Restoring neuronal chloride extrusion reverses cognitive decline linked to Alzheimer's disease mutations

Abstract

Disinhibition during early stages of Alzheimer's disease is postulated to cause network dysfunction and hyperexcitability leading to cognitive deficits. However, the underlying molecular mechanism remains unknown. Here we show that, in mouse lines carrying Alzheimer's disease-related mutations, a loss of neuronal membrane potassium-chloride cotransporter KCC2, responsible for maintaining the robustness of GABAA-mediated inhibition, occurs pre-symptomatically in the hippocampus and prefrontal cortex. KCC2 downregulation was inversely correlated with the age-dependent increase in amyloid-β 42 (Aβ42). Acute administration of Aβ42 caused a downregulation of membrane KCC2. Loss of KCC2 resulted in impaired chloride homeostasis. Preventing the decrease in KCC2 using long term treatment with CLP290 protected against deterioration of learning and cortical hyperactivity. In addition, restoring KCC2, using short term CLP290 treatment, following the transporter reduction effectively reversed spatial memory deficits and social dysfunction, linking chloride dysregulation with Alzheimer's disease-related cognitive decline. These results reveal KCC2 hypofunction as a viable target for treatment of Alzheimer's disease-related cognitive decline; they confirm target engagement, where the therapeutic intervention takes place, and its effectiveness.

Keywords: App NL-G-F/NL-G-F; 5xFAD; Alzheimer’s disease; KCC2; chloride homeostasis; inhibition.

Figure 1

Changes in global and membrane KCC2 expression in the mPFC and hippocampal CA1 regions of 5xFAD and App^NL-G-F mice. (A and B) Confocal images showing KCC2 immunostaining in medial prefrontal cortex (mPFC) LII/III (A) and the CA1 (B) of 5xFAD versus non-transgenic (NonTg) mice (KCC2 in green, DAPI in blue). Scale bar = 50 μm. Insets show KCC2-expressing neurons from ROIs. Scale bar = 10 μm. On the right, examples of KCC2 subcellular profile intensities. The colour-coded maps illustrate the distance from the membrane. The graphs represent the KCC2 intensity as a function of the distance to the membrane (scale bars = vertical, 1000 intensity units; horizontal, 1 μm). (C and E) Global index KCC2 analysis of the mean KCC2 pixel intensity in mPFC LII/III of 2, 4 and 6-month-old 5xFAD versus NonTg mice (C) and the CA1 of 4 and 6-month-old 5xFAD versus NonTg mice (E). (D) Mean KCC2 intensity profiles across the plasma membrane of individually identified 5xFAD (blue) and NonTg (grey) neurons in the mPFC (5xFAD: n₂ = 1163, n₄ = 1537, n₆ = 912 from N₂ = 12, N₄ = 15, N₆ = 10 mice, respectively; NonTg: n₂ = 932, n₄ = 1164, n₆ = 974 neurons from N₂ = 10, N₄ = 11, N₆ = 13 mice, respectively). (F) Similar to D but in the CA1 (5xFAD: n₄ = 625, n₆ = 608 from N₄ = 10 and N₆ = 10 mice, respectively; NonTg: n₄ = 360, n₆ = 523 from N₄ = 8 and N₆ = 7 mice, respectively). (G) Pixel intensity of KCC2 immunostaining in 9-month-old App^{NL-G-F/NL-G-F} versus App^w/w mice in the mPFC (left) and the CA1 (right). (H) Average KCC2 intensity profiles in App^{NL-G-F/NL-G-F} (cyan) versus App^w/w (orange) mice in the mPFC (left) and CA1 (right) (App^{NL-G-F/NL-G-F}: n_mPFC = 742 and n_CA1 = 746 neurons from N_mPFC = 10 and N_CA1 = 10 mice, respectively; App^w/w: n_mPFC = 880, n_CA1 = 594 neurons from N_mPFC = 9 and N_CA1 = 7 mice, respectively). (I) Scheme of the KCC2 membrane analysis of subcellular profile intensity (MASC-π). Circles in C, E and G represent single mice. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. ns = non-significant.

