ARTICLE OPEN
Dysregulation of epithelial ion transport and neurochemical
changes in the colon of a parkinsonian primate
Erika Coletto1,6, Iain R. Tough2,6, Sara Pritchard3, Atsuko Hikima1, Michael J. Jackson1, Peter Jenner1, K. Ray Chaudhuri4,5,
Helen M. Cox 2, Mahmoud M. Iravani 3✉ and Sarah Rose1✉
The pathological changes underlying gastrointestinal (GI) dysfunction in Parkinson’s disease (PD) are poorly understood and the
symptoms remain inadequately treated. In this study we compared the functional and neurochemical changes in the enteric
nervous system in the colon of adult, L-DOPA-responsive, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated common
marmoset, with naïve controls. Measurement of mucosal vectorial ion transport, spontaneous longitudinal smooth muscle activity
and immunohistochemical assessment of intrinsic innervation were each performed in discrete colonic regions of naïve and MPTP-
treated marmosets. The basal short circuit current (Isc) was lower in MPTP-treated colonic mucosa while mucosal resistance was
unchanged. There was no difference in basal cholinergic tone, however, there was an increased excitatory cholinergic response in
MPTP-treated tissues when NOS was blocked with L-Nω-nitroarginine. The amplitude and frequency of spontaneous contractions in
longitudinal smooth muscle as well as carbachol-evoked post-junctional contractile responses were unaltered, despite a decrease in
choline acetyltransferase and an increase in the vasoactive intestinal polypeptide neuron numbers per ganglion in the proximal
colon. There was a low-level inflammation in the proximal but not the distal colon accompanied by a change in α-synuclein
immunoreactivity. This study suggests that MPTP treatment produces long-term alterations in colonic mucosal function associated
with amplified muscarinic mucosal activity but decreased cholinergic innervation in myenteric plexi and increased nitrergic enteric
neurotransmission. This suggests that long-term changes in either central or peripheral dopaminergic neurotransmission may lead
to adaptive changes in colonic function resulting in alterations in ion transport across mucosal epithelia that may result in GI
dysfunction in PD.
npj Parkinson’s Disease             (2021) 7:9 ; https://doi.org/10.1038/s41531-020-00150-x
INTRODUCTION
Parkinson’s disease (PD) is primarily considered to be a movement
disorder resulting from the loss of dopaminergic innervation of
the basal ganglia1. However, numerous non-motor symptoms are
also present, and these may start before, at the same time or
following the onset of motor symptoms2. Gastrointestinal (GI)
dysfunction including dysphagia, gastroparesis, constipation and a
decreased frequency of bowel movements all occur in PD, but it is
the early appearance of a range of non-motor symptoms including
constipation that precedes the onset of motor signs3–6. Constipa-
tion remains a significant problem throughout the course of PD
and adversely affects the quality of life of the patient population7.
Gut function is finely controlled by innervation from both the
peripheral and central nervous systems8. The intrinsic enteric
nervous system (ENS) consists of the myenteric plexus, which
predominantly controls longitudinal and circular muscle function,
and the submucosal plexus, which controls mucosal function and
the smooth muscle activity of the muscularis mucosae8. Although
the transmission in these areas is controlled by a wide range of
neurotransmitters, the main excitatory transmitter is acetylcholine
and the main inhibitory transmitters are vasoactive intestinal
peptide (VIP) and nitric oxide8. Peristaltic movement in the GI tract
and regulation of fluid across the epithelial lining is controlled by
the ENS, and this is crucial for effective peristalsis. While dopamine
(DA) replacement therapy relieves the motor symptoms of PD,
constipation is largely unaffected or worsened9. This suggests that
the basis of GI dysfunction in PD is locally mediated and largely
non-dopaminergic in nature.
GI dysfunction in PD may relate to early pathology in the dorsal
motor nucleus of the vagus (DMV) and the loss of vagal tone10.
Loss of nigral dopaminergic input to the DMV results in alterations
in the peristaltic activity of the lower GI tract11, and in rats
vagotomy prevented reduction of gut motility following paraquat
administration12.
The enteric neurons within the myenteric plexus modulate
motility, while neurons present in the inner submucous plexus
modulate mucosal ion and fluid transport8. Both ganglionated
networks contain sensory, as well as excitatory (e.g. cholinergic)
and inhibitory (e.g. nitrergic and VIPergic) neurons which are
involved in the reflexes that underpin propulsive activity in the
small intestine and colon. More speculatively, based on early α-
synuclein accumulation in the gut in PD13 and the ability of focal
toxin treatment in the gut and inflammatory change to cause cell
loss in the DMV14, there are suggestions that the disease process
in PD may originate in the GI tract and progress retrogradely via
the vagus into the brain15. However, it is not known whether the
onset of gut dysfunction in PD that leads to constipation is
initiated as a consequence of DA loss in the basal ganglia or
whether it is due to functional and neurochemical adaptive
changes at the level of the gut and/or ongoing inflammation.
1Neurodegenerative Diseases Research Group, Institute of Cancer and Pharmaceutical Sciences, Faculty of Life Sciences and Medicine, King’s College London, London SE1 1UL,
UK. 2Wolfson Centre for Age-Related Diseases, IoPPN, King’s College London, Guy’s Campus, London SE1 1UL, UK. 3Department of Clinical and Pharmaceutical Sciences,
University of Hertfordshire, Hatfield AL10 9AB, UK. 4Institute of Psychiatry, Psychology & Neuroscience, King’s College London, London SE5 8AF, UK. 5Parkinson Foundation
International Centre of Excellence, King’s College Hospital, Denmark Hill, London SE5 9RS, UK. 6These authors contributed equally: Erika Coletto, Iain R. Tough. ✉email: m.
iravani@herts.ac.uk; sarah.salvage@kcl.ac.uk
www.nature.com/npjparkd
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In vivo functional alterations of colonic motility have been
evaluated previously in rodent models of central dopaminergic
loss. In the MPTP-, rotenone- and paraquat-treated mice and rats a
delay of colonic transit and constipation have been identi-
fied11,14,15. Increases in contractile activity and impaired relaxation
of the proximal colon have also been reported10,16,17. In 6-OHDA-
treated rats, a significant decrease in the frequency of stools
produced and a delay in colonic transit were observed18,19.
Reports of alterations in neurochemical markers have been more
variable. In the rotenone mouse model no changes were reported
in nitrergic, VIP-ergic or dopaminergic innervation20. However, in
unilaterally lesioned 6-OHDA rats there was an increase in VIP-
ergic and dopaminergic innervation and a decrease in nitrergic
neurons19,21,22. A study by Tian et al.23 described a decrease in
colonic tyrosine hydroxylase (TH) expression in MPTP-treated
mice, whilst in the 6-OHDA rat an increase in the levels of TH and
the dopamine transporter (DAT) was found24. Reports on changes
in the colonic excitatory cholinergic system have also produced
conflicting results. In the rotenone model, ChAT immunoreactivity
was reduced but two 6-OHDA rat studies reported no changes in
ChAT, whereas one suggested a decrease in ChAT immunor-
eactivity in the distal colon18,19,22,25.
