Biochimica et Biophysica Acta 1842 (2014) 127–134

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta

journal homepage: www.elsevier.com/locate/bbadis

Wild type and mutant amyloid precursor proteins influence downstream

effects of proteasome and autophagy inhibition
Valentina Cecarini a,⁎, Laura Bonfili a, Massimiliano Cuccioloni a, Matteo Mozzicafreddo a, Giacomo Rossi b,
Jeffrey N. Keller c, Mauro Angeletti a, Anna Maria Eleuteri a
a
School of Biosciences and Biotechnology, University of Camerino, 62032 Camerino, Italy
b
School of Medical Veterinary Sciences, University of Camerino, 62024 Matelica, Italy
c
Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, LA, USA

a r t i c l e i n f o a b s t r a c t

Article history: Cells rely on complementary proteolytic pathways including the ubiquitin–proteasome system and autophagy to
Received 1 August 2013 maintain proper protein degradation. There is known to be considerable interplay between them, whereby the
Received in revised form 9 October 2013 loss of one clearance system results in compensatory changes in other proteolytic pathways of the cell. Distur-
Accepted 1 November 2013
bances in proteolysis are known to occur in Alzheimer's disease, and potentially contribute to neurophysiological
Available online 8 November 2013
and neurodegenerative processes. Currently, few data are available on how the presence of wild type and mutant
Keywords:
amyloid precursor protein (APPwt and APPmut) potentially alters the reciprocal interplay between the different
Amyloid precursor protein intracellular proteolytic pathways. This study used human SH-SY5Y neuronal cell lines, and SH-SY5Y transfected
Cathepsin B with either APPwt or APPmut (valine-to-glycine substitution at position 717), in order to explore if the presence
Ubiquitin–proteasome system of APPwt or APPmut altered the downstream effects of pharmacological proteasome or autophagy inhibition.
Autophagy The occurrence of APPwt or APPmut was observed to disturb proteasome or autophagy activities upon treat-
ment with proteasome inhibitors or authophagy inhibitors. Interestingly, APPwt and APPmut expression was
observed to significantly and robustly enhance the induction in cathepsin B following the administration of an
established proteasome inhibitor. The presence of APPwt and APPmut also significantly reduced the elevation
in ubiquitinated proteins following proteasome inhibitor treatments. Our data strongly suggest that APP is able
to affect the downstream effects of protease inhibition in neural cells including enhancement of cathepsin B activity,
with these changes in cathepsin B significantly and inversely related to the levels of ubiquitinated protein.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction the regulation of several cellular functions for example cell cycle
progression, DNA repair, apoptosis, gene transcription, signal transduc-
Aging and a wide variety of neurodegenerative conditions, including tion, senescence and immune response [6,7]. Autophagy or self-eating
Alzheimer's disease (AD), are characterized by alterations in the normal is a lysosomal degradation pathway in charge of the recycling of dys-
cellular homeostasis with dysregulation of the proteostasis network. functional organelles or aggregated proteins [8]. Different processes of
Impairments in the functionality of proteolytic pathways favor the accu- autophagy have been characterized based on the modality of cargo rec-
mulation of misfolded and abnormal proteins resulting in the deposi- ognition and delivery to lysosomes: macroautophagy, microautophagy,
tion of toxic aggregates [1,2]. Cells possess two major intracellular and chaperone-mediated autophagy (CMA) [8–11]. Only proteins can
protein degradation systems namely the ubiquitin–proteasome system be delivered to lysosomes via CMA, whereas both macroautophagy
(UPS) and autophagy. For several years they have been thought as and microautophagy concern the degradation of proteins and organ-
distinct and separated complexes whereas recently an increasing elles [10–15].
number of data elucidated their intimate correlation [3–5]. The UPS Alzheimer's disease, the most common form of neurodegenerative
is the major degradation system used by cells to remove short-lived condition, is characterized by intracellular and extracellular accumula-
and misfolded proteins. The UPS-mediated processing of proteins allows tion of toxic protein aggregates. The deposition of these inclusions
further determines alterations in the functionality of the two clearance
systems with a general reorganization of the proteolysis. Additionally,
Abbreviations: APP, amyloid precursor protein; Aβ, amyloid-β; UPS, ubiquitin–pro- in vivo studies indicated that the accumulation of amyloid plaques, a
teasome system; ChT-L, chymotrypsin-like; T-L, trypsin-like; PGPH, peptidylglutamyl- major hallmark of the pathology, can be influenced by both the UPS
peptide hydrolyzing; AP-N, aminopeptidase N; BrAAP, branched-chain amino acids
preferring; 3MA, 3-methyladenine; IHC, immunohistochemistry
system and the autophagy [16,17]. Plaques are a primary cause for pro-
⁎ Corresponding author. Tel.: +39 0737 403247. teolysis inhibition in AD and are mainly constituted of amyloid-beta
E-mail address: [email protected] (V. Cecarini). peptides, Aβ(1–40) and Aβ(1–42). Both peptides derive from the

