Available online at www.sciencedirect.com

www.elsevier.com/locate/brainres

b r a i n r e s e a r c h 1 4 7 1 ( 2 0 1 2 ) 1 0 2 – 1 1 7

0006-8993/$ - see frohttp://dx.doi.org/10

Abbreviations: DA

fluorescent protein;

myristate-13-acetate

deoxynucleotidyl tranCorresponding aut

E-mail address:

Research Report

Dopamine induces apoptosis in APPswe-expressingNeuro2A cells following Pepstatin-sensitive proteolysisof APP in acid compartments

Monica Cagnin, Matteo Ozzano, Natascia Bellio, Ilaria Fiorentino, Carlo Follo, Ciro Isidoron

Department of Health Sciences, Laboratory of Molecular Pathology and Nanobioimaging, Universit �a del Piemonte Orientale ‘‘A. Avogadro’’,

Via Solaroli 17, 28100 Novara, Italy

a r t i c l e i n f o

Article history:

Accepted 21 June 2012

A pathological hallmark of Alzheimer’s disease (AD) is the presence within neurons and

the interneuronal space of aggregates of b-amyloid (Ab) peptides that originate from an

Available online 6 July 2012

Keywords:

Alzheimer’s disease

Parkinson’s disease

Pepstatin A

Chloroquine

Lysosome

nt matter & 2012 Elsevie.1016/j.brainres.2012.06.0

PI, 40,6-diamidino-2-ph

IETD-CHO, acetyl-Ile-Gl

; L685,458, [(2R,4R,5S)-2

nsferase-mediated dUTPhor. Fax: þ39 0321 620421isidoro@med.unipmn.it

a b s t r a c t

abnormal proteolytic processing of the amyloid precursor protein (APP). The aspartyl

proteases that initiate this processing act in the Golgi and endosomal compartments. Here,

we show that the neurotransmitter dopamine stimulates the rapid endocytosis and

processing of APP and induces apoptosis in neuroblastoma Neuro2A cells over-expressing

transgenic human APP (Swedish mutant). Apoptosis could be prevented by impairing

Pepstatin-sensitive and acid-dependent proteolysis of APP within endosomal–lysosomal

compartments. The g-secretase inhibitor L685,458 and the a-secretase stimulator phorbol

ester elicited protection from dopamine-induced proteolysis of APP and cell toxicity. Our

data shed lights on the mechanistic link between dopamine excitotoxicity, processing of

APP and neuronal cell death. Since AD often associates with parkinsonian symptoms,

which is suggestive of dopaminergic neurodegeneration, the present data provide the

rationale for the therapeutic use of lysosomal activity inhibitors such as chloroquine or

Pepstatin A to alleviate the progression of AD leading to onset of parkinsonism.

& 2012 Elsevier B.V. All rights reserved.

1. Introduction

Alzheimer’s disease (AD) is a late-onset neurological disorder

characterized by progressive loss of memory and cognitive

abilities as a result of excessive neurodegeneration in the

hippocampus and cortex (Sabuncu et al., 2011). A pathological

hallmark of AD is the presence in the interneuronal space of

r B.V. All rights reserved.25

enylindole dihydrochlorid

u-Thr-Asp-aldehyde inhib

-benzyl-5-(Boc-amino)-4-h

-biotin nick end labeling;.(C. Isidoro).

amyloid plaques formed by aggregates of b-amyloid (Ab)

peptides that originate from an abnormal proteolytic proces-

sing of the amyloid precursor protein (APP). APP is a large

transmembrane type 1 (cytosolic C-terminal) glycoprotein

coded by a gene located on chromosome 21 and giving rise

to eight alternative transcripts, of which three are mainly

transcribed into the isoforms containing 695, 751 and 770

e; DA, dopamine; FITC, fluorescein isothiocyanate; GFP, green

itor (Ac-IETD-CHO); PI, propidium iodide; PMA, phorbol 12-

ydroxy-6-phenyl-hexanoyl]-Leu-Phe-NH2; TUNEL, terminal

ZVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone

b r a i n r e s e a r c h 1 4 7 1 ( 2 0 1 2 ) 1 0 2 – 1 1 7 103

aminoacids (reviewed in Bekris et al., 2010). APP695 mature

protein differs from the whole length APP770 because it lacks

the 290–364 sequence comprising the Kunitz-Protease Inhibi-

tor peptide.

In the endoplasmic reticulum and during its transport

through the Golgi Complex, nascent APP undergoes co- and

post-translational modifications, including N- and O-glyco-

sylation, phosphorylation and tyrosine sulfation, that lead to

the so-called mature APP (Perdivara et al., 2009). Mature APP

is not permanently resident at the plasma membrane, rather

it is subjected to a continuous retrograde trafficking from the

plasma membrane to intracellular compartments of the

secretory pathway (Vieira et al., 2010), so that at steady state

it is more abundant in the Golgi Complex and in endosomes

(Koo et al., 1996; Xu et al., 1997; Yamazaki et al., 1996).

Moreover, APP is not a stable molecule, as it undergoes

proteolysis through multiple and alternative routes. The

proteolytic pathways involved in the APP processing and

the cellular compartments in which this occurs have been

studied in details (for review see Chow et al., 2010; O’Brien

and Wong, 2011; Thinakaran and Koo, 2008; Zhang et al.,

2011). The order of proteolysis at a, b and g sites determines

whether or not the Ab peptide will be produced: the sequen-

tial action of a- and g-secretases leads to the production of a

soluble APPa fragment (sAPPa), a P3 peptide and an intracel-

lular domain (AICD peptide) at the C-terminus, whereas the

sequential action of b- and g-secretases leads to a soluble

APPb fragment (sAPPb), the Ab peptide (of 40 or 42 aminoa-

cids) and the AICD peptide. Thus, proteolysis at b-site is

alternative to that at a-site and is fundamental for amyloi-

dogenesis. The main protease responsible for such proteoly-

sis is b-APP cleaving enzyme (BACE), a type-1 transmembrane

aspartyl protease mainly localized to endosomes, lysosomes

and the Golgi Complex (Cai et al., 2001; Vassar et al., 1999).

Another protease with potential b-secretase activity is lyso-

somal Cathepsin D, which has been shown able to cleave

in vitro APP and produce Ab (Chevallier et al., 1997; Higaki

et al., 1996), and to be highly expressed in AD brain

(Schechter and Ziv, 2008). However, while BACE-deficient

mice do not produce Ab and show normal phenotype (Luo

et al., 2001; Ohno et al., 2004), Cathepsin D-deficient mice still

produce and accumulate Ab in hippocampal neurons (Saftig

et al., 1996). Amyloidogenic processing of APP has been

proved to occur within the Golgi Complex (Xu et al., 1997)

and the endosomal compartment (Pasternak et al., 2004).

Impairing the internalization of plasma membrane APP

reduces the formation of Ab up to 80% (Koo and Squazzo,

1994), as it does the treatment with drugs that rise the

luminal pH of endosomal–lysosomal compartments

(Schrader-Fischer and Paganetti, 1996).

To what extent the trafficking and processing of APP in vivo

occurs constitutively or is affected by the extracellular sti-

muli, and whether and how neurotransmitters influence the

fate of APP and of cells expressing APP is largely unknown.

Here, we report on the effect of dopamine (DA), a neuro-

transmitter diffused in substantia nigra, striatum and other

brainstem nuclei, in neuroblastoma Neuro2A cells over-

expressing human APP695 (Thinakaran et al., 1996), which

is the isoform mainly expressed in human brain (Kang and

Muller-Hill, 1990). Neuro2A cells express muscarinis receptors

(Edwards et al., 1989) and are prone to cholinergic neuronal

differentiation and neurite development (Kojima et al., 1994).

