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1.  Alterations of Zinc Transporter Proteins ZnT-1, ZnT-4 and ZnT-6 in Preclinical Alzheimer's Disease Brain 
Our previous studies demonstrate alterations of zinc (Zn) transporter proteins ZnT-1, ZnT-4, and ZnT-6 in vulnerable brain regions of subjects with mild cognitive impairment (MCI), early and late stage Alzheimer's disease (AD) and suggest that disruptions of Zn homeostasis may play a role in the pathogenesis of AD. ZnT-1 exports Zn from the cytosol to extracellular compartments, ZnT-4 transports Zn from the cytosol to lysosomes and endosomes, and ZnT-6 sequesters Zn in the trans-Golgi network. A preclinical stage of AD (PCAD) has been described in which subjects show no overt clinical manifestations of AD but demonstrate significant AD pathology at autopsy. To determine if alterations of ZnT proteins occur in PCAD we measured ZnT-1, ZnT-4, and ZnT-6 in the hippocampus/parahippocampal gyrus (HPG) and cerebellum (CER) of 7 PCAD subjects and 7 age matched normal control (NC) subjects using Western blot analysis and immunohistochemistry. Our results show a significant decrease (P < 0.05) of ZnT-1 in HPG of PCAD subjects, along with an increase of ZnT-4 in PCAD CER and ZnT-6 in PCAD HPG, but a significant decrease in PCAD CER compared to NC subjects. Confocal microscopy of representative sections of HPG shows altered ZnTs are associated with neurons immunopositive for MC-1, a monoclonal antibody that identifies neurons early in formation of neurofibrillary tangles. Overall, our results suggest that alterations in Zn transport proteins may contribute to the pathology observed in PCAD subjects before onset of clinical symptoms.
PMCID: PMC3175637  PMID: 19371353
zinc transporter-1; zinc transporter-4; zinc transporter-6; preclinical Alzheimer's disease
2.  Autophagy inhibitor 3-methyladenine regulates the expression of LC3, Beclin-1 and ZnTs in rat cerebral cortex following recurrent neonatal seizures 
Autophagy is a homeostatic process for intracellular recycling of bulk proteins and aging organelles. Increased autophagy has now been reported in experimental models of traumatic brain injury, stroke and excitotoxicity, and in patients with Alzheimer’s disease and critical illness. The role of autophagy in developmental epilepsy, however, is unknown. The present study was to investigate the effects of recurrent neonatal seizure, in the presence and absence of autophagy inhibitor 3-methyladenine (3-MA), on the acute phase gene expression of ZnTs, LC3 and Beclin-1 in rat cerebral cortex and the interaction among them.
Thirty-six Sprague-Dawley neonatal rats at postnatal day 6(P6) were randomly divided into three groups: a recurrent-seizures group (RS, n=12), a 3-MA treated-seizure group (3-MA group, each rat pretreated with 3-methyladenine before seizures, 100nmol/μl/day, i.p., n=12) and a control group (n=12). At 1.5 and 6 hours after the last seizures, the mRNA levels of ZnT1-ZnT3, microtubule-associated protein 1A/1B light chain 3 (LC3) and beclin-1 were detected using the real-time RT-PCR method. The LC3 protein level was examined by Western blotting.
The levels of LC3, beclin-1 and ZnT-2 transcripts in the RS group elevated significantly at 1.5 and 6 hours after the last seizures compared with those in the control and 3-MA groups. At the interval of 1.5 hours, the mRNA level of ZnT-1 increased significantly after the last seizure compared with that in the control group. There was no significant difference in the transcript levels of ZnT-3 among the three groups. Linear correlation analysis showed that the expression of the five genes in the control group exhibited a significant inter-relationship. In the 3-MA group, however, the inter-relationship was only found between beclin-1 and ZnT-1. In the RS group, the inter-relationship was not observed.
The autophagy/lysosomal pathway is immediately activated along with the elevated expression of ZnT1 and ZnT2 in the cerebral cortex after recurrent seizures. 3-MA is involved in the regulation of the autophagy/lysosomal pathway and ZnTs by down-regulating the expression of LC3 and beclin-1.
PMCID: PMC4129681  PMID: 25214972
Zinc transporter 1; Zinc transporter 3; LC3; Beclin-1; Seizure
3.  Altered Expression of ZnT10 in Alzheimer's Disease Brain 
PLoS ONE  2013;8(5):e65475.
There is an increasing body of evidence suggesting that metal homeostasis is dysregulated in the pathology of Alzheimer's disease (AD). Although expression levels of several transporters belonging the SLC30 family, which comprises predominantly zinc transporters, have been studied in the AD brain, SLC30A10 (ZnT10) has not been studied in this context. To determine if dysregulated expression of ZnT10, which may transport both Zn and Mn, could be a factor that contributes to AD, we investigated if there were differences in ZnT10 mRNA levels in specimens of frontal cortex from AD patients and controls and also if brain tissue from the APP/PS1 transgenic (Tg) mouse model showed abnormal levels of ZnT10 mRNA expression. Our results show that ZnT10 is significantly (P<0.01) decreased in the frontal cortex in AD. Furthermore, we observed a significant decrease in ZnT10 mRNA levels in the APP/PS1-Tg mice compared with wild-type controls (P<0.01). Our results suggest that this dysregulation in ZnT10 could further contribute to disease progression.
PMCID: PMC3669266  PMID: 23741496
4.  SLC30A3 Responds to Glucose- and Zinc Variations in ß-Cells and Is Critical for Insulin Production and In Vivo Glucose-Metabolism During ß-Cell Stress 
PLoS ONE  2009;4(5):e5684.
