Thesis
Reference
Proteomic analysis of the substantia nigra in patients with Parkinson's disease
LICKER, Virginie
Abstract
The specific cascade of biological events underlying substantia nigra neurodegeneration in Parkinson's disease (PD) remains elusive. To gain new insights into PD pathogenesis, we conducted some proteomic investigations of nigral autopsy tissues from patients with PD and controls. Our approach highlighted a set of proteins differentially expressed in PD. A majority of them such as CNDP2 or nebulette were novel candidates potentially engaged in PD pathological process. Overall, observed alterations tended to confirm well accepted concepts surrounding PD pathogenesis but also pointed out the involvement of less conventional ones such as ER stress, cytoskeleton or extracellular matrix impairments. This project provides further insights into PD pathogenesis and may ultimately help to delineate new therapeutic targets and biomarkers for the treatment and diagnosis of PD.
LICKER, Virginie. Proteomic analysis of the substantia nigra in patients with Parkinson's disease. Thèse de doctorat : Univ. Genève, 2013, no. Sc. 4532
URN : urn:nbn:ch:unige-333777
DOI : 10.13097/archive-ouverte/unige:33377
Available at:
http://archive-ouverte.unige.ch/unige:33377
Disclaimer: layout of this document may differ from the published version.
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Département des Sciences des Protéines Humaines FACULTE DE MEDECINE Professeur P.R. Burkhard Section des Sciences Pharmaceutiques FACULTE DES SCIENCES
Professeur D.F. Hochstrasser
Proteomic Analysis of the Substantia Nigra in Patients with Parkinson’s Disease
THESE
Présentée à la Faculté des Sciences de l’Université de Genève pour obtenir le grade de Docteur ès Sciences, mention interdisciplinaire
par Virginie Licker
de
Chermignon (VS)
Thèse n°4532
Genève 2013
J’aimerais exprimer toute ma gratitude à ceux qui ont d’une façon ou d’une autre contribué à la réalisation de cette thèse.
Aux membres du Jury, Prof. François Berger et Dr Christian Wider pour avoir accepté de lire et d’évaluer mon travail de thèse.
Aux Prof. Pierre Burkhard et Denis Hochstrasser, directeur et co-directeur de thèse, qui m’ont permis d’évoluer au sein de leur laboratoire. Merci à Pierre de m’avoir encouragée à participer à des congrès internationaux de qualité, ainsi que pour la grande liberté laissée au cours de ces quatre ans qui m’a permis de gagner en indépendance scientifique.
Au Prof. Jean-Charles Sanchez, pour m’avoir aidée à avancer et à me surpasser durant ces quatre années au travers de discussions scientifiques ainsi que par ses commentaires et relectures critiques.
Son dynamisme, son enthousiasme et sa disponibilité auront été des moteurs essentiels.
Au Dr Natacha Turck, pour son soutien autant scientifique que moral, pour ses commentaires pertinents quel que soit le sujet ainsi que pour sa « positive attitude ».
Aux neuropathologues, les Dr. Alexander Lobrinus, Karim Burkhardt et Enikö Kovari dont la collaboration a permis la collecte des échantillons de substance noire. Un merci particulier à Enikö pour sa disponibilité, sa gentillesse et ses conseils, ainsi qu’à Maria Surini pour toute son aide sur la partie IHC.
A Mélanie Côte, ma collègue, pour son aide et savoir-faire apportés tout au long de cette thèse. Ainsi que pour ses incroyables pâtisseries dont mes papilles se souviendront longtemps.
A tous les membres du BPRG pour leur soutien au quotidien.
A mes collègues doctorants, Natalia, Domitille, Didia, Xavier, Hui-Song, Florent, Francesco, ainsi qu’Alex (« &Co ») et Vanessa, ainsi qu’aux nouveaux venus Leire, Florian et Cindy. Merci pour votre soutien scientifique, votre solidarité mais aussi pour tous les bons moments passés ensemble, au labo ou en soirée, congrès et voyage! Qui aurait pu croire qu’un jour Harry, William, Prince Philip ou encore Victoria B s’inviteraient au BPRG…
A Anne et Lisa, co-Queens of Sciences, avec qui j’ai beaucoup ri et partagé les petits soucis du quotidien comme les grandes questions existentielles. Ensemble nous avons appris à relativiser.
Plutôt que de se préoccuper d’un projet voué à « s’effondrer » - les derniers WB, TMT ou IF ne fonctionnant pas, il est parfois préférable de se concentrer sur la subtile différence entre un Waikiki Orange et un Cajun Schrimp. Je n’arrive toujours pas à croire que j’ai réussi à vous faire participer à la course de l’Escalade, je l’écris pour la postérité.
A mes princesses préférées Dany, Loyse, Auré, Vaness R, Jo, Steph, Olivia et la belle Amel. Merci pour tous ces précieux moments entre copines qui m’ont permis de déconnecter de mon doctorat. Merci de m’avoir écoutée et soutenue!! Le voyage à Barcelone fut une véritable bouffée d’oxygène durant ce dernier été « haletant ».
A mes chers amis Louise, Lorric, Vaness P, Caro et David. Merci d’être toujours là depuis si longtemps. Un clin d’œil à deux de mes amis et prédécesseurs qui manquent cruellement au CMU, Lucie et Lorenzo!
A Andrée, pour son inébranlable bonne humeur et gentillesse qui font de ma leçon de piano hebdomadaire un moment de détente incontournable depuis tant d’années.
A Kim, pour m’avoir écoutée et soutenue à certains moments difficiles de ma thèse ainsi que pour être l’un des seuls à connaître mon sujet de thèse.
A mes adorables manoriens Harris, Nat, Anais, Loichot, Béatrice, mais aussi Tatjana. Un remerciement particulier à Béa pour ne douter que rarement du bien-fondé de mes plaintes en tout genre et pour être toujours si bon public.
A Nicolas, pour avoir mis un peu de Cassis, de Mouse, de Muse et de Mousse dans cette dernière phase d’écriture.
A mes parents, mes grands-parents et à mon frère pour leurs encouragements et leur amour. Un merci tout particulier à Greg pour son irrésistible sens de l’humour et de la répartie: rien de tel qu’un bon fou rire pour oublier les soucis de la thèse... à toi de jouer maintenant, courage !
