Intense multidisciplinary research efforts during the last decades have provided detailed knowledge on the molecular pathogenesis of AD, which has been translated into novel promising therapies with putative disease-modifying effects. Several promising drug candidates, such as Aβ immunotherapy and secretase inhibitors, are now being tested in clinical trials. However, because the predicted clinical effect of this type of disease-modifying drugs is a less-pronounced slope in the rate of cognitive deterioration, without any early symptomatologic effect, very large clinical trials with extended treatment periods will be needed to identify a beneficial clinical effect by using rating scales. Thus, biomarker evidence from smaller short-term clinical trials that the drug has the predicted biochemical mode of action directly in patients with AD would be valuable for making a go/no-go decision for expensive Phase III clinical trials. Thus, a great need exists for biomarkers to identify and monitor the biochemical effect of disease-modifying drugs in AD clinical trials.
The main focus with disease-modifying drugs is to inhibit brain Aβ production and aggregation and to increase Aβ clearance from the brain. γ-Secretase inhibitors have previously been shown to reduce Aβ1-40 and Aβ1-42 production and secretion in cells and to reduce soluble Aβ and amyloid plaque burden in mice [10
]. These results have made the γ-secretase complex one of the top targets for developing AD therapeutics. Here we show that the novel Aβ isoforms Aβ1-14, Aβ1-15, and Aβ1-16, together with Aβ1-34, may serve as sensitive biomarkers for γ-secretase inhibition by LY450139 in the CNS of AD patients.
In a previous study using ELISA measurements of CSF Aβ1-40 and Aβ1-42, the expected reduction of the peptides in response to LY450139 treatment was not found [19
]. It was suggested that this lack of changes might be the result of a rapid transport of Aβ from CSF into plasma or that longer treatment duration may be required to identify changes [19
]. In another study using the SILK method to examine whether an effect on Aβ production could be identified with LY450139 treatment, it was shown that Aβ production in the CNS decreased while Aβ clearance remained stable [21
]. The lack of effect on CSF Aβ1-40 and Aβ1-42, despite the reduced Aβ production, may be because the different techniques are measuring different targets. SILK analysis of Aβ turnover requires that all Aβ isoforms are digested with trypsin before analysis, and a cleavage product consisting of Aβ17-28 is then measured by using MS [20
]. Thus, total Aβ (that is, the mean of all of the very different Aβ isoforms that contain the Aβ17-28 sequence) is measured. This means that all longer isoforms detected in the present study (Aβ1-33, Aβ1-34, Aβ1-37, Aβ1-38, Aβ1-40, and Aβ1-42) will contribute to the mass spectrometric signal. The results presented herein suggest that the reduction in Aβ1-34, the generation of which is γ-secretase dependent [10
], may contribute to the overall reduction of Aβ detected in CSF by using the SILK method.
Previous experimental studies on certain cultured cells expressing wild-type human APP have shown that γ-secretase inhibitor can increase in α- and β-secretase cleavage products along with the expected increase in APP C-terminal fragments (C99 APP-CTF) [28
]. Further, recent data have shown that the levels of Aβ1-14, Aβ1-15, and Aβ1-16 are elevated in cell media and CSF from transgenic mice treated with γ-secretase inhibitors and that these shorter isoforms are derived from concerted cleavages of APP by β- and α-secretase, thus reflecting a third metabolic pathway for APP [23
]. Data in this study suggest that these shorter Aβ isoforms may be sensitive novel biomarkers for γ-secretase inhibitor treatment, even at doses that do no affect the CSF levels of Aβ1-40 or Aβ1-42. The increased levels of the isoforms detected after γ-secretase inhibitor treatment might be explained by an increased amount of substrate (that is, β-CTF or C99) (Figure ), for α-secretase, after APP is cleaved by β-secretase.
Here we verify these findings directly in living AD patients (that is, a major increase of the shorter isoforms and a slight increase of α-sAPP, in CSF from AD patients treated with a γ-secretase inhibitor). The unchanged CSF levels of Aβ1-40 and Aβ1-42 are in agreement with previous studies [19
] and suggest that these markers are less sensitive to detect γ-secretase inhibition as compared with Aβ1-14, Aβ1-15, and Aβ1-16, possibly because they may be influenced by other factors, such as brain amyloid load and/or Aβ oligomerization or that they are present at higher levels in CSF and that higher doses are needed to detect effects on these biomarkers in AD patients. Unchanged β-sAPP levels and only slightly elevated α-sAPP levels suggest that γ-secretase inhibition does not result in any major change in the release of these APP fragments to the CSF. It is at this stage unclear whether these fragments are degraded further or if they simply are present in such high concentrations in the CSF that a very high γ-secretase inhibitor dose is needed to detect an effect on CSF α-sAPP and β-sAPP levels.