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

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 Aβ25–35-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 Aβ25–35-induced neuronal death, reversed the Aβ25–35-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 Aβ25–35, relieved the Aβ25–35-induced decrease in the peak amplitude of transient outward K+ currents (IA) and outward-delayed rectifier K+ currents (IDR) at different membrane potentials, and suppressed the steady-state activation and inactivation curves of IA shifted toward the hyperpolarization direction caused by Aβ25–35. These results suggest that Aβ25–35-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 Aβ25–35-induced neuronal damage by recovering the intracellular Zn2+ concentration.


Introduction
Alzheimer's disease (AD) is an age-related neurodegenerative disease characterized by progressive cognitive dysfunction and memory decline [1]. The main histopathological hallmarks of AD include extracellular senile plaques and intracellular neurofibrillary tangles [2]. Amyloid-β (Aβ) protein, the main component of senile Open Access *Correspondence: liuyanq@nankai.edu.cn; liuyanq2@126.com 1 College of Life Sciences, Nankai University, Tianjin 300071, People's Republic of China Full list of author information is available at the end of the article plaques, is believed to play an important role in the pathological process of AD [3]. The neurotoxic effects of Aβ can trigger a deleterious cascade of events, including alterations in neuronal excitability and ion permeability, oxidative stress, inflammatory processes, cell apoptosis, and loss of synapses [4][5][6].
Zinc ions (Zn 2+ ), an essential trace element in the human body, can regulate the function of approximately 10% of human proteins [7][8][9]. However, Zn 2+ is also well known for its neurotoxic effect [10]. Excess intracellular Zn 2+ can stimulate the generation of reactive oxygen species in hippocampal neurons, causing oxidative stress and neuronal death [11]. Some evidence suggests that intracellular Zn 2+ dysregulation may be involved in neurotoxicity caused by the generation of excessive neurotoxic Aβ peptides in AD and mediating age-related cognitive impairment [12,13]. Some autopsy studies have shown an increase in Zn 2+ concentration in amyloid plaques of AD brains [14,15]. In the hippocampal extracellular fluid, Aβ released from synaptic vesicles had a high affinity for Zn 2+ and could rapidly bind to Zn 2+ [16]. After injection of soluble Aβ to the dentate granule cell layer of normal rats, the concentration of Aβ and free Zn 2+ in dentate granule cells increased within 5 min, which subsequently led to the impairment of long-term potentiation and cognition [17][18][19]. Therefore, maintaining intracellular Zn 2+ homeostasis may be a promising strategy for preventing AD progression. As a Zn 2+ -specific chelator, N, N, N′, N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) has been reported to suppress the neurotoxicity induced by soluble Aβ, further showing a close correlation between Zn 2+ and neurotoxicity of Aβ [20]. However, it is still unclear how Zn 2+ influences Aβ neurotoxicity. Therefore, more experimental data are required to further clarify the role of Zn 2+ in the neurotoxicity of Aβ and pathological process of AD.
In the early stages of AD, functional MRI showed neuronal hyperactivation and epileptiform discharges in the hippocampus [21,22], further causing cognitive deficits and memory impairments [23]. In young APP/PS1 transgenic mice, the proportion of hyperactive neurons increased [24]. Acute application of soluble Aβ oligomers on hippocampal slices elevates intrinsic excitability in CA1 pyramidal neurons of wild-type mice [24,25]. These results indicate that soluble Aβ oligomers directly induced neuronal hyperactivity and impaired cognitive function. Further evidence suggests that sodium (Na + ) channel involvement may be related to increases in hippocampal neuron excitability caused by Aβ [26]. Aβ-induced neuronal hyperexcitation was markedly ameliorated by the presence of riluzole, a non-selective antagonist of Na + channels [26]. In fact, voltage-gated Na + channels (Na v ) are crucial for regulating neuronal excitability by initiating and propagating action potentials [27,28]. Among the nine α-subunits of Na v , the Na v 1.1, Na v 1.2, and Na v 1.6 subtypes were mainly expressed in the mammalian central nervous system [29]. The expression of the Na v 1.6 subtype and voltage-dependent Na + current density both significantly increased in Tg2576 mice (Aβ pathology animal model) compared with those in wild-type mice [29]. Similar results were observed in primary cultured pyramidal neurons after incubation with soluble Aβ [30]. Collectively, Na v might be involved in AD development.
In neurons, voltage-gated potassium (K + ) channels (K v ) are crucial regulators of neuronal excitability by controlling membrane repolarization and hyperpolarization [31]. Importantly, K v is a crucial mediator of cell death and cell survival signaling pathways [31]. K v dysfunction is involved in many diseases, such as AD. In rat hippocampal slices, the peak amplitudes of transient outward K + currents (I A ) and outward-delayed rectifier K + currents (I DR ) decreased after acute Aβ incubation [32]. In Aβ-overexpressing cultures, the excitability of neurons increased, accompanied by a decrease in I A current density and K v 4 protein expression [33]. However, restoration of K v 4 protein levels by transgenes could significantly rescue Aβ-induced neuronal hyperactivation and memory deficits [33,34]. In summary, K v is closely related to AD development.
Accordingly, Aβ-induced neuronal deleterious cascades are involved in Zn 2+ dysregulation and changes in the electrophysiological properties of Na v and K v . However, how Zn 2+ dysregulation influences the electrophysiological properties of Na v and K v in Aβ-treated neurons remains unclear. Therefore, in this study, we first established an in vitro model of AD by exposing soluble Aβ [25][26][27][28][29][30][31][32][33][34][35] to primary hippocampal neurons and then detected the effect of TPEN on cell viability and intracellular free Zn 2+ concentration in Aβ 25-35 -incubated hippocampal neurons. Furthermore, we evaluated the electrophysiological properties of the evoked repetitive action potential (APs), Na v and K v in these neurons. We aimed to understand the role of intracellular Zn 2+ dysregulation in Aβ-induced neurotoxicity and hope to provide some basis for preventing and combating AD based on Zn 2+ -specific chelators.

