Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease

Abstract

We investigated why the cerebrospinal fluid (CSF) concentrations of Aβ42 are lower in mild cognitive impairment (MCI) and Alzheimer's disease (AD) patients. Because Aβ38/42 and Aβ40/43 are distinct product/precursor pairs, these four species in the CSF together should faithfully reflect the status of brain γ-secretase activity, and were quantified by specific enzyme-linked immunosorbent assays in the CSF from controls and MCI/AD patients. Decreases in the levels of the precursors, Aβ42 and 43, in MCI/AD CSF tended to accompany increases in the levels of the products, Aβ38 and 40, respectively. The ratios Aβ40/43 versus Aβ38/42 in CSF (each representing cleavage efficiency of Aβ43 or Aβ42) were largely proportional to each other but generally higher in MCI/AD patients compared to control subjects. These data suggest that γ-secretase activity in MCI/AD patients is enhanced at the conversion of Aβ43 and 42 to Aβ40 and 38, respectively. Consequently, we measured the in vitro activity of raft-associated γ-secretase isolated from control as well as MCI/AD brains and found the same, significant alterations in the γ-secretase activity in MCI/AD brains.

Figure 1. Generation of Aβs through stepwise processing of βCTF

At the first step, βCTF is cleaved at the membrane-cytoplasmic boundary (ε-cleavage), producing AICD (APP intracellular domain) 50–99 and 49–99. Counterparts Aβ49 and 48 in turn are cleaved in a stepwise fashion, releasing tri- and tetrapeptides. One product line converts Aβ49 mostly to Aβ40 via Aβ46 and Aβ43. The other product line converts Aβ48 to Aβ38 via Aβ45 and Aβ42. It should be noted that the differences between the amounts of released tri- or tetrapeptide determine the amounts of Aβs produced. Broken lines indicate corresponding Aβs on the two product lines.

Figure 2. Relationships between the levels of Aβ40 and 38, and between those of Aβ43 and 42 in CSF from controls and MCI/AD patients

1. The levels of ln(Aβ40) were proportional to those of ln(Aβ38) (ln(Aβ40) = 0.910 × ln(Aβ38) + 1.642, R = 0.913).

2. The levels of ln(Aβ43) were proportional to those of ln(Aβ42) (ln(Aβ43) = 1.333 × ln(Aβ42) − 4.09, R = 0.979). It should be noted that the levels of both ln(Aβ42) and ln(Aβ43) in MCI [filled triangle (n = 19)]/AD [filled circle (n = 24)] are lower than those in controls [open circles (n = 21)].

Figure 3. Relationships between the levels of Aβ43 and 40, and between those of Aβ42 and 38 in CSF from controls (open circles) and MCI (closed triangle)/AD patients (closed circle)

1. The levels of ln(Aβ43) correlate with those of ln(Aβ40) within controls (R = 0.688), and barely within MCI/AD subjects (R = 0.507/0.736). The plots for MCI/AD were located below the regression line for control (p < 0.001, ANOVA).

2. The levels of Aβ42 correlate with those of Aβ38 within controls (R = 0.723), and barely within MCI/AD (R = 0.500/0.393). The plots for MCI/AD were situated below the regression line for controls (p < 0.001, ANOVA).

Figure 4. Ln(Aβ40/43) versus ln(Aβ38/42) plot

The ratios represent the cleavage efficiency at the final step of each line. Both ratios are largely proportional to each other (y = 0.748 × −2.244, R = 0.936) and plots are located on the line and its close surroundings. This plot clearly distinguishes between control subjects and MCI/AD patients (Aβ40/43 for MCI vs. control, p = 0.000; Aβ38/42 for MCI vs. control, p = 0.000; ANOVA, followed by Dunnett's t-test). Control plots [open circles (n = 21)] are located close to the origin and MCI/AD plots [closed triangles (n = 19) and closed circles (n = 24), respectively] are a little distant from the origin.

Figure 5. Ln(Aβ40/43) versus ln(Aβ38/42) plot based on direct quantification of raft-associated γ-secretase activity

The raft-associated γ-secretase prepared from control and MCI/AD brain specimens was incubated with βCTF for 2 h at 37°C (see Materials and Methods Section). Produced Aβs were quantified by Western blotting using specific antibodies. This plot distinguishes between control subjects and MCI/AD patients (Aβ40/43 for control vs. MCI/AD, p < 0.001; Aβ38/42 for control vs. MCI/AD, p = 0.001; Welch's t-test). MCI/AD plots [closed triangles (n = 10) and closed circles (n = 13), respectively] are as a whole a little distant from the origin, whereas control plots [open circles (n = 16)] are close to the origin.