1 TABLE OF CONTENTS
ABBREVIATIONS ……….……….………. p. 3 ABSTRACT………..…..……….... p. 5 RÉSUMÉ………..…..………... p. 7 CHAPTER I : GENERAL INTRODUCTION
1. Description of Parkinson’s disease…...……….………... p. 11 1.1. Historical background.……….. p. 11 1.1.1. PD in Ancient Times .……… p. 11 1.1.2. The first clinical definition of PD……… p. 12 1.1.3. PD pathology ………..……… p. 13 1.1.4. The miracle of levodopa ………..………..……… p. 14 1.2. Epidemiology: prevalence, incidence and socioeconomic aspects.………. p. 15 1.3. Clinical description.…...………...……… p. 16 1.3.1. Motor and non-motor symptoms………. p. 16 1.3.2. PD progression and rating scales……….. p. 17 1.4. Diagnosis.……… p. 19 1.5. Treatment…….……….……… p. 21 2. Etiopathogenesis of Parkinson’s disease………... p. 24 2.1. PD pathology ………..……… p. 23 2.1.1. The nigrostriatal pathway and the dopaminergic system……… p. 24 2.1.1.1. Anatomy and function of the basal ganglia……….. p. 24 2.1.1.2. PD pathophysiology ………..……….. p. 25 2.1.1.3. Neuropathological hallmarks……….………… p. 27 2.1.2. Beyond the substantia nigra………..……….. p. 29 2.1.3. Braak staging of PD………..……….………….. p. 30 2.1.4. PD progression: a prion-like hypothesis? ……….………..……….. p. 32 2.2. Risk factors and etiological hypotheses PD pathology ……….…… p. 34 2.2.1. Non-genetic risk factors……….………..……….. p. 34 2.2.2. Genetic risk factors……….………..……….. p. 35 2.2.2.1. PD causative genes……….………..………..….. p. 35 2.2.2.2. Susceptibility genes……….………..……….. p. 36 2.3. Pathogenetic mechanisms of PD ……….… p. 37 2.3.1. The specific vulnerability of nigral dopaminergic neurons……… p. 38 2.3.2. Potential mechanisms underlying neurodegeneration……… p. 39 2.3.2.1. Alpha-synuclein, Lewy bodies and protein aggregation………… p. 39 2.3.2.2. Impairment of protein degradation systems……….. p. 40 2.3.2.2.1. Ubiquitin proteasome system……….... p. 41
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2.3.2.2.2. Lysosome and chaperone mediated autophagy………… p. 42 2.3.2.3. Mitochondria and oxidative stress……….….. p. 45 2.3.2.4. Glial reaction and inflammation……….………. p. 47 3. Proteomics and Parkinson’s disease research………. p. 51 3.1. “omics and PD research ……….. p. 51 3.2. Proteomics ……….……….. p. 53 3.2.1. Generalities……….………..…. p. 53 3.2.2. Sample preparation……….………. p. 55 3.2.3. Sample separation……….……… p. 56 3.2.4. Mass spectrometry and bioinformatics……….………. p. 58 3.2.5. Quantitative proteomics……….….………. p. 60 4. Project presentation and aims……… p. 64
5. Bibliography………... p. 66
CHAPTER II: Proteomics in human Parkinson's disease research ……… p.81
CHAPTER III: Neuroproteomics and Parkinson’s disease: don’t forget human samples ……… p. 103
CHAPTER IV: Proteomic profiling of the substantia nigra demonstrates CNDP2
overexpression in Parkinson's disease……….. p. 109 CHAPTER V: Human substantia nigra proteomics: insights into Parkinson’s disease
pathogenesis………..……… p. 123 CHAPTER VI: DISCUSSION, PERSPECTIVES and CONCLUSIONS
Discussion ………. p. 171 Perspectives……… p. 190 Conclusions ………. p. 196 Bibliography ……… p. 197
3 ABBREVIATIONS
2-DE Two-dimensional polyacrylamide gel electrophoresis
α-SYN alpha-synuclein AC Accession number AD Alzheimer’s disease
ALP Autophagy lysosomal pathway ATP Adenosine triphosphate BBB Blood brain barrier BG Basal ganglia BP Biological process CC Cellular component
CID Collision-induced dissociation CMA Chaperone-mediated autophagy CNS Central nervous system
CNDP2 Cytosolic non-specific dipeptidase 2 CP Central proteome
CSF Cerebrospinal fluid DA Dopamine
DAVID Database for Annotation,
visualization and integrated discovery DBS Deep brain stimulation
DLB Dementia with Lewy bodies ECM Extracellular matrix
ESI Electrospray ionization ER Endoplasmic reticulum FDR False discovery rate GO Gene ontology
GWAS Genome-wide association study GPe/i Globus pallidus internal/external IAA Iodoacetamide
ICAT Isotope coded affinity tags IEF Isoelectric focusing IHC Immunohistochemistry L-DOPA Levodopa
LACB Beta-lactoglobulin LB Lewy bodies
LC Liquid chromatography LCM Laser capture microdissection LoC Locus coeruleus
LPS Lipopolysaccharides LTQ Linear trap quadrupole
MSA Multiple system atrophy NO Nitric oxide
NSAID Non-steroidal anti-inflammatory drugs OGE Offgel electrophoresis
OT Orbitrap
MALDI Matrix-assisted laser desorption/ionization MPTP 1 - methyl 4-phenyl 1,2,3,6-
tetrahydropyridine MS Mass spectrometry
MS/MS Tandem mass spectrometry PAGE Polyacrylamid gel electrophoresis PANTHER Protein Analysis Through Evolutionnary
Relationships PD Parkinson’s disease
PET Positron emission tomography pI Isoelectric point
PMF Peptide mass fingerprint PMD Post-mortem delay PrP Prion protein
PTM Post-translational modification RNA Ribonucleic acid
ROS Reactive oxygen species RP Reversed-phase
RR Relative risk
SDS Sodium dodecyl sulfate
SN Substantia nigra pars compacta SNr Substantia nigra pars reticulate STN Subthalamic nucleus
TCA Tricarboxylic acid cycle
TCEP Tris-2-carboxyethyl-phosphine Ub Ubiquitin
UKPDBB United Kingdom Parkinson’s disease brain bank
UPDRS Unified Parkinson’s disease rating scale UPR Unfolded protein response
UPS Ubiquitin proteasome system TOF Time of flight
TMT(-6) Tandem Mass Tags (sixplex) WB Western Blot
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5 ABSTRACT
Parkinson's disease (PD) is the most common neurodegenerative movement disorder, resulting from the massive loss of dopaminergic neurons in the substantia nigra pars compacta (SN) along with the occurrence of Lewy bodies (LB). Despite decades of intensive investigations, the precise etiopathological mechanisms underlying PD pathology remain mysterious, impeding the establishment of curative neuroprotective therapeutic strategies. Although helpful, hypothesis- driven or “candidate-based” approaches might have reached some limits in the understanding of PD pathology, overwhelmed by the impressive complexity and diversity of the processes likely engaged in the disease. Recently, “hypothesis-free” disciplines encompassing high-throughput proteomic technologies have emerged as attractive alternatives, allowing the unbiased and global exploration of molecular pathways at the basis of PD. To gain new insights into PD pathogenesis, we investigated human SN autopsy tissues in patients with PD compared to non-neurological age-matched controls, looking for PD-specific alterations in their protein expression profiles. We analyzed samples using two different but complementary quantitative proteomic workflows, two-dimensional gel electrophoresis (2-DE) and shotgun approach using isobaric sixplexTMT technology.
Taking advantage of the 2-DE potential to resolve protein isoforms together with the high- throughput capability of the shotgun approach, we obtained the most comprehensive picture of the human SN proteome so far. Overall, we identified more than 1800 proteins, with approximately 1200 of them newly associated to human SN. The functional annotation (GO ontology, KEGG pathways) of this large dataset indicated a significant proportion of proteins with neuronal activities (11%) and suggested important roles in the SN for energy metabolic pathways, cytoskeletal organization, proper vesicular transport and trafficking, Ca2+ homeostasis, amino acid cycle, cell-cell junctions or anti-oxidant response. The SN function could thus be particularly sensitive to any perturbation in one or more of these critical processes, which were already linked to PD pathogenesis for most of them.
