Extracellular Zn2 Research Articles

The presence and response to Zn of ZnT family mRNAs in human dental pulp.

Zinc (Zn) is distributed throughout the body and within cells by saturable processes mediated by the transport proteins of the ZnT (SLC30) and ZIP (SLC39) families. The two families function in opposite directions. ZnT transporters mediate cellular zinc efflux or intracellular sequestration. Zn is found in human tooth enamel and dentine at levels that have been related to environmental exposures, such as pollution, disease, and dietary intake. The mechanism by which Zn in the odontoblast is deposited in the hard tissue of the tooth, however, is unknown but is important in determining the physical properties, and hence resilience, of enamel and in the context of the use of tooth zinc level as a biomarker of exposure. We hypothesised that zinc efflux mediated by members of the ZnT family of 10 transporters is a key step in this process and is regulated by zinc availability through effects on mRNA levels. Thus, we determined the profile of ZnT transporter mRNA in a human active-secretory odontoblast-like cell model under conditions of high- and low-extracellular Zn concentration and determined if the same transporter mRNAs were present in human dental pulp. ZnT1, ZnT5 and ZnT9 mRNAs were detected by RT-PCR in both the secretory odontoblast cells and human dental pulp. ZnT2, ZnT3 and ZnT10 mRNAs were not detected, and ZnT4 mRNA was detected in secretory odontoblasts only, which may be indicative of a specialised zinc efflux function during the active secretory phase of tooth development. ZnT1 mRNA was significantly increased in response to extracellular Zn exposure (60 μM) after 24 h. The presence of Zn transporter mRNAs in secretory odontoblasts and dental pulp indicates that the corresponding transport proteins function to deposit zinc in the dental hard tissues. The responsiveness of ZnT1 in odontoblasts to zinc availability is concordant with this being a process that is regulated to maintain cellular Zn homeostasis and that is a mediator of the relationship between environmental Zn exposure and dental Zn deposition. These findings have likely relevance to human dental health through effects of Zn transporter expression level on the hard tissue properties.

Brain zinc dyshomeostasis and glial cells in ischemic stroke

Zinc, an essential trace element, plays an important role in a large number of biological functions. In mammalian brain, whereas the majority of brain zinc is bound to proteins including metallothionein, about 5-15% is stored in presynaptic vesicles of glutamatergic neurons throughout the forebrain, especially in the hippocampus, in a relatively free state. Thus, free zinc (Zn2+) concentration in the brain is considered to be regulated in order to maintain normal brain functions such as learning and memory. On the other hand, brain Zn2+ dyshomeostasis has been recognized as a mechanism for neuronal injury in brain disorders including Alzheimer's disease and brain ischemia. In particular, after transient brain ischemia, Zn2+ accumulates in hippocampal neurons via a zinc transport system, or via release from cytosolic zinc-binding proteins, which results in neuronal cell death. Recently, it has been demonstrated that Zn2+ dyshomeostasis also occurs in glial cells such as microglia, astrocytes and oligodendrocytes after brain ischemia. In oligodendrocytes, ischemic insult triggers intracellular Zn2+ accumulation, resulting in cell death via mitochondrial dysfunction. Increased extracellular Zn2+ inhibits astrocytic glutamate uptake. In addition, extracellular Zn2+ massively released from ischemic neurons primes microglia to enhance production of pro-inflammatory cytokines in response to stimuli that trigger M1 activation. This review aims to describe the impact of brain Zn2+ dyshomeostasis on alterations in glial cell survival and functions in post-ischemic brains.

Amyloid β1-42-Induced Rapid Zn2+ Influx into Dentate Granule Cells Attenuates Maintained LTP Followed by Retrograde Amnesia.