It is clear that there are changes to bowel function in
experimental models of PD in rodents but with conflicting views
on the neurochemical cause or consequence. In addition, there
has been little work carried out in the model closest to man. While
anecdotally we have some evidence of marked bowel dysfunction
following very early stages of MPTP treatment, it has not always
been possible to systematically study gut dysfunction throughout
the life of these primates because the main focus of investigations
in MPTP-treated common marmosets has been movement
disorders. Previously, Chaumette et al.26 identified a reduction in
TH-, and an increase in nNOS-immunoreactive (-ir) neurons in the
MPTP-treated macaques, and we have shown a dysregulation of
nitric oxide signal coupling in the ileum of MPTP-treated common
marmosets, accompanied by a selective reduction in enteric
cholinergic neurons26,27. However, there have been no studies of
the changes in mucosal ion transport in the colon or the
contractility of colonic tissue in isolated preparations, or accom-
panying changes in neuronal populations. As a consequence, this
study investigated the neuro-epithelial signalling in colonic
mucosa and the contractile responses of longitudinal smooth
muscle in the MPTP-treated marmoset. In addition, we investi-
gated changes in colonic neurotransmitters in an attempt to
clarify the current discrepancies that exist.
RESULTS
There was no difference in the basal epithelial resistance values of
colonic mucosa from untreated and MPTP-treated marmosets (Fig.
1a). By contrast, basal Isc levels (which is derived from epithelial
electrogenic ion transport) were significantly lower in mucosa
from MPTP-treated animals compared with naïve controls (Fig. 1b).
In order to investigate whether enteric neuro-epithelial signalling
was altered we utilised the neuronal depolariser, veratridine that
has been used previously to characterise neuro-epithelial mechan-
isms in mouse colon mucosa28 in the absence and presence of
specific antagonists. Pre-treatment with cholinergic antagonists
(atropine and hexamethonium; Atr+ Hex) reduced basal Isc levels
compared with vehicle control (Fig. 2) but there was no significant
difference in this tonic cholinergic activity between untreated and
MPTP-treated groups (Fig. 3a). There were also no significant
differences in Isc responses to the nitric oxide synthase (NOS)
blocker Nω-Nitro-L-arginine (L-NNA) alone, although the increase
in Isc to L-NNA tended to be larger in tissue from MPTP-treated
animals, or in combination with cholinergic antagonists (Fig. 3a)
indicating that the nitrergic as well as cholinergic tones within the
submucosal innervation of colonic mucosa are not significantly
altered. Depolarisation of the colonic submucosal innervation by
veratridine caused initial rapid elevations in Isc (the primary
response; 1°) followed by changes that more often became
reductions in Isc (the secondary response; 2°) and these respective
maxima and minima are presented separately in Fig. 3b. These
complex neurogenic Isc responses were similar in untreated and
MPTP-treated mucosae and were not significantly altered by
cholinergic or nitrergic blockade (or both) compared with vehicle
controls (Fig. 3b). Tetrodotoxin (TTX) reversed the remaining
veratridine-elevated Isc, confirming the neurogenic character of
these biphasic veratridine responses (Fig. 2; pooled data not
shown).
The nitric oxide donor/precursor, L-Arg, reduced Isc levels (after
veratridine) and these responses were increased in MPTP-treated
mucosa (with or without cholinergic blockade), although this was
not statistically significant (Fig. 4a). Internal controls show that
L-Arg responses were abolished predictably by L-NNA pre-
teatment, but not by the cholinergic blockers (Fig. 4a). In contrast,
the muscarinic agonist carbachol (CCh) elevated the Isc levels and
this activity was increased significantly in MPTP-treated tissues
when inhibitory nitrergic mechanisms were blocked with L-NNA
(Fig. 4b). As expected CCh responses were abolished by the
cholinergic antagonists, atropine and hexamethonium and were
not sensitive to L-NNA (Fig. 4b). In summary, significantly
increased post-junctional muscarinic activity was evident in
Fig. 1 Basal resistance and ISC levels in the transverse colon from untreated and MPTP-treated common marmosets. a Basal resistance;
b short circuit current (ISC). Values are mean ± SEM from 4 or 5 specimens, as shown in parenthesis (n= 4 MPTP and n= 5 control).
+P ≤ 0.05
compared to untreated animals (Student’s t-test).
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mucosae where nitrergic inhibition was reduced in the transverse
colon from MPTP-treated animals. A degree of enhanced nitrergic
signalling was also observed (but this was not statistically
significant) in tissue from MPTP-treated colon.
Functional changes in longitudinal muscle contraction
In isolated colon preparations from normal and MPTP-treated
animals there were differences in the pattern of spontaneous
contractions (Fig. 5a). Frequency of spontaneous contraction was
significantly increased in tissues from MPTP-treated animals
(Fig. 5b), whereas the amplitude of the contraction was slightly
reduced although this was not significantly different (Fig. 5c).
Administration of CCh at concentrations ranging from 0.01 to
30 µM led to concentration-dependent contractions of the
descending colon preparations obtained from normal and
MPTP-treated animals (F (7,88) = 7.68, P < 0.001, two-way ANOVA;
Fig. 5d). However, there were no significant differences between
the responses obtained from the normal or MPTP-treated animals
(F (1,88); P= 0.466, P > 0.05, two-way ANOVA; n= 7).
Quantitative analysis of serotonergic and dopaminergic
neurons
Analysis of immunohistochemical staining in the colon for the
pan-neuronal marker HuC/HuD showed no overall difference in
the total number of neurons in the myenteric plexus in proximal
or distal colon in the MPTP-treated animals compared to naïve
controls (Table 1). Analysis of double immunohistochemistry
revealed that 1.0 % (proximal) and 0.6% (distal) of the myenteric
neuronal population in the colon of common marmosets were TH-
ir (Table 1). By contrast, approximately 21% of the myenteric
neuronal population in the colon were serotonergic and
approximately 32% of the neuronal population were immunor-
eactive for substance P (Table 1). Prior treatment with MPTP
produced no long-term change in the number of dopaminergic or
serotonergic or substance P immunoreactive neurons in either the
proximal or distal colonic myenteric plexus.
Fig. 3 The effect of cholinergic and nitrergic antagonists. The
effect of cholinergic and nitrergic antagonists alone (a) and
following subsequent veratridine-induced mucosal responses (b)
on the change in ISC levels in transverse colon from untreated or
MPTP-treated marmosets. Tissues were treated with either atropine
plus hexamethonium (Atr+Hex) alone, L-NNA alone or Atr+ Hex+
L-NNA (a, b). Subsequent veratridine-induced changes in ISC are
divided into an initial 1°, and a slower 2° component after each pre-
treatment (b). Values are change in ISC (mean±SEM). Replicates are
shown in parentheses. *P ≤ 0.05 compared to vehicle treatment
(ANOVA and Dunnett’s test).
Fig. 4 Changes in the short cuircuit current (ISC) in the presence of
L-arginine or carbachol in transverse colon mucosa from naïve or
MPTP-treated animals. a L-Arginine (200 µM) and b carbachol
(10 µM). Values are mean ± SEM from the numbers of animals shown
in parentheses. (a, b) *P ≤ 0.05 compared to vehicle in naïve animals;
#P ≤ 0.05 compared to vehicle in MPTP-treated animals (ANOVA with
Dunnett’s test).
Fig. 2 Typical traces from ISC measurements in the transverse
colon mucosa of an MPTP-treated marmoset. The upper trace is a
vehicle (Veh) control for the lower trace where cholinergic
antagonists, atropine and hexamethonium (Atr+Hex) were added
first. Subsequent changes in ISC in both preparations are evident
after addition of veratridine (Ver), then 20min later L-Arg, followed
by CCh (note this response is blocked by the cholinergic
antagonists) and finally, TTX was added to both preparations.
Values to the left of each trace are the basal ISC levels. Dotted lines
show the extrapolated ISC levels from which drug-induced
maximum or minimum changes were measured between 5 and
20min, as appropriate.
