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Neurotoxicity ResearchNeurodegeneration,Neuroregeneration, NeurotrophicAction, and Neuroprotection ISSN 1029-8428Volume 23Number 4 Neurotox Res (2013) 23:301-314DOI 10.1007/s12640-012-9340-9

Excitotoxicity Through NMDA ReceptorsMediates Cerebellar Granule NeuronApoptosis Induced by Prion Protein 90-231Fragment

Stefano Thellung, Elena Gatta, FrancescaPellistri, Alessandro Corsaro, ValentinaVilla, Massimo Vassalli, Mauro Robello,et al.

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ORIGINAL ARTICLE

Excitotoxicity Through NMDA Receptors Mediates CerebellarGranule Neuron Apoptosis Induced by Prion Protein90-231 Fragment

Stefano Thellung • Elena Gatta • Francesca Pellistri • Alessandro Corsaro •

Valentina Villa • Massimo Vassalli • Mauro Robello • Tullio Florio

Received: 18 April 2012 / Revised: 13 July 2012 / Accepted: 18 July 2012 / Published online: 2 August 2012

� Springer Science+Business Media, LLC 2012

Abstract Prion diseases recognize, as a unique molecular

trait, the misfolding of CNS-enriched prion protein (PrPC)

into an aberrant isoform (PrPSc). In this work, we charac-

terize the in vitro toxicity of amino-terminally truncated

recombinant PrP fragment (amino acids 90-231, PrP90-

231), on rat cerebellar granule neurons (CGN), focusing on

glutamatergic receptor activation and Ca2? homeostasis

impairment. This recombinant fragment assumes a toxic

conformation (PrP90-231TOX) after controlled thermal

denaturation (1 h at 53 �C) acquiring structural character-

istics identified in PrPSc (enrichment in b-structures,

increased hydrophobicity, partial resistance to proteinase

K, and aggregation in amyloid fibrils). By annexin-V

binding assay, and evaluation of the percentage of frag-

mented and condensed nuclei, we show that treatment with

PrP90-231TOX, used in pre-fibrillar aggregation state,

induces CGN apoptosis. This effect was associated with a

delayed, but sustained elevation of [Ca2?]i. Both CGN

apoptosis and [Ca2?]i increase were not observed using

PrP90-231 in PrPC-like conformation. PrP90-231TOX

effects were significantly reduced in the presence of

ionotropic glutamate receptor antagonists. In particular,

CGN apoptosis and [Ca2?]i increase were largely reduced,

although not fully abolished, by pre-treatment with the

NMDA antagonists APV and memantine, while the AMPA

antagonist CNQX produced a lower, although still signifi-

cant, effect. In conclusion, we report that CGN apoptosis

induced by PrP90-231TOX correlates with a sustained ele-

vation of [Ca2?]i mediated by the activation of NMDA and

AMPA receptors.

Keywords Cerebellar neurons � Prion � PrP90-231 �Apoptosis � Calcium � NMDA receptor

Introduction

Prion diseases or transmissible spongiform encephalopa-

thies (TSE) are associated with the conversion of a host-

encoded glycoprotein, named cellular prion protein (PrPC),

into a b sheet-rich and protease-resistant isoform, the

scrapie prion protein (PrPSc). PrPSc accumulation within

central nervous system is an event associated with neuro-

toxicity and transmissibility of TSE (Prusiner 1998; Aguzzi

and Polymenidou 2004), and it was reported that the con-

tinued conversion of PrPC to PrPSc is responsible for prion

neurotoxicity (Mallucci et al. 2003). Spongiform vacuola-

tion of gray matter, neuronal death, and glial proliferation

are the main histopathological alterations characterizing

TSE. Extracellular deposition of PrPSc into amyloid fibrils

and plaques is frequently, but not invariably observed in

proximity of brain areas interested by neuronal loss, sup-

porting the hypothesis of a direct or glial-mediated

neurotoxicity of the misfolded prion protein (Giese et al.

1998; Castilla et al. 2004; Simoneau et al. 2007; Faucheux

Stefano Thellung, Elena Gatta and Francesca Pellistri contributed

equally to this study.

S. Thellung � A. Corsaro � V. Villa � T. Florio (&)

Department of Internal Medicine, Section of Pharmacology and

Centre of Excellence for Biomedical Research (CEBR) School

of Medicine, University of Genova, Viale Benedetto XV, 2,

16132 Genoa, Italy

e-mail: tullio.florio@unige.it

E. Gatta � F. Pellistri � M. Robello

Department of Physics, University of Genova, Genoa, Italy

M. Vassalli

Institute of Biophysics (IBF), National Council of Research

(CNR), Genoa, Italy

123

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DOI 10.1007/s12640-012-9340-9

Author's personal copy

et al. 2009). Nevertheless, PrPSc gain of toxicity does not

rule out the possibility that a significant contribution to

neurodegeneration might also result from reduction of

functioning PrPC, whose activity could be critical in sus-

taining neuronal activity and survival (Sakaguchi et al.

1996; Collinge et al. 1994; Brown et al. 1997; Bounhar

et al. 2001; Rambold et al. 2008; Biasini et al. 2012). PrPSc

amyloid fibrils deposition results from a multi-step process

that passes through the formation of b-structured PrP

monomers, soluble oligomers, and insoluble aggregates

(Baskakov et al. 2002), and the characterization of the

relationship between PrPSc aggregation state and its

neurotoxicity is still debated (Chiesa and Harris 2001).

PrPSc induces neuronal death through both direct and

glial-mediated mechanisms (Muller et al. 1993; Bate et al.

2001; Bate et al. 2004). Noteworthy, it was demonstrated that

pharmacological blockade of NMDA glutamate receptors

inhibits cortical neurons apoptosis induced by PrPSc or

derived peptides, indicating that persistent activation of

glutamate receptors and perturbation of intracellular Ca2?

homeostasis contribute to prion-related neuronal death

(Muller et al. 1993; Peggion et al. Peggion et al. 2011).

Similarly, numerous disease-related amyloidogenic pep-

tides, including b-amyloid, huntingtin, polyglutamines, and

other non-pathogenic proteins, share with PrPSc the property

to induce cell death as a consequence of their interaction with

glutamatergic receptors (Bucciantini et al. 2004; Kelly and

Ferreira 2006; Alberdi et al. 2010; Texido et al. 2011).