Ion transporters of the Slc30A- (ZnT-) family regulate zinc fluxes into sub-cellular compartments. β-cells depend on zinc for both insulin crystallization and regulation of cell mass.
Methodology/Principal Findings
This study examined: the effect of glucose and zinc chelation on ZnT gene and protein levels and apoptosis in β-cells and pancreatic islets, the effects of ZnT-3 knock-down on insulin secretion in a β-cell line and ZnT-3 knock-out on glucose metabolism in mice during streptozotocin-induced β-cell stress. In INS-1E cells 2 mM glucose down-regulated ZnT-3 and up-regulated ZnT-5 expression relative to 5 mM. 16 mM glucose increased ZnT-3 and decreased ZnT-8 expression. Zinc chelation by DEDTC lowered INS-1E insulin content and insulin expression. Furthermore, zinc depletion increased ZnT-3- and decreased ZnT-8 gene expression whereas the amount of ZnT-3 protein in the cells was decreased. Zinc depletion and high glucose induced apoptosis and necrosis in INS-1E cells. The most responsive zinc transporter, ZnT-3, was investigated further; by immunohistochemistry and western blotting ZnT-3 was demonstrated in INS-1E cells. 44% knock-down of ZnT-3 by siRNA transfection in INS-1E cells decreased insulin expression and secretion. Streptozotocin-treated mice had higher glucose levels after ZnT-3 knock-out, particularly in overt diabetic animals.
Zinc transporting proteins in β-cells respond to variations in glucose and zinc levels. ZnT-3, which is pivotal in the development of cellular changes as also seen in type 2 diabetes (e.g. amyloidosis in Alzheimer's disease) but not previously described in β-cells, is present in this cell type, up-regulated by glucose in a concentration dependent manner and up-regulated by zinc depletion which by contrast decreased ZnT-3 protein levels. Knock-down of the ZnT-3 gene lowers insulin secretion in vitro and affects in vivo glucose metabolism after streptozotocin treatment.
PMCID: PMC2683566  PMID: 19492079
5.  Apical Localization of Zinc Transporter ZnT4 in Human Airway Epithelial Cells and Its Loss in a Murine Model of Allergic Airway Inflammation 
Nutrients  2011;3(11):910-928.
The apical cytoplasm of airway epithelium (AE) contains abundant labile zinc (Zn) ions that are involved in the protection of AE from oxidants and inhaled noxious substances. A major question is how dietary Zn traffics to this compartment. In rat airways, in vivo selenite autometallographic (Se-AMG)-electron microscopy revealed labile Zn-selenium nanocrystals in structures resembling secretory vesicles in the apical cytoplasm. This observation was consistent with the starry-sky Zinquin fluorescence staining of labile Zn ions confined to the same region. The vesicular Zn transporter ZnT4 was likewise prominent in both the apical and basal parts of the epithelium both in rodent and human AE, although the apical pools were more obvious. Expression of ZnT4 mRNA was unaffected by changes in the extracellular Zn concentration. However, levels increased 3-fold during growth of cells in air liquid interface cultures and decreased sharply in the presence of retinoic acid. When comparing nasal versus bronchial human AE cells, there were significant positive correlations between levels of ZnT4 from the same subject, suggesting that nasal brushings may allow monitoring of airway Zn transporter expression. Finally, there were marked losses of both basally-located ZnT4 protein and labile Zn in the bronchial epithelium of mice with allergic airway inflammation. This study is the first to describe co-localization of zinc vesicles with the specific zinc transporter ZnT4 in airway epithelium and loss of ZnT4 protein in inflamed airways. Direct evidence that ZnT4 regulates Zn levels in the epithelium still needs to be provided. We speculate that ZnT4 is an important regulator of zinc ion accumulation in secretory apical vesicles and that the loss of labile Zn and ZnT4 in airway inflammation contributes to AE vulnerability in diseases such as asthma.
PMCID: PMC3257720  PMID: 22254085
zinc; zinc transporter; ZnT4; airway epithelium; airway inflammation; asthma; Zinquin; Se-Autometallography (Se-AMG)
6.  Cooperative Functions of ZnT1, Metallothionein and ZnT4 in the Cytoplasm Are Required for Full Activation of TNAP in the Early Secretory Pathway 
PLoS ONE  2013;8(10):e77445.
The activation process of secretory or membrane-bound zinc enzymes is thought to be a highly coordinated process involving zinc transport, trafficking, transfer and coordination. We have previously shown that secretory and membrane-bound zinc enzymes are activated in the early secretory pathway (ESP) via zinc-loading by the zinc transporter 5 (ZnT5)-ZnT6 hetero-complex and ZnT7 homo-complex (zinc transport complexes). However, how other proteins conducting zinc metabolism affect the activation of these enzymes remains unknown. Here, we investigated this issue by disruption and re-expression of genes known to be involved in cytoplasmic zinc metabolism, using a zinc enzyme, tissue non-specific alkaline phosphatase (TNAP), as a reporter. We found that TNAP activity was significantly reduced in cells deficient in ZnT1, Metallothionein (MT) and ZnT4 genes (ZnT1−/−MT−/−ZnT4−/− cells), in spite of increased cytosolic zinc levels. The reduced TNAP activity in ZnT1−/−MT−/−ZnT4−/− cells was not restored when cytosolic zinc levels were normalized to levels comparable with those of wild-type cells, but was reversely restored by extreme zinc supplementation via zinc-loading by the zinc transport complexes. Moreover, the reduced TNAP activity was adequately restored by re-expression of mammalian counterparts of ZnT1, MT and ZnT4, but not by zinc transport-incompetent mutants of ZnT1 and ZnT4. In ZnT1−/−MT−/−ZnT4−/− cells, the secretory pathway normally operates. These findings suggest that cooperative zinc handling of ZnT1, MT and ZnT4 in the cytoplasm is required for full activation of TNAP in the ESP, and present clear evidence that the activation process of zinc enzymes is elaborately controlled.
