Download Enteroglial and neuronal involvement without apparent neuron loss

Journal of General Virology (2007), 88, 2899–2904
Short
Communication
DOI 10.1099/vir.0.82907-0
Enteroglial and neuronal involvement without
apparent neuron loss in ileal enteric nervous system
plexuses from scrapie-affected sheep
Giuseppe Marruchella,1 Ciriaco Ligios,2 Valeria Albanese,1
Maria Giovanna Cancedda,2 Laura Madau,2
Giovanna Lalatta-Costerbosa,3 Maurizio Mazzoni,3 Paolo Clavenzani,3
Roberto Chiocchetti,3 Giuseppe Sarli,4 Luigi De Grossi,5 Umberto Agrimi,6
Adriano Aguzzi7 and Giovanni Di Guardo1
Correspondence
Giovanni Di Guardo
[email protected]
1
Department of Comparative Biomedical Sciences, Faculty of Veterinary Medicine, University of
Teramo, Teramo, Italy
2
Istituto Zooprofilattico Sperimentale della Sardegna, Sassari, Italy
3
Department of Veterinary Morphophysiology and Animal Productions, Faculty of Veterinary
Medicine, University of Bologna, Ozzano Emilia (Bologna), Italy
4
Department of Veterinary Public Health and Animal Pathology, Division of Veterinary Pathology,
Faculty of Veterinary Medicine, University of Bologna, Ozzano Emilia (Bologna), Italy
5
Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, Viterbo, Italy
6
Istituto Superiore di Sanità, Department of Food Safety and Veterinary Public Health, Rome, Italy
7
Institute of Neuropathology, University Hospital of Zurich, Zurich, Switzerland
Received 6 February 2007
Accepted 29 June 2007
The enteric nervous system (ENS) probably plays a dominant role in sheep scrapie pathogenesis,
but little is known about the cell types involved. We investigated the ileal myenteric and
submucosal plexuses of four naturally and four orally experimentally scrapie-affected ARQ/ARQ
Sarda sheep, as well as those of 12 healthy-control Sarda sheep carrying different PrP
genotypes. All scrapie-affected animals, euthanized at clinical-disease end stage, showed PrPd
deposition within enteric glial cells (EGCs) and calbindin-immunoreactive (CALB-IR) and
neuronal nitric oxide synthase (nNOS)-IR neurons. Whole-mount investigations revealed no
significant differences between the densities of total, CALB-IR and nNOS-IR neurons in scrapieaffected versus healthy sheep, irrespective of PrP genotype. Our results suggest that EGCs and
CALB-IR and nNOS-IR neurons are probably involved in the pathogenesis of natural and oral
experimental sheep scrapie. Furthermore, the infectious agent may be less pathogenic towards
ENS neurons than it is towards central nervous system neurons.
Sheep scrapie is the ‘prototype’ of transmissible spongiform encephalopathies (TSEs) or prion diseases, a group of
neurodegenerative disorders affecting humans and animals.
The key pathogenetic event in TSEs is the accumulation
within the central nervous system (CNS) and peripheral
tissues of an abnormal isoform (disease-specific PrP, PrPd)
of the host-encoded cellular prion protein, PrPC (Aguzzi &
Polymenidou, 2004).
throughout the PrP gene (PRNP) (Prusiner, 1998). In
sheep, PRNP codons 136, 154 and 171 are of greatest
importance in modulating susceptibility/resistance to
scrapie (Goldmann et al., 1994). In Suffolk and Sarda
breeds (Westaway et al., 1994; Vaccari et al., 2001), codon
171 plays a major role in disease-susceptibility control,
whereas in Cheviot sheep, the additional influence of
codon 136 has been reported (Goldmann et al., 1994).
Genotype of the host is known to modulate susceptibility/
resistance to TSEs, which is dependent on polymorphisms
The gastrointestinal tract is probably the natural prion
entry site, with the enteric nervous system (ENS) playing,
along with palatine tonsils and Peyer’s patches (PPs), a
crucial role in the early pathogenesis of animal and human
TSEs (Mabbott & MacPherson, 2006). It has been
suggested that ENS plexuses could act as the initial site
A supplementary table showing tissue antigens and details of the
monoclonal and polyclonal antibodies utilized in the study is available
with the online version of this paper.
