Figure 1 _-secretase activity is increased during DNA-damage ...

By Tiffany Willis,2014-02-10 02:29
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Figure 1 _-secretase activity is increased during DNA-damage ...

Letter to the Editor

DNA damage-inducing agent-elicited -secretase activity is dependent on Bax/Bcl-2

    pathway but not on caspase cascades

Dear Editor,

Accumulation of senile plaques composed of amyloid-(A) is a pathological hallmark

    1of Alzheimer’s disease (AD), and A is generated through the sequential cleavage of

    2amyloid precursor protein (APP) by - and -secretases. -secretase excises the

    3ectodomain of APP (-APPs) to leave a 99-amino acid long C-terminal fragment (APP-C99-CTF) in the membrane. γ-Secretase then cleaves this membrane-tethered APP-CTF within the transmembrane domain, releasing A peptides and APP-intracellular domain

    (AICD). As such, - and -secretase are regarded to perform the key steps in the pathogenesis of AD and have become important therapeutic targets in the prevention and treatment of AD. As a result of much effort on identifying the natures of these secretases,

    4presenilin (PS) was found to be the first obligatory component of -secretase. Further

    biochemical analyses revealed that -secretase is composed of multicomponent

    complexes, and another membrane protein, nicastrin (NCT) was identified to be a

    5,6component of the γ-secretase complex by co-immunoprecipitation studies. Moreover,

    two other membrane proteins, anterior pharynx-defective phenotype 1 (APH-1) and PS enhancer 2 (PEN-2) were identified as components of -secretase by two independent

    7,8studies using genetic screening in Caenorhabditis elegans. Finally, it has been shown


    that -secretase activity can be reconstituted by co-expressing human PS, NCT, APH-1,

    9-11and PEN-2 in yeast, a Drosophila cell line, or mammalian cells, providing clear

    evidence that these four proteins compose the minimal constituents of the active γ-

    secretase complex.

     Despite the importance of -secretase as a therapeutic target for AD and the

    enormous progress made in the biochemical characterization of -secretase over recent

    years, relatively few studies have elaborated the endogenous mechanism regulating -

    secretase activity. In this regard, some of previous studies suggest a close relationship

    12between apoptosis and the A-mediated pathogenesis of AD. Galli et al reported

    increased amyloidogenic secretion when cerebellar granule cells were committed to

    13apoptosis by KCl deprivation, and Tesco et al reported a marked increase in total A

    and A levels in Chinese Hamster Ovary (CHO) cells treated with staurosporine or 42

    14etoposide. Ohyagi et al also reported increased level of cellular A during apoptosis 42

    in fetal guinea pig brain cells, suggesting that a death signal regulates the processing of APP by modulating secretase activity. Currently, however, no details are available concerning the mechanism underlying cell death-induced elevation of A production. We

    undertook this study to elucidate the mechanism underlying -secretase modulated

    enhancement of amyloidogenic processing of APP during cell death, based on luciferase

    15reporter gene and in vitro peptide cleavage assay findings.

     Initially, we generated a stable cell line (CHO-C99) coexpressing UAS-controlled luciferase reporter gene and the cDNA for C99 containing GAL4/VP16 transactivating

    16domain (C99-GV) using CHO cells, as previously described. In these cells, -secretase-

    dependent cleavage of C99-GV released the APP intracellular domain containing GV


    transactivation domain (AICD-GV) from the membrane. The released APP domain then translocated to the nucleus and activated the expression of firefly luciferase reporter gene

    under the control of UAS cis-elements.

    Using this cell line, we examined whether DNA-damage inducing agents (DDIAs), i.e., etoposide and camptothecin, can affect -secretase-mediated cleavage of APP. We

    found that etoposide-treated CHO-C99 cells displayed dramatically enhanced -secretase

    activity (Figure 1a). Moreover, this DDIA-induced increase in γ-secretase activity was

    markedly attenuated by NCT specific siRNA. The treatment of a -secretase specific

    inhibitor, inhibitor X (also known as L-685,458; 2 M), also diminished -secretase

    activity to the control level, demonstrating the specificity of DDIA effect on -secretase

    activity (Figure 1a).

    Etoposide-elicited regulation of -secretase activity was found to be a dose dependent response (Supplementary Figure 1a). Treatment of camptothecin, another DNA-damaging agent, for 24 hr after serum starvation for 1 day also activated -secretase in a

    dose-dependent manner (Supplementary Figure 1a), suggesting that -secretase activity

    enhancement is associated with apoptosis-inducing activity of DDIA.