PMCID: PMC3799634  PMID: 24204829
7.  Maximal COX-2 and ppRb expression in neurons occurs during early Braak stages prior to the maximal activation of astrocytes and microglia in Alzheimer's disease 
Neuronal expression of cyclooxygenase-2 (COX-2) and cell cycle proteins is suggested to contribute to neurodegeneration during Alzheimer's disease (AD). The stimulus that induces COX-2 and cell cycle protein expression in AD is still elusive. Activated glia cells are shown to secrete substances that can induce expression of COX-2 and cell cycle proteins in vitro. Using post mortem brain tissue we have investigated whether activation of microglia and astrocytes in AD brain can be correlated with the expression of COX-2 and phosphorylated retinoblastoma protein (ppRb). The highest levels of neuronal COX-2 and ppRb immunoreactivity are observed in the first stages of AD pathology (Braak 0–II, Braak A). No significant difference in COX-2 or ppRb neuronal immunoreactivity is observed between Braak stage 0 and later Braak stages for neurofibrillary changes or amyloid plaques. The mean number of COX-2 or ppRb immunoreactive neurons is significantly decreased in Braak stage C compared to Braak stage A for amyloid deposits. Immunoreactivity for glial markers KP1, CR3/43 and GFAP appears in the later Braak stages and is significantly increased in Braak stage V-VI compared to Braak stage 0 for neurofibrillary changes. In addition, a significant negative correlation is observed between the presence of KP1, CR3/43 and GFAP immunoreactivity and the presence of neuronal immunoreactivity for COX-2 and ppRb. These data show that maximal COX-2 and ppRb immunoreactivity in neurons occurs during early Braak stages prior to the maximal activation of astrocytes and microglia. In contrast to in vitro studies, post mortem data do not support a causal relation between the activation of microglia and astrocytes and the expression of neuronal COX-2 and ppRb in the pathological cascade of AD.
PMCID: PMC1308853  PMID: 16300681
Alzheimer's disease; astrocytes; cell cycle; cyclooxygenase-2; microglia; retinoblastoma protein
8.  Zinc Transporter 8 (ZnT8) Expression Is Reduced by Ischemic Insults: A Potential Therapeutic Target to Prevent Ischemic Retinopathy 
PLoS ONE  2012;7(11):e50360.
The zinc (Zn++) transporter ZnT8 plays a crucial role in zinc homeostasis. It’s been reported that an acute decrease in ZnT8 levels impairs β cell function and Zn++ homeostasis, which contribute to the pathophysiology of diabetes mellitus (DM). Although ZnT8 expression has been detected in the retinal pigment epithelium (RPE), its expression profile in the retina has yet to be determined. Furthermore, the link between diabetes and ischemic retinopathy is well documented; nevertheless, the molecular mechanism(s) of such link has yet to be defined. Our aims were to; investigate the expression profile of ZnT8 in the retina; address the influence of ischemia on such expression; and evaluate the influence of YC-1; (3-(50-hydroxymethyl-20-furyl)-1-benzyl indazole), a hypoxia inducible factor-1 (HIF-1) inhibitor, on the status of ZnT8 expression. We used real-time RT-PCR, immunohistochemistry, and Western blot in the mouse model of oxygen-induced retinopathy (OIR) and Müller cells to evaluate the effects of ischemia/hypoxia and YC-1 on ZnT8 expression. Our data indicate that ZnT8 was strongly expressed in the outer nuclear layer (ONL), outer plexiform layer (OPL), ganglion cell layer (GCL), and nerve fiber layer (NFL), whereas the photoreceptor layer (PRL), inner nuclear layer (INL) and inner plexiform layer (IPL) showed moderate ZnT8 immunoreactivity. Furthermore, we demonstrate that retinal ischemic insult induces a significant downregulation of ZnT8 at the message and protein levels, YC-1 rescues the injured retina by restoring the ZnT8 to its basal homeostatic levels in the neovascular retinas. Our data indicate that ischemic retinopathy maybe mediated by aberrant Zn++ homeostasis caused by ZnT8 downregulation, whereas YC-1 plays a neuroprotective role against ischemic insult. Therefore, targeting ZnT8 provides a therapeutic strategy to combat neovascular eye diseases.
PMCID: PMC3507680  PMID: 23209723
9.  Genetic association of zinc transporter 8 (ZnT8) autoantibodies in type 1 diabetes cases 
Diabetologia  2012;55(7):1978-1984.
Autoantibodies to zinc transporter 8 (ZnT8A) are associated with risk of type 1 diabetes. Apart from the SLC30A8 gene itself, little is known about the genetic basis of ZnT8A. We hypothesise that other loci in addition to SLC30A8 are associated with ZnT8A.
The levels of ZnT8A were measured in 2,239 British type 1 diabetic individuals diagnosed before age 17 years, with a median duration of diabetes of 4 years. Cases were tested at over 775,000 loci genome wide (including 53 type 1 diabetes associated regions) for association with positivity for ZnT8A. ZnT8A were also measured in an independent dataset of 855 family members with type 1 diabetes.
Only FCRL3 on chromosome 1q23.1 and the HLA class I region were associated with positivity for ZnT8A. rs7522061T>C was the most associated single nucleotide polymorphism (SNP) in the FCRL3 region (p = 1.13 × 10−16). The association was confirmed in the family dataset (p ≤ 9.20 × 10−4). rs9258750A>G was the most associated variant in the HLA region (p = 2.06 × 10−9 and p = 0.0014 in family cases). The presence of ZnT8A was not associated with HLA-DRB1, HLA-DQB1, HLA-A, HLA-B or HLA-C (p > 0.05). Unexpectedly, the two loci associated with the presence of ZnT8A did not alter risk of having type 1 diabetes, and the 53 type 1 diabetes risk loci did not influence positivity for ZnT8A, despite them being disease specific.