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G. Marruchella and others
of neuroinvasion for prions, which may subsequently gain
access to the CNS through sympathetic and parasympathetic efferent fibres (Aguzzi & Heikenwalder, 2006).
Nevertheless, no definitive information is available on the
cellular basis for prion transmigration from gut to ENS
plexuses, nor on the ENS cell types colonized by TSE
agents. Likewise, the morphofunctional changes affecting
the ENS cell populations during infection are unknown,
despite the existence of studies addressing CNS neuron
damage and targeting on behalf of TSE agents (Guentchev
et al., 1999).
This study was aimed at characterizing the ENS cells that
are targeted during natural and oral experimental scrapie
infection in Sarda sheep. Major emphasis was placed upon
enteric glial cells (EGCs) and two neuron populations
expressing calbindin (CALB) and neuronal nitric oxide
synthase (nNOS), respectively. In sheep, CALB-immunoreactive (IR) cells account for 20–25 % of myenteric plexus
(MP) and 65–75 % of submucosal plexus (SMP) neurons,
most of them corresponding to cholinergic Dogiel type II
neurons and probably acting as intrinsic primary afferent
neurons (Chiocchetti et al., 2004, 2006). Enteric nNOS-IR
cells, accounting for 31–36 % of MP and 22–24 % of SMP
neurons (Lalatta-Costerbosa et al., 2007), correspond to
Dogiel type I cells, probably acting as inhibitory motoneurons (Pfannkuche et al., 2002).
Another objective was to evaluate whether neuron loss
occurs within ileal ENS plexuses from naturally and orally
experimentally scrapie-affected Sarda sheep. Indeed, whilst
CNS neuron loss is constantly observed in TSEs (Pocchiari,
1994), no data concerning ENS are available.
Ileal MPs and SMPs were obtained from 20 Sarda sheep
carrying different PrP genotypes, which were characterized
as reported elsewhere (Vaccari et al., 2001). Twelve sheep
(three ARQ/ARQ, seven ARR/ARQ and two ARR/ARR
animals), aged 2–4 years and originating from a scrapiefree flock, acted as healthy controls and were slaughtered
according to standard procedures, with tissue samples
(cerebral obex, palatine tonsils and distal ileum) being
collected from them. The remaining animals included four
ARQ/ARQ naturally scrapie-affected and four additional
ARQ/ARQ sheep that had been dosed orally at 20 days of
age with 25 ml 20 % scrapie brain homogenate. These
animals were euthanized, in accordance with approved
protocols, at the terminal stage of disease (between 2 and
5 years), with the same tissues being collected. The diagnosis
of scrapie was confirmed or excluded by immunohistochemistry (IHC) and Western blot (WB) analysis, which
were carried out on the obex, tonsils and distal ileum, in
agreement with published protocols (Ligios et al., 2006).
Ileal samples were fixed in 10 % neutral-buffered formalin,
embedded in paraffin and cut into 5 mm thick sections,
both transverse and tangential, which were placed on
glass slides coated with (3-aminopropyl)triethoxy-silane
(Sigma-Aldrich).
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PrPd IHC was performed with a mouse monoclonal
antibody (mAb) (F99/97.6.1; VMRD, Inc.). A pre-treatment protocol was applied to abolish PrPC immunoreactivity without damaging other epitopes and preserving tissue
morphology (Kovács et al., 2005). Sections were rinsed in
96 % formic acid for 2 min at room temperature and
autoclaved at 100 uC for 20 min in 0.01 M citrate buffer
(pH 6.0). Antibody binding was detected by using a
biotin–avidin method and immune reactions were visualized by 3-39-diaminobenzidine (DAB) chromogen solution (both from Vector Laboratories, Inc.). Control
sections included obex, tonsil and distal ileum from
scrapie-infected and uninfected Sarda sheep. Additional
negative controls were set up by omitting the primary
antibody.
