Rare Individual Amyloid-β Oligomers Act on Astrocytes to Initiate Neuronal Damage

Oligomers of the amyloid-β (Aβ) peptide have been implicated in the neurotoxicity associated with Alzheimer’s disease. We have used single-molecule techniques to examine quantitatively the cellular effects of adding well characterized Aβ oligomers to primary hippocampal cells and hence determine the initial pathway of damage. We found that even picomolar concentrations of Aβ (1–40) and Aβ (1–42) oligomers can, within minutes of addition, increase the levels of intracellular calcium in astrocytes but not in neurons, and this effect is saturated at a concentration of about 10 nM of oligomers. Both Aβ (1–40) and Aβ (1–42) oligomers have comparable effects. The rise in intracellular calcium is followed by an increase in the rate of ROS production by NADPH oxidase in both neurons and astrocytes. The increase in ROS production then triggers caspase-3 activation resulting in the inhibition of long-term potentiation. Our quantitative approach also reveals that only a small fraction of the oligomers are damaging and that an individual rare oligomer binding to an astrocyte can initiate the aforementioned cascade of responses, making it unlikely to be due to any specific interaction. Preincubating the Aβ oligomers with an extracellular chaperone, clusterin, sequesters the oligomers in long-lived complexes and inhibits all of the physiological damage, even at a ratio of 100:1, total Aβ to clusterin. To explain how Aβ oligomers are so damaging but that it takes decades to develop Alzheimer’s disease, we suggest a model for disease progression where small amounts of neuronal damage from individual unsequestered oligomers can accumulate over time leading to widespread tissue-level dysfunction.


Aβ40 and Aβ42 peptide preparation and characterization
Monomeric solutions of HiLyteFluor488 and HiLyteFluor647-labeled Aβ40 or Aβ42 (Anaspec, San Jose, CA) were prepared by dissolving the lyophilized peptides in SSPE buffer (150 mM NaCl, 10 mM Na 2 H 2 PO 4 x H 2 O, 10 mM Na 2 EDTA, adjusted to pH 12 using NaOH) followed by sonication over ice for 30 min (Bandelin Sonorex, Berlin, Germany) and subsequently flash freezing into 5 µL aliquots (1). Prior to each of the incubations, aliquots of each peptide were diluted into SSPE buffer (pH adjusted to 7.4 using HCl) to the desired concentration and placed under conditions for aggregation (e.g. 37 °C, agitation). The concentration of each labeled peptide was measured before mixing the two different fluorophore-labeled samples using cTCCD as previously described (1).
For each experiment Aβ monomers (Aβ40 at 20 µM and Aβ42 at 10 µM) were incubated in SSPE buffer (defined above) at 37 °C with agitation (200 rpm on a rotary shaker). After 1 h of aggregation, the samples were placed at 4 °C and used within 10 h of preparation. Monomeric solutions were kept frozen at -80 °C until use. For experiments with clusterin, the chaperone was added at a 1:1 molar ratio to Aβ (unless otherwise stated) and incubated for 30 min at 25 °C.
For each preparation of Aβ40 or Aβ42, the number and size distributionS of oligomers were determined using the single molecule cTCCD method. The instrumentation and methodology required for this characterization have been described in detail previously (1).

Preparation and labeling of human clusterin
Clusterin was extracted from human serum from Wollongong Hospital (Wollongong, NSW, Australia), as described previously (2). Labeling of clusterin was carried using lysine conjugation of succinimidyl ester-functionalized AlexaFluor647 (Molecular Probes, Grand Island, NY) using previously described protocols (1).

Cell cultures
Mixed cultures of neurons and glial cells were prepared as described previously with modifications, from Sprague-Dawley rat pups 2-4 days post-partum (UCL breeding colony)(3).
Experimental procedures were performed in full compliance with the United Kingdom Animal (Scientific Procedures) Act of 1986. The hippocampus and cortex were removed and placed in ice-cold PBS (Ca 2+ , Mg 2+ -free, Invitrogen, Paisley, UK). The tissue was then minced and trypsinized (0.25% for 5 min at 37 o C), triturated and plated on poly-D-lysine-coated coverslips, and cultured in Neurobasal A medium (Invitrogen, Paisley, UK) supplemented with B-27 (Invitrogen) and 2 mM L-glutamine. Cultures were maintained at 37 o C in a humidified atmosphere of 5% CO 2 and 95% air, and the medium was in each case replaced twice a week and maintained for 12-15 days before experimental use to ensure expression of glutamate and other receptors. Neurons were easily distinguishable from glia using microscopy: they appeared phase bright, had smooth rounded somata and distinct processes, and lay just above the focal plane of the glial layer.

Measurements of [Ca 2+ ] c and ROS
For measurements of [Ca 2+ ] c , cells were loaded for 30 min at room temperature with 5 µM fura-2 AM and 0.005% pluronic acid in a HEPES-buffered salt solution (HBSS) containing 156 mM NaCl, 3 mM KCl, 2 mM MgSO 4 , 1.25mM KH 2 PO 4 , 2mM CaCl 2 , 10mM glucose and 10mM HEPES; the pH of each solution was adjusted to 7.35 with NaOH, and the fluorescence of 488 nM-excitable fura-2 was measured as a function of time.
For measurement of ROS production, dihydroethidium (2 µM HEt) was added into the solutions during the experiments. No pre-incubation ('loading') was used for HEt to limit the intracellular accumulation of oxidized products. Measurements monitored the ratio of two fluorescent wavelengths, representing the oxidized and non-oxidized form, as a function of time (see Microscopy) (4)(5)(6).
In all experiments, we identified the neurons initially with bright field imaging and during the experiments by calcium imaging. Neurons were easily distinguishable from glia: they appeared phase bright, had smooth rounded somata and distinct processes, and lay just above the focal plane of the glial layer. Cells were imaged for up to 30 minutes following the addition of Aβ and large fields of cells containing between 100 and 200 cells were imaged at a time at an image acquisition rate of 10 s -1 .

