Increased K_V2.1 Channel Clustering Underlies the Reduction of Delayed Rectifier K⁺ Currents in Hippocampal Neurons of the Tg2576 Alzheimer’s Disease Mouse

Abstract

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the progressive deterioration of cognitive functions. Cortical and hippocampal hyperexcitability intervenes in the pathological derangement of brain activity leading to cognitive decline. As key regulators of neuronal excitability, the voltage-gated K⁺ channels (K_V) might play a crucial role in the AD pathophysiology. Among them, the K_V2.1 channel, the main α subunit mediating the delayed rectifier K⁺ currents (I_DR) and controlling the intrinsic excitability of pyramidal neurons, has been poorly examined in AD. In the present study, we investigated the K_V2.1 protein expression and activity in hippocampal neurons from the Tg2576 mouse, a widely used transgenic model of AD. To this aim we performed whole-cell patch-clamp recordings, Western blotting, and immunofluorescence analyses. Our Western blotting results reveal that K_V2.1 was overexpressed in the hippocampus of 3-month-old Tg2576 mice and in primary hippocampal neurons from Tg2576 mouse embryos compared with the WT counterparts. Electrophysiological experiments unveiled that the whole I_DR were reduced in the Tg2576 primary neurons compared with the WT neurons, and that this reduction was due to the loss of the K_V2.1 current component. Moreover, we found that the reduction of the K_V2.1-mediated currents was due to increased channel clustering, and that glutamate, a stimulus inducing K_V2.1 declustering, was able to restore the I_DR to levels comparable to those of the WT neurons. These findings add new information about the dysregulation of ionic homeostasis in the Tg2576 AD mouse model and identify K_V2.1 as a possible player in the AD-related alterations of neuronal excitability.

1. Introduction

Alzheimer’s disease (AD) is a devastating neurodegenerative disorder representing the most common cause of dementia in elderly population. The AD pathology consists of the progressive deterioration of cognitive functions, resulting from an irreversible neuronal loss and the pathological derangement of brain circuits and networks, among other mechanisms [1,2]. According to a growing amount of evidence, the dysregulation of brain activity observed in AD is correlated with the alteration of neuronal excitability. In particular, network hyperexcitability and hypersynchrony, and epileptiform activity have been observed in AD mouse models [3,4,5] and proposed as crucial factors causing the malfunction of hippocampal circuitry and subsequent memory impairment [6,7]. Moreover, an increased risk of developing seizures has been observed in the AD patients with mutations in presenilin 1, presenilin 2, and amyloid precursor protein (APP), or with APP duplications [8,9]. Of note, numerous studies have reported that the Amyloid-β_1–42 (Aβ_1–42) peptide, the main component of both the neuritic plaques and intracellular aggregates found in the brain of AD patients, can trigger neuronal hyperactivity [10,11,12]. Accordingly, hyperactive neurons have been found in the hippocampus of different AD transgenic models overproducing the Aβ_1–42 peptide [5,10,13].

Potassium (K⁺) channels are the most abundant and diverse channels in the mammalian brain. Mediating K⁺ fluxes across the plasma membrane (PM), K⁺ channels are crucial determinants of neuronal excitability [14]. Among them, voltage-gated K⁺ channels (K_V) are the largest family of K⁺ channels expressed in both the central and peripheral nervous system, where they participate in different biological processes, including the regulation of action potential conduction, neurotransmitter release, synaptic plasticity, and apoptosis [15].

The K_V2.1 channels are the main α subunits mediating the delayed rectifier K⁺ currents (I_DR) in hippocampal and cortical pyramidal neurons, where they are present as large, high-density clusters on the PM of somata and proximal dendrites [16,17,18,19,20]. Although K_V2.1 channels do not participate in action potential repolarization due to their slow kinetics, they work as homeostatic suppressors of the intrinsic neuronal excitability by regulating Ca²⁺ influx during periods of repetitive high-frequency firing [21,22,23], hence shaping long-term changes in both synaptic efficacy and neuronal phenotype [24]. Despite being controversial in the literature at first, the localization of the K_V2.1 channels has also been ascertained at the axon initial segment of cortical and hippocampal pyramidal neurons, where they may regulate both the frequency and back propagation of the axonal action potential [25,26,27]. Gene mutations altering the K_V2.1 activity have been linked to epilepsy in humans [28,29,30], while K_V2.1 knockout mice display hyperactivity and deficits in spatial learning [31].

The K_V2.1 channels have been extensively investigated for their unique subcellular localization and the ability to sense neuronal activity, which in turn regulates their localization and function through the modulation of their phosphorylation state [32,33]. Indeed, neuronal K_V2.1 channels are extensively modified by phosphorylation as they display numerous phosphorylation sites for different protein kinases in their cytoplasmic domain [34,35,36]. Interestingly, the degree of phosphorylation, which is constitutively high in neurons, crucially affects K_V2.1 biophysical properties and localization [23,34,37,38,39], and is strictly coupled with channel clustering [32,35,40]. It has been extensively demonstrated that the K_V2.1 channels residing within clusters, which represent a large percentage of the channels natively expressed on the neuronal PM, are non-conducting [20,41,42].

