The 28-amino acid form of an APLP1-derived Aβ-like peptide is a surrogate marker for Aβ42 production in the central nervous system

Surrogate markers for the Alzheimer disease (AD)-associated 42-amino acid form of amyloid-β (Aβ42) have been sought because they may aid in the diagnosis of AD and for clarification of disease pathogenesis. Here, we demonstrate that human cerebrospinal fluid (CSF) contains three APLP1-derived Aβ-like peptides (APL1β) that are generated by β- and γ-cleavages at a concentration of ∼4.5 nM. These novel peptides, APL1β25, APL1β27 and APL1β28, were not deposited in AD brains. Interestingly, most γ-secretase modulators (GSMs) and familial AD-associated presenilin1 mutants that up-regulate the relative production of Aβ42 cause a parallel increase in the production of APL1β28 in cultured cells. Moreover, in CSF from patients with pathological mutations in presenilin1 gene, the relative APL1β28 levels are higher than in non-AD controls, while the relative Aβ42 levels are unchanged or lower. Most strikingly, the relative APL1β28 levels are higher in CSF from sporadic AD patients (regardless of whether they are at mild cognitive impairment or AD stage), than those of non-AD controls. Based on these results, we propose the relative level of APL1β28 in the CSF as a candidate surrogate marker for the relative level of Aβ42 production in the brain.

and DELAPAGTGVSREAVSGLLIMGAGGGSL, respectively. Note that the probability score is -10*Log(P), where P is the probability that the observed match is a random event. The experiments were performed three times and values represent means ± SD.

Selection of optimal daughter ions and conditions for quantification by LC/MS/MS analysis.
Parental ions for APL1β25 (A), APL1β27 (B), and APL1β28 (C) were degraded into several daughter ions by MS/MS (collision energy, 50-80 eV). The daughter ions for which the peak heights were relatively high were selected for quantification. For APL1β25/28, the b2, y20, and y21 ions were used, whereas for APL1β27, the b2, y21, and y22 ions were used.

Supplementary figure 4
FA fractions from AD brain tissues contain almost no APL1β.
(A) FA fractions from sporadic AD brain tissues (65 mg) contained very high amounts of Aβ but almost no detectable APL1β. (B, C) Additional presentations of the data of Figure 4A and B. Note that FA fractions extracted from the same amount of sporadic AD (AD1 and AD2) or non-AD (N1 and N2) brain samples (65 mg) were used in the experiment shown in (A) and (C).

Supplementary figure 5
Immunohistochemical detection of senile plaques in serial AD sections.
Both with (E,F, and G) and without (B, C, and D) FA treatment (brain sections were dipped in 100% FA solution for 10 s), paraffin sections of brain tissues were stained with the anti-APL1β antibodies OA601 (C and F) and OA663 (D and G). Senile plaques were not detected with the antibodies, although under the experimental conditions, a considerable number of senile plaques were detected using anti-Aβ antibody 4G8 even in the absence of FA treatment (B). Panels A and H show methenamine silver staining.

Supplementary figure 6
APL1β forms almost no protofibril in vitro.
To further characterize the non-aggregative nature of APL1β, we incubated the APL1β species (B, C, and D) in vitro under conditions where Aβ40 (A) forms amyloid fibrils (Hartley et al., 1999). To observe potential protofibril formation by APL1β, we increased the incubation period 12-fold compared to that necessary for the formation of Aβ40 protofibrils. Following the incubation, we examined amyloid protofibril formation by size exclusion chromatography. We could not detect any APL1β-derived protofibrils (B, C, and D), although we confirmed that Aβ (A) forms protofibrils under these conditions.

Supplementary figure 7
Sulindac sulfide and compound-W, which lower the relative Aβ42 level, do not decrease the relative APL1β28 level.
(A) In addition to S2474, fenofibrate, another PS/γ-secretase modifier, also induces an increase in the relative secretion of APL1β28 to total APL1β in naive SH-SY5Y cells. Blue, red, and yellow bars indicate the ratios of APL1β25, APL1β27, and APL1β28 to total APL1β, 4 respectively. In the conditioned media, the ratio of Aβ42 to total Aβ was elevated (data not shown).
However, sulindac sulfide and Compound-W (CW) decreased the relative generation of Aβ42 to total Aβ in naive SH-SY5Y cells (B), but they did not decrease the relative generation of APL1β28 to total APL1β (C). (D) The relationship between the relative levels of APL1β28 to Aβ42 in the presence of the indicated γ-secretase modulators.

Supplementary figure 8
Displacement of endogenous PS proteins in PS/γ-secretase complex by the exogenous mutant form in stable cell lines.
We prepared K293 cell lines stably expressing both βAPP sw and wt APLP1. From these cell lines, we chose stable clones that expressed exogenous PS1 mutants at levels high enough so that endogenous PS1/2 in the PS/γ-secretase complex was displaced by the exogenous PS1 mutants. To do this, we first chose stable cell lines that overexpress PS1 holoprotein, which is degraded via the ubiquitin-proteasome pathway immediately after translation (Steiner et al., 1998), such that the positive signal is barely detectable in untransfected cells. Subsequently, from these cell lines, we further selected clones in which endogenous PS2 is considerably displaced by the exogenous PS1 mutant proteins. The PS1 holoprotein band (~45 kDa) was clearly observed, whereas the PS2 CTF band (~18 kDa) was barely detectable. Relative levels of APL1β and Aβ species in conditioned media from these PS1 mutant-expressing cells were determined and are shown in Figure 5D.

Supplementary figure 9
Each APL1β species in CSF is degraded in a similar rate.
We investigated whether there is a difference in the rate of degradation for the various APL1β species in CSF between sporadic AD patients (B) and non-demented controls (A). The experiments were performed three times, and representative data are shown. We incubated each APL1β species (2.5 nmol) in the CSF (100 μl) for the indicated time. After boiling for 5 min in SDS-sample buffer, the samples were separated in Tris-Tricine gels and then analyzed by Western blotting with the OA601 antibody. Note that the rate of degradation of each APL1β species did not differ.