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Welcome to the
‘What’s driving Parkinson’s disease?’
research programme
Institute
Institute of
of
Pharmaceutical
Pharmaceutical
Science
Science driving
What’s
Parkinson’s disease?
Contents
1. Quantifying the syndrome
_________________________________________________________________________________________
Hegemony of the global clinical score
5
Profiling phenotype
6
2. Indicative models for the aetiopathogenesis
_________________________________________________________________________________________
Model 1: Laxatives associated with improvement in rigidity, antimicrobials with worsening
8
Model 2: Hypokinesia improves with Helicobacter pylori eradication
9
Model 3: Classical spousal approach to environmental causality – Is it transmissible?
10
Model 4: Circulating leukocyte subtypes as mediators
11
Model 5: Circulating immune-inflammatory markers
12
Model 6: Changes in duodenal enterocytes may flag clinical evolution
13
Model 7: Other Helicobacter species – a zoonosis?
14
Model 8: Underlying viral infection?
15
3. Key established collaboration
_________________________________________________________________________________________
King’s College London Centre for Host Microbiome Interaction with INRA Metagenopolis, Paris
– microbial metagenomics
17
Institute of Pharmaceutical Science– metabolomics
18
For mini-review with original references see:Dobbs SM, Dobbs RJ, Weller C, Charlett A, Augustin A, Taylor D, Ibrahim MA, Bjarnason I. Peripheral
aetiopathogenic drivers and mediators of Parkinson's disease and co-morbidities: role of gastrointestinal
microbiota. J Neurovirol 2015; (Epub ahead of print) doi 10.1007/s13365-015-0357-8.
1.
Quantifying the
syndrome
Hegemony of the global clinical score
I
n Parkinson’s disease (PD), the global score holds sway, but is a blunt
instrument. Simple relevant tests of performance are advocated, but
often with inadequate attention to their sensitivity, specificity and
reliability. The time to walk an individually set distance, return to and sit in a
chair, and the rate of progress at fastening a set of buttons are reliable, but
lack sensitivity to dopaminergic effects. In contrast, our use of gait analysis at
free walking speed has excellent reliability, usefully discriminates for PD and
defines effects of time and intervention.
Dichotomous classification of PD is misconceived. Normality often seems
to drift into abnormality and, moreover, PD belongs to a group of conditions
with overlapping phenotypes. Aetiological clues lying in pre-presentation and
overlap states are obscured by nosological divisions imposed by man.
Continuous physical and cognitive measures are needed, cross-referenced to
the usual clinical presentation threshold.
Defining phenotype by objective quantification of its facets allows for the
possibility of disease facets having different, non-coincident, driving forces.
The aim is a description of the phenotype in terms of the minimum number of
complementary measures. Under our test conditions, measures of three
cardinal signs of PD, brady/hypokinesia, rigidity and tremor, are independent
and complementary, but a forth, postural abnormality, is highly dependent on
brady/hypokinesia and rigidity. (Adjustment is made for pre-determined
demographic / anthropometric characteristics.)
Tremor
seated
Tremor
stance/
walk
Foot
separat’n
Cognitive
efficiency
Mean
stride
length
Simian
posture
Defining phenotype by
objective quantification
allows for facets having
different, noncoincident, driving
forces, and
dichotomous
classification being
misconceived
Extensor
rigidity
Mean
arterial
pressure
Free
walking
speed
Flexor
rigidity
Reaction
time
Sway
Illustration of between-patient relationships of measures
of disease facets.
Facets are represented by discs, single variable associations by
lines ending at corresponding disc perimeters. The closer the
discs, the closer the association. Absence of connecting lines
means lack of association.
5
Profiling phenotype
Our emphasis has
shifted from objective
monitoring of drug
efficacy to exploring
what lies behind the
unexplained variability
in those objective
measures
Gait traces obtained ½ and 2½ h
after a levodopa combination
formulation (Cover image).
Amplitude of waveform (AB, BC, CD)
represents stride length, flattened
peaks and troughs hesitancy
between strides. Lower trace gives
foot separation at mid-swing (x, y, z):
the patient was walking with a
dangerously narrow base before
medication took effect.
Monitoring of flexor and
extensor rigidity monitoring in a
PD patient over 8 years.