0925-4439/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.bbadis.2013.11.002
128 V. Cecarini et al. / Biochimica et Biophysica Acta 1842 (2014) 127–134

processing of the amyloid precursor protein (APP). Recent advances determined by the method of Bradford [24] using bovine serum albu-
have revealed that mutant APP could favor AD development and pro- min (BSA) as standard.
gression regulating the generation of free radicals and oxidative damage
and causing structural alterations in mitochondria, with the production 2.3. Cell viability assay
of defective organelles and a decrease in mitochondrial trafficking
[18–21]. Currently, little is known in terms of how the presence of Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-
wild type or mutant forms of the amyloid precursor protein (APPwt diphenyltetrazolium bromide assay (MTT) [25]. After experimental
and APPmut, respectively) potentially alters the balance between the dif- treatment, MTT was added to the culture medium to a final concentra-
ferent intracellular proteolytic pathways. We recently demonstrated that tion of 0.5 mg/mL and incubated for 2 h at 37 °C. The medium was
the expression of an APP mutant isoform correlates with an increase in replaced with 100 μL DMSO and the optical density was measured at
oxidative stress and a remodeled pattern of protein degradation, with 550 nm in a microtiter plate reader. At least six cultures were utilized
both marked inhibition of proteasome activities and impairment in the for each time point.
autophagic flux [3].
In the present work, we focused our attention on determining if 2.4. Western blotting
the presence of APPwt or APPmut altered the downstream effects of
proteasome/autophagy inhibition on protein ubiquitination and prote- Cell lysates were then resolved by 12% SDS‐PAGE and electroblotted
ase activity. On this purpose, human SH-SY5Y neuroblastoma cells stably onto PVDF membranes. Membranes with transferred proteins were in-
transfected with either wild-type APP gene (APPwt) or 717 valine-to- cubated with primary monoclonal antibodies and successively with
glycine APP-mutated gene (APPmut) were used as experimental model. the specific peroxidase-conjugated secondary monoclonal antibodies
Interestingly, we obtained that the presence of APPwt or APPmut is (Santa Cruz Biotech, Heidelberg). The immunoblot detection was
able to influence the alterations in proteasome or autophagy activity performed with an ECL western blotting analysis system. Each gel was
induced by the treatment with proteasome or authophagy inhibitors. loaded with molecular weight markers, in the range 6.5 kDa to 205 kDa
Additionally, APPwt or APPmut robustly enhanced the induction in (Sigma-Aldrich S.r.l., Milano, Italy). Glyceraldehyde-3-phosphate
cathepsin B and, at the same time, significantly reduced the elevation dehydrogenase (GAPDH) was utilized as control for equal protein
in ubiquitinated proteins following the administration of an established loading: membranes were stripped and re-probed for GAPDH using
proteasome inhibitor. a monoclonal antibody diluted 1:500. Bands were quantified as report-
ed elsewhere [26].
2. Materials and methods
2.5. Measurements of proteasome activities in cell lysates
2.1. Reagents and chemicals
20S proteasome peptidase activities in cell lysates were determined
with fluorogenic peptides: Suc-Leu-Leu-Val-Tyr-AMC was used for ChT-
Substrates for assaying the chymotrypsin-like (ChT-L), trypsin-like
L activity, Z-Leu-Ser-Thr-Arg-AMC for T-L activity, Z-Leu-Leu-Glu-AMC
(T-L), peptidylglutamyl-peptide hydrolyzing (PGPH), aminopeptidase
for PGPH activity, and Z-Gly-Pro-Ala-Leu-Ala-AMC for BrAAP activity.
N (AP-N) activities, proteasome inhibitors (Z-Gly-Pro-Phe-Leu-CHO,
The incubation mixture contained 1 μg of cell lysate, the appropriate
lactacystin and MG132) and the autophagy inhibitor 3-methyladenine
substrate and 50 mM Tris–HCl pH 8.0, up to a final volume of 100 μL.
(3MA) were purchased from Sigma-Aldrich S.r.L. (Milano, Italy). The
Incubation was performed at 37 °C, and after 60 min the fluorescence
substrate Z-Gly-Pro-Ala-Leu-Ala-MCA to test the branched chain
of the hydrolyzed 7-amino-4-methyl-coumarin (AMC) was detected
amino acids preferring (BrAAP) activity was obtained by Biomatik
(AMC, λexc = 365 nm, λem = 449 nm) on a SpectraMax Gemini XPS
(Cambridge, Ontario). Aminopeptidase N (EC 3.4.11.2) for the coupled
microplate reader. The 26S proteasome ChT-L activity was tested using
assay utilized to detect BrAAP activity [22] was purified from pig kidney
Suc-Leu-Leu-Val-Tyr-AMC as substrate and 50 mM Tris–HCl pH 8.0 buff-
as reported elsewhere [23]. SH-SY5Y transfected with APPwt or
er containing 10 mM MgCl2, 1 mM dithiothreitol and 2 mM ATP. BrAAP
APPmut gene were a kind gift of Prof. Uberti Daniela, University of
activity was determined in a coupled test in the presence of aminopepti-
Brescia, Italy. Cathepsin B substrate, Z-Arg-Arg-AMC, and inhibitor,
dase N. The effective 20S proteasome contribution to short peptide cleav-
CA074Me, were obtained from Sigma-Aldrich S.r.L. (Milano, Italy).
age was evaluated with control experiments performed using specific
Cathepsin B antibody was purchased from Millipore (Billerica, MA).
proteasome inhibitors, Z-Gly-Pro-Phe-Leu-CHO and lactacystin (5 μM in
All the other antibodies used in the present work were purchased
from Santa Cruz Biotech (Heidelberg, Germany). Media and chemicals
used for cell cultures were purchased from Euroclone (Milano, Italy).
Membranes for western blotting analyses were purchased from Millipore
(Milano, Italy). Proteins immobilized on films were detected with the
enhanced chemiluminescence (ECL) system (Amersham Pharmacia
Biotech, Milano, Italy).