ZnT8A are not primary pathogenic factors in type 1 diabetes. Nevertheless, ZnT8A testing in combination with other autoantibodies facilitates disease prediction, despite the biomarker not being under the same genetic control as the disease.
Electronic supplementary material
The online version of this article (doi:10.1007/s00125-012-2540-2) contains peer-reviewed but unedited supplementary material, which is available to authorised users.
PMCID: PMC3369141  PMID: 22526605
Autoantibody; FCRL3; HLA; MHC; SLC30A8; Type 1 diabetes; Zinc transporter 8
10.  Serum Zinc in the Progression of Alzheimer's Disease 
Previous studies show significantly decreased levels of zinc transporter 1 (ZnT-1) in the brain of subjects with mild cognitive impairment (MCI) but significantly increased ZnT-1 in late stage AD (LAD). However, the reason for the apparent dichotomy is unclear. Based on in vivo studies that show animals provided a zinc (Zn) deficient diet demonstrate decreased brain ZnT-1, we used inductively coupled plasma-mass spectrometry (ICP-MS) to quantify serum Zn levels from 18 living mild to moderate AD patients (9 men, 9 women), 19 MCI patients (9 men, 10 women) and 16 age-matched normal control (NC) subjects (9 men, 7 women). Zinc levels for all subjects were not significantly different between any of the three subject groups. However, there was a statistically significant decrease of serum Zn (11.7 ± 0.5 μM) in men with MCI compared to women with MCI (13.7 ± 0.6 μM) and NC men (13.9 ± 0.6 μM). Serum Zn levels in probable AD patients were comparable to those in NC subjects. Overall, these data suggest a significant decrease of serum Zn in men with MCI may explain the loss of ZnT-1 observed in previous studies and suggest there may be more pronounced sex differences in MCI than were previously recognized.
PMCID: PMC2683420  PMID: 18997297
Alzheimer's disease; zinc; mild cognitive impairment; zinc transporters; serum
11.  Decreased expression of CD200 and CD200 receptor in Alzheimer's disease: A potential mechanism leading to chronic inflammation 
Experimental neurology  2008;215(1):5-19.
Inflammatory activation of microglia in response to neurodegenerative changes in diseases such as Alzheimer's disease (AD) and Parkinson's disease has been extensively described. These observations have suggested that inflammation could be contributing to disease progression. In this paper, the potential role of CD200 and CD200 receptor (CD200R), whose known functions are to activate anti-inflammatory pathways and induce immune tolerance through binding of CD200 to CD200 receptor (CD200R), was studied in AD. Quantitative studies showed a significant decrease in CD200 protein and mRNA in AD hippocampus and inferior temporal gyrus, but not cerebellum. Immunohistochemistry of brain tissue sections of hippocampus, superior frontal gyrus, inferior temporal gyrus and cerebellum from AD and non-demented cases demonstrated a predominant, though heterogeneous, neuronal localization for CD200. Decreased neuronal expression was apparent in brain regions affected by AD pathology. There was also a significant decrease in CD200R mRNA expression in AD hippocampus and inferior temporal gyrus, but not cerebellum. Low expression of CD200R by microglia was confirmed at the mRNA and protein level using cultured human microglia compared to blood-derived macrophages. Treatment of microglia and macrophages with interleukin-4 and interleukin-13 significantly increased expression of CD200R. Expression of these cytokines was not generally detectable in brain. These data indicate that the anti-inflammatory CD200/CD200R system may be deficient in AD brains. Mechanisms aimed at increasing levels of CD200 and CD200R could have therapeutic potential for controlling inflammation in human neurodegenerative diseases.
PMCID: PMC2765462  PMID: 18938162
Human; In vitro; Gene expression; Immunohistochemistry; Anti-inflammatory
12.  The Znt4 mutation inlethal milk mice affects intestinal zinc homeostasis through the expression of other Zn transporters 
Genes & Nutrition  2006;1(1):61-70.
The lethal milk mouse syndrome is caused by a point mutation in the zinc transporter gene ZnT4 resulting in defective zinc secretion in the milk of homozygous mutant dams. Pups of any genotype fed solely on lm milk die within the first two weeks of neonatal life, displaying zinc deficiency symptoms. Homozygous mutant pups survive when foster nursed by wild type dams and show signs of mild zinc deficiency in adulthood. To further investigate the role of ZnT4 in zinc secretion in the intestinal epithelium, we have studied the expression by real time quantitative PCR of mutant ZnT4 and of other zinc transporters of the Zip and ZnT families, in the jejunum of homozygous lm mice and of the isogenic wild type strain C57BL/ 6J. We report in this paper that expression of the mutant ZnT4 mRNA, carrying a premature translational termination codon (ZnT4/lm), is almost absent in tissues from lm mice, probably as a result of degradation by the Nonsense Mediated mRNA Decay (NMD) Pathway. In the jejunum of mutant mice, we also observed decreased expression of the uptake zinc transporter Zip4, paralleled by increased levels of both metallothionein genes MTI and MTII. Zinc supplementation of lm mice in the drinking water did not result in further decrease of Zip4 expression, but led to full induction of MT mRNAs. These results lead us to conclude that, although in the enterocytes of lm mice the absence of the zinc secretion activity mediated by ZnT4 results in increased intracellular zinc concentration, other zinc efflux activities are able to maintain the level of zinc ions below the threshold necessary for full induction of metallothioneins.