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Quantitative analysis of inhibitory myenteric neuronal
populations
The number of VIP-ir neurons was significantly increased in the
proximal but not in the distal colon in the MPTP-treated
marmosets (Fig. 6a–d). Whilst analysis of the number of neurons
expressing both VIP-ir and nNOS-ir showed no quantitative
changes in either segment of the colon, the number of neurons
expressing VIP-ir or colocalised VIP and nNOS-ir in MPTP-treated
marmosets was significantly increased in the proximal colon
(Fig. 6a–d).
We investigated the distribution of α-synuclein immunoreactiv-
ity in the proximal and distal colon (Fig. 7). Around 40% of the
HuC/HuD-ir neurons in the myenteric plexus were associated with
α-synuclein-ir, the majority of which was found localised
perineuronally (Fig. 7c). There was no change in the total number
of neurons containing α-synuclein in the MPTP-treated animals in
the distal colon, however, there was a significant increase in the
number of α-synuclein-ir soma in the proximal colon (Fig. 7b, d). In
addition, in the proximal but not distal colon the distribution of α-
synuclein also changed from the predominantly perineuronal to a
more diffuse cytoplasmic distribution (Fig. 7a, c).
Quantitative analysis of excitatory cholinergic pathways
The assessment of the number of ChAT containing neurons was
made by double immunofluorescence labelling with HuC/HuD
(Fig. 8). There was a significant decrease in the number of ChAT-ir
neurons in the proximal but not distal colon of MPTP-treated
animals, whilst the total number of myenteric neurons was
unchanged (Fig. 8a–d).
Fig. 5 Frequency and the amplitude of spontaneous contractions in isolated distal colon preparation. a Two representative traces
showing spontaneous contractile response of colon preparations obtained from control (blue trace) and MPTP-treated (red trace) animals.
b The frequency and c amplitude of spontaneous contraction in MPTP-treated (n= 6) and naïve (n= 7) animals; unpaired Student’s t-test.
d The effect of carbachol on the change in tone of isolated distal colon in MPTP-treated (n= 6) and naïve (n= 8) animals); all values are mean
± SEM; Two-way repeated measure ANOVA.
Table 1. The number of 5-HT-, TH- and substance P-ir cells in an area of 1 mm2 relation to the number of Huc/HuD-ir neurons. Each value is mean ±
SEM. There were no significant differences between the number of HuC/HuD-ir cells or other markers in the proximal or distal colon in either control
or tissues obtained from MPTP-treated animals.
Number of immunoreactive neurones/mm2
Markers/regions Huc/HuD-ir 5-HT-ir Huc/HuD-ir Tyrosine hydroxylase-ir Huc/HuD-ir Substance P-ir
Proximal colon - Control 1445 ± 64.4, n= 7 302 ± 15.6, n= 7 1251 ± 44.6, n= 6 12 ± 6.4, n= 6 1495 ± 74.8, n= 5 479 ± 45.6, n= 5
Proximal colon - MPTP 1610 ± 102.4, n= 6 339 ± 24.6, n= 6 1416 ± 64.5, n= 6 12 ± 3.9, n= 6 1595 ± 75.8, n= 4 529 ± 99.3, n= 4
Distal colon - Control 1516 ± 59.5, n= 8 313 ± 13.0, n= 8 1580 ± 125.9, n= 8 9.5 ± 3.4, n= 8 1541 ± 37.5, n= 7 538 ± 45.1, n= 7
Distal colon - MPTP 1484 ± 68.1, n= 5 353 ± 22.8, n= 5 1546 ± 58.9, n= 6 8.4 ± 4.6, n= 6 1699 ± 95.4, n= 4 550 ± 24.8, n= 4
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Quantitative analysis of enteric glial cells
There was an extensive presence of S100β-, GFAP- and SOX-10-ir
inflammatory markers in both proximal and distal colon that rarely
colocalised with HuC/HuD-ir cells. The number of S100β-, GFAP-
and SOX-10-ir cells was determined in the myenteric plexus of the
proximal and distal colon on MPTP-treated and naïve control
marmosets. Whilst compared to the neuronal numbers the
number of SOX-10-ir glia was significantly greater than that of
S100β- or GFAP-ir cells in both the proximal and distal colon,
MPTP treatment did not result in a significant change in their
number or distribution. However, there was a significant increase
in the number of S100β-ir and GFAP-ir in proximal but not distal
colon following MPTP treatment. Moreover, there was some
evidence of GFAP-ir overlap with HuC/HuD-ir in the distal colon,
but overall there was no glial overlap between HuC/HuD-ir cells
Fig. 6 Effect of MPTP treatment on the number of HuC/D-, nNOS-
and VIP-ir, in the proximal and distal colon of naïve control and
MPTP-treated common marmosets. Inset panels (a, e) show HuC/D-
ir; (b, f) show nNOS-ir and (c, g) show VIP-ir in the proximal (a) and
distal (c) colon of naïve control and MPTP-treated common
marmosets. Panels (d, h) show triple immunofluorescence for HuC/
HuD (blue), nNOS (red) and VIP (green) in the myenteric plexus from
proximal (main panel a) and distal (main panel c) colon of naϊve and
MPTP-treated animals. Quantitative analysis of nNOS- and VIP-ir
neurons in MPTP-treated marmosets compared to naϊve controls in
the proximal and distal colon is shown in the main panels (b) and
(d), respectively. The number of immunoreactive neurons are
expressed as mean ± SEM. *P < 0.05 compared to naïve control
(Students t-test). White arrows indicate co-localisation of nNOS and
VIP. The scale bar in panel (aa) indicates 100 µm.
Fig. 7 Effect of MPTP treatment on the number of HuC/D-, VIP-
and α-synuclein-ir in the proximal and distal colon of naïve
control and MPTP-treated common marmosets. Inset panels (a, e)
show HuC/D-ir; (b, f) show VIP-ir and (c, g) show α-synuclein-ir in the
proximal (a) and distal (c) colon of naïve control and MPTP-treated
common marmosets. Panels (d, h) show triple immunofluorescence
for HuC/HuD- (blue), VIP- (green) and α-synuclein-ir (red) in the
myenteric plexus from proximal (main panel a) and distal (main
panel c) colon of naϊve and MPTP-treated animals. Quantitative
analysis of VIP-ir and α-synuclein-ir (a: soma; b: extracellular
surrounds) neurons in MPTP-treated marmosets compared to naϊve
controls in the proximal and distal colon is shown in the main
panels (b) and (d), respectively. The number of immunoreactive
neurons are expressed as mean ± SEM. *P < 0.05 compared to naïve
control (Students t-test). White arrows indicate co-localisation of VIP-
and α-synuclein-ir. The scale bar in panel (aa) indicates 100 µm.
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suggesting the distribution of SOX-10, S100β-ir and GFAP-ir was
glial (Fig. 9).
DISCUSSION
Reduced gut motility and constipation are common problems
associated with changes on colon function in PD. This has been
reproduced in both rodent and primate models of PD where the
nigro-striatal pathway has been destroyed6,18,22,26. However, there
have been no studies connecting the histological changes with
functional alterations at cellular and physiological level. Specifi-
cally, no one to our knowledge has investigated connections
between these changes in the MPTP-treated primate model of PD.
We now show functional, biochemical and physiological changes
in common marmosets in the colon over 1 year after MPTP
treatment suggesting long-term local adaptive change and
possible alteration in central control of gut function.
The majority of neurons in the myenteric plexus of naïve
animals were serotonergic (21%), cholinergic (35%), nitrergic
(40%), substance P-ergic (32%) or VIP-ergic (17%), with the
expression of neurons containing TH-ir being sparse (<1%).
