Amyloid-β production via cleavage of amyloid-β protein precursor is modulated by cell density

Abstract

Mounting evidence suggests that Alzheimer's disease (AD) is caused by the accumulation of the small peptide, amyloid-β (Aβ), a proteolytic cleavage product of amyloid-β protein precursor (AβPP). Aβ is generated through a serial cleavage of AβPP by β- and γ-secretase. Aβ40 and Aβ42 are the two main components of amyloid plaques in AD brains, with Aβ42 being more prone to aggregation. AβPP can also be processed by α-secretase, which cleaves AβPP within the Aβ sequence, thereby preventing the generation of Aβ. Little is currently known regarding the effects of cell density on AβPP processing and Aβ generation. Here we assessed the effects of cell density on AβPP processing in neuronal and non-neuronal cell lines, as well as mouse primary cortical neurons. We found that decreased cell density significantly increases levels of Aβ40, Aβ42, total Aβ, and the ratio of Aβ42: Aβ40. These results also indicate that cell density is a significant modulator of AβPP processing. Overall, these findings carry profound implications for both previous and forthcoming studies aiming to assess the effects of various conditions and genetic/chemical factors, e.g., novel drugs on AβPP processing and Aβ generation in cell-based systems. Moreover, it is interesting to speculate whether cell density changes in vivo may also affect AβPP processing and Aβ levels in the AD brain.

Figure 1. Summary of the proteolytic processing of APP

The early-onset familiar AD gene APP encodes amyloid β-protein precursor, which generates Aβ through the serial proteolytic cleavage by β- and γ-secretase. β-secretase cleavage produces the secreted, ~90kDa protein, sAPPβ, and the β-carboxy-terminal fragment, β-CTF (or C99). sAPPβ is the substrate of an unidentified secretase, which produces N-APP (containing the N-terminal 286 amino acids of APP; ~35kDa) and s-APP55 (~55kDa). C99 can be cleaved by γ-secretase and gives rise to Aβ and AICD (APP intracellular domain). In contrast to this amyloidogenic process by β- and γ-secretase, APP undergoes an alternative cleavage pathway which precludes Aβ generation. This pathway is initiated by α-secretase and produces sAPPα and the carboxy-terminal fragment, α-CTF (or C83). C83 can be further cleaved by γ-secretase to produce P3 and AICD.

Figure 2. Aβ40 and Aβ42 levels vary depending on cell density in H4-APP751 cells

Cells of different numbers were seeded on 6-well plates. Conditioned media and cell lysates were collected after 48h. Cell lysates were applied to BCA assay to calculate protein concentration. Conditioned media were applied to ELISA to calculate Aβ40 and Aβ42 levels, which were then normalized to protein concentration. The Aβ40 and Aβ42 values represented in the figure were normalized values. The values of Aβ40, Aβ42, Aβ(40+42), and the Aβ42:Aβ40 ratio from higher cell densities were compared to those from 100K. A-C At higher seeding densities (200K-400K), the levels of Aβ40 (A), Aβ42 (B), and Aβ(40+42) (C) significantly decreased versus 100K. D. The ratio of Aβ42:Aβ40 was also reduced with increasing seeding densities (n=3 for each treatment group; mean ± S.E.; *p<0.05; **p<0.01).

Figure 3. Aβ40 and Aβ42 levels vary depending on cell density in CHO-APP751 cells

Cells of different numbers were seeded on 6-well plates and harvested after 48h. Cell lysates were applied to BCA assay to calculate protein concentration. Conditioned media were applied to ELISA to calculate Aβ40 and Aβ42 levels, which were then normalized to protein concentration. The values of Aβ40, Aβ42, Aβ(40+42), and the Aβ42:Aβ40 ratios from higher cell densities were compared to the values at 50K. AC At higher seeding densities, the levels of Aβ40 (A), Aβ42 (B), and Aβ(40+42) (C) significantly decreased versus 50K. D. The ratio of Aβ42:Aβ40 was also reduced with increasing seeding densities (n=3 for each treatment group; mean ± S.E.; *p<0.05; **p<0.01).

Figure 4. Aβ40 and Aβ42 levels vary depending on cell density in mouse primary cortical neurons

Mouse primary cortical neurons were seeded on poly-D-lysine/laminin-coated 24-well plates and harvested after 48h. Cell lysates were applied to BCA assay to calculate protein concentration. Conditioned media were applied to ELISA to calculate Aβ40 and Aβ42 levels, which were then normalized to protein concentration. The values of Aβ40, Aβ42, total Aβ, and the Aβ42:Aβ40 ratios from high seeding densities were compared to those from 200K. A-D At higher seeding densities (300K-600K), the levels of Aβ40 (A), Aβ42 (B), Aβ(40+42) (C), and the ratios of Aβ42:Aβ40 (D) significantly decreased compared to those at 200K (n=3 for each treatment group; mean ± S.E.; *p<0.05; **p<0.01).

Figure 5. APP proteolytic processing varies depending on cell density in H4-APP751 cells

A-D. H4-APP751 cells were seeded on 6-well plates with different densities. Cell lysates were collected after 48h and applied to Western blotting analysis. The levels of full-length APP, APP-CTFs (C83 and C99), and their ratios to APP from higher seeding densities were compared to those at 150K. At higher cell densities, full-length APP levels had a trend to decrease versus 150K (A-B). The levels of C83 and C99, as well as their ratios to full-length APP decreased at higher seeding densities (C-D). E. The ratios of Aβ(40+42):APP-C99 were obtained by normalizing the levels of Aβ(40+42) to APP-C99 levels from the same samples, and the ratios from higher seeding densities were compared to those at 150K. The ratios of Aβ(40+42):APP-C99 increased with higher seeding densities (n=3 for each treatment group; mean ± S.E.; *p<0.05; **p<0.01).

Figure 6. APP proteolytic processing varies depending on cell density in CHO-APP751 cells

A-D. CHO-APP751 cells were seeded on 6-well plates with different densities. Cell lysates were collected after 48h and applied to Western blotting analysis. The levels of full-length APP, APP-CTFs (C83 and C99), and their ratios to APP from higher seeding densities were compared to those at 150K. At higher cell densities (200K-400K), full-length APP levels significantly decreased versus those at 150K (A-B). At higher seeding densities, the levels of C83 and C99, as well as their ratios to full-length APP decreased versus 150K (C-D). E. The ratios of Aβ(40+42):APP-C99 were obtained by normalizing the levels of Aβ(40+42) to APP-C99 levels from the same samples. The ratios of Aβ(40+42): APP-C99 initially increased with higher seeding densities (versus 150K), but then began to decrease with seeding densities > 250K (n=3 for each treatment group; mean ± S.E.; *p<0.05; **p<0.01).

Figure 7. Validation of altered APP processing with changes in cell density

A-B, H4-APP751 cells were seeded on 10cm-plates with different densities (0.5M, 1M, 1.5M, 2M, and 3M; equivalent to the densities of 100K, 200K, 250K, 300K, and 400K in 6-well plates, respectively). Cell lysates were collected after 48h and were applied to the BCA assay to determine protein concentration and the fluorometric assay to measure α- or β-secretase activity. The fluorometric readout was normalized to the protein concentration for each sample. Normalized fluorometric values at higher cell densities were compared to those at 100K. At higher seeding densities, the APP processing activity of α- and β-secretase decreased when compared to those at 100K (n=3 for each treatment group; mean ± S.E.; *p<0.05; **p<0.01).