Multiple γ-secretase product peptides are coordinately increased in concentration in the cerebrospinal fluid of a subpopulation of sporadic Alzheimer’s disease subjects

Alcadeins (Alcs) represent a family of neuronal type I membrane proteins (designated as Alc_α, Alc_β, and Alc_γ) that are encoded by independent genes [1]. In neurons, Alc forms a tripartite complex with Alzheimer's amyloid β-protein precursor (APP) via the crosslinking action of the neural adaptor protein X11-like (X11L) [2, 3]. In the absence of X11L, both the free Alc proteins and the free APP are subjected to coordinated proteolytic cleavage through similar mechanisms: APP and Alc are both cleaved by the identical α-secretase at the juxtamembrane region. This cleavage of Alc causes release of N-terminal soluble Alc ectodomain (sAlc) and leaves behind a C-terminal cell-membrane-associated AlcCTF. APP can undergo either an identical α-secretase cleavage (thereby generating a cell-associated APPCTFα) or instead (and unlike Alc) APP can undergo β-secretase cleavage leading to generation of sAPP_β and a cell-associated APPCTFβ [4]. All three CTFs (APPCTFα, APPCTFβ, AlcCTF) are subjected to regulated intramembranous cleavage by the γ-secretase complex, in which presenilin 1 or 2 (PS1, PS2) functions as the catalytic subunit [2]. Mutations in PS1 and PS2 are known to cause early onset familial Alzheimer's disease (FAD). The γ-secretase reaction involving APPCTFα generates the p3 fragment, while the reaction involving APPCTFβ generates the amyloid-β peptide (Aβ) [4]. Cleavage of AlcCTF by γ-secretase liberates a small peptide named p3-Alc (a named selected to be symmetrical with the name of the APP p3 peptide). The p3-Alc peptide is detectable in CSF, while the APP p3 peptide is very labile and difficult to detect in CSF [2, 5].

Most patients with FAD carry one of over 200 pathogenic mutations identified in the coding sequence of PS1 or PS2. These mutations alter intramembranous cleavage of APP so as to increase production of Aβ42, the most aggregation-prone, oligomerogenic, and fibrillogenic species of Aβ [6‐8]. Other patients with FAD may carry pathogenic mutations in the coding sequence of the APP gene, all of which promote the accumulation of Aβ [4]. Furthermore, Down syndrome (DS) patients carry three copies of chromosome 21 which includes the APP gene locus, and therefore, DS patients have a “genetic overdose” of APP, leading them to develop AD by middle age [9]. Therefore, alterations in the generation of Aβ, in both quality and quantity, are considered to be causes of AD pathogenesis in genetic forms of the disease.

In the more common sporadic forms of AD (SAD), the molecular pathogenesis remains unknown. Aβ42 levels are reduced in the CSF of SAD patients [10‐12], but the use of CSF Aβ42 as an in vivo marker for APP metabolism is complicated by its deposition in brain and cerebral vasculature as the disease progresses. Recent evidence suggests that a disturbance in an apolipoprotein E (APOE)-isoform-dependent step in Aβ clearance plays a role in the pathogenesis of SAD [13], although these data could not exclude the possibility that Aβ oligomerization or fibrillization (and not a defect in some clearance pathway alone) may also play a role, since apoE also plays a role in Aβ aggregation (Caesar et al., unpublished observations).

We recently reported that the CSF of subjects with sporadic MCI and early AD showed a relative overrepresentation of a minor p3-Alc_α species, p3-Alc_α38, raising the possibility that γ-secretase dysfunction can exist even in the absence of an FAD-linked genetic mutation [14]. The previous study [14] was performed using immunoprecipitation-mass spectrometry which, as performed, is considered to be a semi-quantitative method. Therefore, we have begun moving toward the development of sensitive ELISAs that will permit convenient, reliable, and sensitive quantitation of total p3-Alc_α and selected minor species (with p3-Alc_α38 being the top priority in that respect). Here we report the application of a recently developed ELISA system (antibody and assay development described elsewhere) [15] that can quantify total p3-Alc_α in the range of 40 to 600 pg/mL. Using this system, we have quantified total p3-Alc_α levels in the CSF of three independent cohorts that consist of subjects with MCI/CDR 0.5 or AD (CDR 1–3), as well as subjects that are either cognitively intact, age-matched controls or suffer from frontotemporal lobar degeneration (FTLD). This latter population served as other neurological disease (OND) controls.

