Copper Enhances Zinc-Induced Neurotoxicity and the Endoplasmic Reticulum Stress Response in a Neuronal Model of Vascular Dementia

Abstract

Zinc (Zn), an essential trace element, is secreted by synaptic vesicles during neuronal excitation and plays several critical roles in neuronal information processing. However, excess Zn ion (Zn²⁺) is neurotoxic and has a causative role in the pathogenesis of vascular dementia. Here, we investigated the molecular mechanism of Zn²⁺-induced neurotoxicity by using immortalized hypothalamic neurons (GT1-7 cells), which are more vulnerable than other neuronal cells to Zn²⁺. We examined the effects of other metal ions on the Zn²⁺-induced neurotoxicity in these cells and found that sub-lethal concentrations of copper ion (Cu²⁺) markedly exacerbated Zn²⁺-induced neurotoxicity. The co-administration of Cu²⁺ and Zn²⁺ also significantly increased the expression of genes related to the endoplasmic reticulum's stress response, including CHOP, GADD34, and ATF4. Similar to Zn²⁺, Cu²⁺ is stored in presynaptic vesicles and secreted during neuronal excitation. Thus, based on our results, we hypothesize here that Cu²⁺ interacts with Zn²⁺ in the synapse to synergistically promote neuronal death and significantly influence the pathogenesis of vascular dementia.

Keywords: ER stress; dementia; ischemia; metal–metal interaction; neurotoxicity; synapse.

Figure 1

Effects of various metals on the neurotoxicity of GT1-7 cells. (A) ZnCl₂, (B) CuCl₂, (C) MnCl₂, (D) NiCl₂, (E) FeCl₂, (F) Fe(NO₃)₃, or (G) AlCl₃ was administered to GT1-7 cells. After 24 h, cell viability was determined using the WST-8 method. Six wells were exposed to the same experimental conditions (n = 6). Data are presented as means ± SD of cell viability. Experiments were replicated at least two times.

Figure 2

Effects of various metals on Zn²⁺-induced neurotoxicity. (A) GT1-7 cells were exposed to 20 μM ZnCl₂, CuCl₂, MnCl₂, NiCl₂, FeCl₂, Fe(NO₃)₃, or AlCl₃. After 24 h, cell viability was determined using the WST-8 method. Six wells were exposed to the same experimental conditions (n = 6). Data are presented as means ± SD of cell viability. Experiments were replicated at least two times. ^*p < 0.05, ^**p < 0.01. (B) GT1-7 cells were exposed to 20 μM CuCl₂, MnCl₂, NiCl₂, FeCl₂, Fe(NO₃)₃, or AlCl₃ with 30 μM ZnCl₂ in the same experimental condition in (A). After 24 h, cell viability was determined using the WST-8 method. Six wells were exposed to the same experimental conditions (n = 6). Data are presented as means ± SD of cell viability. Experiments were replicated at least two times. ^*p < 0.05, ^**p < 0.01.

Figure 3

Effects of Cu²⁺ on Zn²⁺-induced neurotoxicity. Various concentrations of CuCl₂ (0~20 μM) without ZnCl₂ (A) or with 10 μM (B), 20 μM (C), or 30 μM ZnCl₂ (D) were administered to GT1-7 cells. After 24 h, cell viability was determined using the WST-8 method. Six wells were exposed to the same experimental conditions (n = 6). Data are presented as means ± SD. Experiments were replicated at least two times. ^*p < 0.05, ^**p < 0.01 vs. CuCl₂ (0 μM).

Figure 4

Effects of Cu²⁺ on Zn²⁺-induced gene expression. Expression levels of CHOP, GADD34, Arc, Bip, ATF4, EDEM, sXBP1, GRP94, PDI, ZnT-1, MT1, and MT2 were analyzed using real-time RT–PCR. Gene expression levels were normalized with GAPDH. Data are presented as means ± SD (n = 3). Experiments were replicated at least two times. ^*p < 0.05, ^**p < 0.01 compared with control; ^#p < 0.05, ^##p < 0.01 compared with Zn²⁺.

Figure 5

Effects of Cu²⁺ on Zn²⁺–induced CHOP protein expression. The expression of CHOP protein in GT1-7 cells was assayed using western blotting. GT1-7 cells were treated with 30 μM Zn²⁺, 20 μM Cu²⁺, or a mixture of 30 μM Zn²⁺ with 20 μM Cu²⁺ for 4 h. The blot was probed with an anti-CHOP antibody, and the band intensities were analyzed using ImageJ software. The values shown were obtained by dividing the intensity of the respective band by that of the standard band. Data are presented as means ± SD (n = 3). ^**p < 0.01 compared with control; ^##p < 0.01 compared with Zn²⁺. Experiments were replicated at least two times.

Figure 6

Proposed mechanism for the synergistic interaction between Zn²⁺ and Cu²⁺ at the synapse. Zn and glutamate accumulate in synaptic vesicles and are released into synaptic clefts during neuronal excitation. Zn²⁺ regulates Ca²⁺ influx through NMDA-type glutamate receptors, modulates neuronal information, and is implicated in the maintenance of synaptic plasticity and memory formation, similar to Ca²⁺. Zn²⁺ enters target neurons via voltage-dependent Ca²⁺ channels, NMDA-type glutamate channels, and Ca²⁺-permeable AMPA/kainate channels. The increased intracellular Zn²⁺ induces ER stress pathways and triggers apoptotic pathways. The ZnT-1 Zn transporter regulates Zn homeostasis and is localized to post-synaptic membranes that express NMDA-type glutamate receptors. Carnosine is released from glial cells into synaptic clefts, and is thought to regulate excess Zn. ZnT-1, zinc transporter 1; ZnT-3, zinc transporter 3; AMPA-R, AMPA-type glutamate receptor; NMDA-R, NMDA-type glutamate receptor; CAR, carnosine.