In general, despite clinical
optimisation of long half-life antiparkinsonian treatment, withinand between-subject variation in
objectively measured rigidity
remains considerable
6
I
vention, design and development of equipment is bespoke. Devices are
inexpensive, do not require a specialist operator and are useable in a
clinical (even domestic) context. Wherever possible, equipment is small
and unobtrusive. Output is designed to be readily interpreted by the nonspecialist and fed back to the patient. Targets for objective assessment are the
cardinal signs, major associated disability (e.g. cognitive inefficiency, inability to
turn in bed) and events (falls, sleep apnoea,).
A secondary, but important, consideration is commercialization to bring
precise, objective measurement to routine practice. Every physiotherapist
should have a ‘pocket gait analysis laboratory’: it should be a tool of the trade.
The team of senior researcher Dr Clive Weller, with post-doctoral research
associate Dr Luc Maréchal, brings together expertise in real time and storage
telemetry, computer interfacing and deployment of recent advances in
microcontrollers and nanoelectromechanical systems. It is enhanced by
mutually beneficial technical and academic collaboration with Professor Kasper
Althoefer and Dr Trish Nanayakkara, Robotics within Department of Informatics
at KCL.
2.
Indicative models for
aetiopathogenesis
Model 1: Laxatives associated with improvement in
rigidity, antimicrobials with worsening.
There is indicative
evidence of a
continuing role of
gastrointestinal
dysbiosis in
pathogenesis.
G
astrointestinal transit is slowed in PD. We showed that constipation
pre-dates the diagnosis by, on average, 3 decades: a finding upheld
prospectively by the association of infrequent bowel movements and
subsequent diagnosis of PD. Constipation markedly affects quality-of-life in PD.
Aggregates of misfolded protein characteristic of PD, known historically as
Lewy bodies, are found in the enteric nervous system (the little brain), related
sympathetic ganglia, and the parasympathetic vagal nuclei in the brain stem.
There is loss of dopaminergic neurons in the little brain as well as the basal
ganglia.
Our work shows that the flexor-rigidity of PD increases by a clinicallysignificant amount year-on-year before prescribing maintenance laxatives,
plateauing after. A similar pattern is seen with bulk, osmotic and enterokinetic
laxatives, pointing to a common mechanism of action on rigidity. Moreover, a
guanylate cylase-C receptor agonist (linaclotide), which increases
gastrointestinal-secretion, is associated with a large step-down in rigidity.
In contrast, rigidity worsens cumulatively with successive antimicrobial
interventions, irrespective of indication, and over and above the effect of
time. This suggests that a
pathological dysbiosis
resulting from a first
course does not recover
and is exacerbated by
future exposures.
Time trends (95%
confidence interval) in
flexor rigidity in relation to
initiation of laxative
classes.
Based on 1493
measurements in 79 PD
patients.
Cumulative percentage increase in flexor rigidity over baseline following
a first, second and third antimicrobial course.
Based on 396 assessments in 49 PD patients
8
Model 2: Hypokinesia improves with Helicobacter pylori
eradication.
hat peptic ulceration is prodromal paved the way to exploring
Helicobacter pylori as a driver in PD.
In our randomised placebo-controlled trial, biopsy-proven H. pylori
eradication reduced hypokinesia of gait in PD. A longitudinal observational
study showed indication specificity, in that antimicrobials for other indications
did not improve hypokinesia. In the trial, while hypokinesia improved, rigidity
worsened over the year post-eradication, both plateauing over the next. There
was overall clinical benefit. Improved hypokinesia was independent of any antiparkinsonian medication. (Receipt of levodopa an exclusion.) Increased rigidity
may flag acquisition of small-intestinal-bacterial-overgrowth, since H. pylori and
hydrogen-breath-test-positivity for overgrowth are inversely related in PD.
In no disease where H. pylori is causal is it present in all cases. However,
current or past Helicobacter infection may be a necessary though not sufficient
player in developing the full PD syndrome. There is a lack of birth cohort effect
for H. pylori in PD, as in gastric cancer and peptic ulcer where causal links with
H. pylori are generally accepted. Danish population registers show increased
prescription of anti-Helicobacter drugs in the 5 years prior to PD diagnosis.
Dopaminergic agonists can prevent duodenal ulcer relapse in man, but whether
by suppressing H. pylori is unknown.
