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

Abstract

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 (ΔΨ_m), 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 ∆Ψ_m, 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.

Keywords: Ca(2+) channel; Calcium; Excitotoxicity; Ischemia; Metallothionein; Mitochondria; Neuronal cultures; Reactive oxygen species; VSCC; Zinc.

Figure 1. High K⁺/Zn²⁺ exposures induce dose-dependent mitochondrial Zn²⁺ uptake but only mild acute dysfunction

A. High K⁺/Zn²⁺ exposures cause dose-dependent mitochondrial Zn²⁺ accumulation. Cultures were loaded with the low affinity cytosolic Zn²⁺ indicator Newport Green (K_d ~ 1 μM), exposed to 90 mM K⁺ (high K⁺) with Zn²⁺ (25, 75, 300 μM) for 5 min in 0 Ca²⁺ HSS, followed by wash into 0 Ca²⁺ HSS for additional 5 min prior to application of FCCP (1 μM). Left: Representative images: Brightfield image (i) shows appearance of neurons at baseline, and pseudocolor images show Newport Green fluorescence from the same field at baseline (ii), 5 min after high K⁺/300 μM Zn²⁺ exposure (iii), and 5 min after FCCP (iv). (Arrows highlight the same neurons in these images.) Right: Traces show time course of Newport Green ΔF (background subtracted and normalized to baseline [F_x/F₀]; arrows indicate the time points illustrated in the images), and represent means ± standard error of the mean (SEM) of 6 experiments, ≥ 120 neurons. Grey bar indicates time points of comparison (** indicates p < 0.01 by one-way ANOVA with Tukey post hoc). Note the Zn²⁺ exposure concentration-dependent mitochondrial Zn²⁺ accumulation, indicated by the increase in ΔF after FCCP B). These exposures only induce mild mitochondrial dysfunction: Cultures were loaded with the ΔΨ_mito sensitive indicator, Rhod123, or the superoxide preferring ROS indicator, HEt, and exposed to high K⁺/Zn²⁺ for 5 min, followed by wash into 0 Ca²⁺ HSS, as above. After 20 min, FCCP (1 μM) was applied to Rhod123-loaded cultures to induce full loss of ΔΨ_m. Traces show time course of Rhod123 ΔF (left) or HEt ΔF (right), normalized to baseline values (after background subtraction, as above; F_x/F₀), and represent mean ± SEM 6 experiments, ≥ 120 neurons. Grey bars indicate time points of comparison (* indicates p < 0.05, ** indicates p < 0.01, by one-way ANOVA with Tukey post hoc). Note that only 300 μM Zn²⁺ exposure induced discernable effects.

Figure 2. Disruption of cytosolic Zn²⁺ buffering leads to Zn²⁺-dependent mitochondrial dysfunction

Cultures were loaded with the high affinity Zn²⁺ indicator FluoZin-3 (K_d ~ 15 nM), Rhod123 or HEt in 0 Ca²⁺ HSS, then exposed to DTDP (60 or 100 μM; to disrupt cytosolic Zn²⁺ buffering), with FCCP (1 μM) or TPEN (20 μM) applied as indicated. Traces represent mean ± SEM F_x/F₀ values for each indicator and represents ≥ 5 experiments consisting of ≥ 100 neurons. Grey bars indicate time points of comparison (* indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, by two-tailed t-test). A). DTDP induces dose-dependent cytosolic Zn²⁺ release and mitochondrial Zn²⁺ accumulation: FluoZin-3 loaded neurons were exposed to DTDP followed by addition of FCCP, as indicated. Note the dose dependent effects of DTDP, with 100 μM causing both greater cytosolic Zn²⁺ rise and mitochondrial Zn²⁺ loading (as indicated by the FCCP-induced ΔF) than 60 μM. B). Disruption of buffering via DTDP can induce mitochondrial dysfunction: Rhod123- (left) or HEt- (right) loaded neurons were subjected to the indicated DTDP and FCCP exposures. Note that the 100 μM DTDP exposure resulted in substantial loss of ΔΨ_mito within 25 min (as indicated by the minimal response to FCCP) and significant ROS production, while 60 μM DTDP had far smaller effects. C). DTDP effects on mitochondria are Zn²⁺-dependent: Rhod123- (left) or HEt- (right) loaded neurons were exposed to 100 μM DTDP ± Zn²⁺ chelator TPEN (applied 10 min before DTDP), followed by FCCP (only in Rhod123 loaded cultures), as indicated. Note that TPEN largely eliminated the DTDP induced loss of ΔΨ_mito (left) and markedly attenuated the ROS production (right).