Previous studies in untreated macaques showed a similar profile,
although the proportion of VIP-ergic neurons was much lower
than in the marmoset26. Previously Natale et al.29 reported a
reduction in murine enteric dopaminergic neurons 1 week after
MPTP treatment in the duodenum but not in the colon. Whilst we
found no change in the number of TH-ir neurons in the colon, we
have previously reported generalised reduction in the intensity of
TH-ir staining in the ileum but not the actual cell numbers27
suggesting that these neurons may not be as susceptible to the
toxic effects of MPTP as the dopaminergic neurons of the
substantia nigra were, or were better able to repair over time.
However, because TH is a common enzyme in the biosynthesis of
both dopamine and noradrenaline much of the sparse TH
immunoreactivity in the colon is likely to be associated with the
extrinsic sympathetic innervation30, which may explain why
the selective dopaminergic toxin MPTP did not alter the
TH-immunoreactivity in the myenteric plexus of the common
marmosets.
The number of cholinergic neurons in the myenteric plexus was
reduced by 27% in the MPTP-treated marmosets compared to
naïve controls, whereas the number of VIP-ergic neurons almost
doubled. Interestingly, there was no change in the number of
nNOS-ir cells. These findings are contrary to the changes reported
in MPTP-treated macaques where the total neuronal population
(HuC/HuD-ir) and the number of nitrergic neurons increased, but
there were no changes in cholinergic or VIP-ergic cell number26.
There is considerable diversity in the changes in neuronal
populations in the different animal models of PD which may
reflect the species, toxin, time and recovery differences. In the
MPTP-treated mouse there was no change in nNOS-ir or ChAT-ir
neurons 10 days after MPTP treatment17, and there was a
functional recovery of the reduction of dopamine levels in the
ileum over time31. However, recently Sampath et al.32 reported
reduced nNOS expression on all parts of the colon 7 days after
MPTP treatment, with a reduction in dimer formation suggesting
reduced nNOS activity. In unilateral 6-OHDA-lesioned rats the
number of ChAT-ir neurons were reported as decreased or
unchanged, whereas there was a reduction in nitrergic and
increase in VIP-ergic neurons21,22,33, partially agreeing with the
present study. All the models used showed a reduction in nigral
dopamine cell bodies, however, this was greatest in the 6-OHDA-
lesioned rat (>90% loss) and the present study (>70% loss; data
not shown). Whilst it is not possible to make a definitive
conclusion, the data in the 6-OHDA-lesioned rat and the MPTP-
treated marmoset may suggest a central component to these
changes, as no toxin was administered systemically in the rodent
model, so there would be no direct effect on the gut tissue. In
addition, the time after MPTP treatment in the marmosets in the
present study would allow acute toxic damage to the gut to be
reversed as eluded to in the MPTP mouse where recovery of the
neurochemical changes was seen over time31. In the current study
we observed a significant but low-level inflammation exemplified
by the lack of change in SOX-10 glial markers and an increase in
Fig. 8 Effect of MPTP treatment on ChAT / HuC/HuD-ir neurons in the proximal and distal colon. Panels (a, b) show proximal and (c, d)
show distal colon. Quantitative analysis of HuC/HuD- and ChAT-ir neurons in MPTP-treated marmosets compared to naϊve controls in the
proximal and distal colon is shown in panels (b) and (d), respectively. The number of immunoreactive neurons are expressed as mean ± SEM.
**P < 0.01 compared to naïve control (Students t-test). The scale bar in panel (a) indicates 100 µm.
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GFAP- and S100β in the proximal but not in the distal colon. In our
previous study27 we did observe a modest but significant increase
in the number of SOX-10-ir cells in the ileum following MPTP
treatment, but the lack of change in the proximal and distal colon
in this study may suggest a regional pattern in the expression of
inflammation.
The evidence of an inflammatory response in the proximal
colon, albeit mild, is in accord with the reported increase in the
expression of pro-inflammatory and glial marker in PD following
colonoscopy of the ascending colon34, which roughly corresponds
to proximal colon in this study, and in the colon of unilaterally 6-
OHDA-lesioned rats33. However, the magnitude of difference
observed between our study and those in the human PD and 6-
OHDA-lesioned rats is not surprising as in PD the degeneration is
progressive, and in rats the samples were obtained 8 weeks after
lesioning33 while the neuronal cell loss in the MPTP-treated
primate is acute and the samples were taken approximately 1.4
years following MPTP lesioning, thus allowing for some recovery
from an inflammatory response. Indeed, repair is observed only up
to 24 weeks where new neurons appear in the ENS, and only after
5-HT4 receptor stimulation35. Interestingly, Devos et al.34 also
showed the presence of phosphorylated α-synuclein immunor-
eactivity in two PD subjects undergoing colonoscopy in the
ascending colon. Naïve marmosets showed a mainly perinuclear
distribution of α-synuclein in approximately 30% of the neuronal
cells of the ENS, whereas over 1 year after MPTP treatment, the
distribution of α-synuclein was more diffuse. We have previously
shown that MPTP treatment impairs proteasomal enzyme activity
in common marmosets which is intimately linked to the onset of
synucleinopathies36 and more recently, Li et al.37 showed that
32–36 days following MPTP in cynomolgus monkeys, there was a
significant increase in the level of total, phosphorylated and
oligomeric α-synuclein. Lai et al.31 reported that two days
following MPTP treatment in mice, there was an initial reduction
in α-synuclein expression in the myenteric plexus of ileum but
after 2–3 weeks there was a rebound increase with a
corresponding increase in inflammation and pro-inflammatory
cytokine production suggesting that MPTP did not acutely alter
the level of α-synuclein expression but perhaps the increase in the
level of α-synuclein was related to the onset of gut inflammation.
In the present study, the expression and localisation of the α-
synuclein following MPTP was very diffuse and its incidence and
pattern of distribution which corresponded to the pattern of
inflammation in the proximal colon suggest that the expression of
α-synuclein is indeed linked with inflammatory changes.
To our knowledge, monitoring changes in mucosal ion
transport to investigate colonic dysfunction at the cellular level
in a model of PD have not been investigated previously. There was
no effect of MPTP treatment on basal resistance suggesting that
the colonic mucosa was intact and there was no significant
deterioration of epithelial tight junctions. The reduced basal Isc
levels observed in colon of the MPTP-treated marmosets could be
a consequence of reduced neurogenic tone, or reduced secreta-
gogues from non-neuronal resident cells within the lamina
propria. Further investigations will be required to identify the
cellular mechanism(s) that contribute to this difference in basal
activity. Depolarisation of submucosal innervation by veratridine
stimulated complex net changes in Isc that were not significantly
altered following MPTP compared with normal colon mucosa.
Veratridine-activated submucosal neurons, the majority of which
innervate the mucosa and are either sensory, secretomotor or
secretomotor-vasodilator neurons, each of which express and
release different primary (as well as various secondary) neuro-
transmitters8 and may be differentially affected by MPTP
treatment. Previous studies have shown that in mouse distal
colon, the majority of the mucosal veratridine response is non-
adrenergic and non-cholinergic (NANC), and not significantly
altered by cholinergic blockade28. The same appears to be true in
marmoset colon and the neuroeffectors are thus potentially
neuropeptide(s) and/or NO, but further studies are required to
identify the predominant submucosal neurotransmitters. Despite
this complexity, it appears that prejunctional and presynaptic
Fig. 9 Effect of MPTP treatment on SOX-10-, S100β- and GFAP-ir glial cells in the myenteric plexus of the proximal colon of naïve control
and MPTP-treated common marmosets. Panel (a) shows SOX-10-ir, (b) S100β-ir and (c) GFAP-ir. With the exception of GFAP-ir (c) there was no
signal overlap between the immunoreactivity of glial and neuronal cells. The white arrows indicate colocalisation of GFAP-ir and Huc/HuD-ir
cells. The arrows indicate glial/neuronal overlap. Quantitative analysis of glial cells in MPTP-treated marmosets compared to naϊve controls in
the proximal and distal colon is shown in panels (d) and (e), respectively. The number of immunoreactive neurons are expressed as mean ±
SEM. Scale bar in panel (a) indicates 100 µm. *P= 0.02; **P= 0.0016 two-way ANOVA, Sidak multiple comparison test.