Isolation and culture of the primary hippocampal neurons
The primary hippocampal neurons of the rats were cultured as previously described by Beaudoin, et al. [35]. Briefly, early postnatal (P0-P1) Sprague-Dawley rats (either sex) were anesthetized with 50 mg/kg sodium pentobarbital via intraperitoneal injection and then washed with 75% (vol/vol) ethanol. The rats were then decapitated, and their brains were removed and transferred into ice-cold dissociation buffer (HBSS). The hippocampi were dissected and incubated with 0.25% trypsin-EDTA (Invitrogen, UK) at 37 °C for 12 min, with gentle shaking every 5 min. After digestion, the trypsin-EDTA solution was removed, and the hippocampi were dissociated into a single-cell suspension in 10 mL Dulbecco's modified Eagle medium/F12 (Gibco, UK) medium supplemented with 10% fetal bovine serum (Gibco, UK) and 50 μg/mL DNase (Sigma, USA) using a 1-mL pipette with a polished plastic tip. The cell suspension was centrifuged at 100×g for 5 min, and the cells were resuspended in the following plating medium: Dulbecco's modified Eagle medium/F12 medium supplemented with 10% fetal bovine serum, 5 unit/mL penicillin, and 50 µg/mL streptomycin (all from Gibco, UK). The neurons were seeded into 96-well plates or 35-mm culture dishes (pre-coated with 0.1 mg/mL poly-l-lysine for 1 h and washed three times with ddH 2 O before use) at a density of 120cells/ mm 2 in the plating medium. After 4-6 h, the plating medium was replaced with a maintenance medium, i.e., Neurobasal-A medium supplemented with 2% B27, 1% Glutamax, 50 μg/mL streptomycin, and 5 unit/mL penicillin (all from Gibco, UK). To prevent glial overgrowth, we treated the culture with Ara-C (Sigma, USA) at a final concentration of 1-5 μM on day 3. The neurons were cultured in a humidified 5% CO 2 incubator at 37 °C. The maintenance medium was replaced every 3 days. The cultures were grown for 8-12 days in vitro (DIV) before the experiments.

Experimental design
The cultured hippocampal neurons were divided into three groups: control group, Aβ 25-35 group, and Aβ 25-35 + TPEN group. Based on the results of the preliminary experiment in relation to the viability of the hippocampal neurons after the MTT assay, the optimal concentration of TPEN was 100 nM. In the Aβ 25-35 group, the hippocampal neurons were treated with Aβ 25-35 in the maintenance medium at a final concentration of 20 μM for 24 h. In the Aβ 25-35 + TPEN group, the hippocampal neurons were treated with TPEN in the maintenance medium at a final concentration of 100 nM for 30 min before and during exposure to Aβ [25][26][27][28][29][30][31][32][33][34][35] .