In our previous studies, we demonstrated that the products of alternative cleavage of non-APP substrates (known as Alcs) by γ-secretase gave rise to a modified p3-Alc_α peptide profile in media conditioned by transfected cells expressing an FAD-linked mutant PS1 and a similar modified profile was also identified in the CSF of subjects with sporadic MCI (known as CDR 0.5 in Cohort 2 and recently renamed “prodromal AD” in the revised lexicon for dementia syndromes [21]), and mild AD [5, 14]. In order to quantify these peptides reliably and conveniently, we have recently developed an ELISA for p3-Alc_α[15]. This ELISA system quantifies total p3-Alc_α levels, but does not specifically measure the individual species of p3-Alc_α. In the current paper, we have employed this ELISA to quantify total p3-Alc_α levels in the CSF of three independent cohorts of subjects who were categorized as either nondemented controls, sporadic MCI, sporadic AD, or FTLD.

Interestingly, applying our new, quantitative p3-Alc ELISA to CSF for the first time, we were surprised to observe in two cohorts of Japanese subjects the apparent existence of subpopulations of sporadic MCI and AD subjects in whose CSF there was differential elevation of the levels of the reaction products generated by γ-secretase cleavage of multiple substrates; i.e., APP and Alcadein. Since Aβ40 and total p3-Alc_α were highly correlated in these cohorts, the current data support the use of p3-Alc_α as a surrogate for total APP-derived γ-cleaved products. Elevated levels of p3-Alc_α and Aβ were also observed in plasma samples of some female AD patients [15]. However, in CSF, we did not detect any differences in levels between male and female subjects. In another independent cohort study with plasma samples, we confirmed the significant increase of p3-Alc_α levels in MCI and AD patients, but we observed no systematic differences between male and female subjects [22]. Therefore, it is worth noting that the observation of a sex specific increase in p3-Alc_α levels in plasma of female AD patients has not been consistently observed in all cohorts studied.

The increase in p3-Alc_α level could arguably be caused by (1) increased primary α-cleavage by α-secretase; (2) increased intramembranous γ-cleavage by γ-secretase and/or (3) diminished clearance of transmembrane-derived fragments such as p3-Alc_α. Because Aβ, a product of primary β-cleavage of APP by β-secretase, is also increased in this subpopulation, and because we have previously linked PS1 mutations to variant p3-Alc speciation [5], we have argued on the basis of parsimony, that the molecular pathology was more likely attributable to dysfunction of γ-secretase. However, in light of the new data herein, it is possible that both p3-Alc_α speciation and also p3-Alc_α levels may be affected. When these observations are taken together with the model of altered CSF peptide clearance [23], and the evidence that clearance of Aβ from CSF is modulated in an APOE-isoform-specific manner [13], we now must consider it equally likely that altered p3-Alc_α levels and speciation could be attributable to a defect in clearance from CSF of transmembrane domain metabolite peptides.

A stratification of the current (this paper) and prior data [14] according to APOE genotype, followed by re-analysis, is underway. We have attempted a preliminary APOE genotype-dependent analysis using cohort 3 samples. ApoE4 carriers tended to show higher values of both p3-Alc_α and Aβ40 in MCI (CDR 0.5) and AD (CDR 1) patients but not in more advance AD (CDR 2–3) or in FTLD patients (Additional file 3, Figure S2). However, the increase of p3-Alc_α and Aβ40 in APOE4 carriers did not reach statistical significance when compared to the corresponding levels in non-APOE 4 carriers. Because this was a small scale pilot analysis, we consider unresolved the issue of whether APOE4 genotype influences the level of p3-Alc_α in AD. In order to address this issue directly, analysis of the p3-Alc_α levels in the identical samples studied by Castellano et al.[13] is under consideration.

