Mechanisms that synergistically regulate η-secretase processing of APP and Aη-α protein levels: relevance to pathogenesis and treatment of Alzheimer's disease

Abstract

The pathophysiology of Alzheimer's disease (AD) is characterized by the formation of cerebral β-amyloid plaque from a small peptide amyloid-β (Aβ). Aβ is generated from the canonical amyloid-β precursor protein (APP) proteolysis pathway through β- and γ-secretases. Decreasing Aβ levels through targeting APP processing is a very promising direction in clinical trials for AD. A novel APP processing pathway was recently identified, in which η-secretase processing of APP occurs and results in the generation of the carboxy-terminal fragment-η (CTF-η or η-CTF) (Wang et al., 2015) and Aη-α peptide (Willem et al., 2015). η-Secretase processing of APP may be up-regulated by at least two mechanisms: either through inhibition of lysosomal-cathepsin degradation pathway (Wang et al., 2015) or through inhibition of BACE1 that competes with η-secretase cleavage of APP (Willem et al., 2015). A thorough characterization of η-processing of APP is critical for a better understanding of AD pathogenesis and insights into results of clinical trials of AD. Here we further investigated η-secretase processing of APP using well-characterized cell models of AD. We found that these two mechanisms act synergistically toward increasing η-secretase processing of APP and Aη-α levels. Furthermore, we evaluated the effects of several other known secretase modulators on η-processing of APP. The results of our study should advance the understanding of pathophysiology of AD, as well as enhance the knowledge in developing effective AD treatments or interventions related to η-secretase processing of APP.

Figure 1

APP processing. The familial AD gene APP encodes the amyloid-β precursor protein, which undergoes serial proteolytic cleavages and generates two small pathogenic proteins: Aβ and Aη-α. Specifically the cleavage of APP produces Aβ through β- and γ-secretases; or Aη-α through η- and α-secretases. Aβ peptides display several isoforms, e.g., Aβ₃₈, Aβ₄₀, and Aβ₄₂. Aβ₄₂ is prone to aggregation and forms oligomers and then β-amyloid plaque, while Aη-α does not directly appear in the β-amyloid plaque. Abbreviations: APP, amyloid-β precursor protein; CTF, carboxy-terminal fragment; AICD, APP-intracellular domain protein.

Figure 2

Both ALLN and BACE1i increased the pathogenic Aη-α protein levels. Stably-transfected 7PA2 cells were treated with different doses of ALLN or 1 µM BACE1 inhibitor for 24 hrs. Cells were harvested and then cell lysates and conditioned medium were analyzed by WB. A. Cell lysates were subjected to WB and the M3.2 antibody was used for detection of APP-CTFβ/η, Aβ, and Aη-α. B. The pathogenic Aη-α protein levels were quantified and demonstrated via densitometry for samples in A. C. Conditioned medium was subjected to WB for the detection of sAPPβ with the sAPPβ antibody.

Figure 3

Effects of pharmacological agents on Aη-α levels and APP processing. Stably-transfected 7PA2 cells were treated with different pharmacological agents for 24 hrs and harvested. Cell lysates and conditioned medium were analyzed by WB. A-B: Cell lysates were subjected to WB for the detection of APP-CTFβ/η using 6E10 (A) or M3.2 antibody (B), respectively. The η-secretase processing products of APP, CTFη and Aη-α, are underlined. C-D: Conditioned medium was subjected to WB and probed with 6E10 (C) or M3.2 (D) antibody, respectively, to detect Aβ and sAPPα.

Figure 4

ALLN and BACE1i synergistically and robustly increased Aη-α levels. Stably-transfected 7PA2 cells were treated with different pharmacological agents for 24 hrs and harvested. A. Cell lysates were prepared and subjected to WB for the detection of APP processing products through 6E10 antibody. Aη-α levels were increased by 1 µM BACE1i and 10 µM ALLN alone, and more robustly increased by the combined treatment of 1 µM BACE1i and 10 µM ALLN. Short and long exposures (focused on areas around Aη-α) were indicated. B. Cell lysates from cells treated with 10 µM ALLN and different doses of BACE1i (0, 0.125, 0.25, 0.5, and 1 µM) were analyzed by WB for detecting Aη-α levels via the M3.2 antibody. The densitometric measurement for the samples was presented below.