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.
zinc transporter-1; zinc transporter-4; zinc transporter-6; preclinical Alzheimer's disease
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.
Zinc transporter 1; Zinc transporter 3; LC3; Beclin-1; Seizure
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.
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.
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.
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.
zinc; zinc transporter; ZnT4; airway epithelium; airway inflammation; asthma; Zinquin; Se-Autometallography (Se-AMG)
Zinc transporters are solute carrier family members. To date, 10 zinc transporters (ZnTs) and 14 Zrt-, Irt-like proteins (ZIPs) have been identified. ZnTs control intracellular zinc levels by effluxing zinc from the cytoplasm into the extracellular fluid, intracellular vesicles, and organelles; ZIPs also contribute to control intracellular zinc levels with influxing zinc into the cytoplasm. Recently, changes in zinc transporter expression have been observed in some stress-induced diseases, such as Alzheimer’s disease and diabetes mellitus. However, little is known regarding the mechanisms that regulate zinc transporter expression. To address this, we have investigated the effect of a well-established stress response pathway, the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant responsive element (ARE) pathway, on zinc transporter mRNA levels. Exposure to 10−4 M tert-butylhydroquinone (t-BHQ), which activates Nrf2-ARE signaling, for 6 h significantly increases ZnT-1, ZnT-3, and ZnT-6 mRNAs levels, and significantly decreases ZnT-10 and ZIP-3 mRNA levels. These changes are not observed with 10−6 M t-BHQ, which does not activate Nrf2-ARE signaling. Furthermore, t-BHQ exposure does not affect metal responsive element transcription, a cis element that is activated in response to intracellular free zinc accumulation. From these results, we believe that the transcription of ZnT-1, ZnT-3, ZnT-6, ZnT-10, and ZIP-3 is influenced by the Nrf2-ARE signal transduction pathway.
Autophagy has been shown to be involved in the pathophysiology of developmental seizure-induced brain damage. The present study aimed to examine whether E-64d, an autophagy inhibitor, was able to facilitate developmental seizure-induced hippocampal mossy fiber sprouting, in particular sprouting-associated zinc transporter signals. Recurrent seizures were induced by penicillin every other day in Sprague-Dawley rats from postnatal day 21 (P21). Rats were randomly assigned into the control group (CONT), recurrent seizure group (RS) and the seizure plus E-64d group (E64D). The expression levels of beclin-1 and B-cell lymphoma 2 were analyzed at 1.5, 3, 6 and 24 h after the last seizures using western blot analysis. At P51, mossy fiber sprouting and the mRNA expression levels of zinc transporter 2 (ZnT-2), ZnT-4, ZnT-5, ZnT-6, ZnT-7, divalent cation transporter 1, Zrt-Irt-like protein 6 (ZIP-6), ZIP-7, cathepsin D and cathepsin L in the rat hippocampus were assessed using Timm staining and reverse transcription-quantitative polymerase chain reaction analysis, respectively. Reduced hippocampal mossy fiber sprouting were detected in the E-64d-treated rats compared with the non-treated control. In parallel with these observations, there was a marked reduction in the mRNA expression levels of ZnT-4 at P51 in the E-64d-treated rat hippocampus compared with the non-treated seizure group. Linear correlation analysis showed significant inter-relationship among ZIP-7, ZnT-4, ZnT-5, ZnT-7, cathepsin D and cathepsin L. These results indicate that the ZnT-4/ZIP-7/cathepsin signaling pathway serves a crucial function in the neuroprotective effects of E-64d. Thus, E-64d may offer a novel strategy for the development of therapeutic interventions for developmental seizure-induced brain damage.
zinc transporter; mossy fiber spouting; E-64d; developmental; seizure
The human SLC30A8 gene encodes the secretory granule-localised zinc transporter ZnT8 whose expression is chiefly restricted to the endocrine pancreas. Single nucleotide polymorphisms (SNPs) in the human SLC30A8 gene have been associated, through genome-wide studies, with altered type 2 diabetes risk. In addition to a role in the control of insulin release, recent studies involving targeted gene ablation from the pancreatic α cell (Solomou et al., J Biol Chem 290(35):21432-42) have also implicated ZnT8 in the control of glucagon release. Up to now, however, the possibility that increased levels of the transporter in these cells may impact glucagon secretion has not been explored.
