Amyloid β-peptide directly induces spontaneous calcium transients, delayed intercellular calcium waves and gliosis in rat cortical astrocytes

Abstract

The contribution of astrocytes to the pathophysiology of AD (Alzheimer's disease) and the molecular and signalling mechanisms that potentially underlie them are still very poorly understood. However, there is mounting evidence that calcium dysregulation in astrocytes may be playing a key role. Intercellular calcium waves in astrocyte networks in vitro can be mechanically induced after Aβ (amyloid β-peptide) treatment, and spontaneously forming intercellular calcium waves have recently been shown in vivo in an APP (amyloid precursor protein)/PS1 (presenilin 1) Alzheimer's transgenic mouse model. However, spontaneous intercellular calcium transients and waves have not been observed in vitro in isolated astrocyte cultures in response to direct Aβ stimulation in the absence of potentially confounding signalling from other cell types. Here, we show that Aβ alone at relatively low concentrations is directly able to induce intracellular calcium transients and spontaneous intercellular calcium waves in isolated astrocytes in purified cultures, raising the possibility of a potential direct effect of Aβ exposure on astrocytes in vivo in the Alzheimer's brain. Waves did not occur immediately after Aβ treatment, but were delayed by many minutes before spontaneously forming, suggesting that intracellular signalling mechanisms required sufficient time to activate before intercellular effects at the network level become evident. Furthermore, the dynamics of intercellular calcium waves were heterogeneous, with distinct radial or longitudinal propagation orientations. Lastly, we also show that changes in the expression levels of the intermediate filament proteins GFAP (glial fibrillary acidic protein) and S100B are affected by Aβ-induced calcium changes differently, with GFAP being more dependent on calcium levels than S100B.

Figure 1. Calcium transient responses to Aβ-treated and control primary cortical astrocyte cultures for different experimental conditions

(A) The fraction of activated responding cells for the population of each experimental condition expressed as a percentage. (B) Average calcium transient response frequency per activated cell. (C) Representative examples of measured calcium transient responses in individual astrocytes for both Aβ-treated and untreated control conditions. Scale bar, 5 s. Thap, thapsigargin.

Figure 2. Representative examples of the propagation of Aβ-induced spontaneous intercellular calcium waves in astrocyte networks from two experiments

In most cases, waves propagated radially from an initiating focal locus (A, B). In some cases, however, waves travelled linearly in a specific direction across the field of view (C, D). (A, C) These figures show sequential frames from recorded movies as a function of time (with a time stamp in the lower right corner of each frame). Pseudo-colouring encodes the normalized relative concentration of the fluorescence signal displayed by the calcium indicator dye. (B, D) These figures are temporal projections for the propagating waves for the entire movies collapsed on to one frame overlaid on fields of view for each one. Pseudo-colouring in these figures encodes time in seconds. (E) A plot of the distance travelled by waves in microns against time in seconds for several representative experiments. Scale bars, 50 μm.

Figure 3. Changes in immunocytochemical labelling for GFAP (A, B) and S100B (C, D) intermediate filament proteins 12 h after dosing for different experimental conditions

(B, D) Shown are mean fluorescence values, with the error bars representing 95% confidence bounds in A.U. based on image analysis of the micrographs (see the Materials and methods section). Western blot gels were done to confirm protein expression for the different conditions but we did not optimize for or quantify protein levels using this method. Fluorescence data were acquired using the same exposure and gain setting in every case, and the mean fluorescence intensities of the cells were extracted as recorded on the camera for each image. The camera sensor recorded intensity values on a 12-bit scale in A.U., from 0 to 4095. The results shown are means±S.E.M. *P<0.001.