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Thus, the SN proteome characterization constitutes a first step towards a better understanding of the SN function and the specific features making it more vulnerable to neurotoxicity in PD.
By comparing the nigral proteomic profiles of PD versus non-neurological control patients, we discovered a set of proteins displaying significant differences in their relative abundance in PD.
The 2-DE comparative workflow (n=6) resulted in the findings of 32 differentially expressed spots, 14 over- and 18 underexpressed in PD, of which seventeen could be unambiguously identified. The sixplex TMT shotgun quantitative analysis performed on a different set of patients (n=6) yielded 204 differential proteins, 96 over- and 108 underexpressed in PD, among which a few were also found in 2-DE. Our data successfully confirmed the involvement of existing theories including mitochondrial dysfunction, energy metabolism impairment, oxidative stress, cytoskeleton and vesicular defect, synaptic dysfunction, protein homeostasis deregulation or inflammation, previously implicated in PD pathogenesis. They also suggested some less conventional pathogenic pathways such as protein translation defects, endoplasmic reticulum stress, abnormalities in the blood brain barrier or extracellular matrix. Overall, our approach highlighted a majority of novel candidates potentially engaged in PD pathological process, either as a cause or a consequence of it. Of them, we verified the expression levels of cytosolic non specific dipeptidase (CNDP2) and seipin by western blot. We also determined by immunohistochemistry that the expression of several candidates including CNDP2 and nebulette as well as gamma glutamyl hydrolase (GGH) was mainly confined in the neuronal population. The complex proteome alterations observed in the SN of PD patients provide further insights into the underlying pathogenic processes engaged in PD and may ultimately be a source of new therapeutic targets and biomarkers for the treatment and prevention of PD.
7 RÉSUMÉ
La maladie de Parkinson (MP) est l’une des pathologies neuro-dégénératives les plus fréquemment rencontrées chez le sujet âgé. Elle se définit par des troubles moteurs caractéristiques résultant principalement de la perte progressive des neurones dopaminergiques de la substance noire pars compacta (SN). Des agrégats protéiques intra-neuronaux appelés corps de Lewy (LB) sont typiquement observés dans les régions affectées. L’origine et les causes exactes de la MP demeurent encore inconnues, constituant un obstacle majeur au développement de traitements neuroprotecteurs qui permettraient de stopper ou de ralentir le cours de cette maladie incurable. De nouvelles stratégies dont la protéomique fait partie ont récemment émergées, permettant d’explorer la pathogenèse de la MP de façon globale et non biaisée, sans établir d’hypothèses pathogéniques au préalable. Afin de tenter de caractériser les mécanismes neuro-dégénératifs spécifiques à la MP, nous avons analysé des tissus de SN prélevés à l’autopsie à la fois chez des patients parkinsoniens et contrôles, à la recherche d’altérations de leur profil d’expression protéique.
Pour cela, nous avons utilisé deux techniques protéomiques quantitatives complémentaires, l’électrophorèse bidimensionnelle (2-DE) et l’approche dite « shotgun » utilisant des tags isobariques TMT pour le multiplexing de six échantillons (TMT sixplex).
La combinaison de nos deux stratégies protéomiques a permis d’établir le protéome de la SN, avec le set de données le plus complet disponible à l’heure actuelle. Nous avons en effet identifié plus de 1800 protéines dont 1200 environ nouvellement associées à la SN. L’annotation fonctionnelle (gene ontology (GO), KEGG pathways) du protéome nigral a indiqué une proportion significative de protéines impliquées dans des activités neuronales (11%) et permis de relever les rôles prépondérants de certains processus cellulaires dans le fonctionnement de la SN, incluant : les voies d’énergie métaboliques, l’organisation du cytoskelette, le trafic et transport vésiculaire, l’homéostase calcique ou encore les mécanismes de réponse au stress oxidatif. La SN pourrait donc s’avérer particulièrement sensible à une perturbation de l’un ou plusieurs de ces processus, par ailleurs
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souvent mis en cause dans la MP. Ainsi la caractérisation du protéome de la SN constitue une première étape dans la compréhension du fonctionnement de ce noyau et de ses spécificités qui le rendent plus vulnérable à la MP.
En comparant les profils d’expression protéiques nigraux de patients parkinsoniens relativement aux contrôles à l’aide des deux approches protéomiques, un set de protéines différentiellement exprimées dans la MP a pu être identifié. L’analyse 2-DE (n=6) a mis en évidence 32 spots différentiels, 14 surexprimés et 18 sous-exprimés dans la MP, dont 17 ont pu être identifiés.
L’analyse shotgun utilisant le tag TMT effectuée sur un set de patients différents (n=6), a permis de distinguer plus de 200 protéines différentielles, 96 surexprimées et 108 sous-exprimés dans la MP dont quelques-unes commune au 2-DE. Une majorité de ces protéines différentielles a pu être associée à des théories pathogéniques existantes, confirmant ainsi l’implication d’une dysfunction mitochondriale, d’un défaut du métabolisme énergétique, du stress oxidatif, d’altérations au niveau du cytoskelette et du traffic vésiculaire, d’une dysfonction synaptique, de dérégulation de l’homéostase protéique ou encore de mécanismes d’inflammation. Nos données ont également suggéré des voies pathogéniques moins conventionnelles impliquant des défauts dans le processus de traduction protéique, un stress du reticulum endoplasmique ou des anormalités au niveau de la barrière hémato-encéphalique et de la matrice extracellulaire. De façon globale, notre stratégie a permis d’identifier une majorité de nouveaux candidats potentiellement impliqués dans la MP, en tant que cause ou conséquence des processus pathologiques. Parmi eux, nous avons notamment vérifié l’expression de la cytosolic non specific dipeptidase (CNDP2), ferritin light chain et seipin par western blot. Nous avons également pu déterminer que l’expression de certains de ces candidats, tels que la CNDP2 mais aussi la nebulette ou la gamma glutamyl hydrolase était principalement neuronale par des méthodes d’immunohistochimie. En conclusion, l’observation d’altérations protéiques complexes dans la SN des patients parkinsoniens contribue à la compréhension des processus pathologiques engagés dans la MP et pourrait générer de nouvelles cibles thérapeutiques ainsi que des biomarqueurs précoces pour aider au traitement et à la prévention de la MP.
Chapter I
General introduction
11 1. DESCRIPTION OF PARKINSON’S DISEASE
1.1. Historical background
1.1.1. PD in the Ancient times
For thousands of years, Parkinson’s disease (PD) has plagued mankind, as evidenced by the multitude of symptom references throughout history. Traditional Indian Ayurvedic texts dating back to 5000 to 3000 BC already alluded to a nervous disorder called “kampavata”
sharing similarities with PD such as tremor (kampa) [1] and responding to oral administration of Mucuna pruriens seeds (Figure 1) [2]. The
plant, whose therapeutic effect was later attributed to its levodopa content [3], is still used in India [4]. Ancient Chinese medicine sources provided suggestive descriptions of PD around 425 BC, with the first clear clinical case reported by Zhang Zihe during the Jin dynasty (AD 1151-1231) [5]. Traditional Chinese Medicine also advocated the use of a Chinese herb, root and scorpion decoction to treat tremor and rigidity symptoms [5].