2.2. Cell treatment

Cell transfection was performed as previously reported [3]. Cells

were grown in 100-mm tissue culture dishes at an initial concentration
of 2 × 106 cells/dish and were exposed to MG132 (5 μM) or 3MA
(5 mM) for 24 h. After removing the medium and washing with cold
phosphate buffered saline (PBS), cells were harvested in 4 mL of PBS
and centrifuged at 1600×g for 5 min. The pellet was resuspended in a
lysis buffer (20 mM Tris, pH 7.4, 250 mM sucrose, 1 mM EDTA and Fig. 1. Cell viability upon 24 h treatment with MG132 (5 μM) or 3MA (5 mM) was deter-
mined with the MTT assay performed as described in the Materials and methods section.
5 mM β-mercaptoethanol) and passed through a 29-gauge needle at Results are expressed as percentage of viable cells compared to control untreated cells and
least ten times. Lysates were centrifuged at 12,000×g for 15 min and show a 30% decrease of viable cells in the presence of the proteasome inhibitor MG132
the supernatants were stored at −80 °C. Protein concentration was (n = 5).
 V. Cecarini et al. / Biochimica et Biophysica Acta 1842 (2014) 127–134 129

the reaction mixture). The fluorescence values of lysates were subtracted proteolysis was evaluated through control experiments performed
of the values of control assays in the presence of the two inhibitors. using the specific inhibitor CA074Me. Fluorescence values obtained
by analyzing the lysates were then subtracted of the values of control
2.6. Measurements of cathepsin B activity in cell lysates assays in the presence of the inhibitor.

Cathepsin B proteolytic activity was measured following the proto- 2.7. Immunocytochemistry
col described by Tchoupè et al. [27] using the fluorogenic peptide Z-
Arg-Arg-AMC at a final concentration of 50 μM. The mixture, containing For immunocytochemistry, cells were cultured in a Chamber Slide
1 μg of protein lysate, was incubated in 100 mM phosphate buffer system, and after 24 h of incubation with MG132 or 3MA, the mono-
pH 6.0, 1 mM EDTA and 2 mM dithiothreitol for 1 h at 30 °C. The fluo- layers were rapidly fixed in a 50:50 mixture of methanol and acetone
rescence of the hydrolyzed 7-amino-4-methyl-coumarin (AMC, λexc = for 5 min. In monolayers, apoptotic index was highlighted through a
365 nm, λem = 449 nm) was detected on a SpectraMax Gemini TUNEL colorimetric staining (DeadEnd, Promega) according to the man-
XPS microplate reader. The effective cathepsin contribution to the ufacturer's instructions. For evaluation of the apoptotic rate, ten random

Fig. 2. Panel I) T.U.N.E.L. and immunohistochemical results of control SH-SYSY, APPwt and APPmut cell lines after 24 h treatment with MG132 and 3MA. Apoptotic cells are characterized
by black-brownish nuclear stain. Violet stain indicates cathepsin B positive cells (red arrows) whereas brown stain indicates ubiquitin positive cells (brown arrows). Low levels of cathep-
sin B positive cells and ubiquitin positive cells in the SH-SYSY line are observed (A). Higher levels of ubiquitin expression are detected in APPwt and APPmut cell lines (B–C), while cathep-
sin B highest expression is observed in APPmut line (C). 24 h incubation with MG132 triggers the apoptotic pathway in the three cell lines (D, E and F) and enhances ubiquitin expression. A
strong cathepsin B staining in APPmut cells is obtained after MG132 treatment (F). 24 h incubation with 3MA induces in the three cell lines similar low levels of apoptosis and a decrease in
ubiquitinated proteins (G, H, and I) (see text for further details). (IHC with Meyer's hematoxylin nuclear counterstain; original magnification, 400. Bar = 25 μm.). Panel II) Bar graphs
show the percentage of cathepsin B and ubiquitin positive cells calculated in each cell line with respect to the specific untreated control. Data are analyzed as reported in the Materials and
Methods section.
130 V. Cecarini et al. / Biochimica et Biophysica Acta 1842 (2014) 127–134