PMCID: PMC3454819  PMID: 18850221
Copper; Copper transporter; lm syndrome; Metallothionein; zinc deficiency; ZnT4; Zinc Transporter
13.  ZntR-mediated transcription of zntA responds to nanomolar intracellular free zinc 
In E. coli, ZitB and ZntA are important metal exporters that enhance cell viability under high environmental zinc. To understand their functions in maintaining zinc homeostasis, we applied a novel genetically-encoded fluorescent zinc sensor to monitor the intracellular free zinc changes in wild type, ΔzitB and ΔzntA E. coli cells upon sudden exposure to toxic levels of zinc (“zinc shock”). The intracellular readily exchangeable zinc concentration (or “free” zinc) increases transiently from picomolar to nanomolar levels, accelerating zinc-activated gene transcription. After zinc shock, the zitB mRNA level is constant while the zntA mRNA increases substantially in a zinc-dependent manner. In the ΔzitB E. coli strain the free zinc concentration rises more rapidly after zinc shock compared to wild-type cells while a prolonged accumulation of free zinc is observed in the ΔzntA strain. Based on these results, we propose that ZitB functions as a constitutive, first-line defense against toxic zinc influx, while ZntA is up-regulated to efficiently lower the free zinc concentration. Furthermore, the ZntR-mediated transcription of zntA exhibits an apparent K1/2 for zinc activation in the nanomolar range in vivo, significantly higher than the femtomolar affinity for zinc binding and transcription activation previously measured in vitro. A kinetically-controlled transcription model is sufficient to explain the observed regulation of intracellular free zinc concentration by ZntR and ZntA after zinc shock.
PMCID: PMC3408962  PMID: 22459916
intracellular free zinc; zinc sensor; zntA; zitB; zntR; transcriptional response
14.  Zinc transporter expression profiles in the rat prostate following alterations in dietary zinc 
Zinc plays important roles in numerous cellular activities and physiological functions. Intracellular zinc levels are strictly maintained by zinc homeostatic mechanisms. Zinc concentrations in the prostate are the highest of all soft tissues and could be important for prostate health. However, the mechanisms by which the prostate maintains high zinc levels are still unclear. In addition, the response of the prostate to alterations in dietary zinc is unknown. The current study explored cellular zinc levels and zinc transporter expression profiles in the lobes of the prostate during dietary marginal zinc depletion. Rats were given either zinc-adequate (ZA, 30 mg Zn/kg) or marginal zinc-deficient (MZD, 5 mg Zn/kg) diet for 9 weeks. In addition, a subgroup of the MZD rats was supplemented with phytase (1,500 unit/kg diet) to improve zinc bioavailability. We found that both zinc concentrations and ZnT2 expression in the prostate dorsolateral lobes were substantially higher than in the ventral lobes (P<0.05). Marginal zinc depletion significantly decreased ZnT2 expression in the dorsolateral lobes (P<0.05), and phytase supplementation had a trend to increase ZnT2 expression. In addition, of all measured zinc transporters, only ZnT2 mRNA abundance was significantly correlated to the zinc concentrations in the dorsolateral lobe. No correlations were found between zinc transporter expression and zinc concentrations in the ventral lobes. These results indicate that ZnT2 may play a significant role in the maintenance of zinc homeostasis in the prostate.
PMCID: PMC4152227  PMID: 19760107
Zinc transporter; ZnT2; Prostate; Marginal zinc deficiency
15.  Chromosomal Locus for Cadmium Resistance in Pseudomonas putida Consisting of a Cadmium-Transporting ATPase and a MerR Family Response Regulator 
Pseudomonads from environmental sources vary widely in their sensitivity to cadmium, but the basis for this resistance is largely uncharactarized. A chromosomal fragment encoding cadmium resistance was cloned from Pseudomonas putida 06909, a rhizosphere bacterium, and sequence analysis revealed two divergently transcribed genes, cadA and cadR. CadA was similar to cadmium-transporting ATPases known mostly from gram-positive bacteria, and to ZntA, a lead-, zinc-, and cadmium-transporting ATPase from Escherichia coli. CadR was related to the MerR family of response regulators that normally control mercury detoxification in other bacterial systems. A related gene, zntR, regulates zntA in E. coli, but it is not contiguous with zntA in the E. coli genome as cadA and cadR were in P. putida. In addition, unlike ZntA and other CadA homologs, but similar to the predicted product of gene PA3690 in the P. aeruginosa genome, the P. putida CadA sequence had a histidine-rich N-terminal extension. CadR and the product of PA3689 of P. aeruginosa also had histidine-rich C-terminal extensions not found in other MerR family response regulators. Mutational analysis indicated that cadA and cadR are fully responsible for cadmium resistance and partially for zinc resistance. However, unlike zntA, they did not confer significant levels of lead resistance. The cadA promoter was responsive to Cd(II), Pb(II), and Zn(II), while the cadR promoter was only induced by Cd(II). CadR apparently represses its own expression at the transcriptional level. However, CadR apparently does not repress cadA. Homologs of the cadmium-transporting ATPase were detected in many other Pseudomonas species.