T
Helicobacter pylori in PD
may not be a sine qua
non, is definitely not
sufficient, but is
important.
Schematic representation of effect of H. pylori eradication on
hypokinesia & rigidity.
Estimated mean time-trends following successful blinded-active (red) and
open-active (orange), and placebo (blue). At present, the level of evidence
is 1b (Centre for Evidence Based Medicine, Oxford, 2009) since this is the
only trial.
9
Model 3: Classical spousal approach to environmental
causality – Is it transmissible?
PD patients’ spouses
show a
tendency towards
parkinsonism, and share
relative lymphopenia,
abnormal bowel
function
and small-intestinalbacterial-overgrowth.
I
n our case-control study, spouses of PD patients were a short but highly
significant ‘distance-down-the-pathway’ with respect to objective
measures of PD facets. Marked, multifarious, relevant differences
(physiological/psychomotor/ dermatological), between spouses and control
couples are difficult to explain by selective mating or learned/reactive
behaviour.
In a large cohort, patients and spouses were found to have relative
lymphopenia (with particular effect on B cells). There is a proportional increase
in natural killer cell count in PD patients, in CD4+ in spouses. Half of the
patients and a third of spouses have chronic functional bowel abnormality.
Like patients, two-thirds of spouses are hydrogen-breath-test-positive for
small-intestinal-bacterial-overgrowth. The spousal findings are not directly
attributable to H. pylori. Indeed, spouses have a lower frequency of
Helicobacter anti-urease IgG enzyme-linked immunosorbent assay
seropositivity than either PD patients or controls, as though an acquired
dysbiosis had suppressed it.
Crude prevalence rate estimates for frank PD in 34 European studies range
widely, the estimate lying within the binomial exact 95% CI for our entire
spouse cohort in only one study.
A prognostic index for parkinsonism based on
brady-/hypo-kinesia variables in 104 subjects
with PD, 144 without) applied in 20 index
controls (C), their spouses (Cs), 20 patients (P)
with clinically-definite (treated) PD and their
spouses (Ps), cohabiting for half-a-century.
Means (95% C.I.) shown. P and C matched for age
and gender. P<0.0001 for predicted probability in
Ps cf. C + Cs combined.
10
Model 4: Circulating leukocyte subtypes as mediators.
A
majority of PD patients have small-intestinal-bacterial-overgrowth
at presentation, probably a consequence of caeco-ileal reflux.
Blood leukocyte subtypes associated with hydrogen-breathtest positivity for overgrowth (higher natural-killer and total CD4+ counts,
lower neutrophils) are the same as those associated with PD facets. The
higher the natural-killer count, the shorter stride, slower speed and greater
flexor-rigidity (adjusted for patient characteristics).
Cellular associations held after allowing for potentially confounding effect
of hydrogen-breath-test results or Helicobacter status. Indeed, increased
brady/hypokinesia was noted with Helicobacter-positivity, over and above that
explained by natural-killer count, and of a magnitude equivalent to that of a
levodopa challenge.
Association of rigidity with a higher natural-killer count is modulated by
the total CD4+ count. The CD4+ subset includes regulatory T-cells which inhibit
natural-killer effector mechanisms.
Tremor is worse with lower neutrophils: this may reflect neutrophil
sequestration in the gut.
There are biological
gradients of facets of PD
on blood leukocyte
subtype counts
Relationship of PD facets to
leucocyte subtype counts in 38
patients, untreated or on stable
long-t½ PD-medication.
Points shown are average per
person: all values for a longitudinal
outcome used to estimate
regression lines. Relationships
held on including levodopa-takers.
N.B. Effect of natural killer count
on stride length and rigidity
independent: no association over
our test conditions (bottom right).
Relationship of rigidity to CD4+ count to flexor-rigidity, after adjustment
for natural-killer count.
11
Model 5: Circulating immuno-inflammatory markers.
Biological gradients of
facets of PD on
circulating immunoinflammatory markers
W
e have described biological gradients of objective measures of
facets on two systemic markers of inflammation, serum cortisol
and tumour-necrosis-factor-α. Others have subsequently shown
gradients of global motor scores on peripheral blood mononuclear cell
production of cytokines and expression of nuclear factor κB. We found that
serum interleukin-6 increases with age and is elevated in PD by an amount
equivalent to 10 years’ ageing. Others have subsequently shown that a higher
interleukin-6 concentration is predictive of incident PD.
