Transcript Folie 1 - TU Dresden

Critical view on Braak‘s hypothesis.
Has clinical work during the last 10 years supported
the hypothesis?
Oslo, April 25th, 2013
Heinz Reichmann, FRCP, FAAN
Chair Department of Neurology, Technical University of Dresden
PD Progression
Olfactory loss
RBD, constipation
anxiety, depression
Bradykinesia,
Rigidity,
Rest-tremor,
(+/- non-motor-symptoms)
Motor complications:
Non-motor symptoms:
Wearing off/dyskinesias,
gait and balance problems,
axial deformities,
dysarthria/dysphagia
cognitive decline/
dementia, psychosis,
autonomic dysfunction,
sleep–wake-dysregulation
Gene mutations,
alpha-synuclein levels,
imaging abnormalities
Pre-symptomatic
PD Pre-Motor PD
-10y -6y
Early PD
Moderate PD (stable)
0
Onset of clinical PD Onset of clinical PD
premotor phenotype motor phenotype
2y
Advanced PD
5y
10y
15y
Diagnosis
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Genetic Screening
Martin I et al. (2011) Annu Rev Genom Human Genet 12:301-325
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Healy et al. (2008)
Figure 3: Age-specific risk of PD
Risk is estimated with the Kaplan-Meier method for the whole sample
and with the maximum-likelihood estimation (ML) for all patients with
mutations in LRRK2 combined.
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Takahashi, Wakabayashi 2005, Parkinsonism Rel. Dis. 11, S31-S37
Friedrich-Heinrich Lewy
(1885-1950)
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www.neuropathologyweb.org/.../chapter9dPD.html
Halliday G et al. (2011) Movement Disorders 26:1015-1021
The Braak Stages
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Sniffin‘ Sticks
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Idiopathic Hyposmia as a
pre-motor marker of PD
Individual results of testing
normal
borderline
pathological
test not performed
Conversion to IPD
definitive
borderline
UPDRS
II
III
TCS
SPECT
UPDRS III
123I-FP-CIT
(DaTScan)
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Haehner et al. Mov Disord.
2007;22:839-842
Sonografic Analysis of the Mesencephalon
Sommer et al., Figure 2
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Dopaminagonisten
in der dopaminergen Synapse
Dopamine Transporter Scan
Synthese
hemmt
Synthese
Dopamin
Dopamine
Dopamine
DopaminWiederaufnahme
Reuptake
Speicherung
Vesikel
präsynaptischer
D2-SubgruppenRezeptor
DA
intrazellulärer
Metabolismus
hemmt
Freisetzung
Freisetzung
durch Exozytose toxische
Metabolite?
Dopamin
DA
D1-SubgruppenRezeptor
extrazellulärer
Metabolismus
postsynaptischer
D2-SubgruppenRezeptor
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DAT SPECT (123I-FP-CIT)
DAT SPECT normal
EP, unilateral:
DAT SPECT abnormal
Booij J et al. (2001)
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Transcranial sonography in
idiopathic olfactory dysfunction
Individual results of testing
normal
borderline
pathological
test not performed
Conversion to IPD
definitive
borderline
UPDRS
II
III
TCS
SPECT
UPDRS III
123I-FP-CIT
(DaTScan)
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Haehner et al. Mov Disord.
2007;22:839-842
Prevalence of Smell Loss in PD
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Haehner A et al. (2009) Park Rel Disord 15:490-494
Fig. 3. Olfactory loss in the different groups of PD patients as described with the
composite TDI score (sum of odor threshold, odor discrimination, and odor
identification score).
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Haehner A et al. (2009) Park Rel Disord 15:490-494
Fig. 4. Olfactory function separately for three subtypes of PD. Results are
shown separately for odor thresholds, odor discrimination, and odor
identification (means, standard error of means).
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Nasal Cavity Biopsy
Schematic view of the lateral wall of the nasal cavity. Biopsies were
taken from the insertion of the medial turbinate (arrows, see also “MT”
in Figure 2) and from the dorsal septum at the same level (not shown).
Witt et al., (2008)
Modified with permission from Thieme.
