Significance of Zn2+ signaling in cognition: Insight from synaptic Zn2+ dyshomeostasis

Zinc is concentrated in the synaptic vesicles via zinc transporter-3 (ZnT3), released from glutamatergic (zincergic) neuron terminals, and serves as a signal factor (Zn²⁺ signal) in the intracellular (cytosol) compartment as well as in the extracellular compartment. Synaptic Zn²⁺ signaling is dynamically linked to neurotransmission via glutamate and is involved in synaptic plasticity such as long-term potentiation (LTP) and cognitive activity. Zinc concentration in the synaptic vesicles is correlated with ZnT3 protein expression and potentially decreased under chronic zinc deficiency. Synaptic vesicle serves as a large pool for Zn²⁺ signaling and other organelles might also serve as a pool for Zn²⁺ signaling. ZnT3KO mice and zinc-deficient animals, which lack or reduce Zn²⁺ release into the extracellular space by action potentials, are able to recognize novel or displaced objects normally. However, the amount of Zn²⁺ functioning as a signal factor increases along with brain development. Exogenous Zn²⁺ lowers the threshold in hippocampal CA1 LTP induction in young rat. Furthermore, ZnT3KO mice lose advanced cognition such as contextual discrimination. It is likely that the optimal range of synaptic Zn²⁺ signaling is involved in cognitive activity. On the basis of the findings on the relationship between dyshomeostasis of synaptic Zn²⁺ and cognition, this paper summarizes the possible involvement of intracellular Zn²⁺ signaling in cognitive ability.     

Significance of the degree of synaptic Zn<Superscript>2+</Superscript> signaling in cognition

Zinc is a trace nutrient for the brain and a signal factor to serve for brain function. A portion of zinc is released from glutamatergic (zincergic) neuron terminals in the brain. Synaptic Zn²⁺ signaling is involved in synaptic plasticity such as long-term potentiaion (LTP), which is a cellular mechanism of memory. The block and/or loss of synaptic Zn²⁺ signaling in the hippocampus and amygdala with Zn²⁺ chelators affect cognition, while the role of synaptic Zn²⁺ signal is poorly understood, because zinc-binding proteins are great in number and multi-functional. Chronic zinc deficiency also affects cognition and cognitive decline induced by zinc deficiency might be associated with the increase in plasma glucocorticoid rather than the decrease in synaptic Zn²⁺ signaling. On the other hand, excess glutamatergic (zincergic) neuron activity induces excess influx of extracellular Zn²⁺ into hippocampal neurons, followed by cognitive decline. Intracellular Zn²⁺ dynamics, which is linked to presynaptic glutamate release, is critical for LTP and cognitive performance. This paper deals with insight into cognition from zinc as a nutrient and signal factor.     

Significance of Low Nanomolar Concentration of Zn<Superscript>2+</Superscript> in Artificial Cerebrospinal Fluid

Much less attention has been paid to Zn²⁺ in artificial cerebrospinal fluid (ACSF), i.e., extracellular medium, used for in vitro slice experiments than divalent cations such as Ca²⁺. Approximately 2 mM Ca²⁺ is added to conventional ACSF from essentiality of Ca²⁺ signaling in neurons and glial cells. However, no Zn²⁺ is added to it, even though the importance of Zn²⁺ signaling in them is recognizing. On the other hand, synaptic Zn²⁺ homeostasis is changed during brain slice preparation. Therefore, it is possible that not only neuronal excitation but also synaptic plasticity such as long-term potentiation is modified in ACSF without Zn²⁺, in which original physiology might not appear. The basal (static) levels of intracellular (cytosolic) Zn²⁺ and Ca²⁺ are not significantly different between brain slices prepared with conventional ACSF without Zn²⁺ and pretreated with ACSF containing 20 nM ZnCl₂ for 1 h. In the case of mossy fiber excitation, however, presynaptic activity assessed with FM 4–64 is significantly suppressed in the stratum lucidum of brain slices pretreated with ACSF containing Zn²⁺, indicating that hippocampal excitability is enhanced in brain slices prepared with ACSF without Zn²⁺. The evidence suggests that low nanomolar concentration of Zn²⁺ is necessary for ACSF. Furthermore, exogenous Zn²⁺ has opposite effect on LTP induction between in vitro and in vivo experiments. It is required to pay attention to extracellular Zn²⁺ concentration to understand synaptic function precisely.     