    To confirm whether the stimulatory effect of DDIA on -secretase activity is cell type-

    or assay method-specific, we stimulated ANPP cells, which overexpress all 4 -secretase

    17components and Swedish mutant APP in HEK cell, with various concentrations of

    etoposide or camptothecin. In vitro peptide cleavage assays showed the same stimulatory effect of DDIAs on -secretase activity in ANPP cells (Figure 1b). Moreover, Western blotting with APP specific antibody 6E10 (epitope: 1-17 of A sequence in APP C99,

    Signet) showed that APP C99 levels were significantly decreased by DDIA treatment


(lower panel of Figure 1b). These results demonstrate similar DDIA effects on -

    secretase activity regardless of cell type or assay methods.

    Under these conditions, we measured caspase-3 activity as a marker of apoptosis. Treatment of both CHO-C99 and ANPP cell lines with etoposide increased caspase-3 activity in a dose dependent manner (Supplementary Figure 1b). Morphology and DNA fragmentation assay data obtained from etoposide or camptothecin treated cells showed the progress of cell death (Supplementary Figure 1c), indicating that DDIA triggered apoptosis in these cells. We also examined whether DDIA-triggered up-regulation of -

    secretase activity affects S3 cleavage of Notch, which is another -secretase substrate.

    For this, we transiently expressed both Notch1 mutant construct with a deleted extracellular domain (EN1-GV) and UAS-luciferase reporter gene in CHO cells. When we applied etoposide to these cells, -secretase activity was consistently enhanced, as

    observed in APP C99-GV (Supplementary Figure 1d), indicating that DDIA effect was not limited to APP as a -secretase substrate.

    To examine whether stimulation of DDIA-induced -secretase activity affects A

    generation, both secreted and intracellular forms of A and A levels were measured 4042

    from conditioned media (CM) and cell lysates of HBA cells, which overexpress Swedish mutant APP and -secretase (BACE1) in HEK cell, after treating various concentrations of etoposide. Both A and A levels were significantly increased in both CM and cell 4042

    lysates following etoposide treatment (Figure 1c & 1d, respectively). Etoposide-induced A generation was blocked by inhibitor X treatment, indicating that DDIA-induced increase of A generation is dependent on -secretase activity.

    To elucidate the mechanism underlying DDIA-induced -secretase activity, the


    expression of each of the four -secretase components was examined by Western blotting using each specific antibody (Supplementary Figure 1e). No significant increase in the expression level of these components was observed. A slight reduction of immature NCT band was detected, but the reason for this is not clear.

    We next determined whether caspase activation is involved in DDIA-triggered regulation of -secretase. It has been well documented that DDIA treatment can activate

    18caspase, as shown in Supplementary Figure 1b. We treated CHO-C99 cells with a

    potent cell-permeable caspase-3 inhibitor, z-DEVD-fmk (100 M) or a pan-caspase

    inhibitor, z-VAD-fmk (100 M) in the presence of etoposide. The treated caspase

    inhibitors effectively blocked the caspase-3 activities, as expected. However, DDIA-dependent stimulation of -secretase activity was not suppressed by these inhibitors (Figure 1e), indicating that the modulation of -secretase activity triggered by DDIA is

    not a downstream event of caspase cascades.

    19Because Bax can regulate apoptotic process in the upstream of caspase cascades,

    we examined whether -secretase activity is regulated by Bax translocation. When etoposide or camptothecin was added to CHO-C99 cultures with/without furosemide (a Bax translocation inhibitor, 2 mM), the marked reductions in -secretase activity was

    observed in both cases (upper panel of Figure 1f).

    Then, to determine whether Bcl-2/Bax system is essential for DDIA-elicited stimulation of -secretase, CHO-C99 cells were transiently transfected with Bcl-2 cDNA,

    20which antagonizes Bax function. The overexpression of Bcl-2 in CHO-C99 cells

    dramatically blocked -secretase activation triggered by DDIAs (lower panel of Figure 1f), indicating that Bcl-2/Bax dependent death pathway mediates the DDIA-induced


modulation of -secretase activity.

    Although previous reports suggest a close correlation between cell death and the A-

    mediated pathogenesis of AD, few studies have demonstrated how cell death can affect the proteolytic processing of APP. Here, we provide evidence that DDIA-elicited -

    secretase activity is dependent on Bax/Bcl-2 pathway, but not on caspase cascades. Based on these results, we propose that cell death pathways including Bax translocation, triggered by various apoptotic stimuli, critically facilitate the generation of A by

    activating -secretase. Because increased level of A acts as another death signal, a

    feedback loop between cell death and A generation can result in progress of cell death

    process in the sporadic AD brain. Our results suggest that blockade of apoptosis during the early pathologic stage presents as a good therapeutic target for the intervention in the pathogenesis of AD.

    1,21,221SM Jin, HJ Cho, MW Jung, I Mook-Jung*

    1Department of Biochemistry & Cancer Research Institute, College of Medicine, Seoul National University, Seoul, Korea, 110-799.