E. Coletto et al.
7
Published in partnership with the Parkinson’s Foundation npj Parkinson’s Disease (2021)     9 
cholinergic mechanisms are not significantly altered in colonic
tissue from MPTP-treated animals in spite of the 21% loss of
cholinergic cell bodies in the proximal colon. However, muscarinic
epithelial signalling (revealed by CCh addition) is significantly
amplified (perhaps as a compensatory response to the reduced
cholinergic transmission) and NO-mediated responses are
increased in colonic mucosa from MPTP-treated animals. How
this hyper-responsiveness in post-junctional muscarinic and
nitrergic signalling translates into slowed motility and constipation
has yet to be resolved, but increased nitrergic tone (revealed by L-
NNA) might be expected to amplify mucosal anti-secretory effects
(which we observed, Fig. 4a) and slower colonic motility. However,
the immunohistochemical analysis of the myenteric plexus
showed no difference in the number of nNOS immunoreactive
cells per ganglia, but the number of ChAT-ir was significantly
decreased. Although immunoreactivity was not assessed in the
submucosal plexus, a similar decline in the cholinergic neurones in
that region (in line with the decline in the cholinergic neurones of
the myenteric plexus) might explain the alterations in the
epithelial signalling. An earlier study suggested that neuropatho-
logical changes occurring in the GI tract following MPTP are more
limited to the myenteric plexus than the submucosal plexus even
though dopaminergic innervation was similarly reduced26. These
changes may be the result of acute toxicity as no change in
dopamine innervation was seen in the present study over 1.4
years after MPTP treatment. Pathological changes such as the
presence of Lewy bodies do occur in both the submucosal and
myenteric plexuses in PD3,38–40. Therefore, whether other neuro-
chemical phenotypes such as VIP, acetylcholine and nitric oxide in
the submucosal plexus are similarly patterned remains to be
established but any neurochemical changes seen in the myenteric
plexus are also likely to be reflected in the submucosal plexus.
Three to five weeks following MPTP treatment, western blot
analysis suggested α-synuclein levels were reportedly raised in the
ileum of mice31 and colon of monkeys37. Whilst we did not
measure levels of α-synuclein in the present study, we noted that
there was a redistribution of the protein in the neurons of the
proximal but not the distal colon such that α-synuclein was found
to a greater extent in the whole neurons rather than surrounding
the cell body.
The reduction in excitatory innervation and increase in the
number of inhibitory (NOS-ir) neurons conflicts with the increased
frequency and reduced amplitude of the contraction of the
longitudinal muscle seen in colon from the MPTP-treated
monkeys, although the observed changes to these spontaneous
contractions may be explained by the ongoing inflammatory
changes. Interestingly, the amplitude of electrically evoked
contractions in rat colon is reduced, and there is a dysregulation
of contraction ex vivo, following a 6-OHDA lesion, suggesting
some local adaptive changes to centrally mediated effects6,22. The
reduction of ChAT-ir neurons in the myenteric plexus of the MPTP
marmosets in this study would predict that the contractile
response of the longitudinal muscle of colon to exogenous CCh
may be greater due to the phenomenon of denervation super-
sensitivity. However, there was no significant difference in the
concentration-response curves to CCh suggesting that cholinergic
control of longitudinal muscle was not altered significantly.
Further investigations are needed to fully understand the role of
changes in the ENS compared to the CNS in colonic activity.
In conclusion, the most striking finding of this study is that
following MPTP treatment, there are specific adaptive physiolo-
gical changes in the colon ion transport across the mucosal
epithelia. Alterations in ion transport can have several important
implications. These may include altered handling of Na+, Cl−,
HCO3
− and K+ which collectively would influence electrolyte
homoeostasis and water transport across the epithelia. Longer
term such dysfunction might affect the balance and the
proliferation of the GI microbiota (and their metabolites e.g. short
chain fatty acids such as acetate, propionate, and butyrate, which
are synthesised from dietary carbohydrates by colonic bacterial
fermentation) which in turn alter mucosal function and slow colon
motility41. It may be speculated that these changes could cause
constipation, and may be a trigger for accumulation of misfolded
α-synuclein in the gut42. The latter factor has been implicated in
the pathogenesis of Parkinson’s disease via the gut–brain axis43.
METHODS
Animals
Two groups of adult common marmosets (Callithrix jacchus; Harlan UK Ltd.
Loughborough, LE12 9TE, UK; Manchester University and King’s College
London) were used in this study. One group comprised of drug and toxin
naïve controls (n= 8; 4 male and 4 female, 333–407 g, 373 ± 16 g) aged
3.0 ± 0.2 years. The second group (n= 7, 2 males and 5 female, 313–389 g,
359 ± 17 g), aged 3.8 ± 0.4 years had been previously treated with 1-
methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) 1.4 ± 0.4 years prior to
this study. The animals were housed singly or in male (vasectomised)/
female pairs (cage dimensions of height: 166 cm, width: 140 cm, depth:
90 cm), in a room maintained at temperature (24 ± 2 °C), 50% relative
humidity and with a 12-h light/dark cycle. All animals were given 1 meal of
mashed cereal and 1 meal of fresh fruit daily and had ad libitum access to
food pellets and fresh water. Regulated procedures comply with the
ARRIVE guidelines, were carried out in accordance with the UK Animals
(Scientific Procedures) Act 1986, with approval of the King’s College
London Animal Welfare and Ethical Review Board under project licence
PPL 70/7146 and were compliant with the minimal standards as defined by
the European Communities Council Directive (2010/63/EU). The animals’
environment was enriched by the installation of a viewing turret (height:
36 cm, width: 35 cm, depth: 50 cm) on top of the housing cages allowing
the animals to climb above head height of research staff. Also provided
were wooden ladders/perches, hammocks, swings, nesting boxes, multiple
feeding platforms and saw dusted floors for forage feeding.
MPTP treatment
Motor deficits were induced 1.4 ± 0.4 years prior to this study by the
administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochlor-
ide (MPTP; 2.0 mg/kg, sc; S.I.D. for up to 5 days, Sigma, UK)44. Once
recovered from the acute effects of MPTP (approximately 3 months), all of
the animals exhibited stable motor deficits including a marked reduction
of locomotor activity, poor coordination of movement, abnormal and/or
rigid posture and reduced alertness/head-checking movements. The
MPTP-treated animals had previously been used for the behavioural
assessment of response to antiparkinsonian drug treatment. All animals of
this group were subjected to levodopa (8 mg/kg) plus benserazide (15mg/
kg), 3 animals in this group were also subjected to ropinirole (up to 1mg/
kg), an AMPA allosteric modulator and nitric oxide synthase inhibitors. All
animals received a wash out period of at least 2 months prior to this study
and there were no overt contractile differences between tissues obtained
from these animals. Immunohistochemical studies showed >70% TH cell
loss in the substantia nigra pars compacta compared to the naïve
controls27.