Transient fluctuations of intracellular zinc ions in cell proliferation

Zinc is essential for cell proliferation, differentiation, and viability. When zinc becomes limited for cultured cells, DNA synthesis ceases and the cell cycle is arrested. The molecular mechanisms of actions of zinc are believed to involve changes in the availability of zinc(II) ions (Zn²⁺). By employing a fluorescent Zn²⁺ probe, FluoZin-3 acetoxymethyl ester, intracellular Zn²⁺ concentrations were measured in undifferentiated and in nerve growth factor (NGF)-differentiated rat pheochromocytoma (PC12) cells. Intracellular Zn²⁺ concentrations are pico- to nanomolar in PC12 cells and are higher in the differentiated than in the undifferentiated cells. When following cellular Zn²⁺ concentrations for 48 h after the removal of serum, a condition that is known to cause cell cycle arrest, Zn²⁺ concentrations decrease after 30 min but, remarkably, increase after 1 h, and then decrease again to about one half of the initial concentration. Cell proliferation, measured by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, decreases after both serum starvation and zinc chelation. Two peaks of Zn²⁺ concentrations occur within one cell cycle: one early in the G1 phase and the other in the late G1/S phase. Thus, fluctuations of intracellular Zn²⁺ concentrations and established modulation of phosphorylation signaling, via an inhibition of protein tyrosine phosphatases at commensurately low Zn²⁺ concentrations, suggest a role for Zn²⁺ in the control of the cell cycle. Interventions targeted at these picomolar Zn²⁺ fluctuations may be a way of controlling cell growth in hyperplasia, neoplasia, and diseases associated with aberrant differentiation.     

Free zinc concentration in bovine milk measured by analytical affinity chromatography with immobilized metallothionein

A new analytical affinity chromatography method was developed for measuring the free [Zn²⁺] concentration in bovine milk. The column was generated by immobilizing avidin and attaching biotinylated metallothionein (MT) on controlled-pore glass beads. Zinc bound to the MT column at physiological free [Zn²⁺] concentration and was dissociated again in an elution buffer of pH 2. The distributions of extrinsically added⁶⁵Zn and native zinc in different fractions of milk were virtually identical, validating the use of extrinsic labeling in studies of the free [Zn²⁺] concentration in milk. Extrinsically labeled whey fractions were mixed with standard solutions whose free [Zn²⁺] concentrations were calculated by computer model.⁶⁵Zn retained by the column provided an indication of free [Zn²⁺] concentration in the mixture, and by interpolation, in the original milk. The free [Zn²⁺] concentration measured by the affinity chromatography method in the milk of a group of six cows was 90.4±29.7 pM. This value is similar to estimates of free [Zn²⁺] concentrations in other biological fluids by entirely different methods. Measurement of free [Zn²⁺] may be helpful in understanding the physiology and biochemistry of zinc metabolism.     

Enhanced Susceptibility to Spontaneous Seizures of Noda Epileptic Rats by Loss of Synaptic Zn2+

