Abstract
Altered neuronal excitability is emerging as an important feature in Alzheimer's disease (AD). Kv2.1 potassium channels are important modulators of neuronal excitability and synaptic activity. We investigated Kv2.1 currents and its relation to the intrinsic synaptic activity of hippocampal neurons from 3xTg-AD (triple transgenic mouse model of Alzheimer's disease) mice, a widely employed preclinical AD model. Synaptic activity was also investigated by analyzing spontaneous [Ca2+]i spikes. Compared with wild-type (Non-Tg (non-transgenic mouse model)) cultures, 3xTg-AD neurons showed enhanced spike frequency and decreased intensity. Compared with Non-Tg cultures, 3xTg-AD hippocampal neurons revealed reduced Kv2.1-dependent Ik current densities as well as normalized conductances. 3xTg-AD cultures also exhibited an overall decrease in the number of functional Kv2.1 channels. Immunofluorescence assay revealed an increase in Kv2.1 channel oligomerization, a condition associated with blockade of channel function. In Non-Tg neurons, pharmacological blockade of Kv2.1 channels reproduced the altered pattern found in the 3xTg-AD cultures. Moreover, compared with untreated sister cultures, pharmacological inhibition of Kv2.1 in 3xTg-AD neurons did not produce any significant modification in Ik current densities. Reactive oxygen species (ROS) promote Kv2.1 oligomerization, thereby acting as negative modulator of the channel activity. Glutamate receptor activation produced higher ROS levels in hippocampal 3xTg-AD cultures compared with Non-Tg neurons. Antioxidant treatment with N-Acetyl-Cysteine was found to rescue Kv2.1-dependent currents and decreased spontaneous hyperexcitability in 3xTg-AD neurons. Analogous results regarding spontaneous synaptic activity were observed in neuronal cultures treated with the antioxidant 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox). Our study indicates that AD-related mutations may promote enhanced ROS generation, oxidative-dependent oligomerization, and loss of function of Kv2.1 channels. These processes can be part on the increased neuronal excitability of these neurons. These steps may set a deleterious vicious circle that eventually helps to promote excitotoxic damage found in the AD brain.
Highlights
According to recent findings, the Alzheimer’s disease (AD)-related deregulation of brain activity encompasses the simultaneous presence of hyperactive and hypoactive neurons that reorganize their activity in functional clusters.[3]
3xTg-AD mice and hyperexcitability V Frazzini et al occur early on in the disease can promote unbalanced neuronal excitability and activity-dependent regulation as well as network instability and dysfunction. With this conceptual framework as reference, we studied the potential relationship occurring between changes in the levels of oxidative stress, alterations of Kv2.1-dependent K+ currents, and variations in neuronal intrinsic activity in hippocampal neurons obtained from 3xTg-AD mice
With real-time microfluorimetry, we evaluated spontaneous [Ca2+]i transients occurring in soma of 3xTg-AD or Non-Tg hippocampal neurons
Summary
3xTg-AD hippocampal neurons show increased [Ca2+]i spike frequency. Spontaneous activity is an important determinant of information processing in neuronal circuits and in the brain.[5,16,17,18] Neurons develop spontaneous rhythmic and bursting activity at the end of the first week in culture,[19,20] a property that has been exploited to investigate basic aspects of network functioning in vitro. Analysis of amplitudes of spontaneous [Ca2+]i spikes in GxTx-treated 3xTg-AD neurons revealed small but statistically significant increased values (average amplitude value: 119.5 ± 4.207 (S.E.M.) in 113 neurons from three cultures) compared with those obtained in the same neurons before treatment (average fluorescence value was 105.0 ± 3.380 (S.E.M.) from the same 113 neurons; Po0,01; Figure 6d). (d) Kv2.1 immunoblotting indicates no differences in channel expression in the two strains values: 2.189 ± 0.033 (S.E.M.) obtained in 159 neurons from 7 cultures; Figure 8a), thereby further substantiating a selective role of oxidative stress in the modulation of 3xTg-AD neuron activity. Compared with untreated 3xTg-AD neurons, Trolox-treated cultures showed a significant decrease in spiking activity (average spike frequency: 1.304 ± 0.8711 (S.E.M.) obtained in 74 neurons from three cultures; Po0.001; Figure 8a). Compared with untreated Non-Tg cultures, NAC- and Trolox-treated Non-Tg neurons showed decreased spike amplitudes (average fluorescence values in NAC-treated neurons: 105 ± 3.284 (S.E.M.) in 159 cells from seven cultures; and in Trolox-treated neurons: 66.32 ± 2.781 (S.E.M.) in 127 neurons from four cultures; Po0.001, Figure 8b)
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