Determination of cell viability using the MTT assay
We used the MTT assay to assess cell viability. In brief, the culture medium from the 96-well plates was removed and replaced with 90 μL of a fresh maintenance medium after the different treatments. Ten microliters of 5 mg/ mL MTT in HBSS was added to each well, and the plates were incubated at 37 °C for 4 h. The supernatant was discarded and 100 uL DMSO solutions was added to each well. The plates were then incubated at 37 °C for 30 min. The absorbance of each sample was measured at 570 nm using a BIORAD680 plate reader (Thermo, Waltham, MA, USA). The experiments were repeated at least three times, and the results were compared to those of the control group.

Single live-cell confocal imaging
We used live-cell confocal imaging to investigate the intracellular Zn 2+ concentration in the hippocampal neurons. Briefly, the hippocampal neurons were seeded in a 35-mm glass bottom Petri dish (Nest, China). After the corresponding treatments, the neurons were washed twice with HBSS. For intracellular Zn 2+ imaging, the neurons were incubated in HBSS containing 2 mM FluoZin3-AM (Life Technologies, USA) and 0.02% (w/v) pluronic acid (Solarbio) at 37 °C in the dark for 1 h. They were then rinsed and maintained in HBSS. Images were captured using a laser scanning confocal microscope (TCSSP5, Leica, Germany) with a 63 × objective.

Whole-cell patch-clamp recording from the cultured hippocampal neurons
Based on the procedures of Wang, et al. [36], the whole-cell patch-clamp technique was performed to record APs, I Na and K v currents at 22-25 °C. The recording pipettes were pulled using a multistage micropipette puller (P-97, Sutter Instruments, Novato, CA, USA) and a borosilicate capillary glass. The tip resistance of the pipettes was 3-5 MΩ after being filled with the intracellular solution. The hippocampal neurons were then incubated with extracellular solution. We randomly selected hippocampal neurons with a smooth and bright appearance and no visible organelles for recording under an inverted microscope (BX51W1, Olympus, Japan). Signals were filtered, amplified, and digitized using a Multiclamp 700 B amplifier (Molecular Devices, Sunnyvale, CA, USA) and a DigiData 1440A digitizer (Molecular Devices). The data were recorded and analyzed using the pClamp 10.1 software (Molecular Devices). The series resistance was compensated for 85-90%. Recordings were discarded if the series resistance was over 20 MΩ or changed by over 20% during the experiments.
To eliminate the influence of neuronal size, we normalized the currents to the cell membrane capacitance to calculate current densities (pA/pF).

Data analysis and statistics
The experimental results were analyzed using Clampft 10.3 (Molecular Devices), Origin 8.5, and SPSS version 20. Statistical comparisons among the groups were performed using one-way analysis of variance. All data are presented as means ± SEMs. Statistical significance was set at p-values of < 0.05 and extreme significance at p-values of < 0.01.

-treated hippocampal neurons
The evoked APs were examined by using whole-cell current-clamp recordings, and the repetitive firings were evoked by a 500-ms prolonged depolarizing current injection of 50-pA (Fig. 3a). The results showed that Aβ 25-35 treatment markedly increased the frequency of APs (Aβ vs. control, p < 0.01; Fig. 3b). However, TPEN treatment completely reversed the Aβ 25-35 -induced the frequency of APs increase (Aβ + TPEN vs. Aβ, p < 0.05; Aβ + TPEN vs. control, p > 0.05; Fig. 3b). To record Na v currents (I Na ), we held the hippocampal neuron potentials at − 80 mV and evoked the current traces using a 20-ms constant depolarizing pulse from − 80 to + 65 mV in increments of 5 mV (Fig. 4a). Consequently, Aβ 25-35 significantly increased the maximum current density of I Na compared to the control (from − 83.30 ± 5.04 pA/pF to − 121.06 ± 11.55 pA/ pF, p < 0.01; Fig. 4b). Furthermore, the I Na increased at different membrane potentials after exposure to Aβ, which were visible from current-voltage (I-V) curves (Fig. 4c), compared to that after exposure to the control (p < 0.05). However, pretreatment with TPEN not only completely reversed the increase in the maximum I Na current density caused by Aβ 25-35 but also prevented the Aβ 25-35 -induced downward shift of the I-V curves (Aβ + TPEN vs. Aβ, p < 0.05; Aβ + TPEN vs. control, p > 0.05; Fig. 4b, c).