Since PrPSc purification from infected brains is ham-

pered by its insolubility and high propensity to aggregate,

most studies aimed to characterize PrPSc structure, infec-

tivity, and neurotoxicity, and used synthetic PrP-derived

peptides (Forloni et al. 1993; Salmona et al. 2003), or

recombinant full length (Novitskaya et al. 2006) or trun-

cated PrP fragments (Legname et al. 2004).

In this work, we used a recombinant protein matching the

amino acid sequence 90-231 of human prion protein (PrP90-

231) (Corsaro et al. 2002) that corresponds to a protease-

insensitive PrPSc fragment (Chen et al. 1995). PrP90-231 is

purified as a soluble monomer, rich in a-helices and highly

sensitive to proteinase K digestion. After controlled mild

thermal denaturation (53 �C for 1 h), PrP90-231 under-

goes structural alteration that reproduces some aspects of

PrPC-PrPSc misfolding. In particular, the fragment shows

increased b-sheet content, hydrophobicity, and insolubility,

and acquires partial resistance to proteinase K (PK) (Corsaro

et al. 2006). Thermally denatured PrP90-231 gains in vitro

biological activity, including the capability to induce acti-

vation of astrocytes and microglia, and apoptosis in neuronal

cell models (Thellung et al. 2007; Chiovitti et al. 2007;

Corsaro et al. 2009; Thellung et al. 2011; Villa et al. 2011).

Thus, we named the thermally denatured PrP90-231 con-

former as PrP90-231TOX (Corsaro et al. 2012).

Importantly, it was recently reported that PrP90-231TOX

was able to reproduce some cellular effects of Syrian

hamster brain purified PrPSc, such as activation of ionic

conductance through synthetic lipid bilayers (Paulis et al.

2011).

In this paper, we demonstrate that PrP90-231TOX indu-

ces apoptosis in rat cerebellar granule neurons (CGN) in a

structure-dependent manner, while similar to what was

demonstrated using the neuroblastoma cell line SH-SY5Y

(Corsaro et al. 2006; Villa et al. 2006), native PrP90-231

was almost devoid of neurotoxic activity, demonstrating

that the biological activity of this recombinant prion pro-

tein fragment is dependent on its folding state.

The aim of this study was to investigate the role of

glutamatergic activation as a mechanism mediating PrP90-

231TOX-dependent CGN apoptosis, focusing on the alter-

ation of Ca2? homeostasis. Using a live-cell imaging

approach, we observed that PrP90-231TOX produces a

persistent elevation of intracellular Ca2? concentration

([Ca2?]i) that is significantly, although not completely

prevented by the pharmacological blockade of NMDA and

to a lower extent by the inhibition of AMPA/kainate glu-

tamate receptors. Glutamatergic blockade also reduced

PrP90-231TOX-stimulated CGN death suggesting that

NMDA receptor’s hyper activation contributes to the

neurotoxic effects of this prion protein fragment in its

misfolded conformation.

Experimental Procedures

Test Substances

AMPA (receptor for a-amino-3-hydroxy-5-methylisox-

azole-4-propionic acid) and NMDA (receptor for N-methyl

D-aspartate) antagonists CNQX (6-cyano-7-nitroquinoxa-

line-2,3-dione), memantine, and APV (DL-2-amino-

5-phosphonovaleric acid) were purchased from Sigma

Aldrich (Milano, Italy).

Synthesis and Preparation of PrP90-231

PrP90-231 was obtained and purified as previously

described (Corsaro et al. 2002). In order to analyze the

structure dependency of PrP90-231 biological activity, the

protein was incubated for 1 h in 10 mM phosphate buffer,

NaCl-free, pH 7.2, at 4 or 53 �C to obtain native or

refolded PrP90-231, respectively (Corsaro et al. 2006). As

previously demonstrated, PrP90-231 thermal denaturation

at 53 �C induces a three-dimensional folding toward a

b-sheet rich structure and provokes a gain of toxicity in

vitro (Corsaro et al. 2006; Villa et al. 2006). Accordingly,

denatured fragment was named PrP90-231TOX (Corsaro

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et al. 2012), leaving the term ‘‘PrP90-231’’ to indicate the

native non-toxic isoform. CGN treatments were performed

adding the recombinant peptides directly to the culture

medium.

Cerebellar Granule Neuron (CGN) Cultures

CGN were prepared from 8-day-old Sprague–Dawley rats,

Harlan-Nossan, Bresso, Italy as previously reported (Gatta

et al. 2009). One million CGN were plated on 20 mm poly-

L-lysine-coated glass coverslips positioned in 6-multiwell,

and maintained in Basal Eagle’s culture medium, con-

taining 10 % fetal calf serum, 100 lg/ml gentamicin, and

25 mM KCl at 37 �C in humidified, 95 % air/5 % CO2

atmosphere. Ten lM cytosine arabinoside was added to

cultures from day 1 to minimize glial proliferation.

Experiments were performed in cultures between day 6 and

10 after plating (days in vitro, DIV), to allow CGN mat-

uration (Scorziello et al. 1996). Astrocyte contamination

was checked after each individual preparation of CGN by

GFAP cytofluorescence (Bajetto et al. 1999). The presence

of astrocytes was constantly below 5 %.

Circular Dichroism (CD)

PrP90-231 and PrP90-231TOX CD spectra were measured

with a Jasco J-600 spectrophotometer between 190 and

250 nm (1 nm spectral size step, 0.5 nm band width, and

100 nm/min scan rate). Samples (0.5 mg/ml) were diluted

in 10 mM phosphate buffer, pH 7.2, and equilibrated for

10 min before measurements. Spectra were obtained by 10

scans, subtracted of blank (buffer alone) of three inde-

pendent PrP90-231 preparations. Estimated secondary

structure percentage was calculated by the Jasco secondary

structure estimation algorithm (JSSE vers. 1.00.00-1998,

Jasco Corp. Japan) (Corsaro et al. 2011).

Thioflavin T (Th T) Binding

PrP90-231 and PrP90-231TOX (0.5 mg/ml) were incubated

with 10 lM Th T (Sigma Aldrich, Italia) in 20 mM

NaHPO4, 150 mM NaCl, pH 7.0 for 15 min at room tem-

perature. Th T fluorescence (ex/em 385/482 nm) was

monitored using a lambda Bio 10 spectrophotometer

(Perkin-Elmer) (Corsaro et al. 2011). Values of Th T

binding to PrPTOX from three independent preparations

were expressed as percentage of PrP90-231 fluorescence.

