TPEN attenuates amyloid-β25-35-induced neuronal damage with changes in the electrophysiological properties of voltage-gated sodium and potassium channels

Abstract

To understand the role of intracellular zinc ion (Zn²⁺) dysregulation in mediating age-related neurodegenerative changes, particularly neurotoxicity resulting from the generation of excessive neurotoxic amyloid-β (Aβ) peptides, this study aimed to investigate whether N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a Zn²⁺-specific chelator, could attenuate Aβ_25-35-induced neurotoxicity and the underlying electrophysiological mechanism. We used the 3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay to measure the viability of hippocampal neurons and performed single-cell confocal imaging to detect the concentration of Zn²⁺ in these neurons. Furthermore, we used the whole-cell patch-clamp technique to detect the evoked repetitive action potential (APs), the voltage-gated sodium and potassium (K⁺) channels of primary hippocampal neurons. The analysis showed that TPEN attenuated Aβ_25-35-induced neuronal death, reversed the Aβ_25-35-induced increase in intracellular Zn²⁺ concentration and the frequency of APs, inhibited the increase in the maximum current density of voltage-activated sodium channel currents induced by Aβ_25-35, relieved the Aβ_25-35-induced decrease in the peak amplitude of transient outward K⁺ currents (I_A) and outward-delayed rectifier K⁺ currents (I_DR) at different membrane potentials, and suppressed the steady-state activation and inactivation curves of I_A shifted toward the hyperpolarization direction caused by Aβ_25-35. These results suggest that Aβ_25-35-induced neuronal damage correlated with Zn²⁺ dysregulation mediated the electrophysiological changes in the voltage-gated sodium and K⁺ channels. Moreover, Zn²⁺-specific chelator-TPEN attenuated Aβ_25-35-induced neuronal damage by recovering the intracellular Zn²⁺ concentration.

Keywords: Aβ25–35; Channel currents; TPEN; Voltage-gated potassium channels; Voltage-gated sodium channels; Zinc ions.

Fig. 1

Effects of TPEN on the viability of the hippocampal neurons treated with Aβ_25–35. The data are presented as means ± SEMs; ^**p < 0.01 versus the control group; ^##p < 0.01 versus the Aβ group; n = 19. Aβ amyloid-β

Fig. 2

Effects of TPEN on the intracellular Zn²⁺ concentration of the hippocampal neurons treated with Aβ_25–35. a Representative confocal images showing FluoZin3 (green) staining under the different treatments. The scale bar is 50 μm. b Mean fluorescence intensity of FluoZin3 in the different groups. The data shown in b were obtained from three independent experiments, each examining 15–20 neurons for each condition. The data are presented as means ± SEMs; ^**p < 0.01 versus the control group; ^##p < 0.01 versus the Aβ group. Aβ amyloid-β

Fig. 3

Effects of TPEN on the frequency of APs in the hippocampal neurons treated with Aβ_25–35. a Typical example of APs traces obtained in the hippocampal neurons under the different treatments. b The frequency of APs in the different treatments. The data are presented as means ± SEMs; ^**p < 0.01 versus the control group; ^#p < 0.05 versus the Aβ group; n = 8 for the control group; n = 23 for the Aβ group; n = 14 for the Aβ + TPEN group. Aβ, amyloid-β; APs, the evoked repetitive action potential

Fig. 4

Effects of TPEN on the amplitudes and activation properties of I_Na in the hippocampal neurons treated with Aβ_25–35. a Typical example of I_Na traces obtained in the hippocampal neurons (left) and record protocol (right). b Maximum current density of I_Na in the different treatments. c Current voltage (I-V) curves of I_Na in the different treatments. d Activation curves of I_Na in the different treatments. e Half-activation potential of I_Na in the different treatments. f Activation slope factor of I_Na in the different treatments. The data are presented as means ± SEMs; ^*p < 0.05 and ^**p < 0.01 versus the control group; ^#p < 0.05 versus the Aβ group; n = 21 for the control group; n = 16 for the Aβ group; n = 17 for the Aβ + TPEN group. Aβ amyloid-β, I_Na, voltage-gated Na⁺ channel curren