fields of any chamber were examined under a dry-× 40 objective. the margins of the chamber were not considered for evaluation to avoid
TUNEL-positive cells are characterized by a brownish-black nuclear inflation of positive cell numbers.
stain. For double-labeling immunocytochemistry, monolayers were first
incubated with the anti-Ub antibody (1:50, Santa Cruz Biotech., Heidel- 2.8. Statistical analysis
berg, Germany); with the anti-cathepsin B antibody (1:100, Santa Cruz
Biotech., Heidelberg, Germany), and then with biotin-labeled goat Results are expressed as mean values and standard deviation of
anti-rabbit (1:200, Jackson ImmunoResearch, West Grove, PA) second- results obtained from five separate experiments. Statistical analysis
ary antibody. The binding of the antibody was detected with the was performed with one way ANOVA, followed by the Bonferroni test
Elite kit (Vector Laboratories), and the immunoreaction was developed using Sigma-stat 3.1 software (SPSS, Chicago, IL, USA). p-Values b0.05
using two different chromogens: violet (VIP, Vector, Burlingame UK) for and b0.01 were considered to be significant.
cathepsin-B and brown (DAB, Vector) for ubiquitin stain. Lower-power
digitized images were acquired with a BX-60 microscope (Olympus, 3. Results
Melville, NY) equipped with a DEI-470 digital camera (Optronics,
Goleta, CA). For scoring of immunohistochemical positive cells, stained Recent data from our laboratory and other groups evidenced as a
cells were calculated using the mentioned light microscope, a ×40 feature of neurodegenerative disorders the occurrence of an integration
objective, a ×10 eyepiece, and a square eyepiece graticule (10×10 between the two main intracellular clearance systems, proteasome and
squares, with a total area of 62,500 μm2). Ten randomly selected sites autophagy, suggesting for example the activation of compensatory
were chosen for each chamber and arithmetic means were calculated. autophagy in the presence of proteasomal impairments [28–30]. In
Results are expressed as % of IHC positive cells calculated with respect this paper our goal is to better describe the interplay between the two
to their own untreated control. For all parameters considered, cells on proteolytic pathways determining also if the presence of the APP wild-

Fig. 3. Proteasome activities in control SH-SY5Y, APPwt and APPmut cells upon 24 h exposure to the proteasome inhibitor MG132 (5 μM) and the autophagy inhibitor 3MA (5 mM). The
ChT-L, T-L, PGPH and BrAAP activities were measured in cell lysates as described in the Materials and Methods section. 3MA determines a clear and component-dependent activation of the
proteasomal system, with the ChT-L and T-L activities being the most influenced. This activation is mostly evident in SH-SY5Y cells. As expected, MG132 induces a considerable decrease in
proteasome activity (see text for further details). Results are presented as percentage of residual activity toward the respective untreated control. Fluorescence units were subtracted of the
values of control assays in the presence of specific inhibitors. Data points marked with an asterisk are statistically significant compared to their respective non-treated control cells
(*p b 0.05, **p N 0.01).
 V. Cecarini et al. / Biochimica et Biophysica Acta 1842 (2014) 127–134 131