Recent studies indicated that gastrodin, the active component of Gastrodia elata herb, could have a neuroprotective activity through antioxidant [6] and anti-inflammatory [7]
properties. Other references were found in Egyptian papyrus, the Iliad poem by Homer (around 800 BC) or writings of physician Galen (122-200), whose theories about tremor narrated in “De Tremore” influenced James Parkinson. Later, some PD aspects were clearly described, such as
Figure 1 Mucuna pruriens. Mucuna pruriens (also known as cowage or “Atmagupta” in Sanskrit) is an endemic plant found in India as well as Central and South America containing levodopa. Traditional Indian medicine sources suggest its use to treat PD as far as 5000 BC.
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tremor at rest by Sylvius de la Boë (1614-1672) and festination by the French François Boissier de Sauvages (1706-1767). A recently discovered Hungarian text published by Ferenc Papai Pariz (1649-1716) in 1690 was found describing all cardinal signs of PD over 120 years before James Parkinson [8]. Because Hungarian was understood by very few people, the publication unfortunately stayed ignored in the medical literature [8].
1.1.2. The first clinical definition
The first clear clinical description of PD was attributed to the English physician Dr James Parkinson (1755-1824). In 1817, he thoroughly described the neurological syndrome that still bears his name in the original “Essay on The shaking palsy” (Figure 2)[9]. Based on the identification of only six cases, three personally examined and three observed in London’s street, Parkinson provided a detailed description of Paralysis Agitans or Shaking Palsy that he defined as follows:
“Shaking Palsy (Paralysis Agitans): Involuntary tremulus motion, with lessened muscular power, in parts not in action and even when supported; with a propensity to bend the trunk forewards and to pass from a walking to a running pace: the senses and intellect being uninjured”.
Figure 2. Cover of James Parkinson’s original publication «An Essay on the Shaking Palsy” published in 1817. Image from Goetz C G Cold Spring Harb Perspect Med2011
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Over the next 50 years, Parkinson’s work received little attention until the French neurologist Jean-Martin Charcot (1825-1893) further detailed the clinical spectrum of the illness and first referred to it as “maladie de Parkinson” or Parkinson’s disease (PD). Charcot added bradykinesia
“the slowness in execution of movement rather than the real weakness”, or rigidity “the face is masked, the forehead wrinkled, the eyebrows raised, the eyes immobile” to the motor symptoms, but also contributed to differentiate PD from other neurological disorders such as multiple system atrophy (Figure 3). By the end of the 19th century, the modern clinical definition of PD was nearly established.
1.1.3. PD pathology and the substantia nigra
The cause of PD remained unclear for a long time, until Charcot’s student Edouard Brissaud, suggested in 1895 that the anatomical site of neurological dysfunction was situated in the midbrain “locus niger” [10], so named because of the presence of neuromelanin black pigment. In 1912, Lewy discovered intracytoplasmic inclusions referred to as “Lewy bodies” (LB) related to PD in the dorsal nucleus of the vagus and the nucleus basalis of Meynert. A few years later, Trétiakoff emphasized the importance of the substantia nigra (SN) by identifying nigral damage in each of the nine PD cases he had examined [11]. Lewy observations were confirmed
Figure 3. Drawings from Charcot (1888) showing the difference between a typical PD case (left) with a flexed posture and a Parkinsonian variant with no tremor and an extended posture. Image from Goetz C G Cold Spring Harb Perspect Med2011
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and nigral depigmentation was attributed to nerve cell loss. Detailed pathological analyses were continued by Foix and Nicolesco [12] who showed that the more severe lesions were located in the SN, with the most complete description of nigral degeneration finally given by Greenfield and Bosanquet in 1953 [13]. The neurodegeneration observed in the SN, along with the presence of LBs are now recognized as PD major pathological hallmarks.
1.1.4. The miracle of levodopa
From the nineteen to the mid-twentieth century, emerging PD treatments leaded by Charcot and its contemporary William Gowers, remained largely focused on anti-cholinergic drugs (i.e.alkaloids) and dopaminergic activating agents (i.e. rye based ergot products, cannabis) as well as physical therapy. In 1957, Carlsson demonstrated that dopamine (DA) was a neurotransmitter located in the striatum and developed the first reserpine-induced Parkinsonism model that could be reversed by the DA precursor levodopa (L-Dopa) in mice and rabbits [14]. In the 1960s, depletion of DA was documented in PD brains by Ehringer and Hornykiewicz [15]. Subsequently, the landmark studies by Birkmayer and Hornykiewicz [16] and Cotzias [17] demonstrated major improvements of PD symptoms following oral administration of L-dopa. In 1961, the antiakinetic effects of L-dopa were clearly evidenced in PD patients receiving L-dopa by intravenous injection: “Bed-ridden patients who were unable to sit up, patients who could not stand up when seated, and patients who when standing could not start walking performed all these activities with ease after L-dopa. They walked around with normal associated movements and they could even run and jump” [16]. Since then, none of the developed pharmaceutical compounds rivaled L-Dopa, which still remains the premier agent for PD symptomatic treatment.
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1.2. Epidemiology: prevalence, incidence and socioeconomic aspects of PD
PD is the most common neurodegenerative movement disorder after Alzheimers’ disease (AD), affecting adult individuals of all races, gender, geographical locations and even age.
Epidemiological studies of PD are numerous, reflecting the need to assess the burden of the disease in terms of costs related to medical and nursing care as well as the economic impact of workday losses. In large recent prospective population-based cohort studies, most of the past limitations have been overcome. Nowadays, valuable data regarding the prevalence, incidence and potential risk factors of PD can be provided although estimates may still vary depending on methodological parameters such as case-finding strategies or diagnostic criteria [18].
A PD prevalence of 0.3% in the general population and 1-2 % over the age of 60 is commonly accepted based on large population databases [19]. In 2010, approximately 1.2 of the 514 million European people were affected by PD [20], with 15’000 patients living in Switzerland according to Swiss Parkinson. Using stringent diagnostic criteria, the mean incidence of PD is estimated at 14 per 100’000 person-years in Western countries [21]. Early onset of PD is uncommon and its incidence raises beyond the age of 65, with a median rate of 160 per 100’000 person-years [21] that may vary according to gender and ethnic group (Figure 4). A 3:2 male-to- female ratio has been reported [22, 23] and the prevalence of PD might be higher among Caucasian than Africans and Blacks [24]. Overall, variation in PD frequency according to ethnicity might be suggestive of differential environmental exposures and susceptibility genes, but remains a controversial issue.