Zinc homeostasis in the brain is associated with the etiology and manifestation of epileptic seizures. Adult Noda epileptic rats (NER, >12-week-old) exhibit spontaneously generalized tonic-clonic convulsion about once a day. To pursue the involvement of synaptic Zn²⁺ signal in susceptibility to spontaneous seizures, in the present study, the effect of zinc chelators on epileptogenesis was examined using adult NER. Clioquinol (CQ) and TPEN are lipophilic zinc chelotors, transported into the brain and reduce the levels of synaptic Zn²⁺. The incidence of tonic-clonic convulsion was markedly increased after i.p. injection of CQ (30–100 mg/kg) and TPEN (1 mg/kg). The basal levels of extracellular Zn²⁺ measured by ZnAF-2 were decreased before tonic-clonic convulsion was induced with zinc chelators. The hippocampal electroencephalograms during CQ (30 mg/kg)-induced convulsions were similar to those during sound-induced convulsions in NER reported previously. Exocytosis of hippocampal mossy fibers, which was measured with FM4-64, was significantly increased in hippocampal slices from CQ-injected NER that did not show tonic-clonic convulsion yet. These results indicate that the abnormal excitability of mossy fibers is induced prior to epileptic seizures by injection of zinc chelators into NER. The incidence of tonic-clonic convulsion induced with CQ (30 mg/kg) was significantly reduced by co-injection with aminooxyacetic acid (5–10 mg/kg), an anticonvulsant drug enhancing GABAergic activity, which did not affect locomotor activity. The present paper demonstrates that the abnormal excitability in the brain, especially in mossy fibers, which is potentially associated with the insufficient GABAergic neuron activity, may be a factor to reduce the threshold for epileptogenesis in NER.     

Dexamethasone leads to Zn2+ accumulation and increased unbound Zn2+ in C2C12 muscle and 3T3-L1 adipose cells

Skeletal muscle atrophy is associated with increases in circulating glucocorticoid levels and insulin resistance. Zinc accumulates in atrophic muscle, but the relationship between atrophy, insulin resistance, and Zn²⁺ homeostasis remains unclear. In this study, the effect of the glucocorticoid dexamethasone (DEX) on insulin and Zn²⁺ homeostasis was explored. Treatment of differentiated C2C12 skeletal myotubes and 3T3-L1 adipocytes with DEX significantly increased mRNA expression of the metal-binding proteins Mt1 and 2 and altered energy storage as shown by the increased size of lipid droplets in 3T3-L1 cells. In C2C12 cells the total cellular Zn²⁺ was higher after DEX treatment, and in both C2C12 and 3T3-L1 adipocytes, free unbound Zn²⁺ was increased. Insulin treatment led to a gradual increase in free Zn²⁺ in C2C12 cells, and no significant change in DEX-treated cells such that concentrations were similar 10 min after insulin treatment. These data demonstrate that DEX disturbs Zn²⁺ homeostasis in muscle and fat cells. Further study of the molecular pathways involved to identify novel therapeutic targets for treatment of skeletal muscle atrophy is warranted.     

Effect of clay,organic matter and CaCO3 content on zinc adsorption by soils

Summary Zinc adsorption was studied in suspensions of six soils of different physicochemical characteristics in dilute ZnSO₄ solutions. At low concentrations, Zn²⁺ adsorption was described by the Langmuir adsorption equation. The calculated Langmuir adsorption maxima were related positively to clay and carbonate content and negatively with organic matter content of soils. Multiple regression analysis revealed that zinc adsorption maxima can be predicted with good precision from information in soil survey reports. When the added Zn²⁺ exceeded the adsorption maximum, the solid phase of zinc controlling its concentration in solution was either zinc hydroxide or carbonate so long as soil carbonates were present. The values of zinc potential also indicated that soils retain Zn²⁺ more strongly than Zn(OH)₂ or carbonate. Postgraduate student Professor of Soils. Professor of Soils.     

Elevated zinc induces endothelial apoptosis via disruption of glutathione metabolism: role of the ADP translocator

Zinc is the second-most abundant transition metal within cells and an essential micronutrient. Although adequate zinc is essential for cellular function, intracellular free zinc (Zn²⁺) is tightly controlled, as sustained increases in free Zn²⁺ levels can directly contribute to apoptotic endothelial cell death. Moreover, exposure of endothelial cells to acute nitrosative and/or oxidative stress induces a rapid rise of Zn²⁺ with mitochondrial dysfunction and the initiation of apoptosis. This apoptotic induction can be mimicked through addition of exogenous ZnCl₂ and mitigated by zinc-chelation strategies, indicating Zn²⁺-dependent mechanisms in this process. However, the molecular mechanisms of Zn²⁺-mediated mitochondrial dysfunction are unknown. Here we report that free Zn²⁺ disrupts cellular redox status through inhibition of glutathione reductase, and induces apoptosis by redox-mediated inhibition of the mitochondrial adenine nucleotide transporter (ANT). Inhibition of ANT causes increased mitochondrial oxidation, loss of ADP uptake, mitochondrial translocation of bax, and apoptosis. Interestingly, pre-incubation with glutathione ethyl ester protects endothelial cells from these observed effects. We conclude that key mechanisms of Zn²⁺-mediated apoptotic induction include disruption of cellular glutathione homeostasis leading to ANT inhibition and decreases in mitochondrial ATP synthesis. These pathways could represent novel therapeutic targets during acute oxidative or nitrosative stress in cells and tissues.     