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Negative α-Synuclein Staining
Figure 4: Immunohistochemical detection of α-synuclein in the olfactory mucosa of two patients with
Parkinson’s disease (A, #263; B, #264). There is no reactivity of α-synuclein. Scale bar: 50 µm.
Witt et al., (2008)
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Lewy Pathology in the Submandibular
Gland
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Lewy-pathology in the
submandibular gland
normal
a-Syn
Del Tredici K et al. (2010)
a-Syn
Fig. 2 Lewy pathology in the submandibular
gland in Parkinson’s disease. a The normal
submandibular gland consists of multiple
lobules, each of which contains numerous
serous acinar (dark purple) as well as mucous
(m light blue) cells and myoepithelial cells. A
trabecle (t) of connective tissue radiates
inwards from the thin outer capsule of the
organ carrying blood vessels, nerves, and large
secretory ducts. Azan overview staining, 30
mm paraffin section. b Overview of finely
branching nerves bearing LNs (arrows) in the
lobule of the submandibular gland from a male
patient, 75 years of age, with PD stage 4 brain
pathology (case 26, Table 2). c Here, a thin
nerve containing LNs and originating from the
trabecle extends into a glandular lobule, where
its fine-caliber branches innervate the
secretory acinar cells. See also arrows in g.
Micrograph c originates from a 76-year-old
female patient with PD stage 5 brain pathology
(case 30, Table 2). d–j Micrographs from the
same individual as in b. d Ribbon-like LNs in a
thick nerve fiber bundle in the perivascular
connective tissue. e, f Details from d.
Microganglia (f, see also arrow indicating
microganglion in d) within the parenchyma of
the submandibular gland contained LBs. (h)
Thread-like perivascular LNs. j a-synucleinimmunoreactive aggregates (darkened spots)
adjacent to basal portions of the serous acini
probably represent terminal axons. This
aggregated material was confined to serous
acinar cells. The same structures appear at
lower magnification in the lower portion of g.
a-Synuclein immunoreactions, 100 mm
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polyethylene glycol
(PEG) embedded tissue
sections (b–j)
Lewy Pathology in the ENS of the Gut
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a-Synuclein Inclusions in the ENS
• Presence of gastric a-synuclein inclusions could provide first link in susceptible
neurons that extend from the enteric to the central nervous system individuals.
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GI-Tract: Window or Entry Zone
Frequency of bowel movements in 6790 men between 1971 and 1974
• Follow up for incident PD for 24 yrs
• 69 PD with average time to onset 12 yrs
 18,9/ 10.000 person years in men <1 bowel movement/ day
 3,8 / 10.000 person years in men >2 bowel movements/ day
Constipation as an marker of early PD or susceptibility or
environmental factors that may cause PD.
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NMS in PD in comparison with an age-matched control group
NMSQuest study: Non-motor questionnaire for PD patients
(%) 80
•
123 PD patients (mean age 68y, disease duration 6.4y, H&Y 2.5)
•
96 controls (mean age 65y)
70
60
50
40
30
20
PD patients
Controls
10
0
25
Chaudhuri & Odin 2010; Chaudhuri et al 2006
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The Priamo Study: A Multicenter Assessment of Nonmotor Symptoms
and Their Impact on Quality of Life in Parkinson’s Disease
I 1,072 PD patients interviews covering 12 NMS domains and PDQ-39
― Mean age 67y, disease duration 5.1y, H&Y 2.0
― Mean number of NMS per patient: 7.8
― UPDRS motor score was higher in patients with NMSs
70%
TOP 10 NMS symptoms
60%
50%
40%
30%
20%
10%
0%
28
Barone et al 2009
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Prevalence of Nonmotor Symptoms in Parkinson’s Disease:
Study Using Nonmotor Symptoms Questionnaire
I Observational, multicenter, international, cross-sectional study
― 545 PD patients completed the revised NMSQuest
― Mean age 68y, disease duration 7y, H&Y 2.5
― Mean number of NMS per patient (NMSQ-T): 10.3
NMS: Distribution of responses (>30%)
(%)70
60
50
40
N=64
N=40
N=18
30
20
10
0
N=4
29
Martinez-Martin et al 2007
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Lewy Pathology in the ENS of the Gut
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a-Synuclein Inclusions in the ENS
• Presence of gastric a-synuclein inclusions could provide first link in susceptible
neurons that extend from the enteric to the central nervous system individuals.