Perfusion of animals
The animals were euthanised using an overdose of pentobarbital sodium
(60mg/kg; Euthatal, Merial Animal Health Ltd.) between 7.30 and 8.30 a.m.
Upon cessation of foot and corneal reflexes, the thoracic and abdominal
cavities were opened. The animals were transcardially perfused with ice-
cold oxygenated (95% O2 plus 5% CO2) Krebs-Henseleit (KH) solution
(composition mM: NaCl, 118; KCI, 4.7; CaCl2, 2.5; MgSO4, 1.2; NaHCO3, 25;
KH2PO4, 1.2; glucose, 11.1).
Mucosal resistance and short-circuit current (Isc) measurement
Transverse colon (~5 cm taken midway between the caecum and rectum)
was excised and placed in fresh KH buffer prior to microdissection.
Mucosal preparations with intact submucosal innervation were prepared
by removing the overlying smooth muscle and integral myenteric
innervation. Four adjacent mucosal pieces were each mounted between
two halves of the Ussing chambers (exposed area of 0.14 cm2) as described
previously41,45,46. The tissues were bathed in KH (5 ml, both sides) at 37 °C,
E. Coletto et al.
8
npj Parkinson’s Disease (2021)     9 Published in partnership with the Parkinson’s Foundation
aerated with 95% O2/5%CO2 and voltage-clamped at 0mV. After an
equilibration period of 20–30min the stable baseline short-circuit current
(Isc) and transepithelial resistance were noted, and basolateral addition
(unless otherwise stated) of drugs made, recording resultant changes in Isc
continuously. The mucosae were pre-treated for 20min with either; (i) a
mixture of atropine (1 µM) and hexamethonium (200 µM) to block
cholinergic-mediated responses; or (ii) Nω-Nitro-L-arginine (L-NNA; 1
mM) to inhibit nitrergic responses; or (iii) all three inhibitors, with the
fourth mucosal preparation being a vehicle control. Following these pre-
treatments, the neuronal depolarising agent veratridine (30 µM) was added
to establish the mucosal effects of enteric nerve stimulation and the
altered Isc was allowed to stabilise (20min) before adding control agonists,
namely L-arginine (L-Arg, 200 µM), and after a further 20min CCh (10 µM),
and finally in some experiments, tetrodotoxin (TTX, 100 nM) to block any
remaining neural activity. The peak changes in Isc to each of these agents
were recorded at given time points. Data were pooled and changes in Isc
were expressed as mean ± 1 SEM (µA/cm2). Single comparisons utilised
Student’s unpaired t-test, while multiple comparisons were performed
using one-way ANOVA with Dunnett’s post-test, with a P value <0.05 being
considered statistically significant.
Colonic isometric contraction measurement
The descending colon was excised from the rectum to the start of the
descending colon and placed in aerated KH. Three to four 1.5 cm lengths
of the descending colon were cut transversely from the proximal end and
suspended in a 15ml organ baths with a resting tension of 1.0 g-force at
37 °C, as published previously47 with minor modifications. Following a
30min equilibration period and initiation of spontaneous contractions, the
colonic contractile activity was measured using an isometric transducer
connected to LabChart data acquisition system with signal triggers to mark
start of spontaneous and drug-induced contractile responses (AD
Instruments Ltd., Oxford, UK).
Tissue viability was tested initially by administration of CCh (10 µM) to
access the contractile responses. Only tissues that contracted >2 g-tension
were included in the study. To assess whether prior MPTP treatment
affected the receptor/effector coupling or whether there were changes at
the level of smooth muscle, the isolated colonic preparations were
contracted by cumulative addition of various concentrations of CCh
(0.01–30 µM; Sigma Aldrich). The amplitude was estimated by adding
together the tension of individual contractions occurring during a period
of 10min and dividing this sum by the number of contractions during this
period. The rate of spontaneous activity (rate/min) was derived from
dividing the total number of contractions during the 10min
observation time.
Intestinal whole-mount preparation
Sections of the distal colon (1 cm from the rectum) were isolated and
immersed for 15min in phosphate buffered saline (PBS) pH 7.2 containing
1 µM nicardipine as a muscle relaxant (Sigma-Aldrich, UK). They were then
cut along the mesenteric line and washed in PBS to remove luminal
contents. Subsequently, they were stretched on a wax support with the
mucosal layer downwards and fixed for 6/7 h in Zamboni’s fixative (2%
paraformaldehyde containing 0.2% picric acid in 0.1 M PBS) at 4 °C.
Following fixation, the tissues were washed in dimethylsulfoxide (DMSO)
followed by three 10min washes in PBS and then stored at 4 °C in PBS
containing 0.1% sodium azide. Stretched specimens were processed as
longitudinal muscle-myenteric plexus whole-mount preparations (LMMPs)
by dissociating the different intestinal layers (i.e. mucosa, submucosa and
circular muscle) as previously described21.
Immunohistochemistry
To analyse enteric neurons and glial populations in myenteric plexus of the
LMMPs, double staining [HuC/HuD-choline acetyl transferase (ChAT), HuC/
HuD -5-HT, HuC/HuD-substance P (SP), HuC/HuD-tyrosine hydroxylase (TH),
HuC/HuD-glial fibrillary acidic protein (GFAP), HuC/HuD-SOX-10 and HuC/
HuD-S100β)] or triple labelling [HuC/HuD- vasoactive intestinal polypep-
tide (VIP)/neuronal nitric oxide synthase (nNOS), Hu/nNOS, HuC/HuD-α-
synuclein/VIP)] immunofluorescence techniques were performed. Depend-
ing on the antibody characteristics either indirect immunohistochemistry
or labelled streptavidin-biotin (LSAB) methods were used.
The labelled streptavidin-biotin (LSAB)
immunohistochemistry
After staining with primary antibodies, tissues were washed in PBS and
then incubated with biotinylated secondary antibody, diluted in the
blocking buffer, for 1 h at room temperature. Whole-mount preparations
were then incubated overnight with a second primary antibody following
the same procedure for time and temperature. After 24 h, specimens were
washed in PBS and incubated in fluorescent dye streptavidin and suitable
secondary antibody for 3 h, then washed and mounted as described above.
All primary and secondary antibodies used are summarised in Table 2.
Double staining
Prior to immunostaining, whole-mount samples were permeabilised for 1 h
at room temperature in blocking buffer (10% normal goat or donkey
serum, 5–10% PBS and 1% Triton X-100) to reduce non-specific binding.
Whole-mounts were then incubated overnight in mixtures of primary
antibodies diluted in the blocking buffer, at 4 °C. Anti- HuC/HuD antibody
Table 2. Primary and secondary antibodies, their sources and dilutions.