Proteinase K (PK) Resistance Assay

PrP90-231 and PrP90-231TOX (10 lg/100 ll phosphate

buffer, pH 7.3) were digested with increasing concentra-

tions of PK, Sigma Aldrich) (1/1000–1/10 Pk/PrP90-231

w/w ratios) for 30 min at 37 �C. Digestion was stopped by

boiling samples in Laemmli buffer. The amount of cleaved

and uncleaved proteins were detected by immunoblotting,

using the 3F4 anti-PrP monoclonal antibody (Signet,

London UK).

Atomic Force Microscopy (AFM)

Images were obtained using Nanoscope V controller

(Bruker AXS Inc., Madison, WI, USA) equipped with

Multimode head. Proteins were incubated in solution for

1 h at 53 �C and maintained at 37 �C for up to 1 week.

Small aliquots of PrPTOX (30–80 ll of a solution 50 lg/ml)

were deposited on highly oriented pirolite graphite sub-

strates (HOPG, NT-MDT Moscow, Russia), washed with

filtered milliQ water, and rinsed under a gentle nitrogen

blow. Imaging was performed in air environment using

tapping mode (Bracalello et al. 2011).

[Ca2?]i Measurement

CGN were incubated at 37 �C for 40–45 min in 6.0 lM cell-

permeant Oregon Green-acetoxymethyl ester (OG-AM)

(Molecular Probes, Eugene, OR) and then washed several

times with 135 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2,

1.0 mM MgCl2, 5.0 mM HEPES, 10 mM glucose, pH 7.4, at

room temperature. Coverslips were transferred to a record-

ing chamber mounted onto a Nikon Eclipse TE300 inverted

microscope. Cells were continuously perfused with the

appropriate solution and visualized using 1009 objective in

oil (N.A. 1.3) (Pellistri et al. 2008). Fluorescence was

detected using a Hamamatsu digital CCD camera with a

450–490-nm excitation filter, a 505-nm dichroic mirror, and

a 520-nm emission filter (Nikon Italia, Florence, Italy).

Images were acquired with the Simple PCI software (Com-

pix Imaging Systems, Hamamatsu Corp., Sewickley, PA).

Fluorescence intensity was calculated as arbitrary units,

building a scale of the pixel intensity; to this purpose, only

pixels located in the region of interest were considered. The

intensity of OG fluorescence was recorded in at least 120

cells for each treatment.

Cell Survival Assay

Mitochondrial function, as index of cell viability, was

evaluated by measuring the reduction of 3-(4,5-dimeth-

ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT,

Sigma Aldrich). The cleavage of MTT to purple formazan

crystals by mitochondrial dehydrogenases was quantified

spectrophotometrically, as previously reported (Thellung

et al. 2000). In brief, cells were incubated for 1 h with

0.25 mg/ml MTT in serum-free DMEM at 37 �C; after

removal of medium, formazan crystals were dissolved in

Neurotox Res (2013) 23:301–314 303

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dimethylsulfoxide and values of absorbance were measured

spectrophotometrically at 570 nm.

Nuclear Staining

Cells were fixed in 1 % paraformaldehyde (10 min) and

then incubated with 1 lg/ml bisbenzimide (Hoechst 33258,

Molecular Probes) for 30 min, washed three times, and

analyzed for condensed or disrupted nuclei using DM2500

microscope (Leica Microsystems, Wetzlar, Germany)

equipped with a DFC350FX digital camera (Leica Micro-

systems) (Florio et al. 1998). At least 1,000 cells per

coverslip were analyzed. Experiments performed in

duplicate were repeated at least three times.

Annexin-V Binding Test

The test evaluates the apoptosis-induced exposure of phos-

phatidylserine (PS) at the outer face of plasma membrane by

staining cells with the phosphatidylserine-binding protein

annexin-V (Thellung et al. 2011). CGN, plated into glass

bottom Petri dishes, were appropriately treated and then

washed three times with PBS and incubated in 1 ml of

annexin-V binding buffer (NaCl 140 mM, HEPES 10 mM,

CaCl2 2,5 mM, pH 7,4) containing 50 ll of annexin-V

AlexaFluor conjugate 568 (Molecular Probes) for 20 min;

after three washing steps with PBS, 1 ml of serum-free

medium was added to allow live cells observation under

confocal fluorescence microscopy (Bio-Rad MRC 1024 ES).

Statistics

Data were obtained by three independent experiments

conducted in quadruplicate, unless otherwise specified.

Statistical analysis was performed by means of one-way

ANOVA, P [ 0.05 and [ 0.01 were considered statisti-

cally significant and highly significant, respectively.

Results

Structural Characterization of PrP90-231

and PrP90-231TOX

We previously demonstrated that PrP90-231-controlled

thermal denaturation (1 h at 53 �C) induces gain of toxic-

ity (PrP90-231TOX) that correlates with increase of

b-structured regions (Corsaro et al. 2006; Chiovitti et al.

2007). Before the evaluation of the PrP90-231TOX effects

on CGN culture, we analyzed the changes in the biophys-

ical characteristics of PrP90-231 induced by thermal

denaturation. To this purpose, we analyzed the relative

contents of secondary structures in native PrP90-231 and

PrP90-231TOX by CD spectroscopy (Fig. 1a). CD spectra

obtained by three independent preparations of PrP90-231

indicated that b-sheets and b-turns structures are virtually

absent in native PrP90-231 but increased up to 43 % in

PrP90-231TOX, due to a reduction of random coiled com-

ponent of the protein. Conversely, the amount of a-helices

in PrP90-231TOX was basically unchanged.