On the basis of the evidence that amyloid β1-42 (Aβ1-42)-induced Zn2+ influx affects memory acquisition via attenuated long-term potentiation (LTP) induction, here we tested whether Aβ1-42-induced Zn2+ influx affects maintained LTP in freely moving rats, resulting in retrograde amnesia. Both maintained LTP and space memory were impaired by local injection of 250μM ZnCl2 (2μl) into the dentate gyrus, while maintained LTP was impaired by injection of either Aβ1-40 or Aβ1-42 (25μM, 2μl) into the dentate gyrus. Aβ1-40-induced impairment of maintained LTP was rescued by co-injection of CaEDTA, an extracellular Zn2+ chelator, but not by co-injection of ZnAF-2DA, an intracellular Zn2+ chelator, suggesting that maintained LTP is impaired by Aβ1-40 via a mechanism that may involve extracellular Zn2+. In contrast, Aβ1-42-induced impairments of maintained LTP and space memory were rescued by co-injection of either CaEDTA or ZnAF-2DA. Intracellular Zn2+ in dentate granule cells was rapidly increased by Aβ1-42 injection into the dentate gyrus, but not by Aβ1-40 injection. The block of Aβ1-42-induced increase in intracellular Zn2+ by pretreatment with dexamethasone, a metallothionein inducer also rescued Aβ1-42-induced impairment of maintained LTP. The present study indicates that Aβ1-42-induced Zn2+ influx into dentate granule cells, which more readily occurs than free Zn2+-induced Zn2+ influx, attenuates maintained LTP followed by retrograde amnesia. It is likely that controlling Aβ1-42-induced intracellular Zn2+ dysregulation is a strategy for defending AD pathogenesis.

Blockade of Rapid Influx of Extracellular Zn2+ into Nigral Dopaminergic Neurons Overcomes Paraquat-Induced Parkinson's Disease in Rats.

The herbicide paraquat (PQ) has been reported to enhance the risk of developing Parkinson's disease (PD) from epidemiological studies. PQ-induced reactive oxygen species (ROS) are linked with a selective loss of nigrostriatal dopaminergic neurons. Here, we first report a unique mechanism of nigrostriatal dopaminergic degeneration, in which rapid intracellular Zn2+ dysregulation via PQ-induced ROS production causes PD in rats. When the substantia nigra pars compacta (SNpc) of rats was perfused with PQ, extracellular concentrations of glutamate and Zn2+ were increased and decreased, respectively, in the SNpc. These changes were ameliorated by co-perfusion with Trolox, an antioxidative agent. In in vitro slice experiments, PQ rapidly increased extracellular Zn2+ influx via AMPA receptor activation. Both loss of nigrostriatal dopaminergic neurons and increase in turning behavior in response to apomorphine were markedly reduced by coinjection of PQ and intracellular Zn2+ chelator, i.e., ZnAF-2DA into the SNpc. Furthermore, loss of nigrostriatal dopaminergic neurons induced with a low dose of PQ, which did not induce any behavioral abnormality, was completely blocked by coinjection of ZnAF-2DA. The present study indicates that rapid influx of extracellular Zn2+ into dopaminergic neurons via AMPA receptor activation, which is initially induced by PQ-mediated ROS production in the SNpc, induces nigrostriatal dopaminergic degeneration, resulting in PQ-induced PD in rats. Intracellular Zn2+ dysregulation in dopaminergic neurons is the cause of PQ-induced pathogenesis in the SNpc, and the block of intracellular Zn2+ toxicity leads to defending PQ-induced pathogenesis.

Cellular toxicity of zinc can be attenuated by sodium hydrogen sulfide in neuronal SH-SY5Y cell

Although zinc acts as a major regulator of neuronal physiology, its dyshomeostasis may cause neuronal cell death. Hydrogen sulfide (H2S) has been reported to attenuate ischemic brain injury and to suppress Zn2+-induced neuronal cell death, but the underlying mechanisms have not been elucidated. We determined the direct Zn2+-chelating capacity of sodium hydrogen sulfide (NaHS), an H2S donor, using specific Zn2+ fluorescent dyes (Zinpyr-1 and Zinquin) in SH-SY5Y cells. NaHS significantly suppressed the Zn2+-mediated increase in the fluorescence intensities of Zinpyr- 1 and Zinquin in a dose-dependent manner. NaHS significantly inhibited cell death induced by extracellular or intracellular Zn2+ overload. Furthermore, Zn2+-mediated increases in the phosphorylation of glycogen synthase kinase-3β and protein kinase C were highly suppressed by NaHS treatment. These results demonstrate that NaHS has the capacity to chelate extracellular and intracellular Zn2+, and could therefore be used in the protection against Zn2+ neurotoxicity.