Under appropriate stimulation, Neuro2A express tyrosine

hydroxylase and produce DA and L-DOPA (Akahoshi et al.,

2009) and respond to DA excitotoxicity (Castino et al., 2005).

Therefore, Neuro2A cells can be assumed bona fide as a

valuable in vitro model to study the effects of dopamine on

APP processing. The data here reported extend the previous

knowledge on the relationship between neuronal cell toxicity

and endocytosis and processing of APP, and also provide new

evidence on the mechanism of DA excitotoxicity in neuronal

cells over-expressing APP. The latter may have clinical rele-

vance, given that Parkinson’s-like dopaminergic neurodegen-

eration has been observed in the postmortem brain of AD

patients with extrapyramidal signs (Burns et al., 2005;

Jellinger, 2003; Schneider et al., 2002).

2. Results

2.1. Dopamine triggers the intrinsic apoptotic deathpathway in Neuro2A cells over-expressing transgenic HumanAPP

To address whether the abnormal expression of APP renders

dopaminergic neuronal cells susceptible to DA toxicity, we

employed an established in vitro model system represented

by neuroblastoma mouse Neuro2A cells sham-transfected or

stably expressing transgenic human APP695 in the Swedish-

mutant form (Thinakaran et al., 1996). The cells were exposed

to DA and observed under the microscope for gross morpho-

logical alterations and cell loss at increasing time of incuba-

tion. Evidence of toxic effects was noted starting at 16 h of

exposure to DA only in the transfected Neuro2A expressing

APP. By this time, nuclei staining with DAPI of cells adherent

on sterile coverslips revealed chromatin condensation and

fragmentation, typical signs of apoptosis, in samples of

Neuro2A-APP exposed to DA (Fig. 1A). TUNEL staining con-

firmed the occurrence of DNA fragmentation in these sam-

ples (Fig. 1B). A quantitative estimation of DA toxicity was

obtained by cytofluorometry of the hypodiploid (so-called

subG1 peak) cell population, which mirrors late apoptotic

cells, in the cultures exposed or not for 16 h to DA. While

sham-transfected Neuro2A cells showed negligible sensitiv-

ity, Neuro2A-APPswe cells showed high sensitivity to DA

toxicity (Fig. 1C). As an additional quantification and proof

of the apoptosis induced by DA, we estimated by cytofluoro-

metry the presence of phosphatidyl-serine on the outer

leaflet of the plasma membrane (an early marker of apopto-

sis) in sham- and APP-transfected Neuro2A cells treated in

the absence or in the presence of the pan-caspase inhibitor

zVAD-fmk. Data showed that as much as 40% of the APP-

over-expressing cells treated with DA for 16 h were positive

for annexinV (indicative of phosphatidyl-serine exterioriza-

tion) and that pre-incubation with zVAD-fmk completely

abrogated this effect (Fig. 1D). Taken together, these data

demonstrate that chronic DA stimulation, while not toxic to

the sham-transfected counterpart, causes apoptotic cell

death in Neuro2A cells over-expressing transgenic human

APP. Because of the pro-oxidative nature of DA excitotoxicity,

Fig. 1 – Dopamine induces apoptosis in Neuro2A cells over-expressing human APP. (A) Sham- and human APP (Swedish

mutant)-transfected Neuro2A cells were plated and let adhere on coverslips and then treated or not for 16 h with 250 lM

dopamine (DA). At the end, the nuclei of the cells were labeled with DAPI to evidence chromatin alterations. DA induced

chromatin condensation and fragmentation (arrows) in Neuro2A-APPswe cells, but not in the sham-transfected counterpart.

(B) The cells treated as above were processed for TUNEL fluorescent staining to evidence nicked DNA as a sign of apoptosis.

Images show the presence of TUNEL-positive nuclei in a larger proportion in APP-expressing cells than in sham-transfected

counterpart. (C) Sham-transfected and APPswe-expressing cells were plated on Petri dishes and treated or not for 16 h with

250 lM DA. At the end, adherent and suspended cells were recovered, fixed in ethanol and labeled with PI, and finally

analyzed by cytofluorometry to estimate the hypodiploid (SubG1) cell population. In DA-treated cultures, the percentage of

cells containing a SubG1 amount of DNA was in APP-expressing clones 2.5–3.0-fold that in the parental sham-transfected

clone. (D) Sham-transfected and APPswe-expressing cells were plated on Petri dishes and treated or not for 16 h with 250 lM

DA in the absence or the presence of the pan-caspase inhibitor zVAD-fmk. At the end, adherent and suspended cells were

recovered, labeled with AnnexinV-FITC, and analyzed by cytofluorometry to estimate the proportion of apoptotic cells. DA

greatly increased the proportion of Annexin-FITC-positive cells in APP-expressing cells and this effect was completely

abolished by zVAD-fmk. The fluorescent images and the cytofluorograms shown in this figure are representative of four

independent experiments in triple.

b r a i n r e s e a r c h 1 4 7 1 ( 2 0 1 2 ) 1 0 2 – 1 1 7104

we suspected that the apoptotic pathway involved the

lysosome–mitochondrion axis (Castino et al., 2005, 2007).

We checked the integrity of lysosomes with the acidotropic

fluorochrome Acridine Orange, which fluoresces red when

protonated in acidic compartments and green when in

compartments at neutral pH. The images in Fig. 2A show

that endosomes and lysosomes retained their acidity in the

first 8 h of exposure to DA, whereas by 16 h these organelles

lost their integrity in a large number of Neuro2A-APPswe

cells. As quantified by cytofluorometry, at this time 450% of

the cells exposed to DA had lost their staining with red-

emitting Acridine Orange (Fig. 2B). To assess the integrity of

the outer mitochondrial membrane, we employed the fluor-

ochromes Rhodamine-123 and mitotracker, which emit a red

fluorescence when accumulate in the intermembrane space.

These fluorochromes loose their fluorescence when the outer

mitochondrial membrane becomes leaky and the mitochon-

drial membrane potential (DCm) drops. Cytofluorometry data

of Rhodamine-123 staining (upper panel in Fig. 2C) were

compatible with leakage from mitochondria in Neuro2A-

APPswe cells exposed to DA. To further confirm the activation

of the intrinsic apoptotic pathway, the cells were double-

stained with mitotracker and antibodies against the confor-

mational active bax (Castino et al., 2007). In controls, the

mitochondria were red-stained with mitotracker and no bax

oligomerization was evident, whereas in the cells exposed to

DA mitochondria were not labeled with mitotracker and

oligomerization of bax was clearly present (Fig. 2C, lower

panel). We then checked whether the caspase-8 mediated

extrinsic pathway was also activated by DA. To this end, the

cells were pre-incubated or not with IETD-CHO, a specific

inhibitor of caspase-8, and then cell viability was assessed in

cultures exposed for up to 16 h to DA by using CellTracker, a

fluorescent tracer of the mitochondrial metabolic activity.