Systemic immuno-inflammatory activation can increase homocysteine
production. Hyperhomocysteinemia is found in 43% PD patients. It is partially
explained by a lower serum B12 concentration with no complementary effect
of folate. (Methyltetraydrofolate acts as a methyl donor, and cobalamin is a
co-factor, in detoxifying homocysteine by remethylation to methionine by
methionine synthetase.) Hyperhomocysteinemia is not explained by
Helicobacter-status (gastric atrophy being uncommon in PD). Small-intestinalbacterial-overgrowth will increase bacterial utilisation of B12 and provokes a
systemic immuno-inflammatory response. Indeed, significant numbers of
large irregularly-shaped secondary lysosomes in duodenal lining cells
accompany overgrowth in PD, signifying that it is not an innocent bystander.
Using the western blot profile of IgG antibodies against electrophoreticallyseparated H. pylori antigens, the predicted probability of being labelled as
having PD was greatest with cytotoxin-associated-gene-product positivity and
vacuolating-toxin negativity, and urease-B negativity. With this H. pylori
antibody pattern, the odds for having PD were increased five-fold at age 80
years. The predictive ability was not confined to those with current infection.
Association between serum homocysteine and B12
concentrations in PD.
Although, no evidence of frank B12 deficiency in PD, more
patients than expected had a concentration in the ‘equivocal
range’ or below. Increased demand from immunoinflammatory activation may render an ‘equivocal’
concentration pathological. Homocysteine 2 % higher per year
post-diagnosis, and not explained by anti-parkinsonian
treatment.
Electron-micrographs illustrating cloud of irregular lysosomes in a duodenal lining cell
(enterocyte) in a PD patient with small-intestinal-bacterial-overgrowth
12
Model 6: Changes in duodenal enterocytes may flag
clinical evolution.
I
n PD, dysfunction of the cell power houses, mitochondria, is described in
skeletal muscle and blood platelets, as well as in the basal ganglia.
We have found abnormal forms of mitochondria in duodenal lining
cells (enterocytes) in PD. Long thin mitochondria, associated with roughendoplasmic-reticulum indicating functionality, were commonly seen, in the
presence of small-intestinal-bacterial-overgrowth, absence of recent H. pylori
infection. The complex branching seen may result from failure to divide.
With current or recent H. pylori infection, arrays encapsulated in a doublemembrane were found in half of cases, lying among normal mitochondria.
There is a report of similar “mitochondrial” inclusions in cerebral neurons in
Creutzfeldt-Jakob-like-disease. Alternatively, these bodies might be viroplasms
not mitochondria. Our electron microscopists had not previously observed
similar bodies, but subsequently found examples in archived duodenal biopsies
from two patients with human immunodeficiency virus infection.
PD is a disease of
mitochondrial
dysfunction: different
mitochondrial
dysmorphology in
enterocytes
characterises putative
gastrointestinal drivers
Electron-micrographs of duodenal enterocytes from PD
patients.
Above: long thin mitochondria seen to predominate at low
magnification. Complex branching of a mitochondrion is shown
at intermediate magnification.
Below: double-membraned, encapsulated arrays lie amongst
normal mitochondria. Arrays seen longitudinally and in
transverse section at higher magnification.
13
Model 7: Other Helicobacter species – a zoonosis?
arkinson’s disease has been linked with rural living and farm
experience. A study of 6 million death certificates from 26 US states
found the proportional mortality from PD was significantly higher in
livestock farmers than in non-farmers, but lower in crop farmers.
Agrochemicals used in these sectors are different, but transmission of
infections from animal hosts (zoonotic) is a more compelling explanation.