Biphenyl-induced cytotoxicity is mediated by an increase in intracellular Zn2+

Biphenyl is found both in natural and anthropogenic sources and is used as a fungistat in the packaging of citrus fruits. Acute exposure to high levels of biphenyl has been observed to cause skin irritation and toxic effects on the liver and kidneys. However, the mechanisms of cytotoxicity induced by biphenyl are not yet well understood. In the present study, the cytotoxicity of biphenyl was studied by flow cytometry with fluorescent probes. Biphenyl at 100 μM significantly increased cell lethality after 3 h in rat thymocytes. In addition, biphenyl at 100 μM or more elevated intracellular Zn2+ levels. N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), an intracellular and extracellular Zn2+ chelator, but not diethylenetriamine-N,N,N′,N″,N″-pentaacetic acid (DTPA), a membrane-impermeable Zn2+ chelator, attenuated the biphenyl-induced increase in intracellular Zn2+ levels and cell death. These results suggested that biphenyl-induced cytotoxicity caused an increase in intracellular Zn2+ levels, which was dependent on internal Zn2+. Moreover, biphenyl led to an increase in sensitivity to oxidative stress, while TPEN inhibited this biphenyl-induced increase. Our findings revealed that biphenyl caused an increase in the intracellular free Zn2+ concentration, inducing cytotoxicity, cell death, and an increase in sensitivity to oxidative stress.

AMPA-induced extracellular Zn2+ influx into nigral dopaminergic neurons causes movement disorder in rats

On the basis of the findings that the rapid influx of extracellular Zn2+ into nigral dopaminergic neurons causes dopaminergic neurodegeneration, here we report that AMPA causes movement disorder in rats. AMPA markedly increased turning behavior in response to apomorphine 1 and 2 weeks after AMPA injection into the substantia nigra pars compacta (SNpc), while AMPA-induced movement disorder was suppressed by co-injection of intracellular Zn2+ chelators, i.e., ZnAF-2DA and TPEN, suggesting that AMPA-induced movement disorder is due to intracellular Zn2+ dysregulation. Furthermore, AMPA markedly induced loss of nigrostriatal dopaminergic neurons 2 weeks after AMPA injection into the SNpc, while AMPA-induced neurodegeneration was also suppressed in the SNpc and the striatum by co-injection of ZnAF-2DA and TPEN. AMPA rapidly increased nigral intracellular Zn2+ after AMPA injection into the SNpc and this increase was blocked by co-injection of TPEN. These results indicate that AMPA receptor activation rapidly increases influx of extracellular Zn2+ into nigral dopaminergic neurons and causes nigrostriatal dopaminergic neurodegeneration, resulting in movement disorder in rats. The evidence that AMPA-induced intracellular Zn2+ dysregulation causes movement disorder via nigrostriatal dopaminergic neurodegeneration suggests that AMPA receptors, probably Ca2+- and Zn2+-permeable GluR2-lacking AMPA receptors are potential targets for overcoming Parkinson's syndrome.

6-ヒドロキシドパミン誘発ラットパーキンソン病はドパミン作動性神経への細胞外Zn2+流入に起因する

6-ヒドロキシドパミン誘発ラットパーキンソン病はドパミン作動性神経への細胞外Zn2+流入に起因する

Abstract B64: The novel ZIP4 regulation and its role in cancer stem cell-related activities in ovarian cancer