In DA-treated cultures, early signs of mitochondrial suffer-

ance were apparent at 12 h and cell loss was clearly evident at

16 h, and the caspase-8 inhibitor could not rescue cell

viability (Fig. 2D), indicating that the extrinsic apoptotic

pathway was not involved in DA toxicity. To define the

temporal hierarchy of the events involving lysosomes and

mitochondria, we performed a parallel Acridine Orange and

bax/mitotracker staining in a time-course experiment in cells

Fig. 2 – Dopamine affects lysosome and mitochondrion integrity in APPswe-expressing Neuro2A cells. (A) Neuro2A cells

expressing the Swedish mutant of human APP695 were plated on coverslips and exposed to DA for increasing time of

incubation. Lysosome integrity was assessed by Acridine Orange staining. Upon DA treatment, in a large proportion of the cells the

acidic compartments cluster at one pole of the cell and eventually (at 16 h) loose the metachromatic fluorescent dye as a

consequence of membrane rupture. (B) Neuro2A-APPswe cells were plated on Petri dishes and treated with DA for 16 h. At the end,

the cells were recovered, labeled with Acridine Orange and analyzed by cytofluorometry to assay the shift in emitted fluorescence

associated with lysosomal leakage (upper panel). A parallel set of cultures on coverslips was imaged under the fluorescence

microscope (lower panel). (C) Neuro2A-APPswe cells were plated on Petri dishes and treated with DA for 16 h. At the end, the cells

were recovered, labeled with Rho-123 and analyzed by cytofluorometry to assay the shift in emitted fluorescence associated with

mitochondrial leakage (upper panel). A parallel set of cultures on coverslips was labeled with mitotracker, fixed and processed for

bax immunofluorescence (lower panel). The images show the occurrence of mitochondrial permeability and bax oligomerization in

cells exposed to DA. (D) Neuro2A-APPswe cells were plated on coverslips and treated with DA for the time indicated in the absence

or in the presence of the caspase-8 inhibitor IETD-CHO, and at the end of incubation the cells were processed for CellTracker

staining and imaging. (E) Neuro2A-APPswe cells were plated on coverslips and treated with DA for the time indicated in the

absence or in the presence of the caspase-8 inhibitor IETD-CHO. At each time point, the coverslips were processed for Acridine

Orange or bax/mitotracker/DAPI staining and imaged under the fluorescence microscope. (F) Neuro2A-APPswe cells were plated on

coverslips and treated with DA for 16 h. At the end, the cells were labeled with mitotracker, fixed and processed for cathepsin D

(catD) immunofluorescence. The images show a cytosolic diffuse staining of cathepsin D associated with mitochondrial

permeability in cells exposed to DA. The fluorescent images and the cytofluorograms shown in this figure are representative of

three independent experiments in triple. (For interpretation of the references to color in this figure legend, the reader is referred to

the web version of this article.)

b r a i n r e s e a r c h 1 4 7 1 ( 2 0 1 2 ) 1 0 2 – 1 1 7 105

exposed to DA in the absence or in the presence of the

caspase-8 inhibitor IETD-CHO. This experiment (Fig. 2E)

demonstrated that (1) lysosome leakage occurred between 4

and 8 h, while bax activation and mitochondria leakage

occurred at a time 48 h and (2) inhibition of caspase-8

neither preclude, nor altered the sequence of, such events.

b r a i n r e s e a r c h 1 4 7 1 ( 2 0 1 2 ) 1 0 2 – 1 1 7106

A direct link between lysosomal leakage and mitochondria

permeabilization was suggested by the concomitant diffuse

cytosolic staining of lysosomal Cathepsin D, a protease

resident in endosomes and lysosomes, and the absence of

mitotracker staining in DA-treated cells at 16 h (Fig. 2E).

Altogether, these data indicate that DA triggers apoptosis in

dopaminergic Neuro2A cells over-expressing APPswe by

sequential destabilization of lysosomes and mitochondria

and subsequent activation of the intrinsic caspase-cascade.

Next, we investigated on the involvement of APP in the

sensitization of neuronal cells toward DA toxicity.

Fig. 3 – Dopamine induces progressive proteolytic

degradation of transgenic human APP in APPswe-

expressing Neuro2A cells. Neuro2A-swe cells adherent on

Petri dishes were exposed to DA for increasing time of

incubation. At the end, cell homogenates were resolved by

electrophoresis and human APP-related peptides were

identified by western blotting using the anti-N46–60

antibody. A specific band with an estimated apparent

molecular weight of 105–110 kDa was detected only in

samples of the Neuro2A-APPswe clone, as expected. The

intensity of this band, as estimated by densitometry

(average of two independent experiments), declined

progressively with time of incubation with DA.

2.2. Dopamine induces the progressive degradation ofAPPswe

We looked at the fate of APP in transfected Neuro2A cells

exposed to DA for increasing time of incubation. The full

length APP protein was identified with an antibody specific for

human APP (anti-N46–60) directed to the N-terminus (residues

46–60). The APP695-related molecular species potentially

recognized by this antibody are reported in Table 1. Sham-

transfected and APPswe-expressing Neuro2A cells were incu-

bated for up to 8 h with DA (a time at which apoptotic signs

are not yet evident), then the presence of APP-related peptides

were identified in cell homogenates by western blotting. The

antibody revealed the presence of a specific band running at

approximately 110 kDa in APPswe-transfected clones, not in

sham-transfected ones (Fig. 3). A second band, faintly detect-

able in both clones, running at approximately 96 kDa was also

detected. This band could tentatively represent either the

a- or b-APP soluble fragment. However, the fact that it was

present in both clones and that it was not reproduced

in other western blotting (see below) indicates that it is an

occasional contaminant. The amount of cell-associated holo-

APP (as revealed by the anti-N46–60) decayed with time of

exposure to DA. Similar data were obtained in independent

experiments in which APP was identified with antibodies

directed to different epitopes (see below), and therefore

proteolysis of only the N-terminal epitope was excluded.

In a separate study, we found a time-dependent accumulation

of the 4 kDa Ab peptide in the culture medium of DA-treated

APPswe-cells (not shown), suggesting that holo-APP decay

could mirror the amyloidogenic processing of APP.

Table 1 – Antibodies used to detect APP-related molecular spe

Antibody Peptide recognized Approx. M

Anti-N46–60 holo-APP 105–115

sAPPb 94–95

sAPPa 96–97

Anti-AbN1–17 holo-APP 105–115

sAPPa 96–97

C99 (597-695) 12

Ab40–42 4

Anti-C676–695 holo-APP 105–115

C99 (597-695) 12

C83 10

AICD 8

2.3. Dopamine toxicity Is associated with the rapidtranslocation of APP into endosomal–lysosomalcompartments

APP has been shown to undergo amyloidogenic processing

following clathrin-dependent endocytosis driven by the YENPTY

(671–676 in APP695) motif at the C-terminus (Koo and Squazzo,

1994). We therefore looked at the intracellular traffic and

localization of APP as affected by DA treatment. In these

experiments, we followed APP with an antibody (anti-C676–695)

directed to the C-terminus (residues 676–695) that allows to

identify the full length protein and its processed C-fragments

(Table 1). Confocal fluorescence imaging at high magnification

showed the presence of peripheral discrete spots indicative of

the presence of APP on the plasma membrane in control cells

and its rapid (within 30 min) translocation into EEA1-positive

vesicles (Early Endosome Antigen 1 is a marker of early endocytic

vesicles) upon DA treatment (Fig. 4A). Consistent with ongoing

cies.