Although the involvement of K⁺ channels has been poorly examined in the AD pathophysiology, alterations in the K_V2.1 activity were observed in some AD experimental models. In particular, K_V2.1 oxidation in 3xTg hippocampal neurons was supposed to correlate with its increased clusterization and functional downregulation, and to promote neuronal hyperexcitability [43,44]. On the other hand, it was also shown that the injection of the Aβ_25–35 fragments, which are able to mimic the toxicity of the parent, full-length Aβ_1–42, is sufficient to induce a significant upregulation of the K_V2.1 protein expression in the rat brain [45]. Since changes in the K_V2.1 function could be critical for the homeostatic regulation of neuronal excitability, in the present study we investigated any possible modulation of the K_V2.1 protein expression and activity in the Tg2576 mouse model of AD, which expresses a human APP gene carrying the double Swedish mutation (KM670/671NL) and produces elevated levels of Aβ_1–42 [46]. To this aim, we performed (1) electrophysiological experiments on Wild-Type (WT) and Tg2576 primary neurons; (2) Western blotting and immunofluorescence analyses on primary neuronal cultures from Tg2576 and WT mouse embryos; and (3) Western blotting and immunohistochemical analyses on the hippocampus and cerebral cortex of 3-month-old Tg2576 and WT littermates.

2. Materials and Methods

2.1. Animals

All the animals were handled according to the International Guidelines for Animal Research, and the experimental protocols were approved by the Animal Care and Use Committee of the “Federico II” University of Naples. The heterozygous male Tg2576 mice and WT females were purchased from a commercial source (B6; SJLTg(APPSWE) 2576 Kha, model 1349, Taconic Biosciences, Hudson, NY, USA).

2.2. Genotyping: PCR Analysis

Genomic DNA from embryonic brain tissues was isolated by salt precipitation as previously described [47]. Briefly, embryonic brain tissues were harvested during cerebral dissection and then thawed and homogenized with the TRI-reagent (Sigma-Aldrich, Milan, Italy). After adding one volume of chloroform to each sample, the DNA was precipitated with 100% ethanol and centrifuged at 4 °C for 5 min at 16,000× g. The DNA pellet was dried at room temperature and then re-suspended in Tris-EDTA buffer. The following primers were used to amplify the DNA region with the human APP Swedish mutation on both types of genomic DNA: 5′-CTGACCACTCGACCAGGTTCTGGGT-3′ and 5′-GTGGAT AACCCCTCCCCC AGCCTAGACCA-3′ (Eurofins Genomics, Ebersberg, Germany). The DNA was amplified to detect the transgenic genotype as previously described [13].

2.3. Primary Hippocampal Neurons

Primary neuronal cultures were prepared from the hippocampi of embryonic day-15 WT and Tg2576 mice as described by Ciccone et al. [13].The cells were plated on 35 mm culture dishes coated with poly(D)-lysine hydrobromide with a molecular weight >300,000 (Sigma-Aldrich, Milan, Italy) or onto 25 mm glass coverslips (Glaswarenfabrik Karl Hecht KG, Sondheim, Germany), coated with 100 μg/mL poly(D)-lysine hydrobromide with a molecular weight of 30,000–70,000 (Sigma-Aldrich, Milan, Italy) at a density of one embryo hippocampus/1 mL. Cytosine β-D-arabinofuranoside (10 μM, Sigma-Aldrich, Milan, Italy) was added 3 days after plating to inhibit non-neuronal cell growth. The neurons were cultured at 37 °C in a humidified 5% CO₂ atmosphere. The experiments were performed between 10–12 days in vitro (DIV).

2.4. Western Blotting

Total lysates for the immunoblotting analyses were obtained as follows: the primary hippocampal neurons were washed in phosphate buffered saline (PBS) and collected by gentle scraping in ice-cold RIPA buffer containing (in mM): 50 Tris pH 7.4, 100 NaCl, 1 EGTA, 1 PMSF, 1 Na₃VO₄, 1 NaF, 0.5% NP-40, and 0.2% SDS, and supplemented with protease inhibitor cocktail II (Roche Diagnostic, Monza, Italy). The nitrocellulose membranes were incubated with the following antibodies: rabbit K_V2.1 antibody (1:1000, Alomone Labs, Jerusalem, Israel), mouse α-tubulin antibody (1:5000, Sigma-Aldrich, Milan, Italy), and anti-β-actin peroxidase (1:10,000, Sigma-Aldrich, Milan, Italy). The immunoreactive bands were detected with the chemiluminescence system (GE Healthcare, Milan, Italy). The films were developed with a standard photographic procedure, and the quantitative analysis of the bands detected was carried out by densitometric scanning.