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Further Evidence for G.I. Disturbances in PD
Acta Neuropathol 1988; 76:217-221
I Occurence of Lewy-Bodies in Auerbach and Meissner
Plexus
I Predilection of LB in the upper GI-tract
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Colon biopsies in PD
PD
Controls
I 10 untreated Parkinson patients ; all positive for Alpha-Synuclein
I Sigmoidoscopy and Bx: alpha-Synuclein and 3-Nitro-Tyrosin (marker
for mitochondrial stress)
Mov Disord 2012: 27:709-715
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Premotor PD and Colon biopsy
I Alpha-Synuclein positive immunohistochemistry in 3 biopsies 2-5 yrs before
onset
Mov Disord 2012: 27:716-719
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The Dresden Parkinson Model
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Methods and Results
Administration of rotenone intragastrically to one-year-old mice using a gastric
tube
Rotenone could not be measured by HPLC using blood and brain tissue
There was no decrease in complex I activity in muscle and brain
Rotarod test was used to show that there was a significant decrease in the
rodents‘ ability to remain on the rod between 3 months treated mice and controls
A-synuclein aggregation was only detected in treated animals
A-synuclein aggregation was detected in the ENS and after longer periods in the
intermediolateral nucleus in the spinal cord and the dorsal motor nucleus of the
vagus
After three months of treatment a-synuclein could be detected in the SN pars
compacta combined with a 15% decrease in the number of TH-pos neurons
OB and ENS are the only nervous system structures directly exposed to
environmental substances
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Control
Control
Pan-Montojo et al. (2010)
Figure 1 (continued). Locally administered rotenone induces alpha-synuclein phosphorylation, accumulation and
aggregation with gliosis in ENS ganglia. (scale bars 20 um). F, each column represents total number of alpha-synuclein
inclusions/ganglion surface. All graphs show mean +/- s.e.m. G, H, max-projection of staining against GFAP, alpha-synuclein
and DAPI on duodenum sections from control (G) and treated (H) mice. I, J, max-projection of anti-ßIII-tubulin, antiphosphoalpha-synuclein (Ser 129) and DAPI staining on duodenum sections from control (I) and treated (J) www.uniklinikum-dresden.de
animals.
Figure 2. Intracellular and axonal alphasynuclein
increases
in
the
intermediolateral nucleus and the
dorsal horn lamina I layer of the
spinal cord after oral rotenone
Pan-Montojo et al. (2010)
treatment. (scale bars 20 um) A, B, C,
Immunostaining against alpha-synuclein and
choline acetyl transferase (ChAT) in spinal
cord sections showing the intermediolateral
nucleus ChAT+ neurons from 3 months
control mice (A), 1.5 months (B) and 3
months (C) treated mice. Arrow in B,
colocalization of increased intracellular
alpha-synuclein and ChAT+ stainings in the
IML. Arrow in C, large alpha-synuclein
inclusion (|>7.5 mm) inside an IML ChAT+
neuron. D–E, fluorescence intensity colorcoded images from 3 months control (D, D’)
and 3 months treated mice (E, E’) spinal
cord sections stained using DAPI and alphasynuclein and ChAT antibodies. Arrows in D
and E, areas in the proximity of ChAT+
neurons.
F,
mean
fluorescence
quantification of experiment shown in D and
E. Double asterisk, P<0,01. Columns
represent
mean
alpha-synuclein
fluorescence in and around ChAT+ neurons
in
the
IML/mean
alpha-synuclein
fluorescence in the region anterior to the
IML. Graph shows mean 6 +/-s.e.m.. G, H,
DAB-staining against apha-synuclein using
synuclein-1 antibody in the dorsal horn of
the spinal cord www.uniklinikum-dresden.de
from 3 months treated (H)
Treated (DMV)
Control
A-synuclein
Con
Pan-Montojo et al. (2010)
1,5 mo
3mo
Figure 3. Intragastrically administered
rotenone
induces
alpha-synuclein
accumulation, oxidative stress and
inflammation in the dorsal motor
nucleus vagus. (scale bars 20 um). A,
B, double-immunofluorescence staining
against alpha-synuclein and ChAT on
DMV sections from 1.5 months control (A)
and 1.5 months treated (B) mice. Arrows
in B, increased intracellular alphasynuclein in DMV neurons already after
1.5
months.