Importantly, PD affects the working capacity since within a year after diagnosis about one third of the afflicted patients has to retire prematurely [25]. Across epidemiological studies, PD is consistently associated with a marked reduction of life expectancy, mortality rates being nearly doubled compared with age-matched subjects [26, 27]. By the year 2030, the number of PD cases is expected to double along with the aging of the Western population [28]. In Europe, the estimated cost of illness is higher for PD than any other brain disorders, amounting up to
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13.9 billion euros per year [20]. Without any curative treatment, the socioeconomic and financial burdens incurred by PD will continue to grow and will challenge our health care system over the coming decades.
1.3. Clinical description
1.3.1. Motor and non-motor symptoms
The cardinal motor manifestations of PD generally include tremor at rest, slowness of movements (bradykinesia), rigidity and postural instability. Flexed posture and freezing phenomenon (full arrest of movement) have been added to the typical features of parkinsonism, a clinical syndrome with PD as the most common cause. The full spectrum of PD symptoms has been progressively extended far beyond the classical motor picture. Non-motor manifestations in PD patients include sleep and mood disturbances (i.e.depression, anxiety), neurocognitive impairment (i.e.dementia) and autonomous nervous system dysfunction (i.e digestive, sexual impairments), all of them being major sources of functional disabilities and quality of life deterioration [29]. Altogether, PD appears as a complex condition defined by a variable combination of debilitating motor and non-motor impairments, summarized in Table 1. PD is
Figure 4. Prospective population- based incidence studies of Parkinson's disease across the world. Incidence increases markedly in the seventh decade of life. A decrease might occur after 80 years old, which might be real or related to underdiagnosis of PD at that advanced age due to comorbidities. *Study restricted to men. Taken from De Lau et al.
Lancet Neurology, 2006.
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also clinically heterogeneous with different clinical subtypes being recognized based on age of onset, clinical features or progression rate [30].
Table 1. Parkinson’s disease clinical symptoms. Data were adapted from Jankovic et al., 2008 [31] and Massano et al. 2012 [32].
1.3.2. PD progression and rating scales
The onset of PD is often insidious with early signs remaining unnoticed for a long time (Figure 5). Increasing evidence suggests that nonmotor symptoms (i.e. hyposmia, sleep abnormalities, depression, pain) may precede the onset of the earliest noticeable motor manifestations [33]. In the majority of PD patients, rest tremor is the first symptom perceived by the patient and its absence in about 20% of cases [34] is suggestive of a preserved functional integrity of the midbrain neurons (A8) [31]. Alternatively, bradykinesia which best correlates with DA deficiency may indicate the beginning of the disease. Over the following years, PD symptoms and clinical signs gradually worsen, as a result of striatal DA deficiency arising from the progressive loss of nigral dopaminergic neuron projections. At the early disease stages, these signs are usually alleviated with the administration of dopaminergic substituting therapies (e.g., L-dopa and DA agonists). As the disease evolves, disabilities increase with the development of
Motor symptoms Non-motor symptoms
Resting tremor: rythmical oscillations of low frequence and variable amplitude, expressing involuntarily usually in the hands or feet when the muscles are relaxed
Behavioural and psychiatric problems:
depression, anxiety, apathy, hallucinations, fatigue
Bradykineskia/akinesia: slowness of initiation and perfomance (decrease in amplitude) when performing a volontary or spontaneous movement. Other clinical expression: micrographia (progressive smaller handwiriting) , hypomimia (decreased facial expression and eye blinking) or freezing (full arrest of movements)
Cognitive deterioration: mild cognitive impairement to dementia (late PD stage) bradyphrenia (slowness of thoughts)
Rigidity: increased muscle tone or resistance felt when by the examiner during the full range of passive movements.
Sensory symptoms : anosmia (loss of smell sense), pain (shoulder, back)
Postural and gait impairment: Loss of postural reflexes leading to a stooped posture and increasing the fall risk. Slow gait with shuffling steps and decreased arm swings or festination (fast succession of steps)
Autonomic dysfunction: orthostatic hypotension, constipation, urinary and sexual dysfunction Others: i.e. dystonia (involuntary intense muscle contractions) Sleep disorders: REM behaviour disorder, daytime
sleepiness, restless legs syndrome
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symptoms (i.e. flexed posture, freezing, bradykinesia) becoming resistant to L-dopa treatment.
This effect may result from pharmacodynamic tolerance, disappearance of DA receptors and involvement of nondopaminergic transmitter systems. Importantly, in about 75% of the patients surviving more than 10 years, the decline in neurocognitive function evolves towards dementia which is considered as the major long-term cause of disability [35].
Figure 5. Clinical progression of Parkinson’s disease. When about 50-70% of the nigral dopaminergic neurons are lost (thin line), cardinal motor symptoms appear (thick line) and PD becomes fully symptomatic (gray background). During the premotor phase (white background) non-motor symptoms (dotted line) may already be present. Adapted from Lebouvier et al. 2010[36]
Different clinically-based rating scales have been elaborated to assess the full spectrum of PD [37]. The Hoehn & Yahr scale provides gross assessment of the disease progression, ranging from 1.0 (unilateral involvement only) to 5.0 (wheelchair bound or bedridden) [38]. The Unified Parkinson’s Disease Rating scale (UPDRS) was developed to provide a comprehensive instrument that entailed earlier scales already familiar to clinicians and researchers dealing with PD patients [39]. The UPDRS was recently updated to the Movement Disorder Society-UPDRS (MDS-UPDRS) that integrates non-motor symptoms and corrected several flaws and shortcomings of the
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earlier version. [40]. So far, this version has yielded satisfactory clinimetric performances and has been adopted as the official benchmark scale for PD since 2008 [40].
1.4. Diagnosis
PD is still largely diagnosed based on clinical criteria evaluated by patient’s history and physical examination, as there is not definitive laboratory test for diagnosis during life.
Neuropathological confirmation of PD hallmarks - namely the massive depletion of nigral dopaminergic neurons coupled with the occurrence of LB, is still mandatory to make a post- mortem definite diagnosis. Clinically, PD diagnosis is based on the combined presence of cardinal motor signs, additional and exclusion features, as well as a favourable response to levodopa. The use of standard diagnostic criteria, such as those developed by the UK Parkinson’s disease society Brain Bank (UKPDSBB) (Table 2) improves significantly diagnostic accuracy, together with the experience level of the examining physicians [41, 42]. When assessed by movement disorder specialists, idiopathic PD could be accurately diagnosed during patient’s life time with a
predictive positive value of 98.6% [42]. Although the clinical diagnosis of PD can be
straightforward in patients with a typical presentation, it might be challenging in some cases.
Population-based studies suggest that 15% of the patients with a diagnosis of PD do not meet strict clinical criteria for the disease and 20% of patients with PD remain undiagnosed although they receive medical attention [43]. The sources of misdiagnosis are multiple. At the disease onset, early PD symptoms can easily be misinterpreted. Some PD cardinal features can be observed in the elderly as a result of aging or comorbidities (i.e. cancer). PD symptoms are also shared by other forms of parkinsonism with overlapping syndrome, particularly in the early course of the disease [44]. The most difficult entities to differentiate with PD relate to Parkinson- plus syndromes (multiple system atrophy (MSA), progressive supranuclear palsy (PSP), frontotemporal dementia with parkinsonism, dementia with LB (DLB)), essential tremor, drug-
20
induced parkinsonism or vascular parkinsonism [45] as well as Alzheimer’s disease in cases of atypical PD with early dementia. Their occurrence is however much rarer than PD.