2.5. Electrophysiological Recordings

K⁺ currents were recorded from WT and Tg2576 primary hippocampal neurons with the patch-clamp technique in whole-cell configuration using a commercially available amplifier Axopatch200B and Digidata1322A interface (Molecular Devices, San Jose, CA, USA), as previously described [48,49]. In most experiments, the whole-cell configuration of the patch-clamp technique was adopted using glass micropipettes of 2.5–5 MΩ resistance. Currents were filtered at 5 kHz and data were acquired and analyzed using the pClamp software (version 9.0, Molecular Devices, CA, USA). The pipette solution contained (in mM): 140 KCl, 2 MgCl₂, 10 HEPES, 10 glucose, 10 EGTA, and 1 Mg-ATP adjusted at pH 7.4 with KOH. The extracellular solution contained (in mM): 150 NaCl, 5.4 KCl, 3 CaCl₂, 1 MgCl₂, 10 HEPES, and adjusted pH 7.4 with NaOH. A total of 50 nM tetrodotoxin (TTX) and 10 μM nimodipine were added to Ringer’s solution to abolish TTX-sensitive Na⁺, and L-type Ca²⁺ current. Monoclonal K_V2.1 antibody (10 µg mL⁻¹; Neuromab, Davis, CA, USA) was added to the pipette solution to block K_V2.1 currents [22]. The total K⁺ currents (I_K) are composed of the fast-inactivating (I_A) currents and the delayed currents (I_DR). To discriminate K⁺ current components, I_K= I_A + I_DR, appropriate electrophysiological protocols were used [48,49]. In particular, I_DR were isolated by stepping from −80 mV to +40 mV for 250 ms after conditioning pulses at −40 mV, lasting 1.5 s to fully inactivate I_A. Current amplitudes were measured for I_DR at +40 mV at the end of the depolarizing pulse. The steady-state properties of I_DR activation were measured as previously described [49]. The current amplitudes recorded upon depolarizing steps from −100 mV to +40 mV (10 mV) were normalized to peak currents at +40 mV and then fitted to the following form of the Boltzmann equation: G/G_max = 1/(1 + exp[(V−V_1/2)/k]), where V is the pulse voltage, V_1/2 is the half-inactivation voltage, and k is the slope factor. Possible changes in cell size were taken into account by measuring, in each cell, the membrane capacitance, which is directly related to membrane surface area, and by expressing the current amplitude data as current densities (pA/pF). Capacitive currents were elicited by 5 mV depolarizing pulses from −80 mV and acquired at a sampling rate of 50 kHz. The capacitance of the membrane was calculated according to the following equation: C_m = τ_c · I_o/ΔE_m(1 − I_∞/I_o), where C_m is the membrane capacitance, τ_c is the time constant of the membrane capacitance, I_o is maximum capacitance current value, ΔE_m is the amplitude of the voltage step, and I_∞ is the amplitude of the steady state current.

2.6. Immunocytochemistry

Hippocampal neurons were washed twice in cold 0.01 M of PBS (pH 7.4) and fixed at room temperature in 4% of paraformaldehyde for 20 min. Following three washes in PBS, cells were blocked in PBS containing 3% of BSA for 30 min, and then incubated overnight at 4 °C with the rabbit anti-K_V2.1 antibody (1:1000, Alomone Labs, Jerusalem, Israel). The control for the specificity of the antibody against K_V2.1 was performed with its replacement with normal serum as previously described [50]. Then, cells were washed with PBS and incubated with anti-rabbit Cy2-conjugated antibody (1:200; Jackson Immuno Research Laboratories, Inc., West Grove, PA, USA) for 1 h at room temperature under dark conditions. Cells were finally incubated for 5 min with Hoechst 33342. Cover glasses were mounted with a SlowFade Antifade Kit (Molecular Probes, Life Technologies, Milan, Italy) and acquired by a 63× oil immersion objective using a Zeiss inverted 700 confocal microscope.

2.7. Immunohistochemistry

Confocal immunofluorescence procedures were performed as described previously [51,52]. In brief, WT and Tg2576 mice were euthanized at 3 months. Anesthesia was induced with 4% sevoflurane in a mixture of 60% N₂O and 36% O₂ and maintained during intracardiac perfusion with 2% sevoflurane in a mixture of 60% N₂O and 38% O₂. Transcardial perfusion was carried out with 4% paraformaldehyde in PBS. The brains were sectioned coronally (60 μm) on a vibratome. After blocking with Rodent M block (Biocare Medical, Concord, MA, USA), sections were incubated with the following primary antibodies: rabbit polyclonal anti-K_V1.2 (1:200) and mouse monoclonal anti-NeuN (1:1000, Sigma-Aldrich, Milan, Italy). Then, sections were incubated with corresponding fluorescence-labeled secondary antibodies (Alexa488- or Alexa594-conjugated anti-mouse or anti-rabbit IgGs). DAPI was used to stain the nuclei. Images were observed using a Zeiss LSM 700 laser (Carl Zeiss, Jena (Turingia), Germany) scanning confocal microscope. Single images were taken with an optical thickness of 0.7 μm and a resolution of 1024 × 1024.

2.8. Statistical Analyses

Data are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed with unpaired t-test or one-way analysis of variance followed by the Newman–Keuls test.