type form or the APP Val717Gly mutated form may influence such rela- assays performed with primary antibodies directed against ubiquitinated
tionship. On this purpose we treated control SH-SY5Y, SH-SY5Y APPwt proteins and p27, two established markers of proteasomal functionality.
and SH-SY5Y APPmut cells with a well-known proteasome inhibitor, Results shown in Fig. 4 evidence a decrease in the levels of both markers
MG132 (5 μM final concentration), and with 3MA (5 mM final concen- upon 3MA treatment in the three cell lines, in agreement with the data
tration), an inhibitor of the autophagosome formation process [31,32], from the activity assays. As expected, the treatment with the proteasome
for 24 h. Cells were first tested for viability showing a 30% decrease of inhibitor MG132 induced a considerable decrease in proteasome activity
viable cells in the presence of MG132 (Fig. 1). Immunocytochemical and accumulation of its substrates. Interestingly, following the adminis-
analyses indicated an evident positivity to TUNEL staining, character- tration of the proteasome inhibitor, the presence of APPwt and APPmut
ized by black-brownish nuclei, in the three cell lines upon exposure to significantly reduced the elevation in ubiquitinated proteins (Fig. 4).
the proteasome inhibitor (Fig. 2), confirming the ability of MG132 to Results obtained from immunocytochemical analyses were in agreement
trigger the apoptotic pathway [33,34]. Enzymatic assays and western with both evidences on ubiquitin data: in Fig. 2, in fact, panels D, E and F
blotting analyses were then conducted in order to monitor the function- show that 24 h incubation with MG132 was able to enhance ubiquitin
ality and expression levels of components of both pathways upon ex- expression, with the highest levels detected in SH-SY5Y control cells,
posure to the inhibitors. Inhibiting autophagy with 3MA produced a whereas panels G, H, and I illustrate that the treatment with 3MA in-
clear and component-dependent activation of the proteasomal system duced a decrease in the levels of ubiquitinated proteins in the three
(Fig. 3). In particular, not transfected SH-SY5Y cells showed a 50% in- cell lines when compared to the respective untreated cells. Additionally,
crease of the ChT-L activity of the 20S proteasome. An evident increase IHC performed on untreated cells confirmed our previous data on basal
in the 20S ChT-L component was also observed in APPwt and APPmut proteasomal activity and ubiquitin intracellular levels [3]: as a result of
cells, with 3MA inducing a 40% and 25% increase, respectively. A similar an inhibited proteasomal system, in fact, ubiquitinated proteins were
pattern was obtained measuring the ChT-L activity of the 26S protea- considerably higher in transfected cells compared to control SH-SY5Y
some, a structure in charge of the degradation of polyubiquitinated sub- cells (Fig. 2 panels A, B and C).
strates resulted from the binding of the 19S regulatory particle to the We then investigated the way autophagy reacts to the induction
20S catalytic core. Also the T-L proteasomal component resulted particu- of proteasome inhibition. We analyzed the expression levels of three
larly enhanced in not transfected SH-SY5Y and APPwt cells with a 45% known markers of the autophagic pathway: p62, LC3 and beclin-1.
and 30% increase, respectively, compared to the respective untreated The LC3 protein exists in cells in two forms, namely LC3-I (cytosolic)
cells. It is interesting to note that in the presence of the mutant form of and LC3-II (membrane-bound). When autophagy is activated, LC3-I is
APP the increase in proteasome activity is extremely limited. A possible processed and recruited into the autophagosome, where LC3-II is gener-
reason for this reduced activation is that basal activity levels of the UPS ated by site-specific proteolysis and lipidation near the C-terminus.
system in APPmut cells are lower if compared to non-transfected cells Similarly, beclin-1 is involved in the enrolment of membranes to form
thus indicating the presence of a less efficient and less reactive complex autophagosomes, whereas p62 binds to both LC3-II and ubiquitin, and
[3]. The PGPH and BrAAP components only evidenced a slight increase is finally degraded in autophagolysosomes [35,36]. Results shown in
in the presence of the inhibitor and no differences due to the presence Fig. 5 clearly evidence an activation of the autophagic pathway in re-
of APPwt and APPmut were detected. The activation of the proteasomal sponse to MG132-induced proteasome inhibition. In detail, we obtained
system in the presence of 3MA was confirmed by western blotting a decrease in the amount of p62, whose levels inversely correlate with

Fig. 4. Detection of the levels of ubiquitinated proteins and p27 in control SH-SY5Y, APPwt and APPmut cells upon 24 h exposure to the proteasome inhibitor MG132 (5 μM) and the
autophagy inhibitor 3MA (5 mM). In accordance to the data on proteasomal activities, in the three cell lines both markers result up-regulated upon 3MA treatment (see text for further
details). Panels A and B show, respectively, representative immunoblots and densitometric analyses obtained from five separate experiments. Equal protein loading was verified by using
an anti-GAPDH antibody. The detection was executed by an ECL western blotting analysis system. Data points marked with an asterisk are statistically significant compared to their
respective non treated control cells (*p b 0.05, **p b 0.01). (A.U.) arbitrary units.
132 V. Cecarini et al. / Biochimica et Biophysica Acta 1842 (2014) 127–134

Fig. 5. Detection of the autophagy-related proteins LC3-II, beclin-1 and p62 in control SH-SY5Y, APPwt and APPmut cells upon 24 h exposure to the proteasome inhibitor MG132 (5 μM)
and the autophagy inhibitor 3MA (5 mM). Results clearly evidence an activation of the autophagic pathway in response to MG132 with a decrease in the amount of p62 and a simultaneous
increase in the levels of both LC3-II and beclin-1 (see text for further details). Panels A and B show, respectively, representative immunoblots and densitometric analyses obtained from five
separate experiments. Equal protein loading was verified by using an anti-GAPDH antibody. The detection was executed by an ECL western blotting analysis system. Data points marked
with an asterisk are statistically significant compared to their respective non treated control cells (*p b 0.05, **p b 0.01). (A.U.) arbitrary units.

