Introduction inhibits GABA and NMDA receptor currents [37,55],

Brain Research 886 2000 99–107 www.elsevier.com locate bres Interactive report 21 21 L-type Ca channel-mediated Zn toxicity and modulation 1 by ZnT-1 in PC12 cells a a a a b Albert H. Kim , Christian T. Sheline , Min Tian , Toshio Higashi , Robert J. McMahon , b a , Robert J. Cousins , Dennis W. Choi a Department of Neurology and Center for the Study of Nervous System Injury , Washington University School of Medicine, 660 S. Euclid Ave., St . Louis, MO 63110, USA b Food Science and Human Nutrition Department and Center for Nutritional Sciences , University of Florida, Gainesville, FL 32611, USA Accepted 22 September 2000 Abstract 21 21 In view of evidence that Zn neurotoxicity contributes to some forms of pathological neuronal death, we developed a model of Zn neurotoxicity in a cell line amenable to genetic manipulations. Exposure to 500 mM ZnCl for 15 min under depolarizing conditions 2 21 resulted in modest levels of PC12 cell death, that was reduced by the L-type Ca channel antagonist, nimodipine, and increased by the 21 21 21 L-type Ca channel opener, S2-Bay K 8644. At lower insult levels 200 mM Zn 1Bay K 8644, Zn -induced death appeared apoptotic under electron microscopy and was sensitive to the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-CH F Z-VAD; at higher 2 insult levels 1000 mM1Bay K 8644, cells underwent necrosis insensitive to Z-VAD. To test the hypothesis that the plasma membrane 21 transporter, ZnT-1, modulates Zn neurotoxicity, we generated stable PC12 cell lines overexpressing wild type or dominant negative 21 forms of rat ZnT-1 rZnT-1. Clones T9 and T23 overexpressing wild type rZnT-1 exhibited enhanced Zn efflux and reduced 21 vulnerability to Zn -induced death compared to the parental line, whereas clones D5 and D16 expressing dominant negative rZnT-1 exhibited the opposite characteristics.  2000 Elsevier Science B.V. All rights reserved. Theme : Disorders of the nervous system Topic : Neurotoxicity Keywords : Zinc; Cell death; PC12; Bay K 8644; ZnT-1 1. Introduction inhibits GABA and NMDA receptor currents [37,55], 1 21 blocks voltage-gated Na and Ca channels, and poten- The transition metal zinc is a normal constituent of tiates AMPA, glycine, and P receptor currents [16,38]. 2x 21 transcription factors and metalloenzymes [31,51]. In the Given its lack of intrinsic redox activity, Zn has been central nervous system, an additional pool of chelatable considered to be relatively nontoxic [3,46]. However, the 21 Zn exists in a subpopulation of glutamatergic synaptic toxic translocation of presynaptic zinc into postsynaptic 21 vesicles [5,8,13,35] and can be released in a Ca -depen- neurons has now been implicated in the pathogenesis of dent manner by electrical or chemical stimulation [17], selective neuronal death following transient global is- possibly reaching synaptic concentrations in the hundred chemia, prolonged seizures, and trauma [23,42,45,48]. The 21 micromolar range [2]. Zn can modify the behavior of cytotoxicity of exogenously applied zinc has been demon- several membrane receptors and channels [6,15,43]; it strated in vitro and in vivo. In neuronal as well as nonneuronal cells such as thymocytes, prolonged exposure 21 to even 20–80 mM Zn induces cell death [12,26,41]. 1 Published on the World Wide Web on 12 October 2000. 