Cell surface amyloid beta-protein precursor colocalizes with beta 1 integrins at substrate contact sites in neural cells

Abstract

Amyloid beta-protein (A beta), the principal constituent of the senile plaques seen in Alzheimer's disease (AD), is derived by proteolysis from the beta-amyloid precursor protein (beta PP). The distribution and trafficking of cell surface beta PP are of particular interest because some of these molecules are direct precursors of secreted A beta and because the localization of beta PP at the cell surface may be related directly to its physiological functions. Recently, we reported that, in cultured hippocampal neurons, cell surface beta PP is preferentially expressed on axons in a striking discontinuous pattern. In this study, we describe the colocalization of cell surface beta PP and integrins in primary cultured cells. In rat hippocampal neurons, cell surface beta PP was colocalized selectively with alpha 1 beta 1 and alpha 5 beta 1 integrin heterodimers at these characteristic segmental locations. In rat cortical astrocytes, both cell surface beta PP and beta 1 integrin were located at the cell periphery in the "spreading" stage shortly after plating. In "flattened" astrocytes cultured for several days, beta PP was found in punctate deposits called point contacts. In these sites, beta PP was colocalized with alpha 1 beta 1, but not with alpha 5 beta 1 integrin heterodimers, the latter of which were situated at focal contact sites. In both neurons and astrocytes examined after shearing, clathrin and alpha-adaptin were colocalized with beta PP on the surface that directly contacts the substratum. These results are consistent with the putative role of beta PP in cell adhesion and suggests that beta PP either interacts with selected integrins or shares similar cellular machinery to promote cell adhesion.

Fig. 1.

Immunocytochemical colocalization of cell surface βPP and integrins in cultured hippocampal neurons. A hippocampal neuron cultured for 14 d was double-labeled for β1 integrin (a) and cell surface βPP (5A3/1G7)(b). The axonal pattern from both immunostaining reactions was patchy and overlapped entirely. β1 integrin and βPP on the perikaryal surface cannot be compared clearly, because that region of the cell body is not within the plain of focus of the photomicrographs. α1 (c) and α5 (e) subunits of integrins also were colocalized with cell surface βPP (d, f) along neurites. Scale bars, 5 μm.

Fig. 2.

Immunocytochemical colocalization of cell surface βPP and integrins at growth cones. Shown are two examples of colocalization of cell surface βPP (a, c) at growth cones with β1 (b) and α5 (d) integrins in mature hippocampal neurons in culture, as seen by double-labeling. The patchy surface distribution of βPP is highlighted in a, where the arrowheadstrace out the segment of axon devoid of surface βPP immunoreactivity. To examine cell substrate contact sites, we sheared neurons (see Materials and Methods) and stained them with anti-βPP antibodies (5A3/1G7)(e). βPP was localized on neurites (arrows) and on the cell body in a granular pattern. Neuronal cell adhesion molecule (NCAM) staining (f) showed a diffuse, rather than a patchy, pattern on neurites. Scale bars, 5 μm.

Fig. 5.

Immunocytochemical colocalization of cell surface βPP with clathrin and α-adaptin. a, A hippocampal neuron cultured for 10 d labeled with an anti-clathrin antibody (X22) showed a fine punctate staining pattern on neurites, but its distribution was not patchy and occurred predominantly in axons (compare with βPP shown in Fig. 1b). On the other hand, at substrate contact sites visualized in sheared neurons, βPP (antibody 207) (b) and clathrin (c) were specifically colocalized. Within the immunoreactive patches, there is a suggestion of fine granular staining. In sheared astrocytes, βPP (207) (d) also was tightly colocalized with α-adaptin, as demonstrated by antibody AP.6 (e). Scale bars, 5 μm.

Fig. 3.

Immunocytochemical colocalization of cell surface βPP and β1 integrin in sympathetic ganglion neurons cultured for 7 d on type 1 collagen- or laminin-coated glass coverslips in serum-free medium. On both type 1 collagen (a, b) and laminin (c, d), cell surface βPP (a, c) and β1 integrin (b, d) showed the characteristic segmental pattern and tight colocalization by double labeling. Scale bars, 10 μm.

Fig. 4.

Immunocytochemical colocalization of cell surface βPP and integrins in type 1 astrocytes. Type 1 astrocytes were allowed to attach and spread on laminin or fibronectin for 3 hr (spreading stage) and then were labeled with βPP (5A3/1G7) (a) and β1 integrin (b) antibodies. Both molecules were located mainly at the periphery and in the middle of the spreading cells. In cells cultured for 3 d before fixation (“flattened” astrocytes), cell surface βPP (c) and β1 integrin (d) were now colocalized at point contact sites. More specifically, surface βPP (antibody 207)(e) was tightly colocalized with the α1 subunit of integrins (f) when examined in sheared astrocytes. In contrast, the α5 subunit of integrins was localized in focal contact sites, appearing as linear streaks in the sheared cells (h, arrows), and it did not colocalize with βPP (5A3/1G7) (g). At point contact sites, staining with βPP midregion (1G7/5A3) (i) and C-terminal (C7) (j) antibodies in sheared cells showed complete colocalization, suggesting that βPP at the substrate surface represents full-length molecules. Scale bars:a–h, 10 μm; i, j, 5 μm.