Zoonotic-transmission of bacteria of the genus Helicobacter is a strong,
and potentially remediable, candidate. Species, usually resident in domestic
and farm animals, can cause human gastric disease. Several of
these ‘spiral’ non-H. pylori Helicobacter species (NHPH) are
found in dogs and cats, H. suis (the most common Helicobacter,
after H. pylori, to infect humans) in pigs. In our unpublished
preliminary survey, H. suis was present in 60 % of 111 UK pig
stomachs on microscopic examination, and its DNA detected in
stool. As determined by a Ghent University in-house molecular
assay for H. suis, its relative frequency compared with H. pylori
was 10 times greater in 60 of our PD patients, than in 256
patients from UK gastroenterology-services. Risk of death in PD
during follow-up was 8 times greater with H. suis-positivity,
compared with those negative. This contrasts with all-cause
Kaplan-Meier plot of 19 PD-patients with H. suis mortality being less in those with H. pylori, despite excess
DNA detected in gastric biopsy (black line) mortality from gastric cancer. In none of these PD patients
compared with 40 without (grey).
were spiral Helicobacters seen on histopathology.
Little is known, about the epidemiology of zoonotic
Helicobacters. They are hard to culture from human gastric biopsy,
characteristically patchy and sparse on gastric histopathology, in
man, and there is a lack of rigorous molecular diagnostic tests. A
validated molecular screening method for NHPH DNA in stool is
neeeded.
We have described a U-turn in brady/hypokinesia (from wheelchair-bound to being able to walk 3 miles) in a PD-patient, after
Spiral Helicobacter in a PD patient associated
eradicating a spiral Helicobacter.
with chronic antral gastritis
The broad brush of
epidemiology provides
enigmatic
aetiopathogenic clues in
PD
P
.
Other sources of zoonotic non H.
pylori (spiral) gastric Helicobacters
14
Model 8: Underlying viral infection?
aecal overload and small-intestinal-bacterial-overgrowth are
predisposed to by the slow gastrointestinal transit of PD, and may have
detrimental feedback on it, but what initiates that slow transit? Could
there be a viral primer?
Enteroviruses infect via the gastrointestinal tract and are associated with
neurological syndromes. Indeed, there is recent evidence of an enterovirus as
a cause of encephalitis lethargica and post-encephalitic parkinsonism.
Moreover, in our pilot study using faecal samples taken at start of a diarrhoeal
episode, frequency of enterovirus genogroup B RNA appeared high in PD
patients and their spouses.
A viral primer could also be involved in the relative lymphopenia seen in
both PD-patients and their spouses, compared with controls. This robust
finding was not explained by anti-parkinsonian medication, Helicobacter-status
or breath-hydrogen. A relatively benign retrovirus might explain relative
lymphopenia and the slow transit. (Jejunal autonomic denervation is
described with human immunodeficiency virus (HIV) infection.) The
comparatively high frequency of Dientamoeba fragilis (18 % of PD probands
and their spouses cf. 3 % of routine parasitology requests), which we have
found in stool, could flag mild acquired-immunodeficiency. The epidemiology
of IP and HIV are distinct, but parkinsonism is seen in uncomplicated HIVinfection (as well as with opportunistic infections in acquired
immunodeficiency syndrome). Although Lewy bodies are not reported in HIV,
motor dysfunction compatible with basal ganglia damage is found early, and
basal ganglia dopaminergic cell loss is seen without clinical parkinsonism. In
simian immunodeficiency virus-infected monkeys, nigrostriatal dopamine is
halved within 2 months.
There has been no systematic search for candidate or undiscovered viruses
in PD using modern molecular methodology.
F
A viral primer for slow
transit and relative
lymphopenia cannot be
dismissed.
15
3.
Key collaborations
King’s College London Centre for Host Microbiome
Interaction with INRA Metagenopolis, Paris – microbial
metagenomics.
G
erm-free mice move more and take more risks: they have increased
striatal synaptogenesis and dopamine/serotonin turnover. In
specific-pathogen-free mice, non-absorbable antimicrobials
increase exploratory behaviour and hippocampal brain-derived neurotrophic
factor (BDNF). Gavage of caecal contents from a more outgoing mouse strain
into a more timid increases both exploratory behaviour and BDNF, and vice
versa: these effects were independent of vagal integrity. Specific probiotics
cause behavioural change.
Important associations between stool bacterial microbiota and human
health have been identified. Indeed, there is evidence that stool microbial
metagenomics can discriminate better for chronic disease than human
genomics. Modifying the GI microbiota may modify disease. Psychiatric illness in
irritable bowel syndrome, inflammatory bowel disease and autistic spectrum
disorder have been ascribed to dysbiosis. Irritable bowel syndrome with
constipation is common in PD. Autism recalls the restricted behaviour of PD.