Effects of TPEN on the electrophysiological properties of Na v in the Aβ 25-35 -treated hippocampal neurons
To examine the gating properties of Na v, we obtained the activation curve of I Na by fitting the Boltzmann equation: is the half-activation potential and k is the slope factor. The results indicated that there was no significant difference in the activation curve of I Na among all groups ( Fig. 4d-f, p > 0.05).
To examine the kinetics of recovery from inactivation of Na v , we held the hippocampal neuron potentials at − 90 mV and applied a depolarizing pulse of − 10 mV (15-ms duration). The neurons were then treated with a test pulse of − 10 mV (15-ms duration) after a series of − 90-mV intervals varying from 0.5 to 44.5 ms (Fig. 6a). The recovery curve of Na v from inactivation was fitted with the monoexponential equation: I/I Max = 1 − exp(−�t/τ ) , where τ is the time constant. The results indicated that Aβ 25-35 did not alter the recovery characteristics after Na v inactivation. There was no significant difference in the recovery time constant from inactivation of Na v among all groups (Fig. 6b, c).
To explore the steady-state inactivation kinetics of I A , we held the hippocampal neuron potentials at − 90 mV and applied an 80-ms constant depolarizing pulse from − 120 to + 10 mV in increments of 10 mV. The neurons were then treated with a test pulse of 50 mV (80-ms duration) (Fig. 8a). The inactivation curves were fitted using the Boltzmann equation: is the half-inactivation potential and k is the slope factor. Compared to those in the control, the inactivation curves in the Aβ 25-35 group shifted to hyperpolarization (Fig. 8b). Moreover, Aβ 25-35 treatment significantly reduced the V 1/2 and k (Aβ vs. control, p < 0.01; Fig. 8c, d). TPEN treatment reversed the V 1/2 and k decreases caused by Aβ 25-35 (Aβ + TPEN vs. Aβ, p < 0.01; Aβ + TPEN vs. control, p > 0.05; Fig. 8c, d).
To examine the kinetics of recovery from I A activation, we held the hippocampal neuron potentials at − 90 mV and applied a depolarizing pulse of 50 mV (50-ms duration). The neurons were then treated with a test pulse of 50 mV (50-ms duration) following a series of − 90-mV intervals varying from 5 to 290 ms (Fig. 9a). The recovery curve of I A from inactivation was fitted with the monoexponential equation:  Fig. 9b, c).

Effects of TPEN on the electrophysiological properties of I DR in the Aβ 25-35 -treated hippocampal neurons
To investigate the properties of I DR in the hippocampal neurons subjected to the different treatments, we held the hippocampal neuron potentials at − 90 mV and evoked the current traces using a 200-ms constant depolarizing pulse from − 80 to + 100 mV in increments of 10 mV (Fig. 10a) Fig. 10c). The activation curve of I DR was obtained by fitting the Boltzmann equation:  Fig. 10d, e). Additionally, k in the Aβ 25-35 group showed an upward trend; however, there was no significant difference in k among all groups (Fig. 10f ).