Zinc release from thapsigargin/IP3-sensitive stores in cultured cortical neurons

Background

Changes in ionic concentration have a fundamental effect on numerous physiological processes. For example, IP₃-gated thapsigargin sensitive intracellular calcium (Ca²⁺) storage provides a source of the ion for many cellular signaling events. Less is known about the dynamics of other intracellular ions. The present study investigated the intracellular source of zinc (Zn²⁺) that has been reported to play a role in cell signaling.

Results

In primary cultured cortical cells (neurons) labeled with intracellular fluorescent Zn²⁺ indicators, we showed that intracellular regions of Zn²⁺ staining co-localized with the endoplasmic reticulum (ER). The latter was identified with ER-tracker Red, a marker for ER. The colocalization was abolished upon exposure to the Zn²⁺ chelator TPEN, indicating that the local Zn²⁺ fluorescence represented free Zn²⁺ localized to the ER in the basal condition. Blockade of the ER Ca²⁺ pump by thapsigargin produced a steady increase of intracellular Zn²⁺. Furthermore, we determined that the thapsigargin-induced Zn²⁺ increase was not dependent on extracellular Ca²⁺ or extracellular Zn²⁺, suggesting that it was of intracellular origin. The applications of caged IP₃ or IP₃-3Kinase inhibitor (to increase available IP₃) produced a significant increase in intracellular Zn²⁺.

Conclusions

Taken together, these results suggest that Zn²⁺ is sequestered into thapsigargin/IP₃-sensitive stores and is released upon agonist stimulation.     

Zinc Released from Injured Cells Is Acting via the Zn2+-sensing Receptor,ZnR, to Trigger Signaling Leading to Epithelial Repair

A role for Zn²⁺ in accelerating wound healing is established, yet, the signaling pathways linking Zn²⁺ to tissue repair are not well known. We show that in the human HaCaT keratinocytes extracellular Zn²⁺ induces a metabotropic Ca²⁺ response that is abolished by silencing the expression of the G-protein-coupled receptor GPR39, suggesting that this Zn²⁺-sensing receptor, ZnR, is mediating the response. Keratinocytic-ZnR signaling is highly selective for Zn²⁺ and can be triggered by nanomolar concentrations of this ion. Interestingly, Zn²⁺ was also released following cellular injury, as monitored by a specific non-permeable fluorescent Zn²⁺ probe, ZnAF-2. Chelation of Zn²⁺ and scavenging of ATP from conditioned medium, collected from injured epithelial cultures, was sufficient to eliminate the metabotropic Ca²⁺ signaling. The signaling triggered by Zn²⁺, via ZnR, or by ATP further activated MAP kinase and induced up-regulation of the sodium/proton exchanger NHE1 activity. Finally, activation of ZnR/GPR39 signaling or application of ATP enhanced keratinocytes scratch closure in an in vitro model. Thus our results indicate that extracellular Zn²⁺, which is either applied or released following injury, activates ZnR/GPR39 to promote signaling leading to epithelial repair.     

