Changes in Intracellular Calcium and Glutathione in Astrocytes as the Primary Mechanism of Amyloid Neurotoxicity

[Ca²⁺]_c responses to βA are dependent on extracellular Ca²⁺. A, In the absence of external Ca²⁺, cortical astrocytes showed no change in [Ca²⁺]_c after exposure to βA25–35 (50 μm). B, In a coculture exposed to βA25–35 (50 μm) in the absence of external Ca²⁺, no response was seen in either neurons or astrocytes.βA was then washed out and external Ca²⁺ added. Despite the removal of theβA, the addition of Ca²⁺ caused a large increase in [Ca²⁺]_c in the astrocytes but only a very small change in the neurons, reflecting the restoration of basal calcium entry. Once again, the neuronal identity was confirmed by the response to 100 μm glutamate at the end of the experiment.

Intracellular Ca²⁺ stores do not make a significant contribution to the astrocyte [Ca²⁺]_c response to βA. Manipulations that either block components of the IP₃ and ryanodine signaling pathways or that empty ER stores do not significantly alter the astrocyte responses to βA. The application of 50 μm βA25–35 caused [Ca²⁺]_c transients in cortical astrocytes despite the presence of 5 μm U73122 (an inhibitor of PLC; A), 40 μm 2-APB (B), dantrolene (an inhibitor of ryanodine receptors; C), and 1 μm thapsigargin (an inhibitor of ER Ca²⁺ pumps; D), at a concentration that prevented the response to ATP (100 μm).

Mn ²⁺ quench confirms that astrocyte [Ca²⁺]_c transients reflect transient Ca²⁺ influx. Fura-2-loaded hippocampal astrocytes showed typical [Ca²⁺]_c fluctuations (black line) in response to 50 μm βA25–35. A, In the absence of external Mn ²⁺, the fura-2 response excited at 360 nm (gray line) showed no change during the [Ca²⁺]_c transients, confirming that this is close to the isosbestic [Ca²⁺]_c-independent excitation wavelength for fura-2. Bi, Bii, With the addition of 40 μm Mn ²⁺ each [Ca²⁺]_c transient was accompanied by a step quench of the 360 nm fura-2 signal, confirming that each transient reflects a pulsed influx of divalent cations seen in response to βA25–35.

Confocal imaging reveals focal Ca²⁺ influx in response to βA. In a hippocampal coculture loaded with fluo-4, confocal imaging during the exposure to βA shows that the change in [Ca²⁺]_c can originate as a focal change that diffuses through the cell and may be restricted to the subplasmalemmal space. Aa, Time series of confocal images taken during a single [Ca²⁺]_c transient response in an astrocyte. Note that the response begins with a focal rise in [Ca²⁺]_c (arrowhead) followed by the slower spread through the cell. This is illustrated further in Ab, which shows a plot of the signal with time at four different locations in the cell (indicated color-coded on the inset image). The rapid rate of rise at the point of influx contrasts with the much slower increase seen deep in the cytosol of the cell. B, Series of images taken from another astrocyte during a response to βA25–35, again showing that the [Ca²⁺]_c signal may be restricted to the periphery of the cell and fail to propagate through the cell. The first image of the sequence shows the raw data, where as the subsequent images show the ratio of the image sequence with respect to the first image of the sequence.

Responses to βA are blocked by Zn ²⁺ and clioquinol but not by Cu ²⁺. A, Addition of zinc (1 mm ZnCl₂) suppressed the astrocyte response to βA. B, Addition of CuCl₂ (100 μm) had no apparent effect on the βA responses, but the heavy-metal chelator clioquinol (2 μm) (C) suppressed the responses completely.

βA causes Ca²⁺-dependent depletion of GSH in both neurons and astrocytes. MCB was used to image astrocyte and neuronal GSH by digital imaging. Hippocampal cocultures (15–20 DIV) (A, B) and cortical astrocyte cultures (C) were treated for 24 hr with βA25–35 or βA35–25 (50 μm for both) and clioquinol (1 μm) at 37°C in culture medium with (gray columns) or without (black columns) calcium. Mean intensities of MCB–GSH adduct fluorescence (arb.U) are presented. βA25–35 decreased GSH dramatically either in hippocampal astrocytes in coculture with neurons (A) or in cortical astrocytes in monoculture (C). The response was dependent on extracellular calcium and was also suppressed by clioquinol (C). βA35–25 had no effect. Note that neuronal GSH was also significantly reduced (B) and that the reduction was also calcium dependent, although it represents a proportionately smaller response than that of the astrocytes.