Here, we use a recently-developed reverse tetracyline transactivator promoter-regulated ZnT8 transgene to drive the over-expression of human ZnT8 selectively in the α cell in adult mice. Glucose homeostasis and glucagon secretion were subsequently assessed both in vivo during hypoglycemic clamps and from isolated islets in vitro.
Doxyclin-dependent human ZnT8 mRNA expression was apparent in both isolated islets and in fluorescence-activated cell sorting- (FACS) purified α cells. Examined at 12 weeks of age, intraperitoneal glucose (1 g/kg) tolerance was unchanged in transgenic mice versus wild-type littermates (n = 8-10 mice/genotype, p > 0.05) and sensitivity to intraperitoneal insulin (0.75U/kg) was similarly unaltered in transgenic animals. In contrast, under hyperinsulinemic-hypoglycemic clamp, a ~45 % (p < 0.001) reduction in glucose infusion rate was apparent, and glucagon release was significantly (~40 %, p < 0.01) impaired, in transgenic mice. Correspondingly, examined in vitro, glucagon secretion was significantly reduced (~30 %, p < 0.05) from transgenic versus control islets at low, stimulatory glucose concentrations (1 mM, p < 0.05) but not at high glucose (17 mM) glucose (p > 0.05). Over-expression of ZnT8 in glucagonoma-derived αTC1-9 cells increased granule free Zn2+ concentrations consistent with a role for Zn2+ in this compartment in the action of ZnT8 on glucagon secretion.
Increased ZnT8 expression, and a likely increase in intragranular free Zn2+ concentration, is deleterious in pancreatic α cells for stimulated glucagon release. These data provide further evidence that type 2 diabetes-associated polymorphisms in the SLC30A8/ZnT8 gene may act in part via alterations in glucagon release and suggest that ZnT8 activation may restrict glucagon release in some settings.
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.
Copper; Copper transporter; lm syndrome; Metallothionein; zinc deficiency; ZnT4; Zinc Transporter
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.
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.
Alzheimer's disease; astrocytes; cell cycle; cyclooxygenase-2; microglia; retinoblastoma protein
The incidence of Alzheimer's disease (AD) is increasing. Major risk factors for AD are advancing age and diabetes. Lately, obesity has been associated with an increased risk of dementia. Obese and diabetic individuals are prone to decreased circulating levels of zinc, reducing the amount of zinc available for crucial intracellular processes. In the brain, zinc co-localizes with glutamate in synaptic vesicles, and modulates NMDA receptor activity. Intracellular zinc is involved in apoptosis and fluctuations in cytoplasmic Zn2+ affect modulation of intracellular signaling. The ZNT and ZIP proteins participate in intracellular zinc homeostasis. Altered expression of zinc-regulatory proteins has been described in AD patients. Using microarray data from human frontal cortex (BrainCloud), this study investigates expression of the SCLA30A (ZNT) and SCLA39A (ZIP) families of genes in a Caucasian and African-American sample of 145 neurologically and psychiatrically normal individuals. Expression of ZNT3 and ZNT4 were significantly reduced with increasing age, whereas expression of ZIP1, ZIP9 and ZIP13 were significantly increased. Increasing body mass index (BMI) correlated with a significant reduction in ZNT1 expression similar to what is seen in the early stages of AD. Increasing BMI also correlated with reduced expression of ZNT6. In conclusion, we found that the expression of genes that regulate intracellular zinc homeostasis in the human frontal cortex is altered with increasing age and affected by increasing BMI. With the increasing rates of obesity throughout the world, these findings warrant continuous scrutiny of the long-term consequences of obesity on brain function and the development of neurodegenerative diseases.
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.
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.
Zinc transporter; ZnT2; Prostate; Marginal zinc deficiency
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.
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.
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.