It is interesting to note that both the quality and quantity of p3-Alc_α accumulation in CSF may be transient, occurring in MCI and mild AD but not evident in later stages (see ref 14 and this paper). Serial examinations of CSF from the same subjects at different stages of AD will be required in order to establish whether or not such a phenomenon truly exists within the same individual. The Biomarker Core of the Alzheimer’s Disease Neuroimaging Initiative (ADNI) [24] should be a useful resource in pursuing this hypothesis.

Since p3-Alc_α is not incorporated into cerebral or cerebrovascular amyloid, the decrease in p3-Alc_α levels in later stage AD subjects (CDR 2 or more) may be due to progressive neuronal degeneration, thereby eliminating the main cellular source of p3-Alc peptides. This is also consistent with other data suggesting that AD may be divisible into an early Aβ-driven phase (beginning presymptomatically and extending into mild stages of dementia) and a later phase that may be driven by inflammation and/or tauopathy [25]. Consistent with this formulation are the recent reports that fibrillar amyloid burden, as indicated by ¹¹ C]PiB signal, begins accumulating perhaps 10–15 years before symptoms are evident [26] and then plateaus [27]. This reformulation of AD pathogenesis also fits with recent data from Rinne and colleagues, showing that a reduction in the fibrillar amyloid burden caused by ~1.5 yrs of bapineuzumab infusion had no obvious impact on cognition [28].

If the apparent transient elevation of levels of p3-Alc_α and/or Aβ is due, at least in part, to transient γ-secretase dysfunction, the identification of this “spike” of dysfunction could be important for the timing and nature of interventions aimed at this enzyme. For example, elevated CSF p3-Alc_α levels (or the coordinate elevation of CSF Aβ40 and p3-Alc_α levels) could be used as an endophenotype that marks a subpopulation of sporadic MCI/ CDR 0.5/prodomal AD and mild AD subjects that might be especially amenable to γ-secretase modulators [29]. Again, serial CSF examinations of normal elderly and presymptomatic and prodromal AD (such as those performed by the ADNI [24]) will be required in order to determine precisely if and when any CSF p3-Alc_α spike exists and whether the beginning of the p3-Alc_α spike heralds the onset of the Aβ accumulation phase. If so, then periodic determination of a panel of CSF biomarkers (including Aβ42, Aβ40, and p3-Alc_α) in populations at risk might be useful in determining when to initiate clinical trials of Aβ−lowering agents [25]. This concept dovetails well with recent evidence showing that dramatic changes in CSF Aβ42/Aβ40 are observed in some subjects, and these dramatic outlier values can be used to reveal subjects with spontaneous PS1 mutations [30]. Plasma levels of p3-Alc_α were parallel to CSF levels in preclinical stages of disease of subjects (Figure 5). Therefore peripheral sampling may be informative, thereby avoiding the inconvenience of serial CSF sampling, although we have not examined the correlation in MCI/CDR 0.5/prodromal AD and AD subjects. Finally, if the addition of CSF p3-Alc_α determination turns out to contribute useful information about clinical state or pathogenesis, one might consider adding additional γ-secretase reaction products to the panel (e.g., ephrin B [31], ephrin B receptor [32]) in order to establish whether many or all γ-secretase substrates are implicated in this putative stage in the molecular pathogenesis of AD that is characterized by γ-secretase dysfunction, impaired transmembrane domain peptide clearance, or both.

The causes of sporadic AD may be various, and some clinical populations with sporadic mild cognitive impairment (also known as CDR0.5 and prodromal AD) and mild AD showed an increase in the CSF levels of transmembrane domain peptides derived from integral membrane proteins such as Alc and APP. The CSF p3-Alc_α levels paralleled the plasma levels, indicating that peripheral information might reflect the pathological state in brain, at least where γ-secretase malfunction is concerned. This endophenotype may caused by (1) disturbed processing of APP and Alc by γ-secretase; (2) a reduction in clearance mechanism of these peptides; or (3) both. Patients in this category might be especially responsive to drug therapeutics aimed at modulation of γ-secretase function and/or transmembrane domain peptide clearance.

Sam Gandy, Katsuya Urakami, Toshiharu Suzuki are co-senior authors for this study.