A Brief Overview from the Physiological and Detrimental Roles of Zinc Homeostasis via Zinc Transporters in the Heart

Zinc (mostly as free/labile Zn²⁺) is an essential structural constituent of many proteins, including enzymes in cellular signaling pathways via functioning as an important signaling molecule in mammalian cells. In cardiomyocytes at resting condition, intracellular labile Zn²⁺ concentration ([Zn²⁺]_i) is in the nanomolar range, whereas it can increase dramatically under pathological conditions, including hyperglycemia, but the mechanisms that affect its subcellular redistribution is not clear. Therefore, overall, very little is known about the precise mechanisms controlling the intracellular distribution of labile Zn²⁺, particularly via Zn²⁺ transporters during cardiac function under both physiological and pathophysiological conditions. Literature data demonstrated that [Zn²⁺]_i homeostasis in mammalian cells is primarily coordinated by Zn²⁺ transporters classified as ZnTs (SLC30A) and ZIPs (SLC39A). To identify the molecular mechanisms of diverse functions of labile Zn²⁺ in the heart, the recent studies focused on the discovery of subcellular localization of these Zn²⁺ transporters in parallel to the discovery of novel physiological functions of [Zn²⁺]_i in cardiomyocytes. The present review summarizes the current understanding of the role of [Zn²⁺]_i changes in cardiomyocytes under pathological conditions, and under high [Zn²⁺]_i and how Zn²⁺ transporters are important for its subcellular redistribution. The emerging importance and the promise of some Zn²⁺ transporters for targeted cardiac therapy against pathological stimuli are also provided. Taken together, the review clearly outlines cellular control of cytosolic Zn²⁺ signaling by Zn²⁺ transporters, the role of Zn²⁺ transporters in heart function under hyperglycemia, the role of Zn²⁺ under increased oxidative stress and ER stress, and their roles in cancer are discussed.

     

Zn induces hyperpolarization by activation of a K channel and increases intracellular Ca and pH in sea urchin spermatozoa

Zinc (Zn²⁺) has been recently recognized as a crucial element for male gamete function in many species although its detailed mechanism of action is poorly understood. In sea urchin spermatozoa, Zn²⁺ was reported as an essential trace ion for efficient sperm motility initiation and the acrosome reaction by modulating intracellular pH (pH_i). In this study we found that submicromolar concentrations of free Zn²⁺ change membrane potential (Em) and increase the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) and cAMP in Lytechinus pictus sperm. Our results indicate that the Zn²⁺ response in sperm of this species mainly involves an Em hyperpolarization caused by K⁺ channel activation. The pharmacological profile of the Zn²⁺-induced hyperpolarization indicates that the cGMP-gated K⁺ selective channel (tetraKCNG/CNGK), which is crucial for speract signaling, is likely a main target for Zn²⁺. Considering that Zn²⁺ also induces [Ca²⁺]_i fluctuations, our observations suggest that Zn²⁺ activates the signaling cascade of speract, except for an increase in cGMP, and facilitates sperm motility initiation upon spawning. These findings provide new insights about the role of Zn²⁺ in male gamete function.     

Attenuation of abnormal glutamate release in zinc deficiency by zinc and Yokukansan

The mechanism of the abnormal increase in extracellular glutamate concentration in the hippocampus induced with 100 mM KCl in zinc deficiency is unknown. In the present study, the changes in glutamate release (exocytosis) and GLT-1, a glial glutamate transporter, expression were studied in young rats fed a zinc-deficient diet for 4 weeks. Exocytosis at mossy fiber boutons was enhanced as reported previously and GLT-1 protein was increased in the hippocampus. The enhanced exocytosis is thought to increase extracellular glutamate concentration. However, the basal concentration of extracellular glutamate in the hippocampus was not increased by zinc deficiency, suggesting that GLT-1 protein increased serves to maintain the basal concentration of extracellular glutamate. The enhanced exocytosis was attenuated in the presence of 100 μM ZnCl₂, which attenuated the abnormal increase in extracellular glutamate induced with high K⁺ in zinc deficiency. The present study indicates that zinc attenuates abnormal glutamate release in zinc deficiency. The enhanced exocytosis was also attenuated in slices from zinc-deficient rats administered Yokukansan, a herbal medicine, in which the abnormal increase in extracellular glutamate induced with high K⁺ was attenuated. It is likely that Yokukansan is useful for prevention or cure of abnormal glutamate release. The enhanced exocytosis in zinc deficiency is a possible mechanism on abnormal increase in extracellular glutamate in the hippocampus induced with high K⁺.     