Autoantibody; FCRL3; HLA; MHC; SLC30A8; Type 1 diabetes; Zinc transporter 8
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.
intracellular free zinc; zinc sensor; zntA; zitB; zntR; transcriptional response
The present study evaluated the ability and optimal concentration of tetramethylpyrazine (TMP) to induce human umbilical cord-derived mesenchymal stem cells (hUMSCs) to differentiate into neuron-like cells in vitro. Human umbilical cords from full-term caesarean section patients were used to obtain hUMSCs by collagenase digestion after removal of the umbilical artery and vein. The surface antigen expression profile of cultured hUMSCs was monitored by flow cytometry. After amplification, cells of the 5th passage were divided into experimental groups A–C treated with TMP at 4.67, 2.34 and 1.17 mg/ml, respectively, in low glucose-Dulbecco's Modified Eagle's Medium (L-DMEM) (induction medium), while group D (control) was exposed to L-DMEM culture medium only. Differentiation of hUMSCs into neuron-like cells and morphological changes were observed every 0.5 h with an inverted phase contrast microscope for 6 h. After the 6-h induction period, proportions of cells expressing neuronal markers neuron-specific enolase (NSE), neurofilament protein (NF-H) and glial fibrillary acidic protein (GFAP) were detected by immunohistochemistry. The optimal concentration of TMP was selected on the basis of neuron-like cell positive rate. Western blotting and RT-polymerase chain reaction were applied to detect the expression of NSE, NF-H, and GFAP of the group of optimal concentration in each point-in-time. Results showed that most primary cells were adherent 12 h after seeding and first appeared as diamond or polygon shapes. Thereafter, they gradually grew into long spindle-shaped cells and finally in a radiating or swirling pattern. The cells maintained a strong proliferative capacity after continuous passage. Flow cytometry analysis of cultured hUMSCs at the 3rd, 5th and 10th passages expressed CD73, CD90 and CD105, but not CD11b, CD19, CD34, CD45 or human leukocyte antigen-DR. After 6 h of TMP treatment, typical neuron-like cells with many protrusions connected into a net-like pattern were observed in all experimental groups. These neuron-like cells were positive for NSE and NF-H, but negative for GFAP. Among the tested treatment groups, group A with TMP at 4.67 mg/ml had the highest expression of NSE and NF-H. By contrast, no change was found after induction in the control group. The mRNA expression of cells expressing neuronal markers as well as GAPDH was observed, with the relative NSE transcript levels of 0, 1.303±0.031, 1.558±0.025, 1.927±0.019 and 2.415±0.033 after 0, 1, 2, 4 and 6 h of treatment, respectively; the mRNA expression of NH-F was 0, 1.429±0.025, 1.551±0.024, 1.930±0.042 and 1.398±0.014 after 0, 1, 2, 4 and 6 h of treatment, respectively. There was no expression of GFAP before or after induction and all the groups showed high expression of GAPDH at each time point. Protein expression was also observed on cells expressing neuronal markers as well as GAPDH at each time point. The protein expression of NSE was 0, 0.717±0.097, 0.919±0.056, 1.097±0.143 and 1.157±0.055 in proper order; the protein expression of NH-F was 0, 0.780±0.103, 0.973±0.150, 1.053±0.107 and 0.753±0.094 in proper order. There was no expression of GFAP before or after induction, and all the groups showed high expression of GAPDH at each tested time point. Our results demonstrated that TMP can induce hUMSCs to differentiate into neuron-like cells effectively with the optimal concentration of 4.67 mg/ml. After induction, the NSE and NF-H of the neuron-like cells were positive, but the GFAP-2 was negative.
tetramethylpyrazine; human umbilical cord-derived mesenchymal stem cells; differentiation; neuron-like cells
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.