Abstract The role and regulation of ZIP4, a zinc (Zn) transporter, have been studied in the context of extracellular Zn transporting. Factors other than Zn with ZIP4 regulatory effects are essentially unknown. In addition, its role in cancer stem cell (CSC) has not been reported in any type of cancer. RNA-seq analyses were conducted to compare genes differentially expressed in less and more aggressive epithelial ovarian cancer (EOC) cells. ZIP4 expression and its regulation was assessed by immunoblotting, quantitative PCR, or immunofluorescence staining in human ovarian tissues. CSC-related activities were examined to evaluate the role of ZIP4 in human high-grade serous ovarian cancer (HGSOC) cells. RNAi and CRISPR techniques were used to knock down or knock out ZIP4 and related genes. EOC tumors overexpressed ZIP4 when compared with normal and benign tissues. ZIP4 knockout significantly reduced several CSC-related activities in EOC cells, including proliferation, anoikis resistance, colony formation, spheroid formation, drug resistance, and side-population (SP). Only ZIP4-expressing SP highly expressed known CSC markers ALDH1 and OCT4. ZIP4 knockout dramatically reduced tumorigenesis and ZIP4 overexpression increased tumorigenesis in vivo. In addition, the ZIP4-expressing SP population had the tumor initiating activity. Moreover, the oncolipid lysophosphatic acid (LPA) effectively upregulated ZIP4 expression via the nuclear receptor peroxisome proliferator-activated receptor gamma, and LPA’s promoting effects in CSC-related activities in HGSOC cells was at least partially mediated by ZIP4 in an extracellular Zn-independent manner. Our critical data imply that ZIP4 is a new and important cancer stem cell regulator in EOC. Our data also provide an innovative interpretation for the apparent disconnection between low levels of zinc and upregulation of ZIP4 in EOC tissues. Citation Format: Yan Xu, Qipeng Fan, Qingchun Cai, Kenneth P. Nephew. The novel ZIP4 regulation and its role in cancer stem cell-related activities in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B64.

Modulation of P2X7 purinergic receptor activity by extracellular Zn2+ in cultured mouse hippocampal astroglia

The P2X7R protein, a P2 type purinergic receptor functioning as a non-selective cation channel, is expressed in different cell types of the central nervous system in several regions of the brain. The activation of the P2X7R protein by ATP modulates excitatory neurotransmission and contributes to microglial activation, apoptosis and neuron-glia communication. Zinc is an essential micronutrient that is highly concentrated in the synaptic vesicles of glutamatergic hippocampal neurons where free zinc ions released into the synaptic cleft alter glutamatergic signal transmission. Changes in both P2X7R-mediated signaling and brain zinc homeostasis have been implicated in the pathogenesis of mood disorders. Here, we tested the hypothesis that extracellular zinc regulates P2X7R activity in the hippocampus. We observed that P2X7R is expressed in both neurons and glial cells in primary mouse hippocampal neuron-glia culture. Propidium iodide (PI) uptake through large pores formed by pannexins and P2X7R was dose-dependently inhibited by extracellular zinc ions. Calcium influx mediated by P2X7R in glial cells was also reduced by free zinc ions. Interestingly, no calcium influx was detected in response to ATP or 3′-O-(4-Benzoyl) benzoyl ATP (BzATP) in neurons despite the expression of P2X7R at the plasma membrane. Our results show that free zinc ions can modulate hippocampal glial purinergic signaling, and changes in the activity of P2X7R may contribute to the development of depression-like behaviors associated with zinc deficiency.

Is Vulnerability of the Dentate Gyrus to Aging and Amyloid-β1-42 Neurotoxicity Linked with Modified Extracellular Zn2+ Dynamics?

The basal levels of extracellular Zn2+ are in the range of low nanomolar concentrations in the hippocampus and perhaps increase age-dependently. Extracellular Zn2+ dynamics is critical for cognitive activity and excess influx of extracellular Zn2+ into hippocampal neurons is a known cause of cognitive decline. The dentate gyrus is vulnerable to aging in the hippocampus and affected in the early stage of Alzheimer's disease (AD). The reasons remain unclear. Neurogenesis-related apoptosis may induce non-specific neuronal depolarization by efflux of intracellular K+ in the dentate gyrus and be markedly increased along with aging. Extracellular Zn2+ influx into dentate granule cells via high K+-induced perforant pathway excitation leads to cognitive decline. Modified extracellular Zn2+ dynamics in the dentate gyrus of aged rats is linked with vulnerability to cognitive decline. Amyloid-β1-42 (Aβ1-42) is a causative candidate for AD pathogenesis. When Aβ1-42 concentration reaches picomolar in the extracellular compartment in the dentate gyrus, Zn-Aβ1-42 is formed in the extracellular compartment and rapidly taken up into dentate granule cells, followed by Aβ1-42-induced cognitive decline that is due to Zn2+ released from Aβ1-42, suggesting that dentate granule cells are sensitive to extracellular Zn2+-dependent Aβ1-42 toxicity. This paper deals with proposed vulnerability of the dentate gyrus to aging and Aβ1-42 neurotoxicity.