    2Neuroscience & Technology graduate program, Ajou University, Suwon, Korea, 443-479.

    * Corresponding author: I Mook-Jung, Department of Biochemistry & Cancer Research Institute, College of Medicine, Seoul National University, Seoul, Korea, 110-799. Tel: 82 2 740 8245; Fax 82 2 3672 7352; E-mail:



    We thank Dr. Young-Keun Jung (Gwangju Institute of Science and Technology, Gwangju, Korea) for the C99-GV construct, Dr. Sangram Sisodia (University of Chicago) for the ANPP cell line and the EN1-GV construct, and Dr. Gyesoon Yoon

    (Ajou University, Suwon, Korea) for the Bcl-2 construct. This work was supported by KOSEF and AARC (RO1-2004-000-10271-0 and R11-2002-097-05001-2, respectively) and by the 21C frontier functional proteomics project of the Korean Ministry of Science & Technology (FPR05C2-010).

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Titles and legends to figures

Figure 1 DNA-damage inducing agents (DDIAs) increase -secretase activity and A

    generation in a Bcl-2/Bax dependent manner. (a) Etoposide treatment increased -

    secretase activity. Etoposide (100 M; Sigma) was added to CHO-C99 cells for 48 hr. Luciferase assay was performed with 10 g of total protein using Luciferase Reporter Assay System (Promega), as instructed by the manufacturer. Expression levels of luciferase were measured using a Bio-Imaging Analyzer (LAS-3000; Fuji). To confirm the specificity of -secretase activity up-regulation by etoposide, NCT specific siRNA (Dharmacon) or the γ-secretase specific inhibitor, inhibitor X (2 M; Calbiochem), was

    administered. (b) In vitro peptide cleavage assay showing dose-dependent increases in -

    secretase activity by DDIAs. ANPP cells were treated with DDIAs at designated concentrations for 24 hr. The preparations of crude membrane fraction and

    15measurements of -secretase activity in vitro were performed as described previously.

    Ten M of inhibitor X was added to confirm the specificity of the in vitro cleavage assay

    system. To verify in vitro peptide cleavage assay results, the protein levels of APP-CTF (C99) were examined by Western blotting. (c) Etoposide treatment increased A

    accumulation in conditioned media (CM) of HBA cells. Etoposide was treated to HBA cells at the indicated concentrations with/without inhibitor X (5 M). After 48 hrs, CM

    were collected and subjected to the sandwich ELISA using anti-A N-terminal specific

    antibody (capturing antibody) and anti-A or A C-terminal specific antibody 4042

    (detection antibody) according to the manufacturers instruction (Human amyloid

    Immunoassay Kit; Biosource). (d) Etoposide treatment increased intracellular A;? levels.


Total protein extracts were prepared from HBA cells in Figure 1c with RIPA buffer.

    levels in 300 g of total protein were measured as above. (e) Increased -secretase

    activity was independent of caspase-3 activity. CHO-C99 cells were pretreated with a caspase-3 specific inhibitor, z-DEVD-fmk (100 M; Calbiochem) or a pan-caspase

    inhibitor, z-VAD-fmk (100 M; Calbiochem) for 30min and then with etoposide (100

    M) for 48 hr for luciferase activity and 24 hr for caspase-3 activity assay, respectively. Luciferase activity was measured as described in Figure 1a. Caspase-3 activity in 100 g

    of cytosolic protein was measured using a CaspACE? Assay System (Promega), as

    instructed by the manufacturer. (f) Bax inhibitors antagonize the effect of DDIAs on -

    secretase activity. Upper panel; -secretase activity enhancement was inhibited by

    furosemide, which blocks Bax translocation to mitochondria. CHO-C99 cells were pretreated with furosemide (Furo in figure; 2 mM; Sigma) for 30mins, and then etoposide or camptothecin was added for 48 or 24 hr, respectively. Luciferase activity was measured as described in Figure 1a. Lower panel; Bcl-2 overexpression inhibited DDIAs-induced activation of -secretase. CHO-C99 cells were transiently transfected

    with Bcl-2 cDNA or pcDNA 3.1 and then treated with 100 M etoposide or 25 M

    camptothecin for 48 or 24 hr, respectively. Luciferase activity was measured as described in Figure 1a. All results were presented as means ? S.E. and represent three independent experiments. *** P<0.001, ** P<0.01, * P<0.05 vs. vehicle treated controls. ???

    P<0.001, ?? P<0.01 vs. the DDIA treated sample. Open bar: vehicle-treated control, black bar: etoposide-treated cells, hatched bar: camptothecin-treated cells. Eto: etoposide, Cmt: camptothecin, Vcl: vehicle, Inh: inhibitor.


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