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Comparative Study
. 2013 Jan:49:137-47.
doi: 10.1016/j.nbd.2012.08.011. Epub 2012 Aug 24.

Constitutive α- and β-secretase cleavages of the amyloid precursor protein are partially coupled in neurons, but not in frequently used cell lines

Alessio Colombo  1 , Huanhuan Wang  2 , Peer-Hendrik Kuhn  1 , Richard Page  1 , Elisabeth Kremmer  3 , Peter J Dempsey  4 , Howard C Crawford  5 , Stefan F Lichtenthaler  6
Affiliations
Comparative Study

Constitutive α- and β-secretase cleavages of the amyloid precursor protein are partially coupled in neurons, but not in frequently used cell lines

Alessio Colombo et al. Neurobiol Dis. 2013 Jan.

Abstract

Proteolytic cleavage of the amyloid precursor protein (APP) by the two proteases α- and β-secretases controls the generation of the amyloid β peptide (Aβ), a key player in Alzheimer's disease pathogenesis. The α-secretase ADAM10 and the β-secretase BACE1 have opposite effects on Aβ generation and are assumed to compete for APP as a substrate, such that their cleavages are inversely coupled. This concept was mainly demonstrated in studies using activation or overexpression of α- and β-secretases. Here, we report that this inverse coupling is not seen to the same extent upon inhibition of the endogenous proteases. Genetic and pharmacological inhibition of ADAM10 and BACE1 revealed that the endogenous, constitutive α-secretase cleavage of APP is largely uncoupled from β-secretase cleavage and Aβ generation in neuroglioma H4 cells and in neuronally differentiated SH-SY5Y cells. In contrast, inverse coupling was observed in primary cortical neurons. However, this coupling was not bidirectional. Inhibition of BACE1 increased ADAM10 cleavage of APP, but a reduction of ADAM10 activity did not increase the BACE1 cleavage of APP in the neurons. Our analysis shows that the inverse coupling of the endogenous α- and β-secretase cleavages depends on the cellular model and suggests that a reduction of ADAM10 activity is unlikely to increase the AD risk through increased β-secretase cleavage.

Keywords: Alpha-secretase; Alzheimer's disease; Amyloid precursor protein; Beta-secretase.