W (kDa) Notes

Secreted and/or degraded

Secreted and/or degraded

Secreted and/or degraded

C-term after b-secretase cleavage (degraded)

Secreted

C-term after b-secretase cleavage (degraded)

C-term after a-secretase cleavage (degraded)

C-term after g-secretase cleavage (degraded)

Fig. 4 – Dopamine induces the rapid endocytosis of APP. Neuro2A cells expressing the Swedish mutant of human APP were

plated on coverslips and exposed or not to DA for the time indicated. (A) At the end, the cells were processed for

immunofluorescence labeling of EEA1 or APP. Alternatively, the cells were first labeled with Dextran-FITC to trace the

endocytic pathway and then fixed and stained for APP by immunofluorescence. Arrows point to co-staining of APP with

markers of early endosomes and with Dextran-FITC in cells exposed to DA. (B) The cells were processed for

immunofluorescence staining of EEA1 and APP. Images show that after a 4 h treatment with DA the bulk of APP is found in

perinuclear clusters. (C) Neuro2A-APPswe cells were plated on coverslips and exposed to DA for the time indicated and then

processed for immunofluorescence labeling of dynamin and APP. Images show that APP moves toward intracellular

compartments upon exposure to DA, while dynamin consistently remains localized beneath the plasma membrane.

(D) Neuro2A-APPswe cells were transfected with a plasmid coding for the fluorescent chimera Cathepsin D-GFP (CatD-GFP)

and then exposed to DA for 1 or 16 h and, at the end, processed for immunofluorescence labeling of APP. Images show that

colocalization of APP with CD-GFP increases with time of exposure to DA. APP was detected with the anti-C676–695 antibody.

The fluorescent images shown in this figure are representative of three independent experiments. (E) and (F) The cells were

plated on Petri dishes, transfected with a control duplex or a Dynamin I-specific siRNA and then treated or not with DA

for 16 h. At the end, the cells were processed for cytofluorometry analysis of the annexinV-positive (E) and of the subG1

(F) population. Data shown in panels E and F have been reproduced in two independent experiments.

b r a i n r e s e a r c h 1 4 7 1 ( 2 0 1 2 ) 1 0 2 – 1 1 7 107

endocytosis, in DA-treated cells APP colocalized with the endo-

cytosis tracers Dextran-FITC (Fig. 4A) and Lysotracker (not

shown). By 4 h of DA treatment, the bulk of APP appeared

clustered at one pole in the vicinity of the nucleus and showed

reduced colocalization with EEA1 (Fig. 4B). This localization

resembled that of acidic compartments as detected by Acridine

Orange staining (Fig. 2A). DA-induced movement of APP toward

intracellular sites was further assessed using dynamin as a

plasma membrane marker (Fig. 4C). To see whether with time

APP further proceeded downstream the endocytic pathway to

endosomes and lysosomes, we monitored its localization in cells

transiently transfected with a plasmid driving the synthesis of

the endosomal–lysosomal protease Cathepsin D fused with the

green fluorescent protein (CD-GFP). While in control cells no-

colocalization was observed, at 1 h of treatment some organelles

labeled with CD-GFP appeared to also contain APP (Fig. 4D). After

b r a i n r e s e a r c h 1 4 7 1 ( 2 0 1 2 ) 1 0 2 – 1 1 7108

16 h of DA treatment, a nearly complete colocalization of APP

with lysosomal CD-GFP was observed (Fig. 4D). Note that at this

time as many as 50% of the cells detached and most of the cells

still adherent on plastic showed lysosome leakage (Fig. 2). It is

assumed, however, that the size of chimeric CD-GFP exceeds

that allowed to leak out from permeabilized lysosomes (esti-

mated to be of approximately 40 kDa). We asked about the

possible functional link between endocytosis, APP proteolysis

and apoptosis induced by DA. Dynamin, a GTPase involved in

endocytosis and intracellular membrane trafficking, has been

shown to play a pivotal role in APP endocytosis in Neuro2A cells

(Ehehalt et al., 2003). In a separate experiment, we found that

small-interference RNA-mediated knock-down of dynamin pre-

vented the internalization and degradation of APP in Neruo2A-

APPswe cells exposed to DA (not shown). Of note, cytofluoro-

metric analyses demonstrated the complete absence of the

annexinV-positive and subG1 apoptotic population in dynamin-

silenced cultures exposed for 16 h to DA (Fig. 4E and F).

2.4. Pepstatin A inhibits APP proteolysis and protectsfrom DA toxicity

APP processing in endosomal compartments and leading to

Ab peptide production involves the sequential proteolysis by

Fig. 5 – Pepstatin A inhibits dopamine-induced processing of

100 lM Pepstatin A (Pst) and then exposed or not for the time i

was resolved by SDS-gel electrophoresis and APP molecular sp

incubated for 6 h with DA and APP was detected with the anti-N

and APP was detected with the anti-C676–695 or anti-AbN1–17

and 20 h with DA and APP was detected with the anti-AbN1–17

the end of incubation (20 h). The relative intensity of holo-APP (

in two other independent experiments.

a b-secretase and a g-secretase activity (reviewed in Chow

et al., 2010; O’Brien and Wong, 2011; Zhang et al., 2011). Both

these activities are performed by aspartyl-type proteases that

can be found in endosomes (Schechter and Ziv, 2008;

Fukumori et al., 2006; Kinoshita et al., 2003), and therefore

should be effectively inhibited by large spectrum aspartic

protease inhibitors such as Pepstatin A, able to accumulate

within these organelles (Tian et al., 2002; Wolfe and

Haass, 2001). To confirm that DA toxicity was linked

to endosomal–lysosomal proteolysis of APP, we checked

whether this inhibitor could indeed prevent APPswe proteo-

lysis and at the same time save the cells from DA. Neuro2A-

APPswe cells were pre-incubated 12 h with Pepstatin A and

then exposed to DA for increasing time. Holo-APP was

immunodetected in cell homogenates with antibodies direc-

ted to different epitopes and allowing the detection of the

various molecular species as indicated in Table 1. The result

showed that Pepstatin A could prevent the loss of holo-

APPswe imposed by DA (approximately 40% in 6 h, as

detected by anti-N46–60) (Fig. 5A). We extended the incuba-

tion to 16 h, a time at which almost 50% of the cells exposed

to DA die by apoptosis. At this time, Pepstatin A increased the

amount (about 4-fold) of APP detectable in control (untreated)

cells, whichever the epitope recognized by the antibody,

APP. Neuro2A-APPswe cells were pre-incubated 12 h with

ndicated to dopamine (DA). At the end, the cell homogenate

ecies identified by western blotting. (A) The cells were

46–60 antibody. (B) The cells were incubated for 16 h with DA

antibody, as indicated. (C) The cells were incubated for 1, 6

antibody. Control and Pepstatin A-treated cells were taken at

normalized versus actin) bands is reported. Data reproduced

b r a i n r e s e a r c h 1 4 7 1 ( 2 0 1 2 ) 1 0 2 – 1 1 7 109

suggesting that APP constitutively undergoes a slow proteo-

lytic processing (Fig. 5B and C). As assessed by western

blotting with the anti-C676–695 antibody, by 16 h APP was

reduced in DA-treated cells by some 30% (as compared to

control cells), and this loss was fully prevented by Pepstatin A

(Fig. 5B, left panel). In a parallel independent experiment, APP

was detected by western blotting with an antibody directed to

an epitope placed at the N-terminus (residues 1–17) of the Ab

sequence. It was calculated that, as detected with this anti-

body (anti-AbN1–17), DA imposed a loss of APP of approxi-

mately 80%, and again this loss was practically completely

rescued by Pepstatin A (Fig. 5B, right panel). Finally, we

performed a time-course study of the effects of DA and

Pepstatin A on APP as detected with the anti-AbN1–17. This

experiment confirmed the progressive and extensive loss of

APP detectable with this antibody, suggesting that a large

portion (�80%) of APP was processed to produce the Ab

peptide (Fig. 5C). Again, Pepstain A confirmed its ability to

prevent such proteolysis (Fig. 5C).