PMCID: PMC92752  PMID: 11282588
16.  Longitudinal changes in zinc transport kinetics, metallothionein, and zinc transporter expression in a blood-brain barrier model in response to a moderately excessive zinc environment$ 
A blood-brain barrier (BBB) model composed of porcine brain capillary endothelial cells (BCEC) was exposed to a moderately excessive zinc environment (50 µmol Zn/L) in cell culture and longitudinal measurements were made of zinc transport kinetics, ZnT-1 (SLC30A1) expression, and changes in the protein concentration of metallothionein (MT), ZnT-1, ZnT-2 (SLC30A2), and Zip1 (SLC39A1). Zinc release by cells of the BBB model was significantly increased after 12–24 h of exposure, but decreased back to control levels after 48–96 h, as indicated by transport across the BBB from both the ablumenal (brain) and lumenal (blood) directions. Expression of ZnT-1, the zinc export protein, increased 169% within 12 h, but was no longer different from controls after 24 h. Likewise, ZnT-1 protein content increased transiently after 12 h of exposure but returned to control levels by 24 h. Capacity for zinc uptake and retention increased from both the lumenal and ablumenal directions within 12–24 h of exposure and remained elevated. MT and ZnT-2 were elevated within 12 h and remained elevated throughout the study. Zip1 was unchanged by the treatment. The BBB’s response to a moderately high zinc environment was dynamic and involved multiple mechanisms. The initial response was to increase the cell’s capacity to sequester zinc with additional MT and increase zinc export with the ZnT-1 protein. But, the longer term strategy involved increasing ZnT-2 transporters, presumably to sequester zinc into intracellular vesicles as a mechanism to protect the brain and maintain brain zinc homeostasis.
PMCID: PMC2247408  PMID: 18061429
zinc homeostasis; zinc toxicity; blood-brain barrier; zinc transporters; metallothionein
17.  Angiotensin II Requires Zinc and Downregulation of the Zinc Transporters ZnT3 and ZnT10 to Induce Senescence of Vascular Smooth Muscle Cells 
PLoS ONE  2012;7(3):e33211.
Senescence, a hallmark of mammalian aging, is associated with the onset and progression of cardiovascular disease. Angiotensin II (Ang II) signaling and zinc homeostasis dysfunction are increased with age and are linked to cardiovascular disease, but the relationship among these processes has not been investigated. We used a model of cellular senescence induced by Ang II in vascular smooth muscle cells (VSMCs) to explore the role of zinc in vascular dysfunction. We found that Ang II-induced senescence is a zinc-dependent pathway mediated by the downregulation of the zinc transporters ZnT3 and ZnT10, which work to reduce cytosolic zinc. Zinc mimics Ang II by increasing reactive oxygen species (ROS), activating NADPH oxidase activity and Akt, and by downregulating ZnT3 and ZnT10 and inducing senescence. Zinc increases Ang II-induced senescence, while the zinc chelator TPEN, as well as overexpression of ZnT3 or ZnT10, decreases ROS and prevents senescence. Using HEK293 cells, we found that ZnT10 localizes in recycling endosomes and transports zinc into vesicles to prevent zinc toxicity. Zinc and ZnT3/ZnT10 downregulation induces senescence by decreasing the expression of catalase. Consistently, ZnT3 and ZnT10 downregulation by siRNA increases ROS while downregulation of catalase by siRNA induces senescence. Zinc, siZnT3 and siZnT10 downregulate catalase by a post-transcriptional mechanism mediated by decreased phosphorylation of ERK1/2. These data demonstrate that zinc homeostasis dysfunction by decreased expression of ZnT3 or ZnT10 promotes senescence and that Ang II-induced senescence is a zinc and ROS-dependent process. Our studies suggest that zinc might also affect other ROS-dependent processes induced by Ang II, such as hypertrophy and migration of smooth muscle cells.
PMCID: PMC3299759  PMID: 22427991
18.  Zinc transporter-1 concentrates at the postsynaptic density of hippocampal synapses 
Molecular Brain  2014;7:16.
Zinc concentrates at excitatory synapses, both at the postsynaptic density and in a subset of glutamatergic boutons. Zinc can modulate synaptic plasticity, memory formation and nociception by regulating transmitter receptors and signal transduction pathways. Also, intracellular zinc accumulation is a hallmark of degenerating neurons in several neurological disorders. To date, no single zinc extrusion mechanism has been directly localized to synapses. Based on the presence of a canonical PDZ I motif in the Zinc Transporter-1 protein (ZnT1), we hypothesized that ZnT1 may be targeted to synaptic compartments for local control of cytosolic zinc. Using our previously developed protocol for the co-localization of reactive zinc and synaptic proteins, we further asked if ZnT1 expression correlates with presynaptic zinc content in individual synapses.
Here we demonstrate that ZnT1 is a plasma membrane protein that is enriched in dendritic spines and in biochemically isolated synaptic membranes. Hippocampal CA1 synapses labelled by postembedding immunogold showed over a 5-fold increase in ZnT1 concentration at synaptic junctions compared with extrasynaptic membranes. Subsynaptic analysis revealed a peak ZnT1 density on the postsynaptic side of the synapse, < 10 nm away from the postsynaptic membrane. ZnT1 was found in the vast majority of excitatory synapses regardless of the presence of vesicular zinc in presynaptic boutons.
Our study has identified ZnT1 as a novel postsynaptic density protein, and it may help elucidate the role of zinc homeostasis in synaptic function and disease.
PMCID: PMC3975337  PMID: 24602382
ZnT1; Hippocampus; Postsynaptic density; Vesicular Zn2+; PDZ I motif; Dendritic spine
19.  Ligand-Controlled Proteolysis of the Escherichia coli Transcriptional Regulator ZntR▿  
Journal of Bacteriology  2007;189(8):3017-3025.
