Critical view on Braak‘s hypothesis. Has clinical work during the last 10 years supported the hypothesis? Oslo, April 25th, 2013 Heinz Reichmann, FRCP,

Critical view on Braak‘s hypothesis. Has clinical work during the last 10 years supported the hypothesis?Oslo, April 25th, 2013Heinz Reichmann, FRCP, FAANChair Department of Neurology, Technical University of Dresden

www.uniklinikum-dresden.de

Gene mutations,alpha-synuclein levels,imaging abnormalities

-10y -6y 0 2y 5y 10y 15y

Pre-Motor PD Early PD Advanced PDModerate PD (stable)Pre-symptomatic PD

PD Progression

Onset of clinical PDpremotor phenotype

Onset of clinical PDmotor phenotype

Diagnosis

Olfactory lossRBD, constipationanxiety, depression

Bradykinesia,Rigidity,Rest-tremor,(+/- non-motor-symptoms)

Motor complications:Wearing off/dyskinesias, gait and balance problems,axial deformities, dysarthria/dysphagia

Non-motor symptoms:cognitive decline/ dementia, psychosis, autonomic dysfunction, sleep–wake-dysregulation


Martin I et al. (2011) Annu Rev Genom Human Genet 12:301-325

Genetic Screening



Figure 3: Age-specific risk of PDRisk 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.

Healy et al. (2008)


Friedrich-Heinrich Lewy(1885-1950)

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The Braak Stages


Sniffin‘ Sticks


Individual results of testing

normal

borderline

pathological

test not performed

UPDRS II III

TCS SPECT123I-FP-CIT (DaTScan)

Haehner et al. Mov Disord. 2007;22:839-842

Idiopathic Hyposmia as a pre-motor marker of PD

UPDRS III

Conversion to IPD

definitive

borderline

www.uniklinikum-dresden.deSommer et al., Figure 2

Sonografic Analysis of the Mesencephalon


Synthese

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Dopamine Transporter Scan

DopamineReuptake

Dopamine


DAT SPECT normal EP, unilateral: DAT SPECT abnormal

Booij J et al. (2001)

DAT SPECT (123I-FP-CIT)


Individual results of testing

normal

borderline

pathological

test not performed

UPDRS II III

TCS SPECT123I-FP-CIT (DaTScan)

Haehner et al. Mov Disord. 2007;22:839-842

Transcranial sonography in idiopathic olfactory dysfunction

UPDRS III

Conversion to IPD

definitive

borderline


Prevalence of Smell Loss in PD


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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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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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). Modified with permission from Thieme. Witt et al., (2008)

Nasal Cavity Biopsy


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)

Negative α-Synuclein Staining


Lewy Pathology in the Submandibular Gland


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 m 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 -synuclein-immunoreactive 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. -Synuclein immunoreactions, 100 m polyethylene glycol (PEG) embedded tissue sections (b–j)

Del Tredici K et al. (2010)

normal

a-Syna-Syn

Lewy-pathology in the submandibular gland


Lewy Pathology in the ENS of the Gut


-Synuclein Inclusions in the ENS

• Presence of gastric -synuclein inclusions could provide first link in susceptible neurons that extend from the enteric to the central nervous system individuals.


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.

GI-Tract: Window or Entry Zone


NMS in PD in comparison with an age-matched control group

25Chaudhuri & Odin 2010; Chaudhuri et al 2006

NMSQuest study: Non-motor questionnaire for PD patients

• 123 PD patients (mean age 68y, disease duration 6.4y, H&Y 2.5)

• 96 controls (mean age 65y)(%)


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

28 Barone et al 2009

TOP 10 NMS symptoms


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

Prevalence of Nonmotor Symptoms in Parkinson’s Disease:

Study Using Nonmotor Symptoms Questionnaire

29 Martinez-Martin et al 2007

N=4

N=64 N=40 N=18

NMS: Distribution of responses (>30%)(%)


Lewy Pathology in the ENS of the Gut


-Synuclein Inclusions in the ENS

• Presence of gastric -synuclein inclusions could provide first link in susceptible neurons that extend from the enteric to the central nervous system individuals.


I Occurence of Lewy-Bodies in Auerbach and Meissner Plexus

I Predilection of LB in the upper GI-tract

Acta Neuropathol 1988; 76:217-221Acta Neuropathol 1988; 76:217-221

Further Evidence for G.I. Disturbances in PD


PDPD ControlsControls

I 10 untreated Parkinson patients ; all positive for Alpha-SynucleinI Sigmoidoscopy and Bx: alpha-Synuclein and 3-Nitro-Tyrosin (marker

for mitochondrial stress)

Mov Disord 2012: 27:709-715Mov Disord 2012: 27:709-715

Colon biopsies in PD


I Alpha-Synuclein positive immunohistochemistry in 3 biopsies 2-5 yrs before

onset

Mov Disord 2012: 27:716-719Mov Disord 2012: 27:716-719

Premotor PD and Colon biopsy


The Dresden Parkinson Model


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


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, antiphospho-alpha-synuclein (Ser 129) and DAPI staining on duodenum sections from control ( I) and treated (J) animals.

Pan-Montojo et al. (2010)

Control

Control


Figure 2. Intracellular and axonal alpha-synuclein increases in the intermediolateral nucleus and the dorsal horn lamina I layer of the spinal cord after oral rotenone 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 color-coded images from 3 months control (D, D’) and 3 months treated mice (E, E’) spinal cord sections stained using DAPI and alpha-synuclein 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 from 3 months treated (H) and control (G) mice. Arrows in G–H, lamina I layer of the dorsal horn.



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 alpha-synuclein 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 alpha-synuclein 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. Arrow in H, activated microglial cell in the DMV.


Control

Treated (DMV)

Con 1,5 mo 3mo

A-synuclein


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, alpha-synuclein 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 (E) and 3 months treated (F) mice.


Con

Substantia nigra pars compacta

Th-stain in control € and treated mice (F)


Hansen C et JY Li. (2012) Trends in Molecular Medicine 18:248-255


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, Dresden2Department of Neurology, University Hospital Carl-Gustav Carus, Fetscherstr. 74, 01307, Dresden, Germany3Center for Regenerative Therapies Dresden, Tatzberg 47/49, 01307, Dresden, Germany4Max-Planck Institute for Cell Biology and Genetics, Pfotenhauerstr. 108, 01307, Dresden, Germany5Departamento de Anatomía, Histología y Neurociencia, Facultad de Medicina, Universidad Autónoma de Madrid, Arzobispo Morcillo 4, 28029 Madrid, Spain

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.

Further Evidence

Accepted for publications nature.com, Scientific reports


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)


-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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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?


Known environmental factors causingsecondary 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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The Braak Stages


Degeneration of sleep related areas may cause sleep disturbances


REM-Sleep Behavior Disorder

Dream-associated movements in RBD (lacking atonia)

By courtesy Prof. E. Tolosa


RBD in Parkinson‘s disease


Follow-up of patients with idiopathic RBD


Further support for the Braak Hypothesis


Wolters Ech & Braak H (2006) J Neural Transm Suppl 70:309-319


Fig. 1 The flowchart delineating the logistics of this study. A) The S-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

A Critical Appraisal of Braak‘s Staging in PD


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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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 Disease


Thank you for your kind attention

Dresden Opera House

Documents

Critical view on Braak‘s hypothesis. Has clinical work during the last 10 years supported the hypothesis? Oslo, April 25th, 2013 Heinz Reichmann, FRCP,