Next, we checked whether Pepstatin A also exerted a protec-

tive effects against DA toxicity. The cells were pre-incubated or

not with Pepstatin A and then cell viability was assessed in

Fig. 6 – Pepstatin A prevents dopamine-induced activation of int

cells were plated on coverslips (panels A–C) or Petri dishes (pan

exposed to DA as indicated. (A) At the end of the treatment, th

Cell-associated blue fluorescence (indicative of metabolically ac

Pepstatin A increased by 2.5-fold the proportion of viable cells

mitotracker, fixed and processed for bax immunofluorescence a

mitochondrial permeability and bax oligomerization in cells exp

Pepstatin A. (C) Lysosome integrity was assessed by Acridine O

the cells the acidic compartments loose the metachromatic fluor

largely prevented by Pepstatin A. (D) At the end of the treatmen

show that Pepstatin A protected from DA toxicity. (E) Adherent

analyzed by cytofluorometry. (F) Adherent and suspended cells

cytofluorometry. The fluorescent images and the cytofluorogram

independent experiments in triple.

cultures exposed for 16 h to DA by using CellTracker. Pepstatin

A protected, albeit not completely, from DA toxicity (Fig. 6A).

Quantification of the viability-associated fluorescence with the

ImageJ software indicated that 490% of the cells exposed to DA

were metabolically inactivated and that Pepstatin A saved

almost half of this population. Pepstatin A could prevent the

oligomerization of bax and the loss of permeability of mito-

chondria (Fig. 6B) and of lysosomes (Fig. 6C) in a large propor-

tion of the cells exposed to DA. We quantified the protective

effect of Pepstatin A by counting the adherent trypan blue-

excluding cells in cultures after 16 h exposure to DA. The cells

recovered in the cultures at the end of the incubation with DA

amounted to about 50% and to about 80%, respectively, in the

absence and in the presence of Pepstatin A, of the untreated

counterpart (Fig. 6D). By cytofluorometry, almost 40% and 20%

of the cells exposed to DA, respectively, in the absence and in

the presence of Pepstatin A, were labeled with annexinV-FITC,

an early index of apoptosis (Fig. 6E). Thus, consistently Pep-

statin A showed the ability to protect almost 50% of the cells

exposed to DA. This protection was further confirmed looking

at the subG1 peak, which mirrors late events (chromatin

fragmentation) in apoptosis (Fig. 6F).

rinsic apoptosis in Neuro2A-APPswe cells. Neuro2A-APPswe

els D–F), pre-incubated or not with Pepstatin A (Pst) and

e cells were labeled with CellTracker to assess cell viability.

tive mitochondria) was estimated with the ImageJ software.

in the attached population. (B) The cells were labeled with

nd DAPI staining. The images show occurrence of

osed to DA. The latter events were completely prevented by

range staining. Upon DA treatment, in a large proportion of

escent dye as a consequence of membrane rupture, an event

t, adherent trypan-blue excluding cells were counted. Data

and suspended cells were labeled with AnnexinV-FITC and

were labeled with Propidium iodide and analyzed by

s shown in this figure are representative of three

b r a i n r e s e a r c h 1 4 7 1 ( 2 0 1 2 ) 1 0 2 – 1 1 7110

2.5. Chloroquine inhibits APP proteolysis and protectsfrom DA toxicity

The antimalaric drug chloroquine, a lysosomotropic weak

base that impairs endosomal–lysosomal hydrolysis by raising

the luminal pH, has been shown to interfere with the APP

processing (Caporaso et al., 1992; Caporaso et al., 1994) and

Ab peptide-associated toxicity (Liu et al., 2010). We first

checked whether and how chloroquine protected APP from

DA-induced proteolysis at 6 and 16 h of treatment. This

experiment confirmed that APP undergoes basal proteolysis

that could be halted by chloroquine. As assessed by western

blotting with the anti-C676–695 antibody, chloroquine com-

pletely protected APP from DA-induced proteolysis (Fig. 7A).

Of note, in chloroquine-treated samples APP was detected by

the anti-C676–695 as a doublet, the upper band presumably

bearing additional complex-type sugars. An even higher

protection by chloroquine was apparent by detecting APP

with the anti-AbN1–17 antibody. These data are consistent

with our previous finding (Fig. 5) and strongly support the

view that most, if not all, APP is proteolyzed with generation

of the Ab peptide. It is worthy to note that this antibody also

revealed APP as a doublet, but in this case the extra band

migrated faster, showing an apparent molecular weight

diminished of approximately 1.5 kDa (Fig. 7). This second

band was however less protected by chloroquine in the long

incubation with DA (Fig. 7C). Since this band was not detected

with the anti-C676–695, it likely represents a molecular

species that has lost a small peptide at the C-terminus of APP.

Finally, we focused on the link between APP proteolysis and

cell death induced by DA and the protection by chloroquine.

Based on the CellTracker assay, chloroquine afforded a nearly

complete protection from DA toxicity, unequivocally higher than

that of Pepstatin A (compare Figs. 8A and 6A). Consistently,

chloroquine prevented the DA-induced activation of bax in

those cells in which APP was not processed to produce the Ab

peptide, as suggested by immunofluorescence co-labeling with

anti-conformational active bax and anti-AbN1–17 antibodies

Fig. 7 – Chloroquine inhibits dopamine-induced processing of A

30 lM chloroquine (ClQ) and then exposed or not for the time i

was resolved by SDS-gel electrophoresis and APP molecular sp

incubated for 6 h with DA and APP was detected with the anti-C

cells were incubated for 16 h with DA and APP was detected w

with DA and APP was detected with the anti-AbN1–17 antibody

(Fig. 8B). Chloroquine protection of APP from DA-induced

proteolysis was not ascribable to inhibition of endocytosis, since

APP detected with the anti-AbN1–17 antibody reached the

endosomal–lysosomal compartments as demonstrated by its

colocalization with Cathepsin D (Fig. 8C).

2.6. Inhibition of c-secretase activity or phorbol esterstimulation of a-secretase activity protects from dopamine-induced processing of APP and cell toxicity

Finally, we sought to determine if and to what extent the

amyloidogenic processing of APP was indeed causally linked to

DA toxicity in Neuro2A-APPswe cells. The Ab fragment is formed

following g-secretase hydroysis of the C99 peptide generated by

b-secretase cleavage of holo-APP. To inhibit this processing, we

employed the non-competitive inhibitor of g-secretase L685,458

(Tian et al., 2002). As an additional approach to prevent amyloi-

dogenic processing, we stimulated the a-secretase alternative

pathway (Vincent and Govitrapong, 2011) by using a phorbol

ester (Savage et al., 1998). The cells were or not pre-incubated

with L685,458 or PMA and then exposed (or not) to DA for 16 h,

and at the end APP processing and cell toxicity were assessed.

Both drugs were shown able to prevent holo-APP degradation, as

detected by western blotting with either the anti-C676–695 and

the anti-AbN1–17 antibodies (Fig. 9A). DA-toxicity was largely

prevented in cells incubated with either the g-secretase inhibitor

L685,458 or the a-secretase stimulator PMA, as shown by phase-

contrast imaging of the monolayer, CellTracker staining and

bax staining (Fig. 9B). Of note, both these drugs prevented

DA-induced apoptosis in those cells in which amyloidogenic

processing of APP was abrogated, as suggested by immunofluor-

escence co-labeling of active bax and AbN1–17 APP (Fig. 9B).

3. Discussion

Alpha-synuclein-positive Lewy Bodies, accumulation of

hyperphosphorylated tau and neuron loss have been reported

PP. Neuro2A-APPswe cells were pre-incubated 30 min with

ndicated to dopamine (DA). At the end, the cell homogenate

ecies identified by western blotting. (A) The cells were

676–695 or the anti-AbN1–17 antibody, as indicated. (B) The

ith the anti-C676–695. (C) The cells were incubated for 16 h

. Data representative of two independent experiments.