Figure 3. Impaired cytosolic Zn²⁺ buffering markedly enhances the acute impact of Zn²⁺ exposures on mitochondria

Cultures were loaded with Newport Green, Rhod123, or HEt in 0 Ca²⁺ HSS, and exposed to high K⁺/300 μM Zn²⁺ alone (blue), or to high K⁺/50 μM Zn²⁺ with 60 μM DTDP (applied as indicated; red); FCCP (1 μM) was added as indicated. Traces represent mean ± SEM F_x/F₀ values for each dye and represents 6 experiments consisting of ≥ 120 neurons. Grey bars indicate time points of comparison (NS indicates No Significance, * indicates p < 0.05, ** indicates p < 0.01, by two-tailed t-test). A). Low exogenous Zn²⁺ exposure to neurons with impaired buffering results in similar degrees of mitochondrial uptake as much higher Zn²⁺ exposure with intact buffering: Note the similar magnitudes of mitochondrial Zn²⁺ loading caused by the high K⁺/300 μM Zn²⁺ (blue) and the high K⁺/50 μM Zn²⁺/DTDP exposures. B, C). However, the lower Zn²⁺ exposure with impaired buffering results in greater mitochondrial dysfunction: Rhod123 (B) or HEt (C) loaded neurons were exposed as indicated. Note that the 50 μM Zn²⁺/DTDP exposure induced markedly greater loss of Δψ_m and ROS generation than 300 μM Zn²⁺ alone.

Figure 4. Zn²⁺ exposure dose-dependence of mitochondrial Zn²⁺ loading and acute dysfunction in neurons with impaired buffering

Cultures were loaded with Newport Green, Rhod123 or HEt in 0 Ca²⁺ HSS, and exposed to high K⁺ with 10 (blue) or 50 (red) μM Zn²⁺ in 60 μM DTDP. FCCP (1 μM) was added as indicated. Traces represent mean ± SEM F_x/F₀ values for each dye and represents 6 experiments consisting of ≥ 120 neurons. Grey bars indicate time points of comparison (* indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, by two-tailed t-test). A). Zn²⁺ exposure induces dose-dependent mitochondrial Zn²⁺ loading in neurons with disrupted buffering: Note the dose dependency of the cytosolic Zn²⁺ rise and mitochondrial Zn²⁺ uptake, with the 50 μM Zn²⁺ exposure causing far greater Zn²⁺ uptake than the 10 μM Zn²⁺. B, C). Mitochondrial dysfunction reflects the extent of Zn²⁺ accumulation: Rhod123 (B) or HEt (C) loaded neurons were exposed as indicated. Note that the 50 μM Zn²⁺ exposure induced far greater loss of Δψ_m and ROS generation than 10 μM Zn²⁺. Further note that despite causing relatively strong ROS generation, 50 μM Zn²⁺ still only caused modest loss of Δψ_m.

