involvement in T1 needs to be differentiated from the other more common pediatric causes of CTT affliction which are evident on T2 or diffusion weighted images. Identification is crucial as it is a treatable disorder of metal deposition amenable to chelation.

A novel homozygous SLC39A14 variant in an infant with hypermanganesemia and a review of the literature.

Background: Manganese (Mn) is an essential trace metal necessary for good health; however, excessive amounts in the body are neurotoxic. To date, three genes (SLC30A10, SLC39A8, and SLC39A14) have been discovered to cause inborn errors in Mn metabolism in humans. As very rare diseases, the clinical features require further clarification. Methods: A male Chinese patient who mainly presented with hypermanganesemia and progressive parkinsonism-dystonia was recruited for this study. We collected and analyzed clinical information, performed whole-exome sequencing (WES), and reviewed the relevant literature. Results: The motor-developmental milestones of the patient were delayed at the age of 4 months, followed by rapidly progressive dystonia. The patient displayed elevated Mn concentrations in blood and urine, and brain magnetic resonance imaging (MRI) showed symmetrical hyperintensity on T1-weighted images and hypointensity on T2-weighted images in multiple regions. A novel homozygous variant of the SLC39A14 gene (c.1058T > G, p.L353R) was identified. The patient was treated with disodium calcium edetate chelation (Na2CaEDTA). Three months later, mild improvement in clinical manifestation, blood Mn levels, and brain MRI was observed. To date, 15 patients from 10 families have been reported with homozygous mutations of SLC39A14, with a mean age of onset of 14.9 months. The common initial symptom is motor regression or developmental milestone delay, with a disease course for nearly all patients involving development of progressive generalized dystonia and loss of ambulation before treatment. Additionally, hypermanganesemia manifests as Mn values ranging from 4- to 25-fold higher than normal baseline levels, along with brain MRI results similar to those observed in the recruited patient. Nine SLC39A14 variants have been identified. Seven patients have been treated with Na2CaEDTA, and only one patient achieved obvious clinical improvement. Conclusion: We identified a novel SLC39A14 mutation related to autosomal recessive hypermanganesemia with dystonia-2, which is a very rare disease. Patients present motor regression or delay of developmental milestones and develop progressive generalized dystonia. Chelation therapy with Na2CaEDTA appears to effectively chelate Mn and increase urinary Mn excretion in some cases; however, clinical response varies. The outcome of the disease was unsatisfactory. This study expands the genetic spectrum of this disease.

Behavioral and neurochemical studies of inherited manganese-induced dystonia-parkinsonism in Slc39a14-knockout mice.

Inherited autosomal recessive mutations of the manganese (Mn) transporter gene SLC39A14 in humans, results in elevated blood and brain Mn concentrations and childhood-onset dystonia-parkinsonism. The pathophysiology of this disease is unknown, but the nigrostriatal dopaminergic system of the basal ganglia has been implicated. Here, we describe pathophysiological studies in Slc39a14-knockout (KO) mice as a preclinical model of dystonia-parkinsonism in SLC39A14 mutation carriers. Blood and brain metal concentrations in Slc39a14-KO mice exhibited a pattern similar to the human disease with highly elevated Mn concentrations. We observed an early-onset backward-walking behavior at postnatal day (PN) 21 which was also noted in PN60 Slc39a14-KO mice as well as dystonia-like movements. Locomotor activity and motor coordination were also impaired in Slc39a14-KO relative to wildtype (WT) mice. From a neurochemical perspective, striatal dopamine (DA) and metabolite concentrations and their ratio in Slc39a14-KO mice did not differ from WT. Striatal tyrosine hydroxylase (TH) immunohistochemistry did not change in Slc39a14-KO mice relative to WT. Unbiased stereological cell quantification of TH-positive and Nissl-stained estimated neuron number, neuron density, and soma volume in the substantia nigra pars compacta (SNc) was the same in Slc39a14-KO mice as in WT. However, we measured a marked inhibition (85-90%) of potassium-stimulated DA release in the striatum of Slc39a14-KO mice relative to WT. Our findings indicate that the dystonia-parkinsonism observed in this genetic animal model of the human disease is associated with a dysfunctional but structurally intact nigrostriatal dopaminergic system. The presynaptic deficit in DA release is unlikely to explain the totality of the behavioral phenotype and points to the involvement of other neuronal systems and brain regions in the pathophysiology of the disease.

Restriction of Manganese Intake Prevents the Onset of Brain Manganese Overload in Zip14-/- Mice.

As a newly identified manganese transport protein, ZIP14 is highly expressed in the small intestine and liver, which are the two principal organs involved in regulating systemic manganese homeostasis. Loss of ZIP14 function leads to manganese overload in both humans and mice. Excess manganese in the body primarily affects the central nervous system, resulting in irreversible neurological disorders. Therefore, to prevent the onset of brain manganese accumulation becomes critical. In this study, we used Zip14-/- mice as a model for ZIP14 deficiency and discovered that these mice were born without manganese loading in the brain, but started to hyper-accumulate manganese within 3 weeks after birth. We demonstrated that decreasing manganese intake in Zip14-/- mice was effective in preventing manganese overload that typically occurs in these animals. Our results provide important insight into future studies that are targeted to reduce the onset of manganese accumulation associated with ZIP14 dysfunction in humans.