Double-labelling indirect immunofluorescence (DLIIF)
was carried out with a panel of commercial mAbs and
polyclonal (Po)Abs against PrPC (F99/97.6.1 mAb), glial
fibrillary acidic protein, an EGC marker (anti-GFAP
PoAb), neuron-specific enolase, a neuron cell marker
(anti-NSE PoAb), nNOS and CALB (both PoAbs) (see
Supplementary Table S1, available in JGV Online, for
details). The same protocol to abolish PrPC immunoreactivity was employed in DLIIF and then two primary
antibodies were added to sections and incubated overnight
at room temperature. PrPd was detected by an enhanced
biotin–avidin protocol (Vector Laboratories, Inc.) and
sections were incubated with goat anti-mouse and antirabbit biotinylated secondary antibodies for 30 min.
Immune reactions were visualized by Texas red avidin
DCS (D cell sorter) and fluorescein avidin DCS fluorochromes (Vector Laboratories, Inc.). Control sections
included obex, tonsil and distal ileum from scrapieinfected and uninfected Sarda sheep. Additional negative
controls were set up by omitting primary antibodies.
Sections were examined under a Nikon Eclipse 800
microscope equipped with fluorescence and images were
collected with a Nikon DXM 1200 digital camera.
Colocalization analyses were performed by ImageJ software
(National Institutes of Health).
To estimate the density (cells mm22) of total (HuC/D-IR),
nNOS-IR and CALB-IR neurons within ileal MPs, wholemount (WM) preparations were made. We utilized a
protocol originally developed in the rat (Phillips et al.,
2004), adapting it to the sheep ileum, where MPs show a
more variable and less geometric texture (Gabella, 1987)
and MP neurons are often gathered in large ganglia
exhibiting a polygonal or ring-like morphology, with no
apparent directionality (Lalatta-Costerbosa et al., 2007).
Beginning 2 cm oral to the ileocaecal junction, we collected
from each animal 8–10 cm long segments, which were cut
along the mesenteric border, pinned on balsa board and
fixed in 2 % paraformaldehyde plus 0.2 % picric acid in
PBS (pH 7.0) at 4 uC overnight, before being washed three
times in DMSO (10 min each) and stored at 4 uC in PBS
containing sodium azide (0.1 %). Segments were subdivided into six transverse columns, with a 161 cm
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Journal of General Virology 88
Role of the enteric nervous system in scrapie
sample being collected from each column at different
levels, to achieve an adequate representation of the entire
circumference (from anti-mesenteric to mesenteric border)
and length of every segment. WM preparations were
obtained by removing the mucosa, submucosa and circular
muscle layer from each sample. For DLIIF investigations,
MPs were incubated in 10 % normal goat serum in PBS
containing 1 % Triton X-100 for 30 min at room
temperature. An anti-HuC/D mAb was used as panneuron marker (Chiocchetti et al., 2004; Phillips et al.,
2004), whereas CALB-IR and nNOS-IR neurons were
detected with specific PoAbs (see Supplementary Table S1,
available in JGV Online, for details). Samples were
challenged simultaneously with two primary antibodies
for 40 h at 4 uC, washed three times in PBS (10 min each)
and finally incubated with goat anti-mouse and anti-rabbit
secondary antibodies conjugated with Alexa 594
(Molecular Probes) and fluorescein isothiocyanate (FITC)
492 (Calbiochem) fluorochromes, respectively.
small-sized or faintly stained cells, MP neurons were
counted by two independent investigators blind to the
experimental conditions at 640 magnification with a Zeiss
Axioplan microscope equipped with appropriate filters
discriminating the fluorochromes used. Conventionally,
neurons intersected by the upper and lower fields’
hemicircumferences were, respectively, disregarded and
considered (De Souza et al., 1993).
For each of the six samples, the number of total (HuC/DIR), CALB-IR and nNOS-IR cells was counted in 12
microscope fields (each field, 0.28 mm2), which had
previously been determined by means of two orthogonal
coordinates taken from a table of random numbers and
measured on the movable stage of the microscope.