Microscopy
All microscopy protocols used for monitoring intracellular Ca 2+ and ROS have been described previously (5,6). Fluorescence measurements were obtained on an epi-fluorescence inverted microscope equipped with a ×20 Confocal images were obtained using a Zeiss (Oberkochen, Germany) 710 confocal laser scanning microscope and a 40x oil immersion objective. A 488 nm argon laser was used to excite NucView 488 and the resulting fluorescence was measured using a bandpass filter from 510 and 560 nm. Images were acquired at 10 frames s -1 for 30 minutes.

Electrophysiology
All the protocols used are as described previously (7). Acute hippocampal slices were prepared from 26 to 32 day-old male Wistar rats. Experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act of 1986. Animals were sacrificed by dislocation of the neck followed by decapitation. The brains were rapidly removed and placed in ice-cold artificial CSF

Statistical methods
All statistical analysis was performed using both Origin 8 (OriginLab) and Prism 6.00 (GraphPad, La Jolla, CA). Non-parametric tests were performed to avoid assumptions of normality.

Clusterin binds Aβ42 oligomers
cTCCD was used to identify and characterize oligomers formed from mixtures of Aβ42 monomers tagged with either the HiLyteFluor488 or the HiLyteFluor647 fluorophore. In a similar experiment, run concurrently, cTCCD was used to determine the number of oligomer:clusterin complexes in the solution by incubating a solution of Aβ42 tagged with a HiLyteFluor488 fluorophore, with an equimolar quantity of clusterin tagged with an AlexaFluor647 fluorophore. This has been confirmed previously for Aβ40(1). The number of oligomers from the first experiment was, within experimental error, the same as the number of clusterin-associated Aβ species in the second, indicating that effectively all of the oligomers are bound to clusterin ( Figure S3A).

Mechanism of oligomer entry
In order to investigate further the mechanism underlying the Aβ-induced [Ca 2+ ] c transients, the analogous experiments to those discussed above at 25 °C were performed at lower temperatures (~4-10 °C) to reduce the efficacy of ATP-dependent active processes including cellular uptake of extracellular species (8). In these experiments, we observed no change in the amplitude or the frequency of the [Ca 2+ ] c transients relative to those observed in the experiments performed at higher temperatures suggesting that the [Ca 2+ ] c transients and the downstream effects do not depend significantly on active processes such as exocytosis and endocytosis ( Figure S1H).

Single molecule tracking on the cell membrane
Possible mechanisms by which oligomers induce Ca 2+ transients in astrocytes include creating defects in the cell membrane or activating cell-surface ion transporters (9,10). Both of these mechanisms involve interactions of Aβ oligomers with the cell membrane. In order to investigate whether or not pre-incubating the Aβ oligomers with clusterin inhibited the interactions between the oligomers and astrocytic membranes, we used a single-molecule imaging technique that allows us to visualize individual Aβ species on the cell surface (11). We incubated primary astrocytes with a mixture of Aβ42 monomers and oligomers labeled with a HiLyteFluor647 fluorophore and, using this approach, we observed that these Aβ42 species were bound to the cell surface in the absence of clusterin at a low surface density (approximately one monomer or oligomer was found for every 5 µm 2 of surface). When the Aβ42 oligomers were incubated with clusterin prior to adding them to the astrocytes, the number of Aβ42 species observed on the surfaces of the astrocytes was reduced by a factor of approximately two ( Figure S3B). This result suggests that incubation of Aβ oligomers with clusterin prevents them from interacting detrimentally with the cell surface. Taken together, the aforementioned experiments show that picomolar concentrations of Aβ40 and of Aβ42 oligomers act on the cell membranes of astrocytes giving rise to subsequent Ca 2+ influx. The presence of clusterin, however, can suppress this effect by binding the oligomers, thereby preventing the initial interaction between the Aβ oligomers and the cell membranes.

Need for clusterin preincubation
The incubation of Aβ40 and Aβ42 oligomers with clusterin, prior to adding them to cells, inhibited the appearance of the oligomer-induced [Ca 2+ ] c transients in astrocytes ( Figure 6A).
Incubating the cells with clusterin (500 nM) for 15 min before exposing them to oligomeric Aβ42 resulted in only partial inhibition of the Aβ42-induced [Ca 2+ ] c transients (62±4% of astrocytes responded when pre-incubated with clusterin compared to 89±5.7% of astrocytes without an initial incubation of the cells with clusterin, p<0.005). This result suggests that a minimum time is required for the interaction of Aβ oligomers and clusterin in order to enable effective suppression of the former from initiating increases in [Ca 2+ ] c ; indeed, such a necessity for pre-incubation to allow time for binding between chaperones and oligomers has also been observed for a number of other systems (12). The protective effect of clusterin, therefore, appears not to be mediated directly by the effects of clusterin on the cells but by its interaction with Aβ oligomers.  In each of the following experiments, at least 50 cells were examined. 14 (A) The cytosolic Ca 2+ concentration (as quantified by the fura-2 ratio) as a function of time in astrocytes to which 500 nM unconjugated HiLyteFluor647 has been added. Each line represents a single astrocyte.
(B) The cytosolic Ca 2+ concentration (as quantified by the fura-2 ratio) as a function of time in astrocytes to which 500 nM unlabeled Aβ42 (containing, presumably, 19.5 nM oligomers) has been added. Each line represents a single astrocyte.