The increase in b-structures after thermal denaturation

of PrP90-231 was confirmed by evaluating Thioflavine T

binding, a fluorescent molecule that intercalates within

b-stranded areas, providing an additional index of PrP90-

231 refolding. Thioflavine T binding to PrP90-231TOX was

60 % higher than in the natively structured peptide

(Fig. 1b) indicating a net increase of b-stranded regions in

PrP90-231TOX. In order to determine if alteration of PrP90-

231 structure can affect its resistance to proteolysis, a

hallmark of PrPSc, we subjected PrP90-231 and PrP90-

231TOX to enzymatic digestion by increasing concentra-

tions of proteinase K (PK). After digestion, proteins were

subjected to SDS-PAGE and the amount of uncleaved

PrP90-231 and PrP90-231TOX was evidenced as 3F4-immu-

noreactive bands with the apparent molecular weight of

16 kDa (not shown) and quantified by densitometry

(Fig. 1c). PrP90-231 sensitivity to digestion is significantly

reduced after thermal denaturation, an effect that was

particularly evident for a PrP/PK ratio (w/w) of 500/1,

which reduced the amount of uncleaved PrP90-231 to

about 70 %, but almost unaffected the correspondent

PrP90-231TOX immunoreactive band. PrP90-231TOX has

the intrinsic tendency to aggregate and produce oligomers

and fibrils which modifies its physico-chemical and bio-

logical behavior in vitro (Chiovitti et al. 2007). Thus, we

characterized the nature of PrP90-231TOX aggregates pos-

sibly formed in our experimental conditions and which will

be added to CGN in the following experiments. By AFM

measures, we imaged PrP90-231TOX after thermal dena-

turation (the experimental condition able to generate

PrP90-231TOX) (Fig. 1d left panel) and observed the

presence of small globular aggregates, compatible with the

presence of amorphous oligomers but the absence of

structured fibrils. Conversely, after prolonged incubation at

37 �C (up to 1 week), PrP90-231TOX organizes in thin

elastic fibers (Fig. 1d right panel). Based on these data, we

established that PrPTOX structures used in the following

experiments are a mixture of monomers and small oligo-

mers of PrP90-231, while the presence of fibrils could be

considered negligible.

PrP90-231TOX Affects CGN Viability and Causes

Apoptotic Cell Death

An intriguing observation is that PrP90-231TOX and PrPSc

share the ability to affect neuronal cell viability in vitro

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(Muller et al. 1993; Post et al. 2000; Bate et al. 2004;

Corsaro et al. 2006; Villa et al. 2006), indicating that the

molecular determinants responsible for PrPSc neurotoxicity

are also present in recombinant PrP fragments produced for

experimental purposes. The neuroectodermic cell line SH-

SY5Y displays a marked vulnerability to PrP90-231TOX

(Chiovitti et al. 2007; Corsaro et al. 2009; Thellung et al.

2011).

APrP90-231

structure

α -helix

(%)

β -sheet

(%)

β-turn

(%)

Random Coil

(%)

PrP90-231 56.4 0.0 0.0 43.6

57.1 10.7 32.2 0.0

% of Thioflavin T binding

B

PEPTIDE PrP90-231 PrP90-231TOX

PrP90-231TOX

%161%001

% of PK undigested PrP90-231

C

PrP90-231/PK (w/w) 1000/1 500/1 200/1 100/1 10/1

PrP90-231 100% 27.7% 8.2% 0% 0%

TOXPrP90-231TOX 100% 89.9% 50.3% 5.6% 0%

D

Fig. 1 Structural characterization of PrP90-231 and PrP90-231TOX.

a CD analysis of the a-helix, b-turn, b-sheet, and random coil content

of native PrP90-231 and PrP90-231TOX. Percentage of b-turn and

b-strands is significantly increased in PrP90-231TOX. b Evaluation of

thioflavin T binding to native PrP90-231 and PrP90-231TOX. Th T

fluorescence was enhanced by about 60 % in PrP90-231TOX com-

pared to native PrP90-231. Values report the mean ± SEM, from

three independent PrP90-231 preparations. c Evaluation of PK

resistance of PrP90-231 in native conformation and after thermal

denaturation (PrP90-231TOX). PrP fragments were incubated in the

presence of increasing concentrations of PK, resolved by SDS-PAGE,

and probed with anti-prion monoclonal antibody 3F4. Band immu-

noreactivity, corresponding to uncleaved PrP fragments, was

quantified by densitometric analysis. Values are reported as percent-

age of 3F4 immunoreactivity in untreated samples. Native PrP90-231

is significantly and almost completely digested at PrP90-231/PK

ratios of 500/1 and 200/1, respectively; in contrast, PrP90-231TOX

digestion required a PrP90-231/PK ratio superior to 200/1. d Atomic

force microscopy (AFM) analysis of the aggregation state of PrP90-

231TOX solution. PrP90-231 was incubated at 53 �C for 1 h to induce

refolding into PrP90-231TOX, and then incubated at 37 �C for 2 h (leftpanel) and 7 days (right panel). AFM images show that PrP90-

231TOX solution maintained at 37 �C for 2 h produced small globular

aggregates, whereas the formation of structured fibers requires

prolonged incubation and was clearly detectable after 7 days at

37 �C. Scale bar 300 nm

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Here, we demonstrate that this recombinant PrP frag-

ment also exerts significant toxicity in primary cultures

of CGN. First, we compared the effects of PrP90-231

and PrP90-231TOX on CGN survival, to evaluate whether

PrP90-231 cytotoxicity is dependent on the conformational

changes induced by controlled thermal denaturation, as

previously described. To this aim, PrP90-231 in native

conformation or PrP90-231TOX were added to 7 DIV CGN

cultures, and cell viability was evaluated after 2 days by

MTT reduction test (Table 1). CGN exposure to PrP90-231

(1 lM) did not affect cell viability as compared to vehicle-

treated control neurons. In contrast, when cells were treated

with PrP90-231TOX at the same concentration, we observed

a statistically significant time-dependent reduction of CGN

viability.

This result indicates that misfolding of PrP90-231

induced by thermal denaturation causes a gain of toxicity

that affects CGN survival in vitro. PrP90-231TOX effects

were time-dependent being already statistically significant

(-30 %) after 2 days of treatment, and maximal after

4 days (-47 % of cell survival) (Table 1).

In the subsequent pharmacological characterization of

PrP90-231TOX, its activity was evaluated after 2 days of

treatment, in order to act on on-going rather than exhausted

toxicity path. In order to better characterize PrP90-231TOX

neurotoxicity, we analyzed CGN morphology modifica-

tions induced by 2 days of exposure to PrP90-231TOX. As

shown by phase-contrast images (Fig. 2a left panels),

treatment with this PrP fragment produced shrinkage and

condensation of cell bodies and neuritic network rarefac-

tion representing signs of neurotoxicity. We also performed

Hoechst-33258 nuclear staining of CGN before and after

exposure to PrP90-231TOX, to evidence markers of apop-

tosis such as nuclear condensation and/or fragmentation.

After 2 days of treatment, CGN were observed under fluo-

rescence microscopy (Fig. 2a right panels). While viable

cells showed spherical/oval nuclei stained with moderate

blue fluorescence, apoptotic cells evidenced condensed and/

or fragmented nuclei characterized by strong light blue

fluorescence. In these experimental conditions, the number

of apoptotic CGN, after 2 days of treatment with PrP90-

231TOX (1 lM), was two-fold higher than that observed in

untreated control neurons (Fig. 2b).