Human; In vitro; Gene expression; Immunohistochemistry; Anti-inflammatory
Autoantibodies to the islet-specific zinc transporter isoform 8 (ZnT8) are detected in the majority of type 1 diabetes patients prior to and at clinical diagnosis. The presence of ZnT8Ab after diagnosis has not been investigated. This study analyzed the autoantibody response to ZnT8 in regard to age at onset and disease duration. Two new onset type 1 diabetes patient cohorts with different age distributions at onset (2–17 and 15–34 years of age at onset), a longitudinal subset of the younger type 1 diabetes patient cohort (n = 32), and a cohort of GAD65Ab-positive LADA patients (n = 47) was analyzed for the presence of autoantibodies directed to the two major isoforms, ZnT8-Arginine (ZnT8R) and ZnT8-Tryptophan (ZnT8W). The majority of type 1 diabetes patients tested positive for ZnT8Ab to both isoforms. ZnT8Ab titers were significantly higher in the younger type 1 diabetes patients as compared with the older cohort (ZnT8RAb at a median of 148 and 29 U/ml, respectively, p < 0.001) (ZnT8WAb at a median of 145 and 58 U/ml, respectively, p < 0.01). ZnT8RAb and ZnT8WAb titers were significantly lower in the LADA patients (ZnT8RAb at a median of 14 U/ml, ZnT8WAb at a median of 25 U/ml) as compared with either type 1 diabetes cohorts. In our longitudinal analysis of type 1 diabetes patients after clinical diagnosis, ZnT8Ab levels to both isoforms declined significantly during the initial year of disease (ZnT8RAb from a median of 320–162 U/ml, p = 0.0001; ZnT8WAb from a median of 128–46 U/ml, p = 0.0011). The antibody titers further declined during the following 4 years (p < 0.0001). We conclude that ZnT8Ab presents a useful marker for type 1 diabetes, especially in younger patients at disease diagnosis.
Autoimmune diabetes; ZnT8; autoantibodies; longitudinal; radioligand binding assay
The SLC30A8 gene codes for a pancreatic beta-cell-expressed zinc transporter, ZnT8. A polymorphism in the SLC30A8 gene is associated with susceptibility to type 2 diabetes, although the molecular mechanism through which this phenotype is manifest is incompletely understood. Such polymorphisms may exert their effect via impacting expression level of the gene product. We used an shRNA-mediated approach to reproducibly downregulate ZnT8 mRNA expression by >90% in the INS-1 pancreatic beta cell line. The ZnT8-downregulated cells exhibited diminished uptake of exogenous zinc, as determined using the zinc-sensitive reporter dye, zinquin. ZnT8-downregulated cells showed reduced insulin content and decreased insulin secretion (expressed as percent of total insulin content) in response to hyperglycemic stimulus, as determined by insulin immunoassay. ZnT8-depleted cells also showed fewer dense-core vesicles via electron microscopy. These data indicate that reduced ZnT8 expression in cultured pancreatic beta cells gives rise to a reduced insulin response to hyperglycemia. In addition, although we provide no direct evidence, these data suggest that an SLC30A8 expression-level polymorphism could affect insulin secretion and the glycemic response in vivo.
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.
Alzheimer's disease; zinc; mild cognitive impairment; zinc transporters; serum
CYFIP2 is thought to regulate mRNA translation at synapses. Tiwari et al. reveal reduced CYFIP2 expression in post-mortem Alzheimer’s disease brains, and show that CYFIP2 reduction in mice causes abnormal amyloid production, tau hyperphosphorylation, and spatial memory loss. CYFIP2 could represent a molecular ‘hub’ with potential as a therapeutic target in Alzheimer’s disease.
CYFIP2 is thought to regulate mRNA translation at synapses. Tiwari et al. reveal reduced CYFIP2 expression in post-mortem Alzheimer’s disease brains, and show that CYFIP2 reduction in mice causes abnormal amyloid production, tau hyperphosphorylation, and spatial memory loss. CYFIP2 could represent a molecular ‘hub’ with potential as a therapeutic target in Alzheimer’s disease.