Rapid Intracellular Zn2+ Dysregulation via Membrane Corticosteroid Receptor Activation Affects In Vivo CA1 LTP

Involvement of membrane mineralocorticoid (MC) and glucocorticoid (GC) receptors in synaptic Zn2+ dynamics remains unclear. Here, we tested whether synaptic plasticity is affected by rapid intracellular Zn2+ dysregulation via membrane MC and GC receptor activation, in comparison with intracellular Ca2+ dysregulation. In anesthetized rats, extracellular Zn2+ level was increased under local perfusion of the hippocampal CA1 with 500ng/ml corticosterone. In vivo CA1 long-term potentiation (LTP) at Schaffer collateral-CA1 pyramidal cell synapses was attenuated by the pre-perfusion with corticosterone prior to tetanic stimulation, and the attenuation was canceled by co-perfusion with CaEDTA, an extracellular Zn2+ chelator, suggesting that corticosterone-induced increase in extracellular Zn2+ is involved in the subsequent attenuation of LTP. In rat brain slices, corticosterone-induced increases in extracellular and intracellular Zn2+ were blocked in the presence of spironolactone, a MC receptor antagonist that canceled corticosterone-induced attenuation of LTP. Mifepristone, a GC receptor antagonist, which canceled corticosterone-induced attenuation of LTP, also blocked corticosterone-induced increase in intracellular Zn2+, but not extracellular Zn2+. Moreover, corticosterone-induced decrease in phosphorylated CaMKII was restored in the presence of CaEDTA or spironolactone. These results indicate that glucocorticoid rapidly induces the increase in intracellular Zn2+, which occurs via membrane MC and GC receptor activations, and decreases phosphorylated CaMKII level, resulting in attenuating LTP. Membrane MC and GC receptors induce intracellular Zn2+ dysregulation via differential mechanisms. In contrast, glucocorticoid-induced intracellular Ca2+ dysregulation is not crucial for affecting LTP.

Signaling pathways involved in human sperm hyperactivated motility stimulated by Zn2+

To fertilize the egg, sperm cells must reside in the female reproductive tract for several hours during which they undergo chemical and motility changes collectively called capacitation. During capacitation, the sperm develop a unique type of motility known as hyperactivated motility (HAM). The semen contains Zn2+ in millimolar concentrations, whereas in the female reproductive tract, the concentration is around 1 µM. In this study, we characterize the role of Zn2+ in human sperm capacitation focusing on its effect on HAM. Western blot analysis revealed the presence of GPR39-type Zn-receptor localized mainly in the sperm tail. Zn2+ at micromolar concentration stimulates HAM, which is mediated by a cascade involving GPR39-adenylyl cyclase (AC)-cyclic AMP (cAMP)-protein kinase A-tyrosine kinase Src (Src)-epidermal growth factor receptor and phospholipase C. Both the transmembrane AC and the soluble-AC are involved in the stimulation of HAM by Zn2+ . The development of HAM is precisely regulated by cAMP, in which relatively low concentration (5-10 µM) stimulated HAM, whereas at 30 µM no stimulation occurred. A similar response was seen when different concentrations of Zn2+ were added to the cells; low Zn2+ stimulated HAM, whereas at relatively high Zn2+ , no effect was seen. We further demonstrate that the Ca2+ -channel CatSper involved in Zn2+ -stimulated HAM. These data support a role for extracellular Zn2+ acting via GPR39 to regulate signaling pathways in sperm capacitation, leading to HAM induction.