Figure 5. Ca²⁺ attenuates mitochondrial Zn²⁺ accumulation despite exacerbating the consequent dysfunction

Cultures were loaded with Newport Green, Rhod123 or HEt in 0 or 1.8 Ca²⁺ HSS, and exposed to high K⁺ with 300, 50,10 or 0 μM Zn²⁺ (as indicated, along with 10 μM MK-801, to inhibit Ca²⁺-entry via NMDA receptor activation); DTDP (60 μM), FCCP (1 μM) and/or apocynin (500 μM) were added as indicated. Traces represent mean ± SEM F_x/F₀ values for each dye and represents ≥ 5 experiments consisting of ≥ 120 neurons. Grey bars indicate time points of comparison (NS indicates No Significance, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, by two-tailed t-test [A, C] or by one-way ANOVA with Tukey post hoc [B]). A). Presence of Ca²⁺ decreases neuronal and mitochondrial Zn²⁺ uptake: Note that presence of Ca²⁺ attenuated both cytosolic Zn²⁺ rise during the exposure and FCCP-induced mitochondrial Zn²⁺ release. B). Ca²⁺ and Zn²⁺ synergistically induce mitochondrial dysfunction: Neurons loaded with Rhod123 (left) or HEt (right) were exposed to high K⁺/DTDP/MK-801 with 10 μM Zn²⁺ (blue), 1.8 mM Ca²⁺ (red) or with both Zn²⁺ and Ca²⁺ (purple) for 5 min, then washed as indicated. Apocynin was added to HEt-loaded neurons (right) to inhibit contributions from Ca²⁺-dependent NOX activation. Note that despite relatively little effects from Ca²⁺ and Zn²⁺ individually, together they induced significant mitochondrial dysfunction. C). Overwhelming mitochondrial Zn²⁺ loading induces rapid mitochondrial depolarization: Neurons loaded with Rhod123 (left) or HEt (right) in 1.8 Ca²⁺ HSS were exposed to high K⁺/DTDP/MK-801/Ca²⁺, with 50 (blue) or 300 μM (red) Zn²⁺ for 5 min, followed by wash as indicated. Note that 300 μM Zn²⁺ induced greater loss of ΔΨ_m than 50 μM Zn²⁺, despite both inducing similar levels of ROS generation.

Figure 6. Effects of Ca²⁺, Zn²⁺, and disruption of cytosolic Zn²⁺ buffering on mitochondrial morphology

A). Experiment schematic: Neurons loaded with the mitochondrial dye MitoTracker Green (200 nM) were placed in 0 or 1.8 Ca²⁺ HSS, then exposed to DTDP (60 or 100 μM; where indicated), high K⁺/MK-801 with Zn²⁺ (0, 50, or 300 μM) and/or 1.8 mM Ca²⁺, followed by wash into HSS ± DTDP, as described. B). Representative images: Confocal images (1000x) were taken at baseline, 5 min after Zn²⁺ and/or Ca²⁺ exposure, and 10 min after wash. C). Zn²⁺ and Ca²⁺ induce different patterns of morphology change: The length and width of individual mitochondria were measured blindly, and length/width (L/W) ratios calculated and normalized to baseline. Values for baseline, 5 min after exposure, and after 10 min wash are displayed. Traces show mean ± SEM normalized L/W ratio for each time point, each representing ≥ 5 experiments consisting of ≥ 50 mitochondria (* indicates p < 0.05, ** indicates p < 0.01, by one-way ANOVA with Tukey post hoc). Note that while the Ca²⁺ induces a rapid but transient morphologic change, Zn²⁺ triggers more progressive changes (that increase with the degree of Zn²⁺ loading).

Figure 7. Zn²⁺-induced inhibition of mitochondrial respiration: synergy with Ca²⁺ and effects of disrupted buffering