Therefore, for each stain per plexus per animal, a
total area of 10.08 mm2 was evaluated. To avoid missing
All scrapie-affected sheep showed IHC evidence of PrPd
deposition in ileal MPs, SMPs and PPs, as well as in tonsils
and obex. WB analysis confirmed the above results. PrPd
accumulation was more prominent at the MP level, with a
‘pepper granule-like’ pattern strongly compatible with
EGCs. A simultaneous involvement of neuronal perikarya
was observed. No specific immunolabelling was present in
negative-control sections. By DLIIF, PrPd deposition
All quantitative data collected, not grouped for control/
infected status and PrP genotype, were tested for normality
with a Shapiro–Wilk W test. As the Shapiro–Wilk W test
did not demonstrate a normal distribution, comparisons
among healthy controls carrying different PrP genotypes
and between controls – irrespective of PrP genotype – and
scrapie-affected sheep were evaluated with Spearman’s
rank correlation test. Statistical analyses were performed by
using CSS software (StatSoft) and a conventional 5 % level
was used to define statistical significance.
(a)
(b)
(c)
(d)
(e)
(f)
Fig. 1. Naturally scrapie-affected Sarda sheep. Distal ileum (tangential section). (a–c) Specific anti-CALB (a) and anti-PrPd (b)
immunostaining is shown within an SMP neuron. Colocalization of the two signals is shown in white (c). (d–f) Specific antinNOS (d) and anti-PrPd (e) immunolabelling is observed within two neuronal perikarya and nerve fibres from an MP.
Colocalization of the two signals is shown in white (f). DLIIF with anti-CALB, anti-nNOS and anti-PrP antibodies was performed.
Fluorescein avidin DCS (green) and Texas red avidin DCS (red) were used as fluorochromes. Colocalization analyses were
carried out by means of ImageJ software. Bars, 50 mm.
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G. Marruchella and others
(a)
(b)
(c)
Fig. 2. Naturally scrapie-affected Sarda sheep. Distal ileum (tangential section). Specific anti-GFAP (a) and anti-PrPd (b)
immunostaining is shown within the EGCs of a large MP. Colocalization of the two signals is shown in white (c). Fluorescein
avidin DCS (green) and Texas red avidin DCS (red) were used as fluorochromes. Colocalization analyses were carried out by
means of ImageJ software. Bars, 50 mm.
involved EGCs and neurons (NSE-IR cells) of MPs and
SMPs from the eight scrapie-affected sheep. Both CALB-IR
and nNOS-IR neurons (Fig. 1) harboured discrete,
granular PrPd aggregates in cell bodies and fibres, with
no apparent quantitative differences between these cell
populations. In contrast, EGCs exhibited more prominent
and diffuse PrPd accumulations (Fig. 2). Negative-control
sections showed no evidence of PrPd deposition.
No statistically significant differences in the density of
total, CALB-IR and nNOS-IR neurons were observed
within ileal MPs among the different PrP genotypes of the
12 control sheep. Also, no statistically significant differences were found between the eight scrapie-affected and
the 12 control sheep – irrespective of PrP genotype – when
densities of total, CALB-IR and nNOS-IR neurons were
compared (Fig. 3).
deposition and for the progression of infection (Blattler
et al., 1997). EGCs are the morphofunctional equivalent of
CNS astrocytes (Cabarrocas et al., 2003), which harbour
PrPd earlier than CNS neurons (Diedrich et al., 1991) and
probably play a role in prion-induced neuron damage
(Raeber et al., 1997; Jeffrey et al., 2004).