3. Results

3.1. The K_V2.1 Protein Expression Is Increased in the Hippocampus of 3-Month-Old Tg2576 Mice and in Tg2576 Primary Hippocampal Neurons

Starting from the previous report demonstrating that the Aβ_25–35 fragments, which are able to mimic the full-length Aβ_1–42, induced the overexpression of K_V2.1 in the rat brain [45], we tested the hypothesis that the K_V2.1 protein expression could be modulated in the Tg2576 mouse, which overproduces the Aβ_1–42 peptide. To this aim, we performed Western blotting and immunohistochemical analyses on the hippocampus of 3-month-old Tg2576 mice, which start to display alterations in neuronal excitability and early cognitive deficits by this age. The densitometric analysis of Western blotting revealed that the hippocampus of 3-month-old Tg2576 mice displayed higher levels of K_V2.1 protein expression than the WT littermates (Figure 1A). Accordingly, confocal double immunofluorescence analyses showed that the CA1 pyramidal neurons of the Tg2576 mice exhibited a more pronounced K_V2.1 immunofluorescence in comparison with the WT littermates (Figure 1B). In line with previous evidence [22,40,53], the double confocal images also revealed the typical clustered K_V2.1 immunostaining on the PM of the hippocampal CA1 pyramidal neurons of both the WT and Tg2576 mice. Based on the assumption that primary neuronal cultures from Tg2576 mouse embryos, which accumulate Aβ_1–42 with the time in culture, recapitulate some of the main features of the Aβ_1–42 neurotoxicity [54], we also performed Western blotting experiments on hippocampal neurons cultured from Tg2576 and WT mouse embryos. Interestingly, we found that the Tg2576 primary hippocampal neurons also exhibited an increase in the K_V2.1 protein expression in comparison with the WT neurons (Figure 1C).

3.2. Tg2576 Primary Neurons Display Decreased K_V2.1-Dependent I_DR Density

To test the hypothesis that the K_V2.1 overexpression could be accompanied by any alterations in the channel activity, we recorded I_DR in pyramidal neurons cultured from Tg2576 and WT mouse embryos by means of the whole-cell patch-clamp technique. In order to electrophysiologically isolate the I_DR conductance from the total K⁺ currents, we used a previously established protocol [48]. In particular, to record the I_DR component, from a holding potential of −80 mV we applied a conditioning pulse at −40 mV lasting 1.5 s to fully inactivate the fast-inactivating current (I_A) component, followed by 250 ms steps from −80 to +40 mV (Figure 2A, bottom). Of note, the Tg2576 pyramidal neurons displayed a significantly lower I_DR density than the WT neurons (Figure 2A–C). Moreover, the analysis of the activation properties of the I_DR revealed a −10 mV hyperpolarizing shift of the steady-state activation curve in the Tg2576 pyramidal neurons compared with the WT neurons (Figure 2D).

To investigate whether the reduction of the I_DR in the Tg2576 neurons was actually due to the downregulation of the K_V2.1 currents, we assessed the contribution of the K_V2.1 component to both the Tg2576 and WT neuronal I_DR by recording these currents upon the intracellular diffusion of the anti-K_V2.1 monoclonal antibody (10 µg mL⁻¹) through the recording pipette. Figure 3A shows a representative time-course of the effect of the anti-K_V2.1 antibody in the WT and Tg2576 neurons. As previously reported [22], the diffusion of the anti-K_V2.1 monoclonal antibody into the cells drastically reduced the I_DR in the WT neurons (Figure 3A,B), hence indicating that the K_V2.1 currents were the major components of the I_DR in these neurons. By contrast, the anti-K_V2.1 antibody produced a markedly lower effect in reducing the I_DR at 6 min in the Tg2576 neurons than in the WT neurons (Figure 3A–C), hence suggesting that the K_V2.1 current component in the Tg2576 neurons was lower than in the WT neurons. The subtraction of the antibody-resistant currents at 6 min from the initial current illustrated in Figure 3C clearly represents this result. The representative traces of the I_DR and relative current–voltage relationship recorded by 6 min, without (top) and with (bottom) the anti-K_V2.1 monoclonal antibody in the recording pipette, are illustrated in Figure 3D and 3E.

In agreement with the major contribution of the K_V2.1 component to the I_DR in WT neurons, we found that the steady-state parameters of the I_DR activation recorded in the WT neurons upon the intracellular diffusion of the anti-K_V2.1 antibody were significantly different from those recorded in the WT neurons in the absence of the antibody (Figure 3F). This data likely suggested that upon the blockade of the K_V2.1 currents by the anti-K_V2.1 antibody, the contribution of non-K_V2.1 components characterized by different activation properties were unmasked. Interestingly, the left-shift of the voltage dependence of the steady-state activation curve of the I_DR observed in the WT neurons upon the K_V2.1 blockade was similar to that observed in the Tg2576 neurons (Figure 3F), thereby suggesting that a loss of the K_V2.1 currents might underlie the hyperpolarizing shift of the steady-state activation curve observed in the Tg2576 neurons.

Figure 3. Effect of the intracellular diffusion of the anti-K_V2.1 monoclonal antibody on I_DR recorded in the WT and Tg2576 primary hippocampal neurons. (A) Time-dependent inhibitory effect of the anti-K_V2.1 monoclonal antibody (10 µg mL⁻¹) on I_DR recorded in WT and Tg2576 primary hippocampal pyramidal neurons. (B) Quantification of I_DR densities, at +40 mV, before (Control) and after 6 min of the anti-K_V2.1 intracellular diffusion (+K_V2.1Ab). Data are expressed as percentage of WT and Tg2576 internal controls. Values are expressed as mean ± SEM of 3 independent experimental sessions. * p < 0.001 vs. internal controls. (C) Superimposed representative traces of I_DR at +40 mV recorded before and after 6 min of intracellular diffusion of the anti-K_V2.1 antibody (top), and representative traces resulting from the subtraction of the currents resistant to the anti-K_V2.1 antibody from the initial currents (bottom) in WT (left) and Tg2576 (right) neurons. (D) Representative traces of I_DR recorded in control conditions and upon 6 min of intracellular diffusion of the anti-K_V2.1 monoclonal antibody (10 µg mL⁻¹) in WT (left) and Tg2576 (right) primary hippocampal pyramidal neurons. Protocols are shown in the lower part of the panel. (E) I/V relationships for the I_DR recorded in control conditions and upon 6 min of intracellular diffusion of the anti-K_V2.1 antibody in the WT (left) and Tg2576 primary hippocampal neurons (right). (F) Steady-state activation curves of I_DR recorded in WT and Tg2576 hippocampal neurons in control conditions and in WT neurons upon 6 min of intracellular diffusion of the anti-K_V2.1 antibody. Values are expressed as mean ± SEM of 3 independent experimental sessions. * p < 0.001 vs. WT.