Table 2. UK Parkinson's disease society brain bank clinical diagnostic criteria
Establishing an early and correct diagnosis of PD is a prerequisite for predicting prognosis and managing therapeutic interventions of PD. Ancillary diagnostic tests can be used to minimize error rates in differential diagnosis from other parkinsonian syndromes and eventually detect the disease earlier in its course (i.e. pre-motor phase). For example, functional neuroimaging of the nigrostriatal dopaminergic pathway using [18F]-fluorodopa positron emission tomography (PET) or DA transporter (DAT) single photon emission computed tomography (DAT-SPECT) [45-47] measure the decline in striatal dopaminergic nerve terminals in PD patients. Other tests include olfactory testing for hyposmia detection [48], transcranial sonography for hyperechogenicity (iron and ferritine accumulation) pattern identification in SN [49, 50], myocardial scintigraphy for cardiac uptake evalutation [51, 52] as well as genetic testing. While promising these techniques still demonstrate some major limitations (i.e. lack of sensitivity, specificity, high costs) hampering their widespread use as diagnostic tests.
Step 1. Clinical criteria for the diagnosis of a probable PD
● Bradykinesia
● At least one of the following o Muscular rigidity o 4-6 Hz rest tremor
o postural instability not caused by primary visual, vestibular, cerebellar, or proprioceptive dysfunction
Step 2 Exclusion of other forms of parkinsonism Step 3 Supportive prospective positive criteria for PD At least three required:
● Unilateral onset
● Rest tremor present
● Progressive disorder
● Persistent asymmetry affecting side of onset most
● Excellent response (70-100%) to levodopa
● Severe levodopa-induced chorea
● Levodopa response for 5 years or more
● Clinical course of ten years or more
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Research on specific, sensitive and early PD biological markers has been carried out alternatively with a large number of biochemical compounds (i.e. catecholamines, neuropeptides, amino acids, enzymes, IGgs, oxidative stress proteins, mitochondrial proteins...) measured in cerebrospinal fluid (CSF), plasma or urine [53, 54]. Alpha-synuclein (α-SYN), one of the most attractive molecules to investigate as a major component of LB, has been found repeatedly decreased in the CSF of PD patients compared with age-matched controls [55, 56].
Interestingly, this decrease has been correlated with PD severity suggesting a measurable change of the pathology progression, which might be useful to evaluate the efficacy of experimental therapies [55]. However, conflicting results [57], significant overlap of values between groups, insufficient sensitivity and specificity preclude the use of α-SYN as a valid marker at the moment. While promising for some of them, no biomarkers - taken individually or in combination - have achieved the level of certainty necessary for their clinical use [58].
1.5. Treatment
PD is still an incurable disease but medical treatments improve physical ability and life quality. As impaired motor function mainly results from the striatal DA modulation consecutive to nigral neuronal loss, restoring DA balance as well as reducing cholinergic or glutamatergic stimulation were predicted to improve symptoms. The most effective therapy consists in the administration of DA precursor L-dopa in combination with a peripheral dopa decarboxylase inhibitor (i.e. carbidopa) to prevent DA formation in tissues or a catechol-O-methyltransferase (COMT) inhibitor to extend its plasmatic half-life and prolong its action. Other dopaminergic drugs include monoamine oxidase inhibitors B (i.e. selegiline, rasagiline) or DA agonists (i.e.
pramipexole). Non-dopaminergic medications comprise several anticholinergics and a unique antiglutamatergic drug (i.e. amantadine) [59]. These drugs can alleviate effectively PD motor and non motor symptoms, at least in the beginning of the therapy. After about 5 years, various
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invalidating complications usually develop such as dyskinesias, “on-off” fluctuations and drug resistance [59].
When pharmaceutical therapies fail, pallidotomy in rare case or high frequency stimulation of deep brain targets can improve PD symptoms and reduce typical side effects such as dyskinesias. Deep brain stimulation of the subthalamic nucleus (STN) is now an established treatment for carefully screened patients [60, 61](Figure 6). However, although surgery was shown to be safe, the procedure is applicable to a minority of patients and is still associated to a wide range of neurologic and neuropsychological side effects [60, 62]. Finally, the transplantation of dopaminergic cells from fetal mesencephalon to restore physiological DA release has raised a considerable interest. Although reported to provide long-term symptomatic relief in some PD patients [63], it is associated with forms of dyskinesia persisting even after withdrawal of levodopa [64, 65] and do not improve non-dopaminergic PD symptoms (i.e.
freezing, gait dysfunction, dementia…) [66]. Recently, LBs were described in implanted DA neurons, suggesting that they might be affected by PD pathology [67-69]. Collectively, these findings demonstrate that cell-transplantation might not be optimal for the long-term management of PD. Furthermore, no neuroprotective therapies are available to stop the disease progression through the brain. Future research may unmask the disease pathological complex processes to provide a cure for this disabling condition.
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Figure 6. Deep brain stimulation (DBS) for PD. DBS is an established surgical technique used for PD treatment. An electrode is implanted to reach a specific brain targets and connected to an impulse generator delivering electrical stimuli under the skin (left panel). The subthalamic nucleus (STN) (right panel) and the internal globus pallidus are commonly targeted in PD. Through local electrical, chemical or neural- network influence on tissues, DBS modulate the feedback loop responsible for motor control to restore partially its function. Image from Okun et al, NEJM, 2012
24 2. ETIOPATHOGENESIS OF PARKINSON’S DISEASE 2.1. PD pathology
2.1.1. The nigrostriatal pathway and the dopaminergic system 2.1.1.1. Anatomy and function of the basal ganglia
During the last decades, considerable progresses have been made in the understanding of the pathophysiology - referring to changes in neuron electrical activity - underlying movement disorders, especially PD. Important insights into motor function control have come from the establishment of suitable DA depletion model such as the MPTP-treated monkey, as well as electrophysiological recordings of patients subjected to neurosurgical treatment (i.e DBS) for movement disorder diseases. PD as well as other “extrapyramidal” parkinsonian syndromes, is considered to result essentially from dysfunctions in the basal ganglia (BG) circuits. The BG are an important group of interconnected subcortical nuclei,
including the neostriatum (caudate nucleus (CN) and putamen (P)), the external and internal segments of the Globus Pallidus (GB), the subthalamic nucleus, the substantia nigra subdivided into a pars compacta (SN) and a pars reticulata (SNr) and more recently the pedonculopontin nucleus (PPN) and the central complex of the thalamus (Figure 7). These structures participate in a highly organized and complex neuronal network functioning in distinct parallel circuits (i.e. motor, oculomotor, associative, limbic, orbitofrontal) to integrate activities of the various cortical regions [70]. As such, they are associated to a variety of functions including movement control and regulation but also cognitive, emotional and motivational processes leading to action.