the autophagic activity, and an increase in the levels of both LC3-II and cathepsin B. Treating with 3MA, an enhancement in the levels of the
beclin-1 that suggests an up-regulated formation of autophagosomes. enzyme was evident in control SH-SY5Y and APPwt cells (Fig. 2, panels
Recent findings similarly demonstrated adaptive changes in autophagy G and H). This increase can be addressed considering that the antibody
after UPS impairment in Parkinson's disease where SH-SY5Y cells is able to bind both forms of cathepsin B, the proenzyme and the mature
treated with lactacystin showed a significant increase in the expres- form. Conversely, in the western blotting experiment we were able to
sion levels of LC3-I/II and beclin-1 and reduced levels of p62 [28]. separate through gel electrophoresis the two forms and only the active
Changes in the autophagic pathway induced by MG132 were partic- form is shown (Fig. 6).
ularly evident in transfected cells, mainly in those with the APPmut
gene (Fig. 5). 4. Discussion
We then tested if MG132 treatment induced some effect on
the activity of cathepsin B, a lysosomal cysteine protease of the papain The results shown in the present paper contribute to strengthen
family, and if the presence of either APPwt or APPmut could influence the available data on the strict interdependence between the two
this effect. Interestingly, a remarkable activation of the enzyme was main intracellular proteolytic pathways, the UPS and autophagy [3,4],
observed when the proteasome was inhibited (Fig. 6). SH-SY5Y cells and evidence a role for APP in altering the downstream effects of the in-
showed a 2.4-fold increase of cathepsin B activity compared to the re- hibition of proteasome or autophagy on the activity of both pathways.
spective untreated control. This increase was absolutely more evident As for the first point, we clearly demonstrated that the inhibition of
in APPwt and APPmut cells that showed a 5.45- and 6.4-fold enhance- one system promotes a compensatory reaction of the other and that
ment, respectively, compared to their own untreated control. As re- this effect is bidirectional. In details, the inhibition of the autophagic
vealed by the western blotting, MG132 induced in the three cell lines pathway, through the use of 3MA, determines an activation of the
an increased expression of mature cathepsin B, most likely responsible proteasomal system, mainly evident for the ChT-L and T-L components,
for the described up-regulated activity. Immunocytochemical assay, and also confirmed by the corresponding decreased expression of two
performed using an anti-cathepsin B antibody, detected a similar and known proteasomal substrates, ubiquitinated proteins and p27. On
remarkable increase in cathepsin B expression upon MG132 exposure. the other hand, blocking proteasome functionality with MG132 resulted
Panels E and F of Fig. 2, corresponding to APPwt and APPmut cells treated in a strong upregulation of autophagy, as revealed by the changes ob-
with MG132, show an intense violet stain corresponding to high levels of served in the levels of proteins of the autophagic cascade. It is therefore
 V. Cecarini et al. / Biochimica et Biophysica Acta 1842 (2014) 127–134 133

activity are higher in control neuronal cells, whereas cathepsin B basal

activity is upregulated in APPmut cells.
Furthermore, we reported that APPwt and APPmut expression
was able to robustly enhance the induction in cathepsin B and to signif-
icantly reduce the elevation in ubiquitinated proteins following the ad-
ministration of an established proteasome inhibitor. These data strongly
suggest a role for APP in the modulation of the downstream effects of
protease inhibition in neural cells including enhancement of cathepsin
B activity, with these changes in cathepsin B activity significantly and
inversely related to the levels of ubiquitinated protein. The activation
of cathepsin B processing toward its mature form can be therefore con-
sidered a strategy of AD neurons to get rid of the ubiquitinated protein
overload induced by MG132 treatment. Additionally, considering that in
AD models, with or without familial mutations, cathepsin B was able
to reduce Aβ(1–42) levels, thus contrasting its accumulation and aggre-
gation [38,39], increasing its activity could offer a potential strategy in
the treatment of AD.