Fig. 8 – Chloroquine prevents dopamine-induced toxicity in Neuro2A-APPswe cells. Neuro2A-APPswe cells were plated on

coverslips, pre-incubated or not with chloroquine (ClQ) and exposed to DA for 16 h. (A) At the end of the treatment, the cell

viability was assessed by CellTracker staining. Cell-associated blue fluorescence was estimated with the ImageJ software.

Chloroquine increased by 3.5-fold the proportion of viable cells in the attached population. (B) and (C) The cells were fixed

and processed for APP and bax (panel B) or cathepsin D (catD, panel C) immunofluorescence. The images are suggestive of

APP degradation (as revealed by the anti-AbN1–17 antibody) in cells exposed to DA, in concomitance with bax

oligomerization (panel B). Chloroquine inhibits APP processing and loss of cell-associated Ab-reactivity, and prevents bax

oligomerization but not translocation of APP in catD-positive organelles. Data representative of two independent

experiments in triple. (For interpretation of the references to color in this figure legend, the reader is referred to the web

version of this article.)

b r a i n r e s e a r c h 1 4 7 1 ( 2 0 1 2 ) 1 0 2 – 1 1 7 111

in the substantia nigra and extranigral nuclei of AD patients

featuring parkinsonian signs (Burns et al., 2005; Schneider

et al., 2002). Whether the hyper-expression of APP exacer-

bates neuron susceptibility to DA excitotoxicity is not known.

Here, we addressed this issue by studying the molecular and

cellular consequence of DA treatment in Neuro2A cells over-

expressing human APP695swe. We found that DA increased

the recycling of APP determining its translocation and pro-

teolysis within endosomal compartments. These events were

accompanied by rupture of lysosome and mitochondria

integrity and onset of caspase-mediated cell death. We

investigated on the functional relationship between APPswe

processing and neuronal cell death induced by DA. The

Swedish mutation (K595N, M596L in APP695) causes early

onset familial AD and is associated with altered trafficking

and processing of APP (Lo et al., 1994; Lorenzen et al., 2011),

resulting in up to 10-fold higher production of the Ab peptide

(Haass et al., 1995; Sinha and Lieberburg, 1999). Considering

that the Swedish mutation is placed within the b-secretase

consensus sequence, the above findings strengthen the rela-

tionship between cleavage at b-site, excessive generation of

Ab peptide and AD (Thinakaran and Koo, 2008). DA induced

the clustering of endosomes and lysosomes and the rapid

translocation of plasma membrane APPswe into Cathepsin

D-positive acidic compartments. Recently, it has been

shown that, contrary to wild-type APP that can be found in

lysosomes, internalized APPswe localizes to endosomes

(Lorenzen et al., 2011). This would suggest that in our system

DA induced the translocation of APPswe into endosomes. We

asked about the proteases potentially involved in endosomal

processing of APPswe. Cathepsin B was excluded, since this

protease can cleave wild-type APP with production of Ab

peptides, but not APPswe (Hook et al., 2009). Actually, genetic

ablation of Cathepsin B rather increased the accumulation of

Ab peptides in the brain of transgenic mice expressing

APPswe (Mueller-Steiner et al., 2006). The aspartyl-proteases

BACE1 and Cathepsin D were considered as good candidates,

as they have been able to cleave at high rate APPswe, and

with minor efficiency APPwt (Schechter and Ziv, 2008). When

the wide-spectrum aspartyl-protease inhibitor Pepstatin A

was employed, almost 80% of full length APPswe was rescued

from proteolysis induced by DA as detected by the anti-

AbN1–17, indicating that the production of the Ab peptide

was nearly completely abrogated. A similar reduced the

production of Ab peptide was attained when APP internaliza-

tion was abolished by mutation of the YENPTY sequence at

Fig. 9 – L685,458 and PMA prevent APP degradation and cell toxicity induced by dopamine. Neuro2A-APPswe cells were

plated on coverslips or Petri dishes, pre-incubated or not with L685,458 or PMA as indicated, and exposed to DA for 16 h. (A)

At the end, the cell homogenate was resolved by SDS-gel electrophoresis and APP molecular species identified by western

blotting with the anti-C676–695 or the anti-AbN1–17 antibody, as indicated. The relative intensity of holo-APP (normalized

versus actin) bands is reported. Data representative of three independent experiments. (B) At the end of the treatment, the

monolayer in Petri dishes was photographed and the cells grown on coverslips were labeled with CellTracker to assess cell

viability or processed for fluorescence staining with DAPI (nuclei) and anti-bax and anti-AbN1–17 antibodies, as indicated.

The images shown are representative of three independent experiments and demonstrate that both L685,458 and PMA could

protect the cells from DA toxicity.

b r a i n r e s e a r c h 1 4 7 1 ( 2 0 1 2 ) 1 0 2 – 1 1 7112

the C-terminus (Koo and Squazzo, 1994). Internalization of

APP relies on dynamin-driven endocytosis (Ehehalt et al.,

2003). Consistently, siRNA-mediated gene knock-down of

dynamin greatly impaired endocytosis and degradation of

APPswe, and also abolished cell toxicity induced by DA. That

proteolysis of APPswe induced by DA likely occurred in acidic

compartments is supported by the observation that chlor-

oquine, a weak base widely used to rise the luminal pH of

endosomes and lysosomes, also blocked this process, in

agreement with a previous report (Caporaso et al., 1994). In

this context, it is to be noted that in chloroquine-treated

samples an additional band of APPswe, showing an apparent

molecular weight reduced of �1.5 kDa, could be detected.

This band was evidenced by the anti-AbN1–17, not by the

anti-C676–695, antibody, suggesting that in the absence of

chloroquine a small peptide is removed at the C-terminus of

APP. The C-terminus of APPswe is indeed very unstable.

Thinakaran et al. (1996) failed to detect the APP C-terminus

fragment generated by the action of g-secretase, and sug-

gested that this fragment undergoes rapid degradation.

Indeed, whichever antibody we used, we never detected

specific bands below the molecular weight of full length

APPswe. In theory, the sAPPb and the transmembrane C99

fragment generated by BACE could be found in cell homo-

genates. We hypothesize that these species either are rapidly

degraded or extruded from the cell under DA stimulation.

Two additional approaches allowed to exclude the possibility

that DA toxicity was associated with the activation of APP

processing pathways other than the amyloidogenic. In fact,

inhibition of the g-secretase activity or the stimulation of

the a-secretase both afforded complete protection from

DA-induced APP degradation and cell toxicity. It has been

shown that the products of APP proteolysis, including the Ab

peptide, transiently accumulate in exosomes of multivesicu-

lar body-endosomes from which are then released extra-

cellularly (Rajendran et al., 2006). Thus, assuming that

proteolysis of APPswe indeed occurred in multivesicular

body-endosomes, it is conceivable that the products were

promptly exocytosed under DA stimulation. The use of

lysosomal activity inhibitors (i.e., Pepstain A and chloroquine)

also evidenced that a portion of APPswe constitutively under-

goes processing, though in the presence of DA this process

was accelerated and associated with lysosomes and mito-

chondria dysfunction and cell death. Recently, it has been

shown that lysosome leakage may be due to the insertion in

the lysosomal membrane of toxic Ab-42 peptide taken up

b r a i n r e s e a r c h 1 4 7 1 ( 2 0 1 2 ) 1 0 2 – 1 1 7 113

from the extracellular mileau (Liu et al., 2010). In this context,

it is to note that chloroquine could protect Neuro2A-APPswe

cells from Ab42 toxicity by inhibiting the lysosomal mem-

brane insertion of the Ab peptide and thus preventing lyso-

some leakage (Liu et al., 2010). However, the effective toxicity

of the soluble Ab peptide is debatable. Therefore, we consider

the possibility that DA-induced degradation of APP leads to

cell toxicity through the production of pro-oxidative frag-

ments that generate reactive oxygen species.