We found no differences in the density of total, CALB-IR
and nNOS-IR neurons among control sheep with different
PRNP polymorphisms. This suggests that the host’s PrP
genotype neither affects the density and/or the neurochemical code of ileal MP neurons in Sarda sheep, as was
reported previously for nNOS-IR cells (Lalatta-Costerbosa
et al., 2007), nor does it modulate scrapie susceptibility/
resistance by influencing MP features. As no differences
were detected between scrapie-affected and scrapie-negative sheep, our data also suggest that ENS neurons may
This study supports the assumption that ileal ENS plexuses
are involved in the pathogenesis of both natural and oral
experimental scrapie infection in sheep. However, as all
scrapie-affected animals were investigated at clinicaldisease end stage, we cannot exclude the possibility that
ENS plexuses might have also been colonized by centrifugal
spread, from CNS to gut. By IHC, granular PrPd deposits
were detected within ileal MPs and SMPs from scrapieaffected sheep. As reported previously (Heggebø et al.,
2003; Jeffrey et al., 2006), the accumulation pattern was
mainly consistent with EGCs, although neuronal perikarya
and fibres also showed evidence of PrPd deposition.
DLIIF and colocalization analyses confirmed PrPd accumulation in EGCs, CALB-IR and nNOS-IR neurons.
Neurons of ENS plexuses express PrPC (Heggebø et al.,
2000; Shmakov et al., 2000) and may show PrPd deposition
in different TSEs (Andréoletti et al., 2000; McBride et al.,
2001; Sigurdson et al., 2001; van Keulen et al., 2002).
However, PrPd was more consistently detectable within
EGCs, which in humans were also shown to express PrPC
(Shmakov et al., 2000), a crucial prerequisite for PrPd
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Fig. 3. Mean densities of total (HuC/D-IR), CALB-IR and nNOSIR neurons within ileal MPs from the 12 control Sarda sheep
carrying different PrP genotypes and from the eight ARQ/ARQ
(naturally and experimentally) scrapie-affected sheep. No statistically significant differences (P¡0.05) were detected between
healthy-control (filled bars) and scrapie-affected (empty bars)
animals (Spearman’s rank correlation test).
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Journal of General Virology 88
Role of the enteric nervous system in scrapie
interact with the infectious agent in a unique way. Indeed,
it is widely accepted that CNS neuron loss represents a
dominant feature in TSEs (Pocchiari, 1994). Evidence
exists that microglial cells are involved in the pathogenesis
of TSE-associated neuron damage at brain level (Peyrin
et al., 1999; Pasquali et al., 2006; Priller et al., 2006), but
ENS plexuses do not physiologically host a microglial
component, a plausible explanation for the apparent lack
of neuron loss in our scrapie-affected sheep. We cannot,
however, exclude the possibility that qualitative changes
were still affecting ENS neurons.
In conclusion, this study showed that EGCs and neurons,
particularly CALB-IR and nNOS-IR cells, within ileal ENS
plexuses accumulate PrPd during natural and oral
experimental scrapie infection in Sarda sheep, without
apparent neuron loss. This intriguing observation warrants
further study on the complex biological interplay between
prions and neurons.
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Acknowledgements
Scrapie-specific neuronal lesions are independent of neuronal PrP
expression. Ann Neurol 55, 781–792.
The experimentally scrapie-affected sheep were kindly provided by Dr
Ciriaco Ligios, being part of another research project funded by the
European Union. We also thank Mrs Marina Baffoni for outstanding
technical assistance. This work was carried out with research grants
(PRIN 2004, 2006) from Ministero per l’Istruzione, l’Università e la
Ricerca, the Universities of Teramo and Bologna and Fondazione del
Monte di Bologna e Ravenna.
Jeffrey, M., Gonzalez, L., Espenes, A., Press, C., Martin, S., Chaplin, M.,
Davis, L., Landsverk, T., MacAldowie, C. & other authors (2006).
Transportation of prion protein across the intestinal mucosa of scrapiesusceptible and scrapie-resistant sheep. J Pathol 209, 4–14.
Kovács, G. G., Preusser, M., Strohschneider, M. & Budka, H. (2005).
Subcellular localization of disease-associated prion protein in the
human brain. Am J Pathol 166, 287–294.
Lalatta-Costerbosa, G., Mazzoni, M., Clavenzani, P., Di Guardo, G.,
Mazzuoli, G., Marruchella, G., De Grossi, L., Agrimi, U. &
Chiocchetti, R. (2007). Nos-immunoreactivity and NADPH-d
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