In order to obtain a more qualitative and specific index

of apoptosis, we analyzed, by confocal fluorescence

microscopy, the exposure of the phospholipid PS on the

external face of CGN plasma membrane. In control con-

ditions, PS exposure is limited to the cytoplasmic layer of

cell membrane; its redistribution to both sides of cell

membrane is an apoptosis-related event that follows cas-

pase 3 activation and can be evidenced by annexin-V

binding. After 2 days of treatment with PBS or PrP90-

231TOX (1 lM), cells were loaded with ALEXA-Fluor

conjugated annexin-V and analyzed under fluorescence

microscopy (Fig. 3). Control CGN presented only faint and

scattered red fluorescent spots, indicating the absence of

detectable PS exposure (Fig. 3, upper panels). In contrast,

CGN cultures treated with PrP90-231TOX evidenced sev-

eral cells surrounded by continuous fluorescent rings in

correspondence to their borders, caused by annexin-V

binding to PS on the external side of plasma membrane

(Fig. 3, lower panels). Altogether, these results indicate

that PrP90-231TOX fragment is toxic to primary cultures of

CGN through the activation of the apoptotic program.

Sub-chronic Treatment with PrP90-231 Elicits [Ca2?]i

Increase in CGN Cultures

In order to evaluate whether [Ca2?]i increase is involved in

the pro-apoptotic effects of PrP90-231TOX, CGN were

loaded with OG before being exposed to the PrP-derived

peptide (1 lM) in the different conformations. OG fluo-

rescence was continuously measured for 120 s after peptide

administration. However, CGN acute exposure to either

PrP90-231 or PrP90-231TOX did not evoke any significant

increase of [Ca2?]i (data not shown). In order to understand

if early PrP90-231TOX activity could be dependent on its

aggregation state, we induced PrP90-231TOX fibrillar large

aggregates, as described in Fig. 1d. Also in these experi-

mental conditions, no significant variations of intracellular

Ca2? levels were induced by peptide treatment (data not

shown), thus indicating that rapid changes [Ca2?]i are not

involved in prion fragment neurotoxicity.

Since the toxic effects of PrP90-231TOX on CGN were

time-dependent with maximal effects after 4 days of

treatment, we evaluated the possibility that longer exposure

to the toxic peptide was required to determine alterations in

Ca2? homeostasis. Hence, we measured OG fluorescence

after treating CGN with native PrP90-231 or PrP90-231TOX

Table 1 PrP90-231TOX cytotoxicity shows time- and structure-

dependency

Treatment Viability (% of control)

Vehicle 100 ± 3.5

PrP90-231 1 lM (2 days) 94 ± 3.6

PrP90-231TOX 1 lM (2 days) 69.7 ± 5.7**

PrP90-231TOX 1 lM (4 days) 53 ± 4.2**

CGN were treated with PrP90-231 1 lM for 48 h and with PrP90-

231TOX (1 lM) for 48 and 96 h. Cell viability was determined by

MTT test. CGN viability was significantly reduced by PrP90-231TOX

treatment while it remained unaffected by PrP90-231. PrP90-231TOX

affected CGN viability in a time-dependent manner. MTT reduction

values were expressed as percentage of vehicle-treated controls and

represent the mean ± SEM of three independent experiments con-

ducted in quadruplicate. ** p \ 0.01 versus control

306 Neurotox Res (2013) 23:301–314

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(1 lM) for 24, 48, and 72 h, in comparison to PBS-treated

control cells (Table 2).

The results were analyzed considering two parameters:

(i) average level of fluorescence increase compared to

control cells, and (ii) the percentage of cells that showed an

increase of fluorescence compared to controls.

After 24 h of treatment with PrP90-231TOX, mean

fluorescence increased to about 77 % when compared to

control CGN, and such effect lasted up to 72 h. As far as

the percentage of cells showing fluorescence changes, a

clear time-dependent effect was observed and, after 3 days

of treatment with PrP90-231TOX, virtually all neurons

displayed increased [Ca2?]i (Table 2). Analysis of OG

fluorescence also revealed that native PrP90-231 did not

modify [Ca2?]i at any time tested (Table 2), thus indicating

that PrP90-231 refolding, induced by controlled thermal

denaturation, was required for both CGN toxicity and

alteration in Ca2? homeostasis, highlighting a strong rela-

tionship between these events.

NMDA and AMPA/Kainate Receptors Mediate [Ca2?]i

Increase Induced by PrP90-231TOX

Several amyloidogenic proteins, including Ab peptides,

have been described to affect neuron viability through

interactions with NMDA receptors (You et al. 2012). We

have recently demonstrated that HypF-N-soluble oligomers

produces [Ca2?]i increase consisting in both transient and

long lasting components, sustained by NMDA and AMPA/

kainate glutamate ionotropic receptor subtypes (Pellistri

et al. 2008). Hence, we addressed the possibility that

intracellular Ca2? elevation induced by PrP90-231TOX in

CGN might be mediated by NMDA and AMPA/kainate

receptor activation. To this aim, we treated CGN with

PrP90-231TOX in the presence of NMDA-R antagonists

APV and memantine (10 lM), and the AMPA/kainate-R

antagonist CNQX (1 lM), and evaluated [Ca2?]i, by OG

fluorescence test, after 24 h of treatment (Table 3). The

pre-treatment with APV (Table 3) and, to a lesser extent,

A

Con

trol

T

Phase contrast Hoechst T

OX

Sample Condensed nuclei

Counted

cells

PrP

90-2

31

B

Phase contrast Hoechst

(% on total counted)

per coverslip

Control 12.2 ± 0.7 1591

PrP90-231TOX 23,5 ± 0.9* 2347

Fig. 2 PrP90-231TOX induces

CGN death in vitro. a CGN

were plated on glass coverslips

and treated for 2 days with

vehicle (upper) or PrP90-

231TOX (1 lM) (lower). After

treatments, cells were fixed with

-20 �C cold methanol and

incubated with 1 lM Hoechst-

33258 (Hoechst) to stain nuclei.