Figure 3. Effect of the intracellular diffusion of the anti-K_V2.1 monoclonal antibody on I_DR recorded in the WT and Tg2576 primary hippocampal neurons. (A) Time-dependent inhibitory effect of the anti-K_V2.1 monoclonal antibody (10 µg mL⁻¹) on I_DR recorded in WT and Tg2576 primary hippocampal pyramidal neurons. (B) Quantification of I_DR densities, at +40 mV, before (Control) and after 6 min of the anti-K_V2.1 intracellular diffusion (+K_V2.1Ab). Data are expressed as percentage of WT and Tg2576 internal controls. Values are expressed as mean ± SEM of 3 independent experimental sessions. * p < 0.001 vs. internal controls. (C) Superimposed representative traces of I_DR at +40 mV recorded before and after 6 min of intracellular diffusion of the anti-K_V2.1 antibody (top), and representative traces resulting from the subtraction of the currents resistant to the anti-K_V2.1 antibody from the initial currents (bottom) in WT (left) and Tg2576 (right) neurons. (D) Representative traces of I_DR recorded in control conditions and upon 6 min of intracellular diffusion of the anti-K_V2.1 monoclonal antibody (10 µg mL⁻¹) in WT (left) and Tg2576 (right) primary hippocampal pyramidal neurons. Protocols are shown in the lower part of the panel. (E) I/V relationships for the I_DR recorded in control conditions and upon 6 min of intracellular diffusion of the anti-K_V2.1 antibody in the WT (left) and Tg2576 primary hippocampal neurons (right). (F) Steady-state activation curves of I_DR recorded in WT and Tg2576 hippocampal neurons in control conditions and in WT neurons upon 6 min of intracellular diffusion of the anti-K_V2.1 antibody. Values are expressed as mean ± SEM of 3 independent experimental sessions. * p < 0.001 vs. WT.

3.3. Glutamate Recovers the Reduction of the K_V2.1-Mediated I_DR Caused by Increased Channel Clustering

As mentioned above, the majority of the K_V2.1 channels expressed in pyramidal neurons reside within high-density cell surface clusters. Clustered channels are held in a non-conducting state, while non-clustered channels, which are spread diffusely over the cell surface, mediate the majority of the whole I_DR. Based on this aspect, we hypothesized that the reduction of the K_V2.1-mediated I_DR in the Tg2576 neurons could be due to an increase in channel clustering. Further prompted by the evidence that the overexpression of K_V2.1 results in increased channel clustering [55], we performed confocal experiments to assess possible changes in channel clustering in the Tg2576 neurons. To this aim, both the Tg2576 and WT hippocampal neurons were fixed and stained with the anti-K_V2.1 rabbit polyclonal antibody (1:1000, Alomone Labs, Jerusalem, Israel) and subjected to imaging on a confocal microscope. Interestingly, if compared with the WT neurons, the Tg2576 neurons displayed an evident increase in channel clustering. In particular, while microclusters of K_V2.1 channels were present in both the WT and Tg2576 neurons, markedly larger clusters of K_V2.1, usually defined as “macroclusters”, were clearly detectable only in the Tg2576 neurons (Figure 4A).

To functionally investigate whether the increase of channel clustering was actually responsible for the reduction of the K_V2.1 currents previously observed in the Tg2576 neurons, we tested the effect of a declustering stimulus on the K_V2.1 currents in both the WT and Tg2576 neuronal cultures. In particular, based on the evidence provided by the Trimmer group that glutamate is able to induce a calcineurin-dependent K_V2.1 dephosphorylation and the subsequent dispersal of channel clusters [40], we exposed both the WT and Tg2576 neurons to glutamate (10 μM, 10 min). In line with the previous results provided by Mohapatra and coworkers [33], we found that the exposure to 10 μM glutamate for 10 min significantly increased the I_DR in the WT neurons (Figure 4B–E). Notably, glutamate exerted a stimulatory effect on the I_DR also in the Tg2576 neurons, restoring the I_DR density at 10 min to levels similar to those recorded in the WT neurons (Figure 4B–E). Consistent with the higher levels of K_V2.1 channels expressed in the Tg2576 neurons and become available for conduction upon the glutamate-induced declustering, the stimulatory effect of glutamate on the I_DR seemed to be greater in the Tg2576 neurons than in the WT neurons (Figure 4B,C). Interestingly, we also found that the steady-state parameters of the I_DR activation in the Tg2576 neurons upon glutamate exposure were significantly different if compared with those in the Tg2576 neurons under control conditions (Figure 4F). Of note, the shift in the voltage-dependence of activation induced by glutamate restored the steady-state activation curve to values comparable to those of the WT neurons (Figure 4F).