Figure 2

Aβ42 reduces membrane KCC2 in cultured neurons, and its levels are negatively correlated with KCC2 levels in 5xFAD mice. (A) The levels of soluble Aβ42 and Αβ40 in the frontal lobe of 2, 4 and 6-month-old 5xFAD (blue) and non-transgenic (NonTg) (grey) mice. The dashed lines represent the fitted linear regression equations of the Αβ42 or Αβ40 levels versus the age of the mice. (B) Total KCC2 protein levels, quantified with an ELISA, in the frontal lobe of 2, 4 and 6-month-old 5xFAD (blue) and NonTg (grey) mice. The dashed lines represent the fitted linear regression equations of the KCC2 levels versus age. (C) Pearson r correlation of Aβ42 and KCC2 levels in the frontal lobe of 6-month-old mice (dots represent individual mice). (D) Representative confocal images showing KCC2 (grey) and NeuN (red) immunostaining in cultured primary hippocampal neurons incubated with Aβ42 or scramble peptide (Scramble; scale bars = 10 μm). (E) Mean membrane KCC2 intensity of individual neurons (Scramble - CLP257: n = 27 neurons from two coverslips; Scramble + CLP257: n = 25 neurons from two coverslips; Αβ42 - CLP257: n = 56 neurons from four coverslips; Αβ42 + CLP257: n = 50 neurons from four coverslips). (F) Mean KCC2 intensity profiles across the plasma membrane of individually identified neurons. (G) The average area of neurons analysed for membrane KCC2 expression. The R² and P-values of the linear regressions are shown above the graphs. Whiskers in box plots show the 5–95 percentiles. Data in E, F and G are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ns = non-significant.

Figure 3

Impaired chloride transport in 5xFAD mice. (A and D) FLIM images of 5xFAD and NonTg CaMKIIa-positive neurons expressing SuperClomeleon in mPFC (A) and hippocampal (D) slices. Scale bars = 10 μm. (B) Timelapse recording of Cl⁻ accumulation in the cell body of selected NonTg (grey) and 5xFAD (blue) neurons from the ROI in A upon 15 mM KCl extracellular application (dashed line). Insets show examples of fitted photon-distribution histograms during the low KCl condition (left) and upon 15 mM KCl application (right). Scale bars = vertical, 50 photons; horizontal, 5 ns. (C) The mean lifetime of NonTg and 5xFAD neurons during the baseline condition (5 mM KCl) and upon 15 mM KCl application. The distribution of the mean rate of Cl⁻ changes measured in 4-month-old 5xFAD and NonTg mPFC slices. The inset presents the mean slope (±standard deviation, SD) measured in individual neurons from 5xFAD and NonTg mice. Scale bar = 0.1 ns/min. (5xFAD: n = 73 neurons from N = 10 mice; NonTg: n = 46 neurons from N = 7 mice). (E) Similar to B but for NonTg and 5xFAD CA1 neurons within ROIs shown in D. (F) Similar to C but for CA1 neurons from 6-month-old 5xFAD and NonTg mice. Scale bar = 0.1 ns/min. (5xFAD: n = 133 neurons from N = 6 mice; NonTg: n = 122 neurons from N = 5 mice). (G) Graphical illustration of the SuperClomeleon in vivo steady state imaging experimental set-up, and representative images of the mean intensity of YFP and Cerulean merged (left) and split for the areas selected in dashed squares (right) in the prefrontal cortex of 4-month-old 5xFAD and NonTg mice. Scale bars = 20 μm and 10 μm for the selected areas. (H) Images of the sum of the CFP and YFP fluorescence intensity of the selected areas in G and intensity profiles of CFP (blue) and YFP (green) for the lines in cyan. (I) The median FRET ratio of 5xFAD versus NonTg neurons (left; 5xFAD: n = 361 neurons from N = 5 mice; NonTg: n = 638 neurons from N = 5 mice) and the estimated mean depth of imaging (right; mean ± SEM). *P < 0.05; ***P < 0.001. ns = non-significant.