Images obtained by

fluorescence microscopy (rightpanels) show that cell exposure

to PrP90-231TOX induced a

significant increase of

condensed and/or fragmented

nuclei compared to PBS-treated

cells. Phase-contrast pictures

(left panels) show CGN body

shrinkage and neurite network

fragmentation in PrP90-231TOX-

exposed CGN. b The amount of

neuronal death was obtained by

measuring the percentage of

condensed/fragmented nuclei on

total nuclei. Values were

obtained by three independent

experiments performed in

duplicate. * p \ 0.05 versus

control

Neurotox Res (2013) 23:301–314 307

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memantine strongly reduced PrP90-231TOX-induced [Ca2?]i

increase (-79.2 and -44.1 %, respectively). CNQX was

less effective, although was also able to reduce about 50 %

PrP90-231TOX effects (Table 3). Using a combination of

both antagonists, we observed a residual non-significant

effect of PrP90-231TOX (about 10 % of intracellular Ca2?

increase). These results indicate that, although AMPA/

kainate-R opening represent a portion of [Ca2?]i increase

induced by PrP90-231TOX in CGN, the activation of NMDA

is the prevailing mechanism.

NMDA and AMPA/Kainate Receptors Activation

Mediates PrP90-231 Neurotoxicity

Considering the major role of Ca2? homeostasis imbalance in

excitotoxicity, we investigated the possibility that CGN death,

induced by PrP90-231TOX, may result from abnormal increase

of [Ca2?]i subsequent to prolonged activation of NMDA and

AMPA/kainate receptors. We performed viability assays on

CGN treated with PrP90-231TOX in the presence of APV

(10 lM), CNQX (1 lM), or the combination of both

Con

trol

TO

X

Phase contrast Annexin-V Merge

PrP

90-2

31

Fig. 3 CGN death evidences markers of apoptosis. CGN membrane

staining with fluorescent annexin-V indicates apoptosis onset. CGN

were plated on glass coverslips and treated for 2 days with vehicle

and PrP90-231TOX (1 lM). After treatments, CGN were subjected to

live staining and with apoptosis marker annexin-V. In contrast to

vehicle-treated CGN that evidenced a barely detectable scattered

signal from annexin-V, a significant number of cells exposed to

PrP90-231TOX showed annexin-V fluorescent rim

Table 2 PrP90-231TOX elicits [Ca2?]i increase in CGN

PrP90-231TOX (1 lM) PrP90-231 (1 lM)

Treatment time

(h)

% Of cells with fluorescence

increase

% Fluorescence

increase

% Of cells with fluorescence

increase

% Fluorescence

increase

24 84** 77 ± 2** 0 0

48 81** 78 ± 4** 0 0

72 100** 76 ± 4** 0 0

Cells were grown on glass coverslips and treated for 24–72 h with PrP90-231TOX. [Ca2?]i was recorded by OG fluorescence analysis (see

methods). Variations of [Ca2?]i were reported as mean fluorescence increase and as percentage of cells showing fluorescence increase compared

to respective vehicle-treated controls. PrP90-231TOX stimulated [Ca2?]i increase whose intensity reached its maximum level after 24 h of

treatment, although three days of treatment was necessary to detect the phenomenon in the totality of neurons. When treated with native PrP90-

231, CGN did not evidence increase of OG fluorescence compared to vehicle-treated controls. Values represent the mean ± SEM of four

experiments conducted in duplicate recordings performed in at least 120 cells for each treatment. ** p \ 0.01 versus controls

308 Neurotox Res (2013) 23:301–314

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antagonists in comparison with the misfolded PrP fragment

alone (Fig. 4). Cell death was measured by both MTT assay

and nuclear condensation/fragmentation (after Hoechst 33258

staining), as index of apoptosis.

CGN pretreatment with APV significantly reduced

PrP090-231TOX-dependent neurotoxicity in both MTT

(Fig. 4a) and nuclear staining (Fig. 4b) assays. Conversely,

CNQX pretreatment caused a very slight effect that did not

reach the statistical significance, as compared to PrP90-

231TOX-treated cells. Thus, as observed for Ca2? influx,

NMDA activation contributes to PrP-231TOX toxicity much

more than AMPA/kainate. In agreement with this obser-

vation, Fig. 4 also shows that combined APV/CNQX pre-

treatment was not more effective than treatment with

NMDA antagonist alone in preventing PrP90-231TOX

neurotoxicity. This result further supports the hypothesis

that AMPA/kainate contribution to CGN death is entirely

hidden by NMDA activation.

We must consider that a complete blockade of PrP90-

231TOX biological activity could be theoretically obtained

by increasing APV and CNQX concentrations, but con-

centrations exceeding 10 lM for APV, and 1 lM for

CNQX induced per se CGN death (data not shown) and

thus were not used.

Discussion

Prolonged alteration of intracellular Ca2? homeostasis is a

major event responsible for neuronal loss during physio-

logical aging and neurodegenerative disorders, including

Alzheimer’s and Parkinson diseases, amyotrophic lateral

sclerosis, and TSE (Van Den Bosch et al. 2006; Bezprozv-

anny and Mattson 2008; Caudle and Zhang 2009). In par-

ticular, excitotoxicity, through the activation of glutamate

receptors, is involved in neuronal loss and astrocytosis in

experimentally induced scrapie (Scallet and Ye 1997).

Moreover, in vitro studies demonstrated that the blockade of

NMDA receptors protects cortical neurons against apoptosis

induced by PrPSc, partially purified from Syrian hamsters

brains, although it was ineffective in blocking PrPSc repli-

cation (Muller et al. 1993). Such report represented one of the

first evidences that PrPSc toxicity and capability to self-

replicate can be reproduced in vitro but are not mutually

dependent properties. The possibility that infective and

neurotoxic prion species are different entities was recently

proposed and, accordingly, disease progression could

develop in two clearly separate phases: the infection and the

cytotoxic step (Sandberg et al. 2011). Importantly, to study

the cellular and molecular pathways leading to neuronal

death that are activated during TSE, the use of PrPSc from

infected brains has been flanked and supported by the use of

synthetic peptides (Forloni et al. 1993; Thellung et al. 2002;

Chabry et al. 2003; Florio et al. 2003; Salmona et al. 2003;

Ciccotosto et al. 2008) or recombinant full length or trun-

cated PrP fragments (James et al. 1997; Novitskaya et al.

2006). Several advancements have been obtained with

polypeptides encompassing the amino acids 90-231, a por-

tion of PrP that represents the PK-insensitive core of the

pathological protein (approximately corresponding to

PrP27-30 obtained upon PrPSc digestion with protease K)

(James et al. 1997; Swietnicki et al. 1997; Post et al. 2000;

Legname et al. 2004; Corsaro et al. 2006; Chiovitti et al.