To exclude the possibility that the stimulatory effect of glutamate on the I_DR could be unrelated to K_V2.1 declustering, we exposed both the WT and Tg2576 neurons to glutamate (10 μM, 10 min) in the presence of the anti-K_V2.1 antibody in the recording pipette. Remarkably, when K_V2.1 was blocked by the intracellular diffusion of the anti-K_V2.1 antibody, the glutamate-induced upregulation of the I_DR was not observed in the WT or in the Tg2576 neurons (Figure 5A,B). This result clearly shows that the upregulation of the I_DR induced by glutamate was mediated by the specific upregulation of the K_V2.1 currents resulting from the glutamate-induced channel declustering.

3.4. K_V2.1 Clustering Is Increased in Cortical Layer V Pyramidal Neurons of the Somatosensory Cortex of 3-Month-Old Tg2576 Mice

To investigate whether K_V2.1 clustering was increased in the Tg2576 model also in vivo, we performed confocal double immunofluorescence analyses on the somatosensory cortex of both 3-month-old WT and Tg2576 mice, where the K_V2.1 protein expression and function have been extensively characterized [23,56]. Interestingly, the confocal double immunofluorescence analysis revealed that the anti-K_V2.1 antibody depicted a clear and pronounced clustered PM labeling of NeuN-positive neurons in layer V of the somatosensory cortex of both 3-month-old WT and Tg2576 mice (Figure 6A). More important, quantitative analyses showed a significantly higher number of cortical neurons displaying K_V2.1 clustering in the layer V of the Tg2576 mice in comparison with the WT littermates (Figure 6B).

4. Discussion

In the present study, we demonstrated that K_V2.1 protein expression was significantly increased in primary neurons from the Tg2576 mouse, which, carrying the human Swedish double mutation of the APP, accumulates the Aβ_1–42 peptide overtime in culture [54,57]. However, as a consequence of the increased channel clustering, the K_V2.1-mediated currents were markedly reduced in the Tg2576 neurons, resulting in a decrease in the whole I_DR along with clear changes in their voltage-dependence of activation. Moreover, we showed that the K_V2.1 expression and clustering levels were modulated also in vivo in Tg2576 mice.

First, Western blotting analyses on the hippocampal lysates from 3-month-old Tg2576 mice revealed a significant increase in the K_V2.1 protein levels in comparison with the WT hippocampal lysates, hence providing the first evidence, to our knowledge, that the K_V2.1 protein expression is modulated in a transgenic in vivo model of AD. In agreement, immunohistochemical analyses on WT and Tg2576 brain sections showed a more pronounced K_V2.1 immunostaining in the CA1 pyramidal neurons of the Tg2576 mice in comparison with the WT littermates. Of note, Tg2576 mice, a widely used transgenic model of the AD pathology, start by 3 months of age to intracellularly accumulate Aβ_1–42 trimers, which have been implicated in the appearance of precocious cognitive deficits and high seizure susceptibility [58,59,60]. In line with the previous evidence demonstrating that the neuronal cultures from Tg2576 mouse embryos recapitulate some of the main features of the Aβ_1–42 neurotoxicity [54], we found that the K_V2.1 protein expression was also increased in the hippocampal neurons cultured from Tg2576 embryos.

Based on the evidence that K_V2.1 is the main channel α subunit mediating I_DR in hippocampal neurons [16], we investigated whether its overexpression could affect the whole I_DR density in the Tg2576 primary hippocampal neurons. Consistent with the aforementioned literature, our electrophysiological experiments revealed that K_V2.1 was the main channel α subunit mediating I_DR in the WT pyramidal neurons, since the intracellular diffusion of the anti-K_V2.1 monoclonal antibody induced a significant reduction of the I_DR in these neurons. Interestingly, we noticed that the I_DR density was significantly reduced in the Tg2576 neurons compared with the WT neurons. More important, the intracellular diffusion of the anti-K_V2.1 antibody produced a markedly lower effect in promoting the decrease of the I_DR density in the Tg2576 neurons than in the WT neurons, hence suggesting that the percentage of the conducting K_V2.1 channels in the Tg2576 neurons was reduced or functionally compromised. In addition, the analysis of the voltage-dependence of activation revealed significant differences in the steady-state parameters of the I_DR activation in the Tg2576 neurons compared with the WT neurons. Interestingly, the shift of the voltage-dependence of activation to more negative values observed in the Tg2576 neurons was similar to that produced by the intracellular diffusion of the anti-K_V2.1 antibody in the WT neurons. This observation suggested that the left-shift in the voltage-dependence of activation occurring in the Tg2576 neurons could reflect a lower contribution of the K_V2.1 current component to the whole I_DR.