Attenuation of abnormal glutamate release in zinc deficiency by zinc and Yokukansan

The mechanism of the abnormal increase in extracellular glutamate concentration in the hippocampus induced with 100 mM KCl in zinc deficiency is unknown. In the present study, the changes in glutamate release (exocytosis) and GLT-1, a glial glutamate transporter, expression were studied in young rats fed a zinc-deficient diet for 4 weeks. Exocytosis at mossy fiber boutons was enhanced as reported previously and GLT-1 protein was increased in the hippocampus. The enhanced exocytosis is thought to increase extracellular glutamate concentration. However, the basal concentration of extracellular glutamate in the hippocampus was not increased by zinc deficiency, suggesting that GLT-1 protein increased serves to maintain the basal concentration of extracellular glutamate. The enhanced exocytosis was attenuated in the presence of 100 μM ZnCl₂, which attenuated the abnormal increase in extracellular glutamate induced with high K⁺ in zinc deficiency. The present study indicates that zinc attenuates abnormal glutamate release in zinc deficiency. The enhanced exocytosis was also attenuated in slices from zinc-deficient rats administered Yokukansan, a herbal medicine, in which the abnormal increase in extracellular glutamate induced with high K⁺ was attenuated. It is likely that Yokukansan is useful for prevention or cure of abnormal glutamate release. The enhanced exocytosis in zinc deficiency is a possible mechanism on abnormal increase in extracellular glutamate in the hippocampus induced with high K⁺.     

Fluctuation in recoverable nickel and zinc resistant copiotrophic bacteria explained by the varying zinc ion content of Torsa River in different months

Heavy metal content analysis of River Torsa in India did not indicate any alarming level of toxicity for human consumption but revealed variation at the ppb level in different months. The variation in recoverable nickel and zinc resistant copiotrophic (or eutrophic) bacterial counts was explained by the variation of the zinc content (34.0–691.3 ppb) of the river water in different sampling months. Growth studies conducted with some purified nickel and/or zinc resistant strains revealed that pre-exposure of the cells to ppb levels of Zn²⁺, comparable to the indigenous zinc ion concentration of the river, could induce the nickel or zinc resistance. A minimum concentration of 5–10 μM Zn²⁺ (325–650 ppb) was found effective in inducing the Nickel resistance of the isolates. Zinc resistance of the isolates was tested by pre-exposing the cells to 4 μM Zn²⁺ (260 ppb). The lag phase was reduced by 6–8 h in all the cases. Biochemical characteristics and phylogenetic analysis based on 16S rDNA sequence indicated that some of the Torsa River isolates, having inducible nickel and zinc resistance, are members of the genus Pseudomonas, Acinetobacter, Bacillus, Enterobacter, Serratia and Moraxella.     

Zinc Binding to MG53 Protein Facilitates Repair of Injury to Cell Membranes

Zinc is an essential trace element that participates in a wide range of biological functions, including wound healing. Although Zn²⁺ deficiency has been linked to compromised wound healing and tissue repair in human diseases, the molecular mechanisms underlying Zn²⁺-mediated tissue repair remain unknown. Our previous studies established that MG53, a TRIM (tripartite motif) family protein, is an essential component of the cell membrane repair machinery. Domain homology analysis revealed that MG53 contains two Zn²⁺-binding motifs. Here, we show that Zn²⁺ binding to MG53 is indispensable to assembly of the cell membrane repair machinery. Live cell imaging illustrated that Zn²⁺ entry from extracellular space is essential for translocation of MG53-containing vesicles to the acute membrane injury sites for formation of a repair patch. The effect of Zn²⁺ on membrane repair is abolished in mg53^−/− muscle fibers, suggesting that MG53 functions as a potential target for Zn²⁺ during membrane repair. Mutagenesis studies suggested that both RING and B-box motifs of MG53 constitute Zn²⁺-binding domains that contribute to MG53-mediated membrane repair. Overall, this study establishes a base for Zn²⁺ interaction with MG53 in protection against injury to the cell membrane.