21 Short exposures to 150–600 mM Zn destroys cultured Corresponding author. Tel.: 11-314-362-9460; fax: 11-314-362- cortical [58] or cerebellar granule neurons [28], and 9462. E-mail address : wilderspneuro.wustl.edu D.W. Choi. intraparenchymal injection of ZnCl induces neuronal 2 0006-8993 00 – see front matter  2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 9 4 4 - 9 100 A necrosis in the rat hippocampus [25]. With concurrent Val-Ala-Asp-CH F Z-VAD was purchased from Enzyme 2 21 depolarization, the toxicity of extracellular Zn is en- Systems Products. PC12 cells were transfected using hanced such that short exposure to low micromolar con- FuGene 6 Boehringer Mannheim, and stable transfectants 21 centrations of Zn becomes neurotoxic [54]. This de- were selected in medium containing 500 mg ml G418. polarization-induced enhancement likely reflects preferen- 21 tial entry of zinc through L-type voltage-gated Ca 2.2. Constructs channels, leading to toxic elevations of intracellular free 21 21 Zn [Zn ] in the range of 300–500 nM [4,40]. Wild type rZnT-1 cDNA kindly provided by Dr. R.D. i 21 Palmiter and dominant negative rZnT-1 cDNA an 83 bp Likely opposing such toxic elevations of [Zn ] are i EagI fragment deletion; [34] were subcloned into a CMV several mechanisms responsible for maintaining intracellu- 21 promoter-driven mammalian expression vector. After lar Zn homeostasis, including binding to metallothio- mutating the terminal stop codon in both rZnT-1 forms, a neins [1,10,50,53], and export across the plasma membrane fragment encoding a hexameric myc epitope tag gener- mediated by the ubiquitously expressed zinc transporter, ously provided by Dr. R. Kopan was inserted in-frame ZnT-1 [34]. Related zinc transporters appear to bear 21 distal to the altered stop codon. responsibility for transporting Zn into endosomes ZnT- 2 [32] or synaptic vesicles ZnT-3 [33]. Baby hamster 2.3. Toxicity experiments kidney BHK cells [34] and N2A cells [49] overexpres- sing ZnT-1 exhibit resistance to death induced by pro- 21 Immediately before toxic exposures, cells were washed longed Zn exposure. Following transient global ischemia twice using a HEPES-buffered salt solution HSS with the in gerbils, ZnT-1 transcription is upregulated in CA1 1 1 following composition: 130 mM Na , 5.4 mM K , 0.8 pyramidal neurons, consistent with the possibility that 21 21 2 mM Mg , 1.8 mM Ca , 131 mM Cl , 20 mM HEPES ZnT-1 induction may be a cellular strategy to counter 21 pH 7.4 at 258C, 15 mM glucose. Exposures of 15 min to ischemia-induced toxic Zn influx [49]. 21 toxic solutions were conducted at room temperature in We set out to develop a model of Zn influx-induced 1 1 HSS with equimolar substitution of K for Na in cell death in a neuronal cell line. We chose PC12 cells 1 solutions with elevated [K ]. To terminate toxic expo- because they have been widely used for investigating sures, solutions were washed twice with media stock MS, multiple aspects of neurobiology, including neuronal dif- which consists of Eagle’s minimal essential medium plus ferentiation, intracellular signaling pathways, and cell 21 21 mM glucose, and then replaced with MS containing 1 survival [14,56], and specifically express L-type Ca 21 serum 2:1, horse serum:fetal bovine serum before being channels [18,47], the primary route of toxic Zn entry 21 returned to a 378C incubator. A23187 exposures were into neurons. There is one previous report of Zn -induced conducted at 378C in MS after sham washes. Z-VAD was death of PC12 cells, utilizing prolonged 24 h exposure to 21 added to the final replacement solution MS and 1 Zn in the absence of serum, thought to be due to direct 21 serum following exposures and washes. inactivation of NGF by Zn [39]. To eliminate this death 21 mechanism and focus on toxic Zn entry, we utilized 21 2.4. Cell death assays brief 15 min exposure to Zn , followed by return to serum-containing medium, as the presence of serum abro- 21 Cell death was assessed morphologically by phase-con- gated Zn -mediated neurotrophin deprivation-induced trast microscopy and propidium iodide fluorescence. For death [39]. propidium iodide staining, cells were incubated for 30 min at 378C with propidium iodide solution 5 mg ml Molec- ular Probes, and dead cells cells with compromised

2. Materials and methods membrane integrity were visualized by excitation at 488