Although apparently divergent from the higher expression levels observed in the Tg2576 neurons, our functional results are in line with previous evidence clearly indicating that the K_V2.1 channel conductance decreases as the expression density on the cell surface membrane increases. Indeed, the Tamkun group provided solid data demonstrating that the percentage of non-conducting K_V2.1 channels strictly depends on their expression levels and consequent channel density, whereas it appeared to be independent on cluster localization per se [61]. On the other hand, it has been extensively reported that the vast majority of the K_V2.1 channels residing within clusters are held in a non-conducting state [20,41,42], in agreement with the fact that the high concentration of channels within a cluster results in a 5-fold to 10-fold greater density in this restricted space than outside [61]. Prompted by the evidence that the overexpression of K_V2.1 promotes its clusterization, probably by inducing the fusion of smaller clusters to form macroclusters [55], we also hypothesized that the lower K_V2.1-mediated I_DR density observed in the Tg2576 neurons could be due to an increase in channel clustering. In confirmation of this hypothesis, immunocytochemical analyses showed a marked change in the K_V2.1 clustering in the Tg2576 pyramidal neurons. In particular, the K_V2.1 immunostaining revealed that, along with the typical clustered expression detected in both the WT and Tg2576 neurons, a massive presence of macroclusters was detectable only in the Tg2576 neurons.

While the causal relationship between the K_V2.1 overexpression and its clustering has not been fully defined, it was instead clearly demonstrated that K_V2.1 channel clustering strictly depends on its phosphorylation state. Indeed, it was reported that the phosphorylation of specific residues of K_V2.1, identified in mutagenesis studies, drastically affects both the clustered localization and the voltage-dependent gating of the channel [32,34,35,40,62]. Intriguingly, the phosphorylation of the proximal restriction and clustering domain is propaedeutic to the binding of K_V2.1 to the endoplasmic reticulum (ER)-resident VAPA and VAPB (vesicle-associated membrane and protein-associated protein A and B) and the subsequent formation of specialized ER–PM junctions. The function of the K_V2.1-induced ER–PM junctions appeared to be unrelated to K⁺ conductance, hence providing an explanation for the non-conducting state of the K_V2.1 channels residing within clusters [20,42,63,64,65]. Physiologically, K_V2.1 is highly phosphorylated in resting neurons and undergoes a neuronal activity-induced dephosphorylation through a Ca²⁺/calcineurin-dependent mechanism [32,40]. Consistently, stimuli that increase neuronal activity and/or cytoplasmic Ca²⁺ levels induce the dispersal of K_V2.1 clusters and enhance the voltage-sensitivity [32,35,40].

Therefore, we tested the possibility that a declustering stimulus could restore the K_V2.1 currents in the Tg2576 neurons. In particular, based on the results provided by the Trimmer group demonstrating that glutamate stimulation rapidly induces a calcineurin-dependent dephosphorylation of K_V2.1 and its subsequent translocation from clusters to a more diffuse localization [40,63], we measured the I_DR in the Tg2576 neurons upon glutamate application. Interestingly, we found that a brief application of 10 µM glutamate was actually able to increase the I_DR in the Tg2576 neurons. In line with the results provided by the Trimmer group [33,35,66], the same treatment induced an increase in I_DR also in the WT neurons, where the majority of K_V2.1 channels are physiologically clustered. However, the increase of the I_DR induced by glutamate exposure in the Tg2576 neurons was greater than that observed in the WT neurons, arguably due to the larger amount of clustered K_V2.1 channels present in the Tg2576 neurons and become available for conduction upon glutamate-induced declustering. In this regard, it is worth noting that, unlike the results provided by Misonou and co-workers, a study by the Tamkun group showed that the K_V2.1 declustering induced by swinolide A or alkaline phosphatase resulted in no change in current density or voltage-dependence of the channel [41]. Nonetheless, the same authors stated that it cannot be ruled out that distinct declustering stimuli may have different results or that the cellular model they used in their study, namely HEK cells, does not completely mimic a neuronal model.

To exclude any possible non-specific effects of glutamate on I_DR, we recorded the I_DR in both the WT and Tg2576 neurons upon glutamate exposure in the presence of the anti-K_V2.1 antibody in the recording pipette. Importantly, when blocking K_V2.1 with the anti-K_V2.1 antibody, the glutamate-induced upregulation of the I_DR was not observed in the WT or in the Tg2576 neurons, hence indicating that the positive effect of glutamate on the I_DR density was related to K_V2.1 declustering. Interestingly, glutamate application also produced a significant depolarizing shift in the voltage-dependence of activation in the Tg2576 neurons. Of note, this depolarizing shift restored the conductance-voltage relationship to values near to those of the WT neurons. In this respect, it should be noted that molecular studies by the Trimmer group clearly demonstrated that the calcineurin-dependent dephosphorylation of K_V2.1 channels yields hyperpolarizing shifts in their voltage-dependent activation, which seems to be not in line with our results [34,35,40]. However, it should be considered that, while the treatment with glutamate made the K_V2.1 current component newly available, thus significantly increasing its contribution to the whole I_DR, the I_DR registered in the Tg2576 neurons in control conditions had to arguably be attributed to non-K_V2.1 components with different activation properties. In agreement, we observed that the blockade of K_V2.1 in the WT neurons produced a left shift in the steady-state I_DR activation curve, likely suggesting that the loss of the K_V2.1 current component brought out a major contribution of non-K_V2.1 components to the voltage-dependence of activation. Thereby, it is conceivable that the functional effect of K_V2.1 modulation by glutamate on the I_DR activation properties could reflect the restoring of the K_V2.1 current contribution to the whole I_DR in the Tg2576 neurons, with no change in the kinetic properties of the channel. In this respect, it is worth noting that this is the first report, to our knowledge, on the effect of glutamate on the K_V2.1-mediated I_DR in a cellular model where the K_V2.1 channels are both overexpressed and more clustered.