Figure 4

CLP290 enhances membrane KCC2 and restores chloride transport in 5xFAD mice. (A) Confocal images of medial prefrontal cortex (mPFC) slices stained for KCC2 from CLP290 versus vehicle treated 5xFAD mice (KCC2 in green, DAPI in blue). Scale bar = 50 μm. Insets show KCC2-stained neurons from ROIs. Scale bar = 10 μm. Colour-coded maps illustrate the distance from the membrane and graphs represent the subcellular KCC2 intensity profile. Scale bars = vertical, 1000 intensity units; horizontal, 2 μm. (B) On the left, the mean global index KCC2 pixel intensity. In the centre, the mean KCC2 intensity profiles across the plasma membrane of CLP290-treated (filled dark blue dots; n = 919 neurons from N = 9 mice) and vehicle-treated (blue circles; n = 985 neurons from N = 10 mice) 5xFAD mice. On the right, the mean area of neurons selected for the analysis of membrane KCC2 expression. Data are presented as mean ± SEM. (C) FLIM images of CaMKII-positive neurons expressing SuperClomeleon in the CA1 of vehicle- and CLP290-treated 5xFAD mice. Scale bars = 25 μm. (D) Timelapse recording of Cl⁻ accumulation in the cell body of selected vehicle-treated 5xFAD (blue circles) and CLP290-treated 5xFAD (dark blue dots) neurons from the ROI in C upon 15 mM KCl extracellular application (dashed line). Insets show examples of fitted photon-distribution histograms during the low KCl condition (left) and upon 15 mM KCl application (right). Scale bars = vertical, 100 photons; horizontal, 4.8 ns. (E) Distribution of the average rate of Cl⁻ changes (slope) measured in the CA1 of 6-month-old CLP290-treated 5xFAD mice and vehicle-treated 5xFAD and NonTg mice. Inset displays the mean slope (±SD) measured in individual neurons from CLP290-treated 5xFAD mice and vehicle-treated 5xFAD and NonTg mice. Scale bar = 0.1 ns/min. (NonTg + Veh: n = 1028 neurons from N = 6 mice; 5xFAD + Veh: n = 639 neurons from N = 4 mice; 5xFAD + CLP290: n = 675 neurons from N = 6 mice). *P < 0.05; **P < 0.01; ****P < 0.0001. ns = non-significant.

Figure 5

CLP290 augments spatial memory and social behaviour in 5xFAD mice. (A) The chronological order of behavioural testing for the three CLP290 and vehicle-treated experimental groups. (B) Learning curves measured by the path length during the acquisition phase of the Morris water task in non-transgenic (NonTg) vehicle-treated mice and CLP290- and vehicle-treated 5xFAD mice. (C) The average proximity to the platform's previous location during the Morris water task probe trial. (D) Heat maps indicating the average time spent in each spatial bin (bin size = 1 cm²) during the probe trial of the Morris water task. The black circle in the top right quadrant represents the platform's position during acquisition training. (E) The latency to explore the novel arm during the Y-maze spatial memory test. (F) The time spent in the social versus neutral chamber during the unconditioned social interaction test. (G) Heat maps representing the location of the animal's head during the social interaction test. (H) The swim path length during the acquisition phase of the Morris water task (left), and the average proximity to the platform's previous position during the probe trial test (right) of 9-month-old 5xFAD mice receiving long-term (100 mg/kg per day for 5 months) treatment with CLP290 versus vehicle. Inset shows the learning curve for the four first days of the acquisition phase (box-whisker plots with the 5–95 percentile; scale bars = vertical, 2 m; horizontal, 1 day). Circles or dots in C, E, F and H represent single mice. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01. ns = non-significant.

Figure 6

Long-term CLP290 treatment prevents cortical hyperactivity in 9-month-old 5xFAD mice. (A) Traces of local field potential (LFP) activity in retrosplenial cortex (RSC), both raw and filtered in the 60–120 Hz (fast gamma) frequency band and electromyography (EMG) activity from representative 5xFAD mice treated with either vehicle (top) or CLP290 (bottom) for 5 months. The traces depict activity during NREM sleep. (B) Left: LFP wavelet spectrograms of RSC activity during concatenated NREM sleep episodes from the same mice as in A. Right: spectrograms generated from the same data but scaled to highlight the difference in power in the fast gamma band. (C) The mean power of RSC activity in the slow gamma (30–60 Hz), fast gamma and high frequency (120–200 Hz) bands during NREM sleep in mice treated with either vehicle or CLP290 for 5 months, beginning at 4 months of age. LFP recordings from freely moving mice were conducted longitudinally at 6 and 9 months of age. For each figure, power is normalized to the mean of the vehicle-treated group at 6 months of age. (D) Same as C, but for LFP recordings from the hippocampus. Circles or dots in C and D represent single mice. Data are presented as mean ± SEM. ^#P < 0.05 for the interaction between age (6 versus 9 months) and drug treatment (vehicle versus CLP290). *P < 0.05; **P < 0.01.