A). Schematic of experiment: Neurons were exposed to a series of treatments (detailed in B and C, left), incubated for 1 hr, then placed in the Seahorse assay, which measures O₂ consumption rate (OCR) at baseline and after sequential application of oligomycin (Oligo; 1 μM), FCCP (2 μM), and antimycin A & rotenone (AA/Rot; both 1 μM) to characterize various respiratory parameters. Traces (B and C, right) show time course of OCR and represent mean ± SEM of 3 separate experiments, each consisting of 3 – 4 wells of cultured neurons, with arrows indicating time point at which mitochondrial inhibitors were added. Grey bars indicate time points of comparison (* indicates p < 0.05, ** indicates p < 0.01, by one-way ANOVA with Tukey post hoc). B). Ca²⁺ and Zn²⁺ synergistically inhibit mitochondrial respiration: Neurons were placed in 0 or 1.8 Ca²⁺ HSS, exposed to high K⁺/MK-801 with 300 μM Zn²⁺, 1.8 mM Ca²⁺ or both Zn²⁺ and Ca²⁺ as described (left). After 1 hr incubation OCR was measured (right). Note that simultaneous exposure to Zn²⁺ and Ca²⁺ induced significant inhibition of mitochondrial respiration, despite the ions having minimal effects individually. C). Disrupted Zn²⁺ buffering significantly exacerbates Zn²⁺ effects on mitochondrial respiration: Neurons were placed in 0 Ca²⁺ HSS, exposed to DTDP (100 μM; where indicated), high K⁺/MK-801 with Zn²⁺ (300, 10 or 0 μM, as indicated; left). After 1 hr incubation OCR was measured (right). Note the near complete inhibition of mitochondrial respiration by 10 μM Zn²⁺ exposure with DTDP.

Figure 8. Mitochondrial Zn²⁺ accumulation contributes to neuron death

Neurons were exposed to a sequence of 10 min DTDP (100 μM; as indicated in B and C), 5 min high K⁺/MK-801 exposures with Zn²⁺ and/or Ca²⁺ (concentration as shown), washed for 10 min (with DTDP in B and C), transferred to MEM + 25 mM glucose and returned to the incubator for 24 hrs (or for only 2 hrs where indicated in C), prior to assessing cell death via LDH efflux assay. Bars show % cell death (see Material and methods), and represent mean ± SEM of 3 independent experiments, each consisting of 4 wells of cultured neurons (* indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001 by one-way ANOVA with Tukey post hoc [A and B] or by two-tailed t-test [C]). A). Ca²⁺ and Zn²⁺ synergistically induce cell death: Note that while 300 μM Zn²⁺ induced more cell death than 1.8 mM Ca²⁺, its impact was further exacerbated by the presence of Ca²⁺. B). Dose-dependency of Zn²⁺-induced cell death under conditions of strongly disrupted buffering: Note the dose-dependent increase in cell death with increasing Zn²⁺ exposures, that was further enhanced by the presence of Ca²⁺. C). Zn²⁺-induced cell death progresses gradually over hours: Note the significantly greater cell death at 24 hrs compared to 2 hrs.

Figure 9. Delayed Zn²⁺ chelation attenuates mitochondrial ROS generation and neuron death

Neurons were exposed to high K⁺/50 μM Zn²⁺/MK-801, with DTDP (100 μM), and apocynin (500 μM, A only). TPEN was applied as indicated below. Traces (A) represent HEt F_x/F₀ and bars (B) represent % cell death after 24 hr; all values are mean ± SEM and represents ≥ 3 independent experiments. Grey bar (in A) indicates time points of comparison (* indicates p < 0.05, ** indicates p < 0.01, by two-tailed t-test [A] or by one-way ANOVA with Tukey post hoc [B]). A). Delayed Zn²⁺ chelation attenuates Zn²⁺ triggered ROS production: Note the rapid rise in HEt ΔF that was largely attenuated by TPEN (20 μM), added after the Zn²⁺ exposure. B). Zn²⁺ chelation attenuates cell death even when delivered after the Zn²⁺ exposure: Cultured neurons were exposed as described, with TPEN (10 μM) present either for 10 min before, during and 10 min after high K⁺/Zn²⁺ exposure (TPEN pre), or only for 10 min after Zn²⁺ exposure (TPEN post). Cultures were then transferred to MEM + 25 mM glucose and returned to the incubator for 24 hrs, prior to assessing cell death via LDH efflux assay. Note that both the TPEN pre- and post-treatments attenuated neuron death.