An inflammation-associated form of depression is described. Exhibition of the
non-absorbable broad-spectrum antimicrobial, rifaximin, was accompanied
by amelioration of hepatic encephalopathy-associated parkinsonism and of
characteristic imaging features in globus pallidus in 3 patients with cirrhosis
and portosystemic shunting, without change in blood ammonia and
electroencephalogram.
Two approaches to defining the gut microbiome in PD, using 16S
ribosomal RNA gene sequencing (studying 72 patients/72 controls and
38/34, respectively) have been reported, [47, 48] but with no consensus. In
the larger of these almost all PD-patients were on anti-parkinsonian
medication and there was a high frequency of cerebro- and cardio-vascular
co-morbidities and related medications in the controls. Definite
characterisation of the microbiome in PD is needed in terms of gene
function as well as taxonomically.
There is real potential for the collateral benefit of a new generation of
animal models, to test cause/effect hypotheses and aid discovery of
specifically-targeted, disease-modifying medicines. To date, PD models have
relied on surgical, chemical or genetic lesions, and are thus downstream of, or
out-with, environmental driving processes.
Stanislav Dusko Ehrlich
Director Centre for HostMicrobiome Interaction,
King’s College London:
Research Director Emeritus,
Institut National de la
Recherche Agronomique,
France
“One of the most
important pioneers in
the study of the human
microbiome” European
Research Council.
17
Institute of Pharmaceutical Science – metabolomics
M
Peter John Hylands
Director Institute of
Pharmaceutical Science
Professor of Pharmaceutical
Chemistry, Pharmaceutical
Science
Head Department of Pharmacy
and Forensic Science
“During my tenure,
Pharmacy and
Pharmacology at King’s
College London ranked
as 3rd in World (QS
ranking, 2014); 91% of
our research outputs
rated as world-leading/
internationally excellent
(Research Excellence
Framework, 2014); in
unit assessment 3, we
ranked 1st in UK for
research quality and
volume.”
18
etabolomic interest in PD and Alzheimer’s disease has concentrated
on markers of damage (e.g. hypoxia, oxidative stress and
membrane lipid remodelling) in blood and breath, rather than
influence of the metabolome on the disease process.
From an aetiopathogenic standpoint, a key question, is whether the
metabolome regulates systemic inflammation. In mice, short chain fatty
acids, such as butyrate, produced by colonic bacterial fermentation, promote
colonic regulatory T-cells (T-reg) which suppress pro-inflammatory cells. This
suggests a mechanism for the apparent CD4+ (presumably T-reg component)
modulation of rigidity and fits with PD patients’ spouses having a proportional
(potentially protective) increase in total CD4+ count. However, whilst oral
administration of short chain fatty acids to germ free mice, to address their
deficit, increased colonic T-regs, it had no wider effect on mesenteric lymphnode, splenic or thymic T-regs.
Human studies have linked fermentation products to behaviour. However,
there appears to be contradictory evidence. Faecal propionic acid
concentrations have been correlated with anxiety in irritable bowel syndrome
(IBS), but those with constipation (IBS-C) have fewer short chain fatty acids
producing bacteria in stool but no less anxiety.
Mitochondrial dysfunction is described in substantia nigra in PD (complex I)
and multiple-system atrophy (complex IV), and in platelets in PD. Hydrogen
sulphide is known to inhibit mitochondrial complex IV at higher
concentrations. IBS-C patients have more sulfide producers. However, again
there is contradictory evidence. In a rodent model of PD, hydrogen sulphide
was protective against neurodegeneration, not only by reducing oxidative
stress but also by inhibiting neuro-inflammation. This neuroprotection may be
predicated on it being a chemically-lesioned model, and/or be dependent on
H2S dosage.
Others have found that a hydrogen sulphide donor ameliorated naproxeninduced small-intestine enteropathy in rats, and restored naproxen-induced
changes in microbiota. In chemically-induced colitis in mice, the donor reduced
inflammation, and restored microbial biofilm and deficient mucus production.
In vitro, hydrogen sulphide donors promoted formation of biofilms by bacteria
derived from human colon. They inhibited potentially damaging planktonic
bacteria.
As with the microbiome, information relevant to PD is sketchy: our
systematic approach to the disease complex is much needed.