Characteristic features of Alzheimer’s disease are memory loss, plaques resulting from abnormal processing of amyloid precursor protein (APP), and presence of neurofibrillary tangles and dystrophic neurites containing hyperphosphorylated tau. Currently, it is not known what links these abnormalities together. Cytoplasmic FMR1 interacting protein 2 (CYFIP2) has been suggested to regulate mRNA translation at synapses and this may include local synthesis of APP and alpha-calcium/calmodulin-dependent kinase II, a kinase that can phosphorylate tau. Further, CYFIP2 is part of the Wiskott-Aldrich syndrome protein-family verprolin-homologous protein complex, which has been implicated in actin polymerization at synapses, a process thought to be required for memory formation. Our previous studies on p25 dysregulation put forward the hypothesis that CYFIP2 expression is reduced in Alzheimer’s disease and that this contributes to memory impairment, abnormal APP processing and tau hyperphosphorylation. Here, we tested this hypothesis. First, in post-mortem tissue CYFIP2 expression was reduced by ∼50% in severe Alzheimer’s hippocampus and superior temporal gyrus when normalized to expression of a neuronal or synaptic marker protein. Interestingly, there was also a trend for decreased expression in mild Alzheimer’s disease hippocampus. Second, CYFIP2 expression was reduced in old but not in young Tg2576 mice, a model of familial Alzheimer’s disease. Finally, we tested the direct impact of reduced CYFIP2 expression in heterozygous null mutant mice. We found that in hippocampus this reduced expression causes an increase in APP and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) protein, but not mRNA expression, and elevates production of amyloid-β42. Reduced CYFIP2 expression also increases alpha-calcium/calmodulin-dependent kinase II protein expression, and this is associated with hyperphosphorylation of tau at serine-214. The reduced expression also impairs spine maturity without affecting spine density in apical dendrites of CA1 pyramidal neurons. Furthermore, the reduced expression prevents retention of spatial memory in the water maze. Taken together, our findings indicate that reduced CYFIP2 expression triggers a cascade of change towards Alzheimer’s disease, including amyloid production, tau hyperphosphorylation and memory loss. We therefore suggest that CYFIP2 could be a potential hub for targeting treatment of the disease.
amyloid beta; BACE1; mRNA translation; spatial memory; tau phosphorylation
Parkinson’s disease (PD) is characterized by the accumulation of abnormal α-synuclein in selected regions of the brain following a gradient of severity with disease progression. Whether this is accompanied by globally altered protein synthesis is poorly documented. The present study was carried out in PD stages 1-6 of Braak and middle-aged (MA) individuals without alterations in brain in the substantia nigra, frontal cortex area 8, angular gyrus, precuneus and putamen.
Reduced mRNA expression of nucleolar proteins nucleolin (NCL), nucleophosmin (NPM1), nucleoplasmin 3 (NPM3) and upstream binding transcription factor (UBF), decreased NPM1 but not NPM3 nucleolar protein immunostaining in remaining neurons; diminished 18S rRNA, 28S rRNA; reduced expression of several mRNAs encoding ribosomal protein (RP) subunits; and altered protein levels of initiation factor eIF3 and elongation factor eEF2 of protein synthesis was found in the substantia nigra in PD along with disease progression. Although many of these changes can be related to neuron loss in the substantia nigra, selective alteration of certain factors indicates variable degree of vulnerability of mRNAs, rRNAs and proteins in degenerating sustantia nigra. NPM1 mRNA and 18S rRNA was increased in the frontal cortex area 8 at stage 5-6; modifications were less marked and region-dependent in the angular gyrus and precuneus. Several RPs were abnormally regulated in the frontal cortex area 8 and precuneus, but only one RP in the angular gyrus, in PD. Altered levels of eIF3 and eIF1, and decrease eEF1A and eEF2 protein levels were observed in the frontal cortex in PD. No modifications were found in the putamen at any time of the study except transient modifications in 28S rRNA and only one RP mRNA at stages 5-6. These observations further indicate marked region-dependent and stage-dependent alterations in the cerebral cortex in PD. Altered solubility and α-synuclein oligomer formation, assessed in total homogenate fractions blotted with anti-α-synuclein oligomer-specific antibody, was demonstrated in the substantia nigra and frontal cortex, but not in the putamen, in PD. Dramatic increase in α-synuclein oligomers was also seen in fluorescent-activated cell sorter (FACS)-isolated nuclei in the frontal cortex in PD.
Altered machinery of protein synthesis is altered in the substantia nigra and cerebral cortex in PD being the frontal cortex area 8 more affected than the angular gyrus and precuneus; in contrast, pathways of protein synthesis are apparently preserved in the putamen. This is associated with the presence of α-synuclein oligomeric species in total homogenates; substantia nigra and frontal cortex are enriched, albeit with different band patterns, in α-synuclein oligomeric species, whereas α-synuclein oligomers are not detected in the putamen.
Electronic supplementary material
The online version of this article (doi:10.1186/s40478-015-0257-4) contains supplementary material, which is available to authorized users.
α-synuclein oligomers; Parkinson’s disease; protein synthesis; nucleolar stress; ribosomes