A vicious cycle of β amyloid-dependent neuronal hyperactivation

Abstract

β-amyloid (Aβ)-dependent neuronal hyperactivity is believed to contribute to the circuit dysfunction that characterizes the early stages of Alzheimer's disease (AD). Although experimental evidence in support of this hypothesis continues to accrue, the underlying pathological mechanisms are not well understood. In this experiment, we used mouse models of Aβ-amyloidosis to show that hyperactivation is initiated by the suppression of glutamate reuptake. Hyperactivity occurred in neurons with preexisting baseline activity, whereas inactive neurons were generally resistant to Aβ-mediated hyperactivation. Aβ-containing AD brain extracts and purified Aβ dimers were able to sustain this vicious cycle. Our findings suggest a cellular mechanism of Aβ-dependent neuronal dysfunction that can be active before plaque formation.

Fig. 1.. Activity-dependence of the Aβ-dependent neuronal hyperactivation

(A) Top: representative two-photon images of the hippocampal CA1 region of a wild-type mouse in vivo before (left), during the application of 500 nM [AβS26C]₂ (middle) and after 5–10 min of washout (right). The colored dots on the neurons indicate the number of Ca²⁺-transients per minute. Bottom: Ca²⁺-traces of the five neurons circled in the top panel. The grey shaded area indicates the time period of [AβS26C]₂ application. (B) Top: representative two-photon images of the hippocampal CA1 region of an acute slice preparation before (left), during [AβS26C]₂ application (middle) and after washout (right). Bottom: Ca²⁺-traces of the five neurons circled in the top panel. The grey shaded area indicates the period of [AβS26C]₂ application. (C) Same as (A) for a mouse in which glutamatergic transmission was blocked by bath application of D-APV (50 μM) and CNQX (50 μM). (D) Same as (B) for a slice treated with 80 μM bicuculline and an elevated potassium concentration (6.5 mM). The asterisks denote astrocytes. (E) Summary data of the in vivo experiments in (A) (left) and (C) (right). Each dot represents the mean under baseline (BL), [AβS26C]₂ application and washout (WO) conditions. (F). Same as (E) for experiments in (B) and (D). (G) Summary data of the in vitro experiments in which neuronal baseline activity was induced by the superfusion of glutamate (40–60 μM). (H) Summary data of in vitro experiments in which neuronal baseline activity was induced by elevating extracellular K⁺ (to 7.5–8.5 mM). (I) Plot of baseline activity (BL) vs. [AβS26C]₂-dependent relative increase in activity in vivo (DHyper) for individual neurons. The numbers for neurons for each bin of BL activity is indicated in the graph. Red line: linear fit. Scale bars: 5 μm. Error bars show SEM. Wilcoxon signed rank test, *P<0.05; n.s. not significant.

Fig. 2. [AβS26C]₂-dependent suppression of glutamate re-uptake.

(A) Same experimental arrangement as in Fig. 1A, but application of 250 μM DL-TBOA. (B) Summary data for the experiment in (A). Each dot represents the mean number of Ca²⁺-transients per minute for all neurons in one mouse under baseline (BL), TBOA application and washout (WO) conditions. (C) Bar graph showing the normalized number of Ca²⁺-transients. Each point represents the mean number of Ca²⁺-transients in one mouse during application of 500 nM [AβS26C]₂ (left, n = 7 mice), or TBOA (right, n = 7), normalized to baseline. (D and E) pie chart depicting the proportion of silent (blue), normal (white) and hyperactive (orange) neurons in wildtype (D) (n =275 cells from 7 mice) and APP23 x PS45 transgenic (TG) mice (E) (n = 299 cells from 6 mice). (F and G) Overlaid Ca²⁺-traces from all neurons in one wild type mouse (F) and one APP23 x PS45 mouse (G) for baseline (left), TBOA application (middle) and washout (right) conditions. The blue shaded area corresponds to the time of TBOA application. (H) Overlaid Ca²⁺-traces from all neurons (n = 20 cells) in one APP23 x PS45 mouse for baseline (left), [AβS26C]₂ application (middle) and washout (right) conditions. The grey shaded area indicates the period of [AβS26C]₂ application. (I) Bar graph of the normalized activity during the application of TBOA in wildtype (WT, left, solid bars, n = 7) or APP23 x PS45 transgenic (TG, right, open bars, n = 5) mice. Each point represents the mean number of Ca²⁺-transients in one mouse during the application of TBOA, normalized to baseline. (J) Same as (I) for the application of [AβS26C]₂ in WT (n = 6) or TG (n = 6) mice. Error bars show SEM. Wilcoxon signed rank test (D, E) or Wilcoxon rank sum test (F), **P<0.005, *P<0.05; n.s. not significant.

Fig. 3.. [AβS26C]₂-dependent potentiation of synaptic stimulation-evoked glutamate transients.