Figure 7. The Basal ganglia. Coronal picture of the brain showing the main basal ganglia nuclei. Image from Neuroscience, fourth edition by D. Purves
25 2.1.1.2. PD pathophysiology
The motor circuitry is the most relevant for PD pathophysiology as it predicts the effects of nigral degeneration and concomitant DA depletion observed in PD. According to a classical model of the BG, it is composed of two main projection systems, the direct pathway to facilitate desired movement and the indirect pathway to inhibit undesired movement (Figure 8) [70]. The balance between the direct and indirect pathways is differentially modulated by the nigrostriatal pathway, whose cell bodies located in the SN project principally to the putamen. Released DA favors the direct pathway activity (exciting striatal neurons D1 receptors) and reduces indirect pathway activity (inhibiting striatal neuron D2 receptors), resulting in a net decrease in GPi/SNr activity. In PD, SN degeneration and the consecutive striatal DA depletion induce perturbations in the BG loops (Figure 8b). The pathophysiological hallmark of PD is the excessive activity of GPi/SNr leading to thalamo-cortical motor overinhibition, which clinically expresses with akinesia and possibly other PD motor features. The model also provides explanation for levodopa-induced dyskinesias, conversely characterized by a hypoactivity of the GPi/SNr. The resulting increased output to the motor cortex phenotypically expresses as intense involuntary movement manifestations. Thus, the nigrostriatal dopaminergic system plays a central modulatory role to stabilize the motor control circuitry [71]. Based on this model, surgical treatments (i.e. pallidomy, DBS) were designed to reduce excess activities of GPi and STN nuclei occurring in PD, resulting in significant motor improvements. The BG model described here might however be oversimplified and questions have emerged from recent findings that are addressed elsewhere [72]. Importantly, non-motor PD symptoms might also arise from BG dysfunction but our understanding of their underlying pathophysiology is still limited [73].
Advances in the comprehension of BG function will lead to improvement of treatment for hypokinetic diseases as PD or hyperkinetic troubles such as dyskinesia.
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Figure 8. Schematic classical “motor circuit” model of the basal ganglia in normal (a) and PD (b) states.
The putamen receives a glutamatergic (excitatory) input from motor cortical areas to communicate with the GPi/SNpr through a direct inhibitory pathway (D) and a multisynaptic indirect pathway via the GPe and STN (I). In PD (b) compared to normal state (a), DA depletion resulting from SN lesions is associated to i) a reduced facilitation of GABAergic neurons from the striatum through DA D1 receptors in the direct pathway or ii) a decreased inhibition of GABAergic striatal neurons through DA D2 receptors in the indirect pathway, leading to an overinhibition of GPe and disinhibition of the STN. The resulting GPi/SNr overactivity leads to the overinhibition of thalamo-cortical and brainstem motor areas. Black arrows represent inhibitory projections whereas white arrows indicate excitatory projections. The arrows correlates with the increase (thicker) or decrease (thinner) in firing rate activity of specific pathways in PD (b) compared to normal state (a). The color of each box indicates the degree of activity of the brain area compared to the normal level of activity (lighter for a decrease, darker for an increase). The dashed lines labeling the SNc indicate nigrostriatal lesions in PD state. For more clarity, many connections have been omitted. GPe/i, globus pallidus external/internal segment; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; STN, subthalamic nucleus; VL, ventral lateral nucleus of the thalamus.
Image from Current Opinion in neurobiology, 1996.
27 2.1.1.3. Neuropathological hallmarks
Most available evidence suggests that the lesional core of PD pathology is the damage of dopaminergic cells of the ventrolateral region of the substantia nigra pars compacta (SN) which projects primarily to the putamen [74]. As these neurons contain neuromelanin, their loss produces the characteristic pattern of SN depigmentation (Figure 9 A and B) often observed in conjunction with a mild degree of gliosis and the presence of extraneuronal neuromelanin.
Surviving neurons in susceptible regions frequently exhibit proteinaceous inclusions termed Lewy bodies (LB) or Lewy neurites - if located in neuronal processes, which contain misfolded α- SYN and ubiquitin (Ub) (Figure 9C) [75]. Current knowledge on LB structure, formation and composition is still limited and reviewed in chapter 2. LB are not specific for PD and are also found in other forms of parkinsonism termed “synucleopathies” (i.e dementia with LB, multiple system atrophy), in AD, as well as incidentally in aged people [76]. The role of LB in cell death is still controversial.
Figure 9. Neuropathological hallmarks of PD. The nigrostriatal pathway is schematically represented in a normal (A) versus PD (B) patient (red).
Dopaminergic pigmented neuronal loss can be assessed macroscopically by SNpc depigmentation pattern and microscopically in hematoxy-eosin histological stained section. In PD patients, LB containing alpha-synuclein and ubiquitin are found in surviving nigral neurons (C). Image adapted from Dauer et al, 2003 and Dickson et al, 2009
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Inside the SN, neuronal loss appears to be uneven. Neurodegeneration is in fact greater in the nigral calbindin D28K compartments termed nigrosomes (N1>N2>N4>N3>N5) than in the matrix (Figure 10) and follows a stereotyped spatiotemporal progression related to disease duration [77]. This specific pattern of neuronal loss is consistently observed across PD patients, and differs from those resulting from normal aging or other neurodegenerative disorders affecting the SN [74]. On the opposite, cell losses, which occur unevenly among the four other midbrain dopaminergic neuronal populations (medial and medioventral, A8, substantia nigra pars lateralis, central grey substance), vary among individuals and do not seem to correlate with disease duration (Figure 10). These observations indicate that the selective and severe DA neurodegeneration observed in the SN is related to the underlying PD pathological mechanisms, whereas DA neuronal loss occurring in other midbrain regions rather reflect some atypical forms of the disease, ageing or co-morbid degenerative processes [78].
Figure 10. Loss of DA-containing neurons in PD:
regional and intra-nigral patterns. The midbrain is schematically represented at an intermediate transverse level, with a coloric scale indicating the estimated amount of cell loss in PD (least = blue; most
= red) in the different DA-containing neuron subdivisions. Within the SNpc, neuronal loss appears to be consistently greater in the calbindinD28K-poor regions termed nigrosomes (N) comprising 40% of neurons, than in the matrix (M)..A8 = Dopaminergic cell group A8; CGS = central grey substance; CP = cerebral peduncle; M = medial group; N = nigrosome;
RN = red nucleus; SNpd = substantia nigra pars dorsalis; III = exiting fibres of the third cranial nerve.
Image from Damier et al, 2003.
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At a late pathological stage, when about 80% of striatal dopaminergic terminals and 50% of the 450’000 dopaminergic cell bodies in the SN have been lost, the first PD motor symptoms become apparent [66, 79]. The delay in clinical onset is explained by pre- and post-synaptic compensatory mechanisms at work in the nigrostriatal dopaminergic system, incuding increased DA metabolism (i.e. turnover) and density of post-synaptic striatal D2 receptors [80]. After the disease onset, the rate of nigrostriatal terminal loss is estimated at 10% per year according to 18-fluorodopa PET [81], correlating with a worsening of motor symptoms.