1° Antibody Host Dilution Source 2° Antibody Dilution
Neuronal protein
monoclonal HuC/D
Mouse 1:50 16A11: Molecular probes Donkey anti-mouse Alexa fluor 350 IgG (H+ L) 1:100
Vasoactive intestinal polypeptide
polyclonal
Rabbit 1:100 Sc-20727; Santa Cruz
Biotechnology
Horse FITC anti-rabbit IgG (H+ L) 1:100
Neuronal nitric oxide synthase
polyclonal
Rabbit 1:500 AB5380: Millipore Goat FITC anti-rabbit IgG (H+ L) 1:100
Tyrosine hydroxylase polyclonal Rabbit 1:500 P40101; Pel-Freez
Biologicals, USA
Biotinylated goat anti-rabbit-IgG (H+ L) 1:200
Tyrosine hydroxylase polyclonal Chicken 1:400 AB76442, Abcam Goat anti-chicken Alexa fluor 488 IgG (H+ L) 1:100
Choline Acetyltransferase
polyclonal
Goat 1:20 AB144P; Millipore Streptavidin 595 with biotinylated anti-goat IgG (H-L)
made in rabbit
1:100
/ 1:200
SOX-10 monoclonal Rabbit 1:100 ab155279
Abcam
Donkey anti-rabbit Alexa-fluor 594 IgG (H+ L) 1:100
GFAP monoclonal Rabbit 1:200 345860
Merck Chemicals
Donkey anti-rabbit Alexa-fluor 594 IgG (H+ L) 1:100
S100β monoclonal Rabbit 1:200 ab52642
Abcam
Donkey anti-rabbit Alexa-fluor 594 IgG (H+ L) 1:100
α-Synuclein
monoclonal
Mouse 1:800 BD 610787
BD/Transduction
Donkey anti-mouse Alexa-fluor 594 IgG (H+ L) 1:100
E. Coletto et al.
9
Published in partnership with the Parkinson’s Foundation npj Parkinson’s Disease (2021)     9 
was used to identify all myenteric neuronal cell bodies48 (see Table 2).
Whole-mounts were then washed with PBS and incubated with secondary
antibodies diluted in the blocking buffer for 3 h at room temperature. Next,
tissues were washed 3 × 10min in PBS and then mounted on slides using
Mowiol mounting media (final concentrations: Mowiol 40-88 (8%), glycerol
(25%), DABCO (1%), sodium azide (0.1%) in Tris buffer 0.08 M pH 8.5).
Data analyses and statistics
For comparison of basal resistance and short circuit currents (Isc), multiple
readings from different animal groups (control and MPTP) were obtained.
The overall mean ± SEM was determined from the individual average
values of all animals in their respective groups, each n representing a
single animal observation. For the amplitude and the frequency of
spontaneous contractions, the values for the replicates running in parallel
(n= 2–3) were averaged for each animal and presented as n= 1. Thus
each n number represents a mean of multiple observations in each animal
(so that for n= 8 there were between 18 and 24 observations). Data from
the control and MPTP animals were compared using an unpaired, two-
tailed Student’s t-test. Where multiple groups of data in concentration
response curves were compared, a two-tailed, repeated measure two-way
ANOVA followed by Fisher’s LSD multiple comparison post hoc test was
used. For comparison of the effects of drugs on the Isc, a one-way ANOVA
followed by Dunnett’s multiple comparison was carried out, where the
effect of drugs on Isc was compared to the effect of Isc in dH2O. For
comparison of cell counts and immunohistochemical analyses expressed
as total counts/mm2 were means expressed as mean ± SEM and the
differences between naive controls and MPTP samples were compared
using an unpaired, two-tailed Student’s t-test. All statistical analyses were
carried out using Prism 7.0 software (GraphPad, San Diego, CA, USA).
Differences were considered statistically significant at P < 0.05.
Reporting summary
Further information on research design is available in the Nature Research
Reporting Summary linked to this article.
DATA AVAILABILITY
All data and figures will be available upon request.
Received: 7 June 2020; Accepted: 21 October 2020;
REFERENCES
1. Schapira, A. H. & Jenner, P. Etiology and pathogenesis of Parkinson’s disease. Mov.
Disord. 26, 1049–1055 (2011).
2. Titova, N. et al. Nonmotor symptoms in experimental models of Parkinson’s
disease. Int Rev. Neurobiol. 133, 63–89 (2017).
3. Braak, H. et al. Stanley Fahn Lecture 2005: the staging procedure for the inclusion
body pathology associated with sporadic Parkinson’s disease reconsidered. Mov.
Disord. 21, 2042–2051 (2006).
4. Braak, H. et al. Gastric alpha-synuclein immunoreactive inclusions in Meissner’s
and Auerbach’s plexuses in cases staged for Parkinson’s disease-related brain
pathology. Neurosci. Lett. 396, 67–72 (2006).
5. Cloud, L. J. & Greene, J. G. Gastrointestinal features of Parkinson’s disease. Curr.
Neurol. Neurosci. Rep. 11, 379–384 (2011).
6. Pellegrini, C. et al. Effects of L-DOPA/benserazide co-treatment on colonic exci-
tatory cholinergic motility and enteric inflammation following dopaminergic
nigrostriatal neurodegeneration. Neuropharmacology 123, 22–33 (2017).
7. Chaudhuri, K. R. & Schapira, A. H. Non-motor symptoms of Parkinson’s disease:
dopaminergic pathophysiology and treatment. Lancet Neurol. 8, 464–474 (2009).
8. Furness, J. B. The enteric nervous system and neurogastroenterology. Nat. Rev.
Gastroenterol. Hepatol. 9, 286–294 (2012).
9. Pagano, G. et al. Constipation is reduced by beta-blockers and increased by
dopaminergic medications in Parkinson’s disease. Parkinsonism Relat. Disord. 21,
120–125 (2015).
10. Klingelhoefer, L. & Reichmann, H. Pathogenesis of Parkinson disease-the gut-
brain axis and environmental factors. Nat. Rev. Neurol. 11, 625–636 (2015).
11. Anselmi, L. et al. Ingestion of subthreshold doses of environmental toxins induces
ascending Parkinsonism in the rat. NPJ Parkinsons Dis. 4, 30 (2018).
12. Anselmi, L., Toti, L., Bove, C., Hampton, J. & Travagli, R. A. A Nigro-Vagal pathway
controls gastric motility and is affected in a rat model of Parkinsonism. Gastro-
enterology 153, 1581–1593 (2017).
13. Cersosimo, M. G. et al. Gastrointestinal manifestations in Parkinson’s disease:
prevalence and occurrence before motor symptoms. J. Neurol. 260, 1332–1338
(2013).
14. McNaught, K. S. et al. Systemic exposure to proteasome inhibitors causes a
progressive model of Parkinson’s disease. Ann. Neurol. 56, 149–162 (2004).
15. Phillips, R. J. et al. Alpha-synuclein-immunopositive myenteric neurons and vagal
preganglionic terminals: autonomic pathway implicated in Parkinson’s disease?
Neuroscience 153, 733–750 (2008).
16. Greene, J. G., Noorian, A. R. & Srinivasan, S. Delayed gastric emptying and enteric
nervous system dysfunction in the rotenone model of Parkinson’s disease. Exp.
Neurol. 218, 154–161 (2009).
17. Anderson, G. et al. Loss of enteric dopaminergic neurons and associated changes
in colon motility in an MPTP mouse model of Parkinson’s disease. Exp. Neurol.
207, 4–12 (2007).
18. Fornai, M. et al. Enteric dysfunctions in experimental Parkinson’s disease:
alterations of excitatory cholinergic neurotransmission regulating colonic motility
in rats. J. Pharm. Exp. Ther. 356, 434–444 (2016).
19. Zhu, H. C. et al. Gastrointestinal dysfunction in a Parkinson’s disease rat model
and the changes of dopaminergic, nitric oxidergic, and cholinergic neuro-
transmitters in myenteric plexus. J. Mol. Neurosci. 47, 15–25 (2012).
20. Greene, J. G. Causes and consequences of degeneration of the dorsal motor
nucleus of the vagus nerve in Parkinson’s disease. Antioxid. Redox Signal 21,
649–667 (2014).
21. Blandini, F. et al. Functional and neurochemical changes of the gastrointestinal
tract in a rodent model of Parkinson’s disease. Neurosci. Lett. 467, 203–207 (2009).