References
[1] T. Morawe, C. Hiebel, A. Kern, C. Behl, Protein homeostasis, aging and Alzheimer's
disease, Mol. Neurobiol. 46 (2012) 41–54.
[2] K. Dasuri, L. Zhang, J.N. Keller, Oxidative stress, neurodegeneration, and the
balance of protein degradation and protein synthesis, Free Radic. Biol. Med. 62
(2013) 170–185.
[3] V. Cecarini, L. Bonfili, M. Cuccioloni, M. Mozzicafreddo, G. Rossi, L. Buizza, D. Uberti,
M. Angeletti, A.M. Eleuteri, Crosstalk between the ubiquitin–proteasome system and
autophagy in a human cellular model of Alzheimer's disease, Biochim. Biophys. Acta
1822 (2012) 1741–1751.
[4] Q. Zheng, J. Li, X. Wang, Interplay between the ubiquitin–proteasome system and au-
tophagy in proteinopathies, Int. J. Physiol. Pathophysiol. Pharmacol. 1 (2009) 127–142.
[5] C. Park, A.M. Cuervo, Selective autophagy: talking with the UPS, Cell Biochem.
Biophys. 64 (2013) 3–13.
[6] A. Ciechanover, Intracellular protein degradation: from a vague idea thru the lysosome
and the ubiquitin–proteasome system and onto human diseases and drug targeting,
Cell Death Differ. 12 (2005) 1178–1190.
[7] M. Orlowski, The multicatalytic proteinase complex, a major extralysosomal proteo-
lytic system, Biochemistry 29 (1990) 10289–10297.
Fig. 6. Cathepsin B functionality and cathepsin B expression levels in control SH-SY5Y, [8] E. Wong, A.M. Cuervo, Integration of clearance mechanisms: the proteasome and
APPwt and APPmut cells upon 24 h exposure to the proteasome inhibitor MG132 autophagy, Cold Spring Harb. Perspect. Biol. 2 (2010) a006734.
(5 μM) and the autophagy inhibitor 3MA (5 mM). A significant activation of the enzyme, [9] N. Mizushima, B. Levine, A.M. Cuervo, D.J. Klionsky, Autophagy fights disease through
mainly evident in APPwt and APPmut cells, is observed upon proteasome inhibition with a cellular self-digestion, Nature 451 (2008) 1069–1075.
corresponding increased expression of the mature form of cathepsin B (see text for further [10] Z. Yang, D.J. Klionsky, An overview of the molecular mechanism of autophagy, Curr.
details). Panel A shows data on cathepsin B activity measured as described in the Materials Top. Microbiol. Immunol. 335 (2009) 1–32.
and Methods section. Results are presented as percentage of residual activity toward the [11] S. Kaushik, A.M. Cuervo, Chaperone-mediated autophagy: a unique way to enter the
lysosome world, Trends Cell Biol. 22 (2012) 407–417.
respective untreated control. Fluorescence units were subtracted of the values of control
[12] B. Levine, D.J. Klionsky, Development by self-digestion: molecular mechanisms and
assays in the presence of a cathepsin B specific inhibitor. Panels B and C show, respectively,
biological functions of autophagy, Dev. Cell 6 (2004) 463–477.
a representative immunoblot related to cathepsin B expression and the corresponding den- [13] W.W. Li, J. Li, J.K. Bao, Microautophagy: lesser-known self-eating, Cell. Mol. Life Sci.
sitometric analyses obtained from five separate experiments. Equal protein loading was ver- 69 (2012) 1125–1136.
ified by using an anti-GAPDH antibody. The detection was executed by an ECL western [14] D.J. Klionsky, C. P., The mechanism and physiological function of macroautophagy,
blotting analysis system. Data points marked with an asterisk are statistically significant J. Innate Immun. 5 (2013) 427–433.
compared to their respective non treated control cells (*p b 0.05, **p b 0.01). (A.U.) arbi- [15] E. Arias, A.M. Cuervo, Chaperone-mediated autophagy in protein quality control,
trary units. Curr. Opin. Cell Biol. 23 (2011) 184–189.
[16] P. van Tijn, F.J. Dennissen, R.J. Gentier, B. Hobo, D. Hermes, H.W. Steinbusch, F.W. Van
Leeuwen, D.F. Fischer, Mutant ubiquitin decreases amyloid beta plaque formation
in a transgenic mouse model of Alzheimer's disease, Neurochem. Int. 61 (2012)
739–748.
clear that, as components of the intracellular catabolism, autophagy and [17] P. Nilsson, K. Loganathan, M. Sekiguchi, Y. Matsuba, K. Hui, S. Tsubuki, M. Tanaka, N.
UPS are carefully orchestrated and integrated. A deep understanding of Iwata, T. Saito, T.C. Saido, Abeta secretion and plaque formation depend on autophagy,
Cell Rep. 5 (2013) 61–69.
the mechanisms responsible for this integration could certainly be help- [18] P.H. Reddy, Amyloid precursor protein-mediated free radicals and oxidative
ful especially in an attempt to modulate one of the two systems for ther- damage: implications for the development and progression of Alzheimer's disease,
apeutic approaches. For example, considering the occurrence in AD of J. Neurochem. 96 (2006) 1–13.
[19] P.H. Reddy, Amyloid beta, mitochondrial structural and functional dynamics in
serious autophagy and proteasome alterations with a progressive col- Alzheimer's disease, Exp. Neurol. 218 (2009) 286–292.
lapse of the intracellular proteolysis [37], a strategy aimed at compen- [20] Michael H. Yana, Xinglong Wanga, X. Zhu, Mitochondrial defects and oxidative
sating these dysfunctions could ameliorate pathological and cognitive stress in Alzheimer disease and Parkinson disease, Free Radic. Biol. Med. 62 (2013)
90–101.
deficits of AD. [21] X. Wang, B. Su, S.L. Siedlak, P.I. Moreira, H. Fujioka, Y. Wang, G. Casadesus, X. Zhu,
Interestingly, the use of these particular cellular models allowed Amyloid-beta overproduction causes abnormal mitochondrial dynamics via differential
us to establish that the observed downstream effects of proteasome/ modulation of mitochondrial fission/fusion proteins, Proc. Natl. Acad. Sci. U. S. A. 105
(2008) 19318–19323.
autophagy inhibition on the activity of the two degradation systems
[22] M. Orlowski, C. Michaud, Pituitary multicatalytic proteinase complex. Specificity of
are altered by the presence of APPwt or APPmut. In fact, downregulating components and aspects of proteolytic activity, Biochemistry 28 (1989) 9270–9278.
the UPS, autophagy activation was more evident in APPmut cells, con- [23] G. Pfleiderer, Isolation of an Aminopetidase from Kidney Particles, Methods in
versely, reducing autophagy, a higher enhancement of proteasomal Enzymology, Academic Press, New York, NY, 1970.
[24] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram
functionality was detected in SH-SY5Y non-transfected cells. However, quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem.
as previously reported by our group [3], basal levels of proteasome 72 (1976) 248–254.
134 V. Cecarini et al. / Biochimica et Biophysica Acta 1842 (2014) 127–134