2.1.2. Beyond the SN
Neuronal loss and LB formation is however neither confined to the midbrain and the SN, nor restricted to dopaminergic neurochemical system. A small number of dopaminergic neurons can be found outside the mesencephale, and some of them, in the hypothalamus or bone marrow, seem to be spared by the pathological process [82]. In contrast, those found in retina [83] and enteric nervous system [84] are partially damaged, which could account for some of the visual and digestive troubles observed in PD. Widespread neuronal loss is also found in noradrenergic (locus coeruleus), cholinergic (dorsal motor nuclei of the vagus, basalis nucleus of Meynert), serotoninergic (raphe nuclei) neurons, olfactory bulb, autonomic system and finally in the neocortex at late stages of the disease [85]. Clinico-pathological correlations indicate that the neurodegenerative process extension beyond the BG structures may be responsible for numerous signs not attributable to nigrostriatal degeneration such as sleep disorders, dementia, depression and autonomic dysfunction [86]. Thus, although degeneration seems to be more acute amongst nigral dopaminergic neurons, which account for the major motor features, neuropathological studies indicate that PD is a much wider multisystem disorder affecting the central and peripheral nervous system.
30 2.1.3. Braak staging of PD
PD pathology requires years to reach its full extent in the nervous system and the temporal relationships of the lesions are still not well established. By studying brains with PD and incidental Lewy pathology, Braak et al. defined six neuropathological stages of PD, with stage 1 and 2 being pre-motor stages and the next four being motor stages [85, 87]. The authors predicted that PD pathology, assessed by intracellular deposition of aggregated α-SYN, follows a stereotyped and selective caudo-rostral progression within vulnerable structures of the CNS. The disease begins in the dorsal motor nucleus of the vagus nerve, in the olfactory bulb and olfactory anterior nucleus, ascends in the brainstem to reach the raphe nuclei and the locus coeruleus before affecting the SN in stage 3. The presymptomatic phases 1 and 2 concord with the observations than non-motor symptoms such as depression, smell dysfunction, sleep disorders or autonomic disturbances may precede the development of motor features [88, 89]. Then, in stage 3-4, lesions in selective structures of the midbrain and forebrain become progressively more pronounced and the motor disturbances initiate for most individuals. Finally, in the late stages, the disease enters the temporal mesocortex and eventually the neocortex. Clinically, this is in accordance with late-stage PD often characterized by impaired cognition. According to this view, PD pathology does not start in the SN, whose involvement is only a step in a much larger multisytem disorder encompassing severe damage to autonomic, limbic, somatomotor as well as cognitive systems and resulting in charasteristic motor and non-motor manifestations.
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Figure 11. Progression of intraneuronal Lewy pathology in PD. Braak and co-authors proposed a neuroanatomically based staging for sporadic PD, relying on the pathological spread of α-SYN deposits throughout the nervous system. A and B schemes depict the topographic predictable sequence of the lesions occurring during the six defined PD stages, with the gradual increase in severity represented by darker degrees of shading (A, B). Lesions in the SN (stage 3) mark the onset of the symptomatic motor phase. Image A from Braak et al., Cell Tissue Res 2004 and Image B from Doty RL et al., 2012. Nat. Rev.
Neurol.
However, the predictive validity of Braak’s concept of neuropathological staging has been somehow disputed as it does not seem to correlate with PD clinical severity (Hoehn and Yahr stage) and duration [90]. In fact, there is a considerable variability in the temporal sequence and topographical distribution of Lewy pathology among patients. The relationship between Lewy pathology and neuronal dysfunction or death is still uncertain, representing an additional challenge for Braak’s hypothesis. For example, a significant proportion of genetic PD cases caused by LRRK2 mutations does not exhibit Lewy pathology although they demonstrate
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massive nigral degeneration [91]. Conversely, incidental Lewy pathology can be observed in clinically intact cases that may represent a pre-clinical PD stage. It is thus unclear if LB themselves are the pathological entities interfering with normal cell function, if they represent a cytoprotective mechanism such as aggresomes or a failed attempt to eliminate cytotoxic proteins such as misfolded α-SYN. In the particular case of SN, the pattern of cell loss and Lewy pathology significantly correlate with the disease duration and the severity of the motor symptoms [90]. The percentage of LB-bearing nigral cells appears to be stable over time (3.6% in average), suggesting that they are eliminated as the disease progresses when the afflicted neurons die. In the SN at least, LB may be closely related to nigral neuronal loss [92].
To conclude, although Braak’s staging might require further clinical and pathological validation, this hypothesis is still widely accepted as it broadly concurs with clinical observations and might be accurate in about 80% of the cases [93]. A more sensitive procedure might include neurodegeneration patterns in addition to Lewy pathology to define PD stages.
2.1.4. Principle of PD progression: a prion-like hypothesis?
Recent studies suggest that a prion-like cascade could directly be responsible for LB spread within the nervous system in PD, through a neuron to neuron transmission and propagation of misfolded or aggregated α-Syn (Figure 12). Much evidence indicates that α-SYN might behave like the protein prion (PrP) as they share many similarities: i) both can undergo an aberrant conformational change from a native α-helix rich to a β-sheet conformation which promotes their self-aggregation, ii) their misfolded protein form is recognized to be toxic and induce neurodegeneration, iii) their protein aggregates can act as “seeds” to recruit and promote the misfolding of wild-type proteins [94]. This hypothesis has first been highlighted by the recent discovery that fetal mesencephalic cells grafted into the brain of PD patients 11-22 years earlier contained classical LB [67, 69, 95]. This was an unexpected finding as until then, LB had never been found in such young neurons. One possible explanation for this scenario was
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that misfolded α-SYN was transmitted from affected host neurons to healthy transplanted neuron, where it recruited normal α-SYN to misfold. Other findings derived from tissue culture and transgenic animals demonstrated cell-to cell transfer of α-SYN inducing pathological changes and cell death in the recipient [96]. Recently, Luk and co-authors demonstrated the propagation of pathological α-SYN aggregates throughout the CNS of young asymptomatic α-SYN transgenic mice inoculated with mice-derived or synthetic α-SYN fibrils, leading to a Parkinson's disease-like syndrome [97].
Figure 12. Prion-like aggregation and transmission mechanisms in PD. Native prion (A) or α-SYN (B) molecules (green spheres) can both undergo conformational changes leading to misfolded protein forms (red cubes). These abnormal proteins can induce the misfolding of native proteins and when accumulating, the formation of aggregation intermediates such as prion rods or α-SYN fibrils which will finally develop in PrP amyloid plaque or LB respectively. Intracellular protein oligomer/aggregates can reach neighboring cells by different ways schematized in C. They can be released from neurons by exocytosis or after cell death before being taken up by adjacent neuronal cell bodies or terminal axons. At that point, they might be transported by anterograde or retrograde transport and spread throughout the nervous system.
The transmission of LB pathology by a prion-like mechanism through anatomically linked neuronal network might explain the sequential and predictable topographical progression of PD observed by Braak and co-workers. The latter suggested that the pathological process may be initiated by an unknown pathogen from the environment, which could enter the brain through a nasal or digestive route and spread through pathways composed of long unmyelinated axons [98]. Whether the formation of α-SYN aggregates in the olfactory bulb or enteric nervous system is sufficient to initiate PD is still elusive [99].