22. Colucci, M. et al. Intestinal dysmotility and enteric neurochemical changes in a
Parkinson’s disease rat model. Auton. Neurosci. 169, 77–86 (2012).
23. Tian, Y.-M. et al. Alteration of dopaminergic markers in gastrointestinal tract of
different rodent models of Parkinson’s disease. Neuroscience 153, 634–644 (2008).
24. Basel-Vanagaite, L. et al. Deficiency for the ubiquitin ligase UBE3B in a
blepharophimosis-ptosis-intellectual-disability syndrome. Am. J. Hum. Genet. 91,
998–1010 (2012).
25. Murakami, S. et al. Long-term systemic exposure to rotenone induces central and
peripheral pathology of Parkinson’s disease in mice. Neurochem. Res. 40,
1165–1178 (2015).
26. Chaumette, T. et al. Neurochemical plasticity in the enteric nervous system of a
primate animal model of experimental Parkinsonism. Neurogastroenterol. Motil.
21, 215–222. (2009).
27. Coletto, E. et al. Contractile dysfunction and nitrergic dysregulation in small
intestine of a primate model of Parkinson’s disease. NPJ Parkinsons Dis. 5, 10
(2019).
28. Hyland, N. P. & Cox, H. M. The regulation of veratridine-stimulated electrogenic
ion transport in mouse colon by neuropeptide Y (NPY), Y1 and Y2 receptors. Br. J.
Pharm. 146, 712–722 (2005).
29. Natale, G. et al. MPTP-induced parkinsonism extends to a subclass of TH-positive
neurons in the gut. Brain Res. 1355, 195–206 (2010).
30. Resnikoff, H. et al. Colonic inflammation affects myenteric alpha-synuclein in
nonhuman primates. J. Inflamm. Res. 12, 113–126 (2019).
31. Lai, F. et al. Intestinal pathology and gut microbiota alterations in a methyl-4-
phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson’s disease.
Neurochem. Res. 43, 1986–1999 (2018).
32. Sampath, C. et al. Impairment of Nrf2‐ and nitrergic‐mediated gastrointestinal
motility in an MPTP mouse model of Parkinson’s disease. Dig. Dis. Sci. 64,
3502–3517 (2019).
33. Pellegrini, C. et al. Pathological remodelling of colonic wall following dopami-
nergic nigrostriatal neurodegeneration. Neurobiol. Dis. 139, 104821 (2020).
34. Devos, D. et al. Colonic inflammation in Parkinson’s disease. Neurobiol. Dis. 50,
42–48 (2013).
35. Liu, M.-T., Kuan, Y.-H., Wang, J., Hen, R. & Gershon, M. D. 5-HT4 receptor-mediated
neuroprotection and neurogenesis in the enteric nervous system of adult mice. J.
Neurosci. 29, 9683–9699 (2009).
36. Zeng, B.-Y. et al. MPTP treatment of common marmosets impairs proteasomal
enzyme activity and decreases expression of structural and regulatory elements
of the 26S proteasome. Eur. J. Neurosci. 26, 1766–1774 (2006).
37. Li, X. et al. Alpha-synuclein oligomerization and dopaminergic degeneration
occursynchronously in the brain and colon of MPTP-intoxicated parkinsonian
monkeys. Neurosci. Lett. 716, 134640 (2020).
38. Mrabet, S. et al. Gastrointestinal dysfunction and neuropathologic correlations in
Parkinson disease. J. Clin. Gastroenterol. 50, e85–e90 (2016).
39. Wakabayashi, K. et al. Lewy bodies in the visceral autonomic nervous system in
Parkinson’s disease. Adv. Neurol. 60, 609–612 (1993).
E. Coletto et al.
10
npj Parkinson’s Disease (2021)     9 Published in partnership with the Parkinson’s Foundation
40. Wakabayashi, K. et al. Involvement of the peripheral nervous system in synu-
cleinopathies, tauopathies and other neurodegenerative proteinopathies of the
brain. Acta Neuropathol. 120, 1–12 (2010).
41. Tough, I. R., Forbes, S. & Cox, H. M. Signalling of short chain fatty acid receptors 2
and 3 differs in colonic mucosa following selective agonism or coagonism by
luminal propionate. Neurogastroenterol. Motil. 30, e13454 (2018).
42. Felice, V. D. et al. Microbiota-gut-brain signalling in Parkinson’s disease: impli-
cations for non-motor symptoms. Parkinsonism Relat. Disord. 27, 1–8 (2016).
43. Borghammer, P. How does Parkinson’s disease begin? Perspectives on neuroa-
natomical pathways, prions and histology. Mov. Disord. 33, 48–57 (2018).
44. Jackson, M. J. & Jenner, P. G. The MPTP-treated primate, with specific reference to
the use of the common marmoset (Callithrix jacchus). In Animal Models of
Movement Disorders, Vol. I (eds Lane, E.L. & Dunnett S. B.) 371–400 (Springer /
Humana, 2011).
45. Cox, H. M. & Tough, I. R. Neuropeptide Y, Y1, Y2 and Y4 receptors mediate Y
agonist responses in isolated human colon mucosa. Br. J. Pharmacol. 135,
1505–1512 (2002).
46. Tough, I. R. et al. Endogenous peptide YY and neuropeptide Y inhibit colonic ion
transport, contractility and transit differentially via Y(1) and Y(2) receptors. Br. J.
Pharmacol. 164, 471–484 (2011).
47. Pritchard, S. et al. Altered detrusor contractility in MPTP-treated common mar-
mosets with bladder hyperreflexia. PLoS ONE 12, e0175797 (2017).
48. Szabo, S. MPTP, gastroduodenal motility, duodenal ulceration, and Parkinson’s
disease. Dig. Dis. Sci. 35, 666–667 (1990).
ACKNOWLEDGEMENTS
We thank Mr. Carl Hobbs and Professor Patrick Doherty for their help and assistance
with the microscopy. E.C. was funded in part by Parkinson’s Disease Non Motor
Group (PDNMG). PDNMG had no input into the study design, the collection, analysis
and interpretation of data or in the writing of the report and the decision to submit
the article for publication. Contribution of K.R.C. to this paper represents independent
research part funded by the National Institute for Health Research (NIHR) Biomedical
Research Centre at South London and Maudsley NHS Foundation Trust and King’s
College London. The views expressed are those of the author and not necessarily
those of the NHS, the NIHR or the Department of Health.
AUTHOR CONTRIBUTIONS
E.C. (immunohistochemistry), I.R.T. (electrophysiological) and S.P. (organ bath)
performed experiments. E.C. and I.R.T. contributed equally to data generation.
M.J.J. and A.H. prepared animal models. E.C., S.P. and M.M.I. analysed data. M.M.I., H.
M.C., P.J., S.R., C.D.B. and K.R.C. interpreted results of experiments. M.M.I., I.R.T. and E.C.
prepared figures. M.M.I. and S.R. drafted the manuscript. M.M.I., P.J., H.M.C. and S.R.
edited the manuscript. All co-authors approved the submitted version of the
manuscript.
COMPETING INTERESTS
K.R.C. is an Editor in Chief for npj Parkinson’s Disease. The remaining authors declare
no competing interests.
ADDITIONAL INFORMATION
Supplementary information is available for this paper at https://doi.org/10.1038/
s41531-020-00150-x.
Correspondence and requests for materials should be addressed to M.M.I. or S.R.
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© The Author(s) 2021
E. Coletto et al.
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Published in partnership with the Parkinson’s Foundation npj Parkinson’s Disease (2021)     9