Arrowheads
in
B,
autofluorescent punctate inclusion pattern
inside ChAT+ neurons. C, DMV sections
stained with ChAT and DAPI were
sequentially excited with 488 and 561
laser wavelengths. Arrows in C, large
intracellular auto-fluorescent inclusions
inside ChAT+ neurons of the DMV
(arrows). D, E, F, Light microscopy
images of alpha-synuclein staining from
1.5 months control (D), 1.5 months (E)
and 3 months (F) treated mice. Arrows in
E and F, increased staining intensity
inside DMV neuronal soma in treated
mice. Arrowheads in F, increased alphasynuclein
staining
inside
neuronal
processes G, H, average-projection of
triple-immunofluorescence
staining
against ChAT, GFAP, MHC II (clone
M5/114.15.2) and DAPI on sections from
control (G) and treated (H) mice after 3
month treatment. www.uniklinikum-dresden.de
Arrow in H, activated
microglial cell in the DMV.
Substantia nigra pars compacta
Con
Pan-Montojo et al. (2010)
Th-stain in control € and treated mice (F)
Figure 4. Alpha-synuclein accumulation and
neuronal loss in the SNc after 3 but not 1.5
months intragastrical rotenone treatment. (A–C,
scale bars 20 um; E–F, scale bars 200 um). A, B, C,
immunostaining against TH, alpha-synuclein and
DAPI on SNc sections from 1.5 months control (A)
and 3 months (B–C) treated mice. Arrow in B, alphasynuclein small inclusions inside TH+ neurons. Arrow
in C, large alpha-synuclein inclusion (|>8.14 um)
inside a dopamineric neuron in the SN. D,
stereological quantification (n = 3) of TH+ neurons in
the SN from control and treated mice. Asterisk,
P<0.05. Number of neurons was determined based
on the optical fractionator principle using
StereoInvestigator software (MicroBrightField Inc.,
Williston, USA). Each column represents total number
of TH+ neurons in the SN in 1.5 and 3 months control
and treated mice. Graph shows mean +/-s.e.m. E, F,
TH immunostaining on striatum in 1.5 months control
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(E) and 3 months treated (F) mice.
Hansen C et JY Li. (2012) Trends in Molecular Medicine 18:248-255
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Further Evidence
Hemivagotomy and partial sympathectomy delay Parkinson’s disease progression
in mice
Francisco Pan-Montojo1,2, 5, Mathias Schwarz1, Clemens Winkler1, Mike Arnhold2, Gregory O’Sullivan4, Arun Pal4, Margarita Rodrigo-Angulo5,
Gabriele Gille2, Richard H.W. Funk1,3, and Heinz Reichmann2,3
1Institute
for Anatomy, TU-Dresden, Fetscherstr. 74, 01307, Dresden
of Neurology, University Hospital Carl-Gustav Carus, Fetscherstr. 74, 01307, Dresden, Germany
3Center for Regenerative Therapies Dresden, Tatzberg 47/49, 01307, Dresden, Germany
4Max-Planck Institute for Cell Biology and Genetics, Pfotenhauerstr. 108, 01307, Dresden, Germany
5Departamento de Anatomía, Histología y Neurociencia, Facultad de Medicina, Universidad Autónoma de Madrid, Arzobispo Morcillo 4, 28029 Madrid, Spain
2Department
Abstract
Pathological studies on Parkinson’s disease (PD) patients suggest that PD pathology starts at the olfactory bulb (OB) and the
enteric nervous system (ENS) progressing into the central nervous system (CNS). In our previous study, we showed that the local
effect of rotenone on the ENS reproduces this pathological progression in mice affecting only synaptically connected structures,
suggesting transsynaptic and retrograde axonal transport as underlying mechanisms of this progression. Here, we tested this
hypothesis by performing a hemivagotomy or a partial sympathectomy prior to rotenone oral treatment on mice and using primary
enteric and sympathetic neuron co-cultures. For the first time, our results show that the appearance of motor dysfunctions is delayed
in hemi-vagotomized and sympathectomized treated mice when compared to non-operated treated mice. Moreover, we only
observed accumulation of alpha-synuclein in those structures still connected to the ENS. Interestingly, enteric neurons secrete
alpha-synuclein only upon exposure to rotenone and secreted alpha-synuclein can be up-taken by non-neuronal cells or presynaptic
sympathetic neurons. Altogether, these results suggest that pesticide-dependent alterations in the ENS can induce idiopathic PD
pathology and trigger its progression. Moreover, it seems that this progression is based on the transsynaptic and retrograde axonal
transport of alpha-synuclein, playing here the role of a prionic protein.