Proteases play a crucial role in remodeling the bacterial proteome in response to changes in cellular environment. Escherichia coli ZntR, a zinc-responsive transcriptional regulator, was identified by proteomic experiments as a likely ClpXP substrate, suggesting that protein turnover may play a role in regulation of zinc homeostasis. When intracellular zinc levels are high, ZntR activates expression of ZntA, an ATPase essential for zinc export. We find that ZntR is degraded in vivo in a manner dependent on both the ClpXP and Lon proteases. However, ZntR degradation decreases in the presence of high zinc concentrations, the level of ZntR rises, and transcription of the zntA exporter is increased. Mutagenesis experiments reveal that zinc binding does not appear to be solely responsible for the zinc-induced protection from proteolysis. Therefore, we tested whether DNA binding was important in the zinc-induced stabilization of ZntR by mutagenesis of the DNA binding helices. Replacement of a conserved arginine (R19A) in the DNA binding domain both enhances ZntR degradation and abolishes zinc-induced transcriptional activation of zntA. Biochemical and physical analysis of ZntRR19A demonstrates that it is structurally similar to, and binds zinc as well as does, the wild-type protein but is severely defective in binding DNA. Thus, we conclude that two different ligands—zinc and DNA—function together to increase ZntR stability and that ligand-controlled proteolysis of ZntR plays an important role in fine-tuning zinc homeostasis in bacteria.
PMCID: PMC1855835  PMID: 17220226
20.  Zinc transporter 2 (SLC30A2) can suppress the vesicular zinc defect of adaptor protein 3-depleted fibroblasts by promoting zinc accumulation in lysosomes 
Experimental cell research  2007;313(7):1473-1483.
Zinc accumulation in the lumen of cytoplasmic vesicles is one of the mechanisms by which cells can store significant amounts of this essential but potentially toxic biometal. Previous studies had demonstrated reduced vesicular zinc levels in fibroblasts from mutant mice deficient in adaptor protein 3 (AP-3), a complex involved in protein trafficking to late endosomes and lysosomes. We have observed a similar phenotype in the human fibroblastoid cell line, M1, upon small interference RNA-mediated AP-3 knockdown. A survey of the expression and localization of zinc transporter (ZnT) family members identified ZnT2, ZnT3 and ZnT4 as likely mediators of vesicular zinc accumulation in M1 cells. Expression of green fluorescence protein (GFP)-tagged ZnT2 and ZnT3 promoted accumulation of vesicular zinc as visualized using the indicator zinquin. Moreover, GFP-ZnT2 overexpression elicited a significant accumulation of zinc within mature lysosomes, which in untransfected M1 cells contained little or no chelatable zinc, and restored the zinc storage capability of AP-3-deficient cells. These results suggest that ZnT2 can facilitate vesicular zinc accumulation independently of AP-3 function, and validate the M1 fibroblastoid line as a human cell culture system amenable to the study of vesicular zinc regulation using techniques compatible with functional genomic approaches.
PMCID: PMC1885236  PMID: 17349999
AP-3; Hermansky-Pudlak Syndrome; SLC30A; zinc transporter; Zinquin; ZnT
21.  [125I]SD-7015 reveals fine modalities of CB1 cannabinoid receptor density in the prefrontal cortex during progression of Alzheimer's disease 
Neurochemistry international  2011;60(3):286-291.
The cannabinoid type-1 receptor (CB1R) is one of the most abundant members of the G protein-coupled receptor family in the central nervous system. Once activated by their cognate ligands, endocannabinoids, CB1Rs generally limit the timing of neurotransmitter release at many cortical synapses. Prior studies have indicated the involvement of CB1R in neurodegeneration and in various neuronal insults, with an emphasis on their neuroprotective role. In the present study we used a novel selective CB1R radioligand to investigate regional variations in CB1R ligand binding as a factor of progressive Braak tau pathology in the frontal cortex of Alzheimer's disease (AD) patients. The frontal cortex was chosen for this study due to the high density of CB1Rs and their well-characterized involvement in the progression of AD. Post-mortem prefrontal cortex samples from AD patients from Braak stages I to VI and controls were subjected to CB1R autoradiography with [125I]SD-7015 as radioligand. Regional concentration of [125I]SD-7015, corresponding to, and thereby representing, regional CB1R densities, were expressed in fM/g_tissue. The results show that CB1R density inversely correlates with Braak tau pathology with the following tendency: controls
PMCID: PMC4180663  PMID: 22222721
Alzheimer's disease; Braak classification; Endocannabinoid system; Molecular imaging; Biomarker; Human brain autoradiography; CB1R; [125I]SD7015
The Journal of endocrinology  2010;206(2):159-169.
Genetic studies suggest that Zn transporters such as ZnT8 play a role in insulin secretion by pancreatic β-cells; however, little is known about the dynamic roles of Zn trafficking pathways on β-cell physiology. To test the acute effects of the inflammatory cytokines interleukin 1β (IL1β) and tumor necrosis factor α (TNFα) on Zn homeostasis, the mRNA expression profile of Zn transporters of the ZnT and ZIP families was examined. Exposure of MIN6 cells or primary murine islets to IL1β or TNFα altered the mRNA expression profile of Zn transporters; most notable was decreased ZnT8 mRNA levels. siRNA-mediated gene knockdown was used to examine the effects of decreased ZnT8 expression in primary dispersed murine islet cells from C57/BL6 mice and MIN6 cells. ZnT8 knockdown in these murine islets led to reduced glucose stimulated insulin secretion without altering the total cellular insulin content or cell viability at normal or supraphysiological Zn concentrations. The labile Zn content determined by flow cytometry after loading with the Zn-specific sensor FluoZin-3 AM was decreased in MIN6 cells following ZnT8 knockdown or IL1β treatment. These results suggest that an acute decrease in ZnT8 levels impairs β-cell function and Zn homeostasis, and may contribute to inflammatory cytokine-induced alterations in β-cell function.
PMCID: PMC3088990  PMID: 20508080
Diabetes Care  2011;34(8):1760-1765.
We investigated whether measuring autoantibodies against zinc transporter 8 (ZnT8A) and IA-2β (IA-2βA) may improve classification of new-onset type 1 diabetic patients based on detection of autoantibodies against insulin (IAA), GAD (GADA), and IA-2 (IA-2A). In addition, we studied the correlation of IA-2βA and ZnT8A with other biological and demographic variables.