Parkinsonian-like motor signs (including rigidity, tremor,

bradykinesia) have been reported in 13–36% of AD patients

Fig. 10 – Schematic representation of the results. The upper par

and the peptide generated by a, b and c secretase activity. The

potential sites of cleavage by proteases, the sequence specifica

indicated. The lower part of the scheme illustrates the principa

work. DA stimulates the recycling of endocytic vesicles and indu

Here, APP is subject to amyloidogenic processing and the prod

degradation is likely associated with the production of reactive

undergo permeabilization and leakage of cathepsin D. Addition

within lysosomes, and lead to lysosomes leakage. The followin

mitochondrial membranes, the permeabilization of mitochondr

(Scarmeas et al., 2004; Wilson et al., 2000) and appear to be

related to morbidity and mortality. Such parkinsonian symp-

toms are associated with more rapid cognitive decline and

deterioration of physical conditions (Chui et al., 1994;

Mortimer et al., 1992).

The present findings (schematically reproduced in Fig. 10)

strongly support the view that inhibiting aspartyl-protease-

mediated proteolysis of APP within endosomes is a good

strategy to protect neurons in AD patients. Also chloroquine,

an FDA approved drug that can freely pass the blood–brain

barrier, would be a good candidate for this purpose. Indeed, a

t of the scheme shows the transgenic holo-APPswe protein

position of relevant post-translational modifications, the

lly recognized by the antibodies used in this work, are

l findings, and their interpretation, reported in the present

ces the translocation of APP into endosomal compartments.

ucts, including the Ab peptide, are rapidly secreted. APP

oxygen species (ROS) within lysosomes, which eventually

ally, the Ab peptide could be endocytosed and accumulate

g events include sequentially the oligomerization of bax on

ia, the activation of caspases and cell death.

b r a i n r e s e a r c h 1 4 7 1 ( 2 0 1 2 ) 1 0 2 – 1 1 7114

clinical trial with hydroxychloroquine revealed no benefits in

terms of progression of dementia in AD patients treated for

18 months (Van Gool et al., 2001). Based on the data reported

here, we propose the prolonged use of this drug in AD

patients to prevent the progression toward parkinsonism, of

course keeping in mind the potential side effects (Block, 1998;

Good and Shader, 1982).

4. Experimental procedures

4.1. Cells and treatments

Mouse neuroblastoma Neuro2A cells (American Type Culture

Collection, Rockville, MD) and Neuro2A cells stably expres-

sing the transgenic Swedish-mutant APP695 (Thinakaran

et al., 1996) were cultivated under standard culture condi-

tions (37 1C; 95 v/v% air: 5 v/v% CO2) in Dulbecco Modified

Eagle’s Medium (cod. D5671, Sigma-Aldrich, St. Louis, USA)

supplemented with 10% heat-inactivated fetal bovine serum

(cod. DE14-801F, Lonza Group Ltd., Basel, Switzerland), 2 mM

L-glutamine (cod. 35050, Life Technologies Ltd., Paisley, UK),

1 mM sodium pyruvate (cod. S8636, Sigma-Aldrich), 1 w/v% of

non-essential aminoacids (cod. M7145, Sigma-Aldrich) and

1 w/v% of a penicillin–streptomycin solution (cod. P0781,

Sigma-Aldrich). Experiments were carried out during the log

phase of cell growth. Cells (25,000/cm2) were seeded on sterile

plastic dishes or coverslip and allowed to adhere for 24 h

prior to start any treatment. Treatments included 250 mM

dopamine (DA, cod. H8502, Sigma-Aldrich) and, prior to

exposure to DA, 30 mM chloroquine (ClQ, cod. C6628, Sigma-

Aldrich; 30 min in advance), 100 mM Pepstatin A (cod. P5318,

Sigma-Aldrich; 12 h in advance), 30 mM ZVAD (OMe)-fmk

(ZVAD, cod. 260-020-M005, Alexis Laboratories, San Diego,

CA; 1 h in advance), 20 mM IETD-CHO (IETD, cod. A1216,

Sigma-Aldrich; 1 h in advance), 1.5 mM L685,458 (cod.

H-5106, Bachem; 6 h in advance), 5 mM PMA (cod. 8139,

Sigma-Aldrich; 1 h in advance).

4.2. Small-interference RNA silencing of dynamin

Post-transcriptional silencing of dynamin expression was

achieved by the small interference RNA (siRNA) technology.

Duplexes of 27-nucleotide siRNA including two 30-overhan-

ging TT were synthesized by MWG Biotech AG (Washington,

DC). The sequence and use of the siRNA for sham transfec-

tion have been described previously (Trincheri et al., 2007).

The sense strand of siRNA targeting dynamin-1 mRNA was

50–CAG AAC ACA CUG AUG GAA GAA UCG GCC-30. Adherent

cells (plated at 15,000/cm2 in Petri dish) were incubated for

4 h with 400 pmol RNA-duplexes in the presence of 10 ml

Lipofectamine 2000 in 1 ml of Optimem. The cells were then

washed and treated 36 h post-transfection to allow maximal

effect on protein down-regulation.

4.3. Assessment of cell toxicity

At the end of incubation, adherent and suspended cells were

collected, diluted in a solution containing trypan blue and

counted to determine cell loss and occurrence of necrosis

(trypan blue positive cells). For cytofluorometry assessment

of cell death, adherent and suspended cells were collected,

washed in PBS, fixed in ice-cold 70 v/v% ethanol and labeled

with 0.18 mg/ml propidium iodide (PI, cod. P4170, Sigma-

Aldrich) in the presence of RNase A (0.4 mg/ml). Hypodiploid

(SubG1) labeled cells were assumed as apoptotic. In addition,

the presence of phosphatidyl-serine on the plasma mem-

brane, an index of apoptosis, was assessed by cytofluorome-

try in the whole cell population by Annexin-FITC labeling

(cod. ALX-209–256, Alexis Laboratories; 10 min at room tem-

perature) of non-fixed cells. At least 10,000 cells were ana-

lyzed using a FacScan flow cytometer (Becton Dickinson,

Mountain View, CA, USA) equipped with a 488 nm argon

laser. Data were elaborated with the winMDI software.

Apoptosis-associated chromatin alterations were detected

by staining the cells adherent on coverslips with the DNA-

labeling fluorescent dye 40,6-diamidino-2-phenylindole dihy-

drochloride (DAPI, cod. 32670, Sigma-Aldrich). In situ Term-

inal deoxynucleotidyl transferase-mediated dUTP-biotin nick

end labeling (TUNEL) for detection of apoptotic cells was

performed with the ‘‘In situ Cell Death Detection’’ fluorescent

Kit (cod. 1684817, Roche Diagnostics Corporation Indianapo-

lis, IN, USA) (Trincheri et al., 2008). To test cell viability, the

cells adherent on coverslips were labeled with CellTrackerTM

(CellTrackerTMBlue-CMAC 7-amino-4-chloromethylcoumarin)

(cod. C2110, Life Technologies Ltd.), a fluorescent dye that

emits blue fluorescence of intensity proportional to the

mitochondrial respiratory activity. At the end of the treat-

ment, the cells were loaded with CellTracker (5 mM for

20 min), then the cells were washed and incubated for

30 min and imaged under the fluorescence microscope

(Ekkapongpisit et al., 2012).

