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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
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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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