(A) Scheme of the injection of SF-iGluSnFr A184S into the mouse hippocampal CA1 region. (B) Confocal image of a hippocampal slice 21 days post-injection with SF-iGluSnFr (green). Cell bodies are stained with Neurotrace (blue). Scale bar: 100 μm. (C) Sparse labelling of the hippocampal CA1 neurons with SF-iGlu-SnFr. The dashed lines indicate the pyramidal layer (P) of the hippocampal CA1 region. O, stratum oriens; R, stratum radiale. Scale bar: 50 μm. (D) Individual (grey) mean (color) glutamate transients collected in a rectangular region of interest in the stratum radiatum (inset left) after electrical stimulation (arrow head, 100 μs/40V) before (left), during the application of 500 nM [AβS26C]₂ (middle) and after washout (right). The inset indicates the positions of the stimulation and the [AβS26C]₂-application pipettes, respectively. Scale bar: 50 μm. (E) Overlay of the average glutamate transients elicited by synaptic stimulation under baseline (black solid), [AβS26C]₂ application (500 nM, red) and washout conditions (black dashed). (F) Overlay of the average glutamate transients elicited by synaptic stimulation under baseline (black solid), TBOA application (10 μM, blue) and washout conditions (black dashed). (G) Box plot of the amplitude of the glutamate transient after the injection of ACSF (left), [AβS26C]₂ (middle) or TBOA (right). N-numbers are indicated next to the boxes. (H) Top: representative two-photon images of hippocampal CA1 in vivo under baseline conditions (left), during the application of anti-GLT-1 polyclonal antibody (AB, middle) and after washout (right). The colored dots on the neurons indicate the number of Ca²⁺-transients per minute. Bottom: Ca²⁺- traces of the five neurons circled in the top panel. The shaded area represents the time of AB application. Scale bar: 5 μm. (I) Summary data of the experiment in (H) for n = 6 mice. Error bars show SEM. Kruskal wallis test with Dunn-Sidak post-hoc comparison (G) or Wilcoxon signed rank test (I). **P<0.005, *P<0.05; n.s. not significant.

Fig. 4.. Aβ derived from human AD patients induces neuronal hyperactivation.

(A) AD brain extracts were immunoprecipitated with anti-Aβ polyclonal antibody AW7 or pre-immune serum (PI) and IP’s analyzed by immunoblot using a combination of 2G3 and 21F12. Molecular weight markers are indicated on the left. At least two different Aβ species are in AD brain extract: monomers (single arrow) and SDS-stable Aβ dimers (double arrow). Non-specific bands detected are indicated by a solid black line. (B) Mock immunodepleted (AD-ex) and AW7 immunodepleted (ID-ex) brain extracts were analyzed by an MSD-based Aβx-42 immunoassay. To assess the levels of monomeric and soluble aggregated Aβ, samples were pretreated with or without incubation in 5 M GuHCl. The AD extract contained much higher amounts of aggregates than monomer, and both were effectively removed by AW7 immunodepletion. (C) Top: representative two-photon images of hippocampal CA1 in a wild-type mouse in vivo under baseline conditions (left), during the application of AD-ex (diluted 1:10) and after washout (right). The colored dots on the neurons indicate the number of Ca²⁺-transients per minute. Bottom: Ca²⁺-traces of the five neurons circled in the top panel. The green shaded area represents the time of AD extract application. Scale bar: 5 μm. (D) Overlaid Ca²⁺-traces from 5 representative neurons recorded in vivo under baseline (left), AD-ex application (middle) and washout conditions (right). The green shaded are corresponds to the time of AD extract application. (E) Overlaid Ca²⁺-traces from 5 neurons recorded in vitro in a slice treated with bicuculline under baseline (left), AD-ex application (middle) and washout conditions (right). The green shaded are corresponds to the time of AD-ex application. (F) Summary data for the experiment in (D). Each dot represents the mean number of Ca²⁺-transients per minute for all neurons in one mouse under baseline (BL), AD-ex application and washout (WO) conditions. (G) Summary data for the experiment in (E). Error bars show SEM. Wilcoxon signed rank test. ** P<0.005, *P<0.05.

Fig. 5.. Role of human Aβ dimers and vicious cycle of hyperactivation.

(A) Top: representative two-photon images of hippocampal CA1 region of a wild-type mouse in vivo under baseline conditions (left), during the application of 200 ng/ml human Aβ dimer (hAβ-dim, middle) and after washout (right). The colored dots on the neurons indicate the number of Ca²⁺-transients per minute. Bottom: Ca²⁺-traces of the five neurons circled in the top panel. The grey shaded area represents the time of hAβ-dim application. Scale bar: 5 μm. (B) Summary data for the experiment in (A). Each dot represents the mean number of Ca²⁺-transients per minute for all neurons in one mouse under baseline (BL), hAβ-dim application and washout (WO) conditions. (C) Dose- dependency curve of the action of hAβ-dim. The activity during hAβ-dim application, normalized to baseline (hyper ratio), for different dilution steps of the human Aβ dimer for 5 ng/ml (n = 5), 20 ng/ml (n = 5), 50 ng/ml (n = 5) and 200 ng/ml (n = 6) are plotted. (D) Plot of baseline activity (BL) vs. human Aβ dimer-dependent relative increase in activity in vivo (DHyper) for individual neurons. The numbers of neurons for each bin of BL activity is indicated in the graph. Red line: linear fit. (E) Bar graph of the normalized activity during the application of 200ng/ml hAβ-dim (left, n = 6) or human Aβ monomer (hAβ-mon, right, n = 6). Each point represents the mean number of Ca²⁺-transients in one mouse during the application, normalized to baseline. Error bars show SEM. Wilcoxon signed rank test (B) or Wilcoxon rank sum test (D), **P<0.005, *P<0.05. (F) Scheme of the vicious cycle of Aβ-dependent neuronal hyperactivation.