4.4. Primary antibodies used for immunofluorescence andwestern blotting

The following primary antibodies were used: a rabbit poly-

clonal antiserum specific for human Cathepsin D (CD) (Follo

et al., 2007), a polyclonal antibody specific for conformational

active bax (cod. 2772, Cell Signaling Technology, MA, USA), a

mouse monoclonal antibody specific for EEA1 (cod. 610456,

Becton, Dickinson and Company, Franklin Lakes, NJ, USA), a

mouse monoclonal antibody specific for dynamin (cod. 05319,

Millipore, Billerica, MA, USA), a mouse monoclonal antibody

specific for b-actin (cod. A5441, Sigma-Aldrich) and mouse

monoclonal antibody specific for b-tubulin (cod. T5293,

Sigma-Aldrich). APP-related peptides were detected with the

following antibodies (see also Table 1): rabbit polyclonal

anti-C-terminus (cod. 171610, Calbiochem, Merck KGaA,

Darmstadt, Germany); rabbit polyclonal anti-N-terminus

(cod. A8967, Sigma-Aldrich); mouse monoclonal anti-Ab-N-

terminus sequence (cod. 12266, Abcam, Cambridge, UK).

4.5. Immunofluorescence staining

Cells on coverslip were fixed in cold methanol and processed

for immunofluorescence as previously reported (Castino et al.,

2010). Immunocomplexes were revealed with secondary anti-

bodies, either IRIS-2 (green fluorescence)- or IRIS-3 (red fluor-

escence)-conjugated goat-anti-rabbit IgG or goat-anti-mouse

b r a i n r e s e a r c h 1 4 7 1 ( 2 0 1 2 ) 1 0 2 – 1 1 7 115

IgG (cod. 2W5-08, 2W5-07, 3W5-08, 3W5-07, Cyanine Technology

SpA, Turin, I), as appropriate.

4.6. Endocytosis and lysosomal and mitochondrialmembranes integrity

The endocytosis process was monitored using Dextran-FITC

(Life Technologies Ltd., Paisley, UK) as a fluorescent tracer

(Dragonetti et al., 2000). Lysosomal membrane integrity was

assessed with the metachromatic fluorescent dye Acridine

Orange (cod. A6529, Sigma-Aldrich), which emits a red-

orange fluorescence when reside within acidic compartments

(endosomes and lysosomes), and a yellow-green fluorescence

when resides in neutral compartments (cytoplasm). The cells

were incubated with Acridine Orange (15 mg/ml, 15 min), then

washed and rapidly imaged under the fluorescence micro-

scope or analyzed by cytofluorometry (Dragonetti et al., 2000).

Mitochondrial membrane integrity was tested by using Rho-

damine-123 hydrochloride (Rho-123, cod. 610-018-M005,

Alexis Laboratories) or Mitotracker Red (cod. M22425, Life

Technologies Ltd.). The cells were incubated for 10 min at

37 1C with 50 nM Rho-123, then washed and rapidly imaged

under the fluorescence microscope or analyzed by cytofluoro-

metry. Alternatively, the cells on coverslips were incubated

for 15 min at 37 1C with 0.2 ml/ml of Mitotracker solution,

fixed in 3.7% paraformaldehyde for 30 min and permeabilized

with 0.2% Triton X-100 for 20 min, and further processed for

fluorescence staining with anti-bax antibody (Castino et al.,

2007).

4.7. Cathepsin D fluorescent chimera and plasmidtransfection

The cDNA coding for human lysosomal Cathepsin D (Isidoro

et al., 1991) devoid of the stop codon was subcloned in the

multiple cloning site of the plasmids peGFP-N1 (cod. 6085-1,

Clontech Lab., Takara Bio Inc., Shiga, Japan) in order to drive

the synthesis of the fluorescent chimeras CD-GFP

(Ekkapongpisit et al., 2012). The cells were transfected with

the plasmid using the Lipofectamine 2000 Reagent (cod.

11668-019, Life Technologies Ltd.) method as suggested by

the purchaser. Briefly, cells were plated in P35 Petri dish at

15,000/cm2 and let adhere 24 h before to proceed with the

transfection. The DNA–Lipofectamine complexes were pre-

pared in 500 ml of Opti-MEM I Reduced Serum Medium (cod.

11058021, Life Technologies Ltd.) with 5 mg of plasmid and

10 ml of Lipofectamine. After 6 h of incubation, medium of

transfection was removed and replaced with a serum-con-

taining culture medium (10% FBS-DMEM) and the cells were

cultivated for 36 h to allow for maximal protein expression

prior to any treatment.

4.8. Fluorescence microscope imaging

Fluorescently labeled cells were observed under the fluores-

cence microscopes Leica DMI6000 or the confocal Leica

DMIRE2 (Leica Microsystems AG, Wetzlad, Germany). For each

experimental condition, three coverslips were prepared. Five

to ten fields (for a minimum of 100 cells) in each coverslip

were examined independently by two investigators. Selected

images of representative fields are shown. The ImageJ

software freely available at http://rsbweb.nih.gov/ij/ was

employed for quantification of the fluorescent signal.

4.9. Protein expression analysis

Protein expression was evaluated by standard immunoblot-

ting procedure as previously reported (Castino et al., 2007).

Cell homogenates were prepared by freeze–thawing and

ultrasonication in a buffer containing detergents and pro-

tease inhibitors. About 50 mg of cell proteins were denatured

with Laemmli sample buffer, separated by electrophoresis on

a 10% SDS-containing polyacrylamide gel and then electro-

blotted onto nitrocellulose membrane (Bio-Rad, Hercules, CA,

USA). Protein of interest was detected with the specific

primary antibody as detailed above. As an index of homo-

genate protein loading in the lanes was used b-Actin and

b-tubulin. Immunocomplexes were revealed by using a per-

oxidase-conjugated secondary antibody (cod. 170515, 1706516,

Bio-Rad), as appropriate, and subsequent peroxidase-induced

chemiluminescence reaction (cod. NEL103E001EA, PerkinEl-

mer, Waltham, MA, USA). Western blotting data were repro-

duced at least three times independently, unless otherwise

specified. Intensity of the bands was estimated by densito-

metry (Quantity One Software, Bio-Rad; ImageJ software).

4.10. Statistical analysis

All the experiments were performed in triple and data shown

have been reproduced at least three times (unless otherwise

specified). Densitometric data are reported for western blot-

ting shown (difference between replicates was less than 20%

of the absolute value). Quantification data from ImageJ and

cytofluorometry analyses and cell counting data were given

as average7SD. The Student’s t-test (with po0.05 for statis-

tical significance) was employed to compare the results from

different treatments. The Microsoft Excel XLStats software

was used.

Acknowledgments

Research supported by grants from San Paolo (Project Neu-

roscienze 2008.2395), Regione Piemonte (Ricerca Sanitaria

Finalizzata, Torino), Consorzio InterUniversitario per le Bio-

tecnologie (CIB, Trieste). The bio-imaging facility was donated

by Comoli, Ferrari & SpA (Novara, Italy). Thanks are due

to Dr. D.L. Feinstein (University of Illinois, Chicago, USA)

for advices for Ab western blotting, to Dr. V. Bruno

(Mahidol University, Bangkok, Thailand) for discussion, and

to Dr. C. Peracchio for excellent artwork and editorial

assistance.

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