Accepted for publications nature.com, Scientific reports
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Figure 2. Change in 18 F-Fluorodopa Uptake in the Brains of Patients with
Parkinson ’s Disease after Transplantation,as shown in Fluorodopa PET
Scans.
Freed et al
(2001)
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a-Synuclein–positive Lewy bodies in Host Substantia
nigra and Grafted Dopaminergic Neurons.
Li J-Y et al.Nature Medicine
(2008), 14.
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Olanow CW & Prusiner SB (2009)
Manganese-induced Parkinson-Syndrome
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Gatto NM et al. (2009) Env Health Persp 117:1912-1918
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Is the environment causing PD?
I Pestizides
- signifikant association between exposure to pestizides and PD,
but not enough data to claim a causal relationship
- most suspicious are paraquat and rotenone
I Infections
- Helicobacter pylori?: significantly higher incidence of
gastro-duodenal ulcers in PD, Ulcers sometimes 10-20
years before onset of PD
- Substantia nigra shows high microglia concentration
→ remarkably high sensitivity for LPS?
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Known environmental factors causing
secondary Parkinsonism
I Alkaloides:
z.B. Annona muricata: responsible for atypical PD in Guadeloupe
I Infektions
- „von Economo-Encephalitis“ (1916-1917 in Vienna) as
precursor of Encephalitis lethargica Pandemie (1918 - ca.1927),
induced by influenza virus
(„Awakenings“ Oliver Sacks)
I MPTP/MPP+
- End of 70es, formed when a mistake was made to synthesize
heroine
→ the most often used toxic model for PD
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Halliday G et al. (2011) Movement Disorders 26:1015-1021
The Braak Stages
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Degeneration of sleep related areas may cause
sleep disturbances
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REM-Sleep Behavior Disorder
Dream-associated movements in RBD (lacking atonia)
By courtesy Prof. E. Tolosa
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RBD in Parkinson‘s disease
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Follow-up of patients with idiopathic RBD
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Further support for the Braak Hypothesis
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Wolters Ech & Braak H (2006) J Neural Transm Suppl 70:309-319
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A Critical Appraisal of Braak‘s Staging in PD
Fig. 1 The flowchart delineating the logistics of this study. A) The aS-immunoreactive
inclusions were screened in substantia nigra, amygdaloid complex and dorsal motor nucleus of
vagus.
Parkkinen L et al. (2008)
Acta Neuropathol 115:399-407
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Table 1 Applicability of Braak staging and the incidence of
dementia and extrapyramidal signs (EPS) in each stage
Parkkinen L et al. (2008) Acta Neuropathol 115:399-407
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Burke RE et al. (2008) Ann Neurol 64:485-491
Summary
Parkinson‘s Disease seems to follow a clinical pattern with a pre-motor phase
followed by the typical motor impairment
Early signs are in most patients present and consist of loss of olfaction and
constipation
This fits well with the claim that Braak stage 1 is characterised by alpha-synuclein
in the dorasal vagal nc. and the olfactory bulb.
More recent neuropathological work has shown that there is also impairment of
the ENS and the nervous system of the submandibular gland
All these locations are open to the environment, thus it is intriguing to speculate
that a substance from outside causes PD
For this reason we have developed an animal model and could demonstrate that
this model is in perfect agreement with Braak‘s staging
It should be noted, however, that not all autopsied cases with alpha-synuclein
pathology showed any signs of PD, which resembles the situation of Alzheimer‘s
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Disease
Thank you for your kind attention
Dresden Opera House
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