Alternative processing of γ-secretase substrates in common forms of mild cognitive impairment and Alzheimer's disease: evidence for γ-secretase dysfunction

Abstract

Objective: The most common pathogenesis for familial Alzheimer's disease (FAD) involves misprocessing (or alternative processing) of the amyloid precursor protein (APP) by γ-secretase due to mutations of the presenilin 1 (PS1) gene. This misprocessing/alternative processing leads to an increase in the ratio of the level of a minor γ-secretase reaction product (Aβ42) to that of the major reaction product (Aβ40). Although no PS1 mutations are present, altered Aβ42/40 ratios are also observed in sporadic Alzheimer's disease (SAD), and these altered ratios apparently reflect deposition of Aβ42 as amyloid.

Methods: Using immunoprecipitation-mass spectrometry with quantitative accuracy, we analyzed in the cerebrospinal fluid (CSF) of various clinical populations the peptide products generated by processing of not only APP but also an unrelated protein, alcadein (Alc). Alc undergoes metabolism by the identical APP α-secretases and γ-secretases, yielding a fragment that we have named p3-Alc(α) because of the parallel genesis of p3-Alc(α) peptides and the p3 fragment of APP. As with Aβ, both major and minor p3-Alc(α) s are generated. We studied the alternative processing of p3-Alc(α) in various clinical populations.

Results: We previously reported that changes in the Aβ42/40 ratio showed covariance in a linear relationship with the levels of p3-Alc(α) [minor/major] ratio in media conditioned by cells expressing FAD-linked PS1 mutants. Here we studied the speciation of p3-Alc(α) in the CSF from 3 groups of human subjects (n = 158): elderly nondemented control subjects; mild cognitive impairment (MCI) subjects with a clinical dementia rating (CDR) of 0.5; SAD subjects with CDR of 1.0; and other neurological disease (OND) control subjects. The CSF minor p3-Alc(α) variant, p3-Alc(α) 38, was elevated (p < 0.05) in MCI subjects or SAD subjects, depending upon whether the data were pooled and analyzed as a single cohort or analyzed individually as 3 separate cohorts.

Interpretation: These results suggest that some SAD may involve alternative processing of multiple γ-secretase substrates, raising the possibility that the molecular pathogenesis of SAD might involve γ-secretase dysfunction.

Figure 1

Amino acid sequences and cleavage sites of p3-Alc_α and Aβ in human CSF. The amino acid sequences of p3-Alc_α (black-underline) along with the sequences of p3 (gray double-underline) and Aβ40 (black double-underline) of APP. The major primary (black arrowheads) and secondary (black arrowheads) cleavage sites of Alc_α1 are indicated together with those of APP (α, the cleavage site by α-secretase or ADAM 10/17; β, the cleavage site by β-secretase or BACE). Numbers on amino acids indicate their positions. The shaded area indicates the putative transmembrane region. Alc = alcadein; APP = amyloid β protein precursor; CSF = cerebrospinal fluid.

Figure 2

Representative MS spectra of p3-Alc_α peptides and Aβ peptides in human CSF. (A) p3-Alc_α in CSF and (B) Aβ in CSF. The CSF (300μl) were subjected to immunoprecipitation with (A) UT135 or (B) 82E1 antibodies, respectively. The precipitates were analyzed for molecular mass with MALDI-TOF/MS. (A) “34”, p3-Alc_α34; “35”, p3-Alc_α35; “36”, p3-Alc_α36; “37*”, a mixture of p3-Alc_α37 and p3-Alc_α2N+35 (see Hata and colleagues for p3-Alc_α2N+35); “38”, p3Alc_α38. (B) “37”, Aβ37; “38”, Aβ38; “39”, Aβ39; “40”, Aβ40; “42”, Aβ42. Alc = alcadein; CSF = cerebrospinal fluid; MALDI-TOF/MS = matrix-assisted laser desorption ionization–time-of-flight/mass spectrometry.

Figure 3

Comparison of the distribution of the ratio of p3-Alc_α38/total p3-Alc_α in CSF of elderly nondemented subjects, AD subjects and other neurological disease subjects according to cohort. (A) Japanese cohort; nondemented CDR 0 (n = 10), AD CDR 0.5 (n = 9), and AD CDR 1 (n = 12). (B) US1 cohort; nondemented CDR 0 (n = 26), AD CDR 0.5 (n = 20), AD CDR 1 (n = 13), and OND (n = 16). (C) US2 cohort; nondemented CDR 0 (n = 15), AD CDR 0.5 (n = 13), AD CDR 1 (n = 11), and OND (n = 13). (D) Combined subjects of 3 cohorts; nondemented CDR 0 (n = 51), AD CDR 0.5 (n = 42), AD CDR 1 (n = 36), and OND (n = 29). See the Table for raw data of the ratio and subject information. Statistical analysis was performed by a one-way analysis of variance followed by the Tukey Kramer's test (*p < 0.05). AD = Alzheimer's disease; Alc = alcadein; CDR = clinical dementia rating; CSF = cerebrospinal fluid; OND = other neuronal and neurodegenerative diseases except for AD.