The Extracellular Zn2+ Concentration Surrounding Excited Neurons Is High Enough to Bind Amyloid-β Revealed by a Nanowire Transistor.

The Zn2+ stored in the secretory vesicles of glutamatergic neurons is coreleased with glutamate upon stimulation, resulting in the elevation of extracellular Zn2+ concentration (CZn2+ex). This elevation of CZn2+ex regulates the neurotransmission and facilitates the fibrilization of amyloid-β (Aβ). However, the exact CZn2+ex surrounding neurons under (patho)physiological conditions is not clear and the connection between CZn2+ex and the Aβ fibrilization remains obscure. Here, a silicon nanowire field-effect transistor (SiNW-FET) with the Zn2+ -sensitive fluorophore, FluoZin-3 (FZ-3), to quantify the CZn2+ex in real time is modified. This FZ-3/SiNW-FET device has a dissociation constant of ≈12 × 10-9 m against Zn2+ . By placing a coverslip seeded with cultured embryonic cortical neurons atop an FZ-3/SiNW-FET, the CZn2+ex elevated to ≈110 × 10-9 m upon stimulation with α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Blockers against the AMPA receptor or exocytosis greatly suppress this elevation, indicating that the Zn2+ stored in the synaptic vesicles is the major source responsible for this elevation of CZn2+ex. In addition, a SiNW-FET modified with Aβ could bind Zn2+ with a dissociation constant of ≈633 × 10-9 m and respond to the Zn2+ released from AMPA-stimulated neurons. Therefore, the CZn2+ex can reach a level high enough to bind Aβ and the Zn2+ homeostasis can be a therapeutic strategy to prevent neurodegeneration.

Zinc supplementation decreases galactosylation of recombinant IgG in CHO cells.

Trace element composition of culture medium can be altered to modulate glycoform of recombinant glycoproteins. In this study, we show that Zn2+ supplementation at or above 100μM decreases galactosylation of recombinant IgG expressed in Chinese Hamster Ovary cells. This decrease in galactosylation is not due to reduced galactosyltransferase expression. This effect persists upon supplementation of galactose and uridine to the culture, indicating that it may not be due to reduced UDP-Gal availability. Measurements of galactosyltransferase activity in the cell lysate show that activity decreases with increasing Zn2+/Mn2+ ratio. This suggests that one possible explanation of the effect of Zn2+ may be reduced intracellular galactosyltransferase activity due to increase in Zn2+/Mn2+ ratio. Consistent with this, the decrease in galactosylation of IgG could be reversed by supplementation of Mn2+ (a cofactor of galactosyltransferase) which increases intracellular Mn2+ content. Measurement of total intracellular Zn2+ content, however, indicates no significant upregulation of total intracellular Zn2+ content and no significant downregulation of intracellular Mn2+ content with Zn2+ supplementation. One possible explanation could be that cellular detoxification response to higher extracellular Zn2+ concentration might lead to changes in intracellular distribution of Mn2+. In this case, Zn2+ supplementation would be expected to interfere with other known effects of Mn2+. Indeed, the previously reported increase in high mannose glycans upon Mn2+ supplementation in the absence of glucose is reversed by Zn2+ supplementation. This study also suggests the use of Mn2+ supplementation as a strategy to overcome the effect of lot-to-lot variability in trace element concentrations on galactosylation.

Coupling between an electrostatic network and the Zn2+ binding site modulates Hv1 activation.