Of note, we have previously demonstrated that primary hippocampal neurons from the Tg2576 mouse display increased excitability if compared with WT neurons [13]. In the present study, we found that the overexpression of K_V2.1 and its increased clusterization occurred in Tg2576 pyramidal neurons both in vitro and in vivo. Our in vitro experiments also revealed that the increased K_V2.1 channel clustering caused a significant reduction of the K_V2.1-mediated currents and considerable changes in the voltage-dependence of activation of the I_DR in Tg2576 neurons. Therefore, it is likely that similar functional changes may occur in the Tg2576 pyramidal neurons in vivo. More importantly, a downregulation of the K_V2.1-mediated currents might be expected to have a crucial impact on hippocampal and cortical excitability in the Tg2576 mouse. In this regard, a previous study by Frazzini and colleagues [43] revealed that the reduction in the K_V2.1 current density due to channel oxidation was associated with the spontaneous hyperexcitability observed in the neurons from the 3xTg-AD mouse, a transgenic model characterized by elevated levels of oxidative stress in the early phases of the pathology [67]. In particular, they observed increased [Ca²⁺]_i spike frequency in the 3xTg-AD neurons, where the K_V2.1-mediated currents were downregulated, and obtained similar results in the non-transgenic neurons upon the pharmacological blockade of K_V2.1 [43].

Tg2576 mice have been reported to display an ectopic expression of the Neuropeptide Y, a marker of chronic seizures, and an aberrant network activity even before the onset of memory deficits [60,68,69,70], hence emerging as an animal model particularly suitable to study neuronal hyperactivity in association with Aβ_1–42 accumulation. However, I_DR had not been yet investigated in this transgenic model. Nonetheless, in recent studies, the D’Amelio group reported that pyramidal neurons of both dorsal and ventral CA1 of the Tg2576 mouse displayed altered firing and synaptic transmission in the pre-plaque stages of AD [59,71,72]. Intriguingly, based on the results of computational models, they suggested that the persistent perturbation of the I_DR conductance and kinetic properties could be a critical determinant of the firing alterations observed in the Tg2576 ventral CA1 neurons [72].

Finally, considered the non-conducting and structural functions of K_V2.1, further implications unrelated to neuronal excitability could arise from its overexpression in AD. Indeed, immediately facing astrocytic processes and lying on neuronal cell membranes over the ER subsurface cisternae [73], the K_V2.1 clusters play a key role in inducing ER-PM junctions and provide specialized molecular platforms to regulate both somatodendritic Ca²⁺ signaling and ion channel trafficking. In particular, it has recently been shown that K_V2.1 clustering at neuronal ER-PM junctions promotes the clustering of PM L-type Ca²⁺ channels and their coupling to ryanodine receptor ER Ca²⁺ release channels [74], hence allowing the generation of localized Ca²⁺ release events. Moreover, the fact that K_V2.1 clusters induce the formation of -and localize at- ER-PM junctions confers to K_V2.1 the ability not only to trigger a structural remodeling of the ER but also to affect neuronal Ca²⁺ handling by modulating ER Ca²⁺ uptake [75]. Of note, we have recently reported about the occurrence of an ER Ca²⁺ remodeling in the Tg2576 primary neurons. In particular, we showed that the enhanced activity of the Na⁺/Ca²⁺ exchanger 3 promotes an increased ER Ca²⁺ refilling in Tg2576 neurons in comparison with WT neurons [47]. Moreover, we demonstrated that the I_A-mediating K_V3.4 channel subunit, which is overexpressed and functionally upregulated in Tg2576 reactive astrocytes, regulates astrocytic [Ca²⁺]_i transients and ER Ca²⁺ levels [52,76]. The findings reported in the present study bring further information about the ionic dysregulation occurring in the Tg2576 mouse brain, hence suggesting new pathways to explore in studying the alterations of Ca²⁺ signaling and homeostasis in AD and the involvement of potassium channels in these processes.

5. Conclusions

Undoubtedly, the involvement of I_DR, and in particular of the K_V2.1 currents, as well as of many other ionic mechanisms, in the AD-related alterations of neuronal excitability deserves much attention. In fact, how AD brains develop hyperexcitability is still largely unknown. Changes in the excitatory/inhibitory balance [77,78,79] and the increase in intrinsic membrane excitability properties [80] have been suggested as possible explanations, with the Aβ peptide proposed as the main determining factor. In this context, the Aβ-induced alteration of protein expression and function of different K⁺ channels could represent a crucial mechanism promoting hyperexcitability and altered neuronal activity. Considering the pivotal role of the K_V2.1-mediated I_DR in regulating the intrinsic excitability of pyramidal neurons, further studies should be performed to address this issue in AD models. In addition, since the alteration of cytosolic and organellar Ca²⁺ homeostasis is considered a critical event in the AD pathogenesis [81], the impact of the K_V2.1 modulation on these processes should be further investigated in AD models.