2007; Thellung et al. 2011), and corresponding to PrP

cleavage products recovered in the brain of TSE affected

individuals (Chen et al. 1995; Zou et al. 2003).

We previously demonstrated that PrP90-231TOX toxicity

could be mediated by, at least, three mechanisms including

(i) impairment of trophic factors signaling (Corsaro et al.

2009), (ii) alteration of lysosomal integrity (Thellung et al.

2011), and (iii) induction of neurotoxic factors release by

glial cells (Thellung et al. 2007).

In the present work, we demonstrate that PrP90-231 has

structure-dependent toxicity on CGN, causing a sustained

perturbation of cytosolic Ca2? concentration. In agreement

with our previous studies, we demonstrated that native

PrP90-231 does not affect CGC viability but, upon thermal

denaturation, can switch into a neurotoxic configuration,

characterized by high content of b-structures and resistance

to proteolysis. Importantly, the acquisition of a b-sheet-rich

structure also correlates with a higher exposure of hydro-

phobic residues, increasing PrP90-231 capacity to interact

with cells (Corsaro et al. 2006; Chiovitti et al. 2007;

Corsaro et al. 2011).

Table 3 Pharmacological blockade of NMDA and AMPA/Kainate

receptors counteracts [Ca2?]i increase induced by PrP90-231TOX in

CGN

Treatment Fluorescence

increase ( %

over control)

Fluorescence

inhibition (% of

reduction of PrP90-

231TOX effects)

PrP90-231TOX 77 ± 2 100 ± 3

PrP90-231TOX ? MEM 43 ± 3* -44.1 ± 4*

PrP90-231TOX ? CNQX 53 ± 3* -31.2 ± 4*

PrP90-231TOX ? APV 16 ± 3** -79.2 ± 4**

PrP90-

231TOX ? (APV ? CNQX)

10 ± 3** -87 ± 4**

Cells were grown on glass coverslips and treated for 24 h with PrP90-

231TOX (1 lM); when specified, cells were pretreated with APV

(10 lM), CNQX (1 lM), and a mixture. [Ca2?]i was recorded by OG

fluorescence analysis (see ‘‘Experimental Procedures’’ section) and

reported as percentage of fluorescence increase compared to controls

(vehicle-treated cells). Values represent the mean ± SEM of four

experiments conducted in duplicate. Recordings were performed in at

least 120 cells for each treatment. * p \ 0.05 and ** p \ 0,01 versus

PrP90-231TOX

Neurotox Res (2013) 23:301–314 309

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There is a growing bulk of evidence supporting the

hypothesis that both neurotoxicity and Ca2? homeostasis

impairment are not unique properties of disease-related

amyloidogenic proteins, such as b-amyloid or PrPSc, but

could also be induced in vitro by proteins not responsible

for known human diseases (Bucciantini et al. 2004; Buc-

ciantini et al. 2002). HypF-N, the N-terminal domain of the

E. Coli hydrogenase maturation factor, forms prefibrillar

aggregates that demonstrated toxicity in different mam-

malian cell lines through the dysregulation of Ca2?

homeostasis. In particular, primary cultures of CGN

showed both fast and prolonged [Ca2?]i increase, when

exposed to HypF-N aggregates that were mediated by the

activation of NDMA and AMPA/kainate glutamate recep-

tors (Pellistri et al. 2008). Noteworthy, in this previous

study, Ca2? elevation was detected after 24 h of treatment

suggesting that the cerebellar neurons have to be subjected

to potentially lethal Ca2? concentrations for a prolonged

times. Interestingly, HypF-N and PrP90-231TOX share

similar path of fibrillogenesis and both proteins display

aggregation-dependent toxicity: their biological activity is

expressed by prefibrillar soluble oligomers but it is lost

when the fibrillogenic path generates insoluble isoforms

(Bucciantini et al. 2002; Pellistri et al. 2008; Chiovitti et al.

2007). To this regard, images obtained by AFM are par-

ticularly relevant since they show that PrP90-231TOX is

structured by small globular aggregates without detectable

fibrils that are generated only after prolonged thermal

denaturation (up to 1 week). This result strongly indicates

that the formation of PrP90-231TOX fibrils has not yet took

place when the protein is added to CGC cultures and that

PrP90-231 fibrils do not mediate the biological effects

reported here.

In this work, we tested the possibility that PrP90-231TOX

elicits apoptosis in CGN primary cultures through sus-

tained [Ca2?]i elevation mediated by glutamatergic iono-

tropic receptors. Experiments were designed to evidence

time-dependent responses of CGN to recombinant PrP

fragments. Surprisingly, acute treatment with PrP90-

231TOX did not modify the level of [Ca2?]i. The absence of

A

B

100

110

^* ^*

60

70

80

90

** **^*

50MT

T r

ecu

ctio

n (

% o

f co

ntr

ol)

Cont PrPTOX PrPTOX +CNQX

PrPTOX +CNQX/APV

PrPTOX +APV

CNQX/APV

Cont PrPTOX PrPTOX +CNQX

PrPTOX +CNQX/APV

PrPTOX +APV

CNQX/APV

200

250

300

**

^ ^*

50

100

150

ap

op

tosi

s (%

of

con

tol)

^ ^

Fig. 4 PrP90-231TOX-induced cell death is affected by NMDA and

AMPA/Kainate antagonists. a CGN cultures were pretreated for

30 min with APV (10 lM), CNQX (1 lM), or a mixture of both

antagonists before the addition of PrP90-231TOX (1 lM). Cell

viability was evaluated, after 48 h, by MTT test. Controls were

obtained by treating CGN cultures with vehicle, PrP90-231TOX

(1 lM). and antagonists mixture. Data, expressed as percentage of

vehicle-controls, represent the average ± SEM of three experiments

performed in quadruplicate. * p \ 0.05 and ** p \ 0.01 versus

control; ^ p \ 0.05 versus PrP90-231TOX. b CGN cultures, plated on

glass coverslips, were pretreated for 300 with APV 10 lM, CNQX

1 lM, or a mixture of both antagonists before the addition of PrP90-

231TOX (1 lM). After 48 h, cell nuclei were stained with Hoechst-

33258 to evaluate the percentage of condensed/fragmented nuclei.