The voltage sensor (VS) domain in Hv1 proton channels mediates a voltage-dependent and H+-selective "aqueous" conductance (GAQ) that is potently modulated by extracellular Zn2+ Although two conserved His residues are required for Zn2+ effects on GAQ gating, the atomic structure of the Zn2+ coordination site and mechanism by which extracellular Zn2+ stabilizes a closed-state conformation remain unknown. Here we use His mutagenesis to identify residues that increase Zn2+ potency and are therefore likely to participate in first solvation shell interactions with Zn2+ Experimental Zn2+-mapping data were then used to constrain the structure of a new resting-state Hv1 model (Hv1 F). Molecular dynamics (MD) simulations show how protein and water atoms directly contribute to octahedral Zn2+ coordination spheres in Zn2+-bound and -unbound Hv1 F models. During MD simulations, we observed correlated movements of Zn2+-interacting side chains and residues in a highly conserved intracellular Coulombic network (ICN) that contains highly conserved Arg "gating charges" in S4 as well as acidic "counter-charges" in S2 and S3 and is known to control VS activation, suggesting that occupancy of the extracellular Zn2+ site is conformationally coupled to reorganization of the ICN. To test this hypothesis, we neutralized an ICN Glu residue (E153) and show that in addition to shifting GAQ activation to more negative voltages, E153A also decreases Zn2+ potency. We speculate that extracellular gating-modifier toxins and other ligands may use a generally similar long-range conformational coupling mechanism to modulate VS activation in related ion channel proteins.

Extracellular Zn2+ Influx into Nigral Dopaminergic Neurons Plays a Key Role for Pathogenesis of 6-Hydroxydopamine-Induced Parkinson's Disease in Rats.

Parkinson's disease (PD) is a progressive neurological disease characterized by a selective loss of nigrostriatal dopaminergic neurons. The exact cause of the neuronal loss remains unclear. Here, we report a unique mechanism of nigrostriatal dopaminergic neurodegeneration, in which extracellular Zn2+ influx plays a key role for PD pathogenesis induced with 6-hydroxydopamine (6-OHDA) in rats. 6-OHDA rapidly increased intracellular Zn2+ only in the substantia nigra pars compacta (SNpc) of brain slices and this increase was blocked in the presence of CaEDTA, an extracellular Zn2+ chelator, and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist, indicating that 6-OHDA rapidly increases extracellular Zn2+ influx via AMPA receptor activation in the SNpc. Extracellular Zn2+ concentration was decreased under in vivo SNpc perfusion with 6-OHDA and this decrease was blocked by co-perfusion with CNQX, supporting 6-OHDA-induced Zn2+ influx via AMPA receptor activation in the SNpc. Interestingly, both 6-OHDA-induced loss of nigrostriatal dopaminergic neurons and turning behavior to apomorphine were ameliorated by co-injection of intracellular Zn2+ chelators, i.e., ZnAF-2DA and N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). Co-injection of TPEN into the SNpc blocked 6-OHDA-induced increase in intracellular Zn2+ but not in intracellular Ca2+. These results suggest that the rapid influx of extracellular Zn2+ into dopaminergic neurons via AMPA receptor activation in the SNpc induces nigrostriatal dopaminergic neurodegeneration, resulting in 6-OHDA-induced PD in rats.

Zn2+ Modulates Hv1 Proton Channel Gating via Conformational Coupling to an Intracellular Coulombic Network

Gating of the intrinsic ‘aqueous’ H+ conductance (GAQ) in the Hv1 proton channel is sensitive to extracellular Zn2+ and other divalent cations in the micromolar range. Previous studies showed that the extracellularly-accessible residues H140 and H193 are required for Zn2+ to positively shift the GAQ-V relation. However, the molecular mechanism by which extracellular Zn2+ occupancy is coupled to voltage-dependent GAQ gating remains known. We used site-directed mutagenesis and electrophysiology to identify Zn2+-interacting residues and used the experimental data to build new models of the Hv1 voltage sensor (VS) domain in Zn2+-liganded (Hv1F·Zn2+) and unliganded (Hv1F + Zn2+) conformations. Although imidazole nitrogen atoms (H140-Nδ1 and H193-Nδ1) are close enough to simultaneously coordinate a single Zn2+ ion, we did not observe a stable Zn2+ coordination sphere during all-atom molecular dynamics (MD) simulations using the CHARMM36 force field. To simulate electronic interactions between Zn2+ and H140-Nδ1 and H193-Nδ1, we imposed harmonic constraints (5 kcal/mol/Å) and equilibrated the Hv1F·Zn2+ model during a 50 ns MD simulation. 20 ns after releasing constraints, ZN13 spontaneously dissociates from H140-Nδ1 and H193-Nδ1 but remains associated with the carboxyl group of E119. The VS also undergoes backbone reorganization involving the S1, S3 and S4 helices after ZN13 dissociation that suggest Zn2+-dependent conformational changes. Concomitant changes in the organization of an electrostatic network involving intracellular acidic residues (E153 and D174) and S4 ‘gating charge’ Arg side chains may reveal how voltage-dependent gating is altered by Zn2+. In support of this hypothesis, we find that E153 neutralization also alters Zn2+ potency. In summary, extracellular Zn2+ occupancy is coupled to reorganization of an intracellular Coulombic network that is required for voltage-dependent gating in Hv1.

