No endogenous Aβ production in presenilin-deficient fibroblasts

To the editor

Presenilins (PSs) are involved in the cleavage of integral membrane proteins, such as amyloid-β precursor protein (APP), Notch, ErbB-4 and CD44. The question as to whether or not they are proteases is therefore relevant for the broad field of cell biological research. Recently, Armogida et al. reported that small amounts of the Aβ amyloid peptide can be detected in mouse PS-deficient fibroblasts¹. This result challenged the conclusions of previous publications^2,3. However, the assay used by Armogida et al.¹ is not specific for mouse Aβ and we were not able to reproduce their findings using a mouse Aβ-specific ELISA assay (Fig. 1a). When we used a similar immune precipitation/western blotting assay as Armogida et al.¹, we did however detect trace amounts of Aβ peptide, even in the unconditioned culture medium (Fig. 1b). This assay is critically dependent on the monoclonal antibody, WO-2, which was raised against a specific human Aβ epitope. As both rabbit and bovine Aβ, but not mouse Aβ, are identical to human Aβ, they will be equally and efficiently detected by the WO-2 antibody. Thus, bovine Aβ from the serum added to the culture medium or rabbit Aβ (see accompanying letter by Grimm et al.) in the sera used to immunoprecipitate Aβ are potential sources of false-positive results. Although it is possible to incubate fibroblasts for brief periods in serum-free media (see accompanying letter by Petit et al.), it remains uncertain whether the bovine Aβ that accumulated before can be completely depleted from the cells. In any event, the affinity of the WO-2 antibody is too low to detect mouse Aβ from cells that overexpress mouse APP in this assay (Fig. 1c), demonstrating unequivocally that the Aβ detected in the experiments of Armogida et al. cannot be of mouse origin. Thus, the claim that PS-deficient fibroblasts secrete Aβ is not supported by the published data. A more extensive pdf report will be made available on request.

Figure 1: Presenilin-deficient fibroblasts do not synthesize Aβ.

a, Culture media of wild-type (WT) and PS-deficient (PSKO) fibroblasts were assayed for the presence of rodent Aβ in four independent experiments. No Aβ was detected in the PSKO cells. b, Next, total Aβ was immunoprecipitated from the unconditioned medium, the conditioned culture medium (supernatant) and the cell extracts, and detected by western blotting using the WO2 monoclonal antibody, as described by Armogida et al.¹. Positive signals were detected in all lanes, including unconditioned medium, probably reflecting the presence of bovine or rabbit Aβ. c, Finally, neuronal cells were transfected with Semliki Forest Virus (SFV) coding for human APP (Hu APP), mouse APP (Mo APP) or human APP containing the Swedish mutation (Sw APP). Cells were metabolically labelled, and Aβ was immunoprecipitated and visualized by autoradiography (top). The same blot was then probed with WO2. Although human Aβ is clearly detected, mouse Aβ is not (bottom).