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-Alcα 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 ), and mild AD [5, 14]. In order to quantify these peptides reliably and conveniently, we have recently developed an ELISA for p3-Alcα. This ELISA system quantifies total p3-Alcα levels, but does not specifically measure the individual species of p3-Alcα. In the current paper, we have employed this ELISA to quantify total p3-Alcα 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-Alcα were highly correlated in these cohorts, the current data support the use of p3-Alcα as a surrogate for total APP-derived γ-cleaved products. Elevated levels of p3-Alcα and Aβ were also observed in plasma samples of some female AD patients . 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-Alcα levels in MCI and AD patients, but we observed no systematic differences between male and female subjects . Therefore, it is worth noting that the observation of a sex specific increase in p3-Alcα levels in plasma of female AD patients has not been consistently observed in all cohorts studied.
The increase in p3-Alcα 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-Alcα. 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 , 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-Alcα
speciation and also p3-Alcα
levels may be affected. When these observations are taken together with the model of altered CSF peptide clearance , and the evidence that clearance of Aβ from CSF is modulated in an APOE-isoform-specific manner , we now must consider it equally likely that altered p3-Alcα 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  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-Alcα 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-Alcα 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-Alcα in AD. In order to address this issue directly, analysis of the p3-Alcα levels in the identical samples studied by Castellano et al. is under consideration.
It is interesting to note that both the quality and quantity of p3-Alcα 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)  should be a useful resource in pursuing this hypothesis.
Since p3-Alcα is not incorporated into cerebral or cerebrovascular amyloid, the decrease in p3-Alcα 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 . 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  and then plateaus . 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 .
If the apparent transient elevation of levels of p3-Alcα 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-Alcα levels (or the coordinate elevation of CSF Aβ40 and p3-Alcα 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 . Again, serial CSF examinations of normal elderly and presymptomatic and prodromal AD (such as those performed by the ADNI ) will be required in order to determine precisely if and when any CSF p3-Alcα spike exists and whether the beginning of the p3-Alcα 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-Alcα) in populations at risk might be useful in determining when to initiate clinical trials of Aβ−lowering agents . 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 . Plasma levels of p3-Alcα 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-Alcα 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 , ephrin B receptor ) 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.