Nonspecific interaction of prefibrillar amyloid aggregates with glutamatergic receptors results in Ca2+ increase in primary neuronal cells

Abstract

It is widely reported that the Ca(2+) increase following nonspecific cell membrane permeabilization is among the earliest biochemical modifications in cells exposed to toxic amyloid aggregates. However, more recently receptors with Ca(2+) channel activity such as alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), N-methyl D-aspartate (NMDA), ryanodine, and inositol 1,4,5-trisphosphate receptors have been proposed as mediators of the Ca(2+) increase in neuronal cells challenged with beta-amyloid peptides. We previously showed that prefibrillar aggregates of proteins not associated with amyloid diseases are toxic to exposed cells similarly to comparable aggregates of disease-associated proteins. In particular, prefibrillar aggregates of the prokaryotic HypF-N were shown to be toxic to different cultured cell lines by eliciting Ca(2+) and reactive oxygen species increases. This study was aimed at assessing whether NMDA and AMPA receptor activations could be considered a generic feature of cell interaction with amyloid aggregates rather than a specific effect of some aggregated protein. Therefore, we investigated whether NMDA and AMPA receptors were involved in the Ca(2+) increase following exposure of rat cerebellar granule cells to HypF-N prefibrillar aggregates. We found that the intracellular Ca(2+) increase was associated with the early activation of NMDA and AMPA receptors, although some nonspecific membrane permeabilization was also observed at longer times of exposure. This result matched a significant co-localization of the aggregates with both receptors on the plasma membrane. Our data support the possibility that glutamatergic channels are generic sites of interaction with the cell membrane of prefibrillar aggregates of different peptides and proteins as well as the key structures responsible for the resulting early membrane permeabilization to Ca(2+).

FIGURE 1.

Fast response of granule cells to HypF-N in aggregated and monomeric form. a, fluorescence images of granules loaded with Oregon Green at different times before (frame 1) and after (frames 2–5) the addition of prefibrillar HypF-N aggregates aged for 24 h; frame 6, fluorescence intensity of the region of interest (highlighted in white in frames 1–5) as a function of time. Times corresponding to the capture of images 1–5 are indicated on the fluorescence curve. b, histograms of the duration of the fluorescence responses measured in the presence of 24-h (gray) and 48-h aged (dashed) HypF-N aggregates, showing that the distribution for the latter is shifted to shorter times. c and d, addition of native HypF-N (c) and mature HypF-N fibrils (8 days (8d) aged aggregates) (d) does not induce any fast response. In both cases the subsequent addition of early HypF-N aggregates (24 h) induces a fluorescence response, indicative of sample integrity. Arrows correspond to the addition of 0.2 μm HypF-N.

FIGURE 2.

The fast response is because of a Ca²⁺ influx and does not involve voltage-dependent Ca²⁺ channels. a, effect of the external solution on the fluorescence response of a granule cell after the addition of 0.2 μm HypF-N aggregates aged for 24 h. The response was measured in external standard solution (left), in the absence of calcium (middle), and in the presence of 50 μm cadmium (right). b, representative calcium current-voltage relationships measured by the patch clamp technique in the absence of protein (straight line) and after incubation for 24 h with protein aggregates aged for 48 h (dashed line). The data are indicative of the behavior of several observed cells.

FIGURE 3.

NMDA-Rs are involved in the fast response. a, fluorescence response of a granule cell after the addition of 0.2 μm HypF-N aggregates (aged for 24 h) to the external standard solution (black) and in the presence of the NMDA-R antagonist memantine at the concentration of 10 μm (gray). b, fluorescence response of a granule cell in the presence of 100 μm NMDA and 50 μm glycine; the horizontal bar corresponds to the presence of NMDA and glycine in the external solution. The fluorescence intensity is restored to its initial values upon agonist washing out. The data are indicative of the behavior of several observed cells.

FIGURE 4.

Co-localization of HypF-N prefibrillar aggregates and glutamatergic receptors by immunofluorescence confocal microscopy. Granule cells in primary culture were analyzed for co-localization of 24-h aged HypF-N aggregates with AMPA-Rs (a), NMDA-Rs (b), and gp91 phox (c). AMPA-Rs and NMDA-Rs have been labeled with Alexa Fluor 546 (red signal); the gp91 phox has been labeled with Cy3 (red signal); HypF-N aggregates have been labeled with Alexa Fluor 488 (green signal). Full resolution details of the fluorescence signal (red, green, and the corresponding merged image) of the selected areas are shown on the bottom side of the panels. d, scatter plots indicating the co-localization pattern over the selected area of each panel; sampled pixels are plotted as a function of green (y axis) and red (x axis) fluorescence intensity The co-localization of HypF-N with AMPA-Rs results in an evident pixel distribution along the diagonal of the plot (top), whereas a partial localization of HypF-N with NMDA-Rs is shown as a more dispersed pixel distribution (middle). No pixels were located along the diagonal for HypF-N and gp91 phox, corresponding to a low degree of co-localization (bottom).

FIGURE 5.

HypF-N prefibrillar aggregates reduce the density of AMPA receptors at the cell surface. Fluorescence images corresponding to confocal sections of granule cells not treated (a and c) or treated with HypF-N prefibrillar aggregates for 1 h (b and d). After treatment, cells were fixed, counterstained with TRITC-conjugated wheat germ agglutinin to label the cell membrane and immunostained for NMDA-R (a and b) or for AMPA-R (c and d). e, NMDA-R and AMPA-R average fluorescence intensity (a.u.) in the plasma membrane. NMDA-R, n = 20 cells; AMPA-R, n = 30 cells, p << 0.001.

FIGURE 6.

Long term effect of protein aggregates on granule cells. Fluorescence images of granules loaded with Oregon Green are shown as follows. a, in control conditions; b after incubation for 1 day with 0.2 μm HypF-N aged for 48 h; c after incubation for 1 day with 2 μm HypF-N aged for 48 h; d, after incubation for 1 day with 2 μm AcP aged for 48 h. The scale bar is the same for all images.

FIGURE 7.

HypF-N aggregates are internalized in granule cells. Confocal microscopy images corresponding, respectively, to a lower, middle, and upper sections of a granule cell incubated for 1 day in the presence of 48-h aged HypF-N aggregates labeled with Texas Red. The higher fluorescence intensity in the middle section indicates that aggregates are really located inside the cell body. White arrows in the middle frame indicate fluorescence spots corresponding to protein aggregates.