The Zinc Sensing Receptor, ZnR/GPR39, in Health and Disease.

A distinct G-protein coupled receptor that senses changes in extracellular Zn2+, ZnR/GPR39, was found in cells from tissues in which Zn2+ plays a physiological role. Most prominently, ZnR/GPR39 activity was described in prostate cancer, skin keratinocytes, and colon epithelial cells, where zinc is essential for cell growth, wound closure, and barrier formation. ZnR/GPR39 activity was also described in neurons that are postsynaptic to vesicular Zn2+ release. Activation of ZnR/GPR39 triggers Gαq-dependent signaling and subsequent cellular pathways associated with cell growth and survival. Furthermore, ZnR/GPR39 was shown to regulate the activity of ion transport mechanisms that are essential for the physiological function of epithelial and neuronal cells. Thus, ZnR/GPR39 provides a unique target for therapeutically modifying the actions of zinc in a specific and selective manner.

Zn2+-induced disruption of neuronal mitochondrial function: Synergism with Ca2+, critical dependence upon cytosolic Zn2+ buffering, and contributions to neuronal injury

Excitotoxic Zn2+ and Ca2+ accumulation contributes to neuronal injury after ischemia or prolonged seizures. Synaptically released Zn2+ can enter postsynaptic neurons via routes including voltage sensitive Ca2+ channels (VSCC), and, more rapidly, through Ca2+ permeable AMPA channels. There are also intracellular Zn2+ binding proteins which can either buffer neuronal Zn2+ influx or release bound Zn2+ into the cytosol during pathologic conditions. Studies in culture highlight mitochondria as possible targets of Zn2+; cytosolic Zn2+ can enter mitochondria and induce effects including loss of mitochondrial membrane potential (ΔΨm), mitochondrial swelling, and reactive oxygen species (ROS) generation. While brief (5 min) neuronal depolarization (to activate VSCC) in the presence of 300 μM Zn2+ causes substantial delayed neurodegeneration, it only mildly impacts acute mitochondrial function, raising questions as to contributions of Zn2+-induced mitochondrial dysfunction to neuronal injury.Using brief high (90 mM) K+/Zn2+ exposures to mimic neuronal depolarization and extracellular Zn2+ accumulation as may accompany ischemia in vivo, we examined effects of disrupted cytosolic Zn2+ buffering and/or the presence of Ca2+, and made several observations: 1. Mild disruption of cytosolic Zn2+ buffering—while having little effects alone—markedly enhanced mitochondrial Zn2+ accumulation and dysfunction (including loss of ∆Ψm, ROS generation, swelling and respiratory inhibition) caused by relatively low (10–50 μM) Zn2+ with high K+. 2. The presence of Ca2+ during the Zn2+ exposure decreased cytosolic and mitochondrial Zn2+ accumulation, but markedly exacerbated the consequent dysfunction. 3. Paralleling effects on mitochondria, disruption of buffering and presence of Ca2+ enhanced Zn2+-induced neurodegeneration. 4. Zn2+ chelation after the high K+/Zn2+ exposure attenuated both ROS production and neurodegeneration, supporting the potential utility of delayed interventions. Taken together, these data lend credence to the idea that in pathologic states that impair cytosolic Zn2+ buffering, slow uptake of Zn2+ along with Ca2+ into neurons via VSCC can disrupt the mitochondria and induce neurodegeneration.