Circulating autoantibodies were determined by liquid-phase radiobinding assays from 761 healthy control subjects and 655 new-onset (<1 week insulin) diabetic patients (aged 0–39 years) with clinical type 1 diabetes phenotype consecutively recruited by the Belgian Diabetes Registry.
At diagnosis, IA-2βA and ZnT8A prevalences were 41 and 58%, respectively. In IAA-negative, GADA-negative, and IA-2A–negative patients, one IA-2βA–positive and eleven ZnT8A-positive individuals were identified at the expense of eight and seven additional positive control subjects (1%), respectively, for each test. ZnT8A or IA-2βA screening increased (P < 0.001; McNemar) the number of patients with ≥2 antibodies both under (from 78 to 87% for ZnT8A and 82% for IA-2βA) and above age 15 (from 51 to 63% for ZnT8A and 56% for IA-2βA) versus 0% in control subjects. IA-2βA and ZnT8A were preferentially associated with IA-2A, and with younger age at diagnosis. Unlike ZnT8A, IA-2βA levels were positively correlated with HLA-DQ8 and negatively with HLA-DQ2. ZnT8A could replace IAA for classification of patients above age 10 without loss of sensitivity or specificity.
ZnT8A, and to a lesser degree IA-2βA, may usefully complement GADA, IA-2A, and IAA for classifying insulin-treated diabetes under age 40 years.
PMCID: PMC3142046  PMID: 21715527
Nutrition Journal  2005;4:35.
Small increases in zinc (Zn) consumption above recommended amounts have been shown to reduce copper (Cu) status in experimental animals and humans. Recently, we have reported that copper chaperone for Cu/Zn superoxide dismutase (CCS) protein level is increased in tissues of overtly Cu-deficient rats and proposed CCS as a novel biomarker of Cu status.
Weanling male Wistar rats were fed one of four diets normal in Cu and containing normal (30 mg Zn/kg diet) or moderately high (60, 120 or 240 mg Zn/kg diet) amounts of Zn for 5 weeks. To begin to examine the clinical relevance of CCS, we compared the sensitivity of CCS to mild Cu deficiency, induced by moderately high intakes of Zn, with conventional indices of Cu status.
Liver and erythrocyte CCS expression was significantly (P < 0.05) increased in rats fed the Zn-60 and/or Zn-120 diet compared to rats fed normal levels of Zn (Zn-30). Erythrocyte CCS expression was the most sensitive measure of reduced Cu status and was able to detect a decrease in Cu nutriture in rats fed only twice the recommended amount of Zn. Liver, erythrocyte and white blood cell CCS expression showed a significant (P < 0.05) inverse correlation with plasma and liver Cu concentrations and caeruloplasmin activity. Unexpectedly, rats fed the highest level of Zn (Zn-240) showed overall better Cu status than rats fed a lower level of elevated Zn (Zn-120). Improved Cu status in these rats correlated with increased duodenal mRNA expression of several Zn-trafficking proteins (i.e. MT-1, ZnT-1, ZnT-2 and ZnT-4).
Collectively, these data show that CCS is a sensitive measure of Zn-induced mild Cu deficiency and demonstrate a dose-dependent biphasic response for reduced Cu status by moderately high intakes of Zn.
PMCID: PMC1315358  PMID: 16307684
Metallomics  2013;6(1):154-165.
Global transcriptomic analysis, non-invasive real-time flux, nutritional profiling and metallomics reveal cell-centric response to zinc supplementation/depletion in zebrafish uptake epithelia.
Much remains to be understood about systemic regulation of zinc uptake in vertebrates, and adequate zinc status is far from always achieved in animals or human. In addition to absorbing zinc from the diet, fish are able to take up zinc directly from the water with the gills. This provides an elegant system to study zinc uptake, how it relates to zinc status, and the expression of genes for proteins involved in zinc acquisition. A 21-day experiment was conducted in which zebrafish were acclimated to deficient, control or excess zinc concentrations in the water and feed. Deficient provision of zinc reduced whole body zinc, potassium, sodium and calcium levels whilst zinc concentrations in the uptake epithelia (gills and gut) remained unchanged. Excess levels of zinc caused accumulation of zinc in the gills, intestine and carcass, but impaired whole body iron, sodium and calcium concentrations. Fish subjected to zinc deficiency had, surprisingly, a reduced zinc influx across the gill epithelium, even when tested at a high concentration of zinc in the water. Zinc influx in the excess group was indistinct from the control. Expression of genes for metallothionein-2 (Mt2) and zinc transporters-1, -2, and -8 (Znt1, Znt2, Znt8) in uptake epithelia showed in general a direct relationship with zinc supply, while mRNA for Zip4 was inversely related to zinc supply. Transcripts for the epithelial calcium channel (Ecac/Trpv6) showed time-dependent increased expression in the gills of the deficiency group, and a transient decrease of expression during zinc excess. Transcriptome profiling by microarrays showed that in both gills and intestine, the most markedly affected biological functions were those related to cell growth, proliferation and cancer, closely followed by processes of gene transcription and protein synthesis in general. Whilst changes in zinc supply had profound effects in the intestine on genes associated with uptake and metabolism of macronutrients, many of the unique categories of genes preferentially regulated in the gill could be mapped onto signalling pathways. This included pathways for PPAR/RXR, LXR/RXR, ATM, chemokine, and BMP signalling. Overall, the responses of epithelial tissue to zinc deficiency and excess are best explained by local epithelial homeostasis with no evidence of systemic control.
PMCID: PMC4157650  PMID: 24301558

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