[25] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application [32] E. Donohue, A. Tovey, A.W. Vogl, S. Arns, E. Sternberg, R.N. Young, M. Roberge,
to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1983) 55–63. Inhibition of autophagosome formation by the benzoporphyrin derivative verteporfin,
[26] M. Amici, L. Bonfili, M. Spina, V. Cecarini, I. Calzuola, V. Marsili, M. Angeletti, E. J. Biol. Chem. 286 (2011) 7290–7300.
Fioretti, R. Tacconi, G.L. Gianfranceschi, A.M. Eleuteri, Wheat sprout extract induces [33] M. Jullig, W.V. Zhang, A. Ferreira, N.S. Stott, MG132 induced apoptosis is associated
changes on 20S proteasomes functionality, Biochimie 90 (2008) 790–801. with p53-independent induction of pro-apoptotic Noxa and transcriptional activity
[27] T.M.J.R. Tchoupe, F. Gauthier, J.G. Bieth, Photometric or fluorometric assay of cathep- of beta-catenin, Apoptosis 11 (2006) 627–641.
sin B, L and H and papain using substrates with an aminotrifluoromethylcoumarin [34] L. Skalniak, A. Koj, J. Jura, Proteasome inhibitor MG-132 induces MCPIP1 expression,
leaving group, Biochim. Biophys. Acta 1076 (1991) 149–151. FEBS J. 280 (2013) 2665–2674.
[28] Y.F. Shen, Y. Tang, X.J. Zhang, K.X. Huang, W.D. Le, Adaptive changes in autophagy after [35] Y. Cao, D.J. Klionsky, Physiological functions of Atg6/Beclin 1: a unique autophagy-
UPS impairment in Parkinson's disease, Acta Pharmacol. Sin. 34 (2013) 667–673. related protein, Cell Res. 17 (2007) 839–849.
[29] U.B. Pandey, Z. Nie, Y. Batlevi, B.A. McCray, G.P. Ritson, N.B. Nedelsky, S.L. Schwartz, [36] G. Bjorkoy, T. Lamark, S. Pankiv, A. Overvatn, A. Brech, T. Johansen, Monitoring
N.A. DiProspero, M.A. Knight, O. Schuldiner, R. Padmanabhan, M. Hild, D.L. Berry, D. autophagic degradation of p62/SQSTM1, Methods Enzymol. 452 (2009) 181–197.
Garza, C.C. Hubbert, T.P. Yao, E.H. Baehrecke, J.P. Taylor, HDAC6 rescues neurodegen- [37] Y. Ihara, M. Morishima-Kawashima, R. Nixon, The ubiquitin–proteasome system and
eration and provides an essential link between autophagy and the UPS, Nature 447 the autophagic–lysosomal system in Alzheimer disease, Cold Spring Harb. Perspect.
(2007) 859–863. Med. 2 (2012).
[30] U.B. Pandey, Y. Batlevi, E.H. Baehrecke, J.P. Taylor, HDAC6 at the intersection of [38] C. Wang, B. Sun, Y. Zhou, A. Grubb, L. Gan, Cathepsin B degrades amyloid-beta in
autophagy, the ubiquitin–proteasome system and neurodegeneration, Autophagy mice expressing wild-type human amyloid precursor protein, J. Biol. Chem. 287
3 (2007) 643–645. (2012) 39834–39841.
[31] P.O. Seglen, P.B. Gordon, 3-Methyladenine: specific inhibitor of autophagic/lysosomal [39] S. Mueller-Steiner, Y. Zhou, H. Arai, E.D. Roberson, B. Sun, J. Chen, X. Wang, G. Yu, L.
protein degradation in isolated rat hepatocytes, Proc. Natl. Acad. Sci. U. S. A. 79 Esposito, L. Mucke, L. Gan, Antiamyloidogenic and neuroprotective functions of
(1982) 1889–1892. cathepsin B: implications for Alzheimer's disease, Neuron 51 (2006) 703–714.