Discussion
This study showed that TPEN attenuated Aβ 25-35induced neuronal death, reversed Aβ 25-35 -induced intracellular Zn 2+ concentration and the frequency of APs increase, inhibited Aβ 25-35 -induced maximum current density increase in I Na , and relieved Aβ 25-35 -induced decrease in the peak amplitudes of I A and I DR at different membrane potentials. These results suggested that Aβ 25-35 -induced neuronal damage correlated with Zn 2+ dysregulation mediated the electrophysiological changes in Na v and K v .
As mentioned above, hippocampal neuronal hyperexcitability and abnormal neuronal activity contribute to cognitive decline in AD, and excess extracellular Zn 2+ influx is involved in Glu-associated excitotoxicity in AD pathogenesis. Action potential (AP) is the basic characteristic reflecting neuronal excitability on mammalian central nervous system, which is regulated by ion channels in membrane [54]. Some evidence suggests that Na v , a key regulator of neuronal excitability, is involved in AD-related hippocampal pathological hyperactivity [29]. Soluble Aβ may induce neuronal hyperexcitation by increasing the amplitude of Na + currents [26]. However, the connection between Aβ-induced intracellular Zn 2+ dysregulation and changes in Na v properties remains unclear. After observing the protective effect of TPEN on the neurotoxicity caused by Aβ herein, we investigated the involvement mechanism of TPEN neuroprotection aimed at Aβ based on electrophysiological properties. Our study demonstrated that soluble Aβ 25-35 markedly increased the frequency of APs and the maximum current density of I Na , significantly elevated I Na at different membrane potentials. Moreover, soluble Aβ 25-35 induced the inactivation curves to significantly shift to hyperpolarization, indicating that I Na can be inactivated more easily. Taken together, the pathologically related soluble Aβ levels increased the excitability of the primary hippocampal neurons in vitro. However, TPEN treatment largely reversed the changes in the electrophysiological properties of APs and Na v caused by Aβ [25][26][27][28][29][30][31][32][33][34][35] . These results suggested that intracellular Zn 2+ dysregulation may be involved in Aβ-induced changes in Na v , leading to hippocampal excitability impairment.
K v plays a significant role in maintaining the resting membrane potential and regulating cell excitability, becoming a potential therapeutic target for the treatment of neurodegenerative diseases [55]. Based on the current characteristics, K v can be divided into I A and I DR [56]. I A mainly contributes to neuronal repolarization and repetitive firing of the action potential and is characterized by rapid activation and inactivation [32,57]. I DR mainly regulates the process of repolarization in neurons and has the characteristics of delayed long-lasting activation and non-inactivation [32,57]. Inhibiting I A and I DR can increase the excitability of rat hippocampal neurons [32]. Moreover, the expression and functional alterations of K v may be related to the neuronal hyperexcitability caused by Aβ, contributing to AD progress and development [31]. Herein, we observed that the maximum current density and I-V curves of I A and I DR significantly decreased after Aβ 25-35 exposure. Moreover, both the steady-state activation and inactivation curves of I A significantly shifted toward hyperpolarization upon Aβ [25][26][27][28][29][30][31][32][33][34][35] treatment, which implied that the voltage sensitivity of activation and inactivation was reduced. Besides, Aβ [25][26][27][28][29][30][31][32][33][34][35] obviously elevated the recovery time from inactivation, suggesting that I A took a longer time to open again after inactivation. These results indicated that Aβ 25-35 had a significant inhibitory effect on the I A and I DR of the hippocampal neurons, leading to increased hippocampal neuronal excitability. Further, TPEN significantly restored the changes in the electrophysiological properties of I A and I DR caused by Aβ [25][26][27][28][29][30][31][32][33][34][35] , which suggested that Aβ 25-35-induced the excessive influx of intracellular Zn 2+ , changing the electrophysiological characteristics of K v . In fact, the excitability of cultured mouse hippocampal neurons increased in the presence of exogenous Zn 2+ (50 μM) by increasing the firing frequency and inhibiting I A [58]. Furthermore, similar results were found in dopaminergic neurons of the rat substantia nigra and rat cardiomyocytes [59][60][61]. The mRNA levels of K v 1.4 and K v 4.3, which are the major components of I A , markedly decreased in rat cardiomyocytes with a high concentration of intracellular Zn 2+ (100 nM) [61,62]. These observations suggest that the neurotoxicity of Aβ may be, at least partially, attributed to the increase in intracellular Zn 2+ caused by Aβ, which inhibits K v activity; and TPEN could attenuate this excitability impairment via recovering potassium currents.
The existed studies suggest that abnormal Zn 2+ homeostasis be the cause of a variety of health problems [48], for example, in hypoxic-ischemic conditions, TPEN, a specific free Zn 2+ chelator could inhibit neuronal death by modulating apoptosis, glutamate signaling, and voltage-gated K + and Na + channels in neurons [63]. TPEN also could increase the survival rate of retinal ganglion cells and promote considerable axon regeneration after the optic nerve injury [64,65]. Moreover, TPEN induced pancreatic cancer cell death through increasing oxidative stress and restraining cell autophagy [66]. Our study also suggest that maintaining intracellular Zn 2+ homeostasis be also an effective program to alleviate Aβ-induced neuronal damage in AD. And TPEN might represent a potential cell-targeted therapy in Zn 2+ -related diseases. However, most studies including our present study currently focused on cells and animals experiments applying TPEN. To solve some involved human diseases applying TPEN, we should implement some human studies applying TPEN with a step-by-step after more animal experiments.
In conclusion, our study demonstrated that Aβ 25-35induced neuronal death was correlated with intracellular Zn 2+ dysregulation, which markedly changed the electrophysiological properties of Na v and K v , including the obvious increase in Na v activities and noticeable decrease in I A and I DR activities in the primary hippocampal neurons. TPEN attenuated Aβ 25-35 -induced neuronal death by recovering intracellular Zn 2+ concentrations and the electrophysiological properties of Na v and K v . Maintaining intracellular Zn 2+ homeostasis may be an effective program to alleviate Aβ-induced neuronal damage in AD. However, the deep mechanisms of intracellular Zn 2+ or abnormal Zn 2+ homeostasis on the activities of Na v and K v channels changes needs to be further studied. Furthermore, the result in present study only was from in vitro experiment applying cultured neurons, it needs more animals and human studies to conform the role of TPEN, a specific free Zn 2+ chelator in neurodegenerative diseases including AD. If so, TPEN, a specific free Zn 2+ chelator might be developed as drug against neurodegenerative diseases including AD.