Controls were obtained by treating CGN cultures with vehicle, PrP90-

231TOX (1 lM) and antagonists mixture. Results, expressed as

percentage of vehicle-controls, represent the average ± SEM of

three experiments performed in duplicate. * p \ 0.05 and ** p \ 0.01

versus control; ^ p \ 0.05 versus PrP90-231TOX

310 Neurotox Res (2013) 23:301–314

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a fast response contrasts with the marked [Ca2?]i increase

induced by HypF-N and suggest that PrP90-231TOX neither

directly interacts with membrane receptors nor affects

calcium channels, as previously shown using the small

synthetic peptide PrP106-126 (Florio et al. 1996; Florio

et al. 1998; Thellung et al. 2000). In contrast, prolonged

CGN treatment with PrP90-231TOX produced a net long

lasting increase of [Ca2?]i; this effect was not reproduced

by the native form of the peptide indicating that b-sheet

rich, hydrophobic structure of PrP90-231TOX is required to

alter Ca2? homeostasis. The same concentrations of PrP90-

231TOX induced CGN apoptosis, suggesting that [Ca2?]i

increase is not a physiological signaling, but reveals neu-

ronal damage and activation of apoptosis. Hence, we

addressed the possible causative role of glutamate recep-

tors activation in triggering or enforcing PrP90-231TOX

neurotoxicity. By pharmacological blockade of NMDA and

AMPA/kainate receptors, we demonstrated that the acti-

vation of such receptors mediates PrP90-231TOX-induced

CGN death; we show, indeed, that in the presence of APV

CNQX neurons are partially protected from apoptosis. In

the same way, [Ca2?]i increase was prevented by gluta-

matergic receptor blockade.

Combined treatment with both antagonists was also

performed to identify a possible additive effects obtainable

by simultaneous blockade of both AMPA/Kainate on

NMDA receptors. However, AMPA/kainate inhibitor

CNQX that per se produced a slight reduction of PrP90-

231TOX activity, did not show additivity with the strong

protection exerted by the NMDA blocker APV.

Although these results strongly support the hypothesis

that PrP90-231TOX fragment activates a classic excitotoxic

pathway through NMDA and AMPA/kainate receptor

activation followed by sustained calcium increase, the

understanding of molecular events through which PrP90-

231TOX leads to NMDA activation may be particularly

challenging, because of the extended time lapse between

PrP90-231TOX treatment and the detection of a significant

[Ca2?]i increase. Such time gap suggests that the peptide

may not act as a direct agonists on NMDA and AMPA/

kainate receptors. There is evidence that neuronal damage

induced by amyloidogenic peptides and oligomers results

from their capability to modify plasma membrane perme-

ability and form ionic pores causing the imbalance of

calcium homeostasis (Kourie and Culverson 2000; Lin

et al. 1997; Salmona et al. 1997; Demuro et al. 2005). The

almost complete blockade of [Ca2?]i increase exerted by

simultaneous administration of APV/CNQX led us to

exclude that PrP90-231TOX could produce cell death by

cation-permeable channels, although we do not rule out

the possibility that PrP90-231TOX can modify plasma

membrane microviscosity and modify ion distribution

across the membrane. Neuronal death could originate from

the sustained glutamate-mediated calcium elevation and

follow a classic excitotoxic pathway (Scallet and Ye 1997),

or it could be caused by other events, (i.e., intracellular

accumulation and disruption of lysosomal stability) on

which the activation of ionotropic glutamate receptors

plays a permissive role. Basing on studies performed using

amyloid b peptides, we believe that both mechanisms are

conceivable. It was demonstrated that Ab peptides can

activate NMDA-dependent oxidative stress and mitochon-

drial dysfunction (Alberdi et al. 2010; Texido et al. 2011)

and that cell death can be prevented by NMDA antagonists

(Tremblay et al. 2000; Song et al. 2008). On the other hand,

NMDA-dependent [Ca2?]i activation induced by amyloid

peptides has been described to increase neuronal vulnera-

bility to several neurotoxic agents rather than representing

a direct excitotoxic insult (Mattson et al. 1992); at this

regard, it was demonstrated that NMDA blockade could

reduce neuronal sensitivity to Ab1-40, metabolic poisoning

with staurosporine and etoposide, or oxygen deprivation

(Tremblay et al. 2000). Hence, we do not exclude that

PrP90-231TOX fragment can activate NMDA and AMPA

receptors rendering CGN more susceptible to other toxic

agents produced by culturing the neurons. In addition, it

was reported that neuronal uptake of Ab peptides is

affected by the activity of plasma membrane NMDA

receptors. In fact, the NMDA antagonist APV prevents

neuronal uptake of amyloid peptide Ab1-42 (Bi et al.

2002), suggesting that in our cell model also APV and

CNQX could prevent PrP90-231TOX neurotoxicity because

they prevent the internalization of the peptide mediated by

glutamate receptor activation and that calcium entry

reflects CGN alterations induced by internalized PrP90-

231TOX. The possible glial contamination of CGN cultures

could introduce an additional element of complexity in

the correct interpretation of neuronal response to PrP90-

231TOX. Accumulation of PrPSc in astrocytes and the

presence of activated microglia during natural and exper-

imental scrapie suggest the pathophysiological role of glial

cells not only in prion replication, but also in inducing

neuronal death through the release of neurotoxic products

(Giese et al. 1998). We and others demonstrated that PrPSc

and related peptides (including PrP90-231TOX) can induce

activation and proliferation of astrocytes and microglia

resulting in the induction of the release of several cyto/

chemokines and oxygen and nitric radicals (Florio et al.

1996; Hafiz and Brown 2000; Marella and Chabry 2004;

Thellung et al. 2007). Thus, while the release of neurotoxic

factors by activated astrocytes might contribute to PrP90-

231TOX effects, this event could be excluded in our cultures

in which type I astrocytes do not exceed 5 % of total cells.

Thus, we believe that in our experimental conditions, glial

contribution to PrP90-231TOX neurotoxicity, if any, is

minimal. However, the investigation of such issues is

Neurotox Res (2013) 23:301–314 311

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intriguing and will be addressed in future. It must be

pointed out, however, that regardless of the initial mecha-

nisms triggered by the peptide, the key step responsible for

cell death is glutamatergic ionotropic receptors activation,

since CGN apoptosis can be reduced by their blockade.

In conclusion, the results showed in this work evi-

dence the possible central role of ionotropic glutamatergic

receptors in cerebellar granule cells reaction to the extra-

cellular presence of amyloidogenic fragments derived from

PrPSc partial cleavage.

Acknowledgments This study has been supported by grants from

Italian Ministry of University and Research (MIUR-PRIN 2008, and

Accordi di Programma FIRB, Project No. RBAP11HSZS, 2011).

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