Fig. 5

Effects of TPEN on the inactivation properties of Na_v in the hippocampal neurons treated with Aβ_25–35. a Typical example of Na_v inactivation traces obtained in the hippocampal neurons (left) and record protocol (right). b Inactivation curves of Na_v in the different treatments. c Half-inactivation potential of Na_v in the different treatments. d Inactivation slope factor of Na_v in the different treatments. The data are presented as means ± SEMs; ^*p < 0.05 and ^**p < 0.01 versus the control group; ^#p < 0.05 and ^##p < 0.01 versus the Aβ group; n = 18 for the control group; n = 16 for the Aβ group; n = 16 for the Aβ + TPEN group. Na_v, voltage-gated sodium channels; Aβ, amyloid-β

Fig. 6

Effects of TPEN on the recovery of Na_v from inactivation in the hippocampal neurons treated with Aβ_25–35. a Typical example of Na_v recovery traces from inactivation obtained in the hippocampal neurons (left) and record protocol (right). b Recovery curves of Na_v from inactivation in the different treatments. c Time constant of the recovery curves for Na_v in the different treatments. The data are presented as means ± SEMs; n = 13 for the control group; n = 17 for the Aβ group; n = 17 for the Aβ + TPEN group. Na_v, voltage-gated sodium channels; Aβ, amyloid-β

Fig. 7

Effects of TPEN on the amplitudes and activation properties of I_A in the hippocampal neurons treated with Aβ_25–35. a Typical example of I_A traces obtained in the hippocampal neurons (left) and record protocol (right). b Maximum current density of I_A in the different treatments. c Current voltage (I-V) curves of I_A in the different treatments. d Activation curves of I_A in the different treatments. e Half-activation potential of I_A in the different treatments. f Activation slope factor of I_A in the different treatments. The data are presented as means ± SEMs; ^*p < 0.05 and ^**p < 0.01 versus the control group; ^#p < 0.05 and ^##p < 0.01 versus the Aβ group; n = 15 for the control group; n = 17 for the Aβ group; n = 9 for the Aβ + TPEN group. Aβ, amyloid-β; I_A, transient outward potassium current

Fig. 8

Effects of TPEN on the inactivation properties of I_A in the hippocampal neurons treated with Aβ_25–35. a Typical example of I_A inactivation traces obtained in the hippocampal neurons (left) and record protocol (right). b Inactivation curves of I_A in the different treatments. c Half-inactivation potential of I_A in the different treatments. d Inactivation slope factor of I_A in the different treatments. The data are presented as means ± SEMs; ^**p < 0.01 versus the control group; ^##p < 0.01 versus the Aβ group; n = 19 for the control group; n = 12 for the Aβ group; n = 16 for the Aβ + TPEN group. Aβ, amyloid-β; I_A, transient outward potassium current

Fig. 9

Effects of TPEN on the recovery of I_A from inactivation in the hippocampal neurons treated with Aβ_25–35. a Typical example of I_A recovery traces from inactivation obtained in the hippocampal neurons (left) and record protocol (right). b Recovery curves of I_A from inactivation in the different treatments. c Time constant of the recovery curves for I_A in the different treatments. The data are presented as means ± SEMs; ^*p < 0.05 and ^**p < 0.01 versus the control group; ^#p < 0.05 and ^##p < 0.01 versus the Aβ group; n = 24 for the control group; n = 21 for the Aβ group; n = 20 for the Aβ + TPEN group. Aβ, amyloid-β; I_A, transient outward potassium current

Fig. 10

Effects of TPEN on the amplitudes and activation properties of I_DR in the hippocampal neurons treated with Aβ_25–35. a Typical example of I_DR traces obtained in the hippocampal neurons (left) and record protocol (right). b Maximum current density of I_DR in the different treatments. c Current voltage (I–V) curves of I_DR in the different treatments. d Activation curves of I_DR in the different treatments. e Half-activation potential of I_DR in the different treatments. f Activation slope factor of I_DR in the different treatments. The data are presented as means ± SEMs; ^*p < 0.05 and ^**p < 0.01 versus the control group; ^#p < 0.05 and ^##p < 0.01 versus the Aβ group; n = 22 for the control group; n = 15 for the Aβ group; n = 15 for the Aβ + TPEN group. Aβ amyloid-β, I_DR, outward-delayed rectifier potassium current