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Figures

Fig. 1
Fig. 1
BACE1 protein level in different cell types. Western blot analysis of BACE1 protein level in H4, HEK293E (EBNA), HEK293T, differentiated SH-SY5Y, HCT8 and primary cortical neuronal cells. Cellular lysates were blotted for BACE1 using mAb 3D5. Loading control: tubulin.
Fig. 2
Fig. 2
ADAM10 and BACE1 are not coupled in neuroglioma H4 cells. Shown are immunoblots and the densitometric quantification of the indicated proteins and peptides. (A) Cells were transfected with a pool of siRNA specific for ADAM10 (A10 KD). (B) Cells were treated for 24 h with the metalloprotease inhibitor TAPI1 (50 μM) or the BACE1 inhibitor C3 (2 μM). (C) Cells were transfected with a pool of siRNA specific for BACE1 (B1 KD). CTR: pool of control, non-targeting siRNA. Cellular lysates and media were blotted for full length APP (APP FL, mAb 2C11), total secreted APP ectodomain (tot APPs, mAb 22C11), soluble APPα ectodomain (APPsα, mAb 14D6), soluble APPβ ectodomain (APPsβ, pAb 192wt), ADAM10 and BACE1 (mAb 3D5). Loading control: tubulin. Aβ40 level in culture media detected by Meso Scale Discovery (MSD) sandwich immunoassay. Quantifications show mean value of at least six independent experiments (± SEM), *=p<0.05, **=p<0.01 and ***=p<0.001 (compared to CTR).
Fig. 3
Fig. 3
ADAM10 and BACE1 are not coupled in differentiated neuroblastoma SH-SY5Y cells. SH-SY5Y cells were differentiated for three days into neuron-like cells and then treated as indicated. Shown are immunoblots and the densitometric quantification of the indicated proteins and peptides. (A) Cells were transfected with a pool of siRNA specific for ADAM10 (A10 KD). (B) Cells were treated for 24 h with the metalloprotease inhibitor TAPI1 (50 μM). (C) Cells were transfected with a pool of siRNA specific for BACE1 (B1 KD) or (D) treated with the BACE1 inhibitor C3 (2 μM). CTR: pool of non-targeting siRNA. Cellular lysates and media were blotted for full length APP (APP FL, mAb 2C11), total secreted APP ectodomain (tot APPs, mAb 22C11), α soluble APP ectodomain (APPsα, mAb 14D6), ADAM10 and BACE1 (mAb 3D5). β soluble APP ectodomain level (APPsβ) in culture media was detected by specific ELISA assay. Loading control: tubulin. Aβ40 level in culture media detected by Meso Scale Discovery (MSD) sandwich immunoassay. Quantifications were from at least six independent replicates (±SEM), *=p<0.05 **=p<0.01 and ***=p<0.001 (compared to CTR).
Fig. 4
Fig. 4
Characterization of embryonic neuronal cortical cultures and antibody 5G11 specificity. (A) Primary cortical neuronal cultures were fixed at 8DIV and immunostained for neuronal cells (β3 tubulin in red) and glial cells (GFAP in green). Nuclei were stained with the TOPRO reagent in blue. Staining was repeated with at least 3 independent neuronal preparations. (B) Western blot analysis of cell lysates and media from HEK293 cells overexpressing murine full length APP695 (mAPP695), the α soluble ectodomain of APP (APPsα), the β ′ soluble ectodomain (APPsβ ′) or the β ectodomain (APPsβ). CTR: wild type HEK293E cells. Cellular lysates were blotted for full length APP (APP FL, mAb 2C11), while culture media for total secreted APP ectodomain (tot APPs, mAb 22C11), α soluble APP ectodomain (APPsα, mAb 5G11), β soluble APP ectodomain (APPsβ, pAb 192wt). Loading control: tubulin.
Fig. 5
Fig. 5
α and β activity coupling in primary cortical neurons. Shown are immunoblots and the densitometric quantification of the indicated proteins and peptides. (A) ADAM10 conditional knock-out neurons transducted with a lentiviral vector for the CRE recombinase expression to induce gene knock out (A10 KO). (B) cells treated for 24 h with metalloproteinase family inhibitor (TAPI1, 50 μM) and BACE1 inhibitor (C3, 2 μM) and (C) wild type neurons infected with lentiviral vectors to express specific shRNAs against BACE1 sequence (sh1 and sh2 — BACE1 KD). CTR = not transducted neurons. Empty = empty viral vector; Scr = scramble shRNA. Cellular lysates and media were blotted for full length APP (APP FL, mAb 2C11), total secreted APP ectodomain (tot APPs, mAb 22C11), α soluble APP ectodomain (APPsα, mAb 5G11), β soluble APP ectodomain (APPsβ, pAb 192wt), ADAM10 and BACE1 (mAb 3D5). Loading control: tubulin. Aβ40 level in culture media detected by Meso Scale Discovery (MSD) sandwich immunoassay. Quantifications were from at least six independent replicates (±SEM), *=p<0.05 **=p<0.01, ***=p<0.001 (compared to CTR).
Fig. 6
Fig. 6
ADAM10 compensates for BACE1 inhibition in primary cortical neurons. Western blot analysis and densitometric quantification of ADAM10 knock-out primary cortical neurons (A10 KO) treated for 24 h with TAPI1 (50 μM) and C3 (2 μM). CTR: non-transduced neurons. Emp: Empty viral vector. Cellular lysates and media were blotted for full length APP (APP FL, mAb 2C11), total secreted APP ectodomain (tot APPs, mAb 22C11), α soluble APP ectodomain (APPsα, mAb 5G11), β soluble APP ectodomain (APPsβ, pAb 192wt) and ADAM10. Loading control = tubulin. Quantifications were from at least six independent replicates (±SEM), *=p<0.05 **=p<0.01, ***=p<0.001 (compared to CTR).
Fig. 7
Fig. 7
ADAM10 is not required for TAPI1-induced increase in APPsβ level. Western blot analysis and densitometric quantification of primary cortical wild-type (WT) neurons and conditional floxed ADAM10 knock-out neurons transduced with a control virus (Empty (Emp), still expressing ADAM10) or with CRE-virus (A10 KO, ADAM10 knock-out) treated for 24 h with TAPI1 (50 μM) or C3 (2 μM). CTR: non-transduced neurons. Emp: empty viral vector. Cellular lysates and media were blotted for full length APP (APP FL, mAb 2C11), total secreted APP ectodomain (tot APPs, mAb 22C11), α soluble APP ectodomain (APPsα, mAb 5 G11), β soluble APP ectodomain (APPsβ, pAb 192wt) and ADAM10. Loading control = tubulin. Quantifications were from at least six independent replicates (±SEM), *=p<0.05 **= p<0.01, ***=p<0.001 (compared to control CTR).
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